.. ......it. 40‘... ....o I. '00&5.\\i00 .1 . . . III. IIIII- ‘1‘. I'IU‘II I .000000. o. 0'0 1 I .. I . . II «luh I _ I I 00 I I . .. .....vooo 10.0l 90:71.0 ..-0! I V .0 .010... o 2.0..y; o. .I . . .. {.-.-:0 . _ 4 . a ....-..0... ....0.....f.. '\0;w0§v0' 4‘0... .8{.r0...0.s-I..S0'0|0.ooo_b 03000.21oo 2. on!!- o...0.la -. .90 00. .l.0.... .. on 000.. .o 00.00. 0... 0 .....I. ...... .....2 . . .0 .. 0..00.... I- ...... I .Ipu .0.00.0:..003..0.0. .030.- Ilrobo.;l .... .530. V 00000.»...000 0.0...otar3rsv..uo¢.9... ......8... .0 :0... 30o0.... 8... rs .- 0... 0.20 ... .0 .I 0.0.0..- 11:... ...-3-0 .....2 ....-.0 . 0.. .... a......0........l:... . .03... .... .--. .... “O .‘.V .I0. 0.0.1. '0. 00’". '0 . 31y§§m ' 5.000.040.1200. . 0.0 no.9. .00030. . . 0 . 0.. :0.- 00 o 'o 0' I '9. . .. 0.03.09... .0 0.l ..0 000... l o I 0 .. . .0 .0: . .0. . o 0 . I . o 00o .0 o. o u 0 I. ' ..I .0 I .... 03.0 .00000 00.. .Ioliob ...'oo..0...1-00o..v§.l0 ....a... 0000 0.0.0.0.0o..0...0’.020.o....o ..0 0.0300 ......3. . n 0 .3200. .....I ..J... 0..p . . ...... 00 o. . . .. 0.0 ... I... .. 3‘1... .0 u .5... I).. .00 ...Iooo00o"0.'; .- 0. 5000.'0..0..0"..0 I .Y I900..o.0. s... ...-0.0 5.020....200‘: . I. 0.. . . 0.0. .9 I .0 . I. .700. . 1 . ... I. ..I .00. 0...: .I. 0. .. 3 ‘70 I0 .b'oo ...r).0010...00. ‘o\.;obfoa...:0 SO ...?0050: 0000.’0.0 0oorln' o . ...».v ....9..’- .0. I 0:... I 1 .0 .... do . .. ...097.‘ ... o: 0 ..., u 0.. r . 0.)..0.-00._ 00......0 0 0’000 ‘0‘. '.'0sb.f0'.§to.o.00.‘0.l’§ .9... 0.00.0.» ItI.Ro._I.I ...!9ouuoaoo. ...... ...}.3. 00.0.3. 0.. - 0 ..-. a ... 01.... 16.0.5 . ... .300... :0 .... 00" 0.?0o‘l... .\..‘.0Fo-0%.nol.0o 9 I... 0:000:0003... ..ot?’:'..00'0 2...... . . 0 a 0.0.. . .. . . 0. .........0K0.......0 .’| v 1.0.. If. . .0 -... .00.. 0' 0‘ .0....7. .0?..0u)0...0000’70..r\ 300.0 o 0...! 02.90.2000. 030.00....) 20.. , .. .00 .o..1..n... . .. .0... 0...... ... .... .. ....... . .... .0.. fl. .. .V 0. 30000 0P0.0.‘.-.).‘I.vllb.‘. $00.00l" ..‘Q.‘DQO.I0 oJoOot oo.o0..o.r000 5.0.... 0 .9. \o. 3.0. 0...... ... .o-IL. O' .0...-- 00.0 . 0 o . 00:00 . 0 . 0a a . .. ... oonto 00. . o 0.0 "l O 0 0| 0 I. .0 '.00..{.I.0.I loflfo.‘ ...0.0JI.0‘¢ .. 500.0..I. .9 u.- f.o0... I 0... I! ... I... . . 0; o. . .0 .0 .. .......0 . ....... 0.....02: \ _. I 0 ...-u. ....00). 0.. 0110 -'|0. ......r‘n.v..vut vi....v.o0.. .0310 110...}; 0.2.00.9?! .t.-..Is........:.. ... . ....u- . 0.. . .. 0..... .. .00. .. . ... .. ..0- .00 . ...... .. .0... u '0. 00.0. 0000 . 000 0-.79 , 0. c 0‘ 0...... 00.0o. ..V -.\'0.-.130 '00 .90.. 9.00. . . ... 0 I. . . 0 0:...- 0 30-0 ,A .I.I. .0 0.1.! 00 00.0.0.'.. _ . . . c u . D 0 In 0 ... 0|- I 000 00 ...-'20. r002....0..fl'.0000..tdtfi..0....00.....v...0:.o._ 0. 50:91.... . .. ..-... ’23.... ...: ... .. I; .1... .. .-. 2.7.... .. ..0...3I..O. ‘ . . 0 . ...: . 00 90.00.00." I . ...:0000 .0. . ..‘.0b... .0 ..‘hOofilrrunfiooo. 00. 0.0.... .‘0 .0600}!!! 00§o080-O0i0.0 .§,.~..o.. I. . v 2.0.. .0... 0 . ’0 o. u .0... 2. ... s 0.0.... 0.... ..o- I .00.:0.0. 100' 0 :0300000 I. , 00 o . ‘00 .. ‘o‘olo . . I: . co.loo.0o.'00ooI&.Oo’l'l-’ I p.00oo..nh h to .. 0.... 0.8. o 000.200.... Ac. 0 .a _. .o. 0.1 .00. . :00.- \ .0..0..0,II .... . 0 I. . . I .0 0.0 .90. . ...l ... . 009 I . I 0.. 0\ 0 0" V. .9307? s, ..V’.‘ ’0 I... 'Osfl o_|’o'o.0..' .0100”. )7? . 010.10.! .o. a. .. . I: . .9. I o .aot.. . o. . o. . ' . .1. o .I .0 0. . ...I. 00 .sa. . 0 o :0. I o 0 . u \000- ‘ .0 o 00.00.»...0! 01!. 00.05....o...€1.....0101:0' 000'.:. 0... ... .... .p...... .... ...,.........._e,.o ....JI.:0~.... . t . . I ,2. . -.. .. n -.. fi ... ...... ...! . -... ....lu . .. ...: v. 0 0 V 0)..P0 .0.’,'0. .ECQY Iol'9. ‘ #090... o O. . .0.an 0 0 C a. 0.0 0.0...00' . 0-.0 v.00 ...00 o 9. 0.... ...o. I o. . .-V’I. - I 00 ...o o. ......o . 0 . 00 0 VII»... 0 I: ... o o. 0 0 00. 0. II I . u I 0 .;.0 o 0.00. do u .0. I a»... 2.0....' '0 . .\0,..0.n "00 .00,0 Co . 0.5.0.000000 . O 0 000- 00-0 0. :00. o 0 D 0 . I 0. 0 00 .o 0.. 90’. I u 0 .‘0 v.0...- 0.0 I 0.0.. :0. o . - . .. . o 0-0 0.. o u I t. I o o o. o . b 0 ... 0 0 0 o I Q 0' 0 'o . . 0..0.I.. 0o. .0 .0.0._IL.01. 9.90! o....l0.1.0.00-..§.0. .0 .I‘..0$. .0..vb..obat. I... o?0.0....£.. to! .. .... .0. ...... ... ... .. .... ... v ..-... .. . )3 .0 .. ..a. . .... >0. .;0 -0).Ir O"‘0 .. .tu'ol s70\ '0.0o0..i..00‘.‘.0.o.'0o.coor§o 0wl-.I”0I.o..l00 7... r.t.0. 00 .0 .r‘l..|\ aft-..." 0. 0.0.... 0.9.0. . . 0.0 .l .O a o. .0 . 0.. I. .- 0 0.. I. t .0 0. .00... ....I 0 . V 0.0 . . o0 o0'0.0.. 3.300."..0 .50 . ... n0 00".0 00:00:99,317 .o—.......s...~.17. ...: ... ......03900 .0.‘ . .-. 0 ... . 0.0... . . . . .20... ... . 0.... 0.. . no.) . ’0?th n.000.00l.... 00.0 it: I. ol¢-_!.!...0.' 000......I0... ...: 0.. .I.. 0. -..: 0...... . .3 ......o. .0... .u: ..........0 o. .0 . .. .33.. . .0 .0 0.... . .. ....0. a. . . 0.. . . . . . I ... I . ... . I. . . . I . . . I .HW.OII%0 OI ‘0' I .. , IQ'JI O..§00a§.0. 0. H... ... ~0“0o.'90.o.' 000. 90'. 0‘ 00.0..o o.-0o..0o o0. . o 0 .. 0 0 . 0 . 0.. I. 0. 0.0.0 0 .0. O» f ..o 0. ...0n 0.0.0. 0H3. 0-0.0. “IL-0.0.. 0.. . I 0 . I Q 0 0.0 . 0 0 0 . no" I I . a . 0 .— ’00 \. ' I’OO0..OI0.I"O '. .r I’.‘.-bbl.l.ot'a‘l to Illl.’ 0,0 . ...on V O o. .000 I. )0. O .- ... . c o. O. octagon. I 0. no. 1‘- ' . .. .0 .. 00 .0 .~.. 0 t0. 0 ... 0 .. o o. . on. . 0 .0. .000 4.. ....D a . o . l0 0 poo-...! O O. '0. ... . 0:10.2330V921'o00.«at. .... ...}..I .0. ...... .... .....:r..l....007....r..02... .....-P.......:0..0.. 52:90.. ...P. l ..... -..... . r... . 0.. . ... .. ..e. . ... .. Dbl. . 00 ‘..o n.)..\. 0'P0.10 0.0.0.". 0 P . ..0... B\.9o.. 0.3.200: '0 ’o 00—00S‘00 900 ...-0.0.9'. 9.0.00. 0. ...0 to. . . a .- ..I.. . 0 o. 000 can: .... II... In. I i .0 It .Pro\ 01., . 0.0.. 00.0 ..0 0.0 .. 1:0..............0.00.0...._):.00U . .5? . 4.... .0: .. :0 it. v . .-. .... 0 .. ... ...: .. 0.9... ...... L... 0 v 3 II ‘at0 000 0. A 4.. o. VI. ‘90,. u 0.0.0 70..» . Ibo .00.. 4-0. . 0‘ I. . . 0 4 - u. . Iv 0.. . .o 0 o . o o 0 . . 0 o. .00 .- .. . 0 . 00 .0. .. ...0 '0..0‘.I.v0‘.’ Elvtl?! 06'. c . 0.. for? . 0:. 080-. 5.. o. ....J I. 0-..0...0..f. 0 .-.... 0....0 0:0 I109- . ~ I I .. .- 0.8010-..0.!!o....0.1...1of.. ,....0 IT. .. . 0 ... .0 v0. ...... .... :.. .0.. . .0. ... 1... 0:. ..0)......0..0. PI 0".06000' Iooo‘ v... .0...an. 000.'.¢.l. Quouréo .oowo .. 0 .0-0. . .o ...-0 90¢.- ...0 .0... .. a .... 10.12... . I. .....0 .10... .IJ..I. 0. ... ... . .0. .. .00..I.- I08. I .50 . . .I ' . . . 4. o I '. 0’00 '0. . . . .« 0 0r... | |0...9.o-II 0 . . 0 I ' , l 0 . o . 0o 0 . 0 . .. .. . ”I '0 0.0 .0.” 3'0“. {OK-0 .A.0o00.ul. o I 00's,.0fl. ...? ...o. 52.0 to o 3.4:... o .. .00.0 9..» o . 0.. 0.; '5... 5o 0 2 ... ... ...... o n. 0. ..0220... I. 0-..!. > .0 .. .. . . 4 :. o. .0. .. L o o .010. ... .. . ...0 a... :0 ..0...) I"...02..v..o'0“lV.10. . . o 1:. 3‘ I '0. 00. . 00 I . a. . . . .. .. . 0 .0 ... .00. .0. . u l . c 0 .. V '0. 0 . . ...-0.0. s!...!-....Q...?...?.: - . . 0. ....r.... ...; . . .v ) .. 00'003'00- ...-7 3.35.0!’ 00.000.00.400r03loo 0.. 0o .7... 2.0 .‘It. ..?.00. . . ..000.".t.n 300.- ..o oImJ... 03.0. .... 0 o .000 . . . . .II .0 . a! o. .. .0 .0. .40!” .dv' .ia'vno ‘0 \ 0‘ I: c ’4. iv: .II!.. I”. 0’ I .0 '0 0 0000.0 D I ‘0'. o o D','o.ao .- o .0 0. .0 ...-.- \ 0 0 .. . .00. o v. 0 0 0. .. O. . I... I . .o 0 A I .0 . r I . . 0’0. 5‘.‘ 0 9; Ozooho‘orva'l‘: 4.3.0 0000000 ...! I...‘0. ..00.0o..|.0 30.0.0. 9.. '.00'.or~000 .0... .A....v.0 . a. 1.0 ... .v o o0. .. 0.00 o 00.»: o?! 0.0‘0 ..r.u o.....ps.¢.00.0 00.0 lo ...0 ... I 00 I I I ' 4 - . I . . 0 ..90: 00.00. 0- 0.10... 0. o.f"‘-0 0.. 0.. ..0“.90.'I.?v o.'-,....Ilaol...0.. 0..O(..vo..o.07. 00. 0.03....1: . ...... .0 . .... 2...... .0.-... .... . o 1 I .. . .... .50 ..‘I. ... 3.0800x!.00.000l(. ”I I00...II'.-.v-!0I0..OI 00 I, If 'l.’0...0..l’u¢ or 0.00.. or r ...10..¢~.KY 00.00 0.. 012:0- .00‘10....0.0 ... 0......... . .s .. ..IL.. 0.. ...I LI...I . . A . In. Irotol. i...).!00J0.Ai-.>irbaist .0010 0o0.0v00.0o‘ 10 no.1...) I A . 0|! 3: l0..\ .7. .0. 0.3.0. 00.. 0.06“...) I..- I0$0..P.o. 00. .0 0 ... . ... 0 ..o, . .. .. ...: ... .... . . a... 00.70 -...Oo-i. .0. 0 .0 0.."L"000 .0. 000110..‘0o¢\| ' II'OJ '00. 0 'o v 0 ..IVo' '00.}...C.u u€100359l u§0.-.. 0‘- 0.0 ...o.lo...o .(Co.00:00 0 -o ... .... ..JpI . 0 ._ ..0.ov .... o .00.... a... It .70 0.. 0.: .del. ’n‘v‘. . 0(- 0 00.0.0...l0'. 0. 0. ..r . 0? I...orl.. . .0.t.o.-oo.o on... 50. 100.0.oooo‘C4‘0... ’1...o.os’- .91. .- .... 0.. I. 0. II 0 ... .... 0 I. .....0. II...0.00.....I 0.0 0 .o In. n\‘00,, ne.!I"OP . 9.0. ..0005'0 0| 0‘ .)Q\‘o00 0'0..‘.ol 0.0-“0100.3: .80‘ 0‘ ‘..,.'0.0 0'- .050 ..00i..:..1. .. .0 .o 00:... ....Is. . 0 . .00. .0; ... . .4 0.... .0 .o .0 . . 0 ' 0' 3.00 a. to.» .00.: o‘a. . . {09‘ o' O 0 00.0. o. 00'. 0 0|. . 0 to 00 0 It. ‘ .oao- 0r..00..000 . . 0 I to... 00 1900'. .01... o I ..P I. . .0 o 0. . 0 . A 1 I o . o . 0 I . r o .0 o... A . 0.0 a’IoQI no 0 00? 0 o 00 ."I0.'t000-0. I- 0 ..‘I0o..o0.o..|.. . I. .J ’.o.o\00. 1......F .. . 0000 .1. .010 .. ..0. .-.-£2. ...60 0.2.10... .0. .I.... I ._ .00 . x. I .... .- . .o. .02....1. 000 .07.... 0. . ' .r 0000000.... 0.0! .. 0.00 0.. 03-000 . ... . . 3.0... 3011 ...Yrficc... 0...... 0....I... ...!0 0.. . ..r.....II1 9.3.00.0 I. . ..7....?30‘...0 .. . . .. .... .... . 1 .... .. o. .. ..0 .0 ......01. o I; . .n .100... 00 I. .." 0 I! 0 . I0 00- 0.0. a V0001}. I...... o.. 0. . ..i. ...a .... . . ... 2. III. .0. 0. It; I o. .0 .v. ... . .10.! 0 .o 0. 0 40.. . I... 00 ..00 . . ... .u . ...... ... .-. I. .o ..0: .0. .I . . .. 0 0 . 0 ... . ..! .0. I . I 0‘0000 0 00 . . 0. 0. o.t'Il...‘.0 . .000113. 0...‘ _. 2. 0 s o... .9000]! llaoua. 0' 0o...09o0.0.l... .00 o 070 0' Q..o o . . :9 .0 9. . .00. ‘ . . . c». u I... .0 . Izoooo. . . ...... . . . .... . ... o . I... u '0 0... 0 . . "I. O O O. . 0 0.1 ..'!00'. . . 1" ' . 0..H..o.-.000..o.ouov.l' 1.0:. 200.....0‘..;.n.000 o. 0.1.3.9.... 00.. 05“. ...o. .90....toc 1. .. 0.. v. .0. 10. I ... . a .. . .... .5. ...: .l. ......0 .0 . a. .0 V. . .1...’ ‘0 o '0 o 0 . .0... I. ..-...I 0 u..\. ...o..'.o.v. 03.0o.:_l.:u.0..0oov....0‘0(..V..fis.riof..v 0125.00.80 000.9. _« v ...... .-0 .0 00.. I .. I. . .0 ..:.. ...! . .0. 0.. . .13).»... I..0......u. 00'0. .-...0 o.l. 00.0 00. 0'00. .00.... .r..f0l~$.. .- ’.0...-.~. ...... .... 09.....00 .0; 0.!00. ....I. f ...-0.8.0.0. I 0 ...... .3; ... 3... ... 3 . .. . 0.3:! 00.110 .0 .... 00.004000... '1 'III 00000:.. 0 0.03 'o...... :00‘0. no.0 0. 0000‘.~0I2000.0IIIS0' 0 Sp ...’.00 00...? .ou.0...l.'vs. 10‘ .0020! 30~0I.§.'xo00..00702". .000 . I?!‘ ..I 3.0.9: b I. . . . ... ‘ I 0 .0 . . ..0. o. I 0.. |.0 .. b .0 .. '1 . '00 n . II. o .o'!...o.t.o 00 . lirllv 0... ..I ..o ' 0"... o 0.. ... .00 2"... .20 . ...I... 0... I 0(- o o . . 001.071 0 o I 0. o 00 0 030 0 0 .... .....I . 0 _ p. . a 0 00. A . o: 0 00a v .00 0. .00. . .... n .0 0 0|. . I 0 0000. 00 t 1010-... of. ...0000’; .la'.\ 13.3-0- to. . . . ..’.o..o 910....-.0 .00.. 0.0. ...). 5.0.000. Y )Y. ..0..\o.r)..v. . .. .03..00. a... £0... ......5! . l0 0. ..0.0. 0. o.00aIII-000.09.00.00...00000.20 .... ..01‘.S0 ’0', r\.:oo£ 0.- ?.o.. lo. 0.0 I. I-.. .0) I I o. I 0:- .000. .x'I....II0 .00..|3 .40) IP03 0.... o .... .. 0.. f. . .J 000.. In} I 0.0.... . .. xv: I 0. ”on '0 .... 0. I 00 ,I..0 00- . of'..01..0.-0 ....60owna.v ..Pv!-?.0.. ~.vosoo-IJI'..£'I0.0.0.0.0.?Soo. .0 .1. ...-0,072.0... I I: .01...\~...-.. ... 0 00. .0. .-(.o00 0|..04. .. . . 00. .0 ...-0:0..0 , Ola: 0' 0 on... I. 0.004 0 1o.0no00 0 I o". 0 .0000 0. ....0’.o',0.'.0 I o . 0.. .000 I0. ..lclov. AI.- 0- 0 0 0 .‘3: . 0. 3 o .0. 00:.I009 (0.0.70 0 I~00 .000.C,o'.o.o ’ 00 I In I. .u .30. o O. ...-0.0 00.67" I 00,0 o .00 o 03...... .fo; 9’.) . '00.00\ s 00 09.3.0... ..h..00......“o 00.10 00. . 0 0 I ..v . :00. .010 0 0 ...!I .0 I... 0.... n- 0 ‘ uqvll' 9. .-. 00f: 0.‘ I. 0’ 0o 0. :00r—107'I'Il . .014... ... 00001. .0 .0023... ..00...00:..0000 - 00 o. 0.. 0.0.. Z ...‘0. 0 0 0 0 o. a. .10 ..II . . ...'0 o 5004 o I .o u 1.9. .0.. t0..- ‘-| .. . ..0. 0|... . )3..'. l I. 3.6- 0.0.0... I “0...... . ... . ... 3'0. .0 ....3... 0 01 I... I 0....- .1 3. .. s... ... . . .... . I I: y ..I. . .. .... . .... .. . ... I r .. 45...... . . 0 '0000..Q.. 0. ... .000}. 0 .- Le. (0.0.. (..l?’. 30.0? ...-0.3.... ...! .0.000(.3‘0.fl..0...:. .000. . . .. .v . . . . l...l0..~...0 00 3. 0. .. .30 .0 ....u ...-.0? . Ida. .90 .5. - I .00..: I ...! 50- I . .00...0. , 0....- .. .... ...... 0 .13000._. .0 .... 09.00 ... I... 0 0. I I . ... ...- . I ..ofo' .00 .0 00.. ...J ....0. 0".I 1.... 'Sofa‘. '0'... o 0.. '00‘00' ..0000' . . 2;... .0? . ......I . - 00.30.90I 0..- ..I .\ 0.. .00 0.00000 000.... 0.. 0 3. . 0.-0 .. I .70 s... 00.104 .0 Q ‘3‘. 0.00.000... 0001.. o. .I' .l..co.ohool..\0 '9. .0 . ..00lv0:0'0.h. ....o v0...000l.1 I"Oo.-: 0.0.0. 0 0 .0 ..9 V. 0! 0... I000. Lo. 0 . 000 .. 0.. -.0.. o .. Iol'l .'...)...0I.Do‘ v.0 'I-.' '0'. 0'. 9.10 ......0 0 O0 .0... (0 .. . ~ I. L.00.'.0:-D....0l1!..9...‘..1\'0n...........L . .0 . .7 0.0.10.0...Iu In. . I. _ 0. I: 0.... Ix“ ...I _ .. ...-... ,0... '0... . 0.. s 0.1 .03... 0;. 1' 1’ ..Obooxlll o ...... . ..r, .0- 00.0.'.. 000 0. 'll. 400.0 '.l..’. 'fl..o.o00 .l. .o.ll.....a..7 . |00Q 0. §.o.aa.0 .qu 0...... . Ir “ .... .. -30 00 0. .'. I n. on. .0 0.0- o’0. .....0.00 04 ."01 0 O 0.00 . 04. o. .. 0 . . 2.00.0 I. . . ..E t - .\ 0\- Ir. “cum-0.... .. ”MILK-0.0.. I.I.....>r.'.....00. o I. . .0. 0.. 9:10 o 0.... ..l: .1. ...\ 0:30. .0 40.0.... 0 .u....L 0 0 .0 .. . I ... . ...... .1'5 3.90.0?) . . . . ., . . .. . . 0 . . . . .0. .r I . .... .0000! .3 0. no 00 A... . . . . 00!, 0 a V n . C . .....o. 30.930 0. o. .1... . 2.02.... .97.: . 0 0.. ..0 10.. . o o .0 ,. . . 0 00 a. v «I . . . o 20...... . ... o. . . .3 . .0.. .. -.l. . . .00 I! . p .. .0 0.0 . ..Ioo . o 0 0' y “0 4.00 '00 n0."‘ 0.00.:o'u 08-0 .00. II, I'L.o0o0.:o..0.zn...-0.”...0: Do... 9 o0. 0.0 0". 5.0.0.00. . I ..o . 9. . “SQ '0. .I I 0’0 I . 7 0 1.0.0.. .o . 0 .0 u o . .00 . o a 2 . .00 ..0 0’ .0. I 0.07 00.0.0 10 .JI0I.. 00"... Np 0,00 st 0‘ ['00. " 3.4!}DlIép ”0 III-0 . 0 fl ...0 0 . . . 0 0 . 0 . . . . . ..o Pa... .0. a. .. .o 0. .010 .m. .0305. 0.000 . .0 0 0: .0... 0 . I . . .5 . .. ...? . u ...0 .... .r ; r . ..0 1'00. 0.0!... .0080! .I'. ‘0'): .0!co.'.|000"\00fbf||0 00N000II00’. 0 0". a. . o - . 0. £0.90 I 00 .. .0. . . . .0 . I I .000) ., . ... .... o. I. 8. . J3 0.. .00. .‘0.)1-.0. .3 . .5. . . - 00 '00 00300er ‘0 ....-. '70 O\ 00.0 _’._0...0.vJ-0... . I ......u.-.o. . ...0 0 ...: 00‘. ....9. .. 0.. 0 .0 a. . ..0 .0.l:_..a . ... 3 0 .. Ila....ov.'xuuo'900000l)"'ociu,l 00 0.00. o 000 O. .100. 0 0 0 .0000va0 0 0.0... . .0. 0....o to c ‘00 u o o o I. o ...-0.0 .... 0 .0 . 0.0.0 I 0 I . 0 0100. I . 00 0 0 0 I 0 v I ..0 c. 0.. . . . 0. ..0 .103; “q . '0 .I 0. 1. ... 0.0 . .8 r0. .0, . 0 ..III _ .o. '000-0 to' 0 n .. 00l00 ‘o . 9.5. '. . 0 I 0' J .0 0o 0 .I 00' .0 0 I u. . 0|..‘. 0’ I... t .V': 1' 0 .0 9. 0.. ..000 07.0 -.v o 00 . o....00 0 I .0»..\ ..00. I I ...... ... 0V? .- . ' .D .. . 0.. I l I 0‘ . .0 o .0 0 00.0 ...00.‘ .0. .0'0 ’00 I I 0 I I .0 .. '0 0’0. 00 O 0 0 VP 0. 00' .. .. . . I . 0 I 57— I 00 II‘QLII ” . 0' 0I‘tzlg 0 4:. 2.0... . o. o .. I... I o 0 . ... 10.0 I. o.._0_. 0.....00 0 .s?.00 CI... 0 .3. o . 2:. 000 .0 "4.”.00089 o. 0‘ . . 0 0- . o 0- I. .0 . . . . .I ‘ 0 'C"'n . . .\ I '0 .0090. 0.0.0.)». 0.. 0. . 8.0.0000 OI. '0?‘ o o .. 0. . 0 o 00' 0.. I .000 4 0 o i?" 00.. ’ .~ - 0.0.0». 0 ‘_‘ .7 O .I 0..... ... r23 .0 \.3. .... .0 . ... o .. 0' I‘oofis $0.0..OI7 .I ...| .5. 0.. r l. .0... 04. :0..I .0 . . ‘I .. 0. .l .\K.. ..d. o . .....0 0.... 00.03....Jo‘... . . (......u. .o . ~3.0,. I ...! 0...... . . . .....0. 0:3.0930'10. '0. .00; .003 ... .aol'..0. I «0.0 o 0. ... . .0 it. So..0-0 0 . I H ....‘q...... ,4. 1'. ... ... 00.00.. 'i'. 0 0. ol .. .... ......9. .....0 . . . . .- I. .. -. . ... 0. .-. . ... a ' 00' “‘9. .. J! 04... Q-VIOJ‘0 . 0.. 00.0 000' 00\.0|0Io. VI....o0.loOHo-€J III} 0.00.00 ,0. 0.. I . ..0 . . 10.23. .I . .0030... . .0 0.00- 0... o ’ 3.... 00 0 '0 . l0. 0‘... 0 Y I'll ‘ ' ‘3‘101 Q OPo‘ '0 0.9.,. .0l.:. 1 .0 001' “0.0.... . . I. 0 0. I. . I. . . o ...7‘ . . .0 .00.. . oo. 0. ' .‘.000 .0 I .3 u . I. II ' 0| 9 .f ' l 0“ I..vld.oo 1| 2"... C '0': 00001C?.00 0.010‘00010l0.0011o .0 o..u.....?0.uioo..=.0 1190!.” c. 0 0000.0... . ' .Oo.-.: O..'o00 .0. . I 0 . o .. 0.00-0.0 . 0. . o . . . ...02 000.i. .. . .0 Q 0' 0 o . 0. 0 III 0- lo , . .. o .0 . 0 o . 0 . h I 0 '00. I“ .0 O. v.0.[0’.‘.0'0.’0.0 .90- .7.“ o C Q 0 0 0.10400 oi.l,. I I o'l.”-0.C 0 ..o ..v . . 0’0‘020 O 4 In 0.03.0.0... I H’ any... to o 0. .. :00)... 7‘30. 0 . o . . 000 I: 00- 0 0. .....v00 . 1.0 I u 0 0 I II. 0. O I.) o. .. 0‘. .2: 0. . . . .. .00 . . .00 .4 0 . . . . . ..l 0 10.. . . v v . . 00. 0 . 0.000(9 00.04 vaLIQII... . n .o...o.t. ..0q' 0..nI0Q_..0‘...'00.0. 00.99.099.9'0 c.7200}; 0000. ....o...0.00..000.-...< . h....'o’0.u"ov'0. on... 0.! .0 0 00.200 ‘ 0 (00 9.0 . . . ... 0 O . o . 0-0.500'33.‘ ... I c 0 II '0 s 00 . 0.0.0 z . I. .. -. 000-70400. (\- .. 010.0! .. ...-10...... 0.. 0o: . Irro . .000 .V I‘- . . o I . I. . I .. . I1 .. . v0 . 0 . .... . .‘0 Of . . . ... v. 0.04.0 .0 ...»OIo...Iac...t.. (0:10; .....0 0.0.0....» 00.0003. 0?. 0.0.. ?...0.2.0.l!... .92....20..\ .. ..I- 08 . ..2 ... ......Ionor. 0 .‘t . 00.. 0:00.00 J..." . 9.. 9.1 0.0.. .0 . .' .0 . 0‘... 1:50 27.....100 ’OII! .. In} . III 0...? . . . 2!. 0...}... . . - I. I... .. .. . ...! .v .c ...... . I... 0 0. . . .. . :00. 00.2.0.0. .... ... .. ... w . . . . . 0 . I 4 I-.. .0 .rt'r..lu'.t.0.o 000..f. .3)...?D.. .00..~..cv .0-..._0.9o.a...0 100. ... ... . ..0.0 .0014... v 100v l. 00.I.. . 0W0I~ . 0s. .10.. 0'. O .0. I 06 . 0. l I .9 ..‘00 to 0. .0 0 I 0 . .500 OI... 0dr. 'Iotlv b .0, . 0 0— -0. . r ' . V '0 O! ‘0'..'0.\II 0 0..- .1. 0, . 0 ... .... I I... ... .ouohooqno0 ”0000.0..0000..'Crnona-0‘0 00.00 ...-.20... 0 90 . .... .30 o. . 1 . 90 o . 0.00 00.0 I 00 3.0!” . . . . .. I. .000... I O . . . I o. . I ‘ I I r . . I 0 X I... 0- .0 - . . 0..0' -..... . I. 09.. 00!. .300. 0110004 0‘.» 00-... ......SM..... 7 0 o._6.’03 5.! ..Cu 00 . 0. it‘s 9 ... .00... 0 O. o. I... 00¢?- 0... .. In.“ 3.0.. 0.3.11.0... .0 I. .. 29.0.0 .. . ... .3 . 0 l . II .I I . I. . ...... L . ....I‘I 0...? o 010XI.0 .000 .0 I I . 0 0 v . 0.. I. .0 I 0.. .qu. . .00? 0 _ '0 u 0 . v. .0 o O 0 9'. 0r 0 ‘i .1 0‘0". .0 'I‘Poo". .u 0 .00. ..|-0.O"£ 0.0 0.| i)».' a“ I Scr= 'Qr 0.. on o0- 3.00.0.3: 0‘ ...: 0.000 I. I of Q 0 0.0 ... Io. I. 00 . I onl.0'.0;000. 0 L' 0.0 X'II 0 I 00000 .. o 00.0.05. '....00 .. . .o.0.... ...- 0:0... 2 . O u ‘I I .. . .0 .. . a 00 . . Illinuw. J"... o . 0.00... ..013308. . 00 .lo . ..- ....I. .. I: . 0 .0o 0? . .0 0.... 0 0.0.0.0.. .0... .a 5.... ..II ....0 o I .. ... o 0 It .00. . 00 I .0. 0.00 I. I. . {5.35- .0 ...I. n. . I 0 04 . ..0 1:31.530... - 0.4:... 0 ......OP... 0 0.00... .....42!I£0...¢ ...... . .... ...... . ..t. :1... u. I. ...-0.0 r 0 . 0 0‘. o I . 0 o 0 0.. ... 0.;0 01‘ ... 0 0-. .\ I. 9.0.8.000 . ... o .. f: 2‘ 0.0. .. . ‘o "00. J .0 P1000 .00.... .00. ..-. ... . 0.. o, 0.)! .... .0 I 0 _ 00-0. 1 . 0 I o .. . 0 . 0' O V . 0 0 0 '00» 0 II . 0. 0.1000 I 0 .0 0.6:. .00.'I0 0.0.0. -0..I00.0...I 0.0.0.02070Iovov '0:- 0 ‘I‘Io o . 0.2.... ..0 I ago-oil. 0 9.0 o.0 0... . . D I. \ .!. .. ' .0 . .0. I .0 ~ o. .0. 0 .I0.- I u 0 ....00 ..0 0 .00 . 0....l . ., I 0 0 0 . 0 I ’0 v. _ 0 5.1.3100 0000- 0' . .. 0.. ll. 000:05 0.100 .0 0 .0. .0 .vaI' .. ..t... . 0‘. 0 I. . . 0 0.00 ..0' 0 0.0.- 0 0. but -4000! 0 0 0 . ... 0 o 13.»... ...... 0 0.30.9.2. 2... ..0 0.0 .. 8:30.930... .NY... . . . .00 .0 ... ... . 37.3.???” .. . ......s. :.v.....:. 0“”..8..r ...-0 .. .9 o c. . 0 u. . . 0 .08- . - . o 0 2 . -0 L . t ..‘0 ..0-0..0. ...II ... 0'... :‘u. ... 02.0. 1' 3.0.7.0 . ...? fl}... .... Z 0.. 0 29!. 0...... 3.2. . . r... .o .... ..-. .ls. .. -... ....Lo u o .. ... . I. (.... .. .I: . v. 0- 0...... O: . . u 1.00.. .' ......t! .. I... f!.!3!..-o0.97001.3. 0'.-. .. . .00.}! .0. .. II..1¢000....0 .00..0:.ts.:0.0 .. . ....o.?-.. .0. .. 0..\. . '00. . a... . .) .75.- v I 0 .1 O 00" .I I 00'» II I. . \ . 0.0 00! 0 0 '09 0 IL. o.. 0.. ...-r. 0000": o.\ ... .l...o..0Qo coaoII—Qro.ooou o'ooxo 00.. .010 .00. a. .9 0.0.02 0 ..o .u obi .0... oo- f0\. 0 0-00 . 0 0"." 0‘00 0 O'I-'Ioo-I..I’.A.- 0..o 'I .I .00 0'... I .. ......00..I00'..00000‘o009. 0 :IvooLoo- ‘o oo’ou .0000o 7.1.0. ..-. 0.. .0. I. ...-07 .0. o to r 0 o u .....0; .t' .. . 0 . 0 0“ .0 | . 0| 0| .7100, x. ‘0... 0'0 0000: 0- 07 0.0-0.0 0 0 VIA :. 3'10. ‘90 0.0.0. 300 .0. 0 0 020". 5". ono 0.0 0.9-0.0 00.0 0 00.0 . .§ 00 o v 00 00.0” Owl.- 0! 0 00 . 0 II \...0 0t 0 O 0 '00.00 OI I 0|. 7. #0 I I . 0 . 0. o 0. 0.0.01.3...0. 90's... .01.»! .30... . ..oo. 0s...o.~ . 0.... ...!z .. ; ...I...o- 00 ‘I..00 . . P. ..0. I. 0 .\0 a 0 c I 0..- I 0.00 I. 0.. n n.‘ . .. 0 I '5 ..Al. .0 0?: 0.0 0‘: 0:00:09. .on'... .19....0 0100......p.\r.0 ..sI‘.»0000.o.o.o ...-0.0.23.0: 0.... on .. .o..0.. 0.. .. ... . . Q. 0 — 0?.m0...'o .' I 05'00 a: -0300? 00. 0'0’\0o.0 q 0. Cl . no.3; P.L...I|ou b. I'II..3§'-00v'v .039... .0.o. 00.000... 0.0.. ,0.0..\I.¢o..-I‘os 000. 20.01.:0 » . .Iov . . t 0|... 1.0.00... . )10300 {...—000.0. I.. 3 00.. .LN. VA. 000.00 .. 00:... "notoro0. .Mo’».00|'30..0o.0 Ion . ...I. V 0.0.0 0|0..o.0. .-,oyI.II..\.IV 0.510 0.0. 000.3. ...-o o. .9 .01....00 I 0 A . I o b '10 n I . o 0 0 . . , .1 .. .0 . 0‘s -. .I . V . _ . . . . I 0 ..0 . I w . 3.00.. 0 O 0 .0 ...II 000......0 ... -..... - ..0....1t.90.. ...us. .....I.OI.W.?. 0H"... .. $90.:le ....3.0...s.: .0.I....I.o'- .....I. v.:... 1 5.. .. 0 . .0! ...-090...0-03.9.l'o’n00Ib‘t'lf". |0tlvlcl. . I In .0000 0 0 .00 . 0‘ ' O0..I . . . 0 010 n ..A 000 .0.) ..{0\0_ ...:0 .00L. . ... ' . o 09... ...00006 P a-.. 115. IIIII:..vgttrta.’0..opn.v4), .l 0.00.. v.- 00 0 8:8-.. 0 I... 00.00.. 0 0 ... ....It‘. .0. ......l.5...~‘ .... ......» r.....\\.) 0:002:000. .0... . ....).o..... | . . ..,.o: ... ... I... .....l... ..0 0.000(40100 .0 I!06.l. .0..[..I .00000 .l100'... ...“..I0' 300*. C 0-‘II‘ .u 0 0. ' 0 0... 0|. . .0 ‘. 0 o L I.0'00!§.0..00.f1.'002'401001990S 'Iel.0.lo..0..~o....o.0..0-:o0&t00'0.0300.1-0..\0- n u . o. . I . 0.00. 0x0 ...0 .0 . ..0... .0-.00.0 ...-I 00 I. I 0000 .0.O"¢0000010.l. O IO'..-‘ V‘. O I 0 0|. II. I . I .u UOI'II- . H0 0 ’. ' ’0 0 . zitooo' I .70 . -.. . .00 03. (In? 0 nrnol' I! 000 000.0: 00.0 . 0:30-00! .7 Q. 00. . .0 n1 . “I .. . 0.0. 0.0... .. ... . O 0 0.1040. I. . 00 .' 0.0 0 ...! 0o 0. :0... "00.000 0' .0... J '0 0 0.§I.I0I'.f 0' ... .g". ~ . -.. 0 I I 00 . .f . 0. 30.! 00.0 00 >00luu‘0h‘.o\0 C. 0 IL: 0.... .9t9 . fo'V.'. 0 \o .... 1' . . ..Iuv 0.0.3.00 ’\.,‘.D;H..?I.V1000 .... .I §0| 0.. .0“ 00.... ”.0 0-0. 0.. I .. . o 0 .00 .. o o . .0 . 000. . . . .’000 III.) .00.. I 0.0"..0 '.UODI.-Iflbtnlwlon'fi 0'?" «(00"00111‘ 9“. .0-..000u.' o 0. '00.. - 00. . 00" I.."!0~'.0~ 00.0000“- .Ioth‘oo. I”. ,o: .0.” ”QQIo.s-l¢00 ..v’ts 32000100....03002 ......050100 .0I... 021.133 .'.v.:. 00.0... 300 P03. .. o. |.... DIS-.3! 0r000.....0.u.;.al: .I.o 00 0. .‘r ol.'.....u-0' a oolJIH'r0O-\unf.l‘0”\i"ldtl 0“ .o .0. . D . . . . . . . 0 .... 0 I .4 0 It .3970 O. . . l)jo.0.l. I‘Vgtlvviv . 0 .. 0 . . . .51.-.! .0 ....JL . n . 0 0 0“; '5 .0. . .. 0 0". 0 0.... I 00.0. .0 0 '.000 VIIIH‘Ifiu'..000 000000-000C. . 2 \o 9. s '0 t... 0-0.,0 o 0200 tbo to . «I? .00.!o..v0.0 0.'60|.0.’o.9o .0. how .0. 1.! ..0'. .....o! 0031.. 0 o.. 50‘ .. .. I I . 00.. 0 ' II 0 . ~I0100 0 '00 .’|:0£0 ‘0‘- :'.-ID"'00D". I . 00.0 0o . .10....II'I. \ 0.000000.“ 0,00..'0.....OI.0O00. . .15... 7.1.01. ...... ....’..'I...s-.000.. ...... 9.0.0000... ...0- 0r c.0000! 0 .- ‘0 . O. o . o 00" .. - O. a - 00. 0'00. 000”.0I'C'0 .00 ..?">10 ....Foo 0 . .‘n. .. .3130. . 1.0.0.; 00 0 9. .CI 0A,..:. .0: . 0.. 1.... 3070.)... 0 33,0 .1 0.0... . 0 o .30 00...; . . . .0 .0.‘.' ... 0000. 0 P .. .0 ............'0 .o' ..00..I.£.:1..0'...00.. .5...) .3002 {€20.30....P.l:.....’...no0.... ...0 .... . .0....w.0o 0.00.. ol. ..0.. .00.... .3392... a 0.0'00 ....|£_.0!0.00o.. .x....7~$0?0¢|...;.3.002.3... 8.....0.... ...: ... ...7......).0I ...0... ...0 . .I. 0 ....Iv.. O0 4. . I. .10. '0 00.. ' 0.\v0ul. . no 0...“.0 ...o'Itwot'o .....oo“ 0:. .00. z. .30... 00.001t0! 0.5.0...- 0 .80”...! . c0 . 0 .0! 0 .000 ‘0 0 -000 .0. 001. .-Q. i ... 0.3.000. ’0VJH‘WV .‘0 0.5 '..I..o.. .0 3230;00:0330 p. .l- ...00. '0.0.00...J..0.0’0! ...... . 0 Y: . .0 ‘ .5.....00| .0. a... .00 300......900 ..0 I 0“.) 030.30.. 0.000 '00- 00.0.. 0:10 .' cur-.0 :0. I}./0\.9: la. .01...‘ I)- o 00 0..\ . f’0 .00. .0 0’a."0‘0.. .‘Q‘lvfi. I‘ol..§,v.... oi... 0. Oil 0.0..‘700 3.....IEI‘II 010.00 . .0. 1.:- o. .03.. 0010. ...}.I. . 0 0.00.00. “ 0'. 0‘0... 0.... .. . ‘0 0 .00. 0‘050' 00‘01’00 0» ...OIoOQ'“ 0“ IQI“0"0| . l I 0‘ ,0 0!. ~0o0 :43. o~7.?":!o.,o ’00 A...0'§o.0 4.0 “0.5. . 1.“ .0 Q 00 0 g u I .0 . 0| 0 .9. x \ 0 .0 0 ‘0 ' '1 at 00 , ‘0: o o.‘ .00 0 \Iflloo' O. :0.- 0.0.o0.v . ...-’1 ....U I. ,0» .0’0 I0 0. .101? 00.: .L0:~ ..0.0 000 t .. .. . 0 .‘5.0 I o I .r. . '0. o I: 2...: ......- 0.0!: ..l....~.§.00..)0 #500031; I 0.1... . . ... .a I . I... ..0‘! 0. 00 I. ' 0 .... ...-00"... 000..o..30.r.0 ....I . .9!!! 0!. ..I 0..- 0.... .03.... .0 a. o. . a .. :3; J. .0031... . a I 0! 00' ' s I ' 0 . . . . i I I. c O . . ... . ' 0 0 . . o 0. .0 m. . I I. 0 V'|I. '0 .000. ol...0..0..lo I0")00..~..0 0. 0o." 0V0§' Ip.‘ I..." 1.0. 0 0.0 O ..o.0o\b'o v.01... 00000 o‘Co 7000 o. 0 o I .oo... 0 . flouov o ...-'01,? ”0hacu0 .00II I .9. 00.0.80“ 00...0 ”-0.?- to .. .0 . 4 . "~... .0 I... 0.10 0.0.00 In 00 o. .62.. db 0 I ...: \ n ’ . 0. ”cut- I 300.101. 0000"th DIOQIIIII'IA.’ .Illl.l””".. 00"!" 9 t ‘ . . . . .0 . I. o 0 . -.l'o' .0: .0. 07.00. 0.. o, .o . . .. . .. JO 0 .. 0 0. 000 . .I .. .0. O ‘1. .0 .0007. . I . 0 II I 00: 0"f‘ . . ”I I .. . “on. r .0 .' 0.. I .Of'p. I. (00-00 ...r '5. 0‘ I..V.0... : it‘00i. 5!. .1 ... 0 000.. .It 0 .. 00..I'0..?.00M.J.0. 6...... ..uII..IH...d-0¢ ‘00 ITI .0 .5 0...! 0.00).. .0 ..l.-. .0. ... 0 0 0 r.. 8.! 0/0 000 0 U0 . .0 . O...» . U... ’40...“ .o 0!": ’ ...: .0 (I 0.. .00 9 00. .0. '.0 ‘Dl‘l 3.0.0 00'”.$O.5 Io'I-‘av.>d. .q. 0179.00 .00, 5| .0.‘-., .00: o\ov\9 00 .x 00.00‘. Co 00.. II :90 .‘. 0. . I .00 0!. 9.0 v 0.0:: 0.. O. ’0 I0 I I} 0. |' Q .00 o.o . 00! I0 .0 O ' I .‘ I). o I . ...o 0-4, . 0 . -3. 90 t- .. ....- Iv |.0.!0 3‘...» 0 1'... I . I'Dl0.‘,.0 0": .0». .. 00.1.. .000... ... .00992\C0..'0§T. I; 7 ...:00 p 0 Row! . e. .0 0. v. u. . 0: -.. . 0 I! A 00 .. 0 I50: .70 . .03... . ...u . 0. 0 1r -. no . - A . 0 . .'0.0' III . . . . I ..Y. I... ... 00.00.007.70?- !0r..)0001‘5.af.l.|31§03906:. . 0.0 00 . . . . .90... . .I... 0.. ... . o. ...... r . .00 00.0.0. I I . 00.....8 III. ... . I. ..0 0.0 0 0.0. .l . o . . . ‘000\ 0 .l..0 .OI00'I ,..‘000.'00 \ _‘ 01.0... . . I "'o.00 '. 3.40.00“: ’?’lo.0 I . . .00.)... . o o t 00 b..'-.oocl .-.000 .‘Os'. 0.0.. I; ”...-$00Hn‘irttQi-xhi.tog..r’ofl7‘0”,”“00402’....Aa.ol...0:0'00\0‘1’0.10F€£:.oo.f,0r0000.0.... -2370... .(0 .0). ..I. _-<.u.u‘v .100. ...).0033 IVSJH O 0.. - o 00 00.0-. .0 00-000 00’ -.P . '. . .00.\u00.v . ... I f 7. . 300070.10 000?..‘300? 0 . o . ... x 0.0 . ..\-.0\ .. up . 0: ..I .0 000 It I . a . n I -00.-..‘0 0'0 ‘01.. .‘O‘ .I'r.-n‘t '00.‘,‘~Vl\ o 0 o 0" Q' . -30..‘. ...‘7200.z‘.00o‘o'-,0000 05a. 0000. ...-‘0 0 I. 00 .o‘. 90 \.000 I ’0 '0 00 '0. . 0 I ‘0‘ 0o. .Ilo'. 0.0.30.0 . ..- ‘05.! 0 00 0 0:00.91300 :9 .000 00.... . . I» Q I) .4. . 01'. .0. I .. X I, ,’00."I o0 0. '0 l . 0 IO 9 O 1.. 0 0. 0.00 Don. I 0.70,. .’D~ ‘ 00 .. £401..--- G..o|0 0 0.0 “”00”...” ‘0'. 0. 00-....II-V .‘ . .0 l0 .03....‘t1...‘..’\x.i. :00... contociftgp a. 3-01010. .. 03/0010... , “F‘kao.bl0...\hv‘ a... 000D.’I.n.0.33l L‘ H.030 4 . o I. - . . ...-.0. v.0 . . ...; 0.790.? 00 x . .0. . 02-0 .01 . .I (I a . . .10.th ...rllvvnp I: o. 0. . 5- .0 - 0 0 0 .0 II I. O - .‘00000 '0. . ...... fi’or. U .f‘. V‘ "0boo — .00."... . 00 l .'.£60.O- -05!0\IQ0..P§00 In .4 .0.0 . I . 70....0000Q.‘ . O .étp... o0 . 0. u 0": .00 O I 0.30 . .0 . . 1:0 I .. . . II 0 .s.0 .0. 00 '0 .0 so 0.0 . 0 o I ‘I0 0’.I 0000 c '5) 00 0.0 l 0.. I. 5.0. 0...;00r0..0.. I-IoHv.00.0 1H‘.'l than-0:0...” 200.5. 0 D I... O . rihifirl’i .0: . 30:35.30. .0...‘. I .... ‘2: 0.. 8...)... .. 0.5709... . .. :9... up}... ......I..n.s.... 10.. 2 «Ia-.1! . .0 . .or. 9.1.2... 0‘ IV- .vl ..-. on. . o - o \. 0 00 n . . . 0 0 . . 0| . 0) it .050. \I0 ‘0..000£... 00 . 0. . I .40. . . .3 9.... ... .. 00 o. .0. 00. 0.0 r. .000 .0 0., Q. L .. . ... ... I- v . .0-0 .000. ‘00.... "I. . 0'. .1“ 0’0'0-0 0.0” “H.000 |I0O|D0 00‘ '0’! 0‘..- 0 1.001....- O! 0..."! 9'!” ‘L; 00.00., ....‘7‘..!' ." :1o '0‘ I0... I) l.’l 00Mv0". 000. 09.0.0H0'0:’.fl’0?.§u". 0.: .00- .v ‘2! v . I: 07! . 2 ..0. .00 ’ I o . II 0 80 Q 0. 0.0 I 0 up . 03.0 0.0l I 0 .000..u-00..o.0000'.'\0001 o 0".dr’\00100 0'0!" 11". 00.; :0. . rue-.0 .00.»... 0100 o. 0.0.... . 0 H 90.... . 0.. 0 000V“. 20. 0...... 00’0 [to P.0I ..‘I?1. .0.‘.....!.. 0......0 .. 0‘0....E.. C0. 0 .3... 09.00.09... 0. he. 1:06.. ...le . 0.. .0 .r ... ... .3300- ......I. .0400 . . u .... . .... . ....r 2.! -00 (vi..'. . I! 4.0 01 . o0...0?\ O. . '0 ... 0 '0.- . I. 000. . 0 . 0 00.000 0’...- |.I 00 o.‘ 9.30.9010. Euro. . I 0.30.0 9..-. . 5:00p... . o '0 0'... 000.00.. 002000.90- 6021:9321000010 I O D .5 0. _ 3 :33... o. . .024 0 .00? .0. b. .0... 00.. . I ..000 00:. 0.4 ..VI .0 . . ..9 O: ' '11, . 00 . I. . I .. II 0 ’0’ 0.1“... g). '\'o ...M'f1'03'b...‘ Ono ‘,‘olia". 0 ..sz..-.. .0 0-. .’.00 '0:- ..00..o0"00 Oo.'}'2 o f o .00f0o\.0+. ...3'0. .0 ...-0. 0.. ....0 7'30 02.00. 000 . o... .0 . .. :0 I- 0 It :5 . 0.00.00" 0| .0 .0 bl.‘ O I S) ‘ l . tQ. VUQQ Yo 19°00? I04.0.Ih ..OO‘..: '2 «.30 f.'( 00.002.000S3I o0.3o‘..‘¥0}v 00.0, ..Io..o¢ '0? 0.. . .. ., 00.0. o . 0., 0 . .....l‘ .0 ‘L. r.0>0 cl. 40.0....7 0.1 .‘ 0 .0! O- In .0 '0‘ io.....l§l’u."..ol .0.O 0.x..." 0o. !.2.'0 .0, .00.. 0.20.0... .. 06!!‘.I..$ .7...70’Il ..00. 9": 2.! 40.....lb r...0.r\v: 0. I: .00 0 . . .0 0| ... 00. . 0. 0..) 0.00.0.3? I. I 000.. 0". .0 500-310.3010." " .0 0.. 0., i. . ‘Po 9.00.50: .00... ......03’. P 0.0.000. I.- ..08£0 ..Po.‘!..o ...IOSRI .00 0’;‘.¢ -0... 0 .0000 0, f....0.. at . ..f .00....3. 00 -.. I! 0 Il..v L. 0|. 10.0. .0- 100-0.. uroiu I0 00. 0-- .-00}0000 . "‘VI'¢011’0 (0'00'00 . . . 0 II." 3‘ "P 00 ‘- ‘0'!o.’000‘..0 '0 C'Q'I 0‘. not 7’7 . '00.".c ’0..§.0.4o’0.o L .I" $0.'Io 0.0.. ‘.0o 0 .0 35.5.. 0... Q ’0... 0| t"........’ I -' . UV 0. 0. . I‘lo .0: '0) I. If our 0 '0‘. 0 ‘0 . 00' 000 000300 1.0.00) ".0 0‘ 0"..."10' .- '80.l0.. 0. 0 'O .9 79......0. of .t 00...: I 30' .t0'o.0"..070..0. 8050:3003-5081:310 ... l0.. L920 0. I. ... .90... ...!!00 ...n... o. 07 ...n. A I .0. . .0 0.0!... . I). ‘3 . 1?. I 0.0 .0 .to. I. I 00 .00] .0. '0’- O.!'I"0000.0 “.gs'IOolt‘ O '-.o-000/.r0 ..0000.0 0%.). .33.").00‘0 V0.00 O.0.00010.0§00"’f Oil-£00010?- .'0 I ...-I ‘00.. O 0.3....Cotvrfii. 00.3.4000.-. 0000". 0.100.000 II. I: $000.75.!70o...9... . 20001.3, ..1? ...- . 0.... 0o 0.30. . I'Vro.r‘l 2-. II. '.0000. I900. 0.70.600; ‘ con-....IIZl. . 8.. 'IorL- . so 0"'-In' r50... II.'..I:II ... 09:"- 90'3I}! . L ..t: 0:... O ...... 00:... ..rov 000.30....080 o.)7.0{ 19.00.... 0 .9 2 ..Jrl .'. 'Dp I000 I .00.»! IIIr .ul,v..ou'0o'0 I'..O..-.. -(luo'r. 0.3.0....333’33 0 00.13001).0 0.. 0:0. .0. -.Il . u NEW RS ITYLIBRAR IIIIIIIIIIIIIIIIIIIIIIIIIIIIII III II 3 1293 008811 IIIIIIII This is to certify that the dissertation entitled “‘4‘“; I75 Fag-$.53. PxostaglandiLEL-i-nd-ueed Ca++ mobilization in rabbit- in “wt cortical collecting tubule cells and swiss 3T3 cells presented by Ping Shi has been accepted towards fulfillment of the requirements for Ph.D Biochemistry degree in ma... 1. 54% Major professor July, 26, 1993 Date MS U L: an Affirmative Action/Equal Opportunity Institution 0- 12771 ...—... _...__-.'.- . LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution encircwngms-DJ Ca++ MOBILIZATION INDUCED BY PROSTAGLANDIN’Eb IN RABBIT CORTICAL COLLECTING TUBULE CELLS AND SWISS 3T3 CELLS BY Ping Shi A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1993 ABSTRACTS Ca++ MOBILIZATION INDUCED BY PROSTAGLANDIN E2 IN RABBIT CORTICAL COLLECTING TUBULE CELLS AND SWISS 3T3 CELLS BY Ping Shi Prostaglandin E2 (PGEZ) produces a broad range of biological actions in diverse tissues through its binding to specific receptors on plasma membranes. PGE receptors are pharmacologically subdivided into three subtypes, EPl, EP2, and EP3, which are suggested to be coupled to different signal transduction pathways. PGE2 has been shown to mobilize intracellular Ca++ through activation of phospholipase C (PLC) in a number of tissues and cells which possess EPl receptors. Reported here are studies of the effect of PGE2 on Ca‘H' mobilization in rabbit cortical collecting tubule (RCCT) cells and in Swiss 3T3 cells. Also reported is our attempt to clone a cDNA for the PGE receptor involved in Ca++ mobilization using the Xenopus oocyte expression system. PGE2 plays an important physiological role in transport activities in mammalian kidneys. Our study showed that PGEZ mobilizes intracellular Ca'H' in RCC‘I’ cells as determined by digital fluorescence imaging. Ca++ mobilization by RCCT cells occurs in response to the naturally occurring 15-S-PGE2 but not 15-R-PGE2. Moreover, the effect of PGE2 to mobilize Ca++ is partially desensitized by PGEZ, sulprostone, and PGFZa, but not by PGDZ. AH6809, an EPl PGE receptor antagonist was found to inhibit PGEz-induced (2a++ mobilization. These results suggest that PGE2 induces Ca++ mobilization through an EPI- like PGE receptor. Using digital fluorescence imaging in combination with cell-specific staining, we have found that PGEz-induced Ca++ mobilization occurs in principal cells as well as intercalated cells, suggesting possible roles for PGE2 in 11+ and/or anion transport in intercalated cells, in addition to their demonstrated roles in water reabsorption and Na+ flux in principal cells. In Swiss 3T3 cells where PGEZ was known to have a synergistic effect on cell proliferation in the presence of low concentrations of insulin, we found that PGE2 stimulates phosphatidylinositol hydrolysis and intracellular Ca++ mobilization through the same receptor that interacts with PGFZa. Expression cloning of a cDNA for the PGE receptor involved in Ca++ mobilization in 3T3 cells led to the isolation of a protein named GING. A search of GenBank database showed GING is a member of the 14-3-3 protein family. Expression of GING in COS-1 cells potentiates Ca++ mobilization occurring in response to PGEZ, but does not result in a corresponding IPs formation. It is not clear if the potentiating effect of GING on Ca++ mobilization in oocytes and COS-1 cells has any physiological significance or is an artifact involving overexpression of GING in vitro. ACKNOWLEDGEMENTS I thank Dr. William L. Smith for his continued professional guidance, his support and his patience during my graduate training. I thank the members of my thesis committee, Drs. Steve Triezenberg, Shelage Ferguson-Miller, Zachary Burton, and John Linz, for their scientific insight and their encouragement. Special thanks go to Drs. Dave L. DeWitt, Lois Arend, and Maria Burnadowska-Hledin, to whom I am much indebted for their generous help and coaching during the early years of the journey. It also goes to Dr. Gilad Rimon for his comradeship and friendship when working on the arduous cloning project, whose humor brightened up each of those days. I thank all the members of our joint lab, whose effort made the lab a dynamic and stimulating place. Among them I thank particularly Teru Shimokawa, Dave McCoy, Dave LeVier, Odette M. S. Laneuville, and Babara Olsen. I thank people in the department whose technical assistance was indispensable. Among them, John Sell for his time and patience to help me with the ACAS cytometer; Don Herrington for his continued assistance to animal handling. Above all, I thank those of my special Chinese and American friends, who helped to bring out the best in me, who sparkled my life along the years (especially during the darkest part of the "tunnel"), who made my experience here a worthwhile one, and without whom I would not have made it. TABLE OF CONTENTS Page LIST OF TABLES... ................... ........ .......... ...Vii LIST OF FIGURES. .......... . ......... ....................Viii ABBREVIATION.............. ..... ..........................xii CHAPTER I. LITERATURE REVIEW..................................1 II. III. IV. Prostaglandins and Their Physiological Actions in the Kidney...................................2 The family of G Protein-coupled Seven- Transmenbrane-Segment Receptors................14 Receptor-Mediated Inositol Trisphosphate Formation and Subsequent Ca + Mobilization....21 Refenrence........................................33 PROSTAGLANDIN Ez-INDUCED Ca++ MOBILIZATION IN SUBTYPES OF RABBIT CORTICAL COLLECTING TUBULE CELLSOO...O0......O00......00.00.00.000000040 Methods ......................... ..................42 Results....... ............. .......................48 Discussion...... ....... ...........................94 Reference.... ....... . ..... ........................98 PROSTAGLANDIN EZIACTIVATES THE PHOSPHOLIPASE C PATHWAY IN SWISS 3T3 CELLS THROUGH THE SAME RECEPTOR THAT INTERACTS WITH PROSTAGLANDIN’Fba....100 Methads O O O O O O 0000000000 O O O O O O O O O O O I O O O O O O O O O C O O O O 102 Results 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O I O O O O O O O O O O 105 Discussion ......................... . ....... ......140 ReferenceOOOOOOOOOOO...0.0000......00.0.000000000144 EXPRESSION CLONING OF A 14-3-3 PROTEIN THAT POTENTIATES Ca++ MOBILIZATION IN XENOPUS OOCYTES IN RESPONSE TO PROSTAGLANDIN E2...........147 Methods..........................................149 Resu1ts. O O O O O ....... O O O O O O O O O O O O O O O O I O O O O O O O O O O O O 154 DiSCUSSion. O O ........... O O O O O O O I O O O O O O O O O O O O O O O I O 188 Reference 0 I O O o O O O 000000000000 O O O O O O O O O O O O O O O O O O O O 19 3 vi LIST OF TABLES Page The immunostaining and lectin staining of freshly isolated RCCT cells........................61 Responses to PGE2 and AVP in both the principal and intercalated cells.............................76 Responses to PGE2 and AVP in subtype B intercalated cells ............. ......... ........... 77 Ca'H’ mobilization in response to PGE2 and serotonin in transfected COS-1 cells..............182 Ca++ mobilization in response to serotonin in transfected COS-1 cells........................183 vfi LIST OF FIGURES Page Chapter I 1 Prostaglandin biosynthetic pathway..................3 2 The mammalian nephron ........... ...... ..... . ........ 7 3 Structure of the hamster 82 -adrenergic receptor in lipid bilayer..........................19 4 The dual signaling pathway of IP3/Ca++ and DAG/PKC........................ ................ 23 Chapter II 1 Indirect immunostaining of rabbit renal cortex With IgG3(rct-3O)oocoooooooooooooooo000000.057 2. Indirect immunostaining of rabbit renal corteXt With IgG3(DT017)oooooooooooooooooooo ooooooo 59 3. Fluorescence digital image of freshly isolated RCCT cells stained with IgG3Crct-30)...............62 4. Fluorescence digital image of freshly isolated RCCT cells stained with IgG3(DT.17)................64 5. Fluorescence digital image of freshly isolated RCCT cells Stained With FITC-PNAOOOOOOOOOO00.......66 6. Ca++ mobilization in RCCT cells in response to agonists as observed by fluorescence digital imaging......... .......... . ..... ............. ..... .68 7. Representative tracings of individual RCCT cells in response to PGEZ and AVP..................7O vfii 10. 11. 12. 13. 14. 15. 16. 17. Subpopulations of RCCT cells that mobilize Ca++ in response to PGEZ...ococoa-cooooooooooooooooooooo72 Strategy of identifying subtypes of RCCT cells that mobilize Ca++ in response to agonists........74 Dose response to PGE2 in RCCT cells...............78 The stereospecificity of PGEz-induced Ca++ mObilizationOOOOO......OOOOOOOOOOOOOOOO00......0.080 The effect of flurbiprofen on PGEz-induced Ca++.mobilization in RCCT cells...................82 Desensitization of PGEz-induced Ca++ mobilization by various prostaglandins and AVP....0......0......0.00.00.00.00..000000000084 The effect of AH6809 on PGEZ-induced Ca++ mobilization in RCCT cells and 28A cells..........86 The effect of depletion of extracellular Ca++ on PGEZ-induced Ca++ mobilization in RCCT cells. ................... ......‘C... ..... ....88 The effect of TPA on PGEZ- and AVP-induced Ca++ mobilization in RCCT cells............ ....... 90 The effect of pertussis toxin on PGEb-induced Ca++ mobilization in RCCT cells and 28A cells.....92 Chapter III 1. Representative tracings of Ca++ mobilization in confluent quiescent 3T3 cell in response to PGEZand PGanOOOOOOO0....O00.0.0000000000000000110 Dose dependence of the responses to PGEQ and PGFZa in confluent quiescent 3T3 cells. . . . . . . ...... 112 PGEZ- and PGan-induced inositol phosphates formation in confluent quiescent 3T3 cells... ..... 114 The effect of depletion of PKC on PGEZ- and PGFZa-induced inositol phosphates formation ........ 116 The effect of depletion of PKC on PGEZ- and ix 10. 11. 12. 13. 14. 15. PGFZa-induced Ca++ mobilization. . . . . . . . . . . . . . . . . . . . 118 Dose dependence of PGE2- and PGFZa-induced inositol phosphates formation in PKG-depleted 3T3 ceIISOOOOOOOOO0.0......OOOOOOOOOOOOO00.......0120 Time course of PGEz-induced formation of various inositol phosphates in PKG-depleted 3T3 ceIISOOOOOOOO0.0.0......OOOOOOOOOOOOOOOOOO0.0.122 Stimulation of inositol phosphates formation by 15-S-PGE21and 15-R-PGE2 in PRC-depleted 3T3 eel-18.0.00...0.0....O.....0.000.000.0000000000124 The effect of sulprostone on inositol phosphates formation in PRC-depleted 3T3 cells...............126 The effect of AH6809 on PGEz-induced inositol phosphates formation in PKC-depleted 3T3 ceIISOOOOOOO0....OO......OOOOOOOOOO0.0.0.0....128 The effect of pertussis toxin on PGEZ-induced inositol phsophates formation in PRC-depleted 3T3 cell-8000000000oooooooooooooooooo000.000.00.000130 The effect of extracellular Ca++ on PGEf- induced Ca+I mobilization in PRC-depleted 3T3 cells...................C.....................132 Dose dependence of PGE2- and PGFZa-induced Ca++ mobilization in PKC-depleted 3T3 cells.......134 The non-additive effect of maximal doses of P6132 and PGan on Ca++ mobilization and inositol phosphates formation in PRC-depleted 3T3 cells............................136 Effect of pretreatment with PGFZa on Ca++ mobilization in confluent quiescent 3T3 cells.....138 Chapter IV 1. The sib—selection cloning strategy using the oocyte expression system......................160 Ca++ mobilization in response to PGE2 in oocytes injected with a pool of cDNAs.............162 10. 11. 12. 13. Electrophoresis of RNA transcripts transcribed by SP6 and T7 polymerase from the SPE I fragment.......0.0..........OOOOOOOOOOOOOOOOO...0.0164 Traces of Ca++ mobilization in oocytes injected withSP6andT7transcripts.........................166 The nucleotide sequence of GING....................168 The amino acid sequence of GING and its hydropathyplot.............................. ...... 170 Sequence comparison of GING protein and bovine 14-3-3 proteinOOOOOOOOOOOOOOOOO00.0.0.0...00......172 Northern blot analysis of GING in 3T3 cells. ....... 174 In vitro translation of GING using rabbit reticulocyte lysate system. . . . . . . . . . . . . . . . . . ....... 176 Plasmids containing GING and a cDNA for the 5-HT1 serotonin receptoroooooooooooooooooeooo oooooo 178 Traces of Ca++ mobilization in response to agonists in transfected COS-1 cells........... ..... 180 Inotsitol phosphates formation in response to PGEbiand serotonin in transfected COS-1 cells......184 The effect of GING protein on inositol phosphates formation in response to serotonin in ST(+) and.G(-)/G(+) cotransfected COS-1 cells...186 xi AUIBRETVLAIICHQS Abbreviations are: PG, prostaglandin; AVP, arginine vasoppressin; cAMP, adenosine 3',5’-cyclic :monophosphate; RCCT, rabbit cortical collecting tubule; PIPZ, phosphotidylinositol 4,5-phosphate; 1P3, inositol 1,4,5- trisphosphate; IPs, inositol phosphates; PC, phosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; DAG, diacylglycerol; PLC, phospholipase C; PKC, protein kinase C, TPA, 12-0- tetradecanoylphorbol-13—acetate; PTX, pertussis toxin; ER, endoplasmic reticulum; MT, mitochondria; FITC, fluorescein isothiocyanate; FBP, flurbiprofen; DMEM, Dulbecco's modified Eagle medium; FCS, fetal calf serum; SSS, simplified saline solution; MBS, modified Barth's solution. xfi CHAPTER ONE LITERATURE REVIEW The focus of this review is first, on aspects of the biochemistry of prostaglandins and their physiological actions in the kidney; and second, on the rapidly-expanding family of G-protein coupled receptors and their receptor-mediated signal transduction pathways, especially the inositol trisphosphate(IP3) /Ca++ pathway. The concept has been well developed that prostaglandins function as local hormones to elicit their effect through specific cell surface receptors. I will describe how the collecting tubule of kidney has been used as a model system to study the inhibitory action of prostaglandin E2 (PGEZ) on water reabsorption occurring in response to arginine vasopressin (AVP) . Studies in this system have led to the conclusion that there are two G protein- coupled receptors involved in mediating the action of PGE2. Since a large body of biochemical evidence indicates that prostaglandin receptors belong to the G protein-coupled receptor family, an overview of the transmembrane signaling system involving these receptors will be given, with an emphasis on the phospholipase C signaling mechanism involving inositol trisphosphate and Ca++. Prostaglandins and Their Physiological Actions in the Kidney WM Prostaglandins (PG) are a group of oxygenated derivatives of 20-carbon polyunsaturated fatty’ acids. These compounds. and. thromboxane, which. are products resulting from the action of prostaglandin endoperoxide H (PGH) synthase (Fig.1), are collectively known as prostanoids. All the prostaglandins contain a cyclopentane ring, with differences in the oxygen-containing substituents giving rise to PGDZ, PGEZ, PGFZa, and P612. The numeric subscript indicates the number of carbon-carbon double bonds present in the molecules (1). As shown in Fig.1, the prostaglandin biosynthesis occurs in three steps. The first step is the release of the precursor arachidonic acid fromIglycerophospholipids by phospholipases. This is an important regulatory step since the concentration of free arachidonic acid in the cell is too low (<1 pH) for prostaglandin formation to occur, unless it increases in response to hormonal stimuli (2-4) . Arachidonic acid is mainly released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) via the action of phospholipase A2, although some arachidonate is also derived from phosphatidylinositol (PI) by the sequential actions of 2 Figure 1. The biosynthetic pathway of prostaglandins. HORMONE / ’ CELL MEMBRANE PHOSPHOUPIO (PLPCPE). / W CHOLESTEROL ESTER I LipASaS) ARACHIOONIC ACID CYCLOOXYGENASE PG“ 202 I SYNTHASE COOH 0,- ~/‘=\/\/ o PEROXIOASE coon HO / \H ‘ ‘MCOOH SYNTHASES 0 P612 0 0' 9502 '0“ ' VYW OH . 0 OH COOH . ~/"'\/\/ 9 ’Wcoou 110‘ W 3 ' OH ‘FWCOOH PGE2m fl) 5 phospholipase C (PLC) and diacylglycerol lipase (5). The second step in prostanoid biosynthesis is the oxygenation of free arachidonic acid by PGH synthase. PGH synthase is a membrane-bound protein which is associated with the endoplasmic membrane in most cells (6). This enzyme has two catalytic activities: (a) a cyclooxygenase activity which catalyzes a bis-oxygenation reaction, forming the 15- hydroperoxy-prostaglandin endoperoxide PGGZ; and (b) a hydroperoxidase activity which reduces the 15-hydroperoxy moiety of PGG2'to form PGHZ (7). Both activities require heme (5) . An important characteristic of the cyclooxyenase activity is that it is specifically inhibited by aspirin and.a group of related non-steroidal anti-inflammatory drugs including flurbiprofen (FBP) and indomethacin (8). The third step in prostanoid synthesis is the conversion of PGH2 to a biologically active endproduct such as PGEZ, PGFZa PGDZ, PGIZ or thromboxane A2 (TxAz) . Prostaglandin synthesis is cell-specific. A given cell can only form one particular endproduct (9) . The formation of PGDZ is catalyzed by PGH-PGD isomerase (10). The formation of PGE2 requires reduced glutathione and is catalyzed by PGH-PGE isomerase (11) . In contrast to the isomeration, PGFZG synthesis requires a two- electron reduction of PGH2 via PGF reductase (12) . Information about these enzymes is still limited, although their respective activities have been partially purified. PGI2 and TxA2 synthase have been purified to homogeneity (13,14), both 6 enzymes being membrane-bound hemoproteins of the cytochrome P- 450 class. The concentration of prostanoids in plasma is very low (< 103 M) (15). Prostaglandins are degraded into inactive forms during a single pass through the circulation (16) . On the other hand, prostanoid synthesis is not restricted to a central endocrine organ, but rather occurs in most organs and tissues (9, 17) . Therefore, the concept has been developed that prostaglandins are local hormones, which act on parent cells or neighboring cells in an autocrine or paracrine fashion, respectively, to coordinate the actions of circulating hormones that stimulate prostaglandin synthesis (7). Physiological Actions of Prostaglandins in the Kidney. Prostaglandins are involved in the regulation of a wide variety' of cellular’ activities including' :metabolism, secretion, muscle contraction, and cell differentiation and proliferation (18) . One of the model systems in which the role of prostaglandins has been studied.in detail is the process of water and sodium transport in collecting tubule cells of mammalian kidney (19). Fig.2 is a diagram of a nephron. The final segment of the tubule, the collecting tubule, is the critical site where water and sodium reabsorption are regulated. By immunostaining to localize the PGH synthase, and by measuring prostaglandin synthesis in different tubule segments (20,21), it has been found that there are seven different cell types that are Figure 2. The diagram of a mammalian nephron. (G. glomerulus; EA, efferent artirioles; AA, afferent arterioles; IA, intralobular artery; PCT, proximal convoluted tubule; PR, pars recta; DTL, descending thin limb; ATL, ascending thin limb; MTAL, medullary thick ascending limb; CTAL, cortical thick ascending limb; DCT, distal convoluted tubule; CCT, cortical collecting tubule; PCT, papillary collecting tubule; IC, interstitial cells). The darkened areas represent sites of prostaglandin synthesis. CCT CORTEX MEDUllA MC'I' PAPILLA PCT 9 capable of prostaglandin synthesis (darkened areas in Fig.2). Among them, the epithelial cells of the collecting tubule, especially the collecting tubule, have the greatest capacity to form prostaglandins. Moreover, it was shown that PGE2 was the major form of prostaglandin synthesized by dissected rabbit cortical collecting tubules (22,23). Grantham and Orloff were the first to demonstrate the effect of PGE on. tubular function ‘using perfused microdissected cortical collecting tubules (24) . They observed that a low concentration of PGE1 inhibited water reabsorption stimulated by the circulating hormone arginine vasopressin (AVP) , while at higher concentrations, PGEl, by itself, stimulated water reabsorption. When cAMP or forskolin which stimulates cAMP formation was used to pretreat the tubules, no inhibitory effect of PGElion'water flow was seen (24). Since cAMP is believed to be the second messenger that mediates water reabsorption (25), it 'was postulated. that at low concentrations , PGEI inhibited AVP-stimulated cAMP accumulation, and at higher concentrations PGEl stimulated cAMP formation. This hypothesis was later substantiated by Sonnenberg and Smith (26) who studied the inhibitory regulation by PGE2 of AVP-induced cAMP formation in immunodissected rabbit cortical collecting tubule (RCCT) cells. They showed that at low concentrations (5 10'8 M), PGEZ inhibits AVP-induced cAMP accumulation in pure populations of RCCT cells. The inhibitory effect was blocked by pretreatment 10 of cells with pertussis toxin, indicating the involvement of the inhibitory guanine nucleotide-binding regulatory protein (Gi) . Moreover, PGEZ was found to inhibit the AVP-stimulated adenylate cyclase activity directly in membranes isolated from RCCT cells. At higher concentrations (above 10‘7 M) PGEZ stimulates cAMP synthesis, presumably by activating adenylate cyclase via the stimulatory guanine nucleotide-binding regulatory protein (Gs) . Evidence was also obtained that the stimulatory and inhibitory responses are mediated by two subtypes of PGE receptors coupled respectively to G8 and Gi (27) . Two binding activities were detected in membranes of isolated cells. The one with high affinity (Kd=10 nM) only existed in fresh cells but disappeared when RCCT cells were cultured for several days. Curiously, the Kd of the higher affinity receptor was decreased in the presence of GTP (although for most G protein coupled receptors Kd values are increased by GTP). The lower affinity PGE receptor could be detected in RCCT cells cultured for several days (27); the Kd value for binding to this receptor was increased in the presence of GTP analog. The dual effects of PGE2 on cAMP formation are pharmacologically distinct, since sulprostone, a PGE2 analog, is equipotent to PGEZ in its inhibitory effect, but fails to stimulate cAMP formation (26). These results obtained with isolated collecting tubule cells demonstrated that the inhibitory and stimulatory effects of PGE2 on cAMP formation are mediated by two different PGE-specific receptors 11 that are functionally coupled to Gi and G8, respectively. The results obtained with RCCT cells agree with the observations Grantham and Orloff made on microdissected collecting tubules. In addition to cAMP as the major mediator of water reabsorption, Breyer et al. (28) observed with perfused microdissected tubules that PGE2 releases Ca++ from intracellular stores, suggesting that PGE2 might activate a phospholipase C pathway, resulting in activation of protein kinase C (PKC) (as described below). Indeed, they found that staurosporine, an inhibitor of PKC, relieved the inhibitory effect of PGE2 on AVP-stimulated water flow (28). Other studies have also indicated that activation of PKC down- regulates AVP-induced cAMP accumulation in cultured rabbit collecting tubule cells (29). Furthermore, when collecting tubules were pretreated with phorbol ester or diacylglycerol that activate PKC, water flow stimulated by both AVP and cAMP analogs was inhibited (30); these findings suggest that activation of PKC can inhibit water flow at a site distal to cAMP accumulation. When collecting tubules were pretreated with PGEZ, results were obtained which were similar to those obtained with phorbol ester or diacylglycerol treatments (28) . Therefore, the PGEz-induced activation of phospholipase C pathway leading to activation of PKC can be another mechanism by which PGE2 inhibits AVP and cAMP-induced water flow. ass cation of Pros a landi Rece tors. Like other 12 hormones and growth factors, prostaglandins elicit their effects by interacting with specific cell surface receptors. Coleman et al.(31) developed a system for the classification of prostanoid receptors, by comparing the rank order of potency of agonists, and the affinities of receptor-blocking drugs (antagonists) on smooth muscle preparations. They found evidence for five major types of prostanoid receptors for PGDZ, PGEZ, PGFZa, PG12 and TxAz, designated as DP, EP, FP, IP and TP receptors, respectively. For PGEZ, they proposed three subtypes of receptors, which are defined as EP1, EP2, and EP3, based on their coupling to different signal transduction pathways and on their pharmacological profiles. EP1 receptors are coupled to the inositol trisphosphate/diacylglycerol pathway to mobilize intracellular Ca++ and to activate PKC. Its selective agonists include sulprostone and 17-phenyl-w- trinor-PGEZ; its selective antagonists include SC19220 and AH6809. EP2 receptors are coupled to adenylate cyclase to stimulate cAMP formation. Butaprost is a selective agonist. EP3 receptors are coupled to adenylate cyclase to inhibit the cAMP formation, but may also be coupled to the same pathway as EP1 receptors. EP3 selective agonists include sulprostone, enprostil, GR63799 and M828767. In smooth muscle preparations, the EP1 receptors mediate muscle contraction, while the EP2 receptors mediate muscle relaxation. A cDNA for an EP3 receptor has been isolated recently from a mouse lung cDNA library. This EP3 receptor cDNA has an open reading frame 13 encoding 365 amino acid residues (32). As expected, sequence analysis indicates that this receptor belongs to the family of receptors which mediate their actions via G proteins. The in vitro expression of the cDNA, encoding’ the EP1 receptor demonstrated that it is solely coupled to the inhibition of cAMP accumulation. Northern blot analysis showed the tissue most highly expressing EP3 mRNA is kidney, in which PGEZ exerts an inhibitory effect on water reabsoption and sodium reabsorption. Using this cDNA as the probe, a EP2 receptor cDNA was then isolated (33) from a mouse lung cDNA library, encoding a peptide of 513 amino acids. Within the transmembranezdomains, EP2 receptor is 36.2% identical to that of the EP3 receptor. In contrast to EP3, EP2 is only marginally expressed in the kidney. The Family of G Protein-coupled Seven- Transnenbrnne-Segnente Receptors MW]! A transmembrane signaling system which transduces the external stimuli into intracellular signals is very important for intercellular communication in multicellular organisms. Extensive studies in this field since 1960 revealed that one such system consists of three. distinct. components associated. with. the jplasma membrane: (a) cell surface :receptors that. recognize and interact with extracellular signals such as light, odorants, hormones (peptide or autacoid), and neurotransmitters (34); (b) a guanine nucleotide-binding regulatory protein, or G protein that is coupled both *p1346Xrecephad effector (35); and (c) effector enzymes exposed to the cytoplasmic surface including adenylate cyclase which generates the second messenger cAMP (36), phospholipase C (PLC) which forms inositol trisphosphate (1P3) and diacylglycerol (DAG) (37), visual cGMP phosphodiesterase (38) and ion channels (39). To date about 90 kinds of pharmacologically and/or molecularly distinct receptors have been identified as belonging to the family of G protein-coupled receptors (40). The existence of such a transmembrane signaling system makes it possible for an individual cell to adjust its cellular activities to changes 14 15 of the extracellular environment, which is essential for a multicellular organism to coordinate and integrate the functions of the various cells it is composed of. Role O: G proteins. G proteins serve as transducers between receptors and effectors. G proteins are so named because they bind and hydrolyze GTP (35). The first evidence for this came from the work of Rodbell who observed that GTP is required for hormonal activation of adenylate cyclase in isolated plasma membranes (41). This discovery led to the purification and eventually the cloning of the first. G protein, the adenylate cyclase-stimulatory G protein (GQ tar Gilman et al.(42,43). The mechanism of G protein signaling is complicated. All G proteins are trimers consisting of three tightly associated subunits: a, B, and 'y (1:1:1). Upon interaction with an agonist-occupied receptor, a conformational change in the G protein occurs, which results in the exchange of bound GDP for GTP by the a-subunit. This GTP-bound a-subunit subsequently dissociates from the By dimer, acting as the active form that interacts with.and stimulates effector molecules. Bound GTP is slowly hydrolyzed to GDP by the GTPase activity associated with the a-subunit. The resulting a-GDP complex then reassociates with the By-subunits to complete the "G protein cycle”. The fact that one receptor can activate many'G protein molecules and that one activated a-subunit (i.e. (I‘l-GTP) can stimulate many effector molecules leads to amplification of 16 the initial signal (35). Differences in the primary structure of a-subunits distinguish the various G proteins. Among them are G8, which stimulates adenylate cyclase; G- which is involved in u inhibiting adenylate cyclase and stimulating ion channels; GU the visual G protein which activates cGMP phosphodiesterase; Go, which stimulates ion channels; and GP' which activates phospholipases (39). G protein a-subunits can be substrates for ADP-ribosylation.by bacterial toxins such as cholera toxin (for GB) or pertussis toxin (for Si and Go) . ADP-ribosylation of <3 permanently activates this protein, whereas ADP- ribosylation of Gi or Go prevents its activation (35) . The Stgucture of G Protein-coupled Begeptors. The first purification of a G protein-coupled hormone receptor, that of the:fl-adrenergic receptor, was achieved.by Lefkowitz et al. in 1981 (44), and subsequent cloning of its gene and cDNA were reported in 1986 (45). Since then, many receptors of this family have been cloned, largely due to the introduction and improvements of expression cloning techniques (46). To date, over 150 receptors have been isolated and identified. Sequence analysis of these receptors has revealed that they all share sequence homology, and they all have the topography of seven.membrane spanning regions, represented by seven stretches of 20-28 highly hydrophobic amino acids (34). This topography is very similar to that of bacterial 17 rhodopsin, a purple membrane protein of Halobacterium halobium, for which the presence of seven a-helical domains spanning the lipid bilayer has been observed by electron microscopy (47). Based on the hydrophobicity profiles and the analogy to bacteria rhodopsin, a model (Fig.3) was proposed by Dixon and Lefkowitz for the orientation of the fl-adrenergic receptor within the membrane (45). This unique structure of membrane spanning G protein-coupled receptors reflects their role in transmembrane signaling, which involves interacting with the extracellular stimuli and with a G protein located on the cytoplasmic side of the plasma membrane. When comparing sequences of receptors within the G protein-coupled receptor family, it is recognized that the transmembrane regions are the most conserved, while the N- and C-terminal regions and the cytoplasmic loop connecting transmembrane segments V and VI are quite divergent (48). By constructing chimeric molecules comprised of portions of the a- and B- adrenergic receptors, it was found that G protein coupling is interchangeable when the intracellular cytoplasmic loop between transmembrane domain V-VI is exchanged (49) . This finding suggests that the intracellular loop between V-VI plays functional roles in G protein coupling. The sequence conservation in transmembrane regions suggested that these :regions might be involved in the ligand binding. This hypothesis was confirmed by Lefkowitz et a1. (34) in studies of :receptor chimeras. Ligand binding has been found to depend on critical amino acids present in several of the transmembrane 18 domains. These domains are thought to form a cluster to accomodate the incoming ligands. 19 Figure 3. Structure of the hamster Bz-adrenergic receptor as within in the lipid bilayer (boxed). Functionally important amino acids are indicated by different symbols. The seven transmembrane regions are numbered I-IIV from left to right. Abbreviations for the symbols: CHO, N-linked oligosaccharide; PKA, cAMP-dependent protein kinase; BARK, B-adrenergic receptor kinase. - 25 «0.750%. U_Omm2mmo<.ua O O O O O O :nuomolmoilom /\< u 2:; Os; 0 u .3; S... U u ESTES . E 27%; O 3.52.5..5233 I Receptor-mediated Inositol Trisphosphate Formation and Subsequent Ca++ Mobilization hgrmohe-induced Formation of Inositol Irisphgsphahe. Extracellular signals detected by cell surface receptors are translated and amplified into different intracellular second messengers. One pair of messengers is inositol 1,4,5- trisphosphate (1P3) plus diacylglycerol (DAG). They form two limbs of a single signaling pathway, with 1P3 mobilizing calcium from intracellular stores and DAG activating protein kinase C (Fig.4). Together IP3iand DAG initiate a cascade of cellular responses (37). The key step in this signaling system is a receptor- induced activation of phospholipase C (PLC) which catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP§)'to generate both 1P5 and DAG. Ten years ago, Agranoff et al.(50) found that stimulation of platelets with thrombin resulted in a rapid increase in the level of IP3, coinciding with a fall in the level of PIPZ. This was the first direct evidence suggesting that a hormone could stimulate the breakdown of (polyphosphoinositides via a phospholipase. Many, if not all, cell types are now known to exhibit hormone-induced phosphotidylinositol turnover (51-53) . Moreover, there is Iconsiderable evidence that a G protein is involved in this 21 22 receptor-mediated process. It was demonstrated that 1P3 formation was stimulated by the addition of GTP'yS, a non- hydrolyzable GTP analog, to permeablized cells (54). NaAlF4, a compound which persistently activates G protein, also stimulates IP3 formation (55). In fact, two types of G proteins coupled to PLC have been discovered; these include both.pertussis toxin (PTX)-sensitive (56) and PTX-insensitive G proteins (57) . Reconstitution experiments with purified PTX— sensitive G proteins Gi and Go showed that these two G proteins have phospholipase C stimulatory function (58) . Molecular cloning has recently yielded a new G protein called Gq. An antibody to this protein was found to inhibit the phospholipase C activity occuring in response to thromboxane A2 in a PTX-insensitive manner (59) . This finding suggested that Gq is the PTX-insensitive G protein involved in PLC activation. There are several PLCs which hydrolyze phosphatidylinositol derivatives. The PLC isozymes are designated as a, B, 'y, 6, 6 according to their different primary structures (60). All of these PLCs are specific for phosphatidylinositol and/or phosphatidylinositol phosphates (60,61) and do not hydrolyze other phospholipids. In summary, the hormone-induced 1P3 formation requires three membrane components including a receptor, a PLC responsible for the cleavage of IP3 from the precursor lipid, and a G protein that couples the receptor to PLC. 23 Figure 4. The dual signaling pathway of IP3/Ca++ and DAG/PKC. The dashed lines represent the positive and negative feedbacks of PKC on Ca + + signal ing . Abbreviations: PIP2 , phosphatidylinositol 4,5-bisphosphate; 1P3, inositol 1,4,5- trisphosphate; GP. , the G protein involved in phospholipase C activation; DAG, diacylglycerol. meOawwm 442.... A _ \“.'.“."‘..‘.".“I 1.. wEEQ $3303 .. mo / Emecmaooémacié .8 . + \ . . O mu menisci mommoooa \ n u m Eoucmaou¢ma0 . u . Anna .. Tammi mm 3.3:. $5 25 C§++'Signaling;Triggered.hy Inositol Thisphgsphgte (I231. The importance of 1P3 as a signaling molecule is mainly due to its role in controlling the level of intracellular calcium, which itself is a major intracellular messenger. Ca++ is a very important ion in biological systems. In higher organisms, the concentration of free Ca++ in the extracellular pool ([Ca++jo) is very high (1.0-1.5 mM), while the concentration of free Ca++ in the intracellular pool ([Ca++j ) is very low (100-200 nM) (62). For an intracellular 1 ++ messenger like Ca , this distribution of free Ca++ has two advantages. First, the large difference in [Ca++J between the two sides of the plasma membrane results in a large ++ electrochemical force on Ca . Under such conditions, small changes in the permeability of the plasma membrane to Ca++ induced by extracellular stimuli will cause a significant Secondly, the very low level of [CaIIJ- in change in [Ca++]- l l. resting cells makes it possible toiachieve a large fluctuation in [Ca++]i rapidly. The [Ca+”fy is controlled by Ca++-transporting systems 1 in the plasma membrane and membranes of intracellular organelles including the mitochondria and endoplasmic reticulum. The plasma membrane contains three general systems responsible for Ca++’ exchange {between extracellular and intracellular pools. They are Ca++ channels (63), Na+/Ca++ exchangers (64), and Ca++-ATPases (65). The major mechanism 26 ++ for Ca++ entry is the opening of Ca channels. There are several types of these channels which can be operated by changes in membrane potential, by receptors upon binding of ligands, or by second messengers (66) . The extrusion of intracellular Ca++'is achieved.by Ca++ ATPases, whose pumping activity is subject to regulation by calmodulin- and cAMP- dependent protein kinases (67) . In addition to the extracellular source of Ca++, cells also use intracellular sources. The two major calcium stores within mammalian cells are the endoplasmic reticulum (ER) and mitochondria (MT). It is clear now that the ER plays an active role in regulating ++. As will be described below, the intracellular pool of Ca part of the ER pool of calcium is sensitive to 1P5, while the remaining pool of Ca++ can be released by Ca++ ionophores. Ca++ accumulation by the ER is achieved by transport of Ca++ via a Ca++-pumping ATPase. This enzyme has a high Ca++ affinity (Km=1 nM), and can load relatively large amounts of Ca++ into the isolated reticular vesicles in the presence of the trapping anion oxalate (68). Calmodulin- and cAMP- dependent protein kinases (70) have been suggested to be + + pumping . involved in the regulating of the process of Ca Thus, by refilling and releasing the stored Ca++ efficiently, the ER "fine-tunes" cytosolic [Ca++] from 100 to 200 nM in the resting state, to the millimolar range when stimulated by Ca++-mobi1izing agonists. This wide range of change in [Ca++]i permits Ca++ to act as an intracellular regulator. In 27 contrast to the ER, the MT is a low-affinity Ca++ uptake organelle (Km about 10 nM) (71), but it has a large capacity for ca” accumulation (72) since it can coaccumulate phosphate and Ca++ leading to formation of hydroxyapatite deposits in the matrix space. So it has been suggested that the main role of the MT is to absorb and store away the cytosolic Ca++ when there is excess Ca++ influx through the plasma membrane. Evidence has long been accumulated that hydrolysis of phosphoinositides and formation of 1P3 always occur with so called calcium-mobilizing agonists which bind to receptors that induce calcium release from internal stores. Included in this group are muscarinic cholinergic receptors, ail-adrenergic receptors and Vl-vasopressin receptors (73) . The response to the calcium-mobilizing agonists involves initially a release of sequestered Ca++ from the internal stores, which results in a rapid (10-30 sec) increase in [Ca++]i to some maximal value that soon declines. This initial Ca++ transient is then followed by entry of Ca++ from the extracellular medium through the plasma membrane, which keeps the [Ca++]i at a level above the resting level for several minutes. The initial observation that the release of Ca++ from non-mitochondrial calcium stores could be triggered by 1P3 was made by Berridge et a1. (74) about ten years ago, using permeabilized pancreatic cells monitored with a calcium electrode. Similar observations were obtained in many other 28 permeabilized cells. It was shown subsequently that the non- mitochondrial IPg-sensitive calcium pool resides in ER (75); this latter pool accounts for 30-50% of the total non- mitochondrial pool of calcium. A variety of tissues have a high affinity stereospecific binding site for 1P3, but how IP3 interacts with receptors that mediate intracellular Ca++ release remained unclear until recently, when Supattapone et al (76). isolated an IP3-binding protein from cerebellar Purkinje cells which shows abundant 1P3 binding sites. This protein is a tetramer of about 260 Kd subunits. When reconstituted into lipid vesicles, the purified 1P3 receptor protein binds with 1P5 and its derivatives and mediates Ca+*' fluxes (77). A corresponding pharmacological profile of agonist binding and agonist-induced Ca++ release was observed, providing compelling evidence that.IIg binding site and Ca++ channel reside in the same protein. Immunostaining using a specific antibody against this receptor on Purkinje cells revealed that it is localized on the nuclear envelope and on parts of the ER, particularly near the nucleus (78). Eventually, the entire cDNA sequence was obtained from a mouse cerebellum library screened with the monoclonal antibody of this receptor (79), revealing an open reading frame encoding 2,749 amino acids. Comparison of immunolocalization and hydropathy profile suggest that the receptor possesses a very large N-terminal region on the cytoplasmic face of the ER, and a cluster of up to seven transmembrane domains close to the C 29 terminal. As a universal intracellular messenger, Ca++ is involved in the stimulation of a variety of physiological processes, including the contraction of smooth muscle, the breakdown of glycogen in liver, the secretion of enzymes from pancreas, the opening of ion channels in a number of cells, some early events of fertilization; growth factor-induced gene transcription; and information storage by nervous system. Many of these events are short-term responses that are switched off rapidly; but some are long-term events such as cell growth and information storage. Ca++ plays its role in these processes either by directly activating the enzymes involved or by binding to calmodulin, the most abundant high-affinity Ca++- binding protein in most eukaryotic cells. Calmodulin is known to interact with a number of cellular enzymes including adenylate cyclase, cyclic nucleotide phosphodiesterase, phosphorylase b kinase, and Ca++ ATPase of plasma membranes (37,73). One aspect of Ca++ signaling that has attracted considerable attention in recent years is Ca++ oscillations. When cells challenged with Ca++-mobilizing agonists are monitored at the single cell level, oscillatory patterns of response are frequently observed (80). The oscillatory patterns are usually in the form of a constant baseline which is interrupted periodically by calcium spikes. Most oscillations have a period of between 5 and 60 seconds, 30 dependant or independent of the concentration of agonists (81). Each individual cell has a unique pattern and frequency of response, which remains constant after repeated application of a specific agonist; this pattern serves as a cell "fingerprint" (82). In Ca++ oscillations, the Ca++ transient often initiates at a specific subcellular location and then spreads throughout the cells in waves which propagate at a rate of 10-100 um/s (83). These spatial and temporal features of Ca++ mobilization imply that Ca++ may act as an intracellular messenger in a very complex way. The Modulatory Function of Diacylglycegol and ngtein Kinase C. Another product of the hydrolysis of PIPZ is diacylglycerol (DAG), which functions as a second messenger to activate protein kinase C (PKC) (37), forming the second limb of the signaling system (Fig.4). The stimulation of PKC requires the synergistic action of DAG and Ca++k Upon cell activation, cytosolic:Ca++'increases, which mediates the translocation of PKC from the cytosol to the membrane (84) . PKC thus gains access to and binds DAG which remains in the membrane after hydrolysis of PIPZ. Activated PKC exerts its function by phosphorylating specific proteins. Its substrates include receptors, G proteins, phospholipases, Ca++ channels and glycogen synthase. It is proposed that of the two limbs of the PLC signaling pathways resulting from PIPZ hydrolysis, the 31 IP3/Ca‘H’ pathway plays a major role in initiating cellular responses, while the DAG/PKC pathway mainly serves to modulate the IP3/Ca++ pathway, though in some cases it may also contribute directly and synergistically to the responses initiated by IP3/Ca++ (37,85) . PKC is at the center of positive and negative controls, exerting duel actions over various steps of IP3/Ca++ signaling (86) . As cytosolic Ca++ increases, PKC begins to exert a negative feedback by activating the Ca++-transport ATPase and Na+/Ca++ exchange protein, both of which remove Ca'H' from the cytosol (87) , so that the [Ca++]i will not rise too much. PKC also causes inhibition of receptor-mediated PIPZ hydrolysis, blocking further activation of the IP3/Ca++ pathway. There are a number of targets of PKC-mediated phosphorylation. For instance, in liver, activation of PKC selectively inhibits a1- adrenergic receptor-mediated PIP2 hydrolysis, but not vasopressin and angiotensin II receptor-mediated PIP2 hydrolysis; this finding suggests that PKC can phosphorylate the al-receptor (88) . There is also evidence that G proteins and PLC are phosphorylated by PKC (89) . 0n the other hand, PKC can also have positive feedback effects on the IP3/Ca++ pathway. PKC activation leads to the increased formation of PIPZ, which is the substrate for 1P3 formation. The net effect of the feedback effects of PKC on Ca++ signaling probably depends on the degree to which those feedback mechanism are 32 expressed in each cell. Experimentally PKC can be activated by treating cells with tumor-promoting phorbol esters, which mimick the stimulatory effect of the DAG normally only generated from receptor activation (90). One of the most commonly used phorboltesters.is 12-0-tetradecanoylphorbol-13-acetate (TPA). TPA induces persistent activation of PKC because it is very stable to metabolism. With short treatments, TPA elicits a positive effect on PKC by binding to its DAG binding site and immediately activating it. Chronic treatment with TPA, however, depletes cells of PKC, since the persistent activation of this enzyme makes it sensitive to proteolysis which leads to its subsequent degradation (91) . The TPA- induced depletion of PKC, termed down-regulation of PKC, has been observed in many cell types (92, 93). Thus, one way to study the function of PKC is to down-regulate the enzyme by chronic treatment of cells with TPA. It has been shown in a number of cell lines that. depletion of PKC results in potentiation of 1P5 formation occurring in response to Ca++- mobilizing agonists (94), probably due to withdrawal of the normal negative feedback effects that PKC exerts on the IP3/Ca'H' limb of the PLC signaling pathway. 9. 10. 11. 12. 00’ 13. REFERENCE Hinman, J.W. (1972) Ann. Rev. Biochem. 41, 161. Rittenhouse-Simmons, S. (1981) J. Biol. Chem. 256, 4153- 4155. Schwartzman, M., Liberman, E., Raz, A. (1981) J. Biol. Chem. (1981) 256, 2329-2333. Garcia,-Perez, A., Smith, W. L. (1984) J. Clin. Invest. 74, 63-74. Smith, W.L. (1992) Am. J. Physiol. 263, F181-Fl91. Rollins, T. E., Smith, W. L. (1980) J. BIol. Chem. 255, 4872-4875. Smith, W.L. (1989) Biochem. J. 259, 315-324. Ferreira, S.H., Vane, J.R. (1974) Ann. Rev. Pharmacol. 14, 57. Smith W.L. (1986) Ann. Rev. Physiol. 48, 251-262. Pace-Asciak, C.R., Smith, W.L. (1983) In: The_Ehzyh§§ (Boyer, P.D., ed.) Vol.16, pp 543-603. Moonen, P., Buytenhek, M., Nugteren, D.H. (1982) Methods Enzymol. 86, 84-91. Watanabe, K., Iguchi, Y., Iguchi, S., Arai, Y., Hayaishi, Roberts, L.J. (1987) Adv. Prostaglandin Thromboxane Leukotriene Res. 17A, 44-49. DeWitt, D.L., Smith, W.L. (1983) J. Biol. Chem. 258, 3285- 33 34 3293. 14. Shen, R.-F., Tai, H.-H. (1986) J. B. Chem. 261, 11592- 11599. 15. Christ-Hazelhof, E., Nugteren, D.H. (1981) Prostaglandins 22, 739-746. 16. Ferreira, S.H., Vane, J.R. (1967) Nature (London) 216, 868-873. 17. Smith, W.L. (1985) In: B1gghgm1sh;y_gf_h;hghigghig_hgig W (Lands, W.E.M., ed) ,pp. 77-94, Martinus Nijoff, Boston. 18. Samuelssson, B., Granstrom, E., Green, K., Hamberg, M., Hammarstrom, S. (1975) Ann. Rev. Biochem. 44, 669-689. 19. Smith, W.L., Sonnenberg, W.K., Allen, M.L., Watanabe, T., Zhu, J. El-Harith, E.A. In: W (Patrono, C. and Dunn, M.J., eds.), Plenum, New York. 20. Currie, M.G., Needleman, P. (1984) Ann. Rev. Physiol. 46, 327-341. 21. Smith, W.L., Bell, T.G. (1973) Am. J. Physiol. 235, F451- F457. 22. Grenier,F.C., Smith, W.L. (1978) Prostaglandins 16, 759- 772. 23. Kirschenbaum, M.A., Lowe, A.G., Trizna, W., Fine, L.G. (1982) J. Clin. Invest. 70, 63-74. 24. Grantham, J.J., Orloff, J. (1968) J. Clin. Invest. 47, 1154-1161. 25. Handler, J.S., Orloff, J. (1981) Ann. Rev. Physiol. 43, 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 35 611-624. Sonnenberg, W.K., Smith, W.L. (1988) J. Biol. Chem. 263, 6155-6160. Sonnenburg, W.K., Zhu, J. Smith, W.L. (1990) J. Biol. Chem. 255, 8479-8483. Breyer, M.D., Jacobson, H.R., Hebert, R.L. (1990) Kidney International 38, 618-624. Dixon, B.S., Breckon, R., Burke,C., Anserson, R.J. (1988) Am. J. Physiol. 254, C183-C191. Ando, Y., Jacobson, H.R., Breyer, M.D. (1987) J. Clin. Invest. 80, 590-593. Coleman,R.A., Kennedy, 1., Humprey, P.P.A., Bunce, R., LumleY: P- (1990) In: QQmnIBbBn£1!B_Msdisinsl_§hsmi§££1. edited by Transch, C., Sammes, P.G., taylor, J. Oxford: Pergamon, 1990, Vol.3, pp 643-714. Sugimoto, Y., Namba, T., Honda, A., Hayashi, Y., Negishi, M., Ichikawa, A., Narumiya, S. (1992) J. Biol. Chem. 267, 6463-6465. Honda, A., Sugimoto, Y., Namba, T., Watabe, A., Irie, A., Negishi, M., Narumiya, S., Ichikawa, A. (1993) J. Biol. Chem. 268, 7759-7762. Dohlman, H.G., Thorner, J., Caron, M.G., Lefkowitz, R.J. (1991) Ann. Rev. Biochem. 60, 653-688. Gilman, A. G. (1987) Ann. Rev. Biochem. (1987) 56, 615- 649. Iyengar, R., Abramowitz, J., Bordelon,-Riser, M.E., 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 36 Birnbaumer, L. (1980) J. Biol. Chem. 255, 3558-3564. Berridge, M.J.A. (1987) Ann. Rev. Biochem. 56, 159-193. Stryer, L. (1986) Ann. Rev. Neurosci. 9, 87-119. Birnbaumer, L., Brown, A.M. (1990) Ann. Rev. Pharmacol. Toxicol. 30, 675-705. Birnbaumer, L. (1990) FASEB J. 4, 3068-3078. Rodbell, M., Birnbaumer, L., Pohl, S., Krans, H.M.J. (1971) J. Biol. Chem. 246, 1877-1812. Northup,J.K., Sternwise, P.C., Smigel, M.D., Schleifer, L.S., Ross, E.M., Gilman, A.G. (1980) Proc. Natl. Acad. Sol. USA 77, 6516-6520. Harris, B.A., Robishaw, J.D., Mumby, S.H., Gilman, A.G. (1985) Science 229, 96-99. Shorr, R.G.L., Lefkowitz, R.J., Caron, M.G. (1981) J. Biol. Chem. 256, 5820-5826. Dixon, R.A., Caron, M.G., Lefkowitz, R.J., Strader, C.D. et a1. (1986) Nature 321, 75-79. Masu. Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., Nakanishi, S. (1986) Nature 329, 836-838. Henderson, R., Unwin, P.N.T. (1975) Nature 257, 28-32. Dohlman, H.G., Caron, M.G., Lefkowitz, R. J. (1988) Biochemistry 26, 2657-2664. Wong, S.H., Parker, E.M., Ross, E.M. (1990) J. Biol. Chem. 265, 6219-6224. Agranoff, B.W., Murthy, P., Seguin, E.B. (1983) J. Biol. Chem. 258, 2076-2078. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 37 Berridge, M.J. (1983) Biochem J. 212, 849-858. Martin, T.F.J. (1983) J. Biol. Chem. 258, 14816-14822. Rebecchi, M.J., Gershengorn, M.G. (1983) Biochem J. 216, 299-308. Smith, C.D., Lane, B.C., Kusaka, I., Verghese, M.W., Snyderman, R. (1985) J. Biol. Chem. 260, 5875-5878. Strnad, C.F., Parente, J.E., Wong, K. (1986) FEBS Lett. 206, 20-24. Nakamura, T., Ui, M. (1985) J. Biol. Chem. 260, 3584- 3593. Taylor, C.W., Blakeley, D.M., Corps, A.N., Berridge, M.J., Brown, K.D. (1988) Biochem J. 249, 917-920. Kikuchi, A., Kozawa, 0., Kaibuchi, K., Katada, T., Ui, M., Takai, Y. (1986) J. Biol. Chem. 261, 11558-11562. Gutowski, 8., Smrcka, A., Nowak, L., wu, D., Simon, M., Sternwise, P.C. (1991) J. Biol. Chem. 266, 20519-20524. Rhee, S.G., Suh, P.-G., Ryu, S.-H., Lee, S.Y. (1989) Science 244, 546-550. Ryu, S.H., Suh, K.S., Cho, K.Y., Lee, S.Y., Rhee, 5.6. (1987) Proc. natl. Acad. Sci. USA 84, 6659. Carafoli, E. (1987) Ann. rev. Biom. 56, 395-433. Reuter, H. (1984) Ann. Rev. Physiol. 46, 473-484. Renter, H., Seitz, N. (1968) J. Physiol. 198, 46-48. KOStYUR: P-Go (1982) In: HBmhLOnB_ILOn§BQLt_O£_QOlgium. ed. E. Carafoli, pp 41-108. Tsien, R.W. Tsien, R.Y. (1990) Ann. Rev. Cell Biol. 6, 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 38 715-760. Reuter, H., Stevens, C.F., Tsien, R. W., Yellen, G. (1982) Nature 297, 501-504. Constantin, L.L. Franzini-Armstrong, C., Podolsky, R.J. (1965) Science 147, 158-160. Moore, P.B., Kraus-Friedmann, N. (1983) Biochem J. 214, 69-75. Bygrave, F.L., Tranter, C.J. (1978) Biochem. J. 174, 1021-1030. Crompton, M., Sigel, E., Salzmann, M., Carafoli, E. (1976) Eur. J. Biochem. 69, 429-434. Carafoli, E. (1974) Biochem. Soc. Symp. 39, 89-113. Berridge, M.J. (1982) In: Calcium and Cell Function (Cheung, W.Y., ed), Vol. 3, pp 1-36. Academic Press, New York. Streb, H., Irvine, R.F., Berridge, M.J., Schulz, I. (1983) Nature, 306, 67-69. Streb, H., Bayerdorffer, E., Hasse, W., Irvine, R.F., (1984) Schultz I. J. Membrane Biol. 81, 241-253. Supattapone, S., Worley, P.F., Baraban, J.M., Snyder, S.H. (1988) J. Biol. Chem. 263, 1530-1534. Ferris, C.D., Huganir, R.L., Supattapone, S., Snyder, S.H. (1989) Nature 342, 87-89. Ross, C.A. et al. (1989) Nature 339, 468-470. Furuichi, T., Yoshidawa, S., Miyawaki, A., Wada, K., Meada, N., Mikoshiba, K. (1989) Nature 342, 32-38. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 39 Berridge, M.J., Galione, A. (1988) FASEB J. 2, 3074- 3082. Rink, T.J., Jacob, R. (1989) Trends Neurosci. 12, 43-46. Prentki, M; et al. (1988) J. Biol. Chem. 263, 11044-11047. Takamatsu, T., Wier, W.G. (1990) FASEB J. 4, 1519-1525. Kosaka, Y. et a1. (1988) Biochem. biophys. Res. Commun. 149, 946-952. Berridge, M.J. (1984) Biochem. J. 220, 345-360. Nishizuka, Y. (1988) Nature 334, 661-665. Kikkawa, U., Nishizuka, Y. (1986) Ann. Rev. Cell Biol. 2, 149-178. Sibley, D.R., Benovic, J.L., Cron, M.G., Lefkowitz, R.J. (1988) Endocr. Rev. 9, 38-56. O’Brien, R.M., Houslay, M.D., Milligan, G., siddle, K. (1987) FEBS Lett. 212, 281-288. Sando, J.J., Young, M.C. (1983) Proc. Natl. Acad. Sci. USA 80, 2642-2646. Kishimoto, A., Kajikawa, N., Shiota, M., Nishzuka, Y. (1983) J. Biol. Chem. 258, 1156-1164. Young, S., Parker, P.J., Ullrich, A., Stabel, S. (1987) Biochem. J. 244, 775-779. Helper, J.R., Earp, H.S., Harden, T.K. (1988) J. Biol. Chem. 263, 7610-7619. Brown, K.D., Littlewood, C.J., Blakeley, D.M. (1990) Biochem. J. 270, 557-560. CHAPTER TWO PROSTAGLANDIN Ez-INDUCED Ca++ MOBILIZATION IN SUBTYPES OF RABBIT CORTICAL COLLECTING TUBULE CELLS The renal collecting tubule is importantly involved in regulating water and electrolyte balance. PGEZ, the major prostanoid formed by collecting tubule cells (1), plays a key roLe in the modulation of this process. In immunodissected rabbit cortical collecting tubule (RCCT) cells, PGE2 acting at relatively low concentrations (ca.10'8 M) inhibits arginine vasopressin (AVP)-induced cAMP accumulation; at higher concentrations (2 10‘7 M), PGEZ by itself stimulates cAMP formation (2) . These effects appear to be mediated through inhibitory EP3 and stimulatory EP2 receptors, coupled to an inhibitory G protein (Gi) and a stimulatory G protein (G8) , respectively (3). These in vitro studies with isolated RCCT cells provide a biochemical basis for the dual actions of PGE5 on.water reabsorption observed in perfused collecting tubules (4) - In addition to the well-studied cAMP inhibitory and stimulatory pathways, some evidence suggests that PGE2 regulates water and salt transport through a third signaling 40 41 pathway involving increases in the concentration of intracellular Ca++ ([Ca++]i) and the activation of protein kinase C (PKC) (5,6). However, there is noidirect evidence for a third PGE receptor coupled to Ca++ mobilization in the mammalian collecting tubule. The purpose of the present study was to characterize PGEz-induced Ca++ mobilization in RCCT cells. It was demonstrated that PGEZ induced Ca++ mobilization and that this effect. was mediated. via a receptor ‘with properties resembling' those of an EP1 PGE receptor and differing those of EP2 and EP3 PGE receptors. The combination of Ca'H' measurements and immunostaining at the single cell level using fluorescence digital imaging enabled us to establish that this putative EPH PGE receptor is distributed in both principal and type B intercalated collecting tubule cells. MATERIALS AND METHODS ham. Dulbecco's modified Eagle medium (DMEM) , collagenase, fetal calf serum (FCS) and trypsin were purchased from GIBCO. Bovine calf serum (CS) was from Hyclone Laboratories. PGEZ, PGFZa, PGDZ, and 15- (R) -PGE2 were from Cayman Chemical Company. Arginine vasopressin (AVP) , fluorescein isothiocyanate-labeled peanut lectin (FITC-PNA), and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG were from Sigma. Fluo-3/AM was from Molecular Probes, Inc. Pertussis toxin was from List Biological Laboratories. A hybridoma line producing a monoclonal antibody (DT.17) specifically reactive with a cell surface determinant of principal cells was a gift from Dr. Geza Fejes-Toth, Dartmouth Medical School (7) . Other reagents were from common commercial sources . WWW—L‘- f o. .m hybridoma gulture h_e_d_ihm. The monoclonal antibody IgG3(rct-30) , which interacts specifically with a cell surface antigen of RCCT cells, was isolated as follows. The rct-30 hybridoma line was grown in HT-complete medium containing DMEM, 10% FCS, 10% NCTC-135 medium, 2 mM glutamine, 13.6 pg of hypoxanthine/ml and 3.9 ug of thymidine/ml. Before harvest, this medium was replaced by IgG free medium (8) in which cells were allowed to incubate 42 43 for several days to secrete the antibody. This latter medium was collected, filter-sterilized and adjusted to pH 8.0 before being applied to a Protein A-Sepharose column. The column was eluted stepwise with 0.1 M buffers of pH 8.0 (sodium phosphate), and pH 4.5 and pH 3.5 (sodium citrate). IgG3(rct- 30) was eluted at pH 4.5. The eluant was collected and immediately neutralized with 100 pl of 1M Tris (pH 9.5) contained in each collection tube. Fractions containing IgG3(rct-30) were pooled, sterilized by filtration, and diluted with phosphate-buffered saline (PBS: 137 mM NaCl, 8.1 mM Nazi-IP04, 1.5 mM KCl, pH 7.3) . The protein concentration was estimated by measuring the absorbance at 280 nm. Immuhofluorescence shaining sf Lshhih highey sections. Fresh rabbit kidneys were cut into small cubes (ca. 1 cm?) which were frozen rapidly. The frozen pieces were sectioned into thin slices (ca.8 um) and were adhered.ontoiglass slides. After vacuum-drying, these slices were subject to immunostaining. Diluted purified Igcgcrct-30) or medium from DT.17 hybridoma growth were applied to the sections. After a 15 min incubation, the sections were rinsed with simplified saline solution (SSS: 145 mM NaCl, 5 mM KCl, 1 mM NaZHPO4, 0.5 mM MgC12, 5 mM glucose, 10 mM Hepes, 1 mM CaClz, pH 7.4) . and then incubated with a 1:10 diluted FITC-labeled anti-mouse IgG for another 15 min. The stained sections were rinsed with SSS, mounted on glass slides and examined under a fluorescent 44 microscope. Esspshatigh sf cultui'e wells cgahsd with lgG3(rct-30) . Each well of a 24 well culture plate was incubated for 2-4 hours at room temperature with 0.25 ml of IgG3(rct-30) (100-400 pig/ml) . Immediately before use, the antibody solution was aspirated from the wells, which were then washed twice with 1% bovine serum albumin in PBS. Isolation ahg culhhre of RCCT sells. Homogeneous populations of RCCT cells were isolated by immunodissection using IgG3Crct-30) (9). For each isolation, two kidneys were removed from a 8-16 week old male or female New Zealand White rabbit, which had been sacrificed with an overdose of 7.5% sodium pentobarbital injected through the marginal ear vein. The renal cortex was dissected, minced into a fine paste, and transferred into a tube containing 30-40 ml of 0.1% collagenase in SSS. After a 30-40 min incubation at 37°C, the cells were filtered through a 250-um Gelman wire mesh, and resuspended in PBS. The cell suspension was overlayed onto IgG3(rct-30)-coated 24-well culture dishes, which, after a few minutes of incubation, were washed gently with PBS to remove nonadherent cells. The RCCT cells absorbed to the wells were ready for culture in DMEM containing 10% bovine calf serum in a water-saturated 7% C02 atmosphere. Flurbiprofen (FBP) , a PGH synthase inhibitor was included at a concentration of 5 BM in all isolating solutions and in the culture medium. Messusement of Ca"+ hobilisstion in individual RCCT 45 lssllsl, One day-cultured RCCT cells were incubated with 10 uM fluo-3/AM at 37°C for 40 min in SSS containing 0.1% BSA. The cells were then washed.three times with SSS. The fluo-3 loaded cells remained responsive for two or three hours at 24°C without significant loss of the dye. Loaded cells that were immobilized in the culture wells were treated with agonist and scanned using an ACAS 570 interactive laser cytometer with an excitation wavelength of 488 nm and an emission wavelength of 505 nm. The change in fluorescence intensity in the cells was monitored during scanning. The basal and the peak [Ca+*j-Iof responding cells were calculated as described previously (10) with the formula: [Ca++]=(F-Fmin/Fmax-F)xl85%) by treatment of 28A cells with PTX. There was no significant effect of PTX to block Cal“+ mobilization by PGE2 in 28A cells (Fig.17B) . Finally, the effect of PGE2 on the formation of inositol phosphates was examined in confluent (4-5 day old) RCCT cells and in 28A cells. Stimulation with 10‘5 M PGEZ for 30 min had 56 no significant effect on the production of inositol phosphates in RCCT cells (mi-2.8%, n=2). In contrast, bradykinin, a potent Ca++-mobilizing agonist in RCCT cells, caused a 50% increase in those cells under the same condition. Similar results were obtained using 28A cells. 57 Figure 1. Indirect immunofluorescence staining of a section of rabbit renal cortex *with IgG3Crct-30). The staining' was performed as described in the text. The upper figure is a phase contrast photomicrograph (X100) of the stained section shown in the lower photomicrograph. 59 Figure 2. Indirect immunofluorescence staining of a section of rabbit renal cortex with an anti-principal cell antibody Ig63 (DT. 17) . (A) . Phase contrast and fluorescence photomicrographs at low magnifications (X100). (B). phase contrast and fluorescence photomicrographs at higher magnification (X400) , showing the cross section of one collecting tubule, in which two cells were not stained with IgG3(DT. 17) . 61 Table 1. Immunostaining and lectin staining of freshly isolated RCCT cells. RCCT cells were tested for staining with IgG3Crct-30), principal antibody IgG3(DT.17), and intercalated cell marker FITC-PNA. The cells counted are from one isolation, similar results were obtained from another similar isolation. reactive cells cell number IgG3(rct-30) 246 (100%) 247 DT.17 162 (60%) 268 FITC-PNA 76 (28%) 271 62 Figure 3. Fluorescence digital image of freshly isolated RCCT cells stained with IgG3(rct-30). Cells were immunodissected and stained as described in the text. The ring-like staining is typically observed for cell surface antigens. The fluorescence value of pseudo colors are indicated by the color bar on the right. smek mam eme_ - _k__ - __M_ - mme_ - mmm_ -.. MMR_ - ekm_ - m_em - mmrw - ...“. mmmm - fl kmem -.. kkmm - s m_em - mmmm -. Assam - mm3_®} gnu—DD Tat: YET—.T 64 Figure 4. Fluorescence digital images of freshly isolated RCCT cells stained. with. an. anti-principal cell antibody IgG3(DT.17). (A). The basolateral surface of a piece of undigested tubule, showing a mosaic staining pattern for IgG3(DT.17). (B). Individual cells stained with IgG3(DT.17). The dark shades in between the stained cells were unstained cells, whose fluorescence was lower than that of the background of the well. Color Values - 19001 - 1796 - 1693 - 1590 — 1487 - 1384 “ - 1280 Color Values - 19001 - 1856 — 1812 - 1768 - 1724 - 1680 .-- 1637 - 1593 - 1549 - 1505 - 1461 - 1418 - 1374 - 1330 - 1286 - 1242 - 11991 66 Figure 5. Fluorescence digital image of freshly isolated RCCT cells stained with intercalated cell marker FITC-labeled peanut lectin (PNA) .Staining was performed as described in the text. In stained cells, FITC-PNA aggregated on.membrane. This "capping" phenomenon is typically observed with lectin staining. imam eke Nmm - e___ mmm. mmm_ -. ewe. -W. New. - ems. - mem_ - mmm_ -.. emem -1» N_NN - emmm - mmem - mama Leemm mm:_m> Lo_ou 68 Figure 6. Ca++ mobilization in RCCT cells in response to agonists as observed by fluorescence digital imaging. One-day cultured RCCT cells were loaded with fluo-3 and stimulated with 10'5 M PGEZ and 10'5 M AVP consecutively. A. Cells in the buffer. B. cells 10 s after addition of PGE2. C,D: cells 36 s and 52 s after addition of PGE2.1E: cells 78 s after addition of P682 and before addition of AVP. F: cells 10 s after addition of AVP. G,H: cells 36 s and 52 s after addition of AVP. Color Values A¢,- 35631 2 - 3378 g - 3155 i - 2934 f - 2712 _; - 2494 ’i‘- 2268 - 2645 - 1324 - 1502 - 1380 - 1153 - 935 - 714 - 492 - 27a — 494 70 Figure 7. Representative tracings of individual RCCT cells challenged with 10'5 M PGEZ and 10'5 M AVP. Cells loaded with fluo-3 were scanned for their fluorescence using an interactive laser cytometer. The eight cells were from one scanned field. Changes. in [Ca++]i were indicated. by' the changes in fluorescence within each cell. The values on the Y axis are the fluorescence intensity normalized by that of the first scan within each cell. Heterogeneity in responses was shown from this group of cells.+~+m+~+: cells responding neither to PGEZ or AVP.I~~----n---I: cells responding to both PGE; and AVP.o»o: cells responding only to AVP.O"i‘Owo: cells responding only to PGEZ. OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 1 . a 3.2.2:. 35320:: 302.950 Time (s) 72 Figure 8. Subpopulations of RCCT cells which mobilize Ca++ in response to PGEZ. One-day cultured RCCT cells were loaded with fluo-3 and analyzed for the changes in [Ca'H’Ji in response to PGEZ. Cells exhibiting an increase in fluorescence intensity of greater than 10% after challenge with agonist were counted as responding cells. Shown in the figure are the percentage of cells responding to P632 (10'5 M) in eight representative isolations with 100-150 cells analyzed in each isolation. The size of the subpopulation that responded ranged between about 20% to 60%. ................................... — q u 4 . _ . _ . u q a u 1- . d u q u m m m. .m m w m m m w 0 «mod 2 mcficoamo. 2.3 .0 32:09.2. 2: 2345678 1 74 Figure 9. Strategy of identifying subtypes of RCCT cells which mobilize Ca++ in response to agonists. Cells in grey shades are those loaded with fluo-3. Cells with "*" are responsive cells. Cells in white are those whose fluorescence are quenched by treatement of ionomycin and MhClz. Cells in dark shades are those which bind with FITC labeled.antibody or peanut lectin. IDENTIFICATION OF SUBTYPE OF RCCT CELLS THAT RESPOND Agonists Responsive cells identified lonomycin + MnCIZ Cells labeled with FIT C-Ab Cell fluorescence quenched or FIT C-Iectin 76 Table 2. PGEZ and AVP responses in both the principal and intercalated cell. After each tracing of cells challenged with 10"5 M PGEZ and 10'5 M AVP, 2 mM MnClZ was added to cells that were permeabilized with 10‘5 M ionomycin to quench the fluorescence of fluo-3. Then the cells were stained with hybridoma media of principal cell antibody DT.17 for 10 min, washed, and stained with anti-mouse IgG-FITC (1:10 dilution in SSS) for another 10 min. p632”) AVP (+) Fonz”) /1wp(+) n DT.17(+) 58 45 28 134 (43%) (34%) (21%) DT.17(-) 29 32 13 92 (32%) (35%) (14%) Total 87 77 41 226 (39%) (34%) (18%) 77 Table 3. PGEz and AVP responses in subtype B of intercalated cells. 5After each tracing of cells challenged with 105 M PGE2 and 10‘5 M AVP, 2 mM of MnClz was added to the cells that were permeabilized.with 105 M ionomycin to quench the fluorescence of fluo-3. Then FITC-PNA (1: 10 dilution in SSS) was added to stain the cells for 5 min. in two isolations. FITC-PNA(-) FITC-PNA(+) Total n is the number of cells examined 8832“») AVP (+) 2632“) ”we (+) n 85 107 69 213 (40%) (50%) (32%) 25 35 20 58 (43%) (60%) (35%) 110 142 89 271 (41%) (52%) (33%) 78 Figure 10. Dose-response to PGEZ in RCCT cells. The dose effect was examined by challenging groups of cells with different concentrations of PGE2. 1About 100 cells were examined in each group. (A). Dose dependence of the size of responding population. (B). Dose dependence of magnitude of responses in responding cells. NUMBER OF RESPONDING CELLS 12 10 12 10 so- 45' o 1 3 0. 4°“ 0 o u 1 C 2 35' I .- 1 3 30- .1 Ill 0 4 '6 25- ;! . m. ls‘hl . . ' 2 ‘ ‘ '12-; 10.7 10-5 10-5 [P662] L 1- ‘0 1- a 1- 6 1- 4 1- 2 .- 3 W o _ a 160% 0% 1:07. { 106M PGEZ 12 10.91 PGEZ IO 8 6 4 2 o 1 MAGNITUDE OF RESPONDE (PERCENT INCREASE OF FLUORESCENCE) 80 Figure 11. The stereospecificity of PGEz-induced Ca++ mobilization. RCCT cells were challenged with 10'5 M PGE2 or 15-(R)-PGE2. The percentage of cells responding to PGEZ was significantly higher than that to 15-(R)-PGE2. (p<0.02). The results are fromthree separate experiments, with about 100 cells examined for each group in each experiment. 96 OF CELLS THAT RESPOND 15-S-PGE2 ‘4‘ 5' ...' 1 S-R-PG E2 , 82 Figure 12. The effect of flurbiprofen (FBP) on PGEz-induced Ca++ mobilization by RCCT cells. FBP (5x10'5 M) was included in all the isolation solution media and culture media, and the percentage of RCCT cells responding to 10'5 M PGE2 and 10‘5 M AVP was measured in parallel with RCCT cells isolated and cultured in the absence of FBP. *, significantly increased compared with control (non-treated cells) (p<0.02) . The results are from two experiments, with about 100 cells examined for each group in each experiment. - q u 1 u q u q u e w m m m m ozoammm 5:» mime no u AVP/FBP AVP PGEZIFBP PG E2 84 Figure 13. Desensitization of PGEZ-induced Ca++ mobilization by various prostaglandins and AVP. In all the groups except the. control, RCCT «cells ‘were treated. with. desensitizing agonists (105 M) for 30 min, then rinsed and challenged with 10'5 M PGEZ. The size of the responding population in control cells was taken as 100%, to which the response from other groups were compared. *, significantly different from control value (shown by ANOVA analysis) .The result are from three experiments, with about 100 cells examined for each group in each experiment. .— 0 ‘ 5.200 O... ommdufioo mmngmmm m0 8 A)» u» wing-4p); ‘ NAM» A.WA'A.'.AJ - . y.- W a .A ,) N .. . J. k . new .Iw MA - I_KUAV‘J A I E ~‘-.A ,AA. n_>< «Nu—0n. Q DESENSITIZING AGONISTS 86 Figure 14. The effect of AH6809 on PGEz-induced C3++ mobilization by RCCT cells and 28A cells. (A) . RCCT cells were pretreated with 10-5 M AH6809 for 5 min, then rinsed and challenged with 10‘5 M PGEZ. (B). 28A cells were pretreated with 10‘4 M AH6809 for 10 min, then rinsed and challenged with 10'5 M PGEz and CHA consecutively. aoooz<\<:0 (:0 PGEZIAHGBOQ accox<§m0a «wan. PGE2 1 o m m m m m o OZOamwm ._.<:._. maamo “—0 i A ozoamum Siam mime mo .x . B 88 Figure 15. The effect of depletion of extracellular Ca+*'< l7. M m>< D- n. G O .L (atumOn l8 «man. ...—.....O H........... mmmmmmo mmmmmmo 020.“me #5:. flan—mo no $ oz°nmw¢ ...(I... 34m". “—0 «o 92 Figure 17. The effect of PTX on PGEz-induced Ca++ mobilization by RCCT cells and 28A.cells. (A). RCCT cells were treated with PTX (2.5 ug/ml) for 12-16 hours, and then the response to 106 M PGEz‘was examined. The result are from three experiments, with about 100 cells examined for each group in each experiment. (B). 28A. cells were treated. under the same condition used to treat RCCT cells, and the responses to 106 M PGEz, CI-IA and AVP was examined. AVP AVP/PTX PGE2 PG EZIPTX - u u q u a u u u q u 4 q 0 0 0 0 0 0 O 7 6 5 4 3 2 1 3 A OZOamwm ._.<:._. magma ...—O tha>< a>< Xhat one» a: new: HNH agitarxiA I Have a: some R l . Room 53%. ,.. , m ..M.ww_._ . I ..; + MO I . , ...... ... .. ,,.:. tetra. . r. ...-.. : wow»: u tam» vow»- nanniuomne _ L L . . L 5.35 mea.sa 4.3m Hmong percent Increase of IF: tormetlon 138 Figure 15. Effect of pretreatment with PGF2a on Ca++ mobilizationin in confluent quiescent 3T3 cells. Cells were prereated with 10-5 M PGF2a and the subsequent responses to challenges by (A) 10‘5 M PGF2a and (B) 10'5 M PGE2 and 10'5 M bombesin were measured. 599 UN " 2a ‘ i 80 11M +7.; + as B B PG?» PGE’ Bombesm 511 TIM" * $ * 100 TIM 40506‘ DISCUSSION Proliferation of cultured fibroblasts can be induced by a number of hormones that stimulate PIP2 hydrolysis (9,10). PGF2a is known to be a mitogen that elicits its effects through the PLC pathway (4,5). Compared to PGF2a, P6132 is much less potent in stimulating cell growth. In the presence of a low concentration of insulin that does not by itself initiate DNA synthesis, PGE2 induces cell proliferation. It was reported previously that PGE derivatives increase cAMP formation in Swiss 3T3 cells, and this was believed to be the basis of the synergistic effect of PGE8 on initiation of DNA synthesis. The present study demonstrated that PGEZ, as well as PGF2a, can induce PIPZ hydrolysis and subsequent Ca++ release from intracellular stores. This may be a mechanism by which PGE2 exerts its synergistic effect on cell growth. PGEs stimulate adenylate cyclase in a variety of cells. PGEs and PGFs can also activate the PLC system in cells such as MDCK cells, UMR-106 cells, and bovine adrenal chromaffin cells (11,12, 13) . PKC is activated by diacylglycerol generated through the PLC pathway and acts as a negative regulator of this pathway (14). Therefore, when the negative effect of PKC was withdrawn by depletion of PKC, a potentiation of responses 140 141 derived from PLC pathway are expected. The mechanism by which PKC inhibits phosphoinositide hydrolysis in response to mitogens remains to be established. PKC-mediated phosphorylation of receptors, G-proteins, and PLCs were all well.documented (15,16 17). It.is possible that the inhibitory effect of PKC on IPs formation results from phosphorylation at multiple targets in the PLC pathway. TPA-induced down- regulation of PKC has been observed in various cell line, the extent of which differs from cell line to cell line (18). In Swiss 3T3 cells, it was shown that PKC‘was not immunologically detectable after 40 hours of treatment with 300 ng/ml TPA. Numerous examples have been reported of a guanine nucleotide requirement in receptor activation of PLC in cell- free preparations or in permeabilized cells (19,20) . The direct evidence that.G proteins are involved in PLC activation are from Sternweis, who reported recently that an antibody of Ch attenuated the stimulation of PLC by a number of hormones (21). Both PTX-sensitive and PTX-insensitive G proteins involved in activating PLC are found in different types of cells. For instance, chemotactic peptide (f-Met-Leu-Phe)- stimulated PLC activity in neutrophils is abolished by PTX treatment (22). However, in Swiss 3T3 cells, there is only a partial inhibition by PTX of IPs fOrmation in response to vasopressin and bombesin (27% and 23%, respectively) among various mitogens (23) . In our study, overnight incubation with high concentration of PTX (1000 ng/ml) caused a partial loss 142 of responsiveness in IPs formation to PGE2. There might be two possibilities for this observation. One possibility is that PG receptor can interact with more than one type of G protein coupled to PLC, and that not all of these G proteins are substrates for PTX-catalysed ADP ribosylation. Another possibility is that PG receptor interacts with one G protein coupled with PLC, and this G protein is only poorly ADP- ribosylated by PTX. PGE2 and PGF2a are structurally related compounds, which can cross-react with different receptors. It has been found that in some cases, a single tissue contains more than one type of PG receptor, which accounts for the fact that different PGs exert totally different physiological effect on this tissue; while in some cases, there exist only one type of receptor interacting with different PCs with different affinity. For instance, in PGF2a-sensitive tissues such as the rat colon and dog iris (20,21), it has been suggested that PGE2 and PGD2 exert their effect by interacting with a single PGF2a receptor. In the rat UMR-106 cell line, however, evidence has been obtained indicating that PGEZ and PGF2a stimulate PIP2 hydrolysis and release of intracellular Ca++ release by acting through separate receptors (12). Recently, a radioligand binding study using Swiss 3T3 cells (22) revealed a specific binding site for PGF2a. It was shown that PGF2a was 100 fold more potent than PGE2 in competing with the 143 [3111-th for its binding site. Consistent with this finding, results from our experiments suggested that PGEZ activates PLC pathway through a PGF2a receptor. This conclusion is based on two observations. First, the effects of PGE2 and PGF2a on IPs formation and Ca++ mobilization are not additive. Secondly, pretreatment of maximal dose of PGF2a rendered cells refractory to subsequent stimulation of maximal dose of PGF2a and PSI-32, but did not affect the response to bombesin, which also mobilizes Ca++ via mediating 1P3 formation. The unaffected responsiveness to bombesin in PGF2a desensitized cells demonstrated that the IP3-sensitive pool of Ca++ was not depleted by the maximal dose of PGF2a. Therefore, the desensitization of cells to PGEZ by PGF2a did not result from depletion of the Ca++ pool, but probably resulted from desensitization of a single receptor. Another piece of evidence suggesting that this receptor is a PGF2a receptor is that PGEz-induced IPs formation was not inhibited by AH6809, an EP1-selective antagonist. PGE can act through a PGE EP1 receptor to induce Ca++ mobilization, but this receptor does not appear to be present in Swiss 3T3 cells. REFERENCES Samuelsson, E., Goldyne, M., Granstrom, E., Hamberg, M., Hammarstrom, S. and Malmsten, C. (1978) Annu. Rev. Biochem. 47, 997-1029. Powles, T. J., Bockman, R. S., Honn, K. V. and Ramwell, P. (1982) In: The Prostaglandins and Rested Lipids. Alan R. Liss, Inc., New York. Smith, w. L. (1989) Biochem. J. 259. Jimenez de Asua, L., Otto, A. M., Lindgren, J. A. and Hammarstrom, S. (1983) J. Biol. Chem. 258, 8774-8780. Macphee, C. H., Drummond, A. H., Otto, A. M. and Jimenez de Asua, L. (1984) J. Cel, Physiol. 119, 35-40. Rozengurt, E., Collins, M. K. L. and Keehan, M. (1983) J. Cell Physiol. 116, 379-384.! Grynkiewicz, G., Poenie, M. and Tsien, R. (1985) J. Biol. Chem. 260, 3440-3450. Brown, R. D., Littlewood. C. J., Blakeley, D. M. (1990) Biochem. J. 270, 557-560. 9. Vicentini, L.M., Villereal, M.L. (1989) Life Sci. 262, 2269-2276). 10. Tilly, B.C., Moolenar, W.H. (1989) In: 't 1 id and Cell Signalling (Michell, R., Drummond, A.H. and Down, C.P., eds), pp. 485-494, Academic Oress, London 144 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 145 Aboolian, A., Vandermolen, M., Nord, E. P. (1988) Am. J. Physiol. 256, F1135-F1143. Yamaguchi, D. T., Hahn, T. J., Beeker, T. G., Kleeman, C. R., Mullen, S. (1988) J. Biol. Chem. 263, 10745- 10753. Yokohama, D. T., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122. Kikkawa, U., Kishimoto, A., Nishizuka, Y. (1989) Annu. Rev. Biochem. 58, 31-44. Sibley, D.R., Benovic, J.L., Cron, M.G., Lefkowitz, R.J. (1988) Endocr. Rew. 9, 38-56. Katada, T., gilman, A.G., Watanabe, Y., Bauer, S., Jacobs, K.H. (1985) Eur. J. Biochem. 151, 431-437. Rhee, S.G., Suh, P.-G., Ryu, S.-H., Lee, S.Y. (1989) Science 244, 546-550. Adams, J.C., Gullick, W.J. (1989) Biochem. J. 257, 905- 911. Cattaneo, M.G., Vicentini, L.M. (1989) Biochem. J. 262, 665-667. Cockcroft, S., Gomperts, B.D. (1985) Nature 314, 534- 536. Gutowski, S., Smrcka, A., Mowak, L., Wu, D., Simon, M., Sternweis, P.C. (1991) J. Biol. Chem. 266, 20519-20524. Strnad, C. F., Parente, J. E. and Wong, K. (1986) FEBS Lett. 206, 20-24. 23. 24. 25. 26. 146 Taylor, C. W., Blakeley, D. M., Corps, A. N., Berridge, M. J. and Brown, K. D. (1988) Biochem. J. 249, 917-920. Coleman, R.A., Humphrey, P.P.A., kennedy, I., Lumley, P. (1984) Trends Pharmacol. Sci. 5, 303-306. Eglen, R.M., Whiting, R.L. (1989) Br. J. Pharmacol. 98, 1335-1343. Lawrence, R.A., Woodward, D.F. in manuscript. CHAPTER FOUR EXPRESSION CLONING OF A 14-3-3 PROTEIN THAT POTENTIATES Ca++ MOBILIZATION IN XENOPUS OOCYTES IN RESPONSE TO PROSTAGLANDIN E2 The purification of membrane-bound receptors is generally difficult because they are present in low abundance and are difficult to stabilize following solubilization. The first full purification of an adenylate cyclase stimulatory receptor, the B-adrenergic receptor was achieved by Lefkowitz et al (1), which led to subsequent molecular cloning of the cDNA for this receptor (2). For other receptors whose protein purification had not been achieved, cDNA cloning was not possible since no partial protein sequence was available for making oligonucleotide probes for classical cloning methods. This had greatly hindered the progress of receptor studies until 1987, when an innovative approach was developed by Masu et al.(3), who succeeded in cloning the substance K receptor by expressing its activity in Xenopus laevis oocyte. This expression cloning method made it possible to clone receptors without information about their protein sequence. The use Of expression cloning has resulted in the isolation of cDNAs encoding a large number of receptors, most of which belong to 147 148 the G protein-coupled receptor family. The cloning strategy of Masu et al.(3) is conceptually straightforward (Fig.1) . Briefly, a cDNA library is constructed in a lambda phage expression vector containing SP6 or/and T7 promoters for use in in vitro transcription. mRNAs are synthesized from the mixture of cDNAs ligated into the expression vector, and they are then injected into oocytes which translate the mRNA. Functional assays are performed on injected oocytes to test for the presence of the receptor mRNA. A cDNA pool that contains the specific mRNA is identified and this pool can be taken through a series of fractionation steps until a functional defined single clone is obtained. In our attempt to clone a prostaglandin E2 receptor using the Xenopus oocyte expression system, we isolated a clone that potentiated mobilization of intracellular Ca++ in response to PGE2 in oocytes. Surprisingly, sequence analysis of this cDNA showed that it is highly homologous to the 14-3-3 proteins, a family of acidic proteins highly expressed in mammalian brain; but is not homologous to members of the G protein-coupled receptor family. MATERIALS AND METHODS natgzials. Dulbecco's modified Eagle medium, fetal calf serum (FCS) were purchased from GIBCO. xenopus laevis were from Nasco (Fort Atkinson, WI). SP6 and T7 RNA polymerases, RNasin ribonuclease inhibitor, and rabbit reticulocyte lysate in vitro translation kit were Obtained from Promega. 7mG(- 5')pppG was from.Phamacia. RNase free DNase I, No; I were from Mannheim Boehringer. Sequenase DNA sequencing kit was purchased from United States Biological Company. [a4nT1dCTP was from Du Pont-New England Nuclear.:”S-methionine was from Amersham Corp. PGE2 was purchased from Cayman Chemicals. Serotonin was Obtained from Sigma. The cDNA for 5-HT1c serotonin receptor (4) was,a generous gift from Dr. D. Julius, College of Physicians and Surgeons, Columbia University. anstzugti on of cDNA Library. A mixture of complementary' DNAs (cDNAs) was synthesized from. NIHI 3T3 polyA(+) RNA (mRNA) using a specific 30mer oligonucleotide as primer: 5'-CGAGGCCATGGCGGCCGCTTTTTTTTTTTT-3'. This primer contains the rare Not I and Sf; I restriction sites next to the poly(dT) tail so that following incorporation of the cDNA into the lambda ZAP phage vector, cleavage of one of the sites can be used to terminate the subsequent in vitro transcription of cDNA.immediately after the poly(A) tail. After synthesis of 149 150 single- and. double- strand cDNAs, the cDNA 'mixture ‘was modified with EcoRI methylase, and E223 I linkers were blunt- end ligated to both ends. Following cleavage with EcoRI, the resulting cDNAs were inserted into the 3223 I sites located downstream of the SP6 or T7 promoters (they could be ligated in either orientations) in the lambda ZAP vector. Finally, the lambda cDNAs were packaged to yield a cDNA library containing 1.85x104Iclones. Most of the above reaction were performed by protocols described by Maniatis (5). Ln yirro synthesis Of RN . The lambda DNA mixture was isolated and extracted using the method described by Maniatis (5), and cleaved with Not I restriction enzyme. The resultant cDNA mixture was then transcribed in vitro using SP6 and T7 RNA polymerases (which initiated transcriptions from inserts oriented in either direction) . The reaction mixture contained: 10 pl 5x transcription buffer (200 mM Tris-HCl, pH 7.5, 30 mM MgC12, 10 mM spermidine, and 50 mM NaCl), 5.0 pl 0.1 M DTT, 2.0 pl RNasin ribonuclease inhibitor, 5.0 pl 5 mM each ATP, 01?, UTP; 0.5 M GTP, 5.0 pl 7mG(-S’)pppG, 5.0 p1 (Io-20 pg) linear DNA template and add H20 to a final volume of 50 pl. RNA transcripts were then extracted with phenol:CHCl3 and then with CHCl3 alone, and precipitate with ethanol. The pellet was resolved in DEPC water at a concentration of 1 pg/ml. Exprsssion of synthesized RNAs in gssyrs . Ovarian lobes of oocytes were taken from Xenopus laevis and oocytes were isolated under a dissection microscope. The oocytes were then 151 allowed to sit in NBS (modified Barth's solution) overnight at 18°C. Intact oocytes were selected the next day for injection. RNA (50 ng in 50 nl of water) was injected into each oocyte using a micropipette and a micromanipulator under a dissection microscope. The injected oocytes were incubated in MBS for about 40 hours before being assayed for Ca++ mobilization in response to P6132 . yessurement of intracellular Ca++wjp m. The injected oocytes were loaded with the fluorescence dye Fura-Z, by incubation in NBS containing 10 pM fura-Z/AM at 19 degrees with constant shaking for 40-60 min. Dye-loaded oocytes were then washed and kept in MBS until use. Change of fluorescence within the oocytes was monitored with a dual excitation wavelength SPEX spectrofluorometer. Samples containing five oocytes were allowed to stand in the bottom of a crystal spatula inserted into a mini-cuvette, and challenged by the addition of 5 pM PGEZ. During each trace, the oocytes were excited at wavelengths of 340 nm and 380 nm, and the emission was monitored at 505 nm. The fluorescence ratio was obtained by dividing the fluorescence intensity resulting from excitation at 340 nm by the fluorescence intensity resulting from excitation at 380 nm. This value was used to estimate the [Ca++]i(6). Sppfrsspiopation Of cDNA pools. The original cDNA library contained 1.85x104 clones. After determining that cDNAs from this pool was able to confer the PGEz-induced Ca++ 152 mobilization on oocytes, this pool of clones was subdivided into several smaller pools. After a series of subdivisions, a single clone responsible for the Ca++ mobilizing response to PGEZ was obtained . ssgpspsing_pfi_spfls. The cDNA responsible for the Ca++ mobilizing activity of PGE2 was named GING. GING was inserted into the EQQB I sites of M13mp18 and M13mp19 bacteria phage DNA. Single stranded sequencing of both strands of cDNA was performed using the method of Sanger et al. (7). A universal oligonucleotide primer from the vector was used as the first primer for the initial sequencing. Subsequently, a 17mer was synthesized from the known sequence of the cDNA and used as the primer for downstream sequencing. Four primers were used to complete the sequencing of the entire cDNA. Ln V1522 translation of GING RNA. Template RNAs were heated at 67°C for 10 min and immediately cool on ice. This increases the efficiency of translation by destroying secondary structures. Reticulocyte translation reaction mixture were composed of 17.5 pl lysate, 0.5 pl 1 mM amino acid mixture (minus methionine), 2.0 pl 35S-methionine (1,200Ci/mmole) at 10 mCi/ml, 2.2 p1 H20, 0.5 pl RNasin ribonuclease inhibitor (40u/pl), and 1.0 pl RNA substrates (0.5-1 pg/pl) iJIIQO. The reaction mixture was incubated at 37°C for 60 min and then loaded to SDS gel. Trspsgsction of COS-1 cells. To determine if GING can be 153 functionally expressed in mammalian cells (e.g. COS-1 cell), GING was subcloned into the expression vector pSVT7 in two orientations. G(+) is the plasmid in which GING was inserted with its apparent translation start site downstream of the SV40 promoter; G(-) is the plasmid in which GING was inserted in the opposite orientation to serve as a negative control. ST(+) is the plasmid in which the cDNA for S-HTI serotonin receptor was inserted; this later vector served as a positive control. The DEAE-dextran method as described by Shimokawa et al. (9) was used to transfect COS-1 cells. DMEM containing DEAE-dextran (7.5 mg/ml) was filtered and plasmid DNA was added to reach a final concentration of 5 pg/ml. COS-1 cells at 70-80% confluence in the wells of 12 well-tissue culture plates were incubated with 1 m1 of media containing a pSVT7- cDNA plasmid at 37 degrees for one hour. Then 2.3 ml of DMEM/PCS containing 52 pg of chloroquine/ml was added to each well. the samples were incubated for another 5 hours. At the end of transfection period, the cells were washed extensively, and then incubated for 40-48 hours in DMEM containing 10% FCS in a water saturated 7% C02.atmosphere at 37 degrees. flsssprsment of inositol phosphats. As described in the method section of chapter two. Writ Of Ca++ mobilizati n ' ' c s. As described in the method section of chapter two. RESULTS Exprsssipp_slppipg. An initial pool of 1.85x104 lambda ZAP recombinant clones was subfractionated until a pool of 20 cDNA clones was obtained that conferred upon oocytes the ability to mobilize Ca++ in response to PGE2 (Fig.2) . Single clones were picked from this pool and RNA transcripts from each clone were checked individually. One clone containing a 1.7 kb cDNA insert was identified as responsible for the response to PGEZ in oocytes. Based on restriction enzyme analysis, this cDNA, designated GING, was found to be inserted into the lambda ZAP vector with its 5'end next to the SP6 promoter, and its 3’end next to the T7 promoter (Fig.3A). Therefore, SP6 RNA polymerase would be expected to generate a sense transcript, while T7 RNA polymerase should generate an anti-sense transcript (which could serve as a negative control). To obtain sense and anti-sense transcripts, the cDNA was cut out of the vector at the flanking Sps I restriction site (Fig.3B), and RNA transcripts were synthesized using SP6 and T7 polymerase respectively. When the sense (SP6) and anti-sense (T7) transcripts were injected into oocytes separately, the sense transcript rendered oocytes responsive to PGEz, whereas the anti-sense one did not (Fig.4). 154 155 ssgpspss spslysis of GING. Sequence analysis showed that GING contains 1650 nucleotides (Fig.5). Its longest open reading frame extends from an ATG at position 19 to a stop codon at position 720, encoding a polypeptide of 233 amino acids, with a predicted molecular weight of 26,096. The polyadenylation signal AAATAAAA is found at position 1594. The NotI site, CGGCCGCC, which was inserted into the 30mer primer for the synthesis of the cDNA library, was found near the 3' end of GING. The deduced amino acid sequence of GING is shown in Fig.6. The. hydropathy plot, does not show any' prominent hydrophobic domains characteristic of transmembrane spanning regions. In fact, the hydropathy profile suggested that GING encodes a hydrophilic soluble protein. A search of the GenBank Database indicated that the predicted amino acid sequence of the GING protein is 62% identical (73% similar) to the bovine 14-3-3 protein (Fig.7). This protein is a cytosolic protein that is highly rich in brain, but also widely distributed in other tissues (6). uprrnerp plor analysis. As shown in Fig.8, a single 2.0 Kb species was done by Northern blot hybridization of GING with mRNA prepared from 3T3 cells, treated with or without 1 mM PGH synthase inhibitor aspirin. The pretreatment of aspirin did not change the mRNA level of GING. £1 yirro translation of GIN . A rabbit reticulocyte lysate was used for in vitro translation of the RNA 156 transcribed from GING. The translation was performed in both the presence and absence of canine pancreatic microsomal membranes (MM), which can process newly translated proteins. The processing by MM includes glycosylation, signal peptide cleavage, and membrane translocation. Fig.9 shows the SDS gel of the translated products derived from RNA synthesized from GING and from three other control RNAs, with and without the addition of MM. In the presence of MM, (a) a-factor became glycosylated and the mobility of the radioactive products increased from 18.6 Kd to 32.0 Kd; (b) B-lactamase had its signal peptide cleaved and the mobility Ofthe product deceased from 31.5 Rd to 28.9 Kd; and (c) Brome Mosaic Virus (BMV) protein was not processed so that the mobility of the products of 20 Kd, 35 Kd, and 110 Kd were unchanged. The RNA from GING gave rise to a radioactive band of 28 Kd, the mobility of which was not altered by the addition of M. The observed molecular weight is close to the predicted value of 26 Kd. This experiment established that RNA from GING can be translated, and that the translated product is not membrane associated and is not subject to processing by microsomal membranes. Eppsriona; expression of GING in COS—1 cells. Plasmids G(+), G(-) and ST(+) containing cDNAs for sense and anti- sense transcription of GING and sense transcription of the 5- HTH serotonin receptor (Fig.10) were used to transfect COS-1 cells. After expression.for 40-48 hours, the transfected.cells 157 were examined for Ca++ mobilization in response to various agonists at the single cell level. Of ten 'transfection experiments, five showed significant increases in responses to PGEz in G(+) transfected cells compared to G(-) transfected cells. In the other five experiments, no positive results were observed. It is not known whether the negative results were due to the failure of COS-1 cells to express the GING cDNA, or to the lack of an endogenous factor (e.g. an endogenous receptor) present in various batch of cells that is necessary for the function of GING. The results of the five positive transfection experiments are summarized in Table 4. In control COS-1 cells that were transfected with Ging(-), only 711% (n=413) of cells responded to 10 pM PGEZ and 1012% (n=413) of cells responded to 10 pM serotonin; in contrast 32:4% (n=409) of COS-cells transfected with G(+) responded to 10 pM PGEZ, and 4111% (n=172, from three of these five experiments) of COS-1 cells transfected with G(-) responded to 10 pM serotonin. A transfection efficiency of 20-30% is close to that Segre et al. reported about the average transfection efficiency of COS-7 cells transfected with a parathyroid hormone (PTH) receptor cDNA (10). Fig.11 shows the traces of transfected cells in response to serotonin, PGEQ and bradykinin, an agonist which induces an endogenous response in COS-1 cells. To examine if GING is a factor specific for Ca++ mobilization in response to PGE2 or a general factor for Ca++ 158 mobilization in response to other hormones, a series of agonists including PGan, CHA, isoproterenol, PTH and serotonin were tested in G(-) and G(+) transfected cells. None of these agents seemed to potentiate CaJ’+ mobilization in G(+) transfected cells except serotonin. In six of ten experiments, there was a moderate increase of responsiveness to serotonin in G(+) transfected cells compared with G(-) transfected cells. As summarized in Table 5, 1114% (n=528) of cells responded to 10 pM serotonin in G(-) transfected cells, whereas 2614% (n=590) Of cells responded in G(+) transfected cells. The following experiments were formulated to determine if GING protein potentiates Ca++ mobilization in response to PGE2 and serotonin by causing inositol phosphates (IPs) formation. COS-1 cells were transfected with G(-), G(+), and ST(+). IPs were measured as described in method section. As shown in Fig.12, 10 pM serotonin induced a small background increase (<30%) of IPs turnover in G(-), as well as G(+) transfected cells; whereas a significant increase (630%) occurred in response to serotonin in ST(+) transfected cells. PGEZ did not induce IPs formation in either G(-) or G(+) transfected cells. Thus, no effect of GING protein on IPs formation was detected. Surprisingly, when G(+) and ST(+) were introduced into COS-1 cells simultaneously, it was found that IPs formation in response to serotonin was inhibited, compared with control cells transfected with G(-) and ST(+) 159 (Fig.13). The preliminary results of transient expression in COS-1 cell suggest that GING protein potentiates Ca++ mobilization in response to PGEZ and serotonin in mammalian cells as well as in oocytes. Its action did not seem to involve an increase in IPs formation. 160 Figure 1. The sib-selection cloning strategy using the oocyte expression system. Synthesis of cDNA Addition of Linker Ligation to vector Linearization and Transcription Injection of oocyte with cDNA transcripts and measure [Ca“] in Sib selection to yield a specific cDNA clone IV’Y mRNA °\\\ *0: 5C"? A AAAA / / 3 3' r T TTTHHH 5' ‘ (E.CORI methylation) O r‘ AMA TTTT E.CoRI linker m m; T., prmter SpG promoter MAA m m T? “"“V 596 promoter .‘AAA m ""1. T7 Mar “P M A AAA mRNA MM ' In" EZE' - mRNA Sp6 promo or A PGE; ,9? (“If time _ A [55'53-2‘1'2': ‘55 ".-j X PGE! receptor c DNA 162 Figure 2. Ca++ mobilization in response to PGEZ by oocytes injected with (A). water, and (B). RNA synthesized from a positive pool of cDNA containing 20 clones.Ca++ mobilization was measured by spectrofluorometer as described in the text. The spikes in the traces were caused by opening of the shutter. 2. 225' 3:! b b— L Flourcsccncc Ratio e . ' my} .h" .‘fikfi‘V’M 2 fi{ sxlo’6 M PGE? " i l l. . . WWW“ 5' " “" ' W’Vfifi’w q i O 0'" ‘ ‘1'}, ii '1 1.22503 * ' I ‘ ‘ 2.2:: 275-917 ‘9'” Time (sec) 0.225 as r ~ 1 ~ I ' ‘ ‘ b B ‘ O L . ‘ "3 a: * 5x1076MPGEz ‘ F * ‘ a - I ' r: o . I ‘ g NW'W Lu i o l E ‘ u 4 L 1 1.29533 ~ A I ’ ‘ ‘ I ‘ 27.33 238.58 458.” 164 Figure 3. (A). The SEE I fragment from lambda ZAP-GING containing SP6 and T7 promoters at the two ends of the cDNA. (B). Agarose gel of RNA transcripts from the 523 I fragment. Lane 1, synthesized by T7 polymerase; Lane 2, synthesized by SP6 polymerase. T7 transcript (anti-sense) ‘__—.__—_____—___ SPE' NOTI SPE I III:- .32: SP6 promoter T7 Promoter __________—__.> SP6 transcript (sense) 166 Figure 4. Traces of Ca++ mobilization in response to PGE2 in oocytes injected with (A). transcripts synthesized by SP6 polymerase (6+), and. (B). transcripts synthesized. by 'T7 polymerase (G). Ca++' mobilization. 'was ‘measured. by spectrofluorometer as described in the text. .2orm r—--- I e e A a? . 5x10.6M .i 3 .W 3 l PGE2 W “ 2 : . ' " 8 IE}! E i‘ MWJflHE. [i Laura: A I - - 2.22 ‘ 200.38 . 400.38 Time (sec) 24sec ..- - - . V : * B .9 E a I i ‘6 5x10 hd I.) g h * PGE, i d I.) U , § ’ ' W i E {"54“ . Lain as n A - 4U.“ 2. 208.“ Time (sec) 168 Figure 5. The nucleotide sequence of GING. 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 CAGIGQQITC ooaccrecnc AAAATGTGQT senescence ccaorncccc rrcrccnrar rccnaserrr reaarrracc rcccrrncan caccccnrrc aarrcrrnnr areacocaar Tcrncccrca AGACATGCAG rccnacaran crcrancrnc AGAACCACCA cccrsrrarr trrnnnncnn Grannrrtcr AACAGGACTG cncrescnaa ncrtcrnesr ecccrcrcar crrrnnncrr aranrsarrr AAAAGCATGG ccsncrccrc ttscnracst crccccrccr AGCTAAITIG tnrrarrcrt ACAAACCTAA CGGACGéAnT CTGACAGTTG TGGAGCCAGA AAGAAAACAA CAGATGGTTG ACTGGACAAA TCTATTATAA ACAGGAAATG AGCTGCTAGT GTTTAGGTCT TCCCCCGACC TGCAGAACTG TCATGCAGCT GGTGATGGTG GAATCAGTGA CCCTTCCCCC AATTTGACTT TTTCTTTCTT ACAAACAAAC GTGGATTTTA CATAGAGGCT ATCATCATTA GRTGCATCIA TITTCCTTTT AAATTGGGTG TGCTGTAAAC TGCTGGTAAC CCCCTCTGAA GTTCAAFTCT TCGCTCGCTC TACTACTGGA ACTGAAACAC ATTAAAAATG GGIGGAATCA AAGAACGAAA nanccnrccr GGGAGGAGAG AAACIGAGCT cnccrcnrrc AATGAAAGGG ncnacnnGGn GACATTGCGA TGCTCTCAAC BTGCCTGCAG GACACGCTGA GCTACGTGAT QAGAGCAGAA GACGAAAIAA CCTCCCCTTG TCACATTTGG TTTCTITTTT AAACAGTTTT CTGGTCCAGC TTTTCAGCAT GAAATGGAAA AGAAAGATTA ITIAAITGTT TTAGCTTGAG TGTGTTTGGA AGTTCAACAA GOAGGTTAGC GTTCCTCCTC AACCTCTTTT TATCTGACTG AGCATGGAAT CCAAAAAAAA ATGAGGAAfiS CCTTTTATCT GGAGAATAAT GACAAATTAA CAAGTTAATC CAGCAGCTAA GACTACCACA GGCAGCAGAG TGACAGAACT TTTTCCGTAT GTTGGCAAAA GTGAAGAAAG AACCTGACGC TAAAGAAGCG AAGCCAACAA GAAGTTCCCC TCTCAGAATT TTTTTTCTCC CABAAGTTCT TTTAGGTTCT TACTGTATTG TGACATTTGA ATCACACAAT AAATTGAATT GISTTTTGGG ACTCTGCTGA TCCGTGGCTG ATTGAAGGTG CCTCCTCCTC GTTCAGTACG GAGCCGCAGG TAACATTAAA AAAAAGCGGC TAGCQGGGRT GTTGCATATA ananCAIT AGATGGTTCG rsrrercacn cacraccaac GGTATCTGGC ancnoccrca rccrccnnce rcracrnrcn ccaccrrrrs craranaaac raroaaccrc crccaGGAre saaannccar ATTGTCACTG raaarrccrs ccrccccrrr tnnoccnnaa rrncancncr TCTCCGGCCA AAGCCATTAG AGAGGCATAT TTATACCAAT GGAGTTIGTT AGTGTTGCTG CTCATTCTTG GTATGGAAGC GGCCTCCCTC TGTAACTTGA TACAGATCTG CTTAAATAAA CGCCATCGGA 170 Figure 6. (A). The predicted amino acid sequence of the longest open. reading frame: of GING. (B). .Hydropathicity profile of GING. The averaged hydropathicity index of a nonadecapeptide composed of amino acids residue i-9 to i+9 is plotted against i, and i represents amino acid number. "VESNKKUAG NDVELTVEER NLLSVAYKNV IGARRASHRI ISSIEGKEEN KGGEDKLKHI REYRQHVETE LKLICCDILD VLDKHLIPAA NTGESKVFYY KNKGDYHRYL AEFATGNDRK EAAENSLVAY KAASDIANTE LPPTHPIRLG LALNFSVFYY EILNSPDRAC RLAKAAFDDA IAELDTLSEE SYKDSTLINQ LLRDNLTLUT SDHQGDGEEG NKEALQDVED ENG 100 200 1IIIIIIIIIIIIIIIIII‘ILin Axislal- MI .4}. 1: id .1 .I-] «Isiah-e]- l I- ..I'.~.i -. . . 100 200 172 Figure 7. Alignment of the amino acid sequence of bovine 14-3- 3 protein (top) and GING protein (bottom). Pairs of identical residues are connected with two dots. Pairs of similar residues are connected with one dot. 51 29 101 79 151 127 2011 177 HGDREQLLQRARLAEQAERYDDHASAHKAVTELNEPLSNEDRNLLSVAYK 50 i......................NVESNKKVAGNDVELTVEERNLLSVAYK 28 NVVGARRSSWRVISSIEDKTHADGNEKKLEKVKAYREKIEKELETVCNDV 100 NVIGARRASURIISSIEQKEENKGGEDKLKHIREYRQHVETELKLICCDI 78 truism:immravtsmrtmentvmasvésis’zxmswens Is‘o LDVLDKHLIPAANTG..ESKVFYYKNKGDYHRYLAEFATGNDRKEAAENS 126 EAAYKEAFEISKEHHQPTHPIRLGLALNFSVFYYEIQNAPEGACLLAKQA 200 LVAYKAASDIAHTELPPTHPIRLGLALNFSVFYYEILNSPDRACRLAKAA 176 FD A TLNEDSYKDSTLIHQLLRDNLTLHTSDQQDEEAGEGNXA.. 248 FD A ELDTLSEESYKDSTLIH QLLRDNLTLHTSDHOGDGEEONKEALQ 226 174 Figure 8. Northern blot Of 32P labeled GING hybridized with poly(A)+ RNA isolated from lane 1: NIH 3T3 cells; lane 2: NIH 3T3 cells treated with 1 mM aspirin for overnight. 5-10 pg mRNA were used for each lane. The full-length fragment of GING was used as the probe. cflnfimnm\mem mBm 2.0 Kb ——’- ‘I’lll 176 358 Figure 9. -Met labeled products translated in vitro from RNAs by a rabbit reticulocyte lysate system. Each RNA was translated in the presence (indicated by "+") or absence (indicated by "-") of microsomal membrane. Lane 1, no RNA; lanes 2 and 3, Brome Mosaic Virus RNA (BMV); lanes 4 and 5, GING RNA; lanes 6 and 7, a-factor RNA; lane 8 and 9, B- lactamase RNA. $3326 .mmmuomma .+eobome-a . couomurd + IT microsome membrane mcG mEo >2m >2m mcoc RNAS K O 1 1 O... O 35 Kd—r .- 20 Kd—> 178 Figure 10. Plasmids containing GING and a cDNA for the S-HTI serotonin receptor. G(+): GING was inserted into pSVT7 with the transcription start site downstream of SV40 promoter. G(- ): GINwaas inserted intijSVT7 in the orientation opposite to that of G(+) . ST(+): cDNA for 5-HT1 receptor was inserted into pSVT7 with the transcription start site downstream of SV40 promoter. 180 Figure 11. Traces of Ca++ mobilization in response to agonists by transfected COS-1 cells. (A). G(-) transfected cells challenged by 10'5 M serotonin (ST) and 10'5 M bradykinin (BK) consecutively. (B). ST(+) transfected cells challenged by 10'5 M ST. (C). G(-) transfected cells challenged by 105‘M‘PGE2 and 10'5 M BK consecutively. (D). G(+) transfected cells challenged by 10'5 M PGE2 and 10‘5 M BK consecutively. Ca++ mobilization was measured at the single cell level using an interactive laser cytometer as described in the text. Each panel represents traces of cells from one scanned field. 0‘9 111.jllllllTllllililIIIIIIIIII] 0, 50 '00 '59 2.09 25° 30° 0 so we use 200 250 300 352 -. unrealized Floor v5. We (set) - Normalized Fluor vs. time (sec) . o se'm 50200250100 350‘400 a 40 an m Isa'zoo'zro Nomllzed Fluor vs. The (sec) - - Normalized Fluor vs. Time (sec) 182 Table 4. Ca‘H' mobilization in response to PGE2 and serotonin in transfected COS-1 cells. COS-1 cells were transfected with plasmids G(-), G(+) or ST(+) as described in the text. After incubating at 37 degrees for 40-48 hours, cells were loaded with 10 pM fluo-3/AM and examined for increases in [Ca+‘fh when challenged by 10 pM PGEQ or serotonin. Cells with z 10% increases of fluorescence intensity were defined as responsive cells. n is the number of cells examined in five experiments for G(-), G(+) transfected cells, and in three of the five experiments for ST(+) transfected cells. Results were expressed as percentage of cells that respond (meaniS.E.). PGE2 Serotonin GING(-) 7: 1% 1o: 2% n=413 GING(+) 32: 4% N.D. n=409 ST(+) N.D. 41: 1% n=172 184 Figure 12. Formation of inositol phosphates (IPs)in response to PGE2 and serotonin (ST) in G(-) , G(+) and ST(+) transfected COS-1 cells. IPs formation was measured as described in the text after transfected cells were stimulated with 10'5 M agonists for 30 min. The results are expressed as percent increase of in total IPs versus control cells treated with vehicle alone. percent increase of IPs formation 550 500 450 400 150 100 Serotonin 1C] Ging(-) 'Ging (+). I ST(+) PGE2 186 Figure 13. The effect of GING protein on IPs formation in response to serotonin in ST(+) and G(-)/G(+) cotransfected cells. COS-1 cells were cotransfected with 5 pg G(-) and 5 pg ST(+) or 5 pg G(+) and 5 pg ST(+) as described in the text. Total IPs formation was measured as described after transfected cells were stimulated with the indicated concentrations of serotonin for 30 min. 350 - —e— ST(+)/G(-) ——o— ST(+)/G(+) 300 - t: O ’1: E 1 .2 250 - v1 . a . '11-: O a 200- .5 ‘ g 150 - 8' q 100 1—l / I - u - I . o -8 -7 -6 -5 Log [serotonin] DISCUSSION Xenopus oocytes have been extensively used for efficient translation of foreign mRNAs, including mRNAs encoding receptors and ion channels (10, 11). The de novo synthesis of proteins from their mRNAs generate biochemically functional end products, which can be detected.by various biochemical and electrophysiological methods. This makes the oocyte an excellent system for expression cloning. In attempting to clone a receptor for PGEZ with this system, we used the Xenopus oocyte system and employed Ca++ mobilization as our functional assay. We obtained a clone which does not encode a typical G protein-linked receptor, but rather a cytosolic protein belonging to the family of 14-3-3 proteins. PGE2 is an autacoid that elicits effects by interacting with membrane receptors, triggering a series of biochemical events. The fact that GING protein is not a receptor suggests that there exist an endogenous PGEZ receptor in oocytes and that GING can potentiate a downstream effect of receptor activation leading to an increase of [Ca++Ji. In fact, a PGE receptor coupled to activation of adenylate cyclase has been reported to be present in the membranes of follicular cells that surround the oocyte (12). There exist gap junction contacts between oocyte and 188 189 follicular cells that allow passage Of some molecules (13) . It was shown that K+ channels can be activated by PGE2 in a cAMP- mediated manner (12) , indicating that PGE2 stimulates an increase in cAMP in follicular cells, which then leads to phosphorylation of the K+ channels. Phosphorylation of enzymes or ion channels usually involves more than one type of protein kinase, including cAMP-dependent protein kinase, Ca++/phospholipid-dependent protein kinase, and Ca++/calmodulin-dependent protein kinase type II. 14-3-3 protein was first isolated by Grasso and Perez (14) from bovine brain as a "brain-specific” protein ( 12). Later, Boston et al. (15,16) purified the human homolog of the bovine 14-3-3 protein, and localized it to neurons in the human cerebral cortex. Using a radioimmunoassay, they showed that although human brain has the highest concentration of 14-3-3, other tissues also synthesized considerable amounts of this protein (16) . No biological function had been assigned to the 14-3—3 protein until Ichimura et al.(17) isolated a group of bovine 14-3—3 proteins and cloned the cDNA for the n chain from a bovine cerebellum cDNA library. They demonstrated that the purified n chain could activate tyrosine and tryptophan hydroxylase activity in vitro, in the presence of Ca++/calmodulin-dependent protein kinase II. Later, they found that the distribution of 14-3-3 proteins in vertebrate and bovine tissues correlates with the distribution of Ca++- 190 dependent protein kinases (18). More recently, Alastair et al.(19) isolated a group of acidic proteins from sheep brain and found that these proteins inhibit Ca++/phospholipid- dependent protein kinase C. These acidic proteins were found to be homologous to the bovine 14-3-3 proteins. Last year, an intracellular phospholipase A2 was cloned from a human placental cDNA library; the phospholipase A2 was also shown to be a member of the 14-3-3 family (20). Obviously, this family of proteins is widely involved in various cellular activities. Now, our discovery of an effect Of a 14-3-3 protein on Ca++ mobilization in response to PGEZ adds another example of a potential the role for 14-3-3 proteins. The observation by Ichimura et al.(18) that the distribution of 14-3-3 proteins correlates with the distribution of Ca++-dependent protein kinases suggested that 14-3-3 proteins may participate in processes involving Ca++-dependent protein kinases. Based on this observation and the finding that PGE2 increases cAMP formation in follicular cells of oocytes, one explanation for the effect of the GING protein in our system is that GING activates a Ca++/phospholipid-dependent protein kinase, which coupled with the effect of PGE2 to stimulate cAMP-dependent protein kinase, leading to the phosphorylation and activation Of certain components (i.e. enzymes or Ca++ channels) involved in the pathway for the release of intracellular Ca++ or influx of extracellular Ca++. 191 The last question I would like to discuss here is the oocyte system used for expression cloning. Since GING protein is not a receptor that initiates the response to PGEQ, but rather is involved downstream of the receptor to potentiate the response, its effect will largely depend on the level of endogenous receptor. If the level of endogenous receptor is low, there‘will not be enough receptor'toractivate the pathway to initiate the response, thus no effect of GING protein will be observed. According to this assumption, the poor reproducibility of functional assay in oocytes encountered in our cloning work may be due to the large variations of levels of endogenous receptors in different batches of oocytes. In fact, background responses were Observed in 15-20% of oocytes not injected with RNA. Accordingly, the responses to PGE2 between control oocytes and RNA-injected oocytes within those batches could not be resolved due to the limited sensitivity of the system. The situation is similar when GING is in vitro expressed in mammalian cells. The type of cells used for expression of GING has to possess a certain level of endogenous receptors so that the potentiating effect of GING protein in response to the corresponding hormones could be Observed. Cos-1 cells used in our case for GING expression seems to possess a PGE receptor coupled to adynylate cyclase, too, since a small backgrond increase of cAMP can be observed when stimulated with 10‘5 M PGEZ (21). The presence of this native receptor may account for the potentiating effect of 192 GING on Ca++ mobilization in response to PGE2 observed in G(+) transfected COS-1 cells. It is possible, therefore, that the lack of responses to PGE2 in some batches of G(+) transfected cells is due to the lack of enough endougenous PGE receptors mediating cAMP formation. REFERENCE 1. Shorr, R.G., Lefkowitz, R. J., and Caron, M.G. (1981) J. Biol. Chem. 256, 5820-5826. 2. Dixon, R.A.F. et al. (1986) Nature 321, 75-79. 3. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., and Nakanishi, S. (1987) Nature 329, 836-838. 4. Julius, D., MacDermott, A.B., Axel, R., Jessell, T.M. (1988) Science 241, 558-564. 5. Maniatis, T., Fritch E.F., Sambrook, J. (1982) In: Mglssplsr Qloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 6. Grynkiewicz, G., Poenie, M., Tsien, R.Y., (1985) J. Biol. Chem. 260, 3440-3450. 7. Sanger, F., Nicklen, S. and Coulson, A.R., (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 8. Shimokawa, T., Smith, W.L. (1992) J. Biol. Chem. 267, 12387-12392. 9. Juppner, H. et al. (1991) Science 254, 1024-1026. 10. Meyerhof, W., Morley, S., Schwarz, J., Richiter, D. (1988) Proc. Natl. Acad. Sci. USA 85, 714-717. 11. Gundersen, C.B., Miledi, R., Parker, I. (1984) Nature 308, 421-424. 12. Mori, K., Oka, S., Tani, A., Ito, S., Watanabe Y. (1989) 193 13. 14. 15. 16. 17. 18. 19. 20. 21. 194 Biolchem. and Biophy. Res. Comm. 162, 1534-1540. Woodward, R.M., Miledi, R. (1987) Proc. Natl. Acad. Sci. USA 84, 4135-4139. Grasso, A., Roda, G., Hogue-Angeletti R.A., Moore, B.W., and Perez V.J. (1977) Brain Res. 124, 497-507. Boston, P.F., Jackson, P., Kynoch, P.A.M., Thompson, R.J. (1982) J. Neurochem. 38, 1466-1474. Boston, P.F., Jackson, P., Thompson, R.J. (1982) J. Neurochem. 38, 1475-1482. Ichimura,T., Isobe, T., Okuyama, T., Takahashi, N., Araki, K., Kuwano, R. and Takahashi Y. (1988) Proc. Natl. Acad. Sci. USA 85, 7084-7088. Ichimura T., Sugano, H., Kuwano, R., Sunaya T., Okuyama T. and Isobe T. (1990) J. Neurochem. 56, 1449-1451. Aitken, A., Ellis, C.A., Harris, A., Sellers, L.A., Toker. A. (1990) Nature 344, 594. Zupan, L.A., Steffens, D.L., Berry, C.A., Landt, M., Gross, R.W. (1992) J. Biol. Chem. 267, 8707-8710. Honda, A., Sugimoto, Y., Namba, T., Wataba, A., Irie, A., Negishi, M., Narumiya, S. and Ichikawa, A. (1993) J. Biol. Chem. 268, 7759-7762. 3129300881 1493