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

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Sequence of primers used for RT-PCR

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Sequencing

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Decreased BK Ca currents of ACC pyramidal neurons from mCCD rats. a , Voltage ranges from − 60 to + 40 mV with 10 mV increments, typical recordings of BK Ca currents in pyramidal neurons from sham (left) and mCCD (right) rats, BK Ca currents were isolated with paxilline (10 μM). b , The I-V relationship curves showed the differences in the ACC pyramidal neurons from mCCD ( n = 12) and sham (n = 12) rats. c , BK Ca current density at + 40 mV from voltage-clamp recordings of ACC pyramidal neurons from mCCD (n = 12) and sham (n = 12) rats. ** p < 0.01 compared with that of sham group

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Isolation

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Upregulation of BK Ca β4 subunit protein and mRNA in the ACC during neuropathic pain associated anxiety-like behavior. a, The schematic diagram of the behavioral and biochemical experiments. b, Quantification of the mRNA levels of BK Ca channel subunits in the ACC between mCCD (n = 7) and sham ( n = 6) rats on postoperative day 7. c, Quantification of the mRNA levels of BK Ca channels in the insular cortex between mCCD (n = 7) and sham (n = 6) rats on postoperative day 7. d, Quantification of the mRNA levels of BK Ca β4 subunit in the ACC between mCCD (n = 6) and sham (n = 6) rats on postsurgical day 3, 7, 14. e, Representative Western blots for BK Ca α and BK Ca β4 subunit in the ACC obtained on postsurgical day 7. f, Quantification of the protein levels of α and β4 subunits in the ACC between mCCD (n = 3) and sham (n = 3) rats on postoperative day 7. * p < 0.05, ** p < 0.01 compared with that of sham group

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Western Blot

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Postsynaptic and presynaptic upregulation of BK Ca β4 subunit protein in the ACC during neuropathic pain associated anxiety-like behavior. a , Representative Western blots for PSD 95, syntaxin 1A, BK Ca β4 and BK Ca α subunits in the non-PSD and PSD membrane fractions of the ACC in sham and mCCD rats on postoperative day 14; b , BK Ca β4 subunit was significantly enhanced in the PSD and non-PSD fractions of the ACC from mCCD (PSD fraction: 164 ± 37%, n = 6, * p < 0.05; non-PSD fraction: 224 ± 32%, n = 6, * p < 0.05) rats on postoperative day 14 compared with those in sham rats (PSD fraction: 100 ± 32%, n = 6; non-PSD fraction: 100 ± 12%, n = 6). BK Ca α subunit showed no significant change in the PSD fraction between sham (100 ± 17%, n = 6) and mCCD (91.4 ± 14%, n = 6, p > 0.05) rats on postoperative day 14

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Western Blot

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Effects of activating BK Ca channels in the ACC on neuropathic pain and anxiety-like behaviors. a, A schematic diagram of microinjection and the behavioral experiment. b, Bilateral microinjection of NS1619 (10 μM, 0.5 μL) into the ACC reversed the time in the open arms and the open arm entries in mCCD rats on day 7 after surgery. c, Rats infused with NS1619 spent more time in the central areas in open field test in mCCD rats compared with ACSF-treated animals. There was no significant difference in the total distance travelled within the open field for 15 min among mCCD and sham rats (sham + ACSF: n = 6, sham +NS1619: n = 6, mCCD + ACSF: n = 6, mCCD +NS1619: n = 6). d, The pain threshold of the injured hind paw (left) and contralateral feet (right), after bilateral microinjection of NS1619 into the ACC on day 7 after surgery. # p < 0.05, ## p < 0.01 compared with that of mCCD with ACSF group

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: