thapsigargin and tetrabutylammonium

The correlation between changes in cytosolic Ca2+ levels ([Ca2+]i) and the release of vascular relaxing factor(s) was investigated in the endothelium of rabbit aortic valve. ATP, carbachol and thapsigargin increased endothelial [Ca2+]i in rabbit aortic valve loaded with a leakage resistant, fluorescent Ca2+ indicator, fura-PE3. Release of relaxing factors was bioassayed using the 'sandwich' preparation in which contraction was measured in the endothelium-denuded rabbit aorta attached to the endothelial surface of the valve. Addition of ATP, carbachol and thapsigargin induced sustained relaxation of the phenylephrine-induced contraction of the aorta in the 'sandwich' preparation. N(G)-monomethyl-L-arginine (L-NMMA) greatly attenuated the relaxation induced by carbachol, and combined treatment with tetra-n-butylammonium completely inhibited the relaxation. These results suggest that the endothelial relaxing factors released from aortic valve are nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). When the increase in endothelial [Ca2+]i was plotted against the relaxation, the carbachol-induced increase in [Ca2+]i elicited greater relaxation than did ATP or thapsigargin at a given [Ca2+]i. This suggests that various agonists differently modulate the relationship between [Ca2+]i and release of NO.

Topics: Adenosine Triphosphate; Adrenergic beta-Agonists; Animals; Aorta, Thoracic; Calcium; Carbachol; Cytosol; Endothelium, Vascular; Enzyme Inhibitors; In Vitro Techniques; Male; Muscle Relaxation; Nicotinic Agonists; Nitric Oxide; omega-N-Methylarginine; Phenylephrine; Quaternary Ammonium Compounds; Rabbits; Thapsigargin