Replacement Of Extracellular Sodium Research Articles

Relationship between the thyroid hormone transport system and the Na(+)-H+ exchanger in cultured rat brain astrocytes.

The entry of T3 and T4 into rat cultured astrocytes is mediated by a sterospecific saturable transport system. This study examines the effect of inhibiting the Na(+)-H+ exchanger and intracellular acidification on the initial velocity of [125I]T3 and [125I]T4 uptake. The resting intracellular pH (pHi) was approximately 7.15 in astrocytes exposed to CO2/HCO3(-)-free medium buffered with HEPES at pH 7.40 at 22 C. Isoosmotic replacement of extracellular sodium by mannitol or choline decreased the pHi by 0.15 pH unit and reduced uptake by about 20%. Replacing sodium with lithium had no effect on uptake. Amiloride, a specific blocker of the Na(+)-H+ exchanger, reduced pHi, as described above, and inhibited T3 and T4 uptake by about 35%. Acid loading the cells with a NH4+ pulse decreased the pHi by up to 1.2 pH units and the uptake of T3 and T4 by up to 50%. The maximum velocity of uptake was decreased, whereas the Km was unchanged. An isoosmotic increase in the extracellular K+ concentration to 50 mM had no effect on T3 uptake. The initial velocity of T3 uptake by acid-loaded cells was gradually restored by increasing the extracellular Na+ concentration. These results indicate that thyroid hormone transport into rat cultured astrocytes involves a mechanism linked to the activity of the Na(+)-H+ exchanger and the H+ concentration inside the cells.

Action of angiotensin II, 5‐hydroxytryptamine and adenosine triphosphate on ionic currents in single ear artery cells of the rabbit

1. Angiotensin II, 5-hydroxytryptamine (5-HT) and adenosine triphosphate (ATP) evoked a transient inward current in isolated single car artery cells of rabbit held at -60 mV by whole cell voltage clamp in physiological saline using a KCL-containing pipette solution. Under these conditions agonist did not activate a calcium-dependent potassium current. 2. Responses to each agonist were transient and desensitized rapidly. Inward current at -60 mV holding potential was not abolished by blockade of voltage-dependent calcium channels or by buffering intracellular calcium with BAPTA, a calcium chelator, or following depletion of intracellular calcium stores with ryanodine. 3. The shape of the current-voltage relationships and the reversal potentials of the current induced by angiotensin II, 5-HT and ATP were similar under a variety of ionic conditions. Agonist-induced current was unaffected by replacing intracellular chloride with citrate ions or by replacing intracellular sodium with caesium or extracellular sodium with barium or calcium. Replacement of extracellular sodium with Tris shifted the reversal potential in all cases by around 30 mV negatively. 4. These data suggest that angiotensin II, 5-HT and ATP activate similar cationic conductances which are relatively non-selective allowing mono- and divalent cations to cross the smooth muscle cell membrane. These channels may allow the influx of calcium under physiological conditions.

Intracellular calcium and its sodium-independent regulation in voltage-clamped snail neurones.

1. We have used both Ca(2+)-sensitive microelectrodes and fura-2 to measure the intracellular free calcium ion concentration ([Ca2+]i or its negative log, pCai) of snail neurones voltage clamped to -50 or -60 mV. Using Ca(2+)-sensitive microelectrodes, [Ca2+]i was found to be approximately 174 nM and pCai, 6.76 +/- 0.09 (mean +/- S.E.M.; n = 11); using fura-2, [Ca2+]i was approximately 40 nM and pCai, 7.44 +/- 0.06 (mean +/- S.E.M., n = 10). 2. Depolarizations (1-20 s) caused an increase in [Ca2+]i which was abolished by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i was due to Ca2+ influx through voltage-activated Ca2+ channels. 3. Caffeine (10-20 mM) caused an increase in [Ca2+]i in the presence or absence of extracellular Ca2+. The effects of caffeine on [Ca2+]i could be prevented by ryanodine. 4. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a small increase in resting [Ca2+]i and slowed the rate of recovery from Ca2+ loads following 20 s depolarizations. 5. Neither replacement of extracellular sodium with N-methyl-D-glucamine (NMDG), nor loading the cells with intracellular sodium, had any effect on resting [Ca2+]i or the rate of recovery of [Ca2+]i following depolarizations. 6. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCmP) caused a small gradual rise in resting [Ca2+]i. Removal of extracellular sodium during exposure to CCmP had no further effect on [Ca2+]i. 7. Intracellular orthovanadate caused an increase in resting [Ca2+]i and prevented the full recovery of [Ca2+]i following small Ca2+ loads, but removal of extracellular sodium did not cause a rise in [Ca2+]i. We conclude that there is no Na(+)-Ca2+ exchanger present in the cell body of these neurones and that [Ca2+]i is maintained by an ATP-dependent Ca2+ pump.

N-Acetylaspartylglutamate catabolism is achieved by an enzyme on the cell surface of neurons and glia

N-Acetylaspartylglutamate (NAAG) is a nervous system-specific, acidic dipeptide which is released from neurons in a manner consistent with a function in synaptic neurotransmission. The hydrolysis of NAAG to produce glutamate and N-acetylaspartate was analyzed in cell cultures prepared from murine brain cells. Peptidase activity against NAAG was found in cultures which contained both neurons and glia, as well as in cultures of glia alone. Several lines of evidence were obtained in support of the hypothesis that this peptidase activity is predominantly bound to the extracellular face of the plasma membranes of these cells. Glutamate released from NAAG accumulated in the extracellular medium. Extracellular application of peptidase inhibitors effectively reduced NAAG hydrolysis. Peptidase activity was not secreted into the cell culture medium by intact cells, and lysed cells did not release detectable peptidase activity beyond that obtained with intact cells. Replacement of extracellular sodium with choline inhibited peptide uptake while stimulating apparent extracellular NAAG hydrolysis by intact cells in culture. Finally, the steady rise in extracellular glutamate as a consequence of NAAG hydrolysis by these brain cells, including glia, supports the conclusion that glutamate uptake is not tightly coupled to peptidase activity and thus that NAAG serves as a significant source of glutamate in the synaptic space following depolarization-induced peptide release.

Electrophysiology of ammonia transport in renal straight proximal tubules

To test for electrogenic transport of ammonium ions in straight proximal renal tubules, isolated perfused tubules have been exposed to peritubular ammonium ions during continuous recording of cell membrane potential. As a result, 20 mmol/liter NH4+ leads to a rapid, reversible depolarization of the cell membrane by 9.0 +/- 0.3 mV (N = 86). This depolarization is not significantly affected by 10 mmol/liter barium or 0.1 mmol/liter amiloride on both sides of the epithelium, but is significantly blunted by omission of extracellular bicarbonate and CO2 (3.8 +/- 0.4 mV, N = 9), by 1 mmol/liter acetazolamide (4.3 +/- 0.3 mV, N = 11), by 1 mmol/liter peritubular amiloride (4.3 +/- 1.1 mV, N = 7), by 1 mmol/liter SITS (5.7 +/- 0.4 mV, N = 6), and by replacement of extracellular sodium with choline (4.7 +/- 0.5 mV, N = 8). In the presence of both amiloride (1 mmol/liter) and acetazolamide (1 mmol/liter) in the bath, the NH4+ induced depolarization is completely abolished. Furthermore, the combined omission of bicarbonate and addition of 10 mmol/liter barium eliminates the NH4+ induced depolarization. About 50% of the depolarization can be explained by enhanced electrogenic bicarbonate exit due to the intracellular alkalosis. The other 50% is explained by amiloride and barium sensitive electrogenic entry of NH4+ into the cell.

Membrane potential fluctuations and transient inward currents induced by reactive oxygen intermediates in isolated rabbit ventricular cells.

The cellular basis of reactive oxygen intermediate-induced arrhythmias was investigated in isolated rabbit ventricular cells using the whole-cell voltage- and current-clamp techniques. Singlet oxygen and superoxide were generated by the photoactivation of rose bengal. Single ventricular cells exposed to rose bengal (10-100 nM) exhibited spontaneous membrane potential fluctuations at plateau potentials and at the level of the resting membrane potential. The voltage fluctuations induced in the resting potential occasionally triggered repetitive action potential discharges. At the resting membrane potential, the magnitude and dominant frequency of the voltage fluctuations were 1-3 mV and 1.5 Hz, respectively. At plateau potentials, the amplitude of the voltage fluctuations was about 2-5 mV, and the dominant oscillatory frequency was 2.6 Hz. In voltage-clamp experiments, transient inward currents were induced on repolarization after a depolarizing clamp step. Oscillatory currents also occurred occasionally during clamp steps to positive potentials. The peak frequencies of transient inward currents recorded at -20 and -70 mV were approximately 3.7 and 2.3 Hz, respectively, indicating that these currents may underlie the arrhythmogenic membrane potential fluctuations observed in current-clamp experiments. The rose bengal-induced transient inward currents were shown to be dependent on the magnitude and duration of the preceding voltage step. Studies of the voltage dependence of transient inward currents showed that these currents remained inward even at positive potentials (+30 mV), and replacement of extracellular sodium with lithium decreased transient inward current to approximately 10% of its initial value. Thus, the major component of oxidant stress-induced inward current appears to be electrogenic Na-Ca exchange. This oscillatory transient inward current may be responsible for the arrhythmias induced in isolated hearts exposed to reactive oxygen intermediates, and since oxidant stress has been implicated in reperfusion injury, it is possible that similar oscillatory currents may underlie reperfusion-induced arrhythmias.

Glutamate neurotoxicity in spinal cord cell culture

The neurotoxicity of glutamate was investigated quantitatively in mixed neuronal and glial spinal cord cell cultures from fetal mice at 12–13 days of gestation. Five-minute exposure to 10–1000 μM glutamate produced widespread acute neuronal swelling, followed by neuronal degeneration over the next 24 h ( ec 50 for death about 100–200 μM); glia were not injured. Glutamate was neurotoxic in cultures as young as four days in vitro, although greater death was produced in older cultures. By 14–20 days in vitro, 80–90% of the neuronal population was destroyed by a 5-min exposure to 500 μM glutamate. Acute neuronal swelling following glutamate exposure was prevented by replacement of extracellular sodium with equimolar choline, with minimal reduction in late cell death. Removal of extracellular calcium enhanced acute neuronal swelling but attenuated late neuronal death. Both acute neuronal swelling and late degeneration were effectively blocked by the noncompetitive N-methyl- d-aspartate receptor antagonist dextrorphan and by the novel competitive antagonist CGP 37849. Ten micromolar 7-chlorokynurenate also inhibited glutamate neurotoxicity; protection was reversed by the addition of 1 mM glycine to the bathing medium. These observations suggest that glutamate is a potent and rapidly acting neurotoxin on cultured spinal cord neurons, and support involvement of excitotoxicity in acute spinal cord injury. Similar to telencephalic neurons, spinal neurons exposed briefly to glutamate degenerate in a manner dependent on extracellular Ca 2+ and the activation of N-methyl- d-aspartate receptors.

Actions of capsaicin on peripheral nociceptors of the neonatal rat spinal cord‐tail in vitro: dependence of extracellular ions and independence of second messengers

1. We have tested the hypothesis that capsaicin-induced activation, desensitization and impairment of peripheral nociceptor function is mediated by separate mechanisms. This was investigated by use of an in vitro preparation of the neonatal rat spinal cord with the functionally attached tail in which the cord and tail were separately superfused with physiological solution. Activation of peripheral fibres by noxious (capsaicin, bradykinin, 5-hydroxytrptamine, heat, pinch) and innocuous (light brush) stimuli was assessed by recording the depolarization of a spinal ventral root (L3-L5). 2. Brief administration of capsaicin produced dose-related depolarizing responses (EC50 = 280 nM). These responses could be reproduced for many hours following the repeated application of capsaicin at a submaximal concentration. Prolonged application of 0.5-2.0 microM capsaicin induced a selective desensitization to subsequent brief administrations of capsaicin. Prolonged administration at 20-50 microM produced an additional non-selective reduction in responses to all noxious stimuli without changing innocuous brush responses. 3. Removal of extracellular calcium from the tail superfusate did not reduce the response to capsaicin or prevent capsaicin-induced desensitization. However, high concentrations of capsaicin no longer induced a non-specific reduction of responses to other noxious stimuli. The response to a brief administration of capsaicin was unaffected by calcium channel blocking drugs including nifedipine, cadmium or omega-conotoxin. On the other hand high extracellular calcium increased the incidence of the non-selective reduction of responses to all noxious stimuli produced by high concentrations of capsaicin. 4. Replacement of extracellular sodium with choline blocked peripheral nerve conduction but did not prevent the desensitization produced by capsaicin. In addition, high concentrations of capsaicin were less effective in reducing the responsiveness to other noxious stimuli. 5. Neither capsaicin-evoked responses nor capsaicin-induced desensitization were affected by the administration of forskolin, dibutyryl cyclic AMP, nitroprusside, dibutyryl cyclic GMP, beta-12,13 phorbol dibutyrate, trifluoperazine, indomethacin, staurosporine or mepacrine, in the tail superfusate. 6. These data suggest that capsaicin-induced activation, desensitization and impairment of peripheral nociceptors may be separable phenomena. Extracellular calcium is not required for capsaicin-induced activation or desensitization but calcium as well as sodium are important for capsaicin-induced impairment of nociceptive responses. Desensitization may occur independently of peripheral fibre activation and cannot be attributed to a central mechanism. Finally neither capsaicin-induced activation nor desensitization require the participation of a second messenger.

PH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Confluent monolayers of the established opossum kidney cell line were exposed to NH4Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PDm) and membrane resistance (R'm) in bicarbonate-free Ringer. The removal of extracellular NH4Cl leads to an intracellular acidification from a control value of 7.33 +/- 0.08 to 6.47 +/- 0.03 (n = 7). This inhibits the absolute K conductance (gK+), reflected by a decrease of K+ transference number from 71 +/- 3% (n = 28) to 26 +/- 6% (n = 5), a 2.6 +/- 0.2-fold rise of R'm, and a depolarization by 24.2 +/- 1.5 mV (n = 52). In contrast, intracellular acidification during a block of gK+ by 3 mmol/liter BaCl2 enhances the total membrane conductance, being shown by R'm decrease to 68 +/- 7% of control and cell membrane depolarization by 9.8 +/- 2.8 mV (n = 17). Conversely, intracellular alkalinization under barium elevates R'm and hyperpolarizes PDm. The replacement of extracellular sodium by choline in the presence of BaCl2 significantly hyperpolarizes PDm and increases R'm, indicating the presence of a sodium conductance. This conductance is not inhibited by 10(-4) mol/liter amiloride (n = 7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na(+)-conductive channels with 18.8 +/- 1.4 pS (n = 10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl-. The low-conductive channel is 4.8 times more permeable for Na+ than for K+. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).

Non-NMDA receptor-mediated neurotoxicity in cortical culture

The neurotoxicity of 3 non-NMDA glutamate receptor agonists--kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA), and quisqualate--was investigated quantitatively in dissociated murine cortical cultures. Five minute exposure to 500 microM kainate, but not AMPA, produced widespread acute neuronal swelling. Kainate-induced swelling was resistant to 2-amino-5-phosphonovalerate (APV) or replacement of extracellular sodium with choline but attenuated by either kynurenate or low concentrations of quisqualate. Unlike NMDA agonists, kainate or AMPA did not produce much late neuronal loss after a 5 min exposure. In contrast, 5 min exposure to 500 microM quisqualate produced both acute neuronal swelling and widespread late neuronal degeneration. This acute swelling was blocked by APV or by replacement of extracellular sodium by choline, consistent with mediation by NMDA receptors; we speculate that high concentrations of quisqualate may directly activate NMDA receptors or induce the release of endogenous glutamate. Quisqualate-induced late neuronal degeneration may be due to another unexpected process: cellular quisqualate uptake and delayed release, converting brief addition into prolonged exposure. Hours after thorough washout of exogenously added quisqualate, micromolar concentrations could be detected in the bathing medium by high performance liquid chromatography. With lengthy exposure (20-24 hr), all 3 non-NMDA agonists were potent neurotoxins, able to destroy neurons with EC50's of about 20 microM for kainate, 4 microM for AMPA, and 1 microM for quisqualate. Kynurenate and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not APV or L-glutamate diethyl ester, were effective in attenuating the neuronal degeneration induced by these agonists. CNQX was about 3 times more selective than kynurenate against kainate-induced neuronal injury, but CNQX was still nearly equipotent with APV against NMDA-induced injury. Gamma-D-glutamylaminomethyl sulfonate exhibited partial antagonist specificity for AMPA-induced toxicity.

Spermidine uptake by type II pneumocytes: interactions of amine uptake pathways.

Uptake of exogenous spermidine by type II pulmonary epithelial cells in primary culture is inhibited by several amines. The present studies further detail the interaction of those alternative substrates with the pathway for spermidine (SPD) transport. Transport activity was measured on the first day of primary culture in type II cells isolated by elastase digestion, followed by density gradient centrifugation and differential adherence in vitro. Spermidine uptake was inhibited in a concentration-dependent manner by the polyamines putrescine (PUTR) and spermine (SPM), by the drug methylglyoxal bis-(guanylhydrazone) (MGBG), and by the herbicide paraquat (PQ). The order of effectiveness of these competitors was SPM greater than PUTR approximately MGBG much greater than PQ. The kinetics of inhibition by SPM were mixed, with an increase in the apparent Michaelis constant and a reduction in the maximal velocity of the SPD uptake pathway. Cellular uptake of SPD and PUTR was inhibited by replacement of extracellular sodium with choline or lithium (PUTR greater than SPD), but SPM uptake was unaffected. PQ appeared to interact with the polyamine transport pathway with low affinity. Compared with SPD and SPM, PQ was most effective as an inhibitor of PUTR uptake, and like PUTR, uptake of the herbicide was sharply inhibited as extracellular sodium was reduced. These observations suggest the presence of diverse pathways for uptake of exogenous polyamines by type II pulmonary epithelial cells and indicate that PQ probably enters the cells by the sodium-dependent transport system favored by PUTR.

The transport of L-arginine in Chinese hamster ovary cells

The transport of L-arginine has been characterized in Chinese hamster ovary cells (CHO). In the absence of Na + the influx of the amino acid decreased. Both in the presence and in the absence of Na + L-arginine influx was trans-stimulated and cis-inhibited by cationic amino acids. The amino acid entered CHO cells through an apparently non saturable mechanism and a single saturable agency whose Km increased in the absence of Na +. These results indicate that the agency devoted to transport cationic amino acids in CHO cells resembles system y +, the Na +-independent route that transports cationic amino acids in a number of mammalian models, although its activity is lowered by the replacement of extracellular sodium.

THE EFFECT OF LANTHANUM, QUERCETIN AND DINITROPHENOL ON CALCIUM‐EVOKED ELECTRICAL RESPONSES IN HAMSTER EGGS

At room temperature micro-injections of calcium or strontium produced transient hyperpolarizations with an associated rise in input conductance. By contrast micro-injections of potassium, barium, magnesium, cobalt or lanthanum did not produce hyperpolarizations. The reversal potential for the hyperpolarizing response was about -80 mV. Some responses to calcium injections appeared to suffer from an additional transient leak conductance generated by the injected current. In these cases the recovery of the potential and the conductance to normal values was prolonged. The reversal potential of this additional leak pathway was about -10 mV. Experiments designed to investigate the role of active calcium extrusion from the cells showed that extracellular lanthanum or quercetin caused a pronounced slowing of the recovery phase of the potential and conductance response to calcium injection. The metabolic uncoupler dinitrophenol also prolonged the calcium-evoked responses. The replacement of extracellular sodium by lithium or choline produced no alteration in the time course of the calcium-evoked responses, thus suggesting that sodium-calcium exchange exerts no rate control on the recovery phase of those responses.

Intracellular sodium and the differentiation of amphibian embryonic neurones.

1. Experiments have been done to examine the mechanism of the inhibition of neural differentiation produced by inhibiting the sodium pump with cardiac glycosides during the mid-neural fold stages of development of the amphibian embryo. Neural differentiation was assessed quantitatively by counting the number of neurones that undergo primary differentiation in tissue culture, as a proportion of the total number of differentiated cells.2. Inhibition of the sodium pump by lowering the extracellular potassium concentration ([K(o)]) to 0 during the mid-neural fold stages inhibited neural differentiation.3. Raising the extracellular calcium concentration to 10 mM during treatment with strophanthidin protected differentiating neurones from the effects of the sodium pump inhibitor. Lowering [Ca](o) to 0.05 mM potentiated the effect of low doses of glycoside.4. In the presence of high extracellular calcium and 5 x 10(-6) M-strophanthidin the membrane potential of neural plate cells remained close to the levels recorded at the beginning of neurulation; the normal increase in resting potential was not restored.5. Addition of 10 mM-Sr(2+) to the bathing medium also protected nerve cells against the inhibition produced by strophanthidin; Sr(2+) was less effective than Ca(2+).6. Addition of either 10 mM-Mg(2+) or Mn(2+) had no effect on the inhibition of differentiation produced by strophanthidin.7. Addition of Mn(2+) along with high Ca(2+) prevented calcium from exerting its protective effect.8. The eyes of embryos treated with high Ca(2+) together with strophanthidin during neurulation and then allowed to grow into tadpoles developed normally. When Mn(2+) was present together with Ca(2+) and strophanthidin the eyes were disrupted similarly to those of embryos treated with strophanthidin alone.9. Replacement of extracellular sodium with equimolar amounts of choline or lithium prevented the cardiac glycoside from inhibiting neural differentiaion.10. The protection afforded by lowering [Na](o) was abolished when [Ca](o) was simultaneously lowered to 0.05 mM.11. Tadpoles from embryos treated with low extracellular sodium together with strophanthidin during neurulation had normal eyes compared to those treated with strophanthidin alone.12. Measurement of the intracellular sodium concentration ([Na](i)) with sodium-sensitive micro-electrodes put [Na](i) at about 30 mM before the neural folds lift. As the sodium pump is activated (stages 14(1/2)-15)[Na](i) in the neural plate falls; by the end of the mid-neural fold stage it is less than 10 mM.13. Addition of 5 x 10(-6)M-strophanthidin to the bathing fluid before activation of the sodium pump prevented the fall in [Na](i); in embryos where [Na](i) had begun to drop strophanthidin produced a rise to about 30 mM.14. When 10 mM-calcium was present along with strophanthidin [Na](i) fell to about 17 mM during neurulation, despite inhibition of the sodium pump.15. It is concluded that it is unlikely that either abolition of the normal increase in resting potential or a fall in gap junction permeability is responsible for the reduction in neural differentiation produced by blocking the sodium pump during neurulation.16. The results are consistent with the view that strophanthidin achieves its effect by preventing the fall in [Na](i) that occurs during normal neurulation because of activation of the sodium pump. They are discussed in the light of this suggestion.