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.     

Beta-2 adrenergic activation of L-type Ca++ current in cardiac myocytes

The whole-cell patch-clamp and intracellular perfusion techniques were used for studying the effects of a beta-2 adrenergic receptor activation on the L-type Ca current (ICa) in frog ventricular myocytes. The beta-2 adrenergic agonist zinterol increased ICa in a concentration-dependent manner with an EC50 (i.e., the concentration of zinterol at which the response was 50% of the maximum) of 2.2 nM. The effect of zinterol was essentially independent of the membrane potential. The stimulatory effect of zinterol was competitively antagonized by ICI 118,551, a beta-2 adrenergic antagonist. The maximal stimulatory effect of zinterol was comparable in amplitude to the effect of a saturating concentration (1 or 10 microM) of isoprenaline, a nonselective beta adrenergic agonist. Moreover, 3-isobutyl-1-methylxanthine (100 microM), a nonselective phosphodiesterase inhibitor, or forskolin (10 microM), a direct activator of adenylyl cyclase, had no additive effects in the presence of 0.1 microM zinterol. Zinterol had a long lasting action on frog ICa because after washout of the drug, ICa returned to basal level with a time constant of 17 min. An application of acetylcholine (1 microM) during this recovery phase promptly reduced ICa back to its basal level suggesting a persistent activation of adenylyl cyclase due to a slow dissociation rate constant of zinterol from its receptor. Zinterol also increased ICa in rat ventricular and human atrial myocytes, and the maximal effect was obtained at 10 and 1 microM, respectively. In all three preparations, intracellular perfusion with 20 microM PKI(15-22), a highly selective peptide inhibitor of cAMP-dependent protein kinase, completely antagonized the stimulatory effect of zinterol on ICa. We conclude that beta-2 adrenergic receptor activation produces a strong increase in ICa in frog, rat and human cardiac myocytes which is due to stimulation of adenylyl cyclase and activation of cAMP-dependent phosphorylation.     

Comparative study of potential for bisphosphonates to damage gastric mucosa of rats

1. The long-acting beta2-adrenoceptor agonist, salmeterol (10(-9)-10(-5) M), inhibited the IgE-mediated release of histamine from human lung mast cells (HLMC) in a dose-dependent fashion. Additional beta-adrenoceptor agonists were studied and the rank order of potency for the inhibition of histamine release from HLMC was isoprenaline > salmeterol > salbutamol. Approximate EC50 values for the inhibition of histamine release were 10 nM for isoprenaline and 100 nM for salbutamol. An EC50 value for salmeterol could not be calculated because maximal responses to salmeterol were not observed over the concentration range employed. 2. Both salmeterol and isoprenaline inhibited the generation of sulphopeptidoleukotrienes (sLT) more potently and more efficaciously than the release of histamine from immunologically-activated HLMC. Salmeterol (EC50 < 0.1 nM) was more potent than isoprenaline (EC50 0.4 nM) at attenuating sLT generation. 3. The beta-adrenoceptor antagonist, propranolol (1 microM), and the selective beta2-adrenoceptor antagonist, ICI 118,551 (0.1 microM), both caused rightward shifts in the dose-response curve for the inhibition of histamine release by isoprenaline. The antagonism of salmeterol effects by propranolol and ICI 118,551 was more complex. At lower concentrations (< 1 microM) of salmeterol, both antagonists shifted the dose-reponse curve to salmeterol rightward. At a higher concentration (10 microM) of salmeterol, neither ICI 118,551 nor propranolol was an effective antagonist of the salmeterol-mediated inhibition of histamine release. 4. Prolonged exposure (4 h) of HLMC to isoprenaline (1 microM) caused an approximately 50% reduction in the effectiveness of a second exposure to isoprenaline (10 microM) at inhibiting the release of histamine. whereas this pretreatment did not affect the salmeterol (10 microM) inhibition of histamine release. 5. Isoprenaline (10(-9)-10(-5) M) caused a dose-dependent increase in total cell cyclicAMP levels in purified HLMC which paralleled the inhibition of histamine release. Salmeterol (10(-9)-10(-5) M) was considerably less potent than isoprenaline at increasing HLMC cyclicAMP levels. 6. In summary, these data indicate that salmeterol is an effective inhibitor of the stimulated release of mediators from HLMC. The present data also suggest that salmeterol may act to inhibit mediator release from HLMC by beta-adrenoceptor-dependent and independent mechanisms.     

American Pediatric Society John Howland Award 1998: presentation and acceptance

In the dog saphenous vein (DSV), phenylephrine (PE) responses through alpha-1 adrenoceptors receptors are antagonized by both alpha-1 and alpha-2 receptor antagonists. Furthermore, pretreatment with chloroethylclonidine (CEC) eliminates prazosin binding but reduces rauwolscine binding by half (). In new functional experiments, the effects of preincubation with phenoxybenzamine (PBZ), an irreversible alpha adrenoceptor antagonist, on responses to PE and two selective alpha-2 adrenoceptor agonists were evaluated. Also, the ability of prazosin or rauwolscine to prevent irreversible losses of responses to these agonists when coincubated with PBZ was determined. Preincubation in PBZ (10-300 nM) concentration dependently reduced PE Emax and the calculated fraction of residual receptors (q). Preincubation in PBZ (10-300 nM) increased KB values for prazosin (30 and 100 nM) but did not alter the KB value for rauwolscine (50 nM) acting at the residual receptors from control values. Coincubation of PBZ with prazosin partially prevented these PBZ actions (Emax partly restored) on responses to PE, but coincubation of rauwolscine (/=300 nM caused >50% reduction in Emax values of responses but did not alter the EC50 values for either agonist. Coincubation of rauwolscine with PBZ protected responses to alpha-2 agonists against PBZ (1 microM) effects. This study shows that PE initiates contractions at atypical alpha-1 adrenoceptors represented by all sites of PE action. Rauwolscine antagonizes PE actions but does not protect against PBZ inactivation. Typical alpha-2 adrenoceptors are distinguished from the unusual alpha-1 adrenoceptors by their lesser sensitivity to PBZ and their protection by rauwolscine from PBZ.     

Use of a fluorescent analog of CDP-DAG in human skin fibroblasts: characterization of metabolism, distribution, and application to studies of phosphatidylinositol turnover

We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [3H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 microM Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 microM) on beta-adrenergic stimulation of ICa with isoprenaline (Iso, 10 nM or 1 microM). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,ACh by 3 microM ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of IK,ACh by either guanosine-5'-O-(3-thio)triphosphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of IK,ACh in a dose-dependent manner. The specific binding of [3H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [3H]QNB binding, exhibiting equilibrium dissociation constant (Ki) values of 187 nM for Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICa and IK,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.     

The heterocyclic substituted pyridine derivative (+/-)-2-(-3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429): a selective ligand at nicotinic acetylcholine receptors

The present report describes in vitro studies demonstrating that the heterocyclic substituted pyridine compound (+/-)-2-(3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429) is extremely potent in activating human muscle nicotine ACh receptor (nAChR) (EC50 = 59 +/- 17 nM; Emax = 110 +/- 09% vs. nicotine). RJR-2429 is markedly less potent in activating nAChRs in the clonal cell line PC12, with EC50 = 1100 +/- 230 nM and Emax = 85 +/- 20% when compared with nicotine. The activation of a putative alpha 3 beta 4-containing nAChR in PC12 by RJR-2429 reveals a potency intermediate between nicotine and epibatidine (EC50 of 20,000 nM for nicotine and 30 nM for epibatidine). Dose-response curves for agonist-induced ileum contraction indicate that RJR-2429 is equipotent with nicotine, having an EC30 of approximately 2 microM. RJR-2429 binds with high affinity to alpha 4 beta 2 receptor subtype (Ki = 1.0 +/- 0.3 nM), and chronic exposure results in significant up-regulation of the high-affinity [3H]nicotine binding sites. In addition, RJR-2429 does not activate nAChRs present in rat thalamic preparations but is a potent inhibitor of this receptor subtype. It antagonizes nicotine-stimulated ion flux in thalamic synaptosomes with an IC50 of 154 +/- 37 nM. It also is a potent partial agonist at nAChRs mediating dopamine release from rat synaptosomal preparations (EC50 = 2 +/- 1 nM; Emax = 40%; epibatidine and nicotine have EC50 values of 0.4 and 100 nM, respectively). A model for the structure-activity profile of RJR-2429, nicotine and epibatidine was derived by molecular forcefield and quantum mechanics calculations and may provide important clues for the development of ligands selective for nAChR subtypes as probes in the life sciences or as potential therapeutic tools.     

The effect of combretastatin A-4 disodium phosphate in a C3H mouse mammary carcinoma and a variety of murine spontaneous tumors

OBJECTIVE: Human myometrium contains both beta1-adrenergic and beta2-adrenergic receptors. This study was designed to assess the importance of each beta-adrenergic receptor subtype in relaxation of human myometrial muscle strips. STUDY DESIGN: Radioligand binding studies were used to establish the presence of each beta-adrenergic receptor subtype, whereas highly selective beta1-antagonists and beta2-antagonists were used to assess the contribution of beta-adrenergic receptor subtypes to myometrial relaxation after exposure to (-)-isoproterenol. RESULTS: Membranes prepared from myometrium contained 82% +/- 4% beta2-adrenergic receptors. After contraction produced by exposure to potassium chloride (35 mmol/L), isoproterenol produced relaxation with half maximal effect at 0.02 micromol/L and a maximal relaxation of 52% +/- 3%. Beta1-antagonist CGP-20712A had no significant effect, whereas beta2-antagonist ICI-118551 produced a characteristic rightward shift of the isoproterenol concentration-relaxation relationship. CONCLUSIONS: Although both beta1-adrenergic receptors and beta2-adrenergic receptors are present in human myometrial tissue at term, relaxation by nonselective beta-agonist isoproterenol is mediated exclusively by beta2-adrenergic receptors.     

Lipolytic action of Buthus occitanus tunetanus venom: involvement of the beta adrenergic pathway

Scorpion venoms contain active neurotoxins known to act selectively at the level of voltage sensitive Na+ and K+ channels on mammal nervous system. In the present report, we show for the first time that the venom of scorpion Buthus occitanus tunetanus (Bot) contains compounds able to activate another cell function in non excitable cells. Addition of this venom to the culture media of 3T3-L1 adipocytes or freshly dissociated rat adipocytes rapidly increases lipolysis as estimated by glycerol release (approximately 3 to 4 fold over basal values) in a dose-dependent manner (EC50 approximately 12 +/- 1.25 micrograms/ml; n = 3). Bot venom effect was lower and not additive to the effect produced by isoproterenol (IPE) (10 microM), a main lipolytic agent, n = 3. In Sephadex G-50 size exclusion chromatography, the lipolytic activity was excluded and not associated to the included neurotoxic fraction. Furthermore, no lipolytic effect could be detected in the Na+ channel specific toxin II purified from Androctonus australis hector (AaHII) or the K+ voltage-dependent channel toxin from Androctonus mauritanicus mauritanicus (KTx). Propranolol (a non selective beta adrenoreceptor (beta AR) antagonist), alprenolol and pindolol (selective beta 1/beta 2 antagonists) totally inhibited in a dose-dependent manner the lipolytic response to Bot venom (IC50 approximately 1 x 10(-7), 7.5 x 10(-8) and 3 x 10(-7)M, respectively), suggesting that venom stimulated lipolysis through the beta AR pathway. The pharmacological profiles of molecules acting more selectively on beta AR subtypes such as CGP 12177 (beta 1/ beta 2 antagonist with beta 3 agonist properties), CGP 20712A (beta 1 antagonist) and ICI 118551 (beta 2 antagonist) strongly suggest that lipolytic action of venom mainly involves the beta 2/beta 1 AR subtypes.     

Fungal infection of human organs by resistant melanin-synthesizing species is one of the pathogenic factors and one of the real consequences of the accident at Chernobyl power plant

1. Bradykinin (BK) and Lys-BK are peptides which are released at high nanomolar concentrations into the tear-film of ocular allergic patients. We hypothesized that these peptides may activate specific receptors on the ocular surface, especially the corneal epithelium (CE) and thus the CE cells may represent a potential target tissue for these kinins. 2. The purpose of the present studies, therefore, was to determine the presence of and the pharmacological characteristics of bradykinin receptors on normal cultured primary and SV40 virus-transformed human corneal epithelial (CEPI) cells by use of the accumulation of [3H]-inositol phosphates ([3H]-IPs) as a bioassay. 3. Bradykinin (BK) induced a maximal 1.95 +/- 0.24 fold (n = 17) and 2.51 +/- 0.29 fold (n = 26) stimulation of [3H]-IPs accumulation in normal, primary (P-CEPI) and SV40-immortalized (CEPI-17-CL4) cells, respectively. This contrasted with a maximal 3.2-4.5 fold and 2.0-2.9 fold stimulation by histamine (100 microM) and platelet activating factor (100 nM) in both cell-types, respectively. 4. The molar potencies of BK and some of its analogues in the CEPI-17-CL4 cells were as follows: BK (EC50 = 3.26 +/- 0.61 nM, n = 18), Lys-BK (EC50 = 0.95 +/- 0.16 nM, n = 5), Met-Lys-BK (EC50 = 2.3 +/- 0.42 nM, n = 5), Ile-Ser-BK (EC50 = 5.19 +/- 1.23 nM, n = 6), Ala3-Lys-BK (EC50 = 12.7 +/- 2.08 nM, n = 3), Tyr8-BK (EC50 = 19.3 +/- 0.77 nM, n = 3), Tyr5-BK (EC50 = 467 +/- 53 nM, n = 4) and des-Arg9-BK (EC50 = 14.1 +/- 2.7 microM, n = 4). The potencies of BK-related peptides in normal, P-CEPI cells were similar to those found in transformed cells, thus: BK, EC50 = 2.02 +/- 0.69 nM (n = 7), Tyr8-BK, EC50 = 14.6 +/- 2.7 nM (n = 3), Tyr5 = BK, EC50 = 310 +/- 70 nM (n = 4) and des-Arg9-BK, EC50 = 12.3 +/- 3.8 microM (n = 3). 5. The bradykinin-induced responses were competitively antagonized by the B2-receptor selective BK antagonists, Hoe-140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK; Icatibant; molar antagonist potency = 2.9 nM; pA2 = 8.54 +/- 0.06, n = 4; and slope = 1.04 +/- 0.08) and D-Arg0[Hyp3,Thi5,8, DPhe7]-BK (KB = 371 nM; pKB = 6.43 +/- 0.08, n = 4) in CEPI-17-CL4 cells. The antagonist potency of Hoe-140 against BK in normal, P-CEPI cells was 8.4 +/- 1.8 nM (pKi = 8.11 +/- 0.12, n = 4), this being similar to the potency observed in the immortalized cells. 6. This rank order of potency of agonist BK-related peptides, coupled with the antagonism of the BK-induced [3H]-IPs by the specific B2-receptor antagonists, strongly suggests that a B2-receptor subtype is involved in mediating functional phosphoinositide (PI) responses in the CEPI-17-CL4 and P-CEPI cells. 7. In conclusion, these data indicate that the P-CEPI and CEPI-17-CL4 cells express BK receptors of the B2-subtype coupled to the PI turnover signal transduction pathway. The CEPI-17-CL4 cells represent a good in vitro model of the human corneal epithelium in which to study further the role of BK receptors in its physiology and pathology, such as in allergic/inflammatory conditions, potential wound healing and other functions of the cornea.     

Involvement of potentially distinct neurotensin receptors in neurotensin-induced stimulation of striatal [3H]dopamine release evoked by KCl versus electrical depolarization

We intended to determine whether the effect of neurotensin (NT) on K+ and electrically evoked [3H]dopamine (DA) release from rat and guinea-pig striatal slices involved different mechanisms and/or receptors. In the two species, NT and three NT agonists were found to exhibit different relative potencies to enhance K+- and electrically-evoked [3H]DA release. NT(1-13) increased [3H]DA release with EC50 values in the nanomolar range and Emax values in the range of 100% of control. NT(8-13) and Eisai hexapeptide were both as active as NT(1-13) under K+ depolarization, but did not exceed 40% of the NT(1-13) effect under electrical depolarization. In rats, when [3H]DA release was stimulated with two successive K+ depolarizations, in the presence of NT(1-13), the NT effect during the second exposure to K+ was drastically decreased, suggesting that the NT receptor was desensitized. The desensitization process was essentially observed on Emax values, EC50 values being weakly affected. Similar results were obtained in guinea pig. In contrast, with two electrical depolarizations or with two different depolarizations (K+ followed by electrical), the NT effect during the second depolarization was not significantly affected. Concerning NT antagonists, SR 48692 antagonized with IC50 values in the nanomolar range the NT(1-13) stimulated K+-evoked [3H]DA release but did not affect, up to 10(-6) M, the NT(1-13) enhancement of electrically stimulated [3H]DA release. On the contrary, SR 142948A antagonized the NT(1-13) effect on K+- and electrically-evoked [3H]DA release. In conclusion, these results suggest the possible existence of potentially distinct neurotensin receptors differentially involved in the control exerted by NT on DA release under KCl vs electrical depolarization.     

The effect of artemisinine, its derivatives and mefloquine against chloroquine-resistant strains of Plasmodium falciparum in vitro

A 48 h in vitro test of the efficacy of artemisinine, dihydroartemisinine, artemether, mefloquine and chloroquine was carried out against 3 chloroquine-resistant strains of Plasmodium falciparum, strains K1 and T996 from Thailand and LS21 from India. A sigmoid Emax model was fitted to all in vitro inhibition data for each combination of drug and strain. Strains K1 and LS21 were strongly resistant to chloroquine, whereas T996 was partially resistant. Artemisinine, dihydroartemisinine and artemether were active against all strains, with complete growth inhibition at 10(-7) M. Artemether and dihydroartemisinine were both more potent than artemisinine, with 50% effective (EC50) values of 0.57-1.6 nM and 0.36-3.1 nM respectively; the EC50 of artemisinine was 1.5-6.1 nM for the 3 strains. The EC50 values for mefloquine were 46-185 nM. At higher concentrations, strains K1 and LS21 were fully inhibited, while with strain T996 mefloquine did not fully inhibit even at the highest concentration, 1.28 x 10(-6) M. It is concluded that artemisinine and its derivatives were highly effective against the 3 chloroquine-resistant strains, one of which showed borderline resistance to mefloquine.     

Influences of beta-blocking agents on cardiovascular actions induced by endothelin-3 administered intracerebroventricularly in anesthetized rats

Possible involvement that the pressor and positive chronotropic effects of endothelin-3 (ET-3) administered intracerebroventricularly (i.c.v.) participate in the central beta-adrenoceptor has been studied in urethane anesthetized rats. ET-3 administered i.c.v. at dose of 50 pmol elicited the increase in blood pressure and heart rate over a long duration, but a dose of 30 pmol had a minimal effect. These cardiovascular responses to ET-3 were significantly reduced by intravenous pretreatment with pentolinium, ganglionic blocking agent, and phentolamine, alpha-blocker. Also pressor responses to ET-3 were abolished dose-dependently by i.c.v. pretreatment with propranolol at a dose of 1 mumol, nonselective beta-blocker and ICI 118551, beta 2-selective adrenoceptor blocker, but not by i.c.v. pretreatment with metoprolol, atenolol, beta 1-selective blocker, procaine and phentolamine. These results suggest that ET-3 may play a role in cardiovascular regulation through the activation of the sympathetic nervous system involved in the central beta 2-adrenoceptor system.     

Acute effects of the beta 3-adrenoceptor agonist, BRL 35135, on tissue glucose utilisation

1. The acute effects of BRL 35135 (BRL) on tissue glucose utilisation index (GUI) in vivo were investigated in anaesthetized rats by use of 2-deoxy-[3H]-glucose. 2. Intravenous injection of BRL caused a dose-dependent increase in GUI in skeletal muscle, and white and brown adipose tissue; plasma insulin and fatty acid concentrations were also increased. Chronic treatment with BRL added to the diet caused a 34 fold increase in basal GUI of brown adipose tissue (BAT), but had no effect on GUI in other tissues. After chronic treatment, the acute tissue response to an intravenous maximal dose of BRL had disappeared completely in all tissues apart from the soleus muscle. 3. A high dose (20 mg kg-1) of the non-selective beta-antagonist, propranolol, inhibited the acute effect of BRL on GUI in BAT, but failed to affect GUI in muscle. A lower dose (1 mg kg-1) of the antagonist also inhibited the BAT response, but had little or no effect on the response in Type I (working) muscles such as soleus and adductor longus (ADL), and potentiated the response in Type II (non-working) muscles such as tibialis and extensor digitorium longus (EDL). 4. A low dose (1 mg kg-1) of the selective beta 1-antagonist, atenolol, had no effect on the BRL response but the same dose of the selective beta 2-antagonist, ICI 118551, potentiated significantly the effect of BRL on GUI in most muscles without altering plasma insulin levels. 5. It is concluded that: (i) the heterogeneous tissue responses of different muscle fibre types in the presence of P-antagonists indicates that BRL affects muscle GUI directly, in addition to effects mediated by increases in plasma insulin concentration; (ii) the resistance of the BRL response to conventional P-adrenoceptor antagonists implicates an atypical adrenoceptor mediating the GUI response in skeletal muscle, but this may not be identical to the adipose tissue P3-adrenoceptor; (iii) the potentiation of BRL responses by ICI 118551 indicates an inhibitory P2-adrenoceptor-mediated component in the muscle GUI response to BRL.     

Both beta 1- and beta 2-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium

The involvement of beta 1- and beta 2-adrenoceptors in catecholamine-evoked arrhythmias was investigated in isolated human right atrial appendages obtained from 22 patients chronically treated with beta blockers (usually beta 1-selective) and 9 patients not treated with beta blockers. A simple experimental model that assesses the incidence of arrhythmic contractions as a function of heart rate (pacing) is introduced. beta 1-adrenoceptors were activated by (-)-noradrenaline during beta 2-adrenoceptor blockade with 50 nmol/l ICI 118551. beta 2-adrenoceptors were activated by (-)-adrenaline during beta 1-adrenoceptor blockade with 300 nmol/l CGP 20712A. Both (-)-noradrenaline and (-)-adrenaline caused arrhythmic contractions whose incidence was greater at low than at high pacing rates. CGP 20712A (300 nmol/l) blocked the (-)-noradrenaline-evoked contractions in 1/1 atrial strip from 1/1 patient not treated with a beta blocker and 17/17 atrial strips from 15/15 patients chronically treated with beta blockers. ICI 118551 (50 nmol/l) blocked the (-)-adrenaline-evoked contractions in 3/4 atrial strips from 3/4 patients not treated with beta blockers and 17/20 atrial strips from 15/18 patients chronically treated with beta blockers. The incidence of arrhythmic contractions evoked by both (-)-noradrenaline and (-)-adrenaline was higher in chronically beta blocked patients than in non beta blocked patients. We conclude that both beta 1- and beta 2-adrenoceptors mediate atrial arrhythmias and that the generation of these arrhythmias is facilitated by chronic beta 1-adrenoceptor blockade.     

Mutated connexin43 proteins inhibit rat glioma cell growth suppression mediated by wild-type connexin43 in a dominant-negative manner

To explore the role of calcitonin gene-related peptide (CGRP) in rat pregnancy, we determined the density of myometrial CGRP-encoded nerve fibre terminals and examined, in an organ bath, the relaxant effect of the peptide on uterine strips near parturition. Comparisons were made with the uterus and aorta of nonpregnant rats. In the myometrium, CGRP immunoreactive nerve fibers were abundant in nonpregnant rats and scarce at the parturient stage. In the aorta there was no variation in the density of CGRP fibres with gestation. In nonpregnant rats only, CGRP relaxed spontaneous and tetrodotoxin (TTX)-sensitive electrically-evoked uterine contractions (EC50 40 nM, Emax 80%). The effect was antagonized by CGRP[8-37] (pKB 6.47) but was not affected by either blockers of nitricoxid synthase or ATP-sensitive potassium channels. CGRP was also able to relax contractions evoked by direct depolarization of the cells (TTX-insensitive contractions) (EC50, 2 nM, Emax 70%). In aorta contracted with arginine vasopressin, CGRP-induced relaxation was the same in nonpregnant and parturient animals. It was antagonized by CGRP [8-371 (pKB 6.90) and was abolished in presence of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). Amylin neither relaxed the uterus nor the aorta. In pregnant rats, the relaxant effect of CGRP on the uterus was limited on day 21 and was totally absent on day 22 of gestation. We conclude that the primary relaxant effect of CGRP on the uterus occurs at the level of myometrial smooth muscle cells. In the myometrium, gestation decreases CGRP innervation and impairs the relaxant responses to CGRP. Such changes are not observed in vascular tissues like aorta.     

Characterization of the inhibitory prostanoid receptors on human neutrophils

1. We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2-) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl-methionyl-leucyl-phenylalanine (FMLP), respectively. 2. Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ-induced LTB4 release (EC50 0.72 microM and 0.91 microM respectively), and FMLP-induced O2- release (EC50 0.42 microM and 0.50 microM respectively). PGF2 alpha, the TP-receptor agonist, U46619, and the IP-receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2. 3. The EP2/EP3-receptor agonist, misoprostol, and the selective EP2-agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16-times less potent than PGE2, respectively. In contrast, the EP1/EP3-receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 microM. 4. The selective DP-receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 microM) being approximately 50 times more potent than PGD2. BW245C also inhibited O2- release, and this inhibition was antagonized competitively by the DP-receptor blocking drug, AH6809 (pA2 6.6). 5. These data indicate the presence of both inhibitory EP- and DP-receptors on the human neutrophil. The rank order of potency of EP-receptor agonists suggest that the EP-receptors are of the EP2-subtype.     

Opposite modulation of noradrenaline and ATP release in guinea-pig vas deferens through prejunctional beta-adrenoceptors: evidence for the beta 2 subtype

Activation of prejunctional beta-adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of beta-adrenoceptor involved. In [3H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the beta 2-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 microM). Isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin- and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained concentration elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the beta 1-selective antagonist 1-[2-((3-carbamoyl- 4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM). The results confirm the opposite beta-adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional beta-adrenoceptor is of the beta 2-subtype.     

Effects of gavage versus dosed feed administration on the toxicokinetics of benzyl acetate in rats and mice

8-epi-prostaglandin F2 alpha stimulated contraction of human myometrial strips obtained from five different donors at the time of hysterectomy with a pEC50 value of 6.3 +/- 0.5. In paired strips from the same donors the pEC50 value for the selective TP receptor agonist U46619 ([1R-[1a,4a,5b(Z),6a(1E,3S*)]]-7-[6-(3- hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid) was 8.3 +/- 0.4. In strips from four other donors 8-epi-prostaglandin F2 alpha was ineffective whereas the pEC50 for U46619 was 6.9 +/- 0.3. Responses to 8-epi-prostaglandin F2 alpha were unaffected by the selective DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2- cyclohexyl-2-hydroxyethylamino)hydantoin) at 50 nM but were blocked by the selective TP receptor antagonist L670596 ((-)6,8-difluoro-9-p-methylsulfonyl benzyl-1,2,3,4- tetrahydrocarbazol-1-yl-acetic acid) at 50 nM. The pIC50 values obtained when the TP receptor antagonists GR 32191 ([1R- [1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'-biphenyl)-4- yl]methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid), ICI D1542 ((4(Z)-6-[(2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]- 4-(3-pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid), ICI 192605 (4(Z)-6-[(2,4,5-cis)-2-(2-chlorophenyl)-4-(2-hydroxyphenyl)-1,3- dioxan-5-yl]hexenoic acid), L670596 and SQ 29548 ([1S-(1 alpha,2 beta(5Z),3 beta,4 alpha]]-7- [3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7- oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) were added cumulatively to strips pre-contracted with an EC80 concentration of 8-epi-prostaglandin F2 alpha were not significantly different from those obtained when an EC80 concentration of U46619 was used. The effects of 8-epi-prostaglandin F2 alpha on strips pre-contracted with an EC80 concentration of U46619 were not different from those of U46619 itself. It is concluded that in the non-pregnant human myometrium 8-epi-prostaglandin F2 alpha is a medium potency contractile agonist acting predominantly at the TP receptor.     

AT2-antagonist sensitive potentiation of angiotensin II-induced vasoconstrictions by blockade of nitric oxide synthesis in rat renal vasculature

1. Although the actions of angiotensin II (Ang II) on renal haemodynamics appear to be mediated by activation of the AT1 receptor subtype, AT2 binding sites have also been evidenced in the adult kidney vasculature. As NO is known to mask part of the renal effects of vasoconstrictor drugs, we queried whether the Ang II-induced vasoconstrictions could occur via multiple receptor subtypes during inhibition of NO synthesis. We explored the effect of AT1 and AT2 receptor (AT-R) antagonists on Ang II-induced pressure increases during NO synthase or soluble guanylyl cyclase inhibition in rat isolated kidneys perfused in the presence of indomethacin at constant flow in a single-pass circuit. 2. In the absence of NO blockade, the AT1-R antagonist L-158809 (500 nM) antagonized the Ang II-induced vasoconstrictions, while the AT2-R antagonist PD-123319 (500 nM) had no effect. 3. Perfusing kidneys in the presence of either NO synthase inhibitors, L-NAME (100 microM) or L-NOARG (1 mM), or soluble guanylyl cyclase inhibitor, LY-83583 (10 microM), significantly increased both molar pD2 (from 9.40+/-0.25 to 10.36+/-0.11) and Emax values (from 24.9+/-3.1 to 79.9+/-4.9 mmHg) of the concentration-response curve for Ang II-induced vasoconstriction. 4. In the presence of L-NAME, 500 nM L158809 abolished the Ang II-induced vasoconstrictions whatever the concentration tested. On the other hand, 500 nM PD-123319 reversed the left shift of the concentration-response curve for Ang II (molar pD2 value 9.72+/-0.13) leaving Emax value unaffected (91.3+/-7.6 mmHg). 5. In the presence of L-NAME, the potentiated vasoconstriction induced by 0.1 nM and the augmented vasoconstriction induced by 10 nM Ang II were fully inhibited in a concentration-dependent manner by L-158809 (0.05-500 nM). By contrast, PD-123319 (0.5-500 nM) did not affect the 10 nM Ang II-induced vasoconstriction and concentration-dependently decreased the 0.1 nM Ang II-induced vasoconstriction plateauing at 65% inhibition above 5 nM antagonist. 6. Similar to PD-123319, during NO blockade the AT2-R antagonist CGP-42112A at 5 nM decreased by 50% the 0.1 nM Ang II-induced vasoconstriction and at 500 nM had no effect on 10 nM Ang II-induced vasoconstriction. 7. In conclusion, the renal Ang II-induced vasoconstriction, which is antagonized only by AT1-R antagonist in the presence of endogenous NO, becomes sensitive to both AT1- and AT2-R antagonists during NO synthesis inhibition. While AT1-R antagonist inhibited both L-NAME-potentiated and -augmented components of Ang II-induced vasoconstriction, AT2-R antagonists inhibited only the L-NAME-potentiated component.     

Peripheral and central adrenoceptor modulation of the behavioural effects of clozapine in the paw test

1. In rats, the atypical neuroleptic, clozapine, has been found to increase the hindlimb retraction time but not the forelimb retraction time, in the paw test. These parameters have predictive validity for the antipsychotic efficacy and extrapyramidal side-effects of drugs, respectively. The present study analysed to what extent drugs acting on adrenoceptors affect the behavioural effect of clozapine in the paw test. 2. The alpha 1-adrenoceptor agonist, ST 587 but not the peripherally working alpha 1-agonist, methoxamine, decreased the effect of clozapine on the hindlimb retraction time. The alpha 1-antagonist phenoxybenzamine increased this effect of clozapine, and blocked the effect of ST 587 on clozapine at low doses. Only the combination of phenoxybenzamine with clozapine produced an increase in forelimb retraction time. 3. The alpha 2-adrenoceptor agonist, clonidine, decreased the effect of clozapine on the hindlimb retraction time. This effect was neither antagonized by the alpha 2-antagonist rauwolscine nor by the alpha 1-antagonist phenoxybenzamine. Rauwolscine or the peripherally working alpha 2-antagonist L-659,066 did not influence the effect of clozapine on the hindlimb retraction time. The forelimb retraction time was not affected by any of the drug combinations. 4. In contrast to the beta 2-adrenoceptor agonist, clenbuterol, which was ineffective, the peripherally acting beta-agonist, (-)-isoprenaline, increased the effects of clozapine on the hindlimb retraction time. The beta-antagonist, (-)-propranolol as well as the peripherally acting beta-antagonist, nadolol decreased this effect of clozapine. Low doses of the peripherally acting beta 1-antagonist, atenolol, as well as low doses of the beta2-antagonist, ICI-118,551, decreased the effect of clozapine. A low dose of nadolol blocked the effect of (-)-isoprenaline on clozapine. Only the combination of clenbuterol with clozapine produced an increase in forelimb retraction time.5. It is concluded that blockade of central alpha l-adrenoceptors plays an important role in the effect of clozapine on the hindlimb retraction time. Furthermore, the effect of clozapine on the hindlimb retraction time is strongly modulated by peripheral beta 1- and/or beta 2-adrenoceptors. Given the predictive validity of the paw test, the presented data suggest that the alpha 1-adrenoceptor antagonist properties of clozapine are important for its therapeutic effects, but not for its lack of extrapyramidal side-effects.