Na + -Ca 2+ Exchange in Intact Endothelium of Rabbit Cardiac Valve

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Abstract
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. Taken together, these results strongly imply the presence of Na+-Ca2+ exchange as a viable mechanism for Ca2+ transport in intact cardiovascular endothelium and that the Ca2+ entry component is enhanced when [Na+]i is elevated.