Thromboxane A2-mediated shape change: independent of Gq-phospholipase C--Ca2+ pathway in rabbit platelets.

1. Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in washed rabbit platelets to clarify the mechanisms of induction of shape change and aggregation. 2. The TXA2 agonist, U46619 (1 nM to 10 microM) caused shape change and aggregation in a concentration-dependent manner. A forty-times higher concentration of U46619 was needed for aggregation (EC50 of 0.58 microM) than shape change (EC50 of 0.013 microM). The aggregation occurred only when external 1 mM Ca2+ was present, but the shape change could occur in the absence of Ca2+. 3. SQ29548 at 30 nM and GR32191B at 0.3 microM (TXA2 receptor antagonists) competitively inhibited U46619-induced shape change and aggregation with similar potency, showing that both aggregation and shape change induced by U46619 were TXA2 receptor-mediated events. However, ONO NT-126 at 1 nM, another TXA2 receptor antagonist, inhibited U46619-induced aggregation much more potently than the shape change, suggesting the possible existence of TXA2 receptor subtypes. 4. ONO NT-126 (2 nM to 3 microM) by itself caused a shape change without aggregation in a concentration-dependent manner, independent of external Ca2+. Therefore, ONO NT-126 is a partial agonist at the TXA2 receptor in rabbit platelets. 5. U46619 (10 nM to 10 microM) increased internal Ca2+ concentration ([Ca2+]i) and activated phosphoinositide (PI) hydrolysis in a concentration-dependent manner with a similar concentration-dependency. 6. U46619 (3 nM to 10 microM) also activated GTPase concentration-dependently in the membranes derived from platelets. U46619-induced activation of GTPase was partly inhibited by treatment of membranes with QL, an antibody against Gq/11. 7. The EC50 values of U46619 in Ca2+ mobilization (0.15 microM), PI hydrolysis (0.20 microM) and increase in GTPase activity (0.12 microM) were similar, but different from the EC50 value in shape change (0.013 microM), suggesting that activation of TXA2 receptors might cause shape change via an unknown mechanism. 8. U46619-induced shape change was unaffected by W-7 (30 microM), a calmodulin antagonist or ML-7 (30 microM), a myosin light-chain kinase inhibitor, indicating that an increase in [Ca2+]i might not be involved in the shape change. In fact, U46619 (10 nM) could cause shape change without affecting [Ca2+]i level, determined by simultaneous recordings. 9. [3H]-SQ29548 and [3H]-U46619 bound to platelets at a single site with a Kd value of 14.88 nM and Bmax of 106.1 fmol/10(8) platelets and a Kd value of 129.8 nM and Bmax of 170.4 fmol/10(8) platelets, respectively. The inhibitory constant Ki value for U46619 as an inhibitor of 3H-ligand binding was similar to the EC50 value of U46619 in GTPase activity, phosphoinositide hydrolysis and Ca2+ mobilization, but significantly different (P < 0.001 by Student''s t test) from the effect on shape change. 10. Neither U46619 nor ONO NT-126 affected the adenosine 3'',5''-cyclic monophosphate (cyclic AMP) level in the presence or absence of external Ca2+ and/or isobutyl methylxanthine. 11. The results indicate that TXA2 receptor stimulation causes phospholipase C activation and increase in [Ca2+]i via a G protein of the Gq/11 family leading to aggregation in the presence of external Ca2+, and that shape change induced by TXA2 receptor stimulation might occur without involvement of the Gq-phospholipase C-Ca2+ pathway.     

The ether lipid ET-18-OCH3 increases cytosolic Ca2+ concentrations in Madin Darby canine kidney cells.

The effect of the ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) on the intracellular free Ca2+ concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ probe. In Ca2+ medium, ET-18-OCH3 induced a significant rise in [Ca2+]i at concentrations between 10-100 microM with a concentration-dependent delay of 45-175 s. The [Ca2+]i signal was composed of a gradual rise and a sustained plateau. In Ca2+-free medium, ET-18-OCH3 (10-100 microM) induced a Ca2+ release from internal Ca2+ stores with a concentration-dependent delay of 45-175 s. This discharge of internal Ca2+ triggered capacitative Ca2+ entry in a concentration-dependent manner. This capacitative Ca2+ entry was not inhibited by econazole (25 microM), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 microM), nifedipine (10 microM), verapamil (10 microM), diltiazem (10 microM) and cadmium (0.5 microM). Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylat e (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 microM ET-18-OCH3-induced [Ca2+]i rise in a concentration-dependent manner between 1-20 microM, with 20 microM exerting a complete block. The [Ca2+]i rise induced by ET-18-OCH3 (25 microM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 microM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 mM) or the phospholipase A2 inhibitor aristolochic acid (20-40 microM). In Ca2+-free medium, pretreatment with 25 microM ET-18-OCH3 completely depleted the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-sensitive Ca2+ store. In contrast, pretreatment with thapsigargin abolished 0.1 mM ATP-induced [Ca2+]i rise without altering the ET-18-OCH3-induced [Ca2+]i rise. This suggests that ET-18-OCH3 depleted thapsigargin-sensitive Ca2+ stores and also released Ca2+ from thapsigargin-insensitive stores. The thapsigargin-insensitive stores involve mitochondria because the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 microM ET-18-OCH3. ET-18-OCH3 (25 microM) induced a significant Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength confirming that ET-18-OCH3 induced capacitative Ca2+ entry. La3+ (0.1 mM) or Gd3+ (50 microM) abolished the ET-18-OCH3-induced Mn2+ quench and [Ca2+]i rise. Our data imply that ET-18-OCH3 induced a [Ca2+]i rise in MDCK cells by activating PAF receptors leading to an internal Ca2+ release followed by capacitative Ca2+ entry. Phospholipase D and phospholipase A2, but not phospholipase C, might be involved in mediating the capacitative Ca2+ entry. La3+ abolished the ET-18-OCH3-induced [Ca2+]i rise presumably by inhibiting PAF receptors.     

Dissociation of P2 purinoceptor-mediated increase in intracellular Ca2+ level from myosin light chain phosphorylation and contraction in rat aorta.

1. The effects of P2 agonists, adenosine-5'-triphosphate (ATP), alpha, beta-methylene-adenosine-5'-triphosphate (alpha, beta-me-ATP) and adenosine 5-O-(3-thiotriphosphate) (ATP gamma S), on the intracellular free Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction were examined in vascular smooth muscle of rat aorta. 2. ATP (0.1 microM-1 mM), alpha, beta-me-ATP (0.1-100 microM) and ATP gamma S (1-100 microM) induced transient increases followed by sustained increase in [Ca2+]i. The effects of these agonists were concentration-dependent. Compared with the effects of a high concentration of KCl (17.5-72.4 mM), the contractions induced by these P2 purinoceptor agonists were smaller at a given [Ca2+]i. 3. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), ATP gamma S (10 microM) induced large transient increase in [Ca2+]i with only small contraction in Ca(2+)-free solution. In contrast, alpha, beta-me-ATP (10 microM) induced only a very small increase in [Ca2+]i and contraction. 4. ATP (1 mM), alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), added during stimulation with 0.1 microM noradrenaline, induced additional and transient increases in [Ca2+]i which were also not associated with contraction. 5. High K+ (72.4 mM) increased MLC phosphorylation with a similar time course to that of the increase in [Ca2+]i (peak phosphorylation was 56% when [Ca2+]i increased to 100%). In contrast, the time course of the increase in MLC phosphorylation due to ATP (1 mM) did not coincide with that of the large increases in [Ca2+]i; MLC phosphorylation increased to only 31% when [Ca2+]i increased to 163%. The MLC phosphorylation due to alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), measured at peak [Ca2+]i, were only 19% and 14%, respectively, irrespective of a large increase in [Ca2+]i (138% and 188%, respectively). 6. The absence of a clear relationship between P2-purinoceptor-mediated increase in [Ca2+]i (either by Ca2+ influx or Ca2+ release) and MLC phosphorylation or force generation appears to imply that elevation in [Ca2+]i does not contribute to these responses.     

The effects of ATP and alpha,beta-methylene-ATP on cytosolic Ca2+ level and force in rat isolated aorta.

1. The effects of a non-selective P2-receptor agonist ATP and a selective P2x-receptor agonist alpha,beta-methylene-ATP on intracellular free Ca2+ level ([Ca2+]i) and force were examined in rat isolated aorta without endothelium. 2. Both ATP (1-1000 microM) and alpha,beta-methylene-ATP (0.1-100 microM) induced transient increase followed by small sustained increase in [Ca2+]i in a concentration-dependent manner. Compared with the force induced by a high concentration of KCl, the force induced by alpha,beta-methylene-ATP was smaller and that induced by ATP was much smaller at a given [Ca2+]i. 3. An L-type Ca2+ channel blocker, verapamil (10 microM), completely inhibited the high K(+)-stimulated [Ca2+]i and force. Verapamil partially inhibited the transient and sustained increases in [Ca2+]i induced by 10 microM alpha,beta-methylene-ATP and the sustained increase but not the transient increase induced by 1 mM ATP. 4. In the absence of extracellular Ca2+ (with 0.5 mM EGTA) 1 mM ATP caused transient increase in [Ca2+]i while 10 microM alpha,beta-methylene-ATP was ineffective 5. ATP, but not alpha,beta-methylene-ATP, increased the tissue adenosine 3':5'-cyclic monophosphate (cyclic AMP) level. 6. These data suggest that ATP and alpha,beta-methylene-ATP increase [Ca2+]i by an activation of both L-type and non-L-type Ca2+ channels. In addition, ATP, but not alpha,beta-methylene-ATP, increases [Ca2+]i by a release of Ca2+ from an intracellular Ca2+ store. Possible reasons are discussed as to why the increase in [Ca2+]i due to ATP and alpha,beta-methylene-ATP resulted in only a small contraction.     

Effect of the neuroprotective agent riluzole on intracellular Ca2+ levels in IMR32 neuroblastoma cells

Riluzole is an effective neuroprotective drug. Its effect on intracellular free Ca2+ levels ([Ca2+]i) has not been explored. This study examined the effect of riluzole on [Ca2+]i in IMR32 neuroblastoma cells using fura-2 as a Ca2+ probe. Riluzole 0.1-1 mM increased [Ca2+]i in a concentration-dependent manner. Removal of extracellular Ca2+ inhibited the response by 52 +/- 5%. The [Ca2+]i increase induced by 0.2 mM riluzole was unaltered by 0.1 mM La3+ or 10 microM verapamil, but was inhibited by 51 +/- 4% by 10 microM nifedipine. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) reduced the 0.2 mM riluzole-induced Ca2+ release by 44 +/- 3%; this reduction was augmented to 66 +/- 5% by additionally depleting the Ca2+ stores in the Golgi complex with 50 microM brefeldin A. Inhibition of inositol 1,4,5-trisphosphate formation by 2 microM U73122, a phospholipase C inhibitor, did not affect Ca2+ release induced by 0.2 microM riluzole. It was concluded that the neuroprotective agent riluzole increased [Ca2+]i in IMR32 neuroblastoma cells concentration-dependently by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner and also by inducing nifedipine-sensitive Ca2+ influx.     

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.     

Novel action of lignans isolated from Hernandia nymphaeifolia on Ca2+ signaling in human neutrophils

The effects of five lignans (epi-aschantin, epi-magnolin, epi-yangambin, deoxypodophyllotoxin, yatein) isolated from Hernandia nymphaeifolia (Presl.) Kubitzki (Hernandiaceae) on intracellular Ca2+ levels ([Ca2+]i) in human neutrophils were investigated by using fura-2 as a fluorescent probe. In both Ca2+-containing and Ca2+-free media, the lignans (50-100 microM) did not alter basal [Ca2+]i but inhibited the [Ca2+]i increase induced by platelet activating factor (PAF, 10 microM), leukotriene B4 (LTB4, 0.2 microM), and thapsigargin (1 microM) to different extents. In Ca2+-free medium, after depleting stores of Ca2+ with PAF, LTB4 or thapsigargin, addition of 3 mM Ca2+ induced Ca2+ influx. Each of the lignans (50-100 microM) caused 39-89% inhibition of PAF-induced Ca2+ influx; whereas only epi-aschantin was able to inhibit LTB4- and thapsigargin-induced Ca2+ influx by 54-79%. Together, the results suggest that in human neutrophils, these lignans did not alter basal [Ca2+]i but inhibited Ca2+ movement induced by Ca2+ mobilizing agents.     

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.     

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.     

Effects of a newly synthesized K+ channel opener, Y-26763, on noradrenaline-induced Ca2+ mobilization in smooth muscle of the rabbit mesenteric artery.

1. The mechanisms underlying the vasodilatation induced by (-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2, 2-dimethyl-2H-1-benzopyran-3-ol (Y-26763) were investigated by measuring membrane potential, intracellular Ca2+ concentration ([Ca2+]i) and isometric force in smooth muscle cells of the rabbit mesenteric artery. 2. Y-26763 (0.03-1 microM) concentration-dependently hyperpolarized the membrane and glibenclamide (1-10 microM) inhibited this hyperpolarization. Noradrenaline (NA, 10 microM) depolarized the membrane and generated spike potentials. Y-26763 (1 microM) inhibited these NA-induced electrical responses. 3. In thin smooth muscle strips in 2.6 mM Ca2+ containing (Krebs) solution, 10 microM NA produced a large phasic, followed by a small tonic increase in [Ca2+]i and force with associated oscillations. In Ca(2+)-free solution (containing 2 mM EGTA), NA produced only phasic increases in [Ca2+]i and force. In ryanodine-treated strips, NA could not produce the phasic increases in [Ca2+]i and force even in the presence of 2.6 mM Ca2+, suggesting that ryanodine functionally removes the NA-sensitive intracellular storage sites. 4. Nicardipine (1 microM) partly inhibited the NA-induced tonic increases in [Ca2+]i and force but had no effect on either the resting [Ca2+]i or the NA-activated phasic increases in [Ca2+]i and force. By contrast, Y-26763 (10 microM) lowered the resting [Ca2+]i and also inhibited both the phasic and the tonic increases in [Ca2+]i and force induced by NA. All these actions of Y-26763 were inhibited by glibenclamide (10 microM). 5. In ryanodine-treated strips, nicardipine partly, but Y-26763 completely inhibited the NA-induced increases in [Ca2+]i, suggesting that Y-26763 inhibits both the nicardipine-sensitive and -insensitive Ca2+ influxes activated by NA. Y-26763 attenuated the phasic increase in [Ca2+]i and force in a Ca(2+)-free solution containing 5.9 mM K+, but not in one containing 50 mM K+, suggesting that Y-26763 inhibits NA-induced Ca2+ release, probably as a result of its membrane hyperpolarizing action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual effects of mastoparan on intracellular free Ca2+ concentrations in human astrocytoma cells.

1. The effect of mastoparan, a wasp venom toxin, on intracellular free Ca2+ concentration ([Ca2+]i) was examined in human astrocytoma cells. Mastoparan inhibited [Ca2+]i induced by carbachol (100 microM) in a concentration-dependent manner in the absence of extracellular Ca2+, consistent with our previous results showing that mastoparan inhibits phosphoinositide hydrolysis in human astrocytoma cells. 2. In contrast, mastoparan itself increased [Ca2+]i and augmented carbachol-induced increase in the [Ca2+]i in the presence of extracellular Ca2+, suggesting that mastoparan elicited Ca2+ influx from the extracellular medium. The increase appeared to be maximum at extracellular Ca2+ concentrations of 0.1-0.2 mM. The higher concentrations of extracellular Ca2+ depressed the influx. 3. Pertussis toxin did not affect mastoparan-induced inhibition of [Ca2+]i in the absence of extracellular Ca2+, consistent with the previous results that pertussis toxin did not affect mastoparan-induced inhibition of phosphoinositide hydrolysis. 4. Pertussis toxin augmented mastoparan-induced increase in [Ca2+]i in the presence of extracellular Ca2+, suggesting that pertussis toxin substrate(s) seems to be inhibitory for Ca2+ influx induced by mastoparan. 5. Verapamil, nifedipine and diltiazem (each 10 microM), L-type Ca2+ antagonists, did not affect mastoparan-induced Ca2+ influx. However, verapamil (10 microM) slightly inhibited the increase in [Ca2+]i induced by carbachol in the presence of mastoparan. 6. The results obtained in the present study indicate that mastoparan has two opposite effects on [Ca2+]i in human astrocytoma cells and possibly has at least two sites of action.     

Characterization of endothelin receptor subtypes mediating Ca2+ mobilization and contractile response in rabbit iris dilator muscle.

1. We investigated the characteristics of endothelin (ET)-induced contraction and changes in intracellular Ca2+ concentration ([Ca2+]i) using the fura-2-loaded and non-loaded rabbit iris dilator. ET-1 and ET-2 (3-100 nM) and ET-3 (30-100 nM) caused contraction in a concentration-dependent fashion. 2. The selective ETB-receptor agonists, IRL1620 and sarafotoxin S6c produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of potencies for the contraction (pD2 value) was ET-1 = ET-2 > ET-3 >> sarafotoxin S6c = IRL1620. 3. The contractile response to ET-3 was antagonized by pretreatment with BQ-123 (10 nM), a selective ETA receptor antagonist. The contractile responses to ET-1 and ET-2 were antagonized by pretreatment with BQ-123 (10 microM), but not at a concentration of 10 nM. 4. ETs increased [Ca2+]i and sustained muscle contraction. ET-1 (100 nM), ET-2 (100 nM), and ET-3 (1 microM) induced an elevation of [Ca2+]i consisting of two components: first a rapid and transient elevation to reach a peak, followed by a second, sustained elevation; a sustained contraction was produced without a transient contraction. The ETB receptor-selective agonist, IRL1620 (1 microM) and sarafotoxin S6c (1 microM) also induced a rapid and transient elevation of [Ca2+]i to reach a peak and a sustained elevation, together with only a small contraction or no contraction. 5. ET-1 (100 nM) induced a transient increase in [Ca2+]i in a Ca(2+)-free, 2 mM EGTA-containing physiological saline solution (Ca(2+)-free PSS), and a small sustained contraction which was significantly different from that induced by ET-1 (100 nM) in normal PSS. The ET-1-induced increase in [Ca2+]i and sustained contraction were not affected by the voltage-dependent Ca2+ channel blocker, nicardipine (10 microM). The ET-1-induced transient increase in [Ca2+]i was significantly reduced by the sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor, cyclopiazonic acid (30 microM); however, the ET-1-induced sustained contraction was not affected by this agent. 6. The selective ETA receptor antagonist, BQ-123 (100 nM) reduced the ET-3 (100 nM)-induced contraction, but did not affect the transient increase or elevation of the second phase of [Ca2+]i. However, this antagonist at 1 microM did not affect the ET-1 (100 nM)- and ET-2 (100 nM)-induced elevation of [Ca2+]i and contractile response, or the IRL1620-induced elevation of [Ca2+]i. 7. The selective ETB receptor antagonist, BQ-788 (1 microM) reduced the transient increase in [Ca2+]i induced by ET-1 (30 nM), ET-2 (30 nM), ET-3 (100 nM) and IRL1620 (1 microM), but did not affect the sustained elevation of [Ca2+]i and contractile responses produced by ET-1, ET-2 and ET-3. 8. Pretreatment with IRL1620 (1 microM) reduced the increase in [Ca2+]i induced by IRL1620 (1 microM) and sarafotoxin S6c (1 microM), as well as the ET-1 (100 nM)-, ET-2 (100 nM)- and ET-3 (1 microM)-induced elevation of [Ca2+]i, whereas in the presence of IRL1620, ET-1-, ET-2- and ET-3-induced contractions were unaltered. 9. These results suggest that ETA and ETB receptor subtypes exist in the rabbit iris dilator muscle, and that the ETA receptor is divided into: (1) BQ-123-sensitive ETA subtypes activated by ET-1, ET-2 and ET-3, and (2) BQ-123-insensitive ETA subtypes activated by ET-1 and ET-2, which cause the sustained increase of [Ca2+]i and contraction; in contrast, ETB receptor subtypes are activated by ET-1, ET-2, ET-3, IRL1620 and sarafotoxin S6c and cause the transient and sustained increase in [Ca2+]i which is not able to contract the smooth muscle.     

Novel effects of 5,8,11-eicosatriynoic acid, a lipoxygenase inhibitor, on Ca2+ mobilization in Madin Darby canine kidney cells

The effect of 5,8,11-eicosatriynoic acid, a widely used lipoxygenase inhibitor, on Ca2+ fate in Madin Darby canine kidney cells was examined by using fura-2 as a Ca2+ probe. At concentrations between 2-100 microM 5,8,11-eicosatriynoic acid increased [Ca2+]i concentration-dependently with an EC50 of 20 microM . Extracellular Ca2+ removal decreased the Ca2+ signals, indicating that 5,8,11-eicosatriynoic acid triggered Ca2+ release and Ca2+ influx. 5,8,11 -Eicosatriynoic acid (30 microM) induced a [Ca2+]i increase in Ca2+-free medium after pretreatment with carbonylcyanide m-chlorophenylhydrazone (2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor for 20 min. Conversely, 5,8,11-eicosatriynoic acid pretreatment almost abolished the Ca2+ release induced by carbonylcyanide m-chlorophenylhydrazone and thapsigargin. These results suggest that 30 microM 5,8,11-eicosatriynoic acid released Ca2+ from the endoplasmic reticulum, mitochondria and other stores. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 2-50 microM 5,8,11-eicosatriynoic acid for 10 min. in Ca2+-free medium concentration-dependently. Pretreatment with 10 microM La3+ abolished 30 microM 5,8,11-eicosatriynoic acid -induced [Ca2+]i increases, but adding La3+ during the decay phase had no effect. 5,8,11-Eicosatriynoic acid-induced Ca2+ release was not altered 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), but was decreased by 60% by 40 microM aristolochic acid. Several other lipoxygenase inhibitors such as baicalein (50 microM), 5.8.11.14-eicosatetraynoic acid (ETYA; 0.1-0.2 mM), caffeic acid (5-50 microM), esculetin (5-50 microM), alpha-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV-5901; 0.1-0.2 mM) and alpha-pentyl-4-(2-quinolinylmethoxy)-benzenemethanol (L-655238; 80-100 microM) had no effect on [Ca2+]i. Collectively, the data suggest that the lipoxygenase inhibitor 5,8,11-eicosatriynoic acid induced a [Ca2+]i increase in renal tubular cells concentration-dependently, by releasing intracellular Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing extracellular Ca2+ influx in a La3+-sensitive manner.     

Des-Arg9-bradykinin increases intracellular Ca2+ in bronchoalveolar eosinophils from ovalbumin-sensitized and -challenged mice

The effects of the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin and the bradykinin B2 receptor agonist, bradykinin were studied on the intracellular free Ca2+ concentration ([Ca2+]i) in murine bronchoalveolar lavage cells from control and ovalbumin-sensitized mice using fura-2 microfluorimetry. The bronchoalveolar lavage cells of control mice, which were predominantly alveolar macrophages, showed an increase in [Ca2+]i in response to bradykinin (1 microM) but not to des-Arg9-bradykinin (1 microM), indicating the presence of functional bradykinin B2 receptors and the absence of B1 receptors. Such elevation in [Ca2+]i induced by bradykinin was totally inhibited by the selective bradykinin B2 receptor antagonist, D-Arg0-Hyp3-Thi5-D-Tic7-Oic8-bradykinin (HOE-140; 10 microM). In contrast, bronchoalveolar lavage cells from ovalbumin-sensitized and -challenged mice significantly responded to both bradykinin and des-Arg9-bradykinin, indicating the presence of both functional bradykinin B1 and B2 receptors. Eosinophils exhibited higher response to des-Arg9-bradykinin (1 microM; 485% increase in [Ca2+]i) compared to bradykinin (1 microM; 163% increase in [Ca2+]i). This des-Arg9-bradykinin-induced [Ca2+]i increase was markedly inhibited by the selective bradykinin B1 receptor antagonist, Ac-Lys-[D-betaNal7, Ile8]des-Arg9-bradykinin (R-715; 10 microM). Des-Arg9-bradykinin neither modified the basal [Ca2+]i in lymphocytes nor in mononuclear cells from ovalbumin-sensitized and challenged mice, while bradykinin produced a [Ca2+]i increase in both cell types. Our results further support the implication of the inducible bradykinin B1 receptors in airway inflammatory response in ovalbumin-sensitized and challenged mice.     

Effects of a water-soluble forskolin derivative (NKH477) and a membrane-permeable cyclic AMP analogue on noradrenaline-induced Ca2+ mobilization in smooth muscle of rabbit mesenteric artery.

1. Effects were studied of 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative and of dibutyryl-cyclic AMP, a membrane-permeable cyclic AMP analogue on noradrenaline (NA)-induced Ca2+ mobilization in smooth muscle strips of the rabbit mesenteric artery. The intracellular concentration of Ca2+ ([Ca2+]i), isometric force and cellular concentration of inositol 1,4,5-trisphosphate (InsP3) were measured. 2. NA (10 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force in a solution containing 2.6 mM Ca2+. NKH477 (0.01-0.3 microM) attenuated the phasic and the tonic increases in both [Ca2+]i and force induced by 10 microM NA, in a concentration-dependent manner. 3. In Ca(2+)-free solution containing 2 mM EGTA with 5.9 mM K+, NA (10 microM) produced only phasic increases in [Ca2+]i and force. NKH477 (0.01 microM) and dibutyryl-cyclic AMP (0.1 mM) each greatly inhibited these increases. 4. NA (10 microM) led to the production of InsP3 in intact smooth muscle strips and InsP3 (10 microM) increased Ca2+ in Ca(2+)-free solution after a brief application of Ca2+ in beta-escin-skinned smooth muscle strips. NKH477 (0.01 microM) or dibutyryl-cyclic AMP (0.1 mM) modified neither the NA-induced synthesis of InsP3 in intact muscle strips nor the InsP3-induced Ca2+ release in skinned strips. 5. In Ca(2+)-free solution, high K+ (40 and 128 mM) itself failed to increase [Ca2+]i but concentration-dependently enhanced the amplitude of the increase in [Ca2+]i induced by 10 microM NA with a parallel enhancement of the maximum rate of rise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of endothelium-dependent relaxation in the rat aorta by IRL 1620, a novel and selective agonist at the endothelin ETB receptor.

1. The effects of a novel and selective agonist at the endothelin ETB receptor, IRL 1620 (Suc-[Glu9, Ala11,15] endothelin-1 (8-21)), were examined in the isolated aorta of the rat. 2. IRL 1620 (1-300 nM) changed neither the resting tone nor the cytosolic Ca2+ level ([Ca2+]i) of the aorta without endothelium. In the presence of endothelium, however, IRL 1620 increased endothelial [Ca2+]i with little effect on the muscle tone. In the absence of external Ca2+, IRL 1620 still induced a transient increase in endothelial [Ca2+]i. 3. Noradrenaline (100 nM) increased both muscle [Ca2+]i and tension. IRL 1620 (1-300 nM) relaxed the muscle with an increase in endothelial [Ca2+]i only in the presence of endothelium. An inhibitor of nitric oxide synthase, 100 microM NG-monomethyl-L-arginine, inhibited the relaxant effect of IRL 1620 but not the increase in endothelial [Ca2+]i. 4. In resting and noradrenaline-stimulated aorta, the effects of IRL 1620 were inhibited by a selective antagonist of the ETB receptor, IRL 1038 (0.3-3 microM), although a selective antagonist of the ETA receptor, BQ-123 (3 microM), was ineffective. Verapamil (10 microM) did not alter the effects of IRL 1620. 5. A muscarinic receptor agonist, carbachol (1 microM), also induced endothelium-dependent relaxation with an increase in endothelial [Ca2+]i. However, the effects of carbachol were not inhibited by the ETB antagonist, IRL 1038 (3 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nordihydroguaiaretic acid elevates osteoblastic intracellular Ca2+

Nordihydroguaiaretic acid (NDGA) is widely used as a pharmacological tool to inhibit lipoxygenases; however, recent evidence suggests that it increases renal intracellular [Ca2+]i via novel mechanisms. Here the effect of NDGA on Ca2+ signaling in MG63 osteoblastic cells was explored using fura-2 as a Ca2+ indicator. NDGA (2-50 microM) increased [Ca2+]i in a concentration-dependent manner. The signal comprised an initial rise and an elevated phase over a time period of 4 min. Removing extracellular Ca2+ reduced 2-50 microM NDGA-induced signals by 62+/-2%. After incubation with 50 microM NDGA in Ca2+-free medium for several minutes, addition of 3 mM CaCl2 induced an increase in [Ca2+]i. NDGA (50 microM)-induced [Ca2+]i increases were not changed by pretreatment with 10 microM of verapamil, diltiazem, nifedipine, nimodipine and nicardipine. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (1 microM) inhibited 50 microM NDGA-induced [Ca2+]i increases by 69+/-3%. Inhibition of phospholipase C with 2 microM U73122 had little effect on 50 microM NDGA-induced Ca2+ release. Several other lipoxygenase inhibitors had no effect on basal [Ca2+]i. At a concentration that did not increase basal [Ca2+]i, NDGA (1 microM) did not alter 10 microM ATP- or 1 microM thapsigargin-induced [Ca2+]i increases. Alteration of protein kinase C activity with 1 nM phorbol 12-myristate 13-acetate or 2 microM GF 109203X did not affect 50 microM NDGA-induced [Ca2+]i increases. Together, the results show that NDGA increased [Ca2+]i in osteoblasts in a lipoxygenase-independent manner, by releasing stored Ca2+ in a fashion independent of phospholipase C activity, and by causing Ca2+ influx.     

Effects of trimebutine on cytosolic Ca2+ and force transitions in intestinal smooth muscle

The effects of trimebutine maleate on cytosolic free Ca2+ and force transitions in the guinea-pig taenia cecum were studied by fura-2 fluorometry and tension recording. The addition of 80 mM K+ induced a transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) and tension, followed by a sustained increase. Trimebutine (10 microM) suppressed both [Ca2+]i elevation and tension development. The tonic responses were more potently inhibited than the phasic responses. Phasic components gradually increased as the added K+ increased (10-40 mM). The relationship between the peak increases in [Ca2+]i and tension was not affected by trimebutine (10 microM). This means that trimebutine does not affect the Ca2+ sensitivity of contractile elements. In a high K+ and Ca(2+)-free medium, carbachol (10 microM) or caffeine (30 mM) caused transient [Ca2+]i elevation and tension development in the smooth muscle. Trimebutine (10 microM) decreased the amplitude of both responses. Trimebutine (10 microM) inhibited the spontaneous fluctuations in [Ca2+]i and motility of taenia cecum in the presence of tetrodotoxin (TTX; 0.3 microM). These results suggest that trimebutine has two types of inhibitory actions on intestinal smooth muscle; one, the inhibition of Ca2+ influx through voltage-dependent calcium channels, and the other, the inhibition of Ca2+ release from intracellular storage sites.     

Ca(2+)-dependent aggregation of rabbit platelets induced by maitotoxin, a potent marine toxin, isolated from a dinoflagellate.

1. Administration of maitotoxin (MTX), a dinoflagellate toxin, caused aggregation of rabbit washed platelets. The cytosolic Ca2+ concentration ([Ca2+]i), measured by fura-2 fluorescence technique, was also increased by the presence of MTX. Rates of aggregation response and [Ca2+]i-increase were dependent on tested concentrations (3-100 ng ml-1) of the toxin. 2. The MTX-induced platelet aggregation and [Ca2+]i-increase were totally abolished in a Ca(2+)-free solution. The successive administration of Ca2+ in the presence of MTX elicited the aggregation and increase in [Ca2+]i. 3. Ba2+ was capable of substituting for Ca2+ in the MTX-induced platelet aggregation. In the presence of external Ca2+, transition metals, Co2+, Cd2+ and Ni2+, inhibited the aggregation response to MTX. 4. Organic calcium antagonists (verapamil and nifedipine) as well as a cyclo-oxygenase-inhibitor (aspirin) did not apparently inhibit the aggregation response to MTX, except for a high concentration (10(-5) M) of verapamil, while procaine (10 mM) reduced the rate of platelet aggregation. 5. MTX also elicited a release of ATP from platelets, which was abolished in the absence of external Ca2+. 6. In contrast, thrombin 0.5 unit ml-1 could elicit platelet shape change, [Ca2+]i-increase and ATP-release in the absence of external Ca2+. 7. These results suggest that the MTX-induced platelet activation is caused by an enhanced Ca(2+)-influx presumably through voltage-independent Ca2+ channels on the plasma membrane.     

Involvement of thromboxane A2 in the endothelium-dependent contractions induced by myricetin in rat isolated aorta.

1. The present study was undertaken to analyse the mechanism of the contractile response induced by the bioflavonoid myricetin in isolated rat aortic rings. 2. Myricetin induced endothelium-dependent contractile responses (maximal value=21+/-2% of the response induced by 80 mM KCl and pD2=5.12+/-0.03). This effect developed slowly, reached a peak within 6 min and then declined progressively. 3. Myricetin-induced contractions were almost abolished by the phospholipase A2 (PLA2) inhibitor, quinacrine (10 microM), the cyclo-oxygenase inhibitor, indomethacin (10 microM), the thromboxane synthase inhibitor, dazoxiben (100 microM), the putative thromboxane A2 (TXA2)/prostaglandin endoperoxide receptor antagonist, ifetroban (3 microM). These contractions were abolished in Ca2+-free medium but were not affected by the Ca2+ channel blocker verapamil (10 microM). 4. In cultured bovine endothelial cells (BAEC), myricetin (50 microM) produced an increase in cytosolic free calcium ([Ca2+]i) which peaked within 1 min and remained sustained for 6 min, as determined by the fluorescent probe fura 2. This rise in [Ca2+]i was abolished after removal of extracellular Ca2+ in the medium. 5. Myricetin (50 microM) significantly increased TXB2 production both in aortic rings with and without endothelium and in BAEC. These increases were abolished both by Ca2+-free media and by indomethacin. 6. Taken together, these results suggests that myricetin stimulates Ca2+ influx and subsequently triggers the activation of the PLA2 and cyclo-oxygenase pathways releasing TXA2 from the endothelium to contract rat aortic rings. The latter response occurs via the activation of Tp receptors on vascular smooth muscle cells.