PEPTI DES I N T HE B R AI N. T HE NE W E N D O C RI N O- L O G Y OF T HE NE UR O N Nobel Lect ure, 8 Dece mber, 1977 b y R O G E R G U I L L E M I N Laboratories for Neuroendocrinology T h e S al k I nstit ut e, S a n Di e g o, C alif or ni a, U. S. A.

P A R T I

A. The Existe nce of Brai n Peptides Co ntrolli ng Ade nohypophvsial F u nctio ns. Isolatio n a nd Characterizatio n of T heir Pri m ar y M olec ul ar Str uct ures I n t he early 1950s base d o n t he a nato mical observatio ns a n d p hysiological ex peri mentation fro m several gro u ps in the US A an d E uro pe, it beca me ab u n da ntl y clear t hat t he e n docri ne secretio ns of t he a nterior lobe of t he hy po p hysis- well k no w n by t he n to co ntrol all t he f u nctio ns of all t he target e n docri ne gla n ds, (t hyroi d, go na ds, a dre nal cortex) pl us t he overall so matic gro wth of the in divi d ual- were so meho w entirely reg ulate d by so me integra- tive mechanis m locate d in ne uronal ele ments of the ventral hy pothala m us (revie w Harris, 1955). Beca use of t he pec uliar a nato my of t he j u nctio nal regio n bet wee n ve ntral hy pot hala m us (floor of t he 3r d ve ntricle) a n d t he pare nc h y mal tiss ue of t he a nteri or l o be of t he pit uitar y ( Fi g. 1), t he mechanis ms involved in this hypothala mic control of adenohypophysial f unctions were best ex plaine d by pro posing the existence of so me secretory pro d uct(s) by so me ( u nc haracterize d) ne uro nal ele me nts of t he ve ntral h y p o t h a l a m u s , t h e p r o d u c t s o f w h i c h w o u l d s o m e h o w r e a c h t h e a de no hy po p hysis by t he pec uliar ca pillary vessels observe d as if to joi n t he floor of t he hy pot hala m us to t he pit uitary gla n d. T hat co nce pt was defi nitely ascertai ne d i n si m ple ex peri me nts usi ng co mbi ne d tiss ue c ult ures of frag me nts of t he pit uitary gla n d a n d of t he ve ntral hy po- thala m us ( G uille min an d Rosenberg, 1955). The search for characterizing the hypothetical hypothala mic hypophysiotropic factors starte d then. Si mple reasoning an d early che mical confir mation le d to the hy pothesis that these u nk no w n s ubsta nces wo ul d be s mall pe pti des. After several years of pilot st u dies i nvolvi ng bot h biology a n d relatively si m ple c he mistry i n several laboratories i n t he US A, E uro pe a n d Ja pa n, it beca me clear t hat c haracterizi ng these hypothala mic hypophysiotropic substances woul d be a challenge of (originally) uns us pecte d pro portions. Entirely novel bioassays wo ul d have to be de vise d for ro uti ne testi n g of a lar ge n u m ber of fractio ns ge nerate d b y t he c he mical p urificatio n sc he mes; m or e s o b eri n g still w as t h e r e ali z ati o n i n t h e early 1960s that enor mo us a mo unts of hy pothala mic frag ments (fro m sla ughter ho use a ni mals) wo ul d have to be obtai ne d to have available a s ufficie nt q ua ntity of starti ng material to atte m pt a mea ni ngf ul progra m of c he mical isolatio n. T he early pilot st u dies ha d i n dee d s ho w n t he hy pot hala mic s ub-

3 6 4 P e pti d es i n t h e Br ai n. T h e N e w E n d o cri n ol o g y of t h e N e ur o n

l b

Fi g. 1 : a) Dia gra m matic re prese ntatio n of t he pit uitar y gla n d a n d t he i n ner vatio n of t he neurohypophysis by nerve fibers fro m the n.paraventricularis ( P V N) an d supra-opticus (S O N). b) Localize d lesions in the hy pothala m us pro d uce changes in the pit uitary secretion of the vario us a denohy po physial hor mones (increase or decrease c) Diagra m matic re presentation of the hy pothala mo-hy po physial portal syste m. d) Photo microgra ph of the hy pothala mo-hy po physial portal syste m after injection with an o paq ue dye. e) Diagra m- matic re prese ntatio n of t he hy po p hysiotro pic area. f) C ha nges i n pit uitary secretio n of various a dpnohypophysial hor mones (increase , or d e cr e as e ).

sta nces to be extre mely pote nt a n d, o n t he basis of si m ple ass u m ptio ns, to be prese nt i n eac h hy pot hala mic frag me nt o nly i n a fe w na nogra m q ua ntities. Esse ntially o ne, t he n t wo gro u ps of i nvestigators a p proac he d t he proble m wit h e n o u g h c o nsta nc y a n d res ol uti o n t o sta y wit h it f or t he te n years t hat it t o o k t o pr o vi de t he first of its defi niti ve s ol uti o n, i.e. t he pri mar y str uct ure of o ne of t he hy pot hala mic hy po p hysiotro pic factors: My o w n gro u p, t he n at Baylor College of Me dici ne i n Ho usto n, Texas ( wit h a n e piso de at t he Collège de Fra nce i n Paris), or ga nize d t he collectio n o ver se veral years of more t ha n 5 millio n s hee p brai ns, ha n dli n g i n t h e laboratory more than 50 tons of hy pot hala mic frag me nts. Sc hally a n d his collaborators, no w i n t he T ula ne 3 6 6 Physiology or Medicine 1977

U ni versit y Sc hool of Me dici ne i n Ne w Orlea ns, after he ha d left m y laborator y at Baylor, collecte d also very large n u mbers of porci ne hy pot hala mic frag- me nts. Late i n 1968, fro m 300,000 s hee p hy pot hala mi, B urg us a n d I isolate d 1.0 mg of t he first of t hese hy pot hala mic hy po p hysiotro pic pe pti des, t he thyrotro pin releasing factor ( T RF), the molec ule by which. the hy pothala m us reg ulates t hro ug h t he pit uitary t he f u nctio ns of t he t hyroi d gla n d ( G uille mi n et al., 1 9 6 2). T he follo wi ng year, after more tec h nical diffic ulties were overco me, we establis he d t he pri mary str uct ure of ovi ne T RF by mass s pectro metry as t hat of the deceivingly si m ple tri pe pti de p Gl u- His- Pro- N H 2 . T he material of porci ne ori gi n was s ho w n b y Sc hall y a n d his collaborators to be i de ntical. T he s y nt h eti c r e pli c at e, r a pi dl y a v ail a bl e i n u nli mit e d q u a ntiti es, w as s h o w n t o b e hi g hl y pote nt i n all verte brate s pecies a n d partic ularl y i n ma n; it is no w wi d el y us e d t hr o u g h o ut t h e w orl d i n a hi g hl y s e nsiti v e t est of pit uit ar y f u n cti o n a n d a n earl y mea ns of detectio n of pit uitar y t u mors i n ma n. The isolation an d characterization of T RF was the res ult of an enor mo us effort. It was also t he t ur ni n g poi nt w hic h se parate d do ubt -- a n d ofte n co n- f usio n, fro m u nq uestio nable k no wle dge. It was of s uc h he uristic sig nifica nce, t hat I ca n say t hat ne uroe n docri nology beca me a n establis he d scie nce o n t hat e v e nt. T he c haracterizatio n of t he molec ular str uct ure of T RF was ac hieve d i n a n u nco nve ntio nal ma n ner, w hic h will be briefly reco u nte d here. I have wit h B urg us a n d Vale give n a n exte nsive tec h nical revie w of t he w hole series of e ve nts t hat le d to t he c haracterizatio n of o vi ne T R F ( G uille mi n et al., 1971); rea ders i ntereste d i n t he tec h nical as well as hist orical as pects of t hese d e v el o p m e nts will fi n d t h e m i n t h at r e vi e w.

P ublicatio n, Isolatio n a nd Characterizatio n of T R F I n J a n u ar y 1 9 6 9, wit h t h e l at est s u p pl y of hi g hl y p urifi e d o vi n e T R F a v ail a bl e, - 1.0 mg obtaine d fro m 300,000 shee p hy pothala m us frag ments, a mino aci d a n al y si s of 6 N H Cl hy drolyzates of t his pre paratio n reveale d o nly t he a mi no aci ds, Gl u, His, a n d Pro, i n e q ui molar ratios a n d acco u nti n g i n wei g ht for 81 % of t he pre paratio n (t heoretical po n deral co ntri b utio n of His, Pro, a n d Gl u for a tri pe pti de mo noacetate calc ulates to 86 %) ( B urg us & G uille mi n, 1970a). F urt her more t he ultraviolet ( U V), i nfrare d (I R), a n d n uclear mag netic resonance ( N M R) s pectra obtaine d with that pre paration of T RF were co nsiste nt wit h t hose of a poly pe pti de a n d u po n close exa mi natio n most of t he c haracteristics of t h ose s pectra c o ul d be acc o u nte d f or b y t he str uct ural feat ures of t he a mi no aci ds fo u n d i n t he hy drolyzates of T RF. Moreo ver, t he sol ubility pr o perties a n d t he lac k of v olatilit y o bser ve d i n earl y atte m pts t o o btai n mass s pectra or to perfor m gas c hro matogra p hy, as well as ot her a nalytical data, were c o nsiste nt wit h t h ose of a p ol y pe pti de; als o, t he lac k of effect of classical proteolytic e nzy mes, obser ve d earlier, co ul d be relate d to t he partic ular a mi no aci ds observe d. Wit h t he a nalyses of t he more hig hly p urifie d material u n- mista ka bl y s ho wi n g t he a mi no aci ds to acco u nt for t he total wei g ht of t he pre paratio n, a n earlier hy pot hesis t hat T RF co ul d be a hetero meric poly- pe pti de was t herefore aba n do ne d i n favor of t he possibility t hat it mig ht be a cyclic or a protecte d pe pti de, a vie w co m patible wit h fail ure to detect a n N-ter mi n us ( B ur g us et al. 1966b; B ur g us a n d G uille mi n, 1967, 1970a; Sc hall y et al., 1966c, 1968, 1969) or a C-ter mi n us (Sc hall y et al., 1969; B ur g us a n d G uill e mi n, 1 9 7 0 a) as w ell as t h e r esist a n c e of t h e bi ol o gi c al a cti vit y t o pr oteases. The kno wle dge that the a mino aci ds His, Pro, an d Gl u not only occ urre d i n eq ui molar ratio i n porci ne a n d ovi ne T RF b ut i n dee d acco u nte d for al most t he t heoretical total wei g ht of t he molec ule i n t he case of o vi ne T R F, along with the previo us kno wle dge of a lack of an N-ter minal a mino aci d, le d us to re-exa mi ne (t his ha d bee n do ne by Sc hally i n 1968) deri vati ves of sy nt hetic poly pe pti des co ntai ni ng eq ui molar ratios of t hese a mi no aci ds to ser ve at least as possi ble mo dels for t he met ho dolo g y to be use d i n t he c har- acterizatio n of o vi ne T RF. We teste d for T RF acti vity 6 tri pe pti de iso mers co ntai ni ng L- His, L- Pro, a n d L- Gl u sy nt hesize d u po n o ur req uest by Gillesse n et al. (1970) (co ntai ni ng o nly t he pe pti des i n vol vi ng t he a-carboxyl gro u p of gl uta mic aci d). T he tri pe pti des prove d to be devoi d of T RF activity, co n- fir mi n g t h e e arli er r es ults of S c h all y et al. ( 1 9 6 8 , 1 9 6 9). O ur r es p o ns e t o t h es e negative res ults was, ho wever, differe nt fro m w hat ha d bee n t hat of Sc hally et al (see Sc h all y et al., 1 9 6 9). I pro pose d treati ng eac h of t he six tri pe pti des by acetic a n hy dri de i n a n effort to protect t he N-ter mi n us as i n nat ural T R F. T he acet ylatio n mixt ure fro m one, an d only one of the pepti des, na mely H- Glu- His-Pro- O H, yiel de d biolo gical acti vit y q ualitati vel y i n disti n g uis hable fro m t hat of nat ural T R F. It w as a cti v e i n i n vi v o a n d i n vitr o assa ys s pecific f or T R F a n d its acti o n i n vi v o w as bl o c k e d b y pri or i nj e cti o n of t h e a ni m als wit h t h yr o xi n e ( B ur g us et al., 1969a). T he s pecific acti vit y of t he material o btai ne d was lo wer (ca. 1 X 10 - 3 ) t ha n t hat of p urifie d nat ural T RF. T he nat ure of se veral possible reactio n pro d ucts was consi dere d: mono- or diacetyl- derivatives, poly mers of Gl u- His- Pro, a n d cyclic pe pti de deri vati ves or deri vati ves co ntai ni ng pyrogl uta mic aci d ( p Gl u) as t he N-ter mi n us. S ubseq ue ntly we re porte d ( B urg us et al., 1969a, b) t hat t he major pro d uct by weig ht of t his proce d ure was i n dee d p Gl u- His- Pro- O H. T he material was isolate d fro m t he reactio n mixt ure a n d its str uct ure was co nfir me d by mass s pectro metry of t he met hyl ester a n d by its i de ntity to a ut he ntic p Gl u- His- Pro- O H ( Gillesse n et al., 1970) o n T L C, t he I R s p e ctr u m, as w ell as si mil arit y of i ntri nsi c bi ol o gi c al a cti vit y i n vi v o. T his re prese nte d t he first de mo nstratio n of a f ully c haracterize d sy nt hetic molec ule, base d on the kno wn co m position of nat ural T RF, to re pro d uce the biological activity of a hy pot hala mic releasi ng factor. Se veral ot her pro d ucts prese nt i n t he acetylatio n mixt ure, so me possibly having higher s pecific activity than p Gl u- His- Pro- O H, were not characterize d. It is of i nterest t hat acetyl- Gl u- His- Pro- O H obtai ne d by total sy nt hesis ( Gill ess e n et al., 1 9 7 0) w as d e v oi d of T R F a cti vit y i n t h e i n vi v o assa y at d oses u p t o 2 5 0 µ g ( B ur g us et al., 1 9 6 9 1 3). Beca use of t he differe nces bet wee n t he s pecific bi ol o gical acti vities of p Gl u- His- Pro- O H an d nat ural ovine T RF an d the different behavior of these 3 6 8 Physiology or Medicine 1977 t wo co m po un ds in vario us chro matogra phic syste ms, it was evi dent that T RF was not p Gl u- His- Pro- O H as s uc h. It was t he pro posal of B urg us base d o n k no wle dge of t he pri mary str uct ures of ot her biologically active poly pe pti des ( vaso pressi ns, ox ytoci n, gastri ns, etc.) t hat a li kel y ca n di date for t he str uct ure of t he nat ural material wo ul d be p Gl u- His- Pro- N H, a n d its sy nt hesis was a p proac he d t hro ug h t he si m ple proce d ure of met ha nolysis of t he met hyl ester, p Gl u- His- Pro- O Me ( B urg us et al., 1969b, 1970 b, c). T he ester, pre pare d b y treat ment of the pure synthetic p Glu- His-Pro- O H with methanolic H Cl, was p urifie d by partition chro matogra phy an d i dentifie d as p Gl u- His- Pro- O Me, o n t he basis of its be ha vior o n T L C, its I R s pectr u m, a n d by mass s pectro- m etr y ( B ur g us et al., 1 9 7 0 b, c). It h a d bi ol o gi c al a cti vit y i n vitr o a n d i n vi v o no w a p proac hi ng half of t he s pecific activity of isolate d ovi ne T RF. A m mo no- lysis of t he met hyl ester i n met ha nol pro d uce d a material w hic h u po n partitio n chro matography gave a s mall yiel d of a s ubstance pres u mably p Gl u- His- Pro- N H,, occ urri ng i n a Pa uly positi ve zo ne se parate d fro m t he starti ng material, w hic h ha d a s pecific acti vit y i n vi v o or i n vitr o st atisti c all y i d e nti c al t o t h at of ovi ne T RF. A mo ng t he derivatives teste d, t he pro perties of native ovi ne T RF most closel y matc he d t hat of t he a mi de, faili n g to se parate fro m t he s y nt hetic co m po u n d i n fo ur differe nt syste ms of T L C w he n r u n i n mixt ures. T he I R s pectra of se veral of t he more hig hly p urifie d pre paratio ns of t he a mi de,

including p Glu- His-Pro- N H 2 no w pre pare d by total sy nt hesis ( Gillesse n et al., 1970), were al most i de ntical to t hat of ovi ne T RF, s ho wi ng o nly mi nor differe nces i n t wo re gio ns of t he s pectra. T hese ne w obser vatio ns, to get her wit h t he de mo nstratio n t hat t he s pecific activity of t he p Gl u- His- Pro- N H, was n ot statisticall y differe nt fr o m t hat of nat ural o vi ne T R F, le d us t o rec o nsi der ( B ur g us et al., 1969c; 1970b) a n earlier h y pot hesis ( B ur g us et al., 1969b) t hat ovi ne T RF may have a seco n dary or tertiary a mi de o n t he C-ter mi nal proli ne, rat her t ha n corres po n d to t he pri mary a mi de of t he tri pe pti de p Gl u- His- Pr o. A vailability of large a mo u nts of t he sy nt hetic tri pe pti des ma de possible a series of ex peri me nts wit h Desi derio, of Hor ni ng’s gro u p at Baylor, to m o dif y t h e d esi g n of t h e dir e ct pr o b e of t h e t h e n a v ail a bl e l o w r es ol uti o n m ass s pectro meter a n d si m ulta neo usly to obtai n volatile derivatives of t he pe pti des t hat wo ul d gi ve clear mass s pectra o n o nly a fe w microgra ms of t he pe pti des. O nce t his was ac hieve d, evi de nce was obtai ne d ( B urg us et al., 1969c) base d o n lo w a n d hig h resol utio n mass s pectro metry, t hat t he native ovi ne T RF pre paratio n ori gi nall y o btai ne d i n late 1968 ha d bee n all alo n g esse ntiall y

ho mogeneous and had unquestionably the structure p Glu- His-Pro- N H e . Bot h sy nt hetic p Gl u- His- Pro- N H, ( B urg us et al., 1969c; 1970b, c) a n d t he highly p urifie d ovine T RF ( B urg us an d G uille min, 1970a) were intro d uce d by direct pr o be i nt o a l o w res ol uti o n mass s pectr o meter as t he met h yl or trifl u or o- acet yl ( T F A) deri vati ves ( Fi g. 2); all pre paratio ns ga ve volatile materials i n the te m perat ure range of 150-200° C 1 0 - 6 torr). Se veral mass s pectra t a k e n t hr o u g h o ut t h e r a n g e of t h e t h er m al gr a di e nt ( 7 i n t h e c as e of t h e is ol at e d ovine T RF) sho we d frag mentation patterns corres pon ding to a single co m- po ne nt. Alt ho ug h no ne of t he s pectra reveale d a molec ular io n, frag me nts 1 5. 8

7

3 4

m/ e

Lo wer a, b: Lo w resol utio n mass s pectra of met hylate d ovi ne T RF (a) a n d sy nt hetic

P C A- His-Pro- N H 2 ( b) . 3 7 0 Physiology or Medicine 1977 arisi ng fro m t he str uct ures p Gl u, met hyl- p Gl u, His, met hyl- His, Pro, Pro- N H,,

C O N H 2 , p Gl u- His, an d His- Pro- N H, were observe d. The lo w resol ution mass spectra of the correspon ding derivatives of synthetic p Gl u- His- Pro- N H, an d T RF were esse ntially i de ntical. Frag me nts arisi ng fro m u ns ubstit ute d p Gl u or His were o bser ve d i n t he s pectra of b ot h t y pes of deri vati ves. T he ele me ntal co m positio n of all t he fra g me nts, exce pt m/e 2 2 1, t h e i nt e n sit y of w hic h was too weak for it to be observe d o n t he p hoto plate use d, were co nfir me d by hig h resol utio n mass s pectrosco py of t he met hyl derivatives ( B ur g us et al., 1969c; 1970 b). Th us, the str uct ure of ovine T RF as isolate d fro m the hy pothala m us was establishe d as p Gl u- His- Pro- N H, (Fig. 3). H o wever, we di d poi nt o ut ( B urg us et al., 1969c) t hat t he p ossi bilit y was n ot excl u de d t hat, as o p p ose d t o t he isolate d material, the native molec ule of T RF may occ ur as Gln- His- Pro- N H, eit her free or co nj ugate d to a not her str uct ure s uc h as a protei n, w hic h wo ul d n ot be necessar y f or bi ol o gical acti vit y i n vi v o or i n vitr o. At t h e ti m e of t his lect ure we a n d ot hers are still looki ng for a hy pot hetical pro hor mo ne of T RF. T he str uct ure of porci ne T RF was s ho w n i n a series of re ports by Sc hally a n d his c oll a b or at ors t o b e c o m p ati bl e wit h, a n d fi n all y t o b e i d e nti c al wit h, that of p Gl u- His- Pro- N H,; mass s pectro metry was also the metho d of ulti mate pr o of use d b y Nair et al. (1970).

Fi g. 3: T he pri mar y str uct ure of T R F wit h i n dicatio n of t he fra g me ntatio n poi nts i n mass

s pectro metry. R 1 , R 2 , re prese nt t he met hyl derivative pre pare d for mass s pectro metry; i n t he

n a t i v e m o l e c u l e , R 1 = R 2 = H .

It is m ost i nt er esti n g t h at T R F fr o m t w o wi d el y diff er e nt s p e ci es of m a m m als s ho ul d have t he sa me str uct ure a n d a p pare ntly t he sa me s pecific (biological) activity i n si milar assays. It was ra pi dly s ho w n t hat T RF s ho ws no evi de nce of s pecies s pecificity for its biological actio ns, p Gl u- His- Pro- N H, bei ng rea dily a cti v e i n h u m a ns (s e e Fl eis c h er et al., 1 9 7 0; Fl ei s c h er a n d G uill e mi n, 1 9 7 6).

P urificatio n, Isolatio n a nd Characterizatio n of L R F I n t he earl y 1960s, se veral i n vesti gators re porte d ex peri me ntal res ults t hat were best ex plai ne d by pro posi ng t he existe nce i n cr u de aq ueo us extracts of P e pti d es i n t h e Br ai n. T h e N e w E n d o cri n ol o g y of t h e N e ur o n 3 7 1 hy pot hala mic tiss ues of s ubsta nces t hat s pecifically sti m ulate d t he secretio n of l uteinizing hor mone, an d that were probably poly pe pti des ( Mc Cann et al., 1960; Ca m pbell et al., 1961; Co urrier et al., 1961). T he acti ve s ubsta nce was na me d L H-releasi ng factor or L RF. Ra pi dly follo wi ng t hese early observatio ns, pre parati o ns of L R F, acti ve at 1 µ g per d ose i n a ni mal bi oassa ys, were o btai ne d by gel filtration an d ion-exchange chro matogra phy on carboxy methylcell ulose ( G ulle mi n et al., 1963) a n obser vatio n t hat was co nfir me d wit h si milar met ho ds b y se veral i n vesti gat ors (Sc hall y et al., 1968). I n s pite of t he va garies of t he vario us bioassay met ho ds available, several laboratories re porte d pre paratio ns of L RF of i ncrease d pote ncy. Se veral of t hese early p ublicatio ns le d, ho we ver, to contra dictory state ments regar ding p urification an d se paration of L H- releasi n g fact or ( L R F), f ro m a follicle-sti m ulati ng hor mo ne releasi ng factor ( Sc hall y et al., 1968; D h ari wal et al., 1967; G uille mi n 1963). T wo laboratories i n de pe n de ntly re porte d i n 1971 t he isolatio n of porci ne L R F (Sc hall y et al., 1971a) a n d o vi ne L R F ( A m oss et al., 1971), both gro ups co ncl u di ng t hat L RF fro m eit her s pecies was a no napeptide c o nt ai ni n g, o n t h e b asis of a ci d h y dr ol ysis, 1 His, 1 Ar g, 1 S er, 1 Gl u, 1 Pr o, 2 Gl y, 1 L e u, 1 T yr. Earlier res ults with the pyrroli donecarboxylyl pe pti dase pre pare d by Fello ws a n d M u d ge (1970) ha d le d us to co ncl u de ( A moss et al., 1970) t hat t he N- ter mi nal resi d ue of L RF was Gl u i n its cyclize d pyrogl uta mic ( p Gl u) for m, as i n t he case of hy pot hala mic T RF, ( p Gl u- His- Pro- N H,). T he total a mo u nt of t he hi g hl y p urifie d o vi ne L R F t hat we ha d isolate d fro m si de fractio ns of t he T RF progra m a n d t hat was a vailable for a mi no aci d seq ue nci ng was ca. 80 n mol (as meas ure d by q uantitative dansylation). It is to t he cre dit of Sc hall y’s gro u p to ha ve first reco g nize d a n d re porte d ( Mats uo et al., 1971a) t hat porci ne L RF co ntai ne d o ne resi d ue of try pto p ha n ( Tr p), i n a d diti o n t o t he ot her a mi n o aci ds earlier o bser ve d b y aci d h y dr ol ysis. O n t he basis of a series of ex peri me nts i ncl u di ng e nzy matic hy drolysis wit h chy motry psin an d ther molysin an d analysis of the partial seq uences of their deca pe pti de by E d man degra dation- dansylation an d selective tritiation of C-ter mi ni, Mats uo et al. (1 9 7 1 b ) p r o p ose d the seq uence p Gl u- His- Tr p-Ser- Tyr- Gly- Le u- Arg- Pro- Gly- N H, for porcine L RF as that best co m patible with t he partial se q ue nce data. T heir st u dies were carrie d o ut wit h c u. 200 n mol of pe pti de. T hey also state d t hat sy nt hesis of t hat partic ular se q ue nce ha d gi ve n a m at eri al wit h bi ol o gi c al a cti vit y. A f e w w e e ks l at er, w e r e p ort e d t h e s y nt h esis by soli d-phase metho ds of the decapepti de p Glu- His- Trp-Ser- Tyr- Gly- Leu- Arg- Pro- Gly- N H,; after isolation fro m the reaction mixt ure it ha d q uanti- t ati v el y t h e f ull bi ol o gi c al a cti vit y i n vi v o a n d i n vitr o of ovine L RF ( Monahan et al., 1 9 7 1). S hortl y t hereafter, we re porte d ( B ur g us et al., 197 1) t he a mi no-aci d se q ue nce of ovi ne L RF obtai ne d o n 40 n moles of pe pti de by a nalysis of hy drolysis pro d ucts after digestion with chy motry psin or pyrroli donecarboxylyl pe pti dase, using E d man- degra dation follo we d by deter mination of N- an d C-ter mini by a q ua ntitati ve [ 1 4 C]- da nsylatio n tec h niq ue. C o nfir mati o n of t he p ositi o ns of so me of the a mino-aci d resi dues obtaine d by co mbine d gas chro matographic- mass s pectro metric analysis of phenylthiohy dantoin ( P T H) derivatives (Fales 3 7 2 Physiology or Medicine 1977

et al., 1971; Hage n maier et al., 1970) res ulti ng fro m E d ma n degra datio ns was describe d; we also re porte d res ults obtai ne d by degra datio n of t he sy nt he- tic deca pe pti de, si nce t hey co nfir me d a n d clarifie d so me pec uliarities observe d u po n e nzy matic clea vage of t he nati ve pe pti de ( B urg us et al., 1972). T he a mi no aci d seq ue nce of ovi ne L RF was establis he d to be p Gl u- His- Tr p-Ser- Tyr- Gly- Le u- Arg- Pro- Gly- N H,. It is i de ntical to t hat of t he material of p or ci n e ori gi n. Of co nsi derable i nterest was t he obser vatio n t hat t he sy nt hetic re plicate of L RF, no w available in large q uantities, was sho wn to sti m ulate conco mitant secretion of the t wo gona dotro pins L H (l uteinizing hor mone) an d FS H (follicle sti m ulati n g hor mo ne) i n all assa y s yste ms i n vi v o a n d i n vitr o i n w hi c h it was teste d. T his co nfir me d t he earlier res ults obtai ne d wit h t he mi n ute q ua ntities of t he is olate d o vi ne or p orci ne L R F ( A m oss et al., 1971; Sc hall y et al., 1971a). I n ot her wor ds, t he sti m ulatio n of t he release of FS H a p peare d to be i n here nt to t he molec ule of L RF - t h us t hro wi ng co nsi derable do ubt o n e arli er r e p orts ( S c h all y et al., 1 9 6 6; S c h all y et al., 1 9 6 8; I g ar as hi et al., 1 9 6 4) clai mi ng to have obtai ne d pre paratio ns of L RF free of FS H-releasi ng activity. To t he da y of t his lect ure, no soli d e vi de nce has bee n pro d uce d w hic h co ul d be i nter prete d t o i n dicate t he existe nce of a n FS H-releasi n g fact or as a s pecific e ntity, discrete fro m t he deca pe pti de L RF. Re ports by Folkers et al. (1969) clai mi ng p urificatio n of a n FS H-releasi ng factor are diffic ult to a p preciate i n vie w of t he pa ucity of data offere d. Moreover, ot her evi de nce is agai nst t he existe nce of a n FS H-releasi ng factor, se parate fro m L RF: all sy nt hetic a nalogs of L RF ma de so far, wit h no exce ptio n, ca n be s ho w n to release L H a n d FS H wit h t he sa me rati o of s pecific acti vit y w he n relate d t o t he acti vit y of L R F i n t he partic ular assay i nvolve d. T h us, no ne of t hese a nalogs has s ho w n a ny e vi de nce of dissociate d acti vity for releasi ng FS H vs. L H. Also, t here is i n- creasing evi dence that the t wo gona dotro pins ( L H an d FS H) can be de m- onstrate d (i m m unocytoche mistry) mostly in the sa me pit uitary cell ( Moriarty, 1973). It is t h us u nli kel y t hat o ne c o ul d be release d wit h o ut t he ot her as t he y a p pear to be prese nt i n t he sa me secretor y gra n ules. Later o n, b ot h Sc hall y’s gr o u p (1971 b) a n d o ur gr o u p ( Li n g et al., 1973) confir me d the pri mary str uct ure of porcine an d ovine L RF, res pectively, usi n g lar ger q ua ntities of nati ve material.

P urificatio n, Isolatio n a nd Characterizatio n of So matostati n It has bee n ge nerall y acce pte d t hat t he c o ntr ol of t he pit uitar y secreti o n of gro wth hor mone woul d be exerte d by a hypothala mic hypophysiotropic releasi n g factor, as is no w pro ve n to be t he case for t he secretio n of t h yro- tro pin an d the gona dotro pins. The nat ure of the post ulate d hy pothala mic releasi ng factor for gro wt h hor mo ne, ho we ver, re mai ns el usi ve to t his day, m ostl y d ue t o t he diffic ulties a n d a m bi g uities of t he vari o us assa y s yste ms use d so far i n atte m pts at its c haracterizatio n. For i nsta nce t here is no w a gree me nt that the “gro wth hor mone releasing hor mone” ( G H- R H), isolated on the basis of a bioassay a n d c haracterize d by Sc hally et al. (1971c) as H- Val- His- Leu-Ser- Ala- Glu- Glu-Lys- Glu- Ala- O H, was actually a decapeptide frag ment P e pti d es i n t h e Br ai n. T h e N e w E n d o cri n ol o g y of t h e N e ur o n 3 7 3 of t h e N-t er mi n al of t h e of porci ne he moglobi n ( Veber et al., 1971). T he material has ne ver bee n s ho w n to be acti ve i n sti m ulati n g secretio n of i m munoreactive gro wth hor mone. Si milarl y, bi ol o gical acti vit y of a tetra- pe pti de rece ntly re porte d as a gro wt h hor mo ne releasi ng factor of porci ne origi n has not bee n co nfir me d by ot hers, i ncl u di ng o ur o w n laboratory (see G uille mi n, 1973). Searc hi ng to de mo nstrate t he prese nce of t his still hy pot hetical so mato- tro pi n releasi n g factor i n t he cr u de h y pot hala mic extracts use d i n t he isolatio n of T RF (thyrotro pin releasing factor) an d L RF (l uteinizing hor mone releasing factor), we reg ularly observe d t hat t heir a d ditio n i n mi n ute doses . 0 0 1 of a hy pot hala mic frag me nt eq uivale nt) to t he i nc ubatio n fl ui d of dis perse d rat pit uitar y cells i n mo nola yer c ult ures ( Vale et al., 1972a) si g nifica ntl y decrease d t he resti ng secretio n of i m m u noreactive gro wt h hor mo ne by t he pit uitary cells. T his i n hibitio n was relate d to t he dose of hy pot hala mic extract a d de d a n d a p peare d to be s pecific. It was not pro d uce d b y si milar extracts of cerebell u m, a n d t he cr u de hy pot hala mic extracts t hat i n hibit secretio n of gro wt h hor mo ne si m ultaneo usly sti m ulate d secretion of L H an d TS H. The inhibition of gro wth hor mo ne secretio n co ul d not be d u plicate d by a d ditio n to t he assay syste m of [ Arg 8 ]-vaso pressi n, oxytoci n, hista mi ne, vario us polya mi nes, seroto ni n, cate- c hola mi nes, L RF, or T RF. We deci de d to attrib ute t his i n hibitory effect o n t he secretio n of gro wt h hor mo ne to a “so matotro pin-release inhibiting factor” w hi c h w e l at er n a m e d so matostati n. Inhibition of secretion of gro wth hor mone by cr u de hy pothala mic pre para- tio ns ha d bee n re porte d by ot hers ( Kr ulic h a n d Mc Ca n n, 1969). T he active factor possi bl y i n vol ve d i n t hese earl y st u dies base d o n vario us t y pes of assa ys for gro wth hor mone activity ha d not been characterize d. The res ults on the i n hibitio n by t he hy pot hala mic extracts of t he secretio n of i m m u noreactive gro wth hor mone by the monolayer pit uitary c ult ures were so consistent an d easil y q ua ntitate d t hat we deci de d to atte m pt t he isolatio n a n d c haracteri- zatio n of t he hy pot hala mic factor i nvolve d. We realize d t he possible i nterest of s uc h a s ubsta nce i n i n hibiti ng ab nor mally elevate d secretio n of gro wt h hor mo ne i n j uve nile diabetes; also, we co nsi dere d t hat k no wle dge of t he pri mar y str uct ure of a nati ve i n hi bitor of t he secretio n of a pit uitar y hor mo ne co ul d be of si g nifica nce i n o ur efforts at desi g ni n g s y nt hetic i n hi bitors of t he gona dotro pin releasing factor L RF. T he starti ng material was t he c hlorofor m- met ha nol-glacial acetic aci d extract of abo ut 500,000 shee p hy pothala mic frag ments ( B urg us et al., 1971; B urg us et al., 1972) use d i n t he progra m of c haracterizatio n of t he releasi ng factors for t he go na dotro pi ns. T he extract (2 kg) ha d bee n partitio ne d i n t wo syste ms; the L RF concentrate was subjecte d to ion-exchange chro matography on carboxy methyl cell ulose. At that stage, a fraction with gro wth hor mone- release i n hibiti ng-activity was observe d well se parate d fro m t he L RF zo ne; it was f urt her p urifie d by gel filtratio n (Se p ha dex G-25) a n d liq ui d partitio n c hro matogra p hy ( n-b uta nol, acetic aci d, water, 4 : 1: 5). T hi n-layer c hro mato- gra p hy a n d electro p horesis of t he fi nal pro d uct s ho we d o nly traces of pe pti de i m p urities. T he yiel d was 8.5 mg of a pro d uct co ntai ni ng 75 perce nt of a mi no 3 7 4 Physiology or Medicine 1977

a ci ds b y w ei g ht; w e will r ef er t o t his m at eri al b y t h e n a m e so matostatin w hic h was act uall y gi ve n t o it o nl y after it ha d bee n f ull y c haracterize d. Analysis of a mino aci ds obtaine d fro m so matostatin after aci d hy drolysis i n 6 N H Cl- 0. 5 % t hi o gl y c olli c a ci d g a v e t h e m ol ar r ati os Al a ( 0. 9), Gl y ( 1. 1), C y S (0.2), C ys- S S- C ys (1.0), L ys (2.0), As p (1.0), P he (3.3), Tr p (0.5), T hr ( 2. 0), S er ( 0. 8), a n d N H, ( 1. 1). E n z y mi c h y dr ol ysis g a v e t h e r ati os Al a ( 0. 9), Gl y ( 0. 9), L ys ( 2. 0), P he ( 3. 4) a n d Tr p (0.9); As n, T hr, a n d Ser were not well resol ve d, gi vi ng a total of abo ut 3.6 mol / mol of pe pti de. E d ma n de- gra dation of the carboxy methylate d try psin digests of so matostatin an d mass s pectro metry le d fi nally to t he fi nal de mo nstratio n of t he follo wi ng pri mary structure for so matostatin: H- Ala- Gly- Cys-Lys- Asn-Phe-Phe-Trp-Lys-Thr- P he- T hr-Ser- Cys- O H, i n t he oxi dize d for m ( B urg us et al., 1973). T his pe pti de was re pro d uce d by total sy nt hesis usi ng Merrifiel d met ho d (s e e Ri vi er, 1 9 7 4). It h a d t h e f ull bi ol o gi c al a cti vit y of n ati v e s o m at ost ati n i n vi v o a n d i n vitr o ( Brazea u et al., 1973, Vale et al., 1972 b, Brazea u et al., 1974). Of i nterest was t he u nex pecte d observatio n t hat t he pe pti de has t he f ull bi ol o gi c al a cti vit y eit h er i n t h e o xi di z e d f or m ( n ati v e) or r e d u c e d f or m.

B. P urificatio n, Isolatio n a nd Characterizatio n of the E ndorphi ns, Opiate- Like Peptides of Br ai n or Pit uit ar y Ori gi n T he co nce pt a n d t he de mo nstratio n, so me years ago, of t he existe nce i n t he brain of ma m malians of (syna ptoso mal) o piate-rece ptors ( Pert an d Sny der, 1973) le d to t he searc h of w hat has bee n ter me d t he e n doge no us-liga n d(s) of t hese o piate rece ptors. T he ge neric na me e ndorphi ns (fro m endogenous and mor p hi ne) was pro pose d for t hese (t he n hy pot hetical s ubsta nces) by Eric Si mo n a n d will be use d here. So meti me i n t he s u m mer of 1975 I beca me i ntereste d i n t hese early observatio ns. Besi des t he c halle nge of c haracterizi ng a n e n doge no us s ubsta nce as t he liga n d of t he brai n o piate rece ptors, we co ul d not ig nore t hat, like mor p hi ne, t he (t he n hy pot hetical) e n dor p hi ns mig ht sti m ulate t he secretio n of gro wt h hor mo ne; t he nat ure of t he gro wt h hor mo ne releasi ng factor was a n d still re mai ns u nk no w n. I t h us deci de d to e ngage i n t he isolatio n a n d c haracterizatio n of t he e n doge no us liga n d(s) for t he o piate rece ptors. T he isolatio n of t hese e n doge no us liga n ds of t he o piate rece ptors t ur ne d o ut to be a relati vely si m ple proble m to w hic h a sol utio n was pro vi de d i n less t ha n a c o u ple of m o nt hs of eff ort. Dil ute acetic aci d- met ha nol extracts of w hole brai n (ox, pig, rat) were co nfir me d to co ntai n s ubsta nces pres u mably pe pti dic i n nat ure, wit h naloxo ne- reversible, mor phine-like activity in the bioassay using the myenteric- plex us l o n git u di n al m us cl e of t h e g ui n e a pi g il e u m. E vi d e n c e of s u c h bi ol o gi c al a cti vit y i n o ur laboratory was i n agree me nt wit h earlier res ults of H ug hes (1975), Tere ni us & Wa hlstro m (1975), Tesc he mac her et al. (1975), a n d Paster nak et al. (1975). Searc hi ng for a n e nric he d so urce of e n dor p hi ns i n available co n- ce ntrates fro m o ur earlier efforts to war ds t he isolatio n of C RF, T RF, L RF, so matostatin, I recog nize d t hat acetic aci d- met ha nol extracts of porci ne hypothala mus-neurohypophysis contained much greater concentrations of t he mor p hi ne-like activity t ha n extracts of w hole brai n. Fro m s uc h a n extract P e pti d e, i n t h e Br ai n. T h e N e w E n d o cri n ol o g y o f the Neuron 3 7 5 of approxi mately 250,000 frag ments of pig hypothala mus-neurohypophysis we isolate d se veral oli go pe pti des (e ndorphi ns) wit h o pi oi d a cti vit y ( G uill e mi n et al., 1 9 7 6 a; L a z ar us et al., 1 9 7 6; Li n g et al., 1976). T he isolatio n proce d ure involve d s uccessively gel filtration, ion exchange chro matogra phy, liq ui d partition chro matography and high pressure liquid chro matography ( Guille- mi n et al., 1 9 7 6 a; L a z ar us et al., 1 9 7 6; Li n g et al., 1 9 7 6). B y t h at ti m e, h a d a p peare d t he evi de nce for t he isolatio n a n d pri mary str uct ure of Met 5 -e n ke- p hali n a n d Le u 5 -e nke p hali n ( H ug hes, et al., 1975). H ug hes et al. (1975) ha d also ma de t he re mar ka ble o bser vatio n of t he i de ntit y of t he a mi no aci d seq ue nce of Met 5 -e nke p hali n wit h t hat of t he seq ue nce Tyr 6 1 - Met 6 5 of tr o pi n, a p ol y p e pti d e of ill d efi n e d bi ol o gi c al a cti vit y, is ol at e d a n d c h ar a ct eri z e d i n 1964 b y C. H. Li et al. (see Li & C h u n g, 1976). T he pri mar y str uct ure of was esta blis he d ( Li n g et al., 1976; G uille mi n et al., 1976a) b y mass s pectro metry an d classical E d man degra dation of the enzy matically cleaved peptide and is H-Tyr- Gly- Gly-Phe- Met-Thr-Ser- Glu-Lys-Ser- Gln- Thr- Pro- Le u- Val- Thr- O H (Fig. 4 a, b). T he pri mar y str uct ure of

Fi g. 4: Mass s pectra of a-e n d or p hi n after tr y psi n di gesti o n, acetic a n d d e u t e r i o a c e t i c anhy dri de acetylation an d per methylation. T he seq ue nces are: (a) H- Tyr- Gly- Gly- Phe- M e t - T h r - S e r - ; ( b ) : H - S e r - G l n - T h r - P r o - L e u - V a l - T h r - O H . was si milarly establishe d by mass s pectro metry an d by E d man degra dation: has t he sa me pri mary str uct ure as a-e n dor p hi n wit h o ne a d- ditio nal Le u as t he C O O H-ter mi nal resi d ue i n positio n 17. T h us, it was o bvio us t hat Met-e n ke p hali n is t he N-ter mi nal pe nta pe pti de of an d y-en dor phin, which have res pectively the sa me a mino aci d seq uence a s [61-76] a n d [61-77]. a fra g me nt of is olate d earlier o n t he basis of its c he mical c haracteristics ( Bra d b ur y et al., 1975; Li a n d C h u ng, 1976) was s ho w n also to ha ve o piate-like acti vity ( Bra d- b ur y et al., 1976; Lazar us et al., 1976; Cox et al. 1976) a n d has bee n na me d ( Li a n d C h u ng, 1976). Rece ntly we have isolate d, fro m t he sa me starting material of hypothala mus-neurohypophysis origin fro m which we ori gi n all y i s ol at e d a- an d y-en dorphin, t wo pepti des characterize d by a mino aci d co m positio n as a n d No effort was ma de to obtai n t he a mi no aci d seq ue nces of t hese t wo sa m ples. T he sy nt hetic re plicates of t hese t wo poly pe pti des have i de ntical chro matogra phic behavior in several syste ms as the native materials.

P A R T I I

Biological Activities of the Hypothala mic Peptides a nd Sy nthetic A nalogs. Experi me ntal a nd Cli nical St udies As soo n as t he y were o btai ne d i n lar ge q ua ntit y fro m total s y nt hesis, T R F a n d L RF were exte nsively st u die d for t heir biological activities, bot h i n t he laboratory a n d i n cli nical me dici ne. I n dee d, t he observatio n was ra pi dly ma de t hat T RF, a n d later L RF, bot h c haracterize d o nly fro m tiss ues of ovi ne a n d porci ne ori gi n, were biolo gicall y f ull y acti ve i n all s pecies of verte brates st u die d, i ncl u di ng ma n (Fig. 5), t he sa me was to a p ply for t he sy nt hetic re plicate of so matostati n, as c haracterize d fro m o vi ne brai ns ( Fi g. 6). For earl y cli nical st u dies wit h s y nt hetic T R F see Fleisc her et al., 1970 a n d more rece ntly Fleisc her a n d G uille mi n, 1 9 7 6; f or e arl y cli ni c al st u di es wit h s y nt hetic L R F see Ye n et al., 1973; Re bar et al., 1973. Sc hall y’s gr o u p als o p ublis he d exte nsi vely o n cli nical i n vestigatio ns wit h eit her p urifie d nati ve or sy nt hetic T RF a n d L RF (re vie w i n Sc hally et al., 1973). Bot h i n t he case of T RF a n d of L RF c he mists have pre pare d large n u mbers of s y nt hetic a nal o gs of t he pri mar y ( nati ve) str uct ure f or st u dies of c orrelati o n bet wee n molec ular str uct ure a n d biological activity. Also, biologists caref ully scree ne d t hese a nalogs, i n t he ho pe t hat so me of t he m wo ul d pro ve to be a nta go nists of t he nati ve (a go nist) releasi n g factor. T his was of partic ular i nterest i n t he case of t he go na dotro pi n releasi n g factor; a po werf ul a nta go nist of L R F wo ul d be of co nsi derable i nterest as a c he mical mea ns of co ntrolli ng or reg ulati ng fertility, t h us i ntro d uci ng a totally ne w ty pe of s ubsta nces for co ntrace ptio n. I n 1972, o ur laboratory re porte d t he first partial ago nist / a nta go nist a nalo gs of L R F, ( Vale et al., 1972c). T he y all ha d a deleti o n or a s u bstit uti o n of His 2 or Tr p 3 i n t he (ot her wise i de ntical) a mi no aci d seq ue nce of L RF. T hese were a ntago nists of lo w activity a n d of no possible practical val ue as cli nically sig nifica nt i n hibitors of L RF. T hey s ho we d, ho wever, t hat a nalogs as co m petitive a ntago nists of t he deca pe pti de L RF co ul d be pre pare d. P e pti d es i n t h e Br ai n. T h e N e w E n d o cri n ol o g y of t h e N e ur o n 3 7 7

Fi g. 5: Testi n g of t he a bilit y of t he a nteri or pit uitar y t o secrete G H, T S H, pr olacti n ( P R L), L H an d FS H in nor mal h u man s ubjects. Sti m ulation of the secretion of G H is achieve d by i. v. a d mi nistratio n of ar gi ni ne; sti m ulatio n of t he secretio n of TS H a n d P R L, L H a n d FS H is pro d uce d b y i. v. i njectio n of a sol utio n i n sali ne of s y nt hetic T R F (250 µ g) a n d s y nt hetic L R F ( 1 5 0 µ g ) - n o t e t h a t a r g i n i n e i n f u s i o n s t i m u l a t e s s e c r e t i o n o f G H a n d P R L . A l l pituitary hor mone plas ma concentrations measure d by ra dioi m munoassays (fro m Yen et al.).

To t his day, t he most pote nt a ntago nist-a nalogs of L RF still have t he early deleti o n or s u bstit uti o n of His 2 or Tr p 3 of t he a mi no aci d se q ue nce of L RF. Also, a nalogs of T RF a n d L RF wit h i ncrease d pote ncy (o ver t hat of t he native co m po un d) were ex pecte d, searche d for, an d obtaine d (see belo w). a. Biological Activity of Thyrotropi n Releasi ng Factor ( T R F) a nd L utei nizi ng Hor mo ne Releasi ng Factor ( L R F) T he re mar kable obser vatio n was ori gi nall y ma de b y Tas hjia n et al. (197 1) t hat T R F sti m ulates t he secretio n of prolacti n b y t he clo ne d li ne G H 3 o f pit uitary cells. T his was co nfir me d by ot hers a n d exte n de d to s ho w t he ob- ser vatio n to be vali d also wit h nor mal pit uitary tiss ues i n vitr o a n d i n vi v o, i ncl u di ng t he h u ma n pit uitary. T RF ca n t h us be co nsi dere d as i nvolve d i n t he co ntrol of t he secretio n of t h yrotro pi n a n d of prolacti n. Of t he ma n y a na- logs of T RF which have been synthesize d an d st u die d biologically, only one has a si g nifica ntl y i ncrease d s pecific acti vit y o ver t hat of t he nati ve c o m p o u n d. Descri be d b y o ur gr o u p a n d s y nt hesize d b y Ri vier (1971) a fe w years a g o, it is the analog [3 N- Methyl- His]- T RF. Its s pecific activity is a p proxi mately 3 7 8 Physiology or Medicine 1977

Fi g. 6: Effects of t he a d mi nistratio n of s y nt hetic so matostati n i n nor mal h u ma n s ubjects. T here is co m plete i n hibitio n of t he i ncrease i n G H secretio n nor mally pro d uce d by i nf usio n of argi ni ne or oral a d mi nistratio n of L- D O P A, w here so matostati n is a d mi nistere d prior to or conc urrently with the sti m ulating agent. Plas ma concentrations of pit uitary hor mones were measure d by ra dioi m munoassays (fro m Yen et al.).

10 ti mes t hat of t he nati ve molec ule, o n t he secretio n of TS H as well as of prolacti n. Of t he several h u n dre ds of T RF a nalogs sy nt hesize d, no ne has bee n fo u n d so far to be e ve n a partial a nta go nist. T he y are all a go nists wit h f ull i ntri nsic acti vit y b ut varia ble s pecific acti vit y; no tr ue a nta go nist of T R F has bee n re porte d. In contra distinction to the state ment above regar ding analogs of T RF, a ntago nist as well as extre mely pote nt ago nist a nalogs of L RF have bee n pre pare d by a n u mber of laboratories. There are no w available pre parations of a series of w hat we may acc urately call “s u per- L RFs”, a nalogs w hic h have as m uc h as 150 ti mes t he s pecific acti vit y of t he nati ve c o m p o u n d. I n fact, i n certai n assays s uc h as ov ulatio n, t hey may have 1,000 ti mes t he s pecific activity of t he native pe pti de. All t he ago nist-a nalogs or s u per- L RFs possess str uct ural variatio ns aro u n d t wo major mo dificatio ns of t he a mi no aci d se q ue nce of nati ve L R F: T he y all ha ve a mo dificatio n of t he C-ter mi nal gl y ci n e, as ori gi n all y r e p ort e d b y F uji n o et al. ( 1 9 7 4). T h e F uji n o m o difi c ati o n

1 0 co nsists of deletio n of Gl y - N H 2 an d re place ment by pri mary or secon dary 9 a mi de on the (no w C-ter minal) Pr o . I n a d diti o n t o t h e F uji n o m o difi c ati o n, t hey ha ve a n a d ditio nal mo dificatio n at t he Gly 6 p ositi o n b y s u bstit uti o n of o ne of several D-a mi no aci ds as origi nally discovere d i n o ur laboratories ( Mo na ha n et al., 1973). T h e most pote nt of t he L RF-a nalog ago nists pre pare d are [ D- Tr p 6 ]- L RF; des- Glyr 1 0 -[ D- Tr p 6 - Pr o 9 - N- Et]- L R F, [ D- L e u 6 , Pr o 9 - N- Et]- L R F . P e pti d es i n t h e Br ai n. T h e N e w E n d o cri n ol o g y of the Neuron 3 7 9

I n a n i n vitr o assay i n w hic h t he pe pti des sti m ulate release of L H a n d FS H by s urviving a denohy po physial cells in monolayer c ult ures, these analogs of L R F ha ve a s pecific acti vit y 50 t o 100 ti mes greater t ha n t hat of t he s y n- t hetic re plicate of nati ve L R F. T here is n o e vi de nce of diss ociati o n of t he s pecific acti vity for t he release of L H fro m t hat of FS H. All ago nist a nalogs release L H a n d FS H i n t he sa me ratio (i n t hat partic ular assa y s yste m) as does nati ve L RF. Probably beca use of t heir m uc h greater s pecific acti vity, w he n gi ve n i n doses i de ntical i n weig ht to t he refere nce doses of L RF, t he s u per- L RFs are re markably long acting. While the elevate d secretion of L H (or FS H) i n d uce d by L RF is ret ur ne d to nor mal i n 60 mi n utes, i de ntical a mo u nts i n weig ht, of [ D- Tr p 6 - des- Gly 1 0 ]- N- Et- L R F lea d to statisticall y el e v at e d l e v els of L H u p t o 2 4 h o urs i n s e v er al i n vi v o pr e p ar ati o ns, i n cl u di n g ma n. T hese a nalogs are i deal age nts to sti m ulate ov ulatio n ( Vilc hez- Marti nez et al., 1975). Mar ks a n d Ster n (1975) h ave re porte d t hat t hese a nalogs are co nsi derably more resista nt t ha n t he native str uct ures to degra datio n by tiss ue e nzy mes. I njectio n i nto laboratory a ni mals of large doses of t he s u per- L RFs (i.e. doses of several microgra ms /ani mal while mini mal active doses or physio- logical range are in nanogra ms /ani mal) has been recently sho wn by several gro u ps to have profo u n d a nti-go na dotro pic effects, bot h i n males a n d fe males; moreo ver, w he n s uc h large doses of t he s u per- L RFs are i njecte d i n t he early da ys of pre g na nc y i n rats, t he y c o nsiste ntl y lea d t o res or pti o n of t he c o nce pt us (see Ri vier et al., 1977a); mec ha nis ms i n vol ve d i n t hese obser vatio ns ha ve not bee n f ull y clarifie d as yet b ut are best ex plai ne d b y t he c urre nt co nce pts of negative coo perativity bet ween t he pe pti dic liga n ds i n vol ve d a n d t heir rece pt ors at t he se veral tar get- or ga n sites. All of t he a ntago nist L RF-a nalogs as origi nally fo u n d by o ur gro u p ( Vale et al. 1972c) or as later re porte d b y ot hers ha ve deletio n or a D-a mi no aci d s ubstit utio n of His 2 . For reaso ns not clearly u n derstoo d, a d ditio n of t he F uji no mo dificatio n o n t he C-ter mi nal (F uji no et al., 1974) does not i ncrease t he s pecific activity (as a ntago nists) of t he a ntago nist a nalogs. A d mi nistere d si m ulta neo usly wit h L RF t he a ntago nist a nalogs i n hibit L RF i n weig ht ratios r a n gi n g fr o m 3 : 1 t o 1 5 : 1. T h e m ost p ot e nt of t h es e a nt a g o nists i n hi bit a cti vit y of L R F n ot o nl y i n vitr o, b ut als o i n v ari o us t ests i n vi v o. T h e y i n hi bit t h e r el e a s e of L H a n d FS H i n d uce d by a n ac ute dose of L RF; t hey also i n hibit e n do- geno us release of L H-FS H an d th us prevent ov ulation in laboratory ani mals. T he cli nical testi n g of so me of t hese L R F-a nta go nists pre pare d i n o ur la b- orator y has rece ntl y starte d i n colla boratio n wit h Ye n at t he U ni versit y of C alif or ni a i n S a n Di e g o. b. Bi ol o gic al Acti vit y of So matostati n It is no w recog nize d t hat so matostati n has ma ny biological effects ot her t ha n t he o ne o n t he basis of w hic h we is olate d it i n extracts of t he h y p ot ha- la m us, i.e. as a n i n hi bit or of t he secreti o n of gr o wt h h or m o ne ( Brazea u et al., 1973). So matostati n i n hi bits t he secretio n of t h yrotro pi n, b ut not prolacti n, n or m all y sti m ul at e d b y T R F ( V al e et al., 1 9 7 4); it als o i n hi bits t h e s e cr eti o n of gl u c a g o n, i ns uli n ( K o er k er et al., 1974), gastri n, secreti n, b y acti n g directl y o n t he secretory ele me nts of t hese pe pti des. I ha ve rece ntly s ho w n ( G uille mi n, 197610) t hat so matostati n also i n hibits t he secretio n of acetylc holi ne fro m t he (electrically sti m ulate d) mye nteric plex us of t he g ui nea pig ile u m probably at a pres y na ptic l oc us - t h us ex plai ni n g at least i n part t he re p orte dl y i n hi bit or y effects of so matostati n o n g ut co ntractio n, i n vi v o a n d i n vitr o ( Fi g. 7).

P C S E N Z Y M E S

7 : M ulti ple locatio ns of so matostati n a n d m ulti ple effects of so matostati n.

It is also no w well recog nize d t hat so matostati n is to be fo u n d i n ma ny locatio ns ot her t ha n t he hy pot hala m us (Fig. 7), fro m w hic h we origi nally isolate d it. So matostati n has bee n fo u n d i n ne uro nal ele me nts a n d axo nal fibers i n m ulti ple locatio ns i n t he ce ntral nervo us syste m, i ncl u di ng t he s p i n a l c o r d ( s e e H ö k f e l t e t a l . , 1 9 7 6 ) . I t h a s b e e n f o u n d a l s o i n discrete secret or y cells of classical e pit helial a p peara nce i n all t he parts of t he sto mac h, g ut a n d pa ncreas (see L uft et al., 1974; D ubois, 1975) i n w hic h it ha d bee n first rec o g nize d t o ha ve a n i n hi bit or y effect. So matostati n does not i n hibit i n discri mi nately t he secretio n of all poly- pe pti des or pr otei ns. F or i nsta nce, as alrea dy state d, so matostati n does not i n hibit t he secretio n of prolacti n co nco mita nt wit h t hat of t hyrotro pi n w he n sti m ulate d b y a d ose of T R F; t his is tr ue i n vi v o wit h nor mal a ni mals or i n vitr o wit h n or mal pit uitar y tiss ue (see Vale et al., 1974). S o mat ostati n d oes not i n hibit t he secretio n of eit her go na dotro pi n L H or FS H, t he secretio n of calcito ni n, t he secretio n of A C T H i n nor mal a ni mals or fro m nor mal pit uitary tiss u es i n vitr o ; it d oes n ot i n hi bit t he secreti o n of ster oi ds fr o m a dre nal c ortex or go na ds u n der a ny k no w n circ u msta nces (see Brazea u et al., 1973). Re- gar di ng t he secretio n of poly pe pti des or protei ns fro m ab nor mal tiss ues of ex peri me ntal or cli nical so urces, s uc h as pit uitary a de no mas, gastri no mas, ins ulino mas, etc. so matostatin has been sho wn to be inhibitory accor ding to its nor mal patter n of acti vity or bei ng no n discri mi nati ve. T he latter m ust reflect o ne of t he differe nces bet wee n n or mal a n d ne o plastic tiss ue. T his is i n kee pi ng wit h observatio n t hat T RF or L RF ca n sti m ulate release of gro wt h hor mo ne fro m t he pit uitaries of acro megalic patie nts t ho ug h t hat does not ha p pe n wit h nor mal tiss ues. Cli nical st u dies ha ve co nfir me d i n ma n all obser vatio ns obtai ne d i n t he laboratory. T he po werf ul i n hibitory effects of so matostati n o n t he secretio n not o nly of gro wt h hor mo ne b ut also of i ns uli n a n d gl ucago n ha ve le d to exte nsi ve st u dies o ver t he last t hree years of a p ossi ble r ole of s o mat ostati n i n t he ma na ge me nt or treat me nt of j u ve nile dia betes ( Fi gs. 8-9). First of all, t he ability of so matostati n to i n hibit i ns uli n a n d gl ucago n secretio n has pro vi de d a usef ul tool for st u dyi ng t he p hysiological a n d pat hological effects

Fig. 8: Effect of m ulti ple doses of so matostati n decreasi ng t he plas ma levels of gro wt h h o r mo ne, i ns uli n a n d gl ucago n i n a patie nt wit h acro megaly a n d diabetes. (fro m Ye n et al.). 3 8 2 Physiology or Medicine 1977

of these hor mones on h u man metabolis m. Inf usion of so matostatin lo wers plas ma gl ucose levels i n nor mal ma n des pite lo weri ng of plas ma i ns uli n l e v els ( Alf or d et al., 1 9 7 4; G eri c h et al., 1 9 7 4; M orti m er et al., 1 9 7 4). T h es e obser vatio ns pro vi de d t he first clear-c ut e vi de nce t hat gl ucago n has a n i m portant physiological role in h u man carbohy drate ho meostasis. So matostatin itself has no direct effect o n eit her he patic gl ucose pro d uctio n or peri p heral gl ucose utilizatio n, si nce t he fall i n plas ma gl ucose le vels co ul d be pre ve nte d by exoge no us gl ucago n ( Geric h et al., 1974). I n j uve nile-ty pe diabetics, so matostati n di mi nis hes fasti ng hy perglyce mia b y as m u c h as 5 0 % i n t he co m plete a bse nce ofcirc ulati n g i ns uli n ( Geric h et al., 1974). Alt ho ug h so matostati n i m pairs carbo hy drate tolera nce after oral or i ntrave no us gl ucose c halle nges i n nor mal ma n by i n hibiti ng i ns uli n secretio n, carbohy drate tolerance after ingestion of balance d meals is i m prove d in patients with ins ulin- de pen dent diabetes mellit us thro ugh the s u p pression of excessive gl ucago n res po nses ( Geric h et al., 1974). T he co mbi natio n of so ma- tostatin an d a s ubo pti mal a mo unt of exogeno us ins ulin ( which by itself ha d prevente d neither excessive hy perglyce mia nor hy pergl ucagone mia in res ponse to meals) co m pletely prevents plas ma gl ucose levels fro m rising after meal i ngestio n i n i ns uli n- de pe n de nt diabetics ( Geric h et al., 1974). T hro ug h its s u p pression of gl ucagon an d gro wth hor mone secretion, so matostatin has also been sho wn to mo derate or prevent co m pletely the develo p ment of diabetic ketoaci dosis after t he ac ute wit h dra wal of i ns uli n fro m patie nts wit h i ns uli n- de pe n de nt diabetes mellit us ( Geric h et al., 1975). At t he mo me nt, cli nical st u dies wit h so matostati n are procee di ng i n se veral cli nical ce nters t hro ug ho ut t he worl d. Fro m t he foregoi ng descri ptio n of t he ability of so matostati n to i n hibit P e pti d es i n t h e Br ai n. T h e N e w E n d o cri n ol o g y of t h e N e ur o n 3 8 3

t he secretio n of vario us hor mo nes, it wo ul d a p pear t hat it may be of t hera- pe utic use i n certai n cli nical co n ditio ns s uc h as acro megaly, pa ncreatic islet cell t u mors, a n d diabetes mellit us. Wit h regar d to e n docri ne t u mors, it m ust be e m phasize d that while so matostatin will inhibit hor mone secretion by t hese tiss ues, it wo ul d not be ex pecte d to di mi nis h t u mor gro wt h (i n vie w of its l oc us of acti o n relati n g t o t hat of c- A M P -- see Vale et al., 197213). T h us, i n t h es e c o n diti o ns it is u nli k el y t h at s o m at ost ati n will fi n d us e ot h er t h a n as a sy mpto matic or te mporizing measure. I n diabetes mellit us, ho we ver, so matostati n mig ht be of co nsi derable cli nical val ue. First, it has alrea d y bee n de mo nstrate d t hat it ca n ac utel y i m pro ve fasting as well as post pran dial hy perglyce mia in ins ulin-req uiring diabetics, by i n hibiti ng gl ucago n secretio n. Secon d, since gro wth hor mone has been i m plicate d i n t he de velo p me nt of diabetic reti no pat hy, t he i n hibitio n of gro wt h hor mone secretion by so matostatin may lessen this co m plication of diabetes. Finally, thro ugh s u p pression of both gro wth hor mone an d gl ucagon secretion, so matostati n may preve nt or di mi nis h t he severity of diabetic ketoaci dosis a n d fi n d a p plicatio n i n “brittle diabetes.” These o pti mistic ex pectations m ust be c o nsi dere d i n li g ht of t he facts t hat t he m ulti ple effects of s o mat ostati n o n hor mo ne secretio ns a n d its s hort d uratio n of actio n make its cli nical use i m practical at t he prese nt ti me; moreover, its lo ng-ter m effective ness a n d safety ha ve not bee n establis he d as yet. Regar di ng t he cli nical use of so ma- tostati n, see t he rece nt re vie w by G uille mi n a n d Geric h (1976). Wit h t he c o nsi dera ble i nterest i n s o mat ostati n as a part of t he treat me nt of diabetics, “i m prove d” a nalogs of so matostati n have bee n i n t he mi n d of clinicians an d investigators. Analogs of so matostatin have been pre pare d in atte m pts to obtai n s ubsta nces of lo nger d uratio n of activity t ha n t he native for m of so matostati n; t his has not bee n very s uccessf ul so far. Ot her a nalogs have bee n so ug ht t hat wo ul d have dissociate d biological activity o n o ne or more of t he m ulti ple recog nize d targets of so matostati n. Re markable res ults have rece ntly bee n obtai ne d. T he first s uc h a nalog so recog nize d by t he gro u p of the Wyeth Research Laboratories was [ des- Asn 5 ]-so matostati n, a n a nalog wit h a p proxi mately 4 %, 10 % a n d 1 % t he activity of so matostati n to inhibit res pectively secretion of gro wth hor mone, ins ulin an d gl ucagon (Sa- r a nt a kis et al., 1 9 7 6). W hil e s u c h a n a n al o g is n ot of cli ni c al i nt er est, it s h o w e d t hat diss ociati o n of t he bi ol o gical acti vities of t he nati ve s o mat ostati n o n t hree of its rece ptors co ul d be ac hie ve d. So me of t he most i nteresti ng a nalogs wit h dissociate d acti vities re porte d so far were pre pare d a n d st u die d by J. Ri vier, M. Bro w n a n d W. Vale i n o ur laboratories; t hey are [ D-Ser 1 3 ]-so matostati n, [ D- Cys 1 4 ]-so matostatin an d [ D- Tr p 8 , D- C ys 1 4 ]-so matostatin. When co mpared to so matostati n, t his latest co m po u n d has ratios of acti vity s uc h as 300 %, 10 %, 100 % to i n hibit t he secretio ns res pectively of gro wt h hor mo ne, i ns uli n a n d gl ucago n ( Bro w n et al., 1976). T h ese a n d ot her a nalogs are obvio usly of m u c h cli ni c al i nt er est a n d ar e b ei n g s o i n v esti g at e d at t h e m o m e nt i n s e v er al laboratories a n d cli nical ce nters. A n i nter natio nal sy m posi u m was rece ntly de vote d e ntirely to t he biology a n d c he mistry of so matostati n a n d a nalogs (see Ra ptis, 1978). 3 8 4 Physiology or Medicine 1977 c. Biological St udies with the E ndorphi ns

1. Rel ati o n of E ndorphi ns to So far, all mor p hi no mi metic pe pti des isolate d fro m nat ural so urces o n t he basis of a bi oassa y or dis place me nt assa y f or 3 H-o piates on syna ptoso mal pre parations, an d che mically characterize d, have been relate d to a frag ment of t he C-ter mi n us of t he m olec ule of starti n g at T yr 6 1 . I n t h e c as e of Le u 5 -e nke p hali n, t he relatio ns hi p still hol ds for t he seq ue nce Tyr- Gly- Gly- P h e; n o wit h a Le u resi d ue i n p ositi o n 65 has bee n o bser ve d.

3 0 0 2 5 0

Fig. 10: Co nco mita nt elevatio n of plas ma levels of A C T H a n d u po n ac ute ex pos ure to stress a p plie d at ti me 0. ( Pe pti de meas ure d by ra dioi m m unoassays). Soli d line sho ws plas ma corticosterone levels measure d by fluoro metry. 3 8 5

C hr éti e n et al., 1 9 7 7) t h at exists as s uc h a n d as part of a lar ger prec ursor i n discrete pit uitary cells. We ha ve i n dee d rece ntly s ho w n ( G uille mi n et al., 1 9 7 7 b) t h at a n d A C T H are secrete d si m ulta neo usly i n vi v o or i n vitr o i n all circ u msta nces teste d s o far ( Fi g. 10). 3 8 6 Physiology or Medicine 1977

2. Rele ase of Pituitary Hor mones by Endorphins O ne of o ur ori gi nal i nterests i n e n ga gi n g i n t he isolatio n a n d c haracterizatio n of t he e n dor p hi ns was t hat t he o piate-like pe pti des mig ht be i n vol ve d i n t he secretion of pit uitary hor mones, partic ularly gro wth hor mone an d prolactin, lo ng k no w n to be ac utely release d follo wi ng i njectio n of mor p hi ne. We ha ve s h o w n ( Ri vier et al., 1977 b) t hat is a p ot e nt releaser of i m munoreactive gro wth hor mone an d prolactin when a d ministere d t o rats b y i ntracister nal i njecti o n. T hese effects were preve nte d by prior a d mi nistratio n of naloxo ne. T he e n dor p hi ns are not active directly at t he le vel of t he pit uitar y cells; t he y s h o w n o effect, e ve n i n lar ge d oses, w he n a d de d directly to mo nolayer c ult ures of (rat) pit uitary cells. T h us, t he hy po- p hysiotro pic effects of t he e n dor p hi ns, like t hose of t he o piate alkaloi ds, are me diate d b y so me str uct ure i n t he ce ntral ner vo us s yste m a n d are not directl y at t he level of t he a de no hy po p hysis. Si milar res ults have bee n observe d by se veral gro u ps of i n vestigators. We ha ve rece ntly s ho w n t hat i s a pote nt sti m ulator of t he secretio n of vaso pressi n, possibly acti ng at a hy po- t h al a mi c l e v el, si n c e it is n ot a cti v e o n t h e i n vitr o isolated neurohypophysis ( W eit z m a n et al., 1 9 7 7). c. Ne uro nal Actio ns of Endorphins and Enkephalins A mong Brain Regions The existence of en dogeno us pe pti des with o piate-like actions s uggests that these s ubstances may f unction as ne uro mo d ulators or ne urotrans mitters in the C NS. In dee d, recent ionto phoretic st u dies have sho wn that the enke phalins ca n mo dify t he excitability of a variety of ne uro ns i n t he C NS. Most ne uro ns teste d were i n hibite d by t hese pe pti des (Fre derickso n a n d Norris, 1976; Hill et al., 1976; Zieglga nsberger et al., 1976), alt ho ug h Re ns ha w cells res po n de d wit h a n excitatio n ( Davies a n d Dray, 1976). St u dies have rece ntly a p peare d ex plori ng syste matically t he se nsitivity of ne uro ns to t he e n dor p hi ns or re- porting a syste matic regional s urvey of ne urons res ponsive to the pe pti des ( Ni c oll et al., 1 9 7 7). A s ur prisi n g fi n di n g i n t hat st u d y was t he pote nt excitator y effects of t he pe pti des an d nor mor phine on hi p poca m pal pyra mi dal cells (Fig. 11). The re gio nal s pecificit y of t his excitator y actio n co ul d be clearl y de mo nstrate d wit h t he sa me electro de b y recor di n g fro m cells i n t he o verl yi n g cere bral cortex a n d the un derlying thala m us d uring a single penetration. No tachy phylaxis was obser ve d eit her to t he excitatory or i n hibitory actio n of t he pe pti des i n a ny of t he regio ns exa mi ne d, eve n t ho ug h t he pe pti des were ofte n a p plie d re- peate dl y t o t he sa me cell f or peri o ds i n excess of o ne h o ur. To deter mi ne w het her t he res po nses observe d wit h t he pe pti des were relate d to t he acti vatio n of o piate rece ptors t he s pecific o piate a nta go nist, naloxo ne, was a d mi nistere d bot h by io nto p horesis fro m a n a djace nt barrel of t he micro- electro de a n d by s ubc uta neo us i njectio ns. A d mi nistere d by eit her ro ute, naloxo ne a ntago nize d bot h t he excitatio ns a n d t he i n hibitio ns. All t hese effects of o piate-li ke pe pti des o n ne ur o nal acti vit y, ta ke n wit h bioc he mical a n d histoc he mical e vi de nce for t heir existe nce i n brai n, are co nsiste nt wit h t he hy pot hesis t hat t hese pe pti des are ne urotra ns mitters i n Peptides in the Brain. The Ne w Endocrinology of the Neuron 3 8 7

( u nide ntified)

Fig. 11: S u m mary of ne uro nal effects of opioid peptides a nd morp hi ne. I n eac h category t he total n u mber of cells tested a nd t he perce ntage of t his total t hat were i n hibited or excited is s ho w n (fro m Nicol et al.).

the C NS. Moreover, I have recently observe d in collaboration with Gr u m- bat h, Peter nelli a n d Davis t hat p urifie d sy na ptoso mes of ve ntral hy pot hala m us origi n (rat brai n) release large a mo u nts of i m m u noreacti ve when ex pose d to ele vate d [ K Cl] (to be p u blis he d). W he n t he cells of ori gi n of t hese pe pti de-containing fibers have been deter mine d, it may then be possible to procee d wit h st u dies i nto t he effects o n cell ular acti vit y a n d t he secretio n of t he pe pti des i n or der to satisf y more co m pletel y t he criteria for a ne urotra ns- mitter. Cr ucial p oi nts i n s uc h f ut ure a nal yses will be t he q uesti o ns of w het her t he e n dor p hi n a n d e nke p hali n co ntai ni ng fibers are m ut ually i ncl usive syste ms (rece nt st u dies i n dicate t hat t he y are n ot -- R ossier et al., 1977), w het her t he le ngt h of t he pe pti de release d by ne uro nal activity is s ubject to mo d ulatio n, an d whether inter me diate length pe pti des (s uch as the α −, γ −, a n d phins) may partici pate in s uch mo d ulatory changes. Altho ugh the en dor phins a n d may be prohor mones for Met s -e nke p hali n, t here are at prese nt no s uc h ca n di dates for Le u 5 -e nke p hali n. T he res ults prese nte d here i n dicate to us t hat t he cell ular roles of e n dor p hi n a n d e nke p hali n pe pti des ca n not no w be ge neralize d across all brai n re gio ns w here t he y are fo u n d, a n d t hat no si m ple cell ular actio n of a n y pe pti de will yiel d a n i nte grati ve pict ure of t he way in which o piate alkaloi ds pro d uce co m plex analgesic, e u phoric, an d a d dictive res ponses. Involve ment of the en dor phins in the control of a deno- h y p o p h ysial f u ncti o ns is still a s u bject f or f urt her st u d y at t he ti me of writi n g t his re vie w. So far, no direct hy po p hysiotro pic acti vities of t he e n dor p hi n pe pti des have been clearly de monstrate d. 3 8 8 Physiology or Medicine 1977 d. Be h a vi or al effects of e n d or p hi ns The phar macological pro perties of en dor phins have so far been screene d t hro u g h a p plicatio n of tests i n vitr o or i n vi v o pre vio usl y use d to c haracterize o piate ago nists a n d a ntago nists. W he n i njecte d i nto t he cerebros pi nal fl ui d, e n dor p hi ns affect se veral be havioral a n d p hysiological meas ures, i n a d ditio n to res po nses to noxio us a ge nts, a n d eac h of t he pe pti des ex hi bits differe nt dose-effect profiles o n t hese meas ures: i n d uces a marke d catato nic state (Fig. 12) lasti ng for ho urs ( Bloo m et al., 1976) at molar doses 1 /100 t hose at w hic h Met 5 - e n ke p hali n tra nsie ntl y i n hi bits res po nses to noxio us a ge nts ( Bell uzzi et al., 1976; B usc her et al., 1976; L o h et al., 1976). T his p ote nt be ha vi oral effect of a nat urall y occ urri n g s u bsta nce s u g gests its re g ulatio n co ul d ha ve etiolo gical si g nifi c a n c e i n m e nt al ill n ess.

Fi g. 12: T hirt y mi n utes after t he i ntracister nal i njecti o n of ( 1 5 x 1 0 - 9 m o l e ) t his rat ex hi bite d s ufficie nt ri gi d i m mo bilit y to re mai n totall y self-s u p porti n g w he n place d acr oss metal b o o ke n ds w hic h are i n c o ntact o nl y at t he u p per nec k a n d base of t he tail. S uch post ures were maintaine d for prolonge d perio ds. Note the erect ears an d tail, wi dely o pene d eyeli ds an d exten de d lo wer li mbs.

In ter ms of molar dose-effectiveness on the vario us para meters exa mine d, is clearl y t he m ost p ote nt s u bsta nce teste d. Rats gi ve n se ve n dail y i ntracister nal i njecti o ns of 15 x 10 - 9 m o l e o f co nti n ue d to s ho w t he f ull set of res po nses a n d d uratio n of actio n. T he catato nic state i n d uce d b y was not observe d wit h t he ot her e n dor p hi n pe pti des, eve n at co nsi derably hig her doses. At very high doses of a-en dor phin, y-en dor phin or Met 5 -e nke p hali n, tra nsie nt losses of cornea1 reflexes were observe d, an d a-en dor phin see me d more potent in t his re gar d t ha n eit her or Met 5 -e nke p hali n. No sig nifica nt de pressio ns of res po nsive ness to tail- pi nc h or pi n- prick sti m uli were observe d wit h Met 5 -e nke p hali n, a-e n dor p hi n, or y-e n dor p hi n, b ut s uc h effects ( Bra d- b ur y et al., 1976; B usc her et al., 1976; R oss et al., 1976) c o ul d ha ve bee n misse d by t he 5- mi n ute i nter val after i njectio n a n d before testi ng bega n. I n co ntrast to t he sy n dro me i n d uce d by rats given y-en dor phin P e pti d es i n t h e Br ai n. T h e N e w E n d o ni n ol o g y of t h e N e ur o n 3 8 9 s ho we d co nsiste nt elevatio ns i n rectal te m perat ure (abo ut 2.0° C2” C at 30 mi n utes after 281 x 1 0 - 9 mole), an d so meti mes exhibite d so me degree of hy perres po nsivity to se nsory testi ng a n d ha n dli ng, alt ho ug h t here were i n di vi d u al v ari ati o ns i n t his r es p o ns e. All of o ur obser vatio ns s uggest t hat nor mal variatio ns-eit her q ualitati ve or q uantitative-in the ho meostatic mechanis ms reg ulating the post ulate d ( La- zar us et al., 1976) c o n versi o n of as a pro hor mo ne to its se veral e n- dor phin cleavage pro d ucts co ul d constit ute a syste m f un da mentally involve d in maintaining “nor mal” behavior; alterations of the mechanis ms nor mally re g ulati n g ho meostasis co ul d lea d to sig ns a n d sy m pto ms of mental illness. S uch a potential psycho physiological role of en dor phins co ul d logically be testable thro ugh the thera pe utic a d ministration of available o piate antagonists. T his has alrea dy bee n atte m pte d i n se veral cli nical ce nters t hro ug ho ut t he worl d; res ults obtai ne d have bee n i nter prete d differe ntly a n d, i n my o w n mi n d, are m uc h too preli mi nary as yet to warra nt a ny co ncl usio n, positive or negative. T he ulti mate i de ntificatio n of e n dor p hi n- se nsiti ve be ha vioral e ve nts a n d s pecific treat me nt of t heir dysf u nctio nal states may req uire the develo p ment of more s pecific “anti-en dor phins” than those no w available; ot her nat urally occ urri ng brai n pe pti des s uc h as S ubsta nce P, have alrea dy bee n re porte d to be e n dor p hi n a ntago nists i n so me assay syste ms ( G uill e mi n et al., 1 9 7 6 c). T h er e is littl e d o u bt t h at t h e p ot e nti al si g nifi c a n c e i n a n y s uc h st u dies is s o great t hat maj or eff orts i n t his area b ot h i n t he la b- oratory a n d i n cli nical st u dies s ho ul d be s po nsore d a n d p urs ue d wit ho ut fail.

P A R T I I I

Endocrine and Paracrine Secretions of the Brain. Hor mones and Cybernins It has bee n k no w n for so me ti me t hat T RF, L RF, a n d so matostati n origi nally isolate d fro m extracts of t he hy pot hala m us are act ually to be fo u n d i n no n- negligible a mo unts thro ugho ut the central nervo us syste m, incl u ding the s pi nal cor d. T his was de mo nstrate d first by bioassays a n d has bee n a m ply confir med by radioi m munoassays. Extrahy pothala mic T RF, L RF, or so ma- tostati n have not bee n isolate d a n d c he mically c haracterize d as yet. T here is t h us no u nco ntrovertible evi de nce t hat t he perti ne nt s ubsta nces i n extra- hy pot hala mic brai n-extracts are i de ntical to t hose c haracterize d i n t he hy pothala m us. All the c urrent circ u mstantial evi dence is in favor, ho wever, of t he i de ntit y of t he materials i n t heir m ulti ple l ocati o ns ( parallelis m of dose-res po nse c urves i n several bioassays, parallelis m of dis place me nt c urves i n several ty pes of ra dioi m m u noassays, etc. usi ng p urifie d tiss ue extracts a n d p ure sy nt hetic pe pti des as refere nce sta n dar ds). Ubiq uity of t hese pe pti des t hro ug ho ut t he ce ntral nervo us syste m does not i m ply t hat t hey are ra n do mly distrib ute d. Several gro u ps have s ho w n t hat eac h of t hese pe pti des has a uniq ue distrib ution pattern as they have been i dentifie d by i m m unocyto- c he mistry i n axo nal tracts a n d ne uro nal bo dies i n well c haracterize d a nato m- ical f or mati o ns of t he ce ntral ner v o us s yste m ( H ö kfelt et al., 1977). It was t his k no wle d ge of t he m ulti plicit y of locatio n of t he releasi n g factors a n d 3 9 0 Physiology or Medicine 1977 partic ularl y of so matostati n i n t he ce ntral ner vo us s yste m t hat lea d a fe w y e ars a g o t o t h e s ol uti o n of o n e of t h e m ost p u z zli n g dil e m m as i n t his fi el d, a n d t hat sol utio n ha d far-reac hi ng co nseq ue nces: It was diffic ult to reco ncile t he s hort biolo gical half-life of so matostati n (less t ha n fo ur mi n utes), w he n i njecte d i n t he bloo d strea m wit h its well esta blis he d effects o n t he secretio n of gl ucago n a n d i ns uli n, wit h t he hy pot hesis t hat hypothala mic so matostatin co ul d be i n vol ve d i n t he p hysiological co ntrol of t he secretio n of pancreatic gl ucago n a n d i ns uli n. L uft i n Stock hol m a n d I i n de pe n de ntly wo n dere d whether so matostatin co ul d be delivere d to the en docrine pancreas by means ot h er t h a n t h e g e n er al cir c ul ati o n, p ossi bl y b y n er v e fi b ers, k n o w n t o i n n er v at e the islets of Langerhans. The re markable observation was then ma de that in fact t he e n docri ne pa ncreas of all verte brates st u die d so far co ntai n a discrete po p ulation of cell ular ele ments containing so matostatin as sho wn by i m m uno- cytoc he mistry ( L uft et al., 1974; D ubois, 1975). T he so matostati n-co ntai ni ng cells belo ng to t he D-cells of t he e n docri ne pa ncreas, lo ng k no w n to t he mor p hologists to be differe nt fro m t he u-cells co ntai ni ng gl ucago n a n d t he co ntai ni n g i ns uli n, b ut for w hic h no s pecific secretor y pro d ucts ha d bee n recog nize d so far. Moreo ver, i n t hese earl y re p orts a lar ge n u m ber of secretory cells containing i m m unoreactive so matostatin were fo un d thro ugho ut t he gastroi ntesti nal tract a n d it has no w bee n s ho w n t hat so matostati n ca n i n hi bit t he secreti o n of gastri n, of secreti n, of c h olec yst o ki ni n, als o t he secreti o n of p e psi n a n d H Cl b y a cti n g dir e ctl y at t h e l e v el of t h e g astri c m u c os a ( N a k aji, et al., 1975). T RF has rece ntly bee n re porte d i n extracts of t he sto mac h a n d of the d uo den u m ( Le p pal uoto et al. in press). Ne urotensin an d s ubstance P have also bee n locate d i n t he hy pot hala m us a n d t hro ug ho ut t he gastroi n- t esti n al tr a ct i n s p e cifi c c ells a n d i n cr u d e e xtr a cts - as h as b e e n k n o w n i n t h e case of s ubsta nce P, si nce 1931 fro m t he work of Ga d d u m a n d vo n E uler. T here is no w e vi de nce t hat ot her pe pti des ori gi nall y c haracterize d fro m extracts of tiss ues of t he gastr oi ntesti nal tract ca n be f o u n d a n d l ocate d i n t he brai n; t his is t he case for gastri n /c holecystoki ni n, vaso-i ntesti nal pe pti de ( VI P), t he gastric i n hibitory pe pti de ( GI P) ( s e e re vie w by Pearse a n d Takor, 1976); t his is als o tr u e f or t h e e n d or p hi ns a n d e n k e p h ali ns a n d f or s e v er al of t h e s m all pe pti des s uch as bo mbesin, caer ulein, physala mine, isolate d years ago fro m extracts of t he s ki n of se veral s pecies of fr o gs. T here are re mar ka ble a nal o gies a n d ho mologies bet wee n t he a mi no aci d seq ue nces of several of t hese pe pti des of ce ntral ner vo us syste m origi n a n d gastroi ntesti nal origi n, as well as t hose is ol at e d fr o m fr o g s ki n. These pe pti des have been fo un d by i m m unocytoche mistry essentially in t w o t y p es of c ells: 1) T h e y ar e s e e n i n c ell b o di es a n d n er v e fi b ers i. e. a x o n al a n d de n dritic processes of ne uro ns i n brai n, s pi nal cor d, i n s pi nal ga n glia a n d i n t he mye nteric plex us; 2) T hey ar e s e e n als o i n t y pi c al e n d ocri ne cells, f o r i nsta nce i n t he pa ncreatic islets of La nger ha ns, i n t he e nteroc hro maffi n cells of the g ut an d the a drenal me d ulla. Ne uroblasto mas have been re porte d to co ntai n hig h levels of t he vaso-i ntesti nal pe pti de ( VI P) (Sai d a n d Rose n- ber g, 1976). T here is alrea d y a n i nteresti n g u nif yi n g co nce pt to bri n g to get her t hese rat her startli ng observatio ns. M uc h cre dit m ust g o t o A. G. E. Pearse f or his visio nary co nce pt, for m ulate d so me te n years ago, of t he A P U D cells: Pearse observe d that ne urons an d so me en docrine cells pro d ucing poly pe pti de hor mones share d a set of co m mon cytoche mical feat ures an d ultrastr uct ural c haracteristics. A P U D is a n acro ny m referri ng to A mi ne co nte nt a n d /or A mine Precursor Uptake an d Decarboxylation, as co m mon qualities of these cells ( Pearse, 1968). T he A P U D co nce pt post ulate d t hat t hese e n docri ne cells were derive d fro m a co m mon ne uroecto der mal ancestor, the transient ne ural crest. Pearse post ulate d f urt her t hat a still larger n u mber of e n docri ne cells wo ul d be event ually fo un d sharing these co m mon pro perties if one were to ex plore f urt her i n t he a d ult, e n docri ne tiss ues deri ve d fro m t he ne ural crest. Rece nt observatio ns wit h refi ne d tec h niq ues, partic ularly t he work of Le Do uarin on to pical chi meras with chro moso mal markers, have le d Pearse to mo dify t he origi nal A P U D co nce pt, a n d, as we will see, i n a re markable ma n ner. T he ne w evi de nce regar di ng t he m ulti ple so urces of t he several pe pti des me ntio ne d above s ho we d t hat tiss ues were i nvolve d t hat were not of ne ural crest ori gi n; t his is partic ularl y tr ue for t he pe pti de-secreti n g cells of t he g ut. All t hese cells ha ve bee n s ho w n to arise fro m s pecialize d ne uroecto- der m ( Pearse a n d Ta k or, 1976); t hat is, n ot o nl y t he ne ural crest b ut als o t he ne ural t ube, t he ne ural ri d ges a n d t he placo des. The expanded concept no w postulates that all peptide hor mone-producing cells are deri ve d fro m t he ne ural ecto der m, as are all ne uro ns. Wit h s uc h o ntoge nic co m mo nality, it is t h us less s ur prisi ng to recog nize t he prese nce of “gastro-i ntesti nal pe pti des” i n t he brai n, a n d of “ brai n pe pti des” i n t he gastr o-i ntesti nal tract. A C T H, a n d gro wt h hor mo ne of pit uitary origi n, c holecysto- ki ni n, secreti n, or gastri n of gastroi ntesti nal origi n are well recog nize d hor- mo nes w hic h satisf y all t he defi nitio ns of t he wor d, partic ularl y as it i m plies t heir dista nt actio n o n target cells or orga ns far re mo ve d fro m t he so urce of t hese pe pti des. I n t he case of t he release of T R F, L R F, or so matostati n b y hy pothala mic ne urons at the level of the hy pothala mo-hy po physial portal vessels, t hese hy po p hysiotro pic activities ca n also be co nsi dere d as hor mo nal i n nat ure. Let us note i m me diately t hat, w hile t he mea ns of co nveya nce are i n dee d bloo d vessels, t he dista nce travelle d by t he hy po p hysiotro pic pe pti des i n t h es e v ess els is m e as ur e d b y a f e w m m. u ntil t h e y r e a c h t h eir t ar g et pit uit ar y cells. T his is ver y differe nt fro m t he lo n g dista nce tra velle d b y t he classical hor mones mentione d above. Moreover, even in the case of the hy pothala mic pe pti des as i n vol ve d i n t heir hy po p hysiotro pic f u nctio ns, t here is no ge nerally acce pte d e vi de nce t hat t he y e nter t he ge neral circ ulatio n for a n y le n gt h of ti me an d in a physiologically meaningf ul concentration. W he n we co nsi der t hese sa me pe pti des i n parts of t he brai n ot her t ha n t he hy po p hysiotro pic hy pot hala m us, t he sit uatio n is altoget her differe nt: Bot h the optic and the electron microscope, co mbined with i m munocytoche mistry begi n to s ho w evi de nce of very p u nct ual localizatio ns w hic h i m ply si milarly p u nct ual roles; i.e. to be playe d o ver dista nces meas ure d i n a ngstro ms. Berta Sc harrer has for so me ti me describe d w hat s he calls pe pti dergic sy na pses 3 9 2 Physiology or Medicine 1977

( S c h a r r e r , 1 9 7 5 ) . Moreo ver, rece nt st u dies wit h a ntibo dies to so matostati n ha ve yiel de d pict ures w hic h ha ve bee n i nter prete d b y Petr usz et al. (1977) as s ho wi ng t he localizatio n of i m m u noreactive so matostati n i n m ulti ple de n dritic e n di n gs. So me of t hese pict ures are s pectac ular ( Fi g. 13). T heir m ost he uristic i nter pretati o n is t hat eac h dar k p oi nt is t hat of a de n dritic co ntact eit her wit h a not her de n drite or ab utti ng o n a s pecialize d loc us of t he axo n or of t he so ma of reci pie nt ne uro ns. Clearly t hese reci pie nt ne uro ns do not see m to co ntai n i m m u noreactive so matostati n. T he cells of origi n containing an d sen ding the pres u m ptive so matostatinergic ter minals have not bee n c haracterize d as yet. Si milar pict ures have alrea dy bee n observe d i n m ulti ple locatio ns i n t he brai n a n d for several i m m u noreactive pe pti des. I n t his co ntext t hese pe pti des w hic h we calle d hor mo nes earlier do not fit t he defi nitio n of a hor mo ne a ny more; t hey see m to be ca n di dates for t he defi- nitio n of ne urotra ns mitters. Rece ntly Hökfelt (1977) has co ncl u de d t hat so me ne uro ns may co ntai n bot h pe pti des a n d o ne of t he catec hola mi nes, a classical ne urotra ns mitter (1977).

Fig. 13: Pres u m ptive nerve ter minals as so mato- den dritic ter minals aro un d pyra mi dal cells of t he hi p poca m p us rat brai n). I m m u nocytoc he mistry wit h a ntisera to so matostati n (fr o m Petr usz et al.).

It will be ob vio us by no w t hat we are o nly at t he very begi n ni ng of t he p hysiological sig nifica nce of t hese pe pti des i n t he brai n. T he local p u nct ual release t hat we ha ve see n here wit h its m ulti ple locatio ns a n d s hort ra nge of traffic wo ul d make t he m fit w hat Feyrter ha d calle d earlier t he paracri ne secretory syste m. I nteresti ngly e no ug h, Feyrter ha d evolve d his co nce pt of paracrine secretion while st u dying with very si m ple mor phological tools an d P e pti d es i n t h e Br ai n. T h e N e w E n d o cri n ol o g y of the Neuron 3 9 3 a great deal of o bser vatio nal ac u me n t he ver y cells of t he gastroi ntesti nal tract a n d of t he pa ncreas t hat we k no w no w to secrete t he very pe pti des disc usse d i n t his lect ure. I ha ve pro pose d earlier t he ge neric na me cyber ni n for these s ubstances; the ety mology of the wor d i m plying local infor mation. Obvio usly agai n, years of i nvestigatio n are a hea d to u n dersta n d t he me- c ha nis ms i nvolve d i n t he cell biology a n d t he cell p hysiology of t he ne uro ns as t hey sy nt hesize, release, res po n d to a n d metabolize t he ma ny pe pti des disc usse d here. Of t h e m a n y dis c o v eri es a h e a d of us will b e t h os e t h at will l e a d us t o u n d er- st a n d t h e r ol e of e a c h of t h es e p e pti d es i n t h e br ai n, n ot o nl y i n t h eir c ell ul ar p hysiology a n d bioc he mistry, b ut also i n t heir sig nifica nce i n t he hig her f unctions of the central nervo us syste ms. Tho ugh a d mitte dly base d on si m ple a n d e nt h usiastic tele ol o g y, it is diffic ult n ot t o h y p ot hesize t hat t hese pe pti des m ust i n dee d play so me r ol e i n t h e f u n cti o ns of t h e br ai n. O n c e t his si m pl e pro posal is ma de, if we recog nize t hat not o ne wor d abo ut t he existe nce of t hese s ubsta nces i n t he ce ntral ner vo us syste m is to be fo u n d i n a ny of t he classical texts of ne ur o- ps yc hiatr y, o ne ca n n ot b ut be o pti mistic t hat t he earl y observatio ns s u m marize d i n t his lect ure will lea d to profo u n d rea p praisals of t he mec ha nis ms i n vol ve d i n t he f u nctio ns of t he nor mal brai n, b ut also of m e nt al ill n e s s. 3 9 4 Physiology or Medicine 1977

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