Lipid peroxidation and biochemical parameters in umbilical cord blood of well-adapted term newborns

Lipid peroxidation (LPX) can play an important role in the development of pathological changes of foetal and neonatal tissues. We investigated LPX and biochemical parameters in plasma from mixed umbilical cord (m.u.c.) blood and acid-base balance (ABB) parameters in m.u.c. blood of well-adapted full-term newborns. LPX products were estimated as thiobarbituric acid reacting substances (TBARS) and were expressed by using of malondialdehyde (MDA) as a standard solution. Intensity of LPX was estimated in vitro in m.u.c. blood plasma without and with added LPX activator (125 μM L-ascorbate plus 5 μM FeSO₄) and in the incubated plasma (30 min, 37°C) under both conditions. Actual TBARS (3.51 ± 0.49 nmol/mL) were determined in the non-incubated plasma without the added LPX activator. Approximately twice higher TBARS were found in the incubated plasma without the LPX activator (7.29 ± 2.17 nmol/mL) or with it (8.57 ± 2.20 nmol/mL), as well as in the non-incubated plasma after its addition (7.38 ± 1.98 nmol/mL). All analysed biochemical parameters (Fe, total iron-binding capacity, uric acid, proteins, Mg, Ca, phosphate, glucose, K, Na, Cl, ALT, AST, GMT, CK, LD, HBD, AMS, ALP, ACP) and ABB parameters were within their reference ranges. The actual TBARS levels were found being positively correlated with α-hydroxybutyrate dehydrogenase (HBD) activity and negatively with pO₂. These results suggest that LPX in m.u.c. blood plasma might be activated. This activation could probably depend on extent of hypoxia. TBARS and their formation in vitro could be suitable parameters of LPX in m.u.c. blood.     

Basolateral Cl channels in primary airway epithelial cultures

Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.     

Function of the HVCN1 proton channel in airway epithelia and a naturally occurring mutation,M91T

Airways secrete considerable amounts of acid. In this study, we investigated the identity and the pH-dependent function of the apical H⁺ channel in the airway epithelium. In pH stat recordings of confluent JME airway epithelia in Ussing chambers, Zn-sensitive acid secretion was activated at a mucosal threshold pH of ∼7, above which it increased pH-dependently at a rate of 339 ± 34 nmol × h⁻¹ × cm⁻² per pH unit. Similarly, H⁺ currents measured in JME cells in patch clamp recordings were readily blocked by Zn and activated by an alkaline outside pH. Small interfering RNA–mediated knockdown of HVCN1 mRNA expression in JME cells resulted in a loss of H⁺ currents in patch clamp recordings. Cloning of the open reading frame of HVCN1 from primary human airway epithelia resulted in a wild-type clone and a clone characterized by two sequential base exchanges (452T>C and 453G>A) resulting in a novel missense mutation, M91T HVCN1. Out of 95 human genomic DNA samples that were tested, we found one HVCN1 allele that was heterozygous for the M91T mutation. The activation of acid secretion in epithelia that natively expressed M91T HVCN1 required ∼0.5 pH units more alkaline mucosal pH values compared with wild-type epithelia. Similarly, activation of H⁺ currents across recombinantly expressed M91T HVCN1 required significantly larger pH gradients compared with wild-type HVCN1. This study provides both functional and molecular indications that the HVCN1 H⁺ channel mediates pH-regulated acid secretion by the airway epithelium. These data indicate that apical HVCN1 represents a mechanism to acidify an alkaline airway surface liquid.