First and second trimester urinary metabolic profiles and fetal growth restriction: an exploratory nested case-control study within the infant development and environment study.

BACKGROUND: Routine prenatal care fails to identify a large proportion of women at risk of fetal growth restriction (FGR). Metabolomics, the comprehensive analysis of low molecular weight molecules (metabolites) in biological samples, can provide new and earlier biomarkers of prenatal health. Recent research has suggested possible predictive first trimester urine metabolites correlating to fetal growth restriction in the third trimester. Our objective in this current study was to examine urinary metabolic profiles in the first and second trimester of pregnancy in relation to third trimester FGR in a US population from a large, multi-center cohort study of healthy pregnant women. METHODS: We conducted a nested case-control study within The Infant Development and the Environment Study (TIDES), a population-based multi-center pregnancy cohort study. We identified 53 cases of FGR based on the AUDIPOG [Neonatal growth - AUDIPOG [Internet]. [cited 29 Nov 2016]. Available from: http://www.audipog.net/courbes_morpho.php?langue=en ] formula for birthweight percentile considering maternal height, age, and prenatal weight, as well as infant sex, gestational age, and birth rank. Cases were matched to 106 controls based on study site, maternal age (± 2 years), parity, and infant sex. NMR spectroscopy was used to assess concentrations of four urinary metabolites that have been previously associated with FGR (tyrosine, acetate, formate, and trimethylamine) in first and second trimester urine samples. We fit multivariate conditional logistic regression models to estimate the odds of FGR in relation to urinary concentrations of these individual metabolites in the first and second trimesters. Exploratory analyses of custom binned spectroscopy results were run to consider other potentially related metabolites. RESULTS: We found no significant association between the relative concentrations of each of the four metabolites and odds of FGR. Exploratory analyses did not reveal any significant differences in urinary metabolic profiles. Compared with controls, cases delivered earlier (38.6 vs 39.8, p < 0.001), and had lower birthweights (2527 g vs 3471 g, p < 0.001). Maternal BMI was similar between cases and controls. CONCLUSIONS: First and second trimester concentrations of urinary metabolites (acetate, formate, trimethylamine and tyrosine) did not predict FGR. This inconsistency with previous studies highlights the need for more rigorous investigation and data collection in this area before metabolomics can be clinically applied to obstetrics.

Modular organization of cardiac energy metabolism: energy conversion, transfer and feedback regulation.

To meet high cellular demands, the energy metabolism of cardiac muscles is organized by precise and coordinated functioning of intracellular energetic units (ICEUs). ICEUs represent structural and functional modules integrating multiple fluxes at sites of ATP generation in mitochondria and ATP utilization by myofibrillar, sarcoplasmic reticulum and sarcolemma ion-pump ATPases. The role of ICEUs is to enhance the efficiency of vectorial intracellular energy transfer and fine tuning of oxidative ATP synthesis maintaining stable metabolite levels to adjust to intracellular energy needs through the dynamic system of compartmentalized phosphoryl transfer networks. One of the key elements in regulation of energy flux distribution and feedback communication is the selective permeability of mitochondrial outer membrane (MOM) which represents a bottleneck in adenine nucleotide and other energy metabolite transfer and microcompartmentalization. Based on the experimental and theoretical (mathematical modelling) arguments, we describe regulation of mitochondrial ATP synthesis within ICEUs allowing heart workload to be linearly correlated with oxygen consumption ensuring conditions of metabolic stability, signal communication and synchronization. Particular attention was paid to the structure-function relationship in the development of ICEU, and the role of mitochondria interaction with cytoskeletal proteins, like tubulin, in the regulation of MOM permeability in response to energy metabolic signals providing regulation of mitochondrial respiration. Emphasis was given to the importance of creatine metabolism for the cardiac energy homoeostasis.

Profile of Circulatory Metabolites in a Relapsing-remitting Animal Model of Multiple Sclerosis using Global Metabolomics.

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the CNS. Although, MS is well characterized in terms of the role played by immune cells, cytokines and CNS pathology, nothing is known about the metabolic alterations that occur during the disease process in circulation. Recently, metabolic aberrations have been defined in various disease processes either as contributing to the disease, as potential biomarkers, or as therapeutic targets. Thus in an attempt to define the metabolic alterations that may be associated with MS disease progression, we profiled the plasma metabolites at the chronic phase of disease utilizing relapsing remitting-experimental autoimmune encephalomyelitis (RR-EAE) model in SJL mice. At the chronic phase of the disease (day 45), untargeted global metabolomic profiling of plasma collected from EAE diseased SJL and healthy mice was performed, using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry. A total of 282 metabolites were identified, with significant changes observed in 44 metabolites (32 up-regulated and 12 down-regulated), that mapped to lipid, amino acid, nucleotide and xenobiotic metabolism and distinguished EAE from healthy group (p<0.05, false discovery rate (FDR)<0.23). Mapping the differential metabolite signature to their respective biochemical pathways using the Kyoto Encyclopedia of Genes and Genomics (KEGG) database, we found six major pathways that were significantly altered (containing concerted alterations) or impacted (containing alteration in key junctions). These included bile acid biosynthesis, taurine metabolism, tryptophan and histidine metabolism, linoleic acid and D-arginine metabolism pathways. Overall, this study identified a 44 metabolite signature drawn from various metabolic pathways which correlated well with severity of the EAE disease, suggesting that these metabolic changes could be exploited as (1) biomarkers for EAE/MS progression and (2) to design new treatment paradigms where metabolic interventions could be combined with present and experimental therapeutics to achieve better treatment of MS.

Cellular energetics in the preconditioned state: protective role for phosphotransfer reactions captured by 18O-assisted 31P NMR.

Cell survival is critically dependent on the preservation of cellular bioenergetics. However, the metabolic mechanisms that confer resistance to injury are poorly understood. Phosphotransfer reactions integrate ATP-consuming with ATP-producing processes and could thereby contribute to the generation of a protective phenotype. Here, we used ischemic preconditioning to induce a stress-tolerant state and (18)O-assisted (31)P nuclear magnetic resonance spectroscopy to capture intracellular phosphotransfer dynamics. Preconditioning of isolated perfused hearts triggered a redistribution in phosphotransfer flux with significant increase in creatine kinase and glycolytic rates. High energy phosphoryl fluxes through creatine kinase, adenylate kinase, and glycolysis in preconditioned hearts correlated tightly with post-ischemic functional recovery. This was associated with enhanced metabolite exchange between subcellular compartments, manifested by augmented transfer of inorganic phosphate from cellular ATPases to mitochondrial ATP synthase. Preconditioning-induced energetic remodeling protected cellular ATP synthesis and ATP consumption, improving contractile performance following ischemia-reperfusion insult. Thus, the plasticity of phosphotransfer networks contributes to the effective functioning of the cellular energetic system, providing a mechanism for increased tolerance toward injury.

Signaling in channel/enzyme multimers: ATPase transitions in SUR module gate ATP-sensitive K+ conductance.

ATP-sensitive potassium (K(ATP)) channels are bifunctional multimers assembled by an ion conductor and a sulfonylurea receptor (SUR) ATPase. Sensitive to ATP/ADP, K(ATP) channels are vital metabolic sensors. However, channel regulation by competitive ATP/ADP binding would require oscillations in intracellular nucleotides incompatible with cell survival. We found that channel behavior is determined by the ATPase-driven engagement of SUR into discrete conformations. Capture of the SUR catalytic cycle in prehydrolytic states facilitated pore closure, while recruitment of posthydrolytic intermediates translated in pore opening. In the cell, channel openers stabilized posthydrolytic states promoting K(ATP) channel activation. Nucleotide exchange between intrinsic ATPase and ATP/ADP-scavenging systems defined the lifetimes of specific SUR conformations gating K(ATP) channels. Signal transduction through the catalytic module provides a paradigm for channel/enzyme operation and integrates membrane excitability with metabolic cascades.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels.

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (K(ATP)) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with K(ATP) channel-dependent membrane excitability remains elusive. Here, we identify that the response of K(ATP) channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the K(ATP) channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and K(ATP) channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with K(ATP) channels and associated functions.

Directed inhibition of nuclear import in cellular hypertrophy.

Each nuclear pore is responsible for both nuclear import and export with a finite capacity for bidirectional transport across the nuclear envelope. It remains poorly understood how the nuclear transport pathway responds to increased demands for nucleocytoplasmic communication. A case in point is cellular hypertrophy in which increased amounts of genetic material need to be transported from the nucleus to the cytosol. Here, we report an adaptive down-regulation of nuclear import supporting such an increased demand for nuclear export. The induction of cardiac cell hypertrophy by phenylephrine or angiotensin II inhibited the nuclear translocation of H1 histones. The removal of hypertrophic stimuli reversed the hypertrophic phenotype and restored nuclear import. Moreover, the inhibition of nuclear export by leptomycin B rescued import. Hypertrophic reprogramming increased the intracellular GTP/GDP ratio and promoted the nuclear redistribution of the GTP-binding transport factor Ran, favoring export over import. Further, in hypertrophy, the reduced creatine kinase and adenylate kinase activities limited energy delivery to the nuclear pore. The reduction of activities was associated with the closure of the cytoplasmic phase of the nuclear pore preventing import at the translocation step. Thus, to overcome the limited capacity for nucleocytoplasmic transport, cells requiring increased nuclear export regulate the nuclear transport pathway by undergoing a metabolic and structural restriction of nuclear import.

Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement.

Efficient cellular energy homeostasis is a critical determinant of muscle performance, providing evolutionary advantages responsible for species survival. Phosphotransfer reactions, which couple ATP production and utilization, are thought to play a central role in this process. Here, we provide evidence that genetic disruption of AK1-catalyzed ss-phosphoryl transfer in mice decreases the potential of myofibers to sustain nucleotide ratios despite up-regulation of high-energy phosphoryl flux through glycolytic, guanylate and creatine kinase phosphotransfer pathways. A maintained contractile performance of AK1-deficient muscles was associated with higher ATP turnover rate and larger amounts of ATP consumed per contraction. Metabolic stress further aggravated the energetic cost in AK1(-/-) muscles. Thus, AK1-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy, enabling skeletal muscle to perform at the lowest metabolic cost.

ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex.

ATP-sensitive K+ (KATP) channels are unique metabolic sensors formed by association of Kir6.2, an inwardly rectifying K+ channel, and the sulfonylurea receptor SUR, an ATP binding cassette protein. We identified an ATPase activity in immunoprecipitates of cardiac KATP channels and in purified fusion proteins containing nucleotide binding domains NBD1 and NBD2 of the cardiac SUR2A isoform. NBD2 hydrolyzed ATP with a twofold higher rate compared to NBD1. The ATPase required Mg2+ and was insensitive to ouabain, oligomycin, thapsigargin, or levamisole. K1348A and D1469N mutations in NBD2 reduced ATPase activity and produced channels with increased sensitivity to ATP. KATP channel openers, which bind to SUR, promoted ATPase activity in purified sarcolemma. At higher concentrations, openers reduced ATPase activity, possibly through stabilization of MgADP at the channel site. K1348A and D1469N mutations attenuated the effect of openers on KATP channel activity. Opener-induced channel activation was also inhibited by the creatine kinase/creatine phosphate system that removes ADP from the channel complex. Thus, the KATP channel complex functions not only as a K+ conductance, but also as an enzyme regulating nucleotide-dependent channel gating through an intrinsic ATPase activity of the SUR subunit. Modulation of the channel ATPase activity and/or scavenging the product of the ATPase reaction provide novel means to regulate cellular functions associated with KATP channel opening.

Failing energetics in failing hearts.

The perpetual and vigorous nature of heart muscle work requires efficient myocardial energetics. This depends not only on adequate ATP production, but also on efficient delivery of ATP to muscle ATPases and rapid removal of ADP and other by-products of ATP hydrolysis. Indeed, recent evidence indicates that defects in communication between ATP-producing and ATP-consuming cellular sites are a major factor contributing to energetic deficiency in heart failure. In particular, the failing myocardium is characterized by reduced catalytic activity of creatine kinase, adenylate kinase, carbonic anhydrase, and glycolytic enzymes, which collectively facilitate ATP delivery and promote removal of ADP, Pi, and H+ from cellular ATPases. Although energy transfer through adenylate kinase and glycolytic enzymes has been recognized as an adaptive mechanism supporting compromised muscle energetics, in the failing myocardium the total compensatory potential of these systems is diminished. A gradual accumulation of defects at various steps in myocardial energetic signaling, along with compromised compensatory mechanisms, precipitates failure of the whole cardiac energetic system, ultimately contributing to myocardial dysfunction. These advances in our understanding of the molecular bioenergetics in heart failure provide a new perspective toward improving the energetic balance of the failing myocardium.

Compromised energetics in the adenylate kinase AK1 gene knockout heart under metabolic stress.

Rapid exchange of high energy carrying molecules between intracellular compartments is essential in sustaining cellular energetic homeostasis. Adenylate kinase (AK)-catalyzed transfer of adenine nucleotide beta- and gamma-phosphoryls has been implicated in intracellular energy communication and nucleotide metabolism. To demonstrate the significance of this reaction in cardiac energetics, phosphotransfer dynamics were determined by [(18)O]phosphoryl oxygen analysis using( 31)P NMR and mass spectrometry. In hearts with a null mutation of the AK1 gene, which encodes the major AK isoform, total AK activity and beta-phosphoryl transfer was reduced by 94% and 36%, respectively. This was associated with up-regulation of phosphoryl flux through remaining minor AK isoforms and the glycolytic phosphotransfer enzyme, 3-phosphoglycerate kinase. In the absence of metabolic stress, deletion of AK1 did not translate into gross abnormalities in nucleotide levels, gamma-ATP turnover rate or creatine kinase-catalyzed phosphotransfer. However, under hypoxia AK1-deficient hearts, compared with the wild type, had a blunted AK-catalyzed phosphotransfer response, lowered intracellular ATP levels, increased P(i)/ATP ratio, and suppressed generation of adenosine. Thus, although lack of AK1 phosphotransfer can be compensated in the absence of metabolic challenge, under hypoxia AK1-knockout hearts display compromised energetics and impaired cardioprotective signaling. This study, therefore, provides first direct evidence that AK1 is essential in maintaining myocardial energetic homeostasis, in particular under metabolic stress.

Structural plasticity of the cardiac nuclear pore complex in response to regulators of nuclear import.

Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca2+ stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca2+ indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca2+ homeostasis.

Adenylate kinase-catalyzed phosphotransfer in the myocardium : increased contribution in heart failure.

Although the downregulation of creatine kinase activity has been associated with heart failure, creatine kinase-deficient transgenic hearts have a preserved contractile function. This suggests the existence of alternative phosphotransfer pathways in the myocardium, the identity of which is still unknown. In this study, we examined the contribution of adenylate kinase-catalyzed phosphotransfer to myocardial energetics. In the isolated mitochondria/actomyosin system, which possesses endogenous adenylate kinase activity in both compartments, substrates for adenylate kinase promoted the rate and amplitude of actomyosin contraction that was further enhanced by purified adenylate kinase. Inhibition of adenylate kinase activity diminished both actomyosin contraction and mitochondrial respiration, which indicated reduced energy flow between mitochondria and myofibrils. In intact myocardium, the net adenylate kinase-catalyzed phosphotransfer rate was 10% of the total ATP turnover rate as measured by 18O-phosphoryl labeling in conjunction with gas chromatography and mass spectrometry. In pacing-induced failing heart, adenylate kinase-catalyzed phosphotransfer increased by 134% and contributed 21% to the total ATP turnover. Concomitantly, the contribution by creatine kinase dropped from 89% in normal hearts to 40% in failing hearts. These phosphotransfer changes were associated with reduced levels of metabolically active ATP but maintained overall ATP turnover rate. Thus, this study provides evidence that adenylate kinase facilitates the transfer of high-energy phosphoryls and signal communication between mitochondria and actomyosin in cardiac muscle, with an increased contribution to cellular phosphotransfer in heart failure. This phosphotransfer function renders adenylate kinase an important component for optimal myocardial bioenergetics and a compensatory mechanism in response to impaired intracellular energy flux in the failing heart.

Reduced activity of enzymes coupling ATP-generating with ATP-consuming processes in the failing myocardium.

Coupling of ATP-generating with ATP-consuming processes is an essential component in the cardiac bioenergetics responsible for optimal myocardial function. Although a number of enzymatic systems have been implicated in securing proper intracellular energy communication, their integrative response in a failing myocardium has not been determined so far. Therefore, we measured catalytic activities of enzymes responsible for the communication between ATP-generating and ATP-consuming processes in ventricular samples obtained from normal dogs and dogs with tachycardia-induced heart failure. In the failing myocardium, phosphotransfer activities of creatine kinase, adenylate kinase, 3-phosphoglycerate kinase and pyruvate kinase, which collectively deliver ATP and remove ADP from myofibrillar ATPases, were depressed by 30, 21, 44 and 20%, respectively, when compared to normal controls. The activity of hexokinase, an enzyme which directs phosphoryls into the glycolytic phosphotransfer pathway, was unchanged. Also, the activity of glyceraldehyde-3-phosphate dehydrogenase, which may shuttle inorganic phosphate between ATPases and ATP-synthases, was not affected by heart failure. However, the CO2-hydration activity of carbonic anhydrase, which together with creatine kinase, is presumed responsible for removal of protons from ATPases, was diminished by 21%. As these enzymatic systems are collectively required for adequate delivery of high-energy phosphoryl to, and removal of end-products from, cellular ATPases, the cumulative deficit in their flux capacities may provide a bioenergetic basis for impaired contraction-relaxation in the failing heart.

Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function.

Discovered in the cardiac sarcolemma, ATP-sensitive K+ (KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATP channel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+ channel openers to examine the effect of mitochondrial KATP channel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+ transport, and matrix volume. From a mitochondrial membrane potential of -180 +/- 15 mV, K+ channel openers, pinacidil (100 microM), cromakalim (25 microM), and levcromakalim (20 microM), induced membrane depolarization by 10 +/- 7, 25 +/- 9, and 24 +/- 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+ or application of a KATP channel blocker. K+ channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+ channel opener released Ca2+ from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+ concentration and sensitive to KATP channel blockade. In addition, K+ channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome c and adenylate kinase. Thus, in isolated cardiac mitochondria, KATP channel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATP channels in regulating functions vital for the cardiac mitochondria.

Adenylate kinase: kinetic behavior in intact cells indicates it is integral to multiple cellular processes.

Monitoring the kinetic behavior of adenylate kinase (AK) and creatine kinase (CK) in intact cells by 18O-phosphoryl oxygen exchange analysis has provided new perspectives from which to more fully define the involvement of these phosphotransferases in cellular bioenergetics. A primary function attributable to both AK and CK is their apparent capability to couple ATP utilization with its generation by glycolytic and/or oxidative processes depending on cell metabolic status. This is evidenced by the observation that the sum of the net AK- plus CK-catalyzed phosphoryl transfer is equivalent to about 95% of the total ATP metabolic flux in non-contracting rat diaphragm; under basal conditions almost every newly generated ATP molecule appears to be processed by one or the other of these phosphotransferases prior to its utilization. Although CK accounts for the transfer of a majority of the ATP molecules generated/consumed in the basal state there is a progressive, apparently compensatory, shift in phosphotransfer catalysis from the CK to the AK system with increasing muscle contraction or graded chemical inhibition of CK activity. AK and CK appear therefore to provide similar and interrelated functions. Evidence that high energy phosphoryl transfer in some cell types or metabolic states can also be provided by specific nucleoside mono- and diphosphate kinases and by the phosphotransfer capability inherent to the glycolytic system has been obtained. Measurements by 18O-exchange analyses of net AK- and CK-catalyzed phosphoryl transfer in conjunction with 31P NMR analyses of total unidirectional phosphoryl flux show that each new energy-bearing molecule CK or AK generates subsequently undergoes about 50 or more unidirectional CK-or AK-catalyzed phosphotransfers en route to an ATP consumption site in intact muscle. This evidence of multiple enzyme catalyzed exchanges coincides with the mechanism of vectorial ligand conduction suggested for accomplishing intracellular high energy phosphoryl transfer by the AK and CK systems. AK-catalyzed phosphotransfer also appears to be integral to the transduction of metabolic signals influencing the operation of ion channels regulated by adenine nucleotides such as ATP-inhibitable K+ channels in insulin secreting cells; transition from the ATP to ADP liganded states closely coincides with the rate AK-catalyzes phosphotransfer transforming ATP (+AMP) to (2)ADP.

Phosphotransfer reactions in the regulation of ATP-sensitive K+ channels.

ATP-sensitive K+ (K(ATP)) channels are nucleotide-gated channels that couple the metabolic status of a cell with membrane excitability and regulate a number of cellular functions, including hormone secretion and cardioprotection. Although intracellular ATP is the endogenous inhibitor of K(ATP) channels and ADP serves as the channel activator, it is still a matter of debate whether changes in the intracellular concentrations of ATP, ADP, and/or in the ATP/ADP ratio could account for the transition from the ATP-liganded to the ADP-liganded channel state. Here, we overview evidence for the role of cellular phosphotransfer cascades in the regulation of K(ATP) channels. The microenvironment of the K(ATP) channel harbors several phosphotransfer enzymes, including adenylate, creatine, and pyruvate kinases, as well as other glycolytic enzymes that are able to transfer phosphoryls between ATP and ADP in the absence of major changes in cytosolic levels of adenine nucleotides. These phosphotransfer reactions are governed by the metabolic status of a cell, and their phosphotransfer rate closely correlates with K(ATP) channel activity. Adenylate kinase catalysis accelerates the transition from ATP to ADP, leading to K(ATP) channel opening, while phosphotransfers driven by creatine and pyruvate kinases promote ADP to ATP transition and channel closure. Thus, through delivery and removal of adenine nucleotides at the channel site, phosphotransfer reactions could regulate ATP/ADP balance in the immediate vicinity of the channel and thereby the probability of K(ATP) channel opening. In this way, phosphotransfer reactions could provide a transduction mechanism coupling cellular metabolic signals with K(ATP) channel-associated functions.

Suppression of creatine kinase-catalyzed phosphotransfer results in increased phosphoryl transfer by adenylate kinase in intact skeletal muscle.

The kinetics of creatine kinase (CK) and adenylate kinase (AK) activities were monitored in intact diaphragm muscle by 18O phosphoryl oxygen exchange to assess whether these two phosphotransferases provide an interrelated function integral to high energy phosphoryl metabolism. This possibility was examined by quantitating the net rates of CK- and AK-catalyzed phosphoryl transfer in comparison to the total cellular ATP metabolic rate when CK activity in the intact diaphragm muscle was progressively inhibited by 2,4-dinitrofluorobenzene. In noncontracting muscle from untreated rats, net rates of CK- and AK-catalyzed phosphotransfer were equivalent to 88 and 7%, respectively, of the total ATP metabolic rate. These results were compared with reported 31P NMR analyses of total creatine phosphate flux to estimate that each creatine phosphate molecule produced undergoes about 50 unidirectional CK-catalyzed phosphotransfers in transit to an ATP consumption site in the intact muscles. Graded inhibition by 2,4-dinitrofluorobenzene of intracellular CK activity by up to 98% resulted in a progressive shift in phosphotransferase catalysis from the CK to the AK system; the sum of the net rates of phosphoryl transfer by combining the increasing AK and decreasing CK activities continued to approximate the total cellular ATP metabolic rate. These results indicate that in diaphragm muscle CK and AK operate as interrelated cellular high energy phosphoryl transfer systems through which the majority of newly generated ATP is processed prior to its utilization.