Endothelin-1, superoxide and adeninediphosphate ribose cyclase in shark vascular smooth muscle

In vascular smooth muscle (VSM) of Squalus acanthias, endothelin-1 (ET-1) signals via the ETB receptor. In both shark and mammalian VSM, ET-1 induces a rise in cytosolic Ca2+ concentration ([Ca2+](i)) via activation of the inositol trisphosphate (IP3) receptor (IP3R) and subsequent release of Ca2+ from the sarcoplasmic reticulum (SR). IP3R-mediated release of SR Ca2+ causes calcium-induced calcium release (CICR) via the ryanodine receptor (RyR), which can be sensitized by cyclic adeninediphosphate ribose (cADPR). cADPR is synthesized from NAD(+) by a membrane-bound bifunctional enzyme, ADPR cyclase. We have previously shown that the antagonists of the RyR, Ruthenium Red, high concentrations of ryanodine and 8-Br cADPR, diminish the [Ca2+](i) response to ET-1 in shark VSM. To investigate how ET-1 might influence the activity of the ADPR cyclase, we employed inhibitors of the cyclase. To explore the possibility that ET-1-induced production of superoxide (021 might activate the cyclase, we used an inhibitor of NAD(P)H oxidase (NOX), DPI and a scavenger Of O-2(-), TEMPOL. Anterior mesenteric artery VSM was loaded with fura-2AM to measure [Ca2+](i). In Ca2+-free shark Ringers, ET-1 increased [Ca2+](i) by 104 8nmoll(-1). The VSM ADPR cyclase inhibitors, nicotinamide and Zn2+, diminished the response by 62% and 72%, respectively. Both DPI and TEMPOL reduced the response by 63%. The combination of the IP3R antagonists, 2-APB or TMB-8, with DPI or TEMPOL further reduced the response by 83%. We show for the first time that in shark VSM, inhibition of the ADPR cyclase reduces the [Ca2+](i) response to ET-1 and that superoxide may be involved in the activation of the cyclase.