Browsing by Author "Dr. Kenneth Adler, Committee Chair"

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    Lactational Exposure of Atrazine and the role of Prolactin in the Development of the Adult Male TIDA Neuronal System.
    (2004-04-21) Langdale, Christopher Lawrence; Dr. Gregory Cole, Committee Member; Dr. Ida Smoak, Committee Member; Dr. Ralph Cooper, Committee Co-Chair; Dr. Kenneth Adler, Committee Chair
    Previous studies by Stoker et al., (1999b) and Shyr et al., (1986) examined the role of the rat dam's prolactin (Prl) during lactation on subsequent development on the offspring. Shyr identified a critical role for milk-derived Prl in postnatal development of tuberoinfundibular dopamanergic neurons (TIDA) by dosing lactating dams with bromocriptine, a dopamine (DA) agonist, on postnatal day (PND) 2-5, and PND 9-12 to block the release of maternal Prl in the milk. Offspring deprived of milk Prl from PND 2-5 showed a decrease in DA concentration and turnover rate in the median eminence on PND 33-35, demonstrating the importance of Prl to normal TIDA development. Furthermore, this decrease in DA was associated with hyperprolactinemia in male offspring when serum concentrations were measured on PND 30-32. However, no disruption of the TIDA development was observed in dams dosed from PND 9-12, suggesting that PND 2-5 was a sensitive time point for normal development of TIDA neurons. Stoker et al., (1999b) found that exposure of the dam to atrazine during the first 4 days of lactation suppressed maternal suckling-induced prolactin release. Interestingly, this treatment also led to an increased incidence of lateral prostate inflammation in adult offspring. However, prostate inflammation was absent in the male offspring when the atrazine-treated dam was co-treated with ovine prolactin. These observations have led to the hypothesis that decreased maternal prolactin (following atrazine or bromocriptine exposure) will lead to impaired TIDA development and elevated levels of prolactin in the juvenile male offspring (as shown by Shyr et al., 1986 and Stoker et al., 1999b). The two basic assumptions of this hypothesis have yet to be examined and are the focus of this study. First, can an increase in the concentration of serum Prl be determined in male offspring of dams treated with atrazine during early lactation; and second, is there evidence of a decrease in TIDA neuronal activity (DA concentration and turnover) following atrazine exposure? The potential of this herbicide to affect hormones normally present in milk and transiently alter development of the human offspring may be of concern to human health. Lactating females were dosed from PND 1-9, twice a day with 12.5, 25, or 50 mg/kg atrazine or 0.209 mg/kg bromocritine (PND 1-5). Male pups were selected and killed two hours before lights were turned off (14:10 light cycle). Serum Prl and TIDA neuronal development results supported both basic assumptions. An increase in serum Prl in male offspring of both atrazine 12.5 mg/kg and bromocriptine-treated dams were identified on PND 12 and 35, which was also consistent with prior reports by Shyr et al. (1986). The increase in serum Prl also coincided with decreased TIDA neuronal activity on PND 35. However, a non-linear dose response was observed following atrazine exposure to the dam in both brain aminergic and serum Prl concentrations in the offspring. Also, both compounds caused significant changes in the developmental profiles of dopamine and dopaminergic activity in all three brain areas tested. Although the atrazine data revealed a non-linear dose response in the offspring, the fact that the lowest dose of this herbicide produced changes that were similar to the positive control suggests that indeed this complex mode of action is affected by atrazine-treatment. Due to the complexity of the maternal-neonatal unit, subsequent studies examining the offspring at a later age will be required.
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    Role of Protein Kinase C Delta in Airway Mucin Secretion
    (2006-11-19) Park, Jin-Ah; Dr. Kenneth Adler, Committee Chair
    Mucin hypersecretion is a prominent pathophysiologic feature of chronic airway diseases such as cystic fibrosis, asthma and chronic bronchitis. Exacerbated mucin secretion can lead to impaired mucociliary function, increased susceptibility to bacterial pathogens, potentiation of inflammatory responses, and, in extreme cases, death. Despite these deleterious effects, effective therapy to alleviate mucin hypersecretion remains to be developed. The intention of this work was to elucidate the mechanism of mucin secretion in response to secretagogues utilizing normal human bronchial epithelial (NHBE) cells in vitro. NHBE cells were maintained in air/liquid interface and allowed to differentiate into a normal airway epithelium containing mucin secreting (goblet), ciliated and basal cells. Mucin secretion was measured by an enzyme linked immunoabsorbent assay (ELISA) using antibodies recognizing specific mucin proteins (MUC5AC, MUC5B and MUC2) or pan-secreted mucins. In manuscript one, we elucidate some aspects of the mechanism whereby human neutrophil elastase (HNE) enhances mucin secretion from NHBE cells. We found that HNE provokes mucin secretion in a concentration-dependent manner, with maximal stimulation (more than two-fold) occurring within a short (15 minute) time period. The mucins, MUC5AC and MUC5B, but not MUC2, were released in response to 500nM HNE. Myristoylated alanine-rich C-kinase substrate (MARCKS) is rapidly phosphorylated in response to HNE exposure (within 2 minutes), suggesting activation of protein kinase C (PKC) is involved in HNE-induced mucin hypersecretion. PKCδ was the only isoform to translocate from cytoplasm to membrane in response to HNE, and inhibition of PKCδ by a specific pharmacologic inhibitor (rottlerin) attenuated HNE-mediated mucin secretion. Therefore, we concluded that HNE induces mucin secretion via the activation of PKCδ in normal human bronchial epithelial cells in vitro. In manuscript two, we further investigated the role of PKCδ in mucin secretion. Selective activation of PKCδ by bryostatin 1 provoked mucin secretion and induced phosphorylation of MARCKS in NHBE cells. Suppression of PKCδ by rottlerin significantly attenuated HNE-or PMA-mediated mucin secretion as well as phosphorylation of MARCKS. A virally immortalized human bronchial epithelial cell line (HBE-1) was utilized for transient transfections. Transfection of HBE-1 cells with a dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) significantly attenuated PMA-induced mucin secretion and phosphorylation of MARCKS compared to cells transfected with empty vector (pEGFP-N1) alone. These results suggest that PKCδ regulates mucin hypersecretion in human airway epithelial cells.