Possible involvement of arachidonic acid in the initiation of DNA synthesis by rat liver cells

This work 79.01938.01

was supported and NATO grant

by no.

CNR Ih20.

go-ant

no.

I. Cohlan. S Q. Science I I7 I 19%) 535. 2. Giroud. A & Martinet, M. Arch franc _ nediat I? . (1955) 292. 3. Murakami. U & Kameyama. Y. .Arch rnvironhealth IO t 1965) 732. 4. Kochhar. D M. ‘Teratology 7 t 1973) 289. 5. Lewis. A C. Pratt. R M. Pennypacker-. J P & Hassell. J R. Dev biol 63 ( 197X) 31. 6. &plan. A I, Exp cell res 62 t 1970) 331. 7. Dienstman. S R, Biehl. J. Holtzer. S & Holtzer. H. Dev biol 39 (197-11 X3. 8. Hamburger. V & Hamilton. H W. J morphol $8 (1951) 49. 0. Okayama. M. Pacifici. M & Holtzer. H. Proc natl acad sci US 73 (1976) 3223. IO. Yamada. K, Histochemie 23 t 1970) 13. I I. Zani. B. Cossu. G. Adamo. S & Molinaro. M. Differentiation IO t 1978) 95. 12. van der Mark. K & von der Mark. H, J cell biol 73 t 1977, 736. 13. Holtzer. H. Okayama, M. Biehl. J Cy: Holtzsr. S, Experientia 34 t 1978) 781. IJ. Chacko. S. .4bbott. J. Holtzer. S & Holtzer. H. J exp med 130 t 1969) 417. IS. Solurch. M 2s Meier. S. Calcif tissue re\ I3 t 1973) 131. 16. Shapiro. S S & Poon. J P. .4rch biochem biophyb I71 t 1976) 73. J R. Pennypacker. J P& Lewis. .A C. Exp 17. Hassell. cell res I II t 1978) 309. H. Stem cells and tissue homeostasib. pp. IX. Holtzer. l-28. Cambridge University Press. Cambridge (197X). 19. Fell. H B & Dingle. J T. Biochem j X7 t 1963) 303. 20. Hynes. R 0 & Humphrey. K C. J cell biol 62 t 1971) 338. 21 Hassell. J R, Pennypacker. J P, Kleinman. H K. Pratt. R M & Yamada. K M, Cell I7 t 1979) 821. 22. West. C M. Lanza. B. Rosenbloom. I. Lowe. M. Holtzer. H & Avdalovic. N. Cell I7 t 1979) 491. ‘3 Chen. LB. Murray, A, Segal. R N. Bushnell. A & Walbh. M L. Cell I4 t 1978) 377. 24, Furcht. L T. Mosher, D F & Wendelskhafer-Crabb. G. Cell I3 t 197X) 263. IS Podleski. T R. Greenberg. 1. Schlessinger. J &: Yamada. K M, Exp cell res 122 (19791 317. L M et al.. Pure & appl them 51 (19791 26. De Luca. 581. 27. Hogan. B. Nature 277 (1979) 261. 28. Todaro. G J. De Larco. J F Br Sporn. M B. Nature 276 t 1978, 272. 29. Levine. L 8: Ohuchi. K. Natut-e 276 t 1978,271. Received Revised Accepted

March version June

7. 1980 received 25. 1980

June

23.

19X0

Possible involvement of arachidonic acid in the initiation of DNA synthesis by rat liver ceils

.S/r?>!nr~r~~. Calcium-deprived T5 IB rat liver cells initiated DNA synthesis within I h after addition of calcium. The possibility of this DNA-synthetic response having been mediated through arachidonic acid metabolism ti.e.. through an arachidonic acid cascade) is suggested by the fact\ that calcium is known to stimulate phospholipase activity which releases arachidonic acid from membrane phospholipids: that low concentrations t 10-!‘-lOmti mole/l) of arachidonic acid itself elicited the same DNA-synthetic response from the calcium-deprived cells as calcium; and that the stimulatory actions of calcium and arachidonic acid were both blocked b) the endoperoxide synthase inhibitor indomethacin.

Non-neoplastic cells in vitro and in vivo need both calcium ions and a transient increase in their CAMP content near the end of the GI phase of the growth-division cycle to initiate DNA synthesis [Z. 7. IO17, 19-261. Extensive experimentation in this and other laboratories has shown that lowering the extracellular calcium concentration or preventing the CAMP surge does not stop on-going DNA synthesis. but does stop the proliferative development of the cells in late Gl phase [2. 3. 7. IO. I I. cells revert 19-23. 251. These blocked sooner or later to an earlier prereplicative state [IO, I I. IS. 191. unless rescued by a timely addition of calcium [2. 3. 5, 10 20-251. calcium’s intracellular mediator cdmodulin [4] or a prostaglandin (e.g.. PGA or PGE,). anyone of which causes the cells to initiate DNA synthesis within I h [Z-5. 8. 13. 16, 17. 20. 21. 241. Since calcium and its mediator calmodulin might release arachidonic acid from membrane by stimulating phospholipases [6. 9, 761. and since prostaglandins at-e products ofarachidonate

thetic response from calcium-deprived rat liver cells as calcium or its mediator calmodulin. Materials

20

ISi 01

012 Hours Of incubation

lndomethacin cont. in medium (moles/II

Fig. I. (A) The DNA-synthetic response of TSIB rat liver cells in low (0.02)-calcium medium to an increase in the extracellular calcium concentration to 1.25 mM. Cultures were exposed to [3H]TdR 1 h before calcium addition, or between 0 and 0.5 or I and 2 h after calcium addition. Cells were fixed and prepared for autoradiography at the ends of these labeling periods. Note: The DNA-synthetic activity did not change in untreated cultures. (B) The ability of various concentrations of indomethacin to block the DNA response to raising of the extracellular calcium concentration from 0.02 to 1.25 mM at time 0. Indomethacin was added to the cultures I5 min before calcium, [“H]TdR was added at the same time as calcium, and the cells were fixed 2 h afterwards. The points are means & S.E.M. of values in four cultures.

metabolism [9, 15, 181, the rapid DNAsynthetic response to calcium and calmoduiin might be due to the action of one or more of the several products of an arachidonate cascade [ 181. If this be true, exogenous arachidonic acid itself might also trigger DNA synthesis in calcium-deprived cultures.

This communication suggests the feasibility of this idea by showing that arachidonic acid does elicit the same rapid DNA-syn-

rind Methods

Non-neoplastic T51B epithelioid rat liver cells were isolated by Swierenga et al. [?O]. Proliferation of these cells. like that of their counterparts in vivo. is inhibited by lowering the extracellular calcium concentration [24, 20-22, 251. To do this cells were first planted at a density of 0.7~ IO’/cm’ on 25 mm. round plastic coverslips (in 35 mm plastic Petri dishes) in a high (1.8 mM)-calcium medium consisting of 10Cr, (v/v) FBS (fetal bovine serum from Flow Laboratories, Rockville, Md). 90% (v/v) BME (Eagle’s basal medium also from Flow Laboratories) and the antibiotic eentamicin (from Microbioloeical Ass.. Bethesda,-Md). They ‘were then incubated for 24 h at 37°C (in an atmosphere consisting of 95 % air and 5 % CO,) to ensure maximum attarhment and spreadine. after which the high-calcium medium was reulaced bylow (0.02 mM)-cal%um 10% (v/v) FBS. 90’% (v/v) BME medium. Methods for adiusting the calcium concentration in this medium have been described elsewhere r2. 51. Exoeriments began 48 h later when the percen; of cells making DNA-in the low-calcium medium had fallen from the normal level of 60% [24] to 20% or less. At this time appropriate amounts of arachidonic acid [5, 8, I I. 14 eicosatetraenoic acid; Sigma Chemical Co., St Louis, MO.], calcium chloride. or indomethacin (Sigma Chemical Co.), were added. The stock solution of arachidonic acid, prepared by dissolving 100 mg of the acid per 1 ml of hexane, was stored under nitrogen in the dark at -20°C. When needed, 30 ~1 of this solution was removed, the hexane blown off in a current of nitrogen, the solventfree arachidonic acid dissolved to a final concentration of 10m3mole/l in aqueous 0.1 M sodium carbonate, and then appropriately diluted in phosphatebuffered saline before addition to calcium-deprived cultures. The extremely small amounts of sodium and carbonate ions carried over into the cultures were insignificant compared with those in the culture medium. lndomethacin was first dissolved in 2OV (v/v) ethanol, 80% (v/v) water, and then appropriately diluted in phosphate-buffered saline. The greatest amount of ethanol carried over to the cultures was equivalent to 0.00002% (v/v) which did not affect thecells. In some experiments catalytic subunits of type It CAMP-dependent protein kinase were added to the cultures. They were isolated in this laboratory from rabbit skeletal muscle according to Beavo et al. [I]. DNA-synthetic activity was assessed autoradiographically (i.e., from the proportion of cells whose nuclei were labeled during exposure to [3H]thymidine (5 pCi/ml; spec. act. 20 Cilmmole; from Amersham. Arlington Heights, Ill.) according to Boynton et al. [5] and Whitfield et al. [24, 251). a procedure in which the radioactivity is found only in the DNA (and not in the RNA or cold acid-soluble nucleotide fraction) and produces approx. 500 silver grains in the nuclear track emulsion overlying the nucleus of each DNA-synthesizing cell. E.xp Cell Res 129 (1980)

fi,<,:. 2. I’he DN.-\-\!,nthetli re\pon\e Ijt. T‘5 IB rat li\vr cell\ in low ft).OZ hl)-calcium medium to 1.1) 5e\eral concentration5 ol arachidonic acid: (BI addition ofa masimalIy effective concentration t II) !’ moles/II of arachidonic acid at time 0. [“H]TdR wa\ added (.A. CI at time 0. and the cell\ flsed Z h later: (B) for a period of I h preceding the wmple time. IC‘I The nbilit! ot various concentration5 of indomethacin to block the DNA-synthetic response of T5iB rat liver cells in low tO.02 mhlkcalcium medium to addition ofa mauimslly effective concentration ( IO 4 moles/l) of arachidonic acid. Indomethacin ivak added to the cultureb 15 min before arxhidonic acid.

Results

ami Discussion

Around 30% of the cells in T5lB cultures were reversibly blocked in late Gl phase after 48 h of incubation in low (0.02 mM)calcium FBS-BME medium. Raising the calcium concentration from 0.02 to 1.25 mM caused these blocked cells to initiate DNA synthesis within 1 h (fig. 1A; refs [2, 3]), and the proportion of DNA-synthesizing cells remained elevated for at least 6-8 h (see [2, 211). An involvement of arachidonic acid and its derivatives in this DNA-synthetic response to the calcium addition was suggested by its inhibition by low concentrations ( 10~x-lO-s mole/l) of indomethacin (fig. IB). an inhibitor of endoperoxide synthase which converts arachidonic acid into PGH.?, the common precursor of the prostaglandins and thromboxanes [9, 15, 181. Arachidonic acid concentrations between lo-” and IO-‘j mole/l. which did not affect cellular morphology, were as effective as calcium in causing calcium-deprived TS IB liver cells to initiate DNA synthesis (figs 2A, B and 3). However, the DNA-synthetic response was only transient (figs 2B and 3) and the proportion of cells making DNA E.rp Cdl

Re,

129 (I98Ut

declined between 2 and 4 h after the addition of the acid. This DNA-synthetic response, like the response to calcium, was probably triggered by a product of arachidonate metabolism rather than arachidonate itself, because it was blocked by preexposure of the cells to indomethacin at concentrations between lo-’ and lo-” mole/ 1 (figs 2C and 3). However, it should be noted that arachidonate action seemed to be less sensitive to indomethacin than that of calcium: Indomethacin at a concentration of 1OmHmole/l blocked the DNA-synthetic response to calcium addition, but it did not affect the response to lo-” M arachidonic acid. Indomethacin did not block calcium or arachidonate action simply by non-specifically damaging the cells or directly interfering with DNA synthesis. Thus, a different stimulator, the catalytic subunit of type II CAMP-dependent protein kinase [3], elicited a DNA-synthetic response from indomethacin-treated cells when it was added 2 h after arachidonic acid to which these cells could not respond (fig. 3). As noted above, the DNA-synthetic response to arachidonate was shorter and less

Preliminnry

notes

477

deprived liver cells to addition of calcium or calmodulin. the intracellular mediator of calcium action [4, 61. It is possible to speculate from these observations that the later prereplicative development of nonneoplastic cells cycling normally in highcalcium medium might be promoted by a brief influx of calcium ions and a consequent intracellular surge of Ca-calmodulin complexes [20] which would briefly stimulate phospholipase activity [6, 261 and thereby transiently trigger an arachidonic acid cascade.

15 I OL

024 Hours of incubation

Fig. 11. The ability of catalytic subunits from type II CAMP-dependent protein kinase (PK H-C) to elicit a DNA-synthetic response from calcium-deprived TS 1B rat liver cells whose response to arachidonate had been blocked by indomethacin. 0, Cells were exposed to arachidonic acid (IO+ moles/l) in low (0.02 mM)-calcium medium; 0, cells were first exposed to indomethacin (IO-.” moles/l) and then to arachidonic acid (10m9 moles/l) 15 min later (i.e., at time 0); A. indomethacin- and arachidonic acid-treated cells were exposed at 2 h to catalytic subunits (PK IL-C) at a concentration having IO units of activity/ml. One unit is that amount of orotein kinase activitv which transfers I pmole of phosphate from [y3’P]ATP to the histone type VS of Sigma Chemical Co. in 1 min at pH 7.4 and 30°C. Cells were exposed to [3H]TdR as in fig. 2B. The points are means + S.E.M. of values from four cultures.

sensitive to inhibition by indomethacin than the response to calcium. Both these differences might be due to the response to calcium addition being limited in both size and duration by the operation of the calcium homeostatic Ca2’-transport ATPases, while arachidonate action would not be so limited and its stimulatory products would eventually reach excessive levels which would secondarily inhibit DNA synthesis. These observations show that an arachidonic acid cascade is capable of mediating the DNA-synthetic response of calcium-

We gratefully acknowledge the advice and assistance of Dr J. P. MacManus in preparing protein kinase catalytic subunits and the technical assistance of R. J. lsaacs. R. Tremblay and D. J. Gillan, who prepared the illustrations.

References 1. Beavo, J A. Bechtel, J P & Krebs, E G, Methods in enzvmol (ed J C Hardman & B W O’Mallev) vol. 38,-p. 299‘. Academic Press, New York, San Francisco & London (1974). 2. Boynton, A L & Whitfield, J F, J cell physiol 101 (1979) 139. 3. - Exp cell res 126 (1980) 477. 4. Boynton, A L, Whitfield, J F & MacManus, I P. Biochem biophys res commun 95 (1980) 745. 5. Boynton. A L. Whitfield. J F & lsaacs, R 3. J cell physiol 87 (1976) 25. 6. Cheung. W Y, Science 207 (1980) 19. 7. Damluji. R &I Riley, P A, Exp cell bio) 47 (1979) 446. 8. Franks, D J, MacManus, J P & Whitfield. J F. Biochem biophys res commun 44 (1971) 1177. 9. Galli, C, Galli, G & Porcellati, G (ed), Advances in orostaelandin and thromboxane res vol. 3. Raven ‘Press,-New York (1978). 10. Hazelton. B, Mitchell, B & Tupper, J, .I cell biol83 (1979) 487. 11. MacManus. J P. Bovnton. A L & Whitfield, J F. Calcium and cell proliferation. Calcium in normal and oatholoeical bioloeical svstems (ed L 3 Annhilesi): CRC Press Inc. Coca daton. In press. 12. MacManus, J P & Whitfield. J F, EXP cell res 69 (1971)281. 13. Parsons, P G, Austr j exp biol med sci 56 (1978) 297. 14. Rixon. R H & Whitfield. J F, J cell physiol 87 (1976) 343. IS. Samuelsson, B, Hammarstrom, S & Borgeat, P, Advances in inflammation research (ed G Weissmann, B Samuelsson & R Paoletti) vol. I, p. 405. Raven Press, New York (1979). 16. Schimmel. S D & Hallam, T. Biochem biophys res Lommun 92 (1980) 624. Exp

Cd

Res

129 f 1980)

17.

Swierenga. S H H. WhItfield. J F & Kura\aki. S. Proc natl acad \ci US 75 (1978) 6069. 18. Weissmann, G. Samuelshon. A & Paoletti. R. .\dvances in inflammation research ted G Wciqsmann. B Samuelsson & R Paoletti) vol. I. p. ri. Raven Press, New York ( 1979). 19. Whitfield. J F. Handbook exp pharmacol 54/l (1980) 267. 20. Whitfield. J F. Boynton. .A I.. MacManus, J P. Rixon. R H. Sikorska. M & Tsang. B K. Mol cell biochem 27 (1979) 155. II. Whitfield. J F. Boynton. A L, MacManus. J P. Rixon. R H. Sikorska. M. Tsang, B K&Walker. P R. Ann NY acad sci 339 I 1980) 216. 32. Whitfield. J F, Boynton. A L. MacManus. J P. Rixon. R H. Walker. P R & Armato. U. Cyclic nucleotides and the regulation of cell growth (ed M Abou-Sabe) p. 97. Dowden. Hutchinson & Ross. Stroudsburg. Pa t 1976). 23. Whitfield. J F. MacManus. J P. Braceland. B & Gillan, D J. Horm metab res 4 (1972) 304. 24. Whitfield. J F. MacManus. J P. Youdale. T 8: Franks, D J. J cell physiol 82 (1971) 355. 25. Whitfield. J F, MacManus, J P, Rixon, R H, Boynton, A L & Youdale. T. In vitro I2 ( 1976) I. 26. Wong. P Y-K 81 Cheung. W Y. Biochem biophys res commun 90 (1979) 473. Received Revised Accepted

April version July

9. 1980 received 2. 1980

June

30.

1980

Mrrterials

Proteinase activity in macrophage cultures. Effects of heparin and

antithrombin ROLF ULF

SELJELID.’ LINDAHL,’

GUDRUN

BACKSTROM’

[I]. However-. the biological function of heparin remains elusive: in fact. recent resuits indicate that the amounts. if an)‘. of endogenous circulating heparin in human plasma are much too low, to have an) significant effect on the coagulation Status of the blood [?I. In search for the functional role of heparin it would therefore seem reasonable to consider effects on extravasal components. Since mast cells. the producers of heparin. are mainly located in the loose connective tissues. a logical approach would be to look for effects of heparin on such tissues or their constituent cells. The present study demonstrates that cultured histocytes or macrophages. i.e. cells that are located in the near vicinity of mast cells in the tissues, release into the medium one or more peptidases that are inhibited by antithrombin. The inhibition is potentiated by heparin in a manner that resembles the effect on the coagulation mechanism. These findings may suggest a role for heparin in modulating macrophage functions in the connective tissues.

and

‘/tz.s/it~~te ofMedical Biology, UrriL,ersity of Trotnse, N-9001 Trorns0, Narway. and 2Departnlent of Medical and Ph.vsiological Chemistry. University qf Agricultural Sciences, S-751 23 Uppsalrr, Sbl,eden Sunmar.v.

Mouse macrophages in vitro secrete an esterase capable of hydrolysing the chromogenic peptide substrate S-2222. The enzyme activity is inhibited by antithrombin and heparin. Also. the cells produce other hydrolytic activity, mainly associated with the cells capable of decomposing S-2160 and resistant to antithrombin and heparin.

Heparin prevents the coagulation of blood by accelerating the rate at which antithrombin, a plasma protein, inactivates the enzymes of the so-called coagulation cascade

trnd Mlethls

The chromogenic peptide substrates, S-?I60 (ivbenzoyL -phenylalanylL-valylL-arginine-p - nitroanilide hydrochloride) and S-2222 (N-benzoyl-L-isoleucyl-~-glutamyl-glycyl-t.-arginine-p-nitroanilide hydrochloride) were obtained from Kabi Diagnostica AJ3, Stockholm. The substrates are designed to be used in the determination of the blood coagulation enzymes thrombin and factor Xa. respectively, but are not exclusively specific for these enzymes. Heparin isolated from pig intestinal mucosa was ourchased from Inolex Pharmaceutical Division. Park ‘Forest South, Ill., and purified by repeated precipitation with cetvlovridinium chloride from I.2 M NaCl as described 131: The product. with an anticoagulant activity of 165 BP unitslmg, was fractionated with regard to affinity for antithrombin by chromatography on antithrombin-Sepharose 141; the resulting subfractions with high anh low &%ity for antshrombin. respectively. had anticoagulant activities of 257 and I I tiP unitsjmg. Bovine antithrombin was a gift from Dr Ingemar Biiirk. Uppsala. Trypsin (Type III) was purchased from Sigma Chemical Co.. St Louis. MO. Macrophages were harvested from outbred NMRI mice by peritoneal lavage [5]. The cells (about 3X 106