Prostacyclin contributes to inhibition of hypoxic pulmonary vasoconstriction by alkalosis

The mechanism by which extracellular alkalosis inhibits hypoxic pulmonary vasoconstriction is unknown. We investigated whether the inhibition was due to intrapulmonary production of a vasodilator prostaglandin such as prostacyclin (PGI₂). Hypoxic vasoconstriction in isolated salt-solution-perfused rat lungs was blunted by both hypocapnic and NaHCO₃_induced alkalosis (perfusate pH increased from 7.3 to 7.7). The NaHCO₃-induced alkalosis was accompanied by a significant increase in the perfusate level of 6-keto-prostaglandin F_1α (6-keto-PGF_1α), an hydrolysis product of PGI₁. Meclofenamate, an inhibitor of cyclooxygenase, counteracted both the blunting of hypoxic vasoconstriction and the increased level of 6-keto-PGF_1α. In intact anesthetized dogs, hypocapnic alkalosis (blood pH increased from 7.4 to 7.5) blunted hypoxic pulmonary vasoconstriction before but not after administration of meclofenamate. In separate cultures of bovine pulmonary artery endothelial and smooth muscle cells stimulated by bradykinin, the incubation medium levels of 6-keto-PGF_1α were increased by both hypocapnia and NaHCO₃-induced alkalosis (medium pH increased from 7.4 to 7.7). These results suggest that inhibition of hypoxic pulmonary vasoconstriction by alkalosis is mediated at least partly by PGI_2.     

Effect of hemoglobin concentration on maximal O2 uptake in canine gastrocnemius muscle in situ

O2 delivery to maximally working muscle was decreased by altering hemoglobin (Hb) concentration and arterial PO2 (PaO2) to investigate whether the reductions in maximal O2 uptake (VO2max) that occur with lowered [Hb] are in part related to changes in the effective muscle O2 diffusing capacity (DmO2). Two sets of experiments were conducted. In the initial set (n = 8), three levels of Hb [5.8 +/- 0.3, 9.4 +/- 0.1, and 14.4 +/- 0.6 (SE) g/100 ml] in the blood were used in random order to pump perfuse, at equal muscle blood flows and PaO2, maximally working isolated dog gastrocnemius muscle. VO2max declined with decreasing [Hb], but the relationship between VO2max and both the effluent venous PO2 (PvO2) and the calculated mean capillary PO2 (PcO2) was not linear through the origin and, therefore, not compatible with a single value of DmO2 (as calculated by Bohr integration using a model based on Fick's law of diffusion). To clarify these results, a second set of experiments (n = 6) was conducted in which two levels of Hb (14.0 +/- 0.6 and 6.9 +/- 0.6 g/100 ml) were each combined with two levels of oxygenation (PaO2 79 +/- 8 and 29 +/- 2 Torr) and applied in random sequence to again pump perfuse maximally working dog gastrocnemius muscle at constant blood flow. In these experiments, the relationship between VO2max and both PvO2 and calculated PcO2 for each [Hb] was consistent with a constant estimate of DmO2 as PaO2 was reduced, but the calculated DmO2 for the lower [Hb] was 33% less than that at the higher [Hb] (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Architecture and functional aspects of the distal airways of Antarctic seals with different habits,the Weddell Seal (Leptonychotes weddellii) and the Crabeater Seal (Lobodon carcinophagus)

Summary The lung of the deep diving Weddell Seal is characterized by an unusually well developed periacinar dense collagenous connective tissue, and a thick coat of smooth musculature particularly in the distal bronchioli. Both, collagen and smooth musculature appear to be functionally interrelated, the first serving presumably as site of origin or attachment for the latter. The orientation of the bronchiolar smooth muscle cells is complex: there exists a basic pattern of two crisscrossing helical bundles that wind in opposite direction. In addition, longitudinal bundles are frequent both at the inside and the outside of the muscular coat. Furthermore, more or less complete ringshaped bundles occur as well as groups of muscle fibres running radially into the collagenous tissue of the surroundings of a bronchiolus. This complex architecture presumably allows active adjustment to various physiological needs of the Weddell Seal including as extremes both closing and widening of the bronchiolar lumen. Isometric contractions of the smooth musculature may stiffen the wall of the distal airways while diving. In the Crabeater Seal which dives for shorter durations and by far less deeply than the Weddell Seal, both periacinar collagen and bronchiolar smooth musculature are of similar arrangement, however, occur in considerably reduced amounts. A rich supply of autonomie nerve fibres with abundant varicosities controls the smooth muscle cells, which are interconnected by gap junctions and receive their innervation par distance (visceral type of smooth musculature). The majority of varicosities contains small clear vesicles, as is typical for cholinergic nerves, suggesting a strong parasympathetic influence. Other varicosities are presumably of peptidergic type. Mast cells and epithelial endocrine cells may exert additional influence on the musculature.     

Characterization of crystals of genetically engineered human manganese superoxide dismutase

The genetically engineered human manganese superoxide dismutase crystallizes in space group P2(1)2(1)2 with a = 75.51 A, b = 79.00 A, c = 67.95 A. At room temperature the crystals are not stable against radiation, so we cooled them to 90 K and collected a data set to 3 A resolution at this temperature.     

Thiophosphorylation causes Ca2+-independent norepinephrine secretion from permeabilized PC12 cells

Adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was used to examine the role of phosphorylation in the regulation of norepinephrine secretion by digitonin-permeabilized PC12 cells. While most kinases will use ATP gamma S to thiophosphorylate proteins, thiophosphorylated proteins are relatively resistant to dethiophosphorylation by protein phosphatases. Norepinephrine secretion by permeabilized PC12 cells was ATP- and Ca2+-dependent but resistant to calmodulin antagonists. Half-maximum secretion was obtained in 0.75 microM Ca2+. Permeabilized PC12 cells were incubated with ATP gamma S in the absence of Ca2+, the ATP gamma S was removed, and norepinephrine secretion was determined. Preincubation with ATP gamma S increased the amount of norepinephrine secreted in the absence of Ca2+, but it had no effect on the amount released in the presence of Ca2+. After a 15-min preincubation in 1 mM ATP gamma S, there was almost as much secretion in the absence of Ca2+ as in its presence. Inclusion of ATP in the preincubation inhibited the effect of ATP gamma S. Ca2+ stimulated the rate of modification by ATP gamma S as brief preincubations with ATP gamma S in the presence of Ca2+ resulted in higher levels of Ca2+-independent secretion than did preincubations with ATP gamma S in the absence of Ca2+. Similarly, brief preincubations of permeabilized cells with ATP in the presence of Ca2+ resulted in elevated levels of Ca2+-independent secretion. Secretion of norepinephrine from ATP gamma S-treated cells was ATP-dependent. These results suggest that norepinephrine secretion by PC12 cells is regulated by a Ca2+-dependent phosphorylation. Once this phosphorylation has occurred, secretion is still ATP-dependent, but it no longer requires Ca2+.     

Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells

von Willebrand factor (vWf) is secreted from endothelial cells by one of two pathways-a constitutive pathway and a regulated pathway originating from the Weibel-Palade bodies. The molecular form of vWf from each of these pathways differs, with the most biologically potent molecules being released from Weibel-Palade bodies (Loesberg, C., M. D. Gonsalves, J. Zandbergen, C. Willems, W. G. Van Aken, H. V. Stel, J. A. Van Mourik, and P. G. DeGroot. 1983. Biochim. Biophys. Acta. 763:160-168; Sporn, L. A., V. J. Marder, and D. D. Wagner. 1987. Cell. 46:185-190). We investigated the polarity of the two secretory pathways using human umbilical vein endothelial cells cultured on polycarbonate membrane filters which allowed sampling of media from both the apical and basolateral compartments. After metabolic labeling of cells, vWf (constitutively secreted during a 10-min period or released during a 10-min treatment with a secretagogue) was purified from the apical and basolateral chambers and subjected to gel analysis. Approximately equal amounts of vWf were constitutively secreted into both chambers, and therefore this secretory pathway appeared to be nonpolarized. On the contrary, an average of 90% of vWf released from Weibel-Palade bodies after treatment with the calcium ionophore A23187 or PMA appeared in the basolateral chamber, indicating that the regulated pathway of secretion is highly polarized. Thrombin, a secretagogue which promotes disruption of the endothelial monolayer, led to release of vWf from cells with no apparent polarity. The presence of microtubule-depolymerizing agents nocodazol and colchicine inhibited the polarized release of vWf. Ammonium chloride treatment did not disrupt the polarity of the regulated secretory pathway, indicating that maintenance of low pH in intracellular compartments was not required for the polarized delivery of preformed Weibel-Palade bodies to the plasma membrane.     

In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D2 and serotonin 5-HT2 neuroreceptors

Apparent affinities (Ki) of (E)- and (Z)-N-(iodoallyl)spiperone [E)- and (Z)-NIASP) for dopamine D2 and serotonin 5-HT2 receptors were determined in competition binding assays. (Z)-NIASP (Ki 0.35 nM, D2; Ki 1.75 nM, 5-HT2) proved slightly more potent and selective for D2 sites in vitro than (E)-NIASP (Ki 0.72 nM, D2; Ki 1.14 nM, 5-HT2). In vivo, radioiodinated (E)- and (Z)-[125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of (E)-[125I]-NIASP uptake was found for drugs binding to dopamine D2 sites, while drugs selective for serotonin 5-HT2, alpha 1-adrenergic and dopamine D1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-[125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-[125I]-NIASP binds with high selectivity and specificity to dopamine D2 sites in vivo.