Relative and combined effects of estradiol and prolactin on corticosterone secretion in ovariectomized rats

In vivo and in vitro experiments were designed to assess the relationship of the estradiol (E2) and prolactin (PRL) on glucocorticoid secretion in ovariectomized (Ovx) rats. Female rats were Ovx for two weeks and then subcutaneously injected with oil or estradiol benzoate (EB) for 3 days before experimentation. Venous blood samples were collected from right jugular vein at 0, 30, 60, 90, and 120 min after challenge with adrenocorticotropin (ACTH). Adrenal zona fasciculata-reticularis (ZFR) cells from Ovx rats were isolated and incubated with E2 or PRL. In the morning and afternoon, EB enhanced the basal and ACTH-stimulated concentrations of plasma corticosterone (CORT) and PRL. Administration of E2 in vitro increased the basal and ACTH-stimulated release of CORT and production of adenosine 3', 5'-cyclic monophosphate (cAMP) in ZFR cells. E2 enhanced the forskolin-stimulated release of CORT by ZFR cells. However, the 3-isobutyl-l-methylxanthine (IBMX)- or 8-Br-cAMP-stimulated release of CORT was not affected by E2. E2 augmented the lower doses of PRL-stimulated release of CORT and cAMP accumulation as compared with the PRL-treated group alone. Incubation of higher doses of PRL increased the production of cAMP. Administration of nifedipine and R(+) BK8644 (classic L-type Ca2+ channel blocker) significantly attenuated the PRL-stimulated release of CORT. Taken together, these data indicate that E2- and PRL-related increase of CORT in Ovx rats is associated with the increase of cAMP accumulation and calcium influx in ZFR cells. In conclusion, E2 and PRL play a stimulatory role in the co-regulation of CORT secretion.     

Involvement of cAMP but not PKA in the increase of corticosterone secretion in rat zona fasciculata-reticularis cells by aging

The effects and mechanisms of aging on corticosterone secretion in zona fasciculata-reticularis (ZFR) cells of ovariectomized (Ovx) rats were studied. Young (3-month) and old (24-month) female rats were Ovx for 4 days before decapitation. ZFR cells were isolated and incubated with different hormones or reagents at 37 degrees C for 30 min. Aging increased the basal secretion of corticosterone both in vivo and in vitro. The adrenocorticotropin (ACTH)-, forskolin-, 3-isobutyl-l-methylxanthine (IBMX)-, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP)-, and ovine prolactin (oPRL)-stimulated release of corticosterone by ZFR cells was greater in old than in young Ovx rats. H89, an inhibitor of protein kinase A (PKA), decreased the production of corticosterone in ZFR cells from young but not old Ovx rats. Forskolin-, or IBMX-induced production of cAMP was greater in old than in young Ovx animals, which correlated with the increase of corticosterone production by aging. The activity of 11 beta-hydroxylase that converts deoxycorticosterone (DOC, 10(-9) or 10(-8) M) to corticosterone in rat ZFR cells was decreased by age. However, the corticosterone production in response to high dose of DOC (10(-7) M) was indifferent between young and old groups. These results suggest that aging increases corticosterone production in Ovx rats via a mechanism in part associated with an increase of adenylyl cyclase activity and a decrease of phosphodiesterase activity, and then an increase of the generation of cAMP, but not related to either PKA activity or 11 beta-hydroxylase.     

Direct effects of prolactin on corticosterone release by zona fasciculata-reticularis cells from male rats

The role of prolactin (PRL) in the male is not fully defined. The aim of this study was to investigate the function and mechanism of PRL on the production of corticosterone by zona fasciculata-reticularis (ZFR) cells in vitro. The ZFR cells were obtained from male rats under normal, hyperprolactinemic, or hypoprolactinemic situation. PRL stimulated the corticosterone release in a dose-dependent pattern in the ZFR cells from normal male rats. The cellular adenosine 3'-5'-cyclic monophosphate (cAMP) concentration positively correlated with PRL concentration in the presence of forskolin or 3-isobutyl-1-methylxanthine (IBMX). PRL enhanced the stimulatory effects of cAMP mimetic reagents, i.e., forskolin, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), and IBMX on the release of corticosterone. The adenylate cyclase inhibitor (SQ22536) inhibited the corticosterone release in spite of presence of PRL. Nifedipine (L-type calcium channel blocker) did not inhibit corticosterone release. The hyperprolactinemic condition was actualized by transplantation of donor rat anterior pituitary glands (APs) under kidney capsule. By comparison with the cerebral cortex (CX)-grafted group, AP-graft resulted in an increased release of corticosterone, 3beta-hydroxysteriod dehydrogenase (HSD) activity and cAMP production by ZFR cells. Acute hypoprolactinemic status was induced by bromocriptine for 2 days. The results showed the productions of corticosterone were lower in hypoprolactinemic group than in control group, which were persistent along with different ACTH concentrations. These results suggest that PRL increase the release of corticosterone by ZFR cells via cAMP cascades and 3beta-HSD activity.     

Effect of aging on corticosterone secretion in diestrous rats

The roles of age and prolactin (PRL) in regulating glucocorticoid secretion in diestrous rats were investigated. Adrenal zona fasciculata-reticularis (ZFR) cells from young, adult, middle (mid)-aged, and old female rats were isolated. Estrous cycle stage was determined by light microscopy after vaginal smears. Blood samples were collected from right jugular vein at 0, 30, 60, and 120 min after challenge with adrenocorticotropin (ACTH). During the diestrous phase, plasma levels of estradiol and progesterone were lower in mid-aged and old rats than in either young or adult rats. Age-dependent increases of the basal levels of plasma PRL and corticosterone were observed. No difference of ACTH-increased plasma concentrations of corticosterone was observed among young, adult, mid-aged, and old rats. Aging increased the basal, ACTH-, PRL-, forskolin (an adenylate cyclase activator)-, and 3-isobutyl-l-methylxanthine (IBMX, a non-selective phosphodiesterase inhibitor)-stimulated release of corticosterone and production of adenosine 3', 5'-cyclic monophosphate (cAMP) in ZFR cells. However, the 8-Br-cAMP (a membrane-permeable cAMP)-stimulated release of corticosterone was not affected by age. Taken together, these data indicated that aging increased corticosterone secretion in female rats during diestrous phase, which is in part due to an increase in cAMP accumulation. In conclusion, aging and PRL play a stimulatory role in the co-regulation of corticosterone secretion.     

Effects of chronic hypogonadism on corticosterone secretion and cyclic AMP production in male rat adrenocortical cells

AIM: To determine the secretion of corticosterone (CCS) both in vivo and in vitro during different intervals after orchidectomy in male rats. METHODS: Three- and 12-month-old rats had been orchidectomized 0, 3, 6, or 9 months before decapitation. RESULTS: Orchidectomy increased the concentrations of plasma CCS, the basal release of CCS, and the adenosine 3', 5'-cyclic monophosphate (cAMP) production in rat zona fasciculata reticularis (ZFR) cells. The forskolin/3-isobutyl-l-methylxanthine-stimulated releases of CCS and cAMP production by ZFR cells were higher in rats with chronic hypogonadism. The CCS release from ZFR cells of orchidectomized rat was not altered by 8-bromo-cAMP treatment. Orchidectomy enhanced the stimulatory effect of deoxycorticosterone on CCS release in ZFR cells. CONCLUSION: These results suggest that orchidectomy-related increases of CCS secretion in rats are associated with an increase of adenylate cyclase activity, cAMP generation, and 11-beta-hydroxylase activity in ZFR cells.     

Effects of estradiol on aldosterone secretion in ovariectomized rats

The effects and action mechanisms of estradiol on aldosterone secretion in female rats were studied. Replacement of estradiol benzoate (EB) increased the levels of plasma estradiol and aldosterone in ovariectomized (Ovx) rats. The aldosterone release from zona glomerulosa (ZG) cells was higher in EB-treated rats than in oil-treated animals. EB treatment potentiated the responses of aldosterone release to adrenocorticotropic hormone (ACTH), forskolin (FSK), and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Administration of EB in vivo did not alter cAMP production in response to ACTH or FSK. Although angiotensin II (Ang II) increased aldosterone secretion by rat ZG cells, the stimulatory effect of Ang II on the release of aldosterone was not altered by EB treatment. The conversions of [3H]-deoxycorticosterone to [3H]-corticosterone and [3H]-corticosterone to [3H]-aldosterone in EB-treated groups were greater than those in the oil-treated group. These results suggest that estradiol increases aldosterone secretion in part through the mechanisms involving the activation of the post-cAMP pathway, 11beta-hydroxylase and aldosterone synthase activity.     

Direct effects of IL-15 on corticosterone secretion by rat adrenocortical cells

Cytokines might regulate the function of the hypothalamic-pituitary-adrenal axis. IL-15 is a potent non-T-cell-derived cytokine with IL-2-like activities. It has been shown that IL-15 can reverse the inhibition of glucocorticoids on PBMC. In vitro experiments were designed to assess the direct effect of IL-15 on corticosterone (CORT) secretion in the adrenal zona fasciculata-reticularis (ZFR) cells of male rats. Administration of IL-15 dose dependently decreased the basal and adrenocorticotropin-stimulated release of CORT and production of cAMP in ZFR cells. The stimulatory effect of forskolin (an adenylate cyclase activator) on CORT secretion and accumulation of cAMP in ZFR cells was attenuated by the administration of IL-15. However, 8-Br-cAMP (a cAMP analogue)-stimulated release of CORT was not affected by IL-15. Exogenous administration of IL-15 (10(-7) mol/L) significantly attenuated the pregnenolone (the substrate of 3beta-hydroxysteroid dehydrogenase)- or deoxycorticosterone (the substrate of 11beta-hydroxylase)-induced release of CORT. The results indicate that decrease of CORT secretion by IL-15 is in part because of (i) the decrease of adenylate cyclase activity and cAMP production and (ii) the inhibition of 3beta-hydroxysteroid dehydrogenase and 11beta-hydroxylase activities in rat ZFR cells.     

Effects of evodiamine and rutaecarpine on the secretion of corticosterone by Zona fasciculata‐reticularis cells in male rats

Evodiamine (EVO) and rutaecarpine (RUT) are two bioactive alkaloid isolated from Chinese herb named Wu–Chu–Yu. Previous studies have shown that EVO and RUT possess thermoregulation, vascular regulation, anti‐allergic, anti‐nociceptive and anti‐inflammatory activities. The mechanisms of EVO and RUT effect on steroidogenesis are not clear. The goal of this study was to characterize the mechanism by which EVO and RUT affect corticosterone production in rat zona fasciculata‐reticularis (ZFR) cells. ZFR cells were isolated from adrenal glands of male rats and incubated with adrenalcorticotropin (ACTH, 10^?9 M), forskolin (an adenylyl cyclase activator, 10^?5 M), 8‐bromo‐adenosine 3′:5′‐cyclic monophosphate (8‐Br‐cAMP, a permeable cAMP analog, 10^?4 M), or steroidogenic precursors including 25‐hydroxycholesterol, pregnenolone, progesterone, and deoxycorticosterone, 10^?5 M each, in the presence or absence of EVO and RUT respectively (0–10^?3 M) at 37°C for 1 h. The concentrations of corticosterone, pregnenolone and progesterone in the media were measured by radioimmunoassay. After administration of ZFR cells with EVO or RUT (10^?4 M) for 60 and 120 min, Western blot analysis was employed to explore the influence of EVO and RUT on the expression of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). EVO and RUT reduced both basal and ACTH‐, forskolin‐, as well as 8‐Br‐cAMP‐stimulated corticosterone production in rat ZFR cells. The enhanced corticosterone production caused by the administration of four steroidogenic precursors was decreased following EVO or RUT challenge. These results suggest that EVO and RUT inhibit corticosterone production in rat ZFR cells via a mechanism involving: (1) a decreased activity of cAMP‐related pathways; (2) a decreased activity of the steroidogenic enzymes, that is, 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and 11β‐hydroxylase (P450c11), during steroidogenesis; and (3) an inhibition of StAR protein expression. J. Cell. Biochem. 108: 469–475, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of 3,3'-iminodiproprionitrile (IDPN) on corticosteroidogenesis of isolated adrenocortical cells

The neurotoxic agent, 3,3'-iminodiproprionitrile (IDPN), is a disrupter of neurofilament- and intermediate filament-organelle association. In the present study, the effect of IDPN on corticosteroidogenesis was investigated using isolated rat (having few intermediate filaments) and domestic fowl (having abundant intermediate filaments) adrenocortical cells. Cells were incubated with or without steroidogenic agents and precursors and with or without various concentrations of IDPN for 2 hr. IDPN had similar inhibitory potencies (as indicated by the half-maximal inhibitor concentrations (ID50 values] with both rat and domestic fowl cells despite their grossly different intermediate filament content. However, the average ID50 values of IDPN varied with the different steroidogenic agents and precursors used. The average IDPN ID50 values for maximal ACTH- and 8-bromo-cyclic AMP (8-Br-cAMP)-induced corticosterone production were equivalent (49.7 and 45.7 mM, respectively). However, the IDPN ID50 values for maximal ACTH-induced cAMP production, maximal 25-hydroxycholesterol- and pregnenolone-supported corticosterone production, and maximal ACTH- and 8-Br-cAMP-induced protein synthesis varied from 3.7 to 5.4 times the average ID50 values for maximal ACTH- and 8-Br-cAMP-induced corticosterone production. Thus, the inhibitory action of IDPN was not closely linked to the inhibition of ACTH-transmembrane signaling via cAMP, protein synthesis, and steroidogenic enzyme activity. The data suggest that IDPN inhibited corticosteroidogenesis at at a step after cAMP but before cholesterol side-chain cleavage and that the inhibition was not dependent on the presence of intermediate filaments.     

Effects of proopiomelanocortin-derived peptides, methionine-enkephalin and forskolin on the maturation of ovine fetal adrenal cells in culture

The present study examined the effects of peptides derived from the non-ACTH part of proopiomelanocortin (POMC), of met-enkephalin and of forskolin, alone or in combination with ACTH-(1-24), on the development of the ability of ovine fetal adrenal cells to produce both cAMP and corticosteroids in culture. N-POMC-(1-61) amide, gamma 2-melanocyte-stimulating hormone (MSH) and gamma 3-MSH behaved similarly in our system: 1) they increased slightly corticosterone (B) and cortisol (F) production without modification of cAMP output when added alone to the incubation medium, but this effect was observed only after 3 days in culture; 2) they potentiated the steroidogenic response to 10(-11) M ACTH-(1-24) but again only from Day 3 onwards; and 3) a 5-day treatment with the peptides induced fetal adrenal cell maturation resulting in the same enhancement of the B + F production stimulated by 10(-8) M ACTH-(1-24) without modification of the response in both cAMP and pregnenolone (P5). N-POMC-(1-80) and beta-lipotropic hormone (LPH) shared several common features in that, 1) the stimulation of B + F production by each of them alone was always significant and higher than that obtained with the other POMC-derived peptides [except ACTH-(1-24]; 2) they did not potentiate the steroidogenic action of 10(-11) M ACTH-(1-24); and 3) when cells were cultured in their presence for 5 days it resulted in an enhancement of the response to ACTH-(1-24), not only in B + F production but also in cAMP and P5 outputs. No effect of met-enkephalin was observed. The development of cAMP and B + F responses to ACTH-(1-24) provoked by forskolin was very close to that induced by the hormone itself, but forskolin, as opposed to ACTH-(1-24), was unable to induce a desensitization of the cAMP response. These data show that N-POMC-derived peptides can potentiate the acute steroidogenic activity of ACTH-(1-24) on ovine fetal adrenal cells after several days in culture.     

Effects of estradiol benzoate on learning-memory behavior and synaptic structure in ovariectomized mice

There is increasing evidence that estrogen is involved in CNS activity, particularly memory. Several studies have suggested that estrogen improves memory by altering neuronal plasticity, including increased hippocampus CA1 dendritic spine density and enhanced long-term potentiation (LTP). In the present study, we investigated the effects of estrogen on the ultrastructural modifications in cerebral frontal cortex and hippocampus of female ovariectomized mice. One week after ovariectomy (Ovx), ICR female mice received daily injection of estradiol benzoate (EB, 20, 100, 200 microg/kg, s.c.) for 4-5 weeks. Spatial memory was then tested in the water maze, and the overall locomotor activity was monitored in open field. Synaptic morphologic parameters were examined using a graph analyzer. The results from open field did not show any alterations in locomotor activity following Ovx and EB replacement. Both the latency to find the platform and the distance to reach the platform were significantly reduced in Ovx mice by EB at 20 or 100 microg/kg when compared to vehicle treated Ovx mice. The results from synaptic ultrastructural measurement and analysis did not show any differences in hemispheric or hippocampal volumes, the numeric synaptic density, the length of active zones, or the curvature of synaptic interface among Sham, Ovx, and Ovx plus EB replacement mice. However, EB replacement effectively normalized the changes induced by Ovx, reducing the width of the synaptic cleft, enlarging the thickness of postsynaptic density (PSD), and increasing the number of synaptic vesicles in the presynapse in both cerebral cortex Fr1 and hippocampus CA1 areas. These results suggest that the beneficial effects of EB on improving memory behavior of Ovx female mice are associated with the changes of some subtle structural parameters of synapses, including the width of PSD and synaptic cleft rather than some basic and permanent structure in frontal cortex and hippocampus regions.     

Effect of corticosterone on the prolactin response to psychological and physical stress in rats

Both corticosterone and prolactin (PRL) levels increase in response to stress. In these studies we examined the effect of corticosterone on the PRL response to both physical (footshock) and psychological (novel environment) stress. Three groups of rats were used: sham adrenalectomized (SHAM), adrenalectomized (ADX), and adrenalectomized with corticosterone replacement (ADX+CORT). The corticosterone-treated animals received 80 ug corticosterone/ml drinking water. Blood samples were drawn via an indwelling cannula and PRL values determined using radioimmunoassay. ADX rats showed a consistently greater PRL response to being placed on a platform above water (novel environment) or when receiving intermittant footshock than did ADX+CORT rats. The PRL response of the latter group was similar to that of the SHAM animals. These findings indicate that corticosterone levels of an animal can significantly attenuate the magnitude of the PRL response to both physical and psychological stress. These findings further emphasize that the PRL response to stress is dependent not only upon the immediate action of the stressor, but also the prior stress history of the animal.     

Chronic administration of corticosterone impairs LH signal transduction and steroidogenesis in rat Leydig cells

The mechanism involved in the inhibitory actions of chronic corticosterone treatment on Leydig cell steroidogenesis was studied in adult Wistar rats. Rats were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days and another set of rats was treated with corticosterone plus ovine luteinizing hormone (oLH) (100 microg/kg body weight, s.c., daily) for 15 days. Chronic treatment with corticosterone increased serum corticosterone but decreased serum LH, testosterone, estradiol and testicular interstitial fluid (TIF) testosterone and estradiol concentrations. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone and estradiol. Leydig cell LH receptor number, basal and LH-stimulated cAMP production were diminished by corticosterone treatment which remained at control level in the corticosterone plus LH treated rats. Activities of steroidogenic enzymes, 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were significantly decreased in corticosterone treated rats. LH plus corticosterone treatment did not affect 3beta-HSD activity but decreased 17beta-HSD activity, indicating a direct inhibitory effect of excess corticosterone on Leydig cell testosterone synthesis. The indirect effect of corticosterone, thus, assume to be mediated through lower LH which regulates the activity of 3beta-HSD. Basal, LH and cAMP-stimulated testosterone production by Leydig cells of corticosterone and corticosterone plus LH treated rats were decreased compared to control suggesting the deleterious effect of excess corticosterone on LH signal transduction and thus steroidogenesis.     

Prolactin-dependent mitogenesis in Nb 2 node lymphoma cells: effects of immunosuppressive cyclopeptides

Prolactin (PRL)-stimulated ornithine decarboxylase (ODC) activity and subsequent proliferation are inhibited by the cyclopeptides cyclosporine (CsA) and didemnin B (DB) in Nb 2 node lymphoma cells. Similar concentrations of these agents also inhibit 125I-PRL binding, suggesting that their inhibitory effects on these PRL-dependent physiologic responses are mediated at least in part at the level of PRL receptor interactions. The phorbol ester TPA stimulated ODC activity and [3H]thymidine incorporation to 54% and 31% that of a near-optimal mitogenic concentration of PRL (10 ng/ml), suggesting that mitogenesis in these cells is coupled to some degree to the activation of protein kinase C (PKC). The calcium ionophore A23187 increased ODC activity only slightly and actually decreased [3H]thymidine incorporation to a value below the "cells only" controls. The addition of TPA plus A23187 did not further enhance the effects of TPA to elevate ODC activity and [3H]thymidine incorporation. However, A23187 significantly elevated PRL-stimulated ODC activity with a subsequent inhibition of [3H]thymidine incorporation, suggesting a block of entry into S phase. Both cyclopeptides decreased the elevation of ODC activity in G1 phase of cell cycle in response to PRL, suggestive of a site of action for these agents in early G1, a conclusion compatible with their ability to inhibit PRL binding to these cells. Addition of CsA or DB 2 hr after PRL had no effect on PRL-stimulated ODC activity detectable at 6 hr, but addition of either as late as 6 hr still affected the extent of mitogenesis. This is in line with the requirement for PRL to be present in the culture medium for a minimum of 3 to 6 hr to invoke a maximal effect on mitogenesis. Addition of either cyclopeptide after the cells were in S phase had no effect on the extent of [3H]thymidine incorporation. An inhibitor of the cyclooxygenase pathway (indomethacin) enhanced both PRL-stimulated ODC activity and proliferation, whereas inhibition of the lipoxygenase pathway by NDGA attenuated only proliferation, suggesting that in Nb 2 cells, products of the lipoxygenase pathway may contribute to the mechanism of PRL-stimulated mitogenesis. Because Nb 2 lymphoma cells were derived from estrogenized rats, estrogen was tested as a mitogen. By itself it was not mitogenic, but in conjunction with PRL, estradiol-17 beta elevated the ODC response and inhibited proliferation. Inhibitors of PKC known to have minimal effects on RNA synthesis, quercetin and gossypol, totally inhibited both the elevations of ODC activity and [3H]thymidine incorporation in response to PRL in Nb 2 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Attenuation of estradiol on the reduction of striatal dopamine by amphetamine in ovariectomized rats

Amphetamine (AMPH) is a highly addictive drug of abuse which exhibits toxicity to dopaminergic neurons in long‐term abusers. Estrogen seems to show neuroprotection in dopamine (DA) deficit caused by AMPH. The present study was to investigate the effects of estradiol on the levels of striatal DA in ovariectomized (Ovx) rats treated with or without AMPH. Female rats were Ovx for 2 weeks before administration of AMPH (5 mg/kg/day, i.p.) with or without 17β‐estradiol benzoate (EB) (25 µg/kg/day, s.c.) for 7 days. The striatal tissues were collected, homogenized with DA mobile phase, and centrifuged. The concentrations of DA in the supernatants were detected by HPLC. The protein expressions of dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT‐2), and tyrosine hydroxylase (TH) were analyzed by Western blotting. The results indicated that AMPH could attenuate DA level significantly in striatum (P < 0.01). Comparing to control groups, administration of either EB or EB plus AMPH increased DA level (P < 0.01). The protein expression of striatal DAT was significant greater (P < 0.01) in rats treated with AMPH plus EB than AMPH treated animals. These results suggest that the DA levels in striatum can be enhanced by EB via an increase of DAT expression following administration of AMPH. J. Cell. Biochem. 108: 1318–1324, 2009. © 2009 Wiley‐Liss, Inc.     

Steroidogenesis in adrenal glands of young and adult female mice with hyperexpression of the Aguoti gene

Dominant mutation Agouti yellow (AY) leads to ectopic overexpression of the Agouti gene and yellow coat color in mice. Furthermore, the mutation Ay increased adrenal response to emotional stress. The study assessed whether pleiotropic effect of the mutation Ay on adrenals function was dependent on sex and age. 3- and 15-week old female C57B1/6J mice of two agouti-genotypes: Ay/a (ectopic Agouti-gene overexpression) and a/a (absence of Agouti-protein), were investigated. Cyclic AMP level (adenylate cyclase activity) and corticosterone production in adrenal isolated cells stimulated by ACTH and dibutyrul cAMP (db-cAMP) were measured. ACTH increased cAMP accumulation to the same extent in Ay/a- and a/a-mouse adrenal cells of both ages. The dibutyrul cAMP-induced corticosterone production was higher in Ay/a than in a/a-mouse adrenal cells of both ages. The ACTH-induced corticosterone production in 3-week- old Ay/a-m/CQ was lower and in 15-week old Ay/a-mice was higher than in a/a-mice of the respective ages. The ACTH- and db-cAMP-induced steroidogenesis was not changed in Ay/a-mice and decreased in a/a-mice with age. Thus, in females as well as in males, the mutation Agouti yellow did not affect adenylate cyclase activity, increased db-cAMP-induced corticosterone production and disturbed development of adrenal cortex.     

Isoproterenol produces a rapid increase in sialidase activity in rat heart tissue and cardiomyocyte-derived H9c2 cells in culture

The effects of isoproterenol on sialidase activity in rat cardiomyocytes were examined. Administration of isoproterenol to rats (0.2 or 2 mg/kg body weight) produced an increase in sialidase activity in total membrane fraction of heart tissue within 120 min (121+/-13% of the control at 120 min after administration of 0.2 mg isoproterenol/kg, n=5, P<0.05). Sialidase activity in cardiomyocyte-derived H9c2 cells was also increased by treatment with isoproterenol (10 microM) for 60 min. The effect of isoproterenol on sialidase activity was amplified by the addition of 3-isobutyl-1-methylxanthine (IBMX). Sialidase activity in H9c2 cells was elevated by treatment with dibutyryl cAMP plus IBMX without isoproterenol. The content of N-acetylneuraminic acid in cells decreased by 22% after treatment with isoproterenol plus IBMX. These results suggest that sialidase activity in rat cardiomyocytes is regulated by beta-adrenergic stimulators via a cAMP-dependent process. The increased activity of sialidase may account for the reduction of sialic acid content of cells.     

Prolactin interacts with gonadotropin through a suppression of c-AMP production probably as a LH-sensitive adenylate cyclase inhibitor

The interaction between gonadotropin and prolactin (PRL) on ovarian steroidogenesis as well as c-AMP production was studied in rat ovaries. Ovaries obtained from adult female Wistar rats in a morning of proestrus were chopped into 30-40 pieces and subjected to short term incubation studies using various buffers. HCG-stimulated c-AMP, estradiol (E2), progesterone (P) secretions were suppressed in a dose-dependent manner by ovine (o) PRL in a plain Gey-Gey (G-G) buffer. Addition of 3-isobutyl-1-methyl-xanthine (IBMX) increased c-AMP accumulation as well as E2 and P secretions. Deletion of Ca++ from the IBMX buffer stimulated c-AMP production, but suppressed steroid secretion. The inhibitory effect of PRL on E2 and P was not demonstrated in IBMX buffer at any Ca++ concentration examined despite suppression of c-AMP production. In conclusion, it was demonstrated that PRL inhibited gonadotropin-stimulated production of E2 and P by inhibiting c-AMP production. IBMX stimulated accumulation of c-AMP, E2 and P and counteracted with the antigonadal effect of PRL. Ca++ inhibited c-AMP accumulation but stimulated E2 and P secretions. The data suggested that PRL exerts its antigonadal effect through an inhibition of adenylate cyclase action in a manner similar to that of Ca++.     

Estradiol stimulation of pituitary primordia transplanted to the kidney capsule

Summary The purpose of this study was to define better the influence of hormones on the normal and pathological development of the anterior pituitary using Rathke's pouch (RP)-derived model system. RP from either 12- or 15-day fetuses were microsurgically isolated and transplanted beneath the kidney capsule (KC) of intact adult hosts. Eighteen days later the hosts were hypophysectomized. Ten-12 days after hypophysectomy hosts were injected daily with either 1.0 g estradiol benzoate (EB) or 0.1 cc corn oil until necropsy at 80 days. Both 12- and 15-day RP differentiated into large, pars distalis tissues consisting of a variety of granulated and agranulated cell types, as well as large, secretory cysts. Cytodifferentiation was consistently most advanced in 15-day RP-derived grafts. Evidence of secretory granule synthesis, but not exocytosis, was apparent in granulated cells in oil-treated controls and EB-treated 12-day RP-derived grafts. Immunostaining and electron microscopy demonstrated hypertrophied somatotrophs and mammotrophs with numerous profiles of exocytosis in EB-treated 15-day RP-derived grafts. Mammotrophs and somatotrophs were infrequent and not well differentiated in 12-day RP-derived grafts whether EB- or oil-treated, nor in oil-treated 15-day RP-derived grafts. Radioimmunoassay demonstrated highest levels of plasma PRL in EB-treated 15-day RP-derived grafts. Implant invasiveness was noted only in EB-treated 12-day RP-derived grafts when basal laminae were disrupted or absent, and graft cells mixed with connective tissue elements.Results indicate that the Cytodifferentiation of pars distalis cell types derived from KC transplanted RP can be maintained to 80 days. Mammotrophs are especially well differentiated and responsive to EB treatment during this development. However, continued maintenance of a differentiated state by mammotrophs and somatotrophs appears to require the presence of host pituitary and/or end organ hormones as evidenced by the lack of maintenance in oil-treated controls. Furthermore, the loss of well defined tissue boundaries between host and graft tissues of EB-treated 12 day RP animals suggests tumorigenic transformation.Supported in part by Grant No. R01-CA-21426 from the National Cancer Institute, DHEW; California Division ACS Special Grant No. 851; HEW-NIH Grant CA-14089, Los Angeles County/USC Cancer Center; and GRS 53-5104-5283The authors gratefully acknowledge the technical assistance of Vivian Valentin, Alicia Thompson, Linda Melsek, Carolyn Tallent, and Lindsay Gilpin.     

Effects of S-petasin on corticosterone release in rats

Petasites hybridus is used in Chinese herbal medicine. S-petasin is a bioactive compound isolated from leaves or roots of Petasites hybridus. S-petasin has been used to relieve gastrointestinal pain, lung disease, and spasms of the urogenital tract. However, the side effect of S-petasin on endocrine systems are still not clear. This study explored the effects of S-petasin on the release of corticosterone in vivo and in vitro. An intravenous injection of S-petasin (10 microg/kg) decreased both basal and adrenocorticotropin (ACTH)-induced plasma corticosterone concentration in male rats. In vitro, S-petasin (3 x 10(-6) - 10(-4) M) caused a significant reduction of basal and ACTH-stimulated release of corticosterone from the enzymatically dispersed rat zona fasciculata-reticularis (ZFR) cells in a dose-dependent manner. In order to study possible mechanisms, ZFR cells were incubated with S-petasin (10(-5) M) in the presence or absence of forskolin (adenylate cyclase activator, 10(-6) - 10(-4) M), 8-Br-cAMP (a cAMP analogue, 10(-6) 10(-4) M), 25-OH-cholesterol (pregnenolone biosynthesis precursor, 10(-5) M) combined with trilostane (a blocker of 3beta-hydroxysteriod dehydrogenase, 3beta-HSD, 10(-6) M) and deoxycorticosterone (corticosterone biosynthesis precursor, 10(-9) - 10(-6) M) at 37 degrees C for 1h. The concentration of pregnenolone and corticosterone in media were measured by radioimmunoassay. The stimulatory effects of corticosterone secretion induced by forskolin (10(-5) - 10(-4) M), 8-Br-cAMP (10(-5) - 10(-4) M) and deoxycorticosterone (10(-7) - 10(-6) M) were reduced by S-petasin at 10(-5) M. The stimulatory effects of pregnenolone secretion induced by 25-OH-cholesterol combined with or without trilostane was reduced by S-petasin at 10(-5) M. These results suggest that S-petasin inhibits the production of corticosterone from rat ZFR cells in part through decreasing the activities of adenylyl cyclase, P450scc and 11beta-hydroxylase.