Enhanced glucose 6-phosphatase activity in liver of rats exposed to Mg-deficient diet

Total hepatic Mg²⁺ content decreases by >25% in animals maintained for 2 weeks on Mg²⁺ deficient diet, and results in a >25% increase in glucose 6-phosphatase (G6Pase) activity in isolated liver microsomes in the absence of significant changed in enzyme expression. Incubation of Mg²⁺-deficient microsomes in the presence of 1 mM external Mg²⁺ returned G6Pase activity to levels measured in microsomes from animals on normal Mg²⁺ diet. EDTA addition dynamically reversed the Mg²⁺ effect. The effect of Mg²⁺ or EDTA persisted in taurocholic acid permeabilized microsomes. An increase in G6Pase activity was also observed in liver microsomes from rats starved overnight, which presented a ∼15% decrease in hepatic Mg²⁺ content. In this model, G6Pase activity increased to a lesser extent than in Mg²⁺-deficient microsomes, but it could still be dynamically modulated by addition of Mg²⁺ or EDTA. Our results indicate that (1) hepatic Mg²⁺ content rapidly decreases following starvation or exposure to deficient diet, and (2) the loss of Mg²⁺ stimulates G6P transport and hydrolysis as a possible compensatory mechanism to enhance intrahepatic glucose availability. The Mg²⁺ effect appears to take place at the level of the substrate binding site of the G6Pase enzymatic complex or the surrounding phospholipid environment.     

Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg

To clarify the kinetic characteristics and ionic requirements of the tonoplast H⁺-translocating inorganic pyrophosphatase (H⁺-PPiase), PPi hydrolysis and PPi-dependent H⁺ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H⁺-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H⁺-PPiase activity was maximal in the presence of K⁺ (K₅₀ approximately 3 millimolar), with PPi-dependent H⁺ transport being more selective for K⁺ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H⁺-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg²⁺ for activation. At saturating concentrations of free Mg²⁺, H⁺-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi²⁻ but not Mg₂PPi, demonstrating that MgPPi²⁻ was the true substrate of the enzyme. The apparent K_m (MgPPi²⁻) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H⁺ transport (7 micromolar). Free Mg²⁺ was shown to be an allosteric activator of the H⁺-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H⁺ transport. Half-maximal H⁺-PPiase activity occurred at a free Mg²⁺ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg²⁺. In contrast, cytosolic concentrations of K⁺ and MgPPi²⁻ appear to be saturating for H⁺-PPiase activity. We propose that one function of the H⁺-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H⁺-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg²⁺ would be released from the MgATP²⁻ complex, thereby activating the tonoplast H⁺-PPiase.     

Evidence for two distinct Mg2+ binding sites in G(s alpha) and G(i alpha1) proteins

The function of guanine nucleotide binding (G) proteins is Mg²⁺ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5′-triphosphate hydrolysis. It is unclear whether two Mg²⁺ binding sites are present or if one Mg²⁺ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg²⁺-specific fluorophore, to investigate Mg²⁺ binding to α subunits in both conformations of the stimulatory (G_sα) and inhibitory (G_iα1) regulators of adenylyl cyclase. Regardless of the conformation or α protein studied, we found that two distinct Mg²⁺ sites were present with dissimilar affinities. With the exception of G_sα in the active conformation, cooperativity between the two Mg²⁺ sites was also observed. Whereas the high affinity Mg²⁺ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg²⁺ site may involve coordination to the terminal phosphate of the nucleotide.     

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.     

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

Summary ATP-dependent Ca²⁺ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca²⁺-specific electrode and⁴⁵Ca²⁺. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na⁺,K⁺(Mg²⁺)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared,⁴⁵Ca²⁺ uptake correlated with the RNA content and not with the Na⁺,K⁺(Mg²⁺)ATPase activity. The characteristics of nonmitochondrial Ca²⁺ uptake into leaky isolated cells and⁴⁵Ca²⁺ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg²⁺, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca²⁺ concentration of 2 and 2 mol/liter, respectively. Calcium uptake decreased at higher free Ca²⁺ concentration.⁴⁵Ca²⁺ uptake was dependent on monovalent cations (Rb⁺>K⁺>Na⁺>Li⁺>choline⁺) and different anions (Cl^–>Br^–>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–>SCN^–) in both preparations. Twenty mmol/liter oxalate enhanced⁴⁵Ca²⁺ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca²⁺ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca²⁺ concentration to 0.4 mol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca²⁺ concentration in pancreatic acinar cells.     

Ion-translocating ATPases in tendrils ofBryonia dioica Jacq.

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils ofBryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg²⁺ATP-driven transport of⁴⁵Ca²⁺. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca²⁺-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca²⁺-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K⁺, Mg²⁺-ATPase and the Ca²⁺-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.Abbreviations CAM calmodulin - CCCP carbonylcyanidem-chlorophenylhydrazone - IC₅₀ concentration giving 50% inhibition - PM plasma membrane - rER rough endoplasmic reticulum - sER smooth endoplasmic reticulum - FC fusicoccin - U₃+U₃ the two PM-rich upper phases obtained after phase partitioning of microsomal membranes The authors wish to thank the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision) for financial support.     

Involvement of calcium in the stimulation of phosphatidylcholine secretion in primary cultures of rat type II pneumocytes by Escherichia coli lipopolysaccharide

The purpose of this study was to evaluate the mechanism by which Escherichia coli lipopolysaccharide stimulates the secretion of phosphatidylcholine in primary cultures of rat type II pneumocytes. The stimulatory effect of lipopolysaccharide on phosphatidylcholine secretion was additive to those of terbutaline and TPA (protein kinase A and C activators respectively) and this effect was not suppressed by inhibitors of both protein kinases. On the other hand, lipopolysaccharide did not modify the increase on phosphatidylcholine secretion induced by the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin, and enhanced slightly the calcium-ionophore A23187 stimulated phosphatidylcholine secretion. In addition, the stimulatory effect of lipopolysaccharide was suppressed by BAPTA, an intracellular Ca²⁺ chelator, and KN-62, a specific inhibitor of Ca²⁺-calmodulin-dependent protein kinase. These results, together with the lipopolysaccharide-mediated increase in the cytosolic [Ca²⁺], suggest that stimulation of phosphatidylcholine secretion by lipopolysaccharide in type II pneumocytes occurs by a calcium-dependent transduction mechanism via Ca²⁺-calmodulin-dependent protein kinase activation.     

Thermogenic activity of Ca-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca channel

The sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca²⁺ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca²⁺ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH^cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg²⁺ or ruthenium red, conditions that close the ryanodine Ca²⁺ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH^cal values of ATP hydrolysis increased significantly. Neither Mg²⁺ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH^cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca²⁺ channel is opened or closed.     

Vesicularization of the endoplasmic reticulum is a fast response to plasma membrane injury

The endoplasmic reticulum of most cell types mainly consists of an extensive network of narrow sheets and tubules. It is well known that an excessive increase of the cytosolic Ca²⁺ concentration induces a slow but extensive swelling of the endoplasmic reticulum into a vesicular morphology. We observed that a similar extensive transition to a vesicular morphology may also occur independently of a change of cytosolic Ca²⁺ and that the change may occur at a time scale of seconds. Exposure of various types of cultured cells to saponin selectively permeabilized the plasma membrane and resulted in a rapid swelling of the endoplasmic reticulum even before a loss of permeability barrier was detectable with a low-molecular mass dye. The structural alteration was reversible provided the exposure to saponin was not too long. Mechanical damage of the plasma membrane resulted in a large-scale transition of the endoplasmic reticulum from a tubular to a vesicular morphology within seconds, also in Ca²⁺-depleted cells. The rapid onset of the phenomenon suggests that it could perform a physiological function. Various mechanisms are discussed whereby endoplasmic reticulum vesicularization could assist in protection against cytosolic Ca²⁺ overload in cellular stress situations like plasma membrane injury.     

Ca-transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca²⁺ by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K⁺ leading to an enhancement of the Ca²⁺ transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca²⁺ at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K⁺ but not by Li⁺ and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K⁺. These data suggest that the regulation of the SERCA pump by K⁺ and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Effect of clotrimazole on cytosolic Ca2+ rise and viability in HA59T human hepatoma cells

Abstract

Clotrimazole is an antimycotic imidazole derivative that interferes with cellular Ca²⁺ homeostasis. This study examined the effect of clotrimazole on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in HA59T human hepatoma cells. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Clotrimazole induced [Ca²⁺]_i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca²⁺. Clotrimazole-evoked Ca²⁺ entry was suppressed by store-operated channel inhibitors (nifedipine, econazole and SK&F96365) and protein kinase C modulators (GF109203X and phorbol, 12-myristate, 13-acetate). In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone abolished clotrimazole-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished clotrimazole-induced [Ca²⁺]_i rise. At 10–40?µM, clotrimazole inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺. Clotrimazole at 10 and 30?µM also induced apoptosis. Collectively, in HA59T cells, clotrimazole-induced [Ca²⁺]_i rises by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via store-operated Ca²⁺ channels. Clotrimazole also caused apoptosis.     

Membranes of mammary gland : X. Adenosine triphosphate dependent calcium accumulation by golgi apparatus rich fractions from bovine mammary gland

Golgi apparatus rich fractions from lactating bovine mammary gland had an Mg²⁺-dependent, Ca²⁺-stimulated adenosine triphosphatase. These Golgi apparatus fractions also accumulated Ca²⁺ in vitro. Accumulation of Ca²⁺ required ATP and could be abolished by treatment either with low concentrations of deoxycholate followed by ultrasound, or by heating at 100 °C for 10 min. The adenosine triphosphatase activity of Golgi apparatus was strongly stimulated by low concentrations of Ca²⁺ and moderately stimulated by high concentrations of K⁺. This activity was unaffected by Na⁺ and was not inhibited by ouabain. The pH optimum for the Mg²⁺-dependent hydrolysis of ATP was 7.5, the K_m was 5 × 10⁻⁵ M and the activation energy was 6 000 calories/mole. This Mg²⁺-dependent adenosine triphosphatase activity was also found in rough endoplasmic reticulum, smooth microsomes and milk fat globule membrane, the latter membrane being derived directly from the apical plasma membrane. All of these membrane fractions had the ability to specifically accumulate Ca²⁺. Specific accumulation was highest with smooth microsomes and lowest with milk fat globule membrane with Golgi apparatus and rough endoplasmic reticulum being intermediate. These observations provide one plausible explanation for intracellular Ca²⁺ accumulation and secretion into milk. Further, these results help explain the ultrastructural observations of casein micelle formation in secretory vesicles elaborated by Golgi apparatus.     

Environmental cadmium is not sequestered by metallothionein in rainbow trout

Smooth endoplasmic reticulum vesicles from rat liver display an ATP-supported Ca²⁺ transport which is mediated by a (Ca²⁺ + Mg²⁺)-ATPase. During the catalytic cycle the terminal phosphate from ATP is incorporated to form an acid-precipitable reaction product(118 000-M_r in SDS-gel electrophoresis) with stability characteristics of an acylphosphate. Comparative studies with sarcoplasmic reticulum vesicles from fast-twitch skeletal muscle suggest that the 118 000-M_r phosphopeptide may be identified with the phosphorylated reaction intermediate of a Ca²⁺ transport ATPase in endoplasmic reticulum, similar to that in sarcoplasmic reticulum of muscle.     

Calcium-pumping ATPases in vesicles from carrot cells : stimulation by calmodulin or phosphatidylserine, and formation of a 120 kilodalton phosphoenzyme

Ca²⁺-ATPases keep cytoplasmic [Ca²⁺] low by pumping Ca²⁺ into intracellular compartments or out of the cell. The transport properties of Ca²⁺-pumping ATPases from carrot (Daucus carota cv Danvers) tissue culture cells were studied. ATP-dependent Ca²⁺ transport in vesicles that comigrated with an endoplasmic reticulum marker, was stimulated three- to fourfold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase) partially inhibited oxalate-stimulated Ca²⁺ transport activity; however, it had no effect on calmodulin-stimulated Ca²⁺ uptake driven by ATP or GTP. The results would suggest the presence of two types of Ca²⁺-ATPases, an endoplasmic reticulum- and a plasma membrane-type. Interestingly, incubation of membranes with [gamma³²P]ATP resulted in the formation of a single acyl [³²P]phosphoprotein of 120 kilodaltons. Formation of this phosphoprotein was dependent on Ca²⁺, but independent of Mg²⁺. Its enhancement by La³⁺ is characteristic of a phosphorylated enzyme intermediate of a plasma membrane-type Ca-ATPase. Calmodulin stimulated Ca²⁺ transport was decreased by W-7 (a calmodulin antagonist), ML-7 (myosin light chain kinase inhibitor) or thyroxine. Acidic phospholipids, like phosphatidylserine, stimulated Ca²⁺ transport, similar to their effect on the erythrocyte plasma membrane Ca²⁺-ATPase. These results would indicate that the calmodulin-stimulated Ca²⁺ transport originated in large part from a plasma membrane-type Ca²⁺ pump of 120 kilodaltons. The possibility of calmodulin-stimulated Ca²⁺-ATPases on endomembranes, such as the endoplasmic reticulum and secretory vesicles, as well as the plasma membrane is suggested.     

Role of Ca2+,Mg2+-ATPases in Diabetes-Induced Alterations in Calcium Homeostasis in Input Neurons of the Nociceptive System

In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca²⁺ in input neurons of the nociceptive system increases, Ca²⁺ signals are prolonged, while Ca²⁺ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca²⁺,Mg²⁺-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca²⁺,Mg²⁺-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca²⁺,Mg²⁺-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P < 0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P < 0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca²⁺ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities.     

Mechanism of [Ca2+]i rise induced by angiotensin 1–7 in MDCK renal tubular cells

The effect of angiotensin 1–7 (Ang 1–7) 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 1–7 at concentrations of 10–50 µM induced a [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Ang 1–7 evoked store operated Ca²⁺ entry that was inhibited by La³⁺ and aristolochic acid. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin prevented Ang 1–7 from releasing more Ca²⁺. Inhibition of phospholipase C with U73122 abolished Ang 1–7-induced [Ca²⁺]_i rise. Ang 1–7-induced [Ca²⁺]_i rise was abolished by the angiotensin type 1 receptor antagonist losartan, but was not affected by the angiotensin type 2 receptor antagonist PD 123,319. In sum, in MDCK cells, Ang 1–7 stimulated angiotensin type 1 receptors leading to a [Ca²⁺]_i rise that was composed of phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via phospholipase A2-sensitive store-operated Ca²⁺ channels.     

Glucose-6-phosphate dehydrogenase exerts antistress effects independently of its enzymatic activity

G6PD (glucose-6-phosphate dehydrogenase) is the rate-limiting enzyme in the oxidative pentose phosphate pathway that can generate cytosolic NADPH for biosynthesis and oxidative defense. Since cytosolic NADPH can be compensatively produced by other sources, the enzymatic activity deficiency alleles of G6PD are well tolerated in somatic cells but the effect of null mutations is unclear. Herein, we show that G6PD KO sensitizes cells to the stresses induced by hydrogen peroxide, superoxide, hypoxia, and the inhibition of the electron transport chain. This effect can be completely reversed by the expressions of natural mutants associated with G6PD deficiency, even without dehydrogenase activity, exactly like the WT G6PD. Furthermore, we demonstrate that G6PD can physically interact with AMPK (AMPK-activated protein kinase) to facilitate its activity and directly bind to NAMPT (nicotinamide phosphoribosyltransferase) to promote its activity and maintain the NAD(P)H/NAD(P)⁺ homeostasis. These functions are necessary to the antistress ability of cells but independent of the dehydrogenase activity of G6PD. In addition, the WT G6PD and naturally inactive mutant also can similarly regulate the metabolism of glucose, glutamine, fatty acid synthesis, and GSH and interact with the involved enzymes. Therefore, our findings reveal the previously unidentified functions of G6PD that can act as the important physiological neutralizer of stresses independently of its enzymatic activity.     

Diethylstilbestrol-Induced Estrogen Receptor-Dependent [Ca2+]i Rises and Apoptosis in Chinese Hamster Ovary (CHO) Cells

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations ≥ 1∝ increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. In Ca²⁺-free medium, after pretreatment with 50∝ DES, 1∝ thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)-induced [Ca²⁺]_i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca²⁺]_i rises. At a concentration of 5∝, DES increased cell viability. At concentrations of 100–200 μ M, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 μM DES on viability was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′ -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 μ M)-induced [Ca²⁺]_i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 μ M). ICI-182,780 did not affect 5 μ M DES-induced increase in viability but partly reversed 100 μ M DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca²⁺]_i rises by stimulating estrogen receptors leading to Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca²⁺-dependent pathway.     

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.