P2 receptor regulation of [Ca²⁺]_iin cultured mouse mesangial cells

Abstract

Experiments were performed to establish the pharmacological profile of purinoceptors and to identify the signal transduction pathways responsible for increases in intracellular calcium concentration ([Ca(2+)](i)) for cultured mouse mesangial cells. Mouse mesangial cells were loaded with fura 2 and examined using fluorescent spectrophotometry. Basal [Ca(2+)](i) averaged 102 +/- 2 nM (n = 346). One hundred micromolar concentrations of ATP, ADP, 2',3'-(benzoyl-4-benzoyl)-ATP (BzATP), ATP-gamma-S, and UTP in normal Ca(2+) medium evoked peak increases in [Ca(2+)](i) of 866 +/- 111, 236 +/- 18, 316 +/- 26, 427 +/- 37, and 808 +/- 73 nM, respectively. UDP or 2-methylthio-ATP (2MeSATP) failed to elicit significant increases in [Ca(2+)](i), whereas identical concentrations of adenosine, AMP, and alpha,beta-methylene ATP (alpha,beta-MeATP) had no detectable effect on [Ca(2+)](i). Removal of Ca(2+) from the extracellular medium had no significant effect on the peak increase in [Ca(2+)](i) induced by ATP, ADP, BzATP, ATP-gamma-S, or UTP compared with normal Ca(2+); however, Ca(2+)-free conditions did accelerate the rate of decline in [Ca(2+)](i) in cells treated with ATP and UTP. [Ca(2+)](i) was unaffected by membrane depolarization with 143 mM KCl. Western blot analysis for P2 receptors revealed expression of P2X(2), P2X(4), P2X(7), P2Y(2), and P2Y(4) receptors. No evidence of P2X(1) and P2X(3) receptor expression was detected, whereas RT-PCR analysis reveals mRNA expression for P2X(1), P2X(2), P2X(3), P2X(4), P2X(7), P2Y(2), and P2Y(4) receptors. These data indicate that receptor-specific P2 receptor activation increases [Ca(2+)](i) by stimulating calcium influx from the extracellular medium and through mobilization of Ca(2+) from intracellular stores in cultured mouse mesangial cells.

Flow regulation of endothelin-1 production in the inner medullary collecting duct

“…Western blot analysis of P2 receptor expression. Mouse IMCD3 cells were lysed and prepared for Western blot analysis as previously described (42). Protein concentration was determined using the Bradford assay (Bio-Rad).…”

Purinergic signalling in the kidney in health and disease

“…Extracellular ATP increases [Ca 2+ ] i by release from intracellular stores, indicating mediation via P2Y receptors [121,282], although P2X receptor-mediated increase in [Ca 2+ ] i has also been claimed [311]. Cultured mouse mesangial cells expressed P2X2, P2X4, P2X7, P2Y 2 and P2Y 4 receptors; mRNA (but not protein) for P2X1 and P2X3 receptors was also found [311]. Using RT-PCR, P2X1 receptor mRNA was shown to be expressed by an immortalised mouse mesangial cell line (G3) [131].…”

ATP, P2 receptors and the renal microcirculation

“…2 and Fig. 5b) [9,11,59,[67][68][69][70][71][72][73][74]. Low concentrations of ATP (≤1.0µM) vasoconstrict afferent arterioles by activation of L-type Ca 2+ channels.…”

“…Mouse mesangial cells express P2X 2 , P2X 4 , P2X 7 , P2Y 2 and P2Y 4 receptors. mRNA is detected for P2X 1 and P2X 3 receptors in murine mesangial cells but protein is not found by western blot analysis [9]. Considering the wide distribution of renal P2 receptor subtypes, it seems likely that purinoceptors play diverse roles in regulating renal vascular and tubular function.…”