Substance P and Neurokinins

In 1931[1] von Euler and Gaddum reported the presence of a hypotensive substance in extracts of brain and intestine that could stimulate the contraction of various isolated smooth muscles. The active principle in these preparations, a peptide, was later termed substance P. In 1970 [2] Chang and Leeman isolated a sialogogic peptide and soon thereafter characterized it as substance P by multiple biological and chemical criteria.

We have cloned the rat and human genes encoding substance P. Their structure (Fig. 1) is very similar to that of the bovine gene [1], i. e., they have seven exons with the third exon encoding substance P and the sixth exon encoding substance K. The exons (2–6) which have predominantly coding sequences are well conserved (89–99%) in nucleotide sequence while the exons containing predominantly noncoding sequences (1 and 7) are less conserved (57–72%). Although the introns are well conserved in size there is little sequence conservation between the rat and human introns. The region immediately in front of the first exon, i.e., the presumptive promoter region, has an unusually long (360 bases) conserved (84%) region. The precursor proteins encoded by the genes are 130 (rat) or 129 (human) amino acids and differ at only 6 amino acid positions. The 25 amino acids (excluding the Lys-Arg sequences at both ends) which are located between the substance P and substance K peptides are completely conserved between rat and human and have only a single conservative (isoleucine to leucine) substitution in the bovine precursor. This conservation suggests that the intervening peptide has an important function.

We have investigated the expression of the rat preprotachykinin I (PPTI) gene responsible for the production of substance P (SP), substance K (SK) and neuropeptide K (NPK) by using a variety of biochemical and molecular biological strategies. In this report we shall summarize our results concerning the analysis of the rat PPTI gene, molecular cloning of various mRNAs derived from this gene, hybridization analysis of the steady-state expression of the mRNAs in the rat striatum, the structure of three rat preprotachykinins and initial studies on the regulation of striatal PPTI gene expression. We shall not discuss PPTII gene expression, which is responsible for the production of β-neurokinin, a third mammalian tachykinin.

The prominent substance P (SP) projection from the striatum to the substantia nigra is thought to play an important role in the regulation of basal ganglia function and the response to dopaminergic and other pharmacological agents. However, the physiological and pharmacological significance of this striatonigral tachykinin system has been difficult to assess, since no useful measures of SP turnover have been available. Previous studies have shown that the repeated administration of dopamine (DA) antagonist (antipsychotic) drugs decreases the concentration of SP in the substantia nigra [1–5], although the relationship between SP concentration and SP turnover remains obscure. It has recently been demonstrated [6,7] that two mRNAs derived from one preprotachykinin (PPT) gene by alternate RNA splicing encode for either SP alone (αPPT mRNA) or both SP and the newly discovered tachykinin [8,9] substance K (SK) (βPPT mRNA). Thus, the biosynthes is of these two tachykinins could be differentially regulated in response to DA antagonists or other stimuli. In the present experiments, striatonigral SP and SK immunoreactivities were characterized using specific radioimmunoassays (RIAs) coupled to high performance liquid chromatography (HPLC). The acute and chronic effects of the antipsychotic drug haloperidol on the levels of striatonigral PPT mRNA (as well as SP and SK peptide levels) in single rat brain striata were quantitated as a dynamic index of tachykinin turnover.

Substance P (SP) and Substance K (SK, also known as neurokinin alpha) are two mammalian tachykinin peptides which are widely but selectively distributed throughout the central and peripheral nervous systems (1). These peptides are also found in endocrine tissues, in particular, in the anterior pituitary gland and their content within this gland can be regulated by thyroid hormone status (2,3). The present studies were performed to determine if the preprotachykinin (PPT) gene which encodes both SP and SK (4) is expressed within the anterior pituitary gland of adult male rats and if so, whether PPT messenger RNA. (mRNA) abundance can be regulated by thyroid hormone status.

From a phylogenetic point of view, the progress of tachykinin research has been somewhat uneven. The pioneering studies of Erspamer and co-workers (reviewed in [1])were carried out primarily in amphibia and numerous peptides were isolated from the skin of frogs. Eledoisin, however, was purified from the salivary gland of the Mediterranean octopus, Eledone, a highly developed mollusc. Interest in the diversity of the tachykinin family has increased enormously in recent years with the realization that mammalian nervous tissue contains, in addition to substance P, peptides homologous to the amphibian peptide kassinin i.e. neurokinin A, neurokinin Band neuropeptide K (reviewed in [2]). Previous work in the laboratory has led to the development of region-specific radioimmunoassays for substance P [3,4] and neurokinin A [5]. In this study, these assays are used together with HPLC separation to partially characterize the tachykinin-like peptides in tissues from some representative lower vertebrates: Rainbow trout (teleost), dogfish (elasmobranch), Atlantic hagfish (cyclostome) and sea squirt (protochordate).

Tachykinins are rapidly metabolised by tissue preparations and their inactivation is believed to occur through hydrolysis by cell-surface ecto-peptidases [1]. Enzymic and immunocytochemical studies in our laboratory have previously implicated endopeptidase-24.11 (‘ enkephalinase’, EC 3.4.24.11) in the metabolism of substance P [2–4]. The ability of pig striatal synaptic membranes to hydrolyse substance P was abolished by phosphoramidon and by an antiserum to the endopeptidase [2]. Furthermore, substance P was the best substrate identified for the enzyme in vitro [3]. We have also been able to demonstrate immunohistochemically the co-localisation of endopeptidase and substance P in specific brain regions, including globus pallidus and nucleus interpeduncular is [4]. More recently, neurokinins A and B have also been shown to be good substrates for endopeptidase-24.11, implicating this enzyme in their metabolism [5,6].

Substance P is synthesised from two precursors, α and β preprotachykinin (PPT), which are derived from a single gene by differential RNA splicing [1,2]. We have recently elucidated the cDNA sequence of human β-PPT. The human β-PPT polypeptide contains, in addition to a region encoding SP, the sequences of neurokinin A (NkA) and of neuropeptide K (NpK), an N-terminally extended form of NkA first isolated from porcine brain [3]. We are presently searching for novel fragments of α- and β-PPT which may exhibit biological activity. Rat brain contains a 5,500 Mol. Wt. form of NkA-like immunoreactivity which apparently contains antigenic determinants related to both SP and NkA. This peptide may represent an intermediate in the post-translational processing of β-PPT.

In dissociated cultures of neonatal rat nodose ganglion (vagal sensory) neurons, there is a steady net increase of 15–20 pg SP per ganglion equivalent per day. SP content but not neuron survival is enhanced by NGF. SP is released durtng exposure to 50 mmol potassium (K+) or the sensory neurotoxin, capsaicin (10 umol). These neurons in culture exhibit properties analogous to vagal sensory SP-containing neurons in vivo and may prove useful for the study of neuropeptide regulation in this visceral afferent nerve.The factors regulating the synthesis of SP in and its release from vagal sensory neurons are largely unknown. To study this regulation, we have developed cultures of neonatal nodose ganglion (NG) neurons. The cultures synthesize immunoreactive SP in amounts similar to vagal neurons in vivo and release SP in response to capsaicin (CAP), the specific sensory excitatory neurotoxin.

Immunocytochemical studies [1–3] have demonstrated that the guinea pig small intestine is supplied with a complex network of substance P-containing neurons. Extrinsic neurons project to the submucosal blood vessels and to the submucous ganglia. Intrinsic neurons in the submucous ganglia supply the villi and projections from the myenteric plexus supply most regions of the gut. Substance P-immunoreactive cell bodies are most numerous in the myenteric plexus. Recent studies have shown that extracts of guinea pig ileum contain neurokinin A and neuropeptide K [4] and these peptides have been identified in the same population of spinal ganglion cells and capsaicin-sensitive nerve fibres in the guinea pig ureter, inferior mesenteric. ganglion, heart and lung [5]. Biosynthesis in vitro of substance P [6] and neurokinin A [7] has previously been demonstrated in rat dorsal root ganglia and experiments in vivo have shown de novo synthesis of substance P in the rat corpus striatum and its transport to the substantia nigra [8]. This study extends this previous work to demonstrate in vitro biosynthesis of substance P, neurokinin A and neuropeptide K in enteric neurons of the myenteric plexus of the guinea pig ileum.

Substance P (SP) and substance K (SK), two structurally related neuropeptides belonging to the tachykinin family, are derived from a single preprotachykinin (PPT) gene [1,2]. Recent evidence has suggested that these peptides are probably co-localized within the striatonigral tract [3]. Interactions of SP and dopamine (DA) in the basal ganglia are well known although the exact mechanisms are unclear [4]. DA antagonists decrease striatonigral tachykinins and striatal PPT mRNA levels [5–7; Bannon et al., this volume]. However, there are conflicting reports on effects of DA agonists on striatonigral substance P concentrations [7,8]. No data has been published on the effects of DA agonists on the levels of striatonigral SK or PPT mRNA. The present study attempts to clarify the effect of DA agonists on striatonigral SP and SK biosynthesis.

Poly A mRNA extracted from human carcinoid tumour tissue was used to direct protein synthesis in a cell-free translation system, in the presence of rabbit reticulocyte lysate. Approximately 4.5% of radioactively-labelled translation products crossreacted with a SP antiserum.

Chronic treatment of rats with the neuroleptic drug haloperidol, a dopamine receptor antagonist, decreases the concentration of substance P (SP) in the substantia nigra (SN). This drug-induced reduction in peptide concentration was found to be concomitant with the reduction of the mRNA in the striatum which encodes SP. The mRNA encoding substance K (SK), a related tachykinin peptide, was similarly reduced by haloperidol, whereas striatal enkephalin-mRNA was elevated. The results demonstrate that pharmacological blockade of dopamine receptors can differentially affect neuropeptide gene expression.SP-mRNA was localized by in situ hybridization. Brain structures that gave positive hybridization signals were caudate, premammillary area and arcuate nucleus of the hypothalamus, central nucleus of the amygdala, nucleus accumbens, and medial habenula.

The evidence for the existence of multiple classes of receptor for the naturally occurring tachykinins has been reviewed previously [1–4]. Our original proposal for a sub-division of SP-P and SP-E receptors [1] was based on pharmacological data. showing differences in the relative potencies of tachykinins in different peripheral tissue bioassays. These differences have since been confirmed in other laboratories [5,6]. More recently. several different radiolabelled tachykinin derivatives have been used to characterise tachykinin receptors in peripheral tissues and in CNS. Using binding studies [7–10]. Such studies have suggested that there may be three rather than two types of tachykinin receptor binding sites in mammalian tissues. and some of these findings will be reviewed here and by the contributors to this symposium session.

Based on the different rank order of potencies as well as the lack of complete cross-tachyphylaxis observed among different tachykinins and their analogues in several biological assays, we have previously proposed the existence of multiple subtypes of SP receptors in different tissues [1–3]. For the SP-P subtype, both physalaemin (PHY) and SP are active at nanomolar concentrations with eledoisin (E) and kassinin (KAS) more or less equipotent. On the other hand, for the SP-E subtype, KAS, E and neurokinin A (NKA) are active at nanomolar concentrations and they are several hundred times more active than PHY or SP. The distinction between SP-P and SP-E system was further delineated by the development of a SP-P selective agonist, SP-O-methyl ester (SPOMe), which is 100–1000 times less potent than substance P on SP-E systems [4].

Three different tachykinins have been identified in the rat CNS, substance P (SP), neurokinin A (NKA or substance K) (1) and neurokinin B (NKB or neuromedin K) (1,2). Since there is evidence of multiple receptors (3) for the tachykinins particularly in the periphery, much interest has been focused lately to determine whether each tachykinin binds to a corresponding specific binding site in the CNS.

Early attempts at classification of tachykinin receptors were based on the finding that various tachykinins displayed distinct rank order of potencies when tested by different bioassays. Iversen and colleagues [I] identified two profiles of agonist potencies: the SP-P type in which physalaemin and substance Pare slightly more potent than eledoisin and kassinin, and a second profile, designated SP-E type, which is characterized by considerable higher potenices for eledoisin and kassinin than for substance P and physalaemin. Although this classification was found useful and stood the test of time, several lines of evidence suggested the existence of yet a third tachykinin receptor. The discovery of neurokinin A and neurokinin B, which together with substance P constitute the mammalian tachykinin family of peptides, raised the possibility that each tachykinin acts as a preferred ligand for a particular receptor Subtype. Binding studies using radiolabeled substance P, eledoisin and neurokinin A revealed unique patterns of distribution in different regions of the central nervous system and distinct profiles of tachykinin potencies in displaying each tachykinin radioligand. It should be pointed out however that the naturally occuring tachykinins are not highly selective and act on all tachykinin receptors. Also many tissues may possess a mixture of different tachykinin receptors and thus deviate from the classical SP-P and SP-E types.

P-type and K-type tachykinin binding sites exist in variable proportions in urinary bladder of different rodent species with hamster bladder containing predominantly high amounts of K-type sites [1; Burcher and Buck, this volume]. In the present report, 125iodohistidyl₁-SK (ISK) has been employed as the radioligand to examine in detail the K-sites in hamster bladder.

Substance P is one of the most potent vasodilators known [1] and the vasodilatation in some dog, cat and rabbit arteries is endothelium-dependent [2]. Endothelium-dependent relaxation by Substance P has since been shown in rings of dog common carotid arteries [3], pig and dog coronary arteries [4] and in vivo in dog femoral artery [5]. This vasodilatation may be mediated by release of endothelium-derived relaxing factor (EDRF) [2]. In the study described here, we have used the techniques of in vitro radioligand binding and autoradiography [6] to detect the localization of [125I] Bolton-Hunter substance P (BHSP) to sections of blood vessels with the endothelium intact and to similar preparations with the endothelium removed.

In recent years. substance P and related tachykinins have been shown to stimulate inositol phospholipid hydrolysis in a variety of tissues, including rat salivary gland [1], guinea pig ileum longitudinal muscle and rat hypothalamus [2]. In these tissues substance P, eledoisin and substance P methyl ester possess similar nanomolar potencies at inducing this response suggesting that inositol phospholipid breakdown is associated with SP-P receptor activation [2]. However. in slices of rat ileum longitudinal muscle. eledoisin is considerably more potent than substance P at stimulating inositol phospholipid hydrolysis suggesting that in some tissues this response may be mediated by a SP-E receptor [3]. In order to investigate further the association of SP-E receptors with inositol phospholipid breakdown, we examined tachykinin-induced inositol-l-phosphate production in slices of hamster urinary bladder, a tissue whose contractile response exhibits a typical SP-E receptor profile [4].

The three-dim-nsional structure of a peptide deduced from conformational analyses (nuclear magnetic resonance and circular dichroism spectroscopies) and/or from energy calculations should provide an insight into the structural requirements of the binding protein, if this structure has any relevance to the conformation which interacts with the binding protein. A decisive contribution to overcome this problem has been the design of constrained analogues of the peptide which simulate the predicted conformers. Indeed such rigidified analogues, if active, must contribute to a more accurate description of the bioactive conformation[l]. We have established, using NMR and CD spectroscopies, that the three-dimensional structure of SP is strongly influenced by its environment. The main features of the conformation model we have proposed are the flexibility of the N-terminal Arg-ProLys, the a-helical folding of the core of SP, Pro-Gln-Gln-Phe-Phe, and the folding of the C-terminal carboxamide towards the primary amides of Gin5 and Gln6 [2] The validity of this model has been checked by the means of constrained cyclic analogues of SP. Therefore, we have synthesized two groups of disulfide bridged analogues of SP.

The knowledge of binding-site topography is an essential step in the understanding of a receptor-substrate recognition process. However, the direct study of such complex is presently limited in application. Nevertheless, a binding site can be considered as being the imprint of the three-dimensional structure of its substrate. Taking into account the associative properties of the substance P to phospholipids and the aggregation behaviour of the tachykinins, we propose a two-step binding mechanism of these peptides to their receptors. First, the substrate binds to the phospholipids component of the membrane. This is followed by a two-dimensional diffusion of the tachykinins and of its receptor on the surface of the membrane, resulting in the formation of the substrate-receptor complex.

The kinetic effects of conformational selection will depend on the mechanism by which substance P (SP) binds to its receptor. There are two contrasting models which have been proposed for SP: (1) the ‘zipper’ model1; and (2) the ‘lock-and-key’ model2. To understand the binding mechanism in more detail, conformationally constrained analoqs involving the 5–11, 6–11, 5–9, and 5–10 segments were previously designed1,3,4,5 to duplicate the proposed conformation necessary for binding and signal transduction6,7,8,9. We now have prepared several restricted analogs to allow the 5–8 segment of SP free rotation while restricting the 9–11 C-terminal segment. These analogs demonstrate increased activity in the guinea pig ileum when compared to a previous series of analogs which were restricted from the Gln5 (or Gln6) position to the Met11 position1. Also, these analogs did not demonstrate any antagonist activity on the guinea pig ileum against SP or SK at 10 μM.

Substance P has been shown to noncompetitively inhibit nicotinic acetylcholine receptor (nAChR) responses in a number of experimental systems. These include among others, spinal Renshaw cells [1,2], adrenal chromaffin cells [3,4], a neuronal cell line (PCl2) [5,6], and a muscle-like cell line (BC₃HI) [6]. The effect of substance P has been shown to be specific for the nAChR; substance P fails to alter excitation responses by glutamate, aspartate, or muscarinic agoinsts [1,2], and fails to block secretion induced by depolarization by high potassium or veratridine [3,4]. It has recently been shown that the site through which substance P exerts this effect is probably different from sites previously described for the peptide [6].

NE-19550 is an orally active capsaicin analog whose analgesic and anti-inflammatory actions were recently described[1]. Recent studies have shown that the analgesic properties of this compound are not opioid in character, despite its good efficacy in the rodent hot-plate and tail flick assays for antinociception[2]. Further, it has been established that the anti-inflammatory analgesic activity displayed by NE-19550 in the carrageenan rat paw edema assay and other tests does not involve NSAID-like inhibition of prostanoid synthesis[3].

The undecapeptide substance P (SP), a neurotransmitter candidate (1–4), exhibits diverse pharmacological effects in many peripheral tissues as well as in the central nervous system (5). It has been shown that different classes of receptors for a given neurotransmitter are often coupled with distinct second messenger mechanisms to induce their biological effects. In that regard, previous reports have shown that the effects of SP in peripheral tissues (6, 7) and hypothalamus (6) are associated with an increased breakdown of phosphatidyl inositol (PI), suggesting that inositol 1, 4, 5-triphosphate may represent the intracellular second messenger coupled to SP receptors. Mantyh et al. (8) found that the rate of PI hydrolysis was proportional to the number of SP binding sites found in various areas of the rat brain. This suggests that these binding sites correspond to functional SP receptors. It has been shown that different tachykinin receptor sub-types have unique 1igand selectivity patterns and brain distributions (9, 10). However, it is not known if these other classes of tachykinin receptor sites are also coupled to PI turnover. Thus, we have studied the effects of different tachykinins (SP, NKA and NKB) on the PI metabolism in various regions of the brain.

Binding of 125I-Tyr8-substance P (125I-Tyr8-SP) to a plasma membrane enriched fraction (PM) from the circular muscle of canine small intestine was studied. The binding was fast, reversible and saturable with a K_D and B_max of 0.5 ± 0.06 nm and 742 ± 173 fmol/mg protein, respectively. 125Ieledoisin did not bind to PM. These results together with competition studies suggest that PH possesses SP-P receptors but not SP-E receptors.

The tachykinin peptides are potent contractile agents in mammalian urinary bladder. This action appears to be mediated predominant1y by a direct action on smooth muscle since it is generally not altered by atropine, guanethidine, mepyramine or indomethacin [1,2]. As in other smooth muscle preparations, it has been suggested that there is more than one type of tachykinin receptor in mammalian bladder since PHYS is more potent than ELE or SP in rat, guinea-pig, and cat bladder whereas in hamster, mouse, dog, pig, monkey, and human bladder HE and KASS are substantially more potent than SP or PHYS. In view of the recent identification of three distinct types of tachykinin binding sites in the CNS and periphery that may be receptors for each of the three mammalian tachykinins, SP, SK, and NK, we have examined tachykinin binding sites in hamster, rat, and guinea-pig urinary bladder [see 3,4,5].

Substance P (SP) has potent natriuretic and diuretic properties, increases renal blood flow and decreases renin release in dog kidney [1–3]. It has been suggested that some of the effects of SP in kidney result from bradykinin release [3] or increases in renal cAMP levels [4]. More recently it has been shown that SP mediated vasodilatation in renal arteries is endothelium-dependent [5] and is probably mediated by release of endothelium-derived relaxing factor (EDRF) [5,6]. In this study autoradiographic techniques with [ 125I] Bolton-Hunter substance P (BHSP) have been used to determine the localization of BHSP binding sites in dog kidney sections.

There is convincing evidence that substance P (SP) is an excitatory neurotransmitter to the muscle of the mammalian gastrointestinal tract [1]. SP also promotes water and electrolyte secretion across the mucosa of the small intestine of the guinea-pig, in vitro, and this effect is largely due to excitation of the secretomotor neurons in the submucosa [2]. Submucous neurons are surrounded by SP immunoreactive varicosities [3]. Many of these neurons are depolarized by SP via a mechanism similar to that responsible for the slow excitatory synaptic potentials that can be evoked in the same neurons by electrical stimulation of the internodal strands [4,5]. These results have led to the suggestion that SP mediates the slow excitatory synaptic potentials [4].

Autoradiographic binding sites for [125I]-Bolton-Hunter labelled substance P (BHSP) were investigated in the guineapig respiratory tract. Dense binding was observed on the smooth muscle of primary bronchi, secondary bronchi and terminal bronchioles. In the trachea, specific binding sites for BHSP were seen densely clustered over the trachealis muscle. Specific binding of BHSP to guinea-pig lung membranes was displaced by SP > SK > NK, indicating binding to a P-type site. No binding of BHSP was observed to arterioles in the lung. The presence of BHSP binding sites on guinea-pig respiratory smooth muscle is in accordance with the bronchoconstrictor actions of SP.

The presence of substance P (SP) receptors has been demonstrated in adult mammalian CNS in binding studies performed with synaptosomes or membranes using 3H-SP or 125I-Bolton Hunter SP (125I-BHSP) (1,2). Moreover, the precise regional localisation of these sites has been shown by microdissection and by autoradiography (3,4,5,6). On the other hand, 125I-BHSP binding sites were found on intact cells from the brain of embryonic mouse or rat. Particular culture conditions allowed to reveal the occurrence of 125I-BHSP binding sites on neurons. However, these studies did not exclude a localisation of these binding sites on astrocytes as well. The present study was performed to look for the existence of SP receptors on glial cells (astrocytes) in primary culture from new-born mice.

Tritiated and iodinated tachykinin peptide ligands bind to distinctly different types of sites in the CNS. Iritiated substance P (SP) and 125I-Bolton-Hunter reagent-labeled SP bind specifically to sites with higher affinity for SP and physa1aemin (PHYS) than for other tachykinins which has led to these being designated P-type sites. The biochemical regu1ation 0f these sites, their specificity, and their localization to CNS regions that are sensitive to the biological effects of SP suggest that the sites are SP receptors [see 1,2].

The existence of multiple tachykinin receptors is supported by experimental evidence [for recent reviews, 1–4]. For example, substance P (SP) substance K/neurokinin A (NKA), eledoisin (ELE) and neurokinin B (NKB) induce differential effects in bioassays and their respective binding sites are differentially distributed in central and peripheral tissues [1–4]. Thus, the existence of three classes of neurokinin receptors has been proposed [5,6]. The first type preferentially binds SP while the other two demonstrate more affinities for NKA, and ELE and NKB, respectively. However, the presence of these three classes of sites has not been investigated in human brain. We report here on the characterization and autoradiographic distribution of SP, NKA and ELE/NKB binding sites in human brain.

Discovered in 1931 by von Euler and Gaddum [1], substance P was the first neuropeptide to be described as a “substance, which lowers the arterial blood pressure of atropinized rabbits by peripheral vasodilatation, and also stimulates the tone and rhythm of the rabbit’s isolated intestine....”

The responses of cephalic blood vessels to the addition of substance P or substance K were compared in canine cephalic vascular tissues. Both tachykinins relaxed precontracted segments of common carotid and basilar arteries. The addition of SP caused significant homologous desensitization but not desensitization to the subsequent addition of bradykinin, calcitonin generelated peptide or sodium nitroprusside. Comparatively little or no homologous desensitization followed the addition of SP- [4–11], SK or physa1aemin.,SP pretreatment but not pretreatment with SK kassinin physa1aemin or e1edoisin attenuated the vasodi1ating response to substance K. SK did not however, induce tachyphylaxis to the subsequent addition of substance P. Taken together these data suggest that the unique N-termina1 peptide sequence of SP mediates the tachykinin-induced desensitization within canine vascular tissues. The extent to which this mechanism accounts for tachykinin-induced desensitization in other systems remains to be determined

In recent years huge evidence has accumulated indicating that neuropeptides are widely distributed in the lower urinary tract of various animal species(l). The aim of this study was to determine the relative ability of neurokinins (subs tance P, SP; neurokinin A, NKA; neurokinin B, NKB) and tachykinins (kassinin, KASS; eledoisin, ELE; physalaemin, PHYS) to activate motility and produce plasma extravasation in the rat lower urinary tract. The effects of neurokinins (NKs) and tachykinins (TKs) were compared to those of capsaicin, a drug known to stimulate neuropeptide release from primary afferent fibers (2).

Neurokinin A (NKA) and neurokinin B (NKB) are newly discovered mammalian neuropeptides, the chemical structures of which are closely related to tachykinin peptides (1). Both neurokinins have qualitatively the same pharmacological properties, such as contraction of smooth muscles, hypotensive effect and salivation (2). The peptides also possess electrophysiological characteristics like substance P (SP) though the efficacies are not equal (2,3). Furthermore, at least NKA is considered to be involved in pain transmission as is SP (4).

During the last years, huge efforts have been made to upgrade the available tachykinin antagonists. We and several groups have done design studies with purely synthetic permutation series [1,2], others have attempted mathematical predictions based on various physicochemical considerations [3] but in no case antagonists with pA₂-values above 8 were obtained or which satisfied all pharmacological criteria of selectivity and specificity.

Neurokinin A, eledoisin and substance P contracted strips of rabbit main pulmonary artery in a concentration-dependent manner with neurokinin A and eledoisin being 300fold more potent than substance P. Substance P methyl ester was inactive. Neurokinin A and substance P relaxed precontracted vascular strips provided that the endothelium was intact. The rank order of potency of the various agonists suggested the existence of a muscular SP-E type and possibly an endothelial SP-P type tachykinin receptor for the rabbit main pulmonary artery. Porcine coronary artery was devoid of excitatory muscular tachykinin receptors but contained an endothelial SP-P type receptor which, similar to that in the rabbit main pulmonary artery, mediated vasorelaxation. Stimulation of the muscular tachykinin receptor in the rabbit main pulmonary artery resulted in depolarization of the membrane of the smooth muscle cells. In the porcine coronary artery, activation of the endothelial tachykinin receptor produced hyperpolarization of the smooth muscle membrane in all probability through release of endothelium derived relaxing factor. Neurokinin A- and substance P-induced contractions of rabbit main pulmonary artery were not susceptible to inhibition by calcium antagonists and were not affected by pertussis toxin-induced inactivation of a guanine nucleotide binding regulatory protein (N.). It is, therefore, suggested that activation of the smooth muscle tachykinin receptor leads predominantly to a release of calcium from the sarcoplasmic reticulum and that N. is not involved in signal transductioft between the tachykinin receptor and the subsequent steps initiating contraction.

Until recently, substance P was the only member of the neurokinin family of peptides of mammalian origin. Thus, sub-classification of neurokinin receptors as well as synthetic efforts to create a neurokinin antagonist utilized substance P as the prototypic peptide [1]. Within the last three years, however, two additional mammalian peptides have been isolated. sequenced, purified and shown to have biological activities characteristic of the neurokinin family: neurokinin A and neurokinin B [2–4].

Substance P and its homologues belong to a large family of peptides widely distributed in the animal kingdom, from invertebrate to man [1]. The term neurokinin has been suggested [2, 3] to indicate the peptides of mammalian origin (neurokinin P, A and B), while the term tachykinin designates the agents occurring in lower species, for instance physalaemin, eledoisin and kassinin [1, 4].

The characterization of receptors for neurokininsis pursued today by using primarily agonists and by applying the first criterion recommended by Schild [7] in 1973. As shown by the tracing of a precedent paper (D’Orléans-Juste et al. [2]), the effects of neurokinins on isolated vessels, (which represent the most selective pharmacological preparations for neurokinins) are rather persistent and stable: therefore these tissues cannot be used for desensitization experiments. Antagonists for neurokinins are still in an early phase of development and may be used for characterization of one of the three receptor types on which neurokinin act, namely the NK-P, but not for the others [3].

The analysis of the primary structures of three neurokinins, namely neurokinin P (NKP), neurokinin A (NKA) and neurokinin B (NKB) reveals not only major differences in the chemical structure of these peptides at their N-terminal region but also striking similarities of their carboxyl extremities. The N-terminal major difference is represented by the presence of two positively charged residues (ArgI and Lys3) in NKP while NKA contains only one such residue (Lys2) and NKB has none. The C-terminal extremities of the three neurokinins are very similar since all contain the tripeptide Gly-Leu-Met-NH₂.

In smooth muscle, a rise in free intracellular calcium is considered to be of primary importance in determining the degree of actin-myosin interaction, and resultant contraction, although other processes may also operate. Contraction due to hormone-receptor interaction utilises the two main sources of calcium, namely extracellular calcium and stored intracellular calcium, which can be made available by some combination of three mechanisms: (1) namely (a) entry of extracellular calcium via voltage-operated channels (VOCs). (b) Entry of extracellular calcium via receptor-operated channels (ROCs). (c) Release of intracellular calcium from storage sites such as the sarcoplasmic reticulum (SR) within the cell. The extent and efficiency of different agonists in using these three calcium translocation mechanisms varies depending both on the particular type of smooth muscle concerned, and also the agonist-receptor system mediating the response.

There are many instances where these spasmogens applied to smooth muscle preparations have both a direct action on the smooth muscle cells, as well as an indirect action through release of some other mediator [1] . It is obviously of importance when studying a system where an indirect action is apparent, to identify the mediator involved, since analysis of mixed responses is not otherwise possible in receptor classification or mechanistic studies.

We have recently synthesized a neurokinin B analog, [D-Pro2,D-Trp6,8,Nlel0]NB (DPDTNLE-NB), which shows selective competitive antagonism of neurokinin B with minimal agonist activity [1]. Our objective in this study was to use this antagonist and two substance P analog antagonists (Spantide and DPDT-SP) to systematically investigate neurokinin receptors in the guinea pig ileum.

The classification of tachykinin receptors into two subclasses, the SP-P and the SP-E receptors has been well established (1). Recently Laufer et. al. (2) characterized, in the guinea pig ileum, a third tachykinin receptor subclass, designated as SP-N receptor. The SP-N receptor is located on the enteric cholinergic neurons, and madiates the release of acetylcholine. We found that the SP analog (pGlu6,(N-Me)Phe8) SP_6-11 acts as a selective potent agonist for the SP-N receptor (EC₅₀=0.5 nM) while its potency for the SP-P receptor is much lower (EC₅₀=500 nM). Conceivably N-methylation of Phe8 in the hexapeptide sequence of substance P (SP), induces selectivity toward the SP-N receptor. Therefore we set to probe the constraints at the nitrogen of Phe7-Phe8 amide bond by changing the alkyl group at this site.

Substance P, when injected intra-arterially into the small intestine of the anesthetized dog during phasic activity, produces a dose dependant inhibition of the phasic activity. This inhibitory response was abolished by elimination of field stimulated activity by intra-arterial tetrodotoxin, but reduced by atropine treatment (on activity induced by intra-arterial motilin). This suggested that Substance P acted by releasing several inhibitory transmitters, one of which was probably acetylcholine acting on inhibitory autoreceptors on cholinergic nerves [1]. The identity of the other transmitter/ transmitters is unknown.

The concept of multiple neurokinin receptors was first suggested in 1982 [l]; two subtypes were theorized: SP-P and SP-E. Recent work supports the existence of three receptors, one for each known mammalian neurokinin (substance P [SP], neurokinin A [NKA], neurokinin B [NKB]) [2].

The actions of several tachykinins [(substance P (SP), neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), kassinin (KAS), and eledoisin (ELE)] were investigated on transversally cut strips of the pig coronary artery. SP was used as the reference tachykinin.

Many structure-activity studies of C-terminal partial sequences of Substance P (SP) have established that the major potency of this peptide corresponds to the hepta or octa fragments [1,2]. Modifications of such hydrophobic peptides to enhance their activities were attempted through substitutions by sugar moieties or introduction of sulfonium or sulfoxyde groups into the methionine, but did not increase the affinity [3,4].

We have assessed the relative vasodilatory potencies of substance P, deca-substance P and nona-substance P in the canine forelimb. In pentobarbital anesthetized dogs (35 mg/kg, i.v., n=7) the right forelimb was perfused at constant flow via the brachial artery. We measured systemic, forelimb perfusion, skin and muscle small artery and skin and muscle small and large vein pressures. Brachial and cephalic blood flows were measured by timed collections of the venous effluents. The peptides were infused intra-arterially in sequentially increasing dosages of 75, 150, 300, 750 and 1500 ng/min for ten minutes at each infusion rate. Each infusion rate immediately followed the previous one. However, pressures were allowed to return to steady-state values after completion of each peptide infusion before proceeding to the next peptide. The order of peptide infusion was varied for each experiment. Substance P infusion resulted in a 48, 8, 13, 16 and 10% decrease in forelimb perfusion pressure and a 24, 10, 13, 16, and 7% decrease in systemic pressure at the above infusion rates, respectively. Infusion of deca-substance P produced 51, 6, 12, 20 and 15% decreases in forelimb perfusion pressure and 26, 6, 15, 23, and 17% decreases in systemic pressure. Nona-substance P decreased forelimb perfusion pressure 48, 6, 17, 18, and 13% and systemic pressure 15, 3, 11, 17, and 12%. Skin and muscle small and large vein pressures were only transiently altered during peptide infusion. The dilation produced by all three peptides in the forelimb was equally distributed between the skin and skeletal muscle circulations. These data indicate that substance P, deca-substance P and nona-substance P possess similar vasodilatory potencies in the canine forelimb.

Substance P (SP) evokes a vasodepressor effect when injected into the systemic circulation. This action is assumed to be due to a decrease in systemic vascular resistance since SP relaxes the dog isolated carotid artery [1] and increases blood flow in various vascular beds [2]. At doses above the ED₅₀, SP causes greater lowering of systolic than of diastolic blood pressure (BP) suggesting that high doses affect preload as well as after load. At still higher doses SP will evoke a pressor response [3] suggesting that SP is capable of increasing peripheral vascular resistance. The present study was directed at determining the effect of a high dose of SP on cardiovascular dynamics with the aim of determining its action on systemic vascular resistance and cardiac preload.

Antisera to neuropeptides have been used as antagonists of the actions of their respective neuropeptides in the central nervous system [1,2]. If effective, this approach might be particularly useful for determining the roles of neuropeptides for which there are no available antagonists. The present study was prompted by the poor efficacy of substance P (SP) antagonists in the central nervous system. It was considered useful to test SP antiserum in a simple system where the effect of interactions of known concentrations of antiserum and SP may be examined. The aim of this study, therefore, was to determine whether SP antiserum may be used as a chemical antagonist of exogenous and endogenous SP in the guinea-pig isolated ileum.

In an attempt to get immunological tools for studying neurokinins and their receptors, we developed two types of antibodies (Abs): first, both polyclonal and monoclonal antibodies against substance P(SP); secondly, antibodies directed against anti-SP antibodies, called anti-SP anti-idiotypic antibodies, and capable of recognizing the physiological receptors of SP.

On the basis of data obtained in smooth muscles [1] it was assumed that substance P (SP) analogues containing D-amino acids will antagonise the spinal actions of tachykinins. It was however rapidly realised that SP antagonists exert direct effects on the rat spinal cord. In fact, undeca or C-terminal octapeptide analogues of SP containing D-Trp in positions 7 and 9 or 7,9 and 10 cause an initial increase of the blood pressure followed by a long lasting fall to levels below 80 mmHg within 20–30 min when applied intrathecally (i.th.). In a number of experiments, the hypotension was large enough to cause death of the animal [2, 3, 4].

Substance P, an undecapeptide, is present in high concentrations in the dorsal horn of the spinal cord in thin afferent fibers (presumably C-fibers) associated with the transmission of noxious information [1,2]. Administered intrathecally, substance P has been reported to produce a transient hyperalgesia [3] and to induce behavioral responses suggested to be characteristic of noxious stimulation [4]. These pieces of evidence all support the suggestion that substance P has an excitatory role in neural transmission involving spinal nociceptive pathways.

There are similarities in the distribution of Substance P (SP) and enkephalins in the central and peripheral nervous systems. SP and methionine enkephalin (MEK) exhibited opposing effects on nociception, uptake of Ca++ ions by the nervous tissue, release of ACh and modulation of cholinergic function [1–3]. Peripheral branches of certain primary-afferent neurons liberated SP as a neurotransmitter in autonomic post-ganglionic cells and caused long-lasting increase in the excitability of ganghon cells to the primary transmitter, ACh [3]. The male genital system, its glands (seminal vesicles prostate, epididymis) and plexus (vesical, prostatic, cavernous) have rich autonomic and sensory or afferent innervation. However, little information is available about the role of SP in the male genital system.

The study of the roles of substance P and related neurokinins in periphery is particularly complicated by the fact that these peptides are present in a variety of neural and non-neural structures. Thus, substance P immunoreactivity of the adrenal gland is found in chromaffin cells and also in sensory neurons; in the carotid body, in glomic cells and afferent fibers; in the gut, in sensory afferents as well as in intrinsic networks and also in the so-called enterochromaffin cells of the mucosa. Much of the work presented in this section of the symposium refers to attempts to elucidate the specific participation of neurokinins derived from these differential sources on the physiology of peripheral systems. In this chapter, we will summarize some aspects of the organization and physiology of substance P containing peripheral sensory branches in the skin and autonomic ganglia.

A body of in vitro evidence indicates that substance P (SP) or a related tachykinin is involved in the physiologic regulation of peristalsis in the small intestine of the guinea-pig [1]. However, studies of the effect of SP on gastrointestinal motility in the rat in vivo have not been conclusive [2–4). The first aim of the present study was therefore to systematically investigate the time- and dosedependence of the effect of SP and neurokinin A (NKA) on gastrointestinal propulsion in the rat. The second aim was to provide evidence for a role of endogenous tachykinins in the maintenance of gastrointestinal motility in vivo by examlnlng the effect of a tachykinin antagonist on gastrointestinal propulsion.

The classical view of secretion from the adrenal medulla was that of a relatively simple system. Since the early 1930s it has been known that release of acetylchol ine (ACh) from the stimulated splanchnic nerve causes secretion of the catecholamines, adrenaline and noradrenaline, from adrenal chromaffin cells [1]. In addition to these acute effects on catecholamine release, long-term stimulation of chromaffin cells with ACh (in a time course of hours to nays) was later found to produce other biochemical changes in the cell. A well-studied example of this is the ability for long-term stimulation with ACh to induce synthesis of tyrosine hydroxylase (TOH, EC 1.14.16.2), the rate-limiting enzyme in catecholamine synthesis [2]. Thus ACh can produce two types of responses in chromaffin cells. These responses differ in their time courses (one acute and one long-term) and also in their ionic dependences (ACh-stimulated catecholamine release is strictly Ca 2t-dependent [3], while ACh-stimulated induction of TOH is not [4]).

The potent effect of various neuropeptides on vascular functions and their potential role in blood flow regulation have been increasingly recognized in recent years. The hypotensive effect of substance P (SP) was one of its defining characteristics and has been shown to result from peripheral vasodilation (1). Calcitonin gene-related peptide (CGRP) has been shown to be a potent vasodilator in the hamster cheek pouch and in human and rabbit skin (2).

In rat parotid gland protein secretion can be triggered by the activation of α and β-adrenergic, muscarinic, Substance P (SP) and Vasoactive intestinal Peptide (VIP) receptors. Vipergic and B-adrenergic induced secretions seem to be mediated by cAMP (1,2), whereas -adrenergic muscarinic and SP induced secretions would involve inositol trisphosphate (IP₃ ) and calcium (3).

Substance P (SP) is a known potent vasodilator (1, 2, 3). Olgart and his coworkers found SP-like immunoreactivity (SPLI) in nerves of the cat pulp suggesting the presence of SP (4). In addition, Gazelius and Olgart (5) demonstrated that electrical stimulation of the inferior alveolar nerve in cats caused a rapid increase in pulp blood flow which was followed by a prolonged reduction in pulp blood flow. The increase is thought to be mediated by SP released at the pulpal sensory nerve endings (6). However, administration of synthetic SP failed to increase pulp blood flow in cats; only when the vascular tone was elevated by electrical stimulation of the sympathetic nerve did the SP administration cause a pulp blood flow increase (5).

Until the recent discovery of two decapeptides, neurokinin A (NKA) and neurokinin B(NKB), substance P (SP) was the only tachykinin identified in mammalian tissues [1,2,3]. A common precursor for SP and NKA has been identified in bovine striatum [4] and the mRNA coding for these peptides has been found in sensory neurones [5] Using radiolabelled amino acids, co-synthesis of NKA and SP in rat sensory ganglia has been demonstrated [6]. It appears that SP and NKA are co-localized in certain primary afferent neurones while NKB is localized in interneurones or ascending pathways rather than in primary afferents [7]. The role of SP, released from the peripheral terminals of sensory C-fibres in the skin, has been studied in detail and there is convincing evidence for its involvement in antidromic vasodilatation and plasma protein extravasation [8]. It is likely, however, that a part of the neurogenic vascular responses that are now attributed to SP may, in fact, be mediated by other tachykinins.

Neurogenic inflammation refers to the vasodilation and plasma extravasation (PE) that occurs following release of neuromodulators, including tachykinins, from capsaicin-sensitive nerves [1]. Substance P (SP) has been the most widely studied tachykinin, but recently it has been demonstrated that Neurokinins A (NKA) and B (NKB) are also present in the peripheral and central nervous systems, especially in unmyelinated nerves and dorsal horn of the spinal cord [2]. PE appears to be effected by tachykinins acting directly on post-capillary venules as well as through release of mast cell derived amines in rat skin [3].

As ions move across epithelial sheets, they can do so either through the cells (transcellular) or between them (paracellular). Such transfers result in the development of trans-epithelial potential differences (P.D.’s) that are readily observed when these epithelia are set up in Ussing chambers, in vitro [1]. Alterations in the P.D.’s thus developed can be conveniently analyzed in terms of the modulation of either one or both of the above routes of permeation. Although a number of transmitters and hormones have been shown to modulate trans cellular ion movements, relatively few modulators of paracellular pathways have been described; these include, among others, cyclic AMP, Ca 2+, carbachol, pancreozymins, and prostaglandins [2–5]. Luminal addition of several peptides [6–9] produce marked effects on the isolated canine tracheal epithelium. Our studies reported here suggest that these pep tides (SP, tachykinins, bradykinin) could rapidly alter the conductance of the paracellular pathways.

Several investigators have reported that substance P (SP)-immunoreactive nerve fibers are present in the heart and around coronary blood vessels and intracardiac ganglion cells [1–3]. Experiments with capsaicin have provided strong evidence that SP in the heart and coronary vasculature is in primary sensory nerves [4,5]. These SP-containing afferent nerves could be components of pathways mediating cardiac pain and cardiovascular reflexes. Their anatomical localization also supports the suggestion that SP may be released from peripheral sensory nerve endings and affect coronary blood flow and intracardiac ganglia [2,3]. Thusfar, pharmacological studies have shown that SP is a potent coronary vasodilator [1].

It is highly probable that SP and NKA coexist in intrinsic neurones of the enteric nervous system [see 1]. In the present study, the effects of these two tachykinins on rat stomach motility were compared in vivo and in vitro.

Substance P (SP) has been identified within the lumen of the gastrointestinal tract. We have previously demonstrated that electrical stimulation releases SP from the guinea pig myenteric plexus[1]. Vagal stimulation releases SP into the lumen of the feline upper small intestine[2]. The possible physiological role of endoluminal SP remains unclear. The purpose of this study was to investigate if a physiologic stimulus, a high protein meal, provokes the intraluminal release of SP and whether the release is under cholinergic and/or adrenergic control.

Substance P (SP) belongs to the family of tachykinin which are structurally related peptides. They are mainly found in non-mammalian tissues with the exception of SP [1]. Two tachykinin related peptides, neurokinin A (NA), and neurokinin B (NB), have been discovered in mammalian tissues [see 2,3]. These mammalian tachykinins (NA and NB) are also structurally similar to SP with a conserved C-terminal (see Table 1). They also act on various SP-responsive tissues as that of SP, but with different pontencies [3]. The effect of these peptides on smooth muscle contractilities have been studied extensively [3], but information concerning their effects on endocrine secretions is scarce. The first objective of the present experiments was designed to test their effect on gastric somatostatinlike immunoreactivity (SLI) release. In addition, the effect of kassinin (K), another tachykinin which has not been found in rat tissues was also tested. SP has been shown to inhibit both basal and stimulated SLI release [4].

Over the past few years the number of substances shown to be present in vascular nerve fibres has increased dramatically. To early descriptions of efferent perivascular sympathetic fibres containing noradrenaline, afferent systems and other transmitter substances have been added. While purines have been suggested as transmitters in certain circumstances, it is the peptides which have received the greatest attention, particularly VIP, NPY, CGRP and substance P [1–4].

The biochemical agent(s) responsible for the carcinoid flush is still unknown. During 3 episodes of carcinoid flush induced by physical effort the following changes were observed in plasma: a) increase of substance P-like immunoreactivity; b) a minor rise of neurokinin A-like immunoreactivity; c) no change of blood and plasma serotonin. HPLC analysis of plasma extracts collected after the administration of streptozocin, inducing long lasting flushing, showed a marked increase of NKA and of substance P-like material and a minor rise of SP. It is proposed that these compounds take part in the pathogenesis of carcinoid flush.

A case of human adrenal pheochromocytoma, containing elevated concentration of substance P-like immunoreactivity (SP-LI) (217 pmol/g) is described. In the peripheral plasma, however, SP-LI was (8.2 pmol/l) similar to normal controls. HPLC of pheochromocytoma tissue extract revealed the presence of a major immunoreactive fraction eluting as authentic SP. Immunohistochemistry showed that approximately 30% of chromaffin cells were positive for SP-LI. We confirm that SP can be included among the various peptides which may be synthesized by pheochromocytoma.

Maintenance of the luteal phase and progesterone (P₄ ) secretion adequate to support nidation and pregnancy or pseudopregnancy in the proestrous rat requires intact innervation of the uterine cervix [1]. Stimulation of the cervix during copulation, or by experimental means, is conveyed via neural pathways to the hypothalamus [2,3] altering the gonadotropin secretion pattern [4,2] and facilitating the secretion of prolactin (Prl), a luteotropin in the rat, by the hypothalamo-hypophyseal axis [5,6,14]. This results in cessation of estrous cyclicity and stimulation of P₄ secretion by the corpus luteum [7,4,5], thus completing the neuroendocrine copulation reflex (response) [8]. Our previous results have demonstrated an extensive innervation of the uterine cervix by capsaicin- sensitive primary afferent nerves containing markers for substance P [9], and further, that groups of capsaicintreated (CAP) neonatal rats subsequently have a markedly reduced incidence of pregnancy following confirmed matings [10]. This suggested that the neural limb of the neuroendocrine copulation reflex consisted of primary afferent “c” type nerves.

Nerves in the female rat reproductive tract contain a number of neuropeptides in addition to catecholamines and acetylcholine; e.g., neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP) [1] and calcitonin generelated peptide (CGRP) [2]. Peptidecontaining nerves are perivascular, near the epithelium and in nonvascular smooth muscle. All of these substances are vasoactive and can influence nonvascular smooth musecle; some, such as SF, are in primary afferent nerves. Thus, these peptide-containing nerves could have important functions in the female reproductive system. In our continuing studies of the peripheral nervous system’s influence on reproductive tract function.we examined primary afferent nerves for other peptides besides SP; these include CGRP, cholecystokininoctapeptide (CCK-8), and substance K (SK, neurokinin A; hereafter referred to as NKA). We also examined the sensitivity of these nerves to capsaicin, a neurotoxin for primary sensory neurons.

Substance P containing neurons are present in the female reproductive organs. It has been reported that substance Plike- immunoreactivity (SP-LI) is decreased during pregnancy in maternal plasma as well as in umbilical cord plasma. In Sep-Pak CIS plasma extracts we found that SP-LI was not modified during pregnancy and both spontaneous and oxytocin-induced labour. Elevated levels of SP-LI were found in umbilical cord plasma and amniotic fluid. HPLC of maternal and umbilical cord samples indicated the presence of authentic SP. Somatostatin-like immunoreactivity was also increased in umbilical cord plasma and amniotic fluid.

In this keynote address I would like to present some recent results of our study on a substance P (SP) antagonist, namely Span tide (1). The experiments were carried out by M. Yanagisawa and myself using the isolated spinal cord of the newborn rat. Although I am afraid that these results might be more appropriate for presentation in Session 3, they are also related to sensory neurons, which are the subject of this Session 5.

Neurons at many levels of the neuraxis are excited by substance P administered microelectrophoretically from multibarrelled micropipettes. In the spinal cord, the polypeptide is located in abundance in the superficial laminae, and in nonmyelinated primary afferent fibres. It is released into a perfusate on stimulation of the afferent fibres. The release is blocked by the administration of morphine. These facts have led to the hypothesis that substance P may be an excitatory neurotransmitter, released form the terminals of unmyelinated primary sensory neurons. The observation that the excitatory effects are most noticeable on neurons with an excitatory input from peripheral nociceptors has further led to the suggestion that it is an important excitatory transmitter in the pain pathway.

In addition to the neurokinins, previous electrophysiological studies in our laboratory have focussed on the effects of purines including ATP. One of the effects of iontophorectically applied ATP is depression of dorsal horn neurones [1]. This effect is presumably mediated via conversion of ATP to adenosine with subsequent activation of P_l-purinergic (adenosine) receptors. During the course of the studies on the purines and on the neurokinin physalaemin, the serendipitous observation was made that the magnitude of the depressant response to ATP was apparently increased by pre-adminstration of physalaemin. Therefore, a study was done of the effects of neurokinins on the depressant responses to ATP, to AMP and to GABA.

Recent evidence suggests the presence of multiple tachykinins, i.e. substance P (SP), neurokinin A (NKA), neuropeptide K (NPK) and an eledoisin-like peptide (ELE) in capsaicin-sensitive primary afferent neurons [1]. Some of these neurons also contain calcitonin gene-related peptide (CGRP) [2]. Release of SP from collateral branches of primary afferents terminating in prevertebral ganglia [3] appears to be responsible for the generation of a non-cholinergic slow excitatory postsynaptic potential (EPSP) [4,5,6]. Here we present biochemical and electrophysiological studies on guinea-pig sympathetic ganglia.

The local application of noradrenaline (NA), clonidine and ST-91 to the spinal cord by intrathecal (i.t.) injection produces analgesia in a variety of test systems in a number of species (reviewed ref. 1). Alpha-2 adrenergic receptors are implicated in analgesia produced by these agents. The mechanisms by which these agents produce analgesia are not clearly defined. Electrophysiologically, there is evidence for both pre- and postsynaptic effects of NA [1].

Antidromic electrical stimulation of dorsal roots or sensory nerves causes vasodilatation presumably by the release of sensory neuropeptides [see 5]. Various methods have been used to study antidromic vasodilatation (AVD) in the rat, e.g. collection of venous outflow after stimulation of the saphenous [5] or mental nerve [1] or measurement of iodide disappearance rate from the dental pulp [2]. Data obtained by these methods suggest that AVD is modulated by pre junctional opiate receptors [4,6] and may in part involve histamine release from mast cells [1,5]. The involvement of histamine is controversial, as is the inhibitory effect of atropine [1,2,5]. We, therefore, reexamined the pharmacological modulation of AVD using the non-invasive technique of Laser Doppler flowmetry (LDF). Since this technique measures the velocity of moving blood cells, an increase of velocity can be interpreted as an increase in blood flow. The usefulness of LDF for studying blood flow has been demonstrated for various tissues including the skin [3,7].

Capsaicin sensitive neurons have been shown to mediate various autonomic and neuroendocrine reflex responses [1]. Destruction of afferent peptidergic C fibres by treatment of neonatal rats with capsaicin [2] was used to investigate the function of these neurons in (A) stress induced ACTH secretion and (B) in the regulation of adrenaline release by insulin induced hypoglycaemia.

Airway irritants are known to accelerate mucus flow rate in the airways after shortterm exposure in vivo in several species including man. These irritants include cigarette smoke, sulfur dioxide and sulfuric acid. The similarities between the effects of various irritants suggest a nonspecific response (1). The purpose of the present study was to investigate possible mechanisms behind this response.

Of the diverse roles that have been proposed for neuropeptides, modulation of the activity of other neurotransmitter receptors is particularly interesting in view of the coexistence of peptides and conventional neurotransmitters in many neuronal structures (1). An example of peptidergic modu1ation that has been well studied is the inhibition of nicotinic cholinergic receptor function by SP. Reported initially for cat Renshaw cells (2) and the Mauthner fibre-giant fibre synapse of hatchet fish (3) and subsequently in more detail for the clonal cell line PC12 (4), adrenal chromaffin cells (5) and sympathetic ganglion cell s (6), this inhibitory action by SP appears to reflect an enhancement of AcCh receptor desensitization.

The peripheral anterograde transport of substance P (SP) in the cervical vagus nerve was reduced 30–40% in male rats following 2 weeks of ACTH or corticosterone administration. In a different model, chronic shock avoidance stress reduced by 20–40% net SP accumulation in subsequently removed 24h explants of nodose ganglion/vagus nerve. The results suggest that in chronic stress the neuroendocrine axis may interact with SP-containing vagal sensory neurons as part of the long term adaptation response.Within the vagus nerve, about 300–500 pg SP are synthesized in sensory ganglia and exported daily for distal transport. One third or less that amount is transported centrally in vivo. When the vagus is crushed at the-rostral pole of the nodose ganglion (NG), the peripheral transport of SP is reduced to 100 pg daily (1).The factors regulating the synthesis and transport of SP in the vagus are largely unknown. The ACTH-adrenal axis or stress might be among such factors as part of the adaptation response of the autonomic nervous system. To test that hypothesis, the 24h transport of SP in the cervical vagus nerve was measured in rats following two weeks administration of ACTH or corticosterone (C). In a separate series of experiments, as part of a project evaluating the effects of stress on the aging nervous system, Fischer 344 rats were exposed to 4–6 months of snock avoidance stress. Because invasive procedures could not be performed prior to sacrifice, the ligation/transport model was not employed. Rather, explants comprised of the NG and attached distal vagus nerve were removed at sacrifice and maintained for 24h in vitro.

The carotid and aortic bodies transducer changes in arterial PO₂ and PCO₂ into action potential coded nerve signals. There are two types of cells in the carotid body; glomus or type I cells and sustanticular or type II cells. Afferent nerve endings are in direct synaptic contact with type I cells, while the type II cells resemble Schwann cells and envelop glomus cells andadjacent nerve endings (4). Glomus cells seem essential for chemoreception because in their absence afferent nerve endings alone are unable to respond to physiological stimuli (13).

We have studied the mechanism of action of a number of mediators of contractility in the opossum esophagus muscularis mucosa [MM] in vitro. Indirect evidence indicating a physiological role for substance P ESP] has been obtained [1,2]. We have established immumnochemical technlques to investigate this question by the following procedure: 1) Detection, localization, and characterization of SP immunoreactivity [SP-IR] in tissues by immunohistochemistry. 2) Detection, and characterization of SP-IR in tissues by SP-specific RIA. 3) Measurement of SPIR in samples from in vitro studies.

The nucleus tractus spinalis of the trigeminal nerve (DV) is found in the medulla oblongata of vertebrates and functions mainly for skin sensations of the facial area. This nucleus is divided into three parts: the subnucleus oralis (DVo), the interpolaris (DVi), and the caudalis (DVc). The function of the DVc is believed to be related to nociception. If the DVc is related to this function, it is conceivable that there would be a set of specific neurotransmitters for the function; for instance, one of the transmitters could be substance P (SP) [1,2].

In man trigeminal stimulation produces constriction of the pupil: this pupillary effect can be attributed to substance P (SP) release from trigeminal sensory nerve fibers [1]. In fact SP-1ike immunoreactive nerve fibers have been visualized in iris sphincter muscle [2]. This discovery has opened question about the pathophysiological role of SP in the human iris. Our radioimmunological and pupillopharmacological data have been carried out to clarify this problem.

Short-term exposure to airway irritants such as cigarette smoke and ammonia vapour rapidly increases the mucociliary (m.c.) activity in the rabbit maxillary sinus (1,2). These irritants are known to stimulate airway C fibres (3), and it is possible that there is a m.c. defense mechanism in the mucous membrane of the rabbit maxillary sinus mediated by C fibres. Part of the increased m.c. activity is mediated by cholinergic pathways since the response is suppressed by pretreatment with atropine or hexamethonium. It is known that parasympathomimetic drugs such as methacholine accelerate m.c. activity and transport (4,5). However, a m.c. response to C fibre stimulation remains in atropinized animals as evidenced from experiments with capsaicin, antidromic nerve stimulation and airway irritants (1,2,6,7). Thisnon-cholinergic response is mimicked by i.a. injections of substance P (SP) (8). The effect of SP is resistant to both atropine and hexamethonium, but inhibited by (D-Pr02, D-Trp7,9)SP (9). Moreover, the m.c. response to C fibre stimulation is inhibited by pretreatment with capsaicin (13 mg i.a.) or (D-Pro2, D-Trp7,9)SP (1,2,6,7). These findings suggest the existence of a m.c. defense reflex involving capsaicin-sensitive C fibres (afferent pathway) and cholinergic effector neurones (efferent pathway). The final outcome reflects the joint release of both SP (axon reflex) and acetylcholine.

Neurokinin A is a recently discovered mammalian neurokinin [1,2]. Immuno reactivity for neurokinin A is found in the cell bodies of primary afferent neurones [3] and this peptide appears to co-exist with substance P [3,4]. Previous studies have reported that substance P has a preferential excitatory effect on nociceptive neurones in the spinal dorsal horn [5,6]. Therefore, to test the possible functional specificity of neurokinin A, the present study was done in which this peptide was iontophoretically applied to functionally identified dorsal horn neurones.

Substance P (SP) is a putative primary afferent transmitter of nociceptive information [1]. Intrathecal injection of SP produces an increased sensitivity to heat [2,3] and pressure [4] in tests for nociceptive thresholds. Recently, SP was shown to be released from spinal cord by noxious pressure but not noxious thermal stimulation, while somatostatin (SST) was selectively released by noxious thermal stimulation [5] suggesting a modality specific role for these peptides in relation to pain. Previously, antibodies to SP (anti-SP) and SST (anti-SST) have been used to demonstrate the presence of SP and SST immunoreactive fibers in the dorsal horn of the spinal cord [6]. In this study we have used antibodies to SP and SST to determine whether a modality specific analgesia for these peptides could be demonstrated. The present experiments first tested the tail flick latency to heat and the paw withdrawal response to pressure in rats following intrathecal injections of anti SP, anti-SST or normal serum. In a second experiment, the behavioral effects of anti-SP serum were compared to normal serum in rats which were tested prior to injection.

Recent data from our laboatory [l] demonstrated that substance P (SP) and cholecystokinin-octapeptide (CCK) immunoreactivity co-exist in some, but not all, primary afferent neurons in dorsal root ganglia of the rat. Furthermore, four populations of primary afferent perikarya could be distinguished on the basis of size, cytology and peptide content. Within the small dorsal root ganglion cells (cells 12–20 um in diameter), populations were described that contained SP or SP+CCK immunoreactivity. Within the range of intermediate-sized cells (cells 20–45 um in diameter), populations were described which were immunoreactive for CCK or CCK+SP.

Stimulation of the central osmoreceptors when hypertonic solutions were infused into the 3rd cerebral ventricle produced an antidiuretic response, that is vasopressin release, in the waking goats [1] or oxytocin in the anaesthetized rats [2]. The antidiuretic response could also be obtained by electrical stimulation of the peripheral nerves as it was demonstrated in anaesthetized dogs by stimulation of the ulnar nerve [3].

For a number of years, substance P (SP) has been an outstanding candidate for the transmitter of nociceptive primary afferents. Dorsal horn SP derives partly from small dorsal ganglion cells with fine fibres, equivalent to those likely to mediate input from cutaneous nociceptors [1]. Either electrical stimulation of cutaneous nerves at high intensities or noxious cutaneous stimulation elicit release of immunoreactive-SP from the spinal cord [2,3]. Intrathecal administration of tachykinins induces behaviour similar to responses elicited by peripheral irritation and reduce nociceptive response thresholds in some reports. Antagonistic analogues can apparently produce analgesia [4,5]. Local administration of SP, close to dorsal horn sensory neurones, generally elicit excitatory effects [6,7], although complex effects with inhibitory components have also been reported. A more appropriate site for iontophoretic administration of tachykinins, however, is the substantia gelatinosa (SG), where the relevant afferents terminate.

In contrast to many other peptides, substance P was right away purified both from intestine and brain. This may be regarded as a sign of the remarkable intuition that characterized Ulf von Euler’s research throughout his career. In this way substance P never belonged to the many gastrointestinal peptides which to our surprise later on also were discovered in the brain. The research which followed the original discovery of substance P by von Euler and Gaddum (15) has exhibited a wavy pattern with bursts of activity appearing at shorter and shorter intervals, as schematically indicated in Figure 1. Thus, substance P research was slow after the initial studies until the 50’ s, when several groups initiated new efforts, including researchers such as Lembeck, Pernow, Umrath and Zetler, resulting i.a. in an extensive mapping of distribution of substance P activity in various tissues (12) as well as the suggestion that substance P could act as transmitter substance in primary sensory neurons (9). Although interest in substance P remained in the following years, no dramatic events occurred until the beginning of the 70’ s, when Susan Leeman and her collaborators sequenced substance P and showed that it represents a peptide consisting of eleven amino acids. Subsequently this group synthesized substance P, raised antisera and worked out a radioimmunoassay (RIA). This major progress led to intense activity in many laboratories allover the world. The work in Masanori Otsuka’s laboratory was seminal in defining a functional role for substance P and, for example, at the Karolinska Institute Bengt Pernow initiated a new wave of substance P research. In the following years the distribution of substance P was outlined both with radioimmunoassay and immunohistochemistry, and novel aspects of the function of substance P were outlined. We remember particularly Jim Henry’s work on a possible involvement of substance P in pain transmission, and the elucidation by Fred Lembeck and his associates of the role of substance P in the axon reflex.

Substance P (SP) is a member of the tachykinin family of bioactive peptides including physalaemin, eledoisin and kassinin, and SP has putative physiological action in the CNS. In addition to SP, two novel tachykinins have recently been identified in mammalian CNS, namely neurokinin A (substance K, SK) and neurokinin B (1,2). There seem to be differences in the relative potencies of biological activities between the two naturally occurring mammalian tachykinins SP and SK in the peripheral systems. The biological activities of SK more closely resemble those of kassinin than of SP or physalaemin (3–6).

Until recently it has generally been considered that sub stance P (SP) immunoreactive neurons were absent from the mammalian cerebral cortex. There have now been three reports of the existence of SP-like immunoreactivity in the cerebral cortices of rats [1] and monkeys [2,3]. One problem in all these studies, however, was that the antisera used were directed against the C-terminal sequence of the peptide, a sequence that is conserved in the other mammalian tachykinins [4]. In the present report, we provide evidence for the presence of SP-like immunoreactivity in cortical cells, based upon immunocytochemistry using an antiserum directed against other sequences of SP. In addition, we show that there are two populations of putative SP cortical neurons. In one, SP immunoreactivity is colocalized with that for gamma aminobutyric acid (GABA), in the other with immunoreactivity for neuropeptide Y (NPY) and somatostatin (SRIF). Furthermore, in the monkey visual cortex, SP and GABA levels are regulated by visual experience [5].

Substance P (SP), an excitatory neuropeptide, has been shown to inhibit the excitatory actions of acetylcholine at nicotinic receptors in different preparations such as the spinal Renshaw cells (1) and the adrenal chromaffin and PC12 cells (2, 3). This effect of SP is presumably due to an enhanced desensitization of the nicotinic receptor (3,4).

Recent evidence suggests that substance P (SP) may be involved in central regulation of cardiovascular function (1). SP and SP receptors are found in several brainstem structures, among them the nucleus tractus solitarius (NTS) (2, 3), which plays a major role in regulating blood pressure (bp) (4). Afferent fibers of the vagus and glossopharyngeal nerves, some containing SP (5), come from baroreceptors in the carotid sinus and aortic arch, activation of which leads to a depressor effect (fall in bp) (4). Removal of the nodose ganglion leads to a decline in the SP content of those regions of the NTS that receive baroreceptor afferents (6).

In earlier studies made in halothaneanesthetised cats implanted with pushpull cannulae, we demonstrated the occurrence of interactions between the striato-nigral substance P (SP) neurons and the nigro-striatal dopaminegic (DA) neurons (1). Indeed, when SP (10 8M) was locally applied into the SNC, the release of newly synthesised 3H-DA was enhanced in the ipsilateral caudate nucleus (CN) and decreased in the pars compacta of the substantia nigra (SNC). Opposite changes in the release of 3H-DA from nerve terminals and dendrites were found when SP transmission in the SNC was interrupted by a local application of a SP antibody. This led us to conclude that striato-nigral SP neurons exert a tonic excitatory influence on the activity of DA cells and that changes in the dendritic release of DA could be involved in this activation. Although some contradictory results were found depending on the site of the nigra1 application of SP, on the basis of biochemical and behavioral data, other groups reported also that SP activates nigral DA cells in the rat (2,3,4). In addition, when used in high concentration (10 −5M), SP has been shown to stimulate the spontaneous release and to inhibit the potassium-evoked release of 3H-DA previously taken up in striatal slices from the rat (5).

Substance P has been found by immunohistochemistry to be largely distributed throughout the central nervous system in several species. This peptide has also been shown to exert some neuroendocrine effects when injected into the brain, such as stimulation of growth hormone and prolactin secretion [1]. In order to obtain more information about the role of substance P in neuroendocrine function in man, we have proceeded to the immunohistochemical localization of this peptide in the human hypothalamus.

Three distinct types of synaptic glomeruli have been described by one of us in the superficial dorsal horn of rat spinal cord (1,2). These types can be distinguished by the morphology of the central bouton, types of peripheral profiles and distribution in the dorsal horn (1,2). The type I central bouton (C_I) is small, dark and highly scalloped, and prevails in the middle third of lamina II. It is capsaicin sensitive and so is probably of unmyelinated, sensory origin (3). The type IIa glomeruli can be distinguished from type IIb by the presence in the latter of neurofilament bundles (2); both have central endings (C_IIa and C_IIb), that come from myelinated sensory afferents (3), which are lighter, larger and have a smoother surface than the C_I (1,2). In a recent study (4), it was shown that 80% of C_I have FRAP (fluoride resistant acid phosphatase) activity, but no information was available concerning the neurochemical nature of the remaining 20% and of the C_IIa and C_IIb. Based on the above, we decided to study the peptide immunoreactivity in synaptic glomeruli using the bi-specific monoclonal antibodies P₄C₁ (antisubstance P/anti-HRP) (5) and Y₄C₇ (anti-somatostatin/anti-HRP) (6). These antibodies allow a drastic simplification of current immunocytochemical procedures. The incubation with antibodies is reduced to one step. The suppression of link antibodies and PAP complexes results in an intense immunostaining, an increased signal to noise ratio and an excellent penetration of antibodies in the absence of detergents (5). Thus, the ultrastructural preservation is significantly improved. This approach facilitated the correlation of peptide immunoreactive sites with ultrastructural details which define the classification of synaptic glomeruli in the substantia gelatinosa. The precise laminar localization of the FRAP reactive band and somatostatin and substance P-like immunoreactivities was also studied in the superficial dorsal horn of the rat spinal cord.

The bed nucleus of the stria terminalis (BNST) is known to be involved in the mediation of a number of sexually differentiated brain functions in the rat. These include the control of gonadotrophic hormone release [1] and male copulatory behavior [2]. Recently, it was reported that a particular cell group within the BNST, in the caudal part of the medial division, was 36% larger in the male than in the female guinea pig [3].

High Substance P (SP) content in hypothalamus [1] and especially in its anterior part connected with hypophysiotropic area suggests the possibility of this peptide involvement in the regulation of gonadotropin release. Fluctuation of SP and LH-RH contents in the hypothalamus during the estrous cycle in the rat indicate the role of SP in the control of reproduction in female rat [2,3]. The data on the effect of SP on hypophysical endocrine function are rather contradictory. In pituitaries incubated in vitro some activity of SP was demonstrated in the release of luteinizing and folliclestimulating hormones [4]. Administration of SP into the 3rd cerebral ventricle of ovariectomized female rats elevated plasma LH levels [5].

Neuroanatomical and neuropharmacological studies in rats suggest that Substance P (SP) is an excitatory transmitter in a bulbospinal pathway whose cells originate from the ventral medulla [1,2] and the caudal raphe nuclei [3,4] and terminate on sympathetic preganglionic neurons in the intermediolateral nucleus (IML) including those which innervate the adrenal medulla. Iontophoresis of SP onto cells of the IML produces excitation of sympathetic preganglionic neurons in the rat [5,6]. In addition, the intrathecal (i. th.) injection of SP at T₉ spinal level increases plasma concentrations of epinephrine and norepinephrine which can be antagonized by the SP analogue (D-Pro2, D-Phe7, D-Trp9)SP [7]. Furthermore, i.th. injection of a stable analogue, (pGlu5, MePheE8, MeGly9)SP (5-11) in chloraloseurethane anaesthetized animals produced a dose-dependent increase in the mean arterial blood pressure and heart rate that was accompanied by increases in plasma epinephrine and norepinephrine [8 ].

The existence of different types of tachykinin receptors in the central nervous system has been shown in binding studies, as well as by using immunohistochemical or autoradiographic techniques. In the present study the electrophysiological effects of various tachykinins were investigated in the isolated hemisected spinal cord preparation of the neonatal rat. Earlier studies with this preparation had shown that tachykinin antagonists, characterized in peripheral tissues, were neither selective nor potent enough to antagonize responses to tachykinin agonists [1, 2]. We, therefore, investigated the possibility that desensitization experiments could reveal different tachykinin receptors in the mammalian spinal cord. In addition, desensitization experiments were carried out with the same goal in a peripheral organ preparation, the guinea-pig ileum.

Substance P (SP) has undergone extensive investigation since it was discovered in the brain and intestine by Von Euler and Gaddum1. Various findings indicate that SP may act in the central nervous system as a neurotransmitter in sensory neurons.2 Although SP was initially thought to produce hyperalgesia,3 many reports indicate that it has antinociceptive properties.4, 5, 6 The mechanism by which SP elicits its antinociceptive effects seems to resemble that of opioidinduced anlgesia, since SP-induced analgesia is reported to be antagonized by naloxone.4, 6

When the pain transmission in sensitive nerves is impaired or interrupted by a partial or total organic deafferentation (DFT) the following main events occur: a) lowering of SP content into the ipsimetameric dorsal horn, [1,2]; b) an increase in SP receptor number in the same dorsal horn area [3]; c) a supersensitivity to locally-applied SP both in dorsal ipsilateral horn and in peripheral structures (vessels: permeabilization; iris: miosis; gland: secretion) innervated ipsimetamerically by the damaged nerve [3,4,5]; d) generation and rostral dissemination into the spinal cord and brainstem of “quasi epileptic foci” capable of emitting simil-epileptic firings of electrical signs, expression of start of “automatic pains”, then central in nature [6,7,8]; e) arising of phantom pain in amputated humans. Likely aches of the same nature are also responsible for the autotomy in animals, attributed to an attempt to get rid of the deafferentated leg, the apparent source of pain. The DFT, when complete, is characterized by chronic aches having the perverted characteristics of central pain, as well as ipsilateral vasomotor disorders and plasma extravasation (sterile inflammation) in the area where pain projects. In the DFT syndrome the SP empty neuron pathological unit could play a major role. SP secretion (even if in a meagre amount) is expected to provoke pain rostrally and inflammation caudally [9]. The activity in neuraxis of SP in DFT syndrome could depend on: a) a secretion of residual SP in the damaged nerve and/or in some regenerated fibers; b) a secretion from intermetameric SP neurons having consistent compensatory plasticity.

Substance P, eledoisin and physalaemin are undecapeptides that share a common C terminus amino acid sequence and therefore belong to the tachykinin family. The rat hippocampus contains scattered substance P immunoreactive fibres [1], as well as binding sites for radiolabelled physalaemin [2]. The hippocampus also contains numerous bombesin binding sites [3] and densely staining ranatens in immunoreactive fibres [4]. All these observations suggest that tachykinins and bombesin may act in the hippocampus. However, Dodd and Kelly [5] reported that rat hippocampal pyramidal neurones were not responsive to substance P, and although some were excited by bombesin, their response was modest and desensitized rapidly. We have thus re-examined the actions of tachykinins and of bombesin in the hippocampus by assessing their effects on non-pyramidal neurones [6]. Amongst these cells are inhibitory interneurones [7], which respond to neurohypophysial peptides by increased firing [8,9] and to opioid peptides by a decrease in firing rate [10].

There is increasing evidence that kinins are modulators of neuronal function. Immunocytochemical and RIA studies have shown widespread distribution of kinins and substance P in rat brain [1,4]. Several pathways containing these peptides have been traced [11]. Some of the kinins have been designated as neurotransmitters, whereas recently discovered neurokinins are yet to be explored for their neurotransmitter behaviour [10]. Substance P and kinins induce intense depolarization of neurons when applied iontophoretically [6,7,9]. These neuropeptides exhibit structural similarity too. Kinins bind to specific high affinity binding sites, are potent vasodilators and elicit pressor response when injected in the rat brain. Their binding with the respective ligands depends on PH, time dose and ionic concentration [8]. The studies on kinins in the developing brain are rather limited. In spite of their great neurophysiological and neuropharmacological significance, their exact mechanism of action is poorly understood. Hence in the present study, electrophysiological behaviour of different doses of bradykinin (BrK) was studied in the developing rat cerebral cortex by employing computerized EEG. The neurophysiological behaviour of NaloxoneHcl and Ca antagonist Gallopamil (I.P.) was also assessed in these animals.

Central or peripheral administration of substance P (SP) produces a number of effects on behavior. In mice, SP decreases isolation-induced aggressive [1] and nociceptive [2] behaviors. SP alters several measures of spontaneous motor behavior in rats and mice [3]. Intraventricular (ICIT) SP dramatically enhances groaning and scratching behavior in mice [4], but not rats [5], while locomotion, sniffing and hindlimb rearing behavior are enhanced in both species [6,7].

Carbamazepine (CBZ) is known to be a potent anti-epileptic drug. Like lithium it has been reported to prevent the oscillation of manic-depressive psychosis [1][2]. Although the mechanisms of action of both drugs in the brain are not well known, it is possible that they affect some neurotransmitters or neuromodulators in a similar manner. It has been reported that chronic lithium administration increased the substance P (SP) content in the dopamine related brain areas, and co-administration of haloperidol (HAL) prevented this effect [3][4]. We have studied the effect of CBZ, HAL and trihexyphenidyl (TR) alone or in combination on SP content as well as dopamine (DA), serotonin (5HT), glutamate and γ-aminobutyric acid (GABA) in the rat brain.

Acetylcholine (ACh) is released both spontaneously and upon electrical stimulation of the nerve. Both of these processes are dependent upon the influx of extracellular Ca++ ions. Two feedback systems, one positive and the other negative, were postulated for the autoregulation of ACh release from mouse or rat cerebral slices [1-2]. The components of the positive feedback system included muscarinic receptors (Ms), Substance P (SP) and Ca++ uptake by the nerve (Figure 1). If the amount of ACh in the synaptic gap was low, a positive feedback was triggered causing the release of SP either directly or by a disinhibition phenomenon. The released SP increased the uptake of Ca++ and amplified further release of ACh. This amplification of the rate of ACh release proceeded to a certain limit when the negative feedback system was triggered. The components of this negative feedback system were muscarinic receptors (Mi), methionine enkephalin (MEK) and Ca++ uptake by the nerve. High levels of ACh triggered MEK release which depressed Ca++ uptake and the rate of ACh release from the mouse cerebral slices.

The striatum constitutes a pivotal component of the basal ganglia, Which is anatomically and functionally associated with several clinical syndromes like parkinsonism, schizophrenia and Huntington’s chorea. This nucleus is extensively and unilaterally innervated by dopamine (DA) containing fibres originating in the substantia nigra (SN), pars compacta (SNC), and it projects fibres to the globus pillidus, the entopeduncular nucleus and the SN. The striatonigral pathway is a major neuronal route by which motor information originated in the striatum is funnelled. This pathway is, however, heterogeneous since: (1) it contains neuronal fibres utilizing different kind of neurochemical messengers, and (2) it includes different functional units, representing outflow and feed-back loops (for reviews see 1).

The basal ganglia of the mammalian brain consist of a complex neuronal network. A part of this network connects the substantia nigra with the striatum (caudate and putamen). One major nigrostriatal pathway contains dopamine and two striato-nigral pathways contain substance P (SP) and gamma-amino-butyric acid (GABA), respectively. In addition, GABA is found in local striatal interneurons.

The mutant mouse Wobbler (wr) possesses a recessively inherited degeneration of motoneurons and other neurons in the ventral horn, predominantly in the cervical spinal cord. Therefore the Wobbler mouse has been proposed as an animal model of human motoneuron disease, including amyotrophic lateral sclerosis (ALS) and familial infantile spinal muscular atrophy (ISMA). In the human motoneuron diseases as well as in Wobbler, the primary cause of the disease remains unknown. Therefore, before the laboratory experiments began, one of us (LLV) freely speculated on the possibility that substance P (SP), a neuromodulator of primary afferents in the dorsal horn, and motoneurons in the ventral horn of the mammalian spinal cord[l] possessed trophic functions that might be lacking in the Wobbler spinal cord.

Previous studies in our laboratory have shown that injections of the GABA-A agonist muscimol into the median raphe nucleus (MR) of the rat via chronically indwelling cannulae will elicit a dose-dependent increase in locomotor activity (LMA) [5]. The LMA effect of intra-MR muscimol appeared to be dependent upon intact ascending 5HT neurons originating from the midbrain raphe nuclei [5], since it was demonstrated that intracerebroventricular (i.c.v.) and intra-mesencephalic (just dorsal to the interpeduncular nucleus) injections of 5,7-dihydroxytryptamine (5,7-DHT), following pretreatment with the norepinephrine (NE) reuptake blocker desipramine (DMI), blocked the LMA effect of intra-MR muscimol.

The tachykinln family of small bioactive peptides are defined by their similar biological activities and conserved carboxyl-terminal sequence -Phe-X-Gly-Leu-Met-NH₂, where X is an aromatic or branched al iphatic amino acid [1].The amino-termini of the tachykinins shows a much wider variation, and most of the biological activities of this peptide family reside in their similar carboxyl sequences. Substance P (X=Phe) is the most widely known member of the tachykinin family, and was for many years generally believed to be the only mammalian tachykinin. In recent years the discovery [2–4] of the novel mammalian tachykinins substance K and neuromedin K (both X=Val) has shown that mammalian tachykinin systems are more elaborate than previously supposed [9]. The new tachykinins differ from substance Pin their central and peripheral pharmacologies, mediating. different physio1ogica1 funct ions through different receptors. In the present report we have used several different radiolabeled tachyki ni ns to invest i gate the distribution and heterogeneity of tachyki ni n bi ndi ng sites in the rat brain and periphery and the canine gastrointestinal system.

Our present knowledge of the widespread distribution of tachykinins in almost all parts of the nervous system in both mammals and man, their release under various physiological and pathophysiological conditions and their profound biological effects in several organic systems (1) has naturally stimulated a search for u possible involvement of these newly discovered compounds in various clinical disorders.

When the first type of a living organism appeared in Nature, it possessed already the abilities to feed, to reproduce and to defend itself against the environment. But genetic progress soon created many more species and they soon had to develop instruments of defense against each other. On each day of Genesis that brought forth new forms of life, these were equipped with increasingly sophisticated systems of defense (Tab. 1.). Antibiotics were probably the first weapons produced, a capability which was lost in higher species. Certain species developed the art of producing poisons, which are generally regarded not as defensive but as offensive weapons. Hormonal peptides and neuropeptides were already present in bacteria, protozoa, and plants. Like certain amines, which began to serve intercellular communication at a much later stage of development, peptides were already present long before they obtained specific functions in endocrine, nervous or immune systems (32). with the development of vertebrates entirely new systems of defense were set into action: the immune system, the endocrine system and a wide variety of reactions regulated by the nervous system. While some earlier forms of defense were lost during the stages of development, they were replaced by others of a more complex and more efficient nature. Certain links between these systems appeared and were further refined. When Adam accepted the apple sometime rater on, he disregarded the Lord’s advice of intellectual defense, thereby opening his mind to sexual and social life.