Actomyosin energy turnover declines while force remains constant during isometric muscle contraction

Energy turnover was measured during isometric contractions of intact and Triton-permeabilized white fibres from dogfish ( Scyliorhinus canicula ) at 12°C. Heat + work from actomyosin in intact fibres was determined from the dependence of heat + work output on filament overlap. Inorganic phosphate (P_i) release by permeabilized fibres was recorded using the fluorescent protein MDCC-PBP, N-(2-[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3 carboxamide phosphate binding protein. The steady-state ADP release rate was measured using a linked enzyme assay. The rates decreased five-fold during contraction in both intact and permeabilized fibres. In intact fibres the rate of heat + work output by actomyosin decreased from 134 ± s.e.m. 28 μW mg⁻¹ ( n = 17) at 0.055 s to 42% of this value at 0.25 s, and to 20% at 3.5 s. The force remained constant between 0.25 and 3.5 s. Similarly in permeabilized fibres the P_i release rate decreased from 5.00 ± 0.39 mmol l⁻¹ s⁻¹ at 0.055 s to 39% of this value at 0.25 s and to 19% at 0.5 s. The steady-state ADP release rate at 15 s was 21% of the P_i rate at 0.055 s. Using a single set of rate constants, the time courses of force, heat + work and P_i release were described by an actomyosin model that took account of the transition from the initial state (rest or rigor) to the contracting state, shortening and the consequent work against series elasticity, and reaction heats. The model suggests that increasing P_i concentration slows the cycle in intact fibres, and that changes in ATP and ADP slow the cycle in permeabilized fibres.     

Characterization of the cross-bridge force-generating step using inorganic phosphate and BDM in myofibrils from rabbit skeletal muscles

The inhibitory effects of inorganic phosphate (P_i) on isometric force in striated muscle suggest that in the ATPase reaction P_i release is coupled to force generation. Whether P_i release and the power stroke are synchronous events or force is generated by an isomerization of the quaternary complex of actomyosin and ATPase products (AM.ADP.P_i) prior to the following release of P_i is still controversial. Examination of the dependence of isometric force on [P_i] in rabbit fast (psoas; 5-15 °C) and slow (soleus; 15-20 °C) myofibrils was used to test the two-step hypothesis of force generation and P_i release. Hyperbolic fits of force-[P_i] relations obtained in fast and slow myofibrils at 15 °C produced an apparent asymptote as [P_i]∞ of 0.07 and 0.44 maximal isometric force (i.e. force in the absence of P_i) in psoas and soleus myofibrils, respectively, with an apparent K _d of 4.3 m m in both. In each muscle type, the force-[P_i] relation was independent of temperature. However, 2,3-butanedione 2-monoxime (BDM) decreased the apparent asymptote of force in both muscle types, as expected from its inhibition of the force-generating isomerization. These data lend strong support to models of cross-bridge action in which force is produced by an isomerization of the AM.ADP.P_i complex immediately preceding the P_i release step.     

Role of phosphate and calcium stores in muscle fatigue

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (P_i) in the myoplasm ([P_i]_myo) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca²⁺ release contributes to fatigue and in this review we consider how raised [P_i]_myo contributes to this process. Initial evidence came from the observation that increasing [P_i]_myo causes reduced SR Ca²⁺ release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca²⁺ in the SR declines mirroring the decline in SR Ca²⁺ release. In muscle fibres with inoperative creatine kinase the rise of [P_i]_myo is absent during fatigue and the failure of SR Ca²⁺ release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaP_i within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.     

The Smooth Muscle Myosin Seven Amino Acid Heavy Chain Insert's Kinetic Role in the Crossbridge Cycle for Mouse Bladder

The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (−/−) and a heterozygous type (+/−). For skinned muscle, an increase in MgADP or inorganic phosphate (P_i) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and P_i release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca²⁺ concentration in both the +/+ and +/− mouse types. However, the −/− type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the −/− type compared to the +/+ or +/− types. Exogenous P_i led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to P_i release. In addition, the increase in P_i showed similar changes in the +/+ and −/− types whereas in the +/− type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle.     

Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca²⁺ ( k _act ), and the rate of tension redevelopment following a quick release and restretch ( k _tr ), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (P_i) was abruptly increased ( k _Pi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak-to-strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, k _act or k _tr , suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa₅₀ of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca²⁺ sensitivity and the cooperativity of the force-generating mechanism.     

Temperature dependence of the force-generating process in single fibres from frog skeletal muscle

Generation of force and shortening in striated muscle is due to the cyclic interactions of the globular portion (the head) of the myosin molecule, extending from the thick filament, with the actin filament. The work produced in each interaction is due to a conformational change (the working stroke) driven by the hydrolysis of ATP on the catalytic site of the myosin head. However, the precise mechanism and the size of the force and length step generated in one interaction are still under question. Here we reinvestigate the endothermic nature of the force-generating process by precisely determining, in tetanised intact frog muscle fibres under sarcomere length control, the effect of temperature on both isometric force and force response to length changes. We show that raising the temperature: (1) increases the force and the strain of the myosin heads attached in the isometric contraction by the same amount (∼70 %, from 2 to 17 °C); (2) increases the rate of quick force recovery following small length steps (range between −3 and 2 nm (half-sarcomere)⁻¹) with a Q ₁₀ (between 2 and 12 °C) of 1.9 (releases) and 2.3 (stretches); (3) does not affect the maximum extent of filament sliding accounted for by the working stroke in the attached heads (10 nm (half-sarcomere)⁻¹). These results indicate that in isometric conditions the structural change leading to force generation in the attached myosin heads can be modulated by temperature at the expense of the structural change responsible for the working stroke that drives filament sliding. The energy stored in the elasticity of the attached myosin heads at the plateau of the isometric tetanus increases with temperature, but even at high temperature this energy is only a fraction of the mechanical energy released by attached heads during filament sliding.     

Nitric oxide plays a role in the regulation of adrenal blood flow and adrenocorticomedullary functions in the llama fetus

The effect of temperature on isometric tension with and without the regulatory proteins tropomyosin and troponin was studied in bovine myocardium using a thin filament removal and reconstitution protocol. In control bovine myocardium, isometric tension increased linearly with temperature in the range 5–40 °C: isometric tension at 10 and 30 °C was 0.65 and 1.28 times that at 20 °C, respectively, with a Q ₁₀ of about 1.4. In actin filament-reconstituted myocardium without regulatory proteins, the temperature effect on isometric tension was less: isometric tension at 10 and 30 °C was 0.96 and 1.17 times that at 20 °C, respectively, with a Q ₁₀ of about 1.1. The temperature dependence of the apparent rate constants was studied using sinusoidal analysis. The temperature dependence of 2π b (rate constant of delayed tension phase) did not vary significantly with the regulatory proteins under the standard activating condition (5 m m MgATP, 8 m m P_i, 200 m m ionic strength, pCa 4.66, pH 7.00). Q ₁₀ for 2π b in control and actin filament-reconstituted myocardium was 3.8 and 4.0, respectively. There were two phases to the temperature dependence of 2π c (rate constant of quick recovery). In control and thin filament-reconstituted myocardium, Q ₁₀ for 2π c was approximately 5.5 in the low temperature range (≤ 25 °C) and 2.7 in the high temperature range (≥ 30 °C). In actin filament-reconstituted myocardium, Q ₁₀ for 2π c was 8.5 in the low temperature range and 3.6 in the high temperature range. The above results demonstrate that regulatory proteins augment the temperature dependence of isometric tension, indicating that the regulatory proteins may modify the actomyosin interaction.     

Effects of Thyroxine on Myosin Isoform Expression and Mechanical Properties in Guinea-Pig Smooth Muscle

Information on the effects of thyroid hormone on smooth muscle contractile protein expression and mechanical properties is sparse. We have addressed the following questions. (1) Can thyroxine hormone alter myosin isoform composition in smooth muscle? (2) Can a change in myosin isoform composition lead to altered mechanical properties in smooth muscle? (3) Are alterations, if occurring, equal in fast and slow smooth muscle types? Guinea-pigs were treated with thyroxine (T₄) for 12 days. Control animals were given physiological saline solution. Maximal unloaded shortening velocity ( V _max) was measured in chemically skinned, maximally activated muscle preparations from the aorta and the taenia coli. V _max increased following thyroxine treatment, by approximately 20 % in the taenia coli. In the aorta, no significant increase in V _max could be detected. The sensitivity of isometric force to inorganic phosphate (P_i) was increased in the taenia coli following thyroxine treatment. The expression of mRNA (determined with RT-PCR) for the myosin heavy chain with the seven amino acid insert increased by approximately 70 % in the aorta and about 25 % in the taenia coli following thyroxine treatment. Western blot analysis showed an increase in the inserted myosin heavy chain form in the taenia coli. Expression of mRNA for the myosin essential light chains and the corresponding proteins did not change significantly in either muscle type. No alterations in non-muscle myosin heavy chain isoforms could be detected after thyroxine treatment. In conclusion, thyroxine treatment alters the isoform composition of myosin in fast and slow smooth muscles in vivo . This change is sufficient to increase shortening velocity and sensitivity of isometric force to P_i in the fast, but not in the slow, smooth muscle type.     

Effects of old age on human skeletal muscle energetics during fatiguing contractions with and without blood flow

We recently reported lower glycolytic flux (ATP_GLY) and increased reliance on oxidative ATP synthesis (ATP_OX) in contracting muscle of older compared to young humans. To further investigate this age-related difference in the pathways of ATP synthesis, we used magnetic resonance spectroscopy to determine the rates of ATP_OX, ATP_GLY and net phosphocreatine hydrolysis in vivo during maximal muscle contractions under free-flow (FF) and ischaemic (ISC) conditions in the ankle dorsiflexors of 20 young (27 ± 3 years; 10 male, 10 female) and 18 older (70 ± 5 years; 10 male, 8 female) adults. We hypothesized that ATP_GLY would be higher in young compared to old during FF contractions, but that old would be unable to increase ATP_GLY during ISC to match that of the young, which would suggest impaired glycolytic ATP synthesis with old age. Peak glycolytic flux during FF was lower in older (0.8 ± 0.1 m m ATP s⁻¹) compared to young (1.4 ± 0.1 m m ATP s⁻¹, P < 0.001) subjects. During ISC, peak ATP_GLY increased in old to a level similar to that of young (1.4 ± 0.2 m m ATP s⁻¹, 1.3 ± 0.2 m m ATP s⁻¹, respectively; P = 0.86), suggesting that glycolytic function remains intact in aged muscle in vivo . Notably, older adults fatigued less than young during both FF and ISC ( P ≤ 0.004). These results provide novel evidence of unimpaired in vivo glycolytic function in the skeletal muscle of older adults during maximal isometric dorsiflexion, and suggest a potential role for differences in metabolic economy and as a result, metabolite accumulation, in the fatigue resistance of the old.     

Phosphorylated guanidinoacetate partly compensates for the lack of phosphocreatine in skeletal muscle of mice lacking guanidinoacetate methyltransferase

The effects of creatine (Cr) absence in skeletal muscle caused by a deletion of guanidinoacetate methyltransferase (GAMT) were studied in a knockout mouse model by in vivo ³¹P magnetic resonance (MR) spectroscopy. ³¹P MR spectra of hindleg muscle of GAMT-deficient (GAMT–/–) mice showed no phosphocreatine (PCr) signal and instead showed the signal for phosphorylated guanidinoacetate (PGua), the immediate precursor of Cr, which is not normally present. Tissue pH did not differ between wild-type (WT) and GAMT–/–  mice, while relative inorganic phosphate (P_i) levels were increased in the latter. During ischaemia, PGua was metabolically active in GAMT–/–  mice and decreased at a rate comparable to the decrease of PCr in WT mice. However, the recovery rate of PGua in GAMT–/– mice after ischaemia was reduced compared to PCr in WT mice. Saturation transfer measurements revealed no detectable flux from PGua to γ-ATP, indicating severely reduced enzyme kinetics. Supplementation of Cr resulted in a rapid increase in PCr signal intensity until only this resonance was visible, along with a reduction in relative P_i values. However, the PGua recovery rate after ischaemia did not change. Our results show that despite the absence of Cr, GAMT–/– mice can cope with mild ischaemic stress by using PGua for high energy phosphoryl transfer. The reduced affinity of creatine kinase (CK) for (P)Gua only becomes apparent during recovery from ischaemia. It is argued that absence of Cr causes the higher relative P_i concentration also observed in animals lacking muscle CK, indicating an important role of the CK system in P_i homeostasis.     

NaPi-IIa and interacting partners

Regulation of renal proximal tubular reabsorption of phosphate (P_i) is one of the critical steps in P_i homeostasis. Experimental evidence suggests that this regulation is achieved mainly by controlling the apical expression of the Na⁺-dependent P_i cotransporter type IIa (NaPi-IIa) in proximal tubules. Only recently have we started to obtain information regarding the molecular mechanisms that control the apical expression of NaPi-IIa. The first critical observation was the finding that truncation of only its last three amino acid residues has a strong effect on apical expression. A second major finding was the observation that the last intracellular loop of NaPi-IIa contains sequence information that confers parathyroid hormone (PTH) sensitivity. The use of the above domains of the cotransporter in yeast two-hybrid (Y2H) screening allowed the identification of proteins interacting with NaPi-IIa. Biochemical and morphological, as well as functional, analyses have allowed us to obtain insights into the physiological roles of such interactions, although our present knowledge is still far from complete.     

Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres

In the preceding paper, we reported that flexor digitorum brevis (FDB) muscle fibres from S100A1 knock-out (KO) mice exhibit a selective suppression of the delayed, steeply voltage-dependent component of intra-membrane charge movement current termed Q _γ. Here, we use 50 μ m of the Ca²⁺ indicator fluo-4 in the whole cell patch clamp pipette, in addition to 20 m m EGTA and other constituents included for the charge movement studies, and calculate the SR Ca²⁺ release flux from the fluo-4 signals during voltage clamp depolarizations. Ca²⁺ release flux is decreased in amplitude by the same fraction at all voltages in fibres from S100A1 KO mice compared to fibres from wild-type (WT) littermates, but unchanged in time course at each pulse membrane potential. There is a strong correlation between the time course and magnitude of release flux and the development of Q _γ. The decreased Ca²⁺ release in KO fibres is likely to account for the suppression of Q _γ in these fibres. Consistent with this interpretation, 4-chloro- m -cresol (4–CMC; 100 μ m ) increases the rate of Ca²⁺ release and restores Q _γ at intermediate depolarizations in fibres from KO mice, but does not increase Ca²⁺ release or restore Q _γ at large depolarizations. Our findings are consistent with similar activation kinetics for SR Ca²⁺ channels in both WT and KO fibres, but decreased Ca²⁺ release in the KO fibres possibly due to shorter SR channel open times. The decreased Ca²⁺ release at each voltage is insufficient to activate Q _γ in fibres lacking S100A1.     

Crossbridge properties during force enhancement by slow stretching in single intact frog muscle fibres

The mechanism of force enhancement during lengthening was investigated on single frog muscle fibres by using fast stretches to measure the rupture tension of the crossbridge ensemble. Fast stretches were applied to one end of the activated fibre and force responses were measured at the other. Sarcomere length was measured by a striation follower device. Fast stretching induced a linear increase of tension that reached a peak and fell before the end of the stretch indicating that a sudden increase of fibre compliance occurred due to forced crossbridge detachment induced by the fast loading. The peak tension (critical tension, P _c) and the sarcomere length needed to reach P _c (critical length, L _c) were measured at various tensions during the isometric tetanus rise and during force enhancement by slow lengthening. The data showed that P _c was proportional to the tension generated by the fibre under both isometric and slow lengthening conditions. However, for a given tension increase, P _c was 6.5 times greater during isometric than during lengthening conditions. Isometric critical length was 13.04 ± 0.17 nm per half-sarcomere (nm hs⁻¹) independently of tension. During slow lengthening critical length fell as the force enhancement increased. For 90% enhancement, L _c reduced to 8.19 ± 0.039 nm hs⁻¹. Assuming that the rupture force of the individual crossbridge is constant, these data indicate that the increase of crossbridge number during lengthening accounts for only 15.4% of the total force enhancement. The remaining 84.6% is accounted for by the increased mean strain of the crossbridges.     

Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37°C

The mechanisms of muscle fatigue were studied in small muscle bundles and single fibres isolated from the flexor digitorum brevis of the mouse. Fatigue caused by repeated isometric tetani was accelerated at body temperature (37°C) when compared to room temperature (22°C). The membrane-permeant reactive oxygen species (ROS) scavenger, Tiron (5 m m ), had no effect on the rate of fatigue at 22°C but slowed the rate of fatigue at 37°C to that observed at 22°C. Single fibres were microinjected with indo-1 to measure intracellular calcium. In the accelerated fatigue at 37°C the tetanic [Ca²⁺]_i did not change significantly and the decline of maximum Ca²⁺-activated force was similar to that observed at 22°C. The cause of the greater rate of fatigue at 37°C was a large fall in myofibrillar Ca²⁺ sensitivity. In the presence of Tiron, the large fall in Ca²⁺ sensitivity was abolished and the usual decline in tetanic [Ca²⁺]_i was observed. This study confirms the importance of ROS in fatigue at 37°C and shows that the mechanism of action of ROS is a decline in myofibrillar Ca²⁺ sensitivity.     

Neurokinin-1 receptor desensitization to consecutive microdialysis infusions of substance P in human skin

Unloaded shortening velocity ( V ₀) of human triceps surae muscle was measured in vivo by applying the 'slack test', originally developed for determining V ₀ of single muscle fibres, to voluntary contractions at varied activation levels (ALs). V ₀ was measured from 10 subjects at five different ALs defined as a fraction (5, 10, 20, 40 and 60%) of the maximum voluntary contraction (MVC) torque. Although individual variability was apparent, V ₀ tended to increase with AL  ( R ²= 0.089; P = 0.035)  up to 60%MVC (8.6 ± 2.6 rad s⁻¹). This value of V ₀ at 60%MVC was comparable to the maximum shortening velocity of plantar flexors reported in the previous studies. Electromyographic analysis showed that the activities of soleus, medial gastrocnemius and lateral gastrocnemius muscles increased with AL during isometric contraction and after the application of quick release in a similar manner. Also, it showed that the activity of an antagonist, tibialis anterior muscle, was negligible, even though a slight increase took place after the quick release of agonist. Correlation analysis showed that there were no significant correlations between V ₀ and MVC torque normalized with respect to body mass, although the correlation coefficient was relatively high at low ALs. The results suggest that in human muscle, V ₀ represents the unloaded velocity of the fastest muscle fibres recruited, and increases with AL possibly because of progressive recruitment of faster fibres. Individual variability may be explained, at least partially, by the difference in fibre-type composition.     

Functional expression of ionotropic purinergic receptors on mouse taste bud cells

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 μ m ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca²⁺ imaging showed that similarly applied 1 μ m ATP, 30 μ m BzATP (a P2X₇ agonist), or 1 μ m 2MeSATP (a P2Y₁ and P2Y₁₁ agonist) increased intracellular Ca²⁺ concentration, but 100 μ m UTP (a P2Y₂ and P2Y₄ agonist) and α,β-meATP (a P2X agonist except for P2X₂, P2X₄ and P2X₇) did not. RT-PCR suggested the expression of P2X₂, P2X₄, P2X₇, P2Y₁, P2Y₁₃ and P2Y₁₄ among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X₂. The exposure of the basolateral membranes to 3 m m ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 μ m PPADS (a non-selective P2 blocker) and 1 μ m KN-62 (a P2X₇ blocker). These results showed for the first time the functional expression of P2X₂ and P2X₇ on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.     

Mini-dystrophin restores L-type calcium currents in skeletal muscle of transgenic mdx mice

L-type calcium currents ( i _Ca) were recorded using the two-microelectrode voltage-clamp technique in single short toe muscle fibres of three different mouse strains: (i) C57/SV129 wild-type mice (wt); (ii) mdx mice (an animal model for Duchenne muscular dystrophy; and (iii) transgenically engineered mini-dystrophin (MinD)-expressing mdx mice. The activation and inactivation properties of i _Ca were examined in 2- to 18-month-old animals. Ca²⁺ current densities at 0 mV in mdx fibres increased with age, but were always significantly smaller compared to age-matched wild-type fibres. Time-to-peak (TTP) of i _Ca was prolonged in mdx fibres compared to wt fibres. MinD fibres always showed similar TTP and current amplitudes compared to age-matched wt fibres. In all three genotypes, the voltage-dependent inactivation and deactivation of i _Ca were similar. Intracellular resting calcium concentration ([Ca²⁺]_i) and the distribution of dihydropyridine binding sites were also not different in young animals of all three genotypes, whereas i _Ca was markedly reduced in mdx fibres. We conclude, that dystrophin influences L-type Ca²⁺ channels via a direct or indirect linkage which may be disrupted in mdx mice and may be crucial for proper excitation–contraction coupling initiating Ca²⁺ release from the sarcoplasmic reticulum. This linkage seems to be fully restored in the presence of mini-dystrophin.     

The use of caged adenine nucleotides and caged phosphate in intact skeletal muscle fibres of the mouse

The effects of 1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), NPE-caged ADP, NPE-caged phosphate (Pi) and desoxybenzoinyl phosphate (desyl-caged Pi) on mouse skeletal muscle function were studied. All these caged compounds, when microinjected into intact single mouse muscle fibres, reduced the myoplasmic calcium during a tetanus (tetanic [Ca2+]i) and reduced force. Flash photolysis partially reversed this reduction of tetanic [Ca2+]i and force. In fibres fatigued by repeated tetani, flash photolysis of NPE-caged ATP, ADP and Pi, also caused a transient recovery of tetanic [Ca2+]i, and force. Because photolytic release of ATP, ADP and Pi produced comparable effects it seems that the mechanism of action is the reduction in concentration of the caged compound rather than the release of the biologically active molecule. Experiments on mechanically skinned rat skeletal muscle fibres with intact T-tubular/sarcoplasmic reticulum coupling showed that 1 mM NPE-caged ATP had no effect on depolarization-induced force. This result suggests that the depressant effects of the NPE-caged compounds are neither on voltage-activated Ca2+ release from the sarcoplasmic reticulum nor on myofibrillar function. Thus all the caged compounds tested inhibit excitation-contraction coupling in muscle by an unknown mechanism and this limits their value as sources of biologically important molecules. This inhibitory effect was smallest for desyl-caged Pi and under conditions of maximal activation photolytic release of Pi caused a direct inhibition of the contractile proteins. This inhibition amounted to a 1% reduction of maximum force with an increase of [Pi] of about 0.3 mM. The mean rate of force decline under these conditions was 55 s-1, which reflects the rate of cross-bridge cycling during a maximal tetanus.     

On the peripheral and central chemoreception and control of breathing: an emerging role of ATP

Peripheral and central respiratory chemoreceptors are ultimately responsible for maintenance of constant levels of arterial   P _O2  ,   P _CO2  and [H⁺], protecting the brain from hypoxia and ensuring that the breathing is always appropriate for metabolism. The aim of this discussion is to shed some light on the potential mechanisms of chemosensory transduction – the process which links chemosensory mechanisms to the central nervous mechanisms controlling breathing. Recent experimental data suggest that the purine nucleotide ATP acts as a common mediator of peripheral and central chemosensory transduction (within the carotid body and the medulla oblongata, respectively). In response to a decrease in   P _O2  (hypoxia) oxygen-sensitive glomus cells of the carotid body release ATP to activate chemoafferent fibres of the carotid sinus nerve which transmit this information to the brainstem respiratory centres. In response to an increase in   P _CO2/[H⁺]  (hypercapnia) chemosensitive structures located on the ventral surface of the medulla oblongata rapidly release ATP, which acts locally within the medullary respiratory network. The functional role of ATP released at both sites is similar – to evoke adaptive enhancement in breathing. Understanding the mechanisms of ATP release in response to chemosensory stimulation may prove to be essential for further detailed analysis of cellular and molecular mechanisms underlying respiratory chemosensitivity.     

Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 m m lactic acid nor increasing the [K⁺] of the bath solution from 5 to 10 m m had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 m m [K⁺]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca²⁺] ([Ca²⁺]_i). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca²⁺]_i, compared with an increase of ∼20% without cyanide. In conclusion, lactic acid or [K⁺] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca²⁺ release from the sarcoplasmic reticulum.