Vasodilation by the Calcium-mobilizing Messenger Cyclic ADP-ribose*

François-Xavier BoittinDagger , Michelle Dipp§, Nicholas P. KinnearDagger , Antony Galione, and A. Mark EvansDagger ||

From the Dagger  Division of Biomedical Sciences, School of Biology, Bute Building, University of St Andrews, St Andrews, Fife, KY16 9TS, United Kingdom and the  University Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, United Kingdom

Received for publication, May 17, 2002, and in revised form, December 10, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In artery smooth muscle, adenylyl cyclase-coupled receptors such as beta -adrenoceptors evoke Ca2+ signals, which open Ca2+-activated potassium (BKCa) channels in the plasma membrane. Thus, blood pressure may be lowered, in part, through vasodilation due to membrane hyperpolarization. The Ca2+ 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 Ca2+ 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 Ca2+ and by blocking RyRs, cADPR, and BKCa 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 BKCa 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 Ca2+ release via RyRs, which leads to activation of BKCa channels and membrane hyperpolarization.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Ca2+-activated potassium (BKCa) channels and membrane hyperpolarization (1-11). In artery smooth muscle, adenylyl cyclase-coupled receptors, such as beta -adrenoceptors, open BKCa channels by evoking Ca2+ 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.

Previous studies have suggested that activation of PKA may promote BKCa-dependent hyperpolarization by direct phosphorylation of the BKCa channel protein (4, 6, 10). However, the most compelling evidence suggests that BKCa channel activation results from Ca2+ release from the sarcoplasmic reticulum (SR) due to the opening of RyRs proximal to the plasma membrane (1, 2, 4, 5, 8, 11-17). There is evidence to suggest that this may result from direct phosphorylation of RyRs by PKA (4, 12). There is also support for a role for PKA-dependent activation of the SR Ca2+ ATPase by phosphorylation of phospholamban, increased SR Ca2+ load, and thereby increased resting SR Ca2+ release via RyRs in the vicinity of the plasma membrane (4, 13-17). As yet, however, the extent to which each of these three mechanisms contributes to physiological responses remains controversial (4, 5, 10, 12-18).

We have now investigated the role of cyclic adenosine diphosphate-ribose (cADPR), a beta -NAD+ metabolite (19, 20), in mediating vasodilation by BKCa 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 Ca2+ 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 Ca2+ release and consequent activation of BKCa channels in pulmonary artery smooth muscle.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Ca2+ solution of the following composition: 124 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM NaH2PO4, 0.5 mM KH2PO4, 15 mM NaHCO3, 0.16 mM CaCl2, 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 Ca2+ 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 = 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 MgCl2, 1.7 mM CaCl2, 10 mM glucose, 10 mM Hepes, pH 7.45. The pipette solution contained 140 mM KCl, 10 mM Hepes, 1 mM MgCl2, pH 7.4. All experiments were carried out at room temperature (22 °C). Seal resistance was >= 3 gigaohms, series resistance was <= 5 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.

Ca2+ Imaging-- Cells were incubated for 30 min in low Ca2+ solution (see above) containing 5 µM Fura-2 AM, washed, and allowed to equilibrate for 20 min. 5 µM Fura-2 (free acid) was also added to the pipette solution used for intracellular dialysis (see above). Changes in intracellular Ca2+ 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 Ca2+ concentration.

Small Vessel Myography-- Artery rings (2-3 mm in length) were isolated from second order branches of rat pulmonary arteries and mounted onto an automated myograph (AM10, Cambustion Biological, Cambridge, UK) using 50-µm tungsten wire. Tension was set to be equivalent to a pressure of 30 mm Hg. The methods have been described in detail previously (34, 35). The myograph contained physiological salt solution B: 118 mM NaCl, 4 mM KCl, 24 mM NaHCO3, 1 mM MgSO4, 1.2 mM NaH2PO4, 2 mM CaCl2, 5.56 mM glucose. The solution was maintained at 37 °C and bubbled with 75% N2, 20% O2, and 5% CO2 to maintain a pH of 7.4. The endothelium was removed by rubbing the intima of the arteries with braided silk surgical thread. Endothelium removal was confirmed by the failure of 100 µM acetylcholine to relax constrictions induced by 1 µM (5Z, 9alpha , 11alpha , 13E, 15S)-9,11,15-Trihydroxyprosta-5,13-dienoic acid (PGF2alpha ).

Chemicals-- Isoprenaline, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), cADPR, ADP-ribose, iberiotoxin, 8-amino-cyclic adenosine diphosphate-ribose (8-NH2-cADPR), 8-bromo-cyclic adenosine diphosphate-ribose (8-Br-cADPR), heparin, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89), cAMP, ryanodine, PGF2alpha , myo-inositol 1,4,5 trisphosphate (IP3), and cyclopiazonic acid were from Sigma. Fura-2 and Fura-2-AM were from Molecular Probes. Xestospongin C was from Calbiochem. Fura-2, Ryanodine and cyclopiazonic acid were dissolved in Me2SO, final dilution of 1:1000. At this concentration, Me2SO was without effect on the preparations studied here.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cADPR-dependent SR Ca2+ Release via Ryanodine Receptors Activates BKCa 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 Ca2+ 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 -40 to -60 mV, and estimated resting Ca2+ concentrations were in the range 70-150 nM (4, 36). Intracellular application of cADPR (20 µM) evoked a sustained increase in intracellular Ca2+ concentration, as indicated by an increase in the Fura-2 fluorescence ratio (F340/F380) from 0.5 ± 0.1 to 0.8 ± 0.1 (mean ± S.E.; Fig. 1A) and a concomitant hyperpolarization. The increase in intracellular Ca2+ was localized at the cell perimeter, i.e. in close apposition to the plasma membrane. However, the capacity of Fura-2 for binding Ca2+ 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 µM) evoked hyperpolarization from -40 ± 2 to -75 ± 1 mV (n = 28, Fig. 1B), which was reversed by the selective BKCa channel antagonist iberiotoxin (100 nM, n = 7, Fig. 1B). Importantly, no hyperpolarization was observed when intracellular Ca2+ was chelated by intracellular infusion of BAPTA (1 mM, Fig. 1C). Furthermore, cADPR-induced hyperpolarization was slowly reversed when SR Ca2+ stores were depleted by cyclopiazonic acid (10 µM, n = 4, Fig. 1D), blocked following preincubation with 20 µM ryanodine (20 min, n = 4, Fig. 1E), and blocked by the selective cADPR antagonists 8-NH2-cADPR (100 µM, n = 4, Fig. 1F) and 8-bromo-cADPR (100 µM, n = 4, not shown (37)). These findings are summarized in Fig. 1G. Hyperpolarization induced by 1 mM caffeine, which triggers Ca2+ release via RyRs by a mechanism independent of cADPR (22), was insensitive to the cADPR antagonist 8-NH2-cADPR (100 µM; n = 4, not shown) and 8-bromo-cADPR, respectively, (100-300 µM; n = 4, not shown). This is consistent with the view that the cADPR antagonists tested here selectively inhibit SR Ca2+ release by cADPR in pulmonary artery smooth muscle cells but do not block SR Ca2+ release via RyRs or BKCa 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 µM ADP-ribose had no effect on membrane potential (n = 4, not shown). Extracellular application of 20 µM 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-NH2-cADPR (100 µM, n = 4) and 8-bromo-cADPR (<= 300 µM, n = 4), respectively, had any effect on membrane potential.


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Fig. 1.   cADPR hyperpolarises pulmonary artery smooth muscle cells by increasing Ca2+ release from ryanodine-sensitive SR stores. A, effect of intracellular infusion of 20 µM cADPR, via a patch pipette, on the Fura-2 fluorescence ratio (F340/F380) in an isolated rat pulmonary artery smooth muscle cell. B, effect of intracellular infusion of 20 µM cADPR on the membrane potential in an isolated pulmonary artery smooth muscle cell, in the absence and then in the presence of 100 nM iberiotoxin. C, effect of intracellular infusion of 20 µM cADPR in the absence and in the presence of BAPTA (1 mM), respectively. D, effect of intracellular infusion of 20 µM cADPR on the membrane potential in an isolated pulmonary artery smooth muscle cell in the absence and then in the presence of 10 µM cyclopiazonic acid. E, effect of intracellular infusion of 20 µM cADPR in the absence and in the presence of 20 µM ryanodine (20 µM), respectively. F, effect of intracellular infusion of 20 µM cADPR in the absence and then in the presence of the cADPR antagonist 8-NH2-cADPR (100 µM), respectively. G, bar chart showing the control membrane potential (CMP) recorded prior to the onset of the cADPR-induced hyperpolarization and the membrane potential recorded after the hyperpolarization by cADPR (20 µM) had reached its maximum in the absence and in the presence of each of the antagonists tested. In this and subsequent figures, the number of cells tested is indicated above the bars. The columns and vertical bars show the mean ± S.E.

Does Ryanodine Deplete SR Ca2+ Stores?-- In contrast to hyperpolarization by cADPR, ryanodine had no significant effect on the global Ca2+ wave induced by intracellular dialysis of IP3. Thus, intracellular dialysis of 1 µM IP3 increased the Fura-2 fluorescence ratio from 0.60 ± 0.01 to a peak of 2.5 ± 0.1 in the absence of 20 µM ryanodine (n = 5) and from 0.8 ± 0.1 to a peak of 1.8 ± 0.2 in the presence of 20 µM ryanodine (20-min preincubation, n = 5). Conversely, the IP3R antagonist xestospongin C blocked IP3-induced Ca2+ signals (n = 4, not shown) but had no effect on Ca2+ signals or hyperpolarization by cADPR (n = 4, not shown). These findings suggest that ryanodine blocks hyperpolarization by cADPR without significant effect on the ability of the SR to release Ca2+ over the time course of our experiments.

Does 8-Bromo-cADPR Inhibit SR Ca2+ ATPase Activity?-- Preincubation of pulmonary artery smooth muscle cells with 300 µM 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 µM cyclopiazonic acid from 0.37 ± 0.02 to a peak of 0.56 ± 0.03 (n = 10) in the absence of 8-bromo-cADPR and from 0.35 ± 0.03 to a peak of 0.58 ± 0.04 (n = 4) in the presence of 8-bromo-cADPR (300 µM). Thus, it seems unlikely that 8-bromo-cADPR blocks cADPR-dependent hyperpolarization by inhibiting SR Ca2+ ATPase activity. When taken together, the aforementioned findings are consistent with the view that cADPR mediates hyperpolarization by triggering SR Ca2+ release via RyRs by a mechanism independent of the SR Ca2+ ATPase and IP3Rs, respectively, which ultimately leads to BKCa channel activation and hyperpolarization.

cADPR Mediates SR Ca2+ Release and Hyperpolarization by Isoprenaline-- Given our finding that cADPR mediates hyperpolarization by BKCa channel activation, we investigated the role of cADPR in mediating hyperpolarization induced by the activation of beta -adrenoceptors, a family of adenylyl cyclase-coupled vasodilator receptors that are known to mediate smooth muscle relaxation, in part, by activating BKCa channels and membrane hyperpolarization (4-7, 9). Extracellular application of the selective beta -adrenoreceptor agonist isoprenaline (10 µM) induced sustained hyperpolarization from -39 ± 2 to -75 ± 1 mV (n = 20, Fig. 2A), which was reversed by the selective BKCa channel antagonist iberiotoxin (100 nM, n = 5, Fig. 2A). As would be expected from previous studies (6, 7, 9), hyperpolarization by isoprenaline was also blocked by a selective beta -adrenoreceptor antagonist, propanolol (10 µM, n = 4, not shown). In common with cADPR, hyperpolarization by isoprenaline was blocked when intracellular Ca2+ was chelated by BAPTA (1 mM, n = 4, Fig. 2B), underscoring the role of an increase in intracellular Ca2+ concentration. Isoprenaline-evoked hyperpolarization was also blocked when SR Ca2+ release was inhibited with ryanodine (20 µM, n = 6, Fig. 2C). In contrast, intracellular dialysis of heparin, an IP3R antagonist, failed to inhibit hyperpolarization by isoprenaline (n = 3, not shown), which mitigates against a possible role for IP3Rs 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 beta -adrenoreceptor signaling pathway. Hyperpolarization by isoprenaline was abolished by intracellular infusion of 8-NH2-cADPR (100 µM, n = 6, Fig. 2D) and of 8-Br-cADPR (100 µM, n = 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.


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Fig. 2.   Hyperpolarization induced by isoprenaline is mediated by cADPR-dependent Ca2+ release from ryanodine-sensitive SR stores. A, effect of 10 µM isoprenaline on the membrane potential in an isolated pulmonary artery smooth muscle cell in the absence and then in the presence of 100 nM iberiotoxin. Effect of isoprenaline (10 µM) in the absence and then in the presence, respectively, of intracellular infusion of 1 mM BAPTA (B); ryanodine (20 µM) (C); and intracellular infusion of 100 µM 8-NH2-cADPR (D). E, mean hyperpolarization induced by isoprenaline (10 µM) in the absence and in the presence of the antagonists tested. CMP is the membrane potential recorded under control conditions, i.e. prior to the application of isoprenaline and in the absence of the antagonists tested.

cAMP and PKA Mediate cADPR-dependent Hyperpolarization by Isoprenaline-- Since beta -adrenoceptors activate adenylyl cyclase (6, 7, 9), we investigated the relationship between isoprenaline, cAMP, PKA, and cADPR-mediated hyperpolarization. Intracellular infusion of cAMP (10 µM) induced hyperpolarization in pulmonary artery smooth muscle cells from -43 ± 4 mV to -82 ± 5 mV (n = 10), which was reversed by the selective BKCa channel antagonist iberiotoxin (100 nM, n = 6, Fig. 3A). In common with cADPR and isoprenaline, cAMP-dependent hyperpolarization was blocked in cells that had been preincubated with 20 µM ryanodine (n = 5, Fig. 3B) and was abolished by co-infusion with 8-NH2-cADPR (100 µM, n = 11, Fig. 3C) and by co-infusion with 8-Br-cADPR (100 µM, not shown). These findings are summarized in Fig. 3D.


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Fig. 3.   Hyperpolarization induced by cAMP is mediated by cADPR-dependent Ca2+ release from ryanodine-sensitive SR stores. A, effect of intracellular infusion of 10 µM cAMP, via a patch pipette, on the membrane potential in an isolated pulmonary artery smooth muscle cell before and then after extracellular application of 100 nM iberiotoxin. B, effect of intracellular infusion of 10 µM cAMP in the absence and in the presence of ryanodine (20 µM), respectively. C, effect of intracellular infusion of 10 µM cAMP in the absence and in the presence of 100 µM 8-NH2-cADPR, respectively. D, bar chart showing the control membrane potential (CMP) recorded prior to the onset of the cAMP-mediated hyperpolarization and the membrane potential recorded after the hyperpolarization by cAMP (10 µM) had reached its maximum in absence and in the presence of each of the antagonists tested.

Preincubation with H89 (1 µM), a selective PKA inhibitor, abolished hyperpolarization by isoprenaline (n = 5, Fig. 4, A and D). In marked contrast, hyperpolarization by cADPR (20 µM) remained unaffected in the presence of H89 (1 µM), measuring -76 ± 2 mV (n = 28) in the absence of H89 and -78 ± 6 mV in the presence of H89 (1 µM; n = 3; Fig. 4, B and D). In common with isoprenaline, but in contrast to cADPR, cAMP failed to induce membrane hyperpolarization in the presence of 1 µM H89 (n = 6, Fig. 4, C and D). These findings are consistent with cADPR acting downstream of cAMP and PKA activation during beta -adrenoreceptor signaling.


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Fig. 4.   cADPR acts downstream of cAMP and PKA. A, effect of extracellular application of 1 µM isoprenaline in the absence and in the presence of H89 (1 µM). B, effect of intracellular infusion of 20 µM cADPR in the presence of H89 (1 µM). C, effect of intracellular infusion of 10 µM cAMP in the absence and in the presence of H89 (1 µM). D, membrane potential recorded after the hyperpolarization by isoprenaline (1 µM), cADPR (20 µM), cAMP (10 µM), respectively, had reached its maximum in the absence and in the presence of H89 (1 µM).

cADPR-dependent SR Ca2+ Release via Ryanodine Receptors Mediates BKCa-dependent Vasodilation by Isoprenaline-- The role of cADPR in mediating isoprenaline-induced vasodilation was investigated in isolated pulmonary artery rings by tension recording experiments. Isolated pulmonary artery rings, without endothelium, were first constricted with 50 µM PGF2alpha . Subsequent application of isoprenaline (100 nM; IC50) relaxed the PGF2alpha (50 µM)-induced constriction by 68 ± 4% (Fig. 5A, n = 4). Application of 8-Br-cADPR (300 µM), a membrane-permeant cADPR antagonist (37), reversed the isoprenaline-induced relaxation to 36 ± 2% (Fig. 5A, n = 4). Clearly, these findings are consistent with a role for cADPR in mediating dilation by beta -adrenoreceptor activation. Note that in the presence of 50 µM PGF2alpha , isolated pulmonary artery smooth muscle cells were depolarized relative to control, and under these conditions, intracellular dialysis of 20 µM cADPR elicited hyperpolarization consistent with control, from -36 ± 2 mV to -61 ± 12 mV (n = 4, Fig. 5, inset). In contrast to its inhibition of dilation by isoprenaline, 300 µM 8-bromo-cADPR has no effect on constriction by potassium (i.e. by voltage-gated Ca2+ influx) and PGF2alpha , respectively (29).


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Fig. 5.   cADPR mediates dilation induced by isoprenaline in isolated pulmonary artery rings without the endothelium. A, effect of isoprenaline (100 nM) on a rat pulmonary artery ring preconstricted with PGF2alpha (50 µM), in the absence and then presence of 8-Br-cADPR (300 µM). B, effect of isoprenaline (100 nM) on a rat pulmonary artery ring preconstricted with PGF2alpha (50 µM) after blocking ryanodine receptors with ryanodine (20 µM). C, effect of isoprenaline (100 nM) on a rat pulmonary artery ring preconstricted with PGF2alpha (50 µM) after depletion of SR Ca2+ stores with cyclopiazonic acid (CPA, 10 µM). D, as in A, but after block of BKca channels with iberiotoxin (IbTx, 100 nM). The inset shows hyperpolarization by intracellular dialysis of 20 µM cADPR into an isolated pulmonary artery smooth muscle cell preincubated (10 min) with PGF2alpha (50 µM).

After block of RyRs by ryanodine (20 µM) and depletion of SR stores by cylclopiazonic acid (10 µM), isoprenaline (100 nM) relaxed PGF2alpha (50 µM)-induced constriction by 21 ± 5% (n = 4) and 37 ± 4% (Fig. 5 B and C; n = 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 Ca2+ release via RyRs. Support for this viewpoint comes from our finding that preincubation of isolated pulmonary artery smooth muscle cells with ryanodine (20 µM) did not deplete SR Ca2+ stores and that 8-bromo-cADPR was without effect on the Ca2+ transient evoked following inhibition of SR Ca2+ ATPase activity by cyclopiazonic acid (see above). Furthermore, inhibition of SR Ca2+ 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 BKCa channels by preincubation of artery rings with iberiotoxin (100 nM), isoprenaline (100 nM) relaxed PGF2alpha (50 µM)-induced constriction by only 15 ± 3% (n = 4, Fig. 5D). Under these conditions, 8-Br-cADPR (300 µM) was without effect on the isoprenaline-induced dilation (n = 4, Fig. 5D). This finding is again consistent with a role for cADPR in mediating a significant proportion of BKCa-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 BKCa-dependent dilation by isoprenaline that is mediated by mechanisms independent of SR Ca2+ release.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 BKCa channel activation and membrane hyperpolarization (1-11). There is evidence to suggest that PKA may activate these channels by phosphorylating the BKCa channel protein (4, 6, 10) and/or RyRs in the SR (4, 12) or by activating the SR Ca2+ ATPase, increasing the SR Ca2+ load, and thereby increasing resting SR Ca2+ release in the vicinity of the plasma membrane (4, 13-17). However, the extent to which each of these three mechanisms contributes to physiological responses remains controversial (4, 5, 10, 12-18). The present investigation now provides strong support for an alternative mechanism.

Intracellular infusion of low concentrations of cADPR (20 µM) increased cytoplasmic Ca2+ concentration at the perimeter of isolated pulmonary artery smooth muscle cells and induced concomitant membrane hyperpolarization. Hyperpolarization was reversed by the highly selective BKCa channel antagonist iberiotoxin, which demonstrates that BKCa channel activation underpins cADPR-dependent hyperpolarization. Furthermore, SR Ca2+ release via RyRs was shown to be a prerequisite for this response because hyperpolarization by cADPR was abolished by chelating intracellular Ca2+ with BAPTA and by selective block of RyRs with ryanodine. Thus, it seem likely that Ca2+ release via RyRs mediates cADPR-dependent hyperpolarization. Further support for this conclusion may be derived from our finding that ryanodine did not deplete SR Ca2+ stores over the time course of our experiments, as indicated by the fact that ryanodine blocked hyperpolarization by cADPR but not SR Ca2+ release triggered by IP3. It should be noted, however, that there is evidence to suggest that IP3R and RyR activation, respectively, may mobilize Ca2+ from functionally segregated SR compartments (38-41).

A role for the SR was confirmed using the SR Ca2+ ATPase antagonist cyclopiazonic acid. Significantly, cyclopiazonic acid reversed hyperpolarization by cADPR slowly, indicating that block by depletion of SR Ca2+ stores exhibits a degree of use dependence, as one might expect of a process that depends on SR Ca2+ release via RyRs.

Most importantly, however, hyperpolarization by cADPR was blocked by the cADPR antagonists 8-NH2-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 Ca2+ concentration triggered by depletion of SR Ca2+ stores by cyclopiazonic acid, and 8-bromo-cADPR did not trigger an increase in intracellular Ca2+ 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 Ca2+ ATPase activity. Thus, cADPR likely mediates hyperpolarization in artery smooth muscle by inducing SR Ca2+ release via RyRs and consequent activation of BKCa channels in the plasma membrane.

We next investigated the likely physiological role of hyperpolarization by cADPR. Previous studies have demonstrated that BKCa 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 (42-44). 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 Ca2+ release via RyRs has been shown to initiate spontaneous transient outward currents in resting systemic artery smooth muscle, and these BKCa 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 Ca2+ release, by activation of BKCa 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 BKCa 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 beta -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 BKCa channel activation. Thus, iberiotoxin completely reversed hyperpolarization by isoprenaline, as was found to be the case with cADPR. Consistent with a role for SR Ca2+ release via RyRs in this process, hyperpolarization by isoprenaline was blocked by chelation of intracellular Ca2+ 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 IP3R activation had no effect on hyperpolarization by cADPR and isoprenaline, respectively. These findings provide strong support for our proposal that cADPR-dependent SR Ca2+ release via RyRs mediates BKCa channel activation by isoprenaline and mitigates against a role for IP3Rs 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 beta -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 Ca2+ release via RyRs, and hyperpolarization by BKCa channel activation. Neither isoprenaline nor cAMP induced hyperpolarization in the presence of cADPR antagonists, and cADPR antagonists were without effect on RyRs, SR Ca2+ ATPase activity, and/or BKCa channel activation per se. Thus, we find little evidence of a role for PKA-dependent phosphorylation of the BKCa 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 membrane-permeant cADPR antagonist 8-bromo-cADPR reversed by ~50% 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 Ca2+ 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 Ca2+ load in isolated smooth muscle cells over the time course of our experiments. Furthermore, it is unlikely that 8-bromo-cADPR inhibits SR Ca2+ ATPase activity because: 1) 8-bromo-cADPR is without effect on the Ca2+ transient evoked by inhibition of SR Ca2+ ATPase activity by cylcopiazonic acid and does not itself increase intracellular Ca2+ 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 Ca2+ release via RyRs mediates, in part, dilation by beta -adrenoreceptor activation.

Importantly, 8-bromo-cADPR was without effect on residual dilation by isoprenaline after blocking BKCa channels with iberiotoxin. We can conclude, therefore, that 8-bromo-cADPR reverses dilation by isoprenaline by inhibiting BKCa channel activation by SR Ca2+ release because 8-bromo-cADPR does not block BKCa channel activation per se. It is interesting to note, however, that iberiotoxin inhibited dilation by isoprenaline by ~78% 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 ~80% of BKCa-dependent vasodilation by isoprenaline in isolated pulmonary arteries. A further 20% may depend on a mechanism independent of SR Ca2+ release, possibly PKA-dependent phosphorylation of the BKCa 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 BKCa-dependent vasodilation by 11,12-epoxyeicosatrienoic acid (45). In contrast to the effects of 20 µM cADPR, however, we found 20 µM 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 BKCa 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 BKCa-dependent hyperpolarization in the present investigation. Despite this fact, it is possible that inhibition of BKCa 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 Ca2+ release via RyRs in the SR, leading to subsequent BKCa-dependent hyperpolarization and vasodilation. When taken together with previous investigations, it would appear that cADPR-dependent Ca2+ release via RyRs could lead to stimulus-dependent 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) beta -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 BKCa 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.

    ACKNOWLEDGEMENT

We thank Professor Alison Brading for kind help and support.

    FOOTNOTES

* This work was funded by a Wellcome Trust Project Grant (reference number 056423).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Worcester College, University of Oxford, Walton Street, Oxford, OX1 2HB.

|| To whom correspondence should be addressed. Tel.: 44-1334-463579; Fax: 44-1334-463600; E-mail: ame3@st-and.ac.uk.

Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M204891200

    ABBREVIATIONS

The abbreviations used are: PKA, cAMP-dependent protein kinase; BKCa, Ca2+-activated potassium; SR, sarcoplasmic reticulum; cADPR, cyclic ADP-ribose; 8-Br-cADPR, 8-bromo-cADPR; 8-NH2-cADPR, 8-amino-cADPR; RyR, ryanodine receptors; PGF2alpha , (5Z, 9alpha , 11alpha , 13E, 15S)-9,11,15-Trihydroxyprosta-5,13-dienoic acid; IP3, myo-inositol 1,4,5 trisphosphate; IP3R, IP3 receptor; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; H89, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Knot, H. J., Standen, N. B., and Nelson, M. T. (1998) J. Physiol. (Lond.) 508, 211-221[Abstract/Free Full Text]
2. Benham, C. D., and Bolton, T. B. (1986) J. Physiol. (Lond.) 381, 385-406[Abstract]
3. Tanaka, Y. M., Aida, H., Tanaka, K., Shigenobu, K., and Toro, L. (1998) Naunyn-Schmiedebergs Arch. Pharmakol. 357, 705-708[Medline] [Order article via Infotrieve]
4. Jaggar, J. H., Porter, V. A., Lederer, W. J., and Nelson, M. T. (2000) Am. J. Physiol. 278, C235-C256
5. Porter, V. A., Bonev, A. D., Knot, H. J., Heppner, T. J., Stevenson, A. S., Kleppisch, T., Ledderer, W. J., and Nelson, N. T. (1998) Am. J. Physiol. 274, C1346-C1355[Medline] [Order article via Infotrieve]
6. Wang, Y. X., and Kotlikoff, M. I. (1996) Am. J. Physiol. 271, L100-L105[Medline] [Order article via Infotrieve]
7. Satake, N., Shibata, M., and Shibata, S. (1996) Br. J. Pharmacol. 119, 505-510[Abstract]
8. Perez, G. J., Bonev, A. D., Patlak, J. B., and Nelson, M. T. (1999) J. Gen. Physiol. 113, 229-238[Abstract/Free Full Text]
9. Shaul, P. W., Muntz, K. H., and Buja, L. M. (1990) J. Pharmacol. Exp. Ther. 252, 86-92[Abstract]
10. Kume, H., Takai, A., Tokuno, H., and Tomita, T. (1989) Nature 341, 152-154[CrossRef][Medline] [Order article via Infotrieve]
11. Nelson, M. T., Cheng, H., Rubart, M., Santana, L. F., Bonev, A. D., Knot, H. J., and Lederer, W. J. (1995) Science 270, 633-637[Abstract]
12. Valdivia, H. H., Kaplan, J. H., Ellis-Davies, G. C., and Lederer, W. J. (1995) Science 267, 1997-2000[Medline] [Order article via Infotrieve]
13. Linderman, J. P., Jones, L. R., Hathaway, D. R., Henry, B. G., and Watanabe, A. M. (1983) J. Biol. Chem. 258, 464-471[Free Full Text]
14. Lukyanenko, V., Gyorke, I., and Gyorke, S. (1996) Pfluegers Arch. Eur. J. Physiol. 432, 1047-1054[CrossRef][Medline] [Order article via Infotrieve]
15. Raeymaekers, L., Eggermont, J. A., Wuytack, F., and Casteels, R. (1990) Cell Calcium 11, 261-268[Medline] [Order article via Infotrieve]
16. Wellman, G. C., Santana, L. F., Bonev, A. D., and Nelson, M. T. (2001) Am. J. Physiol. 281, C1029-C1037
17. ZhuGe, R., Tuft, R. A., Fogarty, K. E., Bellve, K., Fay, F. S., and Walsh, J. V. (1999) J. Gen. Physiol. 113, 215-228[Abstract/Free Full Text]
18. Lalli, M. J., Shimizu, S., Sutliff, R. L., Kranias, E. G., and Paul, R. J. (1999) Am. J. Physiol. 277, H963-H970[Medline] [Order article via Infotrieve]
19. Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. (1989) J. Biol. Chem. 264, 1608-1615[Abstract/Free Full Text]
20. Lee, H. C. (1997) Physiol. Rev. 77, 1133-1164[Abstract/Free Full Text]
21. Galione, A., Lee, H. C., and Busa, W. B. (1991) Science 253, 1143-1146[Medline] [Order article via Infotrieve]
22. Galione, A., and Summerhill, R. S. (1996) Ryanodine Receptors , pp. 52-70, CRC Press, Inc., Boca Raton, FL
23. Lee, H. C., and Aarhus, R. (1993) Biochim. Biophys. Acta 1164, 68-74[Medline] [Order article via Infotrieve]
24. Cui, Y., Galione, A., and Terrar, D. A. (1999) Biochem. J. 342, 269-273[CrossRef][Medline] [Order article via Infotrieve]
25. Higashida, H., Egorova, A., Higashida, C., Zhong, Z-G., Yokoyama, S., Noda, M., and Zhang, J-S. (2001) J. Biol. Chem. 274, 33348-33354
26. Kuemmerle, J. F., and Makhlouf, G. M. (1995) J. Biol. Chem. 270, 25488-25494[Abstract/Free Full Text]
27. Prakash, Y. S., Kannan, M. S., Walseth, T. F., and Sieck, G. C. (1998) Am. J. Physiol. 274, C1653-C1660[Medline] [Order article via Infotrieve]
28. Wilson, H. L., Dipp, M., Thomas, J. M., Lad, C., Galione, A., and Evans, A. M. (2001) J. Biol. Chem. 276, 11180-11188[Abstract/Free Full Text]
29. Dipp, M., and Evans, A. M. (2001) Circ. Res. 89, 77-83[Abstract/Free Full Text]
30. Makhlouf, G. M., and Murthy, K. S. (1997) Cell. Signal. 9, 269-276[CrossRef][Medline] [Order article via Infotrieve]
31. Yusufi, A. N., Cheng, J., Thompson, M. A., Burnett, J. C., and Grande, J. P. (2002) Exp. Biol. Med. 227, 36-44[Abstract/Free Full Text]
32. Li, P. L., Tang, W. X., Valdivia, H. H., Zou, A. P., and Campbell, W. B. (2001) Am. J. Physiol. 280, H208-H215
33. Li, N., Teggatz, E. G., Li, P. L., Allaire, R., and Zou, A. P. (2000) Microvasc. Res. 60, 149-159[CrossRef][Medline] [Order article via Infotrieve]
34. Dipp, M., Nye, P. C. G., and Evans, A. M. (2001) Am. J. Physiol. 281, L318-L325
35. Mulvany, M. J., and Halpern, W. (1977) Circ. Res. 41, 19-26[Medline] [Order article via Infotrieve]
36. Kirber, M. T., Etter, E. F., Bellve, K. A., Lifhitz, L. M., Tuft, R. A., Fay, F. S., Walsh, J. V., and Fogarty, K. E. (2001) J. Physiol. (Lond.) 531, 315-327[Abstract/Free Full Text]
37. Walseth, T. F., and Lee, H. C. (1993) Biochim. Biophys. Acta 1178, 235-242[Medline] [Order article via Infotrieve]
38. Iino, M., Kobayashi, T., and Endo, M. (1988) Biochem. Biophys. Res. Com. 152, 417-422[Medline] [Order article via Infotrieve]
39. Golovina, V. A., and Blaustein, M. P. (1996) Science 275, 1643-1648
40. Tribe, R. M., Borin, M. L., and Blaustein, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5908-5912[Abstract]
41. Janiak, R., Wilson, S. M., Montague, S., and Hume, J. R. (2001) Am. J. Physiol. 280, C22-C33
42. Lee, S. H., and Earm, Y. E. (1994) Pfluegers Arch. Eur. J. Physiol. 426, 189-198[Medline] [Order article via Infotrieve]
43. Evans, A. M., Osipenko, O. N., and Gurney, A. M. (1996) J. Physiol. (Lond.) 496, 407-420[Abstract]
44. Bae, Y. M., Park, M. K., Lee, S. H., Ho, W. K., and Earm, Y. E. (1999) J. Physiol. (Lond.) 514, 747-758[Abstract/Free Full Text]
45. Li, P. L., Zhang, D. X., Ge, Z. D., and Campbell, W. B. (2002) Am. J. Physiol. 282, H1229-H1236
46. Geiger, J., Zou, A. P., Campbell, W. B., and Li, P. L. (2000) Hypertension 35, 397-402[Abstract/Free Full Text]
47. Li, P. L., Zou, A. P., and Campbell, W. B. (1998) Am. J. Physiol. 275, H1002-H1010[Medline] [Order article via Infotrieve]
48. Coussin, F., Macrez, N., Morel, J. L., and Mironneau, J. (2000) J. Biol. Chem. 275, 9596-9603[Abstract/Free Full Text]
49. Mironneau, J., Coussin, F., Jeyakumar, L. H., Fleisher, S., Mironneau, C., and Macrez, N. (2001) J. Biol. Chem. 276, 11257-11264[Abstract/Free Full Text]
50. Hermann-Frank, A., Darling, E., and Meissner, G. (1991) Pfluegers Arch. Eur. J. Physiol. 418, 353-359[Medline] [Order article via Infotrieve]
51. Neylon, C. B., Richards, S. M., Larsen, M. A., Agrotis, A., and Bobik, A. (1995) Biochem. Biophys. Res. Commun. 215, 814-821[CrossRef][Medline] [Order article via Infotrieve]
52. Colledge, M., and Scott, J. D. (1999) Trends Cell Biol. 9, 216-221[CrossRef][Medline] [Order article via Infotrieve]
53. Pawson, T., and Scott, J. D. (1997) Science 278, 2075-2080[Abstract/Free Full Text]
54. Rubin, C. S. (1994) Biochim. Biophys. Acta 1224, 467-479[Medline] [Order article via Infotrieve]


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