two degradation pathways of endoplasmic reticulum in fish hepatocytes

Biol Cell (1995) 83,69-76 0 Elsevier, Paris

69

Original article

Two degradation pathways of endoplasmic reticulum in fish hepatocytes

Peter VeraniC, Nada Pipan

Institute of Cell Biology, Medical Faculty, 61105 Ljubljana, Lipiteva 2, Slovenia (Received 26 August 1994; accepted 22 February 1995)

Summary - Zebrafish hepatocytes respond to stimulation with estradiol 17J (E2) with an extreme enlargement of the endomembrane system, especially the endoplasmic reticulum (ER), and when stimulation is stopped with a rapid degradation of enlarged endomembranes. Two pathways for degradation of ER were studied: a) the autophagy which was evaluated by stereological measurement; and b) the activity of cytosolic phospholipase B (PL-B) measured by a titration method. After a 30-day treatment with E2 a six-fold increase of the surface density of ER was accompanied by an increase of both autophagy and PL-B activity. 2 days after stopping the stimulation with E2 the ER vesiculated and its surface density decreased to the half value. Interestingly, at the same time autophagic vacuoles (AV) almost disappeared from hepatocytes, while the activity of PL-B reached its maximum at which it persisted for a further 4 days. After 4-6 days without E2 the cistern of ER became flattened again and new AVs reappeared. The data suggest that the regulation mechanisms of endomembrane degradation by PL-B and autophagy do not depend on each other and also that the appearance of AV is strongly related to the shape of ER.

endoplasmic reticulum / autophagocytosis / phospholipase B / degradation

Introduction

It is commonly accepted that the autophagy is the major pathway for degradation of the endoplasmic reticulum (ER) [4, 141. In this process parts of ER cistern are engulfed by sequestrating membranes for which it is still not certain whether they belong to a special part of the ER [lo, 12, 341 or if they are an independent organelle called phagophor [8, 311. After sequestration of a part of the cytoplasm an early autophagic vacuole is formed. It fuses with a lysosome containing enzymes for degradation of the autophagic content. Recent results have suggested that these enzymes may be delivered also by a late endosomal or a prelysoso- ma1 compartment [ 13,291.

Degradation of proteins by autophagy is well accepted [ 11. In contrast, experiments comparing the degradation of membrane lipids in lysosomes and in free cytosol are very rare [5]. The enzyme most representative for degradation of membrane lipids is lysophospholipase, also called phospholipase B (PL-B) because it completes the deacyla- tion of phospholipids and causes the release of glycerol from the membrane sheet [5, 6, 161. Already for more than 25 years has been found that 80% of the liver PL-B activity is located in the cytosol [5]. Considering that 40-60% of cell membranes are composed of lipids [ 181 a prediction could be made that cytosolic phospholipases, by playing an important role in turnover of membrane lipids, also regulate the quantity of endomembranes. Thus a possibility exists that enzymes involved in autophagic degradation cooperate

Abbreviations: AV, autopbagic vacuole; E2, estradiol 178; ER, endoplasmic reticulum; DMF, dimethyl formamide; PL-B, phospholipase B; Q, chloroquine; Nv, numerical density; S,, surface density; V,, volume density.

with cytosolic phospholipases in the process of endomembrane degradation.

In the present work we quantified autophagic vacuoles (AV) and measured the activity of cytosolic PL-B in fish hepatocytes during the enlargement and decrease of endomembranes. The dynamics of endomembrane quantity were enhanced by treatment with estradiol 17J (E2) which stimu- lates the synthesis of lipoproteins vitellogenin [26] and very low density lipoprotein (VLDL) [37], and with a subsequent cessation of the treatment. Zebrafish were chosen as a model because of their extraordinary responsiveness to stimulation with E2 [25,35].

In our work we compared the dynamics of autophagy and cytosolic PL-B during enlargement and diminishing of the endomembrane system and evaluated thus the role of cytosolic PL-B in endomembrane degradation.

Materials and methods

Animals

Male zebrafish (Brachydanio rerio) were raised under laboratory conditions of constant water temperature (24°C) and a 14 h light/l0 h dark rhythm. The animals were fasted for 24 h before killing.

Estradiol treatment

Estradiol 17J (E2) was dissolved in dimethyl formamide (DMF) (2 pg E2/0.01 ml DMF) and added to aquarium water (2 pg E2/1 1 water) [25]. For all the methods used fish were divided into groups. In the first control group fish did not receive E2 or DMF and in the second control group fish were treated with DMF only. In the first experimental group fish received for 30 days E2 dissolved in DMF. In all other groups E2 and DMF

were omitted for 2, 4, 6 and 12 days after 30 days of treatment with this hormone.

Chloroquine treatment

To all groups of fish, where AVs were stereologically measured, chloroquine (Q) was added directly to the water (1 ~mg Q / 100 ml water) 5 h prior to killing. The pH of watei was adjusted to 8.3 to minimise the differences in entrance of Q into the fish body.

Morphometp-

Sampling and preparation of the tissue were done as described [35]. On the technically best ultrathin section, cut from each piece of tissue, six micrographs were prepared. The position of the micrographs was fixed independent of the structure, in accordance with systematic sampling [39]. A coherent multipurpose test system with 84 test lines and 164 end points (P,) was used for analysis. Each group of fish consisted of 3-5 animals. For autophagic vacuoles the volume density (Vv) and numerical density (NV) were measured.

Nv = K /J (N,3 I Vv)“‘2 [38] where K = 1 (distribution fac- tor), p = 1.38 (shape dependent coefficient), N, = number of vacuoles per unit area.

V, = P, / PT [38] where PT = test points end P, = fraction of points on a profile.

For the ER.the surface density (S,) was measured. Sv = 2P, / L, [38] where.P, = intersection points and L, = total test lines.

Biochemical determination ofPL-B

A modification of the method of Larsh et al [21] was used for preparation of the tissue and estimation of the activity of PL-B. The animals were killed by decapitation. The liver was removed and shortly dried on filter paper, weighed and transferred to a glass homogeniser. Ten volumes of cold (4°C) glycerol buffer (12.5% glycerol, 0.1 M potassium phosphate (pH 6.5), 5 mM MgCI,, 2 mM EDTA) were added. The tissue was homogenised for 40 s. 50 fl of homogenate were diluted six-fold. with glycerol buffer and 150 ~1 of lysolecithin~.sdu.tion (20 mM) was added. After 30 min of incubation at 35°C the reaction was stopped by addition of 50 ~1 of 2 N H,SO,. The free ftitty acids were transferred from water medium over isopropyl alcohol to heptan according to Larsh et al [21]. The heptan was carefully removed and titrated under nitrogen with 2 mM NaOH [9]. A 2 mM solution of pahnitic acid in heptane was used as a reference standard. We used two controls: in the first iysolecithin was omitted~ and the second one was without homogenate. Each group of fish consisted of 10 animals. Livers of two animals were homogenised for a sample.

Estimates of statistical significance for stereological and biochemical measurement were performed by the Student’s t-test.

Cytochemistq

PL-B activity A modification of the Nagata and Iwadare [23] method was used to demonstrate the localisation of cytosolic PL-B activity in hepatocytes. Pieces of tissue were fixed in calcium-form01 fixative at 25°C for 30 min [28]. After freezing at -30°C the pieces of tissue were cut with a cryomicrotome:at 30 pm. The sections were incubated for 25 min at 35°C in a reaction medium [28J. After incubation the sections were rinsed twice in 0.9% NaCl solution for 3 min each and the calcium precipitate of fatty acids was con- verted with 0.2% Pb(NO,), solution for 10 min. After washing the tissue was post-fixed in 0.1 M cacodylate buffered 1% 0~0, for 1 h and embedded in Epon.

Acid phosphatase activity Livers were fixed in 0.1 M cacodylate buffered 4% paraformal- dehyde/2% glutaraldehyde mixture for 2 h. After rinsing over- night the tissue was sectioned into 70 pm thick pieees using cryomicrotome. Acid phosphatase activity was localized by the

lead method (31. Liver was post-fixed in 2% 0~0, reduced li;~, K,Fe(CN), [ 171.

For cytochemical analysis each group consisted of 3-S an- mals.

ReSUlt.5

Treatment with E2

After treatment with E2 for 30 days, in male fish-hepatocytes, stereological analysis revealed a six-fold in-ase Of the surface density of ER (fig 1). At the same time the number of dictyosomes also rose. The quantity of other membranes of hepatocytes, among which the mitochondrid membranes formed the main part, remained unchanged (fig I). The gum- ber of autopbagic vacuoles (AV) in hepatocytes was very low in both untreated fish and those treated for 30 days with E2. Quantification of these vacuoles was possible only after treatment of fish with chloroquine (Q). We found that after 5 h of treatment with Q tie quantity of AV viras sufIicient for stereological measurement (fig 2) whereby no visible changes of other compartments were detected in he@atocytes (fig 3). All further results of stereological measueme<t of AV were obtained on fish treated w&Q for 5 h.

During stimulations with E2 the volume density of AV increased significantly buf the numerical density persisted at the same level (figs 4, 5), suggesting that the average diameter of AV increased. However, in hepatocyles of both control and E2 treated fish all steps of AV were found from

+

8’ I r-7 * * I

I ++ *

6 +

CDMFEZ 2 4 6 12

/--.j rest of membranes [ ----.

1 membranes of ER

Fig 1. Stereological values for the surface density (S,) of ER and for the rest of membranes of hepatocytes were calculated in untreated fish (C), after 30 days of treatment with DMF, after 30 days of-treatment with E2 dissolved in DME and at 2,4,6~and 12 days after stopping the treatment with this hormone. Statisti.. caily significant differences in S, of ali endomembranes~compared to untreated fish (+) and to E2-treated fish @) (P < 0.M)

Endoplasmic reticulum in fish hepatocytes 71

l-Q) (Q3h) (Q5h)

I V, (x O.O1pmO)

N NV (X 0.001 pm -3)

Fig 2. The volume density (V,) and numerical density (Nv) of AV were measured in fish which did not receive chloroquine (-Q) and 3 or 5 h after treatment with this lysosomotropic drug.

Fig 3. Hepatocytes of fish after 30 days of treatment with E2 in DMF and 5 h in chloroquine posses a lot of ER which is also the main content of AV (t) (x 10000; Bar = 1 pm).

C DMF E2 2 4 6 12 CDMFE2 2 4 6 12

*

84 * t +

0 4-

Figs 4,5. Stereological values for volume density (V,) (4) and numerical density (NV) (5) of AV measured in fish which did not receive E2 or DMF (C), in those treated with DMF for 30 days, after 30 days of stimulation with E2 and at 2,4, 6 and 12 days after stopping the treatment with E2 in DMF. Chloroquine was added to all fish 5 h before killing. Statistically significant differences compared to untreated fish (t) and to fish treated for 30 days with E2 (*) (P < 0.01).

72 I-’ Veranic. N Pipan

small early AV surrounded by two layers of smooth mem- Activity ot the cytosolic PL-B with its pH optimum az branes (fig 6) to big AV with plenty of different inclusions 6.5 (fig 9) did also increase in hepatocytes after treatment which probably belong to the class of vacuoles called with E2 (fig 10). Histocheinically it was demonstrated that amphysomes (fig 7). The main difference between these the activity of this enzyme mainly appears in ER and to :I vacuoles in control and E2 treated fish was in the content of much lower extent on membranes of mitochondria (fig 1 1 ;I. glycogen, which was more prominent in AV of control fish Some unspecific labelling was found only at the rim of lipid (rig 8) than after treatment with E2 (figs 6,7). droplets (fig 12). At pH 6.5 no activity of PL-B was seen in

Figs 6-8. Early autophagic vacuoles (6) surrounded with two layers of smooth membranes (T) and large amphysomes (*) (7) contaiang different cytoplasmic contents, appear frequently in hepatocytes treated with E2 in DMF f& -30 days and with Q for 5 h. 6; x 22OOB. bar = 1 pm. 7. x 15000, bar = 1 pm). 8. In hepatocytes of fish not treated with E2 some iarge AV (T) were found containinggtycogen (G) which was abundant in surrounded cytoplasm. Fish were treated with Q for 5 h. x 14000, bar = I pm.


2 c

6.0 6.5 7.0 7,5

0

PH CDMFE2 2 4 6 12

Figs 9,10.9. The activity of PL-B in liver homogenate was measured at different pH values. 10. The activity of PL-B was measured at pH 6.5 in hepatocytes of non-treated fish with E2 or DMF (C), treated for 30 days with DMF, treated for 30 days with E2 in DMF or at 2, 4, 6 or 12 days after stopping the 30-day treatment with E2. Statistically significant differences compared to untreated fish (+) and to fish treated for 30 days with E2 (*) (P < 0.01).

vacuoles which could, by their morphology and diameter, be referred to as lysosomes. However, with the cytochemical method of acid phosphatase lysosomes were demonstrated in both control and E2 treated fish (fig 13).

Stopping the treatment with E2

2 days after stopping of treatment with E2 the ultrastructure of hepatocytes was not homogeneous. In some fish the quantity and shape of ER remained the same as in E2 treated fish. In others, the quantity of ER did not change but the cisternae were almost completely vesiculated (fig 14). In hepatocytes of most fish ER was completely vesiculated and its surface density diminished to the half value found in fish treated with E2 (fig 1).

Surprisingly we found that at this early stage, after stopping the treatment with E2, the autophagy decreased to the minimum in all hepatocytes with vesiculated ER regardless of the quantity of ER (fig 4). No early AV were seen and there were only a few late AVs with unrecognisable ingre- dients.

At the time when the autophagy was inhibited, 2 days after stopping the treatment with E2, the activity of PL-B reached its maximum which was significantly higher than in animals treated for 30 days with E2 (fig 10) in spite of the fact that the total quantity of membranes in hepatocytes was lower (fig I). Cytochemical localisation of PL-B was not as homogeneous as in non-treated and 30-day treated animals. The reaction product strongly covered some mem- branous structures, whereas the mitochondria and the nuclear envelope contained evidently less reaction product (fig 15).

During the next 2 days without E2, ER of most hepatocytes became first reticular and then flattened again. The surface density of ER from day 4 to day 12 slowly approached the value found in fish before treatment with E2 (fig 1).

4 days after cessation of the treatment with E2 small early AVs appeared again and their volume density rose quickly till day 6 without E2 when autophagy was most prominent. Afterwards the autophagy slowly decreased during the next 6 days (fig 4).

The activity of PL-B remained at the same level between the 2nd and the 6th day after stopping the treatment with E2 and was diminished again at day 12 (fig 10).

Discussion

The number of AVs was surprisingly low in hepatocytes of zebrafish. Thus, for evaluation of our morphometric results, the application of chloroquine (Q) was crucial. Q is a weak base which accumulates in acid compartments [ 151. By pro- tonation of its own molecule Q increases the pH of acidic autophagic vacuoles beyond the optimum for their enzymes. Because of the inhibition of hydrolytic enzymes which would degrade the content of AV, the life time of these vacuoles increases and so does their number [19]. Q does not interfere with the sequestration, so the increased number of AV is not the result of a stimulated autophagy but presents the AV formed over a longer period. Exposure time for Q was longer than recommended for rat liver [41] or pancreas [42], because we found that both numerical and volume density of AV rose almost linearly from fish which did not receive Q over 3-5 h of treatment with this lyso-

74 P Veraruc, N Plpan


somotropic drug. Treatment as long as 7 h was frequently fatal for fish and was not included in the analysis.

Both autophagy and extralysosomal phospholipases are known to contribute to turnover of endomembranes (for reviews see [6, 111). Our results, which show that the quantity of AV after 30-day stimulation with E2 was much higher than in non-stimulated fish, seem to be in contra- diction with the results of Bahro et al [2]. They found a decreased autophagy after stimulation of the submandibu- lar gland with isoproterenol. Reduced autophagy was found also after partial hepatectomy in rat [27]. It can be concluded that a short-term stimulation of anabolic processes depresses autophagy. In our experiments the situa- tion was quite different, because after 30 days of treatment with E2 the quantity of ER was at its maximal level where it persisted also after a further 10 days of stimulation [35]. A possible reason for the increased autophagy in cells with maximally developed endomembranes is a higher turnover of membrane components and also the regulation of the quantity of membranes at the level which still enables a normal functioning of the cell [32]. Besides the membrane turnover the autophagy is also responsible for degradation of secretory products of the cell. This process is called crinophagy [33] and its meaning for the cell is still uncertain. However, the process was found in several types of cells [32]. During estrogenization of zebrafish the hepatocytes were in an extremely secretory phase where crinophagy could, to a certain extent, contribute to regulation of the synthesis of lipoproteins like vitellogenin and VLDL.

which engulfs parts of the cytoplasm and creates AV. These results are in accordance with a functional dependence of AV on ER [lo].

Reports from other laboratories and our results support the variability of degradation pathways of ER in different cells. In the work of Hehninen and Ericson [14] who studied involution of prostate after castration and in Bolender and Weibels work [4] who measured the removal of membranes in hepatocytes after stopping the treatment with phenobarbital, autophagy was declared as a system which pref- erentially removes ER. The opposite was shown by Romagnoli et al [30] who could not find any increase of the volume density of lysosomes during involution of endomembranes in crown odontoblasts after primary dentinogenesis. They predicted that organelles on the secretory pathway posses an independent system regulating their quantity.

Our results are somewhere in between the reports men- tioned above. Degradation of endomembranes at the early phase, 2 days after cessation of treatment with E2, did not depend on autophagy because there was an obvious decrease in the quantity of AV in hepatocytes. However, after another 4 days without E2 autophagy became very prominent and was probably responsible for the final removal of redundant ER.

The activation of PL-B during stimulation of fish with E2 is probably even more due to a higher turnover of phospholipids in membranes because of their shorter life-time in comparison with proteins [24]. Another reason for activation of PL-B in hepatocytes treated with E2 may be the involvement of phospholipase A in exocytosis [20] which was increased after stimulation of the synthesis of vitellogenin with E2. Vitellogenin is namely a constitutively secreted lipoprotein for which the whole sequence from synthesis to secretion is estimated to take 2 h [7]. This process of exocytosis demands free fatty acids which promote membrane fusion [22]. Free fatty acids are produced in the cytoplasm by the action of phospholipase A which cleaves them from phospholipids producing lysophospholipids as a by-product [40]. Lysophospholipids act destructively on membranes [40] thus PL-B should be activated to degrade this cytotoxic agent.

Activation of PL-B in hepatocytes promptly after stopping the stimulation with E2 indicates that the phospholipases could take a major part in degradation at the time when autophagy was decreased. Higher accumulation of the reaction product of the cytochemical reaction for PL-B localisation on some membranes of cells with vesiculated ER was evidently different in comparison to untreated fish or fish treated with E2 for 30 days where the reaction product was evenly distributed over all endomembranes. We believe that the PL-B in cells with a developing endomembrane system contributes to the process of membrane tum- over, whereas during the involution of the endomembrane system phospholipases actively degrade parts of membranes.

Both degradation processes, with autophagy and PL-B, were strongly influenced by stopping the stimulation of vitellogenin synthesis in fish hepatocytes.

At the 4th day after stopping the treatment with E2, when a new rise in autophagy joins the degradation with PL-B, involution of ER became slower than 2 days before when AVs were extremely rare. This contradic- tion can be explained by a known fact that formation of AVs depends on ER [lo]. The difference in Vv of AV in zebrafish hepatocytes between the second and the sixth day after removal of E2 increased eight-fold so the degradation of ER with AVs and phospholipases could be masked by development of ER because of formation of new AVs.

2 days after cessation of the treatment with E2, AV prac- We hypothesise that degradation of ER in fish hepatocy- tically disappeared from hepatocytes. This change coin- tes after removal of E2 was a two-step process. Within the cided with an almost complete vesiculation of ER. New AV first 2 days, when ER was vesiculated and autophagy were observed only after reconstitution of some normally diminished, degradation of the endomembrane system shaped cistern of ER on the 4th day without E2. Our obser- depended predominantly on phospholipases. After reconsti- vations indicated that vesiculation of ER changes the ability tution of normally shaped ER the autophagy and phospholi- of this compartment to form the sequestrating membrane pases together degraded the excessive ER.

Figs 11-15.11. The reaction product of the cytochemical reaction for localisation of PL-B activity was most abundant in ER (A) and at the rim of lipid droplets. (t) x 30000, bar = 1 mu. 12. The latter seems to be unspecific because the reaction product was found there also when the substrate (lysolecitbin) was omitted (7). x 20000, bar = 1 pm. In figures 11 and 12 the fish were treated with E2 in DMF for 30 days. 13. Lysosomes were labelled with the cytochemical method for acid phosphatase in hepatocytes of non-treated fish with E2. x 20000, bar = 1 pm. 14. In most fish where treatment with E2 was stopped for 2 days, ER was completely vesiculated. x 7500, bar = 1 ,um. 15. The product of the cytochemical reaction for localisation of PL-B activity was concentrated on some vesiculated membranes of fish hepatocytes where treatment with E2 was interrupted for 2 days. x 40000, bar = 1 pm.

16 I’ Veranic, N Pipan

Acknowledgments

The authors would like to thank Prof Dr Rado Komel and Dr Damjana Rozman for lending the equipment for measurement of PL-B activity, Prof Dr Robert Zorec for critical reading and comments,n the manuscript and to M SC Laura Kocmur and Dr Marko Zivin for their help in creating graphs.

References

1 Ahlberg J, Berkenstam A, Henell F, Glaumann H (1985) Degradation of short and long lived proteins in isolated rat liver lysosomes. J Biol Chem 260.5847-5854

2 Bahro M, Dsmmrich J, Pfeifer U (1987) Short-term inhibition of cellular autophagy by isoproterenol in the subman- dibular gland, Virchow Arch 53, 32-36

3 Barka T, Anderson PJ (1962) Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J Histochem Cytochem lo,74 1

4 Bolender RP, Weibel ER (1973) A morphometric study of the removal of phenobarbital-induced membranes from hepatocytes after cessation of treatment. J Cell Biol56,746-761

5 van der Bosch H, Aarsman AJ, Slotbloom AJ, van Deenen LLM (1968) On the specificity of rat-liver lysophospholipase. Biochim Biophys Acta 164,215-225

6 Brockerhoff H, Jensen RG [1974) In: Lipolytic enzymes. Academic Press, New York, San Francisco, London. 194-195

7 Clemens MJ (1974) The regulation of egg yolk protein synthesis by steroid hormones. Prog Biophys Mel Biot 28, 71-107

8 David H, Ellerman J, Uerlings I (1992) Primary phase of hepatocytic autophagocytosis under ischemic conditions. Exp Toxic Path01 44,74-80

9 Dole VP (1956) A relation between non-esteril’icd fatty acids in plasma and the metabolism of glucose. J Clin Znvesr 35,1X&154

10 Dunn WA Jr (1990) Studies on the mechanisms of autophagy: formation of the autophagic vacuole. .I C&E Biol 110, 1923-I933

11 Dunn WA Jr (1994) Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Celt Biol 4. 139-143

12 Furuno K, Ishikawa T, Akasaki K, Lee S, Nishimura Y, T5u1

H, Himeno M, Kato K (1990) Immunocytochemical study of the surrounding envelope of autophagic vacuoles in cultured rat hepatocytes. Exp Cell Res 189,261-268

13 Griffiths G, Hoflack B, Simons K, Melman I, Kornfeld S (1988) The manose 6-phosphate receptor and the biogenesis of lysosomes. Cell 52,329-341

14 Helminen H J, Ericsson JLE (1971) Ultrastructural studies on prostatic involution in the rat. Mechanism of the autophagy in epithelial cells with special reference to the rough- surfaced endoplasmic reticulum. J Ultrastruct Res 36, 708-724

15 Holiemans M, Elfeerink RO, DeGroot PG, Strijland A, Tager JM (1981) Accumulation of weak bases in relation to intralysosomal pH in cultured human skin fibroblasts. Biochim Biophys Acta 643, 140-15 1

16 de Jong JGN, van den Bosch H, Aarsman AJ, van Deenen LLM (I 973) Studies on lysophospholipases. Biochim Biophys Acta 296, 105-l 15

17 Kamovsky MJ (1971) Use of ferrocyanide reduced osmium tetroxide in electron microscopy. J Cell Biol5 1. 145

18 Kotyk A, Jana&k K, Koryta J (1988) In: Riophysical chem- istry oj’membrane functions. J Wiley & Sons, Chichester. New York, Brisbane, Toronto, Singapore, 42-49

19 Kovacs J, Karpati AP (1989) Regression of autophagic vacuoles in mouse pancreatic cells: a morphometric study of the effect of methylamine and chloroquine followed by cyclo- heximide treatment. Cell Biol Int Rep 13, 805-8 11

20 Kurihara H, Kitajima K, Senda T, Fujita H, Nakajima 7 (1986) Multigranular exocytosis induced by phospholipase

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

12

A?-activaLors, melittin and mastoparan, in rat antenor pitug? tary cells. Cell Tissue Res 243, 3 1 l-3 16 Larsh JE Jr, Ottolenghi A, Wheateriy NF (1974) Trichinella spiralis: Phospholipase in chaltenged mice and rats. ,&p Parasitol36, 299-306 Lavoie C, Jolicoeur M, Paiment J (1991) Accumulation oi polyunsaturated free fatty acids coincident with the fusion af rough endoplasxnic reticulum membranes. Biochim Biophys Actu 1070,274-278 Nagata T, Iwadare N (1984) Electron microscopic demonstration of phospholipase B activity in the-liver and the kid- ney of the mouse. Histochemistry 80, 149-l 52 Omura T, Siekievitz P, Palade GE (1967) Turnover of tori- stituents of the endoplasmic reticulum membranes of rat hepatocytes. J Biol Chem 242,2389-2396 Peute J, Huiskamp R, van Oordt PGWJ (1985) Quantitative analysis of estradiol 17J induced changes of the liver nl zebrafish, Brachydanio rerio. Cell Tissue Res 242, 377-382 Peyon P, Baloche S, Burzawa-Gerard E (1993) Synthesis u(’ vitellogenin by eel (Anguilla anguilla L) hepatocytes in primary culture: requirement of i7#-estradiol-priming. Ger: Comp Erzdocrinol9 1,3 18-329 Pfeifer U, Werder, Bergeest H (1979). Inhibited autopha8ic degradation of cytoplasm during compensatory growth oi’ liver cells after partial hepatectomy. Virchows Arch (B) 30, 3 13--333 Pirkle MS, Goven AJ, Foster LA, Kester (1988) Light and electron microscopic demonstration of phospholipase l3 activity in the mouse eosinophil. Histochemistty 88, 181-185 Punnonen EL, Autio S, Marjom&i VS, Reunanen H (I 992 1 Autophagy, cathepsin L transport, and acidification in cul. tured rat fibroblasts. J Histochem Cytochem 40, 1579-1587 Romagnoli P, Mancini G, Galeotti F. Francini E, Pierleoni P (1990) The crown odontoblasts of rat molars from primary dentinogenesis to complete eruption. J Denf Res 69, 1857-l 862 Seglen PO (1987) Regulation of autophagic protein degrada- lion in isolated liver ceils. In: Lysosomtjs: their role in prcb tein breakdown (Glaumann H, Ballard FJ, eds) Academii: Press, London, 369-4 14 Seglen PO, Bohley P (1992) Autophagy and other vacuolar protein degradation mechanisms. Experientiu 48, 158-172 Smith RE, Farquhar MG (1966) Lysosome function in tbc regulation of the secretory process in cells of the anteriw pituitary gland. J Cetl Biol3 1, 319-336 Ueno T, Muno D, Kominami E (1991) Membrane markers of endoplasmic reticufum preserved in autophagic vacuolar membranes isolated from leupeptin-administred rat liver. .I Biol Chem 266, 18995-18999 Verani? P, Pipan N (1992) The relationship between endu- plasm& reticulum and peroxisomes in fish hepatocytes during estradiol stimulation and after cessation of viteliogenc- sis. Period Biol94,29-34 Veranit P, Pipan N (1993) The use of chloroquine in ana- lysing autophagy in fish hepatocytes after cessation of treatment with estradiol 17g. Exp Clin GastroenteroE 3, 184-188 Wallaert (‘, Babin PJ (1992) Effects of 17U-estradiol on trout plasma lipoproteins. Lipids 27, 1032-1041 Weibel ER, Kristler GS, Sherle WR (1966) Practical stereq logical methods for morphometric cytology. J Cell BioE 30. 23-38 Weibel ER (1969) Stereological princip!es of morpbometl~ in electron microscopic cytoiogy. Int Rev Cytol26, 235-307 Weltzien HI! (1979) Cytolytic and membrane-perturbing proprieties of lysophosphatidylcholine. Biochim Biophvr ktd 559,259-287 Wisner-Gehhard AM, Brabec RK, Gray- RH (1979) Mqrpho- metric studies of chloroquine induced autophagy in rat liver Micron 10,65-66 Yucel-Lindberg T, Jansson H, Glaumann H (1991) Proteoly-- sis in isolated autophagic vacuoles from the rat pancreas. Effect of chtoroquine administration. Virchaws Arch {Bf 6 1 i 141 --I45

two degradation pathways of endoplasmic reticulum in fish hepatocytes

Documents