Actions of cardiotoxin on cytosolic free Ca2+ in the presence of a calcium-free environment, calcium channel blockers and the polyamine spermine in vascular smooth muscle and endothelial cells

The effects of calcium-free, calcium channel blockers and polyamines on cardiotoxin (CTX) induced contraction of rat thoracic aorta and bovine pulmonary artery endothelial cells was investigated using both contractility and calcium imaging studies, respectively. Exposure of aortic rings to 15 μM CTX in calcium-free and high calcium (7 mM) conditions respectively, yielded minimal contraction. The selective and non-selective calcium channel blockers, nifedipine and tetrandrine, were observed to inhibit CTX-induced vascular smooth muscle (VSM) contraction. Fura-2 calcium imaging studies showed that exposure of both bovine pulmonary artery endothelial cells to CTX (1-10 μM) in normal Ca²⁺ conditions yielded an increase in |Ca²⁺|i. Fura-2 calcium imaging also showed that pre-incubation of cells in high Ca ²⁺ (7 mM) and Ca²⁺-free media inhibited CTX induced increase in |Ca²⁺|i. Exposure of aortic rings to the cationic polyamine spermine (SPM, 10 μM) did not inhibit CTX-induced contraction. These data suggested that (a) a CTX sensitive internal calcium store does not exist in rat aorta, (b) CTX interacts with the outer plasma membrane at the level of the calcium channels, as well as anionic sites to which Ca²⁺ and other inorganic cations bind, and (c) the polyamine SPM does not compete with CTX for anionic sites on the outer plasma membrane.