Kinetics of Corneal Epithelium Turnover In Vivo

Transcription

1 Investgatve Ophthalmology & Vsual Scence, Vol. 3, No. 0, October 990 Copyrght Assocaton for Research n Vson and Ophthalmology Knetcs of Corneal Epthelum Turnover In Vvo Studes of Lovasrarn Rchard J. Cenedella and Charles R. Fleschner The authors developed a drect chemcal approach for estmatng the rate of turnover of the corneal epthelum n vvo. The method was used to examne the effects of lovastatn, a potent nhbtor of cholesterol bosynthess, on prolferaton and turnover of the epthelum. Corneal DNA was labeled by pulse njecton (IP) of the rat wth 3 H-thymdne, and 3 H-labeled DNA was recovered from perpheral and central corneas over the next 5 days. Only the epthelum became labeled, and the loss of label by cell desquamaton began 3 days after njecton. The loss of 3 H-DNA from the cornea (perpheral plus central regon) followed frst-order knetcs. The half-lfe of the dsappearance was about 3 days. The perpheral cornea became more hghly labeled than the central cornea and began to lose 3 H-DNA before the central cornea. These observatons support the possblty of a hgher mtotc rate n the perpheral regon and the centrpetal movement of a populaton of perpheral epthelal cells n the normal cornea. The half-lves of the dsappearance of 3 H-DNA from perpheral and central corneas measured between days 5 and 5 postnjecton were dentcal, both at 3 days. Complete turnover of the corneal epthelum would, therefore, requre about 2 weeks (4-5 half-lves). Treatment of the rat wth lovastatn had no obvous effects upon the prolferaton or turnover of the corneal epthelum. Although lovastatn nhbted corneal 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key regulatory enzyme of cholesterol synthess, the cornea compensated by nducton of ths enzyme so that there was no net nhbton of cholesterol synthess n the cornea. Invest Ophthalmol Vs Sc 3: ,990 The corneal epthelum undergoes dynamc turnover due to sustaned prolferaton of basal epthelal cells. These basal cells are then dsplaced outward by the next generaton of mtotc cells and eventually lost by desquamaton. ' 2 A slow centrpetal movement of perpheral epthelal cells also contrbutes to the renewal of the epthelum. 3-4 Net turnover of the epthelum has been estmated n vtro by measurng the rate of labelng of the basal cells from 3 H-thymdne ' 2-5 and n vvo by followng the movement of dye-tagged cells. 3 Both expermental approaches provde data whch suggest that the corneal epthelum s completely replaced n about 2 weeks. We are nterested n relatonshps between membrane synthess n epthelal cell populatons and ther turnover. Rapd membrane growth s requred for cells to proceed through the cell cycle. 6 " 9 Because From the Department of Bochemstry, Krksvlle College ofosteopathc Medcne, Krksvlle, Mssour. Supported n part by NIH grant EY02568 and by the Krksvlle College of Osteopathc Medcne. Reprnt requests: Rchard J. Cenedella, Department of Bochemstry, Krksvlle College of Osteopathc Medcne, 800 West Jefferson, Krksvlle, MO cholesterol s requred for membrane formaton and because the cornea appears oblgated to synthesze most of the cholesterol whch t requres for cell growth, 0 we were nterested n the possblty that nhbton of cholesterol synthess n the cornea mght affect prolferaton and turnover of the corneal epthelum n normal and wounded states. To nvestgate these possbltes, we developed a chemcal method for drectly and quanttatvely estmatng rates of prolferaton and turnover of the corneal epthelum n vvo. The approach bascally nvolves pulse labelng prolferatve corneal epthelal cells from a sngle ntrapertoneal (IP) njecton of the rat wth 3 H-thymdne and then measurng the concentraton and dstrbuton of 3 H-DNA n the cornea wth tme after njecton. Knetc analyss of the data permtted calculaton of the net rate of epthelum turnover of both the central and perpheral cornea and the tme requred for mtotc cells to desquamate. Informaton on the possble mgraton of labeled epthelal cells from perpheral to central cornea was also obtaned. Ths method was used to test the nfluence of lovastatn, the prototype of the new generaton of hypocholesterolemc drugs," on prolferaton and turnover of the corneal epthelum. We observed pre- 957 Downloaded From: on 2/07/208

2 958 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / October 990 Vol. 3 vously that lovastatn could block prolferaton of lens epthelal cells n culture and that the block was completely reversed by addton of serum low-densty lpoprotens to the meda. 2 Anmals Materals and Methods Anmal use was approved by the nsttutonal anmal care commttee, and all expermental procedures reported here conformed to the ARVO Resoluton on the Use of Anmals n Research. Male Sprague- Dawley rats (Hlltop, Scottdale, PA), 50 g, were fed Purna rat chow (Ralston Purna, St. Lous, MO) or ground chow contanng 0.% lovastatn for 2 weeks. Cornea Epthelum Turnover The rats (control or lovastatn treated) were njected IP wth 2 nc/g of body weght of 3 H-(methyl) thymdne (2 C/mmol; New England Nuclear, Boston, MA). They were klled by carbon doxde nhalaton at varous tmes over the next 5 days. The corneas were removed and freed of attached scleral tssue by dssecton. The central and perpheral corneas were separated wth a 3-mm dameter trephne. Perpheral cornea accounted for an average 6 % and 60% of the total cornea wet weght n control and treated rats, respectvely. In some cases, corneas were physcally separated nto epthelal and stromal fractons as done prevously. 0 Corneal fractons from ndvdual rats were separately homogenzed n.0 ml of salne contanng 0.4 mg of carrer DNA (salmon, type III; Sgma, St. Lous, MO) usng a 2-ml, ground glass-ground glass hand homogenzer (Radnot Glass, Monrova, CA). The homogenates were combned wth two separate -ml salne washes of the homogenzer, proten was dgested wth protenase K, and the DNA was recovered and assayed for trtum content as descrbed prevously. 3 The 3 H-DNA content of the central, perpheral, and whole cornea was calculated. The dstrbuton of substrate n the cornea for DNA synthess and 6 hr after njecton of 3 H-thymdne was measured by treatng homogenzed corneal fractons wth trchloroacetc acd (TCA). After centrfugaton, the supernatant was collected and the TCA extracted wth ether. The extracted supernatant was quck frozen and lyophlzed. The resdue was redssolved n water and counted. We showed prevously that the TCA-soluble, nonvolatle radoactvty present n the ocular lens after IP njecton of 3 H-thymdne was 3 H-thymdne. 4 The TCA-soluble, volatle radoactvty was 3 H 2 O. Content, Dstrbuton, and Actvty of Corneal 3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG-CoA) Reductase The HMG-CoA reductase concentraton was estmated by mmunofluorescence mcroscopy; 5 t was purfed from rat lver, 6 and antbodes to the purfed enzyme were rased n rabbts. 7 Frozen sectons of rat cornea were ncubated wth rabbt ant-rat HMG-CoA reductase dluted :00 n 0.% albumn, 0. M potassum phosphate, 0.9% NaCl, ph 7.8 (albumn-pbs). After extensve washng wth albumn- PBS, the sectons were ncubated wth fluorescen sothocyanate-conjugated goat ant-rabbt mmunoglobuln G (Sgma) dluted :40 wth albumn-pbs. The staned sectons were examned and photographed usng an Olympus BH-2 mcroscope (Jacobs Instrument Co., Overland Park, KS) wth fluorescence attachment. The HMG-CoA reductase actvty was assayed accordng to Pann et al 8 as prevously descrbed. 0 One unt of reductase actvty was defned as pmol of mevalonate formed per mn at 37 C. Corneal Sterol Synthess In Vvo The rats (control or treated wth lovastatn, 0.% n the det for 4 days) were njected IP wth 50 mc/kg body weght of 3 H 2 O (25 mc/ml, New England Nuclear) between and 4 PM. A blood sample (20-40 /ul) was collected from the tal 2 and 24 hr after njecton to measure the 3 H 2 O content of the plasma. The mean specfc actvty over ths nterval was calculated for each rat by averagng the 2- and 24-hr values. Between 2 and 24 hr, the specfc actvty of plasma water (thus, also body water) decreased by about 25%. The rats were klled 24 hr after njecton. Corneas, lenses, and about 200 mg of lver were collected from each rat, weghed, and saponfed n ethanolc KOH. The dgtonn precptable sterols (DPS) were prepared, recovered, and assayed for 3 H-content as descrbed prevously. 09 All samples (ncludng background and blanks) were counted to a 2-a error of 2.5% or less. The results were expressed as nmol of 3 H of 3 H 2 O ncorporated nto DPS per mg of tssue. Results The epthelum accounted for essentally all of the 3 H-thymdne ncorporated nto DNA by the cornea (Table ). Furthermore, on a unt-weght bass, the perpheral cornea contaned a sgnfcantly hgher concentraton of 3 H-labeled DNA than the central cornea soon after njecton (Table 2). No dfferences were seen on day 5 or later. The lower 3 H-DNA concentraton n the central cornea was apparently not due to a lesser avalablty of substrate for DNA syn- Downloaded From: on 2/07/208

3 No. 0 CORNEAL EPITHELIUM TURNOVER / Cenedello and Fleschner 959 Table. Corneal epthelal versus stromal concentraton of 3 H-DNA Tme (days) postnjecton 2 Epthelum DPM/mg fracton* Stroma * Each set of values s for a par of dvded corneas from an ndvdual rat njected or 2 days earler wth 2 /C/g body water of 3 H-thymdne. The stromal fracton also contaned the endothelum. thess n ths regon. In fact, the concentraton of TCA-soluble, nonvolatle radoactvty ( 3 H-thymdne) was hgher n the central cornea hr after njecton (Table 3). By 6 hr postnjecton no dfferences were seen between central and perpheral cornea. It s apparent from Table 3 that substrate for 3 H-DNA synthess was avalable for only a few hours after njecton, snce the concentraton of TCA-soluble, nonvolatle 3 H-label greatly decreased between and 6 hr n both perpheral and central corneas. The 3 H-DNA content of the perpheral cornea remaned constant untl day 3 after njecton but then decreased by an apparent frst-order process (Fg. ). The half-lfe was about 3.3 days. The 3 H-DNA content of the central cornea remaned constant or slghtly ncreased untl day 5 after njecton-. Then, as wth the perpheral cornea, the 3 H-DNA content dsappeared by frst-order knetcs wth a half-lfe of 3. days (Fg. ). The onset of 3 H-DNA loss from the whole cornea (perpheral plus central fractons) resembled that of the perpheral cornea; e, loss began between days 3-5 postnjecton. The slope of the curve descrbng the loss of 3 H-DNA from the whole cornea was ndstngushable from those of the perpheral or central cornea. Table 2. Perpheral versus central corneal concentraton of 3 H-DNA Tme (days) postnjecton Control (perpheral/central).65 ± ± ± 0.3 ().97 ± ± ±0..8 ±0.06 DPM/mg fracton* Lovastatn-treated (perpheral/central).77 ± ± ± ± ± ± ± 0.09 * Each value s the mean ± SEM of four or fve rats (excepton was day 2 control, 3 rats). The perpheral fracton was 6% of total cornea n control (range, 59-63%) and 60% n control (range, 56-64%). Table 3. Corneal dstrbuton of substrate for DNA synthess wth tme after njecton of 3 H-thymdne Corneal* fracton Perpheral (P) Central (C) p/ct TCA-soluble: nonvolatle 3 H (dpm/mg cornea) +7 hr +6 hr 77 ±34 47 ±2 29 ±67 43 ± ± ±0.07 * Sx rats were njected ntrapertoneally wth 3 H-thymdne (2 /xc/g body water) and klled n groups of three at and 6 hours later. Values are mean ±SEM. t Mean (± SEM) rato n perpheral/central cornea. fx) < 0.05 of dfference between hour and 6. The level of ncorporaton of 3 H-thymdne nto DNA and the dstrbuton of the labeled DNA n the cornea of lovastatn-treated rats were very smlar to that n corneas of control rats (Table 2 and Fg. 2). Also lke controls, the 3 H-DNA content of the perpheral and whole cornea of treated rats began to decrease after day 3. The half-lves of the curves descrbng the dsappearance of 3 H-DNA from all epthelal fractons of treated rats were essentally equal to those of the control anmals (Fgs., 2). 0,000 5OOO # * " ^ N o- X x DAYS POSTINJECTION TI/2 (Days)» TOTAL o PERIPHERAL CENTRAL 3.2 K K N K x > X Fg.. Dsappearance of 3 H-DNA from the cornea of control rats. The concentraton of 3 H-DNA was measured n perpheral and central corneas at to 5 days after njectng (ntrapertoneally) 2 fc/g body water of 3 H-thymdne. Perpheral cornea comprsed 6% of total cornea wet weght. Dsappearance curves were ftted for day 3 to 5 or day 5 to 5 by computer. All correlaton coeffcents (r) were _ Downloaded From: on 2/07/208

4 960 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Ocrober 990 Vol. 3 0, T X N TI/2 * TOTAL 3.08 o o PERIPHERAL DAYS POSTINJECTION (Days) CENTRAL 3.08 Fg. 2. Dsappearance of 3 H-DNA from the cornea of lovastatntreated rats. Rats were treated for 4 days wth 0.% lovastatn n the det. Perpheral cornea comprsed 60% of total cornea wet weght. The concentraton of HMG-CoA reductase proten n the corneal epthelum of lovastatn-treated rats was shown by mmunofluorescence analyss to be clearly ncreased relatve to that of controls (Fg. 3). Also, HMG-CoA reductase actvty measured n mcrosomes from corneas of control and treated rats was 6 and 209 unts/mg proten, respectvely. Lovastatn s washed out of the mcrosomes durng ther preparaton, and thus the actvty of the ncreased enzyme became apparent. The total cholesterol syntheszed over a 24-hour perod by the cornea was smlar for control and treated rats (Table 4). In contrast, the 24-hour cholesterol synthess by the lver of treated rats was almost double that of controls and synthess by lenses of treated rats was sgnfcantly less than that of controls (Table 4). Dscusson Bref exposure n vvo of corneal epthelal cells to 3 H-thymdne should pulse label cells undergong mtoss at ths tme. The fate of ths dscrete populaton of labeled cells was followed by measurng the tme requred for the onset of loss of 3 H-DNA from the cornea and the sustaned rate of loss. Epthelal cells leave the cornea by desquamaton. Labeled cells ( 3 H-DNA) started to dsappear from the whole cornea approxmately 3 days after njectng 3 H-thymdne. Ths ndcated that, once an epthelal cell was dsplaced from the basal layer, about 3 days were requred for t to reach the corneal surface and desquamate. We found that the rate of loss of labeled cells followed apparent frst-order knetcs wth a half-lfe of about 3 days. Thus, 50% of the basal epthelal cells should be replaced about every 3 days. These results show that the corneal epthelum of the rat should completely turnover n 2-5 days (4-5 half-lves). Ths concluson agrees well wth earler results usng autoradographc methods. ' 2 ' 5 Interestng dfferences were observed between the labelng of the perpheral and central cornea. In our experments, the perpheral cornea comprsed about 60% of the cornea's total mass. Early after exposure to 3 H-thymdne (days -3), the concentraton of 3 H- DNA (dpm/mg tssue) was greater n the perpheral cornea. Assumng that the 3 H-DNA content per labeled cell s constant, ths suggested that the densty of mtotcally actve epthelal cells was greater n ths regon, a possblty consstent wth earler suggestons. 4 ' 20 Several observatons also support the possblty that a populaton of epthelal cells mgrated from perpheral to central cornea. The hgher concentraton of 3 H-DNA n the perpheral cornea dsappeared by day 5 after epthelal cell labelng. Thus, some labeled cells could have moved from the perphery to the center between days 3-5. Also, the total 3 H-DNA content of the perpheral cornea began to decrease before t dd n the central cornea (day 3 versus day 5, respectvely) even though the turnover rates of the perpheral and central cornea appeared dentcal (T /2 = 3 days). These observatons are consstent wth the possblty that between days 3-5 after njecton, the central cornea was ganng labeled cells from the perpheral cornea at about the same rate that t was losng labeled cells by desquamaton. The central cornea should have lost about 400 dpm of 3 H-DNA durng ths perod, a level equal to about 5% of the labeled perpheral cells present at day 3 (about 2900 dpm of 3 H-DNA). A sustaned mgraton of labeled cells from perpheral to central regons throughout the expermental perod s nconsstent wth the observaton that the net rates of turnover of the central and perpheral cornea were the same. If the perpheral cornea was losng labeled cells between days 5-5 due to mgraton and desquamaton, then the rate of turnover of the perpheral epthelum should have been sgnfcantly greater than that of the central cornea where cell loss occurred only by desquamaton. Lovastatn s a potent compettve nhbtor of the rate-lmtng enzyme n cholesterol bosynthess, HMG-CoA reductase. 2 After oral dosng t becomes Downloaded From: on 2/07/208

5 96 CORNEAL EPITHELIUM TURNOVER / Cenedello ond Fleschner No, 0 I Fg. 3. Immunofluorescence of HMG-CoA reductase n corneas from normal and lovastatn-treated rats. Frozen sectons of rat corneas were examned for HMG-CoA reductase content by mmunofluorescence mcroscopy usng rabbt ant-rat HMG-CoA reductase as descrbed n the text. (A, left) control rats. (B, rght) rats treated wth lovastatn (0.%, w/w n ground chow) for 3 days. Bar= 0.7 ^m. The HMG-CoA reductase-spectfc actvty of control rat corneas was 6.2 U/mg mcrosomal proten and of treated rat corneas was U/mg mcrosomal proten. wdely dstrbuted and nhbts cholesterol synthess n many tssues.22'23 Treatment of the rat wth lovastatn produced clear effects on the concentraton and actvty of HMG-CoA reductase n the corneal epthelum. Snce the cornea appears dependent on onste synthess to furnsh the cholesterol requred for membrane growth,0 why dd lovastatn not alter turnover of the epthelum? The answer could be that regardless of the drug's presence net cholesterol synthess n the cornea was not nhbted. When cholestable 4. Effects of lovastatn on 24-hr cholesterol synthess n vvo by cornea, lver, and lens nmol H o[3h2o ncorporated nto DPS/mg/24 hr Group* Control Treated No. of rats Cornea Lver Lens 9.22 ± ± ± ± ± ± 0.026f Rats, control, and lovastatn-treated (0.% n the det for 4 days), were njected wth 3 H 2 O, 50 mc/kg BW. The specfc actvty of body water was determned for each rat. Cholesterol as dgtonn-precptable sterol (DPS) was recovered at 24 hr after njecton and assayed for trtum content. t P(l) of dfference from control < 0.05 (student t-test). terol synthess and tssue levels decrease due to nhbton of HMG-CoA reductase, cellular HMG-CoA reductase proten concentratons can dramatcally ncrease through derepresson of transcrpton of the mrna for the enzyme.24 In the rat cornea, the concentraton of HMG-CoA reductase proten apparently ncreased adequately to compensate for the drect nhbtory effects of the drug. The net result was no nhbton of cholesterol synthess, In the lver, whch s extremely senstve to feedback nducton of the enzyme,23 total 24-hour cholesterol synthess almost doubled n the lovastatn-treated group. In contrast to both the lver and cornea, the 24-hour level of cholesterol synthess n the ocular lens of treated rats was sgnfcantly decreased. Durng formaton of cortcal fber cells, the ste of cholesterol synthess n the lens,25 the cell's nucleus and DNA undergo dsntegraton.26 Ths could lmt the capacty of the lens to compensate for nhbton of HMG-CoA reductase by nducton of the enzyme. Treatment of the rat wth lovastatn had no clear effects on the prolferaton or rate of turnover of the corneal epthelum. Because of possble nterspeces dfferences n response to lovastatn, one should be Downloaded From: on 2/07/208

6 962 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Ocrober 990 Vol. 3 cautous n extrapolatng these fndngs to other speces. For example, Kornbrust et al 27 recently reported that HMG-CoA reductase actvty was nduced 80- fold n rat lver after 7 days of treatment but only twofold n rabbt lver after 0 days of treatment wth trple the dose. The authors suggest that the rabbt lver has lttle capacty to nduce HMG-CoA reductase. Whether ths apples to other rabbt tssues s unknown. Key words: cornea, epthelum, turnover, lovastatn, cholesterol Acknowledgments The authors thank Mrs. Nancy Waletzko for her excellent techncal assstance. References. Hanna C and O'Bren JE: Cell producton and mgraton n the epthelal layer of the cornea. Arch Ophthalmol 64:536, Hanna C, Bcknell DS, and O'Bren JE: Cell turnover n the adult human eye. Arch Ophthalmol 65:695, Buck RC: Measurement of centrpetal mgraton of normal corneal epthelal cells n the mouse. Invest Ophthalmol Vs Sc 26:296, Sharma A and Coles WH: Knetcs of corneal epthelal mantenance and graft loss: A populaton balance model. Invest Ophthalmol Vs Sc 30:962, Shapro MS, Thoft RA, Frend J, Parrsh RK, and Gressel MG: 5-Fluorouracl toxcty to the ocular surface epthelum. Invest Ophthalmol Vs Sc 26:580, Bluemnck JG, van Maurk PA, Tertoolen LG, van der Saag PT, and de Laat SW: Ultrastructural aspects of rapd plasma membrane growth n mtotc neuroblastoma cells. Eur J Cell Bol 32:7, Chen HW, Henger HJ, and Kandutsch AA: Relatonshp between sterol synthess and DNA synthess n phytohemagglutn-stmulated mouse lymphocytes. Proc Natl Acad Sc USA 72:950, Cornell RB and Honvtz AF: Apparent coordnaton of the bosynthess of lpds n cultured cells: Its relatonshp to the regulaton of the membrane sterohphospholpd rato and cell cyclng. J Cell Bol 86:80, El-Sayed GN and Cenedella RJ: Relatonshp of cholesterolgeness to DNA synthess and prolferaton by lens epthelal cells n culture. Exp Eye Res 45:443, Cenedella RJ and Fleschner CR: Cholesterol bosynthess by the cornea: Comparson of rates of sterol synthess wth accumulaton durng early development. J Lpd Res 30:079, The Lovastatn Study Group II: Therapeutc response to lovastatn (mevnoln) n nonfamlal hypercholesterolema: A multcenter study. JAMA 256:2829, El-Sayed GN and Cenedella RJ: Relatonshp of cholesterolgeness to DNA synthess and prolferaton by lens epthelal cells n culture. Exp Eye Res 45:443, Cenedella RJ: Agng and rates of lens-cell dfferentaton n vvo, measured by a chemcal approach. Invest Ophthalmol Vs Sc 30:575, Cenedella RJ: Drect chemcal measurement of DNA synthess and net rates of dfferentaton of rat lens epthelal cells n vvo: Appled to the selenum cataract. Exp Eye Res 44:677, Snger II, Kawka DW, Kazazs DM, Alberts AW, Chen JS, Huff JW, and Ness GC: Hydroxymethylglutaryl-coenzyme A reductase-contanng hepatocytes are dstrbuted perportally n normal and mevnoln-treated rat lvers. Proc Natl Acad Sc USA 8:5556, Edwards PA, Lemongello D, and Fogelman AM: Purfcaton and propertes of rat lver 3-hydroxy-3-methlglutaryl coenzyme A reductase. Bochm Bophys Acta 574:23, Hllam RP, Tengerdy RP, and Brown GL: Local antbody producton aganst the murne toxn of Yersna pests n golf ball-nduced granuloma. Infect Immunol 0:458, Pann SR, Sexton RC, and Rudney H: Regulaton of 3-hydroxy-3-methylglutaryl coenzyme A reductase by oxysterol by-products of cholesterol bosynthess: Possble medators of low densty lpoproten acton. J Bol Chem 259:7767, Cenedella RJ: Sterol synthess by the ocular lens of the rat durng postnatal development. J Lpd Res 23:69, Thoft RA and Frend J: The X, Y, Z hypothess of corneal epthelal mantenance. Invest Ophthalmol Vs Sc 24:442, Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez M, Joshua H, Harrs E, Patchett A, Monaghan R, Curre S, Stapley E, Albers-Schonberg G, Henses O, Hrshfeld J, Hoogsteen K, Lesch J, and Sprnger J: Mevnoln: A hghly potent competve nhbtor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowerng agent. Proc Natl Acad Sc USA 77:3957, L AC, Tanaka RD, Callaway K, Fogelman AM, and Edwards PA: Localzaton of 3-hydroxy-3-methylglutaryl CoA reductase and 3-hydroxy-3-methylglutaryl CoA synthase n the rat lver and ntestne s affected by cholestyramne and mevnoln. J Lpd Res 29:78, Mosley ST, Kalnowsk SS, Schafer BL, and Tanaka RD: Tssue-selectve acute effects of nhbtors of 3-hydroxy-3-methylglutaryl coenzyme A reductase on cholesterol bosythess n lens. J Lpd Res 30:4, Luskey KL, Faust JR, Chn DJ, Brown MS, and Goldsten JL: Amplfcaton of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa proten, n UT- cells. J Bol Chem 258:8462, Cenedella RJ: Regonal dstrbuton of sterol and fatty acd synthess n the ocular lens. Exp Eye Res 38:95, Appleby DW and Modak SP: DNA degradaton n termnally dfferentatng lens fber cells from chck embryos. Proc Nat Acad Sc USA 74:5579, Kornbrust DJ, MacDonald JS, Peter CP, Ducha DM, Stubbs J, Germershausen JI, and Alberts AW: Toxcty of the HMG CoA coenzyme A reductase nhbtor, lovastatn, to rabbts. J Pharmacol Exp Ther 248:498, 989. Downloaded From: on 2/07/208