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, 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: 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, Expressing, Activity Assay