Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells.

The molecular mechanism(s) involved in mediating Ca2+ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca2+ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca2+ pools (Ca2+-pool-depleted cells). The rate of Ca2+ entry was determined by measuring the initial increase in free cytosolic [Ca2+] ([Ca2+]i) in Ca2+-pool-depleted, and control (untreated), cells upon addition of various [Ca2+] to the medium. In untreated cells, a low-affinity component was detected with KCa = 3. 4 +/- 0.7 mM (where KCa denotes affinity for Ca2+) and Vmax = 9.8 +/- 0.4 nM [Ca2+]i /s. In thapsigargin-treated cells, two Ca2+ influx components were detected with KCa values of 152 +/- 79 microM (Vmax = 5.1 +/- 1.9 nM [Ca2+]i/s) and 2.4 +/- 0.9 mM (Vmax = 37.6 +/- 13.6 nM [Ca2+]i/s), respectively. We have also examined the effect of Ca2+ and depolarization on these two putative Ca2+ influx components. When cells were treated with thapsigargin in a Ca2+-free medium, Ca2+ influx was higher than into cells treated in a Ca2+-containing medium and, while there was a 46% increase in the Vmax of the low-affinity component (no change in KCa), the high-affinity component was not clearly detected. In depolarized Ca2+-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the KCa of the low-affinity component, without any change in the Vmax. These results demonstrate that Ca2+ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca2+ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca2+.