Kovács, J

Réz, G

English

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NEUTRAL RED INDUCED AUTOPHAGOCYTOSIS IN THE EXOCRINE PANCREATIC CELLS OF THE FROG (RANA ESCULENTA L.)byG. RÉZ and J. KOVÁCSDepartment c f General Zoology o f the Eötvös Lorónd University, Budapest Received on 25th March, 1974IntroductionAutophagosytosis has been widely investigated in recent years (for reviews see E r i c s s ο n 1969, P f e i f e r  1971). It is readily induced by treatment with neutral red in vivo, especially in cell types characteri­zed by a well-developed protein synthesizing apparatus. Thus, the dye was found to induce the formation of autophagic vacuoles in the cytop­lasm of mouse exocrine pancreatic cells, too ( A l o u s i ,  S t  e n g e r  and M o r g a n  1968, R é z  and K o v á c s  1967, 1973, B y r n e  1964, \\ e i s s  1955, E s s n e r  1970), and the detailed morphological investigation of the phenomenon has been carried out (R é z and К  о - v á c s  1971).In the present investigation our aim was to obtain comparative morphological data on neutral red-induced autophagocytosis in the amphibian pancreas.Material and methodsThe first part of the experiments was carried out on summer (August) animals. Twenty-five male frogs (Runa esculenta L.) were given single intraperitoneal injections of 2% neutral red (0.4 mg/g body weight) dissolved in 0.7% saline. They were decapitated 5, 8, 16, 24 and 48 hours after this treatment, 5 frogs at each time. Three frogs killed 24 hours after treatment with corresponding volumes of 0.7% saline solution served as controls.In the second part of the experiments exactly the same treatment was repeated using 28 winter (December) animals. The pancreatic pieces were fixed for light microscopy in Holly’s solution for 1 hour and embedded in paraffin. The sections were stained for basophilia using 2% toluidine blue dissolved in pH 2.7 veronal acetate-HCl buffer solution. For electron microscopy, tissue pieces were fixed for 2 hours in 5% glutaraldehyde buffered with 0.1 M (pH 7.4) sodium cacodylate-НП solution. This was10*followed by a postfixation in buffered 2% osmium tetroxide and embed­ding in Araldit. The ultrathin sections were contrasted with uranvl acetate and lead citrate and examined with a UEMV — 100B electron microscope.14 8  R É Z  — K O V Á C SKig. 1. Autophagic vacuoles in the cytoplasm 24 hours after the neutral red injection. Vacuoles o f increased density (A) and light-type residual bodies (B) are demonstrated.Note the presence o f Golgi-like vesicles round the vacuoles, (x 27000)Results and discussionThe autophagic vacuoles appear in the cytoplasm of the acinar cells 8 hours after the neutral red-treatment. Later on, the number of the vacuoles increases and 24 hours after the injection numerous krinom granules (light microscopic forms of autophagic vacuoles, see R é z and K o v á c s  1973) can also be observed in the cells.The fine structure of the encapsuled organelles (largely rough-sur­faced ER) can clearly be recognized in the interior of the double mem­brane-limited early forms of autophagic vacuoles (Figs 4 and 5). Round them, a large number of Golgi-like vesicles can be observed, many ofN E U T R A L  R ED  IN D U CED  A U TO P H A G O C Y T O SIS I 49Fig. 2. Autophagic vsieuoies (arrows) segregating Golgi material are shown S hours afterthe dye injection, (x 180 )0)which being in close contact with the limiting membrane of the autopha­gic vacuoles (Figs. 1, 4, 5 and 6). Ί he vacuoles are frequently located in the immediate vicinity of the Golgi complex, and even in the Golgi-area itself where they mainly segregate elcmets of the Golgi apparatus (big 2).The advanced forms of autophagic vacuoles can be classed with two main morphologic types. Of the first type, an increasing density of the segregated material is characteristic (Figs. 1 and (5), with a single mem­brane-limited residual body containing highly dense material remaining1 5 0  R É Z  — K O V Á C SFig. 3. A residual body lilted with highly dense material and a newly formed, double membrane-limited autophagic vacuole (A) can be seen in the vicinity o f the Golgi-complex 24 hours after the dye treatment, (x 26400)at the end of the degrative process (Figs. 3 and 6). In the case of the second type the density of the encapsuled material decreases (Figs. 1 and 5) which leads to the formation of usually large-sized vacuoles containing membrane whorls and fragments, vesicles, rod-like crystals and electron- lucent matrix (Fig. 1). These two morphologic types of autophagv pos­sible indicating the existence of vacuoles different in their enzyme con-tent have been described in mouse pancreas, too (R é z and К  о v á c s 1971, 1972).Therefore, we conclude that neutral red-induced autophagocytosis in amphibian pancreas is similar to that described in mammals. However,N E U T R A L  RED  IN D U CED  A U TO P H A G O C Y T O SIS j g jFig. 4. Neutral red for 24 hours. Double membrane-limited autophagic vacuole segregating rough-surfaced KR and Golgi vesicles are demonstrated near the Golgi-complex.(x 33400)while autophagic response to the dye in pancreatic acinar cells of mice and chickens (unpublished data) occurs within 30-60 minutes after the injection, it takes much more time (i. e. 8 hours) in the case o f frogs.The reason for this longer time lag is not understood at present. We could not find any difference between summer and winter animals in reactivity to neutral red-treatment.SummaryNeutral red (0.4 mg/g body weight) was shown to induce massive autophagy in pancreatic acinar cells of t he frog ( Rana esculenta L.) within 8 hours after a single intraperitoneal injection. In the course of the au-1 5 2  R É Z  — K O V Á C SFig. 5. A group o f autophagic vacuoles troni acinar cell oi a lrog treated with the dye for 24 hours. Double membrane-limited newly formed vacuoles (A) and advanced forms are to be seen. Note the presence o f numerous Golgi-like vesicles near the vacuoles.(x 31300)tophagic process, parts of rough-surfaced ER and of the‘Golgi-complex were segregated and degraded. The morphology of this phenomenon was similar to that described earlier in mammalian pancreas.N E U T R A L  RED IN D UCED  A U TO P H A G O C YT O SIS 1Г»3Fig. 6. Advanced vacuole o f increased density, segregating ER cisternae and dense-type residual body, 24 hours after the dye injection, (x 20500)REFERENCESA 1 о u s i, M. A. S t e n g e  t\ It. Л. — M о г g a n, W. S. 10.>S. The fine structure o f pancreatic acinar and hepatic parenchymal cells after neutral red dye injection. Exp. molec. Path. 0: 07 109.B y r n e ,  Л. M. 1904. An elcctroninicroscopical study o f neutral red granules in mouse exocrine pancreas. Quart. Л. micr. Sci. 1 0 5 : 219 — 225.E г i c s s  ο n, Л. L. E. 1969. Mechanism o f cellular autophagy. In: Lysosomes in biology and pathology, 1: 345 — 3S4. Amsterdam - London: North Holland.E s s n e r, E. 1070. Cytochemical studies o f neutral red-induced granules in mouse panc­reatic exocrine cells. VIL-óme? Congr. internat, micr. electr. Grenoble, 3: 407 —408.P f e i f e r ,  Г. 1071. Probleme der cellulären Autophagie. Morphologische, enzymzyto- chemische und quantitative l'ntersuchungen an normalen und alterierten Leberepit- helien der Ratte. Ergehn. Amit. Entwickl.-Geseh. 44: 1 74.R é z. G. — К о v à  es, Л. 1967. A light and electron microscopie study o f the focal cytoplasmic degradation o f the cells o f mouse exocrine pancreas. Mikroskopie 22: 347 (Abstr.)R é z ,  G. — K o v á c s ,  Л. 1071. Morphological examination o f autophagic vacuoles formed in mouse pancreatic exocrine cells under the effect o f neutral red. Ann. T’niv. Sci. Budapest, Sect. Biol. 13: 315 — 326.1 1 ANNALES — Sec do Biologica — Tomus !7.R é z ,  G. — K o v á c s ,  J. 1972. Electron microscopic examination o f autophagy and cytoplasmic degradation induced by cadmium chloride and hyperosmotic sucrose in exocrine pancreatic cells o f mice. Ann. Univ. Sci. Budapest, Sect. Biol. 14: 219 — 234.R é z ,  G. — K o v á c s ,  ·Τ. 1973. Prevention by cyloheximide o f neutral red-inducedformation o f autophagic vacuoles and krinom granules in mouse pancreatic acinar cells. Virchows Arch- Abt. B, Zellpath. 12: 123 — 132.W e i s  s, J. M. 19S5. Intracellular changes due to neutral red as revealed in the pancreas and kidney o f the mouse by the electron microscope. J . Exptl. Med. 107: 213 — 224.] 54  R É Z  — K O V Á C S

Neutral red induced autophagocytosis in the exocrine pancreatic cells of the frog (Rana esculenta)

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Neutral red induced autophagocytosis in the exocrine pancreatic cells of the frog (Rana esculenta)

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