Blocking of cell proliferation, cytokines production and genes expression following administration of Chinese herbs in the human mesangial cells.

In the hope of identifying agents of therapeutic value in immuoglobulin A nephropathy (IgA-N), we tested crude methanol extracts of 15 Chinese herbs for their effect on human mesangial cell proliferation. The results indicated that 4 out of the 15 crude extracts inhibited human cells proliferation activated by IL-1beta and IL-6. The extracts and their median inhibitory concentrations were as follows (in microg/ml): Ludwiga octovalvis (MLS-052), 49.9 +/- 1.8; Rhus semialata (MLS-053), 31.2 +/- 1.6; Tabernaemontana divaricata (MLS-054), 50.0 +/- 2.1; Amepelopsis brevipedunculata (MLS-059), 42.9 +/- 1.1. These findings indicate that human mesangial cells were most sensitive to MLS-053 treatment. These herbs also decreased interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) production. Moreover, IL- 1beta mRNA expression was inhibited by Rhus semialata (R. semialata; MLS-053). It is unlikely that cytotoxicity was involved, because no cell deaths were observable. We hypothesize that the inhibitory mechanisms of these Chinese herbs may be related to the impairments of gene expression and production of cytokines in human mesangial cells. Plans are underway for the isolation of pure compounds from these Chinese herbs and the elucidation of their mechanisms of action.     

Microbial glycosylation of four free anthraquinones by Absidia coerulea

Absidia coerulea transformed four anthraquinones from rhubarb, chrysophanol, physcion, emodin and aloe-emodin to their corresponding glycosylated metabolites. The structures of the products were characterized as chrysophanol 8-O-beta-D-glucoside, physcion 8-O-beta-D-glucoside, emodin 6-O-beta-D-glucoside, and aloe-emodin 1-O-beta-D-glucoside, respectively.     

Role of Ca(2+) on TNF-alpha and IL-6 secretion from RBL-2H3 mast cells

Ca2+ acts as an important second messenger in mast cells. However, the mechanisms involved in the secretion of inflammatory cytokines from activated mast cells are unknown. In this study, we examined the signaling pathway involved in calcium-related cytokine secretion in a mast cell line, RBL-2H3 cells. We report that treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a chelator of intracellular calcium, can inhibit IgE-stimulated TNF-alpha and IL-6 secretion in a concentration-dependent manner with IC50 values of 0.41 and 0.014 microM, respectively. Maximal inhibition of TNFalpha- and IL-6 secretion was 58.5 +/- 3% and 87 +/- 8% in BAPTA-AM, respectively. BAPTA-AM also completely inhibited the IgE-induced TNF-alpha and IL-6 mRNA levels. In activated RBL-2H3 cells, the expression level of NF-kappaB/Rel A protein increased in the nucleus. However, the level of NF-kappaB/Rel A in nucleus was decreased by treatment of BAPTA-AM. In addition, BAPTA-AM completely inhibited the IgE-induced IkappaB kinase beta (IKKbeta) activation and IkappaBalpha phosphorylation. These observations demonstrate that the intracellular Ca2+ may play an important role in IgE-induced TNF-alpha and IL-6 secretion from mast cells via IKKbeta activation.     

CD40 ligand-activated human monocytes amplify glomerular inflammatory responses through soluble and cell-to-cell contact-dependent mechanisms.

Monocytes/macrophages play a critical role in the initiation and progression of a variety of glomerulonephritides. We sought to define the interactions between physiologically activated human monocytes and glomerular mesangial cells (MC) by employing a cell culture system that permits the accurate assessment of the contribution of soluble factors and cell-to-cell contact. Human peripheral blood monocytes, primed with IFN-gamma and GM-CSF, were activated with CD40 ligand (CD40L) or TNF-alpha and cocultured with MC. CD40L-activated monocytes induced higher levels of IL-6, monocyte chemoattractant protein-1 (MCP-1) and ICAM-1 synthesis by MC. Separation of CD40L-activated monocytes from MC by a porous membrane decreased the mesangial synthesis of IL-6 by 80% and ICAM-1 by 45%, but had no effect on MCP-1. Neutralizing Abs against the beta 2 integrins, LFA-1 and Mac-1, decreased IL-6 production by 40 and 50%, respectively. Ligation of mesangial surface ICAM-1 directly enhanced IL-6, but not MCP-1, production. Simultaneous neutralization of soluble TNF-alpha and IL-1 beta decreased MCP-1 production by 55% in membrane-separated cocultures of MC/CD40L-activated monocytes. Paraformaldehyde-fixed CD40L-activated monocytes (to preserve membrane integrity but prevent secretory activity), cocultured with MC at various ratios, induced IL-6, MCP-1, and ICAM-1 synthesis by MC. Plasma membrane preparations from activated monocytes also induced mesangial IL-6 and MCP-1 synthesis. The addition of plasma membrane enhanced TNF-alpha-induced mesangial IL-6 production by approximately 4-fold. Together, these data suggest that the CD40/CD40L is essential for optimal effector function of monocytes, that CD40L-activated monocytes stimulate MC through both soluble factors and cell-to-cell contact mediated pathways, and that both pathways are essential for maximum stimulation of MC.     

Effects of IL-1 and cortisol on beta-adrenergic receptors, cell proliferation, and differentiation in cultured human A549 lung tumor cells

The effects of IL-1 and cortisol, and their interactions on the density of beta-adrenergic receptors (beta AR), cell proliferation, and the adherence of cells to plastic were studied in cultured human A549 lung tumor cells. The density of beta AR, assayed by 125I-pindolol binding, was increased two- to threefold by a 24-h incubation of the cells with IL-1 alpha, IL-1 beta, and TNF-alpha (EC50: 2.7, 8.2, and 24 pM, respectively), although a series of other cytokines and growth factors did not have this effect. Cortisol also increased beta AR density (EC50: 30 nM) and markedly potentiated the effects of IL-1 alpha, IL-1 beta, and TNF-alpha. Neither IL-1 nor cortisol influenced the proportion of cell surface vs internalized beta AR. The IL-1-induced increase in beta AR density was half-maximal after 6 h, was reversible at a similar rate, and was blocked by 1 microM of cycloheximide. The effect of IL-1 on beta AR was specific, as the density of glucocorticoid receptors, measured by 3H-dexamethasone binding, was reduced by IL-1. Both cortisol and IL-1 potentiated the isoproterenol-induced increase in cAMP accumulation. IL-1 inhibited cell proliferation and thymidine uptake, and increased the adherence of A549 cells to the plastic culture flask, as quantified by a cell detachment assay. The effect of IL-1 on cell adherence was not inhibited by cycloheximide. Cortisol decreased cell adherence and prevented the IL-1-induced increase in adherence. The results indicate that multiple effects of IL-1 in a cultured tumor cell line involve different mechanisms, suggesting heterogeneity of IL-1R and/or coupling of IL-1R to distinct, nuclear, and nonnuclear, effector pathways.     

Increased cytosolic calcium. A signal for sulfonylurea-stimulated insulin release from beta cells

The mechanisms by which glyburide and tolbutamide signal insulin secretion were examined using a beta cell line (Hamster insulin-secreting tumor (HIT) cells). Insulin secretion was measured in static incubations, free cytosolic Ca2+ concentration ([Ca2+]i) was monitored in quin 2-loaded cells, and cAMP quantitated by radioimmunoassay. Insulin secretory dose-response curves utilizing static incubations fit a single binding site model and established that glyburide (ED50 = 112 +/- 18 nM) is a more potent secretagogue than tolbutamide (ED50 = 15 +/- 3 microM). Basal HIT cell [Ca2+]i was 76 +/- 7 nM (mean +/- S.E., n = 141) and increased in a dose-dependent manner with both glyburide and tolbutamide with ED50 values of 525 +/- 75 nM and 67 +/- 9 microM, respectively. The less active tolbutamide metabolite, carboxytolbutamide, had no effect on [Ca2+]i or insulin secretion. Chelation of extracellular Ca2+ with 4 mM EGTA completely inhibited the sulfonylurea-induced changes in [Ca2+]i and insulin release and established that the rise in [Ca2+]i came from an extracellular Ca2+ pool. The Ca2+ channel blocker, verapamil, inhibited glyburide- or tolbutamide-stimulated insulin release and the rise in [Ca2+]i at similar concentrations with IC50 values of 3 and 2.5 microM, respectively. At all concentrations tested, the sulfonylureas did not alter HIT cell cAMP content. These findings provide direct experimental evidence that glyburide and tolbutamide allow extracellular Ca2+ to enter the beta cell through verapamil-sensitive, voltage-dependent Ca2+ channels, causing a rise in [Ca2+]i which is the second messenger that stimulates insulin release.     

The in vitro immunomodulatory effects of sulfasalazine on human polymorphonuclear leukocytes, mononuclear cells, and cultured glomerular mesangial cells

Sulfasalazine (SSA) was investigated for its effects on phagocytic activity of normal human polymorphonuclear neutrophils (PMN), proliferation of mononuclear cells (MNC) and cultured glomerular mesangial cells. At concentrations from 25 to 100 microM, it inhibited phagocytic activity of PMN and the 3H-thymidine incorporation of phytohemagglutinin (PHA)-stimulated human MNC in a dose-dependent manner. At comparable concentrations, sulfapyridine and 5-aminosalicylic acid, two of its major metabolites, did not show similar effects. SSA exhibited an inhibitory effect on both mouse and rat mesangial cells but at rather higher concentrations (0.5 mM). Excretion of interleukin (IL)-8 by lipopolysaccharide (LPS)-stimulated PMN was also markedly deterred in a dose-dependent manner but excretion of IL-8 by LPS-stimulated MNC was not interfered by SSA. Production of tumor necrosis factor (TNF)-alpha and IL-1beta by mouse mesangial cells was not blocked by SSA but production of IL-4 by these cells was inhibited by it (>0.1 mM). Inhibition of MNC was not due directly to cytotoxic effect of SSA on these cells as shown by fluorescein diacetate stain. Collectively, SSA inhibits phagocytosis and IL-8 excretion by PMN as well as mitogen-stimulated MNC reaction. On the other hand, at high concentrations, it inhibits glomerular mesangial cells and their IL-4 excretion but not TNF-alpha and IL-1beta excretion. These results can account for minimal nephrotoxic characteristic of SSA and suggest that it may be helpful in the treatment of immune-mediated glomerulonephritis.     

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.     

Prostaglandin E2 production is synergistically increased in cultured human glomerular mesangial cells by combinations of IL-1 and tumor necrosis factor-alpha 1

Both IL-1 alpha and IL-1 beta and TNF-alpha induced a time- and dose-dependent release of authentic PGE2 from cultured human glomerular mesangial cells (HMC). This release became significant only after a 4- to 6-h lag phase, and was abolished by inhibition of protein synthesis, and was not related to cell proliferation. Combinations of IL-1 and TNF-alpha when added simultaneously to HMC resulted in a dose-dependent synergistic increase in PGE2 production. These stimulatory effects were specifically inhibited by anticytokine antibodies and the synergistic effect required the simultaneous presence of both IL-1 and TNF-alpha. Arachidonic acid (AA) release experiments and measurement of cyclooxygenase activity, revealed that while both were increased by IL-1 beta and TNF-alpha alone (IL-1 beta greater than TNF-alpha), combinations of IL-1 beta and TNF-alpha resulted in only additive increases in AA release and cyclooxygenase activity. Taken together, these data suggest that stimulation of PGE2 in HMC, by combinations of these cytokines, is not rate limited by AA release or cyclooxygenase activation, but may be related to the induction of the distal enzymes controlling specific PG synthesis.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

The effect of strong static magnetic field on lymphocytes

We investigated whether static electromagnetic fields (EMFs) at a flux density of 4.75 T, generated by an NMR apparatus (NMRF), could promote movements of Ca2+, cell proliferation, and the eventual production of proinflammatory cytokines in human peripheral blood mononuclear cells (PBMC) as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 mg/ml phytohaemagglutinin (PHA). Our results clearly demonstrate that static NMRF exposure has neither proliferative, nor activating, nor proinflammatory effects on both normal and PHA activated PBMC. Moreover, the concentration of interleukin-1beta, interleukin-2, interleukin-6, interferon, and tumour necrosis factor alpha (TNFalpha) remained unvaried in exposed cells. Exposure of Jurkat cells statistically decreased the proliferation and the proliferation indexes, which 24 and 48 h after exposure were 0.7 +/- 0.29 and 0.87 +/- 0.12, respectively. Moreover, in Jurkat cells the [Ca2+]i was higher than in PBMC and was reduced significantly to about one half after exposure. This is consistent with the decrease of proliferation and with the low levels of IL-2 measured. On the whole, our data suggest that NMRF exposure failed to affect the physiologic behaviour of normal lymphomonocytes. Instead in Jurkat cells, by changing the properties of cell membranes, NMRF can influence Ca2+ transport processes, and hence Ca2+ homeostasis with improvement of proliferation.     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

Thienopyridines: effects on cultured endothelial cells.

In cultured endothelial cells harvested from human umbilical vein (HUVEC) or bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) caused an increase in nitrite generation in HUVEC from basal 227 +/- 37 to 372 +/- 60 or to 325 +/- 33 pmoles per 10(6) cells, respectively, and in BAEC from basal 182 +/- 17 to 378 +/- 18 or to 423 +/- 66 pmoles per 106 cells (n = 6), respectively. Calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) next to 30 min incubation with BAEC increased release of 6-keto-PGF 1alpha from basal level of 9.4 +/- 1.8 to 96.2 +/- 5.1 or to 99.5 +/- 10.2 pmoles per 10(6) cells, respectively. The pretreatment with aspirin (300 microM) cut down this rise to 4.2 +/- 0.1 pmoles per 10(6) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7 +/- 3.3 nM (n = 53), 68.3 +/- 5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cultured endothelial cells calcium ionophore A 23187 (0.1 microM) produced net maximum rise in [Ca2+]i by 157 +/-27 nM (n = 16)[ ECV304], by 107 +/- 58 nM (n=4) [HUVEC], and by 231.0 +/- 41.3 nM (n = 8) [BAEC], respectively, while ticlopidine (30 microM) produced net maximum rise in [Ca2+]i by 30.0 +/- 3.2 nM (n=9)[ECV304], 48.8 +/- 15.6 nM (n = 4)[HUVEC] and 28.4 +/- 5.4 nM (n = 8)[BAEC], respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than that of calcium A 23187 but also its maximum appeared after a lag period that was 2 3 times longer than that for A23187. In ECV304 clopidogrel at concentrations of 10, 30 and 100 microM produced maximum increment of [Ca2+]i by 16.5 +/- 3.8 nM (n = 7), 47.0 +/- 6.9 nM (n = 8) and 67.2 +/- 8.3 nM (n = 8), respectively. Incubation of BAEC with A23187 (microM), ticlopidine or clopidogrel (100 microM) for 2 h did not influence viability of cultured endothelial cells. We claim that thienopyridines, independently of their delayed anti-platelet properties ex vivo do release NO and PGI2 from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be mediated by a rise in [Ca2+]i, however, this possibility has not been proved.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice

Non-selective cation (NSC) channels activated by intracellular Ca2+ ([Ca2+]i) play an important role in Ca2+ signaling and membrane excitability in many cell types. TRPM4 and TRPM5, two Ca2+-activated cation channels of the TRP superfamily, are potential molecular correlates of NSC channels. We compared the functional properties of mouse TRPM4 and TRPM5 heterologously expressed in HEK 293 cells. Dialyzing cells with different Ca2+ concentrations revealed a difference in Ca2+ sensitivity between TRPM4 and TRPM5, with EC50 values of 20.2+/-4.0 microM and 0.70+/-0.1 microM, respectively. Similarly, TRPM5 activated at lower Ca2+ concentration than TRPM4 when [Ca2+]i was raised by UV uncaging of the Ca2+-cage DMNP-EDTA. Current amplitudes of TRPM4 and TRPM5 were not correlated to the rate of changes in [Ca2+]i. The Ca2+ sensitivity of both channels was strongly reduced in inside-out patches, resulting in approximately 10-30 times higher EC50 values than under whole-cell conditions. Currents through TRPM4 and TRPM5 deactivated at negative and activated at positive potentials with similar kinetics. Both channels were equally sensitive to block by intracellular spermine. TRPM4 displayed a 10-fold higher affinity for block by flufenamic acid. Importantly, ATP4- blocked TRPM4 with high affinity (IC50 of 0.8+/-0.1 microM), whereas TRPM5 is insensitive to ATP4- at concentrations up to 1 mM.     

Immunomodulatory potential of thymulin-Zn(2+) in the alveolar epithelium: amelioration of endotoxin-induced cytokine release and partial amplification of a cytoprotective IL-10-sensitive pathway

The immunomodulatory potential of thymulin in the perinatal epithelium is not well characterized. In an in vitro model of fetal alveolar type II epithelial cells, we investigated the exhibition of an anti-inflammatory activity of this peptide hormone. Thymulin selectively ameliorated, in a dose-dependent manner, the endotoxin-induced release of IL-1 beta (IC(50) = 657 ng. ml(-1)), but showed no inhibitory effect on IL-6 and TNF-alpha. Zinc, an anti-inflammatory antioxidant, which is required for the biological activity of thymulin, reduced the secretion of IL-1 beta (IC(50) = 62 microM), TNF-alpha (IC(50) = 1000 microM), and, to a lesser extent, IL-6. This cation (100 microM) amplified the effect of thymulin on IL-1 beta and TNF-alpha (IC(50) < 0.1 ng. ml(-1)), but not on IL-6. Analysis of whether thymulin is up-regulating a counterpart anti-inflammatory signaling loop revealed the involvement of an IL-10-sensitive pathway. These results indicate that thymulin acts as a novel dual immunoregulator by enhancing an anti-inflammatory cytoprotective response and depressing an inflammatory signal, an effect synergistically amplified, in part, by cationic zinc.     

Clomiphene, an ovulation-inducing agent, mobilizes intracellular Ca2+ and causes extracellular Ca2+ influx in bladder female transitional carcinoma cells

BACKGROUND/METHODS: The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca2+ levels ([Ca2+]i) in populations of BFTC human bladder cancer cells was explored by using fura-2 as a Ca2+ indicator. RESULTS: Clomiphene at concentrations between 10 and 75 microM increased [Ca2+]i in a concentration-dependent manner and the signal saturated at 50 microM. The [Ca2+]i signal was biphasic with an initial rise and a slow decay. Ca2+ removal inhibited the Ca2+ signal by about 40-50% in maximum [Ca2+]i. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 50 microM clomiphene in Ca2+-free medium, suggesting that clomiphene induced capacitative Ca2+ entry. In Ca2+-free medium, pretreatment with 50 microM brefeldin A (to disrupt the Golgi complex Ca2+ store), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca2+ pump), and CCCP (to uncouple mitochondria) inhibited 85% of clomiphene-induced intracellular Ca2+ release. Conversely, pretreatment with 50 microM clomiphene in Ca2+-free medium abolished the [Ca2+]i increase induced by brefeldin, thapsigargin or CCCP. The intracellular Ca2+ release was unaltered by inhibiting formation of inositol-1,4,5-trisphosphate (IP3) with 2 mM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; a phospholipase C inhibitor). CONCLUSION: The [Ca2+]i increase induced by 50 microM clomiphene was not affected by 10 microM of nifedipine, verapamil or diltiazem. Collectively, the results suggest that clomiphene releases intracellular Ca2+ in an IP3-independent manner and also activates extracellular Ca2+ influx.