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Protein synthesis in the rat liver acinus after injection of actinomycin D: A radioautographic study
1. lnlroduction I have previously reported radioautogaphic evidence sho ting that normal rat liver cells are not identical with respect to general protein metabolism (LeBouton, 1968). The basis Car the previous study was the structural and functional simple liver acinus (Rappaport, 1958,1963;Rappaport et al., 1950). Basically, the liver acinus IS defmed as that amount of hepatic pxenchyma which secretes its bile into a cornmoo terminal bile ductule located in the center of the parenchymal mass. In addition, a teermim.1 hepatic arteriole and terminal portal venule c.oone parallel to the bile ductule in the middle of the aciow. Blood carried to the center of the a&us by its two afferent terminal vessels drains outwards via sinusoids past the hepatic cell plates (Elias, 1963).to the periphery of the acinus. At the periphery blood flows into term& nal hepatic venoies (centml veins} thcnco out of the liver through the hepatic -&I. Based on histochemistry and pathology Rappaport has divided the acinus into three metabolic zones (Rappapa& 1963). Zone 1 is the one.ttid of paenchyma w&h mmediately surrounds the ienoinal portal wnulc and other terminal vessels in the center of the scinus. Zone 3 is the owthird nearest terminal hepatic venoler at the periphery of
the acinus, leaving zoi;l 2 8s the intermediate onethird. Specifically ther. the previous study (LeBouton, 1968) demonstrated tat after iojcction of leuci&R into rats ceils in acinar :zooe I incorpora:cd more radioactivity faster and lost this radioactivity swner than did cells in either zone 2 or 3. When the above study of protein metabohsr? in the liver acimrs was conpleted it was felt that cells io zone 1 might be synthesizing different p~oteios than cells in the other acioar zonk. If this w&e so then the kinds of messeneer ribonoleic acid fmRNA) in cells of the various s&w zwes would also be different (LoBouton, 1969a). This particular problem was difticolt to solve radio:lurographicallyy until the <.Aperiments of Wilson and Halgland (1967) w !re ooblished on the metabolic blf-life of rat Ii ‘er mRNA. They were abk t,a demonstrate by :~ppropriate Injections of Actioom~chi D followed by anatyyris of the rem&dog polyscxes that there are two bwsd classes OS mRNA in ra !iver ceils with respect tn, durathn of thek me?rrolic half-life. About two.. thirds of the mRNA hive a hal&liie of arou.ld :I br, whereas the remaining one-tidrd have a h&Ii% of near 80 hr. Thus with incieaziog time after Actcnomycin D injection, which inhibits maWA. synthesis (Revel and Hiatt. 1964) the bulk of mltNA which has a short half.life is degraded more rapidly
.
,
ronwed. Subsequenily,W&n, HiU and Hoagland _ (1967) demonstrated a six-fold iocreaaein tha incorporation of leucin@-14C into plasma albumin by nt livers known to contain prlrnarily Ion&lived mRNAdue topriortrealmentwith ActinomycinD. The presentpaperessentiallyrepcatsthe workof WUronet 31.(I9B7) but from a radioautogmphk approachin orderto determinethe qualitativeacinat dbtrlbutionof proteinmetabolismin rat ltvers knownto containmRNAof the type with P long metsbolk hslf.life.
livwa wereper free of bled with a catheter inserted in tlaepartal vein (L&&on, 1963). The right Iatcd lobe wu removedand trimmedinto smaIl blockswhichwere Iinedovcmi&t at S’C. On&rdf of the tii blockswere tixcd h 4 per ant buffered formaldshydc(pi+ 7.2 to7.4) madeby disso?vIog solid parsfonnaldahydc @daliiwkrodtchemicalWotke, Los Angelev,C&f.) in a phosphate huffy (Kulswm and Schultz, 1965). Ths mmaintn~fitwe bloclrowre f’uredin 2.5 per@ phmph~e buffered glutaralidchyde(Kmlsoo md ti&ultz)1965).
3. Reaunl
These au wac iajwmd with AcMotnydtt D to inhibitmRNAsynthe~tJwn&cnkucinofH14hr k&r.
(Elk., 1953; Elias, 1963) consMIng of one layer of cells around portal canals was hi$tly labeled. 3.3. Control on protein JyntheJfJ In ordcdor to see bow much of the label obnwed in
nwbolitcs, the following cOavsolwa8 dons. Again an equal volume of only athnd&ne was
lnjccted.
Cyclohexamirk was thenh#%d 12 hr laterto atop porein synthcsb f&wad in 2 hr by leucin~-~H injection.
and allow the specitic rzdioactitity of the iree amino pool to increase in the presence of the Actinamycin
A:tinomycin D is known to considerably increase the sz%c~Sii radioactivity of the intracellular free ami& acid pwl (Wilson and Hoagland, 1967) and Paten ar,d Ashley (1967) have shown that fixation Of tissue in ~iu:~ldeb~-d~ ca bind free labeled amino acids within the cell resulting in radiaauta Therefore, fa asseess whether an:, ~di~cti~~ seen in the experimental rnimals was due to artifachwdly bound leu~ine.~H, the following cor,tmi was done. Actsomycin D was sdmlnistered to stop mRNA synthesis. Twelve hours later cycle~~~~~ WIS in&ted to itiit protein synthesis
D. Finally leucine-‘H was injecsd 2 hr after the cy&hexamide. This approach did not reveal any labeling patterns within the liver scinus (fig. 4). At higher magnitications the average nunber of reduced silver grains over cells fixed in formaldehyde oi $utaraldehyde ~61: not @niiicantly different and in both czssfl?were only slightly higher than background.
&yAi.z artif..ctr.
4. Diiion As previously stated, Wilson and Hoagland (1967) have demonstrated heterogeneity of rat liver mRNA with respect to metabolic half-life. About 67 per cent
of rat liver mRNA has a half-life of “ear 3 hr while the remaining 33 per cent has a half-life of close to 80 hr. Assumtnn inhibition of mRNA synthesis soon aftei Actinomy~in D injection (Revel and Hiatt, 1944) would &a” thai zi 14 hr after injection of Acthtomvcin D about 4.7 half-lifes of the short lived mRNA would have elapsed compared with approximutely 0.18 half-lives of the more stable variety. If a nomogram is consulted for radionuclides relating elapsed half-lives to per cant radioactivity remaining and assuming random mAJx’A intracellular degradation is similar, then at 14 hr after Actinomycin D injection the of mRNA remaining would be 4 per cent of the short livtd fraction and 88 per cent of the long lived type. Now in a hypothetical liver containing IW mRNA molecules there would initiallv be 67 short lived and 33 long lived molecules. However, I4 hr after Actinomycio 13 treatment the amounts of long lived and short lived mRNA would be 26 and 2.6 molecules respectively. Thus o” a theoretical basis there were ten times as many long lived mRNA molecules as short lived ones in the experimental livers at the time of the leucine.3H injection. The normal control liver (fig. 2) does “of show any apparent differences among cells in the acinus but it would if detailed grain counts were done across the acinus as previously (LeBouton, 1968). I” this case the results would show that cells in acinar zone 1 contain more radioactivity than cells in other zones, but this difference is not “~nnally obvious. Why cells in the limiting plate of the “Onnal control contained high amounts of mdioacdtity is not understood. However, Elias(1953,1%3) has stated that these cells are morphologically different from most liver cells in that they aze usually smu.Uer and stain more intensely with dyes such as win which indicates a high protein content. Perhaps the protein in limiting plate cells is being metabolized eve.” more rapidly than orotetn in cells tn the rest of the liver. Tiecontrol on protein sytKhes!a using only cyclohcxamide (tig. 3) does demonstrate that the majority of injected Ieuci~te-~H which reaches the liver is incorporated into the primpry stmctwe of proteins. The desirabi!ity of leucine-3H as a reliable indicator of protein synthesis has bee” diicussed previously (Lebtond, 1965; LeBouto”, 1968). The control on free leucine-3H demonstrates that under thas experimental conditions there is no
mmunts
.“,
artifactual binding of the anuno acid to celluba constituents (tig. 4). Therefore the radioactivity in the exeerbnental livers is the result of leucine-‘H incorpor&o” into the primary structure of protein and not non-specific binding. Also sinie glutaraldehyde fixed tissues exhibited similar amounts of labcling to those fined with formaldehyde the choice of firative does not appear to be critical in in rziuo experiments such ar this. Therefore, since the injected Ieu~ine-~H was shown by appropriate controls to be nearly exclusiveljj incorporated into protein in rat livers which contained primarily long lived mRNA the conclusion is that the cells in xinar zone I which contained the highest amount of radioactivity probably also possess most of the long lived mRNA found in rat liver. Or. treat. ment with Actinomycin D appears to accentuate the normal subtle difference previously found to exist between liver cells with respect to protein metabolism KeBouton. 1968). Wilson et al. (1967) have conluded that the long lived mRNA probably codes for plasma albumin synthesis, therefore it is very provocative to consider the possibility that cells in acinar zone I xe xncerned with most if not all of the plasma albumin synthesis attributed to the entire liver(Madden an< Whippb. 1940; Miller and Bale, 1954; Peters, 1962: LeBouton. 1967;leBouton 1969b). Arguing @ahtat this proposa! is the report by Hamashims, Hatter and Coons 1’1966) who demonstrated by tluorescconce microscopy that albumin is found primarily in cells near central veins (acinar zone 3). Yet the poss.bility of pinocytosis of olasma by these celk should be considered since the ap&e”s were obtained by punch biopsy at routine cho!qatc~tomy and some time elapsed before the biopsy was frozen. Acknowledgements I gratefully acknowledge the expertise of Mrs. Mirdza Lasmanis in preparation of the r&dioaufograms. Supported by NlH grant GRSG 616 and NSF
~ntGB8306.
Eliiw, H., ,953, Functional “l0rph0logy Of the liver, I”: Remch m the service of medicine. voL 37 G.D.Scsllc and
35s
A.V.LE BOUTON
Miller, L.L. and W.F.IM:,
1954, Synthesis of all ~Iarmr
PttctS. T.. Ir., 1962. The biosynthesis of rat sa-um albumin. II. In.racelluhrphan:mem in the sccrction of newly formed elbumin. 1. Bial. Chem. 237, I t86. Peters.T. and C.A.Arhley. I%7 An artifact in rdiewto mphy Bue hind& of Eec aminua&ids by faceives, 1. Cell BiL 33,53. Rappport, A.M.. 1958, The stmctutnl and functional utii In the human liver, Anat. Rec. 130,673. RRppport. A.M., 1963. Acinv units and the paihophyridlogy ofthe liver, tn:‘z?le lwer. vol. 1. ed.: C,Rouillu (Academic Fwrenl, New YOXic)p. :x5. Rsppapwt, A.M., 2.3. Borowy, WM. Iau@ed, and W.N. LoI.
to
lobudint~ a
to, 1954, Subdivisfori of hex8gondlier [\mctbnal unit, Anat. Rec. 119. I I. Revel. M. and H.H.Hirtt. 1964. TIE stabilihr of Iirar
rkucluml and
phyki. 8s~. 20, 194. n ofconted rsdi& id cmutsion. Rot.
and
the acinus, leaving zoi;l 2 8s the intermediate onethird. Specifically ther. the previous study (LeBouton, 1968) demonstrated tat after iojcction of leuci&R into rats ceils in acinar :zooe I incorpora:cd more radioactivity faster and lost this radioactivity swner than did cells in either zone 2 or 3. When the above study of protein metabohsr? in the liver acimrs was conpleted it was felt that cells io zone 1 might be synthesizing different p~oteios than cells in the other acioar zonk. If this w&e so then the kinds of messeneer ribonoleic acid fmRNA) in cells of the various s&w zwes would also be different (LoBouton, 1969a). This particular problem was difticolt to solve radio:lurographicallyy until the <.Aperiments of Wilson and Halgland (1967) w !re ooblished on the metabolic blf-life of rat Ii ‘er mRNA. They were abk t,a demonstrate by :~ppropriate Injections of Actioom~chi D followed by anatyyris of the rem&dog polyscxes that there are two bwsd classes OS mRNA in ra !iver ceils with respect tn, durathn of thek me?rrolic half-life. About two.. thirds of the mRNA hive a hal&liie of arou.ld :I br, whereas the remaining one-tidrd have a h&Ii% of near 80 hr. Thus with incieaziog time after Actcnomycin D injection, which inhibits maWA. synthesis (Revel and Hiatt. 1964) the bulk of mltNA which has a short half.life is degraded more rapidly
.
,
ronwed. Subsequenily,W&n, HiU and Hoagland _ (1967) demonstrated a six-fold iocreaaein tha incorporation of leucin@-14C into plasma albumin by nt livers known to contain prlrnarily Ion&lived mRNAdue topriortrealmentwith ActinomycinD. The presentpaperessentiallyrepcatsthe workof WUronet 31.(I9B7) but from a radioautogmphk approachin orderto determinethe qualitativeacinat dbtrlbutionof proteinmetabolismin rat ltvers knownto containmRNAof the type with P long metsbolk hslf.life.
livwa wereper free of bled with a catheter inserted in tlaepartal vein (L&&on, 1963). The right Iatcd lobe wu removedand trimmedinto smaIl blockswhichwere Iinedovcmi&t at S’C. On&rdf of the tii blockswere tixcd h 4 per ant buffered formaldshydc(pi+ 7.2 to7.4) madeby disso?vIog solid parsfonnaldahydc @daliiwkrodtchemicalWotke, Los Angelev,C&f.) in a phosphate huffy (Kulswm and Schultz, 1965). Ths mmaintn~fitwe bloclrowre f’uredin 2.5 per@ phmph~e buffered glutaralidchyde(Kmlsoo md ti&ultz)1965).
3. Reaunl
These au wac iajwmd with AcMotnydtt D to inhibitmRNAsynthe~tJwn&cnkucinofH14hr k&r.
(Elk., 1953; Elias, 1963) consMIng of one layer of cells around portal canals was hi$tly labeled. 3.3. Control on protein JyntheJfJ In ordcdor to see bow much of the label obnwed in
nwbolitcs, the following cOavsolwa8 dons. Again an equal volume of only athnd&ne was
lnjccted.
Cyclohexamirk was thenh#%d 12 hr laterto atop porein synthcsb f&wad in 2 hr by leucin~-~H injection.
and allow the specitic rzdioactitity of the iree amino pool to increase in the presence of the Actinamycin
A:tinomycin D is known to considerably increase the sz%c~Sii radioactivity of the intracellular free ami& acid pwl (Wilson and Hoagland, 1967) and Paten ar,d Ashley (1967) have shown that fixation Of tissue in ~iu:~ldeb~-d~ ca bind free labeled amino acids within the cell resulting in radiaauta Therefore, fa asseess whether an:, ~di~cti~~ seen in the experimental rnimals was due to artifachwdly bound leu~ine.~H, the following cor,tmi was done. Actsomycin D was sdmlnistered to stop mRNA synthesis. Twelve hours later cycle~~~~~ WIS in&ted to itiit protein synthesis
D. Finally leucine-‘H was injecsd 2 hr after the cy&hexamide. This approach did not reveal any labeling patterns within the liver scinus (fig. 4). At higher magnitications the average nunber of reduced silver grains over cells fixed in formaldehyde oi $utaraldehyde ~61: not @niiicantly different and in both czssfl?were only slightly higher than background.
&yAi.z artif..ctr.
4. Diiion As previously stated, Wilson and Hoagland (1967) have demonstrated heterogeneity of rat liver mRNA with respect to metabolic half-life. About 67 per cent
of rat liver mRNA has a half-life of “ear 3 hr while the remaining 33 per cent has a half-life of close to 80 hr. Assumtnn inhibition of mRNA synthesis soon aftei Actinomy~in D injection (Revel and Hiatt, 1944) would &a” thai zi 14 hr after injection of Acthtomvcin D about 4.7 half-lifes of the short lived mRNA would have elapsed compared with approximutely 0.18 half-lives of the more stable variety. If a nomogram is consulted for radionuclides relating elapsed half-lives to per cant radioactivity remaining and assuming random mAJx’A intracellular degradation is similar, then at 14 hr after Actinomycin D injection the of mRNA remaining would be 4 per cent of the short livtd fraction and 88 per cent of the long lived type. Now in a hypothetical liver containing IW mRNA molecules there would initiallv be 67 short lived and 33 long lived molecules. However, I4 hr after Actinomycio 13 treatment the amounts of long lived and short lived mRNA would be 26 and 2.6 molecules respectively. Thus o” a theoretical basis there were ten times as many long lived mRNA molecules as short lived ones in the experimental livers at the time of the leucine.3H injection. The normal control liver (fig. 2) does “of show any apparent differences among cells in the acinus but it would if detailed grain counts were done across the acinus as previously (LeBouton, 1968). I” this case the results would show that cells in acinar zone 1 contain more radioactivity than cells in other zones, but this difference is not “~nnally obvious. Why cells in the limiting plate of the “Onnal control contained high amounts of mdioacdtity is not understood. However, Elias(1953,1%3) has stated that these cells are morphologically different from most liver cells in that they aze usually smu.Uer and stain more intensely with dyes such as win which indicates a high protein content. Perhaps the protein in limiting plate cells is being metabolized eve.” more rapidly than orotetn in cells tn the rest of the liver. Tiecontrol on protein sytKhes!a using only cyclohcxamide (tig. 3) does demonstrate that the majority of injected Ieuci~te-~H which reaches the liver is incorporated into the primpry stmctwe of proteins. The desirabi!ity of leucine-3H as a reliable indicator of protein synthesis has bee” diicussed previously (Lebtond, 1965; LeBouto”, 1968). The control on free leucine-3H demonstrates that under thas experimental conditions there is no
mmunts
.“,
artifactual binding of the anuno acid to celluba constituents (tig. 4). Therefore the radioactivity in the exeerbnental livers is the result of leucine-‘H incorpor&o” into the primary structure of protein and not non-specific binding. Also sinie glutaraldehyde fixed tissues exhibited similar amounts of labcling to those fined with formaldehyde the choice of firative does not appear to be critical in in rziuo experiments such ar this. Therefore, since the injected Ieu~ine-~H was shown by appropriate controls to be nearly exclusiveljj incorporated into protein in rat livers which contained primarily long lived mRNA the conclusion is that the cells in xinar zone I which contained the highest amount of radioactivity probably also possess most of the long lived mRNA found in rat liver. Or. treat. ment with Actinomycin D appears to accentuate the normal subtle difference previously found to exist between liver cells with respect to protein metabolism KeBouton. 1968). Wilson et al. (1967) have conluded that the long lived mRNA probably codes for plasma albumin synthesis, therefore it is very provocative to consider the possibility that cells in acinar zone I xe xncerned with most if not all of the plasma albumin synthesis attributed to the entire liver(Madden an< Whippb. 1940; Miller and Bale, 1954; Peters, 1962: LeBouton. 1967;leBouton 1969b). Arguing @ahtat this proposa! is the report by Hamashims, Hatter and Coons 1’1966) who demonstrated by tluorescconce microscopy that albumin is found primarily in cells near central veins (acinar zone 3). Yet the poss.bility of pinocytosis of olasma by these celk should be considered since the ap&e”s were obtained by punch biopsy at routine cho!qatc~tomy and some time elapsed before the biopsy was frozen. Acknowledgements I gratefully acknowledge the expertise of Mrs. Mirdza Lasmanis in preparation of the r&dioaufograms. Supported by NlH grant GRSG 616 and NSF
~ntGB8306.
Eliiw, H., ,953, Functional “l0rph0logy Of the liver, I”: Remch m the service of medicine. voL 37 G.D.Scsllc and
35s
A.V.LE BOUTON
Miller, L.L. and W.F.IM:,
1954, Synthesis of all ~Iarmr
PttctS. T.. Ir., 1962. The biosynthesis of rat sa-um albumin. II. In.racelluhrphan:mem in the sccrction of newly formed elbumin. 1. Bial. Chem. 237, I t86. Peters.T. and C.A.Arhley. I%7 An artifact in rdiewto mphy Bue hind& of Eec aminua&ids by faceives, 1. Cell BiL 33,53. Rappport, A.M.. 1958, The stmctutnl and functional utii In the human liver, Anat. Rec. 130,673. RRppport. A.M., 1963. Acinv units and the paihophyridlogy ofthe liver, tn:‘z?le lwer. vol. 1. ed.: C,Rouillu (Academic Fwrenl, New YOXic)p. :x5. Rsppapwt, A.M., 2.3. Borowy, WM. Iau@ed, and W.N. LoI.
to
lobudint~ a
to, 1954, Subdivisfori of hex8gondlier [\mctbnal unit, Anat. Rec. 119. I I. Revel. M. and H.H.Hirtt. 1964. TIE stabilihr of Iirar
rkucluml and
phyki. 8s~. 20, 194. n ofconted rsdi& id cmutsion. Rot.
and