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Research Article

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

¹University of Wuerzburg ²University of Würzburg ³University of Marburg

Submitted 5 May 2009 ; revised 29 October 2009 ; accepted in final form 29 October 2009

Cyclic AMP and Ca²⁺ are antagonistic intracellular messengers for the regulation of vascular smooth muscle tone: rising levels of Ca²⁺ lead to vasoconstriction, while an increase of cAMP induces vasodilatation. Here we investigated whether Ca²⁺ interferes with cAMP signaling by regulation of phophodiesterases (PDEs) or adenylyl cyclases (ACs). We studied regulation of cAMP concentrations by Ca²⁺ 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 Ca²⁺ with Fura 2 revealed a rise of intracellular Ca²⁺ in response to purinergic stimulation followed by a decline of cAMP. Chelation of intracellular Ca²⁺ and overexpression of Ca²⁺-independent AC4 antagonized this decline of cAMP while pharmacological inhibition of Ca²⁺-activated PDE1 had no effect. Adenylyl cyclase assays with VSMC membranes revealed a significant attenuation of isoproterenol-stimulated cAMP production by the presence of 2 µM Ca²⁺. Furthermore, siRNA knockdown of AC5 and AC6 (the two adenylyl cyclases known to be inhibited by Ca²⁺), significantly reduced the decrease of cAMP upon purinergic stimulation of isoproterenol-prestimulated VSMCs. Taken together, these results implicate a Ca²⁺-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.

vascular smooth muscle; cAMP; FRET; Ca2+