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Metabolite diffusion in giant muscle fibers of the spiny lobster Panulirus argus

Stephen T. Kinsey^1,* and Timothy S. Moerland²

¹ Department of Biological Sciences, University of North Carolina at Wilmington, 601 South College Road, Wilmington, NC 28403-5915, USA
² Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA

View larger version (12K): [in a new window]   Fig. 1. (A) Pulse sequence used to measure D|| and D in lobster abdominal muscle fibers. The solid bars represent excitatory radiofrequency pulses, with the magnetization tip angle (degrees) indicated over each pulse. The stippled bars represent magnetic field gradient pulses. TM is the mixing time, Te, echo time, is the duration of the spatial-encoding gradients, is a short delay, is the approximate diffusion time (corrected as described in text), AQ is the signal acquisition period, and WS1 and WS2 are water suppression sequences that were used in the 1H spectra only. (B) The water suppression sequences are shown diagrammatically (for details, see Kinsey and Ellington, 1996). Diffusion coefficients at a given diffusion time were determined by collecting a series of spectra at different amplitudes of the spatial-encoding gradients. The diffusion time was adjusted by changing the value of . Additional details are given in the text.

View larger version (11K): [in a new window]   Fig. 2. A region of a lobster abdominal muscle 1H-spectrum. The peaks for betaine, arginine/arginine phosphate (at 1.95 p.p.m.) and -CH2/-CH groups yielded linear attenuation plots and sufficient signal-to-noise ratios to allow measurement of D at several diffusion times. Alanine and lactate methyl peaks are also indicated for reference, although diffusion of these metabolites was not examined in the present study.

View larger version (11K): [in a new window]   Fig. 3. Typical attenuation plot of the NMR peak amplitude as a function of the diffusion-weighting factor b in the axial (open squares) and radial (filled squares) directions in P. argus muscle. This example is from a measurement of arginine phosphate diffusion at a diffusion time of 150ms. A steeper attenuation slope is indicative of a higher D, as seen for axial diffusion.

View larger version (18K): [in a new window]   Fig. 4. Time dependence of D|| (open squares) and D (closed squares) for several metabolites in abdominal muscle fibers of P. argus. Diffusion was measured using the 31P-NMR peak for arginine phosphate (A), and the 1H-NMR peaks arising from (B) betaine, (C) arginine/arginine phosphate and (D) -CH2/-CH groups. Values are means ± S.E.M. (N=4, except for -CH2/-CH peak, where N=3).

View larger version (8K): [in a new window]   Fig. 5. The radial root-mean-square (RMS) displacement of arginine phosphate in P. argus fibers as a function of the square root of the diffusion time. The RMS displacement is described by , where t is the diffusion time (ms). The unrestricted RMS displacement was calculated using a D value for AP of 4.054x10-6 cm2s-1 (Ellington and Kinsey, 1998). The effect of restriction is noted by the deviation of the radial RMS displacement values of AP in the fibers (filled squares) from the line depicting the time-dependent displacement in an unrestricted medium. Data points were calculated from the mean values of D shown in Fig. 4A.

View larger version (13K): [in a new window]   Fig. 6. The predicted effect of the thick and thin filament lattice and restriction within the cylindrical sarcolemma on the time dependence of D for arginine phosphate. (A) The effect of the nm-scale thick and thin filament lattice, using a porosity of 0.86, on the relative D of arginine phosphate. Note the short time required for D to reach a steady-state value. (B) The effect of restriction within the sarcolemmal membrane of a cell with a radius of 150 µm on the relative D of arginine phosphate (note: axes are different than A). (C) The predicted time dependence of the absolute D of arginine phosphate in an unrestricted environment (solid line), in the presence of the thick and thin filament lattice (dotted line), and when restricted within the sarcolemmal membrane of a cell with a radius of 150 µm (dashed line). The lower squares depict the raw data collected for D of arginine phosphate (Fig. 4A), and the upper squares show the predicted D of arginine phosphate if the effects of the thick and thin filament lattice and restriction within the sarcolemma are removed. The upper squares therefore predict the pattern of D time dependence imposed by residual, µm-scale barriers. See text for additional details.

View larger version (13K): [in a new window]   Fig. 7. The relationship between D|| at a diffusion time of 100 ms and the reciprocal of the square root of the relative molecular mass Mr. The arginine phosphate and ATP D values were collected using 31P-NMR, while the values for betaine and arginine/arginine phosphate were collected using 1H-NMR. The D values for lactate and alanine are from Kinsey and Ellington (1996). See text for additional details.