Horizontal step-ramp stimuli were used to examine gaze, eye, and head movement dynamics during head-unrestrained pursuit with and without imposed retinal velocity errors (RVE; i.e. open- and closed-loop, respectively) in two rhesus monkeys. In the closed-loop experiment , pursuit was elicited by step-ramp stimuli with a constant velocity of 20--80 deg/s. Each monkey used a combination of eye and head motion to initially fixate and then pursue the target. Additionally, we found that initial eye and head acceleration increased as a function of target velocity. In the open-loop experiment, step-ramp stimuli (40 deg/s) were presented and ~125 ms after pursuit onset, a constant RVE was imposed for a duration of 300 ms. In each monkey, when RVE = 0 deg/s, gaze, eye, and head velocity trajectories were maintained at their current or at a damped velocity. Moreover, the head as well as the eyes mediated the observed increase and decrease in gaze velocity when RVE was +10 and -10 deg/s, respectively. Based on our findings we conclude that the pursuit system uses visual and non-visual signals to drive coordinated eye-head pursuit.

Na+/H+ exchanger (NHE) is a major contributor in controlling intracellular pH. The activity of this protein is allosterically modified by intracellular H+. Histidine residues of the NHE that face the cytoplasm may be involved in determining the intracellular pH set point, with their state of protonation influencing the rate of Na +/H+ exchange. To test this hypothesis, histidine residues in the ubiquitously expressed NHE isoform (NHE1) that are relatively conserved amongst members of the NHE gene family were substituted by site-directed mutagenesis and the mutants were stably transfected into mammalian cells that are deficient in endogenous Na+/H + exchange activity. The pHi sensitivity of each mutant was evaluated by measuring the rate of 22Na + influx as a function of the intracellular H+ concentration. Mutation of the histidines located at the putative cytoplasmic face of the N-terminal transmembraneous domain of NHE1 did not show any significant effect on the pHi sensitivity of the protein. By contrast, substitution of histidines located in the C-terminal cytoplasmic tail activated the exchanger by increasing its sensitivity to H+. These mutants were no longer activated in response to protein kinase C, when compared to wild type. Taken together, these data support the hypothesis that some of the relatively conserved histidine residues in the C-terminal cytoplasmic tail of NHE1 may be involved in determining the pHi "threshold" or "set point" of the transporter.

Acylation stimulating protein (ASP or C3a desArg) is a complement derived product produced by adipocytes and is able to alter their metabolism, stimulating both triglyceride synthesis and glucose transport. This stimulatory activity has been shown to be due to ASP as both plasma derived protein and recombinant protein stimulate glucose transport and triglyceride synthesis. Furthermore, we demonstrated that ASP is functionally distinct from C3a. A two site model for ligand interaction with the receptor is proposed with the carboxy-terminal involved in receptor binding and the disulphide core in stimulating triglyceride synthesis. Functionality of ASP in vivo supported the hypothesis that ASP is involved in plasma triglyceride and glucose clearance postprandially after a fat load. The following data were obtained: (1) Administration of ASP and ASP functional knockouts displayed increased and delayed triglyceride clearance respectively. (2) Administered ASP decreased plasma glucose levels, which was independent from its effects on plasma triglycerides. (3) In ASP functional knockout mice gender differences of greater postprandial lipemia in males and more pronounced reductions of adipose tissue in females were observed. In conclusion the function of ASP was determined as well as structural regions involved. Furthermore, the in vivo physiology of ASP has been determined, with an effect on postprandial metabolism, regulation of adiposity and gender dependent penetrance of the ASP functional knockout phenotype.

This dissertation examines the ovarian development and function in genetically modified mice that lack FSH receptor (FSH-R) signaling. We propose that a complete or partial loss of FSH-R causes ovarian insufficiency resulting in estrogen deficiency and premature aging in female mice. / Targeted disruption of FSH-R caused a gene dose related endocrine and gametogenic abnormality in female mice. The resulting FOllitropin R&barbelow;eceptor K&barbelow;nockO&barbelow;ut (FORKO) mutants were acyclic and infertile due to ovulatory defects, even with very high levels of FSH. Lack of FSH-R signaling in females caused a severe ovarian underdevelopment, producing estrogen deficiency. As a consequence, the null mutants developed obesity and skeletal abnormalities. The expression of nuclear estrogen receptor(s) alpha and beta genes and the corresponding proteins in the ovary and uterus of FORKO mice were maintained intact, as estrogen administration induced uterine growth and decreased accumulation of the adipose tissue. By 12 months of age, 92% of FORKO animals developed ovarian neoplasms of sex cord-stromal type similar to pathology observed in women. Our results showed, for the first time, that the loss of the FSH-R signaling mechanisms predisposes the ovary to molecular and structural changes causing tumor formation. / In contrast to acyclic and infertile FORKO (-/-) females, a phenotype of FORKO mice with a partial (+/-) disruption of the receptor gene exhibits irregular cyclicity and reduced fertility, undergoing early reproductive senescence. Our findings also demonstrate that the loss of a single allele of the FSH receptor gene causes a premature exhaustion of gonadal reserves accompanied by age-related changes in the hypothalamic-pituitary axis. / The study concludes that the FSH receptor signaling offers a protective mechanism, which gradually weakens upon reproductive senescence (menopause in women); therefore this knockout constitutes a unique and promising animal model for studying the physiology and molecular mechanisms of gonadal receptors and hormones.

Recently, various proteoglycans (PGs) have been identified in the lung. The first objective of this thesis was to test the hypothesis that matrix glycosaminoglycans contribute to lung tissue viscoelasticity. Lung parenchymal strips were exposed to specific glycosaminoglycans-degradating enzymes to determine whether the mechanical properties of the tissue were affected. The degradation of heparan sulphate and chondroitin/dermatan sulphate glycosaminoglycans caused significant increases in energy dissipation and dynamic resistance relative to control strips. Hyaluronidase treatment did not alter any of the dynamic or static measures. Since PGs were found to be part of the stress bearing structure, the second part of the thesis aimed at examining whether subjecting the lung to excessive mechanical force can cause alteration in PG composition so as to adapt to the altered stress bearing requirement. To address this hypothesis, the effect of different ventilation regimes on lung tissue mechanics and PGs was examined in an in vivo rat model. After 2 h of mechanical ventilation, lung tissue elastance and resistance were significantly increased in rats ventilated with tidal volume of 30 ml/kg at 0 positive end-expiratory pressure (Vt30PEEP0) as compared to controls (Vt8PEEP1.5). Versican, a basement membrane heparan sulphate PG and biglycan, were all increased in rat lungs ventilated with Vt30PEEP0 as compared to control. These data demonstrated that alterations in lung tissue mechanics with excessive mechanical ventilation are accompanied by changes in all classes of ECM PGs. However, whether the alteration seen in PG composition resulted from excessive mechanical ventilation directly was unclear. In addition the cellular source of these PGs was not determined. Therefore, the aim of the third part of the thesis was to investigate and characterize the effect of mechanical strain on lung fibroblast PG production in vitro. We found cell layer associated versican protein in

A recursive least-squares algorithm was developed to estimate respiratory mechanical parameters with high temporal resolution. This algorithm was used to investigate the time course of bronchoconstriction induced by intravenous histamine injection in the dog. The onset of the response of lung tissue resistance and elastance demonstrated a different time course than airway resistance. This was interpreted in terms of the sequential delivery of the drug first through the pulmonary and then the bronchial circulations. The time course of respiratory mechanical parameters among various alveolar capsules revealed two patterns of inhomogeneity development. The first one was random whereas the second one was progressive with dose. A mathematical derivation elucidated the negative tissue resistance frequently obtained at high levels of constriction. The time courses of respiratory resistance and elastance during bronchoconstriction were transient and scaled with dose. They were reproducible for repeated doses of histamine after indomethacin pre-treatment and were intrinsically modulated by the adrenergic sympathetic system and through the $ rm H sb2$ histamine receptors.

The mechanisms underlying the net fluid/electrolyte gain during pregnancy are not fully understood. Utilizing virgin, pregnant (7, 16 and 21 days gestation), estradiol-17beta- or progesterone-treated female rats, we have examined the role of atrial natriuretic peptide (ANP) and of its receptors in the adaptation of body fluid homeostasis during pregnancy. Pregnancy and progesterone attenuate the ANP-mediated inhibition on aldosterone production in adrenal zona, glomerulosa (ZG) cells. The ribonuclease protection assay and Western analysis revealed that ZG natriuretic peptide guanylyl cyclase-linked (GC) receptors are mainly GC-A and that they are downregulated by pregnancy. In addition, pregnancy downregulates GC-A and GC-B receptors in the uterus; however, it does not modulate GC-linked receptors in the lung. It is concluded that the downregulation of ZG GC-A receptors could lead to a decrease in the aldosterone-suppressant effects of ANP and that the decrease in uterine GC-A and GC-B receptors could lead to a decrease in the tocolytic effects of ANP during pregnancy.

The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent eosinophil chemoattractant in vitro. To determine whether it is active in vivo, 5-oxo-ETE was administered intratracheally to BN rats and pulmonary eosinophils were immunostained with an antibody to major basic protein. 5-Oxo-ETE induced a dramatic increase in eosinophils, which reached maximal levels (5 times control) between 15 and 24 h following administration, and thereafter declined. LTB4 had a similar effect to 5-oxo-ETE but appeared to be somewhat less effective. In contrast, LTD4 and LTE4 were inactive. 5-Oxo-ETE-induced eosinophilia was inhibited by 75% following pretreatment of the animals with antibodies to integrins VLA-4 or LFA-1, but was not significantly inhibited by an antibody to Mac-1, nor after pretreatment with receptor antagonists to LTB4 (LY255283) or PAF (WEB 2170). These observations raise the possibility that 5-oxo-ETE may be an important physiological mediator in inflammatory diseases characterized with eosinophil recruitment, such as asthma.

Autoregulation of renal blood flow is mediated by two mechanisms. The myogenic response operates at $ approx$0.1-0.2 Hz and tubuloglomerular feedback (TGF) operates at $ approx$0.03-0.05 Hz. Atrial natriuretic factor (ANF) dilates pre-glomerular resistance vessels, in which autoregulation occurs, and has been reported to inhibit TGF. We tested the potential actions of ANF on TGF-mediated autoregulation using Sprague-Dawley rats anesthetized with isoflurane. Renal perfusion pressure (RPP) was manipulated by a servo-controlled clamp placed on the aorta between the renal arteries. Time constants for constriction and dilatation were consistent with operation of TGF. ANF did not affect either the magnitude or the time constants of the response. / To test the contribution of TGF resetting to the wide dynamic range of steady state autoregulation experiments, the autoregulatory range was first defined by conventional stepwise reduction of RPP. As ANG II is necessary for TGF resetting, the experiment was repeated in the presence of Losartan, a competitive ANG Il-AT$ sb1$ receptor antagonist. The results do not support the hypothesis that TGF resetting contributes to the wide dynamic range of steady state autoregulation. In fact, the lower limit of autoregulation was extended to a lower RPP. In the Losartan experiment, this shift was not apparent, suggesting that it was ANG II-dependent. (Abstract shortened by UMI.)

Familial hypophosphatemic rickets and its murine model, Hyp, are characterized by a specific defect in renal inorganic phosphate reabsorption. In Hyp mice the defect has been localized to the proximal tubule brush border membrane and a constitutively enhanced protein kinase C has been demonstrated. Since the latter is activated through the protein G pathway, we have studied phosphatidyl inositol 4,5-bisphosphate metabolism (PIP$ sb2$) in normal (+/Y) and Hyp primary cultures of proximal tubules. At confluence, ($ sp3$H) -myo-inositol labelled cultures were stimulated with calcitriol or parathyroid hormone (PTH) for one minute. Cells were extracted and the aqueous phase was chromatographed on ion exchange resin. The inositol phosphate fractions were counted for radioactivity. For calcitriol, maximal PIP$ sb2$ metabolism occurred at 10$ sp{-11}$ M and 10$ sp{-12}$ M for +/Y and Hyp, respectively. For both cultures, PTH produced a maximum at 10$ sp{-8}$ M. Pertussis toxin alone stimulated +/Y but not Hyp cultures and produced no additive stimulation with calcitriol or PTH in either culture.