Rapid increases in respiratory epithelial permeability occur after intratracheal instillation of PMA

We measured pulmonary epithelial permeability by quantifying the disappearance of two water-soluble compounds, [14C]mannitol and [3H]inulin, after their instillation, with and without phorbol myristate acetate (PMA), into gas-filled perfused (50 ml/min) rabbit lungs in situ. Both tracers disappeared in a monoexponential fashion over 30 min with calculated first-order rate constants (control; n = 11) of 0.0008 +/- 0.0002 and 0.0027 +/- 0.0008 min-1 for inulin and mannitol, respectively. The ratio of the rate constants (3.1 +/- 0.5) was not significantly different from the ratio of diffusivities of mannitol:inulin (3.7). Addition of PMA (250 micrograms) significantly (n = 9, P less than 0.05) increased the rate constants for both inulin and mannitol to 0.0024 +/- 0.0007 and 0.0087 +/- 0.0025 min-1, respectively, while not affecting their ratio (4.3 +/- 0.5). Addition of human leukocytes (4-8 X 10(8)/l) to the perfusate did not exacerbate the effect of 250 micrograms PMA (n = 3). The addition of catalase (n = 7) completely inhibited the effect of 250 micrograms PMA. PMA (250 micrograms) did not significantly affect perfusion pressure but increased wet-to-dry weight ratios. Light microscopic histology showed damage to epithelial and endothelial cells after 250 micrograms PMA which was not seen after coinstillation of catalase. Catalase sensitivity of functional and structural effects of PMA suggests that the effect was secondary to production of hydrogen peroxide. Since this effect was noted in lungs not perfused with neutrophils and addition of leukocytes did not exacerbate the increase in permeability, we hypothesize that an undetermined pulmonary cell type was the source of hydrogen peroxide. Finally, we found no evidence for restrictive pores with radii of 0.4-1.4 nm.     

Properties of avian, bovine and porcine erythrocyte membranes

Bovine, porcine and avian EMP were isolated and compared for some physical and chemical properties. Some differences in the compositions of three EMPs were observed. The avian EMP contained less carbohydrate than the bovine and porcine EMPs. Some differences in the monosaccharide distributions for the three preparations were revealed. The profiles obtained by SDS-polyacrylamide gel electrophoresis of the preparation indicated a complex (and different for each preparation) nature of the component polypeptides and glycopeptides.     

Lack of Rapid Desensitization of Ca2+ Responses in Transfected CHO Cells Expressing the Rat Neurotensin Receptor Despite Agonist-Induced Internalization

Abstract: Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization of the neurotensin receptor at the second messenger level. Stimulation with nanomolar concentrations of neurotensin elicited rapid rises in the cytosolic calcium concentration ([Ca²⁺]_i), which remained elevated throughout the peptide application. A significant response was already detected with neurotensin concentrations as low as 0.01 nM. This high efficiency of neurotensin in mediating this calcium response contrasts with the nanomolar affinity of the peptide for its receptor measured in binding experiments. Evidence indicated that the initial elevation of the [Ca²⁺]_i resulted from release of Ca²⁺ from intracellular stores, whereas the sustained response involved an influx of extracellular origin. Return to the basal level was only reached after extensive washing of the peptide or its displacement with the neurotensin receptor antagonist SR48692. After washing, further stimulations were still able to mediate an increase in the [Ca²⁺]_i, indicating an apparent absence of rapid desensitization of the intracellular signaling pathway that mediates calcium mobilization. In contrast with this absence of response desensitization, the neurotensin receptors were found to internalize after stimulation with the peptide. This internalization was maximal after 30 min and accounted for ～70% of the number of neurotensin binding sites located at the cell surface. These results indicate that despite the functional properties of the rat neurotensin receptor present in Chinese hamster ovary cells after transfection, the intracellular signaling pathway triggered by stimulation with neurotensin seems to be resistant to desensitization. This might be related to the high efficiency of the intracellular signaling pathway coupled to the neurotensin receptor observed in these cells. A possible absence of desensitization of the neurotensin receptor itself is also discussed.     

IL-4 inhibits H2O2 production and antileishmanial capacity of human cultured monocytes mediated by IFN-gamma

The effect of IL-4 on the IFN-gamma-induced state of activation of cultured human monocytes was investigated with regard to their ability to produce hydrogen peroxide and their antileishmanial capacity towards the intracellular parasite Leishmania donovani. IL-4 was found to inhibit the IFN-gamma-dependent hydrogen peroxide production of monocytes. Treatment of monocytes with IFN-gamma (200 to 600 U/ml) for 48 h increased the hydrogen peroxide production fourfold above background. Coincubation of the monocytes with IL-4 (1 to 1000 U/ml) and IFN-gamma (200 to 600 U/ml) inhibited this increase by 50 to 100%. IL-4 alone did not modulate the hydrogen peroxide production of monocytes. Pretreatment of monocytes with IL-4 for 20 min to 3 h was already effective in preventing the IFN-gamma response. Addition of IL-4 not later than 6 h after the start of incubation with IFN-gamma was necessary for an optimal inhibitory effect. IL-4 also inhibited the IFN-gamma-induced antileishmanial capacity of monocytes: IFN-gamma (1000 U/ml) induced a 54 +/- 10% reduction in the number of parasites. Monocytes treated with combinations of IL-4 (100 to 1000 U/ml) and IFN-gamma (1000 U/ml) were unable to reduce the parasite numbers. IL-4 alone did not alter the uptake of Leishmania donovani nor induce antileishmanial activity. These results demonstrate that IL-4 disables human cultured monocytes to respond to IFN-gamma activation.