OBJECTIVE

It is not clear how weight loss affects histologic features of liver in patients with nonalcoholic steatohepatitis (NASH). We examined the association between the magnitude of weight loss through lifestyle modifications and changes in histologic features of NASH.

METHODS

We conducted a prospective study of 293 patients with histologically proven NASH who were encouraged to adopt recommended lifestyle changes to reduce their weight over 52 weeks, from June 2009 through May 2013, at a tertiary medical center in Havana, Cuba. Liver biopsies were collected when the study began and at week 52 of the diet and were analyzed histologically.

RESULTS

Paired liver biopsies were available from 261 patients. Among 293 patients who underwent lifestyle changes for 52 weeks, 72 (25%) achieved resolution of steatohepatitis, 138 (47%) had reductions in nonalcoholic fatty liver disease activity score (NAS), and 56 (19%) had regression of fibrosis. At week fifty-two, 88 subjects (30%) had lost ≥5% of their weight. Degree of weight loss was independently associated with improvements in all NASH-related histologic parameters (odds ratios = 1.1-2.0; P < .01). A higher proportion of subjects with ≥5% weight loss had NASH resolution (51 of 88 [58%]) and a 2-point reduction in NAS (72 of 88 [82%]) than subjects who lost <5% of their weight (P < .001). All patients who lost ≥10% of their weight had reductions NAS, 90% had resolution of NASH, and 45% had regression of fibrosis. All patients who lost 7%-10% of their weight and had few risk factors also had reduced NAS. In patients with baseline characteristics that included female sex, body mass index ≥35, fasting glucose >5.5 mmol/L, and many ballooned cells, NAS scores decreased significantly with weight reductions ≥10%.

CONCLUSIONS

A greater extent of weight loss, induced by lifestyle changes, is associated with the level of improvement in histologic features of NASH. The highest rates of NAS reduction, NASH resolution, and fibrosis regression occurred in patients with weight losses ≥10%.

Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes; however, the electrophysiological properties of hiPSC-derived cardiomyocytes have yet to be fully characterized. We performed detailed electrophysiological characterization of highly pure hiPSC-derived cardiomyocytes. Action potentials (APs) were recorded from spontaneously beating cardiomyocytes using a perforated patch method and had atrial-, nodal-, and ventricular-like properties. Ventricular-like APs were more common and had maximum diastolic potentials close to those of human cardiac myocytes, AP durations were within the range of the normal human electrocardiographic QT interval, and APs showed expected sensitivity to multiple drugs (tetrodotoxin, nifedipine, and E4031). Early afterdepolarizations (EADs) were induced with E4031 and were bradycardia dependent, and EAD peak voltage varied inversely with the EAD take-off potential. Gating properties of seven ionic currents were studied including sodium (I(Na)), L-type calcium (I(Ca)), hyperpolarization-activated pacemaker (I(f)), transient outward potassium (I(to)), inward rectifier potassium (I(K1)), and the rapidly and slowly activating components of delayed rectifier potassium (I(Kr) and I(Ks), respectively) current. The high purity and large cell numbers also enabled automated patch-clamp analysis. We conclude that these hiPSC-derived cardiomyocytes have ionic currents and channel gating properties underlying their APs and EADs that are quantitatively similar to those reported for human cardiac myocytes. These hiPSC-derived cardiomyocytes have the added advantage that they can be used in high-throughput assays, and they have the potential to impact multiple areas of cardiovascular research and therapeutic applications.

The peopling of the Americas has been the subject of extensive genetic, archaeological and linguistic research; however, central questions remain unresolved. One contentious issue is whether the settlement occurred by means of a single migration or multiple streams of migration from Siberia. The pattern of dispersals within the Americas is also poorly understood. To address these questions at a higher resolution than was previously possible, we assembled data from 52 Native American and 17 Siberian groups genotyped at 364,470 single nucleotide polymorphisms. Here we show that Native Americans descend from at least three streams of Asian gene flow. Most descend entirely from a single ancestral population that we call 'First American'. However, speakers of Eskimo-Aleut languages from the Arctic inherit almost half their ancestry from a second stream of Asian gene flow, and the Na-Dene-speaking Chipewyan from Canada inherit roughly one-tenth of their ancestry from a third stream. We show that the initial peopling followed a southward expansion facilitated by the coast, with sequential population splits and little gene flow after divergence, especially in South America. A major exception is in Chibchan speakers on both sides of the Panama isthmus, who have ancestry from both North and South America.

1. Outer hair cells from the cochlea of the guinea-pig were isolated and their motile properties studied in short-term culture by the whole-cell variant of the patch recording technique. 2. Cells elongated and shortened when subjected to voltage steps. Cells from both high- and low-frequency regions of the cochlea responded with an elongation when hyperpolarized and a shortening when depolarized. The longitudinal motion of the cell was measured by a differential photosensor capable of responding to motion frequencies 0-40 kHz. 3. Under voltage clamp the length change of the cell was graded with command voltage over a range +/- 2 microns (approximately 4% of the length) for cells from the apical turns of the cochlea. The mean sensitivity of the movement was 2.11 nm/pA injected current, or 19.8 nm/mV membrane polarization. 4. The kinetics of the cell length change during a voltage step were measured. Stimulated at their basal end, cells from the apical (low-frequency) cochlear turns responded with a latency of between 120 and 255 microseconds. The cells thereafter elongated exponentially by a process which could be characterized by three time constants, one with value 240 microseconds, and a second in the range 1.3-2.8 ms. A third time constant with a value 20-40 ms characterized a slower component which may represent osmotic changes. 5. Consistent with the linearity shown to voltage steps, sinusoidal stimulation of the cell generated movements which could be measured at frequencies above 1 kHz. The phase of the movement relative to the stimulus continued to grow with frequency, suggesting the presence of an absolute delay in the response of about 200 microseconds. 6. The electrically stimulated movements were insensitive to the ionic composition of the cell, manipulated by dialysis from the patch pipette. The responses occurred when the major cation was K+ or Na+ in the pipette. Loading the cell with ATP-free solutions or calcium buffers did not inhibit the response. 7. It is concluded that interaction between actin and myosin, although present in the cell, is unlikely to account for the cell motility. Instead, it is proposed that outer hair cell motility is associated with structures in the cell cortex. The implications for cochlear mechanics of such force generation in outer hair cells are discussed.

The dendrites of mammalian pyramidal neurons contain a rich collection of active conductances that can support Na+ and Ca2+ action potentials (for a review see ref. 1). The presence, site of initiation, and direction of propagation of Na+ and Ca2+ action potentials are, however, controversial, and seem to be sensitive to resting membrane potential, ionic composition, and degree of channel inactivation, and depend on the intensity and pattern of synaptic stimulation. This makes it difficult to extrapolate from in vitro experiments to the situation in the intact brain. Here we show that two-photon excitation laser scanning microscopy can penetrate the highly scattering tissue of the intact brain. We used this property to measure sensory stimulus-induced dendritic [Ca2+] dynamics of layer 2/3 pyramidal neurons of the rat primary vibrissa (Sm1) cortex in vivo. Simultaneous recordings of intracellular voltage and dendritic [Ca2+] dynamics during whisker stimulation or current injection showed increases in [Ca2+] only in coincidence with Na+ action potentials. The amplitude of these [Ca2+] transients at a given location was approximately proportional to the number of Na+ action potentials in a short burst. The amplitude for a given number of action potentials was greatest in the proximal apical dendrite and declined steeply with increasing distance from the soma, with little Ca2+ accumulation in the most distal branches, in layer 1. This suggests that widespread Ca2+ action potentials were not generated, and any significant [Ca2+] increase depends on somatically triggered Na+ action potentials.

A Na(+)- and Cl(-)-coupled serotonin (5-hydroxytryptamine, 5HT) transporter is expressed on human neuronal, platelet, placental, and pulmonary membranes. The brain 5HT transporter appears to be a principal site of action of therapeutic antidepressants and may mediate behavioral and/or toxic effects of cocaine and amphetamines. Oligonucleotides derived from consensus transporter sequences were used to identify human placental cDNAs highly related to the rat brain 5HT carrier. Transfection of one of these cDNAs into HeLa cells yields a high-affinity (Km = 463 nM), Na(+)- and Cl(-)-dependent 5HT transport activity which can be blocked by selective 5HT transport inhibitors, including paroxetine, fluoxetine, and imipramine, and which is antagonized by cocaine and amphetamine. Sequence analysis reveals a 630-amino acid open reading frame bearing 92% identity to the cloned rat brain 5HT transporter, with identical predicted topological features and conserved sites for posttranslational modifications. Unlike the rodent, where a single mRNA appears to encode 5HT transporters, multiple hybridizing RNAs are observed in human placenta and lung. Somatic cell hybrid and in situ hybridization studies are consistent, however, with a single gene encoding the human 5HT transporter, localized to chromosome 17q11.1-17q12.

Since the turn of the century, the prefrontal association areas of the cerebral cortex have been thought to be among the last regions of the cortical mantle to develop. We have examined the course of synaptogenesis in the macaque prefrontal cortex by quantitative electron microscopic analysis in 25 rhesus monkeys ranging in age from embryonic day 47 (E47) to 20 years of age. A series of overlapping electron micrographs spanning the whole cortical thickness in each animal provided data on the number, the proportion, and the density of synapses per unit area (NA) and per unit volume (NV) of neuropil. The tempo and kinetics of synapse formation in prefrontal cortex closely resemble those described for sensory and motor areas, particularly during the stages of synapse acquisition and overproduction (Rakic et al., 1986). In young embryos, we describe a precortical phase (E47-E78), when synapses are found only above and below, but not within, the cortical plate. Following that, there is an early cortical phase, from E78 to E104, during which synapses accumulate within the cortical plate, initially exclusively on dendritic shafts. The next rapid phase of synaptogenesis begins at 2 months before birth and ends approximately at 2 months after birth, culminating with a mean density of 750 million synapses per cubic micrometer. This accumulation is largely accounted for by a selective increase in axospine synapses in the supragranular layers. The period of explosive synaptic density is followed by a protracted plateau stage that lasts from 2 months to 3 years of age when synaptic density remains relatively constant. The final period of decline, from 3 years through over 20 years of age, is marked by a slight but statistically significant decline in synaptic density. Concurrent recruitment of synapses with that of sensory and motor areas supports the concept that the initial establishment of cortical circuitry is governed by general mechanisms common to all areas, independent of their specific functional domain. The finding that synaptic density is relatively stable from early adolescence through puberty (the plateau period) is indicative of the importance, in primates, of a consistent and high synaptic density during the formative years when learning experiences are most intense.

The roles of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Na(+)-Ca2+ exchange in Ca2+ removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration ([Ca2+]i) were measured in indo-1-loaded cells. Na(+)-Ca2+ exchange was inhibited by replacement of external Na+ by Li+. To avoid net changes in cell or SR Ca2+ load during a twitch in 0 Na+ solution, intracellular Na+ (Na+i) was depleted using a long pre-perfusion with 0 Na+, 0 Ca2+ solution. SR Ca2+ accumulation was inhibited by caffeine or thapsigargin (TG). Relaxation of steady-state twitches was 2-fold faster in rat than in rabbit (before and after Na+i depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half-time for relaxation of caffeine contractures 15- and 5-fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. Inhibition of the Na(+)-Ca2+ exchange during a twitch increased the [Ca2+]i transient amplitude (delta[Ca2+]i) by 50% and the time constant of [Ca2+]i decline (tau) by 45% in rabbit myocytes. A smaller increase in tau (20%) and no change in delta[Ca2+]i were observed in rat cells in 0 Na+ solution. [Ca2+]i transients remained more rapid in rat cells. Inhibition of the SR Ca(2+)-ATPase during a twitch enhanced delta[Ca2+]i by 25% in both species. The increase in tau after TG exposure was greater in rat (9-fold) than in rabbit myocytes (2-fold), which caused [Ca2+]i decline to be 70% slower in rat compared with rabbit cells. The time course of [Ca2+]i decline during twitch in TG-treated cells was similar to that during caffeine application in control cells. Combined inhibition of these Ca2+ transport systems markedly slowed the time course of [Ca2+]i decline, so that tau was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca2+ transport mechanisms (mitochondrial Ca2+ uptake and sarcolemmal Ca(2+)-ATPase) is similar in these species. We conclude that during the decline of the [Ca2+]i transient, the Na(+)-Ca2+ exchange is about 2- to 3-fold faster in rabbit than in rat, whereas the SR Ca(2+)-ATPase is 2- to 3-fold faster in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on proton and Na+ transport by isolated intestinal and renal brush-border-membrane vesicles were carried out to test for the presence of an Na+/H+-exchange system. Proton transport was evaluated as proton transfer from the intravesicular space to the incubation medium by monitoring pH changes in the membrane suspension induced by sudden addition of cations. Na+ transport was determined as Na+ uptake into the vesicles by filtration technique. A sudden addition of sodium salts (but not choline) to the membrane suspension provokes an acidification of the incubation medium which is abolished by the addition of 0.5% Triton X-100. Pretreatment of the membranes with Triton X-100 prevents the acidification. The acidification is also not observed if the [K+] and proton conductance of the membranes have been increased by the simultaneous addition of valinomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the K+-rich incubation medium. Either valinomycin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone when added alone do not alter the response of the membranes to the addition of Na+. Na+ uptake by brush-border microvilli is enhanced in the presence of a proton gradient directed from the intravesicular space to the incubation medium. Under these conditions a transient accumulation of Na+ inside the vesicles is observed. It is concluded that intestinal and renal brush-border membranes contain a NA+/H+ antiport system which catalyses an electroneutral exchange of Na+ against protons and consequently can produce a proton gradient in the presence of a concentration difference for Na+. This system might be involved in the active proton secretion of the small intestine and the proximal tubule of the kidney.

BACKGROUND

Ranolazine is a novel antianginal agent capable of producing antiischemic effects at plasma concentrations of 2 to 6 micromol/L without reducing heart rate or blood pressure. The present study examines its electrophysiological effects in isolated canine ventricular myocytes, tissues, and arterially perfused left ventricular wedge preparations.

RESULTS

Transmembrane action potentials (APs) from epicardial and midmyocardial (M) regions and a pseudo-ECG were recorded simultaneously from wedge preparations. APs were also recorded from epicardial and M tissues. Whole-cell currents were recorded from epicardial and M myocytes. Ranolazine inhibited I(Kr) (IC50=11.5 micromol/L), late I(Na), late I(Ca), peak I(Ca), and I(Na-Ca) (IC50=5.9, 50, 296, and 91 micromol/L, respectively) and I(Ks) (17% at 30 micromol/L), but caused little or no inhibition of I(to) or I(K1). In tissues and wedge preparations, ranolazine produced a concentration-dependent prolongation of AP duration of epicardial but abbreviation of that of M cells, leading to reduction or no change in transmural dispersion of repolarization (TDR). At [K+]o=4 mmol/L, 10 micromol/L ranolazine prolonged QT interval by 20 ms but did not increase TDR. Extrasystolic activity and spontaneous torsade de pointes (TdP) were never observed, and stimulation-induced TdP could not be induced at any concentration of ranolazine, either in normal or low [K+]o. Ranolazine (5 to 20 micromol/L) suppressed early afterdepolarizations (EADs) and reduced the increase in TDR induced by the selective I(Kr) blocker d-sotalol.

CONCLUSIONS

Ranolazine produces ion channel effects similar to those observed after chronic amiodarone (reduced I(Kr), I(Ks), late I(Na), and I(Ca)). The actions of ranolazine to suppress EADs and reduce TDR suggest that, in addition to its antianginal actions, the drug may possess antiarrhythmic activity.

Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/L-aspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxy-aspartate. In situ hybridization reveals a strikingly high density of GLAST mRNA in the Purkinje cell layer of cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.

The effect of tetraethylammonium ion (TEA) on the voltage clamp currents of nodes of Ranvier of frog myelinated nerve fibers is studied. The delayed K currents can be totally abolished by TEA without affecting the transient Na currents or the leakage current in any way. Both inward and outward currents disappear. In low TEA concentrations small K currents remain with normal time constants. The dose-response relationship suggests the formation of a complex between TEA and a receptor with a dissociation constant of 0.4 mM. Other symmetrical quaternary ammonium ions have very little effect. There is no competition between TEA and agents that affect the Na currents such as Xylocaine, tetrodotoxin, or Ca ions. The pharmacological data demonstrate that the Na, K, and leakage permeabilities are chemically independent, probably because their mechanisms occupy different sites on the nodal membrane. The data are gathered and analyzed by digital computer.

Inherited hypokalaemic alkalosis with low blood pressure can be divided into two groups-Gitelman's syndrome, featuring hypocalciuria, hypomagnesaemia and milder clinical manifestations, and Bartter's syndrome, featuring hypercalciuria and early presentation with severe volume depletion. Mutations in the renal Na-Cl cotransporter have been shown to cause Gitelman's syndrome. We demonstrate linkage of Bartter's syndrome to the renal Na-K-2Cl cotransporter gene NKCC2, and identify frameshift or non-conservative missense mutations for this gene that co-segregate with the disease. These findings demonstrate the molecular basis of Bartter's syndrome, provide the basis for molecular classification of patients with inherited hypokalaemic alkalosis, and suggest potential phenotypes in heterozygous carriers of NKCC2 mutations.

This paper describes the making and testing of calcium-selective microelectrodes for measurement of intracellular [free Ca2+] levels. Pipettes of tip outer diameter down to 0.4 micron were siliconized by a novel and easy method of vapor treatment. The tips were filled with a sensor mixture using the neutral ligand and solvent of Oehme et al. (Oehme, M., Kessler, M. and Simon, W. (1976) Chimia (Aarau) 30, 204-206) but with very hydrophobic cations replacing Na+ in the salt component. This change improved electrode stability and greatly reduced hysteresis in the responses to changing [Ca2+] levels. Lowering the Ca2+ concentration in the internal electrolyte also increased electrode lifetime. Electrodes showed a Nernstian response to [Ca2+] down to 1 micro M free concentration in 0.1 M KCl, and usually a useful response to below 100 nM Ca2+. Selectivity for Ca2+ over Mg2+ and H+ was sufficiently high that typical free intracellular levels of Mg2+ and H+ caused negligible interference. Selectivity for Ca2+ over Na+ was adequate for cells with 10(-2) M free Na+, but higher levels could raise significantly the detection limit for Ca2+. Preliminary measurements of [free Ca2+] have been made in frog skeletal muscle, ferret ventricular myocardium, and early embryos of Xenopus laevis. Relative merits of Ca2+ microelectrodes and optical indicators are discussed.

Calcineurin is a conserved Ca2+/calmodulin-dependent protein phosphatase that plays a critical role in Ca2+ signaling. We describe new components of a calcineurin-mediated response in yeast, the Ca2+-induced transcriptional activation of FKS2, which encodes a beta-1,3 glucan synthase. A 24-bp region of the FKS2 promoter was defined as sufficient to confer calcineurin-dependent transcriptional induction on a minimal promoter in response to Ca2+ and was named CDRE (for calcineurin-dependent response element). The product of CRZ1 (YNL027w) was identified as an activator of CDRE-driven transcription. Crz1p contains zinc finger motifs and binds specifically to the CDRE. Genetic analysis revealed that crz1Delta mutant cells exhibit several phenotypes similar to those of calcineurin mutants and that overexpression of CRZ1 in calcineurin mutants suppressed these phenotypes. These results suggest that Crz1p functions downstream of calcineurin to effect multiple calcineurin-dependent responses. Moreover, the calcineurin-dependent transcriptional induction of FKS2 in response to Ca2+, alpha-factor, and Na+ was found to require CRZ1. In addition, we found that the calcineurin-dependent transcriptional regulation of PMR2 and PMC1 required CRZ1. However, transcription of PMR2 and PMC1 was activated by only a subset of the treatments that activated FKS2 transcription. Thus, in response to multiple signals, calcineurin acts through the Crz1p transcription factor to differentially regulate the expression of several target genes in yeast.

We present the complete sequence of a cDNA encoding the human amiloride-sensitive Na+/H+ antiporter. After functional complementation of a mouse fibroblast mutant by gene transfer, we isolated a 0.8 kb genomic probe from a third-cycle mouse transformant. The probe detects gene amplification in Na+/H+ antiporter "overexpressers" and a single class of mRNA of ca. 5.6 kb in human, mouse, and hamster cells. With this probe we isolated a 4 kb cDNA from a library constructed from a mouse transformant in which the transfected human gene was amplified. This cDNA includes a noncoding leader of 407 bp, a 2682 bp open reading frame, and a 3' noncoding sequence containing a mouse B1 repeated element. The amino acid sequence predicts a protein of Mr = 99,354 with an N-terminal amphipathic domain that contains 10 putative transmembrane-spanning segments and two potential glycosylation sites, followed by a hydrophilic stretch of 395 residues, presumably cytoplasmic. Stable expression of the transfected cDNA in Na+/H+ antiporter-deficient cells restored the key functional features of this transporter: H+i-activated Na+ influx, amiloride sensitivity, and pHi regulation.

1. The membrane currents in Purkinje fibres under voltage clamp conditions have been investigated in the range of potentials at which the action potential plateau occurs. The results show that in this range slow outward current changes occur which are quite distinct from the potassium current activated in the pace-maker range of potentials.2. The time course of current change in response to step voltage changes is non-exponential. At each potential the current changes may be analysed in terms of the sum of two exponential changes and this property has been used to dissect the currents into two components, i(x1) and i(x2), both of which have been found to obey kinetics of the Hodgkin-Huxley type.3. The first component, i(x1), is activated with a time constant of about 0.5 sec at the plateau. At more positive and more negative potentials the time constants are shorter. The steady-state degree of activation varies from 0 at about -50 mV to about 1 at +20 mV. The instantaneous current-voltage relation is an inward-going rectifier but shows no detectable negative slope. In normal Tyrode solution ([K](0) = 4 mM) the reversal potential is about -85 mV.4. The second component, i(x2), is activated extremely slowly and the time constant at the plateau is about 4 sec. The steady-state activation curve varies from 0 at about -40 mV to 1 at about +20 mV. The instantaneous current-voltage relation is nearly linear. The reversal potential occurs between -50 and -20 mV in different preparations.5. It is suggested that these currents are carried largely by K ions, but that some other ions (e.g. Na) also contribute so that the reversal potentials are positive to E(K).6. The relation of these results to previous work on delayed rectification in cardiac muscle is discussed.

Human peripheral T lymphocytes were studied at 20-24 degrees C using the gigaohm seal recording technique in whole-cell or outside-out patch conformations. The predominant ion channel present under the conditions employed was a voltage-gated K+ channel closely resembling delayed rectifier K+ channels of nerve and muscle. The maximum K+ conductance in ninety T lymphocytes ranged from 0.7 to 8.9 nS, with a mean of 4.2 nS. The estimated number of K+ channels per cell is 400, corresponding to a density of about three channels/micron2 apparent membrane area. The activation of K+ currents could be fitted by Hodgkin-Huxley type n4 kinetics. The K+ conductance in Ringer solution was half-maximal at -40 mV. The time constant of K+ current inactivation was practically independent of voltage except near the threshold for activating the K+ conductance. Recovery from inactivation was slow and followed complex kinetics. Steady-state inactivation was half-maximal at -70 mV, and was complete at positive potentials. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+(1.0) greater than Rb+(0.77) greater than NH4+(0.10) greater than Cs+ (0.02) greater than Na+(less than 0.01). Currents through K+ channels display deviations from the independence principle. The limiting outward current increases when external K+ is increased, and Rb+ carries less inward current than expected from its relative permeability. Tail current kinetics were slowed about 2-fold by raising the external K+ concentration from 4.5 to 160 mM, and were 5 times slower in Rb+ Ringer solution than in K+ Ringer solution. Single K+ channel currents had two amplitudes corresponding to about 9 and 16 pS in Ringer solution. Replacing Ringer solution with isotonic K+ Ringer solution increased the unitary conductance and resulted in inward rectification of the unitary current-voltage relation. Comparable effects of external K+ were seen in the whole-cell conductance and instantaneous current-voltage relation. Several changes in the K+ conductance occurred during the first few minutes after achievement of the whole-cell conformation. Most are explainable by dissipation of a 10-20 mV junction potential between pipette solution and the cytoplasm, and by the use of a holding potential more negative than the resting potential. However, inactivation of K+ currents became faster and more complete, changes not accounted for by these mechanisms. K+ efflux through open K+ channels in intact lymphocytes, calculated from measured properties of K+ channels, can account for efflux values reported in resting lymphocytes, and for the increase in K+ efflux upon mitogenic stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Transport of L-lactate across the plasma membrane is of considerable importance to almost all mammalian cells. In most cells a specific H(+)-monocarboxylate cotransporter is largely responsible for this process; the capacity of this carrier is usually very high, to support the high rates of production or utilization of L-lactate. The best characterized H(+)-monocarboxylate transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates, including pyruvate and the ketone bodies acetoacetate and beta-hydroxybutyrate. This carrier is inhibited by alpha-cyanocinnamate derivatives and some stilbene disulfonates and has been identified as a protein of 35-50 kDa on the basis of purification and specific labeling experiments. Other cells possess similar alpha-cyanocinnamate-sensitive H(+)-linked monocarboxylate transporters, but in some cases there are significant differences in the properties of these systems, sufficient to suggest the existence of a family of such carriers. In particular, cardiac muscle and tumor cells have transporters that differ in their Km values for certain substrates (including stereoselectivity for L- over D-lactate) and in their sensitivity to inhibitors. Mitochondria, bacteria, and yeast also possess H(+)-monocarboxylate transporters that share some properties in common with those in the mammalian plasma membrane but are adapted to their specific roles. However, there are distinct Na(+)-monocarboxylate cotransporters on the luminal surface of intestinal and kidney epithelia, which enable active uptake of lactate, pyruvate, and ketone bodies in these tissues. This article reviews the properties of these transport systems and their role in mammalian metabolism.

The potassium-sparing diuretic amiloride has proven to be a useful pharmacological tool for elucidating the molecular basis and physiological regulation of facilitated sodium entry in tissues and cells. There are two general classes of Na+ transport mechanisms which are sensitive to this drug: 1) a conductive Na+ entry pathway found in electrically high resistance epithelia and 2) a Na+-H+ electroneutral exchange system found in certain leaky epithelia such as the renal proximal tubule. This latter system is also found in many different cellular preparations and seems to function in cell proliferation and differentiation, volume regulation, and intracellular pH regulation. In these cells, this exchange pathway becomes operational usually after some external stimuli. Much higher concentrations of amiloride are required to inhibit the exchange pathway than those required to inhibit the Na+ entry pathway. This drug is the most potent and specific inhibitor of Na+ entry found to date and thus affords the opportunity to be used as a label for the isolation of these transport moieties.

Several analogues of the tetraethylammonium (TEA(+)) ion were injected into the giant axon of the squid, and the resultant changes in time course and magnitude of the potassium current (I(K)) were studied. For all the analogues used, three of the ethyl side chains of TEA(+) were left unchanged, while the fourth chain was either lengthened or shortened. Increasing the length of this chain increased binding to the blocking site in the channel by a factor of roughly two for each added CH(2) group. The effect on the rate of entry into the blocking site was relatively slight. Thus the concentration for half-suppression of g(K) decreased by about the same factor of two for each added CH(2). All the analogues caused anomalous or ingoing rectification. The longest chain analogue used, pentyltriethylammonium ion, caused rapid inactivation of g(K), and this inactivation had properties quite similar to g(Na) inactivation. The anomalous rectification and the g(K) inactivation caused by these compounds have the same basic mechanism.

Since the initiation of work on mitochondrial Ca2+ transport in the early 1960s, the relationship between experimental observations and physiological function has often seemed enigmatic. Why, for example, should an organelle dedicated to the crucial task of producing approximately 95% of the cell's ATP sequester Ca2+, sometimes in preference to phosphorylating ADP? Why should there be two separate efflux mechanisms, the Na+ independent and the Na+ dependent, both thought until recently to be driven exclusively either directly or indirectly by the energy of the pH gradient? Does intramitochondrial free Ca2+ concentration control metabolism? Is there evidence for any separate function of the mitochondrial Ca2+ transport mechanisms under pathological conditions? What is the relationship between mitochondrial Ca2+ transport, the mitochondrial membrane permeability transition, and irreversible cell damage under pathological conditions? First, we review what is known about control of metabolism, evidence for a role for intramitochondrial Ca2+ in control of metabolism, the cellular conditions under which mitochondria are exposed to Ca2+, characteristics of the mitochondrial Ca2+ transport mechanisms including the permeability transition, and evidence for and against mitochondrial Ca2+ uptake in vivo. Then the questions listed above and others are addressed from the perspective of the characteristics of the mechanisms of mitochondrial Ca2+ transport.

Transgenic tomato plants overexpressing a vacuolar Na+/H+ antiport were able to grow, flower, and produce fruit in the presence of 200 mM sodium chloride. Although the leaves accumulated high sodium concentrations, the tomato fruit displayed very low sodium content. Contrary to the notion that multiple traits introduced by breeding into crop plants are needed to obtain salt-tolerant plants, the modification of a single trait significantly improved the salinity tolerance of this crop plant. These results demonstrate that with a combination of breeding and transgenic plants it could be possible to produce salt-tolerant crops with far fewer target traits than had been anticipated. The accumulation of sodium in the leaves and not in the fruit demonstrates the utility of such a modification in preserving the quality of the fruit.