Measurement of bile salt aggregation equilibria using kinetic dialysis and spreadsheet modeling.

Bile salts initially self-associate into small aggregates such as dimers and trimers before forming micelles at higher concentrations. The very size of these small aggregates hampers quantitation of their formation. We have devised a method using kinetic dialysis measurements to quantitatively examine aggregation of bile salt at concentrations below 10 mM. Data for rate of dialysis versus concentration were fitted to hypothetical models of aggregation using a personal computer spreadsheet. For sodium taurocholate these data best fit a model of initial dimer and trimer formation with stepwise association constants of 100 and 160, respectively. Addition of larger aggregates to the model did not improve the fit. For sodium taurodeoxycholate the data best fit a model which included not only dimers and trimers, but also tetramers or larger aggregates up to 10-mers with stepwise association constants of 40, 400, and 1700 for dimers, trimers, and tetramers, respectively. These data agree reasonably well with existing literature and suggest that kinetic dialysis with spreadsheet modeling is a useful technique for the study of bile salt aggregation at relatively low concentrations.

H2O2 injury causes Ca(2+)-dependent and -independent hydrolysis of phosphatidylcholine in alveolar epithelial cells.

Oxidants may play a central role in the pathogenesis of adult respiratory distress syndrome, and phospholipase activation is a potential mechanism of oxidant-induced injury of alveolar epithelial cells. Studies were performed in rat alveolar type II epithelial cells (RAEC) after 3 days in culture. As measured by 51Cr and lactate dehydrogenase release, H2O2 caused time- and dose-dependent cytotoxicity to RAEC. RAEC phospholipids labeled with [14C]-stearic acid ([14C]SA) and [3H]arachidonic acid ([3H]AA) released free fatty acids in response to H2O2 in a manner that closely paralleled the cytotoxicity indexes. Analysis of phospholipid subclasses indicated that phosphatidylcholine was preferentially affected. Analysis for putative products of phospholipase activity revealed significant increases in diacylglycerol and phosphorylcholine, expected products of phospholipase C, as well as significant increases in L-alpha-lysophosphatidylcholine and L-alpha-glycerophosphocholine, expected products of phospholipase A2. Increases in phospholipase D activity were not detected. To determine whether H2O2-stimulated phospholipase activity might be Ca2+ stimulated, RAEC were loaded with fura-2/AM, and changes in intracellular Ca2+ concentrations ([Ca2+]i) were monitored by epifluorescent microscopy. Exposure to H2O2 caused elevations in [Ca2+]i, and the time and dose relationships were consistent with the hypothesis that the release of [14C]SA and [3H]AA is related to changes in cellular Ca2+ concentrations. Additionally, pretreatment with MAPTAM, an intracellular chelator of calcium, partially blocked H2O2-mediated [3H]AA liberation. However, experiments in saponin-permeabilized RAEC, in which [Ca2+]i was strongly buffered by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, indicate that H2O2-induced phospholipase activity also has a Ca(2+)-independent component.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of oxidant-mediated cytotoxicity to phospholipid metabolism in endothelial cells.

Exposure to oxidants permeabilizes cell membranes and liberates unesterified fatty acids (UFA) in a variety of cell types, including endothelial cells. Products of phospholipase activity, particularly UFA and lysophosphatides, possess potent detergent-like properties, and we postulated that oxidant injury might be mediated by the accumulation of these toxic phospholipase products. Several radiolabels were incorporated into defined positions in the phospholipids of cultured, confluent bovine pulmonary endothelial cells (BPAEC). The release of radiolabeled fatty acids and the accumulation of cell-associated phospholipase products were measured and compared to a standard cytotoxicity assay (51Cr release) in response to an oxidant stress, in this case 0.1 to 10 mM hydrogen peroxide (H2O2). H2O2 caused time- and dose-dependent 51Cr release as well as liberation of saturated ([14C]stearic acid) and unsaturated ([3H]arachidonic acid) fatty acids and the accumulation of phospholipase A2 and C products. The ability of BPAEC to incorporate UFA into complex phospholipids was shown to be severely impaired in the presence of H2O2. Further studies showed that H2O2 caused depletion of BPAEC adenosine triphosphate (ATP) content to undetectable levels, and that the depletion of cellular ATP by iodoacetic acid induced substantial release of [3H]arachidonic acid but not [14C]stearic acid from BPAEC. This finding suggests that release of UFA in response to an oxidant stress may be due in part to a defect in ATP-dependent reacylation pathways and need not reflect any increase in phospholipase activities. Also unsaturated fatty acids were found to be toxic to BPAEC upon adding them to supernatants of cultured monolayers.

ADP and glucose as possible synergistic partners in the stimulation of liver glycogen synthase activation.

Glucose administered either intravenously or orally causes liver glycogen synthase activation independent of a rise in circulating insulin. In vitro, physiological concentrations of glucose stimulate synthase phosphatase activity but only in the presence of a second effector which reduced the A0.5 for glucose. Caffeine and certain methylxanthines have been in vitro models for a putative natural effector. The present study demonstrates that, in vitro, ADP also reduced the A0.5 for glucose comparable to the effect of caffeine. The maximum stimulation by glucose in the presence of caffeine or ADP was comparable. The effect of ADP was specific among the major nucleoside diphosphates. However, the A0.5 for ADP was greater than the normal liver concentration which does not change in response to either glucose or insulin administration. The effect of ADP appeared distinct from that of the methylxanthines since it was observed that at near saturating concentrations of ADP and of glucose, stimulation was increased by addition of theophylline. Similarly, addition of adenosine, a natural cell constituent, caused increased stimulation. Subsequently, it was shown that adenosine reduced the A0.5 for ADP to a nearly physiological concentration. Thus, while ADP is not the inducible putative effector which has been predicted it may be part of an intracellular amplification system for glycogen synthase activation which increases the sensitivity to an induced effector. The present work suggests that the effective concentration of the natural ligand may be less than originally anticipated. This work also suggests that the putative effector could be structurally related to adenosine. Phosphorylase phosphatase activity known to be stimulated by ADP and glucose is further stimulated by the combination which may be acting in synergy.

The importance of nucleoside triphosphate inhibition of liver glycogen synthase phosphatase activity.

The liver glycogen particle contains constitutive glycogen-synthase phosphatase activity which is inhibited by ATP-Mg in a concentration-dependent manner within the physiological range (I0.5 = 0.1 mM). Therefore, we determined whether other nucleoside triphosphate-magnesium complexes also inhibit synthase phosphatase activity. UTP-Mg, CTP-Mg and GTP-Mg were all found to be inhibitory. The maximum inhibition was 85-90% which was greater than that for ATP-Mg. The I0.5 for UTP-Mg was comparable to that of ATP-Mg but it was greater for CTP-Mg and for GTP-Mg. At in vivo physiological concentrations, both UTP and ATP are possible inhibitors of synthase phosphatase activity. In the presence of a saturating concentration of ATP-Mg, added UTP-Mg increased the inhibition suggesting the presence of at least two distinct nucleotide binding sites. Substitution of calcium for magnesium in an ATP complex had no effect on the I0.5, but increased the maximum inhibition. The present studies also suggest that in the multistep conversion of synthase D to synthase I, ATP-Mg inhibition occurs early in the sequence. Addition of glycogen, a known inhibitor of synthase phosphatase activity, to a reaction mixture containing 3 mM ATP-Mg did not further inhibit synthase phosphatase activity when added at concentrations up to 22 mg/ml. The latter data suggest that the presence of a nucleoside triphosphate may desensitize the phosphatase to glycogen inhibition. ATP-Mg and, to a lesser extent, UTP-Mg and CTP-Mg all stimulated phosphorylase phosphatase activity but GTP-Mg did not.

Regulation of glycogen synthesis in the liver.

The glycogen synthase-mediated reaction is rate-limiting for glycogen synthesis in the liver. Glycogen synthase has been purified essentially to homogeneity and has been shown to be a dimer composed of identical subunits. It is regulated by a phosphorylation-dephosphorylation mechanism, catalyzed by kinases and a phosphatase. The subunits of synthase D, the most phosphorylated form, each contain approximately 17 phosphates. The subunits of synthase I, the least phosphorylated form, each contain 14 phosphates. Thus, during the transition between these two forms, a net of three phosphoryl groups is added or removed. In synthase D, six of the phosphates are alkali-labile. In synthase I, three of the phosphates are alkali-labile. Therefore, all of the phosphorylation sites important in the interconversion of these two forms are alkali-labile (attached to serine or threonine residues). In short-term experiments using isolated hepatocytes, [32P]phosphate was only incorporated into the alkali-labile sites and the phosphate in these sites was shown to turn over rapidly. Glucose addition, which is known to reduce the proportion of synthase in the D form when assayed kinetically, also reduced the [32P]phosphate content. Glucagon addition, which increases the proportion of synthase in the D form, increased it. These changes do not appear to be site-specific. Ingestion or administration of fructose, or galactose, as well as glucose, result in a shift in synthase equilibrium in favor of the less phosphorylated forms. Possible mechanisms by which synthase phosphatase activity may be increased after ingestion of glucose or fructose, and thus shift the equilibrium in favor of the less phosphorylated forms, are discussed. The mechanism by which galactose may stimulate the phosphatase reaction is completely unknown.

The effect of glucose on liver glycogen synthase phosphatase activity in the presence of ATP-Mg.

Glycogen particle synthase phosphatase activity is stimulated by glucose with an A0.5 of approximately 27 mM. The A0.5 is higher than the usual concentrations present in the liver. However, in vitro, certain methylxanthines such as caffeine or theophylline reduce the glucose A0.5 to approximately 10 mM, a concentration well within the normal range of liver glucose concentrations. Methylxanthines do not affect the maximum stimulation by glucose (2.3-fold greater than control rate). The phosphatase reaction also is inhibited by ATP-Mg (I0.5 = 0.1 mM). In the present studies, we have determined the interaction of these effectors. The presence of ATP-Mg at a concentration of 3 mM only slightly reduced the maximal stimulation by glucose. The A0.5 for glucose was unaffected (24 mM). The synergistic effect of caffeine with glucose also was not changed by the presence of ATP-Mg. The A0.5 for glucose was reduced to 11 mM, similar to that in the absence of ATP-Mg. In addition, maximum stimulation by glucose was unchanged. Similar results were obtained when theophylline replaced caffeine. We conclude that the ATP-Mg binding site on either the phosphatase or its substrate, synthase D, does not influence the glucose and methylxanthine binding sites. Effectively, ATP-Mg increased the range over which glucose stimulates the phosphatase activity. In the presence of ATP-Mg, the maximum stimulation by glucose is approximately 7-fold; whereas, in the absence of ATP-Mg it is approximately 2.3-fold. Thus, ATP-Mg may serve to increase the sensitivity of the synthase phosphatase reaction to glucose regulation under in vivo conditions.

Regulation of liver glycogen synthase phosphatase activity by ATP-Mg.

The kinetics of a synthase phosphatase reaction inhibited by ATP-Mg in a liver glycogen particle preparation were complex. In the presence of a physiological concentration of ATP-Mg, synthase phosphatase activity in the glycogen particle follows a biphasic course. Initially, the reaction was inhibited but later the reaction rate accelerated. The reaction was inhibited but the rate was constant in the presence of ATP-Mg with the addition of a physiological concentration of glucose 6-phosphate (Glc 6-P). Therefore, in most subsequent experiments Glc 6-P was added. The concentration of ATP-Mg at which 50% maximal inhibition (I0.5) occurred was approximately 0.1 mM in preparations obtained from rats given glucagon prior to being killed. In preparations from animals given glucose, the I0.5 was increased to 2.0 mM. The maximum inhibition was little changed in preparations from glucose- or glucagon-treated animals. Thus, administration of glucose in vivo reduced the sensitivity of the synthase phosphatase to ATP-Mg inhibition. Complexes of ATP with paramagnetic ions such as Co2+ and Mn2+ were less inhibitory than complexes with diamagnetic ions, including Ca2+ and Mg2+. Magnesium complexes of adenosine tetraphosphate and 5'-adenylimidodiphosphate also were inhibitory. Inhibition was independent of phosphorylase a and not a nonspecific, polyvalent anion effect. The best explanation for the distinctive effects of ATP-Mg in preparations from glucagon- and glucose-treated animals is that the respective treatments promote and stabilize different forms of synthase D or possibly synthase phosphatase with different affinities for ATP-Mg. These forms are interconvertible, as previously suggested, in studies employing EDTA (20).

The mechanism of caffeine-enhanced glucose stimulation of liver glycogen synthase phosphatase activity.

Our report that glucose within its physiological range stimulates glycogen synthase phosphatase activity, provided an appropriate second effector is present, has been expanded. The nature of the stimulatory process, particularly the roles of glucose, and of caffeine which represents the potential second effectors, has been studied. Glucose and caffeine stimulated synthase phosphatase activity in a synergistic manner. With 0.5 mM caffeine the A0.5 for glucose was 11 mM (from 27 mM), whereas in the presence of 30 mM glucose the A0.5 for caffeine was 0.06 mM (from 0.7 mM). At 10 mM glucose the A0.5 for caffeine was 0.1 mM. Glucose stimulation remained non-cooperative, unaffected by the presence of caffeine, whereas the cooperative stimulation of caffeine was unaffected by glucose. Some slight stimulation of synthase activity was observed with caffeine and with glucose over a wide concentration range. However, they did not act synergistically to influence the measurement of synthase activity. Glucose-6-phosphate, which also stimulates synthase phosphatase activity, acted independently, not synergistically with caffeine. All the methylxanthines were tested as potential second effectors in an effort to discover the essential structural elements of the agent. All dimethylxanthines, 3- and 7-methylxanthine and 1-methyl-3-isobutylxanthine enhanced glucose stimulation but none of them alone was stimulatory. Judged from the half-maximal concentrations, in the presence of 10 mM glucose, caffeine was the most potent second effector by a significant margin. The maximum velocity was also greatest with caffeine, whereas that with other methylxanthines was generally lower, and varied. 1-Methylxanthine with increased concentration was slightly inhibitory even in the presence of 10 mM glucose. Xanthine (0.5 mM), itself, strongly inhibited synthase phosphatase activity, an effect not influenced by glucose. Xanthine did not influence the measurement of synthase or phosphorylase phosphatase activity with or without glucose. In general, conditions of methylxanthine-enhanced, glucose stimulation of synthase phosphatase and phosphorylase phosphatase activities differed markedly, confirming that separate, distinct mechanisms are involved.

Metabolic effects of oral fructose in the liver of fasted rats.

Twenty-four-hour-fasted rats were given fructose (4 g/kg) by gavage. Fructose absorption and the portal vein, aorta, and hepatic vein plasma fructose, glucose, lactate, and insulin concentrations as well as liver fructose and fructose 1-P, glucose, glucose 6-P, UDPglucose, lactate, pyruvate, ATP, ADP, AMP, inorganic phosphate (Pi), cAMP, and Mg2+, and glycogen synthase I and phosphorylase alpha were measured at 10, 20, 30, 40, 60 and 120 min after gavage. Liver and muscle glycogen and serum uric acid and triglycerides also were measured. Fifty-nine percent of the fructose was absorbed in 2 h. There were modest increases in plasma and hepatic fructose, glucose, and lactate and in plasma insulin. Concentrations in the portal vein, aorta, and hepatic vein plasma indicate rapid removal of fructose and lactate by the liver and a modest increase in production of glucose. The source of the increase in plasma lactate is uncertain. Hepatic glucose 6-P increased twofold; UDPglucose rose transiently and then decreased below the control level. Fructose 1-P increased linearly to a concentration of 3.3 mumol/g wet wt by 120 min. There was no change in ATP, ADP, AMP, cAMP, Pi, or Mg2+. Serum triglycerides and uric acid were unchanged. Glycogen synthase was activated by 20 min without a change in phosphorylase alpha. This occurred with a fructose dose that did not significantly increase either the liver glucose or fructose concentrations. Liver glycogen increased linearly after 20 min, and glycogen storage was equal in liver (38.4%) and muscle (36.5%).(ABSTRACT TRUNCATED AT 250 WORDS)

The synergistic action of caffeine or adenosine on glucose stimulation of liver glycogen synthase phosphatase activity.

Recent studies indicate that glucose directly stimulates synthase phosphatase activity in vitro but only at high, non-physiological concentrations. Present results demonstrate that at a physiological concentration glucose can be stimulatory, provided that an appropriate second effector is present. Caffeine and adenosine are examples of such effectors which act synergistically with glucose to enhance synthase phosphatase activity. Caffeine but not adenosine enhances glucose stimulation of phosphorylase phosphatase activity. In the absence of glucose, caffeine but not adenosine stimulates both synthase and phosphorylase phosphatase reactions. Thus, glucose regulation of glycogen synthase activation in vivo could require a second effector. Neither the identity nor source of such an effector is known. The putative regulator could be a mediator for a hormone such as insulin. The present work suggests that the chemical nature of the effector might be that of a derivatized purine of which nucleosides are an example.

Direct glucose stimulation of glycogen synthase phosphatase activity in a liver glycogen particle preparation.

In glycogen particle suspensions prepared from fed rats given either glucagon or glucose in order to increase or decrease the phosphorylase a concentration, respectively, glucose stimulation of synthase phosphatase activity was observed. In preparations from glucagon-treated rats, addition of glucose stimulated synthase and phosphorylase phosphatase simultaneously and not sequentially. Synthase phosphatase stimulation was glucose concentration dependent even when phosphorylase a had been rapidly reduced to a low level. The estimated A0.5 for glucose stimulation of synthase phosphatase activity was 27 mM. An A0.5 for glucose stimulation of phosphorylase phosphatase activity could not be estimated since activity was still increasing with concentrations of glucose as high as 200 mM. In preparations from glucose-treated rats which contain virtually no phosphorylase a, glucose stimulation was still apparent but the A0.5 was increased modestly (36 mM). Stimulation of synthase phosphatase activity was specific for glucose. Several other monosaccharides and the polyhydric alcohol sorbitol were ineffective.

Metabolic effects of oral glucose in the liver of fasted rats.

Twenty-four-hour-fasted rats were given glucose (4 g/kg) by gavage. Glucose absorption and portal and peripheral plasma glucose, lactate, and insulin concentrations, as well as liver glucose, UDPglucose, glucose-6-P, lactate, ATP, and inorganic phosphate (Pi), and % glycogen synthase I and % phosphorylase a were measured at 10, 20, 30, 40, 60, and 120 min after the glucose was given. Liver and muscle glycogen also were measured. Ninety-one percent of the glucose load had disappeared from the gut in 2 h. Despite increased plasma glucose and insulin levels the liver continued to produce glucose. Lactate produced in the periphery was the major substrate for gluconeogenesis, and lactate utilization could account for the hepatic glycogen synthesized. Glucose ingestion did not affect lactate production by the splanchnic bed. In the liver glucose-6-P was transiently increased; UDP glucose decreased after glucose administration. ATP and Pi were unchanged. Glycogen synthase was activated by 20 min without a significant change in phosphorylase a. Hepatic glycogen increased linearly after 20 min. Total glucose storage as glycogen was similar in liver (20%) and muscle (19%). We could account for 41% of the glucose absorbed as glycogen, unmetabolized glucose, or glucose metabolites. Most of the remainder probably was oxidized.

Response of liver glycogen synthase and phosphorylase to in vivo glucose and glucose analogues.

Glucose causes a rapid increase in the proportion (%) of glycogen synthase in the active (I) form and a rapid decrease in the proportion of phosphorylase in the active (a) form in both fed and fasted rats. The changes in synthase I and phosphorylase a are more rapid in fasted animals. With graded doses of glucose, the maximal decrease in phosphorylase a occurred at a dose that was considerably smaller than that required to maximally stimulate an increase in % synthase I. Thus, in the intact animal a dissociation between the effects of glucose on the synthase and phosphorylase systems was observed. Sorbitol, mannose, galactose, and arabinose all stimulated an increase in synthase I but did not significantly affect the proportion of phosphorylase in the a form. The % synthase I was not significantly affected by a number of other glucose homologues, pentoses, or three-carbon gluconeogenic substrates. The ketoses fructose and mannoheptulose both caused a striking increase in % phosphorylase a and a decrease in % synthase I, i.e., results opposite to those of glucose. The mechanism by which fructose induces these changes is not known, but the mannoheptulose effects may be accounted for by a rise in liver cAMP concentration.

Liver glycogen synthase and phosphorylase changes in vivo with hypoxia and anesthetics.

Methods for obtaining and processing rat liver for determination of glycogen phosphorylase a and synthase I activity were studied. An extremely rapid and profound increase in phosphorylase was induced by hypoxia. The effect on synthase I was slower and less striking. Using alpha- and beta-adrenergic antagonists, a catecholamine-depleting agent, and a ganglionic blocking agent, it was determined that adrenergic stimulation secondary to the surgical procedure required to obtain the liver was not a significant factor. The anesthetic agent used also had a significant effect on the proportion of phosphorylase in the a form. Seconal anesthesia resulted in lower phosphorylase a levels than did ether or urethan anesthesia.

Influence of fructose on the glycogen synthase and phosphorylase systems in rat liver.

Fructose and glucose, when administered as a single, large intravenous dose (500 mg/kg) produced opposite effects on key regulatory enzymes of glycogen metabolism in intact normal fed animals. Glucose rapidly stimulated glycogen synthase phosphatase activity and increased the proportion of glycogen synthase in the active (I) form as expected; fructose reduced synthase phosphatase activity and the proportion of synthase in the I form. Glucose also stimulated a reduction in the proportion of phosphorylase in the active (a) form, whereas fructose stimulated an increase in the proportion of phosphorylase in thea form. The effect of fructose was not mediated by an increase in cyclic adenylate (cAMP) concentration nor by a conversion of phosphorylase kinase b to phosphorylase kinase a. As expected, the concentration of ATP decreased significantly. The increase in proportion of phosphorylase in the a form may be due to stimulation of phosphorylase kinase b activity by a decrease in the intracellular ATP:Mg++ ratio or by increase in intracellular Ca++ concentration. The mechanism of the fructose-induced change in synthase phosphatase activity and in synthase I activity is unknown.