Direct measurement of intracellular free magnesium in frog skeletal muscle using magnesium-selective microelectrodes

Mg²⁺-selective microelectrodes have been used to measure the intracellular free Mg²⁺ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 μm were backfilled with the Mg²⁺ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg²⁺. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18–24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements. In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than ?82 mV, the intracellular free Mg²⁺ concentration was 3.8±0.41 (S.E.) mM (n=58) at 22°C. No significant change in the intracellular free Mg²⁺ was observed following extensive (approx. 6 h) incubation in Mg²⁺-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg²⁺]_i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na⁺-free solutions. In depolarized muscle fibers (?23±2.7 mV) treated with 100 mM K⁺, the myoplasmic [Mg²⁺] was 3.7±0.45 (S.E.) mM, n=6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg²⁺. These results point out that [Mg²⁺]_i is not modified under these three different experimental conditions.     

Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.     

Fluorescence signals from the Mg2+/Ca2+ indicator furaptra in frog skeletal muscle fibers.

The fluorescent Mg2+/Ca2+ indicator, furaptra, was injected into single frog skeletal muscle fibers, and the indicator's fluorescence signals were measured and analyzed with particular interest in the free Mg2+ concentration ([Mg2+]) in resting muscle. Based on the fluorescence excitation spectrum of furaptra, the calibrated myoplasmic [Mg2+] level averaged 0.54 mM, if the value of dissociation constant (KD) for Mg2+ obtained in vitro (5.5 mM) was used. However, if the indicator reacts with Mg2+ with a two-fold larger KD in myoplasm, as previously suggested for the furaptra-Ca2+ reaction (M. Konishi, S. Hollingworth, A.B. Harkins, S.M. Baylor. 1991. J. Gen. Physiol. 97:271-301), the calculated [Mg2+] would average 1.1 mM. Thus, the value 1.1 mM probably represents the best estimate from furaptra of [Mg2+] in resting muscle fibers. Extracellular perfusion of muscle fibers with high Mg2+ concentration solution or low Na+ concentration solution did not cause any detectable changes in the [Mg2+]-related furaptra fluorescence within 4 min. The results suggest that the myoplasmic [Mg2+] is highly regulated near the resting level of 1 mM, and that changes only occur with a very slow time course.     

Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells

This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+]i; using mag-fura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018+/-0.009 in a Na+-free medium to 0.73+/-0.3 mM.l cells-1.min-1 in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+]e) according to a typical saturation kinetic (Km value for [Na+]e=24 mM; maximal velocity=11 mM.l cells-1.min-1). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP]i led to an [Mg2+]i decrease that amounted to 375+/-105 microM. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.     

Measurements of intracellular Mg2+ concentration in mouse skeletal muscle fibers with the fluorescent indicator mag-indo-1.

Measurements of intracellular free magnesium concentration ([Mg2+]i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg2+] by a factor of approximately 2. Using the in vivo calibration parameters, the mean resting [Mg2+]i was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg2+]i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.     

Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini.

The regulation of the intracellular free Mg2+ concentration ([Mg2+]i) was monitored in rat sublingual mucous acini using dual wavelength microfluorometry of the Mg(2+)-sensitive dye mag-fura-2. Acini attached to coverslips and superfused continuously with a Mg(2+)-containing medium (0.8 mM) have a steady-state [Mg2+]i of 0.35 +/- 0.01 mM. Adjusting the extracellular Mg2+ concentration to 0 and 10 mM or removing extracellular Na+ did not alter the resting [Mg2+]i. Stimulation with the Ca(2+)-mobilizing, muscarinic agonist, carbachol, induced a sustained increase in [Mg2+]i (approximately 50%; t1/2 < 20 s; Kd approximately 1.5 microM), the magnitude and the duration of which were unchanged in Mg(2+)-depleted medium indicating that the rise in [Mg2+]i was generated by Mg2+ release from an intracellular Mg2+ pool. Forskolin, which increases the intracellular cAMP content, produced a small, transient increase in the [Mg2+]i (< 10%). Muscarinic stimulation in a Ca(2+)-free medium blunted the initial increase in [Mg2+]i by approximately 50%, whereas the sustained increase in [Mg2+]i was lost. When the muscarinic-induced increase in [Ca2+]i was blocked by 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the agonist-sensitive intracellular Ca2+ release pathway, both the initial and the sustained phases of the increase in [Mg2+]i were virtually eliminated. Thapsigargin and 2,5-di-(terbutyl)-1,4-benzohydroquinone, which increase [Ca2+]i by inhibiting microsomal Ca(2+)-ATPase, caused a dramatic increase in [Mg2+]i. Stimulation in a Na(+)-free medium or in the presence of bumetanide, an inhibitor of Na+/K+/2Cl- cotransport, blunted the agonist-induced rise in [Mg2+]i (approximately 50%), whereas ouabain, a Na+,K(+)-ATPase inhibitor, had no significant effect. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler, mobilized an intracellular Mg2+ pool as well. The carbachol-induced increase in [Mg2+]i was markedly inhibited by FCCP (approximately 80%), suggesting that the same pool(s) of Mg2+ were primarily involved. The above results provide strong evidence that Ca(2+)-mobilizing agonists increase cytoplasmic free [Mg2+] by releasing an intracellular pool of Mg2+ that is associated with a rise in the [Na+]i.     

Ca2+-dependent modulation of intracellular Mg2+ concentration with amiloride and KB-R7943 in pig carotid artery

It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mm) decreased [Mg2+]i by approximately 12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by approximately 13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mM) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100-200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Carbachol and KCl-induced changes in intracellular free calcium concentration in isolated, fura-2 loaded smooth-muscle cells from the anterior byssus retractor muscle of Mytilus edulis

Intracellular free calcium concentration [( Ca2+]1) was measured in suspensions of fura-2 loaded smooth-muscle cells isolated from the anterior byssus retractor muscle of Mytilus edulis. Successive application of 5mM carbachol (CCh) and 100mM KCl to the cells transiently elevated [Ca2+]1 from the resting value of 124 +/- 4.5nM (mean +/- S.E., n = 14) to 295 +/- 15.3 and 383 +/- 20.5 nM, respectively. The response to CCh was concentration-dependent with an ED50 of 10(-5) M. Under the microscope, 67 +/- 3.0 and 83 +/- 1.3 % of fura-2 loaded cells contracted on the addition of 5mM CCh and 100mM KCl, respectively. In Ca2+ -free sea water, the CCh induced change in [Ca2+]1 was partially suppressed whereas that induced by KCl was completely abolished, suggesting an agonist-evoked release of stored Ca2+.     

Extracellular ATP-induced inward current in isolated epithelial cells of the endolymphatic sac.

Using whole-cell patch-clamp technique and Fura-2 fluorescence measurement, the presence of ATP-activated ion channels and its dependence on intracellular Ca2+ concentration ([Ca2+]i) in the epithelial cells of the endolymphatic sac were investigated. In zero current-clamp configuration, the average resting membrane potential was -66.8+/-1.3 mV (n=18). Application of 30 microM ATP to the bath induced a rapid membrane depolarization by 43.1+/-2.4 mV (n=18). In voltage-clamp configuration, ATP-induced inward current at holding potential (VH) of -60 mV was 169.7+/-6.3 pA (n=18). The amplitude of ATP-induced currents increased in sigmoidal fashion over the concentration range between 0.3 and 300 microM with a Hill coefficient (n) of 1.2 and a dissociation constant (Kd) of 11.7 microM. The potency order of purinergic analogues in ATP-induced current, which was 2MeSATP>ATPgammas>/=ATP>alpha, beta-ATP>ADP=AMP>/=adenosine=UTP, was consistent with the properties of the P2Y receptor. The independence of the reversal potential of the ATP-induced current from Cl- concentration suggests that the current is carried by a cation channel. The relative ionic permeability ratio of the channel modulated by ATP for cations was Ca2+>Na+>Li+>Ba2+>Cs+=K+. ATP (10 microM) increased [Ca2+]i in an external Ca2+-free solution to a lesser degree than that in the external solution containing 1.13 mM CaCl2. ATP-induced increase in [Ca2+]i can be mimicked by application of ionomycin in a Ca2+-free solution. These results indicate that ATP increases [Ca2+]i through the P2Y receptor with a subsequent activation of the non-selective cation channel, and that these effects of ATP are dependent on [Ca2+]i and extracellular Ca2+.     

Basal intracellular free Mg2+ concentration in smooth muscle cells of guinea pig tenia cecum: intracellular calibration of the fluorescent indicator furaptra

Longitudinal muscle strips dissected from tenia cecum of guinea pig were loaded with the Mg2+ indicator, furaptra, and the relation between the fluorescent ratio signal (R) and cytoplasmic free Mg2+ concentration ([Mg2+]i) was studied in smooth muscle cells at 25 degrees C. After the application of ionophores (4-bromo-A23187, monensin, and nigericin), a small immediate offset of R (deltaRjump) was followed by a slow change in R (deltaRslow), which reached a steady level within 2-5 h. The deltaRjump was independent of Mg2+ concentration in solution ([Mg2+]o), and was thought to be unrelated to the change in [Mg2+]i. The direction of the deltaRslow depended on [Mg2+]o with a reversal at approximately 1 mM [Mg2+]o. The intracellular calibration curve was constructed from the steady levels of deltaRslow, and the dissociation constant was 5.4 mM. With the intracellular calibration curve and correction for the deltaRjump, basal [Mg2+], was estimated to be 0.98 +/- 0.05 mM (mean +/- SE, n = 12). When the same calibration was applied to A7r5 cells and rat ventricular myocytes, estimates of basal [Mg2+]i of these cells were 0.74 +/- 0.02 mM (n = 33) and 1.13 +/- 0.06 mM (n = 9), respectively. These results suggest that the basal [Mg2+] level is approximately 1 mM at least in some types of smooth muscle cells, as generally found in striated muscles.     

Low myoplasmic Mg2+ potentiates calcium release during depolarization of frog skeletal muscle fibers.

The role of intracellular free magnesium concentration ([Mg2+]) in modulating calcium release from the sarcoplasmic reticulum (SR) was studied in voltage-clamped frog cut skeletal muscle fibers equilibrated with cut end solutions containing two calcium indicators, fura-2 and antipyrylazo III (AP III), and various concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor calcium transients, whereas fura-2 fluorescence was used to monitor resting calcium. The rate of release (Rrel) of calcium from the SR was calculated from the calcium transient and found to be increased in low internal [Mg2+]. After correcting for effects of calcium depletion from the SR and normalization to SR content, the mean values of the inactivatable and noninactivatable components of Rrel were increased by 163 and 46%, respectively, in low Mg2+. Independent of normalization to SR content, the ratio of inactivatable to noninactivatable components of Rrel was increased in low internal [Mg2+]. Both observations suggest that internal [Mg2+] preferentially modulates the inactivatable component of Rrel, which is thought to be due to calcium-induced calcium release from the SR. This could also explain the observation that, in low internal [Mg2+], the time to the peak of the calcium transient for a 5-ms depolarizing pulse was not very different from the time to the peak of the delta [Ca2+] for a 10-ms pulse of the same amplitude. Finally, in low internal [Mg2+], the calcium transient elicited by a short depolarizing pulse was in some cases clearly followed by a very slow rise of calcium after the end of the pulse. The observed effects of reduced [Mg2+] on calcium release are consistent with a removal of the inhibition that the normal 1 mM myoplasmic [Mg2+] exerts on calcium release in skeletal muscle fibers.     

Intracellular Mg2+ diffusion within isolated rat skeletal muscle fibers

Intracellular free magnesium concentration ([Mg2+]i) was measured in enzymatically isolated rat skeletal muscle fibers using the fluorescent dye mag-indo-1. The change in [Mg2+]i produced by a local intracellular microinjection of magnesium pidolate (magnesium pyrrolidone carboxylate) was measured at a given distance from the injection site. In one series of experiments this protocol was tested on isolated fibers that were completely embedded into silicone grease: under these conditions, the injection produced an increase in [Mg2+]i that reached a steady level some time following the injection. The time-course of the [Mg2+]i change could be well accounted for by a model of longitudinal diffusion. The mean apparent Mg2+ diffusion coefficient (D(app)) was 188+/-9 microm2 s(-1) (n = 16), approximately four times lower than the value measured in vitro. This reduction likely results from the effects of cytoplasmic viscosity and of Mg2+ binding to low affinity static sites. Another series of measurements was performed on fibers that were either partially or completely free of silicone: under these conditions, the time course of the change in [Mg2+]i was in many cases more complex than predicted by simple diffusion.     

The effect of extracellular potassium on the intracellular potassium ion activity and transmembrane potentials of beating canine cardiac Purkinje fibers

We used open tip microelectrodes containing a K+-sensitive liquid ion exchanger to determine directly the intracellular K+ activity in beating canine cardiac Purkinje fibers. For preparations superfused with Tyrode's solution in which the K+ concentration was 4.0 mM, intracellular K+ activity (ak) was 130.0+/-2.3 mM (mean+/-SE) at 37 degrees C. The calculated K+ equilibrium potential (EK) was -100.6+/- 0.5 mV. Maximum diastolic potential (ED) and resting transmembrane potential (EM) were measured with conventional microelectrodes filled with 3 M KCl and were -90.6+/-0.3 and -84.4+/-0.4 mV, respectively. When [K+]o was decreased to 2.0 mM or increased to 6.0, 10.0, and 16.0 mM, ak remained the same. At [K+]o=2.0, ED was -97.3+/-0.4 and Em - 86.0+/-0.7 mV; at [K+]o=16.0, ED fell to -53.8+/-0.4 mV and Em to the same value. Over this range of values for [K+]o, EK changed from - 119.0+/-0.3 to -63.6+/-0.2 mV. These values for EK are consistent with those previously estimated indirectly by other techniques.     

Competition between Li+ and Mg2+ in neuroblastoma SH-SY5Y cells: a fluorescence and 31P NMR study.

Because Mg2+ and Li+ ions have similar chemical properties, we have hypothesized that Li+/Mg2+ competition for Mg2+ binding sites is the molecular basis for the therapeutic action of lithium in manic-depressive illness. By fluorescence spectroscopy with furaptra-loaded cells, the free intracellular Mg2+ concentration within the intact neuroblastoma cells was found to increase from 0. 39 +/- 0.04 mM to 0.60 +/- 0.04 mM during a 40-min Li+ incubation in which the total intracellular Li+ concentration increased from 0 to 5.5 mM. Our fluorescence microscopy observations of Li+-free and Li+-loaded cells also indicate an increase in free Mg2+ concentration upon Li+ incubation. By 31P NMR, the free intracellular Mg2+ concentrations for Li+-free cells was 0.35 +/- 0. 03 mM and 0.80 +/- 0.04 mM for Li+-loaded cells (final total intracellular Li+ concentration of 16 mM). If a Li+/Mg2+ competition mechanism is present in neuroblastoma cells, an increase in the total intracellular Li+ concentration is expected to result in an increase in the free intracellular Mg2+ concentration, because Li+ displaces Mg2+ from its binding sites within the nerve cell. The fluorescence spectroscopy, fluorescence microscopy, and 31P NMR spectroscopy studies presented here have shown this to be the case.     

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.     

Inositol 1,4,5-trisphosphate increases myoplasmic [Ca2+] in isolated muscle fibers. Depolarization enhances its effects

Inositol 1,4,5-trisphosphate (InsP3) has been proposed as an intracellular messenger which mobilizes calcium from the sarcoplasmic reticulum, during excitation-contraction coupling in skeletal muscle. We have measured the myoplasmic free calcium concentration ([Ca2+]i) by means of calcium selective microelectrodes in intact fibers isolated from Leptodactylus insularis microinjected with InsP3. In muscle fibers bathed in normal Ringer, the mean resting [Ca2+]i was 0.11 +/- 0.01 microM (M +/- SEM, n = 30). The microinjection of 0.3, 0.5 and 1 microM InsP3 induced transient increments in the [Ca2+]i to 0.35 +/- 0.02 microM (n = 9), to 0.53 +/- 0.03 microM (n = 11) and 0.94 +/- 0.06 microM (n = 10) respectively. Microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers incubated in low Ca2+ solution induced increments in [Ca2+]i similar to those observed in fibers bathed with normal Ringer. The microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers partially depolarized with 10 mM [K+]o induced transient enhancements of the resting [Ca2+]i that were greater than the transients observed in the normally polarized muscle. In partially depolarized fibers microinjected with 0.3, 0.5 and 1 microM InsP3, the [Ca2+]i was changed to 1.45 +/- 0.14 microM (n = 20), to 3.37 +/- 0.34 microM (n = 7) and to 7.43 +/- 0.70 microM (n = 6) respectively. In all partially depolarized fibers these increments in [Ca2+]i were associated with local contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+ gradient-dependent Mg2+ transport in smooth muscle cells of guinea pig tenia cecum

Thin strips of guinea pig tenia cecum were loaded with the Mg2+ indicator furaptra, and the indicator fluorescence signals measured in Ca2+-free condition were converted to cytoplasmic-free Mg2+ concentration ([Mg2+]i). Lowering the extracellular Na+ concentration ([Na+]o) caused a reversible increase in [Mg2+]i, consistent with the inhibition of Na+ gradient-dependent extrusion of cellular Mg2+ (Na+-Mg2+ exchange). Curve-fitting analysis indicated that the relation between [Na+]o and the rate of rise in [Mg2+], had a Hill coefficient of approximately 3, a [Na+]o at the half-maximal rate of rise of approximately 30 mM, and a maximal rate of 0.16 +/- 0.01 microM/s (mean +/- SE, n = 6). Depolarization with 56 mM K+ shifted the curve slightly toward higher [Na+]o without significantly changing the maximal rate, suggesting that the Na+-Mg2+ exchange was inhibited by depolarization. The maximal rate would correspond to a flux of 0.15-0.4 pmol/cm2/s, if cytoplasmic Mg2+ buffering power (defined as the ratio of the changes in total Mg2+ and free Mg2+ concentrations) is assumed to be 2-5. Ouabain (1-5 microM) increased the intracellular Na+ concentration, as assessed with fluorescence of SBFI (sodium-binding benzofuran isophthalate, a Na+ indicator), and elevated [Mg2+]i. In ouabain-treated preparations, removal of extracellular Na+ rapidly increased [Mg2+]i, with an initial rate of rise roughly proportional to the degree of the Mg2+ load, and, probably, to the Na+ load caused by ouabain. The enhanced rate of rise in [Mg2+]i (up to approximately 1 microM/s) could be attributed to the Mg2+ influx as a result of the reversed Na+-Mg2+ exchange. Our results support the presence of a reversible and possibly electrogenic Na+-Mg2+ exchange in the smooth muscle cells of tenia cecum.     

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.     

Intracellular-free magnesium in the smooth muscle of guinea pig taenia caeci: a concomitant analysis for magnesium and pH upon sodium removal

This study is concerned with the regulation of intracellular-free Mg2+ concentration ([Mg2+]i) in the smooth muscle of guinea pig taenia caeci. To assess an interaction of Ca2+ on the Na(+)-dependent Mg(2+)- extrusion mechanism (Na(+)-Mg2+ exchange), effects of Na+ removal (N- methyl-D-glucamine substitution) were examined in Ca(2+)-containing solutions. As changes in pHi in Na(+)-free solutions perturb estimation of [Mg2+]i using the single chemical shift only of the beta-ATP peak in 31P NMR (nuclear magnetic resonance) spectra, [Mg2+]i and pHi were concomitantly estimated from the chemical shifts of the gamma- and beta- peaks. When extracellular Na+ was substituted with N-methyl-D- glucamine, [Mg2+]i was reversibly increased. This increase in [Mg2+]i was eliminated in Mg(2+)-free solutions and enhanced in excess Mg2+ solutions. ATP content fluctuated little during removal and readmission of Na+, indicating that [Mg2+]i changes were not induced by Mg2+ release from ATP, and that Mg(2+)-extruding system would not be inhibited by fuel restriction. A slow acidification in Na(+)-free solutions and transient alkalosis by a readmission of Na+ were observed regardless of the extracellular Mg2+ concentration. When the extracellular Ca2+ concentration was increased from normal (2.4 mM) to 12 mM, only a marginal increase in [Mg2+]i was caused by Na+ removal, whereas a similar slow acidosis was observed, indicating that extracellular Ca2+ inhibits Mg2+ entry, and that the increase in [Mg2+]i is negligible through competition between Mg2+ and Ca2+ in intracellular sites. These results imply that Na(+)-Mg2+ exchange is the main mechanism to maintain low [Mg2+]i even under physiological conditions.