Metal ion-tetracycline interactions in biological fluids. 2. Potentiometric study of magnesium complexes with tetracycline, oxytetracycline, doxycycline, and minocycline, and discussion of their possible influence on the bioavailability of these antibiotics in blood plasma

The formation constants of the various complexes formed by magnesium with four tetracycline derivatives, namely, tetracycline itself, oxytetracycline, doxycycline, and minocycline, were determined by potentiometry over large pH ranges under experimental conditions pertaining to blood plasma (37 degrees C, NaCl 0.15 mol dm-3). The results were used, together with those previously obtained on the complexation of these tetracyclines with proton and calcium, to assess the influence of the two alkali earth metal ions on the bioavailability of these drugs in blood plasma. Accordingly, simulations of the distribution of the four tetracyclines into their different proton and metal complex species were calculated. The distributions confirm that, in combination with the protein-bound fraction of the tetracyclines, the metal-bound fraction represents more than 99% of these drugs in plasma, the extent of their free fraction commonly being less than 1%.     

Relative roles of Ca2+, Sr2+, Ba2+ and Mg2+ in controlling lens permeability characteristics

The effects of barium, strontium and magnesium upon lens permeability characteristics were studied in the presence and absence of 2 mM calcium in the bathing medium. Permeability characteristics were determined by measuring lens potential, resistance and ⁴²K efflux rates. Barium and strontium at equimolar concentrations to calcium were able to substitute for calcium in controlling lens sodium permeability. Magnesium was ineffective in this respect.Small changes in resistance and ⁴²K efflux rates occurred when calcium was eliminated from bathing solution containing either 2 mM barium or strontium. These changes were interpreted to be the result of an increase in lens permeability to potassium. When 2 mM strontium was added to calcium-containing solution, there was no significant change in the electrical or flux parameters of the lens. However, the addition of 2 mM barium to calcium-containing solution resulted in a 54% increase in lens resistance and a 13 mV depolarization. These observations indicated a barium-induced decrease in lens permeability to potassium, and this was confirmed by an observed decrease in ⁴²K efflux rate constant under similar experimental conditions.The rapid time course of all the observed changes implies that they are the result of changes in the permeability characteristics of membranes lying close to the surface of the lens.     

水稻OsGAP1 C2结构域中对钙离子的结合能力强于对钾离子

钙离子作为植物细胞的第二信使,广泛参与植物应对不同逆境胁迫的信号调控过程。水稻G蛋白促进蛋白1（Oryza sativa GTPase activating protein 1, OsGAP1）包含1个C2结构域,而含C2结构域的蛋白质是一类钙离子结合蛋白质,受钙信号的调控。本研究鉴定了水稻OsGAP1的由5个保守性天冬氨酸残基组成的阳离子结合区域。该区域可结合2个钙离子或者钾离子,且其结合钙离子的强度高于其结合钾离子的强度,但是不能结合镁离子。当将其中2个保守的天冬氨酸残基(Asp-23和Asp-28)突变为丙氨酸后,其对钙离子的结合能力减弱。对OsGAP1 C2结构域阳离子结合区域结合金属离子能力的研究,有助于加深对钙信号调控蛋白质的认识,为其在农业生产中的应用提供理论依据。     

Stimulation by calmodulin of Ca2+ uptake and (Ca2+-Mg2+) ATPase activity in membrane fractions from ox neurohypophyses

Calmodulin stimulated ⁴⁵Ca²⁺ uptake into a plasma membrane enriched fraction from ox neurohypophysial nerve endings and into a microsome fraction. The ⁴⁵Ca²⁺ uptake and the (Ca²⁺-Mg²⁺) ATPase activity in the plasma membrane fraction exhibited similar pCa and calmodulin sensitivities, suggesting that the enzyme activity is the biochemical expression of a high affinity Ca²⁺ pump. Calmodulin thus seems to play a role in regulation of the intracellular free Ca²⁺ concentration in the neurohypophysis.     

Disruption of Ca2+ Homeostasis In Trypanosoma Cruzi By Crystal Violet

ABSTRACT. We have demonstrated previously that crystal violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that crystal violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca²⁺ from the mitochondria. In addition, crystal violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca²⁺ uptake by digitonin-permeabilired epimastigotes. Crystal violet also induced Ca²⁺ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, crystal violet inhibited Ca²⁺ uptake and the (Ca²⁺-Mg²⁺)ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca²⁺ homeostasis by crystal violet may be a key process leading to trypanosome cell injury by this drug.     

Effects of W-7 on catecholamine release and 45Ca2+ uptake in cultured adrenal chromaffin cells.

Effects of N-(6-aminohexyl)-5-chloro-1-naph-thalenesulfonamide (W-7), a calmodulin antagonist, on catecholamine (CA) release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. W-7 inhibited the carbamylcholine (CCh)-evoked CA release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner. The inhibitory effect of W-7 on CCh-evoked CA release was not overcome either by an increase in extracellular calcium or CCh concentration. Although W-7 inhibited the high K⁺-evoked CA release and ⁴⁵Ca²⁺ uptake, potency of the drug was approximately 50–100 fold less than when inhibiting the CCh-evoked CA release and ⁴⁵Ca²⁺ uptake. The inhibitory effects of W-7 were observed both in norepinephrine release and epinephrine release. Moreover, W-7 inhibited the CCh-evoked ⁴⁵Ca²⁺ efflux. These results suggest that the inhibition of CA release by W-7 in adrenal chromaffin cells is mainly due to its inhibition of calcium uptake. W-7 may influence the linkage between acetylcholine-receptor and calcium uptake with higher potency than depolarization-dependent calcium entry.     

Metal ion-tetracycline interactions in biological fluids. Part 6. Formation of copper(II) complexes with tetracycline and some of its derivatives and appraisal of their biological significance

It was previously established by computer simulation that the interactions of tetracyclines with calcium and magnesium play a paramount role in the bioavailability of these antibiotics in blood plasma. More recently, the influence of zinc was found to be insignificant in this biofluid at drug therapeutic levels but clinical data, relative to the gastrointestinal absorption of tetracyclines as well as zinc in the presence of each other, could be interpreted on the basis of the appropriate simulations.The present paper deals first with the experimental determination of the formation constants for the complexes of copper(II) with tetracycline, oxytetracycline, doxycyline, minocycline, chlortetracycline and demethylchlortetracyclin in aqueous NaCl 0.15 mol dm⁻³ at 37 °C.The data was then used for simulating the distribution of copper and each of these drugs in blood plasma as well as in gastrointestinal fluid. No influence can be expected from copper on the bio- availability of tetracyclines in blood plasma, the reverse also being true. For the therapeutic doses under consideration, copper cannot interfere with gastrointestinal absorption of these antibiotics but the presence of the latter tends to favour copper absorption to a determining extent.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.     

A raman study of the interaction of Mg2+, Ca2+, and Ba2+ ions with an acidic model membrane

The interaction of dipalmitoylphosphatidylgly cerol DPPG) liposomes with divalent ions of magnesium, calcium and barium has been investigated with laser-Raman spectroscopy over the temperature range of 0–60°C. The effect of Ca²⁺ ions was also investigated as a function of concentration. At a Ca²⁺/DPPG molar ratio of 0.1, the number of trans carbon to carbon bonds in the hydrocarbon domain of the phospholipid and the lateral order of the hydrocarbon chains was increased both below and above the gel to liquid crystal transition. At higher Ca²⁺ concentrations the number of trans bonds and the lateral order is further increased over the entire temperature range studied, while the transition disappears. Magnesium and barium ions have a much smaller ordering effect on the side-chain packing of DPPG liposomes. At a molar ratio of 0.3, the gel to liquid crystal transition is still discernible for DPPG liposomes in the presence of Ba²⁺ ions, but not in the presence of Mg²⁺ ions.     

Kinetic characterization and substrate requirement for the Ca2+ uptake system in platelet membrane

An ATP-dependent mechanism for Ca²⁺ uptake in human platelet membrane fractions has been identified and characterized. Ca²⁺ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca²⁺ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for free [Ca²⁺] of 0.145 μM. Ca²⁺ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 μM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake ($\text{IC}{}_{50}\text{= 55 μ}\text{M}$), suggesting this ATP-dependent Ca²⁺ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.     

Isolation of plasma membrane and binding of the Ca2+ antagonist nimodipine in Chlamydomonas reinhardtii

Chlorophyll-free plasma membranes of the unicellular green alga Chlamydomonas reinhardtii Dangeard were purified from a microsomal fraction using an aqueous polymer two-phase system of 6.5% (w/w) dextran T500, 6·5% (w/w) polyethylene glycol 3350, 60 mM NaCI, 0 33 M sucrose and 5 mM potassium phosphate (pH 7·8). The plasma membrane fraction contained only 2·4% of the microsomal membrane protein. Specific activity of the plasma membrane marker enzyme, K*, Mg²⁺-ATPase (EC 3.6.1.3). was enriched 9-fold over the microsomal fraction, and 22% of total activity was recovered in the upper, polyethylene glycol-rich phase. Contamination from intracellular membranes was minimal. K*, Mg²⁺-ATPase showed a pH optimum at about 6·5, and addition of 0·05% (w/v) Triton X-100 stimulated the activity 3-fold. [³H]-Nimodipinc was employed to characterize 1,4-dihydropyridine-specific membrane receptors. Two apparent binding sites with different affinities to nimodipine were found in the crude microsomal fraction. The separation of plasma membranes from intracellular membranes revealed that one binding site with higher affinity (K_D= 9 nM) was located on the plasma membrane and a second binding site with lower affinity (K_D= 36 nM) on an intracellular membrane The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments were comparable to those determined by equilibrium experiments. The maximum number of binding sites of the plasma membrane fraction and the intracellular membrane fraction was B_max= 440 and 470 fmol (mg protein)^-1, respectively. [³H]-Nimodipinc binding was inhibited by (±) verapamil and stimulated by D-cis-diltiazem in both fractions. Moreover, ethyle-neglycol-bis(2-aminoethylcther)-N, N'-tetraacctic acid (EGTA) inhibited [³H]-nimo-dipinc binding in the plasma membrane fraction but not in the intracellular membrane fraction This effect was cancelled by the addition of CaCl₂.     

Bioavailability of tetracycline hydrochloride capsules (an in vivo study)]

Blood absorption of tetracycline hydrochloride of various dispersity levels from capsules containing tetracycline alone or in combination with additives, such as magnesium carbonate and calcium salts was studied on humans. It was found that higher dispersity levels of tetracycline hydrochloride powder in capsules was not accompanied by increased blood absorption of the antibiotic. Addition of magnesium carbonate and calcium salts to the antibiotic in the process of capsulation markedly retarded the blood absorption. Clear correlation between the antibiotic dissolution rate in vitro and intensity of its blood absorption in volunteers was shown.     

Plasma Ca2+ concentration limits melatonin night production in two fish species

In freshwater (FW) rainbow trout Oncorhynchus mykiss of spontaneously low plasma calcium concentrations ([Ca²⁺]_pl), plasma melatonin at night was significantly lower than that measured in FW fish with the highest [Ca²⁺]_pl. In brackish water adapted rainbow trout with originally high [Ca²⁺]_pl, plasma melatonin concentration at night was elevated. In cannulated flounder Platichthys flesus , night plasma melatonin increases (ΔMel) corresponded to [Ca²⁺]_pl. It is postulated that in physiological steady-state conditions, melatonin synthesis capacity is coupled to free calcium concentration in plasma of O. mykiss and P. flesus .     

Distributions of H+,K+-ATPase and Cl–,HCO3–-ATPase in micromere-derived cells of sea urchin embryos

In cultured cells derived from isolated micromeres of sea urchin eggs, H+,K+-ATPase activity, which became detectable simultaneously with the initiation of spicule formation, was localized in the plasma membrane and the microsome fractions. Activities of marker enzymes for plasma membrane, 5'-nucleotidase, Na+,K+-ATPase, and adenylate cyclase, were found to be high in the plasma membrane fraction. Considerable activity of rotenone-insensitive NADPH-cytochrome c reductase, a marker enzyme for microsome, was detectable in the microsome fraction. These fractions exhibited barely any appreciable activity of markers for the other organellae. H+,K+-ATPase in plasma membrane probably mediates H+ release from the cells, in which H+ is produced in overall reaction to form CaCO3, the main component of spicules, from Ca2+, CO2 and H2O. Cl-,HCO3(-)-ATPase activity was also found in these two fractions before and after the initiation of spicule formation. After initiation, the skeletal vacuole fraction was obtained from subcellular structures containing spicules. Considerable activity of Cl-,HCO3(-)-ATPase was observed in this fraction, which exhibited a weak activity of UDP-galactose: N-acetylglucosamine galactosyltransferase, a marker enzyme for Golgi body. Cl-,HCO3(-)-ATPase in the skeletal vacuole membrane probably mediates HCO3- transport into the vacuoles to supply HCO3- for spicule formation.     

Ischemia-Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (⁴⁵Ca²⁺) accumulation in the microsomes mediated by Mg²⁺/Ca²⁺ ATPase. Ischemia caused a statistically significant inhibition of presteady-state and steady-state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia-induced inhibition of endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase.     

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.     

Partial purification and characterization of the (Ca2+ + Mg2+)-ATPase from squid optic nerve plasma membrane

A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN₃-insensitive (Ca²⁺ + Mg²⁺)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}{}^1\text{= 0.12 μ}\text{M}$) and the second had a comparatively low affinity ($\text{K}{}^2{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 49.5 μ}\text{M}$). Only the high-affinity component was specifically inhibited by vanadate (K₁ = 35 μM). Calmodulin (12.5 μ/ml) stimulated the (Ca²⁺ + Mg²⁺)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 μM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca²⁺ + Mg²⁺)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca²⁺-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis.     

Kinetic Studies of a (Na++ K++ Mg2+) ATPase in Sugar Beet Roots

Kinetic studies of a dithiothreitol treated membrane ATPase fraction from sugar beet roots led to the following conclusions: 1) In the presence of MgATP, Na⁺ and K⁺ stimulate the ATPase activity in different ways following simple Michaelis-Menten kinetics. Thus separate sites for Na⁺ and K⁺ are suggested. 2) In the absence of K⁺, Na⁺ acts as an uncompetitive modifier raising the apparent K_m and V_max for MgATP. 3) In the absence of Na⁺, K⁺ activates non-competitively with respect to MgATP. Thus K⁺ increases V_max but does not affect the apparent affinity constant. 4) K⁺ and Na⁺ double the rate constants. 5) In the presence of Na⁺ or K⁺, Mg²⁺ in excess acts as a weak inhibitor to Na⁺ and/or K⁺ activity. 6) The temperature-activity dependence in the 5–40°C interval shows biphasic Arrhenius plots with the transition point between 15–18°C. The activation energy is lowered at temperatures > 18°C.