Effects of Acute and Chronic Denervation on Release of Acetylcholinesterase and Its Molecular Forms in Rat Diaphragms

Abstract: Hemidiaphragms were removed from rats at various times after intrathoracic transection of the left phrenic nerve and were incubated in organ baths containing 1.5 ml of oxygenated, buffered physiologic saline solution, with added glucose and bovine serum albumin. After incubation, the acetylcholinesterase (AChE; EC 3.1.1.7) activities of the bath fluid and of the muscle were determined. Innervated left hemidiaphragms were found to release 107 units of AChE over a 3-h period, corresponding to 1.9% of their total AChE activity. Denervation led to a rapid loss of AChE from the muscle coincident with a transient increase in the outpouring of enzyme activity into the bath fluid. Thus, 1 day after nerve transection the left hemidiaphragm contained only 68% of the control amount of AChE activity, but released 140% as much as control. After 3 or 4 days of denervation, the AChE activity of the diaphragm stabilized at 35% of the control value. Release also fell below control by this time, but not as far. One week after denervation the release, 69 units per 3 hr, corresponded to 3.3% of the reduced content of AChE activity in the muscle, indicating that denervation caused an increase in the proportion of AChE released. Sucrose density gradient ultracentrifugation showed that 10S AChE accounted for more than 80% of the released enzyme activity at all times. The results did not rule out the possibility, however, that the released enzyme originally stemmed from 4S or 16S AChE in the diaphragm.     

Attachment and Short-Term Maintenance of Motility and Viability of Entamoeba Histolytica In A Defined Medium*

Organic requirements for attachment to glass, elongation, and motility of Entamoeba histolytica, have been determined. the trophozoite, which has been grown axenically only in highly complex media with reduced oxygen tensions, remains rounded and detached when placed in a Tris-HCl buffered solution containing NaCI, KCI, MgCI₂, and CaCI₂. A maintenance medium in which the amebae could attach to glass, elongate, and remain motile and viable for 12 to 24 h was devised with the addition of cysteine, ascorbic acid, bovine serum albumin, and the vitamin solution of medium NCTC #107. Tris-HCI was the most effective buffer tested and the optimal pH was 6.9 to 7.0. Survival, but not attachment, of the amebae was decreased at osmolalities ranging between 110 and 180 milliosmoles/kg, whereas both functions were decreased above ～260 milliosmoles/kg. Bovine serum albumin, the most effective of the proteins tested, and the vitamin solution helped maintain attachment of some ameba strains, but were not required by other strains. the requirements for cysteine and ascorbic acid were absolute and highly specific. During incubation in the maintenance medium, cell volumes decreased. Sensitivity of the organisms to agglutination by concanavalin A, wheat germ agglutinin, soybean agglutinin and fucose binding protein remained unchanged.     

Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome $\text{c}$ reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

The membrane systems in different fibre types of the triceps surae muscle of cat

Summary The volume and surface area of mitochondria and sarcoplasmic reticulum in fast and slow twitch fibres of the cat triceps surae muscle were determined from thin sections. The width of the Z-line and the array of glycogen granules identified fast and slow twitch fibres.The relative volume occupied by mitochondria was largest in slow twitch gastrocnemius fibres. Fast twitch fibres showed the greatest scatter of mitochondrial content. This corresponds with the fact that motor units of the fast twitch type differ most with respect to resistance to fatigue.The relative volume of the sarcoplasmic reticulum was twice as large in fast as in slow twitch fibres. The volume fraction occupied by longitudinal tubules of the reticulum was the same in fast and slow twitch gastrocnemius fibres but was only half as large in the slow twitch soleus fibres. This difference may be related to post-tetanic potentiation: this property is present in all gastrocnemius fibres but is absent in soleus fibres.The specific tetanic force is 3 to 5 times smaller in slow twitch gastrocnemius than in slow twitch soleus fibres or fast gastrocnemius fibres. There was, however, no detectable morphological difference that might be related to this difference in force.Freeze fractures demonstrated directly that, in soleus fibres, terminal cisternae and longitudinal tubules of the reticulum were scarce as compared to gastrocnemius fibres. The plasma membranes of some gastrocnemius fibres displayed square arrays of 60-nm particles; these arrays were absent in other gastrocnemius fibres and in all soleus fibres. They probably characterize plasma membranes of fast twitch fibres.This study was supported by grants from the Danish Medical Research Council. I wish to thank Mrs. M. Bjærg for valuable technical help     

Ultrastructure of the muscles of the dorsal diaphragm in Locusta migratoria

Summary The alary muscles of Locusta migratoria adults make up the major tissue of the dorsal diaphragm which separates pericardial and perivisceral sinuses in the abdomen. The alary muscles are striated with a sarcomere at rest measuring about 9 m. The Z-line has a staggered-beaded arrangement with A-bands and I-bands readily discernable. Thick myofilaments are surrounded by 10 or more thin filaments. The sarcoplasm has few mitochondria near the area of the Z-line, dyads are present and sarcoplasmic reticulum is poorly developed. Axons which innervate the alary muscle are either contained within invaginated folds of the sarcolemma of the muscle cells or the muscle cells send finger-like projections to envelop the axons. The synaptic terminals contain synaptic vesicles between 40 and 45 nm in diameter and a few electron-dense granules near or less than 170 nm in diameter. Away from synaptic terminals the axon profiles show few or no granules. The axons are accompanied everywhere by well-developed glial cells. This then is not typical neurosecretomotor innervation, however, the presence of electron-dense granules suggests the possibility of peptidergic neurotransmission.     

Long-term culture of human aortas

Summary Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures. Supported in part by the Pangborn Fund and the Graduate School of the University of Maryland. This is publication 443 from the Cellular Pathobiology Laboratory.     

Protein metabolism. 6. Trichostrongylus colubriformis: skeletal protein catabolism in primary and secondary infections of the guinea pig

The urinary excretion of 3-methylhistidine (3-MH) was used as an index of muscle protein catabolism in primary and secondary infections of the guinea pig with Trichostrongylus colubriformis and in uninfected animals fed quantitatively reduced rations. Catabolism, which was depressed in all three groups, was directly related to a fall in food consumption. Possible explanations for the greater depression of catabolism in the primary infection than in the uninfected guinea pigs and its fall in the secondary infection in spite of little change in consumption are briefly discussed. It was concluded that the faster rate of whole-body protein turnover reported earlier in this series on protein metabolism in intestinal nematode infection was not partly due to a faster rate of muscle protein catabolism. It was shown that the urinary excretion of 3-MH could be validly expressed in terms of unit creatinine.     

Subsarcolemmal mitochondria and capillarization of soleus muscle fibers in young rats subjected to an endurance training

Summary Rats, 6 weeks old, were subjected to a program of endurance running for 3, 6 and 12 weeks. 0.5 to 0.8 m thick sections of Epon embedded soleus muscles were studied with morphometric methods.In cross-sections the area occupied by subsarcolemmal mitochondria was independent of the age, but was 53% higher after 12 weeks of training. The mean depth of the zones with subsarcolemmal mitochondria increased only 15% to about 0.9 m. Thus, the subsarcolemmal mitochondria showed a pronounced spreading at the muscle fiber surface in trained muscles. — The number of capillaries per fiber decreased slightly in controls and increased not significantly in trained muscles.It is concluded that the subsarcolemmal mitochondria supply the energy for the active transport of metabolites through the sarcolemma in oxidative muscle fibers, and that they are the limiting factor for endurance performance of the soleus muscle fibers because the changes in the capillarization were only small. It is suggested that the subsarcolemmal and the interfibrillar mitochondria have different functions and may therefore represent different types of mitochondria which can be distinguished by their morphology as well as by their biochemical properties.     

Structural changes in smooth muscle cells during isotonic contraction

Summary Smooth muscle cells of the guinea-pig taenia coli were studied in light and electron microscopy, in condition of mild stretch or of isotonic contraction. During contraction the cells increase in transverse sectional area and their packing density passes from 94,000 · mm^-2 to 18,000 · mm^-2. The percentage increase in transverse sectional area of the taenia is approximately the same as the percentage decrease in length. Measurements of cell transverse sectional area suggest that the individual cells shorten and fatten more than the taenia as a whole. Whereas stretched muscle cells run parallel to each other and show a fairly smooth surface, isotonically contracted cells are twisted and entwine around each other. Their surfaces are covered with myriad processes and folds. Longitudinal, transverse or oblique stripes are seen in light microscopy in the contracted muscle cells and it is suggested that they are related to the characteristics of the cell surface. In electron microscopy a complex pattern of interdigitating finger-like and laminar processes is observed. Caveolae are mainly found on the evaginated parts of the cell surface, dense patches are mainly (but not always) found on the invaginated parts. Desmosome-like attachments between contracted cells are frequent. The collagen fibrils run approximately parallel to the stretched muscle cells; on the other hand, they run obliquely and transversely around the isotonically contracted cells.This work is supported by the Medical Research Council. I thank Miss E.M. Franke and Mr S.J. Sarsfield for excellent technical assistance