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G J Barritt

Publications and source records attributed to G J Barritt.

At least 19 recordsLinked to original sources
PubMed1992

Affinity labelling of the active site of brain phosphatidylinositol 4-kinase with 5'-fluorosulphonylbenzoyl-adenosine.

5'-p-Fluorosulphonylbenzoyl-adenosine (FSO2BzAdo), an affinity labelling analogue of ATP, was used to label the active site of sheep brain phosphatidylinositol 4-kinase (PtdIns 4-kinase). The incubation of PtdIns 4-kinase with concentrations of FSO2BzAdo as low as 50 microM resulted in considerate inactivation of the enzyme. (e.g. 55% less after 60 min with 50 microM FSO2BzAdo). The kinetics of inactivation of PtdIns 4-kinase by FSO2BzAdo suggest a two-step mechanism, in which a rapid reversible binding of FSO2BzAdo to the enzyme is followed by a covalent sulphonation step. The first-order rate constant (k2) for the inactivation of PtdIns 4-kinase was calculated to be 0.063 min-1, and the steady-state constant of inactivation (Ki) to be 200 microM. Preincubation of the enzyme with either ATP plus Mg2+, or PtdIns alone, prior to addition of FSO2BzAdo reduced the degree of inactivation of the enzyme; suggesting that FSO2BzAdo binds within the active site PtdIns 4-kinase. Moreover, since ATP plus Mg2+ provided the greatest protection against inactivation, it is concluded that the main site of labelling of PtdIns 4-kinase by FSO2BzAdo is within the ATP-binding site of the enzyme. Results obtained from chemical modification experiments, which employed pyridoxal 5'-phosphate and tetranitromethane, are consistent with a catalytically-essential lysine being present within the ATP-binding site of PtdIns 4-kinase. Therefore, it is hypothesised that the inactivation of PtdIns 4-kinase by FSO2BzAdo may be due to the labelling of this lysine residue.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

Purification and chemical modification of a phosphatidylinositol kinase from sheep brain.

A membrane-bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500-fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X-100, ammonium sulphate fractionation and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol.min-1.mg-1. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 +/- 10 kDa by HPLC gel filtration in the presence of Triton X-100. Kinetic measurements have shown that the apparent Km value of PtdIns kinase for the utilisation of PtdIns is 22 microM and for ATP 67 microM. Mg2+ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg2+. In addition to adenosine and ADP, the 2'(3')-O-(2,4,6-trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a Ki of 32 microM. Enzymatic activity was found to be stimulated by Triton X-100 but inhibited by deoxycholate.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

Inhibition of Ca2+ inflow causes an abrupt cessation of growth-factor-induced repetitive free Ca2+ transients in single NIH-3T3 cells.

In single NIH-3T3 fibroblasts loaded with fura-2, bombesin induced one of three patterns of increase in the concentration of intracellular free Ca2+ [( Ca2+]i): a single transient increase, a sustained increase, or repetitive transient increases in [Ca2+]i. Foetal-calf serum and ATP also gave these three patterns of response, although a lower proportion of cells gave repetitive Ca2+ transients in response to ATP. An increase in the concentration of bombesin from 1 to 25 nM increased the proportion of cells which exhibited repetitive Ca2+ transients. At 25 nM-bombesin, the proportion of cells which exhibited repetitive Ca2+ transients increased as the extracellular Ca2+ (Ca2+o) concentration was increased from 1 to 5 mM. Removal of Ca2+o by addition of EGTA, or inhibition of Ca2+ inflow by treatment of cells incubated in the presence of Ca2+o with verapamil or an activator of protein kinase C, abruptly terminated repetitive Ca2+ transients, with only one transient observed after the cessation of Ca2+ inflow. Repetitive Ca2+ transients were not observed in cells incubated in the absence of Ca2+o and in the presence of EGTA. Addition of Ca2+o to cells previously incubated in the presence of EGTA caused a resumption of repetitive Ca2+ transients. Addition of thapsigargin alone induced a large transient increase in [Ca2+]i, whereas much smaller transient increases in [Ca2+]i were induced in about 30% of cells tested by caffeine or carbonyl cyanide m-chlorophenylhydrazone (CCCP) plus oligomycin. Thapsigargin or the combination of CCCP plus oligomycin completely inhibited bombesin-induced repetitive Ca2+ transients, whereas caffeine had no effect. It is concluded from the studies of the role of Ca2+o that NIH-3T3 cells differ from other cell types in the anatomical or chemical links between extracellular Ca2+ and the intracellular stores involved in the generation of Ca2+ transients, whereas the results of the experiments with inhibitors indicate that the generation of repetitive Ca2+ transients in NIH-3T3 cells is unlikely to involve Ca(2+)-induced Ca2+ release from caffeine-sensitive stores.

Adenosine Triphosphate↗
PubMed1991

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)

Animals↗
PubMed1991

Low molecular weight GTP-binding proteins in hepatocytes and an assessment of the role of p21ras proteins in the activation of phospholipase D.

A GTP-binding protein with an apparent molecular weight of 25 kDa was detected in hepatocyte extracts using SDS-PAGE and [alpha-32P]GTP. p21ras proteins could only be detected by immunological analysis. The amounts of p21ras proteins present in isolated hepatocytes and in a highly purified preparation of liver plasma membrane vesicles were 0.3 and 4 ng p21ras protein/micrograms membrane protein, respectively. In comparison with the total cell extract, the degree of enrichment of plasma membrane vesicles with p21ras was similar to that of 5'-nucleotidase. The p21ras proteins were tightly associated with the membrane. Treatment of [3H]choline-labelled plasma membranes with an excess concentration of the anti-p21ras antibody Y13-259 failed to inhibit either basal or guanosine 5'-[gamma-thio]triphosphate (GTP[S])-stimulated [3H]choline release. It is concluded that in hepatocytes (a) the majority of p21ras is bound to the plasma membrane and (b) p21ras is not directly involved in the activation by GTP[S] of phospholipase D.

Animals↗
PubMed1990

The roles of phospholipase D and a GTP-binding protein in guanosine 5'-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5'-[beta gamma-imido]triphosphate and guanosine 5'-[alpha beta-methylene]triphosphate, but not adenosine 5'-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5'-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

Animals↗
PubMed1990

NIH-3T3 cells transformed with a ras oncogene exhibit a protein kinase C-mediated inhibition of agonist-stimulated Ca2+ inflow.

1. The ability of bombesin or platelet-derived growth factor (PDGF) to stimulate Ca2+ inflow (assessed by measuring changes in the intracellular free Ca2+ concentration in cells loaded with fura-2) in NIH-3T3 cells transformed with the EJ/T24-Ha-ras-1 oncogene is inhibited when compared with the action of the agonists on wild-type cells. 2. The effects of transformation with the ras oncogene are associated with complete inhibition of the ability of bombesin to release Ca2+ from intracellular stores, a substantial decrease in the number of bombesin receptors, no change in the ability of foetal calf serum or ionomycin to release Ca2+ from intracellular stores and the activation of protein kinase C. 3. The effects of transformation with the H-ras oncogene on the ability of bombesin or PDGF to stimulate Ca2+ inflow were mimicked by a 30 min exposure of wild-type cells to phorbol dibutyrate. This action of phorbol dibutyrate was completely blocked by prior treatment of wild-type cells for 24 h with the phorbol ester. 4. It is concluded that one of the actions of the H-ras oncogene in fibroblasts is to inhibit agonist-stimulated Ca2+ inflow by a mechanism which involves the activation of protein kinase C.

Animals↗
PubMed1990

The liver cell plasma membrane Ca2+ inflow systems exhibit a broad specificity for divalent metal ions.

1. The inflow of Mn2+ across the plasma membranes of isolated hepatocytes was monitored by measuring the quenching of the fluorescence of intracellular quin2, by atomic absorption spectroscopy and by the uptake of 54Mn2+. The inflow of other divalent metal ions was measured using quin2. 2. Under ionic conditions which resembled those present in the cytoplasmic space, Mn2+, Zn2+, Co2+, Ni2+ and Cd2+ each quenched the fluorescence of a solution of Ca2(+)-quin2. 3. The addition of Mn2+, Zn2+, Co2+, Ni2+ or Cd2+ to cells loaded with quin2 caused a time-dependent decrease in the fluorescence of intracellular quin2. Plots of the rate of decrease in fluorescence as a function of the concentration of Mn2+ reached a plateau at 100 microM-Mn2+. 4. The rate of decrease in fluorescence induced by Mn2+ was stimulated by 20% in the presence of vasopressin. The effect of vasopressin was completely inhibited by 200 microM-verapamil. Adrenaline, angiotensin II and glucagon also stimulated the rate of decrease in the fluorescence of intracellular quin2 induced by Mn2+. 5. The rate of decrease in fluorescence induced by Zn2+, Co2+, Ni2+ or Cd2+ was stimulated by between 20 and 190% in the presence of vasopressin or angiotensin II. 6. The rates of uptake of Mn2+ measured by atomic absorption spectroscopy or by using 54Mn2+ were inhibited by about 20% by 1.3 mM-Ca2+o and stimulated by 30% by vasopressin. 7. Plots of Mn2+ uptake, measured by atomic absorption spectroscopy or with 54Mn2+, as a function of the extracellular concentration of Mn2+ were biphasic over the range 0.05-1.0 mM added Mn2+ and did not reach a plateau at 1.0 mM-Mn2+. 8. It is concluded that (i) hepatocytes possess both a basal and a receptor-activated divalent cation inflow system, each of which has a broad specificity for metal ions, and (ii) the receptor-activated divalent cation inflow system is the receptor-operated Ca2+ channel.

Aminoquinolines↗
PubMed1989

The measurement of Ca2+ inflow across the liver cell plasma membrane by using quin2 and studies of the roles of Na+ and extracellular Ca2+ in the mechanism of Ca2+ inflow.

1. Rates of Ca2+ inflow across the hepatocyte plasma membrane in the presence of vasopressin were estimated by using quin2. 2. Plots of the rate of Ca2+ inflow as a function of the intracellular quin2 concentration reached a plateau at about 1.7 mM intracellular quin2. Ca2+ inflow was inhibited by 60% in the presence of 400 microM-verapamil. 3. A plot of the rate of Ca2+ inflow as a function of the concentration of extracellular Ca2+ ([Ca2+]o) was biphasic. The second (slower) phase showed no sign of saturation at values of [Ca2+]o up to 5 mM. It is concluded that, in the presence of vasopressin, Ca2+ flows into the liver cell by two different processes, one of which is not readily saturated by Ca2+o. 4. The effect of the replacement of extracellular NaCl by choline or tetramethylammonium chloride on cellular Ca2+ movement was found to depend on the presence or absence of intracellular quin2. 5. In cells loaded with quin2 and incubated in the presence of choline or tetramethylammonium chloride, a small decrease in the basal intracellular free Ca2+ concentration ([Ca2+]i) was observed, and the increase in [Ca2+]i caused by the addition of vasopressin was considerably diminished when compared with cells incubated in the presence of NaCl. In cells loaded with quin2, replacement of NaCl by choline chloride caused a decrease in Ca2+ inflow in the presence of vasopressin, as measured by using quin2 or 45Ca2+ exchange, whereas no change in Ca2+ inflow was observed in the absence of vasopressin. 6. In cells not loaded with quin2, replacement of NaCl by choline chloride did not alter Ca2+ inflow either in the presence or in the absence of vasopressin. 7. It is concluded that (i) Ca2+ inflow through the basal and receptor-activated Ca2+ inflow systems does not involve the inward movement of Ca2+ in exchange for Na+ or the induction of Ca2+ inflow by intracellular Na+, and (ii) the presence of both intracellular quin2 and extracellular choline or tetramethylammonium chloride (in place of NaCl) inhibits Ca2+ inflow through the receptor-activated Ca2+ inflow system but not through the basal Ca2+ inflow system, and inhibits the release of Ca2+ from intracellular stores.

Aminoquinolines↗
PubMed1989

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.

Animals↗
PubMed1989

Evidence that guanosine 5'-[gamma-thio]triphosphate stimulates plasma membrane Ca2+ inflow when introduced into hepatocytes.

1. Slowly hydrolysable analogues of GTP were introduced into hepatocytes by incubating the cells in the absence of Mg2+ and in the presence of ATP4-. Experiments using guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])indicated that about 50% of the GTP[S] loaded into the cells was subsequently hydrolysed. 2. In cells loaded with GTP[S] and incubated in the absence of added extracellular Ca2+ (Ca2+o), the rate of activation of glycogen phosphorylase observed after addition of 1.3 mM-Ca2+o was 250% greater than the rate observed in unloaded cells. Smaller effects (130%) were observed in cells loaded with either guanyl-5'-yl imidodiphosphate or guanosine 5-[beta-thio]diphosphate (GDP[S]). Cells loaded with adenosine 5'-[gamma-thio]triphosphate showed no increase in glycogen phosphorylase activity on addition of Ca2+o. 3. The effect of a submaximal concentration of GTP[S] on the Ca2+-induced activation of glycogen phosphorylase was additive with that of a half-maximally effective concentration of vasopressin. GTP[S] did not increase the effect of a maximally effective concentration of the hormone. 4. Cells loaded with GTP[S] exhibited an increased initial rate of 45Ca2+ exchange measured at 1.3 mM-Ca2+o. 5. GTP[S] did not affect the amount of 45Ca2+ exchanged by cells incubated at 0.1 mM-Ca2+o or the ability of vasopressin to release 45Ca2+ from these cells. 6. It is concluded that the introduction of slowly hydrolysable analogues of GTP to the liver cell cytoplasmic space stimulates the inflow of Ca2+ across the plasma membrane through a channel similar to that activated by vasopressin.

Adenosine Triphosphate↗
PubMed1988

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.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

Evidence that neomycin inhibits plasma membrane Ca2+ inflow in isolated hepatocytes.

The effects of neomycin on Ca2+ fluxes and inositol polyphosphates in hepatocytes were investigated since it has been proposed that this antibiotic inhibits inositol 1,4,5-triphosphate formation in fibroblasts [D. H. Carney, D. L. Scott, E. A. Gordon and E. F. LaBelle, Cell 42, 479 (1985)]. In hepatocytes incubated at 1.3 mM extracellular Ca2+ (Ca2+o) neomycin (2 mM) inhibited 45Ca2+ exchange both in the presence or absence of vasopressin. At 1.3 mM Ca2+o, but not at higher concentrations of Ca2+o, the antibiotic (2 mM) inhibited the increase in glycogen phosphorylase a activity observed at late but not at early times after addition of vasopressin. The antibiotic also inhibited the increase in phosphorylase activity caused by the subsequent addition of 1.3 mM Ca2+o to cells previously incubated in the presence of vasopressin and in the absence of added Ca2+o. The concentration of the antibiotic (2 mM) which gave half-maximal inhibition of phosphorylase activation by vasopressin had no effect on the activation of phosphorylase by glucagon or the release of Ca2+ from intracellular stores induced by vasopressin. At a concentration of 10 mM, neomycin caused a 50% inhibition of the formation of [3H]inositol polyphosphates induced by vasopressin. It is concluded that neomycin, at concentrations which inhibit phosphoinositide-specific phospholipase C in other types of cells inhibits the inflow of Ca2+ across the plasma membrane but does not inhibit inositol trisphosphate formation in hepatocytes.

Animals↗
PubMed1988

Effects of quinacrine on vasopressin-induced changes in glycogen phosphorylase activity, Ca2+ transport and phosphoinositide metabolism in isolated hepatocytes.

In isolated hepatocytes, quinacrine (150-250 microM) inhibited vasopressin-induced increases in glucose release, glycogen phosphorylase a activity and 45Ca2+ efflux; and glucagon-induced increases in glucose release and cyclic AMP formation. These results indicate that a phospholipase A2 enzyme sensitive to quinacrine is unlikely to be involved in the process by which vasopressin stimulates glycogen phosphorylase activity in the liver cell. In cells labelled with [3H]inositol, much lower concentrations of quinacrine (20-50 microM) inhibited the stimulation by vasopressin of the accumulation of [3H]inositol. The drug had little effect on vasopressin-induced accumulation of [3H]inositol mono-, bis- and tris-phosphates. In the absence of vasopressin, higher concentrations of quinacrine caused a small stimulation of glycogen phosphorylase activity, 45Ca2+ release and the formation of [3H]inositol polyphosphates. Quinacrine did not inhibit the degradation by liver homogenates of inositol 1-phosphate, inositol 4,5-bisphosphate or inositol 1,4,5-trisphosphate. It is concluded that concentrations of quinacrine comparable with those which inhibit phospholipase A2 [G.J. Blackwell, W.G. Duncombe, R.J. Flower, M.F. Parsons and J.R. Vane, Br. J. Pharmac. 59, 353-366 (1977)] inhibit the stimulation by vasopressin of inositol utilization without significantly affecting coupling between hormone receptors and adenyl cyclase or phosphoinositide-specific phosphodiesterase, the action of the phosphodiesterase, and the degradation of inositol triphosphate.

Animals↗
PubMed1987

Evidence that a pertussis-toxin-sensitive substrate is involved in the stimulation by epidermal growth factor and vasopressin of plasma-membrane Ca2+ inflow in hepatocytes.

1. In hepatocytes, epidermal growth factor (EFG) (a) increased the rate of 45Ca2+ exchange in cells incubated at 1.3 mM extracellular Ca2+, (b) increased the activity of glycogen phosphorylase a and the intracellular free Ca2+ concentration (measured with quin2) in a process dependent on the concentration of extracellular Ca2+, and (c) enhanced the increase in glycogen phosphorylase activity which follows the addition of Ca2+ to cells previously incubated in the absence of Ca2+. It is concluded that EGF stimulates plasma-membrane Ca2+ inflow. 2. The effects of the combination of EGF and vasopressin on the rate of 45Ca2+ exchange and on the rate of increase in glycogen phosphorylase activity were the same as those of vasopressin alone. 3. The amount of 45Ca2+ released by EGF from internal stores was about 30% of that released by vasopressin. No detectable increase in [3H]inositol mono-, bis- or tris-phosphate was observed after the addition of EGF to cells labelled with myo-[3H]inositol. 4. In hepatocytes isolated from rats treated with pertussis toxin, the effects of EGF and vasopressin on phosphorylase activity (measured at 1.3 mM-Ca2+) and on the rate of Ca2+ inflow (measured with quin2) were markedly decreased compared with those in normal cells. 5. Treatment with pertussis toxin did not impair the ability of vasopressin to release Ca2+ from internal stores, but decreased vasopressin-stimulated [3H]inositol polyphosphate formation by 50%. 6. It is concluded that the mechanism(s) by which vasopressin and EGF stimulate plasma-membrane Ca2+-inflow transporters in hepatocytes involves a GTP-binding regulatory protein sensitive to pertussis toxin, and does not require an increase in the concentration of inositol trisphosphate comparable with that which induces the release of Ca2+ from the endoplasmic reticulum.

Animals↗
PubMed1987

The stimulation by sodium fluoride of plasma-membrane Ca2+ inflow in isolated hepatocytes. Evidence that a GTP-binding regulatory protein is involved in the hormonal stimulation of Ca2+ inflow.

1. In isolated hepatocytes NaF increased the rate of 45Ca2+ exchange, the cytoplasmic free Ca2+ concentration ([Ca2+]i) (monitored by using quin2), and the activity of glycogen phosphorylase a in a Ca2+-dependent manner. 2. In cells previously incubated in the absence of extracellular Ca2+(Ca2+o), NaF caused a pronounced enhancement in the increases in the activity of glycogen phosphorylase and in [Ca2+]i observed when Ca2+ was subsequently added. The effect of NaF on glycogen phosphorylase activity was inhibited by verapamil and deferoxamine, and was potentiated by AlCl3. 3. The actions of NaF were associated with (a) increases in [3H]inositol polyphosphates, which were slower in onset and about half the magnitude of those induced by vasopressin, in hepatocytes labelled with [3H]inositol, and (b) enhanced rates of O2 utilization and decreased concentrations of ATP. The latter effects were not potentiated by AlCl3. 4. Preincubation of hepatocytes with vasopressin in the absence of added Ca2+o for times up to 30 min did not diminish the ability of a subsequent addition of extracellular Ca2+ to activate glycogen phosphorylase. 5. 12-O-Tetradecanoylphorbol 13-acetate had little effect on 45Ca2+ exchange and did not enhance the activation by Ca2+o of phosphorylase in hepatocytes incubated in the absence of Ca2+o. 6. On the basis of the observation that AlF4- activates GTP-binding regulatory proteins [Sternweiss & Gilman (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4888-4891], it is concluded that the present results provide evidence for the function of a GTP-binding regulatory protein in the mechanism by which hormones stimulate plasma-membrane Ca2+ inflow in the liver cell, and indicate that an increase in [Ca2+]i and the activation of protein kinase C are not part of this mechanism.

Aluminum↗