The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na+ transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na+ transport two- to threefold by increasing Na+-K+ ATPase transport activity and basolateral membrane K+ conductance without increasing the apical membrane amiloride-sensitive Na+ conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na+ absorption with a further increase in Na+-K+ ATPase transport activity and an increase in apical membrane amiloride-sensitive Na+ conductance. The effect of insulin on the Na+-K+ ATPase was the result of an increase in Vmax for extracellular K+ and intracellular Na+, and an increase in affinity of the pump for Na+. Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of α-1 and α-2 isoforms, but not the α-3 isoform of Na+-K+ ATPase, which did not change in the presence of insulin. Insulin-stimulated Na+ transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)3], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na+ transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3–kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3–kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na+-K+ ATPase activation.

Inflammatory changes following infection are central to the clinical manifestation of disease. However, information regarding such changes in animal disease is limited. In canine parvovirus infected puppies we measured the levels of acute phase proteins and changes in leukocyte phenotypes and cell trafficking by flow cytometry. These parameters correlated with conventional assessment of clinical disease in a vaccine efficacy study. Seropositive (CPV-2) 6-week-old puppies given three doses of a CPV-2 containing vaccine developed significant antibody titers and remained healthy after experimental infection with CPV-2b. Unvaccinated controls developed clinical signs and shed virus. Importantly, acute phase proteins became elevated, and lymphopenia, neutropenia and modulation of neutrophil-CD4 were detected in controls but not in vaccinates.

The objective of this study was to investigate the mechanism of uridine 5′-triphosphate (UTP)-dependent inhibition of Na+ absorption in porcine endometrial epithelial cells. Acute stimulation with UTP (5 μM) produced inhibition of sodium absorption and stimulation of chloride secretion. Experiments using basolateral membrane–permeabilized cell monolayers demonstrated a reduction in benzamil-sensitive Na+ conductance in the apical membrane after UTP stimulation. The UTP-dependent inhibition of sodium transport could be mimicked by PMA (1 μM). Several PKC inhibitors, including GF109203X and Gö6983 (both nonselective PKC inhibitors) and rottlerin (a PKCδ selective inhibitor), were shown to prevent the UTP-dependent decrease in benzamil-sensitive current. The PKCα-selective inhibitors, Gö6976 and PKC inhibitor 20–28, produced a partial inhibition of the UTP effect on benzamil-sensitive Isc. Inhibition of the benzamil-sensitive Isc by UTP was observed in the presence of BAPTA-AM (50 μM), confirming that activation of PKCs, and not increases in [Ca2+]i, were directly responsible for the inhibition of apical Na+ channels and transepithelial Na+ absorption.

A rapid and reproducible enzyme linked immunosorbent assay (ELISA) was developed for detection of canine coronavirus (CCV) specific antibodies directed to both the nucleocapsid (NC) and the spike (S) proteins. The coating antigen, a methanol-treated, S-protein enriched preparation, was produced by subjecting infected cells to Triton X-114 detergent followed by phase separation. The sensitivity of this assay was determined by following the course of infection in dogs experimentally infected with CCV. The specificity of the antibody response was determined by Western blot analysis and supported the increased magnitude of the ELISA response and the presence of serum neutralizing (SN) antibody. Due to the sensitivity and specificity of the IgG response detected by this assay it can be used to determine both virus exposure and vaccine efficacy.

No abstract