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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Hypoxic pulmonary vasoconstriction (HPVC) is mediated, in part, via membrane depolarization and inhibition of K+ channels. We recently observed that the naturally occurring steroid dehydroepiandrosterone (DHEA) reversed and prevented HPVC in isolated perfused and ventilated ferret lungs. In the current study, we investigated the effects of DHEA on the major K+ channels of chronically hypoxic human pulmonary smooth-muscle cells (HPSMC). K+ channels were recorded by using the patch-clamp technique in whole-cell and single-channel configurations. Single-channel recordings were performed in inside-out and outside-out excised patches, and in intact HPSMC in cell-attached configuration. Using whole-cell current recording, chronic hypoxia decreased the high-amplitude, high-noise, and charybdotoxin-sensitive Ca2+-dependent K+ channels (KCa). DHEA reversed the effect of chronic hypoxia on KCa, but had no effect on the low-amplitude, low-noise, and 4-aminopyridine-sensitive delayed rectifying K+ channels. In the cell-attached configuration, chronic hypoxia caused a decrease in KCa sensitivity to membrane potential (Em). DHEA reversed the effect of hypoxia on KCa sensitivity to Em and caused a mean of 40-mV left shift in voltage-dependent activation of KCa. DHEA increased KCa activation from both sides of membrane patches of hypoxic HPSMC via a cyclic adenosine monophosphate- and cyclic guanosine monophosphate-independent pathway. We concluded that DHEA is a novel KCa opener of the human pulmonary vasculature.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Chronic hypoxia is associated with high morbidity and mortality secondary to pulmonary hypertension. The mechanisms by which alveolar hypoxia cause pulmonary vascular remodeling and progressive increase in pulmonary vascular resistance are not fully understood. Observations in the animal models suggest that acute (1) and chronic (4) hypoxia cause pulmonary vasoconstriction via depolarizing vascular smooth-muscle cell (SMC) membrane. SMC membrane potential (Em) is a major determinant of vascular tone. The Em of vascular SMC is controlled to a large degree by potassium channels (5, 6). The major types of K+ channels that are described in SMC are the large conductance and Ca2+-dependent K+ channels (KCa), the delayed rectifying K+ channels (Kdr), and the adenosine triphosphate (ATP)-sensitive K+ channels (KATP) (5, 7).
In human pulmonary SMC (HPSMC), we demonstrated that chronic hypoxia causes membrane depolarization in vivo and in vitro (4). The hypoxia-induced membrane depolarization was, in part, due to inhibition of the large conductance and KCa (4). KCa carry ionic currents that mediate membrane hyperpolarization and thus regulate important cellular functions, including vascular relaxation (5). KCa activity is regulated by several physiologic parameters, including membrane voltage, cytosolic Ca2+ concentration ([Ca2+]), and channel phosphorylation (4, 5, 8). The probability of KCa being open is relatively low in resting vascular SMC (5, 9). However, the opening state of the channels is increased by membrane depolarization (5, 8, 9). Thus, it is believed that KCa act as a relaxing negative feedback mechanism after agonist-induced membrane depolarization and extracellular Ca2+ entry. The purified toxin peptides, such as charybdotoxin (CTX) and iberiotoxin, and external tetraethylammonium (TEA) are known inhibitors of KCa (5, 9, 10), but little is known about KCa agonists.
Dehydroepiandrosterone (DHEA, 3
-hydroxy-5-androsten-17-one) and its sulfate ester are major secretory products of the adrenal cortex. The exact physiologic role of
DHEA is unknown. However, epidemiologic studies demonstrate that a high level of DHEA is an independent predictor of a significantly reduced risk for fatal coronary vascular disease in humans (11), and is inversely associated with the development of accelerated coronary allograft
vasculopathy (12). In addition, DHEA was reported to
cause a concentration-dependent inhibition of platelet aggregation in vivo and in vitro (13). This inhibition was associated with reduction in thromboxane B2 production
(13). Deoxycorticosterone, a derivative of the parent molecule pregnenolone and the precursor for DHEA, was reported to cause acute and chronic alteration in membrane
ion transport of aortic smooth muscle (14). In addition,
progesterone, another derivative of pregnenolone, was reported to cause smooth-muscle relaxation (15). We recently
observed that DHEA caused membrane hyperpolarization of ferret pulmonary SMC and reversed the acute hypoxic pulmonary vasoconstriction in isolated ferret lungs
(16). These observations suggest that the steroid-induced
pulmonary vasorelaxation is mediated via membrane hyperpolarization.
In this study, we investigated the effect of DHEA on the chronic hypoxia-induced membrane depolarization of HPSMC. We also determined whether the effects of DHEA on Em are induced via modulating potassium channels.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Patch-Clamp Technique
Currents were recorded with a patch-clamp amplifier (Axopatch 200A; Axon Instruments, Foster City, CA). Currents were low-pass filtered at 1 and 5 kHz and digitized at
5 and 10 kHz for single-channel and whole-cell configurations, respectively, using a Digidata 1200 interface (Axon
Instruments). The data were then stored on a computer
hard disk. For offline analysis of the data, we used the
pClamp program (Axon Instruments). Micropipettes were
made from borosilicate glass tubing (Corning no. 7052, 1.65 mm outer diameter, 1.2 mm inner diameter; Dow Corning Co., Midland, MI) and had a resistance of 2 to 5 M
when filled with pipette solution. Seal resistance was monitored on a Tektronix TDS oscilloscope (Tektronix, Inc.,
Beaverton, OR). The pipette tips were coated with Sylgard to reduce capacitance artifacts. The junction potential between the electrode and bath solution was nulled
before seal formation.
Coverslips containing the cells were placed in a recording chamber that was incorporated onto the stage of a Nikon Diaphot inverted microscope (Nikon, Inc., Melville, NY). Solution bottles were placed in a heater/circulator and warmed to 35°C (VWR Scientific, Philadelphia, PA). The perfusate tubing was jacketed with insulators, thus allowing the cells to be maintained at a temperature of 28 to 32°C. The cells were perfused at constant flow rate of 1.5 to 2.0 ml/min.
Single-Channel Patch Clamp
Single-channel currents were recorded in inside-out, outside-out, and cell-attached configurations (6, 17). Voltage-clamp potentials were applied to membrane patches, and
membrane currents were recorded with an Axopatch amplifier (Axopatch 200A; Axon Instruments). Mean values
for the state of channel open probability (Po) and mean
open times were obtained from a 5- to 15-min steady-state recording, and the channel open probability was expressed as NPo. Average channel activity (NPO) in patches
was determined from recordings by the formula NPO = (
Nj = 1 tj j)/T (4, 6), where PO is the open-state probability
of the channel; T is the duration of the recording in minutes; tj is the time spent, with j = 1, 2, . . . N channels open; and N is the maximal number of functional channels
observed during conditions of high state of PO.
Solutions and Chemicals
Bath solution for whole-cell experiments (in mM). NaCl, 130; NaHCO3, 10; KCl, 4.2; KH2PO4, 1.2; MgCl2, 0.5; CaCl2, 1.5; d-glucose, 5.5; N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes), 10. The pH was adjusted to 7.4 with NaOH. In some experiments, CaCl2 and EGTA concentrations were changed to achieve a desired Ca2+ concentration as indicated in RESULTS. In experiments that required omission of external Ca2+, Ca2+ was replaced by equimolar Mg2+.
Pipette solution for whole-cell experiments (in mM).
KCl, 140; MgCl2, 0.5; ethyleneglycol-bis-(-aminoethyl
ether)-N,N'-tetraacetic acid (EGTA) 0.1 or 10 (for eliciting Ca2+-activated K+ currents [IKCa] and delayed rectifying K+ currents [IKdr], respectively); Mg2-ATP, 5; Hepes,
5. The pH was adjusted to 7.2 with KOH.
Bath and pipette solutions for single-channel experiments (in mM). KCl, 140; MgCl2, 2; EGTA, 3; Hepes, 10; CaCl2, 2.29. The pH was adjusted to 7.4. The estimated free [Ca2+] was 300 nM, as computed by Fabiato's computer program (18). The HPSMC and membrane patches were continuously perfused with this solution. For inside-out pipette solutions and outside-out bath solutions, Ca2+ was replaced with equimolar Mg2+.
The bath solution of the normoxic and hypoxic HPSMC was perfused with physiologic solutions that were bubbled with 5% CO2-95% room air, and 5% CO2-95% N2, respectively. CTX was obtained from Alomone Labs (Jerusalem, Israel). The cyclic nucleotide-dependent protein kinase inhibitor N-(2-[methylamino]ethyl)-5-isoquinlinesulfonamide (H-8) and the cyclic nucleotide antagonists (Rp)-cGMPS and (Rp)-cAMPS were purchased from LC Laboratories (Woburn, MA). TEA, EGTA, verapamil, and 4-aminopyridine (4-AP) were obtained from Sigma Chemical Company, St. Louis, MO. When TEA and 4-AP concentrations exceeded 0.5 mM, the solution pH was adjusted to control values and osmolarity was kept constant by equimolar reduction of NaCl.HPSMC
HPSMC were cultured from explants of the human main pulmonary arteries (four) as previously described (4, 6). 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 culture wells with 0.05 ml chicken plasma (Sigma) plus 0.05 ml chick embryo extract (Sigma). The tissue and eventually the HPSMC were then plated in SMC culture medium, SmGM (Clonetics, San Diego, CA), that was supplemented with 5% fetal bovine serum and 10% bovine calf serum (HyClone, Logan, UT) plus dexamethasone (0.39 mg; Sigma) and antimicrobial agents (gentamicin, 50 µg/ml; amphotericin-B, 25 µg/ml; penicillin, 120 U/ ml; and streptomycin, 0.12 mg/ml; Sigma). All experiments were performed predominantly in primary HPSMC.
To study the effect of chronic hypoxia, control HPSMC were grown from pulmonary arterial explants under hypoxic conditions. The chamber of the incubator was maintained at 5 to 7% O2, 5% CO2, balanced N2. The oxygen level was monitored by an O2/CO2 sensor incorporated into the tissue incubator (Forma Scientific, Mariette, OH). The duration of cell culture for both hypoxic and normoxic HPSMC was about 25 d and was comparable for all groups of experiments.
Statistical Analysis
Values are presented as means ± SE. The data were analyzed using analysis of variance for repeated measures (Scheffe F-test significant at 95%). Results were considered statistically significant when P < 0.05.
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Results |
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Introduction
Materials and Methods
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Discussion
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Whole-Cell Current Experiments
Effect of DHEA on membrane depolarization after
chronic hypoxia in HPSMC.
The effect of DHEA on the
chronic hypoxia-induced change in Em was examined in
three groups of HPSMC using whole-cell recordings. Exposing the cells for 25 d of chronic hypoxia increased Em
from 
58.6 ± 0.8 to 35.5 ± 0.6 mV (n = 25 each, P < 0.01). Treating the hypoxic cells with DHEA (50 µM) hyperpolarized the membrane and reduced Em from 35.5
to 57.8 ± 0.9 mV (n = 25, P < 0.01).
Effect of chronic hypoxia on whole-cell K+ currents.
Net whole-cell K+ currents (IKo) were recorded in bath solutions containing 1.5 mM Ca2+ and pipette solutions containing 0.1 mM EGTA. The currents were evoked by
10-mV step-depolarizations for 500 ms duration from a
holding of 70 to 70 mV. The outward currents were activated at potentials positive to 60 mV, and were time-
and voltage-dependent (Figure 1A). The currents have
two components: high-amplitude, high-noise (IKCa); and
low-amplitude, low-noise (IKdr). We have previously reported that the first component of HPSMC IKo is Ca2+-
and CTX-sensitive, and resistant to 4-AP. The second
component of IKo is CTX-insensitive but can be inhibited
by 4-AP (4, 6). Compared with control normoxic HPSMC,
chronic hypoxia caused a significant reduction in the high-amplitude and high-noise component of HPSMC IKo, or
IKCa (Figure 1B). Treating hypoxic HPSMC with CTX
(200 nM) abolished the high-noise currents that were activated at Em positive to 10 mV (Figures 2 and 3). These
results suggest that in HPSMC chronic hypoxia causes a
30-mV shift in KCa activation (Figure 3). The low-noise
and low-amplitude currents, or IKdr, were not affected by
chronic hypoxia and were insensitive to CTX, but were
blocked by 2 mM 4-AP (Figures 2 and 3).
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Effect of DHEA on IKo in Hypoxic HPSMC.
It was suggested in isolated ferret lungs that treatment with DHEA
reversed hypoxic pulmonary vasoconstriction via membrane hyperpolarization (16). In this group of experiments,
we tested the effect of DHEA (50 µM) on IKo in chronically hypoxic HPSMC. In chronically hypoxic HPSMC,
DHEA significantly increased the high-noise, high-amplitude current without affecting the low-noise and 4-AP-sensitive current, IKdr (Figure 1). Treating hypoxic HPSMC
with CTX (200 nM) abolished the high-noise currents that were activated at Em positive to 10 mV, IKCa, and prevented
the DHEA-induced increase of IKCa (Figure 3). In another
group of experiments we measured the effect of strong
Ca2+ buffering on the DHEA-mediated increase in IKo. To
obtain maximal Ca2+-buffer capacity of the pipette solution, we used nominal Ca2+ buffer plus 10 mM EGTA in
the pipette solution, and the bath solution contained no
added Ca2+. We have previously reported that this protocol
blocks IKCa in HPSMC (6). Strong Ca2+ buffering blocked
the effect of DHEA on IKo in hypoxic HPSMC (Figure 4).
This suggests that DHEA increases KCa activity in hypoxic
HPSMC, and that the DHEA-induced increase in KCa activity is most likely mediated by an approximately 40-mV
shift in voltage-dependent activation of KCa.
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Single IKo Channel Experiments
Effect of DHEA on K+ channels in intact HPSMC. To determine further the type of channel involved in the DHEA-induced membrane hyperpolarization, we investigated the effect of DHEA on single K+ channels in HPSMC membrane patches. Single channels were recorded in inside-out configuration before and after exposing HPSMC to DHEA. Chronic hypoxia decreased the activity of KCa without affecting Kdr (NPO = 0.20 ± 0.005 for control normoxia versus 0.03 ± 0.001 for hypoxic HPSMC, n = 8, P < 0.0001, Figure 5). Treating hypoxic cells with DHEA (50 µM) caused a significant increase in KCa activity (NPO = 0.35 ± 0.013 for hypoxic cells treated with DHEA versus 0.03 ± 0.001 for control hypoxia, n = 8, P < 0.0001, Figure 5). DHEA had no significant effect on Kdr (NPO = 0.002 ± 0.0003 for DHEA-treated cells versus 0.0019 ± 0.00028 for control, n = 6, P > 0.05). The effect of DHEA on KCa activity was reversible after washout (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 12.65 ± 0.19 pA versus 12.5 ± 0.14 pA for control, n = 8, P > 0.05).
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Effect of DHEA on K+ channel sensitivity to Em.
To investigate further the mechanism by which DHEA activates KCa in HPSMC, we determined its effect on the sensitivity of KCa channel to Em. In inside-out excised patches
with perfusate-free [Ca2+] = 300 nM and at an Em range of
20 to 60 mV, DHEA caused a significant increase in KCa
activity at all of the Em values (n = 8, P < 0.0001, Figure
6). The linear fit of ln{NPO /(1 NPO)} versus Em demonstrated that, in chronically hypoxic HPSMC, DHEA caused an approximately 40-mV left shift in voltage-dependent activation of KCa (Figure 6). This was equivalent to about
1.4-fold increase in KCa activity at any Em. These results are
in agreement with the whole-cell current recording presented in Figure 3 and suggest that in HPSMC, DHEA increases KCa activity by increasing the sensitivity of the
channel to Em.
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Role of cGMP and cAMP in the DHEA-mediated KCa activation in intact hypoxic HPSMC. Because the mechanism of vascular agonist-induced increase in KCa activity is reported to be mediated via cyclic neucleotides (8, 19), we investigated the possible role of cGMP or cAMP in the DHEA-mediated KCa activation. We tested the effect of antagonizing the cyclic nucleotides with their Rp isomers, Rp-cGMPS and Rp-cAMPS, on the DHEA-induced increase in KCa activity.
In hypoxic HPSMC, both Rp-cGMPS and Rp-cAMPS reduced baseline activity of KCa (NPO = 0.009 ± 0.0005 for hypoxic HPSMC versus 0.007 ± 0.0006 and 0.0076 ± 0.0006 for Rp-cGMPS and Rp-cAMPS, respectively, n = 6 each, Figure 7). Addition of DHEA (50 µM) to cells pretreated with either of the Rp isomers caused a significant increase in KCa activity (NPO = 0.009 ± 0.001 for control versus 0.09 ± 0.01 and 0.091 ± 0.007 for Rp-cGMPS + DHEA and Rp-cAMPS + DHEA, respectively, P < 0.001, n = 6 each, Figure 7). This suggests that the DHEA-induced activation of KCa is not mediated via generation of cGMP or cAMP.
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Introduction
Materials and Methods
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The principal observations in this study indicate that chronic hypoxia in human pulmonary vascular SMC causes membrane depolarization and decreases the high-amplitude, high-noise, and CTX-sensitive component of IKo, IKCa. The effects of chronic hypoxia on IKo of HPSMC were reversed with the naturally produced hormone DHEA. The mechanism of DHEA-induced HPSMC hyperpolarization is mediated via a cGMP- and cAMP-independent increase in KCa activity.
Effect of Chronic Hypoxia
Although the response of the pulmonary vasculature to acute hypoxic challenge has been studied extensively (20- 23), the effects of sustained or chronic hypoxia on pulmonary vascular SMC IKo and Em are not fully understood. Using single-channel recordings, we previously reported that chronic hypoxia inhibited a large conductance and CTX-sensitive K+ channels in SMC isolated from human pulmonary arterial tissue (4). In the present investigation, we observed that chronic hypoxia lowered the high-noise and high-amplitude component of the net whole-cell outward currents. This component of IKo was activated at an Em positive to 10 mV and was inhibited with CTX. These results suggest that chronic hypoxia inhibited KCa channels by causing a 30-mV shift on channel sensitivity to Em. In addition, the CTX-insensitive component of whole-cell IKo was not affected by chronic hypoxia, but was significantly inhibited by 4-AP. This provides further evidence that chronic hypoxia maintains membrane depolarization in HPSMC by inhibiting KCa channels rather than Kdr. Nossaman and colleagues (24) reported in isolated-perfused rat lungs that the pulmonary vascular response to alveolar hypoxia is potentiated by the KCa antagonist CTX. Cornfield and associates (25) reported in fetal sheep that oxygen-induced pulmonary vasodilatation is mediated through activation of KCa. These authors observed that the fetal pulmonary arterial SMC was depolarized under hypoxic conditions. Subsequent administration of oxygen caused membrane hyperpolarization that was inhibited by CTX (25). The reported data and our observations indicate that KCa channels play an important role in regulating pulmonary vascular tone under hypoxic conditions. Inhibiting KCa after chronic exposure to hypoxia deprives the HPSMC of an effective mechanism to counteract the effect of hypoxia on its membrane potential.
Role of KCa Agonist
We previously reported that DHEA in an isolated ferret
lung preparation reversed the sustained hypoxic pulmonary
vasoconstriction and reversed the effect of TEA-induced
potentiation of hypoxic vasoconstriction (16). These observations suggest that DHEA reversed hypoxic pulmonary
vasoconstriction through activating K+ channels. In the current study, we observed that DHEA hyperpolarized the Em
of chronic hypoxic HPSMC and increased the KCa component of whole-cell IKo. DHEA reversed the effect of chronic
hypoxia on Em by inducing a 40-mV left shift in voltage-dependent activation of KCa. Using single-channel recordings in the cell-attached configuration, DHEA increased
KCa activity in hypoxic HPSMC. The DHEA-induced increase in KCa activity was blocked by a specific KCa inhibitor, CTX. In addition, the effect of DHEA was observed
on either side of the membrane, suggesting that the effect
of DHEA on KCa was not mediated by a surface receptor.
In intact HPSMC in the cell-attached configuration, the
cGMP and cAMP antagonists (Rp-cGMPS and Rp-cAMPS, respectively) did not block the effect of DHEA on KCa. In
addition, in excised inside-out HPSMC membrane patches,
blocking protein kinase mediated phosphorylation of KCa
with H-8 had no significant effect on the DHEA-mediated
KCa activation. These observations suggest that the DHEA-mediated increase in KCa activity was independent of cAMP-
or cGMP-induced phosphorylation. In intact and hypoxic HPSMC, when the current was recorded in cell-attached
configuration, DHEA caused a 21- to 24-mV left shift
in voltage-dependent activation of KCa. This was equivalent to an approximately 1.4-fold increase in KCa activity at
any Em value in the range of 20 to 60 mV. This represents a pathway for KCa activation that has not been described previously, and suggests that DHEA is a novel KCa
channel opener. It is not clear how DHEA increased KCa
activity. DHEA can activate KCa indirectly by modulating
the cytosolic -subunit(s) of the channel, or by interacting
directly with the inner and/or outer parts of the 
-subunit
(26).
The previously described K+ channel openers (cromakalim and pinacidil) are believed to mediate their vasorelaxing effect by activating KATP (7). Limited information is available about KCa openers. Unlike DHEA, soyasaponins, which are isolated from a medicinal herb, can activate KCa only from inside the cell (27). However, their poor membrane permeation limits their therapeutic use (27). Other agents, such as the synthesized imidazopyrazine derivatives, are reported to cause aortic and tracheal smooth-muscle relaxation in part by activating KCa (28). These conclusions were reached because SCA40, the most potent imidazopyrazine derivative, inhibited the effect of an intermediate [KCl] (20 mM) on rat isolated aortic vessels (28). The vasorelaxing effect of SCA40 was not inhibited by the KATP inhibitor glibenclamide but was antagonized by CTX (28). Although these studies were suggestive that SCA40-induced vasorelaxation was mediated through activation of KCa, the investigators provided no electrophysiologic evidence to demonstrate that this agent actually increases the activity of KCa.
Openers of KCa have great therapeutic potential and serve as important investigational tools. KCa have large unitary conductances. Thus, the opening of only a few KCa channels has a significant impact on Em (5, 9). The ability of DHEA to open KCa and relax constricted pulmonary vasculature (16) may provide a novel therapeutic agent for pathologic conditions, such as chronic hypoxia, in which the pulmonary vascular membrane potential is depolarized.
In summary, chronic hypoxia causes a decrease in HPSMC whole-cell IKCa. DHEA, a naturally produced hormone, reversed the chronic hypoxia-induced decrease in the activity of KCa. The DHEA-induced increase in KCa 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 activity is probably mediated via increasing channel sensitivity to Em.
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Footnotes |
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Address correspondence to: Imad S. Farrukh, M.D., Div. of Respiratory, Critical Care and Occupational Pulmonary Medicine, Dept. of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, UT 84132. E-mail: IFARRUKH{at}MED.UTAH.EDU
(Received in original form April 9, 1998 and in revised form August 26, 1998).
Abbreviations: 4-aminopyridine, 4-AP; adenosine triphosphate, ATP; cytosolic Ca2+ concentration, [Ca2+]; charybdotoxin, CTX; dehydroepiandrosterone, DHEA; ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic
acid, EGTA; membrane potential, Em; N-(2-[methylamino]ethyl)-5-isoquinlinesulfonamide, H-8; cultured human pulmonary arterial smooth-muscle cells, HPSMC; Ca2+-activated K+ currents, IKCa; delayed rectifying K+
current, IKdr; K+ currents, IKo; ATP-sensitive K+ channels, KATP; Ca2+-
dependent K+ channels, KCa; delayed rectifying K+ channels, Kdr; cAMP
antagonist, Rp-cAMPS; cGMP antagonist, Rp-cGMPS; smooth-muscle
cell, SMC; tetraethylammonium, TEA.
Acknowledgments: A portion of this work was submitted to the Annual Internal Conference-American Thoracic Society (May 10-15, San Francisco, CA) and was published in an abstract form (Am. J. Respir. Crit. Care Med. 1997; 155: A790).
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