[CA2-INDUCED CA2+ RELEASE IN INTACT HELA-CELLS(] MICRODOMAINS CONTROLAGONIST)

We have monitored specifically the [Ca2+] in the lumen of the endoplas mic reticulum (ER) of intact HeLa cells using-an ER-targeted low Ca2+- affinity aequorin. The steady-state [Ca2+] in the ER was around 600 mu M Histamine induced a concentration-dependent decrease in lumenal [Ca 2+], whose rate increased near one order of magnitude and became ''qua ntal'' when cytosolic [Ca2+] ([Ca2+](c)) was clamped with the Ca2+ che lator BAPTA. This effect was not due to decreased Ca2+ pumping because simultaneous addition of a SERCA inhibitor produced only additive eff ects, Given that inhibition by [Ca2+](c) of the inositol 1,4,5-trispho sphate-gated channels requires a [Ca2+](c) much higher than that obser ved in the bulk cytosol after histamine addition, we conclude that loc al [Ca2+](c) microdomains at the site of release strongly inhibit agon ist-induced Ca2+ mobilization in intact cells, This effect should play a key role in the mechanism controlling cytosolic [Ca2+] oscillations and waves, and therefore in the generation of spatio-temporal Ca2+ pa tterns.