NA-CA2+ EXCHANGE IN INTACT ENDOTHELIUM OF RABBIT CARDIAC-VALVE()

A new method of measuring cytoplasmic free Ca2+ ([Ca2+](i)) of individ ual intact cardiovascular endothelial cells by using imaging fluoresce nce microscopy was designed. Application of agonist to the aortic or p ulmonary 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 subs tituting external Na+ with N-methyl D-glucamine (NMDG) resulted in a [ Ca2+](i) spike, which then returned toward the resting level. Increasi ng intracellular Na+ concentration ([Na+](i)) by application of ouabai n or monensin induced a sustained [Ca2+](i) increase. Na+ substitution by NMDG during the agonist- or monensin-induced [Ca2+](i) increase ga ve rise to a further [Ca2+](i) spike, which subsequently declined to a level higher than that before removal of external Na+. A selective in hibitor 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+-Hexchanger blocker 5-(N,N-hexamethylene) amiloride (HMA) did not abolis h the transient [Ca2+](i) increase caused by Na+ substitution. In summ ary, decreasing the Na+ gradient of the endothelial cells through eith er receptor stimulation (agonist), Na+-K+ pump inhibition (ouabain), p retreatment with Na+ ionophore (monensin), or reversing the Na+ gradie nt through Na+ substitution (NMDG) all increased [Ca2+](i). This raise d [Ca2+](i) was antagonized by agents such as DCB or Mg2+, which are t hought to inhibit Na+-Ca2+ exchange, but not by HMA, an inhibitor of N a+-H+ exchange. Taken together, these results strongly imply the prese nce of Na+-Ca2+ exchange as a viable mechanism for Ca2+ transport in i ntact cardiovascular endothelium and that the Ca2+ entry component is enhanced when [Na+](i) is elevated.