Microsomal Ca2 Research Articles

Energy restriction suppresses microsomal Ca 2+-ATPase activities in various organs in C57BL/6 female mice in both euthermic and torpor states

C57BL/6 female mice were fed a daily control diet ( n = 5, 3.9 g/day, 95 kcal/week) or ER diet ( n = 5, 2.3 g/day, 48 kcal/week) at 1800 h from 6 weeks of age. Telemetry, conducted at 6 months of age, confirmed that all ER mice entered daily torpor (core body temperature less than 31°C) for 6.63 ± 2.34 h/day while control mice were euthermic (> 35° C). In vitro activities of microsomal Ca 2+-ATPase were determined in the brain, liver, salivary gland and kidney from these mice at 6 months of age. Assays were performed at three incubation temperatures of 37°C, 31°C and 25°C. In assays at 37°C, the activities of Ca 2+-ATPase in the brain and salivary gland from ER mice were lower than those in corresponding organs from control mice. The suppression became profound as the incubation temperature decreased. On the other hand, at 37°C Ca 2+-ATPase activities in the liver and kidney from ER mice were not lower than those in corresponding organs from control mice, but decreased significantly at low temperatures. Microsomal Ca 2+-ATPase activities thus appeared to be reduced in ER mice, although it remains unknown whether the present results represent reduced in vivo capacities to regulate cytosolic Ca 2+ concentrations.

Bradykinin inhibits cyclic AMP accumulation in D384-human astrocytoma cells via a calcium-dependent inhibition of adenylyl cyclase

Bradykinin causes a concentration-dependent, transient rise in intracellular Ca 2+ and a sustained inhibition of forskolin-, dopamine- and 5′- N-ethyl-carboxamidoadenosine (NECA)-stimulated cAMP accumulation in D384 astrocytoma cells. Chelation of intracellular calcium abolished bradykinin's inhibitory effect on cAMP accumulation. Chelating extracellular Ca 2+ did not block the initial, but eliminated the sustained inhibition of cAMP accumulation. Increasing Ca 2+ influx by calcium ionophore A23187 caused a concentration-dependent inhibition of stimulated cAMP accumulation. A hydroquinone derivative 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), which inhibits microsomal Ca 2+ sequestration, did not mimic the effect of bradykinin, although it increased [Ca 2+] i even more than A23187 did. The inhibitory effect of bradykinin was not mediated by Ca 2+/CaM-dependent stimulation of phosphodiesterase (PDE). Forskolin-stimulated adenylyl cyclase activity was inhibited by Ca 2+ (10 −7 to 10 −3 M), both in ethyleneglycol-bis-( β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA) washed and native D384 plasma membranes. This effect was not altered by calmodulin (CaM) or CaM-antagonists. Bradykinin treatment, which attenuates cAMP accumulation in intact cells, did not do so in plasma membranes. These findings suggest that bradykinin-induced inhibition of cAMP formation in D384 cells requires mobilization of [Ca 2+] i and subsequent entry of Ca 2+ which directly interacts with a component of the adenylyl cyclase system.

The IP3-sensitive calcium store of HIT cells is located in a surface-derived vesicle fraction

Electron microscopic and biochemical techniques were used to study the cellular localization of the ATP-dependent, IP3-sensitive, Ca 2+ store in the glucose- and phosphatidylinositol(PI) agonist-sensitive hamster insulinoma cell line HIT-T15. Scanning electron microscopy revealed conspicuous shape changes of the microvilli following stimulation of these cells with bombesin or thapsigargin. These changes closely resemble those previously shown to accompany stimulation of hexose transport in adipocytes with insulin [J. Cell. Physiol. 142 (1990) 1-14]. Using a hydrodynamic shearing technique for the isolation of microvilli, two cell surface-derived vesicle fractions were prepared containing 80% of the total cellular Ca 2+-storing activity. In contrast, subcellular fractionation using normal homogenization with a glass/teflon homogenizer yielded the well-known distribution of the Ca 2+-storing activity which is then predominantly recovered within the microsomal fraction. The surface-derived vesicle fraction was clearly distinguished from the microsomal fraction by its high content of Na +/K +-ATPase and an immunoreactive fragment of the GluT-1 glucose transporter isoform which both are not detectable in the microsomal fraction isolated from homogenates from sheared cells. The Ca 2+ uptake properties of the cell surface-derived vesicle fractions including the vanadate, A23187, and thapsigargin sensitivity were found to be identical with those described for the microsomal Ca 2+ stores of various cell types. Inositol 1,4,5-trisphosphate (IP3) at 1 μM induced a maximal release of 35–40% of the stored Ca 2+ from these vesicles.

Depletion and refilling of intracellular Ca2+ stores induce oscillations of Ca2+ current

An agonist-induced Ca2+ influx pathway in vascular endothelium and other nonexcitable cells is closely aligned with the depletion of microsomal Ca2+ stores. The mechanism by which this occurs is unknown. In these studies 2',5',-di(tert-butyl-1,4-benzohydroquinone, a specific inhibitor of the microsomal Ca(2+)-adenosinetriphosphatase, and patch-clamp recordings were used to evaluate the relationship of inward Ca2+ current to the depletion of intracellular Ca2+ stores. The results demonstrate that depletion and refilling of Ca2+ stores control the amplitude of an electrogenic influx pathway in vascular endothelium. Prominent fluctuations in Ca2+ current occur when there is an imbalance between depletion and refilling of the stores. Furthermore, the studies suggest that the Ca2+ influx pathway is spatially in close association with the intracellular store.

Myoinositol Tris-Phosphate-Phytase Complex as an Elicitor in Calcium Mobilization in Plants

Mobilization of Ca 2+ from microsomal /vacuolar fractions was detected when InsP 6-phytase was added after a definite time of hydrolysis which coincides with the time (20-30 min) of optimal production of Ins(2, 4, 5)P 3 bound to phytase. The in vitro constituted Ins(1, 4, 5)P 3or Ins(2, 4, 5)P 3-phytase complex is also effective in releasing Ca 2+. InsP 3-phytase complex releases 45% more microsomal Ca 2+ than that released by free InsP 3 under identical conditions. Other inositol-phytase complexes are ineffective. Furthermore InsP 3-phytase complex is recognized by putative receptor associated with microsomal fraction suggesting that the myoinositol tris-phosphate-Phytase complex can act as an elicitor in Ca 2+ mobilization in plant systems where phytate and phytase occur.

Primary structure and characterization of an Arabidopsis thaliana calnexin-like protein.

A cDNA clone (pTE-83) encoding a protein (CNX1p) related to the microsomal Ca(2+)-binding protein, calnexin, was isolated from an Arabidopsis thaliana expression library. Southern and Northern hybridization indicated that CNX1 is a single-copy gene encoding a message of 1900 nucleotides. The open reading frame encodes a polypeptide with 530 amino acids, a molecular mass of 60.5 kDa, and overall 48% identity to dog calnexin. Both animal calnexin and CNX1p contain a large luminal domain followed by a single potential membrane-spanning domain near the C terminus and a small C-terminal domain exposed to the cytoplasm. The in vitro translation product from the cloned cDNA yielded a polypeptide of 67 kDa that was co-translationally imported into dog microsomes and processed to a 64-kDa product. Antibodies generated against the C-terminal half of the protein cross-react with an identically sized protein present in the microsomal fraction from Arabidopsis. Both the imported and native proteins are cleaved by trypsin to a 59-kDa product indicating that the gene product was indeed correctly processed and translocated into dog microsomes and that the membrane topology of CNX1p resembles that of dog calnexin. The presence of a calnexin-like protein within the plant kingdom indicates that this protein is widespread and involved in processes fundamental to all eukaryotes.

Effects of tauroursodeoxycholic acid on cytosolic Ca2+ signals in isolated rat hepatocytes

Background: Tauroursodeoxycholic acid (TUDCA) is of potential benefit in cholestatic disorders. However, the effects of TUDCA on cytosolic free calcium [(Ca2+)i], which regulates hepatocyte secretion, are unknown. Methods: The effect of TUDCA on (Ca2+)i was investigated in groups of isolated rat hepatocytes by microspectrofluorometry and in single cells by confocal line scanning microscopy. Results: Administration of TUDCA (5–50 μmol/L) induced a nearly fourfold increase of basal levels of (Ca2+)i. After a 15 minute treatment period, the TUDCA (10 μmol/L)-induced change in (Ca2+)i was higher than that of other monodi-, and trihydroxy bile acids at equimolar concentrations. Pretreatment with TUDCA (10 μmol/L) markedly reduced or abolished increases in (Ca2+)i induced by phenylephrine (1 μmol/L), the microsomal Ca2+ translocase inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (25 μmol/L), or taurolithocholic acid (10–25 μmol/L). In Ca2+-free medium, TUDCA caused only a reduced and transient increase in (Ca2+)i. TUDCA (10 μmol/L) induced Ca2+ oscillations in all single cells that responded. However, levels of inositol-1,4,5-trisphosphate (IP3) in hepatocytes were not increased by treatment with TUDCA (10 μmol/L). Conclusions: TUDCA at physiological concentrations potently modulates (Ca2+)i signals in hepatocytes by (1) mobilizing microsomal IP3-sensitive Ca2+ stores by an IP3-independent mechanism, (2) initiating Ca2+ oscillations, and (3) inducing influx of extracellular Ca2+.

Carbachol-Induced Potassium Release in Rat Parotid Acini: Comparison of the Roles of Cytosolic Ca2+ and Protein Kinase C

Carbachol (CCh) stimulated K+ release from rat parotid acini. Treatment with the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) or the intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly suppressed the CCh-induced K+ release. Combined addition of the Ca2+ ionophore ionomycin and the microsomal Ca(2+)-ATPase inhibitor thapsigargin caused a rapid increase in cytosolic Ca2+ concentration ([Ca2+]i) and resulted in a marked release of K+. In the absence of extracellular Ca2+, CCh or a combination of ionomycin and thapsigargin caused a transient release of K+ which correlated well with the transient change in [Ca2+]i. On the other hand, phorbol 12-myristate 13-acetate (PMA) did not potentiate the CCh-induced K+ release, although the CCh-induced amylase release was significantly enhanced in the presence of PMA. Staurosporine, a protein kinase C-inhibitor, did not inhibit the CCh-induced K+ release, which was in contrast with its inhibitory effect on amylase release. These results suggest that the K+ release from rat parotid acini induced by CCh stimulation is mediated by a rapid increase in [Ca2+]i but is not associated with activation of protein kinase C. This signal pathway is different from that for amylase release where activation of protein kinase C plays an important role.

Calcium transport and calcium activated atpase activity in microsomal vesicles of rat gastric mucosa

1.1. Microsomal and plasma membrane vesicles, isolated from rat gastric mucosa, were found to exhibit Ca2+-dependent ATPase activities of 14.1 ± 1.4 and 7.8 ± 1.1 μmol/mg/hr, respectively. The optimum conditions for the microsomal Ca2+-ATPase was pH 6–7, and required Mg2+, while divalent cation such as Cu2+, Zn2+, Fe2+, Ba2+ and Cd2+ had no significant effect.2.2. As in the case of Ca2+, Mg2+-ATPase, the Ca2+ uptake activity of the microsomal membrane required Mg2+. Both processes were stimulated by submicro molar concentrations of Ca2+ and the apparent Km for Ca2+, Mg2+ ATPase and Ca2+ uptake activities were 0.06 μM and 0.02 μM, respectively.3.3. Divalent cations Ba2+ and Fe2+, inhibited both microsomal activities, while Zn2+ and Cd2+ showed no effect on them. However, the monovalent cation K+ did not stimulate Ca2+, Mg2+-ATPase and Ca2+ uptake activities.4.4. The Ca2+ pumping ATPase of rat gastric mucosal microsome cross-reacted with a monoclonal antibody (mAb-5F10) against the human erythrocyte Ca2+ pump. The apparent molecular weight of mucosal Ca2+ pump was 98 kDa.5.5. Close relationship between the kinetic parameters of Ca2+, Mg2+-ATPase and Ca2+ uptake activities, and the cross reaction of 98 kDa protein of mucosal microsome with erythrocyte Ca2+ pump antibody, strongly suggest the expression of Ca2+ pump in rat gastric mucosa.

Thapsigargin Amplifies the Platelet Procoagulant Response Caused by Thrombin

The platelet procoagulant response involves an increase in surface-exposed phosphatidylserine, which allows binding and assembly of enzyme complexes of the coagulation pathway resulting in acceleration of the clotting process. This response essentially requires the presence of extracellular Ca2+, and varies in extent with the type of agonist used. In the present paper we demonstrate that the moderate procoagulant response of human platelets caused by thrombin is strongly amplified by the presence of thapsigargin, an inhibitor of the microsomal Ca(2+)-ATPase. Thapsigargin, like thrombin, has only a weak effect on procoagulant activity. The large increase in procoagulant activity observed with the combined action of these two agonists is associated with increased shedding of microvesicles from the platelet plasma membrane as well as with inhibition of transport of a fluorescent-labeled analog of phosphatidylserine from the outer to the inner leaflet of the plasma membrane by the aminophospholipid translocase. The latter two observations support current concepts regarding the mechanism of development of procoagulant activity. Although the synergistic effect of thapsigargin on thrombin-induced procoagulant activity is at least in part due to the high levels of intracellular [Ca2+] evoked by these agonists, the data clearly indicate that a rise of the intracellular [Ca2+] is insufficient to completely explain this response. The present findings suggest that additional factors control expression of procoagulant activity upon stimulation of platelets by thrombin.

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. II. Effects of thapsigargin on the cellular Ca2+ homeostasis.

Evidence was provided, in the preceding paper (Thuringer & Sauvé, 1992), that the external Ca(2+)-dependent phase of the Ca2+ signals evoked by bradykinin (BK) or caffeine in bovine aortic endothelial cells (BAE), differ in their respective sensitivity to procaine. To examine whether the emptying of the InsP3-sensitive Ca2+ store is the signal for activating the agonist-evoked Ca2+ entry, we have investigated the effects of thapsigargin (TSG), a known inhibitor of the microsomal Ca(2+)-ATPase activity in a variety of cell types, via the activity of calcium-activated potassium channels [K(Ca2+) channels]. In cell-attached experiments, the external application of TSG caused a sustained or oscillatory activation of K(Ca2+) channels depending on both the cells and doses tested. The TSG-evoked channel activity could be reversibly blocked by removing extracellular Ca2+, and strongly decreased by adding 10 mM procaine to the bath medium. In Ca(2+)-free external conditions, TSG did not promote an apparent Ca2+ discharge from internal stores but prevented in a dose- and time-dependent manner the subsequent agonist-evoked channel activity related to the release of internally sequestered Ca2+. These results confirm that TSG and BK release Ca2+ from the same internal stores but with different kinetics. Because the channel response to caffeine was found to be poorly sensitive to procaine, in contrast to that evoked by BK and TSG, it may be concluded that both BK and TSG activate the same Ca2+ entry pathway. Therefore, the emptying of the InsP3-sensitive Ca2+ store is likely to be the main signal for activating the agonist-evoked Ca2+ entry in BAE cells.

Caffeine as a probe for human cytochromes P450: validation using cDNA-expression, immunoinhibition and microsomal kinetic and inhibitor techniques.

The molecular basis for the use of caffeine (CA; 1,3,7-trimethylxanthine) as a probe for specific human cytochromes P450 has been investigated. The CA 1-, 3- and 7-demethylations (to form theobromine, paraxanthine and theophylline, respectively) all followed biphasic kinetics in human liver microsomes. Mean apparent Km values for the high- and low-affinity components of the demethylations ranged from 0.13-0.31 nM and 19.2-30.0 mM, respectively. cDNA-expressed CYP1A2 catalysed all three CA demethylations, and the apparent Km for CA 3-demethylation (the major metabolic pathway in humans) by the expressed enzyme was similar to the Km for the high-affinity liver microsomal CA 3-demethylase. IC50 values for inhibition of the CA demethylations by alpha-naphthoflavone were similar for both expressed CYP1A2 and the high-affinity microsomal demethylases. Moreover, CA was a competitive inhibitor of expressed CYP1A2 catalysed phenacetin O-deethylation, with the apparent Ki (0.080 mM) closely matching the apparent Km (0.082 mM) for CA 3-demethylation by the expressed enzyme. Expressed CYP1A1 was additionally shown to catalyse the 3-demethylation of CA, although activity was lower than that observed for CYP1A2. While these data indicate that CYP1A2 is responsible for the high-affinity component of human liver CA 3-demethylation, two limitations associated with the use of CA as an in vitro probe for CYP1A2 activity have been identified: (i) CA 3-demethylation reflects hepatic CYP1A2 activity only at appropriately low substrate concentrations; and (ii) CA is a non-specific CYP1A substrate and CYP1A1 may therefore contribute to CA 3-demethylase activity in tissues in which it is expressed. An anti-CYP3A antibody essentially abolished the 8-hydroxylation of CA to form trimethyluric acid, suggesting formation of this metabolite may potentially serve as a marker of CYP3A isozyme(s) activity.

Evidence for two pathways of receptor-mediated Ca2+ entry in hepatocytes.

Receptor-mediated Ca2+ entry was studied in fura-2-loaded isolated hepatocytes. Emptying of internal Ca2+ stores by treatment with either the Ca(2+)-mobilizing hormone vasopressin or the inhibitors of the microsomal Ca2+ pump, 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) or thapsigargin, stimulated Ca2+ entry, as indicated by a rise in the cytosolic free Ca2+ concentration after Ca2+ was added to cells suspended in nominally Ca(2+)-free medium. The enhancement of Ca2+ entry was proportional to the degree of depletion of the intracellular Ca2+ pool and occurred also after removal of vasopressin from its receptor. In contrast, the stimulation of Mn2+ entry by vasopressin required the continuous presence of the agonist, since it was prevented by the addition of vasopressin receptor antagonist. This effect was observed under conditions where refilling of the agonist-sensitive pool was prevented by using nominally Ca(2+)-free medium. Unlike vasopressin, tBuBHQ or thapsigargin did not stimulate Mn2+ entry. These results suggest the existence of two pathways for receptor-mediated Ca2+ entry in hepatocytes, a 'capacitative' pathway that is sensitive to the Ca2+ content in the Ins(1,4,5)P3-sensitive Ca2+ pool and does not allow Mn2+ entry, and a second pathway that depends on receptor occupation, seems to require a second messenger for activation, and permits influx of Mn2+.

Vasoconstriction and bronchoconstriction induced by 2,5-Di(tert-butyl)1,4-benzohydroquinone, an endoplasmic reticular Ca2+-ATPase inhibitor in isolated and perfused rat lung

The microsomal Ca(2+)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) induced bronchoconstriction and vasoconstriction in the isolated perfused and ventilated rat lung. These effects were accompanied by increased levels of thromboxane and prostacyclin in the effluent perfusate. The effect of tBuBHQ was inhibited by L-655,240, a thromboxane receptor antagonist, indicating thromboxane-A2-mediated bronchoconstriction and vasoconstriction. Accordingly, the cyclooxygenase inhibitor indomethacin largely blocked the effects of tBuBHQ. The involvement of a phospholipase in the generation of thromboxane A2(TXA2) was supported by dibucaine protection on tBuBHQ effects. The results from this study indicate that tBuBHQ, probably by inhibiting the microsomal Ca(2+)-ATPase, can trigger the arachidonic acid cascade leading to the formation of TXA2, which in turn causes bronchoconstriction and vasoconstriction in rat lung.

Characterization of a microsomal calcium-dependent nitric oxide synthase in activated J774.2 monocyte/macrophages.

The nitric oxide (NO) synthase activity present in murine J774.2 monocyte/macrophages was characterized in terms of its intracellular localization, substrate specificity, and Ca2+ dependency. Traces of constitutive NO synthase activity were found in the microsomal fraction from noninduced J774.2 cells, whereas no NO synthase activity was detected in the cytosol. After 24 h in the presence of bacterial lipopolysaccharide and mouse interferon, NO synthase activity was substantially increased in both fractions with 51-60% of the total activity present in the cytosol. These activities, however, were clearly different, for the microsomal enzyme was Ca2+ dependent, whereas the cytosolic NO synthase was not. Moreover, NG-hydroxy-L-arginine (L-HOArg), L-homo-arginine, and several L-arginine (L-Arg)-containing dipeptides could replace L-Arg as substrates for the Ca(2+)-independent NO synthase, whereas the Ca(2+)-dependent enzyme accepted only L-Arg, L-HOArg, or L-Arg-L-Arg as substrates. Thus, a microsomal Ca(2+)-dependent NO synthase is induced in J774.2 monocyte/macrophages with a substrate specificity different from the inducible Ca(2+)-independent NO synthase as well as the constitutive NO synthase in, for example, endothelial cells. Irrespective of their intracellular localization, therefore, at least three isoforms of NO synthase exist, all of which can accommodate substrates different from L-Arg in size, charge, and hydrophobicity.

Calcium oscillations in parotid acinar cells induced by microsomal Ca(2+)-ATPase inhibition.

Previous studies have demonstrated in single rat parotid acinar cells that the microsomal Ca(2+)-ATPase inhibitor thapsigargin mobilizes Ca2+ specifically from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, activates plasma membrane Ca2+ permeability, and induces intracellular Ca2+ concentration ([Ca2+]i) oscillations that are quite similar to those activated by carbachol. Nevertheless, the IP3-sensitive Ca2+ store remains continuously depleted during thapsigargin-induced oscillations, indicating that this pool is not involved in the oscillation mechanism. To determine the specificity of thapsigargin's effects, in the present study we have examined the effects on [Ca2+]i in single rat parotid acinar cells of two other microsomal Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) and 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ), and compared them with the effects of thapsigargin in the same cells. Our results demonstrate that thapsigargin, CPA, and BHQ all similarly deplete the IP3-sensitive Ca2+ store specifically, activate plasma membrane Ca2+ influx, and induce [Ca2+]i oscillations, strongly suggesting that these agents have a specific inhibitory action on microsomal Ca(2+)-ATPase activity. BHQ, in addition, inhibits plasma membrane Ca2+ influx. The data lend strong support to a model in which the state of Ca2+ filling of the IP3-sensitive store regulates plasma membrane Ca2+ influx. These results suggest either that a Ca2+ pump is involved which is insensitive to structurally dissimilar inhibitors or that a Ca2+ pump is not involved in refilling of the Ca2+ pool involved in [Ca2+]i oscillations in these cells.

Reduction of rat liver endoplasmic reticulum Ca 2+-ATPase activity and mobilization of hepatic intracellular calcium by ciprofibrate, a peroxisome proliferator

Ciprofibrate, a peroxisome proliferating agent, induces cell proliferation in rodent liver during the early periods of exposure. Since Ca 2+ plays an important role in mitogenesis, we have investigated the effects of ciprofibrate on hepatic endoplasmic reticulum (ER) Ca 2+-ATPase, which in part regulates Ca 2+ homeostasis. A single oral dose of 200 mg/kg ciprofibrate to male F344 rats produced a transient decrease in liver microsomal Ca 2+-ATPase activity to 48% of control levels at 24 hr post-exposure. Activity had returned to control levels by 48 and 72 hr after exposure. The decrease in Ca 2+-ATPase activity was not a function of non-specific enzymatic inhibition, since activity of another microsomal enzyme, glucose-6-phosphatase, was not altered in ciprofibrate-exposed rats. Using an ATP-driven 45Ca 2+ accumulation assay, rats exposed to 25, 100 and 200 mg/kg ciprofibrate exhibited a dose-dependent inhibition of liver microsomal Ca 2+ accumulation at 24 hr post-exposure. Analysis of Western immunoblots using a polyclonal antibody to the liver ER Ca 2+-ATPase revealed a marginal increase in Ca 2+-ATPase protein content in microsomes prepared from ciprofibrate-exposed rats compared to controls 24 hr post-exposure. These data indicate that the reduction of Ca 2+-ATPase activity is not attributable to diminished Ca 2+-ATPase protein content in vivo and, therefore, is due to a functional inhibition of the enzyme. Ciprofibrate also produced a concentration-dependent inhibition of rat liver ER Ca 2+-ATPase activity in vitro (IC 50 ≈ 170 μM). In freshly isolated rat hepatocytes, ciprofibrate elevated the free intracellular calcium concentration ([Ca 2+] i) in the presence and absence of extracellular calcium. Collectively, these results suggest that Ciprofibrate mobilizes hepatic [Ca 2+] i via inhibition of the ER Ca 2+-ATPase. These events may lead to an environment of elevated [Ca 2+] i during the early stages of ciprofibrate exposure and may serve to augment Ca 2+-dependent processes, thus playing a pivotal role in the acute mitogenic response.

Mechanisms of activated Ca 2+ entry in the rat pancreatoma cell line, AR4-2J

The characteristics of Ca 2+ entry activated by surface receptor agonists and membrane depolarization were studied in the rat pancreatoma cell line, AR4-2J. Ca 2+ mobilization activated by substance P, bombesin, or muscarinic receptor stimulation was found to involve both Ca 2+ release and entry. In addition, depolarization of the surface membrane of AR4-2J cells with elevated concentrations of K + activated Ca 2+ entry. Ca 2+ entry induced by membrane depolarization was inhibited by the L-channel antagonist, nimodipine, while that due to surface receptor agonists was not inhibited by this agent. The microsomal Ca 2+-ATPase inhibitor, thapsigargin, caused both depletion of the agonist-sensitive intracellular Ca 2+ pool and sustained Ca 2+ influx indistinguishable from that produced by bombesin or methacholine. These results confirm that, unlike the pancreatic acinar cells from which they are presumably derived, AR4-2J cells express voltage-sensitive, dihydropyridine-inhibitable Ca 2+ channels. However, in contrast to previous reports with this cell line, in the AR4-2J cells in use in our laboratory, and under our experimental conditions, surface receptor agonists (including substance P) do not cause Ca 2+ influx through voltage-sensitive Ca 2+ channels. Instead, we conclude that agonist-activated Ca 2+ mobilization is initiated by (1,4,5)IP 3-mediated intracellular Ca 2+ release and that Ca 2+ influx is regulated primarily, if not exclusively, by the state of depletion of the (1,4,5)IP 3-sensitive intracellular Ca 2+ pool.

Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms.

Novel transient biphasic responses of the dog mesenteric artery to phenylephrine hydrochloride (PE, 10 microM) in Ca(2+)-free medium containing 50 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) have been analyzed. The initial component was significantly inhibited by ryanodine (30-100 microM), an agonist enhancing Ca2+ release from the sarcoplasmic reticulum, whereas the second was significantly inhibited by nifedipine (1 microM), and L-type Ca2+ channel antagonist, or EGTA, to chelate Ca2+, and was potentiated by BAY K 8644 (1 microM), an L-type Ca2+ channel agonist. After repletion of Ca2+ stores in normal Krebs solution or in high KCl (60 mM) Krebs, the first component was inhibited by cyclopiazonic acid (CPA, 30 microM), a putative, reversible, and selective microsomal Ca2+ pump adenosinetriphosphatase inhibitor. BAY K 8644 potentiated the second component in the presence of CPA. The inhibition of the first component by CPA suggests that the refilling ultimately requires the CPA-sensitive Ca2+ pump for Ca2+ resequestration. However, the second component may refill by a CPA-independent route opened by BAY K 8644. These results, taken as a whole, indicate that the biphasic PE response in Ca(2+)-free medium may reflect compartmentalization of Ca2+ storage related to the different routes of refilling.

A capacitative Ca2+ influx is required for sustained fluid secretion in sublingual mucous acini

The Ca2+ dependence of muscarinic-induced fluid and electrolyte secretion was studied using rat sublingual mucous gland preparations. During stimulation, secretions from vascularly perfused glands were totally inhibited when perfused with a Ca(2+)-free medium. Fluid secretion correlated with sustained losses of 42K+ and 36Cl- content and sustained increases in 22Na+ content and the intracellular free Ca2+ concentration ([Ca2+]i) in fura-2-loaded acini. The magnitudes of the initial agonist-induced changes in Na+, K+, and Cl- content and [Ca2+]i were unaltered in a Ca(2+)-free medium, whereas extracellular Ca2+ removal resulted in the recovery of these ions during the sustained phase to pre-stimulation levels. The recovery of Cl- content induced by Ca2+ depletion was totally blocked in the presence of bumetanide, an inhibitor of Na(+)-K(+)-2Cl- cotransport, while 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion exchange, did not influence Cl- recovery in a HCO(3-)-containing solution (25 mM NaHCO3, 5% CO2). The stimulated increase in [Ca2+]i was not inhibited by the addition of voltage-activated Ca2+ channel blockers (D 888, nifedipine, and diltiazem) or in a Na(+)-free medium. Studies using the quench of fura-2 by Mn2+ as an index of Ca2+ influx and thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, indicate that a capacitative Ca2+ entry pathway mediates Ca2+ entry during stimulation. The above data demonstrate that Ca2+ uptake, which is dependent on the refill status of the agonist-sensitive intracellular Ca2+ pool, is a prerequisite for sustained muscarinic-induced fluid and electrolyte secretion in the rat sublingual mucous gland.