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Abstract: Normal rat kidney (NRK) fibroblasts exhibit growth-dependent changes in electrophysiological properties and intracellular calcium dynamics. The transition from a quiescent state to a density-arrested state results in altered calcium entry characteristics. This coincides with modulation of the expression of the genes encoding the calcium channels Trpc1, Trpc6 and Orai1, and of the intracellular calcium sensor Stim1. In the present study we have used gene selective short hairpin (sh) RNAs against these various genes to investigate their role in (a) capacitative store-operated calcium entry (SOCE); (b) non-capacitative OAG-induced receptor-operated calcium entry (ROCE); and (c) prostaglandin F2α (PGF2α)-induced Ca2+-oscillations in NRK fibroblasts. Intracellular calcium measurements revealed that knockdown of the genes encoding Trpc1, Orai1 and Stim1 each caused a significant reduction of SOCE in NRK cells, whereas knockdown of the gene encoding Trpc6 reduced only the OAG-induced ROCE. Furthermore, our data show that knockdown of the genes encoding Trpc1, Orai1 and Stim1, but not Trpc6, substantially reduced the frequency (up to 60%) of PGF2α-induced Ca2+ oscillations in NRK cells. These results indicate that in NRK cells distinct calcium channels control the processes of SOCE, ROCE and PGF2α-induced Ca2+ oscillations. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]