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Background Paroxysmal extreme pain disorder (PEPD) is a rare autosomal dominant pain disorder linked to a mutation in the SCN9A gene, which encodes voltage-gated sodium channel Nav1.7. Abnormal pain sensitivity occurs because of changes in the properties of voltage-gated sodium channels. Different mutations in SCN9A and a spectrum of clinical expressions have been described. Case-Diagnosis/Treatment Here we describe a 3-year-old child with a rare clinical picture of PEPD. Extremely painful voiding had been present since the child's birth. The diagnosis was confirmed by the detection of a heterozygous pathogenic mutation in the SCN9A gene, c.554G > A (p.Arg185His) inherited paternally. The same mutation was also found in the girl's father, who has occasionally had some pain in his jaw while yawning since childhood. Significant reduction of the pain was achieved with carbamazepine. Conclusions The case is interesting because the same mutation as that found in the girl and her father has been found in patients with small fiber sensory neuropathy. These data do not correlate with the clinical picture of our case and her father, but intra- and interfamily phenotypic diversity in symptoms associated with a gain-of-function variant of Na(V)1.7 are also described and may explain our case. Keywords Pain * Child * Voiding * Mutation * Sodium channels, Introduction Paroxysmal extreme pain disorder (PEPD) is a rare autosomal dominant pain disorder linked to a mutation in the SCN9A gene, which encodes voltage-gated sodium channel Nav1.7. Abnormal pain sensitivity [...]

The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors. doi/ 10.1085/jgp.201010438

Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the [alpha]- and [gamma]-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the [gamma]-subunit at sites flanking a 43 residue inhibitory tract ([gamma]E144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the [gamma]-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the [gamma]-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the [gamma]-subunit further identified a key 11-met tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native [gamma] inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the [gamma] inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel. ion channels; channel inhibitor; prostasin; amiloride doi: 10.1152/ajprenal.00316.2010.

Earlier, we found that systemic administration of lipopolysaccharide (LPS; 4 mg/kg) hyperpolarized the transepithelial potential difference ([V.sub.t]) of tracheal epithelium in the isolated, perfused trachea (1PT) of the guinea pig 18 h after injection. As well, LPS increased the hyperpolarization component of the response to basolateral methacholine, and potentiated the epithelium-derived relaxing factor-mediated relaxation responses to hyperosmolar solutions applied to the apical membrane. We hypothesized that LPS stimulates the transepithelial movement of [Na.sup.+] via the epithelial sodium channel (ENaC)/[Na.sup.+]-[K.sup.+] pump axis, leading to hyperpolarization of [V.sub.t]. LPS increased the [V.sub.t]-depolarizing response to amiloride (10 [micro]M), i.e., offset the effect of LPS, indicating that [Na.sup.+] transport activity was increased. The functional activity of ENaC was measured in the IPT after short-circuiting the [Na.sup.+]-[K.sup.+] pump with basolateral amphotericin B (7.5 [micro]M). LPS had no effect on the hyperpolarization response to apical trypsin (100 U/ml) in the Ussing chamber, indicating that channel-activating proteases are not involved in the LPS-induced activation of ENaC. To assess [Na.sup.+]-[K.sup.+] pump activity in the IPT, ENaC was short-circuited with apical amphotericin B. The greater [V.sub.t] in the presence of amphotericin B in tracheas from LPS-treated animals compared with controls revealed that LPS increased [Na.sup.+]-[K.sup.+] pump activity. This finding was confirmed in the Ussing chamber by inhibiting the [Na.sup.+]-[K.sup.+] pump via extracellular [K.sup.+] removal, loading the epithelium with [Na.sup.+], and observing a greater hyperpolarization response to [K.sup.+] restoration. Together, the findings of this study reveal that LPS hyperpolarizes the airway epithelium by increasing the activities of ENaC and the [Na.sup.+]-[K.sup.+] pump. endotoxin; lung; airway epithelium; [Na.sup.+] transport; transepithelial voltage doi: 10.1152/ajplung.00123.2010.

The aim of this study is to elucidate the effects of interleukin-6 (IL-6) on the expression and activity of the epithelial sodium channel (ENaC), which is one of the key mechanisms underlying tubular sodium reabsorption. M-1 cortical collecting duct cells were treated with IL-6 (100 ng/ml) for 12 h. Real-time polymerase chain reaction and immunoblotting were employed to examine the mRNA and protein abundance. Transepithelial voltage ([V.sub.te]) and resistance ([R.sub.te]) were measured with an ohm/ voltmeter (EVOM, WPI). The equivalent current was calculated as the ratio of [V.sub.te] to [R.sub.te]. Treatment with IL-6 (n = 5) increased the mRNA abundance of [alpha]-ENaC by 11 [+ or -] 7% (P = not significant), [beta]-ENaC by 78 [+ or -] 14% (P = 0.01), [gamma]-ENaC by 185 [+ or -] 38% (P = 0.02), and prostasin by 29 [+ or -] 5% (P = 0.01), all normalized by [beta]-actin. Treatment with IL-6 increased the protein expression of [alpha]-ENaC by 19 [+ or -] 3% (P = 0.001), [beta]-ENaC by 89 [+ or -] 21% (P = 0.01), [gamma]-ENaC by 36 [+ or -] 12% (P = 0.02), and prostasin by 33 [+ or -] 6% (P = 0.02). The amiloride-sensitive sodium current increased by 37 [+ or -] 5%, from 6.0 [+ or -] 0.4 to 8.2 [+ or -] 0.3 [micro]A/[cm.sup.2] (P < 0.01), in the cells treated with IL-6 compared with controls (P = 0.01). Aprotinin (28 [micro]g/ml), a prostasin inhibitor, reduced the amiloride-sensitive sodium current by 61 [+ or -] 5%, from 6.1 [+ or -] 0.3 to 3.7 [+ or -] 0.2 [micro]A/[cm.sup.2] (P = 0.01). The magnitude of the IL-6-induced amiloride-sensitive sodium current in the presence of aprotinin dropped by 57 [+ or -] 2%, from 8.6 [+ or -] 0.2 to 4.9 [+ or -] 0.2 [micro]A/[cm.sup.2] (P < 0.01 ). This study has identified a novel function of IL-6, namely, IL-6 may activate ENaC. Therefore, renal inflammation mediated by IL-6 likely contributes to impaired pressure natriuresis. distal tubule; prostasin; pressure natriuresis doi: 10.1152/ajpregu.00207.2009.

We evaluated the effects of intracerebroventricular (icv) infusion of [Na.sup.+]-rich artificial cerebrospinal fluid (aCSF), with or without the mineralocorticoid receptor (MR) blocker spironolactone, on epithelial [Na.sup.+] channel (ENaC) subunits and regulators, such as MR, serum/glucocorticoid-inducible kinase 1, neural precursor cells expressed developmentally downregulated 4-like gene, 11[beta]-hydroxylase, and aldosterone synthase, in brain regions of Wistar rats. The effects of icv infusion of the amiloride analog benzamil on brain tissue and CSF [Na.sup.+] concentration ([[Na.sup.+]]) were also assessed. In the choroid plexus and ependyma of the anteroventral third ventricle, ENaC subunits are present in apical and basal membranes. [Na.sup.+]-rich aCSF increased [beta]-ENaC mRNA and immunoreactivity in the choroid plexus and increased [alpha]- and [beta]-ENaC immunoreactivities in the ependyma. [Na.sup.+]-rich aCSF increased [alpha]- and [beta]-ENaC-gold-labeled particles in the microvilli of the choroid plexus and in basolateral membranes of the ependyma. Spironolactone only prevented the increase in [beta]-ENaC immunoreactivity in the choroid plexus and ependyma. In the supraoptic nucleus, paraventricular nucleus, and subfornical organ, [Na.sup.+]-rich aCSF did not affect mRNA expression levels of the studied genes. Benzamil significantly increased CSF [[Na.sup.+]] in the control, but not [Na.sup.+]-rich, aCSF group. In contrast, benzamil prevented the increase in hypothalamic tissue [[Na.sup.+]] by [Na.sup.+]-rich aCSF. These results suggest that CSF [Na.sup.+] upregulates ENaC expression in the brain epithelia, but not in the neurons of hypothalamic nuclei. ENaC in the choroid plexus and ependyma appear to contribute to regulation of [Na.sup.+] homeostasis in the brain. sodium-rich artificial cerebrospinal fluid; benzamil; cerebrospinal fluid sodium concentration doi: 10.1152/ajpregu.00834.2009.

The article presents a complete analysis of the different negative mutations and haploefficiency taking place in [alpha]-II spectrin, STXBP1. These mutations are shown to lead to the onset of west syndrome with severe cerebral hypomyelination, spastic quadriplegia and developmental delay in humans.

The epithelial sodium channel (ENaC) is important for the long-term control of [Na.sup.+] homeostasis and blood pressure. Our previous studies demonstrated that Copper Metabolism Mufti Domain-containing protein 1 (COMMD1; previously known as Murrl), a protein involved in copper metabolism, inhibited amiloride-sensitive current in Xenopus laevis oocytes expressing ENaC (J Biol Chem 279: 5429, 2004). In this study, we report that COMMD1 inhibits amiloride-sensitive current in mammalian epithelial cells expressing ENaC, that the COMM domain of COMMD1 is sufficient for this effect, and that knockdown of COMMD1 increases amiloride-sensitive current. COMMD1 is coexpressed with ENaC in rat kidney medulla cells. COMMD1 increased ubiquitin modification of ENaC and decreased its cell surface expression. COMMD1 abolished insulin-stimulated amiloride-sensitive current and attenuated the stimulation of current by activated serum and glucocorticoid-regulated kinase (SGK1). COMMD1 was found to interact with both SGK1 and Aktl/protein kinase B, and knockdown of COMMD1 enhanced the stimulatory effect of both SGK1 and Aktl on amiloride-sensitive current. COMMDI's effects were reduced in the presence of ENaC proteins containing PY motif mutations, abolished in the presence of a dominant negative form of Nedd4-2, and knockdown of COMMD1 reduced the inhibitory effect of Nedd4-2 on ENaC, but did not enhance current when Nedd4-2 was knocked down. These data suggest that COMMD1 modulates [Na.sup.+] transport in epithelial cells through regulation of ENaC cell surface expression and this effect is likely mediated via Nedd4-2. kidney; SGK1; Aktl; insulin doi: 10.1152/ajprenal.00257.2009.

Proteolytic enzymes cleave the epithelial [Na.sup.+] channel (ENaC) at several positions releasing, in part, the N[H.sub.2] terminus of the [gamma]-subunit. Cleavage increases ENaC activity by increasing open probability; however, the role of polypeptides cleaved from the channel core remains unclear. We find that the cytosolic N[H.sub.2] terminus of [gamma]-ENaC unexpectedly targets to the nucleus being particularly strong in nucleoli. In contrast, the cytosolic COOH terminus targets to the cytoplasm and plasma membrane in a manner similar to full-length subunits. Targeting of the cytosolic N[H.sub.2] terminus of [gamma]-ENaC to the nucleus has functional consequences for coexpression of eGFP-fusion proteins containing this segment of the channel, but not the COOH terminus, decrease ENaC activity in a dose-dependent manner. The mechanism of this negative regulation is associated with a decrease in the functional half-life of ENaC at the plasma membrane. Inspection of the primary amino acid sequence of [gamma]-ENaC reveals possible nuclear localization signals (NLS) conserved at the extreme N[H.sub.2] terminus and just preceding the first transmembrane domain. Disruption of the putative NLS preceding the first transmembrane domain in [gamma]-ENaC but not that at the extreme N[H.sub.2] terminus abolishes both targeting to the nucleus and negative regulation of ENaC activity. These findings are consistent with the release of the N[H.sub.2] terminus of [gamma]-ENaC following cleavage being functionally important for signaling to the nucleus in a manner similar to Notch signaling and release of the cytosolic COOH-terminal tail of polycystin-1. hypertension; aldosterone; polycystin-1; Notch doi: 10.1152/ajprenal.00600.2009.

We examine whether alveolar cells can control release of [O.sup.-.sub.2] through regulated NADPH oxidase (NOX) 2 (NOX2) activity to maintain lung fluid homeostasis. Using FACS to purify alveolar epithelial cells, we show that type 1 cells robustly express each of the critical NOX components that catalyze the production of [O.sup.-.sub.2] (NOX2 or [gp91.sup.phox], [p22.sup.phox], pp67.sup.phox], [p47.sup.phox], and [p40.sup.phox] subunits) as well as Rac1 at substantially higher levels than type 2 cells. Immunohistochemical labeling of lung tissue shows that Rac1 expression is cytoplasmic and resides near the apical surface of type 1 cells, whereas NOX2 coimmunoprecipitates with epithelial sodium channel (ENaC). Since Rac1 is a known regulator of NOX2, and hence [O.sup.-.sub.2] release, we tested whether inhibition or activation of Rac1 influenced ENaC activity. Indeed, 1 [micro]M NSC23766 inhibition of Rac1 decreased [O.sup.-.sub.2] output in lung cells and significantly decreased ENaC activity from 0.87 [+ or -] 0.16 to 0.52 [+ or -] 0.16 [mean number of channels (N) and single-channel open probability ([P.sub.o]) (N[P.sub.o]) [+ or -] SE, n = 6; P < 0.05] in type 2 cells. NSC23766 (10 [micro]M) decreased ENaC N[P.sub.o] from 1.16 [+ or -] 0.27 to 0.38 [+ or -] 0.10 (n = 6 in type 1 cells). Conversely, 10 ng/ml EGF (a known stimulator of both Rac1 and [O.sup.-.sub.2] release) increased ENaC N[P.sub.o] values in both type 1 and 2 cells. N[P.sub.o] values increased from 0.48 [+ or -] 0.21 to 0.91 [+ or -] 0.28 in type 2 cells (P < 0.05; n = 10). In type 1 cells, ENaC activity also significantly increased from 0.40 [+ or -] 0.15 to 0.60 [+ or -] + 0.23 following EGF treatment (n = 7). Sequestering [O.sup.-.sub.2] using 2,2,6,6-tetramethylpiperidineN-oxyl (TEMPO) compound prevented EGF activation of ENaC in both type 1 and 2 cells. In conclusion, we report that Rac1-mediated NOX2 activity is an important component in [O.sup.-.sub.2] regulation of ENaC. doi: 10.1152/ajplung.00230.2009 redox; NOX2; lung slice; fluorescence-activated cell sorting; single-channel patch

Retinal ganglion cells convey information by increasing their firing in response to an optimal visual stimulus or 'trigger feature.' However, one class of ganglion cell responds to changes in the visual scene by decreasing its firing. These cells, termed uniformity detectors in the rabbit retina, are encountered only rarely and the synaptic mechanisms underlying their unusual responses have not been investigated. In this study, patch-clamp recordings of uniformity detectors show that the action potentials underlying the maintained firing arise within 'complex spikes.' Both ON and OFF visual stimuli elicit only inhibitory synaptic input, the immediate effect of which is to suppress the maintained firing. However, this inhibition also alters the properties of the 'renascent' spiking by increasing the amplitude of the spikes within each burst, suggesting that the effect may increase the efficacy of spike propagation and transmission. calcium spike | glycine | Na-channel inactivation | rabbit | suppressed by contrast www.pnas.org/cgi/doi/10.1073/pnas.0909621107

[K.sup.+] plays a catalytic role in AHL [Na.sup.+] reabsorption via [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2), recycling across luminal [K.sup.+] channels, so that luminal [K.sup.+] is not depleted. Based on models of the ascending Henle limb (AHL) epithelium, it has been hypothesized that N[H.sup.+.sub.4] may also catalyze luminal [Na.sup.+] uptake. This hypothesis requires that luminal N[H.sup.+.sub.4] not be depleted, implying replenishment via either direct secretion of N[H.sup.+.sub.4], or N[H.sub.3] in parallel with a proton. In the present work, epithelial models of rat medullary and cortical AHL (Weinstein AM, Krahn TA. Am J Physiol Renal Physiol 298: F000-F000, 2009) are configured as tubules and examined in simulations of function in vitro and in vivo to assess the feasibility of a catalytic role for N[H.sup.+.sub.4] in [Na.sup.+] reabsorption. Modulation of [Na.sup.+] transport is also examined by peritubular [K.sup.+] concentration and by Bartter-type transport defects in NKCC2 (type 1), in luminal membrane [K.sup.+] channels (type 2), and in peritubular [Cl.sup.-] channels (type 3). It is found that a catalytic role for N[H.sup.+.sub.4], which is significant in magnitude (relative to [K.sup.+]), is quantitatively realistic, in terms of uptake via NKCC2, and in terms of luminal membrane ammonia backflux. Simulation of a 90% NKCC2 defect is predicted to double distal [Na.sup.+] delivery; it is also predicted to increase distal acid delivery (principally as N[H.sup.+.sub.4]). With doubling of medullary [K.sup.+], the model predicts a 30% increase in distal [Na.sup.+] delivery, but in this case there is a decrease in AHL acidification. This effect of peritubular [K.sup.+] on proton secretion appears to be akin to type 3 Bartter's pathophysiology, in which there is decreased peritubular HC[O.sup.-.sub.3] exit, cytosolic alkalinization, and a consequent decrease in luminal proton secretion by NHE3. One consequence of overlapping and redundant roles for [K.sup.+] and N[H.sup.+.sub.4], is a blunted impact of luminal membrane [K.sup.+] permeability on overall [Na.sup.+] reabsorption, so that type 2 Bartter pathophysiology is not well captured by the model. NKCC; KCC; NHE3; sodium transport; potassium transport; ammonia; Bartter's syndrome; loop diuretic doi:10.1152/ajprenal.00232.2009

A mathematical model of ascending Henle limb (AHL) epithelium has been fashioned using kinetic representations of [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2), KCC4, and type 3 [Na.sup.+]/[H.sup.+] exchanger (NHE3), with transporter densities selected to yield the reabsorptive [Na.sup.+] flux expected for rat tubules in vivo. Of necessity, this model predicts fluxes that are higher than those measured in vitro. The kinetics of the NKCC and KCC are such that [Na.sup.+] reabsorption by the model tubule is responsive to variation in luminal NaCl concentration over the range of 30 to 130 mM, with only minor changes in cell volume. Peritubular KCC accounts for about half the reabsorptive [Cl.sup.-] flux, with the remainder via peritubular [Cl.sup.-] channels. Transcellular [Na.sup.+] flux is turned off by increasing peritubular KCl, which produces increased cytosolic Cl- and thus inhibits NKCC2 transport. In the presence of physiological concentrations of ammonia, there is a large acid challenge to the cell, due primarily to N[H.sup.+.sub.4] entry via NKCC2, with diffusive N[H.sub.3] exit to both lumen and peritubular solutions. When NHE3 density is adjusted to compensate this acid challenge, the model predicts luminal membrane proton secretion that is greater than the HC[O.sub.-.sub.3]-reabsorptive fluxes measured in vitro. The model also predicts luminal membrane ammonia cycling, with uptake via NKCC2 or [K.sup.+] channel, and secretion either as N[H.sup.+.sub.4] by NHE3 or as diffusive N[H.sub.3] flux in parallel with a secreted proton. If such luminal ammonia cycling occurs in vivo, it could act in concert with luminal [K.sup.+] cycling to facilitate AHL [Na.sup.+] reabsorption via NKCC2. With physiological ammonia, peritubular KCl also blunts NHE3 activity by inhibiting N[H.sup.+.sub.4] uptake on the Na-K-ATPase, and alkalinizing the cell. NKCC; KCC; NHE3; sodium transport; potassium transport; ammonia doi:10.1152/ajprenal.00231.2009

Kinetic models of [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] costransporter (NKCC2) and [K.sup.+]-[Cl.sup.-] cotransporter (KCC4), two of the key cotransporters of the Henle limb, are fashioned with inclusion of terms representing binding and transport of N[H.sup.+.sub.4]. The models are simplified using assumptions of equilibrium ion binding, binding symmetry, and identity of [Cl.sup.-] binding sites. Model parameters are selected to be consistent with flux data from expression of these transporters in oocytes, specifically inwardly directed coupled transport of rubidium. In the analysis of these models, it is found that despite the simplifying assumptions to reduce the number of model parameters, neither model is uniquely determined by the data. For NKCC or KCC there are two- or three-parameter families of 'optimal' solutions. As a consequence, one may specify several carrier translocation rates and/or ion affinities before fitting the remaining coefficients to the data, with no loss of fidelity in simulating the experiments. Model calculations suggest that with respect to NKCC2 near its operating point, the curve of ion flux as a function of cell [Cl.sup.-] is steep, and with respect to KCC4, its curve of ion flux as a function of peritubular [K.sup.+] is also steep. The implication is that the kinetics are suitable for these two transporters in series to act as a sensor for peritubular [K.sup.+], to modulate AHL [Na.sup.+] reabsorption, with cytosolic [Cl.sup.-] as the intermediate variable. The models also reveal the potential for luminal N[H.sup.+.sub.4] to be a potent catalyst for NKCC2 [Na.sup.+] reabsorption, provided suitable exit mechanisms for N[H.sup.+.sub.4] (from cell-to-lumen) are operative. It is found that KCC4 is likely to augment the secretory N[H.sup.+.sub.4] flux, with peritubular N[H.sup.+.sub.4] uptake driven by the cell-to-blood [K.sup.+] gradient. NKCC; KCC; loop diuretic; sodium transport; potassium transport; ammonia doi:10.1152/ajprenal.00230.2009

Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of [alpha][beta][gamma]-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human [alpha][beta][gamma]-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat [alpha][beta][gamma]-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of [alpha][beta][gamma]-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of 13-phenyl1, [N.sup.2]-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH2- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human [alpha][beta][gamma]-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the parachlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal. amiloride; epithelial [Na.sup.+] channel; guanosine 3',5'-cyclic monophosphate; domain mapping doi: 10.1152/ajprenal.00307.2009

Renal blood flow (RBF) autoregulation is mediated by at least two mechanisms, the fast acting myogenic response (-5 s) and slow acting tubuloglomerular feedback (TGF; ~25 s). Previous studies suggest epithelial [Na.sup.+] channel (ENaC) family proteins, [beta]-ENaC in particular, mediate myogenic constriction in isolated renal interlobar arteries. However, it is unknown whether [beta]-ENaC-mediated myogenic constriction contributes to RBF autoregulation in vivo. Therefore, the goal of this investigation was to determine whether the myogenic mediated RBF autoregulation is inhibited in a mouse model of reduced [beta]-ENaC (m/m). To address this goal, we evaluated the temporal response of RBF and renal vascular resistance (RVR) to a 2-min step increase in mean arterial pressure (MAP). Pressure-induced changes in RBF and RVR at 0-5, 6-25, and 110-120 s after step increase in MAP were used to assess the contribution of myogenic and TGF mechanisms and steady-state autoregulation, respectively. The rate of the initial increase in RVR, attributed to the myogenic mechanism, was reduced by ~50% in m/m mice, indicating the speed of the myogenic response was inhibited. Steady-state autoregulation was similar between [beta]-ENaC +/+ and m/m mice. Although the rate of the secondary increase in RVR, attributed to TGF, was similar in [beta]-ENaC +/+ and m/m mice, however, it occurred over a longer period (+ 10 s), which may have allowed TGF to compensate for a loss in myogenic autoregulation. Our findings suggest [beta]-ENaC is an important mediator of renal myogenic constriction-mediated RBF autoregulation in vivo. epithelial [Na.sup.+] channel; ion channel; degenerin; renal blood flow autoregulation; renal injury; myogenic constriction doi: 10.1152/ajprenal.00496.2009

Mitochondrial [Ca.sup.2+] efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an [Na.sup.+]-dependent mechanism mediates mitochondrial [Ca.sup.2+] efflux, the molecular identity of this transporter has remained elusive. Here we show that the [Na.sup.+]/[Ca.sup.2+] exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing [Ca.sup.2+] and Na+ fluorescent imaging, we demonstrate that mitochondrial [Na.sup.+]-dependent [Ca.sup.2+] efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial [Ca.sup.2+] transport was inhibited, moreover, by CGP-37157 and exhibited [Li.sup.+] dependence, both hallmarks of mitochondrial [Na.sup.+]-dependent [Ca.sup.2+] efflux. Finally, NCLX-mediated mitochondrial [Ca.sup.2+] exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial [Na.sup.+]/[Ca.sup.2+] exchanger. mitochondrial calcium exchanger | mitochondrial calcium homeostasis | sodium calcium exchanger | CGP-37157 www.pnas.org/cgi/doi/10.1073/pnas.0908099107

Electrical signaling in biology depends upon a unique electromechanical transduction process mediated by the S4 segments of voltage-gated ion channels. These transmembrane segments are driven outward by the force of the electric field on positively charged amino acid residues termed 'gating charges,' which are positioned at three-residue intervals in the S4 transmembrane segment, and this movement is coupled to opening of the pore. Here, we use the disulfide-locking method to demonstrate sequential ion pair formation between the fourth gating charge in the S4 segment (R4) and two acidic residues in the S2 segment during activation. R4 interacts first with E70 at the intracellular end of the S2 segment and then with D60 near the extracellular end. Analysis with the Rosetta Membrane method reveals the 3-D structures of the gating pore as these ion pairs are formed sequentially to catalyze the S4 transmembrane movement required for voltage-dependent activation. Our results directly demonstrate sequential ion pair formation that is an essential feature of the sliding helix model of voltage sensor function but is not compatible with the other widely discussed gating models. electrical excitability | gating doi/10.1073/pnas.0912307106

Lutken SC, Kim SW, Jonassen T, Marples D, Knepper MA, Kwon TH, Frokiaer J, Nielsen S. Changes of renal AQP2, ENaC, and NHE3 in experimentally induced heart failure: response to angiotensin II [AT.sub.1] receptor blockade. Am J Physiol Renal Physiol 297: F1678-F1688, 2009. First published September 23, 2009; doi: 10.1152/ajprenal.00010.2009.--Heart failure (HF) was induced by ligation of the left anterior descending artery (LAD). Left ventricular end-diastolic pressure (LVEDP) >25 mmHg (at day 23 after LAD ligation) was the inclusion criterion. The rats were divided into three groups: sham-operated (Sham, n = 23, LVEDP: 5.6 [+ or -] 0.6 mmHg), HF (n = 14, LVEDP: 29.4 [+ or -] 1.4 mmHg), and candesartan (1 mg x [kg.sup.-1] [day.sup.-1] sc)-treated HI= (HF + Can, n = 9, LVEDP: 29.2 [+ or -] 1.2 mmHg). After 7 days (i.e., 29 days after LAD ligation) semiquantitative immunoblotting revealed increased abundance of inner medulla aquaporin-2 (AQP2) and AQP2 phosphorylated at [Ser.sup.256] (p-AQP2) in HF. There was also markedly enhanced apical targeting of AQP2 and p-AQP2 in inner medullary collecting duct (IMCD) in HF compared with Sham rats, shown by immunocyto-chemistry. Candesartan treatment significantly reversed the increases in both AQP2 and p-AQP2 expression and targeting. In contrast, there were only modest changes in other collecting duct segments. Semiquantitative immunoblots revealed increased expression of type 3 [Na.sup.+]/[H.sup.+] exchanger (NHE3) and [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2) in kidneys from HF compared with Sham rats: both effects were reversed or prevented by candesartan treatment. The protein abundance of [alpha]-epithelial sodium channel ([alpha]-ENaC) was increased while [beta]-ENaC and [gamma]-ENaC expression was decreased in the cortex and outer stripe of the outer medulla in HF compared with Sham rats, which was partially reversed by candesartan treatment. These findings strongly support an important role of angiotensin II in the pathophysiology of renal water and sodium retention associated with HF. collecting duct: aquaporin; type 3 [Na.sup.+]/[H.sup.+] exchanger; [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter; epithelial sodium channel doi: 10.1152/ajprenal.00010.2009

Svenningsen P, Uhrenholt TR, Palarasah Y, Skjodt K, Jensen BL, Skott O. Prostasin-dependent activation of epithelial [Na.sup.+] channels by low plasmin concentrations. Am J Physiol Regul Integr Comp Physiol 297: RI733-R1741, 2009. First published September 30, 2009; doi: I0.1152/ajpregu.00321.2009.--Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial [Na.sup.+] channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity and undertook this study to investigate the mechanism by which plasmin stimulates ENaC activity. Cy3-1abeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the 'y-subunit, which releases an inhibitory peptide from the extracellular domain. Removal of GPI-anchored proteins from the M-1 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 [micro]g/ml). At a high plasmin concentration of 30 [micro]g/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked plasmin-stimulated ENaC current in single M-1 cells, as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, '[gamma]ENaC and prostasin were colocalized. A monoclonal antibody directed against the inhibitory peptide of [gamma]ENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the [gamma]-subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC. serine protease; sodium retention; kidney; M-1 cells; nephrotic syndrome doi: 10.1152/ajpregu.00321.2009

Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate. A m J Physiol Renal Physiol 297: F1466-F1475, 2009. First published August 12, 2009: doi:10.1152/ajprenal.00279.2009.--Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal [P.sub.i] absorption is one of the pharmacological targets that prevents it. The type II Na-[P.sub.i] cotransporter NaPi-2b is the major transporter that mediates [P.sub.i] reabsorption in the intestine. The potential role and regulation of other Na-[P.sub.i] transporters remain unknown. We have identified expression of the type III Na-[P.sub.i] cotransporter PIT-1 in the apical membrane of enterocytes. Na-[P.sub.i] transport activity and NaPi-2b and PIT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-[P.sub.i] diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-[P.sub.i] transport activity and NaPi-2b, but not PiT-l, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-[P.sub.i] diet, there is an increase in BBM Na-[P.sub.i] transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum [P.sub.i] that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-[P.sub.i] transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia. SLC34A2; PiT-1; hyperphosphatemia; chronic kidney disease; dietary [P.sub.i] doi: 10.1152/ajprenal.00279.2009.

We used patchclamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron [Na.sup.+] reabsorption during water conservation vs. maintenance of Na+ balance. We find that vasopressin significantly increases ENaC activity within 2-3 min by increasing open probability ([P.sub.o]). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18-24 h) and pretreatment of isolated CD with vasopressin (~30 min) resulted in a similar increase in [P.sub.o]. In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to [P.sub.o], increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and [Na.sup.+] each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron [Na.sub.+] reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC. hypertension; [Na.sup.+] reabsorption; aldosterone; cAMP; PKA doi: 10.1152/ajprenal.00371.2009.

The SCN5A-encoded cardiac sodium channel underlies excitability in the heart, and dysfunction of sodium current ([I.sub.Na]) can cause fatal ventricular arrhythmia in maladies such as long QT syndrome, Brugada syndrome (BrS), and sudden infant death syndrome (SIDS). The gene GPD1L encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1), but the function for this enzyme is unknown. Mutations in GPD1L have been associated with BrS and SIDS and decrease [I.sub.Na] through an unknown mechanism. Using a heterologous expression system, we show that GPD1L associated with SCN5A and that the BrS- and SIDS-related mutations in GPD1L caused a loss of enzymatic function resulting in glycerol-3-phosphate PKC-dependent phosphorylation of SCN5A at serine 1503 (S1503) through a GPD1L-dependent pathway. The direct phosphorylation of S1503 markedly decreased [I.sub.Na]. These results show a function for GPD1L in cell physiology and a mechanism linking mutations in GPD1L to sudden cardiac arrest. Because the enzymatic step catalyzed by GPD1L depends upon nicotinamide adenine dinucleotide, this GPD1L pathway links the metabolic state of the cell to [I.sub.Na] and excitability and may be important more generally in cardiac ischemia and heart failure. cardiac arrhythmia; sodium current; ion channel; cell metabolism; glycerol 3-phosphate dehydrogenase 1-like; protein kinase C; cardiac sodium channel [alpha]-subunit doi:10.1152/ajpheart.00513.2009

Recent studies suggest certain epithelial [Na.sup.+] channel (ENaC) proteins may be components of mechanosensitive ion channel complexes in vascular smooth muscle cells that contribute to pressure-induced constriction in middle cerebral arteries (MCA). However, the role of a specific ENaC protein, [beta]ENaC, in pressure-induced constriction of MCAs has not been determined. The goal of this study was to determine whether pressure-induced constriction in the MCA is altered in a mouse model with reduced levels of [beta]ENaC. Using quantitative immunofluorescence, we found whole cell [beta]ENaC labeling in cerebral vascular smooth muscle cells (VSMCs) was suppressed 46% in [beta]ENaC homozygous mutant (m/m) mice compared with wild-type littermates (+/+). MCAs from [beta]ENaC +/+ and m/m mice were isolated and placed in a vessel chamber for myographic analysis. Arteries from [beta]ENaC+/+ mice constricted to stepwise increases in perfusion pressure and developed maximal tone of 10 [+ or -] 2% at 90 mmHg (n = 5). In contrast, MCAs from [beta]ENaC m/m mice developed significantly less tone (4 [+ or -] 1% at 90 mmHg, n = 5). Vasoconstrictor responses to KCl (4-80 mM) were identical between genotypes and responses to phenylephrine ([10.sup.-7] [-10.sup.-4] M) were marginally altered, suggesting that reduced levels of VSMC [beta]ENaC specifically inhibit pressure-induced constriction. Our findings indicate [beta]ENaC is required for normal pressure-induced constriction in the MCA and provide further support for the hypothesis that [beta]ENaC proteins are components of a mechanosensor in VSMCs. epithelial [Na.sup.+] channel; ion channel; degenerin; myogenic constriction; middle cerebral artery

Reduction of repolarization reserve increases the risk of arrhythmia. We hypothesized that inhibition of [K.sup.+] current ([I.sub.K]) to decrease repolarization reserve would unmask the proarrhythmic role of endogenous, physiological late [Na.sup.+] current (late [I.sub.Na]). Monophasic action potentials (MAP) and 12-1ead electrocardiogram were recorded from female rabbit isolated hearts. To block [I.sub.K] and reduce repolarization reserve, E-4031, 4-aminopyridine, and Ba[Cl.sub.2] were used; to block endogenous late [I.sub.Na], tetrodotoxin (TTX) and ranolazine were used. E-4031 (1-60 nM) concentrationdependently prolonged MAP duration ([MAPD.sub.90]) and increased duration of the T wave from [T.sub.peak] to [T.sub.end] ([T.sub.peak] -[T.sub.end]), transmural dispersion of repolarization (TDR), and beat-to-beat variability (BVR) of [MAPD.sub.90]. E-4031 caused spontaneous and pause-triggered polymorphic ventricular tachycardia [torsade de pointes (TdP)]. In the presence of 60 nM E-4031, TTX (0.6-3 [micro]M) and ranolazine (5-10 [micro]M) shortened [MAPD.sub.90], decreased TDR, BVR, and [T.sub.peak]-[T.sub.end] (n = 9-20, P < 0.01), and abolished episodes of TdP. In hearts treated with Ba[Cl.sub.2] or 4-aminopyridine plus E-4031, TTX (0.6-3 [micro]M) shortened [MAPD.sub.90] and decreased [T.sub.peak]-[T.sub.end]. Ranolazine could not reverse the effect of E-4031 to inhibit human ether-a-go-go-related gene (HERG) [K.sup.+] current; thus, the reversal by ranolazine of effects of E-4031 was likely due to inhibition of late [I.sub.Na] and not to antagonism of the HERG-blocking action of E-4031. We conclude that endogenous, physiological late [I.sub.Na] contributes to arrhythmogenesis in hearts with reduced repolarization reserve. Inhibition of this current partially reverses MAPD prolongation and abolishes arrhythmic activity caused by [I.sub.K] inhibitors. late sodium current; repolarization reserve; ventricular tachycardia; monophasic action potential; ion currents

Jesse NM, McCartney J, Feng X, Richards EM, Wood CE, Keller-Wood M. Expression of ENaC subunits, chloride channels, and aquaporins in ovine fetal lung: ontogeny of expression and effects of altered fetal cortisol concentrations. Am J Physiol Regul Integr Comp Physiol 297: R453-R461, 2009. First published June 10, 2009; doi:10.1152/ajpregu.00127.2009. Transition of the epithelium of the fetal lung from fluid secretion to fluid reabsorption requires changes in the expression of ion channels. Corticosteroids regulate expression of several of these channels, including the epithelium sodium channel (ENaC) subunits and aquaporins (AQP). We investigated the ontogenetic changes in these ion channels in the ovine fetal lung during the last half of gestation, a time of increasing adrenal maturation. Expression of the mRNAs for the chloride channels, cystic fibrosis transmembrane conductance regulator (CVTR), and chloride channel 2 (CLCN2) decreased with age. Expression of mRNAs for AQP1, AQP5, and for subunits of ENaC ([alpha], [beta], [gamma]) increased with age. In the fetal sheep the expression of ENaC[beta] mRNA was dramatically higher than the expression of ENaC[alpha] or ENaC[gamma], but expression of ENaC[beta] protein decreased with maturation, although the ratio of the mature (112 kDa) to immature (102 kDa) ENaC[beta] protein increased with age, particularly in the membrane fraction. In contrast, ENaC[alpha] mRNA and protein both increase with maturation, and the mature form of ENaC[alpha] (68 kDa) predominates at all ages. A modest increase in fetal cortisol, within the range expected to occur naturally in late gestation but prior to active labor, increased ENaC[alpha] mRNA but not ENaC[beta], ENAC[gamma], or AQP mRNAs. We conclude that in the ovine fetal lung, appearance of functional sodium channels is associated with induction of ENAC[alpha] and ENaC[gamma], and that ENaC[alpha] expression may be induced by even small, preterm increases in fetal cortisol. fetus; sodium channel; cortisol; lung maturation

The mineralocorticoid aldosterone is a major regulator of sodium transport in target epithelia and contributes to the control of blood pressure and cardiac function. It specifically functions to increase renal absorption of sodium from tubular fluid via regulation of the [alpha] subunit of the epithelial sodium channel ([alpha]ENaC). We previously used microarray technology to identify the immediate transcriptional targets of aldosterone in a mouse inner medullary collecting duct cell line and found that the transcript induced to the greatest extent was the circadian clock gene Period 1. Here, we investigated the role of Period 1 in mediating the downstream effects of aldosterone in renal cells. Aldosterone treatment stimulated expression of Period 1 (Per1) mRNA in renal collecting duct cell lines and in the rodent kidney. RNA silencing of Period 1 dramatically decreased expression of mRNA encoding [alpha]ENaC in the presence or absence of aldosterone. Furthermore, expression of [alpha]ENaC-encoding mRNA was attenuated in the renal medulla of mice with disruption of the Per1 gene, and these mice exhibited increased urinary sodium excretion. Renal [alpha]ENaC-encoding mRNA was expressed in an apparent circadian pattern, and this pattern was dramatically altered in mice lacking functional Period genes. These results suggest a role for Period 1 in the regulation of the renal epithelial sodium channel and more broadly implicate the circadian clock in control of sodium balance., Introduction Sodium (Na) is the principal extracellular cation and plays a vital role in maintaining extracellular fluid volume and therefore blood pressure. The mineralocorticoid hormone aldosterone acts directly on the [...]

The epithelial sodium channel (ENaC) is regulated by epidermal growth factor (EGF). We investigate whether ENaC is regulated by another EGF receptor (EGFR) ligand, transforming growth factor-[alpha] (TGF-[alpha]). We show that chronic (24 h) treatment with TGF-[alpha] inhibits ENaC in Xenopus laevis kidney cells 20 times more strongly than EGF. By using single-channel measurements, we show that TGF-[alpha] significantly reduces the number of ENaC per patch. The open probability ([P.sub.o]) is unchanged by 24-h treatment with TGF-[alpha]. [alpha]-, [beta]-, and [gamma]-ENaC mRNA levels are significantly reduced by TGF-[alpha] or EGF. TGF-[alpha] or EGF reduces [alpha]- and [gamma]-ENaC proteins in the membrane; however, [beta]-ENaC is unchanged. TGF-[alpha] or EGF inhibits ENaC by activating EGFR since the EGFR inhibitor AG1478 blocks the effects of both. The MAPK 1/2 inhibitor U0126 also blocks the effect of TGF-[alpha] or EGF on ENaC, indicating that the MAPK1/2 pathway is involved in the TGF-[alpha]- or EGF-induced inhibition of ENaC. Interestingly, acute treatment (< 1 h) with TGF-[alpha] or EGF does not inhibit ENaC current; it enhances ENaC activity by increasing [P.sub.o]. Pretreatment of the cells with U0126 potentiates the acute TGF-[alpha]- or EGF-induced stimulation of ENaC. This TGF-[alpha]- or EGF-induced increase in sodium current is abolished by a phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, LY294002, suggesting that PI-3 kinase is involved in the activation of sodium transport. In conclusion, chronic treatment with TGF-[alpha] or EGF inhibits ENaC by decreasing the number of channels in the membrane transcriptionally through MAPK1/2 pathways, but acute treatment with TGF-[alpha] or EGF activates ENaC by increasing [P.sub.o] via PI-3 kinase. sodium transport; EGFR; kidney

Hormonal control of transepithelial sodium ([Na.sup.+]) transport utilizes phosphatidylinositide 3'-kinase (PI3K) and Raf--MAPK/ERK kinase (MEK)--ERK-dependent signaling pathways, which impact numerous cell functions. How signals transmitted by these pathways are sorted and appropriately transmitted to alter [Na.sup.+] transport without altering other physiologic processes is not well understood. Here, we report the identification of a signaling complex that selectively modulates the cell surface expression of the epithelial sodium channel (ENaC), an ion channel that is essential for fluid and electrolyte balance in mammals. Raf-1 and the ubiquitin ligase, Nedd4-2, are constitutively-expressed inhibitory components of this ENaC regulatory complex, which interact with, and decrease the expression of, cell surface ENaC. The activities of Nedd4-2 and Raf-1 are inhibited cooperatively by the PI3K-dependent kinase serum-and glucocorticoid-induced kinase 1 (SGK1), and the Raf1-interacting protein glucocorticoid-induced leucine zipper (GILZ1), which are aldosterone-stimulated components of the complex. Together, SGK1 and GILZ1 synergistically stimulate ENaC cell surface expression. Interestingly, GILZ1 and SGK1 do not have synergistic, and in fact have opposite, effects on an unrelated activity, FKHRLl-driven gene transcription. Together, these data suggest that GILZ1 and SGK1 provide a physical and functional link between the PI3K-and Raf-1-dependent signaling modules and represent a unique mechanism for specifically controlling [Na.sup.+] transport without inappropriately activating other cell functions. glucocorticoid-induced leucine zipper protein | Nedd4-2 | Raf-1 | serum-and glucocorticoid-induced kinase 1

A central question in adenohypophyseal cell physiology concerns the role of transmembrane ionic fluxes in the initiation of the hormone secretion process. In the current report, we investigated the effects of the growth hormone (GH) secretagogues ghrelin and GH-releasing peptide-6 (GHRP-6) on the regulation of the functional expression of voltage-gated [Na.sup.+] channels using the tumoral somatotrope GC cell line as a model. Cells were cultured under control conditions or in presence of the GH secretagogues (GHS) for 96 h, and [Na.sup.+] currents ([I.sub.Na]) were characterized in whole cell patch-clamp experiments. GHS treatment significantly increased [I.sub.Na] density in a dose-dependent manner. The effects of GHRP-6 were accompanied by an augment in conductance without changes in the kinetics and the voltage dependence of the currents, suggesting an increase in the number of channels in the cell membrane. Sustained inhibition of L-type [Ca.sup.2+] channel activity decreased 1Na density and prevented the effects of the GHS, whereas long-term exposure to an L-channel agonist increased [I.sub.Na] density and enhanced the actions of GHRP-6, indicating that [Ca.sup.2+] entry through these channels plays a role in the regulation of [Na.sup.+] channel expression. Likewise, GHRP-6 failed to enhance [Na.sup.+] channel expression in the presence of membrane-permeable inhibitors of protein kinases A and C, as well as the [Ca.sup.2+]/calmodulin-dependent kinase If. Conversely, treatment with a cAMP analog or a protein kinase C activator enhanced both basal and GHS-induced secretion of GH measured by enzyme-linked immunoassay, suggesting that GHRP-6 acting through the ghrelin receptor and different signaling pathways enhances [Na.sup.+] channel membrane expression, which favors hormone release from GC somatotropes. calcium channels; GC cells; growth hormone-releasing peptide-6; sodium channels

The [sigma]-receptor, a broadly distributed integral membrane protein with a novel structure, is known to modulate various voltage-gated [K.sup.+] and [Ca.sup.2+] channels through a mechanism that involves neither G proteins nor phosphorylation. The present study investigated the modulation of the heart voltage-gated [Na.sup.+] channel ([Na.sub.v]1.5]) by [sigma]-receptors. The [[sigma].sub.1]-receptor ligands [SKF-10047 and (+)-pentazocine] and [[sigma].sub.1]/[[sigma].sub.2]-receptor ligands (haloperidol and ditolylguanidine) all reversibly inhibited [Na.sub.v]l.5 channels to varying degrees in human embryonic kidney 293 (HEK-293) cells and COS-7 cells, but the [[sigma].sub.1]-receptor ligands were less effective in COS-7 cells. The same four ligands also inhibited [Na.sup.+] current in neonatal mouse cardiac myocytes. In [[sigma].sub.1]-receptor knockout myocytes, the [[sigma].sub.1]-receptor-specific ligands were far less effective in modulating [Na.sup.+] current, but the [[sigma].sub.1]/[[sigma].sub.1]-receptor ligands modulated [Na.sup.+] channels as well as in wild type. Photolabeling with the [[sigma].sub.1]-receptor photoprobe [[sup.125]I]-iodoazidococaine demonstrated that [[sigma].sub.1] receptors were abundant in heart and HEK-293 cells, but scarce in COS-7 cells. This difference was consistent with the greater efficacy of [[sigma].sub.1]-receptor-specific ligands in HEK-293 cells than in COS-7 cells. [sigma]-Receptors modulated [Na.sup.+] channels despite the omission of GTP and ATP from the patch pipette solution, [sigma]-Receptor-mediated inhibition of [Na.sup.+] current had little if any voltage dependence and produced no change in channel kinetics. [Na.sup.+] channels represent a new addition to the large number of voltage-gated ion channels modulated by [sigma]-receptors. The modulation of [Na.sub.v]1.5 channels by [sigma]-receptors in the heart suggests an important pathway by which drugs can alter cardiac excitability and rhythmicity. heart muscle; haloperidol

Neuropathy and myopathy can cause weakness during critical illness. To determine whether reduced excitability of peripheral nerves, rather than degeneration, is the mechanism underlying acute neuropathy in critically ill patients, we prospectively followed patients during the acute phase of critical illness and early recovery and assessed nerve conduction. During the period of early recovery from critical illness, patients recovered from neuropathy within days. This rapidly reversible neuropathy has not to our knowledge been previously described in critically ill patients and may be a novel type of neuropathy. In vivo intracellular recordings from dorsal root axons in septic rats revealed reduced action potential amplitude, demonstrating that reduced excitability of nerve was the mechanism underlying neuropathy. When action potentials were triggered by hyperpolarizing pulses, their amplitudes largely recovered, indicating that inactivation of sodium channels was an important contributor to reduced excitability. There was no depolarization of axon resting potential in septic rats, which ruled out a contribution of resting potential to the increased inactivation of sodium channels. Our data suggest that a hyperpolarized shift in the voltage dependence of sodium channel inactivation causes increased sodium inactivation and reduced excitability. Acquired sodium channelopathy may be the mechanism underlying acute neuropathy in critically ill patients., Introduction Weakness is a frequent neurologic complication of critical illness (1), (2), (3), (4). Disorders of both muscle (myopathy) and nerve (neuropathy) cause weakness during critical illness. Myopathy is due [...]

The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides; it is currently considered an orphan receptor. The sigma-1 receptor pharmacophore includes an alkylamine core, also found in the endogenous compound N,N-dimethyltryptamine (DMT). DMT acts as a hallucinogen, but its receptor target has been unclear. DMT bound to sigma-1 receptors and inhibited voltage-gated sodium ion ([Na.sup.+]) channels in both native cardiac myocytes and heterologous cells that express sigma-1 receptors. DMT induced hypermobility in wild-type mice but not in sigma-1 receptor knockout mice. These biochemical, physiological, and behavioral experiments indicate that DMT is an endogenous agonist for the sigma-1 receptor.

The epithelial [Na.sup.+] channel (ENaC) is a major regulator of salt and water reabsorption in a number of epithelial tissues. Abnormalities in ENaC function have been directly linked to several human disease states including Liddle's syndrome, psuedohypoaldosteronism, and cystic fibrosis and may be implicated in states as diverse as salt-sensitive hypertension, nephrosis, and pulmonary edema. ENaC activity in epithelial cells is highly regulated both by open probability and number of channels. Open probability is regulated by a number of factors, including proteolytic processing, while ENaC number is regulated by cellular trafficking. This review discusses current understanding of apical membrane delivery, cell surface stability, endocytosis, retrieval, and recycling of ENaC and the molecular partners that have so far been shown to participate in these processes. We review known sites and mechanisms of hormonal regulation of trafficking by aldosterone, vasopressin, and insulin. While many details of the regulation of ENaC trafficking remain to be elucidated, knowledge of these mechanisms may provide further insights into ENaC activity in normal and disease states. clathrin-mediated endocytosis; deubiquitination; vesicle recycling

Activation of signal transducer and activator of transcription-3 (Stat3) during cell confluency is related to its regulatory roles in cell growth arrest- or survival-related physiological or developmental processes. We previously demonstrated that this signaling event triggers epithelial dome formation by transcriptional augmentation of sodium hydrogen exchanger-3 (NHE3) expression. However, the detailed molecular mechanism remained unclear. By using serial deletions, site-directed mutagenesis, and EMSA analysis, we now demonstrate Stat3 binding to an atypical Stat3-response element in the rat proximal NHE3 promoter, located adjacent to a cluster of Sp cis-elements (SpA/B/C), within -77/-36 nt of the gene. SpB (-58/-55 nt) site was more effective than SpA (-72/-69 nt) site for cooperative binding of Sp1/Sp3. Increasing cell density had no effect on Sp1/Sp3 expression but resulted in their increased binding to the SpA/B/C probe along with Stat3 and concurrently with enhanced nuclear [pTyr.sup.705]-Stat3 level. Immunoprecipitation performed with the nuclear extracts demonstrated physical interaction of Stat3 and Sp1/Sp3 triggered by cell confluency. Stat3 inhibition by overexpression of dominant-negative Stat3-D mutant in MDCK cells or by small interfering RNA-mediated knockdown in Caco-2 cells resulted in inhibition of the cell density-induced NHE3 expression, Sp1/Sp3 binding, and NHE3 promoter activity and in decreased dome formation. Thus, during confluency, ligand-independent Stat3 activation leads to its interaction with Sp1/ Sp3, their recruitment to the SpA/B/C cluster in a Stat3 DNA-binding domain-dependent fashion, increased transcription, and expression of NHE3, to coordinate cell density-mediated epithelial dome formation. signal transducer and activator of transcription-3; Slc9a3; dome formation; [Na.sup.+]/[H.sup.+] exchanger-3

Voltage-gated [Na.sup.+]] channel currents ([I.sub.Na]) are expressed in several types of smooth muscle cells. The purpose of this study was to evaluate the expression of [I.sub.Na], its functional role, pathophysiology in cultured human (hASMCs) and rabbit aortic smooth muscle cells (rASMCs), and its association with vascular intimal hyperplasia. In whole cell voltage clamp, [I.sub.Na] was observed at potential positive to -40 mV, was blocked by tetrodotoxin (TTX), and replacing extracellular [Na.sub.+] with N-methyl-D-glucamine in cultured hASMCs. In contrast to native aorta, cultured hASMCs strongly expressed SCN9A encoding [Na.sub.v]1.7, as determined by quantitative RT-PCR. [I.sub.Na] was abolished by the treatment with SCN9A small-interfering (si)RNA (P < 0.01). TTX and SCN9A siRNA significantly inhibited cell migration (P < 0.01, respectively) and horseradish peroxidase uptake (P < 0.01, respectively). TTX also significantly reduced the secretion of matrix metalloproteinase-2 6 and 12 h after the treatment (P < 0.01 and P < 0.05, respectively). However, neither TTX nor siRNA had any effect on cell proliferation. L-type [Ca.sup.2+] channel current was recorded, and [I.sub.Na] was not observed in freshly isolated rASMCs, whereas TTX-sensitive [I.sub.Na] was recorded in cultured rASMCs. Quantitative RT-PCR and immunostaining for [Na.sub.v]1.7 revealed the prominent expression of SCN9A in cultured rASMCs and aorta 48 h after balloon injury but not in native aorta. In conclusion, these studies show that [I.sub.Na] is expressed in cultured and diseased conditions but not in normal aorta. The [Na.sub.v]l.7 plays an important role in cell migration, endocytosis, and secretion. [Na.sub.v]1.7 is also expressed in aorta after balloon injury, suggesting a potential role for [Na.sub.v]1.7 in the progression of intimal hyperplasia. vascular smooth muscle; sodium channel; SCN9A; balloon injury

Recent studies have implicated epithelial [Na.sup.+] channels (ENaC) in myogenic signaling. The present study was undertaken to determine if ENaC and/or [Na.sup.+] entry are involved in the myogenic response of the rat afferent arteriole. Myogenic responses were assessed in the in vitro hydronephrotic kidney model. ENaC expression and membrane potential responses were evaluated with afferent arterioles isolated from normal rat kidneys. Our findings do not support a role of ENaC, in that ENaC channel blockers did not reduce myogenic responses and ENaC expression could not be demonstrated in this vessel. Reducing extracellular [Na.sup.+] concentration ([[Na.sup.+]]o; 100 mmol/1) did not attenuate myogenic responses, and amiloride had no effect on membrane potential. Benzamil, an inhibitor of ENaC that also blocks [Na.sup.+]/[Ca.sup.2+] exchange (NCX), potentiated myogenic vasoconstriction. Benzarnil and low [[Na.sup.+]]o elicited vasoconstriction; however, these responses were attenuated by diltiazem and were associated with significant membrane depolarization, suggesting a contribution of mechanisms other than a reduction in NCX. [Na.sup.+] repletion induced a vasodilation in pressurized afferent arterioles preequilibrated in low [[Na.sup.+]]o, a hallmark of NCX, and this response was reduced by 10 [micro]mol/1 benzamil. The dilation was eliminated, however, by a combination of benzamil plus ouabain, suggesting an involvement of the electrogenic [Na.sup.+]-[K.sup.+]-ATPase. In concert, these findings refute the premise that ENaC plays a significant role in the rat afferent arteriole and instead suggest that reducing [[Na.sup.+]]o and/or [Na.sup.+] entry is coupled to membrane depolarization. The mechanisms underlying these unexpected and paradoxical effects of [Na.sup.+] are not resolved at the present time. renal autoregulation; vascular smooth muscle; sodium ion channels; sodium/calcium exchange; benzamil; amiloride; ouabain; sodiumpotassium-adenosinetriphosphatase; epithelial sodium channel

Fejes-Toth G, Frindt G, Naray-Fejes-Toth A, Palmer LG. Epithelial [Na.sup.+] channel activation and processing in mice lacking SGK1. Am J Physiol Renal PhysioI 294: FI298-F1305, 2008. First published April 2, 2008; doi: 10.1152/ajprenal.00579.2007.--Amiloride-sensitive [Na.sup.+] channel activity was examined in the cortical collecting ducts of a mouse line ([SGK1.sup.-/-]) deficient in the serum-and glucocorticoid-dependent protein kinase SGK1. This activity was correlated with changes in renal Na handling and in the maturation of epithelial [Na.sup.+] channel (ENaC) protein. Neither [SGK1.sup.-/-] mice nor paired [SGK1.sup.+/+] animals expressed detectable channel activity, measured as amiloride-sensitive whole-cell current ([I.sub.Na]), under control conditions with standard chow. Administration of aldosterone (0.5 [micro]g/h via osmotic minipump for 7 days) increased [I.sub.Na] to a similar extent in [SGK1.sup.+/+] (378 [+ or -] 61 pA/cell at -100 mV) and in [SGK1.sup.-/-] (350 [+ or -] 57 pA/cell) animals. However, the maturation of ENaC, assessed as the ratio of cleaved to full-length forms of [gamma]-ENaC, was more pronounced in [SGK.sup.+/+] mice. The [SGK1.sup.-/-] animals exhibited a salt-wasting phenotype when kept on a low-Na diet for up to 2 days, losing significantly more Na in the urine than wild-type mice. Under these conditions, [1.sub.Na] was enhanced more in [SGK1.sup.-/-] (94 [+ or -] 14 pA/cell) than in [SGK.sup.+/+] (23 [+ or -] 5 pA/cell) genotypes. Despite the larger currents, the ratio of cleaved to full-length [gamma]-ENaC was lower in the knockout animals. The mice also expressed a smaller amount of [Na.sup.+]-[Cl.sup.-] cotransporter protein under Na-depleted conditions. These results indicated that SGK1 is essential for optimal processing of ENaC but is not required for activation of the channel by aldosterone. epithelial sodium channel; proteolysis; sodium reabsorption; cortical collecting duct

Cardiac sodium channels (voltage-gated [Na.sup.+] channel subunit 1.5) reside in both the plasmalemma and membrane invaginations called caveolae. Opening of the caveolar neck permits resident channels to become functional. In cardiac myocytes, caveolar opening can be stimulated by applying [beta]-receptor agonists, which initiates an interaction between the stimulatory G protein subunit-[alpha] ([G.sub.s][alpha]) and caveolin-3. This study shows that, in adult rat ventricular myocytes, a functional [G.sub.s][alpha]-caveolin-3 interaction occurs, even in the absence of the caveolin-binding sequence motif of [G.sub.s][alpha]. Consistent with previous data, whole cell experiments conducted in the presence of intracellular PKA inhibitor stimulation with [beta]-receptor agonists increased the sodium current ([I.sub.Na]) by 35.9 [+ or -] 8.6% (P < 0.05), and this increase was mimicked by application of [G.sub.s][alpha] protein. Inclusion of anti-caveolin-3 antibody abolished this effect. These findings suggest that [G.sub.s][alpha] and caveolin-3 are components of a PKA-independent pathway that leads to the enhancement of [I.sub.Na]. In this study, alanine scanning mutagenesis of [G.sub.s][alpha] (40THR42), in conjunction with voltage-clamp studies, demonstrated that the histidine residue at position 41 of [G.sub.s][alpha] (H41) is a critical residue for the functional increase of [I.sub.Na]. Protein interaction assays suggest that [G.sub.s][alpha]FL (full length) binds to caveolin-3, but the enhancement of [I.sub.Na] is observed only in the presence of [G.sub.s][alpha] H41. We conclude that [G.sub.s][alpha] H41 is a critical residue in the regulation of the increase in [I.sub.Na] in ventricular myocytes. voltage-gated sodium channel subunit 1.5; caveolae; caveolin; G protein; ventricle

In the microvillar microdomain of the kidney brush border, sodium hydrogen exchanger type 3 (NHE3) exists in physical complexes with the serine protease dipeptidyl peptidase IV (DPPIV). The purpose of this study was to explore the functional relationship between NHE3 and DPPIV in the intact proximal tubule in vivo. To this end, male Wistar rats were treated with an injection of the reversible DPPIV inhibitor Lys [Z(N[O.sub.2])]-pyrrolidide (I40; 60 mg.[kg.sup.-1].[day.sup.-1] ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[Z(N[O.sub.2])]-OH served as controls. [Na.sup.+]-[H.sup.+] exchange activity in isolated microvillar membrane vesicles was 45 [+ or -] 5% decreased in rats treated with I40. Membrane fractionation studies using isopycnic centrifugation revealed that I40 provoked redistribution of NHE3 along with a small fraction of DPPIV from the apical enriched microvillar membranes to the intermicrovillar microdomain of the brush border. I40 significantly increased urine output (67 [+ or -] 9%; P < 0.01), fractional sodium excretion (63 [+ or -] 7%; P < 0.01), as well as lithium clearance (81 [+ or -] 9%; P < 0.01), an index of end-proximal tubule delivery. Although not significant, a tendency toward decreased blood pressure and plasma pH/HC[O.sup.-.sub.3] was noted in 140-treated rats. These findings indicate that inhibition of DPPIV catalytic activity is associated with inhibition of NHE3-mediated NaHC[O.sub.3] reabsorption in rat renal proximal tubule. Inhibition of apical [Na.sup.+]-[H.sup.+] exchange is due to reduced abundance of NHE3 protein in the microvillar microdomain of the kidney brush border. Moreover, this study demonstrates a physiologically significant interaction between NHE3 and DPPIV in the intact proximal tubule in vivo. sodium transport; proton secretion; fluid reabsorption; serine protease

Salt-sensitive (SS) hypertension is a vascular diathesis characterized by reduced cardiovascular and renal nitric oxide bioavailability and local upregulation of ANG II. We have demonstrated that rats infused with ANG II manifest increased cortical cyclooxygenase (COX)-2 expression and activity via NADPH oxidase-derived reactive oxygen species (ROS). In the present studies we used Dahl salt-sensitive (DS) rats to test the hypothesis that hypertensive SS rats have increased cortical COX-2 upregulation, which is mediated by ANG II and ROS. DS rats were placed on either a normal-salt diet (0.5% NaCl) or a high-salt diet (4% NaCl) for 6 wk and treated with either the ANG II type 1 (A[T.sub.1]) receptor blocker candesartan (Can, 10 mg.[kg.sup.-1].[day.sup.-1]) or the SOD mimetic tempol (1 mmol/1). Hypertensive SS rats had a twofold increase in the cortical expression of COX-2 as assessed by Western blot. These changes in COX-2 expression were accompanied by a 10-fold increase in COX-2 mRNA expression and a 2-fold increase in the urinary excretion of PG[E.sub.2]. Treatment with either the A[T.sub.1] receptor blocker Can or the SOD mimetic tempol did not reduce blood pressure but resulted in significant reductions in the cortical expression of COX-2 and the urinary excretion of PG[E.sub.2]. In conclusion, we have demonstrated that local activation of the renin-angiotensin system, via increased ROS generation, mediates COX-2 upregulation in hypertensive SS rats. These studies unveil novel mechanistic pathways that may play a role in the pathogenesis of hypertensive renal injury. cyclooxygenase-2

The aims of the current study were to 1) examine the effects of hypoxia and acidosis on cultured cortical neurons and 2) explore the role of transporters and ion channels in hypoxic injury. Cell injury was measured in cultured neurons or hippocampal slices following hypoxia (1% [O.sub.2]) or acidosis (medium pH 6.8) treatment. Inhibitors of transporters and ion channels were employed to investigate their roles in hypoxic injury. Our results showed that 1) neuronal damage was apparent at 5-7 days of hypoxia exposure, i.e., 36-41% of total lactate dehydrogenase was released to medium and 2) acidosis alone did not lead to significant injury compared with nonacidic, normoxic controls. Pharmacological studies revealed 1) no significant difference in neuronal injury between controls (no inhibitor) and inhibition of [Na.sup.+]-[K.sup.+]-ATP pump, voltage-gated [Na.sup.+] channel, ATP-sensitive [K.sup.+] channel, or reverse mode of [Na.sup.+]/ [Ca.sup.2+] exchanger under hypoxia; however, 2) inhibition of NBCs with 500 [micro]M DIDS did not cause hypoxic death in either cultured cortical neurons or hippocampal slices; 3) in contrast, inhibition of [Na.sup.+]/[H.sup.+] exchanger isoform 1 (NHE1) with either 10 [micro]M HOE-642 or 2 [micro]M T-162559 resulted in dramatic hypoxic injury (+95% for HOE-642 and +100% for T-162559 relative to normoxic control, P < 0.001) on treatment day 3, when no death occurred for hypoxic controls (no inhibitor). No further damage was observed by NHE1 inhibition on treatment day 5. We conclude that inhibition of NHE1 accelerates hypoxia-induced neuronal damage. In contrast, DIDS rescues neuronal death under hypoxia. Hence, DIDS-sensitive mechanism may be a potential therapeutic target. sodium/hydrogen exchanger 1; HC[O.sup.-.sub.3] transporters; disodium 4,4'diisothiocyanatostilbene-2,2'-disulfonate; neuronal protection

Phospholemman (PLM, FXYD1), abundantly expressed in the heart, is the primary cardiac sarcolemmal substrate for PKA and PKC. Evidence supports the hypothesis that PLM is part of the cardiac Na-K pump complex and provides the link between kinase activity and pump modulation. PLM has also been proposed to modulate Na/Ca exchanger activity and may be involved in cell volume regulation. This study characterized the phenotype of the PLM knockout (KO) mouse heart to further our understanding of PLM function in the heart. PLM KO mice were bred on a congenic C57/BL6 background. In vivo conductance catheter measurements exhibited a mildly depressed cardiac contractile function in PLM KO mice, which was exacerbated when hearts were isolated and Langendorff perfused. There were no significant differences in action potential morphology in paced Langendorff-perfused hearts. Depressed contractile function was associated with a mild cardiac hypertrophy in PLM KO mice. Biochemical analysis of crude ventricular homogenates showed a significant increase in Na-K-ATPase activity in PLM KO hearts compared with wild-type controls. SDS-PAGE and Western blot analysis of ventricular homogenates revealed small, nonsignificant changes in Na-K-ATPase subunit expression, with two-dimensional gel (isoelectric focusing, SDS-PAGE) analysis revealing minimal changes in ventricular protein expression, indicating that deletion of PLM was the primary reason for the observed PLM KO phenotype. These studies demonstrate that PLM plays an important role in the contractile function of the normoxic mouse heart. Data are consistent with the hypothesis that PLM modulates Na-K-ATPase activity, indirectly affecting intracellular Ca and hence contractile function. FXYD1; contractile function; intracellular sodium regulation

Dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport and by interacting with vasoactive hormones/humoral factors, such as aldosterone, angiotensin, catecholamines, endothelin, oxytocin, prolactin pro-opiomelancortin, reactive oxygen species, renin, and vasopressin. Dopamine receptors are classified into [D.sub.1]-like ([D.sub.1] and [D.sub.5]) and [D.sub.2]-1ike ([D.sub.2], [D.sub.3], and [D.sub.4]) subtypes based on their structure and pharmacology. In recent years, mice deficient in one or more of the five dopamine receptor subtypes have been generated, leading to a better understanding of the physiological role of each of the dopamine receptor subtypes. This review summarizes the results from studies of various dopamine receptor mutant mice on the role of individual dopamine receptor subtypes and their interactions with other G protein-coupled receptors in the regulation of blood pressure. knockout mice; dopamine receptor

We investigated whether [alpha]-lipoic acid ([alpha]-LA), an antioxidant, attenuates the ischemia-reperfusion (I/R)-induced dysregulation of these transporters. Both renal pedicles of male Sprague-Dawley rats were clamped for 40 min. [alpha]-LA (80 mg/kg) was administered intraperitoneally before and immediately after induction of ischemia. After 2 days, the expression of aquaporins (AQPs), sodium transporters, and nitric oxide synthases (NOS) was determined in the kidney by immunoblotting and immunohistochemistry. The expression of endothelin-1 (ET-1) mRNA was determined by real-time PCR. Activities of adenylyl cyclase and guanylyl cyclase were measured by stimulated generation of cAMP and cGMP, respectively. The expression of AQP1-3 as well as that of the [[alpha].sub.1]-subunit of Na-K-ATPase, type 3 Na/H exchanger, Na-K-2Cl cotransporter, and Na-Cl cotransporter was markedly decreased in response to I/R. The expression of type VI adenylyl cyclase was decreased in I/R-injured rats, which was counteracted by the treatment of [alpha]-LA. AVP-stimulated cAMP generation was blunted in I/R rats and was then ameliorated by [alpha]-LA treatment. [alpha]-LA treatment attenuated the downregulation of AQPs and sodium transporters. The expression of endothelial NOS was decreased in I/R rats, which was prevented by [alpha]-LA. The cGMP generation in response to sodium nitroprusside was blunted in I/R rats, which was also significantly prevented by [alpha]-LA. The mRNA expression of ET-1 was increased, which was recovered to the control level by [alpha]-LA treatment. In conclusion, [alpha]-LA treatment prevents I/R-induced dysregulation of AQPs and sodium transporters in the kidney, possibly through preserving normal activities of local AVP/cAMP, nitric oxide/cGMP, and ET systems. I/R injury; aquaporins; sodium transporters; nitric oxide; endothelin

We previously demonstrated that there are developmental changes in proximal tubule [Na.sup.+]/[H.sup.+] exchanger (NHE) activity. There is a maturational increase in postnatal brush-border membrane (BBM) vesicle NHE3 protein abundance and decrease in NHE8 protein abundance. The purpose of this study was to determine whether thyroid hormone plays a role in the rat renal maturational isoform switch from NHE8 to NHE3 and whether thyroid hormone regulates NHE8. Administration of thyroid hormone to neonatal rats, before the normal postnatal increase in serum thyroid hormone levels at 3 wk of age, resulted in a premature increase in NHE3/[beta]-actin BBM protein abundance and mRNA abundance. Thyroid hormone also caused a premature decrease in BBM NHE8/[beta]-actin protein abundance, whereas there was no change in mRNA expression (standardized to 28s). Rats made hypothyroid from birth were studied at 28 days, after the normal maturational increase in thyroid hormone. In these hypothyroid adult rats, the maturational increase in BBM NHE3 protein abundance and NHE3 mRNA expression was prevented. In contrast, the developmental decrease in BBM NHE8 protein abundance was prevented in hypothyroid adults, but mRNA expression was unchanged in hypothyroid rats. To determine whether the effect of thyroid hormone was due to a direct epithelial effect, we studied normal rat kidney cells in culture. We recently showed that this cell line expresses NHE8, but does not express NHE3. Thyroid hormone caused a decrease in surface expression of NHE8, determined by biotinylation, but total cellular abundance remained unchanged. NHE8 activity, measured as the sodium-dependent rate of intracellular pH recovery from an acid load, was less with thyroid treatment than control. In conclusion, thyroid hormone plays a potential role in the developmental isoform change from NHE8 to NHE3 and decreases NHE8 activity. proximal tubule; Na/H exchanger; development

Epithelial sodium channels (ENaC) are processed by proteases as they transit the biosynthetic pathway. We recently observed that furin-dependent processing of the [alpha]-subunit of ENaC at two sites within its extracellular domain is required for channel activation due to release of a 26-residue inhibitory domain. While channels with [alpha]-subunits lacking the furin sites are not cleaved and have very low activity, channels lacking the furin consensus sites as well as the tract between these sites ([alpha]D206-R231) are active. We analyzed channels with a series of deletions in the tract [alpha]D206-R231 and lacking the [alpha]-subunit furin consensus sites in Xenopus laevis oocytes. We found an eight-residue tract that, when deleted, restored channel activity to the level found in oocytes expressing wild-type ENaC. A synthetic peptide, LPHPLQRL, representing the tract [alpha]L211-L218, inhibited wild-type ENaC expressed in oocytes with an [IC.sub.50] of 0.9 [micro]M, and inhibited channels expressed in collecting duct cells and human primary airway epithelial cells with an [IC.sub.50]s of between ~50 and 100 [micro]M. Analyses of peptides with deletions within this inhibitory tract indicate that eight residues is the minimal backbone length that is required for ENaC inhibition. Analyses of 8-mer peptides with conserved and nonconserved substitutions suggest that [L.sup.1], [P.sup.2], [H.sup.3], [P.sup.4], and [L.sup.8] are required for inhibitory activity. Our findings suggest that this eight-residue tract is a key conserved inhibitory domain that provides epithelial cells with a reserve of inactive channels that can be activated as required by proteases. peptide inhibitors; proteases; furin