Yu-Chung Chiao

The role of cyclic AMP production, calcium channel activation and enzyme activities in the inhibition of testosterone secretion by amphetamine

1 The aim of this study was to investigate the mechanism by which amphetamine exerts its inhibitory effect on testicular interstitial cells of male rats. 2 Administration of amphetamine (10−12–10−6 M) in vitro resulted in a dose‐dependent inhibition of both basal and human chorionic gonadotropin (hCG, 0.05 iu ml−1)‐stimulated release of testosterone. 3 Amphetamine (10−9 M) enhanced the basal and hCG‐increased levels of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation in vitro (P<0.05) in rat testicular interstitial cells. 4 Administration of SQ22536, an adenylyl cyclase inhibitor, decreased the basal release (P<0.05) of testosterone in vitro and abolished the inhibitory effect of amphetamine. 5 Nifedipine (10−6 M) alone decreased the secretion of testosterone (P<0.01) but it failed to modify the inhibitory action of amphetamine (10−10–10−6 M). 6 Amphetamine (10−10–10−6 M) significantly (P<0.05 or P<0.01) decreased the activities of 3β‐hydroxysteroid dehydrogenase (3β‐HSD), P450c17, and 17‐ketosteroid reductase (17‐KSR) as indicated by thin‐layer chromatography (t.l.c.). 7 These results suggest that increased cyclic AMP production, decreased Ca2+ channel activity and decreased activities of 3β‐HSD, P450c17, and 17‐KSR are involved in the inhibition of testosterone production induced by the administration of amphetamine.

Inhibition of Testosterone Production by Propylthiouracil in Rat Leydig Cells

Abstract Propylthiouracil (PTU) is a thioamide drug used clinically to inhibit thyroid hormone production. However, PTU is associated with some side effects in different organs. In the present study, the acute and direct effects of PTU on testosterone production in rat Leydig cells were investigated. Leydig cells were isolated from rat testes, and an investigation was performed on the effects of PTU on basal and evoked-testosterone release, the functions of steroidogenic enzymes, including protein expression of cytochrome P450 side-chain cleavage enzyme (P450scc) and mRNA expression of the steroidogenic acute regulatory protein (StAR). Rat Leydig cells were challenged with hCG, forskolin, and 8-bromo-cAMP to stimulate testosterone release. PTU inhibited both basal and evoked-testosterone release. To study the effects of PTU on steroidogenesis, steroidogenic precursor-stimulated testosterone release was examined. PTU inhibited pregnenolone production (i.e., it diminished the function of P450scc in Leydig cells). In addition to inhibiting hormone secretion, PTU also regulated steroidogenesis by diminishing mRNA expression of StAR. These results suggest that PTU acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450scc function and StAR expression.

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, 3β‐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 3β‐HSD activity. J. Cell. Biochem. 73:563–572, 1999.

Effects of ovarian steroid hormones and thyroxine on calcitonin secretion in pregnant rats

In the present study, the roles of ovarian steroid hormones and thyroxine (T4) in regulating the secretion of calcitonin (CT) in pregnant rats were examined. The levels of plasma progesterone, pre- and post-CaCl2 plasma CT, and recovery time of plasma CT and calcium after calcium challenge were greatest in midterm pregnant rats. The levels of basal plasma progesterone, CT, calcium, and recovery time of plasma CT after calcium challenge were less in late pregnant rats, but basal plasma estradiol was highest in late pregnancy. The concentrations of plasma T4 were gradually decreased in rats during pregnancy. Regardless of the presence of estradiol, administration of progesterone in ovariectomized (Ovx) rats resulted in an increase of plasma T4 as well as the basal and calcium-induced secretion of CT. Administration of estradiol alone did not alter the CaCl2-induced levels but decreased the post-CaCl2 levels of plasma calcium in Ovx rats. The basal levels of plasma CT were decreased in Ovx rats treated with T4. These results suggest that the hypercalcitoninemia in midterm pregnant rats is due to an increased secretion of progesterone. Hypocalcitoninemia in late pregnant rats, however, is due in part to lower plasma calcium.

Regulation of testosterone secretion by prolactin in male rats.

The goal of this study was to characterize the mechanism by which hyperprolactinemia alters testosterone production in rat testicular interstitial cells (TICs). Hyperprolactinemia was induced by grafting 2 anterior pituitary (AP) glands under the subcapsular space of the kidney in experimental rats. Control rats were grafted with brain cortex (CX). Six weeks post‐grafting, rats were challenged with human chorionic gonadotropin (hCG) then, the changes in either plasma testosterone or luteinizing hormone was measured. Additionally, TICs were isolated and challenged in vitro with hCG or prolactin, and the testosterone release measured by radioimmunoassay. Further investigation in signal transduction as intracellular 3′:5′ cyclic adenosine monophosphate (cAMP) production was observed under a regulation of forskolin or SQ22536. After the challenge of hCG or GnRH, the AP‐grafted rats showed a suppressed response in testosterone release as compared to those in the CX‐grafted group. The in vitro data from the AP‐grafted rats compared to the CX‐grafted animals showed a diminished response in testosterone release upon hCG stimulation. Administration of forskolin or SQ22536 disclosed dysfunction of adenylate cyclase in TICs from the AP‐grafted rats. When 8‐Br‐cAMP was incubated with TICs, the testosterone production was lower in the AP‐grafted compared to the CX‐grafted group. These results suggest that in addition to adenylate cyclase dysfunction, inefficiency of post‐cAMP pathways are also involved in the hypogonadism elicited by hyperprolactinemia in rats. J. Cell. Biochem. 74:111–118, 1999.

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)‐stimulated secretion of testosterone and the production of adenosine 3′,5′‐cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin‐releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH‐stimulated LH release as well as hCG‐stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17β‐HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats. J. Cell. Biochem. 73:554–562, 1999.

Direct effects of propylthiouracil on testosterone secretion in rat testicular interstitial cells

The aim of this study was to investigate the mechanism by which propylthiouracil (PTU) exerts its inhibitory effects on the production of testosterone by rat testicular interstitial cells. The plasma testosterone concentration was decreased 60 and 120 min after an intravenous infusion of PTU (10 or 20 mg kg−1), but the concentration of plasma T4 was unaffected by the drug treatment. Exposure of anterior pituitary tissue to PTU (3–12 mM) in vitro did not affect either basal or gonadotropin‐releasing hormone (GnRH)‐stimulated luteinizing hormone (LH) release. PTU (3–12 mM) inhibited both the basal and the human chorionic gonadotropin (hCG, 0.05 iu ml−1)‐stimulated release of testosterone from rat testicular tissue in vitro; at the highest concentration tested (12 mM), it also inhibited the forskolin or 8‐bromo‐adenosine 3′:5′‐cyclic monophosphate (8‐Br‐cyclic AMP)‐stimulated release of testosterone. The 25‐OH‐cholesterol (10−7–10−5 M)‐stimulated release of pregnenolone and testosterone by the testicular interstitial cells was inhibited by PTU (12 mM, P<0.05). The results suggest that the inhibitory actions of PTU on testosterone secretion are exerted, at least in part, at the testicular level through a mechanism which is independent of thyroid status and which involves a reduction in P450scc activity and, hence, in the conversion of cholesterol to pregnenolone.

Inhibition of salmon calcitonin on secretion of progesterone and GnRH-stimulated pituitary luteinizing hormone

The effects of salmon calcitonin (sCT) on the production of progesterone and secretion of luteinizing hormone (LH) were examined in female rats. Diestrous rats were intravenously injected with saline, sCT, human chorionic gonadotropin (hCG), or hCG plus sCT. Ovariectomized (Ovx) rats were injected with saline or sCT. In the in vitro experiments, granulosa cells and anterior pituitary glands (APs) were incubated with the tested drugs. Plasma LH levels of Ovx rats were reduced by sCT injection. Administration of sCT decreased the basal and hCG-stimulated progesterone release in vivo and in vitro. 8-Bromo-cAMP dose dependently increased progesterone production but did not alter the inhibitory effect of sCT. H-89 did not potentiate the inhibitory effect of sCT. Higher doses of 25-hydroxycholesterol and pregnenolone stimulated progesterone production and diminished the inhibitory effects of sCT. sCT did not decrease basal release of LH by APs, but pretreatment of sCT decreased gonadotropin-releasing hormone (GnRH)-stimulated LH secretion. These results suggested that sCT inhibits progesterone production in rats by preventing the stimulatory effect of GnRH on LH release in rat APs and acting directly on ovarian granulosa cells to decrease the activities of post-cAMP pathway and steroidogenic enzymes.

Involvement of Cholecystokinin (CCK) Receptor in the Adaptation of Gastric Emptying Induced by Adrenocorticotropin (ACTH) in Male Rats

It has been shown that gastric emptying is delayed in stressed animals. This investigation was to study the effect of adrenocorticotropin (ACTH), a stress hormone, on the gastric emptying and intestinal transit in rats. Fasted male rats were injected intraperitoneally (i.p.) with saline, ACTH (10 or 20 μg/ml/kg), lorglumide (a selective potent cholecystokinin A receptor antagonist, 10 μg/ml/ kg) or ACTH (20 μg/ml/kg) and lorglumide (10 μg/ml/kg) at 30 min before decapitation. All rats were orally ingested with liquid meal containing radioactive Na_2 ^(51)CrO_4 (0.5 μCi/ml) and 10% charcoal at 15 min before decapitation, and blood samples were collected. Administration of ACTH significantly increased the concentrations of plasma corticosterone, but decreased the levels of plasma cholecystokinin (CCK) and gastric emptying as well as intestinal transit in rats. The decreased gastric emptying, but not intestinal transit, was reversed by the treatment of lorglumide. These results suggested that CCKA receptor was involved in the inhibitory effect of ACTH on the gastric emptying in rats.

Involvement of Cholecystokinin (CCK) Receptor in the Regulation of Dexamethasone on Gastric Emptying in Male Rats

Dexamethasone is a potent glucocorticoid, which has been used in the anti-inflammation therapy. It has been well known that the gastrointestinal (GI) motility is affected by stress. However, the role of glucocorticoids in regulating GI motility is unclear. The purpose of this study was to investigate the effect and action mechanism of dexamethasone on the GI motility. In the first study, the male rats were injected intraperitoneally (i.p.) with saline or dexamethasone (15 or 30 μg/ml/kg) 1 h before decapitation. In the second study, dexamethasone (30 μg/ml/kg) and lorglumide (a selective cholecystokinin A receptor, CCKA, antagonist) were injected intraperitoneally 60 min and 30 min prior to decapitation, respectively. All experimental animals were orally ingested with radioactive Na_2^(51)CrO_4 containing 10% charcoal by a PE-205 tubing into the stomach after a 24 h fast and then decapitated 15 min later. The small intestine was divided equally into ten segments. The radioactivity in stomach and each segment of intestine was counted by a gamma-counter. The ratios of gastric emptying and charcoal transit were calculated. Blood samples were collected. The concentrations of CCK and corticosterone in plasma samples were measured by radioimmunoassay, and those of serum adrenocorticotropin (ACTH) were measured by an enzyme immunoassay (EIA) kit. All data were expressed as mean ± s. e. mean, and analyzed by the analysis of variance (ANOVA). The differences between specific means were analyzed by Dunnetts test. The results demonstrate that dexamethasone dose dependently inhibited the level of serum ACTH and the gastric emptying, and the intestinal transit is also inhibited by dexamethasone at the dose of 15 μg/ml/kg. Plasma CCK and corticosterone concentrations were dose-dependently decreased by dexamethasone injection. Furthermore, after injection of lorglumide, the decreased gastric emptying caused by dexamethasone was restored to the control level. These results suggested that dexamethasone-induced inhibition of gastric emptying and intestinal transit is mediated by an action on CCKA receptor.