Quantal nicotinic transmission, from about half of the preganglionic inputs to the perfused rat superior cervical ganglion, was more potent and better sustained when activity was in short, high frequency bursts, rather than in continuous trains, regardless of train frequency. The advantage of short bursts was in producing post-tetanic potentiation, while limiting both depression of synaptic potential amplitude and increases in failure rate that were evident during long trains. Presynaptic bursting was also more effective in suppressing, via muscarinic and non-cholinergic mechanisms, the postganglionic cell afterhyperpolarization. This suppression allowed cells to fire at higher frequencies during bursts of depolarizing current pulses used to simulate nicotinic epsps. Burst patterning of activity was concluded to enhance ganglionic transmission via both pre and postsynaptic mechanisms. Presynaptic conduction block, possibly associated with accumulation of extracellular K$ sp+$, appeared to be partly responsible for the depression of quantal transmitter release during long trains.

Human subjects were asked to "fixate" an earth-stationary target, in complete darkness, either during or after a brief passive head rotation. During head rotation saccadic eye movements synergistically improved compensatory slow-phase vestibulo-ocular reflex (VOR) stabilization. Visual-vestibular conflict that adaptively attenuated VOR gain caused the combined saccadic and slow-phase response to undercompensate for head rotation. Saccades volitionally generated after the cessation of head rotation (Vestibular Memory-Contingent Saccades, VMCS), successfully acquired the earth-fixed target, indicating that perceived vestibular information has access to the saccade generating mechanism. The above adaptive stimulus contracted the amplitude of VMCSs, suggesting a commensurate modification of the percept of self-movement relative to space. VMCSs displayed a "range effect", slightly biasing all results towards the mid-range amplitude. A schema is proposed implicating the posterior parietal cortex in the perception of vestibular input, the control of gaze and their adaptive modification.

The aim of the study was to investigate the mechanisms of bronchoconstriction induced by dry gas hyperpnea challenge in the rat. The interaction of airway hyperresponsiveness to contractile agonists and the atopic constitution of the animals with responsiveness to hyperpnea challenge was also evaluated. A hyperresponsive strain, Fisher 344, two control normoresponsive strains, Lewis and ACI, and an atopic but normoresponsive rat, Brown Norway (BN) to dry gas hyperpnea challenge were studied. The effects of the NK1 (CP-99994), the NK2 (SR-48968) receptor antagonists, and the selective LTD4 receptor antagonist pranlukast on responses to hyperpnea challenge were examined in BN rats. The animals were anesthetized with xylazine and pentobarbital sodium intraperitoneally and mechanically ventilated with a tidal volume of 8 ml/kg and a frequency of 150 breaths/min for hyperpnea challenge. Pulmonary responses to challenge were measured in spontaneously breathing animals. Dry gas hyperpnea challenge was performed with a dry mixture of 5% CO2-95% O2 for 5 min whereas wet gas was humidified prior to administration. Bronchovascular leakage was assessed by quantitating Evans blue in bronchoalveolar lavage (BAL) fluid. (Abstract shortened by UMI.)

Platelet aggregation is mediated primarily by the binding of fibrinogen to its membrane receptor, GPIIb-IIIa, but such an interaction may not be sufficient to support aggregation. This question could potentially be resolved by reconstituting GPIIb-IIIa into a model membrane system. / A protocol was developed for the generation of liposomes containing purified GPIIb-IIIa. Flow cytometric techniques confirmed that the receptor was present in the lipid bilayer and were used to evaluate the characteristics of fibrinogen binding to the liposomes, which like fibrinogen-platelet interactions exhibited specificity, saturability, time-dependence, and calcium-dependence. / No fibrinogen-specific aggregation of GPIIb-IIIa-liposomes with stir or shear was observed, as determined by flow cytometric cell counting and microscopic examination of particles. In contrast, platelets rapidly formed large aggregates in the presence of fibrinogen. It thus appears that elements other than fibrinogen and GPIIb-IIIa play an important role in platelet aggregation.

$ beta$-Hexosaminidase (Hex) is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G$ sb{ rm M2}$ gangliosidoses which includes Tay-Sachs and Sandhoff diseases. There are two major isoenzymes of Hex, Hex A ($ alpha beta$) and Hex B ($ beta beta$). / Two cell types, apical and narrow cells, in the initial segment of the rat epididymis immunolocalized Hex. Principal cells of the epididymis were intensely reactive for Hex only in the intermediate zone, caput and proximal corpus regions. Clear cells were reactive in the regions of the epididymis where they were found. Electron microscopic immunocytochemistry confirmed the presence of Hex in lysosomes of all reactive cells in the epididymis and of those in Sertoli cells and interstitial macrophages of the testis. / The regulation of Hex in the male reproductive tract was examined in the epididymides of rats at various ages after birth and of adult rats which were orchidectomized and treated with or without testosterone. / To study the impact of absence of Hex in the male reproductive tract, mouse models of human Tay-Sachs (Hexa $-$/$-$) and Sandhoff (Hexb $-$/$-$) diseases, created through gene targeted disruption of the $Hexa ( alpha$-subunit) and $Hexb ( beta$-subunit) genes respectively, were examined. (Abstract shortened by UMI.)

Neural, humoral and mechanical factors affecting cardiac function and the peripheral vasculature were examined to assess the relative importance of peripheral vs cardiac factors in the control of cardiac output during exercise. First, I examined neural vs local regulation of humoral vasoconstrictors endothelin-1 (ET) and NPY, in anesthetized dogs, and showed that plasma ET levels, in contrast to NPY, increase in response to systemic hypotension but not carotid sinus baroreceptor activation. This did not occur with intact vagi suggesting an interaction of neural and humoral factors. In the next two studies I separated peripheral vascular and cardiac neural influences on cardiac output (CO) and right atrial pressure (Pra) responses during graded exercise by comparing cardiac denervated heart transplant patients (HT) with normally innervated subjects. The increase in Pra was higher in HT than normals but stabilized as the CO increased at peak effort. Stimulation of the heart with dobutamine in 2 patients did not increase exercise capacity, suggesting that peripheral not cardiac factors limit exercise in HT patients. The rise in central venous pressure at exercise onset was similar in both groups which demonstrates the importance of the mechanical effect of muscle contraction vs reflex changes in mobilizing blood from the peripheral vasculature at exercise onset. In the fourth study I assessed whether large changes in human body mass, induced by isolated gastric bypass surgery, would affect the heart rate (HR)/oxygen consumption (VO2) relationship during exercise. Peak absolute VO2 was significantly lower in the previously obese group vs obese and control groups despite similar normalized 24-hour energy expenditure. HR was higher in the previously obese at a given submaximal VO2 due to the higher relative VO2, suggesting a significant loss of muscle mass and supporting the idea that HR is a function of the relative VO2. In the fifth study I assessed the influence of

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.