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A new method of measuring cytoplasmic free Ca2+ (Ca2+i) of individual intact cardiovascular endothelial cells by using imaging fluorescence microscopy was designed. Application of agonist to the aortic or pulmonary valve of the rabbit triggered an increase in Ca2+i, which depended on the existence of endothelium on the surface of the valve. Under resting conditions, sudden reversal of the Na+ gradient by substituting external Na+ with N-methyl D-glucamine (NMDG) resulted in a Ca2+i spike, which then returned toward the resting level. Increasing intracellular Na+ concentration (Na+i) by application of ouabain or monensin induced a sustained Ca2+i increase. Na+ substitution by NMDG during the agonist- or monensin-induced Ca2+i increase gave rise to a further Ca2+i spike, which subsequently declined to a level higher than that before removal of external Na+. A selective inhibitor of Na(+)-Ca2+ exchange, 3',4'-dichlorobenzamyl (DCB), abolished the transient Ca2+i increase induced by Na+ substitution, and Mg2+, an inorganic inhibitor of Na(+)-Ca2+ exchanger, markedly reduced this transient Ca2+i increase. On the other hand, the selective Na(+)-H+ exchanger blocker 5-(N,N-hexamethylene)amiloride (HMA) did not abolish the transient Ca2+i increase caused by Na+ substitution. In summary, decreasing the Na+ gradient of the endothelial cells through either receptor stimulation (agonist), Na(+)-K+ pump inhibition (ouabain), pretreatment with Na+ ionophore (monensin), or reversing the Na+ gradient through Na+ substitution (NMDG) all increased Ca2+i. This raised Ca2+i was antagonized by agents such as DCB or Mg2+, which are thought to inhibit Na(+)-Ca2+ exchange, but not by HMA, an inhibitor of Na(+)-H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)
Li et al. (1995) studied this question.