Ionized magnesium in the homeostasis of cells: intracellular threshold for Mg(2+) in human platelets

The role of extracellular magnesium ions in the homeostasis of intracellular ionized magnesium ([Mg(2+)](i)) in human platelets was studied. For media containing 0.00 to 0.60 mmol/l of extracellular ionized magnesium ([Mg(2+)](o)), the mean [Mg(2+)](i) fluctuated between 533 and 760 micromol/l. As the [Mg(2+)](o) was increased to 1.5 mmol/l, the [Mg(2+)](i) increased proportionately and peaked at 1470.1 micromol/l. Additional increase in the [Mg(2+)](o) from 1.50 to 6.00 mmol/l resulted in decreased [Mg(2+)](i) until it equilibrated between 739 and 776 micromol/l. The influx of Mg(2+) at [Mg(2+)](o) of 0.60 and 1.50 mmol/l was studied using verapamil, a calcium channel inhibitor, and ouabain, an inhibitor of the Na/K pump, respectively. The verapamil (25 mmol/l) blocking experiments resulted in a 92.4% inhibition of the Mg(2+) influx into the platelet at a [Mg(2+)](o) of 1.50 mmol/l. Ouabain (0.5 and 2.5 mmol/l) showed an enhancement effect on the influx of Mg(2+) at [Mg(2+)](o) of 0.60 mmol/l and no effect at 1.50 mmol/l. The effect of verapamil indicates that ion channels that are homologous to calcium ion channels may be involved in the influx of Mg(2+) into the platelets. The inhibition of Mg(2+) influx for [Mg(2+)](o) greater than 1.50 mmol/l may illustrate a protective mechanism that attempts to maintain the viability of platelets at abnormally high [Mg(2+)](o). These results suggest that there is an intracellular Mg(2+) threshold of 1500 micromol/l, above which an active mechanism prevents further influx of Mg(2+).     

31P nuclear magnetic resonance and zero-point titration compared for measuring free magnesium concentration in erythrocytes.

Intracellular ionized magnesium concentrations ([Mg2+]i) were measured in erythrocytes by 31P nuclear magnetic resonance (NMR) and zero-point titration in 14 controls and seven patients with renal magnesium loss. The mean intracellular ionized magnesium concentration in controls measured by 31P NMR was 0.20 (SD 0.03) mmol/L cell water, compared with 0.55 (SD 0.12) mmol/L cell water by zero-point titration. Total erythrocyte magnesium content measured with the lysate method was 0.63 mmol/L cell water higher than estimated by 31P NMR, probably because not all magnesium complexes are fully visible to the NMR technique. We found a positive correlation between plasma ultrafiltrable magnesium and [Mg2+]i irrespective of the [Mg2+]i assay used. [Mg2+]i measured with 31P NMR correlated modestly but significantly with [Mg2+]i determined by zero-point titration (r = 0.58, P less than 0.02). Washing erythrocytes before the zero-point titration decreased the ATP content and the cell water fraction, which led to overestimation of [Mg2+]i by zero-point titration. Although absolute values for [Mg2+]i differ with the assay used, both methods determined significantly lower values for [Mg2+]i in patients with isolated renal magnesium loss.     

Activation of Ca2+-dependent K+ channels in human B lymphocytes by anti-immunoglobulin.

Many mammalian cell types exhibit Ca2+-dependent K+ channels, and activation of these channels by increasing intracellular calcium generally leads to a hyperpolarization of the plasma membrane. Their presence in B lymphocytes is as yet uncertain. Crosslinking Ig on the surface of B lymphocytes is known to increase the level of free cytoplasmic calcium ([Ca2+]i). However, rather than hyperpolarization, a depolarization has been reported to occur after treatment of B lymphocytes with anti-Ig. To determine if Ca2+-dependent K+ channels are present in B lymphocytes, and to examine the relationship between intracellular free calcium and membrane potential, we monitored [Ca2+]i by means of indo-1 and transmembrane potential using bis(1,3-diethylthiobarbituric)trimethine oxonol in human tonsillar B cells activated by anti-IgM. Treatment with anti-IgM induced a biphasic increase in [Ca2+]i and a simultaneous hyperpolarization. A similar hyperpolarization was induced by ionomycin, a Ca2+ ionophore. Delaying the development of the [Ca2+]i response by increasing the cytoplasmic Ca2+-buffering power delayed the hyperpolarization. Conversely, eliminating the sustained phase of the [Ca2+]i response by omission of external Ca2+ abolished the prolonged hyperpolarization. In fact, a sizable Na+-dependent depolarization was unmasked. This study demonstrates that in human B lymphocytes, Ca2+-dependent K+ channels can be activated by crosslinking of surface IgM. Moreover, it is likely that, by analogy with voltage-sensitive Ca2+ channels, Na+ can permeate through these ligand-gated Ca2+ "channels" in the absence of extracellular Ca2+.     

Facilitated determination of ionized calcium

Using a calcium-containing heparin preparation for anticoagulation, we determined [Ca2+], the mean concentration of ionized calcium, in whole blood of 120 healthy blood-donors to be 1.23 (SD 0.04) mmol/L. Similarly, for 50 intensive-care patients selected without conscious bias, the correlation between [Ca2+] in serum (mean 1.15, SD 0.10 mmol/L) and in whole-blood samples anticoagulated with the same heparin preparation (mean 1.15, SD 0.09 mmol/L) was very good (r = 0.95). Storing samples anaerobically on ice for as long as 2 h did not alter whole-blood [Ca2+]. On the other hand, various concentrations of calcium-free heparin preparations all induced a significant decrease in measured [Ca2+]. By using whole-blood samples, rather than plasma or serum, for [Ca2+] determination with a calcium-selective electrode, repetitive measurements can be made with simple handling procedures, facilitating rapid implementation of appropriate therapeutic measures for critically ill patients.     

Magnesium relaxes arterial smooth muscle by decreasing intracellular Ca2+ without changing intracellular Mg2+.

Elevations in extracellular [Mg2+] ([Mg2+]o) relax vascular smooth muscle. We tested the hypothesis that elevated [Mg2+]o induces relaxation through reductions in myoplasmic [Ca2+] and myosin light chain phosphorylation without changing intracellular [Mg2+] ([Mg2+]i). Histamine stimulation of endothelium-free swine carotid medial tissues was associated with increases in both Fura 2- and aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and force. Elevated [Mg2+]o decreased myoplasmic [Ca2+] and force to near resting values. However, elevated [Mg2+]o only transiently decreased myosin phosphorylation values: sustained [Mg2+]o-induced decreases in myoplasmic [Ca2+] and force were associated with inappropriately high myosin phosphorylation values. The elevated myosin phosphorylation during [Mg2+]o-induced relaxation was entirely on serine 19, the Ca2+/calmodulin-dependent myosin light chain kinase substrate. Myoplasmic [Mg2+] (estimated with Mag-Fura 2) did not significantly increase with elevated [Mg2+]o. These results are consistent with the hypothesis that increased [Mg2+]o induces relaxation by decreasing myoplasmic [Ca2+] without changing [Mg2+]i. These data also demonstrate dissociation of myosin phosphorylation from myoplasmic [Ca2+] and force during Mg(2+)-induced relaxation. This finding suggests the presence of a phosphorylation-independent (yet potentially Ca(2+)-dependent) mechanism for regulation of force in vascular smooth muscle.     

Cell-mediated mitogenic response induced by leukoagglutinin and Lens culinaris lectin in mouse lymphocytes

The proliferative response of mouse lymphocytes to syngeneic cellular stimulation upon membrane modification with lectins was studied. Brief pretreatment of stimulator cells (mitomycin-C-treated spleen cells) followed by mixed culture with syngeneic cortisone-resistant thymocytes resulted in a significant proliferative response in the thymocytes. This effect was not due to a soluble mediator and was similar to the mitogenic response after Con A-induced membrane modification reported previously. Because of its general characteristics, we refer to this response as cell-mediated mitogenic response (CMMR). Cell contact between stimulator and responder cells was necessary but not sufficient for the induction of the response. The lectins that generated CMMR were T-cell mitogens. CMMR was generated in all the syngeneic combinations tested and even in allogeneic combinations. No detectable cytotoxic activity towards syngeneic targets cells was produced after CMMR. Moreover, CMMR in allogeneic combinations led to the suppression of the generation of specific cytotoxic lymphocytes. Population analysis with antibodies against T or B cells, nylon wool fractionation of stimulator cells, and tests with peritoneal macrophages and with spleen cells from athymic mice revealed that CMMR depends predominantly on the interaction between responder T cells and stimulator Ig-positive lymphocytes.     

Measurement of free calcium ion in capillary blood and serum.

We describe a new calcium ion-selective electrode for measurement of the substance concentration of free calcium ion [Ca2+] in the plasma phase of whole blood and in serum at 37 degrees C. A sample volume of 50 microliter suffices to obtain simultaneous values of pH and [Ca2+]. We found the within-series analytical standard deviation for serum to be 0.013 mmol/litre (CV, 1.1%) and day-to-day precision to be 0.022 mmol/litre (CV, 1.7%). The reference interval for [Ca2+] (at pH 7.40) in serum was found to be 1.184 +/- 0.054 mmol/litre (2 SD) from measurements on sera from 121 healthy blood donors. Measurements on capillary blood from 29 healthy volunteers gave a mean (+/- 2 SD) value for [Ca2+] (at pH 7.40) of 1.22 +/- 0.072 mmol/litre.     

Gentamicin-induced increases in cytosolic calcium in pig kidney cells (LLC-PK1)

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were tested as a model system for assessing the role of calcium in gentamicin-induced nephrotoxicity. Cell viability was evaluated by a vital dye exclusion procedure, and intracellular free calcium [Ca2+]i was measured employing Fura-2 fluorescence. Exposing cell suspensions (10(6)/ml) to concentrations of the drug, which had no apparent effect on viability, produced a rapid and prolonged increase in intracellular [Ca2+]. The perturbation of calcium homeostasis could be blocked by the addition of mepiperphenidol, an inhibitor of the organic cation transport system. We propose that LLC-PK1 cells are an appropriate model to study drug-induced nephrotoxicity. Gentamicin disrupts calcium homeostasis and causes plasma membrane alterations. Since mepiperphenidol blocked the gentamicin-induced Ca2+ increases, the data suggest that aminoglycosides enter the cell via the organic cation transporter.     

Intracellular Mg2+ and magnesium depletion in isolated renal thick ascending limb cells.

Magnesium reabsorption and regulation within the kidney occur principally within the cortical thick ascending limb (cTAL) cells of the loop of Henle. Fluorometry with the dye, mag-fura-2, was used to characterize intracellular Mg2+ concentration ([Mg2+]i) in single cTAL cells. Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat anti-human Tamm-Horsfall and rabbit anti-goat IgG antibodies). Basal [Mg2+]i was 0.52 +/- 0.02 mM, which was approximately 2% of the total cellular Mg. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Mg2+]i, 0.48 +/- 0.02, in the normal range. However, cells cultured in nominally magnesium-free media possessed [Mg2+]i, 0.27 +/- 0.01 mM, which was associated with a significant increase in net Mg transport, (control, 0.19 +/- 0.03 and low Mg, 0.35 +/- 0.01 nmol.mg-1 protein.min-1) as assessed by 28Mg uptake. Mg(2+)-depleted cells were subsequently placed in high Mg solution (5 mM) and the Mg2+ refill rate was assessed by fluorescence. [Mg2+]i returned to normal basal levels, 0.53 +/- 0.03 mM, with a refill rate of 257 +/- 37 nM/s. Mg2+ entry was not changed by 5.0 mM Ca2+ or 2 mM Sr2+, Cd2+, Co2+, nor Ba2+ but was inhibited by Mn2+ approximately La3+ approximately Gd3+ approximately Zn2+ approximately Be2+ at 2 mM. Intracellular Ca2+ and 45Ca uptake was not altered by Mg depletion or Mg2+ refill, indicating that the entry is relatively specific to Mg2+. Mg2+ uptake was inhibited by nifedipine (117 +/- 20 nM/s), verapamil (165 +/- 34 nM/s), and diltiazem (194 +/- 19 nM/s) but enhanced by the dihydropyridine analogue, Bay K 8644 (366 +/- 71 nM/s). These antagonists and agonists were reversible with removal and [Mg2+]i subsequently returned to normal basal levels. Mg2+ entry rate was concentration and voltage dependent and maximally stimulated after 4 h in magnesium-free media. Cellular magnesium depletion results in increases in a Mg2+ refill rate which is dependent, in part, on de novo protein synthesis. These data provide evidence for novel Mg2+ entry pathways in cTAL cells which are specific for Mg2+ and highly regulated. These entry pathways are likely involved with renal Mg2+ homeostasis.     

Persistent calcium elevation correlates with the induction of surface immunoglobulin-mediated B cell DNA synthesis

Surface immunoglobulin (sIg)-mediated stimulation of B lymphocytes induces a tyrosine kinase-dependent sequence of events leading to rapid and large elevations in intracellular ionized calcium ([Ca2+]i). These early biochemical events do not necessarily lead to proliferation of B cells, however, and conversely, the absence of or inhibition of these events does not necessarily prevent cellular proliferation. We now show by digital image analysis of single B cells that conditions which lead to B cell proliferation are associated with low-level but persistent sustained or cyclic elevations in [Ca2+]i. In marked contrast, early and nonsustained elevations in [Ca2+]i are induced in B cells by stimuli that lead to G1 transition but fail to progress to DNA synthesis. Thus, when B cells were stimulated with mitogenic and nonmitogenic anti-IgD antibodies, both of which induce entry of cells into G1 and early calcium transients of comparable magnitude, persistent low-level calcium elevations were only detected in cells stimulated with the mitogenic antibody. Furthermore, persistent calcium elevations were also seen when B cells were stimulated with a multivalent dextran-anti-Ig conjugate which induced very high levels of B cell proliferation in the absence of detectable phosphatidylinositol 4,5-biphosphate hydrolysis or elevations in [Ca2+]i as detected by flow cytometry. Finally, B cells from X-linked B cell-defective mice, which do not proliferate in response to anti-Ig antibody, show marked and early increases in [Ca2+]i, but do not show persistent calcium elevations. These data suggest that the rapid and large increases of [Ca2+]i seen in lymphocytes within seconds after antigen receptor ligation may be associated with entry in G1, whereas low-level but persistent elevations may be the hallmark of a cell destined to synthesize DNA.     

Distinct patterns of transmembrane calcium flux and intracellular calcium mobilization after differentiation antigen cluster 2 (E rosette receptor) or 3 (T3) stimulation of human lymphocytes.

We evaluated CD2 (E rosette) and CD3 (T3)-triggered activation of resting lymphocytes by measuring the intracellular free calcium concentration ([Ca2+]i) of individual cells. The [Ca2+]i of indo-1-loaded cells was measured by flow cytometry and responses were correlated with cell surface phenotype. Stimulation with anti-CD3 antibody caused an increase in [Ca2+]i in greater than 90% of CD3+ cells within 1 min, and furthermore, the response was restricted to cells bearing the CD3 marker. In contrast, stimulation of cells with anti-CD2 antibodies produced a biphasic response pattern with an early component in CD3- cells and a late component in CD3+ cells. Thus, the CD2 response does not require cell surface expression of CD3. In addition, stimulation of a single CD2 epitope was sufficient for activation of CD3- cells, whereas stimulation of two CD2 epitopes was required for activation of CD3+ cells. Both the CD2 and CD3 responses were diminished in magnitude and duration by EGTA. However, approximately 50% of T cells still had a brief response in the presence of EGTA, indicating that the increased [Ca2+]i results in part from intracellular calcium mobilization, and furthermore demonstrates that extracellular calcium is required for a full and sustained response. Our results support the concept that CD2 represents the trigger for a distinct pathway of activation both for T cells that express the CD3 molecular complex and for large granular lymphocytes that do not.     

Calcium ion concentrations and DNA fragmentation in target cell destruction by murine cloned cytotoxic T lymphocytes

To investigate the destruction of target cells by murine CTLs, we examined intracellular Ca2+ concentrations ([Ca2+]i) and DNA fragmentation in target cells. Changes in [Ca2+]i were followed by flow cytometry by loading the cells with indo-1, a Ca2+-binding fluorescent dye, and determining the ration of fluorescence intensities at 405 nm (emission maximum for Ca2+-bound dye) over 480 nm (emission maximum for the free dye). Within minutes after interacting with the cytolytic granule fraction that had been isolated from CTLs, [Ca2+]i in target cells was strikingly increased. A pronounced increase in [Ca2+]i was also observed in target cells when they were specifically recognized by intact CTLs. Since ionomycin, a Ca2+ ionophore, caused a similar increase in [Ca2+]i and lysed cells (provided that extracellular Ca2+ was present), it appears that a sustained high level of [Ca2+]i is cytolytic. In contrast with other cells, CTLs, which have been shown to be refractory to granule-mediated lysis and to be poor targets for other CTLs, did not manifest an elevation in [Ca2+]i when they were similarly loaded with indo-1 and treated with isolated granules. The characteristic cleavage of target cell DNA into nucleosome-sized fragments was also induced by isolated granules as well as by valinomycin, a K+ ionophore, but not by ionomycin. The results support the view that lysis of most target cells by cloned CTLs is due primarily to target cell membrane changes that are fundamentally equivalent to the formation of nonspecific ion channels. The resulting large increase in [Ca2+]i is probably responsible for target cell lysis; and changes in intracellular ion concentrations also appear to be responsible for DNA fragmentation, probably by activating endogenous target cell endonucleases.     

Clinical biochemistry of epilepsy. II. Observations on two types of epileptiform convulsions induced in rabbits with corticotropin.

We propose than an alarm mechanism is operative in animals, designed to regulate neuromuscular irritability by regulating [Ca2+]. Epinephrine or corticotropin (ACTH), injected intramuscularly into animals, causes a hypercitricemia, resulting in decreased [Ca2+]. This increases muscular excitability to facilitate escape. To avoid over reaction, [Cl-] is shifted into the plasma without a concomitant shift of Na+, thus generating an acidosis and an increase in ionization of Ca. Plasma pH, pCO2, total CO2, and [K+] decrease, and [Mg2+] increases. The acidosis, decrease in K+, and increase in [Mg2+] serve to counteract the effect of the decrease in [Ca2+], to protect against tetany. In the rabbit the hypercitricemia observed upon ACTH administration is accompained by a severe hypocalcemia and drop in blood pressure, resluting in tetanic convulsions. This seems to indicate calcitonin release, independent of the hypercitricemia. Thyroidectomized rabbits show only mild hypocalcemia when given ACTH, but develop a severe acidosis and typical grand mal epileptiform seizures. Administration of ACTH and then calcitonin to the goat, an animal resistant to the effects of ACTH alone, simulates the effect observed in the rabbit with respect to changes in blood components and blood pressure. Changes in the blood in the goat and rabbit resemble those in humans before an epileptic seizure. alpha-Melanotropin, containing a portion of the ACTH sequence, reacts in a manner similar to ACTH but more rapidly.     

Endothelial cell Ca2+ increases upon tumor cell contact and modulates cell-cell adhesion.

The signal transduction mechanisms involved in tumor cell adhesion to endothelial cells are still largely undefined. The effect of metastatic murine melanoma cell and human prostate carcinoma cell contact on cytosolic [Ca2+] of bovine artery endothelial cells was examined in indo-1-loaded endothelial cell monolayers. A rapid increase in endothelial cell [Ca2+] occurred on contact with tumor cells, but not on contact with 8-microns inert beads. A similar increase in endothelial cell [Ca2+] was observed with human neutrophils or monocyte-like lymphoma cells, but not with endothelial cells, red blood cells, and melanoma cell-conditioned medium. The increase in endothelial cell [Ca2+] was not inhibited by extracellular Ca2+ removal. In contrast, endothelial cell pretreatment with thapsigargin, which releases endoplasmic reticulum Ca2+ into the cytosol and depletes this Ca2+ store site, abolished the cytosolic [Ca2+] rise upon melanoma cell contact. Endothelial cell pretreatment with the membrane-permeant form of the Ca2+ chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid blocked the increase in cytosolic [Ca2+]. Under static and dynamic flow conditions (0.46 dyn/cm2) bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid pretreatment of bovine pulmonary artery endothelial cell monolayers inhibited melanoma cell adhesion to the endothelial cells. Thus, tumor cell contact with endothelial cells induces a rapid Ca2+ release from endothelial intracellular stores, which has a functional role in enhancing cell-cell adhesion.     

Effect of pentoxifylline on E-rosette formation and on the mitogenic response of human mononuclear cells

Pentoxifylline (Trental) has been shown to increase the intracellular cAMP concentration. In the present study we have examined the capacity of the drug to affect E-rosette formation by T lymphocytes. In addition, the mitogenic response of human lymphocytes to phytohemagglutinin (PHA) and concanavalin A (con A) was studied. The drug exerted a dose dependent inhibitory effect on E-rosette formation and on the proliferative response of lymphocytes to PHA and con A. It is suggested that this inhibitory effect is most probably due to the increased intracellular levels of cAMP induced by the drug.     

Measurement of cytoplasmic free Ca2+ concentration in rabbit aorta using the photoprotein, aequorin. Effect of atrial natriuretic peptide on agonist-induced Ca2+ signal generation.

Addition of norepinephrine, angiotensin II, or histamine leads to a transient rise in the cytoplasmic Ca2+ concentration ([Ca2+]i), as measured with aequorin, in rabbit aortic strips. Each induces a [Ca2+]i transient which peaks in 2 min and then falls either back to baseline (angiotensin II) or to a plateau (norepinephrine and histamine). The [Ca2+]i transient is due to the mobilization of Ca2+ from a caffeine-sensitive, intracellular pool. An elevation of [K+] to 35 mM leads to a monotonic sustained rise in [Ca2+]i which depends entirely on extracellular Ca2+, but an increase to 100 mM leads to a [Ca2+]i transient from the mobilization of intracellular Ca2+. Atrial natriuretic peptide does not alter basal [Ca2+]i nor inhibit the [Ca2+]i transient induced by either histamine or angiotensin II, but blocks that induced by norepinephrine, and blocks the plateau phase induced by either histamine or norepinephrine. The peptide inhibits the contractile response to all three agonists and to K+.     

Interaction of extracellular albumin and intravenous anaesthetics, etomidate and propofol, on calcium signalling in rat airway smooth muscle cells

It has been shown in vitro that general anaesthetics modify airway responsiveness via, at least partially, a direct inhibitory effect on calcium signalling in airway smooth muscle cells. However, in vivo, these anaesthetic compounds bind serum proteins. We have investigated the effect of exposure to extracellular albumin of freshly isolated airway smooth muscle cells on the propofol- and etomidate-induced inhibitory effect on calcium signalling. [Ca2+]i was measured by microspectrofluorimetry in rat isolated tracheal smooth muscle cells using the fluorescent dye indo-1. Propofol (3 x 10(-4) M) and etomidate (10(-4) M) were the lowest 'effective' concentrations that altered the [Ca2+]i response. This alteration consisted of a decrease in both the amplitude of the [Ca2+]i peak (from 358 +/- 13 nM to 65 +/- 15 and 108 +/- 27 nM for propofol and etomidate, respectively) and the percentage of responding cells (from 80% to 37 and 25% respectively) in response to the low concentration of ACh and a decrease in the Ca2+ oscillation frequency (from 9.9 +/- 0.3 min(-1) to 4.7 +/- 0.4 and 6.9 +/- 0.4 min(-1), respectively) in response to the high concentration of ACh. Increasing the concentration of albumin reduced the inhibitory effect of etomidate and propofol on the [Ca2+]i response to ACh. When extracellular albumin concentration was kept constant (20 g/L), increasing the concentration of etomidate by one log restored its inhibitory effect on the calcium signal. This study indicates that increasing the concentration of extracellular albumin reduces the inhibitory effect of intravenous anaesthetics on calcium signalling in airway smooth muscle cells. This report suggests that, in extrapolating in vitro dose-response relationships to those from in vivo conditions, the effect of the concentration of extracellular protein can be estimated.     

Modulation by 1,25-dihydroxycholecalciferol of the acute change in cytosolic free calcium induced by thyrotropin-releasing hormone in GH4C1 pituitary cells.

Receptor-mediated regulation of prolactin synthesis by 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in the pituitary cell strain GH4C1 is dependent on the concentration of extracellular calcium. We have now investigated the actions of 1,25(OH)2D3 on cytosolic free calcium concentrations [( Ca2+]i) in these cells using the fluorescent indicator quin2. Basal resting [Ca2+]i was unchanged in cells treated with 1 nM 1,25(OH)2D3 either acutely (from 0 to 15 min) or for periods of up to 48 h. However, the initial peak of the biphasic change in [Ca2+]i induced by thyrotropin-releasing hormone (TRH) was enhanced more than twofold in cells pretreated for 24 or 48 h with 1,25(OH)2D3. This 1,25(OH)2D3-enhanced calcium response was restricted to the initial phase of TRH action; the secondary plateau phase was unaffected. Neither the affinity nor number of TRH receptors nor the early time course of [3H]MeTRH binding to GH4C1 cells were affected by pretreatment with 1,25(OH)2D3. Because TRH binding was not altered, four sites along the intracellular signal transduction pathway of TRH action were examined. Neither protein kinase C activation nor inositol polyphosphate accumulation were enhanced in response to TRH, in 1,25(OH)2D3 pretreated cells, indicating that phosphatidylinositol hydrolysis was unchanged by pretreatment. A low concentration of ionomycin was used to probe the size of the nonmitochondrial intracellular calcium pool that is sensitive to TRH. Ionomycin was not able to mobilize more calcium from 1,25(OH)2D3 pretreated cells, indicating that TRH-responsive intracellular calcium stores were probably not enhanced by pretreatment. Chelation of extracellular calcium, however, did eliminate enhancement of the TRH response in 1,25(OH)2D3-pretreated cells. We conclude that 1,25(OH)2D3 modulates acute dynamic changes in [Ca2+]i induced by TRH without affecting basal [Ca2+]i. The mechanism of the enhanced response of 1,25(OH)2D3-pretreated cells to TRH appears to depend upon a postreceptor event independent of phosphatidylinositol hydrolysis that involves increased calcium conductance at the level of the plasma membrane. A less likely explanation involves enhancement of intracellular calcium stores in an ionomycin-resistant, EGTA-sensitive, TRH-mobilizable reservoir.     

Release of arachidonic acid from human lymphocytes in response to mitogenic lectins

After exposure to mitogenic lectins in vitro, human mononuclear cells (95% lymphocytes) that had been prelabeled with [14C]arachidonic acid rapidly released a portion of their radioactivity in the medium. Most of the released radioactivity was demonstrated to be free arachidonic acid. Although other sources are not excluded, the most important source of cell-bound radioactivity in the release reaction appeared to be phosphatidylinositol, suggesting that at least part of the response is occurring through an increase in phospholipase A2 activity. By gas liquid chromatography, other fatty acids were also shown to be released, but there was considerable selectivity in the response for arachidonic acid. The response was dependent on the availability of free Ca++ in the medium and was enhanced by serum proteins and unlabeled arachidonic acid. Most of the response appeared to be from the the lymphocytes themselves rather than from contaminating cells. The rapid generation of free arachidonic acid in response to mitogenic lectins suggests a possible role for arachidonic acid metabolites in the activation process.     

Determination of serum ionised calcium by ion-exchange electrode in normal subjects

Serum ionised calcium concentration [Ca2+] was measured with a calcium selective electrode in 65 normal people. A mean value of 1.06 mmol/l (+/- 0.04 S.D.) with an actual range of 0.97 to 1.13 mmol/l was obtained. Serum samples refrigerated at 4 degrees C for 24 h were satisfactory for analysis. Storage of whole blood samples for 6 h at room temperature before separation caused a very small error (+0.04 mmol/l). Mean results and ranges were similar in males and females and there was no significant correlation with age. No significant change in serum [Ca2+] was found following a normal meal. Serum [Ca2+] and total serum calcium showed a very slight correlation (r = 0.35). The method is reproducible and sensitive.