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.     

Effects of purinoceptor agonists on cytosolic Ca2+ concentration in swine tracheal smooth muscle cells in culture.

1. The effects of various purinoceptor agonists on intracellular Ca2+ concentration ([Ca2+]i) in swine tracheal smooth muscle cells in primary culture were examined to investigate the subtype of purinoceptors in these cells. 2. ATP (1 microM to 1 mM) concentration-dependently increased [Ca2+]i which was measured by monitoring the fluorescence signal of fura2. 3. alpha, beta-Me ATP at concentrations higher than 10 microM increased [ca2+]i in the presence of extracellular Ca2+. Responses to the drug were 12 +/- 5 and 61 +/- 4% of responses to ATP (100 microM) at 100 microM and 1 mM, respectively (n = 7). The response to 100 microM ATP was inhibited by 62% in the presence of 1 mM alpha, beta-Me ATP (n = 8), though the drug at concentrations lower than that did not affect the response to ATP. 4. ATP increased [Ca2+]i in the absence of extracellular Ca2+. The response to ATP in this condition was 40% of that in the presence of extracellular Ca2+ (n = 8). 5. Neither cibacron blue 3GA (10 microM) (n = 8) nor suramin (10 and 100 microM) (n = 10) affected the response to ATP (1 microM to 100 microM). 6. The rank order of potency in the absence of extracellular Ca2+ was UTP > ATP > adenosine 5'-o-(3-thiotriphosphate) > > ADP = alpha, beta-methylene adenosine 5'-triphosphate > 2-(methylthio)-adenosine 5'-(tetrahydrogen triphosphate). 7. UTP (1 microM to 100 microM) concentration-dependently increased inositol 1,4,5-triphosphate (IP3) production. 8. These results suggest that the increase in [Ca2+]i induced by purinoceptor agonists is mediated mainly via a nucleotide receptor in swine tracheal smooth muscle cells in primary culture.     

Ryanodine reveals multiple contractile and relaxant mechanisms in vascular smooth muscle: simultaneous measurements of mechanical activity and of cytoplasmic free Ca2+ level with fura-2.

1. The effects of ryanodine on changes in cytoplasmic Ca2+ level ([Ca2+]i) and muscle tension induced by maximum concentrations of phenylephrine (Phe; 1 microM), prostaglandin F2 alpha (PGF2 alpha, 10 microM), caffeine (Caf, 30 mM) and isoprenaline (Iso, 1 microM) were examined in rat aortic strips using fura-2. 2. In normal media, Phe and PGF2 alpha produced a phasic contraction, followed by a tonic one. Caf elicited only a transient contraction. When the preparation was treated with 10 microM ryanodine, an increase in [Ca2+]i was induced accompanied by a nicardipine (1 microM)-resistant contraction which was [Ca2+]o-dependent. 3. In Ca2(+)-free solution, the three stimulants elicited transient increases in [Ca2+]i. Transient contractions to Phe and Caf were accompanied by changes in [Ca2+]i. The transient increase in [Ca2+]i induced by PGF2 alpha was not accompanied by a corresponding contraction. 4. Sustained contractions were induced by Phe and PGF2 alpha in the absence of external Ca2+, while the increase in [Ca2+]i was reduced. A larger maximum contraction was induced by PGF alpha than by Phe. 5. Ryanodine abolished both the Caf- and Phe-induced [Ca2+]i transient increases and the corresponding contractions, but had no substantial effect on the PGF2 alpha-induced [Ca2+]i transient increase. Ryanodine had no influence on the sustained contractions induced by Phe and PGF2 alpha. 6. Iso relaxed both sustained contractions almost completely, without any detectable change in [Ca2+]i. Treatment of the preparation with ryanodine had no effect on the concentration-response curves for Iso in relaxing the 0.1 microM Phe- or 40 mM K(+)-induced precontraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual regulation of cerebrovascular tone by UTP: P2U receptor-mediated contraction and endothelium-dependent relaxation.

1. The mechanisms of vascular tone regulation by extracellular uridine 5'-triphosphate (UTP) were investigated in bovine middle cerebral arterial strips. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored by use of front-surface fluorometry of fura-2. 2. In the arterial strips without endothelium, UTP (0.1 microM-1 mM) induced contraction in a concentration-dependent manner. However, when the endothelium was kept intact, cumulative application of UTP (0.1-100 microM) (and only at 1 mM) induced a modest phasic contraction in arterial strips. This endothelium-dependent reduction of the UTP-induced contraction was abolished by 100 microM N omega-nitro-L-arginine (L-NOARG) but not by 10 microM indomethacin. In the presence of intact endothelium, UTP (30 microM) induced a transient relaxation of the strips precontracted with 30 nM U-46619 (a stable analogue of thromboxane A2), which was completely inhibited by pretreatment with L-NOARG but not with indomethacin. 3. In the endothelium-denuded strips, the contractile response to UTP was abolished by desensitization to either ATP gamma S or ATP (P2U receptor agonists), but not by desensitization to alpha, beta-methylene-ATP (P2x receptor agonist) or to 2-methylthio-ATP (P2Y receptor agonist). Desensitization to UTP abolished the contractile response to ATP. 4. In the endothelium-denuded artery, a single dose application of UTP induced an initial transient, and subsequently lower but sustained increase in [Ca2+]i and force. In the absence of extracellular Ca2+, UTP induced only the initial transient increases in [Ca2+]i and force, while the sustained increases in [Ca2+]i and force were abolished. UTP (1 mM) had no effect on the basic [Ca2+]i-force relationship obtained on cumulative application of extracellular Ca2+ at steady state of 118 mM K(+)-depolarization-induced contraction. 5. We conclude that in the presence of an intact endothelium, UTP-induced relaxation of preconstricted middle cerebral artery is mainly mediated indirectly, by the production of an endothelium-derived relaxing factor, but at high doses of UTP, vascular smooth muscle contraction is mediated directly via activation of P2U purinoceptor and [Ca2+]i elevation without Ca(2+)-sensitization of the contractile apparatus. UTP may thus exert a dual regulatory effect upon cerebrovascular tone, but in cases where the endothelium is impaired, it may also act as a significant vasoconstrictor.     

Activation of rabbit platelets by Ca2+ influx and thromboxane A2 release in an external Ca(2+)-dependent manner by zooxanthellatoxin-A, a novel polyol.

1. Zooxanthellatoxin-A (ZT-A), a novel polyhydroxylated long chain compound, isolated from a symbiotic marine alga Simbiodinium sp., caused aggregation in rabbit washed platelets in a concentration-dependent manner (1-4 microM), accompanied by an increase in cytosolic Ca2+ concentration ([Ca2+]i). 2. ZT-A did not cause platelet aggregation or increase [Ca2+]i in a Ca(2+)-free solution, and Cd2+ (0.1-1 mM), Co2+ (1-10 mM) and Mn2+ (1-10 mM) inhibited ZT-A-induced aggregation. SK&F96365 (1-100 microM), a receptor operated Ca2+ channel antagonist, and mefenamic acid (0.1-10 microM), a non-specific divalent cation channel antagonist, inhibited platelet aggregation and the increase in [Ca2+]i induced by ZT-A. 3. Indomethacin (0.1-10 microM), a cyclo-oxygenase inhibitor, and SQ-29548 (0.1-10 microM), a thromboxane A2 (TXA2) receptor antagonist, inhibited platelet aggregation and the increase in [Ca2+]i induced by ZT-A. 4. Methysergide (0.01-1 microM), a 5-HT2 receptor antagonist, inhibited ZT-A-induced platelet aggregation but did not affect the increase in [Ca2+]i induced by ZT-A. 5. Tetrodotoxin (1 microM), a Na+ channel blocker and chlorpheniramine (1 microM), a H1-histamine receptor antagonist, neither affected ZT-A-induced platelet aggregation nor the increase in [Ca2+]i induced by ZT-A. 6. Genistein (1-100 microM), a protein tyrosine kinase inhibitor, and staurosporine (0.01-1 microM), a protein kinase C inhibitor, also inhibited ZT-A-induced platelet aggregation. 7. The present results suggest that ZT-A elicits Ca(2+)-influx from platelet plasma membranes. The resulting increase in [Ca2+]i subsequently stimulates the secondary release of TXA2 from platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rho-kinase inhibitor inhibits both myosin phosphorylation-dependent and -independent enhancement of myofilament Ca2+ sensitivity in the bovine middle cerebral artery

The role of Rho kinase in Ca2+ sensitization of the contractile apparatus in smooth muscle was investigated in the bovine middle cerebral artery. U46619, a thromboxane A2 analog, induced a greater sustained contraction with a smaller [Ca2+]i elevation than that seen with 118 mm K+. The level of myosin light chain (MLC) phosphorylation obtained in the initial phase of the contraction was higher than that seen with 118 mm K+; thereafter, it gradually declined to a comparable level in the late phase. During the steady state of the U46619-induced contraction, Y27632 (10 microM), a Rho-kinase inhibitor, partially inhibited [Ca2+]i, although it substantially inhibited tension and MLC phosphorylation. Wortmannin (10 microM), an MLC kinase inhibitor, had no significant effect on [Ca2+]i, but it completely inhibited MLC phosphorylation and partially inhibited tension. The wortmannin-resistant tension development was thus not associated with MLC phosphorylation, and this component was completely inhibited by Y27632. In conclusion, U46619 enhanced Ca2+ sensitivity in a manner both dependent and independent of MLC phosphorylation in the bovine middle cerebral artery. Both mechanisms of Ca2+ sensitization can be inhibited by the Rho-kinase inhibitor.     

Effects of pinacidil on contractile proteins in high K(+)-treated intact, and in beta-escin-treated skinned smooth muscle of the rabbit mesenteric artery.

1. The effects of pinacidil were investigated on changes in cellular Ca2+ concentration ([Ca2+]i) and tension in intact and chemically skinned smooth muscle strips of the rabbit mesenteric artery. 2. High K+ (128 mM) produced a large phasic followed by a tonic increase in [Ca2+]i and tension in intact muscle strips. Pinacidil at 10 microM but not 1 microM, inhibited the phasic and tonic contractions induced by 128 mM K+ without a corresponding change in [Ca2+]i. 3. In beta-escin-treated skinned smooth muscle, the minimum Ca2+ concentration that produced contraction was 0.1 microM and the maximum contraction was obtained at 10 microM. Pinacidil at 10 microM but not 1 microM, shifted the pCa-tension relation curve to the right and also inhibited the maximum contraction induced by Ca2+. The concentrations of Ca2+ required for half maximal tension were 0.9 microM in control and 1.5 microM in the presence of 10 microM pinacidil. Calmodulin (2 microM) increased the contraction induced by 0.3 microM Ca2+ (but not by 10 microM Ca2+) in the skinned strips. Pinacidil (10 microM) inhibited the contraction induced by 0.3 microM or 10 microM Ca2+ in the presence of 2 microM calmodulin. 4. Noradrenaline (NA, 10 microM) with guanosine triphosphate (GTP, 3 microM), guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S, 3 microM) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) all enhanced the contraction induced by 0.3 microM Ca2+. Pinacidil (10 microM) inhibited the contraction induced by 0.3 microM Ca2+ more strongly in the presence of the above agents than in their absence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple effects of ryanodine on intracellular free Ca2+ in smooth muscle cells from bovine and porcine coronary artery: modulation of sarcoplasmic reticulum function.

1. The effects of ryanodine and caffeine on intracellular free Ca2+ concentration ([Ca2+]i) were studied by use of fura-2 microfluorometry in single smooth muscle cells freshly dispersed from bovine and porcine coronary artery. 2. Bovine and porcine cells demonstrated similar sensitivities to 10 min of exposure to ryanodine in physiological salt solution (PSS), as determined by comparable dose-dependent decreases in the subsequent [Ca2+]i transient induced by 5 mM caffeine. 3. Ryanodine (10 microM) caused a significant increase in [Ca2+]i to a plateau level 27 +/- 3% and 38 +/- 4% above baseline [Ca2+]i (baseline [Ca2+]i = [Ca2+]i at 0 min) in porcine and bovine cells, respectively, when bathed in PSS. In bovine cells the time required to reach 1/2 the plateau level was only 3 min versus 6 min for porcine cells. 4. The ryanodine-induced plateau increase in [Ca2+]i was 35 +/- 5% above baseline for bovine cells bathed in 0 Ca PSS (PSS including 10 microM EGTA with no added Ca2+), but only 7 +/- 3% above baseline in porcine cells during 10 min exposure to 10 microM ryanodine. In bovine cells [Ca2+]i showed proportional increases when extracellular Ca2+ was increased from the normal 2 mM Ca2+ PSS to 5 and 10 mM. 5. Cells pretreated with caffeine in 0 Ca PSS, which depleted the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, showed no increase in [Ca2+]i when challenged with 10 microM ryanodine. The ryanodine-associated increase in [Ca2+]i, which was sustained in 0 Ca PSS during the 10 min ryanodine exposure in cells not pretreated with caffeine, suggests that ryanodine releases Ca2+ from the sarcoplasmic reticulum, but also inhibits Ca2+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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)     

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 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)     

The inhibitory effect of Mn2+ on the ATP-dependent Ca2+ pump in rat brain synaptic plasma membrane vesicles.

Synaptic plasma membrane (SPM) vesicles take up Ca2+ when both ATP and Mg2+ are added to the reaction medium. Maximal ATP-dependent Ca2+ uptake is obtained with between 3 and 5 mM Mg2+. Higher [Mg2+] results in a slight decrease in the ATP-dependent Ca2+ influx, which at 10 mM Mg2+ is equal to 70% of the maximal uptake. [Mn2+] up to 0.3 mM, can support ATP-dependent Ca2+ uptake as efficiently as equimolar [Mg2+]. Maximal ATP-dependent Ca2+ uptake in the presence of Mn2+ is obtained between 1 and 2 mM but its extent is only 70% of the maximal uptake obtained in the presence of Mg2+. As the [Mn2+] increases the ATP-dependent Ca2+ uptake decreases and at 10 mM Mn2+ it is only 40% of that obtained in the presence of Mg2+. The reduction in ATP-dependent Ca2+ uptake in the presence of Mn2+ is obtained also when Mg2+ is present in the reaction medium. The effect is independent of the [Mg2+] used and depends on the [Mn2+] alone. Studying the relationship between [Mn2+] and [Ca2+] revealed three patterns: at 0.3 mM Mn2+ this ion could replace Mg2+ over the entire [Ca2+] range tested, 5-200 microM, supporting ATP-dependent Ca2+ uptake. When the [Mn2+] was increased to 1.8 mM, it exhibited a competitive behaviour with Ca2+ which resulted in an increase in apparent Km to Ca2+ of the SPM Ca2+ pump from 10.69 microM (SD = 3.49) to 28.88 microM (SD = 21.08). Four millimolar Mn2+ inhibited ATP-dependent Ca2+ uptake to 50% of that obtained in equimolar Mg2+ over the entire [Ca2+], 5-200 microM, tested. No transport of 54Mn2+ by the SPM Ca2+ pump could be detected. Inclusion of Mn2+ in the reaction medium led to excessive phosphorylation of SPM proteins. The excessive levels of phosphorylation persisted also when Ca2+ and/or Mg2+ were present in the reaction media.     

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.     

Tamoxifen-induced Ca2+ mobilization in bladder female transitional carcinoma cells

This study examined the effect of tamoxifen, an anti-breast cancer drug, on Ca2+ handling in bladder female transitional cancer cells. Changes in cytosolic free Ca2+ levels were recorded by using the Ca2+-sensitive dye fura-2. In a dose-dependent manner, tamoxifen induced intracellular free Ca2+ concentrations ([Ca2+]i) increases between 5 and 20 microM with an EC50 of 10 microM. External Ca2+ removal reduced the response by 60+/-6%. Addition of 3 mM Ca2+ caused a [Ca2+]i increase after pretreatment with 10 microM tamoxifen in Ca2+-free medium. In Ca2+-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca2+]i increase induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 1 microM thapsigargin prevented tamoxifen from releasing more Ca2+. Inhibition of phospholipase C-dependent inositol 1,4,5-tris-phosphate formation with 2 microM U73122 did not alter 10 microM tamoxifen-induced Ca2+ release. The [Ca2+]i increase induced by 5 microM tamoxifen was not altered by 10 microM La3+, nifedipine, verapamil, and diltiazem. Collectively, it was found that tamoxifen increased [Ca2+]i in bladder cancer cells by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from external medium.     

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.     

Dual action of palmitoyl trifluoromethyl ketone (PACOCF3) on Ca2+ signaling: activation of extracellular Ca2+ influx and alteration of ATP- and bradykinin-induced Ca2+ responses in Madin Darby canine kidney cells

The effect of the phospholipase A2 inhibitor palmitoyl trifluoromethyl ketone (PACOCF3) on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was examined using fura-2 as the fluorescent Ca2+ indicator. At a concentration of 20 microM, PACOCF3 did not change basal cytosolic free calcium concentrations ([Ca2+]i), but at concentrations of 50-250 microM PACOCF3 induced an increase in [Ca2+]i by activating extracellular Ca2+ entry which was partly suppressed by 50 microM La3+. The effect of PACOCF3 was abolished by removal of extracellular Ca2+. PACOCF3 (10 microM) enhanced both the peak value and the area under the curve of the [Ca2+]i increase induced by 10 microM ATP and 1 microM bradykinin by potentiating extracellular Ca2+ influx without affecting internal Ca2+ release. Several other phospholipase A2 inhibitors had no effect on basal [Ca2+]i or agonist-induced [Ca2+]i increases. Collectively, the results suggest that PACOCF3 alters Ca2+ signaling in renal tubular cells in a manner independent of phospholipase A2 inhibition.     

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.     

Mechanism underlying H2O2-induced inhibition of acetylcholine-induced contraction in rabbit tracheal smooth muscle

The mechanism underlying the inhibition by H2O2 of acetylcholine-induced contraction was investigated in epithelium-denuded strips of rabbit trachea. Acetylcholine (10 microM) generated a phasic, followed by a tonic increase in both the intracellular Ca2+ concentration ([Ca2+]i) and force. Although the acetylcholine-induced tonic contraction was around 9 times the high K+ (80 mM)-induced one, the two stimulants induced similar [Ca2+]i increases (around 0.2 microM), indicating that acetylcholine generates tonic contraction via increases in both [Ca2+]i and myofilament Ca2+-sensitivity. H2O2 (30 microM) (a) enhanced the acetylcholine-induced tonic (not phasic) increase in [Ca2+]i but attenuated both phases of the acetylcholine-induced contraction and (b) enhanced the high K+-induced increase in [Ca2+]i but did not modify the high K+-induced contraction. In beta-escin-skinned strips, application of acetylcholine in the presence of GTP enhanced the contraction induced by 0.3 microM Ca2+ so that its amplitude became similar to that induced by 1 microM Ca2+. H2O2 (30 microM) attenuated the contraction induced by 0.3 microM Ca2+ (alone or in the presence of acetylcholine) but not those induced by higher concentrations of Ca2+ alone (0.5 microM and 1 microM). These results indicate that H2O2 acts directly on contractile proteins in rabbit tracheal smooth muscle to inhibit the contraction induced by low concentrations of Ca2+ (<0.5 microM). An action of H2O2 that increases [Ca2+]i (and thereby masks this reactive-oxygen-induced inhibition of myofilament Ca2+-sensitivity) is apparent in the presence of high K+ but not of acetylcholine. Thus, in rabbit tracheal smooth muscle H2O2 downregulates myofilament Ca2+-sensitivity more potently during acetylcholine-induced contraction than during high-K+-induced contraction, leading to an effective inhibition of the former contraction.