High-energy phosphates and tension production in rabbit tibialis anterior/extensor digitorum longus muscles

Ryschon, T. W., J. C. Jarvis, S. Salmons, and R. S. Balaban.High-energy phosphates and tension production in rabbit tibialisanterior/extensor digitorum longus muscles. J. Appl. Physiol. 82(3): 1024-1029, 1997.The effects ofrepetitive muscle contraction on energy state and tension productionwere studied in rabbit tibialis anterior/extensor digitorum longusmuscles that had been subjected to 90 days of continuous indirectelectrical stimulation at 10 Hz. Anesthetized chronically stimulatedand control rabbits were challenged with 15 min of stimulation at 4 and15 tetani/min.P_i-to-phosphocreatine (PCr) ratio(P_i/PCr) was measured in vivo before, during, andafter acute stimulation by³¹P-magnetic resonancespectroscopy, and tension was recorded at the same time. AlthoughP_i/PCr was low at rest, it wassignificantly higher in chronically stimulated muscle than in controlmuscle (0.20 ± 0.02 vs. 0.05 ± 0.01, P < 0.05). Stimulation of control muscle for 15 min at both 4 and 15 tetani/min induced a significant rise in P_i/PCr, whereas the sameconditions in chronically stimulated muscle did not produce anysignificant departure from initial levels. The tension produced bycontrol muscle fell to 93 ± 3% of its initial value duringstimulation at 4 tetani/min and to 61 ± 7% at 15 tetani/min,respectively. In chronically stimulated muscle, on the other hand,tension was potentiated above its initial level at both stimulationrates (135 ± 15 and 138 ± 11%, respectively) and remainedsignificantly elevated throughout each trial. The ability ofchronically stimulated muscle to sustain high levels of activity withminimal perturbations in P_i/PCr ordecrement in tension is attributable to cellular adaptations thatinclude a well-documented increase in oxidative capacity.

     

Ca2+ influx through alpha1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle

Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (P_i) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of P_i into the cell must equal the rate of efflux, which is expected to be a function of intracellular P_i concentration. We measured ³²P-labeled P_i uptake rates in different muscle fiber types to determine whether they are proportional to cellular P_i content. P_i uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 ± 0.17 µmol·g^–1·h^–1), lower in red gastrocnemius (1.31 ± 0.11 µmol·g^–1·h^–1), and lowest in white gastrocnemius (0.49 ± 0.06 µmol·g^–1·h^–1). Reasonably similar rates were obtained in vivo. P_i uptake rates at plasma P_i concentrations of 0.3–1.7 mM confirm that the P_i uptake process is nearly saturated at normal plasma P_i levels. P_i uptake rate correlated with cellular P_i content (r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of P_i uptake across fiber types implies that other factors are involved in regulating P_i uptake in skeletal muscle. Furthermore, fractional turnover of the cellular P_i pool (0.67, 0.57, and 0.33 h^–1 in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular P_i, likely due to differences in resistance to P_i efflux from the fiber. inorganic phosphate; sodium-inorganic phosphate transporters; PiT-2; inorganic phosphate efflux     

Mechanisms of P(i) regulation of the skeletal muscle SR Ca(2+) release channel

Inorganic phosphate(P_i) accumulates in the fibers of actively working musclewhere it acts at various sites to modulate contraction. To characterizethe role of P_i as a regulator of the sarcoplasmic reticulum(SR) calcium (Ca²⁺) release channel, we examined the actionof P_i on purified SR Ca²⁺ release channels,isolated SR vesicles, and skinned skeletal muscle fibers. In singlechannel studies, addition of P_i to the cis chamberincreased single channel open probability (P_o;0.079 ± 0.020 in 0 P_i, 0.157 ± 0.034 in 20 mMP_i) by decreasing mean channel closed time; mean channelopen times were unaffected. In contrast, the ATP analog,,-methyleneadenosine 5'-triphosphate (AMP-PCP), enhancedP_o by increasing single channel open time anddecreasing channel closed time. P_i stimulation of[³H]ryanodine binding by SR vesicles wassimilar at all concentrations of AMP-PCP, suggesting P_i andadenine nucleotides act via independent sites. In skinned musclefibers, 40 mM P_i enhanced Ca²⁺-inducedCa²⁺ release, suggesting an in situ stimulation ofthe release channel by high concentrations of P_i. Ourresults support the hypothesis that P_i may be an importantendogenous modulator of the skeletal muscle SR Ca²⁺ releasechannel under fatiguing conditions in vivo, acting via a mechanismdistinct from adenine nucleotides.

     

Characteristics of phosphate-induced Ca2+ efflux from the SR in mechanically skinned rat skeletal muscle fibers

The effects of P_i onsarcoplasmic reticulum (SR) Ca²⁺ regulation were studied inmechanically skinned rat skeletal muscle fibers. Brief application ofcaffeine was used to assess the SR Ca²⁺ content, andchanges in concentration of Ca²⁺([Ca²⁺]) within the cytosol were detected withfura 2 fluorescence. Introduction of P_i (1-40 mM)induced a concentration-dependent Ca²⁺ efflux from the SR.In solutions lacking creatine phosphate (CP), the amplitude of theP_i-induced Ca²⁺ transient approximatelydoubled. A similar potentiation of P_i-induced Ca²⁺ release occurred after inhibition of creatine kinase(CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca²⁺ release was almostabolished, whereas P_i-induced Ca²⁺ release wasunaffected. However, introduction of the SR Ca²⁺ ATPaseinhibitor cyclopiazonic acid effectively abolishedP_i-induced Ca²⁺ release. These data suggestthat P_i induces Ca²⁺ release from the SR byreversal of the SR Ca²⁺ pump but not via the SRCa²⁺ channel under these conditions. If this occurs inintact skeletal muscle during fatigue, activation of a Ca²⁺efflux pathway by P_i may contribute to the reporteddecrease in net Ca²⁺ uptake and increase in resting[Ca²⁺].

     

Recovery of free ADP, Pi, and free energy of ATP hydrolysis in human skeletal muscle

We measuredsignificant undershoots of the concentrations of free ADP([ADP]) and P_i([P_i]) and the freeenergy of ATP hydrolysis (G_ATP) belowinitial resting levels during recovery from severe ischemic exercisewith ³¹P-nuclear magneticresonance spectroscopy in 11 healthy sports students. Undershoots ofthe rate of oxidative phosphorylation would be predicted if the rate ofoxidative phosphorylation would depend solely on free[ADP],[P_i], orG_ATP. However,undershoots of the rate of oxidative phosphorylation have not beenreported in the literature. Furthermore, undershoots of the rate ofoxidative phosphorylation are unlikely because there is evidence that a balance between ATP production and consumption cannot be achieved if anundershoot of the rate of oxidative phosphorylation actually occurs.Therefore, oxidative phosphorylation seems to depend not only on free[ADP],[P_i], orG_ATP. Anexplanation is that acidosis-related or other factors control oxidativephosphorylation additionally, at least under some conditions.

     

Estimation of Cytoplasmic Free Mg2+ Levels and Phosphorylation Potentials in Mung Bean Root Tips by In Vivo 31P NMR Spectroscopy

The cytoplasmic [MgATP]/[ATP]_free ratios, free Mg²⁺ concentrations,and phosphorylation potentials in mung bean [Vigna mungo (L.)Hepper] root tip cells were investigated by ³¹P nuclear magneticresonance spectroscopy. ³¹P NMR spectra show well defined peaksdue to G6P, cytoplasmic P_i, vacuolar P_i, ATP, UDP-glucose andnicotinamide adenine nucleotides. The concentrations of phosphorusmetabolites were determined from quantitative ³¹P NMR spectra.The [MgATP]/[ATP]_free ratio was 9.45. Accordingly, about 90%of the cytoplasmic ATP was complexed to Mg²⁺. Utilizing thedissociation constant (K_d) determined for MgATP, the cytoplasmicfree Mg²⁺ concentration was estimated to be 0.4mM. The NMR-derivedphosphorylation potential, [ATP]/([ADP][P_i]), was 960 M^-1. Thesodium azide treatment decreased the [ATP]/[ADP] ratio and thephosphorylation potential, and increased the [Mg²⁺]^free. Metabolicinhibition may have been enhanced by an increase in [Mg^2+freeand a decrease in the free energy change for ATP hydrolysis,which resulted due to a decrease in the ATP level. ¹Present address: National Food Research Institute, TsukubaCity, Ibaraki 305, Japan. (Received February 8, 1988; Accepted June 1, 1988)     

Elevated resting [Ca(2+)](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca²⁺ microelectrodes in myotubes expressing wild-type RyR1 (_WTRyR1) or RyR1 with one of four common MH mutations (_MHRyR1), we measured resting intracellular Ca²⁺ concentration ([Ca²⁺]_i). Changes in resting [Ca²⁺]_i produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the _MHRyR1s had a 2.0- to 3.7-fold higher resting [Ca²⁺]_i than those expressing _WTRyR1. Exposure of myotubes expressing _MHRyR1s to ryanodine (500 µM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 µM) had no effects on their resting [Ca²⁺]_i. However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca²⁺]_i was significantly reduced (P < 0.01). Interestingly, the percent decrease in resting [Ca²⁺]_i in myotubes expressing _MHRyR1s was significantly greater than that for _WTRyR1. From these data, we propose that the high resting myoplasmic [Ca²⁺]_i in _MHRyR1 expressing myotubes is due in part to a related structural conformation of _MHRyR1s that favors "passive" calcium leak from the sarcoplasmic reticulum. ryanodine; FLA 365; bastadin 5; resting intracellular calcium concentration; sarcoplasmic reticulum     

Modeling transmural heterogeneity of K(ATP) current in rabbit ventricular myocytes

To investigate the mechanisms regulating excitation-metabolic coupling in rabbit epicardial, midmyocardial, and endocardial ventricular myocytes we extended the LabHEART model (Puglisi JL and Bers DM. Am J Physiol Cell Physiol 281: C2049–C2060, 2001). We incorporated equations for Ca²⁺ and Mg²⁺ buffering by ATP and ADP, equations for nucleotide regulation of ATP-sensitive K⁺ channel and L-type Ca²⁺ channel, Na⁺-K⁺-ATPase, and sarcolemmal and sarcoplasmic Ca²⁺-ATPases, and equations describing the basic pathways (creatine and adenylate kinase reactions) known to communicate the flux changes generated by intracellular ATPases. Under normal conditions and during 20 min of ischemia, the three regions were characterized by different I_Na, I_to, I_Kr, I_Ks, and I_Kp channel properties. The results indicate that the ATP-sensitive K⁺ channel is activated by the smallest reduction in ATP in epicardial cells and largest in endocardial cells when cytosolic ADP, AMP, PCr, Cr, P_i, total Mg²⁺, Na⁺, K⁺, Ca²⁺, and pH diastolic levels are normal. The model predicts that only K_ATP ionophore (Kir6.2 subunit) and not the regulatory subunit (SUR2A) might differ from endocardium to epicardium. The analysis suggests that during ischemia, the inhomogeneous accumulation of the metabolites in the tissue sublayers may alter in a very irregular manner the K_ATP channel opening through metabolic interactions with the endogenous PI cascade (PIP₂, PIP) that in turn may cause differential action potential shortening among the ventricular myocyte subtypes. The model predictions are in qualitative agreement with experimental data measured under normal and ischemic conditions in rabbit ventricular myocytes. ATP-sensitive K⁺ channel; creatine and adenylate kinase reactions; phosphatidylinositol phosphates; heart; mathematical model     

Light-induced ATP Formation from Acetyl Phosphate and ADP by Broken Spinach Chloroplasts

Spinach chloroplasts catalyzed ATP formation from acetyl phosphateand ADP when exposed to light. No ATP formation was detectablein the dark. In the absence of ADP, chloroplasts did not hydrolyzeacetyl phosphate in the light or dark. Neither high-energy phosphatessuch as creatine phosphate and phosphoenol pyruvate nor inhibitorsof photophosphorylation competitive with P_i, such as ß-naphthylmonophosphate, phenyl phosphate and pyridoxal 5-phosphate, couldsubstitute for acetyl phosphate as a P_i donor. The apparentK_m values for acetyl phosphate and P_i were 0.81 mM and 0.25mM, respectively. The maximal rate of ATP formation with acetylphosphate and P_i were 331 and 521 µmol ATP formed mg chl^–1hr^–1, respectively. The optimum pH value for acetyl phosphate-dependentATP formation was about 8.0. NH₄Cl, dicyclohexylcarbodiimideand triphenyltin chloride inhibited the acetyl phosphate-dependentATP formation. Acid-base transition also could induce subsequentATP formation from acetyl phosphate and ADP. These results suggestthat the acetyl phosphate-dependent ATP formation requires theformation and the utilization of a proton-motive force as ordinaryphotophosphorylation does. ¹ This work was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Science and Culture,Japan to H. S. Part of this work was reported at the 1981 AnnualMeeting of the Japanese Society of Plant Physiologists (Sapporo,May 8, 1981). (Received August 25, 1981; Accepted November 1, 1981)     

Characterization of inorganic phosphate transport in osteoclast-like cells

Osteoclasts possess inorganic phosphate (P_i) transport systems to take up external P_i during bone resorption. In the present study, we characterized P_i transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-B ligand (RANKL). In undifferentiated RAW264.7 cells, P_i transport into the cells was Na⁺ dependent, but after treatment with RANKL, Na⁺-independent P_i transport was significantly increased. In addition, compared with neutral pH, the activity of the Na⁺-independent P_i transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na⁺-independent system consisted of two components with K_m of 0.35 mM and 7.5 mM. The inhibitors of P_i transport, phosphonoformic acid, and arsenate substantially decreased P_i transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K⁺ ionophore, valinomycin, significantly suppressed P_i transport activity. Analysis of BCECF fluorescence indicated that P_i transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K⁺ ion stimulated P_i transport, suggesting that membrane voltage is involved in the regulation of P_i transport activity. Finally, bone particles significantly increased Na⁺-independent P_i transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a P_i transport system with characteristics that are different from those of other Na⁺-dependent P_i transporters. We conclude that stimulation of P_i transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment. Na⁺-dependent phosphate cotransporter; RAW264.7; phosphate uptake     

Oxidative ATP synthesis in skeletal muscle is controlled by substrate feedback

Data from ³¹P-nuclear magnetic resonance spectroscopy of human forearm flexor muscle were analyzed based on a previously developed model of mitochondrial oxidative phosphorylation (PLoS Comp Bio 1: e36, 2005) to test the hypothesis that substrate level (concentrations of ADP and inorganic phosphate) represents the primary signal governing the rate of mitochondrial ATP synthesis and maintaining the cellular ATP hydrolysis potential in skeletal muscle. Model-based predictions of cytoplasmic concentrations of phosphate metabolites (ATP, ADP, and P_i) matched data obtained from 20 healthy volunteers and indicated that as work rate is varied from rest to submaximal exercise commensurate increases in the rate of mitochondrial ATP synthesis are effected by changes in concentrations of available ADP and P_i. Additional data from patients with a defect of complex I of the respiratory chain and a patient with a deficiency in the mitochondrial adenine nucleotide translocase were also predicted the by the model by making the appropriate adjustments to the activities of the affected proteins associates with the defects, providing both further validation of the biophysical model of the control of oxidative phosphorylation and insight into the impact of these diseases on the ability of the cell to maintain its energetic state. computational model; mitochondria; cellular energetics; oxidative phosphorylation; ³¹P-NMR spectroscopy     

Kinetics of creatine kinase in an experimental model of low phosphocreatine and ATP in the normoxic heart

To study the dependence of the forward flux of creatine kinase(CK) on its substrates and products we designed an acute normoxic modelof steady-state depletion of phosphocreatine (PCr) and adenylate in theisovolumic acetate-perfused rat heart. Various concentrations of PCrand ATP were induced by prior perfusion with 2 deoxy-D-glucose in the presenceof insulin. The apparent rate constant(k_f) and theforward CK flux were measured under metabolic and contractile steadystate by progressive saturation-transfer³¹P nuclear magnetic resonance(NMR). At high adenylate content CK flux was constant for a twofoldreduction in PCr concentration ([PCr]); CK flux was 6.3 ± 0.6 mM/s (vs. 6.5 ± 0.2 mM/s in control) because of adoubling of k_f.Although, at the lowest ATP concentration and [PCr], CKflux was reduced by 50%, it nevertheless always remained higher thanATP synthesis estimated by parallel oxygen consumption measurement.NMR-measured flux was compared with the flux computed under thehypothesis of CK equilibrium. CK flux could not be fully predicted bythe concentrations of CK metabolites. This is discussed in terms ofmetabolite and CK isozyme compartmentation.

     

Long-lasting muscle fatigue: partial disruption of excitation-contraction coupling by elevated cytosolic Ca2+ concentration during contractions

The repeated elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) above resting levels during contractile activity has been associated with long-lasting muscle fatigue. The mechanism underlying this fatigue appears to involve elevated [Ca²⁺]_i levels that induce disruption of the excitation-contraction (E-C) coupling process at the triad junction. Unclear, however, are which aspects of the activity-related [Ca²⁺]_i changes are responsible for the deleterious effects, in particular whether they depend primarily on the peak [Ca²⁺]_i reached locally at particular sites or on the temporal summation of the increased [Ca²⁺] in the cytoplasm as a whole. In this study, we used mechanically skinned fibers from rat extensor digitorum longus muscle, in which the normal E-C coupling process remains intact. The [Ca²⁺]_i was raised either by applying a set elevated [Ca²⁺] throughout the fiber or by using action potential stimulation to induce the release of sarcoplasmic reticulum Ca²⁺ by the normal E-C coupling system with or without augmentation by caffeine or buffering with BAPTA. Herein we show that elevating [Ca²⁺]_i in the physiological range of 2–20 µM irreversibly disrupts E-C coupling in a concentration-dependent manner but requires exposure for a relatively long time (1–3 min) to cause substantial uncoupling. The effectiveness of Ca²⁺ released via the endogenous system in disrupting E-C coupling indicates that the relatively high [Ca²⁺]_i attained close to the release site at the triad junction is a more important factor than the increase in bulk [Ca²⁺]_i. Our results suggest that during prolonged vigorous activity, the many repeated episodes of relatively high triadic [Ca²⁺] can disrupt E-C coupling and lead to long-lasting fatigue. skeletal muscle; low-frequency fatigue; ryanodine receptor; skinned fiber     

The depressive effect of Pi on the force-pCa relationship in skinned single muscle fibers is temperature dependent

Increases in P_i combined with decreases in myoplasmic Ca²⁺ are believed to cause a significant portion of the decrease in muscular force during fatigue. To investigate this further, we determined the effect of 30 mM P_i on the force-Ca²⁺ relationship of chemically skinned single muscle fibers at near-physiological temperature (30°C). Fibers isolated from rat soleus (slow) and gastrocnemius (fast) muscle were subjected to a series of solutions with an increasing free Ca²⁺ concentration in the presence and absence of 30 mM P_i at both low (15°C) and high (30°C) temperature. In slow fibers, 30 mM P_i significantly increased the Ca²⁺ required to elicit measurable force, referred to as the activation threshold at both low and high temperatures; however, the effect was twofold greater at the higher temperature. In fast fibers, the activation threshold was unaffected by elevating P_i at 15°C but was significantly increased at 30°C. At both low and high temperatures, 30 mM P_i increased the Ca²⁺ required to elicit half-maximal force (pCa₅₀) in both slow and fast fibers, with the effect of P_i twofold greater at the higher temperature. These data suggest that during fatigue, reductions in the myoplasmic Ca²⁺ and increases in P_i act synergistically to reduce muscular force. Consequently, the combined changes in these ions likely account for a greater portion of fatigue than previously predicted based on studies at lower temperatures or high temperatures at saturating Ca²⁺ levels. force-pCa relationship; phosphate; fatigue     

MITOCHONDRIA: Investigation of in vivo muscle mitochondrial function by 31P magnetic resonance spectroscopy

The most important function of mitochondria is the production of energy in the form of ATP. The socio-economic impact of human diseases that affect skeletal muscle mitochondrial function is growing, and improving their clinical management critically depends on the development of non-invasive assays to assess mitochondrial function and monitor the effects of interventions. ³¹P magnetic resonance spectroscopy provides two approaches that have been used to assess in vivo ATP synthesis in skeletal muscle: measuring P_i ⟶ ATP exchange flux using saturation transfer in resting muscle, and measuring phosphocreatine recovery kinetics after exercise. However, P_i ⟶ ATP exchange does not represent net mitochondrial ATP synthesis flux and has no simple relationship with mitochondrial function. Post-exercise phosphocreatine recovery kinetics, on the other hand, yield reliable measures of muscle mitochondrial capacity in vivo, whose ability to define the site of functional defects is enhanced by combination with other non-invasive techniques.     

Cellular basis for contractile dysfunction in the diaphragm from a rabbit infarct model of heart failure

Abnormal respiratory muscle function isthought to contribute to breathlessness and exercise intolerance inheart failure but little is known about possible alterations in thefunction of such muscle. We have measured tetanic force andintracellular Ca²⁺ concentration([Ca²⁺]_i) in isolated, arteriallyperfused hemidiaphragm preparations from a rabbit coronary arteryligation model of heart failure. Increasing stimulation frequency(10-100 Hz) caused a progressive increase of force and[Ca²⁺]_i in control preparations,whereas force and [Ca²⁺]_i onlyincreased between 10 and 25 Hz stimulation (decreasing at higherfrequencies) in preparations from ligated animals. Cyclopiazonic acidproduced a dose-dependent shift in the relationship between stimulationfrequency and [Ca²⁺]_i in controlpreparations that was similar to the shift observed in the diaphragm ofcoronary-ligated animals. These data indicate that the in vitrocontractile characteristics of the diaphragm are significantly alteredin our model and that altered[Ca²⁺]_i regulation contributes tothe reduced diaphragm strength observed in heart failure.

     

In squid nerves intracellular Mg(2+) promotes deactivation of the ATP-upregulated Na(+)/Ca(2+) exchanger

We investigated the role of intracellular Mg²⁺(Mg_i²⁺) on the ATP regulation ofNa⁺/Ca²⁺ exchanger in squid axons and bovineheart. In squid axons and nerve vesicles, the ATP-upregulated exchangerremains activated after removal of cytoplasmic Mg²⁺, evenin the absence of ATP. Rapid and complete deactivation of theATP-stimulated exchange occurs upon readmission ofMg_i²⁺. At constant ATP concentration, the effectof intracellular Mg²⁺ concentration([Mg²⁺]_i) on the ATP regulation of exchangeris biphasic: activation at low [Mg²⁺]_i,followed by deactivation as [Mg²⁺]_i isincreased. No correlation was found between the above results and thelevels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] measured innerve membrane vesicles. Incorporation ofPtdIns(4,5)P₂ into membrane vesicles activates Na⁺/Ca²⁺ exchange in mammalian heart but not insquid nerve. Moreover, an exogenous phosphatase prevents MgATPactivation in squid nerves but not in mammalian heart. It is concludedthat 1) Mg_i²⁺ is an essentialcofactor for the deactivation part of ATP regulation of the exchangerand 2) the metabolic pathway of ATP upregulation of theNa⁺/Ca²⁺ exchanger is different in mammalianheart and squid nerves.

     

Pinacidil suppresses contractility and preserves energy but glibenclamide has no effect during muscle fatigue

The effects of 10 µM glibenclamide, anATP-sensitive K⁺ (K_ATP) channelblocker, and 100 µM pinacidil, a channel opener, were studied todetermine how the K_ATP channel affects mouse extensor digitorum longus (EDL) and soleus muscle during fatigue. Fatigue waselicited with 200-ms-long tetanic contractions every second. Glibenclamide did not affect rate and extent of fatigue, force recovery, or ⁸⁶Rb⁺ fractional loss. The onlyeffects of glibenclamide during fatigue were: an increase in restingtension (EDL and soleus), a depolarization of the cell membrane, aprolongation of the repolarization phase of action potential, and agreater ATP depletion in soleus. Pinacidil, on the other hand,increased the rate but not the extent of fatigue, abolished the normalincrease in resting tension during fatigue, enhanced force recovery,and increased ⁸⁶Rb⁺ fractional loss in both theEDL and soleus. During fatigue, the decreases in ATP andphosphocreatine of soleus muscle were less in the presence ofpinacidil. The glibenclamide effects suggest that fatigue, elicitedwith intermittent contractions, activates few K_ATP channelsthat affect resting tension and membrane potentials but not tetanicforce, whereas opening the channel with pinacidil causes a fasterdecrease in tetanic force, improves force recovery, and helps inpreserving energy.

     

Ca2+ transients activate calcineurin/NFATc1 and initiate fast-to-slow transformation in a primary skeletal muscle culture

The calcineurin-mediated signal transduction via nuclear factor of activated T cells (NFATc1) is involved in upregulating slow myosin heavy chain (MHC) gene expression during fast-to-slow transformation of skeletal muscle cells. This study aims to investigate the Ca²⁺ signal necessary to activate the calcineurin-NFATc1 cascade in skeletal muscle. Electrostimulation of primary myocytes from rabbit for 24 h induced a distinct fast-to-slow transformation at the MHC mRNA level and a full activation of the calcineurin-NFATc1 pathway, although resting Ca²⁺ concentration ([Ca²⁺]_i) remained unaltered at 70 nM. During activation, the calcium transients of these myocytes reach a peak concentration of 500 nM. Although 70 nM [Ca²⁺]_i does not activate calcineurin-NFAT, we show by the use of Ca²⁺ ionophore that the system is fully activated when [Ca²⁺]_i is 150 nM in a sustained manner. We conclude that the calcineurin signal transduction pathway and the slow MHC gene in cultured skeletal muscle cells are activated by repetition of the rapid high-amplitude calcium transients that are associated with excitation-contraction coupling rather than by a sustained elevation of resting Ca²⁺ concentration. muscle plasticity; NFATc1; resting calcium concentration     

Energetics of muscle contraction: the whole is less than the sum of its parts

Understanding muscle energetics is a problem in optimizing supply of ATP to the demands of ATPases. The complexity of reactions and their fluxes to achieve this balance is greatly reduced by recognizing constraints imposed by the integration of common metabolites at fixed stoichiometry among modular units. ATPase is driven externally. Oxidative phosphorylation and glycogenolysis are the suppliers. We focus on their regulation which involves different controls, but reduces to two principles that enable facile experimental analysis of the supply and demand fluxes. The ratio of concentration of phosphocreatine (PCr) to ATP, not their individual values, sets the range of achievable concentrations of ADP in resting and active muscle (at fixed pH) in different cell types. This principle defines the fraction of available flux of oxidative phosphorylation utilized (at fixed enzyme activities). Then the kinetics of PCr recovery defines the kinetics of oxygen supply and substrate utilization. The second principle is the constancy of PCr and H(+) (lactate) production by glycogenolysis due to the coupling of ATPase and glycolysis. This principle enables glycogenolytic flux to be measured from intracellular proton loads. Further simplification occurs because the magnitude of the interacting fluxes and metabolite concentrations are specified within narrow limits when both the resting and active fluxes are quantified. Thus there is a small set of rules for assessing and understanding the thermodynamics and kinetics of muscle energetics.