Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Calcium regulation of exogenous and endogenous 1-aminocylopropane-1-carboxylic acid bioconversion to ethylene

Ethylene production and overall levels of free and conjugated 1-aminocyclopropane-1-carboxylic acid (ACC) were studied in parenchymatous tissues, excised from clmacteric apples ( Malus domestica Borkh. cv. Granny Smith) and infiltrated with an incubation medium containing 0, 1, 10 or 100 m M Ca²⁺, with or without exogenous ACC (2 m M ). Irrespective of whether exogenous ACC was applied or not, ethylene production was inhibited to the same extent (40%) by an apoplastic effect of 100 m M Ca²⁺. In the absence of external ACC, the inhibition was associated with an increase in total endogenous ACC and may be related to a reduction in the rate of the last step of ethylene pathway. This suggests that the ethylene-forming enzyme (EFE) is localized in the plasma membrane. Low Ca²⁺ concentrations (1 m M ) enhanced basal ethylene synthesis due to influx of Ca²⁺ into the cytosol, while overall concentrations of ACC in the tissue decreased. However, 1 m M Ca²⁺ did not stimulate ACC-dependent ethylene formation. Thus, Ca²⁺ influx may stimulate the translocation of endogenous ACC from synthesis or storage compartment (s) to reactive site(s) of the plasma membrane. The concentration of 10 m M Ca²⁺ had no effect on basal ethylene production and appears to represent a balance point between the stimulating and inhibiting effects of 1 and 100 m M Ca²⁺, respectively, Furthermore, the charge-times of exogenous ACC observed with 0, 1 and 10 m M Ca²⁺ suggest that EFE is located on the inner side of the plasma membrane.     

45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

Acetylcoenzyme A and Acetylcholine in Slices of Rat Caudate Nuclei Incubated with (-)-Hydroxycitrate, Citrate, and EGTA

Abstract: The effects of (-)-hydroxycitrate (OHC) and citrate on the concentration of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh) in the tissue and on the release of ACh into the medium were investigated in experiments on slices of rat caudate nuclei incubated in media with 6.2 or 31.2 m M K⁺, 0 or 2.5 mM Ca²⁺, and 0, 1, or 10 m M EGTA. OHC diminished the concentration of acetyl-CoA in the slices under all conditions used: in experiments with 2.5 m M OHC, the concentration of acetyl-CoA was lowered by 25-38%. Citrate, in contrast, had no effect on the level of acetyl-CoA in the tissue. Although both OHC and citrate lowered the concentration of ACh in the slices during incubations with 6.2 m M K⁺ and 1 m M EGTA, they had different effects on the content of ACh during incubations in the presence of Ca²⁺. The concentration of ACh in the slices was increased by citrate during incubations with 2.5 mM Ca²⁺ and 31.2 or 6.2 m M K⁺, but it was lowered or unchanged by OHC under the same conditions. The release of ACh into the medium was lowered or unchanged by OHC and lowered, unchanged, or increased by citrate. It is concluded that most effects of OHC on the metabolism of ACh can be explained by the inhibition of ATP-citrate lyase; with glucose as the main metabolic substrate, ATP-citrate lyase appears to provide about one-third of the acetyl-CoA used for the synthesis of ACh. Experiments with citrate indicate that an increased supply of citrate may increase the synthesis of ACh. The inhibitory effect of citrate on the synthesis of ACh, observed during incubations without Ca⁺², is interpreted to be a consequence of the chelation of intracellular Ca²⁺; this interpretation is supported by the observation of a similar effect caused by 10 m M EGTA.     

Two forms of fructose 1,6-bisphosphatase from immature wheat endosperm

Fructose 1,6-bisphosphatase (α-D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) from immature wheat endosperm has been resolved into two forms, FBPase-I and FBPase-II. Their specific activities over crude homogenate increased 47- and 77-fold, respectively, by using ammonium sulfate fractionation, DEAE-cellulose chromatography and gel filtration through Sephadex G-200. The pH optimum was 7.6 for FBPase-I and 8.4 for FBPase-II. The two forms were highly specific for the substrate FBP with K_m values of 0.17 and 0.08 m M , respectively, for FBPase-I and FBPase-II at their respective pH optimum and saturating Mg²⁺ concentration. pH had no effect on the K_m value for FBPase-I, but that for FBPase-II increased below optimum pH. Neither of the forms had an absolute requirement for Mg²⁺, although it was essential for maximum activity. Mg²⁺ could not be replaced by Cu²⁺, Ca²⁺, Ba²⁺, Co²⁺ or Ni²⁺. Sulfhydryl reagents inactivated both FBPase-I and FBPase-II. Of the metabolites, only 6-phosphogluconate was inhibitory with 50% inhibition at 2 and 4 m M for FBPase-I and FBPase-II, respectively.     

Uptake and distribution of calcium, magnesium and potassium in cucumber of different age

Uptake and distribution of Ca⁺, Mg²⁺ and K²⁺ were investigated in plants of cucumber ( Cucumis sativus L. var. Cila) which had been cultivated for 12, 19, 32, or 53 days in complete nutrient solution with 1.0 m M Ca²⁺, 2.0 m M Mg²⁺ and 2.0 m M K⁺. The ⁺ concentration was about the same in roots and shoots, while the Ca²⁺ and Mg²⁺ concentrations were low in roots compared to shoots. The K⁺ concentration decreased with increasing leaf age, while the Ca²⁺ and Mg²⁺ concentrations increased, except in older plants with flowers and fruits, where an increased concentration was found in the youngest leaves. This is discussed in connection with increased indoleacetic acid (IAA) synthesis in the shoot. Excision of leaves at different levels from 21-day-old plants, followed by uptake for 24 h from the nutrient solution on days 22 and 23, resulted in no immediate reduction in Ca²⁺ (⁴⁵Ca) uptake. Transport of Ca²⁺ increased to leaves above and below the excision point and total Ca²⁺ uptake remained at the same level as for the intact plant. It is suggested that regulation of Ca²⁺ uptake is primarily achieved in the root while the distribution in the shoot is regulated by the accessability of negative binding sites.     

ROLE OF MALATE TRANSPORT IN REGULATING METABOLISM IN MITOCHONDRIA ISOLATED FROM RABBIT BRAIN

Abstract— Partly purified chromaffin granules were incubated in vitro with Ca²⁺ (with trace amounts of ⁴⁵Ca²⁺) in concentrations ranging from 4 μm to 1 mm. After incubation the granules were washed with media containing EDTA and then subjected to density gradient centrifugation (1.3 to 2.0 m-sucrose solutions) in order to characterize the particles which had taken up ⁴⁵Ca²⁺. By using marker enzymes and various inhibitors of Ca²⁺ uptake into such cell particles as mitochondria it was established that under the conditions of the experiments chromaffin granules took up Ca²⁺ from the incubation medium. To characterize this uptake a simplified density gradient procedure was tested and found to be suitable. The uptake of Ca²⁺ into chromaffin granules was strongly dependent on temperature. It was not activated by ATP. The uptake was linear up to 10 min. At high calcium concentrations (above 200 μm) the rate of uptake levelled off. The uptake at 37°C was 1 nmol Ca²⁺/mg protein/min at a Ca²⁺ concentration of 500 μm. Mg²⁺ had no influence on Ca²⁺ uptake, whereas Sr²⁺ (1 mm) inhibited it. The methods established in this study should prove useful for a further characterization of this Ca²⁺ uptake into chromaffin granules which is likely to represent a useful model for the Ca²⁺ uptake occurring in the intact gland.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

A Reevaluation of the Role of Mitochondria in Neuronal Ca2+ Homeostasis

Abstract: The ability of mitochondrial Ca²⁺ transport to limit the elevation in free cytoplasmic Ca²⁺ concentration in neurones following an imposed Ca²⁺ load is reexamined. Cultured cerebellar granule cells were monitored by digital fura-2 imaging. Following KCI depolarization, addition of the protonophore carbonylcyanide m -chlorophenylhydrazone (CCCP) to depolarize mitochondria released a pool of Ca²⁺ into the cytoplasm in both somata and neurites. No CCCP-releasable pool was found in nondepolarized cells. Although the KCI-evoked somatic and neurite Ca²⁺ concentration elevations were enhanced when CCCP was present during KCI depolarization, this was associated with a collapsed ATP/ADP ratio. In the presence of the ATP synthase inhibitor oligomycin, glycolysis maintained high ATP/ADP ratios for at least 10 min. The further addition of the mitochondrial complex I inhibitor rotenone led to a collapse of the mitochondrial membrane potential, monitored by rhodamine-123, but had no effect on ATP/ADP ratios. In the presence of rotenone/oligomycin, no CCCP-releasable pool was found subsequent to KCI depolarization, consistent with the abolition of mitochondrial Ca²⁺ transport; however, paradoxically the KCI-evoked Ca²⁺ elevation is decreased. It is concluded that the CCCP-induced increase in cytoplasmic Ca²⁺ response to KCI is due to inhibition of nonmitochondrial ATP-dependent transport and that mitochondrial Ca²⁺ transport enhances entry of Ca²⁺, perhaps by removing the cation from cytoplasmic sites responsible for feedback inhibition of voltage-activated Ca²⁺ channel activity.     

Effect of auxins on spermidine uptake into carrot protoplasts

The effect of an auxin, indole-3-acetic-acid (IAA), on spermidine uptake into protoplasts of carrot ( Daucus carota L. cv. Ingrid) was studied. In the presence of 1 m M Ca²⁺, IAA (10⁻⁷ to 10⁻⁴ M ) enchances [¹⁴C]-spermidine uptake into carrot protoplasts, while no stimulation occurs in the absence of Ca²⁺. The time course of the uptake with and without IAA is very rapid and reaches saturation within 1 to 2 min. Preincubation of protoplasts with IAA inhibits the spermidine uptake. La³⁺, known not to penetrate the plasmalemma, exerts the same effect as Ca²⁺, but gives lower uptake values than Ca²⁺. The application of vanadate, an ATPase inhibitor, strongly inhibits IAA-stimulated spermidine uptake, suggesting that an energy-dependent mechanism may be involved in this transport. Neither spermidine nor Ca²⁺ alone stimulate IAA uptake. The synthetic auxin 2,4-dichlorophenoxyacetic acid, yields the same results as IAA with regard to time course of spermidine uptake with and without preincubation while, unlike IAA, no significant effect was observed on the Ca²⁺ -induced increase of spermidine uptake.     

Potentiation of 45Ca Uptake and Acute Toxicity Mediated by the N-Methyl-D-Aspartate Receptor: The Effect of Metal Binding Agents and Transition Metal Ions

Abstract: The activities mediated by the N -methyl-D-aspartate (NMDA) receptor were studied in cultured rat cerebellar granule cells. Micromolar concentrations of the metal binding compounds, EDTA, cysteine, and histidine, as well as serum albumin strongly potentiated receptor activity in the presence of millimolar concentrations of Ca²⁺ and Mg²⁺. The findings indicated that these agents remove an endogenous metal, probably Zn²⁺, which attenuates NMDA receptor-mediated ⁴⁵Ca uptake and toxicity. Several added metal ions were therefore tested at low micromolar concentrations. Zn²⁺ was found to be the most potent inhibitor of NMDA-induced ⁴⁵Ca uptake, followed by Cu²⁺ and Fe²⁺. Co²⁺, Cd²⁺, Fe³⁺, and AI³⁺ had no significant effect, whereas Ni²⁺ potentiated the ⁴⁵Ca uptake but inhibited at much higher concentrations. The potentiating agents that remove the endogenous metal had a particularly dramatic effect in the presence of Mg²⁺, the voltage-dependent suppressor of the NMDA receptor. Mg²⁺ also played an important role in the inhibitory effect of added Zn²⁺. Much lower concentrations of Zn²⁺ were needed to achieve inhibition of NMDA-induced ⁴⁵Ca uptake in the presence of Mg²⁺. Under a variety of conditions, a very good correlation was found between NMDA receptor-mediated ⁴⁵Ca uptake and the magnitude of acute neurotoxicity.     

Elevated Levels of Glucose and L-Fucose Reduce 22Na+ Uptake and Whole Cell Na+ Current in Cultured Neuroblastoma Cells

Abstract: Na⁺ flux was studied in cultured neuroblastoma cells grown in medium containing increased glucose or L - fucose concentrations. Chronic exposure of neuroblastoma cells to 30 m M glucose or 30 m M L-fucose caused a decrease in ouabain-sensitive and veratridine-stimulated ²²Na⁺ uptake compared with cells cultured in unsupplemented medium. The Na⁺ current, determined by using whole-cell configuration of the patch clamp, was also decreased in these cells. Tetrodotoxin (3 μ M ), which blocked whole cell Na⁺ currents, also blocked veratridine-stimulated ²²Na⁺ accumulation. Culturing cells in medium containing 30 m M fructose as an osmotic control had no effect on Na⁺ flux. Specific [³H] saxitoxin binding was not affected by 30 m M glucose or 30 m M L-fucose compared with cells grown in unsupplemented medium, suggesting that the number of Na⁺ channels was not decreased. These studies suggest that exposing cultured neuronal cells to conditions that occur in the diabetic milieu alters Na⁺ transport and Na⁺-channel activity.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Characterization of mitochondrial glutamate dehydrogenase from dark‐grown soybean seedlings

Purified preparations of NAD(H)‐glutamate dehydrogenase (GDH, EC 1.4.1.2.) were assayed to determine the effects of mono‐ and divalent cations, nucleotides and select carbon compounds on NAD(H)‐dependent GDH activity. The amination reaction was stimulated 2‐ to 17‐fold by divalent cations (Ca²⁺ > Cd²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ between 1 and 1000 µ M ), but the reaction was unaffected by monovalent cations (Na ⁺ and K ⁺). The amination reaction was most responsive to changes in Ca²⁺ in a NADH‐dependent manner. The addition of EDTA or EGTA nullified the stimulatory effects of Ca²⁺. Calmodulin alone or in combination with calmodulin antagonists did not affect the amination reaction. Divalent cations (at 1 m M ) inhibited the rate of the deamination reaction by 15 to 25%, while monovalent cations had no effect. ATP inhibited the amination reaction by 10 to 60%, while ADP had little or no effect. ATP or ADP decreased the rate of the deamination reaction 23 to 60 or 20 to 38%, respectively. Many tricarboxylic acid cycle intermediates inhibited the amination reaction, 20 to 50% of the inhibition could be attributed to the chelating capacity of intermediates. Conversely, most of the carbon sources tested did not affect the deamination reaction, the only appreciable differences were increases in activity with sucrose (21%) and glucose (41%) and a decrease in activity with pyruvate (34%). Inhibitors of sulfhydryl groups were used to examine the importance of reduced thiol groups in the amination or deamination reactions. The amination was not dependent on reduced thiol groups, whereas the deamination reaction was dependent on reduced thiol groups.     

Enhancement of Tryptophan Uptake by Divalent Cations in the Absence of Sodium Ions

Abstract: Nations were found to inhibit the uptake of L-tryptophan into synaptosomes with a shallow dose-response curve. Almost maximal inhibition was obtained with 10 mM-Na⁺. The divalent cations Ca²⁺ and Mg²⁺ were shown to be responsible for the increased uptake of L-tryptophan in the absence of Na⁺ ions. Other divalent cations also promoted tryptophan uptake under this condition (Ca²⁺ < Mg²⁺ < Mn²⁺ < Fe²⁺ < Zn²⁺ < Cu²⁺). It was concluded that monovalent chelate complexes were responsible for this enhancing effect. The measured L-tryptophan uptake was the net product of membrane bound and unbound tryptophan. Both bound and unbound tryptophan were increased in the presence of divalent cations. If no divalent cations were added to the incubation medium, Na⁺ ions decreased the unbound tryptophan but were without effect on bound tryptophan. Under these circumstances D-tryptophan had no effect on binding of the L-isomer and affected the transport of 1.-tryptophan only at very high does (100 x conc. L-tryptophan). These results suggest that I -tryptophan binds to a stereospecific transport carrier located in the synaptosomal membrane and that Na⁺ ions prevent the translocation of this carrier amino acid complex from the outer to the inner site of the neuronal membrane.     

Influence of aluminium on phosphate and calcium uptake in beech (Fagus sylvatica) grown in nutrient solution and soil solution

The effects of AICI₃ on uptake of Ca²⁺ and phosphate in roots of intact beech ( Fagus sylvatica L. provenance Maramures) plants were studied in nutrient solution and soil solution. Aluminium reduced the concentrations of Ca, Mg and P in plants and increased that of K. In short term experiments, uptake of Ca²⁺(⁴⁵Ca) was reduced by exposure of the roots to Al. The effect of aluminium on Ca²⁺(⁴⁵Ca) uptake was immediate and primarily of a competitive nature, preventing Ca²⁺ from being adsorbed. Uptake of ³²P-phosphate increased with increasing Al concentration up to 0.1 m M and then decreased at higher Al concentrations. The effect of Al on ³²P-phosphate uptake was most pronounced during the first hours of exposure. Growth of plants for 15 days in soil solution, collected from the upper A horizon of a beech forest soil, had no effect on uptake of Ca²⁺(⁴⁵Ca) and ³²P-phosphate, probably because of a low concentration of labile bound monomeric Al and binding of Al to organic compounds. Soil solution from the deeper B horizon reduced Ca²⁺(⁴⁵Ca) uptake and increased ³²P-phosphate uptake in a manner similar to that with Altreatment in nutrient solution. It is concluded that in soil solution from the deeper regions of the soil, mineral uptake by roots was affected by Al.     

Extracellular Ionic Composition Alters Kinetics of Vesicular Release of Catecholamines and Quantal Size During Exocytosis at Adrenal Medullary Cells

Abstract: The temporal resolution of carbon-fiber microelectrodes has been exploited to examine the plasticity of quantal secretory events at individual adrenal medullary cells. The size of individual quantal events, monitored by amperometric oxidation of released catecholamines, was found to be dependent on the extracellular ionic composition, the secretagogue, and the order of depolarization delivery. Release was observed with either exposure to 60 m M K⁺ in the presence of Ca²⁺ or exposure to 3 m M Ba²⁺ in solutions of different pH, with and without external Ca²⁺. Ba²⁺ was demonstrated to induce Ca²⁺-independent exocytotic release for an extended period of time (>4 min) relative to release induced by K⁺ (∼30 s), which is Ca²⁺ dependent. In all cases, simultaneous changes of intracellular divalent cations, monitored by fura-2 fluorescence, accompanied quantal release and had a similar time course. Exocytosis caused by Ba²⁺ in Ca²⁺-free medium had a larger mean spike area at pH 8.2 than at pH 7.4. When Ba²⁺-induced spikes measured at pH 7.4 were compared, the spikes in Ca²⁺-free medium were found to be broader and shorter but had the same area. Release induced by K⁺ after exposure to Ba²⁺ was comprised of larger quantal events when compared with preceding K⁺ stimulations. Finally, spikes obtained with Ba²⁺ exposure at an extracellular pH of 5.5 had a different shape than those obtained in more basic solutions. These changes in spike size and shape are consistent with the interactions between catecholamines and other intravesicular components.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

Role of external calcium in homeostasis of intracellular pH in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH

The role of external Ca²⁺ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca²⁺ in the medium. The observed Ca²⁺-mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca²⁺ in the medium. Thus, the ability of Ca²⁺ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca²⁺ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca²⁺ channel blockers tested, such as verapamil and LaCl₃, inhibited the observed Ca²⁺-mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m -chlorophenyl hydrazone, inhibited this Ca²⁺-mediated response, whereas monensin, an inhibitor of the Na⁺/H⁺ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca²⁺ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.     

Aluminium Inhibits Muscarinic Agonist-Induced Inositol 1,4,5-Trisphosphate Production and Calcium Mobilization in Permeabilized SH-SY5Y Human Neuroblastoma Cells

Abstract: The effects of aluminium (as Al³⁺) on carbachol-induced inositol 1,4,5-trisphosphate (lnsP₃) production arid Ca²⁺ mobilisation were assessed in electropermeabilised human SH-SY5Y neuroblastoma cells. Al³⁺ had no effect on lnsP₃-induced Ca²⁺ release but appreciably reduced carbachol-induced Ca²⁺ release (lC₅₀ of ∼90 μ M ). Aβ³⁺ also inhibited lnsP₃ production (lC₆₀ of ∼15 μ M ). Dimethyl hydroxypyridin-4-one, a potent Al³⁺ chelator (K₅= 31), at 100 μ M was able to abort and reverse the effects of Al³⁺ on both Ca²⁺ release and lnsP₃ production. These data suggest that, in permeabilised cells, the effect of Al³⁺ on the phosphoinositide-mediated signalling pathway is at the level of phosphatidylinositol 4,5-bisphosphate hydrolysis. This may reflect interference with receptor-G protein-phospholipase C coupling or an interaction with phosphatidylinositol 4,5-bisphosphate.