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F R Simon

Publications and source records attributed to F R Simon.

At least 37 records · Page 2Linked to original sources
PubMed1992

Characterization of the transport of a synthetic bile salt, iodinated cholyl-glycyl-tyrosine, in isolated cultured rat hepatocytes.

The uptake of tri-hydroxy conjugated bile salts by hepatocytes is principally by a sodium-dependent carrier. We examined the uptake kinetics of the high-specific-activity, hydroxylated, conjugated bile salt 125I-labeled cholyl-glycyl-tyrosine, to determine whether this synthetic bile salt was transported by the sodium-dependent bile salt system. 125I-labeled cholyl-glycyl-tyrosine was synthesized, and its transport kinetics were studied in freshly cultured rat hepatocytes. Uptake into hepatocytes was time and temperature dependent and was decreased by the inhibitors diisothiocyanodisulfonic acid stilbene, probenecid and carbonyl cyanide chlorophenyl hydrazone, demonstrating carrier mediation and energy dependence. At concentrations of iodinated cholyl-glycyl-tyrosine less than 10 mumol/L, uptake was 27% +/- 5% sodium dependent, whereas at concentrations from 10 mumol/L to 40 mumol/L uptake was 52% +/- 4% sodium dependent. The apparent affinity for uptake of 125I-labeled cholyl-glycyl-tyrosine was 8 +/- 2 mumol/L, and the maximal velocity was 50 +/- 20 pmol/micrograms DNA/min. Both taurocholate and indocyanine green inhibited uptake of 125I-labeled cholyl-glycyl-tyrosine. Indocyanine green inhibited the uptake of 125I-labeled cholyl-glycyl-tyrosine (Ki = 10 microns) more effectively than taurocholate (Ki = 20 microns). We conclude that 125I-labeled cholyl-glycyl-tyrosine is not a specific probe for either sodium-dependent bile salt or sodium-independent organic anion carriers, but appears to use both systems in a concentration-dependent manner in cultured rat hepatocytes.

Animals↗
PubMed1992

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements.

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

Base Sequence↗
PubMed1991

Impairment of hepatic insulin receptors during chronic ethanol administration.

Chronic alcoholism is frequently associated with impaired intermediary metabolism and insulin resistance. The cellular defects leading to insulin resistance have not been clearly defined but could result from reduced insulin binding or abnormalities in any one of several postreceptor steps. The purpose of the present studies was to measure 125I-insulin binding and internalization kinetics and postreceptor response of the enzyme activity of tyrosine aminotransferase in isolated cultured rat hepatocytes. Four weeks of alcohol ingestion significantly reduced to 47% of control the 125I-insulin binding sites measured either as surface or total (after digitonin permeabilization). In contrast, 125I-epidermal growth factor binding was not significantly changed. Internalization of surface-bound 125I-insulin was decreased, but degradation was not increased, indicating that altered kinetics did not account for the change. Ethanol ingestion markedly reduced in liver cytosol some enzymes regulated by insulin and involved in glucose homeostasis. Basal activities of tyrosine aminotransferase and glucokinase were reduced 51% (P less than 0.01) and 32% (P less than 0.01), respectively. In contrast, phosphoenolpyruvate carboxykinase was unchanged. In short-term cultured hepatocytes from ethanol-fed rats, the maximum response of tyrosine amino-transferase to insulin was reduced 40% (P less than 0.01) without a change in the concentration causing 50% of the maximum response (EC50) compared with controls. In contrast, dexamethasone increased tyrosine aminotransferase to similar maximal levels and with similar EC50, indicating that ethanol did not alter the intracellular response. In conclusion, chronic ethanol ingestion caused significant time-dependent and selective changes in cell surface binding of insulin that was associated with subsequent postreceptor events.

Animals↗
PubMed1990

Identification of taurocholate binding sites in ileal plasma membrane.

Intestinal absorption of bile salts occurs by passive processes throughout the length of the small intestine, whereas active carrier-mediated uptake is localized to the ileum. Although previous studies have extensively characterized brush-border transport of bile acids, their extrusion across the basolateral membrane is less well understood. Because previous reports had failed to show specific bile acid binding sites except with the use of photolabeled bile salt derivatives, we sought to identify and characterize the binding parameters of the physiological bile salt taurocholate in ileal and jejunal plasma membrane subfractions. Brush-border membrane (BBM) and basolateral membrane (BLM) fractions were rapidly and simultaneously isolated from the small intestinal mucosa. BBM fractions were isolated with enrichments of 50- to 54-fold for leucine aminopeptidase, whereas the basolateral membrane enrichment of Na(+)-K(+)-ATPase, its specific marker enzyme, was 22- to 25-fold. Contamination from intracellular organelles was minimal. Binding of [14C]taurocholate was demonstrated in both jejunal as well as ileal plasma membrane fractions. However, only ileal binding demonstrated saturation, reversibility, and susceptibility to proteolytic enzymes. [14C]taurocholate binding to BBM fractions also showed competition with bile acids but was not altered by pH or alkylating agents. In contrast, binding of taurocholate to the basolateral membrane showed optimal pH between 6.5 and 7.5 and was inhibited by thiol and alkylating agents. Kinetic analysis of specific ileal BBM and BLM binding showed the parameters for BBM as 288 +/- 70 microM and 2.4 +/- 0.6 nmol/mg protein and for BLM as 6.6 +/- 0.7 microM and 0.56 +/- 0.01 nmol/mg protein for dissociation constant and maximum binding capacity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Alterations in the functional expression of receptors on cirrhotic rat hepatocytes.

Reduced hepatic uptake and clearance of macromolecules in liver cirrhosis is due to two major factors: increased diffusional barriers, resulting primarily from the deposition of excessive connective tissue in the space of Disse, and hepatocellular dysfunction, manifested by receptor and/or postreceptor defects. To probe the mechanisms underlying hepatocellular dysfunction in liver cirrhosis, we have investigated receptor-ligand interactions for asialoorosomucoid, insulin and epidermal growth factor in hepatocytes isolated from the livers of rats chronically exposed to phenobarbital and carbon tetrachloride for up to 12 weeks. Viable cells were allowed to attach at 37 degrees C and the high-affinity cell surface binding sites for each ligand were assessed at 4 degrees C in the presence of [125I]-ligand. In parallel incubations, digitonin (0.055%) was added to the binding medium to assess total cellular binding sites. Results demonstrated that chronic treatment of rats with phenobarbital increased hepatocyte asialoorosomucoid surface receptor affinity (p less than 0.05) but had no affect on the number of asialoglycoprotein binding sites. Treatment with CCl4 and phenobarbital significantly reduced the number of surface binding sites for asialoorosomucoid (p less than 0.05) and epidermal growth factor (p less than 0.02), although this treatment had no effect on either the binding affinity or the number of binding sites for insulin. The decrease in cell surface binding sites for asialoorosomucoid and epidermal growth factor was not due to a redistribution of the surface sites to intracellular locations, since the total number of cellular binding sites also was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Bradykinin activates protein kinase C in cultured cortical collecting tubular cells.

Bradykinin inhibits vasopressin-stimulated water transport in cortical collecting tubular cells. The biochemical mechanism of this effect was explored by means of primary cultures of rabbit cortical collecting tubular cells. Bradykinin was found to produce a rapid release of calcium from intracellular stores, an increase in sn-1,2-diacylglycerol levels, and a fivefold increase in membrane-bound protein kinase C activity, consistent with stimulation of phospholipase C and activation of protein kinase C in rabbit cortical collecting tubular cells. In addition, bradykinin produced a dose-dependent 46% inhibition of vasopressin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. Pretreatment with the protein kinase C inhibitors, H-7 and staurosporine, reversed the bradykinin-mediated inhibition of vasopressin-stimulated cAMP accumulation. In contrast, pretreatment with either the phospholipase A2 inhibitor, mepacrine, or pertussis toxin did not prevent the inhibitory effect of bradykinin on vasopressin-stimulated cAMP production, suggesting that the effects are not mediated by prostaglandin E2 or activation of a pertussis-toxin sensitive guanine nucleotide regulatory protein (e.g., Gi). Because bradykinin also inhibits isoproterenol-stimulated cAMP formation but does not inhibit either basal-, forskolin-, or cholera toxin-stimulated cAMP accumulation, the site of this inhibition appears to involve the hormone receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory subunit (Gs). The results demonstrate that bradykinin stimulates phospholipase C leading to activation of protein kinase C, which then inhibits vasopressin-stimulated cAMP production at the level of the hormone receptor or coupling of the receptor to Gs in cultured cortical collecting tubular cells.

Animals↗
PubMed1988

Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition.

Administration of high-dose ethinylestradiol to rats decreases bile flow, Na,K-ATPase specific activity, and liver plasma membrane fluidity. By use of highly purified sinusoidal and bile canalicular membrane fractions, the effect of ethinylestradiol administration on the protein and lipid composition and fluidity of plasma membrane fractions was examined. In sinusoidal fractions, ethinylestradiol (EE) administration decreased Na,K-ATPase activity (32%) and increased activities of alkaline phosphatase (254%), Mg2+-ATPase (155%), and a 160-kDa polypeptide (10-fold). Steady-state and dynamic fluorescence polarization was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH) was significantly higher in canalicular compared to sinusoidal membrane fractions. Ethinylestradiol (5 mg/kg per day for 5 days) selectively increased sinusoidal polarization values. Similar changes were demonstrated with the probes 2- and 12-anthroyloxystearate. Time-resolved fluorescence polarization measurements indicated that EE administration for 5 days did not change DPH lifetime but increased the order component (r infinity) and decreased the rotation rate (R). However, 1 and 3 days after EE administration and with low doses (10-100 micrograms/kg per day for 5 days) the Na,K-ATPase, bile flow, and order component were altered, but the rotation rate was unchanged. Vesicles prepared from total sinusoidal membrane lipids of EE-treated rats, as well as phospholipid vesicles, demonstrated increased DPH polarization, as did intact plasma membrane fractions. Liver plasma membrane fractions showed no change in free cholesterol or cholesterol/phospholipid molar ratio, while esterified cholesterol content was increased with high-dose but not low-dose ethinylestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Biochemical localization of hepatic surface-membrane Na+,K+-ATPase activity depends on membrane lipid fluidity.

Membrane proteins of transporting epithelia are often distributed between apical and basolateral surfaces to produce a functionally polarized cell. The distribution of Na+,K+-ATPase [ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37] between apical and basolateral membranes of hepatocytes has been controversial. Because Na+,K+-ATPase activity is fluidity dependent and the physiochemical properties of the apical membrane reduces its fluidity, we investigated whether altering membrane fluidity might uncover cryptic Na+,K+-ATPase in bile canalicular (apical) surface fractions free of detectable Na+,K+-ATPase and glucagon-stimulated adenylate cyclase activities. Apical fractions exhibited higher diphenylhexatriene-fluorescence polarization values when compared with sinusoidal (basolateral) membrane fractions. When 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) was added to each fraction, Na+,K+-ATPase, but not glucagon-stimulated adenylate cyclase activity, was activated in the apical fraction. In contrast, further activation of both enzymes was not seen in sinusoidal fractions. The A2C-induced increase in apical Na+,K+-ATPase approached 75% of the sinusoidal level. Parallel increases in apical Na+,K+-ATPase were produced by benzyl alcohol and Triton WR-1339. All three fluidizing agents decreased the order component of membrane fluidity. Na+,K+-ATPase activity in each subfraction was identically inhibited by the monoclonal antibody 9-A5, a specific inhibitor of this enzyme. These findings suggest that hepatic Na+,K+-ATPase is distributed in both surface membranes but functions more efficiently and, perhaps, specifically in the sinusoidal membranes because of their higher bulk lipid fluidity.

Animals↗
PubMed1988

Selective modulation of hepatic and ileal Na+-K+-ATPase by bile salts in the rat.

Sodium-potassium adenosinetriphosphatase (Na+-K+-ATPase) is modulated by functional demands. We determine whether Na+-K+-ATPase specific activity was changed by oral administration of different bile salts and whether upregulation in the liver is due to increased numbers of catalytic units. In rats after bile duct drainage for 18 h, Na+-K+-ATPase activity was reduced to 50% of control in liver and ileum but unchanged in jejunum and kidney. Increased Na+-K+-ATPase activity after short-term feeding of bile salts was noted only following trihydroxy bile salts, i.e., taurocholate (100 mg/100 g body wt) increased hepatic Na+-K+-ATPase 143% and ileum 138% above control, whereas jejunum and kidney were unchanged. Chronic feeding of trihydroxy bile salts for 4 days increased hepatic Na+-K+-ATPase (214-260%) and alkaline phosphatase (189-274%), whereas 5'-nucleotidase and Mg2+-ATPase activities were unchanged from control. Plasma membrane Na+-K+-ATPase activity significantly increased as early as 4 h after taurocholate administration, whereas homogenate activity did not rise until 16 h; both reached a new steady state between 24 and 48 h. Sixteen hours after bile salt feeding, increased Na+-K+-ATPase activity was blocked by cycloheximide, and in the liver increased enzyme activity (179%) was associated with a comparable change in sodium-dependent [gamma-32P]ATP binding (162%) to liver plasma membrane fractions. These studies show Na+-K+-ATPase activity adapts selectively in liver and ileum following administration of trihydroxy bile salts, and the process involves increased density of Na+-K+ pump sites on the liver plasma membrane.

Animals↗
PubMed1987

Effects of vitamin D-induced chronic hypercalcemia on rat renal cortical plasma membranes and mitochondria.

Increases in intracellular and mitochondrial calcium content that accompany ischemic and toxic acute renal failure have been suggested to mediate renal tubular cell injury and dysfunction, but the mechanism(s) are unknown. We studied the effects of in vivo vitamin D-induced chronic hypercalcemia on rat renal cortical brush-border and basolateral membranes and mitochondria. In the brush-border membrane, hypercalcemia caused significant decreases in alkaline phosphatase-specific activity, total phospholipid molar content, and phosphatidylserine percent molar composition and increases in the cholesterol-to-total phospholipid molar ratio and phosphatidylinositol percent molar composition. In the basolateral membrane, hypercalcemia caused significant decreases in Na+-K+-ATPase-specific activity and total phospholipid molar content and increases in the cholesterol-to-total phospholipid molar ratio and phosphatidylinositol 4,5-bisphosphate percent molar composition. In the mitochondria, hypercalcemia caused a mild increase in the mitochondrial calcium content, but no alterations in succinic dehydrogenase-specific activity, succinate-, ADP-, or uncoupler-induced respiration. Thus hypercalcemia caused alterations in brush-border and basolateral membrane enzyme activity and lipid composition, but no functional changes were detected in mitochondria. These hypercalcemia-induced plasma membrane biochemical alterations may be markers of early cell injury and suggest a role for calcium in causing or predisposing to renal tubular cell injury.

Animals↗
PubMed1986

Maintenance of epithelial surface membrane lipid polarity: a role for differing phospholipid translocation rates.

Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using 32P and either [3H]-glycerol or [2-3H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies, again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 X faster than into the brush border membrane and its relative composition was 2.5 X greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.

Animals↗
PubMed1986

Drug-induced hepatotoxicity.

Drug-induced injury to the liver can mimic any form of acute or chronic liver disease. Acute injury to the liver frequently is due to the action of cytochrome P450, which breaks down drugs into electrophiles or free radicals; these reactive metabolites can covalently bind to protein and unsaturated fatty acids or induce lipid peroxidation, respectively. These events may impair vital functions of the cell, such as maintenance of calcium homeostasis, leading to death; or hypothetically they may elicit a hypersensitivity reaction directed mainly at the liver. Glutathione and tocopherol play critical roles in cellular defense. Cholestatic disease caused by drugs results from a selective disturbance in bile secretion. Agents such as estrogens, chlorpromazine, and monohydroxy bile acids alter the chemical and physical properties of membranes, leading to impaired activity of carriers and pumps for bile acids and electrolytes. Certain drugs produce chronic liver disease that is pathologically identical to chronic active hepatitis, biliary cirrhosis, or alcoholic liver disease.

Acetaminophen↗
PubMed1985

Chronic ethanol increases liver plasma membrane fluidity.

Purified plasma membrane fractions of cultured well-differentiated Reuber H35 hepatoma cells were studied after growth in the presence or absence of ethanol. Growth of cells in the presence of ethanol significantly increased plasma membrane 5'-nucleotidase activity but did not influence sodium-potassium adenosinetriphosphatase activity. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from cells grown in 80 mM ethanol for 3 weeks, compared to controls. Decreased polarization of DPH in plasma membranes was observed after 3-weeks growth of cells in as little as 1 mM ethanol. A 1-h exposure to 80 mM ethanol had no effect. Altered DPH polarization was due to a decrease in the order parameter of the probe. The rotational correlation time of the probe was virtually unchanged. Chronic ethanol treatment of cells did not alter the polarization of the membrane surface probe trimethylammoniodiphenylhexatriene. Plasma membranes from cells grown in 80 mM ethanol had decreased contents of both phospholipid and unesterified cholesterol, but the cholesterol to phospholipid ratio was unchanged. The percentages of sphingomyelin and phosphatidylserine in plasma membrane phospholipids were significantly decreased after ethanol treatment, while the phosphatidylcholine/sphingomyelin ratio was increased by 42%. Vesicles prepared from total plasma membrane lipids of ethanol-treated cells, as well as vesicles prepared from polar lipids alone, showed the same alterations in DPH polarization as did plasma membranes. The importance of ethanol metabolism in the observed plasma membrane changes was demonstrated in two ways.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Renal cortical brush-border and basolateral membranes: cholesterol and phospholipid composition and relative turnover.

A new procedure for the rapid isolation of renal cortical brush-border and basolateral membranes from the same homogenate is described. Brush-border membranes isolated using Mg2+-EGTA precipitation were enriched 18-fold for leucine aminopeptidase and had a recovery of 32.5%. Basolateral membrane fractions were isolated using a discontinuous sucrose gradient and showed an enrichment of 10.7-fold and recovery of 12.8% using (Na+,K+)-ATPase as a marker enzyme. Lipid analysis using two-dimensional TLC separation of phospholipids and gas liquid chromatography for cholesterol showed marked differences in the lipid composition of the brush-border and basolateral membranes. The brush-border membrane had increased sphingomyelin, phosphatidylserine, ethanolamine plasmalogens, and an increased cholesterol-to-phospholipid and sphingomyelin-to-phosphatidylcholine ratio compared to the basolateral membrane. The relative turnover of total membrane and individual phospholipid species using a double isotope ratio method was carried out. Phospholipids were labeled with either phosphorus 32 and 33 or acetate (3H, 1-14C). The relative turnover of phospholipid species and cholesterol differed strikingly. Phosphatidylcholine showed a high turnover, phosphatidylethanolamine and phosphatidylinositol had intermediate values and sphingomyelin, phosphatidylserine and cholesterol had low relative turnover rates. The order of phospholipid class relative turnover was independent of the labeled precursor used. The brush-border membrane had a significantly reduced relative turnover rate for total membrane phospholipids, sphingomyelin and cholesterol compared to the basolateral membrane. These data show marked differences in the lipid composition and relative turnover rates of the phospholipid species of the brush-border and basolateral membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Renal apical membrane cholesterol and fluidity in regulation of phosphate transport.

Renal proximal tubule cells adapt to dietary phosphate (Pi) restriction by increasing Pi transport independent of parathyroid hormone, vitamin D metabolites, or serum Ca2+. To determine the underlying cellular mechanism(s), brush border (BBM) and basolateral membranes (BLM) were isolated from growing male rats fed a synthetic diet containing variable levels of Pi (0.1-1.4%). Dietary Pi restriction was without effect on either BBM or BLM total lipid phosphorus, individual phospholipid species, or BLM Na+-K+-ATPase specific activity. However, dietary Pi restriction (0.1 vs. 1.0%) did cause a significant reduction in BBM but not BLM cholesterol (0.45 vs. 0.41 mumol/mg protein). Brush border membrane cholesterol was inversely correlated with the tubular reabsorption of Pi (r = 0.77, P less than 0.01) over a broad range (99.9-46.2%). Arrhenius analysis of two intrinsic BBM enzymes revealed a significant reduction in the breakpoint temperature for alkaline phosphatase but no change for Mg2+-ATPase. Fluorescence polarization studies showed increased BBM inner core fluidity due to an alteration in neutral lipids but not phospholipid, fatty acid, or protein membrane components. These data demonstrate that the BBM can regulate its cholesterol content independent of the BLM. Furthermore, they suggest that adaptation to dietary Pi restriction involves a reduction in BBM cholesterol, which may be mediated by an increase in membrane fluidity.

Adenosine Triphosphatases↗
PubMed1985

Ischemia induces partial loss of surface membrane polarity and accumulation of putative calcium ionophores.

To determine if ischemia induces alterations in renal proximal tubule surface membranes, brush border (BBM) and basolateral membranes (BLM) were isolated simultaneously from the same cortical homogenate after 50 min of renal pedicle clamping. Ischemia caused a selective decrease in the specific activity of BBM marker enzymes leucine aminopeptidase and alkaline phosphatase, but did not effect enrichment (15 times). Neither specific activity nor enrichment (10 times) of BLM NaK-ATPase was altered by ischemia. Contamination of BBM by intracellular organelles was also unchanged, but there was an increase in the specific activity (41.1 vs. 60.0, P less than 0.01) and enrichment (2.3 vs. 4.3, P less than 0.01) of NaK-ATPase in the ischemic BBM fraction. Ischemia increased BLM lysophosphatidylcholine (1.3 vs. 2.5%, P less than 0.05) and phosphatidic acid (0.4 vs. 1.3%, P less than 0.05). Ischemia also decreased BBM sphingomyelin (38.5 vs. 29.6%, P less than 0.01) and phosphatidylserine (16.1 vs. 11.4%, P less than 0.01), and increased phosphatidylcholine (17.2 vs. 29.7%, P less than 0.01), phosphatidylinositol (1.8 vs. 4.6%, P less than 0.01), and lysophosphatidylcholine (1.0 vs. 1.8%, P less than 0.05). The large changes in BBM phospholipids did not result from new phospholipid synthesis, since the specific activity (32P dpm/nmol Pi) of prelabeled individual and total phospholipids was unaltered by ischemia. We next evaluated if these changes were due to inability of ischemic cells to maintain surface membrane polarity. Cytochemical evaluation showed that while NaK-ATPase could be detected only in control BLM, specific deposits of reaction product were present in the BBM of ischemic kidneys. Furthermore, using continuous sucrose gradients, the enzymatic profile of ischemic BBM NaK-ATPase shifted away from ischemic BLM NaK-ATPase and toward the BBM enzymatic marker leucine aminopeptidase. Taken together, these data suggest that NaK-ATPase activity determined enzymatically and cytochemically was located within ischemic BBM. We propose that ischemia impairs the ability of cells to maintain surface membrane polarity, and also results in the accumulation of putative calcium ionophores.

Animals↗
PubMed1984

Ethynylestradiol impairs bile salt uptake and Na-K pump function of rat hepatocytes.

Ethynylestradiol impairs bile flow and bile salt maximum secretory rate in rats, implying a secretory defect. In addition, Na-K-ATPase activity is decreased in liver surface membranes, suggesting abnormalities at the sinusoidal as well as the canalicular membrane. We investigated whether ethynylestradiol pretreatment affects bile salt uptake and Na-K pump function in isolated rat hepatocytes. Ethynylestradiol-treated cells were functionally intact as assayed with trypan blue exclusion, lactate dehydrogenase release, and oxygen consumption. Initial taurocholate uptake velocity was reduced by 73% in ethynylestradiol-treated hepatocytes [Vmax, 1.0 +/- 0.1 vs. 3.7 +/- 0.2 mumol X min-1 X (10(6) cells)-1; P less than 0.001; Km, 34 +/- 5 vs. 33 +/- 3 microM]. Na-K-ATPase activity in cell homogenates (36 +/- 5 vs. 27 +/- 4 mumol Pi X h-1 X mg prot-1; P less than 0.05), ouabain-suppressible rubidium-86 influx [6.8 +/- 1.1 vs. 4.8 +/- 1.0 nmol K+ X min-1 X (10(6) cells)-1; P less than 0.05], and intracellular potassium concentration (126 +/- 10 vs. 110 +/- 16 mmol/l; P less than 0.05) were reduced after ethynylestradiol. Taurocholate uptake measured at different temperatures between 25 degrees and 37 degrees was linear when plotted according to Arrhenius. The energy of activation was increased by 40% in ethynylestradiol-treated hepatocytes [17 +/- 4 vs. 23 +/- 4 kcal X mol-1 X (10(6) cells)-1; P less than 0.05], consistent with decreased membrane fluidity. These data suggest the possibility that during ethynylestradiol-induced cholestasis a disorder of the sinusoidal domain, caused perhaps by ethynylestradiol-induced alterations in membrane lipid composition, is an important contributing factor.

Animals↗