.Vi R-033q AD- A279 920 NTATION PAGE 4 P10e7a0O0 4.01U I iE N UE~W.9 'a4r .014 1vko,.w l ow(cid:127),' w@,~~e%" P-w A* ov '"t tf '' V.-AmloL ' r.0h0'6. tw M (cid:127) Oe'cq ~1-Os" I4 ".4 41i ww(". IRT OATE_- ' 3. REPOEr TYPE ANO DAY[S COVERED 4. TITLE AND SUIY171. S. FUNDING NUMBERS Corticotropin-releasing factor increased [Ca 2+], via rec re utor-mediated Ca 2 + channels in human epidermoid A-431 6. AUTHOR(S) WID(q Juliann G. Kiang 7. PERFORMING ORGANIZATION NAM.E(S) AND ADOISES 3 EFRMN RANZTO Walter Reeed Army Institute of Research RIPORT NUMBER WashingtontoDC 20307-5100 9. SPONSORING/ MONITORING AGCNCv NAME(S) AND AOORISS(ES) 10. SPONSORING!MONITORING AGENCY REPCRT NUMBER US Army ediTcal Research & Development Go mma nd Ft. Detrick, Frederick, MD) 21702-6012 "APPROVED FOR. PUBLIC RELEASE: DISTRIBUTION -INLIHITED . %SsTR(AMC,T0 0,,U,' 200 wos,. . Cowtcoaisapim-wsWtil fator (CR1) hes bern *awn to 1180=1110 V@Mdff kehP maf i*"d *15, 3mim uetma mius, wad brWa. CbkCum is t"tmvt poy = b*a w job in amy do. p (cid:127)j3cSW 5Upowso to CRF, but thue bet -d bean linkc duhau:t n o how caldum ig mimu inm pms bY whidi CR? Vaewm da=md d0m IU Of dds In* ww to cumasgub chage(cid:127) in etotck free akIum cwcmmio ([Ca÷0) In hum= epd mo d A431 (cid:127)o .awd Wo hu(cid:127)/mt-CMF aNd to vhlatipts ft mechanlsin by w e o oem h t.e" * [C3 Is smar ots at 371C u o6 * 4 &M (na- 32 Whn s.acob waes IsuIdw ith CX, Ce(cid:127)(cid:127)230C bmely. Mh(cid:127)emm derp tad Ina CUP gcoemabdcam wIfth 2 3jeffiajn aaamsefuublel iiO Ofp1L TW5h'uhsim nI s C&2+ depanded am mftemI 63* bot M Na+, Mj2+, or K+. L&34 (10 #&M) and C01- (10 ^M1i nhdbiU(cid:127) tle CIFlUd- d KWa'i=900ms WhsRM WUupsmll MWd 591d401 resWe at apecmoradoi up to I =M dd not a-Hdict CRP<941), a suhetik CRIF senpwo amtuged. end pstW& hula Machad the ems m IC92-L wdced by CIU?.Ww"t neft " 120, Of cfmiteed CO'bi us Witbeyd fmparmd C e anek(cid:127) couped with ppeamwi* ni temdwve 0 pin",. aAthough 430 pU C? Wmihred = Wimedbht -ams in [CO-1, hsted u&acbhak mmd =Oilm cAbe kwh dd not dump widiam 1 0"a "tlui md e pseas or daboa a exte na Caz*. U-7312 (an jahb of ladW kbipbookae psindulm@mI )-, lksida bomun (In omf thetd NsiCd a11i.hamesO)alto(cid:127) did am Moak the macs Ims C(cid:127)n Tlabcyd C R?e. RFI ab cumid [Cal He coels rate wi1*TMB-6oir Wsyanoim, kibbitaso atgscsflmiCa" soeblibsO. Th saWSIRugtt du CRU8u1m1110a a Ca3+ fi&3 msgh CR? t5bg=Sk.OPUd Cs3* thametL 41,1S1JW. 1ECT TERMS IS. NUMBER OF PAGES Ca 2+; Corticotroin-releasing factor, Ca2 + channel; Pertussis • (cid:127)p (cid:127)UOaum toxin; Epithelium 117. SECURITY MA'S;ICATION It. SICUR!TY C.ASSIFICATION 19. SECURITY CLASSIFICA iON 20. UMITATICN C.FAfSS"RA-C Of REPORT OF THIS PAN| Of AS13TRAC'T P65N 7!(cid:127)-04' .80-5500 Staes#da (cid:127)orm 29" (Rev -Z-49)1 I lIIII-'0".; Rpdned from Journal European of Pharmacology European Journal of Pharmacology Molecular Pharmacology Section 267 (1994) 135-142 Corticotropin-releasing factor increases [Ca2"]i via receptor-mediated Ca2+ channels in human epidermoid A-431 cells Juliann G. Kiang Department of ClinicalP hysiology, Division of Medicine, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA (Received 28 April 1993; revised MS received 13 September 1993; accepted 16 November 1993) Accesion For NTIS CRA&I DTIC TAB Unannounced 0 Justification .. ...................... By .................................... .... 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TRIGGLE, Buffalo European Journal of Pharmacology molecutar pharmacology ELSEVIER Molecular Pharmacology Section 267 (1994) 135-142 Corticotropin-releasing factor increases [Ca2 via receptor-mediated 1]i Ca2 channels in human epidermoid A-431 cells 1 Juliann G. Kiang Department of ClinicalP hysiology, Dicision of Medicine, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA (Received 28 April 1993; revised MS received 13 September 1993; accepted 16 November 1993) Abstract Corticotropin-releasing factor (CRF) has been shown to attenuate vascular leakage in injured skin, mucous membrane, muscle, and brain. Calcium is thought to play an important role in many of the physiological responses to CRF, but there has been little characterization of how calcium is involved in process by which CRF protects damaged tissues. The goal of this study was to characterize changes in cytosolic free calcium concentrations ([Ca2+]i) in human epidermoid A-431 cells exposed to human/ rat-CRF and to investigate the mechanisms by which these changes occur. The resting [Ca2+ ]i in normal cells at 37*C was 66 ± 4 nM (n = 32). When cells were treated with CRF, [Ca24]i increased immediately. The increase depended on CRF concentration, with a median effective concentration of 11 pM. This increase in [Ca22 ], depended on external Cal' but not Na +, Mg2+, or K4.La3" (10 AM) and Co2' (10 AM) inhibited the CRF-induced [Ca2+]i increase, whereas verapamil and nifedipine tested at concentrations up to 1 mM did not. a-Helical CRF-(9-41), a synthetic CRF receptor antagonist, and pertussis toxin blocked the increase in [Ca2+]i induced by CRF, which suggests that the entry of extracellular Ca2+ is mediated by receptor-operated Ca2+ channels coupled with pertussis toxin-sensitive G proteins. Although 420 pM CRF stimulated an immediate increase in [Ca2+]i, inositol trisphosphate and cellular cAMP levels did not change within 1 min either in the presence or absence of external Ca2+. U-73122 (an inhibitor of inositol trisphosphate production), amiloride and benzamil (inhibitors of the Na +/Ca2 + exchanger) also did not block the increase in [Ca2+4]i induced by CRF. CRF also increased [Ca2 + ]i in cells treated with TMB-8 or ryanodine, inhibitors of intracellular Ca2+ mobilization. The results suggest that CRF stimulates a Ca2+ influx through CRF receptor-operated Ca2+ channels. Key words: Ca2 +; Corticotropin-releasing factor; Ca2+ channel; Pertussis toxin; Epithelium 1. Introduction present in the final stages of human pregnancy at a level about 20 times the normal level (Linton et al., The endocrine function of corticotropin-releasing 1990a,b; Wolfe et al., 1988). factor (CRF) to stimulate adrenocorticotropin (ACTH) The exposure of tissues to exogenous CRF produces release is well-characterized, and its distribution a variety of effects, including a decreased heart rate throughout the body has been demonstrated. Using and blood pressure (Kiang and Wei, 1986) and a reduc- immunohistological techniques, CRF-like substances tion in the detrimental effects of several kinds of have been detected in many systemic organs including injuries (Kiang and Wei, 1987; Wei and Kiang, 1987; liver, spleen, pancreas, stomach, duodenum, and Wei et al., 1988; Serda and Wei, 1991, 1992; Wei and adrenal medulla (Petrusz et al., 1984). CRF is also Gao, 1991). present in hypothalamus and other parts of brain The mechanism by which CRF reduces the edema (Merchenthaler, 1984). The CRF levels in hypohyseal and protein extravasation characteristic of such injuries portal blood and systemic plasma in human are 400 is not known. It is possible that changes in cytosolic and 75 pg/ml, respectively (Plotsky, 1985). CRF is free calcium ([Ca24]i) play an essential role in CRF protection because other CRF actions have been shown to involve Ca2 '. For example, in human ACTH-secret- * Tel.: (202) 576-3098; Fax: (202) 5760703. ing pituitary adenoma cells and normal rat small ovoid 0922-4106/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0922-4106(93)E0189-Q 136 I G. Kiang I European Journalo f Pharmacology - Molecular PharmacologyS ection 267(1994) 135-142 corticotrophs, Guerineau et al. (1991) found that 100 width 4 nm) with a PTI DeltaScan spectrofluorometer nM CRF increased [Ca2+], by opening both L- and (Photon Technology International, South Brunswick, T-type voltage-gated Ca2+ channels. In corticotroph- NJ). Autofluorescence from cells not loaded with the derived AtT-20 cells, the CRF-induced increase in dye was in the range of 3000-4000 photons/s and was c-fos mRNA is reduced by blocking Ca2+ entry or by subtracted from the fura-2 signal. Fura-2 leaked out of treatment with calmodulin inhibitors (Boutillier et al., A-431 cells at a rate of 0.38 ± 0.01%/rmin (n = 3) at 37 1991). Furthermore, ACTH release stimulated by CRF 'C. To minimize any contribution to the fluorescence is reduced in the absence of external Ca2", by deple- signal resulting from dye in the medium, cells were tion of intracellular Ca2 + stores, or by treatment with washed thoroughly in Hanks' solution before they were calmodulin inhibitors (Won and Orth, 1990). transferred to a cuvette to measure [Ca 2 ]. [Ca2+]i was It has been shown that the epideimal necrosis pro- calculated according to the method of Grynkiewicz et duced by heat injury in rat paw skin (Wei et al., 1987) al. (1985). and human epidermoid A-431 cells (Kiang et al., 1988), a non-endocrine cell line, is reduced by pretreatment lnositoi trisphosphates measurements with CRF. Because Ca2' homeostasis in A-431 cells Inlonosistiotol lt ristrpihspohsophspahteaste s (InsPm)e awseurree mmeenatsured as 3 has been extensively described (Kiang, 1991; Kiang et described by Berridge (1983) and my laboratory (Kiang al., 1992). we used these cells to characterize the effect and McClain, 1993). In brief, cells were grown in 6-well of CRF on [Ca2+]i and investigate the underlying plates and incubated with myo-[3Hlinositol (2 ACi/mi, mechanisms of action. The results of our experiments 0.22 nmol/ml, Dupont/NEN, Boston, MA) for 24 h. show that CRF treatment caused an increase in [Ca-2]i Cells were washed twice with Na Hanks' fsolution that was antagorneiczeepdo rb y a-ahtaegliociaslt .tCheRnF -(9-41), a CRF ten ii ncubeadte dw withit h CcReF ffoorr 55 ss,' 1, 55,, or 110m m in. Tthhee receptor antagonist. reaction was stopped by adding 3 ml ice-cold 4.5% HCIO : Na Hanks' solution (2:1, v/v). Each plate 4 2. Materials and methods was placed on ice for 30 min, and the cells were removed by scraping. Cells were pelleted by centrifuga- 2.1. Cell culture tion (750 x g) and the supernatants were collected. The pH of the supernatants was adjusted to pH 8.0 e(A meri- with KOH before storing at -70'C. An aliquot (100 Human eypieCulureCoidcarionRom -cellsM, D) of supernatant, which contained all of the [3H]in- caown Type Cultuscovere Clectio(n9 , kvim, MD)y w , ositol and [3H]polyphosphoinositol metabolites, was growno nP glass cover slips (9 35 mm, Clay Adams, counted to determine total radioactivity in the cells. Lincoln Park, NJ) incubated at 37°C in a 5% CO2 [3H]InsP was eluted by 100 mM formic acid in 1.0 M 3 atmosphere. The tissue culture medium was Dulbecco's ammonium formate (38 ml) from a Dowex AG 1- X 8 modified Eagle medium supplemented with 0.03% resin column. Radioactivity was determined by mixing glutamine, 4.5 g/1l glucose, 25 mM Hepes, 10% fetal 1 ml of the eluent with 10 ml of aquasol scintillation bovine serum, 50 Ag/mI penicillin, and 50 U/mI cocktail and counted with a scintillation counter. The streptomycin (Gibco Laboratories, Grand Island, NY). amount of InsP was then expressed as a percentage 3 Cells were fed every 3-4 days. Cells from passage of total counts per minute in each well. 28-45 were used for experiments. 2.2. Measurements cAMP measurements Confluent monolayers of cells (2 x 10'/35 mm cul- Intracellular Ca2 measurements ture dish) were washed twice with PBS before incubat- + Confluent monolayers of cells were loaded with 5 ing with PBS for 1 h at 37°C. Incubation was then 1AM fura-2AM plus 0.2% pluronic F-127 (to make cells continued with 3-isobutyl-i-methylxanthine (IBMX, fi- more permeable to the probe) at 37*C for 60 min. Cells nal concentration 1 mM) for 30 min at 37°C, after were washed with Na+ Hanks' solution (in mM: 145 which the medium was removed. Na+ Hanks' solution NaCI, 4.6 KCI, 1.2 MgCI,. 1.6 CaCI,, and 10 HEPES, containing CRF was added into the culture dish for 5 s, pH 7.4 at 24°C) before fluorescence measurements. 1, 5 or 10 min. The medium was discarded, and I ml of The method to determine [CaWWI, has been described 0.1 M HCI was added to each culture dish to stop the previously (Grynkicwicz et al., 1985; Kiang, 1991). reaction by killing the cells. The culture dishes were Briefly. the confluent monolayer of cells was placed in maintained at 4VC for 1-2 h before transferring the a thermostatically controlled cuvette that was kept at a suspensions to test tubes. The samples were boiled for constant temperature of 37°C. The fluorescence signal 10 min and the precipitates removed by centrifugation was measured with the emission wavelength set at 510 at 2000 X g for 10 min. The supernatants were stored nm and dual excitation at 340 nm and 380 nm (slit at -20°C until the cAMP measurement. J.G . Kiang / EuropeanJ ournal of Pharmacology - Molecular Pharmavology Section 267 (1994) 135-142 137 cAMP was determined by radioimmunoassay using 250 the method of Lin et al. (1985). Briefly, the procedure A was as follows. Samples or standards (5 to 2000 fmol/ tube) in 25 to 100 Al were made up to 200 Al with 50 200 mM acetate buffer, pH 6.2, and acetylated with 5 AlI acetic anhydride: triethylamine (1:2, v/v). After 15 c 150 min at room temperature, [1251]succinyl cAMP antiserum, sufficient to bind 30-60% of the radioactive +4-2 ligand, was added. After 4 h at room temperature, t10o0 rabbit serum carrier and the second antiserum (anti- rabbit Ig from sheep, goat, or burro, Meloy Labs) were 50 added. After standing overnight at 4°C, 2 ml of cold 10 mM acetate buffer, pH 6.2, was added, the tubes were centrifuged, and the radioactivity in the pellets counted. 00 50 100 150 Each sample was measured in duplicate. Time (second) 275 2.3. Statistical analysis 2 BB5 All data are expressed as mean ± S.E.M. Analysis of 225 - variance, Bonferroni's inequality, and Student's t-test L 200 were used for comparison of groups. Curve fitting was 175 determined using the Inplot program (GraphPad, San 150 Diego, CA). 125 2.4. Chemicals 10-04 1 0-'2 I 0-" - 0-' [h/,-CRF] (M) Corticotropin-releasing factor (human/rat) and a- helical CRF-(9-41) were purchased from Peninsula Fig. [. CRF increases [Ca2 ]I. Cells were treated with CRF at helabatorie (B welCmAo)n.pt,O thrch efmoiicnusseld different concentrations (n = 3-4). The fluorometer tracing (A) and Laboratories (Belmont, CA). Other chemicals used in a sigmoid-curved fit (B) are presented. The calculated median effec- this study were fura-2/ AM, pluronic acid F-127, 8-(di- tive concentration was II pM. ethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8, Molecular Probes, Eugene, OR), benzamil (Research Biochemicals, Natick, MA), amiloride, verapatnil HCI, not observed (Figs. 2 and 3). When increasing concen- LaC13, CoCI2, pertussis toxin, suramin, (+)-N-methyl- trations of Ca2 + were added back to the buffer, [Ca I] glucamine, ryanodine, IBMX (Sigma Chemicals, St. increased proportionally, suggesting that the CRF-in- Louis, MO). 1-{6-[(17P3-3-methoxyestra-1,3,5(10)-trien- duced increase in [Ca2+]i is a result of Ca2+ entry from 17-yl)amino]hexyl}-I H-tyrrole-2,5-dione (U-73122) was external sources. When cells were incubated in a buffer provided by Upjohn Co.(Kalamazoo, MI). Naloxone without Na+ (replaced with equimolar (+)-N-methyl- was a gift from Dr. Brian Cox. glucamine) or Mg2+, the resting [Ca2+]i increased sig- nificantly. Removal of external Na+ activates the 3. Results 3.1. Effect of CRF on [Ca2+li 250 The resting [Ca2+]i in adherent cells at 37°C in 190. normal Na+ Hanks' solution was 66 ± 4 nM (n = 32). 2 When cells were treated with CRF, there was an 0 immediate increase (within 10 s) in [Ca2+]i dependent on the concentration of CRF (Fig. IA). The median effective concentration (EC) of CRF was 11 pM. A.0 _0._ concentration of 42 pM induced a maximal increase in [Ca2+]o (mM) [Ca2"]i (237 ± 20% control, n = 10) (Fig. IB). The in- cdFig. 2. Effect of external Ca2 * ([Ca"(cid:127) ],,) on the CRF-induced corfe aesxet eirnndaul ceCda b2y+ .C iRn FC waa2s+ -dferepee nbduefnfte ro nc othneta pinriensge nc1e0 iHnacnrekass' es oilnu ti[oCna 2c+o n]it.a iCnienllgs dwifefreer enetx p[Cosae2d+ I t o( n 4=2 3p-M6) . C* RPF < i0n. 05N av÷s. mM EGTA, the CRF-induced increase in [Ca2+]i was 0 external Ca2,. 138 J.G . K~ang / European Journalo f Pharmacology - Molecular PharmacologyS ection 267 (1994) 135-142 ment with CRF in the presence of either verapamil or 160 0 VBjCE nifedipine stimulated a significant additional increase 120t CRF in [Ca2+]i (Table 1). Much higher concentrations of S'2 (cid:127) .verapamil and nifedipine (up to 1 mM) also failed to + so "" inhibit the CRF-induced increase in [Ca2 1i (data not 2" ' (cid:127), shown). These results indicate that the Ca2+ entry Si 40 stimulated by CRF does not occur through voltage- gated Ca2 + channels. The data are consistent with the CCoOnit rol -Ca -Na -MI g High K v1i9e9w2 , thMato oAle-4n3a1a r ceeltl s aal.r,e 1n9o8t6 ) exacnitda blteh at( Ktihane g [Ceat 2a+l.],, Fig. 3. Effect of external ions on CRF-induced increase in [Ca2" ]i. increase is not associated with membrane depolariza- The control buffer contained in mM 145 NaCI, 4.5 KCI, 1.3 MgCI,. tion. 1.6 CaCI,. and 5 HEPES. The Ca-'-free buffer contained 10 mM Because Co2+ and La3+ can function not only to EGTA without Ca2*. The Na'-free buffer contained 145 mM N- block Ca' channels but also to block Na+/Ca2+ methylglucamine to substitute Na'. The Mg2 +-free buffer contained no Mg2*. The high K÷ buffer contained 25 mM K+. Cells were exchange (Trosper and Philipson, 1983; Kaczorowski et placed in each buffer for 5 min before treatment with CRF (42 pM. al., 1989), it was necessary also to assess the effect of = 3-5). * P < 0.05 vs. vehicle control, ** P < 0.05 vs. respective CRF on the Na+/Ca2" exchanger. This antiporter vehicle, two-way ANOVA. and Bonferroni's inequality. system located at the cell membrane translocates Ca , coupled to the movement of Na+ in the opposite direction. The direction of ion transport across the Na+/Ca2' exchanger (Kiang et al., 1992), leading to plasma membrane depends upon the prevailing ionic an increase in the resting [Ca2 ] . The reason for the gradients established by the Na+/K÷-ATPase, the 1 increase resulting from the removal of Mg'- is not Na+/H+ antiporter, and Na÷ channels. Cells were clear. However, CRF still induced the same magnitude treated with 1 mM amiloride or 100 AM benzamil, of [Ca2 ]i increase in cells in absence of either cations inhibitors of the Na+/Ca2+ exchanger. Table 2 shows (Fig. 3). that amiloride and benzamil both increased the resting It has been reported that membrane depolarization [Ca2,1i, indicating that a Na÷/Ca2' exchanger is pre- can increase [Ca2"I] in some cells (Hagiwara, 1983). I sent in these cells. However, CRF treatment still in- therefore sought to determine if the increase in [Ca2+,] creased [Ca2+]i in amiloride- or benzamil-treated cells, is caused by a CRF-induced depolarization of the excluding the possibility that the Na+/Ca2+ exchange A-431 cell membrane. Cells were first bathed in a system is inhibited by CRF. buffer containing 25 mM K' (normal [K+] = 4.5 mM), conditions that will depolarize the membrane of sensi- 3.3. Effect of a-helical CRF-(9-41) tive cells. However, this treatment did not change the resting [Ca2÷],, and CRF induced the same magnitude The increase in [Ca2+], by CRF was inhibited by of [Ca2+]i increase as that measured in cells incubated a-helical CRF-(9-41), a CRF receptor antagonist with normal buffers (Fig. 3). These results indicate that (Rivier et al., 1984). The degree of inhibition depended the increase in [Ca2+]i stimulated by CRF is not a on the concentration of the inhibitor (Fig. 4A), with result of a membrane depolarization, the median inhibitory concentration (IC ) occurring at 50 3.2. Effect of Ca2 + channel blockers Table I We sought to determine the role of extracellular Inorganic Ca2z channel blockers inhibited the CRF-induced in- Ca2÷ in the increase in [Ca2+]i stimulated by CRF. crease in [Ca2 I, The entry of Ca2+ into cells can be blocked by Co2" or [Ca2* l, (tiM) La 3÷ (inorganic Ca2+ channel blockers) in the external Control CRF medium (Hagiwara, 1983). Table I shows that treat- 57± 9 150± 20 ment with either Co2+ or La 3 slightly changed the La-" 63± 4 63± 5 resting [Ca2+]1, but CRF did not increase [Ca2+]i in Co2" 21± 6a 21± 71 the presence of either blocker. These results reinforce Verapamil 82± 27 a 215 ± 84 h the concept that the increase in [Ca2 +]i induced by Nifedipine 99± 161 209±31 b CRF is due to a Ca2+ influx. Cells were treated with Ca2+ channel blockers 10 ;LM for I min Different results were obtained with the organic before 42 pM CRF (n = 5). Ca2÷ channel blockers, verapamil (10 M(cid:127)M) and ae Ps < 0.05 vs. controls without treatment with Ca2' channel blockers. nifedipine (10 AM). Although these agents by them- hP < 0.05 vs. respective control, two-way ANOVA, and Bonferroni's selves induced a change in the resting [Ca2+]i, treat- inequality. J.G. Kiang / European Journalo f Pharmacology - Molecular Pharmacology Sectuon 267(1994) 135-142 139 Table 2 Amiloride, benzamil, U-73122, TMB-8, and ryanodine did not inhibit 250" the CRF-induced increase in (Ca 2 + 20 01 i : Concentration 2 1lO( nCMa) 2 Control CRF . 150- 9 150 ±20a - -57± Amiloride ImM 115±10a 226± 9b N, 100 Benzamil 100 M 82± 5a 167± 14b U-73122 5MuM 173± 12 a ± b 50 264 12 TMB-8 100MpM 114±18 a 283±55 b RCyelalns owdienree treate1d0 0 wAiMth amiloride, be3n5z±am 7il , U-73112024, ±T-1M6Bb- 8, or 01Control ((cid:127)a~ PTX SA ~N ryanodine I min prior to CRF (42 pM) (n - 4-5). Fig. 5. Effect of CRF antagonist, pertussis toxin, suramin, and "P < 0.05 vs. control without any treatment, naloxone on the CRF-induced increase in [Ca2÷ ]i. Cells were treated b P < 0.05 vs. respective control, two-way ANOVA, and Bonferroni's with CRF antagonist (CRFa, 100 nM). suramin (SA, 100 p-g/ml), inequality. naloxone (NX, 50 M.M) for 1 min, or pertussis toxin (PTX. 30 ng/ml) for 24 h before challenging with 42 pM CRF (n = 3-5). * P < 0.05 vs. vehicle control, * * P < 0.05 vs. respective vehicle, two-way ANOVA. 33 nM and complete inhibition at 1 A.M (Fig. 4B). and Bonferroni's inequality. These results suggest that the CRF-induced increase in [Ca2+1i is mediated by receptor-operated Ca2+ channels. Iaeln [ 2+ iopen Ca2+ channels, treatment with pertussis toxin to twould also block the increase, because pertussis toxin mediated by a receptor that coupled to G proteins to has been shown to block Ca2+ channels that are stimulated by receptor-linked Gk/Go proteins (Gilman, 250 1987; Lyengar and Birnbaumer, 1987; Spiegel et al., A 1988). On the other hand, if increases in [Ca 2+]i induced by CRF were directly mediated by a receptor 200 coupled to Ca2+ channels, then treatment with pertus- Ssis toxin should not inhibit the increase. Cells incu- i t5 bated with 30 ng/ml pertussis toxin for 24 h demon- _ strated an increase in [Ca2+], of only 38 ±8%( n = 5, _ 00 : ;(cid:127)(cid:127) (cid:127)r(cid:127) l5 50n ow;" .l PC 0.05, Student's indicated concentrations 1 min prior to challenge with 420 pM CRF test). The results suggest that the CRF-induced in- (n - 5). The fluorometer tracings (A) and the sigmoid-curved fit (B) -rsi are presented. The calculated median inhibitory concentration was crease in [Ca2+ ], is specifically mediated by CRF re- 33 nM. ceptors. 140 J.G, Kiang / European Journalo f Pharmacology - Molecular PharmacologyS ection 267 (1994) 135-142 3.4. Effect of CRF on cAMP cellular Ca2* because (1) the increase did not occur if Ca2+ was omitted from the buffer, and (2) inorganic CRF at 42 pM for 10 min induced a maximal Ca2, channel blockers inhibited the increase. increase in [Ca2+],. However, this concentration did Voltage-gated Ca2+ channels are not involved because not increase cellular cAMP content either in the pres- organic Ca2+ channel blockers such as verapamil and ence or absence of external Ca2+ in the first 10 sec nifedipine failed to block the increase. This is in agree- when the increase in [Ca2+]i was observed. This would ment with the results of Moolenaar et al.(1986) and exclude the involvement of second messenger-operated this laboratory (Kiang, 1991; Kiang et al., 1992). Ca24 channels activated by cAMP. It is important to Changes in resting [Ca2+]i in cells treated with Ca2+ note that a 10 min incubation with CRF increased channel blockers are probably due to their ability to cellular cAMP from 1.0 ± 0.2 pmole/10' cells to 1.4 affect H+-sensitive Ca2` channels (Kiang, 1991). ±0.1 pmole/10' cells (n = 3, P < 0.05, Student's I- The increase in [Ca2+]i induced by CRF was inhib- test). Any future study of the prolonged effect of CRF ited by the CRF receptor antagonist, a-helical CRF- should therefore consider the involvement of cAMP in (9-41). The IC, required to block the activity of CRF 0 any observed increases in [Ca2+]i, because a 10 min (420 pM) was 33 nM. The inhibition was specific be- treatment with 1 mM 8-bromo-cAMP in A-431 cells is cause neither suramin nor naloxone inhibited the capable of increasing [Ca2+]i by 35 ± 5% (Kiang and CRF-induced increase in [Ca2+]i and because this CRF McClain, 1993). receptor antagonist failed to inhibit the increase in [Ca2+]i induced by ATP. Treatment with pertussis 3.5. Effect of CRF on intracellularC a2-+ stores toxin attenuated the [Ca2+I, response to CRF. These results taken together suggest that the Ca2+ channels Three intracellular Ca2 pools have been indenti- involved with CRF activity are CRF receptor-operated fied in A-431 cells: InsP -, monensin-, and ionomycin- and coupled to a pertussis toxin-sensitive G protein. 3 sensitive pools (Kiang and Smallridge, unpublished Because Ca2+ influx can be mediated by second data). My data indicate that the InsP3-sensitive Ca2, messenger-operated Ca2+ channels, increases in [Ca2+]i pool is not involved in the CRF response, because may result from CRF-stimulated increases in cellular CRF did not increase InsP within the same time cAMP. Previously this laboratory showed that an ex- 3 frame (10 s) as that in which the increase in [Ca2+]i ogenous application of 1 mM 8-bromo-cAMP did not was observed. An increase in lnsP was observed only immediately change resting [Ca2+]i in A-431 cells 3 after a 10 min treatment with CRF (from 0.37 ± 0.06% (Kiang et al., 1991), although prolonged incubation total cpm to 0.57 ± 0.07% total cpm, n = 3 for both with 8-bromo-cAMP led to [Ca21]i increases (Rinaldi groups, P < 0.05, Student's t-test). Furthermore, an et al., 1981; Kiang and McClain, 1993). In the present inhibitor of lnsP production, U-73122, did not inhibit study, cAMP did not increase during the same period 3 the increase in [Ca2'÷] induced by CRF (Table 2). Two that [Ca2+]i increased after CRF treatment. That is, inhibitors of intracellular Ca2+ mobilization, TMB-8 (a the [Ca2+]i increase was immediate, whereas cAMP general block for Ca2+ stores, Chiou and Malagodi, failed to increase until 10 mit., suggesting that second- 1975; Mix et al., 1984; McCoy et al., 1988) or ryanodine messenger operated Ca2+ channels are not stimulated (a blocker for non-lnsP -sensitive Ca2+ stores, seef re- by CRF. 3 view Tsien, 1990) were tested to determine whether Intracellular Ca2+ pools also do not appear to be other intracellular pools were associated with the in- involved in the CRF-induced increase in [Ca2 ]Ij.T his crease in [Ca2+]i. Treatment with TMB-8 (100 MM) or view is supported by two observations. First, InsP did 3 ryanodine (100 MLM) should not interfere with an in- not increase during the same period that [Ca2"], increase in [Ca2'+] induced by CRF if the increase were creased after CRF treatment. Second, U-73122 (an derived primarily from external sources. That was in- inhibitor for InsP production), TMB-8 or ryanodine 3 deed the case. Neither TMB-8 nor ryanodine blocked (inhibitors of intracellular Ca2+ mobilization) failed to the increase, which indicates that intracellular Ca2+ inhibit the increase. stores are not involved in the CRF-induced increase in Polyvalent cations have been reported to block [Ca2 i. Na+/Ca2+ exchange (Trosper and Philipson, 1983; Kaczorowski et al., 1989). Such was not the case here because amiloride and benzamil, inhibitors of the 4. Discussion Na+/Ca2+ exchanger, did not inhibit the increase in [Ca2+Ji. This is supported further by the observation The resting [Ca2+]i in adherent A-431 cells was that removal of external Na+ did not block the CRF 66 ± 4 nM. CRF induced an immediate concentration- effect on [Ca2+I,. dependent increase in [Ca2+]i with an EC50 of 11 pM. The physiological significance of changes in [Ca2+]i The increase was apparently due to an influx of extra- has been documented in pituitary cells and in hip- J.G . I~ang / European Journalo f Pharmacology - Molecular PharmacologyS ectiom 267 (1994) 135-142 141 pocampus. It is highly likely that an increase in [Ca2+]1 Gilman, A., 1987, G-proteins: transducers of receptor-generated stimulated by CRF is a common signal for various signals. Annu. Rev. Biochem. 32. 207. properties possessed by CRF. In mouse pituitary AtT- GonRzaelceesp. toFr. Asp.,e ciRfi.cG .f oAr lcfeorntazion. Jn.uRc.l eoTtoidroe s asntidm uLl.aAte. s Hineopspiteoll, p1h9o8s9-. 20 cells, the CRF-induced increase in c-fos mRNA is phate metabolism and Ca2- fluxes in A-431 cells. J.C ell. Physiol. Ca2+-dependent (Boutillier et al., 1991). In dispersed 141.606. rat anterior pituitary cells 10 nM ovine CRF increased Grynkiewicz. G., M. Poenie and R.Y. Tsien. 1985, A new generation ACTH release. When [Ca2-+]i was lowered, ACTH of Ca 2 with greatly improved fluorescence properties. J. Biol. release was attenuated (Won and Orth, 1990). This is Chem. 260, 3440). probably due to the ability of CRF to increase [Ca-]1 GueSrpinoenatua,n eoNu.,s Ja.nBd. cCoortriccuoftfr.o pAin. -rTelaebaasrining afancdt orP-i. ndMucoellda rdc.y to1s9o9l1ic. (Guerineau et al., 1991). In vivo studies show that CRF free calcium transients in corticotrophs. Endocrinology 129. 409. injected into the dentate gyrus of the hippocampus Hagiwara, S.. 1983, Membrane potential-dependent ion channels in enhances memory. Nifedipine and verapamil both an- cell membrane. In: Phylogenic and Developmental Approaches tagonize the memory-enhancing effect of CRF (Lee (Wedoslf. ),H N.Bewad eYr,o rkK: . RGavieetnz.e np.. 6J0. .Rosenthal. R. Rudel and H.U. and Lin, 1991). In this study, CRF at concentrations lyengar. R. and L. Birnbaumer, 1987. Signal transduction by G-pro- between a normal level and a pregnant level induced a teins. In: ISI Atlas of Science: Pharmacology. Vol. 1. p. 213. moderate increase in [Ca2+]i in A-431 cells that was Kaczorowski. G.J., L. Costello. J. Dethmers. M.J. Trumble and R.L. mediated by CRF-receptor operated Ca2+ channels. Vandlen. 1989. Mechanisms of Ca2- transport in plasma mem- The apparent contradiction that nifedipine and vera- bterrainzae tiovens icolef s Npar*e-pCaare2 d freoxmch acnuglteu raedct ivpiittyu. itJa.r yB cioell.l s.C h1e. mC.h a2ra5cY-. pamil antagonized the memory-enhancing effect of 9395. CRF in rat's brain (Lee and Lin, 1991) but did not Kiang, J.G., 1991. Effect of intracellular pH on cytosolic free [Ca2 1, inhibit the CRF-induced increase in [Ca2+]i in A-431 in human epidermoid A-431 cells. Eur. J. Pharmacol. 207. 287. cells can be attributed to the different types of cells Kiang. J.G. and D.E. McClain, 1993. Effect of heat shock, [Ca2- I,. used in these experiments, and cAMP on inositol trisphosphate in human epidermoid A-431 In summary, this paper demonstrates that CRF in- Kiancegl,l sJ. .GA.m, . MJ.. LP.h yKsiooel.n i2g6. 4a (nCde llR .PCh.y sSioml.a l3lr3id).g Ce.1 516919.2. Heat shock creased [Ca2+]i in nonendocrine cells in a concentra- increases cytosolic free Ca 2, concentration via Na'-Ca2- ex- tion-dependent manner. The increase was blocked by change in human epidermoid A 431 cells. Am. J. Physiol. 263 rceamtioonvsa,l poefr tuesxstiesr ntaolx iCn,a +2or, trtreeaattmmeenntt wwitihth a poClRyvFa leannt- Kia(tnhCgae, nllJe .-PGahn..y essaiotnhld.e t3iEz2.e)T.d .C Wr3a0te.si ,i s 1b9l8o6c.k eCdR bFy- envaolkoexdo nbe.r aPdeypctairddei a6 . in4 0u9r.e- tagonist. The data suggest that the increase is due to a Kiang, J.G.. E.T. Wei, and K. Fukujama, 1988, In vivo and in vitro Ca 2 influx through CRF-receptor mediated Ca2+ inhibition of thermal injury to skin by peptides of the corticol- channels. iberin superfamily, Clin. Res. 36. 120A. Kiang, J.G. and E.T. Wei, 1987. Corticotropin-releasing factor in- hibits thermal injury. J. Pharmacol. Exp. Ther. 243, 517. Lee, E.H. and W.R. Lin, 1991. Nifedipine and verapamil block the 5. Acknowledgements memory-facilitating effect of corticotropin-releasing factor in rats. Life Sci. 48. 1333. Lin, M.C., S.K. Beckner and F.J. Darfler. 1985, Characterization of The author thanks Dr. M.C. Lin for cAMP measurement. SPC hormone-sensitive Madin-Dar'by canine kidney cells. Methods Lisa Drouin for lnsPl measurement, and Dr. David E. McClain for Enzymol. 109, 360. his discussion and editorial assistance. This work was supported by Linton. E.A.. D.P. Behan, P.W. Saphier and P.J. Lowry, 1990a, the Department of the Army (ILIR ECN 0027 and RAD 11 STO B). Cortocotropin-releasing hormone (CRH)-binding protein: reduc- Views presented in these paper are those of the author; no endorse- tion in the adrenocorticotropin-releasing a"tivity of placental but ment by the Department of the Army or the Department of Defense not hypothalamic CRF.J. Clin. Endocrinol. Metab. 70, 1574. has been given or should be inferred. Linton, E.A., C.D. W1fe, D.P. Behna and P.J. Lowry, 1990b. Circu- lating corticotropin-releasing factor in pregnancy. Adv. Exp. Med. Biol. 274, 147. McCoy, C.E. A.M. Selvaggio, E.A. Alexander. and J.H. Schwartz, 6. References 1988, Adenosine triphosphate depletion induces a rise in cytosolic free calcium in canine renal epithelial cells. J. Clin. Invest. 82. 1326. Berridge, M.J., 1983, Rapid accumulation of inositol trisphosphate Merchenthaler, 1., 1984, Corticotropin-releasing factor (CRF)-like reveals that agonists hydrolyse polyphosphinositides rather than immunoreactivity in the rat central nervous system. Extrahy- phosphatidylinositol. Biochem. J., 212, 849. pothalamic distribution. Peptide 5 (Suppl. 1), 53. Boutillier. A.L., P. Sassone-Corsi and J.P. L.oeffler, 1991. The pro- Mix, L.L., R.J. Dinerstein, and M.L.. Villereal. 1984. Mitogens and tooncogene c-fos is induced by corticotropin-releasing factor and melittin stimulate an increase in intracellular free calcium con- stimulates proopiomelanocortin gene transcription in pituitary centration in human fibroblasts. Biochem. Biophys. Res. Coin- cells. Mol. Endocrinol. 5. 1301. mun. 119, 69. Chiou, C.Y. and M.H. Malagodi, 1975, Studies on the mechanism of Moolenaar, W.H.. RJ. Aerts, L.G.J. Tertoolen and S.W. Delaat, action of a new Ca2- antagonist, 8-(NN-diethylamino)octyl- 1986, The epidermal growth factor-induced calcium signal in 3.4,5-trimethoxybenzoate hydrochloride in smooth and skeletal A-431 cells. J. Biol. Chem. 261, 279. muscles, Br. J. Pharmacol. 53, 279. Petrusz. P., I. Merchenthaler. P. Ordronneau. J.L. Maderdrut. S.

DTIC ADA279920: Corticotropin-Releasing Factor Increases Ca(2+)sub i via Receptor- Mediated Ca(2+) Channels in Human Epidermoid A-431 Cells

Similar DTIC ADA279920: Corticotropin-Releasing Factor Increases Ca(2+)sub i via Receptor- Mediated Ca(2+) Channels in Human Epidermoid A-431 Cells