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Sodium entry into single striated muscle fibers Brigden, Malcolm Leslie

Abstract

A review of the literature on sodium distribution in muscle has been presented including the evidence for heterogeneous distribution and the possible morphological sites of localization of this sodium. Special attention was paid to influx studies on crustacean muscle. The review reveals that while there is good evidence for a heterogeneous distribution, the morphological sites and their associated concentrations of sodium remain obscure. The experiments described in this thesis were performed on muscle fibers of the giant barnacle Balanus nubilis. Single muscle fibers of Balanus are a unique biological preparation due to their large size and ease of dissection. The major disadvantage to single fiber Balanus preparations is the extent of the extracellular space or cleft system. This cleft system contains approximately half the total fiber sodium in 6% of the fiber volume. The purpose of these experiments was to gain a picture of (22)Na+ entry into these single striated muscle fibers with special emphasis on the role of the extracellular space. A histological study revealed that the cleft system was more extensive than had been reported. From measurements made on light and electron microscope pictures it was concluded that no part of Balanus myoplasm was more than 1 - 2μ from a 0.02μ patent cleft. This study gave a picture of the morphological pathway for (22)Na+ diffusion in the extracellular space. Radioautography and radio isotope counting were two techniques used to examine (22)Na+ exchange between the extracellular space and the myoplasm. Since the extracellular space had a sodium concentration of 10 times the myoplasm, the cleft system should be adequately visualized by radioautography. However, the section thickness (15μ) and the high maximum energy of (22)Na+ (0.540MEV) limited the resolution of the radioautograms to 15 - 17.5μ. This resolution coupled with the extent of the cleft system prevented consistent radioautographic visualization of discrete clefts. Radioautographic analysis of fibers with minimal exposure (0.5 minutes) to (22)Na+ revealed a concentration gradient which could be used to define the extracellular pathway and its diffusion coefficient for Na+. The radius of the fiber cross section was found to be a reasonable approximation of the length of the pathway and the diffusion of (22)Na+ in the extracellular space along the pathway was as rapid as the self diffusion of (22)Na+ in dilute solution. A comparison of fibers that had spent 1.5 minutes in the (22)Na+ bath with similar fibers that had a 0.5 minute sucrose rinse revealed that the 0.5 minute sucrose rinse removed half of the (22)Na+ from the extracellular space. The 1.5 minute fibers and all fibers examined after periods in the (22)Na+ bath revealed a homogenous grain distribution in radioautograms. This data conflicted with the rate of (22)Na+ entry predicted by the 0.5 minute fibers. Inappropriate agitation of the 1.5 minute fibers was responsible for the lack of agreement. A further radioautographic study of (22)Na+ influx with experimental times 5, 20, 60 and 180 minutes was analyzed quantitatively. To substantiate this radioautographic study an influx experiment was done with times 5, 10, 20, 40 and 90 minutes. Each study demonstrated two compartments. An initial rapidly exchanging compartment with a half time of 6 - 10 minutes was identified as the extracellular space. The extracellular space contained approximately half the fiber sodium. Both studies detected a rnyoplasmic influx compartment which was still exchanging when the experiments were terminated. The calculated rate constant for myoplasmic exchange (5.6 x 10(-3)/minute) was in good agreement with the value of Allen and Hinke. In conclusion, a useful technique for the radioautography of soluble substances was developed. Both a morphological and a physiological picture of the pathway for (22)Na+ diffusion in the extracellular space was developed. The size and half time of loading of the extracellular space was verified. The myoplasmic influx component was identified by two methods. From these studies emerges a more comprehensive picture of the extracellular space as a pathway for diffusion in Balanus muscle. The failure to consider this compartment in microinjection or flux studies may result in ambiguous conclusions.

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