Histol Histopathol (1998) 13: 209-220

001: 10.14670/HH-13.209

http://www.hh.um.es

Histology and Histopathology

From Cell Biology to Tissue Engineering

Invited Review

Paracrine control of steroid hormone secretion by chromaffin cells in the adrenal gland of lower vertebrates G. Mazzocchi, G. Gottardo and G.G. Nussdorfer Department of Anatomy, University of Padua, Padua, Italy

Summary. The adrenal glands of lower vertebrates display a notable intermingling between steroidogenic and chromaffin ti ssues, which increases from Pisces to Al'es. As in mammals, adrenal chromaffin cells contain and release, in addition to catecholamines, serotonin and several peptides, which may affect the secretory acti vity of steroidogenic cells in a paracrine manner. Stimulatory molec ul es in c lud e se roto nin , arg inin e-va so tocin. tachykinin s, vasoac ti ve intes tinal peptide , pituita ry adenylate cyclase-acti vating peptide and calcitonin gene­related peptide : inhibitory molec ul es are dopamine, somatotropic hormone- release inhibiting hormone and ga lanin . Epin ephrine and no repinephrine appear to stimulate steroid sec retion in Aves and to inhibit it in Pisces , while their ac tion in Amphibia is controversial. Likewi se . a tri a l na triure ti c peptide exert s an anti­sec re ta gog ue ac ti o n in A mphibia and a ma rk ed secretagogue effect in Pisces and Aves. The effects of opioids (enkephalins and endorphins) have scarcely been in ves ti ga ted a nd th e findin gs obta ined a re hi g hly qu es ti o nabl e. Co mpared with the ama zin g mass of in ves tigations carried out in mammals, studies in lower ve rtebrates a re few, and in large part perform ed in Amphibia and Al'es. It appears that much further work has to be done by comparati ve endocrinolog ists to fully c lari fy the phys iolog ica l relevance of th e functi onal interactions between chromaffin and steroidogenic ce ll s in the adrenal glands of lower vertebrates .

Key words: Adrenal gland . Stero id secretion, Paracrine interactions , Chromaffin ce lls, Lower vertebrates

I. Introduction

Th e intim ate mo rph o log ica l int e rre la ti o nships between corti cal and medull ary chromaffin ce lls in the

Offprint reques ts to: Pro f. Gastone G. Nussdorfer. Department of

Anatomy, Via Gabelli 65, 1·351 21 Padova, Ita ly

ma mm a li a n adrena l g la nd s is today recog ni ze d to possess a grea t fun c ti o nal re leva nce . A la rge bod y o f ev ide nce indic a tes that no t o nl y s te ro id hormo nes may ind uce th e ac ti vit y o f th e e nzy mes in vo lve d in e pin e phrine sy nthe s is (fo r revie w, see C rye r, 1992) . but th a t a lso c hro maffin ce ll s, by sec re tin g a lo t of reg ul a to ry mo lec ul es, may modul ate the function of cortica l steroidogenic ce ll s in a paracrine manne r (for rev iew . see Nu ssdorfe r, 1996).

The peculi ar arrangement of interrenal steroidogenic and chromaffin ti ss ues in the adrenal glands of lower vertebrates may surely favour the paracrine interactions between the two ce ll types . In fac t, the adrenal glands of lower vertebrates di spl ay a marked int e rmin g lin g betw een ste roid oge nic and chrom affin ce ll s, wh ose complex ity increases from Pisces to Aves (for rev iew. see Deane , 1962). Findings are avail able showing that adrenal chromaffin ce ll s of lower vertebrates secrete, as th ose o f mammals, not onl y ca tec holamines, but a ls o se ro to nin and seve ral reg ul a to ry peptid es, whi ch variously affec t the fun cti on of steroidoge ni c ce lls.

As far as we are aware, no comprehensive rev iew articles have been publi shed on the possible parac rine interactions in the adrenal glands of non-mammali an vertebrates . Lesouh aiti er et al. (1 995 ) foc used the ir attention on the neuroendocrine communications in the adre na ls o f Anura. a nd Ha nk e a nd Kl oas ( 199 5 ) provided a survey of the general mec hanisms involved in the regul ation of sec retion of adrenal glands in Pisces and Amphibia .

In the foll owin g sec tio ns o f thi s rev iew, we sha ll summarize and discuss the morphological and functional background of the cortico-medullary interac tions in the lo we r-ve rt e brate adre nal s, a nd th e n th e poss ibl e in vo lve me nt o f 111 0 noa min es (ca tec ho la mines a nd serotonin ) and other reg ulatory peptides sec reted by chromaffin ce ll s in the fin e tuning o f the sec re tory ac ti vity of steroidogenic cells.

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II. The morphological and functional background of the paracrine interactions between cortical and medullary cells in the adrenal glands of lower vertebrates

A. The morphological background

Several review articles are available dealing with the structure of the adrenal glands of lower vertebrates (Deane, 1962; Chester-Jones, 1976: Nussdorfer, 19R6: Chester-Jones et aI., 19R7) .

In CyclostomaTa, presumpti ve steroidogenic cells are located in the pronephros and in the dorsal-vessel region: steroidogenic and chromaffin ti ssues are not mixed. In Chondrichthyes and Osteichthyes, the two ti ssues are in close contact, and adrenals li e nea r or inside th e opisthonephroi and along the wall of the card inal vei ns. In the Amphibia, a very conspicuous intermingling of steroidogenic and chromaffin cells occu rs (Fig. I A): adrenal complex in Urodela is discontinuous and located along the ventral aspect of mesonephroi , while in Anura it is aggregated into a pair of bodies frequently lying within the capsule of meso nephroi. In Rep tilia , the adrenal complex consists of a pair of round or elongated bodies. located near the kidn eys (Chelonia and Cmcodilia), or dorsomedially to the cephalic pole of the go nads (Lacertilia and Ophidia); ste roidogenic and chromaffin ti ss ues are notably intermingled . In Ares , adrenal glands may be an impar median structure or two separate organs always in close contact with each other ( interrenal g land) ; the gland is enclosed by a loose connective capsule, and contains radially arranged cords of steroidoge nic cells among which clusters of chromaffin cells are mingled (Fig. I B) . As clearly results from the above survey, in the adrenal glands of lower vertebrates, with the exception of f ishes, chromaffin cells may relea se their secretory products nea r steroidogenic cells, thereby affecting them in a paracrine manner.

A great deal of evidence indicates that the adrenal glands of lower vertebrates are richl y innerva ted . In addition to catecholaminergic and cholinergic fibers , seve ral peptidergic fibers have been ide ntified by immunocytochemistry (see Section IV ). In mammals, nerve fibers reaching the adrenal cortex may have a two­fold origin: a group of fibers originates from neurons located outside the adrenal gland and reaches it by following blood vessels or splanchnic nerves: a second group ha s its cell body in the adrenal medulla (for review, see Vinson et aI., 1994). Vizi et al. (1992, 1993) demonstrated th e prese nce in the adrenal co rtex of num e rou s varicose axon terminals e ndin g free in close proximity to parenchymal cells, and releas ing catecholamines in respon se to axonal firing. These features, which are remini scent of the hypothalamo­pituitary neurosecretory process, strongly support the possibility of a paracrine non-synaptic modulatory role of catecholamine and other neuropeptides on adreno­cortical cells. Whether this mechani sm is operative in adrenals of lower vertebrates is not known at present.

However, ev idence is available that at least in the frog splanchnic and vagus nerve destruction does not affect the morphology of some intradrenal peptidergic fibers, which would indicate their intraglandular origin.

B. Possible mechanisms involved in paracrine interactions

Theoretica lly. a reg ulatory mol ec ule secretcd by chromaffin cells or re leased by intra-adrenal neurons may act through a direct or an indirect mcchanism . It can directly modulate the secretion of steroidogenic cells by binding to specific receptors located on the ir plasma membrane: if such a mec hani sm is operative, th e regul atory molec ule is able to affect ill I'irro steroid secretion of dispersed and puri fied steroidogcnic cells. Alternatively, a regulatory molecule may act indirectly by e liciting , in a paracrin e or autocrinc mann er, the release by chromaffin cells of other regulat o ry

Fig. 1. Light microg raphs of toluidine blue- stained 0.5 -thick epon sections of the adrenal gland of Rana lemporaria (A) and Gallus gallus domeslicus (8). The arrows indicate chromaffin cells intermingled with steroid secreting cells , which in the frog display a cytoplasm filled with lipid droplets. Asterisk: capillary lumen. x 1.250

._- . __ . -- -.--------------------

--- ------ ------------_ .. . _._---_._-----------_._-- . __ ._ ....

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molec ul es, which in turn may control the fun ct ion of ste roidoge nic ce ll s; if thi s mec hani sm is at play, the reg ul a to ry mo lec ul e ca n affec t th e f un c ti o n of steroidogenic ce lls only when the structural integrity of adre nal ti ss ue is preserved (e .g . sta ti c or dyna mi c incubation of adrenal quarters) .

Obv iously, an intra-adrenal reg ul atory molec ule can be ass umed to exert a parac rine contro l of the function of adrenocortical ce lls of any phys iolog ical relevance only when it mee ts th e fo ll ow in g basic conditi ons . The content of the molecule must be suffic iently elevated in orde r to reac h , upon appropri ate s timul ati on o f it s release, a loca l concent ra tion above its minimal effec ti ve one i ll I' irro. Mazzocchi et al. (1993) prov ided a method to calcul ate this parameter. based on the demonstration that in the fresh mammali an adrenal ti ss ue there is a s tri ct co rres pond e nce be twee n we ight and vo lume (spec ific grav ity, 1.039), and that the interstitial space (as ca lculated by morphometry) is about 3% of the total vo lume. Thus, if e .g. the adrenal content of a molecule is 100 pmol/g . its one-third release will produce a local co nce ntra ti o n of 10-6 M. In fac t , 30 pm ol of th e molecule will be released into about 30 I of interstiti al space (3 % of 1 ml , that is the volume of I g of fresh adrenal ti ssue), producing a local concentration of about I pmollfll or I jlmoli l. This method can be reasonably assumed to be va lid also for non-mammalian adrenals.

III. The involvement of monoamines in the control of the functions of adrenal steroidogenic cells in lower vertebrates

A. Catecholamines

Nussdo rfe r (1 996) has rece ntl y s ur ve yed th e in vestigations dealing with the effects of catecholamines (ep in ephrine, norepinephrine and dopamine) on the sec re ti o n of ma mm a li a n adre noco rti ca l ce ll s . To summari ze: (i) epinephrine and norepinephrine, acting via adenylate cyc lase-coupl ed l3- receptors, direc tl y stimulate mineralocorticoid and probably glucocorticoid sec reti on of ad renocorti cal ce ll s; and (ii ) dopamine , ac tin g via DA I and DA2 rece pt'ors pos iti ve ly and negati ve ly coupl ed with adenylate cyc lase, exe rts a prevalentl y inhibitory effec t on the zona glomerul osa and aldosterone secretion.

I. Epinephrine and norepinephrine

Ad re nal c hro maffin ce ll s of lowe r ve rt e bra tes sy nthesize and release catecholamines, epinephrine and norepinephrine being more abundant than dopamine (for rev iew, see Hanke and Kloas, 1995: Lesouhaitier et aI. , 1995) . Howeve r , th e direc t effec t o f the fir s t two catecholamines on the secretion of adrenocortical ce lls has not been ex tensively in vestigated . Indirect findings indi cate that in the te leos tea n fish Cyprinus carpiu, epin e phrin e and norepin e phrin e inhibit co rti so l product ion by adrenocortical ce ll s (G fe ll et aI. , 1995). Accordin g ly, in th e frog Ra na ridibun da , th ese

catecholamines were found to exert a weak suppress ive effec t on co rti cos te rone and a ldos terone re lease by perifu sed ad renal ti ss ue, the ir half-maximal effec ti ve co nce ntrati ons be ing about 10-5/ 10-4 M for co rti co­sterone and 10-3 M fo r aldosterone (Morra et a!.. 1990). In contrast, Hanke and Kloas (1 994. 1995) reported that in th e urode le Xe nupus lli evis 10-6 M epinephrine redu ced the s timul ating ac ti on of ACTH on adrenal co rti cos te ro ne sec re ti o n a nd e nh anced that o n aldosterone production. Epinephrine and the l3-adreno­ceptor agonist isoprenaline, but not the B-adrenoceptor agoni st guanabenz, were fo und to raise aldosterone and corticos terone production by di spersed fowl interrenal ce lls : minimal and max imal effec ti ve conce ntrations being IO-R M and 10-6 M, respec ti vely (Mazzocchi et aI. , 1997b). Accordingly, these last investigators showed that the l3-adrenoceptor antagoni st I-alprenolol, but not the a- rece ptor ant agoni st phent olamine, suppresses secretory responses to max imal effective concentrations of both epinephrine and isoprenaline. Mazzocchi and assoc iates concluded that epinephrine enhances adrenal steroid secretion in the fo wl , acting, as in mammals, via the activation of the l3-adrenoceptor SUbtype.

2. Dopamine

The studies dea ling with the effect of dopamine give more co ns is te nt res ult s . Morra e t a l. ( 1990 . 1992) showed that dopamine causes a clear-cut inhibition of the basal release of both corticosterone and aldosterone by pe ri fused frog adrenals; ha lf-max imal effec ti ve conce ntrati on being about 10-6 M. The sec retagog ue effect of 10-9 M ACTH is not affec ted by dopamine, while that of 10-7 M angiotensin-II (ANG-I1 ) is impaired ( Mo rra et a l ., 1990 ). Us in g co llagen ase -di spe rsed adre noco rti ca l ce ll s. Morra and co-workers ( 1990) observed that after the second pulse dopamine induces a transient stimulatory effect. The infusion of repeated pulses of a DA I receptor agonist mimiks the stimulating effect of dopamine, while the administration of a DA2 recepto r agoni st inhibit s ste ro id sec reti o n; neith er dopamine nor the two dopamine-receptor agonists affec t cyc li c -A MP fo rmati o n (Morra e t a I. , 1992) . The inhibitory effect of dopamine was found to be assoc iated with a ma rk ed redu c ti o n in th e rel ease o f bo th pros taglandin-E2 and 6-keto-prostaglandin-Fl a by frog adrenal ti ssue (Morra et aI. , 1989). Morra et al. ( 199 1) demonstrated that (i) a short pulse of dopamine evokes a biphasic effec t on phosphatidylinos itol breakdown . a tra ns ie nt in c rease be in g fo ll owed by a s us ta ined inhibition: and (i i) the DA2-receptor agonist apomorphin e li c it s a ma rk ed inhibiti o n of in os it o l ph os phat e produ cti on . Coll ec ti ve ly, these findin gs sugges t that dopamine controls stero id produ cti on by amphibi an adrenoco rti ca l ce ll s actin g via th e DA 1- and DA2-receptor SUbt ypes , whi ch, at va ri ance with those o f mammals (see above), are pos iti ve ly and negati ve ly coupled to both phospholipase A2 and phospholipase C. It is like ly th at und er ph ys io log ical co nditi ons the inhibitory effect prevails.

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B. Serotonin

Co mpe lling ev ide nce ind ica tes th at se ro tonin a nd serotoninerg ic- fibe rs are contained in the mammali an adrenals, where they direc tl y stimul ate mineralocorticoid and g lucocorti co id secretion , acting via phospholipase C-coupled 5-HT 2 and adenylate cyclase-coupled 5-HT 4 receptors (for rev iew, see Nussdorfer, 1996).

T he occurrence of serotonin has been biochemically a nd immun ocytoc hemi call y de monstrated in adre na l chro maffin ce ll s of anurans (De larue e t aI. , 1988 a ,b). Se ro to nin is co-s to re d w ith e p ine phrin e a nd is sy nthes ized by c hro maffin ce ll s from L- trypto ph a n (De larue e t a I. , 1992).

The sec reatagogue effec t o f se ro tonin o n adre no­cortical ce ll s o f lower ve rte brates has been in vestigated almos t exclus ive ly in Rana ridibunda . Serotonin was fo und to ra ise basa l co rti cos te ro ne a nd a ld os te rone secre tion of both peri fu sed adrenal ti ssue and collagen­ase-dispersed adre nocorti ca l ce ll s in a conce ntration­de pe nde nt ma nne r . minim a l a nd max im a l effec ti ve concentrations be ing 10-7 M and 10-6 M . respec tive ly (De la ru e e t a l. . 1988a.b ; Idres e t a l. . 1989). Ind o­methac in , a cyclooxygenase inhibitor, and dantrolene . a bl ocke r of intrace llul ar ca lc ium mobili zatio n , do no t impair the secretagogue ac tion of serotonin (De larue et al. . 1988a,b), which there fore appears to be independent of phospho lipase A2 and phospho lipase C ac ti vation . Idres e t a l. (1 99 1) and C onte sse e t a l. ( 1994 ). us ing spec ific agonists and antagonists of serotonin-receptor SUbtypes 5-HT I , 5-HT2 . 5-HT3 and 5-HT4 ' de mo n­strated that the adre nocorti ca l secretagogue e ffec t of se ro to nin is link ed to th e ac ti va ti o n of the 5-HT4 receptor. Zacopride. a benzamine deri vati ve which is an agonist o f thi s receptor SUbtype, evokes a concentration­re la ted s timul a ti o n of s te ro id secre ti o n . a nd . lik e se ro tonin , enhances cyc lic AMP re lease by peri fu sed adrenal tissue (Idres e t aI., 1991 ). as well as raises the cy toso l ic calci um concentrati on ( [Ca2+] j) in c u Itured ce ll s th rough the acti vation of a T-type ca lcium channel (Contesse e t aI. , 1996). In conclusion . in contrast with ma mm als (see above), se roto nin appears to e nh a nce s te ro id sec re ti o n o f fr og adre no c o rti ca l ce ll s by s timul a tin g. exc lu s ive\[; v ia 5- HT 4 recept o rs. bo th ade nylate cyclase and Ca + intlux.

Before concluding, it must be recalled that serotonin was not show n to affect corticosterone produ cti on by chick adrenal quarters incubated in vitro (Cheung et aI. , 198 7 ), th e re b y s tress in g th a t th e e ffec t of thi s monoamine on adrenocortical ce ll s o f lower-vertebrate spec ies other than frog requires further investigation .

IV. The involvement of regulatory peptides in the control of the functions of adrenal steroidogenic cells of lower vertebrates

A. Hypothalamic pep tides

Se ve ral hypotha la mic peptides have been found in mammalia n adrena l medull a : cortico tropin-re leas ing

hormone (CRH ); arginine-vasopressin (AVP); oxytocin ; so m a to tro pi c ho rmo ne - re lease i nh i bit i ng ho rmo ne (S RIF); and thyrotropin- re leas ing hormone (TRH ) (for rev iew. see Nussdorfe r. 1996). The poss ible functions of these intramedullary peptides have bee n rev iewed by Nussdorfer ( 1996), and can be summarized as fo llows . CRH appears to enhance the release by chromaffin ce ll s of ACTH , the intramedullary CRH-ACTH system being mainly in vo lved in the maintenance and stimulation of the secretion and g rowth of adre na l zonae fasc ic ul ata and reticulari s. AVP. ac ting via phospholipase C-coupled V I receptors, plays an important and direc t ro le in the m a inte na nce a nd s timul a ti o n o f zo na g lo me rul osa growth a nd a ldos te rone sec re ti on. Oxytoc in direc tl y stimulates basal stero id secre tion , but it inhibits ACTH­enhanced g lucocorticoid secreti on in rats. SRI F, acting via spec ific receptors that interfere with the intrace llul ar mechanisms transduc ing ANG -II secretagog ue s ignal , exerts a potent and specific inhibitory action on the zona g lomerulosa. TRH seems to d irectl y inhibit the late steps of glucocorti coid synthes is in the rat.

I . Arginine-vasotocin (AVT)

AVT. the amphibian counterpart of mammalian AVP. has bee n immun ocy toc he mi ca ll y a nd bi oc he mi ca ll y detected in the frog adrenal ti ssue (LarcheI' et al.. 1989). AVT-immuno reac ti v ity ( ir) was found in c hro maffin granules of both nore pinephrine and epinephrine ce ll s . Radio immune assay (RIA ) showed that adrenal content of AVT is about 2.6 pmol/g , which could produce a local concent ration of about 2-3x 10-8 M (see Section II B) . Mesotoc in , the amph ibian counte rpa rt of mamma li an oxytoc in , was not cytoche mica lly detected .

Larcher et al. (1989) showed that AVT concentration de pe nd e ntl y ra ises a ld os te ro ne a nd co rtic os te ro ne re lease by perifused frog adrenals, minimal and max imal effecti ve concentrations be ing 10-10 M and 5x 10-9 M , respecti vely; max imum rise is 4.8 -fo ld fo r a ldos terone and 3 .4- fo ld fo r corti costerone. Kloas and Hanke ( 1990) obtained similar findings in Xenopus laevis, but minimal and maximal effecti ve concentrations are 2x 10- IO M and 2x 10-8 M , and the max imum secretory response is far more elevated (47-fold and 26-fold ri ses for aldos terone and corti cos terone, respec ti ve ly). AVP, oxy toc in and mesotoc in were found to be about 400. 100 and 1500 times less potent than AVT (Larcher et aI. , 1990).

Furth e r s tudi es thre w li g ht o n th e mec ha ni s m underl ying AVT secretagog ue action in the frog . It has bee n o b se r ve d th a t th e V2 -rece pt o r a nta go ni s t [d(CH2)s D ,Phe2 ,Ile4,Al a9-NH2J-AVP (10-6 M). but not the V I -recepto r a ntagoni st 1-( l -merca pt o-4 -phe nyl ­cyc lo hexa ne -ace ti c ac id ) -AVP, s uppresses th e secretagogue effec t of 5x 10-10 M AVT (Larcher et al.. 1992a). These authors also showed that AVT does not c h a nge e ith e r cyc li c A MP o r pros tag la ndin- E2 pro du c ti o n by th e pe rifu se d frog adre na l t iss ue; according ly, indomethac in . although per se decreas ing basa l sec re ti o n , does no t s igni f icantl y a ffec t AVT­stimulated a ldos terone and corti cos terone producti on ,

- _ .. __ ._ .. __ . __ .. - .. ---- --.-_. __ ._---- ----_._--- ----- ----_._-------_. __ . .

---~------------------------------~-.--------------. . ---,. -- ----

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thereby confirming that prostaglandins are not involved in the secretagogue effect of AVT. In contrast, Larcher et al. (l992a) clearly demonstrated that AYT markedly raised inositol phosphate production, which suggests that this peptide acts via V2 receptors positively coupled to phospholipase C. This contention easily explains why AVT exerts additive effect only when combined with adrenocorticotropic factors stimulating adenylate cyclase (e.g. ACTH and serotonin), and why its action is inhibited by dopamine, which depresses phospholipase C activity (see Section l/J B) (Larcher et aI., 1992b). Larcher et al. (1992c) showed that AVT causes an immediate increase in [Ca 2+1j in frog adrenocortical cells, followed by a sustained response. The V2-receptor antagonists block this effect of AVT, which cannot be observed in a calcium-deprived medium: the dihydro­pyridine Ca2+-channel blocker nifedipine does not affect calcium response to AVT. On these grounds , Larcher and associates advanced the hypothesis that AVT exerts a two-fold action on lCa2+]j in frog adrenocortical cells: the initial rise is probably due to the immediate mobilization of intracellular calcium stores mediated by inositol triphosphate-gated channels, while the sustained increase has to be ascribed to the opening of nifedipine­insensitive plasma membrane channels.

2. SRIF

SRIF (up to 10-5 M) was not found to elicit any suppressory action on either basal or ANG-II-stimulated aldosterone release by peri fused frog adrenals (De larue et aI., 1984). On the contrary, Cheung et al. (1988) observed that the i/1 vivo administration to l-day-old domestic cockerels of an anti-SRIF serum raised basal levels of circulating corticosterone, thereby making it likely that endogenous SRIF plays a role in the regulation of adrenocortical function in fowls. More recently, Mazzocchi et al. (1997a) showed that SRIF concentration dependently inhibits aldosterone and corticosterone response of dispersed turkey (Meleagris ga/lopavo) adrenocortical cells to 10-9 M ANG-II. Minimal and maximal effective concentrations are 10-8

M and 10-6 M, and elicit about 20% and 40% inhibition, respectively. As in mammals (see above), basal secretion is not affected. and the inhibitory action of SRIF is abolished by 10-6 M cyclo(7-aminoheptanonyl-Phe-D­Trp-Lys-Thr[Bzll). which suggests the involvement of an SRIF receptor-mediated mechanism. SRIF was not found to affect steroid secretion of fowl adrenocortical cells. which, however, according to Kocsis et al. (l995a,b), are unresponsive to ANG-II.

Before concluding, it must be recalled that urotensin­II, a cyclic dodecapeptide originally isolated from the fish urophysis (the counterpart of the mammalian neurohypophysis) and then from the frog brain (Conlon et aI., 1992a.b), was found to enhance steroid secretion of adrenocortical cells of the dogfish (Conlon et aI., 1992b) and amphibians (Hanke and Kloas, 1994). Urotensin-II exhibits structural similarities with mammalian SRIF and appears to interact with SRIF

receptors, whose activation would therefore mediate the opposite effect in mammal and lower-vertebrate adrenocortical cells (see above). However, Feuilloley et al. (1994) did not observe any appreciable effect of urotensin-II (from 10- 10 to 10-6 M) on either basal or agonist-stimulated steroid release by perifused adrenals of Rana ridibunda.

B. Opioid pep tides

Evidence is available that the three members of this family of peptides (enkephalins, endorphins and dynorphins) are synthesized and secreted by chromaffin cells of the mammalian adrenal medulla, and are able to variollsly affect the secretory activity of the cortex in a paracrine manner (for review, see Nussdorfer, 1996). To summarize. enkephalins exert a direct stimulatory action on both zona glomerulosa and zona fasciculata cells, while f)-endorphin appears to directly inhibit gluco­corticoid secretion. at least under physiological conditions. Dynorphins suppress steroidogenesis in the rat, but the physiological relevance of this effect is questionable.

As far as we are aware, only enkephalins have been detected in adrenal chromaffin cells of lower vertebrates. Kondo and Jui (1984) and Reinecke et al. (1992) provided immunocytochemical evidence of the presence of enkephalin-ir in both chromaffin cells and nerve fibers of the frog adrenals. According to Leboulenger et al. (1983a ,b), both met- and leu-enkephalins are present in about 40 % of adrenomedullary cells of Ra/1a ridibunda and are co-localized with vasoactive intestinal peptide in the chromaffin granules. The investigations on the direct effect of opioid peptides on adrenal steroido­genesis in lower vertebrates are very scarce and their rather controversial results may be summarized as follows.

I. Enkephalins

Leboulenger et al. (,-983a) did not observe any appreciable effect of 10-) M met-enkephalin and leu­enkephalin on either basal or ACTH-stimulated corticosterone production by amphibian adrenocortical cells. Conversely, Hanke and Kloas (1994) reported a clear-cut stimulatory action of JO-6 M met-enkephalin on both corticosterone and aldosterone responses to ACTH.

2. Endorphins

Hanke and Kloas (1994) found that 10-7 M u­endorphin enhanced ACTH-stimulated corticosterone and aldosterone secretion by amphibian adrenals, while Zerani and Gobbetti (1991,1992) described a net inhibitory action of 13-endorphin (about 4x I 0-8 M) on corticosterone and cortisol production of adrenocortical cells of Rana esculenta and Triturus cami/ex. These last investigators also showed that naloxone blocked 13-endorphin secretagogue effect, and proposed that this peptide acts via specific {I-receptors.

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C. Tachykinins

Tac hykinin s a re a g ro up of reg ul a to ry pe ptides belong ing to the neuromedin fa mil y (fo r rev iew, see Malend ow icz and Mark ows ka . 1994). Mamm ali a n tac hykinin s in c lud e ne uro kinin A , ne urokinin B . substance P (s tructurall y re lated to neurokinin A and encoded by the same gene) and neuropeptide K (the N­terminally extended fo rm of neurokinin A) (for review. see Magg io, 1988). Tac hykinin-ir has bee n cy to­c he mi ca ll y and bi oc he mi ca ll y de mo nstra ted in chromaffin cells and nerve fibers of adrenal glands of several mammalian species (for rev iew, see Nussdorfer. 1996). According to Nussdorfe r ( 1996), the direct effects of tachyk inins on mammali an adrenocorti ca l ce ll s are probabl y pharm aco log ica l in nature . Substance P is likely to playa physio log ica l role in the main tenance a nd s timul a ti o n o f sec re ti o n a nd g row th o f rat zo na g lo merul osa , thro ug h an indirec t mec hani sm invo lving the NK I receptor-medi ated enhancement of ca tec holamine re lease by medull ary chromaffin ce ll s . Neuropeptide K appears to indirectl y stimulate gluco­cort icoid secretion of rat adrenal quarters by acti vating the CRH-ACTH system located in the medulla.

Frog interrenal s have been found to conta in three tac hykinin s: substance P, ranakinin (a peptide re lated to sub sta nce P) ; and [Leu 3 .I1 e7]- ne urokinin A (Leboulenger et aI. , 1993; Kodjo et al. . 1995a). Immuno­cy toc hemi stry showed th e presence o f tac hykinin ­pos iti ve ne rve f ibe rs pre fe re nti a ll y appose d o nt o chroma ffin ce ll s. RIA demonstrated an intra-adrenal content of substance P and neurokinin A of abollt 8 and 4.6 pmol/g, which could give rise to local concentrations ra ng in g fro m 5 to 9 x 10-S M (see Sec tio n II B) . Interestingly. the bil ateral transection of splanchnic and vag us nerves does not apparentl y a lter intra-adrenal tachykinin-ir, a findin g ruling out the poss ibility of the extrag landul ar origin of tachykinin-positi ve fibers .

The effects of tachykinins on adrenal steroidogenesis have been studied exclusive ly in Amphibia. According to Leboulenger et al. ( 1993), substance P conce ntrati on de pe nd entl y ra ises corti cos te rone and a ld os te ro ne re lease by perifused adrenal ti ssue of Rona ridibunda; minimal and max imal effecti ve concentrations are 10-7 M and 10-5 M and elicit ri ses of 30-40% and 150- 180%, respec tive ly. In keeping with these findings, Hanke and Kloas (19942 demonstrated that substa.nce P (from 5x 10-7 to 10- M) e nh ances bo th co rti coste ro ne and aldosterone release from peri fu sed adrenals of Xenopus laev is . All th e oth e r a mphibi a n tac hykinin s , a t a concentration of 10-5 M, were fo und to evoke increases in corti cos te rone output rang ing fro m 150 to 200% (Leboul enge r et aI. , 1993) . These in ves ti ga tors a lso o bse rve d th a t sub s tan ce P e l ic it s th e re lease of pros taglandin E2 and prostacycIin by frog adrenals, and that thi s response precedes by 10- 15 min the ri se in co rti cos te roid output. Acco rdin g ly, ind o me th ac in abolishes the secretagogue effect of substance P, which indicates that the mechani sm underlying thi s action of the tachykinin involves the acti vation of the arachidonic

acid cascade. More rece ntl y, Kodjo et al. ( 1995a) con firmed that

ranakinin enhances steroi dogenes is in peri fused frog adrenal ti ss ue . Howeve r, they showed that ranakinin does not affec t basa l sec reti on of di spersed adreno­cortical ce ll s. and hypothes ized the in vo lve ment of an indirect mec hanism of ac tion requiring the integrity of ad re nal ti ss ue . Autoradi og raph y de mo nstra ted th e presence of (l Hl -substance P binding sites exc lu sive ly in adrena l chro maff in ce ll s, and mi cro flu o rim etry ev ide nced a ranakinin-indu ced ri se in [C a2+1i in chro maffin , but no t adrenoco rti ca l ce ll s. Furthe r in vestigations (Kodjo et aI., 1995b) showed that neither the chelation of calcium in the perifusion medium nor nifedipine and lU-conotox in alter calcium-response of chromaffin ce lls to ranakinin; conversely. the incubation of the ce lls with thapsigargin, an inhibitor of calcium­ATPase ac ti vi ty, aboli shes ranakinin effect. Moreover. the phosphol ipase C antagon ist U-73 122 and pertu x i s tox in completely block calcium response of chromaffin ce ll s to ranakinin . In li ght of th ese findin gs . th ese authors suggested that (i) ranakinin-induced increase in [Ca2+]j mu st be asc ribed to mobili zati on of ca lc ium from intrace llul ar stores; and (ii ) ranakinin stimul ates frog adrenal chromaffin ce ll s th rough the ac ti vat ion of phospholipase C via a pertu xin tox in-sensiti ve G protein.

Further studies were carried out to ascertain whi ch rece ptor SUbtype is in vo lved in the medi ati on of the indirect adrenocortical sec retagogue effec t of ranakinin (Kodjo et aI. , \996). It has been found that the selecti ve NK I-receptor antagonists lO-Pro4 ,0-Trp7 .9]-substance p 4- 11 and CP-96 .345 do not affec t the corticos teroid stimul atory ac tion of ranakinin , nor the se lec ti ve NK I ago ni st sub sta nce P6- 11 and th at th e a mphiphili c sub s ta nce P ana log ue lO -Pr0 2 .O -Phe 7 .0-Trp9]­substance P enhance per se corticos teroid secretion. In contrast. the non-peptidic NK I antagonist RP67580 and the NK I/NK2-receptor antagoni st FK-224 significantly suppress the stimul atory e ffec t of ranakinin on both corti cos terone and aldos te rone sec reti on . Kodjo and assoc iates sugges ted that the effects of tac hykinin s on th e frog adrena ls a re mediated by a nove l type of receptor, whi ch diffe rs fro m th e mamm ali an NK I subtype.

D. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP)

VIP and PACAP are 28- and 38-amino ac id-res idue peptides, widely distributed in the mammali an tissues . The N-terminal 28-amino acid sequence of PACAP has a 68% ide ntity with VIP, and the two peptides share a number of phys iolog ica l functi ons, in cluding neuro­transmission, stimulation of pituitary ACTH release , and regul ati on of vasc ul ar tone and gas troint es tin al-trac t ac ti vity (for review, see Fahrenkrug. 1989: Arimu ra and Shioda , 1995). The effects of VIP and PACAP, which arc both synthes ized and secreted by mamm alian adrenal medulla , on the sec reti on and grow th of the adrenal co rtex have bee n rece ntl y rev iewed by Nu ssdorfe r

---------- _. --_._----- _._ .. _---_.---_ .. _--_. __ .- .-.

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( 1996). Intra medull a ry VIP is in vo lved in th e physio logical regulation of zona glomerulosa secretion, the aldos terone secretagogue effec t being both direc t and indirec tl y med iated by the stimulation of catecholamine re lease . VIP may a lso as pec ifi ca ll y ac ti va te ACTH receptors located on both outer and inner adrenocort ical ce lls . but under normal conditions this action is masked by c ircul atin g ACT H, th at toni ca ll y ac ti va tes such receptors. PACAP stimul ates both mineralocorticoid and g lu coco rti co id sec re ti o n indirectl y, by e li c itin g catecholamine re lease by medullary chromaffin cells and by ac ti va ting the intramedull ary CRH-ACTH sys tem. respec ti ve ly: however, the ph ysiologica l relevance of thi s last effec t of PACA P is very doubtful.

I. VIP

Immun ocy toc he mi ca l s tudi es s howe d th e co ­loca li za ti o n of v IP-ir w ith e nk e ph a lin - ir in th e chromaffin granules of adrenomedullary ce ll s of Rona ridib /ll1da. but no v IP-e rg ic f ibe rs we re o bse rve d (Leboulenger et al.. 1983a .b).

Lcbo ul e nge r e t a l . ( 1983a) repo rt ed th a t VIP concentration dependently raises both corticosterone and aldos terone re lease by peri fused frog adrenal ti ss ue. Minimal and max imal effec ti ve concentrations are 10-6

M and 10-) M. and the max imal aldosterone response is much hi gher than the corticosterone one (2-fo ld versus I A- fold 7- ise). The antimicrotubular agent cytochalas in B impairs both basa l and VIP-stimulated steroid secretion of frog adrenals (Leboul enger et aI. , 1984). However, thi s f in d in g may be co nce iva bl y int e rpre ted as an aspecifi c effec t of cy toc halas in B consequ ent to the disruption of the cytoskeleton. which is we ll know n to pl ay an essential role in adrenocortical steroid sy nthesis and release (for rev iew, see Nu ssdorfer. 1986: Hall. 1995: Feuilloley and vaudry. 1996). Ca lcium chelation from perifu sion medium and indometh ac in , a lthough lowering basa l steroid-secretion rate. do not signi ficantly alter corticos terone and ald osterone responses to VIP (Lebo ulenge r e t a I. , 1984). Co ll ec ti ve ly, th ese las t findings would indicate that adrenocortical secretagogue effec t of VIP in th e f rog does no t in vo lve e ith e r pros tag la ndin sy nth es is o r ca lc ium . It co uld be hypothes ized that VIP acts by enhancing cyc lic AMP production: however. a max imal effec ti ve concentration of VIP was fo und to exe rt potenti atin g . more than additi ve . e ffec ts wh e n co mbin ed wi th a max im a l effecti ve concentrati on of serotonin (Leboulenger et aI. , 1988). whi ch exert s it s adrenoco rti ca l sec retagog ue effect by stimul ating adenylate cyc lase (see Section III B).

2. PACA P

Thick and thin PA CA P-pos iti ve nerve fibers were detected in adrenal glands of Rana ridibunda (Yon et al.. 1993. 1994): thi ck va ri cose fibers are located among ad renal ce lls, while thin fibers end in the walls of blood vesse ls . As demonstrated for tac hykinin s . bil ate ral

spl anchnotomy docs not affec t PACAP-ir distribution. which sugges ts the intra-adrenal ori gin of PACAP­pos it ive fibers . According to Yon et al. ( 1993) PACAP co nte nt in fresh frog adrenals, as meas ured by RI A. a tt a in s 0.65 nm o l/g. whi c h co uld produ ce a loca l concentration of about 7x 10-6 M (see Section II B).

Yo n e t a l. ( 1993) re po rt ed th a t fr og- PACA P38 concentration dependently increases corticosterone and aldos terone release by perifu sed frog adrenal glands. Minimal and max imal effec ti ve concentrations are 10-6

M and 10-5 M. and induce ri ses of about I .S-fo ld and 2.5-fo ld . res pec ti ve ly. Di spersed frog adrenocortica l ce ll s di splay similar sec retory responses to 10-6 M frog­PACAP38 (Yon et al.. 1994). These las t in ves tiga tors provided the autoradi ographic demonstration that both adrenocortical and chromaffin ce lls possess bind ing sites fo r l I 25 Il -PACA P27; moreover, they showed that frog­PACA P38 concentration dependently raises cyclic AMP release by frog adrenal quarters, and increases lCa2+h in both adrenoco rti ca l and chro maffin ce ll s. Yon and assoc iates concluded that PACAP directly stimul ates the sec re to ry ac ti vit y o f fr og adre noco rti ca l ce ll s by ac ti va tin g adenylate cyc lase. Howeve r, it mu st be stressed that the above reviewed findings do not rule out the poss ibility of an additional indirect effec t of PACAP, mediated by chromaffin ce lls. In keeping with this last co ntenti o n. Mazzocc hi e t a l . ( 1997c) obse rved th at PACA P is abl e to enhance a ldos te rone and corti co­sterone producti on by fow l adrenal sli ces cont aining chromaffin ce ll s , but not by di spersed adrenocorti ca l ce ll s, m i 11 i mal and ma x i mal effec t ive concentrat ions be ing IO-x M and 10-7 M. respec ti ve ly. The secretory res po ns e to 10- 7 M PACA P (a bo ut 3- fo ld ri se) is suppressed by the PACAP-receptor se lecti ve antagonist PACAP6_3H at a concentration of 10-6 M. Moreover. the PACAP ( 10-7 M)-induced increase in aldos terone and co rri cos te ro ne sec re ti o n is annull ed by 10 -) M /­alprenolol. In light of these findin gs, Mazzocc hi and associat e s ad va nced th e hypo th es is that PACA P stimulates th e sec retory ac ti vity of fow l interrenals indirectl y, by eliciting the release by chromaffin cell s of ca tec ho la min es . whi c h in turn e nhan ce stero id production in a pm'ac rine manner.

E Galanin

Galanin is a 29-arnino ac id peptide widely distributed in th e mamm a li an ce ntra l and periph era l nervo us systems (for rev iew. see Bedecs et aL 1995). It is also contained in ad renal medulla and seems to be directl y in vo lved in the maintenance and stimul ati on of the secretion and growth of inner adrenocortical zones (for rev iew, see Nussdorfer. (996) .

Zentel et al. ( 1990) detec ted by immunocytochemistry galanin-ir in virtually all adrenomedullary cells of ducks and chickens. but not pigeon adrenals, where in contrast galanin-pos it ive nerve fibers are present. More recently, Gas man e t a l . ( 1996) o bse rved th e prese nce of an abundant network of nerve fibers immunoreacti ve to galanin in the adrenals of Runa ridibunda . Furthermore.

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they demonstrated by coupled high performance liquid chromatography and RIA analysis, the existence of a single form of galanin exhibiting the same retention time of frol-galanin. Syn.thetic frog -galanin (from 10-9 to 3x 10- M) was also found to concentration-dependently inhibit both basal and ACTH-, but not ANG-II­stimulated aldosterone and corticosterone release by peri fused frog adrenal slices. These authors concluded that galanin released by nerve terminals in the amphibian adrenals exerts a negative modulation of steroid secretion, probably by inhibiting adenylate cyclase, but not phospholipase C or phospholipase A2 transduction pathway.

F. Calcitonin gene-related peptide (CGRP)

CGRP is a 37-amino acid peptide present in the mammalian central nervous system. as well as in nerve fibers to several peripheral organs (for review, see Owyang and Louie, 1989) . CGRP-ir has been demonstrated in both nerve fibers and chromaffin cells of the mammalian adrenal cortex (for review , see Nussdorfer , 1996) . According to the survey of Nussdorfer (1996), CGRP exerts a direct inhibitory effect on ANG-II-stimulated aldosterone secretion of zona glomerulosa cells, via a CGRP 1 receptor, whose activation interferes with the agonist-stimulated redistribution of intracellular calcium. However, the physiological relevance of this effect is questionable, since it can be observed only at a very high concentra­tion of the peptide. Probably, CGRP enhances in I'ivo mineralocorticoid secretion by zona glomerulosa, via indirect mechanisms overcoming its direct inhibitory effect and probably involving the stimulation of catecholamine release by chromaffin cells.

CGRP-positive nerve fibers have been immunocyto­chemically demonstrated in the adrenal complex of the flat snake (Orezzoli et aI., 1993, 1994) , as well as in the chromaffin cells and nerve fibers of the adrenal glands of several anuran species (Kuramoto, 1987; Reinecke et al.. 1992) . Esneu et a!. (1994) observed that in the RanG ridibunda adrenal gland CGRP-ir is exclusively located in the nerve fibers.

Esneu et a!. (1994) reported that CGRP increases corticosterone and aldosterone secretion by both perifused frog adrenal tissue and dispersed adreno­cortical cells. Frog CGRP is much more effective than rat and human peptides in the perifusion model, maximal effective concentration (about 10-6 M) eliciting a 2.75-fold increase in the release of both corticosterone and aldosterone. Further investigations (Esneu et aI., 1996) characterized the CGRP-receptor SUbtype involved. CGRPI-receptor SUbtype antagonists (human CGRPR_37 and human CGRP j9_37 ) do not alter the secretory respon se to frog-CGRP, while a CGRP2-receptor SUbtype agonist ([acetamidomethyl-Cis2 ,7 J-hu man CGRP) evokes a concentration-related enhancement of corticosterone and aldosterone secretion (EC sO = 1.6x 10-7 M) . Likewise, adrenomedullin and amylin, two members of the CGRP family (for review, see

Nussdorfer, 1996). induce a weak secretagogue effect. Frog-CGRP and CGRP2-agonist significantly rai se cyclic AMP release by frog tissue. These investigators maintain that CGRP directly stimulates adrenal steroid secretion in Anura, via the activation of CGRP2 receptors positively coupled to adenylate cyclase.

G. Natriuretic peptide family

This group of regulatory peptides , originally isolated from atrial myocytes in the late 1970s , at present is known to include three members: atrial natriuretic peptide (ANP), brain or B-type natriuretic peptide (BNP) and C-type natriuretic peptide. An amazing mass of investigations indicates that these peptides exert a potent inhibitory action on aldosterone secretion in mammals , and are all contained and synthesized in adrenal medulla (for review, see Cantin and Genest, 1985; Rosenzweig and Seidman, 199 I: N ussdorfer, 1996).

ANP-positive nerve fibers were found to run near adrenocortical cells in Rana ridibunda (Lihrmann et al.. 1988) . ANP-ir has been immunocytochemically detected in adrenal chromaffin cells of teleostean fishes (Kloas et aI., 1994; Wolfensberger et aI. , 1995), lizards (Wolfensberger et a!., 1995), anurans (Reinecke et aI., 1992; Wolfensberger et aI., 1995) and birds (Wolfensberger et a!., 1995). According to these last investigators, ANP-ir is restricted to 35-40% of chromaffin cells, except in fishes where the number of positive cells attains 80-90%: about 30-550/(' of ANP­positive cells also contain neuropeptide Y-ir. Reinecke et al. (1992) also reported the presence of BNP-ir in the anuran adrenomedullary cells.

The effects of natriuretic peptides on adrenocortical­cell secretion in lower vertebrates have been quite extensively investigated, and the results have varied according to the class and species studied.

I . Pisces

Kloas et al. (1994) demonstrated by quantitative autoradiography [1251]-rat ANP99-126 binding in the adrenal gland of Cyprinus carpio (ECSO , 87±15 pM; Bmax ' 56±11 amol/mm2). Rat-ANP raises basal cortisol secretion (minimal effective concentration, 10-7 M), and more efficiently the acetylcholine-stimulated one (minimal effective concentration, 10-9 M) . Similar results were obtained in the teleostean Plalichrhys ./lesus and Salmo gairdneri, where 2x 10-7 M human ANP increases in virro basal cortisol release by adrenal ti ssue of seawater-adapted animals (Arnold-Reed and Balment, 1991): since no effects were observed when adrenal frolll freshwater-adapted fishes were employed. these authors suggested that ANP Illay playa role in the animal survival in hypertonic media.

2. Amphibia

[125 11-rat ANP99-126 binding sites were demon­strated by autoradiography especially in the outer layer

--------- ------------ -- -------.---. ...

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of adrenal g lands o f Rana temporaria (ECso , 93±1 9 pM ; Bmax ' 1.2 1±0.36 fmol/mm2) (Kloas and Hanke, 1992a) . Lihnnann et al . ( 1988) reported that ANP does not affect basal corticosterone and aldosterone y ie ld by g eri fused adrenal tissue of Rana ridibunda ; however, 10- M ANP s igni ficantl y suppresses maximal ACTI-I- and ANG-II­stimu:ated sec retion of both stero ids (ACTH -stimulated corticosterone and aldosterone release by 25 % and 56% , res pec ti ve ly : A NG- II -s timul ated s te ro id sec re ti o n by about 26-28 %). Kloas and Hanke ( 1992b) o bserved an inhibito ry effec t of A NP o n corticos tero id re lease by Xenopus !aevis adre nals .

3. Aves

Rose nberg e t al. ( 1988 . 1989 ) showed that 10-8 M ANP decreases both basa l (-26%) and ACTH- or cho lera tox in-stimul ated (-84/-94 %) aldosterone production by dispersed chicken ad renocortical ce lls . The loc i o f action of A NP a ppea r to be bo th ea rl y (co n vers io n of cho lestero l to pregne no lone) and late steps (convers ion o f co rti cos te ro ne to a ld os te ro ne) of a ld os te ro ne synthes is. ANP, at vari ance with ACTH. does not change cyc lic AMP release by adrenocorti cal ce ll s, and cyclic G MP a nd th e g uan y la te -cyc lase ac ti va to r sodium nitro pru ssid e exe rt a n a ld os te ro ne anti secre tagog ue ac ti o n s imilar to th at of ANP. Oppos ite res ult s we re obtained by Kocs is e t a l. ( 1995) in the turkey Meleagris gal/opm·o. Turkey adrenocortica l ce ll s are prov ided with a s ing le c lass of spec if ic hig h affinity ANP receptors, whose concentratio n (89,400/ce ll ) is 3.6-times hi gher than that of ANG- II recepto rs. AN P and BNP raise basa l a ld os te ro ne, but no t co rti cos te ro ne sec re ti o n by di spe rsed adre nocorti ca l ce ll s, th e minima l e ffec ti ve conce ntra tio n be ing 10-8 M a nd the effic ie ncy ord er be ing chi cken-ANP > rat- ANP = human-ANP » rat­BNP. A NP a lso enh ances a ldos tero ne res po nse to a maximal effec ti ve concentration of ACT I-I ( I Y l'o ), ANG­II (49 %) and K+ ( 137 %). Cyclic -GMP exert s s imil ar effects as AN P, and ANP increases cyc lic-GMP re lease by adrenal ti ssue , thereby suggesting that the mineralo­co rti co id sec re tagog ue ac ti o n of AN P in vo lves the ac tiva tio n o f g uanylate cyclase . Kocs ic and assoc ia tes try to ex pl ain the ir conflic ting results by stress ing that chi cken and turkey adrenocortica l cells possess di fferent phys iologica l properti es: the fo rmer be ing unresponsive and the lalle r responsive to ANG -II (Rosenberg e t aI. , 1988: Kocs is et aI. , 1995).

H. Other regulatory peptides

Other regulatory peptides are contained in the adrenal g lands of lower vertebrates , but at present no studies are avai lable on the ir effects on in vitro stero id secre tion o f adrenocorti ca l ce ll s .

I . Pancrea ti c po lypeptide family

Thi s group o f 36-amino ac id peptides includes , in addition to pancreatic po lypeptide, po lypeptide YY and

ne urope ptide Y (for rev iew, see Tayl or e t aI. , 1989) . T hey are wide ly distributed in the mammalian ti ssues, including adrenal medulla, and exert multiple important phys io log ica l functi ons, amo ng which is the regulation of adrenal sec re tion (for rev iew, see Malendowicz e t a I. , 1996; Nu ss d o rfe r , 1996) . Po ly pe ptide YY, a t ]I M concentrations, direc tl y depresses aldosterone secretion by rat zona g lo me rul osa ce ll s. Intramedullary ne uro­pe ptide Y is sure ly invo lved in the fine tuning o f zona g lo m e rul osa sec re ti o n und e r ph ys io log ica l a nd patho log ica l co nditio ns requiring e leva ted re lease of aldosterone; the mechanisms are both direct and indirec t , the la tte r in vo lv in g the stimulatio n o f ca tec ho lamine re le ase. Po ly pe ptid e YY- ir has bee n immun oc y to­chemi ca lly de monstra ted in the adrenal complex of the te leostean ee l (Re id e t a l.. 1995). Likew ise , neuropeptide Y-ir has been detected in adre na l chromaffin ce ll s o f the te leostean fi shes (Reid e t a I. , 1995; Wolfe nsberger et a I. , 1995) , anurans (Re inecke et a I. , 1992: Wolfe nsberger et a l.. 1995), li zards (Wolfe nsberger e t a I. , 1995) and birds (Garcia-A rrasas e t a I. , 1992 ; Wolfensberger e t aI. , 1995 ). According to Wolfe nsberger et al. (1 995), abo ut 30-40% of epinephrine ce ll s are pos itive, and many of them also contain ANP-ir.

2. Neurotens in

This 13-amino ac id peptide , wide ly di stributed in the ma mmali an ne rvous sys te m , exert s multipl e phys io ­logica l e ffects. inc luding the regulation of the pituitary­adre na l ax is. Ne uro te ns in is a lso co nt a in ed in th e mamm a li a n adre na l me dulla and direc tl y inhibit s a ld os te ro ne secre tion by zo na g lo m e rul os a ce ll s, probabl y thro ug h it s rece pto r-m edi a ted inte rfe re nce with the tr a n sdu c ti o n mec ha ni s m s of th e ago ni s ts ra is ing [Ca 2+L (fo r rev iew , see G a ng ul y and Dav is , 1994: Nu ss do rfe r , 1996 ). Ne uro te n s in - ir h as been immunocytoche mica ll y de tected in both nor-epinephrine ce ll s a nd ne rve fibers of the adrenal g land of the fia t snake Waglerophis merremii (Orezzoli e t aI. , 1995) .

V. Concluding Remarks

The preceding sections of thi s survey have shown that in the adre nal g lands of lo wer vertebrates chro maffin ce ll s may exert a stimul atory o r inhibito ry parac rine co ntro l o f s te ro id-h o rm o ne sec re ti o n by re leas in g ca tec ho la min es a nd seve ra l reg ul a to r y pe ptid es . Stimul a to r y m o le c ul es include se ro to nin , AVT , tac hy kinin s, V IP, PA CAP a nd CGRP ; inhibit o ry molecules are dopamine, SRIF and gal anin . Epinephrine and no re pin e phrin e possess an inhibito ry ac ti o n in Pisces a nd a stimul ato ry o ne in Aves, th e ir effec t in Amphibia be ing controversial. Likewi se, ANP exerts a stimulatory e ffect in Pisces and Aves, and an inhibitory action in Amphibia . The effect o f opio ids (enkephalins and endorphins) are ques tio nable . In severa l instances the phys iolog ica l re levance of a ll these secretagog ue or antisecre tagogue e ffec ts re mains rather doubtful , s ince they have been obse rved by expos ing ste roidogenic ce ll s

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to very high concentrations of the regulatory molecules . A t present. it seems th at onl y AYT. tachyk inins and PACAP may exert sizeable effec ts on steroidogenic ce lls at concentrati ons in the range of those they could locall y attain upon their release from chromaffin ce lls.

Compared to the huge mass of data available 0 11 this topic in mammals (more than 700 papers) (Nussdorfer. 1996) . studies carried out in lower vertebrates are very sca rcc ( l ess th an 60). Mo reove r. th ey are alm os t exclusive ly concerned w ith Amphibia and Ares . when thc few presentl y avail ab le data ind ica te th at a grea t vari ability occurs in the response of stero idogenic ce ll s to the various regulatory molecules among the di fferent classes of vertebrates.

[n concl usion. it appears that much further work must be done to clarify the functi onal interact ions be tween chromaffin and steroidogenic ce ll s in the adrenal glands of lower vertebrates. and wc shall now take the oppor­tunity to stress some not ye t addressed poi nts, whose elucidation should be the task of futu re in vestigations.

First of all. the steroidogenic or anti stcro idogenic effec ts o f so me regul ator y mo lec ules contai ned in adrenal chromaffin ce lls of lower vertebrates and which have been shown to pl ay important ro les in mammal s (e.g. neuropeptide Y and neul'Otensin ) have not ye t been studied.

The greater part of studies dea ling w ith the presence of regulatory peptides in lower-vertebrate adrenals have been done by immunocytochemica l techniques. Up to now. R[ A measurements of the intra-ad renal content are ava il abl e onl y for f ew reg ul ato r y pep tid es (AYT, tachykinin s and PACA P). and have bee n exc lu si ve ly carried out in Alllphihia . M oreover. the blood levels o f the various regulatory molecules have not been assayed. T his kind of in vestigation, coupled w ith those aimed at evaluating the i n )'i \'{) effec t of se lec ti ve antagonists, could all ow comparati ve endocrinolog ists to ascertain whether or not an in tra-adrenal regulatory peptide plays a phys iolog ica l ro le in the parac rine control of steroid hormone secret ion.

Finall y. mo lec ular bio logy in ves ti ga ti ons of intra­adre nal mRNA ex press ion of the va ri ous regul atory pc ptid es. un de r bo th norm al and cx pc rim c nt al conditions. have not ye t been performed. Parenthetica lly. it must be noted that analogous studi es carried out in mamm als have c lea rl y de mo nstr ated th at so me reg ulatory molecules pl aya relevant ro le onl y in the ph ys iopath o logy of the ad renal g land . when e.g. an excess of stero id hormone prod uction or a fluid and electro lyte imbalance have to be counteractcd.

The settlement of these and many other basic topics w ill not onl y open new frontiers in our know ledge of adrenal cy tophys iology in lower vertebrates , but surely will also improve our understanding of the significance of the morpho log ica l evo luti on of the adrenal g land among the classes of vertebrates.

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