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Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Submitted 7 October 2004 ; accepted in final form 12 December 2004
ANG II induces a rise in cytosolic Ca2+ ([Ca2+]i) in vascular smooth muscle (VSM) cells via inositol trisphosphate receptor (IP3R) activation and release of Ca2+ from the sarcoplasmic reticulum (SR). The Ca2+ signal is augmented by calcium-induced calcium release (CICR) and by cyclic adeninediphosphate ribose (cADPR), which sensitizes the ryanodine-sensitive receptor (RyR) to Ca2+ to further amplify CICR. cADPR is synthesized from 
-nicotinamide adenine dinucleotide (NAD+) by a membrane-bound bifunctional enzyme, ADPR cyclase. To investigate the possibility that ANG II activates the ADPR cyclase of afferent arterioles, we used inhibitors of the IP3R, RyR, and ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca2+]i. In Ca2+-containing buffer, ANG II increased [Ca2+]i by 125 ± 10 nM. In the presence of the IP3R antagonists TMB-8 and 2-APB, the peak responses to ANG II were reduced by 74 and 81%, respectively. The specific antagonist of cADPR 8-Br ADPR and a high concentration of ryanodine (100 µM) inhibited the ANG II-induced increases in [Ca2+]i by 75 and 69%, respectively. Nicotinamide and Zn2+ are known inhibitors of the VSM ADPR cyclase. Nicotinamide diminished the [Ca2+]i response to ANG II by 66%. In calcium-free buffer, Zn2+ reduced the ANG II response by 68%. Simultaneous blockade of the IP3 and cADPR pathways diminished the [Ca2+]i response to ANG II by 83%. We conclude that ANG II initiates Ca2+ mobilization from the SR in afferent arterioles via the classic IP3R pathway and that ANG II may lead to activation of the ADPR cyclase to form cADPR, which, via its action on the RyR, substantially augments the Ca2+ response.
calcium-induced calcium release; ryanodine; vascular smooth muscle; nicotinamide
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