Magnesium–calcium signalling in rat parotid acinar cells: effects of acetylcholine

This study investigated the effects of extracellular Mg²⁺ ([Mg²⁺]_o) on basal and acetylcholine (ACh)-evoked amylase secretion and intracellular free Ca²⁺ ([Ca²⁺]_i) in rat parotid acinar cells. In a medium containing 1.1 mM [Mg²⁺]_o, ACh evoked significant increases in amylase secretion and [Ca²⁺]_i. Either low (0 mM) or elevated (5 and 10 mM) [Mg²⁺]_o attenuated ACh-evoked responses. In a nominally Ca²⁺ free medium, elevated [Mg²⁺]_o attenuated basal and ACh-evoked amylase secretion and [Ca²⁺]_i. In parotid acinar cells incubated with either 0, 1.1, 5 or 10 mM [Mg²⁺]_o, ACh evoked a gradual decrease in [Mg²⁺]_i. These results indicate that the ACh-evoked Mg²⁺ efflux is an active process since Mg²⁺ has to move against its gradient. Either lidocaine, amiloride, N-methyl-d-glucamine, quinidine, dinitrophenol or bumetanide can elevate [Mg²⁺]_i above basal level. In the presence of these membrane transport inhibitors, ACh still evoked a decrease in [Mg²⁺]_i but the response was less pronounced with either [Na⁺]_o removal or in the presence of either amiloride or quinidine. These results indicate marked interactions between Ca²⁺ and Mg²⁺ signalling in parotid acinar cells and that ACh-evoked Mg²⁺ transport was not dependent upon [Na⁺]_o.     

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⁺.     

Magnesium homeostasis in cardiac cells

Several aspects of Mg²⁺ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg²⁺ indicator, FURAPTRA. The concentration of cytosolic Mg²⁺ ([Mg²⁺]_i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg²⁺]_i below electrochemical equilibrium, we manipulated the Na⁺ gradient and assessed the effects on [Mg²⁺]_i. When extracellular Na⁺ was removed, [Mg²⁺]_i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg²⁺]_i, which was dependent upon extracellular Ca²⁺, was observed when intracellular Na⁺ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca²⁺ can modulate [Mg²⁺]_i. In addition, removing extracellular Na⁺ caused a decrease in intracellular pH (pH_i), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca²⁺ and H⁺ interact intracellularly. Since changes in the Na⁺ gradient can also alter pH_i, we questioned whether pH can modulate [Mg²⁺]_i. pH_i was manipulated by the NH₄Cl prepulse method. NH₄ ⁺-evoked changes in pH_i, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg²⁺]_i; [Mg²⁺]_i changed by –0.16 mM/unit pH. These NH₄ ⁺-evoked changes in [Mg²⁺]_i were not caused by movements of Mg₂₊ or Ca²⁺ across the sarcolemma or by changes in cytosolic Ca²⁺. Additionally, pH_i was manipulated by changing extracellular pH (pH_o). When pH_o was decreased from 7.4 to 6.3, pH_i decreased by 0.64 units and [Mg₂₊]_i increased by 0.12 mM; in contrast, when pH_o was raised from 7.4 to 8.3, pH_i increased by 0.6 units and [Mg²⁺]_i did not change significantly. The results of our investigations suggest that Ca ²⁺ and H⁺ can modulate [Mg²⁺]_i, probably by affecting cytosolic Mg²⁺ binding and/or subcellular Mg²⁺ transport and that such redistribution of intracellular Mg²⁺ may play an important role in Mg²⁺ homeostasis in cardiac cells.     

Metabolic Inhibition Strongly Inhibits Na-Dependent Mg Efflux in Rat Ventricular Myocytes

We measured intracellular Mg²⁺ concentration ([Mg²⁺]_i) in rat ventricular myocytes using the fluorescent indicator furaptra (25°C). In normally energized cells loaded with Mg²⁺, the introduction of extracellular Na⁺ induced a rapid decrease in [Mg²⁺]_i: the initial rate of decrease in [Mg²⁺]_i (initial Δ[Mg²⁺]_i/Δt) is thought to represent the rate of Na⁺-dependent Mg²⁺ efflux (putative Na⁺/Mg²⁺ exchange). To determine whether Mg²⁺ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na⁺ gradient, we estimated the initial Δ[Mg²⁺]_i/Δt after metabolic inhibition. In the absence of extracellular Na⁺ and Ca²⁺, treatment of the cells with 1 μM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg²⁺]_i from ∼0.9 mM to ∼2.5 mM in a period of 5-8 min (probably because of breakdown of MgATP and release of Mg²⁺) and cell shortening to ∼50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg²⁺]_i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for ≥90 min. The initial Δ[Mg²⁺]_i/Δt was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59-85 min), a significant decrease in the initial Δ[Mg²⁺]_i/Δt (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na⁺ concentration ([Na⁺]_i) estimated with a Na⁺ indicator sodium-binding benzofuran isophthalate was, on average, 5.0-10.5 mM during the time required for the initial Δ[Mg²⁺]_i/Δt measurements, which is well below the [Na⁺]_i level for half inhibition of the Mg²⁺ efflux (∼40 mM). Normalization of intracellular pH using 10 μM nigericin, a H⁺ ionophore, did not reverse the inhibition of the Mg²⁺ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (∼0.4 mM estimated indirectly in the this study), rather than elevation of [Na⁺]_i or intracellular acidosis, inhibits the Mg²⁺ efflux, suggesting the absolute necessity of ATP for the Na⁺/Mg²⁺ exchange.     

Ca regulates the subcellular localization of adenomatous polyposis coli tumor suppressor protein

Microtubule (MT) plus-end tracking proteins (+TIPs) are involved in the regulation of MT plus-end dynamics and stabilization. It was reported previously that an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by disruption of the plasma membrane stimulates rearrangement of MTs [T. Togo, Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site, J. Cell Sci. 119 (2006) 2780-2786], suggesting that some +TIPs are regulated by Ca²⁺. In the present study, the behavior of adenomatous polyposis coli (APC) following an increase in [Ca²⁺]_i was observed using Xenopus A6 epithelial cell expressing GFP-tagged APC. An increase in [Ca²⁺]_i by cell membrane disruption or by ionomycin treatment induced dissociation of APC without depolymerizing MTs. Inhibition of a tyrosine kinase and GSK-3β suppressed APC dissociation upon an increase in [Ca²⁺]_i. Western blotting analysis showed that Ca²⁺ transients activated GSK-3β through a tyrosine kinase. These results suggest that Ca²⁺ stimulates redistribution of APC through a tyrosine kinase- and GSK-3β-dependent pathway.     

Potentiation of Carbachol-Induced Ca2+ Release by Peroxynitrite in Human Neuroblastoma SH-SY5Y Cells

We have investigated the effect of 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, on carbachol-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human neuroblastoma SH-SY5Y cells by means of single cell imaging of [Ca²⁺]_i. SIN-1 potentiated carbachol-induced [Ca²⁺]_i rise regardless of external Ca²⁺, and the potentiation was completely inhibited by superoxide dismutase, indicating that peroxynitrite may enhance Ca²⁺ release from intracellular stores. On the other hand, SIN-1 reduced carbachol-induced inositol 1,4,5-trisphosphate (IP₃) formation. Genistein, a tyrosine kinase inhibitor, potentiated carbachol-induced rise of [Ca²⁺]_i regardless of external Ca²⁺. These results suggest that peroxynitrite may potentiate the release of Ca²⁺ from intracellular stores through the perturbation of regulation in tyrosine phosphorylation-dephosphorylation system.     

Imipramine inhibition of TRPM‐like plasmalemmal Mg2+ transport in vascular smooth muscle cells

Depression is associated with vascular disease, such as myocardial infarction and stroke. Pharmacological treatments may contribute to this association. On the other hand, Mg²⁺ deficiency is also known to be a risk factor for the same category of diseases. In the present study, we examined the effect of imipramine on Mg²⁺ homeostasis in vascular smooth muscle, especially via melastatin‐type transient receptor potential (TRPM)‐like Mg²⁺‐permeable channels. The intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured using ³¹P‐nuclear magnetic resonance (NMR) in porcine carotid arteries that express both TRPM6 and TRPM7, the latter being predominant. pH_i and intracellular phosphorus compounds were simultaneously monitored. To rule out Na⁺‐dependent Mg²⁺ transport, and to facilitate the activity of Mg²⁺‐permeable channels, experiments were carried out in the absence of Na⁺ and Ca²⁺. Changing the extracellular Mg²⁺ concentration to 0 and 6 mM significantly decreased and increased [Mg²⁺]_i, respectively, in a time‐dependent manner. Imipramine statistically significantly attenuated both of the bi‐directional [Mg²⁺]_i changes under the Na⁺‐ and Ca²⁺‐free conditions. This inhibitory effect was comparable in influx, and much more potent in efflux to that of 2‐aminoethoxydiphenyl borate, a well‐known blocker of TRPM7, a channel that plays a major role in cellular Mg²⁺ homeostasis. Neither [ATP]_i nor pH_i correlated with changes in [Mg²⁺]_i. The results indicate that imipramine suppresses Mg²⁺‐permeable channels presumably through a direct effect on the channel domain. This inhibitory effect appears to contribute, at least partially, to the link between antidepressants and the risk of vascular diseases.     

Mg2+- and ATP-dependent inhibition of transient receptor potential melastatin 7 by oxidative stress

Transient receptor potential melastatin 7 (TRPM7) is a Ca²⁺- and Mg²⁺-permeable nonselective cation channel that contains a unique carboxyl-terminal serine/threonine protein kinase domain. It has been reported that reactive oxygen species associated with hypoxia or ischemia activate TRPM7 current and then induce Ca²⁺ overload resulting in neuronal cell death in the brain. In this study, we aimed to investigate the molecular mechanisms of TRPM7 regulation by hydrogen peroxide (H₂O₂) using murine TRPM7 expressed in HEK293 cells. Using the whole-cell patch-clamp technique, it was revealed that the TRPM7 current was inhibited, not activated, by the application of H₂O₂ to the extracellular solution. This inhibition was not reversed after washout or treatment with dithiothreitol, suggesting irreversible oxidation of TRPM7 or its regulatory factors by H₂O₂ under whole-cell recording. Application of an electrophile, N-methylmaleimide (NMM), which covalently modifies cysteine residues in proteins, also inhibited TRPM7 current irreversibly. The effects of H₂O₂ and NMM were dependent on free [Mg²⁺]_i; the inhibition was stronger when cells were perfused with higher free [Mg²⁺]_i solutions via pipette. In addition, TRPM7 current was not inhibited by H₂O₂ when millimolar ATP was included in the intracellular solution, even in the presence of substantial free [Mg²⁺]_i, which is sufficient for TRPM7 inhibition by H₂O₂ in the absence of ATP. Moreover, a kinase-deficient mutant of TRPM7 (K1645R) was similarly inhibited by H₂O₂ just like the wild-type TRPM7 in a [Mg²⁺]_i- and [ATP]_i-dependent manner, indicating no involvement of the kinase activity of TRPM7. Thus, these data suggest that oxidative stress inhibits TRPM7 current under pathological conditions that accompany intracellular ATP depletion and free [Mg²⁺]_i elevation.     

Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

The efficient retrieval of synaptic vesicle membrane and cargo in central nerve terminals is dependent on the efficient recruitment of a series of endocytosis modes by different patterns of neuronal activity. During intense neuronal activity the dominant endocytosis mode is activity-dependent endocytosis (ADBE). Triggering of ADBE is linked to calcineurin-mediated dynamin I dephosphorylation since the same stimulation intensities trigger both. Dynamin I dephosphorylation is maximised by a simultaneous inhibition of its kinase glycogen synthase kinase 3 (GSK3) by the protein kinase Akt, however it is unknown how increased neuronal activity is transduced into Akt activation. To address this question we determined how the activity-dependent increases in intracellular free calcium ([Ca²⁺]_i) control activation of Akt. This was achieved using either trains of high frequency action potentials to evoke localised [Ca²⁺]_i increases at active zones, or a calcium ionophore to raise [Ca²⁺]_i uniformly across the nerve terminal. Through the use of either non-specific calcium channel antagonists or intracellular calcium chelators we found that Akt phosphorylation (and subsequent GSK3 phosphorylation) was dependent on localised [Ca²⁺]_i increases at the active zone. In an attempt to determine mechanism, we antagonised either phosphatidylinositol 3-kinase (PI3K) or calmodulin. Activity-dependent phosphorylation of both Akt and GSK3 was arrested on inhibition of PI3K, but not calmodulin. Thus localised calcium influx in central nerve terminals activates PI3K via an unknown calcium sensor to trigger the activity-dependent phosphorylation of Akt and GSK3.     

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⁺.     

Extracellular Mg regulates the intracellular Na concentration in rat sublingual acini

The intracellular free Na⁺ concentration ([Na⁺]_i) increases during muscarinic stimulation in salivary acinar cells. The present study examined in rat sublingual acini the role of extracellular Mg²⁺ in the regulation of the stimulated [Na⁺]_i increase using the fluorescent sodium indicator benzofuran isophthalate (SBFI). The muscarinic induced rise in [Na⁺]_i was approximately 4-fold greater in the absence of extracellular Mg²⁺. When Na⁺ efflux was blocked by the Na⁺,K⁺-ATPase inhibitor ouabain, the stimulated [Na⁺]_i increase was comparable to that seen in an Mg²⁺-free medium. Moreover, ouabain did not add further to the stimulated [Na⁺]_i increase in an Mg²⁺-free medium suggesting that removal of extracellular Mg²⁺ may inhibit the Na⁺ pump. In agreement with this assumption, ouabain-sensitive Na⁺ efflux and rubidium uptake were reduced by extracellular Mg²⁺ depletion. Our results suggest that extracellular Mg²⁺ may regulate [Na⁺]_i in sublingual salivary acinar cells by modulating Na⁺ pump activity.     

Ca2+ release and Ca2+ influx in Chinese hamster ovary cells expressing the cloned mouse B2 bradykinin receptor: tyrosine kinase inhibitor-sensitive and -insensitive processes

A cDNA encoding a mouse B₂ bradykinin (BK) receptor was stably transfected in Chinese hamster ovary (CHO) cells. In two resulting transformants, mouse B₂ BK receptor was found to induce a twofold elevation in the inositol-1,4,5-trisphosphate level. In a pertussis toxin-insensitive manner, BK also produced a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). The initial elevation in [Ca²⁺]_i was abolished by thapsigargin pretreatment in Ca²⁺-free medium. The second phase was dependent on external Ca²⁺. The BK/inositol trisphosphate- and thapsigargin-sensitive Ca²⁺ stores required extracellular Ca²⁺ for refilling. Ca²⁺ influx induced by BK and thapsigargin was confirmed by Mn²⁺ entry through Ca²⁺ influx pathways producing Mn²⁺ quenching. Genistein, a tyrosine kinase inhibitor, partially decreased the BK-induced [Ca²⁺]_i increase during the sustained phase and the rate of Mn²⁺ entry. BK had essentially no effect on the intracellular cyclic AMP level. The results suggest that the mouse B₂ BK receptor couples to phospholipase C in CHO cells and that its activation results in biphasic [Ca²⁺]_i increases, by mobilization of intracellular Ca²⁺ and store-depletion-mediated Ca²⁺ influx, the latter of which is tyrosine phosphorylation-dependent.     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

Orexin A-induced extracellular calcium influx in prefrontal cortex neurons involves L-type calcium channels

Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca²⁺]_i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1 ≈1 μM) induced increase of [Ca²⁺]_i in a dose-dependent manner. This elevation of [Ca²⁺]_i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca²⁺ stores by the endoplasmic reticulum inhibitor thapsigargin (2 μM), [Ca²⁺]_i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca²⁺-free condition, orexin A failed to induce any changes of Ca²⁺ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca²⁺]_i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 μM) and D609 (10 μM), respectively. Selective blockade of L-type Ca²⁺ channels by nifedipine (5 μM) significantly suppressed the elevation of [Ca²⁺]_i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca²⁺]_i in neurons from deep layers of PFC, which depends on extracellular Ca²⁺ influx via L-type Ca²⁺ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.     

Are intracellular ionic concentrations accessible using fluorescent probes? The example of Mag-indo-1

The study of the physicochemical properties of Mag-indo-1, a fluorescent probe used for intracellular magnesium measurements, has shown that in a biological environment the deprotonated form of this probe is in simultaneous equilibrium with a protonated form, a protein and a magnesium-bound form. The complex emission fluorescence spectrum emitted by a single living cell was analyzed using a computerized method, allowing the evaluation of the Mag-indo-1 to the cellular fluorescence. This approach used to evaluate intracellular Mg²⁺ concentration has also shown the variability of the important participation of protein-bound Mag-indo-1 to the cellular fluorescence. Thus the widely used ratioing method, unable to take into account this variability, cannot afford a reliable evaluation of [Mg²⁺]. Whatever the technique used for investigation (microfluorimetry, flow cytometry, etc.) the evaluation of [Mg²⁺]_i using the fluorescent probe Mag-indo-1 requires a method able to quantify, in complex fluorescence, the fluorescence intensity of the forms involved in the equilibrium with Mg²⁺.Abbreviations [Ca²⁺]₁ intracellular calcium concentration - [Mg²⁺]_i intracellular magnesium concentration     

Activation of Phosphatidylinositol-linked Novel D<Subscript>1</Subscript> Dopamine Receptor Contributes to the Calcium Mobilization in Cultured Rat Prefrontal Cortical Astrocytes

Recent evidences indicate the existence of an atypical D₁ dopamine receptor other than traditional D₁ dopamine receptor in the brain that mediates PI hydrolysis via activation of phospholipase C_β (PLC_β). To further understand the basic physiological function of this receptor in brain, the effects of a selective phosphoinositide (PI)-linked D₁ dopamine receptor agonist SKF83959 on cytosolic free calcium concentration ([Ca²⁺]_i) in cultured rat prefrontal cortical astrocytes were investigated by calcium imaging. The results indicated that SKF83959 caused a transient dose-dependent increase in [Ca²⁺]_i. Application of D₁ receptor, but not D₂, α₁ adrenergic, 5-HT receptor, or cholinergic antagonist prevented SKF83959-induced [Ca²⁺]_i rise, indicating that activation of the D₁ dopamine receptor was essential for this response. Increase in [Ca²⁺]_i was a two-step process characterized by an initial increase in [Ca²⁺]_i mediated by release from intracellular stores, supplemented by influx through voltage-gated calcium channels, receptor-operated calcium channels, and capacitative Ca²⁺ entry. Furthermore, SKF83959-stimulated increase in [Ca²⁺]_i was abolished following treatment with a PLC inhibitor. Overall, these results suggested that activation of D₁ receptor by SKF83959 mediates a dose-dependent mobilization of [Ca²⁺]_i via the PLC signaling pathway in cultured rat prefrontal cortical astrocytes.     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca²⁺]_i, Ca²⁺ transients and membrane Ca²⁺ current, I_Ca, in cultured murine HL-1 cardiomyocytes. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca²⁺]_i, Ca²⁺ transients and I_Ca. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca²⁺]_i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca²⁺]_i, and inhibited Ca²⁺ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca²⁺]_i, and Ca²⁺ transients and I_Ca. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca²⁺]_i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca²⁺]_i required for excitation-contraction coupling in cardiomyoctyes.     

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.     

Eicosapentaenoic Acid Enhances Intracellular Free Calcium in Cultured Human EndothelialCells

Eicosapentaenoic acid (EPA) caused elevations of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) measured by fura-2 fluorescence in cultured human endothelial cells. The EPA induced increase in [Ca²⁺]_i could still be observed when either cyclooxygenase or lipoxygenase inhibitors were added. These results suggest that EPA itself rather than its metabolites has direct effects on intracellular Ca²⁺ mobilization, causing the elevation of [Ca²⁺]_i.