title: Gq-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.
authors.affiliation: University of Marburg, Institute of Pharmacology and Toxicology, Karl-von-Frisch-Strasse 1, 35033 Marburg, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: von Hayn
authors.forename: Ruth C
authors.initials: RC
authors.lastname: Werthmann
authors.forename: Viacheslav O
authors.initials: VO
authors.lastname: Nikolaev
authors.forename: Leif G
authors.initials: LG
authors.lastname: Hommers
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: Moritz
authors.initials: M
authors.lastname: Bünemann
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000201:enzymology; D015374:Biosensing Techniques; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D011983:Receptors, Purinergic / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014544:Uridine Triphosphate / Q000378:metabolism; D014661:Vasoconstriction; D014664:Vasodilation; D014970:Xanthines / Q000494:pharmacology
chemical_list: C119531:8-methoxymethyl-3-isobutyl-1-methylxanthine; D000067956:Adenylyl Cyclase Inhibitors; D000318:Adrenergic beta-Agonists; D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D011983:Receptors, Purinergic; D014970:Xanthines; D000242:Cyclic AMP; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000262:Adenylyl Cyclases; C406262:adenylyl cyclase 4; C121100:adenylyl cyclase 6; C419510:adenylyl cyclase type V; D007545:Isoproterenol; D014544:Uridine Triphosphate