Bovine adrenal cells secrete interleukin-6 and tumor necrosis factor in vitro

Interleukin-6 (IL-6) and tumor necrosis factor (TNF) are secreted and/or synthesized by the rat and human adrenal cortex. In this study, the release of IL-6 and TNF from bovine adrenal cells was determined. Bovine adrenal glands were collected from an abattoir and dissected into the zona glomerulosa (ZG), zona fasciculata (ZF), zona reticularis (ZR), and medulla. The tissues were enzymatically dispersed to single cells and cultured for 4-6 days. The cells were then exposed (4 h) to angiotensin II (AII), adrenocorticotrophic hormone (ACTH), phorbol dibutyrate (PDB), interleukin-1beta (IL-1beta), interleukin-1alpha (IL-1alpha), and endotoxin (LPS). The IL-6 and TNF content of the incubation medium was determined by bioassays. The release of IL-6 and TNF from the ZG, ZF, ZR, and medulla was increased by PDB, IL-1alpha, IL-1beta, and LPS. In contrast, ACTH and AII increased IL-6 release from the ZG, ZF, and ZR but had no effect on IL-6 release from the medulla. ACTH decreased TNF release from all adrenal cortical zones but had no effect on TNF release from the medulla. Immunohistochemistry utilizing antibodies against TNFalpha demonstrated TNFalpha-containing cells throughout the adrenal gland. The majority of the cells of the ZG, ZF, and ZR contained TNFalpha. However, the cells of the ZG contained more TNFalpha than the cells of the ZR or ZF. Small patches of TNFalpha-containing cells were also found in the adrenal medulla and capsule. These findings support the hypothesis that IL-6 and TNF may have autocrine/paracrine effects on the adrenal gland.     

Stimulation of steroidogenesis by forskolin in rat adrenal zona glomerulosa cell preparations.

The actions of forskolin have been investigated to determine to what extent its effects on steroidogenesis in rat adrenal preparations are dependent on activation of adenylate cyclase. In zona glomerulosa preparations, stimulation of both aldosterone and corticosterone production was obtained at concentrations of forskolin between 1 and 10 mumol/l. The effects of 10 mumol forskolin/l were additive with those of low doses (1 pmol/l) of corticotrophin (ACTH), but not with those of high doses (1 nmol/l) of ACTH. In contrast, in zona fasciculata/reticularis cells, doses of forskolin up to 10 mumol/l produced no significant stimulation of corticosterone production either alone or in the presence of ACTH (1 pmol/l and 1 nmol/l). The response to 1 nmol ACTH/l was attenuated in the presence of forskolin (10 mumol/l) in both zona glomerulosa and zona fasciculata/reticularis cell preparations. Cyclic AMP production increased progressively with dose up to 100 mumol forskolin/l in zona glomerulosa cells, whereas corticosterone production was maximal between 10 and 30 mumol forskolin/l and decreased at 100 mumol forskolin/l. In zona fasciculata/reticularis cells, cyclic AMP production was also increased by forskolin (1 and 10 mumol/l). The stimulation of zona glomerulosa steroidogenesis by forskolin (1-10 mumol/l) and ACTH (1-100 pmol/l) were both reduced by the adenylate cyclase inhibitor, N6-phenylisopropyladenosine (100 mumol/l). The calcium channel inhibitor, nifedipine, only reduced the steroidogenic response to forskolin (3 mumol/l) at doses of 300 mumol/l whereas the response to 8.4 mmol K+/l was inhibited at 10 mumol nifedipine/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoxygenase products as common intermediates in cyclic AMP-dependent and -independent adrenal steroidogenesis in rats

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and ACTH. Mitochondria from these cells respond to intracellular factors generated by AII (cyclic AMP (cAMP)-independent steroidogenesis) and ACTH (cAMP-dependent steroidogenesis), suggesting that the two-signal-transduction mechanisms are linked by a common intermediate. We have evaluated this hypothesis by stimulating mitochondria from the unstimulated zona glomerulosa with a subcellular post-mitochondrial fraction (PMF) obtained from the zona glomerulosa after stimulation with AII or from the fasciculata gland after stimulation with ACTH; the subcellular fractions were also tested on mitochondria from fasciculata cells. PMFs obtained after incubation of adrenal zona glomerulosa with or without AII (0.1 microM) or ACTH (0.1 nM) were able to increase net progesterone synthesis 4.5-fold in mitochondria isolated from unstimulated rat zona glomerulosa. AII-pretreated PMFs from the zona glomerulosa also stimulated steroidogenesis by mitochondria from zona fasciculata cells. Separate experiments showed that inhibitors of arachidonic acid release and metabolism (bromophenacyl bromide, nordihydroguaiaretic acid, caffeic acid or esculetin) blocked corticosterone production in fasciculata cells stimulated with ACTH, suggesting that arachidonic acid could be the common intermediate in the actions of AII and ACTH on steroid synthesis. Evidence to support this concept was obtained from experiments in which the formation of an activated PMF by treatment of zona fasciculata with ACTH was blocked by the presence of the same inhibitors. Moreover, the inhibitory effects of these substances on PMF activation by ACTH were overcome by exogenous arachidonic acid and, in addition, arachidonic acid release was stimulated by ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypophysectomy results in a loss of connexin gap junction protein from the adrenal cortex

To test the hypothesis that gap junctions are dependent on the tropic state of the adrenal gland, the effect of hypophysectomy on connexin 43 (alpha1-Cx43) gap junction protein occurrence and distribution was examined in mice. Gap junction protein occurrence was assessed with immunohistochemical techniques. In the adrenal gland from intact animals, alpha1-Cx43 gap junction protein was detected in the zonae fasciculata(ZF) and reticularis (ZR) while only a few alpha1-Cx43 gap junction plaques were found connecting zona glomerulosa(ZG) cells. Hypophysectomy led to a profound atrophy of the cortex which was more marked in the inner zones (zonae fasciculata and reticularis) than in the zona glomerulosa. There was a time dependent loss of alpha1-Cx43 gap junction protein in the adrenal cortex after hypophysectomy. At 33 day following hypophysectomy there was a two fold decrease in gap junctions in the zona fasciculata while the average gap junction plaque size was not different than the size seen in control animal adrenal glands.. ACTH (1U/gm body weight) treatment in hypophysectomized animals increased the number of gap junction plaques in the zona fasciculata. Hypophysectomy led to diminished alpha1-Cx43 gap junction expression in the zona fasciculata which could be restored by ACTH treatment. Because altering the tropic state of the adrenal glands via hypophysectomy leads to a reduction in gap junction number, it can be suggested that control of gap junction expression in the adrenal gland is hormone dependent and linked to adrenal gland function.     

Serotonin increases interleukin-6 release and decreases tumor necrosis factor release from rat adrenal zona glomerulosa cells in vitro

Interleukin-6 (IL-6) and tumor necrosis factor (TNF) are secreted by rat adrenal zona glomerulosa cells. Serotonin increases the release of aldosterone, corti-costerone, and cortisol from the adrenal cortex. Therefore, the effects of serotonin on IL-6 and TNF release from rat adrenal zona glomerulosa cells were investigated. Cultures of rat adrenal zona glomerulosa cells were enzymatically prepared and cultured for 4–6 d. The cells were then exposed to serum-free RPMl-1640 medium containing vehicle (RPMl medium alone), serotonin, and/or endotoxin, interleukin-1β, or adrenocorticotrophic hormone (ACTH). Following a 5-h incubation, medium was removed from the cells, and IL-6 and TNF content of this medium determined with bioassays. Serotonin (1–1000 nM) increased basal IL-6 release from zona glomerulosa cells, but inhibited basal TNF release from these cells. Endotoxin and interleukin-1β (IL-1β) increased IL-6 and TNF release from zona glomerulosa cells. Serotonin potentiated IL-6 release stimulated by endotoxin and IL-1β, but inhibited TNF release stimulated by these agents. Serotonin potentiated ACTH-stimulated IL-6 release. Serotonin had no effect on IL-6 release from rat anterior pituitary cells. Because IL-6, TNF, and serotonin modify the release of aldosterone and glucocorticoids from adrenal cells, the stimulatory effects of serotonin on aldosterone and glucocorticoid release may be mediated in part by the effects of serotonin on IL-6 and TNF release from adrenal cells.     

Comparative effect of angiotensin II, potassium, adrenocorticotropin, and cyclic adenosine 3',5'-monophosphate on cytosolic calcium in rat adrenal cells

The present study compares changes in cytosolic calcium and steroidogenesis when rat adrenal cells are stimulated with potassium (K+), angiotensin II (AII), ACTH, and (Bu)2cAMP (cAMP). The calcium-sensitive fluorescent dye, quin 2, was used to determine cytosolic calcium concentrations. K+ and AII both induced parallel increases in cytosolic calcium and aldosterone output. Removal of external calcium from the incubation media or addition of nifedipine inhibited the rise in cytosolic calcium in response to these two secretagogues. Inhibition of release of intracellularly-bound calcium by incubating the cells with 8-(N-N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride or dantrolene sodium reduced the rise in cytosolic calcium in response to these two secretagogues by 40-50%. In contrast, neither ACTH nor cAMP altered cytosolic calcium levels in the glomerulosa cells, even though quin 2-loaded cells showed a normal steroidogenic response to these agents. Thus, there was a dissociation between the cytosolic calcium response and steroidogenesis during cAMP stimulation of glomerulosa cells. Fasciculata cells incubated in the presence of increasing concentrations of cAMP, ACTH, K+, or AII failed to demonstrate an increase in cytosolic calcium, although the cells had a normal steroidogenic response to ACTH and cAMP. These results suggest that the responses of fasciculata and glomerulosa cells to secretagogues have different dependencies on calcium. The fasciculata cell has little calcium dependency while the glomerulosa cell has a variable dependency. In the glomerulosa cell, both AII and K+ induced similar responses in steroid output and cytosolic calcium, suggesting an important role for cytosolic calcium as a mediator of the steroidogenic effect of these secretagogues. Furthermore, part of the increase in cytosolic calcium induced by these agents is due to release of intracellularly bound calcium and part from increased calcium flux across the cell membrane. The absence of such dependency with cAMP suggests that an increase in intracellular calcium levels is not required for increased steroidogenesis in glomerulosa cells.     

Properties of rat adrenal zona reticularis cells: production and stimulation of certain steroids.

The outputs of corticosterone, deoxycorticosterone and androstenedione from dispersed, purified rat adrenal zona reticularis and zona fasciculata cells have been measured by radioimmunoassay. Preferential production of deoxycorticosterone by zona reticularis cells was demonstrated by their higher basal deoxycorticosterone : corticosterone ratio when compared with zona fasciculata cells. Adrencorticotrophin (ACTH) stimulated corticosterone output by all cell pools prepared by unit gravity (1 g) sedimentation, zona fasciculata cells being stimulated 130-fold compared with 20-fold for the zona reticularis cells in relation to their basal corticosterone output. In every cell pool, ACTH stimulated the output corticosterone more than it stimulated the output of deoxycorticosterone. In parallel cell preparations, it was shown that ACTH increased the conversion of tracer amounts of radioactive deoxycorticosterone to corticosterone and decreased the conversion of radioactive corticosterone to 11-dehydrocorticosterone. Adrenocorticotrophin did not increase the conversion of radioactive deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone). It is unlikely therefore that ACTH stimulates 11 beta-hydroxylation. Data indicate that the ratio of deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone) is characteristic for each cell type, and that this ratio will be relatively independent of ACTH stimulation or the amount of pregnenolone substrate available. Basal androstenedione outputs were similar for both types of cell, and ACTH stimulation was very small, being slightly greater for zona fasciculata than for zona reticularis cells. The contribution of the zona reticularis cells to the basal output of any steroid by the cells of the inner two zones of the adrenal cortex of the rat was relatively small (20% for deoxycorticosterone and 10% for corticosterone) and was even less after stimulation by ACTH. Unless a specific stimulus can be found, therefore, a significant role for the zona reticularis cannot yet be established.     

Transforming growth factor-beta 1 inhibits aldosterone and stimulates adrenal renin in cultured bovine zona glomerulosa cells.

Transforming growth factors-beta (TFG beta s) are multifunctional peptides that affect proliferation, differentiation, and many other functions in a variety of cell types. In this study we examined the effect of TGF beta 1 on aldosterone and adrenal renin production using cultured bovine adrenal zona glomerulosa cells. Collagenase-dispersed zona glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 72 h, and the medium was replaced with serum-free medium for the next 24 h. The cells during this 24-h period were exposed to TGF beta 1, ACTH, and (Bu)2cAMP (dbcAMP). It was observed that TGF beta 1 at 1 nM 1) inhibited basal aldosterone secretion from 680.0 +/- 40.0 to 270.0 +/- 10.0 pg/10(6) cells.h, 2) inhibited ACTH- and dbcAMP-stimulated aldosterone production, 3) increased levels of active renin in the cells from 17.8 +/- 2.5 to 70.7 +/- 4.4 pg angiotensin-I/10(6) cells.h and prorenin from 270.0 +/- 5.0 to 970.0 +/- 90 pg angiotensin-I/10(6) cells.h, 4) stimulated prorenin in the medium synergistically in combination with ACTH and dbcAMP, and 5) had no significant effect on basal cAMP production, but significantly inhibited the ACTH-stimulated production of cAMP. These observations show that TGF beta 1 is a potent inhibitor of basal and ACTH- and cAMP-stimulated aldosterone production and inhibits ACTH-stimulated cAMP production. Contrary to its effect on aldosterone, TGF beta 1 stimulates the synthesis and release of adrenal renin and prorenin. TGF beta 1 may act as an autocrine or paracrine regulator of aldosterone production.     

The actions of N-terminal fragments of corticotrophin on steroidogenesis in dispersed rat adrenal cells in vitro

The finding that the rat adrenal zona glomerulosa cell shows specific sensitivity to stimulation by alpha-MSH and related peptides has been confirmed both in vivo and in vitro, raising the possibility that alpha-MSH may have a physiological role in the control of glomerulosa function and aldosterone secretion. To define more closely the structural features which confer teh specificity of the glomerulosa response, other ACTH derived peptides have been tested for their specificity of actions on rat adrenal cells in vitro. The peptides tested were ACTH(5-24), ACTH(1-12), ACTH(1-14), ACTH(1-15), ACTH(1-16) and ACTH(1-17). Their actions were compared with those of alpha-MSH and ACTH(1-24). All of the ACTH-derived peptides stimulated glomerulosa corticosterone production with sensitivities similar to that of alpha-MSH; minimum effective concentration was 10 nmol/l. Also, like alpha-MSH, the shorter ACTH peptides stimulated aldosterone production only relatively weakly in these cells from animals on normal sodium intake. Only ACTH(5-24), ACTH(1-16) and ACTH(1-17) stimulated fasciculata/reticularis cells at concentrations up to 1 mumol/1. The actions of all of the shorter peptides were thus unlike those of ACTH(1-24) which stimulates both cell types with approximately equal sensitivity, and which furthermore strongly stimulates aldosterone production. The data suggest that the 18-24 region of the ACTH molecule contains the signal for a fasciculata/reticularis response, and the region 1-13 that for glomerulosa specificity. They confirm the view that, in the rat, alpha-MSH itself may be the specific pituitary glomerulosa-stimulating agent which much experimental work has predicted. They also indicate that synthetic ACTH(1-17) analogues should be used with caution.     

Molecular evidence of organic ion transporters in the rat adrenal cortex with adrenocorticotropin-regulated zonal expression

Experimental evidence suggested that secretion of steroid hormones from adrenocortical cells involves carrier-mediated transport: Cortisol release from, and uptake of p-[3H]aminohippurate into, bovine adrenocortical cells showed properties of the renal p-[3H]aminohippurate/anion exchanger OAT1. Other poly-specific transporters such as organic anion-transporting polypeptides (oatps) and organic cation transporters (OCTs) could also be involved in steroid hormone release. A homology-cloning procedure was established to detect these transporters in rat adrenal gland cDNA. PCR revealed the presence of OAT1, oatp1, oatp2, and oatp3. In situ hybridization localized OAT1 in the outer zona fasciculata, oatp3 in the zona glomerulosa, and oatp1 and oatp2 in the inner zona fasciculata and outer zona reticularis. An OCT2-specific probe produced signals in the zona glomerulosa and outer zona fasciculata. Pretreatment of rats with ACTH increased the expression of OAT1 mRNA that spread to all zones, and hypophysectomy strongly decreased it. A less pronounced regulation was detected for OCT2 and oatp3. Specific antibodies confirmed the localization of OAT1 in the outer zona fasciculata, supporting a possible role of OAT1 in cortisol release. The zonated distribution of transporters furthermore suggest that oatp1-3 and OCT2 may be important for the endocrine function of rat adrenocortical cells.     

Cortisol production by cells isolated from the outer and inner zones of the adrenal cortex of the guinea pig

Cortisol production by cells isolated from the outer (glomerulosa and fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig was evaluated in order to clarify the steroidogenic function of the zona reticularis. Basal cortisol production by the outer zone cells was 5-10 times greater than that by the inner zone cells. Cells from the outer zone increased cortisol production in response to ACTH (ED50, approximately 33 pg/ml), while cells from the inner zone failed to respond to ACTH. Likewise, cells from the outer zone increased cortisol production in response to (Bu)2cAMP, while cells from the inner zone failed to respond. When either pregnenolone or progesterone was added to the incubation mixture, an increase in cortisol production by cells of the inner zone was noted, although the increase was significantly less than that observed for cells from the outer zone. These results suggest that the zona reticularis of the guinea pig adrenal ordinarily produces little or no cortisol, although the enzyme machinery is present and capable of producing a limited amount of cortisol when supplied with the appropriate precursor.     

Biosynthetic ad morphological evidence for inhibition of aldosterone production following administration of ACTH to sheep

The effect of ACTH administration for 1-5 days on the morphology and steroidogenic capability of sheep adrenal tissue has been examined. During this period of treatment there was a gradual decline in the in vitro conversion of 3H-labelled precursors to products of solely zona glomerulosa origin (aldosterone and 18-hydroxycorticosterone) while conversion to products of zona fasciculata origin (17-hydroxyprogesterone, 11-deoxycortisol and cortisol) was stimulated throughout. Conversion to DOC, 18-hydroxydeoxycorticosterone and corticosterone (steroids produced by both the zona glomerulosa and the zona fasciculata) declined after initial stimulation. Within 2--3 days of the commencement of treatment, the zona glomerulosa showed a progressive decrease in cell number associated with disruption of cords and cell separation. Ultrastructurally, it was found that typical zona glomerulosa cells had almost disappeared. The majority of residual cells in this area had a structure intermediate between zona glomerulosa and zona fasciculata cells. The similarity in time-course of the alterations in both the morphological and biosynthetic characteristics suggests that the decline in aldosterone output caused by ACTH administration to sheep results from the loss of adrenal zona glomerulosa cells, predominantly due to selective cellular degeneration.     

Immunohistochemical analysis of cytochrome P-450 17 alpha-hydroxylase in pig adrenal cortex, testis and ovary

Cytochrome P-450 specific for steroid 17 alpha-hydroxylation (P-450(17 alpha] was immunohistochemically observed in pig adrenal cortex, testis and ovary by the biotin-streptavidin method using a specific antibody against P-450(17 alpha) purified from neonatal pig testis. In the adrenal cortex, P-450(17 alpha) was present in the zona fasciculata and reticularis while no immunoreactivity was observed in the zona glomerulosa, confirming the absence of 17-hydroxylated steroid synthesis in the zona glomerulosa. In the testis, P-450(17 alpha) was present exclusively in Leydig cells and immunoreactivity was absent in seminiferous tubules. In the ovary, immunoreactivity was observed only in the theca interna but not in the membrana granulosa of follicles. Among the tissues examined, the relative intensity of immunoreactivity was greatest in the Leydig cells, and progressively less in theca interna cells, outer fasciculata cells and inner fasciculata and reticularis cells.     

Expression and regulation of adenylyl cyclase isoforms in the human adrenal gland

The aim of the present study was to identify which adenylyl cyclase isoforms were expressed in the human adrenal gland and to determine which isoform(s) may be coupled to ACTH action. Our results indicate that, in both glomerulosa and fasciculata zones, adenylyl cyclase 1 was detected in cells at the membrane level, adenylyl cyclases 3 and 2 in both the cytoplasm and the plasma membrane, whereas adenylyl cyclase 5/6 and adenylyl cyclase 4 were found mainly in cytoplasm. The levels of expression of each isoform were similar between the two adrenocortical zones, except for adenylyl cyclase 5/6, which had a lower level of expression in the zona fasciculata. We next evaluated the role of the various adenylyl cyclase isoforms during ACTH-stimulated cAMP production in both glomerulosa and fasciculata cell preparations. Corroborating with previous observations, we found that calcium had a biphasic effect on cAMP production. Interestingly, pertussis toxin treatment increased cAMP production, indicating that, in addition to Gs, ACTH is coupled to a Gi protein. Incubation with the betagamma-subunit sequestrant peptide QEHA decreased cAMP production, as did incubation with inhibitory antibodies against either adenylyl cyclase 2 or adenylyl cyclase 5/6. Inhibitory adenylyl cyclase 3 antibodies interfered with ACTH action only in the zona fasciculata. Altogether these data indicate that adrenocortical cells express one or two isoforms of each class of adenylyl cyclases and, thus, have the ability to produce cAMP in response to various regulatory, intracellular mediators. Importantly, our results indicate that in the human adrenal gland, ACTH acts mainly through adenylyl cyclase 5/6 and adenylyl cyclase 2/4, whereas the effect of ACTH on adenylyl cyclase 3 activity may be a consequence of calcium influx.     

Molecular mechanism of cytochrome P-450-dependent aldosterone biosynthesis in the adrenal cortex.

In the adrenal cortex, the potent mineralocorticoid, aldosterone, is produced in the zoba glomerulosa but not in the zona fasciculata/reticularis. In rodents and humans, two distinct species of P-450(C18) (aldosterone synthase) and P-450(11beta) (11beta-hydroxylase) are expressed in the adrenal cortex. The selective expression of cytochrome P-450 species in different zones contributes to zone specificity of aldosterone synthesis. In the cow and pig, only one molecular species of P-450(11beta) having both 11beta-hydroxylase and aldosterone synthase activity is expressed throughout the adrenal cortex. P-450(11beta) in the zona fasciculata/reticularis catalyzes the formation of corticosterone but not that of aldosterone from 11-deoxycorticosterone; the same enzyme in the zona glomerulosa produces aldosterone from the same substrate, indicating that a local factor in mitochondria is likely to be involved in the selective suppression of the aldosterone synthetic activity of P-450(11beta) in the zona fasciculata/reticularis. The zone specificity of aldosterone synthesis catalyzed by P-450(11beta) in the bovine adrenal cortex appears to be due to differences in interactions between P-450(11beta) and P-450(SCC) in mitochondria in different cortical zones. Thus, two modes exist for aldosterone biosynthesis in mammals: rodent-human and bovine-porcine modes.     

Lipoprotein requirements for secretion of ultraviolet-absorbing corticosteroids by guinea pig adrenocortical cells in vitro: inner versus outer cortices; zona glomerulosa versus zona fasciculata

Most studies of lipoprotein requirements for steroid secretion by the adrenal have examined the mixed cell population of the whole gland; none have examined lipoprotein requirements of guinea pig adrenocortical cells. In this study the effect of exogenous lipoprotein on the ability of cells from each of the different regions of the guinea pig adrenal cortex to synthesize and secrete steroids has been analyzed in vitro, under baseline and ACTH-stimulated conditions. Most studies have assessed the effects of lipoprotein on one or a few selected steroids. In this study the effects of lipoprotein and ACTH were examined both by an assay for fluorogenic steroids and by HPLC analysis of the spectrum of UV-absorbing steroids. Guinea pig outer adrenocortices, containing zona glomerulosa and zona fasciculata, maintained in vitro as fragments or as isolated cells, secreted at least 5 times more steroid than the inner cortex, predominantly zona reticularis, and were dependent upon lipoproteins for their secretion. Corticosterone (B) and cortisol (F) were the predominant products of both zones. Aldosterone (Aldo), 18-hydroxycorticosterone (18-OH B), deoxycorticosterone (DOC), 11 beta-hydroxyandrostenedione (11 beta-OH And), androstenedione (And), and deoxycortisol were less abundant products of the outer cortex, while the inner cortex secreted only very small amounts of these steroids. Each of the outer cortical cell types secreted a distinct spectrum of steroids. Aldo, 18-OH B, and DOC were characteristic of glomerulosa cells, but B was most prominent. Fasciculata cells secreted primarily F, with 11 beta-OH And as their next most prominent product. Low density lipoprotein (LDL) enhanced steroid secretion by glomerulosa cells to a greater extent than that by fasciculata cells, but the stimulation of LDL utilization by ACTH was greatest for fasciculata cells. LDL and ACTH also influenced the pattern of steroids secreted by each cell type. Addition of LDL to glomerulosa cells enhanced secretion of DOC and B, but not that of Aldo or 18-OH B. In fasciculata cell cultures, LDL enhanced secretion of both F and 11 beta-OH And. ACTH, particularly in the presence of LDL, stimulated secretion by glomerulosa cells of Aldo and 18-OH B, as well as that of F, And, and 11 beta-OH And. The combined presence of ACTH and LDL in fasciculata cell cultures preferentially stimulated secretion of F and B.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyclic AMP levels in purified rat adrenal zonae fasciculata and reticularis cells and the effect of adrenocorticotrophic hormone

Cyclic AMP levels were measured in combined cells and supernatant fraction from incubations of dispersed rat adrenal zona fasciculata and zona reticularis cell preparations purified by unit gravity sedimentation. These measurements were correlated with deoxycorticosterone (DOC) and corticosterone outputs from the cells in the presence or absence of ACTH. Similar measurements of cyclic AMP outputs were made for unpurified dispersed, decapsulated rat adrenal cell preparations and they were found to correspond to previously reported measurements made by other workers on such preparations. The response of the purest zona reticularis cells to ACTH in terms of cyclic AMP output was 28-fold lower than that of the purest zona fasciculata cells (compared with a fivefold lower DOC output and a 20-fold lower corticosterone output) and the response to ACTH of the mixed-cell preparations was related to the number of zona fasciculata cells in the preparation, i.e. the greater the proportion of zona fasciculata cells in the preparation the greater the response in terms of both outputs of cyclic AMP and of either of the two steroids measured. This correlation is in accordance with the theory that cyclic AMP may be the secondary messenger for both zona fasciculata and zona reticularis cells of the rat adrenal cortex in mediating the response to an ACTH stimulus.     

Effects of alpha-melanocyte-stimulating hormone on the cyclic AMP and phospholipid metabolism of rat adrenocortical cells

Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bisphosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0.25 nmol/l to 0.25 mumol/l) highly significantly (P less than 0.01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by alpha-MSH (above 0.1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 mumol alpha-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P greater than 0.05) by any dose of alpha-MSH (10 nmol/l, 1 mumol/l and 0.1 mmol/l) used. Therefore, it can be concluded that alpha-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol alpha MSH/l (highly significant above 10 nmol alpha-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0.1 to 1 mumol alpha-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata-reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that alpha-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, alpha-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells.