Direct determination of adp in hypoxic porcine carotid artery using <sup>31</sup>p nmr

Fisher, M. J.; Dillon, Patrick F.

doi:10.1002/nbm.1940010304

Cited by 25 publications

(22 citation statements)

References 15 publications

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“…1). Direct measurement of free ADP by 31 P-NMRS has been performed in a hypoxic coronary artery model using 31 P-NMRS; however, this requires scans to be averaged over a 30-min time period to maximize the signal-to-noise ratio (8). Free-ADP accumulation in skeletal muscle has been suggested to rise to ϳ0.3 mM (4) under high-energy demands, but this accumulation is transient due to the effectiveness of ATP synthesis pathways in skeletal muscle and to the rapid decline in energy demands due to fatigue.…”

Section: Discussionmentioning

confidence: 99%

³¹P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1

Hancock

Brault

Wiseman³

et al. 2005

American Journal of Physiology-Cell Physiology

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. 31 P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1. Am J Physiol Cell Physiol 288: C1298 -C1304, 2005. First published February 2, 2005 doi:10.1152/ajpcell.00621.2004.-Metabolic control within skeletal muscle is designed to limit ADP accumulation even during conditions where ATP demand is out of balance with ATP synthesis. This is accomplished by the reactions of adenylate kinase (AK; ADPϩ ADP 7 AMPϩATP) and AMP deaminase (AMPϩH 2O 3 NH 3ϩIMP), which limit ADP accumulation under these conditions. The purpose of this study was to determine whether AK deficiency (AK Ϫ/Ϫ ) would result in sufficient ADP accumulation to be visible using 31 P-NMRS during the high energy demands of frequent in situ tetanic contractions. To do this we examined the high-energy phosphates of the gastrocnemius muscle in the knockout mouse with AK1 Ϫ/Ϫ and wild-type (WT) control muscle over the course of 64 rapid (2/s) isometric tetanic contractions. Near-complete depletion of phosphocreatine was apparent after 16 contractions in both groups. By ϳ40 contractions, ADP was clearly visible in AK1 Ϫ/Ϫ muscle. This transient concentration of the NMR visible free ADP was estimated to be ϳ1.7 mM, and represents the first time free ADP has been directly measured in contracting skeletal muscle. Such an increase in free ADP is severalfold greater than previously thought to occur. This large accumulation of free ADP also represents a significant reduction in energy available from ATP, and has implications on cellular processes that depend on a high yield of energy from ATP such as calcium sequestration. Remarkably, the AK1 Ϫ/Ϫ and WT muscles exhibited similar fatigue profiles. Our findings suggest that skeletal muscle is surprisingly tolerant to a large increase in ADP and by extension, a decline in energy from ATP. muscle energetics; muscle relaxation; magnetic resonance spectroscopy IN NORMAL MUSCLE DURING EXERCISE the demand for ATP is adequately balanced by ATP supply from oxidative phosphorylation and glycolysis, coupled with the ATP buffering capacity of the creatine kinase reaction, such that ATP is not significantly depleted. Even when ATP demand does exceed ATP supply, and a decline in ATP is observed, the decline in ATP is not matched by a stoichiometric accumulation of ADP because of the action of adenylate kinase (AK), which transfers a phosphate from one ADP to another ADP resulting in ATP and AMP formation. In skeletal muscle the AMP formed from this reaction is rapidly deaminated by AMP deaminase (AMPD) resulting in the formation of inosine 5Ј-monophosphate (IMP). Thus, when the rate of ATP hydrolysis exceeds the ATP supply capacity, there is a stoichiometric accumulation of IMP, due to the coupled reactions of AK and AMPD. Therefore, at high energy demands, the reactions of AK and AMPD both serve to limit ADP accumulation.The importance of limiting ADP accumulation in skeletal muscle is implied by the apparent lack of such a limit on another product of ATP hydrolysis, in...

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Section: Discussionmentioning

confidence: 99%

³¹P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1

Hancock

Brault

Wiseman³

et al. 2005

American Journal of Physiology-Cell Physiology

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“…The lower concentration of Mg ++ in smooth muscle meant that β-ADP had a different chemical shift from γ-ATP, and ADP peaks could be seen under anoxic conditions in vascular smooth muscle. 3 Using the relative concentrations of ADP from these experiments, it was shown that smooth muscle CK is both at equilibrium, and has an equilibrium constant inside cells consistent with its in vitro determination. Given this, the results presented herein are unexpected.…”

Section: See Related Article P 68-73mentioning

confidence: 63%

Equilibrium Enzymes in Regulatory Systems

Dillon

2014

Hypertension

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T he work by Karamat et al 1 contains evidence that creatine kinase (CK) mRNA in vascular smooth muscle has a direct correlation with blood pressure. This result is both interesting and problematic: there is no previous evidence from in vitro experiments that smooth muscle CK is a regulatory enzyme, and there is considerable evidence that the enzyme is at equilibrium inside cells. As the data indicate a regulatory role for the equilibrium enzyme CK in control of blood pressure, the general problem of investigating the role of an equilibrium enzyme in a regulatory system is inherently intriguing.Cytosolic CK has been always considered an equilibrium enzyme, whether in striated or smooth muscle. This assumption was used to calculate the free concentration of ADP in cells, in which the concentrations of ATP, PCr, and creatine could be determined. Phosphate NMR was used to measure the relative free concentrations of ATP, PCr, and Pi, and from the chemical shifts of Pi and β-ATP, the pH and free Mg ++ of the cell could also be determined. Coupled with chemical measurements of creatine, and using in vitro measurements of the CK equilibrium constant, the free concentration of ADP (and thus the free energy of the cell) could be calculated. Measurements of free ADP cannot be made from chemical analyses because ADP liberated from f-actin compromises these measurements.2 ADP could not be seen as a peak in striated muscle phosphate NMR because of both its low concentration and chemical shift similarity to the ATP resonances. The lower concentration of Mg ++ in smooth muscle meant that β-ADP had a different chemical shift from γ-ATP, and ADP peaks could be seen under anoxic conditions in vascular smooth muscle.3 Using the relative concentrations of ADP from these experiments, it was shown that smooth muscle CK is both at equilibrium, and has an equilibrium constant inside cells consistent with its in vitro determination. Given this, the results presented herein are unexpected.All living biological systems have to exist at free energy levels greater than that of the local ambient energy. Systems can only exist at a steady state in this elevated energy state with the constant input of energy exactly balancing the increase in entropy of the system over time. Maintenance of a living system requires that the system be in a nonequilibrium state. However, this requirement does not mean that every part of the system must always be in a nonequilibrium state. In the glycolytic pathway for instance, hexokinase, phosphofructokinase, and pyruvate kinase all have free energies that indicate nonequilibrium states, while all of the other enzymes are at equilibrium. 4 Furthermore, PFK is a regulatory enzyme, exhibiting rate changes produced by nonsubstrate molecules as well as crossover conditions in which the rate can be shown to increase even when the substrate concentration decreases. 5 CK has never been shown to have these properties. Thus, an increase in energy flux tied to CK concentrations can only occur if the flux is rate limited by...

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“…Both the NMR [9, 12]and mechanics [6, 13]experiments employed techniques that our laboratory has used previously.…”

Section: Methodsmentioning

confidence: 99%

“…MgADP has also been proposed as a regulatory agent in the control of smooth muscle contraction [8], where an increase in MgADP is correlated with a decrease in force. 31 P-NMR allows simultaneous measurement of both P i and MgADP within tissues [9], making it possible to ascertain which of these two metabolites has a greater effect on smooth muscle contractions. In addition to the direct measurements of P i , ATP and phosphocreatine (PCr), other metabolites that can be determined simultaneously through mathematical calculations include pH [10], free Mg 2+ [10], total free ADP, MgATP and Mg-free ATP, and MgADP and Mg-free ADP [9]and the free energy of ATP hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

“…These parameters can all be compared to the force developed by muscle in order to determine their relative effects on the force of contraction. Direct measurements of free ADP in vascular smooth muscle (VSM) have allowed calculation of an in vivo equilibrium constant that can be used to infer the concentrations of ADP with a greater degree of confidence than in other intact tissues [9]. Potential regulatory mechanisms can be clarified through our primary purpose in ascertaining the relative influence of P i , MgADP, ATP, PCr, pH, free Mg 2+ , and free energy on the force development of intact VSM.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Influence of Cellular Energy Metabolism on Contractions of Porcine Carotid Artery Smooth Muscle

Dillon

2000

J Vasc Res

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A number of cellular metabolites, including inorganic phosphate and ADP, have been proposed to regulate the contractions of smooth muscle. Hypothesizing that one of these would have a greater influence than the others, parallel experiments using tissue mechanics and ³¹P-NMR allowed comparison of several metabolic components with the generation of force in porcine carotid artery smooth muscle during long-term contractions. P_i, ADP, ATP, PCr, free energy, pH, and free Mg²⁺ were determined from phosphate spectra during a control-hypoxia-postcontrol sequence generated during K⁺ stimulation by replacement of oxygen with nitrogen using either pyruvate or glucose as substrate. Both pH and free Mg²⁺ were significantly lower in control pyruvate-supplied tissues than in glucose-supplied tissues. Mechanical experiments following the same protocol produced variations in force. The pyruvate series produced the greater range of mechanical and metabolic changes. Linear and logarithmic regression analysis found the order of correlation with force to be highest for P_i, followed by pH, free energy, PCr, ATP, ADP, and free Mg²⁺. The results are consistent with models for the regulation of myosin ATPase by free phosphate inhibition. The results are inconsistent with models of ADP as a regulator of smooth muscle force. Perturbations which alter intracellular phosphate, such as creatine loading, may produce side effects on the contractions of vascular smooth muscle.

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Direct determination of adp in hypoxic porcine carotid artery using ³¹p nmr

Cited by 25 publications

References 15 publications

³¹P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1

³¹P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1

Equilibrium Enzymes in Regulatory Systems

Influence of Cellular Energy Metabolism on Contractions of Porcine Carotid Artery Smooth Muscle

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Direct determination of adp in hypoxic porcine carotid artery using 31p nmr

Cited by 25 publications

References 15 publications

31P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1

31P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1

Equilibrium Enzymes in Regulatory Systems

Influence of Cellular Energy Metabolism on Contractions of Porcine Carotid Artery Smooth Muscle

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Product

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Direct determination of adp in hypoxic porcine carotid artery using ³¹p nmr

³¹P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1

³¹P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1