655

PHOSPf IODIESTERASE IN FETAL CORTEX 19. Moore. E. S.. Fine. B . P.. Sntrosook, S . S., Vergel. Z. M.. and Edelmann. C . M.. Jr.: Renal reahsorption of bic:~rhonate in puppies: Effect of intracellul;lr volume contraction on the renal threshold for bicarbonate. Pediat. Res.. 6: 8 5 9 (1972). 20. Parkenson. M. L . . Lubowitz. t l . , White, R. S., and Bricker, N. S.: O n the influence of extracellular fluid volume expansion on bicarbonate rci~bborption in the rill. J. Clin. Invest., 48: 1754 (1969). 2 1 . l'otter, D . . Jnrrah, A.. Sakai. T., Ilarrah, J., and llolliday. M . A , : Char:~ctcr of function and size in kidney during normal growth of rats. Pediat. Res.. 3: 51 (1969). 2 2 . Rector. F. C . . Jr.. Bloomer, 11. A , . and Seldin. D . W.: Effects of K deficiency on reabsorption of bicarhonate in the proximal tubule of the rat. J . Clin. Invest.. 43: 1976 (1964). 23. Rector. F. C.. Jr.. Seldin. D . W., Roberts, A . D.. Jr.. and Smith. J. S.: The role of plasma CO, tension and carbonic anhydrase activity in the renal rcahsorption of bicarhonate. J. Clin. Invest., 39: 1706 (1960). 24. Rohillard, J . E.. Kulvinskas. C.. Sessions. C., Burmeister, L., and Smith. F. G.. Jr.: M;~turationalchnnpcq in the fetal glnmerulnr filtration rate. Amer. J . Obstct. Gynecol.. 122: 601 (1975). 2 5 . Robillard, J . E.. Sessions. C., Kennedy. R., Durmeibter. L . , and Smith,T. G.. Jr.: Renal carbonic anhydrase activity in the chronic fetal lamb. (In preparation.) 26. Rohillard, J. E.. Smilh, F. G., Jr.. Kulvinskas. C., and Bradcn, E.: Carbonic anhydrase function in the fetal kidney [Abstr.]. Pediat. Res.. 7: 188 (1973). 27. Saloman, L . L., and Johnson. J . E.: Enzymatic microdetermination of glucose in blood and urine. Anal. Chem., 31: 453 (1959). 28. Scldin. D . W., and Rector, F. C., Jr.: The generation and maintenance of metabolic acidosis. Kidney Intern., 1: 306 (1972). 29. Siggaard Anderren. 0 . . and Engel, K.: A new acid-base nomogram method for the calculation of the relevant blood acid-base data. Scand. J . Clin. Invest., 12: 177 (1960). 30. Smith, F. G . , Jr., and Schwartz, A , : Response of the intact Iamb fctus t o acidosis. Amcr. J . Ohstet. Gynccol., 106: 52 (1970). 31. Smith, F. G . . Jr., Tinglof, B.. and Adams, F. t1.: Renal phosphate clearance and bicarbonate excretion in the fetal lamb [Abstr.]. J . Pediat., 67: 955 (1965). 32. Solomon, S.: Absolute rates of sodium and potassium reahsorption by proximal tubule of immature rats. Biol. Neonate, 25: 340 (1974). 33. Spitzer, A , . and Brandis, hl.: Functional and morphologic maturation of thc superficial nephrons. J. Clin. Invest., 53: 279 (1974). 34. Spitzer, A.. and Windhager, E . E.: Effect of peritubul;lr oncotic prcs\ure

35. 36. 37. 38. 39. 40.

41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

54. 55. 56.

changer on proximal tubular fluid reabsorption. Amcr. J. Physiol.. 218: 1188 (1970). Stcin, J . )I., and Reineck. 11. J.: Effect of alterations in extracellular fluid volume on segmental sodium trc~nsport.Physiol. Rev., 55: 127 (1975). Sleinmetz, P. R.: Cellular mechanisms of urinary acidification. Phy,iol. Rev., 54: 890 (1974). Suki. W. N.. tlebert, C. S.. Stincbaugh, B. J . , Martinez-hlaldon:ldo, M., and Eknogan. G . : Effects of glucose on hicarbonate reabsorption in thc dog kidney. J . Clin. Invest., 54: 1 (1974). Svenningsen, N. W.: Renal acid-base titration studies in infants with and without metabolic acidosis in the postneonatal period. Petlint. Res.. 8: 659 (1974). Tsoulos. N. G.: Comparison of glucose, fructose and 0, uptakes by fetuses of fed and starved cu,es. Amer. J. ~h~siol.,'221:234 (1971). Van Slyke, D . D.. and Neill, J . M.: The determination of gases in blood and other solutions by vacuum extraction and manometric mearurcment. J. Uiol. Chem., 61: 523 (1924). Vaughn. D..Kirschbaum. T . If., Bersentes. T., Dills, P. V.. Jr.. :lnd Asmli. N. S.: Fetal and nc(inatal respcinsc t o acid loading in tlic sllccp. J . Appl. Physiol.. 24: 135 (1968). Widdowson. E. M.: Growth and composition of the fctus and newborn. In: N. S . Assali: Biology of Gestation. p. 1 (Academic Prcrs. New York, 1968). Xylocaine, Astra Pharmaceutical Products, Inc., Worchester, hlass. llarvord Apparatus, hlillis. Mass. Baxter Laboratories. Glofil, Abhott Laboratories. North Chicago, Ill. Radiometer, London Co.. Cleveland, Ohio. Instrument Laboratory, Lexington, hl;lss. Uuchlcr Instruments. Inc., Fort Lee, N. J. Advanced Instruments. Inc.. Needham Ilcigllt.;, hfass. Beckman Instruments. Inc., P;~loAlto, C a l ~ f . A preliminary report of these findings was presented ;it the 1975 Annual Meeting of tlie American Pediatric Society -The Society for Pediatric Research (Pcdiat Res., 9: 378 (1975). This research was supported by Research Grant No I I D 08953 froni the National In\titute of Child 1le:llth and lluman Development and Rcsc;lrch Grant MA-5652 from the Canadian Medical Research Council. Requests fur reprints should be addresred to: J . E . Rohillard, M.D.. Depnrtment of Pcdi:ltrics, Division of Nephrology. University of lowa Ilospital nnd Clinics, lowa City, lowa, 52240 (USA). Received for publication February 18, 1976. Accepted fnr publication September 30. 1976.

Copyright O 1977 International Pediatric Research Foundation, Inc.

Pediat. Res. 11: 6 5 5 - 6 6 3 (1977)

Cerebral cortex fetus phosphodicstcrase

Cyclic Nucleotide Phosphodiesterase Activities of the Fetal and Mature Human Cerebral Cortex ELLEN S. KANG'"" WITtI T l I E T E C t I N I C A L ASSISTANCE OF D A N I E L L . ELLSWORTH

Di.~'art~trerrt of Bioclre~tri.rtry,St. Jude, Clrilc/rc~tr's Rt~s.srv~rcIr f / o . ~ p i t futrd ~ / , ~ I I PDr~pcrrtttre~~rt of P~~rliotrics, Utrivrrsity of USA Tc~~rt~cssee Cc~trrerfor the Ilenltli Scic,rrci~s,Alettrplris, Tc~rrtrc~ssc~e,

Pliospl~odiesterase activities were exan~inedin the supernatant aritl pellet fractions of a 30,000 x g preparation of brain tissues fro111 11u111anfetuses ant1 young aclults. Differe~~ces in total activity and distribution of the high and low K,,, activity enzynics for atlenosine and guanosine 3',Sf-monophospl~ate (cyclic ARIP and cyclic GRIP, respectively) were found. The n~aturecortex hat1 10 tin~esrriore activity than the fetal brain for cyclic ARIP hydrolysis and 15-20 tinies niore activity for cyclic GRIP hydrolysis. In the fetus, nlore activity for both nucleotides

at both high and low concentrations is associated with the supernatant fraction. \Vith nlaturity, a shift in localization of high K,,, activity for cyclic GRIP aricl low K,,, activities for both nucleotides to the particulate fraction is observed. hlichaelis constants for both niature and inln~aturebrains are sin~ilar.The K,,, values for cyclic ARIP are lo-' and 10-"1 and lo-.' and 10-" RI for cyclic GRIP. The V,,,;,, values differed by a f ~ c t o rof 10 between the high K,,, ant1 the low K,,, fortns for each substrate.

I)ifferc~iceswere obscrvccl I)et\veen tllc fetus a~icl(lie aclult \vl~cn the I~yclrolysisof o n e riucleoticle \!as riicasurecl in (lie p r e w n w of varying a~iiountsof tlic otlier ~iucleotitlc.Low corlc e ~ i t r a t i o ~of~eitlier s ~iucleotitles t i ~ ~ ~ u l a tthe c c l I i ~ c l r o l ~ sof is l o ~ v co~iccntr:itio~is of tlic otlirr in the :itlult \rIicrcas, ill tlic fetus, low co~iccntratio~is of cyclic ARII' inliil)ilecl cyclic GRII' hytlrolysis. A t liiglicr co~icentrationsof eitlier ~iuclcoticlc,tlie :~clclitionof t I ~ e o t l ~ r ovcr r a \title range of c o ~ i c e ~ i t r a f i oresultecl ~is ill i~iliil)ition ~ l i i c lIS ~ esaggcratecl iri tlic fetus.

and the time el;rp\ing hct\vccn tlcath or the expulsion of tlic f e t w ;ind the heginning of tlic po\trnortcni examination for each subject arc listed in Taldc 1. All ;~utopsiesivcrc conducted rotrtinely with tlie removal of the al>dorninal viscera prcccdirig the removal of tlic brain. I'1)I: ;rss;~yswere done o n rcpresen1;llive sections of frontal cortex containing hotli ~vliitcand gray matter. All tissues were ol,t;rincd with the informed consent of tllc rcspon~iblcnest of kin.

I'ho\pIiodiestcrasc activity \v;rs assayed in tlic supernatant and p;~rticul:~tcfraction\ of tissues tvhich were homc>gcnized in 3 \rolurncs of glass-distilled water. freeze-tlia\vcd r;lpidly three tirncs, spun at 30,000 x g (0-4') for 30 nlin, rid rli;rlyzecl overnight in 20 rnhl Tris huffcr, pII 7.5. at -1'. I'IIE activity obscrvcd in the ~rndialyzcdfraction of :I 30,000 x ,q liomogeri:~tc of cortcs frorii a 56-year-old male subject \v;rs 8 0 . 6 nniol/mglrnin Consirlcrnblc evidcncc exists \vliicli suggests tliat tlie t\vo natu- \vlicrcas the dialy7ed fraction \vas 93.8. Tliis increase in ;rctivity rally occurring cyclic nuclcotitlcs, cyclic A h l P ancl cyclic GhII', is ~ x o h a b l y due t o tlic removal of endogenous suhstr:rtcs. are cxtcnsively involvctl iri tlic regulation of the tlevclopmcrit Amounts o f protein trscd for linearity ovcr a 10-tnin periotl wcrc and function o f the nervous systcrn. For instalice. in model 50-1 00 u c in 100 f i l total volurnc for cyclic Ahll' t'I>I: and 25systems. ( I ) the irirluction of c n q m c s involved in ncurotrans- 5 0 p g protein for cyclic G h l P l'I>E. Incutx~tionwas for 1 0 min mission Ii;~sbeen rlcrnonstrateil for the arlcnosinc riuclcotitlc ( 9 , for spccific ;rctivities and 1 min for kinctic studies at 31-30". The 23, 24, 2 0 , 30). ( 2 ) coritrol o f growth and diffcrcnti;~tioriof tliv:~lcrit cation rctluircrncnt M ~ ; I S riict by hln t t ;kt ;I firi;rl co~ieerincuron:~ltissuc :ippe;ir to he Iiiglily rcsponsivc to cliangcs of tlic tration of 0.1 m M a n d the reaction was corid~rctcdin 40 nihl Tri.; intr;rccllular conccritr;~tionsof cyclic Ahll' ( 3 0 , 30, 45). and ( 3 ) I>rrffcr. p l l 7.4. The ttvo-step iwtopic neth hod of Thompson ant1 the pirt;~tivc ~ i c ~ ~ r o t r ; ~ ~ i s r i ielicit i t t c r schanges in the concentra- 12pplcman (44) was used. Substr;~tcconcentrations varied ovcr ;I tions of tllc cyclic ~iuclcotitlcsrluririg ncurotra~ismisaion:is tletcr- r;rrigc of 2 X I OV:' t o 10 * hi. T I i c ~ ~ ) I ~ y l lW;IS i ~ i c ~rscdat firxrl minccl by iontopliorctic stuclics in 110th ccntriil ant1 pcrip1icr;rl concentrations of 0 . 0 to 7.5 rnhl. Final conccntrations of itiiidazprcp;~rations( I 5 , 20). In ;~clclition,cvidcricc is also accuriiulating ole \\.ere 1 0 . 2 0 . :ind JO mhf. /\ctiv;itor cffccfs wcrc stuiliccl wit11 rlrat cyclic Ahll' allel cyclic Ghll' facilitate oppo\ing cll'ccts in ;I and w i t l l o ~ tlic ~ t addition of I 0 p g partially purified protein froni nuriihcr of tissircs inclutling nervous tissuc (13, 38). bovine hrain, ivliich w:ts :I gift from IIr. L. Liu. 'l'hc percentage l'lic coricentration of tlicsc cyclic nirclcotirlcs iri tissues arc of convcrsion of lahclcd substrate t o the 5'-dcriv;rtivc was ilcteriletcrmine~lin part by tlic rate of their hydrolysis by specific niincd in an aliquot of the supernatant after resin precipitation in pliosphoclicsterases. Uriclcr a common stimulus, the intr:~ccllul;tr I3ray's solution in a sl>cctrophotor~ictcr. mct;~holismof rcsponsivc cells coulil vary \vitlcly tlcpcntling o n To tlctcrminc tlic cffcct of diffcrcnccs in I'D[: l~ctivitydue t o tlic functional status of tlic several forriis of pliospliodicstcrase \'ariation in time hettvcen dc:~thand collection of ti\sucs. ccre(I'I>Ii) \vliicli have Ileen clcscrillcrl in m;rrnmali;in tissues. hral cortices froni single rats were frolcn irnn~edi:rtclyand at 3 , In nnitiinls, tlcvcloprncntal st~rdicsby S c l ~ n ~ i ic.1l t trl. (35) :111d 5. a n d 24 lir after death, during \vliicli intcrv:rl the intact carcass \Veiss (40) sho\verl the prcscnce o f I'I>E in tlie brain of tlic of exch ;~nirnal\v;I.; rcfriger;rtcd at 4". A11 tissues were proccsscd ncnl>orn rat \\hicli iricreascrl to maturity levels by 15-23 days. simult;~ncoirslyfor P I I E rnc;tsurcnicnts. Approximately 5 0 % of the total I'IIE ;~ctivity\\.;IS nssociatecl Adcriosiric 3l.5'-cyclic [ S - : ' I l ] ~ ~ l i o s ~ ~was l ~ ; ~purcli;~sed tc from ivitli tlie particulate fraction accortling to the data of Wciss and Scli\var~-hlannand gu;rnosinc 3',5'-cyclic [:'I I-Gjphospliatc was Costa (47). \\liereas Schmitlt ;rncl liis associates found tliat tlle ol>t:~inedfrom Ncw Englirnd Nuclc;~r. Isotopes were further mitjority of tlie i~ctivitywas located in the supernatant at :I ratio p~rrificdby clironiatography on thin layer plates of cellulose in 2of 4 : l for the particul:~tc fractions at all agcs stutlied. pflicsc prop;~rioI,arnmonia, water (7:1:2) hcforc use. All other chernisturlies were performed untlcr espcrimcnt;~lcotitlitions \\liich f i ~ i l c;~ls \\ere of rcirgcnt grade. Protein was detcrmiticd by the to account for the niultiplc forms of PIIIJ since suhstr:~tcwas method of Lowry wit11 hoviric serum ;ilbuniin a s ;I stancl;~rd(25). limitctl to cyclic A h l P at coliceritrations of 10 :' ant1 1 0 ' h l . IINA \v:ts measured lly tlie dil>henyl;imi~icmethod of 13urton Stratla c.1 (11. (30) rcinvcstigatctl the postnatal dc\8elopmcnt of (7). this enzyme in the rat ~rsirigpIiysic;~I sc~>;~r;~tiori 1iic1Iio~I~ iri conjunction with kinetic ;~nalyses..l'Iic large total postnirtal incrc:~scseen in tlic rat ccrel)rurii was fount1 to be due primarily to a (,-fold iricreirsc in the activity of the high I.;,,,enzyme. l'he low Sincc our samples u c r c obtained at alrtopsy and tlic specific I.;,,,crizymc incrcasctl ahout 2.5-fold in activity during tlic snnic diseases. :~gonal conditions, ant1 agcs of our subjects varictl pcriotl. considerably, the stability of the c t i ~ y r n ct o storage effects and a It sllo~rldhe cniphasizcd that age-lunction studics of the brain common b;~sisfor conlparison of cnzyrnc tictivity h:rd t o bc hiivc bceti limited to the nc\vborn and 60-100-day-old rat. Sincc tlctcrmined. maxinium :~lterationsin adaptive rcsponsivcncss t o :I variety o f The specific activities ol~scrvedfor cyclic Ahll' hytlrolysis of stiriiuli arc obscrvcd I , c t ~ . c e nfetal and mature organs, tlic activi- individual rat brains collcctcd after varying periods of stor;~,gcat tics of the kinetically definable forms of P D E \vere examined in 4" :Ire prcsclitcd in Table 2 . No change i r i the spccific activrty of the cerebral cortices of the human fetus and adult. In addition, I'DE was noted in tissues obtained from intirct anirnals rcfrigcrt l ~ ccffcct of a v;~rictyof modul;itors of P D E activity was csarn- atcd for up to 24 lir. This ol~scrvationis in agreement with the incd it1 tlic mature cortcs a n d , in some instances, in the itiirna- p~rhlishcd literature which iridic;~testliat the crude cnzyrnc is ture cortcx. remarkably stable during storage (2). Activity was b;~scd on D N A for the follo\ving reasons. ( I ) hlAI'I~IIIALS AND h1ETIIODS Increase in cellular protein, R N A , lipid, and water content of the cortcx continues tlirougliout the process of niaturation \vllicli CLINICAL h4ATI.RIALS extends through adolesccricc (19). ( 2 ) Ncuroblastic prolifcraTissues for clizynlc studics were collcctcd at autopsy arid kept tion is con~pletcdlly 3 0 ~ v c c k sof gcst:rtional age in man and glial frozen at -20" or -SO" until analysis. Pertinent clinic;~lfc;~turcs proliferation hy the end of the first postn;~talyear (1 1. 49).

I)iffere~ices ill rrspo~isive~icss to ~iiotlulatorsol)servcd I)ct\r.ccr~fetal a ~ i t laclult c~izyriiessuggest the possibility 1Ii:it tliesc eiizyriirs occur as isozy~iies.Sirl)sta~~tiatio~i of this possibilitg brill recluire tllc separation of tlirsc elizyliies in pure for111.

PHOSPIIODIESI'ERASE IN CEIlEBRAL CORI'EX 'I'ablc

I . C'litric~rlit~firtrrrrriot~of srri)jccls

HIGH K M ACTIVITY

LOW KM ACTIVITY

N.3

Sril~jjccr

I 2

3 4 5 6

7

8

Information

1

14-week male fetus, spontaneously :ihorted. twin ofsrthjccr 2 , 2 hrl 14-week female fetus, spontaneously ahortccl. twin ofsrihjccl I , 2 hr' I S-tveek nlalc fctus, sptrritancously nhortcd. ? hrl 18-\vcek m;ile fetus. spontancously ubortccl. 1-3 hr' 19-20-week fetus, sporitat~couslyaborted, several hours1 15-year-old malc, mental retardation, polycystic kidneys, uremia, 12 hrl 18-year-old male, treated acute lyriipliocqtic leukemia. scpqis. 12-24 hr' 19-yc;~r-oldmale, acute heniorrhngc froni traulnatic chest wounrl. 12 hr'

' llours elapsing b~:t\vcencleat11 ancl autopsy. For fetuscs, clapscil tiliie from expulsion of fctus to collection of tissues. T a b l e 7 . Effic.1 of tlcltrjl itr frccjzitrg of cc~rchrctlcorticrrl ri.s.slcc~.sotr pl~o.s~)ltorlic~.srcrtrsr rrcri~Yry Specific activity, nmol/rl~gprotein/ Inin' Interval bct\veen clcath' and freezing of tissues, hr (11 = I)

50 p h l cyclic AhlP

1 rnhl cyclic AhlP

1 4 6 2 0 WK

MATURE

FETAL

CORTEX

CORTEX

I Sprague-Ila\vlcy rats of approximately same weight kille~lby cthcr ;~ncsthcsiabefore removal of brain and freezillg of tissue. Carc;~sscskept intact at 4" for the indicated duration. ' Averages of duplicate determin;~tioris.

T h u s , t h c D N A content of t h e h u m a n cerebral cortex is esscntially constant a f t e r approximately t h e first year of life. Activity per ~ l n i tD N A may. therefore, be considered most rcprescntativc of changes in cellular activity in t h e h u m a n cortex from suhjccts of widely different ages, rcflccting largely ncuroblastic activity in the fetus a n d conibined neuroblastic a n d glial activity after t h e first year of life. COMPARISON Of: TOTAL ACTIVITY AND DISTRII3UTION O F I'IIOSI'IIODIES'fEKASE ACTIVITIES CYC1.IC AhlP PDE ACTIVITY

Differences in distribution a n d total activity a r c found between t h e fetal a n d m a t u r e cortcx. Maturation is accompanied by a 10-fold increase in activity for both t h e high a n d low K,,, efizyrncs. Most of the high K,,, activity is in t h e supernatant fraction in both fct;il 21nd m a t u r e brains. klowcver, m o r c low K,,, activity is prcscnt in t h e pellet in t h e mature cortex, in contrast t o t h e fetal brain, where m o r e is loc:~tedin the supernatant fractitin (see Fig. 1 ) . Activities based on brain weight a r e tabulated in Tables 3 and 4. CYCLIC GhlP PDE ACTIVITY

Differences in total activity a n d distribution a r c found again. Maturation results in a 15-20-fold increase in activity for b o t h high a n d low K,,, cnzyrncs. In the niature brain, most of the high a n d low K,,, activity is located in the pellet fraction, whereas these activities a r c locatcd predornin~lntlyin the supernatant in t h e fetal brain (scc Fig. 2 ) . hllClIAELIS CONSTANTS Nonlinear figures were obtained when t h e d o u b l e reciprocals

of substrate concentration a n d activity o r velocity were plotted

14620 WK FETAL

MATURE CORTEX

CORTEX

Fig. 1 . Ilistribution of cyclic AhlI' ( c / t ~ \ l P )pliosphotliestcr:~scactivity in the fetal and mature human cerebral cortex. Frontal cortex containing both nhitc and gray matter was homogcni~cd in 3 volurncs of gl;~ssilistilled w;iter, freeze-tha\vccl three times, spun at 30.000 x g (0-4") for 30 min, ;lnil di;~lyze~l overnight in 20 rnhl Tris buffer. ph 7.5. at 4". Fifty to 100 p g protein were usccl; total volunie I00 pl; hln++0.1 rnhl in 40 mhl Tris buffcr, p t l 7.4, incuh;~tcd33-36' for 10 min. Reaction tcrminatctl by boiling for 3 min before proiluct. 5'-AhlP, \\'as hy~lrolyzcdwith

Cyclic AhlP p l i o s p h o s p l ~ o t l i ~ s t ~ r ~ i s e lligh K,,, activity (2 rnhl)' Fraction I ~ / from srthj(>c/ pr110llg tis110 . sue/niin DNA/min

I>ow K,,, activity (2 fihl)' rI ~ i t i lsuclniin

I~IIIO~/III~ DNA/rnin

Supernatant I 2

3 4 5 6

7 8 Particulate 3 5 6 7 8 I

Substr;~tcconcentration.

for determin:~tionof t h e Michaclis constants. T w o kinetic forms were a s s u m e d , o n e exhibiting a low K,, or higher affinity a n d the o t h e r a high K,, o r lou.er affinity for each s u b s t r ; ~ t e .(Sce Figure 3 for representative Lineweaver-Burk plot.) Values obtained f o r half-maximum saturation ancl n ~ a x i n ~ velocitics ;~l a r c presented

Table 4 . DisrriOtrriort of c j c l i c GAIP ricri~itiesiit sirpcrrtcitorrt art(/ ccrcbrcil corrcx porticctlltc fractiorts of h~trr~ctr~ Cyclic GhlP phosphodicstcrase -

Iiigh K,,,activity (200 pM)' Fraction from

p~nol/g tissuc/min

strbjecr no.

Supernatant 3

-

-

Low K,,,activity (2 PM)'

prnol/mg DNAIniin

nrnol/g tissuclrnin

nrnol/mg DNA/min

0.074 0.083 0.054 0.1 13 0.150

5 6 7 8

Particulate 3 5

0.020 0.042 0.245 0 . 1 18 0.122

6

7 8

Fig. 3 . Lincwcaver-Burk plot of cyclic AhlP (cAl\lP) hyilrolysis by thc 30,000 x g supernatant fraction of a hornogcnizc~l and frcczcthawed specimen of human cerebral cortex.

' Suhtratc concentration. HIGH KM ACTIVITY

LOW KM ACTIVITY

30.000 a q SUPERNATANT 30,000 a P E L L E T Q

N.3

(3), the cffect of o n e nuclcotidc on the hydrolysis of the other \\*asexamined. In the mature cortex, the hydrolysis of l W 7 M cyclic A M I ' w a s stimulated by the addition of cyclic G M P ovcr the concentration range of 10-7-10-5 M as depicted in Figure 3 . Increasing the concentration of the added nuclcotidc resulted in a progrcsxivc reduction nf stimulation. Whcn cyclic G M P conccntr:~tion.;wcrc increased to LO-J M , hydrolysis of cyclic A M P was inhibited X590%. T h e hydrolysis of higher conccntrations of cyclic A M P (IOV5lo-' M) was progressively inhibited compctitivcly with incrcasing amounts of cyclic G M P . EFFECT OF CYCLIC AMP ON CYCLIC GMI' I'IIOSI'IIODIESTERASE ACTIVITY

14820 W K

MATURE

14620 WK

MATURE

FETAL CORTEX

CORTEX

FETAL CORTEX

CORTEX

Fig. 2. Distribution o f cyclic GhlP ( c C l l l P ) phosphodiestcrasc activity in the fctal and mature human cerchral cortex. Incubation and assay conditions as for cyclic AhlI' phosphodiesterasc using 25-50 pg protein. Suhstratc concentrations, 200 phl and 2 pM. in Table 5 . Lo\ver K,,, v;~lues\\..ere obtained for the high affinity cyclic G h f P enzyrne, a pattern sccn in several tissues and species as reported by others (3). For the low affinity enzymes, the halfsaturation values were higher for cyclic G M P than for cyclic A M P by a factor of 2-3. In particulate fractions at higher substr;~tcconcentrations, these enzymes exhibit slightly greater affinity for cyclic A M P but with maximum hydrolysis of cyclic A M P at a rate substantially lower than for cyclic G M P . No significant differences wcre noted between fetal and mature cortices. EFFECT O F CYCLIC GMP ON CYCLIC AMP I'IIOSPIiODIESTERASE ACTIVITY Since phosphodiestcrnse activity is reported t o be variably affected by the additional presence of the othcr cyclic nucleotide

T h e addition of 10-"10-7 M cyclic A M P inhibited the hydrolM cyclic G M P in the fetus a n d adult. T h e fetus ysis of 2 x exhibits greater sensitivity to inhibition. When the added nucletb M , relief of inhibition was tide is increased from IOFfi t o seen in the adult cortex (Fig. 5 ) . Even at lower substrate concentrations, the addition of lO-!'-IO-' M cyclic A M P also inhibited hydrolysis of cyclic G M P in the fetus. T h e mature brain, in contrast, was slightly s t i n ~ u l a t e dunder the same conditions. When the inhibitor concentrations wcre raised to 10-" t o 10Vi M, the brain of the fetus exhibited progressive rclcasc from inhibition (Fig. 6 ) . Since Ne,2'-0-dibutyryl (db)-cyclic A M P has been observed to mimic some of the effects of cyclic A M P and is believed not t o be hydrolyzed by P D E ( 3 6 ) , the effect of varying conccntrations of this analog o n cyclic G M P hydrolysis \\,as examined in the mature cortcx. A stiniulutory cffect was observed o n cyclic GMI' hydrolysis at high conccntrations by 1 0 - W dd-cyclic A M P which was sustained ovcr increasing conccntrations of the dibutyryl compound to lo-' M . At low conccntrations of cyclic G M P , 10-" M dbcyclic A M P inhibited the hydrolysis of the guanosine nuclcotidc; this inhibition was also sustained ovcr increasing :~rnountsof the added db-cyclic A M P (Fig. 7). These findings are suggestive that db-cyclic A M P may be a n allostcric cffector of the cyclic G M P phospl~odiesteraseswhich is pcrmissivc and stirnulatory for the low affinity enzyme and inhibitory for the high affinity enzyme. These results indicate that the hydrolysis of each cyclic nuclcotide is variously affected by the presence of different concentrations of the other nuclcotidc and significant diffcrcnccs occur as a result of maturation. These findings niay represent differences in the apoprotcins, o r they may represent the sum cffect of a

659

P1lOSPIIODIESTERASE IN CEREBRAL COIIT'EX

Cyclic Gh1P PDE

Cyclic AhlP PDE Low K,,, cnzynic

High K,, enzyme Fraction from sl~bjcctno.

,

(

0

)

v ,,,;,,

K,,,' ( X 1 0 - 9

Low K, enzyme

High K,,, enzyme

v ,,,:,

,,;,,2

,

(X I

)

v

KIII1( X lo-")

VIIInXZ

Supernatant I 5 6

7 8

Particulate I 5 6 7 8

Molar conccntration. N;~~iomolcs per mg pcr min.

7

6

5

4

-LOG MOLAR CONCENTRATION OF cGMP

Fig. 4. Effect of cyclic GhlP ( c C ~ t l P o) n cyclic AMP ( c A I ~ J Pplios) phodicstcrase. Plottcd ;IS percentage of control or baseline hydrolysis. @-@: cyclic AMP conccntration 0.5 ph.1. 0-0: cyclic AhIP conccntration 500 phl. Supernatant fractions used. (S~rbjc,ct8: initial activity with 500 p h l cyclic AhlP was 38.9 nniollnig proteinlmin and with 0.5 p h l cyclic AMP activity was 181 pmollrng proteinlrnin). variety o f factors a s , f o r instance, in the binding protcins and protein kinases which a r e also undergoing developmental change. In the absence of differences of the Michaelis constants o r further purification of the enzymes, the basis of the observed findings is not a p p a r c n t . H o w e v e r , variability of the effect of o n e nucleotidc o n t h e hydrolysis of the o t h e r d o e s not reflect concordance, suggesting that simple competitive inhibition may be but o n e of several mechanisms by which o n e cyclic nucleotide affects the hydrolysis of the o t h e r . EFFECTS O F A NONIONIC DETERGENT, TRITON X-100 In the rat, homogenates of brain reportedly exhibit a latent phosphodiesterase activity which could b e unmasked by the use

- LOG MOLAR

CONCENTRATION OF

CAMP

Fig. 5. Effect of cyclic AhlP (cAAlP) o n cyclic Ghll' (rCi\lP) phosphodicstcr;lse. Values wcrc plotted as percentage of control or basclinc hydrolysis which were as follo\vs: sul>ject2, 37.03 nmollrng protcinllnin and strbjc,ct 8, 34.53 nrnol/nig protcinlmin. @-a: supcrnatant fractions of the fetus (sltbjcct 2 ) when cyclic GMP conccntration was 200 supcrnatant fractions of thc mature cortcx (slrbjcct 8) phl; 0-0: with 100 ph.1cyclic GhlP. o f nonionic detergents ( 1 0 ) . Tlic effect of 0 . 2 % Triton X-100 (v/ v) o n the t w o kinetic forms of the cyclic A M P a n d cyclic G M P P D E of the human cerebral cortex was, tlicrefore, examined. In the m a t u r e cortex, o v e r a range of 5 X 10-H-10-4 M cyclic A M P concentration, Triton X-100 had a slightly inhibitory effect o n cyclic A M P hydrolysis, which dccrcased the V,,, by 55% a t high substrate concentrations a n d the affinity (incrcascd in K, by 2fold) over low substrate concentrations. O v e r comparable conccntration ranges of cyclic G M P , inhibition d u e t o Triton X-100 was again seen and wa: d u e t o reduction of the maximal velocities in both the mature a n d immature brains and in the several fractions examined.

KANG

Irnid:~/olc Iii~shccn demonstrated to s t i ~ n ~ ~ lthe ; ~ t chigh K,,, 1'I)E o f tlic hrain prcpar;~tionsof a nurnl~crof s l ~ c c i e s( 1 2 ) . although 110 cffcct is o l ~ s c r v c do n the low K,,, 1'1111 for a ~ l u n ~ h c r of them ( I ) . In both fctal ;ind rn;~turcI ~ r a i n sconccntr;ltior~s , of i~niil;~zole of 2 0 and 4 0 mhl had ;I stirnulatory cffcct of 25-5076 o n cyclic Ahll' and cyclic Ghll' liydrolysis :kt I~ighs u l ~ s t r ; ~ tconcentrac tions, \vhercas a n inhititory cffcct of 2 5 % was seen at micromo1;1r conccntrations of cyclic Ghll'. A l t h o ~ ~ g ;Il l nlilil inl~il>itory effect was observed at micromol;~rconcentrations of cyclic Ahll' in mature br:~ins,a 2 0 % stimulation of activity was ol>scrvcd ill fctal brains in the presence of 4 0 ~ n h imid;~zole. l

I 9

8

7

6

5

4

-LOG MOLAR CONCENTRATION OF cAMP

I'ig. 0. Lllcct ol cyclic Ahll' ((,,14\11') o n cyclic Cihll' ( c . ( i d l I J ) pliosphoilicslcrasc. Valucs \\ere plotted as pcrcelit;lge of control or hascline protcinl~iii~ifor tllc rn;iture cortcx 1i)~lrolysisant1 \rere 495 p~iiol/~iig for tlie fctal cortcx (.sltt,jrc.l 2 ) . (slt11icc.r6 ) and 336 pmol/liig protciri/nli~~ Supcr~ii~la~lt f r i ~ c t i o ~of~ s1111. Ictus (.----a) ;11111 of tlic ~ii;lt~lrc cortcx (0-0). Cyclic GXII', 0.5 ~ h l .

Activ:~tiorlof hrain I'DE activity by ;I lic;~t-st:~l~lc, nondi;~lyi.:~bIc factor prcscnt in [>rainextracts has hcen ascrihcil to a c;~lciunl binding protcin 1 ~ ya nurn1~c.rof tvorkcrs (14. 2 1 , 22. 42). T'hc possihle regulatory role played hy thi5 f:lctor is not cle;lr since i t is ubiquitous (37), is usually prcscnt in csccss even in tissues where P I I E activity is Ilarely detcctal,lc (37), appears t o activ;~tc only o n e of the scvcr;~l forms of I'D1 o l ~ t a i n e dhy physical sep;~r;~tionmethods (48). ;111d ; I I T ~ C ; L ~tSo s t i ~ i l ~ ~ l;;~~dt e ~ ~ y I ; ~ t e cyclasc activity as \\ell ( 6 ) . Tlic addition of 10 p g I ~ o v i n cactivator protcin, whicll h ; ~ s been shown to stimulate the I'l1l:'s of other species, had n o significant cffcct o n the tis\ucs cs;~rnincd.This could inrlic:~tc tliat saturating amourits of tlic endogenous activator wcrc prcscnt in the samples o r th;~t the preparation of IJovinc ; ~ c t i v ; ~ t o r ~ ~ s c[lid i l not activate [lie l ~ u r n ; ~cnLyrne ri (scc 'l'ablc 0).

These results suggest that Ii~tencyof I'11E activity is not encountered in the prepar;~tionsused in our study. Differences I>ct\vccn o u r results and tlic animal study may represent specics differcnccs. ;~ltIioughI l:~rdman (1 6) observcd :in inhibitory cffcct of 0 . 0 1 % Triton 5 - 1 0 0 o n the activity of the cyclic A h l P I'DE in tllc rat liver \vIiich \v;ls as much as 5 0 % inhibited at high substrate conccntr;~tions.

V:~ri;~bilityof rlrug effects has hccn rlcrnorlstrated for the I'DE's of the taw cyclic nuclcotidcs and is cseniplified by tllc grcilter sclcctivity of thcopliylline for cyclic AhlI' ratllcr t h a ~ i cyclic G h l P I'IIE in the lung of tlle guinea pig (2). In the Ilunlan ccrcbral cortcx, a similar selectivity for the cyclic Ahll' I'D1 is sccn. At 1 m M substrate concentration, cyclic AhlI' hyrlrolysis is markedly i~lliihitcd.\vhcrcas cyclic G M P hydrolysis is relatively un;~ffcctcdby millinlolar ; ~ n l o u n t sof tl~copllylline(see Fig. 8 ) in citllcr the ni:~turco r immature cortes. At micromolar conccntrations of substrate, the hydrolysis of botll nuclcotidcs was in~t for cyclic A M P Ilibited t o a degree comparable to t l i ~ observed at 1 m h l . At all substrate concentr;~tionsstudied, the fetal cortcx was slightly nlore scnsitivc t o the inhibitory cffccts of thcopliylL ~. line than the mature cortex. 9 8 7 6 5 4 cffccts o of thcophyllinc are not limited t o Although the it1 v i ~ ~ -LOG MOLAR CONCENTRATION OF db cAMP I'I1IZ inhibition alone and theophyllinc conccntrations of 1 t o 8 Fig. 7. Effect of rlibutyryl-cyclic AhlP (tlh c.A,llI') 011 cyclic GhlI' nlhl arc rarely achieved clinically, the difference csllibited by the liy~lrolysis;it low affinity enzymes indicate tliat tissue concentrations of the hyilrolysis. Plotted as percentage of coritrol or b;~scli~ic ttvo cyclic nuclcotidcs tvoulil vary considerably in tllc presence of varying suhstratc concentratio~is of cyclic GhlP. 0.1 to 1,000 p % l . Supcrnat;~ritof mature cortcx used. thcopliyllinc.

662

KANG

IOV6 hl conccntrations of the other nuclcotidc. A t low conccntrations of cyclic G M P (IOV7 M), the hydrolysis of cyclic A h l P was stitiiulatcd 2-fold in rat livcr but not iri bovine heart (4). In :I follow-up study o n otlicr tissucs of the rat, a stiriiulatory effcct of 10VH hl conccntration of cyclic G h l P was seen o n particillate p r c p a r ~ ~ t i o nofs the livcr, I)r;~in,kidney, heart, and thymus and o n soluhlc preparations of the livcr and thymus. Increasing the concentration of cyclic GhqP t o 10-"1, o n the otlier hand, was inhibitory (5). In ;I rat hrain nrcnar;~tion.O'Dca c! (11. (251 . obscrvcd tliat although 1 W 7 to iO-iiM cyclib GMI1 was competitively inhtbtted by I()-" t o IOV5 hl cyclic A M P , the hydrolyses of cyclic A h l P was unaffected by comparable amounts of cyclic G h l P . Similar findings tverc reported by Rosen (31) for the frog erythrocyte :it rnillirnolar concentrations of substratc and inhi1)itor. A t more physiologic conccntrations of substr:rtc ant1 inhibitor, Sak;~ic~tc~l. (33) obscrvcd in tlic fat cclls of the Wistar rat that the liyrlrolysis of low concentrations of either cyclic nucleotidc \vus stimulated by the presence of 10-X-10-7 M concentrations of the othcr. llighcr conccntrations of tlic added nucleotidc, l O - V l , \\#ereinhibitory by a noncompetitive rneclianism for cyclic A h I P hydrolysis and coriipctitively for cyclic G M P hydrolysis (33). Tlic physiologic significance of our findings is not readily apparent, p;~rticuI:rrlywitli respect t o tlie high conccntrations of substrate and "inhihitor" interrelationships. If a simplistic view is taken that ICVCIS for each cyclic nuclcotidc may be critical in tissucs, the following interpretation may be made. At the conccntr;~tioriof c;~clinuclcotidc in tissucs, a stirnulatory influence is exerted on each I'DE to cnsurc the maintenance of low, basal conccntrations of cach nucleotidc. In the mature cortcx, the stirnulatory effcct of cyclic G h l P o n cyclic A M P hydrolysis is great" tllali t l ~ ci l ~ l l u e l ~ cofe cyclic Ahll' or1 cyclic Cih.11' clcgradntion. Sincc cortic;~lcells tend to degrade morc cyclic G M P tIi;111A M P at low substratc conccntrations, this is suggestive that the net cffcct of rntlicr comp:~r:tblc tissue lcvcls may result for both nuclcotidcs, assuming that rates of synthesis of each arc similar. I Io\vevcr, tlic apparent greater affinity of these enzymes for cyclic Ghll'at low concentrations (see Table 5) indicates tliat tissuc cyclic G h l P levels would be lower than cyclic A M P if rates of synthesis of the two cyclic nuclcotidcs arc indeed similar. Wllen adcnyl;~tcor g u a n y l ~ ~ cyclasc tc is maximally stimulated, tlic presence of basal amounts of the othcr nucleotidc serves t o raise the tissue concentration of the nuclcotide being synthesized even further, allo\ving a critical level t o be rcnchcd morc quickly for the activation of spccific kinz~ses.Under such maximal stimulation, tlic presericc of tlic otlier nuclcotidc in high conccntrations results in a difference of effect on the specific P D E rcsponsihlc for the hydrolysis of the newly synthesized nuclcotide. When adcnylatc cyclase is stimulatcd, higli lcvcls of cyclic A M P are reached in the prcscncc of higli levels of cyclic G h l P . If gunnyl;~tccyclasc is stimulated, cyclic G M P lcvcls reached are coml)ar;~blet o that observed in the abscncc of added cyclic A M P dcspite tllc prcscncc of 10-Yrnd 10-"4 cyclic A h l P . I f the two cycl~rscscan be cotnparably stimulated in the adult cerebral cortcx, these findings indicatc that higher lcvcls of cyclic G h l P may not be favored by at least one of the factors regulating the conccntr:ttions of the cyclic nuclcotides in the brain. In the fetus, (311 the othcr hand, tlicsc intcrrclationships appear t o favor the sparing of cyclic G M P from dcgrndation. Sincc db-cyclic A M P , which is probably not actively mctabolizcd by the phospl~odicstcrascs,inhibits the hydrolysis of cyclic GhdP catalyzed by cnzymc with low K, and stimulates tlic high K,,, cnzymc, it is possible that one nucleotidc exerts a n allosteric cffcct on the hydrolysis of the othcr at low and high substratc conccntrations. Evidcricc of the separateness of the several forms of P D E has been reported by Russell e! al. (33), Knkiuchi c! 01. (22). and others o n the 11;tsis of kinetic ;~nalysisof physically separated proteins; the fact that thcsc enzymes are under separate control was reported I)y Pastan's group (32). The effects of tlicophylline on cyclic nucleotidc hydrolysis are attributed t o inhibition of

P D E (40), and corrcl:rtiotis bctwccn tlieopliyllinc concentration :itid physiologic resporisivuncss 11;1vcbeen s l l o ~ n(8). O u r o l ~ s c r vation of diffcrenccs in sensitivity of tlie cyclic GhlI' I'DE's t o thcophyllinc suggests that the active sites for c ~ ~ cof l i the G h l P enzymes may he separate. I:urtlicr, the vari;~l,ility of tlic effect of tl~cophylline oli the gu;~nosinc and ;~dcnosinc I'DE's ;rt high substrate conccntrations ;11so tetitls t o support the concept that these sitcs may bc tliffcrcnt.

z

Sincc cyclic Ahll' has been sho\vn t o play a significant role in the diffcrenti;~tionof developing tissucs and hormone production, and rcsponsivencss may he present in n number of tissucs from early stages in man (27), characterization of tlie cnzynics \vhich regulate the lcvcls of the cyclic tiuclcotides in the cercl)ral cortcs of man at various ; ~ g c smay be of itnportance hccaitsc the put;~tive ncurotranstnitters utilize tlicsc nuclcotidcs t o effcct tlicir ncurocndocrinc functions. T h e d a t ; ~reported here, olthough limited to the imm;~turcand nl:~turccortcx, indicatc that total enzyme activity and distribution of I'DE in the ccrcbral cortcs of m a n arc age dependent. In addition, diffcrcnccs in response to stimulators and inl~ibitorscan be found a t both extremes of maturation. This w;~smost notable with respect t o the influence of one nuclcotidc o n the hydrolysis of the other. In the abscncc of differences of the kinetic constants, thcsc finditigs may he tlic result of otlier factors \vliicli arc undergoing changes simultaneously but which arc not directly part of the npoprotcin of the PDE's. 1:urthcr purific:~tionof tlic enzymes will he necessary to explore the possibility of isozytncs. Whether thcsc diffcrcncus arc due to maturation of ncurohlasts, glial prolifcr;~tion,o r to hot11 processes cannot hc dctcrmincd frorn our studics. I:inally, diffcrcnccs noted at low and high substratc conccntrations in tlic presence of tlic other nuclcotide a t hot11 extrcrncs o f maturity together witli diffcrenccs in rcsponsivcncss t o tlieophylline confirm the notion tliat the ;~ctivcsitcs for tlic hydrolysis of the guanosinc and atlcnositic nuclcotidcs may he separate. R E F E R E N C I S A N D NOTES I . Allen. D . 0 . . and CI:lrk. J . n.: Effect of various :~ntilipolyticcompounds on adenyl cycln\e and phosphodiestcra\e activity in i\olated fat cclls. Adv;ln. Enzyme Regul.. 9: W ((1971). 2 . Amer. M.S . , and Krcighbaum, W . E . : Cyclic nuclcoride p h o s p h o d ~ e \ t e r a s s : properties, activators, inhihitors: Structural-activily relatinnships and po\sihle role in drug development. J . Ph:~rm:~ceut.Sci.. 64: 1 ( 1 9 7 5 ) . 3 . Appleman. M . hl., Thompson. W. J . , and Ru\sell. T . R . : Cyclic nuclcotide phosphodiesterases. Advan. Cyclic Nucl. Rcs.. 3: 6 5 ( 1 0 7 3 ) . 4 , 13eavo. J . A , , 1lardm:ln. J. G . . and Sutherlnnd, E . W.: Ilydroly\is of cyclic guano\ine and adeno\ine 3',5'-monophosphatec by rat and bovine ti\\ues. J . I3iol. Chem.. 245: 5649 ( 1 9 7 0 ) . 5 . He:~vo,J . A , , lI:~rdman.J. G . . and Suthcrl;~nd.E . W.:Stirnul;~tionof adennsine 3'.5'-monopho\phare hydrolysis by gunnosine 3'.5'-monophorphatc. J . I3iol. Chem.. 426: 3841 ( 1 9 7 1 ) . 6 . I3rostrom, C. 0 . . llu:~ng, Y . C . , Brcckenridge, 13. M..and Wolff, D . J.: Identification of a c:~lcium-hindingprotein a\ a calcium-dependent regul:~tor of brain adcnylate cyclase. I'roc. Nat. Acad. Sci. U . S . A . , 72: 6 4 ( 1 9 7 5 ) . 7 . Ilurton. K.: Dctcrminalion of D N A concentration with diphenylamine. Mclhods E n ~ y m o l . 1. 2 8 : 163 ( 1 9 6 8 ) . 8 . 13utcher. R . W . . and Suthcrl;lnd, E . W.: Adcno\ine 3'5'-pl~o\phatein hiological m;~terials.I. Purification and properties of cyclic 3'3'-nucleotide phosphodiesterasc and u ~ eof thic cnlyme l o char:lctcri7e adeno\inc 3 ' 5 ' phosphate in human urine. J. Biol. Chem., 237: 1244 ( 1 0 6 2 ) . 9 . Chader. G . J.: llormonal effects o n the neural retina: Induction of glutamine \yntheln\e by cyclic 3',5'-AhlP. Hiochem. Uiophys. R e \ . Commun., 43: 1102 ( 1 9 7 1 ) . 10. Cheung. W . Y . . and Salganicoff. L . : Cyclic 3'.5'-nucleotide pho\phndie\terare: Localization and latent activity in rat hrain. Nature. 214: 9 0 ( 1 9 6 7 ) . 11. Dobbing, J . , and Sands. J.: Timing of ncurohla\t nlulliplic:~tionin developing human hrain. Nature, 226: 63")(1070). 1 2 . Goldbcrg, N . I>., Lusr, W . D . , O ' D e a . K. F., Wei. S . . and O'l'oolc, A . G . : A role of cyclic nuclcotides in hrain metabolism. Advan. D i o c h c n ~ .Psychoph3rmacol.. 3: 6 7 (1970) 1 3 . Goldhcrg, N . D . , O'Dea. R . I:., and Iladdux. M . K.: Cyclic G M P . Advan. Cyclic Nuc. R c s . . ~ : 155 ( 1 9 7 3 ) . 14. Goren, E . N . , and Ro\en. 0.hl.: The effcct of nuclcotides and a nondialyzable factor on the hydrolysis of cyclic A M P by a cyclic nl~cleoride phosphodiesterase from beef heart. Arch. Diochcm. I3iophys.. 42: 7 2 0 (1971). 1 5 . Grccngnrd, P., McAfee, D. A , , and Kebabian. J . W.: On tlle mechanism of

P11OSPIIODIESTERASE I N CEREBRAL CORTEX action of cyclic A M P and its role in synaptic transmission. Advan. Cyclic Nucl. Res., I: 337 (1972). 16. II;~rdman.J. G.: Stimulation of rat liver phocphodiesterase by cyclic GhlP: Alteration by Triton, C a t + and E G T A . Advan. Cyclic Nucl. Res.. I: 574 (1972). 17. Hardman, 1. G . , Beavo, J. A , . Gray, J. P.. Chrisman, T . D., Patterson, W. D . , and Sutherland, E. W.: T h e forrn;~tionand metabolism of cyclic G h l P . A n n . N . Y. Acad. Sci.. 185: 27 (1971). 18. IIerdman, J. G . , and Suthcrland, E . W.: Guanyl cyclase, an enzyme catalyzing the formation of guanosine 3',5'-monophosphate from guanosine triphosphate. J . Biul. Chem., 244: 6363 (1969). 19. Ilimwich, W.:In: W. Himwich: Biochemical Development of the Brain, Vol. 1 (Dekker. Inc., New York, 1973). 20. Hoffer, B. J., Siggins, G . R., Oliver, A . P.. and Bloom, F. E.: Cyclic A M P mediation of norepinephrine inhibition in rat cerebellar cortex: A unique class of synaptic responses. Ann. N. Y. Acad. Sci.. 185: 531 (1971). 21. Kahiuchi, S., and Ynmarahi, R.: Calcium dependent phocphodiestcrnw activity ; ~ n d it5 ;iclivnting f;~ctor (PAP) from brain: Studies on cyclic 3'.5'nucleotide pho\phodiesterase (Ill). Biochem. Biophys. Res. Commun.. 41: 1104 (1970). 22. Kakiuchi. S., Yamazaki, R . , Teshima, Y., Uenishi, K., and Miyamoto, E.: hlultiple cyclic nuclcotide phosphodicsterase activities from rat tissucs and occurrence of a calcium-plus-magnesium-ion-