Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments

Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.     

Ca2+ release in the endoplasmic reticulum of guinea pig peritoneal macrophages

Passive permeability of the endoplasmic reticulum of saponin-treated macrophages to Ca2+ was studied by the filtration method using 45Ca. The Ca2+ release from the endoplasmic reticulum of macrophages was enhanced by the presence of submicromolar concentrations of Ca2+ in the medium. The Ca2+ release was enhanced by caffeine, and suppressed by MgCl2. These phenomena are similar to the Ca2+-induced Ca2+ release reported for the sarcoplasmic reticulum of skeletal muscle. On the other hand, adenine suppressed the Ca2+ release from the endoplasmic reticulum, while it reportedly enhanced the Ca2+-induced Ca2+ release of the skeletal muscle. The threshold concentration of Ca2+ for the Ca2+-induced Ca2+ release was approximately 10(-8) M in the presence of 0.95 mM MgCl2 in macrophages. The spontaneous spreading of macrophages and spontaneous migration of macrophages were inhibited by adenine, and also by caffeine in spite of the enhancement of the Ca2+-induced Ca2+ release.     

Calreticulin, Ca2+, and calcineurin - signaling from the endoplasmic reticulum

Calcium (Ca2+) is a universal signalling molecule involved in many aspects of cellular function. The majority of intracellular Ca2+ is stored in the endoplasmic reticulum and once Ca2+ is released from the endoplasmic reticulum, specific plasma membrane Ca2+ channels are activated, resulting in increased intracellular Ca2+. In the lumen of the endoplasmic reticulum, Ca2+ is buffered by Ca2+ binding chaperones such as calreticulin. Calreticulin-deficiency is lethal in utero due to impaired cardiac development and in the absence of calreticulin, Ca2+ storage capacity within the endoplasmic reticulum and inositol 1,4,5-trisphosphate (InsP3) receptor mediated Ca2+ release from the endoplasmic reticulum are compromised. Over-expression of constitutively active calcineurin in the heart rescues calreticulin-deficient mice from embryonic lethality. This observation indicates that calreticulin is a key upstream regulator of calcineurin in Ca2+-signalling pathways and highlights the importance of the endoplasmic reticulum and endoplasmic reticulum-dependent Ca2+ homeostasis for cellular commitment and tissue development during organogenesis. Furthermore, Ca2+ handling by the endoplasmic reticulum has profound effects on cell sensitivity to apoptosis. Signalling between calreticulin in the lumen of the endoplasmic reticulum and calcineurin in the cytoplasm may play a role in the modulation of cell sensitivity to apoptosis and the regulation of Ca2+-dependent apoptotic pathways.     

GTP mobilization of Ca2+ from the endoplasmic reticulum of islets. Comparison with myo-inositol 1,4,5-trisphosphate.

The effect of the guanine nucleotide GTP on Ca2+ release from the endoplasmic reticulum of digitonin-permeabilized islets was investigated. maximal and half-maximal Ca2+ release were observed at 5 microM- and 2.5 microM-GTP respectively. GTP caused a rapid release of Ca2+ from the endoplasmic reticulum, which was complete within 1 min. GTP-induced Ca2+ release was structurally specific and required the hydrolysis of GTP. The combination of maximal concentrations of GTP (10 microM) and myo-inositol 1,4,5-trisphosphate (IP3) (10 microM) resulted in an additive effect on Ca2+ release from the endoplasmic reticulum. GDP (100 microM), which inhibits GTP-induced Ca2+ release, did not affect IP3-induced Ca2+ release. Furthermore, GTP-induced Ca2+ release was not independent on submicromolar free Ca2+ concentrations, unlike IP3-induced Ca2+ release. These observations suggest that mechanistically GTP-induced Ca2+ release is different from IP3-induced Ca2+ release from the endoplasmic reticulum.     

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes.

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.     

Ca2+ transporting activity of membrane fractions isolated from the post-mitochondrial supernatant of rat liver

The post-mitochondrial supernatant of rat liver contains two vesicular fractions which transport Ca2+ actively. The heavier fraction, sedimenting at 17.500 xg, 20 min, is enriched in plasma membrane markers and apparently contains both a Ca2+ pumping ATPase and a Na+/Ca2+ exchanger. These activities have been attributed to the plasma membrane vesicles. The lighter fraction, sedimenting at 100.000 xg, 60 min, is enriched in endoplasmic reticulum markers, and contains only a Ca2+ pumping ATPase, which can be differentiated from that of the heavier fraction on the basis of the sensitivity to vanadate. The Ca2+ pumping activity of endoplasmic reticulum appears to be regulated by both a cAMP-dependent, and a calmodulin-dependent system. The former system involves a heat-stable protein fraction from the cytosol. The regulation by the cAMP and the calmodulin-dependent systems involves the phosphorylation of several proteins in the endoplasmic reticulum membrane.     

Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine

Two microsomal subfractions from isolated rat pancreatic acini were produced by centrifugation through a discontinuous sucrose density gradient and characterized by biochemical markers. The denser fraction ( SF2 ) was a highly purified preparation of rough endoplasmic reticulum; the less-dense fraction ( SF1 ) was heterogeneous and contained Golgi, endoplasmic reticulum and plasma membranes. 45Ca2+ accumulation in the presence of ATP and its rapid release after treatment with the bivalent-cation ionophore A23187 were demonstrated in both fractions. The pH optimum for active 45Ca2+ uptake was approx. 6.8 for the rough endoplasmic reticulum ( SF2 ) and approx. 7.5 for SF1 . Initial rate measurements were used to determine the affinity of the rough-endoplasmic-reticulum uptake system for free Ca2+. An apparent Km of 0.16 +/- 0.06 microM and Vmax. of 21.5 +/- 5.6 nmol of Ca2+/min per mg of protein were obtained. 45Ca2+ uptake by SF1 was less sensitive to Ca2+, half-maximal uptake occurring at 1-2 microM-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 microM-carbamylcholine, 45Ca2+ uptake was increased in the rough endoplasmic reticulum. The increased uptake was due to a higher Vmax. with no significant change in Km. No effect was observed on 45Ca2+ uptake by SF1 . In conclusion, two distinct non-mitochondrial, ATP-dependent calcium-uptake systems have been demonstrated in rat pancreatic acini. One of these is located in the rough endoplasmic reticulum, but the precise location of the other has not been determined. We have shown that the Ca2+-transporting activity in the rough endoplasmic reticulum may have an important role in maintaining the cytosolic free Ca2+ concentration in resting acinar cells and is involved in Ca2+ movements which occur during stimulation of enzyme secretion.     

Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum

Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.     

Evidence that store-operated Ca2+ channels are more effective than intracellular messenger-activated non-selective cation channels in refilling rat hepatocyte intracellular Ca2+ stores

Liver cells possess store-operated Ca2+ channels (SOCs) with a high selectivity for Ca2+ compared with Na+, and several types of intracellular messenger-activated non-selective cation channels with a lower selectivity for Ca2+ (NSCCs). The main role of SOCs is thought to be in refilling depleted endoplasmic reticulum Ca2+ stores [Cell Calcium 7 (1986) 1]. NSCCs may be involved in refilling intracellular stores but are also thought to have other roles in regulating the cytoplasmic-free Ca2+ and Na+ concentrations. The ability of SOCs to refill the endoplasmic reticulum Ca2+ stores in hepatocytes has not previously been compared with that of NSCCs. The aim of the present studies was to compare the ability of SOCs and maitotoxin-activated NSCCs to refill the endoplasmic reticulum in rat hepatocytes. The experiments were performed using fura-2FF and fura-2 to monitor the free Ca2+ concentrations in the endoplasmic reticulum and cytoplasmic space, respectively, a Ca2+ add-back protocol, and 2-aminoethyl diphenylborate (2-APB) to inhibit Ca2+ inflow through SOCs. In cells treated with 2,5-di-t-butylhydroquinone (DBHQ) or vasopressin to deplete the endoplasmic reticulum Ca2+ stores, then washed to remove DBHQ or vasopressin, the addition of Ca2+ caused a substantial increase in the concentration of Ca2+ in the endoplasmic reticulum and cytoplasmic space due to the activation of SOCs. These increases were inhibited 80% by 2-APB, indicating that Ca2+ inflow is predominantly through SOCs. In the presence of 2-APB (to block SOCs), maitotoxin induced a substantial increase in [Ca2+](cyt), but only a modest and slower increase in [Ca2+](er). Under these conditions, Ca2+ inflow is predominantly through maitotoxin-activated NSCCs. It is concluded that SOCs are more effective than maitotoxin-activated NSCCs in refilling the endoplasmic reticulum Ca2+ stores. The previously developed concept of a specific role for SOCs in refilling the endoplasmic reticulum is consistent with the results reported here.     

Effect of W7 on Ca2+ uptake and Ca2+-ATPase activities of the endoplasmic reticulum of rat liver

In an initial attempt to use calmodulin antagonists as probes to study the role of calmodulin in the modulation of Ca2+ uptake activity in the endoplasmic reticulum of rat liver, we noticed that W7 had a differential effect on the Ca2+ uptake and Ca2+-ATPase activities. To test the specificity of this effect and explore the underlying mechanism, we examined the effects of W7 on Ca2+ accumulation and release by endoplasmic reticulum in both permeabilized hepatocytes and a subcellular membrane fraction (microsomes) enriched in endoplasmic reticulum. W7 reduced the steady-state Ca2+ accumulation in both preparations in a dose-dependent fashion but the half-maximal inhibitory concentrations were different for Ca2+ accumulation (90 microM) and Ca2+-ATPase activity (500 microM). Kinetic analysis indicated that the inhibition of both Ca2+ uptake and Ca2+-ATPase activity by W7 was noncompetitive with respect to Ca2+ and ATP. Addition of W7 did not enhance the rate of Ca2+ efflux from microsomes after Ca2+ influx had been terminated. The effect of W7 was apparently not related to its calmodulin antagonist properties as the phenomenon could not be demonstrated with the other more specific calmodulin antagonists, calmidazolium or compound 48/80. A similar observation with W7 has also been reported with the endoplasmic reticulum of pancreatic islets (B. A. Wolf, J. R. Colca, and M. L. McDaniel (1986) Biochem. Biophys. Res. Commun. 141, 418-425). We concluded that the effects of W7 on microsomal Ca2+ handling were not the result of increased membrane permeability to Ca2+ but rather were due to dissociation of Ca2+ uptake from Ca2+-ATPase activity.     

Kinetic characteristics of Ca2+, Mg2+-ATPases in cells of the submandibular salivary gland of rats

Kinetic properties of Ca2+, Mg2+-ATPases membranes from acinar cells of rat submandibular salivary glands have been investigated. It was found that kinetics of ATP hydrolysis dependent on Ca2+, Mg2+-ATPases corresponds to the first-order reaction during first 2 min. It was found that the initial velocity of the reaction (V0), maximal amount of the reaction product (Pmax) and characteristic time of the reaction (T) comprised 1.8 +/- 0.4 and 1.6 +/- 0.2 mmole Pi/min per 1 mg protein, 7.5 +/- 1.3 and 1.4 +/- 0.2 mmole Pi/mg protein and 4.1 +/- 0.7 min and 1.1 +/- 0.1 for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. High- and low-affinity sites of ATP and Ca2+-binding in Ca2+-ATPases from plasma and endoplasmic reticulum membranes were identified. Negative cooperation in ATP binding to Ca2+-ATPase from plasma membrane and a positive cooperation for Ca2+-ATPase from endoplasmic reticulum has been found. Ca2+ binding to low-affinity sites of both Ca2+-ATPases showed no cooperation, while Ca2+ binding to high-affinity sites showed the positive cooperation. Using the Hill's coordinates we have found the values of the Mg2+ Michaelis constant (K(Mg)) which yielded 3.89 x 10(-5) and 3.80 x 10(-5) mole/l for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. It is supposed that obtained data are important for further studies of molecular and membrane mechanisms involved in the regulation of intracellular calcium signalling and secretion by salivary acinar cells.     

Modulation of active Ca2+ uptake by the islet-cell endoplasmic reticulum.

The possible effects of calmodulin and cyclic AMP on active Ca2+ uptake by the islet-cell endoplasmic reticulum were investigated. Neither calmodulin nor cyclic AMP affected the rate of active Ca2+ uptake, or the steady-state filling capacity of the endoplasmic reticulum when measured in the absence of oxalate. Consistent with these results, calmodulin did not activate the Ca2+-stimulated ATPase activity associated with this cell fraction. During the course of these experiments., it was unexpectedly discovered that the rate of Ca2+ uptake, as well as the steady-state Ca2+ filling capacity of the endoplasmic reticulum, were markedly increased by unidentified factor(s) in the cytosol. This effect could be demonstrated by reconstitution of the membranes in cytosol, or by direct addition of fresh or dialysed cytosol to the Ca2+ uptake assays. The degree of activation by the cytosol indicates that the endoplasmic reticulum may play a prominent role in controlling beta-cell Ca2+ concentrations and that the unidentified activator(s) present in the cytosol may be involved in regulation of this function.     

Subcellular calcium and magnesium mobilization in rat liver stimulated in vivo with vasopressin and glucagon

The total Ca2+ content of the endoplasmic reticulum and the total Ca2+ and Mg2+ content of mitochondria were determined by electron probe microanalysis of rat liver rapidly frozen in vivo following brief (5-15 s) stimulation with vasopressin or prolonged (10-12 min) stimulation with vasopressin + glucagon. Brief vasopressin injection into the anterior mesenteric vein released 1.8 +/- 0.3 (S.D.) mmol of Ca2+/kg dry weight, from the rough endoplasmic reticulum (p less than 0.01), reducing Ca2+ content of the endoplasmic reticulum from 4.4 +/- 0.2 (S.E.) (controls) to 2.6 +/- 0.2 mmol of Ca2+/kg dry weight. Following vasopressin injection, endoplasmic reticulum Ca2+ was also significantly (p less than 0.025) lower than that in brief sham injected animals (3.5 +/- 0.2 mmol/kg dry weight). Mitochondrial Ca2+ was between 1.0 and 2.3 (+/-0.2) mmol/kg dry weight of mitochondrion, under all conditions studied, and no significant differences were observed. Both hormonal and brief sham injection into the anterior mesenteric vein increased mitochondrial Mg2+ from 42 (+/-0.8) to 49 (+/-1.8) mmol/kg dry weight (p less than 0.05). Hormonal stimulation of Mg2+ uptake was further confirmed by injection of vasopressin + glucagon into the jugular vein (to avoid any stimulation of the liver by the anterior mesenteric vein injection itself); mitochondrial Mg2+ increased from 43 (+/-0.9) (10-min sham) to 57 (+/-1.3) mmol/kg dry weight, with 10-min vasopressin + glucagon injection (p less than 0.01). These results demonstrate that hormones can release Ca2+ from the endoplasmic reticulum and modulate mitochondrial Mg2+ content in vivo without causing detectable changes in mitochondrial Ca2+.     

Effect of the antidepressant sertraline on Ca2+fluxes in Madin-Darby canine renal tubular cells

The effect of the antidepressant sertraline on cytosolic-free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether sertraline changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Sertraline at concentrations between 1and 100 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+ implicating Ca2+ entry and release both contributed to the [Ca2+]i rise. Sertraline induced Mn2+ influx, leading to quench of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by suppression of phospholiapase A2 but not by store-operated Ca2+ channel blockers and protein kinase C/A modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors nearly abolished sertraline-induced Ca2+ release. Conversely, pretreatment with sertraline partly reduced inhibitor-induced [Ca2+]i rise, suggesting that sertraline released Ca2+ from endoplasmic reticulum. Inhibition of phospholipase C did not much alter sertraline-induced [Ca2+]i rise. Collectively, in MDCK cells, sertraline induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive Ca2+ channels.     

Vanadyl and vanadate inhibit Ca2+ transport systems of the adipocyte plasma membrane and endoplasmic reticulum

Vanadate and vanadyl have many insulin-mimetic effects on cellular metabolism and also have been shown to alter cellular Ca2+ fluxes. In this report, vanadate and vanadyl, like insulin, are shown to inhibit the plasma membrane (Ca2+ + Mg2+)-ATPase/Ca2+ transport system as well as Ca2+ transport by endoplasmic reticulum from rat adipocytes. Ca2+ transport by the endoplasmic reticulum was inhibited half-maximally (I50) by vanadate and vanadyl at concentrations of 30 and 33 microM, respectively. Inhibition of the plasma membrane Ca2+ transport by vanadate and vanadyl was less sensitive, with I50 values of 144 and 92 microM, respectively. These I50 values for plasma membrane Ca2+ transport were similar when measured under conditions of calmodulin-stimulated and non-calmodulin-stimulated Ca2+ transport. The predominant effect of both ions on the kinetic parameters of Ca2+ transport was a substantial decrease in the Vmax by 43-46% for both transport systems. An increase in intracellular Ca2+ following the inhibition of the (Ca2+ + Mg2+)-ATPase/Ca2+ pump in the plasma membrane and endoplasmic reticulum by these vanadium ions may result, at least in part, in the observed insulin-mimetic alterations in cellular metabolism.     

Calcium sequestration in the liver: Review article

Hepatic parenchymal cells maintain intracellular total and cytosolic free Ca2+ levels by: entry of Ca2+ through channels, extrusion of Ca2+ by an outwardly directed Ca2+ pump, and controlled sequestration into intracellular pools. The mechanism of Ca2+ inflow is poorly characterized. The plasma membrane Ca2+ channels seem to share some of the characteristics of Ca2+ channels in excitable cells, but also differ from them. The outwardly directed plasma membrane Ca2(+)-ATPase is a calmodulin independent, P-type enzyme. Ca2+ uptake into the endoplasmic reticulum is due to the activity of a different Ca2(+)-ATPase, which is similar in molecular weight and shares antigenic determinants with the sarcoplasmic reticulum enzyme. In addition, mitochondria and nuclei also take up calcium. The exact mechanism by which Ca2+ is released from intracellular organelles is not well known. Several mechanisms for Ca2+ release from the endoplasmic reticulum were reported, including IP3 and GTP-induced. The most effective identified way of eliciting Ca2+ release from microsomal fraction is by the oxidation of critical -SH groups. This mechanism is likely to be involved in the rise of cytosolic Ca2+ observed in many situations of hepatocellular injury. In addition to being sequestered into subcellular organelles, some of the intracellular Ca2+ is bound to specific Ca2+ binding proteins. Both calmodulin and members of the annexin family were identified in the liver. Stimulation of the liver with gluconeogenic hormones results in increased Ca2+ entry into the cell, the release of Ca2+ from intracellular pools, and an oscillatory increase in free cytosolic Ca2+ levels. Extensive research is still needed for the elucidation of the exact mechanisms by which these events occur.     

Effect of gossypol on intracellular Ca2+ regulation in human hepatoma cells

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 microM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 microM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 microM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 microM gossypol was not changed by inhibiting phospholipase C with 2 microM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 microM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx.     

Independent effects of the broccoli-derived compound sulforaphane on Ca2+ influx and apoptosis in Madin-Darby canine renal tubular cells

This study explored whether sulforaphane changed basal [Ca2+]i levels in suspended Madin-Darby canine kidney (MDCK) cells by using fura-2 as a Ca(2+)-sensitive fluorescent dye. Sulforaphane at concentrations between 2.5-10 microM increased [Ca2+]i in a concentration-dependent manner. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid but not by Ca2+ channel blockers such as nifedipine, nimodipine, nicardipine, diltiazem, verapamil, econazole and SK&F96365. The Ca2+ signal was abolished by removing extracellular Ca2+. In Ca(2+)-free medium, pretreatment with sulforaphane did not alter the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-induced Ca2+ release suggesting sulforaphane did not induce slow Ca2+ release from endoplasmic reticulum. At concentrations between 1 and 20 microM, sulforaphane induced concentration-dependent decrease in cell viability which was not affected by pre-chelation of cytosolic Ca2+ with BAPTA/AM. Flow cytometry data suggest that 20 (but not 5 and 10) microM sulforaphane induced significant increase in sub G1 phase indicating involvement of apoptosis. Collectively, in MDCK cells, sulforaphane induced [Ca2+]i rises by causing Ca2+ entry through phospholipase A2-sensitive pathways without inducing Ca2+ release from the endoplasmic reticulum. Sulforaphane also induced Ca(2+)-independent cell death that might involve apoptosis.     

The calcium pump of the liver nuclear membrane is identical to that of endoplasmic reticulum.

The envelope membrane of rat liver nuclei contains a P-type Ca(2+)-transporting pump, revealed by the presence of a Ca(2+)-stimulated phosphoenzyme. The level of the nuclear phosphoenzyme in autoradiographed polyacrylamide gels was decreased by lanthanum, as typically observed in the endoplasmic reticulum Ca2+ pump. It was also decreased by thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, two accepted inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. Comparative proteolysis of the phosphorylated enzyme of liver microsomes (endoplasmic reticulum) and nuclear membranes revealed an identical cleavage pattern. In addition, antibodies raised against the endoplasmic reticulum Ca2+ pump cross-reacted with the pump in the nuclear membranes. The findings show that nuclear membranes contain a Ca(2+)-transporting pump closely related to that of the endoplasmic reticulum, if not identical to it. The pump is likely to be involved in the control of nuclear free calcium.