Timothy S. Moerland

Diffusional anisotropy is induced by subcellular barriers in skeletal muscle

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°C) to amplify the contrast between red and white fibers. Apparent diffusion coefficients, D, were measured axially, D∥, and radially, D⟂, 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 ≊ 100 ms. Diffusion was also highly anisotropic, with D∥ being higher than D⟂ for all time points. The time scale over which changes in D⟂ 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. Copyright

High-performance liquid chromatographic assays for free and phosphorylated derivatives of the creatine analogues β-guanidopropionic acid and 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine)

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.

The effects of temperature, pH, and magnesium on the diffusion coefficient of ATP in solutions of physiological ionic strength

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.

Characterization of Metabolic Carbon Flow in Hepatocytes Isolated from Thermally Acclimated Killifish Fundulus heteroclitus

Hepatocytes isolated from temperature-acclimated Fundulus heteroclitus were used in determinations of respiratory rate, maximal activities of regulatory enzymes, and evolution of 14CO2 from labeled glucose and palmitic acid. Oxygen consumption was relatively independent of acclimation temperature between 25 C and 15 C but exhibited greater thermal sensitivity between 15 C and 5 C acclimation temperatures. When determined at the temperature of acclimation, evolution of 14CO2 from labeled palmitate increased from 5 C to 15 C and decreased from 15 C to 25 C. Rates of 14CO2 release from labeled glucose increased slightly between 5 C and 15 C and more dramatically between 15 C and 25 C. The 14C-1-O2/14C-6-O2 ratios from glucose were the same for each acclimation temperature, indicating that the relative participation of the pentose shunt in carbohydrate oxidation remained unchanged with acclimation. The results of labeled substrate utilization experiments indicate that substantial changes in the relative contributions of carbohydrate and lipid to aerobic energy metabolism accompany temperature acclimation in F. heteroclitus. Of enzymes studied, glucose-6-phosphate dehydrogenase, pyruvate kinase, and cytochrome oxidase exhibited compensatory changes in activity. Maximal activities of the glycolytic enzymes phosphofructokinase and pyruvate kinase did not demonstrate changes corresponding to patterns of labeled glucose utilization, indicating that modulation of pathway flux during temperature acclimation may be achieved by means other than alterations in enzyme quantity. Reorganization of metabolic carbon flow in Fundulus hepatocytes may reflect adaptive strategies for freezing avoidance via serum hyperglycemia and seasonal variations in reproductive effort and food resource availability.

Responses of mouse fast and slow skeletal muscle to streptozotocin diabetes: Myosin isoenzymes and phosphorous metabolites

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 by31P-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 μmol g−1 wet weight vs. 4.61±0.20 μmol g−1, respectively), as was creatine phosphate (11.98±0.80 μmol g−1 wet weight vs. 14.22±0.44 μmol 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 fibertype specific. It is suggested that the muscle type-specific effects of STZ-diabetes may be a consequence of differential accumulation of intracellular fatty acids.

Spatial resolution of transdermal water mobility using NMR microscopy.

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.

Parvalbumin characterization from the euryhaline stingray Dasyatis sabina

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 IIs 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.

Magnetic resonance studies of laryngeal tumors implanted in nude mice: effect of treatment with bleomycin and electroporation

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 ADCs, associated with the periphery of the tumors and the central region, may indicate loose structural organization and necrosis resulting from the combination treatment.

Transdermal water mobility in the presence of electrical fields using MR microscopy

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.

Chapter 4 Temperature: Enzyme and organelle

Publisher Summary Fishes as a group have been very successful in exploiting habitats with a wide range of temperatures. Representative teleostean species, for example, inhabit waters ranging from the hot springs of the equatorial region (35 oC and above) to the polar oceans (–1.86 oC). In addition, the life histories of many fish species encompass very considerable seasonal changes in temperature. The obvious success of this group of animals in exploiting such a wide range of thermal habitats should perhaps be puzzling, for the following reasons: First, all chemicophysical processes, including the biochemical reactions of living systems, are sensitive to acute changes in temperature. This sensitivity is often expressed as Q 10 , the ratio of rates at temperatures 10 oC apart, and typical values of this parameter for enzyme-catalyzed reactions range between 2 and 3. Second, most fish cannot maintain a significant thermal disequilibrium with their environment, because the same design features that permit the gills of fish to function effectively in respiratory gas exchange also greatly facilitate thermal exchange with the surrounding water. Although not considered further here, some notable exceptions to this generalization exist. Certain lamnid sharks and large tuna possess vascular retia that function as countercurrent heat exchangers to conserve metabolic heat in the deep musculature, and thermogenic “heater” tissues of billfish serve to maintain cranial temperatures significantly above the ambient level. Apart from these exceptions, however, it generally can be assumed that the mean body temperature of fish is well within one degree of the ambient water temperature.