Interaction of substance P antagonists with substance P receptors on dispersed pancreatic acini

In the present study we examined the abilities of three analogs of substance P, [D-Pro2-, D-Phe7-, D-Trp9]-substance P, [D-Pro2-, D- Trp7 ,9]-substance P and [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to alter substance P-induced changes in pancreatic acinar cell function and to occupy substance P receptors. At 30 microM, each analog of substance P lacked agonist activity and inhibited amylase secretion stimulated by substance P receptor agonists. The inhibition was reversible and specific for peptides that interact with substance P receptors (physalaemin, substance P, eledoisin, kassinin ). The analogs of substance P did not inhibit the actions of cholecystokinin, caerulein, gastrin, carbamylcholine, secretin, vasoactive intestinal peptide, PHI, ionophore A23187 or 8Br -cAMP. At high concentrations, [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P, but not [D-Pro2-, D- Trp7 ,9]-substance P or [D-Pro2-, D-Phe7-, D-Trp9]-substance P, caused a small but significant inhibition of bombesin-stimulated amylase release. For each analog of substance P, the inhibition was competitive in nature in that there was a rightward shift of the dose-response curve for physalaemin-stimulated amylase secretion with no change in efficacy. From Schild plots of the ability of [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to inhibit either substance p- or physalaemin-stimulated amylase release, the slopes were not different from unity. For each analog of substance P, there was a close correlation between its ability to inhibit substance P- or physalaemin-stimulated amylase release and its ability to inhibit binding of 125I-labeled substance P or 125I-labeled physalaemin. [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P was 2-fold more potent than [D-Pro2-, D- Trp7 ,9]-substance P which was 4-fold more potent than [D-Pro2-, D-Phe7-, D-Trp9]-substance P, (i.e., pA2 6.1, 5.9, and 5.2, respectively). For each analog, the dose-response curve for its ability to inhibit physalaemin-stimulated amylase release was superimpossible on the dose-response curve for its ability to inhibit binding of 125I-labeled physalaemin. These results indicate that each of these analogs of substance P is a specific competitive inhibitor of the action of the substance P on dispersed acini from guinea-pig pancreas, and that their abilities to inhibit substance P-induced changes in acinar cell function can be accounted for by their abilities to occupy the substance P receptor.     

Molecular cloning of the murine substance K and substance P receptor genes.

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.     

Are the proposed substance P receptor sub-types,substance P receptors?

Recently, a number of laboratories have postulated the existence of receptor sub-types for substance P. This review is intended to represent a critical appraisal of these reports. In the majority of cases, the evidence for the existence of receptor sub-types has been obtained from observed potency differences of agonists. The problems with this approach are discussed. In addition, information obtained through substance P antagonists, binding studies and investigations of second messenger systems is presented and discussed in relation to the above receptor subdivisions. It is concluded that the present results are consistent with the existence of three receptor sub-types; however, it is suggested that substance P is the natural agonist for only one of these receptors, and that substance K and tuftsin may be the transmitters for the other two receptor sub-types.     

Analgesic effect of antagonists of substance P

[D-Pro²,D-Phe⁷,D-Trp⁹]-SP and [D-Pro²,D-Trp^7,9]-SP havebeen shown to be antagonists of substance P. The hindlimb scratching syndrome of mice, known to be caused by substance P was absent when these peptides were injected into substance P-treated mice. Substance P shortens “tail withdrawal time” from hot water; the two peptides greatly prolonged tail withdrawal time. Antidromic stimulation of the saphenous nerve (rat), known to release substance P and to induce vasodilatation plasma extravasation, was also greatly inhibited by [D-Pro²,D-Phe⁷,D-Trp⁹]-SP. These peptides presumably cause anti-nociceptor effects (analgesia) by inhibition of substance P at receptors and favor the concept that substance P is a sensory neurotransmitter of nociceptive messages.     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

Light microscopic autoradiographic localization of [3H] substance P binding sites in rat thoracic spinal cord

We have used light microscopic autoradiography to look for the distribution of [³H] substance P receptors in the thoracic spinal cord of the rat. High densities of autoradiographic grains were localized to the intermedialateral cell column, the central canal and the substantia gelatinosa of the dorsal horn.     

Delineation of structural domains involved in the subtype specificity of tachykinin receptors through chimeric formation of substance P/substance K receptors.

The mammalian tachykinin receptors belong to the family of G protein-coupled receptors and consist of the substance P, substance K and neuromedin K receptors (SPR, SKR and NKR). We constructed 14 chimeric receptors in which seven transmembrane segments were sequentially exchanged between the rat SPR and SKR and examined the subtype specificity of the chimeric receptors by radioligand binding and inositol phosphate measurements after transfection into COS cells. All chimeric receptors showed maximum responses in agonist-induced inositol phosphate stimulation. Detailed analysis of five receptors with agonist selectivity similar to SPR indicated that the selectivity is mainly determined by the region extending from transmembrane segment II to the second extracellular loop together with a minor contribution of the extracellular N-terminal portion. This conclusion was more directly confirmed by an additional chimeric formation in which the introduction of the above middle portion of SPR into the corresponding region of SKR conferred a high affinity binding to substance P. The tachykinin receptors can thus be divided into two functional domains: the region covering transmembrane segments V-VII and responsible for fundamental recognition of the common tachykinin sequence; and its preceding portion involved in evoking subtype specificity by interacting with the divergent sequences of the peptides.     

The effects of substance P and met5-enkephalin in dog ileum

Substance P initiated tonic contraction of dog ileum when administered in doses from 1 pg to 20 micrograms intraarterially (ED50 = 67 ng). Low doses acted to excite cholinergic postganglionic neurones since atropine or tetrodotoxin (TTX) increased the ED50 of substance P about 25-fold, while hexamethonium and local field stimulation had only a small effect to increase the ED50. Also atropine and tetrodotoxin effects were not additive. Higher doses apparently acted to stimulate smooth muscle directly, but no evidence was obtained that local field stimulation could release substance P to act on smooth muscle. Substance P tachyphylaxis prevented substance P actions on cholinergic nerves, but it did not affect responses to intraaterial acetylcholine or block distal inhibition from proximal distention or field stimulation. Met-enkephalin given intraarterially, was also excitatory in doses from 1 ng to 20 micrograms; the amplitude of tonic and phasic contractions produced was significantly decreased by TTX and atropine but was not diminished by hexamethonium or substance P tachyphylaxis. Partial tachyphylaxis to met-enkephalin was produced but was not diminished by hexamethonium or substance P tachyphylaxis. Partial tachyphylaxis to met-enkephalin was produced without affecting the ED50 for substance P. We conclude that substance P acts in small amounts on receptors in myenteric nerves to release acetylcholine by a mechanism, presumably involving postganglionic cholinergic nerves, while met-enkephalin also apparently may act at least in part through a similar TTX- and atropine-sensitive mechanism. These peptides also caused activation of other receptors, probably on smooth muscle by noncholinergic. TTX-insensitive mechanisms. Also the receptors for each peptide which are located on nerves were distinct and independent since tachyphylaxis could be produced to each without affecting the response to the other.     

Glutamate-stimulated ATP release from spinal cord astrocytes is potentiated by substance P

ATP has recently emerged as a key molecule mediating pathological pain. The aim of this study was to examine whether spinal cord astrocytes could be a source of ATP in response to the nociceptive neurotransmitters glutamate and substance P. Glutamate stimulated ATP release from these astrocytes and this release was greatly potentiated by substance P, even though substance P alone did not elicit ATP release. Substance P also potentiated glutamate-induced inward currents, but did not cause such currents alone. When glutamate was applied alone it acted exclusively through alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptors to stimulate Ca(2+) influx-dependent ATP release. However, when substance P was co-applied with glutamate, ATP release could be elicited by activation of NMDA and metabotropic glutamate receptors. Activation of neurokinin receptor subtypes, protein kinase C and phospholipases A(2), C and D were needed for substance P to bring about its effects. These results suggest that astrocytes may be a major source of ATP in the spinal cord on activation of nerve fibres that release substance P and glutamate.     

An analogue of substance P with broad receptor antagonist activity

[DPro4,DTrp7,9,10]Substance P-4-11 functions as a substance P receptor antagonist in several different systems. Because some analogues of substance P can function as receptor antagonists for bombesin as well as substance P, we tested [DPro4,DTrp7,9,10]substance P-4-11 for its ability to modify the interaction of various pancreatic secretagogues with their receptors in dispersed acini from guinea pig pancreas. [DPro4,DTrp7,9,19]Substance P-4-11 did not stimulate amylase secretion and did not alter the stimulation of amylase secretion caused by secretin, vasoactive intestinal peptide, calcitonin gene-related peptide or carbachol, but did inhibit the stimulation of amylase secretion caused by substance P, bombesin or cholecystokinin. With substance P, bombesin and cholecystokinin, [DPro4,DTrp7,9,10]substance P-4-11 caused a parallel rightward shift in the dose-response curve for stimulation of amylase secretion with no change in the maximal response. Schild plots of these results gave straight lines with slopes that were not significantly different from unity. [DPro4,DTrp7,9,10]Substance P-4-11 inhibited binding of 125I-labeled substance P, 125I-[Tyr4]bombesin and 125I-cholecystokinin octapeptide over the same range of concentrations as that in which it inhibited biologic activity of each of these peptides. Half-maximal inhibition of binding of 125I-substance P occurred with 4 microM, of 125I-[Tyr4]bombesin with 17 microM and of 125I-cholecystokinin octapeptide with 5 microM. With each radiolabeled peptide the value of Ki for inhibition of binding by [DPro4,DTrp7,9,10]substance P-4-11 was not significantly different from the corresponding value of Ki calculated from the appropriate Schild plot. The present results indicate that [DPro4,DTrp7,9,10]substance P-4-11 is a competitive antagonist at receptors for substance P, for bombesin and for cholecystokinin. Thus, these receptors must share a common peptide recognition mechanism even though they interact with agonists that have no obvious structural similarity.     

Molecular characterization of a functional cDNA for rat substance P receptor

This paper describes the amino acid sequence of the rat substance P receptor and its comparison with that of the rat substance K receptor on the basis of molecular cloning and sequence analysis. From a rat brain cDNA library constructed with an RNA expression vector, we identified a cDNA mixture containing a functional substance P receptor cDNA by examining electrophysiologically a receptor expression following injection of the mRNAs synthesized in vitro into Xenopus oocytes. A receptor cDNA clone was then isolated by cross-hybridization with the bovine substance K receptor cDNA. The clone was confirmed by selective binding of substance P to the cloned receptor expressed in mammalian COS cells. The deduced amino acid sequence (407 amino acid residues) possesses seven putative membrane spanning domains and shows a sequence similarity to the members of G-protein-coupled receptors. The rat substance P and substance K receptors are very similar in both size and amino acid sequences, particularly in the putative transmembrane regions and the first and second cytoplasmic loops. This similarity is in marked contrast to the sequence divergence in the amino- and carboxyl-terminal regions and the third cytoplasmic loop. The observed sequence similarity and divergence would thus contribute to the expression of similar but pharmacologically distinguishable activities of the two tachykinin receptors.     

The antagonism of bombesin in the CNS by substance P analogues

The ability of substance P analogues to inhibit the action of bombesin in the CNS was investigated using receptor binding and biological assays. The putative substance P antagonists inhibited binding to central receptors for both substance P and bombesin-like peptides. Spantide, which was the most potent analogue tested, reversed the bombesin induced hypothermia and grooming. Therefore the putative substance P antagonists may also antagonize the actions of bombesin in the CNS     

A comparison of the anatomical distribution of substance P and substance P receptors in the rat central nervous system

A comparison of anatomical distributions of substance P (SP) and substance P receptors in the rat central nervous system was performed. SP was localized by microdissection and radioimmunoassay and SP fibers and cell bodies by immunohistochemistry. Receptors for ¹²⁵I-Bolton Hunter labelled SP (¹²⁵I-BH-SP) were characterized pharmacologically by a slice binding technique in sections that contained primarily striatum. The receptor was saturable and had an equilibrium dissociation constant (K_D) of 0.30 nM and maximum number of binding sites (B_max) of 37.8 fmol/mg protein. Pharmacological characterization using C terminal fragments and naturally occurring analogues of SP reflected characteristics of the receptor which had been shown previously in bioassays and biochemical assays. Comparison of distribution of SP fibers and cell bodies and SP receptors indicated that there is no consistent relationship between the amount of SP receptor and density of SP fibers or cell bodies in a given region of the brain.     

Neurotensin and substance P receptors expressed in Xenopus oocytes by messenger RNA from rat brain

Xenopus oocytes were induced to acquire sensitivity to neurotensin and substance P, by injecting them with a fraction of poly(A)+ mRNA from rat brain. Non-injected oocytes, and oocytes injected with other brain mRNAs, failed to show responses, suggesting that receptors to these peptides were expressed by specific brain mRNAs. Responses to substance P and neurotensin comprised an oscillatory chloride current, and a smooth current having different ionic basis. These currents resembled those seen during activation of muscarinic and serotonergic receptors, but were not blocked by the corresponding antagonists atropine and methysergide. The responses to substance P, and to a lesser extent to neurotensin, showed a long-lasting desensitization. Similarities between the oscillatory currents evoked by the peptides acetylcholine and serotonin suggest that all these receptors may 'link in' to a common intracellular messenger pathway.     

Stereospecificity of SP1 and SP2 substance P receptors

Previous studies with N-terminal fragments of substance P (SP) have suggested the existence of two separate SP receptor populations. SP1 receptors are found in guinea pig ilea and rat colons. SP2 receptors are found in mouse spinal cords and rat salivary glands. We have now found that substitution of Gly9 in substance P's C-terminal hexapeptide leads to an analog (L-Pro9 SP6-11) which selectively and potently stimulates SP2 receptors. In contrast, substitution of the same residue with D-Proline results in a potent and selective agonist for SP1 receptors. The data dramatically confirm the distinction between SP1 and SP2 receptors and demonstrate that the two receptors have distinct stereochemical architectures.     

Calcium- and superoxide anion-mediated mitogenic action of substance P on cardiac fibroblasts

Substance P is released from nerve endings in the heart under pathological conditions like ischemia, but its action on cardiac cells has not been investigated. This study tested the hypothesis that substance P is mitogenic to adult cardiac fibroblasts and delineated the underlying mechanism(s). Substance P, acting via neurokinin-1 (NK-1) receptors, stimulated cellular hyperplasia over a range of 1-10 micromol/l. It elicited no change in net collagen production, total protein synthesis, or cell protein content but increased (45)Ca uptake and superoxide generation. EGTA, N-acetyl-cysteine, and superoxide dismutase attenuated the hyperplastic response to substance P. A combination of substance P and EGTA enhanced superoxide generation without an increase in DNA synthesis, showing that an increase in superoxide production does not result in hyperplasia when extracellular Ca(2+) is chelated. Together, the data suggest that substance P may activate, via NK-1 receptors, a hyperplastic but not hypertrophic response in adult cardiac fibroblasts and that alterations in redox state and Ca(2+) homeostasis may act in concert to mediate its mitogenic action.     

Effects of activators and antagonists of the neuropeptides substance P and substance K on cell proliferation in planarians

Substance P and substance K (Neurokinin A) are mammalian peptides belonging to the tachykinin family. Both have been studied extensively, are widely distributed in both central and peripheral mammalian nervous systems, and seem to be involved in pain reactions and inflammatory responses. We report here that substance P and substance K, as well as Epidermal Growth Factor (EGF), are potent mitogens, at micro and nanomolar concentrations, for planarian cells. This stimulation is inhibited by the substance P and substance K antagonist spantide, while capsaicin, a pungent agent of capsicum peppers that destroys sensory neurons, stimulates cell division, probably through release of substance P. These results, jointly with the reported stimulation of cell division by naloxone and its inhibition by Met-Enkephalin (Bagu?a, 1986), both probably acting on tachykinin release, suggest that target cells, the neoblasts, must have in their cell membranes numerous receptors for growth hormones and neuropeptides analogous to their mammalian counterparts.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

Salivary secretion induced by substance P

Secretion of fluid, ions, and amylase from parotid and submaxillary glands of rat, induced by intravenous injection of substance P (SP), was examined. The action of SP on salivary glands, like physalaemin, resembled that of cholinergic stimulation. While SP-evoked salivary flow from both glands was blocked by atropine, atropine did not modify composition of SP-evoked saliva. The present study suggests that salivary secretion and secretion of ions and amylase evoked by SP are mediated via SP-sensitive cholinergic receptors and specific SP receptors, respectively.     

Reduced CTL response and increased viral burden in substance P receptor-deficient mice infected with murine gamma-herpesvirus 68

One component of the protective host response against mucosal pathogens includes the local production and increased expression of certain neuropeptides and their receptors. The present study further demonstrates this fact by investigating the contribution that substance P receptor expression makes toward immunity against a gamma-herpesvirus infection. Following intragastric inoculation with murine gamma-herpesvirus 68 (gamma HV-68), expression of substance P and its receptor was increased in mucosal and peripheral lymphoid organs in wild-type strains of mice. These results suggested that this receptor/ligand pair might be an important component of the host response against this viral infection. Such a hypothesis was supported by the demonstration that mice, genetically deficient in substance P receptor expression, showed an increased viral burden when compared with syngeneic C57BL/6 mice. Furthermore, substance P receptor-deficient mice showed a reduced CTL response against gamma HV-68, suggesting a mechanism to explain this increased viral burden. Such limitations in the Ag-specific CTL response in substance P receptor-deficient mice could result from lowered expression of IL-12 during viral infection. Consistent with this hypothesis, increases in mRNA encoding IL-12 and secretion of this cytokine into sera of infected, wild-type animals were markedly reduced in substance P receptor-deficient mice. These studies demonstrate that genetic elimination of substance P receptors in mice results in an increased gamma-herpesvirus burden and an altered host response.