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

Abstract

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 beta-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 muM 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.

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…However, only the advent of imaging technologies first by fluorescence (Tsien and Tsien, 1990) and later by FRET (Miyawaki, 2003) allowed the discovery of the full spectrum of spatiotemporal patterns in second-messenger concentrations. These studies revealed not only that concentrations in calcium as well as cAMP can show complex patterns of oscillations but that these may be interlinked by various intracellular mechanisms such as Ca 2ϩ -regulated phosphodiesterases and adenylyl cyclases (Zaccolo and Pozzan, 2003;Landa et al, 2005;Harbeck et al, 2006;Willoughby and Cooper, 2006;Kim et al, 2008;von Hayn et al, 2010;Ni et al, 2011;Werthmann et al, 2011). Oscillations have also been de- 326 scribed for the activity of protein kinase C (Violin et al, 2003).…”

“…Although it has been possible to generate images of primary cells or of functional units such as thyroid follicles or pancreatic islets isolated from mice transgenically expressing second-messenger sensors Calebiro et al, 2009;Mironov et al, 2009;von Hayn et al, 2010;Werthmann et al, 2009Werthmann et al, , 2011, the high degree of background fluorescence calls for several improvements for in vivo microscopy, including red-shifted sensors and FRET analysis by multiphoton and second harmonic generation microscopy (Provenzano et al, 2009). In addition, BRET studies have been performed in cells isolated from transgenic mice expressing luciferaselabeled ␤ 2 -adrenergic receptors and GFP2-labeled ␤-arrestin2 (Audet et al, 2010).…”

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

“…Ca-dependent adenylyl cyclases and PDEs may cause synchronous and antisynchronous changes in cAMP, respectively, and both types of correlation have been observed in different, and sometimes even the same, cells. Depending on the specific isoform, Ca may both inhibit and stimulate cAMP production (Willoughby and Cooper, 2007;von Hayn et al, 2010). In the other direction, cAMP may both induce (e.g., Capiod et al, 1991) and modulate (e.g., Nuttle and Farley, 1996) Ca oscillations and downstream responses.…”

Adenylyl cyclases 5 and 6 underlie PIP3-dependent regulation

“…All plasmids encoding for hAC5, hAC6, and hAC4 [wild-type (wt)] have been described elsewhere (31,32). Phosphatase and tensin homolog (PTEN) was provided by Dr. Dominik Oliver (Philipps University, Marburg, Germany).…”