G^sub q^-mediated Ca^sup 2+^ signals inhibit adenylyl cyclases 5/6 in vascular smooth muscle cells

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