Biomedical Research 3 (5) 525-533, 1982
PANCREATIC POLYPEPTIDE IMMUNOREACTIVE CELLS AND NERVES IN THE CANINE PITUITARY
SHIGEKI FUJII‘, SHIGEAKI BABA1 and TUNEO FUJITA2 1The Second Department of Internal Medicine, Kobe University School of Medicine, Kobe 650, and ZDepart~ merit of Anatomy, Niigata University School of Medicine, Niigata 951, Japan
ABSTRACT . Pancreatic polypeptide (PP)-like immunoreactivity was found to occur in the canine pituitary. Numerous PP-immunoreactive cells were observed in the pars intermedia and a few cells were scattered in the pars tuberalis. Studies using the anti-C terminal fragment of adrenocorticotropic hormone (ACTH) serum suggested the presence of three cell types in the pars intermedia; one type having both PP- and ACTH-like immunoreactivity was predominant, a second type possessing only ACTH-like im- munoreactivity appeared in small clusters, and a third type, which did not react to either antiserum, was seen in small numbers among the other type cells. In the pars tuberalis, only cells with a strong ACTH-like immunoreactivity showed PP-like immunoreactivity, while weakly stained cells did not. In the pars distails, almost no PP-like immunoreactivity was detected, although numerous ACTH-immunoreactive cells were observed. PP-immunoreactive nerve fibers were mainly observed in the hypothalamus and the infundibulum. In the pituitary of canine fetuses, ACTH-like immunoreactivity was observed, but a PP-like substance could not be detected.
Some pituitary hormones and their chemically most representative gut hormones, is present in related peptides have been demonstrated in the a group of cells containing ACTH and Ot- gastro-entero-pancreatic (GEP) tissues by im- melanocyte-stimulating hormone (06-MSH) in munohistochemistry and radioimmunoassay. the mammalian pituitaries (l9, 30). Adrenocorticotropic hormone (ACTH)-like im- We have investigated the morphological munoreactivity has been reported in gastrin- features of PP cells in the GEP endocrine system secreting cells localized in the antropyloric and other neuroendocrine systems. This paper mucosa and in both gastrin and pancreatic will deal with the immunohistochemical and polypeptide (PP) cells in the pancreas (ll“l8). morphological aspects of the PP-immunoreac- These findings were followed by the observations tive cells and nerves in the canine pituitary that growth hormone-like immunoreactivity gland. occurred in the antral gastrin cells and in the pancreatic endocrine cells distributed along the MATERIALS AND METHODS islet periphery in several mammalian species in- Tissue ll/laterials cluding humans (6, 28). Furthermore, )3- endorphin- and leu-enkephalin-like immunoreac- Eight. adult dogs (3 males and 5 females, body tivities were identified in the pancreatic endocrine weight: 9-ll kg) which were kept in individual cells (5, 31) and in enterochromafiin cells in the kennels and given a standard diet (Lovely*R‘, gut (1), respectively. Oriental Yeast, Tokyo) and tap water ad I1'bi'tum Conversely, recent immunohistochemical were sacrificed by exsanguination under sodium studies have revealed that gastrin, one of the thioamylal intravenous anesthesia at a fixed time 526 S. FUJII, S. BABA and T. FUJITA
(from 10:00 to ll:00 a.m.). Eight pituitaries Sra1'm'ng Conrrols with a part of the hypothalamus and the pan- creata were removed as quickly as possible and The specificities of antisera were histologically fixed by immersion in Bouin’s fluid for 12 h at identified by the following methods: 1) normal room temperature. Three fetuses 5-6 cm in non-immunized rabbit serum (NRS, 0.5 %, crown-rump length were taken from an anesthe- DAKO-immunoglobulins) or phosphate buffer tized dog and fixed whole for 24h in Bouin’s saline (PBS, 0.01 M, pH 7.2) was utilized as the fluid. After dehydration and embedding in substituting primary antibody for anti-BPP parafiin, ordinary sections of pituitaries, fetal serum; and 2) immunoabsorption tests of anti- heads (both sagitally), and pancreata were cut BPP serum (diluted 1:2,000) and anti-porcine at 4 ,um. Adjacent sections of 2 pm thickness ACTH serum (1 : 500) incubated for 24 h at 4°C were cut from the pituitary. prior to staining with the following peptides (100 /zg per ml diluted anti-serum): BPP, porcine PP(PPP), synthetic bovine CLIP and synthetic Immimo/1is2‘0c/iemisrry m-MSH. The former two peptides were donated Anti-bovine pancreatic polypeptide (BPP) rabbit by Dr Chance and the latter two were provided serum kindly donated by Dr R. E. Chance, Lilly by Professor Haruaki Yajima, Faculty of Research Laboratories, Eli Lilly, Indianapolis, Pharmaceutical Sciences, Kyoto University, IN, U.S.A. (Lot. 615-R1 10-146-10) was used in a Kyoto, Japan. dilution of 1 : 2,000. Anti-porcine ACTH rabbit serum kindly donated by Dr Yukio Hirata, RESULTS Third Division, Department of Medicine, Kobe University School of Medicine, was fully cross- Marked PP-like immunoreactivity occurred in all reacted with corticotropin-like intermediate lobe the pituitaries of the adult dogs examined. peptide (CLIP) and COOH-terminal fragment of In the adenohypophysis, numerous PP-im- ACTH, but not with other related peptides such munoreactive cells were gathered in the pars as 06-MSH, )3-lipotropin ([3-LPH), or fl-endor- intermedia. The PP-positive cells showed a phine. This antiserum was diluted 1:500 for preferential distribution within the pars inter- use. Both sera, prior to use, were incubated for media. In the lower portion of the pars inter- 30 min at 56°C to inactivate the complements media under the pars nervosa, the immunoreac- possibly contained. tivity was displayed in the great majority of Deparaffinized sections were treated first with endocrine cells (over 95 percent) (Fig. 1), but 3% H202 solution in order to destroy any PP-positive cells were fewer in the upper portion endogenous peroxidase activity, followed by of the pars intermedia extending over the incubation with 0.5% normal swine serum to neurohypophysis (Fig. 2). These cells usually suppress nonspecific binding of immunoglobulins had an elliptic or spindly shape and occasionally to certain substances contained in the tissue. formed an elongated cellular process which had For the demonstration of PP-like immunoreac- a tendency to reach the capillary wall (Fig. 3). tivity, some sections, after an application of PP-positive cells were also scattered in the pars anti-BPP serum for 2 h at 4°C, were subjected to tuberalis (Fig. 9), especially near the infundibular indirect immunoperoxidase technique (24), using stalk. Almost none were seen in the pars distalis horseradish peroxidase conjugated anti-rabbit (Fig. 1). IgG-goat serum (1:40, Lot No. S 692, Miles On the other hand, numerous ACTI—I-im- Lab., U.S.A.). Other sections, after incubation munoreactive cells were observed in the pars with anti-porcine AC-TH serum for 12 h at 4°C, distalis, tuberalis, and intermedia. Their region- were stained with soluble horseradish peroxi- al distribution was similar to that of the PP- dase-rabbit anti-horseradish peroxidase (PAP) positive cells; the cells positive to the anti-ACTH complex (26), employing swine anti-serum to serum occupied the vast majority of the cell rabbit immunoglobulins (1:40, Lot No. 129B, population in the lower portion of the pars DAKO-immunoglobulins, Copenhagen, Den- intermedia (Fig. 5), but only a minority of the mark) and PAP complex (1:50, Lot No. 030, cell population in the upper portion of the pars DAKO-immunoglobulins). Finally, antigen- intermedia. As indicated by comparison of antibody binding sites were visualized with 3,3’- adjacent sections alternately stained for PP and diaminobenzidine-HZOZ mixture (4). the C-terminal of ACTH, the PP-immunoreactive cells were mostly the same as the ACTH-im- munoreactive cells. In the pars intermedia, a PP-LIKE SUBSTANCE IN PITUITARY 527
greater part of the endocrine cells indicated the 1;§jl1.j=_% .=.-.;= .. i I1.1.=1.j1:-=11;=€:-21:1-(é;?.i. 155- =I=f-1.=.i':=I :-I1.= :'.--I: -:-.-I;.:-__1;;.j_.;;5{Zigzag_=;{.1_-.;;;;;s;.;.r;.;-___ l _)<” J / / ii’-'i1':1:I3iI15.E_3.:;é-;.;:;:_::£:E_.;.é.5._-.;.:.__;-2-_i__.1._;__.-I.-::._5::.___.E._;;.1;;.'=_-,-_:;-_.-;:_-;;_1__-_:1;_:_;_ presence of both PP- and ACTH-like immunore- i ‘=£.= i<' >‘=‘>‘.'-'3i>;%§I;:'f'£r!§;£’1'3$ 1.97;,-1; ,-f§}$:?€:.'a1£;=.*./;;1 .-I-I:-;<'i.?3.>.?::'_i=1r='?£€!-L1;1j-I='r;1;-:-.'='-I:-.1-;.=-1:';==.1.= 1- .~ I’, .,-,.. , .g_.._._.?_.,_4,,;,,.;_{,;,;;,,\y=;,,..,_., ,3 ‘H activity (Figs. 4 and 5). However, ACTH-posi- K I » g 11 1-f:;-i;:_= '” I 53 ='11':'='I'= , / 4~’~'-’<'<'»'=1*fi=12113-*?~1§%<¥x~11:-1r/srfnaiiiiaw"-1>1¢::>;=a1;:*-z;-@;=:=9-,.-.,. ,.._.» ,- f>v->_ _,._.;_.,,.,_, . ....,_.._,>_.v_.,.., ._.._ _ _-51:: <1-.-.1 ';.=,<1<;ae€f.z?-7.’-};, .. .>1<;.r§<=-/.::§=-,.1§:,>;€,::‘;._.;:6-1',;> .1.’ ;;;e-‘-=-'.~ > >14;--,1=»,--;.':..-'.='<¢r>/://tie::sa4::;1éi¢b1§4~I=91.. =/-'.~-'>1is,1. .=£$~I<.-5.1;’:av/.2-'-Ir-<\'1:; 51:.-1.2!;11>;-=::.\1!<-=?ii?;=9'-.1-1:-=-=<-:><-:-1-,=.~1-:';='$55>1¢1»1.i.=1-== 1-=;-=1-'.4:.= .: /.:1-.;-1-=-1.; tive but PP-negative cells were often observed .1--;,=i=;;1;-,1,-;;,¢¢.r-23;-_-1;};->_>%*,:.<<,5"’-I‘¢=v'/'1-£'\'.'/.‘)4-:;='=-!i(i}I)))>7-""J;$4i>?'£§1%{I'>7E"$!I-Li5?>QL';:¢'¢\=}/.'v!.\|.;'{?'Q'-Q-1/,¢I‘I-6%’/df'IE{L§!\Y\';§,5{;)=_I§€'_;;-/.1==1. >;1e_».>;./-=<>.-,,4;- ;,-.~;\=1.,,;;.-g\11>_<.;;-;.-.5::-,>.';_.;:;:;.\§','.g=.:¢=;-:,1,1,=,-.;<<-, r~< __ :. L;13.‘-§-i'>'1--351;};?}1!E1.!1\;;“<:ir>1 2'/~‘§.{t>’!r1:/35%;-11 -’./{ii’t>i~1$>->'-=g-"2;§ <:£;?=‘.1'1~=<:-’>,\~=>-5-r15-mm=,-1-11;,-1.-1,':¢_.a ,.;':>1.r.1_-.1.==;;>;,1,-.;..-,-,-_-_=;,=;;---;:-.;_- and a small number of cells with no reactions for .v.~;_,=--: :1: ,/i.>2=_-’>;i=1,r,,;¢»:>;&>-}=,;;};;»>g1¢fé;{=~=_',;-" 541;.’/,:'_<;.;-;..=.,-'¢_<_.;11~> /__.!g'._:j<'$J>\5‘)"\'2<2§;?/{ff//,1‘-).5 ;, -1;-1/11,;-<-1_,<,<;4,-_:-éia‘3§;;;=;1-i;§:fi;;£-yfl»-:1gy.--.-.2-;.1-ti-:I;;-;-5::-I.;<2, .-.>;;..;.,<_-\.;,;;';1.,-_;*_¢,.;;;=;;,1»;;.3=_-.;..;;;;;=.;>;;;;;-I_',§,15'3=.>3;f.1_=1;-.1.;~j-_.-.'=1_1;->_;=.1_!.':1-1;1.-;.-;.>;:1;<-; i 11<.=.g_>f':;.3;.1.>,,=;:.&1‘;"€,»§¢Z i- , r ,.1.; ;-<.;-,-31>-2..-;i==,1:':1f-;,/-. -'f;;>- jr'.’;f-,\,>;11-2i'\1=i§i!f?i!e§:=;=£es;;<§:§%,-:={.1?;£ ii-1,";':;?J;ii~1s2¢é?2r1:.1=>‘>3P?"fz?1§;»é?€{. -C'3i’=,=;,.-=*'-'\~‘.=’?"1‘ ' = .-r.>i1>".'?/';\’?{?5{I;2;.=;-2;;iii;-iéii-i24};::'::-1,-,,-.~.-,=;>_-_- 1-_..=;>; ,-...;_\-. either antiserum were seen among the stained >'.-._-i.1,-.;1»:.'».1,";.--ai;=;>==_:>2<;r:;ié,;-ye;:5,,-.. ~.;:-{){e’%’-'§n21£3:{=’>~f \ SEr7§=I;?)?%$’f<\F§-'\‘Z{;'-§§%Ef=j:Q@$§I§€fg{!f-fi->>1 <.I_‘ 'E’$f /i1=11§., -= f-’2}$,1;';"'.*;-r'>-.»;i,-:-:1';1122.325;-;;,:sgg;:;;g1i;;=.;-;'.=_:j . §.11.13.5A-__ - _. cells (Figs. 6 and 7). In the pars tuberalis, cells I
_ 1' . ,,_ .- __ _|' 1211 _:-.;=§-.-mp .1 -==:-l,;_-_-_._ ._ . _ -- _?_,_,::_€,.-.. __. . . 11 which reacted more strongly to the anti-ACTH .--‘-===.- /. - - ‘ 11;.=.=~,;;J’ ;E._._;.,i.:‘.11 1:e'1\§=j==,§3=_ --1-,_.1===' 1 .1- - -< 1:-;- _ Q _,;1<,|,._'|__ , ii;-1pA.-:-1.. - .3 , §= 951-” -';-:-;~1.1r- '1 ‘;.=i serum possessed simultaneously PP-like im-
11-;;.<;-5: -. :_. -.;:-~--,;,-<,-- ;;=<,.- - ._.,=_,..,-.;.:=-.;-.;- . - .- . *9 ( t- ii .. munoreactivity, and cells more weakly stained __ ' _.\5 , '1 ..:-1-=1.‘-..;=1=1;".; =:11,_.!.=--1 _*'=#-__" _._i1;=1-11 < ,.-'--.11 .1 .. for ACTH usually did not show immunoreac- /J.‘ ,..‘., _. . .. - _ , . ;‘I». “ 4<“¢ 8 ’ 1‘ z’I J 3‘/<-> tivity for PP (Figs. 8 and 9). 5 Q, v u , °' '\ .. , 1 . 1*" 9.2§ * In the neurohypophysis and the hypothalamus, .'=:=11-1 ;.i§i}>.‘ -. -. __ - 5: 1,. =.._.;._=,-_=,-=. __ ' __ I . .1 } < PP-immunoreactive nerve fibers were found in a "-'::.-1.;,=,. __ ' ,. . >-,<=,1-;=1’’:=,1,;;>;§ _ ?_‘;.'1=,___;; ,. _ ; ,1 '1<=1-;/_.~;.-_:_1;_=._ _ _ __=.v ___j;--:;5j5.=-. _. -1.;-(:11. :;_I. _'5';f-j}_3.1_-',1:___ I1 varicose shape to be mainly distributed from the ;;-.,._.4:;=1-;-:-/=e.;.1,-pr.-9;-.;.-.,-:.g-.;.- r.-1-._,_. .r.>,=_1 1 ;.=_.-;.=11. ..,,'_-‘_ =._.;,,-,3»->!:_' . =.== -.-,. 1. ;:11-21i'=?E-¥1jfI-r.-E2:2‘:i:;;>§¢--1;-5:551, 1 - .@_1'1'=-- .- :-_. 1-.11 -1,: i'.‘.'.\‘.'5:‘>>1\')=-1:‘-?;>}'\'-JJK1}!-1.\'\1E-'::E'.;'./_-‘,~.-,i_i<-> '-1-1;-1;‘-.-=/).\§-.-1:='.1-'--1-1=)1-1'1I:-1-f-.-."-1--‘.-1-1-1-.---.---.-,.-,-r-‘.‘/.\-.-./...-:--H.’-,r: =:=:-‘.1;_=1,-5:;-;:_=f<1-1;.r.<;':,.-:>f=§€1;-.:.=;;-;!;:;¢;-:-;-fr;-55::1->=.-,<;_:;.;;_:_-.;;-_a_a;;§_;:;,1_:;.;-.-,.<_i =15.,,..;1-.1;1,',;5.-_1.- 5 - .,5 5 ;._..1:1..; -.; ; -. hypothalamus through the median eminence to =§.< afjs;;g-5;;-;:-:.g1;E-=5=2"-=1-$5?§:j1-=.-;;=?:-*=1=I-:'-;=.15-:1-:=1. -'.1 1
_;:95;§:,:,. :_;.E-2-3:;5).’,-J,‘/_ ;1i;._;_:;l1;;;E._:_,1_-.1.._; _ =’.f;;_-1_=It15:;?.>Ir,-1;‘; ,_ ,, 2 151,11;551-;1:5I:§;:;:?_:i§i-IE1 :_1'-:5;:§;.5';1',-',’;§j.>_=.';:f1.1;I=':' the infundibular stalk (Fig. 10). Almost no
-1=1-1-<1»; z 11rm;-r.¥;1:2-;-1.::§ 1 .-.-,_. . . . - - .- . , - _ . . . .=-'1 PP-positive fibers were observed in the pars "L9'1'<1e?21 1>1;*»C<-1-I-I-=='=1<':=::-1-.<.==11:-1:44! s?1:- = ; §s=':'<'.=:-r~'£r:>‘-'<=.-r’/(,1¢--_-.=_<@;;-‘:./1;;<;.1=3f;I:_ -i=1;-.1-1-1.=>a,== .15135-: 1 ..11-' 131?:=2.i:-1=':-2-Ei'§>1--1-fir . _ _ _ ,€=,'=;.i5.'5,=:1 I =. / .1 nervosa. E1; ;I.13a-1I.i=j-1.-'-:‘1 In the pancreas, PP-positive cells were distrib-
.. .. . uted in both islets and the exocrine tissues but ,35;,,.¢._;{_@,,=;_:5_.§,§j;;,»;¢:;;;".§1,;_.,_=_=;.= r.~.1-::~:-. i1=:'.-.<==;i=.E=.:£=-:r-.'=-=.=.-. ACTH-immunoreactivity could not be detected . :-:=.=&5i§!¢f;'%?%i=*.§,%,, L ‘=1-3£5:-=1'=E'I}If=i-ff;1~ ‘“‘ :-{..':1_.: -~;,-,-.;._:_: ;y:;.:=._1,1,.;.1g,;;.:,-ii? ,7 at all. 5; ;_._\_-.:_E-_-1._1_.\_;-;!A.?\,,-,. A -.'-:-.-,-)31.=-1_:G!}.1.;.;--I;;.-_.,-.-.f;A>1,'_,:.' / '/->5,-;.-;,-.1><>.-=1=;5.=_.;. ,:_=:-;/2;.-J-.= -< 4 ‘:15 5'52:-1.25:5;-1.=='1,{/.1-':‘<-155,?-I31.‘ "' In the three fetuses (5-6 cm in crown-rump . i'-:I.';'P-':-'}.'-:;;_5_= E:\.:§E:. J :-';=Iii.1.1:'?-I-';1w"='=1 -v length) examined in this study, the connection
l .;: ;_E';-'='-1-'1'; :=.1-.=_';.:_.=_I1§1j1-; I ;I E 5 .1 5. ' - between Rathke’s pouch and the oral cavity had / 1.:->,1.:,1..1_§5-1-:.5.'=§ ii 5'1‘ 1'1’1'1 1 1- 3'3 1' -' 1 _' - ii-‘=5:-;'-,‘.==1==zi55.1-1_-j-=_-Q-I 1. -:.=..1._.-_-_:.= = =_ .-. ;;._. - _ already disappeared and the residual lumen was 9 :__:;;.;.-,.,;._-.-;;,.,;._;.1.; 1 '111'5-.'4.1.1_:-_:__:_::-_.-, -_ . . _ . . . formed in the pituitary; the fissura prima and § = -11.-. - I :j-_=.1_:_:: - .1 1 the arrangement of Purkinje cells in a layer were observed in the cerebellum. Many pituitary Fig. 1 Numerous PP-immunoreactive cells in the lower portion of the pars intermedia. The im- endocrine cells reacted to the anti-porcine ACTH munoreaction is not observed in the pars nervosa serum (Fig. ll) but PP-like immunoreactivity (PN) or distalis (PD). RL: residual lumen could not be detected (Fig. 12).
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Fig. 2 PP-immunoreactive cells showing a higher population in the lower portion than in the upper portion of the pars intermedia (PI) 528 S. FUJII, S. BABA and T. FUJITA
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In the staining control tests, application of ACTH serum was blocked with CLIP, but not NRS or PBS instead of anti-BPP serum as the with BPP, PPP, or 06-MSH. first antibody abolished the immuno11eaction. Addition of 100 jug/ml synthetic CLIP or 0!.- MSH to anti-BPP se11um (as an immunoabsorp- DISCUSSION tion test) had no effect. on the immunostaining Pancreatic polypeptide was isolated by Kimmel of PP, but absorption with BPP or PPP com- er al. (10) during the purification of chicken pletely abolished the immunostaining. Anti- insulin as a straight chain peptide composed of I PP-LIKE SUBSTANCE IN PITUITARY 529 I
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36 amino acids. PP has been extracted succes- not only excess pure BPP or PPP but also sively from porcine, ovine, canine, and human synthetic 03-MSH or synthetic bovine CLIP in pancreas, and its amino acid sequences have working dilutions. The results thus obtained been determined (20). Previous reports de- indicated the specificity of the antiserum. The scribed the distribution of PP in the GEP findings in the present study lead us to presume endocrine cells and the central and peripheral that the PP-like substance occurs with ACTH or nervous systems. The present study revealed CLIP in the same cell. It is possible that CLIP that numerous PP-immunoreactive cells are and 06-MSH are derived from ACTH by selective present in an endocrine organ, the pituitary cleavages (2, 23, 25) and coexist in the same cell. gland. If m-MSH immunoreactive cells are indeed It was reported that the pars intermedia con- predominant and ACTH/[i-LPH positive cells sists of two distinct cell types stained by anti-<7» do cluster in the pars intermedia as reported by MSH serum and anti-ACTH/Ii-LPH serum, Halmi er al. (7), our anti-ACTH serum, which respectively, and that the first type was a typical recognized the C-terminus, probably detected predominant. A cell and the latter a B cell mainly CLIP in the pars intermedia. Further resembling the ACTH cell of the anterior lobe detailed examination of adjacent sections en- (7). On the other hand, the greater portion of abled us to distinguish in the pars intermedia the pars intermedia cells were stained by the PP-positive but ACTH- or CLIP-negative cells anti-ACTH serum which reacts fully with the and cells non-immunoreactive to both anti-PP C-terminal fragment of ACTH but not with and anti-ACTH sera. In contrast to the pre- other related peptides such as at-MSH, [3-LPH, vious report by Halmi er al., our observation or fi-endorphin (27). Prior to the present showed the possible presence of three endocrine immunohistochemical studies, an absorption cell types in the pars intermedia. test with anti-BPP serum was performed using In the pars tuberalis, ACTH-immunoreactive 530 S. FUJII, S. BABA and T. FUJITA
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. ‘.2 Fig. 10 PP-immunoreactive varicose nerve fibers in the infundibular stalk (IS). PT: pars tuberalis PP-LIKE SUBSTANCE IN PITUITARY 531
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Fig. 12 No PP-immunoreactive cells are detected yet in the pituitary shown in Fig. 1].. cells showed two distinct grades in immunostain- pars tuberalis. In the pars distalis, many ing, and a few strongly reacting cells usually endocrine cells were stained by the anti-C-termi- coincided with the PP-immunoreactive cells nal fragment of ACTH serum but not by the and many weakly stained cells were not demon- anti-BPP serum. These results are summarized strated with the anti-PP serum, as seen in adjacent in Table 1. sections. There may be two cell types having In the rat antropyloric mucosa, two cell types ACTH- or CLIP-like immunoreactivity in the have been distinguished: G cells containing 532 S. FUJII, S. BABA and T. FUJITA
Table l Population of PP- and ACTH-Iiimzunoreactive Cells in the Canine Adenohypop/zysfs Part of pituitary Pars distalis Pars tuberalis Pars intermedia Cell stained for Upper Lower “PP -
ACTH +t "H" "i" "it
t+: numerous, +: scattered, ~—~: almost non-existent gastrin and G, cells possessing both gastrin- and for mammalian brains (8, 9, 21, 22) or anti-BPP ACTH-like immunoreactivity (14). Larsson serum for invertebrates (29), and therefore, the also reported that gastrin immunoreactivity neuronal PP might be different from the authentic could be detected before the ACTH-like sub- PP originally isolated from the pancreas. The stance became obvious in the rat antral gastrin present study was done with an anti-BPP serum cells during development (13). Our present which crossreacted more with canine PP than study demonstrated that pituitary cells in fetuses an anti-APP serum, and the results suggest that which were rather advanced in development did a peptide structurally related to authentic PP not store PP-like substance, though they already exists in the nervous system of mammals. The had ACTH- or CLIP-like immunoreactivity. physiological role of PP or PP-like substances This finding suggests that the production of PP- in the nervous system has been unsolved until like substance begins in the late phase of now, but. it is interesting that PP-immunoreactive development. nerve fibers tend to terminate in the infun- We also found obvious PP-immunoreactive dibulum where the primary capillary nets of the nerve fibers in the infundibular stalk, median pituitary portal system are formed. eminence, and hypothalamus. Evidence has been accumulated concerning the occurrence of Received for pttbifcarioii 5 Jufy 1982; and in revised form 24 August 1982 PP-like immunoreactivity in the central and peripheral nervous systems in vertebrates (chicken, mouse, rat, and cat) and invertebrates (earthworm and cockroach). In the avian and REFERENCES mammalian central nervous system, PP-immuno- 1. ALUMETS J., HAxANsoN R., SUNDLER F. and reactive neurons exhibited a widespread distri- CHANG K.-J. (1978) Leu-enkephalin-like material bution; nerve cell bodies stained for PP were in nerves and enterochromaffin cells in the gut. seen in several cortical areas (pyriform cortex, en- An immunohistochemical study. Hist0chem:'srr_1> S6, 187-196 torhinal cortex, and amygdala) and in subcortical 2. CRINE P., GossARD F., SEn;>An N. G., BLAN- areas (septum, nucleus accumbens, neostriatum, CHETTE L., LIS M. and Ci-1Ri=:rIEN M. (1979) Con- olfactory tubercule, etc.), immunoreactive nerve comitant synthesis of fl-endorphin and ot- fibers were extremely numerous in subcortical melanotropin from two forms of pro-opiomela- areas (nucleus accumbens, olfactory tubercule, nocortin in the rat pars intermedia. Proc. Natl. interstitial nucleus of the stria terminalis, etc.) Acad. Sci. USA 76, 5085-5089 and moderate in other areas (meso- and neocor- 3. FUJITA T., YU1 R., IWANAGA T., NISHlITSUTSUJl— tex, etc.). In the pars nervosa of the pituitary, Uwo J., Enoo Y. and YANAIHARA N. (1981) PP-immunoreactive nerve fibers were also Evolutionary aspects of “Brain-Gut Peptides”: numerous. In the brain of the cockroach an immunohistochemical study. Peptides 2, Suppl. 2, 123-131 median and lateral neurosecretory cells were 4. GRAHAM R. C. and KARNOVSKY M. J. (1966) The immunoreactive for PP, and their axons, after early stages of absorption of injected horseradish issuing many branches, descended into the peroxidase in the proximal tubules of mouse kid- corpus cardiacum, which is known as a neu- ney: ultrastructural cytochemistry by a new rohemal organ (3). This finding seems to sug- technique. J. Histochem. Cyrochem. 14, 291-302 gest a biological function for the PP-like 5. GRUBE D., Voior K. H. and WEBER E. (1978) substance in the nervous system. Most studies, Pancreatic glucagon cells contain endorphin-like however, have used anti-avian PP (APP) serum immunoreactivity. Hisrocltezmsrry 59, 75-79 PP-LIKE SUBSTANCE IN PITUITARY 533
6. HA1-(ANSON R., ALUMETS J. and SUNDLER F. 19. LARSSON L.-I. and REHFELD J. F. (1981) Pituitary (1978) GH-like immunoreactivity in gastrinoma gastrins occur in corticotrophs and melanotrophs. and in endocrine cells of gut and pancreas. Science 213, 768-770 Scand. J. Gastroenterol. 13, Suppl. 49, 75 20. LIN T. M. (1980) Pancreatic polypeptide: Isola- 7 HALMI N. S., PETERSON M. E., CoLURso G. J., tion, chemistry, and biological function. In LIOTTA A. S. and KRIEGER D. T. (1981) Pituitary Gastrointestinal Hormones (ed. GLASS G. B. J.) intermediate lobe in dog: Two cell types and Raven Press, New York, pp. 275-306 high bioactive adrenocorticotropin content. 2 LQREN I., ALUMETS J., I-IAKANSON R. and Science 211, 72-74 SUNDLER F. (1979) Immunoreactive pancreatic 8 Hox1=ELT T., LUNDBERG J.M., TERENIUS L., polypeptide (PP) occurs in the central and pe- JANcso G. and KIMMEL J. (1981) Avian pancreatic ripheral nervous system: preliminary immuno- polypeptide (APP) immunoreactive neurons in cytochernical observations. Cell Tissue Res. the spinal cord and spinal trigeminal nucleus. 200, 179-186 Peptides 2, 81-87 22 LUNDBERG J.M., I-IGKFELT T., ANGGARD A., 9 HUNT S. P., Eruson P. C., GILBERT R., GOLDSTEIN KIMMEL J., GOLDSTEIN M. and MARKEY K. (1980) M. and KIMMEL J. R. (1981) Presence of avian Coexistence of an avian pancreatic polypeptide pancreatic polypeptide-like immunoreactivity in (APP) immunoreactive substance and catechol- catecholamine and methionine-enkephalin-con- amines in some peripheral and central neurons. taining neurones within the central nervous sys- Acra Plzysiol. Scand. 110, 107-109 tem. Neurosci. Left. 21, 125-130 23 MAINS R. E., EIPPER B. A. and LING N. (1977) 10 KIMMEL J. R., HAYDEN L. J. and POLLOCK H. G. Common precursor to corticotropins and en- (1975) Isolation and characterization of a new dorphins. Proc. Natl. Acad. Sci. USA 74, 3014- pancreatic polypeptide hormone. J. Biol. Client. 3018 250, 9369-9376 24 NAKANE P. K. and PIERCE G. B. (1966) Enzyme- 11 LARSSON L.-I. (1977) Corticotropin-like peptides labeled antibodies; preparation and application in central nerves and in endocrine cells of gut for the localization of antigens. J. Hisroclienr. and pancreas. Lancet II, 1321-1323 Cytoclzem. 14, 929-931 12 LARSSON L.-I. (1978) ACTH-like peptides in 25 Scorr A. P., LOWRY P. J., RATCLIFFE J. G., REEs gastrin and pancreatic polypeptide cells. Scand. L. H. and LANDON J. (1974) Corticotrophin-like J. Gastroenterol. 13, Suppl. 49, 109 peptides in the rat pituitary. J. Endocrinol. 61, 13 LARSSON L.-I. (1978) ACTH-like immunoreactiv- 355-367 ity in the gastrin cell. Independent changes in 26 STERNBERGER L. A. (1974) Intmunocytoclzenzistry, gastrin and ACTH-like immunoreactivity during Prentice Hall, Englewood Cliffs, NJ ontogeny. Histoclremistry 56, 245-251 27 SUEOKA S., MATSUKURA S., Yosnnvn H., YOKOTA 14 LARSSON L.-I. (1978) Gastrin and ACTH-like M. and FUJITA T. (1980) Comparison of the in- immunoreactivity occurs in two ultrastructurally hibitory effects of glucocorticoids on ACTH and distinct cell types of rat antropyloric mucosa. )6’-endorphin-like immunoreactivities secretion Evidence for a non-parallel processing of the by cultured anterior and neuro-intermediate peptides during feeding and fasting. Histocl:enz- pituitary cells of rat. In The 611: International isrry 58, 33-48 Congress of Endocrinology, p. 527 15 LARSSON L.-I. (1978) Distribution of ACTH-like 28 SUNDLER P., ALUMETS J. and HAKANSON R. immunoreactivity in rat brain and gastroin- (1979) Growth hormone-like immunoreactivity testinal tract. Histochemisrry 55, 225-233 in gastrin cells and gastrinomas. H:'stochenn'stry 16 LARSSON L.-I. (1979) Radioirnmunochemical 59, 343-346 characterization of ACTH-like peptides in the 29 SUNDLER P., HAKANsoN R., ALUMETS J. and antropyloric mucosa. Life Sci 25, 1565-1570 WALLES B. (1977) Neuronal localization of pan- 17 LARSSON L.-I. (1980) Immunocytochemical char- creatic polypeptide (PP) and vasoactive intestinal acterization of ACTH-like immunoreactivity in peptide (VIP) immunoreactivity in the earthworm cerebral nerves and in endocrine cells of the (Lumbricus Terrestris). Brain Res. Bull. 2, 61-65 pituitary and gastrointestinal tract by using 30 VANDERHAEGHEN J. J. (1981) Hormones and Cell region-specific antisera. J. HlSl0Cll€l?I. Cyto- Regulation Vol. 5, Elsevier/North-Holland, Am- clrem. 28, 133-141 sterdam, pp. 149-168 18 LARSSON L.-I. (1981) Adrenocorticotropin-like 31 WATKINS W. B., BRUNI J. F. and YEN S. S. C. and or-melanotropin-like peptides in a subpopu1a- (1980) B-Endorphin and somatostatin in the tion of human gastrin cell granules: Bioassay, pancreatic D-cell colocalization by immunocyto- immunoassay, and immunocytochemical evi- chemistry. J. Histoclzenz. Cytochem. 28, 1170- dence. Proc. Natl. Acad. Sci. USA 78, 2990- 1174 2994