2 University Department of Pharmacology, Oxford University, Oxford OX1 3QT; and 1 University Laboratory of Physiology, Oxford University, Oxford OX1 3PT, United Kingdom
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ABSTRACT |
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The hypoxic constriction of isolated pulmonary vessels is composed of an initial transient phase (phase 1) followed by a slowly developing increase in tone (phase 2). We investigated the roles of the endothelium and of intracellular Ca2+ stores in both preconstricted and unpreconstricted intrapulmonary rabbit arteries when challenged with hypoxia (PO2 16-21 Torr). Removing the endothelium did not affect phase 1, but phase 2 appeared as a steady plateau. Removing extracellular Ca2+ had essentially the same effect as removing the endothelium. Depletion of sarcoplasmic reticulum Ca2+ stores with caffeine and ryanodine abolished the hypoxic response. Omitting preconstriction reduced the amplitude of the hypoxic response but did not qualitatively affect any of the above responses. We conclude that hypoxia releases intracellular Ca2+ from ryanodine-sensitive stores by a mechanism intrinsic to pulmonary vascular smooth muscle without the need for Ca2+ influx across the plasmalemma or an endothelial factor. Our results also suggest that extracellular Ca2+ is required for the release of an endothelium-derived vasoconstrictor.
hypoxic pulmonary vasoconstriction; ryanodine; caffeine; rabbit; isolated arteries
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INTRODUCTION |
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HYPOXIA (50 Torr)
constricts pulmonary blood vessels. Hypoxic pulmonary vasoconstriction
(HPV), which tends to divert blood flow away from poorly ventilated
parts of the lung, is the critical and distinguishing characteristic of
the pulmonary circulation because hypoxia relaxes systemic arteries.
However, when alveolar hypoxia is global, e.g., at high altitude or in
diseases such as cystic fibrosis and emphysema, HPV results in
pulmonary hypertension that may lead to pulmonary edema and right heart
failure (33). Although the mechanism underlying HPV
remains obscure, several of its characteristics are well described; for
example, hypoxia constricts isolated small pulmonary arteries
biphasically. An initial transient constriction (phase
1) is followed by a slowly developing and sustained increase in
tone (phase 2) (1, 11, 14, 15, 22, 35).
Phase 1 occurs independently of the endothelium and is
widely thought to be initiated by depolarization of the smooth muscle
cell membrane (19, 21, 39) and Ca2+ influx via
voltage-gated Ca2+ channels (4, 8, 16, 17, 26,
29). In contrast, the full expression of phase 2 requires the release of a vasoconstrictor from the intact endothelium
(14, 15) and may develop at a constant concentration of
free cytoplasmic Ca2+ (22).
Hypoxia also releases Ca2+ from intracellular stores (7, 9, 10, 14, 26, 31, 34). However, to date, little attention has been given to assessing the relative contributions of Ca2+ influx across the cell membrane and Ca2+ release from the sarcoplasmic reticulum (SR) to the two phases of HPV.
We considered three potential mechanisms by which hypoxia may trigger the release of Ca2+ from internal stores: 1) Ca2+-induced Ca2+ release after the influx of Ca2+ through the plasmalemma, 2) SR Ca2+ release evoked by an endothelial vasoconstrictor, and 3) modulation of Ca2+ release from ryanodine-sensitive SR Ca2+ stores. Our results suggest that hypoxia gives a sustained release of Ca2+ from ryanodine-sensitive stores of the SR that requires neither the influx of extracellular Ca2+ nor the presence of the endothelium. The SR may therefore be the most important, immediate source of Ca2+ to which the contractile apparatus is sensitized during phase 2 of HPV.
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METHODS |
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Dissection. Male New Zealand White rabbits (1-2 kg) were killed by cervical dislocation. The heart and lungs were removed together and placed in chilled physiological saline solution A (PSS-A; in mM: 118 NaCl, 4 KCl, 10 NaHCO3, 14 HEPES, 1 MgSO4, 1.2 NaH2PO4, 2 CaCl2, and 5.56 glucose). The pulmonary arteries were dissected out and cleaned of connective tissue. After dissection, the arteries were either used immediately or stored at 4°C in PSS-A. Storage did not alter the responses to hypoxia, KCl, or other vasoconstrictors.
Small-vessel myography. Third-order branches of the pulmonary arterial tree (ID 300-400 µm; 2-3 mm in length) were mounted onto the jaws of an automated myograph (AM10, Cambustion Biological, Cambridge, UK) with 50-µm tungsten wire. The initial tension was set to be equivalent to a pulmonary arterial pressure of 30 mmHg. The technique, protocol, and theory have been described in detail previously (18). The myograph bath contained 8 ml of PSS-B (PSS-A with 24 mM NaHCO3 instead of HEPES) at 37 ± 1°C. The solution was bubbled with 75% N2, 20% O2, and 5% CO2 to maintain a pH of 7.4. For Ca2+-free experiments, CaCl2 was replaced with equimolar MgCl2, and 0.5 mM EGTA was added to the solution.
Experimental protocol.
All arteries were constricted with 75 mM KCl at the start and end of
every experiment. This was done to test their responsiveness and
stability and also to give a standard response against which other
responses could be compared. In the first series of experiments, arteries were preconstricted with 1 µM prostaglandin
F2 (PGF2
) to provide a modest degree of
pretone. For the purposes of data analysis, this level of
preconstriction was taken to be zero. Similar experiments were also
performed in the absence of a preconstrictor, and in these, resting
tension was taken to be zero. The bath chambers were covered by Perspex
hoods and bubbled at a total flow of 125 ml/min with either normoxic
(PO2 154-160 Torr; 75%
N2-20% O2-5% CO2) or hypoxic
(PO2 16-21 Torr; 93% N2-2%
O2-5% CO2) gas supplied by a gas-mixing
flowmeter (Cameron Instruments, Port Aransas, TX).
PO2 was measured with an Oxel O2
electrode and meter (WPI). All drugs were applied to the bath directly.
All solutions were warmed to 37°C and bubbled with 5%
CO2 before they were added to the bath.
Endothelium removal.
In 48 vessels, the endothelium was removed by rubbing the inner surface
with braided silk surgical thread. Removal of the endothelium was
assessed by the failure of 100 µM acetylcholine to relax
constrictions induced by 1 µM PGF2.
Smooth muscle cell isolation. Single smooth muscle cells were isolated from second- and third-order branches of the pulmonary artery with a method adapted from one described previously (3). Arterial rings (2 mm long) were placed in dissociation medium of the following composition (in mM): 110 NaCl, 5 KCl, 15 NaHCO3, 0.16 CaCl2, 2 MgCl2, 0.5 NaH2PO4, 0.5 KH2PO4, 10 glucose, 15 HEPES, 0.49 EDTA, and 10 taurine, adjusted to pH 7.0 after being bubbled with 95% air-5% CO2. The dissociation medium also contained 0.2 mg/ml of papain and 0.02% bovine serum albumin (fraction V, fatty acid and globulin free). The preparation was then stored overnight at 4°C in a refrigerator. The following day, 0.2 mM dithiothreitol (Sigma), which activates papain, was added to the solution, and the preparation was stored at room temperature (22-24°C) for 1 h. It was then transferred to enzyme-free dissociation medium, and the smooth muscle cells were isolated by trituration. Isolated cells were stored in dissociation medium in a refrigerator until required.
Ca2+ imaging. Cells were preincubated for 30 min in dissociation medium containing 5 µM fura 2-AM, 1% pluronic F-127, and 0.02% bovine serum albumin. They were then transferred to an experimental chamber (1 ml volume; open to the air) on a Leica DMIRBE inverted microscope. The cells were allowed to settle for ~10 min and were then warmed to 35 ± 1°C as they were washed for 15 min in PSS-C of the following composition (in mM): 130 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 HEPES, and 10 glucose, pH adjusted to 7.4 with NaOH. The perfusion rate was 5 ml/min. Changes in intracellular Ca2+ were monitored by assessing the fura 2 fluorescence with excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Emitted fluorescence was monitored with a Hamamatsu 4880 image intensifying charge-coupled device camera, recorded, and analyzed with Openlab imaging software (Improvision) on an Apple Macintosh 8600 personal computer. Fluorescence intensity was measured at 0.5-5 Hz, with background subtraction being carried out on-line. Changes in fura 2 fluorescence are reported as the 340- to 380-nm fluorescence ratio and as the estimated intracellular Ca2+ concentration.
Normoxic solutions were obtained by equilibrating PSS-C with 95% air-5% CO2. Hypoxic solutions were obtained by equilibrating PSS-C with 95% N2-5% CO2 (pH adjusted to 7.4) in the absence and presence of 1 mM sodium dithionite. The PO2 of the superfusate was measured close to the cell being studied with an Oxel O2 electrode and meter (WPI). In the presence of sodium dithionite, the gas pressure was adjusted to provide a stable PO2 of 12-17 Torr. This method provided a consistent and reproducible level of hypoxia.Drugs and solutions. Fura 2-AM and pluronic F-127 were obtained from Molecular Probes. All other compounds were obtained from Sigma-Aldrich-Fluka-RBI. All drugs were dissolved in distilled water, PSS-B, or PSS-C, with the exception of ryanodine, which was dissolved in DMSO. Stock solutions in DMSO were diluted at least 1:1,000. At this concentration, the vehicle alone had no effect on the responses to hypoxia or to vasoconstrictors.
Data analysis. Data were compared with a paired, two-tailed Student's t-test, and significance was assumed if P < 0.05.
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RESULTS |
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After preconstriction with PGF2, all 24 vessels
studied constricted biphasically when challenged with hypoxia (Fig.
1A). Phase 1 peaked
after 3-5 min and then declined back to a level above pretone.
Phase 2 then developed, and on return to normoxia, the
vessels relaxed rapidly to the level of preconstriction. After the
preconstrictor had been washed off, all vessels relaxed to baseline,
and all also responded vigorously to a final exposure to 75 mM KCl.
Removal of the endothelium (12 vessels) had little or no effect on
either constriction by KCl or phase 1 of the hypoxic response. Its only obvious effect appeared during phase 2 (Fig. 1B) where it prevented the progressive rise in tension
that is normally seen during this phase (P < 0.02;
Table 1). Only a plateau constriction remained.
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Hypoxia constricts pulmonary arteries in the absence of
extracellular Ca2+.
Ca2+-free PSS abolished the constriction by KCl but did not
change the constriction by PGF2 (P > 0.5; Table 1) and neither did it affect phase 1 of HPV
(P > 0.1; Table 1). However, phase 2 now
exhibited no slowly developing component (P > 0.05;
Table 1). It appeared as a sustained plateau constriction (Fig.
1C) above the level of pretone, similar to that obtained in
the presence of extracellular Ca2+ after removal of the
endothelium (Fig. 1B). After the preconstrictor was washed
off and Ca2+ was readded, a final constriction to KCl
confirmed the continued responsiveness of the preparation. Removing the
endothelium did not affect phase 1 under
Ca2+-free conditions (Fig. 1D), and phase
2 again appeared as a sustained plateau (P > 0.05 for both compared with that with endothelium; Table 1).
Depletion of SR Ca2+ stores with
caffeine and ryanodine abolished constriction to hypoxia.
In the presence of extracellular Ca2+, caffeine (10 mM) and
ryanodine (10 µM) (given 10 min before the hypoxic challenge)
abolished the preconstriction by PGF2 and abolished the
entire constriction to hypoxia both when the endothelium was intact
(Fig. 2A) and after it had
been removed (Fig. 2B). The constriction to KCl remained unaffected.
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Experiments performed in the absence of preconstriction.
To determine the influence of PGF2 on our results, we
repeated the above experiments without any preconstriction. For this,
we used a total of 12 vessels, three being exposed to each set of
conditions (for a summary, see Table 1). In these, we obtained
responses that were strikingly similar to, although generally smaller
than, those obtained in the presence of preconstriction (P < 0.05; compare Fig. 1, A and
B with Fig. 3, A and B; Table 1). A
normal biphasic hypoxic response was observed in the presence of
endothelium (Fig. 3A), and
removal of the endothelium abolished the slowly developing component of
phase 2 (Fig. 3B). It is interesting to note that
this phase 2 plateau was significantly greater than that
observed in the presence of pretone (P < 0.05; Fig.
1B, Table 1).
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Caffeine and ryanodine abolish the hypoxic increase in
intracellular Ca2+ concentration in
isolated pulmonary arterial smooth muscle cells.
We carried out Ca2+ imaging studies of isolated pulmonary
arterial smooth muscle cells to confirm that hypoxia was capable of triggering via a mechanism intrinsic to the cells Ca2+
release from ryanodine-sensitive intracellular stores. In the presence
of extracellular Ca2+, hypoxia increased the fura 2 fluorescence ratio by 27 ± 2% (Fig. 5) and also shortened the cells. Caffeine
and ryanodine (10 mM and 10 µM, respectively; the same concentrations
used on isolated vessels) induced a transient increase in intracellular
Ca2+ and abolished the increase in Ca2+ induced
by hypoxia. In the presence of caffeine and ryanodine, hypoxia
increased the fura 2 ratio by only 1.00 ± 1.80% (Fig. 5,
B and C).
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DISCUSSION |
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HPV in isolated vessels is characterized by an initial transient constriction (phase 1) followed by a slowly developing and sustained increase in tone (phase 2) (1, 2, 14, 15, 22, 35, 38). Similar biphasic responses have also been observed in some isolated lung preparations (32, 36, 37). Phase 1 is generally considered to be intrinsic to the smooth muscle cells and to result from membrane depolarization and Ca2+ influx through voltage-gated Ca2+ channels (4, 8, 16, 17, 19, 21, 26, 29, 39). In contrast, our present results and the work of others show that the full development of phase 2 occurs only when the endothelium is intact (15) and can develop in the presence of a constant cytoplasmic free Ca2+ concentration (22). However, some (2, 11) found that the development of phase 2 is endothelium independent. These contrary findings may possibly be explained by variations in protocol, e.g., the extent to which the endothelium was removed.
In addition to the above, several authors suggest that hypoxia releases Ca2+ from the SR. Thus phase 1 is blocked by thapsigargin and cyclopiazonic acid (1, 7, 10, 30, 31), and the hypoxic response can be inhibited by ryanodine receptor antagonists (7, 11, 29, 31). Furthermore, the protein kinase inhibitor HA-1004, which interferes with the intracellular actions of Ca2+, blocks HPV much more potently than drugs that block voltage-gated Ca2+ channels (5, 9). Indeed, several reports (6, 10, 11, 23, 24, 26, 38) comment on how modest the effect of Ca2+ channel blockers is on hypoxic responses, and some (7, 9, 27, 31) show at least some degree of hypoxic constriction in the absence of extracellular Ca2+. We have therefore assessed the relative contributions of transmembrane Ca2+ influx and intracellular Ca2+ release in HPV to determine whether the primary trigger of Ca2+ release from SR stores is 1) Ca2+-induced Ca2+ release dependent on extracellular Ca2+, 2) SR Ca2+ release in response to an endothelium-derived vasoconstrictor, or 3) the direct release of Ca2+ from ryanodine-sensitive SR stores. Our results support the suggestion that membrane depolarization is not the only, or indeed the primary, mechanism by which hypoxia excites pulmonary arterial smooth muscle (6, 23).
Hypoxia gives a sustained biphasic constriction in the absence of extracellular Ca2+. We have shown that small pulmonary arteries isolated from the rabbit respond to hypoxia in a way that is similar to that of other species: they give a biphasic constriction. We have also shown that removal of the endothelium has little, if any, effect on phase 1 of this response but that it markedly affects phase 2 (compare Refs. 15, 34). However, in our experiments, phase 2 of deendothelialized vessels did not decline back to the basal level of tone as has been observed by others (see Ref. 35 for a review). It fell back to a plateau level of 20-50% of the maximum phase 2 constriction observed when the endothelium was present. The biphasic response described here, with a plateau instead of a rise during phase 2, is strikingly similar to the biphasic response of intracellular Ca2+ described by Robertson et al. (22) in endothelium-intact vessels. The rising component of tension during phase 2 has been ascribed to the action of an endothelium-derived factor that sensitizes the contractile apparatus to intracellular Ca2+ (22), and our results in deendothelialized vessels are entirely consistent with this interpretation. The fact that a plateau constriction during phase 2 has not been previously reported in deendothelialized vessels may be accounted for by the fact that our vessels were very modestly preconstricted. Certainly, an excess of preconstriction, hypoxia, or initial tension all take phase 2 below baseline tension (20, 35). The use of greater amounts of preconstriction may therefore explain why others (2, 11) have not seen a plateau constriction in deendothelialized vessels during phase 2.
The similarity between the effects of buffering extracellular Ca2+ to zero and those of removing the endothelium (Figs. 1, B vs. C, and 3, B vs. C) and the fact that the hypoxic response of deendothelialized vessels is little affected by removing Ca2+ (Figs. 1, B vs. D, and 3, B vs. D) suggest that Ca2+ influx into endothelial cells may be required for hypoxia to release the endothelium-derived factor that sensitizes the smooth muscle contractile apparatus to Ca2+ during phase 2. We were surprised to observe a well-sustained plateau constriction in the absence of extracellular Ca2+ (both with and without the endothelium), for this implies either that there is sufficient steady Ca2+ release from internal stores to balance any loss of Ca2+ across the plasmalemma or that hypoxia completely shuts off Ca2+ extruders. Support for this conclusion may be derived from the fact that ryanodine and caffeine release Ca2+ from ryanodine-sensitive SR stores but do not mimic the effects of hypoxia. This may be explained by the possibility that ryanodine and caffeine deplete and block ryanodine-sensitive SR stores in intact arterial rings without raising Ca2+ sufficiently to induce constriction in intact arteries (13). The fact that we and others (7, 13) have shown that ryanodine and caffeine transiently increase Ca2+ in isolated pulmonary arterial smooth muscle cells may reflect differences in the pharmacokinetics of their action in intact arteries as opposed to those in single isolated smooth muscle cells. When we depleted ryanodine-sensitive SR stores with caffeine and ryanodine, both phases of the hypoxic response were abolished whether or not the endothelium was present. However, caffeine and ryanodine did not affect constriction by KCl in either the presence or absence of hypoxia; the block of SR Ca2+ release by ryanodine is irreversible. Thus these drugs do not interfere with the contractile apparatus, and they do not appear to interfere with depolarization-induced Ca2+ entry, yet they abolish HPV. This suggests that the primary action of hypoxia is to release Ca2+ from ryanodine-sensitive SR stores. The observation that blockers of voltage-gated Ca2+ channels reduce the hypoxic excitation of pulmonary vascular smooth muscle (4, 5, 11, 15, 17, 24, 26-28) may possibly be accounted for by the fact that these drugs may also block Ca2+ release from SR stores (12, 25). Others (11, 20, 23) have found no significant effect of caffeine with ryanodine or of ryanodine alone on the hypoxic response. However, we found that the use of pentobarbital sodium anesthesia, which was used in these investigations, compromises ryanodine receptor signaling (unpublished observations). We killed our animals by cervical dislocation and exsanguination and therefore avoided the use of anesthesia. The only important effect of omitting preconstriction was that both phases of HPV were roughly halved in amplitude (see Table 1). This demonstrates that none of the qualitative responses described here can have resulted from an indirect effect of the level of preconstriction. Also, in agreement with the findings of others (10, 31), our data show that acute hypoxia constricts pulmonary arteries, at least in part, by releasing Ca2+ from ryanodine-sensitive stores that appear to be capable of sustaining constriction for at least 30 min in the absence of extracellular Ca2+. Our results also reinforce the conclusion of Demiryurek et al. (6) and Robertson et al. (23) that HPV does not require the opening of voltage-gated Ca2+ channels in the smooth muscle cell membrane. It is interesting to note that when the ryanodine-sensitive SR Ca2+ stores were depleted in the absence of extracellular Ca2+, hypoxia reversibly relaxed the vessels to below baseline tension. This shows that pulmonary vascular smooth muscle cells had a basal tone in normoxia that did not depend on the presence of the endothelium. The observation that hypoxia can increase the rate of uptake of Ca2+ by internal stores (31) may be relevant here, but if so, it may be ryanodine-insensitive inositol 1,4,5-trisphosphate stores and/or the mitochondria that are responsible.Hypoxia releases SR Ca2+ in isolated pulmonary arterial smooth muscle cells. In agreement with the findings of others (4, 26, 28, 29), we found that hypoxia increases the cytoplasmic free Ca2+ concentration and shortens isolated pulmonary arterial smooth muscle cells. In 70% of all our cells, hypoxia slowly raised the mean fura 2 fluorescence ratio. This reached a peak within 1 min and was then sustained for up to 30 min. The hypoxic increase in the fura 2 fluorescence ratio was always reversed on return to normoxia. In contrast with previous findings (28), Ca2+ oscillations were observed in only two normoxic cells and were induced by hypoxia in only two previously quiescent cells (data not shown).
In agreement with our tension recording studies in intact arteries (see above), depletion of the ryanodine-sensitive SR Ca2+ stores with caffeine and/or ryanodine abolished the hypoxic increase in intracellular Ca2+. These findings reinforce the idea that hypoxia directly releases Ca2+ from the ryanodine-sensitive intracellular stores in rabbit pulmonary arterial smooth muscle cells and that it can do so in the absence of the endothelium. Curiously, the application of caffeine with ryanodine did not constrict isolated pulmonary arteries even though cytosolic Ca2+ increased in the isolated cells. The explanation for this is unclear, but it may result from the different pharmacokinetics of the drugs in the two preparations (see above). Importantly, however, in both of the preparations studied here, block or depletion of ryanodine-sensitive SR stores abolished the response to hypoxia. ![]() |
ACKNOWLEDGEMENTS |
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We thank Chris Hirst for technical assistance and Dr. Antony Galione for helpful discussions.
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FOOTNOTES |
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This work was supported by the Wellcome Trust.
A. M. Evans is a Wellcome Trust Non-clinical Lecturer.
Address for reprint requests and other correspondence and present address of A. M. Evans: Division of Biomedical Sciences, School of Biology, Bute Bldg., Univ. of St. Andrews, St. Andrews, Fife, KY16 9TS, UK (E-mail: ame3{at}st-and.ac.uk).
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.
Received 13 December 2000; accepted in final form 26 February 2001.
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