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Am J Physiol Lung Cell Mol Physiol 276: L220-L228, 1999;
1040-0605/99 $5.00
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Vol. 276, Issue 2, L220-L228, February 1999

K+-channel blockade inhibits shear stress-induced pulmonary vasodilation in the ovine fetus

Laurent Storme1, Robyn L. Rairigh2, Thomas A. Parker2, David N. Cornfield3, John P. Kinsella2, and Steven H. Abman2

1 Service de Medecine Neonatale, Hôpital Jeanne de Flandre, CHRU de Lille, 59110 Lille, France; 2 Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado 80218; and 3 Department of Pediatrics, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455


    ABSTRACT
Top
Abstract
Introduction
Models and methods
Results
Discussion
References

To determine whether K+-channel activation mediates shear stress-induced pulmonary vasodilation in the fetus, we studied the hemodynamic effects of K+-channel blockers on basal pulmonary vascular resistance and on the pulmonary vascular response to partial compression of the ductus arteriosus (DA) in chronically prepared late-gestation fetal lambs (128-132 days gestation). Study drugs included tetraethylammonium (TEA; Ca2+-dependent K+-channel blocker), glibenclamide (Glib; ATP-dependent K+-channel blocker), charybdotoxin (CTX; preferential high-conductance Ca2+-dependent K+-channel blocker), apamin (Apa; low-conductance Ca2+-dependent K+-channel blocker), and 4-aminopyridine (4-AP; voltage-dependent K+-channel blocker). Catheters were inserted in the left pulmonary artery (LPA) for selective drug infusion and in the main pulmonary artery, aorta, and left atrium to measure pressure. An inflatable vascular occluder was placed around the DA. LPA flow was measured with an ultrasonic flow transducer. Animals were treated with saline, high- or low-dose TEA, Glib, Apa, CTX, CTX plus Apa, or 4-AP injected into the LPA. DA compression caused a time-related decrease in pulmonary vascular resistance in the control, Glib, Apa, CTX, CTX plus Apa, and low-dose TEA groups but not in the high-dose TEA and 4-AP groups. These data suggest that pharmacological blockade of Ca2+- and voltage-dependent K+-channel activity but not of low-conductance Ca2+- and ATP-dependent K+-channel activity attenuates shear stress-induced fetal pulmonary vasodilation.

nitric oxide; potassium channels; pulmonary circulation; blood flow; lambs; endothelium-derived relaxing factor; pulmonary hypertension


    INTRODUCTION
Top
Abstract
Introduction
Models and methods
Results
Discussion
References

THE FETAL PULMONARY CIRCULATION is characterized by high resistance and low blood flow. At birth, pulmonary blood flow increases dramatically 8- to 10-fold, and pulmonary vascular resistance (PVR) drops immediately (36). Three main factors contribute to the increase in pulmonary blood flow at birth: ventilation of the lung (39), increased O2 (7), and increased shear stress (1). Vasoactive mediators released from the endothelium, especially nitric oxide (NO), play a major role during the transition at birth (3, 11). Inhibition of NO synthesis can attenuate the postnatal adaptation of the pulmonary circulation, including the vasodilator effect of increased shear stress at birth (3, 20). Although NO stimulates vascular smooth muscle cell guanylate cyclase (6), the mechanism whereby increased guanosine 3',5'-cyclic monophosphate (cGMP) regulates pulmonary vascular tone remains incompletely understood.

Evidence suggests a primary role for ion channel activity, including K+ channels, in the regulation of pulmonary vascular tone and in NO-mediated vasodilation (5, 26). Activation of K+ channels increases K+ efflux, causing smooth muscle cell membrane hyperpolarization and inactivation of voltage-operated Ca2+ channels. Closure of voltage-operated Ca2+ channels decreases smooth muscle cell cytosolic Ca2+ concentrations and causes vasodilation (29). Various types of K+ channels, including ATP, Ca2+, and voltage dependent, are present in smooth muscle cells and play critical physiological roles in vascular regulation (30). K+-channel activity can be modulated by several physiological stimuli such as O2 tension (22), NO (5, 26), or endothelium-derived hyperpolarization factor (EDHF) (10, 27, 28). Furthermore, recent studies (16, 40) have demonstrated that, in the perinatal lung, O2- and ventilation-induced pulmonary vasodilation are partly dependent on the activation of K+ channels. Thus evidence exists that K+ channels may be involved in the regulation of vascular tone in the pulmonary circulation during fetal life. However, the effects of K+-channel activity on shear stress-induced vasodilation in the perinatal lung have not been studied.

Therefore, we hypothesized that stimulation of K+-channel activity can modulate the effects of shear stress on the pulmonary circulation in utero. To test this hypothesis, we studied the effects of K+-channel antagonists on basal fetal pulmonary vascular tone and on the hemodynamic response to partial acute compression of the ductus arteriosus (DA) in chronically prepared late-gestation fetal lambs. Partial compression of the DA increases mean pulmonary arterial pressure (PAP) and pulmonary artery blood flow (1). PVR progressively falls during the first 30-60 min of partial DA compression due to a mechanical increase in blood flow and an increase in sheer stress. A past study (17) showed that a flow- or shear stress-induced pulmonary vasodilation response can be blocked by a nonselective NO synthase antagonist in this model. To study the potential role of K+-channel activity in this response, we tested the effects of diverse K+-channel blockers on fetal PVR under basal conditions and on stimulation during DA compression.


    MODELS AND METHODS
Top
Abstract
Introduction
Models and methods
Results
Discussion
References

Animal Preparation

All animal procedures and protocols used in this study were previously reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Twenty-two mixed breed (Columbia-Rambouillet) pregnant ewes between 124 and 128 days gestation (term, 147 days) were fasted for 48 h before surgery. Ewes were sedated with intravenous pentobarbital sodium (total dose, 2-4 g) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. Ewes were kept sedated but breathed spontaneously throughout the surgery. Under sterile conditions, the fetal lamb's left forelimb was delivered through a uterine incision. A skin incision was made under the left forelimb after local infiltration with lidocaine (2 ml, 1% solution). Polyvinyl catheters (20 gauge) were advanced into the ascending aorta and the superior vena cava after insertion in the axillary artery and vein, respectively. A left thoracotomy exposed the heart and great vessels. Catheters were inserted into the left pulmonary artery (LPA; 22 gauge), main pulmonary artery (20 gauge), and left atrium (20 gauge) by direct puncture through purse-string sutures and secured as described previously (2). An inflatable vascular occluder (outer circumference 10 mm; In Vivo Metric, Healdsburg, CA) was placed loosely around the DA after gentle dissection of adherent connective tissue with cotton-tipped swabs. An ultrasonic flow transducer (size 6; Transonic Systems, Ithaca, NY) was placed around the LPA to measure blood flow. The uteroplacental circulation was kept intact, and the fetus was gently replaced in the uterus. An additional catheter was placed in the amniotic cavity to measure pressure. Ampicillin (500 mg) was added to the amniotic cavity before closure of the hysterotomy. The flow transducer, catheters, and occluder were exteriorized through a subcutaneous tunnel to an external flank pouch. The ewes recovered rapidly from surgery, generally standing in their pens within 6 h. Food and water were provided ad libitum. Catheters were maintained by daily infusions of 2 ml of heparinized saline (20 U/ml). Catheter positions were verified at autopsy. Studies were performed after a minimum recovery time of 48 h.

Physiological Measurements

The flow transducer cable was connected to an internally calibrated flowmeter (T201, Transonic Systems, Ithaca, NY) for continuous measurement of LPA blood flow. The output filter of the flowmeter was set at 100 Hz. The absolute value of flow was determined from the mean of phasic blood flow signals (at least 30 cardiac cycles), with zero blood flow defined as the measured flow value immediately before the beginning of systole (4). Main pulmonary arterial, aortic, left atrial, and amniotic catheters were connected to a blood pressure transducer (TSD104, BIOPAC Systems, Santa Barbara, CA). The pressure and flow signals were continuously recorded and processed on a computer (PowerMac 7100/80 AV) with an analog-to-digital converter system (BIOPAC Systems). Data were sampled at a rate of 200 samples/s. Pressures were referenced to the amniotic cavity pressure. Calibration of the pressure transducers was performed with a mercury column manometer. Heart rate was determined from the phasic pulmonary blood flow signal. PVR in the left lung was calculated as the difference between mean pulmonary arterial and left atrial (LAP) pressures divided by mean left pulmonary blood flow. Blood samples from the main pulmonary artery catheter were used for blood gas analysis and O2 saturation measurements (OSM 3 hemoximeter and ABL 520, Radiometer, Copenhagen, Denmark).

Experimental Design

Three different experimental protocols were included in this study: 1) the effects of K+-channel antagonists on basal pulmonary tone, 2) the effects of K+-channel antagonists on the hemodynamic response to partial DA compression, and 3) the effects of a nonspecific vasoconstrictor, the thromboxane analog U-46619, on basal pulmonary tone and on the hemodynamic response to partial DA compression. The duration of each experiment was 90 min. Physiological variables, including PAP, LAP, aortic pressure (AoP), amniotic pressure, and LPA blood flow were recorded at 10-min intervals. Main pulmonary arterial blood gas tensions and pH were monitored at 0, 60, and 90 min. In the control group, saline was infused throughout the study period. In treated animals, saline was first infused from 0 to 20 min and from 50 to 90 min of the study period; study drugs were infused between 20 and 50 min. All study drugs, including saline, were infused into the LPA at a rate of 0.1 ml/min. Five different K+-channel blockers were used: tetraethylammonium [TEA; low dose (0.2 mg/min), preferential Ca2+-sensitive K+-channel blocker, and high-dose (1 mg/min), which may inhibit Ca2+-sensitive and voltage-sensitive K+ channels at this concentration (33)], glibenclamide (Glib; 1 mg/min; an ATP-sensitive K+-channel blocker), charybdotoxin (CTX; 1 µg/min; a specific high-conductance Ca2+-sensitive K+-channel blocker), apamin (Apa; 10 µg/min; a specific low-conductance Ca2+-sensitive K+-channel blocker), 4-aminopyridine (4-AP; 0.1 mg/min; a voltage-sensitive K+-channel blocker), and the combination of CTX and Apa. Drugs were studied in random order. The selection of these doses was based on extensive preliminary studies, including the demonstration that Glib blocks cromakalim-induced fetal vasodilation (18), and the effects of these agents on O2- and ventilation-induced vasodilation (40). The dose of 4-AP was based on the observation that higher doses (0.6 mg/min) caused severe acidosis and arrythmias. The interval between each study drug infusion was at least 24 h.

Protocol 1: Hemodynamic effects of K+-channel blockers on basal fetal PVR. To investigate the role of K+-channel activity on basal pulmonary tone, we compared the effects of 30-min infusions of different K+-channel blockers on PAP, AoP, flow, and PVR values to the 30-min baseline values.

Protocol 2: Pulmonary hemodynamic effects of K+-channel blockade during DA compression. To investigate the role of K+-channel activity on the hemodynamic response to partial compression of DA, we partially inflated the DA occluder 10 min after the beginning of the infusion of drugs, and DA occlusion was continued for 30 min. The degree of inflation was set to increase mean PAP by 15 mmHg from its baseline value. Mean PAP was kept constant throughout the compression period by small adjustments of the occluder. After 30 min of DA compression, the occluder was deflated.

Protocol 3: Hemodynamic effect of nonspecific pharmacological elevation of basal PVR during DA compression. To ensure that nonspecific vasoconstriction does not alter the hemodynamic response to partial compression of DA, the effects of the thromboxane analog U-46619 were studied under basal conditions (protocol 1) and during DA compression (protocol 2). The dose of U-46619 (0.5 µg/min) selected for study was based on preliminary studies, which showed a 20-30% rise in PVR, to mimic the increase in basal PVR after high-dose TEA and 4-AP (see RESULTS).

Drug Preparation

TEA, 4-AP, CTX, Apa, and U-46619 (all from Sigma, St. Louis, MO) were dissolved in normal saline. The drug concentrations were high-dose TEA, 10 mg/ml; low-dose TEA, 2 mg/ml; 4-AP, 1 mg/ml; CTX, 10 µg/ml; Apa, 100 µg/ml; and U-46619, 5 µg/ml. Glib (Upjohn, Kalamazoo, MI) was dissolved in DMSO and diluted in a physiological salt solution to achieve a final concentration of 1 mg/ml. The pH of this solution was 7.4, and the final DMSO concentration was 1%.

Data Analysis

The results are presented as means ± SE. The data were analyzed with repeated-measures and factorial analyses of variance. Intergroup differences were analyzed with Fisher's least significant test (Stat View 4.1 for Macintosh, Abacus Concepts, Berkeley, CA). A P < 0.05 was considered significant.


    RESULTS
Top
Abstract
Introduction
Models and methods
Results
Discussion
References

Protocol 1: Hemodynamic Effects of K+-Channel Blockers on Basal Fetal PVR

Intrapulmonary infusion of Ca2+-dependent K+-channel blockers, including low-dose TEA (n = 5 animals), CTX (n = 4 animals), and CTX plus Apa (n = 4 animals), did not change mean basal PAP, LPA blood flow, or PVR (Fig. 1) or arterial blood gas, mean AoP, or heart rate (Table 1). High-dose TEA (n = 6 animals) and 4-AP (n = 9 animals) decreased LPA blood flow and increased PVR by 15 and 20%, respectively (P < 0.05; Fig. 2). High-dose TEA and 4-AP did not increase PAP. Arterial O2 saturation and arterial PO2 (PaO2) decreased during 4-AP infusion (P < 0.05; Table 1).


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  		Fig. 1.   Effects of Ca2+-dependent K+-channel antagonists on basal pulmonary vascular tone. Low-dose tetraethylammonium (TEA; 0.2 mg/min), charybdotoxin (CTX), and CTX+apamin (Apa) infusions into left pulmonary artery (LPA) did not change mean pulmonary arterial pressure (PAP), left pulmonary blood flow, and pulmonary vascular resistance (PVR). Data are means ± SE.

                              
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  		Table 1.   Blood gas and hemodynamic measurements during baseline and after 30 min of K+-channel antagonist infusion in control and treated animals



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  		Fig. 2.   Effects of voltage-dependent or nonspecific K+-channel antagonists on basal pulmonary vascular tone. High-dose TEA (1 mg/min) or 4-aminopyridine (4-AP; 0.1 mg/min) infusion into LPA induced increased PVR. Data are means ± SE. * P < 0.05 for treatment vs. baseline period.

Protocol 2: Pulmonary Hemodynamic Effects of K+-Channel Blockade During DA Compression

In control studies (n = 12 animals), partial compression of the DA rapidly increased mean PAP by 33 ± 3% (from 45 ± 1 to 60 ± 1 mmHg). LPA blood flow progressively increased from 90 ± 6 to 195 ± 15 ml/min at 30 min (P < 0.05). PVR in the left lung decreased during the DA compression period by 39 ± 5% (from 0.51 ± 0.03 to 0.30 ± 0.02 mmHg · ml-1 · min; P < 0.05; Fig. 3). During DA compression, the heart rate increased from 154 ± 10 to 174 ± 6 beats/min (P < 0.05), and mean AoP did not change (Table 2). Mean LAP before compression was 2 ± 1 mmHg and did not change during DA compression. PaO2 fell slightly from 18.7 ± 0.8 (baseline) to 17.2 ± 0.06 Torr after 30 min of DA compression (P < 0.05). Arterial pH also decreased slightly from 7.38 ± 0.01 to 7.36 ± 0.01 units after 30 min (P < 0.05; Table 2).


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  		Fig. 3.   Effects of voltage-dependent or nonspecific K+-channel antagonists on response during partial compression of ductus arteriosus (DA). Data are means ± SE. Despite similar increases in PAP, flow was lower and PVR was higher in groups treated with 4-AP and high-dose TEA (1 mg/min) than in control group during DA compression. Furthermore, PVR did not change during DA compression compared with baseline period in treated groups (ANOVA for repeated measures). * P < 0.05 for treated vs. control group.

                              
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  		Table 2.   Blood gas and hemodynamic measurements during baseline and after 30 min of DA compression in control and treated animals

Despite an identical increase in mean PAP during DA compression, the rise in LPA blood flow was attenuated and the decrease in PVR was abolished during infusions of high-dose TEA (n = 6 animals) or 4-AP (n = 8 animals; to inhibit voltage-dependent K+-channels; Fig. 3). The rise in pulmonary blood flow during ductal compression was also attenuated by CTX (n = 4 animals), low-dose TEA (n = 5 animals; to inhibit Ca2+-activated K+-channels; Fig. 4), and CTX plus Apa (n = 4 animals; to inhibit low- and high-conductance Ca2+-activated K+ channels; Fig. 5). Although PVR decreased during DA compression in these groups (Figs. 4 and 5), it was higher than control values at 30 min (P < 0.05). PVR did not change with high-dose TEA and 4-AP treatment during DA compression compared with baseline (Fig. 3). Neither Apa (Fig. 5) nor Glib (Fig. 6) altered pulmonary blood flow or PVR. Mean AoP, LAP, arterial blood gas tensions, and pH did not change in low-dose TEA, CTX, Apa, and CTX plus Apa groups (Table 2). pH and PaO2 decreased at the end of the ductus compression in high-dose TEA and 4-AP groups, respectively (Table 2).


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  		Fig. 4.   Effects of Ca2+-dependent K+-channel antagonists on response to partial compression of DA. Data are means ± SE. Despite a similar increase in PAP, low-dose TEA (0.2 mg/min) or CTX infusion into LPA significantly attenuated rise in flow and decrease in PVR induced by DA compression compared with control group (n = 12 animals). * P < 0.05 for treated vs. control group.


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  		Fig. 5.   Effects of low-conductance or combination of low- and high-conductance Ca2+-dependent K+-channel antagonists on response to partial DA compression. Data are means ± SE. At 30 min of DA compression, flow was lower, and PVR was higher in group infused with Apa + CTX than in control group. Apa infusion alone did not alter vasodilator response to DA compression. * P < 0.05 for treated vs. control group.


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  		Fig. 6.   Effects of ATP-dependent K+-channel antagonism on response to partial compression of DA. Data are means ± SE; n, no. of animals. Glibenclamide did not change hemodynamic effects of DA compression.

Protocol 3: Hemodynamic Effect of Nonspecific Pharmacological Elevation of Basal PVR During DA Compression

Infusion of the thromboxane analog U-46619 (0.5 µg/min; n = 4 animals) increased PVR by 30% (Fig. 7). However, DA compression increased LPA blood flow and decreased PVR after U-46619 treatment (P < 0.05). There was no difference in LPA blood flow and PVR among treated and control groups (Fig. 7).


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  		Fig. 7.   Effects of nonspecific vasoconstriction on response to partial compression of DA. Data are means ± SE; n, no. of animals. U-46619 infusion into LPA decreased flow and increased PVR but did not change PAP. Despite elevation of basal PVR, U-46619 infusion did not alter vasodilator response during DA compression. * P < 0.05 for treated vs. control group.


    DISCUSSION
Top
Abstract
Introduction
Models and methods
Results
Discussion
References

In this study, we hypothesized that activation of K+ channels modulates basal PVR and the effects of shear stress on pulmonary vascular tone during fetal life. To test this hypothesis, we studied the effects of K+-channel blockade on basal pulmonary vascular tone and the vasodilator response due to compression of the DA in the ovine fetus. Although Ca2+-dependent K+-channel antagonists (low-dose TEA and CTX) did not change basal fetal pulmonary blood flow or PVR, these drugs attenuated pulmonary vasodilation during DA compression. Furthermore, treatment with 4-AP, a voltage-dependent K+-channel antagonist, and high-dose TEA, a Ca2+- and voltage-dependent K+-channel blocker, increased basal pulmonary vascular tone and blocked the dilator response during DA compression. To ensure that the inhibition of DA compression-induced vasodilation was not simply due to increased basal PVR, the response to DA compression was measured during treatment with a nonspecific vasoconstrictor, U-46619. We found that the increase in basal pulmonary vascular tone during infusion of U-46619 did not alter the vasodilator response to DA compression. Pulmonary vasodilation during DA compression was not attenuated by ATP-dependent and low-conductance Ca2+-dependent K+-channel blockers. Overall, these results support the hypothesis that activation of both Ca2+- and voltage-dependent K+ channels modulates the effects of shear stress on pulmonary circulation during the fetal life.

This study provides new information regarding mechanisms underlying shear stress-induced vasodilation in the perinatal lung. Flow-mediated vasodilation may play a major role in postnatal adaptation of the pulmonary circulation (17). At birth, the pulmonary circulation undergoes an immediate rise in blood flow induced by ventilation that leads to capillary dilation and distension, along with increased arterial and alveolar O2 tensions (7, 39). The shear stress exerted along the endothelial surface by acute elevation of blood flow may potentially contribute to enhance and sustain the pulmonary vasodilation at birth (17). Pulmonary vasodilation in response to ventilation and increased O2 abruptly increases lung blood flow, which increases shear stress and stimulates flow-dependent dilation in resistance vessels. The propagation of vasodilation to resistance vessels may potentially be an important step to cause further elevation of pulmonary blood flow during the early postnatal period (34). Shear stress stimulates vasoactive mediator release from the endothelium, especially NO, prostacyclin, and EDHF release (2, 15, 17, 21, 35). Cornfield et al. (17) previously showed that NO synthase inhibition blocks fetal pulmonary artery vasodilation during DA compression, suggesting an important role for NO release in this model. As suggested by this current study, activation of non-ATP-dependent K+ channels modulates shear stress-induced vasodilation in the perinatal lung. Furthermore, both Ca2+- and voltage-dependent K+ channels may contribute to basal fetal pulmonary vasular tone because K+-channel blockade with high-dose TEA and 4-AP increased PVR under resting conditions.

Previous studies (16, 40) have demonstrated that O2 and ventilation cause pulmonary vasodilation through activation of non-ATP-sensitive K+ channels at birth. TEA infusion attenuated the increase in LPA blood flow in response to mechanical ventilation and increased O2, whereas Glib, an ATP-sensitive K+-channel antagonist, had no effect. Iberiotoxin, a Ca2+-dependent K+-channel antagonist, decreased O2-induced fetal pulmonary vasodilation. In this study (40), TEA was not found to increase basal fetal pulmonary vascular tone. However, the infusion of TEA was performed 1 h after surgery in the previous study. In the current study, a recovery period of at least 48 h after surgery was provided to ensure hemodynamic stabilization before study. This difference between acute and chronically instrumented fetal lambs may explain the divergent findings.

Other investigators have previously reported that flow-induced vasodilation involves activation of K+ channels in vitro and may involve NO production. Cooke et al. (14) showed that NO release due to shear stress is dependent on activation of Ca2+-sensitive K+ channels of the endothelial cell of rabbit iliac arteries, as demonstrated by inhibition of flow-mediated vasodilation by TEA, CTX, and iberiotoxin. In contrast, Apa, an antagonist of the low-conductance Ca2+-dependent K+ channel, and Glib had no effect. Ohno et al. (31) demonstrated that activation of TEA-sensitive K+ channels stimulated NO release from bovine aortic endothelial cells in response to increased flow. Hutcheson and Griffith (25) found that pulse frequency-related NO release was attenuated by Apa, TEA, and CTX but not by Glib. NO release stimulated by increased viscosity was reduced by TEA, CTX, and Glib but not by Apa. Shear stress has also been shown to activate inward rectifier K+ channels (41). Thus various K+ channels may be involved in the response to shear stress. Heterogeneity in the distribution of ion channels in endothelial or smooth muscle cells from different sources of vessels or species-related differences may partially explain discrepancies between studies (23). Our in vivo studies cannot distinguish between endothelial and smooth muscle cell roles in this response.

These data suggest that high-conductance Ca2+- and voltage-dependent K+ channels can modulate flow-induced vasodilation in the fetal lung. Our laboratory previously reported that inhibition of NO (17) and prostaglandin (1) release also blunts vasodilation in this model. Recent studies (5, 8-10, 12, 19, 25, 26, 42) have demonstrated links between vasodilation caused by NO, prostacyclin, and EDHF and activation of K+ channels in vascular smooth muscle cells. In pulmonary arterial smooth muscle cells, NO-induced vasodilation involves activation of a cGMP-sensitive protein kinase that increases the opening probability of K+ channels, with subsequent decreases in smooth muscle cell Ca2+ content (5, 8, 26, 42). NO may also have non-cGMP mechanisms of vasodilation caused by direct hyperpolarization of smooth muscle cells that causes closure of voltage-dependent Ca2+ channels and vasodilation (38). CTX can inhibit relaxation of rabbit aorta to NO after blockade of soluble guanylate cyclase with methylene blue (9). EDHF and prostacyclin can also hyperpolarize smooth muscle cells through activation of K+ channels (10, 12, 19). Thus blood flow can induce vasodilation through activation of K+ channels in both endothelial and smooth muscle cells. However, our data cannot determine whether shear stress activates K+ channels, leading to enhanced release of some modulator (e.g., NO), causing vasodilation (32), or whether the relaxation response to shear stress was directly mediated by pulmonary arterial smooth muscle cell K+-channel activation.

This study has several limitations. First, the specificity of pharmacological blockade is based on published studies, but the specific effects of the K+-channel blockers during in vivo studies are incompletely identified. A concentration of TEA between 0.2 and 3 mM selectively inhibits Ca2+-dependent K+ channels in vitro (30), and higher concentrations can inhibit both ATP- and voltage-dependent K+ channels. High-dose TEA infusion has been considered as either a preferential Ca2+-dependent (16) or a nonspecific K+-channel antagonist (40). Low-dose TEA infusion likely results in preferential Ca2+-dependent K+-channel blockade, whereas TEA loses its specificity at high doses. In our study, the responses to low-dose TEA and CTX were similar. CTX is a potent antagonist of high-conductance Ca2+-dependent K+ channels. Although CTX inhibits voltage-dependent K+ channels in lymphocytes, CTX acts exclusively on Ca2+-dependent K+ channels in arterial smooth muscle (30). Furthermore, 4-AP is known as a selective inhibitor of voltage-dependent K+ channels but may also inhibit ATP-dependent K+ channels (30). Because Glib, a highly selective ATP-dependent K+-channel antagonist, did not alter the response to DA compression, we speculate that the effects of 4-AP may be related to voltage-dependent K+-channel blockade. The similar responses of high-dose TEA and 4-AP on basal PVR and vasodilation during DA compression support the hypothesis that high-dose TEA can inhibit both Ca2+- and voltage-dependent K+ channels. Our ability to test a higher dose of TEA was limited by adverse effects. Apa infusion had no effect on the response to DA compression. The dose of Apa used in this study (300 or 100-150 µg/kg) could be insufficient; however, Apa has previously been shown to attenuate the effects of alpha -adrenoreceptor agonists in guinea pigs at a dose of 40 µg/kg (13). Glib, at a dose of 1 mg/min, inhibits fetal pulmonary vasodilation to lemakalim, a specific ATP-dependent K+-channel agonist, suggesting that the dose of Glib in this study was adequate (18).

In summary, voltage-dependent K+-channel antagonism increased basal pulmonary vascular tone in the ovine fetus. Both voltage- and Ca2+-dependent K+-channel antagonists blunted the vasodilator response during partial ductus compression. Low-conductance Ca2+- and ATP-dependent K+ channels had no effect. These findings suggest that 1) voltage-dependent K+ channels are involved in the modulation of basal pulmonary vascular tone and 2) shear stress-induced vasodilation is mediated by activation of both voltage- and Ca2+-dependent K+ channels.


    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: S. H. Abman, Pediatric Heart Lung Center, Dept. of Pediatrics, 1056 East 19th Ave., Univ. of Colorado School of Medicine, Denver, CO 80218-1088.

Received 5 June 1998; accepted in final form 16 October 1998.


    REFERENCES
Top
Abstract
Introduction
Models and methods
Results
Discussion
References

1.   Abman, S. H., and F. J. Accurso. Acute effects of partial compression of ductus arteriosus on fetal pulmonary circulation. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H626-H634, 1989[Abstract/Free Full Text].

2.   Abman, S. H., F. J. Accurso, R. M. Ward, and R. B. Wilkening. Adaptation of fetal pulmonary blood flow to local infusion of tolazoline. Pediatr. Res. 20: 1131-1135, 1986[Medline].

3.   Abman, S. H., B. A. Chatfield, S. L. Hall, and I. F. McMurtry. Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1921-H1927, 1990[Abstract/Free Full Text].

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Am J Physiol Lung Cell Mol Physiol 276(2):L220-L228
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