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A P Koretsky

Publications and source records attributed to A P Koretsky.

At least 73 records · Page 4Linked to original sources
PubMed1992

Investigation of cell physiology in the animal using transgenic technology.

Over the past 10 years significant progress has been made in techniques for manipulating the genome of the animal. Production of transgenic mice has led to important insights into the regulation of gene expression, the molecular basis of cancer, immunology, and developmental biology. The tools necessary to generate transgenic mice are becoming widely available, making it possible to study a variety of problems. In this review a description of the strategies being used to address problems of interest in cell physiology using transgenic mice is given. Elucidation of the rules governing the regulation of gene expression now permits the targeted expression of a protein to a particular organ or cell type within an organ. Overexpression of proteins, expression of foreign or mutant proteins, mislocalization of proteins, and directed elimination of proteins are all procedures that can now be used to generate interesting animal models for physiological studies. The applications of these techniques to a variety of problems in normal and abnormal physiology are discussed in this review.

Animals↗
PubMed1992

The role of magnesium in postischemic cardiac dysfunction.

BACKGROUND: The biochemical basis for postischemic myocardial stunning is not fully elucidated. Magnesium is an important regulator of cellular energetic processes and excitation-contraction coupling. We hypothesized that the decrease in function in the postischemic period may be the result of an alteration in magnesium regulation. METHODS: In a Langendorf perfused rabbit heart model, we used 31P nuclear magnetic resonance spectroscopy to noninvasively determine intracellular Mg2+ and high-energy phosphate levels in the preischemic period and after a 30-minute period of normothermic ischemia. We measured adenosine triphosphate (ATP), phosphocreatine, and the phosphocreatine/inorganic phosphate ratio and calculated the free energy of ATP hydrolysis (delta GATP). On reperfusion, hearts were divided into three groups (n = 7 per group)--those receiving unmodified Krebs-Henseleit (control), 192 ng/ml dobutamine, or 5 mmol/L pyruvate. RESULTS: Function (expressed as the rate-pressure product) was approximately 77% of preischemic values in the control group, whereas in both dobutamine and pyruvate groups it returned to preischemic levels. ATP was decreased similarly in all groups in the postischemic period. Phosphocreatine/inorganic phosphate ratio and delta GATP were higher in the pyruvate group compared with the other groups. Intracellular Mg2+ was elevated significantly in the unmodified control postischemic group compared with preischemic, postischemic dobutamine, and pyruvate groups (1.0 +/- 0.12 vs 0.80 +/- 0.08, 0.64 +/- 0.08, and 0.70 +/- 0.05 mmol/L, respectively; p less than 0.05). CONCLUSIONS: We conclude that (1) postischemic "stunned" hearts have elevated Mg2+ levels in association with impaired contractile function, (2) inotropic agents improve contractile function in association with a decline in Mg2+ to preischemic levels despite differing effects on intracellular energetics, and (3) Mg2+ may play an important regulatory role in the heart after ischemia.

Animals↗
PubMed1991

Phosphocreatine protects ATP from a fructose load in transgenic mouse liver expressing creatine kinase.

The effects of an intraperitoneal dose of fructose on hepatic metabolism in transgenic mice expressing creatine kinase in liver were investigated using phosphorus-31 nuclear magnetic resonance (31P-NMR). Transgenic mice were fed diets containing varying amounts of creatine (Cr; 0-12%). It has previously been shown that 31P-NMR spectra of transgenic mice have a peak due to phosphocreatine (PCr), the intensity of which was proportional to the amount of Cr in the diet. No PCr peak was detected in control mice or transgenic mice not fed Cr. In the present study NMR spectra were collected before and for a 1-h recovery period after infusion of 0.15 mmol/10 g body wt fructose. In all mice infusion of fructose resulted in a two- to threefold elevation of phosphomonoesters. In control and non-Cr-fed transgenic mice this was accompanied by a 60% reduction of the inorganic phosphate (Pi) and a 50% fall in ATP. In transgenic mice fed Cr, the extent of reduction of Pi was dependent on the level of PCr and was markedly reduced compared with controls. Falls in Pi of 46, 24, and 6% were detected 12.5 min after fructose infusion in low, intermediate, and high PCr-containing livers, respectively. The presence of PCr also protected hepatic ATP levels from a fructose load. Transgenic mice fed on high or intermediate Cr diets showed no significant loss of ATP. However, livers with low levels of PCr lost ATP during a fructose challenge. From the equilibrium established by creatine kinase, free ADP levels were calculated throughout the fructose dose. Fructose caused a 2.5-fold increase in free ADP. This rise in ADP was independent of the total Cr or whether Pi and ATP were reduced by fructose infusion. These results indicate that an increase in ADP is not sufficient to cause depletion of ATP during a fructose challenge.

Adenosine Triphosphate↗
PubMed1990

Free ADP levels in transgenic mouse liver expressing creatine kinase. Effects of enzyme activity, phosphagen type, and substrate concentration.

ADP is an important regulator of hepatic metabolism. Despite its importance the level of free ADP in the liver remains controversial. Recently, we engineered transgenic mice which express high levels of creatine kinase in liver. The reaction catalyzed by creatine kinase was assumed to be at equilibrium and used to calculate a free ADP level of 0.059 mumol/g wet weight. In this report we test the equilibrium assumption by studying the free ADP level as a function of enzyme activity or substrate content. Over a 5-fold range of creatine kinase activity, from 150-800 mumol/min/g wet weight, there was no change in the free ADP level. The average value of ADP for these mice was 0.061 +/- 0.016 mumol/g wet weight. Similarly, altering hepatic creatine content from 1.6 to 30 mumol/g wet weight had no effect on the calculated total free ADP level. The average value of ADP for the creatine levels was 0.048 +/- 0.015 mumol/g wet weight. Finally, the free ADP level was calculated using the equilibrium with cyclocreatine rather than creatine as substrate. The equilibrium of the reaction with cyclocreatine lies 30 times more toward phosphorylation than does the equilibrium with creatine. A free ADP level of 0.063 +/- 0.031 mumol/g wet weight was calculated using cyclocreatine. This value is not different from that found with creatine. These results show that the equilibrium assumption used to calculate free ADP levels in transgenic mouse liver is valid, and the presence of creatine kinase does not affect ADP levels.

Adenosine Diphosphate↗
PubMed1990

Measurement of cerebral blood flow in rat brain by 19F-NMR detection of trifluoromethane washout.

The washout of trifluoromethane (CHF3) from rat cerebral cortex was monitored by 19F NMR. After 15 min of inhalation of 67% CHF3/33% O2 the fluorine signal detected was in a steady state. The CHF3 was switched off rapidly at the endotracheal tube and the washout detected with 12-s time resolution. Two models were used to extract flow information, a simple exponential fit and a model which accounts for arterial CHF3 recirculation. In both cases, a two-compartment model fit the data significantly better than a one-compartment model. In both models, the faster time component varied with increasing pCO2, but no significant change in the slow component was detected. At control values of pCO2, there was a small difference in washout rate constants derived from the two models. At high pCO2, when tissue washout was comparable to arterial washout of CHF3, the model which accounted for arterial recirculation gave higher flows. Using this two-compartment model with correction for recirculation, a control flow (pCO2 = 35 mm Hg) of 0.73 +/- 0.04 ml/min/g was measured. Increasing plasma pCO2 increased the apparent flow six- to sevenfold with a 4.4% increase in flow per millimeter of Hg change in CO2. These results are qualitatively in agreement with results found by others using the washout of 133Xe. However, this method yields values for flow that are lower than those obtained using 133Xe washout, probably because of diffusion limitations of CHF3.

Animals↗
PubMed1990

Nuclear magnetic resonance determination of flow, lactate, and phosphate metabolites during amphetamine stimulation of the rat brain.

The effects of amphetamine on rat cerebral blood flow, lactate, and phosphate levels were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Proton NMR imaging was used to determine the region of brain being studied. Blood flow was estimated by detecting the washout of trifluoromethane using 19F NMR. A dose of 20 mg/kg D-amphetamine sulphate stimulated flow fourfold over control values for at least 1 h. Even though amphetamine stimulated a large increase in flow, there were no significant changes in levels of inorganic phosphate, ATP, phosphocreatine, pH or Mg2+ as determined by 31P NMR. This was true for doses of 10 and 20 mg/kg D-amphetamine sulphate. No significant changes occurred in 1H NMR detected levels of lactate with a 20 mg/kg dose of amphetamines. Lactate levels increased slightly 30 min after administration of 10 mg/kg D-amphetamine sulphate. The results call into question the central role of changes in levels of phosphates in coupling increased cortical activity to increases in metabolism.

Amphetamine↗
PubMed1990

Absence of pH changes during altered work in the in vivo sheep heart: a 31P-NMR investigation.

Saturation transfer from gamma-ATP to inorganic phosphate was used to assign the intracellular inorganic phosphate resonance of the phosphorus-31 nuclear magnetic resonance spectrum of heart obtained from adult sheep under Halothane anesthesia. The 31P chemical shift of intracellular inorganic phosphate was then used as a probe of myocardial pH. Resting myocardial pH was found to be 7.03 +/- 0.02. The effects of increasing myocardial work on myocardial pH were examined using external pacing and phenylephrine infusion alone or in combination to produce steady-state increases in the rate-pressure product. No alteration in myocardial pH was observed with up to 4-fold increases in rate-pressure product. No changes in high-energy phosphates were observed except at the highest rate-pressure products obtained, where small increases in inorganic phosphate and decreases in the phosphocreatine/ATP ratio were observed. In addition, the transition to a new steady state was studied with a 20-s time resolution after initiation of pacing. Again, no changes in pH or levels of phosphates were detected during the transition to increased work.

Adenosine Triphosphate↗
PubMed1990

NMR detection of creatine kinase expressed in liver of transgenic mice: determination of free ADP levels.

To use the equilibrium established by creatine kinase (CK) to determine hepatic free ADP levels, the transcriptional control elements of the transthyretin gene were used to direct expression of the CK B isozyme to the livers of transgenic mice. Activities of CK ranging from 80-250 mumol per min per g (wet weight) were detected in liver extracts from five founder mice. The CK activity was stably transmitted to subsequent generations. Isozyme gels and immunoblots confirmed that the activity detected in extracts was due to the B isozyme of CK. Immunohistology indicated that the protein was expressed uniformly throughout the liver and was localized primarily to the cytoplasm. 31P NMR spectroscopy was used to detect the metabolic product of the CK reaction, phosphocreatine, demonstrating that the enzyme was active in vivo. The phosphocreatine level fell rapidly during anoxia (t1/2 = 1 min), indicating that the CK reaction was integrated into hepatic energy metabolism. The equilibrium established by CK was used to calculate a hepatic free ADP level of 0.059 +/- 0.004 mumol/g (wet weight). In vivo NMR studies of these mice will be valuable for studying the role of free ADP in regulating liver metabolism.

Adenosine Diphosphate↗
PubMed1989

The B isozyme of creatine kinase is active as a fusion protein in Escherichia coli: in vivo detection by 31P NMR.

A cDNA encoding the B isozyme of creatine kinase (CKB) has been expressed in Escherichia coli from a fusion with lacZ carried by lambda gt11. Western blots indicate that a stable polypeptide with the appropriate mobility for the beta-galactosidase-creatine kinase (beta-gal-CKB) fusion protein cross-reacts with both beta-gal and CKB antiserum. No significant CK activity is detected in control E. coli; however, extracts from cells containing the lambda gt11-CKB construct have a CK activity of 1.54 +/- 0.07 mumol/min per mg protein. The fusion protein appears to provide this activity because immunoprecipitation of protein with beta-gal antiserum leads to a loss of CK activity from extracts. That the enzyme is active in vivo was demonstrated by detection of a phosphocreatine (PCr) peak in the 31P NMR spectrum from E. coli grown on medium supplemented with creatine. As in mammalian brain and muscle, the PCr peak detected was sensitive to the energy status of the E. coli.

Animals↗
PubMed1988

Activation of dehydrogenase activity and cardiac respiration: a 31P-NMR study.

31P-NMR studies were performed to determine the tissue phosphate and oxygen consumption effects of known maneuvers on the activation of pyruvate dehydrogenase during work jumps in the perfused rat heart. In control studies of the glucose-perfused heart, work jumps, with pacing, resulted in a 32% increase in oxygen consumption (QO2) from 1.72 +/- 0.09 to 2.29 +/- 0.12 mmol O2.h-1.g dry wt-1. During this transition no significant change in the high energy phosphates were detected. In contrast, work jumps did cause changes in the phosphates when the activation of pyruvate dehydrogenase was blocked with 2.5 micrograms of ruthenium red per milliliter or maximally stimulated with 11 mM pyruvate before the increase in work. The observed increase in QO2 and inorganic phosphate and calculated increase in ADP are consistent with these phosphates controlling mitochondrial respiration under these conditions. These results suggest that the activation of pyruvate dehydrogenase and/or other dehydrogenases may be an important step in the orchestration of work and QO2.

Animals↗
PubMed1988

Potassium adaptation: 39K-NMR evidence for intracellular compartmentalization of K+.

To investigate the effects of K+ uptake on the intracellular environment, both 39K-nuclear magnetic resonance (NMR) and K+-selective electrodes were used to measure K+ activity with acute K+ loading in control and K+-adapted rats. These results were then compared with tissue K+, measured by flame photometry. There was a lower NMR K+ visibility (ratio of NMR signal to tissue content) in muscle and liver in K+-adapted rats, compared with controls before and after an acute K+ load. This lower K+ visibility in K+-adapted rats was confirmed in liver homogenate with the K+-specific electrode. In liver homogenates from control and K+-adapted rats, addition of RbCl (300 mumol/g) increased the NMR K+ signal more in K+-adapted rats (19 +/- 1.1 mumol/g) than controls (11 +/- 1.0 mumol/g, P less than 0.01). This is consistent with the displacement of K+, by Rb+, from NMR-undetected sites. These results suggest that some 10-15% of intracellular K+ may be within a compartment not detectable by NMR or electrodes and that chronic K+ loading leads to an increased capacity of this compartment.

Adaptation, Physiological↗
PubMed1987

Changes in pyridine nucleotide levels alter oxygen consumption and extra-mitochondrial phosphates in isolated mitochondria: a 31P-NMR and NAD(P)H fluorescence study.

Isolated rat-liver mitochondria were used to study the relation between mitochondrial NADH levels, oxygen consumption (QO2), and extra-mitochondrial phosphates. Alterations in NADH and QO2 were accomplished by incubating mitochondria with different substrates or varying amounts of exogenous ATPase while monitoring QO2 and NAD(P)H fluorescence. Two sets of conditions were studied: (1) in the presence of excess ADP and inorganic phosphate, an increase in NAD(P)H fluorescence was associated with a linear increase in QO2; (2) when QO2 was driven by the steady-state hydrolysis of ATP by exogenous ATPase, increases in QO2 were associated with proportional decreases in NAD(P)H fluorescence. For all substrates tested this relation was linear; however, the slope was substrate dependent. Different substrates were able to maintain different NAD(P)H levels at the same QO2. To investigate this further, effects of changing substrates at constant QO2 on NAD(P)H and extra-mitochondrial phosphates were determined. Addition of glutamate + malate to mitochondria respiring on citrate caused a 50% increase in NAD(P)H fluorescence, a 41% decrease in ADP, and a 30% decrease in inorganic phosphate. Similar changes for the substrate jump, pyruvate + malate to glutamate + malate were found. Finally, it was determined that a linear relation holds between increases in NAD(P)H fluorescence and increases in QO2 when substrates were varied at constant, physiologic levels of extra-mitochondrial ADP. These results indicate that QO2 depends on NAD(P)H levels as well as on extra-mitochondrial phosphates over a wide range of respiratory rates.

Adenosine Diphosphate↗
PubMed1987

Respiratory control in the glucose perfused heart. A 31P NMR and NADH fluorescence study.

The phosphate metabolites, adenosine diphosphate (ADP), inorganic phosphate (Pi), and adenosine triphosphate (ATP), are potentially important regulators of mitochondrial respiration in vivo. However, previous studies on the heart in vivo and in vitro have not consistently demonstrated an appropriate correlation between the concentration of these phosphate metabolites and moderate changes in work and respiration. Recently, mitochondrial NAD(P)H levels have been proposed as a potential regulator of cardiac respiration during alterations in work output. In order to understand better the mechanism of respiratory control under these conditions, we investigated the relationship between the phosphate metabolites, the NAD(P)H levels, and oxygen consumption (Q02) in the isovolumic perfused rat heart during alterations in work output with pacing. ATP, creatine phosphate (CrP), Pi and intracellular pH were measured using 31P NMR. Mitochondrial NAD(P)H levels were monitored using spectrofluorometric techniques. Utilizing glucose as the sole substrate, an increase in paced heart rate led to an increase in Q02 from 1.73 +/- 0.09 to 2.29 +/- 0.12 mmol Q2/h per g dry wt. No significant changes in the levels of Pi, PCr, ATP, or the calculated ADP levels were detected. Under identical conditions, an increase in heart rate was associated with a 23 + 3% increase in NAD(P)H fluorescence. Thus, under the conditions of these studies, an increase in Q02 was not associated with an increase in ADP or Pi. In contrast, increases in Q02 were associated with an increase in NAD(P)H. These data are consistent with the notion that increases in the mitochondrial NADH redox state regulate steady-state levels of respiration when myocardial work is increased.

Adenosine Diphosphate↗
PubMed1987

Determination of renal molar concentrations of phosphorus-containing metabolites in vivo using 31P NMR.

A technique to determine absolute metabolite concentrations of the kidney in vivo using 31P NMR is described. The technique is based on the use of methylphosphonic acid (MPA), which gives rise to a well-resolved peak upfield from in vivo phosphorous metabolite resonances, as an "internal standard." The method involves acquisition of a fully relaxed kidney spectrum with an implanted coil followed by intravenous infusion of MPA (4 ml of 150 mM) for a period of 30 min. The animal is then sacrificed to insure a steady state level of renal MPA and another spectrum is obtained. From these two spectra the ratio of intensities of MPA to beta-ATP was derived. In the method used here, no significant contribution from tissues outside the kidney was observed. In addition, a relatively homogeneous distribution of MPA throughout the kidney was achieved. The amount of MPA per gram wet weight of kidney was also obtained through NMR methods by placing the excised organ in a phosphate-calibrated solenoidal coil. The calibration spectra along with the ratio of intensities for MPA/beta-ATP were used to calculate the number of micromoles of ATP per gram wet weight of kidney. Infusion of a higher concentration of MPA (1.25 M) produced a visible MPA resonance in other organs besides the kidney. Thus, MPA could be useful in determining phosphate metabolite concentrations in other tissues.

Animals↗
PubMed1987

Measurement of tissue potassium in vivo using 39K nuclear magnetic resonance.

39K nuclear magnetic resonance (NMR) spectra were readily obtained, in vivo, from rat muscle, kidney, and brain in 5-10 min with signal-to-noise ratios of approximately 20:1. Quantitation of the K+ signal was achieved by reference to an external standard of KCl/dysprosium nitrate as well as by reference to the proton signal from tissue water. In vitro NMR studies of isolated tissue showed a K+ visibility (NMR K+/total tissue K+) of 96%, 62 +/- 8%, 47 +/- 1.9%, 45 +/- 3.5%, and 43 +/- 2.5% for blood, brain, muscle, kidney, and liver, respectively. Absolute tissue K+ was determined by flame photometry of acid-digested tissue. Changes in tissue K+ status by chronic K+ depletion or acute K+ loading produced changes of 39K NMR signal intensity that were equal to changes of absolute tissue K+. Acidosis, alkalosis, mannitol, or RbCl infusion did not significantly change the NMR K+ signal. These results indicate that the changes in K+ detected by NMR were specifically and accurately detected. To investigate the factors that affect the 39K NMR signal, the effects of liver homogenate on 39K NMR signal intensity were studied. Addition of homogenate produced a 60% loss of signal intensity, suggesting that a large portion of cell K+ may be only 40% visible. Addition of RbCl to undiluted homogenate increased the NMR K+ signal by 11 +/- 2 mumol/g. Addition of H2O or NaCl had no effect, suggesting that Rb+ was replacing K+ in sites of low (less than 40%) NMR visibility. These results demonstrate that 39K NMR experiments can be performed using intact organs. To explain the lack of detectable K+ and changes in K+ NMR visibility, a three compartment model is proposed.

Animals↗
PubMed1987

Determination of pyridine nucleotide fluorescence from the perfused heart using an internal standard.

Mitochondrial reduced nicotinamide adenine dinucleotide (NADH) is a key intermediate in energy metabolism in the heart, which can be qualitatively monitored using nondestructive surface fluorescence techniques. However, this optical technique is subject to artifacts from alterations in tissue absorbance, motion of the heart, and variations in excitation intensity. In this study rapid-scan fluorescence emission spectroscopy was used in conjunction with an internal fluorescence standard to compensate for these optical artifacts. The fluorescence spectra obtained from heart had a maximum at 460 nm and a shoulder at 415 nm. Dilution of heart homogenates resulted in a fluorescent spectrum characteristic of suspensions of mitochondria, indicating that absorption of fluorescence by tissue components produces an inner filter effect. This internal filter was characterized, and isobestic points with regard to O2 were found at 425 and 450 nm. Alterations in the inner filter effect due to changes in tissue oxygenation were eliminated by monitoring the NADH at 425 nm. Motion artifacts and excitation source fluctuations were corrected by loading heart cells with an internal fluorescent standard, 5(6)-carboxy-2',7'-dichlorofluorescein (ClCF). Motion of the heart and changes in excitation intensity altered the fluorescence detected from both NADH and ClCF. The use of the NADH-to-ClCF ratio detected at isobestic wavelengths (425 nm NADH and 520 nm ClCF) gives a relative measure of NADH fluorescence, which adequately compensates for both internal absorbance and motion artifacts.

Animals↗