Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.     

Enhancement of salt responses in frog gustatory nerve by removal of Ca2+ from the receptor membrane treated with 1-anilinonaphthalene-8-sulfonate

Summary The frog tongue was incubated in 1-anilinonaphthalene-8-sulfonate (ANS) solution and the responses of the glossopharyngeal nerve to various chemical stimuli were measured after the ANS solution was washed out. The responses to galactose, quinine and distilled water were unchanged by the ANS treatment. On the other hand, the responses to the salts, except for CaCl₂, were enhanced in greater or lesser degree after the ANS treatment. The order of relative magnitude of the enhanced response to 100mm salts of monovalent cations was Na⁺>NH ₄ ⁺ >K⁺>Li⁺, while that before the treatment was NH ₄ ⁺ >K⁺>Na⁺>Li⁺. The enhancement of the salt responses was also observed after the tongue was treated with 6-p-toluidinonaphthalene-2-sulfonate or 1,2-cyclohexanediaminetetraacetic acid solution.The enhanced responses to the salts were suppressed to the original level before the ANS treatment by addition of CaCl₂ or SrCl₂. The suppression curve satisfied the Langmuir adsorption isotherm when the suppression was postulated to be responsible for the binding of Ca²⁺ or Sr²⁺ to the receptor membrane treated with ANS. The apparent binding constants for Ca²⁺ and Sr²⁺ in the presence of 100mm NaCl were obtained to be 1.2×10⁴ m ^–1 and 6.7×10³ m ^–1, respectively.The ANS treatment modified the temperature dependence of the salt responses. For example, 100mm KCl solution of low temperature induced a large response after the ANS treatment, while that of 20°C induced only small response.It was concluded that the removal of Ca²⁺ from the gustatory receptor membrane in the frog, which was brought about by the ANS treatment, led to the enhancement of the salt responses. The mechanism on the enhancement of the salt response by the Ca²⁺ removal was discussed.     

Evidence that a light-accelerated abrupt change in membrane characteristics of bovine rod outer segment fragments takes place at acidic pH

Changes in the turbidity of suspensions of bovine rod outer segment fragments induced by rhodopsin bleaching were measured in the presence of various concentrations of divalent cations at acidic pH (4.7–5.4). Unlike the situation at neutral pH, the turbidity of the suspensions increased drastically by bleaching at acidic pH. It was found that the extent of turbidity change became maximum at a particular concentration of divalent cations (i.e., 5 mM CaCl₂, 5 mM MgCl₂, or 5 mM mixed divalent cations). However, the turbidity increment in the presence of 5 mM MgCl₂ was greatly enhanced by the addition of a minute amount of CaCl₂. These results evidently show that the membrane characteristic is abruptly changed by bleaching at acidic pH in particular. It is also suggested that there are two kinds of binding sites for Ca ions: one is a Ca²⁺ specific site, and the other is a nonspecific site to which Mg²⁺ can also bind.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

Properties of Ca2+ uptake and release by Golgi membrane vesicles from rat intestine

A previous study of energy-independent in vitro Ca²⁺ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH)₂D₃ repletion (R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology78, 325–332). In the present study, properties of Ca²⁺ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg²⁺ and Sr²⁺, but not by Na⁺ or K⁺. Lowering the external [H⁺] or raising the internal [H⁺] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H⁺] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl₂ or SrCl₂ but not CaCl₂, NaCl, or KCl. Vesicles preloaded with K₂SO₄ failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl₂ was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca²⁺ uptake maintained at equilibrium. Therefore, the remainder of the Ca²⁺ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10^?6m was found for this binding. The bound Ca²⁺ could be rapidly released by external Mg²⁺ or Sr²⁺, but not Ca²⁺, Na⁺, or K⁺. Release of bound Ca²⁺ could also be induced by raising the [H⁺] of the external medium. Failure of external Ca²⁺ to release bound Ca²⁺ suggested that the release induced by external Mg²⁺, Sr²⁺, or H⁺ was not due to competitive displacement of Ca²⁺ from its binding sites. These results indicated that Ca²⁺ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca²⁺ to the vesicle. The effects of H⁺, Mg²⁺, and Sr²⁺ on Ca²⁺ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles.     

Potentiation by ouabain of bradykinin-induced catecholamine secretion and calcium influx into cultured bovine adrenal chromaffin cells: Evidence for involvement of Na+ influx associated with the bradykinin B2-receptor

The effect of bradykinin (BK), in the presence of ouabain, an inhibitor of Na⁺-K⁺ ATPase, on catecholamine (CA) secretion was studied in cultured bovine adrenal chromaffin cells, to determine whether Na⁺, as well as Ca²⁺, is involved in BK-receptor mediated CA secretion. BK (10⁻⁸–10⁻⁵M)-induced CA secretion was markedly potentiated by addition of ouabain (10⁻⁵M), was blocked by a BK-B₂ receptor antagonist, and was decreased in Ca²⁺-free medium. BK-induced increase in ⁴⁵Ca²⁺ influx was also potentiated by addition of ouabain. The cultured cells were first incubated with BK for 30 min in Ca²⁺-free medium in the presence or absence of ouabain and then kstimulated for 15 min with Ca²⁺-medium without BK or ouabain. Prior stimulation of the cells, BK induced ²²Na⁺ influx and increased Ca²⁺-induced CA secretion and these stimulatory effects of BK were potentiated by added ouabain. When the cells were stimulated with BK and ouabain in Na⁺-free sucrose medium, the Ca²⁺-induced CA secretion was greatly reduced. These results indicated that activation of the BK-B2 receptor and inhibition of the Na⁺ pump both increase the intracellular Na⁺ level, resulting in increase in Ca²⁺ influx and CA secretion.     

Metal Ion and Guanine Nucleotide Modulations of Agonist Interaction in G-Protein-Coupled Serotonin1A Receptors from Bovine Hippocampus

1. The serotonin type 1A (5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to GTP-binding regulatory proteins (G-proteins). We have studied the modulation of agonist binding to 5-HT_1A receptors from bovine hippocampus by metal ions and guanine nucleotide.2. Bovine hippocampal membranes containing the 5-HT_1A receptor were isolated. These membranes exhibited high-affinity binding sites for the specific agonist [³H]OH-DPAT.3. The agonist binding is inhibited by monovalent cations Na⁺, K⁺, and Li⁺ in a concentration-dependent manner. Divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺, on the other hand, show more complex behavior and induce enhancement of agonist binding up to a certain concentration. The effect of the metal ions on agonist binding is strongly modulated in the presence of GTP--S, a nonhydrolyzable analogue of GTP, indicating that these receptors are coupled to G-proteins.4. To gain further insight into the mechanisms of agonist binding to bovine hippocampal 5-HT_1A receptors under these conditions, the binding affinities and binding sites have been analyzed by Scatchard analysis of saturation binding data. Our results are relevant to ongoing analyses of the overall regulation of receptor activity for G-protein-coupled seven transmembrane domain receptors.     

RELEASE OF ATP FROM A SYNAPTOSOMAL PREPARATION BY ELEVATED EXTRACELLULAR K+ AND BY VERATRIDINE

A technique was developed which permitted the release of ATP from synaptosomes by elevated extracellular K⁺ or by veratridine to be directly and continuously monitored. The released ATP interacted with firefly luciferin and luciferase in the incubation medium to produce light which could be detected by a photomultiplier. The assay system was specific for ATP, in that similar concentrations of adenosine, AMP or ADP did not produce chemiluminescence. Moreover, the maximum peak of light emission correlated linearly with the concentrations of ATP present in the medium, so that semiquantitative estimates of ATP release could be made. Elevating the extracellular K⁺ concentration produced a graded release of ATP from synaptosomes. Rb⁺ also released ATP but Na⁺, Li⁺ and choline did not. The response to elevated K⁺ was not blocked by tetrodotoxin (TTX), indicating that this effect was not mediated by the opening of Na⁺-channels in synaptosomal membranes. Veratridine (50 μM) caused a graded release of ATP which was larger and more prolonged than that caused by elevated K⁺. The release of ATP by veratridine was blocked by TTX indicating that the opening of Na⁺-channels was involved. Neither veratridine nor elevated K⁺ released ATP from microsomal or mitochondrial fractions, showing that the release of ATP probably did not originate from microsomal, vesicular or mitochondrial contaminants of the synaptosomal preparation. Release of ATP by elevated K⁺ was diminished in a medium lacking CaCl₊ or when EGTA was added to chelate Ca²⁺. In contrast, release by veratridine appeared to be augmented in Ca²⁺-free media or in the presence of EGTA. The K⁺-induced release of ATP, which is Ca²⁺ dependent, closely resembles the exocytotic release of putative neurotransmitters from presynaptic nerve-terminals. On the other hand, the apparent lack of a Ca²⁺ requirement for veratridine's action suggests that this process could originate from other sites, or involve mechanisms other than conventional neurotransmitter release processes.     

Ca++ fluxes in resealed synaptic plasma membrane vesicles

The effect of the monovalent cations Na⁺, Li⁺, and K⁺ on Ca⁺⁺ fluxes has been determined in resealed synaptic plasma membrane vesicle preparations from rat brain. Freshly isolated synaptic membranes, as well as synaptic membranes which were frozen (?80°C), rapidly thawed, and passively loaded with K₂/succinate and ⁴⁵CaCl₂, rapidly released approximately 60% of the intravesicular Ca⁺⁺ when exposed to NaCl or to the Ca⁺⁺ ionophore A 23187. Incubation of these vesicles with LiCl caused a lesser release of Ca⁺⁺. The EC₅₀ for Na⁺ activation of Ca⁺⁺ efflux from the vesicles was approximately 6.6mM. exposure of the Ca⁺⁺-loaded vesicles to 150 mM KCl produced a very rapid (?1 sec) loss of Ca⁺⁺ from the vesicles, but the Na⁺-induced efflux could still be detected above this K⁺ - sensitive effect. Vesicles pre-loaded with NaCl (150 mM) exhibited rapid ⁴⁵Ca uptake with an estimated EC₅₀ for Ca⁺⁺ of 7–10 μM. This Ca⁺⁺ uptake was blocked by dissipation of the Na⁺ gradient. These observations are suggestive of the preservation in these purified frozen synaptic membrane preparations of the basic properties of the Na⁺Ca⁺⁺ exchange process and of a K⁺ - sensitive Ca⁺⁺ flux across the membranes.     

Effects of inorganic solutes on the binding of auxin

The binding of α-naphthaleneacetic acid (¹⁴C-NAA) to pelletable particulates from corn (Zea mays) coleoptiles was found to be influenced by inorganic solutes. La³⁺, Ca²⁺, and Mg²⁺ increased the binding whereas monovalent cations did not. The concentrations of CaCl₂ which increased auxin binding were similar to those which inhibited coleoptile elongation in the presence of auxin. These results are interpreted as suggesting that the alteration of hormonal effectiveness by some inorganic solutes involves alterations in the attachment of the hormone to binding sites in the cell.     

Effects of monovalent cations on Ca uptake by skeletal and cardiac muscle sarcoplasmic reticulum

Ca²⁺ transport by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is sensitive to monovalent cations. Possible K⁺ binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca²⁺ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca²⁺ transport correlated in most cases with their direct effects on SERCA. Choline⁺, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca²⁺ transport. Of the monovalent cations tested, only Cs⁺ significantly affected the Hill coefficient of Ca²⁺ transport (n_H). An increase in n_H from ∼2 in K⁺ to ∼3 in Cs⁺ was seen in all of the forms of SERCA examined. The effects of Cs⁺ on the maximum velocity of Ca²⁺ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K⁺ binding sites of the different forms of the protein.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Role of Internal Potassium in Maintaining Growth of Cultured Citrus Cells on Increasing NaCl and CaCl(2) Concentrations

Shamouti orange (Citrus sinensis L. Osbeck) salt-tolerant cells were grown under low water potential conditions induced by polyethylene glycol (PEG), NaCl, and CaCl₂. On the basis of equal osmotic potentials, PEG was the least inhibitory, NaCl next, and CaCl₂ the most inhibitory. The relation between growth capacity and ion content can be summarized as follows. (a) Internal K⁺ concentration was a major factor which changed in the presence of PEG, NaCl, and CaCl₂ and probably played a key role in determining growth capacity. (b) Internal concentrations of Na⁺, Ca²⁺, or Cl⁻ could not be directly correlated with growth. (C) Internal Mg²⁺ concentration could be significant only in the presence of high external Ca²⁺ concentrations. (d) The contribution of nitrate and phosphate to the internal osmoticum was negligible. The ratio of external (Ca²⁺)/(Na⁺)² concentration is crucial for growth. Ratios above 0.5 × 10⁻⁴ per millimolar gave maximal protection from adverse effects of NaCl. Growth capacity was found to be determined by the combination of (Ca²⁺)/(Na⁺)² ratio and the absolute external concentration of NaCl. However, a correlation between internal K⁺ concentration and growth capacity seemed independent of external NaCl concentration.     

Differential response of two almond rootstocks to chloride salt mixtures in the growing medium

It was examined how essential cations, Ca²⁺ and K⁺, can mitigate the toxic effects of NaCl on two different almond species (Prunus amygdalus Batsch) rootstocks, Garnem (GN15) and Bitter Almond. The tree growth parameters (water potential (Ψ_w), gas exchange, nutrient uptake) and leaf chlorophyll (Chl) content were measured in control and NaCl-treated plants with or without KCl or CaCl₂ supplements. The addition of CaCl₂ and KCl to Bitter Almond trees reduced their dry weight, shoot growth and leaf number although net photosynthetic assimilation rate (A) was not affected. These results indicated that changing of photo-assimilates flux to proline and/or soluble sugars synthesis may help to increase leaf Ψ_w. The Garnem trees also did not respond to the CaCl₂ and KCl addition indicating that the plants are already getting enough of these two cations (C^a2+ and K⁺). In both rootstocks, NaCl in the medium reduced growth attributes, Ψ_w, A, stomatal conductance (g_s), and leaf Chl content. When CaCl₂ and KCl fertilizers were added together with NaCl to Bitter Almond trees, leaf K⁺ and Ca²⁺ contents increased while Na⁺ and Cl^– decreased leading to higher Ca/Na and K/Na ratios, but shoot growth was not improved and even declined compared to NaCl-treated trees. It appears that the addition of salts further aggravated osmotic stress as indicated by the accumulation of proline and soluble sugars in leaf tissues. The addition of KCl or CaCl₂ to NaCl-treated GN15 trees did not increase A, leaf Ψ_w, and shoot growth but improved ionic balances as indicated by higher Ca/Na and K/Na ratios. The reduction in A was mainly due to non-stomatal limitations in GN15, possibly due to the degradation of Chl a, unlike Bitter Almond, for which the reduction of A was due to stomata closure. The improvement in ionic balances and water status of Bitter Almond trees in response to addition of KCl or CaCl₂ was apparently offset by a high sensitivity to Cl^–; therefore, no-chloride salts should be the preferred forms of fertilizers for this rootstock. Both rootstocks were sensitive to soil salinity and cation supplements were of limited value in mitigating the effect of excessive salt concentrations.     

Calcium uptake by excised maize roots and interactions with alkali cations

Ca²⁺ uptake was studied in short-term experiments using 5-day-old excised maize roots. This tissue readily absorbs Ca²⁺, and inhibition by dinitrophenol and low temperature shows that the process is metabolically mediated. The uptake of Ca²⁺, like that of other cations, is influenced by the counter ion, the pH and concentration of the ambient solution, and the presence of other cations. The rate of uptake from various salts decreases in the following order: NO₃⁻ > Cl⁻ = Br⁻ > SO₄²⁻. K⁺ and H⁺ greatly interfere with Ca²⁺ absorption, while Li⁺ and Na⁺ have only slight effects.     

Calcium Ion-Dependent Increase in Thermostability of Dextran Glucosidase from Streptococcus mutans

Dextran glucosidase from Streptococcus mutans (SmDG), which belongs to glycoside hydrolase family 13 (GH13), hydrolyzes the non-reducing terminal glucosidic linkage of isomaltooligosaccharides and dextran. Thermal deactivation of SmDG did not follow the single exponential decay but rather the two-step irreversible deactivation model, which involves an active intermediate having 39% specific activity. The presence of a low concentration of CaCl₂ increased the thermostability of SmDG, mainly due to a marked reduction in the rate constant of deactivation of the intermediate. The addition of MgCl₂ also enhanced thermostability, while KCl and NaCl were not effective. Therefore, divalent cations, particularly Ca²⁺, were considered to stabilize SmDG. On the other hand, CaCl₂ had no significant effect on catalytic reaction. The enhanced stability by Ca²⁺ was probably related to calcium binding in the β→α loop 1 of the (β?α)₈ barrel of SmDG. Because similar structures and sequences are widespread in GH13, these GH13 enzymes might have been stabilized by calcium ions.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis,on inhibition of lipolysis by insulin,and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca²⁺ and Mg²⁺ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca²⁺ + Mg²⁺)-free incubation medium incorporation of L-[¹⁴C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[¹⁴C]-leucine incorporation only in the presence of added CaCl₂ or MgCl₂. Incubation of the cells in the (Ca²⁺ + Mg²⁺)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of ¹²⁵I-labelled insulin to the fat cells was reduced in the absence of Ca²⁺ and Mg²⁺ but was not abolished, even in the presence of EDTA. Ca²⁺ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg²⁺, Sr²⁺ and Ba²⁺.Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

Calcium Induced Modulation of Peripheral-Type Benzodiazepine Receptors in Rat Kidney Membranes

Abstract

The effects of Ca²⁺ ions on ³H-RO 5–4864 binding to the peripheral benzodiazepine receptor were examined. Preincubation of rat kidney membranes with Ca²⁺ at 37°C produced a dose-dependent inhibition of ³H-RO 5–4864 binding. No inhibition was observed in membranes preincubated at 0°C.

The effect of Ca²⁺ was competitive in nature and was fully reversed by the addition of EGTA. At 1 mM, the maximal effect was achieved with CaCl₂, whereas CoCl₂ and CdCl₂ had lesser effects. No other divalent cation salts examined decreased ³H-RO 5–4864 binding to rat kidney membranes. Collectively, these data demonstrate that the affinity of ³H-RO 5–4864 binding to rat kidney membranes is regulated by Ca²⁺ and suggest the presence of cation recognition binding sites coupled to the peripheral benzodiazepine receptor.