Basal Mg2 Research Articles

Gene-nutrient interactions that impact magnesium homeostasis increase risk for neural tube defects in mice exposed to dolutegravir.

In 2018, data from a surveillance study in Botswana evaluating adverse birth outcomes raised concerns that women on antiretroviral therapy (ART) containing dolutegravir (DTG) may be at increased risk for neural tube defects (NTDs). The mechanism of action for DTG involves chelation of Mg2+ ions in the active site of the viral integrase. Plasma Mg2+ homeostasis is maintained primarily through dietary intake and reabsorption in the kidneys. Inadequate dietary Mg2+ intake over several months results in slow depletion of plasma Mg2+ and chronic latent hypomagnesemia, a condition prevalent in women of reproductive age worldwide. Mg2+ is critical for normal embryonic development and neural tube closure. We hypothesized that DTG therapy might slowly deplete plasma Mg2+ and reduce the amount available to the embryo, and that mice with pre-existing hypomagnesemia due to genetic variation and/or dietary Mg2+ insufficiency at the time of conception and initiation of DTG treatment would be at increased risk for NTDs. We used two different approaches to test our hypothesis: 1) we selected mouse strains that had inherently different basal plasma Mg2+ levels and 2) placed mice on diets with different concentrations of Mg2+. Plasma and urine Mg2+ were determined prior to timed mating. Pregnant mice were treated daily with vehicle or DTG beginning on the day of conception and embryos examined for NTDs on gestational day 9.5. Plasma DTG was measured for pharmacokinetic analysis. Our results demonstrate that hypomagnesemia prior to conception, due to genetic variation and/or insufficient dietary Mg2+ intake, increases the risk for NTDs in mice exposed to DTG. We also analyzed whole-exome sequencing data from inbred mouse strains and identified 9 predicted deleterious missense variants in Fam111a that were unique to the LM/Bc strain. Human FAM111A variants are associated with hypomagnesemia and renal Mg2+ wasting. The LM/Bc strain exhibits this same phenotype and was the strain most susceptible to DTG-NTDs. Our results suggest that monitoring plasma Mg2+ levels in patients on ART regimens that include DTG, identifying other risk factors that impact Mg2+ homeostasis, and correcting deficiencies in this micronutrient might provide an effective strategy for mitigating NTD risk.

The role of bafilomycin as a therapeutic agent in the modulation of endo-lysosomal store of rat hepatocytes

Endo-lysosomal system through the process of autophagy is involved in the pathogenesis of many diseases. Acidification of these organelles is carried out by V-type H+-ATPases, which is inhibited by bafilomycin A1. Endosomes and lysosomes are also important Ca2+-storage in a cell. Nіcotіnіc acіd adenіne dіnucleotіde phosphate (NAADP) releases Cа2+ from endo-lysosomes. The main purpose of the study was to found out the effect of bafilomycin A1 and NAADP on stored Ca2+ and on the ATPase activity of rat hepatocytes. The stored Ca2+ was estimated using chlorotetracycline in permeabilized hepatocytes of rats. ATPase activity was determined by level of orthophosphate spectrophotometrically. It was found that bafilomycin A1 reduces stored Ca2+ in permeabilized hepatocytes of rats in the micromolar range of concentration (20 and 0.04 mkM) and averted the effect of NAADP on calcium content. Lower concentrations of bafilomycin A1 (0.001 mkM) did not alter the content of stored calcium, but prevented the influence of NAADP in permeabilized hepatocytes of rats. In the subcellular fraction of rat liver bafi­lomycin A1 (0.001 mkM) increased Ca2+-ATPase and basal Mg2+-ATPase activities and reduced Na+/K+-ATPase acti­vity. Preincubation of the subcellular fraction with bafilomycin A1 completely averts any changes in the activity of estimated ATPases by means of NAADP. It was concluded that the bafilomycin-sensitive store in hepatocytes of rats is NAADP-sensitive endo-lysosomal Ca2+-store. Using of bafilomycin A1 may be useful in treating autophagy-depended diseases.

PECULIARITIES OF THE EFFECTS OF BILE ACIDS ON ATPASE ACTIVITY OF THE COLON MUCOSA IN PATIENTS WITH OVERWEIGHT AND IRRITABLE BOWEL SYNDROME

The aim is to investigate the effect of bile acids on the ATPase activity of the colon mucosa in patients with overweight and irritable bowel syndrome (IBS). Materials and methods: Completely examined 12 patients with IBS and overweight.We estimated the ATPase activity of colon mucous of the patients with IBSspectrophotometrically by determined the content of orthophosphate that was released after ATP hydrolysis. We studied the effect of 3-sulphate of taurolitocholate (TLC-S) on specific activities of Na+/ K+-ATPase, Ca2+-ATPase of endoplasmatic reticulum (EPR),Ca2+-ATPase of plasmatic membrane (PM) and basal Mg2+-ATPase of postmitochondrial subcellular fraction of colon mucous of the patients with IBS. Results: We establishedthe specific activities of Na+/K+-ATPase, Ca2+-ATPase of EPR,Ca2+-ATPase of PM and basal Mg2+-ATPase. Therewere(6.06 ± 1.61), (5.88 ± 1.19), (8.86 ± 1.56) (6.44 ± 2.02)µmol Pi/ mg protein per hour, respectively. TLC-S (50 µM) did not causedany change of Na+/K+-ATPase , as well as Ca2+-ATPasesactivities, but statistically significant increased activity of Mg2+-ATPase of postmitochondrial subcellular fraction of colon mucous of the patients with IBS by 4 fold. Conclusions: TLC-S increased basal Mg2+-ATPase in the postmitochondrial fraction of colon mucous of the patients with overweight and IBS, but had no effect on Na+/K+-ATPase and Ca2+-ATPases activities. It has been suggested that activation of basal Mg2+-ATPase under by TLC-S may indicates the role of the endo-lysosomal system of epitheliocytes of colon mucous in developing of pathology IBS.

Mg2+ regulation of kinase signaling and immune function.

Mg2+ is required at micromolar concentrations as a cofactor for ATP, enzymatic reactions, and other biological processes. We show that decreased extracellular Mg2+ reduced intracellular Mg2+ levels and impaired the Ca2+ flux, activation marker up-regulation, and proliferation after T cell receptor (TCR) stimulation. Reduced Mg2+ specifically impairs TCR signal transduction by IL-2-inducible T cell kinase (ITK) due to a requirement for a regulatory Mg2+ in the catalytic pocket of ITK. We also show that altered catalytic efficiency by millimolar changes in free basal Mg2+ is an unrecognized but conserved feature of other serine/threonine and tyrosine kinases, suggesting a Mg2+ regulatory paradigm of kinase function. Finally, a reduced serum Mg2+ concentration in mice causes an impaired CD8+ T cell response to influenza A virus infection, reduces T cell activation, and exacerbates morbidity. Thus, Mg2+ directly regulates the active site of specific kinases during T cell responses, and maintaining a high serum Mg2+ concentration is important for antiviral immunity in otherwise healthy animals.

Silencing TRPM7 in Mouse Cortical Astrocytes Impairs Cell Proliferation and Migration via ERK and JNK Signaling Pathways

Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel, is highly expressed expressed in the brain and plays a critical role in ischemic neuronal death. Astrocyte, the most abundant cell type in central nervous system (CNS), exerts many essential functions in the physiological and pathological conditions. Here we investigated the expression and functions of the TRPM7 channel in mouse cortical astrocytes. Using reverse transcription (RT)-PCR, immunostaining, western blot and patch clamp recording, we showed that functional TRPM7 channel is expressed in cultured mouse cortical astrocytes. Knocking down TRPM7 with specific siRNA impairs the proliferation and migration of astrocytes by 40.2% ± 3.9% and 40.1% ± 11.5%, respectively. Consistently, inhibition of TRPM7 with 2-aminoethoxydiphenyl borate (2-APB) also decreases astrocyte proliferation and migration by 46.1% ± 2.5% and 64.2% ± 2.4%. MAPKs and Akt signaling pathways have been shown to be implicated in TRPM7-mediated responses including cell proliferation and migration. Our data show that suppression of TRPM7 in astrocytes reduces the phosphorylation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), but not p38 mitogen-activated protein kinase and Akt. In addition, TRPM7, as a cation channel, has been involved in the Ca2+ and Mg2+ homeostasis in several types of cells. In our study, we found that silencing TRPM7 decreases the intracellular basal Mg2+ concentration without affecting Ca2+ concentration in astrocytes. However, an addition of Mg2+ to the growth medium could not rescue the impaired proliferation of astrocytes. Together, our data suggest that TRPM7 channel may play a critical role in the proliferation and migration of astrocytes via the ERK and JNK pathways.

Intracellular free Mg2+ is required to maintain NKG2D expression necessary for controlling EBV infection in XMEN disease (P3028)

Abstract Epstein-Barr virus (EBV) is a B cell-tropic gammaherpesvirus present in latent form in approximately 90% of people worldwide. However, a subset of immunocompromised hosts develop malignancy from uncontrolled EBV infection. While the etiology of such immunodeficiency states is sometimes apparent, as in HIV and post-transplant immunosuppression, the underlying immune deficiency is other cases remains unknown. X-linked immunodeficiency with Mg2+ defect, EBV infection, and neoplasia (XMEN) disease is a novel primary immunodeficiency characterized by deleterious mutations in magnesium transporter 1 (MAGT1), chronic EBV infection and increased susceptibility for EBV-associated lymphomas. We had previously demonstrated that MAGT1 mediates a second messenger role for Mg2+ during T cell activation. Here, we show that loss of MAGT1 also decreases the basal level of intracellular free Mg2+ and abolishes the expression of NKG2D on natural killer cells and cytotoxic T lymphocytes, leading to impaired cytotoxicity against EBV-transformed lymphoblastoid cell lines. Interestingly, both these defects can be restored in vitro and in vivo by exogenous Mg2+ supplementation, leading to decreased EBV-infected cells in vivo. This not only revealed the first specific molecular function of basal free Mg2+ in eukaryotic cells but also identified a novel Mg2+-dependent immune arm of defense against EBV lymphomagenesis that has profound diagnostic and therapeutic implications for XMEN disease.

Two Drosophila Myosin Transducer Mutants with Distinct Cardiomyopathies Have Divergent ADP and Actin Affinities

Two Drosophila myosin II point mutations (D45 and Mhc5) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc5) could stabilize (D45) or destabilize (Mhc5) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1A261T and S1G200D) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1A261T is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin·S1), and reduced ATP-induced dissociation of actin·S1), whereas S1G200D shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin·S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1A261T (dilated) and S1G200D (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1A261T weakens the affinity of S1·ADP for actin, whereas S1G200D increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up2 mutation in Drosophila.

The Arabidopsis Peroxisomal ABC Transporter, Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.

Magnesium ions inhibit Apaf‐1 apoptosome assembly by competition with cytochrome c (Cc)

The disassembly of mammalian cells by apoptosis generally depends on oligomerization of the Apaf‐1:caspase‐9 apoptosome in the cytoplasm, which is evoked by the release of Cc from mitochondria. A soluble fraction (S100) was prepared from the 911 line of immortalized human embryonic retinoblasts or from 911 cells transiently transfected with human Apaf‐1 with carboxy‐terminal V5 epitope and hexahistidine tags. Apoptosome activation was assayed by monitoring the rate of caspase‐3 like activity with a fluorogenic tetrapeptide. Apoptosome activation was absolutely dependent on the addition of Cc and markedly stimulated by the addition of a nucleotide (MgATP or MgdATP). The Km of Cc was 0.6 μM bith a ransfected with human Apaf‐1 with a V5 epitope tag and hexahistidine. The Ki of free Mg2+ was 0.3 mM, which is half the 0.6 mM basal free Mg2+ level of 911 and HeLa cells (Abstract 6098). While MgATP was required for maximal apoptosome activation, excess MgATP did not inhibit prodced activation and had apoptosome activation and had no inhibitory effect Cc‐dependent apoptosome activity. Transfection of 911 cells with Apaf‐1V5His increased apoptosome activation by Cc by approximately 10 fold relative to that of cells transfected with empty vector, which indicates that Apaf‐1in S100 is rate limiting. Mg2+ inhibited apoptosome activity caused by transfected Apaf‐1V5His similarly to that of endogenous Apaf‐1 in S100 from nontransfected 911 cells. Mg2+ inhibition of Cc‐dependent apoptosome activity was highly cooperative with a Hill coefficient of 4 to 7, which indicates that Mg2+ can switch off apoptosome assembly. Apoptosome assembly was observed by sedimentation velocity in glycerol gradients and western analysis of Apaf‐1V5His and procaspase‐9 and the large subunit. Mg2+ prevented apoptosome assembly and autoprocessing of procaspase‐9 to subunits. These results show that Mg2+ potently inhibits apoptosome assembly, presumably by blocking the binding of Cc to monomeric Apaf‐1. Supported by grant GM60383 from NIH.

Early Magnesium Reduction in Advanced Colorectal Cancer Patients Treated with Cetuximab Plus Irinotecan as Predictive Factor of Efficacy and Outcome

Magnesium plays a role in a large number of cellular metabolic reactions. Cetuximab is able to induce hypomagnesemia by interfering with magnesium (Mg(2+)) transport in the kidney. We designed this trial to investigate if Mg(2+) serum level modifications may be related with clinical response and outcome in advanced colorectal cancer patients during treatment with cetuximab plus irinotecan. Sixty-eight heavily pretreated metastatic colorectal cancer patients were evaluated for Mg(2+) serum levels at the following time points: before; 6 hours; and 1, 7, 14, 21, 50, and 92 days after the start of treatment. Basal Mg(2+) median levels were significantly decreased just 7 days after the first anticancer infusion and progressively decreased from the 7th day onward, reaching the highest significance at the last time point (P < 0.0001). Twenty-five patients showed a reduction in median Mg(2+) circulating levels of at least 20% within the 3rd week after the first infusion. Patients with this reduction showed a response rate of 64.0% versus 25.6% in the nonreduced Mg(2+) group. The median time to progression was 6.0 versus 3.6 months in the reduced Mg(2+) group and in that without reduction, respectively (P < 0.0001). Overall survival was longer in patients with Mg(2+) reduction than in those without (10.7 versus 8.9 months). Our results confirm that cetuximab treatment may induce a reduction of Mg(2+) circulating levels and offer the first evidence that Mg(2+) reduction may represent a new predictive factor of efficacy in advanced colorectal cancer patients treated with cetuximab plus irinotecan.

Early magnesium reduction in advanced colorectal cancer patients treated with cetuximab + irinotecan as predictive factor of efficacy and outcome

4106 Background: Magnesium plays a role in a large number of cellular metabolic reactions. Cetuximab is able to induce hypomagnesaemia by interfering with magnesium (Mg2+) transport in the kidney. We designed this trial to investigate if Mg2+ serum levels modifications may be related with clinical response and outcome in advanced colorectal cancer (CRC) patients during treatment with cetuximab plus irinotecan (CTX+CPT11). Methods: 91 pretreated metastatic CRC patients were evaluated for Mg2+ serum levels. The following time-points were analyzed: before 1, 7, 14, 21, 50 and 92 days after the start of treatment. Results: basal Mg2+ median levels were significantly decreased just seven days after the first anticancer infusion and progressively decreased along all the time points, reaching the highest significance at the last time point (P<0.0001). 46 patients showed a reduction in median Mg2+ circulating levels of at least 20% within the third week after the first infusion respect to the basal time point. Patients with this reduction showed a response rate of 68.0% vs 29.5% in the non reduced Mg2+ group. The median time to progression (TTP) was 6.7 vs 3.9 months in the reduced Mg2+ group and in that without, respectively (p<0.0001). Overall survival (OS) was longer in patients with Mg2 reduction than in those without (10.5 vs 7.5 months, p=0.02). Conclusions: our results confirm that cetuximab treatment may induce a reduction of Mg2+ circulating levels and offer the first evidence that early Mg2+ reduction may represents a new predictive factor of efficacy in heavily pretreated advanced CCR patients treated with CTX+CPT11. No significant financial relationships to disclose.

Effect of furosemide on GABAA-induced 36Cl transport and Cl--ATPase activity in synaptic membranes of carp brain (Cyprinus carpio L.)

We studied the effect of furosemide on GABAA-induced 36Cl transport and GABAA-induced Cl--ATPase activity in synaptic membranes of fish brain. At physiological pH 7.4, GABA (0.1–100 µM) stimulated 36Cl influx in synaptoneurosomes and Cl--ATPase activity in synaptic membranes. Furosemide (0.1–0.5 mM) removed the activating effect of the mediator on chloride transport and enzyme activity (I50 equaled 0.16 and 0.12 mM, respectively). In the absence of the mediator, picrotoxin (50 µM) activated the basal 36Cl influx in synaptoneurosomes and the basal Mg2+-ATPase activity of synaptic membranes. Furosemide (1 mM) removed the activating effect of picrotoxin on both biochemical processes. The obtained data demonstrated similar sensitivities of GABAA-induced transport of 36Cl in synaptoneurosomes and of GABAA-induced Cl--ATPase activity in the synaptic membranes to furosemide and indicated the involvement of the ATPase in GABAA-induced processes. The soluble ATPase, recovered by sodium deoxycholate solubilization of the membranes, remained sensitive to GABAA-ergic ligands, which suggested proximity of their binding sites with ATP hydrolysis sites in the protein molecule and their structural coupling.

Properties of Cl--Stimulated Mg2+-ATPase Sensitive to Ligands of Inhibitory Receptors in Brain Plasma Membranes of the Bream Abramis brama

Properties of Cl−-stimulated Mg2+-ATPase in the brain plasma membranes of the bream Abramis brama L. were studied; this enzyme is composed of “basal” Mg2+-ATPase activity that can be stimulated by 40–80% by Cl− ions (Cl−-ATPase). These anions stimulate the basal Mg2+-ATPase starting with 8 mM concentration, their maximal effect being observed at a concentration of 30–100 mM. The Cl−-ATPase activity was found at a low molarity of HEPES-Tris buffer ( 30 mM). The “basal” Mg2+-ATPase activity was detected in the whole studied pH range (5.5–9.0), with maximum at pH 7.2–7.8 values, whereas optimum to reveal Cl−-ATPase was at high and low H+ concentrations (pH 6.0 and 8.5, respectively). At physiological pH values (7.2–7.5) the Cl−-ATPase activity was not revealed, but was detected after preincubation of the enzyme with 10 µM GABA. The “basal” Mg2+-ATPase, like Cl−-ATPase, hydrolyzed ATP with a maximal rate, while CTP, ITP, and ADP only slightly, and did not hydrolyze GTP and AMP. The Cl−-ATPase activity decreased in the presence of divalent cations in the following order: Mg2+ > Co2+ > Mn2+ = Cd2+ > Al3+ = Cu2+, and it was not found in the presence of Ca2+ and Zn2+. Anions of halogen series activated the “basal” Mg2+-ATPase in the descending order: Cl− > Br− > J− > F−. Among other monovalent anions, HCO3− activated the enzyme, NO3− practically had no effect, and SCN− inhibited its activity. Blockers of Cl− transport (ethacrinic acid, furosemide, and SITS) and GABA-receptor ligands (pentobarbital, diazepam, and picrotoxin) suppressed the enzyme activity. Out of SH-reagents, PCMB inhibited the enzyme, while NEM did not affect it. The H+-ATPase blocker oligomycin inhibited the enzyme, while the blocker of Na+,K+-ATPase ouabain and the blocker of Ca2+,Mg2+-ATPase ruthenium red had no effect. The properties of the Cl−-stimulated Mg2+-ATPase of fish brain are discussed in comparison with those of the rat brain Cl−-ATPase. The conclusion is made that the bream brain enzyme differs markedly from Cl−-ATPase (the ATP-dependent Cl−-pump) of mammalian brain.

Free magnesium concentration in salamander photoreceptor outer segments.

Magnesium ions (Mg2+) play an important role in biochemical functions. In vertebrate photoreceptor outer segments, numerous reactions utilize MgGTP and MgATP, and Mg2+ also regulates several of the phototransduction enzymes. Although Mg2+ can pass through light-sensitive channels under certain conditions, no clear extrusion mechanism has been identified and removing extracellular Mg2+ has no significant effect on the light sensitivity or the kinetics of the photoresponse. We have used the fluorescent Mg2+ dye Furaptra to directly measure and monitor the free Mg2+ concentration in photoreceptor outer segments and examine whether the free Mg2+ concentration changes under physiological conditions. Resting free Mg2+ concentrations in bleached salamander rod and cone photoreceptor cell outer segments were 0.86 +/- 0.06 and 0.81 +/- 0.09 mM, respectively. The outer segment free Mg2+ concentration was not significantly affected by changes in extracellular pH, Ca2+ and Na+, excluding a significant role for the respective exchangers in the regulation of Mg2+ homeostasis. The resting free Mg2+ concentration was also not significantly affected by exposure to 0 Mg2+, suggesting the lack of significant basal Mg2+ flux. Opening the cGMP-gated channels led to a significant increase in the Mg2+ concentration in the absence of Na+ and Ca2+, but not in their presence, indicating that depolarization can cause a significant Mg2+ influx only in the absence of other permeant ions, but not under physiological conditions. Finally, light stimulation did not change the Mg2+ concentration in the outer segments of dark-adapted photoreceptors. The results suggest that there are no influx and efflux pathways that can significantly affect the Mg2+ concentration in the outer segment under physiological conditions. Therefore, it is unlikely that Mg2+ plays a significant role in the dynamic modulation of phototransduction.

Intravesicular glucose modulates magnesium2+ transport in liver plasma membrane from streptozotocin-treated rats

Plasma membrane vesicles purified from livers of 4-week-old streptozotocin-injected diabetic rats present an increased basal and cation-stimulated magnesium (Mg)2+ transport as compared with vesicles purified from age-matched nondiabetic animals. Furthermore, diabetic basolateral membranes are unable to accumulate extravesicular Mg2+ in exchange for intravesicular sodium (Na)+. Loading diabetic vesicles with varying concentrations of d-glucose, in addition to Mg2+, renormalizes basal and Na+- or calcium (Ca)2+-induced Mg2+ extrusion in a dose-dependent manner, but does not restore Na+/Mg2+ exchanger reversibility. A similar effect on Mg2+ extrusion is observed when d-glucose is replaced with 2-deoxy-glucose, amylopectin, or glycogen. The loading with 3-methyl-o-glucose or l-glucose, instead, affects basal and Na+-dependent Mg2+ extrusion, but not Ca2+-dependent Mg2+ fluxes. In contrast, loading the vesicles with hexoses other than glucose or varying extravesicular glucose concentration from 5 to 20 mmol/L does not modify basal or cation-stimulated Mg2+ fluxes. Taken together, these data indicate that basal and cation-stimulated Mg2+ transport across the hepatocyte plasma membrane is altered under diabetic conditions as a result of a decrease in intravesicular (intracellular) glucose.

Characterization of Recombinant Heavy Meromyosin of Physarum Polycepharum.

Background & Aims : Myosin II is a typical motor protein and is classified either as non-regulated myosin, for example, skeletal muscle myosin, or as myosin regulated by phosphorylation or Ca-binding. Myosin II from Physarum polycephalum is a Ca-binding myosin. Although expression of myosin II as a recombinant protein is essential for the analysis of its structure, function and regulation, it is not easy. Successful examples are smooth muscle myosin II and Dictyostelium myosin II, both of which are regulated by phosphorylation. Myosin II regulated by Ca-binding has not yet been obtained. Here, I report the expression of heavy meromyosin (HMM), i.e., the motor domain of Physarum myosin II.Method : I used baculovirus expression system. Sf9 cells were infected with the virus constructs.Results and Conclusions : The recombinant HMM showed basal Mg2+-ATPase activity, which was activated by actin to a similar extent to native myosin purified from Physarum. Ca-binding activity of HMM was also comparable to that of the native myosin. In vitro motility assay showed that motor activity of HMM was sensitive to Ca2+. The present study is the first report of expression of the motor domain of a myosin II that is regulated by Ca-binding.

Adenosine modulates Mg(2+) uptake in distal convoluted tubule cells via A(1) and A(2) purinoceptors.

tk;1Adenosine plays a role in the control of water and electrolyte reabsorption in the distal tubule. As the distal convoluted tubule is important in the regulation of renal Mg(2+) balance, we determined the effects of adenosine on cellular Mg(2+) uptake in this segment. The effect of adenosine was studied on immortalized mouse distal convoluted tubule (MDCT) cells, a model of the intact distal convoluted tubule. The rate of Mg(2+) uptake was measured with fluorescence techniques using mag-fura 2. To assess Mg(2+) uptake, MDCT cells were first Mg(2+) depleted to 0.22 +/- 0.01 mM by being cultured in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl(2); next, changes in intracellular Mg(2+) concentration ([Mg(2+)](i)) were determined. [Mg(2+)](i) returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg(2+)](i))/dt, of 137 +/- 16 nM/s. Adenosine stimulates basal Mg(2+) uptake by 41 +/- 10%. The selective A(1) purinoceptor agonist N(6)-cyclopentyladenosine (CPA) increased intracellular Ca(2+) and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated Mg(2+) uptake. On the other hand, the selective A(2) receptor agonist 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS) stimulated Mg(2+) entry in a concentration-dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated Mg(2+) uptake. Selective inhibition of phospholipase C, protein kinase C, or mitogen-activated protein kinase enzyme cascades with U-73122, Ro-31-8220, and PD-98059, respectively, diminished A(2) agonist-mediated Mg(2+) entry. Aldosterone potentiated CGS-mediated Mg(2+) entry, and elevation of extracellular Ca(2+) diminished CGS-responsive cAMP formation and Mg(2+) uptake. Accordingly, MDCT cells possess both A(1) and A(2) purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on Mg(2+) uptake in MDCT cells through separate A(1) and A(2) purinoceptor pathways.

1,25(OH)(2)D(3) stimulates Mg2+ uptake into MDCT cells: modulation by extracellular Ca2+ and Mg2+.

The distal convoluted tubule plays a significant role in renal magnesium conservation. Although the cells of the distal convoluted tubule possess the vitamin D receptor, little is known about the effects of 1alpha,25-dihydroxyvitamin D [1,25(OH)(2)D(3)] on magnesium transport. In this study, we examined the effect of 1,25(OH)(2)D(3) on distal cellular magnesium uptake and the modulation of this response by extracellular Ca2+ and Mg2+ in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCT cells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular Ca2+ and Mg2+ concentrations to diminish peptide hormone-stimulated Mg2+ uptake. Mg2+ uptake rates were determined by microfluorescence in Mg2+ -depleted MDCT cells. Treatment of MDCT cells with 1,25(OH)(2)D(3) for 16-24 h stimulated basal Mg2+ uptake in a concentration-dependent manner from basal levels of 164 +/- 5 to 210 +/- 11 nM/s, representing a 28 +/- 3% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25(OH)(2)D(3)-stimulated(.)Mg2+ uptake (154 +/- 18 nM/s), suggesting that 1,25(OH)(2)D(3) stimulates Mg2+ uptake through gene activation and protein synthesis. Elevation of extracellular Ca2+ inhibited 1,25(OH)(2)D(3)-stimulated Mg2+ uptake (143 +/- 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular Ca2+ of 1,25(OH)(2)D(3)-stimulated Mg2+ uptake (202 +/- 8 nM/s). Treatment with an antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular Ca2+ on 1,25(OH)(2)D(3)-responsive Mg2+ entry. This showed that elevated extracellular calcium modulates 1,25(OH)(2)D-mediated responses through the CaSR. In summary, 1,25(OH)(2)D(3) stimulated Mg2+ uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular Ca2+, acting via the CaSR, inhibited 1,25(OH)(2)D(3)-stimulated Mg2+ entry. These data indicate that 1,25(OH)(2)D(3) has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations.

Caloxin: a novel plasma membrane Ca2+ pump inhibitor

Plasma membrane (PM) Ca2+ pump is a Ca+-Mg2+-ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+ . There are no known extracellularly acting PM Ca2+ pump inhibitors, as digoxin and ouabain are for Na+ pump. In analogy with digoxin, we define caloxins as extracellular PM Ca2+ pump inhibitors and describe caloxin 2A1. Caloxin 2A1 is a peptide obtained by screening a random peptide phage display library for binding to the second extracellular domain (residues 401-413) sequence of PM Ca2+ pump isoform 1b. Caloxin 2A1 inhibits Ca2+-Mg2+-ATPase in human erythrocyte leaky ghosts, but it does not affect basal Mg2+-ATPase or Na+-K+-ATPase in the ghosts or Ca2+-Mg2+-ATPase in the skeletal muscle sarcoplasmic reticulum. Caloxin 2A1 also inhibits Ca2+-dependent formation of the 140-kDa acid-stable acylphosphate, which is a partial reaction of this enzyme. Consistent with inhibition of the PM Ca2+ pump in vascular endothelium, caloxin 2A1 produces an endothelium-dependent relaxation that is reversed by N(G)-nitro-L-arginine methyl ester. Thus caloxin 2A1 is a novel PM Ca2+ pump inhibitor selected for binding to an extracellular domain.

Magnesium transport in the renal distal convoluted tubule.

The distal tubule reabsorbs approximately 10% of the filtered Mg(2+), but this is 70-80% of that delivered from the loop of Henle. Because there is little Mg(2+) reabsorption beyond the distal tubule, this segment plays an important role in determining the final urinary excretion. The distal convoluted segment (DCT) is characterized by a negative luminal voltage and high intercellular resistance so that Mg(2+) reabsorption is transcellular and active. This review discusses recent evidence for selective and sensitive control of Mg(2+) transport in the DCT and emphasizes the importance of this control in normal and abnormal renal Mg(2+) conservation. Normally, Mg(2+) absorption is load dependent in the distal tubule, whether delivery is altered by increasing luminal Mg(2+) concentration or increasing the flow rate into the DCT. With the use of microfluorescent studies with an established mouse distal convoluted tubule (MDCT) cell line, it was shown that Mg(2+) uptake was concentration and voltage dependent. Peptide hormones such as parathyroid hormone, calcitonin, glucagon, and arginine vasopressin enhance Mg(2+) absorption in the distal tubule and stimulate Mg(2+) uptake into MDCT cells. Prostaglandin E(2) and isoproterenol increase Mg(2+) entry into MDCT cells. The current evidence indicates that cAMP-dependent protein kinase A, phospholipase C, and protein kinase C signaling pathways are involved in these responses. Steroid hormones have significant effects on distal Mg(2+) transport. Aldosterone does not alter basal Mg(2+) uptake but potentiates hormone-stimulated Mg(2+) entry in MDCT cells by increasing hormone-mediated cAMP formation. 1,25-Dihydroxyvitamin D(3), on the other hand, stimulates basal Mg(2+) uptake. Elevation of plasma Mg(2+) or Ca(2+) inhibits hormone-stimulated cAMP accumulation and Mg(2+) uptake in MDCT cells through activation of extracellular Ca(2+)/Mg(2+)-sensing mechanisms. Mg(2+) restriction selectively increases Mg(2+) uptake with no effect on Ca(2+) absorption. This intrinsic cellular adaptation provides the sensitive and selective control of distal Mg(2+) transport. The distally acting diuretics amiloride and chlorothiazide stimulate Mg(2+) uptake in MDCT cells acting through changes in membrane voltage. A number of familial and acquired disorders have been described that emphasize the diversity of cellular controls affecting renal Mg(2+) balance. Although it is clear that many influences affect Mg(2+) transport within the DCT, the transport processes have not been identified.