Oxygen-dependent K+ influxes in Mg2+-clamped equine red blood cells.
1. Cl--dependent K+ (86Rb+) influxes were measured in oxygenated and deoxygenated equine red blood cells, whose free [Mg2+]i had been clamped, to examine the effect on O2 dependency of the K+-Cl- cotransporter. 2. Total [Mg2+]i was 2.55 +/- 0.07 mM (mean +/- s.e.m. , n = 6). Free [Mg2+]i was estimated at 0.45 +/- 0.04 and 0.68 +/- 0. 03 mM (mean +/- s.e.m., n = 4) in oxygenated and deoxygenated red cells, respectively. 3. K+-Cl- cotransport was minimal in deoxygenated cells but substantial in oxygenated ones. Cl--dependent K+ influx, inhibited by calyculin A, consistent with mediation via the K+-Cl- cotransporter, was revealed by depleting deoxygenated cells of Mg2+. 4. Decreasing [Mg2+]i stimulated K+ influx, and increasing [Mg2+]i inhibited it, in both oxygenated and deoxygenated red cells. When free [Mg2+]i was clamped, Cl--dependent K+ influxes were always greater in oxygenated cells than in deoxygenated ones, and changes in free [Mg2+]i of the magnitude occurring during oxygenation-deoxygenation cycles had a minimal effect. Physiological fluctuations in free [Mg2+]i are unlikely to provide the primary link coupling activity of the K+-Cl- cotransporter with O2 tension. 5. Volume and H+ ion sensitivity of K+ influx in Mg2+-clamped red cells were increased in O2 compared with those in deoxygenated cells at the same free [Mg2+]i, by about 6- and 2-fold, respectively, but again these features were not responsible for the higher fluxes in oxygenated cells. 6. Regulation of the K+-Cl- cotransporter by O2 is very similar in equine, sheep and in normal human (HbA) red cells, but altered in human sickle cells. Present results imply that, as in sheep red cells, O2 dependence of K+-Cl- cotransport in equine red cells is not mediated via changes in free [Mg2+]i and that cotransport in Mg2+-clamped red cells is still stimulated by O2. This behaviour is contrary to that reported for human sickle (HbS) cells. (+info)
Isoforms of the Na-K-2Cl cotransporter in murine TAL I. Molecular characterization and intrarenal localization.
We have identified several alternatively spliced cDNAs encoding mBSC1, an apical bumetanide-sensitive Na+-K+-2Cl- cotransporter from mouse kidney. Two full-length clones were isolated, designated C4 and C9, predicting proteins of 770 and 1,095 amino acids, respectively. The C4 isoforms are generated by utilization of an alternative polyadenylation site located within the intron between exons 16 and 17 of the mBSC1 gene on chromosome 2; the resultant transcripts predict a truncated COOH terminus ending in a unique 55 amino acid sequence. The predicted C4 and C9 COOH termini differ in the distribution of putative phosphorylation sites for both protein kinase A and C. Independent splicing events involve three previously described cassette exons, which are predicted to encode most of the second transmembrane domain. A total of six different isoforms are expressed, generated by the combinatorial association of three cassette exons and two alternative 3' ends. C9-specific and C4-specific antibodies detect proteins of approximately 150 and 120 kDa, respectively, in mouse kidney. Immunofluorescence and immunohistochemistry indicate expression of both COOH-terminal isoforms within the thick ascending limb of the loop of Henle (TAL). However, staining with the C4 antibody is more heterogeneous, with a decreased proportion of positive cells in the cortical TAL. Functional expression in Xenopus oocytes indicates a dominant negative function for C4 isoforms [companion study, C. Plata, D. B. Mount, V. Rubio, S. C. Hebert, and G. Gamba. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999], and the differential expression of these isoforms may contribute to functional heterogeneity of Na+-K+-2Cl- cotransport in mouse TAL. (+info)
Isoforms of the Na-K-2Cl cotransporter in murine TAL II. Functional characterization and activation by cAMP.
The functional properties of alternatively spliced isoforms of the mouse apical Na+-K+-2Cl- cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of 22Na+ or 86Rb+ uptake. A total of six isoforms, generated by the combinatorial association of three 5' exon cassettes (A, B, and F) with two alternative 3' ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999]. The two 3' ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na+-K+-2Cl- cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na+-K+-2Cl- cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na+-K+-2Cl- cotransport in the mouse TAL by hormones that generate cAMP. (+info)
Developmental expression of sodium entry pathways in rat nephron.
During the past several years, sites of expression of ion transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were defined by mapping the expression of sodium transport pathways during nephrogenesis in the rat. Combined in situ hybridization and immunohistochemistry were used to localize the Na-Pi cotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger (NaCa), the epithelial sodium channel (rENaC), and 11beta-hydroxysteroid dehydrogenase (11HSD). The onset of expression of these proteins began in post-S-shape stages. NKCC2 was initially expressed at the macula densa region and later extended into the nascent ascending limb of the loop of Henle (TAL), whereas differentiation of the proximal tubular part of the loop of Henle showed a comparatively retarded onset when probed for NaPi2. The NCC was initially found at the distal end of the nascent distal convoluted tubule (DCT) and later extended toward the junction with the TAL. After a period of changing proportions, subsegmentation of the DCT into a proximal part expressing NCC alone and a distal part expressing NCC together with NaCa was evident. Strong coexpression of rENaC and 11HSD was observed in early nascent connecting tubule (CNT) and collecting ducts and later also in the distal portion of the DCT. Ontogeny of the expression of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data present a detailed analysis of the relations between the anatomic differentiation of the developing renal tubule and the expression of tubular transport proteins. (+info)
Regulation of intracellular chloride by cotransporters in developing lateral superior olive neurons.
The regulatory mechanisms of intracellular Cl- concentration ([Cl-]i) were investigated in the lateral superior olive (LSO) neurons of various developmental stages by taking advantage of gramicidin perforated patch recording mode, which enables neuronal [Cl-]i measurement. Responses to glycine changed from depolarization to hyperpolarization during the second week after birth, resulting from [Cl-]i decrease. Furosemide equally altered the [Cl-]i of both immature and mature LSO neurons, indicating substantial contributions of furosemide-sensitive intracellular Cl- regulators; i.e., K+-Cl- cotransporter (KCC) and Na+-K+-Cl- cotransporter (NKCC), throughout this early development. Increase of extracellular K+ concentration and replacement of intracellular K+ with Cs+ resulted in [Cl-]i elevation at postnatal days 13-15 (P13-P15), but not at P0-P2, indicating that the mechanism of neuronal Cl- extrusion is sensitive to both furosemide and K+-gradient and poorly developed in immature LSO neurons. In addition, removal of extracellular Na+ decreased [Cl-]i at P0-P2, suggesting the existence of extracellular Na+-dependent and furosemide-sensitive Cl- accumulation in immature LSO neurons. These data show clearly that developmental changes of Cl- cotransporters alter [Cl-]i and are responsible for the switch from the neonatal Cl- efflux to the mature Cl- influx in LSO neurons. Such maturational changes in Cl- cotransporters might have the important functional roles for glycinergic and GABAergic synaptic transmission and the broader implications for LSO and auditory development. (+info)
Na+-K+-2Cl- cotransporter in immature cortical neurons: A role in intracellular Cl- regulation.
Na+-K+-2Cl- cotransporter has been suggested to contribute to active intracellular Cl- accumulation in neurons at both early developmental and adult stages. In this report, we extensively characterized the Na+-K+-2Cl- cotransporter in primary culture of cortical neurons that were dissected from cerebral cortex of rat fetus at embryonic day 17. The Na+-K+-2Cl- cotransporter was expressed abundantly in soma and dendritic processes of cortical neurons evaluated by immunocytochemical staining. Western blot analysis revealed that an approximately 145-kDa cotransporter protein was present in cerebral cortex at the early postnatal (P0-P9) and adult stages. There was a time-dependent upregulation of the cotransporter activity in cortical neurons during the early postnatal development. A substantial level of bumetanide-sensitive K+ influx was detected in neurons cultured for 4-8 days in vitro (DIV 4-8). The cotransporter activity was increased significantly at DIV 12 and maintained at a steady level throughout DIV 12-14. Bumetanide-sensitive K+ influx was abolished completely in the absence of either extracellular Na+ or Cl-. Opening of gamma-aminobutyric acid (GABA)-activated Cl- channel or depletion of intracellular Cl- significantly stimulated the cotransporter activity. Moreover, the cotransporter activity was elevated significantly by activation of N-methyl-D-aspartate ionotropic glutamate receptor via a Ca2+-dependent mechanism. These results imply that the inwardly directed Na+-K+-2Cl- cotransporter is important in active accumulation of intracellular Cl- and may be responsible for GABA-mediated excitatory effect in immature cortical neurons. (+info)
Regulation of thick ascending limb ion transporter abundance in response to altered acid/base intake.
Changes in ammonium excretion with acid/base perturbations are dependent on changes in medullary ammonium accumulation mediated by active NH4+ absorption by the medullary thick ascending limb. To investigate whether alterations in the abundance of medullary thick ascending limb ion transporters, namely the apical Na+/K+(NH4+)/2Cl- -cotransporter (BSC-1), the apical Na+/H+ -exchanger (NHE3), and the Na+/K+ -ATPase alpha1-subunit, may be responsible in part for altered medullary ammonium accumulation, semiquantitative immunoblotting studies were performed using homogenates from the inner stripe of the rat renal outer medulla. After 7 d of NH4Cl (7.2 mmol/220 g body wt per d) loading (associated with increased medullary ammonium accumulation), neither BSC-1 nor Na+/K+ -ATPase protein expression was altered, but NHE3 protein abundance was significantly increased. On the other hand, both BSC-1 and Na+/K+ -ATPase protein abundance was increased significantly in rats fed NaHCO3 (7.2 mmol/220 g body wt per d) for 7 d. Rats fed a high-NaCl diet (7.7 mEq Na+/220 g body wt per d) for 5 d also showed marked increases in both BSC-1 and Na+/K+ -ATPase expression. The expression level of NHE3 protein did not change with either NaHCO3 or high NaCl intake. None of these three transporters showed a significant difference in abundance between the groups fed equimolar (7.2 mmol/220 g body wt per d for 7 d) NaHCO3 or NaCl. It is concluded that outer medullary BSC-1 and Na+/K+ -ATPase alpha1-subunit protein abundance is increased by chronic Na+ loading but not by acid/base perturbations and that outer medullary NHE3 protein abundance is increased by chronic NH4Cl loading. (+info)
Physiological significance of volume-regulatory transporters.
Research over the past 25 years has identified specific ion transporters and channels that are activated by acute changes in cell volume and that serve to restore steady-state volume. The mechanism by which cells sense changes in cell volume and activate the appropriate transporters remains a mystery, but recent studies are providing important clues. A curious aspect of volume regulation in mammalian cells is that it is often absent or incomplete in anisosmotic media, whereas complete volume regulation is observed with isosmotic shrinkage and swelling. The basis for this may lie in an important role of intracellular Cl- in controlling volume-regulatory transporters. This is physiologically relevant, since the principal threat to cell volume in vivo is not changes in extracellular osmolarity but rather changes in the cellular content of osmotically active molecules. Volume-regulatory transporters are also closely linked to cell growth and metabolism, producing requisite changes in cell volume that may also signal subsequent growth and metabolic events. Thus, despite the relatively constant osmolarity in mammals, volume-regulatory transporters have important roles in mammalian physiology. (+info)