Differential effects of exogenous and endogenous estrogen on anxiety as measured by elevated T-maze in relation to the serotonergic system.

The effects of estrogen on anxiety-like behaviors have been widely studied but the mechanisms responsible are still inconclusive. The purpose of the current study was to compare the effects of transient high levels of endogenous estrogen and chronic exogenous estrogen treatment on the anxiety-like behaviors using the elevated T-maze (ETM) test. In addition, serotonin (5-HT) and its metabolite (5-HIAA), serotonin reuptake transporter (SERT) and tryptophan hydroxylase enzyme (TPH) were measured at the end of the study and correlated to the task performances. Female sham-operated rats in proestrous phase (Sham-Pro) and ovariectomized rats treated with or without 17beta-estradiol (10 microg/kg, s.c.; Ovx+E(2) or Ovx) for 4 weeks were used. In the ETM test, the Ovx+E(2) group had reduced inhibitory avoidance responses compared to others, suggesting that exogenous E(2) replacement is anxiolytic, while escape latency was prolonged in the Sham-Pro group suggesting endogenous E(2) is panicolytic. Further, the serotonin turnover rate (5-HIAA/5-HT ratio) in the hippocampus and nucleus accumbens was highest in the Ovx+E(2) group. While the TPH protein in the midbrain of Ovx rats was significantly higher than others, the SERT levels were not significantly different among groups in all measured brain areas. In conclusion, Ovx rats with chronic estrogen administration and Sham-Pro rats with naturally high levels of estrogen, demonstrated anxiolytic behavior by exhibiting different forms of anxiety that related to the changes in the function of serotonergic system.

Osteoblasts express claudins and tight junction-associated proteins.

Osteoblasts were previously reported to form tight junctions, which may play an important role in the regulation of ion transport across the epithelial-like bone membrane. However, the evidence for the presence of tight junction-associated proteins in osteoblasts is lacking. We therefore studied the expression of tight junction-associated genes in primary rat osteoblasts and bone tissues. Quantitative real-time PCR showed that osteoblasts expressed ZO-1, -2, -3, cingulin, occludin, claudin-1 to -12, -14 to -20, -22 and -23. By using western blot analyses of selected claudins, expression of claudin-5, -11, -14 and -15, but not claudin-3, were identified in osteoblasts. A confocal immunofluorescent study in undecalcified tibial sections confirmed that claudin-16 was localized on the trabecular surface, normally covered by osteoblasts and bone-lining cells. In addition, immunohistochemical studies in decalcified tibial sections demonstrated the expression of claudin-5, -11, -14, -15 and -16 in bone-lining cells (inactive osteoblasts). Primary osteoblasts cultured in the Snapwell for 19-26 days were found to form a monolayer with measurable transepithelial resistance of approximately 110-180 Omegacm(2), confirming the presence of barrier functions of the tight junction. It was concluded that osteoblasts expressed several tight junction-associated proteins, which possibly regulated ion transport across the bone membrane.

Prolactin directly enhances bone turnover by raising osteoblast-expressed receptor activator of nuclear factor kappaB ligand/osteoprotegerin ratio.

Hyperprolactinemia leads to high bone turnover as a result of enhanced bone formation and resorption. Although its osteopenic effect has long been explained as hyperprolactinemia-induced hypogonadism, identified prolactin (PRL) receptors in osteoblasts suggested a possible direct action of PRL on bone. In the present study, we found that hyperprolactinemia induced by anterior pituitary transplantation (AP), with or without ovariectomy (Ovx), had no detectable effect on bone mineral density and content measured by dual-energy X-ray absorptiometry (DXA). However, histomorphometric studies revealed increases in the osteoblast and osteoclast surfaces in the AP rats, but a decrease in the osteoblast surface in the AP+Ovx rats. The resorptive activity was predominant since bone volume and trabecular number were decreased, and the trabecular separation was increased in both groups. Estrogen supplement (E2) fully reversed the effect of estrogen depletion in the Ovx but not in the AP+Ovx rats. In contrast to the typical Ovx rats, bone formation and resorption became uncoupled in the AP+Ovx rats. Therefore, hyperprolactinemia was likely to have some estrogen-independent and/or direct actions on bone turnover. Osteoblast-expressed PRL receptor transcripts and proteins shown in the present study confirmed our hypothesis. Furthermore, we demonstrated that the osteoblast-like cells, MG-63, directly exposed to PRL exhibited lower expression of alkaline phosphatase and osteocalcin mRNA, and a decrease in alkaline phosphatase activity. The ratios of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) proteins were increased, indicating an increase in the osteoclastic bone resorption. The present data thus demonstrated that hyperprolactinemia could act directly on bone to stimulate bone turnover, with more influence on bone resorption than formation. PRL enhanced bone resorption in part by increasing RANKL and decreasing OPG expressions by osteoblasts.

Prolactin-stimulated transepithelial calcium transport in duodenum and Caco-2 monolayer are mediated by the phosphoinositide 3-kinase pathway.

Prolactin (PRL) has been shown to stimulate intestinal calcium absorption but the mechanism was still unknown. This study aimed to investigate the mechanism and signaling pathway by which PRL enhanced calcium transport in the rat duodenum and Caco-2 monolayer. Both epithelia strongly expressed mRNAs and proteins of PRL receptors. Ussing chamber technique showed that the duodenal active calcium fluxes were increased by PRL in a dose-response manner with the maximal effective dose of 800 ng/ml. This response diminished after exposure to LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Caco-2 monolayer gave similar response to PRL with the maximal effective dose of 600 ng/ml. By nullifying the transepithelial potential difference, we showed that the voltage-dependent paracellular calcium transport did not contribute to the PRL-enhanced flux in Caco-2 monolayer. In contrast, the calcium gradient-dependent paracellular transport and calcium permeability were increased by PRL. Effects of PRL on Caco-2 monolayer were abolished by PI3K inhibitors (LY-294002 and wortmannin), but not by inhibitors of MEK (U-0126) or JAK2 (AG-490). To investigate whether the PRL-enhanced paracellular transport was linked to changes in the epithelial charge selectivity, the permeability ratio of sodium and chloride (P(Na)/P(Cl)) was determined. We found that PRL elevated the P(Na)/P(Cl) in both epithelia, and the effects were blocked by PI3K inhibitors. In conclusion, PRL directly and rapidly stimulated the active and passive calcium transport in the rat duodenum and Caco-2 monolayer via the nongenomic PI3K-signaling pathway. This PRL-enhanced paracellular calcium transport could have resulted from altered charge selectivity.

Chronic metabolic acidosis upregulated claudin mRNA expression in the duodenal enterocytes of female rats.

Previous investigations showed that chronic metabolic acidosis (CMA) increased the paracellular permeability of ion and neutral hydrophilic molecules in the duodenum of rats and small intestinal-like cell lines. Since proteins of the claudin family have been known to regulate the paracellular transport in several epithelia, an increase in the paracellular permeability during CMA may have resulted from changes in the pattern of claudin expression. The present study aimed to investigate the expression profile of 22 claudins in the duodenum of female Sprague-Dawley rats given 1.5% NH(4)Cl for 21 days to induce CMA. Arterial blood gas analysis revealed plasma pH values of 7.40 in normal rats and 7.31 in acidotic rats. Blood chemistry showed increases in the total plasma calcium, free-ionized calcium and magnesium, indicating a typical adaptive response of animals to CMA. RT-PCR demonstrated mRNA expressions of claudin-1 to -12, -14, -15, -17 to -20, -22 and -23 in duodenum of normal rats. Claudin-16 was not expressed in normal duodenum, but was strongly expressed in the kidney. Claudin-13 expression was seen only in the cecum, colon, liver and kidney of mice. After 21-day CMA, mRNA expressions of claudin-2, -3, -6, -8, -11, -12, -14, -19 and -22 were significantly enhanced, whereas expressions of other claudins were not changed. Confocal laser-scanning microscopy demonstrated that duodenal enterocytes of normal rats expressed claudin-3 protein on the paracellular membrane. The distribution of claudin-3 protein along the paracellular membrane was markedly increased in CMA, especially near the apical surface. Our results, therefore, provided novel evidence that 21-day CMA markedly altered claudin profile in the duodenum of rats by upregulating specific claudin expression.

Anxiolytic property of estrogen related to the changes of the monoamine levels in various brain regions of ovariectomized rats.

Anxiety is a symptom reflecting the dysregulation of monoaminergic neurotransmitters which may be modulated by estrogen. In our current study, we investigated the effects of chronic estrogen administration (10 microg/kg, s.c. for 4 weeks) on anxiety-like behavior using the elevated plus-maze with the corresponding changes of monoamines in the brain regions contributing to anxiety. The behavioral test revealed that estrogen-treated rats (Ovx+E(2)) spent more time in the open arm of the maze as well as a higher time/entry ratio in open arms than ovariectomized (Ovx) rats, indicating an anxiolytic property of estrogen. The increase in open arm time corresponded to an increase in uterine weight, indicated a correlation between the function of estrogen and its anxiolytic effect. Measurements of brain monoamines following estrogen treatment revealed decreases in norepinephrine, dopamine and serotonin in all of the brain regions studied, which also lead to an increase in turnover rates. The concentrations of norepinephrine in caudate putamen, of dopamine in nucleus accumbens, of serotonin in frontal cortex, hippocampus, caudate putamen, nucleus accumbens, and substantia nigra and of the serotonin metabolite, the 5-hydroxyindolacetic acid in substantia nigra of Ovx+E(2) rats were significantly lower than those of Ovx rats. Interestingly, the uterine weight was negatively correlated with the changes of dopamine and serotonin (with the exception of the hippocampus), suggesting a regulatory role of estrogen on these systems. From these data, we concluded that, in fact, there is a relationship between estrogen and monoamines (i.e. serotonin, dopamine) in modulating the anxiety-like behaviors in female rats.