Rat Adrenocortical Cells Research Articles

Proliferogenic effect of neurotensin (NT) and neuromedin-N (NMN) on the rat adrenal cortex: Evidence that angiotensin-II mediates the effect of NMN, but not of NT

NT and NMN, two biologically active peptides acting via the same specific receptor, are both able to stimulate in vivo the proliferative activity of rat adrenocortical cells. The present study aimed to investigate whether the mechanism underlying this effect of NT and NMN may involve an enhanced production of angiotensin-II (ANG-II), a potent adrenocortical proliferogenic factor. Metaphases per adrenal section were counted 120 min after vincristin injection. A bolus administration of ANG-II resulted in a marked increase in the number of metaphase-arrested cells 12, 24 and 48 h after the beginning of the experiment; the concomitant administration of saralasin (SAR), a competitive antagonist of ANG-II, completely blocked the proliferogenic efect of ANG-II. NT-evoked rise in the number of metaphases occurred 48 h after administration and was not influenced by the simultaneous SAR injection. On the contrary, NMN injection induced a burst of adrenocortical cell proliferation within 12 h, and this effect was prevented by SAR. These data suggest that ANG-II mediates the proliferogenic effect of NMN, but not that of NT.

Effect of okadaic acid on utilization of lipoprotein-derived cholesteryl esters by rat steroidogenic cells.

This study examines various functional, biochemical, and structural changes in rat adrenocortical and ovarian granulosa cells that could account for the decline in lipoprotein-supported hormone production after cell treatment with the protein phosphatase inhibitor, okadaic acid. Although the steroidogenic pathway enzymes in these cells are not in themselves affected by okadaic acid, the intracellular transport of cholesterol to important cellular processing sites is defective. That is, okadaic acid does not interfere with the internalization of lipoprotein-derived cholesteryl esters, but the mitochondrial utilization of cholesterol obtained from intracellular cholesterol storage sites is 50% reduced as compared to control cells. Two-dimensional electrophoresis gels from okadaic acid-treated cells demonstrate a number of hyperphosphorylated proteins. Morphological examination of the affected cells reveal completely disrupted Golgi complexes with attendant structures, but otherwise the cells appear unchanged. The results suggest that some necessary sterol transport protein (or cofactor or associated membrane) is adversely phosphorylated by okadaic acid, and is rendered dysfunctional.

Protein kinase C signal transduction pathway in ACTH-induced growth effect of rat adrenocortical cells in primary culture.

ACTH exerts a biphasic effect on the growth of fetal rat adrenocortical cells in primary culture when bromodeoxyuridine (BrdU) incorporation is used as an indicator of proliferation. The immediate inhibitory effect during the first 24 h of ACTH stimulation is not dependent on cyclic AMP (cAMP). Protein kinase C (PKC) inhibitors H-7 and staurosporine blocked this inhibitory effect of ACTH, whereas 12-0-tetradecanoyl phorbol-13-acetate (TPA; a PKC activator) mimicked the ACTH-induced antimitogenic effect. The stimulatory growth effect of ACTH appears after 72 h of treatment. A similar mitogenic effect is also achieved with cAMP derivative 8-bromo cAMP (8-Br cAMP). However, both ACTH- and 8-Br cAMP-induced proliferations could be reduced with H-7. ACTH-induced corticosterone secretion was inhibited 50% with H-7 after 24 h, but 8-Br cAMP-induced secretion was unaffected. However, if the treatments were continued for 72 h, H-7 no longer reduced the steroid secretions. Reduction (50-75%) of cholesterol side-chain cleavage enzyme (P450scc) mRNA expression was also noted with H-7 in ACTH-treated cultures after 6 and 24 h. In contrast, TPA doubled the corticosterone secretion induced by 8-Br cAMP, but did not further increase the ACTH-induced secretion after 24 h. TPA alone, however, was not able to induce steroid secretion or P450scc mRNA expression. The morphological differentiation of fetal rat adrenocortical cells with ACTH or 8-Br cAMP from zona glomerulosa-like cells into zona fasciculata-like cells was not distributed by H-7 nor was it induced by TPA alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of type I and type II isoenzymes of cyclic AMP-dependent protein kinase in steroidogenesis in rat adrenal cells.

The role played by cyclic AMP (cAMP)-dependent protein kinases (PKAs) in rat adrenal steroidogenesis has been investigated using cAMP analogues which show partial selectivity for the type I and type II PKA isoenzymes. These were aminohexylamino-cAMP (AHA-cAMP; selective for site 1 on type I PKA), N6-benzoyl-cAMP (BZ-cAMP; selective for site 2 on PKA types I and II) and 8-thiomethyl-cAMP (TM-cAMP; selective for site 1 on type II PKA). Positive cooperativity exists between the two nucleotide-binding sites, thus the presence of type I PKA was inferred when synergistic increases in corticosteroid production were obtained with AHA-cAMP plus BZ-cAMP and that of type II PKA when synergistic increases were obtained with TM-cAMP plus BZ-cAMP. The effects of AHA-cAMP, TM-cAMP and BZ-cAMP (10-100 mumol/l) on aldosterone production by glomerulosa cell preparations and corticosterone production by fasciculata/reticularis cell preparations were compared. Dose-related stimulation of steroid production was obtained with each cAMP analogue in both types of cell preparation. Experiments were performed using the cAMP analogues in combination at doses which gave minimal stimulation individually. Cells were incubated with AHA-cAMP (66 and 100 mumol/l) or TM-cAMP (15, 30 and 45 mumol/l) in the presence and absence of 15 mumol BZ-cAMP/l. Synergistic responses were obtained with both analogue pairs in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory effect of vasoactive intestinal peptide (VIP) on the secretory activity of dispersed rat adrenocortical cells. Evidence for the interaction of VIP with ACTH receptors

VIP dose-dependently increased basal, but not submaximally ACTH (10 −10 M)-stimulated, aldosterone (ALDO) and corticosterone (B) secretion of dispersed rat capsular and inner adrenocortical cells, respectively. The maximal stimulatory effect (60–70% rise) was obtained with a VIP concentration of 10 −8 M. [4-Cl-D-Phe 6,Leu 17]-VIP, a VIP-receptor antagonist (VIP-A), and corticotropin inhibiting peptide (CIP), an ACTH receptor antagonist (both 10 −6 M), completely annulled VIP (10 −8M)-evoked rises in basal ALDO and corticosterone secretions. The ACTH (10 −10 M)-enhanced (about 5-fold) production of both hormones was completely reversed by CIP (10 −6 M) and only partially reduced (about −30%) by VIP-A (10 −6 M). The hypothesis is advanced that the weak secretagogue effect of VIP on dispersed rat capsular and inner adrenocortical cells may be due to its positive interaction with ACTH receptors.

Effects of gossypol on in vitro bovine oocyte maturation and steroidogenesis in bovine granulosa cells

Abstract Recently, we demonstrated an adverse effect of gossypol on steroidogenesis of bovine luteal, porcine granulosa and rat adrenocortical cells as well as on bovine, rat and hamster early embryonic development. The purpose of this study was to evaluate the effects of varying dosages of gossypol on bovine oocytes and granulosa cells from small and large follicles. Oocytes and bovine granulosa cells from small follicles were harvested from 3- to 8-mm follicles, and bovine granulosa cells from larger follicles (>15 mm) were used. Bovine oocytes were cultured in the presence of 10, 50 and 150 μg/ml gossypol for 26 to 28 h. Both bovine granulosa cells and bovine granulosa cells from larger follicles were treated with 0.1, 0.5, 2.5 and 12.5 μg/ml gossypol in the presence or absence of human chorionic gonadotropin (hCG) for 6 h. Cumulus expansion, meiotic progression of oocytes, progesterone production and intracellular cAMP formation in granulosa cells were measured. Gossypol significantly decreased cumulus expansion and inhibited nuclear maturation of oocytes. In bovine granulosa cells, gossypol inhibited cAMP formation only at 12.5 μg/ml. In bovine granulosa cells from larger follicles, hCG significantly increased both progesterone and cAMP levels. Treatment with gossypol significantly decreased hCG-induced cAMP formation in a dose-dependent manner. Basal cAMP level and progesterone secretion were significantly inhibited by only 12.5 μg/ml gossypol. However, inhibition of hCG-induced progesterone secretion was more dramatic. These results indicate that gossypol is capable of inhibiting cumulus expansion and nuclear maturation of bovine oocytes, and progesterone production and cAMP formation in bovine granulosa cells from larger follicles.

Effects of cyclosporine-A on steroid secretion of dispersed rat adrenocortical cells.

The acute effect of cyclosporine-A (CSA), a potent immunosuppressive agent, on the secretory activity of dispersed rat adrenocortical cells was investigated. The production of the following steroid hormones was assayed by high performance liquid chromatography: pregnenolone (PREG), progesterone (PROG), 11-deoxycorticosterone (DOC), corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18OH-DOC), 18-hydroxycorticosterone (18OH-B) and aldosterone (ALDO); B and ALDO outputs were also measured by radioimmunoassay. Low concentrations of CSA (0.1-0.2 mg/ml) enhanced basal, but not ACTH- or angiotensin-II (ANG-II) 10(-8) M-stimulated, secretions of PREG, non-18-hydroxylated steroids (PROG, DOC and B) and 18-hydroxylated steroids (18OH-DOC, 18OH-B and ALDO) of both zona glomerulosa (ZG) and zonae fasciculata and reticularis (ZF/ZR) cells. Middle concentrations of CSA (from 0.3 to 0.5 mg/ml) did not affect PREG yield, nor did they alter basal and ACTH-stimulated post-PREG output of both ZG and ZF/ZR cells; however, they elicited a marked decrease in ANG-II-enhanced production of 18-hydroxylated steroid by AG cells. Concentrations of CSA higher than 0.5 mg/ml strikingly reduced either basal and agonist-stimulated over-all steroidogenesis of both ZG and ZF/ZR cells. These findings suggest that CSA at low concentrations strongly stimulates the conversion of cholesterol to PREG (i.e. the rate-limiting step of steroidogenesis), while at middle concentrations it did not affect this early step, but specifically interferes with the intracellular events which transduce the stimulatory signal of ANG-II on the late steps of mineralocorticoid production (i.e. the conversion of B to ALDO). At higher concentrations, CSA probably exerts a cytotoxic effect.

Role of adenylate cyclase-cyclic AMP-dependent signal transduction in the ACTH-induced biphasic growth effect of rat adrenocortical cells in primary culture.

ACTH has a biphasic effect on the proliferation of fetal rat adrenocortical cells in primary culture. Dramatic changes occurred during the first 72 h of ACTH stimulation, when incorporation of bromodeoxyuridine was used as an indicator of proliferation. The primary effect of ACTH was the inhibition of proliferation during the first 24 h, which was followed by an intense stimulatory phase during the third day of ACTH treatment. Cycloheximide (a protein synthesis inhibitor) prevented both the inhibitory and the stimulatory effects of ACTH, but did not affect the basal proliferation of unstimulated zona glomerulosa-like cells. Although adrenocortical cells stimulated with cyclic AMP (cAMP) derivatives, 8-bromo cAMP (8-Br cAMP) or dibutyryl cAMP ((Bu)2cAMP), differentiated morphologically into fasciculata-like cells, and secreted corticosterone and 18-OH-deoxycorticosterone, as did ACTH-stimulated cells, neither of the derivatives inhibited proliferation during the first 24 h of treatment. In contrast to ACTH, (Bu)2cAMP had a stimulatory effect on bromodeoxyuridine incorporation during the first 24 h of treatment. 8-Br cAMP did not change proliferation during the 24 h of treatment, but had a stimulatory effect after 72 h, which was not seen with (Bu)2cAMP. Thus, these results suggest that (1) differentiation, steroid hormone synthesis and the mitogenic effect of ACTH are transduced through the cAMP-mediated system, (2) the antimitogenic effect of ACTH is transduced via a cAMP-independent pathway and (3) both antimitogenic and mitogenic effects of ACTH are dependent on protein synthesis.

Mixed parenchymal-stromal populations of rat adrenocortical cells support the proliferation and differentiation of steroidogenic cells

Abstract The mechanisms which regulate adrenocortical steroidogenesis in differentiated parenchymal cells have been studied in great detail. However, the stem cells that are responsible for regeneration of the adult cortex have never been identified or isolated, and their characteristics are unknown. We have developed a tissue culture system that supports the simultaneous proliferation and differentiation of steroidogenic adrenocortical cells. Utilizing density gradient separation, a cell population composed of a mixture of stromal, endothelial and parenchymal cells (MIX) was isolated from the adult rat adrenal cortex. In primary culture, MIX populations formed high saturation density multilayers from which rounded cells emerged. These cells proliferated, contained lipid, and expressed the steroidogenic enzymes δ5,3β-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage cytochrome P-450scc. After selective passaging, rounded MIX-derived cells retained their steroidogenic potential, even in the absence of trophic hormone treatment. In contrast, parenchymal cells obtained from the zonae fasciculata (FASC) and glomerulosa (GLOM) respectively, formed homogeneous monolayers in primary culture, gradually de-differentiated, and no longer responded to trophic hormone treatment after passaging. Thus, primary MIX cultures provided a microenvironment that resulted in the production of adrenocortical cells with stem cell-like qualities. These cultures provide for the first time, a system for the identification of specific inducers that are responsible for both adrenocortical cytogenesis and its associated proliferation and steroidogenic differentiation.

Thyrotropin-releasing hormone inhibits glucocorticoid secretion of rat adrenal cortex: in vivo and in vitro studies.

The bolus iv administration of TRH dose-dependently decreased ACTH-enhanced plasma corticosterone (B) concentration in rats, without affecting the basal one. The effects of TRH on steroid secretion of dispersed rat inner adrenocortical cells were investigated by HPLC. TRH significantly decreased both basal and ACTH-stimulated post-11-deoxycorticosterone (DOC) secretion (i.e. 18-hydroxy-DOC and B) and concomitantly raised DOC and progesterone release, so that the total postpregnenolone yield of our preparations was unaffected. TRH did not alter either basal or ACTH-stimulated pregnenolone production by isolated rat adrenocortical cells. It was concluded that TRH is an inhibitor of glucocorticoid secretion in rats, which electively impairs the late steps of B synthesis (i.e. 11- and 18-hydroxylation) without affecting the earlier steps, including the rate-limiting one of this process.

Thyrotropin-releasing hormone inhibits glucocorticoid secretion of rat adrenal cortex: in vivo and in vitro studies

The bolus iv administration of TRH dose-dependently decreased ACTH-enhanced plasma corticosterone (B) concentration in rats, without affecting the basal one. The effects of TRH on steroid secretion of dispersed rat inner adrenocortical cells were investigated by HPLC. TRH significantly decreased both basal and ACTH-stimulated post-11-deoxycorticosterone (DOC) secretion (i.e. 18-hydroxy-DOC and B) and concomitantly raised DOC and progesterone release, so that the total postpregnenolone yield of our preparations was unaffected. TRH did not alter either basal or ACTH-stimulated pregnenolone production by isolated rat adrenocortical cells. It was concluded that TRH is an inhibitor of glucocorticoid secretion in rats, which electively impairs the late steps of B synthesis (i.e. 11- and 18-hydroxylation) without affecting the earlier steps, including the rate-limiting one of this process.

Vasopressin stimulates cortisol secretion from human adrenocortical tissue through activation of V1 receptors.

It has previously been shown that arginine vasopressin (AVP) exerts a direct stimulatory action on rat adrenocortical cells. In the present study, we have investigated the possible effect of AVP on cortisol secretion by normal human adrenocortical tissue. The occurrence of endogenous AVP in the human adrenal gland has been studied by means of the indirect immunofluorescence technique. The presence of AVP-containing cells was observed in both cortex and medulla. The action of AVP on corticosteroidogenesis has been investigated in vitro using a perifusion system technique coupled to a specific RIA for cortisol. Graded doses of AVP (from 10(-11)-10(-9) M) increased cortisol secretion in a dose-dependent manner (ED50, 4.5 x 10(-11) M). AVP also induced a significant stimulation of cortisol release from acutely dispersed adrenocortical cells. Prolonged administration of AVP (3 h) induced a rapid and transient increase in cortisol output, followed by a gradual decline in cortisol secretion. Repeated pulses of AVP, given at 90-min intervals, resulted in reproducible stimulations of cortisol output. Selective agonists and antagonists have been used to determine the type of receptor involved in the response of adrenocortical cells to AVP. Oxytocin at doses up to 10(-7) M had virtually no effect on cortisol secretion. The stimulatory effect of AVP was blocked by the V1 antagonist [beta-mercapto-beta,beta-cyclopentamethylene propionyl 1, OMe-Tyr2, Arg8]AVP. In contrast, the V2 antagonist [d(CH2)5D-Phe2,Ile4,Ala9-NH2]AVP did not affect the response of the adrenal gland to AVP. The selective V2 agonist [deamino-Cys1,D-Arg8]AVP did not mimic the stimulatory effect of AVP on cortisol secretion. Taken together, these results suggest that AVP, locally released by intracortical cells, may act as a paracrine factor to stimulate adrenal steroidogenesis in man. The effect of AVP on cortisol secretion appears to be mediated through activation of typical V1 receptors.

Glucose-regulated protein-78 expression in the rat adrenal cortex.

We have postulated that steroidogenesis activator polypeptide (SAP) is a product of glucose-regulated protein-78 (grp78) proteolysis on the basis of a number of considerations, including a striking sequence similarity between the carboxyl-terminal region of grp78 and SAP. Since ACTH stimulates the rapid intracellular accumulation of SAP, experiments were conducted to determine whether ACTH might also regulate levels of grp78 mRNA and/or protein. Using a grp78 cDNA probe, Northern analysis of total RNA isolated from hypophysectomized or dexamethasone-suppressed rats revealed that neither treatment had a measurable influence on steady-state levels of grp78 mRNA over a 4-day period. Moreover, immunoblotting with an antiserum directed against a shared grp78/SAP sequence failed to detect a significant change in the grp78 content of adrenal homogenates from dexamethasone-suppressed rats as compared with untreated controls. On the other hand, grp78 in cultured rat adrenocortical cells fell to 50% of that in time-zero controls after 72 h in the absence of ACTH, whereas inclusion of ACTH in the medium blocked this decline. We conclude that while adrenocortical grp78 may be under some measure of trophic ACTH control, the rapid fluctuations reported for SAP are not likely to be driven by large changes in the size of the grp78 pool.

Major Contribution of the Basic Amino Acid Lysine at Position 11 to the Bioactivity of ACTH in Purified Isolated Rat Adrenocortical Cells

The contribution of the positive charge of lysine in position 11 of ACTH to the corticosteroidogenicity was tested in a purified isolated rat adrenal cell assay. ACTH analogues with Lys 11 or Arg 11 had the same bioactivity. Replacement of lysine by norleucine or protection of its positive charge diminished the potency by about a factor of 80. The extent of this reduction has been discussed in relation to the importance of the basic amino acid sequence 15-18 of the peptide. The result suggests a ten times higher contribution of Lys 11 to the bioactivity of ACTH than the surmised potency of the individual amino acids present in the sequence 15-18.

Biphasic effect of ACTH on growth of rat adrenocortical cells in primary culture

The proliferation rate of differentiating fetal rat adrenocortical cells was studied in primary culture. In this system, stimulation with ACTH induces differentiation of zona glomerulosa-like cortical cells into zona fasciculata-like cells. Incorporation of bromodeoxyuridine (BrdU) was studied immunocytochemically by use of anti-BrdU antibody, and the proliferation rate was counted from the monolayer colonies of adrenocortical cells. After 21 days of cultivation in the absence of ACTH, the proliferation rate of zona glomerulosa-like cells was 10%. The rate slowly declined to 1% at the age of 100 days during continuous cultivation in the absence of ACTH. Stimulation with ACTH induced a strong inhibition in the proliferation rate (down to 2% during the first 24 h). Treatment with ACTH during the following 48 h led to an extremely intense proliferation of adrenocortical cells at a proliferation rate of 25%. Continuous treatment with ACTH up to 100 days led to a persistent growth of adrenocortical cells, and a proliferation rate over 2-fold higher than in control cells cultivated in the absence of ACTH. Thus, ACTH is the principal growth-promoting factor also in vitro, as has been found in in vivo studies. This growth effect is mediated by a biphasic course; at the beginning of differentiation the effect is inhibitory and is followed by a persistent stimulation of the growth of adrenocortical cells.

Pancreatic polypeptide stimulates corticosterone secretion by isolated rat adrenocortical cells

Pancreatic polypeptide (PP) dose-dependently enhanced both basal and submaximally ACTH-stimulated corticosterone production by dispersed zona fasciculata/reticularis cells of the rat adrenal gland. Conversely PP did not affect either basal or ACTH- and angiotensin-II-stimulated aldosterone and corticosterone secretion of zona glomerulosa cells. These findings could throw light on the physiological significance of the marked increase in the pancreatic release of PP during stresses.

Stereologic studies on the long-term effect of prazosin on rat adrenal cortex.

Adult female rats were treated with prazosin, a selective alpha 1-adrenoceptor inhibitor, for 60 days in doses of 0.5 mg/kg/day or 0.5 mg/kg/8 h. Prolonged prazosin administration lowered the gain of body weight and significantly decreased systolic blood pressure and heart weight. Prazosin administered every 8 h also lowered serum triglycerides but did not change the total cholesterol level. Selective alpha 1-inhibitor treatment increased the adrenal gland weight and decreased the number of adrenocortical cells; in every 8 h treated group this effect being connected with a marked cell hypertrophy (in all adrenocortical zones). Our results suggest that prazosin induced changes in the structure of the rat adrenal cortex depend on activation of the hypothalamo-pituitary-adrenocortical axis and possibly on the small inhibition of the proliferative activity of rat adrenocortical cells.

Divergent differentiation of rat adrenocortical cells is associated with an interruption of angiotensin II-mediated signal transduction

The zones of the adrenal cortex contain distinct populations of cells which share a common developmental origin and steroidogenic template. In the rat, zona glomerulosa cells respond to angiotensin II (Ang II) with increased Steroidogenesis while zona fasciculata/ reticularis cells do not. We have examined Ang II-mediated signal transduction in homogeneous cellular sub-populations derived from either the zona glomerulosa (GLOM) or the zona fasciculata (FASC). In both of these sub-populations Ang II treatment significantly increased the levels of 3H-labelled inositol phosphates as well as the total mass of inositol 1,4,5-trisphosphate. In contrast, the two cell types exhibited very different Ang II-mediated changes in free intracellular calcium ([Ca 2+] i). Ang II (10 nM), induced [Ca 2+] i increases of > 50 nM in 90% of individual GLOM cells ( 53 58 ), but in only 28% of FASC cells ( 11 39 ). These [Ca 2+] i responses occurred after a transient Ang II stimulation (<1 min), in the presence of verapamil and in the absence of extracellular calcium, indicating an intracellular release. In small groups of 10–30 cells, stimulation with 1, 10 and 100 nM Ang II induced [Ca 2+] i increases of 78, 178 and 215 nM respectively in GLOM cultures compared to only 35, 64, and 65 nM in FASC cultures. Thapsigargin treatment, which releases intracellular calcium in an inositol phosphate independent manner, elicited [Ca 2+] i increases in both populations. Importantly, a calcium ionophore induced elevation of [Ca 2+] i increased Steroidogenesis in both cell types. These results suggest that an interruption of the signaling cascade at the level of intracellular calcium release contributes to the lack of a steroidogenic response to Ang II by the FASC cells. Therefore, in the rat adrenal cortex, divergent differentiation of related cell types may involve alterations within signal transduction pathways distal to initial receptor-mediated events (i.e. inositol phosphate production) and proximal to downstream effector events (i.e. Steroidogenesis).

Glucocorticoid Effects on the Molecular Forms of Adrenocorticotrophic Hormone Secreted by Cultures of Rat Anterior Pituitary Cell Monolayers

The effects of glucocorticoids and corticotrophin-releasing factor (CRF) on the release of various molecular forms of adrenocorticotrophic hormone (ACTH) have been investigated in primary cultures of rat anterior pituitary. The rat cells responded to a 30 min challenge with CRF by secreting increased amounts of ACTH, as assessed both by bioassay, using rat adrenocortical cells, and by radioimmunoassay. Inclusion of a synthetic glucocorticoid, such as dexamethasone (DEX), in the incubation for 5 min prior to, and during the CRF challenge, had no effect on the response as measured by radioimmunoassay. Bioassay, however, indicated profound suppression of the response to CRF. This discrepancy between ACTH immuno- and bioactivity was investigated by fractionating the immunoreactive ACTH species using high-performance liquid chromatography. The lower molecular weight (<15kd) forms (ACTH(1-39) , phosphorylated ACTH(1-39) and glycosylated ACTH(1±39) ) were separated from higher molecular weight (>15kd) forms (i.e. ACTH biosynthetic intermediate and proopiomelanocortin) using C-18 Sep-Pak. The lower molecular weight molecules were further resolved into glycosylated and non-glycosylated ACTH, using an acetonitrile gradient high-performance liquid chromatography with trifluoroacetic acid as an ion-pairer. Neither the proportion of low molecular weight forms of ACTH, nor that of glycosylated ACTH(1-39) secreted in response to CRF, were affected by DEX. Further fractionation of non-glycosylated ACTH, also using acetonitrile gradient high-performance liquid chromatography but with heptafluorobutyric acid as the ion-pairer, yielded peaks corresponding to phosphorylated and non-phosphorylated ACTH(1-39) . DEX significantly increased the proportion of phosphorylated ACTH secreted in response to CRF by 18%. An additional effect of DEX was revealed when Sep-Pak extracts were treated with alkaline phosphatase, prior to analysis. After dephosphorylation, it became clear that the peptides released by CRF-stimulated cells were different if DEX was present in the medium. The peptide released in the presence of DEX (ACTH-S) had a slightly, but consistently, different retention time on high-performance liquid chromatography and very little biological activity. Antibody cross-reactivity studies suggested that ACTH-S was modified in the 1-24 region of the peptide. It is concluded that challenge of anterior pituitary cells in primary culture with CRF, following 5 min previous exposure to DEX, results in a molecular change. The consequence of this is that ACTH immunoreactivity is released, but the molecule has reduced biological activity. This may be part of the mechanism by which fast feedback inhibition of ACTH secretion is effected.

Studies on the intracellular mechanism of action of alpha-melanocyte-stimulating hormone on rat adrenal zona glomerulosa.

The intracellular mechanisms of action of alpha-MSH in rat adrenocortical cells were examined. When rat adrenal capsule (largely glomerulosa) cells were stimulated with a range of concentrations of alpha-MSH there was significant stimulation of aldosterone secretion at 10(-10) mol/l, although cyclic AMP was not increased until high concentrations of alpha-MSH were used (10(-6) mol/l and above). However, cells incubated with ACTH showed an increase in aldosterone secretion at 10(-11) mol/l and levels of cyclic AMP were elevated at 10(-9) mol ACTH/l. When rat adrenal whole capsules were incubated with alpha-MSH, membrane-bound protein kinase C (PKC) activity was increased and cytosolic enzyme activity decreased, showing PKC activation. Stimulation with angiotensin II also induced translocation of PKC activity, but ACTH did not. When [3H]inositol-loaded glomerulosa cells were stimulated with alpha-MSH there was significant generation of [3H]inositol trisphosphate (IP3) at concentrations of alpha-MSH which stimulated secretion of aldosterone. Significantly increased levels of [3H]IP3 were also measured when loaded cells were exposed to angiotensin II. ACTH did not cause any significant stimulation of [3H]IP3 production at any concentration used. These results indicate that activation of PKC and phospholipase C is important in modulating the steroidogenic effect of alpha-MSH.