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R L Tannen

Publications and source records attributed to R L Tannen.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1989

Rocking promotes differentiated properties in LLC-PK cells by improved oxygenation.

Previous studies from our laboratory indicated that in contrast to cells cultured under still conditions, LLC-PK cells cultured under conditions of continuous rocking exhibit pH-modulated ammonia production and also manifest lower levels of lactate dehydrogenase activity. In the present studies, we assessed whether other metabolic features and parameters of differentiation are modified by rocking. Rocked, as contrasted with still, cultures exhibited decreased medium lactate production and increased cellular ATP content consistent with heightened oxidative metabolism. Still cultures did not exhibit either dome formation or sodium-dependent alpha-methylglucoside uptake until the cultures reached full confluency. By contrast, rocked cultures underwent dome formation as well as enhanced alpha-methylglucoside uptake during the growth phase, and these parameters were quantitatively greater than in still culture even after full confluency was achieved. Thus rocking promotes several functions, which can reflect differentiation in LLC-PK cells. To determine whether provision of adequate oxygen accounts for the enhancement of cellular metabolism and differentiated functions by rocking, still cultures grown in a high oxygen concentration of 36% were compared with standard cultures grown in 18% oxygen. The high oxygen environment resulted in the development of pH-modulated ammonia production, a decrease in lactate dehydrogenase activity, lower medium lactate formation, increased ATP levels, more copious dome formation, and increased alpha-methylglucoside uptake. Thus rocking promotes cellular metabolism and differentiated functions by the provision of adequate oxygenation.

Adenosine Triphosphate↗
PubMed1989

Pathways and regulation of ammoniagenesis by the LLC-PK1 cells in culture.

LLC-PK1 kidney epithelial cells grown under the condition of continuous rocking exhibit a variety of differentiated functions of proximal tubular epithelium, including pH-modulated ammoniagenesis. To further determine their value as a model system, we investigated the pathways of ammoniagenesis under both normal conditions and acid-base manipulations. Pulse-chase studies with carbon 14-labeled glutamine demonstrated a marked delay in glutamine conversion to glutamate, indicating that glutamine deamidation is a critical rate-limiting step, and also provided evidence for metabolism of the glutamine carbon skeleton by the tricarboxylic acid cycle. Ammonia and alanine were the predominant nitrogen metabolites of glutamine at all pH conditions, and the stoichiometry suggested that glutamate is metabolized through both glutamate dehydrogenase and glutamate transaminase at pH 7.4. Increased ammonia production in response to a low pH was associated with increased flux through phosphate-dependent glutaminase and the glutamate transamination pathway and was accompanied by a fall in intracellular glutamate and alpha-ketoglutarate concentrations, which was similar to events in the intact kidney. Studies with the inhibitors acivicin and amino oxyacetate suggested that the gamma-glutamyltranspeptidase and glutamine transamination pathways are inconsequential in LLC-PK1 cells. The phosphate-dependent glutaminase pathway appears to play a predominant role in the regulation of ammoniagenesis. The similarity in ammonia metabolism with other in vitro and in vivo models suggests that LLC-PK1 cells will be a useful system for investigating renal ammoniagenesis and the intracellular signals that modulate this process.

Alanine↗
PubMed1987

Effect of potassium on renal acidification and acid-base homeostasis.

In summary, altered potassium homeostasis can modify bicarbonate reabsorption by the proximal tubule and alter acidification by the distal nephron both by affecting proton transport in a primary fashion and by modifying renal ammonia production. With physiologic variations in dietary K+ intake, these processes may serve to sustain concurrent potassium and acid-base homeostasis. However, with pathologic degrees of potassium depletion and hyperkalemia, metabolic alkalosis and acidosis, respectively, can result.

Acid-Base Equilibrium↗
PubMed1987

Renal excretion of divalent ions in response to chronic acidosis: evidence that systemic pH is not the controlling variable.

Although metabolic acidosis produces calciuric, phosphaturic, and magnesiuric effects, the consequences of chronic respiratory acidosis are unclear. To examine the role of systemic pH on renal divalent metabolism, 4-day balance studies were performed in rats with both metabolic acidosis induced by adding 1.5% NH4Cl to the drinking water, and respiratory acidosis produced by exposure to 10% atmospheric CO2 in an environmental chamber, and in controls pair-fed with each group. By the fourth day, blood pH had decreased to an identical degree with both chronic metabolic and respiratory acidosis and averaged 7.28. As anticipated, chronic metabolic acidosis resulted in significant calciuria, magnesiuria, and phosphaturia. However, despite the similar decrement in blood pH, calcium, phosphorus, and magnesium excretion was similar to that in the pair-fed controls with chronic respiratory acidosis. These findings indicate that a low systemic pH, per se, does not account for the modifications in urinary divalent ion handling that accompany chronic metabolic acidosis. However, additional observations suggest that differences in the intracellular pH of the proximal tubular epithelium may be an important regulatory variable.

Acidosis↗
PubMed1986

The clinical spectrum of renal tubular acidosis.

Renal tubular acidosis (RTA) is associated with a large group of disorders that interfere with normal tubular hydrogen-ion handling by any of several mechanisms. This review presents an approach to the pathophysiology, diagnosis, and therapy of RTA by defining three major subgroups: proximal RTA, distal RTA presenting with normal or decreased serum potassium (hypokalemic distal RTA), and distal RTA presenting with increased serum potassium (hyperkalemic distal RTA).

Acidosis, Renal Tubular↗
PubMed1986

Induction of mitochondrial metabolism and pH-modulated ammoniagenesis by rocking LLC-PK1 cells.

In an effort to find a model system in which the regulation of renal ammoniagenesis could be delineated, the LLC-PK1 line of cultured pig kidney epithelial cells was examined. Ammonia production by normally cultured LLC-PK1 cells (monolayers on 75-cm2 flasks, under 6 ml still serum-containing medium) was found to be neither modulated by direct (3 h) nor adapted by chronic (3 day) manipulation of medium pH (production at pH 7.05, 7.40, or 7.60 not significantly different). Considering that the mitochondrial glutamine to alpha-ketoglutarate pathway is the major regulatory site of ammoniagenesis, and that mitochondrial metabolism might be restricted under the normal lactate-generating or hypoxic culture conditions, we examined ammonia production by rocked flasks of LLC-PK1 cells. Flask were rocked continually at a rate of 2.5 oscillations/min, thereby exposing the cells to the atmosphere 40-50% of the time and also maximizing medium O2 tension and nutrient-waste exchange. Mitochondrial enzyme activities in rocked LLC-PK1 cells were shown to be consistently 1.5-fold greater than those in normally cultured cells. Ammonia production by rocked cells was not only directly modulated by short incubations with low and high pH media (production at pH 7.05 greater than 7.40 greater than 7.60) but was adapted by chronic (3 day) manipulations of medium pH (16 h after return to pH 7.40 medium production by pH 7.05- greater than 7.40- greater than 7.60-adapted cells). Thus rocker culture of the LLC-PK1 cell line both induces mitochondrial metabolism and converts cellular ammoniagenesis to a pH-sensitive phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗