Role of a Guanine Nucleotide-binding Regulatory Protein in the ...

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Nov 25, 2015 - Hydrolysis of Hepatocyte Phosphatidylinositol 4,B-Bisphosphate by ... phate; PI-4,5-P2, phosphatidylinositol 4,5-bisphosphate; DAG, 1,2-.
Vol. 260,No. 27, Issue of November 25, pp. 14477-14483.1985 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

0 1985 by The American Societyof Biological Chemists, Inc.

Role of a Guanine Nucleotide-binding Regulatory Protein in the Hydrolysis of Hepatocyte Phosphatidylinositol4,B-Bisphosphate by Calcium-mobilizing Hormones and theControl of Cell Calcium STUDIES UTILIZING ALUMINUMFLUORIDE* (Received for publication, June 14,1985)

Peter F. Blackmore$, Stephen B. Bocckino, Laura E. Waynick, and John H.ExtonQ From the Laboratories for the Studies of Metabolic Disorders,Howard Hughes Medical Institute, and the Department of Physiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Treatment of isolated hepatocytes with NaF produced a concentration-dependent activation of phosphorylase, inactivation of glycogen synthase, efflux of Ca2+,rise in cytosolic free Ca2+([CaZ+li),increase in myo-inositol-1,4,5,-P3 levels, decrease in phosphatidylinositol-4,5-P2 levels, and increase in l,2-diacylglycerol levels. These changes were evident within 1 min and maximum at 2-5 min. Maximum effects on Ca” efflux, [Ca2+]i, glycogensynthase, and phosphorylase were observed with 15 mM NaF, whereas myoinositol-1,4,5-P3 and l,2-diacylglycerol levels were maximally stimulated by 50 n m NaF. The levels of intracellular CAMP were decreased by NaF (up to 10 mM) in theabsence or presence of glucagon (0.1-1 nM) or forskolin (2 PM). The effectsof low dosesof NaF (215 mM) to inhibit basal or glucagon-stimulated CAMP accumulation, mobilize Ca”, activate phosphorylase, and inactivate glycogen synthase were allpotentiated by AlC13. This potentiation was abolished by the A13+ chelator deferoxamine. These results illustrate thatAlF; can mimic the effects of Ca2+-mobilizinghormones in hepatocytes and suggest that the coupling of the receptors for these hormones to the hydrolysis of phosphatidylinositol4,5-P2 to myo-inositol 1,4,5-P3 is through a guanine nucleotide-binding regulatory protein. This is because AlF: is known to modulate the activityof other guanine N., and transducin). nucleotide regulatory proteins(Ni,

There isevidence that a guanine nucleotide-bindingprotein (possibly Nil or analogous to Ni) is involved in the coupling

* This work was supported in partby Research Grant IN-25X from the American Cancer Society and Grant 5R01 AM33291 from the National Institutes of Health, United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This articlemusttherefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Associate Investigator from the Howard Hughes Medical Institute. 3 Investigator from the Howard Hughes Medical Institute. The abbreviations and trivial name used are: Ni, the guanine nucleotide regulatory protein that couples inhibitory receptors to adenylate cyclase; N., the guanine nucleotide regulatory protein that couples stimulatory receptors to adenylate cyclase; deferoxamine, N - [5-[3- [ (5-aminopentyl)hydroxycarbamoyl]propionamido]pentyl] 3 - [ [5- (N-hydroxyacetamido)pentyl] carbamoyl]propionohydroxamic acid monomethanesulfonate (salt); IPB, myo-inositol 1,4,5-triphosphate; PI-4,5-P2, phosphatidylinositol 4,5-bisphosphate; DAG, 1,2-

of various receptors to the hydrolysis of inositol-containing phospholipids and the mobilization of Ca2+.Introduction of nonhydrolyzable analogues of GTP into permeabilized mast cells stimulates histamine release in the presence of calcium (1).These analogues also enhance serotonin release and DAG formation in permeabilized platelets (2, 3) and stimulate PI4,5-P2 breakdown in certain membranes (4-6). There have also been several reports that guanine nucleotides inhibit the binding of calcium-mediated agonists to their receptors (for references, see Ref. 7) and that some of these agonists stimulate guanine nucleotide hydrolysis or exchange in isolated membranes (8,9).In addition, islet-activating protein, a pertussis toxin which inhibits Ni through ADP-ribosylation,suppresses histamine release or PI-4,5-p~breakdown inducedby immunoglobulin E or compound 48/80 in mast cells (10, 11) and inhibits chemotactic peptide-induced arachidonic acid release, PI-4,5-P2breakdown, or calcium mobilizationin neutrophils (12-17). We investigated the possibleroleplayed by a guanine nucleotide-bindingprotein in themobilization of intracellular Ca2+in hepatocytes. It is known that A13+,in thepresence of F-, is a potent modulator of N,, Ni, and transducin (18-23). The active principal is thought to be AlF: (18,20, 23). We thus treated hepatocytes with F- in the presence or absence of A13+to see if this would mimicthe effects of Ca2+-mobilizing hormones on PI-4,5-P2 breakdown and Ca” release (24-26). Since AlF: could conceivably also activate N, and thereby increase CAMP, which can elicit Ca2” mobilization (27, 28), changes in cAMP were also examined. When hepatocytes were stimulated by AlF:, net Ca2+efflux was observedtogether with an increase in free cytosolic Ca2+ ([Ca2+Ii).This increase was most probably mediated by IP3, since this compound was increased after AlF, treatment and has been shown to release Ca2+ fromintracellular stores (for references, see Ref. 29). A decrease in cAMP wasalso observed with low concentrations of F-, which suggeststhat A 1 F ; was either unable to activate N. in the intact cell or that the simultaneous stimulation of N, and another guanine nucleotide-binding protein(s) resulted in no change or a slight decrease in CAMP. The results herein are consistent with the involvement of a guanine nucleotide regulatory protein(s) in the hydrolysis of PI-4,5-P2to DAG and IP3. This guanine nucleotide-binding protein could betermed N, (4) since its function would be to couple the various Ca2+-mobilizinghormone receptors to the diacyl-sn-glycerol; EGTA, ethylene glycol bis(8-aminoethyl ether)N,N,N,N’-tetraacetic acid; [Ca2+Ii,free cytosolic calcium.

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activation of the phosphodiesterase (phospholipase C) which hydrolyzes polyphosphoinositides. EXPERIMENTAL PROCEDURES

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4 Materials-NaF (catalogue no. S299) andAM, (catalogue no. 0 0 140 A573) werefrom Fisher. Vasopressin (synthetic [8-arginine]vasopres7 0 0 =N , sin) was from Sigma. my0-[2-~H]Inositolwas from New England '" u 120 Q Nuclear. Deferoxamine was from Ciba Pharmaceutical Corp. Forsko-I 0 lin was from Calbiochem-Behring. Glucagon was from Lilly. n 600 Preparation of Hepatocytes-Hepatocytes were prepared from fed male rats (200-250 g of body weight) as previously described (30). I c 0.8 Since the addition of F-to Krebs-Henseleit bicarbonate buffer results in the formation of insoluble CaF, (31), Ca" was omitted from the buffer. This omission does not impair the initial effects of hormones E on Ca2+mobilization as previously shown (26, 32). The total Ca2+ - 0.6 concentration in the medium was between 50 and 100 p~ as deterfi mined by atomic absorptionspectroscopy. -CE Methods for Hepatocytes-Methods for the measurement of CAMP, phosphorylase a, glycogen synthase, myo-inositol-1,4,5,-P3,total cell v) 0.4 c Ca2+, [Ca2+Ii,and 1,2-diacylglycerolhave been described previously m (26,27,30,33-37). Cell incubations were performed in eitherduplicate n z or triplicate. Representative experimentsare shown. 0 5 10 15 20 0 5 10 15 20 2 Hepatocyte phosphoinositides were labeled for 90 min with myoNaF (mM) [2-3H]inositol as previously described (26). Phosphoinositides were deacylated by the methylamine method, and thewater-soluble glycero FIG. 2. Dose responseof NaF to increaseDAG (A),decrease derivatives were separatedon1-mlformate form anion-exchange PI-4,5-Pz (B),alter CAMPlevels (0, and increase I P S levels columns (38). (0)in hepatocytes. After 8 min of incubation with NaF, 5-ml aliquots of cell suspensions (45 mg/ml, wet weight) were extracted RESULTS with ch1oroform:methanol (1:2), and theDAG was measured by high pressure liquid chromatography (34). Since changes in PI-4,5-P2were Fig. 1shows the concentration dependence of the effects of transient, thelevels of [3H]PI-4,5-P2and [3H]IP3were measured at 2 NaF on phosphorylase activation and Ca2+ efflux. Both pa- min, when maximal changes were observed. Each value is the mean rameters showed similar concentration dependencies, with f S.E. from triplicate incubations. Data are representative of three maximal changes being observed between 10 and15 mM NaF. experiments.

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FIG. 1. Dose response of NaF to activate hepatocyte phosphorylase and elicit calcium efflux. Hepatocytes (2.0 ml) were incubated a t 37 "C with continuous gassing with 02:COz(955). After 5 min, duplicate 0.5-ml aliquots were removed for measurement of total cell calcium and a 0.5-ml aliquot was removed for phosphorylase a determination. A representative experiment of five is shown, and the data aremeans %.S.E. from triplicate incubations.

cAMP or Caz+,the effects of NaF on cAMP were also measured. The level of cAMP decreased by approximately 30% (Fig. 2C) with 10 mM NaF and then returned toward basal with 20 mM NaF. Thus,the activation of phosphorylase observed in Fig. 1 is not due to an increase in cAMP but is most probably due to anincrease in cytosolic Ca". The latter explanation is supported by the observation that NaF produced a concentration-dependent increase in IP3 (Fig. 2 0 ) due to PI-4,5-Pz breakdown (Fig. 2B). The other product of PI-4,5-Pz hydrolysis by phosphodiesterase is DAG. Sodium fluoride produced a concentration-dependent increase in this lipid (Fig. 2 A ) , which was similar to that in IP3 (Fig. 20). Consistent with the changes in IP3(Fig. 2 0 ) and phosphorylase (Fig. l ) , [Ca2+Iiincreased with 5 mM NaF as shown by the change in Quin 2 fluorescence (Fig. 3A),which was similar to thatseen with 0.02 nM vasopressin. Potentiation of NaF Effects by AZCZ3-The modulation of Ni, N., and transducin activities by NaF requires the presence of a small amount of A13+ (18, 20, 23). Ineffective concentrations of NaF can be made effective by adding A13+,which by itself does not activateguanine nucleotide regulatory proteins (18).We thus examined the ability of A13+ to potentiate the effects of low concentrations of NaF onCa" efflux, phosphorylase activation, [Ca2+Ii,and cAMP levels. Fig. 4 shows the effects of varying concentrations ofA1C13 on Ca" content, phosphorylase activation, and cAMP levels in hepatocytes incubated in the presence and absence of 5 mM NaF. When added alone, A1C13 had no significant effects (Figs. 4 and 5), but produced it a marked potentiation of NaF effects. Maximal potentiation of 5 mM NaF was observed with 1-10 pM Ai3+for all parameters. In subsequent experiments, 10 p M Ai3+was routinely used. Fig. 5 shows the effect of 10 p~ AlC13 on changes induced by NaF in cell Ca2+ content, cAMP levels, phosphorylase a activity, and glycogen synthase activity. Concentrations of NaF which did not cause Ca2+

Hepatic Phosphoinositide Hydrolysis by Sodium Fluoride FIG. 3. Timecourse of NaFand vasopressin to raise [Ca2'li and activate phosphorylase. Hepatocytes were loaded with Quin 2 as previously described (27,30),centrifuged once, and resuspended in low Ca2+-containingmedia for the fluorescence measurements. The basal [Ca2-Iiwas approximately 200 nM. This was increasedto approximately 500 nM with 5 mM NaF d u s 10 uM AlCL (27, 30) (measured a f k i 2.5 min). The arrow indicates the time a t which the various agents were added (0.5 min). Each truce is the mean of triplicate incubations. The effects of two concentrations of vasopressin are included for comparison. In B, the effects of 0.05 nM vasopressin and 5 mM N f l plus 10 WM AlCh on phosphorylase activation are shown for comparison with the fluore cence changes. Each value is the mean f S.E. from triplicate incubations.

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efflux (4 mM and less) were able to elicit Ca" efflux when 10 PM AlCh was present (Fig. 5C). The decrease in cAMP brought about by 1-2 mM NaF was greatly potentiated by 10 PM A13+ (Fig. 5D), but no potentiation by A1Cb was observed at concentrations of NaF higher than 6 mM. The inactivation of glycogen synthase elicited by 1-6 mM NaF was also markedly potentiated by AlC13 (Fig. 5A). The effect ofA1C& on NaF-induced phosphorylase activation was less striking (Fig. 5B) due perhaps to the fact that Ca2+ was increased, but cAMP was decreased. Although not shown, A1C&also potentiated theaccumulation of DAG, [3H]IPz,and [3H]IP3elicited by low concentrations of NaF. The chelator deferoxamine (39) was added to reverse the effects of AP+. Fig. 6 shows the concentration dependence of NaF plus AlC13to activate phosphorylase in thepresence and absence of0.5 mM deferoxamine. Deferoxamine not only completely reversed the potentiation by A1Cl3, but also inhibited the effect of NaF itself. Similar effects were seen on Ca2+ efflux (data notshown). Fig. 7 shows the time courses of NaF and NaF plus AlCh to mobilize cell CaZ+and activate phosphorylase in thepresence and absence of deferoxamine. At all times examined, deferoxamine completely reversed the effects of 5 mM NaF plus A13+on cell Ca2" and phosphorylase. The rate of [Ca2+Iiincrease induced by 5 mM NaF was increased by 10 PM AlC1, (Fig. 3A). The presence of 10 PM AlC1, made 5 mM NaF almost equivalent to theeffects of 0.05 nM vasopressin on [Ca2+]iand phosphorylase (Fig. 3, A and B). Effect on NaF on GlucagonActions-Figs. 2C and 5 0 show that low concentrations of NaF in thepresence or absence of A13+are able to lower CAMP,presumably via activation of Ni or another inhibitory guanine nucleotide-binding protein(s). With higher concentrations of NaF (15-20 mM), cAMP levels returned to control values. When 50 mM NaF was added to hepatocytes, cAMP increased by 45% after 5 min of incubation, and this increase was potentiated by the presence of isobutylmethylxanthine (data notshown). To further confirm the ability of NaF to lower CAMP, hepatocytes were treated with forskolin (Fig. 8A) or glucagon together with different concentrations of NaF in the presence and absence of AP+

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FIG. 4. Dose response of AlCls on NaF-induced changes in phosphorylase activity, cell calcium, and cAMP levels. Hepa-

tocytes were incubated simultaneously with a submaximal dose of 5 mM NaF and various concentrations of AlCl,. After 5 min of incubation, total cell calcium, phosphorylase u activity, and cAMP levels were measured. Each value is the mean k S.E. from triplicate incubations.

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