Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway

Jean-Marc Hyvelin,1 Clare O’Connor,2,3 and Paul McLoughlin1,3

1Department of Physiology, 2Department of Medicine and Therapeutics, Conway Institute of Biomolecular and Biomedical Research and the 3Dublin Molecular Medicine Centre, University College, Dublin 2, Ireland

Submitted 15 September 2003 ; accepted in final form 28 January 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
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Pulmonary arteries (PA) are resistant to the vasodilator effects of extracellular acidosis in systemic vessels; the mechanism underlying this difference between systemic and pulmonary circulations has not been elucidated. We hypothesized that RhoA/Rho-kinase-mediated Ca2+ sensitization pathway played a greater role in tension development in pulmonary than in systemic vascular smooth muscle and that this pathway was insensitive to acidosis. In arterial rings contracted with the {alpha}1-agonist phenylephrine (PE), the Rho-kinase inhibitor Y-27632 (≤3 µM) induced greater relaxation in precontracted PA rings than in aortic rings. In PA rings stimulated by PE, the activation of RhoA was greater than in aorta. Normocapnic acidosis (NA) induced a smaller relaxation in precontracted PA than in aorta. However, in the presence of nifedipine and thapsigargin, when PE-induced contraction was predominantly mediated by Rho-kinase, the relaxant effect of NA was reduced and similar in both vessel types. Furthermore, in the presence of Y-27632, NA induced a greater relaxation in both PA and aorta, which was similar in both vessels. Finally, in {alpha}-toxin-permeabilized smooth muscle, PE-induced contraction at constant Ca2+ activity was inhibited by Y-27632 and unaffected by acidosis. These results indicate that Ca2+ sensitization induced by the RhoA/Rho-kinase pathway played a greater role in agonist-induced vascular smooth muscle contraction in PA than in aorta and that tension mediated by this pathway was insensitive to acidosis. The predominant role of the RhoA/Rho-kinase pathway in the pulmonary vasculature may account for the resistance of this circulation to the vasodilator effect of acidosis observed in the systemic circulation.

vascular smooth muscle; acidosis; RhoA; calcium sensitization; Y-27632


NORMAL GAS EXCHANGE in the lung depends on the appropriate regulation of pulmonary blood flow to ensure matching of regional blood flow to ventilation to provide better gas exchanges. Hypoxic pulmonary vasoconstriction (HPV) is one of the main functional properties of the pulmonary circulation, which enables this matching. A second functional specialization seen in the pulmonary circulation is a resistance to the vasodilator effect of extracellular acidosis (5) in contrast to the potent vasodilator effect of this stimulus in the systemic circulation (1). Reduction in pH and elevation of PCO2 in pulmonary arterial (PA) blood is observed in acute and chronic lung diseases. This resistance to acidosis plays an important role in ventilation-perfusion matching in the lung in such diseases, since a vasodilator response to hypercapnic acidosis would antagonize hypoxic vasoconstriction in poorly ventilated regions of the lung, thus diverting blood to these regions and impairing normal gas exchange.

Sweeney et al. (40, 41) reported that in the PA, normocapnic acidosis (NA) leads to an endothelium-independent relaxation of isolated PA, which is less than that observed in the aorta. These results suggest that in PA smooth muscle, tension development is relatively resistant to reduction in extracellular pH compared with systemic vascular smooth muscle and that this resistance is an intrinsic property of pulmonary arterial smooth muscle cells (PASMC). However, the cellular mechanisms underlying this resistance remain unknown.

Smooth muscle contraction is primarily regulated by phosphorylation of the 20-kDa myosin light chain (MLC20) (20). MLC20 is specifically phosphorylated by Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), which, in turn is activated by the influx of Ca2+ across the plasmalemma or the release of Ca2+ from internal stores. In the systemic circulation, the mechanisms responsible for the vasodilator effect of acidosis have been extensively explored. Ca2+ influx (16, 21), Ca2+ release, and Ca2+ sequestration from internal stores (6, 28, 43), the activity of both the sarcolemmal and sarcoplasmic reticulum Ca2+-ATPase (SERCA) pump (14), and the activity of the Na+-Ca2+ exchanger have been shown to be sensitive to pH in a manner that favors a decrease in the intracellular Ca2+ concentration ([Ca2+]i). Because similar Ca2+ regulatory mechanisms are found in pulmonary vascular smooth muscle, it is likely that they are also sensitive to acidosis and, therefore, may not account for the resistance of pulmonary vascular smooth muscle to acidosis.

Recently, attention has been focused on regulation of force that is independent of changes in [Ca2+]i, so-called Ca2+ sensitization. The signaling pathways involved in Ca2+ sensitization converge on an increase in MLC20 phosphorylation, mediated via inhibition of myosin light chain phosphatase (MLCP) (37). Different mechanisms have been proposed to account for this inhibition. In intact smooth muscle, cell-permeable Rho-inactivating bacterial toxins have been shown to suppress receptor agonist-induced MLC20 phosphorylation and contraction (12, 29). Several RhoA effectors including Rho-kinase have been identified (45). It is now widely accepted that Rho-kinase mediates Rho-dependent MLCP inhibition through Rho-kinase-catalyzed phosphorylation of MLCP (2, 26, 44) and through the phosphorylation and consequently activation of a myosin phosphatase inhibitor protein, CPI-17 (23). On the basis of these observations, RhoA and its downstream effector’s Rho-kinase have emerged as major candidates in agonist-induced Ca2+ sensitization (38). However, there is evidence that the contribution of the RhoA/Rho-kinase pathway in agonist-induced tension development differs between vascular beds (24, 35).

Whereas acidosis has been shown to alter Ca2+ homeostasis, the effects of alteration of pH on the Ca2+ sensitization mechanism remain unknown. In our laboratory previous observations have shown that the relative resistance of PA to acidosis was unaffected by removal of extracellular calcium or by inhibition of the voltage-operated Ca2+ channels (Sweeney M and McLoughlin P, unpublished data). Moreover, Sweeney et al. (41) reported that the PGF2{alpha}-induced contraction that is largely mediated through sensitization of the contractile apparatus (17) was unaffected by acidosis. Together, these results suggest that the Ca2+ sensitization pathway could be insensitive to acidosis and that a greater contribution of the RhoA/Rho-kinase pathway in agonist-induced tension development in PA could account for this resistance.

The present work was thus designed to test the hypothesis that 1) in PA the RhoA/Rho kinase pathway plays a greater role in agonist-induced tension development than in the aorta; and 2) this pathway is insensitive to acidosis and accounts for the resistance of PA to the vasodilator effect of acidosis (Fig. 1).



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Fig. 1. Schematic diagram of the potential sites at which acidosis affects force production and its consequences in the pulmonary artery (PA) and aorta. Arrows at top: relative contribution of the Ca2+-dependent and Ca2+-independent mechanism involved in agonist-induced contraction in PA and aorta. Left: shown are the Ca2+-dependent signaling pathway of the contraction and its modulation by acidosis. Right: shown is the Rho-dependent Ca2+ sensitization pathway, which is believed to be acidosis insensitive and more important in agonist-induced force development in the pulmonary vessels than in systemic vessels. RhoA-GDP, GDP-bound RhoA, inactivated form; GTP-RhoA, GTP-bound RhoA, activated RhoA; MLCP, myosin light chain phosphatase; MLC20, 20-kDa myosin light chain; MLC20-P, phosphorylated 20-kDa myosin light chain; Ca-CaM-MLCK, myosin light chain kinase activated by the complex calcium-calmodulin; A, agonist; R, receptor.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparation. All protocols involving the use of animals were approved by the Institutional Review Panel at University College Dublin and comply with the Report of the American Veterinary Medical Association Panel on Euthanasia (2000). Adult male Sprague-Dawley rats weighing 300–350 g were stunned and then killed by cervical dislocation in compliance. The descending portions of the thoracic aorta and the extrapulmonary artery were collected in an ice-cold HEPES-buffered physiological saline solution (HEPES-PSS, in mM: 130 NaCl, 5.6 KCl, 2.4 CaCl2, 1 MgCl2, 10 HEPES, 11 glucose, pH 7.4 with NaOH), cleaned of adventitial and adherent connective tissues, and cut in 4- to 5-mm-length rings. We carefully removed the endothelium by gently rubbing the intimal surface with the tip of a small forceps.

Isometric contraction measurement. Isometric contraction was measured in rings from the PA and thoracic aortic rings as previously described (42). Briefly, two PA rings and two aortic rings, isolated from the same rat, were mounted between two stainless steel clips in vertical 5-ml organ baths. The baths were filled with PSS (in mM: 123 NaCl, 5.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 20 NaHCO3, 0.9 Na2HPO4, and 5.5 glucose), maintained at 37°C, and equilibrated with 5% CO2-95% air. The upper stainless steel clip was connected to a force transducer (model F30; Hugo-Sachs Electronik, March, Germany). Isometric tension was recorded by an analog-to-digital system (Biopac MP100 WS; Linton Instrumentation, Norfolk, UK) connected to a desktop computer.

The rings isolated from PA and aorta were initially placed under a mean resting tension of 1 and 2 g, respectively, left to equilibrate for 1 h, and washed at 20-min intervals. At the beginning of each experiment, a K+-rich (80 mM) solution obtained by substituting an equimolar of KCl for NaCl in the PSS was repeatedly applied to obtain at least two contractions similar in both amplitude and kinetics. A cumulative concentration-response curve to the {alpha}1-agonist phenylephrine (PE, 10–9–10–5 M) was then constructed to determine the concentration of agonist required to produce 70% of the maximal contraction (EC70). Successful removal of endothelium was confirmed by the inability of acetylcholine (1 µM) to induce relaxation in agonist-induced submaximal contraction. After this run-up procedure, the ring was entered into an experimental protocol. The effects of the pharmacological Rho-kinase inhibitors were studied on rings submaximally contracted with PE. Once a steady contraction was observed, one ring of PA and one ring of aorta were treated with these inhibitors added in a cumulative manner, whereas the second ring of each vessel received only the vehicle (PSS) and was used as a paired time control. The concentration of inhibitor was increased once the maximal relaxant effect of the preceding concentration had been recorded, i.e., after 5–8 min. We produced NA by switching to a modified PSS (NaHCO3 isosmotically replaced by NaCl) equilibrated with 5% CO2-95% air so that the PCO2 was maintained at control values while pH fell. Once a steady contraction was observed, the bath solution was switched to the test condition, i.e., NA, in the continuing presence of the same concentration of agonist. Twenty minutes later, the bath solution was returned to control conditions. The response of a preparation to the intervention in these experiments was quantified as the difference between the average tension during minutes 15–20 after the switch to experimental conditions and the average steady-state control tension during the last 5 min before the switch. We took samples of the bathing fluid throughout all the experiments for the analysis of PCO2 and pH by using an automated blood-gas analyzer (model 278, Ciba Corning). Mean values of pH and PCO2 were 7.38 ± 0.01 and 30.5 ± 0.6 Torr in control conditions and 7.12 ± 0.02 and 30.8 ± 0.8 Torr after the switch to NA conditions.

Cell isolation and Ca2+ measurement. Changes in [Ca2+]i were measured in freshly isolated cells; rings of PA and aorta were cut into small bundles and incubated for 10 min in free-Ca2+ HEPES-PSS. Pieces of tissue were first incubated in free-Ca2+ HEPES-PSS containing 1.6 mg/ml papain and 1.6 mg/ml dithiothreitol for 10–20 min and then incubated in a second enzymatic solution containing 1 mg/ml collagenase and 1 mg/ml soybean trypsin inhibitor for 10–15 min. After this sequence, the enzymatic solution was removed, and the arterial bundles were incubated again in a fresh enzyme-free solution and triturated with a fire-polished Pasteur pipette to release the cells. The cells were stored on glass coverslips at 4°C in PSS containing 1 mM CaCl2 and used on the same day.

To assess the dynamic change in [Ca2+]i of individual myocytes, we used the nonratiometric Ca2+-sensitive fluorophore fluo 3. The cells were loaded with fluo 3 by incubation in PSS containing 1 mM fluo 3-AM for 30 min at room temperature and then washed in PSS for 30 min. The coverslip with the attached cells was then mounted in a perfusion chamber and continuously superfused at room temperature. The recording system included a Nikon Eclipse fitted with epifluorescence system (Image Solution). A single cell per coverslip was tested through a window slightly larger than the cell. The studied cell was illuminated at 470 nm and counted at 510 nm by a photomultiplier (Hamamatsu). We monitored changes in Ca2+ by measuring the variation in fluorescence. For each cell the fluorescence signal was expressed as a percentage of the mean fluorescence recorded 30 s before stimulation. We applied the agonist to the recorded cell by quickly substituting the HEPES-PSS with a new solution containing the agonist at the desired concentration. In a control experiment we verified that no change in fluorescence signal was observed during substitution of HEPES-PSS.

Assessment of RhoA activation. Arterial rings, cleared of adventitial and intimal layers, were mounted in an organ bath. A cumulative concentration response curve to the {alpha}1-agonist PE (10–9–10–5 M) was then constructed to determine the EC70. The rings were washed several times to allow a return to the resting baseline and then submaximally contracted with PE (test ring) or not (control ring), snap-frozen (–70°C), and stored. For each experiment, two PA rings and two aortic rings from two different rats were pooled. Frozen tissue was homogenized with a Polytron in 300 µl of ice-cold homogenization buffer comprising (in mM, except where specified otherwise) 50 Tris·HCl (pH 7.2), 500 NaCl, 10 MgCl2, 1 ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 20 µg/ml each leupeptin and aprotinin, and 1 phenylmethylsulfonyl fluoride (PMSF). Homogenates were clarified by centrifugation at 14,000 g at 4°C for 10 min. Protein concentration was determined with a commercial protein assay kit (BCA Protein Assay, Pierce Biotechnology). A small amount of supernatant (10 µg of protein) was taken to determine the amount of total RhoA by Western blot analysis. Equal amounts of supernatant were used to determine the amount of GTP-bound RhoA. GTP-bound RhoA was measured using rhotekin Rho-binding domain. Briefly, equal amounts of supernatant (150 µg of proteins) were incubated with the recombinant protein rhotekin immobilized onto glutathione-agarose beads (Upstate) for 45 min at 4°C with gentle agitation. The beads were then washed twice with a washing buffer (in nM unless otherwise noted: 50 Tris·HCl, pH 7.2, 150 NaCl, 10 MgCl2, 1% Triton X-100, 1 EDTA, 20 µg/ml each leupeptin and aprotinin, and 1 PMSF). RhoA bound to beads and supernatant were mixed with 2x SDS buffer and boiled for 4 min. Each sample was then analyzed by SDS-15% polyacrylamide gels. Resolved proteins were transferred onto nitrocellulose. To reduce nonspecific binding, we blocked membranes overnight at 4°C in Tris-buffered saline (TBS)-Tween (20 mM Tris·HCl, pH 7.6, 130 mM NaCl, 0.1% Tween) containing 5% nonfat dry milk. After three 10-min washes in TBS-Tween, primary monoclonal antibody 26C4 (Santa Cruz Biotechnology) was used at 1:6,000 dilution for detection of RhoA. Horseradish peroxidase-conjugated secondary goat antibody anti-mouse IgG (Santa Cruz) was used at 1:2,000. Membranes were incubated for 1 h each with primary and secondary antibody diluted in TBS-Tween with 5% nonfat dry milk. Incubations were followed by three 10-min washes in TBS-Tween. The immunoreactive bands were detected by ECL (Amersham) and quantified by densitometric analysis (NIH Image 1.63, National Institutes of Health). The extent of RhoA activation (GTP-RhoA) was expressed as the ratio of the density of the GTP-RhoA band to that of the total RhoA in each sample.

Force determination and permeabilization. Rings isolated from PA and aorta were longitudinally opened. Small muscle strips (~500 µm wide and 5 mm long) were isolated from the media and attached with glue to the tips of two needles, one of which was connected to a force transducer (AE 801; SensoNor, Horten, Norway). Force was recorded on an analog-to-digital system (Biopac MP100 WS, Linton Instrumentation) connected to a desktop computer.

Strips were placed in a well on a Perspex plate filled with HEPES-PSS and stretched to a length that gave the greatest force development in K+-rich (80 mM) solution, ~1.3 resting length. The solution was rapidly changed by moving the preparation to the adjacent well. After equilibration in HEPES-PSS, the strips were contracted twice by immersion in K+-rich (80 mM) solution at 20–22°C. Then the strips were incubated in normal relaxing solution (87 mM KCl, 5 mM MgCl2, 5.2 mM Na2ATP, 10 mM EGTA, 20 mM TES, 10 mM phosphocreatine, and 5 IU/ml creatine phosphokinase), which was set to pH 7.10 with KOH. In all the solutions, NaN3 (1 mM), ryanodine (1 µM), and oligomycin (2 µg/ml) were added to empty the intracellular Ca2+ stores. Strips were permeabilized with 100–150 µM Staphylococcus aureus {alpha}-toxin in solution containing 1 µM free Ca2+ (solution pCa 6.0) for 20–35 min until force reached a plateau. The permeabilized strips were then washed several times with fresh relaxing solution. The efficiency of the permeabilization procedure was assessed by comparison of the amplitude of the K+-rich contraction in intact strips with the response to high free Ca2+ solution (pCa 4.4) recorded after permeabilization. Only permeabilized strips showing responses to pCa 4.4 similar to the K+-rich response were used. After relaxing in fresh relaxing solution containing 10 mM EGTA, the strips were submaximally constricted in a calcium buffer, pCa 6.4. Once a plateau was reached, PE (0.1 µM) plus guanosine triphosphate (10 µM) was added to the bathing solution to induce Ca2+ sensitization. The permeabilization and presence of ryanodine, 10 mM EGTA, and NaN3 ensured that the changes in force under these conditions were not due to a change in Ca2+. The maximal force used to normalize the contractile response was determined by exposure to pCa 4.4 solution.

The free ion concentrations of the physiological solutions were calculated with a computer program adapted from that by Fabiato (10) as previously described (19). In brief, we prepared activating solutions with different free Ca2+ concentration by adding specified amounts of CaCl2 to give a desired pCa. We kept the ionic strength constant to 200 mM by changing the amount of KCl. Solutions of different pH (pH 6.8) were made by a similar procedure. The apparent affinity constants, also obtained from this program, were corrected with respect to the pH and the room temperature. Experiments were done at room temperature (20–22°C).

Lung isolation and perfusion. An isolated ventilated perfused lung preparation was used to assess the effect of experimental interventions on total pulmonary vascular resistance, as previously described (8). In brief, rats were anesthetized (60 mg/kg pentobarbital sodium intraperitoneally) and mechanically ventilated (SAR-830P small-animal ventilator; CWE, Ardmore, PA) at a tidal volume of 1.8 ml and a frequency of 80 breaths/min as previously described (7). The animals were then anticoagulated (1,000 IU heparin intravenously) and killed by exsanguination. The thoracic contents were exposed through a midline sternotomy, and cannulas were inserted into the main PA and left atrium and tied in place. The thoracic contents were then removed en bloc and suspended in a chamber maintained at 37°C while ventilation continued with a warmed and humidified gas mixture of 5% CO2, 21% O2, balance N2. Airway pressure was continuously monitored, and a positive end-expiratory pressure of 2.0 cmH2O was maintained. The lungs were briefly hyperinflated to an airway pressure of 16 cmH20 every 5 min to prevent development of progressive atelectasis.

Lungs were perfused with PSS (in mM: 121 NaCl, 21 NaHCO3, 5.4 KCl, 4 MgSO4, 1 NaH2PO4, 1.8 CaCl2, and 5.6 glucose) with 4% Ficoll and containing 10 mg/l of indomethacin. Perfusion was maintained at a constant flow (0.04 ml·min–1·kg–1) so that changes in arterial perfusion pressure reflected changes in total pulmonary vascular resistance. Venous outflow was maintained constant at 2.0 mmHg to ensure zone 3 conditions at end of expiration. All measurements of arterial perfusion pressure were made at end of expiration. Arterial, venous, and airway pressure were continuously recorded with an analog-to-digital system (Biopac MP100 WS, Linton Instrumentation).

Chemicals. All salts and drugs were supplied by Sigma-Aldrich, with the exception of Y-27632, which was purchased from Tocris, and HA-1077, purchased from Calbiochem.

Data analysis. Responses are reported as means ± SE. For the isometric contraction experiments, n refers to the number of rats from which tissue was obtained. From each rat, both PA and aortic ring were used and compared. The relaxation induced by each increment in concentration of Rho-kinase inhibitors in PA and aorta was corrected for the small loss in tension recorded in the paired, time-control ring. For the intracellular Ca2+, n refers to the number of rats from which smooth muscle cells were isolated. From each rat, 10–20 cells isolated from the PA or the aorta were tested per experimental condition, and the mean of these records was taken as the value for that animal. For the Western blot experiments, two rats were used per experiment. For each pair of rats, the PA rings were pooled and compared with their matched aortic rings. In this case n refers to the number of experiments. Paired or unpaired t-tests were used as appropriate. For multiple comparisons of means across experimental groups, analysis of variance was carried out followed by Student-Newman-Keuls test for pairwise comparisons. Qualitative data (frequency of responding cells) were tested by mean of {chi}2. A value of P < 0.05 was accepted as statistically significant.


    RESULTS
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Effect of Rho-kinase inhibitor on K+- and agonist-induced contraction in arterial vessel. To assess the specificity of Y-27632, a potent Rho-kinase inhibitor (44), on agonist-induced myofilament calcium sensitization, we first investigated its effect on contraction induced by membrane depolarization. Rings from PA and aorta were contracted with high-K+ (80 mM) PSS. Y-27632 up to 3 µM did not affect KCl-induced contraction in both PA and aortic rings. At a concentration of 10 µM, Y-27632 decreased KCl-induced contraction by ~30% in both preparations (data not shown). Thus all subsequent experiments that examined the role of the RhoA/Rho-kinase pathway in agonist-induced contraction used a concentration of Y-27632 up to a maximum of 3 µM.

Subsequently, we investigated the contribution of the Rho-kinase pathway in agonist-induced tension development. Rings were submaximally contracted (70% of the maximal contraction) with the {alpha}1-adrenoceptor agonist PE. The calculated EC70 for PE was similar, 0.10 ± 0.03 µM (n = 8) and 0.11 ± 0.02 µM (n = 8, P = 0.94), respectively, in PA and aortic rings. In all vessels submaximally contracted with PE, Y-27632 (0.01–3 µM) strongly inhibited the force development in a dose-dependent fashion (Fig. 2, left). At each incremental concentration of Y-27632, the relaxation of PE-induced contraction was greater in PA than in aorta. In a subsequent series of experiments, we investigated the effect HA-1077, another Rho-kinase inhibitor (39). HA-1077 strongly inhibited PE-induced contraction in both PA and aortic rings, with a greater effect in PA rings (Fig. 2, right).



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Fig. 2. Effect of Rho-kinase inhibitors on agonist-induced submaximal contraction in isolated arterial rings. Effect of increasing concentration of Y-27632 (left) and HA-1077 (right) on phenylephrine (PE)-induced contraction in isolated PA ({bullet}) and aortic rings ({blacksquare}). Values are means ± SE calculated from 6 preparations. *Significantly different from corresponding values in PA rings (P < 0.05 ANOVA).

 
Effect of Y-27632 on agonist induced contraction in arterial vessel in the presence of nifedipine and thapsigargin. To minimize agonist-induced changes in cytosolic Ca2+, we incubated arterial rings in the presence of the voltage-gated Ca2+ channel inhibitor nifedipine (Nif, 10 µM) and the SERCA inhibitor thapsigargin (TSG, 2 µM), which depletes the intracellular Ca2+ store. Under these conditions, the resting tension was not changed; however, the amplitude of the submaximal contraction induced by PE (EC70) was decreased in both vessels, 72 ± 1% (P < 0.05) of the EC70 control (i.e., without Nif/TSG) and 58 ± 4% (P < 0.05) of the control response, in PA and aorta, respectively (Fig. 3, inset). In both vessels, the Nif/TSG-resistant component was also inhibited by the Rho-kinase inhibitor Y-27632 in a concentration-dependent manner (Fig. 3). As previously observed in control conditions, in the presence of Nif/TSG, Y-27632 up to 3 µM induced a greater relaxation of PE-induced contraction (Fig. 3) in PA than in aorta. These results indicate that, following inhibition of the main sources of intracellular Ca2+, RhoA/Rho kinase-dependent Ca2+ sensitization strongly contributed to PE-induced contraction in both PA and aorta and that inhibition of this pathway induces a greater relaxation in PA.



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Fig. 3. Effect of Y-27632 on the thapsigargin/nifedipine (TSG/Nif)-resistant component of PE-induced contraction in isolated arterial rings. Effect of increasing concentration of Y-27632 on PE-induced contraction in PA ({bullet}) and aortic ({blacksquare}) rings in the presence of TSG (2 µM) and Nif (10 µM). Reductions in tension are expressed as percentage of the submaximal contraction induced by PE (EC70) in the presence of TSG/Nif; values are means ± SE calculated from 6 preparations. Inset: amplitude of PE-induced submaximal contraction (EC70) in control (open bar) and in the presence of TSG/Nif (solid bar) in both PA and aorta; values are expressed as % of the PE-induced submaximal contraction in the control condition. *Significantly different from corresponding value in PA ring (P < 0.05 ANOVA); {dagger}significantly different from control values (P < 0.05, single-group t-test); {ddagger}significantly different from PA (P < 0.05, paired t-test).

 
Effect of Y-27632 on PE-induced Ca2+ rise in PA and aorta smooth muscle cells. To ensure that Y-27632 did not alter Ca2+ homeostasis, we investigated the effect of the Rho-kinase inhibitor on PE-induced Ca2+ rise in myocytes isolated from pulmonary artery (PASMC) and aorta (ASMC).

In PASMC (n = 4) and ASMC (n = 4), the basal fluorescence level was unaffected after exposure (10 min) to Y-27632 (10 µM). Table 1 summarizes the effect of Y-27632 on the PE-induced Ca2+ response in these arterial myocytes. In PASMC, a short (~30 s) application of PE (3 µM) induced cyclic variations (oscillations) of Ca2+ in 45% of tested cells, as previously described in response to other agonists (18), whereas in the other 55% the PE caused a transient increase in Ca2+ followed by a plateau phase, which remained above the baseline as long as the stimulation persisted (Fig. 4A). Pretreatment with Y-27632 (10 µM for 15 min) did not alter the basal Ca2+ activity before stimulation with PE (Table 1). Moreover, neither the percentages of cells that responded with Ca2+ oscillations nor those showing a transient peak followed by sustained plateau were modified. Furthermore, neither the amplitude of the first peak nor the amplitude of the plateau was altered in the presence of Y-27632 (Table 1). In the presence of Nif/TSG, PE (3 µM) failed to induce a rise in [Ca2+] (Fig. 4A). In ASMC, the PE-induced Ca2+ response was biphasic, i.e., a transient peak followed by a sustained phase; an oscillating cytosolic Ca2+ was not observed on these cells. Pretreatment with Y-27632 did not affect the PE-induced Ca2+ rise (Fig. 4B). These results indicate that the Rho-kinase inhibitor Y-27632 up to 10 µM altered agonist-induced contraction independently of any change in Ca2+ homeostasis.


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Table 1. Characteristics of the effects of Y-27632 on PE-induced Ca2+ rise in isolated arterial smooth muscle cells

 


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Fig. 4. Effect of Y-27632 and TSG/Nif on PE-induced cytosolic calcium concentration ([Ca2+]i) in pulmonary arterial (PASMC) and aortic isolated smooth muscle cells (ASMC). Short (30-s) stimulation with PE (3 µM) induced a biphasic [Ca2+]i rise (A, top traces) or [Ca2+]i oscillations (A, bottom) in PASMC. Only biphasic [Ca2+]i responses were observed in ASMC (B). Pretreatment with Y-27632 (10 µM) did not change the PE-induced [Ca2+]i rise, whereas in the presence of TSG (2 µM) and Nif (10 µM) (TSG/Nif) PE failed to induce a detectable [Ca2+]i rise.

 
Effect of PE on RhoA activation in PA and aorta. The previous results suggested a greater contribution of the RhoA/Rho-kinase in tension development in PA. Because Rho-kinase is a downstream effector of the small GTPase protein RhoA, we assessed the extent of activation of RhoA in control rings and rings stimulated with PE. In each sample, the activation of RhoA was expressed as the ratio of GTP-RhoA to the total amount of GTP-RhoA in the same sample. In unstimulated rings, little or no activated RhoA (GTP-RhoA) could be detected in either pulmonary or aortic samples (Fig. 5A). In PA and aortic rings submaximally contracted (70% of the maximum response) with PE (EC70, 0.09 ± 0.03 µM, n = 8 for the PA and 0.08 ± 0.01, n = 8 for the aorta), the activation of RhoA was significantly greater in PA compared with aorta (8.21 ± 1.46% of total RhoA in PA vs. 1.79 ± 0.53% of total RhoA in aorta, n = 4, P < 0.05; Fig. 5B). These results clearly show that in both PA and aorta the {alpha}1-adrenergic agonist PE induces the activation of RhoA. The greater level of activation of GTP-RhoA detected in PA suggests a greater activation of this small GTPase protein in this vessel compared with the aorta.



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Fig. 5. Western blot analysis of RhoA and GTP-RhoA in PA and aortic rings in control conditions and or stimulated with a submaximal concentration (EC70) of PE. Western blot of GTP-RhoA in PA and aorta in control conditions (A) and stimulated with PE (B). Quantitated results of normalized GTP-RhoA (right, A and B) are shown. Values are means ± SE of 3–4 determinations. HeLa 60, whole cell lysate of human epithelioid carcinoma cells used as positive control. *P < 0.05 (paired t-test).

 
We assessed the expression of RhoA in cryosections of endothelium-denuded pulmonary and aortic rings by immunofluorescence. Immunostaining was observed within the smooth muscle cells of the remaining media in both tissue preparations (data not shown).

Effect of NA on the Nif/TSG-resistant component of PE-induced contraction. Having observed that RhoA/Rho-kinase pathway plays a greater role in agonist-induced contraction in PA rings, we then investigated whether this pathway could account for the resistance of PA to the vasodilator effect of acidosis. Our hypothesis predicted that the RhoA/Rho kinase-dependent Ca2+ sensitization would be insensitive to acidosis.

Figure 6 shows reproduction of original experimental records of the response of PA and aortic rings to a switch toNA in control and in the presence of Nif/TSG. Table 2 summarizes the changes in steady-state tension development of pulmonary and aortic rings during NA. In control conditions, NA, in the continuing presence of PE (EC70), caused an initial transient relaxation in PA, which was followed by a partial recovery within 5 min. During steady state, the tension remained significantly lower than in the control condition. Return to normal pH led to a recovery of tension to a value similar to that in the initial control condition. In aortic preparations, the relaxation induced by NA was significantly greater than in PA.



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Fig. 6. Effect of normocapnic acidosis (NA) on PE-induced submaximal contraction (EC70) in arterial rings in control and in the presence of TSG/Nif. Typical recording of PE-induced tension development over time during NA in control conditions and in presence of TSG (2 µM) and Nif (10 µM) in a PA (A) and aortic ring (B).

 

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Table 2. Effect of normocapnic acidosis on steady-state tension development in pulmonary arterial and aortic rings in the absence and in the presence of thapsigargin/nifedipine

 
TSG and Nif were then added into the organ baths, and a similar protocol was repeated. As previously shown, the TSG/Nif-resistant component of PE-induced tension development largely reflects the sensitization of the contractile apparatus. Under these conditions, the switch to NA induced a relaxation of TSG/Nif-resistant component of PE-induced contraction in both PA and TA rings. However, whereas in control conditions NA induced a greater relaxation in aorta compared with PA, in the presence of TSG/Nif the relaxation induced by NA was less and similar in both vessels. These results suggest that the Ca2+ sensitization mechanisms responsible for the Nif/TSG-resistant component of the PE-induced contraction are less sensitive to NA than the control response involving both Ca2+-dependent and -independent pathways.

Effect of NA on PE-induced contraction in absence and presence of Y-27632. We then assessed the effect of Rho-kinase inhibition on NA-induced relaxation. If our hypothesis was correct, then following inhibition of the Rho-kinase-dependent Ca2+ sensitization, NA should have induced a greater relaxation in both PA and aorta, and the magnitude of the relaxation should be similar in both vessels.

Figure 7 shows reproductions of original experimental records of the response of PA and aortic rings to a switch to NA before and during exposure to Y-27632. Table 3 summarizes the changes in steady-state tension development of aortic rings during NA. In control conditions, NA induced a greater relaxation of the PE (EC70)-induced tension development in the aorta compared with PA. The rings were subsequently incubated with Y-27632 (3 µM). In the presence of Y-27632, the resting tension was not altered in either type of vessel; however, the PE (EC70)-induced contraction was decreased in both PA and aorta. Inhibition of the PE-induced submaximal contraction was greater in PA than in the aorta (–82 ± 4% of control response in PA vs. –50 ± 5 of control response in aorta, n = 6, P < 0.05). The switch to NA in the continuing presence of PE and Y-27632 relaxed both preparations to a similar extent (Table 3). These results indicate that following inhibition of RhoA/Rho kinase-dependent Ca2+ sensitization, both PA and aorta are more sensitive to the relaxant effect of NA than in control conditions. Furthermore, both vessel types relaxed to a similar extent, that is, the resistance of the PA to NA had been abolished.



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Fig. 7. Effect of NA on PE-induced submaximal contraction (EC70) in arterial rings in control and presence of Y-27632. Typical recording of PE-induced tension development over time during NA in control conditions and in the presence of Y-27632 (3 µM) in a PA (A) and aortic ring (B).

 

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Table 3. Effect of NA on steady-state tension development in PA and aortic rings in the absence and in the presence of the RhoA/Rho-kinase inhibitor Y-27632

 
Effect of altered pH on PE-induced Ca2+ sensitization in permeabilized smooth muscle. To ascertain that the contraction elicited by Ca2+ sensitization was insensitive to a reduction of intracellular pH, a condition that occurs during NA, we investigated the effect of lowering pH on the PE-induced Ca2+ sensitization in arterial muscle permeabilized with {alpha}-toxin. In such a preparation, contraction induced by increased cytosolic Ca2+ and Ca2+ sensitization of the contractile apparatus could be independently evoked.

We first investigated the effect of Y-27632 on PE-induced Ca2+ sensitization. In {alpha}-toxin-permeabilized PA strips, exposure to submaximal pCa (pCa 6.4) gave rise to a small contraction of 29 ± 4% (n = 3) of the maximal Ca2+-induced contraction (pCa 4.4). Addition of PE (1 µM) plus GTP (10 µM), at constant pCa of 6.4, increased force to 48 ± 4% (n = 3) of the maximal Ca2+-induced contraction. The increase in tension induced by PE was completely reversed by addition of Y-27632 (3 µM) to a value close to that of prior stimulation with the {alpha}1-adrenergic agonist (32 ± 7%, n = 3), indicating that the PE-induced Ca2+ sensitization was RhoA/Rho-kinase dependent, whereas the pCa 6.4-induced contraction was unaffected by Y-27632.

We then assessed the effect of altered pH on the contraction caused by Ca2+ sensitization. Permeabilized PA strips were exposed to pCa 6.4, and then PE (1 µM) plus GTP (10 µM) was added (Fig. 8A). Once steady-state contraction was achieved, the strip was exposed to a pCa 6.4 solution at pH 6.8 in the continuing presence of PE. The PE-induced force was not significantly altered by reduction of pH to 6.8 (41 ± 3% vs. 42 ± 4% at pH 6.8, n = 4). Similar results were obtained in {alpha}-toxin-permeabilized aortic strips (Fig. 8B).



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Fig. 8. Effect of altered pH on PE-induced tension development at constant Ca2+ level in {alpha}-toxin-permeabilized arterial strips. Typical recording of PE-induced Ca2+ sensitization in PA (A) and aortic strips (B). First we recorded a submaximal Ca2+-dependent contraction by immersing the strips in a physiological solution containing 0.4 µM Ca2+ free (pCa 6.4). Then we induced Ca2+ sensitization of the contractile apparatus by adding 0.1 µM PE in the presence of GTP (10 µM). Once steady state was reached, the strip was immersed in a solution of lower pH (pH 6.9) containing the same concentration of PE (0.1 µM). Values of pCa 6.4-induced contraction and PE-induced Ca2+ sensitization at normal and altered pH are shown in insets. Values are expressed as percentage of the maximal contraction induced by solution of pCa 4.4. Values are means ± SE of 4 animals.

 
Effect of NA on PE-induced increase in total pulmonary vascular resistance. Having observed that, in conduit PA vessels, the RhoA/Rho-kinase pathway played a predominant role in {alpha}-adrenergic-mediated vasoconstriction, we addressed the question of whether this pathway played a similar predominant role in the control of pulmonary vascular resistance. In isolated ventilated perfused lungs, PE (600 nM) induced a submaximal increase in the pulmonary artery perfusion pressure (PAP), indicating pulmonary vasoconstriction. The PAP was increased from 6.2 (± 0.4) mmHg to 9.7 (± 0.5) mmHg (n = 8, P < 0.05). Once steady state was achieved, adding Y-27632 (up to 3 µM) strongly inhibited the vasoconstriction development in a dose-dependent fashion (Fig. 9). The relaxation induced by 3 µM of Y-27632 was 90.0 ± 0.5% (n = 4), suggesting that Rho-kinase played a predominant role in the {alpha}-adrenergic receptor-mediated increase in total pulmonary vascular resistance.



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Fig. 9. Effect of Y-27632 on PE-induced increase in pulmonary vascular resistance. Effect of increasing concentration of Y-27632 on PE-induced vasoconstriction in isolated, ventilated, saline-perfused lungs. Values are means ± SE calculated from 4 preparations. Baseline indicates the perfusion pressure before the addition of PE (600 nM). PE indicates the mean pulmonary arterial pressure following the addition of PE (600 nM). *Significantly different from the PE-induced vasoconstriction before addition of Y-27632.

 
In a further series of experiments, we examined the effect of NA on this RhoA/Rho-kinase-mediated pulmonary vasoconstriction. At control pH (normal PSS), addition of PE (600 nM) to the perfusate increased the perfusion pressure by 3.5 (± 0.1) mmHg. Switching to NA did not significantly change the PE-induced increase in pulmonary perfusion pressure (3.4 ± 0.3 mmHg, n = 4, P > 0.05; Fig. 10), demonstrating that the small vessels controlling pulmonary vascular resistance were insensitive to the vasodilator effect of acidosis in a manner similar to that of the larger conduit vessels.



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Fig. 10. Effect of NA on PE-induced increase in pulmonary vascular resistance. Amplitude of PE-induced vasoconstriction in control condition (control), following switch to NA, and after return to control conditions. Values are means ± SE calculated from 4 preparations.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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One of the specific and important functional properties seen in the pulmonary circulation is resistance to the potent vasodilator effect of extracellular acidosis in contrast to the well-known profound relaxant effect of this stimulus in the systemic circulation. In the present study we have observed that the RhoA/Rho-kinase pathway may account for this resistance.

Studies into the role of the Rho-kinase pathway have been facilitated by the development of the potent inhibitor Y-27632, a highly selective inhibitor of the downstream effector of RhoA/Rho-associated kinase (44). In the present study, we investigated the effects of Y-27632 on KCl- and agonist-induced contraction. At concentrations up to 3 µM, Y-27632 was without effect on KCl-induced contraction, whereas at 10 µM we found that it significantly decreased KCl-induced contraction. Robertson et al. (33) have also reported that Y-27632 (10 µM) substantially decreased KCl-induced contraction in PA. Although this effect has been reported in other smooth muscle preparations (25, 44), its mechanism is unexplained. To exclude the possibility that Y-27632 was acting through this unexplained mechanism observed at high concentration (10 µM) during depolarization-induced contractions, in all subsequent experiments that examined the role of the RhoA/Rho-kinase pathway in agonist induced contraction, we used a concentration range of Y-27632 up to 3 µM. These concentrations are within the range in which Y-27632 acts selectively on Rho-kinase (22, 27, 33).

We found that Y-27632 (0.01–3.00 µM) did not affect the passive resting tension before stimulation in either PA or aorta, whereas it potently inhibited agonist-induced contraction, in agreement with previous studies (30, 33, 44). Both PA and aorta submaximally contracted with PE were relaxed by adding Y-27632 (Fig. 1), indicating that Rho-kinase activation is a major pathway in agonist-induced force development in agreement with previous observations made in such vessels (11, 27, 33, 39, 44). However, Y-27632 induced a greater relaxation in PA than in aortic rings, suggesting that the RhoA/Rho-kinase pathway plays a greater role in agonist-induced tension development in the PA than in the aorta. This markedly different contribution of the RhoA/Rho-kinase pathway to tension in PA was also observed after preincubation with Y-27632, as shown in Fig. 7. Furthermore, similar results were obtained using HA-1077, another Rho-kinase inhibitor (39). In smooth muscle preparations, Y-27632 causes relaxation by inhibiting RhoA/Rho-kinase-dependent sensitization of the contractile apparatus. However, in view of the effect on KCl-induced contraction, we undertook additional experiments to confirm that the relaxant activity of Y-27632 was independent of changes in cytosolic Ca2+. First, we examined the contractile response to PE in the presence of Nif, to block Ca2+ entry through voltage-operated Ca2+ channels, and TSG, to deplete sarcoplasmic stores. We confirmed, by fluorescence measurements, that these agents prevented changes in cytosolic Ca2+ in response to {alpha}1-agonist activation (Fig. 4). Under these conditions, tension development in response to PE was reduced in both PA and aorta, but the reduction was significantly less in the PA, in support of our hypothesis that sensitization played a greater role in the pulmonary vessel. Y-27632 caused profound relaxation in vessels, demonstrating that contraction under these conditions was predominantly mediated by the RhoA/Rho-kinase pathway. Furthermore, Y-27632 caused a greater relaxation of the pulmonary vessels, again indicating that the RhoA pathway is more important in the PA than in the aorta. Second, we examined the effect of Y-27632 on the basal and PE-induced increase in cytosolic Ca2+ and found that both were unchanged (Fig. 4). This indicates that at the concentration (up to 3 µM) used in our experiments, Y-27632 did not cause relaxation by reducing intracellular Ca2+.

To assess whether greater RhoA activity was present in PA rings following agonist stimulation, we measured the extent of activation of RhoA (38). We determined the amount of GTP-RhoA with a specific "pull-down assay" (32) using GST-rhotekin, which gives a direct measure of cellular RhoA activity and found that stimulation with PE increased the ratio of GTP-RhoA to total RhoA in both PA and aortic preparations. However, after submaximal stimulation of arterial rings with PE (EC70), the ratio of GTP-RhoA to total RhoA was six times higher in PA compared with aorta. Furthermore, if we expressed the GTP-RhoA/RhoA after stimulation (Fig. 5B) as a multiple of the mean value in control conditions, i.e., in the absence of stimulation (Fig. 5A), it is increased by 10-fold in PA and only by threefold in aorta; the latter value is similar to that previously observed in aortic vascular smooth muscle by Sakurada et al. (34). Together with the effects of Rho-kinase inhibition on tension development, these data indicate that, in PA, the RhoA/Rho-kinase-dependent Ca2+ sensitization contributed importantly to agonist-induced force development and that this pathway played a greater role in the PA than in the aorta.

We then investigated whether this difference in the excitation-contraction coupling mechanism in PA and aortic vessels might explain the resistance of PA to the vasodilator effect of acidosis. We found that NA caused relaxation of isolated PA that was significantly less than that seen in systemic vessels, a result that agrees with that previously reported by Sweeney et al. (40–42). To assess the potential contribution of the RhoA/Rho-kinase pathway to the resistance of PA to the vasodilator effect of extracellular acidosis, we investigated the effect of NA under conditions where the Rho-kinase-dependent Ca2+ sensitization was either dominant or minimized. In the presence of Nif and TSG, that is, in conditions where PE failed to induce any detectable rise in cytosolic Ca2+, extracellular acidosis induced a smaller relaxation than it did in the absence of these blockers, in both PA and aorta, and, furthermore, this relaxation was similar in both vessel types. In other words, the TSG/Nif-resistant component of the PE-induced contraction, which was RhoA/Rho-kinase dependent, was less sensitive to extracellular acidosis than was the contraction in control conditions when tension was also dependent on increased cytosolic Ca2+. Previously, Sweeney et al. (41) reported that the U-46619-induced contraction in PA, which is also independent of cytosolic Ca2+ increases (17, 22), was less sensitive to acidosis than the PE-induced contraction. Conversely, when RhoA-dependent tension development was inhibited by Y-27632 at a concentration that did not affect intracellular Ca2+ homeostasis, the {alpha}1-agonist-induced tension was strongly and similarly inhibited by extracellular acidosis in both PA and aorta. This demonstrates that tension development induced by Ca2+-dependent mechanisms was very sensitive to reduction in extracellular pH.

What then was the mechanism of the small relaxation observed in the presence of TSG/Nif following switch to NA, when Ca2+ sensitization was the dominant mechanism? It is important to note that the presence of TSG/Nif does not completely remove the requirement for intracellular Ca2+. That is, an increase in Ca2+ sensitivity by inhibition of the MLCP activity alone will not lead to contraction; there must also be some ongoing level of MLCK activity to lead to a net increase in the phosphorylation state of myosin (37). This suggests that in the presence of TSG/Nif, the basal cytosolic Ca2+ activity allowed the development of tension in response to activation of the Ca2+ sensitizing mechanisms. Acidosis has been reported to alter the activity of the Na+/Ca2+ exchanger as well as the functioning of the plasmalemma Ca2+-ATPase pump in a manner that favors Ca2+ clearance (4). Such an effect on Ca2+ clearance may occur in the presence of TSG/Nif and may account for the small relaxant effect of acidosis seen in the presence of these agents.

To evaluate more precisely the potential effect of acidosis on the RhoA/Rho kinase-dependent Ca2+ sensitization, we used permeabilized strips so that we could record PE-induced contraction at constant Ca2+ activity. In these permeabilized strips, in the presence of tension produced by a submaximal Ca2+-activating solution, PE caused a further increase in tension, showing that it leads to sensitization of the contractile apparatus in both vessel types. We confirmed that this further increase in tension was RhoA/Rho-kinase dependent by showing that addition of Y-27632 returned tension to the submaximal value observed before addition of PE, in agreement with previous reports (34, 36). The observation that NA failed to decrease PE-induced RhoA/Rho kinase-dependent contraction demonstrates that this pathway was insensitive to acidosis when intracellular Ca2+ activity was held constant.

Together, these results demonstrate that vascular smooth muscle contraction mediated by the RhoA/Rho-kinase-dependent Ca2+ sensitization pathway is not inhibited by acidosis. Furthermore, the predominant role of this pathway in mediating contraction in large PA accounts for the resistance of these vessels to the vasodilator effect of acidosis compared with large systemic arteries, in which the RhoA/Rho-kinase pathway plays a relatively minor role. We must then ask whether the behavior of these large vessels reflects similar properties in small pulmonary arterioles within the gas exchange regions that control vascular resistance in the intact lung. To address this issue we used the isolated, ventilated, saline-perfused lung preparation and demonstrated that the {alpha}1-agonist PE significantly increased pulmonary vascular resistance, as has been previously reported (9). The increase observed was ~60% of that observed in response to hypoxia in isolated, blood-perfused lungs from this strain of rat (Hyvelin JM and McLoughlin P, unpublished data). Y-27632 almost completely reversed this effect, showing that the RhoA/Rho-kinase pathway played a predominant role in {alpha}1-adrenergic-mediated control of total pulmonary vascular resistance. Furthermore, as predicted by our hypothesis, this increase in pulmonary vascular resistance was insensitive to NA.

These findings show that both large PA and small intrapulmonary vessels share this organ-specific response to acidosis. This observation is in good agreement with the previous demonstration that large extrapulmonary arteries constrict in response to hypoxia, a property that distinguishes the control of pulmonary vascular resistance from that of the systemic circulation (7). In contrast, large systemic arteries dilate in response to a similar hypoxic stimulus, a response that parallels the vasodilator effect of hypoxia on systemic resistance vessels (7). Moreover, it has previously been shown that isolated aortic preparations dilate in hypercapnia and acidosis, responses that parallel the vasodilator responses observed in isolated small systemic arteries and the reductions in resistance in isolated vascular beds and intact organisms (3, 8, 13, 15, 31, 40, 46).

The finding that the mechanism of PA resistance to the vasodilator effect of acidosis was a RhoA/Rho-kinase-dependent pathway suggests that this pathway is likely to be important in optimizing gas exchange in the diseased lung. To ensure ventilation-perfusion matching, blood flow must be diverted from poorly to well-ventilated regions; HPV acts to divert blood flow in this manner. However, such regions are also hypercapnic, and if the resultant acidosis caused significant vasodilatation and thus antagonized HPV, the beneficial action of hypoxic vasoconstriction would be lost. Because RhoA/Rho-kinase activation is essential for hypoxia-induced pulmonary arterial contraction (30, 33, 47), the resistance of this pathway to inhibition by acidosis is likely to be important for ventilation-perfusion matching in the diseased lung.

In conclusion, we report that, in the PA, sensitization of the contractile apparatus to Ca2+, mediated by the RhoA/Rho-kinase pathway, plays a greater role in agonist-induced tension development than in the aorta. Our data also demonstrate that this pathway is insensitive to the vasodilator effect of acidosis and thus its predominance in the PA may account for the resistance of this circulation to the vasodilator effect of acidosis compared with systemic arterial beds.


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 ABSTRACT
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This work was supported by the Health Research Board of Ireland.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. McLoughlin, Dept. of Physiology, Univ. College, Earlsfort Terr., Dublin 2, Ireland (E-mail:paul.mcloughlin{at}ucd.i.e.)

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.


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