Cyclophosphamide is an alkylating agent which has deleterious effects on reproduction in both males and females. When administered to male rats in chronic low doses (6 mg/kg/day), cyclophosphamide caused a dose-dependent increase in post-implantation death of the offspring. On day 7 of gestation, two days after implantation, the inner cell mass-derived embryonic tissues were retarded or absent while trophectoderm-derived extraembryonic tissues appeared normal. The preimplantation growth of embryos sired by cyclophosphamide-treated males was affected; as early as day 3 there was a significant decrease in cell number and in DNA synthesis. On day 3, there was no ultrastructural evidence of active cell death, and embryos underwent compaction, despite their decreased cell numbers. On day 4, embryos sired by treated males had less than half the cell number of controls: this decrease was not lineage-specific. A minority of embryos sired by treated males did not cavitate and showed signs of autophagic death on day 4 of gestation. The majority of embryos sired by treated males were able to cavitate and differentiate morphologically to form small blastocysts. Thus the target of cyclophosphamide damage may be a paternal gene more important for cell proliferation than for cell differentiation in the preimplantation rat embryo.

The regulation of the rat epididymal steroid metabolizing enzymes, (DELTA)('4)-5(alpha)-reductase and 3(alpha)-hydroxysteroid dehydrogenase (3(alpha)-HSD), was investigated during sexual maturation. Nuclear (DELTA)('4)-5(alpha)-reductase activity was first detected at day 21 and reached maximal values by day 77. Subsequently, enzymatic activity declined by more than 60% to reach adult plateau levels by day 105. Cytoplasmic 3(alpha)-HSD was already evident in the youngest age group tested (day 7) and reached the characteristic adult plateau levels by day 63. While 3(alpha)-HSD activity correlated closely with androgen-dependent parameters during development, nuclear (DELTA)('4)-5(alpha)-reductase activity correlated neither with the presence of spermatozoa nor with parameters of androgen levels. To investigate the subcellular distribution of (DELTA)('4)-5(alpha)-reductase and 3(alpha)-HSD during development, a discontinuous sucrose gradient method was developed. While 3(alpha)-HSD activity was found solely in cytoplasmic fractions, (DELTA)('4)-5(alpha)-reductase activity was present only in nuclear and microsomal fractions. In contrast to the bell-shaped developmental profile of nuclear (DELTA)('4)-5(alpha)-reductase activity, the activity of microsomal (DELTA)('4)-5(alpha)-reductase increased gradually with age. / To adequately compare the biochemical properties of nuclear and microsomal (DELTA)('4)-5(alpha)-reductase, enzymatic activity was solubilized. The sedimentation coefficient of solubilized (DELTA)('4)-5(alpha)-reductase was greater for the microsomal enzyme than for the nuclear enzyme (11.6S(' )vs(' )10.1S). The relative capacity of steroids to inhibit enzymatic activity and the pH optima were similar for nuclear and microsomal, membrane-bound and solubilized enzyme forms. While the Km for testosterone was similar in nuclear and microsomal fractions (range: 1.75-4.42 x 10('-7)M), the Km for NADPH was about 30-fold greater for the microsomal enzyme than for the nuclear enzyme. / Thus, (1) 3(alpha)-HSD activity is androgen-dependent and found only in the cytoplasmic fraction; (2) (DELTA)('4)-5(alpha)-reductase activity is regulated by a factor(s) not relating directly to either spermatozoa or androgens; (3) (DELTA)('4)-5(alpha)-reductase activity is regulated as a function of age and subcellular distribution; (4) (DELTA)('4)-5(alpha)-reductase activity can be solubilized in an active and stable form with no major alterations in the active site of the enzyme; (5) there apparently exists at least two distinct forms of (DELTA)('4)-5(alpha)-reductase in the epididymis.

This thesis examines the hypothesis that the parotid gland is a target organ for vitamin D. Employing methods previously used to characterize 1,25-dihydroxyvitamin D$ sb{ rm 3}$ (1,25(OH)$ sb{ rm 2}$D$ sb{ rm 3}$) receptors in established target tissues, classical receptors for this sterol were demonstrated in acinar cells of normal rat parotid gland. Submandibular gland, lacrimal gland and pancreatic acinar cells did not contain 1,25(OH)$ sb{ rm 2}$D$ sb{ rm 3}$ receptors. Vitamin D deprivation caused a decrease in the rate of production of pilocarpine-stimulated and auriculotemporal nerve-stimulated parotid saliva, which persisted when serum concentrations of calcium, parathyroid hormone and 1,25(OH)$ sb{ rm 2}$D$ sb{ rm 3}$ were maintained within normal limits, yet was reversed by treatment with vitamin D$ sb{ rm 3}$ (D$ sb{ rm 3}$). The concentration of calcium in pilocarpine-stimulated saliva did not correlate with decreased salivary flow, but appeared to vary with changes in the serum concentration of this ion. Amylase secretion and calcium release by exocytosis were normal in vitamin D-deprived rats. These findings suggested that fluid secretion but not protein secretion by parotid gland was vitamin D dependent, but that 1,25(OH)$ sb{ rm 2}$D$ sb{ rm 3}$ was not the active metabolite for this effect. Examination of the relative abilities of D$ sb{ rm 3}$, 25-hydroxyvitamin D$ sb{ rm 3}$ (25OHD$ sb{ rm 3}$), 24,25-dihydroxyvitamin D$ sb3$ (24,25(OH)$ sb{ rm 2}$D$ sb{ rm 3}$) and 1,25(OH)$ sb{ rm 2}$D$ sb{ rm 3}$ to correct abnormal parotid function in vitamin D-deprived rats revealed that 24,25(OH)$ sb{ rm 2}$D$ sb{ rm 3}$ was the active metabolite, and essential for normal water and electrolyte secretion by parotid gland. Carbachol-stimulated potassium efflux from parotid glands from vitamin D-deprived rats was normal, suggesting that the site of action of 24,25(OH)$ sb{ rm 2}$D$ sb{ rm 3}$ was later in the sequence of fluid secretion. Sucrose density grad

Initiating the muscle differentiation pathway typically results in activation of muscle-specific genes previously maintained in a silenced state. One of the processes controlling changes in gene expression during differentiation is a global demethylation event. The mechanisms involved in this global hypomethylation are not fully understood. / Promoter methylation is one of the normal mechanisms inactivating gene expression. A single CpG site in the 5' flanking region of myogenin is reported to undergo demethylation during C2C12 differentiation. Considering the demethylase feature of MBD2b and its expression profile during C2C12 differentiation, I propose the hypothesis that MBD2b/demethylase is involved in C2C12 differentiation by demethylating the promoter of the myogenin gene as well as other genes involved in myogenic differentiation. / To test this hypothesis, I determined the consequences of up-regulation and down-regulation of MBD2b/demethylase in C2C12 cells. / The state of the myogenin promoter was also altered as determined using a probe recognizing a single HpaII site, which was previously reported to become demethylated during C2C12 differentiation. (Abstract shortened by UMI.)

Trigeminal neuropathic pain is a unique type of neuropathic pain resulting from damage to primary afferents of the trigeminal nerve that innervates the head and neck region. It is characterized by intractable, unremitting pain in the absence of any overt tissue damage, and represents a significant clinical and societal burden. A thorough understanding of the mechanisms driving this condition is required to adequately treat, and ideally cure, this condition. The skin of the lower lip is innervated by several classes of primary afferents, including myelinated Aβ fibers, thinly myelinated Aδ fibers, and unmyelinated C fibers, which together are able to transduce the variety of innocuous and noxious stimuli encountered in the environment. Nociceptive stimuli are largely mediated by the unmyelinated C fibers, which have been further divided into 2 groups based on neurochemical and anatomical criteria, called the peptidergic and non-peptidergic C fibers. Given that nerve lesions initiating neuropathic pain conditions most often occur in the periphery, changes in the peripheral nervous system following nerve injury are particularly relevant in driving this aberrant pain condition. Therefore, the overall objective of this thesis is to explore the peripheral changes in an animal model of trigeminal neuropathic pain. The results of this thesis are presented in three chapters. The pattern of innervation of non-peptidergic C fibers in the skin of uninjured rats has been described, as well as how this fiber population changes following nerve injury. Concomitant ectopic autonomic fibers were observed in close apposition to the regenerating sensory fibers, and correlated with the behavioral phenotype of the animals. GDNF levels in the skin rapidly increased following nerve injury, and provided a possible mechanism for the ectopic parasympathetic fibers and regenerating non-peptidergic C fibers. Ablation of the non-peptidergic C fibers led to specific sprouting of parasympathetic fibers, but no change in behavior, however ablation of non-peptidergic fibers in neuropathic animals led to an exacerbated pain response. These results suggest an important role for non-peptidergic fibers and autonomic sprouting in neuropathic pain, and identifies GDNF as a potential factor for mediating these changes. / La douleur trigeminale neuropathique est une forme unique de douleur qui résulte d'un dommage aux afférents primaires du nerf trijumeau innervant la région de la tête et du coup. Cette douleur constante qui se manifeste en l'absence d'un dommage aux tissus périphériques représente un fardeau social et économique important. Une compréhension rigoureuse des mécanismes qui mènent à cette condition sera nécessaire pour mieux la traiter et préférablement la guérir.La peau de la lèvre inferieure est innervée par des afférents primaires appartenant à différentes classes, incluant les fibres myélinisées Aβ, les fibres légèrement myélinisées Aδ, ainsi que les fibres C non myélinisées. Ensemble, ces fibres peuvent transmettre une variété de stimuli nociceptifs ou inoffensifs tels que rencontrés dans l'environnement. Les stimuli nociceptifs sont majoritairement transmis par les fibres C non myélinisées, lesquelles peuvent être divisées en 2 catégories, peptidergiques ou non peptidergiques, selon des critères neurochimiques et anatomiques. Puisque les lésions nerveuses qui déclenchent la douleur neuropathique se produisent le plus souvent en périphérie, les changements au niveau du système nerveux périphérique sont centraux au développement de la condition douloureuse. Ceci étant dit, l'objectif général de cette thèse est d'explorer les changements périphériques qui se produisent dans un model animal de douleur trigeminale neuropathique.Les résultats de cette thèse sont présentés dans trois chapitres. L'innervation de la peau par les fibres C non peptidergiques chez le rat normal a d'abord été décrite. Ensuite, les changements subis par cette population suivant un dommage au nerf ont été documentés. Suite à un dommage nerveux périphérique, des fibres autonomiques ectopiques ont été observées en proximité des fibres sensorielles en régénération, et ce changement corrélait temporellement avec le phénotype comportemental des animaux. Les niveaux cutanés de GDNF ont rapidement augmenté après le dommage nerveux, fournissant un mécanisme potentiel permettant la régénération des fibres C non peptidergiques et la migration ectopique des fibres parasympathiques. L'ablation des fibres C non peptidergiques a déclenché la pousse des fibres parasympathiques sans changer le comportement des animaux. Par contre, l'ablation de ces fibres chez des animaux neuropathiques a exacerbé la réponse douloureuse de ceux-ci.Ces résultats suggèrent une participation importante des fibres C non peptidergiques et de la migration autonomique dans la douleur neuropathique. De plus, GDNF est rapporté comme étant un facteur pouvant produire ces changements.

No description available.

This thesis is concerned with the relationship between choline delivery and acetylcholine synthesis in the cat superior cervical ganglion. Increasing extracellular choline did not change the amount of acetylcholine either contained in or released by ganglia, except when choline levels were increased by 100-fold in plasma. In the latter case, most of the extra ACh formed was not releasable and may have accumulated as a result of decreased acetylcholine degradation rather than increased acetylcholine synthesis. / Choline transport activity in stimulated ganglia can be monitored using choline analogues and this process was found to have similar, but not identical, characteristics as the high affinity choline transport described for synaptosomes. Under certain conditions, the uptake and acetylation of choline were dissociated and, therefore, choline transport alone does not determine the rate of acetylcholine synthesis. Choline transport is activated following a conditioning stimulus, and under these conditions choline uptake is closely related to acetylcholine synthesis. It is suggested that the relationship between choline uptake and acetylation changes with the functional state or history of cholinergic neurons.

The influence of protein-calorie malnutrition (PCM) on the anti-inflammatory activity (suppression of carrageenan-induced paw edema and pleurisy) of salicylate was studied in Sprague-Dawley male rats. For this purpose, animals were fed a 21% (control) or a 5% (PCM) protein diet ad libitum or a control diet in restricted amounts (pair-fed). A significant increase in the anti-inflammatory activity of salicylate was observed in PCM but not in pair-fed rats. PCM caused a decrease in plasma salicylate (assayed by HPLC) concentration (due to increased synthesis of glycine conjugate and elimination), no change in tissue distribution, and a moderate decrease in serum protein binding of salicylate. PCM exerted inconsistent effects on the activities of liver lysosomal enzymes (activities of (beta)-glucuronidase decreased, of acid phosphatase did not change, and of aryl salfatase increased) and there was no relationship between the lysosome stabilizing and anti-inflammatory activities of salicylate and other aspirin-like drugs (indomethacin and oxyphenbutazone). It was therefore concluded that the PCM-induced increase in the anti-inflammatory activity of salicylate was primarily not due to any changes in its pharmacokinetics or in its effects on lysosomal membranes. The biosynthesis of prostaglandins and leukotrienes was studied because of their important role in inflammatory processes. PCM decreased the biosynthesis by pleural neutrophils of both cyclooxygenase-independent (major products: thromboxane B(,2) and 12-hydroxy-5,8,10-heptadecatrienoic acid) and lipoxygenase-dependent (leukotriene B(,4)) metabolites of arachidonic acid. The biosynthesis of prostaglandins E(,2) and F(,2(alpha)) by renal medulla homogenates and of prostaglandin D(,2) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid by spleen homogenates were also reduced by PCM. At all concentrations of aspirin studied, the amounts of these metabolites were less in preparations from PCM than from control animals. Ho

Hepatic alpha-adrenoceptors have been characterized pharmacologically and biochemically. In isolated rat liver cells, activation of glycogen phosphorylase by adrenergic agonists and its inhibition by subsite selective antagonists are mediated by alpha(,1)-receptors that do not display the phenomenon of receptor reserve. When tested in dilute cell suspensions to minimize uptake and metabolism of ligands, these receptors have properties indistinguishable from those of alpha(,1)-receptors in other tissues. In purified liver plasma membranes, the reversible radioligand {('3)H}prazosin labels a single population of high affinity binding sites with properties similar to the alpha(,1)-receptors mediating the glycogenolytic response of intact cells. Affinity labeling of these sites was achieved by using {('3)H}phenoxybenzamine ({('3)H}POB), synthesized in our laboratory. In intact liver cells {('3)H}POB binds irreversibly to stereoselective sites with properties expected from alpha-receptors. Half-maximal blockade of the glycogenolytic response to adrenaline and half-maximal saturation of binding sites occur at the same, low nanomolar concentrations of POB, at which no labeling of serotonin, histamine or muscarinic receptors can be detected. Further analysis of {('3)H}POB binding in purified plasma membranes indicates selective labeling of the alpha(,1)-receptor subtype. Molecular characterization of detergent-solubilized, {('3)H}POB-labeled alpha(,1)-receptors indicates that it is a protein with a Stokes radius of 6 nm, and a subunit molecular weight of 80,000.

The role of $ beta$-endorphin in the cardiovascular depressor response elicited by clonidine or by electrical stimulation of the nucleus tractus solitarii (NTS) was studied in awake as well as anesthetized rats. The opiate antagonists, naloxone or naltrexone, inhibited the hypotensive and bradycardic effects of clinidine in spontaneously hypertensive rats (SHR), in normotensive and steroid-salt hypertensive Sprague-Dawley (S-D) rats, but not in normotensive Wistar Kyoto (WKY) rats. The brain ACTH/$ beta$-endorphin fibre system originating in the arcuate nucleus was eliminated by neonatal treatment with monosodium glutamate (MSG). MSG-treatment abolished the naloxone-sensitive component of the effects of clonidine in SHR, while the naloxone-resistant effects of clonidine in WKY were not affected. Intra-NTS microinjection of clonidine (5 nmol) decreased blood pressure and heart rate, and these effects were inhibited by previous intra-NTS injection of dl- but not d-naloxone (270 pmol) in both SHR and normotensive S-D rats. Intra-NTS injection of $ beta$-endorphin (0.3 pmol) also reduced blood pressure and heart rate in both rat strains. In SHR, the effects of both clonidine and $ beta$-endorphin were inhibited by ICI 174864, a selective $ delta$ receptor antagonist, but not by $ beta$-funaltrexamine, a selective $ mu$ receptor antagonist (270 pmol, intra-NTS). In contrast, in normotensive S-D rats only the $ mu$ and not the $ delta$ antagonist was an effective inhibitor. In S-D rats made hypertensive by prolonged treatment with a mineralocorticoid and salt, the pattern of inhibition was the same as in SHR. The depressor baroreflex response was elicited by electrical stimulation of the NTS. In SHR, intra-NTS injection of an antiserum to $ beta$-endorphin inhibited, while an antiserum to met-enkephalin potentiated the hypotensive and bradycardic response to NTS stimulation. In similar experiments in WKY rats, the antisera were ineffective. / These findings are interpreted to indicate that the cardiovascular effects of clonidine involve release of $ beta$-endorphin and subsequent stimulation of opiate receptors in the NTS. The most likely source of $ beta$-endorphin is nerve terminals whose cell bodies are in the arcuate nucleus. The released $ beta$-endorphin acts on $ mu$ receptors in normotensive or $ delta$ receptors in hypertensive rats, with no evidence for the additional involvement of peripheral opiate receptors. (Abstract shortened with permission of author.)