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.     

A cAMP-dependent protein kinase inhibitor modulates the blocking action of ATP and 5-hydroxydecanoate on the ATP-sensitive K+ channel.

We studied the blocking mechanism of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the ATP-sensitive K+ channel in the single ventricular myocytes using the inside-out patch clamp technique. The channel activity in response to 5-hydroxydecanoate varied with each membrane patch corresponding to the sensitivity to ATP. In this condition the exogenous application of cAMP or cAMP-dependent protein kinase (PKA) obviously recovered the ATP-sensitive K+ channel activity after channel deactivation. By contrast, in membrane patches exhibited low sensitivity to ATP, endogenous cAMP-dependent protein kinase inhibitor (PKI) depressed the channel activity and restored the inhibitory action of 5-hydroxydecanoate and ATP on the channel. These results suggest that PKA-PKI system is involved in the regulatory mechanism of gating activity of the ATP-sensitive K+ channel and the blocking action of 5-hydroxydecanoate and ATP appears to be exerted by potentiating the inhibitory action of PKI on the channel.     

Modulation of ATP-sensitive potassium channels by cGMP-dependent protein kinase in rabbit ventricular myocytes

This investigation used a patch clamp technique to test the hypothesis that protein kinase G (PKG) contributes to the phosphorylation and activation of ATP-sensitive K(+) (K(ATP)) channels in rabbit ventricular myocytes. Nitric oxide donors and PKG activators facilitated pinacidil-induced K(ATP) channel activities in a concentration-dependent manner, and a selective PKG inhibitor abrogated these effects. In contrast, neither a selective protein kinase A (PKA) activator nor inhibitor had any effect on K(ATP) channels at concentrations up to 100 and 10 microm, respectively. Exogenous PKG, in the presence of both cGMP and ATP, increased channel activity, while the catalytic subunit of PKA had no effect. PKG activity was prevented by heat inactivation, replacing ATP with adenosine 5'-O-(thiotriphosphate) (a nonhydrolyzable analog of ATP), removing Mg(2+) from the internal solution, applying a PKG inhibitor, or by adding exogenous protein phosphatase 2A. The effects of cGMP analogs and PKG were observed under conditions in which PKA was repressed by a selective PKA inhibitor. The results suggest that K(ATP) channels are regulated by a PKG-signaling pathway that acts via PKG-dependent phosphorylation. This mechanism may, at least in part, contribute to a signaling pathway that induces ischemic preconditioning in rabbit ventricular myocytes.     

ATP-sensitive K(+) channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes

The present investigation tested the hypothesis that nitric oxide (NO) potentiates ATP-sensitive K(+) (K(ATP)) channels by protein kinase G (PKG)-dependent phosphorylation in rabbit ventricular myocytes with the use of patch-clamp techniques. Sodium nitroprusside (SNP; 1 mM) potentiated K(ATP) channel activity in cell-attached patches but failed to enhance the channel activity in either inside-out or outside-out patches. The 8-(4-chlorophenylthio)-cGMP Rp isomer (Rp-CPT-cGMP, 100 microM) suppressed the potentiating effect of SNP. 8-(4-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 100 microM) increased K(ATP) channel activity in cell-attached patches. PKG (5 U/microl) added together with ATP and cGMP (100 microM each) directly to the intracellular surface increased the channel activity. Activation of K(ATP) channels was abolished by the replacement of ATP with ATPgammaS. Rp-pCPT-cGMP (100 microM) inhibited the effect of PKG. The heat-inactivated PKG had little effect on the K(ATP) channels. Protein phosphatase 2A (PP2A, 1 U/ml) reversed the PKG-mediated K(ATP) channel activation. With the use of 5 nM okadaic acid (a PP2A inhibitor), PP2A had no effect on the channel activity. These results suggest that the NO-cGMP-PKG pathway contributes to phosphorylation of K(ATP) channels in rabbit ventricular myocytes.     

Activators of protein kinase C depolarize insulin-secreting cells by closing K+ channels.

Carbohydrate stimuli of insulin secretion depolarize the pancreatic B cell and the B-cell line RINm5F by inhibiting ATP-sensitive K+ channels. We examined the possibility that this effect is mediated by activation of protein kinase C. In RINm5F cells, the triose D-glyceraldehyde evoked a rapid increase of the mass of 1,2-diacylglycerol, the endogenous activator of protein kinase C. This effect is mainly due to de novo synthesis of the lipid from glycolytic intermediates, as glyceraldehyde carbon was incorporated into 1,2-diacylglycerol within 1 min of exposure to 14C-labelled glyceraldehyde. The effects of two exogenous activators of kinase C, 4-beta-12-phorbol-myristate 13-acetate (PMA) and 1,2-didecanoylglycerol (DC10) on single K+ channel currents were examined in RINm5F cell-attached membrane patches. Both PMA and DC10 depolarized the cells and decreased the open-state probability of the ATP-sensitive K+ channels. These actions were not due to changes in cellular ATP content, since PMA, like glyceraldehyde, failed to alter cellular ATP. As is the case for glyceraldehyde, PMA and DC10 raised cytosolic free Ca2+ [( Ca2+]i) and stimulated insulin secretion. Both of these effects are inhibited in the absence of external Ca2+. This, and the attenuation of the [Ca2+]i rise by verapamil, suggest that all three stimuli raise [Ca2+]i by promoting Ca2+ influx through voltage-gated channels in turn leading to insulin secretion. As the exogenous activators of protein kinase C mimic the effects of glyceraldehyde, it is proposed that the carbohydrate-mediated production of 1,2-diacylglycerol constitutes the link between metabolism and membrane depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of action potential duration under acute heat stress by I(K,ATP) and I(K1) in fish cardiac myocytes

The mechanism underlying temperature-dependent shortening of action potential (AP) duration was examined in the fish (Carassius carassius L.) heart ventricle. Acute temperature change from +5 to +18 degrees C (heat stress) shortened AP duration from 2.8 +/- 0.3 to 1.3 +/- 0.1 s in intact ventricles. In 56% (18 of 32) of enzymatically isolated myocytes, heat stress also induced reversible opening of ATP-sensitive K+ channels and increased their single-channel conductance from 37 +/- 12 pS at +8 degrees C to 51 +/- 13 pS at +18 degrees C (Q10 = 1.38) (P < 0.01; n = 12). The ATP-sensitive K+ channels of the crucian carp ventricle were characterized by very low affinity to ATP both at +8 degrees C [concentration of Tris-ATP that produces half-maximal inhibition of the channel (K1/2)= 1.35 mM] and +18 degrees C (K1/2 = 1.85 mM). Although acute heat stress induced ATP-sensitive K+ current (IK,ATP) in patch-clamped myocytes, similar heat stress did not cause any glibenclamide (10 microM)-sensitive changes in AP duration in multicellular ventricular preparations. Examination of APs and K+ currents from the same myocytes by alternate recording under current-clamp and voltage-clamp modes revealed that changes in AP duration were closely correlated with temperature-specific changes in the voltage-dependent rectification of the background inward rectifier K+ current IK1. In approximately 15% of myocytes (4 out of 27), IK,ATP-dependent shortening of AP followed the IK1-induced AP shortening. Thus heat stress-induced shortening of AP duration in crucian carp ventricle is primarily dependent on IK1. IK,ATP is induced only in response to prolonged temperature elevation or perhaps in the presence of additional stressors.     

Is ATP Required for K+ Channel Activation in Vicia Guard Cells?

In vivo, K+ entry into guard cells via inward-rectifying K+ channels is indirectly driven by ATP via an H+-ATPase that hyperpolarizes the membrane potential. However, whether activation of the K+ channels of guard cells requires ATP remains unknown. In the present study, both whole-cell and single-channel patch-clamp techniques were used to address this question. Exogenous ATP, ADP, and adenosine-5[prime]-O-(3-thiotriphosphate) applied to the cytoplasm had no effect on whole-cell K+ currents of Vicia faba L. guard cells. Azide, an inhibitor of oxidative phosphorylation, also had no effect. However, an ATP-scavenging system, glucose plus hexokinase, inhibited whole-cell inward K+ currents by 30 to 40%. Single-channel results acquired from cytoplasm-free inside-out membrane patches showed definite activation of inward K+ channels by ATP. Other nucleotides, such as ADP, adenosine-5[prime]-O(3-thiotriphosphate), and GTP, did not increase channel activity in the membrane patches. Inward K+ channel activity in membrane patches preactivated by exogenous ATP was inhibited by glucose plus hexokinase. These results suggest that a low concentration of ATP is required for activation of the inward K+ channels of the guard-cell plasma membrane. The issue of how ATP as a signal regulates these K+ channels is discussed.     

Calcium-dependent inactivation of the ATP-sensitive K+ channel of rat ventricular myocytes

Single-channel currents were recorded from ATP-sensitive K+ channels in inside-out membrane patches excised from isolated rat ventricular myocytes. Perfusion of the internal surface of excised membrane patches with solutions which contained between 5 and 100 microM free calcium caused the loss of K+ATP channel activity which was not reversed when the membranes were washed with Ca-free solution. K+ATP channel activity could be recovered by bathing the patches in Mg.ATP. The loss of K+ATP channel activity provoked by internal calcium was a process which occurred over a time scale of seconds. Channel closure evoked by internal ATP was essentially instantaneous. The speed of K+ATP channel inactivation increased with the concentration of calcium. Neither a phosphatase inhibitor (fluoride ions) nor a proteinase inhibitor (leupeptin) had any effect upon the loss of K+ channel activity stimulated by internal calcium.     

Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles

A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fueled calcium uptake (nanomoles per milligram protein). Furthermore, the calcium uptake fueled by the endogenous glycolytic cascade persisted in the presence of a hexokinase-based ATP trap which eliminated calcium uptake fueled by exogenously added ATP. Thus, it appears that the endogenous glycolytic cascade fuels calcium uptake in PMV via a membrane-associated pool of ATP and not via an exchange of ATP with the bulk solution. To determine whether ATP produced endogenously was utilized preferentially by the calcium pump, the ATP production rates of the endogenous creatine kinase and pyruvate kinase were matched to that of glycolysis and the calcium uptake fueled by the endogenous sources was compared with that fueled by exogenous ATP added at the same rate. The rate of calcium uptake fueled by endogenous sources of ATP was approximately twice that supported by exogenously added ATP, indicating that the calcium pump preferentially utilizes ATP produced by membrane-bound enzymes.     

ATP-sensitive potassium channel traffic regulation by adenosine and protein kinase C

ATP-sensitive potassium (K(ATP)) channels activate under metabolic stress to protect neurons and cardiac myocytes. However, excessive channel activation may cause arrhythmia in the heart and silence neurons in the brain. Here, we report that PKC-mediated downregulation of K(ATP) channel number, via dynamin-dependent channel internalization, can act as a brake mechanism to control K(ATP) activation. A dileucine motif in the pore-lining Kir6.2 subunit of K(ATP), but not the site of PKC phosphorylation for channel activation, is essential for PKC downregulation. Whereas K(ATP) activation results in a rapid shortening of the action potential duration (APD) in metabolically inhibited ventricular myocytes, adenosine receptor stimulation and consequent PKC-mediated K(ATP) channel internalization can act as a brake to lessen this APD shortening. Likewise, in hippocampal CA1 neurons under metabolic stress, PKC-mediated, dynamin-dependent K(ATP) channel internalization can also act as a brake to dampen the rapid decline of excitability due to K(ATP) activation.     

Glucose requirement for postischemic recovery of perfused working heart

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.     

Age-related changes in ATP-producing pathways in human skeletal muscle in vivo.

Energy for muscle contractions is supplied by ATP generated from 1) the net hydrolysis of phosphocreatine (PCr) through the creatine kinase reaction, 2) oxidative phosphorylation, and 3) anaerobic glycolysis. The effect of old age on these pathways is unclear. The purpose of this study was to examine whether age may affect ATP synthesis rates from these pathways during maximal voluntary isometric contractions (MVIC). Phosphorus magnetic resonance spectroscopy was used to assess high-energy phosphate metabolite concentrations in skeletal muscle of eight young (20-35 yr) and eight older (65-80 yr) men. Oxidative capacity was assessed from PCr recovery after a 16-s MVIC. We determined the contribution of each pathway to total ATP synthesis during a 60-s MVIC. Oxidative capacity was similar across age groups. Similar rates of ATP synthesis from PCr hydrolysis and oxidative phosphorylation were observed in young and older men during the 60-s MVIC. Glycolytic flux was higher in young than older men during the 60-s contraction (P < 0.001). When expressed relative to the overall ATP synthesis rate, older men relied on oxidative phosphorylation more than young men (P = 0.014) and derived a smaller proportion of ATP from anaerobic glycolysis (P < 0.001). These data demonstrate that although oxidative capacity was unaltered with age, peak glycolytic flux and overall ATP production from anaerobic glycolysis were lower in older men during a high-intensity contraction. Whether this represents an age-related limitation in glycolytic metabolism or a preferential reliance on oxidative ATP production remains to be determined.     

Functional Coupling between Nucleoside Diphosphate Kinase of the Outer Mitochondrial Compartment and Oxidative Phosphorylation

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.     

Interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation and creatine kinase.

Previous work led to the conclusion that, during oxidative phosphorylation, mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from rat brain was dependent on intramitochondrially compartmented ATP as substrate. The present study demonstrated that, when oxidative phosphorylation was functioning concurrently, mitochondrial creatine kinase could also generate intramitochondrial ATP serving as substrate for hexokinase. In the absence of concurrent oxidative phosphorylation, the kinetics of glucose phosphorylation with ATP generated by creatine kinase were not consistent with the supply of ATP from a saturable intramitochondrial compartment as formed during oxidative phosphorylation. Evidence for intramitochondrially compartmented ATP, generated by creatine kinase, was obtained; this was distinct from compartmented ATP generated by oxidative phosphorylation in terms of kinetics of generation of the compartment and its capacity, sensitivity to release by carboxyatractyloside, and sensitivity to disruption by digitonin. That oxidative phosphorylation did induce a dependence on intramitochondrial ATP as a substrate was further indicated by the observation that, although the initial rate of glucose phosphorylation by mitochondrial hexokinase depended on the extramitochondrial concentration of ATP present at the time oxidative phosphorylation was initiated, a final steady state rate of glucose phosphorylation was attained that was independent of extramitochondrial ATP levels. These and previous results emphasize the probable importance of nucleotide compartmentation in regulation of cerebral glycolytic and oxidative metabolism.     

急性低温/再复温对大鼠心室肌膜电位和钾电流的影响

本文旨在研究急性低温/再复温对大鼠心室肌膜电位和钾电流的影响.膜电位和膜电流分别在全细胞膜片钳的电压钳和电流钳模式下记录.当细胞外灌流液从25℃降低到4℃后,一过性外向电流(transient outward current, Ito)完全消失,膜电位为 60mV时的稳态外向K 电流(sustained outward K current, Iss)和膜电位为-120mV时的内向整流K 电流(inward rectifier K current, IK1)分别降低(48.5±14.1)%和(35.7±18.2)%,同时,膜电位绝对值降低.当细胞外灌流液从4℃再升高到36℃后,膜电位出现一过性超级化,然后恢复到静息电位水平;在58个细胞中,有36个细胞伴随复温出现ATP-敏感性K (ATP-sensitive K , KATP)通道的激活.再复温引起的上述变化可以被Na /K -ATP酶抑制剂哇巴因(100μmol/L)所抑制.再复温引起的KATP通道激活也能被蛋白激酶A抑制剂H-89(100μmol/L)所抑制.在细胞膜电位被钳制在0mV时,当细胞外灌流液温度从25℃降低到4℃后,细胞的体积没有发生明显改变,但当再复温引起KATP通道激活后,细胞很快发生皱缩,同时细胞内部出现许多折光较强的斑点.上述结果表明急性低温/再复温对大鼠心室肌膜电位和K 电流有明显影响,并提示KATP通道激活可能与心肌低温/再复温损伤有关.     

AMP deamination delays muscle acidification during heavy exercise and hypoxia

In silico studies carried out by using a computer model of oxidative phosphorylation and anaerobic glycolysis in skeletal muscle demonstrated that deamination of AMP to IMP during heavy short term exercise and/or hypoxia lessens the acidification of myocytes. The concerted action of adenylate kinase and AMP deaminase, leading to a decrease in the total adenine nucleotide pool, constitutes an additional process consuming ADP and producing ATP. It diminishes the amount of ADP that must be converted to ATP by other processes in order to meet the rate of ADP production by ATPases (because the adenylate kinase + AMP deaminase system produces only 1 ATP per 2 ADPs used, ATP consumption is not matched by ATP production, and the reduction of the total adenine nucleotide pool occurs mostly at the cost of [ATP]). As a result, the rate of ADP consumption by other processes may be lowered. This effect concerns mostly ADP consumption by anaerobic glycolysis that is inhibited by AMP deamination-induced decrease in [ADP] and [AMP], and not oxidative phosphorylation, because during heavy exercise and/or hypoxia [ADP] is significantly greater than the Km value of this process for ADP. The resultant reduction of proton production by anaerobic glycolysis enables us to delay the termination of exercise because of fatigue and/or to diminish cell damage.     

Theoretical phosphorylation rates after addition of a small amount of glucose to intact ascites tumor cells

Changes were measured in glycolytic and respiratory rates during the entire period of glycolysis and respiratory inhibition after addition of 0.08 or 0.15 mM glucose to Ehrlich ascites carcinoma cells in 54 mM phosphate buffer (pH 7.3) at 37°. Glycolytic products fully accounted for the glucose utilized.

Theoretical rates of glycolytic ATP synthesis were calculated from the rates of accumulation of glycolytic products, and rates of oxidative phosphorylation were calculated from respiratory rates, assuming a P:O ratio of 3.0. The maximum in the glycolytic phosphorylation rate curve preceded the minimum in the respiratory phosphorylation rate curve. As a consequence, the total phosphorylation rate curve was biphasic, first rising above, then falling below, and finally returning to the initial, pre-glucose rate. The area under the early rise approximately equalled the area above the later dip and corresponded to between 1 and 2 μmoles of ATP/ml cells. The low rate of change in the ATP content of the cells indicated that most of the change in phosphorylation rate represented changes in both ATP synthesis and ATP utilization.

It is hypothesized that ATP synthesized by glycolysis is more readily available to the ATP-utilizing systems. On addition of glucose, ATP is shifted from a respiratory to a glycolytic reservoir and a period of more rapid ATP utilization associated with a decrease in the level of endogenous substrates involved in the ATP-utilizing reactions ensues; after cessation of glycolysis, the process is reversed, and ATP utilization is slowed for a period while the endogenous substrates increase again.     

Impaired activation of ATP-sensitive K+ channels in endocardial myocytes from left ventricular hypertrophy

ATP-sensitive K(+) (K(ATP)) channels are essential for maintaining the cellular homeostasis against metabolic stress. Myocardial remodeling in various pathologies may alter this adaptive response to such stress. It was reported that transmural electrophysiological heterogeneity exists in ventricular myocardium. Therefore, we hypothesized that the K(ATP) channel properties might be altered in hypertrophied myocytes from endocardium. To test this hypothesis, we determined the K(ATP) channel currents using the perforated patch-clamp technique, open cell-attached patches, and excised inside-out patches in both endocardial and epicardial myocytes isolated from hypertrophied [spontaneous hypertensive rats (SHR)] vs. normal [Wistar-Kyoto rats (WKY)] left ventricle. In endocardial cells, K(ATP) channel currents (I(K,ATP)), produced by 2 mM CN(-) and no glucose at 0 mV, were significantly smaller (P < 0.01), and time required to reach peak currents after onset of K(ATP) channel opening (Time(onset to peak)) was significantly longer (319 +/- 46 vs. 177 +/- 37 s, P = 0.01) in the SHR group (n = 9) than the WKY group (n = 13). However, in epicardial cells, there were no differences in I(K,ATP) and Time(onset to peak) between the groups (SHR, n = 12; WKY, n = 12). The concentration-open probability-response curves obtained during the exposure of open cells and excised patches to exogenous ATP revealed the impaired K(ATP) channel activation in endocardial myocytes from SHR. In conclusion, K(ATP) channel activation under metabolic stress was impaired in endocardial cells from rat hypertrophied left ventricle. The deficit of endocardial K(ATP) channels to decreased intracellular ATP might contribute to the maladaptive response of hypertrophied hearts to ischemia.     

The glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, and pyruvate kinase are components of the K(ATP) channel macromolecular complex and regulate its function

The regulation of ATP-sensitive potassium (K(ATP)) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K(ATP) channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K(ATP) channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K(ATP) channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K(ATP) channel complex) causes channel closure.     

Estimation of effective concentrations of ATP-regenerating enzymes in cilia of Paramecium caudatum

The phosphoarginine shuttle system effectively regenerates ATP in the cilia of Paramecium caudatum. To estimate the effective concentration of ATP‐regenerating enzymes, we attempted to reconstitute certain ATP‐regenerating systems within the cilia of intact cortical sheets using exogenous enzymes and high‐energy substances. The addition of phosphoenolpyruvate, which is one of the substrates in glycolysis, did not increase the ciliary beat frequency, whereas phosphocreatine together with exogenous creatine kinase, effectively increased the ciliary beat frequency. In the presence of 0.6 mg/ml creatine kinase and 0.4 mM phosphocreatine, the ciliary beat frequency was comparable to that produced by the addition of phosphoarginine. This result indicates that the reconstituted phosphocreatine shuttle system can work as an artificial ATP‐regenerating system for ciliary movements. The effective concentration of creatine kinase in the reconstituted phosphocreatine shuttle system was estimated to be about 7.4 μM based on the molecular mass of creatine kinase (MW 81,000). Therefore, the effective concentration of arginine kinase in the cilia of live Paramecium is approximately 10 μM. This estimated concentration of intraciliary arginine kinase is sufficient to maintain a high ATP concentration throughout the cilia of P. caudatum.