Presence Of External Na Research Articles

Na(+)-Ca2+ exchanger-like immunoreactivity and regulation of intracellular Ca2+ levels in fish retinal ganglion cells.

1. We have used two experimental approaches to examine regulation of intracellular calcium ion levels in fish retinal ganglion cells. In the first set of experiments, we ratio-imaged fura-2 emission intensity to estimate the concentration of free intracellular calcium ions ([Ca2+]i) in isolated goldfish retinal ganglion cells depolarized by increases in extracellular levels of potassium ions ([K+]o), in the presence and absence of extracellular sodium ions (Na+). Stepwise increases in [K+]o from 5 mM to as high as 60 mM produced stepwise increases in [Ca2+]i. These increases were sustained in the absence of external Na+, but transient and smaller in the presence of external Na+. The decline of [Ca2+]i in high-K, Na(+)-containing saline could be reversed by application of the ionophore monensin, or by replacement of external Na+ with either N-methyl-D-glucamine or lithium. In Na(+)-containing saline, [Ca2+]i fell to control levels after [K+]o was restored to control levels. 2. In the second set of experiments, we assessed Na(+)-Ca2+ exchanger-like immunoreactivity in goldfish retinal ganglion cells with the use of a polyclonal antiserum directed against Na(+)-Ca2+,K+ exchanger purified from bovine rod outer segments. This antiserum specifically stained the somata, neurites, and growth cones of isolated ganglion cells, the outer segments of rod photoreceptors, and (on Western blots prepared from mechanically isolated rods) protein displaying an apparent molecular mass of 210 kDa. 3. These results provide measurements of changes in [Ca2+]i of retinal ganglion cells depolarized in Na(+)-containing saline, and the distribution and apparent molecular weight of Na(+)-Ca2+ exchanger-like immunoreactivity in teleost retina.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the Na,K-pump function by beta subunit isoforms.

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K-pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.

Calcium influx through neuronal-type nicotinic acetylcholine receptors present on the neuroendocrine cells of the porcine pars intermedia.

The properties of neuronal-type nicotinic acetylcholine receptors (nAChRs) present on the neuroendocrine cells of the porcine pars intermedia of the pituitary were studied in intact single cell using measurements of the free intracellular Ca2+ concentration ([Ca]i) with the calcium-sensitive dye fura 2. Local application of an extracellular solution containing 50 mM K+ or of the selective nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP) depolarised the cells and induced an elevation in [Ca]i. The effect of DMPP on [Ca]i was dose dependent (EC50 = 6 microM), reversibly blocked by d-tubocurarine and strictly dependent on the concentration of extracellular Ca2+. The calcium channel blocker Cd2+ (100 microM) reversibly blocked 80% of the response induced by 50 mM K+, whereas it reduced the DMPP response by only 50%. In the absence of extracellular Na+, DMPP no longer depolarised the cells but still increased [Ca]i. The rise in [Ca]i under these conditions represented 41% of the control response, i.e. in the presence of external Na+. Thus activation of nAChRs induced an elevation in [Ca]i which was in part independent of cell depolarisation. This was confirmed by recording simultaneously, under whole-cell voltage-clamp, a rise in [Ca]i associated with the inward nicotinic current. During prolonged application of the agonist (50 s), the amplitude of the nicotinic current decayed rapidly to a very low plateau level reflecting nAChR desensitisation. However, photometric experiments performed on intact non-dialysed cells revealed the presence of a slowly decaying phase in [Ca]i throughout the application of DMPP. This suggests the persistence of a substantial Ca2+ influx during prolonged exposure to the agonist. Taken together, our results show that stimulation of nAChRs induces an influx of Ca2+ which elevates [Ca]i. This phenomenon is due to activation of voltage-dependent Ca2+ channels and to Ca2+ entry through the nAChR.

Sodium-bicarbonate cotransport current in identified leech glial cells.

1. The membrane current associated with the cotransport of Na+ and HCO3- was investigated in neuropil glial cells in isolated ganglia of the leech Hirudo medicinalis L. using the two-electrode voltage-clamp technique. 2. The addition of 5% CO2-24 mM HCO3- evoked an outward current, which slowly decayed, and which was dependent upon the presence of external Na+. Removal of CO2-HCO3- elicited a transient inward current. Re-addition of Na+ to Na(+)-free saline in the presence of CO2-HCO3- also produced an outward current. Under these conditions an intracellular alkalinization and a rise in intracellular [Na+] were recorded using triple-barrelled, ion-sensitive microelectrodes. Addition or removal of HCO3-, in the absence of external Na+, caused little or no change in membrane voltage, membrane current and intracellular pH, indicating that the glial membrane has a very low HCO3- conductance. 3. Voltage steps revealed nearly linear current-voltage relationships both in the absence and presence of CO2-HCO3-, with an intersection at the assumed reversal potential of the HCO(3-)-dependent current. These results suggest a cotransport stoichiometry of 2HCO3-: 1 Na+. The HCO(3-)-dependent current could be inhibited by diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). 4. Simultaneous recording of current and intracellular pH showed a correlation of the maximal acid-base flux with the transient HCO(3-)-dependent current during voltage steps in the presence of CO2-HCO3-. The maximum rate of acid-base flux and the HCO(3-)-dependent peak current showed a similar dependence on membrane voltage. Lowering the external pH from 7.4 to 7.0 produced an inward current, which increased twofold in the presence of CO2-HCO3-. This current was largely inhibited by DIDS, indicating outward-going electrogenic Na(+)-HCO3- cotransport during external acidification. 5. When external Na+ was replaced by Li+, a similar outward current and intracellular alkalinization were observed in the presence of CO2-HCO3-. The Li(+)-induced intracellular alkalinization was not inhibited by amiloride, a blocker of Na+(Li+)-H+ exchange, but was sensitive to DIDS. These results suggest that Li+ could, at least partly, substitute for Na+ at the cotransporter site. 6. Our results indicate that the Na(+)-HCO3- cotransport produces a current across the glial cell membrane in both directions with a reversal potential near the membrane resting potential, rendering pHi a function of the glial membrane potential.

Transport mechanism of glutamate by hypotonic-treated glial plasmalemmal vesicles from rat hippocampus. Effects of concentration gradients of Na+ and K+ and of ionophores.

Recently, we isolated a novel subcellular fraction of glial plasmalemmal vesicles (GPV), which showed a higher activity of Na(+)-dependent glutamate transport than synaptosomes (Nakamura et al., 1993). In order to study kinetically the glutamate transport mechanism, we measured the reaction under various ionic conditions both inside and outside the vesicles. The vesicles treated hypotonically and preloaded with KCl could take up glutamate in the presence of external Na+. The level of glutamate uptake was dependent on external concentrations of NaCl ([NaCl]o) and competitively inhibited by [KCl]o. However, it was dependent on [KCl]i, and competitively inhibited by [NaCl]i. The activation and inhibition constants of K+ were about 30 mM inside and 20 mM outside, respectively, whereas those of Na+ were 140 mM outside and 4 mM inside, respectively. These results suggest that the transport carrier molecules work asymmetrically to the membranes. Nigericin and monensin, acidic ionophores for K+ and Na+, respectively, inhibited the glutamate uptake. On the other hand, valinomycin, a neutral ionophore for K+, elevated the uptake level, suggesting that the inside-negative membrane potential induced by K+ diffusion enhances the uptake activity. We conclude that glutamate transport by glial cells requires both external Na+ and internal K+ and is regulated by the membrane potential.

Characterization of l-Thyroxine Transport into Hepatocytes Isolated from Juvenile Rainbow Trout (Oncorhynchus mykiss)

Uptake of l-thyroxine (T4) by isolated trout hepatocytes was characterized after 1-min incubation of cells in a Hanks' balanced salts medium containing glucose and [125I]T4. Centrifugation through silicone oil and glycine buffer (pH 10.5) was used to separate cells from the medium and minimize extracellular binding of T4. At a T4 concentration of 0.2 nM, 15% of hepatocyte uptake was due to nonsaturable (passive) diffusion. Saturable T4 uptake obeyed Michaelis-Menten kinetics with an apparent Kt of 0.52 μM and a Jmax of 120 pmol/min/106 cells at 12°. T4 uptake was temperature-dependent. The activation energy was 58.0 kJ/mol between 1 and 6°, and 22.7 kJ/mol between 12 and 24°. T4 uptake was enhanced by increased H+ concentration over the range of 6.0-9.0 pH units and decreased sharply below pH 5.0. Dinitrophenol (1 mM) and potassium cyanide (2 mM) decreased T4 uptake to, respectively, 91 and 66% of controls, indicating a requirement for metabolic energy. The sodium ionophore monensin (10 μM) and the Na+ -H+ exchange inhibitor amiloride (0.5 mM) reduced uptake to 81 and 74%, respectively, of controls, while preincubation with ouabain (0.5 mM) or substitution of LiCl for NaCl in the medium did not modify T4 uptake, indicating that T4 transport does not depend on the presence of external Na+. The sulfhydryl-blocking reagent p-hydroxymercuribenzoate (0.5 mM) depressed T4 uptake to 32% of controls, but N -ethylmaleimide (0.5 mM) was ineffective. Inhibitors of protein binding, bromosulphothalein (0.3 mM) and 8-anilino-1-naphthalene sulfonic acid (1 mM), both decreased T4 uptake to 32 and 36% of controls, respectively, while phloretin (0.1 mM) and 5,5′-diphenylhydantoin, specific inhibitors of carrier-mediated transport, depressed uptake to 8 and 32% of controls, respectively, 3,5,3′-triiodo-l-thyronine (T3) decreased T4 uptake with an apparent Ki of 0.19 μM, indicating that T3 and T4 may share a common transport site(s). Other structural analogues, tested at 0.37 μM, influenced uptake as follows: 3,3′,5′-triiodo-l-thyronine (rT3) to 76% of control; 3,5-diiodo-l-thyronine to 57%; d-thyroxine to 93%; and 3,5,3′-triiodothyropropionic acid to 78%. l-tyrosine and l -phenylalanine (each at 0.2 mM) did not decrease T4 uptake. Colchicine (0.5 mM), an inhibitor of endocytosis, decreased T4 uptake to 75% of control when added with T4 and to 53% when added to cells 3 min prior to addition of T4. We conclude that T4 uptake into trout hepatocytes is a saturable, stereospecific, energy-dependent carrier-mediated transport that does not depend on extracellular Na+ but may involve an endocytotic mechanism. T3 and some other structural analogues of T4 may also be transported by this system.

Recovery of intracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: Role of lactate removal, extracellular sodium and sodium/hydrogen exchange

[ 31P]- and [ 1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO 2/HCO 3 −-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na +, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H + neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the “overshoot” of the alkalinization at 10–12 min of recovery. In a medium containing 70 mM K +, 60 mM Na + and 0.1 mM Ca 2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na + caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na +-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na +. Complexing of extracellular Ca 2+ in the Na +-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na +. In Na + -free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na + was as profound in the presence as in the absence of Ca 2+ showing that accumulation of Ca 2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na + and sensitive to high K +.

Na+/H+ antiporter properties in peripheral blood lymphocytes from normotensive obese and type 2 diabetic patients do not differ significantly from controls

It has been hypothesized that some genetic factors link different conditions characterized by the presence of insulin resistance: among them, obesity, type 2 (non-insulin-dependent) diabetes mellitus and arterial hypertension. A good candidate could be the Na+/H+ exchanger, the increased activity of which is considered a genetic marker of essential hypertension. In this study we looked at whether the Na+ dependence of the Na+/H+ antiporter is modified in obese and type 2 diabetic patients, in the absence of arterial hypertension. The activity of this ion exchanger was measured in peripheral blood lymphocytes by acidifying them in Na+-free buffer and then monitoring the recovery of intracellular pH after Na+ addition. Quiescent lymphocytes were used because they do not have insulin receptors, thus ruling out the effects of the elevated insulin concentrations on the Na+/H+ exchanger activity. Antiport activity, measured as the ability to extrude H+ in the presence of external Na+, showed no differences in normotensive obese and type 2 diabetic patients when compared with healthy subjects. Our data therefore suggest that an altered Na+/H+ exchange activity cannot be considered a common feature of insulin-resistant states.

Release of γ-[ 3H]aminobutyric acid from synaptosomes: effect of external cations and of ouabain

In the present study we have investigated the effect of cations and ouabain on Ca 2+-independent and Ca 2+-dependent release of γ-[ 3H]aminobutyric acid ([ 3H]GABA) from sheep brain synaptosomes. The presence of Na + in the external medium is essential for the Ca 2+-independent release induced by K + or ouabain. Thus, in the absence of Ca 2+, ouabain of K + causes the release of [ 3H]GABA provided that Na + is present in the external medium. Under K +-depolarizing conditions, in a Na + medium, either ouabain or Ca 2+ further increases the [ 3H]GABA release induced by depolarization, but their effects are not additive. The presence of external Na + is not required for the Ca 2+-dependent release of [ 3H]GABA due to K + depolarization, and this release, which occurs in a choline medium, is not modified by ouabain. Under these conditions (choline medium) K +-depolarization dependent release is absolutely dependent on external Ca 2+, which suggests that this release of [ 3H]GABA occurs only by exocytosis, without the carrier-mediated efflux which normally co-exists with exocytosis due to K +-depolarization in a Na + medium. It is likely that the release induced by ouabain or K + involves the membrane carrier which responds to changes in membrane potential.

Intracellular Ca 2+ regulation in CD3 stimulated Jurkat T cells involves H + fluxes

Triggering the CD3/TCR complex of lymphocytes induces a rapid rise in cytosolic free calcium followed by a slowly declining plateau. The level of this plateau depends on external pH, the more alkalinized media leading to higher values. Neither a pH-dependent binding of mAb, nor a pertubation of internal pH can account for this effect. In a sodium-free medium, or in the presence of dimethylamiloride Ca 2+, elevation is accompanied by an acidification of the cells, both of them depend, to the same extent, on external calcium concentration. TPA inhibits CD3-, but not ionomycin-induced Ca 2+ and H + raises, indicating that it acts more probably on Ca 2+ influx, rather than on its efflux. These results suggest that intracellular calcium could be regulated by a Ca 2+/H + ATPase which drives H + in and Ca 2+ out. In the presence of external Na +, H + should return to the medium by the Na +/H + exchanger.

4‐Aminopyridine causes a voltage‐dependent block of the transient outward K+ current in rat melanotrophs.

1. Whole-cell voltage-clamp recordings were made from acutely dissociated melanotrophs obtained from adult rats. 2. In the presence of external Na+ and Ca2+ channel blockers and 20 mM-tetraethylammonium (TEA) depolarizations to -40 mV or more evoked a fast-activating fast-inactivating outward K+ current (IK(f)). Double-pulse experiments showed that steady-state half-inactivation occurred near -37 mV; half-maximal activation of IK(f) occurred at -15 mV. Recovery from inactivation in most cells fitted a single exponential with a time constant of 40-50 ms. 3. When applied either internally or externally, 1-2.5 mM-4-aminopyridine (4-AP) substantially reduced IK(f) but the degree of block was affected by the intensity, duration and frequency of depolarizing commands. 4. Analysis of the steady-state voltage dependence of the block by 4-AP showed that half-maximal blocking occurred at approximately -31 mV. This implied that 4-AP binds to the resting state of the IK(f) channel. 5. Studies of the time dependence for the blocking or unblocking of IK(f) showed that both processes were exponential with mean time constants of 1942 ms (at -70 mV) and 726 ms (at 20 mV), respectively. Recovery from inactivation was apparently unaffected by 4-AP. 6. A four-state sequential model in which 4-AP reversibly binds to the resting state of the channel replicates the frequency dependence of the 4-AP blockade.

Electrophysiology of glutamate and sodium co‐transport in a glial cell of the salamander retina.

1. Müller cells were isolated from salamander retinas and their membrane voltage was controlled with a whole-cell voltage clamp. External D-aspartate, L-aspartate and L-glutamate each induced a membrane current. D-Glutamate, kainate, quisqualate and N-methyl-D-aspartate were more than 100x less effective than L-aspartate. Kynurenic acid had no effect on the current produced by L-glutamate, L-aspartate or D-aspartate. 2. The current induced by an acidic amino acid (AAA) was completely dependent on the presence of external Na+. Neither Li+, Cs+, choline nor TEA+ were able to substitute for Na+. The relationship between external Na+ concentration and current amplitude can be explained if the binding of three Na+ ions enabled transport. The apparent affinity constant for Na+ binding was 41 mM. Altering K+, H+ and Cl- concentrations demonstrated that these ions are not required for transport. 3. The shape of the current-voltage relation did not depend on the external amino acid concentration. The relationship between D-aspartate concentration and current amplitude can be described by the binding of D-aspartate to a single site with an apparent affinity constant of 20 microM. 4. Influx and efflux of AAA were not symmetric. Although influx was electrogenic, efflux did not produce a current. Moreover, influx stimulated efflux; but efflux inhibited influx. 5. Removing external Na+ demonstrated that Na+ carried a current in the absence of an AAA. Li+ was a very poor substitute for Na+. This current may be due to the uncoupled movement of Na+ through the transporter. The relationship between the external Na+ concentration and the amplitude of the uncoupled current can be explained if the binding of two or three Na+ ions enabled the translocation of Na+ in the absence of an AAA. The apparent affinity constant for Na+ binding was approximately 90 mM. 6. The temperature dependence of the AAA-induced current had a Q10 between 8 and 18 degrees C of 1.95. The Q10 is consistent with a rate constant for influx of 10(4) s-1 (at -70 mV and 20 degrees C). The maximum rate of influx was measured following a concentration jump produced by the photolysis of 'caged' L-glutamate. The onset of the observed current was limited by the 1.3 ms resolution of the recording system. Hence, the rate constant for influx must be faster than 10(3) s-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Single inwardly rectifying potassium channels in cultured muscle cells from rat and mouse.

1. Inward unitary currents through inwardly rectifying K+ channels of myotubes derived from newborn rats or from a murine, clonal myoblast cell line were studied in the cell-attached configuration. Open-closed transitions of the channel were observed in the absence of blocking ions. 2. The single-channel conductance was 26.3 +/- 2.9 pS (mean + S.D., n = 14) with 150 mM-K+ pipette solution at room temperature (19-22 degrees C). The channel showed substates of conductance in addition to the main conductance state. A channel with a smaller conductance (8.9 +/- 2.6 pS, n = 4) was also but less frequently observed. 3. The probability of the channel being open is weakly voltage dependent: it decreased from 0.94 to 0.84 as the membrane was hyperpolarized from the resting potential (RP) + 20 mV to RP - 50 mV. 4. The lifetimes of the openings were distributed according to a single exponential. At least three exponentials were required to fit the frequency histogram of the lifetimes of all closed states. The mean open time showed a weak voltage dependence, while the mean closed times had little voltage dependence. 5. In the presence of external Na+, the open probability decreased from 0.89 to 0.43 and the mean open time decreased from 203 to 28 ms (40 mM-K+, 200 mM-Na+ pipette solution) when the patch membrane was hyperpolarized from RP - 40 mV to RP - 110 mV. The mean closed times were not different from those with 150 mM-K+, Na+-free pipette solution and showed little voltage dependence. 6. It is suggested that inactivation of the macroscopic inward currents during hyperpolarization results mainly from a voltage-dependent block by Na+ with relatively slow kinetics.

Effect of Internally Loaded Iodide, Thiocyanate, and Perchlorate on Sodium-Dependent Iodide Uptake by Phospholipid Vesicles Reconstituted with Thyroid Plasma Membranes: Iodide Counterflow Mediated by the Iodide Transport Carrier

Na+-dependent I- transport and I- counterflow were studied using phospholipid vesicles (P-vesicles) made of porcine thyroid plasma membranes and soybean phospholipid by sonication. 1) I- uptake by P-vesicles incubated in the presence of external Na+ was higher than that by P-vesicles incubated in choline+ instead of Na+. The vesicles exhibited Na+-dependent I- uptake. When P-vesicles were internally loaded with I- prior to incubation in Na+, a further increase in Na+-dependent I- uptake was observed, although the concentration of internal I- was very much higher than that outside. In the absence of external Na+, I- uptake by P-vesicles preloaded with I- was comparable to baseline uptake. 2) Na+-dependent I- uptake by P-vesicles not loaded with I- and enhanced Na+-dependent I- uptake by P-vesicles preloaded with I- were both inhibited by either of SCN- and ClO4- added outside the vesicles. 3) When P-vesicles were loaded with SCN- instead of I- and incubated in Na+, I- uptake by these vesicles was also higher than baseline Na+-dependent I- uptake. However, a ClO4- load did not result in an increase in I- uptake. These results indicate that Na+-dependent I- transport including Na+-dependent I- counterflow is specifically mediated by the thyroid I- carrier. SCN- - I- counterflow in addition to I- - I- counterflow occurs dependently on Na+, but ClO4- - I- counterflow does not.

Anion dependence of taurine transport by rat renal brush-border membrane vesicles.

The anionic requirements and the stoichiometric relationships of Na+-taurine cotransport into rat renal brush-border membrane vesicles (BBMV) were evaluated. External Cl- (100 mM) or Br- (100 mM) gradients supported the full overshoot of Na+-taurine symport and yielded similar high-affinity transport systems for taurine uptake. No active uptake of taurine was evident in the presence of external (100 mM) NaF, NaI, Na gluconate, or Na p-aminohippurate (PAH). Na+:taurine stoichiometry was 2.18:1 in the presence of Cl- and 1.60:1 in the presence of Br-. When the external anion gluconate was employed, Na+-dependent taurine uptake was negligible over the whole range of Na+ concentrations examined. Cl-:taurine and Br-:taurine stoichiometries in the presence of external Na+ were 0.97:1 and 0.81:1, respectively. External furosemide (1 mM) or bumetanide (1 mM) did not change taurine accumulation and kinetic parameters. The anionic transport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (5 x 10(-4) M), N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (10(-3) M) and p-chloromercuribenzoate (5 x 10(-4) M) significantly decreased initial rate of taurine uptake by 48, 31, and 31%, respectively. These data suggest that Na+-taurine cotransport into rat renal BBMV is Cl- or Br- dependent and probably operates by means of 2 Na+:1 Cl- or Br-:1 taurine carrier complex. Na+-taurine symport across the rat renal brush-border membrane surface is not affected by diuretics that influence NaCl cotransport but is affected by selected anionic transport inhibitors. An intact anionic binding site may be needed for this translocation process.

Preservation of Sodium-Dependent Iodide Transport Activity By Methimazole and Mercaptoethanol in Phospholipid Vesicles Containing Thyroid Plasma Membranes: with Evidence of Difference in the Action of Perchlorate and Thiocyanate.

The effect of methimazole (MMI) and 2-mercaptoethanol (ME) on I-transport was studied using phospholipid vesicles (P-vesicles) made from porcine thyroid plasma membranes and soybean phospholipids by sonication. 1. When buffer solutions contained either 1 mM MMI or 2 mM ME, I-uptake by P-vesicles in the presence of external Na+ was apparently higher than that in the absence of external Na+. Na+-dependent I- uptake was inhibited by both C1O4- and SCN- added externally. 2. When PM was treated with 4 mM N-ethylmaleimide prior to preparation of P-vesicles, the activity of Na+-dependent I- transport was completely lost even when P-vesicles were incubated in the presence of ME. 3. When neither MMI nor ME was added to buffers, I- uptake in the presence of external Na+ was not at all higher than that in the absence of external Na+. In these instances, however, I- uptake was much higher compared than the baseline uptake in the presence of MMI or ME, and was inhibited by external SCN- and not by C1O4- without relation to external Na+. These data indicate that MMI or ME has two distinct effects on our model system of I- transport. The one is preservation of the Na+-dependent I- transport activity by protecting a sulfhydryl group, and the other is reduction of nonspecific I- binding to P-vesicles. In addition, C1O4- is a more specific inhibitor of thyroid I- transport than SCN-, when non-specific I- oxidation is imperfectly prevented.

Caffeine contracture in guinea-pig ventricular muscle and the effect of extracellular sodium ions.

1. The mechanisms underlying the virtual absence of caffeine contracture in guinea-pig heart in a Na+-rich external solution were reinvestigated in small (50-120 microns thick) bundles of intact and skinned papillary muscle fibres. 2. In Na+-free solution, the peak tension of 30 mM-caffeine contracture corresponded to the maximum tension of the skinned fibres, and was independent of changes in [Ca2+]o and [K+]o. In the presence of external Na+, the peak tension, which was at most several per cent of the maximum, was affected by [Ca2+]o, [Na+]o and [K+]o, and enhanced by Mn2+ and Ni2+. 3. In the absence of Ca2+, replacement of Na+ with K+ allowed caffeine to evoke a large contracture, showing that there was sufficient calcium stored in the cells under Na+-rich conditions. After treatment with 30 mM-caffeine in the Na+-rich, Ca2+-free solution, and upon replacement of all Na+ with Li+, caffeine was still able to produce a large contracture, which was dependent upon Ca2+ pre-loading of the cells before the first caffeine treatment and upon the subsequent duration in the Na+-free solution. 4. Replacement of Li+ with Na+ during the contracture led to rapid relaxation which was delayed by an increase in [Ca2+]o, depolarization by K+, and addition of La3+ and Mn2+. After Na+-induced complete relaxation in the absence of Ca2+, upon removal of the drugs and Na+, subsequent application of caffeine to the cells evoked a large contracture without Ca2+ reloading. 5. In the skinned fibres, 30 mM-caffeine increased the Ca2+ sensitivity of the contractile system and depressed the maximum tension. An increase in Na+ from 8.4 to 58.4 mM altered neither Ca2+ sensitivity nor the rate of tension development in the absence or presence of caffeine. 6. Increase in Na+ affected neither the rate nor the amount of Ca2+ uptake by the sarcoplasmic reticulum (SR) in the absence or presence of caffeine. Increasing Na+ slightly inhibited the caffeine-induced Ca2+ release from the SR, but more than 10 mM-caffeine produced SR Ca2+ depletion. 7. In the presence of a strong Ca2+ buffer, the steady level of Ca2+ uptake by the SR with 1 mM-caffeine was equal to the amount of Ca2+ remaining in the SR just after the application of caffeine, indicating that Ca2+ release was not inactivated.(ABSTRACT TRUNCATED AT 400 WORDS)

Maitotoxin‐Evoked γ‐Aminobutyric Acid Release Is Due Not Only to the Opening of Calcium Channels

The effects of maitotoxin (MTX) on endogenous amino acid release were tested on highly purified striatal neurons differentiated in primary culture. MTX induced a large and concentration-dependent release of gamma-aminobutyric acid (GABA). This effect was abolished when experiments were performed in the absence of external Ca2+, and restored when Ca2+ ions were added after removing the MTX-containing Ca2+-free solution. MTX-induced amino acid release was not affected by 1 microM nifedipine and only slightly inhibited by 1 mM Co2+. MTX also induced a massive accumulation of 45Ca2+ in the neurons which, in contrast to the MTX-evoked GABA release, was totally blocked in the presence of 1 mM Co2+. Whereas 500 nM tetrodotoxin was without significant effect, MTX-evoked GABA release was dependent on the presence of external Na+ and sensitive to nipecotic acid, a GABA uptake inhibitor. It is concluded that, on striatal neurons, MTX induced Na+ influx only in the presence of external Ca2+. The increase in cytoplasmic Na+ ions then triggers the release of GABA.

Sodium‐dependent suppression of gamma‐aminobutyric‐acid‐gated chloride currents in internally perfused frog sensory neurones.

1. The effects of the Na+ electrochemical potential gradient on gamma-aminobutyric acid (GABA)-induced Cl- currents (ICl) in frog sensory neurones were studied, using a suction pipette technique with which internal perfusion can be accomplished under current- and voltage-clamp conditions. 2. Under current clamp, the depolarizing response to GABA decreased in the presence of external Na+. A similar external Na+-dependent reduction in the GABA-induced inward ICl was observed under voltage clamp. The reversal potential of GABA-induced ICl (EGABA) was nearly equal to the Cl- equilibrium potential (ECl), irrespective of the presence or absence of external Na+. 3. Varying the Na+ influx by changing the holding membrane potential (VH) altered the GABA response: the GABA-induced ICl decreased progressively as VH became more negative. 4. The effects of changing the external and internal Na+ concentrations ([Na+]o and [Na+]i) on the GABA-induced ICl were also studied. Increasing [Na+]o at a constant [Na+]i reduced this current while increasing [Na+]i at a fixed [Na+]o facilitated it. 5. A high temperature coefficient of about 3 was estimated with respect to the percentage reduction in GABA-induced ICl due to [Na+]o. 6. These results indicate that the [Na+]o-dependent suppression of GABA-induced ICl was mediated chiefly by the uptake of GABA subserved by a Na-GABA co-transport mechanism. 7. GABA dose-response measurements were made with and without external Na+. The [Na+]o-induced suppression was more pronounced in relative amount at lower concentrations and in absolute amount at intermediate concentrations. Analysis of these data indicates, however, that the Na+-coupled GABA influx kept increasing at GABA concentrations high enough to nearly saturate GABA-induced ICl, and the same saturating level was observed as in the Na+-free case. This indicates that the electrogenic co-transport current was much smaller so that our measurements of GABA-induced ICl' were contaminated very little. Thus, the present method based on recording of GABA-induced ICl was legitimate for the analysis of the Na-GABA co-transport. 8. By analysing the [Na+]o-dependent suppression of GABA-induced ICl, the stoichiometric ratio of the underlying co-transport was estimated to be one: one Na+ ion per GABA molecule. 9. The ICl induced by GABA agonists such as beta-alanine, taurine, l-GABOB (l-gamma-amino-beta-hydroxybutyric acid) and muscimol was not affected by the amount of external Na+ present, suggesting difference in the affinity between receptor and transport carrier.

Inhibition of Na +-dependent GABA uptake in isolated frog sensory nerve cell bodies by extra- and intracellular Li +

The effect of Li + on the Na +-dependent γ-aminobutyric acid (GABA) uptake was investigated by examining the effects of external and internal Li + ([Li] o and [Li] i) on GABA-gated Cl − current ( I Cl) in the frog dorsal root ganglion cells using the suction pipette technique which allows the internal perfusion under voltage-clamp condition. The suppression of GABA responses in the presence of external Na + ([Na] o) was larger at lower than at higher GABA concentration. Replacement of [Na] o with Li + completely removed the Na + suppression, and GABA dose-response curve in Li + external solution agreed well with that in Na +-free (Tris +) external solution. Either increasing [Li] i or internal Na + ([Na] i) at a constant [Na] o equally reduced the Na +-dependent suppression of GABA-gated I Cl. The results indicate that both the [Li] o and [Li] i remove in different manner the [Na] o-dependent suppression of GABA-induced I Cl: i.e. the [Li] o acts as a blocker of Na +-GABA co-transport mechanism while the increase of [Li] i decreases the Na + electrochemical potential gradient across the soma membrane as well as [Na] i does.