Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels

doi: 10.1152/ajpheart.00564.2018

Figure Lengend Snippet: The ryanodine receptor (RyR)1/RyR3 inhibitor dantrolene does not inhibit rat mesenteric collecting lymphatic contractions or the increased CF and decreased EDD/Max elicited by substance P (SP). Representative trace of diameter over time of an isolated rat mesenteric collecting lymphatic vessel before and after the addition of 10 −5 M dantrolene, which remained in the bath ( A ). Continuation of the same trace, showing the addition of 10 −8 M SP in the presence of dantrolene ( B ). The boxes beneath the traces in A and B indicate 2-min time periods during which the quantitative data used for comparisons in C – H were collected. Comparisons between baseline (BL), after addition of dantrolene, and after addition of SP in the presence of dantrolene were performed for CF ( C ), EDD/MaxD ( D ), ESD/MaxD ( E ), AMP/MaxD ( F ), EF ( G ), and FPF ( H ). P values are shown for comparisons that were found to be significantly different ( P

Article Snippet: Primers for all three isoforms amplified cDNA products of the predicted sizes for the amplicons for RyR1, RyR2, and RyR3 described by the manufacturer (Applied Biosystems) ( ).

Techniques: Isolation

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels

doi: 10.1152/ajpheart.00564.2018

Figure Lengend Snippet: Blockade of ryanodine receptors (RyRs) with ryanodine leads to a loss of phasic contractions and limited substance P (SP)-induced contraction. Representative trace of diameter over time of an isolated rat collecting mesenteric lymphatic vessel treated with 10 −5 M ryanodine ( A ). The continuation of the trace shows a later time period in the same experiment with ryanodine, when 10 −8 M SP was added. The boxes beneath the trace represent the time periods during which the quantitative data used for comparisons in B – D were collected. The mean diameter ( B ), mean tone ( C ), and maximum tone ( D ) for the time periods before addition of SP, 0–30 s after SP addition, and 31–150 s after addition of SP were compared using a repeated measures ANOVA model followed by Dunnett’s comparison with control (baseline period). P values for the comparisons are shown. N = 6 lymphatics. Each lymphatic studied was obtained from a unique rat. NS, not significant.

Article Snippet: Primers for all three isoforms amplified cDNA products of the predicted sizes for the amplicons for RyR1, RyR2, and RyR3 described by the manufacturer (Applied Biosystems) ( ).

Techniques: Isolation

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels

doi: 10.1152/ajpheart.00564.2018

Figure Lengend Snippet: Quantitative PCR (qPCR) detection of ryanodine receptors (RyRs) in rat mesenteric collecting lymphatic vessels. A : means show relative expression to GAPDH determined by qPCR and using the 2 -ΔCt method in isolated rat mesenteric collecting lymphatics ( N = 7 rats) and rat brain, skeletal muscle, and cardiac muscle (all N = 2 rats). B : gel of the single amplicon products of expected size from the qPCR reactions. Rat mesenteric collecting lymphatics were harvested (30 lymphatic vessels per rat), and total RNA (50 ng) was reverse transcribed into cDNA and qPCR performed with specific primer/probe sets for Ryr1 (Rn01545085_m1), Ryr2 (Rn01470303_m1), Ryr3 (Rn01486097_m1), and GAPDH (Rn01775763_g1). Reactions with no template were also run as controls (NTC). The gel shown is representative of three separate experiments using mesenteric lymphatic samples obtained from three different rats.

Article Snippet: Primers for all three isoforms amplified cDNA products of the predicted sizes for the amplicons for RyR1, RyR2, and RyR3 described by the manufacturer (Applied Biosystems) ( ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Amplification

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels

doi: 10.1152/ajpheart.00564.2018

Figure Lengend Snippet: Determination of ryanodine receptor (RyR)2 and RyR3 localization in isolated rat mesenteric collecting lymphatic vessels with immunofluorescence labeling and laser confocal microscopy. A . maximum intensity z-projection (37 confocal slices) of a lymphatic vessel labeled with anti-RyR2 AlexaFluor488-conjugated secondary antibodies and DAPI. The RyR2 labeling appeared mostly in hoop-like patterns in the vessel wall, with some labeling in the adventitial layer (1 example denoted by the small white arrow). B : maximum intensity z-projection (85 confocal slices) of a lymphatic immunolabeled for RyR3 and nuclei. A similar hoop-like pattern was observed with RyR3 immunolabeling, with some adventitial layer labeling (small white arrow). No signal was detected in labeling controls with no primary antibody (data not shown). C and E : labeling of RyR2, smooth muscle actin (SMA), nuclei, and a merge of the three channels in a single confocal slice featuring a cross-section of the collecting lymphatic wall. The orientation is with the adventitial layer on the left and the endothelial layer on the right. RyR2 labeling was predominantly found in the smooth muscle layer, as evidenced by partial colocalization with SMA and RyR2 labeling surrounding the nuclei of smooth muscle cells. D and F : labeling of RyR3, SMA, nuclei, and a merge of the three channels in a single confocal slice featuring a cross-section of the lymphatic wall, oriented with the adventitial layer on the left and endothelium on the right. RyR3 was predominantly found in smooth muscle cells, as evidenced by the partial colocalization with SMA. The small arrows in E and F point toward nuclei of endothelial cells (ECs). A and B : representative of N = 8 labeling experiments each. C – F : representative of N = 4 experiments each. The scale bars in C – F represent 5 µm.

Article Snippet: Primers for all three isoforms amplified cDNA products of the predicted sizes for the amplicons for RyR1, RyR2, and RyR3 described by the manufacturer (Applied Biosystems) ( ).

Techniques: Isolation, Immunofluorescence, Labeling, Confocal Microscopy, Immunolabeling

Journal: Molecular neurobiology

Article Title: Control of neuronal ryanodine receptor-mediated calcium signaling by calsenilin

doi: 10.1007/s12035-018-1080-2

Figure Lengend Snippet: Calsenilin and RyR are co-localized and have a direct protein-protein interaction in neuronal tissue Calsenilin (green) and A) RyR2 or RyR3 (red) immunoreactivity in E18 primary cultured cortical neurons B) RyR2 (red) expression in the dentate gyrus and cortical layer VI show a perinuclear staining pattern in regions adjacent to the nucleus indicative of ER staining (Merge) and a high degree of punctate co-localization in the perinuclear region (+PDM) as indicated by arrows and LUT in the last panel. C) Co-localization measurements between calsenilin and RyR2 or RyR3 for neuronal cell types (E18 primary cortical neurons and SH-SY5Y neuroblastoma cells) and 6-week-old C57BL/6 mouse brain sections (dentate gyrus, CA3, CA1, cortical layers II/III, V, VI). The Rr = Pearson’s coefficient and M1 = Mander’s coefficient describe the amount of calsenilin co-localized with the two RyR subtypes, whereas the M2 = Mander’s coefficient describes the amount of RyR2 or RyR3 co-localized with calsenilin. Statistical significance represents the SEM for each replicate; co-localization was tested with the Costes method to ensure true co-localization. D) Western blot shows the presence of calsenilin in the brain-derived ER microsomes incubated with calsenilin protein and subjected to co-immunoprecipitation separation using the RyR2 antibody; the IgG control is shown in lane “IgG”, and molecular weight standards are shown in lane “PSS”. Abbreviations: RyR, ryanodine receptor; DG, Dentate Gyrus; CA1 or CA3, Cornu Ammonis 1 or 3; Ctx, Cortex layer; IgG, Immunoglobulin G; MW, molecular weight; PSS, pre-stained standard.

Article Snippet: After blocking, the cells were incubated with the primary antibodies: DREAM (1:50; sc-9309, Santa Cruz Biotechnologies, Dallas, TX), RyR2 (1:10,000; AB9080, Chemicon Biotechnologies, Temecula, CA), RyR3 (AB9082 (1:5,000; Chemicon Biotechnologies) in incubation buffer (3% normal donkey serum, 1% BSA and 0.05% Triton-X 100 in 0.01 M PBS) overnight at 4 °C.

Techniques: Cell Culture, Expressing, Staining, Western Blot, Derivative Assay, Incubation, Immunoprecipitation, Molecular Weight

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Estrogens Protect Calsequestrin-1 Knockout Mice from Lethal Hyperthermic Episodes by Reducing Oxidative Stress in Muscle

doi: 10.1155/2017/6936897

Figure Lengend Snippet: Assessment of muscle damage and blood levels of CK K + , and Ca 2+ following exposure to heat stress protocol. (a–h) Histological (a–d) and immunofluorescence of EDL fibers labeled with anti-RYR1 antibody (e–h) examination of EDL muscles after exposure to the heat stress protocol in male and female CASQ1-null mice, either untreated or treated with Premarin (males) and leuprolide (females). (i) Quantitative analysis of EDL fibers presenting structural damage and contractures. See also Table S4. (j–l) Blood levels of CK in serum (j), K + , and Ca 2+ in plasma (k and l) following heat stress protocol. Data are given as mean ± SEM; ∗ p

Article Snippet: Briefly, samples were first exposed to a mouse monoclonal primary antibody which recognizes both RYR1 and RYR3 (34C, 1 : 20; Developmental Studies Hybridoma Bank, University of Iowa) and then to a Cy3 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: Immunofluorescence, Labeling, Mouse Assay