Reduction of intracellular Mg caused by reactive oxygen species in rat ventricular myocytes.

The concentration of intracellular free Mg ([Mg]) should be maintained strictly for the regulation of cellular functions. Since reactive oxygen species (ROS) are liable to increase in various pathological conditions and induce cellular damage, we investigated whether ROS affect intracellular Mg homeostasis. We measured [Mg] in ventricular myocytes from Wistar rats using the fluorescent indicator, mag-fura-2.

A novel RyR1-selective inhibitor prevents and rescues sudden death in mouse models of malignant hyperthermia and heat stroke.

Mutations in the type 1 ryanodine receptor (RyR1), a Ca release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH.

The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity.

The activity of the TRPM7 channel is negatively regulated by intracellular Mg2+. We previously reported that oxidative stress enhances the inhibition of TRPM7 by intracellular Mg2+. Here, we aimed to clarify the mechanism underlying TRPM7 inhibition by hydrogen peroxide (H2O2).

Functional expression of TRPM7 as a Ca influx pathway in adipocytes.

In adipocytes, intracellular Ca and Mg modulates physiological functions, such as insulin action and the secretion of adipokines. TRPM7 is a Ca /Mg -permeable non-selective cation channel. TRPM7 mRNA is highly expressed in adipose tissue, however, its functional expression in adipocytes remains to be elucidated.

Effect of a single epidural administration of follicle-stimulating hormone via caudal vertebrae on superstimulation for in vivo and in vitro embryo production in Japanese black cows.

Here, we describe a simplified procedure for embryo production in the Japanese black cow that uses a single caudal epidural injection of follicle-stimulating hormone (FSH). First, we compared the efficiency of superovulation for in vivo embryo production between conventional multiple FSH treatment (control, n = 10) and single epidural administration (epidural, n = 5). The number of transferable blastocysts was similar between control and epidural groups (4.

Modulation of Mg influx and cytoplasmic free Mg concentration in rat ventricular myocytes.

To examine whether TRPM7, a member of the melastatin family of transient receptor potential channels, is a physiological pathway for Mg entry in mammalian cells, we studied the effect of TRPM7 regulators on cytoplasmic free Mg concentration ([Mg]) of rat ventricular myocytes. Acutely isolated single cells were AM-loaded with the fluorescent indicator furaptra, and [Mg] was estimated at 25 °C. After [Mg] was lowered by soaking the cells with a high-K and Mg-Ca-free solution, [Mg] was recovered by extracellular perfusion of Ca-free Tyrode's solution that contained 1 mM Mg.

Generation of exogenous germ cells in the ovaries of sterile NANOS3-null beef cattle.

Blastocyst complementation (BC) systems have enabled in vivo generation of organs from allogeneic pluripotent cells, compensating for an empty germ cell niche in gene knockout (KO) animals. Here, we succeeded in producing chimeric beef cattle (Wagyu) by transferring allogenic germ cells into ovaries using somatic cell nuclear transfer and BC technology. The KO of NANOS3 (NANOS3(-/-)) in Wagyu bovine ovaries produced a complete loss of germ cells.

Physiological pathway of magnesium influx in rat ventricular myocytes.

Cytoplasmic free Mg(2+) concentration ([Mg(2+)]i) was measured in rat ventricular myocytes with a fluorescent indicator furaptra (mag-fura-2) introduced by AM-loading. By incubation of the cells in a high-K(+) (Ca(2+)- and Mg(2+)-free) solution, [Mg(2+)]i decreased from ? 0.9 mM to 0.

Effects of candesartan on electrical remodeling in the hearts of inherited dilated cardiomyopathy model mice.

Inherited dilated cardiomyopathy (DCM) is characterized by dilatation and dysfunction of the ventricles, and often results in sudden death or heart failure (HF). Although angiotensin receptor blockers (ARBs) have been used for the treatment of HF, little is known about the effects on postulated electrical remodeling that occurs in inherited DCM. The aim of this study was to examine the effects of candesartan, one of the ARBs, on cardiac function and electrical remodeling in the hearts of inherited DCM model mice (TNNT2 ΔK210).

Mg(2+)- and ATP-dependent inhibition of transient receptor potential melastatin 7 by oxidative stress.

Transient receptor potential melastatin 7 (TRPM7) is a Ca(2+)- and Mg(2+)-permeable nonselective cation channel that contains a unique carboxyl-terminal serine/threonine protein kinase domain. It has been reported that reactive oxygen species associated with hypoxia or ischemia activate TRPM7 current and then induce Ca(2+) overload resulting in neuronal cell death in the brain. In this study, we aimed to investigate the molecular mechanisms of TRPM7 regulation by hydrogen peroxide (H2O2) using murine TRPM7 expressed in HEK293 cells.

Magnesium homeostasis in cardiac myocytes of Mg-deficient rats.

To study possible modulation of Mg(2+) transport in low Mg(2+) conditions, we fed either a Mg-deficient diet or a Mg-containing diet (control) to Wistar rats for 1-6 weeks. Total Mg concentrations in serum and cardiac ventricular tissues were measured by atomic absorption spectroscopy. Intracellular free Mg(2+) concentration ([Mg(2+)]i) of ventricular myocytes was measured with the fluorescent indicator furaptra.

Mutation of the Mg2+ transporter SLC41A1 results in a nephronophthisis-like phenotype.

Nephronophthisis (NPHP)-related ciliopathies are recessive, single-gene disorders that collectively make up the most common genetic cause of CKD in the first three decades of life. Mutations in 1 of the 15 known NPHP genes explain less than half of all cases with this phenotype, however, and the recently identified genetic causes are exceedingly rare. As a result, a strategy to identify single-gene causes of NPHP-related ciliopathies in single affected families is needed.

[Magnesium and cardiac function].

Free magnesium ion (Mg(2 + )) is involved in numerous processes of cardiac function. However, mechanism of regulation by Mg(2 + ) has not been fully understood. Extracellular Mg(2 + ) can act on the external surface of the cell membrane, whereas intracellular Mg(2 + ) can exert its effects via many different sites : various enzymes, intracellular organella and internal surface of the cell membrane.

Role of Ca(2+) in the rapid cooling-induced Ca(2+) release from sarcoplasmic reticulum in ferret cardiac muscles.

Rapid lowering of the solution temperature (rapid cooling, RC) from 24 to 3°C within 3 s releases considerable amounts of Ca(2+) from the sarcoplasmic reticulum (SR) in mammalian cardiac muscles. In this study, we investigated the intracellular mechanism of RC-induced Ca(2+) release, especially the role of Ca(2+), in ferret ventricular muscle. Saponin-treated skinned trabeculae were placed in a glass capillary, and the amount of Ca(2+) released from the SR by RC and caffeine (50 mM) was measured with fluo-3.

Rapid decrease of intracellular pH associated with inhibition of Na+/H+ exchanger precedes apoptotic events in the MNK45 and MNK74 gastric cancer cell lines treated with 2-aminophenoxazine-3-one.

The effects of Phx-3 on changes in intracellular pH (pHi) in the MKN45 and MKN74 human gastric cancer cell lines were evaluated in order to determine the mechanism for the proapoptotic effects of 2-aminophenoxazine-3-one (Phx-3) on these cells. Phx-3 (100 μM) reduced pHi in MKN45 from 7.45 to 5.

KB-R7943 inhibits Na+-dependent Mg2+ efflux in rat ventricular myocytes.

Na(+)-dependent Mg(2+) efflux activity was studied with the fluorescent Mg(2+) indicator furaptra in the presence of various potential antagonists known to inhibit other transporters and channels. Among the compounds tested, KB-R7943, an inhibitor of Na(+)/Ca(2+) exchange, most potently inhibited the Na(+)/Mg(2+) exchange with half inhibitory concentrations (IC(50)) of 21 μM: (25°C) and 16 μM: (35°C). These IC(50) values were a factor of three to four lower than those of imipramine, a widely used inhibitor of Na(+)/Mg(2+) exchange.

Volume-sensitive outwardly rectifying chloride channel in white adipocytes from normal and diabetic mice.

The volume-sensitive outwardly rectifying (VSOR) chloride channel is ubiquitously expressed and involved in cell volume regulation after osmotic swelling, called regulatory volume decrease (RVD), in various cell types. In adipocytes, the expression of the VSOR channel has not been explored to date. Here, by employing the whole-cell patch-clamp technique, we examined whether or not the VSOR channel is expressed in white adipocytes freshly isolated from epididymal fat pads of normal (C57BL/6 or KK) and diabetic (KKA(y)) mice.

Metabolic inhibition strongly inhibits Na+-dependent Mg2+ efflux in rat ventricular myocytes.

We measured intracellular Mg2+ concentration ([Mg2+]i) in rat ventricular myocytes using the fluorescent indicator furaptra (25 degrees C). In normally energized cells loaded with Mg2+, the introduction of extracellular Na+ induced a rapid decrease in [Mg2+]i: the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) is thought to represent the rate of Na+-dependent Mg2+ efflux (putative Na+/Mg2+ exchange). To determine whether Mg2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na+ gradient, we estimated the initial Delta[Mg2+]i/Deltat after metabolic inhibition.

Differential effects of an expected actin-tropomyosin binding region of heat shock protein 20 on the relaxation in skinned carotid artery and taenia cecum from guinea pig.

To explore the possible role of heat shock protein 20 (HSP20) -linked regulation of actin-myosin interaction in living vascular smooth muscle contraction, we studied the effects of HSP20p and TnIp, synthetic peptides originating from an actin tropomyosin binding region of human heat shock protein 20 [residues 110-121; GFVAREFHRRYR] and that of rabbit cardiac troponin I [residues 136-147; GKFKRPTLRRVR], respectively, on the active stress and phosphorylation level of myosin regulatory light chain (MLC(20)) during relaxation of skinned (cell membrane permeabilized) preparations from "tonic" carotid artery and "phasic" taenia cecum from guinea pig. Active stress of the skinned preparations, resulting from actin-myosin interaction, biphasically decayed following Ca(2+) removal (relaxation). Decay of MLC(20) phosphorylation level by Ca(2+) removal was much faster than active stress in an exponential manner.

Mechanisms of the inhibitory effects of a phenoxazine compound, 2-amino-4,4 alpha-dihydro-4 alpha-7-dimethyl-3H-phenoxazine-3-one, on the contraction of the smooth muscle of the guinea pig taenia cecum.

To elucidate the mechanisms involved in the relaxing effect of 2-amino-4, 4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1) on smooth muscle, we investigated its effects on the contraction of both intact and skinned (cell membrane permeabilized) preparations from the guinea pig taenia cecum. In intact preparations, Phx-1 concentration-dependently suppressed the contraction induced by either acetylcholine (ACh) or high-K(+) with an IC(50) value estimated at around 100 muM. Similar inhibitory actions of Phx-1 on force were observed in intracellular Ca store depleted preparations.

Use of the Ca-shortening curve to estimate the myofilament responsiveness to Ca2+ in tetanized rat ventricular myocytes.

We previously estimated the myofilament responsiveness to Ca(2+) in isolated intact ventricular myocytes, using the steady-state relationship between cytosolic Ca(2+) concentration ([Ca(2+)](i)) and cell-shortening during tetanus (Ca-L trajectory). This method was useful and easy; however, it could not be used for a high dose of Ca sensitizer because the instantaneous plots after the application of Ca sensitizer did not make a fixed point of shortening (we used 5% shortening). Therefore we must produce another method to investigate Ca(2+) responsiveness.

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes.

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.