The possible role of substance P in mediating inflammation in inflammatory bowel disease (IBD) was evaluated by blocking the substance P (NK-1) receptor in an acute animal model. Ethanol and zymosan were administered via the rectum into the colon in anesthetized male Sprague-Dawley rats to induce the model. To assess the role of substance P, 5mg/kg of CP-96,345 was administered subcutaneously thirty minutes prior to induction of IBD and every hour for three hours, at which time testing was begun. In another group of rats, 3 00mug/kg of an antisense oligonucleotide targeted at NK-1 receptor mRNA was administered intraperitoneally twice daily for seven days prior to induction of IBD. Histological sections revealed an infiltration of inflammatory cells in the colons after ethanol/zymosan treatment. Plasma extravasation values in rats treated with ethanol/zymosan were significantly higher than in controls treated only with saline (P < 0.0001) or saline and ethanol (P = 0.0041). In ethanol/zymosan treated rats, those administered CP-96,345 had plasma extravasation values which were significantly less than in ethanol/zymosan treated controls (P < 0.0001). Administration of the antisense targeted at NK-1 receptor mRNA resulted in lower levels of plasma extravasation compared with controls (P < 0.01). NK-1 receptors may be involved in the expression of symptoms as a component of inflammation in IBD.

Properly assembled elastic fibers play an important role in providing skin with the properties of elasticity and resilience to allow for considerable mobility, but the mechanisms involved in elastic fiber assembly remains unknown. Fibulin-5 is an extracellular 66kDa glycoprotein synthesized and secreted by fibroblasts in skin, and has the ability to bind to tropoelastin. This study addresses (1) the role of fibulin-5 in the ultrastructure of elastic fibers in skin, and (2) its function in the cutaneous mechanical properties by using wild type and fibulin-5 null mice. In the first part, skin of fibulin-5 null mice as well as wild type mice was investigated in order to gain insight into the function of fibulin-5 in elastic fiber assembly. Using light and electron microscopy, the dramatic defects of dermal elastic fibers in the absence of fibulin-5 were analyzed. Interestingly, in the immunoelectron microscopy for LOXL-1, an enzyme responsible for the elastin cross-links, fibulin-5 null mouse skin exhibited similar immunoreactivity for LOXL-1 to wild type skin. Moreover, in the wild type skin, fibulin-5 localized to the microfibril-elastin interface at the edges and within the elastic fiber. In the second part of this study, the function of fibulin-5 in skin biomechanics was studied in order to determine its role in skin strength and elasticity. By using tensile tests, fibulin-5 null mouse skin was found to be significantly stiffer and weaker than wild type skin. Taken together, the defective elastic fibers in the absence of fibulin-5 suggest that fibulin-5 is involved in a secondary step of cutaneous elastic fiber assembly and in the mechanical properties of adult mouse skin.

Airway hyperresponsiveness to bronchoconstrictors has been found in asthma and related to the severity of the disease. The factors which result in hyperresponsiveness are not completely understood. A possible mechanism is an imbalance between endogenous bronchoconstrictors and dilators. NO is known to relax tracheal smooth muscle by activating soluble guanylate cyclase and increasing the level of intracellular cyclic guanosine monophosphate (GMP). The first hypothesis tested was that the NO-cyclic GMP-relaxant pathway is involved in the regulation of airway responsiveness. Inhibition of endogenous nitric oxide by N$ sp{ omega}$-nitro-L-arginine (L-NNA) significantly increased airway responsiveness to inhaled methacholine in normoresponsive Lewis rats but less so in hyperresponsiveness Fisher rats. In addition, carbachol increased cyclic GMP levels in tracheal tissues from both strains; this cyclic GMP accumulation in tracheal tissues was also less in Fisher than in Lewis rats and abolished by L-NNA in both strains, indicating that it was mediated by a NO-dependent mechanism. These results suggest that endogenous NO plays a role in regulation of airway responsiveness and contributes to the strain-related difference in airway responsiveness in rats. To investigate the NO-cyclic GMP-relaxant pathway in rat airway, the effect of sodium nitroprusside (SNP, a NO donor) on airway responsiveness to a cholinergic agonist was measured in hyperresponsive Fisher rats and compared with the less responsive Lewis strain. Fisher rats were resistant to SNP as evidenced by less relaxation of carbachol contracted tracheal rings by SNP and less cyclic GMP accumulation induced by SNP in cultured airway smooth muscle cells in Fisher rats compared with Lewis rats, indicating an impaired response to SNP in Fisher airways. / NO is known to be synthesized from L-arginine in a reaction catalyzed by NO synthase (NOS). Liver cytochrome P450 also catalyzes the oxidative cleavage of C=N bonds of compounds containing a -C(NH$ sb2$)NOH function, producing NO in vitro. We hypothesized that the biosynthesis of NO in airway smooth muscle cells could result from P450 enzymes acting on appropriate substrates. NO can be synthesized in a number of lung cell types. However, to date, no constitutive form of NOS activity has been found in airway smooth muscle cells. We next examined the possibility that airway smooth muscle itself might be able to synthesize NO. Formamidoxime, a compound containing the -C(NH$ sb2$)NOH function, was found to produce NO in cultured airway smooth muscle cells. As well, formamidoxime relaxed pre-contracted trachealis and cyclic GMP accumulation in airway smooth muscle cells in culture. These effects were inhibited by P450 inhibitors but not by NOS inhibitors. Thus, an L-arginine-independent pathway for production of NO was demonstrable in airway smooth muscle cells. This NO production was catalyzed by P450 but not by NOS. / In conclusion, my studies have demonstrated an important role for endogenous NO production in determining the airway responsiveness of normal rats to inhaled cholinergic agonists. This mechanism contributes to strain-related differences in airway responsiveness in the rat.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) stimulate signal transduction in target cells by binding to the same G protein-linked receptor (PTHR). The PTHR mediates PTH signaling in kidney and bone, and PTHrP binding to the receptor has been shown to be essential for normal endochondral bone formation in humans and mice. / Expression of the murine PTHR gene is controlled by two promoters that are regulated differently. Whereas the activity of the upstream promoter (P1) is mainly restricted to the kidney, where it provides the bulk of the gene transcription, the downstream promoter (P2) is more widely active. We characterized the upstream regulatory region of the human PTHR gene and showed that its organization is very similar to that of the mouse PTHR gene. RNase protection experiments revealed, however, that the homologue of the mouse renal-specific promoter PI, is only weakly active. This observation led us to identify and characterize a third promoter P3) that is responsible for the expression of more than 80% of human renal transcripts, but is apparently not active in mouse kidney. A study of the tissue distribution of the activity of P1, P2 and P3 showed that, while P2 and P3 are widely active, function of P1 is restricted to renal tissues. This study further revealed the existence of a shorter, differentially spliced variant of P2 derived transcripts. P1 and P3 were found to be developmentally regulated, as no activity was detected at mid-gestation. Hence, expression of the PTHR gene until this stage is driven solely by P2. Furthermore, the shorter P2-specific transcript observed in adult not detected in fetal tissues, suggesting that differential splicing of PTHR mRNA is developmentally regulated as well. / The observation that activity of PI and P3 is developmentally up-regulated, and the presence of P3 within a CpG island, prompted us to examine whether DNA methylation could play a role in regulating PTHR promoter function. Our results show that all three promoters are sensitive to methylation in vitro and that the methylation pattern of Pl is renal specific and is established well before the onset of PTHR gene expression. Our results suggest that while the transcriptional regulation of the PTIHR gene expression during development might be similar in mouse and human, expression in the adult is likely to be controlled by different mechanisms. / We also addressed the presence of structural alterations of the 5 ' regulatory region in patients with pseudohypoparathyroidism type Ib. Genomic southern blot analysis, as well as sequencing of the three upstream untranslated exons did not reveal any deletion or point mutation that could account for the kidney specific loss of PTH response observed in these patients.

The structure of rat hepatic prolactin (PRL) receptors was examined in various subcellular fractions by immunoblotting. In all subcellular fractions, a single 42 kDa species was identified. Tri- and/or tetrantennary complex carbohydrates compose $ sp sim$7 kDa of the weight of the receptor. Enzymatic deglycosylation of the mature receptor does not appreciably reduce ligand binding or antibody recognition of the receptor. However, core glycosylation of the receptor is necessary for the acquisition of binding capacity and antibody recognition. / The intracellular distribution of PRL receptors was determined by Percoll gradient centrifugation and application of the diaminobenzidine (DAB)-shift methodology. There was a sex-dependent distribution of PRL receptors, with females having a 3-fold higher concentration of receptors in early endosomes than males. This discrepancy disappeared when male rats were treated with estrogen. No such sex-dependent distribution of intracellular receptors were discovered for insulin receptors. / Colchicine treatment of estrogen-induced male rats prevented newly synthesized receptors from reaching the cell surface, and allowed the accumulation of at least some of these receptors into an endosomal compartment. These studies suggest that under conditions of colchicine blockade, PRL receptors may access an endosomal compartment by an entirely intracellular route. To date, such a route has only been described for mannose-6-phosphate receptors.

During embryogenesis, patterning is one of the key processes by which the body plan of the embryo becomes established. Patterning of the A-P axis is accomplished by the products of the Hox gene family. In mammals, there are 39 genes in the Hox family that have been grouped into four clusters (A-D) by virtue of their chromosome location. Cross-species studies have confirmed the conservation of the Hox genes during evolution. In vivo, the products of the Hox genes function as transcription factors. The genes have a highly conserved sequence called the homeobox, which translates into a 60 amino acid homeodomain. The homeodomain is a DNA-binding domain made up of three alpha helices and an N-terminal arm which precedes helix 1. Since most HOX homeodomains bind a common core sequence (TAAT), the mechanism by which these proteins achieve specificity in target gene regulation was unknown for some years. This was resolved by the identification of extradenticle (exd). Genetic analysis of zygotic and maternal effect mutations of exd demonstrated that it is a required cofactor for the Drosophila Hox gene products. Examination of exd mutations showed posterior and anterior transformations with alteration in segmental identities. These transformations were similar to those caused by Hox genes. Further analysis of the Hox and exd mutations in vivo, substantiated by in vitro biochemical evidence showed that exd acts in concert with homeotic gene products by cooperatively binding DNA. The mammalian homologue of exd is PBX. PBX functions analogously to its Drosophila counterpart by modulating the DNA-binding specificity of HOX proteins. In mammals, PBX interacts with members of the first 10 paralog group HOX proteins. A second cofactor, MEIS with a high degree of homology to PBX was identified very recently. Both PBX and MEIS belong to the three amino acid loop extension (TALE) family of homeodomain proteins. The homeodomain of these proteins differ from the HOX homeodomains in t

The 9 PAX genes constitute a family of developmental transcriptional regulators characterized by a highly conserved "paired-box" domain. Mutations in 6 of the 9 PAX genes result in autosomal dominant congenital malformations in mice and in humans. PAX2 transcripts are detected in the CNS, throughout the genitourinary tract, eye and ear. In humans, mutations of PAX2 cause Renal-Coloboma Syndrome (RCS), a constellation of renal anomalies, eye defects and, less frequently, hearing loss and mild CNS manifestations. Homozygous PAX2 mutations in mice cause complete renal agenesis and perinatal death. PAX8 transcripts are found in the CNS, kidney and thyroid glands. PAX8 mutations cause congenital thyroid hypoplasia. No renal defects have been detected either in human or murine PAX8 mutants. / In spite of considerable information about PAX2 and PAX8 genes, their precise functions in development are poorly understood. This project was aimed at elucidating the functions of PAX2 and PAX8 in nephrogenesis and is subdivided into two parts: mutational screening of the PAX8 gene (Chapter 4) and delineation of the role of PAX2 in developing kidney (Chapters 5--7). The latter evolved into the major portion of this work. / Familial juvenile nephronophthisis (NPH1) is a rare disease characterized by multiple small cysts at the cortico-medullary junction and end-stage renal failure in pediatric populations. Because of its proximity to the disease locus, we considered PAX8 a candidate gene for NPH1. During analysis of PAX8 exons from patients, we identified a rare non-conservative polymorphic amino acid change, but found no causative mutations. Subsequently, other groups isolated the novel NPH1 gene. Following reports that PAX8 knockout mice have no obvious renal anomaly, we considered the possibility that PAX8 mutations might, nevertheless, affect proximal tubule function. In patients with thyroid hypoplasia and proven PAX8 mutations we found no aminoaciduria, indicating that haploinsufficiency for PAX8 does not alter tubular transport function. / In order to define PAX2 function in the two primordial cell lineages of developing kidney (induced metanephric mesenchyme and ureteric epithelium), we used both a cell culture approach and an in vivo PAX2 mutant mouse model (1Neu). We demonstrated that PAX2 plays a dual role. In mesenchymally-derived HEK293 cells expressing regulatable exogenous PAX2, we showed that PAX2 is responsible for expression of genes involved in differentiation of mesenchyme. Contrary to one published hypothesis, we found that PAX2 does not affect cell division. In ureteric epithelium, however, PAX2 plays a different role, serving as a survival factor, critical for sustaining the ureteric bud stem cell population. Attenuation of PAX2 dosage (1Neu mouse mutants or collecting duct cells transfected with an antisense PAX2 vector) results in increased apoptosis. We demonstrate that the primary renal defect in RCS is reduced nephron number associated with excessive apoptosis and simplified branching of the ureteric bud. We hypothesize that arborization of the uretric bud requires accumulation of sufficient stem cell mass to allow the next round of branching---possibly by lifting the branch point beyond a putative local inhibitory field. / In summary, we establish that inactivation of PAX8 gene expression does not disturb normal kidney development and function. Conversely, PAX2 plays a crucial dual role in the two primordial kidney cell lineages: being a differentiating factor in mesenchyme and a survival factor in ureteric epithelium.

Hox genes encode homeodomain-containing transcription factors that specify positional identity along body axes during animal embryogenesis. Correct spatially and temporally restricted expression of Hox genes is critical for normal embryonic development. The alteration of Hox gene expression may result in homeotic transformation or malformation of the embryo. / Using transgenic assays, we found that a previously mapped 5' retinoic acid response element (RARE) was not required for Hoxd4 gene expression in the central nervous system (CNS), but contributed to a 5' mesodermal enhancer. Neural and mesodermal enhancer activity of the gene were mapped to a 3' 4 kb region (region A). Removal of this region virtually abolished Hoxd4 expression in the CNS and posterorized expression in the somitic mesoderm by one somite. Further deletional analysis of the neural enhancer led to the identification of a 3' RARE. Mutation of this element virtually abolished transgene expression in the CNS. Analysis of permanent transgenic lines showed that this element was required not only for the initiation but also for the maintenance of Hoxd4 expression in the neural tube. In addition, this element is required for the Hoxd4 response to retinoic acid (RA). The second regulatory element we identified (TTTTCTG) is 2 bp downstream to the 3' RARE. It resembles a CDX binding site, but failed to interact with human CDX1 protein. Mutation of this site posteriorized transgene expression in the CNS. Although other regions required for murine Hoxd4 expression in the CNS were also mapped, our results suggest that Hoxd4 lacks an autoregulatory element (ARE). This is in contrast to the regulation of Hoxb4, where expression is initiated by the conserved RARE and subsequently maintained by an ARE. Together with other findings, this suggests that Hox genes sharing similar expression pattern may be regulated in a diverged manner.

The intrahypothalamic distribution of mu, delta and kappa opioid receptor types was examined by in vitro radioautography using the opioid ligands $ sp{125}$I-FK-33 824, $ sp{125}$I-DTLET and $ sp{125}$I-DPDYN, respectively as selective markers. The density and distribution of these receptors in the hypothalamus of normal rats was then compared to that of rats injected with estradiol valerate in order to verify the location of the described increase in $ sp3$H-naloxone binding and to identify the specific opioid receptor type involved. Analysis of opioid receptor changes following long-term exposure to estradiol revealed that mu opioid binding densities were significantly increased in the medial preoptic area of EV-treated animals. Delta and kappa opioid binding densities were unchanged in the medial preoptic area although a slight decrease in delta sites was observed in the suprachiasmatic nucleus. Hypothalamic $ beta$-endorphin concentrations were concomitantly decreased in EV-treated animals, suggesting that observed increases in mu opioid binding were due to a compensatory up-regulation of receptors secondary to loss of $ beta$-endorphin input from the arcuate nucleus. To confirm this interpretation, mu opioid receptor binding was measured in the MPOA of animals treated with monosodium glutamate, which destroys the arcuate nucleus. Results indicated that mu opioid receptor binding densities were inversely proportional to hypothalamic $ beta$-endorphin concentrations in the same animals supporting the existence of a causal relationship between chronic reductions in hypothalamic $ beta$-endorphin concentrations and mu opioid receptor upregulation in the medial preoptic area. / To further document the decreased concentrations of $ beta$-endorphin in the hypothalamus of EV-treated animals, light microscopic immunocytochemistry for $ beta$-endorphin was performed in colchicine treated control and EV-injected rats. Eight weeks following EV treatment, a 60% decrease in the total number of $ beta$-endorphin-immunoreactive neurons was detected in the arcuate nucleus, while neuron numbers for nearby neuronal populations were unchanged. These results were confirmed in biochemical experiments demonstrating reduced hypothalamic $ beta$-endorphin concentrations in the absence of changes in neuropeptide-Y and met-enkephalin in EV-treated rats as compared to controls. Cell counts performed in Nissl-stained material using unbiased stereological methods revealed a reduction in the total number of neurons in the EV-treated group as compared to controls. Furthermore, the estimated number of neurons lost ($ sim$3500) corresponded precisely with the total number of $ beta$-endorphin neurons lost ($ sim$3600) as estimated using quantitative immunocytochemistry. Together, these findings strongly suggest that $ beta$-endorphin neurons are selectively destroyed following long-term exposure to estradiol. Results demonstrated that EV-treated animals co-treated with vitamin E displayed hypothalamic $ beta$-endorphin concentrations similar to controls. In addition, these animals maintained regular estrous cycles and displayed normal ovarian morphology. These findings suggest that estradiol-induced neurotoxicity of $ beta$-endorphin neurons involves the production of free radicals and further supports the notion that the loss of these neurons is important to the induction of chronic anovulation and polycystic ovaries resulting after EV treatment. (Abstract shortened by UMI.)

The anterior pituitary gland is an essential endocrine organ. The primordium of the anterior pituitary gland, Rathke's pouch, is formed by the invagination of somatic ectoderm that lines the roof of the oral cavity. It has been shown that SHH is important in pouch formation, but there is a controversy over where the source of the signal is originating from the neural or oral ectoderm. / The objective of my project was to describe the expression pattern and the role of Shh in the specification of Rathke's pouch in the chick embryo. I analyzed the expression pattern of Pitx2 and Lim3 which are two transcription factors required for pituitary development. Pitx2 was expressed throughout Rathke's pouch and the oral ectoderm during pituitary formation. Lim3 was expressed in Rathke's pouch, with stronger expression on the rostral side of the pouch. / Interfering with the SHH signalling pathway at the time of development of cells that will form the hypophyseal placode altered the expression patterns of Pitx2 and Lim3. This suggests that the SHH signal from the neural ectoderm plays a role in pituitary patterning. (Abstract shortened by UMI.)