Pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO) is enhanced in pulmonary arteries from rats exposed to chronic hypoxia (CH) compared with controls. Furthermore, in contrast to control arteries, relaxation to NO following CH is not reliant on a decrease in VSM intracellular free calcium ([Ca2+]i). We hypothesized that enhanced NO-dependent pulmonary vasodilation following CH is a function of VSM myofilament Ca2+ desensitization via inhibition of the RhoA/Rho kinase (ROK) pathway. To test this hypothesis, we compared the ability of the NO donor, spermine NONOate, to reverse VSM tone generated by UTP, the ROK agonist sphingosylphosphorylcholine, or the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate in Ca2+-permeabilized, endothelium-denuded pulmonary arteries (150- to 300-μm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca2+]i. We further examined effects of NO on levels of GTP-bound RhoA and ROK membrane translocation as indexes of enzyme activity in arteries from each group. We found that spermine NONOate reversed Y-27632-sensitive Ca2+ sensitization and inhibited both RhoA and ROK activity in vessels from CH rats but not control animals. In contrast, spermine NONOate was without effect on PKC-mediated vasoconstriction in either group. We conclude that CH mediates a shift in NO signaling to promote pulmonary VSM Ca2+ desensitization through inhibition of RhoA/ROK.
- nitric oxide
- pulmonary hypertension
- uridine triphosphate
- phorbol 12-myristate 13-acetate
- protein kinase C
the development of chronic hypoxia (CH)-induced pulmonary hypertension is the result of arterial vasoconstriction, vascular remodeling, and polycythemia. The severity of hypoxic pulmonary hypertension may be diminished by various adaptive mechanisms, including enhanced endothelium-derived nitric oxide (EDNO)-dependent vasodilation (12, 27, 29, 30, 42). Although augmented EDNO-reactivity following long-term hypoxia may be a function of elevated endothelial nitric oxide synthase expression as demonstrated by our laboratory (27, 28) and others (6, 21, 36, 42), an additional possibility is that chronic hypoxia increases pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO). Consistent with this possibility, recent studies from our laboratory demonstrate that CH augments pulmonary VSM sensitivity to the NO donor spermine NONOate in isolated, pressurized small pulmonary arteries (17). Furthermore, this enhanced NO-dependent VSM sensitivity following CH is associated with increased protein kinase G (PKG) expression and activity (16). NO-mediated stimulation of PKG elicits relaxation through several mechanisms that result from either a decrease in VSM intracellular free calcium ([Ca2+]i) or a decrease in the sensitivity of the contractile apparatus to Ca2+ (4). Interestingly, augmented NO-dependent vasodilation following CH appears to be independent of decreases in [Ca2+]i compared with vessels from control animals (17), suggesting a more prominent role for PKG-mediated Ca2+ desensitization following CH.
Phosphorylation of the 20-kDa regulatory myosin light chain is a key event in contraction of VSM and reflects the relative activities of the Ca2+/calmodulin-dependent myosin light chain kinase and the Ca2+-independent myosin light chain phosphatase (MLCP). MLCP activity is highly regulated to mediate changes in myofilament Ca2+ sensitization and thus VSM tone. Although several mechanisms have been implicated in mediating Ca2+ sensitization, including PKC-dependent inhibition of MLCP (5, 18, 25), it is well established that MLCP can also be inhibited via activation of the small G protein RhoA. RhoA activates Rho kinase (ROK), which can phosphorylate the regulatory subunit (MYPT1) of MLCP and inhibit its activity (9, 26), thus increasing Ca2+ sensitivity. PKG has been shown to interfere with ROK-induced inactivation of MLCP through direct phosphorylation of MLCP or indirectly by phosphorylation and inactivation of RhoA, thus desensitizing the contractile apparatus to Ca2+ (31, 33). Therefore, we hypothesized that enhanced NO-mediated pulmonary vasodilation following CH is a function of VSM Ca2+ desensitization through inhibition of the RhoA/ROK pathway. This hypothesis was tested in endothelium-denuded, Ca2+-permeabilized small pulmonary arteries from control and CH rats to directly assess potential inhibitory influences of NO on RhoA-, ROK-, and PKC-induced VSM Ca2+ sensitization. We further utilized Western blotting techniques to determine effects of NO on RhoA and ROK activity in intrapulmonary arteries from each group. Our findings suggest that CH shifts NO signaling in pulmonary VSM to mechanisms involving Ca2+ desensitization through inhibition of RhoA and ROK.
All protocols and surgical procedures employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Mexico School of Medicine (Albuquerque, NM).
Male Sprague-Dawley rats (200–250 g body wt, Harlan Industries) were divided into two groups for each experiment. Animals designated for exposure to CH were housed in a hypobaric chamber with barometric pressure maintained at ∼380 mmHg for 4 wk. The chamber was opened three times per week to provide animals with fresh food, water, and clean bedding. On the day of experimentation, rats were removed from the hypobaric chamber and immediately placed in a Plexiglas chamber continuously flushed with a 12% O2-88% N2 gas mixture to reproduce inspired Po2 (∼70 mmHg) within the hypobaric chamber. Age-matched control animals were housed at ambient barometric pressure (∼630 mmHg). All animals were maintained on a 12:12-h light-dark cycle.
Endothelial Disruption and Cannulation of Small Pulmonary Arteries for Dimensional Analysis
Rats were anesthetized with pentobarbital sodium (200 mg/kg ip), and the heart and lungs were exposed by midline thoracotomy. The left lung was removed and immediately placed in ice-cold physiological saline solution (PSS, pH 7.4) containing (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose. The lung was pinned out in iced PSS in a Silastic-coated dissection dish. A fourth-order intrapulmonary artery [150- to 300-μm inner diameter (ID)] of ∼1-mm length and without side branches was dissected free and transferred to a vessel chamber (Living Systems, CH-1) containing aerated PSS. The proximal end of the artery was cannulated with a tapered glass pipette, secured in place with a single strand of silk ligature, and gently flushed to remove any blood from the lumen. After cannulation of the proximal end of the artery, the vessel lumen was rubbed with a strand of moose mane to disrupt the endothelium. Next, the distal end of the vessel was cannulated, and the artery was stretched longitudinally to approximate its in situ length and then pressurized with a column to 12 or 35 mmHg, depending on the experimental protocol. These pressures are estimates of in vivo pressures in conscious rats from each group determined in a previous study from our laboratory (27). The vessel chamber was transferred to the stage of a Nikon Eclipse TS100 microscope, and the preparation was superfused with PSS equilibrated with normoxic gas (10% O2, 6% CO2, balance N2; pH = 7.40 ± 0.02, Po2 = 57 ± 1 mmHg, Pco2 = 31 ± 3 mmHg). A vessel chamber cover was positioned to permit this same gas mixture to flow over the top of the chamber bath. Po2, Pco2, and pH of the superfusate were monitored with a blood-gas analyzer (ABL-5, Radiometer). Bright-field images of vessels were obtained with an IonOptix CCD100M camera and dimensional analysis performed by IonOptix Sarclen software to measure ID. The effectiveness of endothelial disruption was verified by the lack of a vasodilatory response to acetylcholine (1 μM) in UTP-constricted vessels.
Measurement of VSM [Ca2+]i
Pressurized arteries were loaded abluminally with the cell-permeant, ratiometric, Ca2+-sensitive fluorescent indicator fura-2 AM (Molecular Probes). Immediately before loading, fura-2 AM (1 mM in anhydrous dimethyl sulfoxide) was mixed with 0.5 volumes of a 20% solution of pluronic acid in dimethyl sulfoxide, and this mixture diluted in PSS to yield a final concentration of 2 μM fura-2 AM and 0.05% pluronic acid. Arteries were incubated in this solution for 45 min at room temperature in the dark. The diluted fura-2 AM solution was equilibrated with the 10% O2 gas mixture during this loading period. Vessels were rinsed for 20 min with aerated PSS (37°C) following the loading period to wash out excess dye and to allow for hydrolysis of AM groups by intracellular esterases. Fura-loaded vessels were alternatively excited at 340 and 380 nm at a frequency of 10 Hz with an IonOptix Hyperswitch dual-excitation light source, and the respective 510-nm emissions were collected with a photomultiplier tube. Background-subtracted 340/380 emission ratios were calculated with IonOptix Ion Wizard software and recorded continuously throughout the experiment, with simultaneous measurement of ID from bright-field images as described above.
VSM [Ca2+]i was clamped in some experiments by permeabilizing endothelium-disrupted arteries to Ca2+ with the Ca2+ ionophore ionomycin (Sigma), similar to that previously described (19). After demonstration of effective endothelial disruption, vessels were loaded with fura-2 AM in Ca2+-free PSS containing 3 mM EGTA to deplete intracellular Ca2+. After this loading period, vessels were rinsed in Ca2+-free PSS and then permeabilized to Ca2+ by incubation with ionomycin (1 μM), which was present for the remainder of the experiment. Arteries were then equilibrated in PSS with a calculated free Ca2+ concentration of 100 nM [containing (in mM) 129.8 NaCl, 5.4 KCl, 1.6 MgSO4, 19 NaHCO3, 4.5 CaCl2, 5.5 glucose, and 7.1 EGTA] to approximate normal [Ca2+]i (19). This free Ca2+ concentration was calculated using the Kd of EGTA for Ca2+ of 43.7 nM and the Kd of EGTA for Mg2+ of 3.33 mM at 37°C and pH 7.4 (41).
Isolated Vessel Experiments
Because the endothelium is a source of vasoactive factors that may influence reactivity to NO, all experiments were conducted in endothelium-denuded arteries to directly examine mechanisms of NO-mediated VSM relaxation independent of endothelial influences.
Effects of CH on NO-dependent vasodilation and changes in VSM [Ca2+]i.
Effects of CH on pulmonary VSM sensitivity to NO were determined by assessing concentration response curves to the NO donor spermine NONOate (Cayman Chemicals) in UTP-constricted arteries from both control and CH rats. Furthermore, these experiments were performed in vessels pressurized to 12 or 35 mmHg to examine potential influences of altered transmural pressure on NO-mediated pulmonary vasoreactivity. After assessment of endothelial disruption and loading of VSM with fura-2 AM, a cumulative concentration-response relationship to the NO donor spermine NONOate (10−9–10−5 M) was assessed in UTP-constricted arteries. UTP was added at concentrations (2.5–5 μM) required to achieve a stable vasoconstrictor response of ∼30% of baseline ID.
Effects of soluble guanylyl cyclase and PKG inhibition on NO-mediated vasodilatory responses and changes in VSM [Ca2+]i.
To examine the contribution of the soluble guanylyl cyclase (sGC)/PKG signaling pathway to NO-mediated pulmonary vasodilation, we analyzed responses to spermine NONOate in arteries from control and CH rats in the presence of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, Sigma), the PKG inhibitor KT-5823 (Calbiochem), or vehicle (DMSO). Vessels from each group were prepared for experimentation as above. After fura-2 AM loading and 20 min of equilibration, vessels were superfused in the presence of ODQ (50 μM), KT-5823 (10 μM), or vehicle for 30 min before constriction with UTP. The UTP-induced constriction was allowed to stabilize before a cumulative concentration-response relationship to spermine NONOate was assessed as in previous protocols. We have recently shown that these concentrations of ODQ and KT-5823 inhibit vasodilatory responses to NO donors or to the cGMP analog 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP) in isolated saline-perfused lungs (15, 16).
Effects of CH on 8-para-chlorophenylthio-cGMP-dependent vasodilatory responses and changes in VSM [Ca2+]i.
To determine effects of CH on vasodilatory and VSM Ca2+ responses to the membrane-permeable, phosphodiesterase-5-resistant cGMP analog 8-para-chlorophenylthio (8-pCPT)-cGMP (3), endothelium-denuded small pulmonary arteries from control and CH rats were pressurized to 12 mmHg and equilibrated with 10% O2 as before. After fura-2 AM loading and equilibration, vessels from either group were preconstricted with UTP. UTP constriction was allowed to stabilize before a cumulative concentration-response relationship to 8-pCPT-cGMP (10−6–10−4 M) was assessed.
Effects of CH on NO-dependent vasodilation in UTP-, sphingosylphosphorylcholine-, or phorbol 12-myristate 13-acetate-constricted permeabilized arteries.
Potential inhibitory influences of NO on RhoA, ROK, and PKC-induced VSM Ca2+ sensitization were directly assessed in Ca2+-permeabilized arteries from each group. Because VSM [Ca2+]i is clamped by the extracellular Ca2+ concentration in permeabilized vessels, any vasodilatory effect of NO is a function of Ca2+ desensitization. After VSM cell loading with fura-2 AM and permeabilization with ionomycin, arteries were preconstricted (∼30% of baseline ID) with UTP (2.5–5 μM), the ROK activator sphingosylphosphorylcholine (SPC, 10–20 μM), or the PKC agonist phorbol 12-myristate 13-acetate (PMA, 10−10 M). The constriction was allowed to stabilize before a cumulative concentration-response relationship to spermine NONOate (10−9–10−5 M) was performed in these vessels.
Effect of ROK and PKC inhibition on vasoconstrictor responses to UTP, SPC, and PMA in permeabilized arteries.
The contribution of ROK and PKC to UTP-induced vasoconstriction was assessed in endothelium-denuded, permeabilized small pulmonary arteries from control and CH rats. After vessel permeabilization as described above, a concentration-response curve to UTP (10−6 −10−5 M) was generated in the presence of the ROK inhibitor Y-27632 (10 μM, Calbiochem), the PKC inhibitor GF-109203x (1 μM, Biomol), or vehicle (PSS). To verify the specificity of these inhibitors, separate sets of experiments were conducted to examine vasoconstrictor responses to the ROK agonist SPC (10−6–10−4 M) or the PKC agonist PMA (10−12–10−6 M) in the presence of Y-27632, GF-109203x, or vehicle.
Total RhoA and ROKα expression.
To examine potential effects of CH on pulmonary vascular levels of total RhoA and ROKα, intrapulmonary arteries from pentobarbital sodium-anesthetized (200 mg/kg ip) control and CH rats were dissected from accompanying airways and surrounding lung tissue and snap-frozen in liquid N2. Each sample was homogenized in 10 mM Tris·HCl homogenization buffer containing 255 mM sucrose, 2 mM EDTA, 12 μM leupeptin, 1 μM pepstatin A, 0.3 μM aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Samples were centrifuged at 10,000 g for 10 min at 4°C to remove insoluble debris. The supernatant was collected, and sample protein concentrations were determined by the Bradford method (Bio-Rad Protein Assay). Control experiments were conducted using different concentrations of protein to ensure linearity of the densitometry curve. A molecular weight standard (Bio-Rad) was added to each gel, and the optimal concentration of sample protein (15 μg/lane for RhoA and 25 μg/lane for ROKα) was separated by SDS-PAGE (15%/7.5% Tris·HCl gels, Bio-Rad) and transferred to polyvinylidene difluoride membranes. Blots were blocked for 1 h at room temperature with 5% nonfat milk and 0.05% Tween 20 (Bio-Rad) in Tris-buffered saline (TBS) containing 10 mM Tris·HCl and 50 mM NaCl (pH 7.5). Blots were then incubated overnight at 4°C with a mouse monoclonal antibody for RhoA (1:500, Cytoskeleton) or ROKα (1:1,000, BD Biosciences). For immunochemical labeling, all blots were incubated for 1 h at room temperature with goat anti-mouse IgG-horseradish peroxidase (HRP, 1:10,000; Bio-Rad). After chemiluminescence labeling (ECL, Amersham), RhoA (∼23 kDa) and ROKα (∼180 kDa) bands were detected by exposing the blots to chemiluminescence-sensitive film (Kodak). Subsequent to RhoA and ROKα detection, blots were washed in TBS and incubated 1 h with mouse monoclonal antibody for α-actin (1:1,000, Sigma). Immunochemical labeling of α-actin was achieved by incubating blots for 1 h at room temperature with goat anti-mouse IgG-HRP (1:10,000), and blots were then reexposed to chemiluminescence-sensitive film. Quantification of the bands was accomplished by densitometric analysis of scanned images (Sigma-Gel software, SPSS). Bands for RhoA and ROKα were normalized to those of α-actin.
RhoA activity was assessed in intrapulmonary arteries from each group of rats using a Rho activation assay kit (Cytoskeleton) that detects levels of GTP-bound RhoA. Briefly, intrapulmonary arteries were isolated and incubated in a cell culture incubator at 37°C for 30 min in the presence of UTP (10−6 M), UTP (10−6 M) + spermine NONOate (10−5 M), or vehicle (PSS). SPC has been shown to activate ROK independently of RhoA activation (39). To confirm this observation, a separate set of arteries from CH rats was incubated with SPC (10−6 M). The pooled arteries were homogenized as above, and activated RhoA was purified from vessel homogenates by incubation with Rhotekin-Rho binding domain (RBD) glutathione affinity beads (1 mg sample protein/50 μg Rhotekin-RBD beads). The RBD of Rhotekin specifically binds the GTP-bound form of RhoA. The sample and Rhotekin-RBD bead mixture was incubated for 1 h at 4°C and then pelleted by centrifugation at 3,500 g for 5 min. The supernatant was discarded, and the samples were washed with a solution containing 25 mM Tris (pH 7.5), 30 mM MgCl2, 40 mM NaCl, and 150 mM EDTA and repelleted. The pellet was resuspended in Laemmli buffer, and Western blots were performed as described above for RhoA.
Membrane translocation of ROKα.
To assess membrane translocation of ROKα as an index of ROK activity (39), pulmonary arteries from each group were isolated and homogenized as above. The collected supernatant was centrifuged at 100,000 g for 60 min at 4°C. The supernatant was collected as the cytosolic fraction, the microsomal pellet from the high-speed spin was resuspended in homogenization buffer, and sample protein concentrations were determined by the Bradford method (Bio-Rad Protein Assay). Proteins from both the cytosolic and microsomal fraction were separated by SDS-PAGE, and blots were performed as described above for ROKα. Data were expressed as the microsomal fraction divided by total ROKα expression (microsomal plus cytosolic fraction).
Calculations and Statistics
Vasoconstrictor responses were calculated as percentages of baseline ID. Vasodilatory responses were calculated as a percent reversal of UTP-, SPC-, or PMA-induced vasoconstriction. All data are expressed as means ± SE, and values of n refer to the number of animals in each group. A t-test or two-way ANOVA was used to make comparisons when appropriate. If differences were detected by ANOVA, individual groups were compared with the Student-Newman-Keuls test. A probability of P ≤ 0.05 was accepted as significant for all comparisons.
Effects of CH on NO-Dependent Vasodilatory Responses and Changes in VSM [Ca2+]i
Vasodilatory responses to spermine NONOate were augmented in endothelium-denuded, UTP-constricted vessels from CH rats compared with controls at a transmural pressure of 12 mmHg (Fig. 1A). Furthermore, this enhanced vasodilation in CH arteries was associated with a lesser fall in VSM [Ca2+]i (Fig. 1B), suggesting that CH promotes NO-dependent inhibition of myofilament Ca2+ sensitization in small pulmonary arteries. Vasodilatory and VSM [Ca2+]i responses to spermine NONOate were unaltered by elevated intraluminal pressure (35 mmHg) in vessels from either group of rats (Fig. 1, C and D, respectively). Because these preliminary experiments indicated that NO-dependent responses are independent of varying intraluminal pressure, all subsequent protocols were performed at a transmural pressure of 12 mmHg.
Effects of sGC and PKG Inhibition on NO-Mediated Vasodilatory Responses and Changes in VSM [Ca2+]i
The sGC inhibitor ODQ (50 μM) and the PKG inhibitor KT-5823 (10 μM) abolished vasodilatory responses to spermine NONOate (Fig. 2A) and associated decreases in VSM [Ca2+]i (Fig. 2B) in UTP-constricted vessels from control rats. Similar inhibitory effects of these compounds on NO-mediated vasodilation were observed in arteries from CH rats. However, we observed no significant differences in VSM [Ca2+]i between CH vessels treated with vehicle, ODQ, or KT-5823. These findings implicate a primary role for the cGMP-PKG signaling pathway in mediating NO-dependent vasodilation in this preparation.
Effects of CH on 8-pCPT-cGMP-Dependent Vasodilatory Responses and Changes in VSM [Ca2+]i
Similar to effects of CH on reactivity to spermine NONOate, vasodilatory responses to the membrane-permeable, phosphodiesterase-resistant cGMP analog 8-pCPT-cGMP were augmented in vessels from CH rats vs. controls (Fig. 3A). Furthermore, 8-pCPT-cGMP-induced decreases in [Ca2+]i were diminished following CH (Fig. 3B).
Effects of CH on NO-Dependent Vasodilation in UTP-, SPC-, or PMA-Constricted Permeabilized Arteries
To directly assess a role for Ca2+ desensitization in mediating augmented NO-mediated vasodilation following CH, we examined responses to spermine NONOate in UTP-constricted, Ca2+-permeabilized pulmonary arteries from each group of rats. Figure 4 depicts representative traces of 340/380 emission ratios and ID from a permeabilized control artery. Fura-2 ratios increased upon switching from superfusion with Ca2+-free PSS to PSS containing a calculated [Ca2+]i of 100 nM. Administration of UTP provided a stable vasoconstriction independent of a change in [Ca2+]i, demonstrating an effect of UTP to mediate VSM Ca2+ sensitization in this preparation. However, spermine NONOate was without effect on UTP-mediated Ca2+ sensitization in control vessels (Figs. 4 and 5A), suggesting that decreased VSM [Ca2+]i is the predominant mechanism of NO-mediated vasodilation in small pulmonary arteries from control rats. In contrast, UTP-constricted, permeabilized arteries from CH rats demonstrated concentration-dependent vasodilation to spermine NONOate (Fig. 5A). These findings are in agreement with results from Figs. 1–3 and demonstrate an effect of CH to promote NO-dependent VSM Ca2+ desensitization. CH similarly enhanced NO-mediated vasodilation in Ca2+-permeabilized arteries preconstricted with the ROK agonist SPC (Fig. 5B), suggesting CH enhances NO-mediated inhibition of ROK-induced Ca2+ sensitization. In contrast, spermine NONOate had no effect on PMA-constricted arteries in either group, indicating that PKC-induced Ca2+ sensitization is not inhibited by NO in this preparation (Fig. 5C).
Effects of ROK and PKC Inhibition on Vasoconstrictor Responses to UTP, SPC, and PMA in Permeabilized Arteries
To examine the relative contributions of ROK and PKC to UTP-dependent VSM Ca2+ sensitization, concentration-response relationships to UTP were assessed in arteries from each group pretreated with the ROK inhibitor Y-27632 (10 μM) or the PKC inhibitor GF-109203x (1 μM). Y-27632 abolished UTP-induced constriction in permeabilized vessels from both control and CH rats (Fig. 6A), suggesting UTP-induced myofilament Ca2+ sensitization is strictly a function of a Y-27632-sensitive pathway. Similar inhibitory effects of Y-27632 were observed for SPC-mediated Ca2+ sensitization in each group (Fig. 6B), although the inhibitor did not alter vasoconstrictor responsiveness to the PKC agonist PMA (Fig. 6C). In contrast, GF-109203x was without effect on UTP- or SPC-induced constriction in permeabilized arteries from either control or CH rats (Fig. 6, A and B) but largely attenuated vasoconstriction to PMA as expected (Fig. 6C). These data provide evidence for the pharmacological selectivity of Y-27632 and GF-109203x with respect to this preparation and further support a role for CH to augment NO-dependent VSM Ca2+ desensitization in UTP-constricted, Ca2+-permeabilized arteries via inhibition of ROK signaling. Interestingly, vasoconstrictor responses to both UTP and SPC, but not PMA, were augmented in vessels from CH animals compared with controls (Fig. 6, A and B), demonstrating an effect of CH to enhance ROK-dependent Ca2+ sensitization in small pulmonary arteries.
Effects of CH on RhoA Expression and Activity
Although hypoxic acclimation did not alter arterial RhoA expression (Fig. 7A), CH was associated with enhanced basal and UTP-stimulated RhoA activity (Fig. 7B). Consistent with inhibitory influences of NO on UTP-mediated Ca2+ sensitization in CH arteries (Fig. 5A), spermine NONOate abolished UTP-induced RhoA activation in arterial homogenates from CH rats, resulting in levels of activated RhoA below basal (Fig. 7B). Although RhoA activity also tended to be decreased by spermine NONOate in control arteries, this did not reach statistical significance. As predicted, the ROK agonist SPC was without effect on RhoA activity.
Effects of CH on ROK Expression and Membrane Translocation
Levels of ROKα were approximately twofold greater in homogenates of intrapulmonary arteries from CH rats compared with those of control animals (Fig. 8A). In contrast, no differences in membrane-bound/total ROKα expression were observed between groups under basal conditions (Fig. 8B). SPC significantly increased the ratio of membrane-bound/total ROKα in CH vessels, whereas no effect of SPC was indicated in arteries from control animals. In agreement with inhibitory influences of NO on SPC-mediated Ca2+ sensitization in CH arteries (Fig. 5B), spermine NONOate prevented SPC-induced membrane translocation of ROKα in vessels from CH rats but not controls (Fig. 8B).
The present study examined whether enhanced pulmonary VSM sensitivity to NO following hypoxic acclimation is a function of Ca2+ desensitization via inhibition of RhoA/ROK signaling. The major findings from this study are 1) NO-mediated vasodilation is cGMP and PKG dependent in isolated, pressurized, small pulmonary arteries; 2) CH impairs NO and cGMP-induced decreases in pulmonary VSM [Ca2+]i and mediates a shift in NO signaling to promote Ca2+ desensitization; 3) NO-mediated vasodilation is largely independent of VSM Ca2+ sensitization in UTP-constricted pulmonary arteries from control rats; 4) NO inhibits RhoA- and ROK-mediated pulmonary VSM Ca2+ sensitization following CH but is without effect on PKC-induced Ca2+ sensitization; and 5) CH-induced augmentation of RhoA- and ROK-induced pulmonary VSM Ca2+ sensitization is associated with elevated RhoA and ROK activity and increased ROK expression. These data suggest CH alters pulmonary VSM NO signaling to promote Ca2+ desensitization through inhibition of RhoA and ROK.
Earlier studies by Fouty et al. (8) suggest that the inhibitory influence of NO on pulmonary vascular tone in isolated perfused rat lungs is dependent on cGMP formation but independent of PKG. In contrast, our present findings indicate that both sGC and PKG contribute to NO-mediated vasodilation in isolated small pulmonary arteries from control and CH rats. These results are consistent with previous reports from our laboratory in isolated saline-perfused lungs from both control and CH rats (15, 16). The reason for the discrepancy between the current findings and those of Fouty et al. is not clear but may reflect differences in the delivery of PKG inhibitors to the VSM between the two preparations employed or differences in the responses measured (i.e., l-Nω-nitro-l-arginine-induced vasoconstriction in the former study vs. vasodilation to exogenous NO in the current).
Recently, our laboratory has shown that pulmonary arterial PKG expression and activity are increased following CH (16). Whereas enhanced pulmonary VSM reactivity to NO in CH arteries may result in part from greater expression and activity of sGC (22) or PKG-1 (16), it is evident from the current study that such increases in enzyme expression do not correlate with a greater fall in VSM [Ca2+]i. Rather, augmented VSM sensitivity to NO following hypoxic exposure is independent of changes in VSM [Ca2+]i. It is possible the impaired Ca2+ response to NO following CH results from increased degradation of cGMP by phosphodiesterase 5 (15). However, this possibility seems unlikely since similar responses were observed with the cGMP analog 8-pCPT-cGMP (Fig. 2), which is not degraded by phosphodiesterases (3). Together, these data suggest that CH impairs PKG-induced decreases in pulmonary VSM [Ca2+]i and mediates a compensatory increase in PKG-dependent Ca2+ desensitization.
Consistent with data from nonpermeabilized pulmonary arteries (Figs. 1 and 2), experiments in UTP-constricted, Ca2+-permeabilized arteries indicate that NO mediates dilation through a Ca2+ desensitization mechanism in vessels from CH rats but not controls (Fig. 5A). The finding that NO elicits vasodilation independently from Ca2+ desensitization in pulmonary arteries from control rats appears to be at odds with observations that both Y-27632 and the cGMP analog 8-Br-cGMP attenuate phenylephrine-induced contraction of endothelium-denuded rabbit main pulmonary artery rings (31). However, it is not clear whether such inhibitory effects of 8- and Y-27632 were independent of changes in VSM [Ca2+]i. The reasons for the apparent discrepancies between results from the present study and those of Sauzeau and colleagues (31) are not clear but may reflect differences between conduit and small pulmonary arteries or the vasoconstrictors employed.
Ca2+ sensitization can occur when MLCP activity is inhibited through the phosphorylation of either the regulatory subunit by ROK (2, 13, 34) or the catalytic subunit by CPI-17 (43). Cross talk between the two pathways has been implicated in several studies (5, 18, 25), suggesting that CPI-17 can be phosphorylated by ROK (20) and/or the RhoA effector protein kinase N (11). Because UTP can stimulate both PKC (14, 40) and the RhoA/ROK pathway in systemic arteries (31), we examined the possibility that NO-mediated Ca2+ desensitization in UTP-constricted pulmonary arteries is function of inhibition of PKC or RhoA/ROK activity. We observed that constrictions induced by UTP in Ca2+-permeabilized arteries were abolished by Y-27632, but not the PKC inhibitor GF-109203x, suggesting that UTP-mediated Ca2+ sensitization is strictly a function of a Y-27632-sensitive pathway. In agreement with these functional data, RhoA activity assays confirmed a role for NO to inhibit UTP-induced stimulation of RhoA in CH arteries but not controls. Collectively, these results support an effect of CH to induce NO-dependent VSM Ca2+ desensitization in UTP-constricted arteries via inhibition of RhoA.
Although Gopalakrishna et al. (10) have demonstrated that NO can inhibit PKC and phorbol ester binding, spermine NONOate was without effect on PKC-induced Ca2+ sensitization in this preparation (Fig. 5C). Considering evidence that PKC mediates VSM Ca2+ sensitization through CPI-17-dependent inhibition of the catalytic subunit of MLCP, PP1c (35), these findings do not support a role for PKG to directly stimulate MLCP activity in this preparation. Nevertheless, the possibility remains that PKG-dependent phosphorylation of MYPT1 is insufficient to overcome any inhibitory effect of PKC on MLCP activity mediated by CPI-17-dependent attenuation of PP1c.
Similar to responses in UTP-constricted vessels, CH enhanced vasodilation to spermine NONOate in SPC-constricted permeabilized arteries. These functional observations are in agreement with our present findings that NO prevented SPC-induced membrane translocation of ROK in arteries from CH rats. Whereas Y-27632 inhibited SPC-induced constriction in both CH and control arteries, SPC did not induce detectable ROK translocation in arteries from control animals. Although the reason for this lack of ROK translocation is uncertain, one possible explanation is that the elevated expression of ROK in CH arteries provides greater assay sensitivity to detect changes in membrane translocation. Alternatively, SPC could be activating a Y-27632-sensitive, ROK-independent pathway. Although it is uncertain whether PKG directly inhibits ROK or rather interferes with upstream tyrosine kinase signaling of SPC (24, 38), these data suggest that CH unmasks an effect of NO to inhibit ROK-induced Ca2+ sensitization through a RhoA-independent mechanism.
This CH-induced shift in NO/PKG-signaling to promote inhibition of RhoA/ROK-induced Ca2+ sensitization is a novel finding, and although the mechanism by which CH promotes this change in NO signaling remains uncertain, it appears that regulation of VSM tone in pulmonary arteries from CH rats may require less dependence on Ca2+ handling pathways, such as Ca2+ sequestration, influx, or efflux, and greater dependence on mechanisms that regulate Ca2+ sensitivity. Consistent with this possibility, we demonstrated that basal GTP-bound RhoA and total ROK expression were greater following CH. Furthermore, concentration-dependent vasoconstriction mediated by UTP and SPC, but not PMA, was augmented in Ca2+-permeabilized vessels from CH rats compared with controls, suggesting CH enhances RhoA/ROK-dependent Ca2+ sensitization but not PKC-mediated Ca2+ sensitization. These findings are in agreement with studies by Shimoda and colleagues (37), indicating that ET-1-induced increases in [Ca2+]i are reduced following CH, suggesting ET-1 contraction occurs by a mechanism largely independent of changes in [Ca2+]i. Several recent reports have additionally demonstrated the importance of RhoA/ROK-induced Ca2+ sensitization in mediating the sustained increase in pulmonary vasoreactivity (23) and vascular remodeling (7) associated with CH. In contrast to these results, a recent study by Sauzeau et al. (32) demonstrated decreased agonist-mediated contraction in conduit pulmonary arteries from CH rats vs. controls as a result of RhoA downregulation and abolishment of RhoA-mediated Ca2+ sensitization. These discrepancies in the literature may reflect segmental differences in RhoA-ROK signaling between conduit and small pulmonary arteries or differences in the duration of hypoxic exposure (15 days vs. 4 wk).
In conclusion, the present study demonstrates a role for CH to mediate a shift in NO-signaling to promote pulmonary VSM Ca2+ desensitization through inhibition of RhoA/ROK signaling. This switch in NO signaling toward Ca2+ desensitization pathways may have particular importance with respect to the development of hypoxic pulmonary hypertension, considering the emerging role of ROK in regulation of hypoxic vasoreactivity (7, 23), VSM migration, proliferation and apoptosis (1), and basal vascular tone in lungs isolated from CH rats (23). Challenges of future studies include identifying the mechanism by which CH enhances PKG-dependent inhibition of RhoA/ROK in pulmonary VSM, including 1) possible effects of CH to promote access of PKG to its molecular targets (e.g., RhoA/ROK) via regulation of macromolecular complexes, scaffolding proteins, or cytoskeletal elements; 2) altered expression of PKG-1 isoforms that preferentially inhibit RhoA/ROK-induced Ca2+ sensitization; or 3) possible redox influences of CH on regulation of RhoA or ROK activity.
This work was supported by a Scientist Development Grant from the American Heart Association (T. C. Resta), by National Institutes of Health Grants RR-16480 and HL-77876 (T. C. Resta) and HL-58124 and HL-63207 (B. R. Walker), and by a Parker B. Francis Fellowship in Pulmonary Research (T. C. Resta).
The authors thank Pam Allgood and Minerva Murphy for technical assistance.
Present address of N. L. Jernigan: University of Mississippi Medical Center, Dept. of Physiology and Biophysics, 2500 No. State St., Jackson, MS 39216-4505.
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- Copyright © 2004 the American Physiological Society