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Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase activates the serum- and glucocorticoid-inducible kinase 3 (SGK3). The present study explored the role of SGK3 in mast cell function. Mast cells were isolated and cultured from bone marrow (BMMCs) of gene-targeted mice lacking SGK3 ([sgk3.sup.-/-]) and their wild-type littermates ([sgk3.sup.+/+]). BMMC numbers in the ear conch were similar in both genotypes. Stimulation with IgE and cognate antigen triggered the release of intracellular [Ca.sup.2+] and entry of extracellular [Ca.sup.2+]. Influx of extracellular [Ca.sup.2+] but not [Ca.sup.2+] release from intracellular stores was significantly blunted in [sgk3.sup.-/-] BMMCs compared with [sgk3.sup.+/+] BMMCs. Antigen stimulation further led to a rapid increase of a [K.sup.+]-selective conductance in [sgk3.sup.+/+] BMMCs, an effect again blunted in [sgk3.sup.-/-] BMMCs. In contrast, the [Ca.sup.2+] ionophore ionomycin activated [K.sup.+] currents to a similar extent in [sgk3.sup.-/-] and in [sgk3.sup.+/+] BMMCs. [beta]-Hexosaminidase release, triggered by antigen stimulation, was also significantly decreased in [sgk3.sup.-/-] BMMCs. IgE-dependent anaphylaxis measured as a sharp decrease in body temperature upon injection of DNP-HSA antigen was again significantly blunted in [sgk3.sup.-/-] compared with [sgk3.sup.+/+] mice. Serum histamine levels measured 30 rain after induction of an anaphylactic reaction were significantly lower in [sgk3.sup.-/-] than in [sgk3.sup.+/+] mice. In conclusion, both in vitro and in vivo function of BMMCs are impaired in gene targeted mice lacking SGK3. Thus SGK3 is critical for proper mast cell function. allergy; anaphylaxis; calcium ion channels; degranulation; potassium ion channels; [K.sub.ca] 3.1; phosphoinositol-3 kinase doi: 10.1152/ajpcell.00539.2009.

Dendritic cells (DCs) are antigen-presenting cells that provide a link between innate and adaptive immunity. Ca2+-dependent signaling plays a central regulatory role in DC responses to diverse antigens. DCs are a primary target of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a secosteroid hormone, that, in addition to its wellestablished action on Ca2+ homeostasis, possesses immunomodulatory properties. Surprisingly, nothing is known about its effects on DC cytosolic Ca2+ activity. The present study explored whether 1,25(OH)2D3 modifies the intracellular Ca2+ concentration ([Ca2+]i) in DCs. Here we show that mouse DCs expressed K+-independent (NCX1-3) and K+-dependent (NCKX1, 3, 4, and 5) Na+/Ca2+ exchangers. Acute application of LPS (100 ng/ml) to DCs increased [Ca2+]i, an effect significantly blunted by prior incubation with 1,25(OH)2D3. 1,25(OH)2D3 increased the membrane abundance of the NCKX1 protein, up-regulated the K+- and Na+-dependent Ca2+ entry and enhanced the K+-dependent Na+/ Ca2+ exchanger currents. The NCKX blocker 3',4'-dichlorobenzamyl (DBZ) reversed the inhibitory effect of 1,25(OH)2D3 on the LPS-induced increase of [Ca2+]i. Expression of the costimulatory molecule CD86 was down-regulated by 1,25(OH)2D3, an effect reversed by DBZ. In summary, 1,25(OH)2D3 blunts the LPS-induced increase in [Ca2+]i by stimulation of Na+/Ca2+exchanger-dependent Ca2+ extrusion, an effect that contributes to 1,25(OH)2D3-mediated immunosuppression. The results disclose completely novel mechanisms in the regulation of DC maturation and function. [ABSTRACT FROM AUTHOR]

Stimulation of the mast cell IgE-receptor (FcεRI) by antigen leads to stimulation of Ca2+ entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca2+ further activates Ca2+-activated K+ channels, which in turn provide the electrical driving force for Ca2+ entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca2+ and Ca2+-activated K+ channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca2+ concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 μM) and wortmannin (100 nM). Antigen-stimulated Ca2+ entry but not Ca2+ release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca2+ entry was further inhibited by TRPV blocker ruthenium red (10 μM). Ca2+ entry following readdition after Ca+-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca2+-activated K+ channels. These observations disclose PI3-kinase-dependent regulation of Ca2+ entry and Ca2+-activated K+-channels, which in turn participate in triggering mast cell degranulation. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are antigen-presenting cells that provide a link between innate and adaptive immunity. Ca2+-dependent signaling plays a central regulatory role in DC responses to diverse antigens. DCs are a primary target of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a secosteroid hormone, that, in addition to its wellestablished action on Ca2+ homeostasis, possesses immunomodulatory properties. Surprisingly, nothing is known about its effects on DC cytosolic Ca2+ activity. The present study explored whether 1,25(OH)2D3 modifies the intracellular Ca2+ concentration ([Ca2+]i) in DCs. Here we show that mouse DCs expressed K+-independent (NCX1-3) and K+-dependent (NCKX1, 3, 4, and 5) Na+/Ca2+ exchangers. Acute application of LPS (100 ng/ml) to DCs increased [Ca2+]i, an effect significantly blunted by prior incubation with 1,25(OH)2D3. 1,25(OH)2D3 increased the membrane abundance of the NCKX1 protein, up-regulated the K+- and Na+-dependent Ca2+ entry and enhanced the K+-dependent Na+/ Ca2+ exchanger currents. The NCKX blocker 3',4'-dichlorobenzamyl (DBZ) reversed the inhibitory effect of 1,25(OH)2D3 on the LPS-induced increase of [Ca2+]i. Expression of the costimulatory molecule CD86 was down-regulated by 1,25(OH)2D3, an effect reversed by DBZ. In summary, 1,25(OH)2D3 blunts the LPS-induced increase in [Ca2+]i by stimulation of Na+/Ca2+exchanger-dependent Ca2+ extrusion, an effect that contributes to 1,25(OH)2D3-mediated immunosuppression. The results disclose completely novel mechanisms in the regulation of DC maturation and function. [ABSTRACT FROM AUTHOR]

Ca(+)-dependent signaling regulates the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. The activity of DCs is suppressed by glucocorticoids, potent immunosuppressive hormones. The present study explored whether the glucocorticoid dexamethasone influences the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in DCs. To this end, DCs were isolated from mouse bone marrow. According to fura-2 fluorescence, exposure of DCs to lipopolysaccharide (LPS, 100 ng/ml) increased [Ca(2+)](i), an effect significantly blunted by overnight incubation with 10 nM dexamethasone before LPS treatment. Dexamethasone did not affect the Ca(2+) content of intracellular stores, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2 and SERCA3 expression, ryanodine receptor (RyR)1 expression, or Ca(2+) entry through store-operated Ca(2+) channels. In contrast, dexamethasone increased the transcript level and the membrane protein abundance of the Na(+)/Ca(2+) exchanger NCX3. The activity of Na(+)/Ca(2+) exchangers was assessed by removal of extracellular Na(+) in the presence of external Ca(2+), a maneuver triggering the Ca(2+) influx mode. Indeed, Na(+) removal resulted in a rapid transient increase of [Ca(2+)](i) and induced an outwardly directed current as measured in whole cell patch-clamp experiments. Dexamethasone significantly augmented the increase of [Ca(2+)](i) and the outward current following removal of extracellular Na(+). The NCX blocker KB-R7943 reversed the inhibitory effect of dexamethasone on LPS-induced increase in [Ca(2+)](i). Dexamethasone blunted LPS-induced stimulation of CD86 expression and TNF-α production, an effect significantly less pronounced in the presence of NCX blocker KB-R7943. In conclusion, our results show that glucocorticoid treatment blunts LPS-induced increase in [Ca(2+)](i) in DCs by increasing expression and activity of Na(+)/Ca(2+) exchanger NCX3. The effect contributes to the inhibitory effect of the glucocorticoid on DC maturation.