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: The primer sequences used, obtained from Applied Biosystems/Thermo Fisher Scientific, were Rn01545085_m1 (Ryr1), Rn01470303_m1 (Ryr2), Rn01486097_m1 (Ryr3), and Rn01775763_g1 (GAPDH).

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: 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 < 0.05) using Tukey’s multiple comparison test after an initial one-way repeated measures ANOVA. N = 6 isolated lymphatics studied from 6 different rats. CF, contraction frequency; EDD, end-diastolic diameter; EF, ejection fraction; ESD, end-systolic diameter; FPF, fractional pump flow; MaxD, maximal passive diameter; NS, not significant.

Article Snippet: The primer sequences used, obtained from Applied Biosystems/Thermo Fisher Scientific, were Rn01545085_m1 (Ryr1), Rn01470303_m1 (Ryr2), Rn01486097_m1 (Ryr3), and Rn01775763_g1 (GAPDH).

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: The primer sequences used, obtained from Applied Biosystems/Thermo Fisher Scientific, were Rn01545085_m1 (Ryr1), Rn01470303_m1 (Ryr2), Rn01486097_m1 (Ryr3), and Rn01775763_g1 (GAPDH).

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: The primer sequences used, obtained from Applied Biosystems/Thermo Fisher Scientific, were Rn01545085_m1 (Ryr1), Rn01470303_m1 (Ryr2), Rn01486097_m1 (Ryr3), and Rn01775763_g1 (GAPDH).

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

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Aging alters spontaneous and neurotransmitter-mediated Ca 2+ signaling in smooth muscle cells of mouse mesenteric arteries

doi: 10.1111/micc.12607

Figure Lengend Snippet: Transcript levels for ryanodine receptor isoforms in mesenteric artery smooth muscle cells from young and old mice. Data are from 22 with permission.

Article Snippet: Thus prepared, slides were incubated 60 min in primary antibody [Alamone Labs, 1:250] for RyR1 (Cat. #ARR-001), RyR2 (Cat. #ARR-002), or RyR3 (Cat. #ARR-003).

Techniques:

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Aging alters spontaneous and neurotransmitter-mediated Ca 2+ signaling in smooth muscle cells of mouse mesenteric arteries

doi: 10.1111/micc.12607

Figure Lengend Snippet: Representative immunofluorescence images depicting green fluorescence for RyR1 (top rows), RyR2 (center rows) and RyR3 (bottom rows) in 3 separate SMCs from MAs of (A) young and (B) old mice, (C) SMCs incubated with respective blocking peptides, and (D) SMC with primary omitted. ToPro nuclear stain (blue) is included in all images. Scale bars = 20 μm and apply to all panels.

Article Snippet: Thus prepared, slides were incubated 60 min in primary antibody [Alamone Labs, 1:250] for RyR1 (Cat. #ARR-001), RyR2 (Cat. #ARR-002), or RyR3 (Cat. #ARR-003).

Techniques: Immunofluorescence, Fluorescence, Incubation, Blocking Assay, Staining

Journal: Nature Communications

Article Title: Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

doi: 10.1038/s41467-020-20448-x

Figure Lengend Snippet: a qPCR shows the expression of Ryr3 (Cluster 1), Apold1 (Cluster 2), and Col1a1 (Cluster 3) in sinoatrial node (SAN), atrial, and ventricular tissue, respectively ( n = 3 biologically independent animals per group). Dunnett’s multiple comparisons test, data are represented as mean ± s.e.m., adjusted p value was labeled on the top. RA right atrium, LA left atrium, RV right ventricle, LV, left ventricle. b – d Immunostaining shows the location of RYR3 ( b ), APOLD1 ( c ), and COL1A1 ( d ) in mouse SAN tissue, respectively. Representative images are shown from n = 3 biologically independent samples. Scale bar = 50 μm. Zoom images (the box regions in the merged images) show the higher magnification. Scale bar = 10 μm.

Article Snippet: Primary antibodies included HCN4 (Sigma, SAB5200035, 1:50), Connexin 43 (CST, 3512, 1:50), VSNL1 (Gene Tex, GTX115039, 1:50), Collagen I (Abcam, ab21286, 1:50), DLGAP1 (Affbiotech, AF0308, 1:50), UNC80 (BIOSS, BS-12121R, 1:50), APOLD1 (Novus Biologicals, NBP2-58460, 1:50), RYR3 (Novus Biologicals, NBP2-76962, 1:50), Connexin 40 (Invitrogen, 37-8900, 1:50), cTNT (Abcam, ab8295, 1:50).

Techniques: Expressing, Labeling, Immunostaining

Journal: The International Journal of Neuropsychopharmacology

Article Title: The absence of P2X7 receptors (P2rx7) on non-haematopoietic cells leads to selective alteration in mood-related behaviour with dysregulated gene expression and stress reactivity in mice

doi: 10.1017/S1461145711001933

Figure Lengend Snippet: Genes that were up- and down-regulated by the deficiency of P2rx7 in the mouse amygdala

Article Snippet: Ryr3 , 20 192 , Ryanodine receptor 3 (BC116740) , 2.80 , 1.79 ± 0.22 p < 0.0002 , Mm01335482_m1.

Techniques: Real-time Polymerase Chain Reaction, Derivative Assay, Membrane, Variant Assay

Journal: bioRxiv

Article Title: Multiscale activity imaging in mammary gland reveals how oxytocin enables lactation

doi: 10.1101/657510

Figure Lengend Snippet: ( A ) Ryr1 , Ryr2 and Ryr3 expression in lysates prepared from whole mammary tissue (including luminal, basal and stromal cells) dissected from virgin or lactating animals (n = 4 mice). ( B ) Krt14 , Esr1 and Ryr1 levels in freshly sorted luminal and basal cells (n = 3 mice). Graphs show mean ± SEM; * P < 0.05 (Student’s t-test); n.d., not detected. Ryr2 and Ryr3 transcripts were either not detected or detected at very low levels in only a fraction of samples from both luminal and basal cells.

Article Snippet: The following Gene Expression Assays were used in this study: Ryr1 (Mm01175211_m1), Ryr2 (Mm00465877_m1), Ryr3 (Mm01328395_m1), Krt14 (Mm00516876_m1) and Esr1 (Mm00433149_m1).

Techniques: Expressing

Journal: Cardiovascular engineering and technology

Article Title: The ryanodine receptor contributes to the lysophosphatidylcholine-induced mineralization in valvular interstitial cells.

doi: 10.1007/s13239-020-00463-1

Figure Lengend Snippet: TaqMan Probe Accession Information

Article Snippet: RyR3 , Ss03374540_m1.

Techniques:

Journal: Cardiovascular engineering and technology

Article Title: The ryanodine receptor contributes to the lysophosphatidylcholine-induced mineralization in valvular interstitial cells.

doi: 10.1007/s13239-020-00463-1

Figure Lengend Snippet: (A) Immunocytochemistry of paVICs stained for the RyR (left) and no primary antibody control (right); scale = 100 μm. The protein is localized to the cytoplasm and is enriched in perinuclear vesicles that likely represent the endoplasmic reticulum. (B) qRT-PCR results mRNA expression of the three RyR isoforms by paVICs. Expression level is shown relative to GAPDH, and the grey dashed line represents the expression level of RyR1 in porcine skeletal muscle (positive control). Two biological replicates were used for all experiments; 3 technical replicates were used for IF staining, and 4–10 technical replicates were used for qRT-PCR.

Article Snippet: RyR3 , Ss03374540_m1.

Techniques: Immunocytochemistry, Staining, Control, Quantitative RT-PCR, Expressing, Positive Control