Cannabinoid inhibition of guinea-pig intestinal peristalsis via inhibition of excitatory and activation of inhibitory neural pathways.

Since activation of cannabinoid CB1 receptors inhibits gastrointestinal transit in the mouse, this study analyzed the action of the cannabinoid receptor agonist methanandamide on distension-induced propulsive motility. Peristalsis 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. Methanandamide (0.1-3 microM) inhibited peristalsis as deduced from a concentration-related increase in the peristaltic pressure threshold, an action that was prevented by the CB1 receptor antagonist SR141716A (1 microM) per se, which had no effect on peristalsis. The distension-induced ascending reflex contraction of the circular muscle was likewise depressed by methanandamide in a SR141716A-sensitive manner, whereas indomethacin-induced phasic contractions of the circular muscle were left unchanged by methanandamide. The anti-peristaltic action of methanandamide was inhibited by apamin (0.5 microM), attenuated by N-nitro-L-arginine methyl ester (300 microM) and left unaltered by suramin (300 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (150 microM) and naloxone (0.5 microM). It is concluded that methanandamide depresses intestinal peristalsis via activation of CB1 receptors on enteric neurons, which results in blockade of excitatory motor pathways and facilitation of inhibitory pathways operating via apamin-sensitive K+ channels and nitric oxide.     

5-HT7 receptors mediate the inhibitory effect of 5-HT on peristalsis in the isolated guinea-pig ileum

The study was undertaken to investigate the 5-HT receptor mediating the inhibitory effect of 5-HT on peristalsis in the guinea-pig isolated ileum. The facilitatory and inhibitory effects were measured as the decrease and increase, respectively, in the intraluminal pressure required to trigger peristalsis. In the presence of 5-HT(2/3&4) receptor antagonists ketanserin (0.1 micro M), granisetron (1 micro M) and SB-204070 (1 micro M), a cumulative addition (0.1-100 micro M) of 5-HT or 5-carboxamidotryptamine, but not 2-methyl-5-HT produced a concentration-dependent increase in the threshold required to trigger peristalsis. The 5-HT(7) receptor selective antagonist SB-269970-A (0.01-1 micro M) or methiothepin (0.01-0.1 micro M) concentration-dependently antagonised this response to 5-HT. SB-269970-A (1 micro M) and methiothepin (1 micro M) were also able to restore peristalsis in tissues in which peristalsis was inhibited by a prior addition of 30 micro M of 5-HT. The results indicate an involvement of 5-HT(7) receptors in the inhibitory effect of 5-HT on peristalsis in the guinea-pig ileum.     

Purine receptors and guinea-pig trachea: evidence for a direct action of ATP.

1. Adenosine (3-300 microM) and ATP (1-300 microM) each induced concentration-dependent relaxation of guinea-pig isolated tracheal preparations that had been precontracted with methacholine. 2. Ectonucleotidase enzymes on the trachea dephosphorylated ATP to form adenosine which was then further metabolised to inosine and hypoxanthine. 3. Dipyridamole (2 microM) inhibited the metabolism of adenosine but did not inhibit the dephosphorylation of ATP. Nevertheless dipyridamole potentiated the effects of both adenosine and ATP in relaxing tracheal smooth muscle. 4. Although adenosine 5'-[beta,gamma-imido] triphosphate (AMP-PNP), an analogue of ATP, was resistant to catabolism by ectonucleotidases, it was more potent at inducing relaxation than either ATP or adenosine, and was also potentiated by dipyridamole (2 microM). 5. Relaxations induced by ATP and by AMP-PNP were more rapid than those induced by adenosine. 6. We therefore conclude that the intact ATP molecule can itself induce relaxation of the guinea-pig trachea, without first having to be metabolised to adenosine, and furthermore that dipyridamole does not act simply by inhibiting the degradation (or uptake) of adenosine.     

Modulation of peristalsis in the guinea-pig isolated small intestine by exogenous and endogenous opioids.

1. A recording method was developed to measure physiological parameters of the preparatory and emptying phases of peristalsis in vitro. This method enabled measurement of: the compliance of the intestinal wall during the preparatory phase (a reflection of the resistance of the wall to distension); longitudinal muscle contraction during the preparatory phase; the threshold volume required to trigger the emptying phase; the maximal ejection pressure and the average power generated during the emptying phase, which reflects the rate at which the intestine performs work. Modulation of these parameters by exogenous and endogenous opioids acting at mu, kappa and delta opioid receptors was investigated. 2. The compliance of the intestinal wall during the preparatory phase was reduced by the mu opioid receptor agonist, [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin (DAMGO) but not by the kappa agonist, dynorphin, or the delta agonist, [D-penicillamine2, D-penicillamine5] enkephalin (DPDPE). Reflex contraction of the longitudinal muscle during the preparatory phase was inhibited by DAMGO, dynorphin and DPDPE. The threshold volume required to trigger the emptying phase of peristalsis was increased by DAMGO, dynorphin and DPDPE. 3. The maximal ejection pressure generated during the emptying phase was reduced by dynorphin and DPDPE, but not by DAMGO. The average power generated by the intestine when emptying was not altered by any of the agonists. 4. Electrically stimulated contractions of longitudinal muscle in strips of longitudinal muscle-myenteric plexus were not inhibited by DPDPE. Similarly, DPDPE did not significantly inhibit electrically induced contraction of circular muscle in strips of circular muscle-myenteric plexus.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibitory modulation of guinea-pig intestinal peristalsis caused by capsaicin involves calcitonin gene-related peptide and nitric oxide

The effect of capsaicin-induced stimulation of afferent neurons on peristalsis and the possible neural mediators involved in this action were examined in the guinea-pig isolated ileum. The intraluminal pressure threshold for eliciting peristaltic waves was used to quantify facilitation (decrease in threshold) or inhibition (increase in threshold) of peristalsis. Capsaicin (0.1–1 M) caused an initial short-lasting stimulation of peristalsis followed by a prolonged inhibition of peristaltic activity. Capsaicin (1 M) was ineffective when the gut segments had been pretreated with 3.3 M capsaicin, which is indicative of an afferent neuron-dependent action of the drug. In contrast, the abolition of peristalsis caused by a high concentration of capsaicin (33 M) was fully reversible on removal and reproducible on readministration of capsaicin, a feature characteristic of a nonspecific depression of smooth muscle excitability. Baseline peristalsis and the excitatory/inhibitory effect of capsaicin (1 M) on peristalsis remained unaltered by a combination of the tachykinin NK₁ receptor antagonist ( + )-(2S, 3S)-3-(2-methoxybenzylamino)-2-phenyl piperidine (CP-99,994; 0.3 M) and the tachykinin NK₂ receptor antagonist L(-)-N-methyl-N[4-acetylamino-4-phenyl-piperidino-2-(3,4-dichlorophenyl)butyl]-benzamide (SR-48,968; 0.1 M). Further experiments, performed in the presence of a low concentration of atropine (10 nM) showed that the catcitonin gene-related peptide (CGRP) antagonist human -catcitonin gene-related peptide (8–37) [hCGRP (8–37); 10 M] attenuated the delayed inhibitory effect of capsaicin on peristalsis, but did not influence baseline peristaltic activity and the capsaicin-induced facilitation of peristalsis. Blockade of nitric oxide (NO) synthesis by N ^G-nitro-l-arginine methylester (l-NAME, 300 M) facilitated baseline peristaltic activity and reduced the delayed inhibition of peristalsis caused by capsaicin (1 M) without affecting the initial peristalsis-stimulating action of capsaicin. The effects of l-NAME were prevented by l-arginine (1 mM). The data of the current study indicate that capsaicin-sensitive afferent neurons do not participate in the neural pathways subserving peristalsis in the guinea-pig small intestine, but modulate peristaltic activity upon stimulation with capsaicin. The initial stimulant action of capsaicin on peristalsis is independent of tachykinins acting via NK₁ or NK₂ receptors, while the delayed capsaicin-induced depression of peristalsis involves CGRP and NO.     

Enhancement of guinea-pig intestinal peristalsis by blockade of muscarinic M1-receptors.

1. The effects of pirenzepine and hyoscine on the peristaltic reflex were investigated in the guinea-pig isolated small intestine. Peristalsis was induced by raising the intraluminal pressure and the volume of fluid propelled was taken as a measure of the efficiency of peristaltic activity. 2. Low concentrations of pirenzepine (0.1-1 nM) and of hyoscine (0.01 nM) significantly enhanced peristalsis, whereas larger concentrations of both drugs caused inhibition. Pirenzepine was about 6 times less potent than hyoscine in increasing peristalsis, but was about 100 times less potent in inhibiting it. 3. Neither tolazoline (1 microM) nor naloxone (0.3 microM) affected the stimulatory action of pirenzepine on peristalsis. 4. Bicuculline increased the efficiency of peristalsis at concentrations of 1 microM and 10 microM; at 10 nM, bicuculline reduced significantly the increase of peristalsis by pirenzepine. gamma-Aminobutyric acid (GABA) did not affect peristaltic activity, but the stimulatory effect of pirenzepine was abolished in the presence of 100 microM GABA. 5. The results indicate that activation of neuronal M1-receptors causes inhibition of small intestinal peristalsis. Bicuculline-sensitive 'GABAergic' synapses are probably involved in this inhibition.     

An inhibitory action of histamine on the guinea-pig ileum

1. In atropinized, plexus-containing preparations of the longitudinal muscle from the guinea-pig ileum, in which histamine contractions were abolished by mepyramine or diphenhydramine, an inhibitory action of histamine was revealed on the "tetanic spasms" produced by field stimulation.2. The inhibitory action of histamine on the atropine-resistant tetanic spasms, which are due to the excitation of non-cholinergic neurones in Auerbach's plexus (Ambache & Freeman, 1968a, b), was reversible. It is specific for the tetanic spasms, because histamine did not reduce contractions elicited by bradykinin, 5-hydroxytryptamine, prostaglandin E(2), nicotine or dimethylphenylpiperazinium.3. L-Histidinol and 2-mercaptohistamine exerted a considerably weaker inhibitory effect upon the tetanic spasms than histamine. Four other imidazoles tested, L-histidine, murexine, dihydromurexine and imidazolecarboxylcholine, were ineffective; so was the pyrazole ring isomer of histamine, betazole.4. The inhibitory action of histamine persisted after adrenoceptor blockade by phentolamine and pronethalol and after prior reserpinization of the guineapigs.5. The inhibitory action of histamine was also obtained after ganglionic paralysis by hexamethonium or dimethylphenylpiperazinium but was antagonized specifically by nicotine.6. On atropinized preparations of the longitudinal muscle from the guinea-pig descending colon histamine exerted, at most, an insignificant inhibitory effect on the tetanic spasms.     

Stimulant action of pituitary adenylate cyclase-activating peptide on normal and drug-compromised peristalsis in the guinea-pig intestine.

1. Pituitary adenylate cyclase-activating peptide (PACAP) is known to influence the activity of intestinal smooth muscle. This study set out to examine the action of PACAP on normal and drug-inhibited peristalsis and to shed light on its site and mode of action. 2. Peristalsis in isolated segments of the guinea-pig small intestine was elicited by distension through a rise of the intraluminal pressure. Drug-induced motility changes were quantified by alterations of the peristaltic pressure threshold at which aborally moving peristaltic contractions were triggered. 3. PACAP (1-30 nM) stimulated normal peristalsis as deduced from a concentration-related decrease in the peristaltic pressure threshold (maximum decrease by 55%). The peptide's stimulant effect remained intact in segments pre-exposed to apamin (0.5 microM), N-nitro-L-arginine methyl ester (300 microM), naloxone (0.5 microM), atropine (1 microM) plus naloxone (0.5 microM) or hexamethonium (100 microM) plus naloxone (0.5 microM). 4. PACAP (10 nM) restored peristalsis blocked by morphine (10 microM), noradrenaline (1 microM) or N6-cyclopentyladenosine (0.3 microM) and partially reinstated peristalsis blocked by Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine (100 microM) but failed to revive peristalsis blocked by hexamethonium (100 microM) or atropine (1 microM). The peptide's spectrum of properistaltic activity differed from that of naloxone (0.5 microM) and forskolin (0.3 microM). 5. The distension-induced ascending reflex contraction of the circular muscle was facilitated by PACAP (1-30 nM) which itself evoked transient nerve-mediated contractions of intestinal segment preparations. 6. These data show that PACAP stimulates normal peristalsis and counteracts drug-induced peristaltic arrest by a stimulant action on excitatory enteric motor pathways, presumably at the intrinsic sensory neurone level. The action of PACAP seems to involve multiple signalling mechanisms including stimulation of adenylate cyclase.     

The action of ATP in the guinea-pig heart

Experiments were designed to compare the potencies of ATP and adenosine in the isolated guinea-pig heart. Adenosine appeared to be twice as potent as ATP in slowing the heart; however, ATP was broken down to AMP and adenosine in the perfusion fluid. ATP was completely dephosphorylated to adenosine before uptake by the heart. It is concluded that breakdown of ATP to adenosine precedes pharmacological action in the guinea-pig heart in situ, where these compounds appear to be equipotent. The potency of ATP itself remains elusive; however, the results suggest that the adenyl phosphates are pharmacologicall active.     

Pharmacological characterization of ATP receptors mediating vasodilation on isolated rabbit aorta.

1. The effects of adenosine 5'-triphosphate (ATP), 2-methyl-thio-ATP and adenosine on rabbit aorta were examined in isolated preparations precontracted by noradrenaline, both in vessels where the endothelium was present and mechanically removed. 2. In the presence of endothelium, ATP (30 microM-3 mM) induced a relaxation that was reduced by removal of the endothelium. 3. The maximum endothelium-dependent relaxation of ATP was twice the maximum endothelial activity of the potent agonist at P2y-purinoceptors, 2-methyl-thio-ATP. 4. Adenosine which acts on P1 purinoceptors, induced a relaxant effect at 1 mM concentration, both in the vessels with and without endothelium. 5. It is concluded that relaxation by ATP is induced both via the endothelial P2y-purinoceptor and via a "nucleotide" receptor that is located on endothelium and on smooth muscle of rabbit aorta.     

An inhibitory action of tetracyclines on guinea-pig myenteric plexus

In plexus containing preparations of the longitudinal muscle of the guinea-pig ileum, an inhibitory action of tetracyclines on twitch-responses to electrical field stimulation was found. Tetracycline, chlortetracycline, minocycline and doxycycline, but not oxytetracycline (0.02 to 1.6 mmol/l) caused a concentration-dependent presynaptic inhibition of acetylcholine release. The inhibitory effect of the tetracyclines was also obtained after ganglion block by hexamethonium (30 mumol/l). The inhibitory effect of the tetracyclines was not antagonized by piperoxan (2 mumol/l) or yohimbine (1 mumol/l) and was partly reduced by the presence of naloxone (1 to 50 nmol/l). After exposing the preparation the peptidase inhibitors, i.e., to the combination of bestatin (10 mumol/l), captopril (10 mumol/l) and thiorphan (0.3 mumol/l), the inhibitory effect of tetracyclines was significantly increased. From these results it would appear that twitch-inhibition caused by tetracycline, chlortetracycline, minocycline and doxycycline is mainly mediated via the release of endogenous opioids from the myenteric plexus.     

The inhibitory action of zinc sulphate on the contractile activity of guinea-pig ileum.

The present study examines the inhibitory action of zinc sulphate (ZnSO4) on the contractile response of various agonists on guinea-pig isolated ileum. Different doses of agonists were selected to produce similar contractile activity, in order to compare the degree of inhibition produced by ZnSO4. Preincubation of ileum with ZnSO4 1 x 10(-3) or 3 x 10(-3) M for 10 min dose-dependently and significantly prevented the contraction induced by acetylcholine (1.7 x 10(-8) M), 5-HT (2.4 x 10(-6) M), histamine (5.4 x 10(-7) M) and nicotine (1.7 x 10(-6) M) but not by prostaglandin E2 (PGE2, 8.5 x 10(-9) M). The same doses of ZnSO4 reduced the twitch contraction produced by electrical field stimulation. These findings indicate that the contractile activity of PGE2 is mediated by a mechanism different from that of other agonists and of electrical field stimulation. It is likely that the contractile activity of PGE2 is acting through the receptors on the ileal muscle which are not blocked by ZnSO4 pretreatment.     

Characterization of 5-hydroxytryptamine receptors mediating mucosal secretion in guinea-pig ileum.

1. The receptor subtypes through which 5-hydroxytryptamine (5-HT) increases electrolyte secretion across the mucosa of guinea-pig ileum were studied. 2. Flat sheep preparations of guinea-pig mucosa plus submucosa were placed in Ussing chambers and the short circuit current (ISC), an index of net electrogenic electrolyte transport across the mucosa, was measured under voltage clamp conditions. 3. Low concentrations of 5-HT (10-300 nM) evoked monophasic increases in ISC which were significantly reduced by hyoscine (100 nM), tetrodotoxin (TTX, 300 nM) and the 5-HT2 receptor antagonist, ketanserin (3-300 nM). 4. Higher concentrations of 5-HT (1-10 microM) produced biphasic responses which were reduced by hyoscine (100 nM), TTX (300 nM), ketanserin (3-300 nM) and also by the 5-HT3 receptor antagonists, granisetron (1 microM) and ICS 205-930 (100 nM). 5. 2-Methyl-5-HT (1-100 microM) and alpha-methyl-5-HT (30 nM-30 microM), agonists at 5-HT3 and 5-HT2 receptors respectively, also evoked ISC increases. These responses were reduced by hyoscine (100 nM) and abolished by TTX (300 nM) and the respective receptor antagonists, granisetron (1 microM) and ketanserin (30 nM). 6. The 5-HT4 receptor antagonist, SDZ 205-557 (300 nM) had no effect on the response to 5-HT. 7. The TTX-resistant response to 5-HT was not affected by 5-HT2,3 or 4 receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective blocking action of LSD on inhibitory dopamine receptors.

Dopamine-induced postsynaptic responses of both excitory and inhibitory types were recorded from identified neurones of Aplysia ganglion. A 30 sec exposure to 0.1 μm d-lysergic acid diethylamide (LSD) significantly depressed the inhibitory responses to 1 mM dopamine, while a 60 sec exposure to 10 μm LSD did not alter the excitatory responses to 0.1 mm dopamine. Dose-response curves obtained from cells with inhibitory receptors indicate that the mode of LSD interaction was non-competitive.     

Potentiation by endogenously released ATP of spontaneous transmitter secretion at developing neuromuscular synapses in Xenopus cell cultures.

1. Previously we have shown that extracellular application of ATP, a substance co-stored and co-released with acetylcholine (ACh) in the peripheral nervous system, markedly potentiated the frequency of spontaneous synaptic currents (SSCs) produced by ACh. In the present study, we have further characterized the purinoceptor which mediates the potentiation effect of ATP and the role of endogenously released ATP. 2. Pretreatment with a P2-purinoceptor antagonist, suramin (0.3 mM), but not a P1-purinoceptor antagonist, 8-phenyltheophylline (0.1 mM), prevented the potentiating effect of ATP. 3. We studied the role of endogenously released ATP by examining the effect of a specific P2-purinoceptor antagonist on the frequency of spontaneous synaptic events at high-activity synapses (> or = 3 Hz) and found that suramin, but not 8-phenyltheophylline markedly reduced the frequency of SSCs at these high-activity synapses. In addition, desensitizing the P2-purinoceptor with alpha,beta-methylene ATP also produced similar effects to suramin. 4. Extracellular application of the L-type Ca2+ channel blockers, verapamil, nifedipine or diltiazem (10 microM each) reduced SSC frequency of high-activity synapses, while the N-type Ca2+ channel blocker, omega-conotoxin had no appreciable effect. The potentiating effect of ATP was further prevented by pretreatment with the L-type Ca2+ channel blockers. On the other hand, Bay K 8644, which is a depolarization-dependent L-type Ca2+ channel agonist, potentiated SSC frequency at these high-activity synapses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of ATP and related purines in inhibitory neurotransmission to the pig urinary bladder neck

Background and purpose:

As adenosine 5′-triphosphate (ATP) is one of the inhibitory mediators of the bladder outflow region, this study investigates the possible release of ATP or related purines in response to electrical field stimulation (EFS) and the purinoceptor(s) involved in nerve-mediated relaxations of the pig urinary bladder neck.

Experimental approach:

Urothelium-denuded and intact phenylephrine-precontracted strips were mounted in organ baths containing physiological saline solution at 37°C and gassed with 95% O₂ and 5% CO₂ for isometric force recordings.

Key results:

EFS, in the presence of atropine, guanethidine and N^G-nitro-L-arginine, and exogenous purines, produced frequency- and concentration-dependent relaxations respectively. Adenosine 5′-diphosphate (ADP) and adenosine were more potent than ATP in producing relaxation, while uridine 5′-triphosphate, uridine 5′-diphosphate and α,β-methylene ATP were less effective. The non-selective P2 antagonist suramin, and the P2Y₁ and P1 receptor blockers 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium and 8-(p-sulphophenyl)theophylline, respectively, inhibited the responses to EFS and ATP. The P1 agonist''s potency was: 5′-N-ethylcarboxamidoadenosine (NECA)>4-2[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene propanoic acid hydrochloride>2-chloro-N⁶-cyclopentyladenosine>-2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide = adenosine. 4-(-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol, an A_2A antagonist, reduced the relaxations to EFS, adenosine and NECA. In urothelium-intact samples, relaxations to EFS and purines were smaller than in urothelium-denuded preparations. Neuronal voltage-gated Na⁺ channels blockade failed to modify ATP relaxations. At basal tension, EFS- and ATP-induced contractions were resistant to desensitization or blockade of P2X₁ and P2X₃ receptors.

Conclusions and implications:

ATP is involved in the non-adrenergic, non-cholinergic, non-nitrergic inhibitory neurotransmission in the pig bladder neck, producing relaxation largely through muscle A_2A receptors after breakdown to adenosine, and P2Y₁ receptors after breakdown to ADP. Antagonists of these receptors may be useful for urinary incontinence treatment produced by intrinsic sphincteric deficiency.     

Postganglionic muscarinic inhibitory receptors in pulmonary parasympathetic nerves in the guinea-pig.

The location of neuronal inhibitory muscarinic receptors in pulmonary parasympathetic nerves was investigated in vivo and in vitro. The effects of an agonist for neuronal muscarinic receptors (pilocarpine) and an antagonist (gallamine) were tested on contractions of airway smooth muscle induced by pre- and postganglionic cholinergic nerve stimulation. In anaesthetized guinea-pigs, gallamine potentiated constriction of the tracheal tube and smaller airways induced by preganglionic stimulation. Gallamine also potentiated postganglionic stimulation induced by transmural stimulation of the tracheal tube and by 1-1-dimethyl-4-phenylpiperazinium iodide in the rest of the lung. Bronchoconstriction and contraction of the tracheal tube induced by intravenous acetylcholine were not potentiated by gallamine, indicating that postjunctional muscarinic receptors in airway smooth muscle were not involved in the potentiation. The muscarinic agonist, pilocarpine, had the opposite effect to gallamine and inhibited contractions of the tracheal tube induced by both types of nerve stimulation. In vitro, the inhibitory effect of pilocarpine was demonstrated in the tracheal tube preparation stimulated both pre- and postganglionically. The effect of pilocarpine was antagonized by gallamine. Because gallamine and pilocarpine affected the responses to postganglionic stimulation, the inhibitory muscarinic receptors must be located on the postganglionic neurones of the parasympathetic nerves innervating the trachea and the smaller airways. However, these experiments cannot exclude the possibility that muscarinic receptors may also be located on the soma of the ganglion cells.     

Neurotensin receptors on the ileum of the guinea-pig: evidence for the coexistence of inhibitory and excitatory receptors

Neurotensin caused a complex muscular response of the longitudinal muscle-myenteric plexus preparation of guinea-pig ileum: picomoles of neurotensin produced inhibition while larger concentrations caused an inhibitory effect followed by a delayed dose-dependent contraction. The inhibitory phase of the neurotensin-induced muscular activity was not modified by tetrodotoxin but was potently antagonized in a non-competitive manner by apamin, a bee venom toxin. The contractile component was blocked by tetrodotoxin but not by apamin. These toxins were used to dissect the neurotensin muscular response into an inhibitory phase and an excitatory component. It was possible to further characterize the two neurotensin muscular components by their kinetics of desensitization. The inhibitory neurotensin response showed a fast rate of desensitization and presented a relatively low spare receptor capacity. In contrast, desensitization to the excitatory action of neurotensin was much slower, the excitatory receptors apparently having a larger spare receptor capacity. Desensitization to the action of neurotensin was selective for the neuropeptide not altering the contractile activity of substance P, angiotensin II, bradykinin, histamine or acetylcholine. These results strongly suggest the presence of two subsets of neurotensin receptors in the ileum: the inhibitory set probably localized at the postsynaptic effector level and excitatory neurotensin receptors probably of neuronal origin whose function is probably to modulate the release of neurotransmitters. The physiological implications of these two subtypes of neurotensin receptors in the control of gastrointestinal motility are discussed.     

Operational characteristics of somatostatin receptors mediating inhibitory actions on rat locus coeruleus neurones.

1. In order to characterize somatostatin (SRIF) receptor inhibiting spontaneous firing of rat locus coeruleus neurones, and their transduction mechanism(S), extracellular recordings were obtained from a pontine slice preparation of rat brain containing the locus coeruleus (LC). LC neurones were identified by electrophysiological and pharmacological properties; spontaneous firing (characteristically 0.5-5 Hz) was reversibly and concentration-dependently inhibited by exogenously applied noradrenaline. 2. Spontaneous firing of LC neurones was reversibly and concentration-dependently inhibited by SRIF and the N-terminally extended form, somatostatin-28 (SRIF-28), with EC50 values of 15.1 and 19.4 nM, respectively. The synthetic SRIF analogues (octreotide, MK-678, BIM-23027 and L-362,855) also caused concentration-dependent inhibition of LC neurone firing with a rank order of agonist potencies compatible with actions at a receptor resembling the recombinant sst2 receptor. The putative sst3 selective agonist, BIM-23056, was without agonist or antagonist effect. 3. Addition of 100 nM desipramine significantly increased the efficacy of exogenously applied noradrenaline (EC50 values, 2.96 and 0.13 microM, absence and presence of desipramine, respectively) but did not significantly affect SRIF-induced inhibition (EC50 values, 15.6 and 8.0 nM, respectively). Furthermore, application of phenoxybenzamine (3 microM) abolished responses to NA, but did not affect responses to SRIF (EC50 = 14.1 nM). 4. Application of the cyclic AMP analogue, 8-bromoadenosine-cyclic monophosphate (8-Br-cyclic AMP; 500 microM), significantly increased the spontaneous firing rate of all neurones tested (223 +/- 24% over basal rate). Concentration-effect curves for SRIF constructed in the absence and presence of 8-Br-cyclic AMP had similar threshold concentrations, maxima and EC50 values. 5. Incubation of pontine slices in a modified artificial CSF containing 500 ng ml-1 pertussis toxin (PTX) for 18 h prior to extracellular recording affected neither the spontaneous firing of LC neurones, nor the inhibitory responses to muscimol (EC50 2.2 and 1.2 microM, absence and presence of PTX). However, inhibitory responses to SRIF were markedly attenuated. 6. We conclude that the inhibitory actions of SRIF on spontaneous firing of LC neurones are mediated directly by activation of somatodendritic SRIF receptors, and not indirectly by release of noradrenaline. The SRIF receptors involved appear to couple via a pertussis toxin sensitive G-protein, and elicit their response by a mechanism apparently independent of inhibition of cyclic AMP formation. The agonist profile of several selective and novel SRIF analogues suggests the identity of this receptor to be similar to the recombinant sst2 receptor.