Orthograde signal of dihydropyridine receptor increases Ca2+ leakage after repeated contractions in rat fast-twitch muscles in vivo

2021 ◽  
Vol 320 (5) ◽  
pp. C806-C821
Author(s):  
Daiki Watanabe ◽  
Masanobu Wada
Keyword(s):  
Sarcoplasmic Reticulum ◽  
High Intensity ◽  
Ryanodine Receptors ◽  
Inhibitory Effect ◽  
Skinned Fibers ◽  
Inhibitory Effects ◽  
Dihydropyridine Receptors ◽  
Gastrocnemius Muscles ◽  
Fast Twitch

The purpose of this study was to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers and SR microsomes were prepared from the muscles excised 30 min after repeated high-intensity contractions. The mechanically skinned fibers maintained the interaction between dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), whereas the SR microsomes did not. Interestingly, skinned fibers from the stimulated muscles showed increased SR Ca2+ leakage, whereas Ca2+ leakage decreased in SR microsomes from the stimulated muscles. To enhance the orthograde signal of DHPRs, SR Ca2+ leakage in the skinned fiber was measured 1) under a continuously depolarized condition and 2) in the presence of nifedipine. As a result, in either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, the increased SR Ca2+ leakage from the stimulated fibers was alleviated by treatment with 1 mM tetracaine (Tet) but not by treatment with 3 mM free Mg2+ (3 Mg). Tet exerted a greater inhibitory effect on the DHPR signal to RyR than 3 Mg, although their inhibitory effects on RyR were almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RyRs.

scholarly journals Effects of dihydropyridine receptor signal on sarcoplasmic reticulum Ca2+ leakage after muscle contractions in rats

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Daiki Watanabe ◽  
Masanobu Wada
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Recovery Phase ◽  
Inhibitory Effect ◽  
Dihydropyridine Receptor ◽  
Muscle Contractions ◽  
Skinned Fibers ◽  
Transverse Tubular System ◽  
Receptor Signal ◽  
Gastrocnemius Muscles

The purpose of this study is to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage at recovery phase after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers were prepared from the muscles excised 30 min after repeated high-intensity contractions. SR Ca2+ leakage was increased in the skinned fibers from stimulated muscles. Thereafter, SR Ca2+ leakage in skinned fibers was measured (1) under a continuously depolarized condition and (2) in the presence of nifedipine in the sealed transverse tubular system. In either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, 1 mM tetracaine (Tet) treatment, but not 3 mM Mg2+ (3 Mg) treatment, lessened SR Ca2+ leakage in stimulated fibers. Depolarization-induced force in skinned fibers was more greatly decreased by Tet treatment than by 3 Mg treatment (92% reduction in Tet versus 31% reduction in 3 Mg), whereas caffeine-induced force in skinned fibers was similarly decreased by either treatment (73% reduction in Tet versus 75% reduction in 3 Mg). This difference indicates that Tet exerts a greater inhibitory effect on the dihydropyridine receptor (DHPR) signal to ryanodine receptor (RYR) than 3 Mg, although their inhibitory effects on RYR are almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RYRs.

Enhanced sarcoplasmic reticulum Ca2+ release following intermittent sprint training

2000 ◽  
Vol 279 (1) ◽  
pp. R152-R160 ◽  
Author(s):  
Niels Ørtenblad ◽  
Per K. Lunde ◽  
Klaus Levin ◽  
Jesper L. Andersen ◽  
Preben K. Pedersen
Keyword(s):  
Sarcoplasmic Reticulum ◽  
High Intensity ◽  
Vastus Lateralis ◽  
Ryanodine Receptors ◽  
Vastus Lateralis Muscle ◽  
Sprint Training ◽  
High Intensity Training ◽  
Before And After ◽  
Muscle Biopsies ◽  
Intensity Training

To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% ( P < 0.01) after training. The 5-wk sprint training induced a significantly higher ( P < 0.05) peak rate of AgNO3-stimulated Ca2+ release from 709 (range 560–877; before) to 774 (596–977) arbitrary units Ca2+ ⋅ g protein− 1 ⋅ min− 1(after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% ( P < 0.05) increase in total number of RyR. No significant differences in Ca2+ uptake rate and Ca2+-ATPase capacity were observed following the training, despite that the relative density of Ca2+-ATPase isoforms SERCA1 and SERCA2 had increased 41% and 55%, respectively ( P < 0.05). These data suggest that high-intensity training induces an enhanced peak SR Ca2+ release, due to an enhanced total volume of SR, whereas SR Ca2+ sequestration function is not altered.

In vitro inhibitory effects of glucosamine, chondroitin and diacerein on human hepatic CYP2D6

2021 ◽  
Vol 36 (4) ◽  
pp. 259-270
Author(s):  
Boon Hooi Tan ◽  
Nafees Ahemad ◽  
Yan Pan ◽  
Uma Devi Palanisamy ◽  
Iekhsan Othman ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
High Performance ◽  
Inhibitory Effect ◽  
Inhibitory Effects ◽  
Time Curve ◽  
Clinical Drugs ◽  
Inhibition Potencies ◽  
P450 2D6

Abstract Objectives Glucosamine, chondroitin and diacerein are natural compounds commonly used in treating osteoarthritis. Their concomitant intake may trigger drug–natural product interactions. Cytochrome P450 (CYP) has been implicated in such interactions. Cytochrome P450 2D6 (CYP2D6) is a major hepatic CYP involved in metabolism of 25% of the clinical drugs. This study aimed to investigate the inhibitory effect of these antiarthritic compounds on CYP2D6. Methods CYP2D6 was heterologously expressed in Escherichia coli. CYP2D6–antiarthritic compound interactions were studied using in vitro enzyme kinetics assay and molecular docking. Results The high-performance liquid chromatography (HPLC)-based dextromethorphan O-demethylase assay was established as CYP2D6 marker. All glucosamines and chondroitins weakly inhibited CYP2D6 (IC50 values >300 µM). Diacerein exhibited moderate inhibition with IC50 and K i values of 34.99 and 38.27 µM, respectively. Its major metabolite, rhein displayed stronger inhibition potencies (IC50=26.22 μM and K i =32.27 μM). Both compounds exhibited mixed-mode of inhibition. In silico molecular dockings further supported data from the in vitro study. From in vitro–in vivo extrapolation, rhein presented an area under the plasma concentration-time curve (AUC) ratio of 1.5, indicating low potential to cause in vivo inhibition. Conclusions Glucosamine, chondroitin and diacerein unlikely cause clinical interaction with the drug substrates of CYP2D6. Rhein, exhibits only low potential to cause in vivo inhibition.

Endothelin inhibits thick ascending limb chloride flux via ETB receptor-mediated NO release

2000 ◽  
Vol 279 (2) ◽  
pp. F326-F333 ◽  
Author(s):  
Craig F. Plato ◽  
David M. Pollock ◽  
Jeffrey L. Garvin
Keyword(s):  
Intracellular Calcium ◽  
Chloride Transport ◽  
Inhibitory Effect ◽  
Thick Ascending Limb ◽  
Inhibitory Effects ◽  
Chloride Flux ◽  
Nacl Transport ◽  
No Release ◽  
Bq 610

Endothelin-1 (ET-1) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses ET-1 receptors. In many tissues, activation of ETB receptors stimulates release of NO, and we recently reported that endogenous NO inhibits TALH chloride flux ( J Cl). However, the relationship between ET-1 and NO in the control of nephron transport has not been extensively studied. We hypothesized that ET-1 decreases NaCl transport by cortical TALHs through activation of ETBreceptors and release of NO. Exogenous ET-1 (1 nM) decreased J Cl from 118.3 ± 15.0 to 62.7 ± 13.6 pmol · mm−1 · min−1 (48.3 ± 8.2% reduction), whereas removal of ET-1 increased J Cl in a separate group of tubules from 87.6 ± 10.7 to 115.2 ± 10.3 pmol · mm−1 · min−1 (34.5 ± 6.2% increase). To determine whether NO mediates the inhibitory effects of ET-1 on J Cl, we examined the effect of inhibiting of NO synthase (NOS) with N G-nitro-l-arginine methyl ester (l-NAME) on ET-1-induced changes in J Cl. l-NAME (5 mM) completely prevented the ET-1-induced reduction in J Cl, whereas d-NAME did not. l-NAME alone had no effect on J Cl. These data suggest that the effects of ET-1 are mediated by NO. Blockade of ETBreceptors with BQ-788 prevented the inhibitory effects of 1 nM ET-1. Activation of ETB receptors with sarafotoxin S6c mimicked the inhibitory effect of ET-1 on J Cl (from 120.7 ± 12.6 to 75.4 ± 13.3 pmol · mm−1 · min−1). In contrast, ETA receptor antagonism with BQ-610 did not prevent ET-1-mediated inhibition of TALH J Cl (from 96.5 ± 10.4 to 69.5 ± 8.6 pmol · mm−1 · min−1). Endothelin increased intracellular calcium from 96.9 ± 14.0 to 191.4 ± 11.9 nM, an increase of 110.8 ± 26.1%. We conclude that exogenous endothelin indirectly decreases TALH J Cl by activating ETB receptors, increasing intracellular calcium concentration, and stimulating NO release. These data suggest that endothelin acts as a physiological regulator of TALH NO synthesis, thus inhibiting chloride transport and contributing to the natriuretic effects of ET-1 observed in vivo.

Presynaptic inhibition of canine renal adrenergic nerves by acetylcholine in vivo

1979 ◽  
Vol 237 (3) ◽  
pp. H326-H331
Author(s):  
N. W. Robie
Keyword(s):  
Nerve Stimulation ◽  
Neurotransmitter Release ◽  
Intact Animal ◽  
Inhibitory Effect ◽  
Inhibitory Effects ◽  
Renal Vasculature ◽  
Anesthetized Dogs ◽  
Renal Sympathetic

Experiments were performed in anesthetized dogs to determine whether previously reported in vitro inhibition of sympathetic neurotransmitter release by acetylcholine could be demonstrated in the renal vasculature of the intact animal. Vasoconstrictor responses to renal sympathetic nerve stimulation at varying frequencies were compared to intra-arterial injections of norepinephrine before and during intra-arterial infusions of acetylcholine, 2.5--80 micrograms/min. The vasoconstrictor responses to nerve stimulation were inhibited to a greater extent than were responses to norepinephrine during infusions of acetylcholine. The inhibitory effects of acetylcholine on nerve stimulation were dose and frequency dependent. The inhibition was blocked by atropine but not altered by physostigmine. Changes in renal blood flow per se did not contribute to the inhibitory effect of acetylcholine, since another vasodilator agent, sodium acetate, did not affect the nerve stimulation-norepinephrine vasocontriction relationship. Thus, acetylcholine produced inhibition of in vivo renal sympathetic vasoconstrictor responses, and the receptor involved appears to be muscarinic.

A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle

2006 ◽  
Vol 290 (2) ◽  
pp. C539-C553 ◽  
Author(s):  
Jingsong Zhou ◽  
Jianxun Yi ◽  
Leandro Royer ◽  
Bradley S. Launikonis ◽  
Adom González ◽  
...  
Keyword(s):  
Ryanodine Receptors ◽  
Critical Role ◽  
Primary Cultures ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Dihydropyridine Receptors ◽  
Skeletal Muscle Contraction ◽  
Probable Role ◽  
Emission Ratios

To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca2+ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca2+ release. Murine primary cultures were confocally imaged for Ca2+ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca2+ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice ( mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca2+ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 μM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca2+ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca2+] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca2+] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca2+ release channels and/or their susceptibility to Ca2+-induced activation, thereby suppressing the production of Ca2+ sparks.

Cancer-chemopreventive effects of natural sweeteners and related compounds

2002 ◽  
Vol 74 (7) ◽  
pp. 1309-1316 ◽  
Author(s):  
Takao Konoshima ◽  
Midori Takasaki
Keyword(s):  
Mouse Skin ◽  
Stevia Rebaudiana ◽  
Chemical Carcinogenesis ◽  
Inhibitory Effect ◽  
Skin Carcinogenesis ◽  
Inhibitory Effects ◽  
Chemopreventive Agents ◽  
Vitro Assay

To search for possible cancer-chemopreventive agents from natural resources, several natural sweeteners were screened by the in vitro assay indicated by the inhibitory effects of Epstein-Barr virus early antigen (EBV-EA) induction. Of active compounds that showed the remarkable inhibitory effects on the EBV-EA induction, stevioside, from the leaves of Stevia rebaudiana, and mogroside V, from the fruits of Momordica grosvenori, exhibited significant inhibitory effects on the two-stage mouse skin carcinogenesis in vivo induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The inhibitory effect of stevioside is stronger than that of glycyrrhizin, which had been known as an antitumor-promoter in chemical carcinogenesis. Furthermore, stevioside also inhibited mouse skin carcinogenesis initiated by peroxynitrite. These results suggest that stevioside and mogroside V might be valuable as chemopreventive agents for chemical carcinogenesis.

scholarly journals Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca 2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca 2+ Dynamics

2021 ◽  
Vol 128 (1) ◽  
pp. 92-114
Author(s):  
Polina Gross ◽  
Jaslyn Johnson ◽  
Carlos M. Romero ◽  
Deborah M. Eaton ◽  
Claire Poulet ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Close Association ◽  
Ryanodine Receptors ◽  
Left Ventricular ◽  
Induced Mutations ◽  
Adrenergic Stimulation ◽  
T Tubules ◽  
Junctional Sarcoplasmic Reticulum

Rationale: Ca 2+ -induced Ca 2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. Objective: To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. Methods and Results: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mut PG1 JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mut PG1 JPH2 caused asynchronous Ca 2+ -release with impaired excitation-contraction coupling after β-adrenergic stimulation. The disturbed Ca 2+ regulation in mut PG1 JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. Conclusions: The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

scholarly journals Silencing of AURKA augments the antitumor efficacy of the AURKA inhibitor MLN8237 on neuroblastoma cells

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Yan Yang ◽  
Lili Ding ◽  
Qi Zhou ◽  
Li Fen ◽  
Yuhua Cao ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Inhibitory Effect ◽  
Neuroblastoma Cell Line ◽  
Inhibitory Effects ◽  
Rna Transfection ◽  
Interfering Rna

Abstract Background Aurora kinase A (AURKA) has been implicated in the regulation of cell cycle progression, mitosis and a key number of oncogenic signaling pathways in various malignancies including neuroblastoma. Small molecule inhibitors of AURKA have shown potential, but still not as good as expected effects in clinical trials. Little is known about this underlying mechanism. Here, we evaluated the inhibitory effects of AURKA inhibitor MLN8237 on neuroblastoma cells to understand the potential mechanisms responsible for tumor therapy. Methods MLN8237 treatment on neuroblastoma cell line IMR32 was done and in vivo inhibitory effects were investigated using tumor xenograft model. Cellular senescence was evaluated by senescence-associated β-gal Staining assay. Flow cytometry was used to tested cell cycle arrest and cell apoptosis. Senescence-associated signal pathways were detected by western blot. CD133 microbeads and microsphere formation were used to separate and enrich CD133+ cells. AURKA small interfering RNA transfection was carried to downregulate AURKA level. Finally, the combination of MLN8237 treatment with AURKA small interfering RNA transfection were adopted to evaluate the inhibitory effect on neuroblastoma cells. Results We demonstrate that MLN8237, an inhibitor of AURKA, induces the neuroblastoma cell line IMR32 into cellular senescence and G2/M cell phase arrest. Inactivation of AURKA results in MYCN destabilization and inhibits cell growth in vitro and in a mouse model. Although MLN8237 inhibits AURKA kinase activity, it has almost no inhibitory effect on the AURKA protein level. By contrast, MLN8237 treatment leads to abnormal high expression of AURKA in vitro and in vivo. Knockdown of AURKA reduces cell survival. The combination of MLN8237 with AURKA small interfering RNA results in more profound inhibitory effects on neuroblastoma cell growth. Moreover, MLN8237 treatment followed by AURKA siRNA forces senescent cells into apoptosis via suppression of the Akt/Stat3 pathway. Conclusions The effect of AURKA-targeted inhibition of tumor growth plays roles in both the inactivation of AURKA activity and the decrease in the AURKA protein expression level.

Ryanodine receptors mediate high intracellular Ca2+ and some myocyte damage following eccentric contractions in rat fast twitch skeletal muscle

2021 ◽  
Author(s):  
Ayaka Tabuchi ◽  
Yoshinori Tanaka ◽  
Ryo Takagi ◽  
Hideki Shirakawa ◽  
Tsubasa Shibaguchi ◽  
...  
Keyword(s):  
Muscle Damage ◽  
Ryanodine Receptors ◽  
Cytosolic Calcium ◽  
Specific Pattern ◽  
Eccentric Contractions ◽  
Cellular Processes ◽  
Area 5 ◽  
Myocyte Damage ◽  
Fast Twitch

Eccentric contractions (ECC) facilitate cytosolic calcium ion (Ca2+) release from the sarcoplasmic reticulum (SR) and Ca2+ influx from extracellular space. Ca2+ is a vital signaling messenger that regulates multiple cellular processes via its spatial and temporal concentration ([Ca2+]i) dynamics. We hypothesized that: 1) a specific pattern of spatial/temporal intramyocyte Ca2+ dynamics portends muscle damage following ECC, and 2) these dynamics would be regulated by the ryanodine receptor (RyR). [Ca2+]i in the tibialis anterior muscles of anesthetized adult Wistar rats was measured by ratiometric (i.e. ratio, R, 340/380 nm excitation) in vivo bioimaging with Fura-2 pre-ECC and at 5 and 24 hours post-ECC (5 x 40 contractions). Rats received RyR inhibitor dantrolene (DAN; 10 mg/kg i.p.) immediately post-ECC (+DAN). Muscle damage was evaluated by histological analysis on hematoxylin-eosin stained muscle sections. Compared to control (CONT, no ECC), [Ca2+]i distribution was heterogeneous with increased % total area of high [Ca2+]i sites (operationally defined as R > 1.39 i.e., > 1 SD of mean control) 5 hours post-ECC (CONT, 14.0 ± 8.0; ECC5h: 52.0 ± 7.4%, p < 0.01). DAN substantially reduced the high [Ca2+]i area 5 hours post-ECC (ECC5h+DAN: 6.4 ± 3.1%, p < 0.01) and myocyte damage (ECC24h, 63.2 ± 1.0%; ECC24h+DAN, 29.1 ± 2.2%, p < 0.01). Temporal and spatially-amplified [Ca2+]i fluctuations occurred regardless of DAN (ECC vs ECC+DAN, p > 0.05). These results suggest that the RyR-mediated local high [Ca2+]i itself is related to the magnitude of muscle damage while the [Ca2+]i fluctuation is an RyR-independent phenomenon.

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