A maturational shift in pulmonary K+ channels, from Ca2+ sensitive to voltage dependent

Helen L. Reeve1, E. Kenneth Weir2,3, Stephen L. Archer4, and David N. Cornfield5

Departments of 2 Medicine, 5 Pediatrics, and 1 Physiology, University of Minnesota, Minneapolis 55455; 3 Department of Cardiology, Veterans Affairs Medical Center, Minneapolis, Minnesota 55417; and 4 Department of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2B7

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The mechanism responsible for the abrupt decrease in resistance of the pulmonary circulation at birth may include changes in the activity of O2-sensitive K+ channels. We characterized the electrophysiological properties of fetal and adult ovine pulmonary arterial (PA) smooth muscle cells (SMCs) using conventional and amphotericin B-perforated patch-clamp techniques. Whole cell K+ currents of fetal PASMCs in hypoxia were small and characteristic of spontaneously transient outward currents. The average resting membrane potential (RMP) was -36 ± 3 mV and could be depolarized by charybdotoxin (100 nM) or tetraethylammonium chloride (5 mM; both blockers of Ca2+-dependent K+ channels) but not by 4-aminopyridine (4-AP; 1 mM; blocker of voltage-gated K+ channels) or glibenclamide (10 µM; blocker of ATP-dependent K+ channels). In hypoxia, chelation of intracellular Ca2+ by 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid further reduced the amplitude of the whole cell K+ current and prevented spontaneously transient outward current activity. Under these conditions, the remaining current was partially inhibited by 1 mM 4-AP. K+ currents of fetal PASMCs maintained in normoxia were not significantly reduced by acute hypoxia. In normoxic adult PASMCs, whole cell K+ currents were large and RMP was -49 ± 3 mV. These 4-AP-sensitive K+ currents were partially inhibited by exposure to acute hypoxia. We conclude that the K+ channel regulating RMP in the ovine pulmonary circulation changes after birth from a Ca2+-dependent K+ channel to a voltage-dependent K+ channel. The maturational-dependent differences in the mechanism of the response to acute hypoxia may be due to this difference in K+ channels.

ion channels; hypoxia; pulmonary circulation; fetus; adult

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE MECHANISMS RESPONSIBLE for maintenance of the high-tone, low-flow fetal pulmonary vasculature and the rapid increase in pulmonary blood flow that occurs at birth remain incompletely understood (28). Closely regulated and limited pulmonary blood flow is necessary for normal pulmonary vascular development (7, 22, 34). At birth, pulmonary blood flow increases 8- to 10-fold, whereas pulmonary arterial (PA) pressure declines by >50% over the first 24 h of life (9, 13, 14). Although physical factors and vasoactive products elaborated by the pulmonary vascular endothelium have been shown to be involved in the regulation of perinatal vascular tone (3, 10, 11, 15, 30), evidence also suggests an important role for PA smooth muscle cells (SMCs) (11, 12).

The low PO2 environment of the normal fetus may contribute to the high fetal pulmonary vascular resistance by direct effects on the fetal PASMC K+ channels. In adult animals, acute (6, 26, 35) and chronic (29) hypoxia cause inhibition of K+-channel activity and membrane depolarization of PASMCs. The observation that O2-induced fetal pulmonary vasodilation is prevented by blockade of Ca2+-dependent K+ (K Ca) channels suggests a central role for K+ channels in perinatal pulmonary vasodilation (11, 31). Furthermore, inhibition of K Ca channels in cultured fetal PASMCs causes membrane depolarization and an increase in cytosolic Ca2+ concentration (12). A patch-clamp study (11) showed that an increase in PO2 in freshly isolated fetal PASMCs increases K+ efflux through K Ca channels and causes membrane hyperpolarization. In contrast, in adult PASMCs, the O2-sensitive K+ channel appears to be a 4-aminopyridine (4-AP)-sensitive voltage-dependent K+ (K v) channel (6, 35). Although these observations support the importance of PASMC K+ channels in the regulation of perinatal pulmonary vascular tone, they suggest that it is not the same K+ channel that responds to O2 in the fetus and adult.

Maturation-related differences in the capacity of the pulmonary vasculature to respond to changes in PO2 have been previously described (2, 20, 36). For example, a 4- to 6-mmHg increase in PO2 in the late-gestation ovine fetus causes a three- to fourfold increase in pulmonary blood flow (4), whereas in the early-gestation animal, there is no increase in pulmonary blood flow (23). Pulmonary vasodilation caused by endothelium-dependent nitric oxide (NO), a vasoactive mediator that is essential for the normal transition of the pulmonary vasculature (1, 20), also increases with maturation (2). NO causes pulmonary vasodilation, at least in part, through activation of K Ca channels (5). If PASMC K+ channels play a central role in the regulation of perinatal pulmonary vascular tone, then maturation-related changes in PASMC K+-channel activity might also be expected.

To investigate changes in functional expression of K+ channels between the fetal and adult pulmonary circulations, whole cell studies with conventional and amphotericin B-perforated patch-clamp techniques were done in freshly dispersed PASMCs from late-gestation fetal and adult sheep. To study basal physiological K+-channel activity and examine the effects of acute hypoxia on that activity, an environment with a similar PO2 to that seen in the SMCs under study was established. For the fetal PASMCs, the PO2 is normally low (~20 mmHg), whereas in the adult, resistance PASMCs have a PO2 of ~140 mmHg. Thus patch-clamp studies were initially done with cells perfused with hypoxia for the fetus and normoxia for the adult. Because fetal K+-channel activity is known to increase on switching from hypoxia to normoxia (11), studies were also done with normoxic fetal PASMCs to determine whether normoxic currents were sensitive to acute hypoxia.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Whole Cell Patch Clamp

Cell isolation. Approval for the animal studies was obtained for all studies, and experiments were done according to institutional guidelines. Late-gestation fetal (137-144 days, term 147 days) and adult sheep were anesthetized with high-dose pentobarbital sodium, and their lungs were removed. Resistance PAs (5th-7th divisions) were dissected into Ca2+-free Hanks' solution and kept for 30 min at 4°C. The Hanks' solution contained (in mM) 140 NaCl, 4.2 KCl, 1.2 KH2PO4, 0.5 MgCl2, 10 HEPES, and 0.1 EGTA (pH 7.4 with NaOH). The PAs were then transferred to an enzyme solution in Hanks' solution without EGTA containing 1 mg/ml of papain, 0.75 mg/ml of dithiothreitol, and 0.8 mg/ml of albumin for 25 min at 4°C and then for 10-15 min at 37°C. For hypoxic experiments with fetal PASMCs, all enzyme solutions were deoxygenated with N2 to maintain cells in hypoxia. This digestion protocol consistently produced high yields of elongate, viable, relaxed SMCs. After digestion, cells were maintained on ice in Hanks' solution supplemented with 10 mg/ml of glucose (deoxygenated for hypoxic fetal cells).

Whole cell recordings. Gentle trituration produced a suspension of single cells that were pipetted into a perfusion chamber on the stage of an inverted microscope for conventional whole cell (17) and perforated patch-clamp (27) studies. After a brief period to allow partial adherence to the bottom of the recording chamber, cells were perfused with either a hypoxic (fetal PASMCs) or normoxic (fetal and adult PASMCs) solution composed of (in mM) 115 NaCl, 25 NaHCO3, 4.2 KCl, 1.2 KH2PO4, 1.5 CaCl2, and 10 HEPES (pH 7.4 with NaOH). Hypoxic solutions (PO2 ~35 mmHg) were bubbled with 3.5% CO2-balance N2, whereas normoxic solutions (PO2 ~160 mmHg) were bubbled with 20% O2-3.5% CO2-balance N2. O2 levels were measured with a Ciba Corning blood gas analyzer from samples taken from the bath during perfusion. Electrodes (resistance 2-3 MOmega ) were fire polished and filled with a solution composed of (in mM) 140 KCl, 1.0 MgCl2, 10 HEPES and 1.0 EGTA or 5 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; see below) (pH 7.2 with KOH). For perforated patch-clamp studies, amphotericin B (120 µg/ml) was included in the pipette solution. Capacitance was corrected for, and perforation was monitored by changes in membrane potential [observed in current (I) = 0 mode on the patch-clamp amplifier] and changes in series resistance. Recordings were not made until series resistance was <15 MOmega to minimize voltage errors. The small currents recorded in the fetal cells usually made series resistance compensation unnecessary. In the adult cells, series resistance was further minimized by electronic compensation.

Outward currents were recorded from fetal and adult cells with the perforated patch-clamp technique. For these studies, cells were voltage clamped at a holding potential of -70 mV, and currents were evoked by +10- or +20-mV steps to more positive potentials, with test pulses of 100- or 200-ms duration.

A previous study (8) on spontaneously transient outward current (STOC) activity show that the activity can be inhibited by high (1-10 mM) intracellular concentrations of the Ca2+ chelator EGTA. To test the Ca2+ sensitivity of outward currents recorded from hypoxic fetal cells, conventional whole cell recordings were made with 5 mM BAPTA (a Ca2+ chelator with a faster Ca2+-binding time than EGTA) (32) included in the pipette solution. For some of these studies, cells were also voltage clamped at a holding potential of -10 mV to inactivate K v channels and then depolarized to more positive potentials in +10-mV steps. For all patch-clamp studies, currents were filtered at 1 kHz and sampled at 2.5 kHz.

Membrane potentials were recorded with the perforated patch-clamp technique in current-clamp mode at the resting membrane potential (RMP; measured as zero potential) of each cell. Membrane potential stability was always determined for at least 1 min before any recording. Data were recorded and analyzed with pClamp 6.02 software (Axon Instruments, Foster City, CA). Drugs dissolved in the extracellular perfusate were applied to the cells via gravity perfusion at a rate of 2 ml/min. All experiments were performed at 22°C.

Drugs Used

4-AP (inhibitor of K v channels), tetraethylammonium chloride (TEA; inhibitor of K Ca channels), and glibenclamide (inhibitor of ATP-dependent K+ channels) were obtained from Sigma (St. Louis, MO), whereas charybdotoxin (CTX; inhibitor of K Ca channels) was obtained from RBI (Natick, MA). Drug solutions were adjusted to pH 7.4. All drugs were dissolved in normal saline except glibenclamide, which was dissolved in ethanol and then diluted in normal saline. The diluted ethanol vehicle was tested and found to have no effect on K+-channel activity (data not shown).

Statistical Analysis

Intergroup differences were assessed by a factorial analysis of variance with post hoc analysis with Fisher's least significant difference test. Data are expressed as means ± SE. Membrane potential data were compared with Student's unpaired t-test. P values < 0.05 were considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

K+-Channel Activity in Fetal and Adult PASMCs

Basal whole cell K+-current (IK) values were recorded from fetal PASMCs in hypoxia and normoxia and from adult PASMCs in normoxia. IK recorded in fetal PASMCs in hypoxia was small (473 ± 151 pA at +50 mV; n = 11; Fig. 1A) and had the characteristics of STOCs (Fig. 1B) as previously described (14). STOC activity could be recorded with both conventional whole cell and perforated patch-clamp techniques but gradually declined over time under the conventional configuration (data not shown). Pharmacological studies on STOC activity were therefore done with the perforated patch clamp unless otherwise stated. When the cells were held at a steady-state potential of -20 mV, outward STOCs could be inhibited by 100 nM CTX (n = 4) or 5 mM TEA (n = 6), suggesting that they were due to the activation of K Ca channels (Fig. 2). The inhibition of STOCs by TEA could be completely reversed, whereas CTX required longer periods of wash to partially recover activity.


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Fig. 1.   A: average whole cell current-voltage (I-V) plots of outward K+-channel currents recorded from pulmonary arterial smooth muscle cells (PASMCs) with conventional and perforated whole cell patch-clamp techniques. Currents have been corrected for cell capacitance and plotted as current density. Data are means ± SE from fetal PASMCs in hypoxia + 5 mM intracellular BAPTA (n = 4), fetal PASMCs in hypoxia (n = 11), fetal PASMCs in normoxia (n = 5), and adult PASMCs in normoxia (n = 6). All currents were evoked from a holding potential of -70 mV. Significantly different (P < 0.05) from: * adult cells; # normoxic fetal cells. B: actual current traces recorded from hypoxic fetal (left) and normoxic adult PASMCs (right).


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Fig. 2.   Representative recording of spontaneous transient outward currents (STOCS) in a fetal PASMC in hypoxia. Currents were recorded with perforated patch-clamp technique at a steady-state potential of -20 mV. Application of 100 nM charybdotoxin (CTX) is indicated. Break in trace, 15-min washout period.

Because CTX has been shown to block at least two K v channels (16), the Ca2+ sensitivity of the IK activity in hypoxic fetal PASMCs was also tested. With the use of the conventional whole cell patch clamp, cells were dialyzed with the rapid Ca2+ chelator BAPTA (5 mM). Under these conditions, whole cell IK was further reduced (202 ± 44 pA at +50 mV; n = 4 cells; Fig. 1A), and there was no evidence of STOC activity. The remaining outward current (~40% of IK recorded from hypoxic fetal PASMCs with the perforated patch clamp) could be inhibited by 1 mM 4-AP (81.4 ± 2.8% at +50 mV; n = 3; Fig. 3A). Therefore, a small component of hypoxic fetal IK was due to activation of K v channels. Even with BAPTA in the pipette, K Ca-channel activity could be recorded by holding cells at a potential of -10 mV to inactivate K v-channel activity. This channel activity was inhibited by 100 nM CTX (Fig. 3B). Because Cornfield et al. (11) have previously shown, using the perforated patch-clamp technique, that hypoxic fetal IK levels are increased by normoxia, BAPTA-dialyzed cells were exposed to 4 min of normoxia, and the effects on K Ca channels were recorded at potentials positive to -10 mV. Under these conditions, K Ca-channel activity was increased in normoxia (Fig. 3B).


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Fig. 3.   A: representative recording of outward currents recorded from a hypoxic fetal PASMC with conventional whole cell patch clamp with 5 mM BAPTA in pipette solution before (left) and after (right) 1 mM 4-aminopyridine (4-AP). Currents were evoked from a holding potential of -70 mV in +20-mV steps (n = 3 cells). B: currents recorded from a holding potential of -10 mV under the same conditions as in A except with addition of CTX. Currents were evoked in +10-mV steps (n = 4 cells).

IK recorded from normoxic fetal PASMCs was larger (876 ± 261 pA at +50 mV; n = 5: Fig. 1A) than IK recorded from hypoxic PASMCs and without STOC activity but was still inhibited by TEA (data not shown). IK values recorded in normoxia from adult PASMCs were larger than those recorded from either fetal preparation (1,389 ± 214 pA at +50 mV; n = 11 cells; Fig. 1A), with no significant difference in cell capacitance (15.9 ± 1.1 pF, n = 11; 17.8 ± 0.9 pF, n = 5; and 17.7 ± 1.5 pF, n = 11 for hypoxic fetal cells, normoxic fetal cells, and normoxic adult cells, respectively). Outward currents in the adult PASMCs had no STOC activity and were characteristic of delayed rectifier activity (Fig. 1B). At positive membrane potentials, currents could be inhibited by both 1 mM 4-AP (61.1 ± 7.4% at +50 mV; n = 6 cells) and 5 mM TEA (33.3 ± 2.7% at +50 mV; n = 4 cells), suggesting that, as in the fetal cells, both K v and K Ca channels were present but that K v channels were predominant. TEA had little effect on IK recorded at negative membrane potentials, in contrast to 4-AP that completely inhibited currents evoked at these potentials (Fig. 4). Figure 4B shows the percentage of IK inhibited by 5 mM TEA at potentials of -10 and +50 mV in both fetal and adult PASMCs and clearly demonstrates the decrease in contribution of K Ca channels to IK during maturation.


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Fig. 4.   A: average whole cell I-V plots of outward K+ currents recorded from adult PASMCs with perforated patch clamp in normoxia between -50 and -10 mV. Currents were recorded during control and after exposure to 1 mM 4-AP (n = 6 cells) or 5 mM tetraethylammonium (TEA; n = 4 cells). Values are means ± SE. TEA only inhibited currents at potentials more positive than -10 mV. B: percentage of outward K+ current (IK) inhibited by 5 mM TEA at -10 and +50 mV in fetal and adult PASMCs.

RMP in Fetal and Adult PASMCs

Average RMP recorded from fetal cells in hypoxia was -36.5 ± 3 mV (n = 5), with membrane depolarization occurring on exposure to 100 nM CTX (depolarized to -18.8 ± 2 mV; n = 5; Fig. 5) but not to 4-AP (1 mM) or glibenclamide (10 µM; data not shown). Switching the perfusion solution to normoxia hyperpolarized the cell membrane to -49.0 ± 2 mV (n = 5 cells; Fig. 5). Membrane hyperpolarization occurred rapidly (within 5 min of switching solutions). Average RMP recorded from adult PASMCs was the same as that recorded from the normoxic fetus (-49.4 ± 3 mV) and could be depolarized by 1 mM 4-AP (to -30.6 ± 4 mV; n = 5) but was not affected by exposing the cells to TEA or CTX (n = 5; Fig. 5).


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Fig. 5.   Membrane potentials (Em) recorded from fetal and adult PASMCs with perforated patch clamp. Fetal membrane potentials were recorded in hypoxia, after exposure to 100 nM CTX, and after exposure to normoxia. Adult membrane potentials were recorded in normoxia and after exposure to either 4-AP or 100 nM CTX. * P < 0.05 from hypoxic control cells in fetus and normoxic control cells in adult.

Effect of Hypoxia on IK in Fetal and Adult PASMCs

Exposure of normoxic fetal PASMCs to 3 min of hypoxia had little effect on IK (15.1 ± 9% inhibition at +10 mV; n = 4; not significant; Fig. 6). In contrast, IK recorded from adult PASMCs was partially inhibited by acute exposure to hypoxia (33.5 ± 6% inhibition at +10 mV; P < 0.05; n = 5; Fig. 6).


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Fig. 6.   Average whole cell I-V plots of IK normalized to maximum current (I/Imax) at +50 mV recorded from fetal (n = 4; black-triangle) and adult (n = 6; bullet ) PASMCs under normoxia and after 3-min exposure to hypoxia (). * P < 0.05.

    DISCUSSION
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Introduction
Methods
Results
Discussion
References

The mechanism by which hypoxia contributes to the low-flow, high-resistance fetal pulmonary vasculature remains uncertain as does the full mechanism for the dilation of the pulmonary vasculature that occurs at birth. Although the mechanical effects of ventilation and endothelium-dependent dilator factors such as NO play an important role (20), there is also evidence that PASMCs are involved (11, 12). In the adult pulmonary vasculature, hypoxia has been shown to inhibit K v channels in PASMCs to initiate the constriction involved in matching ventilation and perfusion (6, 35). A previous study (11) with fetal PASMCs suggested that the K+ channel controlling RMP and activated by O2 is Ca2+ sensitive. To fully determine the roles of the K v and K Ca channels in both fetal and adult PASMCs, electrophysiological studies of their K+-channel activity were done in both hypoxia and normoxia.

Our data suggest that there are maturational changes in K+-channel expression between the fetus and adult. The K+ channel controlling RMP in the hypoxic environment of the developing fetus appears to be a K Ca channel because membrane potentials recorded from fetal PASMCs were only depolarized by CTX and TEA (K Ca-channel blockers) (Fig. 5). Outward currents in hypoxic fetal PASMCs are characteristic of STOCs. STOCs are likely to be due to the activation of K Ca channels by quantal releases of Ca2+ from the sarcoplasmic reticulum (24). Their activity gradually declined under conditions of cell dialysis with the conventional whole cell patch clamp and could be prevented by chelation of intracellular Ca2+ with high concentrations of BAPTA. The presence of STOCs may reflect the high-tone environment of the fetal pulmonary vasculature because they are also found in other high-resistance beds such as the adult cerebral circulation (24). The physiological significance for STOCs in the developing fetal pulmonary circulation is unknown but may provide a moderating influence that prevents fetal pulmonary vascular resistance from rising too high. The patch-clamp evidence that K Ca-channel activity controls RMP and hence helps to determine pulmonary vascular tone in the fetus is consistent with previous data (12) showing that blockade of K Ca channels in single fetal PASMCs increases cytosolic Ca2+ concentration. It should be noted, however, that in the intact, acutely instrumented fetus, K Ca-channel blockade has not been conclusively shown to affect basal pulmonary vascular tone. This apparent contradiction may be due to the presence of vasoactive mediators such as NO (1) or prostacyclin (18) in the intact pulmonary circulation. These vasodilator agents may antagonize the constrictor effect of K Ca-channel blockade in the whole animal. Indeed, initial patch-clamp studies support this interpretation by showing that NO can overcome the blocking effect of CTX on single K Ca channels (Reeve, unpublished observations).

Although the K Ca channel appears to control RMP, there is some K v-channel activity in hypoxic fetal PASMCs as indicated by partial inhibition of IK by 4-AP (Fig. 3A). In the normoxic adult PASMCs, both K v and K Ca channels contribute to IK, but here K v-channel activity is predominant and controls RMP (Figs. 4 and 5). Because hypoxia has been reported to inhibit one or more K v channels to initiate pulmonary vasoconstriction, the maturation-related increase in the capacity of the pulmonary vasculature to respond to hypoxia may be due to increasing K v-channel activity with age. Exposure to chronic hypoxia can downregulate some K v channels in rat PASMCs (29, 33) and upregulate K Ca-channel activity in human PASMCs (25). Chronic hypoxia is also known to depolarize the RMP of cells to similar values as those recorded from hypoxic fetal PASMCs (29). Because PO2 in the fetus is normally low, it is possible that prolonged exposure to hypoxia downregulates K v-channel activity, resulting in a depolarized membrane potential and hence dominant K Ca-channel activity. It has also been shown in cultured cell lines that prolonged depolarization can downregulate K v-channel expression, providing an additional pathway by which K v-channel activity may be modulated in the constricted fetal pulmonary circulation (19). Furthermore, in systemic hypertension, where basal tone is increased as the blood vessels constrict, there is a compensatory increase in the expression of K Ca channels (21). Importantly, although K v-channel activity was observed in hypoxic fetal PASMCs, it was only recorded when cells were depolarized from a holding potential of -70 mV. At a more depolarized holding potential closer to the RMP of these cells, K v-channel activity was inhibited and only K Ca-channel activity was recorded (Fig. 3).

Because normoxia hyperpolarizes fetal PASMC membrane potential to values similar to those recorded from normoxic adult PASMCs, it could be considered that normoxia simply brings the fetal pulmonary circulation to membrane potentials where K v channels are active. In this case, hypoxia might be expected to inhibit IK recorded from normoxic fetal PASMCs. However, our data do not support this, with little effect of hypoxia observed on IK recorded from fetal PASMCs that had been normoxic for 2-6 h. In contrast, IK values recorded from normoxic adult PASMCs were partially inhibited by acute hypoxia (Fig. 6). On reflection, these data might not be considered surprising. In the whole lung, chronic exposure to hypoxia prevents subsequent pulmonary vasoconstriction to acute hypoxic challenges. Animals must be returned to normoxia for at least 24 h before this acute hypoxic vasoconstriction returns (D. P. Nelson and E. K. Weir, unpublished observations), suggesting that there is some delay in the recovery of the mechanism by which acute hypoxia is sensed. The short exposure of the fetal PASMCs to normoxia before their exposure to acute hypoxia may not have allowed for this conversion to occur. The increase in the proportion of IK sensitive to 4-AP in the adult suggests an increase in K v-channel activity that may account for the larger whole cell currents even between normoxic fetal and normoxic adult cells. Although acute normoxia activates K Ca channels to initiate membrane hyperpolarization, long-term normoxia may allow the upregulation of K v channels and development of the mechanism by which acute hypoxia is sensed.

In summary, these studies show that there are maturational changes in K+-channel activity in the pulmonary circulation and that the change in the channel controlling the RMP between the hypoxic fetal and normoxic adult circulations may parallel the different O2-sensing mechanisms of each bed.

    ACKNOWLEDGEMENTS

H. L. Reeve was supported by National Heart, Lung, and Blood Institute Grant R29-HL-59182-01 and is the 1997 Giles F. Filley Awardee. E. K. Weir and S. L. Archer were supported by Department of Veterans Affairs Merit Review funding. S. L. Archer was also supported by an American Heart Association Grant-in-Aid (National) Award. D. N. Cornfield was supported by American Heart Association Clinician-Scientist Award 93004240 and a University of Minnesota Children's Scholar Foundation Award.

    FOOTNOTES

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.

Address for reprint requests: H. L. Reeve, Research 151, VA Medical Center, 1 Veteran's Dr., Minneapolis, MN 55417.

Received 2 June 1998; accepted in final form 20 August 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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