Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.     

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.     

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+.     

Studies on gel-filtered human platelets: isolation and characterization in a medium containing no added Ca2+, Mg2+, or K+.

We have adapted the gel filtration technique for separation of human platelets from the plasma constituents to permit use of an eluant containing no added Ca2+, Mg2+, and K+, and hence allow direct determination of the intracellular concentrations of these ions in the isolated platelets. The eluant employed is modified Ca2+-free Tyrode's buffer which contains Sr2+ (0.2 mM) as a substitute for Mg2+ and lacks added K+. The functional metabolic, and morphological properties of these isolated platelets have been determined ael-filtered platelets (GFP) to low concentrations of ADP and adrenaline was qualitatively similar to that of platelet-rich plasma (PRP). However, a slower response was observed for the GFP. This rate difference was partially or completely reversed by addition of apyrase to the medium. Analysis of the total adenine nucleotide content and the pattern of 14C incorporation into the metabolic adenine nucleotide pool indicated that isolation in this medium caused to significant change in the ATP and ADP contents or in the adenylate energy change in comparison with the PRP. However, a significant increase in the extent of hypoxanthine production from ATP was noted in GFP isolated in media lacking Mg2+. Inclusion of Mg2+ in the elution media prevented this increased hypoxanthine production. The intracellular concentrations of Ca2+, Mg2+, and K+ of the GFP as determined by atomic absorption analysis were in good agreement with the values obtained for platelets separated from plasma by high-speed centrifugation. Platelet Ca2+ and Mg2+ levels remained stable despite the lack of significant extracellular levels of these ions. However platelet K+ fell to about 30 per cent of its initial value after incubation of 90 minutes at 23 degrees C. and a coincident increase was observed in extracellular K+ concentration. This procedure for platelet isolation may be of particular value for studies on the role of Ca2+, Mg2+, and K+ in platelet physiology and metabolic processes.     

The calcium signal and neutrophil activation

The cytosolic free calcium concentration, [Ca2+]i in phagocytic cells (e.g. neutrophils, human leukemic cell line HL-60) is an important determinant of cellular activity. In resting phagocytes [Ca2+]i is low (approximately 100 nM), but in response to occupation of cell surface receptors, it rises to micromolar levels, thereby activating a variety of cellular functions. The increases in [Ca2+]i consist of two components: an immediate that is independent of extracellular Ca2+, and a more delayed that is abolished by the removal of extracellular Ca2+. These two components reflect the involvement of two subcellular structures in intracellular Ca2+ homeostasis: an intracellular Ca2+ store, referred to as the calciosome; and the plasma membrane. The function of the intracellular Ca2(+)-store depends on a Ca2(+)-pump, functionally and immunologically related to the cardiac sarcoplasmic reticulum Ca2(+)-ATPase, a Ca2(+)-storage protein, similar to muscle calsequestrin, and a Ca2(+)-release channel, which is sensitive to inositol 1,4,5-trisphosphate. The Ca2(+)-regulatory function of the plasma membrane depends on a Ca2+ pump, similar to the erythrocyte-type Ca2(+)-ATPase, and a Ca2+ channel; the activity of the Ca2+ channel is closely coupled to phosphatidylinositol turnover.     

Aspirin potentiates prestimulated acid secretion and mobilizes intracellular calcium in rabbit parietal cells.

The effects of aspirin on gastric acid secretion were studied in isolated rabbit parietal cells (PC). Aspirin (10(-5) M) potentiated histamine-, dibutyryl cyclic AMP (dbcAMP)-, forskolin- and 3-isobutyl-1-methylxanthine-stimulated acid secretion without affecting basal acid secretion. Augmentation of secretagogue-stimulated acid secretion by aspirin was dependent on calcium (Ca2+) since potentiation was blocked by removal of extracellular Ca2+ ([Ca2+]o) or addition of the calcium antagonist lanthanum chloride. Using the Ca2+ probe fura-2, aspirin (10(-6) - 2 X 10(-5) M) rapidly increased intracellular free Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. The source of released Ca2+ was intracellular as demonstrated by depletion of intracellular Ca2+ and [Ca2+]o with EGTA washing. Aspirin did not affect several other signal transduction sites involved in stimulus-secretion coupling, including the H2 receptor, intracellular cyclic AMP (cAMP), inositol 1,4,5, triphosphate (IP3) and H+,K(+)-ATPase. Aspirin decreased PC prostaglandin E2 (PGE2) content by 98%. Exogenous dimethyl PGE2 (dmPGE2) inhibited both histamine-stimulated acid secretion and its enhancement by aspirin. In contrast, dmPGE2 abolished aspirin-induced potentiation of dbcAMP-stimulated acid secretion by augmenting the dbcAMP-stimulated response. These results indicate that aspirin acts at a site beyond the adenylate cyclase/cAMP system and before the proton pump, presumably by releasing Ca2+ from an IP3-independent intracellular storage pool and by inhibiting PGE2 generation.     

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.     

Cytosolic Ca2+ and protein kinase Calpha couple cellular metabolism to membrane K+ permeability in a human biliary cell line.

Cholangiocytes represent an important target of injury during the ischemia and metabolic stress that accompanies liver preservation. Since K+ efflux serves to minimize injury during ATP depletion in certain other cell types, the purpose of these studies was to evaluate the effects of ATP depletion on plasma membrane K+ permeability of Mz-ChA-1 cells, a model human biliary cell line. Cells were exposed to dinitrophenol (50 microM) and 2-deoxyglucose (10 mM) as the standard model of metabolic injury. Whole-cell and single K+ channel currents were measured using patch clamp techniques; and intracellular [Ca2+] ([Ca2+]i) was estimated by calcium green-1 fluorescence. Metabolic stress increased [Ca2+]i, and stimulated translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosolic to particulate cell fractions. The same maneuver increased membrane K+ permeability 40-70-fold as detected by (a) activation of K+selective whole cell currents of 2,176+/-218 pA (n = 34), and (b) opening of apamin-sensitive K+ channels with a unitary conductance of 17.0+/-0.2 pS. PKCalpha translocation and channel opening appear to be related since stress-induced K+ efflux is inhibited by chelation of cytosolic Ca2+, exposure to the PKC inhibitor chelerythrine (25 microM) and downregulation of PKC by phorbol esters. Moreover, K+ currents were activated by intracellular perfusion with recombinant PKCalpha in the absence of metabolic inhibitors. These findings indicate that in biliary cells apamin-sensitive K+ channels are functionally coupled to cell metabolism and suggest that cytosolic Ca2+ and PKCalpha are selectively involved in the response.     

Activation of Ca-permeable cation channels by myocarditis-associated antibody in guinea pig ventricular myocytes.

The pathogenesis of myocarditis and dilated cardiomyopathy is though to involve autoimmunological processes and myocardial calcium overload. Serum containing antiheart antibodies associated with a murine model of myocarditis increased [Ca2+]i in guinea pig ventricular myocytes only in the presence of extracellular Ca2+. The antiheart antibody-positive serum activated Ca(2+)-permeable cation channels that were insensitive to dihydropyridines and membrane stretch. The permeability sequence was Ba2+ > Ca2+ > Na+ approximately K+, and the single-channel conductance to Ba2+ was 12 pS. The channel was activated by extracellular application of the serum during on-cell recording, which suggests that a soluble intracellular messenger may be involved. The antibody-positive serum did not alter voltage-gated Ca2+ currents. We propose that excess Ca entry in myocarditis and dilated cardiomyopathy results from activation of a Ca(2+)-permeable cationic channel by the autoantibodies.     

Cytosolic Ca2+ and phosphoinositide hydrolysis linked to constitutively active alpha 1D-adrenoceptors in vascular smooth muscle

In the present study, we analyzed changes in intracellular Ca2+ levels and inositol phosphate accumulation related to a population of alpha 1d-adrenoceptors in rat aorta resembling constitutively active receptors. Following intracellular Ca2+ store depletion by noradrenaline in Ca2+-free medium and removal of the agonist, restoration of extracellular Ca2+ induced four signals: a biphasic (transient and sustained) increase in [Ca2+]i, inositol phosphate accumulation, and a contractile response in the aorta. The transient increase in Ca2+, the inositol phosphate accumulation, and the contractile response were not observed in aortae incubated with prazosin or BMY 7378 [8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione] (a selective alpha 1d-adrenoceptor ligand), relating the three signals to alpha 1d-adrenoceptor activity. In the presence of nimodipine, only the sustained increase in Ca2+ and the inositol phosphate accumulation were observed, relating both signals to calcium entry through L-channels. The four signals were abolished by Ni2+. In the rat tail artery, where alpha 1d-adrenoceptors are not functionally active, restoration of extracellular Ca2+ after store depletion induced only a sustained increase in [Ca2+]i without inositol phosphate accumulation nor contractile response. Taken together these results suggest that in the aorta, Ca2+ entry is required for the recovery of cytosolic calcium levels and the display of the membrane signals related to the constitutive activity of alpha 1d-adrenoceptors, i.e., inositol phosphate formation and Ca2+ entry through L-type channels, which maintains a contractile response once the agonist has been removed.     

Intracellular free magnesium in human lymphocytes and the response to lectins.

1. Intracellular free [Mg2+] was measured in human peripheral blood lymphocytes using a fluorimetric method based on the dye furaptra. It was necessary to correct for the extracellular leakage of the dye by using either 10 mmol/l EDTA or 0.05 mmol/l Mn2+. 2. As the proliferative response of lymphocytes to mitogenic lectins has been linked to a dependence on extracellular Mg2+, the intracellular [Mg2+] was studied in lymphocytes stimulated with various mitogenic and non-mitogenic lectins. 3. Only lymphocytes treated with phytohaemagglutinin-L, a leucoagglutinin from Phaseolus vulgaris that binds to tri- and tetra-antennary complex glycoproteins, showed a marked increase in intracellular [Mg2+]. This effect was partially inhibited by N-acetylgalactosamine. The stimulation by different lectins of the incorporation of [3H]-thymidine into lymphocytes was not correlated to the changes in intracellular [Mg2+]. 4. The proliferative response of lymphocytes to lectins is therefore not wholly dependent on a rise in intracellular [Mg2+].     

Electrolytes and pH changes in pre-eclamptic rats

BACKGROUND: Intracellular free calcium [Ca2+]i and magnesium [Mg2+]i ions play major roles in the mechanism of vascular smooth muscle (VSM) contraction. Although essential hypertension and abnormal intracellular homeostasis of these ions have long been recognized as major icons in the pathogenesis of pre-eclampsia, the underlying mechanism(s) remain poorly understood. METHODS: Alterations of vascular smooth muscle and platelet intracellular cations [Ca2+]i, [Mg2+]i and [H+]i relative to plasma concentrations of these ions in nitric oxide synthase (NOS) blockade-induced models of pre-eclampsia have been evaluated in the present study. RESULTS: Pregnant rats injected with the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) developed a significantly elevated arterial blood pressure, proteinuria and other clinical parameters characteristic of pre-eclampsia compared to age-matched pregnant and non-pregnant rat controls that received the L-NAME vehicle only. Plasma total calcium concentration was significantly lower in pre-eclamptic rat models compared to normal pregnant rats (10.29+/-0.08 vs 10.67+/-0.18 mg/dl, p<0.05). A significant increase in plasma calcium was observed in pregnant controls compared to non-pregnant rats (10.67+/-0.18 vs 10.14+/-0.09 mg/dl, p<0.01). Plasma Ca2+ levels in pre-eclamptic rats were consistently lower than those of pregnant controls (5.69+/-0.09 vs 5.98+/-0.06 mg/dl, p<0.05). Resting levels of [Ca2+]i was significantly higher in pre-eclamptic rats than in pregnant controls. (351+/-45.2 vs 196+/-23.2 nmol/l, p<0.01). Blood pH was significantly increased in pre-eclamptic rats as compared to pregnant controls (7.16+/-0.02 vs 7.05+/-0.03, p<0.05). There was no significant difference in plasma and intracellular magnesium concentrations between the three rat groups. CONCLUSIONS: These findings suggest that a significantly decreased plasma level of Ca2+ coupled with a concomitant increase in VSM [Ca2+]i concentrations and an altered blood pH are associated with pre-eclampsia in the pregnant rat. Routine monitoring of serum pH, Ca2+ and Mg2+ especially in the late third trimester, may have potential in the early detection of patients at risk for pre-eclampsia, and monitoring the progress of diverse therapeutic regimens during clinical management.     

Amiloride-sensitive pressure-induced myogenic contraction in rat cerebral artery

The inhibitory action of amiloride on the pressure-induced contraction was assessed in isolated rat cerebral artery. The artery was mounted in an arteriograph, and the change in intracellular Ca2+ concentration ([Ca2+]i) and vessel diameter were simultaneously measured. The contractile response elicited by intraluminal pressurization was independent of endothelium, i.e. myogenic in nature, and abolished by nicardipine, a Ca2+ antagonist or by removal of extracellular Ca2+, and was potentiated by 25 mM KCl. Cyclopiazonic acid and thapsigargin, inhibitors of the Ca2+-ATPase pump of the sarcoplasmic reticulum, and a protein kinase C inhibitor calphostin C did not suppress the pressure-induced contraction. Amiloride, a putative stretch-activated cation channel blocker, attenuated with an IC50 (50% inhibitory concentration) of about 3 microM the increase in [Ca2+]i and contractile activity in response to pressure, whereas the drug showed no apparent effect on the contraction produced by high KCl or 9,11-dideoxy-11alpha,9alpha-epoxymethano prostaglandin F2alpha (U46619). Furthermore, amiloride (100 microM) did not significantly affect intracellular pH in the artery. In spite of its multiple pharmacological actions, it seems possible that amiloride is a useful alternative tool at the cellular or tissue level to study the mechanotransduction mechanisms involved in the pressure-induced contraction in rat cerebral artery.     

Halothane increases cytosolic Ca2+ and inhibits Na+/H+ exchange in L6 muscle cells

The effects of the general anesthetic halothane on the concentration of cytosolic free calcium ([Ca2+]i) and cytosolic pH (pHi), were investigated in L6 rat skeletal muscle cells. Basal [Ca2+]i was 169 +/- 8 nM, measured with the fluorescent Ca2(+)-indicator 1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2-(2'-amino-5- methylphenoxy)ethane-N,N,N',N'-tetra-acetate. Halothane (5.7 mM) increased [Ca2+]i to 225 +/- 15 nM in the presence of extracellular Ca2+, and from 137 +/- 6 nM to 179 +/- 9 nM in Ca2+ absence. This increase was dose-dependent. The anesthetic released about 50% of the releasable Ca2+ from intracellular stores. The resting pHi of L6 cells was 7.24 +/- 0.04, measured with the fluorescent pH indicator bis-carboxyethylcarboxyfluorescein. Halothane did not affect resting pHi, but inhibited cytoplasmic alkalinization by hypertonicity or cytoplasmic acidification: (1) The hypertonicity-induced alkalinization via activation of Na+/H+ exchange (to 7.50 +/- 0.08, initial rate 0.10 +/- 0.02 pH U/min) was inhibited with 5.7 mM halothane by 67%. (2) Acid-loaded cells (pHi 6.43 +/- 0.01 in cells) recovered towards neutrality via activation of Na+/H+ exchange (rate 0.47 pH U/min), and halothane inhibited the rate of pHi recovery by 50%. The halothane-mediated inhibition of alkalinizations after hypertonic exposure or acid-loading was also observed in bis-(o-amino-phenoxy)ethane-N,N,N',N'-tetra-acetate-loaded cells in Ca2(+)-free medium. Therefore, halothane increases [Ca2+]i and in parallel inhibits Na+/H+ exchange, compromising the ability of muscle cells to recover from imposed acidification.     

Selective differentiation and proliferation of human eosinophils from umbilical cord blood: calcium fluxes and superoxide ion secretion

Investigation of the physiologic mechanisms involved in the activation of eosinophils is crucial to comprehend their role in the pathogenesis of allergic reactions. To overcome the difficulty of obtaining large numbers of eosinophils, we differentiated in vitro eosinophils from human umbilical cord blood mononuclear cells. These cells responded to fMLP or PAF with an increase in [Ca2+]i, associated with O2 production. Deprivation or chelation of extracellular calcium induced a reduction of fMLP or PAF-induced [Ca2+]i rise and O2- production. Similar results were obtained with extracellular Ni2+ addition. Chelation of intracellular calcium induced an inhibition of fMLP- or PAF-induced [Ca2+]i rise and a decrease in O2- production. Our results indicate that fMLP- and PAF-dependent O2- production in eosinophils requires intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation.     

Corticotropin-releasing hormone excites adrenocorticotropin-secreting human pituitary adenoma cells by activating a nonselective cation current.

The mechanisms of corticotropin-releasing hormone (CRH) induced excitation of ACTH-secreting adenoma cells were investigated using the perforated whole-cell clamp technique and intracellular Ca2+ concentration ([Ca2+]i) measurement. CRH depolarized ACTH-secreting adenoma cells by activating a nonselective cation current that showed slight inward rectification. This channel did not seem to be a member of the Ca(2+)-activated cation currents because it was activated even when the [Ca2+]i was chelated below 50 nM. The activation of the current was induced by protein kinase A-mediated pathways. By [Ca2+]i measurement, CRH increased [Ca2+]i of these cells dependently on voltage-gated Ca2+ current. This CRH-induced [Ca2+]i increase was abolished in Na(+)-free extracellular solution, but was not abolished by the addition of 5 microM tetrodotoxin to the extracellular solution. CRH-induced ACTH secretion from the cultured adenoma cells was also abolished in Na(+)-free extracellular solution, but not in tetrodotoxin-containing extracellular solution. These data indicate that a Na+ current (maybe the nonselective cation current) other than voltage-gated Na+ current plays an important role in CRH-induced [Ca2+]i increase and ACTH secretion. CRH also activated a nonselective cation current in nonadenoma human corticotrophs, suggesting that the activation of a nonselective cation current is a physiological mechanism of CRH-induced excitation in human corticotrophs.     

Phospholipase C activation by Na+/Ca2+ exchange is essential for monensin-induced Ca2+ influx and arachidonic acid release in FRTL-5 thyroid cells.

BACKGROUND: Monensin, a Na+ ionophore, can increase cytosolic Ca2+ ([Ca2+]i) by reversing the Na+/Ca2+ exchange mechanism. This study provided additional insights into the mechanism of this Na+ ionophore-induced increase in [Ca2+]i, and emphasized the critical role of phospholipase C (PLC) in amplifying Na+/Ca2+ exchange-induced Ca2+ influx and subsequent arachidonic acid (AA) release in FRTL-5 thyroid cells. The possible involvement of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and GTP-binding (G) protein in mediating monensin-induced AA release was also explored. METHODS: FRTL-5 thyroid cells were maintained in Coon's modified Ham's F-12 medium supplemented with a 6-hormone (6H) mixture. Cytosolic Ca2+ was measured by using indo-1 AM and a dual-wave-length spectrofluorometer. Release of 3H-labeled inositol trisphosphates and arachidonic acid were determined by a scintillation counter. RESULTS: In Hank's balanced salt solution with Ca2+ (HBSS+), monensin (100 mumol/L) induced a 2.3-fold sustained Ca2+ increase associated with IP3 generation and a 6-fold increase in AA release. Deletion of extracellular Ca2+, or replacement of Na+ by choline chloride in the medium, reduced the [Ca2+]i increase by 77% and completely prevented the monensin-induced rise in AA release. Similar inhibitory effects were observed in cells pretreated with a Na+ channel blocker, or Na+/Ca2+ exchange inhibitors. In HBSS without Ca2+ (HBSS-), monensin induced a 1-fold transient [Ca2+]i increase but did not increase the AA. This Ca2+ increase was not suppressed by U-73122, a PLC inhibitor. In HBSS+, U-73122 did not affect the monensin-induced initial transient peak increase of [Ca2+]i, but reduced the sustained second phase of [Ca2+]i from 400 nmol/L to 250 nmol/L, and completely blocked AA release. A phospholipase A2 (PLA2) inhibitor blocked the monensin-induced AA release without affecting the [Ca2+]i increase. Inhibition of PKC prevented 87% to 94% of the monesin-stimulated AA release. The monensin-induced AA release was also inhibited 94% by pertussis and 51% by a MAP kinase cascade inhibitor. CONCLUSIONS: The results suggest that monensin initiates an increase in [Ca2+]i via a Na+/Ca2+ exchange mechanism that triggers more pronounced and sustained [Ca2+]i increase via activation of PLC and Ca2+ influx. The PLC activation, followed by sustained Ca2+ influx and PKC activation, is a prerequisite for PLA2-mediated processes in monensin-challenged FRTL-5 thyroid cells.     

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.     

Relationship between membrane depolarization and extracellular calcium influx during neutrophil activation

To better define the relationship between membrane depolarization and extracellular Ca2+ influx during neutrophil activation, we compared stimulation by elevating the extracellular K+ concentration, [K+]o, with stimulation by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP). Elevation of [K+]o resulted in uniform depolarization of the entire population of cells. This was associated with an influx of Ca2+ that was temporally delayed and quantitatively less than that induced by fMLP. K+ depolarization also caused increased expression of type 1 (C3b/C4b) complement receptor (CR1) and type 3 (C3bi) complement receptor (CR3), but the increments were less than with fMLP. We then used pertussis toxin to determine if guanosine triphosphate (GTP)-binding proteins were involved in these responses. Toxin inhibited the fMLP-induced membrane depolarization as well as the uptake of extracellular Ca2+ and the expression of both CR1 and CR3 induced by the chemoattractant. This indicates that the fMLP receptor is not directly coupled to an ion channel. The membrane depolarization induced by elevating [K+]o was not inhibited by toxin, but the uptake of Ca2+ and the increased expression of CR1 and CR3 were all significantly inhibited. The toxin failed to block increased CR1 and CR3 expression induced by ionomycin, demonstrating that its effects were not attributable to general toxicity. The results suggest that voltage gating is not the major mechanism by which polymorphonuclear leukocytes (PMNs) increase their permeability to extracellular Ca2+. Initial signals, whether generated by chemoattractants binding to their receptors or by small initial influxes of extracellular Ca2+, must be amplified by pertussis toxin-sensitive steps to fully increase the Ca2+ permeability and optimally activate the cell.     

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+.