Activation of ferret erythrocyte Na⁺–K⁺–2Cl⁻ cotransport by deoxygenation

Abstract

Deoxygenation of ferret erythrocytes stimulates Na+ -K + -2Cl − cotransport by 111% (S.D., 46) compared to controls in air. Half-maximal activation occurs at a P O 2 of 24 mmHg (S.D., 2) indicating that physiological changes in oxygen tension can influence cotransport function. Approximately 25-35% of this stimulation can be attributed to the rise of intracellular free magnesium concentration that occurs on deoxygenation (from 0.82 (S.D., 0.07) to 1.40 mM (S.D., 0.17)). Most of the stimulation is probably caused by activation of a kinase which can be prevented or reversed by treating cells with the kinase inhibitors PP1 or staurosporine, or by reducing cell magnesium content to submicromolar levels. Stimulation by deoxygenation is comparable with that caused by calyculin A or sodium arsenite, compounds that cause a 2-to 3-fold increase in threonine phosphorylation of the cotransporter which can be detected with phospho-specific antibodies. However, the same approach failed to detect significant changes in threonine phosphorylation following deoxygenation. The results suggest that deoxygenation causes activation of a kinase that either phosphorylates the transporter, but probably not on threonine, or phosphorylates another protein that in turn influences cotransporter behaviour. They also indicate that more than one kinase and phosphatase are involved in cotransporter phosphorylation.

The Chloride Transporter Na+-K+-Cl−Cotransporter Isoform-1 Contributes to Intracellular Chloride Increases afterIn VitroIschemia

“…Because NKCC-1 activity is thought to be increased by phosphorylation , it is likely that NKCC-1 phosphorylation during OGD increases [Cl Ϫ ] i . Consistent with this conclusion, Flatman (2005) reported that deoxygenation doubles NKCC-1 activity in ferret erythrocytes. This effect was blocked by a protein kinase inhibitor, indicating that it results from activation of protein kinases.…”

Characterization of the deoxyhemoglobin binding site on human erythrocyte band 3: implications for O2 regulation of erythrocyte properties

“…A major candidate for the O 2 sensor was presumed to be Hb, because the O 2 dependence of the above properties generally follows the O 2 dissociation curve of Hb. 4,12,13 The downstream effector has been more uncertain, but band 3 has represented the prime candidate, because it constitutes the only established binding site of Hb on the membrane. Moreover, multiple observations document that the interaction between Hb and band 3 is O 2 -dependent, where association has been found to occur primarily, if not exclusively, with deoxygenated Hb.…”

Oxygen-sensitive regulatory volume increase and Na transport in red blood cells from the cane toad,Bufo marinus

“…In rainbow trout, the effect of oxygen on transporter activity seems to be mediated through hydroxyl radicals, which stimulate KCC activity, whilst they inhibit the ␤NHE at high P O 2 (Bogdanova and Nikinmaa, 2001;Nikinmaa et al, 2003). The oxygen dependency of NKCC activity in RBCs of some tetrapods such as turkey and ferret and in crucian carp, however, is consistent with modulation by the Hb oxygenation state (Muzyamba et al, 1999;Flatman, 2005;Berenbrink et al, 2006). Shrinkage of B. marinus RBCs was attended by a pronounced amiloride-sensitive RVI response (Fig.·3C, Fig.·4C) that was much faster under deoxygenated conditions (Fig.·3B), reflecting the time course of activation and the magnitude of the Na influxes under the two levels of P O 2 (Fig.·1).…”

“…P O 2 dependency of shrinkage-induced Na influx and RVI Oxygenation-dependent RBC membrane transporters have now been described in nearly all vertebrate classes, ranging from lamprey to mammals (Motais et al, 1987;Nielsen et al, 1992;Gibson et al, 1998;Virkki et al, 1998;Muzyamba et al, 1999;Berenbrink et al, 2000;Berenbrink et al, 2006;Flatman, 2005) (reviewed by Gibson et al, 2000). This also applies to the transporters involved in volume regulation (Jensen, 1995;Nielsen, 1997;Gibson et al, 2000;Brauner et al, 2002;Drew et al, 2004).…”

“…The regulatory volume increase (RVI) in RBCs from mammals, most teleosts and amphibians is mediated by increased NHE activity, resulting in a net gain of inorganic osmolytes followed by osmotically obliged water (Cala, 1980;Parker et al, 1991;Orlov et al, 1994;Romero et al, 1996;Gusev et al, 1997;Weaver et al, 1999) whereas bird RBCs activate the NKCC (Kregenow, 1981;Muzyamba et al, 1999). In addition to shrinkage, the activity of the NHE and NKCC and other RBC ion transporters is modulated by oxygen, catecholamines or acidification [NHE (Motais et al, 1987;Salama and Nikinmaa, 1988;Nielsen, 1997;Virkki et al, 1998) and NKCC Muzyamba et al, 1999;Flatman, 2005;Berenbrink et al, 2006)]. Thus, in various vertebrates, the NHE, NKCC and/or RVI response are inhibited in airequilibrated cells compared with cells at low oxygen partial pressure (P O 2) (Gibson et al, 2000;Brauner et al, 2002;Jensen et al, 2002;Drew et al, 2004).…”