Calcium store depletion activates two distinct calcium entry pathways in secretory cells of the blowfly salivary gland

Ca2+ influx into secretory cells of the intact salivary gland of the blowfly Calliphora erythrocephala elicited by the agonist 5-hydroxytryptamine (5-HT) or the Ca2+ uptake inhibitor thapsigargin was studied by using Fura-2 and digital fluorescence imaging and by recordings of the transepithelial potential. Application of saturating [5-HT] in the absence of Ca2+ (Ca-0(2+)) from the bathing saline did not affect the initial Ca2+ transient but greatly attenuated the subsequent sustained Ca2+ elevation observed in the presence of Ca-0(2+) demonstrating that the latter component of the [Ca2+](i) response is largely dependent on Ca2' entry across the base-lateral plasma membrane. La3+ or Gd3+ (10 mu M) mimicked the effects of the withdrawal of Ca-0(2+), Experimental attempts temporally to uncouple 5-HT stimulation and Ca2+ influx by withdrawal of Ca-0(2+) during agonist application revealed a second Ca2+ entry pathway. This pathway was insensitive to 10 mu M La3+ and produced transient [Ca2+](i) increases whose amplitudes were a function of the [5-HT] during the preceding stimulation and that were selectively suppressed by 50 mu M SK&F 96365. Both (10 mu M) La3+-insensitive [Ca2+](i) transients and (10 mu M) La3+ inhabitable tonic [Ca2+](i) increases could be sequentially activated in the presence of 5-HT or thapsigargin (1 mu M). These results indicate that Ca2+ store depletion by 5-HT or thapsigargin activates two distinct store-operated Ca2+ entry pathways, one of which supports tonic [Ca2+](i) increases. The other is transiently activated, even under conditions that prohibit store refilling and does not significantly contribute to the [Ca2+](i) responses evoked by saturating 5-HT concentrations.