The met-enkephalin analog FK 33-824 directly inhibits ACTH release by the rat pituitary gland in vitro

Systematic administration of the enkephalin analog FK 33-824 was previously shown to stimulate PRL secretion and to inhibit ACTH secretion in man. Naloxone prevented the effect on PRL release, but not on ACTH release. In this study, the direct action of this analog on hormone release by rat anterior pituitary lobes $\text{in}$$\text{vitro}$ were investigated. 1 uM FK 33-824 inhibited basal ACTH secretion by anterior pituitary glands in vitro, while 0.1 uM and 1 uM attenuated the lysine vasopressin stimulated ACTH release. Naloxone did not reverse the inhibitory action of the analog on ACTH release. β-Endorphin (0.01 - 1 uM) did not directly affect ACTH release. Basal and dopamine-induced inhibition of PRL release by anterior pituitary glands was neither influenced by FK 33-824 (0.1 and 1 uM), nor by β-endorphin (0.1 and 1 uM) with or without bacitracin. This study shows that the long-acting met-enkephalin analog FK 33-824 differentially affects PRL and ACTH secretion by the pituitary gland. It seems to stimulate PRL release at a suprapituitary site and this action probably involves u opiate receptors, because naloxone prevents these stimulatory effects. The inhibitory effect of FK 33-824 on ACTH release, however, is mediated via a direct effect at the pituitary level, which does not involve u receptors, as naloxone did not prevent this effect. In this respect, its action differs from that of β-endorphin, which does not directly affect ACTH release by the anterior pituitary gland.     

Inhibition by antiserum to rat growth hormone-releasing factor of growth hormone secretion induced by a met5-enkephalin analog, FK33-824, in rats

A Met5-enkephalin analog, FK33-824 (5, 10 and 20 micrograms/100 g body wt, iv) caused a dose-related increase in plasma growth hormone (GH) in urethane-anesthetized male rats. Pretreatment with cysteamine (30 mg/100 g body wt, sc), a depletor of hypothalamic somatostatin, increased the plasma GH response to FK33-824 (10 micrograms/100 g body wt, iv). Antiserum specific for rat GH-releasing factor (GRF) (0.5 ml/rat, iv) blunted GH release induced by FK33-824 (10 micrograms/100 g body wt, iv) in rats with or without cysteamine pretreatment. These results suggest that GH secretion induced by the opioid peptide is mediated, at least in part, by hypothalamic GRF in the rat.     

Morphologic change and elevation of cortisol secretion in cultured human normal adrenocortical cells caused by mutant p21K-ras protein

In our previous study on the tumorigenesis of human functional adrenal tumors, we observed a high frequency of K-ras point mutations in clinical specimens. Furthermore, we cloned the mutated K-ras gene from the tumors and inserted it into vectors to transfect normal bovine adrenocortical cells to express the mutated K-ras gene. The mRNA level of steroidogenic enzymes such as cholesterol sidechain cleavage enzyme (P450SCC), 17alpha-hydroxylase/17,20-lyase (P450c17), and 3beta-hydroxysteroid dehydrogenase (3betaHSD) in the mutant K-ras stably transfected cells were elevated. Cultured normal adrenocortical cells from donors and patients with adrenocortical tumors were then transfected with mutant K-ras expression plasmids constructed from human adrenal tumors. Stable transfectants grew faster than normal cells. Additionally, morphologic change was observed in the transfected cells. Moreover, when the synthesis of hormones was analyzed, the mRNA of P450SCC, P450C17, and 3betaHSD was found to have increased, and the level of cortisol was 18 to 25 times that in control cells. The increased steroid hormone production in mutant K-ras-transfected cells was reversed by lovastatin, a pharmacologic inhibitor of p21ras function. These results, combined with previous reports of steroidogenic K-ras in bovine adrenocortical cells, suggest that the K-ras oncogene is involved in steroidogenesis in human adrenocortical cells.     

Expression of adrenomedullin mRNA in adrenocortical tumors and secretion of adrenomedullin by cultured adrenocortical carcinoma cells

Immunoreactive-adrenomedullin concentrations and the expression of adrenomedullin mRNA were studied in the tumor tissues of adrenocortical tumors. Northern blot analysis showed the expression of adrenomedullin mRNA in tumor tissues of adrenocortical tumors, including aldosterone-producing adenomas, cortisol-producing adenomas, a non-functioning adenoma and adrenocortical carcinomas, as well as normal parts of adrenal glands and pheochromocytomas. On the other hand, immunoreactive-adrenomedullin was not detected in about 90% cases of adrenocortical tumors (<0.12 pmol/g wet weight (ww)). Immunoreactive-adrenomedullin concentrations ranged from 0.44 to 198.2 pmol/g ww in tumor tissues of pheochromocytomas and were 9.2 ± 1.2 pmol/g ww (mean ± SD, n = 4) in normal parts of adrenal glands. Adrenomedullin mRNA was expressed in an adrenocortical adenocarcinoma cell line, SW-13 and immunoreactive-adrenomedullin was detected in the culture medium of SW-13 (48.9 ± 1.8 fmol/10⁵ cells/24h, mean ± SEM, n = 4). On the other hand, immunoreactive-adrenomedullin was not detectable in the extract of SW-13 cells (<0.09 fmol/10⁵ cells), suggesting that adrenomedullin was actively secreted from SW-13 cells without long-term storage. These findings indicate that adrenomedullin is produced and secreted, not only by pheochromocytomas, but also by adrenocortical tumors. Undetectable or low levels of immunoreactive-adrenomedullin in the tumor tissues of adrenocortical tumors may be due to very rapid secretion of this peptide soon after the translation from these tumors.     

Production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1, by cultured human adrenocortical carcinoma cells

The production and secretion of peptides by adrenocortical tumors have not been well studied. We therefore studied the production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1 in SW-13 human adrenocortical carcinoma cells by radioimmunoassay and Northern blot analysis. Both immunoreactive-adrenomedullin and immunoreactive-endothelin were detected in the culture medium of SW-13 cells (27.7 +/- 1.6 fmol/10 (5) cells/24 h and 11.0 +/- 0.8 fmol/10 (5) cells/24 h, respectively, mean +/- SEM, n = 6). Northern blot analysis showed the expression of adrenomedullin mRNA and endothelin-1 mRNA in SW-13 cells. On the other hand, no significant amount of calcitonin gene-related peptide, corticotropin-releasing hormone, neuropeptide Y, or urocortin was secreted by SW-13 cells. Treatment with ACTH (10(-9)-10(-7) mol/l), angiotensin II (10(-9)-10(-7) mol/l), or dexamethasone (10(-8)-10(-6) mol/l) for 24 h had no significant effects on immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium of SW-13. Treatment with tumor necrosis factor (TNF)-alpha (20 ng/ml) increased significantly both immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium. Interferon-gamma (100 U/ml) increased the immunoreactive-endothelin levels, but not immunoreactive-adrenomedullin levels, whereas interleukin-1 (IL-1)beta (10 ng/ml) increased immunoreactive-adrenomedullin levels, but not immunoreactive-endothelin levels. These findings indicate that SW-13 human adrenocortical carcinoma cells produce and secrete two vasoactive peptides, adrenomedullin, and endothelin-1 and that the secretion of these two peptides is modulated differently by cytokines.     

Effects of ovine corticotropin-releasing hormone and FK 33-824 (met-enkephalin analogue) on the secretions of proopiomelanocortin-derived N-terminal peptide, beta-lipotropin, beta-endorphin and adrenocorticotropin in patients with Addison's disease

The responses of plasma immunoreactive (IR) proopiomelanocortin (POMC)-derived N-terminal peptide (Nt), IR-beta-endorphin (Ep), IR-beta-lipotropin (LPH) and IR-ACTH levels to ovine corticotropin-releasing hormone (CRF) and FK 33-824 (Met-Enkephalin analogue) were studied in nine patients with Addison's disease. The basal plasma levels (mean +/- SE) of IR-Nt, IR-Ep, IR-LPH and IR-ACTH were significantly higher in patients with Addison's disease (4459 +/- 975 pg/ml, 132 +/- 25 pg/ml, 4425 +/- 1030 pg/ml, 553 +/- 89 pg/ml, respectively) than in the normal controls (202 +/- 38 pg/ml, 7 +/- 2 pg/ml, 101 +/- 18 pfi/ml, 53 +/- 16 pg/ml, respectively). Ovine CRF produced rapid and concomitant increases in plasma levels of IR-Nt, IR-Ep, IR-LPH and IR-ACTH. Ep and ACTH levels reached a peak at 30 min. On the other hand, Nt and LPH levels reached a peak at 60 min and these levels gradually decreased up to 120 min. The molar concentrations of these IR-peptides in plasma were changed in close parallel fashion to one another. FK 33-824 produced a pronounced and concomitant fall in IR-Nt, IR-EP, IR-LPH, and IR-ACTH levels. These results support the theory that Nt, Ep, LPH and ACTH are produced simultaneously from POMC as a common precursor in the pituitary gland and are secreted concomitantly under various conditions such as stimulation by CRF and inhibition by FK 33-824 in patients with Addison's disease.     

Effects of a leucine analog on growth hormone processing and secretion by cultured cells

Bovine and rat growth hormones (bGH and rGH, respectively) possess signal peptides that direct the hormone to the secretory pathway and are proteolytically cleaved prior to secretion. Previous in vitro translation studies indicated that incorporation of the polar leucine analog beta-hydroxyleucine into de novo synthesized polypeptides inhibits signal peptide function. To test the effects of this analog on GH secretion by cultured animal cells, transfections of mouse L-cells with a bGH expression plasmid or metabolic labeling of endogenous rGH in anterior pituitary cells was performed in the absence or presence of beta-hydroxyleucine. Transient expression of bGH in mouse L-cells or endogenous expression of rGH in anterior pituitary cells resulted in an accumulation of GH in the culture medium. Treatment with beta-hydroxyleucine resulted in a block in secretion as evidenced by an accumulation of GHs within these cells. Amino-terminal sequencing of the intracellular form of the analog-substituted GHs demonstrated accurate signal peptide cleavage. In contrast, in vitro translations of bGH RNA performed in the presence of beta-hydroxyleucine and microsomal membranes resulted in the inhibition of signal peptide cleavage. The results suggest that beta-hydroxyleucine can uncouple signal peptide processing and protein secretion in cultured cells.     

Synthesis and secretion of thrombospondin by cultured human endothelial cells

Thrombospondin, the major glycoprotein released from alpha-granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160 kilodalton subunits and apparently functions as a platelet lectin. Because cultured human umbilical vein endothelial cells synthesize and secrete a glycoprotein (GP-160) which is a disulfide-bonded multimer of 160 kdalton subunits, the possibility that GP-160 is thrombospondin was investigated. Tritiated GP-160 could be immunoisolated from [3H]leucine- labeled endothelial cell postculture medium using a rabbit antiserum to human platelet thrombospondin. Thrombospondin and GP-160 comigrated in two different two-dimensional electrophoretic systems. Both proteins are disulfide-bonded trimers of acidic 160-kdalton subunits. A competitive radioimmunoassay for binding of 125I-thrombospondin to the rabbit antibodies indicated that 49 micrograms of thrombospondin antigen per 10(6) confluent endothelial cells accumulated in postculture medium over 24 h. Thus, endothelial cells secrete large amounts of a glycoprotein that is identical or very similar to platelet thrombospondin.     

On the mode of action of methionine enkephalin, FK 33-824 and naloxone in regulating the hemolymph glucose level in the fresh water field crab Oziotelphusa senex senex

The possible involvement of opioid system in the regulation of hemolymph glucose level in the fresh water crab Oziotelphusa senex senex Fabricius, was investigated. Opioid agonist and antagonist was also used in addition to methionine-enkephalin itself. Injection of the opioid, methionine-enkephalin and FK 33-824 significantly elevated hemolymph glucose level. In contrast, injection of naloxone in to crab resulted in decrease in hemolymph glucose level. Injection of naloxone prior to injection of methionine-enkephalin blocked the hyperglycemic action of methionine-enkephalin. Injection of methionine-enkephalin, FK 33824 and naloxone produced no significant effect on hemolymph glucose level in eyestalk-less crab. The alterations in the intact crab hemolymph glucose level hypothesised to be due to stimulation of release of hyperglycemic hormone during methionine-enkephalin and FK 33824 treatment and blocking of release of hyperglycemic hormone during naloxone treatment from the eyestalks of crab Oziotelphusa senex senex.     

Direct action of opioid peptides and naloxone on gonadotropin secretion by cultured rat anterior pituitary cells

Most current evidence indicates that opiates act via the hypothalamus to influence pituitary function. There are no detailed studies concerning pituitary site of action. Direct action of opioids on gonadotropin secretion was studied using enzymatically dispersed rat pituitary cells maintained in a monolayer culture. A time course study demonstrated that pretreatment with beta h-endorphin (beta h-End) (10(-7) M) initiated an inhibitory effect on LH release at 24 h, and was more evident at 48 h. A dose dependent decrease in LH release by beta h-End in concentrations of 10(-9) M to 10(-7) M was shown, whereas FSH was unchanged. Equimolar concentrations (10(-7)M ) of methionine enkephaline (Met-Enk) and D-ala2-met-enkephalinamide (DALA) produced a significant decrease in LH. Naloxone (NAL) (10(-5)) enhanced the release of both LH and FSH, and also blocked the inhibitory effect of beta h-End on LH release. These results indicated that opioid peptides act directly on anterior pituitary cells, decreasing the release of LH, but not of FSH. NAL also had a direct effect increasing the release of LH and FSH, and blocking the inhibitory action of beta h-End.     

Melatonin directly stimulates the secretion of progesterone by human and bovine granulosa cells in vitro

Melatonin, at concentrations and periods of exposure reflecting those present during the circadian cycle, was investigated for its influence on steroid production by granulosa cells cultured in serum-supplemented medium. At high (200 pg/ml) but not low (20 pg/ml) physiological concentrations, melatonin significantly stimulated progesterone production by human granulosa cells. This response was independent of the overall level of cell activity and was seen under the different culture conditions associated with different culture media. Exposure to melatonin for 8 h significantly stimulated progesterone secretion to a level similar to that achieved under continuous exposure, and the effect was reduced to control levels during subsequent periods in which no melatonin was added. Melatonin had no consistent effect on aromatase activity in the conversion of stored or serum-available androgen to oestradiol. Melatonin significantly stimulated progesterone production by bovine granulosa cells in vitro, at concentrations similar to those present during the endogenous nocturnal rise (100-400 pg/ml). This response to physiological conditions by human and bovine cells suggests a role for melatonin in the regulation of progesterone production by the ovary.     

Stimulation of progesterone secretion by cultured human granulosa cells with melatonin and catecholamines

Granulosa cells, aspirated from the follicles of patients undergoing treatment for in-vitro fertilization, were cultured in serum-supplemented medium. Adrenaline and noradrenaline stimulated a dose-related increase in progesterone secretion with a maximum stimulation at 10(-5) M, a response that was prevented by the beta-antagonist, propranolol. Adrenaline and hCG showed similar characteristics in their stimulation of progesterone secretion but there was no further increase in progesterone when the 2 compounds were added together. Melatonin stimulated progesterone secretion and, like adrenaline, this stimulation was prevented by propranolol. The ability of both adrenaline and melatonin to increase progesterone secretion was dependent on the degree of follicular development, as determined by peripheral oestradiol concentrations, on the day of laparoscopy. These results suggest that adrenaline and melatonin may have a physiological role in modulating luteal function and that melatonin may act by a beta-adrenergic-related mechanism.     

The inhibitor of phospholipase-A2, AACOCF3, stimulates steroid secretion by dispersed human and rat adrenocortical cells

AACOF3 is a trifluomethylketone analog of arachidonic acid, which inhibits phospholipase-A2 (PLA2). AACOCF3 was found to concentration-dependently increase basal aldosterone and corticosterone secretion by dispersed rat zona glomerulosa and zona fasciculata/reticularis cells, respectively, as well as aldosterone and cortisol production by dispersed human adrenocortical cells. Maximal effective concentration was 10(-5) M, and elicited about 2.5-3.0-fold rises in steroid output. 10(-5) M AACOCF3 also enhanced submaximally (10(-15)/10(-12) M), but not maximally (10(-9) M) ACTH-stimulated hormonal secretion. Quantitative HPLC showed that 10(-5) M AACOCF3 evokes similar increases (from 2.0- to 3.0-fold) in the basal release of the entire spectrum of adrenocortical steroids (i.e. both intermediate and definitive products of steroid synthesis), thereby suggesting that AACOCF3 acts on the early steps of steroid synthesis. Accordingly, when pregnenolone metabolism is prevented by cyanoketone, 10(-5) M AACOCF3 increased by about 8-10-fold the production of this steroid. In conclusion, we have demonstrated a side-effect of AACOCF3, which may become relevant in studies where this chemical is used to inhibit PLA2 in tissues able to convert cholesterol to pregnenolone.     

Regulation of glutamine and pyruvate oxidation in cultured adrenocortical cells by cortisol,antioxidants, and oxygen: Effects on cell proliferation

The regulation of CO₂ production from [U-¹⁴C]glutamine and C₂ of [2-¹⁴C]pyruvate was investigated in cultured bovine adrenocortical cells, and the effect of alterations in the relative rates of oxidation of these substrates on cell proliferation, particularly in the presence of an inhibitor of transamination reactions, was examined. ¹⁴CO₂ production from 2 mM [U-¹⁴C]glutamine and 2 mM [2-¹⁴C]pyruvate was measured in the presence of 100 μM 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. Treatment of primary cultures for 24 h with 50 μM cortisol increased the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate, an effect dependent on prior low cell density. Cortisol treatment also resulted in a prolonged delay in the onset of proliferation from low density, and completely inhibited growth in the presence of 2 mM aminooxyacetate, which reduces mitochondrial utilization of glutamine. The effects on glutamine and pyruvate metabolism and on cell growth, with or without aminooxyacetate, were prevented by simultaneous treatment with the antioxidants dimethyl sulfoxide (10 mM) and butylated hydroxyanisole (100 μM), suggesting the involvement of lipid peroxidation in the action of cortisol, as previously demonstrated for its action on 11β-hydroxylase. During continued proliferation of adrenocortical cells in the absence of cortisol there was also a slower increase in the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate as a function of population doubling level. The rate of this increase was slowed by growth of cells in 2% O₂ rather than the standard 19% O₂, and accelerated by continued growth of cells in the presence of cortisol. The rate of increase in the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate under these three conditions correlated with inhibition of cell growth by aminooxyacetate. In contrast to the complete inhibition of growth in aminooxyacetate demonstrated by cortisol-treated cells, control cells (19% O₂) did proliferate, although growth was limited, whereas cells at 2% O₂ proliferated to a much greater extent. In the absence of aminooxyacetate the rate of growth in primary adrenocortical cell cultures under these three conditions was similar. Lipid peroxidation appears to make cultured adrenocortical cells dependent on glutamine for mitochondrial function and proliferation by inhibiting the utilization of the normal substrate, pyruvate.     

The effect of calcium on the secretion of factor VIII-related antigen by cultured human endothelial cells

Cultured human endothelial cells derived from the umbilical cord vein are able to release factor VIII-related antigen into the culture medium. The experiments described in this paper show the presence of two pathways for the secretion of factor VIII-related antigen from endothelial cells. There is a basal release of this antigen, independent of the presence of extracellular calcium ions. This release can be inhibited by cycloheximide and is therefore directly related to de novo protein synthesis. Besides this basal release, there is an extra release of factor VIII-related antigen that can be stimulated by thrombin, the Ca²⁺-ionophore A23187 or phorbol myristate acetate. As demonstrated by immunofluorescence, the stimulus-inducible release originates from storage granules in the cells. This stimulus-inducible release is dependent on extracellular Ca²⁺ but independent of intracellular cAMP.     

Effect of ACTH and prolactin on dehydroepiandrosterone, its sulfate ester and cortisol production by normal and tumorous human adrenocortical cells

The effect of ACTH and prolactin on the synthesis of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) was studied in cell suspensions of "normal" and tumorous (adenoma) human adrenal cortex. A stimulation of DHEA and no response of DHEAS production by ACTH in "normal" adrenocortical cell suspension was observed. However ACTH stimulated both DHEA and DHEAS synthesis in tumorous adrenocortical cells. Prolactin did not influence either the basal or the ACTH stimulated DHEA and DHEAS production of adrenocortical cells irrespective of their origin. Our results are compatible with the concept that the biosynthesis of DHEA is under ACTH control, while other factor(s) regulate(s) the sulfate pathway of DHEA secretion under normal conditions. In tumorous adrenocortical cells DHEA may be regulated--at least partly--by ACTH. Prolactin seems to have no direct effect on DHEA and DHEAS synthesis. It is postulated that the relationship between serum prolactin and DHEAS (or DHEA) levels observed by several authors might be an extraadrenal effect of prolactin on adrenal androgens.     

Action of the opioid agonist FK 33-824 on porcine small and large luteal cells from the mid-luteal phase: effect on progesterone, cAMP, cGMP and inositol phosphate release.

The present study was designed to investigate the effect of the opioid agonist FK 33-824 on basal and hCG-induced progesterone (P4), cAMP and cGMP secretion and on the phosphoinositide-specific phospholipase C signalling system in separated porcine small (SLCs) and large luteal cells (LLCs). Unit gravity sedimentation was used to produce cultures of small and large luteal cells from corpora lutea (CL) on days 8-10 of the oestrous cycle. In order to examine the effect of FK 33-824 on P4 and cyclic nucleotide release, SLCs and LLCs were incubated in M199 medium at 37 degrees C in 5% CO2:95% air, for 12 h. Small and large luteal cells were treated with hCG (100 ng/ml) alone, FK 33-824 (10(-9) M) alone or were co-treated with FK 33-824 and hCG and with the opioid antagonist, naloxone (NAL, 10(-5) M). FK 33-824 alone did not influence P4 secretion by LLCs and SLCs. However, FK 33-824 completely abolished the stimulatory effect of hCG on P4 secretion by SLCs. The addition of FK 33-824 was followed by a significant increase in cAMP release (p<0.01) by LLCs and a decrease in cGMP secretion by SLCs (p<0.05). The effect of FK 33-824 was blocked by NAL, which strongly suggests that the observed influence of this opioid agonist was achieved through its binding to opioid receptors in luteal membranes. In the presence of hCG, cAMP secretion by both SLCs and LLCs was many-fold higher than in the control group. As regards cGMP output, only LLCs showed elevated secretion of this cyclic nucleotide under the influence of hCG. With the aim of examining the influence of FK 33-824 on phosphatidylinositol hydrolysis, LLCs, SLCs and mixed small and large cells were labelled with [3H]-myo-inositol (100 microCi/ml) for 3 h at 37 degrees C. The cells were then incubated in M199 medium supplemented with 10 mM LiCl, 1% BSA, and antibiotics in the presence and absence of FK 33-824 (10(-9) M) at 37 degrees C for 30 min. Liberated labelled inositol mono-, bis-, and trisphosphates (IPs) were isolated and quantified by affinity chromatography on columns of AG 1-X8 resin, followed by liquid scintillation spectroscopy. Inositol phosphate accumulation in LLCs, SLCs, and mixed small and large cells was not altered by treatment with FK 33-824 at the dose used. In view of these findings we suggest that opioid peptides affect pig corpus luteum steroid secretion, and the response is probably mediated through cyclic nucleotides, but not IPs.