Decreased Expression of Maxi-K+ Channel β1-Subunit and Altered Vasoregulation in Hypoxia by Javier Navarro-Antolín, Konstantin L. Levitsky, Eva Calderón, Antonio Ordóñez, and José López-Barneo Circulation Volume 112(9):1309-1315 August 30, 2005 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Downregulation by hypoxia of maxi-K+ channel β1-subunit mRNA in rat and human arterial myocytes. Figure 1. Downregulation by hypoxia of maxi-K+ channel β1-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 β1- and α-subunit of maxi-K+ channel and HO-1. Ribosome 28S mRNA was used as a control. Quantitative analysis of β1, α, 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 β1 mRNA in the various arterial VSMC types exposed to 1% hypoxia for 24 hours. Bars show the percentage of β1 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). Javier Navarro-Antolín et al. Circulation. 2005;112:1309-1315 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Effects of hypoxia-reoxygenation on the β1-subunit mRNA. Figure 2. Effects of hypoxia-reoxygenation on the β1-subunit mRNA. Effect of various protocols of exposure to hypoxia on the β1, α, 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. Javier Navarro-Antolín et al. Circulation. 2005;112:1309-1315 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Effect of hypoxia on β1-subunit protein levels. Figure 3. Effect of hypoxia on β1-subunit protein levels. A, Top, Expression of maxi-K+ channel β1-subunit present in a trypsinized A7r5 myocyte. Left, Bright-field image. Right, Confocal fluorescence image showing the distribution of β1-subunits. Bottom, Blockade of fluorescence in cells incubated with the β1 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 β1 peptide (70-fold of molar excess with respect to primary antibody). a.u., arbitrary units. Javier Navarro-Antolín et al. Circulation. 2005;112:1309-1315 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Decreased open probability of single maxi-K+ channels in myocytes exposed to hypoxia. 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). Javier Navarro-Antolín et al. Circulation. 2005;112:1309-1315 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Effect of hypoxia on functional and pharmacological properties of single maxi-K+ channels. 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). Javier Navarro-Antolín et al. Circulation. 2005;112:1309-1315 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. Altered maxi-K+ channel-dependent vasoregulation in rat and human arteries after exposure to hypoxia. 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 β1-subunit of maxi-K+ channel and ribosome 28S mRNA (control). Bottom, Decrease of β1-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). Javier Navarro-Antolín et al. Circulation. 2005;112:1309-1315 Copyright © American Heart Association, Inc. All rights reserved.