Constitutively active mutants of the α(1a)- and the α(1b)-adrenergic receptor subtypes reveal coupling to different signaling pathways and physiological responses in rat cardiac myocytes

Abstract

Activation of α1-adrenergic receptors influences both the contractile activity and the growth potential of cardiac myocytes. However, the signaling pathways linking activation of specific α1-adrenergic receptor (AR) subtypes to these physiological responses remain controversial. In the present study, a molecular approach was used to identify conclusively the signaling pathways activated in response to the individual α(1A)- and α(1B)-AR subtypes in cardiac myocytes. For this purpose, a mutant a{1a)-AR subtype (α(1a)-S(290/293)-AR) was constructed based on analogy to the previously described constitutively active mutant α(1b)-AR subtype (α(1b)- S288-294-AR). The mutant α(1a)-S(290/293)-AR subtype displayed constitutive activity based on four criteria. To introduce the constitutively active α1- AR subtypes into cardiac myocytes, recombinant Sindbis viruses encoding either the α(1a)-S(290/293)-AR or α(1b)-S288-294-AR subtype were used to infect the whole cell population with >90% efficiency, thereby allowing the biochemical activities of the various signaling pathways to be measured. When expressed at comparable levels, the α(1a)-S(290/293)-AR subtype exhibited a significantly elevated basal level as well as agonist-stimulated level of inositol phosphate accumulation, coincident with activation of atrial natriuretic factor-luciferase gene expression. By contrast, the α(1b)-S(288- 294)-AR subtype displayed a markedly increased serum response element- luciferase gene expression but no activation of atrial natriuretic factor- luciferase gene expression. Taken together, this study provides the first molecular evidence for coupling of the a{1a)-AR and the α(1b)-AR subtypes to different signaling pathways in cardiac myocytes.