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Pyrimidylpiperazine (Y‐40138), a synthetic derivative of N‐[1‐(4‐{[4‐(pyrimidin‐2‐yl)piperazin‐1‐yl]methyl}phenyl)cyclopropyl] acetamide, is a novel dual regulator of pro‐ and anti‐inflammatory cytokines in vivo. The aim of the present study was to determine the signal transduction mechanisms implicated in vitro.In alveolar epithelial cells, pre‐treatment (30 min) with Y‐40138 reduced LPS‐induced biosynthesis of IL‐1β, IL‐6 and TNF‐α, an effect paralleled by up‐regulating an anti‐inflammatory counter‐loop mediated through IL‐10.This differential regulation of pro‐ and anti‐inflammatory signals was accompanied by an inhibition of the nuclear localization of selective NF‐κB subunits, particularly NF‐κB1(p50), RelA (p65), the major transactivating member of the Relfamily, RelB (p68) and c‐Rel (p75). In addition, Y‐40138 blockaded, in a dose‐dependent manner, the LPS‐induced nuclear activation of NF‐κB.Analysis of the upstream pathway involved in Y‐40138‐dependent retardation of LPS‐induced NF‐κB translocation/activation revealed the involvement of an IκB‐α sensitive pathway. Pre‐treatment with Y‐40138 ameliorated LPS‐induced degradation of IκB‐α in the cytosolic compartment and retarded its phosphorylation, suggesting the involvement of an upstream kinase.Recombinant IL‐10 (0 – 10 ng ml−1) blockaded, in a dose‐dependent manner, LPS‐induced biosynthesis of IL‐1β, IL‐6 and TNF‐α. Furthermore, rhIL‐10 reduced the DNA binding activity of NF‐κB. Immunoneutralization of endogenous IL‐10 by a polyclonal αIL‐10 (5 μg ml−1) reversed the inhibitory effect of Y‐40138 on pro‐inflammatory cytokines and partially restored the DNA binding activity of NF‐κB.These results indicate that Y‐40138 mediated dual immunoregulation of pro‐ and anti‐inflammatory cytokines is IL‐10 sensitive and mediated through the IκB‐α/NF‐κB signal transduction pathway.

1Monolayer cultures of rat fetal distal lung epithelial (FDLE) cells generated larger spontaneous short circuit currents (ISC) when maintained (48 h) at neonatal alveolar PO2(100 mmHg) than at fetal PO2(23 mmHg). When cells were shifted between these atmospheres in order to impose a rise in PO2equivalent to that seen at birth, no rise in ISCwas seen after 6 h but the response was fully established by 24 h.2Studies of basolaterally permeabilised cells revealed a small rise in apical Na+conductance (GNa) 6 h after PO2was raised but no further change had occurred by 24 h. A substantial rise was, however, seen after 48 h.3Reporter gene assays showed that no activation of the α‐ENaC (epithelial Na+channel α‐subunit) promoter was discernible 24 h after PO2was raised but increased transcriptional activity was seen at 48 h.4Studies of apically permeabilised cells showed that a small rise in Na+pump capacity was evident 6 h after PO2was raised and, in common with the rise in ISC, this effect was fully established by 24 h. The rise in ISCthus develops 6‐24 h after PO2is raised and is due, primarily, to increased Na+pump capacity.5The increase in GNathus coincides with activation of the α‐ENaC promoter but these effects occur after the rise in ISCis fully established and so cannot underlie this physiological response. The increased transcription may be an adaptation to increased Na+transport and not its cause.

Hepatocytes from the western painted turtle (Chrysemys picta bellii) are capable of a coordinated metabolic suppression of 88% during 10 h of anoxia at 25 degrees C. The energy dependence and role of proteolysis in this suppression were assessed in labile ([3H]Phe-labeled) and stable ([14C]Phe-labeled) protein pools. During anoxia, labile protein half-lives increased from 24.7 +/- 3.3 to 34.4 +/- 3.7 h, with stable protein half-lives increasing from 55.6 +/- 3.4 to 109.6 +/- 7.4 h. The total anoxic mean proteolytic suppression for both pools was 36%. On the basis of inhibition of O2 consumption and lactate production rates by cycloheximide and emetine, normoxic ATP-dependent proteolysis required 11.1 +/- 1.7 mumol ATP.g-1.h-1 accounting for 21.8 +/- 1.4% of total cellular metabolism. Under anoxia this was suppressed by 93% to 0.73 +/- 0.43 mumol ATP.g-1.h-1. Summation of this with protein synthesis ATP turnover rates indicated that under anoxia 45% of total ATP turnover rate was directed toward protein turnover. Studies with inhibitors of energy metabolism indicated that the majority of energy dependence was found in the stable protein pool, with no significant inhibition occurring among the more labile proteins. We conclude that proteolysis is largely energy dependent under normoxia, whereas under anoxia there is a shift to a slower overall proteolytic rate that is largely energy independent and represents loss mostly from the labile protein pool.

The O2 sensitivity of protein expression was assessed in hepatocytes from the western painted turtle. Anoxic cells consistently expressed proteins of 83.0, 70.4, 42.5, 35.3, and 16.1 kDa and suppressed proteins of 63.7, 48.2, 36.9, 29.5, and 17.7 kDa. Except for the 70.4-kDa protein, this pattern was absent during aerobic incubation with 2 mM NaCN, suggesting a specific requirement for O2. Aerobic incubation with Co2+ or Ni2+ increased expression of the 42.5-, 35.3-, and 16.1-kDa protein bands which was diminished with the heme synthesis inhibitor 4,6-dioxoheptanoic acid. Proteins suppressed in anoxia were also suppressed during aerobic incubation with Co2+ or Ni2+ but this was not relieved by 4,6-dioxoheptanoic acid. The anoxia- and Co2+/Ni2+-induced expression of the 42.5-, 35.3-, and 16.1-kDa protein bands was antagonized by 10% CO; however, with the exception of the 17.7-kDa protein, this was not found for any of the O2- or Co2+/Ni2+-suppressed proteins. Anoxia-induced proteins were compared with proteins expressed during heat shock. Heat shock proteins appeared at 90.2, 74.8, 63.4, 25, and 15.5 kDa and were of distinct molecular masses compared with the anoxia-induced proteins. These results suggest that O2-sensing mechanisms are active in the control of protein expression and suppression during anoxia and that, in the case of the 42.5-, 35.3-, 17.7-, and 16.1-kDa proteins, a conformational change in a ferro-heme protein is involved in transducing the O2 signal.

Land, S. C., R. H. Sanger, and P. J. S. Smith.O2availability modulates transmembrane Ca2+flux via second-messenger pathways in anoxia-tolerant hepatocytes.J. Appl. Physiol.82(3): 776–783, 1997.—Transmembrane Ca2+-flux was studied from single isolated turtle hepatocytes by using a noninvasive Ca2+-selective self-referencing microelectrode. Cells in Ca2+-reduced culture medium demonstrated a vanadate-and lanthanum-inhibitable Ca2+-efflux of 4 × 10−17mol Ca2+⋅ μm−2⋅ s−1continuously over 170 h. This flux diminished with 50 nM phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, and was reinstated on PKC deactivation with sphingosine. Progressive hypoxia resulted in a reversible suppression of Ca2+efflux to 90% of normoxic controls with an apparent Michaelis constant for oxygen of 145 μM. PKC activation was critical in this suppression, as anaerobic administration of sphingosine caused a Ca2+influx and cell rupture. Hypoxia was also associated with an altered pattern of adenosine-mediated control over Ca2+efflux. Adenosine (100 μM) elevated Ca2+efflux twofold in normoxia, but neither adenosine nor the A1-purinoreceptor antagonist 8-phenyltheophylline altered the observed anaerobic suppression. Aerobic administration of 2–10 mM KCN failed to reproduce the anaerobic suppression; however, in conjunction with 10 mM iodoacetate, complete metabolic blockade caused a Ca2+influx and cell rupture. These observations suggest modulatory control by oxygen over transmembrane Ca2+efflux involving second-messenger systems in the hypoxic transition.

O2 availability modulates transmembrane Ca²⁺ flux via second-messenger pathways in anoxia-tolerant hepatocytes. J. Appl. Physiol. 82(3): 776-783, 1997.—Transmembrane Ca²⁺-flux was studied from single isolated turtle hepatocytes by using a noninvasive Ca²⁺-selective self-referencing microelectrode. Cells in Ca²⁺-reduced culture medium demonstrated a vanadate-and lanthanum-inhibitable Ca²⁺-efflux of 4 × 10⁻¹⁷ mol Ca²⁺ · μm⁻² · s⁻¹ continuously over 170 h. This flux diminished with 50 nM phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, and was reinstated on PKC deactivation with sphingosine. Progressive hypoxia resulted in a reversible suppression of Ca²⁺ efflux to 90% of normoxic controls with an apparent Michaelis constant for oxygen of 145 μM. PKC activation was critical in this suppression, as anaerobic administration of sphingosine caused a Ca²⁺ influx and cell rupture. Hypoxia was also associated with an altered pattern of adenosine-mediated control over Ca²⁺ efflux. Adenosine (100 μM) elevated Ca²⁺ efflux twofold in normoxia, but neither adenosine nor the A1-purinoreceptor antagonist 8-phenyltheophylline altered the observed anaerobic suppression. Aerobic administration of 2-10 mM KCN failed to reproduce the anaerobic suppression; however, in conjunction with 10 mM iodoacetate, complete metabolic blockade caused a Ca²⁺ influx and cell rupture. These observations suggest modulatory control by oxygen over transmembrane Ca²⁺ efflux involving second-messenger systems in the hypoxic transition.