Abstract

Agonist occupied α₁-adrenoceptors (α₁-ARs) engage several signaling pathways, including phosphatidylinositol hydrolysis, calcium mobilization, arachidonic acid release, mitogen-activated protein (MAP) kinase activation, and cAMP accumulation. The natural agonist norepinephrine (NE) activates with variable affinity and intrinsic efficacy all adrenoceptors, and in cells that coexpress α₁- and β-AR subtypes, such as cardiomyocytes, this leads to coactivation of multiple downstream pathways. This may result in pathway cross-talk with significant consequences to heart physiology and pathologic state. To dissect signaling components involved specifically in α_1A- and β₂-AR signal interplay, we have developed a recombinant model system that mimics the levels of receptor expression observed in native cells. We followed intracellular Ca²⁺ mobilization to monitor in real time the activation of both G_q and G_s pathways. We found that coactivation of α_1A- and β₂-AR by the nonselective agonist NE or via a combination of the highly selective α_1A-AR agonist A61603 and the β-selective agonist isoproterenol led to increases in Ca²⁺ influx from the extracellular compartment relative to stimulation with A61603 alone, with no effect on the associated transient release of Ca²⁺ from intracellular stores. This effect became more evident upon examination of an α_1A-AR variant exhibiting a partial defect in coupling to G_q, and we attribute it to potentiation of a non G_q-pathway, uncovered by application of a combination of xestospongin C, an endoplasmic reticulum inositol 1,4,5-triphosphate receptor blocker, and 2-aminoethoxydiphenyl borate, a nonselective storeoperated Ca²⁺ entry channel blocker. We also found that stimulation with A61603 of a second α_1A-AR variant entirely unable to signal induced no Ca²⁺ unless β₂-AR was concomitantly activated. These results may be accounted for by the presence of α_1A/β₂-AR heterodimers or alternatively by specific adrenoceptor signal cross-talk resulting in distinct pharmacological behavior. Finally, our findings provide a new conceptual framework to rationalize outcomes from clinical studies targeting α- and β-adrenoceptors.