Contractile properties of muscle fibers from the deep and superficial digital flexors of horses.

Equine digital flexor muscles have independent tendons but a nearly identical mechanical relationship to the main joint they act upon. Yet these muscles have remarkable diversity in architecture, ranging from long, unipennate fibers ("short" compartment of DDF) to very short, multipennate fibers (SDF). To investigate the functional relevance of the form of the digital flexor muscles, fiber contractile properties were analyzed in the context of architecture differences and in vivo function during locomotion. Myosin heavy chain (MHC) isoform fiber type was studied, and in vitro motility assays were used to measure actin filament sliding velocity (V(f)). Skinned fiber contractile properties [isometric tension (P(0)/CSA), velocity of unloaded shortening (V(US)), and force-Ca(2+) relationships] at both 10 and 30°C were characterized. Contractile properties were correlated with MHC isoform and their respective V(f). The DDF contained a higher percentage of MHC-2A fibers with myosin (heavy meromyosin) and V(f) that was twofold faster than SDF. At 30°C, P(0)/CSA was higher for DDF (103.5 ± 8.75 mN/mm(2)) than SDF fibers (81.8 ± 7.71 mN/mm(2)). Similarly, V(US) (pCa 5, 30°C) was faster for DDF (2.43 ± 0.53 FL/s) than SDF fibers (1.20 ± 0.22 FL/s). Active isometric tension increased with increasing Ca(2+) concentration, with maximal Ca(2+) activation at pCa 5 at each temperature in fibers from each muscle. In general, the collective properties of DDF and SDF were consistent with fiber MHC isoform composition, muscle architecture, and the respective functional roles of the two muscles in locomotion.

Dynamic micro-Hall detection of superparamagnetic beads in a microfluidic channel.

We report integration of an InAs quantum well micro-Hall magnetic sensor with microfluidics and real-time detection of moving superparamagnetic beads. Beads moving within and around the Hall cross area result in positive and negative Hall voltage signals respectively. Relative magnitudes and polarities of the signals measured for a random distribution of immobilized beads over the sensor are in good agreement with calculated values and explain consistently the shape of the dynamic signal.

2-deoxy-ATP enhances contractility of rat cardiac muscle.

To investigate the kinetic parameters of the crossbridge cycle that regulate force and shortening in cardiac muscle, we compared the mechanical properties of cardiac trabeculae with either ATP or 2-deoxy-ATP (dATP) as the substrate for contraction. Comparisons were made in trabeculae from untreated rats (predominantly V1 myosin) and those treated with propylthiouracil (PTU; V3 myosin). Steady-state hydrolytic activity of cardiac heavy meromyosin (HMM) showed that PTU treatment resulted in >40% reduction of ATPase activity. dATPase activity was >50% elevated above ATPase activity in HMM from both untreated and PTU-treated rats. V(max) of actin-activated hydrolytic activity was also >50% greater with dATP, whereas the K(m) for dATP was similar to that for ATP. This indicates that dATP increased the rate of crossbridge cycling in cardiac muscle. Increases in hydrolytic activity were paralleled by increases of 30% to 80% in isometric force (F(max)), rate of tension redevelopment (k(tr)), and unloaded shortening velocity (V(u)) in trabeculae from both untreated and PTU-treated rats (at maximal Ca(2+) activation), and F-actin sliding speed in an in vitro motility assay (V(f)). These results contrast with the effect of dATP in rabbit psoas and soleus fibers, where F(max) is unchanged even though k(tr), V(u), and V(f) are increased. The substantial enhancement of mechanical performance with dATP in cardiac muscle suggests that it may be a better substrate for contractility than ATP and warrants exploration of ribonucleotide reductase as a target for therapy in heart failure.

Permeation and interaction of divalent cations in calcium channels of snail neurons.

We have studied the current-carrying ability and blocking action of various divalent cations in the Ca channel of Lymnaea stagnalis neurons. Changing the concentration or species of the permeant divalent cation shifts the voltage dependence of activation of the Ca channel current in a manner that is consistent with the action of the divalent cation on an external surface potential. Increasing the concentration of the permeant cation from 1 to 30 mM produces a twofold increase in the maximum Ca current and a fourfold increase in the maximum Ba current; the maximum Ba current is twice the size of the maximum Ca current for 10 mM bulk concentration. Correcting for the changing surface potential seen by the gating mechanism, the current-concentration relation is almost linear for Ba2+, and shows only moderate saturation for Ca2+; also, Ca2+, Ba2+, and Sr2+ are found to pass through the channel almost equally well. These conclusions are obtained for either of two assumptions: that the mouth of the channel sees (a) all or (b) none of the surface potential seen by the gating mechanism. Cd2+ blocks Lymnaea and Helix Ca channels at concentrations 200 times smaller than those required for Co2+ or Ni2+. Ca2+ competes with Cd2+ for the blocking site; Ba2+ binds less strongly than Ca2+ to this site. Mixtures of Ca2+ and Ba2+ produce an anomalous mole fraction effect on the Ca channel current. After correction for the changing surface potential (using either assumption), the anomalous mole fraction effect is even more prominent, which suggests that Ba2+ blocks Ca current more than Ca2+ blocks Ba current.

Response of upper limb blood flow to handgrip exercise after Blalock-Taussig operation (for tetralogy of Fallot) or subclavian flap operation (for aortic isthmic coarctation).

To evaluate the effects of long-term reductions in perfusion pressure on blood flow responses to increased functional demand, 5 patients (aged 12 to 26 years) without normal aortic to subclavian artery blood flow to 1 arm as a result of surgery to treat congenital heart disease were studied. Five age- and sex-matched healthy (control) subjects were also studied. In the patients, forearm blood flow was not different in the surgical and normal arms at rest (3.6 +/- 0.6 vs 4.0 +/- 0.7 ml/min/100 ml, respectively, mean +/- standard error, difference not significant) despite lower systolic blood pressure in the surgical arm (87 +/- 2 vs 115 +/- 2 mm Hg, p less than 0.05). The increases in heart rate, systolic blood pressure, forearm electromyographic activity (index of muscle fatigue) and postexercise forearm blood flow (index of muscle oxygen deficit) were not different in response to 2.5 minutes of submaximal rhythmic handgrip exercise (50% of maximal force) performed with the surgical versus the normal arms. Peak forearm blood flow elicited by combined ischemia and maximal isometric handgrip exercise was not significantly different in surgical and normal arms in the group as a whole (39 +/- 4 vs 43 +/- 3 ml/min/100 ml, difference not significant), although some bilateral deficit (20 to 38%) was observed in 2 patients. No bilateral differences were observed in the control subjects under any condition. The finding of normal physiologic adjustments to submaximal rhythmic handgrip exercise with the surgical arm suggests that oxygen delivery during exercise was adequate.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for platelet-activating factor receptor subtypes on human polymorphonuclear leukocyte membranes.

Platelet-activating factor (PAF) is a potent phospholipid mediator that acts through specific cell surface receptors. The existence of PAF receptor subtypes has been suggested by functional and radioligand binding studies in a variety of cells and tissues. This report addresses this issue more directly and demonstrates differences between specific PAF receptors in human polymorphonuclear leukocytes (PMNs) and COS-7 cells transfected with the cloned human PAF receptor gene. The presence of more than one receptor in human PMNs is supported by three different studies. First, the Kd from the saturation isotherms for the binding of [3H]WEB 2086 on PMNs was 7-fold larger (Kd = 29.2 nM) than the kinetic Kd (4.2 nM). Second, the pseudo-Hill slope determined from the saturation experiments with PMNs was significantly lower than unity (0.69 +/- 0.05 SEM), and the saturation Kd values for transfected COS-7 (Kd = 9.6 nM) and PMN membranes were significantly different. These results contrasted with those for the transfected COS-7 cells, which showed a Kd from the saturation isotherms similar to that of the kinetic Kd (3.2 nM) and a pseudo-Hill slope that was not different from 1.0. Third, when the radiolabeled ligand [3H]WEB 2086 was increased in concentration from 10 to 50 nM in inhibition experiments with the human PMN membranes, the Ki increased, indicative of binding mainly to receptors with lower affinity. These results suggest that PAF receptor subtypes exist in human PMNs based on distinct radioligand binding characteristics from the human cloned PAF receptor.

Influence of physical training on heart rate variability and baroreflex circulatory control.

Nineteen males (aged 45-68 yr) were studied before and after either a period of regular endurance exercise [walk/jog 3-4 days/wk for 30 +/- 1 (SE) wk, n = 11] or unchanged physical activity (38 +/- 2 wk, n = 8) (controls) to determine the influence of physical training on cardiac parasympathetic (vagal) tone and baroreflex control of heart rate (HR) and limb vascular resistance (VR) at rest in middle-aged and older men. Training resulted in a marked increase in maximal O2 uptake (31.6 +/- 1.2 vs. 41.0 +/- 1.8 ml.kg-1.min-1, 2.56 +/- 0.16 vs. 3.20 +/- 0.18 l/min, P less than 0.05) and small (P less than 0.05) reductions in body weight (81.2 +/- 3.5 vs. 78.7 +/- 4.0 kg) and body fat (23.8 +/- 1.3 vs. 20.9 +/- 1.3%). HR at rest was slightly, but consistently, lower after training (63 +/- 2 vs. 58 +/- 1 beats/min, P less than 0.05). In general, HR variability (index of cardiac vagal tone) was greater after training. Chronotropic responsiveness to either brief carotid baroreflex stimulation (neck suction) or inhibition (neck pressure), or to non-specific arterial baroreflex inhibition induced by a hypotensive level of lower body suction, was unchanged after training. In contrast, the magnitude of the reflex increase in forearm VR in response to three levels of lower body suction was markedly attenuated after training (38-59%; P less than 0.05 at -10 and -30 mmHg; P = 0.07 at -20 mmHg). None of these variables or responses was altered over time in the controls. These findings indicate that in healthy, previously sedentary, middle-aged and older men, strenuous and prolonged endurance training 1) elicits large increases in maximal exercise capacity and small reductions in HR at rest, 2) may increase cardiac vagal tone at rest, 3) does not alter arterial baroreflex control of HR, and 4) results in a diminished forearm vasoconstrictor response to reductions in baroreflex sympathoinhibition.

Autonomic mediation of the pressor responses to isometric exercise in humans.

The purpose of this study was to determine the respective contributions of tachycardia and increases in sympathetic nerve activity (SNA) in mediating the pressor responses to fatiguing vs. nonfatiguing levels of isometric handgrip exercise (IHE) in humans. We performed direct (microneurographic) measurements of muscle SNA from the right peroneal nerve in the leg and recorded arterial pressure (AP) and heart rate (HR) in eight healthy subjects before (control), during, and after 2.5 min of IHE at 15, 25, or 35% of maximal voluntary contraction (MVC). At 15% MVC, AP increased during the initial 1.5 min of IHE (7 mmHg, P less than 0.05) and remained at this level; at 25 and 35% MVC, AP increased throughout IHE (22 and 34 mmHg vs. control, respectively, P less than 0.05). HR increased during the initial 1.5 min of IHE at all three levels (5, 12, and 19 beats/min, respectively, P less than 0.05) but did not increase further over the last minute. At 15% MVC, muscle SNA did not increase above control; during 25 and 35% MVC, muscle SNA did not increase during the 1st min of IHE but increased progressively thereafter (109 and 205% vs. control, respectively, P less than 0.05). The magnitudes of the average increases in AP and muscle SNA over the last minute of IHE were directly related (r = 0.99, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential control of forearm and calf vascular resistance during one-leg exercise.

The purpose of this study was to determine whether blood flow (BF) and vascular resistance (VR) are controlled differently in the nonactive arm and leg during submaximal rhythmic exercise. In eight healthy men we simultaneously measured BF to the forearm and calf (venous occlusion plethysmography) and arterial blood pressure (sphygmomanometry) and calculated whole limb VR before (control) and during 3 min of cycling with the contralateral leg at 38, 56, and 75% of peak one-leg O2 uptake (VO2). During the initial phase of exercise (0-1.5 min) at all work loads, BF increased and VR decreased in the forearm (P less than 0.05), whereas calf BF and VR remained at control levels. Thereafter, BF decreased and VR increased in parallel and progressive fashion in both limbs. At end exercise, forearm BF and VR were not different from control values (P greater than 0.05); however, in the calf, BF tended to be lower (P less than 0.05 at 75% peak VO2 only) and VR was higher (23 +/- 9, 44 +/- 14, and 88 +/- 23% above control at 38, 56, and 75% of peak VO2, respectively, all P less than 0.05). In a second series of studies, forearm and calf skin blood flow (laser-Doppler velocimetry) and arterial pressure were measured during the same levels of exercise in six of the subjects. Compared with control, skin BF was unchanged and VR was increased (P less than 0.05) in the forearm by end exercise at all work loads, whereas calf skin BF increased (P less than 0.05) and VR decreased (P less than 0.05). The present findings indicate that skeletal muscle and skin VR are controlled differently in the nonactive forearm and calf during the initial phase of rhythmic exercise with the contralateral leg. Skeletal muscle vasodilation occurs in the forearm but not in the calf; forearm skin vasoconstricts, whereas calf skin vasodilates. Finally, during exercise a time-dependent vasoconstriction occurs in the skeletal muscle of both limbs.

Hypoxemia raises muscle sympathetic activity but not norepinephrine in resting humans.

The experimental objective was to determine whether moderate to severe hypoxemia increases skeletal muscle sympathetic nervous activity (MSNA) in resting humans without increasing venous plasma concentrations of norepinephrine (NE) and epinephrine (E). In nine healthy subjects (20-34 yr), we measured MSNA (peroneal nerve), venous plasma levels of NE and E, arterial blood pressure, heart rate, and end-tidal O2 and CO2 before (control) and during breathing of 1) 12% O2 for 20 min, 2) 10% O2 for 20 min, and 3) 8% O2 for 10 min--in random order. MSNA increased above control in five, six, and all nine subjects during 12, 10, and 8% O2, respectively (P less than 0.01), but only after delays of 12 (12% O2) and 4 min (8 and 10% O2). MSNA (total activity) rose 83 +/- 20, 260 +/- 146, and 298 +/- 109% (SE) above control by the final minute of breathing 12, 10, and 8% O2, respectively. NE did not rise above control at any level of hypoxemia; E rose slightly (P less than 0.05) at one time only with both 10 and 8% O2. Individual changes in MSNA during hypoxemia were unrelated to elevations in heart rate or decrements in blood pressure and end-tidal CO2--neither of which always fell. We conclude that in contrast to some other sympathoexcitatory stimuli such as exercise or cold stress, moderate to severe hypoxemia increases leg MSNA without raising plasma NE in resting humans.

Skeletal muscle regulatory proteins enhance F-actin in vitro motility.

Using an in vitro motility assay, we have investigated the effects of rabbit skeletal muscle regulatory proteins, troponin and tropomyosin, on the gliding of F-actin filaments or F-actin filaments containing these regulatory proteins. We demonstrate that Ca2+ does not affect the motility of F-actin gliding on HMM, but does in the presence of skeletal muscle tropomyosin and troponin. We conclude that Ca2+ affects motility through troponin because, like F-actin, F-actin-Tm filaments show no Ca(2+)-dependence to their gliding speeds. Furthermore, there is a large enhancement of the gliding speed (about 75%) in the presence of skeletal muscle tropomyosin, troponin + saturating Ca2+ over that seen with F-actin filaments. This enhancement is not due to the action of tropomyosin alone as skeletal muscle tropomyosin without troponin enhances the speed little (about 5%) over that of F-actin. Thus troponin confers Ca2+ sensitivity to the motility and, additionally, potentiates motility greatly along with tropomyosin in the presence of saturating Ca2+. When [HMM] is varied, the decline in speed of F-actin seen at low HMM density is changed little by tropomyosin in the F-actin-Tm filaments. These data show that the skeletal regulatory proteins interact with F-actin to enhance the interaction with HMM particularly in the presence of troponin and saturating Ca2+ and enhance the gliding speed in the in vitro motility assay as they potentiate the ATPase activity in the isolated proteins. This enhancement of speed in the motility assay cannot be ascribed to tropomyosin alone.

Effectiveness of Centruroides scorpion antivenom compared to historical controls.

BACKGROUND: Envenomation by North American scorpions of genus Centruroides is associated with a syndrome of neurotoxicity and respiratory compromise that disproportionately affects rural children. Severe scorpion envenomation is rare, which makes treatment difficult to study using conventional controlled clinical trials; and small-scale placebo-controlled trials conducted in tertiary centers are of limited generalizability to the community setting. Open label studies, although safer and easier to conduct, are of limited value unless a suitable comparator group is used. Historical controls may be appropriate when concurrent controls are not feasible or ethical. METHODS: A successful placebo-controlled, double-blind clinical trial design was adapted for community use in Arizona and Mexico. A comparator population was established by replacement of the placebo group with a retrospective cohort and preservation of criteria for inclusion, exclusion, dosing and endpoint assessment. Study endpoints were selected to demonstrate the clearest possible difference between treatment groups, while minimizing confounders. Results were summarized and endpoints were directly compared between groups and with the prior double-blind study. RESULTS: The clinical syndrome remained evident in 95.9% of the historical cohort (93/97) 4 h after admission, and their cumulative dose of midazolam given between baseline and discharge was 5.29 ± 8.68 mg/kg (range 0-62.8). Among 78 prospectively treated cases, none received midazolam and only 2 (2.8%) remained symptomatic at 4 h. Venom was detectable in the plasma of all antivenom recipients tested, and it dropped by 90% of baseline in 95% of cases studied. CONCLUSIONS: The results of this pragmatic study strongly support the findings of the double-blind, placebo controlled clinical trial of the same antivenom. Recipients of antivenom at rural sites improved at a rate similar to that in the intensive care (ICU) setting, and historical cases resolved at a rate similar to that for untreated ICU controls. Use of antivenom in the primary care setting appeared to be safe and effective and to eliminate the need for intensive care or for transport to a tertiary care center, in all subjects prospectively studied.

Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers.

We have investigated (a) effects of varying proton concentration on force and shortening velocity of glycerinated muscle fibers, (b) differences between these effects on fibers from psoas (fast) and soleus (slow) muscles, possibly due to differences in the actomyosin ATPase kinetic cycles, and (c) whether changes in intracellular pH explain altered contractility typically associated with prolonged excitation of fast, glycolytic muscle. The pH range was chosen to cover the physiological pH range (6.0-7.5) as well as pH 8.0, which has often been used for in vitro measurements of myosin ATPase activity. Steady-state isometric force increased monotonically (by about threefold) as pH was increased from pH 6.0; force in soleus (slow) fibers was less affected by pH than in psoas (fast) fibers. For both fiber types, the velocity of unloaded shortening was maximum near resting intracellular pH in vivo and was decreased at acid pH (by about one-half). At pH 6.0, force increased when the pH buffer concentration was decreased from 100 mM, as predicted by inadequate pH buffering and pH heterogeneity in the fiber. This heterogeneity was modeled by net proton consumption within the fiber, due to production by the actomyosin ATPase coupled to consumption by the creatine kinase reaction, with replenishment by diffusion of protons in equilibrium with a mobile buffer. Lactate anion had little mechanical effect. Inorganic phosphate (15 mM total) had an additive effect of depressing force that was similar at pH 7.1 and 6.0. By directly affecting the actomyosin interaction, decreased pH is at least partly responsible for the observed decreases in force and velocity in stimulated muscle with sufficient glycolytic capacity to decrease pH.

Effect of physiological ADP concentrations on contraction of single skinned fibers from rabbit fast and slow muscles.

To directly assess the possible role of ADP in muscle fatigue, we have studied the effect of physiological MgADP levels on maximum Ca(2+)-activated isometric force and unloaded shortening velocity (Vus) of single skinned fiber segments from rabbit fast-twitch (psoas) and slow-twitch (soleus) muscles. MgADP concentration was changed in a controlled and well-buffered manner by varying creatine (Cr) in solutions, which also contained MgATP, phosphocreatine (PCr), and creatine kinase (CK). To quantify ADP as a function of Cr added, we determined the apparent equilibrium constant (K') of CK for the conditions of our experiments (pH 7.1, 3 mM Mg2+, 12 degrees C): K' = (sigma [Cr]. sigma [ATP])/(sigma [PCr]. sigma [ADP]) = 260 +/- 3 (SE). In this manner, ADP was altered essentially as occurs during stimulation in vivo but without the concomitant changes in pH and P(i), which affect force and Vus. As ADP (and Cr) was increased, force and Vus decreased in both fiber types; at the highest ADP level used, 200 microM, normalized force was 96.6 +/- 1.7% for psoas (n = 6) and 93.7 +/- 2.8% for soleus (n = 6), and Vus was 80.4 +/- 2.4% for psoas and 91.3 +/- 7.7% for soleus. Diffusion-reaction calculations indicated that radial gradients of metabolite concentrations within fibers could not explain the small effects of ADP on fiber mechanics, and experiments verified that metabolite levels were well buffered within fibers by the CK reaction. Exogenous CK was added to bathing solutions at 290 U/ml, threefold above that necessary to maintain Vus independent of CK concentration; in the absence of PCr and exogenous CK, at least a fourfold increased MgATP was necessary to maintain Vus at the control level. Adenylate kinase activity was not detectable; thus myofibrillar adenosine-triphosphatase and exogenous CK activities were the major determinants of nucleotide levels within activated cells. Cr alone (in absence of PCr and exogenous CK) also decreased force and Vus, presumably by a nonspecific mechanism. Over the physiological range, altered ADP had little or no effect on force or Vus in well-buffered conditions. It is therefore likely that other factors decrease force and Vus during muscular fatigue.

Faster force transient kinetics at submaximal Ca2+ activation of skinned psoas fibers from rabbit.

The early, rapid phase of tension recovery (phase 2) after a step change in sarcomere length is thought to reflect the force-generating transition of myosin bound to actin. We have measured the relation between the rate of tension redevelopment during phase 2 (r), estimated from the half-time of tension recovery during phase 2 (r = t0.5(-1)), and steady-state force at varying [Ca2+] in single fibers from rabbit psoas. Sarcomere length was monitored continuously by laser diffraction of fiber segments (length approximately 1.6 mm), and sarcomere homogeneity was maintained using periodic length release/restretch cycles at 13-15 degrees C. At lower [Ca2+] and forces, r was elevated relative to that at pCa 4.0 for both releases and stretches (between +/- 8 nm). For releases of -3.4 +/- 0.7 nm.hs-1 at pCa 6.6 (where force was 10-20% of maximum force at pCa 4.0), r was 3.3 +/- 1.0 ms-1 (mean +/- SD; N = 5), whereas the corresponding value of r at pCa 4.0 was 1.0 +/- 0.2 ms-1 for releases of -3.5 +/- 0.5 nm.hs-1 (mean +/- SD; N = 5). For stretches of 1.9 +/- 0.7 nm.hs-1, r was 1.0 +/- 0.3 ms-1 (mean +/- SD; N = 9) at pCa 6.6, whereas r was 0.4 +/- 0.1 ms-1 at pCa 4.0 for stretches of 1.9 +/- 0.5 (mean +/- SD; N = 14). Faster phase 2 transients at submaximal Ca(2+)-activation were not caused by changes in myofilament lattice spacing because 4% Dextran T-500, which minimizes lattice spacing changes, was present in all solutions. The inverse relationship between phase 2 kinetics and force obtained during steady-state activation of skinned fibers appears to be qualitatively similar to observations on intact frog skeletal fibers during the development of tetanic force. The data are consistent with models that incorporate a direct effect of [Ca2+] on phase 2 kinetics of individual cross-bridges or, alternatively, in which phase 2 kinetics depend on cooperative interactions between cross-bridges.

Compliant realignment of binding sites in muscle: transient behavior and mechanical tuning.

The presence of compliance in the lattice of filaments in muscle raises a number of concerns about how one accounts for force generation in the context of the cross-bridge cycle--binding site motions and coupling between cross-bridges confound more traditional analyses. To explore these issues, we developed a spatially explicit, mechanochemical model of skeletal muscle contraction. With a simple three-state model of the cross-bridge cycle, we used a Monte Carlo simulation to compute the instantaneous balance of forces throughout the filament lattice, accounting for both thin and thick filament distortions in response to cross-bridge forces. This approach is compared to more traditional mass action kinetic models (in the form of coupled partial differential equations) that assume filament inextensibility. We also monitored instantaneous force generation, ATP utilization, and the dynamics of the cross-bridge cycle in simulations of step changes in length and variations in shortening velocity. Three critical results emerge from our analyses: 1) there is a significant realignment of actin-binding sites in response to cross-bridge forces, 2) this realignment recruits additional cross-bridge binding, and 3) we predict mechanical behaviors that are consistent with experimental results for velocity and length transients. Binding site realignment depends on the relative compliance of the filament lattice and cross-bridges, and within the measured range of these parameters, gives rise to a sharply tuned peak for force generation. Such mechanical tuning at the molecular level is the result of mechanical coupling between individual cross-bridges, mediated by thick filament deformations, and the resultant realignment of binding sites on the thin filament.

Calcium current activation kinetics in neurones of the snail Lymnaea stagnalis.

Both the activation kinetics and the magnitude of the Ca current in Lymnaea are strongly dependent on temperature. The Q10 for the reciprocal of the activation time constant is 4.9 +/- 0.2 and the Q10 for the maximum current is 2.3 +/- 0.1. By lowering the temperature to 7-10 degrees C, we have been able to resolve the Ca tail currents. The block of Ca current by Cd2+ is voltage dependent, being more effective at more positive potentials. As determined from the magnitude of the tail currents, the Ca permeability is not maximally activated until the membrane potential is greater than +70 mV. The Ca permeability is half activated in the range 30-35 mV. The open-channel current-voltage relation for the Ca current is in rough agreement with the prediction of the constant-field equation. There is no indication of current saturation at negative potentials for potentials down to -60 mV. The Ca tail current decays with at least two time constants, one 200-400 microseconds and the other 2-4 ms. Although these time constants are not strongly voltage dependent, the ratio of the amplitude of the fast component of the tail current to that of the slow component is much larger at -60 mV than at 0 mV. The time course of the Ba tail current is very similar to that of the Ca tail current. The time course of the activation of the Ca current follows m2 kinetics and does not show evidence for a Cole-Moore-type shift for holding potentials between -50 and -110 mV. During a second positive pulse applied 1 ms after the first, the Ca current activates more rapidly, without the delay characteristic of the Ca current of a single positive pulse. The activation of the Ca current can be represented by a linear sequential model. The simplest model that describes both the turn-on and the turn-off of the Ca current must have at least three closed states, followed by a single open state.

Activation dependence and kinetics of force and stiffness inhibition by aluminiofluoride, a slowly dissociating analogue of inorganic phosphate, in chemically skinned fibres from rabbit psoas muscle.

To examine the mechanism by which aluminiofluoride, a tightly binding analogue of inorganic phosphate, inhibits force in single, chemically skinned fibres from rabbit psoas muscle, we measured the Ca(2+)-dependence of the kinetics of inhibitor dissociation and the kinetics of actomyosin interactions when aluminiofluoride was bound to the crossbridges. The relation between stiffness and the speed of stretch during small amplitude ramp stretches (< 5 nm per h.s.) was used to characterize the kinetic properties of crossbridges attached to actin; sarcomere length was assessed with HeNe laser diffraction. During maximum Ca(2+)-activation at physiological ionic strength (pCa 4.0, 0.2 M gamma/2), stiffness exhibited a steep dependence on the rate of stretch; aluminiofluoride inhibition at pCa 4.0 (0.2 M gamma/2) resulted in an overall decrease in stiffness, with stiffness at high rates of stretch (10(3)-10(4) nm per h.s. per s) being disproportionately reduced. Thus the slope of the stiffness-speed relation was reduced during aluminiofluoride inhibition of activated fibres. Relaxation of inhibited fibres (pCa 9.2, 0.2 M gamma/2) resulted in aluminiofluoride being 'trapped' and was accompanied by a further decrease in stiffness at all rates of stretch which was comparable to that found in control relaxed fibres. In relaxed, low ionic strength conditions (pCa 9.2, 0.02 M gamma/2) which promote weak crossbridge binding, stiffness at all rates of stretch was significantly inhibited by aluminiofluoride 'trapped' in the fibre. To determine the Ca(2+)-dependence of inhibitor dissociation, force was regulated independent of Ca2+ using an activating troponin C (aTnC). Results obtained with a TnC-activated fibres confirmed that there is no absolute requirement for Ca2+ for recovery from force inhibition by inorganic phosphate analogues in skinned fibres; the only requirement is thin filament activation which enables active crossbridge cycling. These results indicate that aluminiofluoride preferentially inhibits rapid equilibrium or weak crossbridge attachment to actin, that aluminiofluoride-bound crossbridges attach tightly to the activated thin filament, and that, at maximal (or near-maximal) activation, crossbridge attachment to actin prior to inorganic phosphate analogue dissociation is the primary event regulated by Ca2+.

Effect of intracellular pH on force development depends on temperature in intact skeletal muscle from mouse.

The cellular mechanism of muscle fatigue is still in debate. Opposite conclusions regarding the role of intracellular pH (pHi) in fatigue have been drawn from skinned fiber vs. isolated perfused muscle studies. Because these experiments are typically performed at different temperatures, we tested the hypothesis that temperature alters the effects of pH on force. Tetanic force of isolated mouse extensor digitorum longus was measured at temperatures between 13 and 25 degrees C in either normocapnia (5% CO2) or hypercapnia (25% CO2). Hypercapnia decreased pHi (monitored by 31P nuclear magnetic resonance spectroscopy) by the same amount at both 15 and 25 degrees C. However, inhibition of force by hypercapnia was greater at the lower temperature. A similar pattern of temperature-dependent inhibition of force by pH was observed in glycerinated fibers from rabbit psoas at maximum Ca2+ activation. We conclude that temperature differences are responsible for disparate conclusions on the role of pHi in muscle fatigue. Based on our results, we suggest that changes in pHi may have little or no role in the loss in force production associated with muscular fatigue at physiological temperatures.

Calmidazolium alters Ca2+ regulation of tension redevelopment rate in skinned skeletal muscle.

To examine if the Ca2(+)-binding kinetics of troponin C (TnC) can influence the rate of cross-bridge force production, we studied the effects of calmidazolium (CDZ) on steady-state force and the rate of force redevelopment (ktr) in skinned rabbit psoas muscle fibers. CDZ increased the Ca2(+)-sensitivity of steady-state force and ktr at submaximal levels of activation, but increased ktr to a greater extent than can be explained by increased force alone. This occurred in the absence of any significant effects of CDZ on solution ATPase or in vitro motility of fluorescently labeled F-actin, suggesting that CDZ did not directly influence cross-bridge cycling. CDZ was strongly bound to TnC in aqueous solutions, and its effects on force production could be reversed by extraction of CDZ-exposed native TnC and replacement with purified (unexposed) rabbit skeletal TnC. These experiments suggest that the method of CDZ action in fibers is to bind to TnC and increase its Ca2(+)-binding affinity, which results in an increased rate of force production at submaximal [Ca2+]. The results also demonstrate that the Ca2(+)-binding kinetics of TnC influence the kinetics of ktr.