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<p>Fluid secretion is essential to organ development and function, yet relatively little is known about the roles of fluid secretion <italic>in vivo</italic>. Early in development, fluid secretion plays important roles during the process of lumen formation and is necessary for organ homeostasis throughout life. A human disease, cystic fibrosis (CF) is caused by loss of cystic fibrosis transmembrane conductance regulator (CFTR) function, a chloride channel and key regulator of vertebrate fluid secretion. CFTR regulates fluid secretion by governing ion transport and osmotic gradients across epithelia. </p><p>To identify the developmental requirements for <italic>cftr</italic> function, we generated <italic>cftr</italic> mutant zebrafish using transcription activator like effector nucleases (TALENs). In <italic>cftr</italic> mutant zebrafish, we observed defects in the specification of left-right (LR) asymmetry. In the zebrafish, LR asymmetry is specified in part by directional fluid flow within a ciliated structure, Kupffer's vesicle (KV). Using live imaging of several transgenic markers in KV, we determined that lumen expansion is impaired in <italic>cftr</italic> mutants, which prevents directional fluid flow necessary for KV function. To examine <italic>cftr</italic> expression, we generated bacterial artificial chromosome (BAC) transgenic zebrafish expressing fluorescent Cftr fusion proteins under the control of the <italic>cftr</italic> promoter. These transgenes express Cftr within the KV epithelium and the protein localizes to the apical membrane. These transgenes rescue the KV function and the specification of LR asymmetry. These studies reveal a new role for <italic>cftr</italic> during KV morphogenesis and function in the zebrafish. </p><p>In the zebrafish pancreas, we found that loss of <italic>cftr</italic> function leads to defects reminiscent of CF including destruction of exocrine tissue and changes in islet morphology. Additionally, we observed exocrine pancreatic destruction by 3 weeks post fertilization (wpf). Analysis of <italic>cftr</italic> BAC expression in the adult and larval zebrafish pancreata revealed that <italic>cftr</italic> is expressed specifically within the ducts, localized to the apical membrane throughout life. Adult <italic>cftr</italic> mutant pancreata developed substantial degeneration of exocrine tissue and experienced reduced growth rates. In contrast, we found that <italic>cftr</italic> is not necessary for the specification or initial development of the larval pancreas. Exocrine and endocrine tissues developed similarly in WT and <italic>cftr</italic> mutant larvae. These results indicate that <italic>cftr</italic>-dependent fluid secretion is important for maintenance of the zebrafish pancreas. Altogether, these studies of <italic>cftr</italic> function in KV and the pancreas demonstrate that fluid secretion is an essential component of lumen morphogenesis and organ function.</p> / Dissertation

Insect haemolynph typically contains very high levels of free amino acids 50 1 00 times that which is normal for mammalian plasma. This study examines the modulatory effects of amino acids on fluid secretion and ion transport by isolated MTs of Rhodnius prolixus and Drosophila melanogaster. The results show that the secretion rates of isolated Malpighian tubules of both Rhodnius and Drosophila are modulated by the presence of specific amino acids in the bathing saline. Some amino acids are stimulatory, some are inhibitory and others have little or no effect. Glutamine appears to be particularly important as a stimulant of fluid secretion. As well, secreted fluid pH and Na +concentration increase and K+ concentration decreases in response to glutamine. Amino acids do not appear to be important as metabolite. in Rhodnius tubules, nor do they act to draw significant amounts of water into the lumen by osmosis. Significant stimulation of fluid secretion can be achieved by physiological levels of particular amino acids, whereas those amino acids that inhibit fluid secretion only do so at concentrations much above those at which they occur naturally in the haemolymph. Amino acids are known to be compatible osmolytes and may be acting to maintain cell homeostasis and thus to sustain fluid secretion. The passive movement of amino acids may result in cell volume changes, and some form of osmosensor is may be coupled to activation of specific kinases to produce the observed increases in fluid secretion. The effects of several kinase inhibitors were therefore examined. The glutamine dependent increase in MT fluid secretion is blocked by two inhibitors of the stress activated protein kinase (SAPK) pathway, SP600125 and dicumoral. Inhibitors of other kinases (PKA, PKC, PKG, PI-3, p38, ERK and MEK), did not block glutamine's effects on fluid secretion rate. Alterations in cytoskeletal structure appear not to be required because cytoskeletal disrupting agents did not block the glutamine dependent inc~ease in fluid secretion, nor was the increase dependent upon protein synthesis. Results of this study are the first to suggest a role for the SAPK pathway in the control of fluid secretion rates by insect Malpighian tubules. / Thesis / Master of Science (MS)

The fetal lung epithelium secretes fluid into the potential pulmonary air-spaces by actively transporting chloride (Cl¯) into the lung lumen. This Cl¯-driven fluid secretion declines with the progression of lung development. Recent studies demonstrate that the A-type γ-aminobutyric acid receptor (GABAAR), a Cl¯ channel, and glutamic acid decarboxylase (GAD65/67), key GABA-synthesizing enzymes, are expressed in adult pulmonary epithelial cells (ECs), forming an autocrine GABAAR signaling system. My thesis study revealed that GABAAR π- and β2- subunits are expressed in high levels in the fetal rat lung epithelium and decline at birth, consistent with pattern of fluid secretion. Immunohistochemistry showed distinct profiles of expression for GABAAR subunits and GAD65/67. Treatment of alveolar ECs with dexamethasone reduced the GABAAR π-subunit expression. These results suggest that the GABAAR signaling in the fetal pulmonary epithelium is developmentally regulated and the GABAAR expression and GABAAR-mediated Cl¯ secretion in pulmonary ECs may be regulated by glucosteroids.

The fetal lung epithelium secretes fluid into the potential pulmonary air-spaces by actively transporting chloride (Cl¯) into the lung lumen. This Cl¯-driven fluid secretion declines with the progression of lung development. Recent studies demonstrate that the A-type γ-aminobutyric acid receptor (GABAAR), a Cl¯ channel, and glutamic acid decarboxylase (GAD65/67), key GABA-synthesizing enzymes, are expressed in adult pulmonary epithelial cells (ECs), forming an autocrine GABAAR signaling system. My thesis study revealed that GABAAR π- and β2- subunits are expressed in high levels in the fetal rat lung epithelium and decline at birth, consistent with pattern of fluid secretion. Immunohistochemistry showed distinct profiles of expression for GABAAR subunits and GAD65/67. Treatment of alveolar ECs with dexamethasone reduced the GABAAR π-subunit expression. These results suggest that the GABAAR signaling in the fetal pulmonary epithelium is developmentally regulated and the GABAAR expression and GABAAR-mediated Cl¯ secretion in pulmonary ECs may be regulated by glucosteroids.

Abdominal surgery inhibits gastrointestinal motility, a phenomenon referred to as postoperative ileus. Since the postoperative ileus disturbs duodenal physiology it is important to minimize the side effects of this condition. Recent experiments in our laboratory show that treatment of anaesthetised rats with parecoxib, a selective cyclooxygenase-2 inhibitor, prevents duodenal postoperative ileus, increases duodenal mucosal bicarbonate secretion and improves other functions as well. One aim of the thesis was to investigate whether removal of luminal chloride affect the parecoxib- and the vasoactive intestinal peptide (VIP)-induced stimulation of duodenal mucosal bicarbonate secretion. The proximal duodenum of anaesthetised Dark Agouti rats was perfused with isotonic solutions containing zero or low Cl- and the effect on luminal alkalinisation determined. The basal as well as the parecoxib-induced increase in alkalinisation, but not that stimulated by VIP, were markedly reduced in the absence of luminal Cl-. One important function of the duodenum is to adjust luminal osmolality towards that in the blood. It is believed that the adjustment of osmolality in the duodenum is achieved by osmosis and diffusion of electrolytes along their concentration gradients and that these processes occur predominately paracellularly. Another aim of the thesis was to examine whether prevention of postoperative ileus affects the duodenal response to luminal hypertonicity. The proximal duodenum of anaesthetised Dark Agouti and Sprague-Dawley rats were perfused with hypertonic solutions of different composition and osmolality and the effects on duodenal motility, alkaline secretion, transepithelial fluid flux, mucosal permeability and the adjustment of luminal osmolality were determined in absence and presence of parecoxib. It is concluded that COX-2 inhibition increases duodenal mucosal bicarbonate secretion by stimulating apical Cl-/HCO3- exchange in duodenocytes. Furthermore, pretreatment of anaesthetised rats with parecoxib improves a number of duodenal functions in both rat strains that contribute to improve the ability to adjust luminal osmolality. The choice of rat strain is another important feature to consider when interpreting the results because the DA strain was more responsive to luminal hypertonicity than the SD strain. Finally, several evidences are provided to suggest that the adjustment of luminal osmolality in the rat duodenum is a regulated process.