Pote
Aimpun
Department of Preventive Medicine and Biometrics
Doctor
of Public Health
2000
Major Advisor: John H. Cross, Ph.D., Department of Preventive Medicine and Biometrics
Thesis Title: A prevalence of intestinal parasites in southern Belize
Abstract
A biomedical survey of stool specimens from 82% of the population (n=672) of 5 villages in Toledo District, Belize were examined by the formalin-ethyl acetate concentration technique for the prevalence of intestinal parasitic infections. Seventy-six percent of the population was infected. The most common infection was hookworm (55%), followed by Ascaris lumbricoides (30%), Entamoeba coli (21%), Trichuris trichiura (19%), Giardia lamblia (12%), and Entamoeba histolytica (6%). The mean age of infected persons was 19 years. The frequency of infections was higher in younger age groups. Females had higher prevalence of hookworm infection than males. The living conditions of 111 surveyed households were characterized as 60% with dirt floor, 43% without toilets, 35% in overcrowded living condition, 10% using stream water and 16% drinking untreated water. A cross-tabulation and logistic regression analysis was used to identify risk and protective factors of the parasites. The risk factors for intestinal parasites were Mayan Ketchi [1.6(1,2.4)], houseworker [2.4(1.2,4.6)], and use of stream water [2.3(1.2,4.5)]. The protective factors were drinking treated water [0.4(0.2,0.9)], and wearing shoes [0.6(0.4,1)]. Prevention and control programs focusing on significant factors associated with parasite infections could save time and money by targeting populations by risk characteristics.
Donald J.
Chabot
Department of Microbiology and Immunology
Doctor of
Philosophy
2000
Major Advisor: Dr. Christopher Broder, Department of Microbiology and Immunology
Thesis Title: Structural and Functional Analysis of HIV-1 Coreceptors: Roles of Charged Residues and Posttranslational Modifications on Coreceptor Activity
Abstract
CXCR4 and CCR5 are chemokine receptors and are coreceptors for human immunodeficiency virus (HIV-1). Host cells must express CD4 and a coreceptor for optimal HIV-1 entry. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex has been under intensive investigation. To define these regions we have employed an alanine-scanning mutagenesis strategy of the extracellular domains of CXCR4 coupled with a highly sensitive reporter-gene assay for HIV-l Env-mediated membrane fusion. Using a panel of 47 different CXCR4 mutations, we have identified several charged residues that appear important for coreceptor activity for X4 Envs: mutations El5A and E32A in the N-terminus, D97A in extracellular loop (ecl)-l, and Rl88A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. Mutation to alanine of one of the six tyrosines present in CXCR4, Y7, decreased coreceptor function. In addition, alanine substitution of any of the four extracellular cysteines alone resulted in conformational changes of varying degrees, while paired cysteine deletions could partially retain structure. Our data supports the notion that all four cysteines are involved in disulfide bond formation.
We have also identified substitutions that greatly enhance or convert CXCR4's coreceptor activity to support R5 Env-mediated fusion: N11A, R30A, D187A, and D193A. Mutation of the aspartic acid at position 187 had the greatest effect on tropism. Mutation of an analogous serine in CCR5 to aspartic acid reduced CCR5 coreceptor activity with R5X4 and some R5 isolates. Mutation of the N11, a putative glycosylation site, had the second greatest effect on CXCR4 tropism. We determined that this site has a large carbohydrate moiety that is responsible for preventing CXCR4 from supporting R5 Env-mediated membrane fusion. Our data suggests the presence of conserved extracellular elements common to both CXCR4 and CCR5 involved in their coreceptor activities. These data will help to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains, and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.
Hugh M. Dainer
Department of Anatomy, Physiology & Genetics
Doctor of
Philosophy
2000
Major Advisor: Rosemary C. Borke, Ph.D., Department of Anatomy, Physiology & Genetics
Thesis Title: Alterations in the Local Axonal Environment Influence Target Reinnervation and Neuronal Survival after Postnatal Axotomy
ABSTRACT
Following peripheral nerve injury in adult animals, Schwann cells (SC) proliferate and provide guidance in the local axonal environment by generating the infrastructure along which regenerating nerves grow. A portion of the SC in the peripheral nerves of postnatal rats undergo apoptosis during normal development, and this apoptosis is augmented at the injury site and the neuromuscular junction (NMJ) following sciatic nerve axotomy. The current work determined that SC apoptosis occurs in the distal but not the proximal nerve segment after postnatal transection of the hypoglossal nerve, suggesting that SC apoptosis is a general, age-related and location-specific response to peripheral nerve injury. Apoptotic SC were found in two strategic locations for guiding axonal outgrowth during peripheral nerve regeneration: a) capping the transected end adjacent to the injury site and b) just proximal to the bifurcation of the nerve into medial and lateral branches. Electron microscopic (EM) analysis identified apoptotic and mitotic SC in close proximity within the distal nerve segment, supporting apoptosis and mitosis as closely related phenomena.
Poor rates of neuronal survival and tongue musculature reinnervation following hypoglossal nerve transection in the postnatal rat may be related to the increased apoptosis of SC in the distal nerve segment after injury. Insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) are neurotrophic factors which influence SC apoptosis in vitro. In an attempt to reduce SC apoptosis after postnatal axotomy, the effects of in vivo IGF-I, bFGF and vehicle treatments were examined in the distal nerve segment after postnatal hypoglossal nerve transection. All three treatments rescued SC, with the inclusion of bovine serum albumin (BSA) in the vehicle solution accounting for some of the decrease in SC apoptosis. While SC apoptosis was reduced with growth factor application, IGF-I treatment was deleterious to tongue musculature reinnervation and neuronal survival compared with bFGF and placebo treatments. Long-term bFGF treatment reduced neuronal survival compared with placebo treatment. These results imply that while SC in developing animals can be rescued by growth factor treatment, SC and motoneurons have not yet established the proper interactive relationship to translate this rescue into improved target musculature reinnervation or long-teml motoneuron survival.
Gerard W.
Dougherty
Department of Pathology
Doctor of
Philosophy
2000
Major Advisor: Mary Lou Cutler, Ph.D., Department of Pathology
Thesis Title: Identification of an in vitro binding protein for and the in vivo phosphorylation sites of the Ras Suppressor Rsu-l
ABSTRACT
The Ras Suppressor Rsu-l is a 33 kDa protein originally discovered as a cDNA that suppressed v-Ras transformation of NIH-3T3 fibroblasts. Rsu-l overexpression in several cell lines (NIH-3T3, U251, PC12, MCF7) increased ERK activity and levels of p21CIP, and inhibited growth rate and decreased JNK activity. Stabilization of p53 and inhibition of CDK2 and cyclin D-associated kinase activity has also been observed in MCF7 cells stably transfected with Rsu-l. In this study we attempted to identify the binding proteins to Rsu-l by the Far Western Cloning method. Preliminary experiments with a fusion protein containing the glutathione-S-transferase (GST) tag and the carboxy-terminus of Rsu-l showed binding to a protein of approximately 30 kDa both in solution and immobilized on nitrocellulose. A K562 human leukemic cell library was screened with 32P-labeled GST-C-terminal Rsu-l. Results indicate that one subclone from approximately one million plaques screened bound to the Rsu-l protein in vitro. This subclone, designated PI-4, expressed a protein of approximately 30 kDa. Sequence analysis of this subclone suggests homology to an as yet uncharacterized gene sequence on the long arm of chromosome 21 and also the WAS protein. Additional studies were done to ascertain the role of Rsu-l phosphorylation on its biological activity. The Rsu-l sequence contains several consensus phosphorylation sites for protein kinase C (PKC), protein kinase A (PKA) and casein kinase II. Previous experiments showed that Rsu-l is phosphorylated in vivo in response to growth factor and TPA, a known activator of PKC. Phosphoamino acid analysis of Rsu-l suggests that in vivo phosphorylation occurs on serine residues. In this study site-directed mutagenesis of individual Rsu-l consensus PKC sites revealed that two serine residues (serine-4 and serine-163) are phosphorylated in vivo in response to TPA. This is supported by evidence that Rsu-l phosphorylation can be specifically blocked by the PKC inhibitor bisindolylmaleimide I (HIM), but not by inhibitors of tyrosine kinase, MEK and PI3K. Stable transfectants of non- phosphorylatable Rsu-l mutants in MCF7 were isolated and tested for growth rate and effect on apoptosis. Previous studies with Rsu-l have demonstrated an enhancement of apoptosis in response to TNF-" and staurosporine in MCF7 cells, accompanied by stabilization of p53 protein levels. Results indicate that non-phosphorylatable mutants of Rsu-l still exhibit growth inhibition but do not enhance apoptosis in MCF7 cells. Together these results support the hypothesis that PKC or a PKC-dependent serine kinase mediates. the proapoptotic effect but not growth inhibitory effect of Rsu-l.
Martha M.
Faraday
Department of Medical and Clinical Psychology
Doctor of
Philosophy
2000
Major Advisor: Neil E. Grunberg, Ph.D., Department of Medical and Clinical Psychology
Thesis Title:
The Role of Sex and Strain in Behavioral and Biologic
Stress Responses of Rats
ABSTRACT
Stress has been implicated in the etiology of many behavioral disorders (i.e., drug abuse, feeding disorders) and disease states (i.e., hypertension, diabetes, depression). Individuals differ, however, in vulnerability to stress- related disease. The goal of this doctoral research was to identify potential behavioral and possibly biochemical markers of stress vulnerability vs. resilience in male and female rats of two strains (Sprague-Dawley and Long-Evans) that might predict eventual development of specific stress-related behavioral disorders or diseases in certain subgroups of humans. The experiment assessed the effects of mild, repeated daily stress on multiple behaviors and biochemical indices within the same subjects to construct a detailed model of potential markers of stress vulnerability vs. resilience.
Specifically, subjects were exposed to no stress or to 20 min/day immobilization stress for three weeks. During this period, behaviors were measured in two domains: 1) body weight, feeding, and locomotion; and 2) acoustic startle reflex and pre-pulse inhibition, passive avoidance, and Morris water maze performance (three measures of cognitive performance). Hormones of the hypothalamo-pituitary-adrenocortical (HPA) axis were measured at the end of the experiment.
The major findings were: 1) the four subgroups (i.e., two strains of male and female rats) manifested behavioral stress responses that varied with the domain assessed (i.e., feeding, body weight, and activity vs. cognitive performance) and imply different stress vulnerabilities; 2) the four subgroups manifested consistent stress responses within each behavioral domain, suggesting that stress vulnerability may be domain-specific; and 3) stressed animals within each of the four subgroups manifested changes in HPA axis hormones consistent with a stress response. Therefore, behaviors, rather than HPA axis hormones, most clearly differentiated apparent subgroup vulnerabilities. In addition, the findings suggest that certain behaviors (i.e., feeding, acoustic startle and pre-pulse inhibition) may have utility as behavioral markers for domain-specific types of stress vulnerability in humans.
Paul Milward
Lea IV, M.S.
Department of Anatomy, Physiology and Genetics
Doctor of
Philosophy
2000
Major Advisor: Howard J. Bryant, Ph.D., Department of Anatomy, Physiology & Genetics
Thesis Title: Modulation of Long-Term Potentiation and Epileptiform Activity in the Rat Dentate Gyrus by the Group II Metabotropic Glutamate Receptor Subtype mGluR3
ABSTRACT
We are the first to report involvement of group n metabotropic glutamate receptors (mGluRs; specifically mGluR3) in modulation of epileptiform activity and long-term potentiation (LTP) in the hippocampal dentate gyrus. By stimulation and/or inhibition of group n mGluRs in rat hippocampal slices, we discovered that: (1) N-acetylaspartylglutamate (NAAG; 50 and 200 :M) blocked LTP of extracellular excitatory post-synaptic potentials (EPSPs) after high-frequency stimulation (100Hz; 2s) of the medial perforant path, (2) the beta-isomer of NAAG ($-NAAG) and ethyl glutamate (100:; group II mGluR antagonist) prevented this blockade, and (3) $-NAAG did not affect EPSPs recorded in a paired-pulse paradigm which argues against a presynaptic effect. These data are the first to indicate competitive effects between $-NAAG and NAAG on mGluR3 receptors. $-NAAG's effects were characterized using cultured cells. In cerebellar granule cells, we found that: (1) $-NAAG did not affect inositol phosphate production stimulated by mGluR group I agonists glutamate, L-CCG- I, and quisqualate, (2) $-NAAG reversed decreases in forskolin-stimulated cAMP caused by the mGluR group II agonist DCG-IV, and (3) $-NAAG did not reverse decreases in forskolin-stimulated cAMP caused by the mGluR group ill agonist L-AP4. These results ruled out group I and ill mGluRs as effectors of $-NAAG. We used cells stably transfected with mGluR2 or mGluR3 to determine that $-NAAG blocked forskolin- stimulated cAMP responses to glutamate, NAAG, the nonspecific group I, II agonist trans-ACPD, and the group II agonist DCG-IV via mGluR3, but not mGluR2. We conclude that $-NAAG is a specific antagonist of mGluR3. We used NAAG and $- NAAG to investigate the mGluR3 receptor contribution to epileptiform activity induced in hippocampal granule cells (considered 'gating' cells for the spread of neuronal activity) using a high-potassium, low-calcium perfusate [Schweitzer, Patrylo, Dudek. J.Neurophys 68:2016]. We found that: (1) burst frequency is decreased by ethyl glutamate and $-NAAG, and (2) NAAG, after induction of spontaneous bursting, has no effect on burst frequency. We conclude that: (1) group II mGluRs modulate epileptiform bursting of granule cells, (2) mGluR3 rather than mGluR2 mediates this modulation, and (3) specific antagonists to mGluR3 may have potential therapeutic effects.
Barry A. Miller
Department of Preventive Medicine and Biometrics
Doctor of
Public Health
2000
Major Advisor: Terry L. Thomas, Ph.D., Department of Preventive Medicine and Biometrics
Thesis Title: The Impact of Sociodemographic Factors on Racial/Ethnic Differences in Tumor Stage and Tumor Size for Cancer of the Female Breast
ABSTRACT
A population-based, case-control study was conducted to determine the importance
of sociodemographic factors in explaining racial/ethnic differences in tumor
stage and size at the time of diagnosis among women with invasive, primary
breast cancer. The study group included 106,607 women newly diagnosed with
breast cancer during the years 1992 through 1996 while residing in any of
the eleven reporting areas in the United States that comprise the Surveillance,
Epidemiology, and End Results (SEER) program of the National Cancer Institute
(NCI).
Descriptive tabulations of the study variables indicated that Japanese and White women tended to be diagnosed at an earlier stage, with smaller diameter tumors, and at a lower tumor grade than other groups. Black and Hispanic women were more likely than other groups to be diagnosed with metastatic disease, with tumors 2 cm or larger in diameter, and with poorly differentiated tumors. In the regression analysis, elevated odds ratios among Black and Hispanic patients for later stage and larger size tumors were reduced by 50% to 60% when sociodemographic factors were added to a model already containing age and geographic area. Tumor grade and hormone receptor status only explained a small amount of the excess odds for distant stage disease among Black and Hispanic women, and did not explain any of the racial/ethnic differences in regional stage disease or larger tumor size. In the analysis of tumor size, odds ratios for Black, Hispanic, Filipino, Chinese, and Korean women remained elevated relative to White women after adjustment for sociodemographic factors, tumor grade, and hormone receptor status. Japanese women, conversely, had consistently lower odds ratios (relative to White women) for every study outcome.
Results from this study suggest that sociodemographic factors account for a significant portion of the observed racial/ethnic differences in the stage of disease and tumor size at the time of diagnosis, but that unmeasured differences in socioeconomic or biological characteristics of breast tumors among some racial/ethnic groups may also exist. The special cancer data base created for this study may now be used to investigate the importance of sociodemographic factors in explaining population patterns for other types of cancer.
Velia Mitro
Molecular and Cell Biology Program
Doctor of
Philosophy
2000
Major Advisor: Dr. William C. Gause, Department of Microbiology and Immunology
Thesis Title: The function of CTLA4 during the in vivo immune response to infectious disease.
ABSTRACT
CD4+ T cells playa key role in the adaptive immune response to foreign antigens. For T cells to be activated, two signals are required. The first signal is delivered through antigen recognition by the T cell receptor. A second, or costimulatory, signal is also required for optimal activation of T cells. CD28 ligation by B7 is a potent mediator of positive costimulation. In contrast, B7 ligation of CTLA4 (CD152), a homologue of CD28, provides a critical downregulatory signal. Recent data has suggested that CTLA4 may also share some stimulatory functions with CD28. Because costimulatory molecule interactions are critical for many immune responses, a greater understanding of CTLA4 function may promote development of immunotherapies where enhancement or inhibition of the immune response would be clinically beneficial. This research was directed at developing a greater understanding of CTLA4 function in the immune response to infectious disease. A murine model of gastrointestinal nematode infection, Heligmosomoides polygyrus, was utilized in this research to investigate the role of CTLA4 after onset of infection, once naive T cells have differentiated to effector T cells. These data support a negative regulatory role for CTLA4 late in the response. Blockade of CTLA4 by in vivo administration of anti-CTLA4 antibody enhanced the polarized Th2 response to H polygyrus, resulting in increased serum concentrations of immunoglobulins, IL-4 secretion, and T and B cell activation. Further evidence of enhanced immune response upon CTLA4 blockade was provided in another nematode model, Trichuris muris. Anti-CTLA4 antibody treatment increased serum immunoglobulin concentrations and T and B cell activation. The treatment also caused immune deviation from Th1 to Th2, as evidenced by decreased IFNm and increased IL-4 secretion. These data are consistent with a model for Th1 versus Th2 cell differentiation which describes the decision as based on the balance between strength of signal and innate response. On a molecular level, the phosphorylation events following CTLA4 blockade were examined, and intracellular binding partners for CTLA4 and CD28 were identified. The dependence of effector T cells upon continued combined CTLA4/CD28 signaling was also explored.
Diana L Schneider
Department of Preventive Medicine and Biometrics
Doctor of
Public Health
2000
Major Advisor: Heidi B. Friedman, Ph.D., Department of Preventive Medicine and Biometrics
Thesis Title: Evaluation of Cervicography Screening for Cervical Cancer in a High-Risk Population
Abstract
Statement of the problem:, CervicographyTM was first described in 1981 as a visual screening system for early detection of cervical neoplasia and cancer. Early studies to assess the validity of cervicography showed the method to have an acceptable sensitivity but an unacceptably low specificity for mass screening. Following revision of the cervicography classification scheme, specificity improved, but at the expense of lowered sensitivity. Most previously published studies have had some methodologic inadequacies which may have affected the outcome.
Methods: Cervigrams were taken for 8460 women who enrolled into a population-based, natural history study of cervical neoplasia in Guanacaste Province, Costa Rica. Cervicography and three cytologic screening tests were the basis for referral for colposcopic examination and directed biopsy. Initial cervicography classification was compared with a referent diagnosis determined by histology and three cytologic tests, cytology, and presence of cancer-associated human papillomavirus types. Cervicography was submitted to additional review and arbitration to achieve an optimal classification. Interobserver agreement was assessed, and the performance of the optimal cervicography result was compared with the referent diagnosis. Sensitivity, specificity, and predictive values were estimated, and results were stratified by characteristics of the woman and visual characteristics of the cervigram image. Digital colposcopic images were interpreted to evaluate the perceived appropriateness of the decision to biopsy and biopsy placement, and the impact of these on sensitivity and specificity.
Results: Moderate agreement on cervigram classification was observed (kappa=0.47 when cervigram results were classified into seven categories and 0.54 when cervigram results were classified into dichotomous categories of referred for colposcopy versus not referred). For the detection of high grade squamous intraepithelial lesion or cancer, optimized cervicography yielded a sensitivity of 55.2% and a specificity of 94.3 %, which was only slightly improved over the initial estimates of 49.3% sensitivity and 95.0% specificity at enrollment. Higher sensitivity was associated with younger age, premenopausal status, the presence of metaplasia, the absence of cervicovaginal atrophy, and improved quality of the acetic acid effect.
Conclusions: Evaluator agreement with cervicography is moderate. The arbitrated cervigram classification improved the performance of cervicography only slightly over a single interpretation. Cytology performed better than cervicography for the detection of high grade squamous intraepithelial lesions, but the two methods performed similarly for the detection of invasive cervical cancer. Cervicography is not recommended for postmenopausal women and/or women ages 50 and older.
Sara Kathleen
Snyder
Molecular and Cell Biology
Doctor of
Philosophy
2000
Major Advisor: Gabriela Dveksler, Molecular and Cellular Biology Program
Thesis Title: Human Pregnancy-Specific Glycoproteins Function as lmmunomodulators In Vitro by Inducing Secretion of IL-10 and IL-6 in Human Monocytes
ABSTRACT
The lack of rejection of the semiallogeneic fetus by the maternal immune system is brought about in part by the maintenance of an anti-inflammatory immune environment at the maternal-fetal interface. The fetoplacental unit produces an arrray of cytokines and other regulatory molecules that assist in the implantation, survival and development of the fetus. Pregnancy specific glycoproteins (PSGs) are a family of highly conserved, secreted proteins abundantly produced by the placenta in various species including human, mouse and rat. PSGs are composed of repeated immunoglobulin (Ig) related domains, and are part of the Ig superfamily. Abnormally low levels of PSGs in maternal serum have been correlated with complications of pregnancy including spontaneous abortion. A peptide derived from the N-terminal domain of human PSG11 has been shown to bind cells of the promonocyte lineage, suggesting a role for PSGs in modulation of macrophage function during pregnancy.
We investigated the ability of three recombinant human PSGs (PSG 1, PSG6 and PSG 11 ), produced using a baculovirus expression system, to regulate the in vitro production of cytokines by human monocytes. Cytokine secretion by monocytes at 24 hours after treatment was measured by quantitative sandwich ELISA. All three PSGs induced dose-dependent secretion of IL-10and IL-6, but not secretion of TNF-", IL-I$ or IL-12. In order to examine the role of the N-terminal Ig-variable-like domain in PSG function, we produced a fusion protein consisting of only the N-terminal domain of PSG6. The PSG6 N-terminal domain was shown to be sufficient for induction of monocyte secretion of IL-I 0 and IL-6, demonstrating that this domain mediates the interaction with a putative PSG receptor on monocytes. As shown by RT-PCR, increased IL-10 and IL-6 secretion was accompanied by an increase in mRNA after PSG6 treatment. PSG6 induction of IL-I 0 and IL-6 secretion was inhibited by the tyrosine kinase inhibitor Herbimycin A, the protein kinase C inhibitor Calphostin C, and the specific protein kinase A inhibitor (Rp)cAMPS, suggesting a possible role for these intracellular signalling molecules in PSG signal transduction in monocytes. Also, the specific phosphodiesterase type IV inhibitor and cAMP elevating agent, rolipram, increased monocyte secretion of IL-10 and IL-6 after treatment with PSG6, indicating that increased production of these cytokines in response to PSGs may be mediated by an increase in cAMP. We also showed that PSGs exhibit cross-species activity in cytokine induction using human PSG treatment of a mouse macrophage cell line, RAW 264.7, and mouse PSG18 N-domain protein treatment of human monocytes, indicating that PSG function may be highly conserved between species. Our results are consistent with a role for PSGs in modulation of macrophage inflammatory responses at the maternal- fetal interface where PSGs are in high concentration.
William D.
Watson
Neuroscience Program
Doctor of
Philosophy
2000
Major Advisor: Ajay Verma, M.D./Ph.D., Department of Neurology
Thesis Title: A Thapsigargin-insensitive Intracellular Calcium Sequestering Compartment in Rat Brain
ABSTRACT
Calcium plays a central regulatory role in the normal function of all cells. Electrical, secretory, and metabolic activities of cells in the brain require fine control over ionized cytoplasmic calcium levels. Intracellular calcium levels are controlled by a diverse set of cytoplasmic and membrane-associated mechanisms including calcium binding proteins, channels, pumps, and exchangers. The endoplasmic reticulum (ER) calcium stores have a major impact on neuronal intracellular signaling. Most of the ER in neurons and glia appears to accumulate calcium by energy driven ion pumps known as sarco/endoplasmic reticulum calcium ATPases (SERCAs), which are potently and selectively inhibited by thapsigargin. However, the ER represents a heterogeneous network of cisternae in which calcium-accumulating subcompartments may be spatially and functionally distinct. We describe here the characterization of a novel calcium accumulating subcompartment of rat brain ER, which is insensitive to thapsigargin. This compartment accumulates calcium in a magnesium and ATP-dependent manner and is distinguished from thapsigargin-sensitive calcium pools with respect to anion permeability I inhibitor sensitivity, sensitivity to calcium mobilizers, and brain anatomical distribution.
David A. Zemo
Department of Anatomy, Physiology and Genetics
Doctor of
Philosophy
2000
Major Advisor: Joseph T. McCabe, Ph.D., Department of Anatomy, Physiology and Genetics
Thesis Title: Osmotic Stress Induces Transcriptional Changes in Vasopressin and Vasopressin 1b Receptor Gene Expression
Abstract
Arginine vasopressin (A VP) plays a critical role in the regulation of mammalian salt and water homeostasis. To further define central nervous system adaptation to osmotic challenges, transcription of A VP and vasopressin 1b receptor (V1BR) genes by magnocellular neurons of the hypothalamus and epithelial cells of the choroid plexus was studied using in situ hybridization. Compared to animals given a single injection of normal saline, animals that received a single injection of hypertonic saline had increases in AVP heteronuclear RNA (hnRNA) after 15 and 30 minutes. Animals given an injection of hypertonic saline followed by a second injection of hypertonic saline (H-H) had an increase in AVP hnRNA levels that were equivalent to the response seen after a single hypertonic saline injection. Levels of AVP hnRNA after H-H were greater than the levels detected in animals given an injection of normal saline followed by a second injection of hypertonic saline (N-H). This is the first study to show repeated exposure to hypertonic saline causes an immediate, robust, and reproducible increase in vasopressin gene transcription. These results suggest there is a correlation between increased neuronal firing rate and vasopressin gene transcription. We also studied long-term-adaptation to an osmotic challenge. Compared to rats maintained on tap water, salt-drinking rats had increased levels of AVP and V1BR mRNAs in the supraoptic and paraventricular nuclei, and in the choroid plexus. The increase in AVP and V1BR mRNAs in the SON and PVN as a result of plasma hyperosmolality may indicate the intranuclear release of AVP has a role in the autoregulation of magnocellular neuron activity. The role of AVP in cerebrospinal fluid formation remains to be elucidated. However, the increase of AVP and V1BR mRNA in the choroid plexus suggests the involvement of AVP in the regulation if brain water content and cerebral edema. Taken together, these results demonstrate coordinated transcriptional control in hypothalamic neurons and epithelial cells of the choroid plexus. Changes in gene regulation for a receptor and its binding peptide occurred in two structures that have crucial, complementary roles in water homeostasis for the periphery and the central nervous system.