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Localization and role of serotonin in the adrenal gland of Podarcis sicula (Reptilia, Lacertidae)
GENERAL AND COMPARATIVE
ENDOCRINOLOGY General and Comparative Endocrinology 132 (2003) 66–76 www.elsevier.com/locate/ygcen
Localization and role of serotonin in the adrenal gland of Podarcis sicula (Reptilia, Lacertidae) Anna Capaldo,* Vincenza Laforgia, Rosaria Sciarrillo, Salvatore Valiante, Flaminia Gay, and Lorenzo Varano Department of Evolutionary and Comparative Biology, University ‘‘Federico II,’’ Via Mezzocannone 8, Naples 80134, Italy Accepted 18 April 2002
Abstract The occurrence of serotonin (5-hydroxytryptamine; 5-HT) in the chromaffin cells of Podarcis sicula adrenal gland was demonstrated by immunocytochemical techniques: ABC and immunogold methods. At LM and EM levels, antiserum against 5-HT revealed serotonin immunoreactivity prevalently in noradrenalin (NA) cells, on and around secretory vesicles; adrenalin (A) cells appeared scarcely stained. The role of serotonin in the regulation of adrenal gland activity was studied in vivo using LM and EM techniques coupled to a specific radioimmunoassay for adrenocorticotropic hormone (ACTH) and corticosterone. 5-HT (0.7 mg/ 100 g body wt)/day for 4 days increased ACTH and corticosterone release; at LM and EM level clear signs of stimulation in the steroidogenic tissue were observed, as evidenced by the variations of lipid/cytoplasm ratio. In the chromaffin tissue, LM observations evidenced a variation of the numeric NA/A cell ratio; at EM level, chromaffin tissue showed intermediate cells with A, NA, and very clear granules with granular elements. The occurrence of these cells might be the result of a process of resynthesis following serotonin-stimulated catecholamine release. These data suggested that serotonin might be involved in the modulation of Podarcis pituitary–adrenal axis, and act as a paracrine factor to modulate corticosteroid production. Ó 2003 Published by Elsevier Science (USA). Keywords: Serotonin; Adrenal gland; Immunocytochemistry; ACTH; Corticosteroids; PNMT
1. Introduction The presence of the biogenic amine serotonin (5-hydroxytryptamine; 5-HT) has been demonstrated by immunocytochemistry and biochemical analysis in all vertebrate species, with the exception of reptiles. The head kidney of several fish species stores, in addition to catecholamines, also 5-HT, immunohistochemically localized in chromaffin cells of both the Atlantic cod and European eel, while in the head kidney of the rainbow trout it appears to be stored in a cell-type other than the chromaffin cell. Positive immunohistochemical labeling for antisera raised against serotonin was also observed in both the systemic and portal hearts of the Atlantic hagfish, M. glutinosa (Reid et al., 1995, 1998). In the interrenal gland of amphibians anurans, serotonin co* Corresponding author. E-mail address: [email protected] (A. Capaldo).
exists with adrenalin within the same secretory vesicles, suggesting that indoleamine and catecholamines may be concomitantly released during splanchnic nerve stimulation (Delarue et al., 1988a, 1992). The presence of serotonin has been demonstrated also in the adrenal gland of birds, where almost all adrenal medullary cells contain 5-HT (Ohmori et al., 1997) and in mammals (Holzwarth and Brownfield, 1985; Kondo, 1985; Kong et al., 1989; Mazzocchi et al., 1998; Nussdorfer, 1996), where the occurrence of 5-HT differs between the various species; in rat and pig adrenal gland 5-HT is synthesized by chromaffin cells, while in the mouse adrenal cortex 5-HT is contained in nerve fibres; in man, serotonin is present in perivascular mast cells (Contesse et al., 2000). Serotonin plays a pivotal role in the regulation of the hypothalamo–pituitary–adrenal axis. In fact, the release of the corticotrophin-releasing-factor (CRF) from the paraventricular nucleus of the hypothalamus is stimulated by serotonin (Dinan, 1996; Holmes et al.,
0016-6480/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0016-6480(02)00531-2
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1982). Moreover, 5-HT can act directly on the adenohypophysis and the adrenal gland by stimulating the release of ACTH and corticosteroids, independently of CRF (Dinan, 1996). In vivo and in vitro studies showed that serotonin stimulates mineralocorticoid and glucocorticoid secretion (Lefebvre et al., 1998; Nussdorfer, 1996) in various species (including human), interacting with a wide variety of receptors in the different species. In humans, the stimulatory effect of 5-HT on adrenocortical cells is mediated through a 5-HT4 receptor subtype positively coupled to adenylyl ciclase and calcium influx (Contesse et al., 2000; Lefebvre et al., 1992), while in the rat the effect of the indoleamine on aldosterone secretion is mediated via activation of 5-HT7 receptors (Contesse et al., 1999). In amphibians, serotonin appears to enhance steroid secretion of frog adrenocortical cells in a concentrationdependent manner, through the activation of 5-HT4 receptors positively coupled to adenylyl ciclase (Delarue et al., 1988b; Idres et al., 1989, 1991); the activation of these receptors in the adrenocortical cells causes stimulation of adenylyl ciclase and subsequently increases calcium influx through a T-type calcium channel (Contesse et al., 1996). Moreover, detailed studies on frog interrenal tissue showed that corticosteroidogenesis is stimulated by acetylcholine, released from splanchnic nerve terminals, as well as by serotonin and VIP, both contained in the chromaffin cells, and that serotonin may interact with acetylcholine and VIP on their target cell to modulate the activity of their congeners. Since these three bioactive signals are released in response to splanchnic nerve stimulation, it is possible that the three neuroregolators may be involved in the precise adaptation of the adrenocortical cell activity in stress condition (Leboulenger et al., 1988). The data on serotonin effects on adrenochromaffin tissue are quite controversial; in fact, 5-HT, which is co-released with catecholamines, increases Naþ dependent Ca2þ efflux from cultured bovine adrenochromaffin cells, stopping catecholamine secretion (Minakuchi et al., 1997). In teleosts and cyclostomes, serotonin administration induces an increase in catecholamine plasma levels, either indirectly via the activation of neural pathways or directly interacting with the adrenochromaffin cells (Bernier and Perry, 1996; Fritsche et al., 1992, 1993; Reid et al., 1998); the release of serotonin, at least in either the rainbow trout or the Atlantic hagfish is not under cholinergic control (Reid et al., 1998). In contrast to the extensive literature available regarding the presence and the involvement of serotonin in the modulation of corticosteroidogenesis in mammals, amphibians, and fish, little is known about its role in reptiles. Therefore, in the present work, immunocytochemical methods were used to study the cellular and subcellular distribution of 5-HT in the adrenal gland of Podarcis sicula, the morphology and physiology of which are well known. In this species chromaffin and
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steroidogenic cells are strongly associated, so to suggest the existence of an high degree of co-regulation between the two cellular types; in fact, as in mammals and birds, glucocorticoids secreted by steroidogenic cells activate the enzyme PNMT (methylating noradrenalin into adrenalin), leading to an increase in the number of adrenalin cells, and a variation in the numeric ratio NA/ A cells (Laforgia and Muoio, 1997; Laforgia and Varano, 1978; Laforgia et al., 1982). Moreover, by analogy with other vertebrates, also Podarcis chromaffin cells contain, in addition to adrenalin and noradrenalin, a lot of secretory products as VIP (Laforgia et al., 1999), NPY (Muoio et al., 1995) and others, involved in the modulation of steroidogenic activity. Therefore the goals of this study have been (1) to localize the presence of 5-HT (2) to investigate its role in the control of the adrenal gland activity, to verify if also in reptiles, that represent an evolutionarily intermediate class, serotonin plays a role similar to that observed in the other vertebrates.
2. Materials and methods 2.1. Animals and housing conditions Adult specimens of P. sicula, (weighing 13–15 g) were captured in the neighbourhood of Naples in December, when adrenals undergo functional stasis (Varano et al., 1969). After capture, the animals were housed in large soil-filled terraria containing heather, and exposed to natural temperature and photoperiod. Water dishes were present in the terraria, and the animals were fed on live fly larvae daily. Captivity lasted 20 days to reverse capture-related stress (Manzo et al., 1994). 2.2. Experimental procedure The animals received intraperitoneal injections of serotonin. Plasma serotonin level has not yet been determined; to ensure that ip administration of 5-HT was reasonable, in a separate series of experiments, serotonin was administered in doses close to the doses used previously in mammals (Nussdorfer, 1996); in fact data on serotonin administration in reptiles are lacking. The effects of the ip 5-HT on plasma levels of ACTH and corticosterone were assessed; based on the results of the dose–response experiments, the right dose of serotonin was chosen for the following experiments. The specimens were divided into five groups, each consisting of 20 animals (10 males and 10 females): Group 1. The animals were given a single intraperitoneal injection of serotonin (5-hydroxytryptamine hydrochloride, Sigma) (0.7 mg/100 g body wt)/day for four consecutive days. 5-HT was dissolved in reptilian physiological solution (NaCl 0.75%) with an injection volume
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of 0.1 ml. Injections were between 8.00 and 8.30 a.m., and the animals were sacrificed 2 h after the last injection. Group 2. The animals were treated as group 1 but sacrificed 24 h after the last injection. Group 3. Untreated control animals. Group 4. Control animals receiving intraperitoneal injections of reptilian physiological solution (NaCl 0.75%) and sacrificed as group 1. Group 5. Control animals receiving intraperitoneal injections of reptilian physiological solution (NaCl 0.75%) and sacrificed as group 2. The animals were anesthetized by hypothermia, chilling them in chipped ice. Blood samples were collected by intracardiac puncture and put into heparinized capillars. They were then centrifuged at 1500 rpm for 10 min to obtain plasma. 2.3. Light microscopy Immediately after collection of blood samples, the animals were killed by decapitation, and their adrenals were fixed in a mixture of 2.5% potassium dichromate, 1% anhydrous sodium sulphate (buffered at pH 4.1 with 5 M acetate buffer), and 10% formaldehyde (Wood, 1963). They were then embedded in paraplast, cut into 7 lm sections with a rotary microtome, affixed to albuminized slides and stained with one of the following solutions: (1) a mixture of eosine aniline blue, buffered at pH 4 with acetate buffer 5 M (Wood, 1963); (2) Giemsa solution, modified according to Pearse (1960); (3) Mallory trichromic stain. Observations were performed using a Zeiss Axioskop microscope; images were captured with a camera attached to an IBM computer running the Kontron Elektronik KS 300 image analysis system and Adobe Photoshop. 2.4. Transmission electron microscopy The adrenal glands were fixed in 2.5% glutaraldehyde in MillonigÕs phosphate buffer at pH 7.4 at 4 °C, rinsed in buffer and postfixed in 1% Os O4 (2 h, 4 °C), dehydrated in ethanol, cleared in propylene oxide, embedded in epoxy resin and polymerized. Ultrathin section (30 nm) were collected on formvar coated copper grids, stained with solutions of uranyl acetate and lead citrate and observed with a Philips EM 301 transmission electron microscope at the Interdepartmental Center for Biological Ultrastructures (Naples). 2.5. Quantitative analysis In 10 specimens from each experimental group, processed for LM, NA/A cell ratio was calculated from cell counts in every tenth longitudinal section from the whole gland of each specimen.
In 10 specimens from each experimental group, processed for EM, 10 low-power micrographs of steroidogenic cells, containing at least four cells for each sample, were examined to evaluate, in each cell, the area occupied by lipid droplets (lipid/cytoplasm ratio). The areas of both lipid droplets and cytoplasms were measured with the help of a planimeter. Variations of lipid contents in control and treated animals were calculated using the formula Lipid=cytoplasm ratio ¼
Lipid droplet area ðlm2 Þ : Cytoplasmic area ðlm2 Þ
2.6. Hormone assay Plasma corticosterone was measured using a sensitive and highly specific radioimmunoassay kit (ICN Biomedicals, COSTA MESA, CA). Before assay, experimental plasma samples were diluted one-third with the kit diluent and heated at 80° for 10 min in order to inactivate corticosterone-binding proteins. Corticosterone titers in nanograms per milliliter were calculated using the standard curve generated in the assay. Standard curves were prepared in buffer with known amounts of radio inert corticosterone (0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 ng/ml) purchased from Amersham (Arlington Heights, IL). The minimum concentration per tube that was distinguishable from zero was 0.176 ng/ml. Cross-reactivities of the corticosterone antiserum with other steroids were 6.1% for deoxycorticosterone; <1% for progesterone and cortisol; <0.1% for aldosterone, 20-a dehydroprogesterone, testosterone, and 11-deoxycortisol; and <0.01% for all the other steroids examined. Inter- and intraassay coefficients of variation were 3.4 and 5.8%, respectively. Plasma ACTH concentrations were measured by a two-site immunoradiometric assay (IRMA) using mouse monoclonal antibodies (Diagnostic Products). Boric acid was added to serum to adequately preserve ACTH. Sensitivity was 0.1 pg/ml, and the inter- and intraassay coefficients of variation were 10 and 6%, respectively. 2.7. Statistical analysis The data obtained from each specimen were averaged per experimental group, and the SD of the mean was calculated. As revealed by the v2 test, the data were not different from normal distribution. After ANOVA, statistical comparison of the data was performed by use of the Multiple Range Test of Duncan. 2.8. Immunocytochemistry The sections were treated by the avidin-biotinylated peroxidase complex (ABC) method (Hsu et al., 1981).
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For each of control groups (3, 4, and 5) the left adrenal glands of ten specimens (30 adrenal glands) were fixed in 2.5% glutaraldehyde in PBS (phosphate buffer saline, pH 7.4) for 2 h, then dehydrated and embedded in paraplast. Rehydrated sections were placed in 3% hydrogen peroxide solution for 5 min to inhibit endogenous peroxidases. The sections were incubated with normal goat serum (NGS) for 20 min, and then incubated with anti-rabbit 5-HT (Biogenesis, Poole, England) (1:300) overnight at 4 °C in a moist chamber. After rinsing in PBS, the sections were incubated for 30 min with biotinylated goat anti-rabbit IgG (1:400) (Vecta-lab ‘‘Elite’’ (ABC) kit, Vector Laboratories, Burlingame, CA) and, after rinsing in PBS, incubated for 30 min with Vectastain Elite ABC reagent. Immunoreactivity was visualized by a reaction with 0.05% 3,30 -diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO) and 0.01% hydrogen peroxide in 0.05 M Tris–HCl buffer, pH 7.2 for 10 min to reveal the brown immunoreactive cells. After the DAB reaction, the sections were counterstained with ematoxylin. For each adrenal gland, four longitudinal sections were examined (120 light micrographs). 2.9. Immunoelectron microscopy Thin sections of adrenals of 10 specimens of each of control groups (3, 4, and 5), mounted on formvar coated nickel grids, were etched in saturated sodium metaperiodate for 1 h, (De Mey et al., 1981), then washed in bidistilled water, exposed to a 1% BSA solution (Sigma bovine serum albumin, essentially globulin free) in TBS (Tris buffered salt) for 30 min, followed by an overnight incubation at 4 °C with anti-rabbit 5-HT (Biogenesis, Poole, England) 1:300 in 1% BSA-TBS. The grids were rinsed several times at room temperature in 1% BSATBS and incubated for 90 min with goat anti-rabbit gold labeled secondary antibody (10 nm, Sigma) (1:50), wa-
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shed several times with 1% BSA-TBS, then with bidistilled water, and stained with uranyl acetate and lead citrate. For each specimen, 10 electron micrographs were examined (300 electron micrographs). For LM and EM immunocytochemistry, controls were made by (1) replacing specific antiserum with normal rabbit serum (2) omitting primary antibody. No immunoreactive structures were observed.
3. Results 3.1. Control specimens 3.1.1. LM observations No differences were observed in the adrenal gland morphology between untreated specimens (group 3) and specimens injected with physiological solution (groups 4, 5). The adrenal gland of P. sicula was formed by chromaffin and steroidogenic tissues. The latter tissue consisted of prismatic cells with a weakly stained cytoplasm, which were arranged in anastomosed cordons of two cell layers intermingled with thin blood vessels (Fig. 1A). The chromaffin tissue was arranged in a dorsal cordon, the most peripherical portion of which consisted of noradrenalin (NA) cells, whereas adrenalin cells (A) were found in its innermost portion and in its digitations penetrating the steroidogenic parenchyma. Small groups of A cells were observed among the steroidogenic cordons (Figs. 1A and B). NA cells were more numerous than A cells with a NA/A ratio of 1.4/1 (Table 1). 3.1.2. EM observations The ultrastructural characters of the adrenal gland were similar in the specimens of groups 3, 4, and 5. The steroidogenic cells contained numerous mitochondria, a smooth endoplasmic reticulum arranged in tubules and
Fig. 1. Light micrographs of P. sicula adrenal gland. Control specimen. Mallory trichromic stain. Steroidogenic tissue is formed by anastomosed cords (A); chromaffin tissue forms a dorsal ribbon, with NA cells (gold yellow) in the outer layers, and A cells (red orange) in the inner layers and islets interspersed between steroidogenic cords (A,B). St, steroidogenic tissue; NA, noradrenalin cells; A, adrenalin cells. Calibration bars, 100 lm.
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Table 1 Numeric NA/A cell ratios following serotonin administration Experimental procedure
NA cells ðmean SEÞ
A cells ðmean SEÞ
NA/A cell ratio
Group Group Group Group Group
380 0:05a; b 760 0:07 719 0:06 638 0:05 736 0:06
1151 0:1a;b 768 0:07 513 0:05 455 0:05 525 0:05
0.3/1 1/1 1.4/1 1.4/1 1.4/1
1 2 3 4 5
(10) (10) (10) (10) (10)
Note. Numbers in parentheses indicate the numbers of animals. a P < 0:01 from group 4. b P < 0:01 from group 3.
vesicles and a large amount of lipid vacuoles accounting for most of the cytoplasm (Fig. 2A) with a lipid/cytoplasm ratio of 0.44 (Table 2). Accumulation of steroids is typical of winter specimens (Varano et al., 1969). The chromaffin cells showed a nucleus variable in shape, an incospicuous endoplasmic reticulum mostly constituted by rough membranes, few mitochondria and a paranuclear Golgi complex. The most typical constituents were the chromaffin granules, which, in A cells, appeared roundish, moderately electron-dense, with a variable halo separating the core from the limiting membrane (Fig. 2B). NA granules were polymorphic and intensely electron-dense with a core closely adhering to the limiting membrane. These granules sometimes appeared enlarged with an eccentric core (Fig. 2C). 3.1.3. Hormone assay Plasma levels of ACTH were: 4:18 0:05 pg/ml in untreated control animals (group 3); 4:14 0:05 pg/ml in group 4, and 4:22 0:05 pg/ml in group 5 (Fig. 3A). Corticosterone plasma levels were 3:87 0:04 ng/ml in specimens of group 3; 3:90 0:04 ng/ml in group 4; and 3:85 0:04 ng/ml in group 5 (Fig. 3B). 3.2. Specimens injected with serotonin 3.2.1. LM observations After 2 h from the 4th serotonin injection, clear signs of stimulation were observed in the steroidogenic tissue, namely dilated blood vessels and hypertrophic cordons formed by swollen cells (Fig. 4A). In the chromaffin tissue, A cells increased in number, also occupying the most peripheral portions of the cordon, usually consisting of NA cells (Figs. 4A and B), Therefore, there was a variation in the NA/A cell ratio, which became 0.3/1 (Table 1). After 24 h, when the signs of stimulation in the steroidogenic tissue were no more evident (Fig. 4C), in the chromaffin tissue the NA/A cell ratio attained values similar to those of control specimens again (1/1) (Figs. 4C and D and Table 1). 3.2.2. EM observations After 2 h from the 4th serotonin injection, the steroidogenic cells showed a cytoplasm characterized by a marked development of the smooth endoplasmic retic-
ulum, the presence of numerous mitochondria with well evident crests, and the almost complete absence of lipid vacuoles (Fig. 5A). The decrease in lipid content was confirmed by the decrease in the lipid/cytoplasm ratio value, which became 0.07 (Table 2). After 24 h, in the steroidogenic cells, which no longer showed signs of intense stimulation, the lipid content appeared similar to that in control specimens, as can be inferred from the lipid/cytoplasm ratio value of 0.39 (Table 2). In the chromaffin tissue, besides A and NA cells, there were chromaffin cells with a cytoplasm showing both A and NA cells at the same time, as well as very clear granules, often closely packed together, containing granular ele in diameter (Figs. ments ranging between 340 and 347 A 5B, 6A). Many chromaffin cells had a low content of catecholamine granules (Fig. 6B). 3.2.3. Hormone assay After 2 h from the 4th serotonin injection, the ACTH plasma value was 5:69 0:01 pg/ml, increasing to 6:50 0:05 pg/ml after 24 h (Fig. 3A); the corticosterone plasma value was 7:25 0:03 ng/ml, increasing to 8:28 0:06 ng/ml after 24 h (Fig. 3B). 3.2.4. Immunocytochemistry Antiserum against serotonin revealed, in the adrenal gland of Podarcis, 5-HT immunoreactivity, that appears marked in the noradrenalin cells, disposed in the outer layers of the chromaffin ribbon; the adrenalin cells appear scarcely stained. The immunoreactivity was restricted to the cytoplasm of positive cells; no fibres immunopositive to 5-HT could be detected. Steroidogenic cells did not appear immunostained (Fig. 7A). Control sections exhibited no staining (Fig. 7B). 3.2.5. Immunoelectron microscopy At EM level, the chromaffin cells appeared immunopositive to serotonin; gold particles were localized on and around secretory vesicles of NA cells (Fig. 8A), while immunolabeling appeared weak on adrenalin granules (figure not shown). Immunoreactive granules were distributed throughout the cytoplasm, showing no preferential distribution; no immunopositivity was found in the steroidogenic tissue. Control sections did not stain for 5-HT (Fig. 8B).
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Fig. 2. Electron micrographs of control specimen interrenal gland. The cytoplasm of steroidogenic cells (A) presents several mitochondria and many lipid droplets. In the adrenalin cells (B), A granules appear rounded, of medium electron density and with a clear halo between the core and the limiting membrane.The noradrenalin cells (C) present polymorphic, strongly electron-dense chromaffin granules. L, lipid droplets; M, mitochondria; A, adrenalin granule; NA, noradrenalin granule. Calibration bars, 1 lm.
4. Discussion The results of the present study indicate that, in P. sicula adrenal gland, serotonin is located in chromaffin
cells. They also demonstrate that 5-HT can influence the activity of the pituitary–interrenal axis. Several studies have revealed that adrenochromaffin cells contain and release not only catecholamines, but
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Table 2 Lipid/cytoplasm ratio values in the steroidogenic cells following serotonin administration Experimental procedure
Lipid droplets area ðmean SEÞ
Cytoplasmic area ðmean SEÞ
Lypid/cytoplasm ratio ¼ lipid area/cytoplasmic area
Group Group Group Group Group
2:8 0:06a ;b 13:6 0:04 15:05 0:05 15:03 0:05 15:07 0:06
37:5 0:04 34:8 0:03 34:1 0:05 34:3 0:05 34:1 0:04
0.07 0.39 0.44 0.44 0.44
1 2 3 4 5
(10) (10) (10) (10) (10)
Note. Numbers in parentheses indicate the number of animals. a P < 0:01 from group 4. b P < 0:01 from group 3.
Fig. 3. ACTH (A) and corticosterone (B) plasma levels in control (groups 3, 4, and 5) and treated (groups 1 and 2) specimens.
also many biogenic amines and regulatory peptides that may modulate the function of adrenocortical cells in a paracrine manner (Bornstein et al., 1997; Mazzocchi et al., 1998). 5-HT has been biochemically and immunocytochemically demonstrated in the adrenal gland of mammals, where it is localized in rat A cells and in NA pig cells (Nussdorfer, 1996), birds (Ohmori et al., 1997), anurans, where 5-HT has been evidenced in A cells (Delarue et al., 1992) and fish, where serotonin is present in many DBH-positive cells within the PCV of Atlantic cod and European eel (Reid et al., 1995, 1998). Our results show that chromaffin cells contain 5-HT in P. sicula as well. By light microscopic immunocytochemical techniques, the cytoplasm of NA cells appears immunopositive to serotonin, while A cells result scarcely stained. Electron microscopic studies confirmed light microscopic immunocytochemistry data, showing 5-HT immunoreactivity on the chromaffin granules of NA
cells, where labeled vesicles are distributed throughout the cytoplasm. At EM level, the immunopositivity to 5HT was weak on A chromaffin granules (figure not shown). The presence of 5-HT in the chromaffin tissue of P. sicula raises the question of the origin of the indoleamine, namely whether 5-HT is synthesized within chromaffin cells or taken up from the circulation. Uptake of 5-HT by adrenalin-storing cells has been observed in the rat (Verhofstad and Jonsson, 1983) and mouse adrenal medulla (Kent and Coupland, 1984), while, in anurans, 5-HT is synthesized by chromaffin cells from L -tryptophan (Delarue et al., 1992). Further studies are needed to determine whether chromaffin cells are able to synthesize serotonin in P. sicula as well. 5-HT markedly affects the adrenal gland of P. sicula. In vivo and in vitro studies have shown that 5-HT stimulates ACTH release (Dinan, 1996) and mineralo-
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Fig. 4. Specimen of group 1 (A,B) and group 2 (C,D). Mallory trichromic stain (A,B,C) and Giemsa stain (D). Steroidogenic cords appear swollen, separated by dilated blood vessels (A). In the chromaffin ribbon (B), A cells appear also in the outer layers of chromaffin tissue, usually filled with NA cells. After 24 h from the last injection, the adrenal gland morphology (C) and the distribution of NA (dark green) and A (light green) cells (D) appear similar to that of control specimen. St, steroidogenic tissue; NA, noradrenalin cells; A, adrenalin cells. Calibration bars, 100 lm (A), 50 lm (B–D).
Fig. 5. Electron micrographs of a steroidogenic cell of group 1 (A) showing the cytoplasm almost devoid of lipid droplets, the smooth endoplasmic reticulum greatly developed and numerous mitochondria, and of a chromaffin cell of group 2 (B) where, in the cytoplasm of the same cell A, NA and very clear granules are present. L, lipid droplet; M, mitochondria; SER, smooth endoplasmic reticulum; NA, noradrenalin granule; A, adrenalin granule; * (CG), very clear granule. Calibration bars, 1 lm.
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Fig. 6. Electron micrographs of chromaffin cells of group 2 specimen. Very clear granules contain granular elements ranging between 340 and 347 A in diameter (A). Many chromaffin cells (B) show a reduced amount of secretory vesicles. NA, noradrenalin granule; A, adrenalin granule; CG, very clear granule. Calibration bars, 1 lm.
Fig. 7. Light micrographs of P. sicula adrenal gland. ABC reaction (A) showing NA cells immunoreactive to 5-HT, while A cells only lightly stained; steroidogenic cells are immunonegative. Control section (B) showing no immunopositivity to 5-HT. NA, noradrenalin cells; A, adrenalin cells; St, steroidogenic tissue. Calibration bars, 100 lm.
and glucocorticoid secretion from adrenal cells (Contesse et al., 1996, 1999, 2000; Delarue et al., 1988b; Idres et al., 1989, 1991; Leboulenger et al., 1988; Lefebvre et al., 1998; Nussdorfer, 1996; Reid et al., 1998). Our results indicate that 5-HT enhances ACTH release and corticosterone production from adrenocortical cells, in P. sicula as well. Ultrastructural features of steroidogenic tissue and the lipid/cytoplasm ratio values revealed a strong stimulation of steroidogenic cells, as is also confirmed by the results of the hormone assays revealing an increase in corticosteroid plasma levels consequent on serotonin administration. In the lizard, capture-related stress causes a drastic increase in corticosteroids, and this effect is reversed after acclimatization in terrarium for some time (Manzo et al., 1994). Therefore, the animals were acclimatized for twenty
days in order to rule out that the increase in corticosteroid plasma levels was due to capture-related stress. Moreover, the influence of handling stress must be considered negligible due to the lack of significant differences in the plasma levels of ACTH and corticosteroids in the untreated specimens (group 3) and in those injected with physiological solution (groups 4, 5). In reptiles, like in mammals, the PNMT enzyme (phenyletanolamine-N-methyl transferase), converting NA into A, is activated by corticosteroids (Laforgia and Muoio, 1997; Laforgia and Varano, 1978; Laforgia et al., 1982). Therefore, the increased corticosteroid plasma levels, resulting from 5-HT administration, enhance PNMT activity, inducing an increase in the number of A cells compared to NA cells and a variation in the NA/A cell ratio.
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Fig. 8. Electron micrographs of NA cells. (A) Immunogold reaction. Noradrenalin granules appear immunopositive to 5-HT. (B) Control section showing absence of immunopositivity to 5-HT. Calibration bars, 0:5 lm.
Literature data on the effects of serotonin on the adrenochromaffin tissue are contrasting. In fact, 5-HT has a role in stopping catecholamine secretion from cultured bovine adrenochromaffin cells (Minakuchi et al., 1997), whereas it causes an increase in catecholamine plasma levels in teleosts and cyclostomes (Bernier and Perry, 1996; Fritsche et al., 1992, 1993). In P. sicula, after 5-HT treatment, besides NA and A cells, the chromaffin tissue shows chromaffin cells with a cytoplasm containing both NA and A granules, as well as very clear granules, closely packed together, containing in granular elements ranging between 340 and 347 A diameter. The occurrence of these cells and of chromaffin cells with a low content of secretory granules suggests that 5HT might have induced catecholamine release from the adrenochromaffin cells, followed by a process of resynthesis. This is confirmed by the presence of secretory granules with variable electron density, which would correspond to subsequent stages of the process of amine deposition. These data suggest that serotonin might have a stimulating action on the hypophyseal–interrenal axis, increasing ACTH and corticosterone release, and hence influencing the adrenochromaffin tissue. Further studies are needed to detect and identify the type of serotonin receptors on the chromaffin and steroidogenic cells, as well as to establish whether the 5-HT action on the adrenal gland is direct or mediated through ACTH release. Immunocytochemical studies revealed that, besides catecholamines, adrenochromaffin cells produce and release a variety of regulatory peptides, which may affect the secretory activity of steroidogenic cells in a paracrine manner (Delarue et al., 1988a, 1992; Mazzocchi et al., 1998; Verhofstad and Jonsson, 1983). In
the adrenal gland of P. sicula, chromaffin and steroidogenic tissues are markedly intermingled, suggesting that chromaffin and steroidogenic cells may interact with each other; in addition, not only steroid hormones may activate the enzymes involved in adrenalin synthesis, but also the chromaffin cells may modulate the function of steroidogenic cells in a paracrine manner. In P. sicula adrenal gland, chromaffin cells contain NPY, Leu-Enkephalin, b-endorphin (Muoio et al., 1995), substance P (De Falco et al., 1999), VIP (Laforgia et al., 1999), inhibin (Manzo et al., 2000); the presence and the effects of serotonin suggest that 5-HT may be involved in the physiological regulation of the adrenal gland, in P. sicula as well.
References Bernier, N.J., Perry, S.F., 1996. Control of catecholamine and serotonin release from the chromaffin tissue of the atlantic hagfish. J. Exp. Biol. 199, 2485–2497. Bornstein, S.R., Ehrhart-Bornstein, M., Scherbaum, W.A., 1997. Morphological and functional studies of the paracrine interaction between cortex and medulla in the adrenal gland. Mic. Res. Technique 36, 520–533. Contesse, V., Hamel, C., Lefebvre, H., Dumuis, A., Vaudry, H., Delarue, C., 1996. Activation of 5-hydroxytryptamine4 receptors causes calcium influx in adrenocortical cells: involvement of calcium in 5-hydroxytryptamine-induced steroid secretion. Mol. Pharmacol. 49, 481–493. Contesse, V., Lefebvre, H., Lenglet, S., Kuhn, J.M., Delarue, C., Vaudry, H., 2000. Role of 5-HT in the regulation of the brain– pituitary–adrenal axis: effects of 5-HT on adrenocortical cells. Can. J. Physiol. Pharmacol. 78, 967–983. Contesse, V., Lenglet, S., Grumolato, L., Anouar, Y., Lihrmann, I., Lefebvre, H., Delarue, C., Vaudry, H., 1999. Pharmacological and molecular characterization of 5-hydroxytryptamine7 receptors in the rat adrenal gland. Mol. Pharmacol. 68, 552–561.
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De Falco, M., Capaldo, A., Valiante, S., Laforgia, V., Varano, L., 1999. Immunocytochemical localization of substance P in the adrenal gland of the lizard Podarcis sicula. E.J.H. 43 (suppl 2), 14. Delarue, C., Becquet, D., Idres, S., Hery, F., Vaudry, H., 1992. Serotonin synthesis in adrenochromaffin cells. Neuroscience 46, 495–500. Delarue, C., Leboulenger, F., Morra, M., Hery, F., Verhofstad, A.J., Berod, A., Denoroy, L., Pelletier, G., Vaudry, H., 1988a. Immunohistochemical and biochemical evidence for the presence of serotonin in amphibian adrenal chromaffin cells. Brain Res. 459, 17–26. Delarue, C., Lefebvre, H., Idres, S., Leboulenger, F., Homo-Delarche, G., Lihrmann, I., Feuilloley, M., Vaudry, H., 1988b. Serotonin stimulates corticosteroid secretion by frog adrenocortical tissue in vitro. J. Steroid Biochem. 29 (5), 519–525. De Mey, J., Moeremans, M., Geuens, G., Nuydens, R., De Brabander, M., 1981. High resolution light and electron microscopic localization of tubulin with IGS (immunogold staining) method. Cell. Biol. Int. Rep. 5, 889–899. Dinan, T.G., 1996. Serotonin and the regulation of hypothalamic– pituitary–adrenal axis function. Life Sci. 58 (20), 1683–1694. Fritsche, R., Reid, S.G., Thomas, S., Perry, S.F., 1993. Serotoninmediated release of catecholamines in the rainbow trout Oncorhynchus mykiss. J. Exp. Biol. 178, 191–204. Fritsche, R., Thomas, S., Perry, S.F., 1992. Effects of serotonin on circulation and respiration in the rainbow trout, Oncorhynchus mykiss. J. Exp. Biol. 173, 59–73. Holmes, M.C., Di Renzo, G., Beckford, U., Gillham, B., Jones, M.T., 1982. Role of serotonin in the control of secretion of corticotrophin releasing factor. J. Endocr. 93, 151–160. Holzwarth, M.A., Brownfield, M.S., 1985. Serotonin coexists with epinephrine in rat adrenal medullary cells. Neuroendocrinology 41, 230–236. Hsu, S.M., Raine, L., Fanger, H., 1981. Use of avidin–biotin peroxidase complex (ABC) in immunoperoxidase techiques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577–580. Idres, S., Delarue, C., Lefebvre, H., Larcher, A., Feuilloley, M., Vaudry, H., 1989. Mechanism of action of serotonin on frog adrenal cortex. J. Steroid Biochem. 34 (1–6), 547–550. Idres, S., Delarue, C., Lefebvre, H., Vaudry, H., 1991. Benzamide derivatives provide evidence for the involvement of a 5-HT4 receptor type in the mechanism of action of serotonin in frog adrenocortical cells. Mol. Brain Res. 10, 251–258. Kent, C., Coupland, R.E., 1984. On the uptake and storage of hydroxytriptamine, 5-hydroxyptophan and catecolamines by adrenal chromaffin cells and nerve endings. Cell Tissue Res. 236, 189– 195. Kondo, H., 1985. Immunohistochemical analysis of the localization of neuropeptides in the adrenal gland. Arch. Histol. Jpn. 48, 453–481. Kong, J.Y., Thureson-Klein, A., Klein, R.L., 1989. Differential distribution of neuropeptides and serotonin in pig adrenal glands. Neuroscience 28 (3), 765–775. Laforgia, V., De Falco, M., Capaldo, A., Varano, L., 1999. Immunohistochemical localization of VIP in the adrenal gland of the lizard Podarcis sicula. Brain Res. 848 (1,2). Laforgia, V., Muoio, R., 1997. Effects of ACTH and corticosteroids on phenylethanolamine-N-methyltransferase (PNMT) expression as determined by immunocytochemical localization in the adrenal gland of the lizard Podarcis sicula. Ital. J. Zool. 64, 301–306. Laforgia, V., Varano, L., 1978. The influence of the interrenal steroidogenic tissue on the chromaffin cells of the adrenal gland of Lacerta s.sicula Raf.: effects of ACTH administration during the winter. Cell. Mol. Biol. 23, 370–390.
Laforgia, V., Varano, L., Cavagnuolo, A., Putti, R., 1982. Modificazioni nella distribuzione dei due tipi di cellule cromaffini nella surrenale di Podarcis s.sicula Raf. dopo somministrazione di ACTH e di cortisone. Annu. Ist. Mus. Zool. Univ. Napoli 25, 103– 112. Leboulenger, F., Benyamina, M., Delarue, C., Netchita€ılo, P., SaintPierre, S., Vaudry, H., 1988. Neuronal and paracrine regulation of adrenal steroidogenesis: interactions between acetylcholine, serotonin and vasoactive intestinal peptide (VIP) on corticosteroid production by frog interrenal tissue. Brain Res. 453, 103–109. Lefebvre, H., Contesse, V., Delarue, C., Feuilloley, M., Hery, F., Grise, P., Raynaud, G., Verhofstad, A.J., Wolf, L.M., Vaudry, H., 1992. Serotonin-induced stimulation of cortisol secretion from human adrenocortical tissue is mediated through activation of a serotonin4 receptor subtype. Neuroscience 47 (4), 999–1007. Lefebvre, H., Contesse, V., Delarue, C., Vaudry, H., Kuhn, J.M., 1998. Serotonergic regulation of adrenocortical function. Horm. Metab. Res. 30 (6–7), 398–403. Manzo, C., Capaldo, A., Laforgia, V., Muoio, R., Angelini, F., Varano, L., 2000. Inhibin in the testis and adrenal gland of the male lacertid, Podarcis sicula Raf.: a light immunocytochemical study. Eur. J. Histochem. 44, 285–293. Manzo, C., Zerani, M., Gobbetti, A., Di Fiore, M.M., Angelini, F., 1994. Is corticosterone involved in the reproductive processes of the male lizard, Podarcis sicula sicula? Horm. Behav. 28, 117– 129. Mazzocchi, G., Gottardo, G., Nussdorfer, G.G., 1998. Paracrine control of steroid hormone secretion by chromaffin cells in the adrenal gland of lower vertebrates. Histol. Histopathol. 13, 209– 220. Minakuchi, K., Houchi, H., Yoshizumi, M., Ishimura, Y., Morita, K., Takasugi, M., Oka, M., Tamaki, T., 1997. Serotonin increases Naþ -dependent Ca2þ efflux from bovine adrenal chromaffin cells in culture. Neurosci. Lett. 223, 17–20. Muoio, R., Laforgia, V., Capaldo, A., Varano, L., 1995. Electron immunocytochemical localization of neuropeptide Y, Leu-Enkephalin and b-endorphin in the catecholamines containing cells of the lizard adrenal medulla. Atti XX Cong. M.E. Rimini, 43. Nussdorfer, G.G., 1996. Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol. Rev. 48, 495–530. Ohmori, Y., Okada, Y., Watanabe, T., 1997. Immunohistochemical localization of serotonin, galanin, cholecystokinin, and methionine-enkephalin in adrenal medullary cells of the chicken. Tissue Cell 29 (2), 199–205. Pearse, A.G.E., 1960. Histochemistry Theoretical and Applied. Little, Brown & Co, Boston. Reid, S.G., Bernier, N.J., Perry, S.F., 1998. The adrenergic stress response in fish: control of catecholamine storage and release. Comp. Biochem. Physiol. C 120, 1–27. Reid, S.G., Fritsche, R., J€ onsson, A.C., 1995. Immunohistochemical localization of bioactive peptides and amines associated with the chromaffin tissue of five species of fish. Cell Tissue Res. 280, 499– 512. Varano, L., Della Corte, F., Galgano, M., 1969. Aspetti ultrastrutturali delle cellule steroidogenetiche dellÕinterrenale di Lacerta s.sicula Raf. in esemplari invernali e primaverili. Atti Soc. pelorit. Sci. fis. mat. nat. 15, 31–38. Verhofstad, A.A.J., Jonsson, G., 1983. Immunohistochemical and neurochemical evidence for the presence of serotonin in the adrenal medulla of the rat. Neuroscience 10, 1443–1453. Wood, J.G., 1963. Identification of and observations on epinephrine and norepinephrine containing cells in the adrenal medulla. Am. J. Anat. 112, 285–304.
ENDOCRINOLOGY General and Comparative Endocrinology 132 (2003) 66–76 www.elsevier.com/locate/ygcen
Localization and role of serotonin in the adrenal gland of Podarcis sicula (Reptilia, Lacertidae) Anna Capaldo,* Vincenza Laforgia, Rosaria Sciarrillo, Salvatore Valiante, Flaminia Gay, and Lorenzo Varano Department of Evolutionary and Comparative Biology, University ‘‘Federico II,’’ Via Mezzocannone 8, Naples 80134, Italy Accepted 18 April 2002
Abstract The occurrence of serotonin (5-hydroxytryptamine; 5-HT) in the chromaffin cells of Podarcis sicula adrenal gland was demonstrated by immunocytochemical techniques: ABC and immunogold methods. At LM and EM levels, antiserum against 5-HT revealed serotonin immunoreactivity prevalently in noradrenalin (NA) cells, on and around secretory vesicles; adrenalin (A) cells appeared scarcely stained. The role of serotonin in the regulation of adrenal gland activity was studied in vivo using LM and EM techniques coupled to a specific radioimmunoassay for adrenocorticotropic hormone (ACTH) and corticosterone. 5-HT (0.7 mg/ 100 g body wt)/day for 4 days increased ACTH and corticosterone release; at LM and EM level clear signs of stimulation in the steroidogenic tissue were observed, as evidenced by the variations of lipid/cytoplasm ratio. In the chromaffin tissue, LM observations evidenced a variation of the numeric NA/A cell ratio; at EM level, chromaffin tissue showed intermediate cells with A, NA, and very clear granules with granular elements. The occurrence of these cells might be the result of a process of resynthesis following serotonin-stimulated catecholamine release. These data suggested that serotonin might be involved in the modulation of Podarcis pituitary–adrenal axis, and act as a paracrine factor to modulate corticosteroid production. Ó 2003 Published by Elsevier Science (USA). Keywords: Serotonin; Adrenal gland; Immunocytochemistry; ACTH; Corticosteroids; PNMT
1. Introduction The presence of the biogenic amine serotonin (5-hydroxytryptamine; 5-HT) has been demonstrated by immunocytochemistry and biochemical analysis in all vertebrate species, with the exception of reptiles. The head kidney of several fish species stores, in addition to catecholamines, also 5-HT, immunohistochemically localized in chromaffin cells of both the Atlantic cod and European eel, while in the head kidney of the rainbow trout it appears to be stored in a cell-type other than the chromaffin cell. Positive immunohistochemical labeling for antisera raised against serotonin was also observed in both the systemic and portal hearts of the Atlantic hagfish, M. glutinosa (Reid et al., 1995, 1998). In the interrenal gland of amphibians anurans, serotonin co* Corresponding author. E-mail address: [email protected] (A. Capaldo).
exists with adrenalin within the same secretory vesicles, suggesting that indoleamine and catecholamines may be concomitantly released during splanchnic nerve stimulation (Delarue et al., 1988a, 1992). The presence of serotonin has been demonstrated also in the adrenal gland of birds, where almost all adrenal medullary cells contain 5-HT (Ohmori et al., 1997) and in mammals (Holzwarth and Brownfield, 1985; Kondo, 1985; Kong et al., 1989; Mazzocchi et al., 1998; Nussdorfer, 1996), where the occurrence of 5-HT differs between the various species; in rat and pig adrenal gland 5-HT is synthesized by chromaffin cells, while in the mouse adrenal cortex 5-HT is contained in nerve fibres; in man, serotonin is present in perivascular mast cells (Contesse et al., 2000). Serotonin plays a pivotal role in the regulation of the hypothalamo–pituitary–adrenal axis. In fact, the release of the corticotrophin-releasing-factor (CRF) from the paraventricular nucleus of the hypothalamus is stimulated by serotonin (Dinan, 1996; Holmes et al.,
0016-6480/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0016-6480(02)00531-2
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1982). Moreover, 5-HT can act directly on the adenohypophysis and the adrenal gland by stimulating the release of ACTH and corticosteroids, independently of CRF (Dinan, 1996). In vivo and in vitro studies showed that serotonin stimulates mineralocorticoid and glucocorticoid secretion (Lefebvre et al., 1998; Nussdorfer, 1996) in various species (including human), interacting with a wide variety of receptors in the different species. In humans, the stimulatory effect of 5-HT on adrenocortical cells is mediated through a 5-HT4 receptor subtype positively coupled to adenylyl ciclase and calcium influx (Contesse et al., 2000; Lefebvre et al., 1992), while in the rat the effect of the indoleamine on aldosterone secretion is mediated via activation of 5-HT7 receptors (Contesse et al., 1999). In amphibians, serotonin appears to enhance steroid secretion of frog adrenocortical cells in a concentrationdependent manner, through the activation of 5-HT4 receptors positively coupled to adenylyl ciclase (Delarue et al., 1988b; Idres et al., 1989, 1991); the activation of these receptors in the adrenocortical cells causes stimulation of adenylyl ciclase and subsequently increases calcium influx through a T-type calcium channel (Contesse et al., 1996). Moreover, detailed studies on frog interrenal tissue showed that corticosteroidogenesis is stimulated by acetylcholine, released from splanchnic nerve terminals, as well as by serotonin and VIP, both contained in the chromaffin cells, and that serotonin may interact with acetylcholine and VIP on their target cell to modulate the activity of their congeners. Since these three bioactive signals are released in response to splanchnic nerve stimulation, it is possible that the three neuroregolators may be involved in the precise adaptation of the adrenocortical cell activity in stress condition (Leboulenger et al., 1988). The data on serotonin effects on adrenochromaffin tissue are quite controversial; in fact, 5-HT, which is co-released with catecholamines, increases Naþ dependent Ca2þ efflux from cultured bovine adrenochromaffin cells, stopping catecholamine secretion (Minakuchi et al., 1997). In teleosts and cyclostomes, serotonin administration induces an increase in catecholamine plasma levels, either indirectly via the activation of neural pathways or directly interacting with the adrenochromaffin cells (Bernier and Perry, 1996; Fritsche et al., 1992, 1993; Reid et al., 1998); the release of serotonin, at least in either the rainbow trout or the Atlantic hagfish is not under cholinergic control (Reid et al., 1998). In contrast to the extensive literature available regarding the presence and the involvement of serotonin in the modulation of corticosteroidogenesis in mammals, amphibians, and fish, little is known about its role in reptiles. Therefore, in the present work, immunocytochemical methods were used to study the cellular and subcellular distribution of 5-HT in the adrenal gland of Podarcis sicula, the morphology and physiology of which are well known. In this species chromaffin and
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steroidogenic cells are strongly associated, so to suggest the existence of an high degree of co-regulation between the two cellular types; in fact, as in mammals and birds, glucocorticoids secreted by steroidogenic cells activate the enzyme PNMT (methylating noradrenalin into adrenalin), leading to an increase in the number of adrenalin cells, and a variation in the numeric ratio NA/ A cells (Laforgia and Muoio, 1997; Laforgia and Varano, 1978; Laforgia et al., 1982). Moreover, by analogy with other vertebrates, also Podarcis chromaffin cells contain, in addition to adrenalin and noradrenalin, a lot of secretory products as VIP (Laforgia et al., 1999), NPY (Muoio et al., 1995) and others, involved in the modulation of steroidogenic activity. Therefore the goals of this study have been (1) to localize the presence of 5-HT (2) to investigate its role in the control of the adrenal gland activity, to verify if also in reptiles, that represent an evolutionarily intermediate class, serotonin plays a role similar to that observed in the other vertebrates.
2. Materials and methods 2.1. Animals and housing conditions Adult specimens of P. sicula, (weighing 13–15 g) were captured in the neighbourhood of Naples in December, when adrenals undergo functional stasis (Varano et al., 1969). After capture, the animals were housed in large soil-filled terraria containing heather, and exposed to natural temperature and photoperiod. Water dishes were present in the terraria, and the animals were fed on live fly larvae daily. Captivity lasted 20 days to reverse capture-related stress (Manzo et al., 1994). 2.2. Experimental procedure The animals received intraperitoneal injections of serotonin. Plasma serotonin level has not yet been determined; to ensure that ip administration of 5-HT was reasonable, in a separate series of experiments, serotonin was administered in doses close to the doses used previously in mammals (Nussdorfer, 1996); in fact data on serotonin administration in reptiles are lacking. The effects of the ip 5-HT on plasma levels of ACTH and corticosterone were assessed; based on the results of the dose–response experiments, the right dose of serotonin was chosen for the following experiments. The specimens were divided into five groups, each consisting of 20 animals (10 males and 10 females): Group 1. The animals were given a single intraperitoneal injection of serotonin (5-hydroxytryptamine hydrochloride, Sigma) (0.7 mg/100 g body wt)/day for four consecutive days. 5-HT was dissolved in reptilian physiological solution (NaCl 0.75%) with an injection volume
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of 0.1 ml. Injections were between 8.00 and 8.30 a.m., and the animals were sacrificed 2 h after the last injection. Group 2. The animals were treated as group 1 but sacrificed 24 h after the last injection. Group 3. Untreated control animals. Group 4. Control animals receiving intraperitoneal injections of reptilian physiological solution (NaCl 0.75%) and sacrificed as group 1. Group 5. Control animals receiving intraperitoneal injections of reptilian physiological solution (NaCl 0.75%) and sacrificed as group 2. The animals were anesthetized by hypothermia, chilling them in chipped ice. Blood samples were collected by intracardiac puncture and put into heparinized capillars. They were then centrifuged at 1500 rpm for 10 min to obtain plasma. 2.3. Light microscopy Immediately after collection of blood samples, the animals were killed by decapitation, and their adrenals were fixed in a mixture of 2.5% potassium dichromate, 1% anhydrous sodium sulphate (buffered at pH 4.1 with 5 M acetate buffer), and 10% formaldehyde (Wood, 1963). They were then embedded in paraplast, cut into 7 lm sections with a rotary microtome, affixed to albuminized slides and stained with one of the following solutions: (1) a mixture of eosine aniline blue, buffered at pH 4 with acetate buffer 5 M (Wood, 1963); (2) Giemsa solution, modified according to Pearse (1960); (3) Mallory trichromic stain. Observations were performed using a Zeiss Axioskop microscope; images were captured with a camera attached to an IBM computer running the Kontron Elektronik KS 300 image analysis system and Adobe Photoshop. 2.4. Transmission electron microscopy The adrenal glands were fixed in 2.5% glutaraldehyde in MillonigÕs phosphate buffer at pH 7.4 at 4 °C, rinsed in buffer and postfixed in 1% Os O4 (2 h, 4 °C), dehydrated in ethanol, cleared in propylene oxide, embedded in epoxy resin and polymerized. Ultrathin section (30 nm) were collected on formvar coated copper grids, stained with solutions of uranyl acetate and lead citrate and observed with a Philips EM 301 transmission electron microscope at the Interdepartmental Center for Biological Ultrastructures (Naples). 2.5. Quantitative analysis In 10 specimens from each experimental group, processed for LM, NA/A cell ratio was calculated from cell counts in every tenth longitudinal section from the whole gland of each specimen.
In 10 specimens from each experimental group, processed for EM, 10 low-power micrographs of steroidogenic cells, containing at least four cells for each sample, were examined to evaluate, in each cell, the area occupied by lipid droplets (lipid/cytoplasm ratio). The areas of both lipid droplets and cytoplasms were measured with the help of a planimeter. Variations of lipid contents in control and treated animals were calculated using the formula Lipid=cytoplasm ratio ¼
Lipid droplet area ðlm2 Þ : Cytoplasmic area ðlm2 Þ
2.6. Hormone assay Plasma corticosterone was measured using a sensitive and highly specific radioimmunoassay kit (ICN Biomedicals, COSTA MESA, CA). Before assay, experimental plasma samples were diluted one-third with the kit diluent and heated at 80° for 10 min in order to inactivate corticosterone-binding proteins. Corticosterone titers in nanograms per milliliter were calculated using the standard curve generated in the assay. Standard curves were prepared in buffer with known amounts of radio inert corticosterone (0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 ng/ml) purchased from Amersham (Arlington Heights, IL). The minimum concentration per tube that was distinguishable from zero was 0.176 ng/ml. Cross-reactivities of the corticosterone antiserum with other steroids were 6.1% for deoxycorticosterone; <1% for progesterone and cortisol; <0.1% for aldosterone, 20-a dehydroprogesterone, testosterone, and 11-deoxycortisol; and <0.01% for all the other steroids examined. Inter- and intraassay coefficients of variation were 3.4 and 5.8%, respectively. Plasma ACTH concentrations were measured by a two-site immunoradiometric assay (IRMA) using mouse monoclonal antibodies (Diagnostic Products). Boric acid was added to serum to adequately preserve ACTH. Sensitivity was 0.1 pg/ml, and the inter- and intraassay coefficients of variation were 10 and 6%, respectively. 2.7. Statistical analysis The data obtained from each specimen were averaged per experimental group, and the SD of the mean was calculated. As revealed by the v2 test, the data were not different from normal distribution. After ANOVA, statistical comparison of the data was performed by use of the Multiple Range Test of Duncan. 2.8. Immunocytochemistry The sections were treated by the avidin-biotinylated peroxidase complex (ABC) method (Hsu et al., 1981).
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For each of control groups (3, 4, and 5) the left adrenal glands of ten specimens (30 adrenal glands) were fixed in 2.5% glutaraldehyde in PBS (phosphate buffer saline, pH 7.4) for 2 h, then dehydrated and embedded in paraplast. Rehydrated sections were placed in 3% hydrogen peroxide solution for 5 min to inhibit endogenous peroxidases. The sections were incubated with normal goat serum (NGS) for 20 min, and then incubated with anti-rabbit 5-HT (Biogenesis, Poole, England) (1:300) overnight at 4 °C in a moist chamber. After rinsing in PBS, the sections were incubated for 30 min with biotinylated goat anti-rabbit IgG (1:400) (Vecta-lab ‘‘Elite’’ (ABC) kit, Vector Laboratories, Burlingame, CA) and, after rinsing in PBS, incubated for 30 min with Vectastain Elite ABC reagent. Immunoreactivity was visualized by a reaction with 0.05% 3,30 -diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO) and 0.01% hydrogen peroxide in 0.05 M Tris–HCl buffer, pH 7.2 for 10 min to reveal the brown immunoreactive cells. After the DAB reaction, the sections were counterstained with ematoxylin. For each adrenal gland, four longitudinal sections were examined (120 light micrographs). 2.9. Immunoelectron microscopy Thin sections of adrenals of 10 specimens of each of control groups (3, 4, and 5), mounted on formvar coated nickel grids, were etched in saturated sodium metaperiodate for 1 h, (De Mey et al., 1981), then washed in bidistilled water, exposed to a 1% BSA solution (Sigma bovine serum albumin, essentially globulin free) in TBS (Tris buffered salt) for 30 min, followed by an overnight incubation at 4 °C with anti-rabbit 5-HT (Biogenesis, Poole, England) 1:300 in 1% BSA-TBS. The grids were rinsed several times at room temperature in 1% BSATBS and incubated for 90 min with goat anti-rabbit gold labeled secondary antibody (10 nm, Sigma) (1:50), wa-
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shed several times with 1% BSA-TBS, then with bidistilled water, and stained with uranyl acetate and lead citrate. For each specimen, 10 electron micrographs were examined (300 electron micrographs). For LM and EM immunocytochemistry, controls were made by (1) replacing specific antiserum with normal rabbit serum (2) omitting primary antibody. No immunoreactive structures were observed.
3. Results 3.1. Control specimens 3.1.1. LM observations No differences were observed in the adrenal gland morphology between untreated specimens (group 3) and specimens injected with physiological solution (groups 4, 5). The adrenal gland of P. sicula was formed by chromaffin and steroidogenic tissues. The latter tissue consisted of prismatic cells with a weakly stained cytoplasm, which were arranged in anastomosed cordons of two cell layers intermingled with thin blood vessels (Fig. 1A). The chromaffin tissue was arranged in a dorsal cordon, the most peripherical portion of which consisted of noradrenalin (NA) cells, whereas adrenalin cells (A) were found in its innermost portion and in its digitations penetrating the steroidogenic parenchyma. Small groups of A cells were observed among the steroidogenic cordons (Figs. 1A and B). NA cells were more numerous than A cells with a NA/A ratio of 1.4/1 (Table 1). 3.1.2. EM observations The ultrastructural characters of the adrenal gland were similar in the specimens of groups 3, 4, and 5. The steroidogenic cells contained numerous mitochondria, a smooth endoplasmic reticulum arranged in tubules and
Fig. 1. Light micrographs of P. sicula adrenal gland. Control specimen. Mallory trichromic stain. Steroidogenic tissue is formed by anastomosed cords (A); chromaffin tissue forms a dorsal ribbon, with NA cells (gold yellow) in the outer layers, and A cells (red orange) in the inner layers and islets interspersed between steroidogenic cords (A,B). St, steroidogenic tissue; NA, noradrenalin cells; A, adrenalin cells. Calibration bars, 100 lm.
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Table 1 Numeric NA/A cell ratios following serotonin administration Experimental procedure
NA cells ðmean SEÞ
A cells ðmean SEÞ
NA/A cell ratio
Group Group Group Group Group
380 0:05a; b 760 0:07 719 0:06 638 0:05 736 0:06
1151 0:1a;b 768 0:07 513 0:05 455 0:05 525 0:05
0.3/1 1/1 1.4/1 1.4/1 1.4/1
1 2 3 4 5
(10) (10) (10) (10) (10)
Note. Numbers in parentheses indicate the numbers of animals. a P < 0:01 from group 4. b P < 0:01 from group 3.
vesicles and a large amount of lipid vacuoles accounting for most of the cytoplasm (Fig. 2A) with a lipid/cytoplasm ratio of 0.44 (Table 2). Accumulation of steroids is typical of winter specimens (Varano et al., 1969). The chromaffin cells showed a nucleus variable in shape, an incospicuous endoplasmic reticulum mostly constituted by rough membranes, few mitochondria and a paranuclear Golgi complex. The most typical constituents were the chromaffin granules, which, in A cells, appeared roundish, moderately electron-dense, with a variable halo separating the core from the limiting membrane (Fig. 2B). NA granules were polymorphic and intensely electron-dense with a core closely adhering to the limiting membrane. These granules sometimes appeared enlarged with an eccentric core (Fig. 2C). 3.1.3. Hormone assay Plasma levels of ACTH were: 4:18 0:05 pg/ml in untreated control animals (group 3); 4:14 0:05 pg/ml in group 4, and 4:22 0:05 pg/ml in group 5 (Fig. 3A). Corticosterone plasma levels were 3:87 0:04 ng/ml in specimens of group 3; 3:90 0:04 ng/ml in group 4; and 3:85 0:04 ng/ml in group 5 (Fig. 3B). 3.2. Specimens injected with serotonin 3.2.1. LM observations After 2 h from the 4th serotonin injection, clear signs of stimulation were observed in the steroidogenic tissue, namely dilated blood vessels and hypertrophic cordons formed by swollen cells (Fig. 4A). In the chromaffin tissue, A cells increased in number, also occupying the most peripheral portions of the cordon, usually consisting of NA cells (Figs. 4A and B), Therefore, there was a variation in the NA/A cell ratio, which became 0.3/1 (Table 1). After 24 h, when the signs of stimulation in the steroidogenic tissue were no more evident (Fig. 4C), in the chromaffin tissue the NA/A cell ratio attained values similar to those of control specimens again (1/1) (Figs. 4C and D and Table 1). 3.2.2. EM observations After 2 h from the 4th serotonin injection, the steroidogenic cells showed a cytoplasm characterized by a marked development of the smooth endoplasmic retic-
ulum, the presence of numerous mitochondria with well evident crests, and the almost complete absence of lipid vacuoles (Fig. 5A). The decrease in lipid content was confirmed by the decrease in the lipid/cytoplasm ratio value, which became 0.07 (Table 2). After 24 h, in the steroidogenic cells, which no longer showed signs of intense stimulation, the lipid content appeared similar to that in control specimens, as can be inferred from the lipid/cytoplasm ratio value of 0.39 (Table 2). In the chromaffin tissue, besides A and NA cells, there were chromaffin cells with a cytoplasm showing both A and NA cells at the same time, as well as very clear granules, often closely packed together, containing granular ele in diameter (Figs. ments ranging between 340 and 347 A 5B, 6A). Many chromaffin cells had a low content of catecholamine granules (Fig. 6B). 3.2.3. Hormone assay After 2 h from the 4th serotonin injection, the ACTH plasma value was 5:69 0:01 pg/ml, increasing to 6:50 0:05 pg/ml after 24 h (Fig. 3A); the corticosterone plasma value was 7:25 0:03 ng/ml, increasing to 8:28 0:06 ng/ml after 24 h (Fig. 3B). 3.2.4. Immunocytochemistry Antiserum against serotonin revealed, in the adrenal gland of Podarcis, 5-HT immunoreactivity, that appears marked in the noradrenalin cells, disposed in the outer layers of the chromaffin ribbon; the adrenalin cells appear scarcely stained. The immunoreactivity was restricted to the cytoplasm of positive cells; no fibres immunopositive to 5-HT could be detected. Steroidogenic cells did not appear immunostained (Fig. 7A). Control sections exhibited no staining (Fig. 7B). 3.2.5. Immunoelectron microscopy At EM level, the chromaffin cells appeared immunopositive to serotonin; gold particles were localized on and around secretory vesicles of NA cells (Fig. 8A), while immunolabeling appeared weak on adrenalin granules (figure not shown). Immunoreactive granules were distributed throughout the cytoplasm, showing no preferential distribution; no immunopositivity was found in the steroidogenic tissue. Control sections did not stain for 5-HT (Fig. 8B).
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Fig. 2. Electron micrographs of control specimen interrenal gland. The cytoplasm of steroidogenic cells (A) presents several mitochondria and many lipid droplets. In the adrenalin cells (B), A granules appear rounded, of medium electron density and with a clear halo between the core and the limiting membrane.The noradrenalin cells (C) present polymorphic, strongly electron-dense chromaffin granules. L, lipid droplets; M, mitochondria; A, adrenalin granule; NA, noradrenalin granule. Calibration bars, 1 lm.
4. Discussion The results of the present study indicate that, in P. sicula adrenal gland, serotonin is located in chromaffin
cells. They also demonstrate that 5-HT can influence the activity of the pituitary–interrenal axis. Several studies have revealed that adrenochromaffin cells contain and release not only catecholamines, but
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Table 2 Lipid/cytoplasm ratio values in the steroidogenic cells following serotonin administration Experimental procedure
Lipid droplets area ðmean SEÞ
Cytoplasmic area ðmean SEÞ
Lypid/cytoplasm ratio ¼ lipid area/cytoplasmic area
Group Group Group Group Group
2:8 0:06a ;b 13:6 0:04 15:05 0:05 15:03 0:05 15:07 0:06
37:5 0:04 34:8 0:03 34:1 0:05 34:3 0:05 34:1 0:04
0.07 0.39 0.44 0.44 0.44
1 2 3 4 5
(10) (10) (10) (10) (10)
Note. Numbers in parentheses indicate the number of animals. a P < 0:01 from group 4. b P < 0:01 from group 3.
Fig. 3. ACTH (A) and corticosterone (B) plasma levels in control (groups 3, 4, and 5) and treated (groups 1 and 2) specimens.
also many biogenic amines and regulatory peptides that may modulate the function of adrenocortical cells in a paracrine manner (Bornstein et al., 1997; Mazzocchi et al., 1998). 5-HT has been biochemically and immunocytochemically demonstrated in the adrenal gland of mammals, where it is localized in rat A cells and in NA pig cells (Nussdorfer, 1996), birds (Ohmori et al., 1997), anurans, where 5-HT has been evidenced in A cells (Delarue et al., 1992) and fish, where serotonin is present in many DBH-positive cells within the PCV of Atlantic cod and European eel (Reid et al., 1995, 1998). Our results show that chromaffin cells contain 5-HT in P. sicula as well. By light microscopic immunocytochemical techniques, the cytoplasm of NA cells appears immunopositive to serotonin, while A cells result scarcely stained. Electron microscopic studies confirmed light microscopic immunocytochemistry data, showing 5-HT immunoreactivity on the chromaffin granules of NA
cells, where labeled vesicles are distributed throughout the cytoplasm. At EM level, the immunopositivity to 5HT was weak on A chromaffin granules (figure not shown). The presence of 5-HT in the chromaffin tissue of P. sicula raises the question of the origin of the indoleamine, namely whether 5-HT is synthesized within chromaffin cells or taken up from the circulation. Uptake of 5-HT by adrenalin-storing cells has been observed in the rat (Verhofstad and Jonsson, 1983) and mouse adrenal medulla (Kent and Coupland, 1984), while, in anurans, 5-HT is synthesized by chromaffin cells from L -tryptophan (Delarue et al., 1992). Further studies are needed to determine whether chromaffin cells are able to synthesize serotonin in P. sicula as well. 5-HT markedly affects the adrenal gland of P. sicula. In vivo and in vitro studies have shown that 5-HT stimulates ACTH release (Dinan, 1996) and mineralo-
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Fig. 4. Specimen of group 1 (A,B) and group 2 (C,D). Mallory trichromic stain (A,B,C) and Giemsa stain (D). Steroidogenic cords appear swollen, separated by dilated blood vessels (A). In the chromaffin ribbon (B), A cells appear also in the outer layers of chromaffin tissue, usually filled with NA cells. After 24 h from the last injection, the adrenal gland morphology (C) and the distribution of NA (dark green) and A (light green) cells (D) appear similar to that of control specimen. St, steroidogenic tissue; NA, noradrenalin cells; A, adrenalin cells. Calibration bars, 100 lm (A), 50 lm (B–D).
Fig. 5. Electron micrographs of a steroidogenic cell of group 1 (A) showing the cytoplasm almost devoid of lipid droplets, the smooth endoplasmic reticulum greatly developed and numerous mitochondria, and of a chromaffin cell of group 2 (B) where, in the cytoplasm of the same cell A, NA and very clear granules are present. L, lipid droplet; M, mitochondria; SER, smooth endoplasmic reticulum; NA, noradrenalin granule; A, adrenalin granule; * (CG), very clear granule. Calibration bars, 1 lm.
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Fig. 6. Electron micrographs of chromaffin cells of group 2 specimen. Very clear granules contain granular elements ranging between 340 and 347 A in diameter (A). Many chromaffin cells (B) show a reduced amount of secretory vesicles. NA, noradrenalin granule; A, adrenalin granule; CG, very clear granule. Calibration bars, 1 lm.
Fig. 7. Light micrographs of P. sicula adrenal gland. ABC reaction (A) showing NA cells immunoreactive to 5-HT, while A cells only lightly stained; steroidogenic cells are immunonegative. Control section (B) showing no immunopositivity to 5-HT. NA, noradrenalin cells; A, adrenalin cells; St, steroidogenic tissue. Calibration bars, 100 lm.
and glucocorticoid secretion from adrenal cells (Contesse et al., 1996, 1999, 2000; Delarue et al., 1988b; Idres et al., 1989, 1991; Leboulenger et al., 1988; Lefebvre et al., 1998; Nussdorfer, 1996; Reid et al., 1998). Our results indicate that 5-HT enhances ACTH release and corticosterone production from adrenocortical cells, in P. sicula as well. Ultrastructural features of steroidogenic tissue and the lipid/cytoplasm ratio values revealed a strong stimulation of steroidogenic cells, as is also confirmed by the results of the hormone assays revealing an increase in corticosteroid plasma levels consequent on serotonin administration. In the lizard, capture-related stress causes a drastic increase in corticosteroids, and this effect is reversed after acclimatization in terrarium for some time (Manzo et al., 1994). Therefore, the animals were acclimatized for twenty
days in order to rule out that the increase in corticosteroid plasma levels was due to capture-related stress. Moreover, the influence of handling stress must be considered negligible due to the lack of significant differences in the plasma levels of ACTH and corticosteroids in the untreated specimens (group 3) and in those injected with physiological solution (groups 4, 5). In reptiles, like in mammals, the PNMT enzyme (phenyletanolamine-N-methyl transferase), converting NA into A, is activated by corticosteroids (Laforgia and Muoio, 1997; Laforgia and Varano, 1978; Laforgia et al., 1982). Therefore, the increased corticosteroid plasma levels, resulting from 5-HT administration, enhance PNMT activity, inducing an increase in the number of A cells compared to NA cells and a variation in the NA/A cell ratio.
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Fig. 8. Electron micrographs of NA cells. (A) Immunogold reaction. Noradrenalin granules appear immunopositive to 5-HT. (B) Control section showing absence of immunopositivity to 5-HT. Calibration bars, 0:5 lm.
Literature data on the effects of serotonin on the adrenochromaffin tissue are contrasting. In fact, 5-HT has a role in stopping catecholamine secretion from cultured bovine adrenochromaffin cells (Minakuchi et al., 1997), whereas it causes an increase in catecholamine plasma levels in teleosts and cyclostomes (Bernier and Perry, 1996; Fritsche et al., 1992, 1993). In P. sicula, after 5-HT treatment, besides NA and A cells, the chromaffin tissue shows chromaffin cells with a cytoplasm containing both NA and A granules, as well as very clear granules, closely packed together, containing in granular elements ranging between 340 and 347 A diameter. The occurrence of these cells and of chromaffin cells with a low content of secretory granules suggests that 5HT might have induced catecholamine release from the adrenochromaffin cells, followed by a process of resynthesis. This is confirmed by the presence of secretory granules with variable electron density, which would correspond to subsequent stages of the process of amine deposition. These data suggest that serotonin might have a stimulating action on the hypophyseal–interrenal axis, increasing ACTH and corticosterone release, and hence influencing the adrenochromaffin tissue. Further studies are needed to detect and identify the type of serotonin receptors on the chromaffin and steroidogenic cells, as well as to establish whether the 5-HT action on the adrenal gland is direct or mediated through ACTH release. Immunocytochemical studies revealed that, besides catecholamines, adrenochromaffin cells produce and release a variety of regulatory peptides, which may affect the secretory activity of steroidogenic cells in a paracrine manner (Delarue et al., 1988a, 1992; Mazzocchi et al., 1998; Verhofstad and Jonsson, 1983). In
the adrenal gland of P. sicula, chromaffin and steroidogenic tissues are markedly intermingled, suggesting that chromaffin and steroidogenic cells may interact with each other; in addition, not only steroid hormones may activate the enzymes involved in adrenalin synthesis, but also the chromaffin cells may modulate the function of steroidogenic cells in a paracrine manner. In P. sicula adrenal gland, chromaffin cells contain NPY, Leu-Enkephalin, b-endorphin (Muoio et al., 1995), substance P (De Falco et al., 1999), VIP (Laforgia et al., 1999), inhibin (Manzo et al., 2000); the presence and the effects of serotonin suggest that 5-HT may be involved in the physiological regulation of the adrenal gland, in P. sicula as well.
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