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E Homsher

Publications and source records attributed to E Homsher.

At least 37 records · Page 2Linked to original sources
PubMed1996

Calcium regulation of thin filament movement in an in vitro motility assay.

The ability of calcium to regulate thin filament sliding velocity was studied in an in vitro motility assay system using cardiac troponin and tropomyosin and rhodamine-phalloidin-labeled skeletal actin and skeletal heavy meromyosin to propel the filaments. Measurements showed that both the number of thin filaments sliding and their sliding speed (Sf) were dependent on the calcium concentration in the range of pCa 5 to 9. Thin filament motility was completely inhibited only if troponin and tropomyosin were added at a concentration of 100 nM to the motility assay solution and the pCa was more than 8. The filament sliding speed was dependent on the pCa in a noncooperative fashion (Hill coefficient = 1) and reached maximum at 5 microns/s at a pCa of 5. The number of filaments moving uniformly decreased from > 90% at pCa 5-6 to near zero in less than 1 pCa unit. This behavior may be explained by a hypothesis in which the regulatory proteins control the number of cross-bridge heads interacting with the thin filaments rather than the rate at which they individually hydrolyze ATP or translocate the thin filaments.

Animals↗
PubMed1995

Regulation of the cross-bridge transition from a weakly to strongly bound state in skinned rabbit muscle fibers.

The regulation of cross-bridge transition from weakly attached to force-bearing states was studied at 10 degrees C in skinned muscle fibers by measuring the rate of force development after a quick release-restretch cycle (ktr), the rate of force decline (kPi) after photogeneration of Pi from caged Pi, and stiffness in the presence and absence of an inhibitor of strong cross-bridge formation, 2,3-butanedione monoxime (BDM). Both BDM and Pi suppressed force more than stiffness. However, reduction of Ca2+ suppressed force and stiffness in a parallel fashion. Both ktr and kPi were reversibly reduced (by 30-35%) in 3 mM BDM, but both were increased by increasing Pi concentration. Reduction of Ca2+ concentration to match the force seen in 3 mM BDM had no effect on kPi but decreased ktr by 85%. These results are inconsistent with cross-bridge models undergoing the transition from a weakly bound to a force-generating state in a single step but are consistent with a model having two steps, one of which is controlled by pCa.

Actins↗
PubMed1993

Factors affecting filament velocity in in vitro motility assays and their relation to unloaded shortening velocity in muscle fibers.

The measurement of fluorescently labeled actin filament movement driven by mechanoenzymes (e.g. myosin) is an important methodology for the study of molecular motors. It is assumed that the filament velocity, Vf, is analogous to the unloaded shortening velocity, Vu, seen in muscle fibers. To evaluate this assumption we compared Vf to literature values for Vu with regard to the effects of [ATP], [ADP], [Pi], pH, ionic strength (10-150 mM) and temperature (15-30 degrees C). Vf and Vu are quantitatively similar with respect to the effects of substrate and product concentrations and temperatures > 20 degrees C. However, Vf is more sensitive to decreases in pH and temperatures < 20 degrees C than is Vu. At ionic strengths of 50-150 mM, Vf and Vu exhibit similar ionic strength dependencies (decreasing with ionic strength). At ionic strengths < 50 mM, Vf is markedly reduced. Thus while Vf is a good analogue for Vu under certain conditions (elevated ionic strength and temperatures > 20 degrees C), under others it is not. The results of motility assays must be cautiously interpreted.

Actin Cytoskeleton↗
PubMed1993

Kinetics of force generation and Pi release in rabbit soleus muscle fibers.

Recent studies have shown that the release of Pi from the AM.ADP.Pi crossbridge occurs in two steps: an initial force-generating isomerization which is followed by the release per se of Pi. These two steps have been shown to be little affected by the presence or absence of Ca2+ but both steps are significantly altered by temperature. In this study, the kinetics of the force generating and Pi release steps of the actomyosin ATPase cycle have been compared in Ca(2+)-activated skinned fibers of rabbit soleus (slow twitch) and psoas (fast twitch) muscle. Pi was rapidly photogenerated within the fiber lattice by laser flash photolysis of caged Pi (1-(2-nitro)phenylethyl phosphate). Pi reduces isometric tension in the steady state, but is less effective in slow muscle than in fast (eg. 14 mM Pi reduces tension by 29 +/- 4.6% in slow muscle and by 47 +/- 5.3% in fast). As in fast muscle, the tension response to a sudden increase in [Pi] in slow muscle has four phases. As in fast muscle, only phase II (an exponential decline in force) appears to be caused by Pi binding to cross-bridges, while the other three phases are probably indirect effects caused by caged Pi photolysis. The amplitude of phase II is consistent with the steady state reduction in force by Pi. The rate of phase II (kpi) is 3.9 +/- 0.33 s-1 at 20 degrees C and 0.28 +/- 0.02 s-1 at 10 degrees C (1 mM Pi).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Reversal of the cross-bridge force-generating transition by photogeneration of phosphate in rabbit psoas muscle fibres.

1. Orthophosphate (P(i), 0.1-2.0 mM) was photogenerated within the filament lattice of isometrically contracting glycerinated fibres of rabbit psoas muscle at 10 and 20 degrees C. The P(i) was produced by laser flash photolysis of the photolabile compound 1-(2-nitrophenyl)ethylphosphate (caged P(i)). Caged P(i) caused a depression of tension that was much smaller than that caused by P(i). 2. Photolysis of caged P(i) produced a decline in isometric force composed of four phases: phase I, a lag phase (e.g. 1-4 ms at 10 degrees C) during which force did not change; phase II, an exponential decline by as much as 20% of the pre-pulse force; phase III, a partial force recovery (0-3% of the pre-pulse force); and phase IV, a further slow (0.5-3 s) decline to the steady value. Phases I, III and IV were largely independent of [P(i)] and are likely to be indirect effects caused by the caged P(i) photolysis. 3. Both the rate and amplitude of phase II depended markedly on [P(i)]. The amplitude of phase II was similar to the reduction of steady-state force by P(i). The rate of phase II increased with increasing temperature and [P(i)]. At high [P(i)] the rate began to saturate, and approached limits of 123 s-1 at 10 degrees C and 194 s-1 at 20 degrees C. 4. The rate of phase II was independent of sarcomere overlap, while the amplitude was proportional to tension at partial filament overlap. A control experiment using caged ATP showed that phase II was not produced by the photolytic by-products or the light pulse. The results suggest that phase II is associated with the force-generating transition of the cross-bridge cycle. 5. Sinusoidal length oscillations at 0.5 and 2 kHz were used to measure muscle stiffness during phase II. Stiffness declined in a single exponential phase, with the same time course as phase II of the tension transient. The change in stiffness was 83 +/- 6% (mean +/- S.E.M., n = 10, 0.5 kHz) of the change in tension when both signals were normalized to their pre-flash values. 6. Analysis of the data shows that two steps are involved in force generation and P(i) release. The non-force exerting AM-ADP-P(i) cross-bridge state first isomerizes to form a force-exerting cross-bridge state (AM'-ADP-P(i)). P(i) is then released to form a second force-generating state, AM'-ADP.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1992

Factors affecting movement of F-actin filaments propelled by skeletal muscle heavy meromyosin.

The measurement of fluorescent-labeled actin filament movement driven by mechanoenzymes (e.g., myosin) is an important methodology for the study of molecular motors. It is assumed that the filament velocity (Vf) is analogous to the unloaded shortening velocity (Vu) seen in muscle fibers. Methods are described to reproducibly quantitate the movement of these filaments and to select uniformly moving filaments and specify their Vf. Use of these techniques allowed comparison of Vf to literature values for Vu with regard to [ATP], [ADP], [Pi], pH, ionic strength (10-150 mM), and temperature (15-30 degrees C). Vf and Vu are quantitatively similar with respect to the effects of substrate and product concentrations and temperatures greater than 20 degrees C. However, Vf is more sensitive to decreases in pH and temperatures less than 20 degrees C than Vu. At ionic strengths of 50-150 mM, Vf and Vu exhibit similar ionic strength dependencies (decreasing with ionic strength). At ionic strengths less than 50 mM, Vf is markedly reduced. Results of experiments using adenosine 5'-O-(3-thiotriphosphate) suggest that increasing the number of weakly bound cross bridges does not seriously affect Vf. Thus, although Vf is a good analogue for Vu under certain conditions (elevated ionic strength and temperatures greater than 20 degrees C), under others it is not. The results of motility assays must be cautiously interpreted.

Actins↗
PubMed1992

Kinetics of force generation and phosphate release in skinned rabbit soleus muscle fibers.

The kinetics of the force generating and Pi release steps of the actomyosin-adenosinetriphosphatase (ATPase) cycle have been compared in Ca(2+)-activated skinned fibers of rabbit soleus (slow twitch) and psoas (fast twitch) muscle. Pi was rapidly photogenerated within the fiber lattice by laser flash photolysis of caged Pi [1-(2-nitro)phenylethyl phosphate]. Pi reduces isometric tension in the steady state but is less effective in slow-twitch muscle than in fast-twitch muscle (e.g., 14 mM Pi reduces tension by 29 +/- 4.6% in slow and by 47 +/- 5.3% in fast). The tension response to a sudden increase in Pi concentration in slow-twitch muscle has four phases, but as in fast-twitch muscle, only phase II (an exponential decline in force) appears to be caused by Pi binding to cross bridges, whereas the other three phases are probably indirect effects caused by caged Pi photolysis. The amplitude of phase II is consistent with the steady-state reduction in force by Pi. The rate of phase II (kappa Pi) is 3.9 +/- 0.33 s-1 at 20 degrees C and 0.28 +/- 0.02 s-1 at 10 degrees C (1 mM Pi). kappa Pi is thus 33 times slower in slow-twitch muscle than in fast at 20 degrees C and 84 times slower at 10 degrees C. In contrast to fast-twitch muscle, in slow muscle kappa Pi is sufficiently slow to partially limit the ATPase turnover rate.

Animals↗
PubMed1990

The effect of phosphate and calcium on force generation in glycerinated rabbit skeletal muscle fibers. A steady-state and transient kinetic study.

The effect of varying concentrations of Pi and Ca2+ on isometric force and on the rate of force development in skinned rabbit psoas muscle fibers has been investigated. Steady-state results show that the three parameters that define the force-pCa relation (Po, pK, and n) all vary linearly with log [Pi]. As [Pi] increases, Po and pK decrease while n increases. The kinetics of force generation in isometrically contracting fibers were studied by laser flash photolysis of caged phosphate. The observed rate of the resulting tension transient, kPi, is 23.5 +/- 1.7 s-1 at 10 degrees C, 0.7 mM Pi, and is independent of [Ca2+] over the range pCa 4.5-7.2. By contrast, kTR, the rate of tension redevelopment following a period of isotonic shortening, is sensitive to [Ca2+] and is slower than kPi (kTR = 13.6 +/- 0.2 s-1 at pCa 4.5, 0.7 mM Pi). The results show that [Ca2+] does not directly affect the Pi release or force-generating steps of the cross-bridge cycle and show that the observed rate of force development depends on how the measurement is made. The data can be interpreted in terms of a model in which strong cross-bridges activate the thin filament, this activation being modulated by Ca2+ binding to troponin.

Actomyosin↗
PubMed1989

Differential recovery of skeletal muscle and peripheral nerve function after ischemia and reperfusion.

Recovery of skeletal muscle function is of prime importance in revascularization of acutely ischemic extremities. In order to study the effect of ischemia and reperfusion on peripheral nerve and skeletal muscle function, temporary unilateral hindlimb ischemia (1 or 3 hr) was induced in 12 rabbits by temporary arterial occlusion of common iliac and femoral arteries and collateral ligation. The control limb was similarly prepared without interruption of blood flow. Both anterior tibialis muscles were attached to force transducers, and the twitch and tetanic tensions were measured at the end of the ischemic interval and during reperfusion (2 hr) by either direct muscle stimulation or stimulation of the deep peroneal nerve. The ischemic limb was reperfused by removal of arterial clamps and reperfusion documented by Doppler flow over the vascular pedicle. The recovery of muscle function of the ischemic/reperfused limb relative to that of the contralateral control limb was used as the index of recovery. By the end of the ischemic interval, no contractile activity was elicited with nerve stimulation for either the 1- or the 3-hr group. After 2 hr of reperfusion, the 1-hr group regained 0.558 +/- 0.116 (mean +/- SEM) of control function with nerve stimulation, while the 3-hr group regained only 0.016 +/- 0.005 of control function (P less than 0.01). In contrast, with direct muscle stimulation, the 1-hr group produced 0.580 +/- 0.015 compared to 0.286 +/- 0.042 (P less than 0.001) after 3 hr of ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

The effect of shortening velocity on the shortening heat and its relationship to the distance shortened.

Irving and Woledge showed using isovelocity (1/2 Vmax) releases that the shortening heat per unit shortening declines with distances shortened. This non-linear behavior of the shortening heat was examined in experiments in which muscles shortened during isovelocity releases at 1/4, 1/2, and 1/1 Vmax. As shortening velocity increases, the reduction in shortening heat production per unit shortening with distance becomes more pronounced. These results could be a consequence of the facts that shortening heat production is load dependent and during shortening at constant velocity, the average force exerted by the muscle shortening a small distance is greater than that exerted by the muscle shortening greater distances. To test this idea, shortening heat production was measured in releases in which the force remained constant during constant velocity shortening. Again, the shortening heat produced per unit shortening declined with distance shortened, and the effect at Vmax was more pronounced than at 1/2 Vmax. These results agree with the two state crossbridge model proposed by Irving and Woledge and suggest that: 1) significant lengths of time are required (minimum of 50 ms at Vmax and 90 ms at 1/2 Vmax) for the energy liberation to reach a steady state in the transition from isometric to shortening contractions, 2) shortening induces shifts in the distribution of crossbridge states whose magnitude increases with shortening velocity, and 3) the non-linearity of shortening heat production is related to the energy imbalance observed during rapid shortening.

Animals↗
PubMed1987

Repriming and reversal of the isometric unexplained enthalpy in frog skeletal muscle.

1. heat production and high-energy phosphate hydrolysis by frog sartorius muscles in 6 s isometric tetani were measured to test the hypothesis that the isometric unexplained enthalpy (u.e.) and labile maintenance heat (l.m.h.) were of similar origin. Muscles were first given a conditioning tetanus to deplete the u.e. and l.m.h. A second (test) 6 s tetanus was given 6-300 s later to ascertain the extent to which l.m.h. and u.e. had recovered (reprimed). 2. The labile maintenance heat repriming was biphasic: 42% of the conditioning l.m.h. reprimed with a time constant of 10 s, the remainder with a time constant of 500 s. 3. The u.e. produced in the test tetanus 6 s after the conditioning tetanus was reduced to 18% of its conditioning value. By 30 s, u.e. had returned to conditioning values even though the amount of high-energy phosphate splitting was 17% less than that in the conditioning tetanus. 4. This observation is supported by measurements revealing that during the 30 s following a 6 s tetanus an amount of enthalpy was absorbed (less heat produced than expected from the measured metabolic changes) whose absolute value (after correction for oxygen consumption) was not different from the amount of unexplained enthalpy liberated during the tetanus. 5. The difference in repriming time course shows that l.m.h. and u.e. are not produced by the same reactions. The data are consistent with the hypothesis that calcium binding to troponin and parvalbumin produce u.e. while calcium binding to troponin and a non-linear time course of ATP hydrolysis produce l.m.h.

Animals↗
PubMed1987

Muscle enthalpy production and its relationship to actomyosin ATPase.

During a maintained tetanus most of the energy liberated is produced by ATP hydrolysis at the cross-bridge. The energy produced by ATP hydrolysis by the sarcoplasmic reticulum is, however, a significant fraction (0.35) of the total. The energy liberation rate depends on a variety of factors (including the shortening velocity, sarcomere length, temperature, fiber type, and duration of contraction) and is, for the most part, consistent with current cross-bridge models. At the beginning of tetanic stimulation, a significant amount of heat (approximately 30 mJ/g) is produced by calcium binding reactions in the sarcoplasm. In the transition from an isometric to a shortening contraction, the cross-bridge cycling rate increases, and as much as 6 mJ/g of unexplained heat is produced. This unexplained heat appears to involve enthalpy changes accompanying a redistribution of cross-bridge intermediates, and it is reversed by high-energy phosphate splitting after the cessation of shortening. The mechanistic significance of these enthalpy changes remains to be elucidated.

Actomyosin↗
PubMed1986

The time course of ATP cleavage by contracting amphibian and mammalian skeletal muscles.

The pattern of energy liberation by tetanically stimulated muscle is reasonably well defined. The energy liberation and ATPase rate are affected by a variety of mechanical factors which, aside from the case of shortening mammalian muscles, are well documented. The amount of ATP consumed by processes associated with the calcium release and sequestration is not trivial, amounting to 30-40% of the energy liberation in an isometric contraction. The energy liberated by isometric and shortening muscles is ultimately accounted for by known chemical reactions. However, there are two instances in which the time course of energy liberation does not correspond to the time course of high energy phosphate utilization. In an isometric tetanus, 30-40 mJ of energy per gram of muscle is produced by reactions probably associated with intracellular circulation of calcium, but not immediately involving ATP hydrolysis. Second, in rapidly shortening muscles, an unknown reaction can produce 6-7 mJ of energy per gram of muscle which is not immediately associated with high energy phosphate splitting. However, immediately after the cessation of shortening, this reaction is reversed by an ATP hydrolysis. Finally, a technique is now available which permits one to perform transient kinetic studies on skinned muscle fibers which are shortening and/or developing force. This development should enable the design of experiments in which the effect of mechanical conditions upon specific steps of the ATPase mechanism can be examined.

Adenosine Triphosphate↗
PubMed1985

Reappraisal of diffusion, solubility, and consumption of oxygen in frog skeletal muscle, with applications to muscle energy balance.

Previously we tested the validity of the one-dimensional diffusion equation for O2 in the excised frog sartorius muscle and used it to measure the diffusion coefficient (D) for O2 in this muscle and the time course of its rate of O2 consumption (Qo2) after a tetanus (Mahler, 1978, 1979, J. Gen. Physiol., 71:533-557, 559-580, 73:159-174). A transverse section of the frog sartorius is in fact well fit by a hemi-ellipse with width divided by maximum thickness averaging 5.1 +/- 0.2. Using the previous techniques with the two-dimensional diffusion equation and this hemi-elliptical boundary yields a value for D that is 30% smaller than reported previously; the revised values at 0, 10, and 22.8 degrees C are 6.2, 7.9, and 10.8 X 10(-6) cm2/s, respectively. After a tetanus at 20 degrees C, Qo2 rose quickly to a peak and then declined exponentially, with a time constant (tau) approximately 15% faster than that reported previously; tau averaged 2.1 min in Rana temporaria and 2.6 min in Rana pipiens. A technique was devised to measure the solubility (alpha) of O2 in intact, respiring muscles, and yielded alpha (muscle)/alpha (H2O) = 1.26 +/- 0.04. With these modifications, the values for O2 consumption obtained with the diffusion method were in agreement with those measured by the direct method of Kushmerick and Paul (1976, J. Physiol. [Lond.]., 254:693-709). Using results from both methods, at 20 degrees C the ratio of phosphorylcreatine split during a tetanus to O2 consumption during recovery ranged from 5.2 to 6.2 mumol/mumol, and postcontractile ATP hydrolysis was estimated to be 13.6 +/- 4.1 (n = 3) nmol/mumol total creatine.

Adenosine Triphosphate↗