THAPSIGARGIN DISCHARGES INTRACELLULAR CALCIUM STORES AND INDUCES TRANSMEMBRANE CURRENTS IN HUMAN ENDOTHELIAL-CELLS

We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca2+-adenosine 5'-triphosphatase (Ca2+-ATPase), on membrane currents and on the intracellular Ca2+ concentration ([Ca2+]i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patch-clamp technique in the whole-cell mode and [Ca2+]i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 mumol/l induced a slow increase in [Ca2+]i to a peak value of 400 +/- 110 nmol/l above a resting level of 120 +/- 35 nmol/l, and then slowly declined to a new steady-state level of 315 +/- 90 nmol/l (n = 33). The thapsigargin-induced increase in [Ca2+]i depended on the extracellular Ca2+ concentration ([Ca2+]o: it declined after removal of extracellular Ca2+, but increased again when [Ca2+]o was augmented, indicating that the response depends on a transmembrane influx of Ca2+ ions. The peak amplitude of the histamine-induced Ca2+ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca2+]i induced by thapsigargin than during the rising phase of the changes in [Ca2+]i. The decline of the Ca2+ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant tau equal to 24.5 +/- 5 s (n = 7). In the presence of thapsigargin, the decline was much slower (n = 8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast tau value of 29.4 +/- 4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of -40 mV. Voltage ramps applied before and during the development of this current indicated that a nonselective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca2+ transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca2+ pump in endothelial cells increases [Ca2+]i. The tonic component of this increase might be partly due to opening of non-selective Ca2+-permeable cation channels activated by depletion of intracellular stores.