Ultrastructural Localization of Acid Phosphatase in - Europe PMC

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Jan 18, 1980 - From the Departments of Pathology and Medicine, Medical University of ... The present study was undertaken to investigate ultrastructural and.
Ultrastructural Localization of Acid Phosphatase in Denervated and Diabetic Striated Muscles S. S. Spicer, MD, M. G. Buse, MD, and M. E. Setser, MA

Catabolism in denervated and diabetic rat skeletal muscle undergoing accelerated protein degradation has been investigated with methods for demonstrating acid phosphatase ultrastructurally. Control muscles displayed strong acid phosphatase activity in lateral sacs and in sparse secondary lysosomes distributed mainly near nuclear poles. Muscles from diabetic rats and, to a lesser extent, 2-day denervated rats, revealed increased secondary lysosomes apparently derived from fusion of mitochondria with acid-phosphatase-reactive vesicles and cisternae. The latter were interpreted as possibly originating from T tubules. Reaction product was also noted in the junctional folds of the motor end plate of a denervated muscle. At the longer post denervation intervals studied, deposits indicative of acid phosphatase were dispersed throughout the sarcoplasm with greater concentration in the I band and appeared more abundant in denervated than in contralateral control muscles. The enzymatic basis for the sarcoplasmic deposits and other deposits was confirmed by their absence from cytochemical controls, which included incubation in substrate-free medium, heat or NaF inactivation of enzyme, and exposure sequentially to PbNO3 and NaH,PO or PbNO3 and 8-glycerophosphate. (Am J Pathol 1980, 99:603-620)

THE PROTEIN content of muscles results from a dynamic balance between rates of synthesis and degradation."2 These processes are both regulated by nutritional and hormonal factors as well as by muscular activity and nervous stimuli. Uncontrolled diabetes, for example, induces a protein-catabolic state with negative nitrogen balance and accelerated gluconeogenesis. Skeletal muscle provides the principal source of the amino acids, which are converted to glucose by the liver and kidneys in the insulin-deficient state.34 The anabolic hormone insulin is the principal regulator of protein turnover in muscles, stimulating protein synthesis and inhibiting protein degradation."7 Denervation or disuse causes muscle atrophy, which results primarily from acceleration of muscle protein catabolism."2'8 Although the regulation of protein catabolism in muscles is poorly understood, some evidence indicates that lysosomal proteases are involved. Thus, conditions that promote muscle protein degradation, eg, prolonged fasting and insulin deficiency, may be associated with increased lysosomal lability.579"0 On the other hand, several nonlysosomal proteases have been reported in skeletal muscle," including a Ca'+-activated protease, which is specific for Z-line From the Departments of Pathology and Medicine, Medical University of South Carolina,

Charleston, South Carolina. Supported by NIH Grants AM-10956, AM-11028, HL-19160, and AM-02001. Accepted for publication January 18, 1980. Address reprint requests to S. S. Spicer, MD, Department of Pathology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29403.

0002-9440/80/0609-0603$01.00 © American Association of Pathologists

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degradation.'2 It has also been suggested that fasting and diabetes result in the activation of muscle alkaline protease, which appears to be bound to myofibrillar proteins.'3 The present study was undertaken to investigate ultrastructural and cytochemical changes in skeletal muscle in two conditions associated with acceletrated protein degradation, denervation atrophy, and severe diabetes. Although acid phosphatase is not known to act directly in muscle protein degradation, it was assessed cytochemically here as an indicator of lysosomal activity in these catabolic states. Skeletal muscle is known, however, to contain relatively few lysosomes. A main objective of this investigation was to ascertain whether or not changes in the number, morphologic changes, and/or distribution of lysosomes could explain the accelerated degradation of myofibrillar proteins throughout the sarcomere. Materials and Methods Male Wistar rats weighing 150-200 g were anesthetized with ether, and the right or left hind leg was denervated by transecting the sciatic nerve in the gluteal area. The surgical incision was closed, and the animals were maintained on a standard diet of Wayne lab blocks and water ad libitum. The rats were killed 1 day to 3 weeks after denervation. Muscles from the opposite leg of the rat upon which the operation was done served as nondenervated control muscles. Diabetes was induced in other comparable rats of the same strain with a single dose of streptozotocin (120 mg/kg intraperitoneally as previously described).'4 At the end of 4 days the plasma glucose level was measured, and only diabetic animals with a concentration greater than 300 mg/100 ml were used in these experiments. Rats not given the injections and maintained under the same conditions served as control animals. In all cases the rats were anesthetized with sodium pentobarbital (20 mg intraperitoneally). The aorta was cannulated, and the legs were perfused with saline at 4 C to wash out blood and then with chilled 2% glutaraldehyde in 0.1 M cacodylate buffer. The gastrocnemius and soleus or quadriceps muscles were excised, minced, and left immersed in the above solution for a total fixation period of 30 minutes. For demonstration of acid phosphatase, cryostat sections 30-40,u in thickness were incubated for 60 minutes at 37 C in Barka-Anderson medium,'5 adjusted to pH 5.0. Some of the minced sections from the animals killed 3 weeks after neurectomy were incubated for 60 minutes in the same medium with the use of cytidine monophosphate substrate. Controls for acid phosphatase cytochemistry included omission of substrate from the medium or inactivation of the enzyme in the cryostat sections by a 10-minute exposure to heat at 100 C or by including 0.01 M NaF in the incubation medium. Since precipitates have been shown to form on a nonenzymatic basis in muscle fibers that have absorbed lead and are then exposed to orthophosphate,'6 additional control procedures were carried out on a portion of several specimens that were also processed for acid phosphatase cytochemistry. These entailed exposure first to 1 mM PbNO3 for 15 minutes, followed by 10 minutes of exposure to 10 or 100 mM NaH2PO4. In a further control test the cryostat sections were exposed to 2.5 mM PbNO3 and then an 11.0 mM solution of the ,B-glycerophosphate employed for the acid phosphatase medium with or without added 0.01 M NaF. Portions of several denervated, diabetic, and control muscles were fixed for 1 hour with the glutaraldehyde solution for routine ultrastructural examination. All specimens were postfixed with 2% osmium tetroxide, dehydrated, and embedded in Epon. A blind analysis of experimental and control micrographs was performed, rating the

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Soleus

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Animal Number

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C DC DC V IV

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Histogram of acid phosphatase activity in the sarcoplasm of 3 week denervated (D) rat muscles and control muscles (C). TEXT-FIGURE 1-Muscles from denervated rat legs displayed more sarcoplasmic acid phosphatase reactivity than did contralateral control legs from the same animal. Micrographs (15-20) from each muscle were scored in a blind analysis by three observers. The dark area of the histogram represents the lowest level of activity and the clear area the highest activity observed in a muscle.

abundance of deposits indicative of acid phosphatase activity on a scale of 0-++++ for various reactive sites (Text-figure 1).

Results Denervated Muscles

Muscles 2 days after denervation and the contralateral control muscles exhibited bodies with strong acid phosphatase activity and morphologic features like those of lysosomes at nuclear poles (Figure 1). Occasionally these reactive structures resembled multivesicular bodies. Collections of small vesicles, and short cisternae with reaction product apparently representing Golgi elements, were encountered infrequently near nuclei and indicated a source of the lysosomal enzyme in denervated and control muscles. Some of the muscles that had been denervated for two days differed from intact muscles in disclosing occasional profiles that could be interpreted as mitochondria with acid phosphatase activity. Fine precipitates indicative of the enzyme occupied a small to large part of these structures (Figure 2). Some muscles two days after denervation differed further from control muscles in showing positive, lysosome-like profiles neighboring mitochondria, particularly where the latter showed acid phosphatase activity (Figure 3). Whether these can be identified as lysosomes or represent degenerated mitochondria is uncertain. In a few animals, 2 days after denervation, dense droplets bordered a number of mitochondria in some but not all specimens, and mitochondria

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in these muscles frequently contained small, often irregular particles of comparable density crowding the cristae (Figure 4). Unlike the aforementioned acid-phosphatase-reactive sites, these bodies appeared equally dense in acid phosphatase control preparations. They were also very dense with and without uranyl acetate and lead citrate staining of the thin section and were, accordingly, interpreted as osmiophilic-lipid-positive rather than acid-phosphatase-positive structures. Deposits of acid phosphatase reaction product filled the lateral sacs of the sarcoplasmic reticulum. These precipitates appeared comparable in control muscles and in those which had been denervated for 2 days or for longer intervals (Figure 1; see also Figures 9 and 11). Fine precipitates were scattered in the sarcoplasm, sparing the Z bands, in an occasional muscle 2 days after denervation, but as a rule these were not present. Seven days after denervation mitochondrial staining was still evident in some animals (Figure 5). Intensely reactive lysosomes and groups of densified small vesicles and short cisternae interpreted as Golgi elements were encountered, usually in the vicinity of a nucleus (Figure 5), and appeared slightly more numerous in the denervated muscle, compared with the contralateral control muscle. However, lysosomes were not encountered frequently enough to allow quantitation of a possible difference. There was no evidence of increased lysosomes in sections studied at other times after denervation. In 1 of 4 animals from this group acid phosphatase reactivity was also seen on the sarcolemma and in occasional T tubules in the denervated leg. The corresponding muscle from the control contralateral muscle in this animal also displayed T tubule reactivity, although the staining was less extensive. In 10-day denervated muscles acid phosphatase activity was seen in a small proportion of the T tubules in each of the 4 animals studied (Figure 6). The sarcolemma displayed deposits in some specimens, and the T tubules invariably disclosed strong reactivity in areas where the sarcolemma stained. Only 1 of the 4 animals showed activity in a few T tubules in the contralateral nondenervated muscle. The denervated muscles also often showed moderate diffuse precipitates in the sarcoplasm (Figure 6). These deposits were more prominent in some of the specimens, especially neighboring reactive sarcolemma, and generally exceeded the light or negligible precipitates found in contralateral control muscles or in muscles denervated for 2 days. Three weeks after denervation fairly heavy precipitates indicative of acid phosphatase lay scattered throughout the sarcoplasm (Figure 7). These deposits appeared uniformly distributed in profiles of some apparently contracted muscles but were concentrated more heavily in the I bands, sparing the Z bands, of other muscles that were presumed from the

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width of the I bands to be in a more relaxed state. The density of the precipitates varied among animals, presumably because of biologic variability and uncontrollable slight differences in fixation and incubation conditions, but the denervated gastrocnemius and soleus muscles consistently displayed heavier sarcoplasmic deposits than the control legs, with only moderate overlap (Text-figure 1). Deposits in lateral sacs were usually lighter or absent in muscles with heavy sarcoplasmic precipitates. The mitochondrial and T tubule staining noted at earlier intervals after denervation were no longer observed. Motor End Plates

Heavy reaction product was noted on the outer surface of the junctional folds of a motor end plate (Figure 8) 7 days after denervation. Vesicles and short cisternae of Golgi complexes with heavy reaction product in adjacent axonal or Schwann cell cytoplasm indicated a possible source of enzyme in the myoneural cleft. This specimen also evidenced heavy reactivity on the outer surface of the sarcolemma and in the T tubules. A motor end plate was encountered in a 3-week denervated muscle that had been processed for demonstration of cytidine monophosphatase (Figure 9). Although nearby lysosomes as well as lateral sacs and sarcoplasm in this muscle disclosed heavy deposits of reaction product, the motor end plate showed little if any evidence of this enzymatic activity. A motor end plate profile from a control animal revealed no acid phosphatase deposits in the myoneural cleft, although lysosome-like structures in the sarcoplasm and in a fibroblast process nearby were positive (Figure 10). Muscles of Diabetic Rats

Muscles from diabetic rats and from control nondiabetic animals disclosed similar acid phosphatase deposits in the lateral sacs (Figure 11). Reaction product filled some T tubules in muscles of diabetic animals (Figure 12) but was absent from muscles of control rats. In contrast to the unreactive mitochondria in muscles of control rats, a moderate number of mitochondria in muscles of diabetic rats revealed staining which ranged widely in intensity and distribution (Figure 13). Occasionally a thin heavily stained cisterna and positive vesicles enveloped part of some mitochondria (Figures 12 and 14). Other mitochondria displayed heavy accumulation of reaction product in the periphery or throughout the profile (Figures 14-19). Evidence of further mitochondrial degeneration was also encountered, eg, densely stained areas suggestive of myelin figures and remnants of mitochondrial membranes (Figures 18 and 19), as well as mitochondria showing dense reactivity resembling that of lysosomes (Figure 19).

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Cytochemical Controls

The acid phosphatase controls in which substrate was omitted from the incubation medium, or in which enzymatic activity was inhibited with NaF or heat, lacked the characteristic enzyme-induced precipitates in lateral sacs, mitochondria, sarcoplasmic reticulum, T tubules, sarcolemma, lysosomes, and sarcoplasm, but occasionally disclosed fine nonenzymatic staining in sarcoplasmic reticulum and mitochondria. Further cytochemical controls were employed to test whether nonenzymatic precipitation or deposition could account for the observed deposits, especially those in the sarcoplasm. Cryostat sections that were exposed sequentially to PbNO3 and NaH2PO4 solutions in the range of the 2.5 mM PbNO3 prevailing in the Barka-Anderson substrate medium (Table 1) showed variable deposits in different regions of the specimen, ranging from random fine deposits to sparse coarse precipitates or no deposition. Higher phosphate levels, however, yielded heavier, fine, randomly distributed precipitates. Sections exposed to PbNO3 followed by glycerophosphate were essentially devoid of deposits. The appearance of these control sections was clearly different from staining attributed to enzymatic activity. Discussion

Acid phosphatase reactivity has classically been associated with cell catabolism and is generally thought to be isolated in membrane bound structures as, for example, lysosomes. Ultrastructural evidence of lysosomes and their phosphatase reactivity in normal and atrophic muscle is contradictory. The normal turnover rate of muscle protein is quite rapid,'7"18 and would seem to require large numbers of lysosomes if acid proteases of lysosomes mediate this protein degradation. However, lysosomes have been described as rare in normal muscle 19,20,21 and the present observations confirm this. In various atrophic situations where protein turnover is enhanced,'7"8'22 the number of lysosomes has been seen to increase.'25 In the diabetic and 2-day denervated rats an increase in secondary lysosomes was noted between muscle fibrils. These lysosomes apparently derived from the fusion of acid phosphatase reactive vesicles or cisternae with mitochondria. Christie and Stoward 25 outlined a process whereby muscle mitochondria in dystrophic muscle became encircled by acid-phosphatase-laden tubules considered sarcoplasmic reticulum. This envelopment apparently led to conversion of mitochondria to acid-phosphatase-positive secondary lysosomes in an autophagic sequence. A similar conversion appears to occur in denervated and diabetic muscle and may be a general

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Table 1-Abundance of Sarcoplasmic Precipitates in Muscles Exposed to Control Solutions

Solution a

1 mMPb2NO315min then 10 mM NaH2PO4 10 min

b

1 mMPbNO315min then 100 mM NaH2PO4 10 min

c

Muscle denervated 3 weeks

Contralateral muscle

0-++

0-++

+++

+++

1 mM PbNO3

0

0

d

100mM NaH2PO4 then 1 mM PbNO3

0

0

e

2.5mM PbNO3 15min then 11.0 mM jB-glycero-PO4 30 min

+

±

f

0.01 M NaF in the solutions in e

+

±

* Different muscle areas varied from showing randomly distributed fine precipitates or sparse, widely separated, coarse deposits to lacking precipitates.

method of disposing of mitochondria in catabolic states. Presumably, these enveloping cisternae or vesicles, or both, fuse with the outer mitochondrial membrane, admitting hydrolases into the mitochondrion. The above mechanism is distinct from that usually envisioned, wherein enzyme is transported only by Golgi-derived vesicles. The stained cisternae around mitochondria in dystrophic muscle have been interpreted as modified elements of sarcoplasmic reticulum.Y Since the lateral sacs contain acid phosphatase, the cisternae could represent extensions of these structures. However, the morphologic features and staining of the cisternae enveloping mitochondria in diabetic muscles resemble more closely the single reactive profile of T tubules rather than the double profile of lateral sacs (Figure 13). Thus, the T tubule may participate in mitochondrial turnover. Decreased numbers 226,27 as well as decreased size 28 of mitochondria have been noted in denervated muscle. Mitochondrial destruction has been reported as early as 2 days after denervation,26 an observation that parallels the appearance of acid-phosphatase-positive mitochondria in some animals in the present study. The lipid deposits noted at this time could be attributed to degradation of mitochondrial membranes. In normal animals the lateral sacs of the sarcoplasmic reticulum are generally the most acid phosphatase reactive site of the sarcomere.

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Adenosine triphosphatase 29 and acid phosphatase 20 have been demonstrated previously in this site. Whether acid phosphatase is produced in the granular reticulum and transported with or without intervention of Golgi vesicles to the lateral sacs or is synthesized by the lateral sacs remains a question. However, ribosomes requisite to the production of protein have not been demonstrated bound to lateral sacs. T tubule staining was often accompanied by sarcolemmal reactivity and was more frequently observed in areas adjacent to the sarcolemma. Acid phosphatase occurred in the T tubules in only a minority of the specimens; it was infrequently seen in control muscles and appeared more consistently in muscles undergoing catabolism, eg, after denervation and in conjunction with diabetes. The significance of this finding needs further elucidation. It has been suggested that acid phosphatase from other cells may be internalized by pinocytotic vesicles into lysosomes of cultured fibroblasts.30 Since the T tubules are in direct contact with the external environment of the muscle cell, an external source of acid phosphatase cannot be ruled out, although no connective-tissue macrophages were observed in this study. The precipitates that were distributed throughout the sarcoplasm appeared to reflect acid phosphatase activity, although diffuse precipitates have been produced in this site nonenzymatically by sequential exposure to lead salts and orthophosphate.'6 The enzymatic basis for sarcoplasmic deposits was indicated by their sparsity in control animals and considerable increase in denervated muscles at three weeks. Moreover, nonenzymatic precipitates produced by the sequential treatment with lead and orthophosphate equaled in abundance the presumed acid phosphatase deposits formed during incubation in complete substrate medium only when the phosphate solution in the sequence contained 10 times more NaH2PO4 than was present as glycerophosphate in the substrate medium. Furthermore, the control employing the sequential exposure to lead solutions showed random distribution of precipitates, compared with the concentration in H and I bands in the acid phosphatase preparation. The finding that a sequential exposure to PbNO3 and glycerophosphate yielded essentially no precipitates with or without NaF in the solution more significantly attests to the enzymatic basis for sarcoplasmic precipitates formed from incubation in complete medium, since Pb and glycerophosphate would be the expected precipitating partners producing nonenzymatic precipitation in the complete substrate medium. To explain the absence of precipitates in NaF or heat-inactivated sections in terms other than enzyme inhibition would require that these inhibitors prevent binding of nonenzymatic Pb-glycerophosphate precipitates. The inhibitors were clearly

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not acting in this capacity, since treatment with Pb followed by glycerophosphate failed to produce precipitates. The biologic significance of sarcoplasmic acid phosphatase remains undetermined, but its greater prevalence in 3-week-denervated muscles would indicate a relationship to a state of predominating catabolic activity. The abundance of precipitates in the sarcoplasm appeared to vary inversely with the intensity of staining in the lateral sacs, suggesting leakage of enzymes from the sacs as a source of the sarcoplasmic reactivity. Acid phosphatase reactivity seen in junctional folds of a motor end plate of a denervated muscle indicates that the enzyme is involved in alteration of this structure after denervation. The Schwann cell or axon may be the source of the enzyme in the junctional folds, as suggested by the staining of the Golgi apparatus in its cytoplasm. References 1. Goldberg AL, Dice JF: Intracellular protein degradation in mammalian and bacterial cells. Annu Rev Biochem 1974, 43:835-869 2. Goldberg AL, St. John AC: Intracellular protein degradation in mammalian and bacterial cells: Part 2. Annu Rev Biochem 1976, 45:747-803 3. Cahill GF, Aoki TT, Marliss EB: Insulin and muscle protein, Handbook of Physiology. Section 7, Endocrinology. Vol 1, Endocrine Pancreas. Edited by DF Steiner, N Freinkel. American Physiological Society, Washington, DC, 1972, pp 563-577 4. Felig P: Amino acid metabolism in man. Annu Rev Biochem 1975, 44:933-955 5. Jefferson LS, Rannels DE, Munger BL, Morgan HE: Insulin in the regulation of protein turnover in heart and skeletal muscle. Fed Proc 1974, 33:1098-1104 6. Fulks RM, Li JB, Goldberg AL: Effects of insulin, glucose, and amino acids on protein turnover in rat diaphragm. J Biol Chem 1975, 250:290-298 7. Jefferson LS, Li JB, Rannels SR: Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus. J Biol Chem 1977, 252:1476-1483 8. Goldspink DF: The effects of denervation on protein turnover of rat skeletal muscle. Biochem J 1976, 156:71-80 9. Bird JWC: Skeletal muscle lysosomes, Lysosomes in Biology and Pathology. Vol 4. Edited by JT Dingle, RT Dean. New York, American Elsevier, 1975, pp 75-109 10. Rannels DE, Kao R, Morgan HE: Effect of insulin on protein turnover in heart muscle. J Biol Chem 1975, 250:1694-1701 11. Barrett AJ (ed): Proteinases in Mammalian Cells and Tissues. Amsterdam, North Holland Publishing Co, 1977 12. Busch WA, Stromer MH, Goll DE, Suzuki AJ: Ca2"-specific removal of Z lines from rabbit skeletal muscle. J Cell Biol 1972, 52:367-381 13. Mayer M, Amin R, Shafrir E: Rat myofibrillar protease: Enzyme properties and adaptive changes in conditions of muscle protein degradation. Arch Biochem Biophys 1974, 161:20-25 14. Crouch R, Kimsey G, Priest DG, Sarda A, Buse MG: Effect of streptozotocin on erythrocyte and retinal superoxide dismutase. Diabetologia 1978, 15:53-57 15. Barka T, Anderson PJ: Histochemical methods for acid phosphatase using hexazonium Pararosanilin as coupler. J Histochem Cytochem 1962, 10:741-753 16. Gillis JM, Page SG: Localization of ATPase activity in striated muscle and probable sources of artifact. J Cell Sci 1967, 2:113-118

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17. Millward DJ: Protein turnover in skeletal muscle: I. The measurement of rates of synthesis and catabolism of skeletal muscle protein using ["C] Na2CO3 to label protein. Clin Sci 1970, 39:577-590 18. Millward DJ: Protein turnover in skeletal muscle: II. The effect of starvation and a protein-free diet on the synthesis and catabolism of skeletal muscle proteins in comparison to liver. Clin Sci 1970, 39:591-603 19. Gordon GB, Price HM, Blumberg JM: Electron microscopic localization of phosphatase activities within striated muscle fibers. Lab Invest 1967, 16:422-435 20. Hudgson P, Pearce GW: Ultramicroscopic studies of diseased muscle, Disorders of Voluntary Muscle. Second edition. Edited by JN Walton. Boston, Little, Brown, 1969, pp 279-317 21. Canonico PG, Bird JWC: Lysosomes in skeletal muscle tissue: Zonal centrifugation evidence for multiple cellular sources. J Cell Biol 1970, 45:321-333 22. Goldberg AL: Protein turnover in skeletal muscle: II. Effects of denervation and cortisone on protein catabolism in skeletal muscle. J Biol Chem 1969, 244:3223-3229 23. Pellegrino C, Franzini C: An electron microscope study of denervation atrophy in red and white skeletal muscle fibers. J Cell Biol 1963, 17:327-349 24. Schiaffino S, Hanzlikova V: Studies on the effect of denervation in developing muscle: II. The lysosomal system. J Ultrastruct Res 1972, 39:1-14 25. Christie KN, Stoward PJ: A cytochemical study of acid phosphatase in dystrophic hamster muscle. J Ultrastruct Res 1977, 58:219-234 26. Manolov S, Ovtscharoff W: Ultrastructural changes in the muscle cells of denervated muscles of rat. Z Mikrosk-Anat Forsch 1974, 88:726-744 27. Gauthier GF, Dunn RA: Ultrastructural and cytochemical features of mammalian skeletal muscle fibers following denervation. J Cell Sci 1973, 12:525-547 28. Miledi R, Slater CR: Some mitochondrial changes in denervated muscle. J Cell Sci 1968, 3:49-54 29. Sommer JR, Spach MS: Electron microscopic demonstration of adenosinetriphosphatase in myofibrils and sarcoplasmic membrane of cardiac muscle of normal and abnormal dogs. Am J Pathol 1964, 44:491-505 30. Hickman S, Neufeld EF: A hypothesis for I-cell disease: Defective hydrolases that do not enter lysosomes. Biochem Biophys Res Commun 1972, 49:992-999

Acknowledgments The authors gratefully acknowledge the technical assistance of Betty Hall, Jane Farrington, and Denie Peeler Ravenel; the editorial assistance of Fran Cameron, and the secretarial assistance of Dot

Smith.

Figures 1-19 illustrate rat muscle processed for acid phosphatase cytochemistry with ,B-glycerophosphate as substrate, except that cytidine monophosphate was employed as substrate for the muscle in Figure 1-Soleus muscle denervated 2 days. Intensely reactive bodies interpreted as Figure 11. lysosomes lie at the pole of a nucleus. Lateral sacs of the sarcoplasmic reticulum (arrow) show deposits indicative of acid phosphatase. Control muscles also show such lysosomal and lateral sac reactivity. Faint staining of mitochondria equals that in cytochemical controls and reflects affinity for lead. The sarcoplasm lacks deposits. (x1 2,500) Figure 2-Two-day denervated soleus muscle. A structure considered, from its morphologic features and location, to be one of several mitochondria aligned longitudinally in the sarcomere shows acid phosphatase reaction product. The staining suggests conversion to a secondary lysosome in a process of mitochondrial autophagy. Figure 3-Soleus muscle 2 days after denervation. Very dense bodies suggestive of (x22,500) lysosomes border mitochondria. Such numerous lysosomes were not observed in control muscles. Figure 4-Gastrocnemius muscle 2 days after denervation. Dense bodies lie beside (x1 2,500) and within the matrix of mitochondria. These structures were equally dense in cytochemical control preparations and presumably consist of osmiophilic lipid. (Counterstained with uranyl acetate and lead citrate, x 1 5,000)

Figure 5-Gastrocnemius muscle 7 days after nerve resection. Large lysosomes around the nucleus and elements of the Golgi apparatus (G) show reactivity. Acid phosphatase tubules or vesicles neighbor a large mitochondria lying next to another profile that appears to represent a degenerating mitochodrion (arrow) with acid phosphatase activity. The lateral sacs of the sarcoplasmic reticulum and the intervening T tubules also show evidence of enzyme activity, as does the outer surface of the sarcolemma. (x 15,000) Figure 6-A soleus muscle 10 days after nerve resection. Reaction product of acid phosphatase is present on the sarcolemma and in T tubules (arrows) and, to a lesser extent, the lateral sacs. The sacroplasm also contains diffusely distributed fine deposits and lipid (L) droplets. (x 1 2,500) Figure 7-Soleus muscle 3 weeks after denervation. Moderately heavy precipitates throughout the sarcoplasm are concentrated in I bands. Lateral sacs (arrow) show minimal reaction product, as appeared to be the case generally in muscles with abundant precipitates in the sarcoplasm. (x18,750)

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Figure 8-A motor end plate of a soleus muscle 7 days after resection shows heavy reactivity on the outer face of the plasmalemma of the junctional folds. Deposits indicative of acid phosphatase reactivity are also seen in the Golgi lamellas and vesicles of the axon or Schwann cell (arrows). (x1 3,750)

Figure 9-A gastrocnemius muscle motor end plate 3 weeks after nerve resection shows no evidence of cytidine monophosphatase reactivity, although lysosomes are heavily stained.

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Figure 10-A motor end plate of a gastrocnemius muscle from the leg opposite a denervated leg. The junctional folds show no acid phosphatase staining, although lysosomes in the sarcoplasm and in a fibroblast process (arrow) are positive. (X1 3,125) Figure 11-In a gastronemius muscle from a control animal untreated and unoperated upon, acid phosphatase is localized in the lateral sacs of the sarcoplasmic reticulum. The sarcoplasm lacks precipitates, and mitochondria possess only the fine nonenzymatic deposits of lead observed in cytochemical controls. (x22,500)

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Figure 12-A quadriceps muscle from a diabetic rat. A mitochondrion with moderate acid phosphatase reactivity lies to the left of one that contains light deposits and is partially enveloped by a stained cisterna reminiscent (long arrow) of a T tubule (short arrow). The reactivity of T tubules at the bottom of the field exceeds that of bordering lateral sacs. (x30,000)

Figure 13-This area from a quadriceps muscle of a diabetic rat shows a mitochondrion in a late stage of degeneration, as indicated by heavy acid phosphatase activity. Staining in the T tubule at the left exceeds that in the bordering lateral sacs. (x30,000) Figure 14-A mitochondrion in a quadriceps muscle of a diabetic rat is partially surrounded by a reactive cisterna and vesicles but has little internal reactivity. (x30,000) Figure 15-A mitochondrion from a gastrocnemius muscle of a diabetic rat is surrounded by acid phosphatase reactivity and appears to have some internal reaction product. (x30,000)

Figure 16-A mitochondrion from a quadriceps muscle of a diabetic rat displays heavy internal deposits of reaction product. (x30,000) Figure 17-This mitochondrion from a quadriceps muscle of a diabetic rat has diffuse enzymatic deposits and appears to be in an advanced stage of autophagic digestion. (X40,000)

Figure 18-A mitochondrion from a gastrocnemius muscle of a diabetic rat shows a myelin-like arrangement of acid-phosphatase-positive material at one end. (x30,000) Figure 19-An area from a quadriceps muscle of a diabetic rat shows extensive mitochondrial degeneration and localization of enzyme in the surrounding sarcoplasmic reticulum. (x 18,750)

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