Biomedical subjects

K Güth

Publications and source records attributed to K Güth.

At least 19 recordsLinked to original sources

PubMed1991

Time-resolved X-ray diffraction studies on stretch-activated insect flight muscle.

The specific feature of stretch activation of the indirect flight muscle of the tropical waterbug Lethocerus was used to correlate mechanical and structural aspects of muscle contraction. The time courses of the changes in intensities of the strongest equatorial reflections, the (10) and (20) and of the first meridional reflection at 14.5 nm-1 were monitored using synchrotron radiation as a high intensity X-ray source. The ratio of the intensities of the equatorial reflections, (I20/I10), which reflects the mass distribution within the filament lattice array, increases by about 10% relative to the Ca(2+)-activated level when a rapid stretch is imposed, compared with a 200% change seen when fibres change from the relaxed to the rigor state, while the spacing of the lattice planes decreases by about 1%. The intensity of the first meridional reflection at 14.5 nm-1 decreases by about 35% during stretch activation with a slightly faster time course than the delayed tension increase. The results suggest that the average structure of cycling crossbridges is different from that present in the rigor state.

Actin Cytoskeleton↗

PubMed1990

Temporal relationship between force, ATPase activity, and myosin phosphorylation during a contraction/relaxation cycle in a skinned smooth muscle.

The temporal relationship between myosin phosphorylation, contractile force and ATPase activity was studied in skinned preparations from the guinea-pig Taenia coli. When free Calcium concentration [( Ca2+]) was increased from pCa (-log[Ca2+]) 9 to pCa 4.5 at low calmodulin concentration (0.05 microM), ATPase activity and myosin light-chain phosphorylation rose quickly, while the increase in force and stiffness was delayed. The time-course of tension increase was faster at higher calmodulin concentrations (5 microM), although the maximal level of phosphorylation was unchanged. Lowering the calcium concentration from pCa 4.5 to pCa 9 at the plateau of contraction caused a rapid decrease in ATPase activity and in myosin phosphorylation, while force and stiffness decayed more slowly. The force decay could be accelerated by inorganic phosphate. These results suggest that, during contraction, force may be produced actively by phosphorylated and ATP-splitting crossbridges, but may be maintained by dephosphorylated crossbridges which cycle slowly. However, force could also be modulated by calmodulin and inorganic phosphate in a manner not involving an alteration in the extent of myosin phosphorylation.

Adenosine Triphosphatases↗

PubMed1990

Effect of ionic strength on crossbridge kinetics as studied by sinusoidal analysis, ATP hydrolysis rate and X-ray diffraction techniques in chemically skinned rabbit psoas fibres.

In order to identify which steps in the crossbridge are affected by changes in ionic strength, we studied the effect of ionic strength on the rate constants and magnitudes of three exponential processes, the ATP hydrolysis rate and isometric tension during maximal activation (pCa 4.52, 5 mM MgATP). Equatorial X-ray diffraction measurements were also carried out in both relaxing and rigor conditions to examine whether the distance between thick and thin filaments changes with ionic strength (range: 100-300 mM). All experiments were carried out at 20 degrees C and at pH 7.0 on chemically skinned rabbit psoas muscle fibres. Isometric tension and muscle stiffness declined significantly as the ionic strength was increased from 150 mM to 300 mM. The concomitant decrease in the ATP hydrolysis rate was much less than tension, resulting in a large increase in the tension cost. Three rate constants of exponential processes, deduced from sinusoidal analysis, did not change appreciably. The magnitude parameters of all three processes diminished as the ionic strength was increased. During relaxation the filament spacing increased by 5% when the ionic strength was increased from 150 mM to 300 mM. After rigor induction, the spacing did not change with ionic strength. We conclude that a change in ionic strength modifies the rapid equilibrium between the detached state and the 'weakly attached' state, and that this causes considerable effect on isometric tension. We also conclude that other steps in the crossbridge cycle are less sensitive to ionic strength, and that the lattice spacing change is unable to account for the considerable effect of ionic strength on isometric tension.

Adenosine Triphosphate↗

PubMed1988

A low cost high intensity flash device for photolysis experiments.

Novel techniques of flash photolysis experiments require high intensity light sources in the near UV. We describe here a simple and inexpensive flash device which may complete with bulky and expensive laser systems if the experiments do not necessitate very short light pulses. Using a particular optical arrangement and stored electrical energy, a variation of the parameters voltage and capacitance led to a difference in light output by a factor of more than two. The system is used to relax both skeletal and smooth muscle fibres in the rigor state by releasing up to 2 mM ATP from 12.5 mM caged-ATP.

Adenosine Triphosphate↗

PubMed1988

The role of monovalent phosphate anions in the crossbridge kinetics of chemically skinned rabbit psoas fibers.

The role of phosphate ions (Pi) in crossbridge kinetics is investigated by parallel measurements of the ATP hydrolysis rate and the tension transients in maximally activated, chemically skinned rabbit psoas fibers. The tension transients are induced by sinusoidal length oscillations, and resulting time courses are analyzed in terms of three exponential processes (A), (B), and (C). The ATP hydrolysis rate is measured by the NADH fluorscence method coupled with enzymatic reactions which rephosphorylate ADP and oxidize NADH. The hydrolysis rate of the standard activation at 20 degrees C is measured at 0.61 mM/sec. The isometric tension, stiffness, and the ATPase rate progressively decrease with increasing concentrations of Pi (0-16 mM). The decrease is most notably observed with tension, followed by stiffness and the ATPase rate. Both the apparent rate constant and the magnitude parameters of exponential process (B) increase with Pi concentration resulting in a large increase in the oscillatory power output. The active species of Pi which causes this effect is found to be the monovalent anion H2PO4-. The effects of Pi on processes (A) and (C) are only marginal. When fibers are oscillated at 1 Hz, no increase in the ATP hydrolysis rate is observed; a small increase is noticed at 10 Hz (1%), and at 100 Hz (6%). We interpret these results in terms of a crossbridge scheme which adds a branch pathway to the conventional hydrolysis cycle: the number of crossbridges entering the branch pathway increases at higher Pi concentrations and in the presence of oscillations.

Adenosine Triphosphatases↗

PubMed1987

Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers.

Intrinsic troponin C (TnC) was extracted from small bundles of rabbit psoas fibers and replaced with TnC labeled with dansylaziridine (5-dimethylaminonaphthalene-1-sulfonyl). The flourescence of incorporated dansylaziridine-labeled TnC was enhanced by the binding of Ca2+ to the Ca2+-specific (regulatory) sites of TnC and was measured simultaneously with force (Zot, H.G., Güth, K., and Potter, J.D. (1986) J. Biol. Chem. 261, 15883-15890). Various myosin cross-bridge states also altered the fluorescence of dansylaziridine-labeled TnC in the filament, with cycling cross-bridges having a greater effect than rigor cross-bridges; and in both cases, there was an additional effect of Ca2+. The paired fluorescence and tension data were used to calculate the apparent Ca2+ affinity of the regulatory sites in the thin filament and were shown to increase at least 10-fold during muscle activation presumably due to the interaction of cycling cross-bridges with the thin filament. The cross-bridge state responsible for this enhanced Ca2+ affinity was shown to be the myosin-ADP state present only when cross-bridges are cycling. The steepness of the pCa force curves (where pCa represents the -log of the free Ca2+ concentration) obtained in the presence of ATP at short and long sarcomere lengths was the same, suggesting that cooperative interactions between adjacent troponin-tropomyosin units may spread along much of the actin filament when cross-bridges are attached to it. In contrast to the cycling cross-bridges, rigor bridges only increased the Ca2+ affinity of the regulatory sites 2-fold. Taken together, the results presented here indicate a strong coupling between the Ca2+ regulatory sites and cross-bridge interactions with the thin filament.

Animals↗

PubMed1987

The effect of inorganic phosphate on the ATP hydrolysis rate and the tension transients in chemically skinned rabbit psoas fibers.

The role of orthophosphate ions (Pi) in crossbridge kinetics was investigated by parallel measurements of the ATP hydrolysis rate and tension transients in maximally activated, chemically skinned rabbit psoas fibers. The hydrolysis rate of the standard activation at 20 degrees C was measured at 1.25 nmole X s-1 X m-1 X fiber-1, which corresponds to the hydrolysis of 3 moles ATP per mole of myosin head per second. The isometric tension, stiffness extrapolated to the infinite frequency, and the ATPase rate progressively decreased when increasing concentrations of Pi (0-16 mM) were added to the activating saline. The decrease was greatest with tension, followed by stiffness and the ATPase rate. Both the apparent rate constant and the magnitude parameters of exponential process (B) increased with Pi concentration resulting in a significant increase in the oscillatory power output. The effects of Pi on processes (A) and (C) were only marginal. When fibers were oscillated at 1 Hz [close to the characteristic frequency of process (A)], no significant increase in the ATP hydrolysis rate was observed. However, a small increase was noticed at 10 Hz [1%, process (B)], and at 100 Hz [6%, process (C)]. We interpret these results in terms of a crossbridge scheme which adds a branch pathway to the conventional hydrolysis cycle. In the proposed scheme, the number of crossbridges entering the branch pathway increases at higher Pi concentrations and in the presence of imposed oscillations at the proper frequency.

Adenosine Triphosphate↗

PubMed1987

Effect of inorganic phosphate on the Ca2+ sensitivity in skinned Taenia coli smooth muscle fibers. Comparison of tension, ATPase activity, and phosphorylation of the regulatory myosin light chains.

Inorganic phosphate (Pi) decreases maximal tension in contracted skeletal and heart muscle fibers. We investigated the effects of 10 mM Pi on the force-calcium relationship in Triton X-100-skinned Taenia coli smooth muscle fibers. Isometric force measurements show that the calcium sensitivity of the force depends on the phosphate concentration. Furthermore 10 mM Pi relaxes the fibers more at intermediate than at high calcium ion concentrations: At pCa 4.5 tension decreases in the presence of 10 mM Pi by approximately 12% but it decreases 70% at pCa 6.17. Removal of phosphate partially reverses the relaxation. Simultaneous determination of actomyosin ATPase activity and force (Güth, K., and J. Junge, 1982, Nature (Lond.), 300:775-776) shows that the ATPase activity does not correlate with the changes in force. In the presence of Pi, tension decreases more than the ATPase activity. The level of phosphorylation of the 20,000-D regulatory myosin light chain is not changed in the presence or absence of 10 mM Pi. The results are discussed in terms of slowly or noncycling myosin crossbridges formed at lower calcium concentrations, which contribute to the force development but not to the ATPase activity. These crossbridges are considered to be dissociated in the presence of phosphate.

Animals↗

PubMed1986

Fast skeletal muscle skinned fibers and myofibrils reconstituted with N-terminal fluorescent analogues of troponin C.

Glycerinated rabbit fast skeletal muscle fibers were chemically skinned with 1% Brij 35 and partially depleted of endogenous troponin C subunit (TnC) by exposure of the fibers to EDTA (Zot, H. G., and Potter, J. D. (1982) J. Biol. Chem. 257, 7678-7683). The TnC-depleted fibers exhibited a decrease in maximal tension that was mostly restored by readdition of TnC or by the addition of the fluorescent 5-dimethylaminonaphthalene-1-sulfonyl aziridine analogue, TnCDanz. TnCDanz is known to undergo an increase in fluorescence intensity when Ca2+ binds to the two low affinity Ca2+-specific regulatory sites of TnC. Steady-state fractional fluorescence and tension changes were measured simultaneously as a function of Ca2+. The Ca2+ sensitivity of the fluorescence curve was about 0.6 log unit greater than the tension curve. This difference in sensitivity could be explained if separate conformational states of TnC, brought about by Ca2+ binding to the Ca2+-specific sites, produce the fluorescence and tension changes. TnC-depleted fibers were also reconstituted with the fluorescent 2-[(4'-iodoacetamido)analino]naphthalene-6-sulfonic acid analogue, cardiac TnCIaans, which undergoes an increase in fluorescence intensity when Ca2+ binds to the single Ca2+- specific regulatory site. The steady-state fractional fluorescence and tension curves for fibers reconstituted with cardiac TnCIaans had nearly the same Ca2+ sensitivity. The steady-state fractional fluorescence of myofibrils reconstituted with TnCDanz was found to have a greater sensitivity to Ca2+ than the simultaneously measured ATPase. In all cases paired fractional fluorescence and activity curves tended to have parallel dependence on Ca2+. These procedures make it possible to study the Ca2+ binding properties of the Ca2+- specific sites in intact myofibrils and skinned fibers; the results presented suggest that the Ca2+ affinity of the Ca2+-specific sites of troponin are reduced in the thin filament compared to that of troponin in solution.

Adenosine Triphosphatases↗

PubMed1986

Calcium-requirement for activation of skinned vascular smooth muscle from spontaneously hypertensive (SHRSP) and normotensive control rats.

The aim of this study was to clarify whether the increased vascular tone in spontaneous hypertension of rats is due to a change of the calcium-sensitivity of the contractile proteins themselves. In skinned rat tail artery rings from SHRSP and WKY rats the calcium-requirement for half maximal activation (3.6 X 10(-6)M Ca++ for both rat strains) as well as relaxation half times (1.45 +/- 0.43 min, SHRSP and 1.63 +/- 0.48 min, WKY) were found to be identical. The extent of myosin phosphorylation in the contracted and in the relaxed state did not differ between SHRSP and WKY. It is concluded that changes at the level of the contractile proteins are not involved in the increased vascular tone of SHRSP essential hypertension.

Animals↗

PubMed1986

Perfusion cuvette for the simultaneous measurement of mechanical, optical and energetic parameters of skinned muscle fibres.

A micro cuvette is described, which allows to measure mechanical, optical and energetic parameters of single skinned muscle fibres or small fibre bundles. Two examples for possible applications are given: The measurement of ATPase activity of single muscle fibres. The detection of fluorescence changes due to Ca binding to fluorescence labeled TnC, which was incorporated into small bundles of rabbit psoas fibres before the experiment.

Adenosine Triphosphatases↗

PubMed1985

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle.

The contraction induced by a Ca2+-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (Fo), immediate elastic recoil (SE), unloaded shortening velocity (Vus), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca2+ -induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. Fo developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. Vus in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3 +/- 8.9% less than Vus induced by Ca2+ (1.6 X 10(-6) M) in the control fibers. Addition of Ca2+ (1.6 X 10(-6) M) to a contraction induced by MLCK-resulted in small increases in both Fo and Vus. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca2+. We conclude that with respect to actin-myosin interaction, MLCK-and Ca2+ -induced contractions are similar.

Adenosine Triphosphatases↗

PubMed1985

Force generated by non-cycling crossbridges at low ionic strength in skinned smooth muscle from Taenia coli.

In skinned smooth muscle preparations of Taenia coli from guinea pig the cyclic actin-myosin-interaction is inhibited to resting levels whereas the force remains high when in the contracted muscle the ionic strength of the incubation solutions is lowered from 65 mM to 18 mM. In accordance with a strongly depressed ATPase activity under these conditions the isotonic shortening velocity is drastically decreased suggesting that crossbridges are not cycling (latch-state). Since the Ca2+ concentration is kept constant (4 X 10(-5) M) and the phosphorylation of the myosin light chain (LC) is found to be unchanged it must be concluded that the formation of latch-bridges is not necessarily dependent on a change in the Ca2+ concentration or the state of phosphorylation.

Adenosine Triphosphatases↗

PubMed1985

The effect of MgATP on forming and breaking actin-myosin linkages in contracted skinned insect flight muscle fibres.

At neutral pH, fully Ca2+ -activated glycerinated dorsal longitudinal fibre bundles from Lethocerus indicus contract under isometric conditions and respond to release by deactivation, i.e. quick release causes a delayed tension fall. At slightly alkaline pH, the release-induced deactivation becomes a transient phenomenon, i.e. a delayed tension fall is followed by a slow tension recovery. This enabled us to study the effect of MgATP concentration on the phases of deactivation and slow recovery. Reduction of the MgATP concentration slows down the tension response to a quick length change and increases the time constants of the delayed deactivation phase and of the slow recovery phase. The rate constants depend on the ATP concentration according to the Michaelis-Menten law yielding apparent dissociation constants (Km) of 2 mM and 0.09 mM and maximal rate constants of 700 s-1 and 20 s-1 for the deactivation phase (crossbridge detachment) and slow recovery phase (crossbridge reattachment) respectively. The rate of MgATP hydrolysis is also hyperbolically related to the MgATP concentration (Km = 0.14 mM, maximal MgATP turnover rate 1.2 s-1. It is concluded that the effect of MgATP on the deactivation phase, in which crossbridges dissociate strain dependent from the actin, is controlled by at least two mechanisms: (1) fast equilibrium transitions within attached crossbridge states which augment MgATP dissociation from crossbridges with discharged elastic elements; and (2) a crossbridge strain-dependent isomerization of the ternary actin-myosin-MgATP complex which determines crossbridge detachment from the actin.

Actins↗

PubMed1984

Skinned smooth muscle: time course of force and ATPase activity during contraction cycle.

The time course of ATPase activity and force has been determined during contraction and relaxation in skinned (hyperpermeable) anterior byssus retractor muscle, ABRM, of Mytilus edulis and compared with corresponding measurements on skinned taenia coli of guinea-pigs. Following a calcium-induced contraction, lowering the [Ca++] to 10(-8) M rapidly reduces ATPase activity within 2 min to resting levels while force declines only to about 30-50% of maximal tension within the same time. Thus slow relaxation is due to a 'catch-like-state' which is common to different kinds of smooth muscles and can be reduced with cAMP in ABRM and by Pi in taenia coli.

Adenosine Triphosphatases↗

PubMed1984

Tension transients in skinned muscle fibres of insect flight muscle and mammalian cardiac muscle: effect of substrate concentration and treatment with myosin light chain kinase.

Glycerinated single fibres from the dorsal longitudinal muscle of Lethocerus maximus were isometrically contracted in MgATP-salines (10 microM Ca2+; 1.5 mM Mg2+; pH 6.7; 22 degrees C and 20 mM PEP; 100 U/ml pyruvate kinase). The ratio of ATPase activity to tension decreased by a factor of 2 after reducing the ATP-concentration from 15 to 0.5 mM. At all ATP-concentrations (0.5-15 mM), the fibres showed tension adjustments in response to small step changes in length characteristic to an actively contracting muscle: i) an elastic phase which did not depend on ATP-concentration ii) a quick phase of stress relaxation with at least two exponential components; iii) a phase of delayed tension generation. An increase in size of the length step and/or a decrease of ATP-concentration slowed the quick phase and the delayed phase. Similar results have been obtained with skinned cardiac muscle (pig right ventricle). To see, how the isolated contractile system is affected by an increase in the light chain phosphorylation, tension transients were studied in skinned right ventricular muscle fibres before and after incubation with ATP gamma S (2 mM), pure myosin light chain kinase (9 micrograms/ml), Calmodulin (1 microM) and Ca2+ (0.8 microM). While isometric tension development elicited by 20 microM Ca2+ in the ATP salt solution was barely affected in presence of the enzyme, the ATPase activity was decreased by about 25% of the control. There was also a marked decrease (about 50%) in the contraction velocity as determined by the recovery of tension following a quick release. Quick stretches cause an immediate increase in tension followed by a rapid fall and a subsequent rise in tension. The velocity of this tension rise decreased by approximately 30% after incubation with myosin light chain kinase.

Adenosine Triphosphate↗

PubMed1983

Force and force transients in skeletal muscle fibres of the frog skinned by freeze-drying.

A freeze-drying method is described by which single skinned skeletal muscle fibres or fibre bundles can readily be obtained. Skinned fibre segments of the ileofibularis and semitendinous muscles of the frog - activated by means of a rapid increase in the Ca-concentration - showed very stable and reproducible contractions. Complete activation occurred at a Ca-concentration of 1.6 X 10(-6) M and the mid-point of the pCa-tension curve occurred at 6.3 X 10(-7) M. Addition of phosphate (less than or equal to 10(-2) M) had a depressing effect on the speed of the Ca-activated tension development as well as on the maximum tension reached. Addition of caffeine (10(-2) M) had no effect on the tension generation, indicating that the sarcoplasmic reticulum, if present, was not active. The force responses due to rapid length changes applied to the Ca-activated fibre preparations were found to be qualitatively similar to the force responses on intact tissue. This skinning technique might be employed on human biopsies, enabling the measurement of physiological parameters such as for example force and shortening velocity.

Animals↗

PubMed1982

Low Ca2+ impedes cross-bridge detachment in chemically skinned Taenia coli.

Muscle force is generated by cycling cross-bridges between actin and myosin filaments. In smooth muscle, cyclic attachment and detachment of cross-bridges is thought to be induced by a Ca2+- and calmodulin-dependent myosin light chain kinase which phosphorylates myosin. The relaxation that occurs after Ca2+ removal is usually ascribed to dephosphorylation of myosin by a phosphatase as non-phosphorylated myosin is unable to form force-generating criss-bridges. Recently, Dillon et al. claimed, however, that dephosphorylation of attached cross-bridges may impede cross-bridge detachment, thus forming so-called 'latch bridges'. Here we present evidence that after a Ca2+- and calmodulin-induced contraction of chemically skinned guinea pig Taenia coli, the rapid removal of Ca2+ impedes the detachment of the myosin cross-bridges from the actin filament; force can then be maintained without energy consumption. The extremely slowly detaching cross-bridges which maintain the force after Ca2+ removal may indeed correspond to the 'latch bridges' mentioned above.

Actins↗