Activation of the cystic fibrosis transmembrane regulator (CFTR) chloride channel by protein kinases has been studied intensively. The mechanism of deactivation by protein phosphatases has received less attention, although it is equally important for physiological regulation of the channel. Characterization and identification of the phosphatases that regulate CFTR has become a priority in CF research because they are potential targets for pharmacotherapies. / The first goal of this project was to characterize the effects of purified phosphatases on single CFTR channels. I found that PP2A, PP2C and alkaline phosphatase all reduced channel activity in excised patches. PP1 and PP2B did not deactivate CFTR, despite having comparable phosphatase activity when assayed biochemically using a standard substrate. Deactivation by exogenous PP2C closely resembled the spontaneous rundown induced by endogenous phosphatase in CHO, BHK, and T84 cells. / Genistein and bromotetramisole (Br-t) have been proposed to activate CFTR by inhibiting phosphatases. The second goal of this project was to assess the role of phosphatases in CFTR activation by these drugs. Genistein did not affect phosphatase activities. By contrast Br-t inhibited all four types phosphatases (PP1, PP2A, PP2B and PP2C). Thus Br-t may activate the channel by inhibiting its dephosphorylation whereas genistein probably acts directly on CFTR. / These studies provide functional evidence that PP2C is the predominant phosphatase regulating CFTR, and clarify the role of phosphatases in CFTR activation by genistein and bromotetramisole.

Plasma membrane Na+/H+ exchangers (NHEs) have been shown to be inhibited by amiloride and its derivatives. Studies have demonstrated that these antagonists act on the transporter by interacting with amino acid residues within certain transmembrane domains (TM4 and 9). Experiments in our laboratory have identified three novel sites involved in inhibitor interactions (PP157--8, E350, and G356). The general focus of this thesis was to study the structure-function relationship of NHE1. One of the aims of this thesis was to assess whether mutations at these novel sites also affected transport kinetics (i.e., Na+ and H+ affinity). The results showed that neither Na+ nor H+ affinities were significantly altered with any of the mutations analyzed. The next objective was to investigate the nature of the interaction between the exchanger and pharmacological agents, by measuring transport activity in the presence of substituted guanidinium antagonists. The results suggest an interaction between L167 and the chlorine moiety at position 3 of the benzoyl group of a novel benzoyl guanidinium compound, while G356 appears to interact with the chlorine moiety at position 4 (rather than position 3) of the benzene ring. / The last objective was to define the membrane topology of NHE1. The methodology involves reintroducing cysteine residues into a cysteine-less mutant NHE1 and assessing their accessibility with thiol-reactive agents. Unfortunately, these results were inconclusive and further optimization of the assay conditions is required.

The anatomy of the diaphragmatic circulation was found to be composed of an internal arterial circle formed by the head to head anastomosis of the phrenic arteries and internal mammary arteries. Branches originating from the internal arterial circle anastomosed head to head with branches of the intercostal arteries (8$ sp{ rm th}$ to 13$ sp{ rm th}$ intercostal space) to form costophrenic arcades all along the muscular fibers of the crural and costal diaphragm. These anastomosis were found to be physiologically functional. Diaphragmatic circulation produced only by the intercostal arteries was able to sustain costal and crural contractility at the fatigue threshold (TTdi of 0.20). However, internal mammary artery perfusion was only able to maintain costal contractility. Left and right hemidiaphragmatic arterial communications (shunting) were inexistant during electrophrenic unilateral and bilateral stimulation. Diaphragmatic venous outflow was produced mostly by the intercostal veins (60% of total diaphragmatic venous outflow) which drained into the azygos trunk. The phrenic veins contributed 25% and the internal mammary veins contributed 15% of total diaphragmatic venous outflow. Diaphragmatic circulation was found to be proportional to the Pdi and was related to the duty cycle by a parabolic function with the highest flow rates being observed at a duty cycle of 0.50, regardless of the Pdi being generated.

We compared both the rate and extent of platelet aggregation with the extent of fibrinogen receptor expression and specific Fg binding to activated platelets, as a function of ADP concentration. Human citrated platelet-rich plasma (diluted ten-fold) was maximally pre-activated by incubating with adenosine diphosphate (ADP) at room temperature and then stirred. Platelet aggregation was determined from the decrease in the total number of particles. The number of fibrinogen receptors or bound Fg was measured from mean fluorescence values obtained with FITC-labelled IgM monoclonal antibody PAC1, and the IgG monoclonal antibody, 9F9, respectively, using flow cytometry. The expression of fibrinogen receptors occurs within seconds of activation. The fraction of platelets with fluorescence values above one critical threshold value increased with increasing activator concentration and correlated linearly with the fraction of platelets recruited into aggregates for ADP (r $>$ 0.9). Aggregation was not rate-limited by fibrinogen receptor expression nor by Fg binding. It appears that each platelet expresses $>$90% of its maximal Fg receptors at a critical ADP concentration i.e, occupancy of ADP receptors. This, in turn, leads to rapid Fg occupancy and platelet aggregation.

The nature of shape change and aggregation of human and rabbit platelets was assessed by light transmission studies of stirred platelet suspensions and phase contrast microscopy. Platelet shape change and aggregation were differently affected by experimental conditions: pCO(,2), pH and anticoagulants, according to choice of aggregating agents. Optimal physiological conditions were established for functional studies. A new class of potent antithrombotic agents, BL-3459 and BL-4162A, were identified as selective low Km cyclic 3':5'-adenosine monophosphate (cAMP) phosphodiesterase inhibitors. These BL-compounds could block platelet shape change and aggregation for all aggregating agents tested. Dose-response studies with adenosine 5'-diphosphate demonstrated that these two consecutive events are separately and oppositely controlled by aggregating agent and inhibitor concentration. A model for the role of cAMP-calcium in regulating these changes is presented. Finally, we studied platelets from a distinct hereditary bleeding disorder, which we designated as the "Montreal Platelet Syndrome". These platelets showed abnormal shape change, low to absent thrombin-induced aggregation; and spontaneous aggregation leading to microaggregates of disc-shaped platelets.

This investigation was primarily concerned with the interaction of the ('125)I-labeled human gonadotropins with testicular tissue from the human and from nonhuman primate species. The binding of the ('125)I-hFSH and ('125)I-hCG (or ('125)I-hLH) to a particulate fraction (P1) of the primate testis was highly specific. Gonadotropin binding was competitively displaced by the synthetic estrogens and an inhibitory factor present in testicular extracts (140,000 x g supernatant). The biochemical properties of the gonadotropin receptor (testicular) interaction of the different primate species were similar in most respects. All tissues had a greater FSH than LH binding capacity with an apparent dissociation constant in the range of 10('-10)-10('-11)M. / An FSH responsive adenylyl cyclase was characterized in human testicular membranes. In the presence of a chemically deglycosylated derivative of FSH it was possible to uncouple the FSH responsive adenylate cyclase system. The synthetic estrogens were also effective inhibitors of the human testicular adenylyl cyclase.

Maturation and aging-associated changes in membrane transport of sheep reticulocytes of the high-K('+) and low-K('+) genotypes were studied. The results indicate that during short term (less than 10 days) in vitro maturation, there is a progressive decrease in ouabain-sensitive Na('+),-K('+)-pump activity (('86)Rb('+) uptake in low-K('+) cells) and Na('+)-ATPase activity (high-K('+) cells). However, during long term (several weeks) in vivo maturation in which HbC was used as a marker of newly formed reticulocytes, kinetic changes, as well as a decline in total activity, were observed. / The effect of metabolic depletion on the maturation-associated loss of two membrane transport functions was also studied using in vitro incubation. Both Na('+)-dependent glycine transport and the Na('+),K('+)-pump, estimated from measurements of the number of ('3)H-ouabain binding sites per cell, were decreased during maturation. ATP also enhanced the decrease in both activities when a 'reconstituted' vesicle system comprised of inside-out vesicles plus cell lysates was incubated at 37(DEGREES)C. Associated with this ATP-dependent loss of activity was an increase in the amount of concomitantly measured ninhydrin-positive material. It is concluded that the loss of certain functions during reticulocyte maturation is retarded by metabolic depletion. / Membrane vesicles of distinct sidedness were prepared from sheep reticulocytes. Using these vesicles the Na('+)-dependent glycine transport system was found to be symmetrical with respect to: (a) the Na('+) dependency of glycine transport, (b) the ability to accumulate glycine against a concentration gradient, (c) the Na('+): glycine stoichiometry, i.e. two Na('+) ions are transported per molecule of glycine, and (d) the apparent Michaelis-Menten constants for Na('+) and glycine.

Properties of sympathetic preganglionic neurones were examined in cat. Following horseradish peroxidase injection into the stellate ganglion, labelled neurones were in spinal segments C(,6)-T(,8) in regions of the intermediolateral nucleus, intermediate grey, central canal and ventral horn. Neurones located in the intermediolateral nucleus had a wide range of physiological properties, and, with the exception of basal activity, the properties studied failed to indicate subgroups. These neurones were also studied according to their sensitivities to substances implicated in synaptic transmission. (gamma)-Aminobutyric acid and glycine had inhibitory effects which were blocked by bicuculline and strychnine, respectively. Glutamate, aspartate and D-L- homocysteic acid exerted excitatory effects. Substance P, thyrotropin-releasing hormone, oxytocin and vasopressin also had excitatory effects; when the physiological properties of responsive vs unresponsive neurones were compared, those excited by oxytocin had shorter latencies of antidromic activation than unresponsive neurones. Thus, subgroups of neurones may be identified on the basis of their responsiveness to some substances and there may be a further breakdown when responsiveness is compared with physiological properties.

The role of capacitance vessels and blood flow distribution (BFD) in the control of venous return and cardiac output were examined. First, saline infusion during exercise in humans maintained plasma volume, reduced heart rate and core temperature, but did not change endurance time. Second, the vascular mechanics of the skin were studied in dogs. The skin has a large venous compliance (C$ sb{ rm v})$ and a long time constant of venous drainage $( tau sb{ rm v}),$ and may act as a blood reservoir during heat stress. A rise in core or skin temperature increases C$ sb{ rm v}$ and decreases venous resistance (R$ sb{ rm v}),$ but does not change $ tau sb{ rm v}.$ The last three studies were performed in dogs on circulatory bypass. The third study examined the mechanisms of increase in venous return during heat stress. Splanchnic (SPL) unstressed volume (V$ sb{ rm u})$ decreases with no change in R$ sb{ rm v},$ C$ sb{ rm v},$ $ tau sb{ rm v}$ or BFD during heat stress. This decrease in V$ sb{ rm u}$ is abolished by ganglionic blockade but not by $ alpha$ or $ beta$-receptor blockade. The fourth study looked at the effects of the baroreflex on capacitance vessels and BFD. A decreases in carotid sinus pressure from 200 to 50 mm Hg increases SPL blood flow and C$ sb{ rm v},$ and decreases SPL R$ sb{ rm v},$ $ tau sb{ rm v}$ and V$ sb{ rm u}.$ The decrease in V$ sb{ rm u}$ is abolished by ganglionic blockade, but is only partially reversed by $ alpha$-receptor blockade. The last study examined the effects of neuropeptide Y (NPY) on capacitance vessels and BFD. NPY decreases SPL V$ sb{ rm u}$ with no change in R$ sb{ rm v},$ C$ sb{ rm v},$ $ tau sb{ rm v}$ or BFD. Thus, NPY may play a role in the control of venous return.

In response to i.v. ANF at 175 ng/kg/min, normal dogs increase sodium excretion ($ Delta$UNaV = 150 uEq/min) independent of changes in GFR and RPF. In contrast, when ANF was infused into chronic caval dogs (TIVC) or cirrhotic dogs (Cir) retaining sodium in the presence of ascites, they divided 50:50 into those who had a marked natriuretic response (responders, R) and those who had no natriuretic response (non-responders, NR). Of 46 TIVC dogs, 22 R had UnaV of 185 + 35 uEq/min and 24 NR had $ Delta$UNaV = 2 + 1 uEq/min. In 19 Cir dogs, 9 R had $ Delta$UNaV = 60 + 10 uEq/min and 10 NR had $ Delta$UNaV = 1.3 +.6 uEq/min. R and NR could not be differentiated in terms of atrial content of ANF, plasma iANF, ANF T1/2, plasma levels of renin and aldosterone, systemic hemodynamics, plasma volume, or papillary plasma flow. All dogs generated plasma and urinary cGMP equally. Renal denervation or vasodilitation did not increase sodium excretion in response to ANF in RN. When NR dogs returned to sodium balance in the presence of ascites, the natriuretic response was restored ($ Delta$UNaV = 90-340 uEq/min) and was not different from R dogs in this phase. Cir dogs studied sequentially in the pre-ascitic phase responded normally to ANF infusion when they were in sodium balance but split 50:50 into R and NR at week 4 during a period of sodium retention, plasma volume expansion, elevated plasma iANF and normal renin and aldosterone. We conclude that the blunting of UNaV in response to ANF is a characteristic of the sodium-retaining kidney, is reversible when sodium balance is restored and occurs at a tubular level, most likely in the medulla.