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Mechanisms of protein turnover in cultured fibroblasts
Esperimental
MECHANISMS
Cell Research
126 (1980) 167-174
OF PROTEIN
CULTURED
J. S. AMENTA
TURNOVER
IN
FIBROBLASTS
and
S. C.
BROCHER
SUMMARY Rat embryo fibroblasts, prelabeled with [“Clleucine showed a 2-fold increase in degradation of cell protein when placed in a serum-free medium. Insulin at a concentration of 100 mlU/ml completely blocked the induced proteolysis. but had no effect upon basal proteolysis of approx. 0.9%/h. Both the induced and basal proteolysis rates were reduced to a sub-basal level of 0.7%/h with IO mM NH,CI. When these cultures were placed in an NH:-free recovery medium. an enhanced rate of catch-up proteolysis was observed during the next 2 h. No catch-up proteolysis was seen when cultures in step-down medium with insulin were transferred to fresh growth medium. Cultures in step-down medium with insulin and NH,CI showed a small catch-up proteolysis when placed in recovery medium comparable to that seen in control culture in growth medium with NH;. Morphologic studies revealed an extensive accumulation of vacuoles in the NH:-treated cells in step-down medium; no accumulation of vacuoles in the insulin-treated cells: and an intermediate vacuolization in cells treated with insulin and NH;. From the biochemical and morphologic observations we propose that cellular proteolysis involves at least three mechanisms: induced autophagy; basal proteolysis occurring in lysosomes; and a neutral proteolytic mechanism. We suggest that insulin inhibits the first at the initial presequestration stage. and NH; inhibits the first two at a final intravacuolar proteolysis step.
The reported effects of insulin on protein degradation in mammalian cells range from no significant change, to a striking depression of proteolysis. Some rationality has been introduced in this matter by the recent recognition that protein degradation in eukaryotic cells is a product of at least two metabolic pathways [ 1, 2, 31. One degradative pathway, normally quiescent, is rapidly induced by placing cells in a step-down medium usually lacking serum. This induced degradation can be inhibited almost immediately by cycloheximide, microtubular poisons, and insulin [l, 3, 4, 51. In contrast, basal protein turnover in these cells is relatively insensitive to these agents.
While the mechanism of basal proteolysis remains an enigma at this time, recent studies suggest that the induced proteolytic pathway is associated with an increase in autophagic activity in the cell [6, 71. This in turn suggests that insulin blocks stepdown proteolysis by interfering at some stage in the autophagic process. This process can be conveniently divided into three sequential stages: sequestration of cellular components into a membranous vacuole; fusion of primary lysosomes with this autophagic vacuole: and hydrolysis of the cellular components within the autophagolysosome. Studies from a number of laboratories have established that NH&I and other salts of
organic bases have the unique property of inhibiting the final hydrolysis step in this sequence [8. 9-121. These agents rapidly block stepdown-induced proteolysis and effect an extensive vacuolization in these cells, in effect acting as a trap for the induced proteolytic pathway. Upon release of this trap, the accumulated cellular protein in these vacuoles is quickly released as acid-soluble and acid-insoluble in a catchup proteolytic response [13]. By utilizing this NH: trap combined with electron microscopy, we have been able to devise a biochemical assay for evaluating the activity of the autophagic pathway and particularly to determine which stage in the autophagic sequence inhibitors may affect. In this report we show by combined morphologic and biochemical techniques that insulin blocks only the induced proteolysis in fibroblast cultures and is without effect on basal proteolysis and that the induced proteolysis, the result of increased autophagic activity in these cells, is inhibited by insulin at the initial sequestration step. Finally, in concurrence with previously reported studies from this laboratory, basal proteolysis includes a small component which. though it involves hydrolysis in secondary lysosomes, appears distinct from the autophagy as defined above and is not inhibited by insulin.
METHODS pi-ocedures for arowine and labeline of rat embrvo fibroblrists in culture h‘gve been previously reported [3, 14-i6]. in bfief, fibroblasts were grown in Eagle’s minimum essential medium supplemented with IO% fetal calf serum (FCS) (growth medium). For labeline. cells were subcultured;” 3 cm dishes in 4 ml rowtkmedium containing 0.8 PCi t-[r4C]leucine and $: 0 bCt ‘. VHlthvmidine oer IO ml of medium and mown b3 i subconfluent stage, usually within 3-4 days’: Cultures were chased in unlabeled medium containing IS mM leucine for 24 h prior to each experiment.
F7.v. I. .-\D.,c.i~r: insulin cont. (mU/mll; o&~rc~. %r”‘Clorotein hvdrolvsedih: 0. growth medium: 52. step-d&n medium. . Effect of insulin on basal and induced proteolysis. Fibroblasts were labeled with [“Clleucine and [:‘H]thymidine. as previously described [3. 161. Cultures were placed in fresh growth medium ( 10% calf serum) or step-down medium (no calf serum) for 4 h and aliquots taken at 2 and 4 h for assaying acidsoluble and total radioactivity. Cell monolayers processed for total protein and radioactivity at the end of 4 h incubation. Proteolysis rate is expressed as % of total radioactivity hydrolysed per h. Pooled estimate of step-down +O.IO% for proteolysis in growth medium and ?O.l3Cr for proteolysis in step-down medium.
Proteolysis
r’rrtes
Crystalline insulin (Sigma Chemical Co., and NH,CI solutions were prepared in IOO-fold concentrations in H,O and added I : 100 to the final concentration in the indicated media. Cells were incubated in experimental media containing either insulin and/or NH,CI. Proteolysis was evaluated by measuring the rate of release of acid-soluble “C into the medium over a 4 h period, sampling at Z and 4 h. in both growth and step-down [serum-free) medium [ 15. 161. In recovery experiments cells were rinsed once with Dtdbecdo’s uhospknte;
medium. chilling the solution for 30 min iii ice, ceflfqi; fugation, followed by placing the clear supematadf iH IO ml of scintillation fluid and counting in a liquid scintillation counter. Monolayers were processed by washing twice with phosphate-buffered saline. adding 8 Cc cold TCA directly to the monolayers and scraping free the cell layer with a rubber policeman. The cell pellets were solubilized in 0.5 ml of 0.5 N NaOH before counting. Cell recoveries. monitored by assaying for “H in cell DNA, were generally greater than 90%.
These procedures have been previously detail [I?]. Rinsed monolayers were fixed aldehyde in Na cacodylate-sucrose buffer. in 0~0, and fixed in propylene oxide-Epon. were cut on a Sorvall MT-I. stained acetate and lead citrate. and examined on electron microscope.
described in in 2 % glutarpost-fixed Sections with uranyl a Phillips 200
Proteia tl~mowr
itzhibition
169
lysis [l2, 131 and that when these cells are returned to a fresh growth medium, I catchup release of both acid-soluble Rnd acidinsoluble lqC occurs. Concurrent morphologic studies indicated that NH: wit t9fectively trapping [‘“C‘Jprotein within the vacuolar system of the cell. Upon release Fip. 2. Abscissu: incubation period (hours); ordinnte: from the NH: block, this [lAC]protein W&S acid-soluble “C in medium (7% of total). rapidly hydrolysed and released into the (N) Effect of NH,CI on basal proteolysis. Fibroblast cultures labeled as described in Methods and medium. This phenomenon is demonstrated incubated for 4 h in either growth medium (0) or in fig. 2. Fibroblasts in fresh growth megrowth medium with 10 mM NH,CI (m). At 4 h (arrorr) all cultures were placed in fresh growth medium. Acdium hydrolysed cell protein at a rate of cumulation of acid-soluble “C in media determined. 1.O%/h during the first 2 h, then dropped to Data from three experiments; each point represents average of nine dishes. Pooled estimate of S.D. was a rate of 0.8%/h for the remaining 6 h of kO.OS% for each 4 h incubation period. Average cell the experiment @ii. 2rr). NH,C1 inhibited protein was 13.3 pg/cm’. (b) Effect of NH,CI on stepdown proteolysis. As in (n), except cultures placed in proteolysis to 0.7%/h during the initial 4 h; step-down medium with and without NH.,CI during however, most of the [‘Clprotein in this initial 4 h incubation. Pooled estimate of S.D. was f0.35 5% for each 4 h incubation period. Average problocked vacuolar proteolysis was released tein was 13. I pg/cm’. in a catch-up period when cells were placed in an NH:-free growth medium (fig. la). We have previously reported that a small RESULTS amount of this catch-up release of label is in Insulin, when added to serum-free medium the form of acid-insoluble Y [ 131, showing at a high concentration, largely inhibited the that the total “C released during the catchinduced proteolysis, yet had no significant up period completely compensates for the effect upon basal proteolysis. Cell cultures NH,-induced inhibition previously obplaced in fresh growth medium showed a served. A similar phenomenon was obproteolysis rate of 1.0%/h (fig. l), while served in cultures in step-down medium similar cultures in step-down medium in- (fig. 2b). These data demonstrated (a) that creased protein degradation 2-fold. Insulin approx. 22% of basal protein turnover in in concentrations from 0.1-10 mIU/ml these cultures was occurring in the vacuolar showed a progressive inhibition of the in- system [ 131; (h) that all of the stepdownduced proteolysis. and at a concentration induced proteolysis was occurring in the of 100 mIU/ml almost completely blocked vacuolar system: (c) that cells both in the stepdown-induced proteolysis. We were growth and step-down medium showed a not able to detect any effect of insulin on non-inhibitied basal proteolysis rate of basal proteolysis at any of these concen0.7%/h; and (ti) that virtually all of the trations. These results were consistent with [‘“Clprotein trapped within the vacuolar previous observations from this laboratory system by NH: was released when cells [3, 161 and with reports of others [l, 5. were placed in an NH:-free chase medium. 17-211. Of particular importance in these studies We have previously shown that cultured was that this trapping by NH,Cl of proteins fibroblasts, when treated with 10 mM designated for proteolysis provided a speNH&I, manifest a reduced rate of proteocific biochemical assay for proteolysis as-
F‘i,y. 2. Ahsci.tstrt incubation period I hours,: ordimrtv acid-soluble ‘-‘C in medium cri of total). ((I) Effect of insulin on NH: trap in basal medium. Fibroblast cultures labeled as described in Methods and incubated for 4 h in either growth medium containing either 100 mU/ml of insulin (0) or insulin and IO mM NH&I(m). At 4 h (~IWOII.) all cultures placed in fresh growth medium. Accumulation of acid-soluble “C in medium determined. Data from three experiments: each point represents average of nine dishes. Average cell protein was 13. I pg/cm’. Pooled estimate of step-down was 10. I 1’7 for each 4 h incubation. fh) Effect of insulin on NH: trap in step-down medium. As in (a). except cultures placed in step-down medium containing either insulin or insulin and NH,CI during initial 1 h incubation. .Average cell protein was 12.8 pg/cm’. Pooled estimate of step-down wah +O. I3 Q for each 4 h incubation.
sociated with the lysosomal system in the cell. Fibroblast cultures incubated in media containing 100 mlU/ml insulin showed proteolysis patterns virtually identical with those previously observed in cultures in growth medium alone. Insulin added to cultures in growth medium had no effect on the proteolysis rate (fig. 3n ). confirming previous results (fig. I). Nor did insulin alter in any way the NH:-induced inhibition and the catch-up proteolysis when cells were placed in NH:-free growth medium. At this high concentration insulin effectively blocked the stepdown-induced proteolysis (fig. 3h), reducing the proteolysis rate to the level observed in cultures maintained in growth medium. NH: again reduced proteolysis to 0.7%/h. Catch-up proteolysis on recovery compensated only for the NH:induced inhibition and not for the insulininduced inhibition of stepdown-induced
proteoly4is. Nor did cell\ iii \tt‘p-~Ikj~4 ti nitdium with insulin \ho\\ an) catch-up PI-Oteolysis when placed in t‘rezh grolktth medium. It appeared that high concentration\ of insulin could almost completely block the autophagy induced by ;I \erum-free medium. but this agent had no effect upon basal proteolysis. either that fraction occur-ring in the vacuolar system or the larger part not occurring in acid vacuoles. We considered the possibilit), that insulin entered the cell. remained attached to intracellular membranes during the reco\‘ery period. and thereby blocked catch-up proteolysis [E]. However. cells incubated with insulin. followed by a 4 h incubation in stepdown medium. showed a normal stepdowninduced proteolysis (data not prehented). demonstrating that the inhibitor!, effect of insulin on induced proteolysi\ wa\ not maintained in cells placed in media lacking insulin supplementation. Our failure to find a catch-up proteoiysis after insulin had blocked the step-down proteolysis suggested that insulin was preventing the initial segregation in the autophagic zequence. rather than blocking either fusion with Iyrosomes and hydrolysis within the acidic vacuoles. The striking morphologic changes induced by NH: were only partially blocked by insulin. Control cells in growth medium contained whorls of membranous material. dense clumps of similar material. some in vacuoles and occasional vacuoles with scanty amorphous debris (fig. 4tr). Cells placed in step-down medium for 4 h showed a slight increase in vacuoles containing both cellular elements and amorphous debris (fig. 4h). These morphologic changes have been previously described in detail [7]. It should be emphasized that in these pal-titular experiments these changes were not striking and it was not possible to con-
Fi,q. 1. ttl) Fibroblasts in fresh growth medium for 4 h. Cells show prominent rough endoplasmic reticulum and numerous dense membranous whorls ttrrrort~). both of which showed marked intercellular variations. Occasional vacuoles, containing small amounts of amorphous cellular debris. were observed in some cells t*). Cells placed in growth medium with insulin were indistinguishable from these. (h) Fibroblasts in
step-down medium for 4 h. Compared with controls in growth medium (uhorr), these cells appeared to have more vacuoles with more entrapped amorphous cellular material. including clumped ribosomes (wrc~,s). (c) Fibroblasts in step-down medium with insulin for 4 h. Cells were indistinguishable from controls in growth medium. Occasional large clear vacuoles with some amorphous material (trrro~.\) could be found in many cells. (d) Fibroblasts in step-down medium with NH,CI for 4 h. Extensive vacuolization within cells, many containing loose membranes ((IIWI(.), loose and clumped ribosomes (*). (P) Fibroblasts in step-down medium containing insulin and NH,CI for 4 h. Vacuolization was also prominent in these cells. though usually less extensive than in (d). These cells could not be distinguished from cells in growth medium with insulin and NH,CI for 4 h. Vacuoles with clumped ribosomes (*) and membrane whorls (urru11 ).
fused
Fix. .i. aegradative pathways fur cell proteins. Autophagic pathway. induced by b&p-down medium. is spbdividod into three steps t--t). The basal pathway t+) involves bQth lysosomes t~cppr,‘/ine) and a neutral mechanism t/o~c,rr lincl) nut affected by insulin. NH,CI inhiblta both pathways invalving lysosomes. (1. Gquestraticm: h, fusian; c. hydrolysis.-. insulin block: %, NH; block: -, basal pathways; -- -, induced WtOphagy.
sistently differentiate between cells in growth medium, in step-down medium, and in similar media containing insulin (fig. 4~). In contrast, cell treated with NH; showed a striking vacuolization, characterized by large clear vacuoles which in some instances contained cellular elements and amorphous material. While fibroblasts in step-down medium appeared to show the greatest degree of NH:-induced vacuolization (fig. 4~0, NH: induced significant vacuoliration in fibroblasts in growth medium, in fibroblasts in growth medium with insulin, and in fibroblasts in step-down medium with insulin (fig. 4~). DISCUSSION
liver
[20, ?I].
Studies
on the mech-
anism of the stepdown-induced proteolysis implicate transitory activation of the autophagic-lysosomal mechanism. Perfused livers, for example, show increased fragility of all secondary lysosomes [20], and increased amounts of protein in the isolated cell fraction containing secondary Iysosomes [6]. We have reported increased numbers of autophagic vacuoles in fibroblasts placed in step-down medium [7, 121. Our findings in this study, that insulin inhibits both the stepdown-induced autophagy and the induced proteolysis in CL& tured fibroblasts, correlates with similar observations on the perfused liver [6]. A similar role for insulin has been proposed for the enhanced degradation of cell protein in insulin-deficient and starving rats [24]. Of particular interest, Khairallah [ZS] has shown that formation of autophagic vacuoles in liver of fasting rats is blocked when rats are fed. That this inhibition correlates with rising insulin levels suggests both that the induced autophagic mechanism plays a significant role in the normal homeostasis of some organs and that insulin may play a physiologic role in its control. Detailed morphologic studies by pfeifer et al. [26. 271
Insdin, proteolysis, titd trirtophrrg~
on this mechanism
With the realization that eukaryotes placed in a nutritional stepdawn medium activate a supplementary proteolytic system, different from basal protein turnover in the cell, we can now produce reasonably consistent responses to various inhibitors of protein degradation. Insulin has little, if any, effect upon degradation when rat fibrablasts are maintained in a growth medium [3, 171, but readily inhibits the enhanced degradation observed when cells are in a stepdown medium. Similar results have been obtained with hepatoma cells [S. 191, isolated liver cells [8. 331, and the isolated per-
that insulin effects a rapid decrease in autophagic vacuoles in the liver, with an average half-life of approx. 10 min. These workers suggest that insulin prevents the formation of new autophagic vacuoles while the rapid degradation of protein within these vacuoles quickly clears those present at the time of the block. Mortimore and coworkers, using primarily cell fractionation techniques, have obtained a similar half-life for these induced vacuoles [6]. It appears that autophagy is a physiologic and certainly pathologic response. which under certain circumstances
in the rat liver
makes
a
significant
indicates
con-
Protein trtrnor~er itd~ibition
tribution to protein turnover and that this cellular response, in culture systems as well as intact animals, can be triggered by a variety of stimuli and modulated by the plasma insulin level. Mechattistns
of’ protein turtim~et
The recognition that NH&I acts as a nontoxic but highly specific reagent to block hydrolysis in secondary lysosomes [7,9-l 1, 13, 281 has furnished a tool by which to dissect the cellular mechanisms for protein turnover. The rapid accumulation of numerous large vacuoles behind this chemical block provides a dramatic morphologic confirmation of this hypothesis. Additionally, the catch-up proteolysis which occurs when this hydrolytic block is released and the concurrent clearing of cellular vacuoles offers a means by which one can evaluate the rate of activity of the lysosomal system in protein turnover [ 12, 131.We have shown that inhibited proteolysis plus the catch-up proteolysis, combined with a small excess of insoluble ‘Y released. matches the total proteolysis observed in comparable control cultures. This would not be an unexpected phenomenon if we were to assume that cell protein, once sequestered into autophagic vacuoles, cannot be returned to the cell
hyalaplasm, but must be hydrelysed and/or released into the medium, The failure TV find any rebound after insulin Black of stepdown induced turnover, along with the small amount of cellular protein trapped in vacuoles by the NH:, indicates that insulin prevents the initial sequestration step in autophagy. Another possible mechanism, that insulin blocked fusion of primary lysosome with autophagosome. was initially considered, particularly in view of the rather extensive NH:-induced vacuolization observed with the electron microscope in these insulin-treated cells. We had ini-
173
tially assumed that since virtually all secondary lysosomes in these cells were derived from autophagy, a sequestration block would prevent the accumulation of the NH:-filled vacuoles. (We have never been able to induce significant heterophagy in these cultures.) However, failure to find the expected catch-up proteolysis when cells were placed in a recovery medium militated aginst this hypothesis. Other lines of evidence suggested the alternative hypothesis, that some cell protein gained entrance into lysosomes by a mechanism other than traditional autophagy. The NH; trap effected vacuolization in cells carefully maintained in the basal state; additionally lO-30% of basal protein turnover occurred in acidic vacuoles (as defined by the NH: trap), and this proteolysis was not affected by insulin or vinblastine [29]. The high rate of turnover of secondary lysosomes [26,27] made it unlikely that we were trapping with NH: a large number of vacuoles present at the beginning of the experiment. Undoubtedly, many of these vacuoles, especially the clear vacuoles with little cellular debris, represent dilitation of primary lysosomes, making it difficult to estimate the rate of protein degradation occurring in vacuoles
on the basis of the morphologic changes alone? The most convincing data, however, w&8provided ky treating f?xsb!ast cultures with cycloheximide: this agent inhibits stepdown-induced proteolysis and, in addition, has a slight inhibitory effect upon basal proteolysis [29]. Using the NH, trap technique, we have found that cycloheximide rapidly blocks proteolysis in virtually all secondary lysosomes. The fact that fibroblasts treated with both cycloheximide and NH:, whether in growth or step-down medium, show almost no cell vacuolization suggests that this agent, unlike insulin and vinblastine. blocks both the initial step in the induced (auto-
173
.4Itlrtlltr lill‘l BtlM~llc~l-
phagic) and the basal \acuolar mechanism. These observations. biochemical and morphologic. are consistent with a model containing three distinct degradative pathways (fig. 5): an induced autophagic mechanism inhibited in the final hydrolysis stage b) NH: and in its initial sequestration step by insulin. a basal neutral mechanism, and finally a basal acidic vacuolar mechanism inhibited by NH;, but insensitive to insulin. At this point we can only speculate what this last mechanism might be: (LI) a fraction of some secretory proteins, already in vacuoles, may be subject to intracellular hydrolysis, as reported for other secretory products in fibroblasts [30]; (h) we have observed that the membranous whorls commonly found in these cells eventually acquire acid phosphatase and are degraded in vacuoles; these structures may not need additional membrane for sequestration via the autophagic mechanism: tc) finally. Locke [3l. 331. studying changes in the protein granules in an insect fat body, suggested that in addition to autophagy. proteins of the hyaloplasm can enter lysosomes by a microphagocytosis along the lysosomal membrane. This mechanism has also been proposed by Trump and co-workers in studies with HeLa cells [33]. At this time we can say only that a small complementary fraction of basal protein turnover occurs in the lysosomal system, and that this process appears to be distinct from the autophagy which under some conditions supplements basal turnover. The remaining largest fraction of basal turnover may yet be found to be related to neutral proteases. both in and out of cellular vacuoles [ 13. 15, 331. REFERENCES I.
Kno~iea. S E & Ballard, F J. Biochem j 156 (19761 609. 2. Poole. B & Wibo. M. J biol them 248 I 1973) 622 I. 3. Amenta. J S. Sargus. M J & Baccino. F M. Biothem j I68 (1977) 223. t 1,’
( , /I H,,,
lh
/ /‘A\ll,
9. IO. II.
12. 13. I.$. IS. 16.
17. 18. 19. IO. 21.
25. 26. 27. 28. ZY. 30. 31. 32. 33. 3-I.
Gtmn. J M. Clarh. M G. Knov.Ic\. b t... Hopgo~~l. M F & Ballard. F J, Nature 26 I IV771 5X. Ward. W F. Cox. J R & Mot-timore. G t. J b1ol them 252 (1977) hYT5. r2menta. J S. Sareu\. hl J. Venkarti\,tn. S ti Shlnoruka. H. J cell ih\siol Y-l ( 197X1 ?’ Ballal-d. F J. Control mechanihm\ 111 c‘ancer IZJ I+ E Ct-ihs. T Ono & J R Sabine) p. 7”). Raven Prebb. New York ( 19761. Seglrn. P 0 & Reith. .A. txp cell re> Ill0 I iY7ht 276. Seglen. P 0. Biozhem biophyh re\ commun 66 (197.0 13. - Use of isolated liver cell\ and kidney, tubules In metabolic studies (ed J M Tager. H D Soling & J R Williamson). North-Holland Publishing Co.. hmsterdam (1976). .4menta. J S. Hlibko. T J. McBee. .A G. Smozuka. H & Brother. S C, Exp cell res I IS I 19781 337. .4menta. J S & Broche;. S C. J cell phy\iol ! 1979). In pres,. r2menta. J S. Sarauh. M J 6i Bacclno. F M. BIOchim biophys act:376 (1977) 253. -J cell phvbiol97 ( 1978) 267. - Intra≪lar protein catabolism ted V furk & N Marks) vol. 2. p. 27. Plenum Pre\>. New York (1977). Hershko. .4. Marmont. P. Shieldb. R & Tomkin\. G M. Nature new biol 232 ( 1971 I 206. Gunn. J M. Ballard. F J 8: Hanson. R W. J biol them 151 ( 1976) 3586. Gelehrter. T D & Emanuel. J R. Endocrinology Y-l ( 1971) 676. Neely, A N. Nelson. P B & Mortunore. G E. B&him biophys acta 45 I ( 1976) 5 I I. Mortimore. G E. Neely. .A N. Cos. J R & Guintvan. R .4. Biochem biophys res commun 5-1( 19731 x9. Goldfins. I D. Joneb. A L. Hradeh. G T. Wong. K Y & Mooney. J S. Science 101 (1978) 760. Grammeltredt. R & Berg. T. Honoe Zevler’y Z phyziol them 317 (1976) 67. ’ ’ . Dice. J F. Walker. C D. B\ne. B & Cordlel. .A. Proc natl acad sci US 75 (19?8) 2093. Khairallah. E A. Personal communication Pfeifer. Ll. .4cta giol med ger 36 (1977) 1691. Pfeifer. U. Werder, E & Bergeist. I. J cell btal 78 (1975) IS?. Warburton. M J & Poole, B. Proc natl acad XI US 7-1(1977) >‘3’7. - .Amenta. J S B Brother. S C. t\n mol nathol (19791. In press. Glimelius. B. Westermark. B & Wa\te\on. .A. ESD cell res IO8 (1977) 23. Locke. M & Collins, J V. J cell biol 26 ( 1965) X57. Locke, M & Sykes. A K. Tissue & cell 7 ( 19751 143. r2rstila. .4 U. Jauregn. H 0. Chenr. J & I‘rumD. B F, Lab invest 21 ( 1971162. Katunuma. N. Rev physiol blochem pharm 72 t 1975) 83.
Received Accepted
July 16. 1979 September 5. 1979
MECHANISMS
Cell Research
126 (1980) 167-174
OF PROTEIN
CULTURED
J. S. AMENTA
TURNOVER
IN
FIBROBLASTS
and
S. C.
BROCHER
SUMMARY Rat embryo fibroblasts, prelabeled with [“Clleucine showed a 2-fold increase in degradation of cell protein when placed in a serum-free medium. Insulin at a concentration of 100 mlU/ml completely blocked the induced proteolysis. but had no effect upon basal proteolysis of approx. 0.9%/h. Both the induced and basal proteolysis rates were reduced to a sub-basal level of 0.7%/h with IO mM NH,CI. When these cultures were placed in an NH:-free recovery medium. an enhanced rate of catch-up proteolysis was observed during the next 2 h. No catch-up proteolysis was seen when cultures in step-down medium with insulin were transferred to fresh growth medium. Cultures in step-down medium with insulin and NH,CI showed a small catch-up proteolysis when placed in recovery medium comparable to that seen in control culture in growth medium with NH;. Morphologic studies revealed an extensive accumulation of vacuoles in the NH:-treated cells in step-down medium; no accumulation of vacuoles in the insulin-treated cells: and an intermediate vacuolization in cells treated with insulin and NH;. From the biochemical and morphologic observations we propose that cellular proteolysis involves at least three mechanisms: induced autophagy; basal proteolysis occurring in lysosomes; and a neutral proteolytic mechanism. We suggest that insulin inhibits the first at the initial presequestration stage. and NH; inhibits the first two at a final intravacuolar proteolysis step.
The reported effects of insulin on protein degradation in mammalian cells range from no significant change, to a striking depression of proteolysis. Some rationality has been introduced in this matter by the recent recognition that protein degradation in eukaryotic cells is a product of at least two metabolic pathways [ 1, 2, 31. One degradative pathway, normally quiescent, is rapidly induced by placing cells in a step-down medium usually lacking serum. This induced degradation can be inhibited almost immediately by cycloheximide, microtubular poisons, and insulin [l, 3, 4, 51. In contrast, basal protein turnover in these cells is relatively insensitive to these agents.
While the mechanism of basal proteolysis remains an enigma at this time, recent studies suggest that the induced proteolytic pathway is associated with an increase in autophagic activity in the cell [6, 71. This in turn suggests that insulin blocks stepdown proteolysis by interfering at some stage in the autophagic process. This process can be conveniently divided into three sequential stages: sequestration of cellular components into a membranous vacuole; fusion of primary lysosomes with this autophagic vacuole: and hydrolysis of the cellular components within the autophagolysosome. Studies from a number of laboratories have established that NH&I and other salts of
organic bases have the unique property of inhibiting the final hydrolysis step in this sequence [8. 9-121. These agents rapidly block stepdown-induced proteolysis and effect an extensive vacuolization in these cells, in effect acting as a trap for the induced proteolytic pathway. Upon release of this trap, the accumulated cellular protein in these vacuoles is quickly released as acid-soluble and acid-insoluble in a catchup proteolytic response [13]. By utilizing this NH: trap combined with electron microscopy, we have been able to devise a biochemical assay for evaluating the activity of the autophagic pathway and particularly to determine which stage in the autophagic sequence inhibitors may affect. In this report we show by combined morphologic and biochemical techniques that insulin blocks only the induced proteolysis in fibroblast cultures and is without effect on basal proteolysis and that the induced proteolysis, the result of increased autophagic activity in these cells, is inhibited by insulin at the initial sequestration step. Finally, in concurrence with previously reported studies from this laboratory, basal proteolysis includes a small component which. though it involves hydrolysis in secondary lysosomes, appears distinct from the autophagy as defined above and is not inhibited by insulin.
METHODS pi-ocedures for arowine and labeline of rat embrvo fibroblrists in culture h‘gve been previously reported [3, 14-i6]. in bfief, fibroblasts were grown in Eagle’s minimum essential medium supplemented with IO% fetal calf serum (FCS) (growth medium). For labeline. cells were subcultured;” 3 cm dishes in 4 ml rowtkmedium containing 0.8 PCi t-[r4C]leucine and $: 0 bCt ‘. VHlthvmidine oer IO ml of medium and mown b3 i subconfluent stage, usually within 3-4 days’: Cultures were chased in unlabeled medium containing IS mM leucine for 24 h prior to each experiment.
F7.v. I. .-\D.,c.i~r: insulin cont. (mU/mll; o&~rc~. %r”‘Clorotein hvdrolvsedih: 0. growth medium: 52. step-d&n medium. . Effect of insulin on basal and induced proteolysis. Fibroblasts were labeled with [“Clleucine and [:‘H]thymidine. as previously described [3. 161. Cultures were placed in fresh growth medium ( 10% calf serum) or step-down medium (no calf serum) for 4 h and aliquots taken at 2 and 4 h for assaying acidsoluble and total radioactivity. Cell monolayers processed for total protein and radioactivity at the end of 4 h incubation. Proteolysis rate is expressed as % of total radioactivity hydrolysed per h. Pooled estimate of step-down +O.IO% for proteolysis in growth medium and ?O.l3Cr for proteolysis in step-down medium.
Proteolysis
r’rrtes
Crystalline insulin (Sigma Chemical Co., and NH,CI solutions were prepared in IOO-fold concentrations in H,O and added I : 100 to the final concentration in the indicated media. Cells were incubated in experimental media containing either insulin and/or NH,CI. Proteolysis was evaluated by measuring the rate of release of acid-soluble “C into the medium over a 4 h period, sampling at Z and 4 h. in both growth and step-down [serum-free) medium [ 15. 161. In recovery experiments cells were rinsed once with Dtdbecdo’s uhospknte;
medium. chilling the solution for 30 min iii ice, ceflfqi; fugation, followed by placing the clear supematadf iH IO ml of scintillation fluid and counting in a liquid scintillation counter. Monolayers were processed by washing twice with phosphate-buffered saline. adding 8 Cc cold TCA directly to the monolayers and scraping free the cell layer with a rubber policeman. The cell pellets were solubilized in 0.5 ml of 0.5 N NaOH before counting. Cell recoveries. monitored by assaying for “H in cell DNA, were generally greater than 90%.
These procedures have been previously detail [I?]. Rinsed monolayers were fixed aldehyde in Na cacodylate-sucrose buffer. in 0~0, and fixed in propylene oxide-Epon. were cut on a Sorvall MT-I. stained acetate and lead citrate. and examined on electron microscope.
described in in 2 % glutarpost-fixed Sections with uranyl a Phillips 200
Proteia tl~mowr
itzhibition
169
lysis [l2, 131 and that when these cells are returned to a fresh growth medium, I catchup release of both acid-soluble Rnd acidinsoluble lqC occurs. Concurrent morphologic studies indicated that NH: wit t9fectively trapping [‘“C‘Jprotein within the vacuolar system of the cell. Upon release Fip. 2. Abscissu: incubation period (hours); ordinnte: from the NH: block, this [lAC]protein W&S acid-soluble “C in medium (7% of total). rapidly hydrolysed and released into the (N) Effect of NH,CI on basal proteolysis. Fibroblast cultures labeled as described in Methods and medium. This phenomenon is demonstrated incubated for 4 h in either growth medium (0) or in fig. 2. Fibroblasts in fresh growth megrowth medium with 10 mM NH,CI (m). At 4 h (arrorr) all cultures were placed in fresh growth medium. Acdium hydrolysed cell protein at a rate of cumulation of acid-soluble “C in media determined. 1.O%/h during the first 2 h, then dropped to Data from three experiments; each point represents average of nine dishes. Pooled estimate of S.D. was a rate of 0.8%/h for the remaining 6 h of kO.OS% for each 4 h incubation period. Average cell the experiment @ii. 2rr). NH,C1 inhibited protein was 13.3 pg/cm’. (b) Effect of NH,CI on stepdown proteolysis. As in (n), except cultures placed in proteolysis to 0.7%/h during the initial 4 h; step-down medium with and without NH.,CI during however, most of the [‘Clprotein in this initial 4 h incubation. Pooled estimate of S.D. was f0.35 5% for each 4 h incubation period. Average problocked vacuolar proteolysis was released tein was 13. I pg/cm’. in a catch-up period when cells were placed in an NH:-free growth medium (fig. la). We have previously reported that a small RESULTS amount of this catch-up release of label is in Insulin, when added to serum-free medium the form of acid-insoluble Y [ 131, showing at a high concentration, largely inhibited the that the total “C released during the catchinduced proteolysis, yet had no significant up period completely compensates for the effect upon basal proteolysis. Cell cultures NH,-induced inhibition previously obplaced in fresh growth medium showed a served. A similar phenomenon was obproteolysis rate of 1.0%/h (fig. l), while served in cultures in step-down medium similar cultures in step-down medium in- (fig. 2b). These data demonstrated (a) that creased protein degradation 2-fold. Insulin approx. 22% of basal protein turnover in in concentrations from 0.1-10 mIU/ml these cultures was occurring in the vacuolar showed a progressive inhibition of the in- system [ 131; (h) that all of the stepdownduced proteolysis. and at a concentration induced proteolysis was occurring in the of 100 mIU/ml almost completely blocked vacuolar system: (c) that cells both in the stepdown-induced proteolysis. We were growth and step-down medium showed a not able to detect any effect of insulin on non-inhibitied basal proteolysis rate of basal proteolysis at any of these concen0.7%/h; and (ti) that virtually all of the trations. These results were consistent with [‘“Clprotein trapped within the vacuolar previous observations from this laboratory system by NH: was released when cells [3, 161 and with reports of others [l, 5. were placed in an NH:-free chase medium. 17-211. Of particular importance in these studies We have previously shown that cultured was that this trapping by NH,Cl of proteins fibroblasts, when treated with 10 mM designated for proteolysis provided a speNH&I, manifest a reduced rate of proteocific biochemical assay for proteolysis as-
F‘i,y. 2. Ahsci.tstrt incubation period I hours,: ordimrtv acid-soluble ‘-‘C in medium cri of total). ((I) Effect of insulin on NH: trap in basal medium. Fibroblast cultures labeled as described in Methods and incubated for 4 h in either growth medium containing either 100 mU/ml of insulin (0) or insulin and IO mM NH&I(m). At 4 h (~IWOII.) all cultures placed in fresh growth medium. Accumulation of acid-soluble “C in medium determined. Data from three experiments: each point represents average of nine dishes. Average cell protein was 13. I pg/cm’. Pooled estimate of step-down was 10. I 1’7 for each 4 h incubation. fh) Effect of insulin on NH: trap in step-down medium. As in (a). except cultures placed in step-down medium containing either insulin or insulin and NH,CI during initial 1 h incubation. .Average cell protein was 12.8 pg/cm’. Pooled estimate of step-down wah +O. I3 Q for each 4 h incubation.
sociated with the lysosomal system in the cell. Fibroblast cultures incubated in media containing 100 mlU/ml insulin showed proteolysis patterns virtually identical with those previously observed in cultures in growth medium alone. Insulin added to cultures in growth medium had no effect on the proteolysis rate (fig. 3n ). confirming previous results (fig. I). Nor did insulin alter in any way the NH:-induced inhibition and the catch-up proteolysis when cells were placed in NH:-free growth medium. At this high concentration insulin effectively blocked the stepdown-induced proteolysis (fig. 3h), reducing the proteolysis rate to the level observed in cultures maintained in growth medium. NH: again reduced proteolysis to 0.7%/h. Catch-up proteolysis on recovery compensated only for the NH:induced inhibition and not for the insulininduced inhibition of stepdown-induced
proteoly4is. Nor did cell\ iii \tt‘p-~Ikj~4 ti nitdium with insulin \ho\\ an) catch-up PI-Oteolysis when placed in t‘rezh grolktth medium. It appeared that high concentration\ of insulin could almost completely block the autophagy induced by ;I \erum-free medium. but this agent had no effect upon basal proteolysis. either that fraction occur-ring in the vacuolar system or the larger part not occurring in acid vacuoles. We considered the possibilit), that insulin entered the cell. remained attached to intracellular membranes during the reco\‘ery period. and thereby blocked catch-up proteolysis [E]. However. cells incubated with insulin. followed by a 4 h incubation in stepdown medium. showed a normal stepdowninduced proteolysis (data not prehented). demonstrating that the inhibitor!, effect of insulin on induced proteolysi\ wa\ not maintained in cells placed in media lacking insulin supplementation. Our failure to find a catch-up proteoiysis after insulin had blocked the step-down proteolysis suggested that insulin was preventing the initial segregation in the autophagic zequence. rather than blocking either fusion with Iyrosomes and hydrolysis within the acidic vacuoles. The striking morphologic changes induced by NH: were only partially blocked by insulin. Control cells in growth medium contained whorls of membranous material. dense clumps of similar material. some in vacuoles and occasional vacuoles with scanty amorphous debris (fig. 4tr). Cells placed in step-down medium for 4 h showed a slight increase in vacuoles containing both cellular elements and amorphous debris (fig. 4h). These morphologic changes have been previously described in detail [7]. It should be emphasized that in these pal-titular experiments these changes were not striking and it was not possible to con-
Fi,q. 1. ttl) Fibroblasts in fresh growth medium for 4 h. Cells show prominent rough endoplasmic reticulum and numerous dense membranous whorls ttrrrort~). both of which showed marked intercellular variations. Occasional vacuoles, containing small amounts of amorphous cellular debris. were observed in some cells t*). Cells placed in growth medium with insulin were indistinguishable from these. (h) Fibroblasts in
step-down medium for 4 h. Compared with controls in growth medium (uhorr), these cells appeared to have more vacuoles with more entrapped amorphous cellular material. including clumped ribosomes (wrc~,s). (c) Fibroblasts in step-down medium with insulin for 4 h. Cells were indistinguishable from controls in growth medium. Occasional large clear vacuoles with some amorphous material (trrro~.\) could be found in many cells. (d) Fibroblasts in step-down medium with NH,CI for 4 h. Extensive vacuolization within cells, many containing loose membranes ((IIWI(.), loose and clumped ribosomes (*). (P) Fibroblasts in step-down medium containing insulin and NH,CI for 4 h. Vacuolization was also prominent in these cells. though usually less extensive than in (d). These cells could not be distinguished from cells in growth medium with insulin and NH,CI for 4 h. Vacuoles with clumped ribosomes (*) and membrane whorls (urru11 ).
fused
Fix. .i. aegradative pathways fur cell proteins. Autophagic pathway. induced by b&p-down medium. is spbdividod into three steps t--t). The basal pathway t+) involves bQth lysosomes t~cppr,‘/ine) and a neutral mechanism t/o~c,rr lincl) nut affected by insulin. NH,CI inhiblta both pathways invalving lysosomes. (1. Gquestraticm: h, fusian; c. hydrolysis.-. insulin block: %, NH; block: -, basal pathways; -- -, induced WtOphagy.
sistently differentiate between cells in growth medium, in step-down medium, and in similar media containing insulin (fig. 4~). In contrast, cell treated with NH; showed a striking vacuolization, characterized by large clear vacuoles which in some instances contained cellular elements and amorphous material. While fibroblasts in step-down medium appeared to show the greatest degree of NH:-induced vacuolization (fig. 4~0, NH: induced significant vacuoliration in fibroblasts in growth medium, in fibroblasts in growth medium with insulin, and in fibroblasts in step-down medium with insulin (fig. 4~). DISCUSSION
liver
[20, ?I].
Studies
on the mech-
anism of the stepdown-induced proteolysis implicate transitory activation of the autophagic-lysosomal mechanism. Perfused livers, for example, show increased fragility of all secondary lysosomes [20], and increased amounts of protein in the isolated cell fraction containing secondary Iysosomes [6]. We have reported increased numbers of autophagic vacuoles in fibroblasts placed in step-down medium [7, 121. Our findings in this study, that insulin inhibits both the stepdown-induced autophagy and the induced proteolysis in CL& tured fibroblasts, correlates with similar observations on the perfused liver [6]. A similar role for insulin has been proposed for the enhanced degradation of cell protein in insulin-deficient and starving rats [24]. Of particular interest, Khairallah [ZS] has shown that formation of autophagic vacuoles in liver of fasting rats is blocked when rats are fed. That this inhibition correlates with rising insulin levels suggests both that the induced autophagic mechanism plays a significant role in the normal homeostasis of some organs and that insulin may play a physiologic role in its control. Detailed morphologic studies by pfeifer et al. [26. 271
Insdin, proteolysis, titd trirtophrrg~
on this mechanism
With the realization that eukaryotes placed in a nutritional stepdawn medium activate a supplementary proteolytic system, different from basal protein turnover in the cell, we can now produce reasonably consistent responses to various inhibitors of protein degradation. Insulin has little, if any, effect upon degradation when rat fibrablasts are maintained in a growth medium [3, 171, but readily inhibits the enhanced degradation observed when cells are in a stepdown medium. Similar results have been obtained with hepatoma cells [S. 191, isolated liver cells [8. 331, and the isolated per-
that insulin effects a rapid decrease in autophagic vacuoles in the liver, with an average half-life of approx. 10 min. These workers suggest that insulin prevents the formation of new autophagic vacuoles while the rapid degradation of protein within these vacuoles quickly clears those present at the time of the block. Mortimore and coworkers, using primarily cell fractionation techniques, have obtained a similar half-life for these induced vacuoles [6]. It appears that autophagy is a physiologic and certainly pathologic response. which under certain circumstances
in the rat liver
makes
a
significant
indicates
con-
Protein trtrnor~er itd~ibition
tribution to protein turnover and that this cellular response, in culture systems as well as intact animals, can be triggered by a variety of stimuli and modulated by the plasma insulin level. Mechattistns
of’ protein turtim~et
The recognition that NH&I acts as a nontoxic but highly specific reagent to block hydrolysis in secondary lysosomes [7,9-l 1, 13, 281 has furnished a tool by which to dissect the cellular mechanisms for protein turnover. The rapid accumulation of numerous large vacuoles behind this chemical block provides a dramatic morphologic confirmation of this hypothesis. Additionally, the catch-up proteolysis which occurs when this hydrolytic block is released and the concurrent clearing of cellular vacuoles offers a means by which one can evaluate the rate of activity of the lysosomal system in protein turnover [ 12, 131.We have shown that inhibited proteolysis plus the catch-up proteolysis, combined with a small excess of insoluble ‘Y released. matches the total proteolysis observed in comparable control cultures. This would not be an unexpected phenomenon if we were to assume that cell protein, once sequestered into autophagic vacuoles, cannot be returned to the cell
hyalaplasm, but must be hydrelysed and/or released into the medium, The failure TV find any rebound after insulin Black of stepdown induced turnover, along with the small amount of cellular protein trapped in vacuoles by the NH:, indicates that insulin prevents the initial sequestration step in autophagy. Another possible mechanism, that insulin blocked fusion of primary lysosome with autophagosome. was initially considered, particularly in view of the rather extensive NH:-induced vacuolization observed with the electron microscope in these insulin-treated cells. We had ini-
173
tially assumed that since virtually all secondary lysosomes in these cells were derived from autophagy, a sequestration block would prevent the accumulation of the NH:-filled vacuoles. (We have never been able to induce significant heterophagy in these cultures.) However, failure to find the expected catch-up proteolysis when cells were placed in a recovery medium militated aginst this hypothesis. Other lines of evidence suggested the alternative hypothesis, that some cell protein gained entrance into lysosomes by a mechanism other than traditional autophagy. The NH; trap effected vacuolization in cells carefully maintained in the basal state; additionally lO-30% of basal protein turnover occurred in acidic vacuoles (as defined by the NH: trap), and this proteolysis was not affected by insulin or vinblastine [29]. The high rate of turnover of secondary lysosomes [26,27] made it unlikely that we were trapping with NH: a large number of vacuoles present at the beginning of the experiment. Undoubtedly, many of these vacuoles, especially the clear vacuoles with little cellular debris, represent dilitation of primary lysosomes, making it difficult to estimate the rate of protein degradation occurring in vacuoles
on the basis of the morphologic changes alone? The most convincing data, however, w&8provided ky treating f?xsb!ast cultures with cycloheximide: this agent inhibits stepdown-induced proteolysis and, in addition, has a slight inhibitory effect upon basal proteolysis [29]. Using the NH, trap technique, we have found that cycloheximide rapidly blocks proteolysis in virtually all secondary lysosomes. The fact that fibroblasts treated with both cycloheximide and NH:, whether in growth or step-down medium, show almost no cell vacuolization suggests that this agent, unlike insulin and vinblastine. blocks both the initial step in the induced (auto-
173
.4Itlrtlltr lill‘l BtlM~llc~l-
phagic) and the basal \acuolar mechanism. These observations. biochemical and morphologic. are consistent with a model containing three distinct degradative pathways (fig. 5): an induced autophagic mechanism inhibited in the final hydrolysis stage b) NH: and in its initial sequestration step by insulin. a basal neutral mechanism, and finally a basal acidic vacuolar mechanism inhibited by NH;, but insensitive to insulin. At this point we can only speculate what this last mechanism might be: (LI) a fraction of some secretory proteins, already in vacuoles, may be subject to intracellular hydrolysis, as reported for other secretory products in fibroblasts [30]; (h) we have observed that the membranous whorls commonly found in these cells eventually acquire acid phosphatase and are degraded in vacuoles; these structures may not need additional membrane for sequestration via the autophagic mechanism: tc) finally. Locke [3l. 331. studying changes in the protein granules in an insect fat body, suggested that in addition to autophagy. proteins of the hyaloplasm can enter lysosomes by a microphagocytosis along the lysosomal membrane. This mechanism has also been proposed by Trump and co-workers in studies with HeLa cells [33]. At this time we can say only that a small complementary fraction of basal protein turnover occurs in the lysosomal system, and that this process appears to be distinct from the autophagy which under some conditions supplements basal turnover. The remaining largest fraction of basal turnover may yet be found to be related to neutral proteases. both in and out of cellular vacuoles [ 13. 15, 331. REFERENCES I.
Kno~iea. S E & Ballard, F J. Biochem j 156 (19761 609. 2. Poole. B & Wibo. M. J biol them 248 I 1973) 622 I. 3. Amenta. J S. Sargus. M J & Baccino. F M. Biothem j I68 (1977) 223. t 1,’
( , /I H,,,
lh
/ /‘A\ll,
9. IO. II.
12. 13. I.$. IS. 16.
17. 18. 19. IO. 21.
25. 26. 27. 28. ZY. 30. 31. 32. 33. 3-I.
Gtmn. J M. Clarh. M G. Knov.Ic\. b t... Hopgo~~l. M F & Ballard. F J, Nature 26 I IV771 5X. Ward. W F. Cox. J R & Mot-timore. G t. J b1ol them 252 (1977) hYT5. r2menta. J S. Sareu\. hl J. Venkarti\,tn. S ti Shlnoruka. H. J cell ih\siol Y-l ( 197X1 ?’ Ballal-d. F J. Control mechanihm\ 111 c‘ancer IZJ I+ E Ct-ihs. T Ono & J R Sabine) p. 7”). Raven Prebb. New York ( 19761. Seglrn. P 0 & Reith. .A. txp cell re> Ill0 I iY7ht 276. Seglen. P 0. Biozhem biophyh re\ commun 66 (197.0 13. - Use of isolated liver cell\ and kidney, tubules In metabolic studies (ed J M Tager. H D Soling & J R Williamson). North-Holland Publishing Co.. hmsterdam (1976). .4menta. J S. Hlibko. T J. McBee. .A G. Smozuka. H & Brother. S C, Exp cell res I IS I 19781 337. .4menta. J S & Broche;. S C. J cell phy\iol ! 1979). In pres,. r2menta. J S. Sarauh. M J 6i Bacclno. F M. BIOchim biophys act:376 (1977) 253. -J cell phvbiol97 ( 1978) 267. - Intra≪lar protein catabolism ted V furk & N Marks) vol. 2. p. 27. Plenum Pre\>. New York (1977). Hershko. .4. Marmont. P. Shieldb. R & Tomkin\. G M. Nature new biol 232 ( 1971 I 206. Gunn. J M. Ballard. F J 8: Hanson. R W. J biol them 151 ( 1976) 3586. Gelehrter. T D & Emanuel. J R. Endocrinology Y-l ( 1971) 676. Neely, A N. Nelson. P B & Mortunore. G E. B&him biophys acta 45 I ( 1976) 5 I I. Mortimore. G E. Neely. .A N. Cos. J R & Guintvan. R .4. Biochem biophys res commun 5-1( 19731 x9. Goldfins. I D. Joneb. A L. Hradeh. G T. Wong. K Y & Mooney. J S. Science 101 (1978) 760. Grammeltredt. R & Berg. T. Honoe Zevler’y Z phyziol them 317 (1976) 67. ’ ’ . Dice. J F. Walker. C D. B\ne. B & Cordlel. .A. Proc natl acad sci US 75 (19?8) 2093. Khairallah. E A. Personal communication Pfeifer. Ll. .4cta giol med ger 36 (1977) 1691. Pfeifer. U. Werder, E & Bergeist. I. J cell btal 78 (1975) IS?. Warburton. M J & Poole, B. Proc natl acad XI US 7-1(1977) >‘3’7. - .Amenta. J S B Brother. S C. t\n mol nathol (19791. In press. Glimelius. B. Westermark. B & Wa\te\on. .A. ESD cell res IO8 (1977) 23. Locke. M & Collins, J V. J cell biol 26 ( 1965) X57. Locke, M & Sykes. A K. Tissue & cell 7 ( 19751 143. r2rstila. .4 U. Jauregn. H 0. Chenr. J & I‘rumD. B F, Lab invest 21 ( 1971162. Katunuma. N. Rev physiol blochem pharm 72 t 1975) 83.
Received Accepted
July 16. 1979 September 5. 1979