The predominant role of IP3 type 1 receptors in activation of store-operated Ca²+ entry in liver cells.

Physiologically, hormone induced release of Ca²+ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP3) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP3 receptors act as channels, releasing Ca²+ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca²+ stores leads to activation of store-operated Ca²+ channels on the plasma membrane which replenishes lost Ca²+ and sustain Ca²+ signalling. There are three isoforms of IP3 receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca²+ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca²+ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca²+ channels, and Ca²+ release activated Ca²+ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP3 receptor but not the others. Here we show that H4IIE liver cells express all three types of IP3 receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I(CRAC)), while type 1 and type 2, but not type 3, participate in observed Ca²+ release in response to receptor stimulation. Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca²+ store linked to the activation of I(CRAC) predominantly expresses type 1 IP3 receptors.

Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins.

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca2+ release activated Ca2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca2+ sensor required for activation of CRAC current (I(CRAC)) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I(CRAC). In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I(CRAC). The results show that cells expressing low Orai1 : STIM1 ratios produce I(CRAC) with strong fast Ca2+-dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I(CRAC) with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca2+, Ba2+ and Sr2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

Increasing cycles of intermittent ischemia can effectively maintain liver function during the acute phase of ischemia reperfusion injury by promotion of bile flow and reduction in bile salt toxicity.

BACKGROUND/AIMS: Intermittent ischemia (INT) can improve liver function following inflow occlusion. The aim was to test whether the number of cycles of INT can be increased without impairing liver function. METHODS: Liver function in the acute phase of ischemia reperfusion injury was assessed by measuring bile flow in rat livers. Phospholipid and bile salts in bile, liver marker enzymes in blood, and liver histology were measured. Aged livers were compared with young livers. RESULTS: Clamping for 45 min reduced postperfusion bile flow to 13% of the initial value compared with 88 +/- 5% for control livers (means +/- SEM, n = 5-8), and substantially reduced the phospholipid:bile salt ratio in bile. Application of 3, 4, 5 and 6 cycles of INT (15 min) restored bile flow to 70 +/- 11, 61 +/- 4, 48 +/- 2 and 35 +/- 3% (p < 0.01) of the initial value, respectively, and restored the phospholipid:bile salt ratio. Multiple cycles of INT were less effective in aged rats. CONCLUSION: Several cycles of INT, through promotion of bile flow recovery and reduction in the cytotoxic actions of bile salts, may provide an effective clinical strategy for increasing clamping time in liver resections.

TRPC1 Ca(2+)-permeable channels in animal cells.

The full-length transient receptor (TRPC)1 polypeptide is composed of about 790 amino acids, and several splice variants are known. The predicted structure and topology is of an integral membrane protein composed of six transmembrane domains, and a cytoplasmic C- and N-terminal domain. The N-terminal domain includes three ankyrin repeat motifs. Antibodies which recognise TRPC1 have been developed, but it has been difficult to obtain antibodies which have high affinity and specificity for TRPC1. This has made studies of the cellular functions of TRPC1 somewhat difficult. The TRPC1 protein is widely expressed in different types of animal cells, and within a given cell is found at the plasma membrane and at intracellular sites. TRPC1 interacts with calmodulin, caveolin-1, the InsP3 receptor, Homer, phospholipase C and several other proteins. Investigations of the biological roles and mechanisms of action of TRPC1 have employed ectopic (over-expression or heterologous expression) of the polypeptide in addition to studies of endogenous TRPC1. Both approaches have encountered difficulties. TRPC1 forms heterotetramers with other TRPC polypeptides resulting in cation channels which are non-selective. TRPC1 may be: a component of the pore of store-operated Ca2+ channels (SOCs); a subsidiary protein in the pathway of activation of SOCs; activated by interaction with InsP3R; and/or activated by stretch. Further experiments are required to resolve the exact roles and mechanisms of activation of TRPC1. Cation entry through the TRPC1 channel is feed-back inhibited by Ca2+ through interaction with calmodulin, and is inhibited by Gd3+, La3+, SKF96365 and 2-APB, and by antibodies targeted to the external mouth of the TRPC1 pore. Activation of TRPC1 leads to the entry to the cytoplasmic space of substantial amounts of Na+ as well as Ca2+. A requirement for TRPC1 is implicated in numerous downstream cellular pathways. The most clearly described roles are in the regulation of growth cone turning in neurons. It is concluded that TRPC1 is a most interesting protein because of the apparent wide variety of its roles and functions and the challenges posed to those attempting to elucidate its primary intracellular functions and mechanisms of action.

Regulation of Orai1/STIM1 mediated ICRAC by intracellular pH.

Ca2+ release activated Ca2+ (CRAC) channels composed of two cellular proteins, Ca2+-sensing stromal interaction molecule 1 (STIM1) and pore-forming Orai1, are the main mediators of the Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores. Previously it has been shown that the amplitude of CRAC current (ICRAC) strongly depends on extracellular and intracellular pH. Here we investigate the intracellular pH (pHi) dependence of ICRAC mediated by Orai1 and STIM1ectopically expressed in HEK293 cells. The results indicate that pHi affects not only the amplitude of the current, but also Ca2+ dependent gating of CRAC channels. Intracellular acidification changes the kinetics of ICRAC, introducing prominent re-activation component in the currents recorded in response to voltage steps to strongly negative potentials. ICRAC with similar kinetics can be observed at normal pHi if the expression levels of Orai1 are increased, relative to the expression levels of STIM1. Mutations in the STIM1 inactivation domain significantly diminish the dependence of ICRAC kinetics on pHi, but have no effect on pHi dependence of ICRAC amplitude, implying that more than one mechanism is involved in CRAC channel regulation by intracellular pH.

Curcumin inhibits activation of TRPM2 channels in rat hepatocytes.

Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca(2+) homeostasis, resulting in a sustained elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)]c) in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca(2+) entry through Transient Receptor Potential Melastatin 2 (TRPM2) channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca(2+)]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

Characterisation of the inhibition of the hepatocyte receptor-activated Ca2+ inflow system by gadolinium and SK&F 96365.

The inhibition of agonist-stimulated divalent cation inflow in hepatocytes by Gd3+ and compound SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride) was investigated. Gd3+ and SK&F 96365 inhibited Ca2+ and Mn2+ inflow stimulated by vasopressin, angiotensin II or phenylephrine. The concentrations of Gd3+ and SK&F 96365 which gave half-maximal inhibition of vasopressin-stimulated Ca2+ inflow were 2 x 10(-7) M and 16 x 10(-6) M, respectively. The action of Gd3+ on vasopressin-stimulated Ca2+ inflow was rapid (less than 10 s in onset) and reversible. Gd3+ had no effect on Mn2+ inflow in the absence of an agonist and no effect on the ability of vasopressin to release Ca2+ from intracellular stores. SK&F 96365 inhibited Mn2+ inflow in the absence of agonists and vasopressin-induced release of Ca2+ from intracellular stores, but at approximately a 5-fold higher concentration than that which inhibited vasopressin-stimulated divalent cation inflow. It is concluded that Gd3+ and SK&F 96365 (at concentrations below 20 microM) inhibit, in a selective manner, divalent cation movement through the putative cation channel of the hepatocyte receptor-activated Ca2+ inflow system. Gd3+ appears to be the most potent inhibitor of this Ca2+ inflow system so far described.

Characterisation of the divalent cation channels of the hepatocyte plasma membrane receptor-activated Ca2+ inflow system using lanthanide ions.

The ability of Gd3+ to inhibit vasopressin-stimulated Ca2+ inflow to hepatocytes was compared with its effect on Mn2+ inflow. In the absence of Gd3+, the stimulation of Mn2+ inflow by vasopressin increased with increasing pH of the extracellular medium. Maximal inhibition of vasopressin-stimulated Ca2+ and Mn2+ inflow by saturating concentrations of Gd3+ was 70 and 30%, respectively. Gd3+ also inhibited thapsigargin-stimulated Ca2+ and Mn2+ inflow with maximal inhibition of 70 and 40%, respectively. It is concluded that vasopressin and thapsigargin each activate two types of Ca2+ inflow processes, one which is sensitive and one which is insensitive to lanthanides. The nature of the pore of the lanthanide-sensitive Ca2+ channel was investigated further using different lanthanides as inhibitors. Tm3+, Gd3+, Eu3+, Nd3+ and La3+ each inhibited vasopressin-stimulated Ca2+ and Mn2+ inflow but had no effect on Ca2+ inflow in the absence of an agonist, or on vasopressin-stimulated release of Ca2+ from intracellular stores. Maximal inhibition of vasopressin-stimulated Ca2+ inflow in the presence of a saturating concentration of each lanthanide ranged from 70-90%. An equation which describes a 1:1 interaction of the lanthanide with a putative binding site in the Ca2+ channel gave a good fit to dose-response curves for the inhibition of vasopressin-stimulated Ca2+ inflow by each lanthanide. Lanthanides in the middle of the series exhibited the lowest dissociation constant (Kd) values. The Kd for Gd3+ increased with increasing extracellular Ca2+ concentration, suggesting competitive inhibition of Ca2+ binding by Gd3+. In the absence of lanthanide, vasopressin-stimulated Mn2+ inflow was substantially reduced when the plasma membrane was depolarised by increasing the extracellular K+ concentration. Changing the membrane potential had little effect on the maximum inhibition by Gd3+ of vasopressin-stimulated Mn2+ inflow. The Kd for inhibition of vasopressin-stimulated Ca2+ inflow by Gd3+, measured at the lowest attainable membrane potential, was about 6-fold lower than the Kd measured at the highest attainable membrane potential. The idea that there is a site in the vasopressin-stimulated lanthanide-sensitive Ca2+ channel composed of carboxylic acid groups which bind Ca2+, Mn2+ or a lanthanide ion is consistent with the data obtained using the different lanthanides.

Effects of inhibitors of diacylglycerol metabolism on protein kinase C-mediated responses in hepatocytes.

In hepatocytes pre-labelled with [3H]glycerol, compound R59022 (6-[2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl]-7- methyl-5H-thiazolo[3,2-alpha]pyrimidin-5-one) and 2-bromooctanoate each increased the amount of radioactivity in diacylglycerols. R59022 mimicked the actions of 12-O-tetradecanoylphorbol 13-acetate in completely abolishing the activation by adrenaline (but not that by vasopressin or glucagon) of glycogen phosphorylase a, and in decreasing the activity of glycogen synthetase. Exogenous dioctanoylglycerol caused a small inhibition of adrenaline-stimulated phosphorylase activity. The concentration of R59022 which gave half-maximal inhibition of adrenaline-stimulated phosphorylase activity was 15 microM. Maximal inhibition was observed within 2 min of addition of R59022. 2-Bromooctanoate activated phosphorylase by a process independent of changes in cyclic AMP and Ca2+, and decreased glycogen synthetase. It is concluded that in hepatocytes (i) diacylglycerols which accumulate as a result of the inhibition of diacylglycerol kinase by R59022 activate protein kinase C and (ii) 2-bromooctanoate increases diacylglycerols but also has other effects on hepatocyte metabolism.

On the source of the vasopressin-induced increases in diacylglycerol in hepatocytes.

In hepatocytes pre-labelled with [3H]glycerol, vasopressin increased by 20% the amount of radioactivity present in diacylglycerols. The effect of vasopressin was partially dependent on Ca2+. The magnitude of the increase in [3H]diacylglycerol was 5-times the sum of the radioactivity present in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. No stimulation by vasopressin of the initial rate of incorporation of radioactivity into diacylglycerols was observed in cells incubated in the presence of 10 mM [3H]glycerol. Treatment of hepatocytes labelled with either [3H]ethanolamine or [3H]choline with vasopressin, ionophore A23187 or phospholipase C increased the amount of radioactivity present in trichloroacetic acid extracts of the cells. The effect of vasopressin was dependent on extracellular Ca2+. It is concluded that in hepatocytes vasopressin increases diacylglycerols by a process which does not principally involve the conversion of phosphoinositides to diacylglycerol or the de novo synthesis of diacylglycerol from glycerol 3-phosphate, but does involve the Ca2+-dependent conversion of phosphatidylethanolamine and phosphatidylcholine to diacylglycerol.

Inhibition of the liver cell receptor-activated Ca2+ inflow system by metal ion inhibitors of voltage-operated Ca2+ channels but not by other inhibitors of Ca2+ inflow.

The properties of the receptor-activated Ca2+ inflow system in the liver cell plasma membrane were compared with those of voltage-operated Ca2+ channels and receptor-operated Ca2+ channels present in other cell types by testing the susceptibility of the Ca2+ inflow system to inhibition by other metal ions and known inhibitors of Ca2+ movement across membranes. Co2+ inhibited Ca2+ inflow through the receptor-activated Ca2+ inflow system, as assessed by measurement of (a) the activation by extracellular Ca2+ (Cao2+) of glycogen phosphorylase in the presence of vasopressin and (b) 45Ca2+ exchange in the presence of the hormone. The concentration of Co2+ which gave half-maximal inhibition was 280 microM. The inhibition by Co2+ was reversed by high Cao2+. Co2+ did not inhibit basal Ca2+ inflow as measured by 45Ca2+ exchange in the absence of vasopressin. Zn2+, Cd2+, Ni2+ and Mn2+ each inhibited Ca2+ inflow through the receptor-activated Ca2+ inflow system. The concentrations of these ions which gave half-maximal inhibition were 10, 50, 220 and 400 microM, respectively. Little inhibition of receptor-activated Ca2+ inflow was observed in the presence of Sr2+ or Ba2+. However, substantial amounts of 90Sr2+ were taken up by hepatocytes. Rates of 90Sr2+ uptake increased from 0.5-8 nmol per min per mg wet wt. when the extracellular concentration of Sr2+ was varied from 0.25 to 2.5 mM. Sr2+ uptake was inhibited 50% by Cao2+ with half-maximal inhibition at 100 microM Cao2+, but was not inhibited by verapamil and was not stimulated by vasopressin. The movement of Ca2+ through the receptor-activated Ca2+ inflow system was not inhibited by high concentrations of each of a number of inhibitors of voltage-operated and receptor-operated Ca2+ channels and intracellular Ca2+ movement. It is concluded that while the susceptibility to inhibition by metal ions of the receptor-activated Ca2+ inflow system in the liver cell plasma membrane is similar to that of voltage-operated Ca2+ channels, there are significant differences between the liver cell receptor-activated Ca2+ inflow system and both voltage-operated Ca2+ channels and some other receptor-operated Ca2+ channels with respect to inhibition by organic compounds.

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes.

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.

Store-operated Ca(2+) inflow in Reuber hepatoma cells is inhibited by voltage-operated Ca(2+) channel antagonists and, in contrast to freshly isolated hepatocytes, does not require a pertussis toxin-sensitive trimeric GTP-binding protein.

The treatment of H4-IIE cells (an immortalised liver cell line derived from the Reuber rat hepatoma) with thapsigargin, 2, 5-di-(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid, or pretreatment with EGTA, stimulated Ca(2+) inflow (assayed using intracellular fluo-3 and a Ca(2+) add-back protocol). No stimulation of Mn(2+) inflow by thapsigargin was detected. Thapsigargin-stimulated Ca(2+) inflow was inhibited by Gd(3+) (maximal inhibition at 2 microM Gd(3+)), the imidazole derivative SK&F 96365, and by relatively high concentrations of the voltage-operated Ca(2+) channel antagonists, verapamil, nifedipine, nicardipine and the novel dihydropyridine analogues AN406 and AN1043. The calmodulin antagonists W7, W13 and calmidazolium also inhibited thapsigargin-induced Ca(2+) inflow and release of Ca(2+) from intracellular stores. No inhibition of either Ca(2+) inflow or Ca(2+) release was observed with calmodulin antagonist KN62. Substantial inhibition of Ca(2+) inflow by calmidazolium was only observed when the inhibitor was added before thapsigargin. Pretreatment of H4-IIE cells with pertussis toxin, or treatment with brefeldin A, did not inhibit thapsigargin-stimulated Ca(2+) inflow. Compared with freshly isolated rat hepatocytes, H4-IIE cells exhibited a more diffuse actin cytoskeleton, and a more granular arrangement of the endoplasmic reticulum (ER). In contrast to freshly isolated hepatocytes, the arrangement of the ER in H4-IIE cells was not affected by pertussis toxin treatment. Western blot analysis of lysates of freshly isolated rat hepatocytes revealed two forms of G(i2(alpha)) with apparent molecular weights of 41 and 43 kDa. Analysis of H4-IIE cell lysates showed only the 41 kDa form of G(i2(alpha)) and substantially less total G(i2(alpha)) than that present in rat hepatocytes. It is concluded that H4-IIE cells possess store-operated Ca(2+) channels which do not require calmodulin for activation and exhibit properties similar to those in freshly isolated rat hepatocytes, including susceptibility to inhibition by relatively high concentrations of voltage-operated Ca(2+) channel antagonists. In contrast to rat hepatocytes, SOCs in H4-IIE cells do not require G(i2(alpha)) for activation. Possible explanations for differences in the requirement for G(i2(alpha)) in the activation of Ca(2+) inflow are briefly discussed.

Maitotoxin activates an endogenous non-selective cation channel and is an effective initiator of the activation of the heterologously expressed hTRPC-1 (transient receptor potential) non-selective cation channel in H4-IIE liver cells.

The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca(2+), Mn(2+) and Na(+), have a high selectivity for Na(+) compared with Ca(2+), and are inhibited by Gd(3+) (half-maximal inhibition at 1 microM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca(2+) concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase-polymerase chain reaction in poly(A)(+) RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca(2+) inflow and a modest enhancement of thapsigargin-initiated Ca(2+) inflow (measured using fura-2) and no enhancement of the highly Ca(2+)-selective store-operated Ca(2+) current (measured using patch-clamp recording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na(+) and Ca(2+), and are inhibited by Gd(3+) (half-maximal inhibition at 3 microM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na(+) compared with Ca(2+); (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca(2+)-selective store-operated Ca(2+) channels.

The nature and mechanism of activation of the hepatocyte receptor-activated Ca2+ inflow system.

Progress in elucidation of the properties of the hepatocyte receptor-activated Ca2+ inflow system (RACIS) has been hampered by difficulties in measuring rates of Ca2+ inflow to hepatocytes. These difficulties have led, for example, to different conclusions about the relationship between the extracellular Ca2+ concentration and the movement of Ca2+ through the RACIS. The hepatocyte RACIS admits Mn2+ and a number of other divalent cations as well as Ca2+. Many of these cations also inhibit the movement of Ca2+ through this system. While the RACIS is inhibited by high concentrations of verapamil and by some other Ca2+ antagonists, it is relatively insensitive to inhibition by organic compounds which inhibit other Ca2+ channels and Ca2+ transporters. There is circumstantial evidence which suggests that the hepatocyte RACIS is an exchange system, possibly one which catalyses Ca(2+)-H+ exchange or the co-transport of Ca2+ and OH-. Other circumstantial evidence suggests that the RACIS is a channel, with some similarities to voltage-operated Ca2+ channels in excitable cells. However, experiments using the patch-clamp technique have not yet detected agonist-stimulated Ca2+ movement across the hepatocyte plasma membrane. The molecular components of the RACIS probably differ from those which facilitate the large inflow of Ca2+ to hepatocytes which occurs in the absence of an agonist. The mechanism by which agonists activate the RACIS has not been elucidated.(ABSTRACT TRUNCATED AT 250 WORDS)

An evaluation of strategies available for the identification of GTP-binding proteins required in intracellular signalling pathways.

Strategies which can be used to elucidate the nature of a GTP-binding regulatory protein (G-protein) involved in an intracellular pathway of interest in the complex environment of the cell are described and evaluated. A desirable strategy is considered to be one in which the first stage indicates a requirement for one or more G-proteins, provides information on whether a monomeric, trimeric or other type of G-protein is involved, and gives some idea of the G-protein sub-class. In the second stage the specific G-protein involved is identified. Approaches available for investigations in the first stage include the use of analogues of GTP and GDP, AlF4-, inhibitors of G-protein isoprenylation, bacterial toxins which covalently modify G-proteins, and the introduction of a purified GDP dissociation inhibitor, GDP exchange and/or GTP-ase activating protein. Identification of the specific G-protein in the second stage can be achieved using anti G-protein antibodies, G-protein-or receptor-derived peptides, antisense G-protein RNA and over-expressed, constitutively-active or dominant-negative G-protein mutants. The correct interpretation of results obtained with GTP and GDP analogues and AlF4- in the first stage is complex and often difficult, and requires a thorough understanding of the functions and mechanisms of activation of G-proteins. Nevertheless, it is important to reach the correct conclusion at this stage since considerable time and expense are usually required for investigations in the second stage.

Effects of adrenaline on a compartment of slowly-exchangeable calcium in the perfused rat heart.

The effects of adrenaline on kinetically-distinct compartments of exchangeable calcium in the spontaneously-beating perfused rat heart were investigated under steady-state conditions using a 45Ca2+ outflow technique together with a nonlinear least-squares curve fitting procedure and 51Cr-EDTA to monitor the loss of freely-diffusible Ca2+ from the extracellular space. In addition to Ca2+ distributed in the vascular (compartment 1) and interstitial (compartment 2) spaces, the minimum number of kinetically-distinct compartments of cellular exchangeable Ca2+ (which include Ca2+ bound to extracellular sites on the sarcolemma) required to fit the data (compartments 3, 4 and 5) was three. A system in which cellular compartment 3 is linked to the vascular space and compartments 4 and 5 are linked to the interstitial space was consistent with the data. For hearts perfused under control conditions, the fractional transfer rates for Ca2+ outflow from cellular compartments 3, 4 and 5 were 0.70, 0.11 and 0.017 min-1, respectively. Adrenaline increased the quantity of exchangeable Ca2+ in compartment 5 and decreased that present in the compartment 4. Evidence that compartment 5 includes mitochondrial exchangeable Ca2+ is discussed. It is concluded that one of the actions of adrenaline on the heart is to increase the quantity of exchangeable Ca2+ in the mitochondria.

Effects of electrical stimulation and an intracellular calcium chelator on calcium movement in suspensions of isolated myocardial muscle cells.

The procedure of Haworth RA, Hunter DR and Berkoff HA (J Mol Cell Cardiol, 1980; 12:715-23) for the isolation of myocardial muscle cells from rat hearts has been modified by the addition of a step which involves centrifugation of the cells through a Percoll gradient. This increased the proportion of rod-shaped cells from 47 +/- 2.2 to 80 +/- 1.3% (mean +/- SEM, n = 7). In the absence of electrical stimulation but in the presence of 1.3 mmol . litre-1 Ca2+ less than 2% of the cells beat spontaneously. This number was not increased by addition of the Ca2+-selective ionophore A23187. A method in which isolated myocytes suspended in a cyclindrical incubation chamber are stimulated to beat by electrical impulses is described. At 1.3 mmol . litre-1 extracellular Ca2+, electrical stimulation increased by 30% the amount of 45Ca2+ exchanged in the period 0.25 to 3 min following addition of 45Ca2+. For myocytes subjected to electrical stimulation, the amount of 45Ca2+ exchanged increased as the concentration of extracellular Ca2+ increased. At 0.5 mmol . litre-1 extracellular Ca2+ verapamil reduced the amount of 45Ca2+ exchanged by 15% while La3+ reduced the amount of 45Ca2+ exchanged by 80%. Incubation of myocytes with the acetoxymethyl ester of the intracellular Ca2+ chelating agent bis (o-aminophenoxy)-ethane-N,N,N'N'-tetraacetic acid (BAPTA) for 45 min led to an inhibition of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of verapamil, dibutyryl cyclic AMP, and extracellular calcium on intracellular free calcium concentrations in myocytes isolated from rat ventricles.

The concentration of free calcium in the myoplasm of myocytes isolated from rat ventricles was measured using quin2. Incubation of myocytes with the acetoxymethylester of quin2 (50 mumol.litre-1) for 45 min was required to give effective loading with quin2. At 1 mmol.litre-1 extracellular calcium free calcium was 210(17) (n = 5) nmol.litre-1. Depolarisation of myocytes by addition of 40 mmol.litre-1 potassium caused a threefold increase in free calcium. Increases in extracellular calcium from 0.25 to 2.0 mmol.litre-1 caused a twofold increase in free calcium in polarised (resting) myocytes and a 2.5-fold increase in cells depolarised with potassium. The ability of verapamil to inhibit the electrically stimulated contraction of myocytes was dependent on the stimulation voltage. Half-maximal inhibition was given by 0.7 and 2 mumol.litre-1 verapamil at 50 and 100 V stimulation respectively. High concentrations of verapamil completely inhibited potassium induced increases in the fluorescence of quin2 loaded cells. Half-maximal inhibition was observed at 0.5 mumol.litre-1 verapamil. The calcium agonist CGP 28392 enhanced potassium induced fluorescence of quin2 loaded cells. Dibutyryl cyclic adenosine monophosphate and adrenaline increased the proportion of rod shaped cells that contracted in response to stimulation by low, but not high, voltage. Dibutyryl cyclic AMP caused a threefold increase in the potassium induced increase in free calcium. It is concluded that verapamil decreases and that calcium agonists, dibutyryl cyclic AMP, and increases in extracellular calcium increase free calcium, as monitored by intracellular quin2.

Does a decrease in subplasmalemmal Ca2+ explain how store-operated Ca2+ channels are opened?

The phenomenon of store-activated Ca2+ inflow (capacitative Ca2+ entry) in which the depletion of Ca2+ in the endoplasmic reticulum (ER) increases the probability of opening of store-operated Ca2+ channels (SOCs) located in the plasma membrane is ubiquitous in 'non-excitable' animal cells and is also found in some 'excitable' cells. At present, neither the structures of SOCs nor the mechanism(s) by which a decrease in Ca2+ in the lumen of the ER activates SOCs are well understood. This paper discusses the hypothesis that a decrease in the concentration of Ca2+ in restricted regions of the subplasmalemmal space (bounded by the plasma membrane and peripheral regions of the ER) is responsible for the activation of SOCs. The hypothesis rests on observations made by others that Ca2+ is a strong feed-back inhibitor of SOCs and of the endoplasmic reticulum (Ca(2+)+Mg2+)-ATPases (SERCAs), and on the concepts (developed previously by others) of a subplasmalemmal space and the directed flow of Ca2+ through SOCs into the lumen of the ER and from there to the deep cytoplasmic space. The way in which the hypothesis might explain the actions of agonists (acting via inositol 1,4,5-trisphosphate) and thapsigargin (an inhibitor of SERCAs) in activating SOCs under physiological conditions is described. The proposed involvement of thapsigargin-insensitive SERCAs, and possible limitations of the hypothesis are discussed.