1 The mechanism of transient contractions induced by the sarcoplasmic ± endoplasmic reticulum calcium ATPase (SERCA) blocker cyclopiazonic acid (CPA) in the presence of L-NAME was investigated in mouse aorta. 2 The contractions elicited by 10 mM CPA required an intact endothelium, were dependent upon external Ca 2+ and were prevented by 10 mM indomethacin, the inhibitor of prostaglandin synthesis, or 1 mM SQ29548, the speci®c prostaglandin H2/thromboxane A2 (PGH2/TXA2) receptor blocker. 3 A blocker of receptor/store operated Ca 2+ channels and voltage gated calcium channels (VGCC), SK&F 96365 (10 mM), completely abolished the contractions, while a speci®c blocker of VGCC nifedipine (1 mM) inhibited them by one third. 4 Dichlorobenzamyl hydrochloride, a blocker of Na + /Ca 2+ exchange e ectively prevented return of tension to baseline value. 5 At higher concentrations (30 ± 100 mM) CPA induced indomethacin-resistant tonic contractions of mouse aorta. The CPA dose response curve for tonic contractions is shifted to the right compared to the transient contractions suggesting that smooth muscle is less sensitive to CPA than endothelium. 6 PGH2/TXA2 receptors in mouse aorta are highly sensitive to the thromboxane analogue U46619 (EC 50 : 1.93 nM). This compound stimulates contractions even in the absence of external Ca 2+ , which are abolished by the Rho-kinase inhibitor HA-1077. 7 The results suggest that 10 mM CPA induced capacitive Ca 2+ entry in endothelial cells stimulating the release of PGH2/TXA2, which subsequently caused smooth muscle contraction dependent on Ca 2+ in¯ux and myo®lament sensitization by Rho-kinase. Higher concentrations of CPA (30 ± 100 mM) directly induced contraction of mouse aortic smooth muscle.
Augmented vasoconstriction contributes to arterial stiffness associated with diabetes. It has been shown that capacitative calcium entry induced by sarcoplasmic-endoplasmic reticulum calcium ATPase blocker cyclopiazonic acid (CPA) in endothelial cells stimulates production of constrictor prostaglandins, which causes contractions of vascular smooth muscle cells. The aim of the work was to study the effect of diabetes on the vasoconstrictor response induced by calcium entry into endothelial and smooth muscle cells. Force was measured in isolated aortae of diabetic ob/ob and control C57BL/6J mice under isometric conditions. Contractions caused by 10 micromol/l CPA in diabetic mouse aortae featured higher amplitudes and longer durations in comparison with nondiabetic aortae. These contractions were abolished by a COX inhibitor indomethacin (10 micromol/l) or a specific thromboxane A2 receptor blocker SQ 29548 (1 micromol/l) and were not observed in denuded aortae. The contractions were sensitive to extracellular Ca (2+) and store-operated channel blockers. All together this suggests that vasoconstriction was caused by thromboxane A2 synthesis in endothelial cells induced by Ca (2+) entry through store-operated channels. Higher concentrations of CPA (30 micromol/l) or thapsigargin (1 micromol/l) elicited indomethacin-resistant tonic contractions of aortae with 2-fold amplitude in diabetic mice compared to their nondiabetic littermates, which were sensitive to store-operated channel blockers, but not to indomethacin, SQ 29548, or denudation. In conclusions, increases in intracellular Ca (2+) cause augmented vasoconstriction in diabetic vasculature through endothelial synthesis of contractile prostaglandins. In addition capacitative Ca (2+) entry is enhanced in diabetic vascular smooth muscle. These mechanisms indicate possible targets for clinical applications.
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