Permeabilized Hepatocytes Research Articles

Evidence against direct involvement of phosphorylation in the activation of carnitine palmitoyltransferase by okadaic acid in rat hepatocytes

The mechanism of activation of mitochondrial overt carnitine palmitoyltransferase (CPT I) by treatment of hepatocytes with okadaic acid (OA) was investigated. Activation was observed when cells were permeabilized with digitonin, but not when a total membrane fraction was obtained by sonication. Both cell disruption methods preserved the activation of phosphorylase observed in OA-treated hepatocytes. Activation of CPT I was also observed in crude homogenates of OA-treated hepatocytes, but it was lost upon subsequent isolation of mitochondria from such homogenates. In all experiments, any activation observed did not depend on the presence or absence of fluoride ions in the permeabilization/homogenization media. When hepatocytes were permeabilized in the absence of fluoride and further incubated with exogenous phosphatases 1 and 2A, the OA-induced activation of CPT was not reversed, whereas the activation of glycogen phosphorylase in the same cells was rapidly reversed. Treatment of hepatocytes with OA, followed by permeabilization and incubation before assay of CPT I, demonstrated that OA had no short-term effect on the sensitivity of CPT I to malonyl-CoA, although the difference in sensitivity between cells isolated from fed and starved rats was fully preserved. Incubation of isolated mitochondria or purified mitochondrial outer membranes with cyclic AMP-dependent or AMP-activated protein kinases, under phosphorylating conditions, did not affect the activity of CPT I or its sensitivity to malonyl-CoA inhibition. Under the same conditions, the use of [32P]ATP resulted in the labelling of several outer-membrane proteins but, unlike [3H]etomoxir-labelled CPT I, none of them was specifically removed from membrane extracts by a specific polyclonal antibody to the enzyme. We conclude that the increase in overt CPT activity observed in permeabilized hepatocytes is not due to direct phosphorylation of CPT I, but may involve interactions between the mitochondrial outer membrane and other membranous or soluble cytosolic components of the cell.

A novel cycle involving fatty acyl-coenzyme A regulates asialoglycoprotein receptor activity in permeable hepatocytes.

Asialoglycoprotein receptors (ASGP-Rs) in permeable rat hepatocytes can be inactivated in the absence of ligand. This cytosol-independent effect is relatively slow (t1/2 approximately 12 min) and is temperature and ATP dependent. Here we show that in the absence of cytosol, the addition of palmitoyl-CoA (Pal-CoA) rapidly (t1/2 < 0.4 min) and quantitatively reactivates the inactivated receptors. Receptor reactivation was half-maximal at approximately 10-12 microM free Pal-CoA at 37 degrees C. Although substantially higher total concentrations were used, much of the added Pal-CoA was cell associated and not free. The effects of Pal-CoA were eliminated by bovine serum albumin at concentrations sufficient to bind all free monomeric fatty acyl-CoA, suggesting that micellar effects are not responsible for the ability to reactivate ASGP-Rs. Also, palmitoyl-carnitine did not substitute for Pal-CoA. The initial ASGP-R inactivation is not affected by treating cells with N-ethylmaleimide or by a KCl wash but is inhibited by sodium orthovanadate or high Ca2+ levels. Myristoyl-CoA (C14) was also able to reactivate inactive ASGP-Rs about as well as Pal-CoA. Fatty acyl-CoAs with chain lengths of C12 (lauroyl) or C18 (steroyl) were < 50% as active. The ligand binding activity of these receptors can subsequently be modulated within minutes by the further addition of ATP or Pal-CoA to achieve additional rounds of ASGP-R inactivation or reactivation, respectively. These in vitro data demonstrate the occurrence of a novel asialoglycoprotein receptor inactivation-reactivation cycle that could regulate receptor activity during endocytosis and receptor recycling.

Imaging of inositol 1,4,5-trisphosphate-induced Ca2+ fluxes in single permeabilized hepatocytes. Demonstration of both quantal and nonquantal patterns of Ca2+ release.

The subcellular organization and function of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores has been investigated in permeabilized hepatocytes using fluorescent probe techniques that monitor InsP3 action at the level of the Ca2+ storage organelles. Chlortetracycline fluorescence was used to follow alterations in luminal Ca2+, and InsP3-activated Mn2+ quench of compartmentalized fura-2 was used as a measure of the distribution and permeability of the InsP3-sensitive channels. Fluorescence imaging of single permeabilized hepatocytes attached to coverslips demonstrated that InsP3-sensitive Ca2+ stores are distributed throughout the cytoplasm, but are not present within the nuclear matrix. When hepatocytes were permeabilized in suspension, InsP3 activation of channel opening and Ca2+ release occurred in a quantal manner, such that the incremental magnitude of the response was determined by the dose of InsP3. Under these conditions dose-dependent steps of InsP3-induced Mn2+ entry into the stores occurred in the absence of changes in cytosolic or luminal Ca2+, providing evidence for a series of separate compartments with different sensitivities to InsP3. Electron microscopy studies revealed that the endoplasmic reticulum was extensively vesicularized when hepatocytes were permeabilized in suspension, whereas essentially normal endoplasmic reticulum structure was retained in cells attached to coverslips. In these attached cells the Ca2+ release and channel opening responses to InsP3 occurred in a nonquantal manner at the single cell level. Submaximal doses of InsP3 gave the same magnitude of response as a maximal InsP3 dose, although the rates of Ca2+ release and Mn2+ permeation through the InsP3-activated channels increased in a dose-dependent manner. Thus, in each cell the entire Ca2+ store was accessible for mobilization by all effective InsP3 concentrations. We conclude that the quantal release properties of the InsP3 receptor are not expressed in attached permeabilized liver cells because there is extensive luminal continuity within the InsP3-sensitive Ca2+ stores. This continuity appears to be disrupted when hepatocytes are permeabilized in suspension.

Phosphorylation of the inositol trisphosphate receptor in isolated rat hepatocytes.

The inositol trisphosphate receptor (IP3R) in brain has been shown to be a substrate for several different protein kinases in vitro. We have studied the phosphorylation of the IP3R in intact cells by using isolated hepatocytes and an antibody to immunoprecipitate the receptor protein from detergent extracts. Stimulation of 32P-labeled hepatocytes with glucagon or N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cAMP) markedly increased phosphorylation of the IP3R. However, no increase was observed in response to angiotensin II, vasopressin, 12-O-tetradecanoyl-phorbol-13-acetate, or epidermal growth factor. The kinetics of phosphorylation in response to glucagon was both rapid and transient. In agreement with previous studies, physiological concentrations of Ca2+ stimulated D-myo-inositol 1,4,5-trisphosphate (IP3) binding to permeabilized hepatocytes (Pietri, F., Hilly, M., and Mauger, J.-P. (1990) J. Biol. Chem. 265, 17478-17485). Pretreatment of cells with db-cAMP had no effect on binding in the absence of added Ca2+ but enhanced binding measured in the presence of basal low concentrations (0.16-0.25 microM) of Ca2+ and decreased the concentration of Ca2+ required for half-maximal stimulation. The effect of db-cAMP was associated with an increase in affinity of the IP3 binding site without a change in maximum number of binding sites. Preincubation of intact hepatocytes with okadaic acid alone produced an increase in basal phosphorylation of the IP3R, and maximal phosphorylation of the receptor was observed in the presence of both okadaic acid and db-cAMP. However, okadaic acid blocked the effect of db-cAMP and inhibited the effect of Ca2+ on IP3 binding. Detergent-solubilized binding sites were already fully activated and insensitive to modulation by Ca2+ or cAMP-dependent protein kinase. It is proposed that the receptor in native membranes is inhibited and that Ca2+ and cAMP-dependent protein kinase may act to relieve this inhibition.

Thiol reagents increase the affinity of the inositol 1,4,5-trisphosphate receptor

Thiol reagents have been shown to increase cytosolic Ca2+ in several cell types. In non-muscle cells, these agents induce Ca2+ spikes by increasing the sensitivity of the intracellular Ca2+ stores to D-myo-inositol-1,4,5-trisphosphate (InsP3). We have investigated the effects of thimerosal and oxidized glutathione on the binding properties of the InsP3 receptor in permeabilized hepatocytes and liver and cerebellar membranes. Thimerosal, at the maximal concentration of 100 microM, decreased the KD for the InsP3 binding to permeabilized hepatocytes and cerebellar membranes from 16 to 3 nM and from 25 to 8 nM, respectively, without affecting the maximal binding capacities. On liver membranes, both thimerosal and high Ca2+ concentrations increased the affinity for InsP3 binding. The Ca2+ and the thimerosal effects were differentiated by kinetic experiments. In low Ca2+ media, two kinetic components were identified and thimerosal decreased the rate of dissociation from both these components without affecting the rate of association. In the high Ca2+ medium, a single kinetic component was found with a very slow rate of dissociation. These data suggest that the InsP3 receptor exists in different states. The high-affinity inactive state induced by high Ca2+ concentrations displays slow rates of association and dissociation. The binding properties of the receptor in its active state can be regulated by thiol reagents which increase the affinity by decreasing the dissociation rate constants. At a resting concentration of 100-200 nM, Ca2+ has two effects: it increases the affinity of the active state of the receptor as thiol reagents do and transforms part of the receptors into the inactive high-affinity state.

Multiple mechanisms by which protein kinase A potentiates inositol 1,4,5-trisphosphate-induced Ca2+ mobilization in permeabilized hepatocytes.

The mobilization of Ca2+ from intracellular stores by Ins(1,4,5)P3 in suspensions of permeabilized rat hepatocytes was potentiated by preincubating intact cells with adenosine 3':5'-cyclic phosphorothioate (cpt-cAMP), or by addition of the catalytic subunit of cyclic-AMP-dependent protein kinase (PKA) after cell permeabilization. This action of PKA involved both an enhancement in Ins(1,4,5)P3 sensitivity and an increase in the size of the Ins(1,4,5)P3-releasable Ca2+ pool. Inclusion of the protein phosphatase inhibitor okadaic acid in the permeabilization medium augmented the effects of PKA. Treatment with PKA catalytic subunit also increased the rate of ATP-dependent Ca2+ sequestration. To determine whether the effects of PKA on the Ca(2+)-release mechanism were secondary to alterations in the Ca2+ load of the Ins(1,4,5)P3-sensitive stores, a method was developed using Mn2+ as a Ca2+ surrogate to examine the permeability properties of the Ins(1,4,5)P3-gated channels independent of Ca2+ fluxes. This approach utilized the ability of Mn2+ to quench the fluorescence of fura-2 compartmentalized within intracellular Ca2+ stores in an Ins(1,4,5)P3-dependent manner, with thapsigargin added to block the ATP-activated Ca2+ pump and to ensure that the Ca2+ stores were fully depleted of Ca2+. The initial rate and extent of Mn2+ quenching of compartmentalized fura-2 was increased in a dose-dependent manner by Ins(1,4,5)P3. PKA activation increased both the initial rate and the extent of Mn2+ quenching at sub-maximal Ins(1,4,5)P3 doses, but there was no effect on the quench rate in the presence of saturating Ins(1,4,5)P3. However, the amount of compartmentalized fura-2 that could be quenched by Mn2+ in the presence of maximal Ins(1,4,5)P3 was increased by PKA. These data suggest two distinct actions of PKA on the Ins(1,4,5)P3-sensitive Ca2+ stores. (1) Modification of the ion-permeability properties of the Ins(1,4,5)P3 receptor/channel through an increase in the sensitivity to Ins(1,4,5)P3 for channel opening. (2) A recruitment of Ca2+ stores from the Ins(1,4,5)P3-insensitive pool. Both actions were independent of the Ca(2+)-loading state of the stores. Imaging studies of single permeabilized hepatocytes showed that the Ins(1,4,5)P3-sensitive stores were distributed throughout the cell and PKA enhanced the rate of Ins(1,4,5)P3-stimulated Mn2+ quench in individual cells, without modifying the subcellular distribution of Ins(1,4,5)P3-sensitive stores.

Effects of digitonin on the intracellular content of rat hepatocytes: implications for its use in the study of intralobular heterogeneity.

Anterograde or retrograde perfusion of rat liver with digitonin selectively permeabilizes the periportal or the perivenous zone of the hepatic lobule. Digitonin perfusion is used to analyze the effluents released by permeabilized hepatocytes or, combined with collagenase perfusion, to obtain cell suspensions enriched in either periportal or perivenous hepatocytes. Despite the wide use of digitonin to study lobular heterogeneity, its affects on rat hepatocytes are not well documented. We therefore analyzed the effects of digitonin perfusion on the intracellular content of rat hepatocytes by combining electron microscopy, histoenzymology, immunohistochemistry, and in situ hybridization. At the concentration currently used for the study of lobular heterogeneity, digitonin perfusion induced a marked cytosolic clarification of permeabilized hepatocytes, while most organelles except mitochondria were well preserved. In the digitonin-altered zones, there was no histochemical detection of non-membrane-bound enzymes (lactate dehydrogenase, glutamate dehydrogenase), whereas membrane-bound enzymes (succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, NADPH dehydrogenase, glucose-6-phosphatase) were still detected. Immunohistochemistry and in situ hybridization revealed significant amounts of several plasma proteins (albumin, alpha 2-macroglobulin, alpha 1-inhibitor 3, alpha 1-acid glycoprotein) and their respective mRNAs in digitonin-permeabilized hepatocytes. The demonstration that digitonin-permeabilized hepatocytes retain many intracellular constituents shows that biochemical analysis of cellular effluents released from digitonin-permeabilized hepatocytes must be interpreted with caution and that the apparent characteristics of cell suspensions obtained by the digitonin-collagenase technique might be significantly altered by contamination with permeabilized hepatocytes from the opposite zone.

Luminal Ca2+ increases the affinity of inositol 1,4,5-trisphosphate for its receptor

Luminal Ca2+ has been proposed to regulate the sensitivity of intracellular Ca2+ stores to InsP3 and perhaps thereby to contribute to the mechanisms responsible for regenerative intracellular Ca2+ signals. Since Ca2+ release from intracellular stores is accompanied by K+ influx into the stores, InsP3-stimulated Ca2+ mobilization can be inhibited by substantially reducing the [K+] of incubation media. By measuring [3H]InsP3 binding to permeabilized hepatocytes in K(+)-deficient media, thereby preventing InsP3-stimulated Ca2+ mobilization, we have examined the effects of Ca2+ within the intracellular stores on equilibrium binding of InsP3 to its receptor. Our results demonstrate a small, but significant (P < 0.05, n = 8-9), effect of luminal Ca2+ on InsP3 binding; the concentration of InsP3 causing half-maximal displacement of [3H]InsP3 (1.25 nM) was 3.5 nM for empty stores and 2.1 nM for stores containing Ca2+. Our results suggest that at least part of the stimulatory effect of luminal Ca2+ on InsP3-stimulated Ca2+ mobilization may result from an effect of luminal Ca2+ on InsP3 binding to its receptor.

Biphasic effects of cytosolic Ca2+ on Ins(1,4,5)P3-stimulated Ca2+ mobilization in hepatocytes

The increase in cytosolic free Ca2+ concentration that follows mobilization of intracellular Ca2+ stores by inositol 1,4,5-trisphosphate Ins(1,4,5)P3 has been reported to modulate the sensitivity of Ins(1,4,5)P3 receptors. We have examined the effects of cytosolic Ca2+ on Ins(1,4,5)P3-stimulated Ca2+ mobilization in permeabilized hepatocytes. Increasing the free Ca2+ concentration in the medium ([Ca2+]m) caused a concentration-dependent increase in the sensitivity of the stores to Ins(1,4,5)P3; the concentration of Ins(1,4,5)P3 that caused half-maximal Ca2+ mobilization (EC50) decreased from 261 +/- 11 nM (n = 3) to 50 +/- 4 nM (n = 8) as [Ca2+]m was increased from approximately 7 nM to 1.6 microM. The EC50 for this effect of Ca2+ was approximately 250 nM. In addition, higher [Ca2+]m (> 600 nM) reduced the extent of Ca2+ release induced by a maximal concentration of Ins(1,4,5)P3; elevating [Ca2+]m to 2.6 microM reduced the proportion of Ca2+ releasable by Ins(1,4,5)P3 by 94 +/- 8% (n = 3). Both effects of Ca2+ were independent of Ca2(+)-stimulated Ins(1,4,5)P3 formation. When elevated [Ca2+]m was returned to control levels, the sensitization of Ins(1,4,5)P3-mediated Ca2+ mobilization reversed completely, whereas the reduction in the size of the Ins(1,4,5)P3-sensitive Ca2+ pool was reversed by only 59 +/- 12% (n = 5) after 20 s and was not further reversed after 100 s. The two distinct effects of Ca2+ on Ins(1,4,5)P3-mediated Ca2+ release combined to control the amount of Ca2+ released by a submaximal concentration of Ins(1,4,5)P3 in a biphasic manner.

Caffeine-stimulated Ca2+ release from the intracellular stores of hepatocytes is not mediated by ryanodine receptors

Caffeine has been much used to examine the possibility that ryanodine receptors similar to those found in skeletal and cardiac muscle may be more widely distributed and perhaps contribute to regenerative Ca2+ signals in electrically inexcitable cells. In permeabilized hepatocytes loaded with 45Ca2+, caffeine (> or = 5 mM) decreased the 45Ca2+ content of the intracellular stores by up to 60%; the effect was substantially reversible and it was not mimicked by the closely related methylxanthine theophylline (20 mM). Ryanodine (5 microM) stimulated a far smaller Ca2+ mobilization (7 +/- 1%). Procaine (1 mM), Ruthenium Red (10 microM) and ryanodine (5 microM) did not affect the Ca2+ release evoked by InsP3 (3 microM) or caffeine (30 mM). We conclude that caffeine can specifically cause Ca2+ release from the intracellular stores of hepatocytes, but the effect is unlikely to be mediated by ryanodine receptors.

The effects of sevoflurane on intracellular Ca 2+ regulation in rat hepatocytes

The effects of sevoflurane, a new volatile anesthetic agent undergoing clinical trial, on the mobilization of intracellular Ca 2+ in isolated rat hepatocytes was studied. This agent produced a dose-dependent release of 45Ca 2+ from internal, non-mitochondrial stores of permeabilized hepatocytes (saponin treated). However, the administration of sevoflurane to aequorin-loaded intact hepatocytes had little or no effect on intracellular [Ca 2+] (i.e., short transient or no increases in luminescence: no toxic effect). These data may indicate that because of the low solubility of sevoflurane, it has a selective effect on endoplasmic reticulum, i.e., mobilizing internal stores of Ca 2+ relative to increasing transmembrane fluxes.

Identification of a Mg(2+)- and guanyl nucleotide-dependent glucagon receptor cycle by use of permeabilized canine hepatocytes.

We have investigated (by use of intact and saponinpermeabilized canine hepatocytes) the roles of Mg2+ and guanyl nucleotides in regulating glucagon-receptor interactions. In contrast to intact cells, saponinpermeabilized hepatocytes bind [[125I]iodo-Tyr10]glucagon according to a single first-order process and exhibit a single apparent dissociation constant for glucagon binding during steady-state incubations. Further analysis of the permeabilized cell system demonstrated (a) the temperature-sensitive action of Mg2+ to enhance the extent and affinity of glucagon-receptor interactions at steady-state, (b) the conversion of Mg(2+)-independent hormone-receptor complexes to Mg(2+)-dependent complexes, (c) the effect of guanyl nucleotides to inhibit specifically the Mg(2+)-dependent component of glucagon-receptor interactions, (d) the more rapid association of glucagon with receptor during cell incubations occurring in the presence of guanyl nucleotides or in the absence of Mg2+, and (e) the ability of guanyl nucleotides to induce both high and low affinity states of glucagon-receptor interactions. Additional experiments identified an effect of cell incubations in the presence of glucagon to limit the subsequent binding of hormone, the ability of GDP, GTP, or guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) to dissociate previously bound glucagon, and a specific requirement for GDP to re-activate the glucagon receptor for additional cycles of hormone binding. A model is presented in which (a) glucagon binds to receptor in a Mg(2+)-independent fashion, (b) glucagon-receptor complexes are converted to a Mg(2+)-dependent state, (c) guanyl nucleotide exchange initiates both an alteration in glucagon-receptor affinity and the subsequent dissociation of hormone, and (d) in the context of the intact cell, G protein-mediated hydrolysis of GTP to GDP is required to reinitialize the system.

Characterization of a rapidly dissociating inositol 1,4,5-trisphosphate-binding site in liver membranes.

The binding of inositol 1,4,5-trisphosphate (InsP3) to a specific receptor induces the release of Ca2+ from an intracellular store. In the liver, the KD of a low affinity state of the receptor (RL) found at low Ca2+ concentration ([Ca2+]) is in close agreement with the EC50 of the InsP3-induced Ca2+ release. We have developed an experimental procedure for measuring the rate of dissociation of this low affinity [32P]InsP3-receptor complex in less than 1 s. When the receptor was in the RL state, two kinetic components, RL1 and RL2, were identified with respective rate constants (k(off)) of 1-2 s-1 and 0.03-0.06 s-1. Increasing the [Ca2+] up to 1 microM transformed the receptor into the high affinity state (RH) and decreased the dissociation rate constant to 2 x 10(-2) min-1. We also investigated the time course of the transformation of the receptor from the high affinity (RH) to the low affinity state (RL) after decreasing the [Ca2+] to less than 10 nM. This reversion was dramatically dependent on temperature: at 4 degrees C, the receptor was locked in the RH state, whereas at 37 degrees C the receptor reverted to the RL state with a half-time of less than 1 s. The reversion from the RH state to the RL one is associated to a recovery of InsP3-induced 45Ca2+ release on permeabilized hepatocytes. The rapid and reversible transformation of the InsP3 receptor from an active to an inactive state may be a key event in the Ca2+ release process in intact cells.

Luminal Ca2+ promoting spontaneous Ca2+ release from inositol trisphosphate-sensitive stores in rat hepatocytes.

1. Spontaneous Ca2+ release from the inositol 1,4,5-trisphosphate (InsP3)-sensitive stores in permeabilized hepatocytes was monitored using Fluo-3 to measure the free [Ca2+] of the medium bathing the cells. 2. Permeabilized cells rapidly sequestered Ca2+, reducing the [Ca2+] to 103 +/- 5 nM. Under conditions that depended critically upon cell density and the amount of Ca2+ in the medium, this was followed by a slow increase in [Ca2+] culminating in a substantial Ca2+ spike representing synchronous discharge from the InsP3-sensitive stores. 3. During the latency preceding the Ca2+ spike, the stores increased their sensitivity to InsP3. This sensitization seemed to be an all-or-none phenomenon. 4. Oxidized glutathione and thimerosal promoted the spontaneous release by sensitizing the InsP3 receptor. 5. An increase in the [Ca2+] within the stores was required for both the increased sensitivity to InsP3 and the subsequent spike. 6. Caffeine (6 mM) antagonized the effect of very low InsP3 concentrations and abolished the Ca2+ spike, without itself releasing Ca2+. 7. Our results suggesting that luminal Ca2+ may sensitive InsP3-sensitive stores leading to spontaneous Ca2+ mobilization will be discussed in the light of a modified version of the two-pool model for explaining cytosolic Ca2+ oscillations.

Oxidized glutathione causes sensitization of calcium release to inositol 1,4,5-trisphosphate in permeabilized hepatocytes.

The effects of GSSG on Ca2+ mobilization by Ins(1,4,5)P3 were studied in permeabilized rat hepatocytes. Incubation with GSSG (2 mM) increased the sensitivity to Ins(1,4,5)P3 for Ca2+ release, with no effect on the size of the Ca2+ pool that could be released with maximal concentrations of Ins(1,4,5)P3. GSSG decreased the EC50 for Ins(1,4,5)P3 from a control value of 578 +/- 23 nM to 137 +/- 21 nM. GSSG had no effect on the metabolism of Ins(1,4,5)P3 in permeabilized cells, and sensitization of Ca2+ release was still observed when the poorly metabolizable analogue inositol 1,4,5-trisphosphorothioate was used. GSSG did not affect the ATP-dependent Ca2+ pump or the extent of loading of intracellular Ca2+ pools. In addition, the enhancement of Ins(1,4,5)P3-sensitivity by GSSG occurred under conditions where the Ca2+ pumps were blocked with thapsigargin or by chelation of medium Ca2+ just before Ins(1,4,5)P3 addition. The effect of GSSG was time- and dose-dependent, maximal effects being observed after 5 min incubation with 2 mM-GSSG. Cystine mimicked the GSSG-induced increase in Ins(1,4,5)P3-sensitivity, and the effects could be reversed by dithiothreitol (DTT). DTT, GSH glutathione and cysteine had no effect when added alone. Other agents known to react with protein thiols, including N-ethylmaleimide, p-chloromercuribenzoic acid and Ag+, did not affect the sensitivity to Ins(1,4,5)P3, but were inhibitors of ATP-dependent Ca2+ uptake. The data suggest that the sensitivity of the intracellular Ca2+ pools to release by Ins(1,4,5)P3 can be modulated by the formation of mixed disulphides with GSSG or other oxidized thiols.

Mechanism of activation of liver glycogen synthase by swelling.

The mechanism linking the stimulation of liver glycogen synthesis to swelling induced either by amino acids or hypotonicity was studied in hepatocytes, in gel-filtered liver extracts, and in purified preparations of particulate glycogen to which glycogen-metabolizing enzymes are bound. High concentrations of KCl, but not of potassium glutamate, were found to inhibit glycogen synthesis in permeabilized hepatocytes. Similarly, physiological concentrations (30-50 mM) of Cl- ions were also found to inhibit synthase phosphatase in vitro, whereas 10-20 mM Cl- ions, a concentration found in swollen hepatocytes, did not inhibit synthase phosphatase. Synthase phosphatase activity was more sensitive to inhibition by Cl- ions at low (0.1%) than at high (1%) concentrations of glycogen. By contrast, 10 mM glutamate and aspartate, a concentration observed in hepatocytes incubated with glutamine or proline, stimulated synthase phosphatase in vitro. Therefore, it is proposed that the fall in intracellular Cl- concentration as well as the increase in intracellular glutamate and aspartate concentrations, that are observed in swollen hepatocytes in the presence of amino acids, are responsible, at least in part, for the stimulation of synthase phosphatase and, hence, of glycogen synthesis.

Restricted efflux of the type II isoform of cyclic AMP-dependent protein kinase in permeabilized rat hepatocytes

Restricted efflux of the type II isoform of cyclic AMP-dependent protein kinase in permeabilized rat hepatocytes

Effects of ryanodine on calcium sequestration in the rat liver

Ryanodine, a highly toxic alkaloid known to react specifically with the Ca 2+ release channels in sarcoplasmic reticulum (SR), was employed to study Ca 2+ sequestration in the liver. Ryanodine at a 200 μM concentration increased cytosolic free Ca 2+ levels and phosphorylase a activity in isolated hepatocytes. These effects may involve microsomal Ca 2+ sequestration, because ryanodine, in the presence of inhibitors of mitochondrial Ca 2+ uptake, at concentrations of 1 nM, 1 μM, 50 μM and 100 μM decreased 45Ca 2+ retention in permeabilized hepatocytes. This inhibition of Ca 2+ retention by ryanodine was not due to inhibition of the microsomal Ca 2+-ATPase. Dantrolene, a compound shown previously to inhibit ryanodine binding in the liver, also decreased 45Ca 2+ retention in permeabilized hepatocytes, and activated phosphorylase a. These results show that ryanodine administration alters calcium sequestration in liver. The possibility of the existence of a ryanodine-sensitive Ca 2+-release channel in liver is discussed.

Menadione inhibits the α1-adrenergic receptor-mediated increase in cytosolic free calcium concentration in hepatocytes by inhibiting inositol 1,4,5-trisphosphate-dependent release of calcium from intracellular stores

In order to establish the mechanism of perturbation of hormonally regulated calcium homoestasis in hepatocytes caused by menadione, the effects of menadione on hepatic α 1-adrenergic receptors and on α 1-adrenergic receptor-mediated increase in cytosolic free calcium concentration were determined. Menadione had no detectable effect on the α 1-adrenergic receptor but significantly inhibited (−)-epinephrine-dependent increases in intracellular free calcium concentration in Quin2 acetoxymethyl ester-loaded hepatocytes. The hormonally induced increase in intracellular free calcium concentration is caused by formation of inositol 1,4,5-trisphosphate (IP 3) which binds to a specific receptor and causes a release of intracellular ATP-dependently sequestrated calcium. The IP 3-stimulated release of calcium from intracellular pools in hepatocytes was inhibited to a great extent after treatment with menadione. This inhibition could also be observed after treatment of hepatocytes with p-benzoquinone and N-ethylmaleimide and could not be reversed by the thiol-reducing reagent dithiothreitol which indicated covalent binding to an essential free sulfhydryl group. The inhibition of IP 3-dependent release of intracellular calcium was accompanied by a large increase in the number of detectable IP 3 receptors without any change in the dissociation constant as determined in permeabilized hepatocytes. The increase in IP 3 receptors caused by menadione could be reversed by dithiothreitol which suggests the involvement of free sulfhydryl groups. It is concluded that the IP 3 receptor plays an important role in the mechanism of menadione-induced perturbation of hormonally regulated calcium homeostasis in rat hepatocytes.

Quantal Ca2+ mobilization stimulated by inositol 1,4,5-trisphosphate in permeabilized hepatocytes

In several cell types, including hepatocytes, submaximal concentrations of Ins(1,4,5)P3 stimulate an initial rapid mobilization of intracellular Ca2+ stores that is followed by either no further Ca2+ release or very much slower release. Further additions of Ins(1,4,5)P3 then evoke further Ca2+ mobilization. Such 'incremental' responses [Meyer & Stryer (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3841-3845] could result from all-or-nothing emptying of stores that differ in their sensitivities to Ins(1,4,5)P3 or from partial emptying of stores that are more uniformly sensitive, but unable to release all of their Ca2+ because the response to Ins(1,4,5)P3 rapidly attenuates. By measuring unidirectional 45Ca2+ efflux from intracellular stores stimulated with Ins(1,4,5)P3 under conditions where they continue to sequester 40Ca2+, we provide evidence suggesting that Ins(1,4,5)P3 stimulates all-or-nothing emptying of stores that differ in their sensitivities to Ins(1,4,5)P3, a quantal response pattern.