MOBILIZATION OF INTRACELLULAR CALCIUM BY SUBSTANCE-P IN A HUMAN ASTROCYTOMA CELL-LINE (U-373 MG)

Variations in intracellular free calcium concentration (Delta[Ca2+](i)) were measured in intact and isolated human astrocytoma cells (U373 MG) loaded with fura-2 acetoxymethylester. Microperfusion of 50 nM substance P (SP), applied for 1 s, increased [Ca2+](i) by 351 nM from a stable basal level of [Ca2+](i) of 26 nM. The peak Delta[Ca2+](i) induced by SP was dose dependent with a threshold of 10(-3) nM, an ED(50) of 1.3 nM and a maximal effect for concentrations of SP greater than 100 nM. The NK1 receptor agonist, [Sar(9)Met(O-2)(11)]SP, mimicked the effect of SP, while the NK2 and NK3 selective receptor agonists, [N1(1O)]NKA(4-10) and senktide, respectively, had no effect. The Delta[Ca2+](i) induced by SP was unaffected by 100 mu M cadmium or by removal of extracellular calcium ions. Caffeine up to 30 mM had no effect on [Ca2+](i). In contrast, thapsigargin increased resting [Ca2+](i) by 92 nM and reduced the Delta[Ca2+](i) induced by SP. A pertussis treatment (500 ng/ml-24 h) did not modify the Delta[Ca2+](i) induced by SP. We conclude that SP, acting on a NK1 receptor, mobilizes cytosolic calcium from an intracellular calcium pool which can be partially depleted by thapsigargin. (C) 1994 Wiley-Liss, Inc.