Increased free Zn2+ correlates induction of sarco(endo)plasmic reticulum stress via altered expression levels of Zn2+‐transporters in heart failure

Zn²⁺‐homoeostasis including free Zn²⁺ ([Zn²⁺]_i) is regulated through Zn²⁺‐transporters and their comprehensive understanding may be important due to their contributions to cardiac dysfunction. Herein, we aimed to examine a possible role of Zn²⁺‐transporters in the development of heart failure (HF) via induction of ER stress. We first showed localizations of ZIP8, ZIP14 and ZnT8 to both sarcolemma and S(E)R in ventricular cardiomyocytes (H9c2 cells) using confocal together with calculated Pearson's coefficients. The expressions of ZIP14 and ZnT8 were significantly increased with decreased ZIP8 level in HF. Moreover, [Zn²⁺]_i was significantly high in doxorubicin‐treated H9c2 cells compared to their controls. We found elevated levels of ER stress markers, GRP78 and CHOP/Gadd153, confirming the existence of ER stress. Furthermore, we measured markedly increased total PKC and PKCα expression and PKCα‐phosphorylation in HF. A PKC inhibition induced significant decrease in expressions of these ER stress markers compared to controls. Interestingly, direct increase in [Zn²⁺]_i using zinc‐ionophore induced significant increase in these markers. On the other hand, when we induced ER stress directly with tunicamycin, we could not observe any effect on expression levels of these Zn²⁺ transporters. Additionally, increased [Zn²⁺]_i could induce marked activation of PKCα. Moreover, we observed marked decrease in [Zn²⁺]_i under PKC inhibition in H9c2 cells. Overall, our present data suggest possible role of Zn²⁺ transporters on an intersection pathway with increased [Zn²⁺]_i and PKCα activation and induction of HF, most probably via development of ER stress. Therefore, our present data provide novel information how a well‐controlled [Zn²⁺]_i via Zn²⁺ transporters and PKCα can be important therapeutic approach in prevention/treatment of HF.     

Novel miR-sc4 regulates the proliferation and migration of Schwann cells by targeting Cdk5r1

Clinical and experimental studies have shown an association between intracellular free Zn²⁺ ([Zn²⁺]_i)-dyshomeostasis and cardiac dysfunction besides [Ca²⁺]_i-dyshomeostasis. Since [Zn²⁺]_i-homeostasis is regulated through Zn²⁺-transporters depending on their subcellular distributions, one can hypothesize that any imbalance in Zn²⁺-homeostasis via alteration in Zn²⁺-transporters may be associated with the induction of ER stress and apoptosis in hypertrophic heart. We used a transverse aortic constriction (TAC) model to induce hypertrophy in young male rat heart. We confirmed the development of hypertrophy with a high ratio of heart to body weight and cardiomyocyte capacitance. The expression levels of ER stress markers GRP78, CHOP/Gadd153, and calnexin are significantly high in TAC-group in comparison to those of controls (SHAM-group). Additionally, we detected high expression levels of apoptotic status marker proteins such as the serine kinase GSK-3β, Bax-to-Bcl-2 ratio, and PUMA in TAC-group in comparison to SHAM-group. The ratios of phospho-Akt to Akt and phospho-NFκB to the NFκB are significantly higher in TAC-group than in SHAM-group. Furthermore, we observed markedly increased phospho-PKCα and PKCα levels in TAC-group. We, also for the first time, determined significantly increased ZIP7, ZIP14, and ZnT8 expressions along with decreased ZIP8 and ZnT7 levels in the heart tissue from TAC-group in comparison to SHAM-group. Furthermore, a roughly calculated total expression level of ZIPs responsible for Zn²⁺-influx into the cytosol (increased about twofold) can be also responsible for the markedly increased [Zn²⁺]_i detected in hypertrophic cardiomyocytes. Taking into consideration the role of increased [Zn²⁺]_i via decreased ER-[Zn²⁺] in the induction of ER stress in cardiomyocytes, our present data suggest that differential changes in the expression levels of Zn²⁺-transporters can underlie mechanical dysfunction, in part due to the induction of ER stress and apoptosis in hypertrophic heart via increased [Zn²⁺]_i- besides [Ca²⁺]_i-dyshomeostasis.     

Overexpression of Slc30a7/ZnT7 increases the mitochondrial matrix levels of labile Zn2+ and modifies histone modification in hyperinsulinemic cardiomyoblasts

BackgroundCellular free Zn²⁺ concentrations ([Zn²⁺]) are primarily coordinated by Zn²⁺-transporters, although their roles are not well established in cardiomyocytes. Since we previously showed the important contribution of a Zn²⁺-transporter ZnT7 to [Zn²⁺]_i regulation in hyperglycemic cardiomyocytes, here, we aimed to examine a possible regulatory role of ZnT7 not only on [Zn²⁺]_i but also both the mitochondrial-free Zn²⁺ and/or Ca²⁺ in cardiomyocytes, focusing on the contribution of its overexpression to the mitochondrial function.MethodsWe mimicked either hyperinsulinemia (by 50-μM palmitic acid, PA-cells, for 24-h) or overexpressed ZnT7 (ZnT7OE-cells) in H9c2 cardiomyoblasts.ResultsOpposite to PA-cells, the [Zn²⁺]_i in ZnT7OE-cells was not different from untreated H9c2-cells. An investigation of immunofluorescence imaging by confocal microscopy demonstrated a ZnT7 localization on the mitochondrial matrix. We demonstrated the ZnT7 localization on the mitochondrial matrix by using immunofluorescence imaging. Later, we determined the mitochondrial levels of [Zn²⁺]_Mit and [Ca²⁺]_Mit by using the Zn²⁺ and Ca²⁺ sensitive FRET probe and a Ca²⁺-sensitive dye Fluo4, respectively. The [Zn²⁺]_Mit was found to increase significantly in ZnT7OE-cells, similar to the PA-cells while no significant changes in the [Ca²⁺]_Mit in these cells. To examine the contribution of ZnT7 overexpression on the mitochondria function, we determined the level of reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) in these cells in comparison to the PA-cells. There were significantly increased production of ROS and depolarization in MMP and increases in marker proteins of mitochondria-associated apoptosis and autophagy in ZnT7-OE cells, similar to the PA-cells, parallel to increases in K-acetylation. Moreover, we determined significant increases in trimethylation of histone H3 lysine27, H3K27me3, and the mono-methylation of histone H3 lysine36, H3K36 in the ZnT7OE-cells, demonstrating the role of [Zn²⁺]_Mit in epigenetic regulation of cardiomyocytes under hyperinsulinemia through histone modification.ConclusionsOverall, our data have shown an important contribution of high expression of ZnT7-OE, through its buffering and muffling capacity in cardiomyocytes, on the regulation of not only [Zn²⁺_]i but also both [Zn²⁺]_Mit and [Ca²⁺]_Mit affecting mitochondria function, in part, via histone modification.     

Calcium-Sensitive Fluorescent Dyes Can Report Increases in Intracellular Free Zinc Concentration in Cultured Forebrain Neurons

Abstract: High concentrations of Zn²⁺ are found in presynaptic terminals of excitatory neurons in the CNS. Zn²⁺ can be released during synaptic activity and modulate postsynaptic receptors, but little is known about the possibility that Zn²⁺ may enter postsynaptic cells and produce dynamic changes in the intracellular Zn²⁺ concentration ([Zn²⁺]_i). We used fura-2 and magfura-2 to detect the consequences of Zn²⁺ influx in cultured neurons under conditions that restrict changes in intracellular Ca²⁺ and Mg²⁺ concentrations. The resulting ratio changes for both dyes were reversed completely by the Zn²⁺ chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, indicating that these dyes are measuring changes in [Zn²⁺]_i. We found that fura-2 was useful in measuring small increases in [Zn²⁺]_i associated with exposure to Zn²⁺ alone that may be mediated by a Na⁺/Ca²⁺ exchanger. Magfura-2, which has a lower affinity for Zn²⁺, was more useful in measuring larger agonist-stimulated increases in [Zn²⁺]_i. The coapplication of 300 µM Zn²⁺ and 100 µM glutamate/10 µM glycine resulted in a [Zn²⁺]_i increase that was ～40–100 nM in magnitude and could be inhibited by the NMDA receptor antagonist, MK-801 (30 µM), or extracellular Na⁺. This suggests that Zn²⁺ influx can occur through at least two different pathways, leading to varying increases in [Zn²⁺]_i. These findings demonstrate the feasibility of measuring changes in [Zn²⁺]_i in neurons.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

β3-adrenergic receptor activation plays an important role in the depressed myocardial contractility via both elevated levels of cellular free Zn2+ and reactive nitrogen species

Role of β₃-AR dysregulation, as either cardio-conserving or cardio-disrupting mediator, remains unknown yet. Therefore, we examined the molecular mechanism of β₃-AR activation in depressed myocardial contractility using a specific agonist CL316243 or using β₃-AR overexpressed cardiomyocytes. Since it has been previously shown a possible correlation between increased cellular free Zn²⁺ ([Zn²⁺]_i) and depressed cardiac contractility, we first demonstrated a relation between β₃-AR activation and increased [Zn²⁺]_i, parallel to the significant depolarization in mitochondrial membrane potential in rat ventricular cardiomyocytes. Furthermore, the increased [Zn²⁺]_i induced a significant increase in messenger RNA (mRNA) level of β₃-AR in cardiomyocytes. Either β₃-AR activation or its overexpression could increase cellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels, in line with significant changes in nitric oxide (NO)-pathway, including increases in the ratios of pNOS3/NOS3 and pGSK-3β/GSK-3β, and PKG expression level in cardiomyocytes. Although β₃-AR activation induced depression in both Na⁺- and Ca²⁺-currents, the prolonged action potential (AP) seems to be associated with a marked depression in K⁺-currents. The β₃-AR activation caused a negative inotropic effect on the mechanical activity of the heart, through affecting the cellular Ca²⁺-handling, including its effect on Ca²⁺-leakage from sarcoplasmic reticulum (SR). Our cellular level data with β₃-AR agonism were supported with the data on high [Zn²⁺]_i and β₃-AR protein-level in metabolic syndrome (MetS)-rat heart. Overall, our present data can emphasize the important deleterious effect of β₃-AR activation in cardiac remodeling under pathological condition, at least, through a cross-link between β₃-AR activation, NO-signaling, and [Zn²⁺]_i pathways. Moreover, it is interesting to note that the recovery in ER-stress markers with β₃-AR agonism in hyperglycemic cardiomyocytes is favored. Therefore, how long and to which level the β₃-AR agonism would be friend or become foe remains to be mystery, yet.     

?-Blocker Timolol Prevents Arrhythmogenic Ca2+ Release and Normalizes Ca2+ and Zn2+ Dyshomeostasis in Hyperglycemic Rat Heart

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca²⁺ handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca²⁺ transients and reduced Ca²⁺ loading of sarcoplasmic reticulum (SR), basal intracellular free Ca²⁺ and Zn²⁺ ([Ca²⁺]_i and [Zn²⁺]_i), and spatio-temporal properties of the Ca²⁺ sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca²⁺]_i-handling regulators, such as Na⁺/Ca²⁺ exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca²⁺]_i and [Zn²⁺]_i, increased intracellular Zn²⁺ hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca²⁺ handling regulators, and prevention of Ca²⁺ leak, and thereby normalization of both [Ca²⁺]_i and [Zn²⁺]_i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.     

Zinc homeostatic proteins in the CNS are regulated by crosstalk between extracellular and intracellular zinc

Release of Zn²⁺ from presynaptic glutamatergic terminals has long been considered the principle challenge necessitating the existence of zinc homeostatic proteins (ZHP) in the mammalian nervous system. It is now known that neural cells also possess an intracellular zinc pool, termed here [Zn²⁺]_i, which functions in a cell signaling context. A major challenge is characterizing the interaction of these two populations of zinc ions. To assess the relationship of this Zn²⁺ pool to cellular ZHP production, we employed immunofluorescence and immunoblot analysis to compare the expression of ZHP's ZnT‐1 and MT‐I/II in olfactory bulb and hippocampus of wild‐type and ZnT‐3 KO mice, which lack synaptic Zn²⁺. In both areas, the respective distribution and concentration of ZnT‐1 and MT‐I/II were identical in ZnT‐3 KO and control animals. We subsequently examined ZHP content in ZnT‐3 KO and WT mice treated with a membrane‐permeable Zn²⁺ chelator. In both olfactory bulb and hippocampus of the KO mice, the ZHP content was significantly reduced 15 h after chelation of [Zn²⁺]_i compared to WT controls. Our findings support the conclusion that ZHP expression is regulated by crosstalk between synaptic and intracellular pools of Zn²⁺. J. Cell. Physiol. 224: 567–574, 2010. © 2010 Wiley‐Liss, Inc.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization

Spreading depolarization (SD) is a feed‐forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn²⁺ release and intracellular accumulation occurs with SD, and elevated intracellular Zn²⁺ ([Zn²⁺]_i) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn²⁺]_i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn²⁺]_i loading prior to SD, by transient ZnCl₂ application with the Zn²⁺ ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post‐synaptic potentials (fEPSPs) in a concentration‐dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn²⁺]_i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn²⁺ release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn²⁺]_i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn²⁺]_i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn²⁺]_i accumulation in a range of brain injury models.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Zn induces hyperpolarization by activation of a K channel and increases intracellular Ca and pH in sea urchin spermatozoa

Zinc (Zn²⁺) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn²⁺ was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pH_i). In this study we found that submicromolar concentrations of free Zn²⁺ change membrane potential (Em) and increase the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn²⁺ response in sperm of this species mainly involves an Em hyperpolarization caused by K⁺ channel activation. The pharmacological profile of the Zn²⁺-induced hyperpolarization indicates that the cGMP-gated K⁺ selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn²⁺. Considering that Zn²⁺ also induces [Ca²⁺]_i fluctuations, our observations suggest that Zn²⁺ activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn²⁺ in male gamete function.     

Discrete Control of TRPV4 Channel Function in the Distal Nephron by Protein Kinases A and C

We have recently documented that the Ca²⁺-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca²⁺ responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca²⁺]_i imaging with immunofluorescence microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of the PKC pathway with phorbol 12-myristate 13-acetate significantly increased [Ca²⁺]_i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with bisindolylmaleimide I diminished cellular responses to elevated flow. In contrast, activation of the PKA pathway with forskolin did not affect TRPV4-mediated [Ca²⁺]_i responses to flow but markedly shifted the subcellular distribution of the channel toward the apical membrane. These actions were blocked with the specific PKA inhibitor H-89. Concomitant activation of the PKA and PKC cascades additively enhanced the amplitude of flow-induced [Ca²⁺]_i responses and greatly increased basal [Ca²⁺]_i levels, indicating constitutive TRPV4 activation. This effect was precluded by the selective TRPV4 antagonist HC-067047. Therefore, the functional status of the TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. Although the PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, the PKC-dependent pathway increases the activity of the channel on the plasma membrane.     

A decrease of both [Ca]e and [H]e produces cell damage in the perfused rat heart

Cell damage of the Langendorff-perfused rat heart in response to a decrease of both [Ca²⁺]_e and [H⁺]_e is described. At pH_e = 7.7, lactate dehydrogenase (LDH) release could be induced during perfusion with media of reduced [Ca²⁺]_e (0.1–0.4 mmol/I). Decreasing pH_e to normal abolished LDH release. The gap junction channel blocker heptanol (2 mmol/I) also reduced enzyme release, and polyethylene glycol (9% PEG₆₀₀₀) totally prevented cell damage. Elevation of buffer capacity of perfusion media or perfusion flow both increased LDH release. Cell damage could also be aggravated by substituting 10 mmol/I of [Na⁺]_e by foreign cations. At [Ca²⁺]_e = 0.1 mmol/I and pH_e = 7.7, [Ca²⁺]_i and [Na⁺]_i of non-lysed cells were markedly increased (in HCO₃/CO₂ buffered media to about 7.0 μmol/I and 36 mmol/I, respectively; in HEPES-buffered media, to about 5.0 μmol/I and 55 mmol/l; physiological values of [Ca²⁺]_i and [Na⁺]_i are around 0.1 μmol/I and 10 mmol/I, respectively), whereas pH_i was not appreciably elevated. In contrast to myocytes in the intact heart, [Ca²⁺]_i of isolated cardiomyocytes under similar conditions was decreased to about 75 nmol/I and LDH release was negligible; pH_i of isolated cardiomyocytes, as in intact myocardium, did not change appreciably. The results indicate that Ca²⁺ overload is produced at lowered [Ca²⁺]_e and [H⁺]_e by an influx of Ca²⁺ through gap junctional leaks.     

Acidosis-Induced Zinc-Dependent Death of Cultured Cerebellar Granule Neurons

Severe acidosis caused death of cultured cerebellar granule neurons (CGNs). Acidosis was accompanied by a progressive increase of the intracellular zinc ions ([Zn²⁺]_i) and decrease of [Ca²⁺]_i. Zn²⁺ chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), prevented the increase of [Zn²⁺]_i and acidosis-induced neuronal death. However, neuronal death was insensitive to blockade of ASIC1 channels with amiloride, as CGNs display considerably lower expression of ASIC1a than other neurons. The antioxidant trolox and menadione significantly protected neurons from acidotic death. Earlier, we demonstrated that menadione rescues neurons from the deleterious effect of inhibition of mitochondrial complex I (Isaev et al. Neuroreport 15:2227–2231, 2004). We speculate that excessive Zn²⁺-dependent production of reactive oxygen species by mitochondrial complex I may be a general motive for the induction of cell death in CGNs under acidotic conditions.     

The effects of Tl<Superscript>+</Superscript> ions on the dynamics of intracellular Ca<Superscript>2+</Superscript> in rat cardiomyocytes

The effects of Т1⁺ ions on the dynamics of intracellular Cа²⁺ in neonatal rat cardiomyocytes have been studied. It was shown for the first time that application of Т1⁺ led to an uncontrolled increase in [Cа²⁺]_i in cells. Moreover, the ability of Т1⁺ to increase [Cа²⁺]_i depended on the Т1⁺ concentration used and the time of exposure to the cells. The increase in [Cа²⁺]_i was related to the entry of Cа²⁺ from the extracellular medium. Thallium did not release Cа²⁺ from intracellular stores. The thallium-induced increase in [Cа²⁺]_i was not inhibited by nifedipine. It is possible that L-channels do not participate in the processes of thalliuminduced increase in [Cа²⁺]_i. It is assumed that the thallium ions-induced calcium overload in cardiomyocytes may contribute to the toxic effect of Т1⁺ on the myocardium.     

Dissection of Calcium Signaling Events in Exocrine Secretion

The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple ion channel and transporter proteins, signaling components, and water transport. Importantly, neurotransmitter stimulated increase in the cytosolic free [Ca²⁺] ([Ca²⁺]_i) is critical for the regulation of salivary gland secretion as it regulates several major ion fluxes that together establish the sustained osmotic gradient to drive fluid secretion. The mechanisms that act to modulate these increases in [Ca²⁺]_i are therefore central to the process of salivary fluid secretion. Such modulation involves membrane receptors for neurotransmitters, as well as mechanisms that mediate intracellular Ca²⁺ release, and Ca²⁺ entry, as well as those that maintain cellular Ca²⁺ homeostasis. Together, these mechanisms determine the spatial and temporal aspects of the [Ca²⁺]_i signals that regulate fluid secretion. Molecular cloning of these transporters and channels as well as development of mice lacking these proteins has established the physiological significance of key components that are involved in regulating [Ca²⁺]_i in salivary glands. This review will discuss these important studies and the findings which have led to resolution of the Ca²⁺ signaling mechanisms that determine salivary gland fluid secretion.     

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca²⁺]_i, Ca²⁺ transients and membrane Ca²⁺ current, I_Ca, in cultured murine HL-1 cardiomyocytes. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca²⁺]_i, Ca²⁺ transients and I_Ca. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca²⁺]_i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca²⁺]_i, and inhibited Ca²⁺ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca²⁺]_i, and Ca²⁺ transients and I_Ca. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca²⁺]_i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca²⁺]_i required for excitation-contraction coupling in cardiomyoctyes.     

The influence of probiotics and probiotic product on respiration of mitochondria and intracellular calcium signal in cells of cardiovascular system

The influence of lactobacilli and new probiotic product on mitochondrial energetics of rat heart mitochondria and on dynamics of intracellular calcium concentration ([Ca²⁺]_i) of cardiomyocytes and rat aortic smooth muscle cells was investigated. Respiration of mitochondra was estimated polarographically. [Ca²⁺]_i was measured using fluorescent calcium indicator Fura 2 AM and calcium imaging system. The application of lactobacilli (5 × 10⁷ CFU/mL) was shown to increase [Ca²⁺]_i in cardiomyocytes, thereby increasing myocardial contractility. On the other hand, application of lactobacilli reduced thapsigargin-induced calcium influx in smooth rat aortic muscle, thus exhibiting some hypotensive effect. It was shown that probiotic product stimulated mitochondria respiration and exerted a mild uncoupling effect on electronic transport and oxidative phosphorylation in mitochondria. In cardiomyocytes and in smooth muscles probiotic product increased [Ca²⁺]_i and consequent increase in contractility of blood vessels and myocardium. It is supposed that the probiotic product can be effectively applied at the endotoxic shock, when contractility of blood vessels in response to vasoconstrictor agents is suppressed.