Multiple Mechanisms of Activating Ca ²⁺ Entry in Freshly Isolated Rabbit Aortic Endothelial Cells

Abstract

In Fura-2-loaded, freshly isolated rabbit aortic endothelial cells the Ca²⁺ entry pathway was investigated using the Mn²⁺-quenching technique. Acetylcholine (ACh) interaction with muscarinic receptors activated Mn²⁺ influx through the plasma membrane. Sarcoplasmic-endoplasmic reticulum Ca²⁺ ATPase blockers such as cyclopiazonic acid (CPA), thapsigargin and BHQ, which block the endoplasmic reticulum Ca²⁺ pump and do not interact with receptors, also activated Mn²⁺ influx. Mn²⁺ influx activated by either ACh or CPA was blocked by the following agents: SKF96365, a receptor-operated Ca²⁺ channel (ROC) blocker; NCDC, a PLC and ROC blocker, and genistein, a tyrosine kinase inhibitor. D600, the L-type Ca²⁺ channel blocker, had no significant effect on Mn²⁺ influx. Caffeine blocked the ACh-induced Ca²⁺ release but had no effect on the ACh-induced Mn²⁺ influx. Similarly dantrolene, which blocked intracellular Ca²⁺ release induced by ACh, did not affect the ACh-activated Mn²⁺ influx. These data suggest that ACh can activate Ca²⁺ influx without depletion of the ACh-sensitive intracellular Ca²⁺ store. It is concluded (1) that in freshly isolated endothelial cells depletion of the intracellular Ca²⁺ store is not necessary for ACh-activated Ca²⁺ influx, and (2) that receptor activation and intracellular Ca²⁺ store depletion may activate the same Ca²⁺ entry pathway through parallel mechanisms.