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It is uncertain if downregulation of β-adrenoceptor signaling pathway is promoted by an enhanced adrenergic tone at an early stage of cardiac disease, or it develops secondary to detrimental local myocardial changes in advanced heart failure. We examined the integrity of β-adrenoceptor signaling pathway upon chronic infusion of isoproterenol, a β-adrenoceptor agonist, at a dose producing no structural left ventricular (LV) remodeling and systolic dysfunction. Subcutaneous isoproterenol infusion (400 μg kg h over 16 days) to guinea pigs using osmotic minipumps produced no change in cardiac weights, LV internal dimensions, myocyte cross-sectional area, extent of interstitial fibrosis, and basal contractile function. Isolated, perfused heart preparations from isoproterenol-treated guinea pigs exhibited attenuated responsiveness to acute β-adrenoceptor stimulation, as evidenced by reduced LV developed pressure increase, less shortening of LV epicardial monophasic action potential and effective refractory period, and less myocardial cyclic adenosine monophosphate elevation, in response to isoproterenol exposure, when compared to saline-treated controls. Pharmacological responses to forskolin, an activator of the adenylate cyclase catalytic subunit, were well preserved in isoproterenol-treated hearts. Downregulation of β-adrenoceptor-mediated effects upon chronic isoproterenol infusion was associated with markedly reduced stimulatory G-protein α-subunit (G) myocardial expression levels. No change in expression levels of β-adrenoceptors, G-protein-coupled receptor kinase 2, inhibitory G-protein α-subunit (G), and Ca1.2 and K7.1 ion channels was determined in isoproterenol-treated hearts. We therefore conclude that sustained adrenergic overstimulation may promote downregulation of myocardial β-adrenoceptor-mediated effects independently of structural LV remodeling and systolic failure, an effect attributed to β-adrenoceptor uncoupling from adenylate cyclase due to reduced G-protein expression. [ABSTRACT FROM AUTHOR]

At 0.1–1 µM, U50488H, a κ-opioid receptor agonist, inhibited the stimulatory effects of 1 µM isoprenaline, a β-adrenoceptor agonist, on the electrically induced intracellular Ca2+ ([Ca2+]i) transient and cAMP accumulation ("cross talk" between κ-opioid receptors and β-adrenoceptors) in the presence of 1 µM ICI118,551, a β2-adrenoceptor antagonist in ventricular myocytes from both normoxic and chronically hypoxic rats. Pretreatment with 20 µg/ml cholera toxin increased the ADP-ribosylation of Gsα subunits and the electrically induced [Ca2+]i transient in both normoxic and chronically hypoxic rats. The effects of cholera toxin were inhibited by 1 µM U50488H and the inhibitory effect of U50488H was attenuated in chronically hypoxic rats. Similarly, 1 µM forskolin also increased the electrically induced [Ca2+]i transient and cAMP accumulation, which were both inhibited by U50488H, in both normoxic and chronically hypoxic rats. The inhibitory effects of 1 µM U50488H were significantly attenuated in chronically hypoxic rats. The results are novel findings, in that the "cross talk" between κ-opioid receptors and β-adrenoceptors is attenuated following chronic hypoxia. The attenuation may be due to decreased responses of Gs-protein and adenylyl cyclase to κ-opioid receptor activation in addition to desensitization of the receptor itself. [ABSTRACT FROM AUTHOR]

We studied the effect of isoproterenol on the Ca-activated K(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca concentration, [Ca]. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential ( V) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from a V of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca] from 0.01 to 1.0 μM. With [Ca] between 0.1 and 1.0 μM, 0.4 μM isoproterenol increased this current by 58.6±17.1%, whereas with [Ca] at 0.01 μM a sixfold smaller increase was observed. With [Ca]≥0.1 μM, 100 μM dibutyryl-adenosine 3′:5: cyclic monophosphate (cAMP) and 1 μM forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 μM propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from a V of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca] from 0.01 to 1.0 μM. Outside-out-whole-cell recordings with [Ca]≥0.1 μM indicated that 100 μM dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca], with a 35-fold larger effect observed with 0.1-0.5 μM Ca compared to 0.01 μM Ca. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by β-adrenoceptor stimulation, that the effect depends strongly on [Ca], and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in β-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined. [ABSTRACT FROM AUTHOR]

Effects of β-adrenergc stimulation on the membrane potential and intracellular Na, K and Cl activities were examined in isolated guinea-pig ventricular muscles using conventional and ion-selective elctrodes. Isoproterenol in concentrations of 30 nM−1 μM produced a transient depolarization followed by a slight hyperpolarization in electrically stimulated or quiescent papillary muscles. The negative logarithm of the concentration producing 50% maximum effect ( pD) for the membrane-depolarizing effect of isoproterenol was smaller than that for the positive inotropic effect, suggesting that a higher level of cAMP accumulation is required to produce the transient depolarization. Whereas the isoproterenol(1 μM)-induced depolarization was not blocked by tetrodotoxin (10 μM), nifedipine (10 μM), Cs (5 mM), Ba (0.3 mM), amiloride (1 mM) or ouabain (10 μM), it was significantly attenuated by anthracene-9-carboxylic acid (1 mM), a Cl-channel blocker. Intracellular K activity increased, whereas intracellular Na activity slightly decreased during the transient depolarization. Intracellular Cl activity significantly decreased during the isoproterenol-induced depolarization of the resting membrane. These results suggest that an inward current resulting from outward Cl movement, rather than inward Na movement, may be involved in the β-adrenoceptor-mediated membrane depolarization. [ABSTRACT FROM AUTHOR]

In isolated buccal segment of the rabbit facial vein, electrical responses produced by perivascular nerve stimulation and exogenously applied noradrenaline (NA) were recorded from the smooth muscle cells using microelectrode. Perivascular nerve stimulation hyperpolarized the smooth muscle cell membrane. The hyperpolarization was converted to depolarization after application of the β-adrenoceptor antagonist, propranolol, and the depolarization was blocked by α antagonists, yohimbine. These responses elicited by nerve stimulation were blocked by tetrodotoxin or guanethidine, but not by atropine. Exogenously applied NA mimicked the responses elicited by nerve stimulation. The amplitude of the β-adrenoceptor-mediated hyperpolarization was increased in low potassium solution, decreased in high potassium solution, but unaltered by low sodium or low chloride solution, i.e., the hyperpolarization may be generated by an increase in potassium conductance of the membrane. An involvement of the apamin-sensitive (Ca-dependent) potassium channel or sodium-potassium ATPase in the hyperpolarization was ruled out. [ABSTRACT FROM AUTHOR]