180

8iochin. "a et BiophFsica Acia, 1135(1992)180-183 I~;92Elsc~,terScience Publishers B.\ All righL~rescr.'ed 11167-4889/92/$05.00

Rapid Report

Hypo-osmolar stimulation of transepithelial CI- secretion

in cultured human T~ intestinal epithelial layers G.T.A. McEwan, C.D.A. Brown, B.H. Hirst and N.L, Simmons G~trob~csti~ul Drug DeficeryRese~h Centre. Departl~wntof Pl~'si~ogical Sci¢~ce~ Unh'ersi~,c~N ~ t l e The Medical5 c h ~ N e ~ t l e upon T~:v~e( UKJ

th,~n Tyne,

(Received4 Match |992)

KeNwords: Intcslinalchloride secrction;T~;H)potunkat~" Intact epithelial monolayers of T~ human colonic adenocarcmoma cells were exposed from the hasolatera[ surfaces to hypo-osmotic media: in responsive tissues this resulted in a transient stimulation of irr,vard shorl-circuit current (SCC) to a peak of 12.9_+ 1.5 (S.E.. n - 10) g A / c m 2 which declined to prcstimulation values of 5CC (2.1 tzA/cmz) wilhin 5 min. Exposure of Tst cells to hypo-osmotie media results in an increase in cytosolie [C.a-'÷]i, dependent on exlracellular Ca-'+ influr, The cell-swellingactivated SCC is abolished upon medium (21- replacement ~nd by 100 ;zM bometanide applied to the basal-surfaces, consistent with the inward SCC resuhing from transepithehal CI - secretion_ lfl0 ~M DIDS (4A'-diisothi~anantostilbene-2.2'disulpbonic acid) also abolished the cell---welling activated increase in SCC; DIDS is without effect upon the VlP-stimulated SCC, suggesting distinct CI- channell are involved in the two responses.

There is considerable interest in the CI- conductive pathways of intestinal epithelial ceils which may mediate transepithelial CI- secretion. In T~4 colon cells. cAMP, Ca -'+ and volume activated CI- conductances have all been demonstrated, ~r,d this is correlated with the existence of separate CI channels with distinct kinetic characteristics [I,3,16,18]. In intact 'I',~ epithelial layers basal short circuit current (SCC) is minimal but secretagogues such as (vasoactive intestinal peptide) VIP or forskolin stimulate an inward transepithelial SCC [4]. The increased SCC is quant~at/vely accounted for by an increased electrogenie CI- secr,ztion [4]. There now seems to be compelling evidence that the small conductance (5-10 pS) cAMP-activated CI channel which is insensitive to inhibition by the stilbene DIDS (4A'-diisothiocyanantostilbene-2,2'-disulphonic acid) accounts for the substantive portion of the cAMP-induced CI- current in Ts~ cells [1,16]; the cAMP-stimulated SCC in intact T ~ epithelia (CI- secretion) is also insensitive to DIDS inhibition [15]. The most-stedied O--channel in epithelial cells is the so-called outward rectifier (30-50 pS) [1,13]. Activ-

Correspondence to: G,T.A. McEwan,GasUuintesrinalDrugDelive~ Rehash Cenlm, Department of PhysiologicalSci~nc¢~|Jniversity of Newc-0Sll¢upon T)n¢. TIle Medi~l School, Framlingmn Place, Newcastleupon Tyne lqE24HH. UK.

it'/of this channel may be induced in quiescent patches by ~trong depolarisation and is blocked by extracellular DIDS [1,!3,16]. Swelling-induced CI- currents arc kinetically similar to the outwardly rectifying CI chann e l but it is still unclear whether the two types of current arc from a single channel displaying different states or whether separate channels extst [13]. It is unknown if either the outward-rectifier or swelling induced CI- channels contribute to ironsepithelial (transapical membrane) CI- current flow ,n intact T~4 epithelia [I]. In Necntrus enterocytes, Giraidez et al. [5] have shown that a volume-activated CI- conductance is present in the apical membrane together with a cAMP-activated C[- conductance. In addition, cell swelling may also stimulate trausepithefial CI secretion in airway epit helium [ 10]. There is also evidence for separate K + channels activated by cell swelling and by secretago~ues in tracheal epithelium [2]: The purpose of the present investigation has been to investigate the location and nature of the volumeactivated eonductances in Ts4 epithelium by investlgalion of the effects of hypo-osmoti¢ media upon transepithelial electrical parameters. Intact epithelial layers were exposed to hypo-osmotic media across their basolateral aspects (Fig. l). Approx. 62% of "I',~ layers responded with an increase in SCC. In responsive layers, an inward SCC increased rapidly to reach peak values of 12.9_+1,5 (S.E., n - 1 0 )

181 a ~ C C respoqse, even w i t h T ~ epithelia f r o m t h e same e u h u r e b a t c h w i t h n o discernable difference in initial electrical p a r a m e t e r s . I n these T m layers w h i c h w e r e unresponsive to hypo-osmotic stimulation, V l P - s t i m u lation o f inward S C C was e n h a n c e d (9.9 ± 1 . 6 / z A / c r n "~, P < 0 . 0 0 5 versus swelling-responsive tissues, M a n n W h i t n e y U-test). T o test the effects o f pharmacological a g e n t s o n t h e cell-swelling activated S C C it was necessary t o c o n f i r m t h a t r e p e a t e d hypo-osmotic exposures gave rise t o a n u n d i m i n i s h e d S C C response. I n four responsive layers t h e initial response t o h y p ~ o s m o t i c m e d i a was 7.7 _+ 2.3 / ~ A / c m 2, whilst a second exposure a f t e r l0 rain in

F A / c m ~, declining t o pre-stimulatinn values within 5 rain (Fig. D. T h e increase in S C C was u n a c c o m p a n i e d by a n increased tlssu¢ c o n d u c t a n c e , indeed a small increase in tissue resistance was h o l e d (transepith¢lia] resistance p r i o r to stimulation was 751 _+46 ~ c m 2, at t h e peak o f t h e S C C respoase it was 8 1 3 ± 5 9 .Qcm'( n = 19, P < 0.(12, paired data, S t u d e n t ' s .t-test; twotailed). I n a separate b a t c h o f Ts4 layers t h e peak m a g n i t u d e o f t h e swelling i.nduced inward S C C (9.8 _+ 0.9 n = [5 / z A / c r n 2) was c o m p a r e d to t h a t observed with l0 n M V I P which was m a i n t a i n e d at 4.7 + 0.8 ( n = 1 5 ) p . A / c m 2. W e n o t e d in s o m e casos ( = 3 8 % ) t h a t hYlY.bosmotie exposure o f Te~ layers did n o t d i c k

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ceils mounted in Ussing c h ~ b a ~ . (A) c~atml data n = 10. =l=S.E, (B) effecl ,,f CI free-media, paired data tram thee ~nalaye~, (C) effect of basolalcml 100 pM bumctanide, paJled data from five mono]ayers, (D) effect of 100/tM DIDS appliad Io both bathing solutions, paired data from five monalaye~. Te~ cells were maintained in ~lial ~h~z~ in a I : 1 mixture of .~ulbac¢o's modified ]'~aglc's medium and Ham's F]2 with 5% new+born calf serum a~d 200 ILlml - 1 of panicillin. 2O0pgml - I streptomycin. T~ cells were grown as epithcllal layers aport AnoCal125 mm culture inserts c.¢~tedwith rat-tail ¢e41agenusing high-denshy seeding, as previously described [7,12). CuRu;es were grown for lypicall'v 2 weeks. in @w~)"plates ~t 3"PC. ~% CO~ -.~E~,Erz..z~,,aicplac~,3; c~,'S 3 d~-~. Cal:~rcd c~;t,~.:lial la~rg "~veremounted in I.]ssiq~ L,~pezh~mb¢l~ maialaingd at 3"/~C.¢ozmected to ~ automatic voltage cIarop via KCl/agar salt-lmidges and rcvei~ible electrodes (Ag/AgCI Jar cuffent passage, calomel for voltage se~ing) ap~ measurements of open-circuit dcclri~l P.d. transephlzellal ~istance and $CC made in modified Krehs sointi~ns [12.LIsotopic =mchtied Krebs" solutions were (A) {all mmal/l) NaCL 70; KO, 5At mannitol, 140; CaClz, 2.8; Mg$O4, 1.2: lqaH ,PO4. 03; KH2PO4, a.~; Tr[s base. 14; HCL 124 glucose. 5 (pH 7A at 37°C) with l% (v/v) donor horse ~rum; (B) Hypo~unic soinlions were as for A except mannital was omitted; (C) L-"l-free ~oluZlons1 ~ ob~dlned by subslitution of NaCI and KC] by meth~P,csulphonate izeutralLs~dby appmp;iale quanthies of NaOH and KOH, final pH being adjusted ,,viih Tds b~'~e.Ca(NO31z reinaced the CI salt. 4A'-diisothiaeya,a ntosdlbc ne2,2'disalphoiac acid (DID$) was f~o~ Sigma. Sto~'k~oZutionswere made in I mM Tds.

182 isosmot ie media gave 6.8 4- 0.7 pA/cmZ; the difference was not statistically significant ( P < 0 . 6 paired data, Mann-Whimey U-test). Fig. l b and c shows the effects o f media C I - replacement with methanesulphanatt: salts and 100 p.M bumetanida on individual responsive epithelial layers. Both conditions abolish the swelling activated increment in inward SCC, consistent with this SCC r ~ u i t i n g from electrogenie C I - secretion. T h e swelling-activated SOU was also abolished by D I D S (100 tzM) (Fig. lc). This aetinn o f DIDS contrasts to the lack o f inh~itian with 100 p M D1DS on the VIP-stimulated C I - secretion (without D I D S , the VIP-stimulated SOU we.s 7.5 4-1.3 ( n = 4) t t A / c m 2, whereas in the presence of DIDS, the V1P-stimulatcd SOU was 13.74-1.9 # A / c a t 2 ) , in confinnatinn of the data o f Tabeharani et al. [15]. T h e increment in transepithalial resistance upon exposure to hypotonic media is m e r e marked in C1-free media, and in the presence o f D I D S (1 rain after the hypotonie switch epitbelial resistance was 774 4- 54 (conh'ol), 1078 + 143 t~ can 2 (CI--free), P < 0.04, n - 3, paired data, Student's t-test and 611 _ 126 (control), 877_+94 .Qcra z (100 p.M DIDS), P < 0 . 0 0 5 , n = 5 , paired data, Student's .,-test). This suggests a conductance increment due to an apical C I - conductance is evident but that cell swelling per se is associated with an increased tsansepithelial resistance, perhaps due to decreased paracellular volume, and that the expected increase in apical conductance, and hence epithelial conductance, in control tissues is obscured by the parallel i n c r e a s e in p a r a c e l h i l a r resistance. F o r

bnmelanide-treated tissues no difference .vilh control tissues is noted for epithelial resistance after exposure to hypotonic media (I rain afler hypotonic exposure transepithelial resistance was 835 + 94 .Q cm 2 (control) and 8 3 0 + 8 8 t2c~. 2 (,3..I m M bttaie~ati;dc), P < 0.84). Fig. 2 sho~s a representative trace of the change in intracelinlar [Ca z+] upon exposure to hypotonic media; it should be noted that the time course for change in [C.a2+]i parallels that observed for activation o f the Cl--seeralian in the intact epithelial layers (Fig. 1). T h e transient response to cell-swelling is markedly reducod in nominally Ca2+-free media suggesting that cell-swelling may activate membrane conductancos that allow C.a2+ influx across the plasma membrane. T h e present data a r e thus consistent with the presence of a volume-activated C I - channel distinct from that activated by c A M P , blocked by extracellulay DIDS, and which is able to participate in transepithelial (21secretion, Le., is present in the apical membrane. T h e observation of a reciprocal relationship between the cell-swelling response and the VIP-stimulated inward SCC in identical batches o f Ts4 epithelia, may indicate that the expression o f Iwo pharmacohigically-distinct C1- channels at the apical membrane is variable and that additional unidentified factors are operative in controlling the tissue response to c A M P and volume stimulL Recent data on the cellnlar loealisation of a DIDS-seasitive C I - channel within Ts4 epithelial cells suggest that differential recruitment of (21- channels from sub-apical vesicles may play a role in ".his phenomenon [14]. "l-he present data ¢~ntrast to that seen

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otherwise solutions as for Fig. I). Rppresentatlse experiment, similar icsu[15were obtained ill fr,'e separate experiments. Te4 cells were grown as sob~onfluent layers on 22 ram (BDH) glass covewslip6.Cells ~ r c loaded with tBe fluorescent indicator, fu~-2, by incubation ilt scram-free growth media containing 5 #M fnra-2/AM (Moleceinr Probes, Oregon, USA) at 37~Cfor 45 mill. ~ cells were then washed and placed in the experimental chamber (volume I ml) through which notations were superfmscd at a ~tc of 5 mlmln - x. Fluorescence measulcmcnts were made using a N~on Diaphot fluorescence microscope mid photon cnuruing system (/'~¢'wcastl¢Phommelrin System~ UIO. Cells r~ere illuminated with excitation light at 350 nm ~nd 38O nm, and emitted light was fdtered IL~nga 5211nm long-pass filter. FlaOlCSCe~ ntio at the two excitation ~ l e n g t h s was ~lh'brated [9l by determining lhe maximum ~ d minimum lluore~c~'nc¢-"ntios of nominalZycalcium-free (approx. I riM) and high calcium (4.6 #M) solutimt~ Calibration cutweswere ~onstructed using a series ,~f known Ca2. activity buffers (Molecular Probes) and/or a dbsociatlon constar.t ,~ 225 nM. No account is made of intraccllalar viscosity, thus measurement represent only relati~'echange in [Ce~* i, 117]-

183 in secretory M D C K epithelia where pharmacologically distinct C I - channels mediating cAMP-stimulated secretion and regulatory volume decrease ( R V D ) are present on opposite epithelial membranes [11]. In this case the vulumc activated CI--conductance reduces transepRhclial C I - secretion [11]. Similar data to those reported here in Tm epithelia a r e observed in tracheal epithelium, although the sensitivity of the cell-swelling stimulated increase in C I - secret/on to D!DS was not determined in the latter [10]. Cell-swelling in T ~ cells is associated with an increase in cytosolic [CaZ+L most probabl~ via an increased transmembrane C a 2+ influx (see above). Thus, in measurements on intact epithelial layers it is not yet possible to Separate C a 2+ and volume activated C I conductaaces, as is also the case in patch clamp studies [13]. Direct activation o f stretch-activated non-specific cation conductances have been implicated in the initiation o f R V D in amphibian kidney [8] and the present data suggest that the apical C I - conductance which is sensitive to D I D S in Ts4 cells may ~ ,~ctivated by r~;Sed [Ca2+]i. Cull s~clling is associated with R V D in hmnan small intestinal cells (Intc~fi;,¢ ,'~37) by increased K ÷ and C I - coeductances |6j. However, in this ca:,¢ the activation of the volume-dependent CI" Conduc~.nce occarred with some delay and was independent of change in [CaZ+]i [6]. K + conductances activated by secretion and cell swelling have been identified in respiratory epithelia [2]; further studies of the K + channels involved in volume regulation and Secretion in Ts4 cells .*herefore need to be made. This work was s u p p o s e d under the L I N K Programme in Selcctwe Drug Delivery a e d Targeting,

funded by S E R C / M R C / D T I and Industry ( S E R C grant GR/F 09747), and by a grant m NLS from the Wellcome Trust (Equipment/030626).

References Anderson. M.P. and Welsh, MJ. (1991) pro¢. Natl. Acad. Sol. USA 88, fifl03-6007. 2 Bull, A-(3., Clapp, W.L. and FrizzelL R.A. (1990) Am. J. Physiol. 258, Cfi7~_~38. 3 Cliff. W.H. and Frlzzeli, ILA. (|990) Pro~. Natl. ~:~d. ScL USA 87, 4956-4960. 4 Dharmsathaphorn, K., MandeL K.G., Mami, H. and McRobcrls, J./~ 0985) L Clla. InvcsL 75, 462-47L 5 Giralde;'~F., Valverd¢, MJL and Scpuk'eda. F.V. (1988)Biochim. Biophys~Acta 942, 353-356. 6 Hazaraa, A. and Ohada, Y (1988) J. Ph~ioL 402, 687-7O2. 7 Hunter. 1, HirsL B.H. and Simmons, N.L (1991) Br. J. C.an~r 64, 43%444. 8 Hu~S, A.M. and Hunter, M. (1990) J'. Physiol.430,13-24. 9 Luckoff. A. (198fi)CItll Calcium 7, 235-242. 10 McCann, J.D., Li, M. and Welsh. MJ. (1989] J. Gem Phs'sioL94, i015-1fl36. ~.1 Simmons, N.L. (1991) Pllugers Arch. 419, 572-578. 12 Simmons, N.L. (1990) Melhods Enzs'mol. 191, 42a-436. 13 Soic, C.IC tad Wia~, .IJ. (199D Am. L Phys[oi. 261, C.65~;-C674. 14 Sorschtr, EJ., Fuller. C.M., Bridges, RJ., "I~ol~, A., Marchase, R.B., Brinkley. B.R., Friz~lL R.A. and Bcnos, DJ. ~'1qq2| Am. J. Physiol. 262, C136~C147. 15 Ta~haratfi, LA., Low, W., Elie, D. anti Hanrahan, J.W. (1990) FEB$ LCR.270, 157-164. 16 W/dtlicombe,J.H. and Wine, JJ. (1991) Trends Biochcm. S~. 16, 474~77. 17 Williams, D.A. and Fay, F.S. (1990) C¢11Calcium 1L 75-83. 18 Worrcll, ILT,, Bull, G.A., Cliff, ~V.H.aad Fr'gzell, R.A. (1989) Am. 1. Physiol. 256, C 1111-C I 119.2.