Pertussis toxin suppresses carbachol-evoked cardiodepression but does not modify cardiostimulation mediated through β1- and putative β4-adrenoceptors in mouse left atria: no evidence for β2- and β3-adrenoceptor function

Abstract

Activation of beta1-, beta2-, beta 3- and putative beta4-adrenoceptors modifies cardiac function. These receptors are usually coupled to Gs protein, but beta2- and beta3-adrenoceptors could also couple to Gi/o proteins. The mouse heart is used increasingly for studies of genetically disrupted or overexpressed proteins, including beta-adrenoceptor subtypes. We therefore investigated in contracting mouse left atria (2 Hz, 37 degrees C) if inactivation of Gi/o proteins with pertussis toxin modifies or uncovers effects mediated through beta-adrenoceptor subtypes. The negative inotropic effects of carbachol in atria exposed to catecholamine or high calcium (6.8 mmol/l) were assumed to be mediated through activation of muscarinic receptors coupled to Gi/o. We report conditions under which incubation of left atria with 200 ng/ml pertussis toxin for 24 h nearly abolished the carbachol responses. Although it has been reported that muscarinic receptor-mediated cardiodepression has an obligatory contribution of nitric oxide, the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (0.1-1 mmol/l) did not modify the negative inotropic effects of carbachol, inconsistent with an involvement of nitric oxide. The positive inotropic effects of (-)-noradrenaline and (-)-adrenaline, mediated through beta1-adrenoceptors, were not affected by pertussis toxin. (-)-Adrenaline did not cause positive inotropic effects attributable to beta2-adrenoceptor-mediation, in the presence of CGP 20712A (300 nmol/l) to block beta1-adrenoceptors, in control atria or atria pretreated with pertussis toxin. The positive inotropic effects of (-)-CGP 12177 (1 micromol/l), a compound with agonist activity at the putative beta4-adrenoceptor, were unaffected by pertussis toxin. The beta3-adrenoceptor-selective agonist BRL 37344 (1 micromol/l), in the presence of (-)-propranolol (200 nmol/l), did not cause positive or negative inotropic effects in control and pertussis toxin-treated atria. In left atria obtained from mice injected with 150 microg/kg i.p. pertussis toxin which abolished carbachol-evoked cardiode-pression, the positive inotropic effects of (-)-adrenaline were antagonised by CGP 20712A. The beta2-adrenoceptor-selective antagonist ICI 118551 (50 nmol/l) did not cause additional blockade of the effects of high (-)-adrenaline concentrations in the presence of CGP 20712A, ruling out the involvement of beta2-adrenoceptors. The results with intraparenteral PTX validate our in vitro PTX method. We conclude that inhibition of murine Gi/o proteins does not alter atrial positive inotropic effects mediated through beta1- and putative beta4-adrenoceptors and does not reveal functional beta2- and beta3-adrenoceptors.

Distribution of mRNA and binding sites of adrenoceptors and muscarinic receptors in the rat heart

“…Also, the expression of α 1A -adrenoceptor mRNA was not regularly recorded and it is conceivable that species differences may exist (for review see Brodde and Michel, 1999). In respect to β 3 -adrenoceptors, while majority of authors (for review see Gauthier et al, 2000) have determined the β 3 -adrenoceptors functionally and also found their mRNA, the others (Kaumann and Molenaar, 1997;Oostendorp and Kaumann, 2000) have found only marginal effects of β 3 -adrenoceptor specific agonist on heart function. Moreover, Evans et al (1996), Berkowitz et al (1995) and Krief et al (1993) were not able to identify β 3 -adrenoceptor mRNA in the heart.…”

Disruption of inhibitory G-proteins mediates a reduction in atrial β-adrenergic signaling by enhancing eNOS expression

“…However, it has also recently been suggested that the contribution of h 2 or h 3 adrenoreceptor signaling to the inotropic and HR response to physiological h-agonists in murine atria may be negligible under these conditions [41]. Furthermore, the h 1 signaling pathway, which may play a dominant role in physiological hadrenergic responsiveness, is not affected by acute Giblockade (for < 24 h) with PTx [43], suggesting the cardioinhibitory effects we see develop over the course of the 3 day incubation period, during which time eNOS gene upregulation occurs.…”

Phosphodiesterase‐4 blunts inotropism and arrhythmias but not sinoatrial tachycardia of (−)‐adrenaline mediated through mouse cardiac β1‐adrenoceptors

“…Spontaneously beating right atria, left atria and the free wall of the right ventricle were rapidly dissected, mounted in pairs and attached to Swema 4‐45 strain gauge transducers in an apparatus containing modified Tyrode's solution at 37 °C. Left atria and right ventricular walls were paced at 2 Hz and stretched as described ( Oostendorp and Kaumann, 2000 ; Heubach et al , 2002 ). Contractile force was recorded through PowerLab amplifiers on a Chart for Windows, version 5.0 recording programme (ADInstruments, Castle Hill, NSW, Australia).…”