Journal: Scientific Reports

Article Title: T-type, but not L-type, voltage-gated calcium channels are dispensable for lymphatic pacemaking and spontaneous contractions

doi: 10.1038/s41598-019-56953-3

Figure Lengend Snippet: PCR to detect VGCC isoforms in mouse LMCs isolated from popliteal ( A ) or IALS ( B ). LMCs were collected by micropipette aspiration after isolation, cleaning and digestion of the respective vessels. Ca v 3.1 and Ca v 3.2, but not Ca v 3.3 isoforms are detected in LMCs from both vessels. Ca v 1.2 shows a strong band in both samples. eNOS is detected in cells collected from PLs ( A ) indicating that there is some contamination by endothelial cells (see Suppl. Fig. for additional information). M = marker; bp = base pairs.

Article Snippet: The vessels were incubated in α-actin primary antibody (Sigma, A2547) (1:200) and one of the primary antibodies for VGCCs (Alomone, Jerusalem, Israel): Ca v 3.1 (#ACC-021), Ca v 3.2 (#ACC-025) (1:100), Ca v 1.2 (#ACC-003) diluted in PBS containing 5% donkey serum (017-000-121, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and 0.1% Triton-X (AC2156825000, Thermo Fisher Scientific).

Techniques: Isolation, Marker

Journal: Scientific Reports

Article Title: T-type, but not L-type, voltage-gated calcium channels are dispensable for lymphatic pacemaking and spontaneous contractions

doi: 10.1038/s41598-019-56953-3

Figure Lengend Snippet: Effects of SM-specific deletion of Ca v 1.2 on popliteal lymphatic contractile function. ( A ) Contractile patterns in a PL from a Ca v 1.2 floxed mouse (without Cre) after tamoxifen administration 12 days prior to the experiment. Contraction patterns are essentially identical to those of PLs from WT mice (See Fig. ). ( B ) Complete absence of large-amplitude, propulsive contractions in a PL from a SM-specific Ca v 1.2 KO mouse. The vessel exhibits only very small amplitude vasomotion, which is mostly independent of the pressure level. ( C ) Expanded y-axis scale showing the vasomotion pattern in another PL from a SM-specific Ca v 1.2 KO mouse. ( D–G ) Summary of contractile parameters for Ca v 1.2 fl/fl (control) and SMMHC-Cre ERT2 ; Ca v 1.2 fl/fl mice studied 10–12 days after tamoxifen administration. With one exception all parameters at all pressures were significantly different between the two groups. The exception was tone at 0.5 cmH 2 O. Note that tone in SM-specific Ca v 1.2 KO vessels is ~3-fold higher than in the corresponding Ca v 1.2 floxed controls at every pressure except 0.5 cmH 2 O.

Article Snippet: The vessels were incubated in α-actin primary antibody (Sigma, A2547) (1:200) and one of the primary antibodies for VGCCs (Alomone, Jerusalem, Israel): Ca v 3.1 (#ACC-021), Ca v 3.2 (#ACC-025) (1:100), Ca v 1.2 (#ACC-003) diluted in PBS containing 5% donkey serum (017-000-121, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and 0.1% Triton-X (AC2156825000, Thermo Fisher Scientific).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: Distribution of store operated calcium channels in Cav +/+ and Cav∼ ∼ tissues. At left are micrographs of Cav +/+ and at right of Cav∼ ∼ tissues, cut with the LM in cross section. The top row of sections shows staining for Cav-1 and the second row shows staining for Orai1 in store operated channels. The third row shows the merged images. Note that Cav-1 Orai 2 stainings in Cav +/+ tissues are punctate at the cell membrane and are well co-localized. In Cav∼ ∼ tissues Cav-1 is absent and Orai 2 staining appears less clearly punctate. Length bars are 10 μM.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) Effects on contraction frequencies of LM segments from two successive exposures of control (Cav +/+ ) and Cav-1 knockout (Cav −/− ) to Ca 2+ Krebs solution with 0.1 mM EGTA. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. When there were significant differences between Cav +/+ and Cav −/− segments the bars for the Cav +/+ tissues are red. ( B ) Effect on contraction amplitudes in the experiments shown in Fig. 1A, with data from the final responses to 10 −5 M CCH. . Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. When there were significant differences between Cav +/+ and Cav −/− segments in spontaneous contraction amplitudes the bars for the Cav +/+ tissues are red but difference in amplitudes of contractions to CCH are in red for Cav −/− tissues. Responses to CCH after exposure to 0.1 mM EGTA are shown by perpendicular hatching and responses after exposure to 1 mM EGTA are shown with diagonal hatching.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Knock-Out

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) Effects on contraction frequencies of LM segments from two successive exposures of control (Cav +/+ ) and Cav-1 knockout (Cav −/− ) to Ca 2+ Krebs solution with 0.1 mM EGTA. In these experiments, either BayK 8644 or nicardipine (both at 10 −6 M) was added with the Ca 2+ -free medium. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. The significantly higher frequencies in Cav −/− tissues are indicated by red bars. ( B ) Effect on contraction amplitudes in the experiments shown in (A) , with data from the final responses to 10 −5 M CCH. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. When there were significant differences between Cav +/+ and Cav −/− segments in spontaneous contraction amplitudes after block of L-type Ca 2+ channels the bars are in red for Cav +/+ tissues.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Knock-Out, Blocking Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) and ( B ) Effects on contraction frequencies ( A ) and amplitudes ( B ) of LM segments, during two successive exposures of control Effects on contraction frequencies ( A ) and amplitudes ( B ) of LM segments, during two successive exposures of control (Cav +/+ ) and Cav-1 knockout (Cav −/− ) to Ca 2+ Krebs solution with 0.1 mM EGTA. Some tissues had received an exposure to 10 μM CCH just prior to the first wash in Ca 2+ solution. There were no significant differences between Cav +/+ and Cav −/− tissues.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Knock-Out

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) and ( B ) Responses to 10 μM CCH of Cav +/+ and Cav −/− tissues exposed to CCH in normal Krebs solution ( A ) and in Ca 2+ -free solution ( B ). Phasic responses were measured at the maximum and tonic responses were measured 2 min. after the addition of CCH. In medium with normal Ca 2+ concentrations there were no significant differences in responses to CCH, but in Ca 2+ -free medium responses of tissues without caveolae were all significantly greater.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) Effects on contraction frequencies of LM segments, during three successive exposures of control (Cav +/+ ) (top panel) and Cav-1 knockout (Cav −/− ) (bottom panel) tissues to Ca 2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca 2+ -free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼ 6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca 2+ -free medium were higher in Cav +/+ tissues than in Cav −/− tissues, only those measured during the third wash with BayK 8644 and the second and third washes with nicardipine were significantly so, indicated by red bars. ( B ) Effects on contraction amplitudes of LM segments, during three successive exposures of control (Cav +/+ ) (top panel) and Cav-1 knockout (Cav −/− ) (bottom panel) tissues to Ca 2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca 2+ -free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼ 6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca 2+ -free medium were higher in Cav +/+ tissues than in Cav −/− tissues, only those measured during the second and third washes with nicardipine were significantly so, indicated by red bars.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Knock-Out

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) This cartoon represents a possible distribution if Ca 2+ handling proteins related to caveolae and Cav-1 based on our findings. The peripheral ER/SR is shown near a caveola, with a restricted space in between them, containing a higher Ca 2+ concentration than in the general cytosol. Sequestered Ca 2+ is also shown associated with the caveolar extracellular membrane and adjacent raft tissue. This is the Ca 2+ which is protected from the Ca 2+ -free medium we used and provides ions for recycling to the restricted space and the ER/SR (and in Cav-1 knockout tissue provides some Ca 2+ for recycling). Owing to space limitations this recycling Ca 2+ is depicted adjacent to the restricted space. Proteins apparently co-localized with Cav-1 including VDCC, PMCA4, myogenic nNOS, and store operated Ca 2+ Channels are shown associated with the caveola membrane. Under the control conditions in Ca 2+ -free medium, the VDCC were the main entry route for Ca 2+ to be recycled, but store operated channels contribute when the VDCC are blocked. The SERCA pump and Leak or Release Sites in SR are also depicted. The Ca 2+ stored in the ER/SR is the other main source of Ca 2+ to maintain recycling and when it is emptied, recycling stops quickly. ( B ) This cartoon depicts the proposed changes in the Cav-1 knockout tissues. The restricted space is lost as well as the higher local Ca 2+ concentration which promotes its recycling to the ER/SR. VDCC, PMCA and store operated Ca 2+ Channels persist, but the myogenic nNOS is missing. The proximity of the remaining Ca 2+ molecules to the ER/SR is lost. There is also less sequestered Ca 2+ available for recycling. The result is more rapid loss of pacing in Ca 2+ -free medium. There is also less rapid clearing of Ca 2+ released or leaked from ER/SR by the PMCA and other clearance mechanisms. The main route for Ca 2+ entry remains the VDCC with a possible contribution from the store operated channel.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Concentration Assay, Knock-Out

Journal: Cells

Article Title: Sympathetic Stimulation Upregulates the Ca 2+ Channel Subunit, Ca V α2δ1, via the β1 and ERK 1/2 Pathway in Neonatal Ventricular Cardiomyocytes

doi: 10.3390/cells11020188

Figure Lengend Snippet: Expression of Ca V α2δ1 ( A ), Ca V α1C ( C ), Ca V β3 ( E ), and Ca V β2 ( G ) in neonatal rat cardiomyocytes. Total proteins were extracted from neonatal rat ventricular cardiomyocytes. Proteins were separated on an 8% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti-Ca V α2δ1, anti-Ca V α1C, anti-Ca V β3, anti-Ca V β2, and anti-GAPDH antibodies overnight and then incubated with HRP-conjugated goat anti-rabbit secondary antibody. Lanes were loaded with 30 μg of proteins. NE: cardiomyocytes treated with 1 µM norepinephrine. Graph showing the total protein expression of Ca V α2δ1 ( B ), Ca V α1C ( D ), Ca V β3 ( F ), and Ca V β2 ( H ) normalized to GAPDH. * p < 0.01 vs. Basal. Statistical analysis was performed using one-way ANOVA.

Article Snippet: Primary antibodies employed include mouse anti-Troponin-T (1:200; Abcam, Cambridge, UK; ab8295), rabbit anti-Ca V α2δ1 (1:200, Alomone, Jerusalem, Israel, ACC-015), and rabbit anti-Ca V α1C (1:200, Alomone, ACC-003).

Techniques: Expressing, Membrane, Incubation

Journal: Cells

Article Title: Sympathetic Stimulation Upregulates the Ca 2+ Channel Subunit, Ca V α2δ1, via the β1 and ERK 1/2 Pathway in Neonatal Ventricular Cardiomyocytes

doi: 10.3390/cells11020188

Figure Lengend Snippet: Neonatal rat cardiomyocytes in basal condition or treated with NE for 24–48 h. WGA-Alexa 647 was added to live cardiomyocytes at room temperature, followed by fixation with 2% Paraformaldehyde for 20 min at room temperature. Cardiomyocytes were then permeabilized with 0.2% Triton-X. Primary antibody: mouse anti-Troponin-T and rabbit anti-Ca V α2δ1 were added for 90 min at room temperature followed by overnight incubation at 4 °C. This was followed by incubation of secondary antibodies: Goat anti-mouse Alexa 555; Donkey anti-rabbit Alexa 488, and DAPI for 90 min at room temperature in the dark. Images were captured by microscope with 20× objective. Images were analyzed using ZEN software. ( A ) Images showing colocalization of WGA and Ca V α2δ1. Green: Ca V α2δ1; Red: WGA; Yellow: Overlap between Ca V α2δ1 and WGA. ( B ) Images showing colocalization of DAPI and Ca V α2δ1. Green: Ca V α2δ1; Red: DAPI; Yellow: Overlap between Ca V α2δ1 and WGA. ( C ) Graph showing Pearson’s correlation coefficient of the overlap between WGA and Ca V α2δ1. ( D ) Graph showing Pearson’s correlation coefficient of the overlap between DAPI and Ca V α2δ1. Pearson’s correlation coefficient (r) of the overlap is determined using ImageJ.

Article Snippet: Primary antibodies employed include mouse anti-Troponin-T (1:200; Abcam, Cambridge, UK; ab8295), rabbit anti-Ca V α2δ1 (1:200, Alomone, Jerusalem, Israel, ACC-015), and rabbit anti-Ca V α1C (1:200, Alomone, ACC-003).

Techniques: Incubation, Microscopy, Software

Journal: Cells

Article Title: Sympathetic Stimulation Upregulates the Ca 2+ Channel Subunit, Ca V α2δ1, via the β1 and ERK 1/2 Pathway in Neonatal Ventricular Cardiomyocytes

doi: 10.3390/cells11020188

Figure Lengend Snippet: Expression of Ca V α2δ1 ( A ) and Ca V β3 ( C ) in neonatal rat cardiomyocytes. Total proteins were extracted from neonatal rat ventricular cardiomyocytes. Cells were treated with 1 µM Norepinephrine (NE) for 24 h or pre-treated with metoprolol tartrate (β1-blocker) for 1 h followed by treatment with NE for 24 h. Proteins were separated on an 8% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti-Ca V α2δ1, anti-Ca V β3, and anti-GAPDH antibodies overnight and then incubated with HRP-conjugated goat anti-rabbit secondary antibody. Lanes were loaded with 30 μg of proteins. NE: cardiomyocytes treated with 1 µM norepinephrine. 24 h + metoprolol tartrate: Cardiomyocytes pre-treated with metoprolol tartrate (β1-blocker) for 1 h, followed by treatment with NE for 24 h. ( B ) Graph showing the total protein expression of Ca V α2δ1 normalized to GAPDH. * p < 0.01 vs. Basal untreated NRVMs; † p <0.01 vs. NE 24 h. (4 cardio-preparations). (D) Graph showing the total protein expression of Ca V β3 normalized to GAPDH. * p < 0.01 vs. Basal untreated NRVMs (4 cardio-preparations). Statistical analysis was performed using one-way ANOVA.

Article Snippet: Primary antibodies employed include mouse anti-Troponin-T (1:200; Abcam, Cambridge, UK; ab8295), rabbit anti-Ca V α2δ1 (1:200, Alomone, Jerusalem, Israel, ACC-015), and rabbit anti-Ca V α1C (1:200, Alomone, ACC-003).

Techniques: Expressing, Membrane, Incubation