Journal: Molecular Pain

Article Title: T-type Ca 2+ channels play a dual role in modulating the excitability of dorsal root ganglia neurons

doi: 10.1177/17448069221132224

Figure Lengend Snippet: T-type Ca 2+ currents of DRG neurons. (a) The distribution of T-type Ca 2+ currents in two groups of DRG neurons (upper panel). T-type Ca 2+ currents recorded in response to step voltage (left panel) and ramp voltage clamps (right panel). LVA: low voltage activated; HVA: high voltage activated. (b) Large (≥10 pA/pF) (left panel) and small (<10 pA/pF) (right panel) T-type Ca 2+ currents recorded at −40 mV command in step voltage clamp were compared between the sham and CCD groups, respectively. (c) Cav3.2 protein expression level of DRG increased in CCD rats. (d) Cav3.3 protein expression level of DRG increased in CCD rats.

Article Snippet: After blocking with 3% fat-free dried milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.6) at room temperature for 1 h, PVDF membranes were incubated with mouse anti-Cav3.2 (1:1000, NBP1-22,444ss, Novusbio, Centennial, USA) or mouse anti-Cav3.3 (1:200, Cat#: ACC-009, Alomone Labs, Israel) and anti-β-actin (1:1000, Abcam, USA) antibodies in 3% fat-free dried milk in TBST overnight at 4°C.

Techniques: Expressing

Journal: Membranes

Article Title: Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice

doi: 10.3390/membranes12060566

Figure Lengend Snippet: Ca currents in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Original traces of Ca currents of a typical wt and Cav3.2 KO myocyte elicited by the pulse protocol displayed on top. ( B ) Current density–voltage relationships of wt (n = 20) and Cav3.2 KO (n = 20) myocytes. Data are expressed as means ± SEM. The current density–voltage relations hardly revealed any T-type Ca channel activity (expected to be maximal at −45 mV) but did reveal significant L-type Ca channel activity (peaking at +5 mV), which was similar in wt and Cav3.2 KO myocytes ( p = 0.67 at +5 mV, unpaired Student’s t -test). Capacitance values of the examined myocytes were 184 ± 13 pF for wt and 169 ± 11 pF for Cav3.2 KO ( p = 0.37). ( C ) The effect of the external application of 100 nM isoprenaline (ISO) on ICaT in a wt myocyte. From a holding potential of −95 mV, depolarizing voltage steps to −45 mV were applied every 3 s to elicit the currents. The arrows indicate the beginning and end of ISO application. Current peaks were finally plotted against time. ( D ) ICaT peaks before, during application after the steady-state was reached, and after the washout of ISO in wt (top) and Cav3.2 KO (bottom) myocytes. There was no significant difference between wt and Cav3.2 KO under control conditions ( p = 0.81, unpaired Student’s t -test) or in the presence of ISO ( p = 0.54). Data are expressed as means ± SEM. Each data point represents a single cell. ** p < 0.01; *** p < 0.001; paired Student’s t -test.

Article Snippet: This was followed by blocking with 10% goat serum (Sigma-Aldrich, Vienna, Austria) and 0.01% azide (Sigma-Aldrich, Vienna, Austria) in PBS for 2 h. Subsequently, the cells were incubated with a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:500) or with one of two selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:1000; anti-Cav3.2 polyclonal antibody, #PA5-106771, source: rabbit, Invitrogen, Paisley, England, 1:200) at 4 °C overnight.

Techniques: Activity Assay

Journal: Membranes

Article Title: Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice

doi: 10.3390/membranes12060566

Figure Lengend Snippet: Immunostaining of T-type Ca channels in ventricular cardiomyocytes isolated from adult wt mice. Cav3.1 ( top ) channel expression and localization was detected using a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, 1:500). Cav3.2 ( bottom ) was detected with the selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, 1:1000; left and right cell; or anti-Cav3.2 polyclonal, #PA5-106771, source: rabbit, Invitrogen, 1:200; middle cell). Secondary antibody: Alexa Fluor 488, #A11008, goat anti-rabbit, Invitrogen, 1:500.

Article Snippet: This was followed by blocking with 10% goat serum (Sigma-Aldrich, Vienna, Austria) and 0.01% azide (Sigma-Aldrich, Vienna, Austria) in PBS for 2 h. Subsequently, the cells were incubated with a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:500) or with one of two selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:1000; anti-Cav3.2 polyclonal antibody, #PA5-106771, source: rabbit, Invitrogen, Paisley, England, 1:200) at 4 °C overnight.

Techniques: Immunostaining, Isolation, Expressing

Journal: Membranes

Article Title: Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice

doi: 10.3390/membranes12060566

Figure Lengend Snippet: Electrically induced and caffeine-induced intracellular Ca transients in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Left: Representative time courses of Fluo-4 fluorescence reporting rises in cytosolic Ca concentration during electrical field stimulation at 0.1 Hz frequency in a single wt and Cav3.2 KO myocyte. The orange lines represent fits of the data with a single exponential function. Right: Comparison of mean Ca peak fluorescence relative to baseline (F/F0) between wt and Cav3.2 KO myocytes. Each data point represents a single cell, and values are expressed as means ± SEM (n= 68 for wt and 59 for Cav3.2 KO myocytes). * p < 0.05 (0.02); unpaired Student’s t -test. The Ca transients decayed with time constants of 0.30 ± 0.02 s in wt and 0.25 ± 0.01 s in Cav3.2 KO myocytes, which were significantly different ( p = 0.04). ( B ) Comparison of mean Ca peak fluorescence (F/F0) before and after the application of 100 nM ISO in wt (n = 20) and Cav3.2 KO (n = 21) myocytes. **** p < 0.0001 (paired Student’s t -test). There was no significant difference between wt and Cav3.2 KO in the presence of ISO ( p = 0.37; unpaired Student’s t -test). The Ca transients under ISO decayed with time constants of 0.14 ± 0.02 s in wt and 0.11 ± 0.02 s in Cav3.2 KO myocytes (not significantly different, p = 0.19). ( C ) Comparison of mean Ca peak fluorescence elicited by an application of 20 mM caffeine, relative to baseline (F/F0), between wt (n = 26) and Cav3.2 KO (n = 35) myocytes. ns—not significant (unpaired Student’s t -test, p = 0.42).

Article Snippet: This was followed by blocking with 10% goat serum (Sigma-Aldrich, Vienna, Austria) and 0.01% azide (Sigma-Aldrich, Vienna, Austria) in PBS for 2 h. Subsequently, the cells were incubated with a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:500) or with one of two selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:1000; anti-Cav3.2 polyclonal antibody, #PA5-106771, source: rabbit, Invitrogen, Paisley, England, 1:200) at 4 °C overnight.

Techniques: Fluorescence, Concentration Assay

Journal: Cells

Article Title: Loganin Ameliorates Painful Diabetic Neuropathy by Modulating Oxidative Stress, Inflammation and Insulin Sensitivity in Streptozotocin-Nicotinamide-Induced Diabetic Rats

doi: 10.3390/cells10102688

Figure Lengend Snippet: Effects of loganin on Ca v 3.2, CGRP expression and glial activation in the spinal dorsal horn in PDN rats. Representative immunofluorescence images: ( A ) Ca V 3.2 (red) with presynaptic markers IB4 (green). Scale bar = 75 µm. ( B ) CGRP (red, neuropeptide) with IB4 (green, labeled afferents terminate in inner lamina II). Scale bar = 100 µm. ( C ) GFAP (red, astrocyte marker) with DAPI (blue, nuclei). Scale bar = 75 µm. ( D ) The inset below shows an enlarged image. ( F ) OX42 (red, microglial marker) with DAPI. Scale bar = 75 µm. ( G ) The inset below shows higher magnification. ( E , H ) mean gray values of GFAP and OX42 staining in spinal dorsal horn ( n = 10 sections from 4 rats, * p < 0.05 vs. CTL group; # p < 0.05 vs. PDN group, ## p < 0.01 vs. PDN group). CTL: control, PDN: painful diabetic neuropathy, GFAP: glial fibrillary acidic protein, CGRP: calcitonin gene-related peptide, IB4: isolectin B4.

Article Snippet: The following items were purchased from the cited commercial sources: streptozotocin (STZ, S0130), nicotinamide (NA, N3376), all-trans-retinoic acid (R2625), N -acetylcysteine (NAC, A9165), 2’,7’-dichlorodihydrofluorescein diacetate (H 2 DCFDA, D6883), TriPure isolation reagents, FITC-conjugated isolectin B4 (L2895), Fluoroshield with DAPI (F6057) and mouse anti-β actin (A5441) from Sigma-Aldrich (St. Louis, MO, USA), glucometer (Rightest, GS700) and glucose test strips from Bionime (San Diego, CA, USA), loganin (19997) and superoxide dismutase activity kits (706002) from Cayman Chemical (Ann Arbor, MI, USA), high-capacity cDNA reverse transcription kits (4368814) from Applied Biosystems (Carlsbad, CA, USA), ChamQ Universal SYBR qPCR Master Mix ( # Q711) from Vazyme (Nanjing, China), rat tumor necrosis factor α (TNF-α, # RTA00) and interleukin-1β (IL-1β, # RLB00) enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN, USA), rat insulin ELISA kits (10-1250-01) from Mercodia AB (Uppsala, Sweden), catalase (E-BC-K031-M) and reduced glutathione (E-BC-K030-M) activity assay kits from Elabscience Biotech (Wuhan, China), Eagle Minimum Essential Medium (EMEM), Ham’s F12 medium, T-PER™ tissue protein extraction reagent, M-PER™ mammalian protein extraction reagent, and Pierce™ BCA protein assay kits from Thermo Fisher Scientific (Waltham, MA, USA), quinazoline (QNZ, S4902) from Selleckchem (Houston, TX, USA), anti-phospho-NF-κB (ser 536 , # 3033), anti-NF-κB ( # 6956), anti-CGRP (ab47027), anti-phospho-JNK (Thr 183 /Tyr 185 , # 4668), and anti-JNK2 ( # 9258) antibodies from Cell Signaling (Danvers, MA, USA), anti-IL-1β (ab9722), anti-phospho-GSK3β (Ser 9 , # 9323), anti-GSK3β ( # 12456), anti-phospho-Akt (ser 473 , # 4060), and anti-Akt ( # 9272) antibodies from Abcam (Cambridge, MA, USA), anti-TNF-α antibody (NBP-19532) from Novus Biologicals (Littleton, CO, USA), anti-insulin receptor substrate-1 (anti-IRS1, AP-0552) from Proteintech (Wuhan, China), anti-phospho-IRS1 (ser307, 17509-1-AP) from ABclonal (Woburn, MA, USA) and anti-Ca V 3.2 antibody (ACC-025) from Alomone Labs (Jerusalem, Israel).

Techniques: Expressing, Activation Assay, Immunofluorescence, Labeling, Marker, Staining