Cells of complex organisms communicate with each other by sending molecular signals. These signals can be classified by their solubility properties. Hydrophilic signals, in the form of peptides or small hydrophilic molecules, interact with extracellular receptors located on the surface of target cells. Binding of ligand to its receptor leads to transduction of an intracellular signal via a second messenger. Lipophilic signals (steroids, vitamin D$ sb3$, thyroid hormone, and retinoids) traverse the plasma membrane and bind to specific intracellular proteins, known collectively as nuclear receptors, rendering them active. These ligand-receptor complexes then regulate the transcription of target genes. In this thesis, transcriptional regulation by two different systems has been studied. First, recent evidence suggests that expression of the parathyroid hormone-related peptide (PTHrP) gene is sensitive to elevated levels of cyclic adenosine monophosphate (cAMP). Second, vitamin D$ sb3$ is activated in the kidney by hydroxylation and functions by diffusing across the plasma membrane of target cells and binding the vitamin D$ sb3$ receptor (VDR). (Abstract shortened by UMI.)

To define the role of mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5; mGPD) in energy balance and intermediary metabolism, we studied female transgenic mice lacking the mGPD gene (mGPD-/-). These mice had higher serum glycerol and triglycerides; lower body weight, blood glucose, and energy expenditure (QO2); and higher glycerol-3-phosphate and lactate/pyruvate ratio in muscle than controls with wild type genotype (WT). When given a high fat/low carbohydrate diet, mGPD-/- mice gain more weight than WT, without the genotype differentially affecting QO2 or calorie intake. On a low fat/high carbohydrate diet, mGPD-/- mice failed to increase QO2 as the WT and gained more weight. After a 30-hour fasting or food restriction to 70% for 10 days, WT lost significantly more weight than mGPD-/- mice, but these latter had lower body temperature and QO2. Thus, mGPD-/- mice exhibit a thrifty phenotype largely resulting from reduced obligatory thermogenesis.

Although an extensive body of literature exists describing the properties of cells found in the primate vestibular nuclei (VN), the rat vestibular nuclei have not been explored in a comparable manner in order to allow for meaningful comparisons. Previous single-unit experiments in rat VN did not track eye movements and were performed in anesthetized or paralyzed preparations. This study characterizes the properties of vestibular-related cells in awake, behaving rats, using standard single-unit methodology, and video oculography to monitor eye movements, thus providing the first description that is comparable to the primate literature. Male, Long-Evans rats were head restrained and sinusoidally rotated at frequencies ranging from 0.1-1.0Hz, reaching a maximum velocity of 100 deg/sec. Eye position sensitivity was assessed by recording cell activity as the rat fixated at different locations. We show the presence of cells that are sensitive only to vestibular stimulation, equivalent to vestibular-only cells in primates. These cells have no eye sensitivity, and show a moderate increase in head velocity sensitivity with increasing stimulus frequency. Additionally, we show the presence of cells with eye movement-related sensitivities that bear a close resemblance to primate eye-head neurons. These results suggest that the rat vestibular nuclei may contain a similar cross section of cells as those found in the primate vestibular nuclei. These results shed light on the type of information in the vestibular nuclei that is available for other upstream systems, and is particularly relevant to spatial orientation, which has been shown to depend on vestibular input.

Renin is an aspartyl protease that plays a critical role in the production of angiotensin peptides and therefore in the modulation of blood pressure. Renin participates not only in the circulating renin-angiotensin system (RAS) but may also be taken up by tissues to control the activity of the local RAS. In this study, we provide evidence for the existence of a high affinity, saturable binding site for human renin (estimated k$ sb{ rm d}$ of 125 pM) and prorenin, the inactive precursor of renin, in the human placenta. The interesting finding that prorenin could bind to the same site as renin suggests that its role in vivo may be to act as a natural antagonist that would limit the local levels of angiotensin II production. It is of further interest to identify the protein regions important in this uptake; here we show that the degree of glycosylation in prorenin and the active site of renin are not essential in this process. In the near future, experiments will involve in-depth studies of the regions implicated in the binding process and long term goals will be to identify and characterize this novel renin and prorenin binding site.

Mammalian prosomatostatin (PSS) is cleaved at an Arg-Lys doublet in order to produce somatostatin-14 (SS-14), and at two singlets, Arg and Lys, in order to release SS-28 and PSS$ sb{ lbrack 1{-}10 rbrack}$ respectively. Furin, PC1, and PC2 are three recently cloned mammalian endoproteases, associated with the Golgi (furin) or targeted towards the secretory granules of the regulated pathway (PC1 and PC2). In order to determine the enzymes involved in the proteolytic processing of PSS and localize the endoproteolytic reactions in specific compartments of the secretory pathway, I compared PSS processing in endocrine (AtT-20, GH3, and GH4C1 pituitary cells) or nonendocrine tumor cell lines (COS-7 kidney cells, PC 12 pheochromocytoma, LoVo colon adenocarcinoma cells). The efficiency of PSS processing to SS-14, SS-28, and PSS$ sb{ lbrack 1{-}10 rbrack}$ was correlated with: (i) secretion through the constitutive or regulated pathway, (ii) mRNA expression for furin, PC1 and PC2, and (iii) exogenous expression of furin, PC1, and PC2 in cells deficient in these proteinases. Coexpression of PC1 and PC2 with rat PSS was accomplished by transient (COS-7 cells) or stable (GH4C1 cells) cotransfections of their cDNAs in the respective cell lines. In the case of furin, recombinant vaccinia viruses expressing human furin or rat PSS were used to coinfect COS-7 or LoVo cells. Furthermore, I examined the effect of granules on the expression of PC2 and on PSS processing, by incubating GH4C1 cells with insulin, EGF, and $ beta$-estradiol, a treatment known to induce granule formation. Cell extracts and secretion media were further analyzed by HPLC and somatostatin specific RIAs. / Conclusions. (i) PSS is capable of monobasic processing within the constitutive secretory pathway. (ii) Such cleavages may be effected by furin or related endoproteases but are relatively inefficient. (iii) PC1 is capable of dibasic cleavage of PSS to SS-14 in both constitutive or regulated secretory cells. (iv) PC2 mediates SS-14 conversion only if expressed in regulated secretory cells. (v) The milieu of secretory cells, and not the granules, is required for PC2 activity. (vi) Furin is a mammalian SS-28 convertase, but not the unique one. (vii) A yet unknown endoprotease is responsible for PSS$ sb{ lbrack 1{-}10 rbrack}$ cleavage.

This thesis addresses the expression, regulation, and functional aspects of NOS in normal and developing skeletal muscles, and their role in contractile dysfunction of respiratory muscles associated with septic shock. Normal skeletal muscles of mammalian species express only ecNOS and nNOS to varying degrees. NOS activity in these muscles is mainly Ca2+/calmodulin-dependent and it associates with fast-twitch muscle fibers in rat and mice, but no such correlation exists in other species. Therefore, NOS activity is not the only factor that specifies contractility of skeletal muscles. In developing skeletal muscles, there is a transient increase in NOS Ca2+-dependent activity and the expressions of cNOS isoforms are upregulated. This coincides with skeletal muscle differentiation and maturation. Despite the negative influence of NOS activity on skeletal muscle contractility, little inhibition is observed on force generated by the developing diaphragm. Therefore, NO may regulate other processes than contraction in developing skeletal muscles. The in-vivo induction of iNOS protein and mRNA in skeletal muscles of septic rat is matched by a parallel induction in GTP-cyclohydrolase-I, the rate-limiting enzyme for BH4 biosynthesis. NOS Ca2+ -independent activity increases several fold mainly in the respiratory muscles. In addition, the expressions of cNOS enzymes are upregulated in septic rat muscles. During 24 hrs of endotoxemia of rats, iNOS is induced by 6 hrs, peak by 12 hrs and disappear by 24 hrs after LPS injection. nNOS and ecNOS expression is upregulated by 6 hrs and remained higher than control values after 24 hrs of LPS injection. The regulation of NOS isoforms is matched by an increase in total and Ca2+/calmodulin-independent NOS activity. Furthermore, peroxynitrite was detected in septic respiratory muscles, and nitrated proteins were detected in these muscles 12 hrs after LPS injection. Submaximal force generated by diaphragm strips was significantly inhibit

Although rodents have been widely used in respiratory research, there are still only limited data comparing respiratory mechanics between different species of small animal. In order to provide further insight into the mechanical behavior of the respiratory systems of different sized small animals, accurate measurements of respiratory impedance (Zrs) were made in four different rodent species and in the developing rat over a broad range of frequencies at various levels of positive end-expiratory pressure (PEEP). PEEP dependencies of airway and tissue properties were interpreted in terms of physiological phenomena such as airway closure and airway-parenchymal interdependence forces. In adult animals, Zrs was fitted to a model including a Newtonian resistance (R) in series with a constant-phase tissue compartment. In general, rodent respiratory parameters obeyed the same scaling laws described in other species, but rabbits had a relatively higher elastance than one would predict from previously published allometric relationships. This is probably due to the rabbit's proportionately smaller airspace volume. R normalized to body weight was lower in smaller species suggesting that they have proportionately wider airways compared to larger animals. By using computer models of the asymmetric airway tree to estimate airway resistance (Raw), we confirmed that the larger of two isomorphic rodent species has relatively higher Raw. Moreover, we showed that both the airway dimensional scaling differences and the asymmetric arrangement of the individual airways are responsible for the relative differences in Raw between smaller and larger animals. Finally, in the developing rat, elastance and resistance normalized to lung weight decreased progressively with age, suggesting that intrinsic changes in the mechanical properties of the respiratory system occur with development. Parenchymal interdependence forces manifested themselves in animals as young as 10 days of age, with PEEP

In the rat, both the activation of peripheral baroreceptors as well as electrical stimulation in the diagonal band of Broca depress the excitability of vasopressin (VP)-secreting neurons in the hypothalamic supraoptic nucleus via GABA$ sb{ rm A}$ receptors. Baroreceptor activation alters the firing of neurons in the diagonal band consistent with a role as a central relay by which baroreceptor activation arrests the activity of supraoptic VP neurons. Neurons in the adjacent median preoptic nucleus (MnPO) also receive baroreceptor-derived input. Anatomical tracing techniques indicate that MnPO neurons project directly to the supraoptic nucleus whereas diagonal band neurons project to the lateral hypothalamic area adjacent to the supraoptic nucleus where they are proposed to contact GABAergic interneurons. This study evaluates the contribution of the diagonal band and MnPO to the central pathway(s) conveying baroreceptor-derived information to supraoptic VP neurons in the rat, using in vivo extracellular electrophysiology combined with neurotoxic (ibotenic acid) lesions. Stimulation in the MnPO inhibited the majority of supraoptic neurons via GBA$ sb{ rm A}$ receptors. Selective destruction of neuronal somata in MnPO failed to alter baroreceptor-induced inhibition of supraoptic VP neurons. By comparison, lesions in the diagonal band of Broca, or lateral hypothalamus, significantly reduced the number of baroresponsive VP neurons. Moreover, lesions in the lateral hypothalamus attenuated diagonal band of Broca-, but not MnPO-evoked inhibition of supraoptic VP neurons. Thus, neurons in the diagonal band, through their connections to GABAergic interneurons located in the lateral hypothalamus, are considered to relay baroreceptor input to supraoptic VP neurons. While stimulation of MnPO inhibits supraoptic neurons via GABA$ sb{ rm A}$ receptors, this innervation does not appear to convey baroreceptor-derived information to the supraoptic nucleus.

The objective of this thesis was to elucidate the neurophysiological and neurochemical bases for modulation of sensory transmission in the spinal cord evoked by the activation of primary afferents in the lightly anaesthetized rat. Effects of prolonged, intense (20 $ times$ threshold), low frequency (4 Hz) stimulation of meridian and non-meridian sites on the thermally evoked nociceptive withdrawal reflexes of the tail and limbs were studied. Threshold was the minimum current required to elicite muscle contraction. / Intense stimulation applied to meridian sites inhibited tail withdrawal. This inhibition persisted beyond one hour after the end of stimulation. Stimulation of non-meridian sites produced a smaller inhibition; this occurred during the conditioning only. Thus, a brief inhibition or both the brief and a persistent, post-stimulation inhibition were produced by stimulation of non-meridian or meridian sites, respectively. Little effect was evoked on limb withdrawal reflexes. / Expression of the post-stimulation effect required 20 $ times$ threshold stimulation with long pulse durations ($ geq$2 ms), low frequency of stimulation (2 Hz-6 Hz) and long train durations (20 or 40 min). The brief effect could be evoked at 10 $ times$ threshold with short pulse durations($ leq$2 ms) at higher frequencies of stimulation (8 Hz) and with short train durations (10 min). / Stimulation of meridian sites evoked both the brief and the post-stimulation effects in chronic spinal transected rats (7-14 days): in acutely spinal transected rats ($ leq$48 h) the brief effect was evoked only. The return of the post-stimulation effect was coincident with the return of bladder function. / Both the brief and post-stimulation inhibition were blocked by the competitive NMDA receptor antagonist 5-amino-2-phosphonovaleric acid (APV). / The wide spectrum opiate receptor antagonist naloxone, or $ mu$-opiate antagonist $ beta$-funaltrexamine, attenuated both the brief and persistent inhibition. The $ delta$- and $ kappa$-antagonists, TIPP($ psi$) and nor-binaltorphimine, attenuated the inhibition during the stimulation. Both drugs blocked the post-stimulation effect and even facilitated withdrawal. In chronically spinal transected rats, naloxone blocked the inhibition. / These data suggest that intense, low frequency activation of primary afferents arising from meridian but not non-meridian sites produces both brief and persistent inhibition of the tail withdrawal reflex. Limb withdrawal reflexes are only minimally inhibited by this activation. It is suggested that the persistent antinociception may be due to long-term plastic changes in inhibitory mechanisms within the CNS because these effects persist long after the end of stimulation and presumably after synaptic inputs from these fibres have ceased. It is also suggested that inhibitory mechanisms are provoked by prolonged activation of high threshold fibres, are dependent on the parameters of stimulation, are extrasegmental in nature and differentially modulate tail vs. limb nociceptive reflexes. Activation of spinal NMDA receptors appears critical for the expression of the persistent antinociception. The inhibition is also differentially mediated by activation of multiple opiate receptors: $ mu$-, $ kappa$- and to a lesser degree $ delta$-receptors mediate the brief effect, while the persistent antinociception is dependent on activation of $ delta$ -and $ kappa$-receptors and to a lesser degree $ mu$-receptors. Data from spinal animals suggest that the mechanisms mediating the inhibitory effects include both spinal and supraspinal components.

Ubiquitin is a highly conserved 8 kDa protein whose many cellular functions are mediated by its covalent ligation to other proteins. Conjugation of ubiquitin is a multistep process involving a ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (UBCs or E2s), and ubiquitin protein ligases (E3s). The multi-ubiquitination of substrates marks them for degradation by the 26S proteasome. / Previously, rat UBC4 isoforms homologous to S. cerevisiae UBC4/UBC5 were cloned and characterized (Wing and Jain, 1995) (Wing et al., 1996). The UBC4-1 isoform is highly expressed in the testis, and the UBC4-testis isoform is induced in round spermatids. This thesis describes the identification and characterization of E3s interacting with these UBC4 isoforms expressed in the rat testis. / First, the isolation and characterization of a novel E3 from rat testis extracts, E3Histone, is described. E3Histone mediates conjugation of ubiquitin to histones in a UBC4-dependent manner. Interestingly, E3Histone was immunodepleted by antibodies against Cdc27, a subunit of the a&barbelow;naphase-p&barbelow;romoting c&barbelow;omplex (APC), an E3 which plays a critical role in the regulation of the cell cycle. However, E3Histone and the APC are distinct complexes. Gel filtration resolved the 600 kDa E3Histone from the 1500 kDa APC. E3Histone interacts preferentially with UBC4, whereas the APC interacts preferentially with UbcH10 and shows specificity for the substrate cyclin. E3Histone and the APC may be members of a newly-recognized family of combinatorial E3s that share some common core subunits, such as CDC27, yet possess distinct subunits that confer upon them their respective E2 and substrate specificities. In addition, E3Histone activity was detected in extracts from various purified germ cells. Induction of UBC4 may lead to the increased ubiquitination of histories and together with E3 Histone may play a role in the chromatin condensation that occurs during spermatid maturation. / Secondly, the characterization of a HECT domain E3, Rat100, is described. UBC4-1 and UBC4-testis were found to transfer ubiquitin to Rat100 in vitro. Immunoblotting showed that Rat100 has a molecular weight of 300 kDa, and that the developmental and cell-specific expression of Rat100 correlates with that of UBC4. The induction of Rat 100 may playa role in the activation of ubiquitin-dependent proteolysis during spermatogenesis.