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Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
Submitted 8 October 2004 ; accepted in final form 19 January 2005
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ABSTRACT |
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-subunits and Kv1, Kv2, and Kv3 
-subunits were expressed in rat dPAs. Exposure to chronic hypoxia decreased mRNA and protein levels of Kv1.1, Kv1.5, Kv1.6, Kv2.1, and Kv4.3 -subunits in dPAs but did not alter gene or protein expression of these channels in aorta. Furthermore, chronic hypoxia did not alter the mRNA levels of -subunits in dPAs. These results suggest that diminished transcription of Kv -subunits may reduce the number of functional Kv channels in dPAs during prolonged hypoxia, causing the decreased Kv current previously observed in PASMCs and leading to pulmonary artery vasoconstriction. voltage-gate K+
In pulmonary arterial smooth muscle cells (PASMCs), the activity of voltage-gated K+ (Kv) channels plays an important role in regulating resting membrane potential (2, 29, 37), which, in turn, modulates the intracellular calcium concentration ([Ca2+]i) because of Ca2+ influx through sarcolemmal voltage-dependent Ca2+ channels. Intracellular Ca2+ levels play a key role in regulating smooth muscle function, and, in PASMCs, a rise in [Ca2+]i is required for both the hypoxic pulmonary vasoconstriction (7, 14, 23, 24) and smooth muscle growth and proliferation (12, 32) characteristic of hypoxic pulmonary hypertension.
Depolarization (31) and reduced Kv channel activity (28, 30) observed in PASMCs following exposure to chronic hypoxia suggest alterations in K+ channel regulation or expression. Because these effects are observed for several hours after return to normoxic conditions and most adaptations to chronic hypoxia are accomplished by changes in gene expression, it is possible that the observed decrease in Kv channel activity is mediated by transcriptional regulation. Few studies have sought to determine the effect of prolonged hypoxia on K+ channel expression. In PASMCs from normoxic animals cultured under hypoxic conditions for 72 h, mRNAs encoding Kv1.1, Kv1.2, Kv1.5, Kv2.1, Kv4.3, and Kv9.3 channel subunits decreased (9, 19, 33), suggesting that hypoxia could repress K+ channel expression. However, the effect of short-term in vitro exposure to hypoxia in cultured cells may not accurately reflect the effects of chronic hypoxia on K+ channel expression in the intact animal, where the duration of exposure and level of hypoxia or changes in pulmonary arterial pressure and circulating factors may alter the response. In intact animals, the protein expression of Kv2.1 and Kv1.5 was shown to decrease in pulmonary arteries isolated from rats exposed to 2–3 wk of hypoxia (16, 20). The decrease in Kv1.5 protein levels was found to be associated with a reduction in mRNA expression; however, the effect on Kv2.1 gene expression was not examined. Although several other Kv channel subtypes are expressed in distal pulmonary arteries (dPAs) (2, 6, 33), a detailed analysis of Kv channel gene expression in intact dPAs from chronically hypoxic rats has been not performed. Thus, in this study, we determined whether exposure of rats to 10% O2 for 3 wk altered dPA expression of regulatory -subunits (Kv1–3) and several pore-forming -subunits of Kv channel families known to exist in vascular smooth muscle, including Shaker (Kv1.1–1.6), Shab (Kv2.1), Shaw (Kv3.1), and Shal (Kv4.3) as well as the -subunit that assembles with Shab family members to form functional heteromeric channels (Kv9.3). To determine whether the effects of chronic hypoxia on Kv channel expression were specific for pulmonary arteries, results obtained in pulmonary arteries were compared with those obtained in aorta.
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METHODS |
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RT-PCR. Total RNA was prepared from deendothelialized fourth-generation or smaller dPA (200–600 µm outer diameter) and aorta (tissue from 3 rats was pooled per sample) by TRIzol (GIBCO BRL) extraction. Isolated total RNA was dissolved in diethyl pyrocarbonate water and stored at –80°C until use. Reverse transcription was performed using the First-Strand cDNA Synthesis Kit (Pharmacia Biotech, Austin, TX) as previously described (34). Three micrograms of the total RNA was reverse transcribed using random hexamers [pd(N)6 primer]. The reaction mixture was incubated for 1 h at 37°C and heated at 90°C for 5 min to inactivate the reverse transcriptase.
PCR was performed using a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA), as previously described (34). Three microliters of the first-strand cDNA reaction mixture was used in a 50-µl PCR reaction consisting of 200 µM each primer, 10 mM Tris·HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 200 µM each dNTP, and 1 unit of Taq DNA polymerase. The sense and antisense PCR oligonucleotide primers (Table 1) chosen to amplify the cDNA were specifically designed from coding regions of various Kv channels as described previously (9, 19, 33).
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-actin was used as a control. The optical density (OD) values in the channel subunit signals, measured by a Kodak ID Image Analysis System, were normalized to the OD values in the -actin signals; the ratios are expressed as arbitrary units for quantitative comparison.
Protein isolation and measurement by Western blotting.
Deendothelialized dPA and aorta were washed with PBS and homogenized using an Ultra-Turrax T-25 at 20,000 rpm in 0.5 ml of HEPES buffer containing 10 mM HEPES, 1 mM DTT, and 1 tablet of protease inhibitor (Complete tablets; Boehringer Mannheim Biochemicals, Indianapolis, IN). Protein concentrations were determined by BCA protein assay, using BSA as a standard. For each sample, 20 µg of total protein were separated by SDS-PAGE on 10% gels. Proteins were then transferred to a Hybond-C extra nitrocellulose membrane (Bio-Rad, Hercules, CA), and the efficiency of the transfer was verified by Ponceau S staining. Membranes were blocked with 5% nonfat dry milk and probed with the affinity-purified polyclonal antibodies specific for various Kv channels (Alomone Laboratories, Jerusalem, Israel and Upstate Biotechnology, Charlottesville, VA), followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG for 1 h. Enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ) was used for detection of the bound antibody. To determine the total amount of protein in each lane, membranes were also probed with, and all protein concentrations normalized to, -actin (Sigma, St. Louis, MO).
Statistical analysis. Data are expressed as means ± SE; n is the number of experiments performed, as indicated in RESULTS and in the figure legends. Statistical comparisons were performed using Student's t-test. Differences were considered to be significant when P < 0.05.
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RESULTS |
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-subunits in rat dPA and aorta.
Consistent with previous reports in cultured PASMCs (33), we found that mRNAs for Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv4.3, and Kv9.3 were expressed in rat dPAs (Fig. 1A). Similarly, aorta also expressed mRNAs for Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv4.3, and Kv9.3 (Fig. 1B). Messenger RNA for Kv1.3 was not expressed in these tissues but was readily detected in rat brain (Fig. 1C), indicating functional primers and PCR conditions for this gene. Exposure to chronic hypoxia significantly reduced the mRNA levels of Kv1.1 (–66.1 ± 5.9%), Kv1.2 (–42.5 ± 3.4%), Kv1.5 (–55.5 ± 2.8%), Kv1.6 (–34.3 ± 3.4%), Kv2.1 (–54.8 ± 6.3%), Kv4.3 (–39.2 ± 6.5%), and Kv9.3 (–37.1 ± 2.6%) in dPAs (Figs. 2 and 3); however, chronic hypoxia had no consistent effect on the mRNA level of Kv1.4 (–0.2 ± 23.6%) and Kv3.1 (3.6 ± 1.1%) (Figs. 2 and 3). The inhibitory effects of hypoxia on Kv channel -subunits appear to be selective for pulmonary arteries, as hypoxia had no significant effect on expression of these channels in the aortas from the same animals (range: –2.1 ± 4.2 to +2.5 ± 4.5%).
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-subunits in rat dPA.
Three Kv -subunits were identified in rat dPAs and aorta: Kv1, Kv2, and Kv3 (Fig. 4). Exposure to chronic hypoxia did not significantly alter the mRNA levels of any of the Kv -subunits examined in rat dPAs (Kv1 = +5.7 ± 0.6%, Kv2 = +2.9 ± 0.2%, and Kv3 = –0.4 ± 5.0%) or aorta (Kv1 = +4.0 ± 0.8%, Kv2 = –3.5 ± 0.6%, and Kv3 = +3.4 ± 0.3%) (Fig. 4).
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-subunits in rat dPA and aorta.
Immunoblot analysis was used to confirm the protein expression of Kv -subunits identified by RT-PCR in rat dPAs and aorta. In both pulmonary arteries and aorta, protein expression of Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, and Kv4.3 was observed (Figs. 5–8). Similar to results obtained with RT-PCR, protein expression of Kv1.3 was not observed in either dPA or aorta (data not shown). We could not test for Kv9.3 protein because an antibody was not available. Exposure to chronic hypoxia significantly reduced the protein levels of Kv1.1 (–52.8 ± 4.9%), Kv1.5 (–49.6 ± 6.6%), Kv1.6 (–39.2 ± 3.5%), Kv2.1 (–61.2 ± 9.7%), and Kv4.3 (–45.6 ± 7.9%) (Figs. 5, 6, and 8), whereas the protein expression of Kv1.2, Kv1.4, and Kv3.1 was not altered by chronic hypoxia (–8.9 ± 4.2, +5.1 ± 7.4, and +0.32 ± 2.9%, respectively) (Figs. 6 and 8). In contrast to the hypoxia-induced reduction in Kv channel protein levels observed in dPAs, exposure to chronic hypoxia had no effect on the expression of any of the Kv channel -subunits in the aorta (Figs. 5–8).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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- (pore-forming) or - (regulatory) subunits could account for the reduction in Kv channel activity observed in our rat model of hypoxic pulmonary hypertension. In the present study, we found that exposure to chronic hypoxia decreased gene and protein expression of several Kv channel subtypes in dPAs of rat. The effect of hypoxia was specific for pulmonary arteries, as similar decreases in Kv channel expression were not observed in aortas isolated from the same animals. Furthermore, the inhibitory effect of hypoxia was restricted to the Kv channel -subunits, as hypoxia negligibly affected the expression of the cytoplasmic regulatory -subunits. Our results also provide a comprehensive exploration of the effects of chronic hypoxia on Kv channel expression in intact rats and indicate that the reduction of PASMC Kv channel activity previously described in this model (28, 30) is due to the pulmonary-specific downregulation of multiple Kv channel -subunits in conjunction with maintained -subunit expression.
Kv channels are hetero- or homomeric tetramers composed of the pore-forming -subunits and the cytoplasmic regulatory -subunits (11). Using RT-PCR and immunoblot analyses, investigators have identified a number of Kv channel - and -subunits expressed in pulmonary vascular smooth muscle (5, 38). We found that endothelium-denuded rat dPAs expressed Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv4.3, and Kv9.3. This agrees with previously published reports in rat cultured PASMCs and freshly isolated pulmonary arteries (9, 19, 33, 38), although some studies find Kv1.3 and not Kv1.4 (3). The reason for this discrepancy is not clear, although given that those results were solely from immunoblot, perhaps antibody strength or specificity could contribute. Our finding of Kv1.4 mRNA was confirmed by immunoblot, and the lack of Kv1.3 mRNA expression was not due to primer or PCR errors, as Kv1.3 in brain was readily detected.
Of the Kv channel subtypes expressed in pulmonary vascular smooth muscle, we found that the expressions of Kv1.1, Kv1.2, Kv1.5, Kv1.6, Kv2.1, Kv4.3, and Kv9.3 were significantly depressed in dPAs from chronically hypoxic rats. These dPAs were denuded of endothelium by a process we have previously demonstrated to render these vessels unresponsive to endothelium-dependent vasodilators (27), indicating disruption of the endothelium. Logically, the downregulation of Kv channel mRNA expression in rat pulmonary arterial smooth muscle during chronic hypoxia would be reflected as a decrease in protein expression. Indeed, this is the case for Kv1.1, Kv1.5, Kv1.6, Kv2.1, and Kv4.3, the protein expressions of which were all decreased in dPAs isolated from chronically hypoxic rats. However, Kv1.2 presented a different scenario, in which protein did not decrease with reduced mRNA expression. This phenomenon is not uncommon as mRNA levels do not always reflect the exact amount of encoded protein. This being the case, protein expression data are more closely correlated with changes in the numbers of functional Kv channels. Unfortunately, we were unable to determine protein expression for Kv9.3 because an antibody was not available.
It would appear that the effect of chronic hypoxia on Kv channel expression is vessel specific. In contrast to the marked reduction in Kv channel mRNA and protein levels observed in dPAs, aortas from the same animals showed no alteration in either mRNA or protein expression of Kv channels. This finding is consistent with maintained expression of Kv channel expression in cultured mesenteric smooth muscle cells subjected to short-term hypoxia (19). The lack of effect of chronic hypoxia on Kv expression in aorta suggests that this response is not generalized and may be restricted to the pulmonary vasculature.
The decrease in Kv channel expression likely explains the reduced Kv channel currents observed in PASMC isolated from chronically hypoxic rats (28, 30). It is interesting that Kv1.4 expression was not altered by exposure to chronic hypoxia. Kv1.4 is typically characterized as a rapidly inactivating, or transient, current (13, 21). Examination of the inactivation kinetics of the whole cell Kv current in normoxic and chronically hypoxic PASMCs demonstrated a faster inactivation following exposure to chronic hypoxia with a greater proportion of the transient, rapidly inactivating current (28). The present data suggest that a decrease in the number of channels conducting non- or slowly inactivating currents, in concert with maintained expression of channels conducting rapidly inactivating currents, could contribute to the faster inactivation kinetics previously observed in PASMCs from chronically hypoxic rats (28, 30).
Another possibility accounting for faster inactivation of Kv currents in PASMCs isolated from chronically hypoxic rats is the lack of effect of chronic hypoxia on Kv1, Kv2, and Kv3 expression. Characterization of cloned Kv channels indicates that the native Shaker-related Kv channels are most likely heteromultimers formed by both - and -subunits, with each -subunit associating with an -subunit (18). The Kv channel -subunits bind to the cytoplasmic side of the -subunits (35), conferring rapid inactivation onto the non- or slowly inactivating Kv channel -subunits (e.g., Kv1.1, Kv1.2, and Kv1.5) (22) and blocking the -subunits as an open channel blocker (8). Thus, maintained Kv -subunit expression, coupled with reduced Kv -subunit expression, might allow for more interaction between Kv - and -subunits and faster current inactivation. It is impossible to delineate at this point which of the two possibilities, maintained expression of Kv1.4 or greater interaction between - and -subunits, is responsible for the change in current inactivation kinetics previously observed.
Little is known about the factors regulating Kv channel gene transcription. The mechanism by which Kv channels are down regulated by chronic hypoxia is unknown but is likely to involve modulation of a transcription factor. For example, hypoxia-inducible factor-1 (HIF-1) has been implicated in the regulation of an increasing number of genes, including vascular endothelial factor, endothelin-1, and nitric oxide synthase-2 (25). Indeed, the reduction in Kv current magnitude observed in PASMCs isolated from chronically hypoxic mice was absent in mice partially deficient for HIF-1 (26), indicating a crucial role of this transcription factor in modulation of Kv channel expression by hypoxia. Unfortunately, these data do not allow determination of whether HIF-1 directly mediates Kv channel expression in PASMCs or influences expression via an intermediate factor. For example, in addition to HIF-1, accumulating evidence suggests that the c-fos and c-jun family of genes can also be induced by hypoxia (4) and may participate in regulation of Kv channel gene expression. Data suggest that overexpression of c-jun significantly decreases whole cell Kv current (36), making c-jun a suitable candidate for the transcriptional downregulation of Kv channel expression during chronic hypoxia. Moreover, the c-jun transcription factor functionally cooperates with HIF-1, potentiating its transcriptional activity (1). Regardless of the exact mechanism of downregulation, decreased transcription of Kv channels is likely to be key in the pathogenesis of chronic hypoxic pulmonary hypertension because the consequent reduction in outward K+ current contributes to a sequence of events that ultimately leads to reduced vascular caliber and elevated pulmonary arterial pressure.
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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- and -subunits in the bovine pulmonary arterial circulation. Am J Physiol Lung Cell Mol Physiol 281: L1350–L1360, 2001.-subunit hKv 1.2. Am J Physiol Heart Circ Physiol 272: H2932–H2941, 1997.-dendrotoxin-sensitive potassium ion channels purified from bovine brain. Biochemistry 31: 11084–11088, 1992.[CrossRef][Medline]
-subunit. Nature 369: 289–294, 1994.[CrossRef][Medline]
deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia. Am J Physiol Lung Cell Mol Physiol 281: L202–L208, 2001. subunits in pulmonary artery smooth muscle cells. J Clin Invest 100: 2347–2353, 1997.[Web of Science][Medline]
- and - complexes of shaker-like potassium channels. Neuron 16: 441–453, 1996.[CrossRef][Web of Science][Medline]
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