U—73122对猪子宫平滑肌细胞和胰腺β细胞株RINm5F的电压依赖…

目的：研究Ｕ－７３１２２对细胞内钙离子浓度和电压依赖性钙通道的作用。方法 用Ｆｕａ－２荧光测定胞浆钙浓度和用穿孔膜片箝记录全细胞钙电流。结果：Ｕ－７３１２２呈剂量相关明显地降低ＲＩＮｍ５Ｆ细胞与子宫平滑肌细胞的去极化诱导的钙电流，并抑制ＫＣｌ诱导的与Ｂａｙ－Ｋ－８６４４诱导的胞浆钙浓度的增加。Ｕ－７３１２２的这种作用对子宫平滑肌细胞要比ＲＩＮｍ５Ｆ细胞强，而一种非磷脂酶Ｃ抑制剂Ｕ－３１２２类似物Ｕ     

The interaction of U-73122 with the histamine H1 receptor: implications for the use of U-73122 in defining H1 receptor-coupled signalling pathways

U-73122, an N-aminosteroid homologue of N-ethylmaleimide (NEM), widely used as an inhibitor of phospholipase C, was found to be a potent inhibitor (IC50 5.5+/-0.5 microM) of the binding of [3H]mepyramine to guinea-pig cerebellar membranes. The succinimido analogue, U-73343, also inhibited the binding of [3H]mepyramine (estimated IC50 24+/-1 microM), but NEM was only a weak inhibitor, even at 10 mM. The interaction of U-73122 and U-73343 with the H1 receptor was effectively irreversible, on the time-scale of the experiment. There is no indication that reaction with a receptor thiol residue is involved in the binding of U-73122, since preincubation of membranes with 2 mM NEM did not significantly increase the IC50 for the inhibition of [3H]mepyramine binding by U-73122. We conclude that U-73122 binds to the histamine H1 receptor in the concentration range in which it acts as an inhibitor or PLC. This compromises the use of U-73122 to provide evidence that an H1 agonist action is mediated via PLC. The tight binding of U-73343 to the receptor appears to be primarily a function of the hydrophobic nature of the compound.     

Histamine increases cytosolic Ca2+ in HL-60 promyelocytes predominantly via H2 receptors with an unique agonist/antagonist profile and induces functional differentiation.

Histamine H1 receptors mediate activation of phospholipase C, with subsequent increases in cytosolic Ca2+ concentration ([Ca2+]i), and H2 receptors mediate accumulation of cAMP. HL-60 promyelocytes possess H2 receptors, but it is not known whether these cells also possess H1 receptors. We studied the effects of histamine on [Ca2+]i and the functional importance of histamine receptors in HL-60 promyelocytes. In these cells, histamine and dimaprit increased [Ca2+]i with EC50 values of 15 microM and 30 microM, respectively. Diphenhydramine inhibited the effect of histamine (100 microM) on [Ca2+]i up to 40%, with an IC50 of 100 nM. Famotidine and cimetidine diminished the effect of histamine (100 microM) up to 75%, with IC50 values of 85 nM and 300 nM, respectively. Diphenhydramine plus famotidine abolished histamine-induced rises in [Ca2+]i. Impromidine, with an IC50 of 100 nM, abolished the effect of histamine (100 microM) on [Ca2+]i. Diphenhydramine, famotidine, cimetidine, and impromidine showed marked noncompetitive antagonism with histamine. Histamine-induced increases in [Ca2+]i were largely due to influx of Ca2+ from the extracellular space. Ca2+ influx was inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). Histamine activated phospholipase C. Histamine induced expression of formyl peptide receptors, which effect was abolished by famotidine. In U-937 promonocytes and in the human erythroleukemia cell lines HEL and K-562, histamine did not induce rises in [Ca2+]i. Our data suggest the following. (i) In HL-60 promyelocytes, histamine increases [Ca2+]i predominantly via H2 receptors and to a lesser extent via H1 receptors. (ii) The agonist/antagonist profile of the H2 receptor-mediated increases in [Ca2+]i differs markedly from that for cAMP accumulation, suggesting the involvement of different H2 receptor subtypes. (iii) In HL-60 promyelocytes, histamine activates nonselective cation channels and induces functional differentiation via H2 receptors.     

Mechanisms involved in the antiplatelet activity of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414)

The effect on human platelets of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414) was tested in vitro by measuring aggregation induced by several agonists, cAMP and cGMP levels, cAMP phosphodiesterase and PKC activities and [Ca2+]i. The RC414 effect on nitric oxide production was also evaluated. RC414 in a dose-dependent manner inhibited aggregation both in platelet rich plasma and in washed platelets. It was particularly effective in platelets challenged by collagen, ADP and thrombin: IC50 values are 0.51 +/- 0.12 microM, 0.98 +/- 0.36 microM and 1.00 +/- 0.15 microM, respectively. RC414 increased cAMP levels, through the specific inhibition of the cAMP high affinity phosphodiesterase (IC50 = 1.73 +/- 0.35 microM). RC414 reduced [Ca2+]i transients and PKC activation induced by thrombin. In addition RC414 was able to increase nitric oxide formation involving the stimulation of constitutive nitric oxide synthase enzyme. In conclusion, RC414 exerts its powerful anti-platelet activity by increasing cAMP intracellular levels and nitric oxide formation.     

Mechanisms of AM404-induced [Ca(2+)](i) rise and death in human osteosarcoma cells

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca2+ levels ([Ca2+]i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. AM404 induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, Ni2+, nifedipine and verapamil. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), AM404-induced [Ca2+]i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca2+]i rise. At concentrations between 10 and 200 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM AM404 was partly reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.     

Endothelin receptor antagonist activity of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy)acetamide hydrochloride (PABSA) in rat aortic smooth muscle cells and isolated rat thoracic aorta

Antagonist activities of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy) acetamide hydrochloride (CAS 188710-94-3, PABSA), a novel endothelin (ET) receptor antagonist, for ETA and ETB receptors were evaluated using rat aortic smooth muscle A7r5 cells and isolated rat thoracic aorta. PABSA concentration-dependently inhibited the ET-1-induced increase in intracellular calcium concentration ([Ca2+]i) mediated via ETA receptors in A7r5 cells with an IC50 of 0.17 nmol/l. PABSA antagonized the ETA receptor-mediated contraction induced by ET-1 in endothelium-denuded rat aorta with a Kb of 0.74 nmol/l. The potency of PABSA in inhibiting ETA receptor-mediated vasocontraction was approximately 40- and 100-fold greater than those of BQ-123, a selective ETA antagonist, and bosentan, a mixed ETA/ETB receptor antagonist, respectively. ETB receptor-mediated endothelium-dependent vasorelaxation induced by ET-3 in the aorta was also antagonized by PABSA, with a Kb of 9.8 nmol/l. In contrast, PABSA affected neither the vasocontraction induced by KCl or norepinephrine nor the vasorelaxation induced by acetylcholine or prostaglandin I2 in the aorta. These results suggest that PABSA is a highly potent and selective ETA receptor antagonist.     

Celecoxib inhibits 5-lipoxygenase

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used in the therapy of inflammatory and painful conditions. Various COX-2-independent pharmacological effects, such as a chemo-preventive and tumor-regressive activity have been suggested, but the respective non-COX-2 targets of celecoxib are still a matter of research. We now demonstrate that celecoxib inhibits 5-lipoxygenase (5-LO), a key enzyme in leukotriene (LT) biosynthesis. Celecoxib suppressed 5-LO product formation in ionophore A23187-activated human polymorphonuclear leukocytes (IC(50) approximately 8 microM). Similarly, celecoxib inhibited LTB(4) formation in human whole blood (IC(50) approximately 27.3 microM). Direct interference of 5-LO with celecoxib was visualized by inhibition of enzyme catalysis both in cell homogenates and with purified 5-LO (IC(50) approximately 23.4 and 24.9 microM, respectively). Related lipoxygenases (12-LO and 15-LO) were not affected by celecoxib. Other COX-2 inhibitors (etoricoxib and rofecoxib) or unselective NSAIDs (non-steroidal anti-inflammatory drugs, diclofenac) failed to inhibit 5-LO. In rats which received celecoxib (i.p.), the blood LTB(4) levels were dose-dependently reduced with an ED(50) value approximately 35.2 mg/kg. Together, celecoxib is a direct inhibitor of 5-LO in vitro and in vivo. These findings provide a potential molecular basis for some of the described COX-2-independent pharmacological effects of celecoxib.     

Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen-activated PK signaling pathways

1 Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long-lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell-free system. 2 Pretreatment of neutrophils with pertussis toxin (1 microg ml(-1)), 50 microM 2'-amino-3'-methoxyflavone (PD 98059), or 1 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) had no effect on ART-stimulated superoxide anion generation. ART (30 microM) did not induce extracellular signal-regulated kinase (ERK) phosphorylation. 3 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) markedly attenuated the ART-stimulated superoxide anion generation (IC50 value of 4.3+/-0.3 microM). Moreover, ART induced p38 mitogen-activated PK (MAPK) phosphorylation and activation. 4 The superoxide anion generation in response to ART was also substantially inhibited in a Ca2+-free medium, and by pretreatment with 1 microM 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) and 100 microM 2-aminoethyldiphenyl borate (2-APB). ART (30 microM) stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and also increased the D-myo-inositol 1,4,5-trisphosphate formation. 5 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) greatly inhibited the ART-stimulated superoxide anion generation (IC50 value of 7.8+/-1.0 nM). ART increased the recruitment of PKC-alpha, -betaI, and -betaII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation. 6 ART induced the phosphorylation of p47phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47(phox), which was inhibited by GF 109203X and SB 203580. 7 These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2+, and PKC signaling pathways in rat neutrophils.     

Calcium mobilization by activation of M(3)/M(5) muscarinic receptors in the human retinoblastoma

Activation of muscarinic acetylcholine receptors (mAChR) is one of the most important signal transduction pathways in the human body. In this study, we investigated the role of mAChR activation in relation to its subtypes in human retinoblastoma cell-lines (WERI-Rb-1) using Ca(2+) measurement, real-time PCR, and Western Blot techniques. Acetylcholine (ACh) produced prominent [Ca(2+)](i) transients in a repeated manner in WERI-Rb-1 cells. The maximal amplitude of the [Ca(2+)](i) transient was almost completely suppressed by 97.3 +/- 0.8% after atropine (1 microM) pretreatment. Similar suppressions were noted after pretreatments with thapsigargin (1 microM), an ER Ca(2+)-ATPase (SERCA) inhibitor, whereas the ACh-induced [Ca(2+)](i) transient was not affected even in the absence of extracellular calcium. U-73122 (1 microM), a PLC inhibitor, and xestospongin C (2 microM), an IP(3)-receptor antagonist, elicited 11.5 +/- 2.9% and 17.8 +/- 1.9% suppressions, respectively. The 50% inhibitory concentration of (IC(50)) values for blockade of a 100 microM ACh response by pirenzepine and 4-DAMP were 315.8 and 9.1 nM, respectively. Moreover, both M(3) and M(5) mAChRs were prominent in quantitative real-time-PCR. Taken together, the M(3)/M(5) subtypes appear to be the major contributor, leading to intracellular calcium mobilization from the internal store via an IP(3)-dependent pathway in the undifferentiated retinoblastoma cells.     

Effect of U-73122 on calcium channel activity in porcine myometrial cells and pancreatic beta-cell line RINm5F

目的：研究Ｕ７３１２２对细胞内钙离子浓度和电压依赖性钙通道的作用．方法：用Ｆｕｒａ２荧光测定胞浆钙浓度和用穿孔膜片箝记录全细胞钙电流．结果：Ｕ７３１２２呈剂量相关明显地降低ＲＩＮｍ５Ｆ细胞与子宫平滑肌细胞的去极化诱导的钙电流，并抑制ＫＣｌ诱导的与ＢａｙＫ８６４４诱导的胞浆钙浓度的增加．Ｕ７３１２２的这种作用对子宫平滑肌细胞要比ＲＩＮｍ５Ｆ细胞强，而一种非磷脂酶Ｃ抑制剂Ｕ７３１２２类似物Ｕ７３３４３也能降低钙电流５０％．结论：Ｕ７３１２２抑制ＲＩＮｍ５Ｆ细胞与猪子宫平滑肌细胞的电压依赖性钙通道和胞浆钙浓度．     

P2-purinoceptor-mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells.

1. The effects of extracellular adenosine 5''-triphosphate (ATP) on smooth muscles are mediated by a variety of purinoceptors. In this study we addressed the identity of the purinoceptors on smooth muscle cells (SMC) cultured from human large coronary arteries. Purinoceptor-mediated increases in [Ca2+]i were measured in single fura-2 loaded cells by applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchange column. 2. Stimulation of the human coronary artery SMC (HCASMC) with extracellular ATP at concentrations of 0.1-100 microM induced a transient increase in [Ca2+]i from a resting level of 49 +/- 21 nM to a maximum of 436 +/- 19 nM. The effect was dose-dependent with an EC50 value for ATP of 2.2 microM. 3. The rise in [Ca2+]i was independent of the presence of external Ca2+, but was abolished after depletion of intracellular stores by incubation with 100 nM thapsigargin. 4. [Ca2+]i was measured upon stimulation of the cells with 0.1-100 microM of the more specific P2-purinoceptor agonists alpha, beta-methyleneadenosine 5''-triphosphate (alpha,beta-MeATP), 2-methylthioadenosine 5''-triphosphate (2MeSATP) and uridine 5''-triphosphate (UTP). alpha, beta-MeATP was without effect, whereas 2MeSATP and UTP induced release of Ca2+ from internal stores with 2MeSATP being the most potent agonist (EC50 = 0.17 microM), and UTP having a potency similar to ATP. The P1 purinoceptor agonist adenosine (100 microM) did not induce any changes in [Ca2+]i. 5. Stimulation with a submaximal concentration of UTP (10 microM) abolished a subsequent ATP-induced increase in [Ca2+]i, whereas an increase was induced by ATP after stimulation with 10 microM 2MeSATP. 6. The phospholipase C (PLC) inhibitor U73122 (5 microM) abolished the purinoceptor-activated rise in [Ca2+]i, whereas pretreatment with the Gi protein inhibitor pertussis toxin (PTX, 500 ng ml-1) was without effect on ATP-evoked [Ca2+]i increases. 7. Receptor activation with UTP and ATP resulted in formation of inositol phosphates with peak levels of inositol 1, 4, 5-trisphosphate (Ins(1, 4, 5)P3) observed 5-20 s after stimulation. 8. These findings show, that cultured HCASMC express G protein-coupled purinoceptors, which upon stimulation activate PLC to induce enhanced Ins(1, 4, 5)P3 production causing release of Ca2+ from internal stores. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, the data indicate that P2y- as well as P2U-purinoceptors are expressed by the HCASMC.     

Effect of triethyltin on Ca2+ movement in Madin-Darby canine kidney cells

The effects of the environmental toxicant, triethyltin, on Ca2+ mobilization in Madin-Darby canine kidney (MDCK) cells have been examined. Triethyltin induced an increase in cytosolic free Ca2+ levels ([Ca2+]i) at concentrations larger than 2 microM in a concentration-dependent manner. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was partly reduced by removing extracellular Ca2+. In Ca(2+)-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, reduced 50 microM triethyltin-induced [Ca2+]i increase by 80%. Conversely, pretreatment with triethyltin abolished thapsigargin-induced Ca2+ release. Pretreatment with U73122 (2 microM) to inhibit phospholipase C-coupled inositol 1,4,5-trisphosphate formations failed to alter 50 microM triethyltin-induced Ca2+ release. Incubation with triethyltin at a concentration (1 microM) that did not increase basal [Ca2+]i for 3 min did not alter ATP (10 microM)- and bradykinin (1 microM)-induced [Ca2+]i increases. Collectively, this study shows that triethyltin altered Ca2+ movement in renal tubular cells by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing Ca2+ influx.     

Effects of kappa- and mu-opioid receptor agonists on Ca2+ channels in neuroblastoma cells: involvement of the orphan opioid receptor.

The effects of micro-, delta- and kappa-opioid receptor agonists, and orphanin FQ/nociceptin (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln), on K+-induced [Ca2+]i increase were examined in SK-N-SH cells. Exposure to K+ (50 mM) resulted in a [Ca2+]i rise, which was blocked (-85%) by furaldipine (1 microM) and increased (63%) by BayK 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl-pyridine-5 -carboxylate) (0.5 microM), indicating the involvement of L-type Ca2+ channels. The kappa-opioid receptor agonists 3,4-dichloro-N-Methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U-50488H) (1-50 microM) and 5,7,8-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benze neacetamide (U-69593) (25 microM), and the mu-opioid receptor agonist sufentanil (100 nM-3 microM) inhibited the amplitude of K+-induced [Ca2+]i increase. The agonist of the orphan opioid receptor, orphanin FQ/nociceptin (1 microM), induced dual excitatory and inhibitory effects on the depolarisation-induced Ca2+ influx. The effects of the opioid receptor agonists were not blocked by the kappa-opioid receptor antagonist nor-binaltorphimine (1 microM), only weakly prevented by naloxone (10-100 microM) and naltrexone (100 microM), and partially prevented by pertussis toxin (100 ng/ml, 24 h). The antagonist of the orphan opioid receptor, [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 (1 microM), prevented the inhibitory effect of U-50488H, sufentanil and orphanin FQ. The present study provides pharmacological evidence for the presence of L-type Ca2+ channels in SK-N-SH cells, that are modulated by opioids through orphan opioid receptor activation.     

Mechanism underlying histamine-induced intracellular Ca2+ movement in PC3 human prostate cancer cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca2+ dye. Histamine at concentrations between 0.1 and 50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1 microM. The [Ca2+]i response comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In the absence of extracellular Ca2+, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 10 microM histamine did not increase [Ca2+]i. After pretreatment with 10 microM histamine in a Ca2+-free medium for several minutes, addition of 3 mM Ca2+ induced [Ca2+]i increases. Histamine (10 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 microM pyrilamine but was not altered by 50 microM cimetidine. Collectively, the present study shows that histamine induced [Ca2+]i transients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca2+ release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca2+ entry.     

Antiplatelet activity of [5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl]guanidine (KR-32570), a novel sodium/hydrogen exchanger-1 and its mechanism of action

The antiplatelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen (10 microg/mL), thrombin (0.05 U/mL), arachidonic acid (100 microM), a thromboxane (TX) A2 mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2, 1 microM) and a Ca2+ ATPase inhibitor thapsigargin (0.5 microM) (IC50 values: 13.8 +/- 1.8, 26.3 +/- 1.2, 8.5 +/- 0.9, 4.3 +/- 1.7 and 49.8 +/- 1.4 microM, respectively). KR-32570 inhibited the collagen-induced liberation of [3H]arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at 50 microM. The TXA2 synthase assay showed that KR-32570 also inhibited the conversion of the substrate PGH2 to TXB2 at all concentrations. Furthermore, KR-32570 significantly inhibited the [Ca2+]i mobilization induced by collagen at 50 microM, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen (10 microg/mL)-induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, TXA2 synthase, the mobilization of cytosolic Ca2+ and NHE-1.     

Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]membrane sulfonamide), an orally active leukotriene antagonist: effects on arachidonic acid metabolism in various inflammatory cells

The LTD4 antagonist, Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methanesulfonamide), was assessed for its ability to modulate arachidonic acid metabolism in several inflammatory cells. In A23187-stimulated rat neutrophils, Wy-48,252 effectively inhibited the conversion of exogenous [14C]arachidonic acid to radiolabeled 5-hydroxyeicosatetraenoic acid (5-HETE) and thromboxane B2 (TxB2) (IC50 = 2 and 9.1 microM, respectively). Synthesis of immunoreactive leukotriene B4 (LTB4) (IC50 = 4.6 microM) and TxB2 (IC50 = 3.3 microM) from endogenous substrate by these cells in the absence of [14C]arachidonic acid was similarly reduced. Wy-48,252 also reduced leukotriene C4 (LTC4) and PGE2 synthesis by zymosan-activated mouse peritoneal macrophages (IC50 = 4.4 and 4.3 microM, respectively). 5-Lipoxygenase (5-LO) catalyzed reactions in human neutrophils, lung mast cells and basophils activated by various stimuli were dose dependently inhibited by Wy-48,252 while PGD2 synthesis by lung mast cells was inhibited at 100 microM. By contrast, 12-LO, 15-LO, phosphodiesterase activity and histamine release from mast cells and basophils were unaffected by Wy-48,252. These data suggested that the LTD4 antagonist, Wy-48,252, also inhibited the synthesis of eicosanoids, a feature that may contribute to its pharmacological actions in vivo.     

Tamoxifen-induced [Ca2+]i rise and apoptosis in corneal epithelial cells

The effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in corneal epithelial cells. This study examined whether tamoxifen altered [Ca2+]i and viability in SIRC corneal epithelial cells. Tamoxifen at concentrations > or = 1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 6 microM. The Ca2+ signal was reduced substantially by removing extracellular Ca2+. Tamoxifen induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to Ca2+ entry inhibitors and protein kinase C modulators. After pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were abolished; conversely, tamoxifen pretreatment abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not change the [Ca2+]i rises. At concentrations of 5-30 microM, tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 15 microM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5-30 microM tamoxifen. Tamoxifen (30 microM did not induce production of reactive oxygen species (ROS). Collectively, in SIRC cells, tamoxifen induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via unknown pathways. Tamoxifen-caused cytotoxicity was partly mediated by a Ca2+-independent apoptotic pathway.     

Stereospecific inhibition of 5-HT-induced increase of intracellular free calcium by (+)- and (-)-desmethoxyverapamil in human platelets.

The concentration of intracellular free calcium [Ca2+]i in human platelets was measured by the quin-2 method. 5-Hydroxytryptamine (5-HT) at 10(-5) M induced a rapid transient increase of [Ca2+]i which was antagonized by 10(-7) M ketanserin or cyproheptadine. The verapamil derivative, desmethoxyverapamil (D888), showed stereospecific inhibition of the 5-HT-induced [Ca2+]i increase. The IC50 for (-)-D888 was approx. 2 X 10(-8) M; (+)-D888 was almost 50 times less potent.     

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca2+ channels in rat pheochromocytoma cells.

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [3H]norepinephrine ([3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an IC50 value of 1.0 +/- 0.2 microM. The DMPP-induced rise in cytosolic free Ca2+ concentration [Ca2+]i was inhibited by CPZ with an IC50 of 1.9 +/- 0.1 microM. The DMPP-induced increase in cytosolic free Na+ concentration [Na+]i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [3H]nicotine to PC12 cells was inhibited by CPZ with an IC50 value of 2.7 +/- 0.6 microM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K+-induced [3H]NE secretion and [Ca2+]i increase were inhibited by CPZ with IC50 of 7.9 +/- 1.1 and 6.2 +/- 0.3 microM, respectively. Experiments with Ca2+ channel antagonists suggest that L-type Ca2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca2+ channels, with the former being more sensitive to CPZ.     

Effect of riluzole on Ca2+ movement and cytotoxicity in Madin-Darby canine kidney cells

Riluzole is a drug used in the treatment of amyotrophic lateral sclerosis; however, its in vitro action is unclear. In this study, the effect of riluzole on intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells was investigated using the Ca2+ -sensitive fluorescent dye, fura-2. Riluzole (100-500 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50 = 150 microM). Some 40 and 50% of this [Ca2+]i increase was prevented by the removal of extracellular Ca2+ and the addition of La3+, respectively, but was unchanged by dihydropyridines, verapamil and diltiazem. In Ca2+ -free medium, thapsigargin - an inhibitor of the endoplasmic reticulum (ER) Caz+ -ATPase--caused a monophasic [Ca2+]i increase, after which the increasing effect of riluzole on [Ca2+]i was attenuated by 70%; in addition, pre-treatment with riluzole abolished thapsigargin-induced [Ca2+]i increases. U73122, an inhibitor of phospholipase C (PLC), abolished ATP (but not riluzole)-induced [Ca2+]i increases. At concentrations of 250 and 500 microM, riluzole killed 40 and 95% cells, respectively. The cytotoxic effect of riluzole (250 microM) was unaltered by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MDCK cells, riluzole rapidly increased [Ca2+]i by stimulating extracellular Ca2+ influx via an La3+ -sensitive pathway and intracellular Ca2+ release from the ER via, as yet, unidentified mechanisms. Furthermore, riluzole caused Ca2+ -unrelated cytotoxicity in a concentration-dependent manner.