Loránd Barthó

Inhibition of neurogenic vasodilatation and plasma extravasation by substance P antagonists, somatostatin and [D-Met2, Pro5]enkephalinamide

The substance P (SP) analogues [D-Pro2, D-Phe7, D-Trp9]SP and [D-Pro2, D-Trp7,9]SP, which have been reported to be SP antagonists, inhibited the vasodilation and plasma extravasation induced by antidromic stimulation of the saphenous nerve or by i.a. infusion of SP. Somatostatin inhibited the vasodilatation and plasma extravasation induced by saphenous nerve stimulation, but had no effect on the vascular responses to i.a. infused SP. The opiate agonist [D-Met2, Pro5]enkephalinamide inhibited the vasodilation evoked by antidromic nerve stimulation in a naloxone reversible manner, but did not change the effect of i.a. infusion of SP. Calcitonin and caerulein had no effect on neurogenic vasodilatation. These results further support the concepts that neurogenic vasodilatation and plasma extravasation are mediated by SP, and that somatostatin and opiates inhibit the release of SP from peripheral sensory nerve endings.

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

1. The possible roles of substance P and opioids in the contractile response of the isolated guinea‐pig ileum to the sensory stimulant drug capsaicin were investigated, and the contractions were found to be inhibited by about 60% in preparations desensitized to substance P. 2. Contractions evoked by stimulation of the mesenteric nerves in the presence of the adrenergic blocking drug guanethidine were inhibited by about 75% after the ileum had been rendered insensitive to substance P. 3. Atropine partially inhibited the effect of capsaicin. The atropine‐resistant component of the contractile response to capsaicin was inhibited by more than 85% in preparations desensitized to substance P and almost abolished by the substance P antagonist, (D‐Pro2,D‐Trp7,9)‐substance P. 4. The opioid peptide (D‐Met2, Pro5)‐enkephalinamide inhibited, whereas the opiate antagonist naloxone enhanced the atropine‐resistant contractions in response to capsaicin. 5. The results indicate that the contractile response of the guinea‐pig ileum to capsaicin and mesenteric nerve stimulation is mediated by release of substance P, presumably from sensory nerve endings in the gut. Substance P appears to act on the smooth muscle both directly and indirectly via cholinergic neurones. It is proposed that opioids modulate the non‐cholinergic response to capsaicin by inhibiting the release of substance P.

Opiate agonists inhibit neurogenic plasma extravasation in the rat

Neurogenic plasma extravasation (NPE) evoked by antidromic stimulation of the saphenous nerve was inhibited by more than 50% by i.p. administration of the synthetic enkephalin analogue (D-Met2, Pro5)-enkephalinamide, 0.3-10 mg/kg and, to a lesser extent, by morphine. The effect of enkephalinamide (10 mg/kg) was reversed by naloxone (1 mg/kg, s.c.). Evidence is presented that the effect of enkephalinamide was not secondary to haemodynamic changes or alteration of mast cell function. It is suggested that opiate agonists inhibit NPE through specific opiate receptors, located peripherally on sensory nerve endings, by inhibiting the release of mediator(s) responsible for NPE.

Evidence for the involvement of substance P in the atropine-resistant peristalsis of the guinea-pig ileum ☆

In the isolated, vascularly perfused guinea-pig ileum, peristalsis could be induced by raising the intraluminal pressure in the presence of atropine. Atropine-resistant peristalsis was greatly inhibited or abolished by the substance P antagonist (D-Pro2, D-Trp7.9)-substance P, substance P desensitization, hexamethonium and by the enkephalin analogue FK 33-824. It is concluded that substance P neurones of the intestine play an important role in the atropine-resistant peristalsis of the guinea-pig ileum.

Activity of SR 142801 at peripheral tachykinin receptors

The pharmacological profile of the novel tachykinin NK3 receptor antagonist SR 142801, ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide), was studied at tachykinin NK1, NK2 and NK3 receptors, in several in vitro bioassays. In the guinea-pig isolated ileum longitudinal muscle preparation, SR 142801 (10 nM-1 microM) caused an insurmountable antagonism of tachykinin NK3 receptor-mediated contractions produced by senktide (apparent pKB = 9.27). The blockade induced by SR 142801 was essentially irreversible, since it was not removed by washout (up to 2 h) and was increased by prolonging the incubation from 15 to 120 min. SR 142801 showed similar antagonist potency at rat tachykinin NK3 receptors (portal vein) and rabbit tachykinin NK2 receptors (pulmonary artery) (pKB = 7.49 and 7.66, respectively), whereas it was distinctly less potent at hamster tachykinin NK2 receptors (trachea; pKB = 6.84) and inactive at guinea-pig tachykinin NK1 receptors (ileum, longitudinal muscle). In the guinea-pig whole ileum SR 142801 (100 nM) did not affect the contraction produced by capsaicin (1 microM). The combined SR 142801 pretreatment and tachyphylaxis of neuronal CGRP (calcitonin gene-related peptide) receptors produced a slight (about 25%), but significant reduction of the response to capsaicin, suggesting that tachykinin NK3 receptors play a minor role in capsaicin-induced neuronal excitation of afferent nerves in the guinea-pig ileum.

Intestinal peristalsis associated with release of immunoreactive substance P.

The release of immunoreactive substance P into the vascular bed of the isolated small intestine of the guinea-pig was investigated. Raising the intraluminal pressure to 5 mbar for 5 min initiated peristalsis and stimulated the release of substance P. The substance P releasing effect of pressure stimulation was reduced by 46% when hexamethonium (240 microM) was added to the perfusion solution. The ganglion stimulant drug dimethylphenylpiperazinium (32 microM) also stimulated the release of substance P; its effect was completely prevented by hexamethonium (240 microM). Intraarterial infusion of capsaicin (22 microM), a neurotoxin known to act on sensory substance P-containing neurones, stimulated the release of substance P and caused intestinal contractions. The motor effect of capsaicin in the gut can thus be explained by release of substance P from sensory nerve endings in the gut. Systemic pretreatment of the guinea-pigs with capsaicin abolished the release of substance P due to capsaicin, whereas that evoked by elevated intraluminal pressure or dimethylphenylpiperazinium was not reduced. This means that substance P released in the course of peristalsis or by dimethylphenylpiperazinium originates from neurones intrinsic to the intestine. These findings indicate that intestinal peristalsis is associated with the release of substance P from enteric neurones. Substance P is likely to be a neurotransmitter involved in the coordination of the peristaltic reflex.

Tachykinin NK1 and NK2 receptor-mediated control of peristaltic propulsion in the guinea-pig small intestine in vitro

The tachykinins substance P and neurokinin A are excitatory cotransmitters of cholinergic enteric neurons, their actions being mediated by NK1, NK2 and NK3 receptors. This study examined which of these receptors are part of the neural circuitry of peristalsis. Peristaltic propulsion in luminally perfused segments of the guinea-pig isolated ileum was elicited by a rise of the intraluminal pressure. The pressure threshold at which peristaltic contractions were triggered was used to quantify drug effects on peristalsis, inhibition of peristalsis being reflected by an increase in the pressure threshold. The NK1, NK2 and NK3 receptor antagonists SR-140333, SR-48968 and SR-142 801 (each at 0.1 microM), respectively, had little effect on peristaltic activity as long as cholinergic transmission was left intact. However, both the NK1 and NK2 receptor antagonist (each at 0.1 microM) abolished peristalsis after cholinergic transmission via muscarinic receptors had been blocked by atropine (1 microM) and peristalsis rescued by naloxone (0.5 microM). When cholinergic transmission via nicotinic receptors was suppressed by hexamethonium (100 microM) and peristalsis restored by naloxone (0.5 microM), only the NK2 receptor antagonist (0.1 microM) was able to attenuate peristaltic performance as deduced from a rise of the peristaltic pressure threshold by 106%. The NK3 receptor antagonist (0.1 microM) lacked a major influence on peristalsis under any experimental condition. It is concluded that tachykinins acting via NK1 and NK2 receptors sustain intestinal peristalsis when cholinergic neuroneuronal and neuromuscular transmission via muscarinic receptors has been suppressed. NK2 receptors help maintaining peristalsis once cholinergic neuroneuronal transmission via nicotinic receptors has been blocked, whereas NK3 receptors play little role in the neural pathways of peristalsis.

Two types of relaxation due to capsaicin in the guinea pig isolated ileum

The relaxant effect of capsaicin was investigated on isolated segments of guinea pig ileum precontracted by histamine. Two types of relaxation could be distinguished. Low concentrations of capsaicin (0.033-3.3 microM) caused partial relaxation (type I) which was prevented by in vitro desensitization to capsaicin or by extrinsic denervation and reduced by tachyphylaxis to calcitonin gene-related peptide. At higher concentrations (10-100 microM) capsaicin caused relaxation (type II) to the baseline; this effect was insensitive to capsaicin desensitization or extrinsic denervation. Neither type of relaxation was inhibited by tetrodotoxin or by alpha- and beta-adrenoceptor antagonists. We suggest that type I relaxation results from a specific effect of capsaicin on extrinsic sensory neurones of the gut while type II represents a non-specific smooth muscle depressant effect. Intrinsic enteric neurones are not involved in these effects.

Peripheral effects of opioid drugs on capsaicin-sensitive neurones of the guinea-pig bronchus and rabbit ear

SummaryThe effect of a potent opioid agonist, [d-Met2, Pro5]-enkephalinamide was investigated on two responses involving capsaicin-sensitive afferent neurones, namely, atropine-resistant contractions of the guinea-pig bronchus evoked by electrical field stimulation and the nociceptor stimulation to intraarterial injections of acetylcholine or capsaicin into the vascularly isolated rabbit ear. The hypotheses to be tested were whether (a) opioid receptor activation may inhibit mediator release from primary afferent neurones and (b) the opioid could exert an analgesic effect at a peripheral site of action. Non-cholinergic contractions of the guinea-pig isolated main bronchi due to electrical stimulation were concentration-dependently inhibited by [d-Met2, Pro5]-enkephalinamide (10 nM–1 μM). This effect was abolished by naloxone (1 μM). Naloxone alone induced no change in the stimulation-evoked contractions of the bronchus, indicating that no endogenous opioid control was present. Substance P and neurokinin A induced bronchial contractions that were not influenced by [d-Met2, Pro5]-enkephalinamide. This indicates that [d-Met2, Pro5]-enkephalinamide inhibits electrically-evoked bronchial contractions by reduced mediator release from capsaicin-sensitive sensory nerve endings, since these contractions are most probably brought about by tachykinins, released from afferent neurones. Capsaicin-induced bronchial contractions were in contrast to electrical stimulation not influenced by [d-Met2, Pro5]-enkephalinamide which suggests a different site of action. The activation of sensory neurones in the rabbit ear by i. a. injection of acetylcholine and capsaicin was not reduced under infusion of [d-Met2, Pro5]-enkephalinamide (1 and 10 μM) or lofentanil (1 and 10 μM). The enhancement of the effect of acetylcholine by infusion of prostaglandin E2 (0.15 μM) also remained unchanged under infusion of 10 μM [d-Met2, Pro5]-enkephalinamide. A peripheral analgesic action of the two opioid agonists studied is therefore not indicated.

Calcitonin gene-related peptide is a potent relaxant of intestinal muscle.

The motor effects of calcitonin gene-related peptide (CGRP) on the guinea-pig ileum (GPI) longitudinal muscle were investigated. CGRP (0.1-100 nM) concentration dependently relaxed ilea precontracted with histamine and inhibited electrically induced cholinergic contractions. At higher concentrations (3-100 nM) CGRP also caused a slight contraction of non-stimulated ilea. Further pharmacological analysis of the CGRP-induced relaxation which seems to be the primary CGRP effect in the GPI, indicated that the effect was due to a direct action on the longitudinal muscle.