Bile acids are transported across the ileal enterocyte brush border membrane by the well characterized apical sodium-dependent bile acid transporter (Asbt) Slc10a2; however, the carrier(s) responsible for transporting bile acids across the ileocyte basolateral membrane into the portal circulation have not been fully identified. Transcriptional profiling of wild type and Slc10a2 null mice was employed to identify a new candidate basolateral bile acid carrier, the heteromeric organic solute transporter (Ost)alpha-Ostbeta. By Northern blot analysis, Ostalpha and Ostbeta mRNA was detected only in mouse kidney and intestine, mirroring the horizontal gradient of expression of Asbt in the gastrointestinal tract. Analysis of Ostalpha and Ostbeta protein expression by immunohistochemistry localized both subunits to the basolateral surface of the mouse ileal enterocyte. The transport properties of Ostalpha-Ostbeta were analyzed in stably transfected Madin-Darby canine kidney cells. Co-expression of mouse Ostalpha-Ostbeta, but not the individual subunits, stimulated Na(+)-independent bile acid uptake and the apical-to-basolateral transport of taurocholate. In contrast, basolateral-to-apical transport was not affected by Ostalpha-Ostbeta expression. Co-expression of Ostalpha and Ostbeta was required to convert the Ostalpha subunit to a mature glycosylated endoglycosidase H-resistant form, suggesting that co-expression facilitates the trafficking of Ostalpha through the Golgi apparatus. Immunolocalization studies showed that co-expression was necessary for plasma membrane expression of both Ostalpha and Ostbeta. These results demonstrate that the mouse Ostalpha-Ostbeta heteromeric transporter is a basolateral bile acid carrier and may be responsible for bile acid efflux in ileum and other ASBT-expressing tissues.

Andersen syndrome (AS) is a rare, inherited disorder characterized by periodic paralysis, long QT (LQT) with ventricular arrhythmias, and skeletal developmental abnormalities. We recently established that AS is caused by mutations in KCNJ2, which encodes the inward rectifier K(+) channel Kir2.1. In this report, we characterized the functional consequences of three novel and seven previously described KCNJ2 mutations using a two-microelectrode voltage-clamp technique and correlated the findings with the clinical phenotype. All mutations resulted in loss of function and dominant-negative suppression of Kir2.1 channel function. In mutation carriers, the frequency of periodic paralysis was 64% and dysmorphic features 78%. LQT was the primary cardiac manifestation, present in 71% of KCNJ2 mutation carriers, with ventricular arrhythmias present in 64%. While arrhythmias were common, none of our subjects suffered sudden cardiac death. To gain insight into the mechanism of arrhythmia susceptibility, we simulated the effect of reduced Kir2.1 using a ventricular myocyte model. A reduction in Kir2.1 prolonged the terminal phase of the cardiac action potential, and in the setting of reduced extracellular K(+), induced Na(+)/Ca(2+) exchanger-dependent delayed afterdepolarizations and spontaneous arrhythmias. These findings suggest that the substrate for arrhythmia susceptibility in AS is distinct from the other forms of inherited LQT syndrome.

1. Electrical activity of neuromuscular junctions of the frog was studied in a medium (Ca-Ringer) whose Na ions had been entirely replaced by Ca.2. Spontaneous miniature end-plate potentials (m.e.p.p.s) of reduced amplitude are recorded in this abnormal ionic environment, and graded end-plate potentials can be elicited by applying depolarizing current pulses to the pre-junctional parts of the nerve.3. Addition of 5 mM tetraethylammonium (TEA) to the Ca-Ringer causes the appearance, in almost all-or-none fashion, of very large e.p.p.s (up to 45 mV in amplitude) in response to nerve stimulation.4. These ;giant' e.p.p.s occur despite the curarizing action of TEA (and its depressing effect on the amplitude of m.e.p.p.s) and they persist after application of tetrodotoxin.5. After several hours exposure to Ca-Ringer, spontaneous end-plate activity gradually declines, and eventually evoked e.p.p. responses fail. On return to normal Na-Ringer, spontaneous end-plate activity is quickly resumed, but the potentials have an abnormal, very wide, amplitude distribution.6. The results are discussed, in conjunction with relevant work on the squid giant synapse, in terms of the ;calcium hypothesis' of transmitter release.

Na+,K(+)-ATPase has distinctly different distributions in mesenchymal cells, where it has an unrestricted distribution over the entire cell surface, compared with polarized epithelial cells, where it is restricted to the basal-lateral membrane domain. The generation of this restricted distribution is important in mesenchyme to epithelia conversion in development and the function of transporting epithelia, but the mechanisms involved are unknown. Here we show that expression of the epithelial CAM uvomorulin in transfected fibroblasts is sufficient to induce a redistribution of Na+,K(+)-ATPase to sites of uvomorulin-mediated cell-cell contacts, similar to that in polarized epithelial cells. This restricted distribution of Na+,K(+)-ATPase occurs in the absence of tight junctions but coincides with the reorganization of the membrane cytoskeleton. The results indicate a direct role for CAMs as inducers of cell surface polarity of selective cytoplasmic and membrane proteins.

The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy. As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca++ concentration and fails if this ion is replaced by Mg++ or Ba++. The removal of Ca++ and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance. Resealing was achieved within 10-15 min by restoring Ca++. Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca++, was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components. Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction. The occluding junctions were also opened by the addition of the ionophore A23187, suggesting that the sealing of the contacts requires high Ca++ on the extracellular side and low Ca++ concentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7.5 degrees C). Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components. The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels; the Na+/Cl- and the cation/cation selectivity were recovered with the same time-course as the electrical resistance. The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article.

Single-channel studies were made using the patch-clamp technique of K channels in dispersed single smooth muscle cells from rabbit longitudinal jejunal muscle and guinea-pig small (less than 0.2 mm o.d.) mesenteric arteries. In isolated inside-out patches from these two types of smooth muscle cell there was a population of K channels which had single-channel conductances of about 100 pS in near physiological K gradients and about 200 pS with symmetrical 126 mM-K solutions. Their conductance and other properties distinguish them from a K channel of smaller conductance which we have previously described in these cells. The relative permeability of the channel with respect to K was 1.4 Tl:1.0 K:0.7 Rb: less than 0.05 Na: less than 0.05 Cs. Cs (1 mM applied to the outside of the membrane) interfered with inward K movement when the membrane was hyperpolarized. Rb conductance of the channel when both sides of the membrane were exposed to 126 mM-Rb was 30 pS. When the Ca concentration on the inside of the membrane ([Ca]i) was about 10(-9) M, K channel opening was rarely observed and then only at strongly positive potentials. At [Ca]i between 10(-9) M and 10(-7) M mean channel open time increased and the probability of channel opening increased steeply; both were further increased by increasing membrane positivity. At [Ca]i between 10(-6) M and 2.5 mM the channel was mainly in the open state and the probability of channel conducting state often declined with increasing membrane positivity. The effects of varying [Ca]i from 10(-7) M to 2.5 mM on the kinetic activity of a single channel was studied largely in mesenteric artery patches containing one active channel. The distribution of open times could be fitted by a single exponential at low (less than 10(-6) M) [Ca]i but a component of fast openings (to less than 1.0 ms) was observed at all potentials at [Ca]i 2.5 mM. Closed time distribution required the sum of three exponentials to fit it all [Ca]i greater than 10(-7) M; at [Ca]i 10(-6) M or greater evidence of a fourth component, probably caused by Ca block of open channels, was obtained. Raising [Ca]i increased the mean duration of the (long) open state and decreased or had no effect on the duration of short, intermediate, and long mean closed states.

Extracellular matrix controls capillary endothelial cell sensitivity to soluble mitogens by binding integrin receptors and thereby activating a chemical signaling response that rapidly integrates with growth factor-induced signaling mechanisms. Here we report that in addition to integrins, growth factor receptors and multiple molecules that transduce signals conveyed by both types of receptors are immobilized on the cytoskeleton (CSK) and spatially integrated within the focal adhesion complex (FAC) at the site of integrin binding. FACs were rapidly induced in round cells and physically isolated from the remainder of the CSK after detergent-extraction using magnetic microbeads coated with fibronectin or a synthetic RGD-containing peptide. Immunofluorescence microscopy revealed that multiple signaling molecules (e.g., pp60c-src, pp125FAK, phosphatidylinositol-3-kinase, phospholipase C-gamma, and Na+/H+ antiporter) involved in both integrin and growth factor receptor signaling pathways became associated with the CSK framework of the FAC within 15 min after binding to beads coated with integrin ligands. Recruitment of tyrosine kinases to the FAC was also accompanied by a local increase in tyrosine phosphorylation, as indicated by enhanced phosphotyrosine staining at the site of integrin binding. In contrast, neither recruitment of signaling molecules nor increased phosphotyrosine staining was observed when cells bound to beads coated with a control ligand (acetylated low density lipoprotein) that ligates transmembrane scavenger receptors, but does not induce FAC formation. Western blot analysis confirmed that FACs isolated using RGD-beads were enriched for pp60c-src, pp125FAK, phospholipase C-gamma, and the Na+/H+ antiporter when compared with intact CSK or basal cell surface preparations that retained lipid bilayer. Isolated FACs were also greatly enriched for the high affinity fibroblast growth factor receptor flg. Most importantly, isolated FACs continued to exhibit multiple chemical signaling activities in vitro, including protein tyrosine kinase activities (pp60c-src and pp125FAK) as well as the ability to undergo multiple sequential steps in the inositol lipid synthesis cascade. These data suggest that many of the chemical signaling events that are induced by integrins and growth factor receptors in capillary cells may effectively function in a "solid-state" on insoluble CSK scaffolds within the FAC and that the FAC may represent a major site for signal integration between these two regulatory pathways. Future investigations into the biochemical and biophysical basis of signal transduction may be facilitated by this method, which results in isolation of FACs that retain the CSK framework as well as multiple associated chemical signaling activities.

The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25 mM glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption, Jv, and to measure the coefficient of osmotic flow, Lp. The clearances from the perfusion fluid of all other test solutes were approximately proportional to Jv. From Lp and rates of clearances as a function of Jv and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 A) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function of Jv. From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any given Jv and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport.

Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na+ toxicity of many crop plants is correlated with overaccumulation of Na+ in the shoot. We have previously suggested that the engineering of Na+ exclusion from the shoot could be achieved through an alteration of plasma membrane Na+ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na+ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na+ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na+ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na+. Plants with reduced shoot Na+ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na+ and grew poorly. Our results demonstrate that the modification of a specific Na+ transport process in specific cell types can reduce shoot Na+ accumulation, an important component of salinity tolerance of many higher plants.

In the CNS, there are multiple isozymes of the sodium and potassium ion-stimulated adenosine triphosphatase (Na,K-ATPase) that have differences in affinity for Na+, ATP, and cardiac glycosides. Three forms of the catalytic subunit (designated alpha 1, alpha 2, and alpha 3) are known to be derived from different genes, but little is known of the cellular distributions of the proteins or their physiological roles. Isozyme-specific monoclonal antibodies permitted the immunofluorescent localization of the 3 Na,K-ATPases in the rat CNS, and markedly different patterns of staining were seen. All 3 isozymes were detected, singly or in combination, in 1 or more neuronal structures, while both alpha 1 and alpha 2 were detected in glia. Many different neuroanatomic structures or cell types stained for more than 1 isozyme. Even when a structure or region stained for more than 1 isozyme, the pattern of staining was frequently dissimilar, suggesting complex differences in gene expression and cellular localization.

Since the initial description of nonalcoholic steatohepatitis (NASH), several sets of pathologic criteria for its diagnosis have been proposed. However, their interprotocol agreement and ability to predict long-term liver-related mortality (LRM) have not been demonstrated. In this study, we examined patients with biopsy-proven nonalcoholic fatty liver disease (NAFLD) for whom liver biopsy slides and clinical and mortality data were available. Liver biopsy samples were evaluated for a number of pathologic features and were classified according to the presence or absence of NASH by (1) the original criteria for NAFLD subtypes, (2) the nonalcoholic fatty liver disease activity score (NAS), (3) the Brunt criteria, and (4) the current study's criteria. All NASH diagnostic criteria and individual pathologic features were tested for agreement and for their independent associations with LRM, which were determined with a Cox proportional hazards model. Two hundred fifty-seven NAFLD patients with complete data were included. The diagnoses of NASH by the original NAFLD subtypes and by the current study's definition of NASH were in almost perfect agreement (κ = 0.896). However, their agreement was moderate with NAS (κ = 0.470 and κ = 0.511, respectively) and only fair to moderate with the Brunt criteria (κ = 0.365 and κ = 0.441, respectively). Furthermore, the agreement of the Brunt criteria with NAS was relatively poor (κ = 0.178). During the follow-up (median = 146 months), 31% of the patients died (9% were LRM). After we controlled for confounders, a diagnosis of NASH by the original criteria for NAFLD subtypes [adjusted hazard ratio = 9.94 (95% confidence interval = 1.28-77.08)] demonstrated the best independent association with LRM. Among the individual pathologic features, advanced fibrosis showed the best independent association with LRM [adjusted hazard ratio = 5.68 (95% confidence interval = 1.50-21.45)].

CONCLUSIONS

The original criteria for NAFLD subtypes and the current study's criteria for NASH were in almost perfect agreement, but their level of agreement with the NAS and Brunt criteria was lower. A diagnosis of NASH by the original criteria for NAFLD subtypes demonstrated the best predictability for LRM in NAFLD patients.

We present an integrated thermokinetic model describing control of cardiac mitochondrial bioenergetics. The model describes the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial Ca(2+) handling. The kinetic component of the model includes effectors of the TCA cycle enzymes regulating production of NADH and FADH(2), which in turn are used by the electron transport chain to establish a proton motive force (Delta mu(H)), driving the F(1)F(0)-ATPase. In addition, mitochondrial matrix Ca(2+), determined by Ca(2+) uniporter and Na(+)/Ca(2+) exchanger activities, regulates activity of the TCA cycle enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. The model is described by twelve ordinary differential equations for the time rate of change of mitochondrial membrane potential (Delta Psi(m)), and matrix concentrations of Ca(2+), NADH, ADP, and TCA cycle intermediates. The model is used to predict the response of mitochondria to changes in substrate delivery, metabolic inhibition, the rate of adenine nucleotide exchange, and Ca(2+). The model is able to reproduce, qualitatively and semiquantitatively, experimental data concerning mitochondrial bioenergetics, Ca(2+) dynamics, and respiratory control. Significant increases in oxygen consumption (V(O(2))), proton efflux, NADH, and ATP synthesis, in response to an increase in cytoplasmic Ca(2+), are obtained when the Ca(2+)-sensitive dehydrogenases are the main rate-controlling steps of respiratory flux. These responses diminished when control is shifted downstream (e.g., the respiratory chain or adenine nucleotide translocator). The time-dependent behavior of the model, under conditions simulating an increase in workload, closely reproduces experimentally observed mitochondrial NADH dynamics in heart trabeculae subjected to changes in pacing frequency. The steady-state and time-dependent behavior of the model support the hypothesis that mitochondrial matrix Ca(2+) plays an important role in matching energy supply with demand in cardiac myocytes.

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl(-) blocker, whereas CLDN16 functioned as a Na(+) channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl(-) permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC.

The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.

The voltage-gated sodium channel Na(V)1.7 is preferentially expressed in peripheral somatic and visceral sensory neurons, olfactory sensory neurons and sympathetic ganglion neurons. Na(V)1.7 accumulates at nerve fibre endings and amplifies small subthreshold depolarizations, poising it to act as a threshold channel that regulates excitability. Genetic and functional studies have added to the evidence that Na(V)1.7 is a major contributor to pain signalling in humans, and homology modelling based on crystal structures of ion channels suggests an atomic-level structural basis for the altered gating of mutant Na(V)1.7 that causes pain.

C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH2-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K+ ions from the internal solution exposes conduction of Na+ and Li+ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear "inactivated" (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K+ ions prevent Na+ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K+ relative to the permeability of Na+, thus altering the ion selectivity of the channel.

Mutations in the gene encoding for the Na(+)-glucose co-transporter SGLT2 (SLC5A2) associate with familial renal glucosuria, but the role of SGLT2 in the kidney is incompletely understood. Here, we determined the localization of SGLT2 in the mouse kidney and generated and characterized SGLT2-deficient mice. In wild-type (WT) mice, immunohistochemistry localized SGLT2 to the brush border membrane of the early proximal tubule. Sglt2(-/-) mice had glucosuria, polyuria, and increased food and fluid intake without differences in plasma glucose concentrations, GFR, or urinary excretion of other proximal tubular substrates (including amino acids) compared with WT mice. SGLT2 deficiency did not associate with volume depletion, suggested by similar body weight, BP, and hematocrit; however, plasma renin concentrations were modestly higher and plasma aldosterone levels were lower in Sglt2(-/-) mice. Whole-kidney clearance studies showed that fractional glucose reabsorption was significantly lower in Sglt2(-/-) mice compared with WT mice and varied in Sglt2(-/-) mice between 10 and 60%, inversely with the amount of filtered glucose. Free-flow micropuncture revealed that for early proximal collections, 78 ± 6% of the filtered glucose was reabsorbed in WT mice compared with no reabsorption in Sglt2(-/-) mice. For late proximal collections, fractional glucose reabsorption was 93 ± 1% in WT and 21 ± 6% in Sglt2(-/-) mice, respectively. These results demonstrate that SGLT2 mediates glucose reabsorption in the early proximal tubule and most of the glucose reabsorption by the kidney, overall. This mouse model mimics and explains the glucosuric phenotype of individuals carrying SLC5A2 mutations.

The affect regulation model of binge eating, which posits that patients binge eat to reduce negative affect (NA), has received support from cross-sectional and laboratory-based studies. Ecological momentary assessment (EMA) involves momentary ratings and repeated assessments over time and is ideally suited to identify temporal antecedents and consequences of binge eating. This meta-analytic review includes EMA studies of affect and binge eating. Electronic database and manual searches produced 36 EMA studies with N = 968 participants (89% Caucasian women). Meta-analyses examined changes in affect before and after binge eating using within-subjects standardized mean gain effect sizes (ESs). Results supported greater NA preceding binge eating relative to average affect (ES = 0.63) and affect before regular eating (ES = 0.68). However, NA increased further following binge episodes (ES = 0.50). Preliminary findings suggested that NA decreased following purging in bulimia nervosa (ES = -0.46). Moderators included diagnosis (with significantly greater elevations of NA prior to bingeing in binge eating disorder compared to bulimia nervosa) and binge definition (with significantly smaller elevations of NA before binge vs. regular eating episodes for the Diagnostic and Statistical Manual of Mental Disorders definition compared to lay definitions of binge eating). Overall, results fail to support the affect regulation model of binge eating and challenge reductions in NA as a maintenance factor for binge eating. However, limitations of this literature include unidimensional analyses of NA and inadequate examination of affect during binge eating, as binge eating may regulate only specific facets of affect or may reduce NA only during the episode.

OBJECTIVE

To determine whether proton MRS (1H MRS) can detect long-term metabolite abnormalities in abstinent methamphetamine users.

BACKGROUND

Methamphetamine is toxic to dopaminergic and serotonergic neurons in rodents; however, little data are available on the toxic effects of methamphetamine on the human brain.

METHODS

1H MRS was performed in 26 abstinent methamphetamine abusers with a history of methamphetamine dependence (median total cumulative lifetime exposure, 3,640 g; median recency of last methamphetamine use, 4.25 months) and 24 healthy subjects without a history of drug abuse. Cerebral metabolite concentrations on 1H MRS were measured in the frontal cortex, frontal white matter, and basal ganglia.

RESULTS

The concentration of N-acetylaspartate ([NA]), a neuronal marker, was reduced significantly (-5 to -6%) in the basal ganglia and frontal white matter of methamphetamine users compared with control subjects. The frontal white matter [NA] correlated inversely with the logarithm of the lifetime methamphetamine use. The methamphetamine users also showed significantly reduced total creatine in the basal ganglia (-8%), and increased choline-containing compounds ([CHO], +13%) and myo-inositol ([MI], +11%) in the frontal grey matter.

CONCLUSIONS

The reduced [NA] on 1H MRS provides evidence for long-term neuronal damage in abstinent methamphetamine users.

Properties of a mechano-electrical transduction channel were studied in enzymatically dissociated chick vestibular hair cells by using a whole-cell recording variation of the patch voltage-clamp technique. The apical hair bundle was stimulated by a glass rod which moved along a one-dimensional axis when stimulated by either a triangular or a trapezoidal command voltage. The motion of the glass rod was monitored optically using a photodiode. In response to triangular stimuli, the hair cell generated a current of triangular wave form with occasional step-like spiky or zigzag-appearing events. Control experiments confirmed that the current was generated only when the hair bundle was displaced towards the tallest stereocilium. The mechano-sensitive current was blocked by streptomycin and by neomycin. The blockage by streptomycin was clearly voltage dependent: the reduction of the current became larger with hyperpolarization of the membrane. This suggests that the positively charged antibiotic molecules plug the mechanically gated channels. From the evidence presented in 3 and 4 above, the mechano-sensitive current recorded here was identified as the mechano-electrical transduction (m-e.t.) current. The permeability of the m-e.t. channel to various monovalent cations was determined from reversal potential measurements. Since a CsCl-EGTA intracellular medium was used, all the permeabilities were calculated relative to PCs. The sequence of permeabilities was Li greater than Na greater than or equal to K greater than or equal to Rb greater than Cs greater than choline greater than TMA greater than TEA. External Ca ions were indispensable for the recording of transduction current and Sr ions could replace Ca ions without loss of the transduction activity. The minimum [Ca]o for stable generation of the m-e.t. current was 20 microM in Cs saline. The addition of 50-200 microM-Ca to the isotonic Ba saline could maintain the m-e.t. current. The m-e.t. current was observed in isotonic Ca and in Sr salines. Isotonic Ba, Mg and Mn salines were enriched with 1-2 mM-Ca in order to generate the m-e.t. current. The permeabilities of the divalent cations relative to Cs were calculated from the reversal potentials, and the sequence of permeabilities among divalent cations was Ca greater than Sr greater than Ba greater than Mn greater than Mg. Step-like m-e.t. currents were observed in Cs saline. The smallest step amplitude with clear resolution had a conductance of 49.7 +/- 4.5 pS (mean +/- S.D., n = 7 cells). This is likely to be an elementary m-e.t. channel conductance.(ABSTRACT TRUNCATED AT 400 WORDS)

The NHE family of ion exchangers includes six isoforms (NHE1-NHE6) that function in an electroneutral exchange of intracellular H(+) for extracellular Na(+). This review focuses on the only ubiquitously expressed isoform, NHE1, which is localized at the plasma membrane where it plays a critical role in intracellular pH (pHi) and cell volume homeostasis. All NHE isoforms share a similar topology: an N-terminus of 12 transmembrane (TM) alpha-helices that collectively function in ion exchange, and a C-terminal cytoplasmic regulatory domain that modulates transport activity by the TM domain. Extracellular signals, mediated by diverse classes of cell-surface receptors, regulate NHE1 activity through distinct signaling networks that converge to directly modify the C-terminal regulatory domain. Modifications in the C-terminus, including phosphorylation and the binding of regulatory proteins, control transport activity by altering the affinity of the TM domain for intracellular H(+). Recently, it was determined that NHE1 also functions as a membrane anchor for the actin-based cytoskeleton, independently of its role in ion translocation. Through its effects on pHi homeostasis, cell volume, and the actin cortical network, NHE1 regulates a number of cell behaviors, including adhesion, shape determination, migration, and proliferation.

Membrane currents through the Ca2+ channel were studied in a hybridoma cell line (MAb-7B) constructed by fusion of S194 myeloma cells and splenic B lymphocytes from the mouse. The whole-cell variation of the patch-electrode voltage-clamp technique was used. When [Ca2+]o = 2.5 mM, [Na+]o = 150 mM and [Na+]i = 155 mM, the current reversed from inward to outward at 20.9 +/- 2.4 mV (mean +/- S.D., n = 62). Both inward and outward currents showed voltage-dependent inactivation with the same membrane potential dependence of steady-state inactivation. The decay time constant of the current decreased from about 27 ms at -44 mV to a saturation value of 16 ms at about -20 mV, and remained at this value even when the current became outward. From the above results both the inward and outward currents were considered to flow through Ca2+ channels. The inward current showed no change when the external Na+ was replaced with Cs+ or tetraethylammonium and increased when [Ca2+]o was increased. Also, the reversal potential became more positive with increasing [Ca2+]o with a slope of 29 mV/decade change of [Ca2+]o. Effects of different divalent cations examined at 10 mM concentration showed the reversal potential to become more positive in the order of Mn2+, Sr2+ approximately equal to Ba2+ and Ca2+ whereas the relative maximum amplitudes of peak inward current were 1.0 for Ca2+, 1.24 for Sr2+, 0.99 for Ba2+ and 0.07 for Mn2+. When [Ca2+]o or [Mg2+]o was reduced by chelators, monovalent cations became capable of carrying inward current through the Ca2+ channel. These monovalent currents share common kinetic properties with the Ca2+ current, as judged from the steady-state inactivation and the decay time constant of the current. The monovalent cation current was blocked by divalent cations in a voltage-dependent manner. The half-blocking concentrations of Ca2+ and Mg2+ at -45 mV were 2.0 X 10(-6) M and 3.0 X 10(-5) M respectively. The same voltage-dependent binding mechanism can explain the outward current carried by monovalent cations at large positive potentials at normal Ca2+ concentrations. The suppression of the monovalent currents by Ca2+ and Mg2+ showed different voltage dependences. The suppression by Ca2+ increased and then decreased as the membrane potential was made negative, whereas the suppression by Mg2+ increased monotonically. This difference can be explained by considering the fact the Ca2+ is permeant and Mg2+ is impermeant through the Ca2+ channel.

In mammals, the perception of pain is initiated by the transduction of noxious stimuli through specialized ion channels and receptors expressed by nociceptive sensory neurons. The molecular mechanisms responsible for the specification of distinct sensory modality are, however, largely unknown. We show here that Runx1, a Runt domain transcription factor, is expressed in most nociceptors during embryonic development but in adult mice, becomes restricted to nociceptors marked by expression of the neurotrophin receptor Ret. In these neurons, Runx1 regulates the expression of many ion channels and receptors, including TRP class thermal receptors, Na+-gated, ATP-gated, and H+-gated channels, the opioid receptor MOR, and Mrgpr class G protein coupled receptors. Runx1 also controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. Thus, Runx1 coordinates the phenotype of a large cohort of nociceptors, a finding with implications for pain therapy.

Electrical properties of the muscle fiber membrane were studied in the barnacle, Balanus nubilus Darw. by using intracellular electrode techniques. A depolarization of the membrane does not usually produce an all-or-none spike potential in the normal muscle fiber even though a mechanical response is elicited. The intracellular injection of Ca(++)-binding agents (K(2)SO(4) and K salt of EDTA solution, K(3) citrate solution, etc.) renders the fiber capable of initiating all-or-none spikes. The overshoot of such a spike potential increases with increasing external Ca concentration, the increment for a tenfold increase in Ca concentration being about 29 mv. The threshold membrane potential for the spike and also for the K conductance increase shifts to more positive membrane potentials with increasing [Ca(++)](out). The removal of Na ions from the external medium does not change the configuration of the spike potential. In the absence of Ca(++) in the external medium, the spike potential is restored by Ba(++) and Sr(++) but not by Mg(++). The overshoot of the spike potential increases with increasing [Ba(++)](out) or [Sr(++)](out). The Ca influx through the membrane of the fiber treated with K(2)SO(4) and EDTA was examined with Ca(45). The influx was 14 pmol per sec. per cm(2) for the resting membrane and 35 to 85 pmol per cm(2) for one spike. From these results it is concluded that the spike potential of the barnacle muscle fiber results from the permeability increase of the membrane to Ca(++) (Ba(++) or Sr(++)).