CLINICAL PERSPECTIVE

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 112/9/1309    most recent CIRCULATIONAHA.104.529404v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Navarro-Antolín, J. Articles by López-Barneo, J. Search for Related Content PubMed PubMed Citation Articles by Navarro-Antolín, J. Articles by López-Barneo, J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB PHENYLEPHRINE Related Collections Animal models of human disease Other hypertension Physiological and pathological control of gene expression Risk Factors for Stroke Other Vascular biology Other Research

(Circulation. 2005;112:1309-1315.)
© 2005 American Heart Association, Inc.

Molecular Cardiology

Decreased Expression of Maxi-K⁺ Channel ß₁-Subunit and Altered Vasoregulation in Hypoxia

Javier Navarro-Antolín, MD, PhD; Konstantin L. Levitsky, PhD; Eva Calderón, MS; Antonio Ordóñez, MD, PhD; José López-Barneo, MD, PhD

From the Laboratorio de Investigaciones Biomédicas (J.N.-A., K.L.L., J.L.-B.) and Unidad de Cirugía y Trasplante Cardíaco (E.C., A.O.), Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Sevilla, Spain.

Correspondence to José López-Barneo, Laboratorio de Investigaciones Biomédicas, Edificio de Laboratorios, 2^a planta, Hospital Universitario Virgen del Rocío, Avenida Manuel Siurot s/n, E-41013 Sevilla, Spain. E-mail jose.l.barneo.sspa{at}juntadeandalucia.es

Received December 13, 2004; revision received May 6, 2005; accepted May 25, 2005.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Hypertension, a major cause of cardiovascular morbidity and mortality, can result from chronic hypoxia; however, the pathogenesis of this disorder is unknown. We hypothesized that downregulation of the maxi-K⁺ channel ß₁-subunit by hypoxia decreases the ability of these channels to hyperpolarize arterial smooth muscle cells, thus favoring vasoconstriction and hypertension.

Methods and Results— Lowering O₂ tension produced a decrease of maxi-K⁺ ß₁-subunit mRNA levels in rat (aortic and basilar) and human (mammary) arterial myocytes. This was paralleled by a reduction of the ß₁-subunit protein level as determined by immunocytochemistry and flow cytometry. Exposure to hypoxia also produced a decrease of open probability, mean open time, and sensitivity to the xenoestrogen tamoxifen of single maxi-K⁺ channels recorded from patch-clamped dispersed myocytes. The number of channels per patch and the single-channel conductance were not altered. The vasorelaxing force of maxi-K⁺ channels was diminished in rat and human arterial rings exposed to low oxygen tension.

Conclusions— These results indicate that a decrease of the maxi-K⁺ channel ß₁-subunit expression in arterial myocytes is a key factor in the vasomotor alterations induced by hypoxia.

Key Words: ion channels • arteries • muscle, smooth • hypoxia • hypertension

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Understanding the molecular pathophysiology of hypertension is of paramount medical interest, because high blood pressure is a well-known risk factor for stroke, congestive heart failure, and coronary or renal diseases.^1–3 Arterial oxygen tension (PO₂) regulates vascular tone, and local and generalized hypoxia are major causes of cardiovascular alterations.^4,5 Hypoxemia resulting from failure of lung gas exchange or by travel to high altitudes produces pulmonary hypertension.⁶ Hypoxia is also an independent risk factor for systemic hypertension in humans and in experimental animals. Moreover, hypertension is one of the major consequences of untreated obstructive sleep apnea syndrome, a frequent disorder characterized by chronic intermittent hypoxemia.^7–9 In recent years, it has been shown that vascular reactivity to acute changes in arterial PO₂ depends on ionic events in the vascular smooth muscle cells (VSMCs).^5,6 However, the mechanisms underlying the modification of VSMC excitability and contractility in hypoxia (either sustained or intermittent) are poorly understood.

Arterial tone depends on the equilibrium between forces causing vasoconstriction and vasodilation. Vasoconstriction is elicited by global increases in cytosolic [Ca²⁺] caused by either Ca²⁺ influx through dihydropyridine-sensitive, voltage-dependent, calcium channels or release of Ca²⁺ from internal stores. Ca²⁺ release is mediated by channels in the sarcoplasmic reticulum, which are opened by inositol trisphosphate synthesized after agonist-induced activation of the G protein–phospholipase-C pathway.^10,11 Increases of cytosolic [Ca²⁺] also induce Ca²⁺ release events from ryanodine receptors, called Ca²⁺ sparks, which in turn activate nearby plasmalemmal Ca²⁺-dependent potassium channels of large conductance (maxi-K⁺ channels). The opening of maxi-K⁺ channels leads to a prominent outward K⁺ current that produces membrane hyperpolarization.^12,13 Hence, Ca²⁺ sparks and maxi-K⁺ channel activation provide a critical negative feedback mechanism that opposes vasoconstriction. The participation of maxi-K⁺ channels in the regulation of arterial tone is demonstrated when they are specifically blocked with iberiotoxin (IbTx), which produces vasoconstriction and inhibits the action of vasodilators.^14–16

Maxi-K⁺ channels are composed of pore-forming -subunits and accessory ß-subunits. Although these channels are expressed in almost all the mammalian cell types, the 4 family members of the ß-subunits identified so far are differentially expressed among the various tissues.^15,17,18 The ß₁-subunit is expressed predominantly in arterial smooth muscle and, in heterologous expression systems, confers to maxi-K⁺ channels an increased Ca²⁺ sensitivity.^17,19 Deletion of the ß₁-subunit gene leads to a decrease in the Ca²⁺ sensitivity of maxi-K⁺ channels and uncoupling of Ca²⁺ sparks to maxi-K⁺ channel activation in VSMCs from mouse cerebral arteries. Animals without the maxi-K⁺ ß₁-subunit have increased mean arterial pressure and cardiac hypertrophy, as seen in many humans after long-standing hypertension.^18,20 Recently, downregulation of the maxi-K⁺ ß₁-subunit has been reported in rats made hypertensive after chronic treatment with angiotensin II,²¹ and a single nucleotide polymorphism in the human maxi-K⁺ ß₁ gene has been found to be associated with low prevalence of diastolic hypertension.²² Altogether, these observations suggest that alterations of ß₁-subunit expression or function could underlie some forms of hypertension.

We hypothesized that in conditions of low PO₂, a major pathogenic cause of hypertension, downregulation of the - and/or ß₁-subunit diminishes the ability of maxi-K⁺ channels to hyperpolarize smooth muscle cells, thus favoring vasoconstriction and high blood pressure. Here, we show that hypoxia decreases the expression of the maxi-K⁺ channel ß₁-subunit in rat and human arterial smooth muscle cells. This is paralleled by inhibition of the maxi-K⁺ channel open probability and a decrease of the vasorelaxing power of the channels. These observations help to explain the molecular mechanism whereby hypoxia might lead to hypertension.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Cultures and Hypoxic Treatments
The procedures followed to maintain clonal rat aortic smooth muscle A7r5 cells in culture as well as to perform primary cultures of rat and human arterial myocytes are based on protocols previously described.^11,23 Details of these methodologies are in the online-only Data Supplement.

Cultured cells were maintained in complete medium in a normoxic (20% O₂) atmosphere (95% air/5% CO₂) at 37°C. Hypoxia exposure was performed in an incubator with oxygen control (Forma Scientific) with 1%, 3%, or 6% O₂ and 5% CO₂, balanced with N₂.

RNA Analysis
The level of expression of the maxi-K⁺ channel subunits in arterial myocytes was evaluated by real-time polymerase chain reaction (PCR) (see supplementary Methods, Figure I, and Table in the online-only Data Supplement).

Flow Cytometry and Immunocytochemistry
Phenotype characterization of myocytes was based on the quantification by flow cytometry of permeabilized cells positive to -smooth muscle actin. The level of expression of maxi-K⁺ ß₁-subunit in A7r5 was also based on the detection by flow cytometry of an extracellular antigen of this subunit. Histological localization of the maxi-K⁺ ß₁-subunit in A7r5 cells was performed by immunohistochemistry. For details, see the online-only Data Supplement.

Patch-Clamp Recordings and Data Analysis
Single-channel recordings were obtained from dispersed rat basilar and aortic myocytes using the inside-out configuration of the patch-clamp technique as adapted to our laboratory.²⁴ Details of the experimental protocol are in the online-only Data Supplement.

Isometric Contraction of Arterial Rings
Rat aortas were obtained from 2-month-old Wistar rats and mammary arteries from patients who had undergone coronary artery bypass graft surgery. The vessels were incubated at either 1% PO₂ (hypoxia) or 20% PO₂ (normoxia) in serum-containing DMEM medium and supplements for 20 hours. Rats exposed in vivo to hypoxia were maintained at 10% PO₂ (hypoxic environment) or at 21% PO₂ (normoxic environment) in modular incubator chambers (Billups-Rothenberg) for 20 hours, and then the aortas were removed. For both in vitro and in vivo experiments, isometric contraction assays were carried out on 3-mm-long arterial rings incubated in Krebs solution in a 4-chambered organ bath (QOB Cibertec). Rings were stretched to a resting tension of 0.5 or 1.5 g for rat aortic and human mammary arteries, respectively, and then equilibrated for 60 minutes in Krebs solution. This time is considered to be long enough to reach a stable "resting" state.¹⁶ Nevertheless, we checked that no differences in agonist-induced contractility existed when rings were incubated for a longer time period (see Figure II in the online-only Data Supplement). In each experiment, at least 2 rings per condition were assayed.

Statistical Analysis
Unless otherwise specified, data are expressed as mean±SEM, with the number (n) of experiments indicated. Statistical analysis was performed by unpaired Student t test. A value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Hypoxia Selectively Downregulates the Maxi-K⁺ Channel ß₁-Subunit mRNA in Arterial Myocytes
The expression of - and ß₁-subunit mRNAs in rat aorta clonal cells (A7r5) and primary cultured myocytes (RASMCs) was initially confirmed by conventional PCR amplifications (Figure 1A, top). In these studies, we noticed that the ß₁-subunit mRNA, but not the -subunit mRNA, decreased in cells exposed to hypoxia (3% O₂ tension) for several hours (Figure 1A, bottom). Upregulation of the O₂-sensitive gene heme-oxygenase 1 (HO-1)²⁵ was used as an internal control. Real-time quantitative PCR was performed to analyze in more detail the expression and regulation by hypoxia of and ß₁ transcripts in comparison with that of HO-1. In rat aortic myocytes, the decrease of ß₁-subunit mRNA level by hypoxia was markedly time dependent and followed a time course parallel to that of HO-1 gene induction by low PO₂ (Figure 1, B and C). Interestingly, in these myocytes (A7r5 and RASMCs), the mRNA encoding the maxi-K⁺ -subunit quantified by real-time PCR was unaltered by hypoxia (Figure 1, B and C). Downregulation of the ß₁-subunit mRNA in hypoxia was also dose-dependent, being more clearly observed at PO₂ 3% (Figure 1, D and E). These PO₂ values are similar to those required to regulate HO-1 and other classic hypoxia-sensitive genes.^5,26 In addition to rat aortic myocytes, downregulation of the ß₁ transcript by hypoxia was also observed in VSMCs from rat basilar and human mammary arteries, although the magnitude of the effect varied from 30% to 60% inhibition depending on the myocyte class (Figure 1F). These data indicate that physiological levels of hypoxia selectively decrease the content of maxi-K⁺ channel ß₁-subunit mRNA in several classes of rat and human arterial VSMCs.

View larger version (52K): [in this window] [in a new window]   Figure 1. Downregulation by hypoxia of maxi-K+ channel ß1-subunit mRNA in rat and human arterial myocytes. A, Agarose gel electrophoresis of 40-cycle PCR products amplified with the primers indicated in the Table in the online-only Data Supplement, using primary cultured RASMCs incubated in normoxia (20% O2) and hypoxia (1% O2) for 24 hours. The transcripts were ß1- and -subunit of maxi-K+ channel and HO-1. Ribosome 28S mRNA was used as a control. Quantitative analysis of ß1, , HO-1, and 28S mRNA levels measured in an aortic myocyte cell line (A7r5) and RASMCs incubated at 1% O2 for the indicated time periods (B and C) or with various PO2 values for 24 hours (D and E). Each data point represents the average of 5 to 10 experiments. F, Downregulation of ß1 mRNA in the various arterial VSMC types exposed to 1% hypoxia for 24 hours. Bars show the percentage of ß1 transcript in hypoxia versus normoxia. Data are mean±SEM of 3 to 8 experiments for each cell type. Asterisks indicate the statistical significance (P<0.05).

The hypoxia-inducible transcription factor (HIF) mediates the transcriptional regulation of numerous O₂-sensitive genes,^5,26 so we tested whether HIF participates in the hypoxic downregulation of the ß₁ transcript. We assayed the effect of dimethyloxalylglycine (DMOG), a competitive inhibitor of prolyl hydroxylases that stabilizes HIF in normoxia.^5,26,27 DMOG induced HO-1 by approximately 2.5-fold but did not alter the mRNA levels of the ß₁-subunit. However, downregulation of the ß₁-subunit was more pronounced in cells exposed sequentially to hypoxia (6 hours, 1% O₂), reoxygenation (18 hours, 20% O₂), and hypoxia (6 hours, 1% O₂) compared with those exposed to sustained hypoxia (30 hours, 1% O₂) (Figure 2). Glutathione ethyl ester, a membrane-permeable quencher of reactive oxygen species,²⁸ had no significant effect on the basal level of ß₁ mRNA expression. However, downregulation of the ß₁ transcript by the hypoxia-reoxygenation-hypoxia treatment (to 44±4% of control, n=3) was significantly attenuated in cells incubated with 1 mmol/L glutathione ethyl ester (mRNA level decreased to 57±4% of control, n=3; P<0.05). Therefore, downregulation of the ß₁-subunit by hypoxia (or by a hypoxia/reoxygenation sequence) could depend, at least in part, on reactive oxygen species production.

View larger version (33K): [in this window] [in a new window]   Figure 2. Effects of hypoxia-reoxygenation on the ß1-subunit mRNA. Effect of various protocols of exposure to hypoxia on the ß1, , and HO-1 mRNA levels measured in A7r5 cells. O2 concentrations (in percent) are given in the inset. Data are mean±SEM of 3 to 4 experiments. Asterisks indicate the statistical significance (P<0.05) with respect to the values in normoxia.

Hypoxia Decreases ß₁-Subunit Protein Expression and Reduces the Activity of Maxi-K⁺ Channels
Antibodies against the ß₁-subunit clearly stained trypsinized A7r5 cells (Figure 3A, top), but this signal almost completely disappeared in cells incubated with the primary and secondary antibodies in the presence of the ß₁ peptide (Figure 3A, bottom). We also estimated the changes of expression of the ß₁-subunit by flow cytometry. The distribution of the quantity of immunodetected ß₁ protein per cell was markedly shifted toward smaller values in hypoxic cells compared with the same distribution estimated from cells maintained in normoxia. The presence of the ß₁ peptide reduced the fluorescence signal by 73±17% (Figure 3B). A parallel analysis with antibodies against the maxi-K⁺ -subunit showed only a slight decrease in the level of expression of this protein (data not shown). Thus, these studies demonstrated a significant decrease of the expression of ß₁ protein in hypoxic VSMCs.

View larger version (63K): [in this window] [in a new window]   Figure 3. Effect of hypoxia on ß1-subunit protein levels. A, Top, Expression of maxi-K+ channel ß1-subunit present in a trypsinized A7r5 myocyte. Left, Bright-field image. Right, Confocal fluorescence image showing the distribution of ß1-subunits. Bottom, Blockade of fluorescence in cells incubated with the ß1 peptide. B, Fluorescence histograms of A7r5 myocytes evaluated by flow cytometry with the same antibodies as used for the confocal image shown in A. A7r5 myocytes were incubated under normoxia (20% O2) or hypoxia (1% O2) for 24 hours as described in Methods. The lower panels show the specificity of the primary antibody. Fluorescence almost disappeared in cells incubated only with the secondary antibody or with the primary and secondary antibodies in the presence of the ß1 peptide (70-fold of molar excess with respect to primary antibody). a.u., arbitrary units.

The functional consequence of hypoxia on maxi-K⁺ channel function was evaluated in patch-clamped dispersed basilar myocytes. These channels were readily identified in inside-out excised membrane patches by their dependence on intracellular [Ca²⁺] and the characteristic large single-channel current amplitude (11.8±0.1 pA at –40 mV in symmetrical 150 [K⁺], n=17).^15–18 Maxi-K⁺ channels showed a clear voltage-dependence (Figure 4A), and in symmetrical 150 K⁺ solutions, they had a linear single-channel current-voltage relationship. Single-channel slope conductance (296±2 pS, n=17 in normoxia) was not altered on cells exposed to low PO₂ (295±2 pS, n=22) (Figure 4B). In fair agreement with the molecular biology data, the average number of maxi-K⁺ channels per patch, an indication of the density of -subunits in the plasmalemma, did not change between cells exposed to normoxia or hypoxia (Figure 4C). Exposure of primary cultured arterial myocytes to sustained hypoxia resulted, however, in clear inhibition of maxi-K⁺ channel activity. Single maxi-K⁺ channels were recorded in inside-out excised patches from primary cultured basilar VSMCs that had previously been maintained in either normoxia or hypoxia for 24 to 30 hours. Channel activation by cytoplasmic [Ca²⁺] (2.5 or 5 µmol/L) within the range predicted for Ca²⁺ sparks²⁹ was markedly reduced in cells previously maintained in a hypoxic environment (Figure 4D). At membrane potentials between –60 and –20 mV (2.5 µmol/L intracellular [Ca²⁺]), values within the range critical for the regulation of voltage-gated L-type Ca²⁺ channels in basilar smooth muscle,¹¹ single-channel open probability decreased 3 to 6 times in hypoxic cells (Figure 4E). Qualitatively similar effects of hypoxia on maxi-K⁺ channel activity were also observed in a few confirmatory experiments performed on dispersed aortic myocytes (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 4. Decreased open probability of single maxi-K+ channels in myocytes exposed to hypoxia. A, Voltage-dependence of a maxi-K+ channel recorded in an inside-out patch held at different voltages with 2.5 µmol/L free Ca2+ in the internal solution. o, open; c, closed. Sampling interval, 0.5 ms. B, Current-voltage relations for single maxi-K+ channel events recorded from myocytes exposed to normoxia and hypoxia. Each data point was obtained from measurements in 7 to 17 cells. C, Estimated average number of channels per patch in normoxic (2.4±0.2, n=74) and hypoxic (2.5±0.2, n=72) cells. D, Representative single maxi-K+ channel recordings at the indicated membrane potentials and intracellular [Ca2+] in patches excised from basilar myocytes cultured in normoxia (20% O2) and hypoxia (1% O2) for 24 to 30 hours. E, Single maxi-K+ channel open probability (ordinate in log scale) at various membrane potentials estimated from myocytes exposed to hypoxia and normoxia. The number of experiments is given in parentheses. Intracellular [Ca2+] was 2.5 µmol/L. Asterisks indicate the statistical significance (P<0.05).

In addition to the changes in open probability, we also studied whether hypoxia altered other functional and pharmacological characteristics of maxi-K⁺ channels, which depend on the expression of the ß₁-subunit. The maxi-K⁺ channel mean open time decreased significantly in myocytes exposed to hypoxia (Figure 5A). In addition, maxi-K⁺ channels from hypoxic myocytes were less sensitive to acute activation by the xenoestrogen tamoxifen (Figure 5, B and C). It has been shown that in the absence of the ß₁-subunit, open probability and the open dwell time of the maxi-K⁺ channels^18,19,21,30 as well as their sensitivity to tamoxifen^21,31 are decreased. Therefore, our observations are consistent with the selective downregulation of the ß₁-subunit mRNA and protein levels observed in hypoxic myocytes.

View larger version (30K): [in this window] [in a new window]   Figure 5. Effect of hypoxia on functional and pharmacological properties of single maxi-K+ channels. A, Open time histograms and single exponential fits obtained from single-channel recordings in excised patches from cells previously exposed to normoxia (20% O2; n=6, 291 events) and hypoxia (1% O2; n=4, 253 events). Representative current traces are shown in the insets. Open dwell time was significantly reduced (P<0.05) in hypoxic myocytes. Intracellular [Ca2+] 2 µmol/L. B, Single maxi-K+ channel recordings at +40 mV (1 µmol/L intracellular Ca2+) in patches from normoxic and hypoxic myocytes before (control) and after acute addition of tamoxifen (10 µmol/L). C, Differential increase of single-channel open probability by tamoxifen (Tam) in normoxic (4.8±1.0-fold, n=6) and hypoxic (1.8±0.7-fold, n=6) myocytes. Asterisk indicates the statistical significance between these 2 conditions (P<0.05).

Altered Maxi-K⁺ Channel-Dependent Vasoregulation in Arterial Rings Exposed to Hypoxia
The effect of hypoxia on ß₁ expression observed in cell cultures was also present in rats subjected to similar experimental protocols. Conventional PCR experiments suggested the decrease of ß₁ mRNA levels in aortas from rats maintained in a hypoxic environment (10% PO₂) for 16 to 20 hours (Figure 6A, top). Real-time quantitative PCR experiments indicated that even in animals exposed to relatively mild hypoxia, the ß₁-subunit transcript decreased to 75% of control levels (Figure 6A, bottom). Then, we tested whether agonist-induced contractility of arteries¹⁶ was altered in animals subjected to hypoxia. The contractile response to phenylephrine (10 µmol/L) of aortic rings from rats maintained in normoxia (21% O₂) was significantly smaller than the response observed in rings from animals kept in hypoxia (10% O₂) before they were euthanized (Figure 6, B and C), thus suggesting that the vasorelaxing power of the maxi-K⁺ channels was decreased in hypoxic arteries.^16,18 The phenylephrine-induced contraction in aortic rings from normoxic animals was markedly accentuated after blockade of maxi-K⁺ channels with IbTx in a dose-dependent manner (Figure 6D). However, the effect of IbTx was much less apparent in rings of arteries removed from hypoxic rats (Figure 6, E and F), which is consistent with the decrease of the sensitivity to this drug in arteries from either ß₁-knockout animals¹⁸ or from rats in which the ß₁-subunit expressed was decreased by chronic administration of angiotensin II.²¹ The relative lack of sensitivity to IbTx of arterial rings from hypoxic animals was also observed when we compared rings from rat aorta and human mammary arteries that after resection were incubated either in normoxia (20% O₂) or in hypoxia (1% O₂) for 16 to 20 hours (Figure 6F). These observations support the conclusion that maxi-K⁺ channels are functionally less active in hypoxic arteries.

View larger version (29K): [in this window] [in a new window]   Figure 6. Altered maxi-K+ channel-dependent vasoregulation in rat and human arteries after exposure to hypoxia. A, Top, Gel electrophoresis of 40-cycle PCR products amplified with the primers indicated in the Table in the online-only Data Supplement using aortas from rats maintained in normoxia (21% oxygen) or hypoxia (10% oxygen) for 16 to 20 hours. The transcripts are ß1-subunit of maxi-K+ channel and ribosome 28S mRNA (control). Bottom, Decrease of ß1-subunit mRNA expression in hypoxic rats (to 74.7±5.9% of control, n=4, P<0.005) as determined by quantitative PCR. B, Contractile response to phenylephrine (Phe, 10–5 mol/L) of aortic rings from rats maintained in normoxia or hypoxia as in A. C, Peak amplitude of Phe-induced contraction in aortic rings from normoxic and hypoxic rats. D, Dose-dependent vasoconstriction induced by IbTx in aortic rings from normoxic rats. E, Contractile response to Phe (10–5 mol/L) in rings of aorta removed from normoxic and hypoxic rats. Arteries were incubated with IbTx (100 nmol/L) for 20 minutes before the experiment. F, Decreased sensitivity to IbTx in rat and human arteries exposed to hypoxia. The data are expressed as percentage of contractile response in the presence or the absence of IbTx. The data shown in C, D, and F are mean±SEM measurements performed in 3 to 5 independent experiments. Asterisks indicate the statistical significance (P<0.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we have obtained molecular and functional data supporting the novel hypothesis that during exposure to hypoxia, downregulation of the ß₁-subunit in arterial VSMCs decreases the activity of maxi-K⁺ channels, thus diminishing their role as a steady vasorelaxing force. We have shown in several arterial myocyte classes (including human VSMCs) that low PO₂ can cause the decrease of ß₁ mRNA and protein expression. Interestingly, in rat aortic myocytes, hypoxia does not significantly change the mRNA level of the maxi-K⁺ channel pore-forming -subunit. It has been reported that the expression of this subunit is increased in aortic myocytes of spontaneously hypertensive rats³² but decreased in pulmonary VSMCs of rats with hypoxic pulmonary hypertension.³³ Our mRNA and protein expression studies are in accordance with the electrophysiological data that we obtained from VSMCs maintained in hypoxia, because the average number of maxi-K⁺ channels per patch and their single-channel conductance (parameters that depend of the -subunit) were not altered, thus further suggesting that in these cells, the effect of hypoxia was selective on the ß₁-subunit.

Normal maxi-K⁺ channels are composed of pore-forming - and accessory ß₁-subunits, and changes in the stoichiometry of these subunits can have a great impact on their function.³⁴ Coexpression of ß₁- with -subunits in heterologous systems decreases several-fold the [Ca²⁺] necessary to activate the maxi-K⁺ channels,^30,35 and in mice lacking the ß₁-subunit gene, the maxi-K⁺ channel Ca²⁺ sensitivity is dramatically decreased.^18,20 Remarkably, the functional modifications observed in maxi-K⁺ channels of myocytes exposed to hypoxia (pronounced decrease in the single-channel open probability at negative membrane potentials without alterations in the single-channel conductance and density) were quite similar to those reported in myocytes of ß₁-knockout animals. The molecular and electrophysiological effects of hypoxia were also fully consistent with the modifications observed at the organ level, because the maxi-K⁺ channel–dependent relaxing force was decreased in both rat and human arteries exposed to low PO₂ for several hours. Altogether, these results support the view that reduced expression of the maxi-K⁺ channel ß₁-subunit contributes to vascular pathophysiology in hypoxia. However, it is important to keep in mind that we have studied downregulation of the maxi-K⁺ channel ß₁-subunit within the first 24 to 48 hours of exposure to hypoxia (a protocol normally used for the study of hypoxia-regulated genes^25–27), whereas the establishment of chronic hypertension in humans results from exposures to hypoxia lasting weeks or months.

Several recent reports have highlighted the importance of the accessory ß₁-subunit for the normal function of the maxi-K⁺ channel and its alteration in pathological states. Decreased sensitivity to Ca²⁺ has recently been reported for maxi-K⁺ channels in vascular myocytes from genetically hypertensive rats,³⁶ and a mutation that enhances maxi-K⁺ channel sensitivity to Ca²⁺ has been reported to occur in association with the low prevalence of diastolic hypertension in humans.²² In addition, the ß₁-subunit is downregulated in animals made hypertensive after sustained administration of angiotensin II²¹ and is also necessary for the acute activation of the channels by estrogens, which reduces the risk of hypertension.^31,37 Nevertheless, regulation of the ß₁-subunit by O₂ tension could have evolved to play, in normal circumstances, an adaptive role. Hypoxia can induce both the inducible isoform of nitric oxide (NO) synthase³⁸ and HO-1, necessary for the endogenous generation of carbon monoxide (CO).²⁵ Because both NO³⁹ and CO⁴⁰ activate maxi-K⁺ channels, downregulation of the ß₁-subunit in hypoxia could act as a counterregulatory mechanism to prevent the excessive hyperpolarization of myocytes and the subsequent alterations of vascular tone and blood flow. Disregulation or overactivation of this response could probably participate in the development of hypertension in humans.

In summary, we show data at the molecular, cellular, and organ levels giving strong support to the hypothesis that hypoxia downregulates the expression of the maxi-K⁺ channel ß₁-subunit and decreases maxi-K⁺ channel activity in arterial smooth muscle. Although chronic vasomotor alterations in humans are caused by multiple factors, the phenomena described here could possibly contribute to the development of hypertension secondary to hypoxia.

	Acknowledgments

 
J. López-Barneo received the Ayuda a la Investigación 2000 of the Juan March Foundation. This study was funded by grant FIS 01/3101, the Lilly Foundation, and the Redes Temáticas de Investigación Cooperativa CIEN and RECAVA. We thank J. Villadiego, A. Castellano, E.M. León-Gómez, V. Rivero-Valdenebro, C. Sáez, B. Sánchez, and G. Gasic for technical help and for comments on the manuscript. The authors declare that no conflict of interest exists with regard to the content of this article.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. MacMahon S, Sharpe N. Future directions for randomized trials of cardiovascular disease prevention in hypertensive patients. J Cardiovasc Pharmacol. 1990; (suppl 7): S96–S99.

2. Kotlikoff M, Hall I. Hypertension: beta testing. J Clin Invest. 2003; 112: 654–656.[CrossRef][Medline] [Order article via Infotrieve]

3. Nelson MT, Bonev AD. The ß1 subunit of the Ca²⁺-sensitive K⁺ channels protects against hypertension. J Clin Invest. 2004; 113: 955–957.[CrossRef][Medline] [Order article via Infotrieve]

4. Wadsworth RM. Vasoconstrictor and vasodilator effects of hypoxia. Trends Pharmacol Sci. 1994; 15: 47–53.[CrossRef][Medline] [Order article via Infotrieve]

5. Lopez-Barneo J, Pardal R, Ortega-Saenz P. Cellular mechanism of oxygen sensing. Annu Rev Physiol. 2001; 63: 259–287.[CrossRef][Medline] [Order article via Infotrieve]

6. Weir EK, Archer SL. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB J. 1995; 9: 183–189.[Abstract]

7. Grote L, Hedner J, Peter JH. Sleep-related breathing disorder is an independent risk factor for uncontrolled hypertension. J Hypertens. 2000; 18: 679–685.[CrossRef][Medline] [Order article via Infotrieve]

8. Peppard P, Young T, Palta M, Skatrud J. Prospective study of the association between sleep disordered breathing and hypertension. N Engl J Med. 2000; 342: 1378–1384.[Abstract/Free Full Text]

9. Fletcher EC. Physiological consequences of intermittent hypoxia: systemic blood pressure. J Appl Physiol. 2001; 90: 1600–1605.[Abstract/Free Full Text]

10. Jackson WF. Ion channels and vascular tone. Hypertension. 2000; 35: 173–178.[Abstract/Free Full Text]

11. del Valle-Rodriguez A, Lopez-Barneo J, Ureña J. Ca²⁺ channel-sarcoplasmic reticulum coupling: a mechanism of arterial myocyte contraction without Ca²⁺ influx. EMBO J. 2003; 22: 4337–4345.[CrossRef][Medline] [Order article via Infotrieve]

12. Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science. 1995; 270: 633–637.[Abstract/Free Full Text]

13. Knot HJ, Standen NB, Nelson MT. Ryanodine receptors regulate arterial diameter and wall [Ca²⁺] in cerebral arteries of rat via Ca²⁺-dependent K⁺ channels. J Physiol. 1998; 508: 211–221.[Abstract/Free Full Text]

14. Brayden JE, Nelson MT. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science. 1992; 256: 532–535.[Abstract/Free Full Text]

15. Kaczorowski GJ, Knaus HG, Leonard RJ, McManus OB, Garcia ML. High-conductance calcium-activated potassium channels; structure, pharmacology, and function. J Bioenerg Biomembr. 1996; 28: 255–267.[CrossRef][Medline] [Order article via Infotrieve]

16. Alioua A, Majan A, Nishimaru K, Zarei MM, Stefani E, Toro L. Coupling of c-Src to large conductance voltage- and Ca²⁺-activated K⁺ channels as a new mechanism of agonist-induced vasoconstriction. Proc Natl Acad Sci U S A. 2002; 99: 14560–14565.[Abstract/Free Full Text]

17. Jiang Z, Wallner M, Meera P, Toro L. Human and rodent maxi-K⁺ channel beta-subunit genes: cloning and characterization. Genomics. 1999; 55: 57–67.[CrossRef][Medline] [Order article via Infotrieve]

18. Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT, Aldrich RW. Vasoregulation by the beta1 subunit of the calcium-activated potassium channel. Nature. 2000; 407: 870–876.[CrossRef][Medline] [Order article via Infotrieve]

19. Cox DH, Aldrich RW. Role of the ß1 subunit in large-conductance Ca²⁺-activated maxi K⁺ channel gating energetics: mechanisms of enhanced Ca²⁺ sensitivity. J Gen Physiol. 2000; 116: 411–432.[Abstract/Free Full Text]

20. Pluger S, Faulhaber J, Furstenau M, Lohn M, Waldschutz R, Gollasch M, Haller H, Luft FC, Ehmke H, Pongs O. Mice with disrupted BK channel ß₁ subunit gene feature abnormal Ca²⁺ spark/STOC coupling and elevated blood pressure. Circ Res. 2000; 87: E53–E60.[Medline] [Order article via Infotrieve]

21. Amberg GC, Bonev AD, Rossow CF, Nelson MT, Santana LF. Modulation of the molecular composition of large conductance Ca²⁺ activated K⁺ channels in vascular smooth muscle during hypertension. J Clin Invest. 2003; 112: 717–724.[CrossRef][Medline] [Order article via Infotrieve]

22. Fernández-Fernández JM, Tomás M, Vázquez E, Orio P, Latorre R, Senti M, Marrugat J, Valverde MA. Gain-of-function mutation in the KCNMB1 potassium channel subunit is associated with low prevalence of diastolic hypertension. J Clin Invest. 2004; 113: 1032–1039.[CrossRef][Medline] [Order article via Infotrieve]

23. Pauly RR, Bilato C, Cheng L, Monticone R, Crow MT. Vascular smooth muscle cell cultures. Methods Cell Biol. 1997; 52: 133–154.[Medline] [Order article via Infotrieve]

24. Ganfornina MD, Lopez-Barneo J. Single K⁺ channels in membrane patches of arterial chemoreceptor cells are modulated by O₂ tension. Proc Natl Acad Sci U S A. 1991; 88: 2927–2930.[Abstract/Free Full Text]

25. Morita T, Perrella MA, Lee ME, Kourembanas S. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP. Proc Natl Acad Sci U S A. 1995; 92: 1475–1479.[Abstract/Free Full Text]

26. Semenza GL. Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology. 2004; 19: 176–182.[Abstract/Free Full Text]

27. del Toro R, Levitsky KL, López-Barneo J, Chiara MD. Induction of T-type calcium channel gene expression by chronic hypoxia. J Biol Chem. 2003; 278: 22316–22324.[Abstract/Free Full Text]

28. Gao L, Mejías R, Echevarría M, López-Barneo J. Induction of the glucose-6-phosphate dehydrogenase gene expression by chronic hypoxia in PC12 cells. FEBS Lett. 2004; 569: 256–260.[CrossRef][Medline] [Order article via Infotrieve]

29. Perez GJ, Bonev AD, Patlak JB, Nelson MT. Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries. J Gen Physiol. 1999; 113: 229–238.[Abstract/Free Full Text]

30. Nimigean CM, Magleby KL. The ß subunit increases the Ca²⁺ sensitivity of the large conductance Ca²⁺-activated potassium channels by retaining the gating in the burst states. J Gen Physiol. 1999; 113: 425–440.[Abstract/Free Full Text]

31. Dick GM, Rossow CF, Smirnov S, Horowitz B, Sanders KM. Tamoxifen activates smooth muscle BK channels through the regulatory ß1 subunit. J Biol Chem. 2001; 276: 34594–34599.[Abstract/Free Full Text]

32. Liu Y, Pleyte K, Knaus HG, Rusch NJ. Increased expression of Ca²⁺-sensitive K⁺ channels in aorta of hypertensive rats. Hypertension. 1997; 30: 1403–1409.[Abstract/Free Full Text]

33. Bonnet S, Savineau JP, Barillot W, Dubuis E, Vandier C, Bonnet P. Role of Ca²⁺-activated K⁺ channels in the remission phase of pulmonary hypertension in chronic obstructive pulmonary diseases. Cardiovasc Res. 2003; 60: 326–336.[Abstract/Free Full Text]

34. Nishimaru K, Eghbali M, Stefani E, Toro L. Function and clustered expression of maxiK channels in cerebral myocytes remain intact with aging. Exp Gerontol. 2004; 39: 831–839.[CrossRef][Medline] [Order article via Infotrieve]

35. Garcia-Calvo M, Knaus HG, McManus OB, Giangiacomo KM, Kaczorowski GJ, Garcia ML. Purification and reconstitution of the high-conductance, calcium-activated potassium channel from tracheal smooth muscle. J Biol Chem. 1994; 269: 676–682.[Abstract/Free Full Text]

36. Amberg GC, Santana LF. Downregulation of the BK channel ß₁ subunit in genetic hypertension. Circ Res. 2003; 93: 965–971.[Abstract/Free Full Text]

37. Valverde MA, Rojas P, Amigo J, Cosmelli D, Orio P, Bahamonde MI, Mann GE, Vergara C, Latorre R. Acute activation of maxi-K channels (hSlo) by estradiol binding to the ß subunit. Science. 1999; 285: 1929–1931.[Abstract/Free Full Text]

38. Jung F, Palmer LA, Zhou N, Johns RA. Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes. Circ Res. 2000; 86: 319–325.[Abstract/Free Full Text]

39. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature. 1994; 368: 850–853.[CrossRef][Medline] [Order article via Infotrieve]

40. Wang R, Wu L. The chemical modification of KCa channels by carbon monoxide in vascular smooth muscle cells. J Biol Chem. 1997; 272: 8222–8226.[Abstract/Free Full Text]

---

 

CLINICAL PERSPECTIVE

Ca²⁺-dependent maxi-K⁺ channels are major regulators of arterial smooth muscle tone and blood pressure. When myocytes contract, the rise of cytosolic Ca²⁺ induces the opening of maxi-K⁺ channels to produce potassium efflux and hyperpolarization. This membrane potential change causes voltage-gated Ca²⁺ channels to close, which decreases Ca²⁺ influx and leads to vasorelaxation. In arterial myocytes, maxi-K channels contain auxiliary ß₁-subunits that increase the sensitivity of the channels for Ca²⁺. Genetically modified mice lacking the ß₁-subunit are prone to vasoconstriction and develop chronic hypertension and cardiac hypertrophy. This raises the possibility that downregulation of the ß₁-subunit could underlie some forms of hypertension in humans. This report shows that exposure to hypoxia for 6 to 24 hours of myocytes dispersed from rat and human arteries produces a selective decrease in the levels of the ß₁-subunit messenger RNA and protein. These molecular changes induce a decrease in the Ca²⁺ sensitivity and open probability of single maxi-K⁺ channels as well as reduction of the action of tamoxifen, an estrogen-derived maxi-K⁺ channel activator that binds to the ß₁-subunit. Moreover, arteries from rats exposed to hypoxia (10% O₂ tension) for 16 to 20 hours are oversensitive to vasoconstrictor agents. The data support the novel hypothesis that ß₁ downregulation in arterial smooth muscle could mediate some of the cardiovascular alterations caused by hypoxia. The maxi-K⁺ channel ß₁-subunit could then be considered a potential target for vasoactive drugs and a possible marker for hypertension.

	Footnotes

 
The online-only Data Supplement is available at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.104.529404/DC1.

This article has been cited by other articles:

				W.-Z. Ying, K. Aaron, P.-X. Wang, and P. W. Sanders Potassium Inhibits Dietary Salt-Induced Transforming Growth Factor-{beta} Production Hypertension, November 1, 2009; 54(5): 1159 - 1163. [Abstract] [Full Text] [PDF]

				M. Tong and R. K. Duncan Tamoxifen inhibits BK channels in chick cochlea without alterations in voltage-dependent activation Am J Physiol Cell Physiol, July 1, 2009; 297(1): C75 - C85. [Abstract] [Full Text] [PDF]

				L. Bautista, M. J. Castro, J. Lopez-Barneo, and A. Castellano Hypoxia Inducible Factor-2{alpha} Stabilization and Maxi-K+ Channel {beta}1-Subunit Gene Repression by Hypoxia in Cardiac Myocytes: Role in Preconditioning Circ. Res., June 19, 2009; 104(12): 1364 - 1372. [Abstract] [Full Text] [PDF]

				S. J. Fountain, A. Cheong, J. Li, N. Y. Dondas, F. Zeng, I. C. Wood, and D. J. Beech Kv1.5 potassium channel gene regulation by Sp1 transcription factor and oxidative stress Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2719 - H2725. [Abstract] [Full Text] [PDF]

				G. Zhao, A. Adebiyi, Q. Xi, and J. H. Jaggar Hypoxia reduces KCa channel activity by inducing Ca2+ spark uncoupling in cerebral artery smooth muscle cells Am J Physiol Cell Physiol, June 1, 2007; 292(6): C2122 - C2128. [Abstract] [Full Text] [PDF]

				P. Ortega-Saenz, A. Pascual, R. Gomez-Diaz, and J. Lopez-Barneo Acute Oxygen Sensing in Heme Oxygenase-2 Null Mice J. Gen. Physiol., October 1, 2006; 128(4): 405 - 411. [Abstract] [Full Text] [PDF]

				N. Tajima, K. Schonherr, S. Niedling, M. Kaatz, H. Kanno, R. Schonherr, and S. H. Heinemann Ca2+-activated K+ channels in human melanoma cells are up-regulated by hypoxia involving hypoxia-inducible factor-1{alpha} and the von Hippel-Lindau protein J. Physiol., March 1, 2006; 571(2): 349 - 359. [Abstract] [Full Text] [PDF]

				M. Senti, J. M. Fernandez-Fernandez, M. Tomas, E. Vazquez, R. Elosua, J. Marrugat, and M. A. Valverde Protective Effect of the KCNMB1 E65K Genetic Polymorphism Against Diastolic Hypertension in Aging Women and Its Relevance to Cardiovascular Risk Circ. Res., December 9, 2005; 97(12): 1360 - 1365. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 112/9/1309    most recent CIRCULATIONAHA.104.529404v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Navarro-Antolín, J. Articles by López-Barneo, J. Search for Related Content PubMed PubMed Citation Articles by Navarro-Antolín, J. Articles by López-Barneo, J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB PHENYLEPHRINE Related Collections Animal models of human disease Other hypertension Physiological and pathological control of gene expression Risk Factors for Stroke Other Vascular biology Other Research