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

Abstract

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 K_Ca = 3.4 ± 0.7 mM (where K_Ca denotes affinity for Ca²⁺) and F_max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ± 79 μM (V_max = 5.1 ± 1.9 nM [Ca²⁺]_is) and 2.4 ± 0.9 mM (V_max = 37.6 ± 13.6 nM [Ca²⁺]_is), 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 V_max of the low-affinity component (no change in K_Ca, the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KC1 in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K_Ca of the low-ffinity component, without any change in the V_max. 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⁺.