Evidence that the contractile response of the guinea-pig ileum to capsaicin is due to substance P release

Summary The mechanism of action of capsaicin on the guinea-pig isolated ileum was studied. Capsaicin (1.5×10^–6mol l^–1) produced a large contractile response of the ileum which exhibited marked tachyphylaxis. The response was reduced by treatment of preparations with atropine, 5×10^–6mol l^–1, and by substance P-autodesensitization, and was abolished by a combination of these treatments. It was concluded that capsaicin released both substance P and acetylcholine from the guinea-pig ileum. Evidence for a cholinergic component in the action of high concentrations of substance P was also obtained and therefore it is proposed that capsaicin releases substance P from neurones in the ileum, and the released substance P in turn stimulates cholinergic neurones to release acetylcholine, as well as producing direct muscle contraction.     

Evidence that axons containing substance P in the guinea-pig ileum are of intrinsic origin

Summary Acid extracts from both normal and extrinsically denervated ileum contained a compound which was indistinguishable from synthetic substance P; this compound was assayed by examining its contractile effect on the longitudinal muscle of segments of ileum in which receptors for acetylcholine and histamine were blocked. Contractions caused by the compound were markedly and selectively antagonized when the ileum was made insensitive to the action of substance P. The activities in the extract and of synthetic substance P were both destroyed by chymotrypsin but were not affected by trypsin or carboxypeptidase B. The concentrations of substance P-like material in normal and extrinsically denervated segments were not significantly different, being equivalent to 0.48 g of substance P per g of external muscle plus myenteric plexus. A compound with substance P-like activity was liberated by stimulation of intramural nerves, either electrically or by dimethylphenylpiperazinium, in both normal and extrinsically denervated segments of ileum. The release of this compound was prevented by tetrodotoxin and its action on the muscle was blocked when the ileum was made insensitive to the action of substance P. Experiments with transmural stimulation showed that excitatory nerve pathways involving substance P neurons extend for less than 4 cm along the intestine.     

Indirect evidence for the inhibition of enteric substance P neurones by opiate agonists but not by capsaicin

There is good evidence indicating that hyoscine-resistant contractions of the guinea-pig ileum evoked by stimulation of the intramural nerves are mediated by substance P (SP). In the present experiments, non-cholinergic neurogenic ileum contractions to field stimulation (100 imp., 5-50 Hz) were inhibited by the opiate agonists morphine and [D-Met2,Pro5]enkephalinamide (10(-6) M) in a naloxone-reversible manner. Neither morphine nor naloxone influenced the musculodirect contracting effect of histamine. Capsaicin, a drug that has been shown to deplete SP from primary afferent neurones, exerted no long-lasting effect on non-cholinergic contractions to field stimulation. Repeated administration of long trains of stimuli (900 impulses) resulted in a progressive decrease of the contractions evoked. Addition of naloxone (3 X 10(-7) M) restored the original height of the responses. The above inhibitory action of opiate agonists and of repeated long-train stimulation was 3-6 times greater at 5 Hz than at 50 Hz. It is concluded that opiate agonists inhibit the release of SP from intramural neurones of the guinea-pit ileum. The decrease in responses to repeated long-train stimulations is mediated, at least in part, by the release of endogenous opioid substance(s).     

Comparison of potency of substance P and related peptides on [3H]-acetylcholine release,and contractile actions,in the guinea-pig ileum

Summary A range of tachykinins including substance P were studied for their ability to contract the guinea-pig ileum longitudinal muscle preparation on brief exposure (20 s) to the peptides, and their ability to evoke the release of [³H]-acetylcholine (ACh) from previously labelled stores within the myenteric plexus. With respect to their immediate spasmogenic activity, none of the peptides differed greatly in potency from substance P. Atropine did not modify the response to the tachykinins suggesting that the release of ACh does not contribute to the contraction resulting from brief exposure to the peptides. In the release studies, all tachykinins used produced a dose-related, calcium-dependent release of [³H]-ACh but the differences in potency were much greater. Eledoisin was the most potent and its evoked release of ACh was unaffected by hyoscine, hexamethonium, guanethidine and naloxone suggesting the release is not mediated via, or modulated by, opiate or autonomic neuronal influences. The two orders of tachykinin potency found suggest that the two processes, initial contraction and ACh release, may be principally mediated via two distinct subclasses of substance P receptor designated SP-P and SP-E respectively.     

Substance P mediates atropine-sensitive response of guinea-pig ileum to serotonin

The action of serotonin (5HT) was investigated on guinea-pig isolated ileum. Low concentrations of 5HT (10(-8) - 2.5 x 10(-7) mol x 1(-1)) produced contractile responses which were abolished by atropine and by methionine enkephalin but not by methysergide. Higher concentrations of 5HT (5 x 10(-7) - 5 x 10(-6) mol x 1(-1)) produced contractions which were inhibited by methysergide. The atropine-sensitive response was also abolished following desensitization of preparations to substance P and by the substance P antagonist [D-Pro2,D-Phe7,D-Trp9]SP. It was concluded that the atropine-sensitive response to 5HT is mediated by substance P.     

Tissue selectivity of substance P alkyl esters: suggesting multiple receptors

Previous studies from this laboratory suggested that two subtypes of substance P receptor may exist, based on the observations that substance P and related peptides did not exhibit complete cross-desensitisation on guinea-pig ileum, and that two distinct rank orders of potency of tachykinins were observed in various test systems. The present study has added support to this hypothesis by extending the screening of tachykinins to further bioassays and by testing novel analogues. In particular, C-terminal alkyl esters of substance P were found to exhibit a high degree of selectivity to one putative receptor subtype. The synthesis of the alkyl esters by esterification of substance P free acid is described.     

The effect of substance P on nerve action potential propagation and cholinergic transmission in the myenteric plexus of the guinea-pig ileum

Summary The effect of substance P on nerve terminals in myenteric plexus of the guinea-pig ileum was investigated. Neurogenic twitches of the myenteric plexus longitudinal muscle strip were recorded. Twitches of the strip portion where excitation involved the most distal parts of cholinergic nerve terminals were more increased by local application of substance P (0.1 and 0.4 nmol/l) than twitches of the portion where excitation involved both distal and proximal parts of nerve terminals. Substance P addition to a portion of the strip conducting nerve action potentials to invade the neighbouring strip portion also augmented twitches of the latter portion so that the interference with the propagation process was considered. The effect of substance P was poorly antagonized by the addition of a substance P antagonist, (D-Arg¹, D-Pro², D-Trp^7,9, Len¹¹)-substance P. Compound nerve action potentials were evoked in strands of fibres of the myenteric plexus by low-frequency train stimulation (1 Hz). The addition of substance P prevented a decrease of the amplitude of responses observed under control conditions. Using high-frequency train stimulation (30 Hz) the amplitude of responses to impulses 2-7 was augmented over that to the first impulse; substance P further increased such facilitation regularly. It seems that substance P might promote nerve action potential invasion of the distal parts of nerve terminals. Send offprint requests to O. Kadlec at the above address     

Involvement of substance P in the excitatory action of GABAA agonists on cholinergic neurons in the guinea-pig ileum

Summary 1. The possible involvement of substance P (SP) in cholinergic contractions induced by GABA_A agonists in the guinea-pig ileum was further investigated. 2. Responses evoked by 3-aminopropane sulphonic acid (3-APS) or muscimol consisted of a rapid phasic contraction followed in 70% of preparations by a tonic contraction, usually smaller in amplitude but considerably longer in duration. Phasic and tonic components were sensitive to bicuculline, neurogenic (cholinergic) in nature and susceptible to desensitization. 3. Capsaicin (0.2 M) pretreatment and SP receptor desensitization caused by 3 different priming SP concentrations (10 nM, 30 nM, 100 nM), depressed both components of the 3-APS-induced response, the magnitude of antagonism being greater for tonic contractions. Similar findings were obtained by using 10 M (d-Pro⁴, d-Trp^7,9)SP-(4–11), even though the degree of antagonism caused by this SP antagonist was consistently lower. 4. These results indicate that depression of SP receptor function achieved by three different procedures decreases cholinergic contractile responses to GABA_A agonists in the guinea-pig ileum. This provides further support for the hypothesis that GABA_A receptor activation evokes both direct and indirect stimulation of enteric cholinergic neurons and that SP and/or a related peptide play an important role in mediating the indirect component of the cholinergic response. Send offprint requests to M. Tonini at the above address     

D-Met2, Pro5]enkephalinamide and dynorphin-(1-13) inhibit the cholinergic contraction induced in the guinea-pig ileum by substance P

In the isolated guinea-pig ileum, the sustained phase of the longitudinal contractile response to substance P is, unlike the initial peak response, mediated by stimulation of cholinergic neurons. This cholinergically mediated response to substance P was inhibited by the specific substance P antagonist, [D-Pro2,D-Trp7,9]substance P, which suggests that substance P stimulates enteric cholinergic neurons through a specific site of action. [D-Met2,Pro5]enkephalinamide and dynorphin-(1-13) also decreased whereas naloxone increased the sustained response to substance P. The results indicate that the enteric cholinergic neurons, which are stimulated by substance P, are also under the control of enkephalin and/or dynorphin neurons.     

Agonist effects of [D-Pro2,D-Phe7,D-Trp9]substance P--evidence for different receptors

[D-Pro2,D-Phe7,D-Trp9]substance P contracted guinea-pig ileum (GPI) indirectly, probably via substance P (SP) receptors on postganglionic parasympathetic neurones. It also depolarised rat superior cervical ganglion cells and contracted the rabbit external jugular vein, suggesting an agonist action on SP receptors in these tissues. In contrast, it had weak antagonist activity on smooth muscle SP receptors in GPI. This suggests that the receptor in GPI smooth muscle may differ from those in the other tissues.     

CP-96,345, a non-peptide antagonist of substance P: I. Effects on the actions mediated by substance P and related tachykinins on the guinea-pig ileum and rabbit jejunum

Summary The effects of a non-peptide antagonist of substance P, CP 96,345, were investigated, in vitro, on the guinea-pig ileum and the rabbit jejunum.Contractions of the guinea-pig ileum, induced by substance P and neurokinin A, were specifically inhibited by the racemate (±)CP-96,345 (pIC₅₀ 7.8 and 7.3, respectively). The inhibition by (±)CP-96,345 of contractions evoked by neurokinin B and by bradykinin (pIC₅₀ 6.1 and 4.9, respectively) was attributed to unspecific effects of the antagonist. The inhibition of substance P-induced contractions of the rabbit jejunum required a 10 times higher concentration of (±)CP-96,345 (pIC₅₀ = 6.8) than was required with the guinea-pig ileum. The plateau phase of contraction of the guinea-pig ileum induced by high concentrations of substance P, neurokinin A or neurokinin B, which is known to be mediated through tachykinin receptors on intrinsic cholinergic neurones, was inhibited by 200 nM (±)CP-96,345 but not by the inactive enantiomer, CP-96,344. This indicates a specific inhibition of these neuronal tachykinin receptors by (±)CP-96,345 Contractions known to be mediated by the release of substance P, such as those evoked by capsaicin and by mesenteric nerve or field stimulation, were partially inhibited by (±)CP-96,345 at concentrations of 200 to 600 nM. Unspecific inhibitory effects of CP-96,345, in concentrations of 1 µM or higher, were observed on histamine-induced contractions, and on the cholinergic twitch response to electrical stimulation, of the guinea-pig ileum. Therefore, an inhibition by CP-96,345 of substance P-related effects can only be regarded as specific if the concentration of the antagonist is below 1 µM. No effect of CP-96,345 (1 µM) was seen on peristalsis in vitro. The peristaltic reflex, in situ, was also not affected by CP-96,345 (1.6 µmolkg^–1, i.v.).The present results demonstrate that, although inhibitory actions of CP-96,345 can be observed on certain motor responses in the guinea-pig small intestine, peristalsis, a physiologically relevant function, remains intact. Send offprint requests to F. Lembeck at the above address     

Direct evidence for a release of acetylcholine from the myenteric plexus of guinea pig small intestine by substance P

Myenteric plexus-longitudinal muscle strips from guinea pig small intestine were labeled with [3H]choline while under continuous field stimulation. Release of newly synthesized [3H]acetylcholine was studied in the presence of substance P afterwards. Substance P evoked release of [3H]acetylcholine in a dose-related fashion. Both tetrodotoxin and [D-Pro2,D-Phe7,D-Trp9]substance P, a synthetic antagonist of substance P, completely blocked this release and provided evidence for a cholinergically mediated mechanism of substance P on enteric neurons.     

ATP release caused by bradykinin, substance P and histamine from intact and cultured smooth muscles of guinea-pig vas deferens

Histamine (60 μM) produced ATP release from segments of guinea-pig vas deferens which was blocked by pyrilamine and triprolidine, H₁-blockers, but not by ranitidine, an H₂-blocker. The evoked-release was inhibited by the mitochondrial inhibitors, carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and oligomycin. Bradykinin (BK) and substance P (SP) also caused substantial and moderate release of ATP, respectively. The BK-evoked release of ATP was inhibited by HOE140, a B₂-antagonist, but not by [Des-Arg¹⁰] HOE140, a B₁-antagonist. On the other hand, VIP, angiotensin II (AII) and cholecystokinin-octapeptide (CCK-8) failed to elicit a measurable release of ATP. Histamine and BK also enhanced the release of ATP from superfused cultured smooth muscle cells. These results suggest that ATP may be released as an autacoid from the smooth muscles in the presence of these chemical mediators. Received: 13 August 1997 / Accepted: 26 November 1997     

Inhibitory effects of procaine on the contractile responses of the guinea-pig Taenia caecum to acetylcholine,substance P and potassium chloride

Summary The effect of procaine on the contractile responses to acetylcholine, substance P and KCl was investigated using the isolated guinea-pig taenia caecum. In normal Tyrode solution (37°C), procaine (10–100 mol/l) caused a parallel shift to the right of only the dose-response curve of acetylcholine (pA₂ value, 5.11). The pA₂ value of procaine against acetylcholine was not significantly affected by increasing the Ca concentration in the bathing solution from 0.9 to 7.2 mmol/l. On the other hand, a high concentration of procaine (10 mmol/l) caused a transient contraction of the taenia caecum, but completely suppressed contractions to all concentrations of the agonists. In K-depolarized preparations, procaine (1–10 mmol/l) shifted the dose-response curve for the CaCl₂-induced contraction to the right. Substance P (3 mol/l)-induced contraction of the taenia caecum incubated with Ca-free EGTA (0.1 mmol/l) solution (20°C) was markedly reduced by procaine (10 mmol/l). Using the single sucrose-gap technique, it was found that procaine (10 mmol/l) produced a membrane depolarization and increases in both amplitude and frequency of spontaneous spike discharge. These potential changes were still observed even after the procaine-induced contraction had disappeared. The spike discharges and contraction caused by procaine were abolished in the presence of a Ca-entry blocker, verapamil (10 mol/l). From these observations, it is concluded that at low concentrations procaine acts as a competitive antagonist of muscarinic receptors in the guinea-pig taenia caecum while high concentrations of procaine may depress the contractile responses to acetylcholine, substance P and KCl by inhibiting the Ca-induced Ca release from the intracellular store site or by reducing the transmembrane Ca influx during depolarization.     

In vitro release of substance P from spinal cord slices by capsaicin congeners

The in vitro release of immunoreactive substance P (I-SP) by capsaicin and three congeners was studied on slices of rat spinal cord upper dorsal horn. Capsaicin and its congeners were all able to stimulate I-SP release, indicating that they act on chemosensitive primary afferents terminating in this region. A positive correlation was found between the I-SP releasing and pain-producing potencies of these compounds. This is in agreement with the concept that primary afferents containing substance P (SP) are involved in the transmission of nociceptive information.     

Nitric oxide induces acetylcholine-mediated contractions in the guinea-pig small intestine

In longitudinal muscle/myenteric plexus preparations of the guinea-pig ileum, exogenous nitric oxide (NO) induced concentration-dependent relaxations. In tissues at basal tone, NO (3 × 10^–6 M) induced a moderate relaxation followed by a pronounced contraction, consisting of a quick and sustained component. Tetrodotoxin (5 × 10^–7 M) abolished both phases of the contraction. Atropine (5 × 10^–7 M) abolished the quick component and reduced the sustained component of the contraction; the latter was further suppressed by the selective NK₁ receptor antagonist CP 96,345. Hexamethonium (5 × 10^–5 M) failed to affect the contractile response to NO. It is concluded that administration of exogenous NO in the guinea-pig ileum can lead to activation cholinergic and to a lesser degree tachykininergic neurones. Correspondence to: L. Barthó at the above address     

Lack of effect of baclofen on substance P and somatostatin release from the spinal cord in vitro

Summary High concentrations of K⁺ increase the release of substance P (SP) and somatostatin (SRIF) from superfused slices of rat spinal cord. This increase is Ca-dependent. Baclofen (100–500 M) does not significantly alter the K⁺-evoked release of SP or SRIF. Stereoisomers of baclofen and GABA, similarly, are without effect. The spinal analgesic action of baclofen does not appear to be due to alterations in the release of SP or SRIF.     

Adenosine analogs do not inhibit the potassium-stimulated release of substance P from rat spinal cord slices

Summary Adenosine agonists produce antinociception when injected directly onto the spinal cord of rats and mice. One mechanism to account for this effect could be inhibition of neurotransmitter release from nociceptive sensory neurons. Consequently, we studied whether these agents could inhibit the potassium stimulated release of one such transmitter, substance P, from rat spinal cord slices. A 2 cm section of lumbar spinal cord was dissected from male Sprague-Dawley rats, chopped into 0.5 × 0.5 mm sections and perfused at 37°C with a modified Krebs bicarbonate buffer containing either 3.5 mM, 30 mM, or 50 mM KCl in the presence and absence of various adenosine analogs. Perfusates, collected every 2 min, were assayed for substance P by radioimmunoassay. Exposure of tissue to 50 mM KC1 produced an approximate three-fold increase in the release of substance P over basal release. This increase in release was calcium dependent. Perfusion of spinal cord tissues with either adenosine (10^–3 M), N⁶-cyclohexyladenosine (10^–5 M or 5 × 10^–5 M), 5-N-ethylcarboxamide adenosine (10^–5 M) or L-N⁶-phenylisopropyladenosine (10^–5 M) did not significantly alter basal or potassium-stimulated release of SP when compared to controls. In contrast to the adenosine agonists, exposure of the spinal cord tissue to 10^–5 M morphine significantly reduced the potassium-stimulated release of substance P. Pretreatment of the slices with 10^–5 M theophylline or 8-phenyltheophylline did not significantly attenuate the inhibition of substance P release produced by morphine. Theophylline alone (10^–5 M) had no significant effect on either basal or potassium-stimulated release of SP. These studies demonstrate that adenosine does not inhibit the release of SP from spinal cord slices and does not appear to mediate the morphine-induced inhibition of SP release. The results suggest that the mechanism of the antinociceptive effects of adenosine at the level of the spinal cord is not via inhibition of substance P release. Send offprint requests to M. R. Vasko at the above address     

Comparison of crude bovine subcortical substance P with synthetic substance P

Summary Synthetic substance P (SP) was compared with the gut-contracting peptide Fa from bovine subcortex by means of thin-layer chromatography, paper electrophoresis, enzymatic digestion, and biological assays. With all four techniques differences in behaviour between Fa and synthetic SP were found, which lead to the conclusion that both active principles are similar but not identical. Both materials could also be distinguished from synthetic physalaemin with the aid of paper electrophoresis and thin-layer chromatography.     

P物质在急性坏死性胰腺炎肠源性胰腺感染中的作用

目的 研究急性坏死性胰腺炎(ANP)时大鼠回肠组织中P物质的表达, 初步探讨P物质在ANP肠源性胰腺感染中的作用.方法 健康成年S-D大鼠,随机分为对照组和ANP组,每组24只.对照组开腹后只翻动胰腺, ANP组大鼠胰胆管恒速逆行注射5%牛黄胆酸钠, 制成ANP大鼠模型.两组大鼠均于8、16h处死取标本,检测P物质在回肠组织中的表达水平和组织学定位,测定肠黏膜通透性(99m Tc-DTPA排泄率),并分别行胰腺组织和小肠内容物细菌培养.结果 ANP组大鼠肠粘膜病理学评分以及胰腺组织细菌培养阳性率较对照组显著升高(P＜0.05);P物质表达于大量浸润的炎症细胞、淋巴细胞表面.结论 ANP时大鼠回肠组织中P物质表达升高; P物质可能与多种细胞共同作用, 加重ANP病情.