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Intracellular calcium regulation in the intact endothelial cells from rabbit aortic or pulmonic valves

University of British Columbia

Intracellular Ca²⁺ signal and nitric oxide (NO) release from the intact endothelial cells of rabbit aortic or pulmonic valves were measured by digital imaging microscopy and NO microsensor, respectively. Vasoactive agents such as agonists, Ca²⁺ ionophore triggered an increase of cytoplasmic free calcium concentration ([Ca²⁺]j) and correspondent increase of NO release. Inhibitors of endoplasmic reticulum (ER) Ca²⁺ ATPase induced a [Ca²⁺]j. increase. SK&F 96365, a receptor-operated cation channel (ROC) blocker, and 2-nitro-4- carboxyphenyl-N, N-diphenyl-carbamate (NCDC), a postulated phospholipase C inhibitor and a ROC blocker, greatly reduce the agonist ATP induced sustained [Ca²⁺]j increase, but not the ER CA²⁺ ATPase inhibitor CPA induced [Ca²⁺]j increase. N2,i a Ca²⁺ entry blocker, blocked both the ATP- and CPA- induced [Ca²⁺]j increases. Divalent cation entry measured as Mn²⁺ quenching of fura-2 fluorescence was inhibited by Ni²⁺ but enhanced by ATP. This enhancement was abolished by pretreatment with NCDC or SK&F 96365. In contrast, the rate of Mn²⁺ quenching was unaffected by CPA. These results demonstrate that ATP stimulates a divalent cation influx through ROC, but interruption of ER Ca²⁺ accumulation does not signal an increased Ca²⁺ entry from the estracellular space. These results can be best explained by a “Superficial Buffer Barrier” (SBB) hypothesis where inhibition of Ca²⁺ uptake into the ER disrupts the Ca²⁺ buffer barrier function of ER, thus increasing the effectiveness of the Ca²⁺ leak in raising [Ca²⁺]j. A voltage gated Ca²⁺ channel (VGC) blocker, diltiazem, did not affect the ATP induced sustained [Ca²⁺]j increase. Depolarization of endothelial cells did not affect the resting [Ca²⁺]j, but blocked the ATP-induced sustained [Ca²⁺]j increase. These results indicate the absence of VGC in intact endothelial cells. Decreasing the Na gradient through either receptor stimulation (agonist), Na⁺,K⁺ pump inhibition (ouabain), Na⁺ ionophore (monensin) or by reversing Na⁺ gradient through Na⁺ substitution all increased [Ca²⁺]j,a implying the presence of a Na⁺ -Ca²⁺ exchange as a mechanism for Ca²⁺ entry on the plasmalemma of intact endothelium. This Ca²⁺ entry component is enhanced when [Na⁺]j is elevated.

Thesis/Dissertation

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2009-04-22

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http://hdl.handle.net/2429/7486

Pharmacology and Therapeutics

University of British Columbia

UBCV

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