The inflammatory cytokines tumor necrosis factor α and interleukin-1β stimulate phosphatidylcholine secretion in primary cultures of rat type II pneumocytes

Tumor necrosis factor and interleukin-1 increase surfactant secretion in type II pneumocytes in a time- and dose-dependent manner. This stimulatory effect was additive to that of lipopolysaccharide, suggesting that cytokines and lipopolysaccharide may exert their actions through different signal transduction pathways. Tumor necrosis factor and interleukin-1 did not modify the increase on phosphatidylcholine secretion induced by the direct protein kinase C activator tetradecanoylphorbol 13-acetate, whereas this effect was inhibited by the protein kinase C inhibitors bisindolylmaleimide (2 × 10-6M) and 1-(5-isoquinolinylsulphonyl)-2-methyl piperazone (10-4M). In addition, the stimulatory effect of tumor necrosis factor and interleukin-1 was not suppressed by the intracellular Ca2+ chelator BAPTA (5 × 10-6M) or by KN-62 (3 × 10-5M), a specific inhibitor of Ca2+-calmodulin-dependent protein kinase. These results suggest that tumor necrosis factor or interleukin-1 stimulate phosphatidylcholine secretion via protein kinase C activation in a Ca2+ -independent manner.     

The imidazoline derivative calmidazolium inhibits voltage-gated Ca2+-channels and insulin release but has no effect on the phospholipase C system in insulin producing RINm5F-cells

The present study shows that the calmodulin antagonist calmidazolium inhibited influx of Ca²⁺ through voltage-gated Ca²⁺-channels in clonal insulin producing RINm5F-cells. The mechanism of inhibition may involve both Ca²⁺-calmodulin-dependent protein kinases and direct binding of calmidazolium to the Ca²⁺-channel. Calmidazolium did not affect uptake of Ca²⁺ into intracellular Ca²⁺-pools, inositol 1,4,5-trisphosphate (InsP₃) formation or action on intracellular Ca²⁺-pools. The calmodulin inhibitor also did not affect glucose utilization or oxidation in RINm5F-cells, speaking against an unspecific toxic effect of the compound. KCl-and ATP-stimulated insulin release from RINm5F-cells was attenuated by calmidazolium, whereas basal hormone secretion was unaffected.     

Involvement of Ca2+ -Calmodulin-Dependent Protein Kinase II in the Intestinal Secretory Action of Escherichia coli Heat-Stable Enterotoxin II

Treating the mouse intestine with the calmodulin antagonist W-7 and KN-93, an inhibitor of Ca²⁺ -calmodulin-dependent protein kinase II (CaMK II), reduced the sensitivity of the host to the action of Escherichia coli heat-stable enterotoxin II (STII). CaMK II activity in mouse intestinal cells increased after exposure to STII. These results indicate that CaMK II is involved in the mechanism of action of STII.     

The mechanism of protriptyline-induced Ca2+ movement and non-Ca2+-triggered cell death in PC3 human prostate cancer cells

Abstract

Protriptyline, a tricyclic anti-depressant, is used primarily to treat the combination of symptoms of anxiety and depression. However, the effect of protriptyline on prostate caner is unknown. This study examined whether the anti-depressant protriptyline altered Ca²⁺ movement and cell viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i. Protriptyline evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Protriptyline-evoked Ca²⁺ entry was inhibited by store-operated channel inhibitors (nifedipine, econazole and SKF96365), protein kinase C activator (phorbol 12-myristate 13 acetate, PMA) and protein kinase C inhibitor (GF109203X). Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydr-oquinone (BHQ) in Ca²⁺-free medium inhibited 60% of protriptyline-evoked [Ca²⁺]_i rises. Conversely, treatment with protriptyline abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 suppressed 50% of protriptyline-evoked [Ca²⁺]_i rises. At concentrations of 50–70?µM, protriptyline decreased cell viability in a concentration-dependent manner; which were not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, in PC3 cells, protriptyline evoked [Ca²⁺]_i rises by inducing phospholipase C-associated Ca²⁺ release from the endoplasmic reticulum and other stores, and Ca²⁺ influx via protein kinase C-sensitive store-operated Ca²⁺ channels. Protriptyline caused cell death that was independent of [Ca²⁺]_i rises.     

Pathways of [Ca2+]i rise evoked by angiotensin II in MDCK renal tubular cells

Abstract

The effect of angiotensin II (Ang II) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in MDCK renal tubular cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Ang II at concentrations of 5–40?µM induced a [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Ang II evoked store-operated Ca²⁺ entry that was inhibited by La³⁺ and Gd³⁺. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) or thapsigargin abolished Ang II-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 abolished Ang II-induced [Ca²⁺]_i rise. Three Ang II analogues [(ASN1,VAL5)-Ang II acetate, (SAR1,THR8)-Ang II acetate, (VAL5)-Ang II acetate] failed to induce a [Ca²⁺]_i rise. Together, in MDCK cells, Ang II induced a [Ca²⁺]_i rise via Ca²⁺ entry through store-operated Ca²⁺ channels and phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum. Moreover, Ang II’s amino acid sequence is important in its stimulatory effect on [Ca²⁺]_i.     

Reversible changes of extracellular pH during action potential generation in a higher plant Cucurbita pepo

A pH-sensitive electrode was applied to measure activity of H⁺ ions in the medium surrounding excitable cells of pumpkin (Cucurbita pepo L.) seedlings during cooling-induced generation of action potential (AP). Reversible alkalization shifts were found to occur synchronously with AP, which could be due to the influx of H⁺ ions from external medium into excitable cells. Ethacrynic acid (an anion channel blocker) reduced the AP amplitude but had no effect on the transient alkalization of the medium. An inhibitor of plasma membrane H⁺-ATPase, N,N’-dicyclohexylcarbodiimide suppressed both the AP amplitude and the extent of alkalization. In experiments with plasma membrane vesicles, the hydrolytic H⁺-ATPase activity was subjected to inhibition by Ca²⁺ concentrations in the range characteristic of cytosolic changes during AP generation. The addition of a calcium channel blocker verapamil and a chelating agent EGTA to inhibit Ca²⁺ influx from the medium eliminated the AP spike and diminished reversible alkalization of the external solution. An inhibitor of protein kinase, H-7 alleviated the inhibitory effect of Ca²⁺ on hydrolytic H⁺-ATPase activity in plasma membrane vesicles and suppressed the reversible alkalization of the medium during AP generation. The results provide evidence that the depolarization phase of AP is associated not only with activation of chloride channels and Cl^? efflux but also with temporary suppression of plasma membrane H⁺-ATPase manifested as H⁺ influx. The Ca²⁺-induced inhibition of the plasma membrane H⁺-ATPase is supposedly mediated by protein kinases.     

Ca<Superscript>2+</Superscript>-CaMKKβ pathway is required for adiponectin-induced secretion in rat submandibular gland

Adiponectin functions as a promoter of saliva secretion in rat submandibular gland via activation of adenosine monophosphate-activated protein kinase (AMPK) and increased paracellular permeability. Ca²⁺ mobilization is the primary signal for fluid secretion in salivary acinar cells. However, whether intracellular Ca²⁺ mobilization is involved in adiponectin-induced salivary secretion is unknown. Here, we found that full-length adiponectin (fAd) increased intracellular Ca²⁺ and saliva secretion in submandibular glands. Pre-perfusion with ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) combined with thapsigargin (TG), an endoplasmic reticulum Ca²⁺-ATPase inhibitor, abolished fAd-induced salivary secretion, AMPK phosphorylation, and enlarged tight junction (TJ) width. Furthermore, in cultured SMG-C6 cells, co-pretreatment with EGTA and TG suppressed fAd-decreased transepithelial electrical resistance and increased 4-kDa FITC-dextran flux responses. Moreover, fAd increased phosphorylation of calcium/calmodulin-dependent protein kinase (CaMKKβ), a major kinase that is activated by elevated levels of intracellular Ca²⁺, but not liver kinase B1 phosphorylation. Pre-perfusion of the isolated gland with STO-609, an inhibitor of CaMKKβ, abolished fAd-induced salivary secretion, AMPK activation, and enlarged TJ width. CaMKKβ shRNA suppressed, whereas CaMKKβ re-expression rescued fAd-increased paracellular permeability. Taken together, these results indicate that adiponectin induced Ca²⁺ modulation in rat submandibular gland acinar cells. Ca²⁺-CaMKKβ pathway is required for adiponectin-induced secretion through mediating AMPK activation and increase in paracellular permeability in rat submandibular glands.     

Mechanisms of resveratrol-induced changes in [Ca2+]i and cell viability in PC3 human prostate cancer cells

Abstract

Resveratrol is a natural compound that affects cellular Ca²⁺ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i and WST-1 was used to measure viability. Resveratrol-evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Resveratrol-evoked Ca²⁺ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca²⁺]_i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca²⁺]_i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca²⁺]_i. Previous studies showed that resveratrol between 10 and 100?µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10?μM) increased cell viability, which was abolished by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10?µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca²⁺]_i by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10?µM also caused Ca²⁺-dependent cell proliferation.     

Regulation of Ca2+ transport by cyclic 3′,5′-AMP-dependent and calcium-calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by cyclic AMP-dependent and by Ca²⁺-calmodulin-dependent protein kinases on a 22 kDa protein, called phospholamban. Both types of phosphorylation have been shown to stimulate the initial rates of Ca²⁺ transport. To establish the interrelationship of the cAMP-dependent and Ca²⁺-calmodulin-dependent phosphorylation on Ca²⁺ transport, cardiac sarcoplasmic reticulum vesicles were preincubated under optimum conditions for: (a) cAMP-dependent phosphorylation, (b) Ca²⁺-calmodulin-dependent phosphorylation, and (c) combined cAMP-dependent and Ca²⁺-calmodulin-dependent phosphorylation. Control vesicles were treated under identical conditions, but in the absence of ATP, to avoid phosphorylation. Control and phosphorylated sarcoplasmic reticulum vesicles were subsequently centrifuged and assayed for Ca²⁺ transport in the presence of 2.5 mM Tris-oxalate. Our results indicate that cAMP-dependent and Ca²⁺-calmodulin-dependent phosphorylation can each stimulate calcium transport in an independent manner and when both are operating, they appear to have an additive effect. Stimulation of Ca²⁺ transport was associated with a statistically significant increase in the apparent affinity for calcium by each type of phosphorylation. The degree of stimulation of the calcium affinity was relatively proportional to the degree of phospholamban phosphorylation. These findings suggest the presence of a dual control system which may operate in independent and combined manners for regulating cardiac sarcoplasmic reticulum function.     

Influence of E. coli lipopolysaccharide binding to rat alveolar type II cells on their functional properties

Summary The interaction between lipopolysaccharide from E. coli0111:B4 and rat alveolar type II pneumocytes and its influence on the functional properties of the cells and their membranes were studied. Type II cells were isolated by a novel procedure involving digestion of the lung connective tissue with elastase and Percoll-gradient centrifugation. Binding of (¹⁴C)lipopolysaccharide to type II cells resulted in a partially reversible, non-specific, high affinity process. (^l4C)Choline incorporation into phosphatidylcholine by type II cells was stimulated by lipopolysaccharide, the maximum effect being observed at 10–20 g/ml. ⁴⁵Ca²⁺ uptake by type II cells was also increased by lipopolysaccharide. Using plasma membranes from lung homogenates an increase of membrane microviscosity versus the amount of lipopolysaccharide was shown. These results indicate that E. coli lipopolysaccharide interacts with alveolar type 11 cells by binding reversibly to particular ingredients of the membrane bilayer and induces a modification of ion permeability and fluidity of the membrane.     

The specific Na(+)/Ca(2+) exchange inhibitor SEA0400 prevents nitric oxide-induced cytotoxicity in SH-SY5Y cells

The Na⁺/Ca²⁺ exchanger (NCX) plays a role in the regulation of intracellular Ca²⁺ levels, and nitric oxide (NO) is involved in many pathological conditions including neurodegenerative disorders. We have previously found that sodium nitroprusside (SNP), an NO donor, causes apoptotic-like cell death in cultured glial cells via NCX-mediated pathways and the mechanism for NO-induced cytotoxicity is cell type-dependent. The present study examined using the specific NCX inhibitor 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) whether NCX is involved in NO-induced injury in cultured neuronal cells. The treatment of neuroblastoma SH-SY5Y cells with SNP resulted in apoptosis and the cytotoxicity was blocked by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor U0126 and the p38 MAP kinase (MAPK) inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP60012. SNP increased Ca²⁺ influx and intracellular Ca²⁺ levels. In addition, SNP increased ERK and p38 MAPK phosphorylation, and production of reactive oxygen species (ROS) in an extracellular Ca²⁺-dependent manner. These effects of SNP were prevented by SEA0400. SNP-induced cytotoxicity was not affected by inhibitors of the Ca²⁺, Na⁺ and store-operated/capacitative channels. Moreover, SNP-induced increase in intracellular Ca²⁺ levels, ROS production and decrease in cell viability were blocked by a cGMP-dependent protein kinase (PKG) inhibitor. These results suggest that Ca²⁺ influx via the reverse of NCX is involved in the cascade of NO-induced neuronal apoptosis and NO activates the NCX through guanylate cyclase/PKG pathway.     

Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart

The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca²⁺) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg^–1 i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca²⁺-uptake activity in diabetic animals. The reduction in SR Ca²⁺-uptake was consistent with a significant decrease in the level of SR Ca²⁺-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca²⁺-uptake was also due to a reduction in the phosphorylation of PLB by the Ca²⁺-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca²⁺-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart. (Mol Cell Biochem 261: 245–249, 2004)     

Role of platelet-activating factor in phosphatidylcholine secretion in primary cultures of rat type II pneumocytes

This study was designed to investigate the effect of platelet-activating factor (PAF) in the secretory response of type II pneumocytes, that are involved in the synthesis and secretion of the pulmonary surfactant. PAF increased phosphatidylcholine secretion in a concentration-dependent manner in the 10(-5) - 10(-10) M range, with a maximum phosphatidylcholine secretion of up to 3.3 fold the basal values (3.4 +/- 0.3% phosphatidylcholine secreted). This effect was prevented by the synthetic PAF-receptor antagonist WEB 2086. A study of the mechanism through which PAF exerts its stimulatory effect was carried out adding different agents that are well known stimulants of phosphatidylcholine secretion. Thus, PAF increased the TPA- and terbutaline-stimulated phosphatidylcholine secretion, that are PKC and PKA activators respectively, suggesting the involvement of both protein kinases in the process. This involvement was further supported by the use of inhibitors of protein kinases and by the stimulation of cAMP production in type II pneumocytes incubated with PAF.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

PACAP stimulates insulin secretion by PAC1 receptor and ion channels in β-cells

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays a crucial role in the endocrine system. The present study aimed to investigate the effect of PACAP38 on insulin secretion and the underlying mechanism in rat pancreatic β-cells. The insulin secretion results showed that PACAP38 stimulated insulin secretion in a glucose- and dose-dependent manner. The insulinotropic effect was mediated by PAC₁ receptor, but not by VPAC₁ and VPAC₂ receptors. Inhibition of adenylyl cyclase and protein kinase A suppressed PACAP38-augmented insulin secretion. Glucose-regulated insulin secretion is dependent on a series of electrophysiological activities. Current-clamp technology suggested that PACAP38 prolonged action potential duration. Voltage-clamp recordings revealed that PACAP38 blocked voltage-dependent potassium currents, and this effect was reversed by inhibition of PAC₁ receptor, adenylyl cyclase, or protein kinase A. Activation of Ca²⁺ channels by PACAP38 was also observed, which could be antagonized by the PAC₁ receptor antagonist. In addition, calcium-imaging analysis indicated that PACAP38 increased intracellular Ca²⁺ concentration, which was decreased by PAC₁ receptor antagonist. These findings demonstrate that PACAP38 stimulates glucose-induced insulin secretion mainly by acting on PAC₁ receptor, inhibiting voltage-dependent potassium channels, activating Ca²⁺ channels and increasing intracellular Ca²⁺ concentration. Further, PACAP blocks voltage-dependent potassium currents via the adenylyl cyclase/protein kinase A signaling pathway.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: II. Mechanism of impairment in ATP-dependent Ca2+ transport

The role of the phosphorylation and dephosphorylation of sarcolemma and that of the alteration of membrane lipids in the endotoxin-induced impairment of the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results indicate that the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma was decreased by 30–35% 4h after endotoxin administration. Phosphorylation of sarcolemma by the catalytic subunit of the cAMP-dependent protein kinase or calmodulin stimulated ATP-dependent Ca²⁺ transport in both groups, however, the phosphorylation-stimulated activities remained significantly lower in endotoxic animals. Dephosphorylation of sarcolemma decreased ATP-dependent Ca²⁺ transport in both groups, yet, the time required to reach maximal dephosphorylation was reduced from 120 to 90 min 4 h post-endotoxin. Analysis of sarcolemmal membranes reveals that phosphatidylcholine and phosphatidylethanolamine contents were decreased while their respective lysophosphatide levels were increased significantly after endotoxin injection. Digestion of control heart sarcolemma with phospholipase A₂ inhibited Ca²⁺ transport and the inhibition was reversible by phosphatidylcholine. The inhibition caused by the in vivo administration of endotoxin was completely reversible by the addition of phosphatidylcholine. Based on these data, it is concluded that endotoxin administration impairs ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment may be due to i) a defective phosphorylation of sarcolemma; ii) a reduced number of Ca²⁺ pumps; iii) an accelerated dephosphorylation of sarcolemma; and iv) an alteration in membrane phospholipid profile in response to phospholipase A activation.     

Effect of diindolylmethane on Ca2+ homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Diindolylmethane at concentrations of 20–50 µM induced [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Diindolylmethane-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca²⁺]_i rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca²⁺]_i rise. At concentrations of 50–100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca²⁺]_i rise in PC3 cells by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via phospholipase A₂-sensitive store-operated Ca²⁺ channels. Diindolylmethane caused cell death in which apoptosis may participate.     

Involvement of Ca<Superscript>2+</Superscript>/calmodulin kinase II (CaMK II) in genistein-induced potentiation of leucine/glutamine-stimulated insulin secretion

Genistein has been reported to potentiate glucose-stimulated insulin secretion (GSIS). Inhibitory activity on tyrosine kinase or activation of protein kinase A (PKA) was shown to play a role in the genistein-induced potentiation effect on GSIS. The aim of the present study was to elucidate the mechanism of genistein-induced potentiation of insulin secretion. Genistein augmented insulin secretion in INS-1 cells stimulated by various energy-generating nutrients such as glucose, pyruvate, or leucine/glutamine (Leu/Gln), but not the secretion stimulated by depolarizing agents such as KCl and tolbutamide, or Ca²⁺ channel opener Bay K8644. Genistein at a concentration of 50 μM showed a maximum potentiation effect on Leu/Gln-stimulated insulin secretion, but this was not sufficient to inhibit the activity of tyrosine kinase. Inhibitor studies as well as immunoblotting analysis demonstrated that activation of PKA was little involved in genistein-induced potentiation of Leu/Gln-stimulated insulin secretion. On the other hand, all the inhibitors of Ca²⁺/calmodulin kinase II tested, significantly diminished genistein-induced potentiation. Genistein also elevated the levels of [Ca²⁺]_i and phospho-CaMK II. Furthermore, genistein augmented Leu/Gln-stimulated insulin secretion in CaMK II-overexpressing INS-1 cells. These data suggest that the activation of CaMK II played a role in genistein-induced potentiation of insulin secretion.     

Expression of calcium-binding protein regucalcin mRNA in the cloned rat hepatoma cells (Hr-II-E) is stimulated through Ca2+ signaling factors: Involvement of protein kinase C

The expression of hepatic Ca²⁺-binding protein regucalcin in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in H4-II-E hepatoma cells. This expression was clearly stimulated in the presence of serum (10% fetal bovine serum). Bay K 8644 (2. 5 × 10^-6 M), a Ca²⁺ channel agonist, significantly stimulated regucalcin mRNA expression in the absence or presence of 10% serum. Dibutyryl cyclic AMP (10^-3 M) did not have a stimulatory effect on the regucalcin mRNA expression. The presence of phorbol 12-myristate 13-acetate (PMA; 10^-6 M) or estrogen (10^-8 M) caused a significant increase in regucalcin mRNA levels in the hepatoma cells cultured in serum-free medium, while insulin (5 × 10^-9 M) or dexamethasone (10^-6 M) had no effect. Bay K 8644-stimulated regucalcin mRNA expression in the hepatoma cells was completely blocked in the presence of trifluoperazine (10^-5 M), an antagonist of calmodulin, or staurosporine (10^-7 M), an inhibitor of protein kinase C. The stimulatory effect of PMA was clearly inhibited in the presence of stauroporine. The present study demonstrates that regucalcin mRNA is expressed in the transformed H4-II-E hepatoma cells, and that the expression is stimulated through Ca²⁺-dependent signaling factors.     

Mitochondrial Ca uptake is inhibited by a concerted action of p38 MAPK and protein kinase D

Angiotensin II elicits cytosolic Ca²⁺ signal that is transferred into the mitochondria. Previously we found in H295R cells that this signal transfer is enhanced by both the inhibition of p38 MAPK and a novel isoform of PKC [G. Szanda, P. Koncz, A. Rajki, A. Spät, Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca²⁺ uptake, Cell Calcium 43 (2008) 250–259]. Now we report that simultaneous activation of these protein kinases (by TNFα and PMA + an inhibitor of the conventional PKC isoforms, respectively) attenuates the transfer of cytosolic Ca²⁺ signal, elicited by depolarisation or store-operated Ca²⁺ influx, into the mitochondria. The Ca²⁺ uptake enhancing effect of the p38 MAPK inhibitor SB202190 is due to the inhibition of p38 MAPK and not to a direct mitochondrial action. Protein kinases reduce mitochondrial [Ca²⁺] by inhibiting the uptake mechanism. The threshold of mitochondrial Ca²⁺ uptake may depend on the activity of p38 MAPK. The silencing of protein kinase D (PKD) also results in enhanced transfer of Ca²⁺ signal from the cytosol into the mitochondria. Our data indicate that Ca²⁺ mobilising agonists, through the simultaneous activation of p38 MAPK, a novel PKC isoform and PKD, exert a negative feed-forward action on mitochondrial Ca²⁺ uptake, thus reducing the risk of Ca²⁺ overload.