0013-7227/90/1266-2831$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 6 Printed in U.S.A.
Mechanism of Prostaglandin E2-Induced Glucose Production in Rat Hepatocytes TETSUYA MINE*, ITARU KOJIMA, AND ETSURO OGATA Division of Gastroenterology and Cell Biology, Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan
ABSTRACT. Effects of prostaglandin E2(PGE2) on glycogenolysis were examined in rat hepatocytes. In a batch incubation system using isolated hepatocytes, PGE2 increased glucose output dose-dependently. The glycogenolytic effect of PGE2 was detected at a concentration of 1CT11 M, and 10~8 M PGE2 elicited the maximum glucose output, which was equal to that by glucagon. PGE2 did not increase cAMP at any dose tested (10~n1O~4 M). Instead, PGE2 increased the cytoplasmic free calcium concentration ([Ca2+]c). When the effect of PGE2 on [Ca2+]c was studied in aequorin-loaded cells, the effect of PGE2 on [Ca2+]c
P
ROSTANOIDS are produced in many tissues and modulate diverse cell functions by exerting specific actions on tissues. Specifically in the liver, Wernze et al. (1) reported that there exist high concentrations of prostanoids in human portal and hepatic veins, which may originate from splanchnic organs as well as liver itself. Rat liver also produces various types of prostaglandins (PGs) (2); in particular, PGE is synthesized in primary culture of rat hepatocytes (3). Furthermore, there are receptors for prostanoids in hepatocytes (4). Despite these observations, the physiological significance of prostanoids in the liver is still a matter of debate because of conflicting results of various studies dealing with the effect of prostanoids on hepatic metabolism. In terms of carbohydrate metabolism, Wheeler and Epand (5) reported that PGE induced glycogenolysis in perfused rat liver. Although Buxton et al. (6) observed that PGE stimulated glycogenolysis, they suggested that in perfused liver, PGE induced glycogenolysis by causing transient hypoxia. It should be mentioned that the doses of PGE used in their experiment were higher than 10~6 M, which may not be physiological. On the other hand, Brass and Garrity (7) showed that 16,16-dimethyl-PGE2, an analog of PGE2, inhibited glucagon-mediated glucose output and cAMP production. In a more recent study, Casteleijn et al. (8) reported that PGE released from Received November 13,1989. * To whom all correspondence and requests for reprints should be addressed.
was detected at 10 12 M, and the magnitude of the response increased in a dose-dependent manner. PGE2 increased [Ca2+]c even in the presence of 1 /uM extracellular calcium, suggesting that PGE2 mobilizes calcium from an intracellular pool. In line with these observations, PGE2 increased the production of inositol trisphosphate. Compared with the action of PGE2, 16,16dimethyl-PGE2, a PGE2 analog, was less potent in stimulating glycogenolysis. These results indicate that PGE2 stimulates glycogenolysis by activating the calcium messenger system. (Endocrinology 126: 2831-2836,1990)
Kupffer and endothelial cells increased glycogenolysis in isolated hepatocytes. In view of these controversial results in the literature, we attempted to examine the effect of PGE2 on glucose production in isolated hepatocytes. We also addressed the mechanism by which PGE2 stimulates glucose output in hepatocytes using both PGE2 and 16,16-dimethyl-PGE2. The results indicate that PGE2 stimulates hepatic glucose production by activating the calcium messenger system.
Materials and Methods Methods Hepatocyte isolation and batch incubation. Hepatocytes were isolated from fed male Wistar rats, weighing about 200 g, using the collagenase perfusion technique of Berry and Friend (9), as previously described (10, 11). Cells were suspended in modified Hanks' solution containing (in millimolar concentrations): NaCl, 137; KC1, 3.5; KH2PO4, 0.44; NaHCO3, 4.2; Na2HPO4( 0.33; CaCl2, 1.0; and HEPES (pH 7.4), 20 (equilibrated with O2 gas). Determination of glucose output. Isolated hepatocytes (4 X 106 cells/ml) were incubated in modified Hanks' solution oxygenated with 100% O2. The glucose concentration in the incubation medium was determined according to the method of Corvera et al. (12). Measurement of phosphorylase-A activity. Measurement of phosphorylase-A activity was carried out by the method of Blackmore and Exton (13), as previously described (14). Cell samples were taken for determination of phosphorylase-A ac-
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PGE2 AND HEPATIC GLYCOGENOLYSIS
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tivity 2 min after PGE2 addition. Phosphorylase-A activity was expressed as a percentage of the maximal phosphorylase-A activity.
Endo • 1990 Vol 126 • No 6
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Measurement of cytoplasmic free calcium concentration by aequorin. Changes in the cytoplasmic concentration of free calcium ([Ca2+]c) were assessed by measuring aequorin bioluminescence. Aequorin was loaded into hepatocytes by making plasma membrane reversibly permeable by the method of Borle et al. (15), as described previously (10, 11). The viability of aequorin-loaded cells was evaluated as follows. First, more than 95% aequorin-loaded cells excluded trypan blue. Second, aequorin-loaded hepatocytes responded to glucagon normally in terms of both glycogenolysis and cAMP production (10). Aequorin bioluminescence was calibrated in terms of free calcium concentration, assuming a magnesium concentration of 1 mM and an even distribution of calcium in cytosol (16). However, in some figures, the magnitude of aequorin luminescence is expressed as electric current. Measurement of cAMP production. Aliquots of cell suspension (4 X 106 cell/ml) were incubated with various agents for 2 min in the presence of 0.5 mM 3-isobutyl-l-methylxanthine. Trichloroacetic acid was then added to the samples to stop the reaction. After the removal of trichloroacetic acid by washing with diethylether, cAMP was measured after succinylation by using a RIA kit (Yamasa, Tokyo, Japan). Measurement of inositol trisphosphate production. Hepatocytes (107 cell/ml) were labeled with [3H]inositol by incubating cells with 10 jiiCi/ml [3H]inositol for 120 min. After the labeling period, cells were washed and incubated in modified Hanks' solution containing 10 mM LiCl for 10 min. Cells were stimulated for 20 sec with PGE2. The reaction was stopped by adding perchloric acid (final concentration, 10%). Cells were homogenized by repetitive aspirations through a 26-gauge needle and centrifuged at 800 X g for 5 min. The supernatant was neutralized with 5 M KOH and applied to an anion exchange column. Inositol phosphates were separated as described by Berridge and Irvine (17). Isomers of inositol trisphosphate were not measured. Materials PGE2and 16,16-dimethyl-PGE2 were kindly donated by Ono Pharmaceutical Co., Ltd. (Osaka, Japan). PGE2 was dissolved in buffer. 16,16-Dimethyl-PGE2 was dissolved in phosphate buffer. Aequorin was purchased from Dr. J. R. Blinks, Mayo Foundation (Rochester, MN). [3H] Inositol was obtained from Amersham International (Buckinghamshire, England). Statistical analyses Student's t test was used for statistical analyses. When appropriate, P < 0.05 was taken as significant.
Results Glucose output induced by PGE2 and 16,16-dimethylPGE2 in isolated hepatocytes In batch incubation system, PGE2 stimulated glycogenolysis in a dose-dependent manner (Fig. 1). The ac-
10"
10"8
10"
[Prostaglandin](M)
FIG. 1. Dose-response relationship for PGE2-and 16,16-dimethylPGE2-induced glucose output in isolated hepatocytes. Aliquots of hepatocyte suspension containing 4 X 106 cells in 1 ml were incubated for 11 min in the presence of various concentrations of PGE2 (•) or 16,16dimethyl-PGE2 (O). Glucose output was determined as described in Materials and Methods. Values are the mean ± SE for six determinations. *, P < 0.05; **, P < 0.01 {us. control). TABLE 1. Effects of PGE2 and glucagon on phosphorylase-A activity Addition
% Increase in phosphorylase-A
PGE2 (lO"9 M) PGE2 (10"8 M) Glucagon (5 x 10" M)
10.2 ± 3.4 14.4 ± 5.0 16.8 ± 3.6
Aliquots of cell suspension (4 x 106 cells/ml) were incubated at 37 C in modified Hanks' solution. Two minutes after the addition of various agents, aliquots were frozen with liquid N2. Phosphorylase-A activity is expressed as a percentage of the maximal activity, and the percent increase from control is presented. Values are the mean ± SE for four determinations. The absolute maximal activity of phosphorylase-A was 55 IU/mg protein.
tion of PGE2 was detected at 10~n M, and the maximum effect was obtained at 10~8 M (Fig. 1). The magnitude of the maximum effect of PGE2 was approximately the same as that of glucagon. Glucose output measured in this condition reflects mainly the breakdown of glycogen (12). As shown in Table 1, both PGE2 and glucagon increased the activity of phosphorylase-A. When 10~9 M PGE2 and 5 X 10"9 M glucagon were added simultaneously, there was additivity between the actions of two agents (Fig. 2). However, when 10~6 M PGE2 was added simultaneously with 5 X 10~9 M glucagon, PGE2 greatly inhibited the action of glucagon. 16,16-Dimethyl-PGE2, a PGE2 analog, was less potent than PGE2 in stimulating glucose output. The maximum effect of 16,16-dimethyl-PGE2,
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PGE2 AND HEPATIC GLYCOGENOLYSIS
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500
150
X
100
200
50
nl 10"
10-"
10"'°
10
10
[Prostaglandin] (M) Glu
PGE2 10'9M
PGE, 10"6M
Glu 5X1O-"M
Glu 5X1O"9M
PGE? 10"'M
PGE? 10 6M
FIG. 2. Effect of PGE2 on glucagon (Glu)-induced glucose output in isolated hepatocytes. Aliquots of hepatocytes suspension containing 4 x 106 cells in 1 ml were incubated for 11 min in the presence of either 10~6 or 10~9 M PGE2 and 5 x 10~9 M glucagon. Glucose output was determined as described in Materials and Methods. Glucose output induced by 5 x 10~9 M glucagon was expressed as 100%. Values are the mean ± SE for six determinations. The absolute value of glucose output induced by 5 X 10~9 M glucagon was 20 nmol/mg protein-11 min. *, P < 0.01 us. glucagon only.
FIG. 3. Dose-response relationship for the elevation of [Ca2+]c induced by PGE2 or 16,16-dimethyl-PGE2 in isolated hepatocytes. Aequorinloaded hepatocytes were stimulated with various concentrations of PGE2 (•) or 16,16-dimethyl-PGE2 (O). The peak [Ca2+]c response is plotted as a concentration of the stimulator. Values are the mean ± SE for eight determinations. *, P < 0.001 us. control.
B
PGE2
PGE,
TABLE 2. Effects of PGE2 and glucagon on cAMP production
cAMP (pmol/ng protein)
Addition None PGE2 (lO"8 M) PGE2 (lO"9 M) Glucagon (5 x 10"
M)
3.7 ± 4.2 ± 3.6 ± 40.3 ±
0.2 0.5 0.2 2.0°
Aliquots of cell suspension (4 x 106 cells/ml) were incubated at 37 C in modified Hanks' solution containing various agents for 2 min in the presence of 0.5 mM 3-isobutyl-l-methylxanthine. cAMP was measured as described in Materials and Methods. Values are the mean ± SE for four determinations. 0 P < 0.001, none vs. glucagon.
which was approximately 20% that of PGE2, was obtained at 10~9 M (Fig. 1). Effects of PGE2-, 16,16-dimethyl-PGE2-, and glucagoninduced cAMP production in isolated hepatocytes In the next set of experiments, changes in cAMP production induced by PGE2,16,16-dimethyl-PGE2, and glucagon were examined. When cAMP production was measured, no response to PGE2 of cAMP was observed at concentrations of 10~9 and 10~8 M (Table 2). As is the case for PGE2, 16,16-dimethyl-PGE2 did not increase
Imin FIG. 4. Comparison of effects of PGE2 on [Ca2+]c in the presence of 1 mM and 1 pM extracellular calcium. Aequorin-loaded hepatocytes were stimulated by 10"9 M PGE2 in the presence of 1 mM (A) or 1 n\l (B) extracellular calcium. When the concentration of extracellular calcium was reduced to 1 fiM, the concentration of calcium was fixed by using Ca2+-EGTA buffer. Values shown are the representative of four experiments with similar results.
cAMP production (data not shown). We also measured cAMP production after 5 or 10 min of incubation with various concentrations of PGE2, but no response of
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PGE2 AND HEPATIC GLYCOGENOLYSIS
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Angiotensin II /
Glucagon
Imin
FIG. 5. Comparison of intracellular calcium pools affected by glucagon, PGE2, and angiotensin-II. Aequorin-loaded cells were first treated with 5 x 10~9 M glucagon (A) or 10~8 M angiotension-H (B); subsequently,
10"8 M PGE2 was added. The concentration of extracellular calcium was 1 nM. The traces presented are representative of at least six experiments with similar results.
~ 60 50
40
Ia 30 in
I 20 10
Endo • 1990 Vol 126 • No 6
quorin-loaded hepatocytes. Figure 3 depicts the doseresponse relationship for the effects of PGE2 and 16,16dimethyl-PGE2 on [Ca2,+]c. The basal concentration of [Ca2+]c was 183 nM on assuming an intracellular magnesium concentration of 1 mM and even distribution of calcium inside the cell. The effect of PGE 2 on [Ca2+]c was detected at 10~12 M. The magnitude of the response increased in a dosedependent manner, and the effect appeared to be saturated at 10~9 M. In contrast to PGE2, 16,16-dimethylPGE2 was less potent in elevating [Ca2+]c than that was PGE. As demonstrated in Fig. 4A, the addition of 10~9 M PGE2 resulted in a prompt increase in aequorin bioluminescence. The action of PGE2 on [Ca2+]c was transient; [Ca2+]c returned to a value close to the resting level within 45 sec. To identify the source of calcium mobilized by PGE2, aequorin-loaded hepatocytes were incubated in modified Hanks' solution containing 1 /uM calcium. In medium containing this concentration of calcium, basal as well as stimulated calcium influx are negligible (18). When cells were stimulated by 10~9 M PGE2 in the presence of 1 juM extracellular calcium, a response of [Ca2+]c was observed, but the peak value was smaller than that observed in cells incubated in modified Hanks' solution containing 1.0 mM calcium (Fig. 4B). To examine whether PGE2 mobilizes calcium from an intracellular pool of calcium affected by either glucagon or angiotensin-II, we added these agents sequentially in the presence of 1 pM extracellular calcium. When hepatocytes were first incubated with glucagon, PGE2 added subsequently induced an additional rise of [Ca2+]c (Fig. 5A). In contrast, when cells were first incubated with angiotensin-II, subsequent addition of PGE2 did not cause any increase in [Ca2+]c (Fig. 5B). Effect of PGE2 on inositol phosphate production
0
10"
12
1 0 -n
10 -io
10"
9
10"
8
[PGE2](M) FIG. 6. Dose-response relationship for PGE2-induced inositol trisphosphate. Hepatocytes (107 cells/ml) were labeled with [3H] inositol by incubating cells with 10 /*Ci/ml [3H]inositol for 120 min. After washing, cells were stimulated for 20 sec with various concentrations of PGE2 in the presence of 10 mM LiCl. Inositol trisphosphate was determined as described in Materials and Methods. Values are the mean ± SE for four determinations. *, P < 0.05; **, P < 0.01 (vs. control).
cAMP was observed (data not shown). In contrast to PGE2 and 16,16-dimethyl-PGE2, 5 x 10~9 M glucagon markedly increased cAMP production. Action of PGE2 and 16,16-dimethyl-PGE2 on cytoplasmic free calcium The effect of PGE2 on [Ca2+]c was assessed by measuring changes in aequorin bioluminescence using ae-
The effect of PGE2 on inositol phosphate production was examined using [3H]inositol-labeled cells. As shown in Fig. 6, 10~10 M PGE2 induced a 1.5-fold increase in [3H] inositol trisphosphate. PGE2 also increased the content of [3H] inositol bisphosphate and monophosphate. The dose-response relationship for the PGE2-induced increase in inositol trisphosphate production reveals that the effect of PGE2 was detected at 10~n M. PGE2 action increased in a dose-dependent manner and was maximal at 10"9 M.
Discussion Results obtained in the present study clearly show that PGE2 stimulates glucose output in isolated rat hepatocytes. It is reported that an increase in glucose output observed in the conditions employed in this study is due largely to breakdown of glycogen (12). In agreement with
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PGE, AND HEPATIC GLYCOGENOLYSIS
this idea, PGE2 augments the activity of phosphorylaseA. It is, therefore, clear that PGE2 affects glycogen metabolism in hepatocytes. These results support the report by Casteleijn et al. (8) that PGE2 released from Kupffer cells acts on parenchymal cells and stimulates glucose production. However, our results differ from the conclusion of Brass and Garrity (7) that PGE2 inhibited, rather than stimulated, glucagon-induced glycogenolysis. Reasons for the discrepancy may be the difference in experimental conditions. Specifically, Brass and Garrity (7) used 16,16-dimethyl-PGE2, and employing concentrations higher than 10~6 M, they showed that 16,16-dimethyl-PGE2 had little stimulatory action and that 16,16-dimethyl-PGE2 inhibited glucagon action. 16,16Dimethyl-PGE2 is much less potent than PGE2 in stimulating glucose production, and the stimulatory effect of 16,16-dimethyl-PGE2 is negligible at high concentrations. Furthermore, a high concentration of PGE2 inhibits glucagon-induced glycogenolysis. It is, therefore, not surprising that 16,16-dimethyl-PGE2 inhibits glucagon action at high concentrations. Hence, when administered at appropriately low concentrations, PGE2 stimulates glycogenolysis. In any event, it is highly likely that PGE2 produced by either Kupffer cells or endothelial cells modulates hepatic glucose metabolism. In the present study we addressed the mechanism by which PGE2 augments glucose production. In various cell systems PGE2 elicits its action via the cAMP messenger system. However, PGE2 does not increase the generation of cAMP. This observation is in agreement with the report by Okamura and Terayama (4). They showed that PGE2, even at a concentration of 10~6 M, has only a minimal stimulatory action (10% above basal) on adenylate cyclase using plasma membrane preparations. Instead of activating the cAMP messenger system, the following observations indicate that PGE2 acts on the calcium messenger system. First, PGE2 increases [Ca2+] c in hepatocytes by causing calcium release from an intracellular pool; second, PGE2 causes production of inositol trisphosphate, a putative mobilizer of calcium; and third, the dose-response relationship of PGE2-induced glucose production correlates well with that of PGE2-induced elevation of [Ca2+]c. In the present study we used aequorin for the measurement of [Ca2+]c. The aequorin method has some limitations. For example, plasma membranes of cells should be made reversibly permeable to load aequorin. Nevertheless, aequorinloaded hepatocytes possess normal responsiveness to agonists, as reported previously (10,11), and the present method thus provides a reasonable way to monitor [Ca2+]c. The idea that PGE2 activates the calcium messenger system is consistent with a recent report by Yokohama and co-workers (19) that PGE2 causes breakdown of
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phosphoinositides in adenal medulla. They further purified the receptor for PGE2 and found that it is associated with G-protein (20). In Swiss 3T3 cells, PGE increases [Ca2+]c by stimulating calcium entry, rather than by causing release from an intracellular pool (21). Therefore, there are at least three types of messenger systems activated by PGE: stimulation of adenylate cyclase, activation of phospholipase-C specific to polyphosphoinositide, and activation of a calcium gating system. It remains to be elucidated whether three different types of messenger systems are linked to three different types of receptors or whether different transducing mechanisms are responsible for such diversity. The results of the present study indicate that PGE2 causes glycogenolysis by inducing phosphoinositide breakdown. In this regard, the mode of action of PGE2 resembles the action of PGF2«, which also activates the calcium messenger system, as described by Athari and Jungermann (22) and Altin and Bygrave (23).
Acknowledgments We thank Ms. Fujimaki and Ms. Kakinuma for their expert technical assistance.
References 1. Wernze H, Titton W, Goerig M 1986 Release of prostanoids into the portal and hepatic vein in patients with chronic liver disease. Hepatology 6:911 2. Morita I, Murota S 1978 Prostaglandin-synthesizing system in rat liver. Eur J Biochem 90:441 3. Robertson RP, Westcott KR, Storm DR, Rice MG 1980 Downregulation in vivo of PGE receptor and adenylate cyclase stimulation. Am J Physiol 239:E75 4. Okamura N, Terayama H 1977 Prostaglandin receptor-adenylate cyclase system in plasma membranes of rat liver and ascites hepatomas and the effect of GTP upon it. Biochim Biophys Acta 465:54 5. Wheeler GE, Epand RM 1975 Prostaglandin Ei: anomalous effects on glucose production in rat liver. Mol Pharmacol 11:335 6. Buxton DB, Fisher RA, Briseno DL, Hanahan DJ, Olson MS 1987 Glycogenolytic and haemodynamic responses to heat-aggregated immunogloblin G and prostaglandin E2 in the perfused rat liver. Biochem J 243:493 7. Brass EP, Garrity MJ 1985 Effect of E-series prostaglandins on cyclic AMP-dependent and -independent hormone-stimulated glycogenolysis in hepatocytes. Diabetes 34:291 8. Casteleijn E, Kuiper J, van Rooij HCJ, Kamps JAAM, Koster JF, van Berkel TJC 1988 Hormonal control of glycogenolysis in parenchymal liver cells by Kupffer and endothelial liver cells. J Biol Chem 263:2699 9. Berry MN, Friend DS 1969 High-yield preparation of isolated rat liver parenchymal cells. J Cell Biol 4:506 10. Mine T, Kojima I, Kimura S, Ogata E 1986 Comparison of the changes in cytoplasmic free calcium concentration induced by phenylephrine, vasopressin and angiotensin II in hepatocytes. Biochem Biophys Res Commun 140:170 11. Mine T, Kojima I, Kimura S, Ogata E 1987 Assessment of the role of Ca2+ mobilization from intracellular pools, using dantrolene, in the glycogenolytic action of a-adrenergic stimulation in perfused rat liver. Biochim Biophys Acta 927:792 12. Corvera S, Huerta-Bahena J, Pelton JT, Hruby JV, Trivedi D, Garcia-Saintz JA 1984 Metabolic effects and cyclic AMP levels
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13. 14. 15. 16. 17. 18.
PGE2 AND HEPATIC GLYCOGENOLYSIS
produced by glucagon, (1-iV-a-trinitrophenylhistidine, 12-homoarginine) glucagon, and forskolin in isolated rat hepatocytes. Biochim Biophys Acta 804:434 Blackmore PF, Exton JH 1985 Assessment of effects of vasopressin angiotensin II, and glucagon on Ca2+ fluxes and phosphorylase activity in liver. Methods Enzymol 109:550 Mine T, Kojima I, Ogata E 1989 Stimulation of glucose production by activin-A in isolated rat hepatocytes. Endocrinology 125:586 Borle AB, Freudrich CC, Snowdowne KW 1986 A simple method for incorporating aequorin into mammalian cells. Am J Physiol 251:323 Snowdowne KW, Bole AB 1984 Changes in cytosolic ionized calcium induced by activators of seretion in GH3 cells. Am J Physiol 246:E198 Berridge MJ, Irvine RF 1984 Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312:315 Mauger JP, Poggioli J, Guesdon F, Claret M 1984 Noradrenalin, vasopressin and angiotensin increase Ca2+ channel in isolated rat liver cells. Biochem J 221:121
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19. Yokohama H, Tanaka T, Ito S, Negishi M, Hayashi H, Hayaishi O 1988 Prostaglandin E receptor enhancement of catecholamine release may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells. J Biol Chem 263:1119 20. Negishi M, Ito S, Yokohama H, Hayashi H, Katada T, Ui M, Hayaishi O 1988 Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins. J Biol Chem 263:6893 21. Yamashita T, Takai Y 1987 Inhibition of prostaglandin E r induced elevation of cytoplasmic free clacium ion by protein kinase Cactivating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts. J Biol Chem 262:5536 22. Athari A, Jungermann K 1989 Direct activation by prostaglandin F2a but not thromboxane A2 of glycogenolysis via an increase in inositol 1,4,5-trisphosphate in rat hepatocytes. Biochem Biophys Res Commun 163:1235 23. Altin JG, Bygrave FL 1988 Prostaglandin F2aand the thromboxane A2 analogue ONO-11113 stimulate Ca2+ fluxes and other physiological responses in rat liver. Biochem J 249:677
Meeting Announcement Symposium on Catecholamines and Other Neurotransmitters and Stress The Fifth International Symposium on Catecholamines and Other Neurotransmitters and Stress will be held at Smolenice Castle, Czechoslovakia from June 24-29, 1991. The program includes invited plenary lectures as well as oral and poster presentations. For additional information, please contact: Dr. Richard Kvetnansky, Secretary General, NINDS, National Institutes of Health, Building 10, Room 5N-214, Bethesda, Maryland 20892, telephone 301-4961314 or write directly to the Meeting Secretariat, Dr. I. Vietor, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Czechoslovakia, telephone 42-7-37 38 00.
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Vol. 126, No. 6 Printed in U.S.A.
Mechanism of Prostaglandin E2-Induced Glucose Production in Rat Hepatocytes TETSUYA MINE*, ITARU KOJIMA, AND ETSURO OGATA Division of Gastroenterology and Cell Biology, Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan
ABSTRACT. Effects of prostaglandin E2(PGE2) on glycogenolysis were examined in rat hepatocytes. In a batch incubation system using isolated hepatocytes, PGE2 increased glucose output dose-dependently. The glycogenolytic effect of PGE2 was detected at a concentration of 1CT11 M, and 10~8 M PGE2 elicited the maximum glucose output, which was equal to that by glucagon. PGE2 did not increase cAMP at any dose tested (10~n1O~4 M). Instead, PGE2 increased the cytoplasmic free calcium concentration ([Ca2+]c). When the effect of PGE2 on [Ca2+]c was studied in aequorin-loaded cells, the effect of PGE2 on [Ca2+]c
P
ROSTANOIDS are produced in many tissues and modulate diverse cell functions by exerting specific actions on tissues. Specifically in the liver, Wernze et al. (1) reported that there exist high concentrations of prostanoids in human portal and hepatic veins, which may originate from splanchnic organs as well as liver itself. Rat liver also produces various types of prostaglandins (PGs) (2); in particular, PGE is synthesized in primary culture of rat hepatocytes (3). Furthermore, there are receptors for prostanoids in hepatocytes (4). Despite these observations, the physiological significance of prostanoids in the liver is still a matter of debate because of conflicting results of various studies dealing with the effect of prostanoids on hepatic metabolism. In terms of carbohydrate metabolism, Wheeler and Epand (5) reported that PGE induced glycogenolysis in perfused rat liver. Although Buxton et al. (6) observed that PGE stimulated glycogenolysis, they suggested that in perfused liver, PGE induced glycogenolysis by causing transient hypoxia. It should be mentioned that the doses of PGE used in their experiment were higher than 10~6 M, which may not be physiological. On the other hand, Brass and Garrity (7) showed that 16,16-dimethyl-PGE2, an analog of PGE2, inhibited glucagon-mediated glucose output and cAMP production. In a more recent study, Casteleijn et al. (8) reported that PGE released from Received November 13,1989. * To whom all correspondence and requests for reprints should be addressed.
was detected at 10 12 M, and the magnitude of the response increased in a dose-dependent manner. PGE2 increased [Ca2+]c even in the presence of 1 /uM extracellular calcium, suggesting that PGE2 mobilizes calcium from an intracellular pool. In line with these observations, PGE2 increased the production of inositol trisphosphate. Compared with the action of PGE2, 16,16dimethyl-PGE2, a PGE2 analog, was less potent in stimulating glycogenolysis. These results indicate that PGE2 stimulates glycogenolysis by activating the calcium messenger system. (Endocrinology 126: 2831-2836,1990)
Kupffer and endothelial cells increased glycogenolysis in isolated hepatocytes. In view of these controversial results in the literature, we attempted to examine the effect of PGE2 on glucose production in isolated hepatocytes. We also addressed the mechanism by which PGE2 stimulates glucose output in hepatocytes using both PGE2 and 16,16-dimethyl-PGE2. The results indicate that PGE2 stimulates hepatic glucose production by activating the calcium messenger system.
Materials and Methods Methods Hepatocyte isolation and batch incubation. Hepatocytes were isolated from fed male Wistar rats, weighing about 200 g, using the collagenase perfusion technique of Berry and Friend (9), as previously described (10, 11). Cells were suspended in modified Hanks' solution containing (in millimolar concentrations): NaCl, 137; KC1, 3.5; KH2PO4, 0.44; NaHCO3, 4.2; Na2HPO4( 0.33; CaCl2, 1.0; and HEPES (pH 7.4), 20 (equilibrated with O2 gas). Determination of glucose output. Isolated hepatocytes (4 X 106 cells/ml) were incubated in modified Hanks' solution oxygenated with 100% O2. The glucose concentration in the incubation medium was determined according to the method of Corvera et al. (12). Measurement of phosphorylase-A activity. Measurement of phosphorylase-A activity was carried out by the method of Blackmore and Exton (13), as previously described (14). Cell samples were taken for determination of phosphorylase-A ac-
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PGE2 AND HEPATIC GLYCOGENOLYSIS
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tivity 2 min after PGE2 addition. Phosphorylase-A activity was expressed as a percentage of the maximal phosphorylase-A activity.
Endo • 1990 Vol 126 • No 6
25 r
Measurement of cytoplasmic free calcium concentration by aequorin. Changes in the cytoplasmic concentration of free calcium ([Ca2+]c) were assessed by measuring aequorin bioluminescence. Aequorin was loaded into hepatocytes by making plasma membrane reversibly permeable by the method of Borle et al. (15), as described previously (10, 11). The viability of aequorin-loaded cells was evaluated as follows. First, more than 95% aequorin-loaded cells excluded trypan blue. Second, aequorin-loaded hepatocytes responded to glucagon normally in terms of both glycogenolysis and cAMP production (10). Aequorin bioluminescence was calibrated in terms of free calcium concentration, assuming a magnesium concentration of 1 mM and an even distribution of calcium in cytosol (16). However, in some figures, the magnitude of aequorin luminescence is expressed as electric current. Measurement of cAMP production. Aliquots of cell suspension (4 X 106 cell/ml) were incubated with various agents for 2 min in the presence of 0.5 mM 3-isobutyl-l-methylxanthine. Trichloroacetic acid was then added to the samples to stop the reaction. After the removal of trichloroacetic acid by washing with diethylether, cAMP was measured after succinylation by using a RIA kit (Yamasa, Tokyo, Japan). Measurement of inositol trisphosphate production. Hepatocytes (107 cell/ml) were labeled with [3H]inositol by incubating cells with 10 jiiCi/ml [3H]inositol for 120 min. After the labeling period, cells were washed and incubated in modified Hanks' solution containing 10 mM LiCl for 10 min. Cells were stimulated for 20 sec with PGE2. The reaction was stopped by adding perchloric acid (final concentration, 10%). Cells were homogenized by repetitive aspirations through a 26-gauge needle and centrifuged at 800 X g for 5 min. The supernatant was neutralized with 5 M KOH and applied to an anion exchange column. Inositol phosphates were separated as described by Berridge and Irvine (17). Isomers of inositol trisphosphate were not measured. Materials PGE2and 16,16-dimethyl-PGE2 were kindly donated by Ono Pharmaceutical Co., Ltd. (Osaka, Japan). PGE2 was dissolved in buffer. 16,16-Dimethyl-PGE2 was dissolved in phosphate buffer. Aequorin was purchased from Dr. J. R. Blinks, Mayo Foundation (Rochester, MN). [3H] Inositol was obtained from Amersham International (Buckinghamshire, England). Statistical analyses Student's t test was used for statistical analyses. When appropriate, P < 0.05 was taken as significant.
Results Glucose output induced by PGE2 and 16,16-dimethylPGE2 in isolated hepatocytes In batch incubation system, PGE2 stimulated glycogenolysis in a dose-dependent manner (Fig. 1). The ac-
10"
10"8
10"
[Prostaglandin](M)
FIG. 1. Dose-response relationship for PGE2-and 16,16-dimethylPGE2-induced glucose output in isolated hepatocytes. Aliquots of hepatocyte suspension containing 4 X 106 cells in 1 ml were incubated for 11 min in the presence of various concentrations of PGE2 (•) or 16,16dimethyl-PGE2 (O). Glucose output was determined as described in Materials and Methods. Values are the mean ± SE for six determinations. *, P < 0.05; **, P < 0.01 {us. control). TABLE 1. Effects of PGE2 and glucagon on phosphorylase-A activity Addition
% Increase in phosphorylase-A
PGE2 (lO"9 M) PGE2 (10"8 M) Glucagon (5 x 10" M)
10.2 ± 3.4 14.4 ± 5.0 16.8 ± 3.6
Aliquots of cell suspension (4 x 106 cells/ml) were incubated at 37 C in modified Hanks' solution. Two minutes after the addition of various agents, aliquots were frozen with liquid N2. Phosphorylase-A activity is expressed as a percentage of the maximal activity, and the percent increase from control is presented. Values are the mean ± SE for four determinations. The absolute maximal activity of phosphorylase-A was 55 IU/mg protein.
tion of PGE2 was detected at 10~n M, and the maximum effect was obtained at 10~8 M (Fig. 1). The magnitude of the maximum effect of PGE2 was approximately the same as that of glucagon. Glucose output measured in this condition reflects mainly the breakdown of glycogen (12). As shown in Table 1, both PGE2 and glucagon increased the activity of phosphorylase-A. When 10~9 M PGE2 and 5 X 10"9 M glucagon were added simultaneously, there was additivity between the actions of two agents (Fig. 2). However, when 10~6 M PGE2 was added simultaneously with 5 X 10~9 M glucagon, PGE2 greatly inhibited the action of glucagon. 16,16-Dimethyl-PGE2, a PGE2 analog, was less potent than PGE2 in stimulating glucose output. The maximum effect of 16,16-dimethyl-PGE2,
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PGE2 AND HEPATIC GLYCOGENOLYSIS
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500
150
X
100
200
50
nl 10"
10-"
10"'°
10
10
[Prostaglandin] (M) Glu
PGE2 10'9M
PGE, 10"6M
Glu 5X1O-"M
Glu 5X1O"9M
PGE? 10"'M
PGE? 10 6M
FIG. 2. Effect of PGE2 on glucagon (Glu)-induced glucose output in isolated hepatocytes. Aliquots of hepatocytes suspension containing 4 x 106 cells in 1 ml were incubated for 11 min in the presence of either 10~6 or 10~9 M PGE2 and 5 x 10~9 M glucagon. Glucose output was determined as described in Materials and Methods. Glucose output induced by 5 x 10~9 M glucagon was expressed as 100%. Values are the mean ± SE for six determinations. The absolute value of glucose output induced by 5 X 10~9 M glucagon was 20 nmol/mg protein-11 min. *, P < 0.01 us. glucagon only.
FIG. 3. Dose-response relationship for the elevation of [Ca2+]c induced by PGE2 or 16,16-dimethyl-PGE2 in isolated hepatocytes. Aequorinloaded hepatocytes were stimulated with various concentrations of PGE2 (•) or 16,16-dimethyl-PGE2 (O). The peak [Ca2+]c response is plotted as a concentration of the stimulator. Values are the mean ± SE for eight determinations. *, P < 0.001 us. control.
B
PGE2
PGE,
TABLE 2. Effects of PGE2 and glucagon on cAMP production
cAMP (pmol/ng protein)
Addition None PGE2 (lO"8 M) PGE2 (lO"9 M) Glucagon (5 x 10"
M)
3.7 ± 4.2 ± 3.6 ± 40.3 ±
0.2 0.5 0.2 2.0°
Aliquots of cell suspension (4 x 106 cells/ml) were incubated at 37 C in modified Hanks' solution containing various agents for 2 min in the presence of 0.5 mM 3-isobutyl-l-methylxanthine. cAMP was measured as described in Materials and Methods. Values are the mean ± SE for four determinations. 0 P < 0.001, none vs. glucagon.
which was approximately 20% that of PGE2, was obtained at 10~9 M (Fig. 1). Effects of PGE2-, 16,16-dimethyl-PGE2-, and glucagoninduced cAMP production in isolated hepatocytes In the next set of experiments, changes in cAMP production induced by PGE2,16,16-dimethyl-PGE2, and glucagon were examined. When cAMP production was measured, no response to PGE2 of cAMP was observed at concentrations of 10~9 and 10~8 M (Table 2). As is the case for PGE2, 16,16-dimethyl-PGE2 did not increase
Imin FIG. 4. Comparison of effects of PGE2 on [Ca2+]c in the presence of 1 mM and 1 pM extracellular calcium. Aequorin-loaded hepatocytes were stimulated by 10"9 M PGE2 in the presence of 1 mM (A) or 1 n\l (B) extracellular calcium. When the concentration of extracellular calcium was reduced to 1 fiM, the concentration of calcium was fixed by using Ca2+-EGTA buffer. Values shown are the representative of four experiments with similar results.
cAMP production (data not shown). We also measured cAMP production after 5 or 10 min of incubation with various concentrations of PGE2, but no response of
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PGE2 AND HEPATIC GLYCOGENOLYSIS
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Angiotensin II /
Glucagon
Imin
FIG. 5. Comparison of intracellular calcium pools affected by glucagon, PGE2, and angiotensin-II. Aequorin-loaded cells were first treated with 5 x 10~9 M glucagon (A) or 10~8 M angiotension-H (B); subsequently,
10"8 M PGE2 was added. The concentration of extracellular calcium was 1 nM. The traces presented are representative of at least six experiments with similar results.
~ 60 50
40
Ia 30 in
I 20 10
Endo • 1990 Vol 126 • No 6
quorin-loaded hepatocytes. Figure 3 depicts the doseresponse relationship for the effects of PGE2 and 16,16dimethyl-PGE2 on [Ca2,+]c. The basal concentration of [Ca2+]c was 183 nM on assuming an intracellular magnesium concentration of 1 mM and even distribution of calcium inside the cell. The effect of PGE 2 on [Ca2+]c was detected at 10~12 M. The magnitude of the response increased in a dosedependent manner, and the effect appeared to be saturated at 10~9 M. In contrast to PGE2, 16,16-dimethylPGE2 was less potent in elevating [Ca2+]c than that was PGE. As demonstrated in Fig. 4A, the addition of 10~9 M PGE2 resulted in a prompt increase in aequorin bioluminescence. The action of PGE2 on [Ca2+]c was transient; [Ca2+]c returned to a value close to the resting level within 45 sec. To identify the source of calcium mobilized by PGE2, aequorin-loaded hepatocytes were incubated in modified Hanks' solution containing 1 /uM calcium. In medium containing this concentration of calcium, basal as well as stimulated calcium influx are negligible (18). When cells were stimulated by 10~9 M PGE2 in the presence of 1 juM extracellular calcium, a response of [Ca2+]c was observed, but the peak value was smaller than that observed in cells incubated in modified Hanks' solution containing 1.0 mM calcium (Fig. 4B). To examine whether PGE2 mobilizes calcium from an intracellular pool of calcium affected by either glucagon or angiotensin-II, we added these agents sequentially in the presence of 1 pM extracellular calcium. When hepatocytes were first incubated with glucagon, PGE2 added subsequently induced an additional rise of [Ca2+]c (Fig. 5A). In contrast, when cells were first incubated with angiotensin-II, subsequent addition of PGE2 did not cause any increase in [Ca2+]c (Fig. 5B). Effect of PGE2 on inositol phosphate production
0
10"
12
1 0 -n
10 -io
10"
9
10"
8
[PGE2](M) FIG. 6. Dose-response relationship for PGE2-induced inositol trisphosphate. Hepatocytes (107 cells/ml) were labeled with [3H] inositol by incubating cells with 10 /*Ci/ml [3H]inositol for 120 min. After washing, cells were stimulated for 20 sec with various concentrations of PGE2 in the presence of 10 mM LiCl. Inositol trisphosphate was determined as described in Materials and Methods. Values are the mean ± SE for four determinations. *, P < 0.05; **, P < 0.01 (vs. control).
cAMP was observed (data not shown). In contrast to PGE2 and 16,16-dimethyl-PGE2, 5 x 10~9 M glucagon markedly increased cAMP production. Action of PGE2 and 16,16-dimethyl-PGE2 on cytoplasmic free calcium The effect of PGE2 on [Ca2+]c was assessed by measuring changes in aequorin bioluminescence using ae-
The effect of PGE2 on inositol phosphate production was examined using [3H]inositol-labeled cells. As shown in Fig. 6, 10~10 M PGE2 induced a 1.5-fold increase in [3H] inositol trisphosphate. PGE2 also increased the content of [3H] inositol bisphosphate and monophosphate. The dose-response relationship for the PGE2-induced increase in inositol trisphosphate production reveals that the effect of PGE2 was detected at 10~n M. PGE2 action increased in a dose-dependent manner and was maximal at 10"9 M.
Discussion Results obtained in the present study clearly show that PGE2 stimulates glucose output in isolated rat hepatocytes. It is reported that an increase in glucose output observed in the conditions employed in this study is due largely to breakdown of glycogen (12). In agreement with
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PGE, AND HEPATIC GLYCOGENOLYSIS
this idea, PGE2 augments the activity of phosphorylaseA. It is, therefore, clear that PGE2 affects glycogen metabolism in hepatocytes. These results support the report by Casteleijn et al. (8) that PGE2 released from Kupffer cells acts on parenchymal cells and stimulates glucose production. However, our results differ from the conclusion of Brass and Garrity (7) that PGE2 inhibited, rather than stimulated, glucagon-induced glycogenolysis. Reasons for the discrepancy may be the difference in experimental conditions. Specifically, Brass and Garrity (7) used 16,16-dimethyl-PGE2, and employing concentrations higher than 10~6 M, they showed that 16,16-dimethyl-PGE2 had little stimulatory action and that 16,16-dimethyl-PGE2 inhibited glucagon action. 16,16Dimethyl-PGE2 is much less potent than PGE2 in stimulating glucose production, and the stimulatory effect of 16,16-dimethyl-PGE2 is negligible at high concentrations. Furthermore, a high concentration of PGE2 inhibits glucagon-induced glycogenolysis. It is, therefore, not surprising that 16,16-dimethyl-PGE2 inhibits glucagon action at high concentrations. Hence, when administered at appropriately low concentrations, PGE2 stimulates glycogenolysis. In any event, it is highly likely that PGE2 produced by either Kupffer cells or endothelial cells modulates hepatic glucose metabolism. In the present study we addressed the mechanism by which PGE2 augments glucose production. In various cell systems PGE2 elicits its action via the cAMP messenger system. However, PGE2 does not increase the generation of cAMP. This observation is in agreement with the report by Okamura and Terayama (4). They showed that PGE2, even at a concentration of 10~6 M, has only a minimal stimulatory action (10% above basal) on adenylate cyclase using plasma membrane preparations. Instead of activating the cAMP messenger system, the following observations indicate that PGE2 acts on the calcium messenger system. First, PGE2 increases [Ca2+] c in hepatocytes by causing calcium release from an intracellular pool; second, PGE2 causes production of inositol trisphosphate, a putative mobilizer of calcium; and third, the dose-response relationship of PGE2-induced glucose production correlates well with that of PGE2-induced elevation of [Ca2+]c. In the present study we used aequorin for the measurement of [Ca2+]c. The aequorin method has some limitations. For example, plasma membranes of cells should be made reversibly permeable to load aequorin. Nevertheless, aequorinloaded hepatocytes possess normal responsiveness to agonists, as reported previously (10,11), and the present method thus provides a reasonable way to monitor [Ca2+]c. The idea that PGE2 activates the calcium messenger system is consistent with a recent report by Yokohama and co-workers (19) that PGE2 causes breakdown of
2835
phosphoinositides in adenal medulla. They further purified the receptor for PGE2 and found that it is associated with G-protein (20). In Swiss 3T3 cells, PGE increases [Ca2+]c by stimulating calcium entry, rather than by causing release from an intracellular pool (21). Therefore, there are at least three types of messenger systems activated by PGE: stimulation of adenylate cyclase, activation of phospholipase-C specific to polyphosphoinositide, and activation of a calcium gating system. It remains to be elucidated whether three different types of messenger systems are linked to three different types of receptors or whether different transducing mechanisms are responsible for such diversity. The results of the present study indicate that PGE2 causes glycogenolysis by inducing phosphoinositide breakdown. In this regard, the mode of action of PGE2 resembles the action of PGF2«, which also activates the calcium messenger system, as described by Athari and Jungermann (22) and Altin and Bygrave (23).
Acknowledgments We thank Ms. Fujimaki and Ms. Kakinuma for their expert technical assistance.
References 1. Wernze H, Titton W, Goerig M 1986 Release of prostanoids into the portal and hepatic vein in patients with chronic liver disease. Hepatology 6:911 2. Morita I, Murota S 1978 Prostaglandin-synthesizing system in rat liver. Eur J Biochem 90:441 3. Robertson RP, Westcott KR, Storm DR, Rice MG 1980 Downregulation in vivo of PGE receptor and adenylate cyclase stimulation. Am J Physiol 239:E75 4. Okamura N, Terayama H 1977 Prostaglandin receptor-adenylate cyclase system in plasma membranes of rat liver and ascites hepatomas and the effect of GTP upon it. Biochim Biophys Acta 465:54 5. Wheeler GE, Epand RM 1975 Prostaglandin Ei: anomalous effects on glucose production in rat liver. Mol Pharmacol 11:335 6. Buxton DB, Fisher RA, Briseno DL, Hanahan DJ, Olson MS 1987 Glycogenolytic and haemodynamic responses to heat-aggregated immunogloblin G and prostaglandin E2 in the perfused rat liver. Biochem J 243:493 7. Brass EP, Garrity MJ 1985 Effect of E-series prostaglandins on cyclic AMP-dependent and -independent hormone-stimulated glycogenolysis in hepatocytes. Diabetes 34:291 8. Casteleijn E, Kuiper J, van Rooij HCJ, Kamps JAAM, Koster JF, van Berkel TJC 1988 Hormonal control of glycogenolysis in parenchymal liver cells by Kupffer and endothelial liver cells. J Biol Chem 263:2699 9. Berry MN, Friend DS 1969 High-yield preparation of isolated rat liver parenchymal cells. J Cell Biol 4:506 10. Mine T, Kojima I, Kimura S, Ogata E 1986 Comparison of the changes in cytoplasmic free calcium concentration induced by phenylephrine, vasopressin and angiotensin II in hepatocytes. Biochem Biophys Res Commun 140:170 11. Mine T, Kojima I, Kimura S, Ogata E 1987 Assessment of the role of Ca2+ mobilization from intracellular pools, using dantrolene, in the glycogenolytic action of a-adrenergic stimulation in perfused rat liver. Biochim Biophys Acta 927:792 12. Corvera S, Huerta-Bahena J, Pelton JT, Hruby JV, Trivedi D, Garcia-Saintz JA 1984 Metabolic effects and cyclic AMP levels
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13. 14. 15. 16. 17. 18.
PGE2 AND HEPATIC GLYCOGENOLYSIS
produced by glucagon, (1-iV-a-trinitrophenylhistidine, 12-homoarginine) glucagon, and forskolin in isolated rat hepatocytes. Biochim Biophys Acta 804:434 Blackmore PF, Exton JH 1985 Assessment of effects of vasopressin angiotensin II, and glucagon on Ca2+ fluxes and phosphorylase activity in liver. Methods Enzymol 109:550 Mine T, Kojima I, Ogata E 1989 Stimulation of glucose production by activin-A in isolated rat hepatocytes. Endocrinology 125:586 Borle AB, Freudrich CC, Snowdowne KW 1986 A simple method for incorporating aequorin into mammalian cells. Am J Physiol 251:323 Snowdowne KW, Bole AB 1984 Changes in cytosolic ionized calcium induced by activators of seretion in GH3 cells. Am J Physiol 246:E198 Berridge MJ, Irvine RF 1984 Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312:315 Mauger JP, Poggioli J, Guesdon F, Claret M 1984 Noradrenalin, vasopressin and angiotensin increase Ca2+ channel in isolated rat liver cells. Biochem J 221:121
Endo • 1990 Vol 126 • No 6
19. Yokohama H, Tanaka T, Ito S, Negishi M, Hayashi H, Hayaishi O 1988 Prostaglandin E receptor enhancement of catecholamine release may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells. J Biol Chem 263:1119 20. Negishi M, Ito S, Yokohama H, Hayashi H, Katada T, Ui M, Hayaishi O 1988 Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins. J Biol Chem 263:6893 21. Yamashita T, Takai Y 1987 Inhibition of prostaglandin E r induced elevation of cytoplasmic free clacium ion by protein kinase Cactivating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts. J Biol Chem 262:5536 22. Athari A, Jungermann K 1989 Direct activation by prostaglandin F2a but not thromboxane A2 of glycogenolysis via an increase in inositol 1,4,5-trisphosphate in rat hepatocytes. Biochem Biophys Res Commun 163:1235 23. Altin JG, Bygrave FL 1988 Prostaglandin F2aand the thromboxane A2 analogue ONO-11113 stimulate Ca2+ fluxes and other physiological responses in rat liver. Biochem J 249:677
Meeting Announcement Symposium on Catecholamines and Other Neurotransmitters and Stress The Fifth International Symposium on Catecholamines and Other Neurotransmitters and Stress will be held at Smolenice Castle, Czechoslovakia from June 24-29, 1991. The program includes invited plenary lectures as well as oral and poster presentations. For additional information, please contact: Dr. Richard Kvetnansky, Secretary General, NINDS, National Institutes of Health, Building 10, Room 5N-214, Bethesda, Maryland 20892, telephone 301-4961314 or write directly to the Meeting Secretariat, Dr. I. Vietor, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Czechoslovakia, telephone 42-7-37 38 00.
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