K+ influx and efflux were measured in normal (HbA) and sickle (HbS) red blood cells to investigate the interaction of swelling, H+ ions and urea with O2 (0 to 150 mmHg O2) in the presence of ouabain and bumetanide (both 100 μM). In HbA cells, K+‐Cl− cotransport was O2 dependent. At low oxygen tensions (PO2s) the transporter was inactive and refractory to low pH, swelling or urea. Cl−‐independent K+ influxes in sickle cells were elevated at low PO2s, as previously reported. Cl−‐dependent K+ influxes were large at both high and low PO2s, whether stimulated by swelling, H+ ions or urea. In the absence of O2, Cl−‐dependent K+ influxes were similar in magnitude to those measured at high PO2s. The minimum for Cl−‐dependent K+ influx was observed at PO2s of about 40‐70 mmHg. K+ efflux from HbS cells was stimulated by the addition of urea (500 mM). The rate constants were of similar magnitude whether measured at high PO2 or in the absence of O2, and were predominantly Cl− dependent under both conditions. In HbS red blood cells, reduction of extracellular Ca2+, addition of 1 mM Mg2+ or nitrendipine (10 μM) to the saline had no effect. Inhibitors of K+‐Cl− cotransport, [(dihydroindenyl)oxy] alkanoic acid (DIOA; 100 μM) or calyculin A (0·1 μM), inhibited influxes by a similar magnitude to Cl− substitution. Results are significant for the pathophysiology of sickle cell disease. Low pH and urea are able to stimulate KCl loss from sickle cells, leading to cellular dehydration, even in regions of low PO2.

. SNAT4 isoform of system A amino acid transporter is expressed in human placenta. Am J Physiol Cell Physiol 290: C305-C312, 2006. First published September 7, 2005 doi:10.1152/ajpcell.00258.2005.-The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na ϩ -coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6 -10 wk) and late (10 -13 wk) first-trimester and full-term (38 -40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta (P Ͻ 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester (P Ͻ 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term. SNAT1; SNAT2; gestational expression; syncytiotrophoblast SYSTEM A IS A UBIQUITOUS Na ϩ -dependent transporter that actively transports small, zwitterionic, neutral amino acids with short, unbranched side chains such as alanine, serine, and glutamine (24). It has a unique ability to transport N-methylated amino acids such as ␣-(methylamino)isobutyric acid (MeAIB) (7). This nonmetabolized amino acid analog has been used extensively to study system A in the placenta (21,27,37).Recent data show that system A is encoded by three different members of the SLC38 gene family (Slc38a1, Slc38a2, and Slc38a4), giving rise to the three subtypes of this Na ϩ -coupled neutral amino acid transporter (SNAT): SNAT1, SNAT2, and SNAT4 (previously referred to as ATA1, ATA2, and ATA3, respectively) (31). SNAT1 was the first isoform to be cloned from rat brain and initially was designated GlnT because of its preference for glutamine as a substrate (49). Cloning of the human homolog as well as SNAT2 and SNAT4 followed (18,19,44,45,50,51). These previous studies showed the three isoforms to be highly homologous: amino acid sequences for SNAT1 and S...

K influx into equine red blood cells (RBCs) was measured using86Rb as a tracer for K under conditions designed to mimic the changes in respiratory blood parameters that occur in vivo during strenuous exercise. The effects on K influx of physiological changes in pH, cell volume, O2 tension ([Formula: see text]), CO2 tension ([Formula: see text]), and bicarbonate and lactate concentrations were defined. Physiological[Formula: see text] exerted a dominant controlling influence on the H+-stimulated Cl-dependent K influx, consistent with effects on the K-Cl cotransporter; [Formula: see text] required for half-maximal activity was 37 ± 3 mmHg (4.9 kPa). Although RBCs were swollen at low pH, results showed explicitly that the volume change per se had little effect on K influx. Lactate had no effect on volume- or H+-stimulated K influxes, nor did bicarbonate or [Formula: see text]affect the magnitude of K influxes after these stimuli or after treatment with protein kinase/phosphatase inhibitors. These results represent the first detailed report of O2 dependence of H+-stimulated K-Cl cotransport in RBCs from any mammalian species. They emphasize the importance of[Formula: see text] in control of RBC K-Cl cotransport.

The effect of urea and its interactions with oxygen tension (PO2), cell volume and inhibitors of protein phosphatases/kinases (PP/PK) on the K influx into equine red blood cells were studied. K influx was measured using 86Rb as a radioactive tracer for K. As in other species, Cl-dependent K influxes were stimulated by urea, with peak fluxes occurring at about 750 mM. This effect was not mediated via changes in cell volume or following formation of cyanate, the hydrolysis product of urea. Stimulation by urea was prevented by pre-treatment with calyculin A (100 nM) at all urea concentrations tested. At low concentrations, urea-stimulated influx was O2 dependent, and sensitive to changes in cell volume and subsequent treatment with calyculin A. By contrast, at high concentrations, urea-stimulated influxes were largely unaffected by these manipulations. Like pharmacological manipulations, e.g. by N-ethylmaleimide, staurosporine and depletion of intracellular Mg by A23187, but unlike cell swelling per se, urea was able to affect transport regardless of PO2. K-Cl cotransport in cells treated with N-ethylmaleimide (1 mM) alone, or with combinations of N-ethymaleimide and calyculin A, was no longer stimulated by addition of urea, rather it was inhibited. Results are consistent with urea acting predominantly as a direct inhibitor of the regulatory PK, with a smaller inhibitory effect downstream of this phosphorylation step possibly on the transporter itself.

The effect of oxidants on K(+)-Cl(-) cotransport (KCC) was investigated in equine red blood cells. Carbon monoxide mimicked O(2). The substituted benzaldehyde, 12C79 (5 mM), markedly increased O(2) affinity. In N(2), however, O(2) saturation was low (<10%) but KCC remained active. Nitrite (NO(2)(-)) oxidized heme to methemoglobin (metHb). High concentrations of NO(2)(-) (1 and 5 mM vs. 0.5 mM) increased KCC activity above control levels; it became O(2) independent but remained sensitive to other stimuli. 1-Chloro-2, 4-dinitrobenzene (1-3 mM) depleted reduced glutathione (GSH). Prolonged exposure (60-120 min, 1 mM) or high concentrations (3 mM) stimulated an O(2)-independent KCC activity; short exposures and low concentrations (30 min, 0.5 or 1 mM) did not. The effect of these manipulations was correlated with changes in GSH and metHb concentrations. An oxy conformation of Hb was necessary for KCC activation. An increase in its activity over the level found in oxygenated control cells required both accumulation of metHb and depletion of GSH. Findings are relevant to understanding the physiology and pathology of regulation of KCC.