Intact Rat Liver Research Articles

Bradykinin Metabolism in the Liver and Lung of the Rat

Bradykinin (BK) in an asanguinous salt solution was perfused through intact rat liver. The perfusate was delivered through the portal vein and was collected from the inferior vena cava. BK concentrations varied from 0.0030 to 38.0 μm. The liver had a large capacity to degrade BK and the system did not approach saturation until perfusate BK concentrations reached 60 μm. The quantitatively predominant BK fragments formed and the amount of each formed, expressed as a percentage of the BK degraded during a single transhepatic passage, were Pro-Pro, 74%; Arg-Pro-Pro-Gly-Phe, 15%; and Arg-Pro-Pro-Gly-Phe-Ser-Pro, 7%; the first is indicative of initial aminopeptidase-P cleavage of BK to yield Arg and des-Arg1-BK and the latter two are indicative of initial angiotension converting enzyme (ACE) cleavage of BK. On the other hand, while the perfused rat lung also had a large capacity to degrade BK, the quantitatively predominant BK fragments formed and the amount of each formed, again expressed as a percentage of BK metabolized during a single transpulmonary passage, were Pro-Pro, 47%; Arg-Pro-Pro-Gly-Phe, 35%; and Arg-Pro-Pro-Gly-Phe-Ser-Pro, 7%. Thus in rat liver the aminopeptidase-P pathway is the major route for BK degradation, whereas in rat lung the aminopeptidase-P and the ACE pathways exhibit nearly equal capacities to degrade BK.

Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments. Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase.

The transfer of sialic acids (Sia) from CMP-sialic acid (CMP-Sia) to N-linked sugar chains is thought to occur as a final step in their biosynthesis in the trans portion of the Golgi apparatus. In some cell types such Sia residues can have O-acetyl groups added to them. We demonstrate here that rat hepatocytes express 9-O-acetylated Sias mainly at the plasma membranes of both apical (bile canalicular) and basolateral (sinusoidal) domains. Golgi fractions also contain 9-O-acetylated Sias on similar N-linked glycoproteins, indicating that O-acetylation may take place in the Golgi. We show here that CMP-Sia-FITC (with a fluorescein group attached to the Sia) is taken up by isolated intact Golgi compartments. In these preparations, Sia-FITC is transferred to endogenous glycoprotein acceptors and can be immunochemically detected in situ. Addition of unlabeled UDP-Gal enhances Sia-FITC incorporation, indicating a substantial overlap of beta-galactosyltransferase and sialyltransferase machineries. Moreover, the same glycoproteins that incorporate Sia-FITC also accept [3H]galactose from the donor UDP-[3H]Gal. In contrast, we demonstrate with three different approaches (double-labeling, immunoelectron microscopy, and addition of a diffusible exogenous acceptor) that sialyltransferase and O-acetyltransferase machineries are much more separated from one another. Thus, 9-O-acetylation occurs after the last point of Sia addition in the trans-Golgi network. Indeed, we show that 9-O-acetylated sialoglycoproteins are preferentially segregated into a subset of vesicular carriers that concentrate membrane-bound, but not secretory, proteins.

In vivo spin trapping of nitric oxide generated in the small intestine, liver, and kidney during the development of endotoxemia: a time-course study.

Spin trapping of nitric oxide (NO.) in vivo in liver, small intestine, kidney, and plasma of intact rats was accomplished using diethyldithiocarbamate (DETC) administered intraperitoneally. DETC combines with Fe2+ to form (DETC)2-Fe and is an excellent trapping agent for nitric oxide. DETC distribution and uptake by the organs of interest was determined and the formation of the active trapping agent (DETC)2-Fe was assayed in the various organs and plasma. The capacity of this spin trap to capture NO. in vivo was demonstrated by administering sodium nitroprusside to the animals. The trapping procedure was then used to assess the course of NO. generation during a 6 h period in animals that had been treated with endotoxin. The rate of NO. generation/gram tissue was determined during the last 15 min of each time period. The results indicate that induction of nitric oxide generation begins earliest in the small intestine, then in the liver, and still later in the kidney and plasma. Nitric oxide production was most intense in the liver and was still increasing at the end of the experiment. Control animals receiving the spin trapping agent showed only little or no evidence of nitric oxide production except for the small intestine. The results show that induction of NO. generation caused by endotoxin begins at different times in different organs.

The metabolism of 22:5(−6) and of docosahexaenoic acid [22:6(−3)] compared in rat hepatocytes

Elevated levels of 22:5(−6), which is the elongated and desaturated product of arachidonic acid, is induced by selective n − 3 fatty acid deficiency, especially in brain cortex. Less elongation and desaturation of 20:4(−6) than of 20:5(−3) has been found in intact rat liver cells in previous studies and is probably the main reason why so little 22:5(−6) is found under adequate nutritional conditions. The present study compares the metabolism of 22:5(−6) with the metabolism of 22:6(−3), the main n − 3 fatty acid in mammals. Freshly isolated rat liver cells were incubated with [1− 14C]22:5(−6) and [1− 14C]22:6(−3). Oxidation and esterification in triacylglycerols, diacylglycerols and phospholipids were studied. The phospholipid classes were separated and the different molecular species identified. Rats with essential fatty acid deficiency were compared with control rats. 22:5(−6) was found to be a good substrate for membrane phospholipid biosynthesis and was conserved well in the phospholipid fraction of the rat liver cells for more than 3 h of incubation. More 22:5(−6) was esterified in the total phospholipid fraction and less was incorporated in triacylglycerols than observed with 22:6(−3) in hepatocytes from control animals. This was not the case in animals with essential fatty acid deficiency. 22:5(−6) was esterified to a greater extent in phosphatidylcholine than 22:6(−3) in control cells but not in essential fatty acid deficiency cells. More 22:5(−6) was coupled with 18:0 in the sn − 1 position of the phospholipid molecular species than 22:6(−3) was in control cells.

Levels of plasma C19 steroids and 5 alpha-reduced C19 steroid glucuronides in primates, rodents, and domestic animals.

In this study, a comparison of circulating levels of androsterone glucuronide and androstane-3 alpha, 17 beta-diol glucuronide in the male and female of several mammalian species was performed. Glucuronidated steroids were not detected in the circulation of the dog, bovine, swine, and rodent. High levels of circulating glucuronidated steroids were measured in the cynomolgus monkey and found to be 10-fold higher than in humans. The determination of tissue levels of unconjugated and conjugated C19 steroids was then performed in intact and castrated rats treated with androgens. Steroid glucuronides were not detected in the plasma, skin, prostate, or liver of either intact or treated rats, although the levels of unconjugated steroids in the plasma and tissues were increased after steroid treatments. Significant levels were detected in the bile, thus suggesting hepatic formation of steroid glucuronides in the rat. It is suggested that the monkey represents the best animal model to date to study the contribution of diphosphoglucuronosyltransferases present in steroid target peripheral tissues to circulating levels of steroid glucuronides.

HYPERTHERMIC STRESS AFFECTS GLUCOCORTICOID RECEPTOR-MEDIATED TRANSCRIPTION IN RAT LIVER

Binding capacity of the cytoplasmic and nuclear glucocorticoid receptor (GR) and the activity of tyrosine aminotransferase (TAT) were examined in the liver of intact and adrenalectomized rats exposed to 41°C whole body hyperthermic stress. In glucocorticoid-deprived animals, stress-induced decrease in the cytoplasmic steroid binding was followed by parallel increases in its nuclear binding and TAT activity, suggesting a stimulation of TAT gene transcription by the GR in the absence of the ligand. In intact animals, however, a diminution of the steroid binding in the cytosol, its unchanged nuclear binding and an impairment of TAT activity were observed upon the stress. The results imply that stress could elicit different structural or functional alterations of unliganded vsliganded GR.

Cohen NS (1996): Intracellular localization of the mRNAs of argininosuccinate synthetase and argininosuccinate lyase around liver mitochondria, visualized by high-resolution in situ reverse transcription-polymerase chain reaction. J Cell Biochem 61:81-96.

Argininosuccinate synthetase and argininosuccinate lyase, two cytoplasmic enzymes of the urea cycle, are released into the soluble phase in the absence of detergent when cells are disrupted. Yet previous biochemical studies, as well as immunocytochemistry at the electron microscope level, have shown that these enzymes are localized around mitochondria in situ. Such intracellular localization of soluble enzymes requires mechanisms to deliver the proteins to the appropriate sites, where they may then be anchored by specific protein-protein interactions. A method was developed to examine the intracellular distribution of the mRNA of argininosuccinate synthetase and argininosuccinate lyase in intact rat liver at the ultrastructural level by in situ reverse transcription and the polymerase chain reaction, using primers targeting regions of the coding sequences of the rat enzymes, digoxigenin-dUTP as the label, and anti-digoxigenin/1nm gold plus silver enhancement as the detection method. The tissue was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl. Examination of the numbers and the location of the silver grains, coupled with morphometric analysis of the electron micrographs, permitted the calculation of the silver “enrichment ratio” for each type of cell structure. These ratios showed that the mRNAs for argininosuccinate synthetase and argininosuccinate lyase were located next to the cytoplasmic side of the mitochondrial membrane and in the nearby endoplasmic reticulum. Most of the silver grains that were observed in the endoplasmic reticulum were within 200 nm of the mitochondria; it was not possible, however, to determine if those grains were actually associated with the reticular membranes. These studies demonstrate that the mRNAs of these two soluble cytoplasmic proteins are localized to the same limited regions where the proteins are situated. Translation of the proteins, therefore, must occur at these specific sites. The targeting of argininosuccinate synthetase and argininosuccinate lyase mRNAs to the immediate vicinity of the mitochondria may be the first step of the mechanisms by which the spatial organization of these soluble proteins in situ is accomplished. The targeting of mRNAs for soluble cytoplasmic proteins of organized metabolic pathways has not been demonstrated previously. These studies also show that in situ reverse transcription and the polymerase chain reaction at the ultrastructural level, which has not been previously reported, can be used to detect specific mRNAs; it should be extremely valuable for the intracellular detection of low-abundance mRNAs. © 1996 Wiley-Liss, Inc.

Effects of isoquinoline derivatives structurally related to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on mitochondrial respiration

Isoquinoline derivatives exert 1-methyl-4-phenylpyridinium (MPP +)-like activity as inhibitors of complex I and α-ketoglutarate dehydrogenase activity in rat brain mitochondrial fragments. We now examine the ability of 19 isoquinoline derivatives and MPP + to accumulate and inhibit respiration in intact rat liver mitochondria, assessed using polarographic techniques. None of the compounds examined inhibited respiration supported by either succinate + rotenone or tetramethylparaphenylenediamine (TMPD) + ascorbate. However, with glutamate + malate as substrates, 15 isoquinoline derivatives and MPP + inhibited state 3 and, to a lesser extent, state 4 respiration in a time-dependent manner. None of the isoquinoline derivatives were more potent than MPP +. 6,7-Dimethoxy-1-styryl-3,4-dihydroisoquinoline uncoupled mitochondrial respiration. Qualitative structure-activity relationship studies revealed that isoquinolinium cations were more active than isoquinolines in inhibiting mitochondrial respiration; these, in turn, were more active than dihydroisoquinolines and 1,2,3,4-tetrahydroisoquinolines. Three-dimensional quantitative structure-activity relationship studies using Comparative Molecular Field Analysis showed that the inhibitory potency of isoquinoline derivatives was determined by steric, rather than electrostatic, properties of the compounds. A hypothetical binding site was identified that may be related to a rate-limiting transport process, rather than to enzyme inhibition. In conclusion, isoquinoline derivatives are less potent in inhibiting respiration in intact mitochondria than impairing complex I activity in mitochondrial fragments. This suggests that isoquinoline derivatives are not accumulated by mitochondria as avidly as MPP +. The activity of charged and neutral isoquinoline derivatives implicates both active and passive processes by which these compounds enter mitochondria, although the quaternary nitrogen moiety of the isoquinolinium cations favours mitochondrial accumulation and inhibition of respiration. These findings suggest that isoquinoline derivatives may exert mitochondrial toxicity in vivo similar to that of MPTP/MPP +.

Intracellular localization of the mRNAs of argininosuccinate synthetase and argininosuccinate lyase around liver mitochondria, visualized by high-resolution in situ reverse transcription-polymerase chain reaction

Argininosuccinate synthetase and argininosuccinate lyase, two cytoplasmic enzymes of the urea cycle, are released into the soluble phase in the absence of detergent when cells are disrupted. Yet previous biochemical studies, as well as immunocytochemistry at the electron microscope level, have shown that these enzymes are localized around mitochondria in situ. Such intracellular localization of soluble enzymes requires mechanisms to deliver the proteins to the appropriate sites, where they may then be anchored by specific protein-protein interactions. A method was developed to examine the intracellular distribution of the mRNA of argininosuccinate synthetase and argininosuccinate lyase in intact rat liver at the ultrastructural level by in situ reverse transcription and the polymerase chain reaction, using primers targeting regions of the coding sequences of the rat enzymes, digoxigenin-dUTP as the label, and anti-digoxigenin/1 nm [corrected] gold plus silver enhancement as the detection method. The tissue was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl. Examination of the numbers and the location of the silver grains, coupled with morphometric analysis of the electron micrographs, permitted the calculation of the silver "enrichment ratio" for each type of cell structure. These ratios showed that the mRNAs for argininosuccinate synthetase and argininosuccinate lyase were located next to the cytoplasmic side of the mitochondrial membrane and in the nearby endoplasmic reticulum. Most of the silver grains that were observed in the endoplasmic reticulum were within 200 nm of the mitochondria; it was not possible, however, to determine if those grains were actually associated with the reticular membranes. These studies demonstrate that the mRNAs of these two soluble cytoplasmic proteins are localized to the same limited regions where the proteins are situated. Translation of the proteins, therefore, must occur at these specific sites. The targeting of argininosuccinate synthetase and argininosuccinate lyase mRNAs to the immediate vicinity of the mitochondria may be the first step of the mechanisms by which the spatial organization of these soluble proteins in situ is accomplished. The targeting of mRNAs for soluble cytoplasmic proteins of organized metabolic pathways has not been demonstrated previously. These studies also show that in situ reverse transcription and the polymerase chain reaction at the ultrastructural level, which has not been previously reported, can be used to detect specific mRNAs; it should be extremely valuable for the intracellular detection of low-abundance mRNAs.

Intracellular localization of the mRNAs of argininosuccinate synthetase and argininosuccinate lyase around liver mitochondria, visualized by high-resolution in situ reverse transcription-polymerase chain reaction.

Argininosuccinate synthetase and argininosuccinate lyase, two cytoplasmic enzymes of the urea cycle, are released into the soluble phase in the absence of detergent when cells are disrupted. Yet previous biochemical studies, as well as immunocytochemistry at the electron microscope level, have shown that these enzymes are localized around mitochondria in situ. Such intracellular localization of soluble enzymes requires mechanisms to deliver the proteins to the appropriate sites, where they may then be anchored by specific protein-protein interactions. A method was developed to examine the intracellular distribution of the mRNA of argininosuccinate synthetase and argininosuccinate lyase in intact rat liver at the ultrastructural level by in situ reverse transcription and the polymerase chain reaction, using primers targeting regions of the coding sequences of the rat enzymes, digoxigenin-dUTP as the label, and anti-digoxigenin/1 nm [corrected] gold plus silver enhancement as the detection method. The tissue was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl. Examination of the numbers and the location of the silver grains, coupled with morphometric analysis of the electron micrographs, permitted the calculation of the silver "enrichment ratio" for each type of cell structure. These ratios showed that the mRNAs for argininosuccinate synthetase and argininosuccinate lyase were located next to the cytoplasmic side of the mitochondrial membrane and in the nearby endoplasmic reticulum. Most of the silver grains that were observed in the endoplasmic reticulum were within 200 nm of the mitochondria; it was not possible, however, to determine if those grains were actually associated with the reticular membranes. These studies demonstrate that the mRNAs of these two soluble cytoplasmic proteins are localized to the same limited regions where the proteins are situated. Translation of the proteins, therefore, must occur at these specific sites. The targeting of argininosuccinate synthetase and argininosuccinate lyase mRNAs to the immediate vicinity of the mitochondria may be the first step of the mechanisms by which the spatial organization of these soluble proteins in situ is accomplished. The targeting of mRNAs for soluble cytoplasmic proteins of organized metabolic pathways has not been demonstrated previously. These studies also show that in situ reverse transcription and the polymerase chain reaction at the ultrastructural level, which has not been previously reported, can be used to detect specific mRNAs; it should be extremely valuable for the intracellular detection of low-abundance mRNAs.

The Mitochondrial K Channel as a Receptor for Potassium Channel Openers

The biochemical properties of the mitochondrial KATP channel are very similar to those of plasma membrane KATP channels, including inhibition by low concentrations of ATP and glyburide (Paucek, P., Mironova, G., Mahdi, F., Beavis, A. D., Woldegiorgis, G., and Garlid, K. D. (1992) J. Biol. Chem. 267, 26062-26069). Plasma membrane KATP channels are highly sensitive to the family of drugs known as K+ channel openers, raising the question whether mitochondrial KATP channels are similarly sensitive to these agents. We addressed this question by measuring K+ flux in intact rat liver mitochondria and in liposomes containing KATP channels purified from rat liver and beef heart mitochondria. K+ channel openers completely reversed ATP inhibition of K+ flux in both systems. In liposomes, ATP-inhibited K+ flux was restored by diazoxide (K1/2 = 0.4 microM), cromakalim (K1/2 = 1 microM), and two developmental cromakalim analogues, EMD60480 and EMD57970 (K1/2 = 6 nM). Similar K1/2 values were observed in intact mitochondria. These potencies are well within the range observed with plasma membrane KATP channels. We also compared the potencies of these K+ channel openers on the plasma membrane KATP channel purified from beef heart myocytes. The KATP channel from cardiac mitochondria is 2000-fold more sensitive to diazoxide than the channel from cardiac sarcolemma, indicating that two distinct receptor subtypes coexist within the myocyte. We suggest that the mitochondrial KATP channel is an important intracellular receptor that should be taken into account in considering the pharmacology of K+ channel openers.

Proton Nuclear Magnetic Resonance Spectroscopy of Lactate Production in Isolated Rat Liver during Cold Preservation

Lactate-edited1H NMR difference spectra have been acquired from intact rat liver tissue following flushing and preservation in ice. A peak, initially at 1.26 ppm, was seen to increase in the liver tissue with preservation time. This peak was assigned to lactate, despite the fact that its chemical shift was initially shifted by approximately −0.1 ppm relative to an externally added standard. The assignment was based on the following: (a) the peak increased over a 24-h ischemic storage period; (b) it was coupled to a signal 2.78 ± 0.02 ppm upfield; and (c) a parallel increase in lactate was noted in perchloric acid extracts of tissue from the same liver. An additional peak, assigned to alanine, was also observed during storage and was also shifted by approximately −0.1 ppm. Inclusion of dimethyl sulfoxide, which readily permeates liver tissue, demonstrated that this chemical shift alteration was a tissue-specific effect. These results demonstrate that1H NMR spectroscopy of intact liver tissue during hypothermic ischemia is possible, though chemical shift assignments should be made with caution.

Mechanisms by Which Fatty‐Acyl‐CoA Esters Inhibit or Activate Glucose‐6‐Phosphatase in Intact and Detergent‐Treated Rat Liver Microsomes

We have studied the effects of fatty-acyl-CoA esters on the activity of glucose-6-phosphatase (Glc6Pase) in untreated and detergent-treated liver microsomes. Fatty-acyl-CoA esters with chain lengths less than or equal to nine carbons do not inhibit Glc6Pase. Medium-chain fatty-acyl-CoA esters (10-14 carbons) inhibit Glc6Pase of untreated microsomes in a dose-dependent manner in the range 1-20 microM. The inhibitory effect is also dependent on the acyl-chain length. The higher the chain length, the stronger the inhibitory effect. It is also dependent on the microsomal protein concentration. The higher the protein concentration, the lower the inhibitory effect. Fatty-acyl-CoA esters with longer chain length (equal to or higher than 16 carbons) inhibit Glc6Pase of untreated microsomes within the range 1-2 microM. However, the inhibitory effect is either partially or totally cancelled, or even changed into an activation effect at higher concentrations. This is due to the release of mannose-6-phosphatase latency. The inhibition is fully reversible in the presence of bovine serum albumin. The mechanism of the Glc6Pase inhibition in untreated microsomes is uncompetitive (Ki for myristoyl-CoA = 1.2 +/- 0.3 microM, mean +/- SD, n = 3). Glc6Pase of detergent-treated microsomes is also inhibited by fatty-acyl-CoA esters, albeit less efficiently. In this case, the mechanism is non-competitive (Ki for myristoyl-CoA = 29 +/- 3 microM).

Reduction pathway of cis-5 unsaturated fatty acids in intact rat-liver and rat-heart mitochondria: assessment with stable-isotype-labelled substrates.

Besides the conventional isomerase-mediated pathway, unsaturated fatty acids with old-numbered double bonds are also metabolized by reduction pathways with NADPH as cofactor. The relative contributions of these pathways were measured in intact rat-liver and rat-heart mitochondria with a novel stable isotope tracer technique. A mixture of equal amounts of unlabelled cis-5-enoyl-CoA and 13C4-labelled acyl-CoA of equal chain lengths was incubated with mitochondria. The isotope distribution of 3-hydroxy fatty acids produced from the first cycle of beta-oxidation was analysed with selected ion monitoring by gas chromatograph-mass spectrometer. 3-Hydroxy fatty acids produced from the reduction pathway of unsaturated fatty acids were unlabelled (m + 0) whereas those produced from saturated fatty acids were labelled (m + 4). The m + 0 content serves to indicate the extent of reduction pathway. Rotenone treatment was used to switch the pathway completely to reduction. The extent of m + 0 enrichment in untreated mitochondria normalized to the m + 0 enrichment of rotenone-treated mitochondria was the percentage of reduction pathway. With this technique, cis-4-decenoate was found to be metabolized completely by the reduction pathway in both liver and heart mitochondria. cis-5-Dodecenoate was metabolized essentially by the reduction pathway in liver mitochondria, but only to 75% in heart mitochondria. When the chain length was extended to cis-5-tetradecenoate, the reduction pathway in liver mitochondria decreased to 86% and that in heart mitochondria to 65%. The effects of carnitine, clofibrate and other conditions on the reduction pathway were also studied. Enrichments of the label on saturated fatty acids and 3-hydroxy fatty acids indicated that the major pathway of reduction was not by the direct reduction of the cis-5 double bond. Instead, it is most probably by a pathway that does not involve forming a reduced saturated fatty acid first.

Sinusoidal (basolateral) bile salt uptake systems of hepatocytes.

Sinusoidal (basolateral) bile sale uptake is mediated by Na(+)-dependent and Na(+)-independent transport systems. Two hepatocellular bile salt uptake systems have been cloned from rat and human livers. The Na(+)-taurocholate cotransporting polypeptides Ntcp and NTCP mediate strictly Na(+)-dependent bile salt uptake into rat and human hepatocytes, respectively. Extensive characterization of Ntcp expression and function in a variety of eukaryotic cell lines, cultured hepatocytes, and intact rat liver indicates that Ntcp can account for most, if not all, Na(+)-dependent bile salt transport functions in rat liver. Whether the same is also true for the human NTCP is less well understood. The Na(+)-independent organic anion transporting polypeptides oatpl (rat) and OATP (human) exhibit a wide and charge-independent substrate specificity. Their transported substrates include sulfobromophthalein, bile salts, estrone-3-sulfates, ouabain, and other neutral steroids, as well as certain amphipathic organic cations. Its broad and charge-independent substrate specificity indicates that oatpl represents the previously suggested hepatocellular "multispecific bile salt transporter.". Oatpl and OATP are also expressed in the kidney and brain. Whether there are additional oatpl- and OATP-related proteins involved in overall hepatic drug and steroid clearance remains to be determined.

Attenuation of glycogenolytic action of activin A in intact rat liver.

Activin A stimulates glucose production by causing glycogenolysis in isolated hepatocytes. To determine the physiological significance of this effect, we examined the effect of activin A on glucose production in the perfused liver. Unlike the effect in isolated cells, activin A did not enhance glucose production nor did it cause radiocalcium efflux in the perfused liver. There was no effect of activin A in the liver perfused in the opposite direction. Although activin A did not promote glucose production, it was recovered from the hepatic vein in a bioactive form. When liver perfusion was performed in partially hepatectomized rats, activin A increased radiocalcium efflux. In isolated hepatocytes, activin A increased inositol phosphates, and the effect of activin A was attenuated by the plasma membrane fraction of hepatocytes. The inhibitory effect of the plasma membrane was abolished by digestion of the membrane with trypsin. These results indicate that the effect of activin A on glucose production is attenuated in the intact liver and that a protein factor(s) in plasma membrane may be involved in the inhibition.

Methylmalonic acid inhibits respiration in rat liver mitochondria.

Methylmalonic acid (MMA), which accumulates and is excreted in urine in mammals during vitamin B-12 deficiency, has been reported to inhibit succinate dehydrogenase, an enzyme involved in the mitochondrial tricarboxylic acid (TCA) cycle in rat liver. The enzyme inhibition by MMA may lead to various metabolic disorders as well as inhibition of mitochondrial energy generation in vitamin B-12-deficient mammals. To clarify the inhibition of succinate dehydrogenase by MMA in intact rat liver mitochondria, the effect of MMA on mitochondrial respiration was studied. When 6 mmol/L MMA was added to the reaction mixture for measuring mitochondrial respiration with succinate as a substrate, MMA was taken up and accumulated by the mitochondria (34-53 mmol/L). The accumulation of mitochondrial MMA was stimulated by the addition of ADP. Methylmalonic acid competitively inhibited State 3 mitochondrial respiration, and the Ki for the acid was 4.2 +/- 0.4 mmol/L. Although the respiratory control ratio decreased with increasing MMA concentration, the acid did not affect the phosphorus/oxygen ratio. Mitochondrial MMA accumulation secondary to vitamin B-12 deficiency inhibits succinate dehydrogenase and may contribute to various metabolic disorders associated with vitamin B-12 deficiency.

Effect of starvation on the activity of the mitochondrial tricarboxylate carrier

The effect of starvation on the activity of the tricarboxylate carrier has been investigated in intact rat liver mitochondria and in a reconstituted system. In both experimental conditions, the rate of citrate transport, when compared to control, is greatly reduced (35–40%) in starved rats. Similar behaviour is shown by the cytosolic lipogenic enzymes. Kinetic analysis of the carrier activity in intact mitochondria and in the proteoliposomal system has showed that during starvation only the V max of this process decreases while there is no change in the K m. No difference in the Arrhenius plot and in the lipid composition has been detected, which indicates that the reduced transport activity in fasted animals is not due to a change in the carrier lipid microenvironment. In starved rats, a reduction of the carrier activity has occurred even after the addition of increasing cardiolipin concentrations to proteoliposomes. These findings thus suggest that starvation-induced decrease of citrate carrier activity could be due to a change of the intrinsic properties of the transport protein.

Glucose Transport and Glucose 6-Phosphate Hydrolysis in Intact Rat Liver Microsomes

Glucose transport was investigated in rat liver microsomes in relation to glucose 6-phosphatase (Glu-6-Pase) activity using a fast sampling, rapid filtration apparatus. 1) The rapid phase in tracer uptake and the burst phase in glucose 6-phosphate (Glu-6-P) hydrolysis appear synchronous, while the slow phase of glucose accumulation occurs during the steady-state phase of glucose production. 2) [14C]Glucose efflux from preloaded microsomes can be observed upon addition of either cold Glu-6-P or Glu-6-Pase inhibitors, but not cold glucose. 3) Similar steady-state levels of intramicrosomal glucose are observed under symmetrical conditions of Glu-6-P or vanadate concentrations during influx and efflux experiments, and those levels are directly proportional to Glu-6-Pase activity. 4) The rates of both glucose influx and efflux are characterized by t1/2 values that are independent of Glu-6-P concentrations. 5) Glucose efflux in the presence of saturating concentrations of vanadate was not blocked by 1 mM phloretin, and the initial rates of efflux appear directly proportional to intravesicular glucose concentrations. 6) It is concluded that glucose influx into microsomes is tightly linked to Glu-6-Pase activity, while glucose efflux may occur independent of hydrolysis, so that microsomal glucose transport appears unidirectional even though it can be accounted for by diffusion only over the accessible range of sugar concentrations.

Synthesis and applicability of a photolabile 7,7-azi analogue of 3-sulfated taurine-conjugated bile salts.

For the identification of proteins involved in hepatobiliary transport, the photolabile derivative 7,7-ASLCT ((7,7-azi-3 alpha-sulfato-5 beta-cholan-24-oyl)-2'-aminoethanesulfonate) was synthesized. 7,7-ASLCT is taken up into liver and excreted into bile completely unmetabolized at a rate between the excretion rate of SLCT ((3 alpha-sulfato-5 beta-cholan-24-oyl)-2'- aminoethanesulfonate) and SCCT ((7 alpha-hydroxy-3 alpha-sulfato-5 beta- cholan-24-oyl)-2'-aminoethanesulfonate). The dependency of flux rate of uptake into freshly isolated hepatocytes on the concentration of 7,7-ASLCT in presence of Na+ (143 mM) and with Na+ depletion (1 mM) is best described by the assumption of two simple transport systems, the kinetic parameters of which are similar to those of SLCT. As studied in the presence of Na+, 7,7-ASLCT and SLCT exhibit a clearly competitive cross-inhibition of uptake with inhibition constants that are similar to the corresponding half-saturation constants. Photoaffinity labeling of isolated hepatocytes using 7,7-ASLCT (400 microM) resulted in the irreversible inhibition of the uptake of 7,7-ASLCT and SLCT to similar extents, confirming the kinetic data that 7,7-ASLCT is a true competing substrate for the uptake of SLCT. Because in intact rat liver 7,7-ASLCT and SLCT mutually inhibit their biliary excretion, the photolabile derivative shares with SLCT the same pathways in uptake and in excretion. Thus, 7,7-ASLCT should be appropriate for the study of hepatobiliary transport of sulfated and taurine-conjugated bile salts by photoaffinity labeling.