Neutral Ca2 Research Articles

GABAA and GABAB Receptors Mediate GABA-Induced Intracellular Ca2+ Signals in Human Brain Microvascular Endothelial Cells

Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABAA receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABAB receptors is still unknown. Preliminary evidence showed that GABAA receptor stimulation can induce an increase in endothelial Ca2+ levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca2+]i that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca2+ release from neutral and acidic Ca2+ stores, respectively, and sustained by store-operated Ca2+ entry. GABAA and GABAB receptors were both required to trigger the endothelial Ca2+ response. Unexpectedly, we found that the GABAA receptors signal in a flux-independent manner via the metabotropic GABAB receptors. Likewise, the full Ca2+ response to GABAB receptors requires functional GABAA receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.

Acute Effects of Cadmium and Copper on Cytochemical Responses in the Polychaete Hediste Diversicolor

This study investigated the cytochemical responses in Hediste diversicolor exposed to the environmental concentrations of cadmium (1µM Cd), copper (1µM Cu) and to their equimolar mixture for 48h. Lysosomal membrane stability (LMS), neutral lipids (NL) level, lipofuscin (LF) content and Ca2+-ATPase activity were determined as early subcellular biomarkers of toxicity. The biomarker battery pointed out a high cytoxicity on the polychaete treated with metals. Excessive induction of neutral lipids and lipofuscin accumulations were observed in polychaete treated with copper than cadmium. In addition, the highest level of neutral lipids and lipofuscin was noted in mixture treated group [Cu+Cd]. The increase of neutral lipids and lipofuscin accumulations were negatively correlated with the lysosomal membrane stability and Ca2+-ATPase activity alterations. Our study also demonstrated that the cytochemical biomarkers responses were very important to identify the cellular toxic effects of metals and promising biological tools in the assessment of biological effects of pollutants in natural marine environment.

A Case Study on the Resolution of the 1‐i‐Butyl‐3‐methyl‐3‐phospholene 1‐Oxide via Diastereomeric Complex Formation Using TADDOL Derivatives and via Diastereomeric Coordination Complexes Formed from the Calcium Salts of O,O′‐Diaroyl‐(2R,3R)‐tartaric Acids

ABSTRACTThe resolution of 1‐i‐butyl‐3‐methyl‐3‐phospholene 1‐oxide was studied applying TADDOL [(−)‐(4R,5R)‐4,5‐bis(diphenylhydroxymethyl)‐2,2‐dimethyldioxolane], spiro‐TADDOL [(−)‐(2R,3R)‐α,α,α′,α′‐tetraphenyl‐1,4‐dioxaspiro[4.5]decan‐2,3‐dimethanol], or the acidic and neutral Ca2+ salts of (−)‐O,O′‐dibenzoyl‐ and (−)‐O,O′‐di‐p‐toluoyl‐(2R,3R)‐tartaric acid as the resolving agent. The absolute configuration of the P‐asymmetric center was determined by circular dichroism spectroscopy and related quantum chemical calculations. In one instance, the single crystal of the diastereomeric complex incorporating i‐butyl‐3‐phospholene oxide and spiro‐TADDOL was subjected to X‐ray analysis, which suggested a feasible hypothesis for the efficiency of the resolution process under discussion that may be an example for the “solvent‐inhibited” resolution.

Resolution of 1-n-propoxy-3-methyl-3-phospholene 1-oxide by diastereomeric complex formation using TADDOL derivatives and calcium salts of O,O′-dibenzoyl-(2R,3R)- or O,O′-di-p-toluoyl-(2R,3R)-tartaric acid

1-n-Propoxy-3-methyl-3-phospholene 1-oxide was prepared in optically active form by extending resolution methods applying (−)-(4R,5R)-4,5-bis(diphenylhydroxymethyl)-2,2-dimethyldioxolane (‘TADDOL’) and (−)-(2R,3R)-α,α,α′,α′-tetraphenyl-1,4-dioxaspiro[4.5]decan-2,3-dimethanol (‘spiro-TADDOL’), as well as the acidic and neutral Ca2+ salts of (−)-O,O′-dibenzoyl- and (−)-O,O′-di-p-toluoyl-(2R,3R)-tartaric acid. In one case, the diastereomeric complex could be identified by single crystal X-ray analysis. The absolute P-configuration of the enantiomers of the phospholene oxide was also determined by CD spectroscopy.

Neutral carrier-type Ca2+ sensors based on sol-gel-derived membranes incorporating diether-amide derivatives

We have synthesised Ca2+-selective neutral carriers for sol-gel-derived membranes encapsulating and covalently binding them. These ion-sensing membranes showed excellent EMF responses to Ca2+ activity changes with very short response times. Especially, the membranes covalently binding the neutral carriers did not deteriorate for at least 2 months. These sol-gel-derived membranes were selective for Ca2+ over Na+ and K+. The Ca2+-selective electrode is, therefore, promising for applications to clinical analysis in future.

Obtaining Nernstian Response of a Ca2+-Selective Electrode in a Broad Concentration Range by Tuned Galvanostatic Polarization

Linear Nernstian response is obtained for a neutral ionophore-based Ca(2+)-selective electrode down to 10(-10) M CaCl2 by means of galvanostatic polarization. The densities of the applied cathodic current were tuned for particular concentrations of Ca2+. The procedure included recording the potential at zero current, followed by measurements when current is passed through the electrode, and then again at zero current. The respective chronopotentiometric curves included negative ohmic drop immediately after turning the current on, the polarization domain, and positive ohmic drop when the current was turned off, followed with the relaxation domain. The potentials immediately after the positive ohmic drops were used as analytical signals. These potentials make a straight line with Nernstian slope when currents are tuned (optimized) for each particular concentration. An iteration procedure is proposed which allows for simultaneous optimization of the current density and accessing analyte concentration in the sample.

Wheat coleoptile endonuclease Wen2 is dependent on S-adenosyl-L-methionine and sensitive to DNA methylation status

Endonuclease WEN2 with an apparent molecular mass 21.5 kD was isolated from subcellular vesicular fraction obtained from aging apoptotic coleoptiles of 8-day-old etiolated wheat seedlings and partially characterized. Similar to wheat endonuclease WEN1 of the same origin described earlier, the WEN2 enzyme is a neutral Ca2+,Mg2+,Mn2+-dependent endonuclease. Both enzymatic activities were found also in nuclei from the same cells. Mn2+ activates WEN2 more efficiently than Mg2+ or Ca2+. High ionic strength, Zn2+, and EDTA inhibit the enzyme completely. In the presence of Mg2+, elevated WEN2 activity was observed at pH between 5.5 and 7.7 and at 37 degrees C, and without Mg2+ added it was observed in narrower pH range (from pH 6.8 to pH 7.7). The enzyme is active even at high temperature (65 degrees C). WEN2 splits preferentially unmethylated, but WEN1 - methylated lambda phage DNA. Double-stranded DNA is a preferential substrate to be hydrolyzed with WEN2. S-Adenosyl-L-methionine (SAM) significantly activates endonuclease WEN2 (the optimal SAM concentration is 0.3 mM). Contrary to strong stimulating action on WEN1, the competitive inhibitors of the DNA methylation reaction (SAM analogs S-adenosyl-L-homocysteine and S-isobutyladenosine) at concentration 0.3 mM increase WEN2 activity slightly. It is suggested that WEN2 may take part in apoptotic DNA degradation. Thus, in plants there are endonucleases that recognize methylation status of substrate DNAs and are modulated by the methyl group donor, SAM, in different fashions. Therefore, all this may indicate the presence of a restriction-modification (R-M) system in higher plants.

Lactoferrin binding to the rat asialoglycoprotein receptor requires the receptor's lectin properties

Lactoferrin binds to rat hepatic lectin 1 (RHL1), the major subunit of the asialoglycoprotein (ASGP) receptor, with high affinity, by a galactose-independent mechanism. To better understand the molecular basis of this novel interaction, we compared the binding of lactoferrin and asialo-orosomucoid (ASOR) to isolated rat hepatocytes and to purified ASGP receptors as a function of pH, Ca2+ and receptor acylation. Binding of 125I-lactoferrin and 125I-ASOR to isolated rat hepatocytes at 4 °C decreased sharply at pH < 6, following similar titration curves. Binding of 125I-lactoferrin and 125I-ASOR to hepatocytes was Ca2+-dependent. Binding increased progressively at ≥ 300 μM CaCl2, in the presence of 1 mM EDTA. Monensin treatment of hepatocytes, which causes hepatocytes to accumulate inactive ASGP receptors, reduced surface binding of 125I-lactoferrin and 125I-ASOR by 46 and 49%, respectively, with only a 16% loss of immunodetectable receptor protein from the cell surface. Finally, deacylation of purified ASGP receptors in vitro with 1 M hydroxylamine abolished receptor lectin activity as reflected by the loss of 125I-ASOR binding as well as the complete loss of specific 125I-lactoferrin binding. Treatment with 1 M Tris had no effect on binding of either ligand. We conclude from these data that galactose-independent lactoferrin binding to the ASGP receptor requires the receptor's carbohydrate-recognition domain to be in an active configuration. An active configuration is promoted by neutral pH and Ca2+, and also requires the receptor subunits to be acylated.

Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells.

Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age-induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage-sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10-100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N-methyl-D-aspartate receptor activation. Amlodipine also inhibited free radical-induced damage to lipid constituents of the membrane in a dose-dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose- and time-dependent manners by antioxidants (U-78439G, alpha-tocopherol, and melatonin), nitric oxide synthase inhibitors (N-nitro-L-arginine and N-nitro-D-arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage-sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.

Isolation and Characterization of NUC70, a Cytoplasmic, Hematopoietic Apoptotic Endonuclease

Endonucleolytic DNA fragmentation is the common end point and the prevailing indicator of apoptosis. We have identified a 70-kDa endonuclease (NUC70) that is activated in drug-induced apoptosis of human hematopoietic cells. We purified NUC70 to homogeneity and generated a rabbit polyclonal antibody to distinguish it from previously identified nucleases. Biochemical characterization of isolated NUC70 demonstrates that it is Ca2+/Mg2+-dependent and active over a pH range of 6-8. When incubated with isolated HeLa nuclei, NUC70 was capable of generating internucleosomal DNA fragmentation. This endonucleolytic activity was inhibited by Zn2+, aurintricarboxylic acid, N-ethylmaleimide, spermine, and iodoacetamide. Western immunoblots using the anti-NUC70 antibody and DNA-SDS-polyacrylamide gel electrophoresis assays indicate that NUC70 expression and activity is restricted to human hematopoietic cells. No such activity was detected in human epithelial cell lines or murine hematopoietic cells. We also observed no difference in levels of NUC70 expression between apoptotic and nonapoptotic cells, suggesting that activation of NUC70 may be by posttranslational modification. We demonstrate that NUC70 activity is diminished in cells pretreated with the caspase inhibitors z-DEVD-fmk, z-VAD-fmk, and Z-CH2-Asp-DCB. Time course studies of cytoplasmic and nuclear endonuclease activities during apoptosis show that NUC70 is a cytoplasmic endonuclease that is translocated to the nucleus after the initiation of apoptosis. We confirmed this with immunostaining studies using anti-NUC70 antibody. These results demonstrate that NUC70 is an endogenous cytoplasmic endonuclease that is activated during apoptosis in a caspase-dependent mechanism.

Selective cobalamin malabsorption and the cobalamin-intrinsic factor receptor.

The disease is characterised by cobalamin (Cbl) deficiency in children 0-5 years old, causing failure to thrive, infections, megaloblastic anaemia, neuropathy, and mild general malabsorption; slight proteinuria is common. Cbl injections produce remission, but Cbl malabsorption and proteinuria persist. About 250 cases have been reported. Dogs also have it. The heredity is autosomal and recessive. The physiological and pathological absorption mechanisms are described: Cbl liberated from food by digestion is first bound to haptocorrin, but in the intestine it is transferred to intrinsic factor. In the ileum the complex attaches to a receptor on the enterocytes; this requires neutral pH and Ca2+. The receptor is a membrane-bound glycoprotein consisting of multiple subunits. The receptor-ligand complex is endocytosed and degraded in lysosomes, and the vitamin is transferred to transcobalamin which carries it to tissues. The same receptor is strongly expressed in the kidneys, but urine also contains its activity which can be assayed for diagnosis. The basic lesion is an error in the ileal receptor. In the affected dogs the synthesised receptor is retained intracellularly. Urine and ileal biopsies from human cases contained little receptor but it had conserved affinity for the ligand. Recently examined Arab patients did not excrete reduced amounts of the receptor. Apparently, the disease has subsets, such as different structural errors in the receptor and possibly faulty transport inside the enterocyte. The cause of the proteinuria is unknown but kidney damage due to severe Cbl deficiency and an error in a multiligand renal receptor are among the possibilities.

Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pig ovaries.

The porcine antral follicles, 3-6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca2+/Mg2+-dependent endonuclease and DNA ladder formation, estimated by electrophoresis, were noted in granulosa cells but not in cumulus cells. Extremely low activity of the endonuclease and no DNA ladder formation were observed in both types of cells obtained from healthy follicles. Moreover, apoptotic cells were observed histochemically among granulosa cells only. A good correlation (r = 0.987) between the endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle was found. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs.

Ca2+/Mg2+-Dependent Endonuclease but not Ca22+-Dependent, Mg2+-Dependent or Cation-lndependent Endonuclease is Involved in Granulosa Cell Apoptosis of Pig Atretic Follicles.

The antral follicles, 4-6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca2+/Mg2+-dependent endonuclease by which the chromatin DNA is degraded at internucleosomal sites in the cells undergoing apoptosis was noted only in granulosa cells but not in cumulus cells. Low activity of the Ca2+/Mg2+-dependent endonuclease was observed in both types of cells obtained from healthy follicles. Low activities of neutral Ca2+-dependent endonuclease, neutral Mg2+-dependent endonuclease and acidic cation-independent endonuclease were demonstrated in all cases. A good correlation (r=0.892) between the Ca2+/Mg2+-dependent endonuclease activity of granulosa cells and the progesterone/estradiol-17β ratio of follicular fluid in each follicle, which provides an index of follicular atresia in pig ovaries, was found. These results suggest that the Ca2+/Mg2+-dependent endonuclease, and not the Ca2+-dependent endonuclease, the Mg2+-dependent endonuclease or the cation-independent endonuclease, is involved in granulosa cell apoptosis of the atretic antral follicles in pigs.

Signal Transmission of Granulosa Cell Apoptosis in the Atretic Antral Follicles in the Pig Ovaries

The porcine antral follicles, 4-6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, apoptotic cells were observed histochemically among granulosa cells only. In atretic follicles, high activity of neutral Ca2+/Mg2+-dependent endonuclease by which the chromatin DNA is degraded at internucleosomal sites in the cells undergoing apoptosis was noted only in granulosa cells but not in cumulus cells. Low activity of the Ca2+/Mg2+-dependent endonuclease was observed in both types of cells obtained from healthy follicles. Low activities of neutral Ca2+-dependent endonuclease, neutral Mg2+-dependent endonuclease and acidic cation-independent endonuclease were demonstrated in all cases. A good correlation between the Ca2+/Mg2+-dependent endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle, which provides an index of follicular atresia in pig ovaries, was found. In vitro study showed that the specific inhibitors for interleukin-β converting enzyme (ICE) like cysteine proteases could stop the apoptotic process induced by specific monoclonal antibody to granulosa cell membrane protein in primary cultured granulosa cells. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs, and ICE like protease (s) is involved in intracellular signal transmission of granulosa cell apoptosis of the atretic antral follicles in pigs.

The Ionophore ETH 129 as Ca2+ Translocator in Artificial and Natural Membranes

We have investigated the ability of the neutral jonophore ETH 129 to translocate Ca2+ across artificial and biological membranes. ETH 129 induces Ca2+ transport across planar lipid bilayer. The zero-current membrane potential in a gradient of Ca2+ concentration exhibits Nernst behavior. The dependence of the membrane conductance on ionophore and Ca2+ concentration indicates that three ionophore molecules are needed to transfer one Ca2+ across the hydrophobic region of the membrane. In mitochondria the neutral Ca2+ ionophore can move Ca2+ inside in response to a negative membrane potential under conditions in which the endogenous uniporter is blocked by ruthenium red. This electrophoretic transport of Ca2+ by ETH 129 occurs at a concentration much lower than the one previously reported with the neutral Ca2+ ionophore ETH 1001. Using sea urchin eggs, we have also shown that the efficiency of ETH 129 in inducing egg activation, as revealed by cortical granules exocytosis, is four orders of magnitude higher than that of the commonly used Ca2+ ionophore A21387. ETH 129 is a very efficient and useful tool for use in the investigation of Ca2+-dependent biological processes.

Enzymatic Properties of Two Calcium-stimulated ATPases in the Neutral and Acidic pH Regions Found in the Membrane Fraction of Human Milk.

Two calcium-stimulated ATPases at optimal pH values of 5.0 and 7.0, which are designated as acid and neutral Ca(2+) -ATPase, respectively, were found in the membrane fraction of human milk, and their enzymatic properties were studied. For maximal activity, neutral Ca(2+)-ATPase required 0.45 mM Ca ion, while acid Ca(2+) -ATPase required 207 mM Ca ion. Neutral Ca(2+) -ATPase activity was not enhanced by adding the Mg ion at more than 0.1 mM. Among the nucleotides, neutral Ca(2+) -ATPase showed a higher substrate specificity to GTP, CTP, ITP, and UTP than to ATP, while ATP was the best substrate for acid Ca(2+) -ATPase. Neutral and acid Ca(2+) -ATPases had apparent Km values of 0.361 and 0.192 mM, and Vmax of 186 and 178 μmol ATP hydrolyzed/mg of protein per min, respectively. Both Ca(2+) -ATPases were potently inhibited by fluoride, lanthanide, vanadate, and p-chloromercuribenzoate, and inactivated by EDTA, EGTA, and CDTA, but were unaffected by N-ethylmaleimide, NaN3, ouabain, oxidized glutathione, or oligomycin, and were inactivated by heating at 60°C for 10 min. These enzymes were concentrated in the membrane fraction of the cream and skim milk membrane.

Effects of Calmodulin and Calmodulin Antagonists on Neutral Ca(2+)-ATPase in the Membrane Fraction of Human Milk.

Effects of Calmodulin and Calmodulin Antagonists on Neutral Ca(2+)-ATPase in the Membrane Fraction of Human Milk.

Intracellular neutral carrier-based Ca2+ microelectrode with subnanomolar detection limit

Intracellular neutral carrier-based Ca2+ microelectrode with subnanomolar detection limit

Intracellular neutral carrier-based Ca2+ microelectrode with subnanomolar detection limit.

In intracellular electrolyte solutions a Ca2+-selective microelectrode based on the synthetic electrically neutral carrier N,N,N',N'-tetracyclohexyl-3-oxapentanediamide (ETH 129) shows an improved detection limit when compared with the so far widely used Ca2+ microelectrodes based on the neutral carrier ETH 1001. Detection limits are found at pCa = 9.2 in Ca2+ buffers containing an intracellular background of K+ (125 mM). Selectivity studies in mixed solutions show a preference of Ca2+ over Na+ of 6 X 10(5), over K+ of 1.6 X 10(6), and over Mg2+ of 5 X 10(6). The microelectrode does not suffer from significant interference by inorganic and organic inhibitors and by lipophilic cations and anions. The low detection limit is unchanged at least during the first eight hours of continuous contact with Ca2+ solutions. The EMF drift during the first hour of use is between 5 and 10 mV and is then reduced to about 1 mV/h. The changes in EMF induced between solution of pCa = 7 and pCa = 8 are reproducible within 24.7 +/- 0.4 mV (SD, n = 8, about 3 h). These electrode characteristics were found for single-barrelled microelectrodes of one micrometer diameter front-filled with a PVC-containing membrane phase. In the absence of poly(vinyl chloride) in the membrane phase irregular EMF response curves were obtained throughout. Preliminary punctures of ferret ventricular muscle cells indicate that the Ca2+ electrode response is not disturbed by the contact of a cytosolic milieu.

Design of ionophores for ion-selective microsensors.

Requirements for a reliable use of liquid membrane microelectrodes are discussed in terms of stability, response time, and lifetime on the basis of membrane technological considerations. The selectivity of H+, Li+, Na+, K+, Mg2+, Ca2+, and Cl- microelectrodes is critically evaluated using the Nikolskii-Eisenman formalism. Recent progress in the design of new ionophores is presented. A novel neutral carrier-based Ca2+-selective microelectrode with a detection limit of about 5 X 10(-10) M Ca2+ at a background of 125 mM K+ has been realized. An neutral carrier-based microelectrode for H+ with extended pH range of the sample solution is now available. Promising developments in the field of Li+-, Mg2+-, and Cl--selective ionophores are discussed.