Antagonism of the cannabinoid CB-1 receptor protects rat liver against ischaemia-reperfusion injury complicated by endotoxaemia.

BACKGROUND/AIM: Endotoxaemia can complicate hepatic ischaemia-reperfusion (IR) injury. Endocannabinoids appear to modulate the haemodynamic alterations and cytokine response induced by lipopolysaccharide (LPS). Thus, we aimed to determine the effect of the endocannabinoid CB1-receptor antagonist Rimonabant in a model of hepatic IR injury complicated by endotoxaemia. METHODS: Sprague-Dawley rats pre-treated with Rimonabant 3 or 10 mg/kg or vehicle underwent partial hepatic IR and lipopolysaccharide (LPS) injection at reperfusion. Liver injury was evaluated by serum alanine aminotransferase (ALT) and necrotic-cell count. The inflammatory response was investigated by assessing hepatic neutrophil infiltration, tumour necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), interleukin 6 (IL6), and suppressor of cytokine signalling (SOCS) 1 and SOCS3 gene expression by real-time polymerase chain reaction (RT-PCR). Systolic blood pressure and hepatic blood flow were measured as haemodynamic parameters. Finally, lipid peroxidation, glutathione status, and immunoreactive CB1 receptor expression in the liver were also determined. RESULTS: Liver injury and neutrophil infiltration occurring in the late-phase of LPS-enhanced IR were significantly reduced by CB1-receptor antagonism. Rimonabant-treated rats showed significantly higher gene expression of IFNgamma, IL6, SOCS1 and SOCS3 in "early" reperfusion, while that of TNFalpha was reduced. These findings were associated with increased STAT3 phosphorylation. Furthermore, CB1-receptor antagonism significantly improved the oxidative injury and haemodynamic alterations occurring during reperfusion in untreated rats. Finally, CB1-receptor immunoreactivity was upregulated early after reperfusion. CONCLUSIONS: This study demonstrates that CB1-receptor antagonism protects the liver against LPS-enhanced IR injury by interfering with the inflammatory response that causes the late, neutrophil-dependent phase of reperfusion injury, although the prevention of the transient endotoxin-related hypotension occurring early during reperfusion may be also involved.

Effect of the cannabinoid CB1 receptor antagonist rimonabant on nociceptive responses and adjuvant-induced arthritis in obese and lean rats.

BACKGROUND AND PURPOSE: Obesity is a risk factor for several inflammation-based diseases including arthritis. We investigated the anti-nociceptive and anti-inflammatory effects of the cannabinoid CB1 receptor antagonist rimonabant in lean and diet-induced obese female rats with arthritis induced by complete Freund's adjuvant (CFA) injected in the right hind-paw. EXPERIMENTAL APPROACH: The effect of oral rimonabant was assessed in rat paws on thermal hyperalgesia, mechanical allodynia, oedema, global arthritis score, nitrite/nitrate levels and ankle widths. KEY RESULTS: After 7 but not after 14 days, the inflammatory response to CFA was significantly higher in obese than lean rats; however, the nociceptive response (thermal hyperalgesia and mechanical allodynia) was similar. Oral rimonabant (3 or 10 mg kg-1, once a day for 1 week from day 7 after CFA) only reduced the global arthritic score and joint width in obese rats, with no effect on the paw oedema. It also markedly reduced thermal hyperalgesia and mechanical allodynia in both lean and obese rats, with a greater effect in the latter. CONCLUSION AND IMPLICATIONS: Rimonabant appears to be a potent inhibitor of sensorial hypersensitivity associated with CFA-induced arthritis in obese rats, in which the inflammatory reaction is more severe than in lean rats. It may thus have therapeutic potential in obesity-associated inflammatory diseases, particularly in the treatment of the pain associated with arthritis.

Early stages of N-2-fluorenylacetamide-induced hepatocarcinogenesis in male and female rats and effect of gonadectomy on liver neoplastic conversion and neoplastic development.

The early stages of N-2-fluorenylacetamide [(2-FAA) CAS: 59-96-3]-induced liver carcinogenesis in inbred F344 male and female rats and the effect of gonadectomy on liver carcinogen biotransformation capability and hepatocarcinogenesis were studied. Feeding of 2-FAA induced more altered hepatocellular foci characterized by exclusion of cellular iron and gamma-glutamyl-transferase activity in male rats than in female rats. At 6-22 weeks after cessation of carcinogen exposure, only males developed liver neoplastic nodules. Liver cytochrome P450, aryl hydrocarbon hydroxylase, and uridine-5'-diphosphateglucuronosyltransferase activity toward p-nitrophenol, but not phenolphthalein, were greater in males than in females. Gonadectomy of males reduced the activities that were greater than in females, whereas none was significantly altered by gonadectomy of females. Gonadectomy of male rats prior to 2-FAA feeding suppressed the induction of both altered foci and neoplastic nodules, whereas in female rats gonadectomy prior to 2-FAA feeding enhanced the induction of foci. Gonadectomy of males after administration of 2-FAA slightly enhanced the persistence of foci at 6 and 12 weeks after removal of carcinogen, but it did not affect their persistence by 22 weeks post carcinogen or the incidence of neoplastic nodules. However, only the males that were gonadectomized after receiving 2-FAA developed hepatocellular carcinomas at 22 weeks. Gonadectomy of females after receiving 2-FAA did not affect the persistence of foci, and no liver neoplasms developed. Thus gonadectomy of male rats, which reduced liver carcinogen metabolism, when done before carcinogen feeding had the greatest effect on hepatocarcinogenesis.

Beta3-adrenoceptor is the predominant beta-adrenoceptor subtype in human myometrium and its expression is up-regulated in pregnancy.

To assess whether pregnancy might influence the functionality and expression of human myometrial beta(2)- and beta(3)-adrenoceptors (beta(2)- and beta(3)-AR), we performed functional, binding, Western blot, and molecular biology experiments in human nonpregnant and near-term pregnant myometrium. Inhibition of spontaneous contractions induced by a beta(3)-AR agonist, SR 59119A, was significantly greater in pregnant, compared with nonpregnant, myometrial strips (E'(max) = 61 +/- 5% vs. 44 +/- 5% for pregnant and nonpregnant myometrium, respectively), whereas salbutamol, a beta(2)-AR agonist, was significantly less efficient in pregnant, compared with nonpregnant, myometrium (E(max) = 29 +/- 4 vs. 54 +/- 8%). Although two populations of binding sites corresponding to beta(2)- and beta(3)-AR were identified in both nonpregnant and pregnant myometrium, we found a clear predominance of the beta(3)-AR subtype. Moreover, beta(3)-AR binding sites were up-regulated 2-fold in myometrium at the end of pregnancy. Both beta(2)- and beta(3)-AR mRNA were expressed in human nonpregnant and pregnant myometrium. Contrary to beta(2)-AR, the expression of the beta(3)-AR transcripts and immunoreactive proteins was increased in pregnant, compared with nonpregnant, myometrium. Such compelling data suggest a predominant role for beta(3)-AR in the regulation of human myometrium contractility, especially at the end of pregnancy, which might have important consequences for the clinical management of preterm labor.

Arylcarbamate derivatives of 1-piperidineethanol as potent ligands for 5-HT4 receptors.

A series of carbamate derivatives (7) of 2-(1-piperidinyl)ethyl 4-amino-5-chloro-2-methoxybenzoates, which have been described as potent agonists and antagonists of 5-HT4 receptors, were synthesized. They were evaluated using radioligand binding assays with [3H]GR 113808, a 5-HT4 receptor selective ligand, in the rat striatum and the electrically stimulated myenteric plexus longitudinal muscle of the guinea pig. In contrast to the previously described ester derivatives, a drop in the affinity for 5-HT4 receptors was observed and the compounds were inactive as agonists in the guinea pig ileum preparation. Unexpectedly, the ortho-substituted carbamates 8b,c (R' = H, RO = MeO or EtO, R" = H) had nanomolar affinity for 5-HT4 receptors (Ki = 8.9 +/- 0.5 and 2.6 +/- 0.4 nM, respectively). As reported previously, the cis- or trans-3,5-dimethyl substitution of piperidine (8n,o) was particularly favorable (Ki = 1.1 +/- 0.6 nM for both isomers). 8c is an antagonist equipotent to the 5-HT4 receptor antagonist SDZ 205-557 (1).

New esters of 4-amino-5-chloro-2-methoxybenzoic acid as potent agonists and antagonists for 5-HT4 receptors.

A number of benzoates derived from 4-amino-5-chloro-2-methoxybenzoic acid and substituted 1-piperidineethanol were synthesized and found to be potent 5-HT4 receptor agonists in the electrically-stimulated myenteric plexus and longitudinal muscle of the guinea pig ileum and the rat esophagus muscle. Monosubstitution of the piperidine ring with Me, OH, NH-Ac, or CONH2 groups gave compounds equipotent to 7a (ML 10302), a 5-HT4 receptor agonist previously reported to have nanomolar affinity. 7a,k were as potent as serotonin (5-HT) but had maximal responses which were only 60-80% of that of 5-HT, suggesting a partial agonist profile for these compounds. Binding assays were performed with [3H]GR 113808 in the rat striatum, and several of these compounds were found to have nanomolar affinity for 5-HT4 receptors (7a, Ki = 1.07 +/- 0.5 nM; 7k, Ki = 1.0 +/- 0.3 nM). The introduction of two methyl groups on the piperidine ring brought about a dramatic change in the pharmacological profile of 2-[(cis- and trans-3,5-dimethylpiperidinyl)ethyl]-4-amino-5-chloro-2- methoxybenzoate, 7g,h. 7g (Ki = 0.26 +/- 0.06 nM) inhibited the relaxant action of 5-HT in the rat esophagus muscle with a pA2 value of 8.6. The advantage of the ester function was demonstrated by comparing the activity of several such compounds at 5-HT4 receptors with those of the corresponding amidic derivatives. This difference was less marked when the basic moiety was sterically constrained as in the quinuclidine and tropane moieties. Structural analyses of 7a,g were performed by determining their X-ray crystal structures and by molecular modeling (SYBYL). A relatively limited number of minimum energy conformers was found for both compounds. They were characterized by the cis folded conformation of the ethyl chain and by the orientation of the lone pair of the nitrogen atom pointing out of the molecule as seen in conformationally-constrained benzamides such as zacopride and renzapride. A hypothetical model for the 5-HT4 receptor with two sites for the binding of agonist and antagonist molecules was proposed.

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.

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.

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-.

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.

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.

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.

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.

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 human near-term myometrial beta 3-adrenoceptor but not the beta 2-adrenoceptor is resistant to desensitisation after sustained agonist stimulation.

1. In order to compare the beta(2)- and beta(3)-adrenoceptor (beta-AR) desensitisation process in human near-term myometrium, we examined the influence of a pretreatment of myometrial strips with either a beta(2)- or a beta(3)-AR agonist (salbutamol or SR 59119A, respectively, both at 10 microm, for 5 and 15 h) on the relaxation and the cyclic adenosine monophosphate (cAMP) production induced by these agonists. 2. To assess some of the mechanisms potentially implicated in the beta-AR desensitisation process, we studied the influence of such treatment on the number of beta(2)- and beta(3)-AR binding sites, the beta(2)- and beta(3)-AR transcripts expression and the phosphodiesterase 4 (PDE4) activity. 3. Salbutamol, but not SR 59119A, concentration-response curve (CRC) was shifted by a 15 h salbutamol preincubation, with a significant difference in -log EC(20) values (6.31+/-0.13 vs 5.58+/-0.24, for control and 15 h salbutamol pretreatment, respectively, P<0.05). Neither salbutamol nor SR 59119A CRCs were modified after a 15 h preincubation with SR 59119A. 4. A 15 h exposure of myometrial strips to salbutamol significantly reduced the salbutamol-induced (0.60+/-0.26 vs 1.54+/-0.24 pmol mg(-1) protein, P<0.05), but not the SR 59119A-induced, cAMP production. No decrease in cAMP production was observed after a 15 h SR 59119A exposure. 5. A 15 h salbutamol exposure of myometrial strips significantly reduced the beta(2)- but not the beta(3)-AR binding site density, whereas no decrease in the number of beta(2)- and beta(3)-AR binding sites was observed after a 15 h SR 59119A treatment. 6. Neither PDE4 activity nor the beta(2)- and beta(3)-AR mRNA expression levels were affected by salbutamol or SR 59119A treatments. 7. Our results indicate that beta(3)-AR, but not beta(2)-AR, are resistant to the agonist-induced desensitisation. In our model, beta(2)-AR desensitisation is mediated by a decreased number of beta(2)-AR that was not explained by transcriptional regulation of the receptor.

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.

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.

Activities of several phase I and phase II xenobiotic biotransformation enzymes in cultured hepatocytes from male and female rats.

Hepatocytes were isolated from adult male and female rats and maintained in monolayer culture for up to 24 hr. The degree of preservation of representative phase I and phase II xenobiotic biotransformation enzymes was studied in these cells immediately after isolation, after attachment in culture, and after 24 hr in culture. Regarding phase I pathways, hepatocytes during 24 hr lost 50% of cytochrome P-450, but maintained high mixed function oxidase activities; 75% of aryl hydrocarbon hydroxylase and 65% of benzphetamine demethylase activities were preserved in hepatocytes from males, whereas in hepatocytes from females 70 and 50% of these activities, respectively, were maintained. Of phase II pathways, glutathione transferase activity after 24 hr, tested toward 1,2-dichloro-4-nitrobenzene as substrate, was diminished in male hepatocytes to 20% of the initial liver activity and in female cells, to 35%, whereas the activity tested toward 1-chloro-2,4-dinitrobenzene as substrate was stable. UDP-glucuronosyltransferase activities, tested toward p-nitrophenol and phenolphthalein as substrates, were slightly increased during 24 hr of culture of hepatocytes to levels higher than in liver before perfusion. The level of UDP-glucuronic acid, the endogenous substrate for the enzyme, was reduced after isolation to only 6% of the initial liver value, and then increased during culture to a level approximately 60% of normal. Thus, the changes in xenobiotic biotransformation enzymes and associated constituents in cultured hepatocytes were not uniform, although biotransformation capability remained reasonably intact.

Phenylethanolaminotetralines compete with [3H]dihydroalprenolol binding to rat colon membranes without evidencing atypical beta-adrenergic sites.

[3H]Dihydroalprenolol ([3H]DHA) specific binding (determined by the difference in the presence and absence of 20 microM (-)isoprenaline) to rat colon membranes was saturable (Bmax = 39.6 fmol/mg protein), of high affinity (Kd = 0.87 nM) and stereospecific (IC50 330 and 3510 nM for (-)- and (+)isoprenaline, respectively); the Hill coefficient was close to one, indicating binding homogeneity. [3H]DHA (0.6 nM) specific binding was potently inhibited (Ki range 1.9-3.3 nM) by the non-selective beta-adrenoceptor antagonists pindolol, alprenolol, but not by the non-adrenergic compounds 5-hydroxytryptamine, 8-hydroxydipropylaminotetraline, methysergide, dopamine and verapamil (Ki greater than 10,000 nM). The selective beta 1- and beta 2-adrenoceptor antagonists CGP 20,712A and ICI 118,551 resulted in biphasic competition binding curves, whose low and high affinity components were compatible with two populations of binding sites accounting for about 75 (beta 2) and 25% (beta 1) of total sites. The relative competing potencies of reference adrenergic agonists also suggested a prevalence of beta 2-adrenergic sites. The new agonists phenylethanolaminotetralines (PEATs), highly selective for the atypical beta-adrenoceptors whose abundance in rat colon has been confirmed by comprehensive functional studies, had variable affinity for the [3H]DHA-labelled sites depending on chirality, but with no substantial correlation with their pharmacological potency. Only 40% of [3H]DHA binding, at a concentration about 10 times its Kd for high affinity sites (beta 1 and beta 2), was prevented by saturating concentrations of isoprenaline. Under this condition, the representative PEAT, SR 58611A, highly potent and selective for atypical beta-adrenoceptors in functional tests, and its pharmacologically inactive enantiomer, both inhibited the residual binding equipotently. In conclusion, [3H]DHA binding did not detect atypical beta-adrenoceptor sites in rat colon membranes, most probably because of its weaker affinity for them than for the coexisting beta 1 and beta 2 sites. PEAT stereoisomers proved essential for assessing both the stereospecificity and the functional significance of this atypical binding and to compare their affinity for [3H]DHA-labelled sites and pharmacological potency.

Biochemical and pharmacological activities of SR 144190, a new potent non-peptide tachykinin NK2 receptor antagonist.

(R)-3-(1-[2-(4-benzoyl-2-(3,4-difluorophenyl)-morpholin-2-yl)- ethyl]-4-phenylpiperidin-4-yl)-1-dimethylurea (SR 144190) is a new non-peptide antagonist of tachykinin NK2 receptors. SR 144190 potently and selectively inhibited neurokinin A binding to NK2 receptors from various species, including humans. In in vitro functional assays, it was a potent, selective and competitive antagonist of NK2 receptors with apparent affinities (pA2 values) between 9.08 and 10.10. In vivo, SR 144190 blocked [Nle10]neurokinin A-(4-10)-induced bronchoconstriction in guinea pigs (ID50 = 21 micrograms kg-1 i.v. and 250 micrograms kg-1 i.d.) and [beta Ala8]neurokinin A-(4-10)-induced urinary bladder contraction in rats (ID50 = 11 micrograms kg-1 i.v. and 190 micrograms kg-1 i.d.). It prevented citric acid-induced cough and airway hyperresponsiveness to acetylcholine in guinea pigs (1 mg kg-1 i.p.) as well as castor oil-induced diarrhoea in rats (0.01-10 micrograms kg-1 s.c. or p.o). Finally, it blocked the turning behaviour induced by intrastriatal injections of [Nle10]neurokinin A-(4-10) in mice (ID50 = 3 micrograms kg-1 i.v. and 16 micrograms kg-1 p.o.).