Different roles of inositol 1,4,5-trisphosphate receptor subtypes in prostaglandin F2α-induced calcium oscillations and pacemaking activity of NRK fibroblasts

Abstract

We investigated the role of inositol 1,4,5-trisphosphate (IP3)-receptor isoforms in the prostaglandin F2α (PGF2α)-induced calcium oscillations and pacemaking activity of normal rat kidney (NRK) fibroblasts. Reverse transcription polymerase chain reaction (RT-PCR) studies revealed that NRK fibroblasts express only the IP3-receptor subtypes IP3R1 and IP3R3. Quantitative RT-PCR analysis demonstrated that their expression levels varied as a function of the growth status of NRK cells; NRK cells made quiescent (Q) by serum deprivation expressed significantly higher levels of subtypes 1 and 3 than cells grown to density-arrest (DA). Using Ca2+-imaging techniques, we show that the frequency of PGF2α-induced calcium oscillations in DA-cells is lower than in Q-cells. To study whether these differences in the frequency of calcium oscillations relate to the relative amounts of IP3-receptor subtypes expressed by the cells, we knocked down the genes for either IP3-receptor subtype by using an shRNA approach. Knockdown of the IP3R1 gene significantly decreased the frequency of the PGF2α-induced calcium oscillations in both Q- and DA-cells. It also reduced the frequency of the repetitive firing of calcium action potentials by DA-cells. In contrast, knockdown of the IP3R3 gene caused an increase in the frequency of both processes, suggesting a role for this receptor subtype as an anti-Ca2+-oscillatory unit in NRK fibroblasts. Our findings indicate that the reduction in the frequency of PGF2α-induced calcium oscillations in DA-cells compared with Q-cells results from the reduced expression ratio of IP3R1 versus IP3R3 receptors in DA-cells. Moreover, these data provide direct evidence that the frequency of IP3-dependent calcium oscillations determines the periodicity of action potential firing by density-arrested NRK fibroblasts.