External Sodium Concentration Research Articles

Identification and pharmacological characterization of histamine-gated chloride channels in the fall armyworm, Spodoptera frugiperda

Histamine-gated chloride channels (HACls) mediate fast inhibitory neurotransmission in invertebrate nervous systems and have important roles in light reception, color processing, temperature preference and light-dark cycle. The fall armyworm, Spodoptera frugiperda is a main destructive pest of grain and row crops. However, the pharmacological characterization of HACls in S. frugiperda remain unknown. In this study, we identified two cDNAs encoding SfHACl1 and SfHACl2 in S. frugiperda. They had similar expression patterns and were most abundantly expressed in the head of larvae and at the egg stage. Electrophysiological analysis with the two-electrode voltage clamp method showed that histamine (HA) and γ-aminobutyric acid (GABA) activated inward currents when SfHACls were singly or collectively expressed with different ratios in Xenopus laevis oocytes. These channels were ≥2000-fold more sensitive to HA than to GABA. They were anion-selective channels, which were highly dependent on changes in external chloride concentrations, but insensitive to changes in external sodium concentrations. The insecticides abamectin (ABM) and emamectin benzoate (EB) also activated these channels with the EC50 to SfHACl1 lower than that to SfHACl2. And the EC50s of ABM and EB to the co-expressed channels gradually increased with increase in the injection ratio of SfHACl2 cRNA. Homology models and docking simulations revealed that HA bound to the large amino-terminal extracellular domain of SfHACl1 and SfHACl2 by forming 4 and 2 hydrogen bonds, respectively. The docking simulations of ABM and EB had similar binding sites in the transmembrane regions. Overall, these findings indicated that HACls act as targets for macrolide, and this study provides theoretical guidance for further derivatization of abamectin insecticides.

Plasma-membrane electrical responses to salt and osmotic gradients contradict radiotracer kinetics, and reveal Na+-transport dynamics in rice (Oryza sativa L.).

A systematic analysis of NaCl-dependent, plasma-membrane depolarization (∆∆Ψ) in rice roots calls into question the current leading model of rapid membrane cycling of Na+ under salt stress. To investigate the character and mechanisms of Na+ influx into roots, Na+-dependent changes in plasma-membrane electrical potentials (∆∆Ψ) were measured in root cells of intact rice (Oryza sativa L., cv. Pokkali) seedlings. As external sodium concentrations ([Na+]ext) were increased in a step gradient from 0 to 100mM, membrane potentials depolarized in a saturable manner, fitting a Michaelis-Menten model and contradicting the linear (non-saturating) models developed from radiotracer studies. Clear differences in saturation patterns were found between plants grown under low- and high-nutrient (LN and HN) conditions, with LN plants showing greater depolarization and higher affinity for Na+ (i.e., higher Vmax and lower Km) than HN plants. In addition, counterion effects on ∆∆Ψ were pronounced in LN plants (with ∆∆Ψ decreasing in the order: Cl- > SO42- > HPO 4 2- ), but not seen in HN plants. When effects of osmotic strength, Cl- influx, K+ efflux, and H+-ATPase activity on ∆∆Ψ were accounted for, resultant Km and Vmax values suggested that a single, dominant Na+-transport mechanism was operating under each nutritional condition, with Km values of 1.2 and 16mM for LN and HN plants, respectively. Comparing saturating patterns of depolarization to linear patterns of 24Na+ radiotracer influx leads to the conclusion that electrophysiological and tracer methods do not report the same phenomena and that the current model of rapid transmembrane sodium cycling may require revision.

Reactive oxygen species increase neuronal excitability via activation of nonspecific cation channel in rat medullary dorsal horn neurons.

The caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn; MDH) receives direct inputs from small diameter primary afferent fibers that predominantly transmit nociceptive information in the orofacial region. Recent studies indicate that reactive oxygen species (ROS) is involved in persistent pain, primarily through spinal mechanisms. In this study, we aimed to investigate the role of xanthine/xanthine oxidase (X/XO) system, a known generator of superoxide anion (O2·−), on membrane excitability in the rat MDH neurons. For this, we used patch clamp recording and confocal imaging. An application of X/XO (300 µM/30 mU) induced membrane depolarization and inward currents. When slices were pretreated with ROS scavengers, such as phenyl N-tert-butylnitrone (PBN), superoxide dismutase (SOD), and catalase, X/XO-induced responses decreased. Fluorescence intensity in the DCF-DA and DHE-loaded MDH cells increased on the application of X/XO. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), significantly decreased X/XO-induced depolarization. X/XO elicited an inward current associated with a linear current-voltage relationship that reversed near −40 mV. X/XO-induced depolarization reduced in the presence of La3+, a nonselective cation channel (NSCC) blocker, and by lowering the external sodium concentration, indicating that membrane depolarization and inward current are induced by influx of Na+ ions. In conclusion, X/XO-induced ROS modulate the membrane excitability of MDH neurons, which was related to the activation of NSCC.

Properties of sodium-driven bacterial flagellar motor: A two-state model approach

Bacterial flagellar motor (BFM) is one of the ion-driven molecular machines, which drives the rotation of flagellar filaments and enable bacteria to swim in viscous solutions. Understanding its mechanism is one challenge in biophysics. Based on previous models and inspired by the idea used in description of motor proteins, in this study one two-state model is provided. Meanwhile, according to corresponding experimental data, mathematical relationship between BFM membrane voltage and pH value of the environment, and relationship between internal and external sodium concentrations are given. Therefore, with model parameter values obtained by fitting theoretical results of torque-speed relation to recent experimental data, many biophysical properties of bacterial flagellar motor can be obtained for any pH values and any external sodium concentrations, including the rotation speed, stall torque (i.e. the torque generated by BFM), rotation dispersion, and rotation randomness. In this study, the single-stator BFM will be firstly analyzed, and then properties of multiple-stator BFM are addressed briefly.

Blood pressure and amiloride-sensitive sodium channels in vascular and renal cells

Sodium transport in the distal nephron is mediated by epithelial sodium channel activity. Proteolytic processing of external domains and inhibition with increased sodium concentrations are important regulatory features of epithelial sodium channel complexes expressed in the distal nephron. By contrast, sodium channels expressed in the vascular system are activated by increased external sodium concentrations, which results in changes in the mechanical properties and function of endothelial cells. Mechanosensitivity and shear stress affect both epithelial and vascular sodium channel activity. Guyton's hypothesis stated that blood pressure control is critically dependent on vascular tone and fluid handling by the kidney. The synergistic effects, and complementary regulation, of the epithelial and vascular systems are consistent with the Guytonian model of volume and blood pressure regulation, and probably reflect sequential evolution of the two systems. The integration of vascular tone, renal perfusion and regulation of renal sodium reabsorption is the central underpinning of the Guytonian model. In this Review, we focus on the expression and regulation of sodium channels, and we outline the emerging evidence that describes the central role of amiloride-sensitive sodium channels in the efferent (vascular) and afferent (epithelial) arms of this homeostatic system.

Effect of aldosterone on kinetics of intracellular sodium in cortical portion of collecting ducts in rat kidney

We studied the effect of protein kinase C inhibitor RO-31-8220 (10(-7) M) on rapid nongenomic effect of aldosterone in cells of isolated segment of distal region of collecting duct in rat kidney. Experiments with fluorescent dye Na-Green showed that the inhibitor abolished the modulating effect of aldosterone (10 nM) on intracellular sodium concentration at external sodium concentration of 14 mM. Aldosterone decreased by half the initial rate of the changes in internal sodium concentration in both 10-day and mature rats (p<0.05). Similarly to sodium channel blocker amiloride (10(-5) M), RO-31-8220 abolished rapid nongenomic effect of aldosterone on the rate of the changes in internal sodium concentration.

An empirical model to simulate sodium absorption in roses growing in a hydroponic system

Modelling nutrient absorption can contribute to the management of hi-tech cultivation systems in greenhouse horticultural production. Nevertheless, previous studies to understand the kinetics of nutrient absorption, rarely take into consideration the accumulation of salt ions in the nutrient solution. In this project we develop and validate an empirical model for sodium uptake concentration in hydroponic rose ( Rosa spp. cv. Kardinal) production. Model development and validation was conducted using a series of experiments in both greenhouse and growth chamber conditions. The model framework takes into account plant developmental stage and external sodium concentration. While model calibration data were collected at levels of sodium up to 40 mol m 3 NaCl as root environment salt concentration, model validation was carried out at lower ranges. The proposed model not only shows a high predictive capability, but also provides useful output parameters such as electrical conductivity, which is the main parameter currently monitored for managing nutrient solution in greenhouse cultivation. Incorporated as part of a larger DSS, our model can be used to improve nutrient solution management in production regions that do not have economically valid alternatives other than the use of poor quality (saline) irrigation water.

Growth and photosynthetic responses to salinity of the salt-marsh shrub Atriplex portulacoides.

Atriplex (Halimione) portulacoides is a halophytic, C(3) shrub. It is virtually confined to coastal salt marshes, where it often dominates the vegetation. The aim of this study was to investigate its growth responses to salinity and the extent to which these could be explained by photosynthetic physiology. The responses of young plants to salinity in the range 0-700 mol m(-3) NaCl were investigated in a glasshouse experiment. The performance of plants was examined using classical growth analysis, measurements of gas exchange (infrared gas analysis), determination of chlorophyll fluorescence characteristics (modulated fluorimeter) and photosynthetic pigment concentrations; total ash, sodium, potassium and nitrogen concentrations, and relative water content were also determined. Plants accumulated Na(+) approximately in proportion to external salinity. Salt stimulated growth up to an external concentration of 200 mol m(-3) NaCl and some growth was maintained at higher salinities. The main determinant of growth response to salinity was unit leaf rate. This was itself reflected in rates of CO(2) assimilation, which were not affected by 200 mol m(-3) but were reduced at higher salinities. Reductions in net photosynthetic rate could be accounted for largely by lower stomatal conductance and intercellular CO(2) concentration. Apart from possible effects of osmotic shock at the beginning of the experiment, salinity did not have any adverse effect on photosystem II (PSII). Neither the quantum efficiency of PSII (Phi(PSII)) nor the chlorophyll fluorescence ratio (F(v)/F(m)) were reduced by salinity, and lower mid-day values recovered by dawn. Mid-day F(v)/F(m) was in fact depressed more at low external sodium concentration, by the end of the experiment. The growth responses of the hygro-halophyte A. portulacoides to salinity appear largely to depend on changes in its rate of photosynthetic gas exchange. Photosynthesis appears to be limited mainly through stomatal conductance and hence intercellular CO(2) concentration, rather than by effects on PSII; moderate salinity might stimulate carboxylation capacity. This is in contrast to more extreme halophytes, for which an ability to maintain leaf area can partially offset declining rates of carbon assimilation at high salinity.

Nonequivalence of Membrane Voltage and Ion-Gradient as Driving Forces for the Bacterial Flagellar Motor at Low Load

Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential ( V m) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V m in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V m using the ratio of intracellular/extracellular dye concentration. We found V m = −140 ± 14 mV in Escherichia coli at external pH 7.0 ( pH ex), decreasing to −85 ± 10 mV at pH ex 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V m and intracellular sodium concentration. We were able to vary the SMF between −187 ± 15 mV and −53 ± 15 mV by varying pH ex in the range 7.0–5.0 and extracellular sodium concentration in the range 1–85 mM. Rotation rates for 0.35- μm- and 1- μm-diameter beads attached to Na +-driven chimeric flagellar motors varied linearly with V m. For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.

The effect of external sodium concentration on sodium–calcium exchange in frog olfactory receptor cells

During the response of vertebrate olfactory receptor cells to stimulation, Ca(2+) enters the cilia via cyclic nucleotide-gated channels and is extruded by Na(+)-Ca(2+) exchange. The rise in Ca(2+) concentration opens a Ca(2+)-activated Cl(-) conductance which carries most of the inward receptor current. The dependence of Ca(2+) extrusion upon external Na(+) concentration was studied by using the falling phase of the Ca(2+)-activated Cl(-) current following a brief exposure to the phosphodiesterase inhibitor IBMX to monitor indirectly the decay in intraciliary Ca(2+) concentration. External Na(+) concentration was reduced by partial substitution with guanidinium, an ion which permeates the cyclic nucleotide-gated channel but does not support Na(+)-Ca(2+) exchange. The time constant describing the decay in current following IBMX stimulation was surprisingly little affected by substitution of external Na(+), being substantially retarded only when its concentration was reduced to a third or less of its normal value in Ringer solution. When the cilia were returned to Ringer solution after a period in reduced-Na(+) solution, the time constant for the final decay of current was similar to that seen when returning immediately to IBMX-free Ringer solution. This observation suggests that Ca(2+) extrusion via Na(+)-Ca(2+) exchange dominates the falling phase of the response to IBMX, which can therefore be used to assess exchanger activity. Rate constants derived from the time constants for current decay at different external Na(+) concentrations could be fitted by the Hill equation with a K(d) of 54 +/- 4 mm and Hill coefficient of 3.7 +/- 0.4. The cooperativity of the dependence upon external Na(+) concentration indicates that at least three Na(+) ions enter for each exchanger cycle, while the high affinity for external Na(+) contrasts with the photoreceptor and cardiac exchangers. The functional importance of this observation is that the relative insensitivity of the Na(+)-Ca(2+) exchanger to external Na(+) concentration allows normal response termination even following partial dilution or concentration of the olfactory mucus.

Multiple Consequences of Mutating Two Conserved β-Bridge Forming Residues in the Translocation Cycle of a Neuronal Glutamate Transporter

Glutamate transporters remove this neurotransmitter from the synapse in an electrogenic process. After sodium-coupled glutamate translocation, the cycle is completed by obligatory outward translocation of potassium. In the crystal structure of an archaeal homologue, two conserved residues form a beta-bridge, which points away from the binding pocket. In the neuronal glutamate transporter EAAC1, the equivalent residues are asparagine 366 and aspartate 368. Substitution mutants N366Q and D368E, but not N366D and D368N, show glutamate-induced inwardly rectifying steady-state currents, but their apparent substrate affinity is dramatically decreased. Such currents, which reflect electrogenic net uptake of substrate are not observed with the reciprocal double mutant N366D/D368N. Remarkably, the double mutant exhibits slow substrate-induced voltage-dependent capacitative transient currents. These currents apparently reflect the reversible sodium-coupled glutamate translocation step, because the interaction of the double mutant with potassium is largely impaired. Moreover, when the analogous double mutant in the glutamate transporter GLT-1 is reconstituted into liposomes, a slow exchange of radioactive and unlabeled acidic amino acids is observed. Our results suggest that it is the interaction of asparagine 366 and aspartate 368 that is important during the glutamate translocation step. On the other hand, the side chains of these residues themselves are required for the subsequent potassium relocation step.

Fluorescence Measurement of Intracellular Sodium Concentration in Single Escherichia coli Cells

The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na +] in) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H +) or sodium ions (Na +), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H +- and Na +-driven components that functions as a sodium motor. Changing external sodium concentration ([Na +] ex) in the range 1–85 mM resulted in changes in [Na +] in between 5–14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na +] in and [Na +] ex, and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2–3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.

Arginine 445 Controls the Coupling between Glutamate and Cations in the Neuronal Transporter EAAC-1

The substrate-binding sites in membrane transporters are alternately accessible from either side of the membrane, but the molecular basis of how this alternate opening of internal and external gates is achieved is largely unknown. Here we present data indicating that, in the neuronal electrogenic sodium- and potassium-coupled glutamate transporter EAAC-1, the substrate-binding site and one of the gates, or a residue controlling the gating process, are in close physical proximity. Arginine 445, located only two residues away from a residue implicated in glutamate binding (Bendahan, A., Armon, A., Madani, N., Kavanaugh, M. P., and Kanner, B. I. (2000) J. Biol. Chem. 275, 37436-37442), has been mutated to serine (R445S). Upon expression in oocytes, measurements of l-[(3)H]-glutamate transport under voltage clamp reveal that the charge/flux ratio for l-glutamate at -60 mV is approximately 30-fold higher than that of the wild type. Also, with d-aspartate, R445S exhibits an approximately 15-fold increase in this ratio. In contrast to the wild type, the reversal potential of the substrate-dependent currents in R445S shifts to more negative potentials when either the external sodium or potassium concentration is decreased. These findings indicate that these two cations are the main current carriers in the R445S mutant. Introduction of a methionine or a glutamine, but not a lysine, at position 445 gives rise to a phenotype similar to R445S. Therefore, it seems that the elimination of a positive charge in the vicinity of the substrate-binding site converts the transporter into a glutamate-gated cation-conducting pathway.

Correction for the adverse influence of sodium–potassium cotransport on apparent sodium–lithium countertransport activity in human erythrocytes

Introduction: Erythrocyte sodium–lithium countertransporter (SLC) has traditionally been characterised as the sodium-stimulated lithium efflux from lithium-loaded erythrocytes. Concurrent activity of the sodium–potassium cotransporter (NKCC) can be expected to lead to imprecise estimates of the activity of the SLC. In the present study, we have characterised this methodological problem and have shown that it can be corrected with the inclusion of bumetanide in the physiological salt solution. Methods: Lithium efflux was studied in lithium-loaded erythrocytes from 35 healthy, normotensive subjects. Erythrocytes were divided into two identical samples (A and B) and lithium efflux characteristics in both samples studied simultaneously by incubating aliquots from each in 10 media of differing external sodium concentrations. Efflux media employed for A and B were the same except for 0.02 mM bumetanide in the media used in B. Results: Increased external sodium was associated with increasing lithium efflux both in the absence and presence of bumetanide; efflux rates were consistently lower in media containing bumetanide ( P<.05 in all cases). As external sodium increased, bumetanide-sensitive lithium efflux decreased in a manner that correlated inversely with external sodium concentration ( r=−.77, P<.01). A small, nonsignificant increase in SLC activity was observed between measurements made under control conditions (median [range] in mmol Li/l RBC h; 0.272 [0.098–0.491]) and those made in the presence of bumetanide (0.286 [0.135–0.650]; P=.064). Bumetanide did not influence maximal rate of turnover or the affinity constant for external sodium. In contrast, the ratio of these variables was lower when determined in the absence than in the presence of bumetanide (5.5 [1.5–14.6] vs. 6.9 [2.8–24.2], respectively; P<.05). Discussion: This work shows that a component of lithium efflux mediated by the NKCC changes substantially with alterations in external sodium, resulting in a variable contribution of this second transport pathway to apparent SLC activity. To eliminate this variability, bumetanide should be included in all media when studying SLC, and the relationship to external sodium concentration determined.

Transport of (22)Na(+) and( 45)Ca(2+) by Xenopus laevis oocytes expressing mRNA from lobster hepatopancreas.

This paper describes the development of a functional assay system to express crustacean epithelial electrogenic 2Na(+)/1H(+) antiporters in Xenopus laevis oocytes. Subsequent publications will use this assay method to establish nucleotide and amino acid sequence information about this transporter by functionally screening an hepatopancreatic cDNA library. In this method, oocytes were injected with hepatopancreatic mRNA (50 ng) isolated from Homarus americanus, while control oocytes received injections of an equivalent volume of distilled water. Three to five days post-injection, oocytes were incubated in media containing either (22)Na(+) or (45)Ca(2+) for specific time intervals and the rates of ion transfer into the oocytes were monitored under a variety of experimental conditions. Uptakes of both radiolabelled cations were stimulated by mRNA injection. mRNA-stimulated (22)Na(+) uptake was significantly (P < 0.05) inhibited by addition of calcium, amiloride, or by an antiporter-specific monoclonal antibody to the external medium. mRNA-stimulated (45)Ca(2+) uptake was significantly (P < 0.05) inhibited by addition of sodium, amiloride, cadmium, zinc, or by the antiporter-specific monoclonal antibody (also inhibitory for (22)Na(+) transport) to the external medium. The kinetics of (22)Na(+) influx in mRNA-injected oocytes were sigmoidal functions of external sodium concentration, exhibiting a Hill Coefficient (n) of approximately 3.0. Both calcium and amiloride significantly (P < 0.05) reduced sigmoidal sodium influx kinetics by alterations in the J(max) (amiloride) or K(Na) (calcium) of the transporter. Size fractionation of hepatopancreatic mRNA resulted in a single fraction that was most stimulatory for sodium and calcium transport and which likely contains the antiporter transcript. The results of this study provide the basis for using (22)Na(+) and (45)Ca(2+) transport assays of lobster mRNA-injected oocytes to functionally screen an hepatopancreatic cDNA library for clones that will provide full length nucleotide and amino acid sequences of the invertebrate electrogenic 2Na(+)/1H(+) antiporter protein.

Alterations in electrical and mechanical activity in Langendorff-perfused guinea pig hearts exposed to decreased external sodium concentration with or without hypotonic insult

In order to examine electrical and mechanical effects of hyponatremia and hypotonicity, relevant to those in patients with ‘water intoxication’ syndrome, Langendorff-perfused guinea pig hearts were exposed to reduced NaCl concentrations (hypotonic [NaCl] 0-reduction) under the monitoring of left ventricular developed pressure (LVDP) and epicardial ECG. In some hearts, hyponatremia (from 140 to 80 mEq/l) was compensated for by adding mannitol to maintain osmolarity at a constant level (isotonic [NaCl] 0-reduction) or tetraethylammonium chloride to maintain both osmolarity and chloride concentrations at a constant level (isotonic [Na +] 0-reduction). Progressive isotonic [NaCl] 0-reduction increased LVDP, which was abolished in the presence of KB-R7943, a novel inhibitor of Na +/Ca 2+-exchange. LVDP was reduced in hypotonic [NaCl] 0-reduction in which myocardial water content was increased. PQ interval and QRS duration were prolonged with both hypotonic and isotonic [NaCl] 0-reduction and these changes tended to be more pronounced with hypotonic than with isotonic [NaCl] 0-reduction. Similar ECG changes were also evident with isotonic [Na +] 0-reduction. Gd 3+ (1–5 μM), a blocker of stretch-activated nonspecific cation channels, had no substantial effects on the electrical or mechanical changes seen with hypotonic [NaCl] 0-reduction. In conclusion, isotonic [NaCl] 0-reduction produced a positive inotropism by modulating Na +/Ca 2+-exchange, whereas hypotonic [NaCl] 0-reduction led to negative inotropism, due in part to hypotonic myocardial swelling. In addition, [Na +] 0-reduction, irrespective of the concomitant [Cl −] 0 or osmotic changes, depressed atrioventricular as well as intraventricular conduction.

Integumentary L-histidine transport in a euryhaline polychaete worm: regulatory roles of calcium and cadmium in the transport event.

Integumentary uptake of L-[(3)H]histidine by polychaete worms (Nereis succinea) from estuarine waters of Oahu, Hawaii was measured in the presence and absence of calcium and cadmium using a physiological saline that approximated the ion composition of 60 % sea water. In this medium 1 micromol L(-1) cadmium significantly increased (P<0.01) the uptake of 10 micromol L(-1)L-[(3)H]histidine, while 1 micromol L(-1) cadmium plus 25 micromol L(-1)L-leucine significantly decreased (P<0.01) amino acid uptake. L-[(3)H]histidine influx was a sigmoidal function (n=2. 21+/-0.16, mean +/- s.e.m.) of [L-histidine] (1?50 micromol L(-1)) in the absence of cadmium, but became a hyperbolic function with the addition of 1 micromol L(-1) cadmium. A decrease of calcium concentration from 6 to 0 mmol L(-1) (lithium substitution) significantly increased (P<0.01) amino acid influx in the presence and absence of cadmium. Calcium significantly reduced (P<0.01), and cadmium significantly increased (P<0.01), L-[(3)H]histidine influx J(max), without either divalent cation affecting amino acid influx K(t). Variation in external sodium concentration (0?250 mmol L(-1)) had no effect on 10 micromol L(-1)L-[(3)H]histidine influx, but amino acid entry was a sigmoidal function of both [cadmium] (n=2.34+/-0.44) and [lithium] (n=1.91+/-0.39) in the absence of calcium. A model is proposed for transapical L-[(3)H]histidine influx by a transporter that resembles the classical sodium-independent L-system carrier protein that is regulated by the external divalent cations calcium and cadmium.

Synergistically Interacting Dopamine D1 and NMDA Receptors Mediate Nonvesicular Transporter-Dependent GABA Release from Rat Striatal Medium Spiny Neurons

Given the complex interactions between dopamine D1 and glutamate NMDA receptors in the striatum, we investigated the role of these receptors in transporter-mediated GABA release from cultured medium spiny neurons of rat striatum. Like NMDA receptor-mediated [(3)H]-GABA release, that induced by prolonged (20 min) dopamine D1 receptor activation was enhanced on omission of external calcium, was action potential-independent (tetrodotoxin-insensitive), and was diminished by the GABA transporter blocker nipecotic acid, indicating the involvement of transporter-mediated release. Interestingly, lowering the external sodium concentration only reduced the stimulatory effect of NMDA. Blockade of Na(+)/K(+)-ATPase by ouabain enhanced NMDA-induced but abolished dopamine-induced release. Moreover, dopamine appeared to potentiate the effect of NMDA on [(3)H]-GABA release. These effects of dopamine were mimicked by forskolin. mu-Opioid receptor-mediated inhibition of adenylyl cyclase by morphine reduced dopamine- and NMDA-induced release. These results confirm previous studies indicating that NMDA receptor activation causes a slow action potential-independent efflux of GABA by reversal of the sodium-dependent GABA transporter on sodium entry through the NMDA receptor channel. Moreover, our data indicate that activation of G-protein-coupled dopamine D1 receptors also induces a transporter-mediated increase in spontaneous GABA release, but through a different mechanism of action, i.e., through cAMP-dependent inhibition of Na(+)/K(+)-ATPase, inducing accumulation of intracellular sodium, reversal of the GABA carrier, and potentiation of NMDA-induced release. These receptor interactions may play a crucial role in the behavioral activating effects of psychostimulant drugs.

Na(+)-Ca2+ exchange induces low Na+ contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase.

Contractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum.

Hyperpolarization-activated inward potassium and calcium-sensitive chloride currents in beating pacemaker insect neurosecretory cells (dorsal unpaired median neurons)

Hyperpolarization-activated inward currents were studied in single adult cockroach Periplaneta americana pacemaker neurosecretory cells, identified as dorsal unpaired median neurons using the whole-cell patch-clamp technique. Under current clamp, injection of negative current produced a hyperpolarization of the cell membrane with a sag in the membrane potential toward the resting value. Under voltage clamp, the whole-cell current–voltage relationship exhibited an unexpected biphasic aspect. The global hyperpolarization-activated inward current could be dissociated by means of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid and tetraethylammonium chloride sensitivity, ionic selectivity, voltage dependence and activation threshold as inward potassium and calcium-sensitive chloride currents. The inward potassium current was activated around −80 mV. The reversal potential followed the potassium equilibrium potential when the extracellular potassium concentration was raised. This current was not dependent on the external sodium concentration and was sensitive to 10 mM tetraethylammonium chloride or 5 mM barium chloride. The hyperpolarization-activated inward calcium-sensitive chloride current was activated in a range of potential 20 mV more positive than the potassium current. The estimated reversal potential (−71 mV) was very close to the equilibrium potential for chloride ions (−73 mV). Intracellularly applied 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and external application of 1 mM zinc chloride, calcium-free saline or high concentrations of intracellular 1,2-bis-(2-aminophenoxy)ethane- N, N, N′, N′-tetra-acetate blocked the inward chloride current. Current-clamp experiments indicated that the inward potassium current accounted for inward rectification of dorsal unpaired median neurons. Our findings report, for the first time in pacemaker neurosecretory cells, the co-existence of two distinct hyperpolarization-activated inward currents which have specialized function in pacemaker activity.