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R S Ochs

Publications and source records attributed to R S Ochs.

At least 19 recordsLinked to original sources
PubMed1999

Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation.

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) release in muscle. The site of intracellular Ca2+ release, the ryanodine receptor (RyR), is established as the route of Ca2+ movement from the sarcoplasmic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetrically in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryanodine), during differentiation in vitro. The undifferentiated L6 cells showed little response to caffeine. However, a substantial caffeine-induced calcium release (caffCR) was evident by Day 3 of differentiation, and was nearly maximal by Day 7 of differentiation. By contrast, ryanodine failed to stimulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryanodine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrations of ryanodine, known to inhibit Ca2+ release, only began to affect caffCR at Day 4, indicating that cells were insensitive to both ryanodine stimulation and ryanodine inhibition prior to this time. Most of the results could be obtained both in permeabilized and intact cells. Using intact cells, we measured the time course of K+ -dependent (i.e., depolarization-induced) Ca2+ release. This time course matched caffeine and not ryanodine-induced Ca2+ release suggesting the action of caffeine was not due to Ca2+ release unrelated to excitation-contraction coupling. These findings suggest that ryanodine binding sites on the RyR may not be functional at early stages of muscle development, that ryanodine sensitivity is a poor indicator of Ca2+ flux through the RyR, or that other proteins are involved in Ca2+ release under certain circumstances.

Animals↗
PubMed1998

Control of calcium in skeletal muscle excitation-contraction coupling: implications for malignant hyperthermia.

The missing link in our understanding of excitation-contraction coupling (ECC) in skeletal muscle is the mechanism by which Ca2+ increases in the cytosol to trigger contraction. We discuss here a general background of intracellular Ca2+ handling, some characteristics of the major proteins involved in Ca2+ flow during ECC, and mechanisms currently believed to explain the increase in Ca2+ upon stimulation of muscle cells. These mechanisms include the calcium-induced calcium release, the direct coupled mechanism in which a plasma membrane and sarcoplasmic reticulum membrane protein interact, and mechanisms involving Ca2+ secretagogues that are known to elicit increases in calcium in other cells, inositol trisphosphate, and cyclic ADP ribose. We also consider possible roles for proteins associated with the principal calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor. Finally, we discuss malignant hyperthermia, a disease associated directly with aberrant control of muscle cell calcium release.

Adenosine Diphosphate Ribose↗
PubMed1997

Calcium mediated proteolysis enhances calcium release in skinned L6 myotubes.

The mechanism for calcium (Ca2+) release in heart and skeletal muscle during excitation-contraction coupling is currently unknown. A widely held hypothesis is that a small amount of Ca2+ enters the cell and elicits a larger intracellular release of Ca2+ from the sarcoplasmic reticulum (SR), termed "Ca2+-induced Ca2+-release" (CICR). In addition to its role in excitation-contraction coupling, Ca2+ is also known to activate the cysteine protease calpain, which has been recently found to specifically cleave the ryanodine receptor in vitro. The authors investigated the question of whether Ca2+ sensitive protease activation could account for an apparent CICR. The authors first reproduced the phenomenon of CICR using detergent treated L6 myotubes ("skinned cells"). Leupeptin, a cysteine protease inhibitor, reduced the initial velocity and extent of Ca2+ release from the SR; a similar result was obtained when skinned cells were treated with iodoacetate, a sulfhydryl alkylating agent. Dithiothreitol enhanced both the rate and extent of Ca2+ release. Caffeine-induced Ca2+-release was unaffected by the thiol protease inhibitors or activators. This suggests that a cysteine protease may be responsible, in part, for CICR in vitro. The authors also found that vesicles exposed to Ca2+ to induce CICR were unable to fully reaccumulate Ca2+ a second time. Yet, when caffeine released comparable amounts of Ca2+, the initial Ca2+ level was fully restored. Similarly, leupeptin protected the vesicles from the reaccumulation deficit induced by Ca2+. The authors' findings suggest that proteolysis activated by a Ca2+-sensitive protease may account for the direct in vitro demonstration of CICR; such an effect may more likely reflect a role in apoptosis than excitation-contraction coupling.

Animals↗
PubMed1992

Effects of EGF on the mass of inositol 1,4,5-trisphosphate and SN(1,2)-diacylglycerol in freshly isolated rat hepatocytes: comparison with vasopressin.

We measured the masses of inositol 1,4,5 trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG) in hepatocytes in response to both epidermal growth factor (EGF) and vasopressin. EGF at 25 nM did not alter Ins(1,4,5)P3 content of hepatocytes. However, the combination of 100 nM EGF concentration and incubation with lithium did increase Ins(1,4,5)P3 content. This increase was only one tenth of that elicited by vasopressin in parallel incubations. This finding resolves a controversy concerning the ability of EGF to increase Ins(1,4,5)P3 in hepatocytes, and argues against a role for phosphoinositide hydrolysis in EGF action in hepatocytes. Both EGF and vasopressin caused a rapid (30 s) increase in DAG content. A delayed increase in DAG content, that was maximal after several minutes, was observed only for vasopressin. The rapid increase in DAG content implies an activation of protein kinase C for both EGF and vasopressin.

Animals↗
PubMed1991

Vasopressin stimulates pyruvate utilization through a Ca(2+)-dependent mechanism and lactate formation by a protein kinase C-dependent mechanism in isolated rat hepatocytes.

Vasopressin stimulates lactate production by hepatocytes from fed rats, an effect which has been attributed exclusively to Ca2+ activation of glycogenolysis. We provide evidence here for two further actions of vasopressin which affect lactate formation by rat hepatocytes. In the presence of 50 mM glucose, vasopressin inhibited lactate production by hepatocytes. The inhibition was relieved by the presence of alpha-cyano-4-hydroxycinnamate (alpha-CHC), which blocks mitochondrial pyruvate transport. This suggests that vasopressin stimulates pyruvate utilization in the presence of a high concentration of glucose. Epidermal growth factor (EGF), which also increases lactate formation by hepatocytes, did not similarly decrease lactate accumulation in the presence of high glucose, suggesting no stimulation of lactate and pyruvate utilization by this hormone. In cells depleted of Ca2+, vasopressin also stimulated lactate formation. Although vasopressin did not cause the apparent translocation of protein kinase C between cell spaces, phospholipase C treatment of hepatocytes did duplicate vasopressin stimulation of lactate formation, provided fatty acid oxidation was suppressed by the simultaneous presence of the inhibitor palmixorate. We conclude that three actions of vasopressin affect lactate and pyruvate formation: the calcium-linked activations of glycogenolysis and mitochondrial pyruvate utilization, and a stimulation of glycolysis likely mediated by protein kinase C.

Adenosine Triphosphate↗
PubMed1991

A radiochemical enzymatic endpoint assay for GTP and GDP.

We present here a radiochemical enzymatic endpoint assay for the guanine nucleotides GTP and GDP that is suitable for use with cell extracts. The major coupling enzyme used is phosphoenolpyruvate carboxykinase purified from chicken liver. The ancillary coupling enzyme, aspartate aminotransferase, was used to generate a low steady-state concentration of oxalacetate. GTP was determined by the overall conversion of [U-14C]aspartate into [14C]phosphoenolpyruvate. This reaction was also scaled-up as a preparative method for [U-14C]phosphoenolpyruvate. This was used with the same coupling enzymes in reverse to measure GDP by the formation of [14C]aspartate. The assay method was applied to isolated rat hepatocytes. The total GTP and GDP concentrations found were within the range reported by others for rat liver. The advantages of this assay are its sensitivity, specificity, and applicability to large numbers of samples.

Animals↗
PubMed1991

A computerized metabolic map.

We have developed a computer representation of metabolic pathways that incorporates metabolic reactive spaces, species distinctions, and reversibility considerations. Furthermore, the distinction is made between pathway substrates and cosubstrates. Reactions are represented as entries in a database; the code was developed under dBASE III+. Two views onto the database have been developed: a spanning tree which performs a depth search and a stepwise navigational approach which offers reaction possibilities one at a time. In addition, metabolic pathways of any desired starting point are output as a Warnier diagram.

Databases, Bibliographic↗
PubMed1990

Epidermal growth factor and 12-O-tetradecanoylphorbol 13-acetate stimulate lactate production and the pentose phosphate pathway in freshly isolated rat hepatocytes.

Epidermal growth factor (EGF) and tetradecanoylphorbol acetate (TPA) rapidly stimulated the production of lactate by hepatocytes isolated from fed rats. Our results indicate that enzymes of both glycolysis and the pentose phosphate pathway are involved in these actions. EGF stimulated CO2 release from the 1-position of glucose, and caused a small but significant increase in pyruvate kinase activity. In addition, EGF caused a rise in fructose 1,6-bisphosphate and fructose 2,6-bisphosphate concentrations, indicating activation of phosphofructokinase. TPA did not alter the concentrations of these sugar phosphates, but did cause an increased lactate production and CO2 production from the 1-position of glucose similar to EGF. Furthermore, the EGF stimulation of lactate formation was independent of the presence of medium Ca2+. Phenylephrine stimulation of this process, in parallel incubations, was entirely dependent upon the presence of Ca2+ in the medium. We conclude that EGF stimulates glycolysis and the pentose phosphate pathway in isolated hepatocytes from fed rats. The duplication of these actions by TPA suggests that protein kinase C is a mediator of EGF action in hepatocytes.

Animals↗
PubMed1989

Mechanism for the inhibitory and stimulatory actions of proteins on the activity of phospholipase A2.

The influence of proteins on phospholipase A2 was found to depend strongly on the enzyme assay system. We have used three different systems to measure phospholipase A2 which represent the different assay conditions used by a number of previous investigators. Two distinct stimulatory and two distinct inhibitory effects of proteins were observed. (1) A number of proteins - such as albumin, gamma-globulin and lysozyme - were found to inhibit phospholipase A2 activity only at very low substrate concentrations. This 'substrate depletion' was recently proposed as the mode of action for lipocortin. We therefore suggest that substrate depletion is not sufficiently specific to serve as a physiological regulatory mechanism and that the observed inhibition by lipocortin and other proteins more recently reported to mimic it are unlikely to be of physiological significance. (2) Use of liposomes at higher concentrations led to a nonlinear time-course. In this assay system, albumin (and other protein) stimulation can be accounted for as relief of product inhibition. (3) With high concentrations of phospholipids in the presence of cholate (mixed micelles), the behavior of proteins in the assay was complex. The assay time-course appeared linear in the absence of added protein, but at concentrations of added albumin up to 1 mg/ml, stimulation of phospholipase A2 activity was observed. Concentrations greater than this led to diminution of enzyme activity to the original activity. No effect whatever was observed when lysozyme was substituted for albumin. Since this biphasic result was not observed with liposomes, we suggest that the product whose inhibition is being relieved is the lysophosphatidylcholine, and not the free fatty acid. The inhibitory effect at high albumin concentrations is probably the result of removal of free fatty acids from the micelle: fatty acids are known to cause stimulation of phospholipase A2 by providing a negative charge to the lipid/water interface. (4) A different type of phospholipase A2 stimulation was apparent with melittin. This was found to be more specific than generally believed: we found no melittin stimulation of pancreatic phospholipase A2, yet confirmed a several-fold stimulation of bee venom phospholipase A2. We also found that high (millimolar) concentrations of calcium suppressed the melittin stimulation of bee venom phospholipase A2, and that a cationic detergent mimicked the stimulation by melittin. (5) We conclude that the effects of proteins on phospholipase A2 studied here can all be explained by proteins binding to substrate or product rather than enzyme-protein interactions.

Animals↗
PubMed1986

Mechanism for the oleate stimulation of gluconeogenesis from dihydroxyacetone by hepatocytes from fasted rats.

Oleate stimulates glucose production and concomitantly decreases lactate and pyruvate production by rat hepatocyte suspensions incubated with dihydroxyacetone as substrate. The actions of oleate could be blocked by D-(+)dodecanoylcarnitine, which inhibits transport of the fatty acid into the mitochondria and the subsequent oxidation. beta-Hydroxybutyrate, but not acetoacetate, also stimulated glucose synthesis and inhibited lactate and pyruvate production. Furthermore, both beta-hydroxybutyrate and oleate stimulated oxygen consumption to the same extent. This suggests that oleate stimulates glucose production by the provision of energy subsequent to mitochondrial beta-oxidation of the fatty acids. The content of ATP itself did not appear to be responsible for the effects of oleate. Crossover analysis of the gluconeogenic intermediates implicated a site of oleate action between fructose 1,6-bisphosphate and fructose 6-phosphate, suggesting phosphofructokinase and/or fructose-bisphosphatase as possible regulatory sites. Coupled with the finding that intracellular citrate accumulates upon addition of oleate or beta-hydroxybutyrate, but not acetoacetate, the results suggest that citrate inhibition of phosphofructokinase accounts for the redirection of carbon flow from lactate and pyruvate formation and towards that of glucose.

Animals↗
PubMed1986

The fatty acid synthase gene in avian liver. Two mRNAs are expressed and regulated in parallel by feeding, primarily at the level of transcription.

The rates of synthesis of fatty acid synthase and the levels of its mRNA are high in livers of chicks, ducklings, or goslings fed high-carbohydrate mash diets and low in livers of starved birds, indicating pretranslational regulation of fatty acid synthase activity. Determination of the step(s) at which the nutritional state regulates the fatty acid synthase mRNA level was the objective of this study. Total RNA extracted from gosling or duckling liver contains two discrete fatty acid synthase transcripts, one of about 12,200 nucleotides and the other about 10,800 nucleotides. Both mRNAs are transcribed from the same gene because there is only one fatty acid synthase gene/haploid genome. A combination of 1) comparison of restriction fragment lengths in genomic DNA and cloned fatty acid synthase cDNAs, 2) differential hybridization of cloned cDNAs to the two mRNAs, and 3) sequence analysis indicates that the longer mRNA is a 3'-extension of the shorter one. The half-lives for fatty acid synthase mRNAs in fed ducklings and in starved ducklings were estimated from the rate at which mRNA level approached steady state during starvation or refeeding. The amount of fatty acid synthase mRNA in total liver RNA increased rapidly when starved ducklings were fed a high-carbohydrate mash diet, reaching an apparent steady state of 10 times the initial level after 9 h. The kinetics of accumulation suggested a half-life of 4-6 h for fatty acid synthase mRNA in fed ducklings. When fed ducklings were starved, fatty acid synthase mRNA decayed with a half-life of about 3 h. Therefore, the half-life for fatty acid synthase mRNA appeared to be little affected by feeding or starvation. The levels of both mRNAs changed in parallel indicating that half-lives of the two mRNAs were not regulated differentially. Transcription of the fatty acid synthase gene, as measured in isolated nuclei, increased about 10-fold when starved ducklings were refed for 24-30 h. Most of the increase in transcription occurred within 45 min after feeding was initiated. However, when fed ducklings were starved, the initial decrease in fatty acid synthase mRNA level occurred more rapidly than the decrease in transcription of the fatty acid synthase gene, indicating some degree of post-transcriptional regulation. Nevertheless, after 48 h of starvation, both mRNA level and transcription were decreased to the same extent. Nutritional state, therefore, regulates the transcription of two fatty acid synthase mRNAs from a unique gene. In addition, transient regulation occurs at an as yet undefined post-transcriptional step.

Animals↗
PubMed1984

Glutamine metabolism of isolated rat hepatocytes. Evidence for catecholamine activation of alpha-ketoglutarate dehydrogenase.

Effects of norepinephrine on gluconeogenesis and ureogenesis from glutamine by hepatocytes from fasted rats were assessed. Comparisons were made to asparagine metabolism and to the effects of NH4Cl and dibutyryl cyclic AMP. With asparagine as substrate, aspartate content was very high but norepinephrine, dibutyryl cyclic AMP, or NH4Cl had little effect on gluconeogenesis or ureogenesis. Metabolism of asparagine could be greatly enhanced by the combination of oleate, ornithine, and NH4Cl. However, even under these conditions, asparatate content remained high, and norepinephrine and dibutyryl cyclic AMP had little influence on glucose or urea synthesis. With glutamine as substrate, aspartate content was much lower, but was greatly elevated by norepinephrine, dibutyryl cyclic AMP, or NH4Cl. Each of these effectors strongly stimulated glucose and urea formation from glutamine. NH4Cl stimulation was accompanied by an increased glutamate and decreased alpha-ketoglutarate content. This suggests the mechanism for NH4Cl stimulation is a near-equilibrium adjustment to ammonia by glutamate dehydrogenase and aspartate aminotransferase rather than a principal involvement of glutaminase. Although both norepinephrine and dibutyryl cyclic AMP lowered alpha-ketoglutarate to the same extent, norepinephrine more rapidly increased aspartate content and led to a smaller accumulation of glutamate than did dibutyryl cyclic AMP. Moreover, only norepinephrine led to a rapid increase in succinyl-CoA concentration. The catecholamine effect could not be explained by specific changes in cytosolic or mitochondrial redox states. The results suggest that alpha-ketoglutarate dehydrogenase is a site of catecholamine action in rat liver. Since purified alpha-ketoglutarate dehydrogenase is known to be Ca2+ stimulated and Ca2+ flux is involved in catecholamine action, these findings also suggest that mitochondrial Ca2+ is elevated by catecholamines.

Acyl Coenzyme A↗
PubMed1983

Catecholamine stimulation of hepatic gluconeogenesis at the site between pyruvate and phosphoenolpyruvate.

Phosphoenolpyruvate carboxykinase has been implicated by Rognstad (Rognstad, R. (1979) J. Biol. Chem. 254, 1875-1878) as the rate-limiting step for gluconeogenesis from lactate on the basis of a linear Dixon plot (reciprocal rate of gluconeogenesis versus concentration of inhibitor, mercaptopicolinate). We have confirmed this result with isolated hepatocytes incubated in the absence, but not the presence, of bovine serum albumin. Nonlinear plots are likely the result of mercaptopicolinate binding to the albumin. Both norepinephrine and dibutyryl cyclic AMP decreased the slopes and intercepts of the Dixon plots, but a linear relationship was still obtained. When aminooxyacetate inhibited transaminase reactions sufficiently to depress gluconeogenesis, the resulting mercaptopicolinate inhibition plot was still linear in the presence or absence of norepinephrine. Thus, linearity in the Dixon plot does not assure that the enzyme at the site of inhibition is the rate-limiting step for a pathway. Flux through phosphoenolpyruvate carboxykinase does not appear to be hormonally regulated by changes in oxalacetate concentration since this compound was unchanged by norepinephrine or dibutyryl cyclic AMP. Ca2+ enhanced norepinephrine stimulation of gluconeogenesis from asparagine and glutamine and of ureogenesis from glutamine, indicating both mitochondrial and cytosolic sites of action for this hormone. The effects of catecholamines and cyclic AMP were most clearly distinguished by their influence on glutamate concentration when glutamine was the substrate. Dibutyryl cyclic AMP increased, but norepinephrine decreased glutamate. It is possible that decreased glutamate concentration is a reflection of a catecholamine-directed oxidation of mitochondrial NADPH.

Aminooxyacetic Acid↗
PubMed1980

Aminooxyacetate inhibits gluconeogenesis by isolated chicken hepatocytes.

Although the pathway for glucose synthesis from lactate in avian liver is not thought to involve transamination steps, inhibitors of transamination (aminooxyacetate and L-2-amino-4-methoxy-trans-3-butenoic acid) block lactate gluconeogenesis by isolated chicken hepatocytes. Inhibition of glucose synthesis from lactate by aminooxyacetate is accompanied by a large increase in the lactate-to-pyruvate ratio. Oleate largely relieves inhibition by aminooxyacetate and lowers the lactate-to-pyruvate ratio. In parallel studies with rat hepatocytes, oleate did not overcome aminooxyacetate inhibition of glucose synthesis. The ratios of lactate used to glucose formed were greater than 2 with both rat and chicken hepatocytes, were increased by aminooxyacetate, and were restored toward 2 by oleate. Thus, in the absence of oleate, lactate is oxidized to provide the energy needed to meet the metabolic demand of chicken hepatocytes. Excess cytosolic reducing equivalents generated by the oxidation of lactate to pyruvate are transferred from the cytosol to the mitosol by the malate-aspartate shuttle. Aminooxyacetate inhibits the shuttle and, consequently, glucose synthesis for want of pyruvate.

Acetates↗
PubMed1980

Glucagon and N6,O2'-dibutyryl adenosine 3':5'-monophosphate inhibition of lipogenesis and phosphofructokinase activity of hepatocytes from meal-fed rats.

Glucagon and N6,O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) inhibit net glucose utilization, lactate plus pyruvate accumulation and fatty acid synthesis by isolated hepatocytes prepared from meal-fed rats. A crossover in the metabolite profile of the glycolytic intermediates occurs between fructose-6-phosphate and fructose-1,6-bisphosphate, suggesting either inhibition of phosphofructokinase or activation of fructose diphosphatase, or both. Direct assay of the enzymes in cell-free extracts of the hepatocytes indicates that dibutyryl cyclic AMP inhibits phosphofructokinase but has no effect upon fructose diphosphatase. The assay for phosphofructokinase was modified by the use of ITP in place of ATP for the phosphate donor as the ATP-linked assay is complicated by an apparent time-dependent activation of the enzyme. These findings strongly suggest that cyclic AMP inhibition of phosphofructokinase explains in part cyclic AMP inhibition of aerobic glycolysis and lipogenesis by rat liver hepatocytes.

Adenosine Triphosphate↗