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The effects of cyclic 3′,5′-AMP on the lysosomes of newborn rat hepatocytes
JOURNALOF ULTRASTRUCTUREAND MOLECULARSTRUCTURERESEARCH
97 s
210-215 (1986)
The Effects of Cyclic 3',5'-AMP on the Lysosomes of Newborn Rat Hepatocytes O. B. KOTOULAS Department of Anatomy, Histology, and Embryology, University of Ioannina Medical School, loannina 45332, Greece Received November 21, 1986, and in revisedform July 7, 1987 Certain effects of cyclic 3',5'-AMP administration on the lysosomes of newborn rat hepatocytes were studied using biochemical assays, electron microscopy, and quantitative morphometry. Cyclic A M P produced accelerations of postnatal hyaloplasmic glycogen breakdown and lysosomal glycogen breakdown, and an increase in activity of the lysosomal enzyme acid a-l,4-glucosidase (maltase). Ergotamine, a known antagonist of the effects of cyclic AMP, produced inhibitions of postnatal hyaloplasmic glycogen breakdown and lysosomal glycogen breakdown. Cyclic A M P increased while ergotamine decreased the volume of lysosomes. The results support the postulate that the catabolism oflysosomal glycogen is controlled by those agents that regulate the catabolism ofhyaloplasmic glycogen (O, B. Kotoulas and M. J. Phillips, Amer. J. Pathol. (1971) 63, 1-17; O. B. Kotoulas et al., Amer. J. PathoL (1971) 63, 23-36). Control is mediated by changes in the activity of the lysosomal enzyme acid a-l,4-glucosidase. Lysosomes actively participate in the degradation of hepatocellular glycogen. © 1986AcademicPress, Inc.
The regulation of the breakdown of glycogen inside lysosomes is rather obscure. Previous studies suggested that the lysosomal enzyme acid a-l,4-glucosidase (maltase) may control this process (Hers, 1963; Rosenfeld, 1964; Kotoulas et aL, 1971). Glucagon increases the activity of acid glucosidase in newborn rat liver while insulin decreases this activity (Kotoulas, 1981; Kotoulas, 1984). The actions of glucagon and insulin could be mediated by alterations in the level of cyclic 3',5'-AMP (Exton et aL, 1966; Sutherland et aL, 1968; Glinsmann and Mortimore, 1968). Therefore, the question must be asked whether this agent has an effect on the activity of the enzyme and the breakdown of the lysosomal glycogen. In this paper, the cyclic 3'-5'-AMP-induced change in the activity of the newborn rat liver acid a-l,4-glucosidase is determined and the cyclic AMP-induced ultrastructural changes in the lysosomes of the hepatocytes are described. Also, the ultrastructural changes that occur after the administration ofergotamine, a known antagonist of the effects of cyclic AMP, are
described (Sutherland and Rail, 1960; Moran, 1966; Sutherland et aL, 1968). MATERIALS A N D METHODS
Animals and Handling of Tissues Newborn rats were obtained from 10 pregnant Wistar females. The average litter contained 10 newborns. The sex of the newborns was not determined. The newborns were sacrificed at birth or kept at 35°C and Sacrificed after 6 hr, unless otherwise specified. Part of the liver was processed for electron microscopy and the rest was immersed in liquid nitrogen and used for enzyme assays.
Chemicals Cyclic adenosine-3',5'-phosphate (cyclic AMP) (Cat. No. 10870) and glycogen (Cat. No. 23540) were obtained from Serva. Ergotamine tartrate, Lot 67033, was from Sandoz. The reagents for determining glucose and for electron microscopy were obtained as before (Kotoulas, 1981).
Biochemical Methods The acid a-1,4-glucosidase assay was performed on homogenates of liver tissue in a 10% dilution with distilled water as before (Kotoulas, 1981). Usually 100 X of homogenate were used for the assay. The total activity of the enzyme was determined according to 210
0889-1605/86 $3.00 Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
211
CYCLIC AMP AND LYSOSOMES Hers (1963) and Lejeune et al. (1963) with glycogen as substrate.
Electron Microscopy and Morphometric Analysis Electron microscopy was performed as described previously (Kotoulas, 1981). The sections were examined in Philips 300 and Hitachi IIB electron microscopes (Department of Pathology, McGill University, Montreal, Canada) and partly in a Philips 75 electron microscope (Department of Clinical Therapeutics, University of Athens, Greece). Morphometric analysis was made as described previously (Kotoulas and Phillips, 1971). From each group three experimental animals and three controls from the same litters were used. From each liver, five blocks were prepared. Two randomly taken micrographs from each block were used for morphometry.
Experimental Design, Dose, Route, and Mode of Administration Ten animals were sacrificed at birth. The rest of the animals were divided into two groups according to the agent used, cyclic AMP or ergotamine. Ten cyclic AMPtreated animals and ten controls from the same litters were sacrificed at the age of 6 hr. Cyclic AMP was administered intraperitoneally in 0.2 ml of a 0.3% solution of the compound in 0.9% NaCI. The animals were injected at birth and 3 hr after birth (a dose of 100 mg/kg each time). Control animals were injected at birth and 3 hr after birth with 0.9% NaC1. Ten ergotamine-treated animals and their normal controls were sacrificed at the age of 6 hr. Ergotamine was administered intraperitoneally in 0.1 ml of a 0.05% solution. The animals were injected at birth and every hour thereafter (a dose of 8.3 mg/kg each time). RESULTS
Biochemical Results F o u r n e w b o r n animals were used in a prel i m i n a r y e x p e r i m e n t in order to d e t e r m i n e the response o f the a n i m a l to repeated in: jections o f cyclic A M P . T h e agent was adm i n i s t e r e d at b i r t h o r at b i r t h a n d 3 h r a f t e r
TABLE I GLUCOSIDASE ACTIVITY AT THE AGE OF 6 HR AFTER ADMINISTERING A DOSE OF 100 MG/KG CYCLIC A M P 0 AND 3 HR AFTER BIRTH (/.tMOLE GLUCOSE/HR/MG PROTEIN)
Control Cyclic AMP P
0.274 +_ 0.085 (lO) 0.598 +- 0.210 (lO) <0.05
Note. Results are means +. standard deviations. Numbers in parentheses represent the number of observations. Controls were injected at 0 and 3 hr with carrier only. birth. T h e a n i m a l w h i c h w as i n j e c t e d at b i r t h a n d its c o n t r o l w e r e s a c r i f i c e d at t h e age o f 3 hr. T h e a n i m a l w h i c h w a s i n j e c t e d at b i r t h a n d 3 h r af t er b i r t h a n d its c o n t r o l w e r e s a c r i f i c e d at t h e age o f 6 hr. N o i n c r e a s e i n the liver acid a- 1,4-glucosidase activity was o b s e r v e d at t h e age o f 3 h r i n t h e a n i m a l r e c e i v i n g o n l y o n e i n j e c t i o n w i t h cyclic A M P (control 0.135, experimental 0.149 ~ m o l e / h r / m g protein). A m a r k e d increase was note d at t h e age o f 6 h r i n t h e a n i m a l r e c e i v i n g two injections with cyclic A M P (control 0.223, e x p e r i m e n t a l 0 . 4 6 6 ~ m o l e / h r / m g protein). The level o f activity o f acid glucosidase, at t h e age o f 6 h r i n cyclic A M P - t r e a t e d animals and their controls, was d e t e r m i n e d in a more definitive experiment. The a d m i n i s t r a t i o n o f c y c l i c A M P r e s u l t e d in a s i g n i f i c a n t i n c r e a s e o f t h e a c t i v i t y o f t h e en z y m e . I n 10 t r e a t e d a n i m a l s t h e e n z y m e act i v i t y w a s 0 . 5 9 8 +_ 0 . 2 1 0 g m o l e / h r / m g p r o t e i n w h i l e i n 10 c o n t r o l s it w a s 0 . 2 7 4 _+ 0 .0 8 5 ~ t m o l e / h r / m g p r o t e i n ( T a b l e I).
TABLE 1I VOLUME FRACTIONS OF CYTOPLASMIC COMPONENTS OF THE RAT HEPATOCYTE AT BIRTH
Percentage of cytoplasmic volume~ Glycogen
Lysosomes
Glycogen in autophagic vacuoles
50.0 +_ 3.3
0.35 ± 0.08
0.15 +. 0.03
Percentage of lysosomal volume occupied by glycogen 43
a Results are means +__standard errors. For the morphometric analysis three newborn animals were used. From each liver five blocks were prepared and from each block two randomly taken micrographs were used, The results were computed from a total of 30 micrographs and an area of 5100 ~tm2.
212
O.B. KOTOULAS
213
CYCLIC AMP AND LYSOSOMES TABLE III EFFECTS ON HEPATOCYTES OF ADMINISTERING CYCLIC A M P TO NEWBORN RATS AT THE AGE OF 6 rm
Percentage of cytoplasmic volumea Treatment Controlb
Cyclic AMPc P
Glycogen
Lysosomes
12.6 _+ 2.1 8.0 + 1.5 <0.05
1.29 _+ 0.11 1.97 _+ 0.18 <0.05
Percentage of lysosomal volume ocGlycogen in autophagic vacuoles cupied by glycogen 0.23 +- 0.04 0.14 + 0.03 <0.05
18 7
Results are means + standard errors. For the morphometric analysis three experimental animals and three controls from the same litters were used. From each liver five blocks were prepared and from each block two randomly taken micrographs were used. bResults were computed from a total of 30 micrographs and an area of 5200 # m 2. c Results were computed from a total of 30 micrographs and an area of 5350 gm2.
Morphologic Results Both qualitative and quantitative changes were studied. The term lysosomes refers to lysosomes and related particles including autophagic vacuoles. In the tables o f m o r p h o m e t r i c analysis the term glycogen refers to hyaloplasmic glycogen unless otherwise specified (Kotoulas and Phillips, 1971). Group I: Cyclic AMP administered. The ultrastructure o f control hepatocytes during the first postnatal hours was as previously (Kotoulas and Phillips, 1971; Kotoulas, 1984). At birth vast stores o f hyaloplasmic glycogen were present. L y s o s o m e s and autophagic vacuoles were rare and small. A large part, i.e., 43%, o f the total lysosomal v o l u m e was occupied by glycogen (Table II). At the age o f 6 hr the hyaloplasmic glycogen was reduced. L y s o s o m e s were well developed. These organelles occurred pred o m i n a n t l y at the j u n c t i o n o f glycogen and glycogen-free areas. It was estimated that at least 8 5% o f the lysosomal v o l u m e belonged
to lysosomes o f the autophagic type. Autophagic vacuoles contained moderate a m o u n t s o f glycogen (Fig. 1). M o r p h o m e t r i c analysis showed that 18% o f the lysosomal v o l u m e was occupied by glycogen (Table III). In the cyclic A M P - t r e a t e d animals at the age o f 6 hr the depletion o f h y a l o p l a s m i c glycogen was a d v a n c e d and in some cells complete. Lysosomes were n u m e r o u s and often occurred at the margins o f the stores o f glycogen as in the control animals. At least 8 5% o f the total lysosomal v o l u m e was occupied by lysosomes o f the autophagic type. The autophagic vacuoles contained very small a m o u n t s o f glycogen (Fig. 2). M o r p h o m e t r i c analysis showed that 7% o f the total lysosomal v o l u m e was occupied by glycogen (Table III). M i t o c h o n d r i a in various stages o f disintegration were often seen inside autophagic vacuoles. Some o f the lysosomes contained a m o r p h o u s or m e m b r a n o u s electron-dense material and had the appearance o f residual bodies. Group II: Ergotamine administered. The n o r m a l controls at the age o f 6 hr were ob-
FIG. 1. Portion of a control rat hepatocyte at the age of 6 hr. The animal was injected with carrier only. A lysosome-autophagicvacuole (ly) is present. Moderate amounts of glycogen(gly)are seen in the hyaloplasm and within the lysosome-autophagicvacuole. Uranyl acetate-Reynold's lead citrate. × 41 600. FIG. 2. Portion of a cyclic AMP-treated rat hepatocyte at the age of 6 hr. The animal was injected at 0 and 3 hr after birth with a dose of 100 mg/kg each time. Large lysosome-autophagic vacuoles (ly) are present. Glycogen is negligible both in the hyaloplasm and within the lysosomes. Uranyl acetate-Reynold's lead citrate. × 41 600.
214
o. B. KOTOULAS TABLE IV EFFECTS ON HEPATOCYTES OF ADMINISTERING ERGOTAMINE TO NEWBORN RATS AT THE AGE OF 6 HR
Percentage of cytoplasmic volumea Treatment
Glycogen
Lysosomes
Control b Ergotaminec P
11.6 _+ 2.2 31.0 _+ 2.8 <0.05
1.35 _+ 0.20 0.58 + 0.11 <0.05
Percentage of lysoGlycogen in somal volume ocautophagic vacuoles cupied by glycogen 0.24 + 0.07 0.22 + 0.07 >0.5
18 38
a Results are means __+standard errors. For the morphometric analysis three experimental animals and three controls from the same litters were used. From each liver five blocks were prepared and from each block two randomly taken micrographs were used. b Results were computed from a total of 30 micrographs and an area of 4950 ttmL c Results were computed from a total of 30 micrographs and an area of 5400 tim2.
served to h a v e the s a m e ultrastructural features described for the control animals o f G r o u p I. Treating the animals with erg o t a m i n e resulted in inhibited depletion o f h y a l o p l a s m i c glycogen. In the e r g o t a m i n e treated a n i m a l s at the age o f 6 hr the lysos o m e s were small. These organelles usually occurred at the j u n c t i o n o f glycogen a n d glycogen-free areas. At least 85% o f the total lysosomal v o l u m e was occupied by lysos o m e s o f the autophagic type. T h e autophagic vacuoles contained relatively large a m o u n t s o f glycogen. T h e results o f the m o r p h o m e t r i c analysis are shown in T a b l e IV. DISCUSSION P r e v i o u s studies showed that glucagon or adrenalin produces an acceleration o f the n o r m a l postnatal b r e a k d o w n o f b o t h hyalop l a s m i c a n d l y s o s o m a l glycogen in rat hepatocytes. Insulin or glucose inhibits this b r e a k d o w n (Kotoulas a n d Phillips, 1971; K o t o u l a s et al., 1971). T h e effects on the h y a l o p l a s m i c glycogen could be explained on the basis o f changes in activity o f the e n z y m e phosphorylase. T h e effects on the l y s o s o m a l glycogen were suggested to be due to changes in activity o f the l y s o s o m a l enz y m e acid a-l,4-glucosidase. Indeed, glucagon a n d adrenalin were f o u n d to increase acid glucosidase activity in the liver o f rats a n d rabbits, respectively. Insulin was f o u n d to decrease this activity (Hers, 1963; R o -
senfeld, 1964; Kotoulas, 1981; Kotoulas, 1984). F r o m the data presented in this study, it is a p p a r e n t that cyclic A M P produces an acceleration o f the postnatal b r e a k d o w n o f glycogen in b o t h the h y a l o p l a s m a n d the lysosomes o f hepatocytes. T h e effect o f cyclic A M P on h y a l o p l a s m i c glycogen can be explained on the basis o f an activation o f the e n z y m e phosphorylase (Dawkins, 1963). Cyclic A M P increases the activity o f the e n z y m e acid glucosidase which can degrade lysosomal glycogen. E r g o t a m i n e which is k n o w n to antagonize the action o f cyclic A M P , produces an inhibition o f the breakd o w n o f h y a l o p l a s m i c a n d l y s o s o m a l glycogen. P r e l i m i n a r y o b s e r v a t i o n s suggested that the a d m i n i s t r a t i o n o f e r g o t a m i n e results in a decrease in acid glucosidase activity. In five e r g o t a m i n e - t r e a t e d rats the activity o f e n z y m e was lower, i.e., 0.150 + 0.070 # m o l e g l u c o s e / h r / m g protein, t h a n that in five controls, i.e., 0.245 + 0.080 # m o l e g l u c o s e / h r / m g protein. T h e results were m e a n s __+ standard deviations. T h e difference was statistically significant (P < 0.05). It is not k n o w n whether the effects o f cyclic A M P on acid glucosidase i n v o l v e d activation or synthesis o f the enzyme. Cyclic A M P could act in either way (Greengard, 1969; M e a n s et al., 1984; H e i d e m a n n et al., 1985; Milsted et al., 1985). This is presently u n d e r study a n d will be the subject o f a subsequent report.
215
CYCLIC AMP AND LYSOSOMES
Since exogenous cyclic A M P mimics the effect of glucagon and adrenalin on acid glucosidase and lysosomal glycogen, it is likely that these hormones exert their effect by causing an elevation in the level of nucleotide. Insulin may act to lower the level of nucleotide. Several hormones including glucagon, adrenalin, and insulin produce some of their effects by causing an alteration in the intracellular level of cyclic A M P (Sutherland et al., 1968). These findings provide further support for the hypothesis previously made that breakdown of lysosomal glycogen is controlled by those agents that regulate the breakdown ofhyaloplasmic glycogen (Kotoulas and Phillips, 1971; Kotou1as et al., 1971). It is of interest to note that glucagon, adrenalin, and insulin also influence other aspects ofautophagocytosis such as the change in the volume of lysosomes and Golgi apparatus, and that this effect could be mediated through alterations in the cyclic AMP level (Rosa, 1971; Kotoulas and Phillips, 1971; Pfeifer, 1978; Kotoulas, 1981). An increase of the lysosomal volume by cyclic A M P and a decrease by ergotamine were noted in this work, too. In considering the role of lysosomes in degrading the hepatocellular glycogen, it must be remembered that the lysosomal pathway may be important in cases where there is a demand for the massive liberation of free glucose (Rosenfeld, 1964). Under our experimental conditions the administration of cyclic A M P results in a marked hyperglycemia (Kotoulas and Phillips, 1971). In this case cyclic A M P induces both pathways for glycogen degradation, phosphorolytic in the hyaloplasm and hydrolytic in the lysosomes. Lysosomes can selectively degrade glycogen as well as various other cytoplasmic components and exhibit different rates of removal from the cytoplasm, for different
components (Pfeifer, 1978; Glaumann et al., 1985), The findings of this study and previous ones constitute good evidence for active participation o f lysosomes in the overall breakdown of cellular glycogen in the newborn rat hepatocytes (Kotoulas et al., 1971).
REFERENCES DAWKINS,M. J. (1963) Ann, N. Y. Acad. Sci. 111, 203211, EXTON, J. H., JEFFERSON,L. S., JR., BUTCHER,R. W., ANDPARK,C. R. (1966)Amer. J. Med. 40, 709-715. GLAUMANN,H., AHLBERG,J., BERKENSTAM,m., AND HENELL, F. (1985) in Proceedings, Annual Meeting of the ScandinavianSociety of Electron Microscopy, Link~pin, Sweden. GLINSMANN, W. H., AND MORTIMORE, G. E. (1968) Amer. J. Phys. 215, 553-559. GREENGARD,O. (1969) Biochem J. 115, 19-24. HEIDEMANN, S. R., JOSHI, H. C., SCHECHTER, A., FLETCHER,J. R., ANDBOTHWELL,M. (1985) J. Cell. Biol. 100, 916-917. HERS, H. G. (1963) Biochem. J. 86, 11-16. KOTOULAS,O. B. (1981)Pathol. Res. Pract. 172, 138147. KOTOULAS,O. B. (1984) Bull. Assoc. Anat. (Nancy) 68, 189-197. KOTOULAS,O. B., HO, J., ADACHI,F., WEIGENSBERG, B. I., AND PmLLWS, M. J. (1971) Amer. J. Pathol. 63, 23-36. KOTOULAS,O. B., ANDPHILLWS,M. J. (1971) Amer. J. Pathol. 63, 1-17. LEJEUNE, N., THINES-SEMPOUX,D., AND HERS, H. G. (1963) Biochem. J, 86, 16-21. MEANS, A. R., SLAUGHTER,G. R., AND PUTKEY,J. A. (1984) J. Cell. Biol. 99, 226S-231S. MILSTED,A., Cox, R. P., ANDNILSON,J. H. (1985) J. Cell. Biol. 101, 319a. [Abstract] MORAN, N. C. (1966) Pharmacol. Rev. 18, 503-512. PFE1FER,U. (1978) J. Cell. Biol. 78, 152-167. ROSA, F. (1971) J. Ultrastruct. Res. 34, 205-213. ROSENFELD,E. L. (1964) in WHELAN,W. J., ANDCAMERON, M. P. (Eds.), Ciba Foundation Symposium, pp. 176-192, Churchill, London. SUTHERLAND,E. W., AND PALL, T. W. (1960) Pharmacol. Rev. 12, 265. SUTHERLAND,E. W., ROBISON, A,, AND BUTCHER,R. W. (1968) Circulation 37, 279-306.
97 s
210-215 (1986)
The Effects of Cyclic 3',5'-AMP on the Lysosomes of Newborn Rat Hepatocytes O. B. KOTOULAS Department of Anatomy, Histology, and Embryology, University of Ioannina Medical School, loannina 45332, Greece Received November 21, 1986, and in revisedform July 7, 1987 Certain effects of cyclic 3',5'-AMP administration on the lysosomes of newborn rat hepatocytes were studied using biochemical assays, electron microscopy, and quantitative morphometry. Cyclic A M P produced accelerations of postnatal hyaloplasmic glycogen breakdown and lysosomal glycogen breakdown, and an increase in activity of the lysosomal enzyme acid a-l,4-glucosidase (maltase). Ergotamine, a known antagonist of the effects of cyclic AMP, produced inhibitions of postnatal hyaloplasmic glycogen breakdown and lysosomal glycogen breakdown. Cyclic A M P increased while ergotamine decreased the volume of lysosomes. The results support the postulate that the catabolism oflysosomal glycogen is controlled by those agents that regulate the catabolism ofhyaloplasmic glycogen (O, B. Kotoulas and M. J. Phillips, Amer. J. Pathol. (1971) 63, 1-17; O. B. Kotoulas et al., Amer. J. PathoL (1971) 63, 23-36). Control is mediated by changes in the activity of the lysosomal enzyme acid a-l,4-glucosidase. Lysosomes actively participate in the degradation of hepatocellular glycogen. © 1986AcademicPress, Inc.
The regulation of the breakdown of glycogen inside lysosomes is rather obscure. Previous studies suggested that the lysosomal enzyme acid a-l,4-glucosidase (maltase) may control this process (Hers, 1963; Rosenfeld, 1964; Kotoulas et aL, 1971). Glucagon increases the activity of acid glucosidase in newborn rat liver while insulin decreases this activity (Kotoulas, 1981; Kotoulas, 1984). The actions of glucagon and insulin could be mediated by alterations in the level of cyclic 3',5'-AMP (Exton et aL, 1966; Sutherland et aL, 1968; Glinsmann and Mortimore, 1968). Therefore, the question must be asked whether this agent has an effect on the activity of the enzyme and the breakdown of the lysosomal glycogen. In this paper, the cyclic 3'-5'-AMP-induced change in the activity of the newborn rat liver acid a-l,4-glucosidase is determined and the cyclic AMP-induced ultrastructural changes in the lysosomes of the hepatocytes are described. Also, the ultrastructural changes that occur after the administration ofergotamine, a known antagonist of the effects of cyclic AMP, are
described (Sutherland and Rail, 1960; Moran, 1966; Sutherland et aL, 1968). MATERIALS A N D METHODS
Animals and Handling of Tissues Newborn rats were obtained from 10 pregnant Wistar females. The average litter contained 10 newborns. The sex of the newborns was not determined. The newborns were sacrificed at birth or kept at 35°C and Sacrificed after 6 hr, unless otherwise specified. Part of the liver was processed for electron microscopy and the rest was immersed in liquid nitrogen and used for enzyme assays.
Chemicals Cyclic adenosine-3',5'-phosphate (cyclic AMP) (Cat. No. 10870) and glycogen (Cat. No. 23540) were obtained from Serva. Ergotamine tartrate, Lot 67033, was from Sandoz. The reagents for determining glucose and for electron microscopy were obtained as before (Kotoulas, 1981).
Biochemical Methods The acid a-1,4-glucosidase assay was performed on homogenates of liver tissue in a 10% dilution with distilled water as before (Kotoulas, 1981). Usually 100 X of homogenate were used for the assay. The total activity of the enzyme was determined according to 210
0889-1605/86 $3.00 Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
211
CYCLIC AMP AND LYSOSOMES Hers (1963) and Lejeune et al. (1963) with glycogen as substrate.
Electron Microscopy and Morphometric Analysis Electron microscopy was performed as described previously (Kotoulas, 1981). The sections were examined in Philips 300 and Hitachi IIB electron microscopes (Department of Pathology, McGill University, Montreal, Canada) and partly in a Philips 75 electron microscope (Department of Clinical Therapeutics, University of Athens, Greece). Morphometric analysis was made as described previously (Kotoulas and Phillips, 1971). From each group three experimental animals and three controls from the same litters were used. From each liver, five blocks were prepared. Two randomly taken micrographs from each block were used for morphometry.
Experimental Design, Dose, Route, and Mode of Administration Ten animals were sacrificed at birth. The rest of the animals were divided into two groups according to the agent used, cyclic AMP or ergotamine. Ten cyclic AMPtreated animals and ten controls from the same litters were sacrificed at the age of 6 hr. Cyclic AMP was administered intraperitoneally in 0.2 ml of a 0.3% solution of the compound in 0.9% NaCI. The animals were injected at birth and 3 hr after birth (a dose of 100 mg/kg each time). Control animals were injected at birth and 3 hr after birth with 0.9% NaC1. Ten ergotamine-treated animals and their normal controls were sacrificed at the age of 6 hr. Ergotamine was administered intraperitoneally in 0.1 ml of a 0.05% solution. The animals were injected at birth and every hour thereafter (a dose of 8.3 mg/kg each time). RESULTS
Biochemical Results F o u r n e w b o r n animals were used in a prel i m i n a r y e x p e r i m e n t in order to d e t e r m i n e the response o f the a n i m a l to repeated in: jections o f cyclic A M P . T h e agent was adm i n i s t e r e d at b i r t h o r at b i r t h a n d 3 h r a f t e r
TABLE I GLUCOSIDASE ACTIVITY AT THE AGE OF 6 HR AFTER ADMINISTERING A DOSE OF 100 MG/KG CYCLIC A M P 0 AND 3 HR AFTER BIRTH (/.tMOLE GLUCOSE/HR/MG PROTEIN)
Control Cyclic AMP P
0.274 +_ 0.085 (lO) 0.598 +- 0.210 (lO) <0.05
Note. Results are means +. standard deviations. Numbers in parentheses represent the number of observations. Controls were injected at 0 and 3 hr with carrier only. birth. T h e a n i m a l w h i c h w as i n j e c t e d at b i r t h a n d its c o n t r o l w e r e s a c r i f i c e d at t h e age o f 3 hr. T h e a n i m a l w h i c h w a s i n j e c t e d at b i r t h a n d 3 h r af t er b i r t h a n d its c o n t r o l w e r e s a c r i f i c e d at t h e age o f 6 hr. N o i n c r e a s e i n the liver acid a- 1,4-glucosidase activity was o b s e r v e d at t h e age o f 3 h r i n t h e a n i m a l r e c e i v i n g o n l y o n e i n j e c t i o n w i t h cyclic A M P (control 0.135, experimental 0.149 ~ m o l e / h r / m g protein). A m a r k e d increase was note d at t h e age o f 6 h r i n t h e a n i m a l r e c e i v i n g two injections with cyclic A M P (control 0.223, e x p e r i m e n t a l 0 . 4 6 6 ~ m o l e / h r / m g protein). The level o f activity o f acid glucosidase, at t h e age o f 6 h r i n cyclic A M P - t r e a t e d animals and their controls, was d e t e r m i n e d in a more definitive experiment. The a d m i n i s t r a t i o n o f c y c l i c A M P r e s u l t e d in a s i g n i f i c a n t i n c r e a s e o f t h e a c t i v i t y o f t h e en z y m e . I n 10 t r e a t e d a n i m a l s t h e e n z y m e act i v i t y w a s 0 . 5 9 8 +_ 0 . 2 1 0 g m o l e / h r / m g p r o t e i n w h i l e i n 10 c o n t r o l s it w a s 0 . 2 7 4 _+ 0 .0 8 5 ~ t m o l e / h r / m g p r o t e i n ( T a b l e I).
TABLE 1I VOLUME FRACTIONS OF CYTOPLASMIC COMPONENTS OF THE RAT HEPATOCYTE AT BIRTH
Percentage of cytoplasmic volume~ Glycogen
Lysosomes
Glycogen in autophagic vacuoles
50.0 +_ 3.3
0.35 ± 0.08
0.15 +. 0.03
Percentage of lysosomal volume occupied by glycogen 43
a Results are means +__standard errors. For the morphometric analysis three newborn animals were used. From each liver five blocks were prepared and from each block two randomly taken micrographs were used, The results were computed from a total of 30 micrographs and an area of 5100 ~tm2.
212
O.B. KOTOULAS
213
CYCLIC AMP AND LYSOSOMES TABLE III EFFECTS ON HEPATOCYTES OF ADMINISTERING CYCLIC A M P TO NEWBORN RATS AT THE AGE OF 6 rm
Percentage of cytoplasmic volumea Treatment Controlb
Cyclic AMPc P
Glycogen
Lysosomes
12.6 _+ 2.1 8.0 + 1.5 <0.05
1.29 _+ 0.11 1.97 _+ 0.18 <0.05
Percentage of lysosomal volume ocGlycogen in autophagic vacuoles cupied by glycogen 0.23 +- 0.04 0.14 + 0.03 <0.05
18 7
Results are means + standard errors. For the morphometric analysis three experimental animals and three controls from the same litters were used. From each liver five blocks were prepared and from each block two randomly taken micrographs were used. bResults were computed from a total of 30 micrographs and an area of 5200 # m 2. c Results were computed from a total of 30 micrographs and an area of 5350 gm2.
Morphologic Results Both qualitative and quantitative changes were studied. The term lysosomes refers to lysosomes and related particles including autophagic vacuoles. In the tables o f m o r p h o m e t r i c analysis the term glycogen refers to hyaloplasmic glycogen unless otherwise specified (Kotoulas and Phillips, 1971). Group I: Cyclic AMP administered. The ultrastructure o f control hepatocytes during the first postnatal hours was as previously (Kotoulas and Phillips, 1971; Kotoulas, 1984). At birth vast stores o f hyaloplasmic glycogen were present. L y s o s o m e s and autophagic vacuoles were rare and small. A large part, i.e., 43%, o f the total lysosomal v o l u m e was occupied by glycogen (Table II). At the age o f 6 hr the hyaloplasmic glycogen was reduced. L y s o s o m e s were well developed. These organelles occurred pred o m i n a n t l y at the j u n c t i o n o f glycogen and glycogen-free areas. It was estimated that at least 8 5% o f the lysosomal v o l u m e belonged
to lysosomes o f the autophagic type. Autophagic vacuoles contained moderate a m o u n t s o f glycogen (Fig. 1). M o r p h o m e t r i c analysis showed that 18% o f the lysosomal v o l u m e was occupied by glycogen (Table III). In the cyclic A M P - t r e a t e d animals at the age o f 6 hr the depletion o f h y a l o p l a s m i c glycogen was a d v a n c e d and in some cells complete. Lysosomes were n u m e r o u s and often occurred at the margins o f the stores o f glycogen as in the control animals. At least 8 5% o f the total lysosomal v o l u m e was occupied by lysosomes o f the autophagic type. The autophagic vacuoles contained very small a m o u n t s o f glycogen (Fig. 2). M o r p h o m e t r i c analysis showed that 7% o f the total lysosomal v o l u m e was occupied by glycogen (Table III). M i t o c h o n d r i a in various stages o f disintegration were often seen inside autophagic vacuoles. Some o f the lysosomes contained a m o r p h o u s or m e m b r a n o u s electron-dense material and had the appearance o f residual bodies. Group II: Ergotamine administered. The n o r m a l controls at the age o f 6 hr were ob-
FIG. 1. Portion of a control rat hepatocyte at the age of 6 hr. The animal was injected with carrier only. A lysosome-autophagicvacuole (ly) is present. Moderate amounts of glycogen(gly)are seen in the hyaloplasm and within the lysosome-autophagicvacuole. Uranyl acetate-Reynold's lead citrate. × 41 600. FIG. 2. Portion of a cyclic AMP-treated rat hepatocyte at the age of 6 hr. The animal was injected at 0 and 3 hr after birth with a dose of 100 mg/kg each time. Large lysosome-autophagic vacuoles (ly) are present. Glycogen is negligible both in the hyaloplasm and within the lysosomes. Uranyl acetate-Reynold's lead citrate. × 41 600.
214
o. B. KOTOULAS TABLE IV EFFECTS ON HEPATOCYTES OF ADMINISTERING ERGOTAMINE TO NEWBORN RATS AT THE AGE OF 6 HR
Percentage of cytoplasmic volumea Treatment
Glycogen
Lysosomes
Control b Ergotaminec P
11.6 _+ 2.2 31.0 _+ 2.8 <0.05
1.35 _+ 0.20 0.58 + 0.11 <0.05
Percentage of lysoGlycogen in somal volume ocautophagic vacuoles cupied by glycogen 0.24 + 0.07 0.22 + 0.07 >0.5
18 38
a Results are means __+standard errors. For the morphometric analysis three experimental animals and three controls from the same litters were used. From each liver five blocks were prepared and from each block two randomly taken micrographs were used. b Results were computed from a total of 30 micrographs and an area of 4950 ttmL c Results were computed from a total of 30 micrographs and an area of 5400 tim2.
served to h a v e the s a m e ultrastructural features described for the control animals o f G r o u p I. Treating the animals with erg o t a m i n e resulted in inhibited depletion o f h y a l o p l a s m i c glycogen. In the e r g o t a m i n e treated a n i m a l s at the age o f 6 hr the lysos o m e s were small. These organelles usually occurred at the j u n c t i o n o f glycogen a n d glycogen-free areas. At least 85% o f the total lysosomal v o l u m e was occupied by lysos o m e s o f the autophagic type. T h e autophagic vacuoles contained relatively large a m o u n t s o f glycogen. T h e results o f the m o r p h o m e t r i c analysis are shown in T a b l e IV. DISCUSSION P r e v i o u s studies showed that glucagon or adrenalin produces an acceleration o f the n o r m a l postnatal b r e a k d o w n o f b o t h hyalop l a s m i c a n d l y s o s o m a l glycogen in rat hepatocytes. Insulin or glucose inhibits this b r e a k d o w n (Kotoulas a n d Phillips, 1971; K o t o u l a s et al., 1971). T h e effects on the h y a l o p l a s m i c glycogen could be explained on the basis o f changes in activity o f the e n z y m e phosphorylase. T h e effects on the l y s o s o m a l glycogen were suggested to be due to changes in activity o f the l y s o s o m a l enz y m e acid a-l,4-glucosidase. Indeed, glucagon a n d adrenalin were f o u n d to increase acid glucosidase activity in the liver o f rats a n d rabbits, respectively. Insulin was f o u n d to decrease this activity (Hers, 1963; R o -
senfeld, 1964; Kotoulas, 1981; Kotoulas, 1984). F r o m the data presented in this study, it is a p p a r e n t that cyclic A M P produces an acceleration o f the postnatal b r e a k d o w n o f glycogen in b o t h the h y a l o p l a s m a n d the lysosomes o f hepatocytes. T h e effect o f cyclic A M P on h y a l o p l a s m i c glycogen can be explained on the basis o f an activation o f the e n z y m e phosphorylase (Dawkins, 1963). Cyclic A M P increases the activity o f the e n z y m e acid glucosidase which can degrade lysosomal glycogen. E r g o t a m i n e which is k n o w n to antagonize the action o f cyclic A M P , produces an inhibition o f the breakd o w n o f h y a l o p l a s m i c a n d l y s o s o m a l glycogen. P r e l i m i n a r y o b s e r v a t i o n s suggested that the a d m i n i s t r a t i o n o f e r g o t a m i n e results in a decrease in acid glucosidase activity. In five e r g o t a m i n e - t r e a t e d rats the activity o f e n z y m e was lower, i.e., 0.150 + 0.070 # m o l e g l u c o s e / h r / m g protein, t h a n that in five controls, i.e., 0.245 + 0.080 # m o l e g l u c o s e / h r / m g protein. T h e results were m e a n s __+ standard deviations. T h e difference was statistically significant (P < 0.05). It is not k n o w n whether the effects o f cyclic A M P on acid glucosidase i n v o l v e d activation or synthesis o f the enzyme. Cyclic A M P could act in either way (Greengard, 1969; M e a n s et al., 1984; H e i d e m a n n et al., 1985; Milsted et al., 1985). This is presently u n d e r study a n d will be the subject o f a subsequent report.
215
CYCLIC AMP AND LYSOSOMES
Since exogenous cyclic A M P mimics the effect of glucagon and adrenalin on acid glucosidase and lysosomal glycogen, it is likely that these hormones exert their effect by causing an elevation in the level of nucleotide. Insulin may act to lower the level of nucleotide. Several hormones including glucagon, adrenalin, and insulin produce some of their effects by causing an alteration in the intracellular level of cyclic A M P (Sutherland et al., 1968). These findings provide further support for the hypothesis previously made that breakdown of lysosomal glycogen is controlled by those agents that regulate the breakdown ofhyaloplasmic glycogen (Kotoulas and Phillips, 1971; Kotou1as et al., 1971). It is of interest to note that glucagon, adrenalin, and insulin also influence other aspects ofautophagocytosis such as the change in the volume of lysosomes and Golgi apparatus, and that this effect could be mediated through alterations in the cyclic AMP level (Rosa, 1971; Kotoulas and Phillips, 1971; Pfeifer, 1978; Kotoulas, 1981). An increase of the lysosomal volume by cyclic A M P and a decrease by ergotamine were noted in this work, too. In considering the role of lysosomes in degrading the hepatocellular glycogen, it must be remembered that the lysosomal pathway may be important in cases where there is a demand for the massive liberation of free glucose (Rosenfeld, 1964). Under our experimental conditions the administration of cyclic A M P results in a marked hyperglycemia (Kotoulas and Phillips, 1971). In this case cyclic A M P induces both pathways for glycogen degradation, phosphorolytic in the hyaloplasm and hydrolytic in the lysosomes. Lysosomes can selectively degrade glycogen as well as various other cytoplasmic components and exhibit different rates of removal from the cytoplasm, for different
components (Pfeifer, 1978; Glaumann et al., 1985), The findings of this study and previous ones constitute good evidence for active participation o f lysosomes in the overall breakdown of cellular glycogen in the newborn rat hepatocytes (Kotoulas et al., 1971).
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