Date of Award
8-1981
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Robert H. Fitts
Second Advisor
James B. Courtright
Third Advisor
Nelson J. Horseman
Abstract
Sarcoplasmic Reticulum (SR)
For the past three decades, the SR has drawn considerable attention primarily for two reasons: (I) physiologically it plays a critical role in muscle contraction controlling intracellular calcium levels, and (II) from the biochemical and biophysical point of view, this membrane system provides one of the simplest and most effective ways to study the molecular nature of active cation transport. As a result, its structure, function energetics, and role in muscle contraction have at least in part been revealed.
Following nerve excitation, muscle contraction is initiated by the propagation of action potentials along the sarcolemma and down the transverse tubules (T-tubules). By an unknown mechanism, the signals from the T-tubules induce calcium release from the terminal cisternae region of the SR. The released calcium ions increase the sarcoplasmic calcium concentration to a critical level causing contractile protein activation and muscle contraction. During relaxation, the SR actively sequesters calcium from the sarcoplasm decreasing sarcoplasmic calcium concentration. It has been generally agreed that two calcium ions are sequestered per ATP molecule hydrolyzed (1, 2, 3).
The SR membrane is composed of 3 main proteins identified as an ATPase, a high affinity calcium binding protein, and calsequestrin. The ATPase,with a molecular weight of about 100,000 daltons, plays a major role in calcium transport (4). During calcium transport the aspartate moiety of the ATPase is phosphorylated by ATP forming an acyl phosphate compound (5). During this phosphorylation step, calcium ions are translocated from the outside to the inside of the membrane and calcium is then released from the enzyme into the lumen. Simultaneously, the phosphorylated intermediate is dephosphorylated (4) and this step is accelerated by magnesium ions (6, 7). This process can be reversed allowing ATP synthesized from ADP and Pi if a high calcium concentration gradient exists (8, 9).
The high affinity calcium binding protein, with a molecular weight of about 55,000 daltons, is located inside the lumen and shows a high affinity but low capacity for calcium binding (10, 11). Its physiological role is not yet known. Calsequestrin is a highly acidic protein and shows a high capacity and low affinity for calcium binding. Its molecular weight is somewhat controversial and varies with different determination methods (11, 12, 13, 14) from 44,000 to 67,000 daltons. It plays a calcium-buffering role in the SR lumen and might be involved in the release of calcium at the terminal cisternae (15).
Most of the SR studies have evaluated fragmented SR (FSR). The FSR is obtained from the microsomal fractions between 20,000 and 45,000 G (4). Oxalate or phosphate is usually added to the assay medium to facilitate the calcium transport since it induces a calcium oxalate or calcium phosphate precipitation inside the vesicles.
The majority of the SR lipid is phospholipid (80%) and a general feature of the SR membrane is a lower content of total and neural lipids, and a high degree of unsaturated fatty acids. The presence of phospholipids is required for the activity of the SR ATPase (16).
Problems Related to the Present Studies
Mammalian skeletal muscles are made up of different fiber types (17). In rodents, the slow twitch red, type I fibers have a moderately high respiratory capacity, a low glycogenolytic capacity, and a low myosin ATPase activity. The fast twitch red, type IIA fibers have a high respiratory capacity, a high glycogenolytic capacity and high
myosin ATPase activity. The fast twitch white, type IIB fibers have a low respiratory capacity, a high glycogenolytic capacity, and high myosin ATPase activity (18).
It is also known that fast muscles with a shorter contraction time (CT) and one-half relaxation time (½RT) have a greater SR content, a higher ca2+-ATPase activity, and a higher rate of calcium uptake than slow muscles (19, 20).
Skeletal muscles show fiber type-specific adaptations in response to alterations in motor activity. The changes involve mitochondrial and cytosolic enzymes, the actomyosin ATPase, substrate utilization, and contractile properties (18, 21, 22, 23, 24, 25, 26).
Changes in SR function in response to different motor activity patterns have not been fully evaluated.
Recently the effect of thyroid hormone on muscle function has received considerable attention. Excess thyroid hormone is known to alter the functional capacity of the SR as well as the contractile properties. However, it is unknown whether the hormone is acting directly on the muscle or via the nerve (27, 28).
Present Studies
The studies described herein were undertaken to evaluate the effect of use and disuse on SR function and determine the role of the SR in regulating muscle function.
Exercise training and hindlimb immobilization were used to evaluate use and disuse respectively. The effect of excess thyroid hormone on SR function was also evaluated.
In Chapter 1, SR studies utilizing FSR and crude homogenate techniques are described and the SR function of the different fiber types compared. The functional studies included an evaluation of calcium uptake kinetics, total calcium uptake, and the determination of SR ATPase activity (2, 20). The functional studies are accompanied by structural studies including 1-D acrylamide gel electrophoresis (29, 30), and 2-D isoelectric focusing gel electrophoresis (31).
Generally, the results of these studies show changes in motor activity to be fiber type-specific. The reduced neural activity with hindlimb immobilization caused SR changes opposite to those induced by exercise training. Thyroid hormone action on SR appears to be confined to slow muscles. The relationship between twitch duration and SR function was maintained in the exercise training and thyrotoxic models, but in the disuse model the relationship was disturbed in part due to the large decrease in twitch and tetanic tension.