Finite temperature string by K-means clustering sampling with order parameters as collective variables for molecular crystals: application to polymorphic transformation between ß-CL-20 and ε-CL-20.

Polymorphic transformation of molecular crystals is a fundamental phase transition process, and it is important practically in the chemical, material, biopharmaceutical, and energy storage industries. However, understanding of the transformation mechanism at the molecular level is poor due to the extreme simulating challenges in enhanced sampling and formulating order parameters (OPs) as the collective variables that can distinguish polymorphs with quite similar and complicated structures so as to describe the reaction coordinate. In this work, two kinds of OPs for CL-20 were constructed by the bond distances, bond orientations and relative orientations. A K-means clustering algorithm based on the Euclidean distance and sample weight was used to smooth the initial finite temperature string (FTS), and the minimum free energy path connecting ß-CL-20 and ε-CL-20 was sketched by the string method in collective variables, and the free energy profile along the path and the nucleation kinetics were obtained by Markovian milestoning with Voronoi tessellations. In comparison with the average-based sampling, the K-means clustering algorithm provided an improved convergence rate of FTS. The simulation of transformation was independent of OP types but was affected greatly by finite-size effects. A surface-mediated local nucleation mechanism was confirmed and the configuration located at the shoulder of potential of mean force, rather than overall maximum, was confirmed to be the critical nucleus formed by the cooperative effect of the intermolecular interactions. This work provides an effective way to explore the polymorphic transformation of caged molecular crystals at the molecular level.

A Novel Rhynchophylline Analog, Y396, Inhibits Endothelial Dysfunction Induced by Oxidative Stress in Diabetes Through Epidermal Growth Factor Receptor.

Aims: Endothelial dysfunction appears in early diabetes mellitus partially because of epidermal growth factor receptor (EGFR) abnormal activation and downstream oxidative stress. The aim of this study was to determine whether Y396, a synthesized analog of rhynchophylline, could protect against endothelial dysfunction in diabetes and the underlying molecular mechanism. Results: Y396 could directly target the EGFR and inhibit its phosphorylation induced by high glucose and EGF, downstream translocation to the nucleus of E2F1, and its transcriptional activity and expression of Nox4. Diabetes-induced endothelium malfunction was ameliorated by Y396 treatment through EGFR inhibition. Downstream oxidative stress was decreased by Y396 in the aortas of type 1 diabetes mellitus mice and primary rat aorta endothelial cells (RAECs). Y396 could also ameliorate tunicamycin-induced oxidative stress in the aorta and RAECs. In addition, we again determined the protective effects of Y396 on high-fat diet/streptozotocin-induced type 2 diabetes mellitus. Innovation: This is the first study to demonstrate that Y396, a novel rhynchophylline analog, suppressed high-glucose-induced endothelial malfunction both in vivo and in vitro by inhibiting abnormal phosphorylation of EGFR. Our work uncovered EGFR as a novel therapeutic target and Y396 as a potential therapy against diabetes-induced complication. Conclusion: Y396 could directly bind with EGFR, and inhibit its phosphorylation and downstream E2F1 transcriptional activity. It could also preserve tunicamycin-evoked endothelial dysfunction and oxidative stress. It could protect against diabetes-induced endothelium malfunction in vivo through EGFR inhibition and downstream oxidative stress. Antioxid. Redox Signal. 32, 743-765.

ZYZ-803, a novel hydrogen sulfide-nitric oxide conjugated donor, promotes angiogenesis via cross-talk between STAT3 and CaMKII.

Endothelial angiogenesis plays a vital role in recovery from chronic ischemic injuries. ZYZ-803 is a hybrid donor of hydrogen sulfide (H2S) and nitric oxide (NO). Previous studies showed that ZYZ-803 stimulated endothelial cell angiogenesis both in vitro and in vivo. In this study, we investigated whether the signal transducer and activator of transcription 3 (STAT3) and Ca2+/CaM-dependent protein kinase II (CaMKII) signaling was involved in ZYZ-803-induced angiogenesis. Treatment with ZYZ-803 (1 µM) significantly increased the phosphorylation of STAT3 (Tyr705) and CaMKII (Thr286) in human umbilical vein endothelial cells (HUVECs), these two effects had a similar time course. Pretreatment with WP1066 (STAT3 inhibitor) or KN93 (CAMKII inhibitor) blocked ZYZ-803-induced STAT3/CAMKII activation and significantly suppressed the proliferation and migration of HUVECs. In addition, pretreatment with the inhibitors significantly decreased ZYZ-803-induced tube formations along with the outgrowths of branch-like microvessels in aortic rings. In the mice with femoral artery ligation, administration of ZYZ-803 significantly increased the blood perfusion and vascular density in the hind limb, whereas co-administration of WP1066 or KN93 abrogated ZYZ-803-induced angiogenesis. By using STAT3 siRNA, we further explored the cross-talk between STAT3 and CaMKII in ZYZ-803-induced angiogenesis. We found that STAT3 knockdown suppressed ZYZ-803-induced HUVEC angiogenesis and affected CaMKII expression. ZYZ-803 treatment markedly enhanced the interaction between CaMKII and STAT3. ZYZ-803 treatment induced the nuclear translocation of STAT3. We demonstrated that both STAT3 and CaMKII functioned as positive regulators in ZYZ-803-induced endothelial angiogenesis and STAT3 was important in ZYZ-803-induced CaMKII activation, which highlights the beneficial role of ZYZ-803 in STAT3/CaMKII-related cardiovascular diseases.

Neuroprotective Effect of SCM-198 through Stabilizing Endothelial Cell Function.

Leonurine, also named SCM-198, which was extracted from Herba leonuri, displayed a protective effect on various cardiovascular and brain diseases, like ischemic stroke. Ischemic stroke which is the leading cause of morbidity and mortality, ultimately caused irreversible neuron damage. This study is aimed at exploring the possible therapeutic potential of SCM-198 in the protection against postischemic neuronal injury and possible underlying mechanisms. A transient middle cerebral artery occlusion (tMCAO) rat model was utilized to measure the protective effect of SCM-198 on neurons. TEM was used to determine neuron ultrastructural changes. The brain slices were stained with Nissl staining solution for Nissl bodies. Fluoro-Jade B (FJB) was used for staining the degenerating neurons. In the oxygen-glucose deprivation and re-oxygenation (OGD/R) model of bEnd.3 cells treated with SCM-198 (0.1, 1, 10 µM). Then, the bEnd.3 cells were cocultured with SH-SY5Y cells. Cell viability, MDA level, CAT activity, and apoptosis were examined to evaluate the cytotoxicity of these treatments. Western blot and immunofluorescent assays were used to examine the expression of protein related to the p-STAT3/NOX4/Bcl-2 signaling pathway. Coimmunoprecipitation was performed to determine the interaction between p-STAT3 and NOX4. In the transient middle cerebral artery occlusion (tMCAO) rat model, we found that treatment with SCM-198 could ameliorate neuron morphology and reduce the degenerating cell and neuron loss. In the in vitro model of bEnd.3 cell oxygen-glucose deprivation and reoxygenation (OGD/R), treatment with SCM-198 restored the activity of catalase (CAT), improved the expression of Cu-Zn superoxide dismutase (SOD1), and decreased the malondialdehyde (MDA) production. SCM-198 treatment prevented OGD/R-induced cell apoptosis as indicated by increased cell viability and decreased the number of TUNEL-positive cells, accompanied with upregulation of Bcl-2 and Bcl-xl protein and downregulation Bax protein. The results were consistent with SH-SY5Y cells which coculture with bEnd.3 cells. The forthcoming study revealed that SCM-198 activated the p-STAT3/NOX4/Bcl-2 signaling pathway. All the data indicated that SCM-198 protected against oxidative stress and neuronal damage in in vivo and in vitro injury models via the p-STAT3/NOX4/Bcl-2 signaling pathway. Our results suggested that SCM-198 could be the potential drug for neuroprotective effect through stabilizing endothelial cell function.

An inhibitor of 11-ß hydroxysteroid dehydrogenase type 1 (PF915275) alleviates nonylphenol-induced hyperadrenalism and adiposity in rat and human cells.

BACKGROUND: Nonylphenol (NP) is an environmental endocrine-disrupting chemical (EDC) detected in human cord blood and milk. NP exposure in developmental periods results in hyperadrenalism and increasing 11ß-hydroxysteroid dehydrogenase I (11ß-HSD1) activity in an adult rat model. Alleviating 11ß-HSD1 activity is therefore a logical and common way to treat hyperadrenalism. PF915275 (PF; 4'-cyano-biphenyl-4-sulfonic acid (6-amino-pyridin-2-yl)-amide) is a selective inhibitor for 11ß-HSD1. This study aimed to determine whether PF915275 could alleviate the hyperadrenalism induced by NP. In addition to a rat model, the effects of NP and PF915275 were measured in human preadipocytes. METHODS: For the in vivo rat model, female adult rats exposed to NP during the developmental period were divided into two treatment groups, with one receiving oral DMSO solution and the other receiving PF915275 once per day for 4 weeks. After the final treatment, the rats from each group were sacrificed for analysis. For the in vitro human model, human preadipocytes received 2 regimens of NP treatment. One treatment regimen occurred before differentiation (to mimic the sensitive developmental period; P exposure), and the other included continuous exposure from preadipocytes to fully differentiated adipocytes (to mimic the growing and adult periods, respectively; C exposure). Protein and RNA were extracted from rat tissues and the preadipocytes for western blot and real-time PCR analysis. RESULTS: In the rat model, PF915275 alleviated NP-induced effects by interfering with adipogenesis pathways, including enhancing PPARα expression, decreasing PPARγ expression, and reducing both 11ß-HSD1 protein and mRNA expression levels. Additionally, PF915275 reduced the effects of the adrenal corticoid synthesis pathway by reducing StAR expression and 11ß-hydroxylase and aldosterone synthase activities. With short-term exposure, NP enhanced PPARγ and FASN mRNA expression levels and reduced PPARα expression, whereas PF915275 alleviated these effects. With C exposure, the NP-induced accumulation of intracellular lipids was reduced by PF915275 treatment, which was mediated by decreased PPARγ mRNA and protein expression levels and increased PPARα protein expression. CONCLUSIONS: The effects of NP and PF915275 treatment in both rat and human cell models are similar. Rats may be an appropriate model to study the effects of NP in humans, especially during the developmental period.

Stimulatory Effect of Intermittent Hypoxia on the Production of Corticosterone by Zona Fasciculata-Reticularis Cells in Rats.

Hypoxia or intermittent hypoxia (IH) have known to alter both synthesis and secretion of hormones. However, the effect of IH on the production of adrenal cortical steroid hormones is still unclear. The aim of present study was to explore the mechanism involved in the effect of IH on the production of corticosterone by rat ZFR cells. Male rats were exposed at 12% O2 and 88% N2 (8 hours per day) for 1, 2, or 4 days. The ZFR cells were incubated at 37 °C for 1 hour with or without ACTH, 8-Br-cAMP, calcium ion channel blockers, or steroidogenic precursors. The concentration of plasma corticosterone was increased time-dependently by administration of IH hypoxia. The basal levels of corticosterone production in cells were higher in the IH groups than in normoxic group. IH resulted in a time-dependent increase of corticosterone production in response to ACTH, 8-Br-cAMP, progesterone and deoxycorticosterone. The production of pregnenolone in response to 25-OH-C and that of progesterone in response to pregnenolone in ZFR cells were enhanced by 4-day IH. These results suggest that IH in rats increases the secretion of corticosterone via a mechanism at least in part associated with the activation of cAMP pathway and steroidogenic enzymes.

Nonylphenol-induced hyperadrenalism can be reversed/alleviated by inhibiting of 11-ß hydroxysteroid dehydrogenase type 1.

We previously observed that nonylphenol (NP) exposure during development resulted in increases in body weight and hyperadrenalism in adult male offspring. The mechanism of hyperadrenalism includes the primary activation of the adrenal gland and the conversion of inactive glucocorticoids to active glucocorticoids by 11ß-HSD1. The inhibition of 11ß-HSD1 is investigated as a new therapeutic approach. This study examined the effect of PF915275 (a selective 11ß-HSD1 inhibitor) on hyperadrenalism and adipogenesis in male rats exposed to NP during development. The results showed that treatment with the 11ß-HSD1 inhibitor PF915275 reversed/alleviated NP-induced hyperadrenalism via the following mechanisms: (1) decreasing serum corticosterone, 11ß-hydroxylase, and aldosterone synthase levels; (2) significantly increasing PPARα protein and mRNA expression. In adipose tissue, NP significantly increased PPARγ mRNA expression, whereas PF915275 significantly decreased the level of mRNA expression; and (3) the expression of key regulators/enzymes in the adipogenesis metabolic pathway was also modulated.

Effect of swimming on the production of aldosterone in rats.

It has been demonstrated that exercise is one of the stresses known to increase the aldosterone secretion. Both potassium and angiotensin II (Ang II) levels are shown to be correlated with aldosterone production during exercise, but the mechanism is still unclear. In an in vivo study, male rats were catheterized via right jugular vein (RJV), and divided into four groups namely water immersion, swimming, lactate infusion (13 mg/kg/min) and pyruvate infusion (13 mg/kg/min) groups. Each group was treated for 10 min. Blood samples were collected at 0, 10, 15, 30, 60 and 120 min from RJV after administration. In an in vitro study, rat zona glomerulosa (ZG) cells were challenged by lactate (1-10 mM) in the presence or absence of Ang II (10(-8) M) for 60 min. The levels of aldosterone in plasma and medium were measured by radioimmunoassay. Cell lysates were analyzed by immunoblotting assay. After exercise and lactate infusion, plasma levels of aldosterone and lactate were significantly higher than those in the control group. Swimming for 10 min significantly increased the plasma Ang II levels in male rats. Administration of lactate plus Ang II significantly increased aldosterone production and enhanced protein expression of steroidogenic acute regulatory protein (StAR) in ZG cells. These results demonstrated that acute exercise led to the increase of both aldosterone and Ang II secretion, which is associated with lactate action on ZG cells and might be dependent on the activity of renin-angiotensin system.

Recovery from developmental nonylphenol exposure is possible for female rats.

Nonylphenol (NP) is an environmental endocrine-disrupting chemical that has been detected in human cord blood and milk. Developmental exposure to NP is unavoidable and can lead to hyperadrenalism, a syndrome that resembles Cushing's disease and has a life-long impact on the affected individual. In this study, we investigated the recovery of female rats from developmental exposure to NP and the effects of such exposure on future generations. Female rats were time-mated, and rats in the experimental group (NP group) were administered NP in drinking water (2µg/mL) throughout gestation and lactation. Pregnant females in the control group were given water only (Veh group). The resulting litters were recognized as the first-generation F1 offspring. The F1 females were time-mated with non-sibling F1 males within the same treatment group. NP was not administered after the F0 generation. The treatment procedures for F3 offspring were identical to those for the F2 generation. The experimental results showed that the observed characteristics of the F3 NP generation had reverted to normal and resembled those of the F3 Veh generation. Thus, our study indicated that developmental exposure to NP resulted in a life-long impact on the exposed individual, but that recovery to the "normal" state was possible if further NP exposure was prevented.

In utero and neonate exposure to nonylphenol develops hyperadrenalism and metabolic syndrome later in life. I. First generation rats (F(1)).

Nonylphenol (NP) is an endocrine disruptor (ENDR). It is a chemical associated with the production and degradation of nonylphenol ethoxylates (NPE). NPE is widely used as nonionic surfactants. Previously, we observed that NP increased the production of corticosterone and aldosterone from zona fasciculata-reticularis, and zona glomerulosa cells, respectively. By the "fetal origins adult diseases" (Barker hypothesis), we examined the possible impact of NP exposure during developmental (in utero and neonatal) period with focus on disturbed adrenal function and related hyperadrenal syndrome, i.e. Cushings syndrome/metabolic syndrome. In this study, female rats drink NP water during pregnancy and lactation conferred F(1) generation: (1) increase the corticosterone, aldosterone concentration in plasma; (2) increase 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity in liver and adipose tissue; (3) increase aldosterone synthase activity in adrenal for adult offspring. Furthermore, it can increase body weight, adrenocorticotropin (ACTH) concentration in plasma, 11ß-HSD1 protein expression in liver, steroidogenic acute regulatory (StAR) protein expression and 11ß-hydroxylase activity in adrenal for male adult offspring. In summary, NP exposure during developmental period bestowed F(1) generation with hyperadrenalism and its consequence of metabolic syndrome.

Effects of nonylphenol on aldosterone release from rat zona glomerulosa cells.

Alkylphenol ethoxylate, which consists of approximately 80% nonylphenol ethoxylate (NPE), is a major nonionic surfactant. Nonylphenol (NP), the primary degradation product of NPE, has been reported to interfere with reproduction in fish, reptiles, and mammals by inducing cell death in the gonads and by affecting other reproductive parameters. However, the effects of NP on rat adrenal zona glomerulosa cells (ZG) and the underlying mechanisms remain unclear. In this study, we explored the effects of NP on aldosterone release. ZG cells were incubated with NP in the presence or absence of the secretagogues angiotensin II (ANG II), potassium, 8-Br-cAMP, 25-OH-cholesterol, corticosterone or cyclopiazonic acid (CPA). After performing radioimmunoassay (RIA) and Western blot analysis, we found that (1) NP stimulated aldosterone release in cells induced by ANG II, KCl, 8-Br-cAMP, 25-OH-cholesterol, corticosterone, and CPA; (2) NP triggered the release of higher amounts of pregnenolone in cells treated with vehicle and 25-OH-cholesterol+trilostane than in cells treated with other compounds; and (3) the stimulatory effect of NP seemed to be mediated through steroidogenic acute regulatory protein (StAR) and aldosterone synthase activity. These observations suggest that the effects of NP are mediated via increased free Ca(2+) in the cytoplasm.

A radioimmunoassay for rat ghrelin: evaluation of method and effects of nonylphenol on ghrelin secretion in force-fed young rats.

Antiserum YJC 13-31 against the rat ghrelin conjugated to bovine serum albumin (BSA) was produced in the rabbit and a double antibody radioimmunoassay (RIA) for ghrelin has been developed. Characterization results of this antiserum revealed no cross-reaction with human growth hormone and somatostatin. Weak cross-reactions with insulin (0.1%), rat growth hormone (0.1%) and glucagon (0.3%) were observed, which scarcely interfered the assay system. The sensitivity of this RIA was 5 pg per assay tube. With the rat serum samples, the within-assay precision was 7.1% and the between-assay precision was 12.3%. The RIA was also available to detect the ghrelin in rat tissue extracts with good parallelism to the rat ghrelin standard. In application, the serum ghrelin and corticosterone levels in weaned rats were measured by RIA. Gavage of saline was sufficient to raise serum ghrelin from 2.6 +/- 0.18 to 6.7 +/- 0.7 ng/ml (P < 0.01). Gavage with nonylphenol (NP) suppressed the elevation of serum ghrelin levels in a dose-dependent manner. Besides, gavages of saline elevated the serum levels of corticosterone from 108.8 +/- 13.5 to 188.7 +/- 23.5 ng/ml (P < 0.01) but the elevation effects of corticosterone from gavages were overcome by NP in the low dose of 50 mg/kg. It can be speculated that ingestion of NP is harmful to young animals during growth and environmental adaptation.

Effects and mechanisms of nonylphenol on corticosterone release in rat zona fasciculata-reticularis cells.

Alkylphenol ethoxylate, consisting of ∼80% nonylphenol ethoxylate (NPEO), is a major group of nonionic surfactant. The primary degradation product of NPEO, nonylphenol (NP), interferes with reproduction, induces cell death in gonads, and leads to changes in other reproductive parameters. With such apparent stress, NP is believed to induce stress response mechanism, i.e., adrenal cortical hormone. However, the effects and action mechanisms of NP on rat adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study explored the effects of NP on corticosterone release. ZFR cells were incubated with NP in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-cyclic 3',5'-adenosine monophosphate (8-Br-cAMP), forskolin (FSK), 25-hydroxyl cholesterol (25-OH-cholesterol), pregnenolone, progesterone, or deoxycorticosterone at 37°C for 1 h. The concentrations of corticosterone or pregnenolone in the spent media were measured by radioimmunoassay. The expressions of steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc) protein, and 11ß-hydroxylase in the cells were measured by Western blot. The data demonstrated that (1) NP stimulated corticosterone release induced by ACTH, 8-Br-cAMP, FSK, 25-OH-cholesterol, pregnenolone, progesterone, or deoxycorticosterone; (2) NP significantly increased pregnenolone release in the control, 25-OH-cholesterol, trilostane, and 25-OH-cholesterol + trilostane groups; (3) NP-stimulated corticosterone release was estrogen receptor dependent, but mediated by nitric oxide and p38 mitogen-activated protein kinase pathway independent; and (4) NP did not affect StAR, 11ß-hydroxylase, or P450scc protein expression. These results suggest that NP acts directly on rat ZFR cells to stimulate corticosterone release and that the stimulation mechanism of NP mediates through post-cAMP corticosterone manufacture enzymes, i.e., P450scc and 11ß-hydroxylase.

[Genetic polymorphism of the IL8 gene and its associations with milk traits and SCS in Chinese Holstein].

The polymorphism of Interleukin-8 (IL8) gene were investigated for 610 Chinese Holstein cows of 30 bull families from a dairy farm in Shanghai using Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) technique with a mixed animal model to verify the effects of the polymorphisms on some milk productive performance, tested day milk yield, tested day fat percentage, tested day milk protein percentage, 305 d corrected milk yield, 305 d milk fat yield, 305 d milk protein yield, and somatic cell score (SCS). The aim was to explore the significant molecular marker in practical dairy production. Three genotypes were identified and the genotypic frequencies of KK, KA, and AA were 0.187, 0.451, and 0.362, respectively. The gene frequencies of K and A were 0.412 and 0.588. The results showed highly significant (P < 0.01) association of IL8 mutations with tested day milk yield, 305 d milk protein yield, 305 d corrected milk yield and 305 d milk fat yield, SCS and tested day milk protein percentage (P < 0.05). However, no association (P > 0.05) with tested day milk fat percentage was recorded. The cows with KK genotype had higher tested day milk yield, 305 d milk protein yield, 305 d corrected milk yield and 305 d milk fat yield than those with AA and KA genotypes (P < 0.01). The least square mean of SCS for KK was significantly lower than that with AA and KA genotypes (P < 0.01). AA genotype was significant lower in tested day milk protein percentage than KK and KA genotypes (P < 0.05). The IL8 gene genetic diversity has a great genetic effect on milk traits and mastitis resistance and could be a useful genetic marker for Chinese Holstein breeding.

Effects of dehydroepiandrosterone on aldosterone release in rat zona glomerulosa cells.

The present study was to investigate the effects and action mechanisms of dehydroepiandrosterone (DHEA) on steroidogenesis in rat adrenal zona glomerulosa cells (ZG). ZG cells were incubated with DHEA in the presence or absence of angiotensin II (AngII), a high concentration of potassium, 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone, deoxycorticosterone, corticosterone, A23187, or cyclopiazonic acid (CPA) at 37 degrees C for 1 h. The concentration of aldosterone or pregnenolone in the culture medium was then measured by radioimmunoassay (RIA). The cells were used to determine the cellular cAMP content. The data demonstrated that: (1) DHEA inhibited AngII-, high concentration of KCl-, forskolin-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone-, deoxycorticosterone-, corticosterone-, A23187-, or CPA-stimulated aldosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA noncompetitively inhibited aldosterone synthase but showed uncompetitive inhibition of P450scc. These results suggest that DHEA acts directly on rat ZG cells to diminish aldosterone secretion by inhibition of a post-cAMP pathway or by acting on intracellular Ca2+ mobilization. In addition it affects the function of post-P450scc steroidogenic enzymes.

Mechanisms of inhibition of dehydroepiandrosterone upon corticosterone release from rat zona fasciculata-reticularis cells.

We have demonstrated that dehydroepiandrosterone (DHEA) acts directly on rat zona fasciculata-reticularis (ZFR) cells to diminish corticosterone secretion by an inhibition of post-cAMP pathway, and decreases functions of steroidogenic enzymes after P450(scc) as well as steroidogenic acute regulatory (StAR) protein expression. However, the mechanisms by which DHEA engages with environmental messenger signals which translate into interfering StAR protein expression are still unclear. This study explored the effects of DHEA on the phosphorylation/activation of extracellular signal-regulated kinases (ERKs). ERK activation resulted in enhancing phosphorylation of steroidogenic factor-1 (SF-1) and increased StAR protein expression. ZFR cells were incubated in the presence or absence of adrenocorticotropin (ACTH), forskolin (FSK), 25-OH-cholesterol, U0126, and H89 at 37 degrees C. The concentration of corticosterone released into the media was measured by radioimmunoassay (RIA). The cells were used to extract protein for Western blot analysis of ERKs or StAR protein expression or immunoprecipitation of SF-1 analysis. The results suggested that (1) ERK pathway of rat ZFR cells might be PKA dependent, (2) ERK activity was required for SF-1 phosphorylation to upregulate steroidogenesis in rat ZFR cells, and (3) DHEA did not affect ERK phosphorylation, however, it attenuated forskolin-stimulated SF-1 phosphorylation to affect StAR protein expression.

Effects of crude adlay hull acetone extract on corticosterone release from rat zona fasciculata-reticularis cells.

Adlay is a grass crop which has been used in traditional Chinese medicine and also as a nourishing food. It has been shown to posses anti-allergic, antimutagenic and hypolipemic effects. However, the effects and action mechanisms of crude adlay hull acetone extract (AHA) on adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study explored the effects of AHA on corticosterone release. ZFR cells were incubated with AHA in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-cyclic 3': 5'- adenosine monophosphate (8-Br-cAMP), forskolin (FSK), 25-hydroxy cholesterol (25-OH-cholesterol), pregnenolone, progesterone or deoxycorticosterone. The concentrations of corticosterone or pregnenolone in the media were measured by radioimmunoassay (RIA). The cells were used to measure the expression of steroidogenic acute regulatory (StAR) protein by Western blot. The present data demonstrated that: (1) AHA inhibited ACTH-, 8-Br-cAMP-, forskolin-, 25-OH-cholesterol-, pregnenolone-, progesterone- or deoxycorticosterone-stimulated corticosterone release; (2) AHA (800 microg/ml) caused more pregnenolone release in control group, but not in 25-OH-cholesterol, trilostane or 25-OH-cholesterol+trilostane group; (3) kinetic study showed an uncompetitive inhibition model of AHA to P450 side chain cleavage enzyme (P450scc); (4) kinetic study showed a noncompetitive inhibition model of AHA to 11beta-hydroxylase; and (5) AHA inhibited the expression of StAR protein. These results suggest that AHA acts directly upon rat ZFR cells to diminish corticosterone release. These results indicate the inhibitory mechanism of AHA mediates through an inhibition of the activities of the post-cAMP corticosterone synthesis enzymes, i.e. 3beta-HSD, 21-hydroxylase, 11beta-hydroxylase, and inhibition of StAR protein expression.

Effects of S-petasin on cyclic AMP production and enzyme activity of P450scc in rat zona fasciculata-reticularis cells.

Petasites hybridus is used in Chinese herbal medicine. S-petasin is a bioactive compound isolated from leaves or roots of P. hybridus, which has been used to relieve gastrointestinal pain, lung disease, and spasms of urogenital tract. We have demonstrated that S-petasin inhibited corticosterone release from rat zona fasiculata-reticularis cells. However, the mechanism and molecular effects of S-petasin on zona fasiculata-reticularis cells are still unclear. This study explored the effects of S-petasin on cellular adenosine 3':5'-cyclic monophosphate (cAMP) production, the functions of steroidogenic enzymes including cytochrome P450 side-chain cleavage enzyme (P450scc), 11beta-hydroxylase, and the expression levels of steroidogenic acute regulatory protein or P450scc. In this experiment, zona fasciculata-reticularis cells were incubated with S-petasin in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), forskolin, 25-OH-cholesterol, deoxycorticosterone at 37 degrees C for 0.5, 1 or 3 h. The media were used to measure the concentration of corticosterone or pregnenolone by radioimmunoassay. The cells were used to measure the content of cAMP by radioimmunoassay and extracted protein for Western blot or messenger RNA (mRNA) for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Our data demonstrated that (1) S-petasin inhibits ACTH- or forskolin-stimulated cellular cAMP production, (2) S-petasin increased the Michaelis constants of P450scc and 11beta-hydroxylase and (3) S-petasin decreased the expression levels and mRNA of steroidogenic acute regulatory protein. In summary, the actions of S-petasin mediate the inhibition of cAMP formation, decrease the activities of key enzymes P450scc and 11beta-hydroxylase, and reduce mRNA of steroidogenic acute regulatory protein and expression of steroidogenic acute regulatory protein.

Effects of dehydroepiandrosterone on corticosterone release in rat zona fasciculata-reticularis cells.

The decline of plasma dehydroepiandrosterone (DHEA) and maintenance of glucocorticoid levels with increasing age contribute to excess body fat accumulation, hyperglycaemia, hyperlipidaemia, hyperinsulinaemia and cancer. Although opposing actions of DHEA and corticosterone have been proposed in a rat model, the effects and action mechanisms of DHEA on rat adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study addressed the effects of DHEA on corticosterone release, cellular cAMP production, the functions of steroidogenic enzymes and the expression levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc). ZFR cells were incubated with DHEA in the presence or absence of adrenocorticotropin (ACTH), 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone or deoxycorticosterone at 37 degrees C for 30 min, 1 h or 5 h and the concentration of corticosterone or pregnenolone measured subsequently in the media by RIA. The cells were used to measure the content of cAMP by RIA and to extract protein for Western blot or mRNA for RT-PCR analysis. The data demonstrated that (1) DHEA inhibited ACTH-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone- or deoxycorticosterone-stimulated corticosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA increased the K(m) of 11beta-hydroxylase but not P450scc; (4) DHEA affected the expression levels of StAR protein but not of P450scc. These results suggest that DHEA acts directly on rat ZFR cells to diminish corticosterone secretion by inhibition within the post-cAMP pathway, by inhibiting steroidogenic enzymes downstream from P450scc and by inhibiting StAR expression.

Stimulatory effects of hyperprolactinemia on aldosterone secretion in ovariectomized rats.

BACKGROUND: To evaluate the effects of hyperprolactinemia on aldosterone secretion and its mechanisms of action in ovariectomized (OVX) rats. METHODS: Hyperprolactinemia was induced by the transplantation of rat anterior pituitary (AP) glands under the kidney capsule for 6 weeks in female rats. Control rats underwent cerebral cortex (CX) transplantation. Four weeks after transplantation, the rats were OVX 2 weeks before decapitation. After decapitation, the trunk blood was collected, and the adrenal glands of CX- and AP-grafted rats were prepared as zona glomerulosa (ZG) cells for in vitro study. RESULTS: Plasma prolactin and aldosterone in the rats were increased by AP gland transplantation. In the in vitro study, the basal aldosterone secretion by the adrenal ZG cells was higher in AP-grafted rats than in CX-grafted rats. The AP-grafted group showed increased responsiveness to angiotensin II (10(-8) M), KCl (8 x 10(-3) M), or 8-bromo-adenosine 3',5'-cyclic monophosphate (8-br-cAMP; 10(-4) M, a membrane-permeable analogue of cAMP) with regard to aldosterone secretion as compared with the CX-grafted group. N-(2-[p-Bromocinnamylamine]ethyl)-5-isoquinolinesulfonamide (H89; 10(-6), 10(-5) M, a protein kinase A inhibitor) or tetrandrine (10(-5) M, a blocker for both L-type and T-type Ca2+ channels) induced a greater suppression of aldosterone secretion in the AP-grafted group than in the CX-grafted group. No significant differences between the CX- and AP-grafted groups were observed, however, with regard to the adrenocorticotropichormone (10(-9) M)-, forskolin (10(-5) M, an adenylyl cyclase activator)-, or nifedipine (10(-5) M, an L-type Ca2+ channel blocker)-induced responsiveness of aldosterone secretion. In addition, there was no difference in the expression of desmolase (i.e., cytochrome P450 side-chain cleavage enzyme) in ZG cells between AP- and CX-grafted rats. The conversions of 25-OH-cholesterol into pregnenolone in the presence of trilostane (an inhibitor of 3beta-hydroxysteroid dehydrogenase) and corticosterone into aldosterone, as well as the expression of the steroidogenic acute regulatory protein in ZG cells, were greater in AP-grafted rats than in CX-grafted rats. CONCLUSIONS: These results suggest that hyperprolactinemia increases basal, angiotensin II- and KCl-stimulated aldosterone secretion by ZG cells in OVX rats through activation of T-type Ca2+ channels, the post-cAMP and protein kinase A pathway, cytochrome P450 side-chain cleavage enzyme, and aldosterone synthase, as well as by causing increased expression of steroidogenic acute regulatory protein in ZG cells.