Gq-mediated Ca2+ signals inhibit adenylyl cyclases 5/6 in vascular smooth muscle cells

University of Würzburg, Institute of Pharmacology and Toxicology, Würzburg, Germany Submitted 5 May 2009 ; accepted in final form 29 October 2009 cAMP and Ca 2+ are antagonistic intracellular messengers for the regulation of vascular smooth muscle tone; rising levels of Ca 2+ lead to vasoconstriction, whereas an increase of cAMP induces vasodilatation. Here we investigated whether Ca 2+ interferes with cAMP signaling by regulation of phophodiesterases (PDEs) or adenylyl cyclases (ACs). We studied regulation of cAMP concentrations by Ca 2+ signals evoked by endogenous purinergic receptors in vascular smooth muscle cells (VSMCs). The fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps allowed the measurement of cAMP levels in single-living VSMCs with subsecond temporal resolution. Moreover, in vitro calibration of Epac1-camps enabled us to estimate the absolute cytosolic cAMP concentrations. Stimulation of purinergic receptors decreased cAMP levels in the presence of the β-adrenergic agonist isoproterenol. Simultaneous imaging of cAMP with Epac1-camps and of Ca 2+ with Fura 2 revealed a rise of intracellular Ca 2+ in response to purinergic stimulation followed by a decline of cAMP. Chelation of intracellular Ca 2+ and overexpression of Ca 2+ -independent AC4 antagonized this decline of cAMP, whereas pharmacological inhibition of Ca 2+ -activated PDE1 had no effect. AC assays with VSMC membranes revealed a significant attenuation of isoproterenol-stimulated cAMP production by the presence of 2 µM Ca 2+ . Furthermore, small interfering RNA (siRNA) knockdown of AC5 and AC6 (the two ACs known to be inhibited by Ca 2+ ), significantly reduced the decrease of cAMP upon purinergic stimulation of isoproterenol-prestimulated VSMCs. Taken together, these results implicate a Ca 2+ -mediated inhibition of AC5 and 6 as an important mechanism of purinergic receptor-induced decline of cAMP and show a direct cross talk of these signaling pathways in VSMCs. cAMP; adenylyl cyclases; fluorescence resonance energy transfer; vascular smooth muscle cells; purinergic receptors Address for reprint requests and other correspondence: M. Bünemann, Univ. of Marburg, Institute of Pharmacology and Toxicology, Karl-von-Frisch-Strasse 1, 35033 Marburg, Germany (e-mail moritz.buenemann{at}staff.uni-marburg.de ).