Department of Internal Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany
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ABSTRACT |
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Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation, thus optimizing gas exchange. NADPH oxidase-related superoxide anion generation has been suggested as part of the signaling response to hypoxia. Because protein kinase (PK) C activation can occur during hypoxia and PKC activation is known to be critical for NADPH oxidase stimulation in different cell types, we probed the role of PKC in hypoxic vasoconstriction in intact rabbit lungs. Control vasoconstrictor responses were elicited by angiotensin II (ANG II) and the stable thromboxane analog U-46619. Portions of the experiments were performed while NO synthesis and prostanoid generation were blocked with NG-monomethyl-L-arginine and acetylsalicylic acid to avoid confounding effects due to interference with these vasoactive mediators. The PKC inhibitor H-7 (10-50 µM) caused dose-dependent inhibition of HPV, but this agent lacked specificity because ANG II- and U-46619-induced vasoconstrictions were correspondingly suppressed. In contrast, low concentrations of the specific PKC inhibitor bisindolylmaleimide I (BIM; 1-15 µM) strongly inhibited the hypoxic vasoconstriction without any interference with the responses to the pharmacological agents. Superimposable dose-inhibition curves were also obtained for BIM when lung NO synthesis and prostanoid generation were blocked throughout the experiments. Under either condition, BIM did not affect normoxic vascular tone. The PKC activator farnesylthiotriazole (FTT), ascertained to stimulate rabbit NADPH oxidase by provocation of alveolar macrophage superoxide anion generation in vitro, caused rapid-onset, transient pressor responses in normoxic lungs. After FTT, the hypoxic vasoconstrictor response was totally suppressed, in contrast to the largely maintained pressor responses to ANG II and U-46619. The lungs became refractory even to delayed hypoxic challenges after FTT application. In conclusion, these data support the concept that activation of PKC is involved in the transduction pathway forwarding pulmonary vasoconstriction in response to alveolar hypoxia.
hypoxia; isolated lung; nitric oxide; pulmonary hypertension; rabbit
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INTRODUCTION |
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HYPOXIC PULMONARY VASOCONSTRICTION (HPV) represents an essential physiological principle that matches lung perfusion to ventilation, thus optimizing pulmonary gas exchange (for a review, see Refs. 15, 28). Since the first description of this mechanism by von Euler and Liljestrand in 1946 (30), this field of research has attracted the attention of many scientists because of its broad clinical implication. Nevertheless, the O2 sensor mechanism(s) and the cell type(s) responsible for O2 sensing as well as for the transduction pathway(s) leading to contraction of the vascular smooth muscle cells are not yet fully clarified. It has been demonstrated that protein kinase (PK) C is activated in pulmonary arterial smooth muscle cells during hypoxia (5). PKC activation may thus participate in the response of lung cells to hypoxia. Moreover, we and others provided evidence that 1) changes in nitric oxide (NO) synthesis contribute to the regulation of HPV (1, 9) and 2) NAD(P)H oxidase is involved in hypoxia sensing (10, 25), with signaling events via superoxide anion generation and subsequent hydrogen peroxide formation (19, 23, 31). Recently, Marshall et al. (16) demonstrated that superoxide release by precapillary smooth muscle cells, the predominant site of HPV, is increased during hypoxia and that this superoxide is produced by NADPH oxidase. In stimulated leukocytes, PKC-dependent phosphorylation of the cytosolic p47-phox component, with subsequent translocation to the plasmalemmal cytochrome, is known to be a critical step in the activation of NADPH oxidase (3, 4, 11, 20, 26).
Against this background, the present study addressed the hypothesis that PKC may be involved in the hypoxic vasoconstrictor response in intact rabbit lungs. Both inhibitors and activators of PKC were employed, and the effects on HPV were compared with those on pharmacologically induced vasoconstriction [responses to angiotensin II (ANG II) and a stable thromboxane analog (U-46619)] to test for specificity. Moreover, portions of the study were performed while NO synthesis and prostanoid generation were blocked to exclude confounding effects on the generation of these agents that are also centrally enrolled in pulmonary vasoregulation. In essence, the data provide strong evidence of a role for PKC activation in hypoxic vasoconstriction in intact rabbit lungs.
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MATERIALS AND METHODS |
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Reagents. Bisindolylmaleimide I (BIM), farnesylthiotriazole (FTT) and NG-monomethyl-L-arginine (L-NMMA) were provided by Calbiochem (Bad Soden, Germany), and acetylsalicylic acid (ASA) was obtained from Bayer (Leverkusen, Germany). U-46619 was from Paesel+Lorei (Frankfurt am Main, Germany). H-7, phorbol 12-myristate 13-acetate (PMA), and [Asn1,Val5]ANG II acetate salt were purchased from Sigma (Deisenhofen, Germany). Hanks' balanced salt solution free of Ca2+ and Mg2+ was purchased from GIBCO (Paisley, UK), and sterile polypropylene tubes were from Falcon Becton Dickinson (Heidelberg, Germany). Dihydroethidine was obtained from Molecular Probes, and phoshate-buffered saline (PBS) with glucose, pyruvate, Ca2+, and Mg2+ was from BioWhittaker (Serva, Heidelberg, Germany). The perfusate was provided by Serag-Wiessner (Naila, Germany). All other biochemicals were purchased from Merck (Munich, Germany).
Lung isolation, perfusion, and ventilation. The model of isolated perfused rabbit lungs has been previously described (32). Briefly, pathogen-free rabbits of either sex (body weight 2.2-3.2 kg) were deeply anesthetized by a combined intravenous application of ketamine (30-50 mg/kg) and xylazine (6-10 mg/kg) and anticoagulated with heparin (1,000 U/kg body wt). The lungs were excised while being perfused with Krebs-Henseleit buffer through cannulas in the pulmonary artery and left atrium. The buffer contained 125.0 mM NaCl, 4.3 mM KCl, 1.1 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgCl2, and 275 mg glucose/100 ml; NaHCO3 was adjusted to result in a constant pH range of 7.37-7.40. After the lungs were rinsed with at least 1 liter of buffer fluid for the washout of blood, the perfusion circuit was closed for recirculation (total system volume 350 ml). Meanwhile, the flow was slowly increased from 20 to 150 ml/min, and left atrial pressure was set at 1.5-2.0 mmHg to ensure zone III conditions throughout the lung at end expiration. The alternate use of two separate perfusion circuits allowed repeated exchange of buffer fluid. In parallel with the onset of artificial perfusion, ventilation was changed from room air to a mixture of 5.3% CO2-21.0% O2-balance N2 (tidal volume, 30 ml; frequency, 30 strokes/min). A positive end-expiratory pressure of 1 cmH2O was chosen (zero referenced at the hilum). The isolated perfused lungs, freely suspended from a force transducer for continuous monitoring of organ weight, were placed in a temperature-equilibrated housing chamber. The whole system (perfusate reservoirs, tubing, and housing chamber) was heated to 38.5°C. Pressures in the pulmonary artery, left atrium, and trachea were measured by means of small-diameter tubing threaded into the perfusion catheters and trachea and connected to pressure transducers. Lungs included in the study were those that 1) had a homogeneous white appearance with no signs of hemostasis, edema, or atelectasis; 2) revealed constant mean pulmonary arterial pressure (PAP) and peak ventilation pressure in the normal range; and 3) were isogravimetric during the initial steady-state period of at least 20 min.
Hypoxic maneuvers and pharmacological challenges. The technique of successive hypoxic maneuvers in buffer-perfused rabbit lungs has been previously described (32). Briefly, a gas mixing chamber (KM 60-3/6MESO, Witt, Witten, Germany) was employed for step changes in the ventilator O2 content {21% vol/vol [alveolar PO2 (PAO2) ~ 160 mmHg; baseline condition] to 3% vol/vol (PAO2 ~ 23 mmHg; hypoxic condition)}. CO2 at 5.3% (vol/vol) was used throughout, and the percentage of N2 was balanced accordingly. During normoxic and hypoxic ventilation, the perfusate was almost totally equilibrated with PAO2. O2 tension of the pulmonary effluent was not affected by any of the drug interventions. Buffer returning from the perfusate reservoir to the lungs passed through a membrane oxygenator (M8Exp, Jostra, Hirrlingen, Germany). With this device, the partial pressure was set at ~40 mmHg for both CO2 and O2 in the postoxygenator buffer fluid entering the pulmonary artery. Sequential hypoxic maneuvers of 10-min duration, interrupted by 15-min periods of normoxia, were performed. The effects of various pharmacological agents on pressure responses provoked by alveolar hypoxia (3% O2) were determined within such a sequence of repetitive hypoxic maneuvers. Each agent was added to the buffer fluid 5 min before a hypoxic challenge, commencing the addition after the second hypoxic maneuver was accomplished. Cumulative dose-effect curves were established. For comparison, the influence of these agents on U-46619- and ANG II-elicited pressor responses was tested. In these experiments, a mode of repetitive bolus applications of the stable thromboxane analog or ANG II was employed [addition to the perfusate at 0.5 (U-46619) or 40 nM (ANG II) every 25 min] as previously described (9, 10). In each lung preparation, the response to the second vasoconstrictor provocation in a sequence of challenges was set at 100% (=reference response). The strength of the following vasoconstrictor responses was related to this reference response. Control experiments were performed with use of the vehicle only. Lung weight was continuously monitored, but the total weight gain was <3 g in all experiments.
Measurement of alveolar macrophage respiratory
burst. Freshly isolated rabbit lungs, suspended from a
tracheal cannula inserted under sterile conditions, were lavaged with
four 50-ml washes with endotoxin-free saline, with a total recovered
volume of 180-190 ml. The lavage fluid was centrifuged at 200 g for 10 min at room temperature, and
the cell pellet was washed once in Hanks' balanced salt solution
without Ca2+ and
Mg2+ in polypropylene tubes.
Measurement of the respiratory burst on a single-cell level was
performed by flow cytometric analysis as previously described (13, 24).
Briefly, alveolar macrophages were resuspended in phosphate-buffered
saline (PBS; with 1 mM Ca2+, 1 mM
Mg2+, 1 g/l of glucose, and 40 mg/l of sodium pyruvate) at a density of 2.5 × 105 cells/ml. The cells were
loaded with 0.28 µg/ml of dihydroethidine for 10 min at 37°C.
Stimulation of respiratory burst was performed by the addition of
106 M PMA in DMSO for 15 min at 37°C. The same amount of DMSO (1% vol/vol) was added to the
control samples. During oxidative burst, dihydroethidine is oxidized to
ethidium bromide, which can be measured by its red fluorescence at 630 nm with the 488-nm line of an argon-ion laser for excitation. Alveolar
macrophages were gated by their forward- and right-angle light-scatter
and green autofluorescence (530 nm) properties (FACStar Plus
cell sorter equipped with a 5-W argon-ion laser operating at 300 mW;
Becton Dickinson, Mountain View, CA). Ten thousand events of each
sample were acquired and analyzed for red fluorescence intensity. For measuring the influence of FTT, this agent was admixed to the buffer
fluid 5 min before stimulation with PMA.
Statistics. Data are means ± SE.
Analysis of variance for repeated measures was performed, with the
Student-Newman-Keuls post hoc test to detect statistical differences of
the drug effects on the strength of hypoxia- and pharmacologically
induced vasoconstrictions; significance was assumed when
P was 0.05.
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RESULTS |
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Under baseline conditions, mean PAP values were 6.5 ± 0.3 (SE) mmHg (n = 32 lungs). A 3% hypoxic challenge (PAO2 ~ 23 mmHg) consistently provoked a rapid increase in PAP, with pressure elevations of 2.4 ± 0.5 mmHg (n = 16 lungs). The vasoconstrictor responses to repetitive hypoxic maneuvers were well reproducible within the same lung (Fig. 1). The pressor responses were increased in experiments with prior blockage of the cyclooxygenase pathway and of NO synthesis due to the admixture of ASA (1 mM) and L-NMMA (400 µM) to the perfusion fluid throughout the experiments; the mean hypoxia-elicited pressure elevation was then 6.7 ± 2.8 mmHg (n = 8 lungs). Reproducibility of the hypoxic vasoconstrictions under these conditions of prostanoid and NO blockage was ensured (Fig. 2B). Similarly, repetitive bolus applications of ANG II and U-46619 provoked well-reproducible vasoconstrictor responses (Figs. 1 and 2).
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Admixture of the PKC inhibitor H-7 (10-50 µM) to the perfusate dose dependently suppressed HPV to 28.1 ± 9.3% of the reference response (Fig. 1). However, superimposable dose-inhibition curves were obtained for ANG II- and U-46619-induced vasoconstrictions. H-7 did not affect the normoxic vascular tone or the posthypoxic relaxation (return of PAP to baseline after cessation of the hypoxia challenge).
The PKC inhibitor BIM inhibited the hypoxia-induced vasoconstrictor response in a dose-dependent manner, to ~45% of the reference response at 15 µM (Fig. 2A). Virtually superimposable dose-effect curves were obtained in lungs in which prostanoid and NO syntheses were consistently blocked due to the presence of ASA and L-NMMA (Fig. 2B). In contrast, the ANG II- and U-46619-elicited pressor responses were not affected by BIM in this concentration range in either the absence or presence of ASA and L-NMMA. Inhibition of HPV by BIM was even more prominent with the use of higher concentrations of this agent (25 and 50 µM). However, the effects on HPV were then partially unspecific because in this higher dosage range, some inhibition of U-46619- and ANG II-induced vasoconstriction was also noted (data not shown). BIM did not alter the vascular tone under normoxic conditions, and it did not interfere with the posthypoxic vasorelaxation.
To probe the efficacy and to establish dose-effect curves of the PKC activator FTT in cells of rabbit lung origin, this agent was first admixed to alveolar macrophages for measurement of respiratory burst. Compared with the maximum response elicitable by phorbol ester stimulation, nearly identical efficacy was obtained for 100 µM FTT, with 50 µM FTT provoking an approximately half-maximal response (Fig. 3).
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When administered in perfused lungs, FTT provoked a rapid yet transient increase in baseline PAP, with pressure elevations commencing within 40-45 s after buffer admixture of this agent and pressure peaks at 2.1 ± 0.5 (10 µM FTT; n = 4 lungs) and 15.8 ± 2.8 mmHg (30 µM FTT; n = 4 lungs); no immediate vasoconstrictor response was observed in response to 1 µM FTT (Fig. 4). The FTT-elicited pressure elevations were not accompanied by any significant increase in lung weight. A preceding FTT administration did, however, interfere with the vasoconstrictor response to a subsequent hypoxia challenge. One micromolar FTT, not yet affecting baseline PAP, inhibited the subsequent HPV by ~20%, and ~80% and even total inhibition were observed after the administration of 10 and 30 µM FTT (Fig. 5). Posthypoxic relaxation was not affected in the presence of 1 and 10 µM FTT. As evident from the original record displayed in Fig. 4, total inhibition of the HPV response elicited by 30 µM FTT persisted for at least 30 min after buffer admixture of this agent. In contrast to HPV, the ANG II- and U-46619-provoked pressor responses were not inhibited by 1 µM FTT and only partially inhibited by the higher FTT doses (Fig. 5).
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DISCUSSION |
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In the present study, dose-dependent inhibition of HPV by H-7 was noted; however, this agent lacked specificity because ANG II- and U-46619-induced vasoconstrictions were correspondingly suppressed. In contrast, low concentrations of BIM strongly inhibited the hypoxic vasoconstriction without any interference with the responses to the pharmacological agents. This also held true with superimposable dose-inhibition curves when lung NO synthesis and prostanoid generation were blocked throughout the experiments. The PKC activator FTT elicited an immediate, transient pressor response in normoxic lungs. After FTT, the hypoxic vasoconstrictor response was totally suppressed, in contrast to the largely maintained pressor responses to ANG II and U-46619. Collectively, these data support the concept that an activation of PKC is involved in the transduction pathway in HPV.
H-7, one of the longest-known PKC inhibitors, was previously described to blunt both HPV and KCl-induced vasoconstriction in perfused rat lungs (22). In isolated rat pulmonary arteries, H-7 did not inhibit the early but did inhibit the late vasoconstrictor response to hypoxia (12). The present study confirmed the inhibitory effect of H-7 on HPV in intact rabbit lungs; however, no selectivity for this type of pressor response was noted because the ANG II- and U-46619-evoked PAP elevations were blocked with superimposable dose-inhibition curves. This lack of specificity of H-7 may be explained by its poor selectivity for PKC (21). Similarly to H-7, staurosporin was previously noted to block both hypoxic- and ANG II-elicited vasoconstrictor responses in intact lungs with virtually identical dose-inhibition curves (29).
In contrast, low concentrations of the highly specific PKC inhibitor BIM (27), which has been shown to block most of the PKC isoforms (7, 17, 27), markedly suppressed the hypoxic vasoconstriction but did not interfere at all with the pressor responses to both ANG II and U-46619. Moreover, superimposable dose-inhibition curves were obtained in lungs in which both NO synthesis and prostanoid generation were blocked throughout the experiments. These data strongly suggest that the molecular target of BIM is specifically involved in the signaling response to hypoxia and that this effect is not related to any impact of BIM on the generation of NO and on vasodilatory or vasoconstrictive prostanoids. Such control experiments are particularly important with respect to NO because this agent is continuously generated in intact lungs, and its synthesis immediately drops, preceding the PAP increase, at the onset of alveolar hypoxia (1, 9); inhibition of lung NO generation exaggerates HPV (1, 9).
Inhibition of PKC by BIM did not affect the baseline state of normoxic vasodilation. This was, however, lost within seconds after admixture of the PKC activator FTT to the perfusion fluid. In leukocytes, FTT promotes phosphorylation of the cytosolic p47-phox subunit of the NADPH oxidase complex, which is then translocated to the plasma membrane as part of the multicomponent oxidase complex (6, 8). The central role of p47-phox phosphorylation by PKC for NADPH oxidase activation and stimulation of superoxide generation is known for leukocytes (4, 11, 26), and corresponding evidence was forwarded for pulmonary arterial endothelial cells (33). In the present study, we first ascertained that FTT stimulates rabbit lung cell NADPH oxidase by incubation of alveolar macrophages in vitro and fluorescence-activated cell-sorter analysis of superoxide generation. With the use of the same concentration range in the intact lungs, dose-dependent pressor responses were provoked by FTT under conditions of normoxia. This is in line with previous observations (18, 22) on pulmonary arterial vasoconstriction in response to PKC activators such as phorbol ester and mezerein in perfused lungs and isolated pulmonary arterial segments. Taken together, these studies indicate that the stimulation of PKC in intact lungs provokes a vasoconstrictor response, the magnitude of which may even surpass the acute pressor response elicited by a step change from normoxia to hypoxia.
Most interestingly, lungs undergoing a preceding FTT challenge became refractory to subsequent HPV maneuvers. This was not only true for hypoxic challenges performed within 5 min after FTT application, under conditions of ongoing vasoconstrictor response to FTT itself, but also for delayed challenges performed 30 min post-FTT when PAP had fully returned to baseline. Moreover, this feature was again specific for the hypoxia-elicited pressor response because the ANG II- and U-46619-provoked vasoconstrictor events were only partially and significantly less affected compared with HPV. The finding of a refractory state is reminiscent of the well-known observation in leukocytes that a preceding PKC activation by phorbol ester is followed by a period of dramatically reduced responsiveness to subsequent challenges with PKC-dependent NADPH oxidase stimulation (2, 14).
The present findings suggest that PKC activation is involved in hypoxia sensing and/or signal transduction of HPV. Further investigations targeting putative hypoxia-sensing cells and the involved PKC isoforms may help to clarify the role of PKC in HPV. One pathway that should be included in such studies is a possible direct activation of the NADPH oxidase complex by PKC because this may contribute to the hypoxia-sensing mechanism (16, 31). However, techniques for direct monitoring of NADPH oxidase activity in the hypoxia-sensing cells in intact lungs are required for further elucidation of this issue.
In conclusion, to our knowledge, this is the first description of a PKC inhibitor that specifically inhibits hypoxic vasoconstriction without affecting pharmacologically induced pressor responses. Moreover, the PKC activator FTT was found to provoke vasoconstriction during normoxia, and in post-FTT lungs, the pressor response to hypoxia was again specifically inhibited. These data are strongly suggestive of a role for PKC in the signaling cascade forwarding the pulmonary vasoconstrictor response to acute alveolar hypoxia.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. L. Snipes (Giessen, Germany) for linguistic editing of the manuscript and K. Quanz for excellent technical assistance.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft SFB 547, Project B7.
A portion of the thesis of R. Voswinckel was incorporated into this report.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: F. Grimminger, Dept. of Internal Medicine, Justus-Liebig-Univ. Giessen, Klinikstrasse 36, 35392 Giessen, Germany.
Received 22 June 1998; accepted in final form 14 October 1998.
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