Acid phosphatase activity in the neutral red granules of mouse exocrine pancreas cells

Transcription

1 343 Acid phosphatase activity in the neutral red granules of mouse exocrine pancreas cells By JENNIFER M. BYRNE (From the Cytological Laboratory, Department of Zoology, University Museum, Oxford) With 2 plates Summary The Gomori test for acid phosphatase was performed on pancreatic tissue from mice injected with neutral red. On an electron microscopical level, acid phosphatase activity was found to be localized in the neutral red granules. It is suggested that the neutral red granules are of lysosomal derivation. Introduction AN electron microscopical study of the neutral red granules of mouse exocrine pancreas (Byrne, in press) shows that there is a striking morphological resemblance between the neutral red granules and the lysosomes (de Duve and others, 1955; Novikoff, Beaufay, and de Duve, 1956) of various cells, particularly those of liver cells (de Duve, 1959; Novikoff, 1959; Novikoff and Essner, i960). The closest resemblance is to the lysosomal derivatives, 'autophagic vesicles', 'residual bodies', 'phagosomes', and 'cytolysomes' described in various tissues (Straus, 1958; Novikoff and Essner, 1962; Beaufay, 1963; Behnke, 1963; de Duve, 1963). Since the identification of a lysosome depends on enzymatic and not on morphological criteria (de Duve and others, 1955; de Duve, 1959; Novikoff, 1961), the Gomori (1952) test for acid phosphatase was performed on pancreatic tissue from mice injected with neutral red, and investigated by both light and electron microscopy., Material and methods Adult mice weighing between 25 and 35 g were taken from a stock provided with a continuous supply of food and water. The mice were injected as before (Byrne, in press) with a 1 % solution of neutral red chloride in distilled water. The dosage was that prescribed by Morgan (1953a), namely g of neutral red per g of mouse body weight. The mice were killed 6 h after injection and small pieces of pancreas squashed in saline under a coverslip to check that the cells showed neutral red granules. Gomori's test for acid phosphatase (Gomori, 1952) was performed on tissue from mice injected with neutral red solution, and from a control series of mice injected with distilled water alone. Small pieces of pancreas were fixed overnight in cold formaldehyde-calcium [Quart. J. micr. Sci., Vol. 105, pt. 3, pp , 1964.]

2 344 Byrne Acid phosphatase activity (Baker, 1944) or in cold formaldehyde-calcium to which had been added 5% of sucrose (Holt and Hicks, 1961). The tissue was washed in cold distilled water and kept in cold sucrose / gum acacia solution (Holt, 1959). For light microscopy, 10 to 15 /x sections were cut on the freezing microtome, with sucrose / gum acacia as support. The sections were placed flat on slides in a little distilled water, and the sections thoroughly dried. The sections were incubated at 37 0 C in o-1 M sodium /J- glycerophosphate medium at ph 5-0 (Gomori, 1952) for 1, 2, 3, 4, 5, and 6 h. Tissue from animals injected with neutral red and also from those which had received distilled water alone was incubated for the same lengths of time in a medium in which o-i M sodium fluoride (a specific inhibitor of acid phosphatase) was added to the incubating medium (Holt, 1959). The final reaction-product was made visible by dipping the slides in dilute ammonium sulphide solution. For electron microscopy, 40 \L sections of tissue were cut in sucrose / gum acacia on the freezing microtome, or small pieces of tissue sliced with a razor blade. The sections or slices were incubated in Gomori's medium at ph 5-0 for 5, 10, 15, and 30 min. Sections were also incubated for the same lengths of time in medium to which sodium fluoride had been added. The sections were washed, fixed in Palade's buffered osmium tetroxide (Palade, 1952) for 1 h, dehydrated, and embedded in araldite, according to Luft's technique (Luft, 1961). The blocks were sectioned on a Huxley ultramicrotome, and the sections examined either unstained or after staining with Millonig's lead tartrate solution (Millonig, 1961) or with Reynolds's lead citrate solution (Reynolds, 1963). Some of the material was examined in an Akashi transcope (TRS 50 E 1) electron microscope and some in a modified (Meek, 1958) Siemens Elmiskop 1 electron microscope, operated at 60 KV, with the double condenser. Results Light microscopy Pancreas from mice injected with neutral red was incubated in Gomori's medium at ph 5-0 for periods up to 6 h. No reaction-product was found in the region where the neutral red granules occur. After 6 h the nucleus showed a positive reaction. Material from mice injected with distilled water alone showed a positive reaction in the nucleus after incubation for 4 h. No other site of reaction was found with incubation periods up to 6 h. No reaction was found in material incubated in medium to which sodium fluoride had been added. Electron microscopy Pancreatic tissue taken from mice injected with neutral red and incubated for 5 min in Gomori's medium at ph 5-0 shows under the electron microscope deposits of lead phosphate localized in inclusions measuring up to 3-2 fx in greatest dimension. The majority measure between 0-27 p and 2-0 //.. These

3 in the neutral red granules of mouse exocrine pancreas cells 345 inclusions showing acid phosphatase activity are generally found in the cytoplasm near the luminal end of the nucleus. Their structure varies considerably (figs. 1, 2), but they commonly consist of an aggregate comprising electronlucent vacuoles rimmed with electron-dense material, and a mass of membranous material (fig. 1, A, B). The membranous material is often arranged concentrically (fig. 2, A, B). The whole is enclosed in a single membrane. The reaction product is deposited on the electron-dense material, particularly on the parallel or concentrically arranged membranous material (figs. 1 and 2). No reaction-product was found in the vacuoles or on the enclosing membrane. No reaction-product was found on the membranes of the endoplasmic reticulum, on the mitochondria, or on the zymogen granules, except in obviously damaged cells at the edge of the block. The inclusions showing acid phosphatase activity are never found in pancreatic tissue from animals injected with distilled water alone. Such tissue, when incubated in Gomori's medium for the same length of time, does not show any such localized deposit of lead phosphate. After longer incubation times (10 to 30 min) reaction-product is found on the membranes of the endoplasmic reticulum, on the nuclear membrane, and on the zymogen granules. This is probably an unspecific deposition. Material incubated in the medium to which sodium fluoride has been added shows no granular deposit. Discussion Under the light microscope acid phosphatase activity was found only in the nucleus. It is interesting that tissue from mice injected with distilled water showed a reaction in the nucleus earlier than tissue from mice injected with neutral red. In view of the electron microscopical results the absence of any reaction-product in the region occupied by the neutral red granules is surprising, unless the amount of precipitated reaction-product is below the threshold of resolution of the light microscope. On an electron microscopical level, the inclusions showing acid phosphatase activity are of the same size and distribution as the neutral red granules seen in the mouse exocrine pancreas cell under the light microscope. They are never found in tissue from animals injected with distilled water. Although their detailed structure, as seen with the electron microscope, is so varied, they closely resemble the inclusions identified as neutral red granules in earlier work (Byrne, in press). The inclusions are not as strongly osmiophil as was found in earlier work, but this is probably because of the differences in fixation. Prior fixation in formaldehyde may block some uptake of osmium. Novikoff (1961) showed that in cells from vertebrate tissues acid phosphatase is often localized in granules concentrated in the Golgi area. These granules are the lysosomes (Novikoff, Beaufay, and de Duve, 1956; de Duve, 1959). Lysosomes show a great variety of morphological forms, but the presence of acid phosphatase is taken to be indicative of a lysosome or lysosomal derivative (de Duve and others, 1955; Novikoff, 1961). The

4 346 Byrne Acid phosphatase activity presence of acid phosphatase activity in the neutral red granules suggests that they are lysosomes or lysosomal derivatives. A certain correlation exists between the presence of acid phosphatase activity in an inclusion and its ability to stain with neutral red or other vital dyes. Ogawa and Okamoto (i960) and Ogawa, Mizuno, and Okamoto (1961) found that the granules in the cytoplasm of cultured astrocytes and fibroblasts which stain in life with neutral red, methylene blue, toluidine blue, and azure B, show acid and alkaline phosphatase and esterase activity. They conclude that these granules are lysosomes. Koenig (1962) found that glycolipoprotein granules in neural cytoplasm stain with neutral red and methylene blue. He suggests that these granules are lysosomes on the grounds of their enzyme content. Lane (1963) and Meek and Lane (in press) using Helix aspersa neurones find acid phosphatase activity in the cortices of granules which have the same distribution as the 'blue' and 'y e ll w> granules, which are known to take up neutral red, Nile blue, methylene blue, and brilliant cresyl blue (Chou, 1957). Lane (in press) finds granules which take up neutral red and which show acid phosphatase activity in tentacular cells of H. aspersa. Brachet (1957) also suggests (from unpublished results) that it is likely that lysosomes are identical with vacuoles which can be demonstrated in life with basic dyes such as neutral red or toluidine blue. Beaufay, quoted in de Duve (1959), finds that in fractionated, vitally stained liver cells part of the dye is retained in the particulate fractions, the highest concentration being in the fraction richest in lysosomes. Lysosomes are known to act as intracellular phagocytes. Cells that are forced to engulf large amounts of foreign substances for the digestion of which they are not equipped tend to accumulate such substances in the lysosomes. The lysosomes of the parenchymal cells (Novikoff and Essner, 1962) and of the Kupffer cells (Baudhuin and Wattiaux, 1963) of rat liver become enormously enlarged by the detergent triton WR Vital dyes may be treated in the same way. In their role as intracellular phagocytes the lysosomes may accumulate dye within themselves, becoming enlarged in the process. The osmiophilia of the inclusions and their membranous components suggests that the dye may be accumulated in association with phospholipid. On a light microscopical level, Morgan (19536) found that the neutral red granules were positive for the acid haematein test for phospholipid (Baker, 1946, 1947). Lysosomes are not normally seen in pancreatic tissue, but the number within each cell may perhaps be greatly increased as a response to the presence of the dye. Novikoff (1961) considers that the concentration of lysosomes near the Golgi apparatus, which has been seen in a number of vertebrate tissues by both light and electron microscopy, suggests a developmental or functional FIG. I (plate), A, B, pancreas from mouse injected with neutral red and killed after 6 h. The tissue has been incubated for 5 min in Gomori's medium at ph 50. cm, cell membrane; er, endoplasmic reticulum; nrg, neutral red granule.

5 Fie. : J. M. BYRNE

6 FIG. 2 J. M. BYRNE

7 in the neutral red granules of mouse exocrine pancreas cells 347 interrelationship between the Golgi apparatus and the lysosomes. In a few cells (NovikofF and others, 1962) acid phosphatase activity is found in the Golgi lamellae. In view of this, it is perhaps significant that the neutral red granules are confined to the part of the cell near the Golgi apparatus. Recently, Robbins and Marcus (1963) have studied the supravital staining of HeLa cells with acridine orange. In a preliminary electron microscopical study they find that in vitally stained material 'transformations of multivesicular bodies into abnormal myelin figures are extremely numerous. These transformations represent the acridine orange particles.' They find that the acridine orange particles show acid phosphatase activity. This suggests a strong similarity between the neutral red granules and acridine orange particles. It is significant that Morgan (1958) found that acridine orange was the only one of several dyes of a similar molecular weight to neutral red that gave similar results in the vital staining of pancreas. The present results indicate that the neutral red granules of mouse exocrine pancreas are of lysosomal origin. The cycle of neutral red granule formation must then be envisaged as a process of segregation of dye in the lysosomes (possibly in combination with phospholipid), resulting in their enlargement. This process reaches a peak about 8 h after injection of the dye (Morgan, 1953a; Byrne, in press), and is followed by the removal of the dye from the cell. The electron micrographs of the material from mice injected with neutral red published here were taken by Dr. S. Bradbury, to whom I should like to express my most grateful thanks. I am also grateful to Dr. J. R. Baker, F.R.S., for his advice and encouragement during the course of this work, and for performing the injections of neutral red, and to Professor J. W. S.Pringle, F.R.S., for accommodating me in his Department. This work was partly carried out during the tenure of a Medical Research Council Scholarship. All the electron micrographs published here were taken with a Siemens Elmiskop 1 electron microscope in the Department of Human Anatomy, Oxford. Both this microscope and the Akashi transcope were provided by the Wellcome Trustees. References Baker, J. R., Quart. J. micr. Sci., 85, Ibid., 87, Ibid., 88, 463. Baudhuin, P., and Wattiaux, R., quoted in de Duve, Beaufay, H., quoted in de Duve, quoted in de Duve, Behnke, O., J. Cell Biol., 18, 251. Brachet, J., Biochemical cytology, p. 52. New York (Academic Press). FIG. 2 (plate), A, B, pancreas from mouse injected with neutral red and killed after 6 h. The tissue has been incubated for 5 min in Gomori's medium at ph 5'0. er, endoplasmic reticulum; m, mitochondrion; nrg, neutral red granules; s, zymogen granule.

8 348 Byrne Acid phosphatase activity Byrne, J. M., Quart. J. micr. Sci. (In press.) Chou, J. T. Y., Quart. J. micr. Sci., 98, 47. Duve, C. de, In Subcellular particles, edited by T. Hayashi. New York (Ronald Press) Sci. Amer., 208, no. 5, 64. Pressman, B. C, Gianetto, R., Wattiaux, R., and Appelmans, F., Biochem. J., 60, 604. Gotnori, G., Microscopic histochemistry. Chicago (University of Chicago Press). Holt, S., Exp. Cell Res., suppl. 7, 1. and Hicks, R. M., J. biophys. biochem. Cytol., 11, 31. Koenig, H., Nature, 195, 782. Lane, N. J., Quart. J. micr. Sci., 104, 401. Ibid. (In press.) Luft, J. H., J. biophys. biochem. Cytol., 9, 409. Meek, G. A., Proc. Internat. Conf. Electron Microscopy, 4, Berlin, 1, 187. and Lane, N. J., J. roy. micr. Soc. (In press.) Millonig, G., J. biophys. biochem. Cytol., II, 736. Morgan, W. S., 1953a. Quart. J. micr. Sci., 94, Ibid., 94, Nature, 181, Novikoff, A. B., In Subcellular particles, edited by T. Hayashi. New York (Ronald Press) In The Cell, 2, edited by J. Brachet and A. E. Mirsky. New York (Academic Press). Beaufay, H., and Duve, C. de, J. biophys. biochem. Cytol., suppl. 2, 179. and Essner, E., i960. Amer. J. Med., 39, J. Cell Biol., 15, 140. Essner, E., Goldfischer, S., and Heus, M., In The interpretation of ultrastructure Symposium of the International Society for Cell Biology, 1, edited by R. J. C. Harris. London (Academic Press). Ogawa, K., and Okamoto, M., i960. J. histochem. cytochem., 8, 351. Mizuno, N., and Okamoto, M., Ibid., 9, 20Z. Palade, G. E., J. exp. Med., 95, 285. Reynolds, E. S., J. Cell Biol., 17, 208. Robbins, E., and Marcus, P. I., J. Cell Biol., 18, 237. Straus, W., J. biophys. biochem. Cytol., 4, 541.