We assessed the sensitivity of phospholipase D (PLD) activity in vascular smooth muscle to cytosolic Ca2+ by increasing cytosolic Ca2+ levels independently of agonist stimulation. When rat tail artery was preloaded with the Ca2+ indicator fluo 3 pentaacetoxymethyl ester, the addition of high extracellular K+, caffeine, or norepinephrine rapidly enhanced cytosolic Ca2+ levels. Neither increased extracellular K+ nor caffeine addition increased phosphatidylethanol production, indicating that cytosolic Ca2+ elevation alone did not stimulate PLD. In contrast, norepinephrine stimulated phosphatidylethanol production in this tissue. In strips of tail artery permeabilized with alpha-toxin and incubated in solutions containing free Ca2+ concentrations observed during physiological stimulation (pCa 6.4), PLD was not stimulated, whereas incubation with guanosine 5'-O-(3-thiotriphosphate) at pCa 7.0 activated this enzyme. Aluminum fluoride (AlF4-) stimulated PLD, and this activity was insensitive to pertussis toxin after stimulation by either norepinephrine or AlF4-. These results indicate that PLD in vascular smooth muscle is activated by norepinephrine via stimulation of a pertussis toxin-insensitive G protein and not via an increase in intracellular Ca2+ levels.
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