(24,36). Only recently, however, has a role for cADPR been investigated in vascular smooth muscle (VSM). In membrane preparations of aorta (13, 53), renal microvessels (39), coronary arteries (31, 52, 55), and pulmonary artery (16), evidence for ADPR cyclase activity has been demonstrated. Measurement of changes in [Ca 2ϩ ] i in response to cADPR has been made in permeabilized renal VSM cells (39). To our knowledge, there have been no Ca 2ϩ studies examining the activation of the cADPR pathway in intact fresh afferent arterioles and no studies exploring the effect of ANG II on this particular pathway in VSM of any origin.The ADPR cyclase of VSM has several unique properties that distinguish it from the CD38 ADPR cyclase of nonvascular cells. In contrast to the CD38 enzyme of sea urchin eggs, aplysia, and HL-60 cells, in which Zn 2ϩ enhances the activity of the enzyme, Zn 2ϩ inhibits the cyclase of rat aortic VSM cells (13). Nitric oxide (NO) inhibits the VSM enzyme, whereas it is stimulatory in macrophages, neurons, pancreatic cells, and sea urchin eggs (52). Recently, it has been shown that oxidative stress increases [Ca 2ϩ ] i in fresh bovine coronary VSM cells through a pathway that involves cADPR (55) and that NO inhibits ADPR cyclase in coronary artery VSM (52).Only one study investigated the effect of ANG II on the activity of ADP-ribosyl cyclase (27). This laboratory found that in membrane preparations of neonatal but not older cardiac myocytes, ANG II increased cyclase activity in a dose-dependent fashion (27). The mechanism by which ANG II stimulated an increase in ADPR cyclase activity is unknown, but these investigators speculated that a G protein-coupled process is involved (27). Because of the crucial importance of afferent arterioles in regulating glomerular filtration and sodium balance, we investigated the effects of ANG II on Ca 2ϩ signaling in freshly isolated afferent arterioles using inhibitors of IP 3 R, RyR, and ADPR cyclase. METHODSAll studies were performed in compliance with the guidelines and practices of the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.

In afferent arteriolar vascular smooth muscle cells, ANG II induces a rise in cytosolic Ca(2+) ([Ca(2+)](i)) via inositol trisphosphate receptor (IP(3)R) stimulation and by activation of the adenine diphosphate ribose (ADPR) cyclase to form cyclic ADPR, which sensitizes the ryanodine receptor (RyR) to Ca(2+). We hypothesize that ANG II stimulation of NAD(P)H oxidases leads to the formation of superoxide anion (O(2)-*, which, in turn, activates ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca(2+)](i). ANG II rapidly increased [Ca(2+)](i) by 124 +/- 12 nM. In the presence of apocynin, a specific inhibitor of NAD(P)H oxidase assembly, the [Ca(2+)](i) response was reduced to 35 +/- 5 nM (P < 0.01). Tempol, a superoxide dismutase mimetic, did not alter the [Ca(2+)](i) response to ANG II at a concentration of 10(-4) M (99 +/- 12 nM), but 10(-3) M tempol reduced the response to 32 +/- 3 nM (P < 0.01). The addition of nicotinamide, an inhibitor of ADPR cyclase, to apocynin or tempol (10(-3) M) resulted in no further inhibition. Measurement of superoxide production with the fluorescent probe tempo 9-AC showed that ANG II caused an increase of 48 +/- 20 arbitrary units; apocynin or diphenyl iodonium (an inhibitor of flavoprotein oxidases) inhibited the response by 94%. Hydrogen peroxide (H(2)O(2)) was studied at physiological (10(-7) M) and higher concentrations. In the presence of H(2)O(2) (10(-7) M), neither baseline [Ca(2+)](i) nor the response to ANG II was altered (125 +/- 15 nM), whereas H(2)O(2) (10(-6) and 10(-5) M) inhibited the [Ca(2+)](i) response to ANG II by 35 and 46%, respectively. We conclude that ANG II rapidly activates NAD(P)H oxidases of afferent arterioles, leading to the formation of O(2)-*, which then stimulates ADPR cyclase to form cADPR. cADPR, by sensitizing the RyR to Ca(2+), augments the Ca(2+) response (calcium-induced calcium release) initiated by activation of the IP(3)R.

It is unknown if endothelin-A and -B receptors (ET(A)R and ET(B)R) activate the production of superoxide via NAD(P)H oxidase and subsequently stimulate the formation of cyclic adenine diphosphate ribose (cADPR) in afferent arterioles. Vessels were isolated from rat kidney and loaded with fura 2. Endothelin-1 (ET-1) rapidly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) by 303 nM. The superoxide dismutase mimetic tempol, the NAD(P)H oxidase inhibitor apocynin, and nicotinamide, an inhibitor of ADPR cyclase, diminished the response by approximately 60%. The ET(B)R agonist sarafotoxin 6c (S6c) increased peak [Ca(2+)](i) by 117 nM. Subsequent addition of ET-1 in the continued presence of S6c caused an additional [Ca(2+)](i) peak of 225 nM. Neither nicotinamide or 8-bromo- (8-Br) cADPR nor apocynin decreased the [Ca(2+)](i) response to S6c, but inhibited the subsequent [Ca(2+)](i) response to ET-1. The ET(B)R blockers BQ-788 and A-192621 prevented the S6c [Ca(2+)](i) peak and reduced the ET-1 response by more than one-half, suggesting an ET(B)R/ET(A)R interaction. In contrast, the ET(A)R blocker BQ-123 had no effect on the S6c [Ca(2+)](i) peak and obliterated the subsequent ET-1 response. ET-1 immediately stimulated superoxide formation (measured with TEMPO-9-AC, 68 arbitrary units) that was inhibited 95% by apocynin or diphenyl iodonium. S6c or IRL-1620 increased superoxide by 8% of that caused by subsequent ET-1 addition. We conclude that ET(A)R activation of afferent arterioles increases the formation of superoxide that accounts for approximately 60% of subsequent Ca(2+) signaling. ET(B)R activation appears to result in only minor increases in superoxide production. Nicotinamide and 8-Br-cADPR results suggest that ET-1 (and primarily ET(A)R) causes the activation of vascular smooth muscle cell-ADPR cyclase.

Variations in Ca2+ are directly correlated with clinically significant changes in myocardial contractility.

No abstract