calcimycin and 2-5-di-tert-butylhydroquinone

Ca(2+) regulates cell differentiation and morphogenesis in a diversity of organisms and dysregulation of Ca(2+) signal transduction pathways leads to many cellular pathologies. In Dictyostelium Ca(2+) induces ecmB expression and stalk cell differentiation in vitro. Here we have analyzed the pattern of ecmB expression in intact and bisected slugs and the effect of agents that affect Ca(2+) levels or antagonize calmodulin (CaM) on this expression pattern. We have shown that Ca(2+) and CaM regulate ecmB expression and pstAB/pstB cell differentiation in vivo. Agents that increase intracellular Ca(2+) levels increased ecmB expression and/or pstAB and pstB cell differentiation, while agents that decrease intracellular Ca(2+) or antagonize CaM decreased it. In isolated slug tips agents that affect Ca(2+) levels and antagonize CaM had differential effect on ecmB expression and cell differentiation in the anterior versus posterior zones. Agents that increase intracellular Ca(2+) levels increased the number of ecmB expressing cells in the anterior region of slugs, while agents that decrease intracellular Ca(2+) levels or antagonize CaM activity increased the number of ecmB expressing cells in the posterior. We have also demonstrated that agents that affect Ca(2+) levels or antagonize CaM affect cells motility and regeneration of shape in isolated slug tips and backs and regeneration of tips in isolated slug backs. To our knowledge, this is the first study detailing the pattern of ecmB expression in regenerating slugs as well as the role of Ca(2+) and CaM in the regeneration process and ecmB expression.

Topics: Calcimycin; Calcium; Calcium Signaling; Calmodulin; Dictyostelium; Egtazic Acid; Extracellular Matrix Proteins; Gene Expression Regulation, Developmental; Hydroquinones; Macrolides; Protozoan Proteins; Regeneration; Sulfonamides; Verapamil

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca(2+)-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-kappaB (NF-kappaB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca(2+)-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca(2+)-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC-alpha and -beta activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-kappaB-specific DNA-protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-kappaB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-kappaB activation. These data suggest that persistent inhibition of ER Ca(2+)-ATPase by TG would influence calcium release from ER Ca2+ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-kappaB activation that induces iNOS expression and NO production.

Topics: Animals; Calcimycin; Cell Line; Endoplasmic Reticulum; Enzyme Activation; Hydroquinones; Ionomycin; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Thapsigargin

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Topics: Animals; Binding, Competitive; Brefeldin A; Calcimycin; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Carbazoles; Cattle; Cells, Cultured; Chromaffin Cells; Dose-Response Relationship, Drug; Down-Regulation; Egtazic Acid; Enzyme Inhibitors; Hydroquinones; Indoles; Ionophores; Marine Toxins; Oxocins; Protein Subunits; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Saxitoxin; Scorpion Venoms; Sodium; Sodium Channels; Thapsigargin; Time Factors; Tritium; Veratridine

Central to the pathophysiology of stenosis following balloon angioplasty and arterial bypass surgery is proliferation of vascular smooth muscle cells (VSMCs). To investigate the role of calcium (Ca2+) in VSMC proliferation, the effect of thapsigargin, Ca2+ ionophore A23187, ionomycin, cyclopiazonic acid and di-tert-butylhydroquinone (all of which raise intracellular Ca2+ levels) on the proliferation of cultured human VSMCs was observed.. Cultured VSMCs from human saphenous vein were treated with calcium-modulating drugs and proliferation was assessed by determining [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation and cell number.. Over a 48-h exposure, thapsigargin inhibited VSMC replication (median 50 per cent maximal inhibitory concentration 2 nmol/l) whereas the other drugs were much less effective. Short-term exposure (5, 10, 30 and 60 min) to thapsigargin elicited a significant dose-dependent inhibition of VSMC replication whereas, again, the other drugs were without significant effect.. Thapsigargin-sensitive intracellular Ca2+ pools play a key role in controlling VSMC proliferation and specialized means of administering thapsigargin may constitute a possible approach to preventing stenosis.

Topics: Adult; Aged; Calcimycin; Calcium; Cell Division; Cells, Cultured; Enzyme Inhibitors; Female; Humans; Hydroquinones; Indoles; Ionomycin; Male; Middle Aged; Muscle, Smooth, Vascular; Saphenous Vein; Thapsigargin; Varicose Veins

The aim of this study was to investigate the effect of an internal calcium ATPase inhibitor, TBQ, on human sperm capacitation and the acrosome reaction during incubation in a calcium-depleted media. Sperm were isolated into and incubated for up to 6 hr in media depleted of Ca2+ and two Ca(2+)-containing media controls. At set time intervals, sperm in each media group were treated with 100 microM TBQ for 5 min. Afterwards, sperm were induced to acrosome react using the divalent cation ionophore, A23187, as a measure of sperm fertilizing potential. It was established, using the Chlortetracycline assay, that incubation of sperm in a Ca(2+)-depleted media inhibited or delayed sperm capacitation resulting in fewer spontaneous or A23187-induced acrosome reacted sperm. However, incubation of sperm in a Ca(2+)-depleted media did not appear to inhibit sperm motility. The treatment of sperm with TBQ during their incubation in Ca(2+)-depleted media was found to have very little effect resulting in low numbers of capacitated and acrosome reacted sperm. The results from this study suggest that human sperm have an obligatory requirement for extracellular calcium during capacitation and the acrosome reaction, but may require either very little extracellular Ca2+ to maintain motility or possess internal Ca2+ stores sufficient for their requirements. In addition, TBQ did not increase the number of capacitated and acrosome reacted sperm during incubation in a Ca(2+)-depleted media suggesting that the TBQ-effect of accelerating sperm capacitation is dependent on presence of extracellular Ca2+.

Topics: Acrosome; Calcimycin; Calcium; Calcium-Transporting ATPases; Egtazic Acid; Enzyme Inhibitors; Female; Fertilization; Humans; Hydroquinones; Male; Sperm Capacitation; Sperm-Ovum Interactions; Spermatozoa

The biochemical transductional events involved in NO synthesis are not fully understood. These studies, therefore, were undertaken to elucidate the role of intracellular calcium and protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Thapsigargin (TG), Ca(2+)-ATPase inhibitor of endoplasmic reticulum, had modest activity on NO synthesis by itself, whereas phorbol ester, PKC activator, alone had no effect. When TG was used in combination with phorbol ester, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of phorbol ester was shown in the first 6 h after TG treatment. In addition, the ability of TG with phorbol ester on NO synthesis could be mimicked by another chemically unrelated inhibitor of Ca(2+)-ATPase, 2,5-di-(t-butyl)-1, 4-benzohydroquinone, and Ca2+ ionophore, A23187. This increase of NO synthesis was reflected as increased amount of NO synthase (NOS) mRNA, as determined by Northern blotting. Intracellular Ca2+ transient by TG was not affected in the presence or absence of extracellular Ca2+, indicating that TG must be effective on cytosolic Ca2+ pool. In addition, chelation of intracellular Ca2+ by acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), an intracellular Ca2+ chelating agent, blocked TG- or TG + PMA-induced NO production. PKC inhibitors such as staurosporine or polymyxin B reduced only the synergistic cooperative effect of TG with phorbol ester without affecting TG-induced NO production. In addition, when the cells were pretreated with phorbol ester before TG treatment, there was no synergy between TG and phorbol ester, indicating that PKC is not directly involved in the expression of NOS but involved in "triggering" signal. Secretion of NO corresponded with tumor cell killing, but TG plus phorbol ester-activated macrophages failed to kill tumor cell targets in the presence of Ng-monomethyl-L-arginine. Collectively, these data illustrate that mobilization of intracellular Ca2+ provides a "priming" signal for induction of NOS gene expression by itself and it also requires PKC as a "triggering" signal for macrophage tumoricidal activity.

Topics: Animals; Calcimycin; Calcium; Calcium-Transporting ATPases; Chelating Agents; Cytotoxicity, Immunologic; Drug Synergism; Egtazic Acid; Humans; Hydroquinones; Interferon-gamma; Lipopolysaccharides; Macrophage Activation; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Nitric Oxide; Protein Kinase C; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

Thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, depletes intracellular Ca2+ stores and induces a sustained Ca2+ influx without altering phosphatidyl inositol levels. TG plus phorbol myristate acetate (PMA) but not TG alone induced IL-2 in Jurkat T cells, suggesting that TG had no effect on protein kinase C (PKC). However, TG induced increases in IL-2R alpha protein as well as IL-2R alpha mRNA in Jurkat T cells in a dose-dependent manner. A similar increase in IL-2R alpha by TG was also observed in human peripheral T cells. Further, like PMA, TG markedly induced NF kappa B in Jurkat T cells. However, TG and PMA exhibited a synergistic action on IL-2R alpha expression, suggesting that TG and PMA induce IL-2R alpha through distinct pathways. PMA- but not TG-induced IL-2R alpha is inhibited by the PKC inhibitor H7, whereas TG- but not PMA-induced IL-2R alpha was inhibited by cholera toxin, forskolin and 1,9-dideoxy forskolin. In toto, these results suggest that TG induces IL-2R alpha in human T cells through a PKC-independent pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Base Sequence; Calcimycin; Calcium; Calcium-Transporting ATPases; Cholera Toxin; Colforsin; Drug Synergism; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Humans; Hydroquinones; Isoquinolines; Leukemia-Lymphoma, Adult T-Cell; Molecular Sequence Data; Neoplasm Proteins; NF-kappa B; Piperazines; Protein Kinase C; Receptors, Interleukin-2; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Staurosporine; T-Lymphocytes; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin

Both protein kinase C and cytosolic Ca2+ are involved in the regulation of exocytosis in a number of cell types. However, the relative importance of each of these for apical exocytosis in the hepatocyte is unknown. To investigate this, we studied the effects of protein kinase C and Ca2+ agonists on horseradish peroxidase excretion in the isolated perfused rat liver. Vasopressin increased both horseradish peroxidase concentration and net horseradish peroxidase excretion in bile, and these effects were abolished by the protein kinase C inhibitor H-7. The protein kinase C activator phorbol dibutyrate also increased both net excretion and the concentration of biliary horseradish peroxidase. In contrast, the Ca2+ ionophore A23187 and the Ca2+ mobilizing agent 2,5'-di(tertbutyl)-1,4-benzohydroquinone both had minimal effects on horseradish peroxidase concentration and inhibited the rate of horseradish peroxidase excretion. These results suggest that protein kinase C stimulates apical exocytosis in the hepatocyte, whereas increased Cai2+ per se does not influence exocytosis and inhibits excretion only transiently by reducing bile flow.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Bile; Calcimycin; Calcium; Cytosol; Exocytosis; Horseradish Peroxidase; Hydroquinones; In Vitro Techniques; Isoquinolines; Liver; Male; Perfusion; Phorbol 12,13-Dibutyrate; Piperazines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Vasopressins

Treatment of T lymphoblasts with stimuli that mobilize [Ca2+]i, such as ionophores (ionomycin and A23187) and endoplasmic reticulum Ca(2+)-ATPase inhibitors (thapsigargin, 2,5-di-(tert.-butyl)-hydroquinone and cyclopiazonic acid), activated T cell binding to extracellular matrix (ECM) proteins. T lymphoblast adhesion to ECM proteins stimulated by ionomycin, thapsigargin, or PMA was inhibited by an anti-beta 1 integrin mAb (4B4), confirming the role of beta 1 integrins in regulated T cell-ECM interactions. Study of the alpha integrin subunit specificity of activated lymphoblast-fibronectin interactions demonstrated that alpha 5 beta 1 was the major integrin receptor regulating binding to fibronectin. These results indicate that intracellular Ca2+ mobilization plays a major contributory role in the activation of T cell beta 1 integrins.

Topics: Antibodies, Monoclonal; Antioxidants; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Adhesion; Cells, Cultured; Collagen; Endoplasmic Reticulum; Fibronectins; Humans; Hydroquinones; Indoles; Integrin beta1; Integrins; Ionomycin; Laminin; T-Lymphocytes; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin

Bradykinin causes a concentration-dependent, transient rise in intracellular Ca2+ 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 Ca2+ did not block the initial, but eliminated the sustained inhibition of cAMP accumulation. Increasing Ca2+ 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 Ca2+ sequestration, did not mimic the effect of bradykinin, although it increased [Ca2+]i even more than A23187 did. The inhibitory effect of bradykinin was not mediated by Ca2+/CaM-dependent stimulation of phosphodiesterase (PDE). Forskolin-stimulated adenylyl cyclase activity was inhibited by Ca2+ (10(-7) to 10(-3) M), both in ethyleneglycol-bis-(beta-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 [Ca2+]i and subsequent entry of Ca2+ which directly interacts with a component of the adenylyl cyclase system.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Astrocytoma; Bradykinin; Brain Neoplasms; Calcimycin; Calcium; Calmodulin; Colforsin; Cyclic AMP; Dopamine; Egtazic Acid; Humans; Hydroquinones; Inositol 1,4,5-Trisphosphate; Neoplasm Proteins; Pyrrolidinones; Rolipram; Signal Transduction; Tumor Cells, Cultured

Autophagic sequestration of endogenous lactate dehydrogenase or electroinjected [3H]raffinose in isolated rat hepatocytes was strongly suppressed by the Ca2+ chelator EGTA, unless the cells had previously been electroloaded in the presence of high concentrations of Ca2+ (1.2 mM). The extracellular Ca2+ chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and the intracellular Ca2+ chelator BAPTA/tetra(acetoxymethyl)-ester (BAPTA/AM) both inhibited autophagy to the same extent as did EGTA. Inhibitors of Ca(2+)-activated protein kinases (KN-62, H-7, W-7) had little or no effect on autophagy, indicating that the Ca2+ requirement of autophagy was not mediated by such kinases. Agents that elevate cytosolic Ca2+ by releasing Ca2+ from intracellular stores, like thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and the ionophores A23187 and ionomycin, inhibited autophagy strongly, implicating depletion of sequestered rather than of cytosolic intracellular Ca2+ as a common mechanism of inhibition. Lysosomal (propylamine-sensitive) protein degradation, known to be largely autophagy-dependent, was inhibited by thapsigargin and tBuBHQ. Thapsigargin had no effect on cellular ATP levels, but all agents tested (thapsigargin, tBuBHQ, ionophores) inhibited protein synthesis. Our results suggest that autophagy, like protein synthesis, is dependent on the presence of Ca2+ in some intracellular storage compartment.

Topics: Animals; Autophagy; Calcimycin; Calcium; Calcium-Transporting ATPases; Chelating Agents; Hydroquinones; In Vitro Techniques; Ionomycin; Liver; Male; Protein Kinase Inhibitors; Rats; Rats, Wistar; Terpenes; Thapsigargin

The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells. As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R. D. (1991) J. Biol. Chem. 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal. Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols. Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins. Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols. Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation. A working model for the degradation pathway is proposed.

Topics: Animals; Antioxidants; beta-Galactosidase; Calcimycin; Calcium; CHO Cells; Cholesterol; Cricetinae; Endoplasmic Reticulum; Homeostasis; Hydroquinones; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Ionomycin; Kinetics; Mevalonic Acid; Models, Biological; Recombinant Fusion Proteins; Signal Transduction; Sterols; Terpenes; Thapsigargin; Time Factors; Transfection

The biosynthesis of phosphatidylserine (PtdSer) by the serine base-exchange enzyme system, in Jurkat T-lymphocytes, was inhibited in intact cells maintained in low-Ca(2+)-containing buffer (< 10 microM-Ca2+) by using Ca2+ ionophores (A23187 or ionomycin). The rise in cytosolic Ca2+ concentration under these experimental conditions was only due to the release of Ca2+ from intracellular compartments, suggesting that the inhibition of PtdSer synthesis was correlated with the emptying of intracellular Ca2+ pools. This was further studied in saponin-permeabilized cells, in which PtdSer synthesis was found to be inhibited by EGTA, Ca2+ ionophores (A23187 or ionomycin) and Ca(2+)-ATPase inhibitors [thapsigargin or 2,5-di-(t-butyl)-benzohydroquinone]. Since Ca(2+)-ATPase inhibitors impaired refilling of the Ca2+ stores with Ca2+, and since in CD3-activated Jurkat T-cells the Ca2+ stores remained empty after 1 h of treatment with anti-CD3 monoclonal antibodies, we suggest that PtdSer synthesis is mainly regulated by the level of Ca2+ in the intracellular compartments and that the Ca(2+)-dependent serine base-exchange system responsible for PtdSer synthesis is probably located within or close to a Ca(2+)-storage organelle.

Topics: Antibodies, Monoclonal; Calcimycin; Calcium; CD3 Complex; Cell Membrane Permeability; Cells, Cultured; Humans; Hydroquinones; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Ionomycin; Lymphocyte Activation; Phosphatidylserines; T-Lymphocytes; Terpenes; Thapsigargin

The effects of increases in cytosolic Ca2+ on hepatocyte bile secretion are unknown. A number of agents that alter levels of cytosolic Ca2+ in the hepatocyte also produce hepatic vasoconstriction and activate protein kinase C, which complicates interpretations of their effects on bile secretion. To better understand the role of cytosolic Ca2+ in bile secretion, we examined the effect of the Ca2+ ionophore A23187 (0.1 mumol/L), the Ca2+ agonist vasopressin (10 nmol/L) and the Ca(2+)-mobilizing agent, 2,5-di(tert-butyl)-1,4-benzohydroquinone (25 mumol/L) on cytosolic Ca2+ in isolated hepatocytes and on bile flow in the isolated perfused rat liver, using vasodilators and inhibitors of protein kinase C and Ca2+ influx. Single-pass perfused livers were used, and cytosolic Ca2+ was measured by luminescent photometry in isolated hepatocytes loaded with the Ca(2+)-sensitive photoprotein aequorin. After A23187 perfusion, a sustained 74% +/- 10% (mean +/- S.D.) decrease in bile flow and a sustained 271% +/- 50% increase in perfusion pressure was observed. Simultaneous pretreatment with the vasodilator papaverine (25 mumol/L) and the protein kinase C inhibitor H-7 (50 mumol/L) abolished the pressure increase but not the decrease in bile flow, whereas pretreatment with Ni2+ (25 mumol/L) to block the influx of extracellular Ca2+ markedly reduced both the pressure increase and the decrease in bile flow. Vasopressin produced a transient (mean = 6 min) 75% +/- 4% decrease in bile flow and a sustained 7% +/- 4% increase in perfusion pressure. Pretreatment with H-7 alone corrected the vasopressin-induced pressure increase but also failed to eliminate the decrease in bile flow, whereas pretreatment with Ni2+ decreased the magnitude of the decrease by two-thirds without affecting the increase in perfusion pressure, 2,5'-di(tert-butyl)-1,4-benzohydroquinone produced a transient 65% +/- 20% decrease in bile flow and a transient 56% +/- 15% increase in perfusion pressure. In isolated hepatocytes, bromo-A23187, the nonfluorescent form of the ionophore, produced a sustained 56% +/- 32% increase in the cytosolic Ca2+ signal, whereas vasopressin resulted in a transient 241% +/- 75% increase and 2,5-di(tert-butyl)-1,4-benzohydroquinone resulted in a sustained 149% +/- 66% increase. The ionophore-induced increase in Ca2+ was abolished completely by pretreatment of the hepatocytes with Ni2+, whereas the vasopressin-induced increase was reduced by 38%.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Aequorin; Animals; Bile; Calcimycin; Calcium; Cell Separation; Cytosol; Hydroquinones; In Vitro Techniques; Liver; Male; Pressure; Rats; Rats, Inbred Strains; Vasopressins

Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca2+ in a Ca(2+)-free extracellular medium as revealed by changes in the absorbance of the Ca2+ indicator arsenazo III. The process is completed in 2 min and the amount of Ca2+ released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca2+ that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progressive loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.5. However, this decrease does not significantly prevent 45Ca2+ accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to 1, does not promote a significant release of 45Ca2+ from 45Ca(2+)-preloaded microsomes. Finally, experiments with Fura-2-loaded hepatocytes reveal that agents specifically releasing Ca2+ from non-mitochondrial stores (vasopressin and 2,5-di-tert-butyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCCP specifically releases Ca2+ from mitochondrial stores without significantly affecting active Ca2+ sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca2+ in liver cells.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Arginine Vasopressin; Arsenazo III; Biological Transport, Active; Calcimycin; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fura-2; Hydroquinones; Liver; Male; Microbial Collagenase; Microsomes, Liver; Mitochondria, Liver; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence

The mechanisms through which Ca2+ mobilization in rat hepatocytes results in the loss of total activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase [Zammit & Caldwell (1990) Biochem. J. 269, 373-379] were investigated. The loss of total activity was shown to be paralleled by an equal loss of immunoreactive HMG-CoA reductase protein after exposure of hepatocytes to optimal concentrations of vasopressin plus glucagon for 40 min. This loss of enzyme protein was due to an inhibition of enzyme synthesis; the rate of degradation was unaffected. Other Ca(2+)-mobilizing conditions (phenylephrine, glucagon, vasopressin added singly and A23187) also resulted in graded inhibition of synthesis of HMG-CoA reductase. These effects were accentuated by omission of Ca2+ from the cell incubation medium, suggesting that it is the depletion of an intracellular InsP3-sensitive pool of Ca2+ to which synthesis of HMG-CoA reductase is sensitive. In agreement with this we found that t-butylhydroxybenzoquinone, which inhibits the activity of the Ca(2+)-ATPase of the endoplasmic-reticular membrane, mimicked the action of Ca(2+)-mobilizing hormones. However, taurolithocholate, which transiently mobilizes Ca2+ from the same pool, was ineffective. All these effects on HMG-CoA reductase were accompanied by parallel inhibition of 35S incorporation from [35S]methionine into total protein, suggesting that inhibition of reductase synthesis formed part of a generalized response of the hepatocyte to Ca2+ mobilization. Inhibition of the rate of synthesis of HMG-CoA reductase was, however, more responsive to Ca2+ mobilization in the absence of added Ca2+ from the extracellular medium. The concentrations of vasopressin required to elicit the inhibition of synthesis of HMG-CoA reductase were of the same order as those that elicited activation of glycogen phosphorylase in hepatocytes.

Topics: Animals; Calcimycin; Calcium; Dose-Response Relationship, Drug; Female; Glucagon; Hydroquinones; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kinetics; Liver; Phenylephrine; Rats; Rats, Inbred Strains; Taurolithocholic Acid; Vasopressins