The potentiating effect of genistein on the relaxation induced by isoproterenol in rat aortic rings.

In rat aortic rings, the mechanism of potentiating effect of genistein, a tyrosine kinase inhibitor, on the relaxation induced by isoproterenol was examined. Pretreatment of the aortic rings by genistein, but not by daidzein, an inactive analogue of genistein, potentiated the relaxation induced by isoproterenol. Genistein also potentiated the relaxation induced by forskolin, an activator of guanylyl cyclase, and dibutyryl cyclic AMP. In addition, theophylline, an inhibitor of phosphodiesterase, potentiated the relaxation induced by isoproterenol and forskolin. Theophylline partly inhibited the potentiation of isoproterenol-induced relaxation by genistein while it completely inhibited the potentiation of forskolin-induced relaxation by genistein. Iberiotoxin, an inhibitor of Ca-activated K (KCa) channels, partly inhibited the isoproterenol-induced relaxation and the potentiating effect of genistein on the relaxation induced by isoproterenol. Quinacrine (an inhibitor of phospholipase A2), alpha-naphthoflavone (an inhibitor of cytochrome P-450 enzymes), and 8-methoxypsoralen (an inhibitor of cytochrome P-450 enzymes), partly inhibited the potentiating effect of genistein on the isoproterenol-induced relaxation, but metyrapone (an inhibitor of cytochrome P-450 enzymes), indomethacin (an inhibitor of cyclooxygenase), and AA861 (an inhibitor of 5-lipoxygenase) did not. These results suggest that the potentiation of isoproterenol-induced relaxation by genistein may be related to the activities of phosphodiesterase, KCa channels, and cytochrome P-450 enzymes.     

The inhibitory effects of iberiotoxin and 4-aminopyridine on the relaxation induced by beta 1- and beta 2-adrenoceptor activation in rat aortic rings.

1. In rat aortic rings contracted by phenylephrine, the relaxation induced by isoprenaline was partly inhibited by iberiotoxin, (ibTX), tetraethylammonium, 4-aminopyridine (4-AP) and 1,9-dideoxyforskolin, but not by glibenclamide. 2. In the presence of 4-AP, 1,9-dideoxyforskolin failed to inhibit further the relaxant response to isoprenaline. Cromakalim-induced relaxation was inhibited by glibenclamide. 3. In the absence of endothelium, ibTX and 4-AP still inhibited the relaxant response to isoprenaline. 4. The inhibitory effect of ibTX on the relaxant response to isoprenaline was eliminated by pretreatment with ICI-118,551, a beta 2-adrenoceptor antagonist, but not by atenolol, a beta 1-adrenoceptor antagonist. 5. The inhibitory effect of 4-AP on the relaxation induced by isoprenaline was abolished by atenolol, but not by ICI-118,551. 6. The inhibitory effect of ibTX on the isoprenaline-induced relaxation in the presence of atenolol was completely abolished by MDL 12,330A, an adenylate cyclase inhibitor. Further, the inhibitory effect of 4-AP on the isoprenaline-induced relaxation in the presence of ICI-118,551 was markedly reduced by MDL 12,330A. 7. The relaxation induced by dibutyryl cyclic AMP was partly inhibited by 4-AP but not by ibTX. However, in the presence of KT5720, an inhibitor of cyclic AMP-dependent protein kinase, ibTX failed to inhibit further the relaxation induced by isoprenaline. 8. These results suggest that, in rat aortic rings, KCa channels are involved in the relaxation induced by isoprenaline. In addition, KCa channels are mainly activated by beta 2-adrenoceptors through cyclic AMP-dependent pathways. Further, the inhibition of isoprenaline-relaxation by 4-AP may be related to the activation of beta 1-adrenoceptors and cyclic AMP formation.     

Demonstration of the beta 2-adrenoceptor intrinsic efficacy of AY-28,925 using the PGF2 alpha-contracted guinea-pig trachea

The abilities of AY-28,925, labetalol and medroxalol to relax the PGF2 alpha-contracted isolated guinea-pig trachea have been investigated to compare their activities at beta 2-adrenoceptors. Maximum relaxation induced by AY-28,925 was significantly greater than that induced by either labetalol or medroxalol. This relaxation occurred in a concentration-dependent manner over a range of concentrations consistent with the previously determined affinity of AY-28,925 for beta-adrenoceptors. ICI-118,551 inhibited AY-28,925-induced relaxation in a concentration-dependent manner with a pA2 value similar to that determined for ICI-118,551 inhibition of the selective beta 2-adrenoceptor agonist salbutamol, but not the selective beta 1-adrenoceptor agonist norepinephrine. The Schild plot slope for ICI-118,551 inhibition of AY-28,925 or salbutamol did not differ significantly from unity, while that for inhibition of isoproterenol (a non-selective beta-adrenoceptor agonist) did. It is concluded that AY-28,925 is a more efficacious relaxant of tracheal smooth muscle than either labetalol or medroxalol, and that this relaxant activity is the result of its greater intrinsic efficacy at the beta 2-adrenoceptor.     

Beta 1- and beta 2-adrenoceptor antagonist activities of ICI-215001, a putative beta 3-adrenoceptor agonist.

1. The present study was undertaken to characterize the beta 3-adrenoceptor agonist activity of ICI-215001 and to determine whether it exhibits additional activities on beta 1- and beta 2-adrenoceptors in isolated spontaneously beating atrium, trachea and ileum of guinea-pig. 2. In guinea-pig atrium, isoprenaline, a non-selective beta-adrenoceptor agonist, caused concentration-dependent, positive chronotropic effects that were inhibited by atenolol, a selective beta 1-antagonist. ICI-215001 also competitively antagonized the increase in heart rate caused by isoprenaline. 3. ICI-215001 exhibited low intrinsic activity at increasing the beating rate of atrium and no activity on resting or induced tone of tracheal strips. 4. In strips of guinea-pig trachea, contracted submaximally with carbachol, isoprenaline, caused concentration-dependent relaxations. Both ICI-118551, a selective beta 2-adrenoceptor antagonist, and ICI-215001 competitively inhibited the relaxations caused by isoprenaline. 5. In isolated strips of guinea-pig ileum longitudinal smooth muscle contracted with histamine, isoprenaline and ICI-215001 caused relaxations which were inhibited by alprenolol, a beta-adrenoceptor antagonist with modest affinity for beta 3-adrenoceptors, but were resistant to ICI-118551 and atenolol. 6. These results indicate that ICI-215001 exhibits beta 3-adrenoceptor agonist activity as demonstrated by relaxations mediated via atypical beta-adrenoceptors in the longitudinal smooth muscle of guinea-pig ileum. Further, the studies demonstrate that ICI-215001 can act as an antagonist at beta 1- and beta 2-adrenoceptors in situations where its intrinsic agonist activity is low.     

Comparison of the beta-adrenoceptor affinity and selectivity of cetamolol, atenolol, betaxolol, and ICI-118,551

The objective of the present study was to compare the quantitative differences in the beta 1- vs. beta 2-adrenoceptor affinity and selectivity of cetamolol and its enantiomers to the reference compounds atenolol, betaxolol, and ICI-118,551, using isolated tissues obtained from the dog, guinea pig, and rat. Cetamolol antagonized the beta-adrenoceptor-mediated responses induced by isoproterenol, epinephrine, norepinephrine, and salbutamol, in tissues from both the dog and guinea pig, in a concentration-dependent manner. For a given tissue, the beta-adrenoceptor antagonist activity of cetamolol (measured as a pA2 or pKB value) was independent of the agonist used. In the dog tissues, cetamolol was more potent at inhibiting responses in the coronary artery (beta 1-adrenoceptors) than in the saphenous vein (beta 2-adrenoceptors). In the guinea pig tissues, the potency of cetamolol was approximately the same in the trachea (mixed beta 1- and beta 2-adrenoceptors) and atria (predominately beta 1-adrenoceptors), but lower in the soleus muscle (beta 2-adrenoceptors). Studies with the S-(-) and R-(+) enantiomers of cetamolol demonstrated that the S-(-) enantiomer was approximately 100-fold more potent at beta 1-adrenoceptors than the R-(+) enantiomer. In rat brain, cetamolol displaced [3H]-dihydroalprenolol bound to homogenates of cortex (beta 1-adrenoceptor binding sites) and cerebellum (beta 2-adrenoceptor binding sites). The potency of cetamolol at beta 1-adrenoceptors was found to be similar to that of betaxolol but greater than that of atenolol. However, the magnitude of the beta 1-adrenoceptor selectivity displayed by atenolol and betaxolol was greater than that displayed by cetamolol. In contrast, ICI-118,551 was found to possess potent and selective affinity for beta 2-adrenoceptors.     

Inhibitory effect of sodium nitroprusside on the relaxing action of isoproterenol in isolated rat urinary bladder

1. Isoproterenol caused relaxation of rat detrusor muscles contracted by electrical stimulations. Sodium nitroprusside (SNP) and methylene blue inhibited the relaxation induced by isoproterenol without affecting forskolin-induced relaxation.2. In the presence of theophylline, zaprinast still potentiated isoproterenol-relaxation. In the presence of butoxamine, a selective β₂-adrenoceptor inhibitor, but not metoprolol, a selective β₁-adrenoceptor inhibitor, SNP still inhibited isoproterenol-relaxation.3. SNP inhibited the relaxation to dobutamine, a β₁-adrenoceptor agonist, but not that to terbutaline, a β₂-adrenoceptor agonist. The relaxation to dobutamine was also inhibited by methylene blue. Further, in the presence of theophylline, zaprinast still potentiated dobutamine-relaxation.4. Isoproterenol increased tissue level of cGMP, which was inhibited by propranolol.5. These results suggest a possibility that, in rat detrusor muscles, isoproterenol causes relaxation due to increases in the level of both cAMP and cGMP.     

Pharmacological characterization of beta2-adrenoceptor in PGT-beta mouse pineal gland tumour cells

1. The adrenoceptor in a mouse pineal gland tumour cell line (PGT-beta) was identified and characterized using pharmacological and physiological approaches. 2. Adrenaline and noradrenaline, adrenoceptor agonists, stimulated cyclic AMP generation in a concentration-dependent manner, but had no effect on inositol 1,4,5-trisphosphate production. Adrenaline was a more potent activator of cyclic AMP generation than noradrenaline, with half maximal-effective concentrations (EC50) seen at 175+/-22 nM and 18+/-2 microM for adrenaline and noradrenaline, respectively. 3. The addition of forskolin synergistically stimulated the adrenaline-mediated cyclic AMP generation in a concentration-dependent manner. 4. The pA2 value for the specific beta2-adrenoceptor antagonist ICI-118,551 (8.7+/-0.4) as an antagonist of the adrenaline-stimulated cyclic AMP generation were 3 units higher than the value for the betaI-adrenoceptor antagonist atenolol (5.6+/-0.3). 5. Treatment of the cells with adrenaline and forskolin evoked a 3 fold increase in the activity of serotonin N-acetyltransferase with the peak occurring 6 h after stimulation. 6. These results suggest the presence of beta2-adrenoceptors in mouse pineal cells and a functional relationship between the adenylyl cyclase system and the regulation of N-acetyltransferase expression.     

Characterization of beta 3-adrenoceptor-mediated relaxation in rat abdominal aorta smooth muscle

The present study was carried out to characterize beta-adrenoceptor subtypes mediating relaxation of rat abdominal aorta smooth muscle. (-)-Isoprenaline and a nonconventional beta(3)-adrenoceptor agonist, (+/-)-[4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((+/-)-CGP12177A), induced concentration-dependent relaxation of (-)-phenylephrine (0.3 microM) preconstricted spiral preparations. Pretreatment with a combination of (+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP20712A, a selective beta(1)-adrenoceptor antagonist) and (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI-118,5511, a selective beta(2)-adrenoceptor antagonist) (0.1 microM for each) produced a 14-fold rightward shift of the concentration-response curve for (-)-isoprenaline; however, the relaxation in response to (+/-)-CGP12177A was unaffected by the blockade of beta(1)- and beta(2)-adrenoceptors. In the presence of CGP20712A and ICI-118,551 (0.1 microM for each), the concentration-response curves for (-)-isoprenaline and (+/-)-CGP12177A were shifted to the right by a nonselective beta(1)-, beta(2)- and beta(3)-adrenoceptor antagonist, (+/-)-bupranolol (3 and 10 microM). These results clearly suggest that beta(3)-adrenoceptors are involved in beta-adrenoceptor-mediated relaxation of rat abdominal aorta smooth muscle.     

Potentiation of the relaxing action of isoproterenol by forskolin in rabbit aortic rings: The involvement of β2-adrenoceptors

• 1.1. Forskolin, an activator of adenylate cyclase, potentiated the relaxing response to isoproterenol in rabbit aortic rings precontracted by phenylephrine (PE).
• 2.2. The potentiating effect of forskolin was inhibited by propranolol, a β-adrenoceptor inhibitor, but not by methylene blue, a guanylate cyclase inhibitor.
• 3.3. The relaxing response to terbutaline, a β₂-adrenoceptor agonist, but not lower concentrations of dobutamine, a β₁-adrenoceptor agonist, also was potentiated by forskolin. Forskolin, however, potentiated the relaxing response to high concentrations of dobutamine, which activates both β₁- and β₂-adrenoceptors.
• 4.4. Yohimbine, an α₂-adrenoceptor inhibitor, glyburide, an ATP-sensitive K⁺ channel inhibitor, iberiotoxin, a Ca²⁺-activated K⁺ channel inhibitor, or endothelium-removal failed to affect the potentiating effect of forskolin.
• 5.5. Dibutyryl cyclic AMP (cAmp) also potentiated the relaxing response to terbutaline.
• 6.6. These results suggest that in rabbit aortic rings forskolin causes the apparent potentiation of isoproterenol-induced relaxation by mainly affecting the relaxing response due to the activation of β₂-adrenoceptors by the forskolin-induced increase in the level of cAMP.

     

Evidence for a cooperation between adenosine A2 receptors and beta 1-adrenoceptors on cardiac automaticity in the isolated right ventricle of the rat.

1. The effects of the adenosine receptor agonists, 5'-N-ethyl-carboxamidoadenosine (NECA) and 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS-21680) on ventricular automaticity induced by a local injury in the isolated right ventricle of the rat were studied. 2. In concentrations ranging from 0.1 to 100 nM, NECA significantly increased ventricular automaticity. This effect was more reproducible when the adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was present at 5 nM, a concentration that blocks A1 adenosine receptors. 3. The excitatory effect of NECA was not observed when DPCPX was present at a concentration of 10 microM, which antagonizes both A1 and A2 adenosine receptors, as well as when rats were reserpinized. 4. In reserpinized rats, NECA increased ventricular automaticity in the presence of isoprenaline and the beta 2-adrenoceptor antagonist, ICI-118,551, but not in the presence of the beta 1-adrenoceptor antagonists, bisoprolol or atenolol. 5. The A 2s-selective adenosine receptor agonist, CGS-21680 (0.1 nM-10 microM) was devoid of excitatory effect on ventricular automaticity. Binding studies of this compound to the rat ventricular membranes revealed that in the preparation there was no specific binding. 6. These results suggest that the excitatory effect of NECA on ectopic ventricular automaticity is dependent on endogenous catecholamines and is mediated by an A2 adenosine receptor which is in some way 'linked' to the beta 1-adrenoceptor. These A2 receptors do not appear to be of the A2a adenosine receptor subtype.     

beta(2) and beta(3)-adrenoceptor inhibition of alpha(1)-adrenoceptor-stimulated Ca(2+) elevation in human cultured prostatic stromal cells

Prostatic beta-adrenoceptors inhibit alpha(1)-adrenoceptor-stimulated contractility. This study examines the effects of beta-adrenoceptor stimulation upon phenylephrine-induced elevations of intracellular Ca(2+)([Ca(2+)](i)) in human cultured prostatic stromal cells, and contractility of human prostatic tissue. Human cultured prostatic stromal cells were used for [(3)H]-cAMP accumulation studies or were loaded with 5-oxazolecarboxylic acid, 2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(2-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benzofuranyl)-, (acetyloxy)methyl ester (FURA-2AM, 10 microM) for Ca(2+) imaging studies. The beta-adrenoceptor agonist isoprenaline increased the accumulation of [(3)H]-cAMP (pEC(50)+/-S.E.M. 6.58+/-0.11) in human cultured prostatic stromal cells, an effect antagonized by the beta(2)-adrenoceptor antagonist (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551), but not by the beta(1)-adrenoceptor antagonist, atenolol. Isoprenaline (3 microM), the adenylyl cyclase activator, forskolin (20 microM) and the phosphodiesterase-4 inhibitor, rolipram (10 microM) inhibited the elevation of [Ca(2+)](i) elicited by phenylephrine (20 microM). The effect of isoprenaline could be blocked by ICI 118,551 (100 nM), the adenylyl cyclase inhibitor cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine (MDL 12,330A, 20 microM) and the K(Ca) channel blocker, iberiotoxin (100 nM), but not by atenolol (1 microM) or the K(ATP) channel blocker, glibenclamide (3 microM). Agonists selective for beta(1)-(xamoterol and prenalterol), beta(2)-(procaterol and salbutamol) and beta(3)-((+/-)-(R(*), R(*))-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid, BRL37344) adrenoceptors inhibited the elevation of [Ca(2+)](i) elicited by phenylephrine (20 microM) with a rank order of BRL37344> or =xamoterol> or =isoprenaline>procaterol> or =prenalterol>salbutamol. The xamoterol effect was reversed by ICI 118,551 (100 nM), but not by 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol (SR59230A, 100 nM) or atenolol (1 microM). The BRL37344 effect was reversed by SR59230A (100 nM), but not by atenolol (1 microM) or ICI 118,551 (100 nM). Both xamoterol and BRL37344 inhibited phenylephrine-induced tissue contractility. This study shows that both xamoterol and BRL37344 are effective inhibitors of phenylephrine-induced effects in human cultured prostatic stromal cells and in prostatic tissue.     

Increased nitroglycerin-induced relaxation by genistein in rat aortic rings.

The effect of genistein, a tyrosine kinase inhibitor, on nitroglycerin-induced relaxation was examined in rat aortic rings contracted by phenylephrine. In rat aortic rings, genistein (10(-5) M and 3x10(-5) M), a tyrosine kinase inhibitor, but not daidzein, an analogue of genistein, increased relaxation induced by nitroglycerin in a concentration-dependent manner. Iberiotoxin, an inhibitor of Ca2+ -activated K+ channels, inhibited the relaxation induced by nitroglycerin, but it did not affect the effect of genistein. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, did not affect the relaxation induced by nitroglycerin. Theophylline, an inhibitor of cyclic AMP-dependent phosphodiesterase, increased the relaxation induced by nitroglycerin, and genistein (10(-5) M) failed to affect the relaxation induced by nitroglycerin in the presence of theophylline. Genistein also inhibited the activity of cyclic AMP-dependent phosphodiesterase. In addition, 6-[4-(4'-pyridyl)amino phenyl]-4,5-dihydro-3(2H)-pyridazinone hydrochloride, an inhibitor of cyclic GMP-inhibitable cyclic AMP phosphodiesterase, inhibited the relaxation induced by nitroglycerin. These results suggest that, in the rat aortic rings, genistein inhibits cyclic AMP-dependent phosphodiesterase activities, resulting in the increase of the relaxation induced by nitroglycerin.     

Pharmacological characterization of the β-adrenoceptor that mediates the relaxant response to noradrenaline in guinea-pig tracheal smooth muscle

Pharmacological characteristics of beta-adrenoceptors (beta-ARs) mediating noradrenaline-induced relaxation were investigated in guinea-pig tracheal smooth muscle. The inhibitory effects of several types of beta-AR antagonists on noradrenaline-induced relaxation against histamine contraction were scrutinized with Schild plot analysis. The concentration-response curve for noradrenaline obtained in the absence of phentolamine and uptake inhibitors was competitively antagonized by all of the beta-AR antagonists used in this study (propranolol, bupranolol, atenolol, butoxamine and ICI-118,551). However, their pA2 values were markedly less than the expected values for beta1-AR and beta2-AR. On the other hand, pA2 values of ICI-118,551 (6.85) determined in the presence of phentolamine suggested a contribution of a beta1 -AR rather than beta2 -AR. In the presence of phentolamine and uptake inhibitors (desipramine and deoxycorticosterone), the Schild plot for atenolol was a better fit, with two distinct straight lines. The pA2 values of atenolol provided by the regression were: approximately 7.0, which corresponds to the expected beta1-AR value, and approximately 6.5, which was 3 times less than the expected value for beta1 -AR, and thus the possible presence of two classes of beta1 -AR (beta1(Low) and beta1(High)) was suggested. This view was also supported by Schild plot analysis for propranolol, which fit two straight lines each with a slope of 1.0. The present findings indicate that beta1 -ARs contributing to noradrenaline-elicited relaxation in guinea-pig tracheal smooth muscle exhibit diverse pharmacological characteristics and may be subdivided into at least two classes with distinct affinities for atenolol. Under physiological conditions, beta1(Low) rather than beta1(High) seems to play a more significant role in noradrenaline-regulated airway smooth muscle tone.     

Sympathetic nerve stimulation activates both beta 1- and beta 2-adrenoceptors of SA and AV nodes in anesthetized dog hearts.

We investigated blocking effects of the selective beta 1-adrenoceptor blocker atenolol (0.1-100 micrograms/kg, i.v.), the selective beta 2-adrenoceptor blocker ICI 118,551 (1-1000 micrograms/kg, i.v.) and the combination of the two drugs on positive chronotropic and dromotropic responses to norepinephrine (NE) released by stimulation of the sympathetic nerves in anesthetized, neurally decentralized, open-chest dogs after atropine was given. Stimulation of the intracardiac sympathetic nerves to the SA nodal region or to the AV nodal region selectively increased heart rate or decreased AV conduction time, respectively. ICI 118,551 inhibited the chronotropic or dromotropic response to each stimulation in a dose-dependent manner, but its inhibition of the dromotropic response was less than that of the chronotropic response. Atenolol similarly inhibited either the positive chronotropic or dromotropic response to each stimulation in a dose-related manner. The combination of atenolol and ICI 118,551 attenuated the responses to each stimulation more than atenolol alone. These data indicate that sympathetic nerve stimulation activates both beta 1- and beta 2-adrenoceptors of the SA and AV nodes and that the proportion of beta 2-adrenoceptor-mediated effects on the AV node is less than that on the SA node. These results suggest that neurally released NE in part controls physiological functional cardiac responses mediated through beta 2-adrenoceptors, in addition to the responses predominantly mediated through beta 1-adrenoceptors.     

Evidence for different interactions between beta(1)- and beta(2)-adrenoceptor subtypes with adenylyl cyclase in the rat brain: a concentration-response study using forskolin.

The aim of this study was to investigate beta(1)- and beta(2)-adrenoceptor signalling systems in the rat brain studying the synergistic effects between beta-adrenoceptor agonists and forskolin- induced activation of adenylyl cyclase. Experiments were performed in slices from cerebral cortex and cerebellum because they contain mainly beta(1)- and almost exclusively beta(2)- adrenoceptors, respectively. Five beta-adrenergic agonists were used, clenbuterol, flerobuterol, isoproterenol, salbutamol, and tulobuterol. All agonists stimulated cyclic AMP accumulation in the cerebral cortex but flerobuterol was inactive in the cerebellum. Forskolin amplified the generation of cyclic AMP. Forskolin potentiation was observed in glial cells but not in neurons and was not dependent on the number of beta-adrenoceptors. In return the amplitude of the potentiation was highly dependent on the intrinsic activity of the agonist in the cerebral cortex whereas it was constant whatever the agonist tested in the cerebellum. To analyse this difference we developed a modelling approach using a concentration-response study. Isoproterenol and forskolin stimulations of cyclic AMP production were studied either alone or in combination with increasing concentrations of forskolin and isoproterenol, respectively. In the cerebral cortex isoproterenol and forskolin were both able to potentiate the cyclic AMP accumulation induced by the other compound, whereas, in the cerebellum, isoproterenol was unable to increase the stimulation induced by forskolin. The results support the hypothesis that beta(1)- and beta(2)-adrenoceptors display distinct mechanisms of action in the signalling system by which they stimulate the accumulation of cyclic AMP.     

The coupling of canine ventricular myocyte beta2-adrenoceptors to L-type calcium current

To establish the functional coupling of beta adrenoceptor (beta AR) subtypes of beta 1AR and beta 2AR to L-type calcium current (ICaL), we investigated the non-selective agonist isoproterenol (ISO), and the relatively selective beta 2AR agonists zinterol (ZIN) and salbutamol (SAL) on ICaL in isolated canine ventricular myocytes in the presence and absence of CGP 20712A (CGP) and atenolol (AT), selective beta 1AR antagonists, and ICI 118,551 (ICI) a selective beta 2AR antagonist. Peak ICaL was determined using "patch type" microelectrodes and whole cell voltage clamp. ISO (0.5 microM) increased ICaL maximally 3.5 +/- 0.67 fold. ZIN (10.0 microM) and SAL (10.0 microM) increased ICaL maximally 1.5 +/- 0.2 (n = 5) fold and 1.4 +/- 0.1 (n = 5) fold, respectively. These effects were fully inhibited by CGP (0.3 microM) and AT (1.0 microM), inhibitors of beta 1AR but not by ICI (0.1 microM) a beta 2AR inhibitor. ZIN at relatively lower concentrations (< or = 0.1 microM) did not increase ICaL. CGP (0.3 microM) but not AT and ICI inhibited ICaL in the absence of beta AR agonists. CGP inhibition of ICaL was absent in the presence of forskolin (FK, 1.0 microM) that increases cAMP levels and ICaL by directly stimulating the adenylate cyclase. These indicate that none of the antagonists affect ICaL through an action downstream of beta AR. CONCLUSION: beta-adrenergic agonists increase ICaL via beta 1AR but not beta 2AR in canine ventricular myocytes.     

Cardiac electrical responses to catecholamines are differentially mediated by beta 2-adrenoceptors in anesthetized dogs.

We investigated the beta 2-adrenoceptor-mediated effects of atrial and ventricular effective refractory period (ERP), SA node pacemaker activity, and AV conductivity induced by sympathetic nerve stimulation or epinephrine infusion in anesthetized dogs. A beta 2-adrenoceptor antagonist, ICI 118,551 up to 100 micrograms/kg, i.v., inhibited the positive chronotropic and dromotropic responses to sympathetic stimulation but did not shorten the atrial or ventricular ERP, ICI 118,551 also attenuated the positive chronotropic and dromotropic responses and the shortening of atrial ERP in response to epinephrine but not the shortening of ventricular ERP. A selective beta 1-adrenoceptor antagonist, atenolol, inhibited each electrical cardiac response to sympathetic stimulation and epinephrine infusion in a similar manner. These results suggest that beta 2-adrenoceptor-mediated electrical cardiac responses to endogenous catecholamines also exist in addition to the predominant beta 1-adrenoceptor-mediated responses, and that the order of the proportion of beta 2-adrenoceptor-mediated cardiac effects was SA node pacemaker activity much greater than AV conductivity = atrial ERP much greater than ventricular ERP in the dog heart.     

kappa-opioid receptor stimulation inhibits cardiac hypertrophy induced by beta1-adrenoceptor stimulation in the rat

To test the hypothesis that kappa-opioid receptor stimulation inhibits cardiac hypertrophy induced by beta1-adrenoceptor stimulation, we determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on cardiac hypertrophy induced by isoprenaline, a selective beta-adrenoceptor agonist, in neonatal ventricular myocytes upon blockade of beta2-adrenoceptor. Hypertrophy of cardiomyocytes was determined by increases in (i) total protein content; (ii) [3H]leucine incorporation; and iii) cell size. 10 micromol/l isoprenaline increased all three parameters. The effects were abolished by 2 micromol/l propranolol, a beta-adrenergic receptor antagonist, or 300 nmol/l CGP20712A, a beta1-adrenoceptor antagonist, but not by 100 nmol/l ICI118,551, a beta2-adrenoceptor antagonist. The effects were also abolished by Rp-cAMPs 100 micromol/l, a protein kinase A inhibitor and not by pertussis toxin 5 mg/l. The effects of isoprenaline in the presence or absence of ICI118,551 were also abolished by 1 micromol/l U50,488H. The inhibitory effects of U50,488H were abolished by 1 micromol/l nor-binaltorphimine, a selective kappa-opioid receptor antagonist. U50,488H also abolished the increases in the amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by 10 micromol/l isoprenaline in the presence or absence of ICI118,551, an effect also abolished by nor-binaltorphimine. In conclusion the results show that kappa-opioid receptor stimulation abolished both the cardiac hypertrophy and enhanced amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by beta1-adrenoceptor stimulation.     

Negative inotropic effects of isoprenaline on isolated left atrial assays from aged transgenic mice with cardiac over-expression of human beta(2)-adrenoceptors

The action of isoprenaline has been evaluated in an isolated, left atrial assay, from aged transgenic mice with cardiac-specific over-expression of the beta(2)-adrenoceptor. In the assay, isoprenaline produced a negative inotropic concentration-response curve that was not altered by incubation with CGP-20712A (1 microM), a beta(1)-adrenoceptor antagonist. However, after incubation with ICI-118,551 (300 nM), a selective beta(2)-adrenoceptor antagonist, isoprenaline produced a positive inotropic concentration-effect curve that was located to the left of the negative inotropic curve. This suggests that the negative inotropic effect was mediated by a homogenous population of negatively-coupled beta(2)-adrenoceptors. In the presence of CGP-20712A (300 nM), the positive curve was shifted to the right, suggesting that the positive inotropic effect was mediated, at least in part, by beta(1)-adrenoceptors. These results differ substantially from those previously obtained in young transgenic mice. An outline of an explanatory model, based on a concept of over-expressed receptors 'stealing' G-proteins, is suggested.