Journal: PLoS Genetics

Article Title: Absence of the ER Cation Channel TMEM38B /TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

doi: 10.1371/journal.pgen.1006156

Figure Lengend Snippet: Secreted proteins such as collagen are synthesized within the ER, which contains the major intracellular Ca 2+ store. In response to extracellular stimuli, Ca 2+ is released from the ER lumen into the cytoplasm via IP 3 and ryanodine receptors. Cytoplasmic Ca 2+ that does not bind signaling proteins involved in gene expression is transported back into the ER via SERCA pumps. The continuous fluctuations in free [Ca 2+ ] to and from the ER are indirectly regulated by TRIC channel activity, which mediates transmembrane K + flux to maintain electroneutrality across the ER membrane. In fibroblasts and osteoblasts, ER-resident Ca 2+ -binding chaperones, including BiP, CyPB, PDI and calreticulin (CRT), directly interact with collagen alpha chains (red polypeptides) to facilitate or catalyze specific modifications required for assembly and folding. In the absence of TRIC-mediated K + flux, Ca2 + -dependent “cycling” of these chaperones between inactive, sequestered forms and active, substrate-interacting forms, is dysregulated. Thus, absence of TRIC-B alters ER luminal [Ca 2+ ], affecting synthesis and secretion of collagen, as well as interactions of collagen-specific chaperones with each other and with their substrate, resulting in abnormal collagen assembly and post-translational modification. BiP, GRP78; CyPB, cyclophilin B; CRT, calreticulin; IP3R, inositol triphosphate receptor; LH1, lysyl hydroxylase 1; PDI, protein disulfide isomerase; SERCA2b, sarcoplasmic-endoplasmic reticulum Ca 2+ -ATPase isoform 2b; TRIC-B, trimeric intracellular cation channel subtype B.

Article Snippet: Gene transcript levels were quantitated by real-time RT-PCR following reverse-transcription using a High Capacity cDNA Archive Kit and Taqman Assays on Demand (Life Technologies, TMEM38A , Hs00225325_m1; TMEM38B , Hs00216531_m1; COL1A1 , Hs00164004_m1; FKBP10 , Hs01000263_m1; PLOD1 , Hs00609368_m1; PLOD2 , Hs01118190_m1; PLOD3 , Hs01126617_m1; P4HB/PDI , Hs00168586_m1; HSPA5 , Hs99999174_m1; PPIB , Hs00168719_m1; ATP2A/SERCA2 , Hs00544877_m1; ITPR1 , Hs00181881_m1; RYR1 , Hs00166991_m1; GAPDH , Hs99999905_m1; ACTB , Hs99999903_m1; B2M , Hs99999907_m1).

Techniques: Synthesized, Expressing, Activity Assay, Binding Assay, Modification

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: Anatolian Journal of Cardiology

Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

doi: 10.5152/AnatolJCardiol.2022.1223

Figure Lengend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).

Article Snippet: ID: Mm00465877_m1, Mm01201431_m1, Mm00446971_m1, and Mm01197698_m1).

Techniques: Expressing

Journal: Anatolian Journal of Cardiology

Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

doi: 10.5152/AnatolJCardiol.2022.1223

Figure Lengend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a protein expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a protein expression. n = 6. No statistically significant comparison.

Article Snippet: ID: Mm00465877_m1, Mm01201431_m1, Mm00446971_m1, and Mm01197698_m1).

Techniques: Expressing