Kenneth A. Volk

Inhibition of the Epithelial Na+ Channel by Interaction of Nedd4 with a PY Motif Deleted in Liddle’s Syndrome

The epithelial Na+ channel (ENaC) plays a critical role in Na+ absorption in the kidney and other epithelia. Mutations in the C terminus of the β or γENaC subunits increase renal Na+ absorption, causing Liddle’s syndrome, an inherited form of hypertension. These mutations delete or disrupt a PY motif that was recently shown to interact with Nedd4, a ubiquitin-protein ligase expressed in epithelia. We found that Nedd4 inhibited ENaC when they were coexpressed in Xenopusoocytes. Liddle’s syndrome-associated mutations that prevent the interaction between Nedd4 and ENaC abolished inhibition, suggesting that a direct interaction is required for inhibition by Nedd4. Inhibition also required activity of a ubiquitin ligase domain within the C terminus of Nedd4. Nedd4 had no detectable effect on the single channel properties of ENaC. Rather, Nedd4 decreased cell surface expression of both ENaC and a chimeric protein containing the C terminus of the β subunit. Decreased surface expression resulted from an increase in the rate of degradation of the channel complex. Thus, interaction of Nedd4 with the C terminus of ENaC inhibits Na+ absorption, and loss of this interaction may play a role in the pathogenesis of Liddle’s syndrome and other forms of hypertension.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.

rENaC Is the Predominant Na + Channel in the Apical Membrane of the Rat Renal Inner Medullary Collecting Duct

The terminal nephron segment, the inner medullary collecting duct (IMCD), absorbs Na+ by an electrogenic process that involves the entry through an apical (luminal) membrane Na+ channel. To understand the nature of this Na+ channel, we employed the patch clamp technique on the apical membrane of primary cultures of rat IMCD cells grown on permeable supports. We found that all ion channels detected in the cell-attached configuration were highly selective for Na+ (Li+) over K+. The open/closed transitions showed slow kinetics, had a slope conductance of 6-11 pS, and were sensitive to amiloride and benzamil. Nonselective cation channels with a higher conductance (25-30 pS), known to be present in IMCD cells, were not detected in the cell-attached configuration, but were readily detected in excised patches. The highly selective channels had properties similar to the recently described rat epithelial Na+ channel complex, rENaC. We therefore asked whether rENaC mRNA was present in the IMCD. We detected mRNA for all three rENaC subunits in rat renal papilla and also in primary cultures of the IMCD. Either glucocorticoid hormone or mineralocorticoid hormone increased the amount of alpha-rENaC subunit mRNA but had no effect on the mRNA level of the beta-rENaC or gamma-rENaC subunits. From these data, taken in the context of other studies on the characteristics of Na+ selective channels and the distribution of rENaC mRNA, we conclude that steroid stimulated Na+ absorption by the IMCD is mediated primarily by Na+ channels having properties of the rENaC subunit complex.

SGK is a primary glucocorticoid-induced gene in the human.

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

The ClC-3 Cl-/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl−/H+ antiport current (IClC-3) in HEK293 cells. The outward rectification and time dependence of IClC-3 closely resemble an endogenous HEK293 cell acid-activated Cl− current (IClacid) seen at extracellular pH ≤ 5.5. IClacid was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. IClacid was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (Erev) of IClC-3 between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl− and H+ transport become uncoupled at low pH. At pH 4.0 Erev was completely Cl− dependent (55.8 ± 2.3 mV/decade). Several findings linked ClC-3 with native IClacid; 1) RNA interference directed at ClC-3 message reduced native IClacid; 2) removal of the extracellular “fast gate” (E224A) produced large currents that were pH-insensitive; and 3) wild-type IClC-3 and IClacid were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10–500 μm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type IClC-3 and from native IClacid by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native IClacid. Low pH uncouples Cl−/H+ transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl−/H+ antiporters and perhaps for pH regulation in highly acidic intracellular compartments.

ClC-3 and IClswell are Required for Normal Neutrophil Chemotaxis and Shape Change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and IClswell, are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and IClswell are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScan™ and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3-/- PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3-/- and Clcn3+/+ PMNs demonstrated a similar regulatory volume decrease, indicating that the IClswell response to hypotonic challenge was intact in these cells. We further demonstrated that IClswell is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and IClswell have unique roles in regulation of PMN chemotaxis; IClswell through direct effects on PMN volume and ClC-3 through regulation of IClswell.

Vascular nitric oxide and superoxide anion contribute to sex-specific programmed cardiovascular physiology in mice

Intrauterine environmental pertubations have been linked to the development of adult hypertension. We sought to evaluate the interrelated roles of sex, nitric oxide, and reactive oxygen species (ROS) in programmed cardiovascular disease. Programming was induced in mice by maternal dietary intervention (DI; partial substitution of protein with carbohydrates and fat) or carbenoxolone administration (CX, to increase fetal glucocorticoid exposure). Adult blood pressure and locomotor activity were recorded by radiotelemetry at baseline, after a week of high salt, and after a week of high salt plus nitric oxide synthase inhibition (by l-NAME). In male offspring, DI or CX programmed an elevation in blood pressure that was exacerbated by N(omega)-nitro-l-arginine methyl ester administration, but not high salt alone. Mesenteric resistance vessels from DI male offspring displayed impaired vasorelaxation to ACh and nitroprusside, which was blocked by catalase and superoxide dismutase. CX-exposed females were normotensive, while DI females had nitric oxide synthase-dependent hypotension and enhanced mesenteric dilation. Despite the disparate cardiovascular phenotypes, both male and female DI offspring displayed increases in locomotor activity and aortic superoxide production. Despite dissimilar blood pressures, DI and CX-exposed females had reductions in cardiac baroreflex sensitivity. In conclusion, both maternal malnutrition and fetal glucocorticoid exposure program increases in arterial pressure in male but not female offspring. While maternal DI increased both superoxide-mediated vasoconstriction and nitric oxide mediated vasodilation, the balance of these factors favored the development of hypertension in males and hypotension in females.

Sertraline exposure leads to small left heart syndrome in adult mice

Background:Sertraline, a selective serotonin reuptake inhibitor (SSRI), is the most commonly prescribed therapy for maternal depression. Epidemiologic studies have linked SSRI exposure with decreased fetal growth, altered autonomic regulation, and cardiac malformations. We hypothesized that SSRI exposure decreases left-ventricular (LV) volumes and increases adult sympathetic nervous system activation, resulting in increased adult heart rates.Methods:C57BL/6 mice received saline or sertraline (5 or 15 mg/kg/day i.p.) on postnatal days 1–14. Adult phenotypes were assessed at 5 mo.Results:Sertraline-exposed mice had smaller LV internal diameters in diastole (control 4.0 ± 0.1 mm, SSRI 3.7 ± 0.1 mm, P < 0.05), decreased stroke volumes (control 46 ± 2.6 µl, SSRI 37 ± 2.3 µl, P < 0.05), higher heart rates (control 530 ± 13 beats per minute (bpm), SSRI 567 ± 6 bpm, P <0.05), and increased urinary excretion of noradrenaline (control 174 ± 29.4 ng/ml, SSRI 276 ± 35.1 ng/ml, P < 0.05). These changes were associated with increased cerebral serotonin transporter (5-HTT) expression.Conclusion:Neonatal sertraline exposure causes long-term changes in cardiac morphology and physiology. We speculate that early-life SSRI exposure impairs cardiomyocyte growth and central serotonin signaling, leading to a small left heart syndrome in adult mice.

Genotype-specific alterations in vascular smooth muscle cell function in cystic fibrosis piglets☆

BACKGROUND The most common CF-causing mutations interfere with CFTR trafficking from the endoplasmic reticulum (CFTR-F508del) or prematurely terminate transcription (CFTR-null). We suspected that genotype-specific patterns of CFTR expression would have differential effects on smooth muscle cell calcium signaling and hence vascular tone. We hypothesized that compared to wild-type or CFTR-null aorta, aorta from CFTR-F508del (dF) piglets will have reduced endoplasmic reticulum calcium mobilization and decreased vasoconstriction. METHODS Aortic reactivity was assessed by myography, and ratiometric calcium imaging was performed in isolated vascular smooth muscle cells. RESULTS Aorta from dF piglets had reduced myogenic tone (P<0.001) and decreased constriction to KCl (P<0.05). Combined inhibition of ryanodine and IP3 receptors decreased wild-type and CFTR-null responses to levels seen in dF aorta. Compared to wild-type cells, dF-expressing smooth muscle cells had reduced calcium transients, while CFTR-null cells had decreased baseline intracellular calcium concentrations. CONCLUSIONS Expression of CFTR-F508del interferes with smooth muscle cell calcium handling and decreases aortic responsiveness.

Neonatal SSRI Exposure Programs a Hypermetabolic State in Adult Mice

Background. Selective serotonin reuptake inhibitor (SSRI) therapy complicates up to 10% of pregnancies. During therapy, SSRIs exert pleiotropic antidepressant, anorexigenic, and neurotrophic effects. Intrauterine SSRI exposure has been modeled by neonatal administration to developmentally immature rodents, and it has paradoxically elicited features of adult depression. We hypothesized neonatal SSRI exposure likewise programs a rebound hypermetabolic state in adult mice. Methods. C57BL/6 pups were randomized to saline or sertraline (5 mg/kg/d) from P1–P14. Because estrogen increases tryptophan hydroxylase 2 (TPH2) expression, a subset of female mice underwent sham surgery or bilateral ovariectomy (OVX). Metabolic rate was determined by indirect calorimetry. Results. In both male and female mice, neonatal SSRI exposure increased adult caloric intake and metabolic rate. SSRI-exposed female mice had significantly decreased adult weight with a relative increase in brain weight and melatonin excretion, independent of ovarian status. Cerebral cortex TPH2 expression was increased in SSRI-exposed male mice but decreased in OVX SSRI-exposed female mice. Conclusions. SSRI exposure during a critical neurodevelopmental window increases adult caloric intake and metabolic rate. Ovarian status modulated central TPH2 expression, but not adult energy balance, suggesting programmed neural connectivity or enhanced melatonin production may play a more important role in the post-SSRI hypermetabolic syndrome.