Stimulated Corticosterone Production Research Articles

Does the Glucocorticoid Stress Response Make Toads More Toxic? An Experimental Study on the Regulation of Bufadienolide Toxin Synthesis.

Chemical defense is a crucial component of fitness in many organisms, yet the physiological regulation of defensive toxin synthesis is poorly understood, especially in vertebrates. Bufadienolides, the main defensive compounds of toads, are toxic to many predators and other natural enemies, and their synthesis can be upregulated by stressors, including predation risk, high conspecific density, and pollutants. Thus, higher toxin content may be the consequence of a general endocrine stress response in toads. Therefore, we hypothesized that bufadienolide synthesis may be stimulated by elevated levels of corticosterone (CORT), the main glucocorticoid hormone of amphibians, or by upstream regulators that stimulate CORT production. To test these alternatives, we treated common toad tadpoles with exogenous CORT (exoCORT) or metyrapone (MTP, a CORT-synthesis inhibitor that stimulates upstream regulators of CORT by negative feedback) in the presence or absence of predation cues for 2 or 6 days, and subsequently measured their CORT release rates and bufadienolide content. We found that CORT release rates were elevated by exoCORT, and to a lesser extent also by MTP, regardless of treatment length. Bufadienolide content was significantly decreased by treatment with exoCORT for 6 days but was unaffected by exposure to exoCORT for 2 days or to MTP for either 6 or 2 days. The presence or absence of predation cues affected neither CORT release rate nor bufadienolide content. Our results suggest that changes in bufadienolide synthesis in response to environmental challenges are not driven by CORT but may rather be regulated by upstream hormones of the stress response.

Mechanism of Toona sinensis-stimulated adrenal steroidogenesis in primary rat adrenal cells

Toona sinensis is a well-known nutritious plant food widely distributed in Asia. The effects of aqueous leaf extracts of T. sinensis (TSL-1) on steroidogenesis was examined using primary rat adrenal cells. TSL-1 stimulated corticosterone production in a time- and concentration-dependent manner. Cells were then challenged with forskolin, dibutyryl cyclic AMP (dbcAMP), pregnenolone, progesterone or deoxycorticosterone in the presence or absence of TSL-1 for 6 h to determine the mechanism. TSL-1 could enhance forskolin- and dbcAMP-induced steroidogenesis and promote pregnenolone-, progesterone- and deoxycorticosterone-induced steroidogenesis. Moreover, TSL-1 induced steroidogenic acute regulatory protein (StAR) mRNA and protein expression in rat adrenal cells and increased StAR promoter activity in CHO-K1 cells. Thus, the stimulatory effect of TSL-1 was presumed to be mainly due to methyl and ethyl gallates. Therefore, TSL-1-stimulated corticosterone production requires the participation of cAMP-dependent PKA, steroidogenic enzymes and the expression of StAR, which regulates the rate-limiting step in steroidogenesis.

Production of corticosteroid hormones in vitro by adrenals in rats with streptozotocin-induced diabetes.

We studied baseline and ACTH-stimulated in vitro production of corticosteroids by rat adrenals. Production of the basic corticosteroids pregnenolone (early precursor in corticosteroid synthesis), progesterone (intermediate precursor in synthesis of gluco- and mineralocorticoid hormones), and corticosterone (major glucocorticoid hormone in rodents) in animals with streptozotocin-induced diabetes was enhanced by 1.8-2.0 times in comparison with the control animals. Addition of ACTH to the incubation medium stimulated pregnenolone production by the adrenals equally in the control and experimental (diabetic) groups, while the increase in corticosterone production was less pronounced in the experimental group. Stimulation of corticosterone production in response to ACTH after saturation of the incubation medium with pregnenolone was also less pronounced in diabetic rats.

The β-Human Chorionic Gonadotropin-Related Peptide LQGV Exerts Anti-Inflammatory Effects through Activation of the Adrenal Gland and Glucocorticoid Receptor in C57BL/6 Mice

The systemic inflammatory response syndrome is a complex host response to a variety of clinical insults, generally leading to severe pathology. The human chorionic gonadotropin β-chain-related tetrapeptide leucine-glutamine-glycine-valine (LQGV) reduces hemorrhagic and LPS-induced systemic inflammatory response syndrome, but its mechanisms of action are not yet fully understood. Through the combination of in vivo, in vitro, and ex vivo approaches, we demonstrate that LQGV actively stimulates corticosterone production in mice and thereby suppresses in vivo TLR4-directed inflammation upon LPS administration. Blocking in vivo glucocorticosteroid receptor signaling reduced the prosurvival effect of LQGV. Also, upon multiple TLR activation by heat-killed Listeria monocytogenes, splenocytes from LQGV-treated mice produced significantly less TNF-α and IL-6, which was absent after in vitro blockage of the glucocorticosteroid receptor. Using adrenal gland and adrenal cell line cultures, we show that LQGV stimulates corticosterone production. Moreover, by using specific pharmacological inhibitors of the adrenocorticotropic hormone (ACTH) and luteinizing hormone receptors as well as of cAMP signaling, we demonstrate that LQGV stimulates the ACTH receptor. These data show that the β-human chorionic gonadotropin-related tetrapeptide LQGV stimulates adrenal glucocorticosteroid production through activation of the ACTH receptor with consequent glucocorticoid receptor activation and immunosuppression in C57BL/6 mice.

Calcitonin directly increases adrenocorticotropic hormone-stimulated corticosterone production in the hen adrenal gland

The present study was performed to elucidate whether the receptor for calcitonin (CT) exists in the adrenocortical cells of hens and to determine the effect of CT on adrenocorticotropic hormone (ACTH)-stimulated corticosterone production in its cell. The binding site of CT in the membrane fraction of the adrenal gland in hens was determined using a [125 I]CT binding assay system. The binding properties in the adrenal gland satisfied the criteria of a receptor-ligand interaction in terms of specificity, reversibility, and saturation. When the cortical cells were incubated in vitro with chicken ACTH in the presence of CT, greater corticosterone production was observed. The result suggested that CT acts directly on the adrenocortical cells via its receptor binding and increases responsiveness of ACTH on corticosterone production in the laying hen.

Ectopic Agouti protein overexpression increases stimulated corticosterone production without effect on adenylate cyclase activity in mouse adrenal cells

The antagonism of Agouti protein (AP) and Agouti-related protein on melanocortin receptors suggests an inhibitory role in the regulation of steroidogenesis. However, we have previously demonstrated that ectopic AP overexpression increased restraint-induced corticosterone release and adrenal reactivity to ACTH in mice. A high steroidogenic response to ACTH may be a consequence of a stimulatory AP action on the adenylate cyclase (AC) and/or intracellular steroidogenic enzymes. The aim of the present study was to estimate the effect of ectopic AP overexpression on the activity of AC and steroidogenic intracellular enzymes. ACTH and forskolin were used for AC stimulation, and dibutyryl cAMP and progesterone were used for stimulation of intracellular steroidogenic enzymes in isolated adrenal cells in male C57Bl/6J mice of two Agouti genotypes: A(y)/a (ectopic AP overexpression) and a/a (absence of AP in all tissues). ACTH and forskolin increased cAMP accumulation to the same extent in both A(y)/a and a/a mouse adrenal cells (P<0.001; ANOVA), but resulted in higher corticosterone production in A(y)/a mice (P<0.001 for ACTH and P<0.01 for forskolin; ANOVA). Dibutyryl cAMP- and progesterone-induced corticosterone production was higher in A(y)/a mice than in a/a mice (P<0.001 for dibutyryl cAMP and P<0.01 for progesterone; ANOVA). Ectopic AP overexpression increased stimulated corticosterone production and intracellular steroidogenic enzyme reactivity to cAMP without an effect on AC activity.

Effects of leptin and leptin fragments on corticosterone secretion and growth of cultured rat adrenocortical cells

Compelling evidence indicates that leptin, acting via specific receptors (Ob-Ra and Ob-Rb) modulates adrenocortical-cell secretion. However, the results are controversial, inasmuch as either secretagogue or antisecretagogue effects have been reported. Hence, we decided to study the effects of a 96-h incubation with leptin and leptin fragments 116-130, 150-167, 138-167, 93-105, 22-56 and 26-39 (10(-8) and 10(-6) M) on the secretion and growth of cultured rat adrenocortical cells. Reverse transcription-polymerase chain reaction showed that control cultures expressed both Ob-Ra and Ob-Rb isoforms. As expected, ACTH (10(-8) M) raised corticosterone secretion and lowered proliferation rate of cultured cells. Native leptin elicited ACTH-like effects, while fragment 116-130 was ineffective. Leptin fragments 150-167 and 26-39 stimulated corticosterone production, and fragments 138-167 and 22-56 inhibited it. Fragment 93-105 exerted a dose-dependent biphasic effect on corticosterone secretion (i.e. stimulation and inhibition at the concentration of 10(-8) and 10(-6) M, respectively). Leptin fragment 26-39 enhanced proliferation of cultured cells, while fragments 138-167 and 22-56 were ineffective. Fragments 150-167 and 93-105 displayed proliferogenic and antiproliferogenic effects at the concentration of 10(-8) and 10(-6) M, respectively. Taken together, these findings allows us to conclude that native leptin and its fragments interact differently with Ob-Rs or interact with different Ob-R isoforms, thereby variously modulating secretion and growth of cultured rat adrenocortical cells.

An oxidized metabolite of linoleic acid stimulates corticosterone production by rat adrenal cells.

Oxidized derivatives of linoleic acid have the potential to alter steroidogenesis. One such derivative is 12,13-epoxy-9- keto-10-(trans)-octadecenoic acid (EKODE). To evaluate the effect of EKODE on corticosterone production, dispersed rat zona fasciculata/reticularis (subcapsular) cells were incubated for 2 h with EKODE alone or together with rat ACTH (0, 0.2, or 2.0 ng/ml). In the absence of ACTH, EKODE (26 microM) increased corticosterone production from 5.3 +/- 2.3 to 14.7 +/- 5.0 ng. 10(6) cells. h(-1). The stimulatory effect of ACTH was increased threefold in the presence of EKODE (26.0 microM). Cholesterol transport/P-450scc activity was assessed by measuring basal and cAMP-stimulated pregnenolone production in the presence of cyanoketone (1.1 microM). EKODE (13.1 and 26.0 microM) significantly increased basal and cAMP-stimulated (0.1 mM) pregnenolone production. In contrast, EKODE decreased the effect of 1.0 mM cAMP. EKODE had no effect on early or late-pathway activity in isolated mitochondria. We conclude that EKODE stimulates corticosterone biosynthesis and amplifies the effect of ACTH. Increased levels of fatty acid metabolites may be involved in the increased glucocorticoid production observed in obese humans.

OXIDIZED PRODUCTS OF LINOLEIC ACID STIMULATE ADRENAL STEROIDOGENESIS

Adrenal steroidogenesis is under complex control, and clinical observations suggest that not all regulators have been identified. We postulated that fatty acid oxidation products found in the diet or formed in the body could affect steroidogenesis. Linoleic acid is a prominent constituent of animal fat and is readily oxidized. We found that several products of linoleic acid oxidation affect production of aldosterone and corticosterone by isolated cells from rat adrenals. We characterized one linoleic acid derivative by gas chromatography/mass spectrometry. It is 12,13-epoxy-9-oxo-10(trans)-octadecenoic acid (“EKODE”). At concentrations between 1 and 30 µM, EKODE stimulated production of aldosterone by zona glomerulosa cells, but at concentrations above 50 µM, it was inhibitory. In zona fasciculata cells, EKODE stimulated corticosterone production at concentrations of 5 µM or greater, and there was no evidence of inhibition at high concentrations. Stimulation of steroidogenesis was observed after 15 min of incubation and continued for at least 2 hrs. The potential relevance of our findings to the hypertension of obesity is discussed.

Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10 −8 M. In contrast, orexins did not affect either maximally ACTH (10 −9 M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10 −6 M) annulled corticosterone response of ZF/R cells to ACTH (10 −9 M), but not to orexins (10 −8 M). Orexins (10 −8 M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10 −5 M) abolished corticosterone responses to both ACTH (10 −9 M) and orexins (10 −8 M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.

Hypothalamic-pituitary-adrenal activation by the bacterial superantigen staphylococcal enterotoxin B: role of macrophages and T cells.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen which stimulates T cells bearing the V beta 8 motif on the T-cell receptor. This stimulation is MHC class II dependent, and in vivo results in a rapid and pronounced T-cell cytokine response. Based on previous evidence that SEB stimulates corticosterone production in BALB/c mice, which possess a high percentage of V beta 8+ T cells, we explored the effects of SEB on the hypothalamic-pituitary-adrenal (HPA) axis and identified the peripheral immunologic cellular requirements for these effects. Administration of SEB stimulates corticosterone in a dose-dependent manner, with peak production of corticosterone occurring by 2 h after intraperitoneal challenge with 50 micrograms SEB. Challenge with staphylococcal enterotoxin A, which activates V beta 3+ and V beta 11+ T cells (deleted during ontogenesis in BALB/c mice), did not increase ACTH or corticosterone production. Furthermore, SEB challenge increased plasma ACTH, which accounted for the increased plasma corticosterone, and increased the expression of c-fos in the PVN region of the hypothalamus. In vivo elimination of macrophages did not prevent the corticosterone response to SEB, suggesting that pituitary-adrenal activation does not require macrophages. However, when mice were pretreated with the T-cell immunosuppressant cyclosporin A, the significantly increased ACTH and corticosterone production in response to SEB was dramatically attenuated. These results demonstrate that bacterial superantigens can stimulate the HPA axis, and that functional T cells may play an obligatory role in this effect.

Atrial Natriuretic Peptide Stimulates Aldosterone Production by Turkey (Meleagris gallopavo) Adrenal Steroidogenic Cells

The inhibitory action of atrial natriuretic (ANPs) on mammalian aldosterone synthesis is well documented. In addition, other work indicates that ANP and an analogue of its second messenger, 8-Br-cGMP, inhibit aldosterone production by chicken adrenal steroidogenic cells. However, the interaction between angiotensin II (AII) and ANP in the regulation of avian aldosterone production is poorly understood because chicken adrenal steroidogenic cells, the commonly used in vitro avian model, are comparatively unresponsive to AII. By contrast, turkey (Meleagris gallopavo) adrenal steroidogenic cells are sensitive to AII. Thus, in the present study, the action of ANPs and related peptides and their interaction with other stimulators of aldosterone production were investigated using freshly isolated and briefly cultured turkey adrenal steroidogenic cells. Surprisingly, several ANPs [rat (r), human (h), chicken (c)], and rat brain natriuretic peptide (rBNP) were as efficacious as [Ile5]AII for stimulating aldosterone production (2 hr) in freshly isolated cell suspensious but were less potent than [Ile5]AII (ED50 of ANPs -5-10 nM; (Ile5]AII ED50 -0.1nM). In addition, chicken ANP enhanced maximal aldosterone production induced by [Ile5]AII (1 nM). K+ (25 nM), and hACTII-(1-39) (I nM: maximal enhancement of the action of these secretagogues was +49% +137% and +15%, respectively (P < 0.05; n = 3). Furthermore, other ANPs and related peptides [rBNP and bovine aldosterone secretion inhibiting factor (bASIF) enhanced maximal [He5]AII-induced aldosterone production; the order of maximal enhancement was rBNP (I 180%) > hANP/rANP (+50%) > bASIF (+25%) (P < 0.05; n = 3). Moreover, 8-Br-cGMP was also effective (+ 31%). In addition, ANPs and related peptides stimulated cGMP production (2 hr) with relative potencies and efficacies that were similar to their relative potencies and efficacies for stimulating aldosterone production, thus suggesting that cGMP mediated the action of ANPs and related peptides in turkey adrenal steroidogenic cells. However,like [Ile5]AII and K+ ANPs and related peptides and 8-Br-cGMP did not stimulate corticosterone production, thus suggesting that these agents stimulated the conversion of the existing intracellular pool of corticosterone to aldosterone. Evaluation of specific [125I-Tyr28]rANP binding to intact, freshly isolated cells indicated the presence of high-affinity ANP receptors [dissociation constant (Kd) = 1.46 ± 0.23 nM; cellular concentration = 89.400 ± 2600 sites/cell (means ± SEM; n = 3)]. Competitive binding studies with briefly cultured (12 hr) cells indicated that these receptors were specific for ANPs and related peptides. In addition, the order of competition potencies was consistent with the order of ED50s for the stimulation of cGMP and aldosterone production. Collectively, these findings suggest that ANP, through the generation of cGMP positively modulates aldosterone production by turkey adrenal steroidogenic cells. In addition, the present study suggests that the turkey adrenal steroidogenic cell may be a unique model to elucidate the effector molecules of ANP action on aldosterone synthesis.

Effect of age on cholesterol uptake and utilization by rat adrenals: I. Internalization of lipoprotein-derived cholesteryl esters

Previous studies from this laboratory have documented a progressive age-related decline in trophic hormone (or second messenger cAMP) stimulated corticosterone production in isolated adrenocortical cells. In the current study, we examined the possibility that the aging process exerts this effect by interfering with an early step in the delivery of lipoprotein-derived cholesteryl esters to the cell. As such, we monitored the ability of two different rat adrenocortical cell model systems (intact perfused adrenal glands and primary cultures of adrenocortical cells from 5- and 18- to 20-month-old rats) to internalize lipoprotein cholesteryl esters, and to convert the newly internalized cholesteryl esters to corticosterone production. The results indicate that lipoprotein (hHDL 3 and rHDL) cholesteryl ester internalization (by both the endocytic and ‘selective’ pathways) is comparable in adrenocortical cells of the young and old rats. However, despite this, both the mass of corticosterone produced and the ratio of newly internalized (radiolabeled) cholesteryl ester incorporated into corticosterone is dramatically reduced in cells of the older animals. Thus, the lipoprotein uptake pathway appears to be intact in adrenals of older rats, but the intracellular processing of internalized cholesteryl ester is defective.

Inhibition of aldosterone synthesis in rat adrenal cells by nicotine and related constituents of tobacco smoke.

Previous work has shown that nicotine and related constituents of tobacco smoke inhibit selected P450 enzymes in the glucocorticoid and sex steroid synthetic pathways. Because aldosterone synthesis is also cytochrome P450 dependent, we hypothesized a similar inhibitory action on aldosterone production. In this study we examined the effects of nicotine, anabasine (a related alkaloid), and cotinine (the major metabolite of nicotine) on in vitro aldosterone synthesis. Freshly isolated rat adrenal cells were assayed for corticosterone and aldosterone production in the basal state and after stimulation with ACTH or angiotensin-II (ANG-II). The addition of nicotine, anabasine, and cotinine in concentrations up to 100 microM did not inhibit stimulated corticosterone production. However, there was a potent dose-dependent inhibitory action of all three tobacco compounds on aldosterone production. The relative inhibitory potency was: cotinine > anabasine > nicotine. When employed at a concentration of 100 microM, the three compounds inhibited ACTH-stimulated aldosterone synthesis by 75%, 44%, and 21%, respectively. ANG-II-stimulated aldosterone synthesis was inhibited by 92%, 78%, and 62%, respectively. The plasma cotinine concentration range attained in tobacco smokers is between 1-10 microM. When tested with [3H]corticosterone and [3H]progesterone as exogenous substrates, 1-10 microM cotinine caused a significant dose-dependent inhibition of ACTH- and ANG-II-stimulated aldosterone synthesis. Cotinine substantially blocked the conversion of corticosterone to 18-hydroxycorticosterone, implicating the 18-hydroxylase or corticosterone 18-methyloxidase-I (CMO-I) step as the major site of inhibition. In summary, our results indicate that tobacco compounds cause direct and specific inhibition of aldosterone synthesis, primarily at the CMO-I step. This enzymatic blockade would be expected to result in activation of the renin-angiotensin system in vivo. We postulate that chronic stimulation of the renin-angiotensin system by this mechanism might contribute to the cardiovascular damage that occurs with long term tobacco use.

Defensins are mitogenic for epithelial cells and fibroblasts.

Defensins are a family of structurally homologous peptides contained within phagocytic cells. Although these peptides are best known for their broad spectrum antimicrobial properties, they also inhibit ACTH (corticotropin) stimulated corticosterone production, chemoattract monocytes, and lyse mammalian cells. We now report that these peptides are potent mitogens in vitro in the same concentration range that they display potent antimicrobial activity in vitro. These concentrations are in the same range as those expected to be present in vivo during the wound healing process. All defensins tested were stimulatory for epithelial cells and fibroblasts and acted synergistically with insulin. These are the first data to disclose the strong growth-promoting effects of this unique family of peptides and point to another basic mechanism whereby the macrophage and neutrophil may participate in a variety of trophic, physiologic, and pathologic processes.

Distribution of β-endorphin-like and adrenocorticotropin-like activities in various tissues of the winter flounder, Pseudopleuronectes americanus

1. Different tissues of the winter flounder were extracted with an acidic medium (1 N acetic acid in 0.1 N HCl), 2. ACTH-like activity judged by the ability to stimulate corticosterone production by isolated rat adrenal cells, and opiate receptor binding activity, were present in extracts of the pituitary and brain. 3. Opiate receptor binding activity was absent from the gonadal extract but present in extracts of the heart, liver, interrenal and intestine. 4. β-Endorphin-like immunoreactivity was present in all of the afore-mentioned tissues. 5. Flounder pituitaries were also extracted with Tris-Cl buffer (pH 7.8) and the extract subjected to affinity chromatography on Con A-Sepharose to remove glycoproteins and ultrafiltered to remove high molecular weight materials. 6. When the resulting fraction (mol. wt < 10,000 but >2000) was gel filtered on Sephadex G-10, the unretarded peak contained ACTH-like bioactivity, opiate receptor binding activity and β-endorphin-like immunoreactivity.

Effects of corticostatin-I on rat adrenal cells in vitro

We have examined the mechanism by which corticostatin-I (CS-I) acts to attenuate ACTH-induced steroidogenesis in rat adrenal cells. CS-I inhibited ACTH-induced corticosterone production in a dose-dependent manner, without any effects on the basal corticosterone level in adrenal cells. When the cells were stimulated by 100 pg ACTH/ml, the minimum effective concentration of CS-I was 100 ng/ml, and 0.3-1.0 micrograms CS-I/ml produced a 50% reduction of the stimulated corticosterone production. The inhibitory effect of CS-I on ACTH-stimulated corticosterone production became apparent within 15 min of incubation, and the effect was reversed quickly by the removal of CS-I from the media. CS-I had no effect on angiotensin II-stimulated aldosterone production by adrenal zona glomerulosa cells. CS-I also did not affect cyclic AMP- or forskolin-stimulated corticosterone production. In an in-vitro binding study using 125I-labelled CS-I, CS-I showed considerable specific binding to rat adrenal cells, and the binding competed with ACTH in a dose-dependent manner. These experiments suggest that CS-I competes with ACTH on their binding sites and exerts an inhibitory effect on the adrenal cells.

Adrenocorticotropin-like and opiate-like materials in the brain of the red grouper Epinephelus akaara (Teleostei: Serranidae)

1. 1. Grouper ( Epinephelus akaara) brains were heated and then extracted with a mixture of water, hydrochloric acid and acetone. An acid acetone powder was formed by adding the brain extract into a large volume of chilled acetone and subsequently recovering the precipitate. 2. 2. The precipitate was dissolved and subjected to salt fractionation and gel nitration on Sephadex G-10. 3. 3. The resultant unretarded fraction stimulated corticosterone production in isolated rat adrenal cells and exhibited opiate receptor binding activity. 4. 4. A fraction retarded on Sephadex G-10 possessed opiate receptor binding but not corticosteroidogenic activity. 5. 5. The data suggest the elaboration of ACTH-like and opiate-like materials by the grouper brain.

β-endorphin-like and adrenocorticotropin-like materials in heart tissues of the rat, gerbil, hamster and guinea pig

1. 1. Heart tissues of several rodent species including the rat, gerbil ( Meriones unguiculatus), hamster ( Mesocricetus auratus) and guinea pig ( Cavia porcellus) were extracted with an acetone-water-HC1 mixture. An acid acetone powder was obtained by adding a copious volume of acetone to the extract. 2. 2. Rat heart acid acetone powder was subjected to ion exchange chromatography on CM-cellulose. Gerbil heart acid acetone powder was subjected to salt fractionation, gel filtration on Sephadex G-10 and then ion exchange chromatography on CM-cellulose. Hamster and guinea pig heart acid acetone powders were subjected to gel filtration on Sephadex G-25. 3. 3. The fractions were assayed for the ability to stimulate corticosterone production in isolated rat adrenal decapsular (zona fasciculata, zona reticularis and medulla) cells, to displace D-ala 2-D-leu 5-(tyrosyl-3,5- 3H) enkephalin from binding to rat brain membranes, and to inhibit 125I-human β-endorphin from binding to its antibodies. 4. 4. The widespread occurrence of β-endorphin-like immunoreactivity among the rat heart CM-cellulose fractions may reflect different species of β-endorphin. The fraction with the highest β-endorphin-like immunoreactivity and opiate receptor binding activity was strongly adsorbed on CM-cellulose. 5. 5. In hamster and guinea pig hearts, β-endorphin-like immunoreactivity and opiate receptor binding activity were distributed among high molecular weight and low molecular weight fractions. 6. 6. In gerbil hearts, opiate receptor binding activity was present in fractions unretarded on Sephadex G-10 (i.e. with a molecular weight greater than 700) as well as in the retarded fractions (i.e. with a molecular weight smaller than 700). 7. 7. Steroidogenic activity resided in both high and low molecular weight fractions in hamster and guinea pig hearts. In rat hearts the majority of Steroidogenic activity was adsorbed on CM-cellulose. In gerbil hearts Steroidogenic activity was also present.

Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.