bk β1  (Alomone Labs)

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Empagliflozin Induces Vascular Relaxation in Rat Coronary Artery Due to Activation of BK Channels

doi: 10.2147/DMSO.S419125

Figure Lengend Snippet: Empagliflozin induces vasodilation in KCl-constricted coronary arteries.

Article Snippet: The PVDF membranes were incubated with specific primary antibodies, with β-actin (ab20272, Abcam, 1:5000), BK-α (APC-021, Alomone Labs, 1:500), BK-β1 (APC-036, Alomone Labs, 1:1000), Sirt1 (ab110304, Abcam, 1:2000) and Nrf2 (SAB5700720, Sigma, 1:1000).

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: BK Ca -β1 expression is reduced in the carotid arteries of rat balloon-injured model. Representative images of H&E (A) and immunohistochemical staining of BK Ca −α and BK ca −β1 (B) in injured and sham-operated carotid arteries of rat balloon-injured model at 14 days post injury. Scale bar = 100 μm. (C) Quantified mRNA levels of BK ca −α and BK ca −β1 analyzed by qRT-PCR. Western blot images (D) and quantification (E) for indicated proteins. Eukaryotic initiation factor 5 (eIF5) was used as an internal control. Data are presented as means ± SEM ( n = 4∼6 for each group, ns, not significant, * P < 0.05, ** P < 0.01 vs. sham).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: BK Ca -β1 expression is positively associated with VSMC differentiation markers in vitro . Western blot images (A) and quantification (B) of BK ca −α, BK ca −β1, and VSMC differentiation markers expression in VSMCs of passage 1 and passage 6. Western blot images (C) and quantification (D) of BK ca −α, BK ca −β1, and VSMC differentiation markers expression in VSMCs after PDGF-BB treatment (25 μg/L). Western blot images (E) and quantification (F) of BK ca −α, BK ca −β1, and VSMC differentiation markers expression in VSMCs treated with TGF-β (2.5 μg/L). VSMCs were subjected to serum-starvation for 24 h and then treated with the indicated stimulation for 48 h. Passages 3-5 of VSMCs were used in (C) through (F) . Eukaryotic initiation factor 5 (eIF5) was used as an internal control. Data are presented as means ± SEM ( n = 5 for each group, ns, not significant, * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Expressing, In Vitro, Western Blot, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: Decreased BK ca −β1 expression is mediated by elevated PDGF-BB in vivo . (A) Quantified mRNA levels of PDGF-B and TGF-β analyzed by qRT-PCR in injured and sham-operated carotid arteries. Western blot images (B) and quantification (C) of BK ca −α and BK ca −β1 in the carotid arteries of mice injected with saline or PDGF-BB via tail vein. Data are presented as means ± SEM ( n = 6 for each group, ns, not significant, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Expressing, In Vivo, Quantitative RT-PCR, Western Blot, Injection, Saline, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: BK Ca -β1 is essential for VSMCs to maintain a differentiated phenotype. (A) Representative micrographs of VSMCs treated with BK ca −β1 siRNA or scramble siRNA transfection. (B) Representative immunofluorescent images of F-actin of VSMCs stained with phalloidin (red). Nuclei were stained with DAPI (blue). Scale bar = 25 μm. (C) The percentage of spindle-like or polygonal-like cells in each group. Western blot images (D) and quantification (E) of BK ca −β1, α-SMA, and SM22α. Representative images (F) and quantification (G) of the areas of collagen gel in each group. The size of the collagen gel in each well was calculated compared to the total area of the well. Data are presented as means ± SEM ( n = 5∼6 for each group, *** P < 0.001 vs. scramble).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Transfection, Staining, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: BK Ca -β1 knockdown induces proliferative, migratory, and synthetic phenotype of VSMCs. (A) Proliferation curves of VSMCs transfected with BK ca −β1 or scrambled siRNA. Representative images (B) and quantification (C) of migration assay. (D) Quantified mRNA levels of inflammatory factors analyzed by qRT-PCR. Representative images (E) and quantification (F) of activities of MMP-9 and MMP-2 tested using gelatin zymography. Data are presented as means ± SEM ( n = 5∼6 for each group, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. scramble).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Knockdown, Transfection, Migration, Quantitative RT-PCR, Zymography

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: Decreased BK ca −β1 expression is correlated with VSMC dedifferentiation and atherosclerosis in human. Western blot images (A) and quantification (B) of BK ca −α and BK ca −β1 expression in human primary aortic VSMCs after PDGF-BB treatment (25 μg/L). Western blot images (C) and quantification (D) of BK ca −α and BK ca −β1 expression in human primary aortic VSMCs treated with TGF-β (2.5 μg/L). VSMCs were subjected to serum-starvation for 24 h and then treated with the indicated stimulation for 48 h. Passages 3–5 of VSMCs were used. (E) Representative western blot images of the protein levels of BK ca −α and BK ca −β1 in lysates of human carotid endarterectomy artery (CEA) and control internal mammary arteries (IMA). (F) Quantification was performed by calculating the ratio between the levels of BK ca −β1 and BK ca −α. Eukaryotic initiation factor 5 (eIF5) was used as an internal control. Data are presented as means ± SEM (n = 3∼5 for each group, ns, not significant, * P < 0.05, ** P < 0.01 vs. control).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Aldosterone treatment increases Ca v 1.2 expression but does not change depolarization-induced vasoconstriction responses of rat mesenteric arteries. (A) Representative traces of tension changes ( top recordings ) in response to potassium chloride (KCl) additions (from 10 to 60 mM KCl) of rat MA rings treated with aldosterone (ALDO 10 nM, 24 h, red trace ) or without it (Control, black trace ). The concentration-response graph ( below ) shows vasoconstriction responses of MA rings treated ( red circles ) or not ( empty circles ) with ALDO (10 nM, 24 h). Depolarization-induced contraction was expressed as a percentage of maximal contractile response induced by 60 mM KCl. Data are shown as mean ± SEM of n = 7 MA rings from N = 7 rats for each experimental group. (B) Representative immunoblot image and scatterplot with mean ± SEM ( below ) of Ca v 1.2 protein expression from homogenates of control MAs ( empty circles ), ALDO-treated MAs ( red circles ), and ALDO-treated MAs co-incubated with RU 28318, a selective MR antagonist (1 μM, red/black circles ). Data are shown as mean ± SEM of n = 6 independent experiments for each group. Each experiment was performed with a pool of 3–4 MA segments from three rats. * P < 0.01 vs. untreated MAs. # P < 0.05 vs. ALDO-treated MAs.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Expressing, Control, Concentration Assay, Western Blot, Incubation

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: SERCA pump counterbalances depolarization-induced Ca 2+ entry in ALDO-treated MASMCs. (A) Representative Ca 2+ influx recordings ( F / F 0 ) of Fluo 4-loaded MASMCs from control ( empty circles ) or ALDO-treated ( red circles ) cells preincubated with thapsigargin (TGN) 100 nM for 10 min ( white/black and red/black circles , respectively) to block SERCA pump activity, or in its absence. Cells were kept in Na + and Ca 2+ free solution. After 2-min of frame-scan recording, cells were perfused with Na + free solution (to block Na + /Ca 2+ exchanger activity) containing 1.8 mM CaCl 2 plus 20 mM KCl to induce LTCC-mediated Ca 2+ influx. Changes in cytoplasmic Ca 2+ were recorded with a laser scanning confocal microscope (Zeiss, LSM 700) equipped with an ×63 oil immersion objective (N.A. 1.2). (B) Scatterplot with mean ± SEM illustrates the amplitude of depolarization-induced Ca 2+ entry (Δ F / F 0 ) of Fluo 4-loaded MASMCs under the different experimental conditions. The amplitude of Ca 2+ influx was determined at minute 8 of the recording in control MASMCs ( n = 17 cells/ N = 4 rats, empty circles ), ALDO-treated MASMCs ( n = 13 cells/ N = 5 rats, empty circles ), control MASMCs + TGN ( n = 15 cells/ N = 6 rats, white/black circles ) and ALDO-treated MASMCs + TGN ( n = 13 cells/ N = 5 rats, red/black circles ), respectively. *** P < 0.001 vs. control cells; ### P < 0.001 vs. ALDO-treated cells; &&& P < 0.001 vs. control + TGN cells. (C) Representative immunoblot image and scatterplot with mean ± SEM ( below ) of SERCA pump protein expression from homogenates of control MAs ( empty circles ), ALDO-treated MAs ( red circles ), and ALDO-treated MAs co-incubated with 1 μM RU28318, a selective MR antagonist ( red/black circles ). Data are shown as mean ± SEM of n = 5 experiments for each group. Each experiment was performed with a pool of 3–4 MA segments from three rats. SERCA pump expression levels were normalized to the expression of GAPDH for each independent experiment. * P < 0.05 vs. control group. # P < 0.05 vs. ALDO-treated group. (D) Scatterplot with mean ± SEM of Atp2a2 relative mRNA levels determined by real-time qPCR from control MAs ( empty circles , n = 8), ALDO-treated MAs ( red circles , n = 8), and ALDO-treated MAs co-incubated with 1 μM RU28318 ( red/black circles, n = 3). * P < 0.05 vs. control group.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Control, Blocking Assay, Activity Assay, Microscopy, Western Blot, Expressing, Incubation

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Aldosterone treatment increases Ca 2+ spark frequency in MASMCs. (A) Representative pseudo-colored confocal images of Ca 2+ sparks ( top ) and normalized ( F / F 0 ) fluorescence profiles ( bottom ) from Fluo 4-loaded MASMCs treated or not with aldosterone (ALDO 10 nM, 24 h) and preincubated or not with Nifedipine (1 μM, 10 min). The fluorescence profile was calculated in the region indicated by the green bar. (B) Scatterplot with mean ± SEM illustrates Ca 2+ spark frequency in Fluo 4-loaded MASMCs from control ( empty circles , n = 35 cells/ N = 5 rats); ALDO-treated arteries ( red circles , n = 29 cells/ N = 4 rats); pre-incubated with Nifedipine (NIF, white/black circles, n = 18 cells/ N = 4 rats; and red/black circles, n = 11 cells/ N = 3 rats, respectively). ** P < 0.01 vs. control cells. # P < 0.05 and ### P < 0.001 vs. ALDO-treated group.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Fluorescence, Control, Incubation

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Aldosterone treatment increases the frequency and the amplitude of STOCs in MASMCs without modifying BK channel subunit expression. (A) Representative traces of STOCs recorded at a holding potential of –40 mV from MASMCs in the absence (CONTROL, black trace ) or after 24 h-treatment with aldosterone 10 nM (ALDO, red trace ). Scatterplots with mean ± SEM illustrate STOC frequency ( B , normalized with respect to cell capacitance, in events/s/pF), STOC amplitude ( C , normalized with respect to cell capacitance, in pA/pF), and STOC area-under-the-curve ( D , in pA.s) in control MASMCs ( n = 119 events/ n = 12 cells/ N = 4 animals, empty circles ) and ALDO-treated cells ( n = 333 events/ n = 12 cells/ N = 5 animals, red circles ). * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control group. (E) Histogram distribution of normalized STOC amplitudes in control ( n = 119 events/ n = 12 cells/ N = 4 rats, white bars ) and ALDO-treated MASMCs ( n = 333 events/ n = 12 cells/ N = 5 rats, red bars ) indicates the increase in the amplitude of STOCs above 3.6 pA/pF in ALDO-treated cells. (F,G) Representative immunoblot images and scatterplot with mean ± SEM of BK Ca channel α subunit expression ( n = 4 control samples, empty circles ; n = 4 ALDO-treated samples, red circles ), and β1 subunit expression ( n = 5 control samples, empty triangles ; n = 5 ALDO-treated samples, red triangles ). Each sample was prepared with a pool of 3–4 MA segments from three rats. Values were normalized with respect to GAPDH expression.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Expressing, Control, Western Blot

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Enhanced acetylcholine-mediated vasorelaxation of ALDO-treated MAs. Relaxation responses of MAs treated (ALDO, red circles ) or not (CONTROL, empty circles ) with aldosterone (10 nM, 24 h) to increasing concentrations of acetylcholine (ACh, from 0.001 to 1 μM). MA rings were pre-contracted with KCl 60 mM and exposed to cumulative ACh concentrations ( n = 6 MA rings/ N = 6 rats, for each experimental condition). Relaxation is shown as a percentage with respect to the contraction response produced by 60 mM KCl, which was taken as 100%. Data are shown as mean ± SEM. * P < 0.05 vs. non-linear data fit of control arteries.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Control, Produced

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Proposed model for ALDO-mediated upregulation of the functional unit that controls Ca 2+ dynamics at the SR-PM nanodomain of MASMCs. In normal conditions (CONTROL, left ) K + -mediated membrane depolarization induces a Ca 2+ influx via Ca v 1.2 (LTCCs). This Ca 2+ entry is buffered by the SERCA pump toward the luminal SR Ca 2+ reservoirs activating RyRs (Ca 2+ sparks) and BK Ca channels (STOCs). Ca v 1.2, SERCA pump, RyRs and BK Ca channels work as a functional unit in the PM-SR nanodomain regulating [Ca 2+ ] cyt , luminal SR Ca 2+ levels, and opposing vasoconstriction. The treatment of MAs with aldosterone (ALDO, right ) increases Ca v 1.2 protein expression and induces higher Ca 2+ entry in MASMCs. However, the depolarization-induced vascular contraction was not enhanced because of the upregulation of this functional unit, which involves increased expression and activity of SERCA pump controlling abnormal Ca 2+ influx at the PM-SR nanodomain, increasing SR Ca 2+ content, Ca 2+ spark and STOC frequencies, opposing to depolarization-induced vasoconstriction and enhancing ACh-mediated vasorelaxation.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Functional Assay, Control, Membrane, Expressing, Activity Assay