Chromaffin cells and interrenal tissue in the head kidney of the grouper,Epinephilus tauvina (Teleostei,Serranidae): a morphological (optical and ultrastructural) study

This work analyses the distribution, histology and ultrastructure of chromaffin cells (CCs) and interrenal tissue (It) in the head kidney of Epinephilus tauvina. Histological examination revealed that chromaffin cells are found in small groups under the endothelium of the posterior cardinal vein (PCV) and are mostly closely associated with the interrenal tissue. Ultrastructure examination confirmed the existence of two main chromaffin cell types, distinguished by different types of secretory granules. The first type was characterized by the presence of vesicles with round, strongly electron dense core granules, which were eccentrically located. Such cells were interpreted as being noradrenaline cells. Meanwhile, cells with vesicles that were completely electron lucent or that contained small less dense eccentric granules were identified as adrenaline cells. Nerve endings were invaginated into the chromaffin cells through synaptic junctions. Interrenal tissue consisted of nests, cords, or strands of cells in contact with the posterior cardinal vein (PCV) and interposed with haematopoietic tissue. Ultrastructure analysis revealed only one interrenal cell type, which contained abundant smooth endoplasmic reticulum (sER) and numerous mitochondria with tubulo‐vesicular cristae, characteristics of steroid‐producing cells. The interrenal tissue cells have different cytological aspects that can be linked to a steroidogenic cell cycle allowing a periodical renewal of organelles.     

Ultrastructure of the cortical cell of the interrenal gland of the American bullfrog (Rana catesbeiana)

Summary The cortical cell of the interrenal gland of the American bullfrog, Rana catesbeiana, was examined in the electron microscope. These cells occur in small groups and cords and are quite irregular in shape. The cortical cell is reminiscent of adrenal cortical cells from other vertebrates. Liposomes are variable in size and density. Smooth endoplasmic reticulum is scant in amount and predominantly of the fine tubular type. Mitochondria have vesicular cristae, a dense matrix, and occasionally have blebs, vacuoles, and myelin-like whorls at their surfaces. Intimate morphological relationships are found among the Golgi apparatus, lysosomes, and liposomes, and among Golgi vacuoles, mitochondria, and liposomes. In addition microfibrils are a prominent feature of the cortical cell. The biochemical events of steroidogenesis in amphibia and other vertebrates are discussed in relationship to the organellar interrelations found in the bullfrog interrenal cortical cells. Based on the available chemical and morphological information a scheme is proposed of movement of the steroidal intermediates through the cell that tentatively identifies the localizations of the various enzyme systems involved in corticosteroidogenesis from acetate to corticosterone and aldosterone.Supported in part by N.I.H. Grant RR 06138. Health Sciences Advancement Award.     

Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex

We have previously determined that atrial natriuretic factor (ANF) is a potent inhibitor of steroid secretion in cultured bovine zona glomerulosa and fasciculata cells. The present report describes a comparison of the effect produced by ANF on aldosterone, deoxycorticosterone and progesterone secretions by zona glomerulosa cells and on cortisol, corticosterone and progesterone secretions by zona fasciculata cells. The equipotent inhibitory action of ANF on the stimulated secretion of these steroids in both cell types indicates a common site of action prior to progesterone synthesis at which ANF inhibits the steroidogenic pathway.     

Adrenocortical response in the duck exposed to corticosteroid administration and salt loading

The functional zonation in the adrenocortical tissue of the duck was experimentally investigated after chronic administration of corticosteroids of different types (corticosterone, desoxycorticosterone, dexamethasone) and salt loading (chlorides of sodium and potassium). The cytomorphology of the interrenal cells belonging to subcapsular and central zones was explored by light- and electron microscopy and by biochemical analysis of plasma corticosterone. Corticoid-induced involution of the interrenal tissue, or hyperactivity elicited by salt loading were evident in both subcapsular and central regions of the gland. In the duck the adrenocortical tissue appears to be devoid of steroidogenic functional differentiation. The modifications of different cellular organelles and inclusions can be explained in the light of known concepts about corticosteroid metabolism. The depression of plasma corticosterone titre in corticosteroid-treated animals agrees with the image of cytological inhibition. The lowered corticosterone value in KCl-loaded ducks signifies increased peripheral metabolism of this hormone. The hypercorticosteronemia in NaCl-loaded ducks is probably related to activation of the nasal gland under osmotic stress.     

Interrenal function during amphibian metamorphosis: in vitro biosynthesis of radioactive corticosteroids from (4(14)C)-progesterone by interrenal in Xenopus laevis tadpoles

Whole kidneys of Xenopus laevis tadpoles (containing interrenal cells) or the outer postero-dorsal part of adult kidney (containing a few or no interrenal cells) were incubated with (4(14)C)-progesterone. Methylene chloride extractable radioactivity of tissue homogenates in their incubation medium were purified by sequential paper chromatography and identification of some isolated fractions was attempted. A significant conversion of progesterone was only observed when interrenals were present in incubation; however, possible steroidogenic activity of the other tissue types present in the kidney cannot be ignored. At all the developmental stages of tadpoles (from stage 45 to stage 66) and in juveniles, the same steroids were isolated. Hence the metabolic pathways for corticosteroidogenesis in Xenopus laevis remained unchanged during metamorphosis and several weeks after the end of this process. Aldosterone was identified. Corticosterone and 11-dehydrocorticosterone were also synthesized but not completely purified. Their yields were always larger than those of aldosterone. Cortisol was never detected.     

Functional significance of interrenal cell zonation in the adrenal gland of the duck (Anas platyrhynchos)

Slices of whole adrenal gland tissue, incubated in vitro in the presence of ACTH for 1 h and 2 h produced corticosterone and aldosterone in constant ratio (16:1). Tangential slices taken from the region immediately below the connective tissue capsule and slices taken from deeper regions of the gland consisted primarily of cells conforming to the distinct structural characteristics of the subcapsular zone (SCZ) and inner zone (IZ) tissues respectively. When samples were incubated in the presence of ACTH for 1 h and 2 h, the interrenal cells of the SCZ produced relatively more aldosterone than cells taken from the IZ of the gland. The corticosterone: aldosterone ratio for the IZ after 1 h (68:1) and after 2 h (102:1) were ten times greater than the ratios for the SCZ after 1 h (7:1) and after 2 h (10:1). The SCZ slices were not more than 60 cells thick and consisted of cells arranged in cords. These cells contained irregular nuclei, mitochondria with shelf-like cristae and a moderate abundance of smooth endoplasmic reticulum. In contrast, the production of large amounts of corticosterone by the cells of the IZ was associated with tissue containing more vascular space than the SCZ and the cells contained large round nuclei surrounded by an abundance of smooth endoplasmic reticulum and the mitochondria had tubular rather than shelf-like cristae.     

Characterization of cDNAs encoding cholesterol side chain cleavage and 3beta-hydroxysteroid dehydrogenase in the freshwater stingray Potamotrygon motoro

The interrenal gland (adrenocortical homolog) of elasmobranchs produces a unique steroid, 1α-hydroxycorticosterone (1α-B). The synthesis of this and most other steroids requires both cholesterol side chain cleavage (CYP11A) and 3β-hydroxysteroid dehydrogenase (HSD3). To facilitate the study of elasmobranch steroidogenesis, we isolated complementary DNAs encoding CYP11A and HSD3 from the freshwater stingray Potamotrygon motoro. The P. motoro CYP11A (2182 bp total length) and HSD3 (2248 bp total length) cDNAs harbor open reading frames that encode proteins of 542 and 376 amino acids (respectively) that are similar (CYP11A: 39–61% identical; HSD3: 36–53% identical) to their homologs from other vertebrates. In molecular phylogenetic analysis, P. motoro CYP11A segregates with CYP11A proteins (and not with related CYP11B proteins) and P. motoro HSD3 segregates with steroidogenic HSD3 proteins from other fishes. CYP11A and HSD3 mRNA is found only in interrenal and gonadal tissues, indicating de novo steroidogenesis is restricted to these tissues. Because 1α-B is thought to act in the elasmobranch response to hydromineral disturbances, we examined the effect of adapting P. motoro to 10 ppt seawater on mRNAs encoding steroidogenic genes. The P. motoro response to this salinity challenge does not include interrenal hypertrophy or an increase in the levels of interrenal CYP11A, HSD3 or steroidogenic acute regulatory protein (StAR) mRNA. This study is the first to isolate full length cDNAs encoding elasmobranch CYP11A and HSD3 and the first to examine the regulation of steroidogenic genes in elasmobranch interrenal cells.     

Competitive protein-binding radioassay of corticosterone in the plasma of the frog, Rana esculenta L. (author's transl)]

1. Plasma corticosterone levels were measured in the plasma of the edible frog, Rana esculenta, by a competitive protein-binding radioassay method using baboon plasma as CBG source. 2. This technique was sensitive enough to make the assessment of corticosterone levels in 50 microliter plasma samples possible. The assay sensitivity threshold reached 0.5 ng per tube and the corticosterone rate assessment was correct between 0 and 5 ng. The specificity was tested, using 12 different steroids (fig. 2) : baboon CBG had very slight avidity for aldosterone, the second circulating steroid in frog plasma. 3. Using this technique, we have shown that plasma corticosterone underwent seasonal variations. Plasma corticosterone levels, in animals captured in nature during February and June, were 1.51 +/- 0.06 microgram/100 ml (n = 60) and 2.76 +/- 0.14 microgram/100 ml (n = 36), respectively, as appeared in table III. 4. It appeared that the interrenal gland of the frog was not totally dependent on pituitary ACTH, since total hypophysectomy reduced, but did not suppress, corticosterone secretion (table III).     

The Hemodynamically-Regulated Vascular Microenvironment Promotes Migration of the Steroidogenic Tissue during Its Interaction with Chromaffin Cells in the Zebrafish Embryo

Background

While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role.

Methodology and Principal Findings

In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio). Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak) signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a) or matrix metalloproteinase 2 (mmp2), two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through mechanosensitive or Fn-pFak dependent mechanisms.

Conclusions and Significance

These results demonstrate that hemodynamically regulated Fn-pFak signaling promotes the migration of steroidogenic cells, ensuring their interaction with chromaffin cells along both sides of the midline during interrenal gland development.     

Co-localization of vasoactive intestinal peptide (VIP) and enkephalins in chromaffin cells of the adrenal gland of amphibia. Stimulation of corticosteroid production by VIP

Recent studies have shown that biologically active peptides and monoaminergic neurotransmitters coexist in certain neuronal cell populations. Using the immunofluorescence technique, we have examined the localization of enkephalins, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase in the adrenal gland of the frog Rana ridibunda. Most chromaffin cells which stained for tyrosine hydroxylase contained VIP-like immunoreactivity, whereas methionine- (Met-) and leucine- (Leu-) enkephalin-like immunoreactivity was detected in about 40% of the cells revealed by the anti-tyrosine hydroxylase serum. No VIP- or enkephalin-like immunoreactive nerve fibres were observed. Since in the frog, the chromaffin cells are in close contact with the adrenocortical (interrenal) tissue, a possible action of VIP and opiates on corticosteroidogenesis has been investigated. At doses 10(-6) and 10(-5) M, 20-min infusions of synthetic porcine or chicken VIP elicited a significant increase in corticosterone and aldosterone production by perifused frog adrenals, in a dose-dependent manner. As compared to ACTH, VIP was several orders of magnitude less effective in stimulating corticosteroid production. Morphine, Met- and Leu-enkephalins (10(-5) M) had no effect on spontaneous secretion of corticosteroids. In addition, Met- and Leu-enkephalins (10(-5) M) did not alter the production of corticosterone induced by ACTH. THese results suggest that VIP contained in the chromaffin cells of the frog adrenal gland may exert a local action in stimulating corticosteroid production by the interrenal tissue.     

Development and fibronectin signaling requirements of the zebrafish interrenal vessel

Background

The early morphogenetic steps of zebrafish interrenal tissue, the teleostean counterpart of the mammalian adrenal gland, are modulated by the peri-interrenal angioblasts and blood vessels. While an organized distribution of intra-adrenal vessels and extracellular matrix is essential for the fetal adrenal cortex remodeling, whether and how an intra-interrenal buildup of vasculature and extracellular matrix forms and functions during interrenal organogenesis in teleosts remains unclear.

Methodology and Principal Findings

We characterized the process of interrenal gland vascularization by identifying the interrenal vessel (IRV); which develops from the axial artery through angiogenesis and is associated with highly enriched Fibronectin (Fn) accumulation at its microenvironment. The loss of Fn1 by either antisense morpholino (MO) knockdown or genetic mutation inhibited endothelial invasion and migration of the steroidogenic tissue. The accumulation of peri-IRV Fn requires Integrin α5 (Itga5), with its knockdown leading to interrenal and IRV morphologies phenocopying those in the fn1 morphant and mutant. fn1b, another known fn gene in zebrafish, is however not involved in the IRV formation. The distribution pattern of peri-IRV Fn could be modulated by the blood flow, while a lack of which altered angiogenic direction of the IRV as well as its ability to integrate with the steroidogenic tissue. The administration of Fn antagonist through microangiography exerted reducing effects on both interrenal vessel angiogenesis and steroidogenic cell migration.

Conclusions and Significance

This work is the first to identify the zebrafish IRV and to characterize how its integration into the developing interrenal gland requires the Fn-enriched microenvironment, which leads to the possibility of using the IRV formation as a platform for exploring organ-specific angiogenesis. In the context of other developmental endocrinology studies, our results indicate a highly dynamic interrenal-vessel interaction immediately before the onset of stress response in the zebrafish embryo.     

Origins of the differences in function of rat adrenal zona glomerulosa cells incubated as intact tissue and as collagenase-prepared cell suspensions

While in vitro incubation of dispersed cell preparations of adrenal cell types has been widely used as an experimental model, few studies have addressed the possibility that the enzymic and mechanical treatments involved may affect tissue functions. Using rat adrenal whole capsule tissue, consisting of glomerulosa cells still attached to the connective tissue capsule together with some fasciculata cells, and dispersed glomerulosa cell preparations formed by a variety of enzymic and incubation treatments, striking differences have been demonstrated between the functions of the various preparations in vitro. Under ACTH stimulation, whole capsules produced (ng per pair ± s.e.) 405 ± 35 ng aldosterone, 650 ± 60 ng 18-hydroxycorticosterone (18-OH-B) and 850 ± 90 ng corticosterone. In cells dispersed by collagenase incubation followed by repeated pipetting and filtration, aldosterone and 18-OH-B yields under ACTH stimulation fell to values less than 10% of those produced by whole tissue, whereas corticosterone values were unchanged. Omitting the filtration step gave a less well marked decline in aldosterone and 18-OH-B to 50% of intact tissue values. When the tissue was not dispersed after collagenase incubation, aldosterone and 18-OH-B outputs were similar in the two preparations. The decline in aldosterone and 18-OH-B is not attributable to loss in cell–cell contact alone, since short term culture of collagenase dispersed cells on contracting collagen discs did not restore the capacity to produce these steroids, and a decline in their output also occurred in similar culture of intact capsule tissue. In acute incubations, hyaluronidase had similar effects to collagenase, whereas trypsin, papain and a bacterial protease evoked aldosterone release during the preincubation period, but did not affect subsequent yields of aldosterone and 18-OH-B in incubations of dispersed (but not filtered tissue) in the presence of ACTH. Chymo-trypsin had no effect on preincubation but eliminated subsequent response to ACTH in all incubation conditions. Together with previously published data on the effects of trypsin, the results support the view that in intact rat adrenal glomerulosa tissue, aldosterone and 18-OH-B are sequestered into intracellular stores in the form of novel steroid-protein complexes. These are hydrolysed by trypsin and other preoteases with consequent release of steroid, but are virtually eliminated by conventional methods of cell suspension preparations, using collagenase preincubation with subsequent mechanical dispersal and filtration.     

Evolutionary trends in adrenal gland of anurans and urodeles

Morphological, histological, ultrastructural, and developmental research on the adrenal gland of several species of anurans and urodeles belonging to different families is presented. Urodeles show a large variability in adrenal glandular structure without a clear taxonomic pattern, although increased compactness of the gland and mingling of steroidogenic and chromaffin cells are found only in some neourodeles. In anurans the glandular pattern may be divided into two subtypes: one more medial and diffuse, which is observed in frogs of the more primitive families; the other more lateral and aggregated, as seen in the more advanced families. The adrenal gland therefore increases in its compactness and aggregation of chromaffin and steroidogenic tissues in the transition from primitive to advanced families, both in urodeles and anurans. Until the end of metamorphosis, morphogenesis of the gland is similar in all amphibians studied. This process is extended after metamorphosis in the advanced anurans, in order that the gland may reach its definitive position.     

Morphogenesis of the adrenal gland of Triturus cristatus carnifex during larval development and metamorphosis

During the morphogenesis of the adrenal gland of Triturus cristatus, a cranio-caudal differentiation is observed together with a migration of the two cell types composing the adrenal gland: the steroidogenic cells and the chromaffin cells. During the cranio-caudal differentiation the two cell type gradually occupy an increasingly posterior position on the mesonephros until they are distributed, in the adult, along the whole kidney. The migration brings the cells from dorsal or dorso-lateral position, with respect to the venous vessels, to the ventral surface of the kidney, an arrangement typical of the adult.     

Ontogenic corticosteroidogenesis of the domestic fowl: response of isolated adrenocortical cells

Ontogenic adrenocortical function of the domestic was investigated using adrenocortical cells isolated from embryonic chicks (18, 19, 20, and 21 days old) and male and female posthatch birds (1 day, 1 week, and 3 weeks old). Production of the predominant corticosteroids secreted by the chicken adrenal gland, corticosterone, cortisol, and aldosterone, was measured by radioimmunoassay after 2-hr incubation of cells with or without steroidogenic agents. Approaching hatch, basal and maximal ACTH-(1-24) (ACTH)-induced corticosteroid production increased steadily and peaked around 1 day posthatch (5-18 times and 3-9 times, respectively, the production values at 18 days embryonic life). Thereafter, corticosteroid production values decreased steadily to 3 weeks posthatch. Corticosterone predominated over the ages studied: Maximal ACTH-induced corticosterone production averaged 52 and 115 times the production values of aldosterone and cortisol, respectively. In addition, maximal ACTH-induced aldosterone production was roughly 2.2 times greater than cortisol production over the ages studied except for a short-lived, disproportionately greater aldosterone production at 1 day posthatch. In addition to perihatch and age-related differences in cellular corticosteroid production, there were also differences in cellular sensitivity to steroidogenic agents as indicated by the differences in half-maximal steroidogenic concentration values (ED50 values) of the steroidogenic agents. Sensitivity to ACTH increased 2.7 times from Day 18 of embryonic life to 1 day posthatch and then decreased steadily to 3 weeks posthatch. In addition, sensitivity to 8-bromo-cAMP (8-Br-cAMP) increased abruptly at 1 day posthatch (nearly 3 times) but then remained constant thereafter. However, a consistent change in cellular sensitivity to 25-hydroxycholesterol was not observed until 3 weeks posthatch (an increase in sensitivity of 3 times that at Day 18 of embryonic life). These data of cellular sensitivity suggest that there were distinct development and maturational alterations in the cellular loci at which ACTH, 8-Br-cAMP, and 25-hydroxycholesterol acted. Thus, during the transition from embryonic to postembryonic life of the domestic fowl, there are alterations in adrenocortical cell steroidogenic capacity and in the function of some cellular loci comprising the corticosteroidogenic pathway.     

Structure-activity relationships of monomeric and dimeric synthetic ACTH fragments in perifused frog adrenal slices

The effect of synthetic monomeric and dimeric ACTH fragments on spontaneous and ACTH(1-39)-evoked steroidogenesis in frog interrenal tissue was studied in vitro. Infusion of ACTH fragment 11-24 (10(-6) M) or its dimeric conjugates, attached either by their N-terminal, Glu(11-24)2, or their C-terminal amino acid, (11-24)2Lys, had no effect on the spontaneous release of corticosteroids. The monomer ACTH(11-24) and the dimer Glu(11-24)2 were also totally devoid of effect on the steroidogenic response to ACTH(1-39) (10(-9)M). In contrast, the (11-24)2Lys conjugate (10(-6)M) significantly decreased ACTH-induced stimulation of corticosterone and aldosterone (-63 and -62%, respectively). The dimeric conjugate of the fragment ACTH(7-24), linked through the C-terminal ends, (7-24)2Lys (10(-6)M), was also completely devoid of effect on basal steroidogenesis but caused a marked decrease of ACTH-evoked corticosterone and aldosterone release (-72 and -80%, respectively). Conversely, infusion of the dimer (1-24)2Lys gave rise to a dose-related stimulation of corticosterone and aldosterone release. The time-course of the steroidogenic response to the dimer was similar to that of ACTH(1-24). The 1-24 conjugate was 70 times less potent than the monomers ACTH(1-24) and ACTH(1-39). These results suggest that amphibian adrenocortical cells contain only one class of ACTH receptor which recognizes the 11-24 domain of ACTH with an affinity which depends on the presence of a strong potentiator segment, located at the N-terminus end of ACTH(1-39). Since the ACTH-dimers are thought to induce cross-linking of the receptors, our results suggest that aggregation of ACTH receptors causes a down-regulation of the receptors.     

Of 11 candidate steroids,corticosterone concentration standardized for mass is the most reliable steroid biomarker of nutritional stress across different feather types

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Effects of TMB-8 and dantrolene on ACTH- and angiotensin-induced steroidogenesis by frog interrenal gland: evidence for a role of intracellular calcium in angiotensin action

The influence of intracellular calcium on the steroidogenic response of adrenocortical tissue to ACTH and angiotensin has been studied in the frog, using a perifusion system technique. The release of corticosterone, aldosterone and prostaglandins in the effluent medium was monitored by specific radioimmunoassays. TMB-8 and dantrolene, two potential blockers of calcium mobilization from intracellular pool(s), were tested. Dantrolene (5 X 10(-5) M) significantly reduced basal and angiotensin-induced corticosterone and aldosterone production but had little effect on ACTH-evoked steroid release. Conversely TMB-8 (10(-4) M) profoundly depressed spontaneous as well as ACTH- and angiotensin II-induced corticosteroid secretion, suggesting that this compound may affect not only calcium mobilization from the endoplasmic reticulum pool but also calcium influx. Adrenal glands perifused with both dantrolene and calcium-free medium showed no response to angiotensin II. Conversely, in calcium-free conditions and in the presence of dantrolene, angiotensin II still caused an increase in prostaglandin synthesis. Taken together, these results indicate that 1) dantrolene is a more specific agent than TMB-8 in inhibiting calcium mobilization from intracellular pool(s); 2) ACTH increases corticosteroidogenesis without inducing mobilization of intracellular calcium; 3) angiotensin II stimulates both the efflux of calcium from the endoplasmic reticulum and the influx of calcium through the plasma membrane; 4) calcium is required after prostaglandin production in the steroidogenic response of frog interrenal gland to angiotensin II.     

Effects of cytochalasin D on the distribution of actin and ACTH-induced steroidogenesis in cultured embryonic adrenal gland cells from the Pekin duck (Anas platyrhynchos)

Cultured steroidogenic cells derived from the adrenal glands of duck embryos were used to study changes in the distribution of actin associated with the corticotropic responsiveness. Actin-containing components were identified by rhodamine-phalloidin staining. The actin in most of the unstimulated cells occurred as stress fibers that either ran parallel throughout the cell or were present as domains of parallel fibers at angles to one another. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH, the cells released approximately equal amounts of corticosterone and aldosterone. Incubation of the cells in buffer containing cytochalasin D caused the cells to lose their stress fibers, and the actin became distributed at the periphery in what appeared to be fragments of stress fibers and clumps of fibrous material in the central cytoplasm. Although cytochalasin D did not affect the basal output of corticosterone and aldosterone, the 1–24 ACTH-induced rates of both hormones were suppressed significantly. After the cells had been washed in unadulterated buffer, the normal distribution of actin stress fibers was restored and the cells responded normally when incubated in buffer containing 1–24 ACTH. These results suggest that the actin components of the cytoskeleton are important determinants of corticotropin-induced steroidogenic responsiveness.