Passive Ca(2+) overload in H9c2 cardiac myoblasts: assessment of cellular damage and cytosolic Ca(2+) transients

Increase of resting Ca(2+) levels and amplitude of vasopressin-induced Ca(2+) transients were observed when cells in serum-free medium were exposed to 5mM Ca(2+) for 2h. Small effect on cell viability was also observed. A rapid cytotoxic effect was developed in the presence of 10mM Ca(2+) and absence of serum. However, cells exposed to 10mM Ca(2+) in the presence of serum were protected from damage for at least 2days. Resting Ca(2+) levels and cytosolic Ca(2+) transients in serum-containing medium with 10mM Ca(2+) displayed lower increases and a tendency to recover control values. When serum was absent, cells preincubated with 10mM Ca(2+) were more sensitive to thapsigargin-induced damage than cells preincubated with lower Ca(2+). The sensitivity was similar when serum was present. Tolerance to high Ca(2+) in the presence of serum was linked to potentiation of the mitochondrial Ca(2+) entry to decrease the sarcoplasmic reticulum Ca(2+) overload.     

Angiotensin II stimulates cardiac L-type Ca(2+) current by a Ca(2+)- and protein kinase C-dependent mechanism

Angiotensin II (ANG II) evokes positive inotropic responses in various species. However, the effects of this peptide on L-type Ca(2+) currents (I(Ca)) are still controversial. We report in this study that the effects of ANG II on I(Ca) differ depending on the mode of patch-clamp technique used, standard whole cell (WC) or perforated patch (PP). No significant effects of ANG II (0.5 microM) were observed when WC in cells dialyzed with high EGTA was used. However, when the intracellular milieu was preserved using PP, ANG II induced a significant 77 +/- 6% increase in I(Ca) (-2.2 +/- 0.3 in control and -3.9 +/- 0.6 pA/pF in ANG II, n = 8, P < 0.05). When WC was used in cells dialyzed with low Ca(2+) buffer capacity (EGTA 0.1 mM), ANG II was able to induce an increase in I(Ca) (-3.5 +/- 0.3 in control vs. -4.8 +/- 0.4 pA/pF in ANG II, n = 13, P < 0.05). This increase was prevented when the cells were also dialyzed with the protein kinase C (PKC) inhibitor chelerythrine (50 microM) or calphostin C (1 microM). The above results allow us to conclude that strong intracellular Ca(2+) buffering prevents the physiological actions of ANG II on cardiac I(Ca), which are also dependent on activation of PKC.     

Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes

BackgroundLowering blood glucose levels by increasing glucose uptake in insulin target tissues, such as skeletal muscle and adipose tissue, is one strategy to discover and develop antidiabetic drugs from natural products used as traditional medicines.PurposeOur goal was to reveal the mechanism and activity of acacetin (5,7-dihydroxy-4′-methoxyflavone), one of the major compounds in Agastache rugose, in stimulating glucose uptake in muscle cells.MethodsTo determine whether acacetin promotes GLUT4-dependent glucose uptake in cultured L6 skeletal muscle cells, we performed a [¹⁴C] 2-deoxy-_D-glucose (2-DG) uptake assay after treating differentiated L6-GLUT4myc cells with acacetin.ResultsAcacetin dose-dependently increased 2-DG uptake by enhancing GLUT4 translocation to the plasma membrane. Our results revealed that acacetin activated the CaMKII-AMPK pathway by increasing intracellular calcium concentrations. We also found that aPKCλ/ζ phosphorylation and intracellular reactive oxygen species (ROS) production were involved in acacetin-induced GLUT4 translocation. Moreover, acacetin-activated AMPK inhibited intracellular lipid accumulation and increased 2-DG uptake in HepG2 cells.ConclusionTaken together, these results suggest that acacetin might be useful as an antidiabetic functional ingredient. Subsequent experiments using disease model animals are needed to verify our results.     

Lipocalin-type prostaglandin D(2) synthase stimulates glucose transport via enhanced GLUT4 translocation

Previously, we demonstrated that lipocalin-type prostaglandin D(2) synthase (L-PGDS) knockout mice become glucose intolerant and display signs of diabetic nephropathy and accelerated atherosclerosis. In the current study we sought to explain the link between L-PGDS and glucose tolerance. Using the insulin-sensitive rat skeletal muscle cell line, L6, we showed that L-PGDS could stimulate glucose transport approximately 2-fold as well as enhance insulin-stimulated glucose transport, as measured by 2-deoxy-[(3)H]-glucose uptake. The increased glucose transport was not attributed to increased GLUT4 production but rather the stimulation of GLUT4 translocation to the plasma membrane, a phenomenon that was lost when cells were cultured under hyperglycemic (20 mM) conditions or pretreated with wortmannin. There was however, an increase in GLUT1 expression as well as a 3-fold increase in hexokinase III expression, which was increased to nearly 5-fold in the presence of insulin, in response to L-PGDS at 20 mM glucose. In addition, adipocytes isolated from L-PGDS knockout mice were significantly less sensitive to insulin-stimulated glucose transport than wild-type. We conclude that L-PGDS, via production of prostaglandin D(2), is an important mediator of muscle and adipose glucose transport which is modulated by glycemic conditions and plays a significant role in the glucose intolerance associated with type 2 diabetes.     

Phosphoinositide 3-kinase facilitates antigen-stimulated Ca(2+) influx in RBL-2H3 mast cells via a phosphatidylinositol 3,4,5-trisphosphate-sensitive Ca(2+) entry mechanism

This study presents evidence that phosphoinositide 3-kinase (PI3K) plays a concerted role with phospholipase Cgamma in initiating antigen-mediated Ca(2+) signaling in mast cells via a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3))-sensitive Ca(2+) entry pathway. Exogenous PI(3,4,5)P(3) at concentrations close to its physiological level induces instantaneous Ca(2+) influx into RBL-2H3 cells. This PI(3,4,5)P(3)-induced intracellular Ca(2+) increase is independent of phospholipase C activity or the depletion of internal stores. Moreover, inhibition of PI3K by LY294002 or by overexpression of the dominant negative inhibitor Deltap85 suppresses the Ca(2+) response to the cross-linking of the high affinity receptor for IgE (FcepsilonRI). Concomitant treatment of RBL-2H3 cells with LY294002 or Deltap85 and 2-aminoethyl diphenylborate, a cell-permeant antagonist of D-myo-inositol 1,4,5-trisphosphate receptors, abrogates antigen-induced Ca(2+) signals, whereas either treatment alone gives rise to partial inhibition. Conceivably, PI(3,4,5)P(3)-sensitive Ca(2+) entry and capacitative Ca(2+) entry represent major Ca(2+) influx pathways that sustain elevated [Ca(2+)]i to achieve optimal physiological responses. This study also refutes the second messenger role of D-myo-inositol 1,3,4,5-tetrakisphosphate in regulating FcepsilonRI-mediated Ca(2+) response. Considering the underlying mechanism, our data suggest that PI(3,4,5)P(3) directly stimulates a Ca(2+) transport system in plasma membranes. Together, these data provide a molecular basis to account for the role of PI3K in the regulation of FcepsilonRI-mediated degranulation in mast cells.     

Epithelial Ca(2+) channel expression and Ca(2+) uptake in developing zebrafish

The purpose of the present work was to study the possible role of the epithelial Ca(2+) channel (ECaC) in the Ca(2+) uptake mechanism in developing zebrafish (Danio rerio). With rapid amplification of cDNA ends, full-length cDNA encoding the ECaC of zebrafish (zECaC) was cloned and sequenced. The cloned zECaC was 2,578 bp in length and encoded a protein of 709 amino acids that showed up to 73% identity with previously described vertebrate ECaCs. The zECaC was found to be expressed in all tissues examined and began to be expressed in the skin covering the yolk sac of embryos at 24 h postfertilization (hpf). zECaC-expressing cells expanded to cover the skin of the entire yolk sac after embryonic development and began to occur in the gill filaments at 96 hpf, and thereafter zECaC-expressing cells rapidly increased in both gills and yolk sac skin. Corresponding to ECaC expression profile, the Ca(2+) influx and content began to increase at 36-72 hpf. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater caused upregulation of the whole body Ca(2+) influx and zECaC expression in both gills and skin. Colocalization of zECaC mRNA and the Na(+)-K(+)-ATPase alpha-subunit (a marker for mitochondria-rich cells) indicated that only a portion of the mitochondria-rich cells expressed zECaC mRNA. These results suggest that the zECaC plays a key role in Ca(2+) absorption in developing zebrafish.     

Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa²⁺ concentration([Ca²⁺]_i)and also stimulated inositol 1,4,5-trisphosphate(IP₃) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP₃ generation and[Ca²⁺]_iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa²⁺-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca²⁺. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca²⁺]_iincrease, IP₃ generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca²⁺]_iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca²⁺]_i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca²⁺]_iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa²⁺ in pertussis toxin-sensitiveG protein-mediated MAPK activation.

     

Structural insights into the molecular mechanism of Ca(2+)-dependent exocytosis

The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions. Recent structures of the SNARE complex, synaptotagmin III, nSec1, domains of NSF and its adaptor SNAP, along with Rab3 and some of its effectors, provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models. Ultimately, this knowledge of the structures of higher-order complexes and their dynamic behavior will allow us to obtain a full understanding of the vesicle fusion protein machinery.     

Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini.

Eukaryotic cells respond to various stimuli by an increase or decrease in levels of phosphoproteins. Phosphotyrosine levels on eukaryotic cellular proteins are tightly regulated by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases, EC 3.1.3.48). Studies on permeabilized mast cells suggest that the enabling reaction for exocytosis might involve protein dephosphorylation. In the present studies, a recombinant form of rat brain PTPase (rrbPTP-1) has been used to examine the potential role of PTPases in Ca(2+)-dependent amylase secretion from permeabilized rat pancreatic acini. Additionally, the concentrations and subcellular distributions of endogenous PTPase activity in rat pancreas were determined. The results from these experiments indicate that addition of exogenous PTPase stimulated Ca(2+)-dependent amylase secretion from pancreatic acinar cells and that endogenous PTPase activity was associated with the postgranule supernatant, zymogen granules, and in particular zymogen granule membranes. Our data suggest that protein tyrosine dephosphorylation is potentially involved in regulated secretion at a site(s) distal to receptor-mediated elevation of intracellular second messengers.     

Stimulation of tyrosine phosphorylation by progesterone and its 11-OH derivatives: dissection of a Ca(2+)-dependent and a Ca(2+)-independent mechanism

Progesterone has previously been shown to exert non-genomic effects on human spermatozoa by opening plasma membrane ion channels and by stimulating protein tyrosine phosphorylation. Here we examined how these two activities are influenced by 11-hydroxyl substitution of the steroid molecule either in the alpha- or in the beta-configuration. Both the 11alpha-OH and the 11beta-OH derivatives of progesterone were more effective than progesterone in stimulating tyrosine phosphorylation, although 11alpha-OH-progesterone was a markedly weaker Ca(2+)-influx inducing agonist than the other two steroids. In Ca(2+)-containing medium, the agonist activity of the 11alpha-OH derivative was weaker than that of the 11beta-OH derivative, and it was completely abolished by genistein, whereas that of progesterone and its 11beta-OH derivative was inhibited only partly by this drug. In contrast, when applied in Ca(2+)-free medium, the 11alpha-OH derivative was the strongest of the three agonists tested, and the effects of all the three steroids were completely abolished by genistein. These data show that the structural motifs of steroid molecules that are responsible for the stimulation of tyrosine phosphorylation are different from those mediating the steroid action on Ca2+ influx through plasma membrane channels. The synthesis of selective agonists of both activities may lead to the development of new pharmacological agents to be used in the treatment of steroid-dependent pathologies.     

Ca(2+)-dependent proteolysis in muscle wasting

Skeletal muscle wasting is a prominent feature of cachexia, a complex systemic syndrome that frequently complicates chronic diseases such as inflammatory and autoimmune disorders, cancer and AIDS. Muscle wasting may also develop as a manifestation of primary or neurogenic muscular disorders. It is now generally accepted that muscle depletion mainly arises from increased protein catabolism. The ubiquitin-proteasome system is believed to be the major proteolytic machinery in charge of such protein breakdown, yet there is evidence suggesting that Ca(2+)-dependent system, lysosomes and, in some conditions at least, even caspases are involved as well. The role of Ca(2+)-dependent proteolysis in skeletal muscle wasting is reviewed in the present paper. This system relies on the activity of calpains, a family of Ca(2+)-dependent cysteine proteases, whose regulation is complex and not completely elucidated. Modulations of Ca(2+)-dependent proteolysis have been associated with muscle protein depletion in various pathological contexts and particularly with muscle dystrophies. Calpains can only perform a limited proteolysis of their substrates, however they may play a critical role in initiating the breakdown of myofibrillar protein, by releasing molecules that become suitable for further degradation by proteasomes. Some evidence would also support a role for lysosomes and caspases in muscle wasting. Thus it cannot be excluded that different intracellular proteolytic systems may coordinately concur in shifting muscle protein turnover towards excess catabolism. Many different signals have been proposed as potentially involved in triggering the enhanced protein breakdown that underlies muscle wasting. How they are transduced to initiate the hypercatabolic response and to activate the proteolytic pathways remains largely unknown, however.     

Caffeine-induced Ca(2+) release increases AMPK-dependent glucose uptake in rodent soleus muscle

Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because alpha-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-beta (ACCbeta) Ser221 and immunoprecipitated alpha(1)-AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated alpha(2)-AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCbeta and alpha(1)-AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 microM), the CaM-competitive inhibitor KN-93 (10 microM), or the SR Ca(2+) release blocking agent dantrolene (10 microM) all inhibited ACCbeta phosphorylation and alpha(1)-AMPK phosphorylation, suggesting that SR Ca(2+) release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca(2+)-activated CaMKK may control alpha(1)-AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle.     

Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores.     

Phenethyl isothiocyanate stimulates glucose uptake through the Akt pathway in C2C12 myotubes

Phenethyl isothiocyanate (PEITC) is an aromatic isothiocyanate present in cruciferous vegetables. Several studies have shown that isothiocyanates regulate various intracellular signaling pathways, and thereby show anti-inflammatory and detoxifying activities. However, little is known about the effects of PEITC on glucose metabolism. In this study, we examined whether PEITC promotes glucose utilization in mouse skeletal muscle cells, C2C12 myotubes. PEITC induced glucose uptake, glucose transporter 4 (Glut4) translocation to the plasma membrane, and activation of Akt and ERK in C2C12 cells. Inhibition of Akt suppressed PEITC-induced Glut4 translocation and glucose uptake, whereas ERK inhibition did not. Furthermore, PEITC increased phosphorylation of ErbB2 and ErbB3. Treatment with a pan-ErbB inhibitor reduced Akt activation and the subsequent glucose uptake induced by PEITC. These results indicate that PEITC promotes glucose utilization through the ErbB/Akt pathway in C2C12 myotubes. PEITC may therefore serve as a dietary constituent with beneficial effects on the carbohydrate metabolism.

Abbreviations: PEITC: phenethyl isothiocyanate; Glut4: glucose transporter 4; PI3K: phosphatidylinositide 3-kinase; Nrf2: erythroid?2-related factor; ARE: antioxidant response element; HO?1: heme oxygenase?1; NRG: neuregulin     

Role of PDE3B in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis in primary rat adipocytes

In adipocytes, phosphorylation and activation of PDE3B is a key event in the antilipolytic action of insulin. The role of PDE4, another PDE present in adipocytes, is not yet known. In this work we investigate the role of PDE3B and PDE4 in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis. Inhibition of PDE3 (OPC3911, milrinone) but not PDE4 (RO 20-1724) lowered insulin-induced glucose uptake and lipogenesis, especially in the presence of isoproterenol (a general beta-adrenergic agonist), CL316243, a selective beta3-adrenergic agonist, and pituitary adenylate cyclase-activating peptide. The inhibitory effect of OPC3911 was associated with reduced translocation of GLUT-4 from the cytosol to the plasma membrane. Both OPC3911 and RO 20-1724 increased protein kinase A (PKA) activity and lipolysis. H89, a PKA inhibitor, did not affect OPC3911-mediated inhibition of insulin-induced glucose uptake and lipogenesis, whereas 8-pCPT-2'-O-Me-cAMP, an Epac agonist which mediates PKA independent cAMP signaling events, mimicked all the effects of OPC3911. Insulin-mediated activation of protein kinase B, a kinase involved in insulin-induced glucose uptake, was apparently not altered by OPC3911. In summary, our data suggest that PDE3B, but not PDE4, contributes to the regulation of insulin-induced glucose uptake, GLUT-4 translocation, and lipogenesis presumably by regulation of a cAMP/Epac signalling mechanisms.     

Isoleucine, a potent plasma glucose-lowering amino acid, stimulates glucose uptake in C2C12 myotubes

To examine which branched-chain amino acids affect the plasma glucose levels, we investigated the effects of leucine, isoleucine, and valine (0.3 g/kg body weight p.o.) in normal rats using the oral glucose tolerance test (OGTT, 2 g/kg). A single oral administration of isoleucine significantly reduced plasma glucose levels 30 and 60 min after the glucose bolus, whereas administration of leucine and valine did not produce a significant decrease. Oral administration of valine significantly enhanced the plasma glucose level at 30 min after the glucose administration and leucine had a similar effect at 120 min. At each measurement timepoint, the insulin levels of the treated groups were lower than that of the control group. We then investigated the effects of leucine, isoleucine or valine at the same concentration (1 mM) on glucose metabolism in C(2)C(12) myotubes in the absence of insulin. Glucose consumption was elevated by 16.8% in the presence of 1 mM isoleucine compared with the control. Conversely, 1 mM leucine or valine caused no significant changes in glucose consumption in the C(2)C(12) myotubes. The 2-deoxyglucose uptake of C(2)C(12) myotubes significantly increased upon exposure to 1-10 mM isoleucine and 5-10 mM leucine. However, isoleucine caused no significant difference in glycogen synthesis in C(2)C(12) myotubes, although leucine and valine caused a significant increase in intracellular glycogen compared with the control. The isoleucine effect on glucose uptake was mediated by phosphatidylinositol 3-kinase (PI3K), but was independent of mammalian target of rapamycin (mTOR). These results suggest that isoleucine stimulates the insulin-independent glucose uptake in skeletal muscle cells, which may contribute to the plasma glucose-lowering effect of isoleucine in normal rats.