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National Institute for Medical Research, London, UK. tcarter@nimr.mrc.ac.uk

1. The role of the InsP3 receptor and its interaction with Ca2+ in shaping endothelial Ca2+ spikes was investigated by comparing InsP3-evoked intracellular Ca2+ release with hormonally evoked Ca2+ spikes in single endothelial cells. 2. InsP3 was generated by flash photolysis of intracellular caged InsP3. InsP3 at 0.2 microM or higher released Ca2+ from stores with a time course comprising a well-defined delay, a fast rise of free [Ca2+] to a peak where net flux into the cystosol is zero, and a slow decline to preflash levels. InsP3-evoked Ca2+ flux into unit cytosolic volume was measured as the rate of change of free [Ca2+]i during the fast rise, d[Ca2+]i/dt (mol s-1 l-1). 3. The mean delay decreased from 433 ms at 0.2 microM to 30 ms at 5 microM. At very high InsP3 concentrations, 78 microM, the delay was shorter, < 10 ms. At low InsP3 concentration the delay was reduced by approximately 30% by prior elevation of free [Ca2+]i, supporting a co-operative action of free [Ca2+] and InsP3 in activation. 4. Both Ca2+ flux and peak free [Ca2+]i increased with InsP3 concentration within each cell. Maximal activation was at > 5 microM, 50% maximum Ca2+ flux was at 1.6 microM InsP3 and the Hill coefficient was between 3.6 and 4.3. A large variation of Ca2+ flux and peak [Ca2+]i was found from cell to cell at the same InsP3 concentration. 5. Strong inhibition of InsP3-evoked flux was produced by an immediately preceding response, with complete inhibition at peak free [Ca2+]i due to the first pulse. InsP3 sensitivity returned over 1-2 min, with 50% recovery at approximately 25 s. The recovery of InsP3 sensitivity may determine the minimum interval between hormonally evoked spikes. 6. Ca2+ flux due to a pulse of InsP3 terminated rapidly, in the continued presence of InsP3, producing a well-defined peak [Ca2+]. A reciprocal relation was found between the duration and the rate of Ca2+ flux, such that high Ca2+ flux was of brief duration. The rate of termination of flux measured as the reciprocal of the 10-90% rise time of free [Ca2+]i showed a linear correlation with Ca2+ flux over a large range in all cells. A systematic deviation from linearity at low InsP3 concentration showed a greater rate of termination at low InsP3 concentration than at high for the same flux. 7. Elevating cytosolic free [Ca2+] by 0.1-2.5 microM strongly inhibited Ca2+ release by InsP3, and buffering free [Ca2+] to low levels greatly prolonged Ca2+ release. Both results support the idea that Ca2+ flux quickly produces locally high free [Ca2+] which inhibits the receptor and terminates Ca2+ release. 8. Hormonally evoked Ca2+ spikes showed a similar reciprocal relation between rise time and Ca2+ flux, seen in the initial Ca2+ spike evoked by extracellular ATP in porcine aortic endothelial cells and by acetylcholine in rat aortic endothelial cells in situ, supporting the idea that the same mechanism of cytosolic Ca2+ inhibition determines the duration of hormonally and InsP3-evoked Ca2+ spikes.

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