Calcium efflux and influx in f-met-leu-phe (fMLP)-activated human neutrophils are chronologically distinct events

The kinetics of efflux of calcium mobilized from intracellular stores following activation of human neutrophils with the synthetic chemotactic tripeptide, fMLP (1 μm), as well as that of the subsequent store-operated influx of this cation, has been measured by radiometric procedures using ⁴⁵Ca. These procedures enabled distinction between net efflux and influx of ⁴⁵Ca. Preincubation of neutrophils in medium containing ⁴⁵Ca as the sole source of Ca²⁺, followed by activation with fMLP, resulted in a rapid efflux of the cation, which coincided with its release from intracellular stores. Efflux terminated at ≈ 30 s after addition of fMLP to neutrophils and resulted in the loss of 42 ± 3% (P< 0.005) of cell-associated ⁴⁵Ca. Net influx of ⁴⁵Ca, which was insensitive to the voltage-dependent Ca²⁺ channel blockading agent, verapamil (20 μm), could only be detected at 30–60 s after the addition of fMLP to neutrophils, and proceeded for about 5 min, resulting in intracellular concentrations of Ca²⁺ which were 27 ± 3% (P< 0.05) higher than preactivation levels. These results demonstrate that the efflux of cytoplasmic Ca²⁺ mobilized from intracellular stores during activation of neutrophils by fMLP, and the subsequent influx of extracellular Ca²⁺ to replete these stores, are chronologically distinct events in fMLP-activated neutrophils.     

Accelerated calcium influx and hyperactivation of neutrophils in chronic granulomatous disease

The relationship between activation of NADPH-oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from four subjects with chronic granulomatous disease (CGD). Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, and net efflux and influx of Ca2+ by radiometric procedures. Exposure of normal neutrophils to the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM) was accompanied by an abrupt increase in cytosolic Ca2+ coincident with membrane depolarization and efflux of the cation. These events terminated at around 30 s after the addition of FMLP and were followed by membrane repolarization and store-operated influx of Ca2+, both of which were superimposable and complete after about 5 min. Activation of CGD neutrophils was also accompanied by an increase in cytosolic Ca2+, which, in spite of an efficient efflux response, was prolonged in relation to that observed in normal cells. This prolonged increase in cytosolic Ca2+ in activated CGD neutrophils occurred in the setting of trivial membrane depolarization and accelerated influx of Ca2+, and was associated with hyperactivity of the cells according to excessive release of elastase and increased activity of phospholipase A2. Treatment of CGD neutrophils with the type 4 phosphodiesterase inhibitor, rolipram (1 microM) restored Ca2+ homeostasis and attenuated the increase in elastase release. These findings support the involvement of NADPH-oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses and granuloma formation which are characteristic of CGD.     

Exposure of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-activated human neutrophils to the Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine is associated with impaired calcium efflux and potentiation of primary granule enzyme release.

The effects of pathologically relevant concentrations (0.38 to 12.5 microM) of the proinflammatory, Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine (1-hp) on Ca2+ metabolism and intracellular cyclic AMP (cAMP) in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM)-activated human neutrophils, as well as on the release of myeloperoxidase (MPO) and elastase from these cells, have been investigated in vitro. Ca2+ fluxes were measured by the combination of a fura-2/AM-based spectrofluorimetric method and radiometric procedures, which together enable distinction between net efflux and influx of the cation, while radioimmunoassay and colorimetric methods were used to measure cAMP and granule enzymes, respectively. Coincubation of neutrophils with 1-hp did not affect intracellular cAMP levels or the FMLP-activated release of Ca2+ from intracellular stores but did retard the subsequent decline in the chemoattractant-induced increase in the concentration of cytosolic free Ca2+. These effects of 1-hp on the clearance of Ca2+ from the cytosol of activated neutrophils were associated with decreased efflux of the cation from the cells and increased release of MPO and elastase, while the delayed store-operated influx of the cation into the cells was unaffected by the pigment. The plasma membrane Ca2+-ATPase rather than a Na+-Ca2+ exchanger appeared to be the primary target of 1-hp. These observations suggest that the proinflammatory interactions of 1-hp with activated human neutrophils are a consequence of interference with the efflux of cytosolic Ca2+ from these cells.     

Capacitative Ca2+ influx and a Ca2u+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells

We have investigated the effect of genistein on the hormone-stimulated Ca²⁺ influx and on a 28 pS nonselective cation channel in mouse pancreatic acinar cells using the Ca²⁺ indicator fluo-3 and the patch-clamp technique. The identity of the Ca^2u+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca²⁺-dependent nonselective cation channel as a potential pathway for Ca²⁺ influx. Capacitative Ca²⁺ entry was induced by depletion of intracellular Ca²⁺ stores with 500 nM acetylcholine or with the Ca²⁺ ATPase inhibitor 2,5-di-tert- butylhydroquinone. In the presence of 100 M genistein, Ca²⁺ release was unimpaired, whereas Ca²⁺ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca²⁺-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28 pS nonselective cation channel does not contribute to Ca²⁺ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca²⁺ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca²⁺ entry mechanism in mouse pancreatic acinar cells is unclear.     

Function and stimulus-specific effects of phorbol 12-myristate 13-acetate on human polymorphonuclear neutrophils: Autoregulatory role for protein kinase C in signal transduction

Exposure of polymorphonuclear neutrophils (PMNs) to phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent (1–10 ng/ml) inhibition of granule exocytosis induced with the receptor-specific ligands,N-formylmethionyl-leucyl-phenylalanine (FMLP), pepstatin A, 5(S),12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB₄), and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), PMA exerted a marginal inhibitory effect on calcium ionophore A23187-induced PMN degranulation, and the PMA analog, 4-phorbol 12, 13-didecanoate (4-PDD), was inactive. However, PMA potentiated AGEPC, pepstatin A, FMLP, LTB₄, and A23187-stimulated Superoxide anion (O ₂ ^– ) production. The mobilization of intracellular sequestered calcium (Ca²⁺) by the receptor-specific ligands, as reflected by a rise in the cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) in PMNs loaded with the CA²⁺-sensitive dye, Fura-2, was suppressed by PMA. A protein kinase C (PKC) inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) reversed the PMA-mediated inhibition of PMN degranulation and intracellular CA²⁺ mobilization. However, another, but less potent PKC inhibitor,N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), had no effect on the inhibition of PMN activation by PMA.     

Agonist-induced calcium flux,phosphoinositide metabolism,aggregation and enzyme secretion in human neutrophils

Formyl-methionyl-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and leukotriene B₄ (LTB₄) are potent activators of human neutrophils. Using human neutrophils prelabelled with the fluorescent indicator dye, Quin 2, or with [³²P]-orthophosphate, we examined the effects of these stimuli on intracellular free calcium concentration, [Ca²⁺]i, and, on various indices of phosphoinositide metabolism, including [³²P]-phosphatidic acid (PtdA) formation. The concentration-dependence of the observed changes in [Ca²⁺]i or [³²P]-PtdA were then compared to stimulus-induced aggregation and enzyme release (-N-acetylglucosaminidase (NAG) and lysozyme).FMLP, PAF and LTB₄ caused a concentration-dependent elevation of [Ca²⁺]i, aggregation and enzyme release. However, unlike FMLP and PAF, LTB₄ (2.5 M) did not cause significant formation of [³²P]-PtdA. The concentration response curves for agonist-induced elevation of [Ca²⁺]i lie to the left of those for aggregation and enzyme release. FMLP and PAF also caused an elevation of [Ca²⁺]i at concentrations lower than those required to elicit [³²P]-PtdA formation.These observations suggest that [Ca²⁺]i elevationper se cannot mediate human neutrophil functional responses to FMLP, PAF and LTB₄. Consequently there may exist other mediator(s) that act in concert with [Ca²⁺]i or are triggered by [Ca²⁺]i elevation to promote human neutrophil activation. Both the elevation of [Ca²⁺]i and the formation of these putative mediator(s) in response to LTB₄ apparently occur independently of inositol phospholipid hydrolysis.     

In vitro effects of Ω-3 fatty acids on neutrophil intracellular calcium homeostasis and receptor expression for FMLP and LTB4

Diets enriched in-3 fatty acids exert antiinflammatory properties by suppressing some neutrophil (PMN) functions. Changes in cytosolic Ca²⁺ concentration, [Ca²⁺]_i, are important for PMN activation and are in part regulated by membrane Ca²⁺ ATPases. Since membrane proteins are influenced by their lipid environment, we investigated the in vitro effects of the-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the [Ca²⁺]_i of PMNs in response to f-Met-LeuPhe (FMLP), leukotriene B₄ (LTB₄), and ionomycin. The resting [Ca²⁺]_i of PMNs (in high Ca²⁺ environment) was increased after pretreatment (37C, 2 h) with DHA, but not with EPA, or the other fatty acids, oleic acid (OA), or linolenic acid (LA). The stimulated [Ca²⁺]_i by either FMLP or LTB₄ was suppressed in a high Ca²⁺ environment after pretreatment with either EPA or DHA but not with OA or LA. The stimulated [Ca²⁺]_i rise by ionomycin was augmented after pretreatment with DHA but not with EPA, OA, or LA. Pretreatment of PMNs with either EPA or DHA reduced the receptor expression for both FMLP and LTB₄. Since -3 fatty acids inhibit the expression of receptors for two activators of PMNs, FMLP and LTB₄, as well as the [Ca²⁺]_i rise in response to those two stimuli, we propose that the antiinflammatory properties of EPA and DHA may be attributed, at least in part, to alteration in membrane activation of phagocytes.     

Effect of nisoldipine on priming and activation of the human neutrophil respiratory burst

Nisoldipine inhibits calcium (Ca⁺⁺) influx in human neutrophils: Preincubation with the dihydropyridine, nisoldipine (1.5 M) resulted in a 30% decrease in^[45]Ca⁺⁺ influx during formyl-methionineleucine-phenylalanine (FMLP) stimulation in primed as well as resting cells. Although the drug does not inhibit Ca⁺⁺ dependent effector functions elicited by FMLP, e.g. superoxide (O ₂ ^– ) production, it inhibits FMLP priming, a phenomenon that is independent, of extracellular Ca⁺⁺. Nisoldipine, exhibited a narrow dose response with an ED₅₀ of ca. 1 M and total inhibition of primedO2/– response at 1.5 M. Nisoldipine (1.5 M) also abolished the incremental rise of Ca⁺⁺ _i in primed neutrophils stimulated with FMLP. The dissociation of nisoldipine inhibitory effects on cell effector function and Ca⁺⁺ transport were corroborated in studies with neutrophils stimulated with influenza virus and phorbol myristate acetate (PMA), stimuli which do not exhibit an extracellular Ca⁺⁺-dependence in their elicited responses. Unlike in FMLP-stimulated cells, nisoldipine reduced influenza virus and PMA initiated respiratory burst, indicating that this drug has inhibitory effects on neutrophil function independent of its effect on Ca⁺⁺ metabolism. Possible sites of action are postulated at phospholipase A₂ or calmodulin-regulated activities. Caution is thus required in interpreting the effects of dihydropyridine on cell function, when the drug is used at micromolar concentration.This work was supported in part by NIH grants NIAID 20064, NIHDK 37105 and training grant HL07501.     

Role of extracellular calcium in neutrophil responsiveness to chemotactic tripeptides

Cellular stores of Ca²⁺, but not extracellular Ca²⁺, are required for effective FMLP stimulation of neutrophil Of production and degranulation. Neutrophils transferred from Ca²⁺-containing to Ca²⁺-free medium gradually lose their responsiveness to FMLP, such that after 40 min in the Ca²⁺-free environment they have lost 60–70% of their initial responsiveness to FMLP, The loss in responsiveness is reflected both in an increase in lag interval and decrease in velocity of O ₂ ^– synthesis. The rate of decline in responsiveness to FMLP is greatly accelerated when neutrophils incubated in the presence of A23187 and Ca²⁺-free medium, while the rate of loss of responsiveness to FMLP is not affected by EGTA but the extent of loss is increased. Gradual recovery of FMLP-induced Of generation occurs when cells are transferred from Ca²⁺-free to Ca²⁺-containing medium. PMA-induced neutrophil of generation is not influenced by the presence or absence of extracellular Ca²⁺. It is our view that the rise or fall of neutrophil responsiveness reflects repletion or depletion of cellular stores of Ca²⁺ essential for stimulus-effector coupling and that the role of extracellular Ca²⁺ is subservient to maintenance of these stores.     

Protein Phosphatase Inhibitors Exert Specific and Nonspecific Effects on Calcium Influx in Thapsigargin-Treated human Neutrophils

C2-ceramide but not inhibitors of phosphatase types 1 and 2A (okadaic acid, calyculin A, tautomycin) blocked store-regulated Ca²⁺ entry induced in human neutrophils by thapsigargin. This contrasts with previous results showing that both types of compounds inhibit Ca²⁺ influx in fmet-leu-phe-treated cells. In present studies, phosphatase inhibitors increased the rate of secondary Ca²⁺ influx in a temperature-dependent manner. Their mechanism of action appeared to be independent of phosphatase inhibition since the inactive congeners, norokadaone and tetraacetyl okadaic acid, also potentiated Ca²⁺ influx at similar concentrations. When Ca²⁺ stores were predischarged by thapsigargin, okadaic acid but not norokadaone acted synergistically with fMLP to inhibit subsequent Ca²⁺ entry. Results suggest that blockade of Ca²⁺ influx in neutrophils is mediated by a phosphorylation reaction that is prolonged by phosphatase inhibitors. The requisite phosphorylation occurs in fMLP-activated cells but may be absent in cells incubated with thapsigargin.     

Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na+/Ca2+ exchange mechanism

Törnquist , K. 1992. Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na⁺/Ca²⁺ exchange mechanism. Acta Physzol Scand 144 , 341–348. Received 28 April 1 991 , accepted 30 October 1991. ISSN 0001–6772. Endocrine Research Laboratory, University of Helsinki, Minerva Foundation Institute for Medical Research, Helsinki, Finland. The effect of extracellular Na⁺ on cytosolic free Ca²⁺ and on influx and efflux of Ca²⁺ was investigated in FRTL-5 thyroid cells. Stimulating the cells with the purinergic agonist ATP induced a rapid efflux of ⁴⁵Ca²⁺ from cells loaded with ^4aCa²⁺. Replacement of extracellular Na⁺ with choline⁺, significantly decreased the adenosine triphosphate-induced efflux of ⁴⁵Ca²⁺. Furthermore, adenosine triphosphate-induced uptake of ⁴⁵Ca²⁺ was increased when extracellular Na⁺ was replaced with choline⁺, compared with the uptake seen in Na⁺ buffer. Replacing extracellular Na⁺ with choline⁺, increased resting levels of cytosolic free Ca²⁺ from 50 ± 2 nM (mean ± SE) to 81 ± 3 nM (P < 0.05) in Fura 2 loaded cells. In cells preincubated with 1 mM ouabain for 30 min, resting cytosolic free Ca²⁺ increased to 73 ± 3 nM (P < 0.05). In a Na⁺ buffer, the adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ was 872 ± 59 nM, compared with 1070 ±63 nM in choline' buffer (P < 0.05). The plateau level of cytosolic free Ca²⁺ in response to adenosine triphosphate was 130±16 nM in Na⁺ buffer, compared with 209±9 nM in choline⁺ buffer (P < 0.05). Readdition of Na⁺ to the plateau phase decreased cytosolic free Ca⁺² to 152 ± 5 nM. Stimulating the cells with 10 μM of the Na^+-selective monovalent ionophore monensin increased cytosolic free Ca²⁺ from 53 ± 9 nM to 12416 nM (P < 0.05). This increase in cytosolic free Ca²⁺ was dependent on both extracellular Na⁺ and extracellular Ca²⁺ If cells were first stimulated with monensin, and then with adenosine triphosphate, the transient increase in cytosolic free Ca²⁺ was 1027 ± 24 nM (P < 0. 05 , compared with control cells). The results thus indicate, that FRTL-5 cells have a functional Na⁺/Ca²⁺ exchange mechanism and that this mechanism is of importance in restoring adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ to resting cytosolic free Ca²⁺ levels.     

Ryanodine modulation of45Ca efflux and tension in rabbit aortic smooth muscle

The effects of ryanodine on isometric tension development, net cellular Ca content and⁴⁵Ca efflux were measured in the isolated rabbit aorta. Pretreatment of the tissue with 10 M ryanodine for 30 min reduced the norepinephrine (NE)-induced tension of the aortic rings bathed in the absence of extracellular Ca²⁺ in parallel with a reduction in the NE-stimulated⁴⁵Ca efflux. Ryanodine alone caused a delayed moderate increase in⁴⁵Ca efflux, slowly increasing the fractional loss from 0.02/min to 0.03/min, without any increase in tension. The increased⁴⁵Ca efflux was accompanied by a decrease in the net cellular Ca content. When ryanodine and NE were administered in sequence during⁴⁵Ca efflux, we found a quantitative correlation between the ryanodine induced loss of⁴⁵Ca and the inhibition of the NE-induced⁴⁵Ca release. We conclude that the inhibition of the NE-induced contraction by 10 M ryanodine is the result of depletion of Ca from intracellular stores rather than inhibitions of Ca²⁺ release.Abbreviations used Ca total calcium - Ca²⁺ free calcium - NE norepinephrine - SR sarcoplasmic reticulum - PSS physiological salt solution     

Basolateral K+ efflux is largely independent of maxi-K+ channels in rat submandibular glands during secretion

The involvement of large-conductance, voltage- and Ca²⁺-activated K⁺ channels (maxi-K⁺ channels) in basolateral Ca²⁺-dependent K⁺-efflux pathways and fluid secretion by the rat submandibular gland was investigated. Basolateral K⁺ efflux was monitored by measuring the change in K⁺ concentration in the perfusate collected from the vein of the isolated, perfused rat submandibular gland every 30 s. Under conditions in which the Na⁺/K⁺-ATPase and Na⁺-K⁺-2Cl^– cotransporter were inhibited by ouabain (1 mmol/l) and bumeta-nide (50 mol/l) respectively, continuous stimulation with acetylcholine (ACh) (1 mol/l) caused a transient large net K⁺ efflux, followed by a smaller K⁺ efflux, which gradually returned to the basal level within 10 min. These two components of the K⁺ efflux appear to be dependent on an increase in cytosolic Ca²⁺ concentration. The initial transient K⁺ efflux was not affected by charybdotoxin (100 nmol/l) or tetraethylammonium (TEA) (5 mmol/l) but the smaller second component was strongly and reversibly inhibited by charybdotoxin (100 nmol/l) and TEA (0.1 and 5 mmol/l). The initial K⁺ efflux transient induced by ACh was inhibited by quinine (0.1–3 mmol/l), quinidine (1–3 mmol/l) and Ba²⁺ (5 mmol/l), but not by verapamil (0.1 mmol/l), lidocaine (1 mmol/l), 4-aminopyridine (1 mmol/l) or apamin (1 mol/l). Ca²⁺-dependent transient large K⁺ effluxes induced by substance P (0.01 mol/l) and A23187 (3 mol/l) were not inhibited by TEA (5 mmol/l or 10 mmol/l). A23187 (3 mol/l) evoked a biphasic fluid-secretory response, which was not inhibited by TEA (5 mmol/l). Patch-clamp studies confirmed that the whole-cell outward K⁺ current attributable to maxi-K⁺ channels obtained from rat submandibular endpiece cells was strongly inhibited by the addition of TEA (1–10 mmol/l) to the bath. It is concluded that maxi-K⁺ channels are not responsible for the major part of the Ca²⁺-dependent basolateral K⁺ efflux and fluid secretion by the rat submandibular gland.     

Analogue computation of transient changes of intracellular free Ca2+ concentration with the low affinity Ca2+ indicator furaptra during whole-cell patch-clamp recording

The measurement of cytosolic free Ca²⁺ ion concentration ([Ca²⁺]) with low affinity Ca²⁺ indicators has advantages for kinetic studies of cytosolic [Ca²⁺] transients when compared with more commonly used high affinity Ca²⁺ indicators. Their dynamic range and linearity are better suited to measurement of high localised transient concentration changes that exist near sites of influx or release, and the additional buffering introduced by the indicator is minimised. The fluorescent indicator furaptra (magfura-2) has low affinity for Ca²⁺ (approx. 50 M) and can be used conveniently with single wavelength excitation at 420 nm with the procedure described by Konishi et al. [6]. The application of this protocol in whole-cell patch-clamp recording permits calibrated measurements of [Ca²⁺] during an experiment with minimal distortion of the time course and amplitude of [Ca²⁺] transients. A simple and inexpensive analogue circuit is described for direct computation of [Ca²⁺] from furaptra fluorescence with single wavelength excitation and emission during whole-cell recording. Data are shown which compare furaptra and fluo-3 estimates of the time course and amplitude of [Ca²⁺] changes in vascular endothelia, Purkinje neurones and hepatocytes.     

Calcium exchange in the resting and electrically stimulated canine myocardium

⁴⁵Ca²⁺ exchange was studied in small pieces of canine left ventricular free wall. The loss of⁴⁵Ca²⁺ from⁴⁵Ca²⁺ equilibrated tissue into ice-cold (4°C) wash medium could be best fitted with a model containing a minimum of 3 compartments.⁴⁵Ca²⁺ uptake into and⁴⁵Ca²⁺ efflux from the most slowly exchanging compartment (compartment 3) at 37°C allowed it to be subdivided into two fractions; a rapidly exchanging fraction (t _1/21.25 min) and a slowly exchanging fraction (t _1/250 min). The total Ca²⁺ content of compartment 3 was enhanced by isoproterenol but little affected by caffeine. The slowt _1/2 for exchange of the Ca²⁺ in compartment 3 at 4°C and its increased Ca²⁺ content following isoproterenol treatment suggest that this compartment contains some Ca²⁺ of intracellular origin. In addition, the finding that the rapidly exchanging part of compartment 3 could be preserved by cooling the tissue to 4°C shows that rapidly exchanging Ca²⁺ compartments can be studied in superfused cardiac preparations using this technique. Action potentials, elicited by electrical stimulation of the tissue, caused large changes in the Ca²⁺ content of compartment 3 (up to 170 M/kg) indicating that this postulated intracellular compartment may play a significant role in the normal contraction-relaxation cycle.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

Flufenamate and Gd3+ inhibit stimulated Ca2+ influx in the epithelial cell line CFPAC-1

The relevant influx pathway for stimulated Ca²⁺ entry into epithelial cells is largely unknown. Using flufenamate (Flu) and Gd³⁺, both known pharmacological blockers of non-selective cation currents in other epithelial preparations, we tested whether the stimulated Ca²⁺ entry in CFPAC-1 cells was inhibited by these agents. Transmembraneous Ca²⁺ influx into CFPAC-1 cells was stimulated by either ATP (10^–4 and 10^–5 mol/l), carbachol (CCH, 10^–4 mol/l) or thapsigargin (TG, 10^–8 mol/l). Three different experimental approaches were used. (1) Because the plateau phase of an agonist-induced [Ca²⁺]_i transient reflects Ca²⁺ influx into these cells, we investigated the influence of Flu and Gd³⁺ on the level of the stimulated [Ca²⁺]_i plateau. (2) The fura-2 Mn²⁺-quenching technique was used to visualise divalent cation entry and monitor its inhibition. (3) During the refilling period after agonist-induced discharge of the intracellular pools the putative influx inhibitors Flu and Gd³⁺ were given and subsequently the filling state of the agonist-sensitive intracellular stores tested. The results from the first experimental approach showed that both Flu and Gd³⁺ were potent inhibitors of the stimulated Ca²⁺ entry in CFPAC-1 cells. Flu reversibly decreased the ATP-induced [Ca²⁺]_i plateau in a concentration dependent manner, with an IC₅₀ value of 33 mol/l (n = 6). Similar results were obtained for the CCH-(n = 5) and the TG-induced (n = 5) [Ca²⁺]_i plateau. Gd³⁺ concentration dependently inhibited the stimulated Ca²⁺ plateau. A complete block of the ATP-induced [Ca²⁺]_i plateau was seen at 0.5 mol/l (ATP 10^–5 mol/l, n = 8). The second approach showed that Flu (10^–4 mol/l) completely inhibited the ATP- (10^–5 mol/l, n = 3), CCH-(10^–4 mol/l, n = 4) and TG-(10^–8 mol/l, n = 3)-induced fura-2 Mn²⁺ quench. Gd³⁺ also inhibited the fura-2 Mn²⁺-quenching rate (n = 9). The third approach showed that Flu (n = 6) and Gd³⁺ (n = 8) inhibited the refilling of the ATP-sensitive intracellular Ca²⁺ store. These results show that inhibitors of non-selective cation currents in other epithelial preparations are potent inhibitors of stimulated Ca²⁺ influx in CFPAC-1 cells. Whether this inhibitory effect concerns a non-selective cation channel remains to be established.     

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in HT29 colonic carcinoma cells

Cl^– secretion in HT₂₉ cells is regulated by agonists such as carbachol, neurotensin and adenosine 5-triphosphate (ATP). These agonists induce Ca²⁺ store release as well as Ca²⁺ influx from the extracellular space. The increase in cytosolic Ca²⁺ enhances the Cl^– and K⁺ conductances of these cells. Removal of extracellular Ca²⁺ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca²⁺ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=6) inhibited ATP (0.1 mmol·l^–1) induced increases in whole-cell conductance (G _m). When Cl^– and K⁺ currents were inhibited by the presence of Cs₂SO₄ in the patch pipette and gluconate in the bath, ATP (0.1 mmol·l^–1) still induced a significant increase in G _m from 1.2±0.3 nS to 4.7±1 nS (n=24). This suggests that ATP induces a cation influx with a conductance of approximately 3–4 nS. This cation influx was inhibited by flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=9). When Ba²⁺ (5 mmol·l^–1) and 4,4-diisothiocyanatostilbene-2-2-disulphonic acid (DIDS, 0.1 mmol·l^–1) were added to the KCl/K-gluconate pipette solution to inhibit K⁺ and Cl^– currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol·l^–1) reduced the membrane current (I _m) significantly from 86±14 pA to 54±11 pA (n=13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5–10 mmol·l^–1 1,2-bis-(2-aminoethoxy)ethane-N,N,N,N-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA). The zero-current membrane voltage (V _m) and I _m (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30–120 s after membrane rupture. V _m depolarised significantly from –33±2 mV to –12±1 mV, and I _m fell significantly from 17±2 pA to 8.9±1.0 pA (n=71). This negative current, representing a cation inward current, was activated when Ca²⁺ stores were emptied and was reduced significantly (I _m) when Ca²⁺ and/or Na⁺ were removed from the bathing solution: removal of Ca²⁺ in the absence of Na⁺ caused a I _m of 5.0±1.2 pA (n=12); removal of Na⁺ in the absence of Ca²⁺ caused a I _m of 12.8±3.5 pA (n=4). The cation inward current was also reduced significantly by La³⁺, Gd³⁺, and flufenamate. We conclude that store depletion induces a Ca²⁺/Na⁺ influx current in these cells. With 145 mmol·l^–1 Na⁺ and 1 mmol·l^–1 Ca²⁺, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.     

Calcium signaling in B cells: Regulation of cytosolic Ca increase and its sensor molecules,STIM1 and STIM2

Calcium signals are crucial for diverse cellular functions including adhesion, differentiation, proliferation, effector functions and gene expression. After engagement of the B cell receptor, the intracellular calcium ion (Ca²⁺) concentration is increased promoting the activation of various signaling cascades. While elevated Ca²⁺ in the cytosol initially comes from the endoplasmic reticulum (ER), a continuous influx of extracellular Ca²⁺ is required to maintain the increased level of cytosolic Ca²⁺. Store-operated Ca²⁺ entry manages this process, which is regulated by an ER calcium sensor, stromal interaction molecule (STIM). STIM proteins sense changes in the levels of Ca²⁺ stored within the ER lumen and regulates the Ca²⁺-release activated Ca²⁺ channel in the plasma membrane. This review focuses on the signaling pathways leading to Ca²⁺ influx and the role of Ca²⁺ signals in B cell functions.     

Calcium uptake in preterminal central synapses: Importance of mitochondria

Summary Energy dependent ⁴⁵Ca²⁺ uptake in the synaptosomal preparation from guinea pig cortex has been investigated. ⁴⁵Ca²⁺ uptake was stimulated by ATP, the absolute value of uptake being dependent on the extent of synaptosomal disruption caused by osmotic shock. A quantitative comparison of microsomal and mitochondrial ATP-dependent ⁴⁵Ca²⁺ uptake showed that only mitochondria had a large enough capacity to account for the Ca uptake levels observed in the synaptosomal preparation. ATP-stimulated ⁴⁵Ca²⁺ uptake in mitochondria, intact and shocked synaptosomes was inhibited by atractyloside, DNP, oligomycin and ruthenium red but unaffected by antimycin A and rotenone. This was interpreted as evidence that mitochondria were responsible for ATP-dependent synaptosomal Ca²⁺ uptake, the increase in uptake seen on osmotic lysis being due to the deocclusion of intraterminal mitochondria. Synaptosomal and mitochondrial ⁴⁵Ca²⁺ uptake was also stimulated by the mitochondrial respiratory substrate glutamate; this uptake was sensitive to antimycin A, DNP, rotenone and ruthenium red but insensitive to atractyloside or oligomycin thus indicating it was of mitochondrial origin. No change in glutamate-dependent ⁴⁵Ca²⁺ uptake was seen on osmotic lysis of the synaptosomes as the expected increase due to the release of occluded mitochondria was counterbalanced by the damaging effect of hypo-osmotic shock on the glutamate-stimulated ⁴⁵Ca²⁺ uptake process.