The object of this study was to investigate the scope of cognitive control over vestibulo-ocular reflex (VOR) performance during rotational vestibular stimulation. Human subjects, with head fixed to the body, were rotated in the dark through 20$ sp circ$ at 40$ sp circ$/s while trying to "look" at an earth-fixed target which was viewed straight ahead just before extinguishing the lights. During this rotation an apparent error of performance was systematically introduced by displacing this target at constant velocity through 12$ sp circ$, either in the same direction as the subject (Diminishing paradigm), or in the opposite direction (Augmenting paradigm). After cessation of rotation the target was re-illuminated and the subject allowed to see the final positional "error" of his/her oculomotor performance. During each trial they were asked either to try and CORRECT or NOT to CORRECT for the extrinsically induced "errors". In a subsequent, series of experiments, 2 hrs of synchronous rotation of the subject and the surrounding visual scene was used to produce adaptive attenuation of the VOR ($ approx$26%). The central component of the second experimental series was performance of the gaze stabilization test described above, conducted on the adapted subjects. (Abstract shortened by UMI.)

Recent studies have shown that a variety of K$ sp+$ channels can be enhanced by somatostatin (SST; inward and delayed rectifier, muscarinic regulated K$ sp+$ channel, Ca$ sp{2+}$-activated K$ sp+$ channel, ...). Since a somatostatin receptor (SSTR) is heterogeneous, it is not clear which SSTR subtype (five are identified) is involved in regulating each K$ sp+$ channel, nor whether the regulation is and if so to what extent receptor subtype specific. In this study we characterized electrophysiologically the effect of somatostatin-14 (SST-14) in monkey kidney cells (COS-7) stably expressing one of the five recently cloned SSTR subtypes, human somatostatin receptor subtype 4, using whole cell voltage clamp technique at room temperature (20-23$ sp circ$C). / The outward current produced by a sequence of depolarizing steps from a holding potential of $-$80 mV was enhanced by $>$1 nM SST-14. Both the endogenous and SST-14 enhanced outward current had a high threshold ($ sim -$40 mV). The reversal potentials of their tail currents changed with a change of (K$ sp+ rbrack sb{ rm o}$ as expected for a current carried predominantly by K$ sp+$ ions. It was blocked by TEA (10-20 mM) and Ba$ sp{2+}$ (1 mM), but not by charybdotoxin (100 nM) and carbachol (50 $ mu$M) in the extracellular solution suggesting that it is largely due to a delayed rectifier K$ sp+$ channel. / A smaller inward current at potentials negative to $ sim -$60 mV was also observed and was enhanced by SST-14 in a similar concentration range. It was blocked by Ba$ sp{2+}$ (1 mM) and Cs$ sp+$ (1 mM) suggesting that SST-14 also enhances an inward rectifier K$ sp+$ current. Both outward and inward currents enhanced by SST-14 were pertussis toxin and cAMP sensitive.

The organum vasculosum of the lamina terminalis (OVLT) is a central osmoreceptor which regulates neurohypophysial hormone release through glutamatergic synapses onto hypothalamic magnocellular neurosecretory cells (MNC's). Previous studies have shown that OVLT neurons can express low threshold spikes which, in other neurons, contribute to rhythmic firing. Electrophysiological recordings were therefore obtained in the medial OVLT in superfused explants of rat hypothalamus, which was found to contain neurons projecting to MNC's in the supraoptic nucleus. The OVLT neurons recorded revealed the presence of tonic firing at varying frequencies (0-40 Hz), as well as two distinct forms of bursting activity. Among these, clustered firing featured the highest frequencies (up to 100 Hz), and was observed in 31% of the neurons studied. The effects of two conditioning protocols on synaptic function were investigated by examining excitatory postsynaptic potentials (EPSP's) in MNC's during electrical stimulation of the OVLT. Trains of 100 Hz stimulation lasting 1 s were found to induce post-tetanic potentiation and long lasting potentiation ($>$1 hr). Patterned stimulation mimicking clustered firing evoked only short-term ($<$30 s) increases in EPSP amplitude capable of increasing the probability of spike discharge in MNC's. Activity patterns adopted by OVLT neurons may therefore be important for the regulation of neurohypophysial hormone release and body fluid homeostasis in vivo.

Earlier lesion and imaging studies in humans, as well as electrophysiological recordings in other animals, have suggested an important role for the thalamus during slow wave sleep (SWS). This structure has been shown to undergo disfacilitation and active inhibition during SWS, changing its firing pattern and thus altering sensory afference to the cortex during this state. / Regional cerebral blood flow (rCBF) decreases are reported in the human thalamus in association with delta (1-4 Hz) and, even further, with spindle (12-15 Hz) wave activity during SWS. These results support the physiological changes purported to occur in the thalamus during this state. We also found rCBF decreases in association with delta in the brainstem reticular formation, cerebellum, anterior cingulate and orbito-frontal cortex. These changes, together with the thalamic decreases, might provide the physiological substrate for the progressive attenuation of sensory awareness and motor activity that occur during SWS. Regional CBF increases as a function of delta activity were found mainly in the visual and auditory cortices, possibly supporting dream-like imagery during SWS.

The Interferon Regulatory Factors (IRFs) are a family of interferon-inducible proteins which play distinct roles in diverse processes such as pathogen response, cytokine signalling, cell growth regulation and hematopoietic development. The objective of this research was to investigate the mechanisms by which IRF-1 and IRF-2 regulate gene expression and cell growth. Structure-function analyses of the IRF-2 protein demonstrate that transcriptional repression by IRF-2 is contained within the first 125 N-terminal amino acids and correlates directly with IRF-2 DNA binding. Overexpression of functional IRF-2 deletion mutant proteins in NIH3T3 cells results in oncogenic transformation and tumorigenesis, suggesting that IRF-2 oncogenicity correlates directly with transcriptional repression. Similar structure-function analyses localize IRF-1 transactivation to the C-terminus. Like IRF-1, hybrid constructs which fuse the DNA binding domain of IRF-1 and IRF-2 to the transactivation domain of NF-kappaB RelA(p65) are transcriptional activators. Inducible expression of IRF-1 and IRF/RelA in NIH3T3 cells results in reduced cellular growth and induction of apoptosis. Furthermore, expression of the PKR, STAT1(p91), and WAF1 growth regulatory proteins are elevated following induction of IRF-1 or IRF/RelA, correlating transactivation function and tumor suppressor activity of IRF-1 or IRF/RelA. By RNA fingerprinting, the secretory leukocyte protease inhibitor (SLPI) was identified as the first gene whose expression is downregulated by IRF-1 or IRF-1/RelA. A region in the SLPI promoter was identified that bound IRF-1, suggesting a direct mechanism for IRF-1 regulation of SLPI expression.

Although cardiac hypertrophy is a compensatory mechanism of the stressed heart, it ultimately leads to cardiac dysfunction. GATA-4, a member of the GATA family, plays an essential role in this process, nevertheless, its mechanism of action is still largely unknown. We have isolated a new evolutionarily conserved protein, ENDO1, that belongs to a new subfamily of PHD finger proteins. In adult mouse, ENDO1 was highly expressed in the kidney, lung, spleen, heart and brain. In ventricular cardiomyocytes, endogenous ENDO1 was expressed in the nucleus and cytoplasm. Overexpression analysis of antisense and sense GATA-4 suggests that GATA-4 downregulates endogenous ENDO1 in cardiomyocytes at the mRNA and protein levels. The protein levels of ENDO1 were enhanced in cardiomyocytes stimulated with phenylephrine, a hypertrophy stimulus. Structure-function analysis revealed that ENDO1 inhibits the activity levels of the BNP promoter. Our results indicate that the ENDO1 protein may play a significant role in cardiac development and differentiation.

The Na,K-ATPase or sodium pump is an integral membrane protein found in the plasma membrane of virtually all animal cells. It exists as an alphabeta heterodimer for which several isoforms have been described. During its reaction cycle, the sodium pump extrudes three Na+ ions from the cell in exchange for two extracellular K+ ions using the energy of hydrolysis of one ATP molecule. The electrochemical sodium gradient generated provides the driving force for secondary solute transport. Structure/function analysis of the catalytic subunit, alpha, provides strong evidence for interactions between the actuator domain which comprises the cytoplasmic N-terminus and the M2--M3 loop, and the M4--M5 loop containing the ATP binding and phosphorylation sites. This thesis describes two major aspects of Na,K-ATPase structure and function. First, the role of the unique N-terminus of the ubiquitous al isoform of the rat Na,K-ATPase in E1/E2 conformational transitions is investigated as this region extends beyond that of the well-characterized and related SERCA pump for which high resolution structures have been obtained. Furthermore, the N-terminus is the region of greatest primary sequence diversity among the otherwise homologous alpha1, alpha2 and alpha3 isoforms. The results provide strong evidence for a self-regulatory domain within the N-terminus that modulates conformational transitions via novel intramolecular interactions within the N-terminus and between the N-terminus and regions within the cytoplasmic M2--M3 and M4--M5 loops. Another aspect of this thesis concerns a comparative study of the isoform-specific ligand affinity differences. For this study, the alpha2 and alpha3 isoforms were compared with the ubiquitous alpha1 isoform. The results demonstrate that alpha3-specific ligand affinities can be explained by its distinct reactivities with alkali cations, as well as by differences in the rates of partial reactions. The distinct kinetics of a

Steroid receptors mediate their effects on gene transcription. by interacting with cis-acting enhancer regions known as response elements (REs). Recent evidence from the long terminal repeat of the Mouse Mammary Tumour Virus (MMTV) suggests that glucocorticoid response elements (GREs) may consist of directly-repeated half-sites separated by nine base pairs (GRE-D9). Evidence is provided that GRE-D9 can bind to GR homodimers in vitro, and that, when present in multiple copies, can mediate transactivation of a reporter gene in vivo. / Heat shock protein 90 (hsp90) has been implicated in modulating the ligand-inducibility of the estrogen receptor (ER) in vivo. In order to better characterize the functional significance of the interaction between these two proteins, chimeric ERs consisting of the GAL-4 DNA binding domain fused to the ligand binding domain (LBD) of the ER were constructed. Although these chimeras are ligand-dependent transactivators, they bind DNA in the absence of estradiol and do not interact with hsp90 in vitro. Furthermore, data are presented which are consistent with a model in which transcriptional interact with the ER LBD in a ligand-dependent manner. Introduction of the destabilizing Gly400Val substitution in the ER L33D results in a chimera which is ligand-dependent for both DNA binding and transactivation. These partially destabilized chimeras are shown to interact with hsp90 in vitro in a ligand-dependent manner, whereas chimeras derived from wild-type ER show only a transient association with hsp90, suggesting that hsp90 serves as a molecular chaperone for the ER. A functional model for ER/hsp90 interaction is discussed.

Chromogranin-A (CgA) and parathyroid hormone (PTH) are the two major secretory products of the parathyroid gland. While CgA is believed to be important for cell secretory activity, its precise function(s) remain to be fully elucidated. The effects of the active metabolite of vitamin D, 1,25-dihydroxyvitamin D$ sb3$ (1,25(OH)$ rm sb2D sb3)$ were investigated on parathyroid gland CgA mRNA in the rat. Parathyroid CgA mRNA levels increased in a dose-dependent manner in response to injections of 1,257(OH)$ rm sb2D sb3$ whereas PTH mRNA levels decreased. CgA mRNA levels were less sensitive to 1,25(OH)$ rm sb2D sb3$ than PTH mRNA levels. Having established in vivo regulation of CgA by 1,25(OH)$ rm sb2D sb3,$ the potential role of altered expression of CgA in the pathophysiology of secondary hyperparathyroidism (2$ sp circ$HPT) was explored using the uremic rat model. Five-sixth (5/6) nephrectomized rats maintained on a normal diet had normal levels of serum calcium and phosphate but reduced levels of 1,25(OH)$ rm sb2D sb3.$ In these uremic rat parathyroid glands, whereas PTH mRNA levels were four-fold higher, CgA mRNA levels were 50% lower that those in sham-operated rats. In uremic rats maintained on a high phosphorus diet, 5/6 nephrectomy resulted in decreased serum calcium and 1,25(OH)$ rm sb2D sb3$ levels. These animals had elevated parathyroid CgA and PTH mRNA levels. Thus, in vivo, increased serum 1,25(OH)$ rm sb2D sb3$ concentrations stimulate, and decreased serum levels inhibit parathyroid CgA mRNA expression. However, this latter effect depends upon the calcium status of the animal. (Abstract shortened by UMI.)

The ultradian rhythm of growth hormone (GH) secretion from the anterior pituitary gland is ultimately controlled by the complex interaction of two hypothalamic hormones, a stimulatory GH-releasing factor (GRF), and an inhibitory hormone, somatostatin (SRIF). / In the present study, we used the long-acting SRIF analog, octreotide, as a probe in both the normal and mutant dwarf rat to (1) further clarify the temporal nature of the SRIF/GRF interplay in GH regulation in vivo, and (2) define possible mechanisms of action of SRIF in generating the ultradian rhythm of GH secretion characteristic of the normal male rat. Administration of octreotide to free-moving, chronically cannulated adult male rats resulted in an almost complete obliteration of spontaneous GH pulses for 3 hours, with gradual recovery observed 3-6 hours after injection. Rats pretreated with octreotide i.v. and subsequently challenged with GRF exhibited reduced GH responsiveness to exogenous GRF at 1 hour post treatment. In contrast, preexposure to octreotide for 3 hours resulted in a 2-3 fold enhancement in GH responsiveness to GRF compared to controls pretreated with normal saline to normal saline-pretreated controls. In contrast, we report that preexposure to octreotide (n = 6) in a strain of dwarf rats, which shows a selective reduction in pituitary GH synthesis and storage, failed to significantly enhance GRF-induced GH release. / Our findings suggest that SRIF pretreatment promotes the accumulation of pituitary GH stores in a readily releasable pool so that subsequent GRF challenges can exert an accentuated effect on pituitary somatotrophs. (Abstract shortened by UMI.)