Toxicity of arsenic species is dependent on chemical oxidation state. Inorganic arsenic in the trivalent state, arsenite or As(III), is more biologically active than pentavalent arsenic, arsenate or As(V), and is more toxic by most measures. As(V), however, is more stable and prevalent in the environment. One consequence of environmental exposure is peripheral vascular disease, which is primarily due to vascular changes, but toxicity to the erythrocyte has not been evaluated. To understand toxicity and the implications of arsenic oxidation state, human erythrocytes were utilized to model the uptake, biotransformation (metabolism) and toxicity of sodium arsenate, As(V). It was first established that biotransformation, both in vivo and in vitro, would not be limited by uptake of As(V) into the cell. Evidence suggested that reduction was accomplished by at least two separate pathways. All reductive metabolism was dependent on the presence of reduced thiols including both non-protein thiols (glutathione; GSH) and protein thiols (ProSH). These pathways are: (1) chemically mediated reduction by GSH and (2) protein mediated reduction. It was established that the protein-dependent pathway required a reduced protein thiol and also required the presence of GSH. This points to reduction through a redox coupling to form a protein mixed disulfide (ProSSG). Toxicity to the erythrocyte was evaluated by determining total cell death, morphologic changes and effects on the energy cofactor adenosine triphosphate (ATP). Based on these three parameters, the erythrocyte was more susceptible to As(V) and not As(III) as other tissues are. The morphologic effects on the cell were also consistent with ATP depletion. These changes were characterized by formation of morphologically altered cells that are unable to deform in circulation effectively and occlude the microcirculation. This could contribute to vascular tissue damage associated with arsenic-induced circulatory disorders. In summary, the erythrocyte is able to take in As(V) which is detrimental to the ability of the cell to perform its intended function. Biotransformation to As(III) would therefore be a detoxifying event, and understanding the factors involved in biotransformation will help to understand human susceptibility to arsenic-induced vascular disease.

The three α₂-adrenergic receptors (α₂-ARs) were studied in both recombinant and endogenous systems. It was my general hypothesis that the three α₂-adrenergic receptor (AR) subtypes exhibit differential tissue and subcellular localization and couple to physiological pathways to directly elicit functional responses. It was our hope, that in identifying differences between the α₂-ARs we may elucidate their physiological roles and provide a potential means for the rational development of therapeutic drugs or models of such pathologies as atherosclerosis, hypertension and glaucoma. Subtype selective antibodies have been generated and these have provided a tool with which to study the receptors on a molecular and cellular level. Utilizing a number of biochemical, pharmacological, molecular and cellular techniques, I have identified differences between the three receptors with respect to the tissues in which they are expressed, the subcellular domains in which they localize and their patterns of sequestration and down-regulation. In addition, I have demonstrated the expression and co-localization of the α₂-ARs in a variety of native tissues, and a number of functional responses these receptors mediate. These functional responses may have ramifications relevant to tissue healing mechanisms as well as may play a role in a number of pathologies.

Menopause is accompanied by neuronal hypertrophy and increased neurokinin B (NKB) gene expression in the human infundibular (arcuate) nucleus. We have hypothesized that these changes are secondary to withdrawal of gonadal steroids. In the present study, a rat model is used to determine if orchidectomy and hormone replacement alter the size and gene expression of arcuate NKB neurons. Compared to intact animals, gonadectomy significantly increased the mean profile area and number of neurons/section expressing NKB mRNA in the rat arcuate nucleus. Both the increase in NKB neuron size and number of neurons was prevented by implantation of estradiol- or testosterone-containing capsules. These data provide strong support for the hypothesis that the hypertrophy and increased numbers of neurons expressing NKB gene transcripts in postmenopausal women are secondary to ovarian failure. Studies were also conducted to determine if the conditions that induce neuronal hypertrophy and enhanced gene expression in the arcuate nucleus are accompanied by other morphological changes. To achieve this end, a fixed slice preparation was used to examine the morphology of neuroendocrine neurons in the rat arcuate nucleus. In comparison to intact controls, arcuate neuroendocrine neurons in the orchidectomized group had significantly larger somatic profile areas and exhibited significant increases in dendrite length, dendrite volume, terminal branch number, and spines per unit length of dendrite. These results provide evidence for hormonal regulation of dendritic morphology of arcuate neuroendocrine neurons in adult mammals. To assess the role of testosterone withdrawal in inducing these dendritic changes, hormone replacement studies were conducted. Testosterone treatment of castrate animals prevented increases in dendrite length and volume when compared to untreated castrate rats. Testosterone treatment also prevented the castration induced increase in the number of branches per dendrite. Finally, immunohistochemical studies indicate that approximately 22% of arcuate neuroendocrine neurons contain estrogen receptors, while 16% contain androgen receptors, indicating that these neurons can respond directly to changes in steroid hormone levels. These findings clearly demonstrate a role for testosterone in modulating the dendritic morphology of arcuate neuroendocrine neurons in adult rats.

These studies were conducted to determine the chemical identity of heparin binding proteins in semen that are related to fertility of bulls. The first study describes the isolation and identification of a 31,000 dalton fertility-associated antigen (FAA). FAA was found to have significant primary structure homology to a recently described novel DNase I-like protein. The physiological significance of the similarities between FAA and a protein homologous to DNase I is unknown at this time as no function has been described for the DNase I-like protein. The second study describes the isolation and identification of a 24,000 dalton seminal heparin binding protein. It was found to be similar, if not identical, to tissue inhibitor of metalloproteinase-2 (TIMP-2). TIMP-2 regulates matrix metalloprotease activity and therefore, potentially plays a key role in the structural makeup of the extracellular matrix. These findings suggest that regulation of enzymatic activity in seminal fluid is in large part a function of heparin binding proteins that have been correlated to fertility of bulls, one being a potentially novel extracellular nuclease and a second acting as a specific inhibitor of metalloprotease activity. In addition to the ability to modulate capacitation of sperm, seminal heparin binding proteins likely are key players in protecting sperm and male reproductive tract tissues from enzymatic hydrolysis. The proteins identified in this dissertation represent novel additions to the previously described seminal heparin binding protein families. Clearly, these data indicate a growing complexity of seminal fluid and implicate a novel Dnase I-like protein and TIMP-2 in affecting cellular events related to fertility potential of males.

The gonadal steroid withdrawal of menopause is associated with neuronal hypertrophy and increased tachykinin gene expression in the hypothalamic infundibular nucleus. Previous studies have shown that secretion of hypothalamic b -endorphin is modified by gonadal steroids, and there are consistent age-related changes in b -endorphin neurons in rodents. Therefore, in situ hybridization was used to determine if the expression of POMC mRNA, the precursor for b -endorphin, is altered in the hypothalamus of postmenopausal women. The number of POMC mRNA-containing neurons/section in the infundibular nucleus was reduced by 65% in postmenopausal women. In contrast, there was no significant difference in the number of neurons expressing POMC gene transcripts in the retrochiasmatic region. Our findings support the hypothesis that the activity of hypothalamic POMC neurons is decreased in the infundibular nucleus of postmenopausal women. In a second study, we examined the effects of hormone replacement therapy (HRT) on the hypothalamus of young, ovariectomized cynomolgus monkeys. HRT dramatically suppressed tachykinin gene expression while having no detectable effects on POMC neurons. These results provide strong support for the hypothesis that alterations in tachykinin neurons in postmenopausal women are secondary to estrogen withdrawal. Conversely, postmenopausal changes in POMC gene expression may reflect hypothalamic aging. Finally, we found no evidence that HRT, in doses designed to mimic currently prescribed regimens, produces signs of estrogen toxicity in the primate infundibular nucleus. Degenerative changes, including neuron loss, have been reported in the arcuate nucleus of aging rodents, and hypothalamic aging has been shown to contribute to reproductive decline in these species. In addition, in the infundibular nucleus of postmenopausal women, there is an age-associated decline in proopiomelanocortin gene expression. To evaluate the possibility of neuron loss associated with reproductive aging, unbiased stereological methods were used to compare the total number of infundibular neurons between groups of premenopausal and postmenopausal women. The mean neuronal volume was increased by 40% in postmenopausal women but there was no change in the total number of neurons. These data suggest that the neuronal hypertrophy observed in the postmenopausal human hypothalamus is not a pathological process secondary to degeneration of adjacent infundibular neurons.

Gap junction channels connect the cytoplasms of adjacent cells and provide a pathway for the exchange of materials between cells. The nature of the materials exchanged is determined by the biophysical characteristics of the channels. The functional gap junction channel is composed of paired hemichannels (connexons) from each cell. Connexons are hexamers of protein subunits called connexins (Cx). Of the 15 connexin genes found in the mammalian genome, the products of only two, Cx40 and Cx43 have been localized in vascular smooth muscle cells (SMC) (1;2). We have been interested in identifying the role of gap junctions in cardiac rhythmic activity and vascular function. Like many other cell types, mammalian heart and blood vessels express multiple gap junction connexins (3). These connexins may form heteromeric channels. A7r5 cells, a cell line derived from embryonic rat aortic smooth muscle cells, provide a good model because they express both connexins 40 and 43. From the previous studies in this laboratory, Moore and Burt reported the presence of channels with a wide range of unitary conductance with major peaks at 75, 110 and 145 pS. One explanation for the wide range of unitary conductance could be the presence of heteromeric Cx40 and Cx43 channels. Thus, the goal of this study was to investigate gating behaviors of gap junction channels in A7r5 cells to determine whether heteromeric Cx40/43 channels are formed. I will demonstrate that Cx40 and Cx43 form heteromeric channels with unique unitary conductances, voltage-dependent gating properties and enhanced sensitivity to halothane induced closure.

During the past decade precision-cut tissue slices have begun to be utilized for toxicity and metabolism studies. These studies have primarily involved a single organ type. In this study, a new preparation of rat renal cortical slices was validated and used to investigate the toxicity of chlorotrifluoroethylene and its cysteine and glutathione conjugates. An additional level of complexity was added by utilizing a sequential incubation system in which rat renal cortical slices were directly incubated in the medium from liver slice incubations. Once the new renal slice preparation and sequential incubation system had been validated, these new methods were used to study the mechanism of toxicity of chlorotrifluoroethylene and it metabolites. The hypothesis being tested in these studies is that sequential biotransformation in the liver and the kidney is required for CTFE nephrotoxicity. In these studies I developed a sequential incubation system with precision-cut rat liver slices as the drug activating system and renal cortical slices as the target tissue for toxicity. Utilizing the sequential incubation system, I found that first incubating liver slices with CTFE and then transferring kidney slices to this liver slice incubation medium causes toxicity in the kidney slices. I also found that this toxicity correlates well with the toxicity observed with kidney slice incubations with the cysteine and glutathione conjugates of CTFE. By incubating slices with inhibitors of the various enzymes in the proposed metabolic pathway of CTFE, it was determined that glutathione conjugation in the liver and subsequent degradation by gamma-glutamyltranspeptidase are important steps in toxicity of CTFE. Although previous research with inhibitors of β-lyase have indicated that β-lyase is an essential enzyme in the bioactivation of CTFE, inhibition of the pyridoxal phosphate cofactor of this enzyme in renal slices did not reduce the toxicity of conjugates of CTFE. There was no reduction in toxicity when dipeptidases were inhibited when transport via the organic anion transporter or neutral amino acid transporter were inhibited. These data indicate that the glutathione conjugate of CTFE is formed in the liver and that the subsequent metabolism of this glutathione conjugate in the kidney is required for nephrotoxicity.

The blood-brain barrier (BBB) is the regulated interface between the peripheral circulation and the central nervous system (CNS), formed primarily by the cerebral microvascular endothelium. Tight junctions (TJ) between BBB endothelial cells restrict paracellular diffusion from blood to brain. Expression and localization of TJ proteins are modulated by multiple pathways. Disruption of TJ by disease or drugs can impair BBB function and compromise the CNS. Therefore, understanding how BBB TJ are affected by various factors is useful for the prevention and treatment of neurological diseases. Smoking is associated with increased risk of disease, including neurological disease. The effects of nicotine on endothelial cells are profound, and nicotinic acetylcholine receptors (nAChR) have been characterized in peripheral endothelial cells. However, relatively little is known about the specific effect of nicotine on the BBB. The hypothesis of this study is that nicotine increases BBB permeability by alteration of TJ via a nAChR-mediated pathway. To test this hypothesis, studies were undertaken to (1) explore the effects of nicotine on the expression and distribution of TJ proteins in cerebral microvessels; (2) determine the effect of nicotine on BBB permeability in an intact animal model; and (3) investigate the role of nAChR in the effects of nicotine on the BBB. Rats were given nicotine at a dosage designed to mimic the exposure experienced by heavy smokers. While nicotine did not alter the expression of the TJ proteins investigated, changes in their distribution were observed. Nicotine was found to increase BBB permeability to sucrose without significant changes in its initial volume of distribution. These data suggest that nicotine increases the permeability of the BBB via modulation of TJ proteins. Positive immunoreactivity was found for nAChR subunits α3, α5, α7, and β2, but not α4, β3, or β4. Nicotine was co-administered with the nicotinic antagonists mecamylamine, which readily crosses the BBB, and hexamethonium, which does not. Both of these antagonists attenuated the effect of nicotine on BBB permeability, indicating that this effect is receptor-mediated and that receptors within the CNS are probably not involved. Taken together, these data suggest a novel role for endothelial nAChR in regulation of BBB TJ and permeability.

The loss of bone mineral density (BMD) plays a major role in the increased incidence of osteoporosis in aging women and, consequently, strategies to maintain BMD are critical to quality of life for these women. The role of vitamin D in the accrual and maintenance of bone mineral and its relationship to the incidence and severity of osteoporosis is not well understood. By measuring serum and intake levels this study investigates the relationship of vitamin D to baseline BMD and changes in regional and total body BMD over 1 y. The role of sex hormone binding globulin (SHBG) is also investigated. Because SHBG binds with both estrogen, an antiresorptive agent, and testosterone, a bone formation agent, lower serum SHBG concentrations may promote a greater bioavailability of estrogens and androgens, which could decrease resorption, stimulate formation and increase BMD. Women who were 3-10 y postmenopausal, aged 55 ± 5.1 y, and taking or not taking hormone replacement therapy (HRT) were randomized into exercise and non exercise groups: (1) No HRT, no exercise; (2) HRT, no exercise; (3) No HRT, exercise; and (4) HRT, exercise. The number of subjects per group at the end of one year was 25, 19, 27 and 20, respectively. The thrice weekly exercise regimen, consisting mainly of weight lifting and weight bearing activities, lasted for 1 y. Vitamin D deficiency was found in 3% of the subjects, Serum 25(OH)D₃ concentrations had inverse relationships with changes in BMD in the femoral neck (P < 0.05) and trochanter (P = 0.07). When subjects were grouped according to HRT status, BMD at baseline and one 1 y was never positively related to serum 25(OH)D₃ concentrations in HRT users, Subjects having greater than 80 nmol/L 25(OH)D₃ had significantly decreased concentrations of serum osteocalcin and urinary deoxypyrodinoline (Dpd) crosslinks (P < 0.05). Exercise had no effect on serum content of 25(OH)D₃. Serum concentrations of SHBG were not significantly related to BMD at any site, nor did they show a decrease with exercise even when HRT status was taken into account. Significant inverse relationships (P < 0.05) were found between SHBG, sex hormone indices (Estrone/SHBG; Estradiol/SHBG) and bone turnover markers, osteocalcin and Dpd crosslinks/creatinine.

Various modified methods have been proposed in response to criticisms regarding the practical applicability of allometric scaling, which is one of the most widely used approaches in predicting human drug clearance based on data from animal species. The major problems encountered among allometric methods in predicting human drug clearance are addressed in this dissertation. In chapter 2, a large data set for allometric scaling (n = 138) was collected from the literature and was categorized according to the following criteria: oral or systemic clearance; elimination routes; protein or non-protein chemicals; low, intermediate, or high metabolic clearance. Some significant observations have been made regarding the applicability of allometric scaling according to the pharmacokinetic and physical-chemical properties of the drugs examined. Of special note, several potential rules were developed for when one could expect large vertical allometry. In chapter 3, a new model for predicting human clearance was developed. The new model was shown to provide better predictability than any other current approach. In particular, the new model for the first time predicts the occurrence of large vertical allometry noted in humans. In chapter 4, a general equation was derived, which directly describes the mathematical relationship between predicted pharmacokinetic (PK) parameters in humans and the body weights of animals and the values of their corresponding measured PK parameters. This relationship clearly illustrates the species or body weight-dependency of the prediction performance by allometric scaling. Finally, real data from the literature demonstrated the species-dependency predicted from the equation. In chapter 5, the functionality of the correction factors, maximum life-span potential (MLP) and brain weight (BrW) in allometry is mathematically described for the first time. It was found that corrections by MLP or BrW are equivalent to a multiplication of certain constants by the predicted values in humans from simple allometry and has nothing to do with any measured values of PK parameters in any animal species. The role of correction factors (MLP and BrW) or "rule of exponents" in species scaling was evaluated.