Cyclic ADP-Ribose Is the Primary Trigger for Hypoxic Pulmonary Vasoconstriction in the Rat Lung In Situ
Abstract—Hypoxic pulmonary vasoconstriction (HPV) is unique to pulmonary arteries, and it aids ventilation/perfusion matching. However, in diseases such as emphysema, HPV can promote hypoxic pulmonary hypertension. We recently showed that hypoxia constricts pulmonary arteries in part by increasing cyclic ADP-ribose (cADPR) accumulation in the smooth muscle and, thereby, Ca2+ release by ryanodine receptors. We now report on the role of cADPR in HPV in isolated rat pulmonary arteries and in the rat lung in situ. In isolated pulmonary arteries, the membrane-permeant cADPR antagonist, 8-bromo-cADPR, blocked sustained HPV by blocking Ca2+ release from smooth muscle ryanodine-sensitive stores in the sarcoplasmic reticulum. Most importantly, we showed that 8-bromo-cADPR blocks HPV induced by alveolar hypoxia in the ventilated rat lung in situ. Inhibition of HPV was achieved without affecting (1) constriction by membrane depolarization and voltage-gated Ca2+ influx, (2) the release (by hypoxia) of an endothelium-derived vasoconstrictor, or (3) endothelium-dependent vasoconstriction. Our findings suggest that HPV is both triggered and maintained by cADPR in the rat lung in situ.
Since it was first described, hypoxic pulmonary vasoconstriction (HPV) has been recognized as the critical and distinguishing characteristic of pulmonary arteries1 ; systemic arteries dilate in response to hypoxia. Physiologically, HPV contributes to ventilation-perfusion matching in the lung. However, when alveolar hypoxia is global, as it is in disease states such as emphysema and cystic fibrosis, it results in pulmonary hypertension and, eventually, right heart failure.2 In isolated pulmonary arteries, HPV is biphasic. A transient constriction (phase 1) is followed by slow tonic constriction (phase 2). It was thought that phase 1 was initiated by a reduction in membrane K+ conductance in the smooth muscle3 4 and voltage-gated Ca2+ influx.5 6 In addition, the primary mediator of phase 2 of HPV was thought to be an endothelium-derived vasoconstrictor.7 8 Contrary to this, we and others discovered that hypoxia promotes phases 1 and 2 by releasing Ca2+ from ryanodine-sensitive stores in the sarcoplasmic reticulum (SR) by a mechanism intrinsic to the smooth muscle.9 10 11
Recently, we showed that the β-NAD+ metabolite cyclic ADP-ribose (cADPR),12 13 which increases Ca2+ release by ryanodine receptors,14 plays a role in this process. Thus, hypoxia increases cADPR accumulation and SR Ca2+ release in pulmonary artery smooth muscle, leading to constriction in isolated rabbit pulmonary arteries.15
In the present investigation, we demonstrate that HPV is triggered and maintained by cADPR in isolated rat pulmonary arteries and in the rat lung in situ.
Materials and Methods
Male Wistar rats (250 to 350 g) were anesthetized with 4% enflurane and exsanguinated. The heart and lungs were removed and placed in chilled physiological saline solution A containing (in mmol/L): 118 NaCl, 4 KCl, 24 NaHCO3, 1 MgSO4, 1.2 NaH2PO4, 2 CaCl2, and 5.56 glucose (pH 7.4). Pulmonary arteries were dissected free and used immediately.
Small Vessel Myography
Third-order branches of the pulmonary artery (internal diameter, 300 to 400 μm; 2 to 3 mm in length) were mounted on the jaws of a myograph (AM10, Cambustion Biological) using 50 μm tungsten wire. Initial tension was equivalent to 30 mm Hg, and the chamber (8 mL) was maintained at 37±1°C. The technique, protocol, and theory have been described previously.16 The endothelium was removed by rubbing the intima with braided silk surgical thread. Endothelium removal was confirmed by the failure of 100 μmol/L acetylcholine to relax constriction by 1 μmol/L prostaglandin F2α (PGF2α).
Arteries were constricted by high K+ (75 mmol/L) before and after each experiment to test the responsiveness and stability of the preparation and to give a standard response for comparative studies (9±3 mN/mm; n=42). Resting tension was taken to be zero. Experimental chambers were covered and bubbled (150 mL/min) with normoxic gas at 154 to 160 Torr (75% N2, 20% O2, and 5% CO2). When required, we switched to hypoxic gas at 47 to 52 Torr (89% N2, 6% O2, and 5% CO2) or 16 to 21 Torr (93% N2, 2% O2, and 5% CO2). Gas was supplied by a gas-mixing flowmeter (Cameron Instruments). All drugs were applied to the bath directly. All solutions were warmed to 37°C and bubbled with 5% CO2 to maintain pH 7.4.
Male Wistar rats (250 to 350 g) were anesthetized by 4% enflurane, injected with 1000 IU of heparin intravenously, and killed by exsanguination (approved by Home Office). A polyethylene catheter was inserted into the pulmonary artery through the right ventricle and tied in place. Another catheter was inserted into the left atrium. The carcass was placed on a plexiglas sheet over a water bath (38°C). The lung was perfused (0.06 mL · min–1 · g–1) with 10 mL of physiological saline solution B containing (in mmol/L): 118 NaCl, 4 KCl, 1.2 NaH2PO4, 1 MgSO4, 24 NaHCO3, 2 CaCl2, and 5.56 glucose and 4% Ficoll (albumin substitute) at pH 7.4. Pulmonary vascular perfusion pressure was measured by a pressure transducer that was located on a side arm of the pulmonary arterial catheter. From the left atrial catheter, the venous outflow entered a reservoir and was recirculated. The lungs were inflated (by a tracheotomy) 30 times per minute with normoxic gas (75% N2, 20% O2, and 5% CO2). When required, we switched to hypoxic gas containing either 6% O2 (balanced with 89% N2 and 5% CO2) or 2% O2 (balanced with 93% N2 and 5% CO2). Gas was supplied by a gas-mixing flowmeter (Cameron Instruments). Maximum ventilation pressure was <15cm H2O.
All compounds were from Sigma. 8-Bromo-cADPR and caffeine were dissolved in distilled water or physiological saline solution. Ryanodine was dissolved in DMSO. The minimum dilution of DMSO was 1:10000, which had no effect on the arteries.
HPV Is Abolished by Ryanodine and Caffeine
Isolated pulmonary artery rings constricted biphasically by hypoxia (16 to 21 Torr; Figure 1A⇓). Phase 1 peaked after 3 to 5 minutes at 63±7% of the constriction by 75 mmol/L K+ (n=4), and it then declined back to a level above pretone. Phase 2 of HPV then developed to a maximum of 39±9% (n=4) after 40 minutes of hypoxia. Removal of the endothelium had no effect on phase 1 of HPV. However, during phase 2, the progressive rise in tension was lost (Figure 1B⇓), leaving a maintained plateau constriction that measured 13±4% (n=4).
Whether the endothelium was present (Figure 1C⇑) or absent (Figure 1D⇑), preincubation with caffeine (10 mmol/L) and ryanodine (10 μmol/L) abolished the constriction by hypoxia (16 to 21 Torr). In contrast, in the presence of ryanodine, caffeine, and hypoxia, the constriction by 75 mmol/L K+ was similar to control (9±2 mN/mm in the presence of the endothelium and 10±2 mN/mm in its absence; n=4). Thus, hypoxia initiates and maintains acute HPV in isolated rat pulmonary arteries by triggering Ca2+ release from ryanodine-sensitive SR stores.
8-Bromo-cADPR Blocks Phase 2 but Not Phase 1 of HPV
The effect of a membrane-permeant cADPR antagonist, 8-bromo-cADPR,17 was different from that of caffeine or ryanodine. Figure 2A⇓ shows the constriction of an intact pulmonary artery ring by hypoxia (16 to 21 Torr). Phase 1 peaked at 62±6% and phase 2 measured 43±7% after 40 minutes (n=6). Figure 2B⇓ shows the response in the absence of the endothelium. Phase 1 peaked at 59±7% and the plateau constriction measured 13±2% after 40 minutes (n=6). Figure 2C⇓ shows the effect of preincubating (10 minutes) an intact pulmonary artery with 300 μmol/L 8-bromo-cADPR. Phase 1 was unaltered at 63±8%. In contrast, phase 2 was abolished (n=6). The effect of 300 μmol/L 8-bromo-cADPR was similar in de-endothelialized artery rings (Figure 2D⇓). Phase 1 measured 61±6%, and the plateau constriction was abolished. This suggests that cADPR-dependent SR Ca2+ release maintains acute HPV in isolated rat pulmonary artery smooth muscle.
However, full development of phase 2 of HPV requires the release of an endothelium-derived vasoconstrictor.7 Thus, 8-bromo-cADPR may also have inhibited the release or action of the vasoconstrictor. Because 8-bromo-cADPR had no effect on constriction by K+, we were able to test this possibility. Arteries were preconstricted with 20 mmol/L K+ to a level (2±0.8 mN/mm; n=6) equivalent to the endothelium-independent, 8-bromo-cADPR-sensitive plateau constriction by hypoxia (2±0.6 mN/mm; n=6). Under these conditions and with the endothelium (Figure 2E⇑), phase 1 peaked at 51±5% and phase 2 peaked at 40±6%, inclusive of the constriction by 20 mmol/L K+ (n=4). Thus, phases 1 and 2 of HPV per se were slightly smaller when evoked in the presence of K+–induced preconstriction. In the absence of the endothelium (Figure 2F⇑), phase 1 measured 50±4% and the plateau constriction measured 24±4%, inclusive of the constriction by 20 mmol/L K+ (n=4). Figure 2G⇑ shows the effect of 300 μmol/L 8-bromo-cADPR on constriction by hypoxia (16 to 21 Torr) in an intact artery after preconstriction with 20 mmol/L K+. Phase 1 measured 52±6%, inclusive of the constriction by 20 mmol/L K+. Strikingly, the slow tonic constriction associated with phase 2 HPV was also observed; it measured 38±5% after 40 minutes. Figure 2H⇑ shows the effect of 300 μmol/L 8-bromo-cADPR on HPV in a de-endothelialized artery preconstricted with 20 mmol/L K+. Phase 1 measured 59±8%, inclusive of the constriction by 20 mmol/L K+. In contrast, no plateau constriction by hypoxia was observed over and above the constriction by 20 mmol/L K+. Therefore, 8-bromo-cADPR blocks SR Ca2+ release to hypoxia in the smooth muscle, but it does not block the release or action of the endothelium-derived vasoconstrictor(s).
Figure 3⇓ shows that when the level of hypoxia was reduced from 16 to 21 Torr to 47 to 52 Torr, the phase 1 constriction was reduced from 61±6% to 31±5%, respectively (n=4). The phase 2 constriction was also reduced from 43±6% to 22±4%, respectively (n=4). Preincubation with 8-bromo-cADPR (300 μmol/L) had no effect on the phase 1 constriction by 47 to 52 Torr hypoxia, which measured 29±5% (n=4), and the phase 2 constriction was abolished as before (Figure 3⇓).
Cyclopiazonic Acid Blocks Phase 1 but Not Phase 2 of HPV
The fact that phase 1 of HPV remained unaffected in the presence of 8-bromo-cADPR but was abolished by ryanodine and caffeine suggested the involvement of a mechanism independent of cADPR but dependent on ryanodine-sensitive SR Ca2+ stores. Therefore, we investigated the possibility that hypoxia triggered phase 1 of HPV by inhibiting SR Ca2+ ATPase activity, leading to an increase in the net SR Ca2+ efflux. We used the selective Ca2+ ATPase antagonist cyclopiazonic acid. Figure 4A⇓ shows constriction by hypoxia in an intact artery. Phase 1 peaked at 60±7% and phase 2 measured 39±4% after 40 minutes (n=6). Figure 4B⇓ shows constriction by hypoxia in an artery without the endothelium. Phase 1 peaked at 59±7% and the residual phase 2 plateau measured 14±4% after 40 minutes (n=6). Preincubation (10 minutes) with 10 μmol/L cyclopiazonic acid abolished phase 1 of HPV in the presence (Figure 4C⇓) and absence (Figure 4D⇓) of the endothelium (n=4).
In contrast to ryanodine and caffeine (Figure 1C⇑) and to 8-bromo-cADPR (Figure 2C⇑), cyclopiazonic acid had no effect on phase 2 of HPV. In intact arteries after 40 minutes of hypoxia, phase 2 measured 39±8% in the absence and 39±6% in the presence of 10 μmol/L cyclopiazonic acid (Figures 4A⇑ and 4C⇑; n=4). In the absence of the endothelium, the plateau constriction measured 13±4% in the absence and 13±4% in the presence of 10 μmol/L cyclopiazonic acid (Figures 4B⇑ and 4D⇑; n=4). Thus, inhibition of SR Ca2+ ATPase activity with cyclopiazonic acid has no effect on the induction or magnitude of phase 2 of HPV in pulmonary artery rings.
8-Bromo-cADPR Inhibits HPV in the Rat Lung In Situ
From the findings described above, 3 components of HPV in isolated pulmonary arteries can be separated pharmacologically: the constriction mediated by inhibition of SR Ca2+ ATPase (component 1), by cADPR-dependent Ca2+ release from ryanodine-sensitive SR stores in the smooth muscle (component 2), and by an endothelium-derived vasoconstrictor (component 3). Because components 1 and 3 were insensitive to 8-bromo-cADPR, we were able to investigate the contribution of cADPR to HPV in the perfused and ventilated rat lung in situ. Figure 5A⇓ shows that perfusion pressure increased from 7±1 mm Hg (n=6) with alveolar normoxia (20% O2) to 14±1 mm Hg (n=6) with alveolar hypoxia (2% O2). On return to normoxia, the perfusion pressure declined to 8±1 mm Hg (n=4), after which a second exposure to alveolar hypoxia (2% O2) increased the perfusion pressure to 15±1 mm Hg (n=6). The increase in perfusion pressure was dependent on the degree of alveolar hypoxia. Figures 5B⇓ and 5C⇓, respectively, show that the perfusion pressure increased from 7±1 mm Hg to 14±1 mm Hg when the alveoli were supplied with 2% O2, and from 7±1 mm Hg to 11±1 mm Hg with 6% O2 (n=4). The increase in perfusion pressure with 2% and 6% O2, respectively, was abolished by preincubation (10 minutes) with 300 μmol/L 8-bromo-cADPR (Figures 5B⇓ and 5C⇓; n=4).
Concentration-Dependence and Selectivity of 8-Bromo-cADPR
Figure 6A⇓ shows the effect of cumulative application of 8-bromo-cADPR (1 to 100 μmol/L) on the plateau constriction by hypoxia (16 to 21 Torr) in an isolated pulmonary artery ring without the endothelium (ie, the constriction maintained by Ca2+ release from ryanodine-sensitive SR stores). The plateau constriction was inhibited with a threshold for inhibition of 3 μmol/L, and complete reversal was obtained with 100 μmol/L 8-bromo-cADPR (n=4). Figure 6B⇓ shows that cumulative application of 8-bromo-cADPR (1 to 300 μmol/L) inhibited the increase in perfusion pressure by alveolar hypoxia (2% O2) in the rat lung with a threshold of 30 μmol/L, and complete reversal with 300 μmol/L 8-bromo-cADPR (n=4). Figure 6C⇓ shows the concentration-inhibition curves for each preparation. The IC50 for inhibition of HPV in isolated arteries and in the rat lung was 30 μmol/L and 55 μmol/L, respectively.
Curiously, preincubation with 8-bromo-cADPR blocked the initiation of HPV in an all-or-none manner but with a clear threshold concentration. In intact pulmonary artery rings (Figure 7A⇓), phase 1 of HPV measured 62±6% in the absence and 64±6% in the presence of 1 μmol/L 8-bromo-cADPR. Phase 2 measured 42±5% in the absence and 40±5% in the presence of 1 μmol/L 8-bromo-cADPR. In contrast, after preincubation with 3 μmol/L 8-bromo-cADPR (Figure 7B⇓) phase 1 measured 61±5% (compared with 63±5% in its absence) and phase 2 (40±4% in absence) was abolished (n=4).
In the perfused and ventilated rat lung, hypoxia (2% O2) increased perfusion pressure from 7±1 mm Hg to 15±1 mm Hg before and from 7±1 mm Hg to 14±1 mm Hg after preincubation (10 minutes) with 3 μmol/L 8-bromo-cADPR (Figure 7C⇑). In marked contrast, the increase in perfusion pressure by hypoxia, from 7±1 mm Hg to 14±1 mm Hg, was abolished after preincubation (10 minutes) with 10 μmol/L 8-bromo-cADPR (Figure 7D⇑; n=4). The block of HPV by 8-bromo-cADPR was not reversed on washing (≤2 hours) in either preparation (Figures 7B⇑ and 7D).
The effect of 8-bromo-cADPR was selective for HPV over vasoconstriction by K+ and PGF2α, respectively. Figure 8A⇓ shows the constriction of an intact pulmonary artery ring by 20 mmol/L K+ (4±0.6 mN/mm) and by 75 mmol/L K+ (10±1 mN/mm). After preincubation (10 minutes) with 300 μmol/L 8-bromo-cADPR, constriction by 20 and 75 mmol/L K+ measured 4±0.3 and 10±1 mN/mm, respectively (n=4). Figure 8B⇓ shows constriction of an intact artery ring by 1 μmol/L PGF2α (3±0.5 mN/mm) and by 3 μmol/L PGF2α (8±1 mN/mm). After preincubation (10 minutes) with 300 μmol/L 8-bromo-cADPR, constriction by 1 and 3 μmol/L PGF2α remained unaffected, at 3±0.7 and 8±1 mN/mm, respectively (n=4).
Findings in the rat lung in situ were similar. Figure 8D⇑ shows the increase in perfusion pressure by 20 mmol/L K+ (from 7±1 to 10±1 mm Hg) and 75 mmol/L K+ (from 7±1 to 19±2 mm Hg). After preincubation (10 minutes) with 300 μmol/L 8-bromo-cADPR, 20 mmol/L K+ increased perfusion pressure from 7±1 to 10±1 mm Hg, and 75 mmol/L K+ increased perfusion pressure from 7±1 to 19±2 mm Hg (n=4). Figure 8B⇑ shows the increase in perfusion pressure by 1 μmol/L PGF2α (from 7±1 to 10±1 mm Hg) and by 3 μmol/L PGF2α (from 7±1 to 17±1 mm Hg). Preincubation (10 minutes) with 300 μmol/L 8-bromo-cADPR had no effect, because 1 and 3 μmol/L PGF2α increased perfusion pressure from 7±0.4 to 10±1 mm Hg and from 7±1 to 17±1 mm Hg, respectively (n=4).
Previously, we showed that the level of enzyme activities for the synthesis and metabolism of cADPR in pulmonary artery smooth muscle is inversely related to artery diameter,15 as is the magnitude of the hypoxic constriction.18 We also showed that hypoxia increases cADPR accumulation in pulmonary artery smooth muscle,15 leading to SR Ca2+ release and constriction.15
To advance our proposal that cADPR promotes HPV, we further investigated the effect of 8-bromo-cADPR, a membrane-permeant cADPR antagonist, on acute HPV in isolated rat pulmonary arteries and in the perfused and ventilated rat lung in situ.
We first showed that phases 1 and 2 of HPV in isolated rat pulmonary arteries were abolished after depletion of ryanodine-sensitive SR Ca2+ stores with ryanodine and caffeine. In contrast, phase 1 of HPV remained unaffected in the presence of 8-bromo-cADPR. Thus, a cADPR-independent O2-sensing mechanism must initiate ryanodine-sensitive SR Ca2+ release during phase 1 of HPV.9 15 Conversely, cyclopiazonic acid abolished phase 1 of HPV. This suggests that phase 1 of HPV in isolated arteries is mediated by the inhibition of SR Ca2+ ATPase activity, yielding net Ca2+ efflux from SR stores. When taken together with the fact that removing extracellular Ca2+ has no effect on the phase 1 constriction,9 this finding brings into question the proposal that phase 1 of HPV relies heavily on the activation, by hypoxia, of capacitative Ca2+ entry in the smooth muscle.19
The maintained constriction associated with phase 2 of HPV in intact and de-endothelialized pulmonary artery rings was blocked by ryanodine, caffeine, and 8-bromo-cADPR. In contrast, it remained unaltered in the presence of the SR Ca2+ ATPase antagonist cyclopiazonic acid. Thus, HPV in isolated rat pulmonary arteries must be maintained by cADPR-dependent SR Ca2+ release alone and not by inhibition of SR Ca2+ ATPase.
When the endothelium was present, phase 2 of HPV in intact pulmonary artery rings was also abolished by 8-bromo-cADPR, but it was recovered when arteries were preconstricted with K+ (ie, by voltage-gated Ca2+ influx). In marked contrast, block by 8-bromo-cADPR of the plateau constriction by hypoxia in pulmonary arteries without the endothelium was not reversed by K+-induced preconstriction. Thus, we can conclude with some confidence that 8-bromo-cADPR blocked phase 2 of HPV in isolated arteries by inhibiting Ca2+ release from ryanodine-sensitive SR stores in the smooth muscle and that it did so without affecting (1) depolarization-induced Ca2+ influx or constriction by Ca2+ per se, (2) the release of the endothelium-derived vasoconstrictor, or (3) the increase in myofilament Ca2+ sensitivity promoted by the released vasoconstrictor.8 20 The release of physiological concentrations of the endothelium-derived vasoconstrictor during phase 2 of HPV7 8 18 20 is therefore unable to induce sufficient myofilament Ca2+ sensitization8 20 to promote constriction in isolated pulmonary arteries in the absence of maintained cADPR-dependent SR Ca2+ release.
These findings suggest that there are at least 3 discrete components to HPV in isolated rat pulmonary arteries: (1) inhibition of the smooth muscle SR Ca2+ ATPase, (2) activation by cADPR of SR Ca2+ release from ryanodine-sensitive SR stores, and (3) the release of endothelium-derived vasoconstrictor(s). Furthermore, we showed that 8-bromo-cADPR was without effect on components 1 and 3, but it abolished component 2. Therefore, we were able to investigate the relative importance of these key processes in triggering and maintaining HPV in the perfused and ventilated rat lung in situ. When added to the perfusate at concentrations ≥10 μmol/L, 8-bromo-cADPR blocked the initiation of acute HPV. Thus, cADPR-dependent SR Ca2+ release seems to be the primary trigger for acute HPV in the rat lung. Because phase 1 of HPV in isolated arteries remains unaffected in the presence of 8-bromo-cADPR, we can conclude that the mechanisms underpinning this phase of constriction (ie, inhibition of the SR Ca2+ ATPase) do not contribute greatly to HPV in the lung. It is surprising, therefore, that inhibition of the SR Ca2+ ATPase by hypoxia triggers such a pronounced constriction in isolated pulmonary arteries. One explanation for this could be that the change in O2 tension around the artery is faster in the myograph chamber than it is when associated with alveolar hypoxia in the lung. As a result, the former but not the latter may induce a transient fall in smooth muscle ATP levels,21 possibly due to delayed accommodation with respect to the energy state of the smooth muscle.
Our findings in isolated arteries also showed that 8-bromo-cADPR had no effect on the release or action of the endothelium-derived vasoconstrictor(s). Therefore, we can conclude that the release of physiological concentrations of the vasoconstrictor by hypoxia is unable to promote HPV in the lung in the absence of cADPR-dependent SR Ca2+ release in the smooth muscle of pulmonary arteries.
Previous studies have shown that nitric oxide production by the endothelium may first increase and then decline in response to hypoxia and that nitric oxide synthase inhibitors augment acute HPV.22 23 24 25 26 However, in the presence of 8-bromo-cADPR, hypoxia had no influence on resting tension in isolated arteries and had no effect on resting perfusion pressure in the rat lung. Nitric oxide may therefore act as a secondary modulator of HPV.
Our proposals are supported by the fact that, once triggered, HPV in the lung was completely reversed by concentrations of 8-bromo-cADPR that were unable to block phase 1 of HPV or the endothelium-dependent component of phase 2 of HPV in isolated arteries. Further support comes from the fact that the IC50 (55 μmol/L) for inhibition by 8-bromo-cADPR of HPV in the lung closely matched the IC50 (30 μmol/L) for inhibition by 8-bromo-cADPR of the ryanodine-sensitive plateau constriction by hypoxia in isolated arteries without the endothelium. The small difference between the measured IC50 in each preparation is likely due to differences in pharmacokinetics.
Surprisingly, 8-bromo-cADPR was able to block the initiation of HPV in isolated pulmonary arteries and in the rat lung at concentrations an order of magnitude lower than the concentrations required to completely reverse HPV after it had been initiated. This is all the more intriguing because, in contrast to its concentration-dependent reversal of HPV, preincubation with 8-bromo-cADPR blocked the initiation of HPV in an all-or-none manner but with a clear threshold concentration. This all-or-none block is reminiscent of the block by α-bungarotoxin of transmission at the neuromuscular junction. Thus, skeletal muscle fibers respond maximally to nerve stimulation when 45% of the nicotinic acetylcholine receptors are blocked. However, paralysis develops rapidly once any more than 45% of receptors are blocked.27 A similar “margin of safety” may be built into HPV, such that in pulmonary artery smooth muscle, a certain proportion of ryanodine receptors must be activated by cADPR to breach a given threshold for the initiation of a regenerative, global Ca2+ wave in the smooth muscle and, therefore, constriction. It would follow that activation by cADPR of fewer ryanodine receptors than is required to breach this threshold would be insufficient to trigger HPV. However, more detailed investigations will be required to confirm this proposal.
Finally, it is interesting to note that previous studies have found that the constriction by low concentrations of PGF2α can be inhibited by depletion of ryanodine-sensitive SR stores,9 although 8-bromo-cADPR had no effect on constriction by PGF2α or by K+ in the present study. It seems likely, therefore, that PGF2α may, unlike hypoxia, access ryanodine-sensitive SR Ca2+ stores through a cADPR-independent mechanism (eg, calcium-induced calcium release by ryanodine receptors or IP3 induced SR Ca2+ release).
In conclusion, we propose that cADPR acts as the primary trigger for acute HPV in isolated rat pulmonary arteries and in the rat lung in situ. Our findings argue against a significant role for membrane depolarization and voltage-gated Ca2+ influx3 4 5 6 in acute HPV. The development of selective cADPR antagonists may therefore provide new and important therapeutic agents for the treatment of hypoxic pulmonary hypertension.
This work was supported by the Wellcome Trust. Dr A. Mark Evans is a Wellcome Trust Nonclinical Lecturer.
Original received December 21, 2000; revision received April 16, 2001; accepted May 25, 2001.
- © 2001 American Heart Association, Inc.
Von Euler US, Liljestrand G. Observations on the pulmonary arterial blood pressure in the cat. Acta Physiol Scand. 1946;12:301–320.
Voelkel NF. Mechanisms of hypoxic pulmonary vasoconstriction. Am Rev Respir Dis. 1986;133:1186–1195.
Post J, Hume J, Archer S, et al. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am J Physiol. 1992;262:C882–C890.
Osipenko ON, Evans AM, Gurney AM. A novel O2-sensing potassium channel may mediate hypoxic pulmonary vasoconstriction. Br J Pharmacol. 1997;120:1461–1470.
Cornfield D, Stevens T, McMurty I, Abman S, Rodman D. Acute hypoxia causes membrane depolarization and calcium influx in fetal pulmonary artery smooth muscle cells. Am J Physiol. 1993;265:L53–L56.
Salvaterra C, Goldman W. Acute hypoxia increases cytosolic calcium in cultured pulmonary arterial myocytes. Am J Physiol. 1993;264:L323–L328.
Kovitz KL, Aleskowitch JT, Sylvester JT, Flavahan NA. Endothelium-derived contracting and relaxing factors contribute to hypoxic responses of pulmonary arteries. Am J Physiol. 1993;260:L516–L521.
Robertson TP, Aaronson PI, Ward JPT. Hypoxic vasoconstriction and intracellular Ca2+ in pulmonary arteries: evidence for PKC-independent Ca2+ sensitization. Am J Physiol. 1995;268:H301–H307.
Dipp M, Nye PCG, Evans AM. Hypoxia induces sustained sarcoplasmic reticulum calcium release in rabbit pulmonary artery smooth muscle in the absence of calcium influx. Am J Physiol. In press.
Jabr RI, Toland H, Gelband CH, Wang XX, Hume JR. Prominent role of intracellular Ca2+ release in hypoxic vasoconstriction of canine pulmonary artery. Br J Pharmacol. 1997;122:21–30.
Gelband CH, Gelband H. Ca2+ release from intracellular stores is an initial step in hypoxic pulmonary vasoconstriction of rat pulmonary artery resistance vessels. Circulation. 1997;96:3647–3654.
Lee HC, Walseth TF, Bratt GT, Hayes RN, Clapper DL. Structural determination of a cyclic metabolite of NAD with intracellular calcium-mobilizing activity. J Biol Chem. 1989;264:1608–1615.
Walseth T, Aarhus R, Zeleznikar R, Lee HC. Determination of endogenous levels of cyclic ADP-ribose in rat tissues. Biochem Biophys Acta. 1991;1094:113–120.
Galione A, Lee HC, Busa WB. Ca2+-induced Ca2+ release in sea urchin egg homogenates: modulation by cyclic ADP-ribose. Science. 1991;253:1143–1146.
Wilson HL, Dipp M, Thomas JM, Lad C, Galione A, Evans AM. ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor: a primary role for cADPR in hypoxic pulmonary vasoconstriction. J Biol Chem. 2000;276:11180–11188.
Mulvany MJ, Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res. 1977;41:19–26.
Sethi JK, Empson RM, Bailey VC, Potter BV, Galione A. 7-Deaza-8-bromo-cyclic ADP-ribose, the first membrane-permeant, hydrolysis-resistant cyclic ADP-ribose antagonist. J Biol Chem. 1997;272:16358–16363.
Leach RM, Robertson TP, Twort CHC, Ward JPT. Hypoxic vasoconstriction in rat pulmonary and mesenteric arteries. Am J Physiol. 1994;266:L223–L231.
Robertson TP, Hague D, Aaronson PI, Ward JPT. Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat. J Physiol. 2000;525:669–680.
Robertson TP, Dipp M, Ward JPT, Aaronson PI, Evans AM. Inhibition of sustained hypoxic pulmonary vasoconstriction by Y-27632 in isolated intrapulmonary arteries and perfused lung of the rat. Br J Pharmacol. 2000;131:5–9.
Leach RM, Sheehan DW, Chacko VP, Sylvester JT. Energy state, pH, and vasomotor tone during hypoxia in precontracted pulmonary and femoral arteries. Am J Physiol. 2000;278:L294–L304.
Nye PCG, Robertson BA, Warren H. Inhibition of nitric oxide production enhances hypoxic pulmonary vasoconstriction of the isolated rat lung. J Physiol. 1990;422:94P.
Brashers VL, Peach MJ, Rose CE Jr. Augmentation of hypoxic pulmonary vasoconstriction in the isolated perfused rat lung by in vitro antagonists of endothelium-dependent relaxation. J Clin Invest. 1988;82:1495–1502.
Johns RA, Linden JM, Peach MJ. Endothelium-dependent relaxation and cyclic GMP accumulation in rabbit pulmonary artery are selectively impaired by moderate hypoxia. Circ Res. 1993;65:1508–1515.
Shaul PW, Wells LB. Oxygen modulates nitric oxide production selectively in fetal pulmonary endothelial cells. Am J Respir Cell Mol Biol. 1994;11:432–438.
Higenbottam T, Cremona G. Acute and chronic hypoxic pulmonary hypertension. Eur Respir J. 1993;6:1207–121.
Colquhoun D. Mechanisms of drug action at the voluntary muscle endplate. Annu Rev Pharmacol. 1975;15:307–325.