Extracellular regulation of fibroblast multiplication: A direct kinetic approach to analysis of role of low molecular weight nutrients and serum growth factors

The principles of enzyme kinetic analysis were applied to quantitate the relationships among serum-derived growth factors, nutrients, and the rate of survival and multiplication of human fibroblasts in culture. The survival or multiplication rate of a population of cells plotted against an increasing concentration of a growth factor or nutrient in the medium exhibited a hyperbolic pattern that is characteristic of a dissociable, saturable interaction between cells and the ligands. Parameters equivalent to the K_m and V_max of enzyme kinetics were assigned to nutrients and growth factors. When all nutrient concentrations were optimized and in steady state, serum factors accelerated the rate of multiplication of a normal cell population. The same set of nutrients that supported a maximal rate of multiplication in the presence of serum factors supported the maintenance of non-proliferating cells in the absence of serum factors. Therefore, under this condition, serum factors are required for cell division and play a purely regulatory iole in multiplication of the cell population. The quantitative requirement for 18 nutrients of 29 that were examined was significantly higher (P < 0.001) for cell multiplication in the presence of serum factors than for cell maintenance in the absence of serum factors. This indicated specific nutrients that may be quantitatively important in cell division processes as well as in cell maintenance. The quantitative requirement for Ca²⁺, Mg²⁺, K⁺, Pi, and 2-oxocarboxylic acid for cell multiplication was modified by serum factors and other purified growth factors. The requirement for over 30 other nutrients could not clearly be related to the level of serum factors in the medium. Serum factors also determined the Ca²⁺, K⁺, and 2-oxocarboxylic acid requirement for maintenance of non-proliferating cells. Therefore, when either Ca²⁺, K⁺, or 2-oxocarboxylic acid concentration was limiting, factors in serum played a role as cell “survival or maintenance” factors in addition to their role in cell division as “growth regulatory” factors. However, with equivalent levels of serum factors in the medium, the requirement for Ca²⁺, K⁺, and 2-oxocarboxylic acids was still much higher for multiplication than for maintenance. Kinetic analysis revealed that the concentrations of individual nutrients modify the quantitative requirement for others for cell multiplication in a specific pattern. Thus, specific quantitative relationships among different nutrients in the medium are important in the control of the multiplication rate of the cell population. When all nutrient concentrations were optimal for multiplication of normal cells, the multiplication response of SV40-virus-transformed cells to serum factors was similar to that of normal cells. When serum factors were held constant, transformed cells required significantly less (P < 0.001) of 12 of the 26 nutrients examined. Therefore, the transformed cells only have a growth advantage when the external concentration of specific nutrients limits the multiplication rate of normal cells. Taken together, the results suggest that the control of cell multiplication is intimately related to external concentrations of nutrients. Specific growth regulatory factors may stimulate cell proliferation by modification of the response of normal cells to nutrients. Transforming agents may confer a selective growth advantage on cells by a constitutive alteration of their response to extracellular nutrients.     

Effect of low extracellular Ca2+ on growth spreading area,cytoplasmic Ca2+ concentration,and intracellular pH in normal and transformed human fibroblasts

The transformation of certain cells reduces the requirement of extracellular Ca²⁺ for growth. The SV-40 transformed human lung fibroblasts, WI-38 VA13, require less Ca²⁺ than normal WI-38 cells. Spreading area of normal cells decreases when cultured in 10 μM Ca²⁺ medium. Intracellular calcium concentration ([Ca²⁺]_i), of the normal and transformed cells cultured in 10μM and 2 mM Ca²⁺ media was measured by the fluorescence microscope technique using fura-2 as a probe. The [Ca²⁺], is measured in the resting state and during mobilization by serum or bradykinin stimulation. The lowering of extracellular calcium concentration results in a decrease in the resting state [Ca²⁺],_i of both normal and transformed cells. Although the total decrease in [Ca²⁺]_i is the same for both cell, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca²⁺ reduces the number of cells responsive to the serum or bradykinin stimulation and decreases the peak [Ca²⁺]_i value in both cells. In addition, we investigated, using BCECF as a fluorecent probe, the intracellular pH (pH_i) of normal and transformed cells maintained at low and normal Ca²⁺. The low Ca²⁺ condition makes pH_i acidic in normal cells but not in transformed cells. The acidification of the normal cell is accompanied by a decrease in the spreading area of the cells. The decrease of the cell attacment, followed by the reduced spreading area, induced the acidic pH_i. These results suggest that the reduced Ca²⁺ requirement of transformed cells for growth is related to the mechanism of pH_i regulation rather than Ca²⁺ homeostasis and, possibly, to the anchorage-independent growth, which is a unique feature of transformed cells. © 1993 Wiley-Liss, Inc.     

Calcium effects on epidermal growth factor receptor-mediated endocytosis in normal and SV40-transformed human fibroblasts

Lowering of extracellular Ca²⁺ levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus₄₀(SV₄₀)-transformed WI38 cells do not exhibit this Ca²⁺-dependent arrest. One possibility for this difference in Ca²⁺ requirement is that extracellular or surface membrane-bound Ca²⁺ may be required for growth factor receptor-mediated endocytosis and this Ca²⁺ requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca²⁺ in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV₄₀ WI38 cell. The binding of [¹²⁵I]EGF to the cell surface is not significantly altered by lowering of Ca²⁺ to 10^?5-M levels in either the normal or transformed cell. At this Ca²⁺ level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca²⁺ deprivation. Degradation of the EGF-receptor complex is also sensitive to Ca²⁺. A twofold reduction in the rate of release of acid-soluble ¹²⁵I occurs in the normal but not the transformed cell under conditions of lowered medium Ca²⁺. In contrast, 2-chloro-10-3-aminopropyl phenothiazine (CP), an inhibitor of the Ca²⁺-dependent regulator protein calmodulin, causes an inhibition of [¹²⁵I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of [¹²⁵I]EGF binding to the cell surface receptor of the transformed cell but not the normal cell.     

Mg2+-sensitive alterations in Ca2+ regulation associated with cell transformation

The intracellular Ca²⁺ content of nontransformed Balb/c3T3 cells is two to three times higher than that of a spontaneously transformed derivative. Depriving either cell type of extracellular Mg²⁺ causes a 2- to 3-fold increase in their Ca²⁺ content over a 24-hr period. Restoring Mg²⁺ to the medium decreases the Ca²⁺ content of the cells to their original values in about the same time. The increase in Ca²⁺ content is not blocked by cycloheximide suggesting that normal rates of protein synthesis are not required to produce this effect. Mg²⁺ deprivation also decreases the initial rate of Ca²⁺ efflux from the transformed cells and increases the size of the slowly exchanging fraction of Ca²⁺ to the levels found in the nontransformed cells. Since Mg²⁺ deprivation normalizes the appearance and growth behavior of the transformed cells, the possible intermediary role of Ca²⁺ in this normalization was studied. Large changes in extracellular Ca²⁺ produced large changes in the Ca²⁺ content of the transformed cells with little change in appearance or thymidine incorporation rate. Ca²⁺ deprivation did inhibit thymidine incorporation in early passage nontransformed cells; however with repeated passage, this effect decreased, as did the Ca²⁺ content of these cells. The possible role of Mg²⁺ in regulating cellular Ca²⁺ content and distribution is discussed, as is the relation of Ca²⁺ content and distribution to the development of the transformed state.     

Epidermal growth factor modulates extracellular Ca requirement for multiplication of normal human skin fibroblasts

Epidermal growth factor (EGF) at 10 ng/ml reduces by over 50-fold the extracellular Ca²⁺ required for multiplication of normal human skin fibroblasts. Therefore, a Ca²⁺-related process may play a central role in the mechanism by which EGF exerts its effect on cell multiplication.     

Epidermal growth factor modulates extracellular Ca2+ requirement for multiplication of normal human skin fibroblasts.

Epidermal growth factor (EGF) at 10 ng/ml reduces by over 50-fold the extracellular Ca²⁺ required for multiplication of normal human skin fibroblasts. Therefore, a Ca²⁺-related process may play a central role in the mechanism by which EGF exerts its effect on cell multiplication.     

Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferation

Abstract. Bone tissue in the adult is continuously being remodelled, and overall bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation. Adequate osteoblastic proliferation is essential for both appropriate formation and for regulation of resorption, and thereby the maintenance of bone remodelling equilibrium. Objectives: Here, we have investigated the roles of melastatin‐like transient receptor potential 6 and 7 (TRPM6, TRPM7), which are calcium (Ca²⁺) and magnesium (Mg²⁺) conducting channels, during proliferation of human osteoblasts. Results: Genetic expression of TRPM6 and TRPM7 was shown in human osteoblast‐like MG‐63, SaOS and U2‐OS cells, and reduction of extracellular Mg²⁺ or Ca²⁺ led to a decrease of cell proliferation. Concomitant reduction of both ions further accentuated reduction of cell proliferation. Expression of TRPM7 channels was increased under conditions of reduced extracellular Mg²⁺ and Ca²⁺ levels whereas expression of TRPM6 was not modified, suggesting compensatory mechanisms afforded by TRPM7 in order to maintain intracellular ion homeostasis. Pre‐incubation of cells in reduced extracellular Mg²⁺ conditions led to activation of Ca²⁺ and Mg²⁺ influx. Reduction of TRPM7 expression by specific siRNA prevented latter influx and inhibited cell proliferation. Conclusions: Our results indicate that extracellular Mg²⁺ and Ca²⁺ deficiency reduces the proliferation of human osteoblastic cells. Expression and activity of TRPM7 is modulated by extracellular Mg²⁺ and Ca²⁺ availability, indicating that TRPM7 channels are involved in intracellular ion homeostasis and proliferation of osteoblasts.     

Increase in Serum Ca2+/Mg2+ Ratio Promotes Proliferation of Prostate Cancer Cells by Activating TRPM7 Channels

TRPM7 is a novel magnesium-nucleotide-regulated metal current (MagNuM) channel that is regulated by serum Mg²⁺ concentrations. Changes in Mg²⁺ concentration have been shown to alter cell proliferation in various cells; however, the mechanism and the ion channel(s) involved have not yet been identified. Here we demonstrate that TRPM7 is expressed in control and prostate cancer cells. Supplementation of intracellular Mg-ATP or addition of external 2-aminoethoxydiphenyl borate inhibited MagNuM currents. Furthermore, silencing of TRPM7 inhibited whereas overexpression of TRPM7 increased endogenous MagNuM currents, suggesting that these currents are dependent on TRPM7. Importantly, although an increase in the serum Ca²⁺/Mg²⁺ ratio facilitated Ca²⁺ influx in both control and prostate cancer cells, a significantly higher Ca²⁺ influx was observed in prostate cancer cells. TRPM7 expression was also increased in cancer cells, but its expression was not dependent on the Ca²⁺/Mg²⁺ ratio per se. Additionally, an increase in the extracellular Ca²⁺/Mg²⁺ ratio led to a significant increase in cell proliferation of prostate cancer cells when compared with control cells. Consistent with these results, age-matched prostate cancer patients also showed a subsequent increase in the Ca²⁺/Mg²⁺ ratio and TRPM7 expression. Altogether, we provide evidence that the TRPM7 channel has an important role in prostate cancer and have identified that the Ca²⁺/Mg²⁺ ratio could be essential for the initiation/progression of prostate cancer.     

Magnesium-Dependent stimulation of protein synthesis by the insulin mimic,pervanadate

The insulin mimic, peroxide of vanadate (pervanadate), stimulated ³⁵S-methionine incorporation into Xenopus oocyte protein in a Mg²⁺-dependent manner. Reducing the extracellular Mg²⁺ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg²⁺ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg²⁺ to 0.01 mM inhibited about 50% of the insulinstimulated component of methionine incorporation. Mg²⁺ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn²⁺ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg²⁺. Reducing extracellular Ca²⁺ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 m?I droplet of buffer containing the Mg²⁺ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg²⁺ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg²⁺. In contrast, apparent extracellular Mg²⁺ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg²⁺, but not Ca²⁺, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg²⁺, and this change in membrane transport may be an early event in signalling the increase in translation. © 1995 Wiley-Liss, Inc.     

The role of magnesium in regulating CCK-8-evoked secretory responses in the exocrine rat pancreas

This study investigates the effect of magnesium (Mg²⁺) on the secretory responses and the mobilization of calcium (Ca²⁺) and Mg²⁺ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10^–10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg²⁺ [Mg²⁺]_o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg²⁺]_o Similar effects of perturbation of [Mg²⁺]_o on amylase secretion and ⁴⁵Ca²⁺ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca²⁺ concentration [Ca²⁺]_i in zero and normal [Mg²⁺]_o compared to a much reduced response in elevated [Mg²⁺]_o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB₂ cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca²⁺]_i. In magfura-2-loaded acinar cells CCK-8 (10^–8 M) stimulated an initial transient rise in intracellular free Mg²⁺ concentration [Mg²⁺]_i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na⁺ was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg²⁺ resulted in an elevation in [Mg²⁺]_i. Upon stimulation with CCK-8, [Mg²⁺]_i. decreased only slightly compared with the response obtained in normal [Mg²⁺]_o. CCK-8 caused a net efflux of Mg²⁺ in pancreatic segments; this effect was abolished when extracellular sodium [Na⁺]_o was replaced with either NMDG or choline. The results indicate that Mg²⁺ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca²⁺ mobilization. Moreover, the movement of Mg²⁺ in pancreatic acinar cells is dependent upon extracellular Na⁺.     

Insulin restores expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats

The effects of extracellular Mg²⁺ on both dynamic changes of [Ca²⁺]_i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca²⁺ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca²⁺]_i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca²⁺]_i (150 nM) for several hours. The initial [Ca²⁺]_i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca²⁺]_i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg²⁺ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg²⁺, caused an increase in basal [Mg²⁺]_i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg²⁺, 1 M thapsigargin induces, in 10 mM Mg²⁺, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca²⁺]_i and a lower Ca²⁺ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca²⁺]_i which was inhibited by high extracellular and intracellular Mg²⁺.     

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2–3 nmol Mg²⁺/mg protein or ~10% of the total cellular Mg²⁺ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg²⁺ release, a quantitatively similar amount of Ca²⁺ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg²⁺:1Ca²⁺. Calcium induced Mg²⁺ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg²⁺:1Ca²⁺ exchange ratio. The uptake of Ca²⁺ for the release of Mg²⁺ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP⁺) electrode or ³H-TPP⁺. Collapsing the Δψ by high concentrations of TPP⁺ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca²⁺-induced Mg²⁺ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca²⁺ uptake and Mg²⁺ release. These data demonstrate the operation of an electroneutral Ca²⁺/Mg²⁺ exchanger which represents a novel pathway for Ca²⁺ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.     

Magnesium depletion causes growth inhibition,reduced expression of cyclin D1, and increased expression of P27KIP1 in normal but not in transformed mammary epithelial cells

In this study, we have evaluated the effects of extracellular magnesium restriction on the growth and cell cycle parameters of normal (HC11) and transformed (MCF-7) breast epithelial cell lines. Cells were incubated in medium with different concentrations of Mg²⁺ (from 0.5 to 0 mM) and the growth rates were determined by [³H]-thymidine incorporation and cell counting. The growth of the HC11 cells was drastically inhibited by Mg²⁺ depletion whereas the MCF-7 cells were only slightly inhibited (about 50% and 15%, respectively, after incubation in 0.05 mM Mg for 48 h). Cell cycle analyses showed a decrease in the percentage of cells in the S phase when both cell lines were incubated at low Mg²⁺ concentration. However, while the percentage of cells in both the G0/G1 and G2/M phases was increased in the HC11 cells, only the percentage of cells in the G2/M phase was increased in the MCF-7 cell line. Extracellular magnesium depletion was associated with increased expression of the cyclin-dependent kinase inhibitor p27^Kip1 and decreased expression of cyclin D1 in the HC11 but not in the MCF-7 cells. We also demonstrated that Mg²⁺ depletion does not inhibit kinase activities in the normal HC11 cells and that Mg²⁺-restricted HC11 cells are still responsive to the epidermal growth factor (EGF)- and insulin-mediated stimulation of cell growth. These data suggest that normal but not transformed mammary epithelial cells are inhibited by extracellular Mg²⁺ restriction and that this effect might be mediated by changes in the levels of expression of both cyclin D1 and p27^Kip1. J. Cell. Physiol. 180:245–254, 1999. © 1999 Wiley-Liss, Inc.     

Effects of salicylate and zinc deficiency on the serum concentrations of magnesium,calcium, and parathyroid hormone

Oral application of 700 mg/kg salicylic acid to pregnant and nonpregnant female rats caused an increase of serum Mg²⁺ and a decrease of serum Ca²⁺ concentration. The salicylate effect was drastically enhanced by Zn deficiency. The increase in serum Mg²⁺ is probably caused by the nephrotoxicity of salicylate. The decrease of serum Ca²⁺ concentration is combined with an increase of parathyroid hormone concentration in serum. Probably, salicylate and Zn deficiency inhibit Ca²⁺ mobilization by parathyroid hormone in bone.     

Alkalinization Stimulates Ca2+-Dependent Spreading During the Activation of Resting Lens Epithelial Cells In Primary Culture

Lens epithelium, when attached to its natural substratum, the lens capsule, can be maintained in culture for more than 2 weeks in a simple HEPES- and EDTA-buffered salt solution (HBS). In HBS, the epithelium shows the same characteristic phenomena of locomotion, initial retraction and respreading which in MEM plus serum precedes the inception of DNA synthesis. These phenomena have been shown to be dependent on extracellular Ca²⁺. 0·05 mM Ca²⁺ is necessary for maintaining cell-to-cell contacts of the in vivo epithelium. Higher concentrations of Ca²⁺ cause the epithelium to retract initially. In contrast, Mg²⁺ greatly favours cell-substratum interactions leading to the formation of lamellopodia and an initial spreading of the epithelium. After some hours in culture the epithelium changes markedly in response to extracellular Ca²⁺ and Mg²⁺; it respreads and flattens in the presence of Ca²⁺, while Mg²⁺ becomes less effective in maintaining cell-to-substratum contacts. Mg²⁺-dependent initial spreading is promoted at pH values near 7·0 but the Ca²⁺-dependent respreading requires an alkalinization of the salt solution.     

Activation of the Ca2+ “receptor” on the osteoclast by Ni2+ elicits cytosolic Ca2+ signals: Evidence for receptor activation and inactivation,intracellular Ca2+ redistribution,and divalent cation modulation

Earlier studies have demonstrated that a high (mM) extracellular Ca²⁺ concentration triggers intracellular [Ca²⁺] signals with a consequent inhibition of bone resorptive activity. We now report that micromolar concentrations of the divalent cation, Ni²⁺, elicited rapid and concentration-dependent elevations of cytosolic [Ca²⁺]. The peak change in cytosolic [Ca²⁺] increased monotonically with the application of [Ni²⁺] in the 50–5,000 μM range in solutions containing 1.25 mM-[Ca²⁺] and 0.8 mM-[Mg²⁺]. The resulting concentration-response function suggested Ni²⁺-induced activation of a single class of binding site (Hill coefficient = 1). The triggering process also exhibited a concentration-dependent inactivation in which conditioning Ni²⁺ applications in the range 5–1,500 μM-[Ni²⁺] inhibited subsequent responses to a maximally effective [Ni²⁺] of 5,000 μM. Ni²⁺-induced cytosolic [Ca²⁺] responses were not dependent on extracellular [Ca²⁺]. Thus, when 5,000 μM-[Ni²⁺] was applied to osteoclasts in Ca²⁺-free, ethylene glycol bis-(aminoethyl ether) tetraacetic acid (EGTA)-containing medium (≤5 nM-[Ca²⁺] and 0.8 mM-[Mg²⁺]), cytosolic [Ca²⁺] responses resembled those obtained in the presence of 1.25 mM-[Ca²⁺]. Prior depletion of intracellular Ca²⁺ stores by ionomycin prevented Ni²⁺-induced cytosolic [Ca²⁺] responses, suggesting a major role for intracellular Ca²⁺ redistribution in the response to Ni²⁺. The effects of Ni²⁺ were also modulated by the extracellular concentration of the divalent cations, Ca²⁺ and Mg²⁺. When these cations were not added to the culture medium (0 μM-[Ca²⁺] and [Mg²⁺]), even low [Ni²⁺] ranging between 5 pM and 50 μM elicited progressively larger cytosolic [Ca²⁺] transients. However, the response magnitude decreased at higher, 250–5,000 μM-[Ni²⁺], resulting in a “hooked” concentration-response curve. Furthermore, increasing extracellular [Mg²⁺] or [Ca²⁺] (0–1 mM) diminished the response to 50 μM-[Ni²⁺], a concentration on the rising phase of the “hook.” Similar increases (0–10 mM) in extracellular [Mg²⁺] or [Ca²⁺] increased the response to 5,000 μM-[Ni²⁺], a concentration on the falling phase of the “hook”. These findings are consistent with the existence of a membrane receptor strongly sensitive to Ni²⁺ as well as the divalent cations, Ca²⁺ and Mg²⁺. Receptor occupancy apparently activates intracellular Ca²⁺ release followed by inactivation. Furthermore, repriming is independent of intracellular Ca²⁺ stores, suggesting that such inactivation operates at a transduction step between receptor occupancy and intracellular Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

Mag-indo1 Affinity for Ca, Compartmentalization and Binding to Proteins: The Challenge of Measuring Mg Concentrations in Living Cells

A physicochemical study of the Mag-indo1 binding to Ca²⁺ in solution showed that: (i) the characteristic fluorescence spectra of Ca²⁺-bound and Mg²⁺-bound Mag-indo1 are identical; (ii) two successive equilibria occur for increasing Ca²⁺ concentrations; and (iii) the value of the dissociation constant of the first one, as determined by using a probe dilution protocol, amounts to 780 nM. In order to investigate the fluorescence level of Mag-indo1 trapped in cell organelles, fluorescence spectra of Mag-indo1-loaded fibroblasts were recorded before and after a digitonin permeabilization. Their resolution into cation-bound, protein-bound, and free Mag-indo1 characteristic spectra allowed measurement of the fluorescence intensities of these species. The intensities emitted from whole cells were compared to those emitted from organelles (assumed to be endoplasmic reticulum according to a DiOC₆ loading). The cation-bound Mag-indo1 fluorescence resulted partially (20 to 50%) from the cytosol for 30% of the cells, and totally from compartments for 70% of the cells. We found a concentration value of 500 nM for compartmentalized Ca²⁺ and concluded that the Mag-indo1 binding to Ca²⁺ is likely to affect drastically the Mg²⁺ concentration measurements in cells. Moreover, we showed that the amount variation of protein-bound Mag-indo1 also affects Mg²⁺ measurements when using the two-wavelength ratio method.     

Magnesium-dependent induction of phagocytosis-associated chemiluminescence of adherent human polymorphonuclear leukocytes by non-opsonized zymosan

A severe dysfunction in the cellular response of human polymorphonuclear leukocytes (PMNL) to non-opsonized zymosan was observed under a deficiency of extracellular Mg²⁺. The phagocytosis-association native (luminol-independent) luminescence (NL), as well as luminol-dependent luminescence (LDL) (detected simultaneously and discriminated by spectral methods), was strongely inhibited. Apart from a general decrease of total light production, a Mg²⁺-concentration-dependent delay of the maximum of NL and LDL was observed. A disorder in recruitment of activated membrane-bound NADPH-oxidase of PMNL is suggested. The presence of extracellular Ca²⁺ did not compensate for the Mg²⁺ deficit. In the presence of Mg²⁺ only a slight Ca²⁺-dependent reduction of NL was obtained, but Ca²⁺ seemed to selectively promote LDL. This may indicate a positive influence of Ca²⁺ on the myeloperoxidase release from the cells. Experiments with the metalions-chelating agents EDTA and EGTA, which complex Mg²⁺ to differing extents, confirmed the important role of Mg²⁺ in PMNL-activation by non-opsonized zymosan.     

Divalent regulation and intersubunit interactions of human Connexin26 (Cx26) hemichannels

Control of plasma membrane connexin hemichannel opening is indispensable, and is achieved by physiological extracellular divalent ion concentrations. Here, we explore the differences between regulation by Ca²⁺ and Mg²⁺ of human connexin26 (hCx26) hemichannels and the role of a specific interaction in regulation by Ca²⁺. To effect hemichannel closure, the apparent affinity of Ca²⁺ (0.33 mM) is higher than for Mg²⁺ (1.8 mM). Hemichannel closure is accelerated by physiological Ca²⁺ concentrations, but non-physiological concentrations of extracellular Mg²⁺ are required for this effect. Our recent report provided evidence that extracellular Ca²⁺ facilitates hCx26 hemichannel closing by disrupting a salt bridge interaction between positions D50 and K61 that stabilizes the open state. New evidence from mutant cycle analysis indicates that D50 also interacts with Q48. We find that the D50-Q48 interaction contributes to stabilization of the open state, but that it is relatively insensitive to disruption by extracellular Ca²⁺ compared with the D50-K61 interaction.     

Calcium Binding to Plasma Membranes from Normal and SV40 Transformed Hamster Lymphocytes

Abstract

The calcium binding properties of isolated plasma membranes from normal and SV40 transformed hamster lymphocytes were compared over the Ca²⁺ concentration range of 10^?5M to 5 × 10^?3M and at physiological ionic strength. At all Ca²⁺ concentrations, normal membranes bound more Ca²⁺ than tumor membranes; at blood Ca²⁺ levels (1–2 mM) plasma membranes of normal cells bind twice as much as membranes from tumor cells. Normal plasma membranes demonstrated positive cooperative Ca²⁺ binding whereas tumor membranes displayed non-interacting Ca²⁺-binding sites. Ca²⁺ binding to both membranes was insensitive to Mg²⁺ (0.1 to 2.5 mM). A pH shift from 7 to 6 resulted in a 70% decrease of normal membrane-bound Ca²⁺ compared to a 40% decrease observed with tumor membranes. Extracellular surface Ca²⁺ binding to intact cells was also studied after a 72-hour equilibration of cells with ⁴⁵Ca²⁺ and with ethylene-glycol-bis-(β-amino-ethyl ether) N, N′-tetraacetate chelation as marker for surface Ca²⁺. Tumor cell surface Ca²⁺ binding was only 10% of that observed with quiescent lymphocytes. Normal lymphocytes stimulated to divide with phytohemagglutinin also showed a decreased level of surface Ca²⁺ (50%). However, plasma membranes isolated from non-dividing and phytohemagglutinin-stimulated lymphocytes exhibited equivalent Ca²⁺ binding.