Cytochemical observations of two distinct acid phosphatase-reactive structures in anterior latissimus dorsi muscle of the chicken

Synopsis Acid phosphatase activity has been localized cytochemically in the avian anterior latissimus dorsi muscle in two distinct structures, the sarcoplasmic reticulum and membrane-limited dense bodies. Cross and longitudinal sections confirmed that the reaction product was membrane-bound. Acid phosphatase activity was observed in the longitudinal tubules of the sarcoplasmic reticulum in the region of the I band and in dense bodies located along the length of the fibre just beneath the sarcolemma and between the myofibrils. This dual localization is discussed in relationship to previous cytochemical and zonal centrifugation evidence for more than one acid phosphatase-containing organelle in skeletal muscle.     

Cytochemical studies on peroxisomes in rat and guinea pig myocardium

The enzymatic activity and distribution of peroxisomes (microbodies) in rat and guinea pig hearts were studied cytochemically, by means of oxidation of 3-3'-diaminobenzidine (DAB) and by using B-glycerophosphate and cytidine-5'-monophosphate as substrates. Peroxisomes were localized in proximity to mitochondria and sarcoplasmic reticulum and measured from 0.2 micrometers to 0.5 micrometers in diameter in both animal species. DAB positive bodies were seen both at pH 9.0 and pH 5.0 in rat myocardial cells. However, in guinea pig myocardial cells the reaction was observed only at pH 9.0, or very faintly at pH 5.0. Acid and alkaline phosphatases were not demonstrated in the peroxisomes. Lipid droplets were surrounded by a ring of dense granular reaction product for enzymes, such as acid and alkaline phosphatase, and lipofuscin granules were limited by acid phosphatase or DAB reaction products. The pathophysiological function of peroxisomes is discussed.     

Distribution of Acid Phosphatase in Paramecium caudatum: Its Relations with the Process of Digestion

SYNOPSIS. The distribution of acid phosphatase was investigated at the ultrastructural level in Paramecium caudatum. Acid phosphatase occurs in endoplasmic reticulum, Golgi apparatus, food vacuoles, autophagic vesicles, vacuolar and dense bodies. Some slight deposits are also seen in the mitochondria.
These observations point out that this hydrolase activity is related to digestive processes. The enzyme, originating from the endoplasmic reticulum and Golgi apparatus reaches the food vacuole or autophagic vesicle likely via the reticulum. The digestion of the bacteria or of the enclosed organelle gives rise to electronopaque material which is later found in dense bodies. These dense bodies are likely secondary lysosomes and it is possible that they may fuse with the young food vacuole or with autophagic vesicles.     

Effects of colchicine on myofilament arrangement and the lysosomal system in skeletal muscle

Summary Colchicine was intraperitoneally administered chronically to adult male Sprague-Dawley rats. The ultrastructural study of hind-limb muscles revealed that myofilament desorientation resulted. Bundles of myofilaments were found coursing perpendicular or oblique to the longitudinal axis of the muscle fiber. It is concluded that a colchicine-sensitive factor is involved in maintaining normal orientation of myofibrils in mature muscle. Also found in the sarcoplasm of the colchicine treated animals were complex spheromembranous bodies. These bodies enveloped mitochondria or other organelles and appeared to be derived from the sarcoplasmic reticulum. The lysosomal nature of these bodies is indicated by the localization of acid phosphatase activity in them. Acid phosphatase activity was also displayed in the sarcoplasmic reticulum. The spheromembranous bodies seem to be part of a sarcotubulo-lysosomal system in skeletal muscle.This study was supported in part by N.I.H. Grant RR-5576.The author gratefully acknowledges the technical assistance of Mrs. Patricia Driscoll.     

Ultrastructural Localization of Acid Phosphatase in Starved Tokophrya infusionum*

SYNOPSIS. Young organisms of Tokophrya infusionum starved for several hr, are best suited for a study of the fine structure of this organism including the distribution of its organelles. Acid phosphatase was localized by a combined electron microscopy and cytochemical approach using modified Gomori methods. The enzyme was found in small dense bodies, spheroid vesicles, missile-like bodies, rough-surfaced endoplasmic reticulum, residue and autophagic vacuoles. The small dense bodies are thought to be primary lysosomes since electron micrographs show a) a continuity between the membrane of the rough-surfaced endoplasmic reticulum and that of the dense bodies and b) a connection between the contents of both structures when the dense bodies form from the endoplasmic reticulum.     

Intracellular enzymes and their localization in slender and stumpy forms of Trypanosoma brucei rhodesiense

Acid phosphatase and cathepsin D activity is greater in stumpy than in slender forms, especially when estimations are made on a ‘light lysosomal’ fraction. Acid phosphatase is localized in the region of the flagellar pocket in slender forms but is present in lysosomes, phagolysosomes, autophagosomes and rough endoplasmic reticulum of stumpy forms. In stumpy forms dense bodies develop the macrocrystalline structure of peroxisomes. Intense enzyme activity in stumpy forms appears to be associated with absorption of lipid from the plasma by a mechanism similar to but of greater intensity than that described in atheromatous rabbit aorta.     

Degeneration and regeneration in denervated tonic and phasic skeletal muscle: Morphology and acid phosphatase cytochemistry

Following denervation, ultrastructural alterations were observed in the tonic, anterior (ALD) and phasic posterior (PLD) latissimus dorsi muscles of the chicken. In the ALD muscle these changes were characteristic of both degeneration and regeneration, while in the PLD muscle, the changes were characteristic only of degeneration. Acid phosphatase positive structures, which included dense bodies in the ALD and PLD as well as T-tubules in the PLD, were observed intact with no evidence of release of enzyme into the sarcoplasm. No evidence of an increase in the number of autophagic vacuoles was found. The morphological evidence presented in this communication does not support the hypothesis that lysosomes are involved in denervation atrophy through autophagy of muscle cell constituents.     

Localization of acid phosphatase in lamellar bodies of tannic acid treated alveolar type II cells

Summary Acid phosphatase was demonstrated in well preserved lamellar bodies of rats' alveolar type II cells. The highly ordered lamellar organization was preserved by using tannic acid in the tissue procession protocol. Acid phosphatase reaction products were observed in the amorphous regions of the lamellar bodies adjacent to the limiting membranes and in the central core regions. No reaction product was observed in the lamellar areas. 85%±5% of the lamellar bodies were positively reactive, unrelated to their size. Multivesicular bodies were only partially reactive (approx. 50%), except for those attached to lamellar bodies which all had reaction product.     

Localization of acid phosphatase in lamellar bodies of tannic acid treated alveolar type II cells

Acid phosphatase was demonstrated in well preserved lamellar bodies of rats' alveolar type II cells. The highly ordered lamellar organization was preserved by using tannic acid in the tissue procession protocol. Acid phosphatase reaction products were observed in the amorphous regions of the lamellar bodies adjacent to the limiting membranes and in the central core regions. No reaction product was observed in the lamellar areas. 85% +/- 5% of the lamellar bodies were positively reactive, unrelated to their size. Multivesicular bodies were only partially reactive (approx. 50%), except for those attached to lamellar bodies which all had reaction product.     

Cytochemical detection of phosphatase and arylsulphatase activities in the midgut of Centropages typicus (Copepod, Calanoid)

Ultrastructural study of the midgut of Calanoid Copepods revealed the presence of several cell types in all species. In a previous report we described and assigned a function to each of these cell types. In order to affirm the validity of those assignments we undertook an investigation of enzymatic activity especially of phosphatase and arylsulphatase. By cytochemical methods, alkaline phosphatase activity was detected in R-, R'-D- and B-cells, with labelling being observed on the apical plasmic membrane level in all four, and in B-cells on the pinocytotic vesicle membranes. Acid phosphatase and aryl-sulphatase activities were only detectable in B-cells; the most frequently labelled structures were located in the vacuolar system, dictyosomes and Golgi vesicles, although Golgi structures occasionally reacted to acid phosphatase. Nome of the dense bodies observed in B-cells reacted to arylsulphatase. Similarly they were unevenly labelled during acid phosphatase tests. Hence it may be assumed that dense bodies are not involved in hydrolases. It is possible that these enzymes originated from vesicles generated by the Golgi saccules surrounding and joined to the vacuoles, thus bypassing the lysosome I stage.     

A phospholamban protein phosphatase activity associated with cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic phospholamban protein phosphatase activity, which is also effective in dephosphorylating phosphorylase a. The phosphatase associated with sarcoplasmic reticulum membranes was solubilized with Triton X-100 and subjected to chromatography on Mono Q HR 5/5 and polylysine-agarose. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. Thermal denaturation of the enzyme resulted in progressive and coincident loss of both phospholamban and phosphorylase a phosphatase activities. Enzymic activity was partially inhibited by protein phosphatase inhibitor 1. Migration of the enzyme during sucrose density gradient ultracentrifugation corresponded to a globular protein with an apparent Mr of 46,000. This enzyme preparation could dephosphorylate both the calcium-calmodulin-dependent as well as the cAMP-dependent sites on phospholamban. Thus, dephosphorylation of phospholamban by this sarcoplasmic reticulum-associated phosphatase may participate in modulating sarcoplasmic reticulum function in cardiac muscle.     

Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites

To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.     

Histochemical study of calcium on T-tubule membranes and in the sarcoplasmic reticulum, in frog twitch muscle fibres at rest and during activity

The trigger calcium hypothesis of signal transmission between T-tubules and terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in twitch muscle fibres implies the presence of calcium along T-tubule membranes at rest and its release upon excitation. To test this hypothesis, calcium was immobilised using a fixing and precipitating solution of glutaraldehyde in phosphate buffer at pH 8.0 and the calcium was substituted for by lead. Simultaneous tension recordings revealed the occurrence of contractions or a burst of twitches upon perfusion with the fixative. Procaine or tetrodotoxin (TTX) was used to inhibit this activity. In fibres without fixative-induced activity, precipitates were observed along T-tubules and in adjoining parts of TC. In activated fibres, tubular and TC precipitates were absent. These results are consistent with the trigger calcium hypothesis. In fibres activated by depolarisation, calcium returned to TC after passing successively through different parts of the SR.     

Histochemical study of calcium on T-tubule membranes and in the sarcoplasmic reticulum,in frog twitch muscle fibres at rest and during activity

Summary The trigger calcium hypothesis of signal transmission between T-tubules and terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in twitch muscle fibres implies the presence of calcium along T-tubule membranes at rest and its release upon excitation. To test this hypothesis, calcium was immobilised using a fixing and precipitating solution of glutaraldehyde in phosphate buffer at pH 8.0 and the calcium was substituted for by lead. Simultancous tension recordings revealed the occurence of contra tions or a burst of twitches upon perfusion with the tixative. Procaine or tetrodotoxin (TTX) was used to inhibit this activity. In fibres without fixative-induced activity, precipitates were observed along T-tubules and in adjoining parts of TC. In activated fibres, tubular and TC precipitates were absent. These results are consistent with the trigger calcium hypothesis. In fibres activated by depolarisation, calcium returned to TC after passing successively through different parts of the SR.     

DISTRIBUTION OF PHOSPHATASES IN THE GOLGI REGION AND ASSOCIATED STRUCTURES OF THE THORACIC GANGLIONIC NEURONS IN THE GRASSHOPPER, MELANOPLUS DIFFERENTIALIS

The neuronal perikarya of the grasshopper contain sudanophilic lipochondria which exhibit an affinity for vital dyes. These lipochondria are membrane-delimited and display acid phosphatase activity; hence they correspond to lysosomes. Unlike those of most vertebrates, these lysosomes also hydrolyze thiamine pyrophosphate and adenosine triphosphate. Like vertebrate lysosomal "dense bodies," they are electron-opaque and contain granular, vesicular, or lamellar material. Along with several types of smaller dense bodies, they are found in close spatial association with the Golgi apparatus. The Golgi complexes are frequently arranged in concentric configurations within which these dense bodies lie. Some of the smaller dense bodies often lie close to or in association with the periphery of dense multivesicular bodies. Further, bodies occur that display gradations in structure between these multivesicular bodies and the dense lysosomes. Acid phosphatase activity is present in the small as well as the larger dense bodies, in the multivesicular bodies, and in some of the Golgi saccules, associated vesicles, and fenestrated membranes; thiamine pyrophosphatase is found in both the dense bodies and parts of the Golgi complex. The close spatial association of these organelles, together with their enzymatic similarities, suggests the existence of a functional or developmental relationship between them.     

Demonstration of acid phosphatase-containing vacuoles in hyphal tip cells of Sclerotium rolfsii.

A lysosomal system was demonstrated in hyphal tip cells of Sclerotium rolfsii by light and electron microscopy observations of the sites of acid phosphatase activity visualized by a modified Gomori lead nitrate method. The cytochemical reaction product was found to be present in numerous vacuoles, each aout 0.5 mum in diameter, which were seen as chains of spheres when viewed with the light microscope. They usually did not occur in the first 30 to 40 mum of the hyphal tip cell, but were concentrated in a zone extending from 30 to 200 mum from the hyphal apex. As shown by the electron microscope, the vacuoles were sometimes interconnected by narrow channels. Acid phosphatase reaction product was also occasionally localized in vacuoles of the older hyphal cells, but never in apical vesicles, lipid bodies, or microbodies. It is proposed that this vacuolar system may orginate from the endoplasmic reticulum.     

The fine structural localization of testicular phosphatases in man: The control testis

Summary Electron microscopic cytochemistry was used to determine the localization of five phosphatase enzymes—glucose-6-phosphatase, inosine diphosphatase, thiamine pyrophosphatase, acid phosphatase, and adenosine triphosphatase—in control human testes. Glucose-6-phosphatase occurred in the endoplasmic reticulum and nuclear envelope of Sertoli cells, Leydig cells and primitive spermatogonia, but was not observed in more advanced spermatogenic cells. The presence of glucose-6-phosphatase activity paralleled the presence of glycogen in spermatogenic cells, i.e., both occurred in type AL and AD spermatogonia but not in type AP or B spermatogonia or in more advanced spermatogenic cells. Inosine diphosphatase activity was found in the endoplasmic reticulum, nuclear envelope, and Golgi complex of Sertoli cells and all spermatogenic cells except late spermatids. Additionally, inosine diphosphatase activity was localized at the junctions between Sertoli cells and late spermatids, but was not associated with any other plasma membrane. Thiamine pyrophosphatase reaction product was found in the Golgi bodies of Sertoli cells and in spermatogenic cells through immature spermatids. Neither inosine diphosphatase nor thiamine pyrophosphatase was observed in the Golgi bodies of spermatids during acrosomal formation. Acid phosphatase activity was found in lysosomes of spermatogonia, spermatocytes, and spermatids, in lysosomes of Leydig cells, and in lysosomes, lipofuscin bodies, and Golgi cisternae of Sertoli cells. It is thought that Sertoli lysosomes play a role in the phagocytosis of degenerating germ cells; however, the role of spermatogenic or Leydig lysosomes is unknown. Adenosine triphosphatase activity occurred at the interfaces between two spermatogonia, and between Sertoli cells and spermatogonia, but was not observed in the spaces between two Sertoli cells, two spermatocytes, two spermatids, or between Sertoli cells and spermatocytes, or between Sertoli cells and spermatids.Supported in part by a grant from the U.S. Atomic Energy Commission (AT-(40-1)-4002).     

Cytochemical demonstration of amine-storing vacuoles and lysosomes in the chicken thrombocytes

A combined electron microscopic and cytochemical study of the thrombocytes of the chicken has clearly identified the amine-storing organelles and lysosomes. A chromaffin positive-reaction product was observed on the inner surface and the granules of the large electron-lucent vacuoles. No acid phosphatase activity was localized in these amine-storing vacuoles. However, the acid phosphatase activity was observed in the small vesicles, the primary lysosomes, and in the large electron dense inclusions with myelin which may be secondary lysosomes. The results of this study suggest that the large empty vacuoles, with one or two very dense osmiophilic peripherally-situated granules, in the chicken thrombocytes are comparable to the vesicles with electron dense materials called "dense bodies" in mammalian thrombocytes.     

The organization of a tunicate heart

Cardiac myoepithelial cells contain three myofibrils adjacent to the lumen. Zonula adhaerentes connect myofibrillar ends; desomosomes and peg-in-socket connections occur laterally with zonulae occludenfes at the pericardial border. Conduction and contraction are vertebrate cardiac muscle type. T-tubules are absent ; atypical sarcoplasmic vesicles run between myofibrils and beneath the sarcoplasmic membrane. Multivesiculate vacuoles, on cytoplasmic lobes protruding into the lumen from sarcomeric positions, induce reversal of heartbeat by a secretory feedback mechanism. Auto-UV-fluorescent dense bodies aooarentlv assemble thick and thin filaments. Both pericardial cavity borders are pinocytotic. Exaggerated interdigitation of adjacent plasma membranes and thick basement membrane of pericardial cells contribute mechanically to heart action.     

Ultrastructural localization of lysosomal enzymes in the egg cortex of Brachydanio

The localization of acid phosphatase (E.C. 3.1.3.2), inorganic trimetaphosphatase (E.C. 3.6.1.2), and aryl sulfatase (E.C. 3.1.6.1) in the cortex of unactivated and activated eggs of Brachydanio was examined by ultrastructural cytochemistry. Using a lead capture method, activity for all three acid hydrolases was demonstrated in organelles of the cortex before and after egg activation. Acid phosphatase (AcPase) reaction product was consistently present in primary lysosomes, secondary lysosomes, multivesicular bodies, and yolk bodies. AcPase activity was absent from mitochondria, profiles of the endoplasmic reticulum, coated pits of exocytosed cortical granules, and coated vesicles. Although most cortical granules of the mature, unactivated egg were unreactive for this enzyme, a few showed AcPase reaction product. It is not clear whether the AcPase-positive granules might be an immature form of cortical granules or a subpopulation of these organelles with lysosomal properties. Most cisternae of the Golgi apparatus did not stain for AcPase; however, reaction product was occasionally localized in a single cisterna as well as several small vesicles at the inner face of the Golgi. The intensity of the reaction product and the pattern of distribution of trimetaphosphatase (Tm-Pase) activity was very similar to that of AcPase. However, TmPase was never observed in cortical granules. Cortices of unactivated and activated eggs showed less overall aryl sulfatase (ArSase) activity when compared with AcPase and TmPase. The presence of ArSase reaction product in lysosomes and multivesicular bodies confirmed the acid hydrolytic nature of these organelles. AcPase and TmPase, and to a lesser extent ArSase, are adequate markers of a cortical lysosomal system in the danio egg.(ABSTRACT TRUNCATED AT 250 WORDS)