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PubMed1982

Calcium uptake and catecholamine secretion by cultured bovine adrenal medulla cells.

The uptake of 45Ca2+ and secretion of catecholamines by primary cultures of adrenal medulla cells were studied. Nicotine, veratridine, potassium, and Ionomycin stimulate both the accumulation of 45Ca2+ and the secretion of catecholamines. Nicotinic antagonists block 45Ca2+ uptake induced by nicotine, tetrodotoxin blocks 45Ca2+ uptake induced by veratridine, and D600 or secretion induced by Ionomycin. The EC50 for nicotine is 3 microM for catecholamine secretion and 10 microM for 45Ca2+ uptake, while the EC50s for veratridine-stimulated uptake and secretion are approximately the same (75 microM). Kinetic studies show that the uptake of Ca2+ is rapid and appears to precede the secretion of catecholamines, and that the rate of uptake declines rapidly. The 50 mM-K+ show saturation kinetics with respect to external calcium concentrations at about 2 mM. On the other hand, the uptake of 45Ca2+ stimulated by nicotine does not become saturated at external calcium concentrations of 10 mM although the secretion of catecholamines reaches a maximum at external calcium concentrations of 2 mM. The data suggest that depolarizing agents such as veratridine and 50 mM-K+ stimulate 45Ca2+ entry through voltage-sensitive calcium channels, while nicotinic agonists stimulate calcium entry through the acetylcholine receptor ion channels as well as through voltage-sensitive calcium channels.

Adrenal Medulla↗
PubMed1991

Phorbol myristate acetate inhibition of phospholipase C activation by carbachol in slices of rat brain cortex is a delayed and indirect effect.

We examined the effect of phorbol esters on phospholipase C activation in rat brain cortical slices and membranes. There was little effect of concurrent addition of phorbol 12-myristate 13-acetate (PMA) with carbachol on phosphoinositide breakdown due to carbachol over a 1-h incubation of brain slices. However, if slices were preincubated for 3 h with 1 microM PMA or 200 microM sphingosine before addition of carbachol, there was a 35-50% inhibition of phosphoinositide breakdown. There was also a marked loss of protein kinase C (PKC) activity from both cytosol and membranes after a 3-h exposure to PMA. The loss in responsiveness to the muscarinic agonists in slices was not reflected in carbachol-stimulated phospholipase C activation using isolated membranes. However, the decrease in carbachol-induced phosphoinositide breakdown seen in slices after a 3-h exposure to PMA was abolished if the extracellular K+ concentration was elevated from 5.9 to 55mM. Because elevation of the K+ level induces depolarization and increases Ca2+ entry, we examined the effect of ionomycin, a Ca2+ ionophore. Ionomycin potentiated the effects of carbachol on phosphoinositide breakdown but was unable to reverse the effects of a 3-h incubation with PMA. Because apamin, an inhibitor of Ca2(+)-dependent K+ channels, mimicked the effects of exposure to PMA for 3 h, it is possible that these channels are involved in muscarinic cholinergic regulation of phosphoinositide breakdown in rat brain slices. These results support the hypothesis that prolonged PMA treatment in rat brain cortex has no direct effect on phospholipase C activation by muscarinic cholinergic stimulation.

Animals↗
PubMed1993

Calcium potentiates cyclic AMP stimulation of pineal arylalkylamine N-acetyltransferase.

Pineal arylalkylamine N-acetyltransferase (N-acetyltransferase) controls large daily changes in melatonin production. It is generally thought that the activity of this enzyme is controlled by norepinephrine acting exclusively via elevation of cyclic AMP. However, norepinephrine also elevates pineal intracellular Ca2+ concentration ([Ca2+]i), and it is not known whether Ca2+ is involved in regulating N-acetyltransferase activity other than through its established role in cyclic AMP production. In this study, the issue of whether Ca2+ enhances the effects of cyclic AMP on N-acetyltransferase activity was investigated. The effects of cyclic AMP protagonists (isobutylmethylxanthine, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and adenosine 3',5'-cyclic monophosphothioate, Sp-diastereomer) were examined in combination with [Ca2+]i protagonists (A23187, ionomycin, and phenylephrine). All [Ca2+]i protagonists potentiated the effects of cyclic AMP protagonists. For example, ionomycin potentiated the effects of low concentrations of 8-bromoadenosine 3',5'-cyclic monophosphate, and A23187 potentiated the effects of isobutylmethylxanthine without altering cyclic AMP accumulation. These findings indicate that Ca2+ and cyclic AMP probably act physiologically in a coordinated manner to stimulate N-acetyltransferase activity; these second messengers could act directly at one or more sites or through indirect actions mediated by kinases.

1-Methyl-3-isobutylxanthine↗
PubMed2006

Unconventional neuroprotection against Ca2+ -dependent insults by metalloporphyrin catalytic antioxidants.

We evaluated whether both inert and catalytically active metalloporphyrin antioxidants, meso-substituted with either phenyl-based or N-alkylpyridinium-based groups, suppress Ca(2+)-dependent neurotoxicity in cell culture models of relevance to cerebral ischemia. Representatives from both metalloporphyrin classes, regardless of antioxidant strength, protected cultured cortical neurons or PC-12 cultures against the Ca(2+) ionophores ionomycin or A23187, by suppressing neurotoxic Ca(2+) influx. Some metalloporphyrins suppressed excitotoxic Ca(2+) influx indirectly induced by the Ca(2+) ionophores in cortical neurons. Metalloporphyrins did not quench intracellular fluorescence, suggesting localization to the plasma membrane interface and/or interference with Ca(2+) ionophores. Metalloporphyrins suppressed ionomycin-induced Mn(2+) influx, but did not protect cortical neurons against pyrithione, a Zn(2+) ionophore. In other Ca(2+)-dependent paradigms, Ca(2+) influx via plasma membrane depolarization, but not through reversal of plasmalemmal Na(+)/Ca(2+) exchangers, was modestly suppressed by Mn(III)meso-tetrakis(4-benzoic acid)porphyrin (Mn(III)TBAP) or by an inert analog, Zn(II)TBAP. Mn(III)TBAP and Zn(II)TBAP potently protected cortical neurons against long-duration oxygen-glucose deprivation (OGD), performed in the presence of antagonists of NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and L-type voltage-gated Ca(2+) channels, raising the possibility of an unconventional mode of blockade of transient receptor protein melastatin 7 channels by a metalloTBAP family of metalloporphyrins. The present study extends the range of Ca(2+)-dependent insults for which metalloporphyrins demonstrate unconventional neuroprotection. MetalloTBAPs appear capable of targeting an OGD temporal continuum.

Animals↗
PubMed1992

Thimerosal blocks stimulated but not basal release of endothelium-derived relaxing factor (EDRF) in dog isolated coronary artery.

1. The effect of an acetly-coA lysolecithin acyltransferase inhibitor, thimerosal, on the release of endothelium-derived relaxing factor (EDRF) was examined in the greyhound isolated coronary artery. 2. Thimerosal (1-10 microM) relaxed fully, ring segments of coronary artery which were contracted with the thromboxane A2-mimetic, U46619 (30 nM). The response was endothelium-dependent, slow in both onset and time to reach maximum. The maximum relaxation to the highest concentration of thimerosal (10 microM) was maintained for 10-20 min before the tissue slowly regained active force (1-2 h) to the same or higher level as that prior to the addition of thimerosal. At this time the endothelium-dependent relaxation responses to acetylcholine (ACh), substance P (SP), bradykinin (BK) and the calcium ionophores, ionomycin and A23187 were abolished. The endothelium-dependent contractions to the nitric oxide synthase inhibitors, NG-nitro-L-arginine (L-NNA; 10-100 microM) and NG-monomethyl-L-arginine (L-NMMA: 10-100 microM), however, were unaffected. 3. Thimerosal (10 microM) did not affect the relaxation curve to sodium nitroprusside (SNP) nor the contraction curve to the thromboxane A2-mimetic, U46619. 4. Both the relaxation response to thimerosal and the selective block of the relaxation responses to stimulated EDRF release were unaffected by either indomethacin (10 microM) or superoxide dismutase (150 u ml-1). 5. L-NNA (100 microM) significantly blocked the relaxation curves to thimerosal and A23187 but not that to SNP.6. Abolition of stimulated EDRF-mediated responses with thimerosal was unlikely to result from maximal and maintained stimulation of EDRF release even when active U46619-induced force had returned to pre-thimerosal levels, since the relaxation curves to glyceryl trinitrate (GTN) and SNP were markedly attenuated in the presence of SNP and GTN respectively when active force was restored with endothelin-1 (ET-1).7. Melittin (1 microM), ionomycin (1 microM) and A23187 (1 microM) each had selective effects on stimulated but not basal EDRF responses, similar to those of thimerosal.8. We propose that stimulated but not 'basal' release of EDRF is dependent on the release of arachidonic acid or one of its non-cyclo-oxygenase metabolites, possibly by Ca2'-dependent activation of phospholipase A2.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1996

Synergistic inhibition of thrombin-induced platelet aggregation by the novel nitric oxide-donor GEA 3175 and adenosine.

1. The influence of the novel nitric oxide-donor GEA 3175 on thrombin- and ionomycin-stimulated human platelets was investigated. The effect of GEA 3175 was compared with that of adenosine, an activator of platelet adenylyl cyclase. 2. GEA 3175 inhibited thrombin-induced secretion of ATP but did not affect aggregation; similar results were obtained with adenosine. 3. Thrombin-stimulated rises in the cytosolic free Ca2+ concentration, [Ca2+]i, were dose-dependently inhibited by GEA 3175 and adenosine. GEA 3175 and adenosine maximally reduced the initial rise in [Ca2+]i by 41% and 35%, respectively. 4. Simultaneous exposure to GEA 3175 and adenosine nearly abolished both the functional responses (i.e. aggregation and degranulation) and the rises in [Ca2+]i in thrombin-stimulated platelets. 5. Aggregation and increases in [Ca2+]i triggered in platelets by the Ca(2+)-ionophore ionomycin were only marginally affected by a combination of GEA 3175 and adenosine. 6. GEA 3175 potently increased the guanosine 3':5'-cyclic monophosphate (cyclic GMP) content in platelets but did not affect adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. Adenosine did not increase either the cyclic AMP or the cyclic GMP levels in platelets. However, adenosine and GEA 3175 combined significantly elevated the platelet cyclic AMP content. 7. The results show that simultaneous exposure to GEA 3175 and adenosine promotes potent anti-aggregatory properties in platelets in vitro. The findings suggest that blockage of the cytosolic Ca(2+)-signal, which is probably mediated by an amplified cyclic nucleotide response, is an important event during the synergistic inhibition of thrombin-induced aggregation.

Adenosine↗
PubMed1999

Jeg-3 human choriocarcinoma-induced immunosuppression: downregulation of interleukin-2, interleukin-2 receptor alpha-chain, and its Jak/Stat signaling pathway.

PROBLEM: The mechanisms of the immunosuppressive and immunosuppression-inducing capacities of Jeg-3 human choriocarcinoma cell line supernatants (HCSs) are not yet completely understood. The influence on interleukin (IL)-2, IL-4 and interferon (IFN)-gamma production; IL-2 receptor (IL-2R) alpha-, beta-, and gamma-chain; and the signaling pathway molecules Janus kinase (Jak)1, Jak3, signal transducers and activators of transcription (Stat)1, Stat3, and Stat5 should be investigated. METHOD OF STUDY: For assessment of IL production, whole peripheral venous blood from healthy donors was stimulated with phorbol-myristate-acetate and ionomycine. Secretion of ILs was blocked with monensine. Intracellular ILs were analyzed by flow cytometry. For IL-2R and signaling pathway molecule analysis, peripheral blood lymphocytes were stimulated with phytohemagglutinin (PHA). IL-2R chains were measured by flow cytometry, and Jaks/Stats by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. RESULTS: Phorbol-myristate-acetate and ionomycine strongly increase the percent-age of IL-2+ cells; an additional 50% HCSs significantly suppresses the percentage to, or below the level of unstimulated cells. IFN-gamma production is strongly decreased by HCSs in some cases, but not in others. PHA stimulates IL-2R alpha-, beta-, and gamma-chain expression and their signaling pathway molecules Jak1, Jak3, Stat1, Stat3, and Stat5. 50% HCS downregulates the alpha-chain and slightly upregulates the beta-chain. Jak1, Jak3, Stat1, Stat3, and Stat5 expression is suppressed approximately to, or below the level of unstimulated cells. CONCLUSIONS: HCS forcefully blocks the production of IL-2; the IL-2R alpha-chain; and Jak1, Jak3, Stat1, Stat3, and Stat5 expression. The observed phenomena might be caused by downregulation of an IL-2R regulation gene, and might play a key role in the expansion of choriocarcinoma, and possibly in the survival of the fetal allograft.

Adult↗
PubMed2005

A novel artemisinin derivative, 3-(12-beta-artemisininoxy) phenoxyl succinic acid (SM735), mediates immunosuppressive effects in vitro and in vivo.

AIM: To study the immunosuppressive activity of SM735 {[3-(12-beta-artemisininoxy)] phenoxyl succinic acid}, a synthetic artemisinin derivative with nonsteroidal anti-inflammatory drug structure, with the aim of finding potential immunosuppressive agents. METHODS: Concanavalin A (ConA), lipopolysaccharide (LPS), and mixed lymphocyte reaction (MLR), were used to induce the proliferation of splenocytes, and [3H]-thymidine incorporation was used to evaluate the proliferation of splenocytes. Cytokine production was promoted with ConA, LPS, or PMA plus ionomycin, and was detected with the enzyme-linked immunosorbent assay. Dinitrofluorobenzene (DNFB) and sheep red blood cells (SRBC) were used to induce delayed-type hypersensitivity and quantitative hemolysis of SRBC (QHS) mouse models, as criteria for the evaluation of in vivo immune activity. RESULTS: SM735 strongly inhibited the proliferation of splenocytes induced by ConA, LPS, or MLR, with IC(50) values of 0.33 micromol/L, 0.27 micromol/L, and 0.51 micromol/L, respectively. When compared with a CC(50) value of 53.1 micromol/L, SM735 had a favorable safety range. SM735 dose-dependently inhibited proinflammatory cytokine production [including interleukins (IL)-12, interferon (IFN)-gamma and IL-6] induced by LPS or PMA plus ionomycin. Upon ConA stimulation, SM735 suppressed IFN-gamma in a dose-dependent manner, but did not affect IL-2 secretion. SM735 also strongly suppressed both T-cell-mediated delayed-type hypersensitivity (DTH) and B-cell-mediated QHS reactions. CONCLUSION: SM735 had strong immunosuppressive activity in vitro and in vivo, suggesting a potential role for SM735 as an immunosuppressive agent, and established the groundwork for further research on SM735.

Animals↗
PubMed1991

Noradrenaline-evoked cation conductance recorded with the nystatin whole-cell method in rabbit portal vein cells.

1. Noradrenaline-evoked currents were studied with the perforated patch technique (nystatin in the patch pipette) in freshly dispersed rabbit portal vein cells. 2. With potassium-containing solutions noradrenaline produced an inward current at -50 mV and an outward current (IK(Ca] at 0 mV. In potassium-free conditions noradrenaline elicited two distinct membrane currents which could be differentiated according to their sensitivity to the presence of caffeine in the bathing solution. One current was blocked whereas the second response was unaffected by caffeine. 3. The reversal potential (Er = -2.0 mV) of the caffeine-sensitive current was altered when external chloride was replaced with more permeant anions but Er was not changed when external sodium was replaced by Tris. Therefore the caffeine-sensitive current appears to be a calcium-activated chloride current (ICl(Ca]. 4. Er (+6.0 mV) of the caffeine-insensitive current was not altered by anion substitution but was changed when external sodium was replaced by Tris and barium ions. Thus the caffeine-insensitive conductance is a non-selective cation current (Icat). 5. When external NaCl was replaced by BaCl2, Er was shifted to more positive potentials which suggests that the cation conductance is more permeable to barium than to sodium. Icat was not affected by 10(-6) M-nifedipine. 6. The steady-state current-voltage relationship was linear between -50 and +50 mV for ICl(Ca) but the cation conductance mechanism displayed pronounced inward rectification and little outward current flowed across the membrane at positive potentials. 7. Caffeine (which releases calcium from internal stores) and the calcium ionophore ionomycin, which are expected to increase intracellular calcium concentration, evoked ICl(Ca) but not Icat. Thus ICl(Ca) but not Icat can be activated directly by an increase in intracellular calcium concentration. 8. When calcium was removed from the bathing solution the amplitude of Icat was not altered at early times in Ca(2+)-free conditions but was abolished after 10 min. Icat was readily activated by noradrenaline in the presence of ionomycin, which inactivated ICl(Ca). A permissive role for calcium in the generation of Icat is suggested. 9. The times between the application of noradrenaline and the onset of the chloride and cationic conductances were similar (0.75 and 0.9 s respectively). In contrast the rise time (3.9 s) and half-decay time (11 s) of Icat were much longer than the corresponding values of ICl(Ca) (respectively 1.9 and 2 s). In high-barium solution noradrenaline sometimes evoked a response that lasted for up to 10 min.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Calcium release-activated calcium current in rat mast cells.

1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward calcium release-activated calcium current (ICRAC) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to -100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to -200 mV. 6. The selectivity of ICRAC for Ca2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the calcium-sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N-methyl-D-glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 7. The current amplitude was not greatly affected by variations of external Mg2+ in the range of 0-12 mM. Even at 12 mM Mg2+ the current amplitude was reduced by only 23%. 8. ICRAC was dose-dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Mechanisms of vasoconstriction induced by endothelin-1 in smooth muscle of rabbit mesenteric artery.

1. The mechanism underlying the vasoconstriction induced by endothelin-1 (ET-1) was investigated by measuring the intracellular concentration of Ca2+ ([Ca2+]i), isometric force and phosphorylation of the myosin light chain (MLC) in endothelium-denuded unskinned and beta-escin-treated skinned smooth muscle from resistance vessels of the rabbit mesentery. The role of protein kinase C (PKC) in the action of ET-1 was studied in skinned smooth muscle using a synthetic peptide, PKC19-36, which corresponds to the autoinhibitory domain of PKC. 2. ET-1 (> 0.1 nM) induced slowly developing, maintained increases in [Ca2+]i and force. Nicardipine completely blocked the ET-1-induced increase in [Ca2+]i. BQ-123 (an inhibitor of the ETA receptor) blocked the ET-1-induced contraction but IRL 1620 (Suc-[Glu9,Ala11,15]-ET-1(8-21), an agonist of the ETB receptor) failed to induce contraction. 3. In ionomycin- and 70 mM K(+)-treated strips, ET-1 shifted the [Ca2+]i-force relationship to the left and enhanced the maximum amplitude of contraction induced by 2.6 mM Ca2+. In skinned smooth muscle treated with ionomycin, Ca2+ (0.1-3 microM) increased both force and MLC phosphorylation, in a concentration-dependent manner. ET-1 with GTP shifted both the Ca(2+)-force and Ca(2+)-MLC phosphorylation relationships to the left without significant changes in the maximum responses. ET-1 with GTP did not change the relationship between force and MLC phosphorylation. Similar effects were observed with phorbol 12,13-dibutyrate (PDBu, an activator of PKC). These results indicate that the sensitivity of MLC phosphorylation to Ca2+ is enhanced both by ET-1 with GTP and by PDBu. 4. PKC19-36 (an inhibitor of PKC) modified neither the contraction nor MLC phosphorylation induced by 0.3 microM Ca2+ but blocked the PDBu-induced enhancement of both these Ca(2+)-induced responses. However, PKC19-36 only partly inhibited the enhancement produced by ET-1 with GTP on the Ca(2+)-induced responses. PKC19-36 did not modify the relationship between force and MLC phosphorylation in the presence either of ET-1 with GTP or of PDBu. By contrast, BQ-123, neomycin and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) each abolished the ET-1-induced enhancement of the contraction induced by 0.3 microM Ca2+. 5. These results suggest that ET-1 acts on the ETA receptor and increases Ca2+ influxes through an activation of the dihydropyridine-sensitive Ca2+ channel, causing a long-lasting and maintained contraction in resistance vessels of the rabbit mesentery.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

Carbachol triggers RyR-dependent Ca(2+) release via activation of IP(3) receptors in isolated rat gastric myocytes.

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.

Animals↗
PubMed2006

Cross-talk between G(s)- and G(q)-coupled pathways in regulation of interleukin-4 by A(2B) adenosine receptors in human mast cells.

Human mast cells express functional A(2A) and A(2B) adenosine receptors. However, only stimulation of A(2B), not A(2A), leads to secretion of interleukin (IL)-4, an important step in adenosine receptor-mediated induction of IgE synthesis by B-cells. In this study, we investigate intracellular pathways that link stimulation of A(2B) receptors to IL-4 up-regulation in HMC-1 mast cells. Both A(2A) and A(2B) receptors couple to G(s) proteins and stimulate adenylate cyclase, but only A(2B) stimulates phospholipase Cbeta through coupling to G(q) proteins leading to activation of protein kinase C and calcium mobilization. Inhibition of phospholipase Cbeta completely blocked A(2B) receptor-dependent IL-4 secretion. The protein kinase C inhibitor 2-{8-[(dimethylamino)-methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide (Ro-32-0432) had no effect on A(2B) receptor-mediated IL-4 secretion but inhibited phorbol 12-myristate 13-acetate-stimulated IL-4 secretion. In contrast, chelation of intracellular Ca(2+) inhibited both A(2B) receptor- and ionomycin-dependent IL-4 secretion. This Ca(2+)-sensitive pathway probably includes calcineurin and nuclear factor of activated T cells, because A(2B) receptor-dependent IL-4 secretion was blocked with cyclosporin A or 11R-VIVIT peptide. G(s)-linked pathways also play a role in the A(2B) receptor-dependent stimulation of IL-4 secretion; inhibition of adenylate cyclase or protein kinase A attenuated A(2B) receptor-dependent IL-4 secretion. Although stimulation of adenylate cyclase with forskolin did not increase IL-4 secretion on its own, it potentiated the effect of Pasteurella multocida toxin by 2-fold and ionomycin by 3-fold. Both forskolin and stimulation of A(2B) receptors up-regulated NFATc1 protein levels. We conclude that A(2B) receptors up-regulate IL-4 through G(q) signaling that is potentiated via cross-talk with G(s)-coupled pathways.

Adenosine-5'-(N-ethylcarboxamide)↗
PubMed1998

Suppression of interleukin-2 by the putative endogenous cannabinoid 2-arachidonyl-glycerol is mediated through down-regulation of the nuclear factor of activated T cells.

2-Arachidonyl-glycerol (2-Ara-Gl) recently was identified as a putative endogenous ligand for cannabinoid receptor types CB1 and CB2 by competitive binding. More recent immune function assays demonstrated that 2-Ara-Gl possessed immunomodulatory activity. Because several plant-derived cannabinoids inhibit interleukin-2 (IL-2) expression, 2-Ara-Gl was investigated for its ability to modulate this cytokine. The direct addition of 2-Ara-Gl to mouse splenocyte cultures suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 secretion and steady state mRNA expression in a dose-dependent manner. 2-Ara-Gl also produced a marked inhibition of IL-2 promotor activity as determined by transient transfection of EL4.IL-2 cells with a pIL-2-CAT construct. 2-Ara-Gl at 5, 10, 20, and 50 microM suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 promotor activity by 18%, 28%, 39%, and 54%, respectively. To further characterize the mechanism for the transcriptional regulation of IL-2 by 2-Ara-Gl, the DNA-binding activity of transcription factors, nuclear factor of activated T cells (NF-AT), nuclear factor for immunoglobulin kappa chain in B cells (NF-kappa B/Rel), activator protein-1(AP-1), octamer, and cAMP-response element binding protein was evaluated by electrophoretic mobility shift assay in mouse splenocytes. In addition, a reporter gene expression system for p(NF-kappa B)3-CAT, p(NF-AT)3-CAT, and p(AP-1)3-CAT was used in transiently transfected EL4.IL-2 cells to determine the effect of 2-Ara-Gl on promoter activity for each of the specific transcription factors. 2-Ara-Gl reduced both the NF-AT-binding and promoter activity in a dose-dependent manner and, to a lesser degree, NF-kappa B/Rel-binding and promoter activity. No significant effect was observed on octamer- and cAMP-response element-binding activity. AP-1 DNA-binding activity was not inhibited by 2-Ara-Gl, but a modest inhibition of promoter activity was observed.

Adjuvants, Immunologic↗
PubMed2004

Vanadate is a potent activator of endothelial nitric-oxide synthase: evidence for the role of the serine/threonine kinase Akt and the 90-kDa heat shock protein.

We investigated the molecular mechanisms of sodium vanadate (vanadate)-induced nitric oxide (NO) production. Exposure of bovine lung microvascular cells (BLMVEC) to vanadate increased the release of biologically active NO in endothelium/smooth muscle cocultures, as measured by the accumulation of its surrogate marker, cGMP. This release was sensitive to NO synthase (NOS) inhibition and was greater than that observed with ionomycin. Although calcium chelators (BAPTA, EGTA) inhibited basal and ionomycin-induced NO production, they failed to inhibit vanadate-induced NO release. Moreover, in the absence of calcium/calmodulin, cell lysates from vanadate-treated cells exhibited greater NOS activity compared with control cells. Vanadate activates the phosphoinositide3-kinase (PI3-K)/Akt pathway, which is known to increase endothelial NOS (eNOS) activity by direct phosphorylation of Ser-1179. Treatment of BLMVEC with vanadate resulted in phosphorylation of both Akt and endothelial NOS. In addition, wortmannin, a PI3-K inhibitor, blocked both the vanadate-induced phosphorylation of eNOS and the increase in cGMP accumulation. Similarly, adenovirus-mediated gene transfer of an activation deficient form of Akt (AA-Akt) blocked the release of NO brought about by vanadate. To further investigate the mechanism of action of vanadate, eNOS was immunoprecipitated and its association with proteins that alter eNOS activity was tested. Immunoblots demonstrated that the eNOS-caveolin interaction remained unaffected by vanadate, whereas vanadate promoted recruitment of the 90-kDa heat shock protein (hsp90) to eNOS. We conclude that vanadate causes NO release via a mechanism that involves Akt-induced eNOS phosphorylation and increased binding of the activator protein hsp90 to eNOS.

Animals↗
PubMed2002

PEPT1-mediated cefixime uptake into human intestinal epithelial cells is increased by Ca2+ channel blockers.

Ca2+ channel blockers like nifedipine have been shown to increase the oral bioavailability of beta-lactam antibiotics, such as cefixime, in humans. The molecular mode of action of Ca2+ channel blockers on beta-lactam absorption, however, has not yet been defined. Using the Caco-2 human intestinal epithelial cell line, we assessed whether alterations in intracellular free Ca2+ ion (Ca2+in) concentrations by Ca2+ channel blockers or by Ca2+ ionophores affect [14C]cefixime absorption. Reduction of Ca2+in levels by Ca2+ channel blockers (nifedipine, verapamil, diltiazem, or bepridil) at concentrations of 100 microM led to 35 to 50% increases in the cellular uptake of 1 mM [14C]cefixime. Increases in Ca2+in levels by Ca2+ ionophores, on the other hand, led to 40% reductions in [14C]cefixime absorption. Nifedipine increased the V(max) of cefixime transport by 67%, whereas the K(m) of cefixime transport remained unaffected. By measuring the pH in Caco-2 cells loaded with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein, we show that cefixime transport mediated by the intestinal H+-coupled peptide transporter PEPT1 leads to intracellular acidification. This acid load was reduced by nifedipine, although the Ca2+ channel blocker increased the level of H+ and cefixime cotransport. Increases in Ca2+in levels by ionomycin enhanced the decline in intracellular pH induced by cefixime alone, although ionomycin reduced the level of H+ and cefixime cotransport. In conclusion, our studies demonstrate that alterations of Ca2+in levels, e.g., by Ca2+ channel blockers, affect pH regulatory systems, such as apical Na+ and H+ exchange, and thereby alter the H+ gradient that serves as the driving force for uptake of beta-lactams into intestinal epithelial cells.

Caco-2 Cells↗
PubMed2003

The adenovirus e3 promoter is sensitive to activation signals in human T cells.

The group C adenoviruses typically cause acute respiratory disease in young children. In addition, a persistent phase of infection has been observed in which virus may be shed for years without producing overt pathology. Our laboratory recently reported that group C adenovirus DNA can be found in tonsil and adenoid T lymphocytes from the majority of pediatric donors (C. T. Garnett, D. Erdman, W. Xu, and L. R. Gooding, J. Virol. 76:10608-10616, 2002). This finding suggests that immune evasion strategies of human adenoviruses may be directed, in part, toward protection of persistently or latently infected T lymphocytes. Many of the adenoviral gene products implicated in prevention of immune destruction of virus-infected cells are encoded within the E3 transcription unit. In this study, the E3 promoter was evaluated for sensitivity to T-cell activation signals by using a promoter reporter plasmid. Indeed, this promoter is extremely sensitive to T-cell activation, with phorbol myristate acetate (PMA) plus ionomycin increasing E3-directed transcription 100-fold. By comparison, in the same cells E1A expression leads to a 5.5-fold increase in transcription from the E3 promoter. In contrast to induction by E1A, activation by PMA plus ionomycin requires the two E3 NF-kappaB binding sites. Interestingly, expression of E1A inhibits induction of the E3 promoter in response to T-cell activation while increasing E3 promoter activity in unactivated cells. Collectively, these data suggest that the E3 promoter may have evolved the capacity to respond to T-cell activation in the absence of E1A expression and may act to upregulate antiapoptotic gene expression in order to promote survival of persistently infected T lymphocytes.

Adenoviridae↗
PubMed2006

Wnt-5a/Ca2+-induced NFAT activity is counteracted by Wnt-5a/Yes-Cdc42-casein kinase 1alpha signaling in human mammary epithelial cells.

Wnt-5a has been shown to influence the metastatic behavior of human breast cancer cells, and the loss of Wnt-5a expression is associated with metastatic disease. We show here that NFAT1, a transcription factor connected with breast cancer metastasis, is activated by Wnt-5a through a Ca2+ signaling pathway in human breast epithelial cells. This activation was simultaneously counteracted by a Wnt-5a-induced Yes/Cdc42 signaling pathway. The observation that inhibition of the Wnt-5a/Yes/Cdc42 signal prolonged the duration of ionomycin-induced NFAT1 activation revealed the general importance of this pathway. The Wnt-5a-induced inhibition of NFAT1 did not require glycogen synthase kinase 3beta, JNK, or Pak1 activity or modulation of the cytoskeleton. Instead, we observed that Wnt-5a induced a complex formation of NFAT1/casein kinase 1alpha, even upon treatment with ionomycin, which was blocked upon inhibition of the Wnt-5a/Yes/Cdc42 signaling pathway. Our results explain why Wnt-5a/Ca2+-induced NFAT activity is hard to detect and suggest a novel mechanism by which Wnt-5a can suppress tumor-specific, agonist-induced NFAT activity and thus the metastatic behavior of breast cancer cells.

Calcium↗