Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21224
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ABSTRACT |
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Effects of acute
hypoxia on intracellular Ca2+ concentration
([Ca2+]i) and cell length were recorded
simultaneously in proximal and distal pulmonary (PASMCs) and femoral
(FASMCs) arterial smooth muscle cells. Reducing
PO2 from normoxia to severe hypoxia
(PO2 < 10 mmHg) caused small but
significant decreases in length and a reversible increase in
[Ca2+]i in distal PASMCs and a small decrease
in length in proximal PASMCs but had no effect in FASMCs, even though
all three cell types contracted significantly to vasoactive agonists.
Inhibition of voltage-dependent K+ (KV) channel
with 4-aminopyridine produced a greater increase in
[Ca2+]i in distal than in proximal PASMCs. In
distal PASMCs, severe hypoxia caused a slight inhibition of
KV currents; however, it elicited further contraction in
the presence of 4-aminopyridine. Endothelin-1 (1010 M),
which itself did not alter cell length or
[Ca2+]i, significantly potentiated the
hypoxic contraction. These results suggest that hypoxia only has small
direct effects on porcine PASMCs. These effects cannot be fully
explained by inhibition of KV channels and were greatly
enhanced via synergistic interactions with the endothelium-derived
factor endothelin-1.
vasoconstriction; calcium; voltage-dependent potassium channel; pulmonary arterial smooth muscle cell
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INTRODUCTION |
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ACUTE REDUCTION in alveolar PO2 causes reversible hypoxic pulmonary vasoconstriction (HPV) in intact animals and isolated perfused lungs (13). Despite extensive research, the exact mechanism of this response remains unclear. Previous work indicated that HPV required extracellular Ca2+ and L-type Ca2+ channels (36, 37, 53) but was not mediated by vasoactive substances such as angiotensin, histamine, serotonin [5-hydroxytryptamine (5-HT)], or leukotriene (34, 37, 48). HPV has been demonstrated in isolated pulmonary arteries (4, 6, 27, 30, 32, 33, 45, 46), indicating that HPV may be mediated through a direct effect on these vessels. Hypoxic responses may depend on size and location, with small distal "resistant" arteries thought to be more responsive than large proximal "conduit" vessels (3, 32, 33).
In recent years, new information has been obtained about the direct effects of hypoxia on pulmonary arterial smooth muscle. Harder et al. (19) were the first to report that hypoxia caused membrane depolarization of smooth muscle in small pulmonary arteries. This result was confirmed subsequently in pulmonary arterial smooth muscle cells (PASMCs) (15, 43, 44, 64). Exposure of PASMCs to hypoxia also increased intracellular Ca2+ concentration ([Ca2+]i) and myosin light chain phosphorylation and caused contraction (10, 11, 15, 33, 38, 43, 47, 56, 66). Moreover, patch-clamp studies demonstrated that hypoxia inhibited delayed rectifier voltage-dependent K+ (KV) currents in PASMCs (2, 44, 64). These hypoxic effects might be specific for PASMCs because they were not observed in myocytes of systemic arteries (33, 56, 64). Because KV channels are important regulators of resting membrane potential in PASMCs (2, 43, 49, 64), it was proposed that the initiation of HPV involves inhibition of KV channels, which leads to membrane depolarization, activation of L-type Ca2+ channels, Ca2+ influx, increase in [Ca2+]i, and cell contraction (2, 59, 64). Other studies, however, showed that hypoxia released Ca2+ from intracellular stores independent of Ca2+ influx (15, 47), membrane depolarization (17, 43, 45), or inhibition of KV channels (15). Thus the role of KV channels in initiating HPV requires further elucidation.
Besides its direct effects on PASMCs, hypoxia may act indirectly through the release of factors derived from the endothelium; however, the role of the endothelium in HPV remains controversial. Some reports (22, 29, 45, 46) showed that removal of the endothelium completely or partially abolished HPV, whereas others (6, 40, 65) found little or no effect of endothelium removal. Recent evidence suggests that endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor, may mediate HPV. Plasma levels of ET-1 were elevated during acute hypoxia (25), and infusion of ET-1 antagonists prevented acute HPV in the rat, dog, pig, and fetal lamb (7, 23, 42, 57, 61) and blocked or reversed pulmonary hypertension associated with prolonged exposure to hypoxia (8). Moreover, hypoxic contractions of isolated pulmonary arteries from rats and pigs were endothelium dependent (30, 31, 46, 58) and in the pig could be blocked by ET-1 antagonists (31, 50).
In this study, we examined the direct effects of hypoxia on [Ca2+]i and length in PASMCs from adult pigs to determine whether hypoxia could directly activate myocytes from vessels exhibiting predominantly endothelium-dependent hypoxic response (14, 22, 29). Specific experiments were designed to characterize the effects of hypoxia on proximal and distal PASMCs and femoral arterial smooth muscle cells (FASMCs) and to examine the involvement of KV channels in and the modulatory effect of ET-1 on the hypoxic responses. Our results demonstrate that hypoxia causes small contractions and increases in [Ca2+]i in distal PASMCs under severe hypoxia but has little or no effect in proximal PASMCs or FASMCs. Hypoxic responses of distal PASMCs could not be explained completely by the inhibition of 4-aminopyridine (4-AP)-sensitive K+ channels but was enhanced significantly by a low concentration of ET-1, which by itself did not alter cell length or [Ca2+]i.
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MATERIALS AND METHODS |
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Cell Preparation
Porcine PASMCs were isolated as described by Clapp and Gurney (9). In brief, male and female pigs (~35 kg) were anesthetized with ketamine (20 mg/kg im) followed by pentobarbital sodium (12.5 mg/kg iv) and exsanguinated through the femoral arteries. Proximal (5- to 10-mm-ID) and small (0.2- to 0.7-mm-ID) intralobar pulmonary arteries were excised and placed in modified Krebs-Ringer bicarbonate solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2 CaCl2, 25 NaHCO3, and 11.1 glucose, 100 U/ml of penicillin, and 0.1 mg/ml of streptomycin bubbled with 21% O2-5% CO2 to achieve a pH of 7.4. The adventitia was carefully removed with forceps under a dissecting microscope. The endothelium was removed by opening the vessel longitudinally and rubbing the intimal surface with a cotton swab. The cleaned vessels were washed and cut transversely into small pieces, transferred to a vial with 10 ml of dissociation medium containing (in mM) 0.16 CaCl2, 110 NaCl, 5 KCl, 2 MgCl2, 10 HEPES, 10 NaHCO4, 0.5 KH2PO4, 0.5 NaH2PO4, 10 glucose, 0.04 phenol red, 0.49 EGTA, and 10 taurine, 3.2 U/ml of papain (type IV), 0.02% bovine serum albumin (BSA; type V), 100 U/ml of penicillin, and 0.1 mg/ml of streptomycin, pH titrated to 7.4 with NaOH, and stored overnight at 4°C to allow complete penetration by the enzyme. The next morning, papain was activated by adding 0.1 mM dithiothreitol and raising the temperature to 37°C. After 30 min of digestion, the tissue was transferred to fresh dissociation medium. Gentle trituration with a wide-bore pipette yielded long relaxed single smooth muscle cells, which were stored at 5-7°C for use within 8 h.FASMCs were isolated with the same method with a minor modification. After overnight incubation and 30 min of papain digestion, the tissues were transferred to dissociation medium containing collagenase (400 U/ml, type I; Sigma) for an additional 10 min of digestion. The myocytes were dispersed in fresh medium by trituration.
Measurement of [Ca2+]i and Cell Shortening
Freshly isolated myocytes were placed in a laminar flow cell chamber on the stage of a Nikon Diaphot inverted microscope and incubated with 2.5 µM indo 1-AM, a membrane-permeant Ca2+ fluorescent dye, for 30 min at room temperature (~22°C) under an atmosphere of 21% CO2-5% CO2. The cells were then washed by superfusion with Krebs bicarbonate solution for 30 min at 35°C to remove extracellular indo 1 and allow complete deesterification of cytosolic indo 1-AM.[Ca2+]i of isolated single myocytes was
measured by exciting indo 1 at 365 nm via a ×40 fluorescence
oil-immersion objective (Fluor ×40, Nikon, Japan). Emission
fluorescence at 405 (F405) and 495 (F495) nm
were detected by two photomultiplier tubes, and the signals were
amplified with a dual-emission fluorometer (Biomedical Instrumentation
Group, University of Pennsylvania, Philadelphia, PA). Photobleaching of
indo 1 was minimized by using a neutral density filter (ND-3, Omega
Optics, Brattleboro, VT) and an electronic shutter (Vincent Associates,
Rochester, NY). The shutter was opened for 35 ms every 2 s, and
the fluorescence signals during the open period were integrated with a
sample-and-hold circuit. Hence, in a recording period of 1 h, the
cells were actually exposed to 0.1% of excitation light for a total of
63 s. The protocols were executed, and data were collected online
with a Labmaster analog-to-digital interface (Axon Instruments, Foster
City, CA) and the pClamp software package (Axon Instruments).
[Ca2+]i was calculated with the equation of
Grynkiewicz et al. (18): [Ca2+]i = KdB(R Rmin)/(Rmax
R), where R is the ratio of
(F405
F405,bg) to
(F495
F495,bg), where
F405,bg and F495,bg are the background
fluorescence values at 405 and 495 nm, respectively. Rmin
and Rmax are the fluorescence ratios measured in situ in myocytes permeabilized with the Ca2+ ionophore 4-bromo
A-23187 (10 µM) with external solutions containing 10 mM
EGTA and 10 mM Ca2+, respectively. B = F495,EGTA/F495,Ca, where
F495,EGTA and F495,Ca are the fluorescence
values of EGTA and Ca2+,
respectively, at 495 nm, and the dissociation constant
(Kd) of indo 1 is 288 nM (60).
F405,bg and F495,bg were
measured by quenching indo 1 with MnCl2.
Cell length was measured alone in the studies of vasoconstrictor agents or simultaneously with [Ca2+]i in the hypoxia experiments. The myocytes were monitored continuously with red light with a charge-coupled device camera TV system (Dage, Michigan City, IN). Images were captured and stored every 2 min with a video frame grabber (Data Translation, Marlborough, MA). Cell length was measured off-line with image analysis software (Mocha, Jandel Scientific, San Rafael, CA).
Measurement of KV Currents
The myocytes were bathed in modified Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. They were whole cell voltage clamped with patch pipettes (tip resistance of 3-5 MControl of PO2
PO2 of the superfusate was controlled by an open laminar flow cell chamber (51), which prevented contact of the superfusate with atmospheric air by means of a laminar counterflow argon column. Krebs bicarbonate solutions were bubbled with an argon-balanced gas mixture containing 16% O2 plus 5% CO2 (normoxia), 5% O2 plus 5% CO2 (moderate hypoxia), 0% O2 plus 5% CO2 (severe hypoxia), or 0% O2 plus 5% CO2 plus 0.3 mM sodium dithionite (anoxia). The superfusate was delivered to the cell chamber at a flow rate of 2.5 ml/min by a peristaltic pump via stainless steel tubing. With this system, PO2 values measured inside the chamber with a microelectrode (Microelectrode, Bedford, NH) were 117 ± 1, 36 ± 2, 8 ± 2, and 0 mmHg during normoxia, moderate hypoxia, severe hypoxia, and anoxia, respectively. The temperature of the chamber was maintained at 35°C with a heat exchanger coupled to a circulator.Experimental Protocols
Protocol 1: Contractile responses to vasoconstricting agents. The myocytes were equilibrated under control conditions for 30 min and then exposed to continuous superfusion of KCl (10 mM), phenylephrine (PE; 10 µM), 5-HT (10 µM), or A-23187 (10 µM) for 15 min. Each myocyte was exposed to only one vasoconstricting agent. Cell length was measured before and during 15 min of vasoconstrictor exposure. This time was always sufficient for achievement of maximal responses.
Protocol 2: Hypoxic responses in PASMCs and FASMCs. In this and the following protocols, [Ca2+]i and cell length were measured simultaneously. After equilibration under normoxic conditions for 30-35 min for deesterification and 10 min of baseline [Ca2+]i and cell length recording, proximal and distal PASMCs and FASMCs were exposed to severe hypoxia for 15 min followed by 30 min of normoxia. The cells were then exposed sequentially to 4-AP (10 mM), a blocker of KV channels, and KCl (100 mM) for 5 min, allowing 30 min for recovery between exposures. To determine the O2 dependence of the hypoxic responses, the same protocol was applied to additional distal PASMCs exposed to moderate hypoxia or anoxia instead of severe hypoxia. Cells were discarded if significant changes occurred during the baseline recording period.
Protocol 3: Hypoxic responses and KV channels.
After equilibration under normoxic conditions, distal PASMCs were
exposed to 10 mM 4-AP for 10 min followed by 15 min of severe hypoxia
in the continued presence of 4-AP. After 30 min of recovery, KCl (100 mM) was given to verify the responsiveness of the cell. Control
experiments were performed in a separate group of myocytes that were
exposed to 4-AP without subsequent exposure to hypoxia. In a set of
separate experiments, the effect of hypoxia on KV currents
was examined. The myocytes were held at holding potential of 70 mV
and depolarized to test potentials ranging from
60 to +30 mV in
+10-mV step increments for 1 s once every 10 s.
Current-voltage relationships generated before and during severe
hypoxia were constructed for comparison.
Protocol 4: Hypoxia and Ca2+sensitivity of cell shortening. After equilibration under normoxic conditions, distal PASMCs were exposed to either continued normoxia or severe hypoxia. Seven minutes after the onset of exposure, the Ca2+ ionophore 4-bromo A-23187 was administered at 5-min intervals in progressively increasing concentrations (0.3, 1, and 3 µM).
Protocol 5: ET-1 and hypoxic responses.
After equilibration under normoxic conditions for 45 min, distal PASMCs
were first exposed to ET-1 (1010 M) for 10 min followed
by 15 min of severe hypoxia in the continued presence of ET-1. After 30 min of recovery, KCl (100 mM) was given to verify the myocyte responsiveness.
Chemicals and Drugs
Papain, collagenase, BSA, and other chemicals were purchased from Sigma (St. Louis, MO). Indo 1-AM was purchased from Molecular Probes; 4-bromo A-23187 was purchased from Calbiochem (La Jolla, CA). Stock solutions of indo 1-AM (1 mM) and 4-bromo A-23187 (10 mM) were made up fresh in dimethyl sulfoxide and diluted 1:400 and 1:1,000, respectively, in Krebs-Ringer bicarbonate solution. 4-AP was prepared fresh, with pH adjusted to 7.4 before use. High KCl solution was prepared by equimolar replacement of NaCl.Data Analysis
Data are expressed as means ± SE. Changes in [Ca2+]i during hypoxia and anoxia or drug challenges were determined from the averaged [Ca2+]i transients measured in a group of cells. For statistical analysis, values of individual Ca2+ transients were averaged over a period of 1 min. One- or two-factor analysis of variance (ANOVA) with repeated measures was used to test for the significance of changes in the variables occurring over time. When significant variance ratios were obtained by one-factor ANOVA, Dunnett's test was used to identify the means that were significantly different from baseline. Student's t-test for paired analysis was also used for some comparisons. Values were considered significantly different when P < 0.05. ![]() |
RESULTS |
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Contractile Responses to Vasoconstricting Agents
Averaged resting cell length was 77.8 ± 2.0 µm in distal PASMCs (n = 107), 55.8 ± 1.2 µm in proximal PASMCs (n = 114), and 80.4 ± 2.4 µm in FASMCs (n = 81). Proximal PASMCs were significantly shorter than the other myocytes. Maximal concentrations of KCl (100 mM), PE (10 µM), 5-HT (10 µM), and A-23187 (10 µM) caused significant contractions in all three cell types (Table 1). Within the cell types, there were no differences among contractile responses induced by the four agonists. Among the cell types, contractile responses expressed as absolute shortening (
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Hypoxic Responses in PASMCs and FASMCs
Figure 1 shows the time course of PO2, [Ca2+]i, and normalized cell length (L/L0) in FASMCs (n = 7) and proximal (n = 10) and distal (n = 12) PASMCs exposed to severe hypoxia for 15 min. During the normoxic control period, PO2 was stable at levels > 100 mmHg, and [Ca2+]i averaged 73.1 ± 9.2, 75.3 ± 10.9, and 66.5 ± 8.2 nM in FASMCs, proximal PASMCs, and distal PASMCs, respectively. During hypoxia, PO2 fell rapidly in all myocytes, achieving levels < 10 mmHg within 5 min. In FASMCs, [Ca2+]i (75.6 ± 13.0 nM) and cell length (
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O2 Dependence of Hypoxic Responses
Two other groups of distal PASMCs were exposed to moderate hypoxia (n = 12) or anoxia (n = 13). As shown in Fig. 3A, moderate hypoxia had no effect on [Ca2+]i, which averaged 59.1 ± 8.4 and 69.0 ± 13.9 nM before and during hypoxia, respectively; however, moderate hypoxia did cause a slight decrease in cell length (
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Hypoxia and Ca2+Sensitivity of Cell Shortening
To examine the effect of hypoxia on the Ca2+sensitivity of cell shortening, distal PASMCs were exposed at 5-min intervals to progressively increasing concentrations (0.3, 1, and 3 µM) of the Ca2+ ionophore 4-bromo A-23187 during normoxia or severe hypoxia. Under both conditions, 4-bromo A-23187 caused significant concentration-dependent increases in [Ca2+]i and decreases in L/L0. The relationship between mean
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Hypoxic Responses and KV Channels
To examine whether inhibition of KV channel activates PASMCs, 4-AP was applied to proximal and distal PASMCs. Inhibition of KV channels with 10 mM 4-AP increased [Ca2+]i in 24 of 33 distal PASMCs and 6 of 14 proximal PASMCs. TheTo examine whether hypoxia inhibits KV channels,
KV currents were recorded with whole cell patch-clamp
techniques. Cell capacitance of distal PASMCs (n = 20)
was 21 ± 2 pF. Depolarization to test potentials ranging between
30 and +30 mV activated KV currents, which were
characterized by low noise and slow inactivation kinetics. Hypoxia
caused a small reversible inhibition of KV currents in whole cell patch-clamped distal PASMCs (Fig.
5A). After exposure to
severe hypoxia, the current-voltage relationship of the
KV current was shifted downward significantly
(P < 0.01 by two-factor repeated-measures ANOVA), with
an average inhibition of the KV current of 22 ± 10%
at
20 mV and 17 ± 7% at +20 mV.
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To further investigate whether KV channels are required for
hypoxic responses, distal PASMCs were first exposed to 4-AP for 10 min
to inhibit KV channels followed by 15 min of severe hypoxia in the continued presence of 4-AP (Fig.
6). 4-AP caused
L/L0 to decrease 4.4 ± 1.2%
(P < 0.05) and
[Ca2+]i to increase from 85.2 ± 17.8 to
108.7 ± 17.0 nM. Subsequent hypoxia caused a further significant
reduction in cell length (10.0 ± 2.4%; P < 0.05) and raised [Ca2+]i to 140.5 ± 25.6 nM. Compared with myocytes that were exposed to 4-AP only, the
reduction in L/L0 was significantly
greater after exposure to severe hypoxia (P < 0.05 by
two-factor repeated-measures split-plot ANOVA). Although the
[Ca2+]i responses of the two groups were not
significantly different, [Ca2+]i in the
hypoxic group was persistently higher during the hypoxic period.
Moreover, 5 of 13 cells exhibited both a clear increase in
[Ca2+]i and a marked cell shortening on
exposure to hypoxia in the presence of 4-AP. Therefore, inhibition of
KV channels did not abolish the hypoxic responses in distal
PASMCs.
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ET-1 Priming and Hypoxic Responses
To examine the interaction between ET-1 and hypoxia, distal PASMCs were exposed to a low concentration (10
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DISCUSSION |
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This study was designed to examine the direct effects of hypoxia
on porcine PASMCs. We found that 1) hypoxia caused a
small contraction in proximal and distal PASMCs but not in FASMCs and small increases in [Ca2+]i in distal PASMCs
but not in proximal PASMCs or FASMCs; 2) the magnitude of
hypoxic responses in distal PASMCs was greatest at PO2 < 10 mmHg; 3) severe
hypoxia caused a slight inhibition of KV channels in distal
PASMCs; 4) inhibition of KV channels in distal
PASMCs with 4-AP did not always abolish the hypoxic response; and
5) hypoxic contraction, but not
[Ca2+]i responses, in distal PASMCs were
significantly enhanced in the presence of 1010 M ET-1,
which by itself did not alter cell length or
[Ca2+]i. These results suggest that hypoxia
can directly stimulate contraction and increase
[Ca2+]i in PASMCs from vessels that exhibit a
predominantly endothelium-dependent hypoxic response. However,
these responses are small and cannot be fully explained by inhibition
of KV channels of PASMCs, and their full expression may
rely critically on factors derived from the endothelium.
Our contraction experiments demonstrated that porcine FASMCs and proximal and distal PASMCs shortened significantly on exposure to KCl, PE, 5-HT, or A-23187, indicating that receptor-linked transduction pathways and pathways acting through membrane depolarization and Ca2+ influx were intact in these cells. The magnitude of contraction (12-38%) was comparable to that previously observed in pulmonary and systemic arterial myocytes under similar conditions (33, 66).
Hypoxia caused contraction in distal and proximal PASMCs but not in FASMCs and elevated [Ca2+]i in distal PASMCs but not in proximal PASMCs or FASMCs. The lack of hypoxic contraction and [Ca2+]i response in FASMCs is consistent with previous observations in systemic arterial myocytes (32, 33, 56). These results cannot be explained by functional impairment because, as stated above, FASMCs responded significantly to vasoactive agonists acting through a variety of mechanisms. Our finding that distal PASMCs were more responsive to 4-AP and hypoxia and less responsive to A-23187 than proximal PASMCs is consistent with several reports suggesting that myocyte characteristics vary with location and size of the pulmonary artery from which they are obtained. For example, in the cat, HPV occurred in myocytes or segments from pulmonary arteries < 600 µm; however, myocytes or segments from arteries > 800 µm failed to contract and even relaxed during hypoxia (32, 33, 56). Cornfield et al. (10) showed that hypoxia increased [Ca2+]i in cultured PASMCs from distal but not from main pulmonary arteries of fetal lambs. Similarly, Archer et al. (3) found that hypoxia produced a sustained contraction in small pulmonary resistance vessels from the rat but elicited an initial contraction, which was followed by relaxation, in large conduit vessels. Differences in hypoxic responses were attributed to a differential distribution of phenotypically distinct myocytes. Proximal cells were generally larger and expressed mainly KCa channels, whereas distal cells were usually shorter with predominantly KV channels (1, 3). The differences we observed in the 4-AP responses between proximal and distal PASMCs are consistent with their findings; the discrepancy between cell length observed in our study could be related to species. However, the significance of the phenotypical difference between proximal and distal PASMCs remains unsettled because consistent hypoxic responses have also been described in proximal pulmonary arteries of intact pigs (21) and segments or cells from main pulmonary arteries of rats (47, 65) and the hypoxic responses of rat main and distal pulmonary arteries were found to be quantitatively similar (30).
Hypoxic contractions in pulmonary myocytes were accompanied by either
no significant change (proximal PASMCs) or only small changes (distal
PASMCs) in [Ca2+]i, suggesting that hypoxia
may have altered myofilament sensitivity to Ca2+. As shown
in Fig. 4, the relationships between
[Ca2+]i and
L/L0 elicited by exposure to varying
concentrations of the calcium ionophore 4-bromo A-23187 were similar
during normoxia and hypoxia. If the magnitude of myocyte shortening in
these experiments was determined primarily by the strength of
myofilament contraction, these results suggest that hypoxia did not
alter Ca2+ sensitivity.
To further characterize the hypoxic responses in distal PASMCs, we determined the effect of different PO2 values on [Ca2+]i and L/L0 (Fig. 3). Although decreasing PO2 from >100 to 0 mmHg elicited proportional decreases in L/L0 and increases in [Ca2+]i, the changes in both variables were small and occurred mainly at a PO2 < 10 mmHg. These findings differ from the larger contractions and increases in [Ca2+]i observed in PASMCs of cats, dog, fetal lambs, and rats at a PO2 of 25-50 mmHg (10, 15, 33, 38, 43, 47) and the robust responses to mild or moderate hypoxic challenges observed in intact or isolated perfused pig lungs (52, 54).
Previous studies in isolated porcine pulmonary arteries suggested that the endothelium plays an important role in the hypoxic responses of this tissue. Holden and McCall (22) reported vigorous contraction of porcine main pulmonary arteries after prolonged (4- to 6-h) preconditioning of the tissue at a PO2 of 40 mmHg, but the responses were greatly attenuated after endothelial denudation. Kovitz et al. (29) could not elicit endothelium-independent hypoxic contraction in porcine pulmonary arteries with IDs of 1-12 mm. Recently, Liu et al. (31) found that HPV occurred in porcine pulmonary arteries with IDs of 0.2-0.3 mm only if the endothelium was intact. In contrast, Ogata et al. (40) reported that contractions of small porcine pulmonary arteries (2-mm OD) to hypoxia (PO2 = 15 mmHg) were enhanced by removal of the endothelium. We cannot explain this discrepancy. Our observation that PASMCs obtained from distal porcine pulmonary arteries with IDs of 0.2-0.7 mm did not respond to moderate hypoxia and exhibited only small responses to severe hypoxia suggests that myocytes from arteries that exhibit predominantly endothelium-dependent hypoxic responses can respond directly to hypoxia, but additional factors may be required for the full expression of HPV.
It has been proposed that hypoxia causes pulmonary vasoconstriction by reducing activities of smooth muscle KV channels, resulting in membrane depolarization and Ca2+ influx via L-type Ca2+ channels (2, 59, 64). This hypothesis is based on findings in pulmonary arterial myocytes that 1) hypoxia caused membrane depolarization (3, 19, 44, 64), 2) hypoxia inhibited KV currents (2, 3, 64), 3) pharmacological inhibition of KV channels caused membrane depolarization and an increase in [Ca2+]i (3, 49, 63), and 4) hypoxic responses were blocked by Ca2+ channel antagonists and potentiated by Ca2+ channel agonists (19, 36, 37, 46, 53). Given the small hypoxic contractions and [Ca2+]i responses we observed in distal PASMCs, we performed two experiments to determine whether KV channels played a similar role in these cells.
To determine whether hypoxia inhibited KV channels, we measured voltage-current relationships in distal PASMCs using whole cell patch-clamp techniques. Because the internal solution was Ca2+ free and contained high concentrations of ATP and EGTA, outward currents resulted primarily from KV channels rather than from ATP-dependent K+ or KCa channels. As shown in Fig. 5, hypoxia caused only a slight inhibition of these currents (e.g., 17 ± 7% at +20 mV), consistent with the small magnitude of its effects on cell length and [Ca2+]i. In comparison, hypoxia caused a >50% inhibition of KV currents measured at +40 mV under similar conditions in PASMCs from rats (2, 64).
To determine whether blockade of KV channels prevents HPV,
we examined the effect of 4-AP, an inhibitor of KV
channels, alone and in combination with hypoxia on cell length and
[Ca2+]i in distal PASMCs. Hypoxia caused a
further significant reduction in L/L0
in the presence of 4-AP because a clear divergence in L/L0 in 4-AP-treated and untreated
cells was observed starting at the onset of hypoxia (Fig. 6). Moreover,
the additional 3% reduction in L/L0
in myocytes exposed to hypoxia is comparable to the hypoxic contraction
elicited in the absence of 4-AP (Fig. 1). The small hypoxic
[Ca2+]i responses of distal PASMCs did not
achieve a significant difference compared with those in control
myocytes. However, [Ca2+]i was
persistently higher in myocytes during the hypoxic challenge, and clear
hypoxia-induced responses were observed in several 4-AP-treated cells.
Because 10 mM 4-AP completely inhibits KV channels
(2, 49), these results suggest that in distal
PASMCs, hypoxic contraction can be elicited via mechanism(s) other than
the inhibition of KV channels, although a contributing role
of KV channels in the responses cannot be excluded.
The inability of 4-AP to inhibit HPV in some distal PASMCs, in fact, is
consistent with several previous studies. In isolated perfused lungs
and distal and proximal pulmonary arteries isolated from rats, complete
inhibition of KV channels with 10 mM 4-AP caused
significant enhancement instead of inhibition of HPV (3, 20). In rat PASMCs, intracellular dialysis of an antibody
against a KV channel subtype (Kv1.5) inhibited
KV currents and caused membrane depolarization but failed
to inhibit the hypoxia-induced [Ca2+]i
response (15). These findings suggest that hypoxic
responses could be activated even when KV channels were
unavailable. Moreover, several studies suggested that initiation of HPV
depends on Ca2+ release from intracellular stores. For
example, hypoxia-induced elevation in [Ca2+]i
in PASMCs could be abolished or significantly reduced by
inhibiting Ca2+ release from the sarcoplasmic reticulum
(SR) with ryanodine or depleting SR Ca2+ stores with
thapsigargin or caffeine (15, 26,
47, 56). Removal of extracellular
Ca2+ inhibited the sustained but not the transient phase of
the hypoxic Ca2+ responses (15,
47), and a hypoxia-induced increase in
[Ca2+]i preceded membrane depolarization
after KCa and Cl channels were blocked
(43). Because intracellular Ca2+ is known to
inhibit KV channels (16, 17), it
has been proposed alternatively that hypoxia first activates
Ca2+ release from the SR, leading to the inhibition of
KV channels, membrane depolarization, and Ca2+
influx via L-type Ca2+ channels (15,
43).
Besides direct effects on PASMCs, hypoxia may elicit HPV by stimulating or inhibiting release of factors derived from endothelium. It has been suggested that an unidentified endothelium-derived constricting factor(s) is responsible for the late-phase contraction induced by hypoxia in isolated rat and porcine arteries (14, 29, 45, 58). ET-1, a potent pulmonary vasoconstrictor, is an attractive candidate for this role because it is synthesized by pulmonary endothelial cells and released in greater quantity during hypoxia (28, 41). ET-1 antagonists can prevent acute HPV in isolated proximal and distal intrapulmonary porcine arteries (31, 50) and in intact animals (7, 23, 24, 42, 57, 61) including the pig (23, 24).
In this context, our observation that a low concentration of ET-1
(1010 M) did not alter length or
[Ca2+]i in distal PASMCs during normoxia but
markedly enhanced contractile responses to hypoxia (Fig. 7) suggests
that even basal release of ET-1 from endothelial cells may have
significant synergistic effects on the direct contractile response of
PASMCs to hypoxia. Similar results were observed in porcine pulmonary
arterial microvessels (150- to 200-µm ID) in that hypoxia caused
significant vasoconstriction only after they were primed with
10
10 M ET-1 (Liu Q and Sylvester JT, unpublished
data). This synergism is consistent with the findings of
Turner and Kozlowski (55) that an initial priming of rat
PASMCs with ET-1 (10
10 M) allowed the expression of
membrane depolarization induced by hypoxia (55). They
proposed that an initial "priming" depolarization induced by ET-1
may confer a sensitivity to hypoxia by activating delayed rectifier
KV channels, which can then be closed by hypoxia, leading
to further depolarization. Moreover, threshold concentrations of
ET-1 (3 × 10
10 and 10
9 M) were found
to potentiate vasoconstriction induced by norepinephrine and 5-HT in
coronary arteries (62). However, the potentiation of HPV
by ET-1 in this study might not be due to a greater increase in
[Ca2+]i because the Ca2+ response
to hypoxia was not significantly enhanced in the presence of ET-1
despite a large Ca2+ spike that occurred at the onset of
hypoxia in one cell. Enhancement of contraction without alteration in
[Ca2+]i suggests that ET-1 increased
myofilament Ca2+ sensitivity. This effect of ET-1 has
been previously demonstrated in systemic vessels (5,
39) and is consistent with the findings that the
endothelial mediator of late-phase hypoxic contraction in isolated
pulmonary arteries may act by increasing myofilament Ca2+
sensitivity via a protein kinase C-independent mechanism
(45, 58). Our results, however, did not prove
that ET-1 is the unidentified endothelial factor responsible for
endothelium-dependent HPV or that it is the only such factor because
prestimulation with other agonists has been shown to enhance HPV in
both isolated lungs and vessels (35, 46), and
some investigators (12, 14) have not been
able to demonstrate the involvement of ET-1 in HPV in isolated vessels.
Therefore, the exact mechanism(s) through which ET-1 priming enhances
hypoxic vasoconstriction remains to be elucidated.
In conclusion, our results indicate that hypoxia can increase [Ca2+]i and cause contraction in freshly isolated porcine distal PASMCs; however, the small magnitude of these direct effects and their low sensitivity to a reduced PO2 suggest that they do not account completely for HPV, which may require synergistic interactions between endothelium and smooth muscle for the full expression.
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ACKNOWLEDGEMENTS |
---|
This study was supported by National Heart, Lung, and Blood Institute Grants HL-52652 (to J. S. K. Sham) and HL-51912 (to J. T. Sylvester) and an American Heart Association Established Investigator Award (to J. S. K. Sham).
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: J. S. K. Sham, Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: jsks{at}welchlink.welch.jhu.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. §1734 solely to indicate this fact.
Received 19 March 1999; accepted in final form 3 March 2000.
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