Relations Between Intracellular Ions and Energy Metabolism Under Acidotic Conditions: A Study with Nigericin in Synaptosomes, Neurons, and C6 Glioma Cells

Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na⁺]_i., [K⁺]_i, and [Ca²⁺]_i, and pH under conditions when [H⁺]_i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K⁺ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca²⁺], and [Na⁺]., The increase in [Na⁺], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K⁺ and subsequent depolarization lead to an increase in Na⁺ influx and stimulation of the Na⁺/K⁺ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H⁺] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K⁺], depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia.     

Gramicidin-D-stimulated influx of monovalent cations into plant roots

Summary Gramicidin D and nigericin were found to stimulate K⁺ influx into oat roots. Valinomycin and nonactin had little effect on K⁺ influx. The region of the root most sensitive to gramicidin was the elongation zone. Monocot roots were more sensitive to gramicidin than dicot roots. At 0.2 mM KCl, gramicidin stimulated K⁺ influx by 4- to 8fold over a 30-min absorption period. Although a gramicidin response is detectable within one minute, maximum stimulation occurred after a slight (approximately 2-min) lag period. The gramicidin effect was much greater at 0.2 mM KCl than at 20 mM KCl. Respiratory inhibitors reduced the gramicidin-stimulated K⁺ influx by 50–80%. The results are discussed in terms of possible mechanisms of action of the various ionophores on ion transport in roots.This work was supported by National Science Foundation Grants GB-5549 and GB-12674 and by the Illinois Agriculture Experiment Station.     

Ionophoretic stimulation of K+-ATPase of oxyntic cell microsomes

The K⁺ -selective ionophores valinomycin, trinactin, nigericin, and gramicidin stimulated the K⁺-ATPase of oxyntic cell microsomes as much as 5 fold. Gramicidin altered the K⁺ activation kinetics: while the V_max greatly increased, the K_a for K⁺ shifted only slightly. Activation also occurred with lyophilization, repeated freezing and thawing and nonionic detergents. The results are consistent with one population of membranes being freely permeable to K⁺ (source of the K⁺-dependent activity in absence of ionophore) and another population being impermeable to K⁺. The K⁺-ATPase in these latter vesicles would be activated by the appropriate ionophore or membrane disruptive procedure.     

Ionophore-induced efflux of sodium and potassium ions from sea urchin eggs

The efflux of K⁺ and Na⁺ from sea urchin eggs during Ca²⁺ ionophore A23187-induced parthenogenesis was studied in a K⁺ and Na⁺-free artificial seawater using extracellular ion-specific electrodes. We have probed this model system with monovalent cation-specific ionophores to determine if they affect K⁺ efflux in the unfertilized egg and whether any changes in ionophore sensitivity are observed during egg activation. In 500 mM choline chloride, 10 mM CaCl₂, 50 mM MgCl₂, 10 mM Tris-Cl pH 8.0, A23187 induced a rapid efflux of K⁺ and Na⁺ from the eggs after a short lag time (10–15 seconds). After the burst, the rate of K⁺ efflux remained higher than the pre-activation rate, but was lower than during the burst phase, while the rate of Na⁺ efflux became nearly zero. Monovalent cation-specific ionophores (valinomycin, gramicidin and nigericin) had no effect on K⁺ efflux from the unfertilized eggs in our model system. However, once the egg was activated by A23187, each of the above ionophores caused a prolongation of the burst phase for many minutes. These results show that the unfertilized egg plasma membrane (using our artificial conditions) is not susceptible to the monovalent cation-specific antibiotics and suggest that either the inserted cortical granule membrane or the developing fertilization envelope interacts with these ionophores to cause the change in rate-limiting step for K⁺ efflux observed egg activation.     

Demonstration of Voltage-Dependent and TTX-Sensitive Na+-Channels in Human Melanocytes

The electrophysiological properties of cultured human melanocytes were investigated using the whole-cell configuration of the patch-clamp technique. Depolarizations to membrane potentials more positive than -30 mV resulted in the rapid development (<1 ms to peak) of an inward current. The maximum peak current was observed at +10 mV and reached an average amplitude of about 270 pA. During the depolarizations, the current inactivated with a time constant of about 2 ms. The current was abolished by the addition of 0.3 μM tetrodotoxin, a blocker of voltage-gated Na⁺-channels, and disappeared when Na⁺ was omitted from the extracellular medium. In addition, the melanocytes contain at least two types of outward K⁺-current. The first type, observed in every cell, was highly sensitive (K_i 1 mM) to the K⁺-channel blocker TEA, required depolarizations beyond zero to be activated and did not inactivate. The second type was less regularly observed (10% of the cells). This current activated at more negative voltages (–20 mV), was resistant to TEA (20 mM) but was blocked by 2 mM 4-aminopyridine and inactivated rapidly during depolarizations. We conclude that human melanocytes are equipped with voltage-dependent Na⁺-channels, a delayed rectifying K⁺-current and a K⁺-current similar to the A-current in neurones.     

An ATPase from dog gastric mucosa: changes of outer pH in suspensions of membrane vesicles accompanying ATP hydrolysis

A microsomal Mg-ATPase from the gastric mucosa of dog, cat and frog has a K_m for ATP in the region 20–25 μM and by the value of this coefficient can be differentiated from the mitochondrial Mg-ATPase. The microsomal Mg-ATPase from dog gastric mucosa can be stimulated by gramicidin, nigericin and valinomycin in a KCl medium. This Mg-ATPase seems to be located in the ion impermeable membrane of microsomal vesicles and ATP hydrolysis driven changes of the outer pH can be observed. The data are consistent with the ATP hydrolysis driven entry of H⁺ ions across the vesicle membrane.     

Transport of Ammonium and Methylammonium Ions by Azotobacter vinelandii

Ammonium and methylammonium are rapidly taken up by cultures of Azotobacter vinelandii respiring in the presence of succinate. The rate of methylamine uptake increased with external pH from 5.5 to 7.5 but increasing the pH further to 8.5 had little effect on activity, indicating that methylammonium cation rather than uncharged methylamine is the permeant species. The kinetics of methylammonium entry followed the Michaelis-Menten relationship, yielding a K_m of 25 μM and a V_max of 3.8 nmol/min per mg of cell protein. At saturating concentrations ammonium was taken up at rates 30-fold higher than those for methylammonium. Ammonium was a competitive inhibitor of methylammonium uptake and gave an inhibition constant of 1 μM. Ammonium derivatives were inhibitors of methylammonium entry in order of effectiveness: hydrazine > methylhydrazine > formamidine > guanidine > dimethylamine > ethylamine; amides and amino acids did not block uptake. Likewise, metal cations inhibited in the order Tl⁺ > Cs⁺ > Rb⁺, whereas Na⁺, K⁺, and Li⁺ produced no significant effect. Methylammonium uptake was blocked in cells exposed to an uncoupler, p-trifluorome-thoxycarbonyl cyanide-phenyl hydrazone or gramicidin D, but not with dicyclo-hexylcarbodiimide or arsenate. Valinomycin stimulated methylammonium entry into cells in a K⁺-free medium but prevented entry in the presence of 10 mM K⁺. Monensin and nigericin had little effect on transport. These results indicate that methylammonium and ammonium ions enter A. vinelandii electrogenically via a specific transporter.     

The diabetogenic agent alloxan increases K+ permeability by a mechanism involving activation of ATP-sensitive K+-channels in mouse pancreatic β-cells

The effects of the diabetogenic agent, alloxan, on membrane potential, input resistance and electrical activity of normal mouse pancreatic -cells were studied. Tetraethylammonium (TEA), quinine and Glyburide were used to block K⁺-channels and to elucidate the mechanisms underlying alloxan's effects on -cell membrane potential. Exposure of the islet to alloxan (75–100 M) in the presence of glucose (11 mM), produced a rapid (15 sec), transient inhibition of electrical activity, often accompanied by hyperpolarization of the membrane, and this was followed by recovery of the burst pattern. This early effect of alloxan was followed after approximately 15 min by a complete inhibition of electrical activity and hyperpolarization. The inhibition accompanied by hyperpolarization was associated with a decrease in input resistance, indicating increased K⁺-conductance. Both the transient and delayed effects of alloxan were blocked by glucose (33 mM), quinine and glyburide but not by other conditions which induced continuous electrical activity such as elevated external [K⁺] (10 mM), ouabain, K⁺ removal, or TEA (20 mM). The transient inhibition induced by alloxan may be due to a direct competition with glucose transport/metabolism since it did not occur when alpha-keto isocaproic acid (KIC) was used to induce electrical activity. The delayed inhibition may reflect indirect effects of accumulation of this agent or its metabolites within the cell. Since both effects of alloxan are blocked by glyburide they appear to involve activation of the ATP-sensitive K⁺-channel (K-ATP).     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.     

Effects of K+ Channel Blockers and Nitric Oxide on the Electrical and Contractile Activities of Smooth Muscles of the Rabbit Main Pulmonary Artery

Using a sucrose-bridge technique, we studied electrical and mechanical responses of smooth muscle ring strips of the rabbit main pulmonary artery to applications of blockers of voltage-operated (including Ca²⁺-dependent) K⁺ channels, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), as well to application of nitric oxide (NO); nitroglycerin (NG) was used as a donor of the latter. All experiments were carried out under conditions of blockade of the adreno- and cholinoreceptors in the preparation. Both TEA and 4-AP evoked dose-dependent effects: depolarization of smooth muscle cells (SMC) and their contraction. Simultaneous addition of TEA and 4-AP to the normal superfusate (Krebs solution) resulted in intensification of depolarization and initiated generation of action potentials (AP); contractions became rather intensive and possessed a tetanic pattern. Addition of NG to TEA- and 4-AP-containing Krebs solution effectively suppressed AP generation and contractions, whereas the depolarization level underwent only mild modifications. These findings show that Ca²⁺-dependent high-conductance K⁺ channels (K_Ca channels) and 4-AP-sensitive voltage-operated K⁺ channels (K_V channels) are involved in the formation of the resting membrane potential (RMP) in SMC of the rabbit main pulmonary artery. The impact of the K_Ca channels is greater than that of the K_V channels. We suppose that the effects of NO on SMC are related to inhibition of the activity of high-threshold voltage-operated L-type Ca²⁺ channels and, probably, to lowering of the sensitivity of the contractile SMC apparatus to Ca²⁺.     

Effect of potential-dependent potassium uptake on calcium accumulation in rat brain mitochondria

The effect of potential-dependent potassium uptake at 0–120 mM K⁺ on matrix Ca²⁺ accumulation in rat brain mitochondria was studied. An increase in oxygen consumption and proton extrusion rates as well as increase in matrix pH with increase in K⁺ content in the medium was observed due to K⁺ uptake into the mitochondria. The accumulation of Ca²⁺ was shown to depend on K⁺ concentration in the medium. At K⁺ concentration ?30 mM, Ca²⁺ uptake is decreased due to K⁺-induced membrane depolarization, whereas at higher K⁺ concentrations, up to 120 mM K⁺, Ca²⁺ uptake is increased in spite of membrane depolarization caused by matrix alkalization due to K⁺ uptake. Mitochondrial K _ATP ⁺ -channel blockers (glibenclamide and 5-hydroxydecanoic acid) diminish K⁺ uptake as well as K⁺-induced depolarization and matrix alkalization, which results in attenuation of the potassium-induced effects on matrix Ca²⁺ uptake, i.e. increase in Ca²⁺ uptake at low K⁺ content in the medium due to the smaller membrane depolarization and decrease in Ca²⁺ uptake at high potassium concentrations because of restricted rise in matrix pH. The results show the importance of potential-dependent potassium uptake, and especially the K _ATP ⁺ channel, in the regulation of calcium accumulation in rat brain mitochondria.     

Stimulation of human cheek cell Na+/H+ antiporter activity by saliva and salivary electrolytes: amplification by nigericin

Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na⁺ uptake (Na⁺/H⁺ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na⁺/H⁺ antiporter. Using the standard conditions of 1 mmol/1 Na⁺, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K⁺) and gramicidin (Na⁺) increased EIPA-sensitive Na⁺ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K⁺-H⁺) increased EIPA-sensitive Na⁺ uptake by 654% (p < 0.01), with maximal Na⁺ uptake achieved by 1 min and at an ionophore concentration of 50 mol/l, with an EC ₅₀ value 6.4 mol/l. Preincubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37°C, also significantly stimulated EIPA-sensitive Na⁺ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K⁺-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 mol/1 nigericin in the assay, produced maximum EIPA-sensitive Na⁺ uptake. After pre-incubation with water, 25 mmol/1 K⁺-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na⁺ uptake was saturable with respect to external Na⁺ with K_m values of 0.9, 1.7, and 1.8 mmol/l, and V _max values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 mol/1 nigericin in the assay, Na⁺ uptake was inhibited by either increasing the [K⁺]_o in the assay, with an ID ₅₀ of 3 mmol/l. These results indicate that nigericin can facilitate K⁺ _i exchange for H⁺ _o and the attending re-acidification of the cheek cell amplifies IINa+ uptake via the Na⁺/H⁺ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na⁺ uptake is therefore dependent, in part, on the intracellular K⁺ _i.     

Purification and characterization of malate dehydrogenase from the phototrophic bacterium,Rhodopseudomonas capsulata

Malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37) has been purified about 480-fold from crude extract of the facultative phototrophic bacterium, Rhodopseudomonas capsulata by only two purification steps, involving Red-Sepharose affinity chromatography. The enzyme has a molecular mass of about 80 kDa and consists of two subunits with identical molecular mass (35 kDa). The enzyme is susceptible to heat inactivation and loses its activity completely upon incubation at 40°C for 10 min. Addition of NAD⁺, NADH and oxaloacetate, but not l-malate, to the enzyme solution stabilized the enzyme. The enzyme catalyzes exclusively the oxidation of l-malate, and the reduction of oxaloacetate and ketomalonate in the presence of NAD⁺ and NADH, respectively, as the coenzyme. The pH optima are around 9.5 for the l-malate oxidation, and 7.75–8.5 and 4.3–7.0 for the reduction of oxaloacetate and ketomalonate, respectively. The K_m values were determined to be 2.1 mM for l-malate, 48 μM for NAD⁺, 85 μM for oxaloacetate, 25 μM for NADH and 2.2 mM for ketomalonate. Initial velocity and product inhibition patterns of the enzyme reactions indicate a random binding of the substrates, NAD⁺ and l-malate, to the enzyme and a sequential release of the products: NADH is the last product released from the enzyme in the l-malate oxidation.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Measurement and significance of the membrane potential in Methanoba cterium bryantii

The membrane potential (Δψ) of Methanobacterium bryantii was 133–142 mV as measured from the distribution of ⁸⁶Rb⁺ in valinomycin-treated cells, and was considerably higher than that obtained using triphenylmethylphosphonium in the presence of tetraphenylboron. The Δψ measured using the Rb⁺/valinomycin method was sensitive to certain ionophores including gramicidin, nigericin, carbonyl cyanide m-chlorophenylhydrazone and 3,3′,4′,5-tetrachlorosalicylanilide. It was also dissipated by 1 mM tetraphenylphosphonium and was abolished in heat-treated or permeabilized cells. The Δψ could be varied by adjusting the extracellular potassium concentration in valinomycin-treated cells. Monensin-treated cells possessed a significantly increased Δψ, as monitored by the Rb⁺ / valinomycin method. Tetraphenylphosphonium cation (1 mM) abolished methane synthesis, intracellular ATP and Δψ, supporting a role for Δψ in ATP and CH₄ synthesis. However, lower concentrations of the lipophilic cation (50 μM) greatly elevated both the intracellular ATP concentration and Δψ but decreased the rate of CH₄ synthesis by almost 50%. Thus, tetraphenylphosphonium cation exerts a primary inhibitory effect on CH₄ synthesis which cannot be attributed to the loss of Δψ or ATP.     

Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K+ transport in a higher plant cell

Fusicoccin (FC) has long been known to promote K⁺ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H⁺ pumping stimulated in FC, could help drive K⁺ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V _m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K⁺ (⁸⁶Rb⁺) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K⁺ movement across the membrane could be modified in FC. We have explored a role for K⁺ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (⁸⁶Rb⁺) flux and in electrical current under voltage clamp was followed, using the K⁺ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K⁺-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K⁺ channel current contributed appreciably to charge balance maintainingV _m, and adding 10 mM TEA to block the current depolarized (positive-going)V _m; TEA also reduced⁸⁶Rb⁺ efflux by 68–80%. Following treatments with 10 M FC, both K⁺ channel current and⁸⁶Rb⁺ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced⁸⁶Rb⁺ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K⁺ (Rb⁺), despite membrane potentials which were 30–60 mVpositive of the K⁺ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K⁺ channels. They offer evidence of an energy-coupled mechanism for K⁺ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K⁺ transport capacity of the cells, independent of any changes in membrane potential.Abbreviations and symbols E _K equilibrium potential for K⁺ - FC fusicoccin - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - G _m membrane (slope) conductance atV _m - I-V current-voltage (relationship) - apparent rate constant for exchange - K _i ⁺ , K ₀ ⁺ intracellular, extracellular K⁺ (concentration) - TEA tetraethylammonium chloride - V _m free-running membrane potential (difference)     

Salt tolerance inNitellopsis obtusa

Summary The mechanism of salt tolerance was studied using isolated internodal cells of the charophyteNitellopsis obtusa grown in fresh water. When 100 mM NaCl was added to artificial pond water (0.1 mM each of NaCl, KC1, CaCl₂), no cell survived for more than one day. Within the first 30 minutes, membrane potential (E_m) depolarized and membrane resistance (R_m) decreased markedly. Simultaneously, cytoplasmic Na⁺ increased and K⁺ decreased greatly. At steady state the increase in Na⁺ content was roughly equal to the decrease in K⁺ content. The Cl^– content of the cytoplasm did not change. These results suggest that Na⁺ enters the cytoplasm by exchange with cytoplasmic K⁺. Both the entry of Na⁺ and the exit of K⁺ are assumed to be passive and the latter being caused by membrane depolarization. Vacuolar K⁺, Na⁺, and Cl^– remained virtually constant, suggesting that rapid influx of Na⁺ from the cytoplasm did not occur.In 100 mM NaCl containing 10 mM CaCl₂, membrane depolarization, membrane resistance decrease and changes in cytoplasmic [Na⁺] and [K⁺] did not occur, and cells survived for many days. When cells treated with 100 mM NaCl were transferred within 1 hour to 100 mM NaCl containing 10 mM CaCl₂, Em decreased, Rm increased, cytoplasmic Na⁺ and K⁺ returned to their initial levels, and cells survived. Two possible mechanisms for the role of Ca²⁺ in salt tolerance inNitellopsis are discussed; one a reduction in plasmalemma permeability to Na⁺ and the other a stimulation of active Na⁺-extrusion.     

Extracellular potassium controls responsiveness of the noradrenergic cAMP system in the rat retina to fluphenazine

The effects of fluphenazine (FLU) on the noradrenaline (NA) induced cAMP-synthesis in intact rat retinae were studied as a function of extracellular K⁺- and Ca²⁺-ions. Thus NA-induced cAMP levels were measured after incubating intact rat retinae with 50 μM NA in the presence or absence of FLU and in the presence of 1 or 10 mM theophylline. Results were: (1) Experimental condition a: standard NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, at 10 mM theophylline, at 10 μM FLU and at 2 and 0 mM K⁺. FLU does not affect the NA-response at 2 mM K⁺ significantly; however, it inhibits the NA-response at 0 mM K⁺ in this condition. (2) Experimental condition b: NA-responses were measured after incubating retinae at 0.125 mM Ca²⁺, 10 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU replaces a Ca²⁺ function probably connected with the synthesis part of the NA-cAMP system and NA-responses in this low Ca²⁺ condition are consequently enhanced by FLU; however, FLU inhibits the NA-response at 0 mM K⁺ in this condition. (3) Experimental condition c: NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, 1 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU enhances the NA-response by further inhibition of the degradation part of the NA-cAMP system; FLU inhibits the NA-response at 0 mM K⁺ in this condition. (4) The inhibitions of the NA-responses by FLU at 0 mM K⁺ in all three conditions a, b and c showed an apparent K_m of 1 μM. (5) Low concentrations of K⁺ (0.4–0.8 mM) maintain the property of FLU to enhance the NA-responses at condition b (0.125 mM Ca²⁺) and at condition c (1 mM theophylline). Results suggest that the activation of NA-receptor coupled adenylate cyclases (NA-AC-ases) by NA, resulting in activation of phosphodiesterase activity by the NA-elevated cAMP-levels, is sustained by (a) membraneous factor(s) connected to the NA-receptor. This (these) factor(s) is (are) switched off in the absence of K⁺. Evidence has been presented, that Ca²⁺ and FLU do not have access to this intramembraneous factor-enzyme activating moiety of the NA-cAMP system at 0 mM K⁺. Between 0.4 and 0.8 mM K⁺ the factor-enzyme-NA-receptor complex is still intact.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

l-proline dehydrogenase of Triticum vulgare germ: Purification,properties and cofactor interactions

The enzyme catalysing the l-proline-dependent reduction of NAD⁺has been purified over 600-fold from wheat germ acetone powder extracts. l-Proline, 3,4 dehydro-dl-proline, thiazolidine-4-carboxylate were the only substrates utilized readily. The K_m for l-proline was 1·0 mM and for NAD⁺ 0·8 mM. The enzyme was highly specific for NAD⁺ with NADP⁺ and NADPH acting as effective competitive inhibitors with a K_i of 1·8 and 0·4 μM, respectively. All ribonucleoside triphosphates tested were good non-competitive inhibitors, in particular UTP. The purified enzyme could reduce pyrroline-5-carboxylate, either chemically synthesized or generated in a linked assay system from ornithine by a highly-purified ornithine transaminase. In the latter case both NADH and NADPH were utilized equally well as the reductant. With chemically synthesized dl-pyrroline-5-carboxy-late as the substrate. NADPH was used at only 25% the rate of NADH, and NADPH strongly inhibited the oxidation of NADH.