Peter W. Flatman

Regulation of Na-K-2Cl cotransport by phosphorylation and protein-protein interactions.

The Na-K-2Cl cotransporter plays important roles in cell ion homeostasis and volume control and is particularly important in mediating the movement of ions and thus water across epithelia. In addition to being affected by the concentration of the transported ions, cotransport is affected by cell volume, hormones, growth factors, oxygen tension, and intracellular ionized Mg(2+) concentration. These probably influence transport through three main routes acting in parallel: cotransporter phosphorylation, protein-protein interactions and cell Cl(-) concentration. Many effects are mediated, at least in part, by changes in protein phosphorylation, and are disrupted by kinase and phosphatase inhibitors, and manoeuvres that reduce cell ATP content. In some cases, phosphorylation of the cotransporter itself on serine and threonine (but not tyrosine) is associated with changes in transport rate, in others, phosphorylation of associated proteins has more influence. Analysis of the stimulation of cotransport by calyculin A, arsenite and deoxygenation suggests that the cotransporter is phosphorylated by several kinases and dephosphorylated by several phosphatases. These kinases and phosphatases may themselves be regulated by phosphorylation of residues including tyrosine, with Src kinases possibly playing an important role. Protein-protein interactions also influence cotransport activity. Cotransporter molecules bind to each other to form high molecular weight complexes, they also bind to other members of the cation-chloride cotransport family, to a variety of cytoskeletal proteins, and to enzymes that are part of regulatory cascades. Many of these interactions affect transport and may override the effects of cotransporter phosphorylation. Cell Cl(-) may also directly affect the way the cotransporter functions independently of its role as substrate.

The magnesium dependence of sodium‐pump‐mediated sodium—potassium and sodium—sodium exchange in intact human red cells

1. The magnesium content of human red blood cells was controlled by varying the magnesium concentration in the medium in the presence of the ionophore A23187. The new magnesium levels attained were very stable, which allowed the magnesium dependence of the sodium pump to be investigated.

Cotransporters, WNKs and hypertension: important leads from the study of monogenetic disorders of blood pressure regulation.

Major advances are being made in identifying the structure and behaviour of regulatory cascades that control the activity of cation-Cl(-) cotransporters and certain Na(+), K(+) and Cl(-) channels. These transporters play key roles in regulating arterial blood pressure as they are not only responsible for NaCl reabsorption in the thick ascending limb and distal tubule of the kidney, but are also involved in regulating smooth muscle Ca(2+) levels. It is now apparent that defects in these transporters, and particularly in the regulatory cascades, cause some monogenetic forms of hypertension and may contribute to essential hypertension and problems with K(+) homoeostasis. Two families of kinases are prominent in these processes: the Ste-20-related kinases [OSR1 (oxidative stress-responsive kinase 1) and SPAK (Ste20/SPS1-related proline/alanine-rich kinase)] and the WNKs [with no lysine kinases]. These kinases affect the behaviour of their targets through both phosphorylation and by acting as scaffolding proteins, bringing together regulatory complexes. This review analyses how these kinases affect transport by activating or inhibiting individual transporters at the cell surface, or by changing the surface density of transporters by altering the rate of insertion or removal of transporters from the cell surface, and perhaps through controlling the rate of transporter degradation. This new knowledge should not only help us target antihypertensive therapy more appropriately, but could also provide the basis for developing new therapeutic approaches to essential hypertension.

Regulation of Na+-K+-2Cl− cotransport by protein phosphorylation in ferret erythrocytes

1 Na+‐K+‐2Cl− cotransport in ferret erythrocytes was measured as the bumetanide‐sensitive uptake of 86Rb. 2 The resting cotransport rate was high but could be increased threefold by treating erythrocytes with calyculin A, a potent inhibitor of serine/threonine phosphatases. Twenty nanomolar was sufficient to maximally and rapidly (within 4 min) stimulate transport. 3 The effects of several kinase inhibitors were tested. High concentrations of K‐252a, K‐252b, calphostin C and hypericin caused less than 20 % inhibition. Staurosporine (IC50, 0.06 μm) and 4‐amino‐5‐(4‐methylphenyl)‐7‐(t‐butyl)pyrazolo[3,4‐d]pyrimidine (PP1; IC50, 2.5 μm) were more potent but still only partially (40–50 %) inhibited transport, an effect mimicked by reducing ionized intracellular Mg2+ concentration to submicromolar levels. Genistein may inhibit all transport at a sufficiently high dose (IC50, 0.36 mM) perhaps by directly inhibiting the transporter. 4 Staurosporine, PP1 and the removal of Mg2+ all prevented subsequent stimulation by calyculin A, and all inhibited calyculin‐stimulated transport by 20–30 %. The effects of staurosporine, PP1 and Mg2+ removal were not additive. 5 The phosphatase that dephosphorylates the cotransporter is probably Mg2+ (or possibly Ca2+ or Mn2+) sensitive and not the target for calyculin A. The data suggest that this phosphatase is inhibited by phosphorylation, and that it is the regulation of this process which is affected by calyculin A and the kinase inhibitors tested here. Phosphorylation of the phosphatase is probably regulated by members of the Src family of tyrosine kinases.

Changes in protein expression in the rat medial vestibular nuclei during vestibular compensation.

The molecular mechanisms of neural and synaptic plasticity in the vestibular nuclei during ‘vestibular compensation’, the behavioural recovery that follows deafferentation of one inner ear, are largely unknown. In this study we have used differential proteomics techniques to determine changes in protein expression in ipsi‐lesional and contra‐lesional medial vestibular nuclei (MVN) of rats, 1 week after either sham surgery or unilateral labyrinthectomy (UL). A systematic comparison of 634 protein spots in two‐dimensional electrophoresis gels across five experimental conditions revealed 54 spots, containing 26 proteins whose level was significantly altered 1 week post‐UL. The axon‐guidance‐associated proteins neuropilin‐2 and dehydropyriminidase‐related protein‐2 were upregulated in the MVN after UL. Changes in levels of further specific proteins indicate a coordinated upregulation of mitochondrial function, ATP biosynthesis and phosphate metabolism in the vestibular nuclei 1 week post‐UL. These may reflect the metabolic energy demands of processes such as gliosis, neuronal outgrowth and synaptic remodelling that occur after UL. Our findings suggest novel roles for axon elaboration and guidance molecules, as well as mitochondrial and metabolic regulatory proteins, in the post‐lesional physiology of the MVN during vestibular system plasticity.

Stimulation of Na+-K+-2Cl− cotransport by arsenite in ferret erythrocytes

1 Na+‐K+‐2Cl− cotransport activity was measured in ferret erythrocytes as the bumetanide‐sensitive uptake of 86Rb. 2 The Na+‐K+‐2Cl− cotransport rate was stimulated by treating erythrocytes with sodium arsenite but not by sodium arsenate (up to 1 mM). Stimulation took an hour to develop fully. Arsenite had no effect on bumetanide‐resistant 86Rb uptake. 3 In cells stored for 3 days or less, cotransport stimulation by arsenite could be described by assuming arsenite either acts at a single site (EC50, 60 ± 14 μM, mean ± s.e.m., n= 3) or that it acts at both high‐ (EC50, 35 ± 9 μM, mean ± s.e.m., n= 3) and low‐ (EC50 > 2 mM) affinity sites. 4 Stimulation by 1 mM arsenite was greatest on the day of cell collection (rate about 3 times that of the control), even exceeding that produced by 20 nM calyculin A, and declined during cell storage. Addition of calyculin A to arsenite‐stimulated cells resulted in further stimulation of Na+‐K+‐2Cl− cotransport, suggesting that arsenite and calyculin act synergistically. This was most apparent in stored cells. 5 Stimulation by 1 mM arsenite was not affected by treating cells with the mitogen‐activated protein kinase inhibitors SB203580 (20 μM) and PD98059 (50 μM), but was both prevented and reversed by the kinase inhibitors staurosporine (2 μM), 4‐amino‐5‐(4‐methylphenyl)‐7‐(t‐butyl)pyrazolo[3,4‐d]pyrimidine (PP1, 50 μM) and genistein (0.3 mM), and with a combination of 10 μM A23187 and 2 mM EDTA (to reduce intracellular Mg2+ concentration). Only treatment with EDTA and A23187 prevented stimulation by the combination of 1 mM arsenite and 20 nM calyculin, whereas no treatment was able to fully reverse this stimulation once elicited. 6 Our data are consistent with arsenite stimulating (perhaps indirectly) a kinase that phosphorylates and activates the Na+‐K+‐2Cl− cotransporter.

Hypertrophy in the Distal Convoluted Tubule of an 11β-Hydroxysteroid Dehydrogenase Type 2 Knockout Model

Na(+) transport in the renal distal convoluted tubule (DCT) by the thiazide-sensitive NaCl cotransporter (NCC) is a major determinant of total body Na(+) and BP. NCC-mediated transport is stimulated by aldosterone, the dominant regulator of chronic Na(+) homeostasis, but the mechanism is controversial. Transport may also be affected by epithelial remodeling, which occurs in the DCT in response to chronic perturbations in electrolyte homeostasis. Hsd11b2(-/-) mice, which lack the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) and thus exhibit the syndrome of apparent mineralocorticoid excess, provided an ideal model in which to investigate the potential for DCT hypertrophy to contribute to Na(+) retention in a hypertensive condition. The DCTs of Hsd11b2(-/-) mice exhibited hypertrophy and hyperplasia and the kidneys expressed higher levels of total and phosphorylated NCC compared with those of wild-type mice. However, the striking structural and molecular phenotypes were not associated with an increase in the natriuretic effect of thiazide. In wild-type mice, Hsd11b2 mRNA was detected in some tubule segments expressing Slc12a3, but 11βHSD2 and NCC did not colocalize at the protein level. Thus, the phosphorylation status of NCC may not necessarily equate to its activity in vivo, and the structural remodeling of the DCT in the knockout mouse may not be a direct consequence of aberrant corticosteroid signaling in DCT cells. These observations suggest that the conventional concept of mineralocorticoid signaling in the DCT should be revised to recognize the complexity of NCC regulation by corticosteroids.

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

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 PO2 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.

Blood pressure and sodium: Association with MRI markers in cerebral small vessel disease:

Dietary salt intake and hypertension are associated with increased risk of cardiovascular disease including stroke. We aimed to explore the influence of these factors, together with plasma sodium concentration, in cerebral small vessel disease (SVD). In all, 264 patients with nondisabling cortical or lacunar stroke were recruited. Patients were questioned about their salt intake and plasma sodium concentration was measured; brain tissue volume and white-matter hyperintensity (WMH) load were measured using structural magnetic resonance imaging (MRI) while diffusion tensor MRI and dynamic contrast-enhanced MRI were acquired to assess underlying tissue integrity. An index of added salt intake (P = 0.021), pulse pressure (P = 0.036), and diagnosis of hypertension (P = 0.0093) were positively associated with increased WMH, while plasma sodium concentration was associated with brain volume (P = 0.019) but not with WMH volume. These results are consistent with previous findings that raised blood pressure is associated with WMH burden and raise the possibility of an independent role for dietary salt in the development of cerebral SVD.

Glucocorticoids Induce Nondipping Blood Pressure by Activating the Thiazide-Sensitive Cotransporter

Blood pressure (BP) normally dips during sleep, and nondipping increases cardiovascular risk. Hydrochlorothiazide restores the dipping BP profile in nondipping patients, suggesting that the NaCl cotransporter, NCC, is an important determinant of daily BP variation. NCC activity in cells is regulated by the circadian transcription factor per1. In vivo, circadian genes are entrained via the hypothalamic–pituitary–adrenal axis. Here, we test whether abnormalities in the day:night variation of circulating glucocorticoid influence NCC activity and BP control. C57BL6/J mice were culled at the peak (1:00 AM) and trough (1:00 PM) of BP. We found no day:night variation in NCC mRNA or protein but NCC phosphorylation on threonine53 (pNCC), required for NCC activation, was higher when mice were awake, as was excretion of NCC in urinary exosomes. Peak NCC activity correlated with peak expression of per2 and bmal1 (clock genes) and sgk1 and tsc22d3 (glucocorticoid-responsive kinases). Adrenalectomy reduced NCC abundance and blunted the daily variation in pNCC levels without affecting variation in clock gene transcription. Chronic corticosterone infusion increased bmal1, per1, sgk1, and tsc22d3 expression during the inactive phase. Inactive phase pNCC was also elevated by corticosterone, and a nondipping BP profile was induced. Hydrochlorothiazide restored rhythmicity of BP in corticosterone-treated mice without affecting BP in controls. Glucocorticoids influence the day:night variation in NCC activity via kinases that control phosphorylation. Abnormal glucocorticoid rhythms impair NCC and induce nondipping. Night-time dosing of thiazides may be particularly beneficial in patients with modest glucocorticoid excess.Blood pressure (BP) normally dips during sleep, and nondipping increases cardiovascular risk. Hydrochlorothiazide restores the dipping BP profile in nondipping patients, suggesting that the NaCl cotransporter, NCC, is an important determinant of daily BP variation. NCC activity in cells is regulated by the circadian transcription factor per1. In vivo, circadian genes are entrained via the hypothalamic–pituitary–adrenal axis. Here, we test whether abnormalities in the day:night variation of circulating glucocorticoid influence NCC activity and BP control. C57BL6/J mice were culled at the peak (1:00 AM) and trough (1:00 PM) of BP. We found no day:night variation in NCC mRNA or protein but NCC phosphorylation on threonine53 (pNCC), required for NCC activation, was higher when mice were awake, as was excretion of NCC in urinary exosomes. Peak NCC activity correlated with peak expression of per2 and bmal1 (clock genes) and sgk1 and tsc22d3 (glucocorticoid-responsive kinases). Adrenalectomy reduced NCC abundance and blunted the daily variation in pNCC levels without affecting variation in clock gene transcription. Chronic corticosterone infusion increased bmal1, per1, sgk1, and tsc22d3 expression during the inactive phase. Inactive phase pNCC was also elevated by corticosterone, and a nondipping BP profile was induced. Hydrochlorothiazide restored rhythmicity of BP in corticosterone-treated mice without affecting BP in controls. Glucocorticoids influence the day:night variation in NCC activity via kinases that control phosphorylation. Abnormal glucocorticoid rhythms impair NCC and induce nondipping. Night-time dosing of thiazides may be particularly beneficial in patients with modest glucocorticoid excess.