Cell Tiss. Res. 162, M I-550 (197~) C by Springer.Verlag 197~

Ultrastructure of the Adrenal Medulla of Normal and Insulin~Trea Led Hamsters

I . Benedeczky &nd P. Somogyi

Tst Tn8titntc of Pathology, Semmelweis Uedica.l School, Budapest, Hungary

Received June 9, 1 97~

Sr .... t.murlJ· Frne structural cl"laract.eriniCII of the chromaffin cella both in normal and in. all lin·administered hamster adl'ellnl gland were studied.

Exocytosis OCC\lrS in.5 per cent of nonsUmnlnted cella especiAJJy on the apical eell.uJ"f,~. At the same LIme the occurrence oln great number of closely nttMhed secretory granula was eonaprcuoulon the lateml plurnJl. membrane in ~he untreated haUllter n.drena l medulla.

Following inlulin trcmt.mcnt (10 IU/loo g/body weight), ChOtBCteOltlC WiUI the develop-men~ of large intercellular vacuoles between the inlersl pll~S!lla membrane, in which electron-denseaeeretory matcrill l WIIS frequently present. On the bash; of thia observation it ia suggested that in the ClIItI of IIISl1lin"induced hormone secretion, exooytosia preferentially occun Oll the IQt.eral plasma lIlemhmne, and may play all import.ont role in th" discharge of aeeretory materials f rorn the cells.

Key ~: Adrenal m~llLlla - Golden hamater - Illsuli ,\ treatment - Induced eJl:O· eytosis - Hormone discharge.

Introductio n

The tel"m exocytosis was introduced by De Duve in 1963 and since then a number of experiments hayegiven'evidence for the occurrence of thil:; phenomenon m endocrine glands as well D.S in several other tissues (sce Smith and W inkler, 1972 ; BenooeC'Zkyand Smith , 1972; "R6hlich et al., 197 1; Masw' et al., 1972). Exocytosis hBEI been extensively studied in the adrenal medulla by means of I'arious methods, t hose of biochcluiloMy (Banks, 1966 ; Reli c, lOBB; Blaschko, et (u., 1967 ; Schneider et al., 1067) pharmacology, (Douglas o.nd Poisner, 1966 ; K irshner et al., 1967 ; Viveros et al., 1969) and morphology (Coupland , 1965 ; Diner, 1967 ; Benedeczky and Smith, 1972). Today it is generally accepted that the process IS of basic significance m the se

54' L Benedeczky and P . Somogyi

Since in the litera.t.ure t.here were no convent.ional electron microscopic observa-t ions on stimulated homster adrenal medulla available, the aim of this worl, was to study the fine structumi changes of in sulin-treated chromaffin cells, particularly in respect of the development of exocytotic profiles.

~Jatc l'i a ls and iHethods Adult golden h !l. llllIl.el'll (100- 120 I) of both &eX. were used: 10 animal, wt!~ \lsed ,18

controls, /l 1,d otuer groups of l a an imal. were 84(lrificed lit 2, 3, 4, and 6 ht afte r insulin trellt. ment. The an imllla were not fed 101' 24 hr before injection of ,n,u!;n, but wator "'1II1111owed Ad lIb. The inaulin "'la injeeted Lp. in. dote of 10 I U/ IOO g{body weight.. To pr.vent oonvu lsionll, 1- 2 ml ~,. dextrose soln t ion IIere give •• i.p. 90min l'Iftcr ndmini.trntion of in llllin . The II.nimn.J& were ki lled 2, 3, 4, and 6 hr following In.ulin treatment.

Two lI1othods were used to fix the adrenal medullary ~lane ; immersion of the gland in fiutive solution 01' perfu.iOll in si tu with fjxa~ive IIOlution followed by iw.mllraion . Fot fixation by immersion, the !lnimA]' "ere deeapltll.ted, nnd the Adrenal 810.nd. removed Immodiately.

Before fi:t>o. tklll by perfllllon, the o.n imo.ls were anettheti?.ed with ether. Perfu~ ion fluid \Vu introduced n needle into th~ left ventrleie of the hea rt 'llId \YU' allowed to eaco.pe through a cut in the right ~\uriele. '£lle animal. wel'~ perfused wi th SIIline (NaG!, 0.9% wt/,'ol) lOt' 2 min to remO\'e most of I·he blood, and then With llnt ive IOlution (2. ~ "" glutllrllldehydo and 4% formaldehyde) for 30 min . After perfusion, the gland. were immerw(\ overnight in the same solution.

The material fixed by perlnsion !'Ind/or immenion wu washed for 10 mill in 0.1 1>1 phosphate buffer. (pH 7,4) cOllt.lining i;UCrose (7.5 "" wt/"ol).

Aft.er WIshing, the Ilidehyde.fixed tjll$ue wu cut into cubed bloek:s immened in 06mium tetroxide (2% wt/voll, and dissolved in Palacle '. (1952) "eronal buffer for 2 hours ut 4" C. The blocks were then dehydrated in !'Ilcohol (30%, 70% , 95%, 100%) and embedded in Durcupau ACM. Ultrathin seetions were cut with II n LKB Illtrotome IInd . tained with a. saturated aqueon • .solution of urllnyl nce t!'lte for 30 min followed by Reynold'lI ( 1963) lead ci trote for 2 min. Sections "'ere exnm incd in Il. JE1I.I 7 A electron microscope.

Resul ts

1. UUrastruclun 0/ NOI'fIW Adrll!Ml Mt.dulla 0/ Goldll!n Hammy A gret\ t propol"~ioll of th e clll'Ol"UaJfin cells i8 located around t he 8inusoid!

(Fig. I). In t he lumen of t he s inusoid , platelets are oft.en vi~lble . Tho cytoplasm of the endothe.lial cells is flattened and t he basnl lamina. fu sed with the basement melubrane of t.h o chromaffi n cells Distri bution of secretory granules in the ce1l8 IS not uniform. The gra n ules arc gcneNl.lly concent rated a l the apical poles of t.he cells ; thus their /lum ber is low around t he nucleus and in the area of the Golgi ilopparatus MOflt of t he granules are spherical and their membranes easy to detect around the highly electt·on·denso cen~el" . There are a great number of" closely at· tach ed " secretory granules a long t.ll e plasUla membranes, on the apical poles, and on t he lateral sides of the cells. I n 0. great proportion of chromalfin cells exocytosis in not observable (Fig I) . Among 600 nonstimulated conlrol cells only 29 cells (appro.l. 6% of the examincd cells) showed exocytotic profiles 011 the cell memo branes. In these cells where exocytosis occurs (Fig. 2), there arc often fou r to five exocytotic profiles on a relatively short. sect ion of the apical plasma membrane. A fe w c>:ocytotlc profiles occur also on t he lateral plasma membrane of the cell .

2. 'J'ke Ultrastnl(;tuf"e o/ the Adrenal .il1edtdla Following Administratwn o/ insulm Three hours after insulin t reatment, ultrastructul'al changes related to stlmu·

lated secretion were v isible in a grCt\.t proport ion of medulla ry cells. Exocytosis

FIg. I. AdrenornedullMy eella of normAl hAm,ter, located around the aonu~d (8). Sec~tory gr. nules ('I) .... lIenen.llr ooneentn.t«I It apIcal poles of eel" (A). There are. grut number of .. clowly ItLAdoed" secretory graoulee along the apical .. nd lateral pI ... nla rr.embral"lCll ("rrowl. E"oeytCMi.I is nOt detectable on these cella. Note nllc!en, (N) And mitochondnn. (M).

X 18000

I. Benedeet.ky and p , Somogyi

YII.I. Chrolllf.ffin ce111 in lhe no.timulnted c:ont.JoI odrenal .\o.\c • tinlQ()t(i ($). Exo-cytou. (8) e1~rly and .. ,.ily detHtAb1e. both on the .piti"ll (A ) ",net latenl cell roembrtl"ON

(L). x 18000

Ultrll5tructure of In,u l in·S~imullltW Adrenal MednHn '" Wi\S frequent in t hese stimu lated ch romaffin cells, particul!lrly 011 the lateI'll.! plasma. membrane (Fig. 3). T he omega shaped invaginations of the exocyrotic profil es werc often extremely d ilat ed , given rise to v!\clloles. However, the electl·on· dense material of the extruded grallules was usua.lly detectable in t he lumen of the vacuoles. At the same time it was surprising that a. conspicuous increase of exocytosis could not be obsen:ed a t the a-picaJ poles of stimulated chromaffin cells (Fig. 4). [n some instances exocytosis was found a lso on t,he lat eI'll I surface of cells containing llOrepincphrine.

Six hours after insulin trea tment t he vacuoles fOI'med after exocytosis were still llUmCI'OUS and t heir lumina were constantly dilated , but, the elech·on·dense material of secretory granules was no longer obscrvable (Fig. 5). The number of secretory granules decreased in the cytoplasm whercas t he amOtlnt of the rough . surfaced ~lldoplilsm i c reticuhl111 increasEd.

DIscussion Alt hough a number of aut hors (Diner 1967 ; Grynszpan.Winograd, 1971 ;

Benedeczky a nd Smith, 1972) provided morphologic examples of e:tocytosis in t he hamster adrenal medulla, the stimulated gland-as far as; we know-has not been examined so far by conventional electron microscopy . The studies of Yates (1964) \I'ere concerned with Lhe effect of insulin administra.tion on the Syrian hamster but at that. time exocytosil> had not yet been described in endocrine tissues and the author suggested that tho release of cat echolami nes waf; not dependent upon the complete disintegration of secretory granules. I n view of r ecent data on exocytosis the question a.rises whet·her or not t.hel'e is a. mO"phologically detecta blc correlat.ion between stimulated secretion and exocyto~is in ~he hamster adl"Cll(l1 medulla! Before answering t he above question it is considercd necessar y to analyze briefly the fine str uct ural features of the nonstimulated adrena l lIleduBa.. In ~hi s regard the folloll'ing observations were made:

1_ Frequency of cxocyt.osis was low in the nonstimulated (normal) adrenal medulla l'l' cells (approx . 5%).

2. Numerous exoc}"tosis ma.y occur at the same t lme on t he surface of a given chromaffin cell.

3. A great num ber of secretory granules are in close morphologic contact wit h t he plasma membranes.

The incident,al occurrence of exocytosis in the nonstimulat-ed hamster adrena l medulla is not surprising. According to physiologiea-! da.ta the so·called "resting secretion " of t.he adrenal medul la is very small (0 .1 p.g/min body weight kg: Eul~r, 1956; H oltz et al., 1952 ; Ma lmajec, 1952). It is a lso possible th at exocyt.osis, as a.-single extrusion process, is able to perfOrlll this function satisfactorily, The fact, however, that 11.1. the same time, a. lal'ge number of secretory grn-n\l les is attached t-o the plasma membranes suggests that· hormone discharge can take place also in other ways.

According to Smit h and Winkler (1972) t here may be "a high frequeucy of random collisions bet,ween the vesicles and the inner side of t.he plasma membrane as a result of Brownian motion."

)) ('oH. TIoo-. 11 ...

Fig. 3. E~oc.l' tOOlft (If) WAS f re4"e,,~ '" .. g~M p,oJl

Ult l·"Al rue-llIl"C of .l nAlIlin Sti lllll l~ t &d Adl·enul )ledll tl~

Ilig 4. Chrom(lffin cells, 3 hours "fler Insulin (10 IU/loo g/body "'c'ihtJ ~dmin ls~rllt jOll. 111 U group of eel I. o

... , r , Benmenky and 1'. 8olJlogyi

."g. S. CllI'omuff in ceJh~, 6 h01l11l alter lIaulin t.l'eau nen1.. ExocytoUc VACUOIe$ (E I') " 'ere titill Ilumerou.l bilL e1ectl'OlI·dell.5e 'nAter;:! ] of ace~l.Ory g~nnle. '''" 110 longer obtervable. Note

ttb undant:e of rough'III,inced enrioplllSmie reticul um (rer). X 18000

Ultrastructure of Insulin·Stimulated Adrena.l Medultn '" I n consequence of t.he coll is.ion t.he membrane of the granu les may fuse with

t he plasm a. membrane, and the Iusion , gives an opportuni ty fOl" t.he release of secretory material. If the fusion is short (1 ms or less} the release of the low moleo· ular weight substan ces takcs place preferentially and t he secretory granules become relatively rich in protei n (e,g .,dopamine f3, hydl·oxy lase : Smith and Winkler, 1972). All of this suggest-s tllat exocytosis is not the exclusive way of hormone release. In the nonst imulated hamster adrenal medulla therefore both cxocytosis and t he close morphologic contact of secretory gra.nules to t.he cell membranes (tight junction) may represent a fo.·m of hormone secretion.

I n ordel' to study ", hethe.· the ultra.structurnl organizntion of act ively secreting cells changes in the stimulat.ed gland, golden h amsters were subjected to insulin t reatment. Since the eatechol ll,mine.mobilizing effect of insulin is welllnJOwn, an increase ill t he frequency of exocytosis 'I'M expected to occur fi rst of all on t he apical surfaces of chrollla.ffill cells. Cont.rary to th is, however, an increased inci· d ence of exocytosis was lIot observed on th e apical cdl membrane, but. on th e lateral one. It was conspicuous that. at. the s ite of exocytosis, large "acuoles also developed . On the bas is of these the questioll ari£es as to why the exocytotic profi les prefel"entially d evelop on th e In.toral plasma. membranes. An answer can be suggested jf we a.ssume that during an increased and long lasting secret ion, (i.e. , induced by insulin ) th e apical pole of chromaffin cells becomes unable t.o perform the discharge of secretory materiB!. r n consequ ence, i t i ~ obvious that t be large surface of la teral plasma membranes furn ishes an excellent opportunity for hormone IiberaLion and so they become the nlain poillt fOI" t he events of exocytosis. Oil the basis of morphologic observations it is rat her diffi cult t o explain th e signif. icance of large exocytot ic vacuoles on the lateral cell ~ lIrfa.ce under intensive secretion in the hamster adrenal medulla.

Our preliminary mOI·phometric measurements (Belledec7.1ty et al. , 1973) showed that th e concell~ratioll of secretory granules in ~he cytoplasm decreased . T his indica.tes that t he insulin·induced hormone mobilization also causes a.n absolute d ecrease in t he number of secretion granules. I n other words, th eir disappearance occurs by means of a complete d isintegration process, that is, by exocytosis.

References Abrahams. S. J ., Holt.l;PlIln. E.: Secrction nnd endocytosis in insulin.stimulated nIt Ildreno.l

meduUa cells. J. CeU BioI. ~&, 540-558 (t973) Banks. P. : The release of adenosine triphosphate C/l.t:.a.boI ites during the eecretion of catechol.

amine. by bovine adl'flOAI medulla. Biochem. J. 101, ~36-541 (1966) Benede~ky, I ., (Alk6s, A.; Ultrns tfucturlJ.1 chllngcs of the pll\.llma membrnne in the norma l

and insulin stimulated hnrnster'e Wrena.1 med\l ll a. . Conf. of Hung. Elec~r. Mieroscop. Soc. Bo.la.tonfiired VIIl. 78-79 (1913)

.BenedeC"l:ky. L, Smith, A. D. : U1 tl1l ltructural stud ies on the adreIl ~ 1 medulla of golden ha mater: origin Gnd fate of secretory granules. Z. Zel1forsch. 124,367- 380 (1972)

BlilSChlto, H., Coll'lline, R. S., Schneider, Y. H ., Silver. M., Smith, A. D.: Secretion oi 11 chromaffin granule protein, chroO'logranin. from the "drenal glAnd nfter spl:mchnic ltimu· latlOl\. Ne.ture (Lond.) 2Ht, ~-M ( 1967)

Couplo.nd, R. E.: Electron microscopio obscrv"tion& on the Iltn, ctnl'e of the r"~ adrenal medullA. L The 1I1traatrnctllre and o.·ganilUltion or chromaffin cells in the normal ad rennJ rucdull ... J. AMt. (Lol\d.) 99, 231_2M (1960)

10 Cell. :rl" . Res.

550 L Benedeczky a.nd P. :-':()',n r\i'T" ,

De Du ve, C.: Endocytosis, In: Lysosomes. Foundn,tion Symposium,) (De Renck, A, V. S, ,-IDd Ci1merO)), eds,) 'London: Churchill 1963

Diner, 0.: L' des granules de [;.t mednllo-surrennle chez le hn,mster. C. R. Acad. Sci. (Paris) 26i>, (1967)

Douglas, \V. W., Poisner, A. ~1.: On the rdittlon between ATP l1.drenn,] chl'omaffin cells: extnlsion of ATP (unhydwlised) J, Physiol. (Lond.) 183, 249-25G (196G)

Elller, U, S. von: Noradrena,line. SpringfieJd (111.): Thomas, C. C, 19/56

and secretion in the

Gladst.one, G. P., V[m Heynillger, W. E.: SUl,phylococcal leucidins. Brit. J. expo Path. 3S, 123-1.37 (1\:)67)

Gl'ynszJ)(1,n-Winogl'lld, 0.: i\1ol'phologic(l,l t1.spects of exooytosis in the a.dl'eon.l mednlh. PhiL Tm .. ns. B 2Gl, 291-292 (19'71) Helle, K.: Some chemical Hnd

ftclrenal chJ'omaffi n griLli n Ics, Holt%, P., Engelhal'dt, A.,

del' Nebennieremjlaiks

nrl"'l"""l-,,,, of the solubJe prot-ein fraction of bovine, 2,298-310 (19CiG)

Schllmann, H. J.: Del' Adrennhn- und Arterenolgeh!l!t

~bgege bencn Inkretes. Nan nyn· Schm iede bergs (1952)

lInd elekt,l'ischer Spln,nchniktlsl'eiwng expo P,"Lth. Pharmilcol 215, 58-67

Kirshner, N" Sage, H .. J., Smith, VV, J" Kirshner, A. G,; Mechanism of secreLioll from tbe adrcnnl JUedul!n. 2. ReJease of cn,t,echoillmines I.md stDmge vesicle protein in response to chcmica,] scimnhiion. Molee, Phannncol 3, 254-265 (1967)

rvrllhnejac, J., Chn.rdon, G" Gross, A., Ncverre, G.: Action du C.733i sur les secretions sym· pathomimetiques de 111 ghtnde surrena.16. Arch. intern, 90,429-435 (1952)

iUnsur, S. J., Holtzm,m, K, \Vf!.lter, R: Honnone·stilli\llated in the tOll.d urina.ry bladder. J. CeJl BioI. :)g, 211-219 (1972)

Norma.n, T. C.: The neUl"osecretory system of the ildlllt Calliphora e:J·yllvroceplwJa. 1. The fine structure of the cu.rdiacuH1 wit.h some obsen'!\.tions on adjacent organs. Z. Zell-fm'sch. (j'j, 461-501

Pnln.de, G. E.: A sendy of n:G\.tion fol' electron microscopy. J. expo IvIecl. V3, 285-299 (1952) E. S.: The use of ICl1d citl'nte at pH as an stain in electron

microscopy. J. Cell BioI. 17, 20-212 (1963) R.oh!icb, P., Anderson, P., Uvnes, B.: Electron observ,\,tion on COJrnTJ,ound 18/80

indu(;ed degrannlation in ti'l-t mast ce!L Evidence for seqnentiul exocyt.osis. J. BioL 51, 4G5-483 (1971)

Schneider, F. H., Smith, A. D., \Ninklel', H.: Secretion from the ,tdrennl medulla; biochemical evidence for Brit. J. Phi\.rmacol. 31, 94-104 (19(7)

Smith, A, D.; and Secretion of Hormones. The Scientific Basis of lVIedicine Annual Reviews 74-102

Smith, A. Winkler, : Fundument",] mechanism in tbe relMse of c'Jtecholn.mines. In: CIl,techolnmines (Bll1schko, H. and rvIllScholl, K, cos.), Handbook of Experimenta,[ PhMJ1lit-

cology, vol. 33, p. 538-617. York: Springer 19'72 Smith, D., Smith, D. S., 'Vinkler, H., : Exocytosis in the adrenal medulla. de-

monstrated Science 79-82 (1973) Viveros, O. H., L., R J., Kir.shner, N.: Qw:mtal secretion from tldrennl

medulla: a,ll 01' none release of stol'l1,ge vesicle content, Science 16;:), 91J-913 (1969) Y o,tes, R. D.: Fine structun:t-l "Iterations of adreno-medullary cells of the syrian hamster

following intrn.peritone"l injections of insulin. Tex. Biol. Med. 22,756-763 (1964)

ultrastruc