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Our objective was to examine the topology-, gestation- and labor-related changes of estrogen receptor (ER)alpha, progesterone receptor (PR), oxytocin receptor (OTR) and thrombospondin-1 (TSP1) mRNA in pregnant baboon myometrium. ER alpha, PR, OTR and TSP1 mRNAs extracted from the lower uterine segment and fundal myometrium of pregnant baboons not in labor between 121 and 180 days of gestational age (n=9) and in established spontaneous labor between 164 and 193 days of gestational age (n=5) were analyzed by Northern blot. There were no topology-, gestation- or labor-related changes of ER alpha and PR mRNA in or between the lower uterine segment or/and the fundus. OTR mRNA was the same in the lower uterine segment and the fundus from baboons not in labor and non-labor fundal, but not lower uterine segment, myometrial OTR mRNA increased with gestation (R(2)=0.81, P<0.05). Fundal OTR mRNA rose significantly compared with the lower uterine segment during spontaneous labor. TSP1 mRNA increased significantly in both the fundus and lower uterine segment during labor. TSP1 mRNA in the lower uterine segment during spontaneous labor was higher than in the fundus during spontaneous labor. In conclusion, fundal and lower uterine segment ER alpha and PR mRNA remained unchanged in late gestation and spontaneous labor. The increased OTR mRNA may serve as a mechanism to increase uterine sensitivity to OT during late gestation. The higher fundal OTR mRNA compared with the lower uterine segment provides polarity which assists fetal expulsion by uterine contractions during labor. The significance of increased TSP1 mRNA during labor may relate to homeostasis and merits further study.

A proliferative effect of insulin-like growth factor-I (IGF-I) was previously shown in pancreatic islets. However, the mechanism under which IGF-I actions are exerted in insulin-secreting cells is not clear. The rat insulinoma cell line, RINm5F, was shown to have both IGF-I receptors and IGF-Il/mannose-6-phosphate receptors. IGF-I binding to cell surface receptors stimulated phosphorylation of 97 kDa and 93 kDa subunits of the IGF-I receptor and incorporation of [3H]thymidine into RINm5F cells. Both the IGF-I-induced protein phosphorylation and [3H]thymidine incorporation were abolished in the presence of the tyrosine kinase inhibitor, genistein. Under basal conditions, IGF-I did not induce insulin release or changes in cytosolic free Ca2+ concentration. Immunoprecipitation of proteins from RINm5F cells, using phosphotyrosine antibodies, followed by western blotting using antibody against IRS-1 revealed no distinct band of phosphorylated insulin receptor substrate (IRS)-1. Instead, tyrosine-phosphorylated IRS-2 was detected and stimulated by IGF-I when western blotting was performed using antibody against IRS-2. These results indicate that IRS-1 is not likely to be involved in IGF-I signalling in RINm5F cells. Hence, IGF-I stimulated DNA synthesis in RINm5F cells was associated with phosphorylation of IGF-I receptors and IRS-2.

In the present study we characterized two labor-induced genes, DSCR1 (Down syndrome candidate region 1) and TCTE1L (murine t-complex like), which were identified by suppression subtractive hybridization in the pregnant ovine myometrium. DSCR1 and TCTE1L cDNA sequences were retrieved from a custom-made labor-myometrial cDNA library by hybridization screening. The characterized cDNA sequences include 5'-untranslated region (UTR), coding region and 3'-UTR, which are 12 bp, 351 bp and 1716 bp for TCTE1L, and 64 bp, 594 bp and 1539 bp for DSCR1 respectively. The two cDNA sequences encode proteins of 116 and 197 amino acids for TCTE1L and DSCR1 respectively. Northern analysis further confirmed the significant increases of myometrial DSCR1 and TCTE1L mRNA associated with spontaneous term labor (n=6) compared with gestation-matched controls not in labor (n=6). The abundance of DSCR1 and TCTE1L mRNA was attenuated when myometrial contraction was inhibited by Nimesulide (n=6), a specific prostaglandin H synthase 2 inhibitor. Fetal occupancy greatly upregulated DSCR1 and TCTE1L mRNA in the gravid horn during betamethasone-induced premature labor (n=6) compared with the non-gravid horn not in labor (n=3). Estradiol upregulated TCTE1L mRNA, but had no effect on DSCR1 mRNA expression in the non-pregnant sheep myometrium. Progesterone alone had no effect on both DSCR1 and TCTE1L mRNA expression, however progesterone antagonized estradiol's stimulating effect on myometrial TCTE1L mRNA expression in ovariectomized non-pregnant sheep. Upregulation of DSCR1 and TCTE1L in both betamethasone-induced premature labor and spontaneous term labor and inhibition of their expression by Nimesulide suggest a functional role of these two genes in myometrial activation associated with onset of labor. Mechanical stretch, labor and steroids differentially regulated DSCR1 and TCTE1L mRNA in the pregnant and non-pregnant sheep myometrium.

Aldosterone, which plays a key role in the regulation of blood pressure, is produced by zona glomerulosa (ZG) cells of the adrenal cortex. Exaggerated overproduction of aldosterone from ZG cells causes primary hyperaldosteronism. In ZG cells, calcium entry through voltage-gated calcium channels plays a central role in the regulation of aldosterone secretion. Previous studies in animal adrenals and human adrenal adrenocortical cell lines suggest that the T-type but not the L-type calcium channel activity drives aldosterone production. However, recent clinical studies show that somatic mutations in L-type calcium channels are the second most prevalent cause of aldosterone-producing adenoma. Our objective was to define the roles of T and L-type calcium channels in regulating aldosterone secretion from human adrenals. We find that human adrenal ZG cells mainly express T-type Ca_V3.2/3.3 and L-type Ca_V1.2/1.3 calcium channels. TTA-P2, a specific inhibitor of T-type calcium channel subtypes, reduced basal aldosterone secretion from acutely prepared slices of human adrenals. Surprisingly, nifedipine, the prototypic inhibitor of L-type calcium channels, also decreased basal aldosterone secretion, suggesting that L-type calcium channels are active under basal conditions. In addition, TTA-P2 or nifedipine also inhibited aldosterone secretion stimulated by angiotensin II- or elevations in extracellular K⁺. Remarkably, blockade of either L- or T-type calcium channels inhibits basal and stimulated aldosterone production to a similar extent. Low concentrations of TTA-P2 and nifedipine showed additive inhibitory effect on aldosterone secretion. We conclude that T- and L-type calcium channels play equally important roles in controlling aldosterone production from human adrenals.

Activated signaling proteins regulate diverse processes, including the differentiation of the pancreatic islet cells during ontogeny. Here we uncover the in vivo phosphorylation status of major growth factor-activated signaling proteins in normal adult mice and during pancreatic islet regeneration. We report elevated phospho-mitogen-activated protein kinase (phospho-MAPK), phospho-c-Jun-NH₂-terminal kinase (phospho-JNK), and phospho-p38 MAPK expression during pancreatic regeneration. Immunoblotting experiments demonstrated elevated phosphorylation of p52 Src-homology/collagen (SHC) in the ductal network as well, substantiating the activation of this pathway. Furthermore, protein kinase B (PKB/Akt), a key signaling protein in the anti-apoptotic pathway, was phosphorylated to a greater extent in the ductal network from regenerating pancreas. We observed fibroblast growht factor (FGF)10 and platelet-derived growth factor (PDGF)AA expression in embryonic as well as regenerating adult pancreas. Epidermal growth factor (EGF) and PDGFAA stimulated MAPK and Akt phosphorylation, while FGF10 stimulated MAPK but not Akt phosphorylation in a time-dependent manner in freshly isolated cells from the adult ductal network. These data suggest that a heightened level of expression and stimulation of key signaling proteins underlie the expansion and differentiation processes that support pancreatic ontogeny and regeneration.

It has been documented that stress or glucocorticoids have conflicting effects on memory under different conditions. However, it is not fully understood why stress can either impair or enhance memory. Here, we have examined the performance of six age groups of Wistar rats in a water maze spatial task to evaluate the effects of stress under different conditions. We found that the impairment or enhancement effect of an 'elevated platform' (EP) stress on memory was dependent on previous stress experience and on age. EP stress impaired memory retrieval in water maze naive animals, but enhanced rather than impaired memory retrieval in young water maze stress-experienced animals. Furthermore, exogenously applied corticosterone or foot shock stress before water maze training prevented the impairment of memory retrieval that should be induced by treatment with corticosterone or foot shock before the 'probe trial'. Again, memory retrieval was enhanced in young animals under these conditions, and this enhancement can be prevented by the glucocorticoid receptor antagonist RU 38486. Thus, glucocorticoid receptor activation not only induced impairment of memory but also increased the capacity of young animals to overcome a later stress. The present findings suggest that the effect of stress on memory can be switched from impairment to enhancement dependent on both stress experience and age.

Macrophage migration inhibitory factor (MIF) is an essential regulator of the macrophage responses to endotoxin. MIF also has the ability to override the anti-inflammatory actions of glucocorticoids during an immune response, and is thus an important pro-inflammatory factor. The presence of MIF in cells of the anterior pituitary has been described, and high levels of MIF in other rapidly proliferating tIssues have also been demonstrated. It has been hypothesised that MIF release from these cells is influenced by the hypothalamo-pituitary-adrenal axis, and that ACTH and MIF are released simultaneously to exert counter-regulatory effects on cortisol. However, another intracellular role for MIF has also been suggested as it has been shown that MIF exerts an effect on the inhibitory cell cycle control protein p27 through an interaction with Jab1, a protein implicated in p27 degradation. We studied MIF expression in different normal and adenomatous human pituitary samples using immunohistochemistry and RT-PCR. There was evidence of co-immunoprecipitation of MIF with Jab1, suggesting an interaction of the two proteins. Our results showed that there is increased expression of MIF protein in the nuclei of all pituitary adenomas compared with normal tIssue (P=0.0067), but there was no statistically significant difference in nuclear MIF expression between the different adenoma types. Nuclear MIF expression correlated positively with p27 and its phosphorylated form in normal tIssue (P=0.0028 and P<0.0001); however, this relationship was not seen in the adenoma samples. Cytoplasmic expression of MIF was found to be variable both in normal and adenomatous samples, with no consistent pattern. MIF mRNA was demonstrated to be present in all tumour and normal samples studied. Somatotroph tumours showed higher MIF mRNA expression compared with normal pituitary or other types of adenomas. In conclusion, MIF is expressed in cell nuclei in pituitary adenomas to a greater extent than in normal pituitary tIssue. We speculate that it may play a role in the control of the cell cycle, but whether its higher level in adenomas is a cause or a consequence of the tumorigenic process remains to be clarified.