Force: velocity relationship in single isolated toad stomach smooth muscle cells

The relationship between force and shortening velocity (F:V) in muscle is believed to reflect both the mechanics of the myosin cross-bridge and the kinetics of its interaction with actin. To date, the F:V for smooth muscle cells has been inferred from F:V data obtained in multicellular tissue preparations. Therefore, to determine F:V in an intact single smooth muscle cell, cells were isolated from the toad (Bufo marinus) stomach muscularis and attached to a force transducer and length displacement device. Cells were electrically stimulated at 20 degrees C and generated 143 mN/mm2 of active force per muscle cross-sectional area. At the peak of contraction, cells were subjected to sudden changes in force (dF = 0.10-0.90 Fmax) and then maintained at the new force level. The force change resulted in a length response in which the cell length (Lcell) rapidly decreased during the force step and then decreased monotonically with a time constant between 75 and 600 ms. The initial length change that coincided with the force step was analyzed and an active cellular compliance of 1.9% cell length was estimated. The maintained force and resultant shortening velocity (V) were fitted to the Hill hyperbola with constants a/Fmax of 0.268 and b of 0.163 Lcell/s. Vmax was also determined by a procedure in which the cell length was slackened and the time of unloaded shortening was recorded (slack test). From the slack test, Vmax was estimated as 0.583 Lcell/s, in agreement with the F:V data. The F:V data were analyzed within the framework of the Huxley model (Huxley. 1957. Progress in Biophysics and Biophysical Chemistry. 7:255-318) for contraction and interpreted to indicate that in smooth muscle, as compared with fast striated muscle, there may exist a greater percentage of attached force-generating cross-bridges.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Cross-bridge elasticity in single smooth muscle cells

In smooth muscle, a cross-bridge mechanism is believed to be responsible for active force generation and fiber shortening. In the present studies, the viscoelastic and kinetic properties of the cross-bridge were probed by eliciting tension transients in response to small, rapid, step length changes (delta L = 0.3-1.0% Lcell in 2 ms). Tension transients were obtained in a single smooth muscle cell isolated from the toad (Bufo marinus) stomach muscularis, which was tied between a force transducer and a displacement device. To record the transients, which were of extremely small magnitude (0.1 microN), a high-frequency (400 Hz), ultrasensitive force transducer (18 mV/microN) was designed and built. The transients obtained during maximal force generation (Fmax = 2.26 microN) were characterized by a linear elastic response (Emax = 1.26 X 10(4) mN/mm2) coincident with the length step, which was followed by a biphasic tension recovery made up of two exponentials (tau fast = 5-20 ms, tau slow = 50-300 ms). During the development of force upon activation, transients were elicited. The relationship between stiffness and force was linear, which suggests that the transients originate within the cross-bridge and reflect the cross-bridge's viscoelastic and kinetic properties. The observed fiber elasticity suggests that the smooth muscle cross-bridge is considerably more compliant than in fast striated muscle. A thermodynamic model is presented that allows for an analysis of the factors contributing to the increased compliance of the smooth muscle cross-bridge.     

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.     

Dinucleoside monophosphates. I. Optical properties and conformation in solution with one base charged.

A least squares analysis of the titration properties of several dinucleoside monophosphates enables calculation of the pK's for protonation. These pK's are used to resolve the spectral properties of dinucleoside monophosphates with one base charged from the apparent spectral properties of a dinucleoside monophosphate in aqueous solution. This method is applied to dinucleoside monophosphates containing adenosine and/or cytidine. Results of CD, nmr, and CD-temperature dependence measurements are presented. The results indicate that singly protonated dimers of these nucleosides stack as do their unprotonated analogs. It is suggested that this is true for all dimers with one base charged.     

Effect of pH alteration on growth and glucose oxidation in myoblast cultures

The growth of chick heart cells in culture declines when the cells reach confluency. The decline in growth rate is associated with both a decrease in the pH of the bicarbonate-CO₂ buffered medium and a reduced capacity for glucose oxidation by the pentose phosphate pathway. The pH of proliferating cultures supplemented with either 14 mM NaHCO₃ or with a mixture of organic buffers (pK 7.4) was increased by 0.3 pH unit over that of the controls. The rate of glucose oxidation by the pentose phosphate pathway in confluent cultures supplemented with NaHCO₃ or organic buffer increased by 60% 24 h after pH correction. This was associated with an increase in glucose uptake from the medium. We conclude that pH elevation in confluent heart cell cultures stimulates both growth and the capacity for glucose oxidation by the pentose phosphate pathway. The data also provide further evidence for a relationship between activity of the pentose phosphate pathway and cell growth.