We hypothesized that the ventral anteroventral third ventricle region (vAV3V), which mainly comprises the organum vasculosum laminae terminalis (OVLT) and extends ∼100 mum into the median preoptic nucleus, mediates salt-induced sympathoexcitation and hypertension via AT1 receptor activation. We utilized ibotenic acid lesions of the vAV3V to determine a role of neurons in this area in pressor responses to intracerebroventricular (icv) administration of hypertonic saline (0.3M NaCl, 2 mul/min for 10 min), ouabain (0.3 and 0.6 mug) and angiotensin II (Ang II; 10 and 30 ng), and in two rat models of hypertension in which mechanisms of salt-sensitive hypertension are mimicked: chronic icv hypertonic, saline (0.8 M NaC1, 5 mul/h icv for 2 weeks) to increase CSF [Na+] and chronic subcutaneous (sc) ouabain (50 mug/d sc for 3 weeks) to mimick increases in central ouabain-like compound content. 125I-Sar1Ile8-Ang II binding to AT1 receptors was determined by in vitro receptor autoradiography to determine if changes in AT1 receptor density accompany the development of salt-induced hypertension in Dahl salt-sensitive (S) rats. In conscious vAV3V lesioned Wistar rats with systemic vasopressin receptor blockade, pressor and tachycardic responses to 0.3 M NaCl and ouabain, were significantly attenuated by 26--32% whereas responses to Ang II were unaffected. Resting mean arterial pressure (MAP) in sham-operated rats that received chronic hypertonic saline infusions or ouabain treatment was significantly higher (17--19%) than in aCSF or placebo treated controls. vAV3V lesions abolished the chronic hypertonic saline or ouabain induced increases in MAP and the enhanced depressor responses to ganglionic blockade with hexamethonium. AT1 binding in the OVLT, the suprachaismatic nucleus and the paraventricular nucleus did not differ between Dahl S and R rats on regular salt diet, and was decreased similarly on high salt diet. Within the subfornical organ AT1 receptor binding did not differ between Dahl S and R rats on regular salt diet and decreased in both strains on the high salt diet, but decreased more in hypertensive Dahl S versus the normotensive Dahl R rats (50% versus 23%). In conclusion, the vAV3V area appears to play a crucial role in mediating salt-induced hypertension, possibly via sympathetic activation, but autoradiography did not provide evidence for differential AT1 stimulation in the vAV3V.

Background. The mechanism of cisplatin-induced nephrotoxicity is not well understood. The distal tubules are affected in both human and animal studies, although the majority of cisplatin-induced renal damage is in proximal tubules. Platinum (Pt) forms intra- and interstrand cross-links with DNA in cancer cells. Hypothesis. A mechanism of cisplatin-induced cytotoxicity in MDCK cells relates to its ability to bind to DNA and interfere with its synthesis. Methods. The canine distal renal tubular epithelial cell line, MDCK was used as an in vitro model to investigate the mechanism of cisplatin-induced nephrotoxicity. The intracellular Pt accumulation and Pt binding with DNA were assayed by atomic absorption spectrophotometry. DNA synthesis was measured by BrdU labeling and fluorescence microscopy at the concentrations from 0 to 100 muM. The alkaline comet assay with 10 Gy radiation was used to measure Pt DNA interstrand cross-links after a one hour cisplatin exposure from 0 to 100 muM at both zero and sixteen hour time points. According to the principles of alkaline comet assay, the tail moment is inversely related with the amount of Pt interstrand cross-links. (Abstract shortened by UMI.)

Adverse reactions to sulfonamides were initially described more than 50 years ago. It is not yet clear whether these side effects are due to immunological reactions or drug toxicity. More than 50% of AIDS patients develop adverse reactions to sulfamethoxazole, a sulfonamide drug commonly used for the treatment of infections in these individuals. In order to study the possible role of the humoral immune response in adverse reactions to SMX in HIV-seropositive patients, I designed an ELISA to determine the classes, subclasses and the quantities of the immunoglobulins directed against SMX. Twenty eight HIV-seropositive individuals with a history of adverse reactions to SMX, 20 HIV-seropositive individuals with no such history, and 39 healthy infants and adults were evaluated for the presence of anti-SMX antibodies. To evaluate the cellular immune response in vitro, lymphocyte proliferation assays were performed in the presence of SMX or structurally related compounds. The lymphocytes from HIV-seropositive patients did not proliferate or produce antibody when incubated with these compounds. Only one immunocompetent individual who had a known hypersensitivity to SMX demonstrated high lymphocyte proliferation to SMX-PLL, but not to structurally related compounds. The mechanism of the development of side effects to SMX is complex, and likely involves both toxic and immunologic events. It may be that high blood levels of SMX may lead to an immune response which produces symptoms only when specific antibody levels have exceeded a certain threshold. The detection of antibodies to SMX may be useful in predicting and managing the adverse reactions to this compound in HIV-seropositive individuals. (Abstract shortened by UMI.)

Susceptibility to acetaminophen-induced hepatotoxicity was found to vary widely in an outbred colony of Swiss Webster mice based on differences in serum levels of the hepatic enzyme alanine aminotransferase among males. A selective breeding program produced inbred mouse strains which were either susceptible (APS strain) or nonsusceptible (APN strain) to the hepatotoxic effects of acetaminophen. Hepatic enzyme activities associated with the cytochrome P450 isoform CYP1A2 showed a statistically significant increase in APS versus APN mice. Further examination of hepatic Cyp1a1 and Cyp1a2 gene expression revealed that mRNA and specific protein levels were significantly elevated in animals from the susceptible group. The cosegregation of elevated basal gene expression for both Cyp1a subfamily genes in animals selected for susceptibility to acetaminophen-induced hepatotoxicity suggested a common heritable basis for regulation of basal expression. Caffeine 3-demethylation can be used as an index of CYP1A2 activity in vivo and was found to co-segregate with acetaminophen susceptibility in APS mice. Serum levels of caffeine and the 3 demethylated metabolite were compared among six common inbred strains (A/J, P/J, BALB/cJ, C3H/HeJ, AKR/J and SWR/J) and the APN strain. Significant variations were found between a number of different strains, including the APN strain, indicating a genetic basis for variation in this trait. Hepatic Cypla2 gene expression levels were significantly higher in C3H/HeJ, relative to APN male mice. The striking differences observed between the APN and C3H/HeJ mice suggested that these strains would be suitable for a genetic analysis of the factors affecting caffeine 3-demethylation and, by extension, its use as an index of CYP1A2 expression. Conflicting data from caffeine assays have suggested a uni-, bi- or tri-modal distribution of CYP1A2 activity in human populations, although no linkage of a genetic polymorphism affecting basal expression to the CYP1A2 locus has been demonstrated. In order to investigate the genetic determinants of variable caffeine 3-demethylation in the mouse, quantitative phenotypic data, in the form of caffeine 3-demethylation indices, were obtained for the progeny of an F2 intercross of C3H/HeJ X APN. The phenotypically extreme animals were genotyped at marker loci which achieved complete coverage of the genome. Interval mapping was employed to search for statistically significant linkages between trait data and marker genotypes across the genome. Two statistically significant quantitative trait loci (QTLs) were mapped to chromosomes 1 and 9, and statistically suggestive linkage was found for a QTL on chromosome 4. The QTL on chromosome 9 showed highly significant linkage to the Cyp1a2 gene, while no obvious candidate genes known to be involved in caffeine metabolism have been mapped to chromosomes 1 or 4. Pharmacogenetic studies of variable xenobiotic metabolism in humans and experimental animal models have historically focused on monogenic polymorphisms of the enzymes of metabolism and detoxification. Methods to permit genetic analysis of complex quantitative traits have not previously been applied to the study of variations in drug metabolism.

Background. In North America, the prevalence of chronic obstructive pulmonary disease (COPD) amongst elderly patients is increasing, and COPD exacerbation is now a common cause for Emergency Department (ED) patient visits. Over 900 patient visits were logged at the Ottawa General and Civic Hospital's Emergency Departments in 1996 because of COPD exacerbation. Fifty-two percent of these ED patient visits for COPD resulted in a discharge home. Although exacerbations of COPD occur commonly, the optimal therapy of this condition remains unknown. It was felt that a state of clinical equipoise exists which justifies the need for a randomized, double-blind, placebo-controlled trial examining whether oral glucocorticoids should be used to treat patients with COPD exacerbation who are discharged home from the ED. This thesis will report on the results of the pilot study which was undertaken to establish the feasibility of a larger, definitive clinical trial. Objectives. The primary objective of the pilot study was to ensure that a larger clinical trial will be feasible. Secondary objectives of the pilot study were: to assess the rate of patient relapse in order to determine whether this clinical variable could serve as the primary outcome variable for the definitive study, to verify the sample size estimates for the larger trial, to evaluate patient referral rates and trial recruitment, and to verify the enrollment process and allow for pre-testing of the data collection and data analysis process. (Abstract shortened by UMI.)

We demonstrate the presence of myristoylated alanine-rich C kinase substrate (MARCKS), another protein kinase C (PKC) substrate, in platelets and its phosphorylation during phorbol 12-myristate 13-acetate (PMA) or thrombin stimulation. Both MARCKS phosphorylation and serotonin release from permeabilized and intact platelets stimulated by either PMA or thrombin, respectively, have the same concentration-dependency and were blocked by a peptide (MPSD) with the amino acid sequence corresponding to the phosphorylation site domain of MARCKS. Under these conditions, pleckstrin and myosin light chain phosphorylation in response to either PMA or thrombin simulation was not modified. A similar peptide (Ala-MPSD) in which the four serine residues of MPSD were substituted by alanines was ineffective. The present results provide the first evidence that MARCKS may play a role in platelet secretion. Moreover, the fact that pleckstrin phosphorylation has a different concentration-dependency than that of MARCKS or serotonin release and was not modified when MARCKS phosphorylation and serotonin release were inhibited, suggests that pleckstrin is either not directly involved in platelet secretion or it might only be involved upstream in the cascade of events leading to exocytosis. (Abstract shortened by UMI.)

The cortical collecting duct (CCD) is a major site of intrarenal prostaglandin E2 (PGE2) synthesis. By indirect immunofluorescence using isoform specific antibodies, we have localized COX-1 and -2 immunoreactivity to all cell types of the murine M-1 CCD cell line. By, immunohistochemistry, both COX-1 and COX-2 were localized to the intercalated cells of the collecting duct on paraffin embedded mouse kidney sections. When COX enzyme activity was measured in the M-1 cells, both indomethacin (COX-1 and -2 inhibitor) and the specific COX-2 inhibitor NS-398 effectively blocked PGE2 synthesis. These results demonstrate that COX-2 is a major contributor to the pool of PGE2 synthesized by the CCD. PGE2 exerts predominantly diuretic and natriuretic effects upon the CCD. Our results which document the expression of COX-2 in the CCD provide a mechanism through which the newly developed class of COX-2 specific inhibitors could exert side effects with respect to the regulation of fluid and electrolyte homeostasis. (Abstract shortened by UMI.)

The shape of the dose-response curve (DRC) has been hypothesized to reflect mechanisms of resistance (Stewart et al. Invest. New Drugs 14:115, 1996). Active resistance (AR) would give rise to a shoulder on a DRC, while passive resistance (PR) would result in a reduced slope or terminal plateau. We obtained DRCs for human small cell lung cancer cells (SBC-3), and for human osteosarcoma cells (Saos-2) exposed to cisplatin (1 hour CP [0.02--40 mug/ml]). We then fit the data to curve models using non-linear regression techniques. We repeated this technique with SBC-3 cells transfected with a gamma-glutamylcysteine synthetase (gamma-GCS) gene, and with Saos-2 cells transfected with a wild-type p53 gene in order to observe their effects on cisplatin resistance. We also repeated these experiments in Saos-2/p53 transfectants whose wild-type p53 production was effectively eliminated, and in SBC-3 and SBC-3/GCS transfectants whose glutathione production was inhibited. Results. Curve modeling of the transfected and parent SBC-3 cell lines suggest that there is a significant difference when the gamma-GCS gene is transfected into this cell line, with changes occurring in the LD50 and the slope after 1 hour exposure (p < 0.001 in both cases). A statistically significant difference is also detected between the transfected and parent Saos-2 cells, where the wild-type p53 gene results in a decrease in the terminal plateau, and a subsequent decrease in PR (p < 0.001). Reversal of the effects of transfection results in an approximate return of the response curve shape to that of the parent cell lines. Conclusions. Our results suggest that drug resistance mechanisms exert an effect on the shape of the dose-response curve.

HIV-1 protease inhibitor therapy is associated with a novel lipodystrophy syndrome characterized by truncal adiposity, peripheral fat atrophy, dyslipidemia, and type 2 diabetes. The increase in truncal fat may be due to increase in adipocyte number as a result of enhanced preadipocyte differentiation. We show that addition of 10 mug/ml ritonavir to standard differentiation medium enhanced 3T3-L1 preadipocyte differentiation as measured by a 30% increase in triacylglycerol (TG) accumulation and a 50% increase in glycerol 3-phosphate dehydrogenase (GPDH) activity. Although ritonavir partially inhibited protein expression of peroxisome proliferator activated receptor gamma (PPARgamma), CCAAT/enhancer binding protein alpha (C/EBPalpha), and the aP2 gene (which encodes the adipocyte lipid binding protein), it resulted in higher levels of the active form of adipocyte determination and differentiation-dependent factor-1 (ADD-1), also known as sterol regulatory element binding protein 1 (SREBP-1). The enhancing effects of ritonavir on late events of 3T3-L1 preadipocyte differentiation may be mediated by ADD-1/SREBP-1 which has been shown to directly activate transcription of several genes encoding lipogenic enzymes. Preliminary results suggest that ritonavir preferentially enhances differentiation of human preadipocytes derived from abdominal omental but not subcutaneous adipose tissue in primary culture.

Antioxidant utilization potentially is an excellent index of oxidative stress. These studies assessed the effects of two doses of CsA (10 mg/kg/day and 20 mg/kg/day for 14 days) on antioxidant status in numerous tissues, and on the function and structure of testis and kidney in male Wistar rats. Animals were placed on either vitamin E sufficient or deficient diets and injected subcutaneously with either CsA or vehicle. Vitamin E levels in various tissues were measured using the very sensitive technique of gas chomatography and mass spectrometry (GC-MS) to determine the effect of CsA on vitamin E status and the protective extent of vitamin E on CsA toxicity. Oxidative damage was also assessed by measuring levels of GSH and protein sulfliydryl (PSH) content using colorimetric methods. In addition, frozen sections of kidney and testes were subjected to standard Hematoxylin and Eosin staining to examine these tissues for structural alterations following CsA treatment. (Abstract shortened by UMI.)