Binding of alpha- and beta gamma-subunits of Go to beta 1-adrenoceptor in sealed unilamellar lipid vesicles.

First, we describe a preparation of sealed unilamellar lipid vesicles. When this preparation was subjected to sucrose density gradient centrifugation, two rather uniform fractions emerged, one consisting of lighter lipid-rich vesicles with average diameters ranging over 150-200 nm (fraction I), the other consisting of heavier vesicles with average diameters ranging over 30-70 nm (fraction II). When the lipid mixture containing dimyristoylglycerophosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoylglycerophosphoethanolamine at molar ratios of 54:35:10:1 was reconstituted with alpha- and beta gamma-subunits of Go-proteins purified to homogeneity from bovine brain, the lipid-rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a beta 1-adrenoceptor preparation purified from turkey erythrocyte membranes was reconstituted, it was found nearly completely in the smaller heavier vesicle fraction II where it was incorporated inside-out. On co-reconstitution of either alpha o or beta gamma alone with beta 1-adrenoceptors, some of these subunits appear together with beta 1-adrenoceptors in the small vesicle fraction II, but much more alpha o was bound to the receptor in the presence of beta gamma-subunits. The observations reported are novel and surprising in several respects: firstly, they suggest that beta gamma-subunits can bind to the non-activated beta 1-receptor where they may serve as an anchor for alpha-subunits. Secondly, the binding of alpha o- and beta gamma-subunits to the beta 1-adrenoceptors enhances the basal GTPase activity of alpha o. Thirdly, since the binding domains of the beta 1-adrenoceptor for G-proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G-proteins with the beta-receptor can occur at the aqueous membrane interface as was postulated originally by M. Chabre [Trends Biochem. Sci. 12, 213-215 (1987)] for the transducin-rhodopsin interactions. Finally, the binding of Go-subunits from bovine brain to a beta 1-adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.     

Adrenoceptors modulate depressor responses of endothelin-1 into the superior colliculus of rats

Endothelin-1 (ET-1) (10 pmol) microinjected into the superficial layer of superior colliculus induces decreases in blood pressure (control, 108 +/- 5 mmHg, n=6; ET-1, 71 +/- 4 mmHg, n=5). The effects on blood pressure induced by endothelin-1 were significantly (p<0.05) reduced by pre-administration into the superior colliculus of the alpha1-adrenoceptor agonist phenylephrine (1 nmol) (46 +/- 5%, n=5), beta1-adrenoceptor antagonist acebutolol (5 nmol) (51 +/- 6%, n=5) or beta1/beta2-adrenoceptor antagonist propranolol (3.4 nmol) (51 +/- 11%, n=5). In contrast, endothelin-1-induced effects were increased (p<0.05) by microinjections into the superior colliculus of prazosin (2.4 nmol) (49 +/- 7%, n=5), an alpha1-adrenoceptor antagonist; dobutamine (4 nmol) (51 +/- 9%, n=5), a beta1-adrenoceptor agonist or isoprenaline (1 nmol) (49 +/- 6%, n=5), a beta1/beta2-adrenoceptor agonist. No involvement of alpha2- or beta2-adrenoceptors has been detected. Therefore, ET-1 induces decreases in blood pressure with selective involvement of alpha1- and beta1-adrenoceptors.     

Reconstitution of beta 1-adrenoceptor-dependent adenylate cyclase from purified components.

In continuation of our efforts to reconstitute from purified components into lipid vesicles the signal transmission chain from beta 1-adrenoceptors to adenylate cyclase, we now report on the total reconstitution of the hormone-dependent adenylate cyclase. In these reconstitution experiments we have employed the purified adenylate cyclase (C) from bovine brain and rabbit heart, the stimulatory GTP-binding protein (GS) purified from turkey erythrocytes and rabbit liver and the beta 1-adrenoceptor (R) from turkey erythrocytes. Several detergents were compared with respect to their suitability to allow reconstitution of subunits into phospholipid vesicles. While octyl-polyoxyethylene (octyl-POE) was almost as potent as lauroyl-sucrose for preparation of vesicles containing GS.C, the latter detergent was clearly superior for vesicles enabling productive R.GS and R.GS.C coupling. The catalytic subunit from either bovine brain or rabbit heart was equally efficient in reconstitution. However, GS from turkey erythrocytes and rabbit liver revealed significant differences in RGS and RGS.C containing vesicles. While isoproterenol-induced activation of GS by GTP gamma S was first order in both instances, kon with turkey GS was 0.12 min-1, whereas kon with rabbit liver GS was 0.6 min-1. Moreover, GTP gamma S activation of erythrocyte GS was significantly more dependent on the presence of hormone than that of liver GS, confirming observations made on the native membrane-bound system. Compared with stimulation by isoproterenol (GTP gamma S) (4-fold), stimulation by isoproterenol/GTP was modest (1.3- to 1.6-fold).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dobutamine as selective beta(1)-adrenoceptor agonist in in vivo studies on human thermogenesis and lipid utilization.

The use of dobutamine as selective beta(1)-adrenoceptor agonist in in vivo studies on human thermogenesis and lipid utilization was investigated in 20 men. At 2.5, 5, and 10 microg x kg(-1) x min(-1), dobutamine induced significant increases in energy expenditure, lipid oxidation, and lipolysis. The beta(1)-adrenoceptor antagonist atenolol (bolus: 42.5 microg/kg, infusion: 1.02 microg x kg(-1) x min(-1)) blocked all dobutamine-induced effects on thermogenesis and lipid utilization. All parameters remained at levels comparable to those during saline infusion. The dose of atenolol used did not inhibit beta(2)-adrenoceptor-specific changes in energy expenditure, lipid oxidation, and lipolysis during salbutamol infusion (85 ng x kg(-1) x min(-1)). This indicates that atenolol was specific for beta(1)-adrenoceptors and did not camouflage concomitant beta(2)-adrenoceptor stimulation during dobutamine infusion. Therefore, we conclude that dobutamine can be used as a selective beta(1)-adrenoceptor agonist at dosages 相似文献   

Further characterization of beta3-adrenoceptors in the guinea pig gastric fundus: stereoselectivity, beta-adrenoceptor alkylation, and structure-activity relationship

The stereoselectivity of beta3-adrenoceptors, the effect of a beta-adrenoceptor alkylating agent, and the structure-activity relationship at beta3-adrenoceptors were investigated on the guinea pig gastric fundus. Isomeric activity ratios ((+)/(-)) for isomers of isoprenaline and noradrenaline were 20.9-fold and 43.7-fold, respectively, and were less than those obtained for activation of beta1- and beta2-adrenoceptors in the guinea pig atria and trachea, respectively. The concentration-response curves to the catecholamines ((-)-isoprenaline, (-)-noradrenaline, and (-)-adrenaline), the selective beta3-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium), and the nonconventional partial beta3-adrenoceptor agonist (+/-)-CGP12177A ((+/-)-[4-[3-[(1,1-dimethylethyl) amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride) were resistant to blockade by (+/-)-pindobind (10 microM), the beta-adrenoceptor alkylating agent. Furthermore, (+/-)-nadolol, which belongs to the aryloxypropanolamine class and has beta1- and beta2-adrenoceptor antagonistic characteristics, displays agonistic activity at beta3-adrenoceptors. These results indicate that pharmacological characteristics of the beta3-adrenoceptors of guinea pig gastric fundus differ from those of beta1- and beta2-adrenoceptors. (-)-Noradrenaline and (-)-adrenaline were more potent than dopamine and (-)-phenylephrine, respectively. In addition, dobutamine was 22-fold more potent than dopamine. These results suggest that the 4-hydroxyl group at the catechol ring and the beta-hydroxyl group and the large moiety on the alkylamine chain characterized efficacy at beta3-adrenoceptors.     

Subunit interactions of GTP-binding proteins.

Fluorescence energy transfer [cf. F?rster, T. (1948) Ann. Phys. 6, 55-75] was tested for its suitability to study quantitative interactions of subunits of G0 with each other and these subunits or trimeric G0 with the beta 1-adrenoceptor in detergent micelles or after reconstitution into lipid vesicles [according to Feder, D., Im, M.-J., Klein, H. W., Hekman, M., Holzh?fer, A, Dees, C., Levitzki, A., Helmreich, E. J. M. & Pfeuffer, T. (1986) EMBO J. 5, 1509-1514]. For this purpose, alpha 0- and beta gamma-subunits and trimeric G0 purified from bovine brain, the beta gamma-subunits from bovine rod outer segment membranes and the beta 1-adrenoceptor from the turkey erythrocyte were all labelled with either tetramethylrhodamine maleimide or fluorescein isothiocyanate under conditions which leave the labelled proteins functionally intact. In the case of alpha 0- and beta gamma-interactions, specific high-affinity binding interactions (Kd approximately 10 nM) and nonspecific low-affinity binding interactions (Kd approximately 1 microM) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10 microM guanosine 5'-O-[gamma-thio]triphosphate and 10 mM MgCl2 where only low-affinity binding interactions remained. Interactions between alpha 0- and beta gamma-subunits from bovine brain or from bovine retinal transducin did not differ much. The beta gamma-subunits from bovine brain were found to bind with high transfer efficiency and comparable affinities to the hormone-activated and the nonactivated beta 1-receptor reconstituted in lipid vesicles: Kd = 100 +/- 20 and 120 +/- 20 nM, respectively; however, beta gamma-subunits from transducin appeared to bind more weakly to the beta 1-adrenoceptor than beta gamma-subunits from bovine brain. Separated purified homologous alpha 0- and beta gamma-subunits from bovine brain interfered mutually with each other in binding to the beta 1-adrenoceptor presumably because they had a greater affinity for each other than for the receptor. These findings attest to the suitability of fluorescence energy transfer for studying protein-protein interactions of G-proteins and G-protein-linked receptors. Moreover, they supported the previous finding [Kurstjens, N. P., Fr?hlich, M., Dees, C., Cantrill, R. C., Hekman, M. & Helmreich, E. J. M. (1991) Eur. J. Biochem. 197, 167-176] that beta gamma-subunits can bind to the nonactivated beta 1-adrenoceptor.     

Agonist occupancy of a single monomeric element is sufficient to cause internalization of the dimeric beta2-adrenoceptor

A range of studies have indicated that many rhodopsin-like, family A G protein-coupled receptors, including the beta(2)-adrenoceptor, exist and probably function as dimers. It is less clear if receptors internalize as dimers and if agonist occupancy of only one element of a dimer is sufficient to cause internalization of a receptor dimer into the cell. We have used a chemogenomic approach to demonstrate that this is the case. Following expression of the wild type beta(2)-adrenoceptor, isoprenaline but not 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone, which does not have significant affinity for the wild type receptor, caused receptor internalization. By contrast, 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone, but not isoprenaline that does not have high affinity for the mutated receptor, caused internalization of Asp(113)Serbeta(2)-adrenoceptor. Following co-expression of wild type and Asp(113)Serbeta(2)-adrenoceptors each of isoprenaline and 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone caused the co-internalization of both of these two forms of the receptor. Co-expressed wild type and Asp(113)Serbeta(2)-adrenoceptors were able to be co-immunoprecipitated and 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone produced internalization of the wild type receptor that was not prevented by the beta-adrenoceptor antagonist propranolol that binds with high affinity only to the wild type receptor. These results demonstrate that agonist occupancy of either single binding site of the beta(2)-adrenoceptor dimer is sufficient to cause internalization of the dimer and that antagonist occupation of one of the two ligand binding sites is unable to prevent agonist-mediated internalization.     

Functional and structural characterization of the two beta 1-adrenoceptor forms in turkey erythrocytes with molecular masses of 50 and 40 kilodaltons

We have previously described a specific protease in turkey erythrocytes that converts the larger 50-kDa (P50) form of the beta 1-adrenoceptor to a smaller 40-kDa (P40) form [Jürss, R., Hekman, M., & Helmreich, E. J. M. (1985) Biochemistry 24, 3349-3354]. Further functional and structural characterization studies of the two forms are reported here. When purified P50 and P40 receptors were compared with respect to their relative capabilities to couple in lipid vesicles with pure stimulatory G-proteins (Gs-proteins) prepared from turkey erythrocytes or rabbit liver, a faster and larger activation of Gs-proteins was observed in response to l-isoproterenol and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with P40 than with P50 receptor. The kon values for P40 were 0.47 min-1 in the case of liver Gs and 0.22 min-1 in the case of erythrocyte Gs, whereas the corresponding values for P50 were 0.34 min-1 and 0.12 min-1, respectively. The binding properties of P50 and P40 forms of the receptor were not different, and desensitization of turkey erythrocytes on exposure to l-isoproterenol did not activate the protease. We furthermore ascertained that only the larger form with a molecular mass of 50 kDa carries the N-linked carbohydrates, which are removed on proteolytic conversion to the 40-kDa form and have either a triantennary or a tetraantennary nonfucosylated complex-type structure containing terminal sialyl residues.     

Use of electrofocusing for the analysis and purification of turkey erythrocytes beta 1-adrenoceptors

We show that following one cycle of alprenolol affinity chromatography of turkey erythrocyte beta 1-adrenoceptors, electrofocusing on polyacrylamide gels in digitonin, followed by electroelution, results in complete receptor purification. The overall yield from the electrofocusing-electroelution step of turkey erythrocyte beta-adrenoceptor is 75 +/- 3%. In addition, we are able to demonstrate that receptor-binding assays can be performed directly on the polyacrylamide gel, using 125I-cyanopindolol. This method can be employed for minute quantities of receptor which is an advantage when one wishes to characterize rapidly the beta-adrenoceptor in its native state from tissues that may be available only in limited amounts. We also report, for comparison, on the behavior of the turkey erythrocyte beta 1-adrenoceptor on immobiline polyacrylamide gels and the ability to purify only partially the receptor on these gels.     

Potential involvement of a propranolol-insensitive atypical beta-adrenoceptor the vasodilator effect of cyanopindolol in the human pulmonary artery.

The aim of our study was to examine whether non beta(1)-/beta(2)-adrenoceptors participate in the relaxation of the human pulmonary artery. For this purpose the vasodilatory effect of the non-conventional partial beta-adrenoceptor agonist cyanopindolol was examined. Cyanopindolol (1-300 microM), studied in the presence of the beta(1)-/beta(2)-adrenoceptor antagonist propranolol, relaxed the human pulmonary artery preconstricted with serotonin 1 microM in a concentration-dependent manner (maximally by about 80%). This effect was diminished by bupranolol 10 microM (an antagonist of beta(1)-beta(3)-adrenoceptors and the low affinity state of the beta(1)-adrenoceptor) and CGP 20712 10 microM (known to antagonize the low-affinity state of the beta(1)-adrenoceptor at high concentrations). In further experiments, the effect of beta-adrenoceptor ligands on the serotonin-induced vasoconstriction was examined. The concentration-response curve for serotonin was not affected by cyanopindolol 30 microM, bupranolol 10 microM and CGP 20712 10 microM but shifted to the right by cyanopindolol 100 and 300 microM; the serotonin 5-HT(2A) receptor antagonist ketanserin 0.3 microM abolished the maximum contraction elicited by serotonin. In conclusion, the present study reveals that the vasodilatory effect of cyanopindolol in the human pulmonary artery consists of two components, i.e. activation of a propranolol-insensitive atypical beta-adrenoceptor and antagonism against 5-HT(2A) receptors.     

Analysis of responses to sympathetic nerve stimulation in the feline pulmonary vascular bed

The adrenergic receptor subtypes mediating the response to sympathetic nerve stimulation in the pulmonary vascular bed of the cat were investigated under conditions of controlled blood flow and constant left atrial pressure. The increase in lobar vascular resistance in response to sympathetic nerve stimulation was reduced by prazosin and to a lesser extent by yohimbine, the respective alpha 1- and alpha 2-adrenoceptor antagonists. Moreover, in animals pretreated with a beta-adrenoceptor antagonist to prevent an interaction between alpha- and beta 2-adrenoceptors, responses to nerve stimulation were reduced by prazosin, but yohimbine had no significant effect. On the other hand, in animals pretreated with a beta-adrenoceptor antagonist, yohimbine had an inhibitory effect on responses to tyramine and to norepinephrine. Propranolol had no significant effect on the response to nerve stimulation, whereas ICI 118551, a selective beta 2-adrenoceptor antagonist, enhanced responses to nerve stimulation and injected norepinephrine. The present data suggest that neuronally released norepinephrine increases pulmonary vascular resistance in the cat by acting mainly on alpha 1-adrenoceptors and to a lesser extent on postjunctional alpha 2-adrenoceptors but that this effect is counteracted by an action on presynaptic alpha 2-receptors. The present studies also suggest that neuronally released norepinephrine acts on beta 2-adrenoceptors and that the response to sympathetic nerve stimulation represents the net effect of the adrenergic transmitter on alpha 1-, alpha 2-, and beta 2-adrenoceptors in the pulmonary vascular bed.     

Chronotropic response to (+/-)-CGP12177 in right atria of stressed rats

Foot-shock stress changes the sensitivity of the rat right atria to beta1- and beta2-adrenoceptor (AR) agonists. We investigated whether the same stress protocol also changes the atrial sensitivity to the non conventional agonist, (+/-)-CGP12177. Concentration-response curves to (+/-)-CGP12177, a beta1- and beta2-adrenoceptor antagonist with agonist properties at the putative beta4-adrenoceptors, were obtained in the absence and presence of propranolol (200 nM or 2 microM), CGP20712A 10 nM plus ICI118,551 50 nM, or CGP20712A (1 microM or 3 microM), in right atria from rats submitted to three daily foot-shock sessions (120 mA pulses of 1.0 s duration applied at random intervals of 5-25 s over 30 min) and killed after the third session. The pD2 for (+/-)-CGP12177 was not influenced by foot-shock stress. The stimulant effect of (+/-)-CGP12177 was resistant to blockade by 200 nM and 2 microM (+/-)-propranolol, and to combined blockade by CGP20712A and IC1118,551. However, in right atria from stressed rats given 200 nM propranolol, the concentration-response curve to the agonist was shifted 2.0-fold to the right. CGP20712A shifted the concentration-response curve to (+/-)-CGP12177 to the right by 4.6- (1 microM) and 19-fold (3 microM) in atria of control rats, and by 2.2- (1 microM) and 43-fold (3 microM) in atria of stressed rats. Maximum response to CGP12177 was not affected by propranolol or CGP20712A in concentrations ranging from 0.1 nM to 10 microM. These results show that the chronotropic effect of (+/-)-CGP12177 is mediated by atypical beta4-adrenoceptors. In constrast with to beta1-and (or) beta2-AR, this receptor is resistant to the effects of foot-shock stress, suggesting that the putative beta4-AR is a different receptor from a low affinity state of beta1-adrenoceptor, as previously proposed, unless both proposed isoforms of beta1-adrenoceptor show independent stress-induced behavior.     

Molecular mechanisms of G-proteins coupling with membrane receptors and second messenger systems

G-proteins transmit the signals from hormone receptors onto intracellular effector systems which take part in production of the second messengers such as cAMP, IP3, DAG and Ca2+. Molecular mechanisms of G-protein participation in the coupling of the seven-domain receptors to adenylate cyclase, phospholipase C and channels for Ca2+ and K+ ions are discussed in this paper. G-protein is a heterotrimers built of alpha-, beta- and gamma-subunits, which dissociate onto alpha- and beta gamma-subunits during interaction with hormone-receptor complex. alpha-subunit as well as beta gamma-dimmer may interact with effector system that leads to acceleration or slowing down of second messengers formation. Molecular mechanisms of such regulatory signal diversification are described. Seven-domain receptors possess very high recognition specificity of G-proteins. It is defined by combination of both alpha- and beta gamma-subunits in the G-protein structure. There is well-defined interaction specificity of G-protein alpha-subunit with effector systems. Combinations of different beta- and gamma-subunits involved in complex formation define interaction specificity of G-protein beta gamma-complex with effector systems. The highest interaction specificity of receptors with G-proteins and G-proteins with effector systems is found during triple complex formations: receptor--G-protein--effector. Such specificity is stronger in living cells than in membrane preparations. It can be an evidence of intracellular factors influence on the processes of interaction of the proteins involved in transmembrane regulatory signal transduction.     

Presynaptic beta(2)-adrenoceptors mediate nicotine-induced NOergic neurogenic dilation in porcine basilar arteries

We previously reported that nicotine-induced nitric oxide (NO)-mediated cerebral neurogenic vasodilation was dependent on intact sympathetic innervation. We hypothesized that nicotine acted on sympathetic nerve terminals to release norepinephrine (NE), which then acted on adrenoceptors located on the neighboring nitric oxidergic (NOergic) nerve terminals to release NO, resulting in vasodilation. The adrenoceptor subtype in mediating nicotine-induced vasodilation in isolated porcine basilar arterial rings denuded of endothelium was therefore examined pharmacologically and immunohistochemically. Results from using an in vitro tissue bath technique indicated that propranolol and preferential beta(2)-adrenoceptor antagonists (ICI-118,551 and butoxamine), in a concentration-dependent manner, blocked the relaxation induced by nicotine (100 microM) without affecting the relaxation elicited by transmural nerve stimulation (TNS, 8 Hz). In contrast, preferential beta(1)-adrenoceptor antagonists (atenolol and CGP-20712A) did not affect either nicotine- or TNS-induced relaxation. Results of double-labeling studies indicated that beta(2)-adrenoceptor immunoreactivities and NADPH diaphorase reactivities were colocalized in the same nerve fibers in basilar and middle cerebral arteries. These findings suggest that NE, which is released from sympathetic nerves upon application of nicotine, acts on presynaptic beta(2)-adrenoceptors located on the NOergic nerve terminals to release NO, resulting in vasodilation. In addition, nicotine-induced relaxation was enhanced by yohimbine, an alpha(2)-adrenoceptor antagonist, which, however, did not affect the relaxation elicited by TNS. Prazosin, an alpha(1)-adrenoceptor antagonist, on the other hand, did not have any effect on relaxation induced by either nicotine or TNS. The predominant facilitatory effect of beta(2)-adrenoceptors in releasing NO may be compromised by presynaptic alpha(2)-adrenoceptors.     

The interaction of the enantiomers of carvedilol with alpha 1- and beta 1-adrenoceptors

The stereoselectivity of carvedilol, a novel beta-adrenoceptor antagonist and vasodilator with one asymmetric carbon atom, was examined at alpha 1- and beta 1-adrenoceptors in vitro and in vivo. (-)-(S)-Carvedilol is a potent, competitive antagonist of the beta 1-adrenoceptor-mediated positive chronotropic response to isoproterenol in guinea pig atrium, with a dissociation constant (KB) of 0.4 nM. (+)-(R)-Carvedilol was more than 100-fold less potent than the (-)-S-enantiomer as an antagonist of beta 1-andrenoceptors, having a KB of approximately 45 nM. Consistent with these findings (-)-(S)-carvedilol (0.1 mg/kg, i.v.) produced a 25-fold rightward shift in the beta 1-adrenoceptor-mediated positive chronotropic response to isoproterenol in pithed rats, whereas the (+)-R-enantiomer had no beta 1-adrenoceptor blocking activity in vivo at this dose. In contrast to the marked degree of stereoselectivity observed at beta 1-adrenoceptors, both (-)-(S)- and (+)-(R)-carvedilol produced equal antagonism of the alpha 1-adrenoceptor-mediated vasoconstrictor response to norepinephrine in rabbit aorta, with KB values of 14 and 16 nM, respectively. Furthermore, in the pithed rat, the alpha 1-adrenoceptor-mediated pressor dose-response curve to cirazoline was shifted approximately 6-fold to the right by both the (+)-R- and (-)-S-enantiomers of carvedilol at a dose of 1 mg/kg, i.v. In anesthetized spontaneously hypertensive rats, (-)-(S)-carvedilol was 6-fold more potent as an antihypertensive than (+)-(R)-carvedilol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional characterization of beta-adrenoceptor subtypes in rabbit right atria

The advent of radioligand binding studies has allowed the classification of receptor subtypes in various tissues. However, the presence of a receptor subtype in a heterogenous tissue does not insure that the receptor has a significant physiological role. beta 1- and beta 2-Adrenoceptors have been reported to coexist in the rabbit right atria. The purpose of the present investigation was to determine the physiological role of beta-adrenoceptor subtypes in catecholamine-induced chronotropic responses in the rabbit right atria through comparison of data from functional and radioligand binding studies. Rank order of potency was determined using isoproterenol, epinephrine and norepinephrine for both chronotropic and inotropic responses in the rabbit right atria and right ventricular papillary muscles, respectively. These studies indicated that the beta 1-adrenoceptor was primarily responsible for catecholamine-induced responses. Next, the beta 1-selective antagonist, atenolol, was found to inhibit the chronotropic responses of the nonselective beta-agonist, isoproterenol, and the beta 2-selective agonist, terbutaline, to the same extent. These data indicate that terbutaline produces its chronotropic effects in the rabbit right atria through stimulation of beta 1-, not beta 2-adrenoceptors. Finally, competition studies for [125I]iodocyanopindolol and the relatively selective beta 1- and beta 2-adrenoceptor antagonists (ICI 89406 and ICI 118551, respectively) indicated that the ratio of beta 1- to beta 2-adrenoceptor subtypes is 6:1. It is concluded that while both receptors may be present in the rabbit right atria, the beta 1-adrenoceptor is the predominant subtype both in density and physiological significance, while the beta 2-adrenoceptor plays little, if any role, in the chronotropic responses induced by catecholamines.     

Pharmacological evidence for beta2-adrenoceptor in right atria from stressed female rats.

The purpose of the present study was to demonstrate a physiological response to TA2005, a potent m-adrenoceptor (beta2-AR) selective agonist, in right atria isolated from stressed female rats under the influence of the estrous cycle. We obtained concentration-response curves to the agonist in the presence and in the absence of selective antagonists in right atria isolated from female rats submitted to three daily foot-shock sessions (30 min duration, 120 pulses of 1.0 mA, 1.0 s, applied at random intervals of 5-25 s) and sacrificed at estrus or diestrus. Our results showed that the pD2 values of TA2005 were not influenced by estrous cycle phase or foot-shock stress. However, in right atria from stressed rats sacrificed during diestrus, the concentration-response curve to TA2005 was biphasic, with a response being obtained at concentrations of 0.1 nM, whereas during estrus no response was observed at doses lower than 3 nM. ICI 1118,551, a beta2-AR antagonist, abolished the response to nanomolar concentrations of TA2005 in right atria from stressed rats at diestrus, with no changes in agonist pD2 values in right atria from control rats (7.47 +/- 0.09, p > 0.05) but a 3-fold decrease in pD2 values of TA2005 in right atria from foot shock stressed rats (7.90 +/- 0.07, p < or = 0.05). Concentration-response curves to TA2005 in the presence of ICI118,551 were best fitted by a one-site model equation. The beta1-AR antagonist, CGP20712A, shifted to the right only the second part of the concentration-response curves to the agonist, unmasking the putative beta2-AR-mediated response to the agonist in tissues isolated from stressed rats at diestrus. Under this condition, concentration-response curves to the agonist were best fitted by a two-site model equation. pD2 and maximum response of TA2005 interaction with beta1- and putative beta2-adrenoceptor components were calculated. Schild analyses gave a pK(B) value for CGP20712A that was typical for the interaction with beta1-AR in each experimental group. pK(B) values for ICI118,551 could not be obtained in stressed rats sacrificed at diestrus since Schild plot slopes were lower than 1.0. In right atria from control rats, ICI118,551 pK(B) values were similar to reported values for the interaction of the antagonist with beta1-AR. These results confirm that a heterogenous beta-AR population mediating the chronotropic response to catecholamines can be demonstrated in right atria from foot shock stressed female rats sacrificed at diestrus. The stress-induced response seems to be mediated by the beta2-AR subtype. Right atria from rats sacrificed during estrus are protected against stress-induced alterations on the homogeneity of beta-AR population.     

S1319: a novel beta2-andrenoceptor agonist from a marine sponge Dysidea sp.

In the course of screening of potential leads for beta2-receptor agonists, we found a novel beta2-adrenoceptor selective agonist, S1319, from a marine sponge Dysidea sp. The active compound was isolated and structurally characterized as 4-hydroxy-7-[1-(1-hydroxy-2-methylamino)ethyl]-1,3-benzothiazole-2(3H)-o ne, a new member of the beta2-adrenoceptor agonist. This is the first example of a sponge-derived beta2-adrenoceptor agonist.     

Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase. Structural alterations in the beta-adrenergic receptor revealed by photoaffinity labeling

Preincubation of turkey erythrocytes with isoproterenol results in an impaired ability of beta-adrenergic agonists to stimulate adenylate cyclase in membranes prepared from these cells. The biochemical basis for this agonist-induced desensitization was investigated using the new beta-adrenergic antagonist photoaffinity label [125I]p-azidobenzylcarazolol ([125I]PABC). Exposure of [125I]PABC-labeled turkey erythrocyte membranes to high intensity light leads to specific covalent incorporation of the labeled compound into two polypeptides, Mr approximately equal to 38,000 and 50,000, as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Incorporation of [125I]PABC into these two polypeptides is completely blocked by a beta-adrenergic agonist and antagonist consistent with covalent labeling of the beta-adrenergic receptor. After desensitization of the turkey erythrocyte by preincubation with 10(-5) M isoproterenol, the beta-adrenergic receptor polypeptides specifically labeled by [125I]PABC in membranes prepared from desensitized erythrocytes were of larger apparent molecular weight (Mr approximately equal to 42,000 versus 38,000, and 53,000 versus 50,000) compared to controls. When included during the preincubation of the erythrocytes with isoproterenol, the antagonist propranolol (10(-5) M) inhibited both agonist-promoted desensitization of the adenylate cyclase and the altered mobility of the [125I]PABC-labeled receptor polypeptides. These data indicate that structural alterations in the beta-adrenergic receptor accompany the desensitization process in turkey erythrocytes.     

Effect of prolonged 5-hydroxytryptamine uptake inhibition by paroxetine on cortical beta 1 and beta 2-adrenoceptors in rat brain

The effects of prolonged (21 day) oral administration of the antidepressants paroxetine (0.9 to 8.9 mg/kg/day) and amitriptyline (2.7 to 27 mg/kg/day), on rat brain cortical beta 1- and beta 2-adrenoceptor numbers and affinities were investigated using [3H]-CGP 12177. Although amitriptyline, 27 mg/kg, caused a significant (p less than 0.05) 20% reduction in the number of beta 1-adrenoceptors, paroxetine, at doses up to 8.9 mg/kg p.o., did not influence binding of [3H]-CGP 12177 to cortical beta 1- or beta 2-adrenoceptors. This study with paroxetine provides further evidence that the down-regulation of central beta 1-adrenoceptors in rat brain after repeated administration is not a property of all antidepressant drugs.