Tight control of exogenous SERCA expression is required to obtain acceleration of calcium transients with minimal cytotoxic effects in cardiac myocytes.

Collateral effects of exogenous sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA) expression were characterized in neonatal rat and chicken embryo cardiac myocytes, and the conditions required to produce acceleration of Ca(2+) transients with minimal toxicity were established. Cultured myocytes were infected with adenovirus vector carrying the cDNA of wild-type SERCA1, an inactive SERCA1 mutant, or enhanced green fluorescence protein under control of the cytomegalovirus promoter. Controls were exposed to empty virus vector. Each group was tested with and without phenylephrine (PHE) treatment. Under conditions of limited calf-serum exposure, the infected rat myocytes manifested a more rapid increase in size, protein content, and rate of protein synthesis relative to noninfected controls. These changes were not accompanied by reversal to fetal transcriptional pattern (as observed in hypertrophy triggered by PHE) and may be attributable to facilitated exchange with serum factors. SERCA virus titers >5 to 6 plaque-forming units per cell produced overcrowding of ATPase molecules on intracellular membranes, followed by apoptotic death of a significant number of rat but not chicken myocytes. Enhanced green fluorescence protein virus and empty virus also produced cytotoxic effects but at higher titers than SERCA. Expression of exogenous SERCA and enhancement of Ca(2+) transient kinetics could be obtained with minimal cell damage in rat myocytes if the SERCA virus titer were maintained within 1 to 4 plaque-forming units per cell. Expression of endogenous SERCA was unchanged, but expression of exogenous SERCA was higher in myocytes rendered hypertrophic by treatment with PHE than in nontreated controls.

Dolichyl phosphate metabolism in brain. Developmental increase in polyisoprenyl phosphate phosphatase activity.

The enzymatic dephosphorylation of endogenous and exogenous dolichyl phosphate (Dol-P) by membrane preparations from calf brain has been reported previously (Burton, W.A., Scher, M.G., and Waechter, C. J. (1981) Arch. Biochem. Biophys. 208, 409-417). The study presented here shows that polyisoprenyl phosphate phosphatase activity increases with age in crude microsomal fractions from white (glial-enriched) and grey (neuronal-enriched) matter of pig brain. A kinetic analysis of grey matter phosphatase activity revealed that the apparent Km values for Dol-P are similar for membranes from adults (0.13 mM) and prenatal subjects (0.12 mM), but the Vmax is significantly higher in adults. In direct contrast to this result, the specific activity of dolichol kinase is nearly 2-fold higher in membranes from pig brain grey matter of prenatal subjects as compared to identical preparations from animals 90 days of age. While the changes in kinase and phosphatase activity are approximately 2-fold, reciprocal changes in these activities would enhance shifts in the metabolic balance of a phosphorylation-dephosphorylation process. In another aspect of this study on polyisoprenyl phosphate phosphatase in nervous tissue, membrane preparations from calf brain are shown to dephosphorylate S- and R-Dol-P. Similar apparent Km values were obtained for S-Dol-P and R-Dol-P, but the Vmax for the dephosphorylation reaction was found to be 1.8-fold greater for S-Dol-P. These results extend the description of polyisoprenyl phosphate phosphatase activity in brain and provide information on developmental changes in phosphatase and kinase activities involved in Dol-P metabolism in nervous tissue.

Gluconeogenic enzymes in fibroblasts from infants dying of the sudden infant death syndrome (SIDS).

The activities of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC), and total malic enzyme (ME) were determined in cultured fibroblasts from 74 infants diagnosed as having died from the Sudden Infant Death Syndrome (SIDS) and 36 infants who died from known causes. In addition, the glycemic state of infants was measured by determining the 'maximum' glucose present in plasma and cerebral spinal fluid (CSF) based on the following formula: glucose (maximum) = glucose + 1/2 (lactate). There were no statistical differences between SIDS and control infants in any of the three enzymatic activities measured in fibroblasts or in the estimated 'maximum' glucose values in plasma or CSF. The present results do not support the involvement of hypoglycemia as a cause of death in SIDS.

Comparison of the oxidation of glutamine, glucose, ketone bodies and fatty acids by human diploid fibroblasts.

The contribution of glutamine, glucose, ketone bodies and fatty acids to the oxidative energy metabolism of human diploid fibroblasts ws studied. The rate of glutamine oxidation by fibroblasts was 98 nmol/h per mg cell protein compared to 2 nmol/h per mg cell protein or less for glucose, acetoacetate, D-3-hydroxybutyrate, octanoic acid and palmitic acid. Glucose inhibited glutamine oxidation by 85%, while the other substrates had no effect. Therefore, these cells meet their energy requirement almost solely by anaerobic glycolysis and glutamine oxidation.

Effect of glucose on aspartate and glutamate synthesis by human diploid fibroblasts.

The quantity of aspartate, but not of glutamate, synthesized by human diploid fibroblasts (HDF) was inversely proportional to the glucose concentration. Forty hours after confluent cells were refed with media containing 1.5 mM or 55 microM glucose, the aspartate concentration was 8 microM in medium high in glucose and 28 microM in medium low in glucose; glutamate was 350 microM at both glucose levels. The label incorporation from [U-14C] glutamine (0.8 C/ml) into aspartate after 40 hours in 1.5 mM glucose medium was less then 1000 DPM/ml and into glutamate 540,000 DPM/ml. The respective label incorporation into aspartate in 55 microM glucose medium was 118,000 DPM/ml and into glutamate 450,000 DPM/ml. In media with 1.5 mM glucose, label incorporation into both aspartate and glutamate was observed from [6-14C] glucose, [3-14C] pyruvate, and [1-14C] pyruvate. Since [1-14C] pyruvate labeled aspartate in 1.5 mM glucose medium, whereas [U-14C] glutamine did not, the observations support a cystolic pathway of aspartate synthesis from glycolytic intermediates in the presence of glucose. However, when the glucose concentration is decreased, glutamine appears to be the primary carbon source for aspartate synthesis. Therefore, both the quantity and the pathway of aspartate synthesis in HDF are functions of the glucose concentration. It is postulated that increased accumulation of aspartate in the media of HDF at confluency may be explained by decreased availability of acetyl CoA or of NADH.

Activities of enzymes required for the conversion of 4-carbon TCA cycle compounds to 3-carbon glycolytic compounds in human diploid fibroblasts.

Phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase activities were localized in the mitochondrial (particulate) fraction of human diploid fibroblasts (HDF). Malic enzyme, malate dehydrogenase (MDH) and glutamate oxalacetate transaminase (GOT) were distributed between the mitochondrial and cytosolic (soluble) fractions, the latter two enzymes being more equally distributed than malic enzyme, which was largely cytosolic. The specific activities of mitochondrial PEPCK and cytosolic malic enzyme, MDH and GOT increased during cell growth. The results support an active conversion of 4-carbon tricarboxylic acid cycle intermediates to 3-carbon glycolytic intermediates in HDF.

Lactate: a major product of glutamine metabolism by human diploid fibroblasts.

Human diploid fibroblasts metabolize up to 13% of the glutamine in tissue culture medium to lactate. Four microCi of glutamine-U-14C were added to media containing 5 mM or 65 microM glucose or medium containing no added glucose, but supplemented with purine and pyrimidine nucleosides (HGTU). Aliquots of the media were taken at daily intervals and were assayed for glucose, lactate, pyruvate, malate, citrate, aspartate, glutamine, and glutamate. The label incorporation into these compounds was determined, except for glutamine and glucose. The distribution of label from glutamine-U14C in 5 mM glucose medium by day 4 was lactate (10.2%), glutamate (15.2%), citrate (1.9%), pyruvate (2.0%), malate (1.1%), and aspartate (< 0.1%). The accumulation of label in lactate and glutamate occurred continuously during the growth cycle. Malate, citrate, and aspartate accumulation occurred primarily in confluent cultures. The label in aspartate was seen only in stationary phase cells or when the glucose concentration was decreased to 65 microM or less; net aspartate accumulation was increased twofold in low glucose media. These data demonstrate an actively functioning pathway for the conversion of 4-carbon TCA-cycle intermediates to 3-carbon glycolytic intermediates in human diploid fibroblasts.