Glycolysis in the rat kidney shortly after ischemia and administration of calmodulin inhibitors, AMP and NAD

It is shown in experiments on rats that the early postischemic period after 1- and 1.5-hour ischemia of kidneys is characterized by a decrease in the damage of the glycolytic system site which induces glucose-6-phosphate transformation into lactate and by an increase in the inhibition intensity of the initial hexokinase reaction of glycolysis. In the postischemic period after more prolonged (2-, 3-hour) ischemia the damage of the glycolytic system develops also at the site of glucose-6-phosphate transformation into lactate. Administration either of the nucleotide complex (NAD and AMP) or calmodulin inhibitors (aminazine and zinc sulphate) to rats prior to two-hour occlusion of kidneys vessels promotes a decrease in the inhibition of the glycolytic system activity in the postischemic period. At the same time the separate and combined application of zinc sulphate and triftazin (the most intensive calmodulin inhibitor) is not efficient. The positive effect of NAD, AMP and aminazine on the state of the glycolytic kidney system in the postischemic period correlates with the improvement of the blood microcirculation processes in them.     

Effect of NAD and ADP on glycolysis in the kidneys and liver of rats during ischemia

Experiments on albino rats have shown that kidney ischemia and its simulation by the anaerobic incubation of postmitochondrial kidney homogenate fraction without a substrate induce a considerable damage of the glycolytic system at the stage of the glucoso-6-phosphate transformation into fructoso-1.6-diphosphate and a less pronounced damage in the fructoso-1.6-diphosphate transformation into lactate. Administration of adenosine diphosphate (ADP) and nicotinamide adenine dinucleotide (NAD) to rats before kidney vessel occlusion or their addition to the postmitochondrial fraction before the anaerobic incubation without a substrate decreased a degree of the glycolytic system damage. The damage of the glycolytic system and protective action of NAD are also detected under simulation of liver ischemia. Possible mechanisms of the ischemic damage in the glycolytic liver and kidney tissue system are discussed.     

Fructose-1,6-bisphosphate as a metabolic substrate in hog ileum smooth muscle during hypoxial

Exogenously applied fructose-1,6-bisphosphate has been reported to be effective in preventing some damage to the small intestine during ischemia. To determine whether exogenously applied fructose-1,6-bisphosphate protects ileum smooth muscle from damage from hypoxia and from reoxygenation, we examined the effect of fructose-1,6-bisphosphate on the ability of hog ileum smooth muscle to maintain isometric force during hypoxia and to generate isometric force after reoxygenation in the presence of 5 mM glucose. After 180 min of hypoxia, tissues incubated with 20 mM fructose-1,6-bisphosphate maintained significantly greater levels of isometric force than tissues incubated in the absence of exogenous substrate (23% of pre-hypoxia force compared to 16%). During the first contraction following reoxygenation there was a significantly greater force generation in tissues incubated with 20 mM fructose-1,6-bisphosphate during the hypoxia period compared to tissues with no exogenous substrate included during the hypoxia period (29% of pre-hypoxia force compared to 19%). However, glucose always was a better metabolic substrate compared to fructose-1,6-bisphosphate under all experimental conditions. The presence of fructose-1,6-bisphosphate during hypoxia likely improved tissue function by fructose-1,6-bisphosphate entering the cells and acting as a glycolytic intermediate, since during a 120 min period of hypoxia, unmounted ileum smooth muscle metabolized 1,6-¹³C-fructose-1,6-bisphosphate to 3-¹³C-lactate. This conversion of 1,6-¹³C-fructose-1,6-bisphosphate to 3-¹³C-lactate was inhibited by the addition of 1 mM iodoacetic acid, a glycolytic inhibitor. We conclude that exogenously provided fructose-1,6-bisphosphate does provide modest protection of ileum smooth muscle from hypoxic damage by functioning as a glycolytic intermediate and improving the cellular energy state.This work was supported in part by NIH (HL48783 to CDH), NSF (Instrumentation Grant 8908304), and the Department of Physiology of the University of Missouri. T. Juergens was supported by the School of Medicine and the Department of Physiology of the University of Missouri.     

Existence of extrathyroidal conversion inhibitor (IEC) in starved animals and its influence on thyroid hormones deiodination in liver and kidney in vitro

To find out whether an inhibitor of extrathyroidal conversion of iodothyronines is present in sera of starved animals, pig liver and kidney homogenates were incubated with T4, T3 or rT3 and dithiotreitol in the presence of evaporated diethyl ether extracts of sera obtained from fed and starved (1-12 days) rabbits. Sera extracts of short-term (1-4 days) starved rabbits caused a significant inhibition of T4 to T3 conversion (54% on day 3) and T4 to rT3 deiodination (52% on day 2) in liver homogenates. Extracts of sera from long-term (8 and 12 days) starved animals diminished only liver T4 to T3 conversion on day 8 and had no influence on liver T4 to rT3 conversion. 5'-deiodination of rT3 (to 3,3'-T2) in liver was gradually decreased by extracts of sera from animals starved during 2-12 days. Liver rT3-5-deiodination (to 3',5'-T2) was significantly impaired on day 4 and totally depressed by long-term starvation. In vitro T3 to 3,3'-T2 conversion in liver was markedly (59-103%) increased by ether extracts of sera from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term nor from long-term fasted rabbits but T4-5-deiodination (to rT3) was reduced by sera extracts of short-term fasted animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of multi-enzyme complexes in the integration of cell metabolism

General properties of enzymes and structurally ordered multienzyme complexes as controllable systems are discussed: the spatial isolation of working sites and sites of control and the realization of control mechanisms with the participation of "external" factors which provide the optimal functioning of the controllable system in the biological system of higher level of complexity. The basic mechanisms of the control of soluble enzymes are isosteric and allosteric mechanisms which directed to the maintenance of cellular homeostasis. The mechanism of functioning of a multienzyme complex as a whole which is realized with the participation of second messengers is classified as a mechanism for tracing of the signals from higher levels of the control of metabolism (from nervous, hormonal and immune systems). When discussing the control of functioning of the multienzyme complexes, special attention was paid to the complex of glycolytic enzymes formed on the structural proteins of skeletal muscles and on the membranes. An order of assembly of the complex of glycolytic enzymes is proposed. The possible localization of this complex in myofibrils is discussed.     

Microvascular and myocardial contractile responses to ischemia: influence of exercise training.

We hypothesized that exercise training preserves endothelium-dependent relaxation, lessens receptor-mediated constriction of coronary resistance arteries, and reduces myocardial contractile dysfunction in response to ischemia. After 10 wk of treadmill running or cage confinement, regional and global indexes of left ventricular contractile function were not different between trained and sedentary animals in response to three 15-min periods of ischemia (long-term; n = 17), one 5-min bout of ischemia (short-term; n = 18), or no ischemia (sham-operated; n = 24). Subsequently, coronary resistance vessels ( approximately 106 +/- 4 microm ID) were isolated and studied using wire myographs. Maximal ACh-evoked relaxation was approximately 25, 40, and 60% of KCl-induced preconstriction after the long-term, short-term, and sham-operated protocols, respectively, and was similar between groups. Maximal sodium nitroprusside-evoked relaxation also was similar between groups among all protocols, and vasoconstrictor responses to endothelin-1 and U-46619 were not different in trained and sedentary rats after short-term ischemia or sham operation. We did observe that, after long-term ischemia, maximal tension development in response to endothelin-1 and U-46619 was blunted (P < 0.05) in trained animals by approximately 70 and approximately 160%, respectively. These results support our hypothesis that exercise training lessens receptor-mediated vasoconstriction of coronary resistance vessels after ischemia and reperfusion. However, training did not preserve endothelial function of coronary resistance vessels, or myocardial contractile function, after ischemia and reperfusion.     

Glycolytic oscillations in isolated rabbit ventricular myocytes

Previous studies have shown that glycolysis can oscillate periodically, driven by feedback loops in regulation of key glycolytic enzymes by free ADP and other metabolites. Here we show both theoretically and experimentally in cardiac myocytes that when the capacity of oxidative phosphorylation and the creatine kinase system to buffer the cellular ATP/ADP ratio is suppressed, glycolysis can cause large scale periodic oscillations in cellular ATP levels (0.02-0.067 Hz), monitored from glibenclamide-sensitive changes in action potential duration or intracellular free Mg2+. Action potential duration oscillations originate primarily from glycolysis, since they 1) occur in the presence of cyanide or rotenone, 2) are suppressed by iodoacetate, 3) are accompanied by at most very small mitochondrial membrane potential oscillations, and 4) exhibit an anti-phase relationship to NADH fluorescence. By uncoupling energy supply-demand balance, glycolytic oscillations may promote injury and electrophysiological heterogeneity during acute metabolic stresses, such as acute myocardial ischemia in which both oxidative phosphorylation and creatine kinase activity are inhibited.     

The heart as a working model to explore themes and strategies for anoxic survival in ectothermic vertebrates

Most vertebrates die within minutes when deprived of molecular oxygen (anoxia), in part because of cardiac failure, which can be traced to an inadequate matching of cardiac ATP supply to ATP demand. Cardiac power output (PO; estimated from the product of cardiac output and central arterial pressure and an indirect measure of cardiac ATP demand) is directly related to cardiac ATP supply up to some maximal level during both normoxia (ATP supply estimated from myocardial O(2) consumption) and anoxia (ATP supply estimated from lactate production rates). Thus, steady state PO provides an excellent means to examine anoxia tolerance strategies among ectothermic vertebrates by indicating a matching of cardiac glycolytic ATP supply and demand. Here, we summarize in vitro measurements of PO data from rainbow trout, freshwater turtles and hagfishes to provide a reasonable benchmark PO of 0.7 mW g(-1) for maximum glycolytic potential of ectothermic hearts at 15 degrees C, which corresponds to a glycolytic ATP turnover rate of about 70 nmol ATP g(-1) s(-1). Using this benchmark to evaluate in vivo PO data for hagfishes, carps and turtles, we identify two cardiac survival strategies, which in conjunction with creative waste management techniques to reduce waste accumulation, allow for long-term cardiac survival during anoxia in these anoxia-tolerant species. Hagfish and crucian carp exemplify a strategy of evolving such a low routine PO that routine cardiac ATP demand lies within the range of the maximum cardiac glycolytic potential. Common carp and freshwater turtles exemplify an active strategy of temporarily and substantially decreasing cardiac and whole body metabolism so that PO is below maximum cardiac glycolytic potential during chronic anoxia despite being quite close to this potential under normoxia.     

Histological and ultrastructural regulation in rabbit endometrial explants by estrogen in serum-free culture

A repertoire of hormonal signals including estrogen regulate the growth, differentiation, and functioning of diverse target tissues, including the ovary, the mammary gland, and skeletal tissue. A serum-free culture system derived from rabbit endometrium explants has been devised and is reported here to explore estrogen action in vitro. The system involves aseptically harvesting the uterus from a virgin rabbit, dissecting the endometrium, explanting it into 1- to 2-mm(3) pieces weighing approximately 1-2 mg each, and incubating these pieces in serum-free Medium-199. The culture is carried out for a period of 4 d in a humidified CO(2) incubator at 37 degrees C with 5% CO(2). The effect of extraneously added estrogen (1 microg/ml) was investigated by histological and ultrastructural procedures. It was observed that estrogen could induce specific changes, such as abundant mitochondria, rough endoplasmic reticulum, golgi complex, and intracellular collagen deposition, in both the epithelial and the fibroblast cell components of the explanted tissue. The study, therefore, indicates that the proposed system is an ideal tool for exploring and demonstrating estrogen responsiveness under in vitro conditions.     

Regulation of concentrations of glycolytic enzymes and creatine-phosphate kinase in “fast-twitch” and “slow-twitch” skeletal muscles of the chicken

The distinctive contractile and metabolic characteristics of different skeletal muscle fiber types are associated with different protein populations in these cells. In the present work, we investigate the regulation of concentrations of three glycolytic enzymes (aldolase, enolase, glyceraldehyde-3-phosphate dehydrogenase) and creatine-phosphate kinase in “fast-twitch” (breast) and “slow-twitch” (lateral adductor) muscles of the chicken. Results of short-term amino acid incorporation experiments conducted both in vivo and with muscle explants in vitro showed that these enzymes turnover at different rates and that aldolase turns over 2 to 3 times faster than the other three enzymes. However, these differences in turnover rates were difficult to detect in long-term double-isotope incorporation experiments, presumably because extensive reutilization of labeled amino acids occurred during these long-term experiments. Mature muscle fibers synthesize these four cytosolic enzymes at very high rates. For example, 11 to 14% of the total labeled leucine incorporated into protein by breast muscle fibers was found in the enzyme aldolase. Results of short-term amino acid incorporation experiments also showed that the relative rates of synthesis of the three glycolytic enzymes were about fourfold higher in mature “fast-twitch” muscle fibers than in mature “slow-twitch” ones while the relative rates of synthesis of creatine-phosphate kinase were similar in the two fiber types. The relative rates of synthesis of these four enzymes and cytosolic proteins in general were found to be very similar in immature muscles of both types. More profound changes in the relative rates of synthesis of major cytosolic proteins, including the glycolytic enzymes, occurred during postembryonic maturation of fast-twitch fibers than occurred during maturation of slow-twitch fibers. Our work demonstrates that (1) the synthesis of creatine-phosphate is independently regulated with respect to the synthesis of the glycolytic enzymes in muscle fibers; and (2) the approximate fourfold higher steady-state concentrations of glycolytic enzymes in fast-twitch muscle fibers as compared with slow-twitch fibers are determined predominantly by regulatory mechanisms operating at the level of protein synthesis rather than protein degradation. Our demonstration that more profound changes in the relative rates of synthesis of major cytosolic proteins occur during maturation of fast-twitch fibers as compared with slow-twitch fibers is discussed in terms of the mode(s) of fiber-type differentiation proposed by others.     

Estimation of age-related changes in the regulation of peripheral blood flow in humans

The age-dependent features in the state of skin microvascular bed has been studied with laser Doppler flowmetry in healthy volunteers of different age groups. To reveal the reaction of skin blood flow in response to short-term ischemia, the occlusive test has been carried out. To estimate the contribution of rhythmic components to blood flow signal, continuous wavelet-transform spectral analysis was used. Age-dependent increase of pulse-wave amplitude and decrease of respiratory wave amplitude reflecting age-dependent changes in functioning of arteriolar and venular links of microvascular bed have been observed at rest. In response to short-term ischemia the age-dependent reduction of reserve resources has been revealed in functioning of arteriolar link of microvascular bed. The reduction of activity of myogenic, neurogenic and endothelial regulation systems have been shown at rest in ageing.     

Metabolic regulation during early frog development. Identification of proteins labeled by 32P-glycolytic intermediates

When 32P-labeled phosphoenolpyruvate is injected into Xenopus laevis oocytes, a 50-60-kDa protein of subunit size Mr 29,000 is rapidly labeled, followed by a second (monomeric) protein of 66 kDa concomitant with the loss of label from the first protein. We have identified these proteins as, respectively, the glycolytic enzymes phosphoglyceromutase and phosphoglucomutase. The phosphoglyceromutase is labeled at a histidine and the phosphoglucomutase at a serine, presumably at their active sites during the gluconeogenic transformation of phosphoenolpyruvate into glycogen. The transfer of the 32P label from phosphoenolpyruvate to these two enzymes also occurs in in vitro lysates made from full-grown Xenopus oocytes, eggs, or early embryos, but with a slower time course. Lysates prepared from leg muscle show labeling of the phosphoglyceromutase, but not the phosphoglucomutase, when incubated with [32P]phosphoenolpyruvate. This last result is expected in tissues showing metabolic flux largely in the glycolytic direction. The data indicate that in full-grown oocytes and embryos metabolic flux occurs largely in the gluconeogenic direction.     

Short-term regulation of glycolysis by insulin and dexamethasone in cultured rat hepatocytes

Evidence for a direct metabolic effect of insulin in isolated liver preparations is scarce. The stimulation of glycolysis by insulin previously demonstrated in monolayer cultures of adult rat hepatocytes [(1982) Eur. J. Biochem. 126, 271-278] was further investigated. The degree of stimulation varied with the age of the culture and amounted to 250%, 200%, 500% and 200% of the control value using cells at the culture age of 2 h, 24 h, 48 h, and 72 h, respectively. Half-maximal dose of insulin was 0.1 nM. Maximal stimulation was reached within 5 min and lasted for at least 4 h. Dexamethasone acted both as a long-term and short-term modulator. Long-term pretreatment of the cells with dexamethasone proved necessary to permit insulin action. In addition to this permissive action, pretreatment with dexamethasone reduced the insulin-independent basal glycolytic rate. In short-term experiments dexamethasone decreased the basal glycolytic flux, however, it did not affect the absolute increase in glycolysis brought about by insulin. The half-maximal dose of dexamethasone was 10 nM. The stimulatory effects of insulin may in part be attributed to the activation of pyruvate kinase. Insulin produced a left-shift of the substrate saturation curve, decreasing the K0.5 value for phosphoenolpyruvate.     

AMPK and ACCchange with fasting and physiological condition in euthermic and hibernating golden-mantled ground squirrels (Callospermophilus lateralis)

AMP-activated protein kinase (AMPK) is a cellular energy sensor that responds to low endogenous energy by stimulating fatty acid oxidation (through inactivation of acetyl-CoA carboxylase (ACC)) and food intake. Fasting generally stimulates phosphorylation of AMPK (pAMPK) and ACC (pACC), but it is unclear how AMPK and ACC react to a long-term fast (i.e. hibernation). We performed Western blots for total and pAMPK and pACC on tissues from a species of hibernator (Callospermophilus lateralis) after short-term summer fasting (1-5 days) and long-term winter fasting (3 months). Winter animals were sacrificed during hibernation at low body temperature (torpid, T(b)~5°C) or at normal high T(b)(euthermic, T(b)~37°C). We found a general increase in pAMPK in most tissues (liver, muscle, and white adipose tissue (WAT), but not hypothalamus) and pACC in all tissues after a short-term summer fast. Response of AMPK and ACC to a long-term winter fast differed by tissue-in liver, there was no difference in total or pAMPK or pACC between groups, but in muscle, WAT and BAT, euthermic GMGS had lower relative abundance of pAMPK and pACC than torpid animals. Therefore, AMPK may be an important energy sensor at all points in hibernator's circannual cycles of food intake and T(b).     

Activation of glycolysis by insulin with a sequential increase of the 6-phosphofructo-2-kinase activity, fructose-2,6-bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes

The involvement of 6-phosphofructo-2-kinase, fructose 2,6-bisphosphate [Fru(2,6)P2] and pyruvate kinase in the insulin-dependent short-term activation of glycolysis was studied in primary cultures of rat hepatocytes. The short-term influence of insulin on these parameters was dependent on the insulin concentration used for the long-term culture. Cells were cultured either with 10 nM or 0.1 nM insulin for 48 h, and are referred to as 'insulin cells' and 'control cells', respectively. Insulin cells exhibited a high level of Fru(2,6)P2. Addition of insulin to insulin cells led to an immediate stimulation of glycolysis (two-fold) and activation of pyruvate kinase. The concentration of Fru(2,6)P2 and activity of 6-phosphofructo-2-kinase remained constant. Control cells exhibited a very low level of Fru(2,6)P2 and low activity of 6-phosphofructo-2-kinase directly after the medium change. However, both parameters increased during a 1-2-h incubation in the absence of insulin. Although the level of Fru(2,6)P2 thus changed up to tenfold the glycolytic rate remained at a constant value. Addition of insulin to control cells led to a 5-8-fold stimulation of glycolysis but only after a 30-90-min lag phase. During this lag period insulin strongly increased sequentially the 6-phosphofructo-2-kinase, the level of Fru(2,6)P2 and the pyruvate kinase activity. The activation of the latter enzyme slightly preceded the onset of the insulin-stimulated glycolysis. Addition of insulin to control cells, which were preincubated for 3 h in the absence of insulin and in which the Fru(2,6)P2 level had risen insulin-independently, led to an immediate increase in glycolysis without a lag phase. It is concluded that in this insulin-sensitive cell system: the changes of glycolytic flux did not correlate with changes in the level of total Fru(2,6)P2 either in insulin or in control cells; an increase in the Fru(2,6)P2 concentration was not obligatory for the insulin-dependent stimulation of glycolysis in insulin cells; activation of pyruvate kinase and thus glycolysis by insulin did not proceed unless the Fru(2,6)P2 level had been elevated above a threshold level. The lack of correlation between total Fru(2,6)P2 levels and the glycolytic flux and the apparent existence of a threshold concentration for Fru(2,6)P2 suggest a permissive action for this effector in enzyme interconversion.     

Permissive action of triiodothyronine on the long-term increase of glycolysis by insulin in cultured rat hepatocytes

The long-term influence of triiodothyronine (T3) and insulin on glycolysis, some glycolytic/gluconeogenic enzymes and insulin responsiveness and sensitivity was investigated in rat hepatocytes cultured for 48 h without T3, with 10 microM T3, with 10nM insulin and with insulin plus T3. From 48 h-51 h basal glycolysis ([14C]lactate formation from [14C]glucose) was measured in the absence and short-term insulin-stimulated glycolysis in the presence of 100 nM insulin. 1) T3 addition for 48 h had no significant influence on basal or on insulin-stimulated glycolysis. 2) Insulin addition for 48 h increased basal glycolysis to 300%, and insulin-stimulated glycolysis to 160%. 3) T3 plus insulin addition for 48 h elevated basal glycolysis to 560% and insulin-stimulated glycolysis to 230%. 4) The 48-h treatment with T3 did not change glucokinase (GK) and pyruvate kinase (PK) activity, yet it increased phosphoenol-pyruvate carboxykinase (PEPCK) activity to 150%. 5) The 48-h treatment with insulin as well as T3 plus insulin enhanced GK to 200% and PK to 140% and decreased PEPCK to 65%. 6) The long-term effect of T3 on glycolysis was maximal at initial concentrations of 100 nM. 7) The long-term treatment with T3 did not alter the short-term responsiveness or sensitivity of glycolysis for insulin, neither in cells from euthyroid nor from hypothyroid rats. The present results allow the conclusion that T3 had a permissive effect on the long-term increase of glycolysis by insulin, and that T3 exerted this function by altering neither the cellular content of key enzymes nor the short-term insulin responsiveness and sensitivity.