Human atrial ß(1L)-adrenoceptor but not ß3-adrenoceptor activation increases force and Ca(2+) current at physiological temperature.

BACKGROUND AND PURPOSE: It has been proposed that BRL37344, SR58611 and CGP12177 activate ß3-adrenoceptors in human atrium to increase contractility and L-type Ca(2+) current (I(Ca-L)). ß3-adrenoceptor agonists are potentially beneficial for the treatment of a variety of diseases but concomitant cardiostimulation would be potentially harmful. It has also been proposed that (-)-CGP12177 activates the low affinity binding site of the ß1-adrenoceptor in human atrium. We therefore used BRL37344, SR58611 and (-)-CGP12177 with selective ß-adrenoceptor subtype antagonists to clarify cardiostimulant ß-adrenoceptor subtypes in human atrium. EXPERIMENTAL APPROACH: Human right atrium was obtained from patients without heart failure undergoing coronary artery bypass or valve surgery. Cardiomyocytes were prepared to test BRL37344, SR58611 and CGP12177 effects on I(Ca-L). Contractile effects were determined on right atrial trabeculae. KEY RESULTS: BRL37344 increased force which was antagonized by blockade of ß1- and ß2-adrenoceptors but not by blockade of ß3-adrenoceptors with ß3-adrenoceptor-selective L-748,337 (1 µM). The ß3-adrenoceptor agonist SR58611 (1 nM-10 µM) did not affect atrial force. BRL37344 and SR58611 did not increase I(Ca-L) at 37°C, but did at 24°C which was prevented by L-748,337. (-)-CGP12177 increased force and I(Ca-L) at both 24°C and 37°C which was prevented by (-)-bupranolol (1-10 µM), but not L-748,337. CONCLUSIONS AND IMPLICATIONS: We conclude that the inotropic responses to BRL37344 are mediated through ß1- and ß2-adrenoceptors. The inotropic and I(Ca-L) responses to (-)-CGP12177 are mediated through the low affinity site ß(1L)-adrenoceptor of the ß1-adrenoceptor. ß3-adrenoceptor-mediated increases in I(Ca-L) are restricted to low temperatures. Human atrial ß3-adrenoceptors do not change contractility and I(Ca-L) at physiological temperature.

Functional domains of the mouse beta3-adrenoceptor associated with differential G protein coupling.

Alternative splicing of mouse beta3-adrenoceptor transcripts produces an additional receptor isoform (beta3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (beta3a-adrenoceptor). We have shown that the beta3b-adrenoceptor couples to both Gs and Gi, whereas the beta3a-adrenoceptor couples only to Gs. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant beta3-adrenoceptors. In Chinese hamster ovary cells expressing beta3-adrenoceptors truncated at the splicing point, cAMP accumulation with CL316243 [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] increased by 59% following pretreatment with pertussis toxin, suggesting that the C-terminal region of the beta3a-adrenoceptor inhibits coupling to Gi. We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing beta3a-adrenoceptor, beta3b-adrenoceptor, and the truncated beta3-adrenoceptor. Treatment with beta3a-Tp10 (1 microM) caused cAMP responses to CL316243 in the beta3a-adrenoceptor to become pertussis toxin-sensitive and display a 30% increase over control, whereas the other peptides did not affect any receptor. Mutation at a potential tyrosine phosphorylation site (Tyr392Ala beta3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant beta3b-adrenoceptor became unresponsive to CL316243 while retaining an extracellular acidification rate response to SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate]. Our findings suggest that the beta3a-adrenoceptor cannot couple to Gi because of conformational changes induced by a protein(s) that interacts with residues in the C-terminal tail or because this protein(s) affects the intracellular localization of the beta3a-adrenoceptor.

Synthesis of 4-(1-oxo-isoindoline) and 4-(5,6-dimethoxy-1-oxo-isoindoline)-substituted phenoxypropanolamines and their beta1-, beta2-adrenergic receptor binding studies.

Phenoxypropanolamines with 1-oxo-isoindoline and 5,6-dimethoxy-1-oxo-isoindoline groups at the para position were synthesized. beta1, beta2-Adrenergic receptor binding affinities for the synthesized compounds were tested and compared with propranolol and atenolol. It was found that the incorporation of para-amidic functionality within the 1-oxo-isoindoline ring and 5,6-dimethoxy-1-oxo-isoindoline ring system led to a high degree of cardioselectivity in the phenoxypropanolamines. Two of the compounds and possessed beta1-adrenergic receptor affinity comparable with that of atenolol and both showed a better cardioselectivity than atenolol. Both and are undergoing further pharmacological evaluation.

Effect of carvedilol on Ca2+ movement and cytotoxicity in human MG63 osteosarcoma cells.

Carvedilol is a useful cardiovascular drug for treating heart failure, however, the in vitro effect on many cell types is unclear. In human MG63 osteosarcoma cells, the effect of carvedilol on intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Carvedilol at concentrations greater than 1 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50=15 microM). Carvedilol-induced [Ca2+]i rise was reduced by 60% by removal of extracellular Ca2+. Carvedilol-induced Mn2+-associated quench of intracellular fura-2 fluorescence also suggests that carvedilol induced extracellular Ca2+ influx. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of carvedilol on [Ca2+]i was inhibited by 50%. Conversely, pretreatment with carvedilol to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not carvedilol-induced, [Ca2+]i rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, did not alter carvedilol-induced [Ca2+]i rise. Separately, overnight treatment with 0.1-30 microM carvedilol inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, carvedilol increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum and other stores via a phospholipase C-independent manner. Carvedilol may be cytotoxic to osteoblasts.

Intrinsic sympathomimetic activity of (-)-pindolol mediated through a (-)-propranolol-resistant site of the beta1-adrenoceptor in human atrium and recombinant receptors.

The beta-blocker (-)-pindolol produces intrinsic sympathomimetic activity manifested clinically by cardiostimulation, but the beta-adrenoceptor subtype, which mediates these effects, is unknown. Recent work indicates the existence of a (-)-propranolol-resistant site of the cardiac beta(1)-adrenoceptor and we propose that it mediates the cardiostimulation evoked by (-)-pindolol. We compared the interaction of (-)-pindolol both with human atrial myocardium and with recombinant beta(1)-adrenoceptors. The effects of (-)-pindolol on paced human atrial trabeculae were studied in the presence of 3-isobutyl-1-methylxanthine (IBMX; 20 microM). (-)-Pindolol caused small negative and positive inotropic effects at nanomolar and micromolar concentrations respectively, which were unaffected by N(G)-monomethyl-L-arginine (L-NMMA, 10 microM), inconsistent with an involvement of nitric oxide. (-)-Pindolol, in the presence of (-)-propranolol, increased atrial contractile force and cAMP through recombinant beta(1)-adrenoceptors with identical potency (-logEC(50)M=6.5). The positive inotropic effects of (-)-pindolol were resistant to blockade by L-748,337 (100 nM), a beta(3)-adrenoceptor antagonist. (-)-CGP12177, known to act through the (-)-propranolol-resistant site of the beta(1)-adrenoceptor, also increased with similar potency atrial contractile force (-logEC(50)M=7.6) and cAMP at recombinant beta(1)-adrenoceptors (-logEC(50)M=7.7). (-)-Pindolol blocked the effects of (-)-CGP12177 in human atrium and recombinant beta(1)-adrenoceptors with similar equilibrium dissociation constants (pK(B)=6.5 and 6.3). Thus, stimulant potency and blocking potency of (-)-pindolol against (-)-CGP12177 agree. In contrast, (-)-pindolol was 200-400 times more effective at blocking the effects of a catecholamine than the effects of (-)-CGP12177 in both human atrium (pK(B)=9.1) and at recombinant beta(1)-adrenoceptors (pK(B)=8.6). We conclude that the cardiostimulant effects of (-)-pindolol in human atrial myocardium are mediated through a (-)-propranolol-resistant site of the beta(1)-adrenoceptor with low affinity for (-)-pindolol. In contrast, (-)-pindolol blocks the effects of catecholamines through a high-affinity site of the beta(1)-adrenoceptor. beta(3)-Adrenoceptors are not involved in the atrial effects of (-)-pindolol.

Apricot extract inhibits the P-gp-mediated efflux of talinolol.

Within the framework of developing strategies to enhance the intestinal absorption of P-glycoprotein (P-gp) substrates, the modulatory effect of a standardized apricot extract on P-gp-related efflux carriers was investigated in the Caco-2 system, Ussing chambers and the rat in situ perfusion model using talinolol as a model substrate. Using the Caco-2 system, polarity in transport of talinolol could be observed, the absorptive transport being much lower than the secretory transport (P(app-abs) = 1.08 +/- 0.29 x 10(-6) cm/s and P(app-secr) = 11.74 +/- 0.80 x 10(-6) cm/s). Inclusion of apricot extract (1%) in the apical medium resulted in a statistically significantly diminished polarity (P(app-abs) = 4.88 +/- 0.96 x 10(-6) cm/s and P(app-secr) = 9.39 +/- 0.58 x 10(-6) cm/s, p < 0.05). In addition, the inhibitory effect of apricot extract on P-gp related efflux mechanisms was shown to be concentration (0% approximately 0.1% < 0.3% < 1%) and pH dependent. Experiments performed with the Ussing chambers resulted in similar observations. In the rat in situ perfusion model, inclusion of apricot extract (1%) in the perfusion medium resulted in a threefold increase of the amount of talinolol appearing in the collected blood compared to the reference condition (23.6 +/- 5.53 pmol/cm. min and 7.13 +/- 1.08 pmol/cm. min, respectively; p < 0.05). Coadministration of this standardized apricot extract might be a safe and useful strategy to enhance the intestinal absorption of P-gp substrates. The nature and structure of the compound(s) responsible for this inhibiting effect on P-gp-related efflux carriers remain to be elucidated, as well as the exact mechanism by which apricot extract exerts its inhibitory function.

Nipradilol induces vasodilation of canine isolated posterior ciliary artery via stimulation of the guanylyl cyclase-cGMP pathway.

We examined the effect of nipradilol on contraction of the posterior ciliary artery induced by high potassium or norepinephrine and on cyclic GMP (cGMP) levels in the posterior ciliary artery of dogs. Nipradilol caused dose-dependent relaxation of KCl-and norepinephrine-induced contractions of posterior ciliary artery. The relaxant effect of nipradilol on norepinephrine-contracted ciliary artery was significantly greater than that on KCl-contracted ciliary artery. In KCl-contracted ciliary artery, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 10(-4) M) did not alter the relaxant effect of nipradilol, whereas 1H-1,2,4-oxadiazolo-4,3-a-quinoxalin-1-one (ODQ, 10(-6) M) significantly inhibited this effect. Ethacrynic acid at 10(-5) M, sulfasalazine at 10(-4) M and S-decylglutathione at 10(-4) M (glutathione S-transferase inhibitors) did not inhibit the relaxant effect of nipradilol. In addition, nipradilol produced dose-dependent increases in cGMP levels in the canine posterior ciliary artery. These findings indicate that nipradilol-induced vasorelaxation in the canine posterior ciliary artery occurs via stimulation of the guanylyl cyclase-cGMP pathway.

Differential effects of short and prolonged exposure to carvedilol on voltage-dependent Na(+) channels in cultured bovine adrenal medullary cells.

We examined the effects of short and prolonged exposure to carvedilol, an antihypertensive and beta-adrenoceptor blocking drug, on voltage-dependent Na(+) channels in cultured bovine adrenal medullary cells. Carvedilol (1-100 microM) reduced (22)Na(+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels. Carvedilol also suppressed veratridine-induced (45)Ca(2+) influx and catecholamine secretion in a concentration-dependent manner similar to that of (22)Na(+) influx. Prolonged exposure of the cells to 10 microM carvedilol increased [(3)H]saxitoxin ([(3)H]STX) binding, which reached a plateau at 12 h and was still observed at 48 to 72 h. Scatchard analysis of [(3)H]STX binding revealed that carvedilol increased the B(max) value (control, 14.9 +/- 0.9 fmol/10(6) cells; carvedilol, 23.8 +/- 1.2 fmol/10(6) cells) (n = 3, P < 0.05) without altering the K(d) value, suggesting a rise in the number of cell surface Na(+) channels. The increase in [(3)H]STX binding by carvedilol was prevented by cycloheximide, an inhibitor of protein synthesis, whereas carvedilol changed neither alpha- nor beta(1)-subunit mRNA levels of Na(+) channels. The carvedilol-induced increase of [(3)H]STX binding was abolished by brefeldin A and H-89, inhibitors of intracellular vesicular trafficking of proteins from the trans-Golgi network and of cyclic AMP-dependent protein kinase (protein kinase A), respectively. The present findings suggest that short-term treatment with carvedilol reduces the activity of Na(+) channels, whereas prolonged exposure to carvedilol up-regulates cell surface Na(+) channels. This may add new pharmacological effects of carvedilol to our understanding in the treatment of heart failure and hypertension.

Strategies used by rhizobia to lower plant ethylene levels and increase nodulation.

Agriculture depends heavily on biologically fixed nitrogen from the symbiotic association between rhizobia and plants. Molecular nitrogen is fixed by differentiated forms of rhizobia in nodules located on plant roots. The phytohormone, ethylene, acts as a negative factor in the nodulation process. Recent discoveries suggest several strategies used by rhizobia to reduce the amount of ethylene synthesized by their legume symbionts, decreasing the negative effect of ethylene on nodulation. At least one strain of rhizobia produces rhizobitoxine, an inhibitor of ethylene synthesis. Active 1-aminocyclopropane-1-carboxylate (ACC) deaminase has been detected in a number of other rhizobial strains. This enzyme catalyzes the cleavage of ACC to alpha-ketobutyrate and ammonia. It has been shown that the inhibitory effect of ethylene on plant root elongation can be reduced by the activity of ACC deaminase.

Aspirin impairs reverse myocardial remodeling in patients with heart failure treated with beta-blockers.

OBJECTIVES: We hypothesized that aspirin (ASA) might alter the beneficial effect of beta-blockers on left ventricular ejection fraction (LVEF) in patients with chronic heart failure. BACKGROUND: Aspirin blunts the vasodilation caused by both angiotensin-converting enzyme (ACE) inhibitors and beta-blockers in hypertensive patients and in patients with heart failure. Several studies suggest that ASA also blunts some of beneficial effects of ACE inhibitors on mortality in patients with heart failure. To our knowledge, there have been no data evaluating the possible interaction of ASA and beta-blockers on left ventricular remodeling in patients with heart failure. METHODS: We retrospectively evaluated patients entered into the Multicenter Oral Carvedilol Heart failure Assessment (MOCHA) trial, a 6-month, double-blind, randomized, placebo-controlled, multicenter, dose-response evaluation of carvedilol in patients with chronic stable symptomatic heart failure. Multivariate analysis was performed to determine if aspirin independently influenced the improvement in LVEF. RESULTS: Over all randomized patients (n = 293), LVEF improved 8.2 +/- 0.8 ejection fraction (EF) units in ASA nonusers and 4.5 +/- 0.7 EF units in ASA users (p = 0.005). In subjects randomized to treatment with carvedilol (n = 231), LVEF improved 9.5 +/- 0.9 EF units in ASA nonusers and 5.8 +/- 0.8 EF units in ASA users (p = 0.02). In subjects randomized to treatment with placebo (n = 62), LVEF improved 2.8 +/- 1.2 EF units in ASA nonusers and 0.5 +/- 1.4 EF units in ASA users (p = 0.20). Aspirin did not significantly affect the heart rate or systolic blood pressure response in either the placebo or carvedilol groups. The effect of ASA became more significant on multivariate analysis. The change in LVEF was also influenced by carvedilol dose, etiology of heart failure, baseline heart rate, EF and coumadin use. The detrimental effect of ASA on the improvement in LVEF was dose-related and was present in both placebo and carvedilol groups, although the effect was statistically significant only in the much larger carvedilol group. CONCLUSIONS: Aspirin significantly affects the changes in LVEF over time in patients with heart failure and systolic dysfunction treated with carvedilol. The specific mechanism(s) underlying this interaction are unknown and further studies are needed to provide additional understanding of the molecular basis of factors influencing reverse remodeling in patients with heart failure.

Identification and coupling to adenylate cyclase of three different [(3)H]CGP 12177 binding sites in Caco-2 cell membranes.

In the present investigation the identification of beta -adrenoceptor (beta -ARs) subtypes in the Caco-2 cell line was performed using radiometric assays. beta -ARs were measured using increasing concentrations of the highly specific beta -AR antagonist (-)[(3)H]CGP 12177 (0.06-4 nM), whereas the beta(1)- and beta(2)-AR subtypes discriminated through selective binding assays using the highly selective unlabelled antagonists CGP 20712A and ICI 118551. Atypical beta -ARs were measured using an incubation system formed by higher concentrations (0.6-20 nM) of (-)[(3)H]CGP 12177. beta - Atypical binding site concentrations (69 +/- 5 fmol mg ml(-1)of membrane protein) were higher than beta(1)-ARs (7 +/- 1) and beta(2)-ARs (24 +/- 2), respectively. The different beta -AR subtype affinities were characterized by binding inhibition experiments and the adrenergic agonists displaced the radioligand from its specific binding sites in the following order of potency: isoproterenol > clenbuterol > dobutamine > SR 58611A; for antagonists the order of potency was: propranolol approximately = ICI118551 approximately = CGP20712A. For atypical beta -ARs the order was: SR 58611A > clenbuterol > dobutamine > isoproterenol for agonists and propranolol > CGP 20712A > ICI 118551 for antagonists. As far as in vitro functional studies are concerned, beta -AR subtypes were shown to be coupled to adenylyl cyclase as their stimulation produced cAMP in an amount significantly higher than basal values. cAMP production after stimulation with dobutamine, clenbuterol, isoproterenol, and SR 58611A was measured using a cAMP radioassay kit. The order of efficacy suggested that the stimulation of beta(2)-ARs was the most effective in inducing the activation of cell signalling mechanisms. The identification of functional beta -ARs in a cancer cell line represents the first step in the study of the possible adrenergic control of cellular activities (e.g. proliferation and/or differentiation), which could suggest the use of this cancer cell line as a model for the study of cell activity or possibly new therapeutic strategies.

Evidence for cooperative binding of (-)Isoproterenol to rat brain beta1-adrenergic receptors.

In the present study, the effects of the thiol oxidising agent diamide upon the properties of rat brain beta1-adrenergic and human platelet serotonin2A receptor recognition sites have been investigated using [3H](-)CGP-12177 (in the presence of ICI-118551) and [3H]LSD as ligands. (-)Isoprenaline inhibited [3H](-)CGP-12177 binding with nH values of 0.87, 0.67, and 0.56 for added Mg2+ concentrations of 0, 2.5, and 25 mM, respectively. Pretreatment with diamide increased the nH to above unity for the inhibition of the binding by (-)isoprenaline, without a concomitant effect on the inhibition of the binding by (-)propranolol. In contrast, diamide reduced the affinity of human platelet serotonin2A-receptors for antagonists, but did not consistently induce nH values above unity for the inhibition of antagonist binding by serotonin. These results suggest that cooperative interactions reported for cardiac muscarinic receptors may also occur for beta1-adrenergic receptors in the rat brain.

Metabolism of carvedilol in dogs, rats, and mice.

The excretion and biotransformation of carvedilol [1-[carbazolyl-(4)-oxy]-3-[(2-methoxyphenoxyethyl)amino]-2-p ropanol], a new, multiple-action, neurohormonal antagonist that exhibits the combined pharmacological activities of beta-adrenoreceptor antagonism, vasodilation, and antioxidation, were investigated in dogs, rats, and mice. Carvedilol was absorbed well, and biliary secretion was predominant in each species. Carvedilol was metabolized extensively in each species, and elimination of unchanged compound was minor in bile duct-catheterized rats and dogs. In dogs, glucuronidation of the parent compound and hydroxylation of the carbazolyl ring, with subsequent glucuronidation, were the major metabolic pathways. Rats showed the simplest metabolite profile; the primary metabolites were formed by hydroxylation of the carbazolyl ring, with subsequent glucuronidation. Mice displayed the most complicated metabolite profile; glucuronidation of the parent compound and hydroxylation of either the carbazolyl or phenyl ring, with subsequent glucuronidation, were the major metabolic routes. O-Dealkylation was a minor pathway in all species examined.

In-vitro metabolic interaction of bunitrolol enantiomers in rabbit liver microsomes.

We have examined the 4-hydroxylation of bunitrolol in rabbit and rat liver microsomes. Significant species differences (rabbit < rat of both sexes) and sex (male > female of both species) were observed in the formation of 4-hydroxybunitrolol from racemic bunitrolol (10 microM). The 4-hydroxylation of bunitrolol racemate and enantiomers showed biphasic kinetics, a low-Km system and a high-Km system, in liver microsomes from rabbits of both sexes. There were significant differences in Km and Vmax values [(+) > (-)] for 4-hydroxylations of (+)-bunitrolol and (-)-bunitrolol in the low-K(m) system. Furthermore, the rate of clearance (Vmax/Km) was 20- to 200-fold for the low-Km system compared with the high-Km system, indicating that enzymes in the low-Km system play a major part in the rabbit liver microsomal bunitrolol metabolism. Inhibition studies using cytochrome P450 inhibitors such as quinidine, quinine, and alpha-naphthoflavone or polyclonal antibodies raised against rat P450-2D and -1A enzymes did not make clear which P450 enzymes are involved in bunitrolol 4-hydroxylation in rabbit liver microsomes. The 4-hydroxylase activity of (+)-bunitrolol was slightly higher than that of (-)-bunitrolol in separated incubations containing male rabbit liver microsomes and an enantiomer concentration of 10 microM. However, the 4-hydroxylation of (+)-bunitrolol (10 microM) was markedly suppressed in the presence of its antipode (10 microM), whereas (-)-bunitrolol 4-hydroxylation was not affected by the presence of its antipode, resulting in a change of the stereoselectivity from (+) > (-) for enantiomer to (+) < (-) for racemate. The difference in the Michaelis constants in the low-Km system, where the Km value of (-)-bunitrolol is one-eighth that of (+)-bunitrolol, is thought to cause the change in the stereoselectivity in rabbit liver microsome-mediated bunitrolol 4-hydroxylation.

Triiodothyronine and amiodarone effects on beta 3-adrenoceptor density and lipolytic response to the beta 3-adrenergic agonist BRL 37344 in rat white adipocytes.

The beta-adrenergic effects of catecholamines are potentiated by thyroid hormones in adipose tissue. Amiodarone (AM) is structurally similar to thyroid hormones and was used to explore the mechanism of the triiodothyronine (T3) effect on beta-adrenergic receptors (beta-ARs) in adipose tissue. AM decreases the expression of some T3 sensitive genes in various tissues and antagonizes the effect of T3 on its nuclear receptors. In this study, the T3, AM and AM + T3 effects on the beta 1- and beta 3-AR density were assessed on rat white adipocytes by radioligand binding using [3H]CGP 12177 after characterization of these subtypes by displacement of [3H]CGP 12177 binding by isoproterenol, BRL 37344 and noradrenaline. BRL 37344 was used to study beta 3-AR lipolysis. White adipocytes from hyperthyroid rats had increased responsiveness (Emax x 2) and sensitivity (+ 38%) to BRL 37344, while those given AM alone had decreased values. Moreover, AM antagonized the T3 effect on lipolysis. The beta 1-binding characteristics (receptor density [Bmax]: 45 +/- 4 fmol/mg of proteins; dissociation factor [Kd]: 0.96 +/- 0.10 nM) were not modified by either compound. Finally, T3 significantly increased beta 3-AR density (587 +/- 69 versus 363 +/- 25 fmol/mg of proteins) and Kd (38 +/- 2 versus 23 +/- 3 nM), while AM alone had no effect and did not antagonize the T3 effect on beta 3-AR number. In conclusion, the hyperthyroid state in the rat potentiated the lipolytic response of white adipocytes to a specific beta 3-agonist and increased the beta 3-AR density without changing in beta 1-AR number and affinity. Furthermore, the lack of antagonism between AM and T3 on beta 3-AR expression suggests that T3 does not work directly on the beta 3-AR gene. Moreover, AM induced a functional tissular hypothyroid-like effect and its antilipolytic effect probably occurred at a postreceptor level.

Isoproterenol interacts differently with beta-adrenoceptors in astrocytes and neurons isolated from the adult rat brain.

The interaction of isoproterenol with beta-adrenoceptors has been investigated in astroglial and neuronal cells isolated from adult rat cerebral cortices. Using the non-selective beta-adrenergic antagonist (3H)CGP-12177 as a ligand, binding experiments revealed that both types of cells exhibit beta-adrenoceptors. However the analysis of the isoproterenol displacement curve indicated that only neuronal cells contained the high affinity conformational state of the beta-adrenoceptor.

Isoproterenol and selective agonists stimulate similar atypical beta-adrenoceptors in rat adipocytes.

We have demonstrated previously that (-)isoproterenol triggers lipolysis in rat epididymal fat cells by stimulating both classical (beta 1, beta 2) and atypical beta-adrenoceptors. The contribution of the classical beta-adrenoceptors can be blocked by addition of 3 nM CGP12177(di-4-3[(1,1-dimethylethyl)amino]-(2-hydroxylpropoxy )1,3-dihydro-2H-benzimidazol-2-one hydrochloride). At higher concentrations, CGP12177 triggers lipolysis also, but by stimulating atypical beta-adrenoceptors only. To find out whether (-)isoproterenol and CGP12177 stimulate similar atypical beta-adrenoceptors, we compared their interaction with recognised beta 3-adrenoceptor antagonists: CGP20712 (1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl- 4-trifluoromethyl-2-imidazolyl)phenoxy]-propan-2-ol) (beta 1-selective), ICI118551 [erythro-1-(7-methylindan-4-yloxy)-3- (isopropylamine)-butan-2-ol] (beta 2-selective) and the stereoisomers as well as the racemic mixture of propranolol (non-beta 1/beta 2-subtype selective) and of metoprolol (beta 1-selective). There was a highly significant relationship (r = 0.93) between the potencies of these antagonists for inhibiting the lipolytic response to (-)isoproterenol (in the absence of classical beta-adrenoceptor stimulation) and CGP12177. In both cases, propranolol and metoprolol showed also the same degree of stereoselectivity. These findings suggest that (-)isoproterenol and CGP12177 stimulate the same type and/or form of atypical beta-adrenoceptors in rat epididymal adipocytes.

Are 5-HT receptors or beta-adrenoceptors involved in idazoxan-induced food and water intake?

Idazoxan (10 mg/kg, i.p.) produces an unexpected increase in food intake in freely-feeding rats which has been linked to its high affinity for non-adrenoceptor idazoxan binding sites. In this study, a dose-related antagonism of idazoxan-induced food intake by the beta-adrenoceptor antagonist (-)-propranolol (5-20 mg/kg, i.p.), which also blocks 5-HT1 (5-hydroxytryptamine1) receptors has been demonstrated. (+)-Propranolol (10, 20 mg/kg, i.p.) did not attenuate idazoxan-induced feeding. (-)-Propranolol (10 mg/kg, i.p.) but not the (+)-enantiomer (10 mg/kg, i.p.) also significantly inhibited the food intake, induced by the 5-HT1A agonist 8-OH-DPAT (0.25 mg/kg, i.p.). Idazoxan-induced feeding was not altered by the selective beta-adrenoceptor antagonists betaxolol (beta 1; 5 mg/kg, i.p.) and ICI 118,551 (beta 2; 5 mg/kg, i.p.) but was potentiated by the 5-HT receptor antagonist metergoline (5 mg/kg, i.p.). The anomalous findings with metergoline may reflect its action at different sub-types of 5-HT receptor. The water intake induced by idazoxan and the peripherally-active alpha 2-adrenoceptor antagonist L-659,066 was also blocked in a stereoselective manner by propranolol (10 mg/kg) but not significantly by either metergoline (5 mg/kg, i.p.), the beta 1-adrenoceptor antagonist betaxolol (5 mg/kg, i.p.) nor by the beta 2-adrenoceptor antagonist ICI 118,551 (5 mg/kg, i.p.). These results suggest that the food intake induced by idazoxan (and perhaps mediated by non-adrenoceptor idazoxan binding sites) may involve the 5-HT system, although further studies, using antagonists acting selectively at the different sub-types of 5-HT receptor, are required to confirm this.(ABSTRACT TRUNCATED AT 250 WORDS)

Labelling of rat brain beta-adrenoceptors: (3H)CGP-12177 or (125I)iodocyanopindolol?

Binding of (125I)iodocyanopindolol (ICYP) and (3H)CGP-12177 to rat brain homogenates was characterized and compared. ICYP was shown to bind to both beta-adrenergic and serotonin1B (5HT1B) receptors whereas (3H)CGP-12177 only labelled the first ones. The addition of 10 microM serotonin (5HT) prevented ICYP binding to 5HT receptors and under these experimental conditions both ligands labelled a similar total number of beta-adrenoceptors in the different rat brain regions. ICYP displayed a higher affinity for cerebellar (mainly beta 2-subtype) than for cerebral cortex beta-adrenoceptors (mainly beta 1-subtype) suggesting a subtype selectivity. A multiple displacement binding approach using CGP-20712A, a beta 1-subtype ligand, as competitor revealed a 2.6 fold selectivity of ICYP for the beta 2-adrenoceptor subtype. On the other hand, (3H)CGP-12177 binds only to beta-adrenoceptors and is not subtype selective in the rat brain homogenate. Considering both its high specificity and its lack of subtype selectivity (3H)CGP-12177 seems to be a more suitable ligand than ICYP to non-selectively label beta-adrenoceptors in rat brain.

Human beta 1- and beta 2-adrenergic receptor binding and mediated accumulation of cAMP in transfected Chinese hamster ovary cells. Profile of nebivolol and known beta-adrenergic blockers.

The interaction of nebivolol and its SRRR and RSSS enantiomers, and of known beta-adrenergic blockers, with human beta 1- and beta 2-adrenergic receptors expressed separately in Chinese hamster ovary cells in culture (CHO-Hu beta 1 and CHO-Hu beta 2), was investigated. We studied [3H]CGP-12177 binding to the intact cells and the accumulation of cAMP induced by isoproterenol. Each of the receptor subtypes displayed saturable [3H]CGP-12177 binding on intact cells with sub-nanomolar affinity. The density of beta 1- and beta 2-adrenergic receptor sites was 1.1 x 10(6) receptor binding sites per CHO-Hu beta 1 cell and 0.2 x 10(6) receptor binding sites per CHO-Hu beta 2 cell, respectively. The beta-adrenergic antagonists CGP 20712-A, ICI 118-551 and propranolol showed the same binding properties as beta-adrenergic receptors in previously described tissues or cells. The potencies of these compounds in inhibiting beta-adrenergic receptor mediated accumulation of cAMP corresponded well with their binding affinities. d-Nebivolol (SRRR) and nebivolol showed combined high affinity and selectivity for inhibition of beta 1-adrenergic receptor coupled accumulation of cAMP in CHO-Hu beta 1 cells (0.41 and 0.42 nM for d-nebivolol and nebivolol, respectively). l-Nebivolol (RSSS) was 1460 times less potent than d-nebivolol in CHO-Hu beta 1 cells. The binding affinities of d-nebivolol and nebivolol for human beta 1-adrenergic binding sites correlated well with their potencies in inhibiting beta 1-adrenergic receptor coupled accumulation of cAMP. CHO cells transfected with human beta 1- and beta 2-adrenergic receptors are a valid model system for studying the interaction of compounds with human beta-adrenergic receptors.