A hypo-osmotically induced increase in intracellular Ca2+ in lactating mouse mammary epithelial cells involving Ca2+ influx

Using a fluorescent Ca2+-sensitive dye, we studied the effect of hypo-osmotic stress on the intracellular free Ca2+ concentration ([Ca2+](i)) in acini freshly isolated from lactating mouse mammary gland. The basal [Ca2+](i) of mammary acini was unaffected by a 50% (v/v) dilution of suspensions with isotonic or hypertonic buffer, or after ionic (iso-osmotic) dilution (external Ca2+ was 3 mM). Hypo-osmotic dilution (50%) elicited a rapid increase in [Ca2+](i) comprising a large, transient elevation, followed by a maintained plateau phase. No hypo-osmotically induced rise in [Ca2+](i) was observed in the absence of extracellular Ca2+. Neither microtubule disassembly using nocodazole nor actin disruption with cytochalasin D prevented hypo-osmotically evoked stimulation of [Ca2+](i). Pre-incubation of acini with nifedipine did not prevent hypo-osmotically induced stimulation of [Ca2+](i), whereas a non-specific cation channel blocker, gadolinium, partially inhibited the increases in [Ca2+](i) induced by hypo-osmotic stress. Furthermore, the transient component was still apparent, and not diminished in magnitude, after [Ca2+](i) had been elevated by mobilisation of Ca2+ from intracellular stores using thapsigargin. The results demonstrate that hypo-osmotic stress generates an increase in [Ca2+](i) in lactating mammary epithelial cells, the major, transient component of which appears to be due to influx of extracellular Ca2+.