?‘HE , J O U R N A L OF BIoLoQICAL CHEMISTRY

Printed m I J S A. Vol. 25i. No. 21, Issue of November IO, pp 12611-12615, 19x2

LLC-PK, Cells Derived from Pig Kidneys Have a Defect in Cyclooxygenase*

(Received for publication, June 7, 1982)

Meyer D. Lifschitz With the technical assistance of Shashi Rattan, Margie Newberry, Betty Waldron, and Pearl de la Cruz

From the Department of Medicine, University of Texas Health Science Center a t S a n Antonio and Audie L. Murphy Veterans Administration Hospital, Sun Antonio, Texas 78284

Two epithelial cell lines derived from mammalian kidneys, the MDCK, cell from dog kidney and the LLC- PK1 cell from pig kidney, were studied in terms of prostaglandin synthesis. The MDCKl cell line regularly produced prostaglandin E and 6-keto-PGF1, under basal conditions and following augmentation of pros- taglandin synthesis by the addition of arachidonic acid or with stimulation of phospholipase by the calcium ionophore A23187. Addition of butyrate to the growth medium, a maneuver previously shown to induce new cyclooxygenase, augmented basal and A23187-stimu- lated-prostaglandin E production by MDCK, cells.

In contrast, the LLC-PK1 cell line produced virtually no prostaglandins in any of these conditions. To eval- uate the possible defect in prostaglandin synthesis present in the LLC-PK1 cell line, studies were per- formed in which radioactive arachidonic acid uptake into membrane lipids and subsequent release by acti- vation of phospholipase were evaluated. Results with LLC-PK1 cells were similar to those found with the MDCKl cells. Thus, neither of these steps was defective in the LLC-PKl cell line. Since the radioactive arachi- donic acid released by the LLC-PKI cell line remained almost completely as arachidonic acid, the next step in prostaglandin synthesis, i.e. cyclooxygenase, is likely to be the defective step in limiting this cell’s ability to produce prostaglandins. In contrast to previous studies in mastocytoma cells and the present findings with MDCKl cells, addition of butyrate to the growth me- dium did not induce cyclooxygenase activity in LLC- PKI cells.

Prostaglandin synthesis has been found in virtually all nucleated cells studied to date. Different cell types, however, can produce prostaglandins in different amounts and of differ- ent types. Because of this laboratory’s interest in transepithe- lid transport, we have been studying two lines of kidney epithelial cells, the LLC-PKI cell line derived from the pig kidney (1) and the MDCKl cell line’ derived from the dog kidney (2). Both of these cell lines actively transport electro-

* Portions of these studies were supported by funds from the National Aeronautics and Space Administration Grant NAS 9-16366, the Veterans Administration Medical Research Service, and the National Institutes of Health Program Project Grant AM-17387. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby

solely to indicate this fact. marked “aduertisement” in accordance with 18 U.S.C. Section 1734

’ These cells are referred to as MDCKl cells because a recent publication has demonstrated differences in prostaglandin production between MDCK cells obtained from the ATCC and La Jolla, Califor- nia (12).

lytes from luminal (medium) to contraluminal surface and in the process form domes or hemicysts, which represent the accumulation of actively transported fluid trapped between the cell and the culture dish (3). In the process of character- izing prostaglandin production by these two cell lines, we observed that the MDCKl cell line readily produced PGE2 under basal conditions, but LLC-PK1 cells did not. Stimula- tion of acylhydrolase (phospholipase) by addition of the cal- cium ionophore A23187 augmented prostaglandin synthesis in MDCKl cells, but did not in LLC-PK, cells. Addition of butyrate to the growth medium augmented basal and iono- phore-stimulated PGE synthesis by MDCK, cells, but not in LLC-PKI cells, possibly due to induction of cyclooxygenase activity (4, 5) in the MDCKl cell line. Subsequent studies demonstrated that the defect in prostaglandin synthesis of LLC-PKI cells was not related to uptake of arachidonic acid from the medium or to release of arachidonic acid by activa- tion of acylhydrolase. Thus, a defect in the subsequent step in prostaglandin synthesis, cyclooxygenase, is likely to exist in this cell line.

MATERIALS AND METHODS

Cell Culture Techniques-LLC-PK1 cells and MDCK, cells were originally obtained from the American Type Culture Collection, Rockville, MD. Cells were regularly grown in 6 cm Corning plastic dishes in a 37 “C incubator in 5% COa, 95% air. MDCKl cells were regularly grown in Dulbecco’s modified Eagle’s medium with 1 g/liter of glucose plus 10% fetal calf serum and 1% antibiotics. LLC-PK, cells were grown in similar medium with the exception that the glucose concentration was 4.5 g/liter. Studies were performed with cells just reaching confluency between the 5th and the 30th passages.

Release of Prostaglandins into the Medium with Time and follow- ing Addition of Arachidonic Acid-To evaluate the release of pros- taglandin E into the medium with increasing time, dishes of LLC-PKI and MDCK, cells grown to confluency were used. The medium was changed and then aliquots of the medium were obtained a t subsequent time periods for prostaglandin E analysis. Additional dishes were studied in which arachidonic acid was added at M (final concen- tration) and similar timed collections were obtained. The medium was frozen for subsequent analysis.

Release of Prostaglandin E a n d fi-keto-PGF,, from LLC-PKI and MDCK, Cells following Stimulation with the Calcium Ionophore A23I87”To stimulate prostaglandin synthesis, both cell lines were studied under control conditions and conditions in which acylhydro- lase was stimulated by addition of the calcium ionophore A23187 (4 X IO”’ M). In addition, studies were performed with both the addition of A23187 and indomethacin (0.25 X 10“’ M ) . Three dishes, all derived from the previous passage of cells, were grown to confluency and studied at the same time. In all 3 dishes, the medium was changed a t zero time to either fresh medium, fresh medium including A23187, or fresh medium with A23187 and indomethacin. Four hours later, medium was collected and frozen for subsequent PGE and 6-keto- PGF,, analysis.

’ The abbreviation used is: PC, prostaglandin.

12611

12612 Defective Cyclooxygenase in LLC-PKI Cells

Effect of Butyrate Added to the Growth Medium on Prostaglandin E Release from MDCKl and LLC-PKI Cells-Since previous studies in mastocytoma cells had shown that addition of butyrate to the growth medium had induced cyclooxygenase formation, studies were designed to evaluate the effect of butyrate on PGE release by MDCK, and LLC-PKI cells. Four experiments of 8 dishes each were conducted with each cell type. Equal numbers of cells were inoculated into each dish and the usual growth medium employed. When the cells were within 2 to 3 days of confluency, butyrate was added to the growth medium a t a final concentration of 2 mM to half of the dishes. Forty- eight to 72 b later, the medium was changed to fresh medium without butyrate. The calcium ionophore A23187 (4 x 10"' M ) was added to half the dishes and 4 h later all medium was collected and frozen for subsequent PGE analysis. Thus, 4 groups of cells, with and without butyrate in the growth medium and with and without ionophore were evaluated.

Evaluation of Radioactive Arachidonic Acid Uptake by MDCKl and LLC-PKI Cells-To evaluate whether a defect in arachidonic acid uptake could explain the lack of prostaglandin synthesis by LLC- PKI cells, this group of studies was designed to evaluate the uptake of radioactive arachidonic acid from the medium. Studies with LLC- PKI cells and MDCK, cells were performed at the same time. A total of 4 experiments each containing 6 dishes of each cell type were performed. Once the cells had grown to confluency, the medium was changed to fresh medium containing 60,OOO-80,000 cpm [I4C]arachi- donic acid. After 24 h, aliquots of the medium and cells were counted separately by p scintillation spectrometry.

Evaluation of Acylhydrolase Activity in LLC-PKI Cells-To eval- uate the possibility that a defect in acylhydrolase (phospholipase) might be responsible for the inability of LLC-PKI cells to produce prostaglandins, this series of studies was performed. Cells were pre- labeled with [I4C]arachidonic acid as described above. The medium was changed to either fresh medium or fresh medium containing the calcium ionophore A23187 (4 X M ) . Two dishes of each cell type were included in each experiment as controls and 2 dishes of each cell type in each experiment received the calcium ionophore. The calcium ionophore was used as a means of activating intracellular acylhydro- lase. Four hours later, medium aliquots were obtained and the cells removed from the dish. The amount of 14C label found in the medium and in the cells was determined separately by /3 scintillation spectrom- etry.

Thin Layer Chromatography of Radioactive Products Released from MDCK, Cells and LLC-PKI Cells-To evaluate the possibility that LLC-PK, cells might be producing prostaglandins other than PGE2 or PGIr, these studies were performed. Cells were prelabeled with ["C]arachidonic acid as described above. Both MDCKl and LLC-PKI cells were studied. The I4C label released into the medium was evaluated. Radioactivity released into the medium was deter- mined on a fractional basis by counting an aliquot of the medium to evaluate recovery. The majority of the medium was extracted with chloroform and analyzed using thin layer chromatography procedures as described below.

Radioreceptor Assay Methodology for Prostaglandin E-PGE was measured in 1-ml volumes of medium by hepatic receptor assay as previously used in this laboratory (6). Because these receptors were relatively indifferent to PGE, and PGE2, the results are ex- pressed as PGE. The standard curve ranges from 62 pg to IO00 pg.

Radioimmunoassay of 6-keto-PGFI,,-Radioimmunoassay for 6- keto-PGFl, was performed on 0.25-ml volumes of medium. Samples were labeled with approximately 1,OOO cpm ["H]6-keto-PGFI, for estimation of recovery. The pH of the samples was then adjusted to 3.0 with 0.1 normal HCI and extracted with 20 ml of CHClp twice. The CHCla extraction volumes (which contained prostaglandins) were flash evaporated to dryness. The 6-keto-PGFI, was resuspended in phosphate saline buffer (pH 3.0) and applied to a Sep-Pak CIH car- tridge. (The CIH cartridge had previously been activated by rinsing with 10 ml of tetrahydrofuran and 10 ml of phosphate saline buffer and then flushed with air). The total sample was then expressed through the Sep-Pak cartridge by attaching a plunger to the syringe and gradually pushing air through the syringe and the Sep-Pak cartridge. This allowed the 6-keto-PGFI, to bind to the Sep-Pak cartridge. After all the sample has been expressed through the car- tridge and flushed with air, the eluate was discarded. The 6-keto- PGFI. was then eluted from the cartridge into a glass scintillation vial with 3 ml of 100% methanol. The methanol was blown to dryness under nitrogen and the sample stored in 1 ml of 100% ethanol under nitrogen in the refrigerator until assay. Recovery of radioactive 6- keto-PGFI, through this procedure averaged greater than 70% The

sample was then chromatographed on a Water's high pressure liquid chromatography system with a reverse phase column using 28% acetonitrile as the solvent system to separate 6-keto-PGF,, from other prostaglandin products. A routine radioimmunoassay using chemical 6-keto-PGFI, obtained from The Upjohn Co. and ['H]G-keto-PGF,, from New England Nuclear was then performed in phosphate- buffered saline (pH 7.3) with 1% gelatin. Known and unknown amounts of sample plus trace were added to all assay tubes along with phosphate-buffered saline. Subsequently, antibody was added to yield a final dilution of 1 to 20,000. After chemical and trace 6-keto-PGFI,, buffer, and antibody had been added, the tubes were vortexed and then incubation in a covered rack at 37 "C in a shaking water bath for 1 h. Tubes were then removed from the water bath and placed in an ice bath. Separation of bound from free was effected with 200 pl of dextran-coated charcoal. An aliquot of the supernatant was removed and counted in a Beckman liquid scintillation spectrometer. Samples were all run in duplicate and in either two or three dilutions. The standard curve regularly ranged from 10 to 1,OOO pg. Because of the degree of sensitivity of the standard curve, calculations of samples were corrected for the chemical amount of 6-keto-PGFI, included in the radioactive trace. In 20 different samples run on 4 different days, recovery of added known amounts of 6-keto-PGFI, averaged 111.1 k 14.6%.

Thin Layer Chromatography of [14C/Arachidonic Acid and I t s Prostaglandin Products-The medium was extracted after adjusting the pH to 3.5 to 4.0 with acetic acid. 20 ml of chloroform was used twice for 3-5 ml of media. The chloroform layers were added together and evaporated to dryness. The residue was then redissolved in chloroform, heptane, acetic acid, and ethanol (100:100:230), and evaporated again under nitrogen to dryness. Each time, care was taken to ensure that the evaporation vessel wall had been carefully rinsed. Samples were then redissolved into 200 pl of benzene, trans- ferred to a small polypropylene tube, covered with parafilm, and placed in ice. This material was then ready to be spotted on thin layer chromatography plates.

After heat activation, silica gel thin layer chromatography plates were cooled in a desiccator. Radioactive and chemical arachidonic acid, nonradioactive prostaglandin Fra, ET, D,, A2, thromboxane Bs, and 6-keto-PGFI. were prepared in benzene in a manner similar to that described for the samples. 25 p1 of the sample and a known amount of radioactive arachidonic acid were placed in scintillation vials for determining isotopic recovery. 25 p1 of the samples were then spotted using a gas-tight syringe onto the thin layer plate using a continuous stream of nitrogen to dry the spot. The plate was devel- oped in hexane, ethyl acetate, acetic acid, Hr0 (30:54:12:60). The region of the plate with the cold prostaglandin and arachidonic acid standards was sprayed with 10% phosphomolybdic acid. The plate was then placed in a hot air oven for 15 min to allow for reaction of the acid spray with the standards. Regions (corresponding to the chemical standards) were scraped from the sample areas into scintil- lation vials, counting solution added, and the radioactivity determined in a Beckman p scintillation spectrometer.

RESULTS

The amount of prostaglandin E released into the medium with increasing time is shown in Fig. 1. There was no release of PGE from LLC-PKI cells. In contrast, MDCKl cells re- leased increasing amounts of PGE with time. Also shown in this figure are the findings from identical cell lines pretreated with arachidonic acid M. Again, there was almost no increase in PGE release from LLC-PK, cells, whereas there was a considerable increase in that released from MDCK, cells.

In order to evaluate whether this difference in prostaglandin production from these two cell lines was only related to different basal levels of prostaglandin synthesis, we aug- mented basal prostaglandin synthesis by addition of the cal- cium ionophore A23187. This agent has previously been known to activate prostaglandin synthesis mainly by activat- ing acylhydrolase. Both PGE and 6-keto-PGF1, (a breakdown product of PGL) were measured in the medium from these studies (Fig. 2). A23187 markedly increased both PGE and 6- keto-PGFl, release from MDCKl cells and this increase in prostaglandin release was inhibited by simultaneous treat-

Defective Cyclooxygenase in LLC-PK, Cells 12613

o MDCK, Cells MDCK,Cells t 5 ~ 1 0 - ~ M AA

o LLC -PK, Cells LLC-PK, C ~ I I S ~ ~ X I O - ~ M AP

J r - 1

I , , I I I

0 15 30 60 90 120 Minutes

FIG. 1. This figure depicts the release of PGE into cell cul- ture medium with increasing periods of time. Each value rep- resents the mean of duplicate determinations. There was a marked increase in PGE released from MDCK, cells (open circles) but not PGE released from LLC-PKI cells (open squares). In similar experi- ments, when chemical arachidonic acid was added at M, there was a marked increase in PGE released from MDCK, cells (filled circles), but no consistent increase in PGE released from LLC-PKI cells (filled squares)

Conlrol ' A23187' A23187I Indomethacln

30 1

O t Control 1A23187'A23187

1 Indornelhacln lndornethccln

FIG. 2. This figure depicts results from both MDCKl (top) and LLC-PKI cells (bottom), which were stimulated to produce prostaglandins by the addition of the calcium ionophore A23187. Lines connect results from experiments performed at the same time from cells inoculated into dishes from a single previous passage of cells. Clearly, the calcium ionophore markedly stimulated release of prostaglandins of both types from MDCKI cells and this increase in prostaglandin release was inhibited by indomethacin. In contrast, LLC-PKi cells did not produce either type of prostaglandin product following addition of the calcium ionophore.

ment with A23187 and indomethacin. In contrast, LLC-PK, cells demonstrated no increase in either PGE or 6-keto-PGF,, release into the medium with stimulation by A23187. Similar results were found with these cell lines when medium calcium concentration was raised approximately 10-fold (data not shown).

Since previous studies (4, 5) had demonstrated that the addition of butyrate to the growth medium of mastocytoma cells had augmented prostaglandin production, presumably by inducing cyclooxygenase, studies were designed to evaluate the effect of butyrate on PGE released into the medium by MDCK, and LLC-PK1 cells. Results of 4 paired experiments with each cell type are shown in Fig. 3. All 4 pairs of dishes of MDCKi cells grown with butyrate had an augmented level of PGE released into the medium when compared with MDCKl cells grown without butyrate. Following the addition of the calcium ionophore A23187, there was an increase in PGE released into the medium from MDCKl cells grown with or without butyrate, but in each case the value obtained from MDCKl cells grown with butyrate was higher than values obtained from MDCK, cells grown without butyrate. In con- trast, LLC-PK1 cells grown with butyrate did not augment their basal level of PGE released into the medium and addition of ionophore to LLC-PKI cells did not augment PGE release into the medium whether the cells were grown with or without butyrate in the growth medium.

Because of the marked difference in prostaglandin produc- tion under basal and stimulated conditions between LLC-PK1 and MDCKI cells, the following series of studies were designed to address the possible biochemical defect in prostaglandin production in LLC-PKl cells. To evaluate whether there was a defect in uptake of arachidonic acid, [14C]arachidonic acid was added to the culture medium and 24 h later the amount of isotope remaining in the media versus that found in the cells was evaluated. The results of a representative study are shown in Fig. 4. Both MDCKl and LLC-PKI cells were able to take up over 80% of the isotope from the medium and incorporate it into the cells within 24 h. In 6 paired experi- ments with both cell lines, MDCK, cells took up 90 f 7% of the isotope and LLC-PKl cells took up 81 f 7%. Thus, there was no major defect in the ability of the LLC-PKI cell to take up arachidonic acid from the medium.

The next set of studies were designed to evaluate acylhy-

MDCKl Cells

No Butyrote Butyrate

I

L L C - PKI Cells

i: ; j0I 20 odLi k

O ' Control a2&; Canlroi A23187

FIG. 3. This figure depicts results from both MDCKI (top) and LLC-PKI cells (bottom) grown with and without butyrate in the growth medium. Each value represents the mean of duplicate determinations. Symbols of the same shape depict studies performed in a paired manner. Addition of butyrate to the growth medium augmented basal and ionophore-stimulated PGE release from MDCK] cells. In contrast, neither ionophore, butyrate, or butyrate and ionophore augments PGE release from LLC-PKI cells.

12614 Defective Cyclooxygenase in LLC-PKI Cells

P 2 -. Cells gz GO- m

60 z: l MDCK, gs 40- 2ia o LLC - PK,

0

3 20- x+3 8 4 o / M&m

-- 1 Control A23187

FIG. 4. This figure depicts the amount (%) of radioactive [%]arachidonic acid found in MDCKl and UC-PK, cells and their respective media at 24 h after addition of the is&ope. Also depicted are results following addition of the c&ium ionopbore A23187 to both cell lines. A23187 increased the rate of isotope released from both cell lines as indicated by the decrease in isotope found in the cells and the increase in isotope found in the medium.

TABLE I

Results of thin layer chromatography of “‘C-labeled products of arachidonic acid from MDCK, and LLC-PK, cells

Results are expressed as a percentage of the amount of isotope located in each area of the plate. The results are the mean values of duplicate experiments. The percentage of isotope added to each plate recovered in the various areas counted on the plate was always greater than 75%.

Arachadonic Standards MDCK, LLC-PK, acid without

cells

Arachidonic acid PGA, PGD? and PGEz PGF2, and TXBZ &keto-PGF,, Origin

43 70 70 22 12 7 16 8 10 9 3 6 5 2 1 5 4 1

drolase activity in LLC-PK, cells. In cells previously labeled with [?Z]arachidonic acid, control dishes were treated with diluent whereas experimental dishes were exposed to A23187 for 4 h. The amount of 14C label released from the cells into the medium following A23187 was evaluated in both cell lines. It can be seen in Fig. 4 that activation of acylhydrolase with A23187 augmented isotopic release from MDCK, cells into the medium approximately 4-fold. The increase in isotopic release from LLC-PK, cells was similar during this time period. In 6 paired experiments with both cell lines, A23187 augmented release of [“Clarachidonic acid in MDCKl cells from 2 f 2 to 8 + 3% and in LLC-PK, cells from 2 f 1 to 6 & 3%. Both of these changes were significant (p < 0.01). Thus, there was no defect in either arachidonic acid uptake or arachidonic acid release from LLC-PK1 cells.

In an effort to determine whether arachidonic acid released from LLC-PK, cells could be converted to prostaglandins, studies were performed in which cells were prelabeled with [‘4C]arachidonic acid and allowed to release the radioactivity over 2 h. The radioactivity released was chromatographed on thin layer chromatography plates to determine whether the isotope was all arachidonic acid or whether prostaglandin products were found. Results are shown in Table I. In studies with MDCK, cells, over 50% of the isotope released into the medium was regularly found in the area which co-chromato- graphed with prostaglandin standards. In contrast, of the isotope released by LLC-PKI cells, only 25% co-chromato- graphed with prostaglandin standards and 70% remained as arachidonic acid.

DISCUSSION

A variety of cells grown in tissue culture have been studied in the past few years and have been shown to produce pros-

taglandins. Previous studies have shown that the MDCK, cell can produce prostaglandin E (7). A recent study of LLC-PKI cells found observations consistent with those in this report in that the LLC-PK1 cell under basal conditions did not produce prostaglandins (8). Since arachidonic acid is normally able to be produced from linoleic acid, many artificial media both with and without calf serum should have the necessary sub- strate for subsequent prostaglandin synthesis. The lack of prostaglandin synthesis under basal conditions by the LLC- PKI cell prompted us to evaluate what the defect was in prostaglandin synthesis in this cell line.

Based on the studies with both added arachidonic acid, acylhydrolase stimulation by A23187, and by increasing me- dium calcium concentration, we have been able to demon- strate a marked increase in prostaglandin E and 6-keto-PGF,, release by MDCK, cells. The absence of any increase in prostaglandin release by LLC-PK1 cells is confirmatory of the absence of prostaglandin production under basal conditions and suggests that even under stimulated conditions, prosta- glandin production by this cell line is minimal to absent.

The accepted biochemical sequence for prostaglandin pro- duction requires both arachidonic acid incorporation into the cell and subsequent release, usually from membrane lipids, by acylhydrolase prior to incorporation of oxygen by cyclooxy- genase (9). The studies in which radioactive arachidonic acid was taken up by the two cell lines demonstrate clearly that both LLC-PK1 and MDCK, cells can avidly take up arachi- donic acid from the medium and thus demonstrate that this is not the defect in LLC-PK, cells. In addition, the studies with the calcium ionophore A23187 demonstrate that both cell lines are able to release previously incorporated arachi- donic acid from membrane lipids and in the case of MDCK, cells make this arachidonic acid available for subsequent prostaglandin synthesis. The small amount of radioactive prostaglandin products found on thin layer chromatography and the large amount of remaining arachidonic acid in studies with LLC-PKI cells confirms the virtual absence of prosta- glandin production by this cell line. This is in marked distinc- tion to the findings with the MDCKl cell line which produced considerable radioactive material which co-chromatographed with prostaglandin standards under identical conditions. The finding with measurement of PGE and 6-keto-PGF,, release into the medium (Figs. 1 and 2) are also consistent with this conclusion. Thus, the LLC-PKI cell line can incorporate ar- achidonic acid into membrane lipids and, following stimula- tion of acylhydrolase, can release arachidonic acid from mem- brane lipids, but produces almost no prostaglandins. This leads to the conclusion that the defect in prostaglandin syn- thesis in this cell line is at the level of cyclooxygenase. Since the majority of the radioactive arachidonic acid in Table I from the LLC-PKI cells remains as arachidonic acid, this suggests that there is not another major product of arachi- donic acid formed by this cell line.

Because a series of recent studies have demonstrated that addition of butyrate to the growth medium of cells in culture can induce a variety of enzymes (10) including cyclooxygenase (4, 5), the effect of butyrate on MDCK, and LLC-PKI cells was evaluated, Following the addition of butyrate to the growth medium of MDCKl cells, both basal and ionophore- stimulated PGE release were augmented when compared to cells grown without butyrate. These are the results one would predict if there were an increase in the amount of activity of cyclooxygenase present and as such confirm the previous findings of others (4, 5). In contrast, there was no definite increase in PGE released into the medium from LLC-PKI cells under basal or A23187stimulated conditions. Thus, these findings are consistent with the possibility that cyclooxygen-

Defective Cyclooxygenase in LLC-PKI Cells 12615

ase activity is virtually absent from the LLC-PK1 cell line. These studies do not allow one to determine whether the

cyclooxygenase is absent, defective, or inhibited in some man- ner. A defect in cyclooxygenase has recently been found in a clone of L1210 mouse leukemia cells (11). It is unlikely that in the present studies there is an inhibitor in the incubation medium since the media for both cell lines and the experi- ments comparing the two cell lines were virtually identical. Conceivably, the LLC-PK1 cell line itself produced an inhibi- tor of cyclooxygenase, but studies were not performed to address this issue. If such an inhibitor were produced, it would necessarily act quite quickly since the differences in PGE production shown in Fig. 1 were evident by 15 min.

The characterization of a cell line which does not regularly produce prostaglandins could be of considerable interest for those interested in cell injury or the role of prostaglandins in cellular disease states. Having a cell which does not produce prostaglandins would allow one to compare findings in this cell line with cell lines which do produce prostaglandins, and thus potentially one could evaluate the role of prostaglandin products in leading to protection or cell damage from injurious agents.

In conclusion, two cell lines, the MDCK, and LLC-PKI cell lines, were compared in terms of their ability to produce prostaglandins. The MDCK, cell line produces prostaglandins following addition of arachidonic acid, stimulation with the calcium ionophore A23187, with increased calcium concentra- tion in the medium, and following addition of butyrate to the growth medium. In contrast, the LLC-PK, cell line produced

no measurable prostaglandins under any set of stimulated conditions. The defect in the LLC-PKI cell line prostaglandin synthetic process appears not to be related to either arachi- donic uptake or release, but rather to a defect in cyclooxygen- ase per se.

Acknowledgments-The secretarial assistance of Helen Nunn is gratefully appreciated. Chemical prostaglandins were kindly supplied by Dr. John Pike, The Upjohn Co., Kalamazoo, MI.

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