Electron microscopic cytochemical localization of nucleoside phosphatases in normal and chronic granulocytic leukaemic human neutrophils

Summary Using electron microscope cytochemistry and cells separated on Ficoll-Hypaque, Mg²⁺-dependent ATPase, ADPase and 5-nucleotidase were predominantly localized as ectoenzymes on normal human granulocytes. Large deposits of ATPase final reaction product and more finely granular deposits of 5-nucleotidase final reaction product were firmly attached to the outer surface of cell plasma membranes. The final reaction product from ecto-ADPase was, however, only loosely associated with the plasma membrane. In addition, finer deposits of ADPase final reaction product were seen in specific granules and in background cytoplasm. No nucleotidase phosphatase activity was localized to the alkaline phosphatase-containing granules (phosphasomes) recently described by Rustinet al.In granulocytes from patients with chronic granulocytic leukaemia, ecto-ATPase had a patchy distribution on the plasma membranes. There was considerable heterogeneity between cells with regard to ADPase and 5-nucleotidase localization. In some cells, ADPase was seen only as an ectoenzyme and in a few it was present in specific granules, but in others it was seen at both sites, while in some cells no activity was detected. 5-Nucleotidase localization was normal in some cells but lacking from many. No correlation was found between enzyme heterogeneity and the degree of morphological cell maturity.     

Cytochemical markers of bladder carcinogenesis

Summary Enzyme cytochemical and immunocytochemical techniques at the light and electron microscope levels were used to study the distribution of potential markers of chemical transformation in rodent bladders. In rat tumours induced byin vivo treatment with methylnitrosourea, alkaline phosphatase localization was normal on the external surface of the plasma membranes of some cells but abnormal in others where reaction product was seen only on intracellular membranes. 5-Nucleotidase localization was abnormal in all cells, being seen on endoplasmic reticulum and nuclear membranes only, while in normal bladders only ectoenzyme localization was seen. Heterogeneity of alkaline phosphatase and 5-nucleotidase localization was seen on the plasma membranes of these tumours after 15 days in organ culture. Some cells produced enzyme and others did not; in other cells only parts of the membrane reacted heavily, while other regions were negative.In transformed cell cultures and tumours of mouse bladder derived byin vitro treatment of explants with dimethylbenz (a) anthracene, a bimodal pattern of alkaline phosphatase localization was seen. Cells had either normal ectoenzyme reaction product or abnormal intracellular membrane reaction product. 5-Nucleotidase and ADPase were lost after transformation while cAMP-phosphodiesterase was retained as an ectoenzyme. Mg.ATPase and a cAMP-independent, calcium-insensitive protein phosphatase were induced in transformed cell cultures. An epithelial antigen was detected in the cytoplasm of both normal and transformed cells associated with reticular cytoplasmic ground substance, plasma membrane vesicles and cytoskeletal elements.     

Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes

Adenosine diphosphatase (ADPase) activities were studied in human polymorphonuclear leukocytes with a recently developed radio-assay. The neutrophils were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for ADPase activity and for principal organelle marker enzymes. ADPase activity was distributed between the plasma membrane, specific granule and soluble fractions. The plasma membrane and specific granule activities had similar kinetic and inhibitor properties but the cytosolic enzyme was clearly different. Studies with the non-penetrating inhibitor diazotized sulphanilic acid and measurements of latent activity indicate that plasma membrane ADPase activity is located on the external aspect to the cell. Its possible role in inhibiting platelet aggregation is discussed. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mU/mg protein) of ADPase activity, in contrast to those of alkaline phosphatase, were similar in all three groups. This result, together with fractionation experiments and inhibition studies strongly suggests that ADPase activity is not attributable to neutrophil alkaline phosphatase.     

Subcellular localization and properties of pyridoxal phosphate phosphatases of human polymorphonuclear leukocytes and their relationship to acid and alkaline phosphatase

Using a novel fluorimetric assay for pyridoxal phosphate phosphatase, human polymorphonuclear leucocytes were found to exhibit both acid an alkaline activities. The neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrigfugation. The alkaline pyridoxal phosphate phosphatase showed a very similar distribution to alkaline phosphatase an was located solely to the phosphasome granules. Fractionation experiments on neutrophils treated with isotonic sucrose containing digitonin and inhibitor studies with diazotised sulphanilic acid and levamisole further confirmed that both enzyme activities had similar locations and properties. Acid pyridoxal phosphate phosphatase activity was located primarily to the tertiary granule with a partial azurophil distribution. Fractionation studies on neutrophils homogenised in isotonic sucrose containing digitonin and specific inhibitor studies showed that acid pyridoxal phosphate phosphatase and acid phosphatase were not the result of a single enzyme activity, Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (munits/mg protein) of alkaline pyridoxal phosphate phosphatase an alkaline phosphatase varied widely in the three groups and the alterations occurred in a parallel manner. The specific activities of acid pyridoxal phosphate phosphatase and of acid phosphatase were similar in the three groups. These results, together with the fractionation experiments and inhibition studies strongly suggest that pyridoxal phosphate is a physiological substrate for neutrophil alkaline phosphatase.     

Subcellular localization and properties of lipase activities in human polymorphonuclear leukocytes

A fluorimetric assay for lipase activity has been optimized for measurement of the enzyme in human neutrophils. Activity was maximal at acid (4.5) and alkaline (9.5) pH, although there was also a neutral peak of activity at pH 6.5. Neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The gradient fractions were assayed for acid, neutral and alkaline lipase activity and for the principal organelle marker enzymes. Neutral lipase showed a unimodal distribution with an equilibrium density of 1.19 g . cm-3, corresponding to the distribution of particulate leucine aminopeptidase. Acid and alkaline lipase activities showed very similar distribution profiles to each other with both soluble components and a broad peak of particulate activity. The broad modal density of 1.19-1.22 g . cm-3 suggests that acid and alkaline lipase activities could be localised to more than one population of cytoplasmic granule. Fractionation experiments with neutrophils homogenised in sucrose medium containing digitonin confirmed the localisation of neutral lipase and leucine aminopeptidase to the same cytoplasmic granule, and suggested that at least part of the acid lipase activity was localised to the specific granule. No lipase activity could be attributed to the alkaline phosphatase-containing granule. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity of acid, neutral and alkaline lipase, and leucine aminopeptidase, in contrast to that of alkaline phosphatase, were similar in the three patient groups.     

The cytochemical localization of adenyl cyclase activity in rat sublingual gland.

Adenyl cyclase activity in mucous acinar cells and serous demilune cells of the rat sublingual gland was localized cytochemically. After incubation with adenylyl-imidodiphosphate (AMP-PNP) as substrate, deposits of reaction product are found along the cell membranes bordering the secretory surfaces of serous demilune cells. These are the membranes which participate directly in secretion by fusing with the granule membranes. The granule membranes of the demilune cells do not reveal reaction product, but the membranes of the granules which are fused with and become part of the cell membrane do show deposits. Thus, it appears that the cell membranes which fuse with granule membranes during secretion are associated with a high level of adenyl cyclase activity. In support of this, the luminal membranes of the mucous acinar cells which do not fuse with granule membranes during secretion are not associated with detectable amounts of adenyl cyclase activity.     

The ultrastructural localization of adenosinetriphosphatase and alkaline phosphatase activity in eosinophil leukocytes

Summary The previously undescribed localization of reaction products of adenosinetriphosphatase and of alkaline phosphatase in eosinophil leukocytes was demonstrated by cytochemical studies of the rat intestine. Alkaline phosphatase reaction product was found only in minimal amounts on the plasma membrane but was distinct on the nuclear membranes and outer compartment of mitochondria but not on the cristae. The Golgi membranes and the endoplasmic reticulum reacted but less intensely. The specific granules showed no alkaline phosphatase activity.The adenosinetriphosphatase reaction, on the other hand, was found on the plasma membrane, vesicular or tubular profiles of the endoplasmic reticulum and on the matrix of the specific granules. The crystalloid of the granules did not show any reaction.Recipient of a postdoctoral fellowship from the muscular distrophy association of Canada.     

Cytochemical studies on the localization and functional properties of calcium in anterior pituitary cells

Summary The localization of calcium and its functional properties in anterior pituitary cells were studied using a potassium pyroantimonate technique. In all kinds of secretory cells, the precipitates of the calcium-pyroantimonate complex were distributed on the limiting membrane of the secretory granule. They were present also in the cytoplasmic matrix, the mitochondrial matrix, small smooth vesicles, coated vesicles, and in the nuclear euchromatin area. The precipitates were usually seen at the contact region between the limiting membranes of two adjacent secretory granules, or between the granule limiting membrane and the plasma membrane. When the tissues were incubated in the medium containing A23187 (10 M) for 5 min, the deposits on the granule limiting membrane were increased in number and those on the mitochondrial matrix were decreased; the reaction products almost disappeared on the limiting membranes of the secretory granules after membrane fusion following single or multigranular exocytosis induced by A23187-treatment. In addition, small vesicles in the capillary endothelium contained reaction precipitates. Based on these results we propose a hypothetical model for the relationship between the localization of calcium and secretory activity.This study was supported by grants from the Japan Ministry of Education     

Modulation of the beta-adrenergic-response in cultured rat heart cells

Summary Subcellular fractionation studies in resting human neutrophils indicated a bimodal distribution for cytochrome b. A. major peak of cytochrome b co-sedimented with gelatinase under different experimental conditions. This localization was partially overlapped with specific granules (using lysozyme and lactoferrin as specific granule markers), but clearly resolved from azurophilic granules, plasma membrane, mitochondria, as well as from a novel alkaline phosphatase-rich intracellular organelle. A minor localization of cytochrome b was found in fractions enriched in both the plasma membrane marker 5-nucleotidase and alkaline phosphatase. A significant portion of ubiquinone cell content co-fractionated with the gelatinase-containing granules. After phorbol myristate acetate (PMA)-cell stimulation, cytochrome b was mobilized to fractions showing respiratory burst activity and enriched in 5-nucleotidase activity. This mobilization paralleled secretion of gelatinase and lysozyme to the extracellular medium. Furthermore, neutrophil stimulation with fluoride in the absence of cytochalasin B induced release of gelatinase and generation of superoxide anion with only minimal release of lysozyme. Preincubation of cells with the anion channel blocker 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) prevented lysozyme release, but had only a minor effect on the release of gelatinase and did not inhibit the superoxide anion generation elicited by N-formyl-methionyl-leucyl-phenylalanine or PMA. These results suggest a main location of cytochrome b in mobilizable gelatinase-containing granules, which can constitute a subpopulation of specific granules. Furthermore, these findings show that the gelatinase-containing granule is functionally involved in the respiratory burst in neutrophils and that membrane fusion between plasma membrane and the gelatinase-containing granule occurs during activation of cells.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - FMLP N-formyl-methionyl-leucyl-phenylalanine - PMA 4-phorbol, 12-myristate, 13-acetate     

Light microscope and electron microscope alkaline phosphatase cytochemistry of rat bone marrow leukocytes.

An enzyme cytochemical method yielding an osmiophilic reaction product, visible at both the light and electron microscope levels, has been applied to the study of alkaline phosphatase in rat bone marrow cells. The enzyme is present in both eosinophils and, in much smaller amounts, in neutrophils. In both cases it is present on the plasma membrane, and in eosinophils intracellular aggregations of reaction product are also seen. The specific granules in both cell types fail to react and the enzyme is first detectable at the promyelocyte stage. Thus the enzyme is demonstrable before specific granule formation begins in the neutrophil, indicating that they are not a significant site of alkaline phosphatase activity in the rat.     

ASSOCIATION OF THE ALKALINE PHOSPHATASE OF RABBIT POLYMORPHONUCLEAR LEUKOCYTES WITH THE MEMBRANE OF THE SPECIFIC GRANULES

The localization of alkaline phosphatase in the specific granules of rabbit polymorphonuclear leukocytes was investigated. The results obtained suggest very strongly that alkaline phosphatase is a component of the granule membrane. The enzyme remains attached to the membrane upon disruption of the granules by the use of detergents or by hypotonic shock and subsequent extraction with sodium sulfate, and can be isolated together with fragments of the granule membrane by isopycnic equilibration. Treatment of the granules with high amounts of Triton-X-100, sodium deoxycholate, or hexadecyltrimethylammonium bromide releases the enzyme in soluble form. In polymorphonuclear leukocyte homogenates, lysis of the granules is needed in order to render alkaline phosphatase fully accessible to substrates. This suggests that the catalytic site of the enzyme is exposed at the inner face of the granule membrane.     

Ultrastructural patterns of the alpha-naphthyl butyrate esterase activity in normal and leukaemic peripheral blood leucocytes

Summary The activity of -naphthyl butyrate esterase was investigated at the ultrastructural level in normal human peripheral blood and in a few cases of hairy cell leukaemia, B-chronic lymphocytic leukaemia and acute monocytic leukaemia. A membrane reactivity was detected in most normal monocytes and lymphocytes. The activity in monocytes was very strong and was inhibited by NaF. It was NaF-resistant and less intense in lymphocytes. The reaction product was localized in the cytoplasm only in a small percentage of lymphocytes.In lymphocytes and monoblasts from pathological samples the pattern of reactivity was similar to that found in their normal counterparts, except for a lower intensity. The hairy cells showed a discrete distribution of the NaF-resistant reaction product on their cell surface.The different patterns of enzyme distribution are discussed critically.     

Localization of four phosphatases in rat liver sinusoidal cells

Summary In the present study we have localized neutral phosphatase, acid phosphatase, alkaline phosphatase and 5 nucleotidase in the sinusoidal cells of rat liver using enzyme cytochemistry at light and electron microscopical level.Neutral phosphatase was present in the endoplasmic reticulum and nuclear envelope of parenchymal cells and of sinusoidal endothelial, Kupffer and fat-storing cells. The intensity of the neutral phosphatase reaction was stronger in sinusoidal than in parenchymal cells. Sinusoidal cells were devoid of cytochemically demonstrable alkaline phosphatase. Abundant acid phosphatase was present in the many lysosomes of endothelial and Kupffer cells. Substanually less acid phosphatase-positive lysosomes were found in fat-storing cells. 5 nucleotidase was present on the cell membrane of fat-storing cells, on 90% of all Kupffer cells and on the microvilli of parenchymal cells.We have further shown that combined staining for 5 nucleotidase and for endogenous peroxidase, offers a histochemical tool to discriminate between the three main sinusoidal cell types in normal rat liver.     

Acid hydrolases in the perinuclear secretion granules of the rat preputial gland. A light and electron microscope study

Synopsis Four acid hydrolases in the secretory cells and the sebum of the preputial sebaceous gland of the rat were incestigated cytochemically. A strong -glucuronidase activity was found to occur in the matrix of the perinuclear secretion granules, whereas the granule crystalloids were unreactive. The distribution of acid phosphatase at the light microscope level was similar, though the intensity of the reaction was lower and the number of positive granules smaller. By electron microscopy, the final reaction product of acid phosphatase occurred in patches at the periphery of the granule matrix, as well as in the vesicles adjoining the Golgi stacks, from which the perinuclear granules seemed to arise. In the sebum, the two hydrolases occurred in the background material between the unstained crystalloid masses. There was noN-acetyl--glucosaminidase or aryl sulphatase activity in the gland. The perinuclear granules appear to be secretory lysosomes which, after discharge from the disaggregating cell, release their acid hydrolases into the sebum.     

Cytochemical localization of phosphatase activity in vascular bundles and contiguous tissues of the leaf ofZea mays L.

Summary The cytochemical localization of phosphatase activity has been carried out on small and intermediate vascular bundles and contiguous tissues of the leaf ofZea mays L. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP, and with ADP and -GP. Reaction product (lead deposits) was observed on the plasma membrane of all cell types. It was invariably heavier on the plasma membranes of the bundle-sheath cells, vascular-parenchyma cells, and the thin-walled sieve tubes and their associated companion cells than on those of the mesophyll cells. Within the bundles, the heaviest lead deposits frequently were found on the plasma membranes of the thin-walled sieve tubes and the least amount (often lacking) on those of the thick-walled sieve tubes. Formation of reaction product was suppressed by NaF, vanadate, and molybdate but not by PCMBS (p-chloromercuribenzene sulfonic acid). The results of the substrate-specificity and inhibitor-sensitivity studies indicate that a nonspecific acid phosphatase was probably responsible for the deposition of the reaction product and not the plasma membrane H⁺-ATPase. These results, in addition to an evaluation of the pertinent literature, lead us to conclude that H⁺-ATPase activity has yet to be demonstrated unequivocally in association with the plasma membrane of phloem cells with lead precipitation procedures. Nevertheless, the differences in amounts of reaction product generally associated with the plasma membranes of the thick- and thin-walled sieve tubes of the maize leaf indicate that the two types of sieve tube differ from one another physiologically.     

Electron cytochemistry of developing and mature eosinophils in the bone marrow of the fowl and the duck

Synopsis Enzyme cytochemical studies have been carried out on eosinophils in the fowl and the duck. Peroxidase was found in all regions of the Golgi apparatus, the rough endoplasmic reticulum and the perinuclear cisternae of the early cells. In fowl eosinophil granules irregular deposits of peroxidase and arylsulphatase final reaction product were found, but the acid phosphatase deposits were even. In the duck in contrast, peroxidase was demonstrated in the external part of the granule only. Acid phosphatase and arylsulphatase were found in both the interna and the externa of the duck eosinophil granules. An ammoniacal silver nitrate reaction for the presence of the histone arginine was also studied. Silver deposits were found occupying all regions of the granules of eosinophils from both species of bird.The presence of the hydrolytic enzymes acid phosphatase and arylsulphatase in avian eosinophil granules supports the theory that these structures are lysosomal in nature and that they correspond with mammalian eosinophils in this respect.     

Fine structural localization of alkaline phosphatase in the fracture callus of the rat

Summary The fine structural localization of nonspecific alkaline phosphomonoesterase in the different cells constituting the fracture callus in the rat was studied by incubating sections of glutaraldehyde-fixed callus tissue of variable age in media containing -glycerophosphate and either lead or calcium ions. The specificity of the reactions were tested by exposing the tissues to inhibitors of alkaline phosphatase.The results showed presence of final product on the plasma membranes and associated structures (subplasmalemmal endocytotic vesicles) of fibroblasts, pre-osteoblasts, osteoblasts, and cartilaginous cells in the callus. With the calcium method, reaction product was demonstrated in vesicular elements of the Golgi apparatus in osteoblasts and chondrocytes. Precipitates indicating presence of alkaline phosphatase activity were also observed on the membranes bordering cytoplasmic projections and fragments of cytoplasm located adjacent to enzyme-containing cells. Furthermore, the globule-shaped bodies in the matrix (Bonucci-bodies) showed evidence of alkaline phosphatase activity.The evidence obtained supported the view that alkaline phosphatase plays a role in calcification. It is suggested that transfer of cellular alkaline phosphatase to the sites of initial calcification in the extracellular matrix occurs by way of pinched off vesicular fragments of the cytoplasm and plasma membrane of osteogenic enzyme-producing cells; these structures appear to move awy from their cells of origin to form the Bonucci bodies in the matrix.     

Lateral diffusion of luminal membrane components during secretion in parotid acinar cells of the rat

Summary The movements of the molecular components of the luminal plasma membrane during exocytotic secretion in parotid acinar cells were examined. For immunocytochemical study, we used an antiserum of dipeptidyl peptidase IV as a marker for the components of the luminal plasma membrane of acinar cells. In unstimulated acinar cells, dipeptidyl peptidase IV immunoreactivity is restricted to the luminal plasma membrane. However, after secretion was stimulated with a -adrenergic agonist, isoproterenol, immunostaining became detectable on the membrane of discharged granules. Freeze-fracture images showed that the density of intramembrane particles on the P-fracture leaflets of discharged granule membranes is much higher than that of undischarged granule membranes during secretion. These results suggest that in parotid acinar cells of the rat, the components of the luminal plasma membrane move laterally, during secretion, to the membranes of discharged granules.     

Cytochemical studies on the localization and functional properties of calcium in anterior pituitary cells

The localization of calcium and its functional properties in anterior pituitary cells were studied using a potassium pyroantimonate technique. In all kinds of secretory cells, the precipitates of the calcium-pyroantimonate complex were distributed on the limiting membrane of the secretory granule. They were present also in the cytoplasmic matrix, the mitochondrial matrix, small smooth vesicles, coated vesicles, and in the nuclear euchromatin area. The precipitates were usually seen at the contact region between the limiting membranes of two adjacent secretory granules, or between the granule limiting membrane and the plasma membrane. When the tissues were incubated in the medium containing A23187 (10 microM) for 5 min, the deposits on the granule limiting membrane were increased in number and those on the mitochondrial matrix were decreased; the reaction products almost disappeared on the limiting membranes of the secretory granules after membrane fusion following single or multigranular exocytosis induced by A23187-treatment. In addition, small vesicles in the capillary endothelium contained reaction precipitates. Based on these results we propose a hypothetical model for the relationship between the localization of calcium and secretory activity.     

Ultrastructural and cytochemical observations on the granulocytes of the sturgeon, Acipenser brevirostrum (Chondrostei)

Granulocytes from cranial granulopoietic tissue were studied under the electron microscope, and cytochemistry carried out oncranial and peripheral blood granulocytes of two sturgeons, Acipenser brevirostrum . Ultrastructurally, eosinophils and basophils had homogeneous electron-dense granules similar to those of teleosts and some higher vertebrates. Neutrophils contained two granule types: small elongated fibrillar granules and large (<3.8μm long) usually homogeneous granules.
Neutrophil fibrillar granules were positive for alkaline phosphatase (ALP), acid phosphatase (ACP), α-naphthyl acetate esterase (ANAE), acetyl-l-tyrosine-α-naphthyl esterase (ATNE) and periodic acid Schiff (PAS) reaction. The large homogeneous granules were negative for all enzymes, and were only PAS positive. Eosinophils had granular, cyanide-, azide- and aminotriazole-resistant peroxidase (PO) and were ACP, ATNE, tosyl-l-lysine-α-naphthyl esterase (TLNE) and Luxol fast blue positive.
Ultrastructure and cytochemistry are discussed in relation to other vertebrates, and eosinophils identified as the main phagocytic leucocyte.