GABAergic modulation of ventilatory response to acute and sustained hypoxia in obese Zucker rats.

OBJECTIVE: To determine whether altered central and/or peripheral gamma-aminobutyric acid (GABA)ergic mechanisms acting in GABA(A) receptors contribute to the abnormal ventilatory response to acute and sustained hypoxia in obese Zucker rats. METHODS: In all, 10 lean and 10 obese Zucker rats were studied at 12 weeks of age. Ventilation (V(.-)(E)), tidal volume (V(T)), and breathing frequency (f) during room air breathing and in response to sustained (30 min) hypoxic (10% O(2)) challenges were measured on three separate occasions by the barometric method following the randomized blinded administration of equal volumes of DMSO (vehicle), bicuculline methiodide (B(M), 1 mg/kg, peripheral GABA(A) receptor antagonist), or bicuculline hydrochloride (B(HCl), 1 mg/kg, peripheral and central GABA(A) receptor antagonist). RESULTS: Administration of B(M) and B(HCl) in lean animals had no effect on ventilation either during room air breathing or 30 min of sustained exposure to hypoxia. Similarly, B(M) failed to alter ventilation in obese rats. In contrast, B(HCl) significantly (P<0.05) increased V(.-)(E) and V(T) during room air breathing and 10-30 min of hypoxic exposure in obese rats. During 5 min of acute hypoxic exposure, V(T) remained elevated with B(HCl) in obese rats, but the V(.-)(E) appeared not to be increased with B(HCl) due to a decrease in f. CONCLUSION: Thus, endogenous GABA modulates both ventilation during room air breathing and ventilatory response to sustained hypoxia in obese, not in lean, Zucker rats by acting specifically on GABA(A) receptors located within the central, not peripheral, nervous system. However, endogenous GABA does not modulate ventilation but the pattern of breathing during acute hypoxia in obesity in a different manner from that during sustained hypoxia.

Aging effects on the secretion of corticosterone in male rats.

BACKGROUND: The effects of aging on zona fasciculatareticularis (ZFR) cell function in male rats were studied. METHODS: Male rats 3, 6, and 22 months of age were divided into three groups, and collagenase-dispersed ZFR cells were isolated and incubated with adrenocorticotropin (ACTH), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), ovine prolactin (oPRL), deoxycorticosterone (DOC), or 3-isobutyl-l-methylxanthine (IBMX) at 37 degrees C for 1 hour. Corticosterone concentrations in cell media and cAMP production in ZFR cells were measured by radioimmunoassay. Protein expression of PRL receptor in ZFR cells were analyzed by Western blot. RESULTS: The basal levels of plasma and medium corticosterone were higher in 22-month-old than in 3-month-old rats. In contrast, the release of corticosterone in response to ACTH, 8-Br-cAMP, and DOC was lower in 22-month-old than in 3- and 6-month-old rats. Aging decreased the oPRL-stimulated release of corticosterone but increased the protein expression of PRL receptor in ZFR cells. The basal levels of intracellular cAMP increased with age. However, the ACTH-stimulated production of intracellular cAMP decreased in 22-month-old compared with 3- or 6-month-old rats. The increment of cAMP accumulation in ZFR cells after administration of IBMX was greater in 22-month-old than in 3- or 6-month-old rats. CONCLUSIONS: These results suggest that the aging effects on the production of corticosterone in rat ZFR cells is associated with change of the generation of cAMP, the activity of 11 beta-hydroxylase and the protein expression of PRL receptor.

Effects of estradiol on corticosterone 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.

Effects of thyroid hormones on the release of calcitonin gene-related peptide (CGRP) by rat prostate glands in vitro.

It has been well known that calcitonin (CT) and calcitonin gene-related peptide (CGRP) are derived from the CT/CGRP gene which is localized in chromosome 11. CGRP is a 37-amino acid neuropeptide expressed predominantly in the nervous system and is one of the most potent endogenous vasodilatory peptides that have been found. Only few reports described the distribution of CGRP in reproductive organs. Moreover, the hormonal regulation of CGRP secretion is still not clear. The present study was designed to examine the presence of CGRP in rat prostates and the direct effect of thyroxine (T4) on the release of CGRP by rat prostate glands in vitro. Male rats were thyroidectomized (Tx) or sham Tx for two weeks before decapitation. The ventral prostate glands were either extracted by phosphate buffer saline or bisected and preincubated with Locke's solution containing 10 mM glucose, 0.03% bacitracin, and 0.05% Hepes at 37 degrees C for 90 min. The hemi-prostate tissues were then incubated with Locke's medium containing T4 (0 to approximately 10(-7) M) for 1 hr. After incubation, the medium was collected, and the prostate tissues were weighed. The concentration of CGRP in both medium and prostate tissue extracts were measured by a specific radioimmunoassay (RIA) developed in our laboratory. Incubation of T4 at 10(-9) M was effective to increase the release of CGRP in rat prostate glands. Incubation of rat prostate glands with T4 at 10(-7) M resulted in a maximal release of CGRP (270% of the basal). These results suggest that thyroid hormones increase CGRP release by acting directly on rat prostate glands.

Inhibition of aldosterone production by testosterone in male rats.

In vivo and in vitro experiments were designed to assess the effect of testosterone on aldosterone secretion in male rats. Orchidectomized rats were injected subcutaneously with oil or testosterone propionate ([TP] 2 mg/kg) for 7 days. Intact rats were injected with oil only. The results indicate that the plasma aldosterone level was higher in orchidectomized versus intact and TP-replaced rats. In the in vitro study, testosterone caused a marked decrease of aldosterone secretion by zona glomerulosa (ZG) cells, but failed to alter the accumulation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Testosterone significantly decreased the corticotropin (ACTH)-stimulated production of aldosterone and accumulation of cAMP in rat ZG cells. The conversion of corticosterone to aldosterone and of 25-OH-cholesterol to pregnenolone, as well as angiotensin II (ANG II)-stimulated production of aldosterone, were decreased by testosterone. These results suggest that testosterone inhibits the basal and ANG II- and ACTH-stimulated release of aldosterone, via inhibition of aldosterone synthase activity and cytochrome P-450 side-chain cleavage (P450scc) activity, and ACTH-stimulated cAMP accumulation in rat ZG cells.

Age-related differences in corticosterone secretion in female rats.

The effects of age on steroidogenesis in rat zona fasciculata-reticularis (ZFR) cells were studied. Young, adult, and middle-aged rats were ovariectomized (Ovx) and received replacement therapy with oil or estradiol benzoate ([EB] 25 microg/mL/kg). Rat ZFR cells were incubated with corticotropin (ACTH), prolactin (PRL), or forskolin at 37 degrees C for 1 hour. The effects of age on the activity of steroidogenic enzymes of ZFR cells were measured by the amount of intermediate steroidal products separated by thin-layer chromatography. Plasma levels were higher for PRL (54% to 254%) and corticosterone (179% to 257%) in middle-aged versus young rats. In oil-treated Ovx rats, basal and ACTH-stimulated corticosterone release by ZFR cells were also greater in middle-aged compared with young rats. Replacement with EB in Ovx rats increased the ACTH-stimulated release of corticosterone. Administration of ovine PRL in vitro resulted in a dose-dependent increase of corticosterone production. In oil-treated middle-aged rats, ovine PRL-stimulated corticosterone release was higher than in young rats. Forskolin-induced production of cyclic adenosine 3',5'-monophosphate (cAMP) was greater in middle-aged versus young rats and correlated with the increase of corticosterone production. The activity of steroidogenic enzymes in rat ZFR cells was unchanged by age. These results suggest that the age-related increase of corticosterone production in female rats is associated with the stimulatory effect of PRL on ZFR cells and is due in part to an increase of cAMP generation.

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.

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(beta-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.

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.

Pharmacological effects of propylthiouracil on corticosterone secretion in male rats.

BACKGROUND: We investigated the direct effects of propylthiouracil (PTU) on corticosterone secretion both in vivo and in vitro. METHODS: Male rats were divided into 4 groups and then injected subcutaneously with saline, PTU, PTU plus thyroxine (T4), or T4 once daily for 2 weeks. After 2 weeks, rats were decapitated or received adrenocorticotropic hormone (ACTH), intravenously. Zona fasciculata-reticularis (ZFR) cells from normal, saline-, PTU-, PTU plus T4-, or T4-treated rats were incubated with ACTH, forskolin, 8-Br-cAMP, deoxycorticosterone (DOC) +/- PTU (1, 2, or 5 mg/mL) at 37 degrees C for 2 hours. Corticosterone concentrations in plasma and cell media, and 3':5'-cyclic adenosine monophosphate (cAMP) production in ZFR cells were determined by radioimmunoassay. The effects of PTU on the activities of steroidogenic enzymes in ZFR cells were measured by the amounts of intermediate steroidal products separated by thin-layer chromatography. RESULTS: The basal and ACTH-stimulated levels of plasma corticosterone in PTU-treated rats were lower as compared to saline-treated animals. Both basal and ACTH-stimulated corticosterone secretion were inhibited by PTU > 2 mg/mL in rat ZFR cells. The cAMP production induced by forskolin was lower in PTU, PTU plus T4, or T4-treated rats than in saline-treated animals. Chronic administration of PTU or PTU plus T4 inhibited the 3 beta-hydroxysteroid dehydrogenase, 21 beta-hydroxylase, and 11 beta-hydroxylase activities. Administration of PTU (1, 2, and 5 mg/mL) suppressed the basal, ACTH, 8-Br-cAMP, forskolin, and DOC-stimulated corticosterone secretion in rat ZFR cells. Likewise, PTU > 2 mg/mL inhibited the ACTH and 8-Br-cAMP-stimulated levels of intracellular cAMP in rat ZFR cells. CONCLUSIONS: These results suggest that PTU counteracts both basal and ACTH-induced adrenal steroidogenesis through their attenuation of the activity of 11 beta-hydroxylase and cAMP production in rat ZFR cells.

Acute effects of thyroid hormones on the production of adrenal cAMP and corticosterone in male rats.

The acute effects of thyroid hormones on glucocorticoid secretion were studied. Venous blood samples were collected from male rats after they received intravenous 3,5,3'-triiodothyronine (T3) or thyroxine (T4). Zona fasciculata-reticularis (ZFR) cells were treated with adrenocorticotropic hormone (ACTH), T3, T4, ACTH plus T3, or ACTH plus T4 at 37 degrees C for 2 h. Corticosterone concentrations in plasma and cell media, and also adenosine 3',5'-cyclic monophosphate (cAMP) production in ZFR cells in the presence of 3-isobutyl-1-methylxanthine, were determined. The effects of thyroid hormones on the activities of steroidogenic enzymes of ZFR cells were measured by the amounts of intermediate steroidal products separated by thin-layer chromatography. Administration of T3 and T4 suppressed the basal and the ACTH-stimulated levels of plasma corticosterone. In ZFR cells, both thyroid hormones inhibited ACTH-stimulated corticosterone secretion, but the basal corticosterone was inhibited only with T3 > 10(-10) M or T4 > 10(-8) M. Likewise, T3 or T4 at 10(-7) M inhibited the basal- and ACTH-stimulated levels of intracellular cAMP. Physiological doses of T3 and T4 decreased the activities of 3 beta-hydroxysteroid dehydrogenase, 21-hydroxylase, and 11 beta-hydroxylase. These results suggest that thyroid hormones counteract ACTH in adrenal steroidogenesis through their inhibition of cAMP production in ZFR cells.

Glucocorticoids and aging.

In this review we analyze the morphologic changes, hypothalamic-pituitary-adrenal (HPA) axis functions, glucocorticoid (GC) receptors, and steroidogenic enzyme activities in both animals and humans during aging. In rodent studies, older animals tend to show: 1) hypertrophy of adrenal zona fasciculata (ZF) cells; 2) neuronal loss in the hypothalamic area; 3) loss of GC receptors in the hippocampus; 4) raised circulating adrenocorticotropic hormone (ACTH) and GC levels, and increased release of corticotropin-releasing hormone from the hypothalamus; 5) reduced suppression of endogenous GC secretion after administration of dexamethasone; 6) decreased attenuation of response to chronic stress; and 7) increased activity of P450scc and 21-hydroxylase. According to the GC cascade hypothesis, stress and GCs facilitate the aging process in rats. Stress induces downregulation of GC receptors in the hippocampus, then impairs GC feedback on stress-induced HPA axis activation. Finally, an increase in the basal level of corticosterone and extended GC secretion following stress occurs. Because activation of the hippocampus decreases HPA axis function, the unrestrained elevation of GC concentration and the reduction in the level of GC receptors in the hippocampus may gradually weaken the feedback mechanisms and halt the response to stress. In humans, there are conflicting reports of HPA axis function during aging, so it is difficult to make a final conclusion regarding the relationship between aging and HPA axis function.

Inhibition of corticosterone secretion by thyroxine in male rats.

The effects of thyroxine (T4) on the secretion of corticosterone both in vivo and in vitro in male rats were studied. Rats were thyroidectomized (Tx) or sham Tx. The Tx rats were subcutaneously with T4 (20 micrograms/kg) or saline once daily for two weeks. In an in vitro experiment, adrenal glands were incubated with ACTH, T4, or ACTH plus T4 in the presence or absence of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) at 37 degrees C for 60 min. Medium and ether-extracted plasma samples were analyzed for corticosterone by radioimmunoassay (RIA). The accumulation of cyclic adenosine monophosphate (cAMP) in adrenal tissues after incubation with IBMX was measured by RIA. The levels of plasma corticosterone in Tx rats were significantly increased as compared with euthyroid rats. T4 replacement in Tx rats restored plasma corticosterone to euthyroid level. Administration of T4 in vitro resulted in an inhibition of both basal and ACTH-stimulated release of corticosterone. Both basal and ACTH-stimulated generations of cAMP in adrenal tissues were decreased by T4. These results suggest that T4 inhibits the spontaneous and ACTH-stimulated secretion of corticosterone by acting directly at adrenal glands via a decrease in cAMP production.

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 3beta-hydroxysteroid dehydrogenase (3beta-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 3beta-HSD, P450c17, and 17-KSR are involved in the inhibition of testosterone production induced by the administration of amphetamine.

Oxidative modification of DNA bases in rat liver and lung during chemical carcinogenesis and aging.

The extent of DNA modification in cancerous rat live and lung tissues was investigated and compared to their respective normal tissues. Liver tumors were induced by 2-fluorenylacetamide (2-FAA) or N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA), and lung tumors were induced by sodium nitrite plus trimethylamine. In the DNA samples isolated from these tissues, two pyrimidine-derived and four purine-derived modified DNA bases were identified and quantified by gas chromatography/mass spectrometry with selected-ion monitoring. These compounds were characterized as 5-hydroxyuracil (5-OHUra), thymine glycol (TG), 4,6-diamino-5-formamidopyrimidine (FapyAde), 2,6-diamino-4-hydroxy-5- formamidopyrimidine (FapyGua), 8-hydroxyadenine (8-OHAde), and 8-hydroxyguanine (8-OHGua). Elevated amounts of modified DNA bases were found in most cancerous tissues when compared to the controls. Chemicals used for tumor induction were responsible for inducing DNA lesions that could be promutagenic in vivo and could lead to various types of mutations. When endogenous oxidative damage to DNA during aging was examined, a roughly 2-fold increase of thymine glycol, 8-OHAde and 8-OHGua was found in aged (12 months) rat liver tissues compared to young tissues (1 month). The same results were also found in lung tissues, except that the amount of thymine glycol exhibited more than a 10-fold increase in aged tissues when compared to young tissues. The association of the modified bases with the processes of aging and carcinogenesis deserves further investigation.