NO causes perinatal pulmonary vasodilation through K+-channel activation and intracellular Ca2+ release

Connie B. Saqueton, Robert B. Miller, Valerie A. Porter, Carlos E. Milla, and David N. Cornfield

Division of Pediatric Pulmonary and Critical Care Medicine, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota 55455


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Evidence suggests that nitric oxide (NO) causes perinatal pulmonary vasodilation through K+-channel activation. We hypothesized that this effect worked through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channel that requires release of intracellular Ca2+ from a ryanodine-sensitive store. We studied the effects of 1) K+-channel blockade with tetraethylammonium, 4-aminopyridine, a voltage-dependent K+-channel blocker, or glibenclamide, an ATP-sensitive K+-channel blocker; 2) cyclic nucleotide-sensitive kinase blockade with either KT-5823, a guanylate-sensitive kinase blocker, or H-89, an adenylate-sensitive kinase blocker; and 3) blockade of intracellular Ca2+ release with ryanodine on NO-induced pulmonary vasodilation in acutely prepared late-gestation fetal lambs. N-nitro-L-arginine, a competitive inhibitor of endothelium-derived NO synthase, was infused into the left pulmonary artery, and tracheotomy was placed. The animals were ventilated with 100% oxygen for 20 min, followed by ventilation with 100% oxygen and inhaled NO at 20 parts/million (ppm) for 20 min. This represents the control period. In separate protocols, the animals received an intrapulmonary infusion of the different blockers and were ventilated as above. Tetraethylammonium (n = 6 animals) and KT-5823 (n = 4 animals) attenuated the response, whereas ryanodine (n = 5 animals) blocked NO-induced perinatal pulmonary vasodilation. 4-Aminopyridine (n = 5 animals), glibenclamide (n = 5 animals), and H-89 (n = 4 animals) did not affect NO-induced pulmonary vasodilation. We conclude that NO causes perinatal pulmonary vasodilation through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channels and release of Ca2+ from ryanodine-sensitive stores.

nitric oxide; potassium channel; newborn; pulmonary hypertension; smooth muscle cells; oxygen sensing


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HIGH RESISTANCE AND LOW BLOOD FLOW characterize the fetal pulmonary circulation. At birth, pulmonary arterial blood flow increases 8- to 10-fold (8, 13, 14) and pulmonary arterial pressure decreases by 50% within the first 24 h of life (15). Factors that contribute to the postnatal adaptation of the pulmonary circulation include establishment of an air-fluid interface, ventilation of the lung, an increase in blood oxygen content, and elaboration of vasoactive substances from the endothelium (2, 10, 13, 14, 16, 23).

Endothelium-derived nitric oxide (EDNO) modulates pulmonary vascular tone under basal conditions in the fetus and during transition of the pulmonary circulation at birth (2, 10). Inhibition of EDNO synthesis elevates pulmonary vascular resistance in utero and attenuates postnatal adaptation of the pulmonary circulation (2, 10, 17). Furthermore, EDNO mediates the increase in pulmonary blood flow that results from several birth-related stimuli, including ventilation, elevation of oxygen tension, and increased shear stress (2, 10, 21, 22).

Previous studies from our laboratory provide support for the notion that K+-channel activation plays a key role in perinatal pulmonary vasodilation and that EDNO may act through K+-channel activation in the perinatal pulmonary circulation. First, oxygen causes fetal pulmonary vasodilation through activation of a protein kinase-sensitive K+ channel (12). Second, the observation that tetraethylammonium (TEA), a K+-channel antagonist, attenuated, whereas glibenclamide (Glib), a blocker of the ATP-sensitive K+ channel, had no effect on perinatal pulmonary vasodilation (32) further supports the hypothesis that TEA-sensitive K+ channels modulate the transition from fetal to neonatal pulmonary circulation. Interestingly, K+-channel blockade and EDNO synthase inhibition attenuated pulmonary vasodilation in response to sequential ventilation with low and high inspired oxygen fractions (FIO2) (10) in remarkably similar patterns.

Although nitric oxide (NO) causes vasodilation through activation of soluble guanylate cyclase (4), the mechanism whereby increased cGMP levels cause vasorelaxation remains incompletely understood. There are reports that NO acts on K+ channels in arterial smooth muscle cells (SMCs) either directly (5) or through a cGMP-sensitive kinase (3, 29). Recent evidence (7, 24, 27) suggested that NO causes vasodilation in part through activation of a Ca2+-activated K+ (K Ca) channel by local release of Ca2+ from an intracellular Ca2+ store.

In SMCs, free cytosolic Ca2+ is the major determinant of the contractile state (19, 33). The primary source of Ca2+ is influx across the plasma membrane. Ca2+ can also be released from the intracellular sarcoplasmic reticulum (SR) Ca2+ store through ryanodine receptors (7, 24). Ryanodine receptors are Ca2+-activated release channels named for their ability to be blocked by the plant alkaloid ryanodine. The Ca2+ released from the intracellular SR stores in SMCs is not sufficient to cause contraction. Rather, the Ca2+ released can cause activation of KCa channels, which, in turn, causes membrane hyperpolarization and closure of voltage-operated Ca2+ channels (7, 24, 27). This prevents entry of extracellular Ca2+ into the cytosol, resulting in a decrease in cytosolic Ca2+ concentration and relaxation of SMCs (33). Therefore, we hypothesized that NO causes perinatal pulmonary vasodilation through 1) cGMP-dependent kinase-mediated activation of a KCa channel and 2) release of intracellular Ca2+ from a ryanodine-sensitive store.

To test these hypotheses, we studied the effect of K+-channel inhibition, cyclic nucleotide-dependent kinase inhibition, and Ca2+-release inhibition on inhaled NO (INO)-induced perinatal pulmonary vasodilation. Acutely prepared, late-gestation fetal lambs were treated with N-nitro-L-arginine (L-NNA), a competitive inhibitor of NO synthase, to prevent the production of endogenous NO. In separate experimental protocols, TEA, a preferential KCa-channel antagonist (12, 28, 32), 4-aminopyridine (4-AP), a voltage-dependent K+ (KV)-channel antagonist (28), Glib, an ATP-sensitive K+ (KATP)-channel blocker (11), KT-5823, a guanylate kinase blocker (12), H-89, an adenylate kinase antagonist (12), ryanodine, a blocker of intracellular Ca2+ release (27), or saline was infused continuously into the left pulmonary artery (LPA). Pulmonary and systemic hemodynamics were then monitored in response to sequential ventilation with an FIO2 of 1.00, followed by ventilation with an FIO2 of 1.00 and INO at 20 parts/million (ppm) for 20 min.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical Preparation

All procedures and protocols performed in this study conformed with the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892] and were approved by the Animal Care and Use Committee of the University of Minnesota (Minneapolis, MN) and the Veterans Affairs Medical Center (Minneapolis). Eleven mixed-breed pregnant ewes between 136 and 141 days gestation (term = 147 days) were obtained. One ewe and its fetus were used as time controls, leaving 10 fetuses available for the experimental protocols. The ewes were fasted for 24 h before surgery and sedated with intravenous pentobarbital sodium (800-1,000 mg), which crosses the placenta to induce fetal anesthesia. The ewes were further anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. The ewes breathed spontaneously during surgery and the study period. A midline uterine incision was made, and the fetal lamb's left forelimb was exteriorized. A skin incision was performed within the axilla after infiltration with lidocaine (1%, 2-3 ml). Polyvinyl catheters were inserted into the axillary artery and vein and advanced into the aorta and superior vena cava, respectively. A left thoracotomy was made to expose the heart and great vessels. Catheters were introduced by direct puncture into the main pulmonary artery (MPA) and LPA as previously described (2, 10-12, 32). The catheters were guided into position with a 14- or 16-gauge Gelco introducer (Angiocath, Travesol, Deerfield, IL) and secured with a purse-string suture. The LPA catheter was inserted at the bifurcation of the MPA and ductus arteriosus and advanced into the LPA proximal to the takeoff of the lobar arteries. The MPA catheter was placed between the ductus arteriosus and the pulmonic valve. Catheter position was confirmed at autopsy. An ultrasonic flow transducer (T201 flowmeter, Transonic Systems, Ithaca, NY) was placed around the LPA to measure blood flow. Flow probes were selected to make adequate contact with but cause minimal compression of the artery (as judged by the intraoperative phasic flow signal). Accuracy of the flow probes was confirmed by ex vivo calibration with a variable rate roller pump (Masterflex pump, Cole-Parmer Instrument, Chicago, IL). After instrumentation, the fetus and uterus were returned to the abdominal cavity, and the exposed surfaces were bathed in warm towels. Fetal temperature was maintained at 39°C. The fetus was allowed to recover for a minimum of 1 h before initiation of the study (Fig. 1). The umbilical circulation remained intact throughout the study.


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Fig. 1.   Schematic diagram of experimental protocol. After instrumentation, animals were given a minimum of 1-h recovery period. Endogenous nitric oxide (NO) production was blocked by administration of N-nitro-L-arginine (L-NNA) into left pulmonary artery (LPA). Preventilation values were then obtained. Animals were sequentially ventilated with inspired O2 fraction (FIO2) of 1.00 for 20 min followed by ventilation (Vent) with FIO2 of 1.00 and 20 parts/million (ppm) inhaled NO for 20 min. Vent was discontinued, and animals were provided a minimum of 30 min to recover. After hemodynamic parameters returned to preventilation values, study drugs were administered into LPA. Animals were subsequently ventilated with FIO2 of 1.00 for 20 min followed by Vent with FIO2 of 1.00 and 20 ppm inhaled NO for 20 min. Prep, preparation; TEA, tetraethylammonium; 4-AP, 4-aminopyridine; Glib, glibenclamide.

Physiological Measurements

MPA pressure (MPAP), aortic pressure (AoP), and LPA blood flow were continuously recorded with an analog-digital computer system (MacLab, AD Instruments, Medford, MA). Pressure transducers (Radnoti, Monrovia, CA) were placed at the level of the right atrium and referenced to zero against atmospheric pressure. Heart rate was determined from the phasic blood flow tracings. Fetal blood gas tensions and pH were sampled from the MPA and analyzed in a Corning 288 blood gas analyzer (Corning, NY). The values recorded in Tables 1 and 2 were obtained preventilation (i.e., after study drug infusion but immediately before ventilation), during ventilation, and during INO administration.

                              
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Table 1.   Effect of K+-channel blockers on fetal blood gases and hemodynamics in response to sequential ventilation


                              
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Table 2.   Effect of kinase blockers and ryanodine on fetal blood gases and hemodynamics in response to sequential ventilation

Drug Preparation

L-NNA (Sigma) was suspended in HCl, and the pH was corrected to 7.4 with NaOH. TEA (Sigma), H-89 (Calbiochem), ryanodine (Alomone), and 4-AP (Sigma) were dissolved in saline. Glib (Calbiochem) was dissolved in 50% dextrose and 0.1 N NaOH, heated, stirred, and pH balanced. KT-5823 (LC Services) was dissolved in <0.1% DMSO, with the balance saline.

Experimental Design

After a 60-min recovery period (Fig. 1), serial MPA blood gas tensions and pH were monitored at 20-min intervals. L-NNA (10 mg/ml) was continuously infused into the LPA at a rate of 0.1 ml/min for 30 min (total dose 30 mg) immediately after the recovery period. To ensure that endogenous NO release was blocked, acetylcholine was administered via the LPA at 2.5 µg/min for 5 min (total 12.5 µg). This dose generally increases pulmonary blood flow by 20-25 ml/min (1). If there were any pulmonary vasodilation in response to acetylcholine (i.e., pulmonary blood flow > 5 ml/min), the same dose of L-NNA was repeated. Generally, a single dose of L-NNA was sufficient to inhibit pulmonary vasodilation. After 20 min of L-NNA infusion, pancuronium bromide (0.5 mg; Gensia Laboratories, Irvine, CA) was administered into the superior vena cava to prevent spontaneous fetal respiration. The head was removed from the uterus, a tracheostomy was performed, and a 4.5-mm endotracheal tube was placed. Ventilation was initiated with a volume ventilator (Siemens model 900B) with the following initial settings: rate, 20 breaths/min; tidal volume, 40 ml; positive end-expiratory pressure, 4 cmH2O; inspiratory time, 0.5 s; and FIO2, 1.00. Tidal volume was adjusted to maintain a peak inspiratory pressure of 35 cmH2O. Ventilator rate and inspired oxygen concentration were adjusted to maintain fetal pH and carbon dioxide tension at preventilation values. Sodium bicarbonate was administered to maintain pH >=  7.30. After 20 min of ventilation with FIO2 of 1.00, INO (800 ppm in nitrogen; Ohmeda, Cherry Hills, NJ) at 20 ppm was added to the inspiratory limb of the ventilator circuit. Twenty parts/million of INO was chosen because of its proven physiological efficacy in vivo (9, 20). Physiological measurements were continued for an additional 20 min. This represents the control period. After pulmonary blood flow was allowed to return to the preventilation values, the experimental protocols were begun. In separate protocols, outlined in Experimental Protocols, study drugs were administered via the LPA at 0.1 ml/min for 20 min. The animal was then ventilated at an FIO2 of 1.00 followed by ventilation at an FIO2 of 1.00 with 20 ppm NO. As noted in Fig. 1, more than one study drug was used in each protocol. However, the animals were allowed to recover back to preventilation conditions between study drugs. At the conclusion of the study, the animals were euthanized with a barbiturate-potassium solution (Beuthanasia, Schering-Plough Animal Health, Kenilworth, NJ), and the catheter position was confirmed. The fetuses were not weighed because fixed dosages of the drugs (listed under each protocol) were given as intrapulmonary infusions, thus minimizing systemic effects. However, the average weight of the fetus at this gestational age was ~4 kg (32).

Experimental Protocols

Protocol 1: K+-channel blockade. TEA (20 mg at 10 mg/ml; n = 6 animals), 4-AP (17 mg at 8.5 mg/ml; n = 5 animals), or Glib (30 mg at 15 mg/ml; n = 5 animals) was administered via the LPA over 20 min. Ventilation was then initiated as described in Experimental Design. LPA blood flow, hemodynamic values, and fetal blood gas tensions were obtained during each study condition. Previous work (11, 12) from our laboratory confirmed that this dose of Glib was sufficient to block the pulmonary vasodilation caused by lemakalim, an ATP-sensitive K+-channel opener. The effects of TEA and 4-AP on lemakalim-induced pulmonary vasodilation were not tested because lemakalim can cause prolonged perinatal pulmonary vasodilation (11).

Protocol 2: Kinase inhibition. The guanylate-sensitive kinase-specific blocker KT-5823 (220 µg at 110 µg/ml; n = 4 animals) or the adenylyl-sensitive kinase-specific blocker H-89 (110 µg at 55 µg/ml; n = 4 animals) was administered via the LPA over 20 min. Ventilation was then initiated as previously described. LPA blood flow, hemodynamic values, and fetal blood gas tensions were obtained during each study condition. Previous work from our laboratory (12) confirmed that these doses of KT-5823 and H-89 were sufficient to cause inhibition of the perinatal pulmonary vasodilation caused by 8-bromo-cGMP. Vehicle controls were performed for KT-5823.

Protocol 3: Intracellular Ca2+-release blockade with ryanodine. Ryanodine (150 µg at 75 µg/ml; n = 5 animals) was administered via the LPA over 20 min. Ventilation was then initiated as previously described. LPA blood flow, hemodynamic values, and fetal blood gas tensions were obtained during each study condition.

Data Analysis

The data on the variables measured are expressed as means ± SE. To evaluate the individual effects of each of the drugs studied and because of the repeated measures in the study protocol, the data were analyzed by ANOVA for repeated measures.

Contrasts between the least-squares means observed during each treatment period among the study drugs were obtained with Scheffé's method so as to identify significant differences between the drugs under a given condition. In addition, similar methodology was used to contrast the responses observed for each of the drugs under the different treatment conditions. An alpha  value of 0.05 was used as the cutoff for significance. All analyses were performed with the SAS statistical package (SAS Institute, Cary, NC).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Control. Measurements were performed after L-NNA infusion and immediately before initiation of ventilation (n = 10 animals). Under these conditions, LPA flow was 30 ± 4 ml/min (Fig. 2), and MPAP and AoP were 52 ± 3 and 50 ± 3 mmHg, respectively (Table 1).


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Fig. 2.   Effect of K+-channel inhibition on sequential Vent with FIO2 of 1.00 and Vent with FIO2 of 1.00 and 20 ppm inhaled NO. In presence of pharmacological blockade of endogenous NO production with L-NNA, LPA flow increased in each experimental group during Vent with FIO2 of 1.00 and was not affected by K+-channel inhibition. Vent with FIO2 of 1.00 and 20 ppm inhaled NO caused a significant increase in LPA flow in each study group. Compared with control group, treatment with TEA, a preferential Ca2+-activated K+-channel blocker, attenuated increase in LPA blood flow associated with Vent and inhaled NO (P = 0.0003). Glib, an ATP-sensitive K+-channel blocker, and 4-AP, a voltage-dependent K+-channel blocker, had no effect on NO-induced pulmonary vasodilation. Significant difference (P < 0.05) from: * control group; dagger  preventilation value; × Vent with FIO2-alone value.

After ventilation with an FIO2 of 1.00, LPA flow significantly increased to 215 ± 24 ml/min (P < 0.0001 vs. preventilation; Fig. 2). MPAP, AoP, and heart rate did not change. The PO2 increased from preventilation level (P < 0.05; Table 1), whereas pH and PCO2 remained unchanged.

With administration of INO at 20 ppm and continued ventilation with an FIO2 of 1.00, LPA flow increased further (to 365 ± 29 ml/min; P < 0.0001 vs. ventilation with FIO2-alone value; Fig. 2). MPAP, AoP, and heart rate did not change. The PO2 increased further, but this increase was not significantly different from ventilation with an FIO2 of 1.00 alone. The PCO2 and pH remained unchanged.

Protocol 1: K+-Channel Blockade

Under preventilation conditions, LPA flows and fetal blood gas tensions in the K+-channel blockade-treated animals were not different from control levels. Administration of TEA caused an increase in MPAP and AoP (P < 0.05 vs. control level; Table 1). Hemodynamic parameters were not significantly different from the control levels in the Glib- and 4-AP-treated animals.

With ventilation and an FIO2 of 1.00, LPA flow increased from preventilation values in all groups (P < 0.0001; Fig. 2). MPAP, AoP, heart rate, and fetal blood gas tensions were not significantly different from control levels (P > 0.1; Table 1). MPAP and AoP in the TEA-treated animals were not different from preventilation values. PO2 increased significantly from preventilation levels in the TEA- and 4-AP-treated animals (P < 0.01) but not in the Glib-treated groups (P = 0.1; Table 1).

With administration of INO at 20 ppm and continued ventilation with an FIO2 of 1.00, LPA flow increased further in the Glib- and 4-AP-treated animals (P < 0.01) but not in the TEA-treated animals (P = 0.3 vs. ventilation with FIO2-alone level; Fig. 2). Moreover, LPA flow was significantly lower (P = 0.0003) compared with the control level under this condition. MPAP decreased significantly from the preventilation level in the TEA-treated animals (P = 0.05; Table 1), whereas AoP, heart rate, and PCO2 did not significantly change. The pH increased in the 4-AP group compared with the preventilation level (P < 0.05). Compared with ventilation with FIO2-alone level, PO2 did not increase significantly (P > 0.10) in all groups except the Glib group where the increase was significant (P = 0.05; Table 1).

Protocol 2: Kinase Inhibition

Under preventilation conditions, LPA flow, hemodynamics, pH, and PO2 in the KT-5823- and H-89-treated animals were not significantly different from control values. PCO2, however, was higher in the kinase-inhibited animals (P < 0.05 vs. control value; Table 2).

With ventilation at an FIO2 of 1.00, LPA flow increased in all groups (P < 0.03 vs. preventilation level; Fig. 3). However, LPA flow in the KT-5823-treated animals was significantly lower than the control value (P = 0.03; Fig. 3) compared with that in the H-89 group where the flow was comparable to the control value (P = 0.9). MPAP, AoP, heart rate, pH, and PCO2 were not different from the control or preventilation values. PO2 increased from preventilation values in the KT-5823- and H-89-treated animals (P < 0.01; Table 2).


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Fig. 3.   Effect of kinase inhibition on sequential Vent with FIO2 of 1.00 and Vent with FIO2 of 1.00 and 20 ppm inhaled NO. In presence of pharmacological blockade of endogenous NO production with L-NNA, LPA flow increased in each experimental group during Vent with FIO2 of 1.00. Compared with control value, treatment with KT-5823 attenuated perinatal pulmonary vasodilation during Vent with FIO2 of 1.00 (P = 0.03). Vent with FIO2 of 1.00 and 20 ppm inhaled NO caused a significant increase in LPA flow in control and H-89 groups compared with Vent with FIO2-alone value. Compared with control value, treatment with KT-5823, a guanylyl kinase antagonist, attenuated increase in LPA blood flow associated with Vent and inhaled NO (P < 0.0001). H-89, an adenylyl kinase antagonist, had no effect on NO-induced pulmonary vasodilation. Significant difference (P < 0.05) from: * control group; dagger  preventilation value; × Vent with FIO2-alone value.

With administration of INO at 20 ppm and continued ventilation at an FIO2 of 1.00, LPA flow increased further in the H-89-treated animals (P = 0.006 vs. ventilation with FIO2-alone level; Fig. 3). In the KT-5823- treated animals, LPA flow was not significantly different from the LPA flow with ventilation with FIO2-alone value (P = 0.4; Fig. 3). LPA flow was significantly lower in the KT-5823-treated animals compared with the control value (P < 0.0001; Fig. 3). Hemodynamic parameters were not different from the control or ventilation with FIO2-alone values. pH increased in the H-89-treated animals compared with preventilation value (P = 0.01; Table 2). PO2 significantly increased in all groups (P < 0.05 vs. preventilation value) but was not significantly increased compared with ventilation with FIO2-alone value (P > 0.1; Table 2).

Protocol 3: Intracellular Ca2+-Release Blockade With Ryanodine

Under preventilation conditions, LPA flow, hemodynamic parameters, and fetal blood gas tensions in the ryanodine-treated animals were not different from the control values (Table 2, Fig. 4).


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Fig. 4.   Effect of intracellular Ca2+-release blockade with ryanodine (Rya) on sequential Vent with FIO2 of 1.00 and Vent with FIO2 of 1.00 and 20 ppm inhaled NO. In presence of pharmacological blockade of endogenous NO production with L-NNA, LPA flow increased in control and Rya-treated groups during Vent with FIO2 of 1.00. Compared with control value, treatment with Rya had no effect on perinatal pulmonary vasodilation during Vent with FIO2 of 1.00. Vent with FIO2 of 1.00 and 20 ppm inhaled NO caused a significant increase in LPA flow in control group. Compared with control value, treatment with Rya blocked increase in LPA blood flow associated with Vent and inhaled NO (P < 0.0001 vs. control group; P = 0.9 compared with Vent with FIO2-alone value). Significant difference (P < 0.05) from: * control group; dagger  preventilation value; × Vent with FIO2-alone value.

With ventilation at an FIO2 of 1.00, LPA flow significantly increased from the preventilation level in the ryanodine-treated animals (P < 0.0001; Fig 4). MPAP, AoP, heart rate, pH, and PCO2 did not change significantly from preventilation values nor were they significantly different from control values (P > 0.1; Table 2). PO2 in the ryanodine-treated animals was significantly lower than the control value (P = 0.01; Table 2).

With administration of INO at 20 ppm and continued ventilation at an FIO2 of 1.00, LPA flow in the ryanodine-treated animals did not change (P = 0.9 vs. ventilation with FIO2-alone value; Fig. 4). This was significantly lower than what was observed in the control group (P < 0.0001). Hemodynamic parameters, pH, and PCO2 did not significantly change and were not different from control values (Table 2). PO2 in the ryanodine-treated animals was significantly lower than the control value (P = 0.01; Table 2) but was still significantly higher than the preventilation value (P = 0.0006; Table 2).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NO derived from the pulmonary endothelium plays a key role in perinatal pulmonary vasodilation. Although NO causes vasodilation through an increase in cytosolic cGMP (4), the subcellular mechanism whereby cGMP causes relaxation of the pulmonary vascular SMCs remains incompletely understood. In this study, we tested the hypothesis that NO causes perinatal pulmonary vasodilation through activation of a KCa channel by local release of Ca2+ from an intracellular Ca2+ store.

Although endogenous NO production was pharmacologically inhibited, the effect of either K+-channel blockade; ryanodine, a blocker of Ca2+ release from the SR; or kinase inhibition on the perinatal pulmonary vasodilation caused by INO was studied. We report that blockade of the KCa channel attenuated the NO-induced pulmonary vasodilation, whereas ryanodine and KT-5823, a guanylate kinase inhibitor, blocked NO-induced pulmonary vasodilation. Inhibition of either the KATP or KV channels or adenylate kinase had no effect on NO-induced perinatal pulmonary dilation. These results support the hypothesis that NO causes perinatal pulmonary vasodilation by activating KCa channels through a cGMP-sensitive protein kinase-mediated pathway that requires release of Ca2+ from a ryanodine-sensitive store.

These results provide details into the subcellular mechanisms whereby NO causes perinatal pulmonary vasodilation. Although previous studies (3, 5, 26, 29) have demonstrated that NO causes K+-channel activation, this is the first report demonstrating that NO causes perinatal pulmonary vasodilation through activation of a KCa but not a KV or KATP channel. Further mechanistic details derive from the observation that guanylate kinase activity, but not adenylate kinase activity, is necessary for NO to cause perinatal pulmonary vasodilation. These results provide support for the notion that NO causes KCa-channel activation through a cGMP-sensitive kinase-mediated pathway and not through direct action on the KCa channel.

The present study also provides evidence that subcellular Ca2+ release from ryanodine-sensitive pools in the SR is required for NO-induced perinatal pulmonary vasodilation. After treatment with ryanodine, NO did not cause perinatal pulmonary vasodilation. This observation fits well with previous studies (7, 24, 25, 27) that demonstrated that KCa channels are activated by subcellular Ca2+ release from ryanodine-sensitive pools in the SR. The data reported in these studies suggested that in the perinatal pulmonary circulation, NO acts to increase cGMP concentration in the cytosol, which activates cGMP-dependent kinase to phosphorylate the KCa channels and/or the ryanodine-sensitive Ca2+ pool. Phosphorylation of the ryanodine-sensitive Ca2+ stores results in a local increase in Ca2+ in the region of the KCa channel and activates nearby sarcolemmal KCa channels, resulting in K+ efflux, membrane hyperpolarization, closure of voltage-operated Ca2+ channels, and vasodilation. Activation of only a small number of high-conductance KCa channels would be necessary to effect a significant change in pulmonary arterial SMC membrane potential (6, 7, 24, 27).

Interestingly, the frequency of the local elevations in Ca2+ concentration, termed Ca2+ sparks (6), has been shown to be inversely proportional to the contractile state of the vasculature (24, 27). Decreasing spark frequency or blocking the sparks results in vasoconstriction (34), whereas increasing the frequency of sparks results in vasodilation. Thus NO may cause vasodilation via an increase in spark frequency. Such a construct is strengthened by the observation of Porter et al. (27) that cyclic nucleotides modulate the frequency of these sparks. If this mechanism is operative, then fetal pulmonary arterial SMCs would possess a spontaneous transient outward current, which Reeve et al. (28) have previously demonstrated. Taken together with the clear evidence that NO causes vasodilation by increasing cGMP concentration, it seems reasonable to conclude that NO causes perinatal pulmonary vasodilation through a pathway that entails a cyclic nucleotide-mediated increase in local Ca2+ release.

These findings fit well with previous reports (12, 28) that demonstrated a maturational shift in the K+-channel setting of the resting membrane potential in pulmonary arterial SMCs from the KCa channel in the fetus and newborn to the KV channel in the adult. In the perinatal pulmonary circulation, when the response of the pulmonary circulation to an acute increase in oxygen tension and NO is biologically imperative, the KCa channel sets the resting membrane potential (12). In the adult pulmonary circulation, several reports (35, 36) indicated that the KV channel regulates the resting membrane potential and is inactivated by an acute decrease in oxygen tension, allowing for hypoxic pulmonary vasoconstriction to prevent hypoxemia. Such a developmental shift in the ion channel that responds to a change in oxygen tension in the pulmonary circulation might represent the mechanism whereby the pulmonary circulation of the normal newborn infant is adapted to respond to an acute increase in oxygen tension and NO, whereas the pulmonary circulation of the adult is adapted to respond to an acute decrease in oxygen tension.

This represents the first effort to address these questions with an integrative physiological approach. The current findings demonstrate that in vitro findings are applicable in vivo. However, the findings are limited by the specificity of the pharmacological probes used. In the absence of specific K+-channel agonists, we were obliged to assume that the effects of the pharmacological K+-channel antagonists are directly applicable in vivo. To mitigate against this concern, we tested whether the drugs were acting as predicted whenever the pharmacological tools were available. Moreover, the concentrations used in these studies were based on extrapolations from in vitro work. It is not clear that either the effects or concentrations of these drugs can be directly applied. With regard to the use of TEA rather than a more specific KCa-channel blocker such as iberiotoxin or charybdotoxin, previous reports (25, 28, 31) have shown that the dose of TEA used works specifically at the KCa channels. Moreover, because of the technical difficulty of the surgical preparation and the need to use more than one study drug per protocol, it was important to use agents such as TEA with a shorter duration of action than the toxins (18, 28). Furthermore, these agents may possess nonspecific effects that confound our interpretation of the data. For example, there is increasing evidence that endothelial cells possess KCa channels (30). Use of K+-channel antagonists may have affected the endothelial cells in an unanticipated manner. With respect to ryanodine administration, there are no pharmacological probes currently available to specifically test whether the ryanodine-sensitive stores had been blocked. However, the lack of a response to the addition of INO does suggest biological relevance.

In summary, we report that NO causes perinatal pulmonary vasodilation through activation of a KCa channel. The present study provides data that the KCa channel is activated through cGMP-sensitive kinase and requires Ca2+ release from a ryanodine-sensitive intracellular store. We speculate that if K+-channel activation does play a central role in the postnatal adaptation of the pulmonary circulation, then an alteration in K+-channel activity may lead to the altered perinatal pulmonary vascular reactivity that is the hallmark of persistent pulmonary hypertension of the newborn. The implication of the present study is that in addition to alterations in endothelium-derived vasoactive products such as NO and endothelin, infants with persistent pulmonary hypertension of the newborn may have alterations in K+-channel activity or in the signal transduction pathway that leads to K+-channel activation.


    ACKNOWLEDGEMENTS

We thank Jerry Manning and Jean Herron for expert technical assistance and Pamela Vavra for outstanding editorial assistance.


    FOOTNOTES

This work was supported in part by an American Heart Association Clinician-Scientist Award (to D. N. Cornfield), a Minnesota Medical Foundation Award (to D. N. Cornfield), and an American Heart Association Minnesota Chapter Affiliate Grant-in-Aid (to V. A. Porter).

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 and other correspondence: D. N. Cornfield, Box 742, Division of Pediatric Pulmonology and Critical Care, Univ. of Minnesota Medical School, 420 Delaware St. S.E., Minneapolis, MN 55455 (E-mail: cornf001{at}tc.umn.edu).

Received 8 December 1998; accepted in final form 17 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS 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., 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].

3.   Archer, S. L., J. M. C. Huang, V. Hampl, D. P. Nelson, P. J. Schultz, and E. K. Weir. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 91: 7583-7587, 1994[Abstract].

4.   Arnold, W. P., C. K. Nittal, S. Katsuki, and F. Murad. Nitric oxide activates guanylate cyclase and increases guanosine 3': 5'-cyclic monophosphate levels in various tissue preparations. Proc. Natl. Acad. Sci. USA 74: 3203-3207, 1977[Abstract].

5.   Bolotina, V. M., S. Najibi, J. J. Palacino, P. J. Pagano, and R. A. Cohen. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850-853, 1994[Medline].

6.   Bolton, T. B., and Y. Imaizumi. Spontaneous transient outward currents in smooth muscle cells. Cell Calcium 20: 141-152, 1996[Medline].

7.   Brayden, J. E., and M. T. Nelson. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 256: 532-535, 1992[Medline].

8.   Cassin, S., G. S. Dawes, and B. B. Ross. Pulmonary blood flow and vascular resistance in immature foetal lambs. J. Physiol. (Lond.) 171: 80-89, 1964.

9.   Cornfield, D. N., and S. H. Abman. Inhalational nitric oxide in pulmonary parenchymal and vascular disease. J. Lab. Clin. Med. 127: 530-539, 1996[Medline].

10.   Cornfield, D. N., B. A. Chatfield, J. A. McQueston, I. F. McMurtry, and S. H. Abman. Effects of birth-related stimuli on L-arginine-dependent pulmonary vasodilation in ovine fetus. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H1474-H1481, 1992[Abstract/Free Full Text].

11.   Cornfield, D. N., J. A. McQueston, I. F. McMurtry, D. M. Rodman, and S. H. Abman. Role of ATP-sensitive potassium channels in ovine fetal pulmonary vascular tone. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1363-H1368, 1992[Abstract/Free Full Text].

12.   Cornfield, D. N., H. L. Reeve, S. Tolarova, E. K. Weir, and S. Archer. Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel. Proc. Natl. Acad. Sci. USA 93: 8089-8094, 1996[Abstract/Free Full Text].

13.   Dawes, G. S., and J. C. Mott. The vascular tone of the foetal lung. J. Physiol. (Lond.) 164: 465-477, 1962.

14.   Dawes, G. S., J. C. Mott, J. G. Widdicombe, and D. G. Wyatt. Changes in the lungs of the new-born lamb. J. Physiol. (Lond.) 121: 141-162, 1953.

15.   Emmanouilides, G. C., A. J. Moss, E. R. Duffie, and F. H. Adams. Pulmonary arterial pressure changes in human newborn infants from birth to 3 days of age. J. Pediatr. 65: 327-333, 1964.

16.   Enhorning, G., F. Adams, and A. Norman. Effects of lung expansion on the fetal lamb circulation. Acta Paediatr. Scand. 55: 441-451, 1966[Medline].

17.   Fineman, J. R., J. Wong, F. C. Morin, L. M. Wild, and S. J. Soifer. Chronic nitric oxide inhibition in utero produces persistent pulmonary hypertension in newborn lambs. J. Clin. Invest. 93: 2675-2683, 1994[Medline].

18.   Galvez, A., G. Gimenez-Gallego, J. P. Reuben, L. Roy-Constancin, P. Feigenbaum, G. J. Kaczorowski, and M. L. Garcia. Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J. Biol. Chem. 265: 11083-11090, 1990[Abstract/Free Full Text].

19.   Karaki, H., H. Ozaki, M. Hori, M. Mitsui-Saito, K. Amano, K. Harada, S. Miyamoto, H. Nakazawa, K. Won, and K. Sato. Calcium movements, distribution, and functions in smooth muscle. Pharmacol. Rev. 49: 157-230, 1997[Abstract/Free Full Text].

20.   Kinsella, J. P., W. E. Truog, W. F. Walsh, R. N. Goldberg, E. Bancalari, D. E. Mayock, G. J. Redding, R. A. Delemos, S. Sardesai, D. C. McCurnin, S. G. Moreland, G. R. Cutter, and S. H. Abman. Randomized multicenter trial of inhaled nitric oxide and high-frequency oscillatory ventilation in severe, persistent pulmonary hypertension of the newborn. J. Pediatr. 131: 55-62, 1997[Medline].

21.   McQueston, J. A., D. N. Cornfield, I. F. McMurtry, and S. H. Abman. Effects of oxygen and exogenous L-arginine on EDRF activity in fetal pulmonary circulation. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H865-H871, 1993[Abstract/Free Full Text].

22.   Moore, P., H. Velvis, J. R. Fineman, S. J. Soifer, and M. A. Heymann. EDRF inhibition attenuates the increase in pulmonary blood flow due to O2 ventilation in fetal lambs. J. Appl. Physiol. 73: 2151-2157, 1992[Abstract/Free Full Text].

23.   Morin, F., E. Eagan, and W. Norfleet. Development of pulmonary vascular response to O2. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H542-H546, 1988[Abstract/Free Full Text].

24.   Nelson, M. T., H. Cheng, M. Rubart, L. F. Santana, A. D. Bonev, H. J. Knot, and W. J. Lederer. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633-637, 1995[Abstract].

25.   Nelson, M. T., and J. M. Quayle. Physiological roles and properties of potassium channels in arterial smooth muscle. Am. J. Physiol. 268 (Cell Physiol. 37): C799-C822, 1995[Abstract/Free Full Text].

26.   Peng, W., J. R. Hoidal, and I. S. Farrukh. Regulation of Ca2+-activated K+ channels in pulmonary vascular smooth muscle cells: role of nitric oxide. J. Appl. Physiol. 81: 1264-1272, 1996[Abstract/Free Full Text].

27.   Porter, V. A., A. D. Bonev, H. J. Knot, T. J. Heppner, A. S. Stevenson, T. Kleppisch, W. J. Lederer, and M. T. Nelson. Frequency modulation of Ca2+ sparks is involved in regulation of arterial diameter by cyclic nucleotides. Am. J. Physiol. 274 (Cell Physiol. 43): C1346-C1355, 1998[Abstract/Free Full Text].

28.   Reeve, H. L., E. K. Weir, S. L. Archer, and D. N. Cornfield. A maturational shift in K+ channels, from Ca2+ sensitive to voltage dependent. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1019-L1025, 1998[Abstract/Free Full Text].

29.   Robertson, B. E., R. Schubert, J. Hescheler, and M. T. Nelson. cGMP dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am. J. Physiol. 265 (Cell Physiol. 34): C299-C303, 1993[Abstract/Free Full Text].

30.   Rusko, J., L. Li, C. van Breeman, and D. J. Adams. Calcium-activated potassium channels in native endothelial cells from rabbit aorta: conductance, Ca2+ sensitivity and block. J. Physiol. (Lond.) 455: 601-621, 1992[Abstract].

31.   Storme, L., R. L. Rairigh, T. A. Parker, D. N. Cornfield, J. P. Kinsella, and S. H. Abman. K+ blockade inhibits shear stress-induced pulmonary vasodilation in the ovine fetus. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L220-L228, 1999[Abstract/Free Full Text].

32.   Tristani-Firouzi, M., E. B. Martin, S. Tolarova, E. K. Weir, S. L. Archer, and D. N. Cornfield. Ventilation-induced pulmonary vasodilation at birth is modulated by potassium channel activity. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H2353-H2359, 1996[Abstract/Free Full Text].

33.   Van Breemen, C., and K. Saida. Cellular mechanisms regulating (Ca++)i smooth muscle. Annu. Rev. Physiol. 51: 315-329, 1989[Medline].

34.   Vandier, C., M. Delpech, and P. Bonnet. Spontaneous transient outward currents and delayed rectifier K+ current: effects of hypoxia. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L145-L154, 1998[Abstract/Free Full Text].

35.   Weir, E. K., H. L. Reeve, D. N. Cornfield, M. Tristani-Firouzi, D. A. Peterson, and S. L. Archer. Diversity of response in vascular smooth muscle cells to changes in oxygen tension. Kidney Int. 51: 462-466, 1997[Medline].

36.   Yuan, X. J., J. Wang, M. Juhasgova, V. A. Golovina, and L. J. Rubin. Molecular basis and function of voltage-gated K+ channels in pulmonary artery smooth muscle cells. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L1-L15, 1998[Abstract/Free Full Text].


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