Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

The goal of the present study was to testthe hypothesis that local Ca²⁺ release events(Ca²⁺ sparks) deliver high local Ca²⁺concentration to activate nearby Ca²⁺-sensitiveK⁺ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca²⁺ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca²⁺ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa²⁺ concentration ([Ca²⁺]_i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 10⁶). A Ca²⁺spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca²⁺ spark, BKchannel activity increases 6 × 10⁵-fold to 6 × 10³-fold, which corresponds to ~30 µM to 4 µM sparkCa²⁺ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca²⁺sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca²⁺ spark sites are in closeproximity to the BK channels and that local[Ca²⁺]_i reaches micromolar levels to activateBK channels.

     

IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500–5,000 ng/ml) alone increased cytosolic Ca²⁺ level ([Ca²⁺]_i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca²⁺]_i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5–50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca²⁺]_i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca²⁺]_i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca²⁺]_i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca²⁺ signaling and degranulation. mast cell; immunoglobulin E; cytochalasin D; Y-27632; wortmannin     

Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C

Localized Ca²⁺ transients resulting from inositoltrisphosphate (IP₃)-dependent Ca²⁺ releasecouple to spontaneous transient outward currents (STOCs) in murinecolonic myocytes. Confocal microscopy and whole cell patch-clamptechniques were used to investigate coupling between localizedCa²⁺ transients and STOCs. Colonic myocytes were loadedwith fluo 3. Reduction in external Ca²⁺([Ca²⁺]_o) reduced localized Ca²⁺transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca²⁺ transients and STOCs showed increasedcoupling strength between Ca²⁺ transients and STOCs when[Ca²⁺]_o was reduced. Gd³⁺ (10 µM) did not affect Ca²⁺ transients but increased STOCamplitude and frequency. Similarly, an inhibitor of Ca²⁺influx,1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C(PKC) inhibitor, GF-109203X, also increased the amplitude and frequencyof STOCs but had no effect on Ca²⁺ transients. Phorbol12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequencybut did not affect Ca²⁺ transients. 4-Phorbol (1 µM)had no effect on STOCs or Ca²⁺ transients. Single channelstudies indicated that large-conductance Ca²⁺-activatedK⁺ (BK) channels were inhibited by aCa²⁺-dependent PKC. In summary 1)Ca²⁺ release from IP₃ receptor-operated storesactivates Ca²⁺-activated K⁺ channels;2) Ca²⁺ influx through nonselective cationchannels facilitates activation of PKC; and 3) PKC reducesthe Ca²⁺ sensitivity of BK channels, reducing the couplingstrength between localized Ca²⁺ transients and BK channels.

     

Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts

Physiological and pathologicalCa²⁺ loads are thought to be takenup by mitochondria via a process dependent on aerobic metabolism. Wesought to determine whether human diploid fibroblasts from a patientwith an inherited defect in pyruvate dehydrogenase (PDH) exhibit adecreased ability to sequester cytosolicCa²⁺ into mitochondria.Mobilization of Ca²⁺ stores withbradykinin (BK) increased the cytosolicCa²⁺ concentration([Ca²⁺]_c)to comparable levels in control and PDH-deficient fibroblasts. Innormal fibroblasts transfected with plasmid DNA encodingmitochondrion-targeted apoaequorin, BK elicited an increase inCa²⁺-dependent aequorinluminescence corresponding to an increase in the mitochondrialCa²⁺ concentration([Ca²⁺]_mt)of 2.0 ± 0.2 µM. The mitochondrial uncoupling agent carbonyl cyanidep-(trifluoromethoxy)phenylhydrazoneblocked the BK-induced [Ca²⁺]_mtincrease, although it did not affect the[Ca²⁺]_ctransient. Basal[Ca²⁺]_cand[Ca²⁺]_mtin control and PDH-deficient cells were similar. However, confocalimaging of the potential-sensitive dye JC-1 indicated that thepercentage of highly polarized mitochondria was reduced from 30 ± 1% in normal cells to 19 ± 2% in the PDH-deficient fibroblasts. BK-elicited[Ca²⁺]_mttransients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability totake up cytosolic Ca²⁺. Thus cellswith compromised aerobic metabolism have a reduced capacity tosequester Ca²⁺.

     

Ca2+ dependence and pharmacology of large-conductance K+ channels in nonlabor and labor human uterine myocytes

Two populations,Ca²⁺-dependent(BK_Ca) andCa²⁺-independentK⁺ (BK) channels of largeconductance were identified in inside-out patches of nonlabor and laborfreshly dispersed human pregnant myometrial cells, respectively.Cell-attached recordings from nonlabor myometrial cells frequentlydisplayed BK_Ca channel openings characterized by a relatively low open-state probability, whereas similar recordings from labor tissue displayed either no channel openings or consistently high levels of channel activity that oftenexhibited clear, oscillatory activity. In inside-out patch recordings,Ba²⁺ (2-10 mM),4-aminopyridine (0.1-1 mM), andShaker B inactivating peptide("ball peptide") blocked theBK_Ca channel but were much lesseffective on BK channels. Application of tetraethylammonium toinside-out membrane patches reduced unitary current amplitude ofBK_Ca and BK channels, withdissociation constants of 46 mM and 53 µM, respectively.Tetraethylammonium applied to outside-out patches decreased the unitaryconductance of BK_Ca and BKchannels, with dissociation constants of 423 and 395 µM,respectively. These results demonstrate that the properties of humanmyometrial large-conductance K⁺channels in myocytes isolated from laboring patients are significantly different from those isolated from nonlaboring patients.

     

Neuropeptide regulation of human dermal microvascular endothelial cell ICAM-1 expression and function

There isincreasing evidence that sensory nerves may participate in cutaneousinflammatory responses by the release of neuropeptides such assubstance P (SP). We examined the direct effect of SP on human dermalmicrovascular endothelial cell (HDMEC) intercellular adhesion molecule1 (ICAM-1) expression and function. Our results indicated that,although cultured HDMEC expressed mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R), SP initiated a rapid increase in HDMECintracellular Ca²⁺ levels,primarily by the activation of NK-1R. Immunohistochemistry studieslikewise demonstrated that HDMEC predominantly expressed NK-1R. Theaddition of SP to HDMEC resulted in a rapid increase in cellular ICAM-1mRNA levels, followed by a fivefold increase in ICAM-1 cell surfaceexpression. This functionally resulted in a threefold increase in⁵¹Cr-labeled binding of J-Ylymphoblastoid cells to HDMEC. In vivo studies demonstrated a markedincrease in microvascular ICAM-1 immunostaining 24 and 48 h afterapplication of capsaicin to the skin. These results indicate thatneuropeptides such as SP are capable of directly activating HDMEC toexpress increased levels of functional ICAM-1 and further support therole of the cutaneous neurological system in modulating inflammatoryprocesses in the skin.

     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange

In fura 2-loaded N1E-115 cells, regulationof intracellular Ca²⁺ concentration([Ca²⁺]_i) following a Ca²⁺ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa⁺ dependent and inhibited by 5 mM Ni²⁺. Incells with normal intracellular Na⁺ concentration([Na⁺]_i), removal of bath Na⁺,which should result in reversal of Na⁺/Ca²⁺exchange, did not increase [Ca²⁺]_i unlesscell Ca²⁺ buffer capacity was reduced. When N1E-115 cellswere Na⁺ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa⁺/Ca²⁺ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca²⁺]_ioccurred despite normal cell Ca²⁺ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa⁺/Ca²⁺ exchanger (net efflux ofCa²⁺) by observing increases (~ 6 mM) in[Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could onlybe observed when a continuous ionomycin-induced influx ofCa²⁺ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na⁺dependent and blocked by 5 mM Ni²⁺ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na⁺/Ca²⁺ exchanger that is capableof regulating [Ca²⁺]_i after release ofCa²⁺ from cell stores.

     

Ca2+ and protein kinase C activation of mucin granule exocytosis in permeabilized SPOC1 cells

Mucin secretion by airway goblet cells is under the control ofapical P2Y₂, phospholipaseC-coupled purinergic receptors. In SPOC1 cells, the mobilization ofintracellular Ca²⁺ by ionomycin orthe activation of protein kinase C (PKC) by phorbol 12-myristate13-acetate (PMA) stimulates mucin secretion in a fully additive fashion[L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis.Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17):L201-L210, 1997]. This apparent independence between PKC andCa²⁺ in the stimulation of mucinsecretion was tested in streptolysin O-permeabilized SPOC1 cells. Thesecells were fully competent to secrete mucin whenCa²⁺ was elevated from 100 nM to3.1 µM for 2 min following permeabilization; theCa²⁺EC₅₀ was 2.29 ± 0.07 µM.Permeabilized SPOC1 cells were exposed to PMA or 4-phorbol atCa²⁺ activities ranging from 10 nMto 10 µM. PMA, but not 4-phorbol, increased mucin release at allCa²⁺ activities tested: at 10 nMCa²⁺ mucin release was 2.1-foldgreater than control and at 4.7 µM Ca²⁺ mucin release was maximal(3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 µMthan at 10 nM Ca²⁺. Hence, SPOC1cells possess Ca²⁺-insensitive,PKC-dependent, and Ca²⁺-dependentPKC-potentiated pathways for mucin granule exocytosis.

     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

Effect of intracellular pH on acetylcholine-induced Ca2+ waves in mouse pancreatic acinar cells

We have used fluo3-loaded mouse pancreatic acinar cells to investigate the relationshipbetween Ca²⁺ mobilization andintracellular pH (pH_i). TheCa²⁺-mobilizing agonist ACh (500 nM) induced a Ca²⁺ release in theluminal cell pole followed by spreading of the Ca²⁺ signal toward the basolateralside with a mean speed of 16.1 ± 0.3 µm/s. In the presence of anacidic pH_i, achieved by blockade of theNa⁺/H⁺exchanger or by incubation of the cells in aNa⁺-free buffer, a slowerspreading of ACh-evoked Ca²⁺ waveswas observed (7.2 ± 0.6 µm/s and 7.5 ± 0.3 µm/s,respectively). The effects of cytosolic acidification on thepropagation rate of ACh-evokedCa²⁺ waves were largely reversibleand were not dependent on the presence of extracellularCa²⁺. A reduction in the spreadingspeed of Ca²⁺ waves could also beobserved by inhibition of the vacuolarH⁺-ATPase with bafilomycinA₁ (11.1 ± 0.6 µm/s), whichdid not lead to cytosolic acidification. In contrast, inhibition of theendoplasmic reticulum Ca²⁺-ATPaseby 2,5-di-tert-butylhydroquinone ledto faster spreading of the ACh-evokedCa²⁺ signals (25.6 ± 1.8 µm/s), which was also reduced by cytosolic acidification or treatmentof the cells with bafilomycin A₁.Cytosolic alkalinization had no effect on the spreading speed of theCa²⁺ signals. The data suggestthat the propagation rate of ACh-induced Ca²⁺ waves is decreased byinhibition of Ca²⁺ release fromintracellular stores due to cytosolic acidification or toCa²⁺ pool alkalinizationand/or to a decrease in the proton gradient directed from theinositol 1,4,5-trisphosphate-sensitiveCa²⁺ pool to the cytosol.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Physiology of a Microgravity Environment: Selected Contribution: PKC activation inhibits Ca2+ signaling in tracheal epithelial cells kept in simulated microgravity

Microgravity has been shown to alterprotein kinase C (PKC) activity; therefore, we investigated whethermicrogravity influences mechanically stimulated Ca²⁺signaling and ATP-induced Ca²⁺ oscillations, both of whichare modulated by PKC. Rabbit tracheal epithelial outgrowth cultures orsuspended epithelial sheets were rotated in bioreactors to simulatemicrogravity. Mechanical stimulation of a single cell increased thecytosolic Ca²⁺ concentration in 35-55 cells of bothoutgrowth cultures and epithelial sheets kept at unit gravity (G) or insimulated microgravity (sµG). In outgrowth cultures,12-O-tetradecanoylphorbol-13-acetate (TPA; 80 nM), a PKCactivator, restricted Ca²⁺ "waves" to about 10 cells inunit G and to significantly fewer cells in sµG. TPA only slightlyreduced the spread of Ca²⁺ waves in epithelial sheets keptin sµG but did not inhibit Ca²⁺ waves of sheets kept inunit G. In both cell preparations from both conditions, TPA inhibitedATP-induced Ca²⁺ oscillations; however, the effect was morepronounced in cells kept in sµG. These results suggest that PKCactivation is more robust in cells subjected to sµG.

     

Comparison of inhibitory activities of zinc oxide ultrafine and fine particulates on IgE-induced mast cell activation

The effects of ultrafine and fine particles of zinc oxide (ZnO) on IgE-dependent mast cell activation were investigated. The rat mast cell line RBL2H3 sensitized with monoclonal anti-ovalbumin (OVA) IgE was challenged with OVA in the presence or absence of ZnO particles and zinc sulfate (ZnSO₄). Degranulation of RBL2H3 was examined by the release of β-hexosaminidase. To understand the mechanisms responsible for regulating mast cell functions, the effects of ZnO particles on the levels of intracellular Zn²⁺, Ca²⁺, phosphorylated-Akt, and global tyrosine phosphorylation were also measured. IgE-induced release of β-hexosaminidase was obviously attenuated by ultrafine ZnO particles and ZnSO₄, whereas it was very weakly inhibited by fine ZnO particles. The intracellular Zn²⁺ concentration was higher in the cells incubated with ultrafine ZnO particles than in those with fine ZnO particles. Consistent with inhibitory effect on release of β-hexosaminidase, ultrafine ZnO particles and ZnSO₄, but not fine ZnO particle, strongly attenuated the IgE-mediated increase of phosphorylated-Akt and tyrosine phosphorylations of 100 and 70 kDa proteins in RBL2H3 cells. These findings indicate that ultrafine ZnO particles, with a small diameter and a large total surface area/mass, could release Zn²⁺ easily and increase intracellular Zn²⁺ concentration efficiently, thus decreasing FcεRI-mediated mast cell degranulation through inhibitions of PI3K and protein tyrosine kinase activation. Exposure to ZnO particles might affect immune responses, especially in allergic diseases.     

Control of Ca2+ wave propagation in mouse pancreatic acinar cells

We haveinvestigated control mechanisms involved in the propagation ofagonist-induced Ca²⁺ waves inisolated mouse pancreatic acinar cells. Using a confocal laser-scanningmicroscope, we were able to show that maximal stimulation of cells withacetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initialCa²⁺ release of comparable amountswith both agonists at the luminal cell pole. SubsequentCa²⁺ spreading to the basolateralmembrane was faster with ACh (17.3 ± 5.4 µm/s) than with bombesin(8.0 ± 2.2 µm/s). The speed of bombesin-inducedCa²⁺ waves could be increased upto the speed of ACh-induced Ca²⁺waves by inhibition of protein kinase C (PKC). Activation of PKCsignificantly decreased the speed of ACh-inducedCa²⁺ waves but had only littleeffect on bombesin-evoked Ca²⁺waves. Within 3 s after stimulation, production of inositol1,4,5-trisphosphate [Ins(1,4,5)P₃]was higher in the presence of ACh compared with bombesin, whereasbombesin induced higher levels of diacylglycerol (DAG) than ACh. Thesedata suggest that the slower propagation speed of bombesin-inducedCa²⁺ waves is due to higheractivation of PKC in the presence of bombesin compared with ACh. Thehigher increase in bombesin- compared with ACh-induced DAG productionis probably due to activation of phospholipase D (PLD). Inhibition ofthe PLD-dependent DAG production by preincubation with 0.3% butanolled to an acceleration of the bombesin-induced Ca²⁺ wave. In further experiments,we could show that ruthenium red (100 µM), an inhibitor ofCa²⁺-inducedCa²⁺ release in skeletal muscle,also decreased the speed of ACh-induced Ca²⁺ waves. The effect ofruthenium red was not additive to the effect of PKC activation. Fromthe data, we conclude that, following Ins(1,4,5)P₃-inducedCa²⁺ release in the luminal cellpole, secondary Ca²⁺ release fromstores, which are located in series between the luminal and the basalplasma membrane, modifies Ca²⁺spreading toward the basolateral cell side byCa²⁺-inducedCa²⁺ release. Activation of PKCleads to a reduction in Ca²⁺release from these stores and therefore could explain the slower propagation of Ca²⁺ waves in thepresence of bombesin compared with ACh.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

Stretch-induced Ca(2+) release via an IP(3)-insensitive Ca(2+) channel

Various mechanicalstimuli increase the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). A part of the increase in [Ca²⁺]_i isdue to the release of Ca²⁺ from intracellular stores. Wehave investigated the effect of mechanical stimulation produced bycyclical stretch on the release of Ca²⁺ from theintracellular stores. Permeabilized VSMC loaded with ⁴⁵Ca²⁺ were subjected to 7.5% average (15%maximal) cyclical stretch. This resulted in an increase in⁴⁵Ca²⁺ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP₃),ryanodine, and nicotinic acid adenine dinucleotide phosphate channels(NAADP) with 50 µg/ml heparin, 50 µM ruthenium red, and 25 µMthio-NADP, respectively, did not block the increase in⁴⁵Ca²⁺ efflux in response to cyclical stretch.However, 10 µM lanthanum, 10 µM gadolinium, and 10 µMcytochalasin D but not 10 µM nocodazole inhibited the increase in⁴⁵Ca²⁺ efflux. This supports the existence of anovel stretch-sensitive intracellular Ca²⁺ store in VSMCthat is distinct from the IP₃-, ryanodine-, and NAADP-sensitive stores.

     

Direct neurite-mast cell communication in vitro occurs via the neuropeptide substance P.

Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. However, whether mast cell activation occurs as a direct response to neuronal activation or requires an intermediary cell is unclear. Addressing this issue, we used an in vitro coculture approach comprising cultured murine superior cervical ganglia and rat leukemia basophilic cells (RBLs; possesses properties of mucosal-type mast cells). Following loading with the calcium fluorophore, Fluo-3, neurite-RBL units (separated by <50 nm) were examined by confocal laser scanning microscopy. Addition of bradykinin, or scorpion venom, dose-dependently elicited neurite activation (i.e., Ca2+ mobilization) and, after a lag period, RBL Ca2+ mobilization. Neither bradykinin nor scorpion venom had any direct effect on the RBLs in the absence of neurites. Addition of a neutralizing substance P Ab or a neurokinin (NK)-1 receptor antagonist, but not an NK-2 receptor antagonist, dose-dependently prevented the RBL activation that resulted as a consequence of neural activation by either bradykinin or scorpion venom. These data illustrate that nerve-mast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating via NK-1 receptors, is an important mediator of this communication. Our findings have implications for the neuroimmune signaling cascades that are likely to occur during airways inflammation, intestinal hypersensitivity, and other conditions in which mast cells feature.     

Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse

The myoplasmic free Ca²⁺concentration([Ca²⁺]_i)was measured in intact single fibers from mouse skeletal muscle withthe fluorescent Ca²⁺ indicatorindo 1. Some fibers were perfused in a solution in which theconcentration of Na⁺ was reducedfrom 145.4 to 0.4 mM (low-Na⁺solution) in an attempt to activate reverse-modeNa⁺/Ca²⁺exchange (Ca²⁺ entry in exchangefor Na⁺ leaving the cell). Undernormal resting conditions, application oflow-Na⁺ solution only increased[Ca²⁺]_iby 5.8 ± 1.8 nM from a mean resting[Ca²⁺]_iof 42 nM. In other fibers,[Ca²⁺]_iwas elevated by stimulating sarcoplasmic reticulum (SR)Ca²⁺ release with caffeine (10 mM)and by inhibiting SR Ca²⁺ uptakewith2,5-di(tert-butyl)-1,4-benzohydroquinone(TBQ; 0.5 µM) in an attempt to activate forward-modeNa⁺/Ca²⁺exchange (Ca²⁺ removal from thecell in exchange for Na⁺ influx).These two agents caused a large increase in[Ca²⁺]_i,which then declined to a plateau level approximately twice the baseline[Ca²⁺]_iover 20 min. If the cell was allowed to recover between exposures tocaffeine and TBQ in a solution in whichCa²⁺ had been removed, theincrease in[Ca²⁺]_iduring the second exposure was very low, suggesting thatCa²⁺ had left the cell during theinitial exposure. Application of caffeine and TBQ to a preparation inlow-Na⁺ solution produced a large,sustained increase in[Ca²⁺]_iof ~1 µM. However, when cells were exposed to caffeine and TBQ in alow-Na⁺ solution in whichCa²⁺ had been removed, a sustainedincrease in[Ca²⁺]_iwas not observed, although[Ca²⁺]_iremained higher and declined slower than in normalNa⁺ solution. This suggests thatforward-modeNa⁺/Ca²⁺exchange contributed to the fall of[Ca²⁺]_iin normal Na⁺ solution, but whenextracellular Na⁺ was low, aprolonged elevation of[Ca²⁺]_icould activate reverse-modeNa⁺/Ca²⁺exchange. The results provide evidence that skeletal muscle fibers possess aNa⁺/Ca²⁺exchange mechanism that becomes active in its forward mode when [Ca²⁺]_iis increased to levels similar to that obtained during contraction.