Luciano Manara

In vitro inhibition of intestinal motility by phenylethanolaminotetralines: evidence of atypical β‐adrenoceptors in rat colon

1 The new compounds phenylethanolaminotetralines (PEAT), unlike the reference β‐adrenoceptor agonists isoprenaline (Iso), ritodrine (Ri) and salbutamol (Sal), produced half‐maximal inhibition of spontaneous motility of rat isolated proximal colon at substantially lower concentrations (EC50 2.7–30 nm) than those inducing β2‐adrenoceptor‐mediated responses (relaxation of guinea‐pig isolated trachea and rat uterus) and had virtually no chronotropic action (EC50 > 3 × 10−5 m.) on the guinea‐pig isolated atrium (a β1‐adrenoceptor‐mediated response). 2 The nonselective β‐adrenoceptor antagonists alprenolol and propranolol prevented the inhibition of rat colon motility by the PEAT with low and different potencies (pA2 values around 7.5 and 6.5 respectively). Conversely alprenolol and propranolol had a higher and similar potency (pA2 values around 9.0) in preventing typical β1‐ or β2‐responses (increase in atrial frequency by Iso or tracheal relaxation by Ri or Sal). 3 The selective β‐adrenoceptor antagonists CGP 20712A (β1) and ICI 118,551 (β2) either alone or in combination, did not prevent rat colon motility inhibition by the representative PEAT SR 58611A, which was also fully resistant to α‐adrenoceptor, acetylcholine, dopamine, histamine, opioid and 5‐hydroxytryptamine antagonists. 4 These results indicate that the PEAT are a new class of β‐adrenoceptor agonists and suggest that their preferential intestinal action may be accounted for by selectivity for atypical β‐adrenoceptors, abundant in the rat colon and distinct from the currently recognized β1 and β2 subtypes.

In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum

We investigated the effect of the cannabinoid agonist (+)WIN‐55212‐2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or co‐incubated, reduced twitch responses to a similar degree (85%). (+)WIN‐55212‐2 concentration‐dependently inhibited twitch responses (IC50 73 nm), but had no additive effect with atropine or TTX. The cannabinoid CB1 receptor antagonist SR 141716 (pA2 8.2), but not the CB2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN‐55212‐2. Atropine but not (+)WIN‐55212‐2 or TTX prevented carbachol‐induced tonic contraction.  These results provide functional evidence of the existence of prejunctional cannabinoid CB1‐receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB1 receptors naturally expressed in the human gut.

Morphine is most effective on gastrointestinal propulsion in rats by intraperitoneal route: Evidence for local action

Abstract A very low dose of morphine (37.5 μg/kg) entirely delivered into the peritoneal cavity of rats, consistently reduced the transit of a forced charcoal meal through the small intestine (to about 30% of control), but failed to elicit such action in naloxone-pretreated animals or if administered either intravenously or intracerebroventricularly. The same dose of tritium labeled morphine, injected i.v., resulted in brain and small intestine morphine levels respectively 2.7 times higher and 3.9 times lower than in the corresponding tissues of rats injected i.p.. These findings suggest that activation of opiate specific sites located in the gastrointestinal tract can per se be primarily responsible for the antipropulsive effects of morphine.

Functional identification of rat atypical β‐adrenoceptors by the first β3‐selective antagonists, aryloxypropanolaminotetralins

1 We have assessed the relative abilities of compounds belonging to the new aryloxypropanolaminotetralin (APAT) class and of the reference β‐adrenoceptor‐blocking agent, alprenolol, to antagonize functional responses in vitro and in vivo involving atypical (β3) or conventional (β1 and β2) β‐adrenoceptors. 2 The range of pA2 values for three representative APATs against inhibition of spontaneous motility in the rat isolated colon by the selective β3‐adrenoceptor agonist, SR 58611A (8.1–8.8), was well above similarly calculated values for non‐competitive antagonism of guinea‐pig trachea relaxation by salbutamol (β2, 6.5–6.9) and the atrial chronotropic response by isoprenaline (β1, 6.7‐7.3). Alprenolol, however, was substantially more potent in antagonizing atrial (pA2, 8.2) and tracheal (pA2, 8.9) responses than SR 58611A mediated inhibition of colonic motility (pA2, 6.8). 3 Several APAT isomers with different configurations at the chiral carbons, when tested on isolated organs, presented stringent stereochemical requirements for β3‐selectivity, including high antagonist potency‐ratios between active and inactive enantiomers. 4 In vivo, the inhibition of colonic motility and the thermogenic response of brown adipose tissue elicited in rats by the selective β3‐adrenoceptor agonists SR 58611A and BRL 37344 respectively were substantially diminished by the representative APAT, SR 59230A, at oral doses (≤5 mg kg−1) well below those half maximally effective (ID50) for preventing β1‐(isoprenaline tachycardia ≥80 mg kg−1) or β2‐(salbutamol bronchodilatation, 44 mg kg−1) mediated responses. Alprenolol, as expected, was a less potent and nonselective antagonist of the putative β3‐responses. 5 These findings support APATs as the first potent, orally effective selective antagonists at β3‐adrenoceptors, and provide final unambiguous evidence that β3‐adrenoceptors underlie inhibition of colonic motility and brown adipose tissue thermogenesis in rats

Quaternary narcotic antagonists' relative ability to prevent antinociception and gastrointestinal transit inhibition in morphine-treated rats as an index of peripheral selectivity

Single doses of naloxone (0.025 to 0.5 mg/kg) or of one of four quaternary narcotic antagonists (i.e. nalorphine allobromide, nalorphine methobromide, naloxone methobromide or naltrexone methobromide, 1 to 60 mg/kg) were given s.c. to rats before morphine, 5 mg/kg i.v. In the absence of antagonists morphine reduced G.I. transit of a charcoal meal to about 15% of drug-free controls and consistently delayed nociceptive reactions (55 degrees C hot plate) in all animals. Doses of antagonists slightly reducing morphine antinociception (centrally effective = A) and restoring G.I. transit to about 50% of drug-free rats (peripherally effective = B) were estimated. The A:B ratio, indicating peripheral selectivity, was at least 8 for any of the quaternary antagonists given 10 min before morphine, but prolonging this interval may have resulted in a lower figure (i.e. less peripheral selectivity) because of reduced A and increased B. This was definitely so for naltrexone methobromide (A:B, greater than 60 at 10 min, about 1 at 80 min) and was not apparent for nalorphrine methobromide according to available data, which for nalorphrine allobromide and to a lesser extent for naloxone methobromide showed only an increase in B at intervals longer than 10 min. Both morphine-induced antinociception and inhibition of G.I. transit were reduced by naloxone at the lower doses tested and were fully prevented at the higher. These findings indicated that, unlike naloxone, the investigated quaternary narcotic antagonists are interesting prototype drugs for selective blockade of opiate receptors outside the CNS, although certain critical aspects, possibly biological N-dealkylation to the corresponding tertiary antagonists, condition peripheral selectivity.

Modulation of gastric emptying and gastrointestinal transit in rats through intestinal cannabinoid CB1 receptors

We studied the delay in gastric emptying and gastrointestinal transit induced by the cannabinoid receptor agonists (+)-WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) and CP 55,940 ((-)-cis-3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol), as prevented by the selective cannabinoid CB(1)-receptor antagonist SR141716 ((N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide)) in rats after systemic or central drug administration. Oral SR141716 showed comparable potency (ID(50) range 1.0-3.9 mg/kg) in antagonizing gastric emptying and gastrointestinal transit delay by (+)-WIN 55,212-2 or CP 55,940. Gastric emptying and gastrointestinal transit delay after intracerebroventricular (i.c.v.) (+)-WIN 55,212-2 was prevented by oral or i.c.v. SR141716, but i.c.v. SR141716 did not significantly reduce the effect of i.p. (+)-WIN 55,212-2. Pertussis toxin prevented the delaying action of i.c.v. (+)-WIN 55,212-2 on both gastric emptying and gastrointestinal transit, but had no effect on (+)-WIN 55,212-2 i.p. These findings are consistent with a primary role of peripheral cannabinoid CB(1) receptor mechanisms in gastrointestinal transit delay by specific agonists.

Role of tachykinins in castor oil diarrhoea in rats

We set out to ascertain the role of tachykinins, neurokinin A and substance P, in castor oil‐induced diarrhoea in rats as disclosed by the inhibitory effect of the non‐peptide NK1‐ and NK2‐receptor antagonists, SR 140333 and SR 48968, respectively. SR 48968 (0.02 to 20 μg kg−1, s.c. or p.o.), and the opioid receptor agonist loperamide (1–10 mg kg−1, p.o.), dose‐dependently prevented castor oil effects: % inhibition vs castor oil, diarrhoea 0 to 100, increase in faecal mass 7 to 90 and water content 16 to 90. SR 140333 (0.02 to 20 μg kg−1, s.c.) and the platelet activating factor antagonist SR 27417 (5 to 500 μg kg−1, p.o.) did not prevent the increase in faecal water content, but reduced faecal mass (35 to 66%) and diarrhoea (0 to 57%). The R‐enantiomers of tachykinin NK1 and NK2 receptor antagonists, SR 140603 and SR 48605 (both at 2 or 20 μg kg−1, s.c.) had no effect other than reducing faecal mass at the highest dose tested. SR 48968 (20 μg kg−1, p.o.) but not loperamide (10 mg kg−1, p.o.) given 24 h before castor oil, still slightly but significantly reduced by 30% the increase of faecal mass output; both treatments significantly reduced (30 to 70%) the effect of castor oil on faecal water content, although the incidence of diarrhoea was only slightly less than in controls. In castor oil‐treated rats, naloxone (2 mg kg−1, s.c.) completely blocked the antidiarrhoeal action of loperamide (10 mg kg−1, p.o.) but not of SR 48968 (20 μg kg−1, p.o.); a similar result was obtained on faecal mass and water content. Castor oil strongly increased the occurrence of manometrically recorded propulsive giant contractions (500 to 1000% over control values) of transverse and distal colon, this effect being significantly prevented (80 to 100%) by SR 48968 and loperamide and partially by SR 140333 (35% distal colon, 70% transverse colon). In castor oil free rats, loperamide but not SR 48968 or SR 140333 significantly reduced by 50% the gastrointestinal transit of a charcoal test meal, as well as 24 h faecal mass output. Consistently, loperamide, unlike the tachykinin receptor antagonists, had a dramatic effect on manometric recordings of intestinal motility, reducing all kinds of colonic contractions. Our findings suggest that castor oil diarrhoea in rats entails activation of NK1 and NK2 receptors by endogenous tachykinins, whose antagonists may have a potential as antidiarrhoeal agents free from the constipating action of opioids.

SR 57227A: a potent and selective agonist at central and peripheral 5-HT3 receptors in vitro and in vivo.

SR 57227A (4-amino-(6-chloro-2-pyridyl)-1 piperidine hydrochloride) is a novel compound with high affinity and selectivity for the 5-HT3 receptor. The compound had affinities (IC50) varying between 2.8 and 250 nM for 5-HT3 receptor binding sites in rat cortical membranes and on whole NG 108-15 cells or their membranes in vitro, assayed under various conditions with [3H]S-zacopride or [3H]granisetron as radioligand. Like reference 5-HT3 receptor agonists, SR 57227A stimulated the uptake of [14C]guanidinium into NG 108-15 cells in the presence of substance P (EC50 = 208 +/- 16 nM) and contracted the isolated guinea-pig ileum (EC50 = 11.2 +/- 1.1 microM), effects that were antagonised by the 5-HT3 receptor antagonist tropisetron. The agonist effect of SR 57227A was also observed in vivo, as the compound elicited the Bezold-Jarisch reflex in anesthetised rats (ED50 = 8.3 micrograms/kg i.v.), an effect that was blocked by tropisetron and R,S-zacopride, but not by methysergide. When injected unilaterally into the mouse striatum, SR 57227A, like 2-methyl-5-HT, elicited contralateral turning behaviour which was antagonised by ondansetron. Furthermore, microiontophoretic application of SR 57227A markedly inhibited the firing rate of rat cortical neurones, an effect antagonised by tropisetron. Finally, in contrast to reference 5-HT3 agonists, SR 57227A bound to 5-HT3 receptors on mouse cortical membranes after systemic administration (ED50 = 0.39 mg/kg i.p. and 0.85 mg/kg p.o.). These results suggest that SR 57227A is a potent agonist at peripheral and central 5-HT3 receptors, both in vitro and in vivo. In view of the dearth of 5-HT3 receptor agonists which are capable of crossing the blood-brain barrier, SR 57227A may be useful in the characterisation of the neuropharmacological effects produced by the stimulation of these receptors.

β3‐adrenoceptors and intestinal motility

Summary— Early substantial evidence of the low susceptibility to β‐adrenoceptor antagonists of non α‐adrenergic responses reducing gut motility and tone was reluctantly accepted as indicating a third β‐receptor subtype different from the β1 and β2. This applied likewise to lipolysis until new selective “lipolytic” β‐agonists poorly effective at established β‐receptors were introduced. Shortly afterwards these “lipolytic” as well as certain newer and even more selective β‐adrenoceptor agonists were shown to be potent inhibitors of intestinal motility. The latter are the “gut‐specific” phenylethanolaminotetralins whose availability as pure isomers attested to the stringent stereochemical requirements for selectivity at non‐β1, non‐β2 β‐adrenoceptors. Acceptance of the functionally based concept of a β3‐adrenoceptor was boosted on structural grounds by molecular biology studies. Sequence analysis indicated the existence in humans and rodents of genes coding for a third subtype of β‐receptor that, when expressed in transfected heterologous cells, had a pharmacological profile distinct from the previously established subtypes. Finally, aryloxypropanolaminotetralins have been prepared as the first selective antagonists of β3‐adrenoceptors, thus providing unambiguous conclusive evidence of the distinctive functional features of those abundant in the rat colon. The therapeutic potential in gastroenterology of the newer compounds targetable on the β3‐adrenoceptor is suggested by their potent intestinal action in vivo in animal models without any of the cardiovascular or other unwanted effects of conventional β3‐adrenoceptor agonists and antagonists, and by the clinically confirmed importance of β‐adrenergic control of motor function throughout the alimentary canal. However, open questions include the incidence of species‐related differences in β3‐adrenoceptors, and as yet there are no data on gastrointestinal functions in humans under the influence of drugs designed to act selectively at these receptors.

Functional evidence of atypical β3‐adrenoceptors in the human colon using the β3‐selective adrenoceptor antagonist, SR 59230A

The role of β3‐adrenoceptors in human colonic circular smooth muscle was assessed in vitro by use of the β2‐selective antagonist SR 59230A. Isoprenaline, in the presence of the selective β‐adrenoceptor antagonists CGP 20712A (β1) and ICI 118551 (β2), both at 0.1 μ, concentration‐dependently relaxed the preparation (pEC50 = 5.22). This effect was potently and competitively antagonized by SR 59230A with a pA2 of 8.31, while its R,R enantiomer SR 59483A gave an apparent pKB of 6.21. Relaxation was likewise produced by CGP 12177A (pEC50 = 6.05), but not by BRL 37344. Although only one of these β3‐selective agonists was effective, the remarkably high potency of SR 59230A as a stereospecific antagonist of non‐β1 non‐β2 relaxation of human colonic muscle by isoprenaline provides strong functional evidence of β3‐adrenoceptors in that tissue.