15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and 2-5-di-tert-butylhydroquinone

The purpose of this study was to test whether the elevated intracellular Ca++ level ([Ca++]i) resulting from store-operated Ca++ entry was associated with vascular smooth muscle contraction. Cyclopiazonic acid (CPA), a selective inhibitor of sarcoplasmic reticulum Ca(++)-ATPase, concentration-dependently (1-10 microM) elevated [Ca++]i in rat aorta, as indicated by an increase in the fura-2 340/380 ratio. Simultaneous measurement of contraction demonstrated that 1 and 10 microM CPA induced insignificant and variable amounts of contraction, respectively. Verapamil (10 microM) had relatively little effect on the 1 and 10 microM CPA-elevated [Ca++]i. In contrast, Ni++ (0.1 mM), in the presence of verapamil, abolished the 1 microM CPA-elevated [Ca++]i. Ni++ (0.1 mM) also partially decreased the 10 microM CPA-elevated [Ca++]i and, furthermore, abolished the associated contraction. A higher Ni++ concentration (1 mM) abolished the 10 microM CPA-elevated [Ca++]i that remained after verapamil and 0.1 mM Ni++. Phorbol dibutyrate (10 nM), a protein kinase C activator, potentiated contractions to 1 and 10 microM CPA in the presence of verapamil. Ni++ (0.1 mM) abolished the enhanced contractions, and decreased the elevated [Ca++]i. These results suggest that 1) elevated [Ca++]i due to store-operated Ca++ entry is dissociated from contraction; 2) the elevated [Ca++]i is restricted to at least two noncontractile compartments that can be differentiated by their relative sensitivities to blockade by low (0.1 mM) and higher (1 mM) Ni++ concentrations, and 3) [Ca++]i elevation within the compartment sensitive to blockade by 0.1 mM Ni++ can be coupled to contraction via protein kinase C activation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta; Calcium; Hydroquinones; Indoles; Male; Nickel; Phorbol 12,13-Dibutyrate; Protein Kinase C; Rats; Rats, Sprague-Dawley; Vasoconstriction; Verapamil

1. The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca(2+)-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca(2+)-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2. Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic. However, ET-3 did not affect Ca2+ movements induced by 1 microM ADP. Addition of Tg (0.5 to 5 microM) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3. ET-3 did not modify the Ca2+ response to another Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 microM Tg, which became similar to that induced by 15 microM tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3. 4. ET-3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+-ATPase inhibitors. The small but significant inhibitory effect of ET-3 leads us to propose that endothelin-3 acts as a modulator of platelet activation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Antioxidants; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Calcium Channel Agonists; Calcium-Transporting ATPases; Endothelins; Fatty Acids, Unsaturated; Fura-2; Humans; Hydrazines; Hydroquinones; In Vitro Techniques; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Receptors, Endothelin; Receptors, Thromboxane; Terpenes; Thapsigargin; Thrombin; Thromboxane A2; Vasoconstrictor Agents