Effect of bicarbonate on neonatal serum ionized magnesium in vitro.

Sodium bicarbonate is used to treat metabolic acidosis, or to induce metabolic alkalosis in sick neonates. The aim of this study was to quantify the decrease in serum concentration of ionized magnesium ([Mg2+]) when sodium bicarbonate is added to neonatal serum in vitro. Sodium bicarbonate was added to 30 cord serum samples of term infants to reach incremental concentrations of 0 to 2.0 mEq/L and [Mg2+] was measured. Serum [Mg2+] decreased significantly with the addition of sodium bicarbonate. At incremental sodium bicarbonate concentrations of 1.0 to 2.0 mEq/L, which is within the range of the desired aim in the treatment of metabolic acidosis, the magnitude of the decrease in serum [Mg2+] was approximately 0.084 to 0.158 mmol/L (18% to 34%) from the average baseline value. The addition of sodium bicarbonate causes a significant decrease in [Mg2+]. From this in vitro study we speculate that fast infusion of sodium bicarbonate in human neonates may potentially cause a clinically significant decrease in serum [Mg2+].     

Magnesium net fluxes and distribution in rabbit myocardium in irreversible contracture

Distribution of magnesium (Mg) in heart muscle was studied by measuring fluxes of Mg and transmembrane potentials as a function of perfusate [Mg2+] after a massive increase in permeability of the sarcolemma was induced in the Langendorff prepared heart from the Nembutal-anesthetized rabbit. After onset of 0 mM [Ca2+] perfusion which produced excitation-contraction (E-C) uncoupling and mechanical arrest, action potentials recorded from subepicardial cells showed an increase in duration and decrease in amplitude, which progressed until no transmembrane potentials could be observed. Restoration of physiological salt solution perfusion after 15 min of [Ca2+]-free perfusion caused an irreversible contracture that was associated with 1) efflux of potassium (K) and myoglobin, 2) perfusate [Mg2+]-dependent flux of Mg, and 3) transmembrane potentials of 0 mV. The magnitude of net efflux of K and myoglobin during contracture was unaffected by perfusate [Mg2+]. During the first 2 min of contracture, net efflux of Mg (mumoles per gram wet muscle +/- SE) was 1.37 +/- 0.09 and 0.48 +/- 0.19 during 0 mM and 2.5 mM [Mg2+] perfusion, respectively; but a net influx of 0.56 +/- 0.23 occurred during 5 mM [Mg2+] perfusion. Total sarcoplasmic [Mg] may correspond to perfusate [Mg2+] of 3.6 mM, which was found by interpolation to prevent any net flux of Mg during contracture. 3.6 mM may, therefore, represent the upper limit of the intracellular free-ionized Mg concentration in rabbit heart.     

Effects of magnesium on contractile activation of skinned cardiac cells.

1. In the presence of a slight buffering of the free [Ca2+] with 0.050 mM total EGTA cyclic contractions were induced by a Ca2+-triggered release of Ca2+ on skinned (sarcolemma-free) segments of single cardiac cells from rat ventricle. The threshold of the free [Ca2+] trigger was elevated when the free [Mg2+] was increased. 2. At a suprathreshold free [Ca2+] increasing the free [Mg2+] resulted in a decrease in frequency and in an increase in amplitude of the phasic contractions. Addition of caffeine at a specified interval after a cyclic contraction produced a larger contraction when free [Mg2+] was higher. It was concluded that an increase of free [Mg2+] increased the capacity and the rate of binding for Ca2+ by the sarcoplasmic reticulum (SR). 3. Small skinned fibres of skeletal muscle which were perfused with 10 mM caffeine yielded results similar to those obtained in skinned cardiac cells. It was concluded that the mechanism of action of free Mg2+ was similar in both preparations, but that the SR of skeletal muscle had a higher capacity and rate of binding for Ca2+ than the cardiac SR. 4. With a strong buffering of the free [Ca2+] with 4-0 mM total EGTA, a smaller tonic tension was developed for a given pCa in the presence of a higher free [Mg2+]. This result was nearly identical in skinned cells from cardiac and skeletal muscle tissue. 5. A decrease of the [MgATP2-] produced a tension in the skinned cardiac cells that were perfused in Ca2+ free media. The maximum tension was observed for [MgATP2-] 10(-5-50)M as in skinned fibres of skeletal muscle. A further decrease of [MgATP2-] resulted in a decrease of tension.     

Metabolic factors contributing to altered Ca2+ regulation in skeletal muscle fatigue

AIM: Skeletal muscle fatigue is characterized by a failure to maintain force production or power output during intense exercise. Many recent studies on isolated fibres have used brief repetitive tetanic contractions to mimic fatigue resulting from intensive exercise and to investigate the underlying cellular mechanisms. Such studies have shown that characteristic changes in Ca2+ regulation occur during fatiguing stimulation. This includes prolongation of the 'Ca2+-tails' which follow each period of tetanic stimulation and a progressive rise in resting [Ca2+]. More importantly, the final stage of fatigue is associated with a rapid decrease in tetanic [Ca2+]i and force. These fatigue-induced changes in sarcoplasmic reticulum (SR) Ca2+ regulation are temporally associated with alterations in the intracellular levels of phosphate metabolites and a causal relationship has often been proposed. The aim of this review is to evaluate the evidence linking changes in the levels of phosphate metabolites and altered Ca2+ regulation during fatigue. RESULTS: The following current hypotheses will be discussed: (1) the early changes in Ca2+ regulation reflect alterations in the intracellular levels of phosphate metabolites, (2) inhibition of the SR Ca2+ release mechanism (e.g. caused by ATP depletion and increased [Mg2+]) contributes to the decrease in tetanic [Ca2+]i during the final stages of fatigue and (iii) delayed entry of inorganic phosphate ions (Pi) into the SR, followed by precipitation of calcium phosphate (Ca-Pi), can explain the fatigue-induced decrease in tetanic [Ca2+]i. CONCLUSION: There is strong evidence that changes in phosphate metabolite levels contribute to early changes in SR Ca2+ regulation during fatigue and that inhibition of the SR Ca2+ release mechanism can partially explain the rapid decrease in tetanic [Ca2+]i during the final stages of fatigue. While precipitation of Ca-Pi may occur within the SR during fatigue, there is currently insufficient evidence to establish whether this contributes to the late decline in tetanic [Ca2+]i.     

Low extracellular Mg2+ concentrations suppress phagocytosis in vitro by alveolar macrophages from rats.

The aim of this study was to assess the effects of extracellular Mg2+ concentrations on phagocytosis in vitro by alveolar macrophages from rats. Phagocytosis was suppressed in the presence of low, but not high, Mg2+ concentrations. Vanadate, a Mg(2+)-ATPase inhibitor, suppressed phagocytosis. In the presence of a low Mg2+ concentration or vanadate, the cytosolic free Ca2+ concentration ([Ca2+]i) increased, but the cytosolic free Mg2+ concentration did not. These results suggest that low extracellular Mg2+ concentrations and vanadate suppress phagocytosis by rat alveolar macrophages by increasing [Ca2+]i.     

The effect of local changes in potassium and bicarbonate concentration on hypothalamic blood flow in the rabbit.

Blood flow has been measured locally in the hypothalamus of anaesthetized rabbits by measuring the clearance of small volumes (5-20 mul.) of a mock cerebrospinal fluid solution containing 133Xe. The effect of varying the [K+] or [HCO-3] of the 133Xe-containing solution on local hypothalamic blood flow has been investigated. 2. There was an increase in local hypothalamic blood flow if the 133Xe-containing solution was HCO3--free; raising the [HCO--3,] of the solution to 40 mM caused a fall in local blood flow. 3. There was an increase in local hypothalamic blood flow when 133Xe was injectedin a mock cerebrospinal fluid containing 10 or 20 mM-[K+]. There was no significant change in blood flow if a K+-free or a 40 mM [K+] solution was used. 4. The decrease in hypothalamic blood flow caused by injecting a 40 mM-[HCO3] solution could be reversed by the addition of 20 mM [K+] to the solution. There was no further increase in blood flow if 20 mM-[K+] was added to a HCO3--free solution. 5. It is concluded that local blood flow in the hypothalamus changes as a result of variation in local [K+] as well as local [HCO-3]. The changes in blood flow in the brain which accompany neuronal activity could be mediated by variation in local [K+].     

Trifluoperazine, a calmodulin inhibitor, blocks secretion in cultured chromaffin cells at a step distal from calcium entry

Trifluoperazine, a calmodulin antagonist, inhibited the secretory response of cultured bovine adrenal medullary chromaffin cells to acetylcholine (10(-4) M) or a depolarizing concentration of [K+] (56 mM KCl) in a dose-related fashion. The ID50s of this effect were 2 x 10(-7) M and 2.2 x 10(-6) M for acetylcholine and high [K+], respectively. A decrease in external [Ca2+] concentration of the incubation medium from 4.4 to 0.275 mM resulted in an increase in the percentage of inhibition produced by trifluoperazine on the acetylcholine-evoked secretory response from 20.7 to 96.5%, respectively. However, trifluoperazine inhibited the acetylcholine-evoked catecholamine output by a similar absolute magnitude for all [Ca2+] concentrations tested with the exception of 4.4 mM [Ca2+]. Trifluoperazine, unlike the [Ca2+] channel blocker Ni2+, in concentrations (10(-6)-10(-5) M) that were found to inhibit significantly [K+]-induced amine output did not modify [K+]-induced 45Ca uptake or 45Ca efflux. However, trifluoperazine at a concentration of 2.5 x 10(-5) M was found to produce a small decrease in the 45Ca efflux curve and a decrease in the [K+]-evoked 45Ca uptake of 30 +/- 14% (n = 6). In addition, 2.5 x 10(-6) M trifluoperazine, a concentration which was found to suppress high [K+]-induced amine release by 64 +/- 5%, did not inhibit the 45Ca2+-Ca2+ exchange mechanism. These results demonstrate that trifluoperazine, an antipsychotic agent with anticalmodulin activity, blocks catecholamine release from cultured chromaffin cells at a step distal from calcium entry and, consequently, suggests a role for calmodulin in the secretory process of these cells.     

Evidence for Na+/Ca2+ exchange in the rectal gland of Squalus acanthias

Previously we have shown that stimulation of in vitro perfused rectal gland tubules (RGT) of the dog-fish Squalus acanthias by adenosine 3',5'-cyclic monophosphate (cAMP), (as a cocktail comprising 0.1 mmol/l dibutyryl-cAMP, 10 micromol/l forskolin and 0.1 mmol/l adenosine, hereafter termed STIM) leads to an increase in cytosolic Ca2+ ([Ca2+]i) and that this assists Cl- secretion by enhancing basolateral K+ conductance. In the present study we examined the mechanism of the cAMP-induced increase in [Ca2+]i. [Ca2+]i was measured using the fura-2 technique in isolated in vitro perfused RGT. As before, STIM enhanced [Ca2+]i. This elevation of [Ca2+]i was prevented completely when STIM was added in the presence of the Na+2Cl-K+ cotransport inhibitor furosemide (0.5 mmol/l). This suggests that the increase in [Ca2+]i induced by STIM is caused by a concomitant increase in cytosolic Na+ ([Na+]i) and not by the activation of second messenger cascades. Furosemide prevents this increase in [Na+]i and hence the elevation of [Ca2+]i. Moreover, the plateau phase of the [Ca2+]i transient produced by carbachol (CCH, 0.1 mmol/l) was augmented strongly when bath Na+ was reduced to 5 mmol/l. These data suggest that the level of [Ca2+]i is determined by Na(+)-dependent Ca2+ export, most likely via a Na+/Ca2+ exchanger. The increase in [Na+]i accompanying stimulation of Cl- secretion reduces the rate of Ca2+ export leading to an elevation of [Ca2+]i, as does a reduction in bath Na+ which augments the [Ca2+]i plateau produced by CCH.     

Subcellular localization of glutamate-stimulated intracellular magnesium concentration changes in cultured rat forebrain neurons using confocal microscopy

Glutamate can stimulate increases in intracellular magnesium concentration ([Mg2+]i) and induce neurotoxicity, both independent of Ca2+ changes. Although Mg2+ is essential within the cell, very little is known about how it is regulated, especially in neurons. Therefore we used the fluorescent indicator, magindo-1 and confocal microscopy to examine possible intracellular pools of Mg2+ in cultured neurons that can be dynamically regulated by glutamate. The magindo-1 fluorescence signal was present throughout the cell body and extends into the neuronal processes. The magindo-1 405 nm/490 nm ratio signal was similar in the cytoplasm and nucleus, suggesting that resting [Mg2+]i is uniform across the neuron. The addition of 100 microM glutamate/10 microM glycine in an extracellular Ca2+- and Na+-free buffer stimulated an increase in [Mg2+]i in both the nuclear and cytoplasmic regions of similar magnitude and duration. This glutamate exposure also stimulated a [Mg2+]i increase in neuronal processes which was inhibited by the N-methyl-D-aspartate receptor antagonist, MK-801 (10 microM). The glutamate-stimulated [Mg2+]i increase in both the cell body and neuronal processes was dependent on the extracellular Mg2+ concentration. These findings suggest glutamate-stimulated [Mg2+]i changes may not only impact cytoplasmic processes, but also directly trigger nuclear events involved, for example, in neuronal injury.     

Comparison between measured and calculated ionised concentrations in Mg2+ /ATP, Mg2+ /EDTA and Ca2+ /EGTA buffers; influence of changes in temperature, pH and pipetting errors on the ionised concentrations.

The apparent dissociation constants (Kapp) and total ligand concentrations ([Ligand]T) from extensive published and unpublished macroelectrode measurements for Mg2+/ATP, Mg2+/EDTA and Ca2+/EGTA buffers have been recalculated. These calculations were made feasible by the introduction of an Excel program which reduced the time of calculation for Kapp and [Ligand]T from over an hour to under five minutes. These estimations of Kapp and [Ligand]T allowed, not only a comparison between measured and calculated ionised magnesium and calcium concentrations ([Mg2+] and [Ca2+]) for Mg2+/ATP, Mg2+/EDTA and Ca2+/EGTA buffers but also a comparison amongst calculated values. Calculated [X2]1 values always differed from measured, and calculated values differed amongst themselves by factors of at least 2. These variations cast doubts on the published absolute values for intracellular [Mg2+] estimated by 31P-NMR and the resting values for [Ca2+] in cells. The allowable range for [X2+] in the buffers and consequently for Kapp and [Ligand]T has not been defined, which introduces uncertainties into published absolute values for [X2+]. This paper shows that an upper limit of +/- 10% deviation from the mean value for [X2+] is attainable. This requires the temperature to be maintained within +/- 0.5 degrees C, pH within +/- 0.01 units and pipetting errors of less than 0.25%. Until internationally defined buffer standards are available, the lack of correlation between measured and calculated [X2+] means that measurement of Kapp and [Ligand]T and hence [X2+] is more reliable than calculation.     

Observations on the potential across the rumen of the sheep.

1. The electric potential difference between rumen contents and jugular venous blood was measured in anaesthetized sheep. In order to investigate the effect on the potential of changing the ionic concentrations within the rumen, the digesta were removed from the rumen and various salt solutions were substituted. The reticulo-rumen sac was isolated before the experiment by ligation of the oesophagus and the reticulo-omasal junction. 2. The observation of Dobson & Phillipson (1958) that the rumen contents are normally of the order of 30 mV negative to the blood was confirmed. 3. For potassium concentrations between 25 and 100 mM the potential at constant [Na+] varied linearly with log [K+]. With sulphate as the anion, the slope for a 10-fold concentration change was 39.7 +/- 3.0 mV when [Na+] was around 50 mM. The slope showed a tendency to increase when [Na+] was lowered, and to decrease when [Na+] was raised. 4. When chloride was substituted for sulphate, both the slope and the absolute size of the potential were slightly reduced. 5. When the sodium concentration was varied at constant [K+], the potential increased as an approximately linear function of [Na+]. At around 10 mM-K the mean slope was 0-32 +/- 0.07 mV/mM; at the highest potassium concentrations it fell to 0-13 +/- 0 05 mV/mM. 6. In most of these experiments isotonicity was maintained with sucrose. The results of a few tests in which Li+ was substituted for Na+ or K+ suggested that the rumen epithelium behaves in a relatively inert fashion towards this ion.     

The intracellular concentration of free magnesium in extensor digitorum longus muscles of the rat

Magnesium-sensitive microelectrodes were used to measure the intracellular concentration of free Mg2+, [Mg2+]f, in rat extensor digitorum longus muscles in vitro at 30 degrees C. The intracellular activities of Na+ and K+ were also determined so that allowance could be made for the interference from these ions with the Mg2+ electrode response. The mean value for [Mg2+]f based on twenty-six measurements in twelve muscles was 0.47 mM.     

PHI+], a novel Sup35-prion variant propagated with non-Gln/Asn oligopeptide repeats in the absence of the chaperone protein Hsp104

BACKGROUND: The [PSI+] element of the budding yeast is an aggregated form of the translation release factor Sup35 that is propagated and transmitted cytoplasmically in a manner analogous to that of mammalian prions. The N-terminal of Sup35, necessary for [PSI+], contains oligopeptide repeats and multiple Gln/Asn residues. RESULTS: We replaced the Gln/Asn-rich prion repeats of Sup35 with non-Gln/Asn repeats from heterologous yeast strains. These non-Gln/Asn repeat Sup35s propagated a novel [PSI+] variant, [PHI+], that appeared de novo 103 times more frequent than [PSI+]. [PHI+] was stably inherited in a non-Mendelian fashion, but not eliminated upon the inactivation of Hsp104, unlike known [PSI+] elements. In vitro, non-Gln/Asn repeat domains formed amyloid fibres that were shorter and grew more slowly than did Gln/Asn-rich prion domains, while [PHI+] aggregates were smaller than [PSI+] aggregates in vivo. CONCLUSIONS: These findings suggest the existence of an alternative, Hsp104-independent pathway to replicate non-Gln/Asn variant Sup35 prion seeds.     

The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa.

The neoglycoproteins alpha-D-mannose-bovine serum albumin (mannose-BSA) and N-acetyl-alpha-D-glucosamine-BSA (glucNAc-BSA) were shown to rapidly increase intracellular free calcium ([Ca2+]i) in human spermatozoa. The increase in [Ca2+]i induced by these neoglycoproteins accounts for the known ability of these compounds to induce the acrosome reaction in human spermatozoa. Our data support the hypothesis that mannose-BSA, but not progesterone, activates T-type Ca2+ channels in human spermatozoa for the following reasons: (i) the capacity of mannose-BSA to increase [Ca2+]i was inhibited by the specific T-type Ca2+ channel blocker mibefradil (IC50 = 10(-6) mol/l) while progesterone was not inhibited by 10(-5) M mibefradil; (ii) the effect of mannose-BSA to elevate [Ca2+]i was inhibited more potently by Ni2+ (IC50 = 0.1 mmol/l) than Cd2+ (IC50 = 0.5 mmol/l), whereas the effect of progesterone to elevate [Ca2+]i was inhibited equally by Ni2+ and Cd2+ (IC50 = 0.25 mmol/l); (iii) the effects of mannose-BSA and progesterone to increase [Ca2+]i were greater than additive. These data support the idea that mannose-BSA and progesterone were activating distinct Ca2+ channels, one of which was a T-type Ca2+ channel activated by mannose-BSA whereas the Ca2+ channel that was activated by progesterone has yet to be defined at the molecular level.     

Myoplasmic Mg2+ concentration in Xenopus muscle fibres at rest, during fatigue and during metabolic blockade.

Intracellular free Mg2+ concentration ([Mg2+]i) was measured in isolated single fibres of Xenopus muscle using the fluorescent Mg2+ indicator furaptra. In resting muscle the [Mg2+]i was 1.7 mM in a Mg(2+)-free Ringer solution. There was no significant change in [Mg2+]i over 2 h in Mg(2+)-free Ringer solution. Elevating extracellular [Mg2+] to 40 mM for 5 min caused a small rise (0.13 mM) in [Mg2+]i. There was no detectable rise in [Mg2+]i after 5 min in Na(+)-free Ringer solution. These results suggest that the membrane is relatively impermeable to Mg2+ and that there was no detectable Na(+)-Mg2+ exchange over 5 min. When muscle fibres were fatigued by repeated tetani continued until force declined to about 40% of control, [Mg2+]i showed characteristic changes. During the early period of fatigue when force first showed a small decline and then became almost stable, [Mg2+]i was unchanged; during the final period of fatigue when force declined more rapidly, [Mg2+]i increased by 0.8 mM. Recovery of [Mg2+]i took about 30 min. Recovery of force was complex: tetanic force first declined (post-contractile depression) and then slowly recovered to control. Since the minimum force occurred at about the time when [Mg2+]i had recovered, it seems unlikely that post-contractile depression is caused by elevated [Mg2+]i. Rigor, produced by inhibiting oxidative phosphorylation and glycolysis, was associated with a larger increase (1.6 mM) in [Mg2+]i than fatigue. The rise in [Mg2+]i during fatigue and metabolic blockade could be explained as release of Mg2+ normally bound to ATP. A model of the metabolic changes and the resulting increase in [Mg2+]i explains our results reasonably well.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Effects of alpha- and beta-adrenergic stimulation on free magnesium concentrations in platelets from healthy and obese individuals.

We performed this study to investigate whether alpha- and beta-adrenergic agonists are able to regulate intracellular free magnesium concentrations [Mg2+]i in platelets from healthy and obese individuals. Twenty-six informed-consent men (14 healthy and 12 obese) were enrolled in the study. We measured fasting plasma glucose, insulin, epinephrine and norepinephrine. Platelet [Mg2+]i at the baseline and after stimulation with clonidine or isoproterenol was measured by fluorescent probe mag-fura-2. In platelets from healthy subjects, alpha-adrenergic stimulation by clonidine led to a dose-dependent decrease in [Mg2+]i (basal: 245 +/- 39 microM; clonidine 5 pg/mL: 109 +/- 27 microM, p < 0.05; clonidine 10 pg/mL: 77 +/- 26 microM, p < 0.01), while no significant change in platelet [Mg 2+]i was detected in obese men. Furthermore, the co-incubation with clonidine (10 pg/mL) and yohimbine (50-100 pg/mL) completely abated the effect of clonidine on [Mg2+]i in platelets from healthy individuals. Analysis of the time course for platelet magnesium showed that the intracellular magnesium loss induced by clonidine (10 pg/mL) was time-dependent. Conversely, the beta-adrenergic agonist isoproterenol was able to produce a significant rise in [Mg2+]i in platelets from healthy individuals (basal: 234 +/- 40 microM; isoproterenol 2.5 pg/mL: 594 +/- 44 microM, p < 0.05: isoproterenol 5 pg/mL: 681 +/- 56 microM, p < 0.01), while no such finding was detectable in platelets from obese patients. When platelets from healthy subjects were co-stimulated with isoproterenol (5 pg/mL) and propranolol (10-20 pg/mL), the ionophoric effect of the beta-adrenergic agonist was completely reverted. The time course of isoproterenol (5 pg/mL) effect on platelet [Mg2+]i showed that the ionophoric effect of isoproterenol was time-dependent. In conclusion, (1) the stimulation of alpha-adrenergic receptor by clonidine is able to induce a significant dose- and time-dependent fall in platelet [Mg2+]i; (2) the stimulation of beta-adrenoceptors by isoproterenol lead to a signifcant time- and dose-dependent rise in platelet [Mg2+]; (3) the ionic effect of alpha- and beta-adrenergic stimulation is not detectable in obese subjects, in whom is probably present a reduced sensitivity to the ionic effect of adrenergic agonists.     

Effect of external magnesium on intracellular free sodium: Na+ flux via Na+/Mg2+ antiport is masked by other Na+ transport systems in rat cardiac myocytes.

Mg2+ efflux from heart cells on a Na+/Mg2+ antiport has been postulated, but the Na+ flux component of the antiport has not been demonstrated. The study aimed to establish if the Na+ flux component could be measured by following changes in [Na+]i with SBFI during conditions known to reverse the antiport (5 mmol/L Mg2+(o), Na+(o)- & Ca2+(o)-free): and after minimising the activity of other Na+ transport pathways. Resting [Na+]i was 8 +/- 0.7 mmol/L (mean +/- S.E., n = 39 cells) in normal Tyrode's solution. [Na+]i decreased below the normal level in all cells (a decline of 4-5 mmol/L, n = 21) during perfusion with 5 mmol/L Mg2+(o) (Na+(o)- & Ca2+(o)-free). Controls using 1 mmol/L Mg2+(o) showed similar declines in [Na+]i, but the fall was greatest when Na+(o) was replaced by K+(o) (decline of 6 mmol/L) rather than the tetramethylammonium ion (TMA+). The rate of decrease in [Na+]i during perfusion with 5 mmol/L Mg2+(o) (Na+(o)- & Ca2+(o)-free) was slowed by 20 microM ouabain (n = 5) or by elevation of pHo to pH 9 (n = 7) so that [Na+]i remained close to the initial value. The decrease of [Na+]i was not affected by 10 microM imipramine (n = 15). These data suggest that the Na+ efflux component of the Na+/Mg2+ antiport is masked in Na+(o)- and Ca2+(o)-free conditions by other Na+(i) efflux pathways.     

Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes

Mg-ATP-dependent 45Ca2+ uptake and Ca2+-ATPase activity have been examined in isolated microsomes obtained by differential centrifugation and in purified subcellular fractions obtained by Ficoll-sucrose density centrifugation in the presence of mitochondrial inhibitors. Mg-ATP-dependent 45Ca2+ uptake increased with increasing EGTA-buffered free [Ca2+], reaching a maximum of 2 nmol 45Ca2+ X 15 min-1 X mg prot-1 at 2 mumol/1 [Ca2+] in the incubation medium. Half-maximal 45Ca2+ uptake was at approximately 0.2 mumol/1 [Ca2+]. Maximal Ca2+ -Mg2+ -ATPase activity was 130 nmol X 15 min-1 X mg prot-1 at 2 mumol/l [Ca2+], with an apparent Km of approximately 0.3 mumol/l [Ca2+]. The Ca2+ ionophore A23187 (10(-6) mol/l), the mercurial compounds mersalyl (10(-5) mol/l) and CH3ClHg (10(-3) mol/l), as well as La3+ (10(-4) mol/l), vanadate (10(-4) mol/l), and saponin (50 micrograms/mg prot), abolished Mg-ATP-promoted 45Ca2+ uptake. In the absence of Mg2+, ATP did not provoke 45Ca2+ uptake. Using the purified smooth membrane fraction (F1) from the Ficoll-sucrose density gradient (enrichment of Na+-K+-ATPase specific activity by ninefold and of NADH-cytochrome c reductase by threefold as compared with total tissue homogenate), Mg-ATP-dependent 45Ca2+ uptake correlated better with Na+-K+-ATPase (r = 0.97) than with the smooth endoplasmic marker NADH-cytochrome c reductase (r = 0.52). No correlation was found with RNA, the marker for rough endoplasmic reticulum. We conclude that pancreatic plasma membranes contain a Ca2+-Mg2+-ATPase that represents the Ca2+ extrusion system from acinar cells. It is also possible that vesicular membrane structures associated with the plasma membrane, or endocytotic plasma membrane vesicles, take up Ca2+ and represent an intracellular Ca2+ pool.     

Effect of extracellular magnesium on secretagogue-evoked amylase secretion in the isolated rat parotid gland segments.

This study investigates the effect of perturbation of extracellular magnesium [Mg2+]o on basal and acetylcholine (ACh), noradrenaline (NA) and phenylephrine (PHE)-evoked amylase secretion from isolated rat parotid gland segments. Both zero (0 mM) and elevated (5 mM and 10 mM) [Mg2+]o can significantly (P < 0.05) inhibit basal and secretagogue-evoked amylase secretion compared to the responses obtained in normal (1.1 mM) [Mg2+]o. The inhibitory effect of zero [Mg2+]o was more pronounced compared 10 mM [Mg2+]o. A concentration of 5 mM, [Mg2+]o was less effective at a secretagogue concentration of 10(-5) M but more pronounced in inhibiting amylase secretion when the concentration of the secretagogue was reduced to 10(-6) M. The results indicate that both hypo and hypermagnesaemia are associated with reduced salivary amylase secretion and both conditions may be associated with 'the dry mouth syndrome'.