1 Department of Physiology and Pharmacology, Institute of Comparative Medicine, University of Georgia, Athens, Georgia 30602-7389; and 2 Department of Asthma, Allergy, and Respiratory Science, Guy's King's and St. Thomas' School of Medicine, King's College London, London SE1 9RT, United Kingdom
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ABSTRACT |
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The main aim of
this study was to determine the effects of endothelium removal on
tension and intracellular Ca2+
([Ca2+]i) during hypoxic pulmonary
vasoconstriction (HPV) in rat isolated intrapulmonary arteries (IPA).
Rat IPA and mesenteric arteries (MA) were mounted on myographs and
loaded with the Ca2+-sensitive fluorophore fura PE-3.
Arteries were precontracted with prostaglandin F2, and
the effects of hypoxia were examined. HPV in isolated IPA consisted of
a transient constriction superimposed on a second sustained phase. Only
the latter phase was abolished by endothelial denudation. However,
removal of the endothelium had no effect on
[Ca2+]i at any point during HPV. The
endothelin-1 antagonists BQ-123 and BQ-788 did not affect HPV, although
constriction induced by 100 nM endothelin-1 was abolished. In MA,
hypoxia induced an initial transient rise in tension and
[Ca2+]i, followed by vasodilatation and a
fall in [Ca2+]i to (but not below) prehypoxic
levels. These results are consistent with sustained HPV being mediated
by an endothelium-derived constrictor factor that is distinct from
endothelin-1 and that elicits vasoconstriction via Ca2+ sensitization.
pulmonary circulation; arteries; hypoxia
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INTRODUCTION |
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THE MECHANISM(S) UNDERLYING hypoxic pulmonary vasoconstriction (HPV), a unique and vital homeostatic mechanism that matches ventilation to perfusion in the event of localized alveolar hypoxia (32), remain unresolved. Because HPV can be observed in isolated pulmonary arteries (8, 10, 18, 22, 24, 25), the oxygen sensor and subsequent constrictor mechanism(s) must reside in either the smooth muscle, endothelium, or both. In rat intrapulmonary arteries (IPA), the response to acute hypoxia, usually in the presence of a small degree of agonist-induced tone, is biphasic in nature (9, 10, 18, 19, 22-26), consisting of an initial transient rise in tension (phase I) superimposed on a more slowly developing sustained constriction (phase II). Under similar experimental conditions, an almost identical transient phase I constriction has been observed in a variety of isolated systemic arteries (6, 22, 37), whereas the second phase of the response in systemic arteries to hypoxia is vasodilatation (22). Because the phase II constriction is peculiar to pulmonary arteries, we have argued that phase II is the more physiologically important process for sustained HPV (2, 33). In support of this contention, we have reported that the selective Rho kinase antagonist Y-27632 inhibited the hypoxic pressor response in perfused rat lungs yet only inhibited the second phase of HPV in rat isolated arteries (25).
We have reported that the sustained phase II constriction of HPV is endothelium dependent (22). This is in agreement with other reports that the full expression of sustained HPV requires an intact endothelium (10, 13, 18), although this is not a universal finding (4, 16). We have also reported that the generation of tension during phase II appears to involve Ca2+ sensitization of the contractile apparatus, which is protein kinase C independent (24) and which appears to involve activation of Rho kinase (25). However, there have been no substantive studies as to whether the removal of the endothelium affects the rise in pulmonary vascular smooth muscle intracellular [Ca2+] ([Ca2+]i) that is observed during hypoxia. In the present study, we used the Ca2+-sensitive fluorophore fura PE-3 coupled with the small vessel myograph, to study the influence of the endothelium on changes in tension and [Ca2+]i during HPV in rat IPA. For comparison, the relationship between [Ca2+]i and tension in rat mesenteric arteries during acute hypoxia has also been examined under the same experimental conditions. We report here the novel finding that the coupling of the increase in [Ca2+]i to tension development during sustained HPV in isolated rat IPA is dependent on the presence of the endothelium. We also report that the expression of sustained HPV in rat IPA does not involve endothelin-1. These results are consistent with sustained HPV being mediated via the production of a yet-to-be-identified endothelium-derived constrictor factor that acts via increasing the sensitivity of the contractile apparatus to Ca2+ (11, 27).
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MATERIALS AND METHODS |
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IPA isolation and mounting. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication no. 85-23, revised 1996). Male Wistar rats (250-350 g) were anesthetized with pentobarbital sodium (55 mg/kg ip) and killed by cervical dislocation, as approved by the local UK Home Office Inspector. The mesentery, heart, and lungs were rapidly excised and placed in a cold physiological salt solution (PSS) containing (in mM): 118 NaCl, 24 NaHCO3, 1 MgSO4, 0.435 NaH2PO4, 5.56 glucose, 5 sodium pyruvate, 1.8 CaCl2, and 4 KCl. Small intrapulmonary arteries [200-500 µm internal diameter (ID)] and mesenteric resistance arteries (MA, 250-400 µm ID) were then dissected free of connective tissue and mounted in a small vessel myograph (Cambustion AM10; Cambustion, Cambridge, UK), as previously described in detail (22), and gassed with 95% air/5% CO2.
Estimation of [Ca2+]i. To study changes in [Ca2+]i, we loaded IPA and MA with the Ca2+-sensitive fluorophore fura PE-3, via incubation of the vessels with the acetoxymethyl ester of fura PE-3 (fura PE-3 AM, 3 µM) for 2 h at room temperature. Similar loading protocols have previously shown that subsequent measurements are of smooth muscle [Ca2+]i, with negligible contributions to the fluorescence signal from the endothelium (15). IPA were continually gassed with 95% air/5% CO2 during this procedure. After loading, the vessels were washed with PSS, the myograph bath temperature was raised to, and maintained at, 37°C, and the myograph was transferred to the stage of an inverting fluorescence microscope (Olympus IMT2; Olympus, London, UK). We assessed changes in [Ca2+]i by calculating the ratio of the light emitted through a >500-nm emission filter when the vessel was illuminated at 340 and 380 nm, respectively (F340/380, Cairn spectrophotometer; Cairn Research, Newnham, UK).
Hypoxic protocol.
IPA and MA were equilibrated with four exposures to 80 mM KCl-PSS
(KPSS, 2-min duration, isotonic replacement of NaCl by KCl), as
described previously (22, 24). As we have previously
shown, a small degree of agonist-induced tone is required to facilitate the hypoxic response in rat IPA and MA (22, 24). The
vessels were therefore exposed to 3 µM prostaglandin
F2 (PGF2
) for 20 min before and during
the hypoxic challenge. Hypoxia was induced by gassing with 1%
O2/94% N2/5% CO2 for 45 min,
after which time the vessels were reoxygenated for 20 min, washed with PSS, and subsequently reexposed to KPSS to ensure that hypoxia had no
detrimental effect on the contractile function of the vessels. In some
experiments, oxygen tension in the myograph chamber was continually
monitored via a dissolved oxygen meter (Diamond General electrode, Ann
Arbor, MI; Strathkelvin meter, Glasgow, UK). During the hypoxic
challenge, the chamber PO2 was typically
18-20 mmHg, compared with control PO2 of
135-145 mmHg.
Removal of IPA endothelium.
IPA were subjected to the equilibration procedure described above
and subsequently exposed to 10 µM PGF2 for 10 min, after which 1 µM acetylcholine was added to the myograph chamber. After washing with PSS, endothelium denudation was accomplished by the
gentle rubbing of the luminal surface of IPA with a human forearm hair.
Endothelial disruption was confirmed by the abolition of the
vasodilator response to acetylcholine (1 µM) in arteries preconstricted with PGF2
(10 µM). To ensure that this
procedure had not damaged the smooth muscle, we exposed denuded IPA to
KPSS. IPA that did not generate at least 95% of the tension measured during the final exposure to KPSS of the equilibration procedure were excluded.
Antagonism of endothelin-1 during HPV. IPA were exposed to two hypoxic challenges, 90 min apart, with the endothelin-1 antagonists BQ-123 and BQ-788 (both 10 µM) added in combination 30 min before the second hypoxic challenge. The combination of these antagonists abolished the robust contractile response to endothelin-1 (100 nM, n = 3, data not shown).
Data presentation and statistical analysis. Tension is presented as a percentage of the maximum constriction (TK) obtained to the final exposure to 80 mM KPSS during the equilibration procedure. In endothelium-denuded arteries, TK was taken as the maximum constriction obtained to 80 mM KPSS postdenudation. Changes in [Ca2+]i are represented in terms of the change in the fura PE-3 ratio. Mean changes are expressed as a percentage of the maximum ratio change seen either during the final 80 mM KPSS, during the equilibration procedure, or during the postdenudation exposure in arteries where the endothelium had been removed (F340/380) as described previously (24, 26). Results are expressed as means ± SE. Means were compared by repeated-measures ANOVA or paired or unpaired Student's t-test (SigmaStat, Jandel). A value of P < 0.05 was deemed significant.
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RESULTS |
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Effect of hypoxia on tension and
[Ca2+]i in IPA.
Hypoxia elicited a biphasic response in
tension and [Ca2+]i in IPA preconstricted
with 3 µM PGF2 (Figs. 1 and
2). The first phase (phase I)
of HPV consisted of a transient contraction, which peaked within
2-4 min of the onset of hypoxia (an increase of 57.9 ± 5.2%
TK), followed by a relaxation that reached a
nadir of 12.6 ± 1.7% TK above the
PGF2
-induced tone within 10-15 min. There was a
concomitant transient increase in [Ca2+]i
(peak rise of 32.8 ± 2.5% F340/380) that had a
similar time course to that of tension (phase II) and that
fell to a level that was 14.1 ± 1.9% F340/380 above
that obtained before induction of hypoxia. We then observed a more
slowly developing increase in tension, which reached a level of
20.4 ± 2.4% TK after 40 min of hypoxia.
However, this increase in tension was not associated with any further
change in [Ca2+]i, since this remained
constant at ~14% F340/380 throughout this period.
Reoxygenation resulted in a rapid return of both tension and
[Ca2+]i to the initial
PGF2
-induced values (Figs. 1 and 2).
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Effect of endothelial denudation on tension and
[Ca2+]i during HPV.
Endothelial denudation caused an increase in the effect of
PGF2 (3 µM) on tension (endothelium-intact vs.
-denuded, 11.3 ± 1.5% vs. 28.2 ± 4.5%
TK, P < 0.05), though the
increase in [Ca2+]i did not reach
significance (endothelium-intact vs. -denuded, 12.3 ± 2.3% vs.
24.3 ± 6.3% F340/380, P > 0.05).
This increased vasoconstrictor sensitivity to PGF2
was
presumably due to the loss of endothelium-derived relaxing factor(s).
Because the level of pretone has profound effects on the subsequent
hypoxic constrictor response (28, 33), we reduced the
concentration of PGF2
used with endothelium-denuded IPA
(typically to 0.5-1 µM) to match the level of pretone to
that observed in the endothelium-intact arteries (pretone in denuded
arteries, 14.4 ± 0.9% TK; Figs. 1 and 2).
Under these conditions, endothelium denudation was found to be without
effect on the phase I constriction in IPA (phase I peak = 60.0 ± 3.1% TK).
However, the phase II constriction was abolished (peak rise
in tension during phase II in control vs. denuded arteries,
20.4 ± 2.4% vs.
3.4 ± 3.4% TK,
P < 0.05). Endothelial denudation did not affect
hypoxia-induced changes in [Ca2+]i at any
point during the hypoxic challenge (Fig. 2). The phase I
peak rise in [Ca2+]i in control and denuded
arteries was 32.8 ± 2.5% and 29.6 ± 1.7%
F340/380, respectively (P > 0.05). The
phase II peak rise in [Ca2+]i in
control and denuded arteries was 14.1 ± 1.9% and 14.3 ± 2.5% F340/380, respectively (P > 0.05).
It is noteworthy that the dissociation between tension and
[Ca2+]i was observed even if pretone in the
denuded IPA was not matched to the control by reducing the
PGF2
concentration. In endothelium-denuded IPA where 3 µM PGF2
was used to preconstrict the arteries, phase II consisted of a dilation (17.5 ± 5.2%
TK reduction from prehypoxic tension,
n = 6), whereas the phase II elevation in [Ca2+]i (18.3 ± 4.5%
F340/380 above prehypoxic
[Ca2+]i, n = 5) was similar
to that in endothelium-intact arteries (see above).
Effect of endothelin-1 receptor antagonism on HPV.
Several reports exist that are supportive of a role for
endothelin-1 in HPV (e.g., Refs. 29, 30).
However, our results are not consistent with a role for endothelin-1 in
HPV in rat IPA. The effect of endothelin-1 receptor antagonism on HPV
in rat IPA is shown in Fig. 3. When used
in combination, the endothelin A (ETA) and ETB
receptor antagonists BQ-123 and BQ-788 (both 10 µM), had no effect on
HPV in rat IPA, whereas the response to 100 nM endothelin-1 was
abolished (n = 3, data not shown).
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Effect of hypoxia on tension and
[Ca2+]i in MA.
The response of rat MA to acute hypoxia is shown in Fig.
4. Upon induction of hypoxia in MA
preconstricted with PGF2 (5 µM), there was a
transient, simultaneous increase in tension and
[Ca2+]i (peak rise in tension, 48.3 ± 5.8% TK; peak rise in
[Ca2+]i, 38.6% ± 5.6%
F340/380; P < 0.05, for both responses).
In contrast to endothelium-intact IPA, the transient constriction was
followed by a marked vasodilatation (prehypoxic tension, 18.0 ± 5.5% TK; relaxation from prehypoxic tension,
15.6 ± 5.3% TK; P < 0.05), which rapidly reversed upon reoxygenation. This dilatation was independent of any reduction in [Ca2+]i,
since following phase I, [Ca2+]i
fell to, but not below, prehypoxic levels (prehypoxic
[Ca2+]i, 13.3 ± 3.9%
F340/380; phase II
[Ca2+]i after 40-min hypoxia,
1.0 ± 3.7% F340/380; P > 0.05) and was unaltered upon reoxygenation.
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DISCUSSION |
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We have reported that the progressive rise in tension during phase II of HPV in rat IPA was not associated with any concomitant rise in [Ca2+]i, which remained at a constant elevated level (24). We hypothesized that phase II of HPV involves an endothelium-dependent sensitization of the smooth muscle contractile apparatus to Ca2+ (24). In agreement with this hypothesis, we have recently reported that the Rho kinase inhibitor Y-27632 selectively attenuates phase II of HPV and abolishes the hypoxic pressor response in perfused lungs (25). The present results now provide the first direct evidence that phase II of HPV involves an endothelium-dependent sensitization of the smooth muscle contractile apparatus to Ca2+. Our data also show that phase II is not inhibited by simultaneous blockade of ETA and ETB receptors, indicating that endothelin-1 is not involved in this endothelium-dependent Ca2+ sensitization process in our model of HPV. These results therefore suggest that the pulmonary endothelium releases an as-yet unidentified constrictor factor in response to hypoxia, which elicits Ca2+ sensitization.
Isometric tension and [Ca2+]i were simultaneously monitored in rat IPA by means of the Ca2+ fluorophore fura PE-3 coupled with a small vessel myograph. Consistent with several other reports, HPV in rat IPA was biphasic in nature, consisting of an initial transient first phase (phase I), superimposed on a more slowly developing sustained constriction (phase II) (9, 10, 18, 19, 22-26). In agreement with our earlier observations (22), we found that, whereas phase I was endothelium independent, phase II was completely dependent on the presence of an intact endothelium. Analogous biphasic responses to hypoxia have been described in a number of pulmonary vascular preparations, and a similar endothelium dependency of the full generation of sustained HPV has also been observed (10, 13, 18), although it is noted that this finding is not universal (4, 16).
Consistent with our previous findings, hypoxia caused a biphasic rise in [Ca2+]i (24, 26). During phase I, the increase in [Ca2+]i paralleled the increase in tension. However, during phase II, [Ca2+]i remained stable, while tension continued to rise. The pivotal finding of the present study was that removal of the endothelium had no effect on the phase II elevation in [Ca2+]i, whereas the phase II constriction was converted into a vasodilation. Therefore, removal of the endothelium resulted in an uncoupling of tension development from the rise in [Ca2+]i. In addition, an analogous dissociation of tension development and [Ca2+]i was observed in small MA with an intact endothelium, since after an initial transient rise [Ca2+]i returned to the pretone level, whereas tension fell. This would imply that one of the crucial differentiating factors in the response of these two artery types to an acute hypoxia resides in the pulmonary endothelium.
The endothelium dependency of phase II could be explained by a hypoxia-induced inhibition of the basal release of a dilator factor(s) such as nitric oxide or prostacyclin. However, there is insubstantial support for this possible mechanism, and it is more likely that phase II of HPV in rat IPA is mediated via an endothelium-derived constrictor factor (2). One obvious candidate for this endothelium-derived constrictor factor is endothelin-1, especially since endothelin-1 receptor antagonists appear to show much promise for the treatment of chronic hypoxia-associated pulmonary hypertension (5). Several reports have demonstrated a contributory role for endothelin-1 in the mechanism underlying HPV in a variety of preparations (3, 17). Conversely, other investigators have found endothelin-1 antagonists to be without effect on HPV (19, 36). Moreover, exogenous endothelin-1 or endothelin-3 have been shown to attenuate HPV when added during an acute hypoxic challenge (7, 12). In the present study, endothelin-1 did not appear to be the endothelium-derived constrictor factor involved in phase II, since combined blockade of ETA and ETB receptors was without effect on HPV. It is interesting to note that hypoxia elicits the release of contractile factor(s), distinct from endothelin-1, from pulmonary endothelial cells (11) and perfused rat lung (27) and that the latter "factor(s)" appears to induce contraction in pulmonary but not mesenteric arteries via Ca2+ sensitization. The possibility that these factors mediate the endothelium-dependent phase II contraction of HPV remains speculative but is worthy of more detailed investigation.
In both intact MA and endothelium-denuded IPA, hypoxia caused an initial transient rise in [Ca2+]i and tension followed by a sustained relaxation. In IPA, the initial transient constriction appears to be mediated via release of Ca2+ from intracellular stores (9, 10, 14, 26, 35), which may result in activation of capacitative Ca2+ entry (26), whereas the mechanism underlying phase I in MA remains unresolved. The subsequent relaxation in both artery types was not associated with a fall in [Ca2+]i below the pretone level. Indeed [Ca2+]i remained elevated above pretone in endothelium-denuded IPA. The pulmonary-selective elevation of [Ca2+]i by hypoxia, which was independent of the presence of the endothelium, is consistent with the recent elegant work proposing a role for cADP ribose-mediated Ca2+ release from the sarcoplasmic reticulum in the sustained rise in [Ca2+]i during HPV (9, 10, 35).
A number of reports have described a similar dissociation between tension and [Ca2+]i during acute hypoxia in a variety of "systemic" arteries including cerebral, mesenteric, and coronary arteries (1, 31). We have recently reported that acute hypoxia elicits an increase in NAD(P)H levels in IPA (21). Because NAD(P)H fluoresces when excited at 340 nm, it is possible that some portion of the rise in the F340/380 signal during hypoxia is due to an increase in NAD(P)H. We previously calculated that increases in NAD(P)H fluorescence during hypoxia accounted for ~25% of the sustained increase in F340/380 in rat isolated IPA (21). Because the F340/380 remain unchanged during hypoxia in MA, it is possible that there was actually a fall in [Ca2+]i that was masked by an increase in NAD(P)H fluorescence during hypoxia. We have recently reported that, under identical experimental conditions to those used in this study, the rise in NAD(P)H fluorescence elicited by hypoxia in MA was only ~20% of that observed in IPA and, as such, would have only a marginal effect on the fura signal (34). These observations would therefore appear to be consistent with a minimal contribution of NAD(P)H to the F340/380 signal during hypoxia in MA and, in turn, support Ca2+ desensitization in the hypoxic dilation observed in rat isolated MA. The mechanism(s) underlying this apparent desensitization of the contractile apparatus to [Ca2+]i remain unknown, but increases in inorganic phosphate concentration and cytoskeletal alterations have been proposed. Our results suggest that IPA are able to counteract this general tendency of smooth muscle to relax to hypoxia by virtue either of an endothelial factor that is unique to the pulmonary vasculature or of a unique sensitivity of the IPA vascular smooth muscle to a factor that may be released ubiquitously throughout the vasculature.
An important point related to the above is that full development of sustained HPV requires both a rise in [Ca2+]i and an increase in endothelium-mediated Ca2+ sensitivity; suppression of the sustained rise in smooth muscle [Ca2+]i also suppresses the phase II vasoconstriction, even in the presence of an intact endothelium (20, 26). In all our studies and those of several other groups, removal of the endothelium entirely abolishes the sustained phase II of HPV (reviewed in Ref. 2), and this is consistent with the apparently critical requirement for Rho kinase-mediated Ca2+ sensitization in the hypoxic pressor response of isolated lungs (24). However, Dipp and Evans (9) have reported that, although endothelium-denuded IPA lack the slowly developing component of phase II, a steady elevated tone above baseline is retained, which they hypothesized was due to the residual effect of a rise in [Ca2+]i in the absence of endothelium-dependent Ca2+ sensitization (9). Neither this nor other reports showing only partial suppression of phase II (2) are inconsistent with our model, which would predict that a sufficiently large hypoxia-induced rise in [Ca2+]i, perhaps promoted by the particular experimental conditions, would be able to cause some degree of vasoconstriction even without Ca2+ sensitization.
In summary, this investigation supports a role for Ca2+ sensitization during the phase II constriction of HPV in isolated rat IPA and provides the first evidence that the full expression of the sustained constrictor response to acute hypoxia has an absolute dependency on the presence of an intact endothelium. We hypothesize that sustained HPV in rat IPA requires both an increase in smooth muscle [Ca2+]i and the action of an as-yet unidentified endothelium-derived constrictor factor that induces a sensitization of the contractile apparatus to [Ca2+]i, possibly via the RhoA/Rho kinase pathway.
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ACKNOWLEDGEMENTS |
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We are grateful to the Wellcome Trust (Grants 043357, 062554) and the British Heart Foundation (Grant PG96044).
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Robertson, Dept. of Physiology and Pharmacology, Inst. of Comparative Medicine, Univ. of Georgia, Athens, GA 30602-7389 (E-mail: troberts{at}vet.uga.edu).
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.
First published February 28, 2003;10.1152/ajplung.00422.2002
Received 10 December 2002; accepted in final form 14 February 2003.
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