The effect of dibutyryl cyclic AMP on the excretion of taurocholic acid from isolated rat liver cells

Dibutyryl cyclic AMP (50-1000 microM) was found to increase the initial rate of efflux of taurocholic acid from isolated rat hepatocytes. Efflux of the bile acid was inhibited by sodium, and in the absence of sodium dibutyryl cyclic AMP failed to stimulate the rate. Increasing the concentration of calcium from 0 to 1.2 mM had no effect on the initial rate of taurocholic acid efflux from the cells, but the absence of calcium markedly reduced the effect of dibutyryl cyclic AMP. The results suggest that changes in the fluxes of sodium and calcium are involved in the effect of the cyclic nucleotide on taurocholic acid efflux from the cells.     

The effect of ionophore A23187, verapamil, and dibutyryl cyclic AMP on bile acid synthesis in isolated rat hepatocytes

The effect of the divalent cation ionophore A23187 and the calcium channel-blocker verapamil on bile acid synthesis in isolated hepatocytes in the presence and absence of dibutyryl cyclic AMP was studied. Both A23187 (1 microM) and verapamil (0.04 mM) caused a small (approximately 15-20%) but consistent decrease in total bile acid synthesis in the cells. When hepatocytes were incubated with dibutyryl cyclic AMP (1 mM) production of total bile acid was increased by about 25%, and this effect was unchanged by A23187 but abolished by verapamil. The relative proportions of the individual bile acids produced were not affected by either A23187 or verapamil. Dibutyryl cyclic AMP (1 mM) lowered the ratio of the amount of conjugated cholic acid to conjugated chenodeoxycholic + beta-muricholic acid formed in the cells by about 50%. Neither A23187 nor verapamil was able to prevent this change. These results suggest that the stimulatory effect of dibutyryl cyclic AMP on total bile acid synthesis is dependent on mobilisation of calcium from intracellular stores, but its effect on the relative proportions of bile acid formed via the cholic acid versus the chenodeoxycholic acid pathway is independent of calcium movement.     

Plasma membrane vesicles from isolated hepatocytes retain the increase of amino acid transport induced by dibutyryl cyclic AMP in intact cells

We have investigated the effect of cyclic AMP on hepatic amino acid transport by measuring the uptake of L-alanine in plasma membrane vesicles purified from hepatocytes incubated without or with dibutyryl cyclic AMP. The application of an Na+ gradient to vesicles from hepatocytes exposed to dibutyryl cyclic AMP, compared to control, resulted in a greater transient accumulation of L-alanine, whereas in the presence of a K+ gradient a similar slow equilibration of L-alanine was observed. Kinetic analysis of L-alanine influx revealed that the increased uptake resulted from an increased capacity (Vmax) with no change in the affinity (Km) of the carriers for L-alanine. These results strongly suggest that dibutyryl cyclic AMP induces stable changes at the membrane level probably by increasing the number of amino acid carrier molecules.     

Effects of calcium ionophore (A-23187) on glucose oxidation and iodide transport in dog thyroid slices.

A calcium ionophore (A-23187, 20 mug/ml) stimulted 14C-1-glucose oxidation in dog thyroid slices to an extent equivalent to that obtained by the optimal concentration of dibutyryl cyclic AMP (1mM). Furthermore, the ionophore augmented the stimulation by dibutyryl cyclic AMP much more than the simple additive effect. The ionophore also enhanced the effect of TSH, but to a lesser extent. Under conditions where organic binding was blocked, T/M ratio of radioiodine concentration was lowered in slices by the ionophore; the findings similar to those obtained with TSH and dibutyryl cyclic AMP. The ionophore exhibited a slightly depressive effect on the basal cyclic AMP level. The elevation by TSH of cyclic AMP levels was also slightly depressed by the ionophore, but statistically insignificant in most cases. These results indicate that calcium ion may play an important role in the TSH regulation of iodide transport and glucose metabolism in the thyroid, in some cases by augmenting the effects of cyclic AMP.     

Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.     

The effect of theophylline and dibutyryl cyclic AMP on the uptake of radioactive calcium and phosphate ions by boar and human spermatozoa.

Radioactive calcium uptake by suspensions of washed boar and human spermatozoa was inhibited by the mitochondrial uncoupling agent carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Theophylline + dibutyryl cyclic AMP also inhibited calcium uptake in the presence or absence of FCCP. Uptake of low concentrations of calcium (0 . 1 mM) was inhibited by the calcium ionophore A23187, but at high calcium concentrations the ionophore stimulated calcium uptake. These observations are explained in terms of a mechanism for the regulation of calcium uptake in spermatozoa based on competing mitochondrial and plasma membrane pumps. Uptake of 32P was also inhibited. These effects provide evidence that cyclic AMP plays a role in the transport of ions across the plasma membrane of spermatozoa.     

Insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes. Synthesis of a high affinity component of transport.

Insulin and glucagon stimulate amino acid transport in freshly prepared suspensions of isolated rat hepatocytes. The kinetic properties of alpha-amino[1-14C]isobutyric acid (AIB) transport were investigated in isolated hepatocytes following stimulation by either hormone in vitro. In nonhormonally treated cells (i.e. basal state), saturable transport occurred mainly through a low affinity (Km approximately equal to 40 mM) component. In insulin or glucagon-treated hepatocytes, saturable transport occurred through both a low affinity component (similar to that observed in the basal state) and a high affinity (Km approximately equal to 1 mM) component. At low AIB concentrations (less than 0.5 mM), insulin and glucagon at maximally stimulating doses increased AIB uptake about 2-fold and 5-fold, respectively. The high affinity component induced by either hormone exhibited the properties of the A (alanine preferring) mediation of amino acid transport. This component required 2 to 3 h for maximal expression, and its emergence was completely prevented by cycloheximide. Half-maximal stimulation was elicited by insulin at about 3 nM and by glucagon at about 1 nM. Dibutyryl cyclic AMP mimicked the glucagon effect and was not additive to it at maximal stimulation. Maximal effects of insulin and glucagon, or insulin and dibutyryl cyclic AMP, were additive. We conclude that insulin and glucagon can modulate amino acid entry in hepatocytes through the synthesis of a high affinity transport component.     

Calcium metabolism in rat hepatocytes.

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.     

Effects of dibutyryl cyclic AMP on the transport of alpha-methyl-D-glucoside and alpha-aminoisobutyric acid in separated tubules and brush border membranes from rabbit kidney.

The effect of dibutyryl cyclic AMP on the transport of alpha-methyl-D-glucoside and alpha-aminoisobutyric acid in separated tubules and purified brush border membranes from rabbit kidney was investigated using a rapid filtration procedure. Dibutyryl cyclic AMP stimulated the uptake of alpha-methyl-D-glucoside and alpha-aminoisobutyric acid by separated renal tubules in agreement iwth prior studies utilizing renal slices (Rea, C. and Segal, S. (1973) Biochim. Biophys. Acta 311, 615--624; Weiss, I.W., Morgan, K. and Phang, J.M. (1972) J. Biol. Chem. 247, 760--764). However, in contrast to previous reports, no preincubation of the tissue with dibutyryl cyclic AMP was required for stimulation of transport to be manifest. Dibutyryl cyclic AMP stimulated oxygen consumption by separated tubules suggesting that stimulation of transport may occur by a linkage with renal oxidative metabolism. Dibutyryl cyclic AMP increased the uptake of alpha-aminoisobutyric acid into purified renal brush border membranes. However the uptakes of alpha-methyl-D-glucoside, proline, leucine and phosphate into brush border membranes were significantly inhibited.     

Bile salt alteration of ion transport across jejunal mucosa.

The effect of conjugated dihydroxy and trihydroxy bile salts on electrolyte transport across isolated rabbit jejunal mucosa was studied. Both taurochenodeoxycholic acid and taurocholic acid increased the short-circuit current (Isc) in bicarbonate-Ringer solution but not in a bicarbonate-free, chloride-free solution. Taurochenodeoxycholic acid was significantly more effective than taurocholic acid in increasing Isc. The presence of theophylline prevented the taurochenodeoxycholic acid- and taurocholic acid-induced increase in Isc. Transmural ion fluxes across jejunal mucosa demonstrated that 2 mM taurochenodeoxycholic acid decreased net Na+ absorption, increased net Cl- secretion and increased the residual flux (which probably represents HCO3- secretion). These studies support the hypothesis that cyclic AMP may be a mediator of intestinal electrolyte secretion.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Effects of dibutyryl cyclic amp on the transport of α-methyl-d-glucoside and α-aminoisobutyric acid in separated tubules and brush border membranes from rabbit kidney

The effect of dibutyryl cyclic AMP on the transport of α-methyl-d-glucoside and α-aminoisobutyric acid in separated tubules and purified brush border membranes from rabbit kidney was investigated using a rapid filtration procedure. Dibutyryl cyclic AMP stimulated the uptake of α-methyl-d-glucoside and α-aminoisobutyric acid by separated renal tubules in agreement with prior studies utilizing renal slices (Rea, C. and Segal, S. (1973) Biochim. Biophys. Acta 311, 615–624; Weiss, I.W., Morgan, K. and Phang, J.M. (1972) J. Biol. Chem. 247, 760–764). However, in contrast to previous reports, no preincubation of the tissue with dibutyryl cyclic AMP was required for stimulation of transport to be manifest. Dibutyryl cyclic AMP stimulated oxygen consumption by separated tubules suggesting that stimulation of transport may occur by a linkage with renal oxidative metabolism. Dibutyryl cyclic AMP increased the uptake of α-aminoisobutyric acid into purified renal brush border membranes. However the uptakes of α-methyl-d-glucoside, proline, leucine and phosphate into brush border membranes were significantly inhibited.     

Effect of taurine on ATP-dependent calcium transport in guinea-pig cardiac muscle

The effect of taurine on ATP-dependent calcium transport was examined in guinea-pig cardiac ventricle homogenates and in microsomal preparations enriched in sarcoplasmic reticulum. Taurine (5?50 mM) did not affect ATP-dependent calcium binding or uptake in either of these preparations or alter the rate of decay of calcium uptake activity. Taurine (20 mM) also did not affect the oxalate-dependent calcium uptake stimulation noted in the presence of cyclic AMP-dependent protein kinase and cyclic AMP. The mechanism by which taurine alters cardiac function remains to be elucidated.     

Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs.

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.     

Characterization of cloned mouse Na+/taurocholate cotransporting polypeptide by transient expression in COS-7 cells

The mouse Na+/taurocholate cotransporting polypeptide transiently expressed in COS-7 cells caused sodium-dependent uptake of [3H]taurocholic acid with Km and Vmax values of 18 microM and 102 pmol/mg protein/min, respectively. This Km value is comparable to that for rat NTCP and higher than that for human NTCP. Substrate specificity was evaluated by measuring inhibitory effects of unlabeled bile acids on [3H]taurocholic acid transport.     

Similar and opposite effects of dibutyryl cyclic AMP and insulin on glucose metabolism in adipose tissue

The effects of N⁶-2′-O-dibutyryl cyclic AMP on glucose metabolism and lipolysis in fragments of rat epididymal adipose tissue were studied. Measurements were made of glucose uptake, conversion of glucose carbon to CO₂ and tissue fatty acids and glyceride-glycerol, lactate production, and glycerol release. Low concentrations of dibutyryl cyclic AMP (0.1–0.5 mM) increased all parameters of glucose metabolism and inhibited glycerol release in tissue from both normally fed and fasted rats. Higher concentrations of dibutyryl cyclic AMP (3–5 mM) diminished glucose utilization and greatly accelerated lipolysis. Insulin, 50 μunits/ml, accelerated glucose metabolism in the presence of either low or high concentrations of dibutyryl cyclic AMP though the effect of insulin was greatly reduced by 3 mM dibutyryl cyclic AMP. Tissue exposed to concentrations of dibutyryl cyclic AMP which inhibited glucose metabolism (5 mM), then rinsed and reincubated without dibutyryl cyclic AMP, displayed increased glucose utilization. The results of these experiments emphasize the need for caution in interpretation of the effects of dibutyryl cyclic AMP on adipose tissue metabolism and the need for further research to elucidate the role of cyclic AMP in the regulation of glucose metabolism.     

Inositol lipid turnover and adenosine 3,5 cyclic monophosphate in the salt-secreting rectal gland of the dogfish (Scyliorhinus canicula)

The rectal gland of the dogfish is rich in inositol lipids. Total phospholipids from the gland contained 9.1 mol% of phosphatidylinositol (PtdIns), 1.0 mol% of phosphatidylinositol 4-phosphate (PtdIns4P) and 0.9 mol% of phosphatidylinositol 4,5-biphosphate (PtdIns4,5P2). [32P]Orthophosphate was readily incorporated into PtdIns, phosphatidic acid (PtdA) and especially into PtdIns4P and PtdIns4,5P2 in salt gland slices incubated in elasmobranch Ringer with glucose and no other additions over a 2 hr period. The calcium ionophore A23187 stimulated incorporation into PtdIns and PtdA, but not into PtdIns4P or PtdIns4,5P2. Oxygen uptake by rectal gland slices was maximally stimulated by 0.08mM forskolin, 2.5mM 8-chlorophenylthio cyclic AMP, 2.0mM dibutyryl cyclic AMP and 0.25mM theophylline. Stimulated oxygen uptake was inhibited by 0.1mM ouabain in all cases. Incorporation of [32P]orthophosphate into PtdIns, PtdA, PtdIns4P and PtdIns4,5P2 was inhibited by 0.08mM forskolin and 2.0mM dibutyryl cyclic AMP over a 2 hr period. The results are discussed in relation to the control of salt secretion by the rectal gland.     

Amylase release from rat parotid glands. II. Calcium kinetics.

The kinetics of 45Ca2+ uptake, efflux, and calcium potentiation of amylase release by slices of rat parotid glands were examined. Pretreatment of the tissue with 11.25 mM 45Ca2+ medium increased the total tissue 45calcium content. Lanthanum (1 mM) decreased tissue uptake, blocked the slow components of exchange and appeared to inhibit transcellular calcium movement. Neither dibutyryl cyclic AMP nor caffeine caused consistently significant effects on 45Ca2+ kinetics, or total 45calcium content. Carbamylcholine increased the initial rate of 45Ca2+ uptake, but had no effect on total uptake. Elevation of the extracellular Ca2+ concentration to 11.25 mM during stimulation of amylase release resulted in an initial decrease in the rate of amylase release followed by a potentiation of release which developed slowly, requiring 40--50 min to reach the maximal response. The inability to detect release-related changes in either calcium influx or mobilization, and the lengthy times and high Ca2+ concentrations required to achieve calcium potentiation suggests that calcium does not couple amylase release.     

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.