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Am J Physiol Lung Cell Mol Physiol 291: L976-L982, 2006. First published June 30, 2006; doi:10.1152/ajplung.00512.2005
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Rho kinase activation maintains high pulmonary vascular resistance in the ovine fetal lung

Thomas A. Parker,1 Gates Roe,1 Theresa R. Grover,1 and Steven H. Abman2

Pediatric Heart Lung Center and Sections of 1Neonatology and 2Pulmonary Medicine, Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado

Submitted 5 December 2005 ; accepted in final form 19 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Mechanisms that maintain high pulmonary vascular resistance (PVR) in the fetal lung are poorly understood. Activation of the Rho kinase signal transduction pathway, which promotes actin-myosin interaction in vascular smooth muscle cells, is increased in the pulmonary circulation of adult animals with experimental pulmonary hypertension. However, the role of Rho kinase has not been studied in the fetal lung. We hypothesized that activation of Rho kinase contributes to elevated PVR in the fetus. To address this hypothesis, we studied the pulmonary hemodynamic effects of brief (10 min) intrapulmonary infusions of two specific Rho kinase inhibitors, Y-27632 (15–500 µg) and HA-1077 (500 µg), in chronically prepared late-gestation fetal lambs (n = 9). Y-27632 caused potent, dose-dependent pulmonary vasodilation, lowering PVR from 0.67 ± 0.18 to 0.16 ± 0.02 mmHg·ml–1·min–1 (P < 0.01) at the highest dose tested without lowering systemic arterial pressure. Despite brief infusions, Y-27632-induced pulmonary vasodilation was sustained for 50 min. HA-1077 caused a similar fall in PVR, from 0.39 ± 0.03 to 0.19 ± 0.03 (P < 0.05). To study nitric oxide (NO)-Rho kinase interactions in the fetal lung, we tested the effect of Rho kinase inhibition on pulmonary vasoconstriction caused by inhibition of endogenous NO production with nitro-L-arginine (L-NA; 15–30 mg), a selective NO synthase antagonist. L-NA increased PVR by 127 ± 73% above baseline under control conditions, but this vasoconstrictor response was completely prevented by treatment with Y-27632 (P < 0.05). We conclude that the Rho kinase signal transduction pathway maintains high PVR in the normal fetal lung and that activation of the Rho kinase pathway mediates pulmonary vasoconstriction after NO synthase inhibition. We speculate that Rho kinase plays an essential role in the normal fetal pulmonary circulation and that Rho kinase inhibitors may provide novel therapy for neonatal pulmonary hypertension.

Y-27632; fasudil; nitric oxide; persistent pulmonary hypertension of the newborn; newborn

IN THE FETAL LUNG, elevated pulmonary vascular resistance (PVR) causes right-to-left shunting of blood across the ductus arteriosus and limits pulmonary blood flow. Although increased oxygen tension, shear stress, and endothelium-dependent agonists such as ACh dilate the fetal pulmonary circulation, the vasodilator response is transient (2, 3, 5). Even with sustained exposure to many dilating stimuli, the "fetal phenotype" of high PVR and limited pulmonary blood flow is reestablished. At birth, PVR falls dramatically with the initiation of air breathing and exposure to increased oxygen. In contrast to the fetus, the fall in PVR at birth is sustained, establishing a "postnatal phenotype." Mechanisms that underlie high basal PVR in the fetal lung and differentiate it from the postnatal lung are poorly understood.

Recent advances in vascular biology have identified the small GTPase RhoA and its effector protein Rho kinase as key regulators of vascular tone (40). Activation of Rho kinase reduces expression of endothelial nitric oxide synthase (eNOS), the key enzyme responsible for nitric oxide (NO) generation, in vascular endothelial cells (7, 41). In vascular smooth muscle cells, Rho kinase phosphorylates and inactivates myosin light chain phosphatase (MLCP), thereby stabilizing myosin light chain and promoting sustained contraction (21, 22, 39). Rho kinase is activated in physiological states characterized by both acute and chronic vasoconstriction, including essential hypertension and myogenic reactivity (15, 24, 26, 30, 35, 36). Moreover, the capacity of Rho kinase inhibitors to reduce vasoconstriction in these settings underscores its role in the pathogenesis of systemic hypertension. New pharmacological inhibitors targeting the Rho kinase pathway have recently been developed to treat coronary and cerebral vasospasm, the latter resulting from subarachnoid hemorrhage (20, 24, 32).

Several recent studies suggest that Rho kinase activation may also play a central role in adult models of sustained pulmonary vasoconstriction, particularly in response to hypoxia (13, 28, 29). Hypoxia acutely increases Rho kinase activity in isolated pulmonary artery smooth muscle cells (44), and pharmacological inhibitors of Rho kinase reverse the pressor response to hypoxia in isolated intrapulmonary arteries and perfused lungs from rats (29, 31). In addition, long-term treatment with Rho kinase inhibitors reduces pulmonary artery pressures (PAP) and pulmonary vascular remodeling in monocrotaline- and hypoxia-induced models of more chronic pulmonary hypertension (1, 13).

Based on its importance in acute and sustained vasoconstriction in other vascular beds and the demonstration of its role in adult pulmonary hypertension, we hypothesized that Rho kinase activation contributes to the sustained elevation of PVR in the fetus. To address this hypothesis, we studied the pulmonary hemodynamic effects of two pharmacological inhibitors of Rho kinase, Y-27632 and fasudil (HA-1077), in chronically catheterized fetal lambs. We also studied the interaction between Rho kinase and NO in fetal lambs by testing the effects of Y-27632 on the pulmonary vasoconstrictor response evoked by nitro-L-arginine (L-NA), an NO synthase (NOS) inhibitor. We report that Rho kinase activation contributes to the sustained increase in PVR in the normal fetal lung and blocks the pulmonary vasoconstrictor response to NOS inhibition. These studies suggest a novel role for the Rho kinase signal transduction pathway in maintaining high PVR before birth.


   METHODS
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 METHODS
RESULTS
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Pregnant, mixed-breed (Columbia-Rambouillet) ewes were used in this study. All procedures and protocols were reviewed and approved by the Animal Care and Use Committee of the University of Colorado Health Sciences Center and followed the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996.

Fetal Surgical Preparation

Surgery was performed at 126 ± 2 days gestation (term=147 days) after ewes had fasted for 24 h. Animals were given intramuscular penicillin G (600,000 units) and gentamycin (80 mg) immediately before surgery. Ewes were sedated with intravenous ketamine (8 ml) and diazepam (2 ml) and intubated and ventilated with 1–2% isofluorane for the duration of the surgery. Under sterile conditions, a midline abdominal incision was made, and the uterus was externalized. A hysterotomy was made, and the left fetal forelimb was exposed. Polyvinyl catheters (20 gauge) were placed in the left axillary artery and vein and advanced in the ascending aorta and superior vena cava, respectively. A left thoracotomy and pericardial incision were made, and the heart and great vessels were exposed. Using a 16-gauge intravenous placement unit (Angiocath; Travenol, Deerfield, IL), a 22-gauge catheter was placed through purse string sutures in the left pulmonary artery (LPA) to allow for selective drug infusions. Using a 14-gauge intravenous placement unit (Angiocath; Travenol), 20-gauge catheters were placed in the main pulmonary artery (MPA) and left atrium. After gentle, blunt dissection of the bifurcation of MPA, a flow transducer was placed around the LPA to measure blood flow to the left lung. A catheter was placed in the amniotic cavity to serve as a pressure referent. The uterus was sutured, and a dose of ampicillin (500 mg) was given in the amniotic cavity. The catheters and flow transducer cable were externalized to a flank pouch on the ewe after the abdominal wall was closed. Postoperatively, ewes were allowed to eat and drink ad libitum and were generally standing within 1 h. All animals are treated with scheduled buprenorphine (0.6 mg) for 48 h postoperatively and then as indicated (based on veterinary assessment of pain). All catheters were gently flushed daily with 1–2 ml of heparinized normal (0.9%) saline to maintain catheter patency.

Drug Preparation

A solution of L-NA (30 mg/ml) was made immediately before each study by first dissolving the drug in 1 N HCl and then neutralizing the solution to pH = 7.4 with NaHCO3. Y-27632 (catalog no. 688000; Calbiochem Biochemicals, San Diego, CA) and fasudil (HA-1077; catalog no. H-2330; LC Laboratories) were dissolved in normal saline and infused in 1-ml aliquots.

General Study Design

Ewes were allowed to recover from surgery for a minimum of 48 h before the initiation of physiological studies. During physiological studies, pulmonary artery, aortic, and left atrium pressure were measured by connecting the externalized catheters to computer-driven pressure transducers (MP100A; Biopac Systems, Santa Barbara, CA). Pressure transducers were calibrated using a mercury column manometer before each study. Pressure measurements were referenced to simultaneously recorded amniotic pressure. The flow transducer was connected to an internally calibrated flowmeter (Transonics Systems, Ithaca, NY) to measure left pulmonary blood flow (QLPA). Before infusion of any drugs, a 20-min period of stable baseline hemodynamics was established during infusion of normal saline. Because normal saline was used as the vehicle for both Rho kinase inhibitors, hemodynamic responses were compared with baseline variables. Hemodynamic variables, which included pulmonary artery, aortic, and left atrial (LAP) pressures and QLPA, were measured continuously for the duration of each study protocol. Left lung PVR was calculated using the formula (PAP – LAP)/QLPA. Heart rate (HR) was determined from phasic pressure tracings. Arterial blood gas measurements included pH, PCO2, PO2 (ABL 500; Radiometer, Copenhagen, Denmark), and oxygen saturation and Hb (OSM3 Hemoximeter; Radiometer).

Protocol 1: the dose-response effects of intrapulmonary infusion of Y-27632 on baseline fetal pulmonary hemodynamics. The purpose of this protocol was to determine whether a selective inhibitor of Rho kinase, Y-27632, would cause pulmonary vasodilation. Y-27632 was infused directly in the LPA of chronically instrumented fetal lambs (n = 6), and the response to a range of doses (1.5, 5, 15, and 50 µg/min for 10 min) was tested. The dosing range of Y-27632 was based on in vitro data suggesting a similar dosing profile to ACh, a receptor-mediated endothelium-dependent vasodilator (9, 14). Four out of six animals received all four doses of Y-27632; two out of six received three doses (1 did not receive the 50-µg dose and another did not receive the 500-µg dose). Doses were tested in random order. Animals were allowed to recover for at least 2 h between infusions, and no more than two studies were performed on any animal on a given day.

Protocol 2: effects of intrapulmonary infusion of HA-1077 on baseline fetal pulmonary hemodynamics. The purpose of this protocol was to confirm the specificity of the pulmonary hemodynamic effects of selective Rho kinase inhibition noted in protocol 1 by using a second, pharmacologically distinct Rho kinase antagonist, HA-1077 (fasudil). Brief infusions of HA-1077 (500 µg) were administered in the LPA in a separate set of chronically instrumented fetal lambs (n = 3), as described under protocol 1. This dose of HA-1077 was considerably lower than the oral dose used in a previously published rodent study (1).

Protocol 3: effect of Rho kinase blockade on the pressor response to L-NA in the fetal pulmonary circulation. The purpose of this protocol was to study the interactions between NO and Rho kinase by determining whether pharmacological blockade of the Rho kinase pathway would attenuate the vasoconstrictor response to NOS inhibition caused by L-NA administration. After a period of stable baseline hemodynamics was established, Y-27632 (15 µg/min for 10 min) and L-NA (1.5–3 mg/min for 10 min) were sequentially infused in the LPA of fetal lambs (n = 5). The dose of L-NA was based on previous studies demonstrating a rapid increase in PVR and blockade of ACh vasodilation (4). The response was compared with individual infusions of both L-NA and Y-27632.

Statistical Analysis

Statistical analysis was performed using the GraphPad Prism version 3.00 for Windows, GraphPad Software (San Diego, CA). Hemodynamic variables over time were compared using repeated-measures ANOVA with Newman-Keul's post hoc testing. Differences between groups in protocol 3 were compared by two-way repeated-measures ANOVA with Bonferroni post hoc testing. Data are presented as means ± SE. Significance was set as P < 0.05.


   RESULTS
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Protocol 1: Dose-response Effects of Intrapulmonary Infusion of Y-27632 on Baseline Fetal Pulmonary Hemodynamics

Brief infusions of Y-27632 caused potent pulmonary vasodilation in the chronically prepared fetal lamb. The dose-related effects of Y-27632 on mean PAP (mPAP), QLPA, and PVR are shown in Fig. 1. Y-27632 increased QLPA at each dose tested, and the magnitude of increase was dose dependent, with the highest dose tested (500 µg) increasing QLPA ~3.5-fold (P < 0.05). Similarly, PVR fell in a dose-dependent manner, with the maximal response to the 500-µg dose. None of these doses lowered mean systemic pressure, PAP, or LAP or changed HR. Pre- and postinfusion aortic pressure and arterial blood gas values are shown in Table 1. Other than a modest reduction in PCO2 level after the 15- and 150-µg dose, blood gas variables were not changed during the infusions. When expressed as percent change from baseline, the 500-µg dose caused a greater fall in PVR than either the 15- or 50-µg doses (P < 0.05; Fig. 2).


Figure 1
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  		Fig. 1. Maximal pulmonary hemodynamic response (solid bars) compared with baseline (checkered bars) to range of doses of Y-27632. mPAP, mean pulmonary artery pressure; QLPA, left pulmonary artery blood flow; PVR, pulmonary vascular resistance. *P < 0.05 vs. baseline for each individual dose. Y-27632 did not change mPAP but increased flow and lowered PVR for each dose tested.

 

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  		Table 1. Aortic blood pressure and arterial blood gas tensions before and after LPA infusion of Y-27632

 

Figure 2
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  		Fig. 2. Dose-dependent fetal pulmonary vasodilator response, expressed as %change in baseline PVR, for Y-27632. *P < 0.05 vs. response to both 15- and 50-µg doses.

 
Despite the brief duration of drug infusion, Y-27632 caused a sustained pulmonary vasodilator response (Fig. 3). Although the Y-27632 infusion was completed in 10 min, the vasodilator response was sustained for at least 50 min (P < 0.05).


Figure 3
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  		Fig. 3. Pulmonary vasodilator response to the selective Rho kinase inhibitor Y-27632 (150 µg infused over 10 min; gray bar) is sustained for 50 min in late-gestation fetal lambs. Infusion increased QLPA and lowered PVR without changing mPAP. *P < 0.05 vs. baseline (time 0) values.

 
Protocol 2: Effects of Intrapulmonary Infusion of HA-1077 on Baseline Fetal Pulmonary Hemodynamics

As observed with Y-27632, HA-1077, an unrelated Rho kinase inhibitor, caused potent pulmonary vasodilation in fetal lambs (Fig. 4). HA-1077 caused a progressive twofold increase in QLPA that peaked 20 min after the start of the infusion (99 ± 5 vs. 207 ± 31 ml/min, P < 0.05). HA-1077 transiently lowered mPAP (42 ± 1 vs. 40 ± 1, P < 0.01), but systemic blood pressure was not changed. HA-1077 decreased PVR by 51 ± 11%, and the pulmonary vasodilator response was sustained for 30 min (P < 0.05). LAP and arterial blood gas tensions did not change in response to HA-1077.


Figure 4
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  		Fig. 4. Pulmonary hemodynamic response to selective Rho kinase inhibitor HA-1077 (fasudil; 50 µg/min for 10 min; gray bar) in late-gestation fetal lambs. Infusion caused a 2-fold increase in QLPA and lowered PVR by 51 ± 11%. mPAP also fell slightly. *P < 0.05 vs. baseline (t=0) values.

 
Protocol 3: Effect of Rho kinase Blockade on the Pressor Response to L-NA in the Fetal Pulmonary Circulation

Under control conditions, selective NOS inhibition with L-NA acutely reduced QLPA by 35 ± 12% and caused a sustained pressor response, increasing mPAP and aortic pressure above baseline for at least 60 min (P < 0.05). Pretreatment with Y-27632 caused a twofold increase in QLPA (77 ± 16 vs. 153 ± 24 ml/min, L-NA vs. L-NA + Y-27632, P < 0.05) at the start of the L-NA infusion (time 0), but PVR was not statistically different between groups. Pretreatment with Y-27632 completely blocked the fall in QLPA and the pressor response caused by L-NA. After pretreatment with Y-27632, QLPA rose during the L-NA infusion and remained higher than baseline through the 20-min time point. Figure 5 shows changes in PVR over time in response to L-NA, either alone or after pretreatment with Y-27632. PVR was higher at 10 min (P < 0.05) in the L-NA alone protocol compared with the Y-27632 + L-NA protocol. The response to Y-27632 without L-NA (protocol 1) is shown for comparison. L-NA had no effect on PVR after pretreatment with Y-27632, as shown by the comparison between the Y-27632 alone and Y-27632 + L-NA protocols (Table 2).


Figure 5
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  		Fig. 5. Pulmonary vascular response to infusion of nitro-L-arginine (L-NA; 1.5–3 mg/min for 10 min; dark gray bar) in fetal lambs before (L-NA alone) and after (L-NA + Y-27632) pretreatment with Rho kinase inhibitor Y-27632. In pretreatment protocol, Y-27632 (15 µg/min for 10 min) is given immediately before L-NA (light gray bar). Pretreatment with Y-27632 completely blocks the pulmonary vasoconstrictor response to L-NA. Response to Y-27632 alone is provided for comparison. *P < 0.05 between treatment protocols (L-NA alone vs. either L-NA + Y27632 or Y-27632 alone).

 

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  		Table 2. Hemodynamic response to L-NA (infusion) with and without pretreatment with Y-27632. * Indicates P < 0.05 vs. Baseline. {dagger} Indicates P < 0.05 vs. L-NA Alone

 
Given the higher baseline QLPA after pretreatment with Y-27632 (time 0), the response to L-NA was also analyzed by expressing PVR as the percent change from baseline. Figure 6 shows that L-NA increased PVR by 127 ± 73% in the control group but that pretreatment completely blocked this effect (P < 0.05).


Figure 6
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  		Fig. 6. Pulmonary vascular response to infusion of L-NA, expressed as %change from baseline PVR. Pretreatment with Y-27632 (L-NA + Y-27632) completely abolished the 127% rise in PVR induced by L-NA. *P < 0.05 vs. L-NA.

 

   DISCUSSION
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 METHODS
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We found that two distinct and selective pharmacological inhibitors of Rho kinase acutely lower resting pulmonary vascular tone in chronically prepared fetal lambs. These findings are the first to invoke the Rho kinase signal transduction pathway in the maintenance of physiologically elevated PVR in the fetus. In addition, we found that Rho kinase antagonism abolished the acute pulmonary vasoconstrictor response to a selective NOS inhibitor. These studies suggest that the persistent elevation of pulmonary vascular tone that complicates a number of neonatal lung diseases associated with endothelial dysfunction and loss of NO modulation may result from disinhibition of the Rho kinase signal transduction pathway.

Rho kinase is a serine-threonine kinase that is activated by RhoA after G protein-coupled receptor binding of a number of extracellular stimuli (16). Activated Rho kinase inactivates MLCP, either via direct phosphorylation of the MYPT1 subunit or indirectly via phosphorylation of the regulatory protein CPI-17 (17, 22, 23). Inactivation of MLCP increases calcium sensitivity of the actin-myosin contractile apparatus and promotes sustained vasoconstriction (21, 38, 39). Although Belik et al. (6) recently reported high specific activities of the regulatory and catalytic subunits of MLCP in the lungs of fetal rats, a potential role for Rho kinase activation in the regulation of pulmonary vascular tone in the fetus has not been previously studied. Prior investigation of pulmonary vascular tone in the fetal and transitional pulmonary circulation has primarily focused on the roles of individual vasoactive mediators. Those studies clearly demonstrate that endogenous NO modulates PVR in the fetal lung and that exogenous NO agonists cause marked fetal pulmonary vasodilation (3, 4). In addition to NO, several physiological stimuli and a number of other mediators, including endothelin, angiotensin, platelet-activating factor, and atrial natriuretic peptide, all have the capacity to regulate vascular tone in the fetal lung as well (18, 19, 25). However, the unique or common intracellular signaling mechanisms by which these many vasoactive agents effect changes in pulmonary vascular tone have been largely unexplored in the fetal lung. We undertook the current study because of the potential that the state of activation of Rho kinase might serve as a downstream effector of several important mediators and thereby coordinate the vasoactive response to multiple inputs. Our findings implicate Rho kinase as a central signal transduction pathway in the modulation of PVR in the normal fetal lung, potentially acting to coordinate the response to several individual mediators. Importantly, our study did not seek to determine which of the several potential upstream physiological stimuli or vasoactive mediators are responsible for Rho kinase activation, nor do our findings rule out the possibility that other signal transduction pathways, such as protein kinase C (PKC) or mitogen-activated protein kinase, also contribute to regulation of fetal and neonatal pulmonary vascular tone. These important questions will require further investigation.

Several previous studies in adult models have established a role for Rho kinase activation in experimental pulmonary hypertension. In chronically hypoxic adult rats, pharmacological inhibition of Rho kinase acutely lowers PAP and reverses pulmonary vasoconstriction induced by NOS inhibition (29). Rho kinase inhibitors reduce acute hypoxia-induced constriction of normal isolated rat pulmonary artery segments (31) and isolated mouse lungs (13). In addition, prolonged treatment with fasudil or Y-27632 reduces the severity of experimental pulmonary hypertension and pulmonary vascular remodeling in rats caused by exposure to monocrotaline or sustained hypoxia, respectively (1, 13). Together, these studies support the concept that activation of the Rho kinase signal transduction pathway modulates the vasoconstrictor response to pathological stimuli in the adult pulmonary circulation. Our study extends these findings to the normal fetal pulmonary circulation, which under physiological conditions is in a state of high resistance. The partial pressure of oxygen in the healthy fetus is 20–25 torr, a state of relative hypoxia comparison with the neonate and adult. This might result in physiological activation of Rho kinase in the normal fetal lung, increasing vascular tone directly by increasing calcium sensitization of vascular smooth muscle cells or indirectly from inhibition of endothelial NOS. Future studies are needed to address the role of relative hypoxia in the physiological activation of the Rho kinase cascade in the fetal lung.

In this study, we also found that treatment of fetal lambs with Y-27632 prevents the marked pulmonary vasoconstrictor response to an infusion of L-NA, which inhibits the production of NO. This finding may have important implications for our understanding of neonatal disease states characterized by pulmonary hypertension. Previous studies have demonstrated important reciprocal interactions between NO and Rho kinase. For example, Rho kinase activation in vascular endothelial cells suppresses endothelial NOS expression and NO production (7, 41). In addition, NO-dependent cGMP activation in vascular smooth muscle cells stimulates MLCP and antagonizes Rho kinase (9, 10, 33, 34). Release of NO modulates PVR in the fetus and is critical to the normal fall in PVR in response to birth-related stimuli (4, 12). Moreover, previous studies demonstrate reduced eNOS expression and NO production in the lamb model of persistent pulmonary hypertension of the newborn (PPHN; see Refs. 37 and 43). Our study suggests that Rho kinase activation contributes to the pressor response of the fetal lung to diminished NO production and raises the possibility that Rho kinase activation may underlie high PVR in PPHN. Based on the potential interactions between Rho kinase and NO at the level of both the endothelial and vascular smooth muscle cell (9, 33, 41), additional studies will be necessary to further delineate their complex interactions, which underlie our physiological observations.

The demonstration of basal Rho kinase activation in the normal fetal lung offers a potential explanation for the transient nature of the fetal pulmonary vasodilator response to several stimuli. For example, although both ACh and increased oxygen tension acutely increase pulmonary blood flow and lower PVR in the fetus, the vasodilator response is not sustained and PVR returns to high baseline levels, even when exposure to the vasodilator stimulus is continued (5). Mechanisms that underlie this vascular behavior and that differentiate the fetal pulmonary circulation from that of the normal newborn, in which the pulmonary vasodilation induced by birth-related stimuli is dramatic and sustained, are unknown. Based on our present study, we speculate that prenatal activation of Rho kinase limits the pulmonary vasodilator response to agents such as ACh and oxygen and that a postnatal reduction in Rho kinase activation may allow the more sustained fall in PVR that is necessary for a normal transition.

Because of potential concerns regarding selectivity and dosing, we used two pharmacological agents, Y-27632 and HA-1077, to inhibit Rho kinase. Studies indicate that Y-27632, a pyridine derivative, binds to and inhibits the Rho associated protein kinase p160ROCK with a nearly 200-fold greater affinity than other tested protein kinases, such as PKC or cAMP-dependent protein kinase (42). Similarly, the inhibitory affinity of HA-1077 (fasudil), a structurally distinct isoquinoline derivative, for Rho kinase is 30-fold that for PKC (11, 42). We believe that the demonstration of similar physiological findings with two separate inhibitors strengthens our findings. Although we cannot rule out the possibility that these agents had vasoactive effects on the placental, or even maternal, circulation, we saw no changes in fetal blood gas monitoring to indicate impaired placental function. In previous studies, treatment with Rho kinase inhibitors given for a variety of indications, including experimental pulmonary hypertension, has caused systemic hypotension. In our study, systemic blood pressure did not fall during treatment with either Y-27632 or HA-1077. Several differences in our study design compared with previous studies may explain the selective effect of these agents for the pulmonary circulation. First, we selectively infused these agents directly in the LPA to optimize delivery to the lung circulation. Second, we used much lower doses compared with previous studies. In the report by Nagaoka et al. (29), intravenous doses of 1, 3, and 10 mg/kg of Y-27632 were given. In contrast, we used a dosing range of ~5–15 µg/kg. Whether systemic or prolonged treatment would have adverse systemic hemodynamic effects would require further studies. Potential therapeutic application of these agents to neonates with pulmonary hypertension might require more localized delivery to the lung, either by inhalation or tracheal instillation (28).

We conclude that activation of the Rho kinase signal transduction pathway plays an important role in modulation of vascular tone in the fetal lung. In neonatal conditions associated with pulmonary hypertension, particularly in the setting of impaired NO signaling, targeting of the Rho kinase pathway may have therapeutic benefit.


   GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-04206 (to T. A. Parker).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. A. Parker, Dept. of Pediatrics, P.O. Box 6508, MS F441, Aurora, CO 80045 (e-mail: parker.thomas{at}tchden.org)

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.


   REFERENCES
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