Functional assessment of neuronal cannabinoid receptors in the muscular layers of human ileum and colon.

BACKGROUND & AIMS: The notion that specific receptors account for the ability of natural and synthetic cannabinoids to alter physiological functions, prompted this study aimed at assessing their functional presence in the human gut. METHODS: The effects have been studied of cannabinoids and selective antagonists of their receptors on chemically or electrically evoked contractions in preparations of human intestinal smooth muscle in vitro. RESULTS: Atropine prevented the contractions of longitudinal and circular muscle strips of ileum and colon induced by carbachol or electrical field stimulation; tetrodotoxin abolished only the latter which suggests they do involve activation of cholinergic neurons. The synthetic cannabinoid (+)WIN 55,212-2 had no effect on carbachol contractions, but in a concentration-dependent fashion prevented those elicited by electrical field stimulation - which were insensitive to the putative endogenous cannabinoid anandamide - more potently in longitudinal than in circular strips. The selective CB1 receptor antagonist SR141716, which had no effect in the absence of (+)WIN 55,212-2, competitively antagonised its inhibition of electrical field stimulation contractions, unlike the selective CB2 antagonist SR144528. CONCLUSIONS: Cannabinoid CB1 receptors are functionally present in the human ileum and colon; their pharmacological activation apparently results in inhibition of excitatory cholinergic pathways subserving smooth muscle contraction.

Functional assessment of beta adrenoceptor subtypes in human colonic circular and longitudinal (taenia coli) smooth muscle.

BACKGROUND AND AIMS: The subtype and species related heterogeneity of beta adrenoceptors prompted a functional reappraisal of these molecular targets of motility inhibition in the human colon. METHODS: Relaxation of muscle strips was measured in vitro. RESULTS: The following agonists had decreasing relaxing potency (effective concentration range 10(-8)-10(-4) mol/l): (-)isoprenaline (non-selective), terbutaline (beta(2) selective), CGP 12177 (beta(3) selective, also beta(1), beta(2) antagonist), and SR 58611A (beta(3) selective). Isoprenaline and terbutaline were more potent on circular than taenia strips; CGP 12177 and SR 58611A weakly and partially relaxed taenia but had little effect on circular strips. The potency of isoprenaline on circular strips was greatly reduced by the beta(1) selective antagonist CGP 20712 (10(-7) mol/l), and less so by ICI 118551 (10(-7) mol/l, beta(2) selective). CGP 20712 and ICI 118551 together (both 3 x 10(-6) mol/l) had no effect on taenia relaxation by SR 58611A and rendered isoprenaline and terbutaline virtually inactive on circular strips, although not on taenia, which was relaxed at higher than control concentrations and maximally by isoprenaline. Propranolol, a beta(1), beta(2) non-selective antagonist, at high concentrations (10(-5) mol/l) prevented taenia relaxation by CGP 12177 and SR 58611A; its quantitative antagonism of isoprenaline (in common with that of CGP 12177 used as an antagonist) was competitive in circular strips but not on taenia. CONCLUSIONS: beta(1), beta(2), and beta(3) adrenoceptors are functionally detectable in the human colon; agonist stimulation of any one type relaxed taenia but only isoprenaline was fully effective at the beta(3) subtype.

Functional, biochemical and molecular biological evidence for a possible beta(3)-adrenoceptor in human near-term myometrium.

The possible existence of a beta(3)-adrenoceptor (beta(3)-AR) in human near-term myometrium was investigated by in vitro functional and biochemical studies and analysis of mRNA expression. SR 59119A and SR 59104A and CGP 12177 (two selective agonists and a partial agonist, respectively, of the beta(3)-AR), salbutamol and terbutaline (beta(2)-AR agonists) each produced a concentration-dependent relaxation of the myometrial spontaneous contractions. There were no differences in pD(2) values for the relaxing potencies of terbutaline, salbutamol, CGP 12177 and SR 59119A. The rank order for their relaxing efficacies was SR 59119A>SR 59104A>terbutaline approximately salbutamol approximately CGP 12177 (E(max)=52+/-7%, 42+/-12% and approximately 30% respectively). Propranolol, a beta(1)- and beta(2)-AR antagonist, and ICI 118551, a beta(2)-AR antagonist (both at 0.1 microM), did not affect the SR 59119A-induced relaxation whereas SR 59230A, a selective beta(3)-AR antagonist (1 microM), significantly reduced the maximal relaxing effect of SR 59119A. SR 59119A and salbutamol induced a significant increase in cyclic AMP levels that was antagonized by SR 59230A but not by propranolol for SR 59119A, and by propranolol but not by SR 59230A for salbutamol. The beta(3)-AR mRNA was positively expressed in myometrium preparations in a reverse transcription polymerase chain assay. The results presented provide the first evidence for the existence of the beta(3)-AR subtype in human near-term myometrium and suggest that the effects of SR 59119A might be mediated through an increase in cyclic AMP level.

Inhibition by SR 59119A of isoprenaline-, forskolin- and VIP-induced relaxation of human isolated bronchi.

In the human isolated bronchus (HIB) it has been shown that beta(3)-adrenoceptor stimulation fails to induce relaxation of airway smooth muscle. It has however been reported in human ventricular endomyocardial biopsies that beta(3)-adrenoceptor stimulation induced a marked negative inotropic effect which could be linked to Gi protein activation. The aims of this study were: (1) to determine in HIB (internal diameter 1-2 mm) whether the selective beta(3)-adrenoceptor agonist SR 59119A (N[7-methoxy-1,2,3, 4-tetrahydronaphthalen-(2R)methyl]-(2R)-2-hydroxy-2-(3-chloroph eny l)e thanamine hydrochloride) was able to inhibit adenylate-cyclase-mediated airway smooth muscle relaxation induced by isoprenaline, forskolin or vasoactive intestinal peptide (VIP) and (2) to investigate the role of the Gi protein in this interaction. SR 59119A (0.1 microM and 1 microM) induced a shift to the right of concentration response curve for isoprenaline (-0. 15+/-0.06 and -0.54+/-0.21 log unit, P<0.05 and P<0.01 respectively), forskolin (-0.12+/-0.02 and -0.30+/-0.05 log unit, P<0.001), and VIP (-0.42+/-0.12 log unit, P<0.01 with SR59119A 10(-6)M). The inhibitory effect of SR 59119A was (1) abolished by an incubation of HIB with pertussis toxin (1 microg/ml, during 15 h in Krebs-Henseleit solution, at 21 degrees C), which is known to inactivate the Gi protein and (2) increased after an incubation of HIB with the pro-inflammatory cytokine IL-1beta (10 ng/ml, during 15 h in Krebs-Henseleit solution, at 21 degrees C), which is known to up-regulate Gi protein expression. Our results suggest that the selective beta(3)-adrenoceptor agonist SR59119A might inhibit the cAMP-dependent relaxation of human isolated bronchus through Gi protein-mediated inhibition of adenylate cyclase.

In vitro functional evidence of different neurotensin-receptors modulating the motor response of human colonic muscle strips.

1. The newly developed non-peptide neurotensin (NT)-receptor antagonists SR 48692 and SR 142948 were used to challenge NT responses of human colonic circular smooth muscle strips in vitro. The presence of NT1 and NT2 receptor transcripts in this tissue was tested by reverse transcriptase polymerase chain reaction (RT - PCR) analysis. 2. NT potently and dose-dependently contracted muscle strips, with significant regional differences in potency and efficacy between the transverse and distal colon: EC50, 3.6 and 7.5 nM; the maximal effect was 70 and 55% of 0.1 mM carbachol. Colonic responses to NT in both segments were virtually the same in the presence of atropine (1 microm), levocabastine (10 microM) or tetrodotoxin (1 microM). 3. SR 142948 (10 nM - 1 microM) competitively antagonized NT responses in the transverse and distal colon with similar affinities: pA2 values 8.71 and 8.45, slopes 0.98 and 0.99. SR 48692 (10 nM - 10 microM) antagonized the NT response competitively in the distal colon (pA2 6.55, slope 0.79) and non-competitively in the transverse colon (pA2 8.0, slope 0.51). Neither compound had any agonist effect. 4. The fact that the specific antagonists prevented NT-evoked atropine- and tetrodotoxin-insensitive mechanical responses of colonic muscle strips is highly consistent with the presence in these tissues of non-neuronal NT receptors, whose heterogeneity in the transverse segment is supported by the non-competitive antagonism of SR 48692. The finding of NT1 receptor transcript in both transverse and distal colon suggests its identity with the lower affinity site disclosed functionally by SR 48692 in these segments.

In vitro inhibition of human colonic motility with SR 59119A and SR 59104A: evidence of a beta3-adrenoceptor-mediated effect.

The new beta3-adrenoceptor is present in the gastrointestinal tract of various species. This study aimed to show that this receptor modulates human colonic motility in vitro. We used circular muscle strips from the human colon suspended in single organ baths containing Krebs solution and subjected to an initial 1.5-2 g tension. We measured the effects of different beta3-adrenoceptor agonists, including SR 59104A (N-[(6-hydroxy-1,2,3,4-tetrahydronaphthalen-(2R)-2-yl)methyl]-(2 R)-2-hydroxy-2-(3-chlorophenyl)ethanamine hydrochloride), SR 59119A (N-[(7-methoxy-1,2,3,4-tetrahydronaphthalen-(2R)-2-yl)methyl]-(2R) -2-hydroxy-2-(3-chlorophenyl)ethanamine hydrochloride), BRL 37344 (R,R + S,S) [4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino] propyl] phenoxy] acetic acid), and of isoprenaline and salbutamol in the absence or in the presence of propranolol alone or in combination with the beta3-adrenoceptor antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydro-naphthalen-1- ylamino]-(2S)-2-propanol oxalate) on amplitude of spontaneous contractions. To evaluate a possible beta2-adrenoceptor-mediated effect, we studied the action of these compounds on human isolated bronchi. On the human isolated colon, SR 59119A, SR 59104A and isoprenaline reduced the initial amplitude of spontaneous contractions by 60%. The curves obtained in the presence of antagonists suggested an action mediated by beta3-adrenoceptor stimulation, since propranolol did not antagonize the action of SR 59119A and SR 59104A, whereas the combination of propranolol and SR 59230A significantly displaced the concentration-response curve of these agonists to the right. This study provides pharmacological evidence of modulation of human colonic motility, and especially of the amplitude of spontaneous contractions, by the atypical beta-adrenoceptor, the beta3-adrenoceptor.

In vitro characterization of tachykinin NK2-receptors modulating motor responses of human colonic muscle strips.

1. Human in vitro preparations of transverse or distal colonic circular smooth muscle were potently and dose-dependently contracted by neurokinin A (EC50, 4.9 nM), the tachykinin NK2-receptor selective agonist [beta-Ala8]neurokinin A (4-10) ([beta-Ala8]NKA (4-10)) (EC50, 5.0 nM), neurokinin B (EC50, 5.3 nM) and substance P (EC50, 160 nM), but not by the tachykinin NK1-receptor selective agonist [Sar9Met(O2)11] substance P, or the NK3-receptor selective agonists, senktide and [MePhe7] neurokinin B. No regional differences between transverse and distal colon were observed in response to [beta-Ala8]NKA (4-10). 2. Atropine (1 microM) and tetrodotoxin (1 microM) did not significantly inhibit responses to [beta-Ala8]NKA (4-10), neurokinin A, substance P or neurokinin B. 3. The newly developed non-peptide antagonists for tachykinin NK2-receptors SR 48968, SR 144190 and its N-demethyl (SR 144743) and N,N-demethyl (SR 144782) metabolites, were used to challenge agonist responses, as appropriate. SR 144190 and the metabolites all potently and competitively antagonized the response to [beta-Ala8]NKA (4-10), with similar potency (Schild plot pA2 values 9.4, 9.4 and 9.3, slope = 1). SR 48968 antagonism was not competitive: the Schild plot slope was biphasic with a high (X intercept approximately 9.3) and a low (X intercept 8.4, slope 1.6) affinity site. Co-incubation of SR 48968 (10, 100 nM) and SR 144782 (10 nM) produced additive effects; in this experimental condition, SR 48968 apparent affinity (pKB) was 8.2. In addition, SR 144782 (0.1 microM) antagonized responses to neurokinin A, substance P and neurokinin B, with pKB consistent with its affinity for tachykinin NK2-receptors. The potent and selective NK1 and NK3-receptor antagonists, SR 140333 and SR 142801 (both 0.1 microM), failed to inhibit contractions induced by SP or NKB. 4. In conclusion, the in vitro mechanical responses of circular smooth muscle preparations from human colon are strongly consistent with the presence of non-neuronal tachykinin NK2-receptors, but not tachykinin NK1- or NK3-receptors. Our findings with SR 48968 suggest the existence of two tachykinin NK2-receptor subtypes, that it seems to distinguish, unlike SR 144190 and its metabolites. However, the precise nature of SR 48968 allotopic antagonism remains to be elucidated, since allosteric effects at the tachykinin NK2-receptor might well account for the complexity of the observed interaction.

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 coincubated, 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.

Role of tachykinins in castor oil diarrhoea in rats.

1. 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. 2. SR 48968 (0.02 to 20 micrograms 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 micrograms kg-1, s.c.) and the platelet activating factor antagonist SR 27417 (5 to 500 micrograms 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%). 3. The R-enantiomers of tachykinin NK1 and NK2 receptor antagonists, SR 140603 and SR 48605 (both at 2 or 20 micrograms kg-1, s.c.) had no effect other than reducing faecal mass at the highest dose tested. 4. SR 48968 (20 micrograms 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. 5. 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 micrograms kg-1, p.o.): a similar result was obtained on faecal mass and water content. 6. 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). 7. 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. 8. 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.

Negative modulation of nitric oxide production by neurotensin as a putative mechanism of the diuretic action of SR 48692 in rats.

1. We investigated the effect of the non-peptide neurotensin (NT) antagonist SR 48692 on renal function in rats and the involvement of nitric oxide (NO) in the diuretic action of this compound. 2. In fed animals, SR 48692 dose-dependently (0.5 to 12.5 mg kg-1, p.o., 0.03 to 1 mg kg-1, i.p. and 0.1 to 1 microgram/rat, i.c.v.) increased urine output and urinary excretion of Na+, K+ and Cl- and reduced urine osmolality. The diuretic activity was also evident in water-deprived, fasted animals and in fasted, water-loaded rats. 3. NT (0.1 microgram/rat, i.c.v.) had no effect on urine output in fed rats, but reduced the diuretic action of SR 48692 (1 microgram/rat, i.c.v.). The opposite result was obtained in fasted, water-loaded animals: NT dose-dependently (0.01 and 0.1 microgram/rat, i.c.v.) inhibited diuresis and this effect was significantly inhibited by i.c.v. SR 48692. In this experimental condition, SR 48692 did not further increase the on-going diuresis. 4. The NO synthesis inhibitor N(1)-nitro-L-arginine methyl ester (L-NAME; 30 mg kg-1, i.p.) alone had no effect on urine output in fed rats but prevented the diuretic action of i.c.v. or i.p. SR 48692; L-arginine (1 g kg-1, i.p.) but not D-arginine (1 g kg-1, i.p.) restored the SR 48692-dependent increase in diuresis, L-NAME had no effect on furosemide-stimulated diuresis. 5. Systemically administered L-NAME or i.c.v. NT in fasted, water-loaded rats significantly reduced water diuresis but this effect was no longer seen in animals given i.p. L-arginine. Rats receiving i.c.v. NT, whose diuresis was significantly reduced, also excreted less nitrates and nitrites in urine. 6. Increased diuresis after central or systemic administration of SR 48692 to fed rats was paralleled by increased urinary excretion of nitrates and nitrites, this being consistent with peripheral enhancement of NO production after NT-receptor blockade by SR 48692. The increase in diuresis after furosemide also involved an increase of nitrates and nitrites in urine, but this effect was about half that attained with an equipotent diuretic dose of SR 48692. 7. In fed rats, the NO donor isosorbide-dinitrate, reduced systolic blood pressure (unlike SR 48692 which did not affect blood pressure) but also dose-dependently (1 and 5 mg kg-1, i.p.) stimulated urine output. 8. The overall effects of SR 48692 strongly support a link between the actions of endogenous NT, AVP and peripheral NO production in the modulation of renal excretion of water, Na+, K+ and Cl-.

Prevention of platelet-activating factor-induced gastrointestinal lesions in rats by the new specific antagonist N-(2-dimethylaminoethyl)-N-(3-pyridinylmethyl)[4-(2,4,6-triisopropylphe nyl) thiazol-2yl]amine.

SR 27417 (CAS 136468-36-5, N-(2-dimethylaminoethyl)-N-(3-pyridinylmethyl)[4-(2,4,6-triisop ropylphenyl) thiazol-2-yl]amine), a highly potent platelet-activating factor (PAF) receptor antagonist, was tested for its ability to prevent macroscopic and histologically assessed gastrointestinal (GI) lesions in rats induced by PAF as compared to the reference compound apafant. Both compounds were orally effective but SR 27417 prevented the gut lesioning effects of PAF at lower doses than apafant. In addition, a dose of apafant (1.5 mg/kg) that showed almost maximal effect when given 30 min before PAF, had lost most of its protective action by 3 h, while SR 27417 at a comparably effective dose (0.5 mg/kg) retained substantial ability to prevent gut lesions in all the GI tract segments investigated, 18 h after administration. These findings suggest that SR 27417 is a potent and long lasting inhibitor of PAF-induced gastrointestinal lesions in rats.

Functional evidence of atypical beta 3-adrenoceptors in the human colon using the beta 3-selective adrenoceptor antagonist, SR 59230A.

The role of beta 3-adrenoceptors in human colonic circular smooth muscle was assessed in vitro by use of the beta 3-selective antagonist SR 59230A. Isoprenaline, in the presence of the selective beta-adrenoceptor antagonists CGP 20712A (beta 1) and ICI 118551 (beta 2), both at 0.1 microM, 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 beta 3-selective agonists was effective, the remarkably high potency of SR 59230A as a stereospecific antagonist of non-beta 1 non-beta 2 relaxation of human colonic muscle by isoprenaline provides strong functional evidence of beta 3-adrenoceptors in that tissue.

Functional identification of rat atypical beta-adrenoceptors by the first beta 3-selective antagonists, aryloxypropanolaminotetralins.

1. We have assessed the relative abilities of compounds belonging to the new aryloxypropanolaminotetralin (APAT) class and of the reference beta-adrenoceptor-blocking agent, alprenolol, to antagonize functional responses in vitro and in vivo involving atypical (beta 3) or conventional (beta 1 and beta 2) beta-adrenoceptors. 2. The range of pA2 values for three representative APATs against inhibition of spontaneous motility in the rat isolated colon by the selective beta 3-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 (beta 2, 6.5-6.9) and the atrial chronotropic response by isoprenaline (beta 1, 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 beta 3-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 beta 3-adrenoceptor agonists SR 58611A and BRL 37344 respectively were substantially diminished by the representative APAT, SR 59230A, at oral doses (< or = 5 mg kg-1) well below those half maximally effective (ID50) for preventing beta 1-(isoprenaline tachycardia > or = 80 mg kg-1) or beta 2-(salbutamol bronchodilatation, 44 mg kg-1) mediated responses. Alprenolol, as expected, was a less potent and nonselective antagonist of the putative beta 3-responses. 5. These findings support APATs as the first potent, orally effective selective antagonists at beta 3-adrenoceptors, and provide final unambiguous evidence that beta 3-adrenoceptors underlie inhibition of colonic motility and brown adipose tissue thermogenesis in rats.

The non-peptide tachykinin NK1- and NK2-receptor antagonists SR 140333 and SR 48968 prevent castor-oil induced diarrhea in rats.

Castrol-oil induced diarrhea in rats was potently prevented by compounds SR 140333 and SR 48968, the first a tachykinin NK1- and the second a NK2-receptor antagonist. SR 48968 was more effective and also reduced fecal water content.

Promotion by SR 48692 of gastric emptying and defaecation in rats suggesting a role of endogenous neurotensin.

We investigated the influence of the nonpeptide neurotensin receptor antagonist, SR 48692, administered orally, on gastric emptying and on acute defaecation. SR 48692 dose-dependently (ED50 approximately 0.7 microgram kg-1) increased gastric emptying of a food suspension, but it had no effect on that of a non-caloric meal. SR 48692 also dose-dependently promoted defaecation and increased faecal water content. We suggest that antagonism of endogenous neurotensin accounts for the gastric emptying and defaecation promoting action of SR 48692.

Drug-induced defaecation in rats: role of central 5-HT1A receptors.

1. We investigated the acute effects of 5-hydroxytryptamine (5-HT), and of the 5-HT1A receptor agonists, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), buspirone and SR 57746A, on rat faecal pellet output and water content. 2. 5-HT, 8-OH-DPAT, buspirone and SR 57746A, a new selective 5-HT1A receptor agonist, displaced [3H]-8-OH-DPAT from specific binding sites in rat hippocampus membranes (Ki, nM; 1.8, 1.2, 15, 3.1 respectively) and stimulated rat defaecation dose-dependently. SR 57746A and buspirone induced 1 g dry weight of faeces at 1.3 and 6.1 mg kg-1, p.o. (AD1) respectively. 8-OH-DPAT and 5-HT stimulated defaecation after s.c. injection (AD1, 0.07 and 7.5 mg kg-1, respectively). All these agents increased faecal water content. 3. The putative 5-HT1A receptor antagonist, pindolol, injected s.c. or i.c.v., significantly reduced the defaecation induced by systemically administered 8-OH-DPAT, buspirone or SR 57746A, but not 5-HT. 4. Pretreatment with p-chlorophenylalanine (i.p.) or 5,7-dihydroxytryptamine (i.c.v.), according to protocols designed to cause either generalized or CNS-limited 5-HT depletion respectively, also reduced the defaecation induced by buspirone or SR 57746A. 5. No specific 5-HT1A binding sites could be labelled by incubating rat colon membranes with [3H]-8-OH-DPAT, and in vitro preparations of rat colon segments showed no response to 8-OH-DPAT or SR 57746A up to 5 microM. 6. After eight days' repeated daily treatment, complete tolerance developed to the stimulant effects of SR 57746A and buspirone on faecal water content, but not on faecal pellet output. This suggests that faecal mass excretion and water exchange through the gut wall are affected by independent mechanisms.7. The present findings support the involvement of central 5-HTIA receptors in intestinal propulsion and regulation of luminal fluid content, presumably accounting for the drug-induced defaecation in rats.

In vitro characterization of the non-peptide tachykinin NK1 and NK2-receptor antagonists, SR140333 and SR48968 in different rat and guinea-pig intestinal segments.

We investigated the potent non-peptide tachykinin receptor antagonists SR140333 and SR48968 for their ability to prevent the contraction of isolated intestinal tissues elicited by the non-selective agonists substance P (SP) and neurokinin A (NKA), or by [Sar9,Met(O2)11]SP and [beta-Ala8]NKA-(4-10) that are selective agonists for NK1 and NK2 receptors, respectively. In guinea-pig ileum, containing mainly NK1-receptors: SR140333 caused a pseudo-irreversible blockade of contractions induced by either SP (KB, 0.01 nM) or [Sar9,Met(O2)11]SP (KB, 0.03 nM); SR140333 but not SR48968, dose-dependently (IC50, 0.06 nM) antagonized the contractions elicited by capsaicin. In rat duodenum, containing mainly NK2 receptors, SR48968 caused a parallel rightward shift of the concentration-response curves of [beta-Ala8]NKA-(4-10) (pA2, 9.5), but not of NKA. In rat esophageal tunica muscularis mucosae, SR48968 non-competitively antagonized [beta-Ala8]NKA-(4-10) and NKA. SR48968 and SR140333 thus appear to be potent tachykinin receptor antagonists, selective for intestinal receptors respectively of the NK2 and NK1 type. The results also suggest that rat esophagus might contain a NK2-receptor subtype different from that of rat duodenum.

Beta 3-adrenoceptors and intestinal motility.

Early substantial evidence of the low susceptibility to beta-adrenoceptor antagonists of non alpha-adrenergic responses reducing gut motility and tone was reluctantly accepted as indicating a third beta-receptor subtype different from the beta 1 and beta 2. This applied likewise to lipolysis until new selective "lipolytic" beta-agonists poorly effective at established beta-receptors were introduced. Shortly afterwards these "lipolytic" as well as certain newer and even more selective beta-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-beta 1, non-beta 2 beta-adrenoceptors. Acceptance of the functionally based concept of a beta 3-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 beta-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 beta 3-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 beta 3-adrenoceptor is suggested by their potent intestinal action in vivo in animal models without any of the cardiovascular or other unwanted effects of conventional beta-adrenoceptor agonists and antagonists, and by the clinically confirmed importance of beta-adrenergic control of motor function throughout the alimentary canal. However, open questions include the incidence of species-related differences in beta 3-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.