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.

No description available.

Reactive airways dysfunction syndrome is a form of irritant-induced asthma that has been documented after acute exposure to chlorine (Cl2) gas. Animal models of Cl2 exposure indicate that the airway epithelium is a target. gammadelta T-Cells are present in the mucosal surface of the airways and may control the growth and differentiation of the airway epithelial cells. However, the role of these cells in the airway response to Cl 2 exposure has not been elucidated. / Aim. To study the role of gammadelta T-cells in the response to Cl2 exposure with respect to inflammation and lung function. / Methods. C57B1/6J (wild type) and TCR-delta -/- mice exposed to Cl2 (400ppm) for 5 minutes were mechanically ventilated for measurement of responses to i.v. methacholine (MCh) at 1, 3, and 5 days after exposure. Bronchoalveolar lavage was performed to determine epithelial and leukocyte counts, and protein content. Tissues were harvested for PCNA immunoreactivity to evaluate the rate of repair of the epithelium. / Results. Wild type mice developed a greater degree of airway hyperresponsiveness to MCh at 1 day post exposure to Cl2 compared with TCR-delta-/- mice. Epithelial cell counts in BAL after Cl2 exposure were greater in TCR-delta -/- mice, and the pattern of inflammation differed in wild type and TCR-delta-/- mice; macrophages showed a later peak and granulocyte numbers were lower in TCR-delta-/- than in wild type mice. Both groups of mice had increased levels of total protein content in BAL after Cl2 exposure that resolved after 3 and 5 days, respectively. TCR-delta -/- mice have a lower rate of epithelial regeneration as shown by PCNA immunoreactivity. / Conclusion. The severity of airway injury after Cl 2 appears to be greater in TCR-delta-/- mice but the lack of TCR-delta seems to abrogate the changes in airway responsiveness to i.v. methacholine.

The N-terminal 236 residues of Ssa4p, a hsp70 in budding yeast, are sufficient to target GFP to nuclei in ethanol-treated cells; this transport is mediated by the karyopherin-beta Nmd5p. Ssa4p(1-236)-GFP nuclear accumulation upon ethanol exposure depends on the cell surface sensors Wsc1p and Mid2p as well as protein kinase C, an activator of the cell integrity MAPK cascade. I have analyzed the distribution of Ssa4p(1-236)-GFP and Nmd5p-His6-HA in deletion mutants of the cell integrity MAPK cascade and demonstrate that this pathway controls ethanol-induced nuclear accumulation of Ssa4p(1-236)-GFP. Protein phosphorylation regulates protein trafficking, and I have studied this modification for Ssa4p(1-236)-GFP, Nmd5p and the nucleoporin Nsp1p. My results suggest that the phosphorylation status of Nmd5p may change when cells are treated with ethanol, and threonine was identified as the putative target residue(s) for modification. Furthermore, Snf1p kinase is required for stress-induced nuclear accumulation of Ssa4p(1-236)-GFP; my data are in line with the idea that Snf1p kinase phosphorylates a threonine residue present at a consensus Snf1p site in Nmd5p. In summary, my studies link the stress-induced nuclear accumulation of Ssa4p(1-236)-GFP to the cell integrity MAPK pathway and Snf1p kinase.

The release of vasopressin (VP) from magnocellular neurosecretory cells (MNCs) of the supraoptic (SON) and paraventricular (PVN) nuclei is essential to hydromineral homeostasis. This release is controlled by several physiological stimuli, including changes in the osmotic pressure of the extracellular fluid, and in core body temperature. The osmotic control of VP release is mediated by specific and highly sensitive 'osmoreceptors'. Indeed, VP-releasing neurons in the SON are directly osmosensitive, and this osmosensitivity is mediated by stretch-inhibited cation channels. The molecular identity of these channels, however, remains unknown. The thermal control of VP release, on the other hand, is largely unexplained. In this thesis, we demonstrate that the mouse SON is a valid model for investigating the molecular basis of osmotransduction. We show that hyperosmotically-induced increases in membrane conductance are blocked by ruthenium red (RR), a non selective blocker of TRPV channels. In addition, SON neurons were found to express an N-terminal splice variant of TRPV1, but not full-length TRPV1. Unlike their wild-type counterparts, SON neurons in Trpv1 knockout (Trpv1-/-) mice could not generate RR-sensitive increases in membrane conductance and depolarizing potentials in response to hyperosmotic stimulation. Moreover, Trpv1-/-mice showed a pronounced serum hyperosmolality under basal conditions and severely compromised VP responses to osmotic stimulation in vivo. These results suggest that the Trpv1 gene may encode a central component of the osmoreceptor. Furthermore, we demonstrate that VP neurons are intrinsically thermosensitive. In these neurons, thermal stimuli spanning core body temperatures activate a RR-sensitive non selective cation current. Interestingly, VP neurons isolated from Trpv1 -/-mice are significantly less thermosensitive. These results suggest that channels encoded by the Trpv1 gene can confer thermosensitivity in the physiological range. Overall, these data suggest that products of the Trpv1 gene in VP neurons may represent a molecular point of convergence for the detection of osmotic and thermal stimuli.