Osmotic activation of the Na+H+ antiport in protein kinase C-depleted lymphocytes

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OSMOTlCACllVATlONOF THENat/H+ ANTIPORTIN PROTEINKINASEC-DEPLETED LYMPHCCYTES Sergio Brinstein*q, Esther fl&*

endowdon 6. Mills5

*Department of Call Biology, The Hospital for Sick Chilcken, 555 Univarsity Ave., %epartment of Btochemlstry, Univarslty of Torontoand %Iivlsion of Oncology,Toronto @neroI Hospital Toronto, Caneda,M58 IX8 Received

November

8,

1985

SUMMARY.TheNat /Ht antiportof rat thymlc lymphaq&sis activatedwhenprotein ktnaseCIs stimulatedby phorbolasters.Asimilar activationof thaanttport Is obtained whanthe callsm treatedwith hypertonicsolutions.Wet&ad thapossibilitythat proteinkinaseCalso mediates theosmoticactivationof Na*/H* exchange.ProteinkinaseCwasdepleted by prelncubatton of thymocytasfor 24 hr In thepresana? of highconcentrations of phorbolester. Disappearance of theenzymewasa%essed by direct measurement of phosphotrmsferase activity, andby theI@sof biologicalresponses to phorbolesters.TheNat/H* antiport in proteinkin&se C-depleted callswasnotstimulatedbyadditionof phorbolastar,but raqmndsd normallyto hypertonictreatment.Theresultsindicatethat theosmoticactivationof countertrmsportdoesnot rtqulre stlmulatlonof protelnklnassC. 0 1986 Academic Press, Inc.

Anamlloride-sensitive,e1ectroneutral Nat/H* exchanger hasbeendetected in theplasma membranes of mostmammaltan cell types(seeI and2 for revlew). Thlsantiport is thou&t to be essentialfor pHt*regulation(3), andtrmsapithallalion transport(4) andmayplay a role in the initietion of mitogenesis (5). In restinglymphocytes theantiport is nearlyg&scant at physiological pHi, but beames activatedby a&fitionof tumor-promotlngphorbolastars(6). Theprimary targatfor phorbol esters,whichwe structural analogs of dtacylglycerol,Is theCa2*- andphospholipld-dependmt proteinkinasaC ( 7,8). Severallinesof evidsnce suggest that in lymphocytes theactlvatim of Na+/H+exchangs by phorbolestersis mediated by stlmulatlonof protetnkinaseC a) the concentrations of TPAregulredfor activationof countertransport aresimilar to thosereportedto activatethekinase;b) only thosephorbolderivativesthat ax&rate kin&seactivity haveaneffect onNa+/H+exchange (6) mdc) theantlport isalsostlmulabxtby diacylglycerol(9), theputative physiological activatorof proteinklnasaC. Inerythrocytes(10,1I)andeplthellalcells(12).eswellesInF/mphocyles(9,13),t~ Nat/Ht antiportcanalsobestimulatedat normalpHi Ly osmoticcell shrinking, In this instance,

pHt: cytoplasmlcpH; TPA:l2-0-tetr&canoylphorbol 13,xetate; BCECF: 2’,7 bis(cerboxyethyl)-5,6 carboxyfluorescein. WBREVIATIONS:

0006-291X/86 Copyright All rights

$1.50 0 1986 by Academic Press, qf’ reproduction in any form

Inc. reserved.

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Nat /Ht exchangs appears to play a role in volume regulation by inducingreswelling of shrunken cells ( 1 I ). The osmottc activattm of the antiport begs a remarkable resemblmce to that praknxd bv phorbol esters: both respormes ere attributeble to en alkaline shift in the pHt @endsnm of transport (9,131. Moreaver, both are blocked by &pletim of ATPor by additionof triflwperazine or N-ethylmaleimide t 13). Thesesimilarities led to the suggestionthat the osmotic activation of the antiport might also bedue to activation of protein phospharyldim, perheps m&ted ty protein kinaseC(13). It was recently reported that prolongtxl incubatlm of flbmblasts with phorbol esters causes 8 decrease in the number of phtrbol ester-binding sites ( 14) end in the activity of pmtein kinese C ( IS). Further, the cells bccae unresponsive to the btoiogical affects normally elicited by phorbol esters In untreated cells ( 16). Theseprotein kinase C-depleted cells provide a useful modelto test the involvement of this enzyme in the osmotic activation of Nat /Ht exchange. Disappearanceof the osmotic reqonse alongwith the kinase would be indicetive of a mle of this enzyme in cell volume fqulatim.

We here report that down-regulation of protein kinase C by

phorbol esters can also be inducedin rat thymic lymphooytes, md thet the antiport in klnase-depleted cells fails to respond to actlvatlm by TPA. In contrast, the osmotic actlvatim persists in these cells. MATERiAlSANDMETHOOS Meterlab Htstone type Ill-S, TPAandphosphatldylserlne wereobtalned frtxn SigmaChsmicel 00. N-methyl-D-glucamine was from Aldrtch. MediumRPtll 1640 (HCO3--free), and 100X amcentratedmtibiotic-antimycotic mixture were from BiBcO, [gamma-32PI-ATP was from ICN BCECFac&xymethylester was purchased from Molecular Probes. Amllorlde was ths kind gift of Merck Fro& Canada Net -solutlm contained(in mtl): 140 N&l, 1 KCl, I MgCl2, 1 CaCl2, 10glucosemd20Hepes(pH 7.3). Nat-freesolutimcontained 140 mM N-methyl-D-glucamine chloride instead of N&l, but was otherwise identical. T-letho& Thymocytes were is&tad from maleWtstar rats ( 1SO- 200 g) , aunted and maintainedin Hepes-bufferad RPM1 1640 as previously dasaibad (9,131. For overni(lht incubations, the mediumwas supplementedwith 100 U/ml penicillin, 100 ug/ml streptomycin and 0.25 ug/ml fungizone. For pHt maesutwwnts the C&S were sedimented,resuspendedat 40 x 106/m1 md lc&d with BCECFby Incubatim with the parent acetoxymethylester (2-3 pg/ml) la 30 min at 37oc. pHt wee determined fluorimetrically at 37% using a Perkin-Elmer 650-40 spectroflwrimeter and calibrated with nigericin KC1as described (9,131. For pmteln kinasa C activity determinations, the cells were sedimented,washed and lysed by resuspenslm In a medium containing 0. I mfl EDTA, 1 mM EBTA,0.5% Trlton X- 100,O.S mil phenylmethylsulfonylflucri~, IO mM mercaptmthenol and 20 mfl Tris.HCl, pH7.5. The suspensim wea homogenizedby 20 strokes in a Damce-type homogenizer,andthen cantrifu@ at 4% for 30 mln at 48,000 x g Thesupernatant was fractianatad with (NH412 So, as described by Niedelet al ( 17). Thefraction precipttatad between 33% and 70X saturatlm, which cmtalns all the protein kinaee C in the extract ( IS, I 71, was mat I -3 mg protein/ml in 8 medium conteining 0. I mM EDTA,50 mM mercqtc@thmol, 1 mM EOTA,25X glywrol and 20 mtl TrisHCl, pH 7.5. The fracttmattm step serves to arncentrate the kinasemd to eliminate low molecular weight effectore present in the crude extract ( IS, 17). Far aanperison, in some experiments the unfractictnetedextract was used dtrectly for kinme activity measurements. n kim C divity wtn &termtsd try msasuriq urd inmrporalim of 32Pi from Pr?& PI-ATP into histonetype Ill-S. The[gemmawereperformedfor 3 mlnat3O%usiq

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5-20 ugy2oteinin a total volumeof 250 pl of medtum contatning0.5- 1.OuC1 [gamma- PI-ATP, t 00 ptl ATP,20 mMmaytesium acetate,t mMCsCT2.50ug historm ad 20 mMTrisHCl, pH7.5. Thedeterminations wereperfcrmedin triplicate in the presence and absence of TPA( 1OV7M,final) andphcsphatid&erins(24 pg per assay),to definethe protein kinaseC-m&&d phcsphorylation.Thereacttmwasterminatedby additlmof 1 ml of ice-cold 25I trichloroeceticacid Thepracipitatadhistmewasthenseparated by filtration m 0.45 pmHA Mfllipore filters, folknvedby four washes, eachwith 3 mlof 5%trichloroaceticacid Thefilters werecountedby liquidscinttllatlm. RESULTS ANDDISCUSSION TableI summarizes measurements of protein(histone)kinsseactivity in extractsobtained fromcellsincubatedfor24hrinthepresenceandabsenceof2x10m7M TPA. Thecellswere extrsctedwith Triton X- 100andfractionatedwith (NH412SO4asdescribed bv Nledelet al ( 17) endRodriguez-PeRa andRomgurt ( 151,to removephcepholipicls mdsoiubieeffecters. The BSS[YS wersperformedin thepresence andebsence of phcephatidylserine andTPA,to definethe fractionof thekirmserztivity attributableto protetnkineeeC. In controlcells,proteinklnaseC accounts for approximately40%of thetotal phosphotrmsfsrese activity. In contrast,mty a very small,statisticallyinsignificantfractim of theactivity is dueto proteinkinaseC in TPA-treated cells. Thisreducttmis notdueto carryover of TPAend/orphospholipid by theTPA-treetedcells, sincethe basalactivity ( -PL in Tablet ) is not increased.Moreover,thetotal phosphotransferese rctivfty ( +PLin Table1) isdeceased in the treatedcells,consistent with diseppeaance of protein kinaseCactivity. Qualitativelysimilar resultswereobtained whenthe unfraction&f Triton X- 100extract wasusedfor theassw+s, indicetingthat theTPA-induced &creasein activity is not artlfactualh/generated by thefrectlonatimprocedure.These dataresemble thosereportedfcr 3T3 cells( 15) endereamsistentwith functionaldown-regulation of proteinkinaseCuponprolongsd incubationwith phcrbolesters.

Table 1

Determinetimd protelnkin@seCactivityin

amtroldTPA-trdsdcslls

-PL (pmo132P~mqLn/mgpro!eln)APL

cilntrol

880t

TPA-tredsd

657t118

124

1403t

120

714t265

523 57

Ttyna@es(20~106/ml) efncubdedfor24hra3~in~-bufferedmadiumRPMl F MTPA Altsrwsshlng,thecsllswerelyssdInTrltonX-100ands 1640wiU1wwltha~t2x10prolelnkinsseGr~fractim~isolded~~NH4)2SOqfract~ianas~ibed(15;see

MethorN). Proteinktnwe wesmamurat for 3 minet 3oeC in ths (-PL) ofphas~hstidylssrineand TPAapdnscribedunder Msthadn phosghsQi~lserineandTPA-inducedklnaseactivity,I.a~inkinaseC. ofslxd&ermlnetions 10

pressnca ( +PL) or dsencu ~PLrderstothe Theddswemecmst

SE

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Further 8vid8nc8for the disappearanceof function8l protein kinma C ~8s obtainadby meesuring the effects of TPAm l&*/H+ cumtertr8nsport.

Theactivity of the mtiport ~8s

Assessedby measuring pHt , since ectivetim h8s beenreported to result in cytopla~mic 8lk8liniZ8M

(6,9). As shown in Fig I , cells incubetedin the absenceof TPAfor 24 hr respond to

theaddition of the phorbol ester with 8lk8liniZ8tim (top trace, Fig IA), 8s has beenreported for freshly isolated cells (6). Th8tthis dk8linizatim is dw to stimulstim of the Nat/H* mtipart is evid8ncedby its requiremmt for 8xt8rn8l N8+ (the respond is absent in N-methyl-D-glaamine+ solutim; bottmn trace, Fig 1A) md by its sensitivity toamiloride, en inhibitor of the antiport (middle trace, Fig iA). ThepHf of TPA- traeted cells ~88 typically * 0. I units low@ than thnl of untre8ted controls (in three preparations pHi 8veqed 7.11 f 0.03 [SEMI in control cells and6.98 f 0.05 in pharbol ester-treated cells). This indic8te8that the TPA-inducedalkalinizstim did not persist efter 24 hr. This could be

8WibUt8d

to

w8shout of the phorbol 8&r during the BCECF-io8dingand

reapert8im procedures, or alt8rn8tively to dis8ppeeranceof function81protein kin8se C. That the latter explanstim is likely correct is indicstad by the experiment shown in Fig IC. Additionto the chronically phorbol ester- treated cells of I Om7M TPA, 8 arncentratim that produces III8Xim8i stimulation in control cells (61, f8iled to elicit 8 cytop18smic8lk8lini7atim. An alk8liniiatim

PHI

Fig I. Elf&s of TPA cmdhypertonicity m piii in control and protein kinss C-dcpiebd rat thymlch/mphon/tes. T ocqteswere pf8incubobi far 24 hr at 37% in the presence (C-D)or

9 M TPA.Thewiiswerethen~,rasuspendedadlaadedwithBCECF abaenm(A-B)ofZxIObyincubetion withtheparant8wtoxymethyiester. Aftermeackiitlmel w8sh mdresqmneim. the calls were usui for pHi dstaminstim asdmribsd u&r Mathat~ A) Cmtrol culls wav sqmdal in either Ne+-medium, withor without 00pII allaride, or in N-methyl-D-giucunlne+

medium (NM@1. WhereIndia&d,IO-3M TPA(finei)wueadded tothecuvette;B) Wtrol cells weresuspended in Nat OTN-methyl-D-glumnine+ media (&tonic). Where indicated, the medte were! mab hypertonic ( 450 f 5 modI) by addltim of 8 small voiums of amcmtrated NaCi or N-methyl-D-giutxninechlcride, respectivety. Simils r8sultswareabt&wd when N-me&G-D-gltxuninechla’itbwesusadtomake theNa+-medium hypertonk(mtshown;sea ref. 13); C) Ceils preincubatal for 24 hr with TP prkr to BCECFloading and ding wara ape&d in Na+-medium. Where indicated, IO- # M TPAwasadded Finally, thesoiutim was mate hypedanic try alditton of camWr8WWI; 0) Cellspreina~beted in TPAfor 24hr were suspendedin Nat-medium with or without 200 pM amiloride or in N-methyl-D-giucunirw’ medium. Wtwre indiated. the solutions were mwb hypertmic with tha approprlats solute as above. Thetraxw a% repwmbtiw of three ssparsts experiments. The time SEsleapplies to&D. Tempw%turez 37%: 11

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(Fig 1C; s~8 discussion below), indicating thet

theentlport remains functlmel end responsive to stlmulatlon followingecklitlon of the phorbol ester. Thus, this biologicaleffect of the phorbol ester receptor(s) disappears concomitmtly with the dzcrmse in protein kinese C activity. As shown in Fig 16, increasing the osmolarity of the mediumfrom 290 f 5 mc6M to 450 f 5 mosfl by additionof NeCl resulted in ectivetion of the entiport in untm

cells. As reported for

fresh cells ( 131, the resulting alkelinizetirm is eliminated when extracellular Nat is replaced by N-methyl-D-glucmnine*

(Fig lB)cr byadditienofemiloride(not

stimulation of Nat/H+ countertrmsport.

illustrated), indictsting

A distinct alkelinization wee also observed when cells

chronically treeted with TPAwere challengedosmotically (Fig ID). Theelkalinizetim was similerly Ne+-&pendent mdamiloride-sensitive (Fig ID, middleend bottom tracas), indicating activation of the Nat/H* mtiport. The magrituds of the osmotic respmse wes simils in untreuted

(apHi= 0.26 kO.02 ) end in TPA-treetedcells (APHI = 0.20 f 0.03). The present results indicott! that the osmotic activation of the Na+/H+ exchangeram be accomplishedin cells largely c&voidof functional protein kinase C, ruling out 8 role of this enzyme In the stimulation of the antlport during volume regulation. This wncluslon is consistent wtth recent observations that migration ofqtoplasmic kinme Cto the membrane, which is indicative of activation of this enzyme ( 18). was obtainedwhen the cells were tre&d with TPA but not with hypertonic solutions (Orinstein et al, submitted for publication). Moreover. osmotic shrinking did not &crease the cellular content of phosphati~linosito14,5 bisphosphate or increase the levels of inositol trisphosphate, indicating thet phospholipaseC doesnot liberate diecylglycerol from phosphoinositidesun&r thesecanditions. it mustbeconc1t&ithatatle&twodistiti mechenisms cm activete the Net/H+ mtiport in lymphocytes. phosphoryletim of the exchanger or an wcillary protein by protein kinsse C, and an unidentified prcoess triqpered by osmotic cell shrinking, which neither stimulates phospholipaseC activity nor requires the presence of functional protein klneseC. Thetwo pathways must shere the final step, inasmuch BSthe phorbol ester- endosmotically-inducsd responses are not additive (9).

This amclusim resembles that

obtainedfor the growth factor-induced stimulation of the antiport, which also eppears to be unrelated to protein kinase C ( 16).

ACKNOWLEDGMENTS Supported by the National Cmcer institute (Cmab) andthe MedicalResearch Council (Cane@. S. Orinstein 1sthe reclplent of e Medical Research Council Scfentlst award.

REFERENCES I. Amman, P. S. ( 1985) Annu. Rev. Physiol. 47,545-560. 2. Mahnensmith, R. L., mdAmnsm, P. S. ( 1985) Circ. Res. 56,773-788. 3. Boron, W. F. ( 1983) J. Membrene Biol. 72, I- 1. 4. Aronson, P. S. ( 1983) Am. J. Physiol. 245, F647-F659. 5. L’Allemein, 6, Fren&i, A., Cregoe, E., endPouyssqur, J. ( 1984) J. 8101.Chem. 259, 4313-4319. 12

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6. Brinstein, S., Cohen,S., Boetz,J. D., Rothstein, A., andOelfand,E. W. ( 1985) Pram.Nat. Acad. Scl. (USA) 82,1429-1433. 7. Nishizuka, Y. ( 1984) Science225,1365- 1370. 8. Bed@, M. J. ( 1984) Biochem.J. 220,345-360. 9. Orinstein, S., Cohen,S., &I&, J. D., and Rothstein, A ( 1965) 3. Cell Biol. IO 1,269-276. 10. Parker, J. C., andC&ranows. V. ( 1984) J. Oen.Physiol. 84,379-40 I. 1 I. Cala, P. N. ( 1985) Fed.Proc. 44,2500-2507. 12. Spring, K. R., and Ericsun, A. C. ( I9821 J. Membrane Biol. 69, 167- 176. 13. Qrinstein, S., Cohen,S., and Rothstein, A. ( 1985) J. E&n.Physiol. 85,765-787. 14. Collins, N. K. L., andR&!engurt, E. ( 1984) J. Cell. Physlol. I 18, 133- 142. 15. Rodriguez-Pena,A, and Rozengurt, E. ( 1984) Biochem.Biophys. Res. Cunmun. 120, 1053- 1059. 16. Vara, F., a&Rozengurt, E. ( 1985) Biochem.Biophys. Res. Cummun. 130,646-653. 17. Niedel,J. E., Kuhn, L. J., andVmdenbsrk, (3.R. ( 1983) Proc. Nat. Aad Sci. USA80,36-40. 18.Kraft,A.S.,andAnderson.W.8.(1983)Nature301,621-624.

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