Division of Respiratory, Critical Care and Occupational Medicine, Department of Internal Medicine, The University of Utah Health Science Center, Salt Lake City 84132; and Pharmadigm, Inc., Salt Lake City, Utah 84109
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ABSTRACT |
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In the present study, we investigated the
effects of the naturally occurring hormone dehydroepiandrosterone
(DHEA) on hypoxic pulmonary vasoconstriction (HPVC) in isolated ferret
lungs and on K+ currents in
isolated and cultured ferret pulmonary arterial smooth muscle cells
(FPSMCs). Severe alveolar hypoxia (3%
O2-5%
CO2-92% N2) caused an initial increase
in pulmonary arterial pressure (Ppa) that was followed by a
reversal in pulmonary hypertension. Maintaining alveolar hypoxia caused
a sustained secondary increase in
Ppa. Pretreating the lungs with
the K+-channel inhibitor
tetraethylammonium (TEA) caused a small increase in baseline
Ppa, potentiated HPVC, and
prevented the reversal of HPVC during the sustained alveolar hypoxia.
Treating the lungs with DHEA caused a near-complete reversal of HPVC in
control lungs and in lungs that were pretreated with TEA. DHEA also
reversed the KCl-induced increase in
Ppa. In FPSMCs, DHEA caused an
adenosine 3',5'-cyclic monophosphate- and guanosine
3',5'-cyclic monophosphate-independent increase in activity
of the Ca2+-activated
K+
(KCa) current. In a
cell-attached configuration, DHEA caused a mean shift of 22 mV
in the voltage-dependent activation of the
KCa channel. We conclude
that DHEA is a novel
KCa-channel opener of the
pulmonary vasculature.
pulmonary circulation; pulmonary hypertension; calcium-activated potassium-channel agonist; potassium-channel opener; potassium currents; tetraethylammonium
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INTRODUCTION |
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HYPOXIC AND PRIMARY pulmonary hypertension cause substantial morbidity and mortality. Current therapy for pulmonary hypertension is ineffective, in part, because of inadequate understanding of basic mechanisms that regulate pulmonary vascular tone. Alveolar hypoxia was reported to cause membrane depolarization of pulmonary arterial smooth muscle cells (SMCs) (14, 27, 29, 35). Several types of membrane ion channels were reported to be involved in mediating SMC depolarization and hypoxic pulmonary vasoconstriction (15). Post et al. (29) demonstrated in canine pulmonary arterial SMCs that lowering PO2 caused membrane depolarization by decreasing Ca2+-dependent whole cell outward currents. Yuan et al. (35) reported in rat pulmonary arterial SMCs that sodium dithionite-induced hypoxia inhibited a voltage-gated K+ channel. More recently, it was reported that alveolar hypoxia inhibited a delayed rectifier K+ (Kdr) channel (2) or ATP-dependent K+ (KATP) channel (31) of rat small pulmonary arterial SMCs. Previous observations by Peng and colleagues (26, 27) in human pulmonary SMCs and previous reports (6, 30, 35) on animal pulmonary vasculature suggested that Ca2+-activated K+ (KCa) channels can act as a negative feedback mechanism to counteract the acute effect of hypoxia and other agonist-induced membrane depolarizations. In vascular SMCs, the membrane potential (Em) is a major determinant of vascular tone. The Em of vascular SMCs is controlled to a large degree by KCa, Kdr, and KATP channels (6, 8, 28).
KCa channels are large-conductance channels and carry ionic currents that mediate membrane hyperpolarization and thus regulate important cellular functions including vascular relaxation. KCa-channel activity is regulated by several physiological parameters including Em, cytosolic Ca2+ concentration ([Ca2+]i), and channel phosphorylation (6). The probability of the KCa channel being open is relatively low in resting vascular SMCs. However, the opening state of the channels is increased by membrane depolarization. Thus it is believed that the KCa channel acts as a relaxing negative feedback mechanism after agonist-induced membrane depolarization (6, 27). The purified toxin peptides such as charybdotoxin (CTX), iberiotoxin, and external tetraethylammonium (TEA) are known inhibitors of the KCa channel (7). However, little is known about KCa-channel agonists.
Dehydroepiandrosterone (3-hydroxy-5-androsten-17-one; DHEA) and its
sulfate ester are major secretory products of the adrenal cortex. The
exact physiological role of DHEA is unknown. However, epidemiologic
studies demonstrated that in humans a high level of DHEA is an
independent predictor of a significantly reduced risk for fatal
coronary vascular disease (3) and is inversely associated with the
development of accelerated coronary allograft vasculopathy (16). In
addition, DHEA was reported to cause a concentration-dependent in vivo
and in vitro inhibition of platelet aggregation (17). The observed
platelet-aggregation inhibition was associated with a reduction in
thromboxane B2 production (17). Farrukh et al. (10) previously reported that administration of
corticosteroid caused a marked inhibition of lipid peroxide- or
thromboxane A2-mediated pulmonary
vasoconstriction. Progesterone, a derivative of the parent molecule
pregnenolone and the precursor of DHEA, was reported to cause smooth
muscle relaxation (5). In addition, deoxycorticosterone, another
derivative of the parent molecule pregnenolone, was reported to cause
both an acute and a chronic alteration in membrane ion transport of
aortic SMCs (19). These observations suggest that the steroid-induced
pulmonary vasorelaxation is mediated via membrane hyperpolarization.
In this study, we investigated the effect of DHEA on hypoxic pulmonary vasoconstriction in an isolated, ventilated, and perfused ferret lung preparation. We also determined in cultured ferret pulmonary arterial SMCs (FPSMCs) whether the effects of DHEA on pulmonary vascular tone were induced via modulating K+ channels.
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METHODS AND MATERIALS |
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Isolated Lung Preparation
The isolated lung preparations were performed as previously described (12) and as approved by the Institutional Animal Care and Use Committee at the University of Utah. Briefly, male ferrets (Marshall Farm, North Rose, NY) weighing 3.5-4 kg were maintained on formula and water ad libitum. On the day of the experiment, the ferrets were anesthetized with an intraperitoneal injection of pentobarbital sodium (25 mg/kg). The chest was opened, and the animal was given 3,000 units of heparin by intracardiac injection and killed by rapid exsanguination from the left ventricle. Right and left parasternal incisions were made along the costal cartilages to remove the sternum and open the chest widely. Stainless steel cannulas were secured in the left atrium and pulmonary artery with umbilical tape. The ligature around the pulmonary artery was also passed around the aorta, preventing loss of perfusate into the systemic circulation. The open chest was covered with moist gauze and plastic wrap to conserve heat and humidity.The lungs were perfused with a mixture of 70 ± 5 ml of autologous blood and 70 ± 5 ml of Krebs-Henseleit buffer in a recirculating manner at a constant rate of 85 ml/min. The chemical composition of the Krebs-Henseleit buffer was (in mM) 137 NaCl, 4.7 KCl, 2.5 CaCl2 · 2H2O, 1.3 KH2PO4, 1.2 MgSO4, 11 dextrose, and 18 NaHCO3. The temperature of the perfusate was kept between 37 and 38°C. The pH was maintained at 7.35-7.40 by adding sodium bicarbonate to the reservoir as needed. The venous reservoir was placed below the lowermost portion of the lung.
The lungs were ventilated via a tracheotomy with a Harvard animal respirator (Harvard Apparatus, Millis, MA) with either a normoxic gas mixture of 5% CO2, 21% O2, and 74% N2 or a hypoxic gas mixture of 5% CO2, 3% O2, and 92% N2. The animals were ventilated with a tidal volume of 30 ml at a rate of 12 breaths/min and with 2 cmH2O positive end-expiratory pressure.
Measurements. Pulmonary arterial, left atrial, and tracheal pressures were monitored with Gould Statham P23ID pressure transducers connected to the inflow circulation and recorded on a Gould polygraph (model RS 3400, Gould, Valley View, OH). Arterial blood gases and pH were measured on a radiometer MK2 blood gas analyzer at 37°C.
Interventions. TEA (Sigma Chemical, St. Louis, MO) was dissolved in modified Krebs-Henseleit buffer and added directly to the perfusion reservoir. DHEA (Pharmadigm, Salt Lake City, UT) was dissolved in 0.5 ml of dimethyl sulfoxide (DMSO) and added to the perfusion reservoir to achieve the desired final concentration. In pilot experiments, this amount of DMSO had no effect on hypoxic pulmonary vasoconstriction.
FPSMC Culture
FPSMCs were cultured from explants of ferret pulmonary arteries as previously described (11). Briefly, the arteries were separated from their adventitia and endothelium and then minced into 1- to 3-mm2 pieces with sterile scalpel blades. The tissues were mounted in tissue culture wells with 0.05 ml of chicken plasma (Sigma Chemical) plus 0.05 ml of chick embryo extract (Sigma Chemical). The tissue, and eventually the FPSMCs, were then plated with the same SMC culture media, SmGM (Clonetics, San Diego, CA), that was supplemented with 5% fetal bovine serum (HyClone, Logan, UT) and 10% bovine calf serum (HyClone) plus dexamethasone (0.39 mg; Sigma Chemical) and antimicrobial agents (50 µg/ml of gentamicin, 25 µg/ml of amphotericin B, 120 U/ml of penicillin, and 0.12 mg/ml of streptomycin; Sigma Chemical). Experiments were performed in primary FPSMCs. FPSMCs were assessed by light microscopy, electron microscopy, and indirect immunofluorescent staining ofElectrophysiological Measurements
Single cells were voltage clamped, and membrane currents were recorded with cell-attached inside-out and outside-out configurations of the patch-clamp technique (13, 26). The patch-clamp micropipettes were made of borosilicate glass capillaries and had a resistance of 5-10 MSolutions and Chemicals
The pipette and bath solutions were composed of (in mM) 140 KCl, 2 MgCl2, 3 ethylene glycol-bis(Statistical Analysis
Values are means ± SE. The data were analyzed with analysis of variance for repeated measures (Scheffé's F-test significance at 95%) and Student's t-test. Results were considered statistically significant when P < 0.05. ![]() |
RESULTS |
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Effect of Severe Alveolar Hypoxia on Pulmonary Arterial Pressure
In a group of animals (n = 4), we tested the effect of severe hypoxia (3% O2) on pulmonary arterial pressure (Ppa). During an initial stabilizing period, the lungs were ventilated with a normoxic gas mixture (5% CO2-21% O2-74% N2) for 20 min. Switching the ventilation to a hypoxic gas mixture (5% CO2-3% O2-92% N2) caused a sharp increase in Ppa [change in (
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Effect of K+-Channel Blocker (TEA) on Ppa During Severe Alveolar Hypoxia
It was suggested in isolated ferret lungs that the secondary pulmonary vasodilatation during severe hypoxia was most likely mediated by opening K+ channels (34). Therefore, we tested the effect of pretreating the lungs (n = 3) with TEA (10 mM) on the vascular response to severe alveolar hypoxia. TEA, when applied externally, has a relatively high-affinity blockade of the KCa channel [dissociation constant (Kd) = 150-300 µM] (6, 28). In vascular smooth muscle, high concentrations of TEA (5-20 mM) can also inhibit the Kdr channel (6, 28). Pretreating the lungs with TEA (10 mM) caused a small baseline increase in Ppa (
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Effect of DHEA on Ppa Responses During Severe Alveolar Hypoxia
Administration of DHEA to human volunteers to achieve serum concentrations of 0.1, 0.2, and 0.3 mM had no reported side effects (17). In two groups of experiments, we tested the effect of DHEA on the acute and the delayed severe hypoxia-induced pulmonary vasoconstriction. In the first group of ferrets, DHEA (0.14 or 0.2 mM; n = 3 each) was administered at about the peak of the acute hypoxic pulmonary vasoconstriction. Treating the lung with DHEA caused an acute and complete reversal of the hypoxic pulmonary vasoconstriction and prevented the secondary hypoxic vasoconstriction (Fig. 3). Treating the hypoxic lungs with vehicle (DMSO) alone had no effect on the hypoxic pulmonary vasoconstriction. We also tested the effect of DHEA on lungs that were pretreated with 10 mM TEA before exposure to hypoxia. As illustrated earlier (Fig. 2), pretreating the lungs with TEA potentiated the hypoxic pulmonary vasoconstriction and blocked the secondary pulmonary vasodilatation during the sustained severe hypoxia. After the sustained hypoxic pulmonary vasoconstriction was established, the addition of DHEA (0.14 or 0.2 mM; n = 3 ferrets each) dramatically reversed the effects of TEA treatment plus alveolar hypoxia (Fig. 2). The vasodilating effects of 0.14 and 2.0 mM DHEA were maintained throughout the 75 and 150 min, respectively, of sustained hypoxia. Because the effects of both TEA and hypoxia are believed to be mediated via SMC membrane depolarization, the observations in this group of experiments suggested that the DHEA-induced pulmonary vasodilatation was mediated via membrane hyperpolarization.
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Effect of DHEA on KCl-Induced Pulmonary Vasoconstriction
To investigate further the mechanism of DHEA-induced pulmonary vasodilatation, we tested the effect of DHEA on KCl-induced pulmonary vasoconstriction. Infusion of 30 mM KCl into the pulmonary arteries of ferret lungs (n = 3) caused an initial transient hyperacute increase in Ppa that was followed by a sustained increase in Ppa (Fig. 4). Posttreating the lungs with DHEA (0.14 mM) completely reversed the effect of KCl on Ppa and reduced the Ppa below baseline (Fig. 4). This observation further supports the hypothesis that DHEA-induced pulmonary vasodilatation is mediated via SMC membrane hyperpolarization.
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Effect of DHEA on Em in Intact FPSMCs
The effect of DHEA on the resting Em was examined in primary FPSMCs by using whole cell current-clamp recordings. Treating the cells with DHEA (50 µM) caused significant baseline membrane hyperpolarization (Em =
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Effect of DHEA on K+ Channels in Intact FPSMCs
To determine whether the DHEA-induced reversal of hypoxic pulmonary vasoconstriction was mediated by membrane hyperpolarization, we investigated the effect of DHEA on the major K+ channels in intact cells. The two major components of the K+ current (IK) of pulmonary arterial SMCs have previously been characterized (28). The first component or Kdr current [IK(dr)] is a low-amplitude and low-noise current that is voltage and time dependent and insensitive to Ca2+ or low concentrations of TEA but can be inhibited with high-dose TEA (>2 mM) and 4-aminopyridine (4-AP). The second component or Ca2+-activated K+ current [IK(Ca)] is a high-amplitude and large-noise current that is voltage dependent. IK(Ca) is sensitive to Ca2+, CTX, and low concentrations of TEA (26-28). Single channels were recorded in a cell-attached configuration before and after FPSMCs were exposed to DHEA. DHEA (50 µM) exposure caused a significant increase in KCa-channel activity (NPo = 0.147 ± 0.007 vs. 0.029 ± 0.001 for control cells; P < 0.0001; n = 10 cells; Fig. 6). DHEA had no significant effect on the Kdr channel (NPo = 0.026 ± 0.002 vs. 0.022 ± 0.001 for control cells; P > 0.05; n = 10 cells). The effect of DHEA on KCa-channel activity was reversible after washout (data not shown). The DHEA-induced increase in channel activity was not associated with a change in unitary current (mean single unitary current after DHEA was 9.6 ± 0.16 vs. 9.4 ± 0.1 pA for control cells; P > 0.05; n = 10 cells).
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To investigate further whether the action of DHEA was selective for the KCa channel, we tested its effect on outward K+ currents (IKo) from FPSMCs that were pretreated with a selective KCa-channel inhibitor, CTX (2, 26, 35). In a cell-attached configuration, CTX (100 nM in the pipette solution) blocked the KCa channel and prevented the DHEA-induced activation of the KCa channel (Fig. 6A). In the presence of CTX, DHEA had no effect on the low-conductance K+ currents (Fig. 6A). This indicates that DHEA selectively activates the KCa channel. In another group of experiments, we tested the effect of 4-AP and glibenclamide on the DHEA-induced increase in KCa-channel activity. 4-AP (2 mM) blocked the low-amplitude or Kdr channel but had no effect on the DHEA-induced increase in KCachannel activity (NPo = 0.037 ± 0.001 for control baseline activity vs. 0.19 ± 0.007 for DHEA + 4-AP-induced activity; P < 0.0001; n = 6 cells each; Fig. 6B). To determine whether the KATP channel is involved in the DHEA-induced increase in IKo, we tested the effect of the KATP-channel inhibitor glibenclamide (6, 8) on the DHEA-induced activation of the KCa channel. Glibenclamide (50 µM) had no effect on the DHEA-mediated increase in KCa-channel activity (NPo = 0.037 ± 0.001 for control baseline activity vs. 0.177 ± 0.006 for DHEA + glibenclamide-induced activity; P < 0.05; n = 10 cells; Fig. 6B).
Role of Guanosine 3',5'-Cyclic Monophosphate and Adenosine 3',5'-Cyclic Monophosphate in DHEA-Mediated KCa-Channel Activation in Intact FPSMCs
Because the mechanism of vascular agonist-induced increase in KCa-channel activity is reported to be mediated via guanosine 3',5'-cyclic monophosphate (cGMP) or adenosine 3',5'-cyclic monophosphate (cAMP) (26, 30), we investigated the possible role of cGMP and/or cAMP in DHEA-mediated KCa-channel activation. We tested the effect of antagonizing the cyclic nucleotides with Rp-cGMPS, Rp-8-pCPT-cGMPS, and Rp-cAMPS on the DHEA-induced increase in KCa-channel activity. FPSMCs were incubated with one of the Rp isomers (1 × 10Rp-cGMPS, Rp-8-CPT-cGMPS, and Rp-cAMPS reduced baseline activity of the KCa channel (NPo = 0.029 ± 0.001 for mean control activity vs. 0.007 ± 0.0016, 0.008 ± 0.0003, and 0.011 ± 0.001 for Rp-cGMPS-, Rp-cAMPS-, and Rp-8-pCPT-cGMPS-induced activity, respectively; P < 0.0001; n = 6 cells each; Fig. 7). The addition of DHEA (50 µM) to cells pretreated with one of the Rp isomers caused a significant increase in KCa-channel activity (NPo = 0.029 ± 0.001 for mean control activity vs. 0.15 ± 0.001, 0.16 ± 0.008, and 0.171 ± 0.009 for Rp-cGMPS + DHEA-, Rp-cAMPS + DHEA-, and Rp-8-pCPT-cGMPS + DHEA-induced activity, respectively; P < 0.0001; n = 6 cells each; Fig. 7). This suggests that the DHEA-induced activation of the KCa channel is not mediated via the generation of cGMP or cAMP.
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To test further whether the DHEA-induced K+ activation was mediated via cyclic nucleotide phosphorylation, we studied the effect of the cyclic nucleotide-dependent protein kinase inhibitor H-8 on the DHEA-induced increase in KCa-channel activity. In inside-out excised patches, applying H-8 (0.45 µM) to the inner surface of the membrane caused a significant decrease in baseline KCa-channel activity (NPo = 0.034 ± 0.003 vs. 0.078 ± 0.006 for control baseline activity; P < 0.001; n = 6 patches; Fig. 8). In the presence of H-8, DHEA caused a significant increase in KCa-channel activity [NPo = 0.329 ± 0.018 for DHEA (50 µM) + H-8 vs. 0.078 ± 0.006 for control baseline activity; P < 0.0001; n = 6 patches; Fig. 8]. The effects of H-8 and DHEA were reversible after the patches were washed with the bath solution (Fig. 8).
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Effect of DHEA on K+-Channel Sensitivity to Em
To investigate further the mechanism by which DHEA activates the KCa channel, we determined its effect on the sensitivity of KCa channels to Em. In a cell-attached configuration with a perfusate Ca2+ concentration of 300 nM and at an Em range of
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DISCUSSION |
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The principal observation in this study is that severe alveolar hypoxia in ferret lungs causes a triphasic vascular response: an initial acute pulmonary vasoconstriction that is followed by a transient vasorelaxation and then a progressive and sustained vasoconstriction. The transient vasorelaxation is mediated via activating TEA-sensitive K+ channels. DHEA, a naturally produced hormone, reverses the acute hypoxic pulmonary vasoconstriction and prevents the development of the secondary increase in Ppa during sustained alveolar hypoxia. The mechanism of DHEA-induced pulmonary vasorelaxation is mediated, in part, via an increase in KCa-channel activity.
Effect of Severe Alveolar Hypoxia
The response of the pulmonary vasculature to alveolar hypoxia is dependent on the degree of hypoxia. Previous work from our laboratory (12) and by other investigators (32) demonstrated that lowering alveolar oxygen to 5-7% caused a sustained increase in Ppa. In this investigation, a further decrease in alveolar O2 to 3% caused the observed triphasic changes in pulmonary vasculature (Fig. 1). It was also reported that ventilating the pulmonary vasculature or isolated lungs with 5% CO2-95% N2 further decreased arterial PO2 and caused a biphasic response of a brief initial vasoconstriction followed by a predominant vasodilatation (32, 34). The multiphasic response to acute and severe hypoxia was also observed in isolated pig lungs (31) and isolated proximal large and small rat pulmonary arteries that were precontracted with vascular agonists (4, 18, 23). Intrapulmonary ferret and porcine arterial rings were reported to have predominant biphasic responses to acute and severe hypoxia, characterized by an initial vasoconstriction followed by a predominant vasodilatation (20, 33). The discrepancies in the reported observations indicate that hypoxic pulmonary vasoconstriction is species and arterial size specific.The mechanism of hypoxia-induced secondary pulmonary vasodilatation is not fully understood. Recent observations suggested that the secondary relaxation is endothelium independent and is unaffected by inhibitors of endothelium-derived relaxing factors, by inhibitors of the cyclooxygenase pathway, or by blockers of adrenergic receptors (18, 23). In the present study, we observed that pretreating ferret lungs with TEA potentiated the hypoxic pulmonary vasoconstriction and blocked the secondary vasodilatation during severe alveolar hypoxia. Previous reports from our laboratory (26, 28) and observations by other investigators (1, 2, 30) indicate that, in pulmonary vascular cells, intermediate and high concentrations of TEA inhibit both IK(Ca) and IK(dr). Thus we conclude that in isolated ferret lungs the mechanism of the severe alveolar hypoxia-induced transient pulmonary vasodilatation is probably mediated via activating TEA-sensitive currents. These results are different from the observations reported by Wiener et al. (33), who reported in ferret intrapulmonary arteries that the hypoxia-induced vasodilatation can be blocked, in part, by either an inhibitor of the KATP channel (glibenclamide or tolbutamide) or TEA. A combination of the two types of inhibitors completely blocked the vasodilating effect of hypoxia. They (33) concluded that the vasodilatation during severe hypoxia was mediated by activation of both KATP- and TEA-sensitive currents. In contrast, Jin et al. (18) observed in isolated rat proximal pulmonary arterial rings that the hypoxia-induced arterial vasodilatation was not blocked by glibenclamide. They concluded that KATP channels are not involved in the arterial dilatation during hypoxia. One possible explanation for the apparent discrepancy in the observations is the different degree of hypoxia used in each protocol. Because the pulmonary vasculature responds differently to varying degrees of hypoxia, it is feasible that different IK are inhibited as the PO2 of the perfusate changes. In addition, the response of isolated pulmonary arterial rings to hypoxia may differ from the responses of the pulmonary vasculature in isolated whole lungs to alveolar hypoxia.
Role of KCa-Channel Agonist
The second important observation of the present investigation is that the naturally occurring steroid DHEA reversed acute hypoxic pulmonary vasoconstriction, prevented the development of secondary hypoxic vasoconstriction, and reversed the effect of TEA-induced potentiation of hypoxic vasoconstriction. The mechanism of DHEA-induced pulmonary vasodilatation is most likely mediated via activation of the KCa channel. This conclusion is supported by our physiological and electrophysiological observations. In the isolated ferret lungs, DHEA reversed the KCl-induced increase in Ppa and reversed the effect of TEA on hypoxic pulmonary vasculature. It is not fully clear to us why, in the isolated ferret lung experiments, TEA did not affect the DHEA-induced pulmonary vasodilatation. One explanation is that external TEA causes a flickery block, and the rate constants for blocking and unblocking the KCa channel were reported to be 380 and 73 mM/ms, respectively (21). Thus it is possible that applying a potent activator (like DHEA), which has the ability to activate KCa channels from inside and outside the membrane (Figs. 6-8), can reverse the effect of TEA, probably by affecting its Kd to the KCa channel or by modulating the interaction between theIt is unlikely that the DHEA-mediated pulmonary vasorelaxation is
mediated via inhibition of L-type
Ca2+ channels. L-type
Ca2+-channel blockers are known to
inhibit the KCa channel (6, 28). Contrary to this, DHEA caused FPSMC membrane hyperpolarization and
an increase in KCa-channel
activity. Accordingly, we do not believe that the DHEA-induced
vasorelaxation could be mediated via inhibiting L-type
Ca2+ channels. With the use of the
patch-clamp technique in a cell-attached configuration, DHEA increased
the KCa-channel activity of
FPSMCs. The DHEA-induced increase in
KCa-channel activity was
blocked by a specific
KCa-channel inhibitor, CTX,
and was not affected by 4-AP or glibenclamide, inhibitors of the
Kdr and
KATP channels, respectively. It is
not clear how DHEA increases
KCa activity. In an intact
FPSMC, cell-attached configuration, cGMP and cAMP did not block the
effect of DHEA on the KCa
channel. In addition, in excised inside-out FPSMC membrane patches,
blocking protein kinase-mediated phosphorylation of the
KCa channel with H-8 had no
significant effect on the DHEA-mediated
KCa-channel activation. These observations suggest that the DHEA-mediated increases in KCa-channel activity were
independent of cAMP- or cGMP-induced phosphorylation. In an intact
FPSMC current recording in a cell-attached configuration, DHEA caused a
21- to
24-mV (or leftward) shift in the voltage-dependent
activation of the KCa
channel. This was equivalent to an ~1.4-fold increase in
KCa-channel activity at any
Em value in the
range of
20 to +60 mV. These observations suggest that the
DHEA-mediated increase in the
KCa channel is mediated by
increasing the sensitivity of the channels to
Em. This
represents a pathway for
KCa-channel activation that
has not been previously described and suggests that DHEA is a novel KCa-channel opener.
Although we have strong evidence to indicate that DHEA acts as a
KCa-channel agonist in
FPSMCs, the mechanism of DHEA-mediated pulmonary vasodilatation in
hypoxic ferret lungs needs further investigation. Additional
experiments are needed to explore the effect(s) of DHEA on isolated
microvascular endothelial cells and SMCs.
The previously described K+-channel openers cromakalim and pinacidil are believed to mediate their vasorelaxing effect via activating KATP channels (8). Limited information is available about KCa-channel openers. Soyasaponins, which were isolated from a medicinal herb, activate KCa channels from inside the cell (24). However, their poor membrane permeation limits their therapeutic use (24). Other agents like the synthesized imidazopyrazine derivatives were reported to cause aortic and tracheal smooth muscle relaxation, in part, via activating the KCa channel (22, 25). These conclusions were reached because SCA40, the most potent imidazopyrazine derivative, inhibited the effect of an intermediate KCl concentration (20 mM) on rat isolated aortic vessels (22). The vasorelaxing effect of SCA40 was not inhibited by the KATP-channel inhibitor glibenclamide but was antagonized by CTX (25). Although these studies suggested that SCA40-induced vasorelaxation was mediated via activation of the KCa channel, the investigators provided no electrophysiological evidence to demonstrate that this agent actually increases the activity of the KCa channel.
Openers of KCa channels have great therapeutic potential and serve as important investigational tools. KCa channels have large unitary conductances; thus the opening of only a few KCa channels has a significant impact on Em (6, 26, 28). The ability of DHEA to open the KCa channel of pulmonary SMCs and relax constricted pulmonary vasculature may provide a novel therapeutic agent for pathological conditions such as chronic hypoxia in which the pulmonary vascular Em is depolarized. The pulmonary vasorelaxing effects of DHEA were observed at concentrations (0.1-0.2 mM) that were previously shown to have no systemic side effects in healthy human volunteers (17). If this holds true in future clinical trials, unlike the other available vasodilators, DHEA will have the advantage of being a potent pulmonary vasodilator without significant systemic side effects. Nonetheless, because KCa channels are ubiquitous to pulmonary and systemic circulations, further studies are needed to characterize the effect(s) of DHEA on the systemic circulation.
In summary, severe alveolar hypoxia caused an initial acute pulmonary vasoconstriction that was followed by a transient vasorelaxation and then a progressive and sustained vasoconstriction. The transient vasorelaxation was mediated via activating a TEA-sensitive K+ channel. DHEA, a naturally produced hormone, reversed the acute hypoxic pulmonary vasoconstriction and prevented the development of the secondary increase in Ppa during sustained hypoxia. DHEA also reversed the effects of KCl and TEA on the pulmonary vasculature. In FPSMCs, DHEA increased the activity of the KCa channel. The DHEA-induced increase in KCa-channel activity was not mediated via cAMP or cGMP and was not blocked by the protein kinase inhibitor H-8. The mechanism of DHEA-mediated increase in KCa-channel activity was probably mediated via increasing channel sensitivity to Em.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Insitute Grant HL-50153.
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FOOTNOTES |
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A portion of this work was presented at the Annual International Conference, American Thoracic Society in May 1996 in New Orleans, LA, and was published in abstract form (Am. J. Respir. Crit. Care Med. 153: A586, 1996).
Present address of U. Orlinska: ZENECA Pharmaceuticals, Wilmington, DE.
Address for reprint requests: I. S. Farrukh, Division of Respiratory, Critical Care and Occupational Pulmonary Medicine, Dept. of Internal Medicine, Univ. of Utah Health Science Center, Salt Lake City, UT 84132.
Received 16 May 1997; accepted in final form 14 October 1997.
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