title: Adenosine-A2a receptor down-regulates cerebral smooth muscle L-type Ca2+ channel activity via protein tyrosine phosphatase, not cAMP-dependent protein kinase.
abstract: 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 Cav1.2b 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 alpha1c 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.
authors.affiliation: Department of Neurosurgery, University of Maryland School of Medicine, 22 South Greene St., Baltimore MD 21201-1595, USA.
authors.forename: Katrina
authors.initials: K
authors.lastname: Murphy
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Gerzanich
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.forename: Svetlana
authors.initials: S
authors.lastname: Ivanova
authors.forename: Yafeng
authors.initials: Y
authors.lastname: Dong
authors.forename: Gloria
authors.initials: G
authors.lastname: Hoffman
authors.forename: G Alexander
authors.initials: GA
authors.lastname: West
authors.forename: H Richard
authors.initials: HR
authors.lastname: Winn
authors.forename: J Marc
authors.initials: JM
authors.lastname: Simard
mesh_terms: D000818:Animals; D001488:Basilar Artery / Q000187:drug effects / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002560:Cerebrovascular Circulation / Q000187:drug effects / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D043705:Receptor, Adenosine A2A; D018047:Receptors, Purinergic P1 / Q000502:physiology*
chemical_list: D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D043705:Receptor, Adenosine A2A; D018047:Receptors, Purinergic P1; D017868:Cyclic AMP-Dependent Protein Kinases; D017027:Protein Tyrosine Phosphatases