Uncoupling of Phospholipase C from Receptor Regulation of [Ca2+]

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Printed in U. S. A. Uncoupling of Phospholipase C from Receptor Regulation of [Ca2+]i in. Tg4 Colonic Cells by Prolonged Exposure to Phorbol Dibutyrate*.
THE

JOURNALOF BIOI.OGICAI. CHEMISTRY

Vol. 266, No. 27, Issue of September 25, pp. 17904-17911,1991 Printed in U. S.A .

Uncoupling of PhospholipaseC from Receptor Regulation of [Ca2+]i in Tg4Colonic Cells by Prolonged Exposure to Phorbol Dibutyrate* (Received for publication, April 18, 1991)

Leslie Reinlib$QT#, Fadia Hornaidan**, Alastair Watson$, David S. BredtS, Geoffrey W. G. Sharp**, David Zahnisers, and Mark Donowitz$Q From the $Departmentsof Medicine (Gastroenterology Diuision), Physiology and Neurosciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the §Departments of Medicine and Physiology, Tufts UniversitySchool of Medicine and New England Medical Center, Boston, Massachusetts 0211 I , the (Diuision of Intramural Clinical and Biological Research, National Institute on Alcohol Abuse and Alcoholism,Rockuille, Maryland 20852, and the **Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York14853

The Tg4colonic cell line,a cultured C1- secretory cell, creasedbasallevels of inositol1,4,5-trisphosphate. elevates intracellular free Ca2+ ([Ca2+li)in a concentra- Protein kinaseC apparently is not involved directlyin tion-dependent manner when exposed to carbacholor the mechanism that leads to these effects. histamine. As determined with a fluorescence microscope imaging system, exposureof Tg4cells to 100 I.LM carbachol or histamineresultedinanimmediate [CaZ+li rise of approximately 50-80 nM in all cells. The T,, human colonic cancer cell line isa model epithelial Preincubation of monolayers for1h or longer with 0.4 C1- secretory cell. Monolayers respond to physiologic secreI.LMphorbol 12,13-dibutyrate (PDB) reduced thenumtagogues (Beuerlein et al., 1987; Cartwright et al., 1985; ber of cells which responded to histamine or carbachol Dharmsathaphorn et al., 1984, 1985, 1989; Madaraand and reduced the magnitude of the increase in the re- Dharmsathaphorn, 1985; Reinlib et al., 1989), including carsponding cells. This effect reached its maximum after bachol and histamine, which act through elevation of intra2 h and persisted for at least 24 h of PDB incubation. cellular free Ca2+ ([Ca‘+],)’ (Dharmasathaphorn et al., 1989; Binding of quinuclidinyl benzilate,a cholinergic recep- Merritt and Rink,1987; Wollheim and Biden, 1986), with an tor antagonist, indicated that down-regulation of ex- increase in short-circuit current and transcellular C1- secreternal receptors was not an explanation for this effect. tion. In TR4 cells the carbachol-induced [Ca2+], rise is dependExamination of phospholipase C activity in TS4 cell ent on bothcalcium stores and extracellular Ca2+ (Reinlib et membranes showed increased basal activity in PDBal., 1989). Histamine alsoshows a biphasic Ca2+ response treated compared with control cells. Measurement of although a larger part of the [Ca2+Irresponse appears to be inositolphosphatesgeneratedbyintactcellsusing rny~-[~H]inositol incorporation or receptor bindingas- from an extracellular source (Dharmsathaphorn et al., 1989). says showed that 2 h of incubation with PDB elevated A role in TS4cell C1- secretion for protein kinase C, which can be stimulated by phorbol esters, has recently been sugbasal levels of inositol 1,4,5-trisphosphate and prevented any further carbachol-induced generation of gested (Beuerlein et al., 1987; Vongkovit et al., 1989). Since inositol trisphosphate. Probably as a consequence, both the simultaneous additionof phorbol 12-myristate 13-acetate and Ca2+-dependentsecretagogues depressed C1- secretion in total cell calcium and Ca2+ ionophore-releasable calTx,cells without affecting the normal rise of [Ca”],, protein cium were decreased after 2h of PDBincubation. kinase C was postulated to inhibit agonist-induced secretion Membrane-associated protein kinase C activity was elevated after a 2 h exposure to PDB but was below at a step distal to [Ca*+’];elevation (Vongkovit et al., 1989). Prolonged incubationof fibroblasts with phorbol esters has the level of detection after 24 h with PDB. Protein kinase C antagonists neither duplicated nor blocked been shown to down-regulate protein kinase C (Issandou and the uncoupling of carbachol receptors induced by long Rozengurt, 1989; Rodriguez-Pena andRozengurt, 1984). Prolonged phorbol ester exposure has now been applied to T,, term treatment with PDB. The results suggest that prolonged PDB incubation caused uncouplingand ele- cells to investigate the relationships among[Ca2+],,phosphovation of phospholipase C activity from cholinergic andlipase C, and protein kinase C. In this study the effects of PDB and the role of protein kinase C on [Ca”], in T,, cells histaminergicreceptorregulationresultingininwere investigated at the single cell level using Fura-2 and a * This work was supported in partby a Cystic Fibrosis Foundation fluorescence microscope imaging system. In addition, measCenter grant to Johns Hopkins UniversitySchool of Medicine, Na- urements were made of phospholipase C activity and the levels tional Institutes of Health Grants R01 DK26523 and DK31667 and of cytosolic inositol phosphates under the same conditions PO1 DK39428 (Center for Gastrointestinal Research on Absorptive that [Ca2+Ii was monitored. PhospholipaseC wasactivated by andSecretoryProcesses),andthe MeyerhoffDigestiveDiseases Center. The costsof publication of this article were defrayed in part prolonged PDB incubation while protein kinase C decreased by the paymentof page charges. This article must therefore he hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 Supported by Career DevelopmentAward I074 8-1 from the Cystic Fibrosis Foundation anda career development award from the National Foundation for Ileitis and Colitis. To whom correspondence should be sent: Division of Intramural Clinical and Biological Research, National Institute on Alcohol Abuse and Alcoholism, 12501 Washington Ave., Rockville, MD 20852. Tel.: 301-443-5800.

The abbreviations used are: [Ca’+],, intracellular free ca2+ concentration; CCCP, carbonyl cyanide m-chlorophenylhydrazone; EGTA, [ethylenehis(oxyethylenenitrilo)]tetraaceticacid; HEPES, 4(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Fura-2 AM, Fura2 acetoxymethylester; PDB, phorbol 12,13dbutyrate; InsPx, inositol trisphosphate; Ins(1,4,5)P:,, inositol 1,4,5-trisphosphate; InsP1, inositolmonophosphate;InsP2,inositolhisphosphate;InsP4, inositol tetraphosphate; QNB, quinuclidinyl henzilate.

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Phospholipase C Uncoupling in Ta4Cells

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subtracted automatically from the experimental reading. Ro was calculated at 3-s intervals. After each experiment Rmaxwas determined for each coverslip by the additionof 10 p~ ionomycin in thepresence of 2 mM Ca2+, and Rmi, was determined by addition of 8.3 mM EGTA. Calculation of [Ca"], was made using the methoddescribed above. EXPERIMENTALPROCEDURES Treatment of Cells with Phorbol 12,13-Dibutyrate-TR4 monolayers Chemicals-Cell culture media were purchasedfrom GIBCO. were incubated in the presence or absenceof 0.4 p~ PDB for 0.5-24 Ins(1,4,5)Pa was obtained from Amersham Corp., and ["HI h a t 37 "C in1ml of culture medium (ethanol concentration= 0.02%). In~(1,4,5)P,~, myo-[:'H]inositol, [3H]polyethyleneglycol, and [3H]quin- The monolayer was washed three times with 1 ml of Buffer A a t uclidinyl benzilate (QNB)were from Du Pont-New EnglandNuclear. 20 "C prior to further use. When [Ca"]i measurements were perIonomycin was from Calbiochem. Fura-2 and Fura-2 AM were from formed, Fura-2 AM was included in the medium for the final 90 min Molecular Probes,Inc.(Eugene,OR).EDTA (disodium salt) was of PDB incubation, 60 min of which was carried out at 20 "C. from J. T. Baker,Inc.Liquiscint Protein Kinase C Assay-After incubation of T,, monolayers in was fromNational Diagnostics the absence or presence of 0.4 p~ PDB, protein kinaseC was assayed (Mannville, NJ). All other chemicals were from Sigma. in either whole cell homogenates or membrane andcytosol fractions Cell Culture-T, cells, used to passage number 69, were cultured using an activity assay based on phosphorylation of histone IIIS, as on glutaraldehyde-cross-linked rat tail collagen-coated glass coverslips as described (Dharmsathaphorn and Pandol, 1984; Reinlib et modified from the methodof Cohen etal.' Each coverslip of cells was al., 1989). Monolayerswere maintained in1:l Dulbecco-Vogt modified washed three times in 1 ml of Buffer A a t 20 "C and scraped into0.5 ml of 5 mM Tris, pH 7.4, 5 mM EDTA, 0.01% leupeptin, 0.28 mM Eagle's and Ham's F-12 media with 15 mM HEPES, pH 7.5, 1.2 g/ liter NaHCO:], 40 mg/liter penicillin, 8 mg/liter streptomycin and 5% phenylmethylsulfonyl fluoride, 26 pg/ml aprotinin, 1 pg/ml phosnewborn calf serum and studied between 4 and 14 days after conflu- phoramidon. The suspension of broken cells was homogenized five ence. The cells were a complete monolayer, as examined by thin times with a Teflon pestle drill. When homogenate was used, the histologic sections. In rare areas, cells appeared stratified (Reinlib et solution also contained 0.3% Triton X-100 and was centrifuged in a Beckman Microfugefor3 min a t top speed to removeinsoluble al., 1989). Fura-2 Loading and Single Cell Analysis-T, cells were loaded particulate. In other studies the homogenate included 2 mM dithiowithFura-2,and [Ca"], was studiedin singlecells as described threitol and 0.3% Triton X-100 and after30 min was centrifuged a t 100,000 X g for 30 min in a Beckman Airfuge and separated into previously (Grynciewicz et al., 1985; Reinlib et al., 1989). Coverslips of cells were incubated at 20 "C for 60 min in 1 ml of culture medium cytosol (supernatant) and membrane (particulate) fractions. A 0.45containing 10 p~ Fura-2 AM followed bya30-min incubation a t ml aliquot of each fraction was mixed for 10 min on ice with 0.5 ml 37 "C. Before study, the monolayer was washed three times with1 ml of a slurry of packed DE52 (Whatman) equilibratedpreviously with of Ringer's-HCO:j, supplemented with 10 mM HEPES, pH 7.40, and 20 mM HCl and washed repeatedly in 20 mM Tris, pH 7.4, 0.5 mM 10 mM glucose (Buffer A). The coverslip was mounted in a water- EGTA, 5 mM dithiothreitol (Buffer B). The slurry was centrifuged tight Dvorak perfusion chamber, with a total volume of 0.25 ml, and for 4 min in a Microfuge a t approximately 12,000 X g and washed was maintained a t 30 "C. Cells were visualized through a 63X Zeiss three times in Buffer B. Protein kinase C was released from the gel Planeofluor lens (numerical aperture 1.3), and emitted fluorescence by a 10-min incubation in 0.4 ml of Buffer B plus 200 mM NaCl. DE52 was removed by centrifugation and the supernatant immedi(480-520 nm) was collectedviaa DAGE 66 SIT camera (DAGE, Indianapolis, IN). The excitation light was controlled by a MicroVax ately assayed for phosphorylation of histone IIIS as follows. 10 pl of I1 computer (Digital ElectronicsCorp., Maynard, MA) which rotated sample was incubated for 10 mina t 30 "C in a 100-pl volume containbandpass filters of 350 and 380 nm in a motorized wheel in front of ing 40 pg of histone IIIS, 2 mM Tris, p H 7.4, 10 mM magnesium the 75-watt xenonsource. The time requiredfor exposure to a pair of acetate, 0.5 mM EGTA, 0.55 mM CaCI2 (51 p~ free Ca"), 5 p~ [ y excitation filters and data acquisition was approximately 1 s. Data =P]ATP (4.5 pCi) in the absence or presence of 1 pg/ml phosphatiwere determined for 8-frame averaged images a t 10-s intervals aldylserine and 10 p M PDB. The assay was stopped by the addition of though some experiments were performed using l-s, nonaveraged 1 ml of ice-cold 10% trichloroacetate and0.2% NaPPi followed by 10 intervals. Data were acquired and stored as theaverage fluorescence min of boiling. The sample was filtered through a presoaked GF/A intensity within 7.8-pm2areas within eachcell, and calculationswere filter (Whatman) and was washed twice with 5 ml of trichloroacetatemade by a MicroVax I1 computer with 100 megabytes of memory and NaPPi. The filterswere air dried overnight andassayed in a scintillation counter. Protein kinase C activity was considered as phosphaIP-512 imaging boards(Imaging Technology, Inc., Woburn, MA). Approximately eight cells/field were measuredsimultaneously by tidylserine plus PDB-stimulatable activity above that occurring in these methods, using previously defined selection criteria (Reinlibet the presence of Ca'+ alone andwas expressed per mg of protein. al., 1989). Calculations of [Ca"], were made following Grynciewicz et QNB Binding Assay-After exposure to control or PDB-containing al. (1985). The K,, for Fura-2 was confirmed as 224 nM at 30 "C in solutions of 37 "C, particulate fractionsof T,, cell homogenates were the fluorescence microscope. The average R, (F1 350/F1 385) values prepared for carbachol receptor binding, using the cholinergic recepfor 1p~ Fura-2 dye standards in the absence (RmJ or presence (Rmax) tor antagonist QNB, by scraping the washed monolayers into 5 mM of 2 mM Ca2+were 0.73 & 0.01 and 7.50 k 0.47, respectively ( n = 16). HEPES, pH 7.5, 5 mM EDTA, 0.28 mM phenylmethylsulfonyl fluo[Ca'+], changes of less than 10 nM were considered indistinguishable ride, 1 pg/ml phosphoramidon. The suspension was homogenized 10 from system noise and calculated as zero change. All values were timesin a Teflonpestledrill homogenizer. Thesuspension was corrected forbackground and cellularautofluorescence, measured centrifuged a t 8,000 X g for 10 min and the pellet discarded. The separately. Cells containing punctate fluorescence were rare and not membrane fraction was pelleted by centrifugation at 150,000 X g for studied when found. Thus, asshown previously (Reinlib etal., 1989), 60 min and resuspended in50 mM Tris, p H 7.4. Membranes (0.3-0.5 the dye did not appear to accumulate subcellularly. As single cells mg of protein/tube) were incubated in 50 mM Tris, pH7.4, with 0.05were studied and the peak [Ca"], occurred a t variable times after 3 nM [3H]QNB, in a total volume of 0.5 ml, for 2 h at room tempersecretagogue exposure,themeanpeak [Ca"], inthedatatables ature. Reactions were terminated by filtration through GF/B glass differed slightly from the peak responses illustrated in thefigures. filters(Whatman)using a Brandel cell harvester(Brandel Inc., For fluorometry of whole monolayers, T,, cells were seeded onto Gaithersburg, MD),washed three times with5 ml of 50 mM Tris, pH glass coverslips coated with rat tail collagen. The coverslips were 7.4, and radioactivitywas determined in a liquid scintillation counter. glued toplasticsupportswith silicone rubberadhesive(General Nonspecific binding was defined as binding in the presence of excess Electric Co., Waterford, NY) and studied 7-15 days after seeding. atropine (1 pM), and specific binding was determined by subtracting Cells were loaded with Fura-2 by incubation of the cells a t 23 "C for nonspecific from total binding. The KI, and the maximum densityof 60 min in "Namedium" containing (in mM) 130 NaCl, 5 KC1, 2 binding sites (Bmax) for ['HIQNB binding were calculated by nonlinCaCL, 1 MgS04, 1 NaH2P0,, 25 glucose, 20 HEPES, pH 7.30, plus 5 ear least square regression analysis as described (Bevington, 1969). p M Fura-2 AM followed bya 30-minincubation at 37 "C.After Determination of Total Cell Calcium-To evaluate the effect of washing three times with Na medium, the cells on the coverslips were PDB on total cell calcium, plasma emission spectroscopy was carried mounted a t 45 in a glass cuvette. Fluorescence was measured in an out on Te4 monolayers. Monolayers were incubated for 2 h a t 37 "C SLMspectrofluorometer (model S P F 500C, SLM,Urbana, IL) in the presence or absenceof 0.4 p M PDB andwashed three times in equipped with a cuvette stirrer and kept at 37 "C. Wavelengths were Buffer A and then once in buffer A that contained no added Ca2+ computer controlled a t excitations 340 k 1 nm and 380 & 1 nm, and emission was monitored a t 505 k 20 nm. Autofluorescence, deterM. E. Cohen, J. Wesolek, J. McCullen, S. Pandol, R. P.Rood, G. mined from a filter seeded with cells but not loaded with dye, was W. G. Sharp, and M.Donowitz, manuscript in preparation.

over a 24-h period. The results demonstrate anuncoupling of phospholipase C from receptor regulation after PDB incubation.

Phospholipase C Uncoupling in TB4Cells

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(Muscholl, 1970). The cells were then scraped into 2 ml of ice-cold

video camera system. After the addition of 100 p~ carbachol tocontrol cells, [Ca2+Iirosequickly to apeak andthen sion was centrifuged at 100,000 X g for 5 min, and the supernatant declined slowly over thenext few minutes (Fig. l A ) , as was frozen in liquid N,until the Ca'+ determination. In parallel,cells were treated identically except that 0.5 mg/ml ["HJpolyethyleneglycol reported previously (Reinlib et al., 1989). All the T,, cells studied under control conditions in Ringer's-HC03, 10 mM (10 pCi/ml; molecular weight 900) was included in the incubation solution for 1 h to assess the volume of, and correct for Cay+ in, the glucose, 10 mM HEPES, pH 7.4, responded in this manner extracellular space. Data are expressed as mg of Ca"+/mgof cell (Table I). The effects of incubation with PDB on basal protein. [Ca2+]iin TSqcells and on the elevation in [Ca"Ii caused by Determination of Inositol Phosphate Levels and Phospholipase C carbachol were studied at intervals up to 24 h (Fig. 1B).One Activity-Inositol phosphates were assayed in cultures incubated in 1.5 ml of inositol-free medium (Raben et al.,1987) supplemented with min of phorbol ester exposure had no effect on basal [Ca'+Ji 5 pCi/ml myo-[:'H]inositol for 50-60 h a t 37 "C. In half of the cultures, (data not shown) or on the changes caused by carbachol in 0.4 p~ PDB was included inthe mediumfor thefinal 2h of terms of peak [Ca2+Ii rises, plateau level, and duration of incubation. LiCl was also present in the wash and carbachol-contain-[Ca2+];elevation (Fig. lB), as earlier suggested (Beuerlein et ing solutions. The monolayers were washed once in Ringer's-HC03 al., 1987). In the absence or presence of PDB (0.4 p M ) for 1 containing 10 mM glucose, 10 mM Lic1, and 10 mM HEPES, pH 7.4. min, 100% of the monitoredcells responded to carbachol with The solution was drawn off, and monolayerswere incubated a t 30 "C arisein [Ca"];. Prolonged PDB exposuredecreased the in the same buffer in eitherthe absence or presence of 100 p~ carbachol. After 30 min the solutionwas withdrawn and the reaction percentage of cells which responded to carbachol and also decreased the magnitude of the response in those cells which stopped by theaddition of 2 ml of acidified methanol(MeOH/ concentrated HC1,50:0.3, v/v). InsP, Imp2, and InsP,' were separated did increase [Ca2+Ji (TableI and Fig. 1B).After 1 h of PDB and assayed as described (Downes and Mitchell, 1981; Raben et al., treatment, a rise in [Ca'+], occurred in 80% of cells, and the 1987) by anion-exchange column chromatography on Bio-RadAG 1- magnitude of the increase in responding cells was only 60% X8 resin (formate form). The 0.1 M:0.2 M (formate/ammonium formate) eluate was defined as InsP1; 0.1 ~ : 0 . 4M as Imp,; 0.1M:l M as of the response in control cells (Table I). Two h of PDB InsP,,; and 0.1 ~ : 1 . M 5 as InsP4. Two-ml fractionswere collected, 10 exposure decreased to 43% the cells that elevated [Ca"Ii in ml of scintillation mixture added, and radioactivity determined in a response to carbachol with respondingcells elevating [Ca'+], liquid scintillation counter. only to 37% of the control response (Table I). The effect of Total free Ins(1,4,5)Pa a t specified times after carbachol addition PDB on the carbachol-induced [Ca"]i elevation continued for in nonradiolabeled T H 4 monolayers was also measured witha receptor 24 h, at which time only 40% of cellselevated [Ca2+Iiin binding assay. This assay was also used to measure phospholipase C response to carbachol, with the magnitudeof the increase in activity incrude membranes prepared fromTa monolayers. TH, crude membranes were prepared by washing two T75 flasksof cells with 1 responding cells being only 44% of the control response. In Fig. l B , time courses are shown for the carbachol-induced mM EDTA, 1 mM P-mercaptoethanol, 20 mM HEPES, pH 7.50,lpg/ ml phosphoramidone, 40 pg/ml phenylmethylsulfonyl fluoride. The increase in [Ca2+Ii averaged for all cells and for responding cells were scraped into 10 ml of the same buffer, homogenized three cells alone. Basal [Ca2+Ii levels did notvary significantly from times for 10 s in an Ultra-turrax tissue disrupter, and centrifuged for the control value of 86 f 5 nM when cells were pretreated 45 min at 100,000 X g. The pelletwasresuspended in a minimal with PDB for 2 h and for up to 24 h. With 2 h of PDB volume of the homogenizing buffer and used for phospholipase C treatment, basal [Ca2+Iiwas 81 k 6 nM (Table I), and basal generation assays. For the assay, rat cerebellarmicrosomalmem0 . 3 mM LaCI:', 5 M HCl and drill homogenized 10 times. The suspen-

branes, which containthe specific Ins(1,4,5)P2 receptor (5), were prepared from adult, male Sprague-Dawleyrats killed by decapitation. The cerebellum was homogenized with an Ultra-turrax tissue disrupter (Tekmar Co., Cincinnati, OH) at top setting, 10 s, in 30 volumes of ice-cold 50 mM Tris, pH 7.7, 1 mM EDTA, 1 mM P-mercaptoethano], pelleted by centrifugation at 20,000 x g, and resuspended in 30 volumes of the same buffer. The latter stepwas repeated three times, and the final pellet was resuspended in the samebuffer a t 1.2 mg of protein/ml. TX4monolayers were preincubatedintheabsence or presence of PDB, washed with 1 ml of Buffer A, and exposed to Buffer A plus 100 p~ carbachol a t 30 "C for specified time intervals. Then thesolution was drawn off and the reaction stopped by addition of 0.5 ml of ice-cold 1 M trichloroacetate. In the case of T x crude ~ membranes,thegeneration of was stopped by theaddition of a n equal volume of 2 N trichloroacetate. The scraped cells or membranepellets were homogenized threetimesand centrifuged a t 100,000 X g for 10 min, and the supernatants were extracted four times with water-saturatedether.Theaqueous layers were used undiluted to quantify the amount of Ins(1,4,5)Pn present as follows. 100 p1 of sample was incubated in a total volume of 500 p1 of 50 mM Tris, pH 9.0, 1 mM EDTA, 1 mM P-mercaptoethanol with 1 nM ['HI Ins(1,4,5)P:,(5 pCi) and 60pg of rat cerebellar membranes. Displacement of [:'H]In~(1,4,5)P:~ from the rat brain membrane was determined after 10 min by centrifuging the assay mixtureat topspeed in a Beckman Microfuge and determining the radioactivity in thepellet. Computation of cytosolic Ins(1,4,5)P3 was made by comparison with a standard curve after correctingfor nonspecific binding not removed by 2 p M cold In~(l,4,5)P:~. RESULTS

Effect of PDB Preincubation on the [Ca"], Response to Carbachol and Histamine-Carbachol, a muscarinic secretagogue thatoperatesthroughcalcium-dependentpathways, was studied for its effects on [Ca"], in single TS4cells. Single Tx, cells were monitored usingafluorescencemicroscope

- 2 o ~ " " " " " ' " " " " " " " ' ~ -20 0 50 la, 150

200

2%

Time ( s e c )

FIG. 1. Time course of

M carbachol-induced A[Ca2+], in cells and effects of PDB preincubation. Cells grown on glass coverslips were loaded with Fura-2 and studied individually in the fluorescence microscope as under "Experimental Procedures." Up to eight cells/coverslip were analyzed simultaneously by measuring fluorescence a t fixed locations in individual cells at 10-s intervals. Data in panel A are means k S.E., expressed as A[Ca'+]; where the [Ca'+], immediately before carbachol addition was set to 0. In panel B only the mean values are plotted. Panel A , untreated cells, all responding to carbachol( n = 75 cells). Panel B , cells pretreated with0.4 p~ PDB for A, 1 min ( n = 6 cells, all responding); 0 and 0, 2 h ( n = 30; 43% responding); or and 0, 24 h ( n= 47,36% responding). Open symbols ( 0 , O ) represent the average of all cells; solid symbols (0,m) are the average of responding cells only.

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TABLEI [Ca'+l, changes induced by carbachol and histamine incontrol and 0.4 P M PDB-pretreated Tx4cells Maximal A[Ca'+], represents the maximal increase from the basal [Ca'+], found for each cell immediately before the addition of carbachol or histamine and includes only cells in which [Ca"]i > 10 nM. TRIcells were either not treated or pretreated with 0.4 PM PDB for 1-2 h. Four to eight single cells were monitored simultaneously for [Ca"], from a t least two cultures. R, was determined at the endof each experiment by perfusing cells with 10 PM ionomycin, 10 1 M CCCP, 4 mM CaC12 for 10 min with all cells elevating [Ca"], to a level that saturated the intracellular Fura-2, indicating that the dye was unaffected by the PDB treatment. Student's unpaired t tests were performed to determine the significance between "no treatment" and "1 h" and "2 h of PDB" values. Values in parentheses are the numberof cells studied. Data are meanf S.E. Addition

No treatment

Basal [Ca"], (nM) Carbachol (100 p ~ ) Maximal A[Ca"]i (nM) % Cells responding R(,:ionomycin, CCCP, 4 mM ca2+ Histamine (100 p M ) Maximal A[Ca"],47(nM) % Cells responding R,>:ionomvcin. CCCP. 4 mM Ca" 'I

1 h of PDB

P

86 f 5 (96) 81 f 5 (75) 100% 6.0 f 0.7 (75)

49 f 5 (5) 80%

f 4 (20) 9519% % 4.5 f 0.5 (21)

36 f 2 (8) 100%