Phospholipase C isoform expression in rat renal preglomerular microvascular smooth muscle cells

Abstract

Expression of phospholipase C (PLC) isoforms varies in different tissues across species, and has not been thoroughly investigated in rat renal preglomerular microvascular smooth muscle cells (PVSMCs). The goals of this study were to compare mRNA expression of PLC isoforms in rat PVSMC and to confirm protein expression of these isoforms. PVSMCs (from interlobular artery and afferent arteriole) were cultured by the explant method. Total RNA and protein were extracted at passages 4–6. Quantitative real time RT‐PCR revealed PLC isoform mRNA expression according to the following rank order: δ1 = β4 = γ2 > β3 = β1 > γ1 = δ3 = δ4 ≫ β2. mRNA expression for the PLC isoforms ranged from 105 to 108 copies/ng total RNA (n=5–7 samples/isoform), indicating a 1000‐fold difference in expression of PLC β2 and the most highly expressed isoforms (δ1, β4 and γ2). Western blot analysis verified that PLC β3, β1 and γ1 existed in rat PVSMCs at the protein level. PLC β2 protein could not be detected. It was difficult to detect PLC β4 protein, although its mRNA level was high. We have not yet assessed protein expression of PLC δ1 and γ2. Preliminary experiments utilized siRNA to produce ≥80% decreases in PLC γ1 and PLC β3 protein levels in these cells. We conclude that at least the β3, β1 and γ1 isoforms of PLC exist in rat PVSMCs, and that siRNA should be a useful tool in investigating the roles of specific PLC isoforms in PVSMC function. (Supported by DK71152)