Myocardial subcellular fractionation studies on cardiomyopathic Syrian hamsters.

Myocardial homogenates from control animals and from hamsters with hereditary cardiomyopathy were subjected to analytical subcellular fractionation and enzymic microanalysis. Animals without ventricular hypertrophy or overt heart failure were used in this study. The principal subcellular organelles were characterised by density gradient centrifugation. Apart from evidence of enhanced lysosomal and peroxisomal fragility, probably secondary to the intracellular oedema, the intracellular organelles investigated in this study were unaffected by the myopathic process. Highly significant increases in 5'-nucleotidase activity, a marker for the sarcolemma, and an increased equilibrium density of this organelle were found in the myopathic tissue. Ultrastructural studies revealed patchy myocytolysis associated with lysosomes and with more extensive invaginations of the sarcolemma. It is suggested that a primary defect in membrane composition, leading to increased cation permeability, is the underlying abnormality in the myopathic hamster.

Cytochemical localization of glucose-6-phosphatase activity in the central nervous system of the rat.

The histochemical detection of glucose-6-phosphatase (G-6-Pase) in neurons of the CNS has been confirmed at the level of electron microscope. Both glucose-6-phosphate (G-6-P) and alpha-glycerophosphate (alpha-gP) can be used as substrates to localize the reaction product of this enzyme, which we have found in all cell types of the cerebral cortex, cerebellum and brain stem. The reaction was most prominent in large neurons, such as the Purkinje cells of the cerebellum and the pyramidal cells of the cerebral cortex. This is due to their extensive content of rough and smooth endoplasmic reticulum, the ultrastructural sites of G-6-Pase activity. It was possible to measure quantitatively the hydrolysis of G-6-P and alpha-gP in brain homogenates and also in microsomal fractions, the biochemical correlate of the cytochemically demonstrable activity. These results call for a reappraisal of the previous biochemical evidence, which negates the existence of brain G-6-Pase, and consequently a reassessment of current concepts pertaining to the metabolic regulation of brain glucose.

Collagen types in neuromuscular diseases.

The striking proliferation of connective tissue characteristic of the muscular dystrophies can be attributed predominantly to an increase in endomysial and perimysial type III collagen. Carriers of muscular dystrophy occasionally revealed a slight increase in anti-type III collagen fluorescence, but no abnormalities in collagen disposition were observed in foetuses "at risk" for DMD. In contrast, the proportion of collagen types in neurogenic atrophies appeared normal although anti-type IV and V staining, which delineated the basement membrane, was very intense around atrophied fibres, as was also the case in small fibres in myopathic diseases. The detection of staining with anti-type III, IV and V collagens in splits which are sometimes observed in hypertrophied fibres in the muscular dystrophies supports the suggestion that abnormalities in collagen production, perhaps involving a defective modulation of myoblast-fibroblast expression, may be involved in the pathogenesis of these diseases.

Localization of the pool of G4 acetylcholinesterase characterizing fast muscles and its alteration in murine muscular dystrophy.

The distributions of acetylcholinesterase and its molecular forms within muscles of normal and dystrophic 129/ReJ mice were established by a concomitant cytochemical and biochemical study performed on 1-mm serial sections of three predominantly fast muscles, i.e., anterior tibialis, extensor digitorum longus, and sternomastoid, as well as the slow-twitch soleus. This comparative study showed the following main findings. 1) In every muscle of both normal and dystrophic mice a) the three asymmetric forms were confined to the motor zone where they systematically codistributed with the endplates, and b) all globular forms, including G4, were concentrated at the motor zone from which they extended over the entire muscle length along a concentration gradient. 2) In the normal muscles, the perijunctional sarcoplasmic cytochemical reaction exhibited by individual fibers was grouped into a well-defined cojunctional acetylcholinesterase compartment in which the endplates were embedded. The overall intensity of the cojunctional cytochemical reaction was either high or low according to whether the muscle was predominantly fast or slow. 3) This cojunctional acetylcholinesterase compartment varied in close parallelism with G4 and thus appeared as the cytochemical correlate of the G4 molecules concentrated around the endplates. In particular, as the shape of the motor zone progressively increased in complexity along with the intricacy of the muscle fiber organization, from sternomastoid to extensor digitorum longus to anterior tibialis, so did both the relative volume occupied by the cojunctional acetylcholinesterase compartment and the proportion of G4. 4) The motor zone of the normal fast-twitch muscles characteristically differed from that of the soleus by the presence of a G4-rich environment around the endplates, which was cooperatively provided by the surrounding fibers. 5) In dystrophic muscles, this cojunctional G4-rich compartment was lost: the cojunctional cytochemical compartment was no longer discernable, while G4 was reduced to a minimal low level similar to that characteristic of the normal soleus.

Asymmetric and globular forms of AChE in slow and fast muscles of 129/ReJ normal and dystrophic mice.

Acetylcholinesterase activities and molecular forms were studied in normal and dystrophic 129/ReJ mice, focusing on four predominantly fast-twitch muscles and the slow-twitch soleus. The asymmetric and globular forms were analyzed separately so that the effect of dystrophy on each form could be determined. This comparative study showed the following. (1) In the normal condition, each muscle exhibited a distinct distribution of the molecular forms. (2) The diversity among the fast muscles resulted mainly from variations in the proportions of the three globular forms; in contrast, these muscles showed a constant and precise A12/A8/A4 ratio. (3) The slow-twitch soleus clearly differed from the other muscles in its low acetylcholinesterase activity and distinct distribution of the molecular forms, characterized by a low level of G4 and a peculiar ratio among its asymmetric forms, resulting from a relative increase of the A8 and A4 forms. (4) In dystrophic mice, the diversity of the acetylcholinesterase distribution was lost; all the fast muscles displayed profiles exhibiting the characteristics typical of the soleus. The fast-twitch extensor digitorum longus, sternomastoid, and plantaris converged towards an identical set of acetylcholinesterase molecules. (5) In contrast, the acetylcholinesterase activity and molecular forms of the soleus were only slightly affected by the disease. These results reveal that the dystrophy modifies both categories of molecular forms of acetylcholinesterase in a very precise manner. Such complex changes, which are highly reproducible in a variety of different muscles, are unlikely to result from nonspecific reactions secondary to the disease.

Cytochemical detection of nucleoside diphosphatase activity in the central nervous system of the rat.

The cytochemical localization of nucleoside diphosphatase and thiamine pyrophosphatase occurs within the "mature face" of the Golgi apparatus and over the neurilemma in neurons of the cerebellum, the cerebral cortex and the brain stem. The hydrolytic reaction product of the brain enzyme differs from that of the liver in that it is not found in the endoplasmic reticulum or nuclear envelope. Hydrolysis of IDP, UDP or GDP is not greater than that of ADP or CDP in brain homogenates, in contrast to that found in the liver. The NDPase activity of brain homogenates is optimal at pH 7.2, stimulated by heavy metals and inhibited by uranyl nitrate. Thick section cytochemistry suggests that the reaction product is restricted to a network of polygonally shaped compartments. NDPase activity on the neurilemma may reflect the role of this enzyme in the synthesis of glycoproteins involved in neuronal surface recognition.

Decreased G4 (10S) acetylcholinesterase content in motor nerves to fast muscles of dystrophic 129/ReJ mice: lack of a specific compartment of nerve acetylcholinesterase?

Acetylcholinesterase (AChE; EC 3.1.1.7) activity and the distribution of its molecular forms were studied in the nervous system of normal and dystrophic 129/ReJ mice, including the sciatic-tibial nerve trunk and motor nerves to slow- and fast-twitch muscles. In normal mice, motor nerves to the slow-twitch soleus exhibited a low AChE activity together with a low level of G4 (10S form) as compared with nerves of the predominantly fast-twitch plantaris and extensor digitorum longus. In contrast, in dystrophic mice, the AChE activity as well as the G4 content of nerves to the fast-twitch muscles were low, displaying an AChE content similar to that of the nerve of the soleus muscle. In the sciatic-tibial nerve trunk, the AChE activity decreased along the nerve in an exponential mode, at rates that were similar in both conditions. However, in dystrophic mice, the AChE activity was reduced throughout the nerve length by a constant value of approximately 180 nmol/h/mg protein. Further analyses indicated that AChE in this nerve trunk was distributed among two compartments, a decaying and a constant one. The decay involved exclusively the globular forms. The activity of A12 (16S form) remained constant along the nerve and was similar in both normal and dystrophic mice. In addition, according to the equation describing the decay of AChE, the reduction in enzymatic activity observed in the dystrophic mice affected mainly G4 in the constant compartment. Brain, spinal cord, sympathetic ganglia, and serum, which were also examined, showed no remarkable differences between the two conditions in their G4 content. The AChE abnormalities that we found in nervous tissues of 129/ReJ dystrophic mice were confined to the motor system.

Absorption and distribution of sodium [2-14C]barbital in tissues of normal and dystrophic mice.

The absorption and distribution of [2-14C]barbital after oral administration was studied in various tissues, including skeletal muscle, of normal and dystrophic mice. There appeared to be a more rapid gastric emptying in the mutant homozygote as reflected in lower levels of the drug recuperated from the gastrointestinal tract. This resulted in initially higher plasma and tissue concentrations of barbital in the dystrophic mice. Two hours after oral administration, this kinetic profile was reversed so that less barbital remained in the tissues of the dystrophic mouse. The tissue:plasma concentration ratios were consistently, but not significantly, higher in all tissues of the dystrophic animals. Analysis of the half-life of the drug in both groups suggests that there is an increase in the distribution volume of barbital in the dystrophic mice. The phenomenon of more rapid absorption of the barbiturate seems to be more consistent as the symptoms of the disease progress. The altered absorption and disposition of barbital in various tissues of the dystrophic mouse support the concept that a generalized multisystemic disorder may be crucial to the pathogenesis of murine muscular dystrophy, in contradistinction to a purely myogenic origin.