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Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. We hypothesize that antenatal inflammation causes physiological abnormalities of the ASM that predisposes asthma. This study determined the short-term effects of antenatal inflammation on the developing ASM. Fourteen pregnant ewes were randomly assigned to one of three groups. Fetal lambs were exposed to intra-amniotic injections of lipopolysaccharide (LPS, n = 4) or saline (controls; n = 5) at 127 days' gestational age (GA). Preterm lambs were surgically delivered at 129 days' GA and received intensive care for 7 days before euthanasia. Naïve fetal controls ( n = 5) were delivered and euthanized at 136 days' GA. ASM force to acetylcholine was measured in bronchial rings and normalized to ring length (tension) and ASM cross-sectional area (stress). Airway narrowing (% volume) to acetylcholine was assessed in bronchial segments. Fetal controls were structurally and functionally similar to saline-exposed lambs. Compared with saline, LPS-exposed lambs had increased macrophages in lung tissue ( P = 0.0002) and interleukin-8 in alveolar wash ( P = 0.003). LPS exposure increased ASM thickness ( P = 0.005), airway narrowing ( P = 0.003), ASM tension ( P = 0.0002), and contractile stress ( P < 0.0001). Notably, LPS-exposed lambs were more dependent on mechanical ventilation, and both LPS ( P < 0.001) and ventilation ( P = 0.012) were independent factors in increasing ASM stress. Only LPS independently increased ASM thickness ( P = 0.045). Results indicate that antenatal exposure to LPS and subsequent mechanical ventilation promotes intrinsic changes to the ASM that enhances bronchoconstriction. If persistent into postnatal life, these developmental abnormalities may contribute to the known association between chorioamnionitis and asthma. NEW & NOTEWORTHY Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. Using an ovine model of antenatal inflammation, we demonstrate thickening and increased contraction of the premature ASM layer. If such physiological abnormalities persist throughout postnatal life, this represents a predisposition to an asthma diagnosis.

Following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess Ca2+ a likely trigger for loss of neurons, compact myelin and function. Treatment using inhibitors of specific Ca2+ channels has shown promise in preclinical studies, but clinical trials have been disappointing and combinatorial approaches are needed. We assessed efficacy of multiple combinations of three Ca2+ channel inhibitors at reducing secondary degeneration following partial optic nerve transection in rat. We used lomerizine to inhibit voltage gated Ca2+ channels; oxidised adenosine-triphosphate (oxATP) to inhibit purinergic P2X7 receptors and/or 2-[7-(1H-imidazol-1-yl)-6-nitro-2,3-dioxo-1,2,3,4-tetrahydro quinoxalin-1-yl]acetic acid (INQ) to inhibit Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Only the three Ca2+ channel inhibitors delivered in combination significantly preserved visual function, as assessed using the optokinetic nystagmus visual reflex, at 3 months after injury. Preservation of retinal ganglion cells was partial and is unlikely to have accounted for differential effects on function. A range of the Ca2+ channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration. Each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin. Nevertheless, limiting decompaction of myelin was not sufficient for preservation of function in our model. Multiple combinations of Ca2+ channel inhibitors reduced formation of atypical node/paranode complexes; outcomes were not associated with preservation of visual function. However, prevention of lengthening of the paranodal gap that was only achieved by treatment with the three Ca2+ channel inhibitors in combination was an important additional effect that likely contributed to the associated preservation of the optokinetic reflex using this combinatorial treatment strategy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Secondary degeneration of nerve tissue adjacent to a traumatic injury results in further loss of neurons, glia and function, via mechanisms that may involve oxidative stress. However, changes in indicators of oxidative stress have not yet been demonstrated in oligodendrocytes vulnerable to secondary degeneration in vivo. We show increases in the oxidative stress indicator carboxymethyl lysine at days 1 and 3 after injury in oligodendrocytes vulnerable to secondary degeneration. Dihydroethidium staining for superoxide is reduced, indicating endogenous control of this particular reactive species after injury. Concurrently, node of Ranvier/paranode complexes are altered, with significant lengthening of the paranodal gap and paranode as well as paranode disorganisation. Therapeutic administration of 670 nm light is thought to improve oxidative metabolism via mechanisms that may include increased activity of cytochrome c oxidase. Here, we show that light at 670 nm, delivered for 30 minutes per day, results in in vivo increases in cytochrome c oxidase activity co-localised with oligodendrocytes. Short term (1 day) 670 nm light treatment is associated with reductions in reactive species at the injury site. In optic nerve vulnerable to secondary degeneration superoxide in oligodendrocytes is reduced relative to handling controls, and is associated with reduced paranode abnormalities. Long term (3 month) administration of 670 nm light preserves retinal ganglion cells vulnerable to secondary degeneration and maintains visual function, as assessed by the optokinetic nystagmus visual reflex. Light at a wavelength of 670 nm may serve as a therapeutic intervention for treatment of secondary degeneration following neurotrauma.

Partial injury to the central nervous system (CNS) is exacerbated by additional loss of neurons and glia via toxic events known as secondary degeneration. Using partial transection of the rat optic nerve (ON) as a model, we have previously shown that myelin decompaction persists during secondary degeneration. Failure to repair myelin abnormalities during secondary degeneration may be attributed to insufficient OPC proliferation and/or differentiation to compensate for loss of oligodendrocyte lineage cells (oligodendroglia). Following partial ON transection, we found that sub-populations of oligodendroglia and other olig2+ glia were differentially influenced by injury. A high proportion of NG2+/olig2-, NG2+/olig2+ and CC1-/olig2+ cells proliferated (Ki67+) at 3 days, prior to the onset of death (TUNEL+) at 7 days, suggesting injury-related cues triggered proliferation rather than early loss of oligodendroglia. Despite this, a high proportion (20%) of the NG2+/olig2+ OPCs were TUNEL+ at 3 months, and numbers remained chronically lower, indicating that proliferation of these cells was insufficient to maintain population numbers. There was significant death of NG2+/olig2- and NG2-/olig2+ cells at 7 days, however population densities remained stable, suggesting proliferation was sufficient to sustain cell numbers. Relatively few TUNEL+/CC1+ cells were detected at 7 days, and no change in density indicated that mature CC1+ oligodendrocytes were resistant to secondary degeneration in vivo. Mature CC1+/olig2- oligodendrocyte density increased at 3 days, reflecting early oligogenesis, while the appearance of shortened myelin internodes at 3 months suggested remyelination. Taken together, chronic OPC decreases may contribute to the persistent myelin abnormalities and functional loss seen in ON during secondary degeneration.

Evidence was presented previously that rabbit erythroid cells possess a low-affinity Fe2+ transport system which operates via the Na+/Mg2+antiport [Biochim. Biophys. Acta 1282 (1996) 163]. This was investigated further by measurements of Mg2+ efflux as well as Fe2+ uptake by the cells and by examining the inhibitory effects of valinomycin, diethylstilbestrol (DES) and protein kinase inhibitors. Mg2+ efflux and Fe2+ uptake were measured using rabbit reticulocytes and mature erythrocytes incubated in isotonic KCl or NaCl solutions. Both processes were slower in mature cells than reticulocytes. Mg2+ efflux into KCl solution was much lower than into NaCl solution but was stimulated by addition of Fe2+ to the solution. The rate of Fe2+-stimulated Mg2+ efflux closely followed that of Fe2+ uptake in a one-to-one molar ratio. Valinomycin, DES and the protein kinase inhibitors all inhibited Fe2+ uptake from KCl solution. Valinomycin also inhibited Fe2+-stimulated Mg2+ efflux into KCl solution but markedly stimulated the efflux into NaCl. Maximal inhibition of Fe2+ uptake from KCl solution required the presence of K+, Rb+ or Cs+ ions with which valinomycin forms strong complexes. The results could not be explained on the basis of changes in cell membrane potential or cell volume. By contrast, the increase in Mg2+ efflux into NaCl solution produced by valinomycin was accompanied by cell shrinkage and production of a more negative membrane potential, either of which may be responsible for the effect. The inhibition produced by the protein kinase inhibitors indicate that phosphorylation of the transporter or an associated protein by protein tyrosine kinase is probably required to activate the transporter.

The aim of this investigation was to determine the mechanism of action of the nitrosophenylpyridine derivative of nifedipine ("nitrosonifedipine", NN) on Fe(II) transport into erythrocytes. Nifedipine is rapidly degraded to NN by daylight. We used rabbit erythrocytes, NN, and several chemically related substances, and examined their effects on the transfer of iron and other transition metals (cadmium, cobalt, manganese, nickel, zinc) into and out of the cells. NN mediated the transfer of iron and zinc but not the other metals into the cell cytosol. The transfer of Fe(II) was not affected by changes in cell membrane potential and could not be ascribed to free radical production. Two hydroxamic acid compounds chemically related to NN also stimulated iron and zinc uptake, but no evidence was obtained for cell-induced transformation of NN to them. In vivo, NN is probably converted to a lactam derivative. This compound was found to have no effect on iron uptake by the cells. It is concluded that NN has a relatively high specificity for the transport of iron compared with other transition metals, and small changes in its structure markedly affect this action. Also, because the lactam to which NN is converted in vivo is inactive, it is unlikely that nifedipine will affect iron metabolism after therapeutic administration.

Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

1. Earlier studies have shown that Fe2+ transport into erythroid cells is inhibited by several transition metals (Mn2+, Zn2+, Co2+, Ni2+) and that Fe2+ transport can occur by two saturable mechanisms, one of high affinity and the other of low affinity. Also, the transport of Zn2+ and Cd2+ into erythroid cells is stimulated by NaHCO3 and NaSCN. The aim of the present investigation was to determine whether all of these transition metals can be transported by the processes described for Fe2+, Zn2+ and Cd2+ and to determine the properties of the transport processes. 2. Rabbit reticulocytes and mature erythrocytes and reticulocytes from homozygous and heterozygous Belgrade rats were incubated with radiolabelled samples of the metals under conditions known to be optimal for high- and low-affinity Fe2+ transport and for the processes mediated by NaHCO3 and NaSCN. 3. All of the metals were transported by the high- and low-affinity Fe2+ transport processes and could compete with each other for transport. The Km and Vmax values and the effects of incubation temperature and metabolic inhibitors were similar for all the metals. NaHCO3 and NaSCN increased the uptake of Zn2+ and Cd2+ but not the other metals. 4. The uptake of all of the metals by the high-affinity process was much lower in reticulocytes from homozygous Belgrade rats than in those from heterozygous animals, but there was no difference with respect to low-affinity transport. 5. It is concluded that the high- and low-affinity 'iron' transport mechanisms can also transport several other transition metals and should therefore be considered as general transition metal carriers.

Iron uptake by rabbit reticulocytes and mature erythrocytes was investigated using 4 incubation systems: 1. Fe-transferrin in NaCl at pH 7.4, 2. Fe-transferrin in sucrose at pH 5.9, 3. Fe(II)-sucrose in sucrose at pH 6.5, and 4.Fe(II)-sucrose in KCl at pH 7.0. These systems were compared with respect to their magnitude and response to many membrane transport inhibitors and modifying agents. Iron uptake via the first 3 systems had many similar features that were quite distinct from those of iron uptake in the fourth system. On the basis of these results, it is concluded that erythroid cells contain two iron transport mechanisms, one with high affinity and relatively low capacity for iron transport, which can be studied using incubation systems 1-3, and the other of low affinity but high capacity (incubation system 4). High-affinity transport is present only in immature erythroid cells, is relatively sensitive to inhibition by N-ethylmaleimide (NEM), N,N1- dicyclohexylcarbodiimide (DCCD), and 7-chloro-4-nitrobenz-2-oxa-1,3 diazole (NBD), and is probably the mechanism by which iron, released from transferrin within endosomes, is transported across the endosomal membrane into the cytosol. DCCD and NBD are also inhibitors of the endosomal H(+)-ATPase, which is in keeping with the hypothesis that this ATPase functions as the iron transporter in endosomal membranes. However, the more-specific inhibitor of this enzyme, bafilomycin A1, inhibited iron uptake only in incubation system 1, where its action can be attributed to inhibition of endosomal acidification. Hence, it is unlikely that the ATPase also functions as the iron transporter. The low-affinity uptake mechanism is sensitive to inhibition by amiloride, valinomycin, quinidine, imipramine, quercetin, and diethylstilbestrol (to all of which high-affinity transport is relatively resistant), and is present in mature erythrocytes as well as reticulocytes.

The effects of five Ca2+ channel antagonists on iron uptake by erythroid cells were investigated using rabbit reticulocytes and erythrocytes, and transferrin-bound iron and non-transferrin-bound iron (Fe(II)). All of the antagonists except nifedipine inhibited iron uptake, but only at relatively high concentrations (10-100 microM). Nifedipine markedly stimulated the uptake of Fe(II) but not transferrin-bound iron, but only after it had been photodegraded to its nitrosophenylpyridine derivative. This compound was found to mediate Fe(II) exchange between the cytosol and extracellular medium in both directions with both reticulocytes and erythrocytes, but not by the known iron transport processes. The effect could be reversed by washing the cells with ice-cold NaCl solution. It appeared to be relatively specific for Fe(II) since photodegraded nifedipine had little effect on the uptake of Fe(III) or Mn2+. It is suggested that the nitrosopyridine derivative of nifedipine can act as an Fe(II) ionophore and may be of use as an adjuvant in chelator therapy with desferrioxamine in conditions of iron overload.

Rabbit erythroid cells can take up non-transferrin-bound iron by a high-affinity and a low-affinity transport mechanism (Hodgson et al. (1995) J. Cell. Physiol. 162, 181-190). The latter process, which is present in mature erythrocytes as well as reticulocytes, was investigated in this study using rabbit reticulocytes and erythrocytes. Iron uptake was optimal in isotonic KCI (pH 7.0), was shown to be much greater for Fe(II) than Fe(III), to be saturable with a Km value of approx. 15 microM Fe(II), temperature-dependent and inhibited by inhibitors of cell energy metabolism, by Na+ and many divalent cations and by several known inhibitors of membrane cation transport mechanisms. Uptake was more rapid with rabbit than with rat or human erythrocytes. The Fe(II) transport process was much more sensitive to inhibition by Mg2+ than by Ca2+ and the inhibition by both Mg2+ and Na+ was of competitive type. Cells depleted of intracellular Mg2+ by the use of the ionophore A23187 had low rates of Fe(II) uptake. High rates of uptake could be achieved by replenishment of intracellular Mg2+, and the Mg(2+)-dependent uptake of Fe(II) was inhibited by the same reagents which reduced the uptake by untreated cells. Many features of the Fe(II) transport process are very similar to those of the previously described Na+/Mg2+ antiport. These features, plus the stimulation of Fe(II) uptake by intracellular Mg2+ and inhibition by extracellular Mg2+ or Na+, strongly suggest that the iron is transported into the cells by the antiport.

1. The mechanisms of manganese transport into erythroid cells were investigated using rabbit reticulocytes and mature erythrocytes and 54Mn-labelled MnCl2 and Mn2-transferrin. In some experiments iron uptake was also studied. 2. Three saturable manganese transport mechanisms were identified, two for Mn2+ (high and low affinity processes) and one for transferrin-bound manganese (Mn-Tf). 3. High affinity Mn2+ transport occurred in reticulocytes but not erythrocytes, was active only in low ionic strength media such as isotonic sucrose and had a Km of 0.4 microM. It was inhibited by metabolic inhibitors and several metal ions. 4. Low affinity Mn2+ transport occurred in erythrocytes as well as in reticulocytes and had Km values of approximately 20 and 50 microM for the two types of cells, respectively. The rate of Mn2+ transport was maximal in isotonic KCl, RbCl or CsCl, and was inhibited by NaCl and by amiloride, valinomycin, diethylstilboestrol and other ion transport inhibitors. The direction of Mn2+ transport was reversible, resulting in Mn2+ efflux from the cells. 5. The uptake of transferrin-bound manganese occurred only with reticulocytes and depended on receptor-mediated endocytosis of Mn-Tf. 6. The characteristics of the three saturable manganese transport mechanisms were similar to corresponding mechanisms of iron uptake by erythroid cells, suggesting that the two metals are transported by the same mechanisms. 7. It is proposed that high affinity manganese transport is a surface representation of the process responsible for the transport of manganese across the endosomal membrane after its release from transferrin. Low affinity transport probably occurs by the previously described Na(+)-Mg2+ antiport, and may function in the regulation of intracellular manganese concentration by exporting manganese from the cells.