title: Calcium signaling pathways utilized by P2X receptors in freshly isolated preglomerular MVSMC.
abstract: This study tested the hypothesis that P2X receptor activation increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in preglomerular microvascular smooth muscle cells (MVSMC) by evoking voltage-dependent calcium influx. MVSMC were obtained and loaded with the calcium-sensitive dye fura 2 and studied by using single-cell fluorescence microscopy. The effect of P2X receptor activation on [Ca(2+)](i) was assessed by using the P2X receptor-selective agonist alpha,beta-methylene-ATP and was compared with responses elicited by the endogenous P2 receptor agonist ATP. alpha,beta-Methylene-ATP increased [Ca(2+)](i) dose dependently. Peak increases in [Ca(2+)](i) averaged 37 +/- 11, 73 +/- 15, and 103 +/- 21 nM at agonist concentrations of 0.1, 1, and 10 microM, respectively. The average peak response elicited by 10 microM alpha,beta-methylene-ATP was approximately 34% of the response obtained with 10 microM ATP. alpha,beta-Methylene-ATP induced a transient increase in [Ca(2+)](i) before [Ca(2+)](i) returned to baseline, whereas ATP induced a biphasic response including a peak response followed by a sustained plateau. In Ca(2+)-free medium, ATP induced a sharp transient increase in [Ca(2+)](i), whereas the response to alpha,beta-methylene-ATP was abolished. Ca(2+) channel blockade with 10 microM diltiazem or nifedipine attenuated the response to alpha,beta-methylene-ATP, whereas nonspecific blockade of Ca(2+) influx pathways with 5 mM Ni(2+) abolished the response. Blockade of P2X receptors with the novel P2X receptor antagonist NF-279 completely but reversibly abolished the response to alpha,beta-methylene-ATP. These results indicate that P2X receptor activation by alpha,beta-methylene-ATP increases [Ca(2+)](i) in preglomerular MVSMC, in part, by stimulating voltage-dependent Ca(2+) influx through L-type Ca(2+) channels.
authors.affiliation: Louisiana State University School of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: White
authors.forename: J D
authors.initials: JD
authors.lastname: Imig
authors.forename: T T
authors.initials: TT
authors.lastname: Kim
authors.forename: B C
authors.initials: BC
authors.lastname: Hauschild
authors.forename: E W
authors.initials: EW
authors.lastname: Inscho
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D004110:Diltiazem / Q000494:pharmacology; D066298:In Vitro Techniques; D007678:Kidney Glomerulus / Q000098:blood supply*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009532:Nickel / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000378:metabolism*
chemical_list: D002121:Calcium Channel Blockers; D018048:Receptors, Purinergic P2; D009532:Nickel; D000255:Adenosine Triphosphate; D004110:Diltiazem; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium