Discriminating between capacitative and arachidonate-activated Ca(2+) entry pathways in HEK293 cells.
We have recently questioned whether the capacitative or store-operated model for receptor-activated Ca(2+) entry can account for the influx of Ca(2+) seen at low agonist concentrations, such a those typically producing [Ca(2+)](i) oscillations. Instead, we have identified an arachidonic acid-regulated, noncapacitative Ca(2+) entry mechanism that appears to be specifically responsible for the receptor-activated entry of Ca(2+) under these conditions. However, it is unclear whether these two systems reflect the activity of distinct entry pathways or simply different mechanisms of regulating a common pathway. We therefore used the known selectivity of the Ca(2+)-stimulated type VIII adenylyl cyclase for Ca(2+) entry occurring via the capacitative pathway (Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444) to attempt to discriminate between these two entry mechanisms in HEK293 cells. Consistent with the earlier reports, we found that thapsigargin induced an approximate 3-fold increase in adenylyl cyclase activity that was unrelated to global changes in [Ca(2+)](i) or to the release of Ca(2+) from internal stores but was specifically dependent on the induced capacitative entry of Ca(2+). In marked contrast, the arachidonate-induced entry of Ca(2+) completely failed to affect adenylyl cyclase activity despite producing a substantially greater rate of entry than that induced by thapsigargin. These data demonstrate that the arachidonate-activated entry of Ca(2+) occurs via an entirely distinct influx pathway.
Shuttleworth
T J
TJ
Department of Pharmacology, University of Rochester School of Medicine,Rochester, New York 14642, USA. tshut@pharmacol.rochester.edu
Thompson
J L
JL
D000262:Adenylyl Cyclases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016718:Arachidonic Acid / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D008954:Models, Biological; D011994:Recombinant Proteins / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology*
D011994:Recombinant Proteins; D005576:Colforsin; D016718:Arachidonic Acid; D019284:Thapsigargin; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8; D002118:Calcium