Search

Parvalbumins (PVs) from Antarctic notothenioid fishes display a pattern of thermal adaptation that likely reflects evolutionary changes in protein conformational flexibility. We have used ancestral sequence reconstruction and homology modeling to identify two amino acid changes that could potentially account for the present thermal sensitivity pattern of Antarctic fish PVs compared with a PV from a theoretical warm-adapted ancestral fish. To test this hypothesis, ancient PVs were resurrected in the lab using PV from the notothenioid Gobionotothen gibberifrons as a platform for introducing mutations comparable to the reconstructed ancestral PV sequences. The wild-type PV (WT) as well as three mutant expression constructs were engineered: lysine 8 to asparagine (K8N), lysine 26 to asparagine (K26N) and a double mutant (DM). Calcium equilibrium dissociation constants (K(d)) versus temperature curves for all mutants were right-shifted, as predicted, relative to that of WT PV. The K(d) values for the K8N and K26N single mutants were virtually identical at all temperatures and showed an intermediate level of thermal sensitivity. The DM construct displayed a full conversion of thermal sensitivity pattern to that of a PV from a warm/temperate-adapted fish. Additionally, the K(d) versus temperature curve for the WT construct revealed greater thermal sensitivity compared with the mutant constructs. Measurements of the rates of Ca(2+) dissociation (k(off)) showed that all mutants generally had slower k(off) values than WT at all temperatures. Calculated rates of Ca(2+) binding (k(on)) for the K8N and K26N mutants were similar to values for the WT PV at all temperatures. In contrast, the calculated k(on) values for the DM PV were faster, providing mechanistic insights into the nature of potentially adaptive changes in Ca(2+) binding in this PV. The overall results suggest that the current thermal phenotype of Antarctic PVs can be recapitulated by just two amino acid substitutions.

Polyphenism is the expression of multiple, discrete phenotypes from one genotype, and understanding the environmental factors that trigger development of alternative phenotypes is a critical step toward understanding the evolution of polyphenism and its developmental control. While much is known about the ecology of the well-known carnivore/omnivore polyphenism in spadefoot toad tadpoles, the environmental cues for the development of the specialized carnivore phenotype are not completely clear. We examined 27 different experimental treatments in two spadefoot toad species and used over 1,000 tadpoles in an attempt to elucidate those cues. While only 44 carnivores developed in these treatments, they were concentrated at cooler water temperatures and a diet that included fairy shrimp. However, while a diet of fairy shrimp promoted carnivore development, it was not necessary for inducing carnivore development at lower and intermediate water temperatures. Evidence also suggested a role for social inhibition that limited the proportion of interacting tadpoles that become carnivores. Tadpoles of Spea multiplicata grew larger at cooler temperatures and larger when their diets included fairy shrimp, whereas tadpoles of S. bombifrons grew larger at warmer temperatures and when their diets did not include fairy shrimp. These results indicate that carnivore induction can occur through different cues and that our current model for carnivore development is too limited. Finally, we argue that the carnivore/omnivore spadefoot system is neither a polyphenism nor a polymorphism but is a continuously distributed plasticity.

Understanding the evolution of phenotypic plasticities and the connections among the environment, genotype, and phenotype requires detailed understanding of the proximate mechanisms regulating morphological differences between phenotypes. Spea multiplicata tadpoles can develop into two different phenotypes, i.e. carnivores and omnivores, which differ in many morphological and behavioral traits. One of the major differences is enlargement of the jaw and tail musculature in carnivores relative to those of omnivores. We investigated pattern of muscle enlargement by measuring differences in myofiber number and cross-sectional area between the phenotypes during early and mid-development. The data show that both hyperplasia and hypertrophy underlie the carnivores' enlargement of both the orbitohyoideus jaw muscle (OH) and the tail muscle (TL). Carnivores had more OH and TL myofibers than did omnivores at all ages, but the rate of myofiber addition differed, by approximately 9 and 17 myofibers per day respectively. Carnivores also had larger OH and TL myofibers than did omnivores, at many of the ages studied, and the rate of myofiber cross-sectional area increase (log-transformed myofiber cross-sectional area plotted against age in days) was significantly greater for carnivores than for omnivores in the internal, but not the peripheral, regions for both the OH and TL muscle., (2009 Wiley-Liss, Inc.)

The Atlantic stingray, Dasyatis sabina found along the Gulf of Mexico and southeastern Atlantic coasts, is a euryhaline species of elasmobranch. This species is able to osmotically compensate for changing environmental salinity by altering plasma and intracellular solutes, including urea and counteracting methylamines (betaine and TMAO). Parvalbumin (PV) is an intracellular protein that facilitates muscle relaxation by sequestering calcium. Determining the effects that in situ concentrations of urea (146 mM), betaine (62 mM), and TMAO (11 mM) have on PV function in marine and freshwater adapted populations of D. sabina could provide insight into intracellular correlates of euryhaline tolerance for this species. PV from marine and freshwater populations of D. sabina was identified and purified by SDS-PAGE, western blot analysis, and full amino acid sequence analysis. Both populations exhibited two PV isoforms, PV I (approximately 12.18 kDa mw) and PV II (11.96 kDa mw). PV dissociation constants (K(D)) were determined in the presence and absence of physiological concentrations of urea, betaine, and TMAO by fluorescence spectroscopy using the fluorescent Ca(2+) indicator fluo-3 which competes with PV for Ca(2+). Functional studies revealed PV I showed no significant changes in calcium binding from in situ muscle conditions, except in the presence of betaine. In contrast, PV II's ability to bind calcium was increased relative to physiological conditions in the presence of each osmolyte independently. Thus, it appears that organic osmolytes have isoform specific effects on PV function.

Calcium dissociation constants (KD) were measured as a function of temperature for parvalbumin, a small acidic protein expressed abundantly in fast-twitch muscle, from the Arctic cod (Boreogadus saida) and compared to values previously determined for Antarctic and temperate zone teleosts. Estimates of KD were derived independently from fluorometric titrations and calorimetry. In addition, the primary structure of B. saida parvalbumin was determined. Calcium KDs for parvalbumin from B. saida were fundamentally similar to those for parvalbumins from Antarctic species (6.68+/-0.59 nM and 7.77+/-0.72 nM at 5 degrees C, respectively), but significantly different from temperate zone species (1.35+/-0.28 nM at 5 degrees C). However, estimates of KD for B. saida parvalbumin at 5 degrees C closely matched values for temperate zone fish at 25 degrees C (6.54+/-0.56 nM), recapitulating the prior observation that calcium affinity of parvalbumin is conserved at the native temperature of teleost fish. Full sequence of B. saida parvalbumin was generated using reverse-phase HPLC and RACE-PCR. The Arctic parvalbumin showed 83% homology to a carp parvalbumin. None of the 16 total substitutions between the two parvalbumins resided in the cation binding sites of the protein, indicating that the structural locus of the thermal sensitivity of function lies outside the active regions.

Parvalbumin (PV) is a soluble calcium-binding protein that is especially abundant in fast-twitch muscles of fish and other lower vertebrates. Despite its prevalence in ectothermic taxa, few data address the effects of temperature on PV binding function. In this study, calcium dissociation constants (KD) were measured as a function of temperature (0-25 degrees C) for PV from two Antarctic (Gobionotothen gibberifrons and Chaenocephalus aceratus) and two temperate zone fish species (Cyprinus carpio and Micropterus salmoides). Measurements by fluorometric competitive binding assay show that KD values for PVs from the Antarctic species were significantly higher at all assay temperatures and were less sensitive to temperature relative to carp and bass. However, estimates of KD are fundamentally similar for PVs from the Antarctic and temperate zone species when examined at their native physiological temperature. Variation in pH and ionic strength within a physiologically relevant range had only modest effects on KD. Thermodynamics of calcium binding to PV from G. gibberifrons and C. carpio was measured by isothermal microcalorimetry. When measured at 15 degrees C, the Gibbs free energy change (deltaG) was significantly greater for calcium binding to PV from G. gibberifrons than from carp (-43.4+/-1.5 kJ mol(-1) and -46.6+/-3.0 kJ mol(-1), respectively), and the relative contribution of entropy to deltaG for calcium binding to PV from the Antarctic species was about twice that of carp (deltaS=16.0+/-0.8 J degrees C(-1) mol(-1) for G. gibberifrons; deltaS=7.5+/-0.8 J degrees C(-1) mol(-1) for C. carpio).

The mummichog, Fundulus heteroclitus, is an intertidal fish that exhibits little change in swimming ability despite large and rapid variations in environmental parameters. We therefore tested the hypothesis that this nearly constant function is due to Fundulus myosin being intrinsically insensitive to changes of temperature, ionic strength and pH. In vitro motility assays were used to quantify the speed of unregulated actin filaments on myosin purified from F. heteroclitus glycolytic skeletal muscle. Filament speed was 2.07+/-0.17 microm s(-1) at 26 degrees C, ionic strength (Gamma/2) of 0.08 M Gamma/2 and pH 7.4. Speed increased as temperature increased over the range of 5-36 degrees C with an activation energy (E (a)) of 94.0+/-7.0 kJ mol(-1)) and an enthalpy (DeltaH (double dagger)) of 91.5+/-7.0 kJ mol(-1) at 20 degrees C. A linear relationship between temperature and ATPase activity was also obtained with actin-activated myosin Mg(2+)-ATPase assays over the temperature range 5-35 degrees C with E (a=)59.9+/-2.4 kJ mol(-1) and DeltaH (double dagger)=57.4+/-2.4 kJ mol(-1) at 20 degrees C. There was little or no effect of ionic strength on filament speed over the range 0.19 M Gamma/2-0.54 M Gamma/2. Speed increased significantly at lower ionic strengths and was 7.9-fold higher at 0.08 M Gamma/2 than at 0.19 M Gamma/2. Speed increased with pH with a 16-fold increase between pH 6.7 and 7.4. These results indicate that changes in physiological parameters that include temperature, pH and ionic strength affect the function of unregulated F. heteroclitus myosin, and thus other factors must be responsible for the mummichog's swimming performance being comparatively insensitive to environmental variation.

The time- and orientation-dependence of metabolite diffusion in giant muscle fibers of the lobster Panulirus argus was examined using (31)P- and (1)H-pulsed-field gradient nuclear magnetic resonance. The (31)P resonance for arginine phosphate and the (1)H resonances for betaine, arginine/arginine phosphate and -CH(2)/-CH groups were suitable for measurement of the apparent diffusion coefficient, D. Diffusion was measured axially, D(//), and radially, D( perpendicular ), in fibers over diffusion times of 20 to 300 ms. Diffusion was strongly anisotropic, and D(//) was higher than D( perpendicular ) at all times. Radial diffusion decreased with time until a steady-state value was reached at a diffusion time of approximately 100 ms. Changes in D( perpendicular ) occurred over a time scale that was consistent with previous measurements from fish and mammalian muscle, indicating that diffusion is hindered by the same types of barriers in these diverse muscle types. The time dependence indicated that the sarcoplasmic reticulum is the principal intracellular structure that inhibits mobility in an orientation-dependent manner in skeletal muscle. The abdominal muscles in P. argus are used for anaerobic, burst contractions during an escape maneuver. The fact that these muscle fibers have diameters that may exceed hundreds of microns in diameter, and nearly all of the mitochondria are localized near the sarcolemmal membrane, suggests that barriers that hinder radial diffusion of ATP equivalents may ultimately limit the rate of post-contractile recovery.

Recently, a new type of cancer treatment has been introduced that combines pulsed electric fields (PEF) with anticancer drugs. The proposed mode of action is that PEF create transient pores in the membranes which allow entry of drugs into the cells. This method increases cytotoxicity of some anticancer drugs like bleomycin (BLM) by 2-3 orders of magnitude, which, in turn, reduces systemic drug dosage without decreasing efficacy. In the present study, magnetic resonance imaging (MRI) was used to determine changes in apparent water self-diffusion coefficients (ADC) and spin-lattice (T(1)) and spin-spin (T(2)) relaxation times that occur in an animal laryngeal tumor (HEp-2 cells) model with BLM delivered by PEF. A Bruker 14 Tesla (600 MHz) wide-bore spectrometer with micro-imaging capability was used to generate all the data. Mice carrying approximately 8 mm tumors were treated with several combinations of drug and PEF. All measurements were made on tumor samples excised from mice 24 and 48 hours after treatment with (i) saline, intratumor injection (i.t.), (ii) BLM, i.t., (iii) saline with PEF, and (iv) BLM, i.t., followed by PEF. Although T(1) does not differ between the controls (i, ii, and iii) and full treatment (iv) 6.72 +/- 0.20 s vs. 6.31 +/- 1.7 s, T(2) for (iv) at 24 hours is significantly different from the controls 52.4 +/- 0.91 ms vs. 46.5 +/- 1.54 ms. T(2) differences between treatment and controls disappear at 48 hours. ADC increases significantly from 24 to 48 hours (7.31 +/- 0.16 x 10(-6) to 8.28 +/- 0.28 x 10(-6) cm(2)/sec, p = 0.05). Longer T(2) values may reflect early apoptosis and tumor death when the tumor is structurally less dense. Higher ADC's, associated with the periphery of the tumors and the central region, may indicate loose structural organization and necrosis resulting from the combination treatment.

Magnetic resonance microscopy of skin from hairless rats under the influence of electrical fields was conducted for two cases: 1) low voltage constant electrical fields and 2) high-voltage short pulse, electrical fields. Under conditions of the low voltage and low current iontophoresis, i.e., 0 to 20 V, and 0 to 0.5 mA/cm2, it was found that the skin structure, as observed by magnetic resonance microscopy, did not significantly change until 20 Volts were applied across the 0.1 cm thick skin. Under these conditions, the viable epidermis appeared to swell, and this result corresponded to observations from scanning electron microscopy and other research from the literature. High voltage electrical fields, i.e., 220 V 1 ms pulses repeated once per second, appeared to hydrate the stratum corneum as is consistent with published literature on electroporation. In the case of iontophoresis, water self-diffusion coefficients in the epidermis and hair follicle regions at all voltages were affected by the electrical field. Statistical analysis at the 95% confidence level for the comparison of the average differences between diffusion coefficients with the electrical field on and with the electrical field off for pair matched pixels for the viable epidermis show that for 5 V (p = 0.00377), 10 V (p = 0.0108), 20 V (p = 0.0219) regimes there are statistically significant (p < or = 0.05) changes due to the applied electric field. The same analysis for the hair follicle region at 5 V (p = 6.89 x 10(-7)), 10 V (p = 1.42 x 10(-5)), 20 V (p = 3.23 x 10(-3)) also show statistically significant changes (p < or = 0.05). When the electroporation pulse was applied, the water diffusion coefficients increased by about 30% to 6.6 x 10(-6) cm2/s +/- 2.4 x 10(-7) cm2/s and 8.3 x 10(-6) cm2/s +/- 3.7 x 10(-7) cm2/s, for the epidermis and hair follicle regions, respectively. Significant differences were noted between diffusion coefficients in the viable epidermis and the hair follicles for all cases.

The time- and orientational-dependence of phosphocreatine (PCr) diffusion was measured using pulsed-field gradient nuclear magnetic resonance (PFG-NMR) as a means of non-invasively probing the intracellular diffusive barriers of skeletal muscle. Red and white skeletal muscle from fish was used because fish muscle cells are very large, which facilitates the examination of diffusional barriers in the intracellular environment, and because they have regions of very homogeneous fiber type. Fish were cold-acclimated (5 degrees C) to amplify the contrast between red and white fibers. Apparent diffusion coefficients, D, were measured axially, D(axially) and radially, D(radially), in small muscle strips over a time course ranging from 12 to 700 ms. Radial diffusion was strongly time dependent in both fiber types, and D decreased with time until a steady-state value was reached at a diffusion time approximately 100 ms. Diffusion was also highly anisotropic, with D(axially) being higher than D(radially) for all time points. The time scale over which changes in D(radially) occurred indicated that the observed anisotropy was not a result of interactions with the thick and thin filament lattice of actin and myosin or restriction within the cylindrical sarcolemma, as has been previously suggested. Rather, the sarcoplasmic reticulum (SR) and mitochondria appear to be the principal intracellular structures that inhibit mobility in an orientation-dependent manner. This work is the first example of diffusional anisotropy induced by readily identifiable intracellular structures.

The structure of polyacrylamide gels was studied using proton spin-lattice relaxation and PFG diffusion methods. Polyacrylamide gels, with total polymer concentrations ranging from 0.25 to 0.35 g/ml and crosslinker concentrations from 0 to 10% by weight, were studied. The data showed no effect of the crosslinker concentration on the diffusion of water molecules. The Ogston-Morris and Mackie-Meares models fit the general trends observed for water diffusion in gels. The diffusion coefficients from the volume averaging method also fit the data, and this theory was able to account for the effects of water-gel interactions that are not accounted for in the other two theories. The averaging theory also did not require the physically unrealistic assumption, required in the other two theories, that the acrylamide fibers are of similar size to water molecules. Contrary to the diffusion data, T1 relaxation measurements showed a significant effect of crosslinker concentration on the relaxation of water in gels. The model developed using the Bloch equations and the volume averaging method described the effects of water adsorption on the gel medium on both the diffusion coefficients and the relaxation measurements. In the proposed model the gel medium was assumed to consist of three phases (i.e., bulk water, uncrosslinked acrylamide fibers, and a bisacrylamide crosslinker phase). The effects of the crosslinker concentration were accounted for by introducing the proton partition coefficient, Keq, between the bulk water and crosslinker phase. The derived relaxation equations were successful in fitting the experimental data. The partition coefficient, Keq, decreased significantly as the crosslinker concentration increased from 5 to 10% by weight. This trend is consistent with the idea that bisacrylamide tends to form hydrophobic regions with increasing crosslinker concentration. Copyright 1998 Academic Press.

Thermal acclimation results in dramatic changes in the fractional volume of mitochondria within skeletal muscle of teleost fish. We investigated the hypothesis that changes in mitochondrial volume represent a compensatory response to temperature-induced changes in intracellular diffusion coefficients (D) of the high-energy phosphate compounds ATP and creatine phosphate (PCr). Using 31P nuclear magnetic resonance spectroscopy, we determined DPCr and DATP in goldfish (Carassius auratus) skeletal muscle at 25 degrees C and 5 degrees C: DPCr was 3.28 +/- 0.18 x 10(-6) cm2s-1 at 25 degrees C and 2.00 +/- 0.90 x 10(-6) cm2s-1 at 5 degrees C: DATP was 2.13 +/- 0.16 x 10(-6) cm2s-1 at 25 degrees C and was estimated to be 1.30 x 10(-6) cm2s-1 at 5 degrees C. There was no evidence for an effect of acclimation temperature or fiber type on DATP or DPCr. A mathematical reaction-diffusion model was used to calculate profiles of [ATP], [PCr] and the free energy of ATP hydrolysis (delta GATP) in activated goldfish muscle fibers at 5 degrees C and 25 degrees C. The results showed spatial and temporal constancy of [ATP], [PCr] and delta GATP in red fibers at both temperatures, regardless of changes in acclimation temperature or mitochondrial density. The model also showed spatial and temporal constancy of [ATP] in white fibers at 5 degrees C and 25 degrees C, but gradients in [PCr] and delta GATP developed in white fibers under all conditions of temperature and acclimation temperature. These gradients were attenuated in cold-acclimated animals by cold-induced increases in mitochondrial density. However, the model shows that the proximal stimulus for temperature-induced changes in mitochondrial volume density in muscle is not a disruption in intracellular diffusion of high-energy phosphates.

High resolution images were obtained using high-field nuclear magnetic resonance microscopy of in vitro preparations of hydrated hairless rat skin. The major anatomical features observed were comparable to those seen by electron microscopy and include the stratum corneum, the viable epidermis, sebaceous glands, the cell layers surrounding hair follicles (the outer and inner root sheaths), and regions of subcutaneous fatty deposits. Calculated diffusion maps demonstrated that signal intensity is sufficient to obtain quantitative water mobility data from the viable epidermis and the hair follicle/sebaceous gland regions. Images from skin immersed in D2O clearly distinguish signal contributions that arise from fat from those which arise from water, and indicate that the calculated diffusion maps include only proton mobility from water in skin. These results may lead to further applications for using quantitative nuclear magnetic resonance microscopy for examining transdermal transport processes of in vitro skin preparations.

Intracellular diffusive transport of adenosine triphosphate (ATP) is critical to cellular metabolism. Physical models predict that diffusion coefficients (D) of small molecules are functions of temperature and viscosity of the diffusive environment. Therefore, changes in body temperature, commonly experienced by poikilotherms, are expected to result in changes in the rate of intracellular ATP transport. However, it has been postulated that changes in the electrical charge of ATP may influence the interaction between ATP and the cytosol and that the temperature sensitivity of DATP may deviate from the predicted relationship. To investigate the effects of changes in electrical charge on the temperature sensitivity of DATP, we measured DATP under various conditions of temperature, pH, and pMg2+. Changes in pH and pMg2+ were used to alter the net charge of ATP, and DATP was measured in solutions of physiological ionic strength. Results showed a positive correlation between DATP and temperature; DATP = 1.75 +/- 0.09, 3.68 +/- 0.14, and 4.64 +/- 0.13 (mean +/- S.E.M.) x 10(-6) cm2/s at 5 degrees C, 25 degrees C, and 40 degrees C, respectively. Changes in pH and pMg2+ did not significantly influence DATP, and the change in DATP with respect to temperature was similar to that predicted on the basis of changes in temperature and viscosity of the aqueous medium.

The diffusion coefficient for O2 (Do2) and the solubility constant for O2 (alpha O2) were measured at 15 degrees C in oxidative muscle from striped bass (Morone saxatilis) that had been acclimated to 5 degrees and 25 degrees C. This design allowed us to test the hypothesis that changes in composition of the tissue that are known to occur during thermal acclimation may affect O2 movement. Our measurements permitted calculation of the diffusion constant for O2 (Ko2) through the tissue, which is a primary determinant of capacity for O2 flux. Under isothermal conditions, alpha O2 was 3.59 +/- 0.20 x 10(-2) and 6.64 +/- 0.27 x 10(-2) ml O2.cm-3.atm-1 in tissues from 25 degrees- and 5 degrees C-acclimated animals, respectively. Because O2 is more soluble in lipid than aqueous phase, higher alpha O2 in tissues from cold-acclimated animals can be accounted for by the 13-fold increase in lipid content that is known to occur in oxidative muscle of striped bass during acclimation from 25 degrees to 5 degrees C. When measured under similar isothermal conditions, Do2 showed no significant difference between animals acclimated to warm or cold temperature; Do2 through tissues from 25 degrees- and 5 degrees C-acclimated animals was 2.50 +/- 0.18 and 2.57 +/- 0.40 cm2/s, respectively. Because alpha O2 increases, the calculated KO2 (DO2. alpha O2) is greater in tissue from cold- than from warm-acclimated fish. At physiological temperature, elevated lipid content in oxidative muscle of cold-acclimated striped bass should result in enhanced intracellular movement of O2 and at least partially offset the expected decrease in DO2 at cold temperature.

A condition similar to insulin-dependent diabetes mellitus (IDDM) was induced in male CD-1 mice by injection of streptozotocin (STZ). Five weeks after treatment, the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles were isolated for analysis. Phosphorous metabolites were quantified by 31P-NMR and HPLC, native myosin was characterized electrophoretically, and activities of metabolic enzymes were measured spectrophotometrically. Relative to control animals, STZ-diabetes resulted in a significant 32% decrease in the FM1 isoform of myosin in EDL and a 24% decrease in IM myosin of SOL. Mass-specific activities of phosphofructokinase, citrate synthase, and cytochrome oxidase were significantly lower in SOL from STZ-diabetic mice than in controls by 23, 18, and 36%, respectively. Intracellular ATP was significantly lower in SOL from STZ-diabetic mice than in controls (3.44 +/- 0.20 mumol g-1 wet weight vs. 4.61 +/- 0.20 mumol g-1, respectively), as was creatine phosphate (11.98 +/- 0.80 mumol g-1 wet weight vs. 14.22 +/- 0.44 mumol g-1). In contrast to results from SOL, there were no significant changes in phosphorus metabolites or enzyme activity in EDL. These results show that the effects of IDDM on levels of phosphorus containing metabolites and maximal activities of key regulatory enzymes in muscle are markedly fiber-type specific. It is suggested that the muscle type-specific effects of STZ-diabetes may be a consequence of differential accumulation of intracellular fatty acids.

Pulsed field gradient (PFG) spin echo 31P NMR can be used to measure diffusion coefficients of phosphorus-containing metabolites in vivo. In biological spin echo spectra, the ATP resonances are phase modulated by J-coupling between the three phosphorus atoms. This phase modulation may severely decrease the apparent signal intensity of the ATP peaks. In this paper, we describe the use of homonuclear decoupling during spin evolution to suppress the effects of J-coupling in biological spin echo spectra. Phosphorous spectra of ATP and creatine phosphate (PCr) in solution and goldfish (Carassius auratus) skeletal muscle demonstrate the effectiveness of homonuclear decoupling in improving the effective signal-to-noise ratio of ATP. In addition, diffusion coefficients of ATP and PCr determined in goldfish skeletal muscle show that PFG homonuclear decoupled spin echo (HDSE) NMR provides accurate measures of diffusion coefficients.

Measurements of the intracellular diffusion coefficients (Di) of ATP and creatine phosphate (PCr) in stable, isolated preparations of skeletal muscle were made by means of pulsed field gradient (PFG) 31P NMR. Experiments used a PFG NMR probe specifically designed for small, superfused biological samples. This provided a magnetic field gradient in the z axis of up to 195 G/cm with minimal eddy currents. DiATP and DiPCr in white (fast, glycolytic) skeletal muscle from goldfish (Carassius auratus) were determined to be 2.48 +/- 0.33 and 3.49 +/- 0.33 x 10(-6) cm2/s, respectively, at 25 degrees C and a diffusion time of approximately 19 ms. For comparison with Di values, diffusion coefficients of ATP and PCr also were measured in solutions of ionic composition similar to that of fish muscle cytosol. The in vitro diffusion coefficients of ATP and PCr were 3.54 +/- 0.11 and 5.28 +/- 0.08 x 10(-6) cm2/s, respectively, at 25 degrees C.

We investigated whether the creatine kinase-catalyzed phosphate exchange between PCr and gamma ATP in vivo equilibrated with cellular substrates and products as predicted by in vitro kinetic properties of the enzyme, or was a function of ATPase activity as predicted by obligatory "creatine phosphate shuttle" concepts. A transient NMR spin-transfer method was developed, tested, and applied to resting and stimulated ex vivo muscle, the soleus, which is a cellularly homogeneous slow-twitch mammalian muscle, to measure creatine kinase kinetics. The forward and reverse unidirectional CK fluxes were equal, being 1.6 mM.s-1 in unstimulated muscle at 22 degrees C, and 2.7 mM.s-1 at 30 degrees C. The CK fluxes did not differ during steady-state stimulation conditions giving a 10-fold range of ATPase rates in which the ATP/PCr ratio increased from approximately 0.3 to 1.6. The observed kinetic behavior of CK activity in the muscle was that expected from the enzyme in vitro in a homogeneous solution only if account was taken of inhibition by an anion-stabilized quaternary dead-end enzyme complex: E.Cr.MgADP.anion. The CK fluxes in soleus were not a function of ATPase activity as predicted by obligatory phosphocreatine shuttle models for cellular energetics.

Mice were treated for 7-12 wk with the creatine analogue beta-guanidinopropionic acid (beta-GPA). Treatment reduced total creatine to approximately 5% of control values in soleus (SOL) and extensor digitorum longus (EDL) muscles. In both muscles from treated mice, phosphorylated beta-GPA accumulated and resting [ATP] decreased by approximately 50%. Relative to controls, cytochrome oxidase and citrate synthase activities increased significantly in EDL from treated mice, but not in SOL; creatine kinase activity decreased significantly in SOL, but not in EDL. Measurements of poststimulation energy metabolism show that the energy cost to maintain tension in SOL and EDL from treated mice was approximately 50% of that in control muscle. Relative to controls, first-order rate constants of poststimulation O2 demand were 2- and 3.6-fold greater in SOL and EDL, respectively, from treated mice. Increased economy of SOL and EDL from treated mice is consistent with previously reported changes in myosin isoenzymes. Increases in rate constants of O2 utilization in creatine-depleted muscle are inconsistent with the hypothesis that cytoplasmic or mitochondrial creatine kinase is rate limiting for cellular respiration.

The development and locomotion of the amoeboid sperm of the nematode, Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly-disassembly-reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHi was 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHi to 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re-establishment of the pH gradient, and re-initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHi occurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH-sensitive MSP-membrane interaction.

In this paper we describe features of an NMR probe designed for the study of small, superfused muscles. We also present the results of an empirical study of the performance characteristics of several configurations of small solenoid coils, ca 2 mm diameter. Our data show that optimal use of the available volume of sample becomes the prime consideration in coil design at this scale. In contrast to large biological samples, for such small coils the equivalent resistance associated with the sample is minor relative to the resistance of the RF coil itself. Thus, substantial improvements in the S/N ratio can be obtained by adopting coil configurations that are inferior electrically, but which can sample a greater volume of tissue.

Phosphorus NMR spectroscopy and HPLC analyses were made on isolated rat and mouse muscles selected for different volume fractions of the major known fiber types. We tested the hypothesis that muscle cell types at rest have intrinsically different contents of PCr, ATP and Pi. The Pi content was low and the PCr and ATP contents were high in muscles with large contents of type 2b and 2a fibers, and vice versa in muscles with large volume fraction of types 1 and 2x fibers. From the profile of these metabolites we could distinguish only two classes of fibers in the murine muscles and predict well the composition of cat muscles. For the first class, types 2a and 2b fibers, the intracellular concentrations were: ATP 8 mM; total Cr 39 mM; PCr 32 mM; Pi 0.8 mM; ADP 8 microM. For the second class, type 1 and 2x fibers, these quantities are: ATP 5 mM; TCr 23 mM; PCr 16 mM; Pi 6 mM; ADP 11 microM. Thus our results establish a new and apparently general criterion upon which to distinguish skeletal muscle cells, one based on the resting content of bioenergetically important metabolites.

We tested the proposition that muscle cell types have different contents of phosphocreatine (PCr), ATP, and Pi by 31P NMR spectroscopy and HPLC analyses of adult rat and mouse muscles containing various volume fractions of different fiber types. There was a 2-fold difference in the PCr content between muscles with a high volume fraction of fiber types 1 and 2x versus those with fast-twitch (types 2a and 2b) fiber types. Pi content was low, and PCr and ATP contents were high in muscles with large contents of type 2b and 2a fibers; the reverse was true in muscles with a large volume fraction of type 1 and 2x fibers. There is a large range in the Pi/PCr ratios in normal resting muscles, from less than 0.05 in type 2 to 0.51 in type 1 fibers, depending upon the distribution of their component fiber types. In all muscles, the peak area resulting from the beta phosphate of ATP constituted approximately 13% of the sum of all peak areas observable in the 31P spectrum. Fiber types 2a and 2b were not distinguishable, and the content of type 2x fibers was similar to type 1 fibers. From the profile of these metabolites, we could distinguish only two classes of fibers. For type 2a and 2b fibers, the intracellular concentrations were 8 mM ATP, 39 mM total creatine, 32 mM PCr, 0.8 mM Pi, and 8 microM ADP. For type 1 and 2x fibers, these quantities were 5 mM ATP, 23 mM total creatine, 16 mM PCr, 6 mM Pi, and 11 microM ADP. Thus our results establish an additional criterion upon which to distinguish skeletal muscle cells, one based on the resting content of bioenergetically important metabolites. These results also provide the basis for estimating skeletal muscle fiber-type composition from noninvasive NMR spectroscopic data.

Creatine and phosphocreatine are substrates for creatine kinase which is a key enzyme involved in energy transfer within the cell. Analogues of creatine have been fed to animals to determine the role this enzyme plays in energy metabolism, but progress in interpretation has been hampered by the lack of quantitative techniques to determine tissue content of these compounds. We describe the separation and quantitation of substituted guanidino compounds and their phosphorylated forms by high-performance liquid chromatography. First, a cation-exchange column is used to assay free creatine and its unphosphorylated analogues, and then phosphocreatine and its phosphorylated analogues as well as adenylate content (AMP, ADP, ATP) are assayed on an anion-exchange column. These methods have proven successful in measuring the chemical contents of these compounds in neutralized perchloric acid extracts of mammalian skeletal muscles. The sensitivity of this method ranges from 50 to 200 pmol, which is adequate to provide information from tissue extracts of 5- to 10-mg samples.

A creatine analogue, beta-guanidinopropionic acid (beta-GPA), was administered in the food (2% wt/wt) and the water (0.5% wt/vol) of male CD-1 mice. Uptake of the phosphorylated analogue and depletion of phosphocreatine in hindlimb muscle was monitored by 31P nuclear magnetic resonance and was found to be complete within 7 wk. After this time, the isomyosin composition of soleus, extensor digitorum longus (EDL), and ventricle was analyzed by pyrophosphate gel electrophoresis. The analogue was found to induce significant alterations in the type of myosin expressed in soleus and EDL. Normal soleus contains both intermediate (IM) and slow (SM) myosins, and treatment reduced the relative content of IM by approximately 50%. In EDL, treatment decreased fast isomyosin FM3 by 60% compared with controls. Sodium dodecyl sulfate-gel electrophoresis also showed a decrease of parvalbumin in EDL by approximately 50%. Treatment had no significant effect on the isomyosin composition of heart ventricle. Levels of physical activity and concentrations of serum glucose and thyroxine of treated mice were not significantly different from controls. These results indicate a role for intracellular energetics in mediating adaptive changes in the phenotype of muscle in mature animals.

The effects of acute and long-term changes in temperature upon catalytic and calcium regulatory function of red (slow oxidative) and white (fast glycolytic) muscle from striped bass (Morone saxatilis) were determined. Acclimation to 5 degrees C or 25 degrees C had no significant effect on catalytic function (ATPase activity) or regulatory sensitivity (Ca++-activation) of myofibrils from either muscle type. Substantial differences between red and white muscle were found in the intrinsic thermal sensitivity of maximally-activated Mg++-Ca++ myofibrillar ATPase. Arrhenius plots of myofibrillar ATPase from white muscle show one significant breakpoint at 29 degrees C, with activation energies (Ea) of 2.3 and 23.4 kcal mole-1 at temperatures above and below this transition, respectively. Arrhenius plots of myofibrillar ATPase from red muscle show two transitions occurring at 22 and 9 degrees C, with Ea of 7.6 kcal mole-1 above 22 degrees C and 18.3 kcal mole-1 between 9 and 22 degrees C. Activation energies for myofibrils from red muscle increase substantially to approximately 107.3 kcal mole-1 below the 9 degrees C breakpoint. Differences in the intrinsic thermal sensitivity of red and white muscle catalytic function are apparently due to interaction of actomyosins and calcium regulatory proteins which are specific to each muscle type. The results suggest that capacity for sustained swimming in striped bass, which is powered exclusively by red muscle, will be severely impaired at cold temperature unless compensations occur above the level of contractile proteins.