Chloride modulation of insulin release, 86Rb+ efflux, and 45Ca2+ fluxes in rat islets stimulated by various secretagogues

Substitution of extracellular Cl- by impermeant isethionate (5 mM residual Cl-) caused a monophasic inhibition of glucose-stimulated insulin release, accompanied by an initial transient increase and a secondary lasting decrease in 86Rb+ efflux from perifused islets. Cl- reintroduction restored insulin release with an overshoot above control values and successively produced a small decrease and a large increase in efflux. Theophylline potentiated the insulinotropic effect of glucose more markedly at low Cl- than at normal Cl-, but did not restore a normal rate of 86Rb+ efflux. Lowering the concentration of Cl- did not alter the effect of glucose, tolbutamide, or arginine on 86Rb+ efflux, but simply shifted the efflux rates to lower values. The first phase of glucose-stimulated insulin release was not modified, but the second phase was inhibited. The insulinotropic effect of tolbutamide was augmented at low Cl- and that of arginine (at 7 mM glucose) was not affected. In incubated islets, the stimulation of insulin release by glyceraldehyde was barely inhibited when Cl- was substituted by isethionate and the marked decrease of the effect of glucose could be prevented by glutamine. In a glucose-free, low Cl- medium, the insulinotropic effect of leucine, arginine, and lysine was inhibited; this inhibition was reversed by glutamine, but not by theophylline. Lowering the concentration of Cl- had no effect on 45Ca2+ influx or efflux in the absence of glucose, did not alter the increase in influx and efflux during the first 5 min of glucose stimulation, but impaired both influx and efflux during the second phase. Leucine-induced 45Ca2+ uptake was inhibited at low Cl- and this inhibition was prevented by glutamine. In conclusion, islet cells possess a Cl- -activated modality of K efflux, which does not seem to play a role in the stimulus-secretion coupling. Since Cl- substitution by an impermeant anion does not inhibit the stimulation of insulin release by all agents, the role of Cl- ions does not appear to be restricted to a chemiosmotic mechanism of exocytosis. No single mechanism explains the multiple changes in B-cell function resulting from the decrease in Cl- concentration, but it is proposed that some of them could result from modifications of intracellular pH.     

Resistance of mTAL Na+-dependent transporters and collecting duct aquaporins to dehydration in 7-month-old rats

BACKGROUND: Aging is associated with a defect in urinary concentration in both human and experimental animals. The purpose of these studies was to examine the urinary concentrating ability, the expression of kidney water channels [aquaporins (AQP1 to AQP3)], and medullary thick ascending limb (mTAL) Na+-dependent transporters in old but not senescent versus young animals in response to water deprivation. METHODS: Two-month-old and 7-month-old rats were placed in metabolic cages and deprived of water for 72 hours. Kidney tissues were isolated and examined for the expression of AQP1 to AQP3 and mTAL, peptide-derived polyclonal antibody specific to kidney apical Na+-K+-2 Cl- cotransporter (BSC1), Na+/H+ exchanger isoform 3 (NHE3), and Na+ pump using semiquantitative immunoblotting and Northern hybridization. RESULTS: After 72 hours of water deprivation, urine osmolality increased from 1269 to 3830 mOsm/kg H2O in 2-month-old rats, but only from 1027 to 2588 mOsm/kg H2O in 7-month-old rats. In response to water deprivation, AQP2 and AQP3 expression increased significantly in the cortex and medulla of 2-month-old rats but remained unchanged in the medulla or slightly increase in the cortex of 7-month-old animals. AQP1 expression was not altered by dehydration in both groups. The protein abundance of mTAL BSC1, NHE3, and Na+ pump increased significantly in young but remained unchanged in 7-month-old rats subjected to water deprivation. CONCLUSION: Age-related decrease in urinary concentrating ability is an early event, developed before the onset of senescence. This defect results from reduced responsiveness of cortical AQP2 and AQP3 and a blunted response of medullary AQP2 and mTAL BSC1, NHE3, and Na+ pump to dehydration in aging kidneys.     

Expression of the K+ channel Kir7.1 in the developing rat kidney: role in K+ excretion

BACKGROUND: Coordinated expression of ROMK (luminal K+ channel in the thick ascending limb and the collecting duct) and Na+,K+-ATPase has been demonstrated to be involved in the postnatal development of renal K+ excretion; however, the developmental expression of the basolateral K+ channel Kir7.1 is unknown. The purpose of this study was to elucidate the possible involvement of Kir7.1 in the maturation of renal K+ excretion. METHODS: Developmental changes in the renal K+ excretion under the condition of K+ overload was investigated by collecting urine from neonatal rats infused with K+ (KCl solution). RNase protection analysis was used to elucidate the expression of Kir7.1 and Na+,K+-ATPase mRNA in pre- and postnatal rats, and the expression of Kir7.1 and ROMK mRNA at 7, 14, and 21 days. Western blotting of Kir7.1, and immunohistochemistry of Kir7.1 and ROMK were used to determine their protein expression. RESULTS: The ratio of urinary K+ excretion to K+ load increased between 7 and 14 days after birth. In addition, half excretion time of K+ load gradually decreased through the experimental period of 7 and 21 days. Na+,K+-ATPase mRNA levels showed a peak of up-regulation at birth that remained elevated. ROMK1 mRNA levels significantly increased between 7 and 14 days. On the other hand, Kir7.1 mRNA and protein levels significantly increased between 14 and 21 days. Kir7.1 protein in the thick ascending limb was first recognized at 7 days, whereas its expression in the distal convoluted tubule and the cortical collecting duct was found in 21-day-old neonates. CONCLUSION: Our results suggest that Kir7.1 is involved in the development of renal K+ excretion between 14 and 21 days after birth under the condition of K+ overload.     

Mechanisms of PKC-dependent Na+ K+ ATPase phosphorylation in the rat kidney with chronic renal failure

The present work was designed to study Na+ K+ ATPase alpha1-subunit phosphorylation in rats with chronic renal failure (CRF) in comparison with normal rats. Na+ K+ ATPase alpha1-subunit phosphorylation degree was measured by binding the McK-1 antibody to dephosphorylated Ser-23 in microdissected medullary thick ascending limb of Henle (mTAL) segments. In addition, the total Na+ K+ ATPase alpha1-subunit expression and activity were also measured in the outer renal medulla homogenates and membranes. CRF rats showed a higher Na+ K+ ATPase activity, as compared with control rats (18.95 +/- 2.4 vs. 11.21 +/- 1.5 micromol Pi/mg prot/h, p < 0.05), accompanied by a higher total Na+ K+ ATPase expression (0.54 +/- 0.04 vs. 0.27 +/- 0.02 normalized arbitrary units (NU), p < 0.05). When McK-1 antibody was used, a higher immunosignal in mTAL of CRF rats was observed, as compared with controls (6.3 +/- 0.35 vs. 4.1 +/- 0.33 NU, p < 0.05). The ratio Na+ K+ ATPase alpha1-subunit phosphorylation/total Na+ K+ ATPase alpha1-subunit expression per microg protein showed a non-significant difference between CRF and control rats in microdissected mTAL segments (2.11 +/- 0.12 vs. 2.26 +/- 0.18 NU, p = NS). The PKC inhibitor RO-318220 10(-6) M increased immunosignal (lower phosphorylation degree) in mTAL of CRF rats to 128.43 +/- 7.08% (p < 0.05) but did not alter McK1 binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10(-6) M and dopamine 10(-6) M decreased immunosignal in CRF rats, corresponding to a higher Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23 (55.26 +/- 11.17% and 53.27 +/- 7.12% compared with basal, p < 0.05). In mTAL of CRF rats, the calcineurin inhibitor FK-506 10(-6) M did not modify phosphorylation degree at Ser-23 of Na+ K+ ATPase alpha1-subunit (100.21 +/- 3.00% compared with basal CRF). In control rats, FK 506 10(-6) M decreased the immunosignal, which corresponds to a higher Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.     

Diabetes decreases Na+-K+ pump concentration in skeletal muscles, heart ventricular muscle, and peripheral nerves of rat

Na+-K+-ATPase or the Na+-K+ pump is essential for some specific properties of muscle and nerve tissue such as contractility and excitability. Previous studies have shown conflicting variations in Na+-K+-ATPase activity or Na+-K+ pump concentration of muscle cells in experimental diabetes. Our study demonstrates that early untreated diabetes in rats induced by injection of streptozocin is associated with decreases in [3H]ouabain binding-site concentration of 24-48% in various skeletal muscles and 16% in peripheral nerves as well as a decrease in K+-dependent 3-O-methylfluorescein phosphatase activity of 21% in the heart ventricle. These effects could be prevented by insulin treatment. They probably represent a decrease in the concentration of Na+-K+ pumps. There was no evidence for more than one population of Na+-K+ pumps in intact samples of skeletal muscle and nerves from normal, diabetic, and insulin-treated animals. The decrease in Na+-K+ pump concentration in nerve cells may be due to atrophy of the axons. In skeletal muscles, myocardium, and peripheral nerves, the observed decrease in Na+-K+ pump concentration may be important for the pathophysiology of diabetes. We emphasize that quantification of Na+-K+-ATPase or the Na+-K+ pump in muscle and nerve tissue from diabetic animals should preferably be performed with either intact samples or crude homogenates of whole tissue.     

Parathyroid hormone regulation of NA+,K+-ATPase requires the PDZ 1 domain of sodium hydrogen exchanger regulatory factor-1 in opossum kidney cells

It was demonstrated that expression of murine sodium hydrogen exchanger regulatory factor (NHERF-1) lacking the ezrin-binding domain blocks parathyroid hormone (PTH) regulation of Na+,K+-ATPase in opossum kidney (OK) cells. The hypothesis that the NHERF-1 PDZ domains contribute to PTH regulation of Na+,K+-ATPase was tested by comparison of PTH regulation of Na+,K+-ATPase in wild-type OK (OK-WT) cells, NHERF-deficient OKH cells, OK-WT transfected with siRNA for NHERF (NHERF siRNA OK-WT), and OKH cells that were stably transfected with full-length NHERF-1 or constructs with mutated PDZ domains. OKH cells and NHERF siRNA OK-WT showed decreased expression of NHERF-1 but equivalent expression of ezrin and Na+,K+-ATPase alpha1 subunit when compared with OK-WT cells. PTH decreased Na+,K+-ATPase activity and stimulated phosphorylation of the Na+,K+-ATPase alpha1 in OK-WT cells but not in NHERF-deficient cells. Rubidium (86Rb) uptake was equivalent in OK-WT, OKH, and OKH cells that were transfected with all but the double PDZ domain mutants. PTH decreased 86Rb uptake significantly in OK-WT but not in OKH cells. PTH also significantly inhibited 86Rb uptake in OKH cells that were transfected with full-length NHERF-1 or NHERF-1 with mutated PDZ 2 but not in OKH cells that were transfected with mutated PDZ 1. Transfection with NHERF expressing both mutated PDZ domains resulted in diminished basal 86Rb uptake that was not inhibited further by PTH. PTH stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation in OK-WT but not in NHERF-deficient cells. Transfection of OKH cells with NHERF constructs that contained an intact PDZ1 domain restored PTH-stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation. These results demonstrate that NHERF-1 is necessary for PTH-mediated inhibition of Na+,K+-ATPase activity and that the inhibition is mediated through the PDZ1, not PDZ2, domain.     

Cell volume and ionic transport systems after cold preservation of coronary endothelial cells

BACKGROUND: Hypothermia-induced changes in cell volume and ionic transport systems of coronary endothelial cells may play a role in the development of coronary artery disease in cardiac transplant recipients. METHODS: Coronary endothelial cells were incubated in University of Wisconsin solution or culture control medium for up to 48 hours at 4 degrees C. Parallel control cultures were incubated at 37 degrees C. Na/K-ATPase and Na/K/Cl cotransport activities were determined as ouabain- and furosemide-sensitive 86Rb+ uptake, respectively. Cell volume changes and cell death were analyzed by a FACScan flow cytometer and the release of lactate dehydrogenase, respectively. RESULTS: Coronary endothelial cells stored in University of Wisconsin solution up to 6 hours showed an increased Na/K-ATPase activity compared to control cells, whereas no changes were observed in Na/K/Cl cotransport activity or cell volume. Long-term preservation (24 and 48 hours) was associated with a partial loss of cell viability, as demonstrated by lactate dehydrogenase release, and dramatic alterations in ionic transport system activities. CONCLUSIONS: University of Wisconsin solution seems to prevent coronary endothelial cells Na/K/Cl cotransport activity changes during cold preservation, which could alter cell volume regulation and cause cell injury.     

The action of pinacidil in the isolated human bladder

The need for effective symptomatic treatment of patients with detrusor hyperactivity is widely recognized. In search of new principles of decreasing bladder contraction we have studied the effects of pinacidil on the isolated human bladder. Pinacidil is a recently developed antihypertensive agent classified as a K+ channel opener, and is believed to depress smooth muscle activity by this action. Pinacidil concentration-dependently depressed contractions elicited by carbachol, low concentrations of K+ (less than 60 mM) and electrical stimulation. In addition it caused a concentration-related increase in the efflux of 86Rb from preloaded detrusor cells. The effects on 86Rb efflux could be inhibited by tetraethylammonium chloride and procaine, but not by apamin, agents known to block K+-channels. The results support the view that part of the pinacidil effect on the human bladder is caused by an opening of K+-channels, efflux of K+ and subsequent hyperpolarization of the detrusor cells. Clinical testing of this new therapeutic principle for treatment of bladder hyperactivity seems justified.     

Biphasic modulation of K+ permeability in pancreatic islets during acute stimulation with glucose

The regulation of 86Rb+ efflux (marker of K+ permeability) during acute secretagogic stimulation with glucose was studied with cultured as well as freshly isolated pancreatic islets from rats and freshly isolated islets from mice. A perifusion system with minimal dead-space and "flow-through" characteristics conductive to abrupt, steep increases in ambient glucose was combined with multiple samplings of effluent to achieve high temporal resolution. Under these conditions, acute increases in perifusate glucose concentration from 4 to 16.7 mM or from 1 to 27.8 mM effected a biphasic change of the 86Rb+ fractional efflux rate. A rapid reduction of 86Rb+ efflux was interrupted by an evanescent increase in 86Rb+ outflow, which appeared to be temporally coincident with the initiation of the first phase of stimulated insulin release. It is suggested that the glucose-induced biphasic oscillations in K+ permeability may contribute to the well-known initial biphasic changes in beta-cell membrane potential and insulin release during the inception of beta-cell stimulus secretion coupling.     

Differential effects of diabetes on rat choroid plexus ion transporter expression

Though diabetes is a disease with vascular complications, little is known about its effects on the blood-brain barrier or the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB is situated at choroid plexuses located in the lateral, third, and fourth ventricles. Choroid plexuses are the primary site of cerebrospinal fluid (CSF) production and express numerous ion transporters. Previous studies have shown a perturbation of ion transport in the periphery and brain during diabetes. In this study, we investigated the effect of diabetes on ion transporters in the choroid plexuses of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of STZ (60 mg/kg in citrate buffer, confirmed by glucose analysis: 601 +/- 22 mg/dl diabetic rats, 181 +/- 46 mg/dl age-matched controls); and at 28 days, rats were killed, choroid plexuses harvested, and protein extracted. Western blot analyses were carried out using antibodies for ion transporters, including Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit. The efflux of the K(+) analog (86)Rb(+) from choroid plexus was also studied. Diabetic rats showed an increase in expression of the Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit, as compared with age-matched controls, a decrease in Na(+)-H(+) exchanger expression, and no change in Na(+)-K(+)-ATPase beta1- or beta2-subunit. The net effect of these changes was a 66% increase in (86)Rb(+) efflux from diabetic choroid plexus compared with controls. These changes in expression may affect choroid plexus ion balance and thus significantly affect CSF production in diabetic rats.     

Renal outer medullary potassium channel knockout models reveal thick ascending limb function and dysfunction

The renal outer medullary potassium channel (ROMK) is an adenosine triphosphate-sensitive inward-rectifier potassium channel (Kir1.1 or KCNJ1) highly expressed in the cortical and medullary thick ascending limbs (TAL), connecting segment (CNT) and cortical collecting duct (CCD) in the mammalian kidney, where it serves to recycle potassium (K⁺) across the apical membrane in TAL and to secrete K⁺ in the CNT and CCD. ROMK channel mutations cause type II Bartter’s syndrome with salt wasting and dehydration, and ROMK knockout mice display a similar phenotype of Bartter’s syndrome in humans. Studies from ROMK null mice indicate that ROMK is required to form both the small-conductance (30pS, SK) K channels and the 70pS (IK) K channels in the TAL. The availability of ROMK^−/− mice has made it possible to study electrolyte transport along the nephron in order to understand the TAL function under physiological conditions and the compensatory mechanisms of salt and water transport under the conditions of TAL dysfunction. This review summarizes previous progress in the study of K⁺ channel activity in the TAL and CCD, ion transporter expression and activities along the nephron, and renal functions under physiological and pathophysiological conditions using ROMK^−/− mice.     

Regulation of potassium channel Kir 1.1 (ROMK) abundance in the thick ascending limb of Henle's loop

The renal outer medullary potassium channel (ROMK) of the thick ascending limb (TAL) is a critical component of the counter-current multiplication mechanism. In this study, two new antibodies raised to ROMK were used to investigate changes in the renal abundance of ROMK with treatments known to strongly promote TAL function. These antibodies specifically recognized protein of the predicted size of 45 kD in immunoblots of rat kidney or COS cells transfected with ROMK cDNA. Infusion of 1-deamino-(8-D-arginine)-vasopressin (dDAVP), a vasopressin V2 receptor-selective agonist, for 7 d into Brattleboro rats resulted in dramatic increases in apical membrane labeling of ROMK in the TAL of dDAVP-treated rats, as assessed by immunocytochemical analyses. Using immunoblotting, a more than threefold increase in immunoreactive ROMK levels was observed in the outer medulla after dDAVP infusion. Restriction of water intake to increase vasopressin levels also significantly increased TAL ROMK immunolabeling and abundance in immunoblots. In addition, dietary Na(+) levels were varied to determine whether ROMK abundance was also affected under other conditions known to alter TAL transport. Rats fed higher levels of sodium, as either NaCl or NaHCO(3) (8 mEq/250 g body wt per d), exhibited significantly increased density of the 45-kD band, compared with the respective control animals. Moreover, in rats fed a low-NaCl diet (0.25 mEq/250 g body wt per d), a 50% decrease in band density for the 45-kD band was observed (relative to control rats fed 2.75 mEq/250 g body wt per d of NaCl). These results demonstrate that long-term adaptive changes in ROMK abundance occur in the TAL with stimuli that enhance transport by this segment.     

Cyclosporin inhibits nitric oxide production in medullary ascending limb cultured cells

Background: Nitric oxide (NO) has been shown to play a role in cyclosporin (CsA) nephrotoxicity, but its mechanism of action is still unclear. As inducible NO synthase (iNOS) mRNA has been found to be expressed in rat medullary thick ascending limb (mTAL) cells, we investigated the effects of CsA on NO production in a model of mouse cultured mTAL cells. Materials and methods: The experiments were carried out on sub-cultured cells derived from isolated mTAL microdissected from the kidney of C57BL/6 mice. The identification of the iNOS mRNA in mTAL microdissected segment and cultured cell was confirmed by RT-PCR and RsaI digestion. Nitrite (NO2^-) released by mTAL cells was determined using the modified Griess reagent method and taken as an index of nitric oxide production. The cultured cells were treated with various concentrations of CsA and different signal transduction regulators to assess the effect and possible pathway(s) of action of CsA on NO production in mTAL cells. Results: The basal production of NO by mTAL cells increased by 1.8-fold following incubation with bacterial lipopolysaccharide (LPS). Both aminoguanidine and L-NAME inhibited NO production. CsA (10-300 ng/ml) also inhibited NO production in a dose-dependent manner and prevented its increase induced by LPS. Phorbol 12-myristate 13-acetate (PMA), a PKC stimulator, enhanced slightly the production of NO under basal conditions and prevented the inhibitory action of CsA on NO production. These results suggest that the NO secreted by mouse cultured mTAL cells is dependent on the PKC pathway. Conclusion: These results show that CsA may downregulate the production of NO by cultured mTAL cells expressing iNOS mRNA and that the PKC pathway is involved in this process.     

Improvement of leucocytic Na+ K+ pump activity in uremic patients on low protein diet.

Leucocytic Na+ K+ pump activity was assessed in 20 patients with advanced renal failure. Na+ K(+)-ATPase activity was reduced when compared with the values obtained from normal subjects (101.8 +/- 48.6 versus 165.13 +/- 8.9 microM of Pi hr-1.g-1; P less than 0.001) and the mean 86Rb uptake by U 937 cells was depressed by 38% after the addition of patients' sera. Subsequently, patients were put on a diet providing 0.3 g protein/kg body weight daily and supplemented with ketoacids. After three months of dietary treatment Na+ K(+)-ATPase activity increased to 142 +/- 48.3 (P less than 0.01) and reached normal values at the sixth month (162.8 +/- 54.70 microM of Pi hr-1.g-1; P less than 0.001) whereas 86Rb uptake increased by 23 percent when compared to initial values. These data suggest that among the different mechanisms which have been advanced to explain the defects in the Na+ pump observed in uremic patients, circulating inhibitors deriving from alimentary protein intake may affect cation transport.     

Effects of cardiac glycosides on sodium pump expression and function in LLC-PK1 and MDCK cells

BACKGROUND: The decreases in proximal tubule sodium reabsorption seen with chronic renal failure and volume expansion have been ascribed to circulating digitalis-like substances (DLS). However, the circulating concentrations of DLS do not acutely inhibit the sodium pump to a degree consistent with the observed changes in proximal tubule sodium reabsorption. METHODS: We examined how cell lines that simulated proximal (LLC-PK1) and distal tubule (MDCK) cells responded to acute (30 min) and long-term (up to 12 hours) Na+,K+-ATPase inhibition with DLS. RESULTS: In LLC-PK1, but not MDCK cells, low concentrations of ouabain decreased 86Rb uptake profoundly in a time and dose dependent manner. In LLC-PK1 cells grown to confluence, transcellular 22Na flux was markedly reduced in concert with the decreases in 86Rb uptake. Similar findings were observed with marinobufagenin (MBG) and deproteinated extract of serum derived from patients with chronic renal failure. However, inhibition of the Na+,K+-ATPase with low extracellular potassium concentrations did not produce any of these effects. Western and Northern blots detected no change in alpha1 Na+,K+-ATPase protein and message RNA, respectively, in LLC-PK1 cells treated with ouabain for 12 hours. However, the decrease in enzymatic activity of Na+,K+-ATPase of these cells was comparable to observed decreases in 86Rb uptake. Differential centrifugation as well as biotinylation experiments demonstrated a shift of the Na+,K+-ATPase from the plasmalemma with prolonged ouabain treatment. CONCLUSIONS: The results show that binding of cardiac glycosides by proximal (but not distal) tubular cells results in internalization of Na+,K+-ATPase with the net effect to amplify inhibition of the Na+,K+-ATPase. As the circulating concentrations of DLS increase with chronic renal failure and volume expansion, we suggest that this phenomenon explains some of the decreased sodium reabsorption by the proximal tubule seen in these conditions.     

The serum and glucocorticoid-inducible kinase SGK1 and the Na+/H+ exchange regulating factor NHERF2 synergize to stimulate the renal outer medullary K+ channel ROMK1

Mineralocorticoids stimulate Na(+) reabsorption and K(+) secretion in principal cells of connecting tubule and collecting duct. The involved ion channels are ENaC and ROMK1, respectively. In Xenopus oocytes, the serum and glucocorticoid-sensitive kinase SGK1 has been shown to increase ENaC activity by enhancing its abundance in the plasma membrane. With the same method, ROMK1 appeared to be insensitive to regulation by SGK1. On the other hand, ROMK1 has been shown to colocalize with NHERF2, a protein mediating targeting and trafficking of transport proteins into the cell membrane. The present study has been performed to test whether NHERF2 is required for regulation of ROMK1 by SGK1. Coexpression of neither NHERF2 nor SGK1 with ROMK1 increases ROMK1 activity. However, coexpression of NHERF2 and SGK1 together with ROMK1 markedly increases K(+) channel activity. The combined effect of SGK1 and NHERF2 does not significantly alter the I/V relation of the channel but increases the abundance of the channel in the membrane and decreases the decay of channel activity after inhibition of vesicle insertion with brefeldin. Coexpression of NHERF2 and SGK1 does not modify cytosolic pH but leads to a slight shift of pK(a) of ROMK1 to more acidic values. In conclusion, NHERF2 and SGK1 interact to enhance ROMK1 activity in large part by enhancing the abundance of channel protein within the cell membrane. This interaction allows the integration of genomic regulation and activation of SGK1 and NHERF2 in the control of ROMK1 activity and renal K(+) excretion.     

Clinical profiles and erythrocyte Na+ transport abnormalities of four major types of primary hypertension in Spain

The interaction of three different Na+ transport systems (Na+-K+ pump, Na+-K+ cotransport and Na+-Li+ countertransport), with internal Na+ and the passive Na+ leaks, were measured in erythrocytes from 72 Spanish, essential hypertensive patients and 30 normotensive controls. According to the observed abnormalities in Na+ transport pathways, 93.1% of the patients were classified into the following subsets: 12 (16.7%) exhibited a decreased apparent affinity of Na+-K+ pump for internal Na+ (Pump "-" hypertensives); 20 (27.7%) showed a decreased apparent affinity of Na+-K+ cotransport for internal Na+ (Co "-" hypertensives); 27 (37.5%) showed an accelerated Na+-Li+ countertransport (Counter "+" hypertensives); and 5 (6.9%) exhibited an increased rate constant of passive Na+ leaks (Leak "+" hypertensives). Finally, 5 patients (6.9%) did not show any abnormality in their Na+ transport systems and 3 exhibited more than one. Moreover, distinctive clinical features were recognize in Co "-" and Counter "+" subsets. Blood pressure values were lower in the former and, conversely, Counter "+" hypertensives showed a higher prevalence of moderate or severe hypertension (65.5% vs. 32.6%; P = 0.0059) and higher values of stimulated plasma renin activity (1.63 +/- 0.52 vs. 0.81 +/- 0.15; P = 0.0443). Our results confirm the heterogeneity of Na+ transport abnormalities in essential hypertension and suggest that these subsets of hypertensives could represent clinical entities.     

Erythrocyte Na+ and K+ transport systems in children with Bartter syndrome: increase in passive sodium permeability

Na+ and K+ intracellular content was studied in five children with Bartter syndrome and their age and race-paired controls. Na+ and K+ pump (ouabain sensitive) fluxes, Na+-K+ co-transport (furosemide sensitive), and rate constants of passive Na+ and K+ permeability were determined in each patient and control and also in six parents. The results show that in Bartter syndrome, there is a significant increase in the rate constant of passive Na+ permeability without any change in passive K+ permeability. This increase in the rate constant of passive permeability might explain at least partially the increased intracellular Na+ concentration also found in these patients. Moreover, the maximal rate of ouabain sensitive Na+ efflux was increased slightly, and co-transport fluxes were variable. Parents of patients had normal erythrocyte fluxes.