Stephen R. Max

Enzymatic adaptation in ligaments during immobilization

Ligaments are a composite of fibroblasts and collagen in a proteoglycan matrix. Seventy-five percent of the organic solid is collagen and 23% is proteoglycan. Fibroblasts are responsible for the overall composition of the ligament, that is the synthesis and the degrada tion of macromolecular components. Like muscle and bone, ligaments are dynamic, undergoing hypertrophy with exercise and atrophy with immobilization. This paper reviews the structure and composition of liga ments and discusses the cellular events responsible for atrophy of ligaments with immobilization. As an exper imental model, one knee of New Zealand White rabbits was immobilized with a pin. After 2, 4, and 8 weeks of immobility, the medial collateral ligaments were isolated and enzyme analysis was performed. Gross and micro scopic changes were apparent after 2 weeks. As for enzyme changes, lactic dehydrogenase and malic de hydrogenase decreased in activity. The lysosomal hy drolases responsible for glycosaminoglycan degrada tion increased in activity, suggesting that enzymatic adaptations mediate the physical and chemical changes in the ligament. The cells switch from an anabolic synthetic state to a catabolic, degradative state during immobility. It would seem from the biochemical view point that, whenever possible, cast-bracing and func tional splints may be preferable to rigid plasters in many sports-related ligamentous injuries.

Lysosomes and disuse atrophy of skeletal muscle.

The specific activities of the acid hydrolases β-glucosidase, β-galactosidase, β-N-acetylhexosaminidase, arylsulfatase, and acid phosphatase have been measured during the time course of disuse atrophy of rat gastrocnemius muscles. These enzymes are considered to be lysosomal in origin. The specific activity of β-glucosidase increased to 180% of the control level by the second day following the production of disuse, and to 400% of the control level by the 12th day. The specific activities of β-galactosidase, β-N-acetylhexosaminidase, arylsulfatase, and acid phosphatase were increased markedly by the 12th day. All enzyme activities returned to control levels by the 15th day. The early increase in the activity of β-glucosidase indicates that lysosomal enzymes may an important role in the etiology of disuse atrophy.

Simple procedure for rapid isolation of functionally intact mitochondria from human and rat skeletal muscle

A simple procedure for the rapid isolation of functionally intact skeletal muscle mitochondria is described. The method involves homogenization of muscle in a medium comprising sucrose (0.25 M) containing 50,000 units of heparin/liter, followed by differential centrifugation. Mitochondria so isolated are functionally and morphologically intact.

Disuse atrophy of skeletal muscle: loss of functional activity of mitochondria.

Summary Mitochondria isolated from rat gastrocnemius muscles subjected to disuse atrophy displayed a marked loss of respiratory control at a very early stage in the wasting phenomenon. This abnormality was reversed by bovine serum albumin. It is postulated that the effect was caused by free fatty acids. Mitochondrial dysfunction may be of significance in the initiation of disuse atrophy.

GM3 (Hematoside) Sphingolipodystrophy

Abstract To characterize further disorders involving storage of glycosphingolipids, we studied an infant who had poor physical and motor development, coarse facies, macroglossia, gingival hypertrop...

Effect of 8-Bromo-cAMP and Dexamethasone on Glutamate Metabolism in Rat Astrocytes

Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP)+1 μM dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP+DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hanks salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original [U-14C]glutamate was recovered in the medium as radioactive glutamine, 2–3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and, (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate.

Developmental patterns of glycolytic enzymes in regenerating skeletal muscle after autogenous free grafting

Extensor digitorum longus muscles of rats were removed and injected with a solution of Marcaine plus hyaluronidase. After incubation in Marcaine solution for 10 min, the muscles were grafted into their original beds. The grafts and the contralateral control muscles were removed from the rats at 0, 1-5, 7, 11, 36, and 69 days postoperatively. The muscles were then frozen in dry ice and isopentane and subsequently homogenized and centrifuged. The supernatant was analyzed for a number of enzymes, the regenerative patterns of which can be classified into 3 groups: (1) early increase in activity: hexokinase, glucose-6-phosphate dehydrogenase; (2) early decrease in activity with failure to recover to control levels: phosphorylase, phosphofructokinase, alpha-glycerophosphate dehydrogenase; and (3) early decrease followed by return to control levels: lactate dehydrogenase, pyruvate kinase, creatine phosphokinase, adenylate kinase. These patterns are not identical to those reported for embryogenesis of muscle. The data are discussed with regard to correlative histological studies of muscle regeneration.

Skeletal muscle glucocorticoid receptor and glutamine synthetase activity in the wasting syndrome in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

This study demonstrated specific changes in rat skeletal muscles after a single oral dose (100 micrograms/kg) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The development of the wasting syndrome was characterized by marked body weight loss, as well as atrophy of plantaris and gastrocnemius muscles. Fourteen days after administration of TCDD, gastrocnemius muscle cytosolic glucocorticoid receptor binding, measured at a single saturating concentration of [3H]triamcinolone acetonide, was significantly diminished, while plantaris muscle glutamine synthetase activity was strikingly elevated, indicating that specific biochemical alterations occur in skeletal muscle in the wasting syndrome.

Metabolic studies of skeletal muscle regeneration

Metabolic studies were carried out on regenerating skeletal muscle in the rat following intramuscular injection of the myotoxic agent Marcaine plus hyaluronidase or after mincing and autoimplantation. Both after mincing and following injection of Marcaine, the ability to oxidize glucose-6-14C and palmitate-1-14C was lost, while the oxidation of glucose-1-14C was markedly enhanced. The loss of oxidative metabolism was accompanied by decreased muscle activity of monoamine oxidase, suggesting diminished numbers of mitochondria. In addition, in Marcaine-treated rats, the activitities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase were markedly elevated and returned to the control level in parallel to the enhanced metabolism of glucose-1-14C. The elevation of these enzymes is consistent with activation of the hexose monophosphate shunt. Substrate utilization and total monoamine oxidase activity returned to control values in the Marcaine-treated muscles, while no recovery was observed following mincing and implantation. In addition, the activity of β-galactosidase, a lysosomal marker enzyme, was enhanced in both experimental systems. β-galactosidase activity returned to control values in Marcaine-treated muscles but not in minced muscles, within the time-course of these experiments. We conclude that the Marcaine plus hyaluronidase preparation is more suitable for metabolic studies in regenerating muscle than is mincing and autoimplantation.

Effects of neuromuscular activity on choline acetyltransferase and acetylcholinesterase

Changes in neuromuscular activity are known to modify neuromuscular transmission. In this report, we describe changes in the activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) in response to decreased and increased activity of skeletal muscles in the rat. Disuse atrophy of gastrocnemius muscles was produced by surgical pinning of the hind limb. Overuse hypertrophy of the plantaris muscle was produced by severing the tendon of the gastrocnemius and soleus muscles. At various times after production of disuse or overuse, the activities of CAT and AChE were measured by radiometric assay. In disused muscles, CAT activity per muscle decreased markedly (50% of control by day 7), while AChE activity per muscle decreased to a smaller extent (80% of control by day 7). The Km of CAT with respect to acetyl CoA did not change in homogenates of atrophic muscles (1 × 10−4m), suggesting that the loss of activity was not due to alteration of enzyme protein, but rather to decreased amounts of enzyme. Overuse of muscle resulted in an increase in the activities of CAT (120% of control by day 8) and AChE (142% of control by day 8). Thus, use and disuse may influence neuromuscular transmission by regulating the production of enzymes involved in the synthesis and degradation of acetylcholine.