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Adenosine-A2a Receptor Down-Regulates Cerebral Smooth Muscle L-Type Ca²⁺ Channel Activity via Protein Tyrosine Phosphatase, Not cAMP-Dependent Protein Kinase

Departments of Neurosurgery (K.M., V.G., H.Z., S.I., Y.D., J.M.S.), Physiology (J.M.S.), Pathology (J.M.S.), and Anatomy & Neurobiology (G.H.); University of Maryland School of Medicine, Baltimore, Maryland and Department of Neurological Surgery (A.W., R.W.), University of Washington, Seattle, WA

Adenosine acting via A2a receptors (A2aR) is a potent cerebral vasodilator that relaxes vascular smooth muscle cells (VSMCs) by a mechanism attributed to activation of cAMP-dependent protein kinase (cAK). We examined effects of adenosine and its mechanism of action on L-type Ca²⁺ channels in native VSMCs from rat basilar artery. Reverse transcription-polymerase chain reaction and immunofluorescence imaging confirmed transcription and expression of A2aR, and in situ hybridization confirmed presence of mRNA for L-type Ca_v1.2b channels. In patch-clamp experiments, adenosine down-regulated Ca²⁺ channel currents in a concentration-dependent manner, with receptor-subtype-specific antagonists [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) versus 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine] showing that this was caused by action of A2aR. Down-regulation of channel currents was mimicked by stimulation of cGMP-dependent protein kinase (cGK; 8-Br-cGMP) and by inhibition of tyrosine kinase (AG-18) but not by stimulation of cAK [forskolin and 8-bromo-cAMP (8-Br-AMP)]. Down-regulation of currents by the A2aR agonist 2-[p-(2-carboxyeth yl)phenylethylamino]-5'-N-ethyolcarboxamidoadenosine (CGS-21680) was blocked by inhibiting protein tyrosine phosphatase (PTP; orthovanodate and dephostatin), but not by inhibiting cGK (KT-5823 and H-7). Western blots of lysate or of immunoisolated Ca²⁺ channels from arterial segments incubated with CGS-21680 showed 1) increased phosphorylation of vasodilator-stimulated phosphoprotein that was blocked by inhibiting cAK (KT-5720), consistent with activation of cAK by A2aR; and 2) decreased tyrosine phosphorylation of immunoisolated 1c subunit of the Ca²⁺ channel. Our data show that cAK, although activated, was not germane to down-regulation of Ca²⁺ channel activity by A2aR, and they delineate a novel signaling mechanism involving reduced tyrosine phosphorylation of Ca²⁺ channels by A2aR probably caused by PTP activation.

Address correspondence to: Dr. J. Marc Simard, Department of Neurosurgery, University of Maryland School of Medicine, 22 South Greene St., Baltimore MD 21201-1595. E-mail: msimard{at}surgery1.umaryland.edu

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