We have identified receptor protein tyrosine kinases (PTKs) that are expressed and/or activated during kidney development. mRNA from fetal rat kidneys in late gestation (embryonic day 21), was used to prepare cDNA template for polymerase chain reaction amplification with primers based on conserved regions of PTKs, and products were subcloned and sequenced. Among 346 clones, we identified epidermal growth factor receptor (EGFr), Tie 2, platelet derived growth factor receptor (PDGFr)-alpha, PDGFr-beta, Flk-1, Flt-4, fibroblast growth factor receptor (FGFr)-1, FGFr-3, FGFr-4, Met, and RYK//Nbtk-1. PTK expression was studied by immunoprecipitation and immunoblotting of kidney membrane proteins with specific antibodies. EGFr, PDGFr-alpha, FGFr-1, FGFr-3, Met, and in some cases Tie-2 protein expression was greater in fetal kidneys, as compared with kidneys from 12 week adult rats, (controls). Flk-1, PDGFr-beta, and FGFr-4 proteins were expressed comparably; Flt-4 was not detected. As a reflection of receptor PTK activity, we assessed endogenous tyrosine phosphorylation, and in vitro autophosphorylation. EGFr and PDGFr-alpha displayed activity in fetal, but not adult kidneys. FGFr-3 and Flk-1 were active in some fetal kidneys; the other PTKs were not active. Thus, in late gestational rat kidney, there are distinct patterns of receptor PTK expression and activity. EGFr, PDGFr-alpha, FGFr-3 and Flk-1 are among PTKs that are activated, and they may mediate perinatal development of renal epithelial, interstitial, or vascular structures.

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which in some models is partially mediated by eicosanoids. Recently, it has been demonstrated that the C5b-9 complex activates a high molecular mass cytosolic phospholipase A$ sb2$ (cPLA$ sb2)$ in cultured rat GEC, which leads to release of arachidonic acid (AA) and eicosanoids, and may contribute toward plasma membrane injury. In this study, we investigated if C5b-9 can also activate other PLA$ sb2$ isoforms. In GEC stably overexpressing sPLA$ sb2$ (Type II PLA$ sb2,$ 14 kD) (produced by transfection), the C5b-9-stimulated increase in free $ sp3$H-AA, ($ sim$2 fold above basal) was not significantly different from neo (control) GEC. In contrast, in GEC overexpressing cPLA$ sb2,$ C5b-9 stimulated a $ sim$9 -fold increase in free AA, as compared to unstimulated cells. This result suggests that C5b-9 activates cPLA$ sb2,$ but not the sPLA$ sb2$ enzyme, to release free AA. In further studies, we addressed the regulation of cPLA$ sb2$ in C5b-9-stimulated GEC. The present model of cPLA$ sb2$ activation involves the combined effects of: (1) an increase in cytosolic calcium concentration, which induces translocation of cPLA$ sb2$ from the cytosol to the plasma membrane, and may be mediated by the amino terminal "CaLB" (Ca$ sp{+2}$-dependent lipid binding) domain, and (2) phosphorylation by MAP kinase. To test the role of the CaLB domain, release of AA was monitored in GEC stably transfected with three constructs; cPLA$ sb2$ wild type (wt), cPLA$ sb2 Delta$CaLB (i.e., cPLA$ sb2$-wt with deleted CaLB domain), and neo (control) GEC. In GEC overexpressing cPLA$ sb2$-wt, C5b-9 stimulated a marked increase in free AA (as above). In contrast, in GEC overexpressing the cPLA$ sb2 Delta$CaLB enzyme, the C5b-9 induced increase in free $ sp3$H-AA was comparable to neo, despite expression of cPLA$ sb2 Delta$CaLB at levels similar to cPLA$ sb2$-wt. The results indicate tha

The multiple indicator dilution technique was employed to examine the in vivo kinetics of oxygen transport in the liver, known for its very permeable capillary bed, and the brain, known for its very tight capillary bed. $ rm sp{18}O sb2$-saturated $ sp{51}$Cr-labeled erythrocytes, labelled albumin, sucrose and water (the tracers for the study substance, vascular, interstitial, and cellular references respectively) were simultaneously injected into the target organs. Timed anaerobic samples were collected at the outflow and analyzed by mass spectrometry for relative $ rm sp{18}O sb2$ enrichment, and for $ gamma$ and $ beta$-radioactivity. Distributed-in-space models of capillary transport were used to analyze the data. / In the liver, oxygen transport and distribution were influenced by the temperature and hematocrit of the perfusate, and shown to be compatible with a flow-limited process. In the brain, flow varied significantly between animals, in concert with parallel changes in oxygen consumption. Analysis indicated that transfer of both $ rm sp{18}O sb2$ and $ sp3$H-water indicators from blood to brain is barrier limited, and it is concluded that low tissue oxygen concentration in the brain is due to limited endothelial permeability to oxygen.

From the onset of internal pipping (i.e. embryonal breathing of air cell gas) until hatching, the chorioallantoic membrane (CAM) and the lungs work simultaneously to serve the metabolic needs of the embryo. (1) The carbon dioxide and oxygen exchange rates (V̊O2 and V̊CO2) through the lungs and the CAM were separately, but simultaneously, measured during the last two days of incubation (day 20--21), while ventilation (V̊E) was calculated from the measurements of pressure oscillations in the air cell. When the embryo's total metabolic rate was increased, V̊E was linearly proportional to lung V̊O2 and V̊CO2 and not to the embryo's total metabolic rate. (2) Tracheal pressure and changes in lung volume were quantified through mechanical ventilation of the embryo. The curled up posture of the embryo, the eggshell and its membranes did not represent a significant mechanical constraint to V̊E. (3) V̊E, lung V̊O2 and V̊CO 2 were measured while the CAM compartment was exposed to either 10% O2, 100% O2 or 5% CO2. Total V̊O2 was also measured under these conditions. There is a clear V̊E-sensitivity to CO2 and a rather weak V̊E-sensitivity to changes in arterial oxygenation present at this stage of development.

The estrogen receptor (ER) is a ligand dependent transcriptional activator that belongs to the steroid/nuclear receptor superfamily. To probe the structure and function of the ER ligand binding domain (LBD), we developed a genetic screen in yeast Saccharomyces cerevisiae using a library of reverse ERs screened with a low affinity estrogen agonist, 2-methoxyestrone. Mutants isolated from this screen demonstrated altered ligand binding and/or transactivation properties. One of these mutants, L536P, showed high levels of constitutive activity in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. This suggests that substitution of a proline at position 536 in the wild type ER (HEGO) induces a reversible conformational change in the region of AF-2 that partially mimics activation of the receptor by hormone binding. / Activation of transcription by the ER requires the recruitment of different classes of coactivators. L536P interacted with coactivators in the absence of hormone and this constitutive interaction can be abolished by antiestrogens. We conclude that constitutive activity of L536P-HEGO is manifested to in part from constitutive coactivator binding. We also demonstrated that different classes of coactivators do not recognize the ER LBD in the same manner and can compete for binding to the ER LBD suggesting that different classes of coactivators recognize distinct but overlapping binding sites. Interestingly, coexpression of RIP140 blocked enhanced transactivation by HEGO observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. / Using a yeast two-hybrid system with the ER LBD as bait, we isolated a novel estrogen receptor cofactor (ERC) that interacts with the ER LBD in a hormone dependent manner. The primary structure of ERC consists of 4 ankyrin repeat domains, 2 LXXLL motifs and an ATP/GTP binding domain. ERC is highly expressed in ovary, testes, and spleen, with moderate levels in heart, brain, and placenta. ERC repressed ER transactivation in several cell lines in the presence of estradiol suggesting that ERC may function as a novel estrogen dependent repressor of the ER. Immunohistochemistry and confocal microscopy localized ERC to the cytoplasm with partial nuclear staining. Recently, synphilin-1, a novel protein that has been implicated in the pathogenesis of Parkinson's disease was isolated and is identical to ERC. A role for ERC in intracellular signaling through a membrane bound ER in the brain is currently being investigated.

This thesis examines the importance of the length-force relationship and the three-dimensional shape of the diaphragm with regard to its inspiratory function. As well as it reports on the manner in which fatigue and aminophylline affect the length-force properties of the diaphragm. First, I studied the effect of fatigue on diaphragm contractility as a function of sarcomere length using an in vitro rat diaphragm strips. Results indicated that fatigue resulted in disproportionately greater reduction of tetanic force at short sarcomere lengths. Second, I reconstructed the three-dimensional shape of the diaphragm to determine if in vitro results are physiologically relevant in humans. I estimated the changes in fibre length and shape that occurs with lung inflation from residual volume to total lung capacity in normal subjects. Results suggested that the inspiratory function of the human diaphragm can be entirely attributed to its length-force relationship rather than changes in shape under conditions of twitch phrenic nerve stimulations. Finally, I confirmed that fatigue caused a greater percent reduction of transdiaphragmatic pressure at high lung volume in response to single supramaximal shocks delivered bilaterally to the phrenic nerves at high lung volume; and demonstrated that aminophylline potentiated human diaphragm contractility more at high than at low lung volumes, both under fresh and fatigue conditions. I propose an explanation for the effect of fatigue and aminophylline on diaphragm contractility at different sarcomere lengths based on known actions of these factors and muscle shortening on excitation-contraction coupling mechanisms of skeletal muscles.

Taurine is an amino acid found in high concentration (20-30 mM) in mammalian heart. Treatment of rats with the transport antagonist guanidinoethyl sulfonate (GES) depletes cardiac taurine ($>$70%). Electrocardiograms were recorded weekly in restrained unanaesthetized rats. GES treatment caused a selective prolongation of the QT interval which was correlated with the degree of myocardial taurine depletion (r$ sp2$ = 0.92, p $<$ 0.001). Ventricular muscle action potential durations (APD$ sb{95}$) from taurine-depleted hearts were significantly prolonged compared to control (88 +/- 8 ms vs. 66 +/- 9 ms; p $<$ 0.001). Taurine supplements given to depleted rats reversed the taurine depletion and the QT prolongation; no effect was seen in controls. No action potential differences between control and "reversed" rats were seen. Superfusion of GES or taurine at concentrations of 0.2-10 mM had no effect on action potential characteristics of control or taurine-depleted hearts. When hamsters were GES-treated no cardiac biochemical or QT changes were seen.

The effect of interleukin-3 (IL-3), a candidate hemopoietic growth factor, on prenatal hemopoiesis is unclear. Microinjection of IL-3 directly into mouse fetuses (day 13) via the yolk sac, allowed us to evaluate the effects on morphogenetic events and more specifically on fetal liver populations using quantitative in vitro clonal assays for hemopoietic precursors. Control studies, required to distinguish stress effects of surgical laparotomy and microinjection, clearly revealed that the fetal liver is a sensitive organ responding with limited tissue disorganization, reduced cellularity and erythropoietic activity as identified 24 h after experimental intervention. Microinjection of 15 units of IL-3 promoted significant expansion of the depleted fetal liver hemopoietic cell populations and had stimulatory effects on connective tissue mast cells, absolute cell numbers including hemopoietic precursors (erythroid, granulocyte, macrophage, megakaryocyte) compared to controls. These studies suggest that fetal liver cells acquire a responsiveness to IL-3 early in development and that, IL-3 has a positive stimulatory effect on fetal liver cell populations, promoting the recovery of normal liver hemopoiesis.

To investigate the role of gonadal steroids in the hypothalamic-pituitary-adrenal (HPA) response to stress, we studied adrenocorticotrophin (ACTH) and corticosterone (B) responses to 20 min restraint stress in cycling female rats, and in ovariectomized (OVX) rats replaced with physiological levels of estradiol (E2) and progesterone (P). In cycling rats, we found significantly higher peak ACTH and B responses to stress during proestrous compared to the estrous and diestrous phases. No differences were found in either basal ACTH and B levels across the cycle phases. In a separate study, OVX rats were maintained on low, physiological levels of E2 and P with silastic implants for three days, and injected either with oil (O$ sp prime$), 10 ug of E2 (E$ sp prime$) 24 h before stress testing, or with E2 and 500 ug P 24 h and 4 h, respectively, prior to stress (EP$ sp prime$). These treatments mimicked endogenous profiles of E2 and P occurring during diestrous, proestrous, and late proestrous-early estrous phases, respectively. In response to stress, ACTH levels were higher in the E$ sp prime$ group compared to the EP$ sp prime$ and O$ sp prime$ groups. Within the 20 min stress period, ACTH levels and plasma B levels in the E$ sp prime$ group were significantly higher after the onset of stress, compared to the EP$ sp prime$ and O$ sp prime$ groups. $ beta$-endorphin-like immuno-reactive responses to restraint stress were also significantly higher in the E$ sp prime$ group. There was no effect of E2 on ACTH clearance. These results indicate that the HPA axis in the female rat is most sensitive to stress during proestrous.

The outwardly rectifying chloride channel is found in most vertebrate cells however its physiological role is uncertain. Patch clamp, short-circuit current, and electronic cell sizing techniques were used to investigate the role of the outward rectifier in transepithelial chloride secretion and cell volume regulation, the two main functions that have been proposed for this channel in epithelia. Patch clamp studies of the human cell lines PANC-1 and T$ sb{84}$ showed that the chloride channel blockers IAA-94 and NPPB decrease the open probability of the outward rectifier, with half-maximal inhibition at 15 $ mu$M and 23 $ mu$M, respectively. At these concentrations the blockers did not affect cAMP-induced short-circuit current. They did inhibit the regulatory volume decrease (RVD) which occurs after hypotonic cell swelling, but only at much higher concentrations. Moreover, the commonly-used inhibitor DIDS, which blocks the outward rectifier in the 10-20 $ mu$M range, had no effect on the RVD when tested at 100 $ mu$M. The results indicate that the outwardly rectifying Cl channel does not mediate a significant fraction of transepithelial Cl secretion across T$ sb{84}$ cells. Although the data do not exclude a role for the outward rectifier in cell volume regulation, the selectivity and pharmacological properties of the swelling-induced anion conductance in T$ sb{84}$ cells is more similar to the ClC-2 channel than to the outward rectifier.