Vasodilation by the calcium-mobilizing messenger cyclic ADP-ribose.

In artery smooth muscle, adenylyl cyclase-coupled receptors such as beta-adrenoceptors evoke Ca(2+) signals, which open Ca(2+)-activated potassium (BK(Ca)) channels in the plasma membrane. Thus, blood pressure may be lowered, in part, through vasodilation due to membrane hyperpolarization. The Ca(2+) signal is evoked via ryanodine receptors (RyRs) in sarcoplasmic reticulum proximal to the plasma membrane. We show here that cyclic adenosine diphosphate-ribose (cADPR), by activating RyRs, mediates, in part, hyperpolarization and vasodilation by beta-adrenoceptors. Thus, intracellular dialysis of cADPR increased the cytoplasmic Ca(2+) concentration proximal to the plasma membrane in isolated arterial smooth muscle cells and induced a concomitant membrane hyperpolarization. Smooth muscle hyperpolarization mediated by cADPR, by beta-adrenoceptors, and by cAMP, respectively, was abolished by chelating intracellular Ca(2+) and by blocking RyRs, cADPR, and BK(Ca) channels with ryanodine, 8-amino-cADPR, and iberiotoxin, respectively. The cAMP-dependent protein kinase A antagonist N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked hyperpolarization by isoprenaline and cAMP, respectively, but not hyperpolarization by cADPR. Thus, cADPR acts as a downstream element in this signaling cascade. Importantly, antagonists of cADPR and BK(Ca) channels, respectively, inhibited beta-adrenoreceptor-induced artery dilation. We conclude, therefore, that relaxation of arterial smooth muscle by adenylyl cyclase-coupled receptors results, in part, from a cAMP-dependent and protein kinase A-dependent increase in cADPR synthesis, and subsequent activation of sarcoplasmic reticulum Ca(2+) release via RyRs, which leads to activation of BK(Ca) channels and membrane hyperpolarization.

A variety of transmitters relax smooth muscle by increasing cAMP levels and thereby activating cAMP-dependent protein kinase A (PKA). 1 In arteries, trachea, human airway, and lymphatic vessels, PKA-dependent smooth muscle relaxation has been shown to be mediated, in part, by opening of Ca 2ϩactivated potassium (BK Ca ) channels and membrane hyperpolarization (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). In artery smooth muscle, adenylyl cyclasecoupled receptors, such as ␤-adrenoceptors, open BK Ca channels by evoking Ca 2ϩ signals proximal to the plasma membrane, leading to smooth muscle cell hyperpolarization and a consequent reduction in blood pressure through vasodilation (1-5, 7-9, 11). Identifying the precise mechanisms involved is therefore essential to our understanding of how blood pressure may be regulated by PKA-dependent signaling and may provide fundamental insights into the causes of essential hypertension.
We have now investigated the role of cyclic adenosine diphosphate-ribose (cADPR), a ␤-NAD ϩ metabolite (19,20), in mediating vasodilation by BK Ca channel activation in pulmonary artery smooth muscle. Previous investigations have identified cADPR as a primary regulator of RyR function in a variety of preparations, including arterial smooth muscle cells (19 -23), and have demonstrated a role for cADPR-dependent SR Ca 2ϩ release in mediating contraction in both cardiac (24,25) and smooth muscle (26 -33). Despite the wealth of information linking RyR activation to vasodilation by adenylyl cyclase-coupled receptors, however, little attention has been paid to the role of cADPR in this process. We show here that hyperpolarization and dilation by adenylyl cyclase-coupled receptors is mediated, in part, by cADPR-dependent SR Ca 2ϩ release and consequent activation of BK Ca channels in pulmonary artery smooth muscle.

EXPERIMENTAL PROCEDURES
Cell Isolation-Single smooth muscle cells were enzymatically isolated from second order branches of the pulmonary artery of male Wistar rats (150 -300 g, sacrificed by cervical dislocation). Briefly, arteries were incubated (1 h, 22°C) in a low Ca 2ϩ solution of the following composition: 124 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 0.5 mM NaH 2 PO 4 , 0.5 mM KH 2 PO 4 , 15 mM NaHCO 3 , 0.16 mM CaCl 2 , 0.5 mM EDTA, 10 mM glucose, 10 mM Taurine, 10 mM Hepes, pH 7.4, 0.5 mg/ml papain (Fluka), and 1 mg/ml bovine serum albumin (Sigma). Then, 0.25 mg/ml 1,4-dithio-DL-threitol (Fluka) was added to the solution followed by a further 30-min incubation. The tissue was placed in enzyme-free low Ca 2ϩ solution, and single smooth muscle cells were isolated by gentle trituration. Cells were placed, as required, onto a glass coverslip in the experimental chamber.
Electrophysiological Recordings-Membrane potential in single pulmonary artery smooth muscle cells was measured in the whole-cell configuration of the patch clamp technique in current-clamp mode (I ϭ 0) using an Axopatch 200B amplifier (Axon instruments, Foster City, CA). Cells were bathed in physiological salt solution of the following composition (physiological salt solution A): 130 mM NaCl, 5.2 mM KCl, 1 mM MgCl 2 , 1.7 mM CaCl 2 , 10 mM glucose, 10 mM Hepes, pH 7.45. The pipette solution contained 140 mM KCl, 10 mM Hepes, 1 mM MgCl 2 , pH 7.4. All experiments were carried out at room temperature (22°C). Seal resistance was Ն3 gigaohms, series resistance was Յ5 megaohms, and pipette resistance was 2-4 megaohms. Fetchex and Fetchan software (Axon instruments, Foster City, CA) were used to perform data acquisition and analysis.
Ca 2ϩ Imaging-Cells were incubated for 30 min in low Ca 2ϩ solution (see above) containing 5 M Fura-2 AM, washed, and allowed to equilibrate for 20 min. 5 M Fura-2 (free acid) was also added to the pipette solution used for intracellular dialysis (see above). Changes in intracellular Ca 2ϩ were monitored by assessing Fura-2 fluorescence, using excitation wavelengths of 340 nm (F340) and 380 nm (F380), respectively, and an emission wavelength of 510 nm. Emitted fluorescence was monitored using a Hamamatsu 4880 image-intensifying CCD camera and recorded and analyzed using Openlab imaging software (Improvision) on an Apple Macintosh G4 personal computer. Fluorescence intensity was measured at 0.25-5 Hz with background subtraction being carried out on-line. Changes in Fura-2 fluorescence are reported as the F340/F380 ratio and as the estimated intracellular Ca 2ϩ concentration.

cADPR-dependent SR Ca 2ϩ Release via Ryanodine Receptors
Activates BK Ca Channels and Hyperpolarization-Using Fura-2 fluorescence imaging techniques and the whole-cell configuration of the patch clamp technique, we measured, in parallel, changes in the intracellular Ca 2ϩ concentration and resting membrane potential evoked by intracellular dialysis of cADPR in isolated rat pulmonary artery smooth muscle cells. In agreement with previous investigations in arterial smooth muscle, resting membrane potentials were within the range Ϫ40 to Ϫ60 mV, and estimated resting Ca 2ϩ concentrations were in the range 70 -150 nM (4,36). Intracellular application of cADPR (20 M) evoked a sustained increase in intracellular Ca 2ϩ concentration, as indicated by an increase in the Fura-2 fluorescence ratio (F340/F380) from 0.5 Ϯ 0.1 to 0.8 Ϯ 0.1 (mean Ϯ S.E.; Fig. 1A) and a concomitant hyperpolarization. The increase in intracellular Ca 2ϩ was localized at the cell perimeter, i.e. in close apposition to the plasma membrane. However, the capacity of Fura-2 for binding Ca 2ϩ resulted in marked attenuation of cADPR-dependent hyperpolarization. Thus, subsequent investigations of the transduction pathway leading to hyperpolarization utilized electrophysiological techniques alone. In the absence of Fura-2, intracellular dialysis of cADPR (20 M) evoked hyperpolarization from Ϫ40 Ϯ 2 to Ϫ75 Ϯ 1 mV (n ϭ 28, Fig. 1B), which was reversed by the selective BK Ca channel antagonist iberiotoxin (100 nM, n ϭ 7, Fig. 1B). Importantly, no hyperpolarization was observed when intracellular Ca 2ϩ was chelated by intracellular infusion of BAPTA (1 mM, Fig. 1C). Furthermore, cADPR-induced hyperpolarization was slowly reversed when SR Ca 2ϩ stores were depleted by cyclopiazonic acid ( (37)). These findings are summarized in Fig. 1G. Hyperpolarization induced by 1 mM caffeine, which triggers Ca 2ϩ release via RyRs by a mechanism independent of cADPR (22), was insensitive to the cADPR antagonist 8-NH 2 -cADPR (100 M; n ϭ 4, not shown) and 8-bromo-cADPR, respectively, (100 -300 M; n ϭ 4, not shown). This is consistent with the view that the cADPR antagonists tested here selectively inhibit SR Ca 2ϩ release by cADPR in pulmonary artery smooth muscle cells but do not block SR Ca 2ϩ release via RyRs or BK Ca channel activation per se. Note that we found no measurable contamination of cADPR-containing solutions with ADP or ADP-ribose and, in contrast to the effects of cADPR, intracellular infusion of 20 M ADP-ribose had no effect on membrane potential (n ϭ 4, not shown). Extracellular application of 20 M cADPR was without effect on membrane potential in isolated pulmonary artery smooth muscle cells, and neither intracellular nor extracellular application of the cADPR antagonists 8-NH 2 -cADPR (100 M, n ϭ 4) and 8-bromo-cADPR (Յ300 M, n ϭ 4), respectively, had any effect on membrane potential.
Does Ryanodine Deplete SR Ca 2ϩ Stores?-In contrast to hyperpolarization by cADPR, ryanodine had no significant effect on the global Ca 2ϩ wave induced by intracellular dialysis of IP 3 . Thus, intracellular dialysis of 1 M IP 3 increased the Fura-2 fluorescence ratio from 0.60 Ϯ 0.01 to a peak of 2.5 Ϯ 0.1 in the absence of 20 M ryanodine (n ϭ 5) and from 0.8 Ϯ 0.1 to a peak of 1.8 Ϯ 0.2 in the presence of 20 M ryanodine (20-min preincubation, n ϭ 5). Conversely, the IP 3 R antagonist xestospongin C blocked IP 3 -induced Ca 2ϩ signals (n ϭ 4, not shown) but had no effect on Ca 2ϩ signals or hyperpolarization by cADPR (n ϭ 4, not shown). These findings suggest that ryanodine blocks hyperpolarization by cADPR without significant effect on the ability of the SR to release Ca 2ϩ over the time course of our experiments.
Does 8-Bromo-cADPR Inhibit SR Ca 2ϩ ATPase Activity?-Preincubation of pulmonary artery smooth muscle cells with 300 M 8-bromo-cADPR was without effect on the transient increase in Fura-2 fluorescence ratio triggered upon SR store depletion by cyclopiazonic acid. The Fura-2 fluorescence ratio was increased by 20 M cyclopiazonic acid from 0.37 Ϯ 0.02 to a peak of 0.56 Ϯ 0.03 (n ϭ 10) in the absence of 8-bromo-cADPR and from 0.35 Ϯ 0.03 to a peak of 0.58 Ϯ 0.04 (n ϭ 4) in the presence of 8-bromo-cADPR (300 M). Thus, it seems unlikely that 8-bromo-cADPR blocks cADPR-dependent hyperpolarization by inhibiting SR Ca 2ϩ ATPase activity. When taken together, the aforementioned findings are consistent with the view that cADPR mediates hyperpolarization by triggering SR Ca 2ϩ release via RyRs by a mechanism independent of the SR Ca 2ϩ ATPase and IP 3 Rs, respectively, which ultimately leads to BK Ca channel activation and hyperpolarization.
cADPR Mediates SR Ca 2ϩ Release and Hyperpolarization by Isoprenaline-Given our finding that cADPR mediates hyperpolarization by BK Ca channel activation, we investigated the role of cADPR in mediating hyperpolarization induced by the activation of ␤-adrenoceptors, a family of adenylyl cyclasecoupled vasodilator receptors that are known to mediate smooth muscle relaxation, in part, by activating BK Ca channels and membrane hyperpolarization (4 -7, 9). Extracellular application of the selective ␤-adrenoreceptor agonist isoprenaline (10 M) induced sustained hyperpolarization from Ϫ39 Ϯ 2 to Ϫ75 Ϯ 1 mV (n ϭ 20, Fig. 2A), which was reversed by the selective BK Ca channel antagonist iberiotoxin (100 nM, n ϭ 5, Fig. 2A). As would be expected from previous studies (6,7,9), hyperpolarization by isoprenaline was also blocked by a selective ␤-adrenoreceptor antagonist, propanolol (10 M, n ϭ 4, not shown). In common with cADPR, hyperpolarization by isoprenaline was blocked when intracellular Ca 2ϩ was chelated by BAPTA (1 mM, n ϭ 4, Fig. 2B), underscoring the role of an increase in intracellular Ca 2ϩ concentration. Isoprenalineevoked hyperpolarization was also blocked when SR Ca 2ϩ re-lease was inhibited with ryanodine (20 M, n ϭ 6, Fig. 2C). In contrast, intracellular dialysis of heparin, an IP 3 R antagonist, failed to inhibit hyperpolarization by isoprenaline (n ϭ 3, not shown), which mitigates against a possible role for IP 3 Rs in this process. As RyRs mediate isoprenaline-evoked hyperpolarization and cADPR mimicked the effects of isoprenaline, we examined the possible role of cADPR in the ␤-adrenoreceptor signaling pathway. Hyperpolarization by isoprenaline was abolished by intracellular infusion of 8-NH 2 -cADPR (100 M, n ϭ 6, Fig. 2D) and of 8-Br-cADPR (100 M, n ϭ 6, not shown), respectively. The powerful effects of these two selective cADPR antagonists strongly suggest a key role for cADPR in mediating isoprenaline-induced membrane hyperpolarization in arterial smooth muscle cells. These findings are summarized in Fig. 2E.
After block of RyRs by ryanodine (20 M) and depletion of SR stores by cylclopiazonic acid (10 M), isoprenaline (100 nM) relaxed PGF 2␣ (50 M)-induced constriction by 21 Ϯ 5% (n ϭ 4) and 37 Ϯ 4% (Fig. 5 B and C; n ϭ 4), respectively. In each case, the inhibition of dilation by isoprenaline was equivalent to that obtained in the presence of 8-bromo-cADPR. These findings are therefore consistent with the view that cADPR mediates the component of vasodilation by isoprenaline that is dependent on SR Ca 2ϩ release via RyRs. Support for this viewpoint comes from our finding that preincubation of isolated pulmonary artery smooth muscle cells with ryanodine (20 M) did not deplete SR Ca 2ϩ stores and that 8-bromo-cADPR was without effect on the Ca 2ϩ transient evoked following inhibition of SR Ca 2ϩ ATPase activity by cyclopiazonic acid (see above). Furthermore, inhibition of SR Ca 2ϩ ATPase activity by cyclopiazonic acid induced transient constriction of isolated arteries (Fig. 5C), whereas 8-bromo-cADPR has no effect on resting tone (28,29).
Following block of BK Ca channels by preincubation of artery rings with iberiotoxin (100 nM), isoprenaline (100 nM) relaxed PGF 2␣ (50 M)-induced constriction by only 15 Ϯ 3% (n ϭ 4, Fig.  5D). Under these conditions, 8-Br-cADPR (300 M) was without effect on the isoprenaline-induced dilation (n ϭ 4, Fig. 5D). This finding is again consistent with a role for cADPR in mediating a significant proportion of BK Ca -dependent dilation by isoprenaline. Clearly, however, iberiotoxin inhibited a greater proportion of isoprenaline-induced dilation than did 8-bromo-cADPR, ryanodine, or cylclopiazonic acid. Thus, there may be a component of BK Ca -dependent dilation by isoprenaline that is mediated by mechanisms independent of SR Ca 2ϩ release.

DISCUSSION
As mentioned previously, vasodilators that activate adenylyl cyclase-coupled receptors relax arterial smooth muscle by increasing cAMP levels and activating PKA. Vasodilation by PKA-dependent signaling is mediated, in part, by BK Ca channel activation and membrane hyperpolarization (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). There is evidence to suggest that PKA may activate these channels by phosphorylating the BK Ca channel protein (4, 6, 10) and/or RyRs in the SR (4,12) or by activating the SR Ca 2ϩ ATPase, increasing the SR Ca 2ϩ load, and thereby increasing resting SR Ca 2ϩ release in the vicinity of the plasma membrane (4,(13)(14)(15)(16)(17). However, the extent to which each of these three mechanisms contributes to physiological responses remains controversial (4,5,10,(12)(13)(14)(15)(16)(17)(18). The present investigation now provides strong support for an alternative mechanism. Intracellular infusion of low concentrations of cADPR (20 M) increased cytoplasmic Ca 2ϩ concentration at the perimeter of isolated pulmonary artery smooth muscle cells and induced concomitant membrane hyperpolarization. Hyperpolarization was reversed by the highly selective BK Ca channel antagonist  iberiotoxin, which demonstrates that BK Ca channel activation underpins cADPR-dependent hyperpolarization. Furthermore, SR Ca 2ϩ release via RyRs was shown to be a prerequisite for this response because hyperpolarization by cADPR was abolished by chelating intracellular Ca 2ϩ with BAPTA and by selective block of RyRs with ryanodine. Thus, it seem likely that Ca 2ϩ release via RyRs mediates cADPR-dependent hyperpolarization. Further support for this conclusion may be derived from our finding that ryanodine did not deplete SR Ca 2ϩ stores over the time course of our experiments, as indicated by the fact that ryanodine blocked hyperpolarization by cADPR but not SR Ca 2ϩ release triggered by IP 3 . It should be noted, however, that there is evidence to suggest that IP 3 R and RyR activation, respectively, may mobilize Ca 2ϩ from functionally segregated SR compartments (38 -41).
A role for the SR was confirmed using the SR Ca 2ϩ ATPase antagonist cyclopiazonic acid. Significantly, cyclopiazonic acid reversed hyperpolarization by cADPR slowly, indicating that block by depletion of SR Ca 2ϩ stores exhibits a degree of use dependence, as one might expect of a process that depends on SR Ca 2ϩ release via RyRs.
Most importantly, however, hyperpolarization by cADPR was blocked by the cADPR antagonists 8-NH 2 -cADPR and 8-bromo-cADPR, respectively, whereas hyperpolarization by caffeine remained unaffected. Furthermore, 8-bromo-cADPR was shown to be without effect on the increase in intracellular Ca 2ϩ concentration triggered by depletion of SR Ca 2ϩ stores by cyclopiazonic acid, and 8-bromo-cADPR did not trigger an increase in intracellular Ca 2ϩ in its own right.
The findings described above are consistent with the view that the cADPR antagonists tested here block the action of cADPR without blocking the activation of RyRs per se and without inhibiting SR Ca 2ϩ ATPase activity. Thus, cADPR likely mediates hyperpolarization in artery smooth muscle by inducing SR Ca 2ϩ release via RyRs and consequent activation of BK Ca channels in the plasma membrane.
We next investigated the likely physiological role of hyperpolarization by cADPR. Previous studies have demonstrated that BK Ca channels contribute little to the resting potential in isolated pulmonary artery smooth muscle cells, and spontaneous transient outward currents are not observed under resting conditions (42)(43)(44). Taking this into consideration, it was not so surprising that the cADPR antagonists tested here had no effect on the resting membrane potential in isolated pulmonary artery smooth muscle cells nor on resting tone in isolated pulmonary arteries. It should be noted, however, that SR Ca 2ϩ release via RyRs has been shown to initiate spontaneous transient outward currents in resting systemic artery smooth muscle, and these BK Ca channel currents have been shown to determine the resting tone in smooth muscle of pressurized systemic arteries (for review, see Ref. 4). It may not be surprising, therefore, if future studies demonstrate that cADPR-dependent SR Ca 2ϩ release, by activation of BK Ca channels, regulates resting smooth muscle tone in certain vascular beds. By contrast, in the pulmonary vasculature it seemed most likely that receptor-dependent regulation of cADPR synthesis would serve pulmonary artery dilation by BK Ca channel activation.
Given that cADPR synthesis is up-regulated by cAMP in cardiac muscle (25), it appeared plausible that cADPR may mediate hyperpolarization by adenylyl cyclase-coupled receptors, such as ␤-adrenoceptors. As shown previously in smooth muscle from a variety of tissues (for review, see Ref. 4), isoprenaline-induced hyperpolarization in isolated pulmonary artery smooth muscle cells was abolished by selective block of BK Ca channel activation. Thus, iberiotoxin completely reversed hyperpolarization by isoprenaline, as was found to be the case with cADPR. Consistent with a role for SR Ca 2ϩ release via RyRs in this process, hyperpolarization by isoprenaline was blocked by chelation of intracellular Ca 2ϩ with BAPTA and by inhibition of RyR function with ryanodine, respectively. Significantly, hyperpolarization by isoprenaline was also blocked by the cADPR antagonists 8-amino-cADPR and 8-bromo-cADPR. In marked contrast, block of IP 3 R activation had no effect on hyperpolarization by cADPR and isoprenaline, respectively. These findings provide strong support for our proposal that cADPR-dependent SR Ca 2ϩ release via RyRs mediates BK Ca channel activation by isoprenaline and mitigates against a role for IP 3 Rs in this process.
Consistent with the above, hyperpolarization by intracellular dialysis of cAMP was also blocked by iberiotoxin, BAPTA, ryanodine, and 8-amino-cADPR, respectively. However, our findings with respect to the selective PKA antagonist H89 were quite different. H89 blocked hyperpolarization by both isoprenaline and cAMP but was without effect on hyperpolarization by cADPR. Thus, it would appear that cADPR is a downstream element in this signaling cascade. We propose, therefore, that hyperpolarization by ␤-adrenoceptors in pulmonary artery smooth muscle cells results, at least in part, from activation of adenylyl cyclase, increased cytoplasmic cAMP levels, and activation of PKA, leading to activation of ADP-ribosyl cyclase, increased cADPR synthesis, consequent SR Ca 2ϩ release via RyRs, and hyperpolarization by BK Ca channel activation. Neither isoprenaline nor cAMP induced hyperpolarization in the presence of cADPR antagonists, and cADPR antagonists were without effect on RyRs, SR Ca 2ϩ ATPase activity, and/or BK Ca channel activation per se. Thus, we find little evidence of a role for PKA-dependent phosphorylation of the BK Ca channel protein, RyRs, or phospholamban in mediating hyperpolarization by adenylyl cyclase-coupled receptors in isolated pulmonary artery smooth muscle cells under the conditions of our experiments.
In isolated artery rings without endothelium, the membranepermeant cADPR antagonist 8-bromo-cADPR reversed by ϳ50% vasodilation evoked by isoprenaline. This is consistent with the view that cADPR mediates, in part, dilation by isoprenaline. Block of RyRs with ryanodine and depletion of SR Ca 2ϩ stores by cyclopiazonic acid inhibited dilation by isoprenaline by between 50 and 60%. As mentioned previously, neither 8-bromo-cADPR nor ryanodine appeared to have a significant effect on SR Ca 2ϩ load in isolated smooth muscle cells over the time course of our experiments. Furthermore, it is unlikely that 8-bromo-cADPR inhibits SR Ca 2ϩ ATPase activity because: 1) 8-bromo-cADPR is without effect on the Ca 2ϩ transient evoked by inhibition of SR Ca 2ϩ ATPase activity by cylcopiazonic acid and does not itself increase intracellular Ca 2ϩ concentration and 2) cyclopiazonic acid induces constriction of pulmonary arteries, whereas 8-bromo-cADPR does not. These findings therefore provide strong support for our view that cADPR-dependent SR Ca 2ϩ release via RyRs mediates, in part, dilation by ␤-adrenoreceptor activation.
Importantly, 8-bromo-cADPR was without effect on residual dilation by isoprenaline after blocking BK Ca channels with iberiotoxin. We can conclude, therefore, that 8-bromo-cADPR reverses dilation by isoprenaline by inhibiting BK Ca channel activation by SR Ca 2ϩ release because 8-bromo-cADPR does not block BK Ca channel activation per se. It is interesting to note, however, that iberiotoxin inhibited dilation by isoprenaline by ϳ78% as compared with 50 -60% reversal by 8-bromo-cADPR, ryanodine, and cyclopiazonic acid, respectively. It would appear, therefore, that cADPR-dependent signaling is responsible for ϳ80% of BK Ca -dependent vasodilation by isoprenaline in isolated pulmonary arteries. A further 20% may depend on a mechanism independent of SR Ca 2ϩ release, possibly PKA-dependent phosphorylation of the BK Ca channel protein (4,6,10).
Given our findings, it is interesting to note that previous studies in coronary artery smooth muscle have suggested that increased synthesis of ADP-ribose, a cADPR metabolite, may mediate BK Ca -dependent vasodilation by 11,12-epoxyeicosatrienoic acid (45). In contrast to the effects of 20 M cADPR, however, we found 20 M ADP-ribose to be without effect on membrane potential in pulmonary artery smooth muscle cells. A further variation on the theme comes from the proposal that nitric oxide may mediate vasodilation by inhibiting cADPR formation (46). It is possible, therefore, that the mechanism by which pyridine nucleotide signaling promotes relaxation of artery smooth muscle may vary in a manner dependent on the nature of the vasodilator and/or vascular bed.
A recent report has also demonstrated that concentrations of cADPR in the mM range may inhibit BK Ca channel activation (47). The physiological significance of this observation is open to question, as the concentrations used were 50 times greater than those found to mediate BK Ca -dependent hyperpolarization in the present investigation. Despite this fact, it is possible that inhibition of BK Ca channel activity by cADPR may 1) serve as a negative feedback loop with respect to hyperpolarization and/or 2) contribute to vasoconstriction by cADPR.
In summary, we have shown that adenylyl cyclase-coupled receptors may mediate vasodilation by cADPR-dependent Ca 2ϩ release via RyRs in the SR, leading to subsequent BK Ca -dependent hyperpolarization and vasodilation. When taken together with previous investigations, it would appear that cADPR-dependent Ca 2ϩ release via RyRs could lead to stimulusdependent relaxation or contraction in arterial smooth muscle (26 -29). Given that RyR subtypes 1, 2, and 3 are present in vascular smooth muscle (48 -51), this paradox may be explained if: 1) ␤-adrenoreceptor signaling targets, via protein kinase A anchoring proteins (AKAPs; Refs. 52-54), PKA-dependent cADPR synthesis to a particular RyR subtype in the "peripheral" SR that is in close apposition to BK Ca channels in the plasma membrane; 2) cADPR-dependent vasoconstriction results from the activation of a discrete RyR subtype localized in the central SR proximal to the contractile apparatus; and 3) the peripheral and central SR represent functionally segregated compartments. Further investigations will, however, be required to confirm these proposals.