Jiann-Jong Chen

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.

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 prolactin on aldosterone secretion in rat zona glomerulosa cells

Acute effects and action mechanisms of prolactin (PRL) on aldosterone secretion in zona glomerulosa (ZG) cells were investigated in ovariectomized rats. Administration of ovine PRL (oPRL) increased aldosterone secretion in a dose‐dependent manner. Incubation of [3H]‐pregnenolone combined with oPRL increased the production of [3H]‐aldosterone and [3H]‐deoxycorticosterone but decreased the accumulation of [3H]‐corticosterone. Administration of oPRL produced a marked increase of adenosine 3′,5′‐cyclic monophosphate (cAMP) accumulation in ZG cells. The stimulatory effect of oPRL on aldosterone secretion was attenuated by the administration of angiotensin II (Ang II) and high potassium. The Ca2+ chelator, ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA, 10−2 M), inhibited the basal release of aldosterone and completely suppressed the stimulatory effects of oPRL on aldosterone secretion. The stimulatory effects of oPRL on aldosterone secretion were attenuated by the administration of nifedipine (L‐type Ca2+ channel blocker) and tetrandrine (T‐type Ca2+ channel blocker). These data suggest that the increase of aldosterone secretion by oPRL is in part due to (1) the increase of cAMP production, (2) the activation of both L‐ and T‐type Ca2+ channels, and (3) the activation of 21‐hydroxylase and aldosterone synthase in rat ZG cells. J. Cell. Biochem. 72:286–293, 1999.

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 inhibitory effect of digitalis on progesterone release from rat granulosa cells

Digoxin (10−7 – 10−5 M) or digitoxin (10−7 – 10−5 M) decreased the basal and human chorionic gonadotropin (hCG)‐stimulated release of progesterone from rat granulosa cells. Digoxin (10−5 M) or digitoxin (10−5 M) attenuated the stimulatory effects of forskolin and 8‐bromo‐cyclic 3′ : 5′‐adenosine monophosphate (8‐Br‐cAMP) on progesterone release from rat granulosa cells. Digoxin (10−5 M) or digitoxin (10−5 M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450scc) activity (conversion of 25‐hydroxyl cholesterol to pregnenolone) in rat granulosa cells but did not influence the activity of 3β‐hydroxysteroid dehydrogenase (3β‐HSD). Neither progesterone production nor P450scc activity in rat granulosa cells was altered by the administration of ouabain. Digoxin (10−5 M) or digitoxin (10−5 M), but not ouabain, decreased the expression of P450scc and steroidogenic acute regulatory (StAR) protein in rat granulosa cells. The present results suggest that digoxin and digitoxin decrease the progesterone release by granulosa cells via a Na+,K+‐ATPase‐independent mechanism involving the inhibition of post‐cyclic AMP pathway, cytochrome P450scc and StAR protein functions.

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.

Progesterone attenuates the inhibitory effects of cardiotonic digitalis on pregnenolone production in rat luteal cells.

Previous studies have shown that digoxin decreases testosterone secretion in testicular interstitial cells. However, the effect of digoxin on progesterone secretion in luteal cells is unclear. Progesterone is known as an endogenous digoxin‐like hormone (EDLH). This study investigates how digitalis affected progesterone production and whether progesterone antagonized the effects of digitalis. Digoxin or digitoxin, but not ouabain, decreased the basal and human chorionic gonadotropin (hCG)‐stimulated progesterone secretion as well as the activity of cytochrome P450 side chain cleavage enzyme (P450scc) in luteal cells. 8‐Br‐cAMP and forskolin did not affect the reduction. Neither the amount of P450scc, the amount of steroidogenic acute regulatory (StAR) protein, nor the activity of 3β‐hydroxysteroid dehydrogenase (3β‐HSD) was affected by digoxin or digitoxin. Moreover, in testicular interstitial and luteal cells, progesterone partially attenuated the reduction of pregnenolone by digoxin or digitoxin and the progesterone antagonist, RU486, blocked this attenuation. These new findings indicated that (1) digoxin or digitoxin inhibited pregnenolone production by decreasing the activity of P450scc enzyme, but not Na+–K+‐ATPase, resulting in a decrease on progesterone secretion in rat luteal cells, and (2) the inhibitory effect on pregnenolone production by digoxin or digitoxin was reversed partially by progesterone. In conclusion, digoxin or digitoxin decreased progesterone production via the inhibition of pregnenolone by decreasing P450scc activity. Progesterone, an EDLH, could antagonize the effects of digoxin or digitoxin in luteal cells. J. Cell. Biochem. 86: 107–117, 2002.

Bacterial lipopolysaccharide activates protein kinase C, but not intracellular calcium elevation, in human peripheral T cells

The increase of intracellular free calcium concentration ([Ca2+]i) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human peripheral T cells. Bacterial lipopolysaccharide (LPS) is nonmitogenic in human T cells. However, in the presence of monocytes, LPS becomes mitogenic to proliferate T cells. The aim of this study was to define the incompetency of LPS on two mitogenic signals in human peripheral T cells. T cells were isolated from human peripheral blood. [Ca2+]i and pHi were determined by loading the cells with the fluorescent dyes, Fura‐2 acetoxymethyl ester (Fura‐2/AM) and 2′,7′‐bis(2‐carboxyethyl)‐5‐(and 6)carboxyfluorescein acetoxymethyl ester (BCECF/AM). PKC activity was determined by protein kinase assay and cell proliferation was estimated from the incorporation of [3H]‐thymidine. The results indicated that (1) LPS (10 μg/ml) stimulated PKC activity significantly within 5 min, reached a plateau at 30 min, and maintained that level for at least 2 h; and (2) LPS stimulated cytoplasmic alkalinization but did not affect the levels of [Ca2+]i and [3H]‐thymidine incorporation into T cells. Moreover, the combination of calcium ionophore A23187 with LPS significantly stimulated [3H]‐thymidine incorporation into T cells. Thus, the results demonstrate that LPS failed to proliferate T cells, probably because of a lack of the machinery necessary to stimulate the mitogenic signal on [Ca2+]i elevation. J. Cell. Biochem. 76:404–410, 2000.