Biomedical subjects
F R Simon
Publications and source records attributed to F R Simon.
Overview of clinical aspects of bile salt physiology.
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Studies on the differences in NaK-ATPase and lipid properties of liver plasma membranes in long sleep and short sleep mice.
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Ethanol treatment increases triacylglycerol and cholesteryl ester content of cultured hepatoma cells.
Well-differentiated Reuber H35 rat hepatoma cells in culture maintain a variety of biochemical functions characteristic of hepatocytes [Deschatrette, J., and M. C. Weiss. 1974. Biochimie. 56: 1603-1611]. To demonstrate the suitability of this system as a model for exploring mechanisms of ethanol hepatotoxicity, the following were investigated: 1) ethanol metabolism in whole cells and cell extracts and 2) effects of ethanol exposure on cellular lipid content. Cultures of H35 cells exposed to 10 mm ethanol metabolized the ethanol at rates similar to those reported in rat liver. Under these conditions, soluble alcohol dehydrogenase activity accounted for greater than 87% of total ethanol metabolism. H35 cells exposed to 240 mm ethanol for 3 days contained four times more triacylglycerol and cholesteryl ester than control cells. Total phospholipid and unesterified cholesterol levels were unaffected by ethanol. Neutral lipid content of Chinese hamster ovary cells was unchanged after ethanol exposure. The increased triacylglycerol content of ethanol-treated H35 cells appeared to result from an accelerated rate of conversion of long chain fatty acids into triacylglycerol. Several lines of evidence indicated that alcohol dehydrogenase-mediated ethanol oxidation was critical in promoting increased triacylglycerol content of cultured cells. Since 240 mm ethanol blocked cellular proliferation, long term effects of ethanol were studied at a level of 10 mm, which allowed a nearly normal growth rate. After 7 weeks of continuous exposure, 10 mm ethanol-treated H35 cells contained five times more triacylglycerol than paired controls. The well-differentiated H35 cell appears to be an excellent in vitro model system for studying both short-term and long-term effects of ethanol on liver cells.-Polokoff, M. A., M. Iwahashi, and F. R. Simon. Ethanol treatment increases triacylglycerol and cholesteryl ester content of cultured hepatoma cells.
Regulation of bile salt transport in rat liver. Evidence that increased maximum bile salt secretory capacity is due to increased cholic acid receptors.
Expansion of the bile salt pool size in rats increases maximum excretory capacity for taurocholate. We examined whether increased bile salt transport is due to recruitment of centrolobular transport units or rather to adaptive changes in the hepatocyte. Daily sodium cholate (100 mg/100 g body wt) was administered orally to rats. This treatment was well tolerated for at least 4 d and produced an 8.2-fold expansion of the bile salt pool. This expanded pool consisted predominently (99%) of cholic and deoxycholic acids. Significantly increased bile salt transport was not observed until 16 h after bile acid loading, and maximum elevations of transport capacity to 2.3-fold of control required approximately 2 d. In contrast, maximum sulfobromophthalein excretion rates increased 2.2-fold as early as 4 h and actually fell to 1.5-fold increase at 4 d. We studied the possibility that this adaptive increase in bile salt secretory transport was due to changes in canalicular surface membrane area, lipid composition, or increased number of putative carriers. Canalicular membrane protein recovery and the specific activities of leucine aminopeptidase, Mg(++)-ATPase and 5'-nucleotidase activities were unaltered by bile salt pool expansion. The content of free and esterified cholesterol and total phospholipids was unchanged in liver surface membrane fractions compared with control values. In contrast, sodium cholate administration selectively increased specific [(14)C]cholic acid binding sites twofold in liver surface membrane fractions. Increased numbers of [(14)C]cholic acid receptors (a) was associated with the time-dependent increase in bile salt transport, and (b) was selective for the taurine conjugate of cholate and (c) was reduced by chenodeoxycholate. Changes in bile acid binding sites 16 h following taurocholate and chenodeoxycholate and the lack of change with glycocholate was associated with comparable changes in bile salt transport. In conclusion, selective bile salts increase bile salt transport in the liver through an adaptive increase in the density of putative bile acid carriers in liver surface membrane.
Bile secretory failure: recent concepts of the pathogenesis of intrahepatic cholestasis.
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Effect of the position of the phenolic group in morphinans on their affinity for opiate receptor binding.
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Reversal of ethinyl estradiol-induced bile secretory failure with Triton WR-1339.
The effects of Triton WR-1339 and phenobarbital on ethinyl estradiol bile secretory failure were examined to determine the mechanism responsible for decreased bile salt excretion. When administered to ethinyl estradiol-treated rats, Triton WR-1339 restored bile salt independent bile flow and maximum taurocholate transport, whereas phenobarbital corrected bile flow only. Ethinyl estradiol decreased the activities of Na(+)-K(+)-ATPase, 5'-nucleotidase, while increasing the activities of Mg(++)-ATPase and alkaline phosphatase. In contrast to these heterogeneous changes in surface membrane enzyme activities, the number and affinity of [(14)C]cholic acid carriers were not altered. When administered in vivo or added directly to surface membrane fractions Triton WR-1339 restored the activities of Na(+)-K(+)-ATPase and Mg(++)-ATPase of rats treated with ethinyl estradiol through a process that did not require protein synthesis (unaffected by cycloheximide). Phenobarbital also restored the activity of Na(+)-K(+)-ATPase to control levels, but, unlike Triton WR-1339 it did not correct the defect responsible for reduced bile salt secretion. Ethinyl estradiol increased the concentration of cholesterol esters in surface membrane fractions. When administered to ethinyl estradiol-treated rats, Triton WR-1339 restored cholesterol ester concentrations to normal, whereas phenobarbital did not. These combined data suggest that decreased or altered bile salt carriers or reduced sodium driving forces resulting from impaired activity of Na(+)-K(+)-ATPase are not responsible for decreased bile salt excretion in ethinyl estradiol-treated rats. It is proposed that the diverse changes in surface membrane function, which are associated with ethinyl estradiol bile secretory failure, may be the result of a generalized alteration in membrane lipid structure.
Regulation of hepatic sodium plus potassium-activated adenossine triphosphatase activity by glucocorticoids in the rat.
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Mechanism of renal potassium conservation in the rat.
The mechanisms responsible for renal potassium (K) conservation during dietary potassium deficiency are poorly understood. This study was undertaken to investigate the time course of potassium conservation as well as the roles of distal sodium (Na) delivery, the distal delivery or sodium plus a nonpermeable anion, mineralocorticoid hormone, renal tissue potassium content, and Na-K-ATPase activity in renal potassium conservation. After 72 hours of a low-potassium diet, basal potassium excretion was negligible. After 24 hours, and even more so after 72 hours of potassium restriction, the kaliuretic response to increasing distal delivery of sodium or sodium plus a nonpermeable anion was impaired. After 24 hours of a low-potassium diet, plasma aldosterone levels fell from 180 +/- 25 to 32 +/- 9 pg/ml (P less than 0.001). Mineralocorticoid hormone given in the first 24 hours of a low-potassium diet resulted in a greater potassium loss (1564 +/- 125 muEq) than it did in controls on the same diet not receiving mineralocorticoid hormone (1032 +/- 83 muEq, P less than 0.005). In contrast, after 72 hours of diet, large doses of mineralocorticoid hormone failed to cause a kaliuresis in either anesthetized or conscious rats. After both 24 and 72 hours, outer medullary Na-K-ATPase was increased. At 72 hours, cortical, medullary, and papillary tissue potassium concentrations were significantly depressed. Acute administration of potassium repleted tissue potassium levels and restored basal and saline-stimulated potassium excretion to normal. Although potassium excretion was markedly depressed after 24 hours of the low-potassium diet, 42K microinjection studies of the distal nephron did not suggest any increase in potassium reabsorption. Following 72 hours of diet, potassium reabsorption increased significantly from 26 +/- 2% to 41 +/- 2% (P less than 0.001). We conclude that renal potassium conservation is at first primarily related to a decrease in potassium secretion, which is most likely mediated by falling levels of mineralocorticoid hormone. After 72 hours of the potassium-deficient diet, however, potassium conservation becomes independent of mineralocorticoid hormone, distal delivery of sodium, and Na-K-ATPase. The decreased tissue potassium content appears to be the primary mediator of both the increase in potassium reabsorption by the distal nephron and of renal potassium conservation at this time.
Rate limitation of (Na+ + K+)-stimulated adenosinetriphosphatase by membrane acyl chain ordering.
A somatic cell mutant (CR1) of the Chinese hamster ovary cell line (CHO-K1) that is defective in the regulation of cholesterol biosynthesis can be grown under conditions in which plasma membranes from these cells display various cholesterol contents and acyl chain order parameters. The (Na+ + K+)-stimulated adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from these cells was shown to vary in activity by a factor of 10 as the order parameter was varied, and the activity exhibited an exponential dependence on this parameter. Under these conditions the number of Na+,K+-ATPase molecules was shown to remain constant by affinity labeling with [gamma-32P]ATP in the absence of Na+. Control experiments showed that alteration in cholesterol content without change in order parameter did not result in altered enzyme activity. It is concluded that, under our conditions, the rate of catalysis by the Na+,K+-ATPase is determined by the order parameter. These studies suggest a physical mechanism by which variation of membrane lipid composition or other factors that determine membrane lipid acyl chain order parameter can result in variation in membrane enzyme activity.
Regulation of hepatic transport of bile salt. Effect of protein synthesis inhibition on excretion of bile salts and their binding to liver surface membrane fractions.
The overall transport of bile salts across the hepatocyte is characterized as a carrier-mediated process whose rate-limiting step is biliary secretion. Specific bile salt binding proteins have been identified in liver surface membrane fractions and were postulated to represent the initial interaction in bile salt translocation across both the sinusoidal and canalicular membranes. To test this hypothesis, cycloheximide was administered to rats to inhibit hepatic protein synthesis. 16 h after cycloheximide administration [14C]leucine incorporation into hepatic protein was inhibited by 93% at 1 h and 47% at 12 h. However, values of liver function tests were not increased, although serum albumin, serum alanine amino-transferase, and alkaline phosphatase were significantly decreased. Light and electron microscopy did not demonstrate necrosis or fat accumulation. The latter demonstrated minimal disorganization of rough endoplasmic reticulum and occasional lamellar whorls. 16 h after cycloheximide administration bile salt independent bile flow, basal bile salt excretion, and basal bile flow were unaltered, but the maximum bile salt transport capacity was reduced to 62% of control and 24 h later to 38%. Decreased bile salt transport was reversible, for it returned to control values after 48 h, when hepatic protein synthesis was also normal. Maximum bromosulfophthalein (BSP) transport, on the other hand, was reduced after 16 h to only 85% of control. Both bile salt and BPS maximum transport capacities decreased with time during inhibition of protein synthesis, apparently following first order kinetics. It was estimated that their half-lives are 20 h for bile salt transport and 55 h for BSP transport. These different turnover rates suggest that cycloheximide does not decrease active transport through generalized hepatic dysfunction or alteration of high energy sources possibly required for transport. The maximum number of [14C]cholic acid binding sites in liver surface membrane fractions was determined by an ultrafiltration assay. They were reduced to 68% of control after 16 h of cycloheximide and to 25% after 24 h. This reduction in the number of binding sites is apparently selective, for the activities of the liver surface membrane enzymes (Na+-K+)ATPase, Mg++-ATPase, and 5'-nucleotidase were not significantly changed. The associated alterations in bile salt transport and the maximum number of binding sites after cycloheximide administration suggests that these receptors may be the bile salt carriers.
Qualitative alteration in hepatic microsomal cytochrome P-450 apoproteins associated with bile duct ligation, and the administration of ethinyl estradiol, phenobarbital and 3-methylcholanthrene.
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Alterations of hepatic Na+,K+-atpase and bile flow by estrogen: effects on liver surface membrane lipid structure and function.
Administration of the synthetic estrogen ethinyl estradiol (17alpha-ethinyl-1,3,5-estratriene-3,17beta-diol) decreases hepatic Na(+),K(+)-ATPase (ATP phosphohydrolase; EC 3.6.1.3) activity and bile flow to 50% and alters the composition and structure of surface membrane lipid in rats. Although the content of phospholipids was not changed by treatment, free cholesterol (130%) and cholesterol esters (400%) were increased in liver surface membrane fractions. These observations correlate with changes in membrane viscosity, as shown by electron spin resonance probes. Both rotational correlation time, using the isotropic probe methyl (12-nitroxyl)stearate, and the order parameter, determined by the anisotropic probe 5-nitroxylstearic acid, were significantly increased in liver surface membrane fractions from rats treated with ethinyl estradiol. Administration of Triton WR-1339, a nonionic detergent that corrects hepatic and serum lipid changes caused by ethinyl estradiol treatment, restored toward normal elevated membrane lipids and viscosity as well as Na(+),K(+)-ATPase activity and bile flow. Although restoration of normal liver surface membrane structure and function may be due to reversal of abnormal lipid composition, detergents also may directly alter membrane enzyme activity. Addition of Triton WR-1339 in vitro increased Na(+),K(+)-ATPase activity and reduced membrane viscosity of surface membranes from rats treated with ethinyl estradiol. Triton had no effect on either parameter in normal membrane preparations. Studies of membrane structure and function both in vivo and in vitro suggest that alterations in lipid composition may alter Na(+),K(+)-ATPase function and bile flow.
Hepatic alkaline phosphatase isoenzymes: isolation, characterization and differential alteration.
Although it is generally believed that hepatic alkaline phosphatase is localized to liver plasma membranes, 63% is present in the cytosol fraction after ultracentrifugation of rat liver homogenates. Divalent cation requirements, heat inactivation, pH optima, Km and chemical inhibition characteristics of partially purified alkaline phosphatase enzymes prepared from membrane and cytosol fractions suggested different structural forms. Furthermore, bile duct obstruction and ethinyl estradiol administration preferentially increased membrane-bound alkaline phosphatase activity, while cytosol activity was unaltered. In contrast, phenobarbital treatment decreased membrane-bound alkaline phosphatase and increased cytosol activity. These studies support the presence of two forms of hepatic alkaline phosphatase in rat liver which are regulated by different control mechanisms.
Stimulation of hepatic sodium and potassium-activated adenosine triphosphatase activity by phenobarbital. Its possible role in regulation of bile flow.
Since phenobarbital administration produces a profound increase in bile flow without changing bile acid secretion, we examined whether this drug increases the activity of hepatic sodium-potassium-activated ATPase [Na+-K+)-ATPase], the postulated regulating enzyme in the secretion of bile salt independent bile flow. After freeze-thawing to increase substrate accessibility, (Na+-K+) ATPase activity was determined by ouabain inhibition of total ATPase activity. Its activity was highest in isolated liver surface membrane fractions enriched in bile canalicult. Phenobarbital administration significatly increased (Na+-K+)-ATPase activity in both liver surface membrane fractions as well as liver homogenates. This enhanced activity is apparently selective for other membrane phosphatases and the enzyme activity in other tissues is either unaltered or decreased. Kinetic analysis of (Ka+-K+)-ATPase indicates that phenobarbital treatment increased maximum velocity and half-maximum activation constant was unchanged, consistent with activation of latent molecules or an increased number of enzyme molecules. The latter process seems more likely because cycloheximide prevented phenobarbital induction and activators were not demonstrated in vitro. Examination of the full time course of phenobarbital induction to determine whether phenobarbital increased synthesis or decreased degradation was consistent with increased synthesis since the apparent degradation rates were similar with or without phenobarbital treatment. The apparent half-life for (Na+-K+)-ATPase was estimated to be approximately 2.5 days, consistent with liver surface membrane protein turnover. The correlation of changes in bile flow with (Na+-K+)-ATPase was examined under several experimental situations. Phenobarbital caused a parallel increase in each during the 1st 2 days of greatment: thereafter other factors become rate limiting for flow, since enzyme activity doesn't reach a new steady state until 4-days. Consistent with increased sodium-potassium exchange, bile sodium was unchanged while potasium concentrations were significantly reduced. Changes in both bile flow and (Na+-K+)-ATPase induced by phenobarbital are independent of thyroid hormone. These studies support the postulate that (Na+-K+)-ATPase is an important factor in regulation of bile flow. In addition, phenobarbital enhancement of both bile flow and (Na+-K+)-ATPase is dependent upon de novo protein synthesis.
Effects of ethinyl estradiol on hepatic microsomal proteins and the turnover of cytochrome P-450.
The effect of ethinyl estradiol, a steroid commonly used in birth control pills and possibly associated with impaired drug metabolism in humans, on the activity of and turnover of components of the hepatic microsomal mixed-function oxidase system was studied in male rats. After 5 days of ethinyl estradiol, 5 mg/kg/day, there was a significant decrease in the activity of ethylmorphine-N-demethylase and in cytochrome P-450, cytochrome b2, and NADPH cytochrome c reductase. Cytochrome P-450 apoproteins were identified within an SDS-polyacrylamide gel system, and the rate of turnover of cytochrome P-450 apoproteins was studied by double-isotope labeling techniques. After 5 days of ethinyl estradiol administration, the rate of degradation of cytochrome P-450 apoprotein was reduced (half-life of 50 hr compared to 24 hr in control), and their relative rate of synthesis was likewise reduced, indicating that a new steady state of protein turnover associated with reduced synthesis rate had been reached. This was confirmed by studies of the effect of ethinyl estradiol on the level of microsomal cytochrome P-450 over a 10-day period.
Identification and characterization of a bile acid receptor in isolated liver surface membranes.
It is generally assumed that hepatic transport of bile acids is a carrier-mediated process. However, the basic mechanisms by which these organic anions are translocated across the liver cell surface membrane are not well understood. Since carrier-mediated transport involved binding of the transported molecule to specific receptor sites, we have investigated the possibility that bile acid receptors are present in liver surface membranes. Isolated liver surface membranes were incubated at 4 degrees C with [14C]cholic acid and [14C]taurocholic acid, and membrane-boudn bile acid was separated from free by a rapid ultrafiltration technique through glass-fiber filters. Specific bile acid binding is rapid and reversible and represents approximately 80% of the total bile acid bound to liver surface membranes. Taurocholic acid binding is independent of the medium pH, while cholic acid binding demonstrates an optimum at pH 6.0. Analysis of equilibrium data for both cholic and taurocholic acid binding indicates that specific binding is saturable and consistent with Michaelis-Menten kinetics, while nonspecific binding is nonsaturable. Apparent maximal binding capacity and dissociation constant values indicate a large capacity system of receptors that have an affinity for bile acids comparable to that of the hepatic transport mechanism. Scatchard analysis of the saturation kinetics as well as inhibition studies suggest that bile acids bind to a single and noninteracting class of anion that competes with bile acids for hepatic uptake, also inhibits cholic acid binding. In contrast, no inhibition was demonstrated with indocyanine green and probenecid. Specific bile acid binding is enriched and primarily located in liver surface membranes and found only in tissues involved in bile acid transport. Specific bile acid binding is independnet of Na+, Ca2+, and Mg2+ and does not require metabolic energy. In addition, thiol groups and disulfide are not required for activity at the binding site. However, specific bile acid binding is markedly decreased by low concentrations of proteolytic enzymes and is also decreased by the action of neuraminidase and phospholipases A and C. These results are consistent with the existence of a homogeneous bile acid receptor protein in liver surface membranes. The primary surface membrane location of this receptor, its binding properties, and its ligand specificity suggest that bile acid binding to this receptor may represent the initial interaction in bile acid transport across liver surface membranes.