ClJRRENTS IN MODRRN tuOLOCY 3 (1969) 4-S. NORTHkOLLAND PUSLlSHlNG COMPANY, AMSTERDAM

RELATIONS AflD EXTENVDF THE ZONE OF INTENSIFIED PROTEIN METABOLISM IN THE LlVER ACINUS

Received L3 Jut% ,969

1. INTRODUCTiON

A recent study demonstrated tbat a group of cells

within the simple liver winus metabolizes protein ?lt a faster rate than other liver ceUs(lcBouton, :968e). The data from this pre\iour study were obtained by arbitrarily dividing 3H Ieucinelabelled radiorutograms of the simple liver acinus into three metabolic zones after Rappaport (1963). This approach served to local-

ize highest protein metabolism to the cclb of arbitrary

metab4ic zone I but produced no significant informa- tion pertaining to the relationship of this group of cells to the two other arbitrary metabolic :was add to tbc terminal hepatic vasculatore (Rappaport, 1963).

Therefore, radioautogmms from liven of rats in-

jected with 38 leucine were critically evaluated to de

termine the microanatomical domain af this higher metabolic zone as seen in two-dimensional histological sections.

2. METHODS AND MATERIALS

Radioautograms were prepared f:om livers of rate injected with %i leucine as dezibed previously (LeBcuton, 1968a).

Results of the prior investip,ation showed that&e ~.rea of intensified protein metabolism (zone I) con.

taioed more radioactivity than other areas at 15 mio after radioisotope injection but that all zones were equally radioactive 30 min after injection of 3H kucine. Therefox, thee two time itawls were chosen for analy& h the following three locations: Terminal hepatic venuk to terminal hepotic vent& (acinus) analysis. Radioautogmma were scanned under low magnification (100 X) until an area was found where two terminal bepatic venuler (WV) were

separated by 25 to 35 cells end there was evidence of

a terminal portal venuk (TPV) midway between the two THVs (fii I). Tbe number of silver prains/ceU wes then determined at 1000 X for eacn cell in the straightest plate of cells urnnesting the two THVs. Portd canal to lerminal hepatic venuk mdyai% Under low mqnifiiation areas of liver were found where a portal canal (PC) wes separated from an adjacent THV by 20 to 25 liver cells (4 1). The amount of silver grains/c4l was then recorded at 1000 X over the straightest plate of cells between the PC and THV. Pcwtal cmnl through nod&point to tednal hepatic venuk amlysk A nodal point (NP) has been ds- scribed by MaU(l904) as the area of amutomosis between three TPVs (tig. 1). Thk point coincides with ow corner of the ckssii hexagonal liver lobule (Kiernan. 1833). Thus, liver sections were seanned at LOU X until an area was found where an imeginary line could be drawn starting from I FC, continuing

along the length of a TW (one side of i classic hexa- gonal lobule). through the respective NP and on up to the THV of the adjacent classic lobule (B;!. 1). At 1000 X the number of silver greins/ceU was then scored over the 40 to SO cells located along this imagblnry line.

In order to reduce irregularities due to irdividual ceUuiu vaAion6 the data from !he abov:: three analyses were treated in the followinl: ,nanner. In fig. 2 the fuel point on each graph denwee the combmed l”eC4@ Srainr/ccU Oye, % fust three CeUZi Of that particular analyair. The next point represents the combined average gains/cell over cells two, three and four; while the next point is the average grains/ cell over combined cells three, four end fwe, end so on. This type of triplet ave@n& overlapping the previous triple1 by two cells was used for each graph Of mpIe%+ntetiie eIk&‘8e8 6een in fiS. 2.

Four determinalions were made for each analysis and background (2 to 3 grains/cell) was not subtracted.

3. RESULTS

In every case, the results from the four deter- minatiotx of each approach and time interval were consistent. The distribution of silver twin? over ceUs spread from THV to THV is seen in tig. 2 (top,. At

15 min after injectton an area about tive or rix cells wide and corresponding to the location of a TPV has incorporated almost SE5 more of the label than cells further removed from the TW. By 30 min after in- jection the initial hi&ly labeled cells have Iwt close to half of their radioactivity with the result that sbnus’r all cells are equally labelled.

The spread of .gah~.+ll obtained by cou~tiug from

6 A.V.lE BOUTON

t t THV* TWX

THV analysis, bottom.

a FC to a THV is expressed in fig. 2 (middle). During the tirst 15 min the four or five cells nearest the PC were most radioactive. In the ensuing 15 mic the highly labeled periportal cells lost much of their IaSd 80 that k/ 30 tin after injection the) contained djghtly less label 1San other cells.

The number of grains/;zU extending from a F’C through an NP and up to a THV is shown in fig. 2 Chottomj. The fust eight to ten cells along the axis of &eTPV contained mme label 15 min after injection than cells located abng this line but more distant from the PC Zy the next 15 min all cells contained essentiaUy the same radioactivity. regardless of their proximity to the PC.

4. DISCUSSION

Discusllg the various metabolic ones in the Liver acinus Rappap& (1963) suggests that the micrcs wwironment of c&s adjacent to PCs is sirr.ilar to thal found around THVr However, the results prerentcd here show that periportal cells are metaboiically similar to cells around TWs, at least with respect to general protein metabolism. Thus, the microenvironment of zone I around TWII extends to also surround PCs. ‘These obscrvationhve ban Jiagrar?uned in fig I in an attempt lo emphasize this point. This jnterpreta- tion has an anatomical basis in the wart of Hare and

Bri:nm (1966) using microinjection techniques and Kernret al.(l!k%) with microradiography. In both papers anall inlet venules(Eliar, 1963) were ken coming from portal vessels and wpplyiig the paren- chyma adjacent to portal canals.

In his diagram of advanced pericentral Meerosis, Rappport (1953) indicates three extendons of necrotic tissue radiating from the TRV towards nearby PCs. The diagarn shows that the necrotic areas do not reach the postal canal but instead leave a small cuff of normal tissue between the nwrotic processes and PCs. With respect to the pnrnt study, this cuff of remind tissue is considered to be the extension of zone 1 from the area around TPVs.

It is interesting that this extension contains the only subpopula!ion of parcnchymal cells which con- * ~ntly appear different from the majority of hepatic . .euchymal cells. This group of cells surrounds KS, appear narrower and stain more strongly with routine histological rtainr (Elias, 1953). Eltas( 1953) has named these cells the p&portal limiting plate. In ad- dition, gUaraldehyd~fiicd specimens stained with hematoxylin and ewin contain baaophilic material in zone I cells which is fkcly dispersed giving a hyaline appearance while the basophilic mat&l in cella

around THVs is coarse and clumpy. This observation

has ultrastructural correlates since Rappaport (1963)

has shown that the rough endoplasmic reticulum in

zone l cells is evenly distributed whereas the same material in cells near ‘MWr is unevenly concentrated into discrete dumps.

The zone of intensified protein metabolism is about eight cells wide, ten cells long and extends four to fie cells out from PCs ffie. 1). Thus zone I IS m- proximately one-third the‘dist&ce between a TI’+ and adjacent THV but only about one-fourth the disteoce between a PC and a THV. These are values from IWD dimensional sections and therefore give no informa- tion as to the depth “I third dimension of tbis meta- bolic area. The dutaoce between adjacent TPVs along the length of PCs has not measured. Assuming that aU

TPVs come off the PC at right angles, are the TPVs close enough to each other so that their surrounding zones of increawd protein metabolism become con- fluent “1 are they spaced farther apart so that each zone I is unique to its own TPV and ooly the exten-

sion of zone I around PCs serves as e connecting

sleeve between areas of increavd protein metabolism’!

Actually, the TPVs pursue arched looping paths through theliver(Rappaport, 1963) which makes any approach to this problem of tbe threedimensional ex- tent of zone I verv difficult. This information will have to be ascertainedbefore any estimate ten be made of

the percentage of total liver parenchyma localized in this MI of high protein metabolism.

One basic assumption is that the composition of sinuwidal blood with respect to nutrients ztnd waste productr is not 11 same at different points along the sinuroid(Ra”Daoort. 19631. This altarelton of sinus . . . . . r

oidal blood is presumed to be gradual, yet the change in rate of genemel cellular protein metabolism is quite

abrupt II seen in the 15 mio curves in fiti. 2. ‘This is not to infer that cells outside zone I ere metabolically deprived, but only that their general protein meta- bolism is not operating at an increased rat<. Certainly

the level of protein metabolii in cells outride zone 1 is adequate to handle intracellular protein require-

ments, probably repreacnted by endugenous enzyme turnover.

The abrupt change in rate of protein metabolism becomes even more pronounced if instead of slow per- eolation of blood down the sinusoid other progressions

are cunddered. For example, due to inlet and outlet

sphincters the sinusoids may alternativel:y fdl and

empty with blood (K&&y, 1936, 1939; Knisely

et al., 1957; Wakim, 1942). Also reticulo-endotheiial cells bulging into the sinusoidal lumen in conjection with precapihry arteriole sphincters can control the quality of blwd from purely arterial to purely portal, to any mixture of the tw”(McCuskey, 1066).

Another view holds that blood flow ciao be uneven and progress in pulsaGle spurts (Rappaport et al., 1966).

Whatever the actual mode of blood flow through the sinusoid the fwt remains that cells in zone I maintain an increased rate of protein metabolism. Either zone I

cells metabolize proteins that are identical to those found in cells of the other zones. but at a faster rate, or they metabolize species of protehs not found in other htoatlc cells. If this is so. zone I cells should con- tain different species of mRNA and hence c”:resp”nd- ing structural genes in their genomes would be trsns

cribed. Accepting the assumption that enzyme metabolirm

in ceils other than in zone I is adequate for cel!ular needs the obvious comllary is that cndogeoous en-

zyme metabolism in zone I cells is most assuredly suf-

ficient. Reference to fig. 2 shows that zone I cells rapidly incorporate and lose radioactivity 5” that by 30 min after injection they contain label at almost the same level as uther cells not in zone 1. Since the only other kind of protein exPected in liver cells “Szone I

is newly synthesized plasma protein such as a:‘umin, which has B I5 to 20 min turnover time (LeBoutcn, 1968b; hiiller et a!., 1964; Perers, 1962). acinar zowz I could prove to be an especially active site ( .“plnsma protein synlhes~s.

8 A.V.LI

Krdsely, M.H., F.Hardiq and H.Dcbpcktr. 1957. Hepatr suhihincters. Bti summaw of urwcnt day knowl&e.

L+Sou,on, A.V.. PXlab, Pr~~r~~~-pwJwcf relationship be. tween intrahopatic albumin and p&ml albumin, Biochem. J. 1%. 503.

MaU. F.P., 1906, A stwly of Le sbucblwJ unit of u1e liver, Am. I. Anal. 5,727.

McCuskey, R.S., 1966, A dynamic ald stltif scUdy of hepatic artcrioler s”d kepalic rpbb,c,e,s, Am J. Anat 119,455.

Milhr. L.L.. H.R.Hanavan N.Tit,bac.bi and A.Chwdhury, ,964, Dominm,:~irif,~heLerinbiorynUKriro~ plasma priltiw with spectl referrncc ta ,he pluma mu- caprotcins Oewnucoid), unr,o,dw~,n md f,b,,“o~e,,, Ad”. Chem. 44, L7.

Peten, T.. Jr., 1962.7he biosynthesis of rat serum albumin II. LntraccuuJaI pi?:“omena in the rcretion of newly

formed albumin: J. Biel Chem. 237,11%. Rappnpo~t, A.M.. 1963, Acina “nils and ,be patbopbysi~

logy of th. lie,, in: fbe Liver. od C.Rar,Sq vo,. , <Academic Press, hew Yak) p. 266.

RappapolS A.M.., R.G.Blrk, C.C.Lueu, J.H.Ridw, and CN.Bert, ,966, Normal and Pa,ho,ogie micr.xircula,ion of ,he Ming mammlli liver, Rcr. Inlerm,. HoparoL 16, 813.

Rwer, J.T., J.E.Leathcn and C.Boatri$t 1966. Microradio y;yshy of the rabbits bopatic microcind~tion The simikrity of the hepatic porta, am, pdnmary utertal ckc”la,b”+ AnaL Rec. 15 . 103.

Wakbn, K.G. and F,CMum, 1942, lntnkepatic circulation ofblood, Anat. Rec. 82.233.