Possible relationship between the activity of the adrenal gland and the subcommissural organ in the lizard Lacerta s. sicula raf.

Summary In order to study the possible functional relationship between the adrenal gland and the subcommissural organ (SCO) in the lizard Lacerta s. sicula Raf., ACTH was administered to some specimens of this species in January when both the adrenal gland and the subcommissural organ have a very low activity. In comparison to untreated controls, the adrenals of animals treated with ACTH showed clear signs of stimulation, presenting enlarged blood vessels, very few lipid droplets, numerous polymorphic mitochondria and abundant tubular smooth endoplasmic reticulum. In addition, a distinct increase in secretory material was observed in the subcommissural cells of specimens treated with ACTH. These cells showed large cisternae of the rough endoplasmic reticulum filled with granular material in the basal region, numerous secretory granules of two types in the apical region and a reduced number of microvilli on the free cell surface. These findings, together with the results of preceding studies, lead the authors to the consideration that steroid hormones might play a role in the regulation of the secretory activity of the SCO.     

The anatomy of the parotoid gland in Bufonidae with some histochemical findings. II. Bufo alvarius.

The gross and microscopic anatomy of the venom producing parotoid glands of Bufo alvarius has been studied by light and electron microscopy. Histochemical reactions for the presence of venom constituents and of components in biochemical pathways in the synthesis and release of venom were performed. The gland is composed of numerous lobules. Each lobule is an individual unit with a lumen surrounded by a double cell layer. Microvilli of the outer layer interdigitate with microvilli of the inner layer. Cells of the outer layer resemble smooth muscle cells, are rich in adenosine triphosphatase and glucose-6-phosphatase, and contain numerous pinocytotic vesicles, glycogen granules and various organelles. These organelles include "crystalloids" of what seem to be highly organized agranular reticulum. These outer layer cells probably function in some aspects of venom synthesis, active cellular transport and contraction in the discharge of the secretory product. The inner cell layer demonstrates a positive chromaffin reaction, contains steroid material, various organelles, some pinocytotic vesicles and glycogen granules, and appears devoid of a plasmalemma on its inner surface. This layer is probably involved in venom formation and release via an apocrine type of secretion. Bufo alvarius parotid gland shows significant morphological and histochemical differences from that of B. marinus and more nearly resembles a typical steroid producing organ.     

Release of aldosterone and catecholamines from the interrenal gland of Triturus carnifex in response to adrenocorticotropic hormone (ACTH) administration

The influence of adrenocorticotropic hormone (ACTH) on the interrenal gland of Triturus carnifex was investigated by in vivo administration of synthetic ACTH. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the circulating serum levels of aldosterone, noradrenaline (NA), and adrenaline (A). In June and November, ACTH administration increased aldosterone release (from 281.50 +/- 1.60 pg/ml in carrier-injected newts to 597.02 +/- 3.35 pg/ml in June; from 187.45 +/- 1.34 pg/ml in carrier-injected animals to 651.00 +/- 3.61 pg/ml in November). The steroidogenic cells showed clear signs of stimulation, together with a reduction of lipid content in June and an increase of lipid content in November. Moreover, ACTH administration decreased the mean total number of secretory vesicles in the chromaffin cells in June (from 7.73 +/- 0.60 granules/microm2 in carrier-injected animals to 5.91 +/- 0.40 granules/microm2) and November (from 7.78 +/- 0.75 granules/microm2 in carrier-injected newts to 4.87 +/- 0.40 granules/microm2). In June, however, when T. carnifex chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/microm2; A: 0.32 +/- 0.13 granules/microm2), ACTH decreased NA content (5.52 +/- 0.32 granules/microm2) increasing NA release (from 639.82 +/- 3.30 pg/ml in carrier-injected to 880.55 +/- 4.52 pg/ml). In November, when both catecholamines, NA (3.92 +/- 0.34 granules/microm2) and A (3.84 +/- 0.33 granules/microm2), are present in the chromaffin cells, ACTH administration reduced A content (1.02 +/- 0.20 granules/microm2), enhancing adrenaline secretion (from 681.30 +/- 3.62 pg/ml in carrier-injected newts to 1,335.73 +/- 9.03 pg/ml). The results of this study indicate that ACTH influences the steroidogenic tissue, eliciting aldosterone release. The effects on the chromaffin tissue, increase of NA or A secretion, according to the period of chromaffin cell functional cycle, may be direct and/or mediated through the increase of aldosterone release. Finally, the lack of an increase of A content in the chromaffin cells, or A serum level, following ACTH administration in June might suggest an independence of PNMT enzyme on corticosteroids.     

Localization of macrophage migration inhibitory factor (MIF) to secretory granules within the corticotrophic and thyrotrophic cells of the pituitary gland.

BACKGROUND: Macrophage migration inhibitory factor (MIF) was one of the first lymphokine activities to be discovered and was described almost 30 years ago to be a soluble factor(s) produced by activated T lymphocytes. In more recent studies, MIF has been "rediscovered" to be an abundant, pre-formed constituent of the anterior pituitary gland and the macrophage, and to be a critical component in the host response to septic shock. Pituitary-derived MIF enters the circulation after infectious or stressful stimuli and appears to act to counterregulate glucocorticoid suppression of cytokine production. MATERIALS AND METHODS: Immunoelectron microscopy utilizing a combination of anti-MIF and anti-pituitary hormone-specific antibodies was used to study the ultrastructural localization of MIF within the anterior pituitary gland. Pituitaries were obtained from resting, unstimulated mice and from mice 16 hr after endotoxin administration. The release of MIF also was investigated in vitro by examining the effect of corticotropin-releasing hormone (CRH_ on the AtT-20, corticotrophic cell line. RESULTS: MIF localizes to granules present exclusively in ACTH and TSH secreting cells. Within each cell type, a subset of granules was found to contain both MIF and ACTH, or MIF and TSH. The pituitary content of MIF-containing granules decreased significantly after experimentally induced endotoxemia. In seven pituitaries examined 16 hr after LPS injection, the number of MIF-positive granules diminished by 38% in corticotrophic cells and by 48% in thyrotrophic cells when compared with controls (p < 0.05). CRH was observed to be a potent MIF secretagogue in vitro, inducing the release of MIF from corticotrophic cells at concentrations lower than that required for ACTH release. CONCLUSION: These data provide ultrastructural information that identify MIF to be a novel anterior pituitary hormone, support earlier studies showing a time-dependent release of pituitary MIF during endotoxemia, and suggest an important, systemic role for MIF in the stress response to infection and other stimuli.     

Sensitivity of the fetal rat pituitary-adrenal system to corticotropin-releasing factor in organ culture.

Six groups of adrenal glands from 17-day fetal rats were explanted to organ culture for 2 days. In one group, adrenal gland was cultured alone, and in the remaining five groups adrenal gland was cultured with pituitaries from fetuses ranging in age from 14 to 18 days. In each of the groups, half of the cultures had corticotropin-releasing factor (CRF) added to the medium. A histometric parameter utilized the size of adrenocortical cells as an indicator of sensitivity of the pituitary-adrenal system. When 17-day adrenal gland was cultured alone, addition of CRF did not cause any enlargement of cortical cells. When the adrenal gland was cultured with two 14-day pituitaries, cortical cells were enlarged. Addition of CRF to this culture induced no further change. With two 15-day pituitaries in the presence of CRF, cortical cells were slightly larger than those in the absence of CRF. With 16- to 18-day pituitaries, a marked hypertrophy of cortical cells was induced, and the addition of CRF caused further acceleration in their enlargement. These results suggest that, in organ culture, 14-day pituitary can release some adrenocorticotropic hormone (ACTH) with or without additional CRF. Older pituitaries (16- to 18-day) can apparently release an amount of ACTH in the presence of CRF that is greater than their own spontaneous ACTH secretion.     

Evidence for 6-keto-PGF1 alpha in adrenal cortex of the rat and effects of 6-keto-PGF1 alpha and PGI2 on adrenal cAMP levels and steroidogenesis.

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF1 alpha, the hydrolysis metabolite of PGI2, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF1 alpha was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF2 alpha or PGE2. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI2 metabolite. Studies in vitro, using isolated cortical cells exposed to 6-keto-PGF1 alpha (10(-6)-10(-4)M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI2 (10(-6)-10(-4)M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering cortico-sterone production, ACTH (5-200 microU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI2 produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused in situ with PGI2 showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF1 alpha is present in the rat adrenal cortex, its precursor, PGI2, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Immunomodulatory ionophore copolymers, T150R1 and T130R2, induce corticotropin from anterior pituitary cells.

T150R1 is a synthetic copolymer with Na+ ionophore activity. We demonstrated previously that T150R1, when injected into mice, produces rapid thymic involution with depletion of cortical thymocytes. Elevated serum ACTH and corticosterone levels, as well as abrogation of the effects of T150R1 on the thymus by adrenalectomy and hypophysectomy, suggested a pituitary-mediated mechanism. In this work, we investigated the ability of T150R1, and of the related ionophore copolymer T130R2, to stimulate ACTH in vitro from the mouse anterior pituitary cell line AtT-20. Copolymer-induced ACTH release was dose-, time-, and temperature-dependent. Hormone induction peaked at 30 degrees C for T150R1 and 37 degrees C for T130R2. The temperature dependence of ACTH release paralleled that of ionophore activity measured in red blood cells, providing evidence that the ability to induce ACTH is related to the ionophore property of the copolymers. Peak ionophore activity and hormonal release occurred at the temperatures when the copolymers form partially soluble complexes which interact optimally with cell membranes. Cotreatment with exogenous phospholipase C inhibited the effects of T150R1, which suggests that the enzyme either blocks the insertion of T150R1 into the cell membrane or that the phospholipase C-induced increase in intracellular calcium inhibits the ionophore activity of T150R1. These data support an ionophore mechanism for copolymer-induced ACTH release in which changes in the physicochemical structure of the copolymers may affect their interaction with cell membranes. The data also suggest that direct stimulation of pituitary ACTH accounts for at least some of the in vivo immunomodulatory effects of T150R1.     

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.     

The relationship between adrenal vascular events and steroid secretion: the role of mast cells and endothelin.

The actions of ACTH on the adrenal cortex are known to be 2-fold. In addition to increased steroidogenesis, ACTH also causes marked vasodilation, reflected by an increased rate of blood flow through the gland. Our studies, using the in situ isolated perfused rat adrenal preparation, have shown that zona fasciculata function and corticosterone secretion are closely related to vascular events, with an increase in perfusion medium flow rate causing an increase in corticosterone secretion, in the absence of any known stimulant. These observations give rise to two important questions: how does ACTH stimulate blood flow; and how does increased blood (or perfusion medium) flow stimulate steroidogenesis? Addressing the first question, we have recently identified mast cells in the adrenal capsule, and shown that Compound 48/80, a mast cell degranulator, mimics the actions of ACTH on adrenal blood flow and corticosterone secretion. We have also demonstrated an inhibition of the adrenal vascular response to ACTH in the presence of disodium cromoglycate, which prevents mast cell degranulation. We conclude, therefore, that ACTH stimulates adrenal blood flow by its actions on mast cells in the adrenal capsule. Addressing the second question, we looked at the role of endothelin in the rat adrenal cortex. Endothelin 1, 2 and 3 caused significant stimulation of steroid secretion by collagenase dispersed cells from both the zona glomerulosa and the zona fasciculata. A sensitive response was seen, with significant stimulation at an endothelin concentration of 10(-13) mol/l or lower. Endothelin secretion by the in situ isolated perfused rat adrenal gland was measured using the Amersham assay kit. Administration of ACTH (300 fmol) caused an increase in the rate of immunoreactive endothelin secretion, from an average of 28.7 +/- 2.6 to 52.6 +/- 6 fmol/10 min (P less than 0.01, n = 5). An increase in immunoreactive endothelin secretion was also seen in response to histamine, an adrenal vasodilator, which stimulates corticosterone secretion in the intact gland, but has no effect on collagenase-dispersed cells. From these data we conclude that endothelin may mediate the effects of vasodilation on corticosterone secretion, and this mechanism may explain some of the differences in response characteristics between the intact gland and dispersed cells.     

The role of high density lipoproteins in rat adrenal cholesterol metabolism and steroidogenesis

Addition of rat or human high density lipoproteins (HDL) or human low density lipoproteins (LDL) to rat adrenocortical cells in vitro was found to enhance steroid production and increase cell cholesterol content. These effects of HDL were not observed in cultured mouse Y-1 adrenal cells, suggesting that rat adrenal cells possess a specific mechanism for uptake of HDL cholesterol not found in Y-1 cells. The effects of HDL were most marked on cells previously stimulated with adrenocorticotropin (ACTH) and depleted of their endogenous cholesterol stores. Such cells were prepared either by treatment in vivo with 4-aminopyrazolopyrimidine or in vitro with ACTH (10(-7) M) in lipoprotein-poor media. Steroid production by treated cells exhibited a saturable dependence on media HDL concentration. In addition to enhancing ACTH stimulated steroid production, addition of HDL also resulted in a saturable concentration-dependent increase in cell cholesterol content. Both aminoglutethimide and cycloheximide were found to inhibit HDL-enhanced steroid production. Finally, addition of HDL to short term incubations (5 1/2 h) of ACTH-treated cells caused no change in the rate of incorporation of 14C-acetate into cholesterol or corticosterone. These results indicate that rat adrenocortical cells possess a specific, saturable, ACTH-dependent mechanism for uptake of HDL cholesterol. Moreover, cellular uptake of HDL cholesterol exceeded by at least 4-fold the amount of cholesterol associated with HDL apoprotein degraded by the cells, suggesting that utilization of HDL cholesterol does not require endocytosis and lysosomal degradation of the entire HDL particle.     

Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands.

nurr77 and nurr-1 are growth factor-inducible members of the steroid/thyroid hormone receptor gene superfamily. In order to gain insight into the potential roles of nur77 in the living organism, we used pharmacologic treatments to examine the expression of nur77 in the mouse adrenal gland. We found that nur77 and nurr-1 are induced in the adrenal gland upon treatment with pentylene tetrazole (Ptz; Metrazole). This induction is separable into distinct endocrine and neurogenic mechanisms. In situ hybridization analysis demonstrates that nur77 expression upon Ptz treatment in the adrenal cortex is localized primarily to the inner cortical region, the zona fasciculata-reticularis, with minimal induction in the zona glomerulosa. This induction is inhibitable by pretreatment with dexamethasone, indicating involvement of the hypothalamic-pituitary-adrenal axis in the activation of adrenal cortical expression. When mice were injected with adrenocorticotrophic hormone (ACTH), nur77 expression in the adrenal gland spanned all cortical layers including the zona glomerulosa, but medullary expression was not induced. Ptz also induces expression of both nur77 and nurr-1 in the adrenal medulla. Medullary induction is likely to have a neurogenic origin, as nur77 expression was not inhibitable by dexamethasone pretreatment and induction was seen after treatment with the cholinergic neurotransmitter nicotine. nur77 is also inducible by ACTH, forskolin, and the second messenger analog dibutyryl cyclic AMP in the ACTH-responsive adrenal cortical cell line Y-1. Significantly, Nur77 isolated from ACTH-stimulated Y-1 cells bound to its response element whereas Nur77 present in unstimulated cells did not. Moreover, Nur77 in ACTH-treated Y-1 cells was hypophosphorylated at serine 354 compared with that in untreated cells. These results, taken together with the previous observation that dephosphorylation of serine 354 affects DNA binding affinity in vitro, show for the first time that phosphorylation of Nur77 at serine 354 is under hormonal regulation, modulating its DNA binding affinity. Thus, ACTH regulates Nur77 in two ways: activation of its gene and posttranslational modification. A promoter analysis of nur77 induction in Y-1 cells indicates that the regulatory elements mediating ACTH induction differ from those required for induction in the adrenal medullary tumor cell line PC12 and in 3T3 fibroblasts.     

Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells

The pituitary cell line, AtT-20, synthesizes adrenocorticotropic hormone (ACTH) as a glycoprotein precursor that is cleaved into mature hormones during packaging into secretory granules. The cells also produce an endogenous leukemia virus (MuLV) that is glycosylated after translation similar to the glycosylation of the ACTH precursor. Our evidence suggests that the envelope glycoprotein and some precursor ACTH get to the cell surface in a vesicle different from the mature ACTH secretory granule. Viral glycoproteins and ACTH precursor are released from the cells much sooner after synthesis than mature ACTH. Isolated secretory granules do not contain significant amounts of the envelope glycoprotein or ACTH precursor. Exposing cells to 8Br-cAMP stimulates release of mature ACTH four to five fold, but has little effect on the release of the ACTH precursor or the viral glycoproteins. We propose that the viral glycoproteins and some of the ACTH precursor are transported by a constitutive pathway, while mature ACTH is stored in secretory granules where its release is enhanced by stimulation.     

The ACTH-interrenal axis in the freshwater stickleback,Gasterosteus aculeatus form leiurus

Summary The ACTH-interrenal axis of the freshwater stickleback has been examined with the fish in a variety of physiological conditions. A morphometric analysis of ACTH cell ultrastructure in spring animals revealed that the only change from the winter condition was a significant decrease in the amount of perinuclear RER. The interrenal gland responded to metopirone treatment by an increase in both nuclear and cell size, although only a high dose of metopirone could degranulate the ACTH cells. Morphometry of the ACTH cells from metopirone-treated animals showed a significant increase in the amount of RER and a significant decrease in the number of free ribosomes and secretory granules, compared with control animals maintained in freshwater. Such ultrastructural changes may be expected of a cell that is stimulated to increase its secretion of polypeptide hormone. The ACTH-interrenal axis also responded to 70% seawater, as this treatment increased the interrenal cell and nuclear sizes.     

Regulation of adrenal scavenger receptor-BI expression by ACTH and cellular cholesterol pools.

Scavenger receptor BI (SR-BI) mediates selective uptake of high density lipoprotein (HDL) cholesteryl ester in the liver and adrenal gland. Adrenal SR-BI is increased both in adrenocorticotropic hormone (ACTH)-treated mice and also in apolipoprotein A-I knock-out (apoA-I0) mice which have depleted adrenal cholesterol stores. The goal of the present study was to determine whether adrenal cholesterol stores and ACTH have independent effects on SR-BI expression in adrenal gland. Adrenal SR-BI levels were 5-fold higher in apoA-I0 than wild-type mice when killed under low stress condition, and plasma ACTH levels were similar in both strains. After male apoA-I0 or wild-type mice were treated with dexamethasone to suppress ACTH release, adrenal SR-BI protein levels were decreased in both groups but remained 13-fold higher in apoA-I0 than in wild-type mice. By contrast, uncontrolled stress or supplemental ACTH treatment increased SR-BI levels but narrowed the difference in SR-BI expression between apoA-I0 and wild-type. Cholesterol depletion by beta-cyclodextrin in cultured Y1-BS1 adrenal cells also led to a rapid 2- to 3-fold increase in SR-BI mRNA and protein levels, in association with a significant depletion of cellular free cholesterol.These results indicate that depletion of adrenal cholesterol stores can act independently from ACTH to increase SR-BI expression, but in vivo this effect is diminished under high ACTH conditions. Both stimuli may increase selective uptake via increased SR-BI as a means of replenishing cholesterol stores for steroid hormone synthesis.     

The ultrastructure of functional mouse adrenal cortical tumor cells in vitro.

Cells derived from a transplantable mouse adrenal cortical tumor maintain their differentiated function in vitro and secrete steroids in response to ACTH and other stimulatory agents. The cell line has been widely employed for various biochemical investigations but there have been few attempts to correlate this work with morphologic data. This communication describes the electron microscopic appearance of the tumor transplant in vivo and primary cultures derived from it at various intervals after the cells are placed in culture. Tumor cells in vivo bear considerable resemblance to normal adult mouse adrenal cortical cells. Organelles generally considered to be directly involved in steroid biosynthesis (mitochondria, smooth endoplasmic reticulum and lipid droplets) are not drastically altered. Certain modifications of the vasculature and cell membrane, seemingly related to steroidogenesis, are present in both the tumor and normal adrenal cortex. Within 2 days after the tumor cells are introduced to culture, their cytoplasm assumes a more simplified appearance. Smooth endoplasmic reticulum is less conspicuous and free ribosomes and polysomes are very abundant. Mitchondrial inner membranes are reorganized from a saccular arrangement in the cells in vivo into distinct lamellar cristae. The tumor cells now resemble undifferentiated embryonic adrenal cells, or cultured adrenal cells from various mammalian sources which have dedifferentiated in the absence of ACTH. In their morphologically unspecialized state, the normal cells are incapable of functional responses to ACTH. In contrast, the cultured, dedifferentiated tumor cells respond within minutes to this hormone and can demonstrate 5-20 fold increases in their basal steroid output. These data suggest that substantial steroidogenic activity can occur although the characteristic appearance of adrenal mitochondria is absent.     

P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells

P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to alpha granules of platelets and Weibel-Palade bodies of endotheial cells. Upon stimulation of these cells, P-selectin is translocated to the plasma membrane where it functions as a receptor for monocytes and neutrophils. To investigate whether the mechanism of targeting of P-selectin to granules is specific for megakaryocytes and endothelial cells and/or dependent on von Willebrand factor, a soluble adhesive protein that is stored in the same granules, we have expressed the cDNA for P-selectin in AtT-20 cells. AtT-20 cells are a mouse pituitary cell line that can store proteins in a regulated fashion. By double-label immunofluorescence, P-selectin was visible as a punctate pattern at the tips of cell processes. This pattern closely resembled the localization of ACTH, the endogenous hormone produced and stored by the AtT-20 cells. Fractionation of the transfected cells resulted in the codistribution of P-selectin and ACTH in cellular compartments of the same density. Immunoelectron microscopy using a polyclonal anti-P-selectin antibody demonstrated immunogold localization in dense granules, morphologically indistinguishable from the ACTH granules. Binding experiments with radiolabeled monoclonal antibody to P-selectin indicated that there was also surface expression of P-selectin on the AtT-20 cells. After stimulation with the secretagogue 8-Bromo-cAMP the surface expression increased twofold, concomitant with the release of ACTH. In contrast, the surface expression of P-selectin transfected into CHO cells, which do not have a regulated pathway of secretion, did not change with 8-Br-cAMP treatment. In conclusion, we provide evidence for the regulated secretion of a transmembrane protein (P-selectin) in a heterologous cell line, which indicates that P-selectin contains an independent sorting signal directing it to storage granules.     

Relationship between fetal adrenal morphology and anterior pituitary function

A comparative study of adrenal morphology between normal fetuses and those with anencephaly or congenital adrenal hyperplasia (CAH) was performed in order to examine the hypothesis that fetal adrenal mass and structure are adrenocorticotrophin (ACTH)-dependent throughout gestation. Combined adrenal weight in 102 normal fetuses was used to establish a reference range for the gestational ages of 15-27 weeks. During this period, mean adrenal weight showed a 6-fold linear increase. In 38 anencephalic fetuses of similar gestation age, adrenal weight was below the normal range and did not show a rise. Three fetuses with CAH (18, 22 and 30 weeks gestation) had adrenal weights considerably above the normal range. Adrenal cortical thickness was significantly increased in CAH fetuses, largely as a consequence of cell hypertrophy, whereas decreased cortical thickness in the anencephalic group represented cellular hypoplasia. Conspicuous secretory granules in the cytoplasm was the electron-micrographic feature of the adrenal gland in the 22-week fetus with CAH. These observations are consistent with close dependency of fetal adrenal growth and development upon fetal pituitary function from an early age, mediated primarily through ACTH.     

The Ultrastructure of Functional Mouse Adrenal Cortical Tumor Cells in vitro

Cells derived from a transplantable mouse adrenal cortical tumor maintain their differentiated function in vitro and secrete steroids in response to ACTH and other stimulatory agents. The cell line has been widely employed for various biochemical investigations but there have been few attempts to correlate this work with morphologic data. This communication describes the electron microscopic appearance of the tumor transplant in vivo and primary cultures derived from it at various intervals after the cells are placed in culture. Tumor cells in vivo bear considerable resemblance to normal adult mouse adrenal cortical cells. Organelles generally considered to be directly involved in steroid biosynthesis (mitochondria, smooth endoplasmic reticulum and lipid droplets) are not drastically altered. Certain modifications of the vasculature and cell membrane, seemingly related to steroidogenesis, are present in both the tumor and normal adrenal cortex. Within 2 days after the tumor cells are introduced to culture, their cytoplasm assumes a more simplified appearance. Smooth endoplasmic reticulum is less conspicuous and free ribosomes and polysomes are very abundant. Mitochondrial inner membranes are reorganized from a saccular arrangement in the cells in vivo into distinct lamellar cristae. The tumor cells now resemble undifferentiated embryonic adrenal cells, or cultured adrenal cells from various mammalian sources which have dedifferentiated in the absence of ACTH. In their morphologically unspecialized state, the normal cells are incapable of functional responses to ACTH. In contrast, the cultured, dedifferentiated tumor cells respond within minutes to this hormone and can demonstrate 5–20 fold increases in their basal steroid output. These data suggest that substantial steroidogenic activity can occur although the characteristic appearance of adrenal mitochondria is absent.     

Histochemical,ultrastructural and hormonal studies on the pars distalis of the echidna (Tachyglossus aculeatus)

There were no consistent significant differences between the concentrations of luteinizing hormone (LH) and adrenocorticotrophin (ACTH) in the rostral compared with the caudal zone of the echidna pars distalis. This suggests that LH is secreted by cells containing S-type granules (probably corresponding to secretory vesicles 200-300 nm diameter) which are distributed throughout the gland. Some of the cells containing vesicles 100-200 nm diameter, seen in small numbers in both zones of the gland, may be responsible for the secretion of ACTH. The concentration of pituitary LH is in the range of that found in eutherian mammals, but the concentration of ACTH is lower than that reported for other vertebrates, and this may be linked causally with the remarkably low rate of corticosteroid secretion in the echidna. The absence of significantly increased levels of pituitary LH and ACTH in a chronically orchidectomized and adrenalectomized animal adds to other evidence which suggests that mechanisms involving a negative feedback of steroid hormones on the hypothalamo-hypophysial axis may not be fully developed in the echidna.