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R L Tannen

Publications and source records attributed to R L Tannen.

At least 19 recordsLinked to original sources
PubMed1995

Prostaglandin F2 alpha- and 12-O-tetradecanoylphorbol-13-acetate-induced alterations in the pathways of renal ammoniagenesis.

The mechanisms whereby prostaglandin F2 alpha (PGF2 alpha) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit ammoniagenesis and the reason why they behave differently at pH 7.4, were examined with (15N)glutamine to assess the metabolic pathways and 2'-7'-bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescein, acetoxymethylester (BCECF-AM) to evaluate Na+/H+ antiporter activity. LLC-PK1 cultures were incubated for 1 h in a Krebs-Hensleit bicarbonate buffer of pH 7.4 and pH 6.8 supplemented either with 5-15N- or 2-15N-labeled glutamine, followed by the assessment of (15N)ammonia and (15N)amino acid formation. Exposure of cells to either PGF2 alpha or TPA completely inhibited the low pH-induced increases in (15N)ammonia formation from incubations with 5-15N, reflecting reduced flux through the mitochondrial phosphate-dependent glutaminase, and from (2-15N)glutamine, reflecting reduced flux through the mitochondrial glutamate dehydrogenase pathway. They also qualitatively reversed the acute acidosis-induced changes in (15N)alanine formation and (15N)glutamate accumulation in the media. By contrast only TPA, but not PGF2 alpha, modified glutamine metabolism at pH 7.4. Na+/H+ antiporter activity was assessed under both acidified and basal (pH 7.4) conditions by measuring changes in intracellular pH in cells loaded with BCECF. TPA and PGF2 alpha both stimulated Na+/H+ antiporter activity comparably under acidified conditions. When cells were studied at pH 7.4, TPA but not PGF2 alpha stimulated the Na+/H+ antiporter and increased steady-state intracellular pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1994

Chronic hypoxia impairs the differentiation of 3T3-L1 fibroblast in culture: role of sustained protein kinase C activation.

The effect of hypoxia on 3T3-L1 cell differentiation was examined in confluent cultures incubated with differentiation medium (DM) followed by incubation in growth medium (GM). Control cultures remained in GM throughout the incubation period. Eight days after the incubation, cells were assessed either for changes in morphology by staining with Oil Red O/hematoxylin or harvested to measure protein kinase C activity. Morphological examination of stained cells showed almost complete differentiation of normoxic cells to adipocytes when exposed to DM. By contrast hypoxia caused a dramatic inhibition of differentiation under similar media conditions with only 34 +/- 4% of cells accumulating fat deposits. Cultures sustained in GM under normoxic or hypoxic conditions were devoid of any fat deposits, reflecting an undifferentiated phenotype. Normoxic cells exposed to DM exhibited a significantly lower membrane to cytosolic ratio of protein kinase C in comparison with cells maintained in GM, which is consistent with differentiated and undifferentiated phenotypes, respectively. In comparison with normoxic cells incubated in DM, cells exposed to hypoxia under similar media conditions exhibited a significantly higher membrane to cytosolic ratio of protein kinase C, indicating sustained activation of the enzyme. In addition, cells in differentiation medium exposed to hypoxia in the presence of the protein kinase C inhibitors staurosporine or H7 exhibited a significant increase in the number of fat accumulating cells when compared with hypoxic controls. These studies indicate that chronic hypoxia impairs the differentiation of 3T3-L1 cells to adipocytes in association with the sustained activation of protein kinase C, which appears to play a role in mediating this process.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

The intensity of acidosis differentially alters the pathways of ammoniagenesis in LLC-PK1 cells.

Utilizing [5-15N] and [2-15N]-labeled glutamine and gas chromatography mass spectrometry methodology, we examined the pathways of ammoniagenesis under basal and acute acidotic conditions of pH 7.0 and pH 6.8, respectively. LLC-PK1 cultures were incubated for one hour with gentle rocking in a bicarbonate buffer of pH 7.4, pH 7.0, or pH 6.8 supplemented either with [5-15N] or [2-15N] glutamine at 37 degrees C in a 5% CO2/95% air incubator atmosphere. Incubation of cultures with [5-15N] glutamine at pH 7.4 resulted in a substantial amount of 15N ammonia formation which was not significantly altered by incubations at pH 7.0. By contrast, exposure to pH 6.8 significantly increased 15N ammonia formation in comparison with its production at pH 7.0 or 7.4. However, 15N ammonia production from [2-15N] glutamine was significantly stimulated at pH 7.0 and was further increased at pH 6.8. Incubation of the cells with [2-15N] glutamine resulted in a substantially lower amounts of 15N ammonia formation than produced with [5-15N] glutamine. Alanine formation from [2-15N] glutamine increased significantly at pH 7.0; but in contrast to 15N ammonia formation, pH 6.8 had no additional stimulatory effect on 15N alanine formation. Cells incubated with [2-15N] glutamine resulted in a significant decrement in 15N glutamate production at both pH 7.0 and 6.8 when compared with pH 7.4. 15N aspartate formation was unaltered by the changes in media pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1994

PGF2 alpha activation of Na/H antiporter and ammoniagenesis in parent/variant LLC-PK1 cells.

A novel variant of the LLC-PK1 cell line was used to examine directly the mechanism whereby PGF2 alpha and TPA inhibit renal ammoniagenesis. The variant cells, which exhibit a growth pattern and morphology similar to the parent cell line, were isolated by a self selection process utilizing long-term cultures of parent cells maintained under conditions of continuous gentle rocking of the media fluid. Incubation of both parent and variant LLC-PK1 cells for one hour in a glutamine supplemented Krebs-Hensleit media of low pH (pH 6.8) increased ammonia and alanine production in comparison to the basal rates at pH 7.4. The phorbol ester TPA and also PGF2 alpha inhibited the low pH-induced increases in ammonia and alanine formation in parent cells; however, neither TPA nor PGF2 alpha inhibited ammonia or alanine metabolism in variant cells. TPA and PGF2 alpha activated PKC similarly in the parent and variant cells as demonstrated by a significant increase in membrane bound enzyme activity. BCECF labeling of cells indicated that the parent and variant cells possess an amiloride sensitive Na+/H+ antiporter of comparable activity. Exposure of parent cells to PGF2 alpha or TPA resulted in the activation of Na+/H+ antiporter activity. By contrast, neither compound stimulated antiporter activity in variant cells. These studies strongly suggest that PKC mediated activation of the Na+/H+ antiporter accounts for the inhibition of ammonia production produced by both PGF2 alpha and TPA. In addition, this novel variant of LLC-PK1 cells should provide a valuable tool to investigate various normal and pathophysiological functions involving mediation by PKC and/or Na+/H+ antiporter activity.

Alanine↗
PubMed1994

Metabolic substrates alter attachment and differentiated functions of proximal tubule cell culture.

Proximal tubules cultured in vitro gradually lose their differentiated functions. Because standard culture media lacks several substrates important for renal proximal tubule oxidative metabolism, whether a mixture of substrates including butyrate, alanine, and lactate (BAL) would modify growth and/or differentiated function of proximal tubular cells in culture was examined. Tubules cultured in media supplemented with 2 mM butyrate, alanine, and lactate exhibited enhanced attachment but did not exhibit an altered growth rate. Higher levels of phosphoenolpyruvate carboxykinase and leucine-amino peptidase were sustained, although these activities were still diminished in comparison with that in fresh tubules. Sodium-dependent glucose uptake and dome formation--other reflections of epithelial cell differentiated function--also were enhanced. These studies demonstrate that the substrates used to culture proximal tubules can modify both their attachment and their manifestation of differentiated function in culture.

Alanine↗
PubMed1992

Hypoxia-mediated impaired differentiation by LLC-PK1 cells: evidence based on the protein kinase C profile.

We recently reported that mild hypoxia in LLC-PK1 cells, grown in standard fashion under a still layer of overlying medium at 5% CO2/18% O2 environment, result in decreased oxidative metabolism and impaired differentiated functions in comparison to adequately oxygenated cultures maintained either under a higher oxygen (36% O2) environment or conditions of continuous rocking of the media fluid. In the present study, subcellular distribution of a regulatory enzyme protein kinase C (PKC) was examined between hypoxic still and normoxic rocked LLC-PK1 cells. Subconfluent cultures of hypoxic LLC-PK1 cells exhibited significantly lower and predominantly membrane-bound PKC activity in comparison to mostly cytosolic localization of this enzyme in normoxic rocked cells. One hour of exposure of adequately oxygenated-rocked LLC-PK1 cells with the phorbol ester TPA, a dedifferentiating agent that did not effect the cell ATP content, resulted in significant inhibition of dome formation and sodium-dependent glucose transport activity, a partial loss of pH-responsive ammoniagenesis, and almost complete translocation of protein kinase C activity from cytosol to the membrane pool; all of which resembles the behavior of hypoxic still cultured cells. In addition, acute re-oxygenation of hypoxic still cultures by rocking the media fluid for one hour resulted in an increase in cell ATP content to the cellular levels of ATP observed in normoxic rocked cells. However, all the parameters of differentiation were unaffected by re-oxygenation. These studies support the notion that hypoxia can act in some primary fashion, independent of its effects on energy metabolism, to impair cellular differentiation in LLC-PK1 cells. They also raise the possibility that activation of protein kinase C may act as an important mediator in this process.

Ammonia↗
PubMed1992

Signal transduction events whereby PGF2 alpha inhibits the ammoniagenic response to acute acidosis.

Subconfluent cultures of LLC-PK1 cells were incubated for 1 h in Krebs-Henseleit buffer (KHB) of pH 7.4 or 6.8 to investigate the signal transduction events associated with prostaglandin F2 alpha (PGF2 alpha) inhibition of ammonia metabolism. Exposure of these cultures to PGF2 alpha (0.1 ng/ml) inhibited the acute low pH stimulation of ammonia production and to a lesser degree alanine formation in a manner analogous to the response exhibited with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment with an inhibitor of protein kinase C [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, i.e., H-7] or utilization of cultures with downregulated protein kinase C activity abolished the inhibitory response to PGF2 alpha. Exposure to PGF2 alpha for 10 min in KHB of pH 6.8 resulted in an activation of protein kinase C, as demonstrated by a significant increase in membrane-bound enzyme activity. Incubation of the cells with PGF2 alpha in KHB of pH 6.8 also resulted in a significant increase in inositol trisphosphate formation. Treatment of the cultures with verapamil in calcium-containing medium or removal of calcium from the incubating medium resulted in a significant loss of the PGF2 alpha inhibitory response on both ammonia and alanine production. Furthermore, under conditions of calcium-free incubation, PGF2 alpha had no significant effect on protein kinase C activity. Because both PGF2 alpha- and TPA-induced inhibition of ammoniagenic response to acute acidosis was prevented by amiloride, the underlying mechanism may involve protein kinase C-mediated changes in intracellular pH. These results indicate that the activation of protein kinase C plays a key role in mediating PGF2 alpha inhibition of ammoniagenesis.

Acidosis↗
PubMed1991

Pathways of acute pH regulation of ammoniagenesis in LLC-PK1 cells: study with [15N]glutamine.

The present study utilized [15N]glutamine labeled at amide (5-N) and amino (2-N) groups to analyze the metabolic fate of glutamine nitrogen in basal and in acute pH regulation of ammoniagenesis. One-hour incubation of LLC-PK1 cultures in a media of pH 7.4, 7.0, or 7.6 containing either [5-15N]glutamine or [2-15N]glutamine resulted in parallel alterations in glutamine consumption in response to acute acid-base maneuvers. Incubation with [5-15N]glutamine resulted in substantial enrichment and production of ammonia at pH 7.4, which was unaffected by the changes in media pH, and in no significant enrichment of alanine, aspartate, and glutamate. In contrast, significant enrichment and production of 15N-labeled ammonia, alanine, aspartate, and glutamate were detected from cultures incubated with [2-15N]glutamine. Ammonia formation, from incubation with [2-15N]glutamine, was stimulated significantly by a low pH and inhibited by high pH. Alanine production was altered in a fashion similar to ammonia formation, whereas aspartate production was unaltered and glutamate formation significantly decreased by a low pH. Furthermore, a low pH significantly increased the production of alpha-ketoglutaramate in a fashion qualitatively similar to alanine production. In contrast to our prior conclusions based on total metabolite production, these studies indicate that although ammonia formation at pH 7.4 is predominantly generated from the mitochondrial phosphate-dependent glutaminase pathway, the increased ammonia formation in acute acidosis is a result of increased flux through glutamate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1990

Relationship between proliferation and glucose metabolism in primary cultures of rabbit proximal tubules.

Primary cultures of rabbit proximal tubules, which revert to a glycolytic profile as reflected by increased activity of pyruvate kinase (PK) paralleled by increased glucose consumption and lactate formation, were utilized to explore the relationship between glycolytic metabolism and proliferation. Tubules placed in serum-free, hormonally defined Dulbecco's modified Eagle's medium with 5 mM glucose exhibited logarithmic growth beginning on day 3 in culture. The increase in PK activity lagged approximately 1 day behind, suggesting that the reversion to glycolysis is a consequence of rather than a prerequisite for cellular proliferation. Tubules cultured in 0.5 mM as contrasted with 25 mM glucose exhibited heightened proliferation reflected by an increase in protein content and cell number on day 5 in culture. The heightened proliferation was accompanied by increased PK activity. On day 9, after confluency had been achieved, no differences in protein content or PK activity were detected between tubules cultured in different glucose concentrations. These findings indicate that a low glucose concentration is mitogenic for renal proximal tubules and that the proliferative process in some fashion up-regulates the activity of the glycolytic enzyme PK. Furthermore, because accelerated growth proceeds in the presence of glucose restriction, the energy from glycolysis is not required for the proliferative process.

3-O-Methylglucose↗
PubMed1990

Relationship between intracellular pH and ammonia metabolism in LLC-PK1 cells.

Previous studies from our laboratory have confirmed that cultures of LLC-PK1 cells exhibit pH-responsive alterations in ammonia metabolism produced by changes in media bicarbonate concentration. To further elucidate the mechanism of ammonia regulation, studies were carried out using parallel cultures of still and rocked LLC-PK1 cells subjected to acute alterations in media pH by either metabolic or respiratory acid-base manipulations. When media pH was altered by modifying PCO2 levels, the response of ammonia and alanine production by rocked culture was identical to the changes observed with metabolic acid-base maneuvers. Furthermore, both metabolic and respiratory acute acidosis resulted in a fall of intracellular alpha-ketoglutarate concentrations in these cells. In contrast, standard still cultures subjected to acute acidosis/alkalosis by metabolic and respiratory manipulations did not exert any significant change in ammonia and alanine production or in intracellular alpha-ketoglutarate concentration. Measurements of intracellular pH (pHi) by the 5,5-[2-14C]dimethyloxazolidine-2,4-dione method in rocked cells demonstrated changes in pHi parallel to media pH changes induced by both metabolic and respiratory acid-base maneuvers. Despite the absence of pH-responsive ammonia-genesis in still cultured cells the pHi values were altered in a fashion similar to their rocked counterparts, indicating the lack of an effect of the pHi signal on ammonia metabolism.

Acidosis↗
PubMed1990

Prostaglandin F2 alpha inhibits the ammoniagenic response to acute acidosis in LLC-PK1 cells.

A kidney epithelial cell line, LLC-PK1, which does not synthesize prostaglandins, provides an ideal in vitro model system to investigate the effect of prostaglandins in the regulation of renal ammoniagenesis. Previous studies from our laboratory have demonstrated significant increases in glutamine-dependent ammonia and alanine production by rocked cultures of LLC-PK1 cells subjected to either acute metabolic or respiratory acidosis. In the study presented here, experiments were conducted to investigate the role of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) in the response of ammonia metabolism to acute metabolic acidosis by LLC-PK1 cells. A low dose of PGF2 alpha (0.1 ng/mL) dramatically inhibited the stimulatory effect of a low pH (pH 6.8) on ammonia production. In contrast, the inhibition of cytosolically generated alanine was less dramatic and averaged only 20% of the effect on ammonia production. Furthermore, PGF2 alpha increased cellular alpha-ketoglutarate concentration, suggesting an increase in intramitochondrial pH. Thus, the cellular mechanism of PGF2 alpha action appears to involve either interference with the cytosolic pH signal or its translation to the intramitochondrial compartment. The inhibitory response of PGF2 alpha on pH-stimulated ammoniagenesis was progressively lost at higher concentrations. Both low-dose (0.1 ng/mL) and high-dose (10 ng/mL) PGF2 alpha had no significant effect on the basal rates of ammonia and alanine production at pH 7.4. PGE2, on the other hand, did not exhibit any significant response on ammonia or alanine production at either pH 6.8 or 7.4 when given in a wide range of doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1990

Biochemical pathways and modulators of renal ammoniagenesis.

The renal proximal tubule contains a variety of biochemical pathways, which can metabolize glutamine, the major substrate for renal ammoniagenesis. The intramitochondrially located phosphate-dependent glutaminase (PDG) pathway, rather than the various cytosolic pathways, appears to play the predominant role in regulating the rate of renal NH3 production. Acute acidosis stimulates NH3 production by activating alpha-ketoglutarate dehydrogenase and secondarily glutamate dehydrogenase; whereas the adaptation to chronic metabolic acidosis results primarily from enhanced glutamine transport into the mitochondria and possibly increased activity of PDG. There is no adaptation of ammoniagenesis to chronic respiratory acidosis, because the proximal tubular intracellular pH is not decreased. Alkalosis suppresses NH3 formation but the precise mechanism is not clarified. Ammoniagenesis can be modulated independent of acid-base status by a variety of factors including potassium homeostasis, TCA cycle intermediates, hormones which increase cAMP, prostaglandin F2 alpha, insulin, growth hormone, angiotensin II, corticosteroids, aldosterone, and tubular flow rate.

Acidosis, Renal Tubular↗
PubMed1989

Response of ammonia metabolism to acute acidosis: insights from cultured renal epithelium.

LLC-PK1 kidney epithelial cell lines cultured under the condition of continuous rocking exhibit both acute and adaptive changes in glutamine-dependent ammonia metabolism in response to acid-base manipulations in the media pH. Pulse-chase studies with 14C-glutamine as well as studies with various metabolic inhibitors of the ammoniagenic pathways suggest that glutamine in LLC-PK1 cells is metabolized via mitochondrial pathway and that intramitochondrial phosphate-dependent glutaminase pathway plays a predominant role in the regulation of ammoniagenesis to acute acidosis. Furthermore, the measurements of intracellular pH under both basal and acute low pH conditions resulted in the estimation of intracellular pH, which paralleled the alterations in media pH. Therefore, a change in intracellular pH appears to act as a direct signal for alterations in ammonia metabolism in LLC-PK1 cells. Thus, LLC-PK1 cultures provide an excellent model system to investigate renal ammoniagenesis and the intracellular signals that modulate this process.

Acidosis↗
PubMed1989

Carbohydrate metabolism by primary cultures of rabbit proximal tubules.

Renal proximal tubular epithelia were used to assess the factors responsible for the induction of glycolysis in cultured cells. Primary cultures of rabbit proximal tubules, which achieved confluency at 6 days, exhibited hormonal responsiveness and brush-border characteristics typical of proximal tubular cells. Beginning at day 4, these cultured cells exhibited increased glycolytic metabolism reflected by enhanced glucose uptake and lactate production, along with parallel increases in activity of the glycolytic enzymes, pyruvate kinase and lactate dehydrogenase. The gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FDP), were downregulated, and the cultured cells exhibited lower oxygen consumption rates than fresh tubules. Cells grown on a rocker, to mitigate hypoxia, exhibited a metabolic and enzymatic profile similar to cells grown under still conditions. ATP levels in cultured cells were higher than in fresh tubules. Furthermore, pyruvate kinase activity was higher in cells grown in media containing 0.5 as contrasted with 25 mM glucose. The enhanced glycolytic metabolism exhibited by cultured proximal tubular cells appears to be a characteristic of proliferation and is not a response to hypoxia, the Pasteur effect, or environmental glucose.

Animals↗