PubMed Health⌕ Search

Biomedical subjects

K F LaNoue

Publications and source records attributed to K F LaNoue.

At least 37 records · Page 2Linked to original sources
PubMed1996

Differentiation-dependent expression of CA V and the role of carbonic anhydrase isozymes in pyruvate carboxylation in adipocytes.

The incorporation of radioactivity from 14C-labeled compounds into metabolic intermediates and total lipids was examined in 3T3 adipocytes. The heterocyclic sulfonamide carbonic anhydrase inhibitor (SCAI) 6-ethoxyzolamide (ETZ) caused a decrease (42+/-7% of control, IC50 = 2.2+/-1.1 x 10(-7) M) in the incorporation of [14C] bicarbonate into several Krebs cycle intermediates in 3T3-F442A adipocytes. This decrease in pyruvate carboxylase-mediated [14C] carbon fixation was associated with a reduction in fluorometrically determined [citrate] and [malate]. The ability of ETZ to decrease both the incorporation of radioactivity into and the concentrations of Krebs cycle intermediates was not of sufficient magnitude to lower [ATP], but was associated with a decrease in de novo lipogenesis from [14C]glucose. De novo lipogenesis was also inhibited to a similar extent by trifluormethanesulfonamide, an aliphatic SCAI, which suggests that the effects are mediated by carbonic anhydrase. ETZ did not inhibit de novo lipogenesis from [14C]glutamine (12.38+/-1.068 nmol/mg protein, ETZ; 12.5+/-0.846 nmol/mg protein, DMSO). This suggests that ETZ inhibition of lipogenesis involves an inhibitory effect on pyruvate carboxylase as opposed to acetyl CoA carboxylase, because the incorporation of glutamine into lipids does not involve pyruvate carboxylase. Decreased de novo lipogenesis was also observed by incubating cultures in media that contained 1 mM bicarbonate (atmosphere:100% humidified air) rather than 25 mM bicarbonate (atmosphere: 95% humidified air/5% CO2). This suggests that exogenous CO2/bicarbonate may be required to sustain maximal rates of de novo lipogenesis. Because these results implied that CA V, the mitochondrial isoform of carbonic anhydrase, might be present in adipocytes, CA V levels were measured by immunoblotting. Mitochondrial preparations of adipocytes and liver were found to contain similar concentrations of CA V. Unlike adipocyte CA III, CA V concentrations were not significantly different in lean and obese Zucker rats. However, CA V levels were ninefold higher in differentiated 3T3-F442A adipocytes compared to undifferentiated adipoblasts. Our data indicate that CA V is relatively abundant in adipocyte mitochondria and exhibits differentiation-dependent expression like pyruvate carboxylase and the cytosolic isozymes CA II and CA III. The possible roles of CA II and CA V in pyruvate carboxylation are discussed.

3T3 Cells↗
PubMed1995

Beta-adrenergic responsiveness of adenylate cyclase in human adipocyte plasma membranes in obesity and after massive weight reduction.

The aim of this study was to find out how beta-adrenergic responsiveness of adipocytes is altered in obesity and by weight loss and to investigate what mechanisms lead to potential alterations in responsiveness. Crude plasma membranes were prepared from adipocytes of massively obese and normal-weight individuals, as well as previously obese patients that had lost an average of 38% of their initial weight after bariatric surgery. Stimulation of adenylate cyclase by isoproterenol varied considerably in fat cell plasma membranes from different individuals. Crude fat cell plasma membranes from obese patients were less responsive to isoproterenol than those from normal-weight subjects, whereas those from postgastroplasty patients were hyperresponsive. The response was correlated negatively with cell size and positively with beta-adrenergic receptor density and with the ratio of beta-receptors and stimulatory G-proteins (Gs). There was no correlation with Gs content. However, differences in receptor density between small and large cells or normal-weight, obese, and post-bypass patients could not explain the observed differences in responsiveness to isoproterenol between the different groups.

Adenylyl Cyclases↗
PubMed1995

Hormone-regulated Ca2+ channel in rat hepatocytes revealed by whole cell patch clamp.

An inward current responsible for hormone regulated Ca2+ entry has been identified in cultured rat hepatocytes using whole cell patch clamp. Addition of 20 nM vasopressin or of 100 microM ATP induced the inward current, which could be observed more clearly after blocking an outward K+ current. This large outward K+ current, which appeared after addition of vasopressin or ATP, could be blocked either by replacing K+ with Cs+ in the external medium and in the pipette solution, or by simply including 0.5 microM apamin in the K(+)-containing external medium. The outward current appears to be carried by a Ca2+ activated K+ channel. In the presence of apamin, hepatocytes pretreated with vasopressin in a Ca(2+)-free media reveal an inward current on addition of external Ca2+ (5 mM). The current could also be elicited by addition of vasopressin when cells are preincubated in the presence of 5 mM external Ca2+. No current is seen on addition of Ca2+ in the absence of vasopressin. Initially, the inward current was ca 200-300 pA at -60 mV, but it declined rapidly over 3 min to ca 20 pA. The current approached zero, as an asymptote at positive potential, and appeared to be somewhat inwardly rectifying. Additions of 5 mM Mn2+ or 5 mM Ba2+ in place of Ca2+ produced little or no current. An inhibitor of ER Ca(2+)-ATPase, thapsigargin, could also trigger the cascade of events leading to plasma membrane conductance of Ca2+. The data suggest that hormone-stimulated Ca2+ entry into hepatocytes is mediated by a Ca(2+)-release activated channel highly specific for Ca2+. This is the first demonstration of such a channel in hepatocytes, though similar ones have been described in mast cells, in vascular endothelial cells and T-lymphocytes.

Adenosine Triphosphate↗
PubMed1995

Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts.

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.

Acetates↗
PubMed1995

Evidence for regulated coupling of A1 adenosine receptors by phosphorylation in Zucker rats.

Studies were designed to find the molecular basis for previous observations that lipolysis is less active and A1 adenosine receptor signaling is more active in adipocytes from obese than from lean Zucker rats. With quantitative immunoblot procedures for detection, Gi alpha 1 and Gs alpha 45 levels were found anomalously low in obese compared with lean membranes (50 and 30%, respectively), but other G alpha subunit levels were normal. However, the sensitivity of the receptor-Gi protein to GTP was about 5- to 10-fold higher in obese compared with lean membranes when assessed from 1) the ability of GTP to inhibit forskolin-stimulated adenylyl cyclase in the presence of an adenosine receptor agonist and 2) the ability of a nonhydrolyzable guanine nucleotide analogue to alter A1 adenosine receptor agonist binding. Alkaline phosphatase treatment of isolated adipocyte membranes from obese but not lean animals decreased guanine nucleotide sensitivity of agonist binding. Surprisingly, solubilized adipocyte A1 adenosine receptors from all animals exhibited the same high sensitivity to guanine nucleotides as that of intact obese membranes, and this high sensitivity could be decreased 20-fold by treatment with alkaline phosphatase. These data suggest that protein phosphorylation may regulate coupling of the A1 adenosine receptor in rat adipocyte membranes.

Adenosine↗
PubMed1995

Cyclic AMP and glycerol concentrations in freeze-clamped human omental and subcutaneous adipose tissues.

OBJECTIVE: To compare intracellular concentrations of cyclic AMP, GTP, ATP, AMP and glycerol in omental and abdominal subcutaneous adipose tissues. DESIGN: Samples of omental and/or abdominal subcutaneous adipose tissues of 14 women undergoing elective surgery were freeze-clamped, deproteinized with perchloric acid and neutralized. All subjects were obese since it was not possible to freeze-clamp lean individuals. MEASUREMENTS: The concentrations of ATP and GTP were determined by ion-paired high performance liquid chromatography and AMP and glycerol enzymatically. Cyclic AMP was determined by radioimmunoassay. Intracellular nucleotide concentrations were calculated from tissue weights, water contents and relative amounts of sodium and potassium. RESULTS: The intracellular concentration of cyclic AMP in omental adipose tissue (31 mumol/l) was twice as high as that in subcutaneous fat while those of ATP, GTP and AMP were similar at the two sites. Glycerol concentrations were higher in subcutaneous (472 mumol/l) than in omental (325 mumol/l) adipose tissue. CONCLUSION: The results suggest higher relative stimulation of lipolysis by cyclic AMP in omental adipose tissue than in the subcutaneous region in situ. However, probably because of differences in blood flow and possibly in the lipolytic machinery, this is not reflected as a higher glycerol concentration.

Adenosine Monophosphate↗
PubMed1994

Quantitation of inhibitory G-proteins in fat cells of obese and normal-weight human subjects.

Antisera were raised in rabbits against synthetic peptides corresponding to sequences of the guanine nucleotide binding proteins Gi1, Gi2, Gi3 and Go. These and previously described antisera were used to identify different G-proteins in Western blots of human adipocyte plasma membranes and to quantify them using purified recombinant alpha subunits as standards. Go was shown to be absent or << 15 pmol/mg of protein. A band stained by a previously characterized Go antiserum is suggested to be due to nonspecific staining of Gi1. Gi1 and Gi2 were the major G-proteins. Gi1 was present at concentrations of 52 and 18 pmol/mg of protein in lean and obese subjects, respectively, and the concentration was negatively correlated with the body mass index. Gi2 concentrations averaged 64 pmol/mg of protein and there was no correlation to the body mass index. Gi3 levels were much lower (<< 13 pmol/mg of protein) and the presence of this protein could not be demonstrated with certainty. The concentrations of Gi1 and Gi2 are thus over two orders of magnitude higher than those of the receptors whose effects they mediate. The low concentration of Gi1 in adipocyte plasma membranes of obese subjects could in part explain the attenuated inhibitory responses of adenylate cyclase in isolated fat cells in obesity.

Adipocytes↗
PubMed1994

Adenosine receptor prodrugs: synthesis and biological activity of derivatives of potent, A1-selective agonists.

5'-Ester derivatives of the potent adenosine agonists N6-[4-[[[[4-[[[(2-acetylaminoethyl)amino]carbonyl]methyl] anilino]carbonyl]methyl]phenyl]adenosine (N-AcADAC; 1) and N6-cyclopentyladenosine (CPA; 2) were prepared as prodrugs. Both alkyl esters or carbonates (designed to enter the brain by virtue of increased lipophilicity) and 1,4-dihydro-1-methyl-3-[(pyridinylcarbonyl)oxy]esters designed to concentrate in the brain by virtue of a redox delivery system were synthesized. In the 5'-blocked form, the adenosine agonists displayed highly diminished affinity for rat brain A1-adenosine receptors in binding assays. The dihydropyridine prodrug 29 was active in an assay of locomotor depression in mice, in which adenosine agonists are highly depressant. The behavior depression was not reversible by peripheral administration of a non-central nervous system active adenosine antagonist. In an assay of the peripheral action of adenosine (i.e., the inhibition of lipolysis in rats), the parent compounds were highly potent and the dihydropyridine prodrug was much less potent.

Adenosine↗
PubMed1994

Abnormal A1 adenosine receptor function in genetic obesity.

Obesity is increasingly recognized as an important health problem in developed, industrialized countries. As a large proportion of the variance in individual adiposity is based on genetic factors (1-3), recent efforts have focused on identifying genes involved in regulating the percentage of body fat in a given individual. This effort is helped by the existence of rodent models of genetic obesity. Many strains of mice and at least three rat strains have been identified thus far that exhibit inherited obesity accompanied by a similar set of endocrine abnormalities (4). Although the symptoms of the disease are similar in different strains, different genes appear to be involved in causing the syndrome, as the mutation responsible for the obesity maps to different chromosomal sites in the different strains. Efforts to find the products of the mutated genes over the past 30 years have generally been unsuccessful. However, the available data imply that many obesity mutations may involve genes that code for proteins in a single signal transduction pathway or one particular cascade of covalent modification. Reasonable theories are plentiful about the identity of such a pathway, but current studies in the laboratories of the authors suggest that the A1 adenosine receptor signaling pathway may be involved. Evidence of abnormal A1 receptor function has been obtained from studies of Zucker rats and obese (ob/ob) mice (5-7). These strains are obese because of a single recessive mutation. Measurements of adenylyl cyclase activity and regulation in isolated adipocytes and isolated plasma membranes suggest that the receptor is unusually and tonically active in obese rats. Because signaling from this receptor inhibits lipolysis in white and brown fat, induces insulin resistance in skeletal muscle (8, 9), but increases insulin sensitivity in adipose tissue (10, 11), the possibility arises that the excessive activity of the A1 adenosine receptor may induce obesity. Data from human volunteers are also compatible with the possibility that the activity of the receptor is unusually high in vivo in obese individuals (12).

Adenylyl Cyclases↗
PubMed1993

A method of determining electrical potential gradient across mitochondrial membrane in perfused rat hearts.

The electrical potential gradient across the mitochondrial membrane (delta psi m) in perfused rat hearts was estimated by calculating the equilibrium distribution of the lipophilic cation tetraphenylphosphonium (TPP+), using measured kinetic constants of uptake and release of TPP+. First-order rate constants of TPP+ uptake were measured during 30-min perfusions of intact rat hearts with tracer amounts (5.0 nM) of tritium-labeled TPP+ ([3H]TPP+) in the perfusate. This was followed by a 30-min washout, during which the first-order rate constant of efflux was estimated. Values of [3H]TPP+ outside the heart and total [3H]TPP+ inside the heart at equilibrium were calculated. From this information and separately estimated time-averaged plasma membrane potentials (delta psi c) it was possible to calculate free cytosolic [3H]TPP+ at equilibrium. It was also possible to calculate free intramitochondrial [3H]TPP+ at equilibrium as the difference between total tissue [3H]TPP+ minus free cytosolic TPP+ and the sum of all the bound [3H]TPP+. Bound [3H]TPP+ was determined from [3H]TPP+ binding constants measured in separate experiments, using both isolated mitochondria and isolated cardiac myocytes under conditions where both delta psi m and delta psi c were zero. Delta psi m was calculated from the intramitochondrial and cytosolic free TPP+ concentrations using the Nernst equation. Values of delta psi m were 144.9 +/- 2.0 mV in hearts perfused with 5 mM pyruvate and 118.2 +/- 1.4 mV in hearts perfused with 11 mM glucose, in good agreement with delta psi m obtained from isolated rat heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effects of cardiac work on electrical potential gradient across mitochondrial membrane in perfused rat hearts.

The myocardium responds to alterations in cardiac work by changing its rate of O2 consumption. This reflects an increase in the oxidative synthesis of ATP to meet the contractile demand for ATP. However, the biochemical mechanisms responsible for increased ATP synthesis are not fully understood. To localize the flux-controlling reaction(s) in the pathway of ATP synthesis, the effects of substrates and cardiac work on mitochondrial membrane potential (delta psi m), total tissue NADH-to-NAD+ ratio, and high-energy phosphate metabolites were examined in perfused rat hearts. Delta psi m was measured using the equilibrium distribution of tetraphenylphosphonium (33). Cytosolic phosphorylation potential, total tissue NADH-to-NAD+ ratio, and delta psi m were higher in hearts perfused with pyruvate than in those perfused with glucose. Increasing cardiac work induced a four-fold increase in O2 consumption, which was accompanied by 1) decreased or unaltered cytosolic ADP concentration, 2) increased tissue NADH-to-NAD+ ratio, and 3) decreased delta psi m. The results indicate that both NADH-generating reactions and the ATP synthase-catalyzed reaction are important in causing the increase in respiration that accompanies increased work. Because the activation of ATP synthase by cardiac work occurred in the absence of increases in delta psi m, ADP, and Pi, it is possible that the work-related acceleration in ATP synthesis may be due to modification of the kinetic properties of the ATP synthase.

Animals↗
PubMed1992

Differences in 2-oxoglutarate dehydrogenase regulation in liver and kidney.

In response to acidosis, renal ammoniagenesis is stimulated, enhancing urinary buffering power, while hepatic ammoniagenesis and ureagenesis decrease, so as to spare bicarbonate consumed in the urea cycle. 2-Oxoglutarate (2-OG) levels can regulate ammoniagenesis in kidney and gluconeogenesis in liver and kidney. Since the activity of 2-oxoglutarate dehydrogenase (2-OGDH) has an important influence on cellular levels of 2-OG, this study evaluated the effects of pH on 2-OGDH in liver and kidney and found that: (1) the isolated enzyme from both organs has the same pH-sensitivity; (2) 2-OGDH flux measured in intact mitochondria was inhibited by increasing H+ in liver, but stimulated in kidney; (3) transport of 2-OG into the mitochondria was not rate-limiting; (4) liver mitochondrial 2-OGDH exhibited a strong preference for 2-OG generated within the mitochondria from glutamate-oxaloacetate transaminase (GOT), suggesting that channelling between GOT and 2-OGDH occurs. Since complexation between 2-OGDH and GOT occurs in vitro, we propose that the degree of complexation is higher in liver than in kidney, such that most of the 2-OGDH may be complexed to GOT in liver. In the liver the inherent H(+)-sensitivity of 2-OGDH is masked by the pH-sensitivity of GOT and the glutamate-aspartate carrier.

Animals↗
PubMed1992

A 31P NMR study of mitochondrial inorganic phosphate visibility: effects of Ca2+, Mn2+, and the pH gradient.

The effects of external pH, temperature, and Ca2+ and Mn2+ concentrations on the compartmentation and NMR visibility of inorganic phosphate (Pi) were studied in isolated rat liver mitochondria respiring on succinate and glutamate. Mitochondrial matrix Pi is totally visible by NMR at 8 degrees C and at low external concentrations of Pi. However, when the external Pi concentration is increased above 7 mM, the pH gradient decreases, the amount of matrix Pi increases, and the fraction not observed by NMR increases. Raising the temperature to 25 degrees C also decreases the pH gradient and the Pi fraction observed by NMR. At physiologically relevant concentrations, Ca2+ and Mn2+ do not seem to play a major role in matrix Pi NMR invisibility. For Ca2+ concentrations above 30 nmol/mg of protein, formation of insoluble complexes will cause loss of Pi signal intensity. For Mn2+ concentrations above 2 nmol/mg of protein, the Pi peak can be broadened sufficiently to preclude detection of a high-resolution signal. The results indicate that mitochondrial matrix Pi should be mostly observable up to 25 degrees C by high-resolution NMR. While the exact nature of the NMR-invisible phosphate in perfused or in vivo liver is yet to be determined, better success at detecting and resolving both Pi pools by NMR is indicated at high field, low temperature, and optimized pulsing conditions.

Animals↗
PubMed1992

Error in the calibration of the MgATP chemical-shift limit: effects on the determination of free magnesium by 31P NMR spectroscopy.

The measurement of free intracellular magnesium (Mg2+) using the 31P chemical shifts of ATP requires the use of appropriate calibration solutions to determine the chemical-shift limits delta ATP alpha beta and delta MgATP alpha beta. Solutions containing excess Mg2+ contain significant amounts of Mg2ATP and yield positive errors in the value of delta MgATP alpha beta. For physiological applications this may overestimate free intracellular Mg2+ by as much as 300%. This error may be minimized if appropriate mole ratios of Mg2+/ATP are used to calibrate delta MgATP alpha beta.

Adenosine Triphosphate↗
PubMed1992

Kinetic studies of ATP synthase: the case for the positional change mechanism.

The mitochondrial ATP synthases shares many structural and kinetic properties with bacterial and chloroplast ATP synthases. These enzymes transduce the energy contained in the membrane's electrochemical proton gradients into the energy required for synthesis of high-energy phosphate bonds. The unusual three-fold symmetry of the hydrophilic domain, F1, of all these synthases is striking. Each F1 has three identical beta subunits and three identical alpha subunits as well as three additional subunits present as single copies. The catalytic site for synthesis is undoubtedly contained in the beta subunit or an alpha, beta interface, and thus each enzyme appears to contain three identical catalytic sites. This review summarizes recent isotopic and kinetic evidence in favour of the concept, originally proposed by Boyer and coworkers, that energy from the proton gradient is exerted not directly for the reaction at the catalytic site, but rather to release product from a single catalytic site. A modification of this binding change hypotheses is favored by recent data which suggest that the binding change is due to a positional change in all three beta subunits relative to the remaining subunits of F1 and F0 and that the vector of rotation is influenced by energy. The positional change, or rotation, appears to be the slow step in the process of catalysis and it is accelerated in all F1F0 ATPases studied by substrate binding and by the proton gradient. However, in the mammalian mitochondrial enzyme, other types of allosteric rate regulation not yet fully elucidated seem important as well.

Animals↗
PubMed1992

Developmental differences in the response of cytosolic free calcium to potassium depolarization and cardioplegia in cardiac myocytes.

Several biochemical and functional characteristics of immature myocardium suggest a diminished capacity to regulate intracellular Ca2+ during stress. In particular, cellular calcium overload has been postulated as an important pathogenetic mechanism accounting for suboptimal functional recovery following cardioplegia in immature myocardium. Using intracellular Fura-2 fluorescence as Ca2+ indicator, we measured cytosolic free calcium ([Cai]) in single myocytes and cell suspensions derived from both juvenile (4 weeks post-partum) and mature (6-12 months post-partum) New Zealand white rabbits. Resting [Cai] in juvenile heart cells (26 +/- 3 nM) were approximately 50% of that found in adult myocytes (55 +/- 5 nM). In addition, on exposure to increasing concentrations of extracellular potassium ([Kex]), adult but not juvenile myocytes exhibited increases in [Cai]. These two observations underscore developmental differences in intracellular Ca2+ homeostasis. Of particular clinical relevance is the [Cai] response to cardioplegia containing 16 mM [Kex]: neither group demonstrated the expected [Cai] increase in response to potassium depolarization. The lack of [Cai] response to cardioplegia was most likely due to the high levels of Mg2+ (32 mM) contained in cardioplegic solutions. We conclude that cellular calcium overload does not occur following exposure to cardioplegia alone. Accordingly, these findings do not account for recognized developmental differences in functional recovery from "myocardial protection".

Age Factors↗