Chronic hypertension impairs flow-induced vasodilation and augments the myogenic response in fetal lung

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

1 Department of Neonatology, CHRU de Lille, France; and 2 Department of Pediatrics, Pediatric Heart Lung Center, University of Colorado School of Medicine, Denver, Colorado 80218-1088


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that altered vasoreactivity in perinatal pulmonary hypertension (PH) is characterized by abnormal responses to hemodynamic stress, including the loss of flow-induced vasodilation and an augmented myogenic response. Therefore, we studied the acute hemodynamic effects of brief compression of the ductus arteriosus (DA) in control fetal lambs and in lambs during exposure to chronic PH. In both groups, acute DA compression decreased pulmonary vascular resistance (PVR) by 20% at baseline (day 0). After 2 days of hypertension, acute DA compression paradoxically increased PVR by 50% in PH lambs, whereas PVR decreased by 25% in controls. During the 8-day study period, PVR increased during acute DA compression in PH lambs, whereas acute DA compression continued to cause vasodilation in controls. Brief treatment with the nitric oxide (NO) synthase inhibitor nitro-L-arginine (L-NA) increased basal PVR in control but not PH lambs, suggesting decreased NO production in PH lambs. Chronic hypertension increased the myogenic response after L-NA in PH lambs, whereas the myogenic response remained unchanged in controls. The myogenic response was inhibited by nifedipine in PH lambs, suggesting that the myogenic response is dependent upon the influx of extracellular calcium. We conclude that chronic PH impairs flow-induced vasodilation and increases the myogenic response in fetal lung. We speculate that decreased NO signaling and an augmented myogenic response contributes to abnormal vasoreactivity in PH.

myogenic response; nitric oxide; pulmonary circulation; persistent pulmonary hypertension of the newborn; smooth muscle; calcium channels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AT BIRTH, the pulmonary circulation undergoes marked vasodilation, which allows blood flow to increase nearly 10-fold and enables the lung to assume its postnatal role in gas exchange (13, 33). Several stimuli, including drainage and absorption of fetal lung liquid, rhythmic distension of the lung, increased PO2, and altered production of several vasoactive products, including nitric oxide (NO), are known to contribute to pulmonary vasodilation at birth (4, 17, 40). The effects of several birth-related stimuli, including increased blood flow or shear stress, increased PO2, and ventilation, are partly dependent upon activation of NO synthase (NOS; see Refs. 4, 10, 28, 39). The increase in NO production accounts for nearly 50% of the abrupt fall in pulmonary vascular resistance (PVR) at birth in fetal lambs (4, 10).

In addition to its role as a vasodilator at birth, NO production also modulates fetal pulmonary vascular tone in response to diverse stimuli, including acute changes in hemodynamic forces (1, 10). In fetal lambs, acute compression of the ductus arteriosus (DA) abruptly increases blood flow and pressure and causes acute pulmonary vasodilation through the shear stress-induced release of NO (1, 10). However, despite maintenance of constant pressure, pulmonary blood flow progressively falls and PVR increases over time (1). Thus pulmonary blood flow in utero is normally maintained within narrow limits despite increases in vascular perfusion pressure resulting from active vasoconstriction. This pressure- or stretch-induced vasoconstriction, known as the myogenic response, was first described by Bayliss (8) and has been characterized in several systemic vascular beds (12, 30). However, whether high myogenic tone and active myogenic responses play critical roles in vasoregulation of the normal fetal pulmonary circulation or contribute to abnormal vascular function in chronic hypertension are unclear. Recently, inhibition of NOS activity has been shown to not only block shear stress-induced pulmonary vasodilation but also cause a striking and paradoxical vasoconstrictor response (36). These findings suggest that NO not only plays a critical role in mediating vasodilation in response to hemodynamic stimuli at birth but that NO signaling also opposes high myogenic tone in the pulmonary circulation in utero (36). Furthermore, it has been suggested that myogenic tone contributes to high PVR and the inability of several stimuli to cause sustained pulmonary vasodilation in the normal fetus (1, 2, 36, 37).

Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome that is associated with diverse neonatal cardiopulmonary diseases, ranging from asphyxia, sepsis, meconium aspiration, respiratory distress syndrome, and others, or can be idiopathic (26). Physiologically, PPHN is characterized by the failure of the pulmonary circulation to dilate at birth. This leads to sustained elevation of PVR, causing extrapulmonary right-to-left shunting of blood across the DA and foramen ovule and severe hypoxemia (24, 26). PPHN contributes significantly to high morbidity and mortality in hypoxemic newborns, but its pathogenesis and pathophysiology are poorly understood.

Because NO contributes significantly to normal regulation of vascular tone in the perinatal pulmonary circulation, it has been suggested that PPHN may be partly due to decreased NO production (4, 34, 41). To better understand the etiology and pathophysiology of PPHN, our laboratory and others have developed an experimental model of chronic intrauterine pulmonary hypertension caused by partial compression or ligation of the DA in the late-gestation ovine fetus (6, 31). In this model, chronic DA compression causes sustained intrauterine pulmonary hypertension without increased pulmonary blood flow or hypoxemia (6). After delivery, PVR remains elevated with extrapulmonary right-to-left shunting and hypoxemia, despite mechanical ventilation with hyperoxia (6). Past studies have also demonstrated blunted endothelium-dependent vasodilation and downregulation of endothelial NOS expression (29, 34, 41). Mechanisms by which impaired NO signaling alters pulmonary vasoreactivity and contributes to the failure of PVR to fall at birth are incompletely understood.

Therefore, we hypothesize that, in addition to impaired NO production, altered pulmonary vasoreactivity in PPHN is characterized by an unmasked or increased myogenic response in the pulmonary circulation. To test this hypothesis, we performed serial studies of the acute hemodynamic effects of brief DA compression in chronically prepared fetal lambs with chronic pulmonary hypertension and control animals. We report that chronic pulmonary hypertension in utero rapidly impairs flow-induced vasodilation, blocks basal NO production, and augments a potent myogenic response. In addition, we found that treatment with nifedipine abolished the myogenic response in lambs with severe pulmonary hypertension, suggesting that the myogenic response is dependent on activation of voltage-gated calcium channels.


    METHODS
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ABSTRACT
INTRODUCTION
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. Fourteen 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 in the ascending aorta and the superior vena cava after insertion in the axillary artery and vein. A left thoracotomy exposed the heart and great vessels. Catheters were inserted in the left pulmonary artery (LPA), main pulmonary artery, and left atrium by direct puncture through purse string sutures, as previously described (3). An inflatable vascular occluder (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 (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 infused in the amniotic cavity before closure of the hysterotomy. The flow transducer cable, catheters, and vascular 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 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 (Transonic Systems) for continuous measurements of LPA blood flow. The output filter of the flowmeter was set at 100 Hz. Absolute values of flows were determined from means of phasic blood flow signals from at least 30 cardiac cycles with zero blood flow defined as the flow value immediately before the onset of systole. Main pulmonary artery, aortic, left atrial, and amniotic catheters were connected to blood pressure transducers (TSD 104; Biopac System, Santa Barbara, CA). The pressure and flow signals were continuously recorded and processed on a computer using an analog-to-digital converter system (Biopac). The 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 daily 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 artery and left atrial pressures divided by mean left pulmonary blood flow. Blood samples from the main pulmonary artery catheter were used for blood gas analysis and oxygen saturation measurements.

Experimental Design

After at least 48 h of recovery from surgery, animals were randomly assigned to either the control (n = 7) or the chronic pulmonary hypertension (n = 7) group. The duration of the study was 8 days. Chronic pulmonary hypertension was induced by inflating the vascular occluder by slowly infusing a small volume of saline in the cuff around the DA, causing a partial compression of the DA (1, 24). The degree of inflation was set to increase mean pulmonary artery pressure (PAP) by 15 mmHg from its baseline value. Mean PAP was kept constant throughout the study period by readjusting the degree of inflation of the occluder during the first 2 h of DA compression and then by maintaining a constant inflation pressure within the occluder. Briefly, a 10-ml saline syringe was connected to the occluder. The piston was pressed continuously by a spring, the length of which was set to obtain the target PAP. Mean PAP was measured at least two times daily to readjust the pressure in the occluder, if necessary. In the chronic pulmonary hypertension group, DA compression before each acute study was released by completely deflating the occluder 20 min before the beginning of each experiment. In control animals, the vascular occluder was not chronically inflated, but serial measurements of hemodynamic and arterial blood gas tensions were recorded (as described below).

Four different protocols were included in this study. These include 1) serial measurements of the acute hemodynamic response to partial DA compression in pulmonary hypertension and control fetal lambs (protocol 1); 2) serial determinations of the hemodynamic effects of nitro-L-arginine (L-NA), an NOS inhibitor, on basal pulmonary vascular tone (protocol 2); 3) serial assessments of the acute hemodynamic response to partial DA compression after L-NA treatment (for assessment of the myogenic response; protocol 3); and 4) studies of the acute hemodynamic response to partial DA compression after treatment with nifedipine (a voltage-operated calcium channel blocker; protocol 4). Comparisons were made between serial measurements from the control and the chronic pulmonary hypertension (PPHN) study groups.

Protocol 1: Serial measurements of the acute hemodynamic response to partial DA compression in pulmonary hypertension and control fetal lambs. To study serial changes in the hemodynamic response during acute partial compression of the DA, we partially inflated the DA occluder for 10 min after recording baseline measurements. The degree of inflation was set to increase mean PAP by 15 mmHg from baseline values. Mean PAP was kept constant throughout the compression period by readjusting the degree of inflation of the occluder, as needed. After 10 min of DA compression, the occluder was released rapidly (deflated). Mean PAP, left atrial pressure, mean aortic pressure (AoP), amniotic pressure, and left pulmonary blood flow were recorded at 5-min intervals starting 20 min before compression (baseline) and for 10 min after the DA compression period. The duration of each experiment was 40 min.

Protocol 2: Serial determinations of the hemodynamic effects of NOS inhibition on basal pulmonary vascular tone. To determine serial changes in the acute effects of NOS inhibition on basal hemodynamic variables, L-NA (30 mg over 10 min) was infused in the LPA after the completion of protocol 1 (i.e., 40 min after the beginning of the experiment). This dose was selected from published studies that have demonstrated that this dose of L-NA inhibits ACh, oxygen, and flow-induced pulmonary vasodilation (4, 10, 36, 39). Hemodynamic parameters were recorded at 5-min intervals, starting from the beginning of L-NA infusion and for 30 min. The duration of each experiment was 30 min.

Protocol 3: Serial assessment of the acute hemodynamic response to partial DA compression after L-NA treatment. To investigate the effects of L-NA on the hemodynamic response to acute partial DA compression, we partially inflated the DA occluder at the end of the second part of the experiment. The DA occluder was partially inflated for 10 min, and the degree of inflation was set to increase PAP by 15 mmHg from its baseline value. Mean PAP was kept constant throughout the compression period. After 10 min of DA compression, the occluder was deflated. Hemodynamic parameters were recorded at 5-min intervals for 10 min during and 10 min after the compression period. The duration of each experiment was 20 min.

Protocol 4: Studies of the acute hemodynamic response to partial DA compression after nifedipine treatment. To investigate the effects of voltage-operated calcium channel blockade on the hemodynamic response to acute partial DA compression, we partially inflated the DA occluder after treatment with nifedipine, a voltage-operated calcium channel blocker. Nifedipine (0.8 mg) was infused for 10 min in the LPA. Preliminary studies showed that this dose does not affect basal pulmonary vascular tone in the fetus. After completion of drug infusion, the DA occluder was partially inflated for 10 min. As described above, 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. After 10 min, the occluder was deflated. Hemodynamic parameters were measured at 5-min intervals during and after the compression period.

Each study was performed after at least 48 h of recovery. The first day of study was designated as "day 0." Study protocols were performed at day 0 (before initiating chronic DA compression in the pulmonary hypertension group), at 48 h (days 2, 4, 6, and 8 for protocol 1), or at 96 h (days 4 and 8 for protocols 2 and 3) intervals. Protocol 4 was performed in control and hypertensive lambs on day 9. At each study, arterial blood gas tensions and pH were measured during the baseline period, at the end of the first DA compression period (30 min), after L-NA infusion at 70 min, and at the end of the second DA compression period (80 min). One animal in each group was excluded from the study at days 6 and 8 because of technical problems with the DA or fetal demise.

Drug Preparation

L-NA and nifedipine solutions were freshly prepared just before the study. L-NA (Sigma Chemical, St. Louis, MO) at the dose of 30 mg was dissolved in 1 M HCl and diluted in normal saline (1 ml final volume). NaOH (1 M) was added in small increments to titrate the pH of the solution to 7.40. Nifedipine (0.8 mg; Sigma Chemical) was dissolved in 0.1 ml of 100% ethanol and diluted in normal saline (1 ml final volume). Each drug (L-NA and nifedipine) was infused at a rate of 0.1 ml/min.

Data Analysis

The results are presented as means ± SE. Data were analyzed by using repeated-measures and factorial ANOVA (Stat View 4.1 for Macintosh; Abacus Concepts, Berkeley, CA). Post hoc analysis was performed with Newman-Keuls testing. A P value <0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Protocol 1: Serial Determinations of the Acute Hemodynamic Response to Partial DA Compression in Hypertensive and Control Animals

Day 0 studies. The acute hemodynamic response to partial compression of the DA was similar in the control and pulmonary hypertension groups when studied on day 0 (before initiating chronic DA compression in the hypertension group). In the control group, brief compression of the DA rapidly increased mean PAP by 35% (P < 0.05 vs. baseline) and LPA blood flow by 82% (P < 0.05) at 10 min (Fig. 1). In the pulmonary hypertension group, mean PAP increased 32%, and LPA blood flow increased by 74% (P < 0.05 for each parameter; Fig. 1). During acute DA compression, PVR decreased 20 ± 5% below baseline values in both groups (P < 0.05; Figs. 1 and 2). Heart rate increased from 149 ± 13 (baseline) to 169 ± 9 beats/min and from 154 ± 11 (baseline) to 176 ± 14 beats/min in the control and pulmonary hypertension groups, respectively (P < 0.05 for baseline vs. peak values within each group). Mean left atrial pressure was 2 ± 1 at baseline and did not change during the study period. Values for mean AoP, arterial blood gas, and pH parameters were not different between study groups at baseline (Table 1) and did not change significantly during the DA compression period.


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Fig. 1.   Day 0 studies of the pulmonary vascular response during acute ductus arteriosus (DA) compression before and after nitro-L-arginine (L-NA) treatment in control and pulmonary hypertension [persistent pulmonary hypertension of the newborn (PPHN)] groups. As shown, acute DA compression increased mean pulmonary artery pressure (PAP) to the same degree in control and PPHN animals (top; at 20-30 min), causing a rise in left pulmonary artery (LPA) blood flow (middle) and a fall in pulmonary vascular resistance (PVR; bottom). Treatment with the nitric oxide synthase (NOS) inhibitor L-NA elevated PAP and PVR to the same degree in both groups (at 40-70 min). Acute DA compression after L-NA treatment blocked the fall in PVR and caused a marked increase in PVR (bottom; at 70-80 min; see text for details). *P < 0.05 vs. baseline values within each study group for PVR measurements; n, no. of animals.



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Fig. 2.   Serial changes in PVR from baseline values during acute DA compression in control and PPHN animals (protocol 1). As shown, PVR fell nearly 25% in control animals throughout the 8-day study. In contrast, acute DA compression increased PVR from day 2 to 8 in the PPHN animals. *P < 0.05 vs. baseline values within each study group.


                              
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Table 1.   Blood gas and hemodynamic variables at baseline (20 min after the release of the chronic ductus compression in pulmonary hypertensive group) in control and PPHN groups

Day 2 studies. After 2 days of hypertension, release of the DA occluder decreased mean PAP from 62 ± 4 to 54 ± 5 mmHg in the hypertensive group (Fig. 3). Reinflation of the DA occluder rapidly increased mean PAP by 33 ± 4% in the control group and by 31 ± 5% in the pulmonary hypertension group [P = not significant (NS) between groups; Fig. 3]. However, although LPA blood flow increased nearly twofold in the control group (P < 0.05), there was no change in LPA blood flow in the chronic pulmonary hypertension group. PVR increased in the chronic pulmonary hypertension group but not in the control group (Fig. 3). During acute DA compression, heart rate increased in both groups (from 145 ± 11 to 167 ± 12 beats/min in the control group and from 151 ± 11 to 178 ± 14 beats/min in the chronic pulmonary hypertension group; P < 0.05, treatment vs. baseline within each group). Left atrial pressure, mean AoP, and arterial blood gas parameters did not change during the study period.


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Fig. 3.   Day 2 studies of the pulmonary vascular response during acute DA compression in control and PPHN groups (protocol 1). In contrast to the fall in PVR in control animals, DA compression caused a marked increased in PVR in the PPHN group. *P < 0.05 vs. baseline values within each study group.

Day 4 studies. After 4 days of DA compression, release of the DA occluder reduced mean PAP from 64 ± 2 to 55 ± 4 mmHg (Fig. 4). After recovery from chronic DA compression, acute inflation of the DA occluder increased mean PAP by 41 ± 4 and by 36 ± 5% in the control and pulmonary hypertension groups, respectively. Although LPA blood flow increased by more than twofold in the control group (104 ± 14%; P < 0.05 vs. baseline), there was no change in LPA blood flow during acute DA compression in the pulmonary hypertension group (Fig. 4). During the DA compression period, PVR decreased from 0.57 ± 0.03 to 0.41 ± 0.05 mmHg · ml-1 · min-1 in the control group (P < 0.05) but increased from 0.62 ± 0.03 to 0.99 ± 0.1 mmHg · ml-1 · min-1 in the chronic pulmonary hypertension group (P < 0.05; Figs. 2 and 4). During acute DA compression, heart rate increased in both groups, but there was no difference between study groups. Mean left atrial pressure, mean AoP, and arterial blood gas parameters did not change during or after the compression period and were not different between groups.


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Fig. 4.   Day 4 studies of the pulmonary vascular response during acute DA compression before and after L-NA treatment in control and PPHN groups. As shown, release of chronic DA compression decreased PAP in the PPHN group (top; 0-30 min). In the control animals, acute DA compression increased mean PAP (top; at 20-30 min) and LPA blood flow (middle) and reduced PVR (bottom). In contrast, flow did not change (middle), and PVR was increased markedly (bottom) during acute DA compression in the PPHN group. Treatment with L-NA increased PVR in the control but not the PPHN group (bottom; 50 min). Acute DA compression elevated PVR to the same degree in both groups (bottom; 80 min). *P < 0.05 vs. baseline values within each study group for PVR measurements.

Day 6 studies. In the chronic pulmonary hypertension group, release of the DA occluder decreased mean PAP from 68 ± 2 to 55 ± 4 mmHg (Fig. 5). Subsequently, the DA occluder was partially inflated to increase mean PAP by 35 ± 3 and by 36 ± 6% in the control and chronic pulmonary hypertension groups, respectively (Fig. 5). LPA blood flow increased by 108 ± 16% (from 85 ± 5 to 177 ± 21 ml/min), and PVR decreased by 29 ± 7% (from 0.56 ± 0.05 to 0.4 ± 0.07 mmHg · ml-1 · min-1) during acute DA compression in the control group (P < 0.05). In contrast, LPA blood flow did not change in the pulmonary hypertension group during acute DA compression (Fig. 5). Unlike the controls, PVR increased by 50 ± 8% during acute DA compression in the chronic pulmonary hypertension group (Fig. 5; bottom). Mean left atrial pressure, mean AoP, and arterial blood gas parameters did not change during the study period and were not different between groups.


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Fig. 5.   Day 6 studies of the pulmonary vascular response during acute DA compression before L-NA in control and PPHN groups (protocol 1). As shown, acute DA compression in the absence of NOS inhibition decreased PVR in the control group but increased PVR in PPHN animals. *P < 0.05 vs. baseline values within each study group for PVR measurements.

Day 8 studies. In the chronic pulmonary hypertension group, acute release of the DA decreased mean PAP from 71 ± 4 to 60 ± 6 mmHg. Partial compression of the DA rapidly increased mean PAP by 37 ± 3% in the control group (from 49 ± 2 to 67 ± 3 mmHg; P < 0.05) and by 30 ± 6% in the chronic pulmonary hypertension group (from 60 ± 6 to 78 ± 6 mmHg). LPA blood flow increased by 89 ± 12% (from 109 ± 5 to 206 ± 18 ml/min) in the control group (P < 0.05) but did not change with acute DA compression in the chronic pulmonary hypertension group. During acute DA compression, PVR decreased from 0.44 ± 0.03 to 0.32 ± 0.04 mmHg · ml-1 · min-1 in the control group (P < 0.05), but PVR increased from 0.62 ± 0.06 to 0.95 ± 0.10 mmHg · ml-1 · min-1 in the chronic pulmonary hypertension group (Figs. 2 and 6). Mean left atrial pressure, mean AoP, and arterial blood gas parameters did not change during the study.


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Fig. 6.   Day 8 studies of the pulmonary vascular response during acute DA compression before and after L-NA treatment in control and PPHN groups. As shown, release of chronic DA compression decreased PVR in the PPHN group, but PVR remained elevated above control values (at 20 min). In control animals, acute DA compression reduced PVR (at 30 min). In contrast, PVR was increased markedly during acute DA compression in the PPHN group. Treatment with L-NA increased PVR in the control but not the PPHN group (50 min). Acute DA compression after L-NA treatment elevated PVR in the control animals, but the PVR achieved in controls was less than that achieved in the PPHN group (80 min). *P < 0.05 vs. baseline values within each study group.

Protocol 2: Serial Determinations of the Hemodynamic Effects of NOS Inhibition on Basal Pulmonary Vascular Tone

Day 0 Studies. At day 0, L-NA treatment increased PVR in both study groups from 0.53 ± 0.07 (baseline) to 0.72 ± 0.06 mmHg · ml-1 · min-1 and from 0.54 ± 0.05 (baseline) to 0.78 ± 0.04 mmHg · ml-1 · min-1 in the control and pulmonary hypertension groups, respectively [P < 0.05 for baseline vs. treatment within each group (Fig. 1) and at 40-70 min (Fig. 7)]. Mean AoP increased after L-NA treatment by 20% in both groups (P < 0.05 vs. baseline). Values for heart rate, left atrial pressure, arterial blood gas values, and pH did not change during this study period and were similar between groups.


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Fig. 7.   Serial changes in PVR after treatment with the NOS inhibitor L-NA in control and PPHN lambs (protocol 2). In controls, L-NA treatment increased PVR throughout the study but had little effect on basal PVR in the PPHN group. *P < 0.05 vs. baseline values within each study group.

Day 4 studies. In the control group, L-NA infusion increased PVR from 0.52 ± 0.05 to 0.78 ± 0.07 mmHg · ml-1 · min-1 (P < 0.05 vs. baseline) and decreased LPA blood flow (P < 0.05). In contrast, L-NA treatment did not change PVR or LPA blood flow in the chronic pulmonary hypertension group (at 40-70 min; Fig. 4). The increase in PVR after L-NA treatment, as expressed as percentage change from baseline, was greater in control animals than in the chronic pulmonary hypertension group (Fig. 7). In contrast to the pulmonary circulation, mean AoP increased after L-NA treatment in both groups. Heart rate, left atrial pressure, pH, and arterial blood gas parameters remained unchanged during this study period, and these parameters were not different between groups.

Day 8 studies. L-NA infusion increased mean PAP from 48 ± 4 to 59 ± 5 mmHg (P < 0.05; treatment vs. baseline values) and increased PVR from 0.45 ± 0.04 to 0.62 ± 0.03 mmHg · ml-1 · min-1 in the control group (P < 0.05; at 40-70 min; Fig. 6). In contrast, L-NA did not change mean PAP, LPA blood flow, or PVR in the chronic pulmonary hypertension group (Figs. 6 and 7). Mean AoP increased from 44 ± 3 to 55 ± 4 mmHg (P < 0.05) in the control group and from 49 ± 3 to 57 ± 3 mmHg (P = 0.05) in the pulmonary hypertension group. Heart rate, left atrial pressure, pH, and arterial blood gas parameters remained unchanged during this study period.

Protocol 3: Serial Determinations of the Acute Hemodynamic Response to Partial DA Compression After L-NA Treatment

Baseline studies (day 0). Partial DA compression after L-NA treatment rapidly increased mean PAP from 58 ± 3 to 76 ± 2 mmHg in the control group (P < 0.05) and from 58 ± 2 to 72 ± 2 mmHg in the chronic pulmonary hypertension group (P < 0.05 and P = NS for comparisons of mean PAP during compression between groups; at 70-80 min; Fig. 1). The increase in PVR during DA compression after L-NA treatment was not different between groups. After deflation of the DA occluder, PVR fell to baseline values within each group. Mean AoP, left atrial pressure, pH, and blood gas parameters did not change during the study period.

Day 4 studies. After L-NA treatment, partial DA compression increased mean PAP by 30 ± 3 and by 30 ± 5% in the control and pulmonary hypertension groups, respectively (at 70-80 min; Fig. 4). LPA blood flow did not change during DA compression after the L-NA period in either group. PVR progressively increased in both groups (P < 0.05, treatment vs. baseline within each group; P = NS between study groups). The percentage change of PVR during DA compression was not different between control and pulmonary hypertension groups (Fig. 8). Mean AoP, left atrial pressure, pH, and arterial blood gas parameters did not change during the study in either group.


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Fig. 8.   Serial changes in PVR during acute DA compression after NOS inhibition in control and PPHN lambs (protocol 3). The rise in PVR during DA compression increased progressively in the PPHN group but did not change in control animals. *P < 0.05 vs. day 0 values within each study group and for comparison between groups at day 8.

Day 8 studies. Partial compression of the DA after L-NA treatment rapidly increased mean PAP by 32 ± 5% in the control group and by 36 ± 4% in the chronic pulmonary hypertension group (P = NS between groups). LPA blood flow did not change during acute DA compression in either group. PVR progressively increased from 0.62 ± 0.03 to 0.78 ± 0.03 mmHg · ml-1 · min-1 in the control group (P < 0.05; baseline vs. compression values) and from 0.71 ± 0.07 to 1.06 ± 0.07 mmHg · ml-1 · min-1 in the chronic pulmonary hypertension group (P < 0.05 for baseline vs. compression values; at 70-80 min; Fig. 6). The increase in PVR during DA compression was greater in the chronic pulmonary hypertension group than in the control group (Fig. 8). Mean AoP, left atrial pressure, pH, and arterial blood gas parameters did not change during this study period in either group.

Protocol 4: Acute Hemodynamic Response to Partial DA Compression After Inhibition of Voltage-Operated Calcium Channels in Control and Hypertensive Lambs

At day 9, mean PAP decreased from 71 ± 4 to 61 ± 5 mmHg after acute release of the DA occluder in the chronic pulmonary hypertension group. Nifedipine infusion did not change mean PAP, LPA blood flow, or PVR in either study group. Partial compression of the DA after nifedipine treatment rapidly increased mean PAP by 38 ± 5% in the control group (from 44 ± 3 to 61 ± 3 mmHg; P < 0.05) and by 33 ± 6% in the chronic pulmonary hypertension group (from 52 ± 6 to 69 ± 6 mmHg; P < 0.05). The percent change in PAP during acute DA compression was not different between study groups. After nifedipine treatment, DA compression increased LPA blood flow by 122 ± 17% (from 102 ± 8 to 226 ± 24 ml/min) in the control group (P < 0.05) but did not change LPA flow in the chronic pulmonary hypertension group (from 98 ± 9 to 126 ± 19 mmHg · ml-1 · min-1; P = NS). As observed on the previous DA compression on day 8, PVR decreased from 0.41 ± 0.03 to 0.27 ± 0.03 mmHg · ml-1 · min-1 in the control group (P < 0.05; baseline vs. compression values). In contrast to the marked increase in PVR observed during DA compression in the pulmonary hypertension group on day 8, nifedipine treatment completely blocked the rise in PVR (Figs. 9 and 10). Mean AoP, left atrial pressure, pH, and arterial blood gas parameters did not change during this study period in either group. However, nifedipine increased heart rate from 157 ± 12 to 190 ± 18 beats/min in control animals and from 169 ± 16 to 199 ± 16 beats/min in the chronic pulmonary hypertension group (P < 0.05; Table 1).


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Fig. 9.   Effects of nifedipine treatment on the hemodynamic response to acute compression of the DA in control and PPHN groups after 9 days (protocol 4). In the PPHN group, the DA occluder was deflated immediately before treatment with nifedipine. Acute DA increased mean PAP in both groups (top) but increased LPA flow only in the control group (middle). During DA compression after nifedipine treatment, PVR decreased in control animals but not in the PPHN group (bottom).



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Fig. 10.   Effects of nifedipine treatment on the percent change in PVR in control and PPHN animals (protocol 4). Comparisons are made from day 8 (no nifedipine) and day 9 (nifedipine) within each group. As shown, acute DA compression decreased PVR by 25% on day 8; this response was not changed after nifedipine treatment. In the PPHN group, acute DA compression increased PVR by 50% above baseline values. The rise in PVR was abolished after nifedipine treatment in the PPHN group. *P < 0.001, treated vs. baseline for the PPHN group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we tested the hypothesis that chronic intrauterine pulmonary hypertension impairs the pulmonary vascular response to changes in hemodynamic forces, as assessed by serial studies of acute compressions of the DA. In normal fetal lambs, acute compression of the DA causes progressive pulmonary vasodilation, which is blocked by NOS inhibition, suggesting that NO mediates shear stress-induced vasodilation. After NOS inhibition or in the pulmonary hypertension lambs, LPA blood flow increases rapidly during the first few minutes but decreases rapidly toward baseline levels (36, 37). In addition, acute DA compression after NOS inhibition causes a marked pulmonary vasoconstrictor response, suggesting that NOS blockade unmasks a potent myogenic response in the normal fetal lung. In contrast to controls, exposure to chronic pulmonary hypertension impairs flow-induced vasodilation and enhances the myogenic response during acute DA compression in the absence of NOS inhibition. Overall, we found that, in the late-gestation fetus: 1) flow-induced pulmonary vasodilation was absent and pressure-induced vasoconstriction was unmasked after 2 days of pulmonary hypertension; 2) the vasoconstrictor response after NOS blockade was diminished by 4 days of pulmonary hypertension; and 3) pressure or stretch-induced vasoconstriction, i.e., the myogenic response, was enhanced after 8 days of chronic pulmonary hypertension. In addition, we have also demonstrated that nifedipine treatment abolished this myogenic response, demonstrating that influx of extracellular calcium through voltage-operated channels is critical for stretch-induced vasoconstriction in the hypertensive lung. These results support the hypothesis that chronic pulmonary hypertension abolishes flow-induced vasodilation and increases the myogenic response in the perinatal lung.

These findings are the first demonstration that an increased myogenic response contributes to abnormal vasoreactivity in chronic pulmonary hypertension in vivo. Past experimental studies of chronic pulmonary hypertension in newborn and adult animals have demonstrated several potential mechanisms of abnormal vascular tone (5). Such mechanisms include impaired production of endogenous vasodilators, including NO and prostacyclin, increased production of vasoconstrictors such as endothelin (ET)-1, or abnormal smooth muscle cell responsiveness to vasoactive stimuli (5, 20, 21, 29, 34, 35, 41). Previous work in this model of perinatal pulmonary hypertension has also demonstrated decreased soluble guanylate cyclase expression and activity (35), increased cGMP-specific (or, type 5) phosphodiesterase activity (15), and increased ET-1-mediated vasoconstriction (20, 21). Thus past studies suggest that chronic pulmonary hypertension in utero alters the balance of endogenous vasoactive substances in favor of increased tone (5). This present study suggests that an enhanced myogenic response also contributes to abnormal vasoreactivity in chronic pulmonary hypertension. In addition to the role of NO, the altered response to acute hemodynamic stress may be partly due to decreased production or activity of other dilators, including prostacyclin or an endothelium-derived hyperpolarizing factor, or increased production of vasoconstrictor products, such as ET-1, thromboxane, or leukotrienes. However, recent studies suggest that cyclooxygenase products, such as prostacyclin or thromboxane, are unlikely to play a significant role in modulation of the myogenic response (43). Recent studies have suggested an important role for cytochrome P-450 metabolites of arachidonic acid, including 20-hydroxyeicosatetraenoic acid, in pressure-induced vasoconstriction of cerebral and renal arteries in vitro (14, 23, 38). This mechanism has not yet been studied in the fetal lung circulation. In addition to the loss of the braking effect of NO production on the myogenic response in the normal fetus, chronic hypertension also augments the myogenic response, thereby contributing to abnormal vasoreactivity due to enhanced stretch-induced vasoconstriction. The effects of prolonged hypertension on the acute response to hemodynamic forces are partly due to decreased endogenous NOS activity (29, 34, 41), which unmasks the underlying myogenic response (36, 37). However, the presence of an enhanced myogenic response in this model suggests an additional mechanism that underlies abnormal vasoreactivity in perinatal pulmonary hypertension.

The myogenic response is defined as the ability of vascular smooth muscle to constrict during an increase in intravascular pressure and to dilate upon lowering intravascular pressure (8, 12, 30). The myogenic response plays a key role in autoregulation of blood flow to the brain, kidney, and other systemic vascular beds, but little is known about the presence and physiological roles of myogenic responses in the pulmonary circulation. Past studies have described stretch-induced vasoconstriction in the pulmonary circulation, but these findings were based exclusively on in vitro studies of isolated arteries (9, 25). More recently, studies in chronically prepared fetal lambs have demonstrated that a potent myogenic response exists in the pulmonary circulation in vivo (36). In the normal fetus, acute DA compression rapidly increases PAP and pulmonary artery blood flow, and PVR rapidly decreases (1). However, despite maintaining PAP constant over time, PVR steadily increases above baseline values after 1 h. These time-dependent changes in PVR during acute DA compression likely represent the effects of the following two opposing stimuli: 1) flow-induced vasodilation because of an abrupt increase in shear stress and 2) pressure- or stretch-induced vasoconstriction resulting from activation of a strong myogenic response that opposes the vasodilator response. Shear stress-induced pulmonary vasodilation is primarily mediated by NO release (10). DA compression after NOS inhibition causes PVR to increase in the normal fetal lamb, suggesting that NO synthesis blockade unmasks the myogenic response (36).

The myogenic response may play important physiological roles in the maintenance of high PVR and time-dependent autoregulation of pulmonary blood flow in the normal fetus. Several physiological and pharmacological stimuli cause fetal pulmonary vasodilation, but vasodilation is often transient despite continued exposure to the dilator stimulus (5). For example, increases in fetal arterial PO2 cause a two- to threefold increase in blood flow after 1 h, but, despite maintaining high arterial PO2, blood flow falls progressively over time (7). Similar patterns have also been observed during prolonged treatment with pharmacological vasodilators, including ACh, bradykinin, tolazoline, and dilator prostaglandins (5). Mechanisms that oppose pulmonary vasodilation in the fetus are uncertain, but we speculate that this time-dependent process represents an autoregulatory mechanism caused by stretch-induced activation of a strong myogenic response. Although treatment with a selective ETA receptor blockers improves blood flow during prolonged DA compression, the myogenic response cannot simply be explained by increased ET-1-mediated vasoconstriction (2). Interestingly, endothelium-independent agonists, such as 8-bromo-cGMP, atrial natriuretic peptide, and inhaled NO, cause more sustained fetal pulmonary vasodilation during prolonged treatment than endothelium-dependent agonists (22). These findings have led to the speculation that the time-dependent loss of vasodilation may represent an inability of the fetal lung to sustain NO production in response to endothelium-dependent stimuli (5).

Thus the fetal pulmonary circulation is characterized by the presence of a strong myogenic response that is increased by chronic pulmonary hypertension. Mechanisms by which hypertension augments the myogenic response in the developing lung are uncertain but are likely to primarily be the result of altered calcium handling in vascular smooth muscle cells, secondary to changes in potassium ion channel activity, intracellular signaling, and autocrine or paracrine factors (11, 14, 16, 18, 23, 27, 38). In this study, inhibition of voltage-operated calcium channels inhibited the myogenic response, as has been observed previously for the myogenic response in systemic vessels (19, 32, 42). Dihyhdropyridines abolish pressure- or stretch-induced increases in intracellular calcium concentration in smooth muscle from isolated rat mesenteric arteries (42). In addition, activation of voltage-operated calcium channels enhances the myogenic response in skeletal muscle arteries (19). Finally, voltage dependence of the L-type channel predicts that the 20- to 35-mV depolarization resulting from stretch of vascular smooth muscle increases the open probability of voltage-gated calcium channels by 10- to 15-fold (32). It is possible, however, that nifedipine may have blocked the myogenic response in hypertensive lambs by an alternate mechanism, but their effects in this model are most likely the result of inhibition of calcium channels.

Our results further support the hypothesis that the myogenic response is mediated by activation of voltage-operated calcium channels and that calcium influx is likely a key downstream event in the signaling pathway. These data do not rule out an "upstream" role of other types of channels that could regulate voltage-operated calcium channels through depolarization. Vasoconstriction in response to increased intravascular pressure is mediated by changes in the activation states of potassium and calcium ion channels, resulting in depolarization of vascular smooth muscle cells and an influx of calcium. Activation of phosopholipases C and A2 and protein kinase C and increased production of lipid mediators are associated with the development of myogenic tone (11, 14, 16, 18, 23, 27).

In conclusion, we report that chronic hypertension dramatically alters the response of the fetal pulmonary circulation to abrupt changes in hemodynamic forces, as characterized by the loss of flow-induced vasodilation and enhancement of a potent myogenic response. These observations are likely due in part to marked impairment of endogenous NOS activity, as reflected by the loss of L-NA-mediated vasoconstriction and the finding that NOS inhibition blocks flow-induced vasodilation. In addition, hypertension likely directly alters the vascular smooth muscle cell's response to stretch stress or pressure-induced vasoconstriction, causing further augmentation of the myogenic response resulting from increased activity of voltage-operated calcium channels. We speculate that chronic hypertension decreases NO production, inhibiting the normal vasodilator response to birth-related stimuli and causing paradoxical vasoconstriction to acute hemodynamic stress. In addition, hypertension alters smooth muscle function, causing an enhanced myogenic response. We further speculate that interventions that specifically target mechanisms underlying the myogenic response may improve the treatment of patients with severe pulmonary hypertension.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the March of Dimes Research Foundation (to S. H. Abman) and National Heart, Lung, and Blood Institute Specialized Center of Research Program Grants HL-46481 (to S. H. Abman) and RO1 HL-68702 (to S. H. Abman).


    FOOTNOTES

Address for reprint requests and other correspondence: S. H. Abman, Pediatric Heart Lung Center, B-395, Dept. of Pediatrics, The Children's Hospital, 1056 E. 19th Ave., Denver, CO 80218-1088 (E-mail: steven.abman{at}uchsc.edu).

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. Section 1734 solely to indicate this fact.

Received 20 July 2001; accepted in final form 27 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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

2.   Abman, SH, and Accurso FJ. Sustained fetal pulmonary vasodilation during prolonged infusion of atrial natriuretic factor and 8-bromo-guansosine monophosphate. Am J Physiol Heart Circ Physiol 260: H183-H192, 1991[Abstract/Free Full Text].

3.   Abman, SH, Accurso FJ, Ward RM, and Wilkening RB. Adaptation of fetal pulmonary blood flow to local infusion of tolazoline. Pediatr Res 20: 1131-1135, 1986[Abstract].

4.   Abman, SH, Chatfield BA, Hall SL, and McMurtry IF. Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth. Am J Physiol Heart Circ Physiol 259: H1921-H1927, 1990[Abstract/Free Full Text].

5.   Abman, SH, Kinsella JP, Parker TA, Storme L, and Le Cras TD. Physiologic roles of NO in the perinatal pulmonary circulation. In: Fetal and Neonatal Pulmonary Circulations, edited by Weir EK, Archer SL, and Reeves JT.. Armonk, NY: Futura, 1999, p. 239-260.

6.   Abman, SH, Shanley PF, and Accurso FJ. Failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs. J Clin Invest 83: 1849-1859, 1989[ISI][Medline].

7.   Accurso, FJ, Alpert B, Wilkening RW, Petersen RG, and Meschia G. Time-dependent response of fetal pulmonary blood flow to an increase in fetal oxygen tension. Respir Physiol 63: 43-52, 1986[ISI][Medline].

8.   Bayliss, WM. On the local reactions of the arterial wall to changes of internal pressure. J Physiol (Lond) 28: 200-231, 1902.

9.   Belik, J. Myogenic response in large pulmonary arteries and its ontogenesis. Pediatr Res 36: 34-40, 1994[Abstract].

10.   Cornfield, DN, Chatfield BA, McQueston JA, McMurtry IF, and Abman SH. Effects of birth-related stimuli on L-arginine-dependant pulmonary vasodilation in ovine fetus. Am J Physiol Heart Circ Physiol 262: H1474-H1481, 1992[Abstract/Free Full Text].

11.   D'Angelo, G, and Meininger GA. Transduction mechanisms involved in the regulation of myogenic activity. Hypertension 23: 1096-1105, 1994[Abstract].

12.   Davis, MJ, and Hill AM. Signaling mechanisms underlying the vascular myogenic response. Physiol Rev 79: 387-423, 1999[Abstract/Free Full Text].

13.   Dawes, GS, Mott JC, and Widdicombe JG. Changes in the lungs of the newborn lamb. J Physiol (Lond) 121: 141-162, 1953[ISI].

14.   Gebremedhin, D, Lange AR, Lowry TF, Taheri MR, Birks EK, Hudetz AG, Narayanan J, Falck JR, Okamoto H, Roman RJ, Nithipatikom K, Campbell WB, and Harder DR. Production of 20-HETE and its role in autoregulation of cerebral blood flow. Circ Res 87: 60-65, 2000[Abstract/Free Full Text].

15.   Hanson, K, Ziegler JW, Rybalkin SD, Miller J, Abman SH, and Clarke WR. Chronic pulmonary hypertension increases fetal lung cGMP phosphodiesterase activity. Am J Physiol Lung Cell Mol Physiol 275: L931-L941, 1998[Abstract/Free Full Text].

16.   Harder, DR. Pressure-dependent membrane depolarization in cat middle cerebral artery. Circ Res 55: 197-202, 1984[Abstract].

17.   Heymann, MA, and Soifer SJ. Control of fetal and neonatal pulmonary circulation. In: Pulmonary Vascular Physiology and Pathophysiology, edited by Weir EK, and Reeves JT.. New York: Dekker, 1989, p. 33-50.

18.   Hill, MA, Falcone JC, and Meininger GA. Evidence for protein kinase C involvement in arteriolar myogenic reactivity. Am J Physiol Heart Circ Physiol 259: H1586-H1594, 1990[Abstract/Free Full Text].

19.   Hill, MA, and Meininger GA. Calcium entry and myogenic phenomena in skeletal muscle arterioles. Am J Physiol Heart Circ Physiol 267: H1085-H1092, 1994[Abstract/Free Full Text].

20.   Ivy, DD, Kinsella JP, and Abman SH. Physiologic characterization of endothelin A and B receptor activity in the ovine fetal pulmonary circulation. J Clin Invest 99: 1179-1186, 1994[Abstract/Free Full Text].

21.   Ivy, DD, Parker TA, Ziegler JW, Galan HL, Kinsella JP, and Abman SH. Prolonged endothelin A receptor blockade attenuates chronic intrauterine pulmonary hypertension. J Clin Invest 99: 1179-1186, 1997[Abstract/Free Full Text].

22.   Ivy, DD, Ziegler JW, Kinsella JP, and Abman SH. Endothelin blockade augments fetal pulmonary vasodilation. J Appl Physiol 81: 2481-2487, 1996[Abstract/Free Full Text].

23.   Kaley, G. Regulation of vascular tone. Role of 20-HETE in the modulation of myogenic reactivity. Circ Res 87: 4-5, 2000[Free Full Text].

24.   Kinsella, JP, and Abman SH. Recent developments in the pathophysiology and treatment of persistent pulmonary hypertension of the newborn. J Pediatr 126: 853-864, 1995[ISI][Medline].

25.   Kulik, TJ, Evans JN, and Gamble WJ. Stretch-induced contraction in pulmonary arteries. Am J Physiol Heart Circ Physiol 255: H1191-H1198, 1988.

26.   Levin, DL, Heyman MA, Kitterman JA, Gregory GA, Phibbs RH, and Rudolph AM. Persistent pulmonary hypertension of the newborn. J Pediatr 89: 626-630, 1976[ISI][Medline].

27.   Li, C, and Xu Q. Mechanical stress initiated signal transductions in vascular smooth mucle cells. Cell Signal 12: 435-445, 2000[ISI][Medline].

28.   McQueston, JA, Cornfield DN, McMurtry IF, and Abman SH. Effects of oxygen and exogenous L-arginine on EDRF activity in the fetal pulmonary circulation. Am J Physiol Heart Circ Physiol 264: H865-H871, 1993[Abstract/Free Full Text].

29.   McQueston, JA, Kinsella JP, Ivy DD, McMurtry IF, and Abman SH. Chronic pulmonary hypertension in utero impairs endothelium-dependent vasodilation. Am J Physiol Heart Circ Physiol 268: H288-H294, 1995[Abstract/Free Full Text].

30.   Meininger, GA, and Davis MJ. Cellular mechanisms involved in the vascular myogenic response. Am J Physiol Heart Circ Physiol 263: H647-H659, 1992[Abstract/Free Full Text].

31.   Morin, FC. Ligating the ductus arteriosus before birth causes persistent pulmonary hypertension in the newborn lamb. Pediatr Res 25: 245-250, 1989[Abstract].

32.   Nelson, MT, Patlak JB, Worley JF, and Standen NB. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am J Physiol Cell Physiol 259: C3-C18, 1990[Abstract/Free Full Text].

33.   Rudolph, AM. Fetal and neonatal pulmonary circulation. Annu Rev Physiol 41: 383-395, 1979[ISI][Medline].

34.   Shaul, PW, Yuhanna IS, German Z, Chen Z, Steinhorn RN, and Morin FC. Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 272: L1005-L1012, 1997[Abstract/Free Full Text].

35.   Steinhorn, RH, Russell JA, and Morin FC. Disruption of cGMP production in pulmonary arteries isolated from fetal lambs with pulmonary hypertension. Am J Physiol Heart Circ Physiol 268: H1483-H1489, 1995[Abstract/Free Full Text].

36.   Storme, L, Rairigh RL, Parker TA, Kinsella JP, and Abman SH. In vivo evidence for a myogenic response in the fetal pulmonary circulation. Pediatr Res 45: 425-431, 1999[Abstract].

37.   Storme, L, Rairigh RL, Parker TA, Kinsella JP, and Abman SH. Acute pulmonary hypertension in utero impairs endothelium-dependent vasodilation. Pediatr Res 45: 575-581, 1999[Abstract].

38.   Sun, CW, Alonso-Galicia M, Taheri MR, Falck JR, Harder DR, and Roman RJ. NO-20-HETE interaction in the regulation of K+ channel activity and vascular tone in renal arterioles. Circ Res 83: 1069-1079, 1998[Abstract/Free Full Text].

39.   Tiktinsky, MH, and Morin FC. Increasing oxygen tension dilates fetal pulmonary circulation via endothelium-derived relaxing factor. Am J Physiol Heart Circ Physiol 265: H376-H380, 1993[Abstract/Free Full Text].

40.   Velvis, H, Moore P, and Heymann MA. Prostaglandin inhibition prevents the fall in pulmonary vascular resistance as the result of rhythmic distension of the lungs in fetal lambs. Pediatr Res 29: 538-542, 1991[Abstract].

41.   Villamor, E, Le Cras TD, Horan M, Halbower AC, Tuder RM, and Abman SH. Chronic hypertension impairs endothelial NO synthase in the ovine fetus. Am J Physiol Lung Cell Mol Physiol 272: L1013-L1020, 1997[Abstract/Free Full Text].

42.   Wesselman, JPM, Van Bevel E, Pfaffendorf M, and Spaan JAE Voltage-operated calcium channels are essential for the myogenic responsiveness of cannulated rat mesenteric small arteries. J Vasc Res 33: 32-41, 1996[ISI][Medline].

43.   Zenge, JP, Rairigh RL, Grover TR, Storme L, Parker TA, and Abman SH. NO and prostaglandin interactions during hemodynamic stress in the fetal ovine pulmonary circulation. Am J Physiol Lung Cell Mol Physiol 281: L1157-L1163, 2001[Abstract/Free Full Text].


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