Molecular and cellular aspects of muscle contraction. General discussion part I.
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Actins↗
Biomedical subjects
H Sugi
Publications and source records attributed to H Sugi.
Effect of deuterium oxide on actomyosin motility in vitro.
Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D(2)O) and water (H(2)O) to examine the effect of D(2)O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D(2)O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H(2)O assay solution, but the maximum velocity (4.1+/-0.5 microm/s, n=11) at pD 8.5 in D(2)O was about 60% of that (7.1+/-1.1 microm/s, n=11) at pH 8.5 in H(2)O. The K(m) values of 95 and 80 microM and V(max) values of 3.2 and 5.1 microm/s for the D(2)O and H(2)O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis-Menten equation. The K(m) value of actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) was decreased from 50 microM [actin] in H(2)O to 33 microM [actin] in D(2)O without any significant changes in V(max) (9.4 s(-1) in D(2)O and 9.3 s(-1) in H(2)O). The rate constants of ADP release from the acto-S1-ADP complex measured by the stopped flow method were 361+/-26 s(-1) (n=27) in D(2)O and 512+/-39 s(-1) (n=27) in H(2)O at 6 degrees C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D(2)O results from a slowing of the release of ADP from the actomyosin-ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D(2)O.
Actin Cytoskeleton↗
Targeting antigen-specific receptors on B lymphocytes to generate high yields of specific monoclonal antibodies directed against biologically active lower antigenic peptides within presenilin 1.
We describe a targeting technique that selects antigen-specific receptors on B lymphocytes using antigen driven selective production of monoclonal antibodies which are directed against functional peptide sequences within the presenilin 1 molecule that is believed to be related to the early-onset of familial Alzheimer's disease. Three different peptide sequences of presenilin 1 were constructed, one including the region around the amino acid position 300, where the putative cleavage site exists and the other two present in the N- and C-terminal regions of that site. The efficiency in production of the desired monoclonal antibodies was at least 5-40-fold that obtained with the poly(ethylene glycol) (PEG)-mediated method. In addition, monoclonal antibodies directed against each of the peptide sequence displayed a high specificity for the corresponding peptide, in contrast to the lack of success using the PEG method. Also, the selection of surface immunoglobulin receptors on B lymphocytes by the peptides of interest was confirmed by immunofluorescent analysis. Here we demonstrate that targeting B lymphocytes results in the successful and efficient production of highly specific monoclonal antibodies against the lower antigenic peptide sequences.
Animals↗
The Annual Meeting on Muscle Contraction and Cell Motility. Tokyo, Japan, 6-8 January 2001.
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Adenosine Triphosphate↗
A novel method to study contraction characteristics of a single cardiac myocyte using carbon fibers.
To facilitate cardiac muscle research, we developed a novel method by which the force and length of a single ventricular myocyte can be recorded with a pair of carbon graphite fibers attached firmly to both ends. One fiber was stiff, whereas the other fiber was compliant to allow the recording of force and shortening during twitch contractions. The image of the compliant carbon fiber was projected onto a pair of photodiodes, and their output was fed to a piezoelectric transducer after variable amplifications to alter the effective compliance of the carbon fiber. Thus contraction of the myocyte was induced under virtually isometric conditions as well as under auxotonic conditions. We obtained a bell-shaped relation between the compliance under an auxotonic load and the work output of the myocyte, which was directly related to myocyte performance in the heart. Because it is easy to attach myocytes to the experimental apparatus, the present method would allow us to study cardiac muscle mechanics at the cellular and molecular levels.
Animals↗
A novel screening for inhibitors of a pleiotropic drug resistant pump, Pdr5, in Saccharomyces cerevisiae.
Yeast is an excellent model system of eukaryotes for the study of molecular mechanisms of ATP-binding cassette transporters. Pdr5 protein is a yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Here, we described a novel drug screening system designated to detect compounds that inhibit the function of Pdr5. An indicator strain with increased drug sensitivity was constructed with an ergosterol-deficient background (delta syr1/erg3 null mutation). The sensitivity of the indicator strain (delta syr1/erg3 delta pdr5 delta snq2) to the Pdr5 substrates, cycloheximide and cerulenin, was increased 16-fold and 4-fold against wild type, respectively. The screening system is mainly based on the growth inhibition of the PDR5-overexpressed indicator strain with the combination of a sample and cycloheximide or cerulenin. The effect of an mdr inhibitor, FK506 on the screening system was clearly detected even at a low concentration (approximately 0.5 microg/ml). In addition, accumulation of rhodamine 6G in the cells was detected as a result of Pdr5 inhibition by FK506. These results indicated that the screening system is useful for a sensitive screening of Pdr5-specific inhibitors with low toxicity.
ATP-Binding Cassette Transporters↗
Effect of extracellular [Ca(2+)] on Ca(2+) release from sarcoplasmic reticulum in rat ventricular myocytes.
The effect of external [Ca(2+)] ([Ca(2+)](o)) on Ca(2+) release from the sarcoplasmic reticulum (SR) was examined with rested-state twitches in rat ventricular myocytes. The magnitude of transient rise of intracellular [Ca(2+)] ([Ca(2+)](i)) relative to the resting one, F/F(o), as measured with fluo-3, was 1.75+/-0.07 (mean+/-SEM, n=9) and 1.86+/-0.13 (n=9) at 0.3 and 1.8 mM [Ca(2+)](o), respectively; the difference was insignificant. The time from onset to peak and the rate of rise of the [Ca(2+)](i) transient were 0.107+/-0.017 s (n=9) and 18.8+/-3.38 F/F(o)/s, respectively, at 0.3 mM [Ca(2+)](o), they were 0.064+/-0.005 s (n=9) and 31.1+/-0.03 F/F(o)/s, respectively, at 1.8 mM [Ca(2+)](o). The difference in the corresponding values at the two [Ca(2+)](o) was significant (t-test, p<0.05). The half decay time of the [Ca(2+)](i) transient was 0.217+/-0.016 s (n=8) at 0.3 mM [Ca(2+)](o) and was similar to the value of 0.230+/-0.022 s (n=8) at 1.8 mM [Ca(2+)](o), indicating that the rate of decrease of [Ca(2+)](i) is independent of the [Ca(2+)](o). The duration of action potential was similar at 0.3 and 1.8 mM [Ca(2+)](o) as examined with papillary muscle. The results suggest that a lowering of [Ca(2+)](o), i.e., reducing the Ca(2+) influx, slows the rate of Ca(2+) release from the SR fully loaded with Ca(2+) with little effect on the total amount of the Ca(2+) release. An instantaneous relationship between the [Ca(2+)](i) and the myocyte shortening at 0.3 and 1.8 mM [Ca(2+)](o) suggested that the time course of unloaded contraction is related not only to the magnitude, but also to the rate of rise of [Ca(2+)](i).
Animals↗
Intracellular [Ca(2+)] transients in Ca(2+) wave in single rat ventricular myocyte.
Ca(2+) release from the sarcoplasmic reticulum (SR) in heart muscle grades depending on Ca(2+) influx in the physiological twitch; Ca(2+( wave results from regenerative Ca(2+) release from the SR. To examine if the Ca(2+) release from the SR in the Ca(2+) wave takes a duration similar to the physiological one, a transient rise of intracellular [Ca(2+)] ([Ca(2+)](i) transient) was recorded during both a propagating Ca(2+) wave and an electrically evoked twitch with single rat ventricular myocytes, using a laser scanning confocal microscope. Care was taken to record the fluo-3 fluorescence from a segmental region with little lateral movement, especially during a propagating Ca(2+) wave. During a typical Ca(2+) wave, the time-to-peak (TP) and the half-width (HD) of the averaged [Ca(2+)](i) transient were 161 and 253 ms respectively, but they were 76 and 145 ms during an electrically evoked twitch. The difference in the duration between the two types of [Ca(2+)](i) transients could not be accounted for by modification of duration of [Ca(2+)](i) transient by possible asynchronous Ca(2+) release from the SR during a Ca(2+) wave, suggesting that the regenerative Ca2+) release from the SR in the Ca2+) wave occurs more slowly than the physiological one in rat ventricular myocytes.
Animals↗
Protein kinase A increases the rate of relaxation but not the rate of tension development in skinned rat cardiac muscle.
To clarify the contribution of cross-bridge kinetics to the contraction profile of cardiac twitch during beta-adrenergic stimulation, we studied the rate of tension development and relaxation following laser flash photolysis of caged compounds in rat-skinned ventricular trabeculae before and after treatment with the catalytic subunit of protein kinase A (PKA, 0.5 U/microl, 40 min). Tension development following nitrophenyl (NP)-EGTA photolysis was fitted with a single exponential function. The rate constant increased with an increase in postphotolysis steady tension, and the relation between the rate constant and the tension was not influenced by PKA. The rate of relaxation following diazo-2 photolysis was fitted with a double exponential function. The rate of both initial rapid and subsequent slow relaxation was independent of the extent of relaxation. PKA increased the rate of initial rapid relaxation by about twofold, but showed no significant effect on the rate of subsequent slow relaxation. These results suggest that in beta-receptor stimulated rat cardiac muscle, the increased rate of tension development and the facilitated relaxation rate during twitch can be partly explained as being due to the combined effects of decreased Ca(2+) affinity of troponin C and increased cycling rate of cross-bridges (subtractive combination for tension development and additive combination for tension relaxation).
Animals↗
Possible contribution of titin filaments to the compliant series elastic component in horseshoe crab skeletal muscle fibers.
In horseshoe crab skeletal muscle fibers, the extension of SEC at the maximum isometric force P0 is about 6% of the slack fiber length L0 (sarcomere length, 7 microns), i.e., about 210 nm per half-sarcomere, being too large to be explained by the cross-bridge and the thin filament elasticities. Cinematographic studies of isometrically contracting myofibril bundes indicate that the highly compliant SEC mostly originates from the "elastic" thick filament misalignment in each A-band during isometric force generation. Possible contribution of the titin filaments to the "elastic" thick filament misalignment is discussed.
Animals↗
Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog.
The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (, Biophys. J. 73:2033-2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K(m) for Cy3-EDA-ATP and the K(d) for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 microM and 3.8 microM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 microM and 50 microM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 +/- 0.007 s(-1) (n = 32) at 10 degrees C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s(-1) (isometric) to 0.14 s(-1) at 20 degrees C with increasing shortening velocity up to 0.1 myofibril length/s (V(max), the shortening velocity under no load was approximately 0. 2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.
Adenosine Triphosphate↗
The Annual Meeting on Muscle Contraction and Cell Motility. Osaka, Japan, 6-8 January 2000.
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Animals↗
The Annual Meeting on Muscle Contraction and Cell Motility. Nagoya, Japan, 6-8 January 1999.
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Animals↗
A novel synthetic anti-acute pancreatitis agent, IS-741.
A novel synthetic drug, IS-741, inhibited cell adhesion in vitro and neutrophil in vivo. Thus, IS-741 inhibited the magnification of pancreatic lesion as well as progression to multiple organ failure in acute pancreatitis models. Furthermore, IS-741 at identical plasma concentrations equally improved the survival rates in animals of various species with severe acute pancreatitis. Based on these observations, it was considered that IS-741 inhibited tissue destruction by neutrophil after inhibiting neutrophil infiltration into the pancreas or other important organs in acute pancreatitis. It was also considered that IS-741 demonstrated various anti-acute pancreatitis effects by interrupting a vicious cycle of inflammation. Therefore, IS-741 is expected to become a useful drug for treating acute pancreatitis and multiple organ failure in clinical settings.
Acute Disease↗
Effect of IS-741 on cell adhesion between human umbilical vein endothelial cells and HL-60 cells.
The effect of IS-741 (N-[(2-ethylsulfonylamino)-5-trifluoromethyl-3-pyridyl] cyclohexanecarboxamide monohydrate) on a model for pancreatitis has been previously reported. Recent patho-histological observations of remedial tests using rats found that the IS-741 administered group showed a low degree of tissue infiltration by inflammatory cells (polymorphonuclear leukocytes). We therefore examined cell adhesion, which is the first step in tissue infiltration by activated neutrophils, and investigated the effect of IS-741 on cell adhesion between human umbilical vein endothelial cells (HUVEC) and human promyelo-leukemia cell line (HL-60) cells during lipopolysaccharide stimulation in vitro. IS-741 significantly inhibited the adhesion of HL-60 cells to HUVEC. Further investigation of IS-741 on individual cells revealed that IS-741 mainly affected HL-60 cells. Investigation of the inhibitory effect of IS-741 at the molecular level (targeting adhesion molecules) also revealed that IS-741 had no effect on the appearance of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1) on HUVEC, which supports the theory that IS-741 is mainly effective on HL-60 cells, even at the molecular level. However, the inhibition of adhesion was noticed in experiments in which an anti-ICAM-1 or anti-VCAM-1 antibody was added to the adhesion test system. Therefore, IS-741 is likely to affect adhesion molecules which belong to the beta1 or beta2 integrin family.
Antibodies↗
Evidence for two distinct cross-bridge populations in tetanized frog muscle fibers stretched with moderate velocities.
When tetanized frog skeletal muscle fibers are subjected to moderate-velocity stretches (< 1 L0/s), the tension developed above the level of isometric tension starts to decay after a sudden reduction of stretch velocity by more than 40-50%, though the fibers are still being stretched. We analysed the decay of tension response caused by the sudden reduction of stretch velocity, by applying three different types of stretch to a tetanized fiber, i.e., a 1.5% stretch with velocity V1 (stretch-1), a 1.5% stretch with velocity V2 < V1 (stretch-2), and a 3% stretch consisting of stretch-1 and stretch-2 applied in succession (stretch-3) and comparing the corresponding tension responses, TR-1, TR-2, and TR-3. It was found that TR-3 to stretch-3 resulted from algebraical summation of TR-1 to the preceding stretch-1 and TR-2 to the subsequent stretch-2. These results can be accounted for by assuming two distinct cross-bridge populations in stretched fibers.
Animals↗
Comparison of unitary displacements and forces between 2 cardiac myosin isoforms by the optical trap technique: molecular basis for cardiac adaptation.
To provide information on the mechanism of cardiac adaptation at the molecular level, we compared the unitary displacements and forces between the 2 rat cardiac myosin isoforms, V1 and V3. A fluorescently labeled actin filament, with a polystyrene bead attached, was caught by an optical trap and brought close to a glass surface sparsely coated with either of the 2 isoforms, so that the actin-myosin interaction took place in the presence of a low concentration of ATP (0.5 micromol/L). Discrete displacement events were recorded with a low trap stiffness (0.03 to 0.06 pN/nm). Frequency distribution of the amplitude of the displacements consisted of 2 gaussian curves with peaks at 9 to 10 and 18 to 20 nm for both V1 and V3, suggesting that 9 to 10 nm is the unitary displacement for both isoforms. The duration of the displacement events was longer for V3 than for V1. On the other hand, discrete force transients were recorded with a high trap stiffness (2.1 pN/nm), and their amplitude showed a broad distribution with mean values between 1 and 2 pN for V1 and V3. The durations of the force transients were also longer for V3 than for V1. These results indicate that both the unitary displacements and forces are similar in amplitude but different in duration between the 2 cardiac myosin isoforms, being consistent with the reports that the tension cost is higher in muscles consisting mainly of V1 than those consisting mainly of V3.
Animals↗
Evidence for bidirectional functional communication between myosin subfragments 1 and 2 in skeletal muscle fibers.
It has been shown that, in glycerinated skeletal muscle fibers, polyclonal antibody directed against myosin subfragment 2 (S-2) eliminates Ca(2+)-activated isometric force development, while Mg-ATPase activity of the fibers remains unchanged. To further explore possible involvement of myosin S-2 in muscle contraction, we studied the effect of anti-S-2 antibody on rigor linkage formation in the fibers. It was found that the antibody inhibited development of rigor force in a dose- and time-dependent manner, without changing the relation between muscle fiber stiffness and force. If, however, the antibody was applied after development of rigor force, it had no effect on both the stiffness and force. These results strongly suggest bidirectional functional communication between myosin subfragments 1 and 2 in the fibers.
Actins↗