Different roles of inositol 1,4,5-trisphosphate receptor subtypes in prostaglandin F(2alpha)-induced calcium oscillations and pacemaking activity of NRK fibroblasts.
We investigated the role of inositol 1,4,5-trisphosphate (IP(3))-receptor isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-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 IP(3)-receptor subtypes IP(3)R1 and IP(3)R3. 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 Ca(2+)-imaging techniques, we show that the frequency of PGF(2alpha)-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 IP(3)-receptor subtypes expressed by the cells, we knocked down the genes for either IP(3)-receptor subtype by using an shRNA approach. Knockdown of the IP(3)R1 gene significantly decreased the frequency of the PGF(2alpha)-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 IP(3)R3 gene caused an increase in the frequency of both processes, suggesting a role for this receptor subtype as an anti-Ca(2+)-oscillatory unit in NRK fibroblasts. Our findings indicate that the reduction in the frequency of PGF(2alpha)-induced calcium oscillations in DA-cells compared with Q-cells results from the reduced expression ratio of IP(3)R1 versus IP(3)R3 receptors in DA-cells. Moreover, these data provide direct evidence that the frequency of IP(3)-dependent calcium oscillations determines the periodicity of action potential firing by density-arrested NRK fibroblasts.
Almirza
W H M
WH
Department of Cell Biology, Radboud University Nijmegen, The Netherlands.
Peters
P H J
PH
van Meerwijk
W P M
WP
van Zoelen
E J J
EJ
Theuvenet
A P R
AP
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015237:Dinoprost / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007668:Kidney / Q000166:cytology; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D034622:RNA Interference; D051381:Rats
D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D015237:Dinoprost; D002118:Calcium