Chien-Chen Lu

Effects of estradiol on aldosterone secretion in ovariectomized rats.

The effects of estradiol benzoate (EB) on steroidogenesis in rat zona fasciculata‐reticularis (ZFR) cells were studied. Female rats were ovariectomized (Ovx) for 2 weeks and then injected subcutaneously with oil or EB for 3 days before decapitation. ZFR cells were isolated and incubated with adrenocorticotropin (ACTH) or prolactin (PRL) for 1 h. Corticosterone concentrations in plasma and cell media, and adenosine 3′,5′‐cyclic monophosphate (cAMP) production in ZFR cells were determined by radioimmunoassay. The effects of EB replacement in vivo on the activities of steroidogenic enzymes in ZFR cells were measured by the amounts of intermediate steroidal products separated by thin‐layer chromatography. Replacement of EB in vivo resulted in a dose‐dependent increase of plasma PRL and corticosterone in Ovx rats. The basal, ACTH‐, and PRL‐stimulated release of corticosterone by ZFR cells was greater in EB‐ than in oil‐treated animals. Forskolin‐induced production of cAMP was greater in the EB‐replaced rats than in oil‐treated animals, which correlated with the increase of corticosterone production. The 3‐isobutyl‐l‐methylxanthine (IBMX) plus ACTH‐, IBMX plus PRL‐, and forskolin plus PRL‐stimulated productions of cAMP were higher in EB‐ than in oil‐treated rats. The enzyme activities of postpregnenolone were not affected by EB replacement in Ovx rats. These results suggest that the EB‐related increase of corticosterone production in Ovx rats is associated with an increase of cAMP generation and the stimulatory effect of PRL on ZFR cells. J. Cell. Biochem. 77:560‐568, 2000.

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.

Disturbed Small Intestinal Motility in the Late Rat Pregnancy

We investigated whether various periods of pregnancy might disturb rat gastrointestinal motility. When the proestrus of female rats occurred, they were housed with male rats. Motility studies were conducted on day 7 (first period), day 14 (second) and day 21 (third) of pregnancy, respectively. After the orogastric feeding of radiochromium marker, rats were sacrificed 15 min later. Gastric emptyings of pregnant rats measured at various periods did not differ from the nonpregnant diestrus controls. The geometric center represented intestinal transits in the first, second and third periods of pregnancy and controls were (mean ±SEM) 4.54 ± 0.25, 4.47 ± 0.17, 3.61 ± 0.27 and 4.98 ± 0.13, respectively (p < 0.01) while their plasma progesterone levels were 15.6 ± 2.6, 18 ± 1.4, 7.1 ± 0.5 and 8.6 ± 0.4 ng/ml, respectively (p < 0.01). This shows that late pregnancy inhibits small intestinal transit, whereas gastric emptying remains unchanged. Altered progesterone during pregnancy is not a main mediator to disturb intestinal transit.

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.

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.

Effects of aging on erythropoietin secretion in female rats

The secretion of erythropoietin (EPO) in old (22 25 months), middle (mid)-aged (15-17 months), adult (6-7 months), and young (3 months) female rats was studied. Rats of different ages were bled by cardiac puncture for measuring plasma EPO, estradiol, and progesterone levels. The metabolic clearance rate of EPO was determined by a single-injection method. The effects of ovariectomy and replacement of estradiol (E) and/or progesterone (P) on plasma EPO concentration were also investigated. Analysis of direct effects of E and/or P on EPO from kidney tissues was carried out in an in vitro study. Plasma EPO levels were higher in both mid-aged and old rats than in young rats (P < 0.01). The metabolic clearance rate of EPO was also greater in adult, mid-aged and old rats than in young rats (P < 0.01). Ovariectomy increased rat plasma EPO concentration which might be inhibited by E (P < 0.01), P (P < 0.01), or E and P (P < 0.05) replacement. Neither E (10(-10) or 10(-9) M) nor P (10(-8) or 10(-7) M) alone altered EPO release from kidney tissue in vitro. Combination of E (10(-10) M) and P (10(-8) x or 10(-7) M) stimulated the EPO release from kidney tissue (P < 0.01). These data suggest that the mechanism of the secretion of EPO by kidneys in female rats during aging is ovarian steroid hormones independent.

Age-related differences in the secretion of calcitonin in female rats

The mechanism that causes hypercalcitonemia in female rats and is associated with aging was investigated. Young (3 mo), adult (8 mo), middle-aged (12 mo), and old (21 mo) rats were infused with CaCl2 and were bled from a jugular catheter after a CaCl2 challenge. To mimic some of the hormonal changes caused by aging, the anterior pituitary (AP)-grafted ovariectomized rats with hyperprolactinemic syndrome were used to mimic the physiological status of aging. The rat thyroid gland was incubated with or without ovine prolactin (oPRL; 40 or 80 ng/ml) at 37 degreesC for 30 min. Old rats possessed the lowest levels of plasma estradiol and progesterone yet had the highest levels of plasma prolactin and calcitonin (CT) compared with young, adult, and middle-aged rats. The basal release of thyroid CT in vitro in thyroid glands gradually increased with age. Compared with cortex (CX)-grafted rats, the AP-grafted rats possessed higher levels of plasma PRL, basal and CaCl2-induced levels of plasma CT, and the release of thyroid CT in thyroid glands. After stimulation with oPRL, the in vitro release of thyroid CT increased in both CX- and AP-grafted rats. These results suggest that the hypersecretion of CT in old rats is due at least in part to hyperprolactinemia.The mechanism that causes hypercalcitonemia in female rats and is associated with aging was investigated. Young (3 mo), adult (8 mo), middle-aged (12 mo), and old (21 mo) rats were infused with CaCl2 and were bled from a jugular catheter after a CaCl2 challenge. To mimic some of the hormonal changes caused by aging, the anterior pituitary (AP)-grafted ovariectomized rats with hyperprolactinemic syndrome were used to mimic the physiological status of aging. The rat thyroid gland was incubated with or without ovine prolactin (oPRL; 40 or 80 ng/ml) at 37°C for 30 min. Old rats possessed the lowest levels of plasma estradiol and progesterone yet had the highest levels of plasma prolactin and calcitonin (CT) compared with young, adult, and middle-aged rats. The basal release of thyroid CT in vitro in thyroid glands gradually increased with age. Compared with cortex (CX)-grafted rats, the AP-grafted rats possessed higher levels of plasma PRL, basal and CaCl2-induced levels of plasma CT, and the release of thyroid CT in thyroid glands. After stimulation with oPRL, the in vitro release of thyroid CT increased in both CX- and AP-grafted rats. These results suggest that the hypersecretion of CT in old rats is due at least in part to hyperprolactinemia.