Essential role of extracellular charged residues of the human CCK(1) receptor for interactions with SR 146131, SR 27897 and CCK-8S

We hypothesized that charge-charge interactions may be important for the binding of the human cholecystokinin type 1 (CCK(1)) receptor-specific non-peptide full agonist SR 146131, (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-[2-(4-(2-chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid) and the natural octapeptide CCK-8S to the CCK(1) receptor. Alanine replacement studies of positively charged residues in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expressed in monkey kidney epithelial COS-7 cells. Two residues, Lys(115) and Lys(187), were implicated in SR 27897 binding. Only the replacement of Lys(115), Arg(197) and Arg(336) significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues, but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, although these molecules probably occupy different binding sites on the CCK(1) receptor, we show that a small non-peptide agonist, SR 146131, can stimulate the dual signaling pathways mediated by the CCK(1) receptor.     

Pharmacological characterization of rebamipide: its cholecystokinin CCK1 receptor binding profile and effects on Ca2+ mobilization and amylase release in rat pancreatic acinar cells

We previously reported that rebamipide (2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid) generated oscillations of intracellular Ca2+ concentration ([Ca2+]i) probably through the activation of cholecystokinin type 1 (CCK1) receptors in rat pancreatic acinar cells. Therefore, in the present study, we aimed to establish the pharmacological characteristics of rebamipide in rat pancreatic acinar cells. CCK-8S and rebamipide inhibited [125I]BH-CCK-8S binding to rat pancreatic acinar cell membranes with IC50 values of 3.13 nM and 37.7 microM, respectively. CCK-8S usually evoked [Ca2+]i oscillations at concentrations lower than 50 pM, and it induced biphasic [Ca2+]i increases at higher concentrations. In contrast to CCK-8S, rebamipide only induced [Ca2+]i oscillations at all the concentrations we used in this study. In addition, rebamipide was shown to inhibit high concentrations of CCK-8S-induced biphasic increases in [Ca2+]i, suggesting that rebamipide might be a partial agonist at cholecystokinin CCK1 receptors. Although rebamipide induced [Ca2+]i oscillations by activating the cholecystokinin CCK1 receptors, rebamipide did not cause amylase release and only inhibited CCK-stimulated amylase release reversibly and dose-dependently. However, rebamipide did not inhibit carbachol-, vasoactive intestinal polypeptide (VIP)-, and forskolin-induced amylase releases. These data indicate that rebamipide functions as a partial agonist for Ca2+ -mobilizing action, and it is also an antagonist for the amylase-releasing action of CCK.     

Contrasting roles of leu(356) in the human CCK(1) receptor for antagonist SR 27897 and agonist SR 146131 binding

A new highly specific, potent non-peptide agonist for the cholecystokinin subtype 1 receptor (CCK(1)), SR 146131 (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid) was recently described [Bignon, E., Bachy, A., Boigegrain, R., Brodin, R., Cottineau, M., Gully, D., Herbert, J.-M., Keane, P., Labie, C., Molimard, J.-C., Olliero, D., Oury-Donat, F., Petereau, C., Prabonneaud, V., Rockstroh, M.-P., Schaeffer, P., Servant, O.Thurneyssen, O., Soubrié, P., Pascal, M., Maffrand, J.-P., Le Fur, G., 1999. SR 146131: a new, potent, orally active and selective non-peptide cholecystokinin subtype I receptor agonist: I. In vitro studies. J. Pharmacol. Exp. Ther. 289, 742-751]. From binding and activity assays with chimeric constructs of human CCK(1) and the cholecystokinin subtype 2 receptor (CCK(2)) and receptors carrying point mutations, we show that Leu(356), situated in transmembrane domain seven in the CCK(1) receptor, is a putative contact point for SR 146131. In contrast, Leu(356) is probably not in contact with the CCK(1) receptor specific antagonist SR 27897 (1-[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl indoyl]acetic acid), a compound structurally related to SR 146131, since its replacement by alanine, histidine or asparagine gave receptors having wild-type CCK(1) receptor SR 27897 binding affinity. Previous mutational analysis of His(381), the cognate position in the rat CCK(2) receptor, had implicated it as being involved in subtype specificity for SR 27897, results which we confirm with corresponding mutations in the human CCK(2) receptor. Moreover, binding and activity assays with the natural CCK receptor agonist, CCK-8S, show that CCK-8S is more susceptible to the mutations in that position in the CCK(1) receptor than in the CCK(2) receptor. The results suggest different binding modes for SR 27897, SR 146131 and CCK-8S in each CCK receptor subtype.     

Bimatoprost and its free acid are prostaglandin FP receptor agonists.

Bimatoprost (17-phenyl-prostaglandin F(2alpha) ethyl amide) has been reported not to exert its actions via prostaglandin receptors. Here, bimatoprost displaced [3H]prostaglandin F(2alpha) from FP receptors (K(i)=6310+/-1650 nM). Bimatoprost rapidly mobilized intracellular Ca(2+) ([Ca(2+)](i)) via cloned human FP receptors expressed in human embryonic kidney cells (EC(50)=2940+/-1663 nM) and via native FP receptors in 3T3 mouse fibroblasts (EC(50)=2200+/-670 nM). Furthermore, AL-8810 ((5Z, 13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20-pentanor-5,13-prostadienoic acid), an FP receptor antagonist, blocked the bimatoprost-induced [Ca(2+)](i) mobilization.     

Alteration of the [Ca(2+)](i)-force relationship during the vasorelaxation induced by a Ca(2+) channel blocker SR33805 in the porcine coronary artery

The mechanism of vasorelaxation induced by SR33805 was investigated by simultaneously monitoring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and force, and by determining level of myosin light chain (MLC) phosphorylation in the medial strip of the porcine coronary artery. SR33805 inhibited the sustained increases in [Ca(2+)](i) and force (IC(50); 3.2+/-1.0 and 49.4+/-27.5 nM, respectively) induced by 118 mM K(+)-depolarization. There was about a 10 fold difference in the inhibitory potency between [Ca(2+)](i) and force. SR33805 completely inhibited the [Ca(2+)](i) elevation induced by a thromboxane A(2) analogue, U46619 and histamine, at concentrations (1 microM) higher than those required for the complete inhibition of K(+)-depolarization induced [Ca(2+)](i) elevation. SR33805 had no effect on the [Ca(2+)](i) elevation induced by histamine or caffeine in the absence of extracellular Ca(2+). SR33805 caused a leftward shift of the [Ca(2+)](i)-force relationship of the contraction induced by cumulative application of extracellular Ca(2+) during 118 mM K(+)-depolarization. The relationship between [Ca(2+)](i) and MLC phosphorylation also shifted to the left by SR33805, while the relationship between MLC phosphorylation and force remained unaffected. In conclusion, SR33805 caused an apparent leftward shift of the [Ca(2+)](i)-force relationship, accompanied by a greater degree of MLC phosphorylation for a given level of [Ca(2+)](i). The mechanism of this leftward shift, however, still remains to be elucidated.     

Pharmacological evaluation of the role of cytochrome P450 in intracellular calcium signalling in rat pancreatic acinar cells

We have investigated whether the cytochrome P450 system is involved in Ca(2+) signalling in rat pancreatic acinar cells. Intracellular free [Ca(2+)] ([Ca(2+)](i)) was measured in collagenase-isolated cells using fura-2 microspectrofluorimetry and imaging. The imidazole P450 inhibitor ketoconazole (5 - 50 microM) inhibited [Ca(2+)](i) oscillations induced by cholecystokinin octapeptide (CCK). However, ketoconazole also raised baseline [Ca(2+)](i) when applied in the absence of CCK. These effects were mimicked by 5 - 50 microM SKF96365, an imidazole widely used as an inhibitor of Ca(2+) entry. The non-imidazole P450 inhibitor proadifen (SKF525A) inhibited CCK-induced [Ca(2+)](i) oscillations at a concentration of 10 - 50 microM. Proadifen alone caused intracellular Ca(2+) release at 25 or 50 microM, but not at 10 microM. Octadecynoic acid and 1-aminobenzotriazole, structurally-unrelated non-imidazole P450 inhibitors, did not alter baseline [Ca(2+)](i) or CCK-evoked oscillations. We compared cumulative CCK dose-response relationship in control cells and in cells where P450 had been induced by prior injection of animals with beta-naphthoflavone. Only minor differences were apparent, with induced cells showing some decrease in responsiveness at moderate and higher concentration of CCK (30 pM - 3 nM). Direct assessment of depletion-activated Ca(2+) entry showed no clear differences between control and induced cells. In conclusion, we could find no compelling evidence for a role of P450 in controlling Ca(2+) signalling generally, or Ca(2+) entry in particular, in pancreatic acinar cells. Induction of P450 is therefore probably toxic to acinar cells via a Ca(2+)-independent mechanism.     

A novel effect of rebamipide: generation of [Ca(2+)](i) oscillations through activation of CCK(1) receptors in rat pancreatic acinar cells

The protective effect of 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid (rebamipide) on gastric mucosa is well established. Here we demonstrate that rebamipide acts on pancreatic acinar cells to generate oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the activation of cholecystokinin subtype 1 (CCK(1)) receptors. At concentrations higher than 5 microM, rebamipide induced [Ca(2+)](i) oscillations in individual fura-2-loaded pancreatic acinar cells. The frequency of oscillations increased with increasing concentrations of rebamipide, while the latency between stimulation of cells and initiation of [Ca(2+)](i) oscillations decreased with increasing concentration. The [Ca(2+)](i) oscillations evoked by rebamipide were inhibited by the CCK(1) receptor antagonist L-364,718 but not by atropine or the CCK(2) receptor antagonist L-365,260 indicating that rebamipide is a nonpeptide CCK(1) receptor agonist.     

Effect of betulinic acid on intracellular-free Ca(2+) levels in Madin Darby canine kidney cells

The effect of betulinic acid, an anti-tumor and apoptosis-inducing natural product, on intracellular-free levels of Ca(2+) ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was examined by using fura-2 as a Ca(2+) dye. Betulinic acid caused significant increases in [Ca(2+)](i) concentration dependently between 25 and 500 nM with an EC(50) of 100 nM. The [Ca(2+)](i) signal was composed of an initial gradual rise and a plateau. The response was decreased by removal of extracellular Ca(2+) by 45+/-10%. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) abolished 250 microM betulinic acid-induced [Ca(2+)](i) increases. Conversely, pretreatment with betulinic acid only partly inhibited thapsigargin-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 250 nM betulinic acid in Ca(2+)-free medium for 5 min. This [Ca(2+)](i) increase was not altered by the addition of 20 microM SKF96365 and 10 microM econazole. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 microM) abolished 250 nM betulinic acid-induced Ca(2+) release. Pretreatment with 10 microM La(3+) inhibited 250 nM betulinic acid-induced [Ca(2+)](i) increases by 85+/-3%; whereas 10 microM of verapamil, nifedipine and diltiazem had no effect. In Ca(2+) medium, pretreatment with 2.5 nM betulinic aid for 260 s potentiated 10 microM ATP and 1 microM thapsigargin-induced [Ca(2+)](i) increases by 33+/-3% and 45+/-3%, respectively. Trypan blue exclusion revealed that acute exposure of 250 nM betulinic acid for 2-30 min decreased cell viability by 6+/-2%, which could be prevented by pretreatment with 2 microM U731222. Together, the results suggest that betulinic acid induced significant [Ca(2+)](i) increases in MDCK cells in a concentration-dependent manner, and also induced mild cell death. The [Ca(2+)](i) signal was contributed by an inositol 1,4, 5-trisphosphate-dependent release of intracellular Ca(2+) from thapsigargin-sensitive stores, and by inducing Ca(2+) entry from extracellular medium in a La(3+)-sensitive manner.     

SR146131, a cholecystokinin-A receptor agonist,antagonizes prepulse inhibition deficits produced by dizocilpine and DOI

Abstract Rationale. Converging evidence has demonstrated that cholecystokinin (CCK) inhibits mesolimbic brain dopamine (DA) function via activation of CCK-A (CCK-1) receptors. These effects of CCK have stimulated interest in the potential use of CCK agonists as antipsychotic drugs. Most research on the antipsychotic-like drug effects of CCK has used CCK or CCK analogues that nonselectively activate both CCK-A and CCK-B (CCK-2) receptors, which may produce opposite effects. SR146131, a CCK-A selective nonpeptide agonist, has recently been developed (Sanofi-Synthelabo). Objective. To determine whether SR146131 exhibits antipsychotic-like qualities in the prepulse inhibition (PPI) paradigm. Methods. We performed experiments to determine whether SR146131 (vehicle, 0.01, 0.1, 1.0 mg/kg) would attenuate PPI deficits induced by amphetamine (2.0 mg/kg), an indirect dopamine agonist, and dizocilpine (0.1 mg/kg), a noncompetitive N-methyl-D-aspartate (NMDA) antagonist. Since SR146131 demonstrated significant effects on PPI disrupted by the noncompetitive NMDA antagonist, an effect associated with drugs that inhibit serotonin (5HT)2A transmission, we also tested the effects of SR146131 on PPI disruption produced by 2,5-dimethoxy-4-iodoamphetamine (DOI, 0.5 mg/kg), a direct 5HT2A agonist. Results. SR146131 did not significantly affect startle magnitude, baseline PPI, or amphetamine-induced PPI deficits. However, it dose-dependently antagonized dizocilpine and DOI-induced PPI deficits. Conclusions. The lack of an effect of SR146131 on amphetamine-induced disruption of PPI suggests that a selective nonpeptide CCK-A agonist may not produce antipsychotic-like effects on dopamine transmission. However, the unexpected effects of SR146131 on dizocilpine and DOI-induced PPI deficits are consistent with the effects of drugs that inhibit transmission in the 5HT2A receptor system, including atypical antipsychotic drugs. Possible mechanisms underlying these findings are discussed. Electronic Publication     

Choline induces Ca2+ entry in cultured sympathetic neurones isolated from rat superior cervical ganglion

Choline has been shown to be a specific agonist at alpha7 nicotinic acetylcholine receptors, which are the most Ca(2+) permeable of the ionotropic receptor channels. Whole-cell patch recording combined with the measurement of intracellular free Ca(2+) concentration ([Ca(2+)](i), using Indo1, in cultured rat superior cervical ganglion neurones demonstrated that application of choline induced a slowly desensitizing inward current and increased [Ca(2+)](i). The effect was dose dependent with an EC(50) of 1.6 mM and an n(H) of 1.19. The relationship between the elevation of [Ca(2+)](i) (Delta[Ca(2+)](i)) and charge transfer analysed under various recording conditions showed that the Delta[Ca(2+)](i) induced by choline resulted from an influx of Ca(2+) through nicotinic acetylcholine receptors. The effect of choline on the membrane current and Delta[Ca(2+)](i) was not affected by either short application or pretreatment with alpha-bungarotoxin (50 nM) and methyllycaconitine (1 nM), two alpha7 nicotinic receptors antagonists. These results indicate that activation of non-alpha7 nicotinic acetylcholine receptors by choline significantly increases the Ca(2+) concentration in rat superior cervical ganglion neurones.     

Proliferative effects of cholecystokinin in GH3 pituitary cells mediated by CCK2 receptors and potentiated by insulin

1. Proliferative effects of CCK peptides have been examined in rat anterior pituitary GH3 cells, which express CCK2 receptors. 2. CCK-8s, gastrin(1-17) and its glycine-extended precursor G(1-17)-Gly, previously reported to cause proliferation via putative novel sites on AR4-2J and Swiss 3T3 cells, elicited significant dose dependent increases of similar magnitude in [3H]thymidine incorporation over 3 days in serum-free medium of 39 +/- 10% (P < 0.01, n = 20), 37 +/- 8% (P < 0.01, n = 27) and 41 +/- 6% (P < 0.01, n = 36) respectively. 3. CCK-8s and gastrin potentially stimulated mitogenesis (EC50 values 0.12 nM and 3.0 nM respectively), whilst G-Gly displayed similar efficacy but markedly lower potency. L-365,260 consistently blocked each peptide. The CCK2 receptor affinity of G-Gly in GH3 cells was 1.09 microM (1.01;1.17, n = 6) and 5.53 microM (3.71;5.99, n = 4) in guinea-pig cortex. 4. 1 microM G-Gly weakly stimulated Ca2+ increase, eliciting a 104 +/- 21% increase over basal Ca2+ levels, and was blocked by 1 microM L-365,260 whilst CCK-8s (100 nM) produced a much larger Ca2+ response (331 +/- 14%). 5. Insulin dose dependently enhanced proliferative effects of CCK-8s with a maximal leftwards shift of the CCK-8s curve at 100 ng ml(-1) (17 nM) (EC50 decreased 500 fold, from 0.1 nM to 0.2 pM; P < 0.0001). 10 microg ml(-1) insulin was supramaximal reducing the EC50 to 5 pM (P = 0.027) whilst 1 ng ml(-1) insulin was ineffective. Insulin weakly displaced [125I]BHCCK binding to GH3 CCK2 receptors (IC50 3.6 microM). 6. Results are consistent with mediation of G-Gly effects via CCK2 receptors in GH3 cells and reinforce the role of CCK2 receptors in control of cell growth. Effects of insulin in enhancing CCK proliferative potency may suggest that CCK2 and insulin receptors converge on common intracellular targets and indicates that mitogenic stimuli are influenced by the combination of extracellular factors present.     

Effect of MEN 10755, a new disaccharide analogue of doxorubicin, on sarcoplasmic reticulum Ca(2+) handling and contractile function in rat heart

1. The use of anthraquinone antineoplastic agents is limited by their cardiac toxicity, which is largely due to activation of the sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor). MEN 10755 is a new disaccharide analogue of doxorubicin. We have evaluated its effects on SR function and its toxicity in isolated working rat hearts. 2. In rat SR vesicles, doxorubicin stimulated [(3)H]-ryanodine binding by increasing its Ca(2+)-sensitivity. At 1 microM Ca(2+), ryanodine binding increased by 15.3+/-2.5 fold, with EC(50)=20.6 microM. Epirubicin produced a similar effect, i.e. 9.7+/-0.6 fold stimulation with EC(50)=11.1 microM. MEN 10755 increased ryanodine binding by 1.9+/-0.3 fold (P:<0.01 vs doxorubicin and epirubicin), with EC(50)=38.9 microM. 3. Ca(2+)-induced Ca(2+) release experiments were performed by quick filtration technique, after SR loading with (45)Ca(2+). At 2 microM Ca(2+), doxorubicin (50 microM) increased the rate constant of Ca(2+) release to 82+/-5 s(-1) vs a control value of 22+/-2 s(-1) (P:<0.01), whereas 50 microM MEN 10755 did not produce any significant effect (24+/-3 s(-1)). 4. Ca(2+)-ATPase activity and (45)Ca(2+)-uptake were not significantly affected by doxorubicin, its 13-dihydro-derivative, epirubicin, MEN 10755 and the 13-dihydro-derivative of MEN 10755, at concentrations < or =100 microM. 5. In isolated heart experiments, administration of 30 microM doxorubicin or epirubicin caused serious contractile impairment, whereas 30 microM MEN 10755 produced only minor effects. 6. In conclusion, in acute experiments MEN 10755 was much less cardiotoxic than equimolar doxorubicin or epirubicin. This result might be accounted for by reduced activation of SR Ca(2+) release.     

Mechanism of trypsin-induced endothelium-dependent vasorelaxation in the porcine coronary artery

1. To investigate the mechanism underlying the trypsin-induced endothelium-dependent relaxation, cytosolic Ca(2+) concentration ([Ca(2+)](i)) and tension development of smooth muscle were simultaneously monitored in the porcine coronary artery, and [Ca(2+)](i) of in situ endothelial cells were monitored in the porcine aortic valvular strips, using fura-2 fluorometry. 2. During the contraction induced by 30 nM U46619, a thromboxane A(2) analogue, 100 nM trypsin induced a rapid transient significant decrease in both [Ca(2+)](i) (from 67.9+/-5.1 to 15.7+/-4.4%) and tension (from 97.5+/-9.2 to 16.8+/-3.5%) of smooth muscle only in the presence of endothelium (100% level was assigned to the level obtained with the 118 mM K(+)-induced contraction). [Ca(2+)](i) and the tension thus returned to the levels prior to the application of trypsin by 5 and 10 min, respectively. 3. The initial phase of this relaxation was partly inhibited by 100 microM N(omega)-nitro-L-arginine (L-NOARG), and was completely inhibited by L-NOARG plus 40 mM K(+) or L-NOARG plus 100 nM charybdotoxin and 100 nM apamin, while the late phase of the relaxation was inhibited by L-NOARG alone. 4. Trypsin induced a transient [Ca(2+)](i) elevation in the endothelial cells mainly due to the Ca(2+) release from the intracellular stores, at the concentrations (1 - 100 nM) similar to those required to induce relaxation. 5. In conclusion, trypsin induced an elevation in [Ca(2+)](i) mainly due to Ca(2+) release in endothelial cells, and thereby caused endothelium-dependent relaxation. The early phase of relaxation was due to nitric oxide and hyperpolarizing factors, while the late phase was mainly due to nitric oxide in the porcine coronary artery.     

Apelin increases contractility in failing cardiac muscle

Apelin, a ligand for apelin-angiotension receptor-like 1 (APJ), has recently been shown to be a potent positive inotropic agent in normal hearts. In humans, levels of apelin have been shown to rise in early-stage heart failure and to fall in late-stage heart failure. In this study, we tested the hypothesis that apelin augments contraction directly in failing rat cardiac muscle. Right ventricular heart failure secondary to pulmonary hypertension was induced by exposing the rats to hypoxia (10% O(2) inhaled air) for 14-16 weeks. Trabeculae were dissected and mounted between a force transducer and a motor arm, superfused with Krebs-Henseleit (K-H) solution (pH 7.4, 22 degrees C), and loaded with fura-2. Both force development and [Ca(2+)](i) transient amplitude increased in a dose-dependent manner in the presence of Apelin-12 (10 approximately 70 nM, [Ca(2+)](o)=0.5 mM) in failing muscles as compared to control (36+/-7% vs. 7.4+/-5% at 70 nM, P<0.05). Also, [Ca(2+)](i) transients increased up to 18.4+/-9.5% as compared to control (4.5+/-1.9%, P<0.05). The increases in contraction in the presence of apelin were also maintained over a range of external Ca(2+) (0.5-2.0 mM). Steady-state force-[Ca(2+)](i) relation of the failing muscles reveals decreased maximal Ca(2+)-activated force (F(max)) (51.45+/-5.3 vs. 98.5+/-11.5 mN/mm(2), P<0.001), with no changes in Ca(2+) required for 50% of maximal activation (Ca(50)) (0.45+/-0.07 vs. 0.30+/-0.04 muM, P>0.05) and Hill coefficient (4.60+/-0.73 vs. 3.17+/-0.92, P>0.05). Apelin (70 nM) had no effect on the steady-state force-[Ca(2+)](i) relation in failing muscles (F(max): 63.03+/-3.5 mN/mm(2); Ca(50): 0.50+/-0.08 microM; Hill coefficient: 4.73+/-0.89). These results indicate that apelin exerts a selective positive inotropic action in failing myocardium. The increased force development is the result of increased [Ca(2+)](i) transients rather than changes in myofilament calcium responsiveness.     

Effect of xanthine oxidase-catalyzed reactive oxygen species generation on secretagogue-evoked calcium mobilization in mouse pancreatic acinar cells.

In the present study we have employed fura-2 loaded isolated mouse pancreatic acinar cells to monitor the effect that xanthine oxidase (XOD)-catalyzed reactive oxygen species generation presents on Ca(2+) mobilization by the secretagogue cholecystokinin octapeptide (CCK-8). Our results show that perfusion of pancreatic acinar cells with CCK-8 at a physiological concentration (20 pM) induced low frequency oscillations in intracellular free calcium concentration ([Ca(2+)](i)) at a rate of 1 per minute; this oscillatory pattern was completely inhibited by the introduction in the perifusion medium of 20 mU/mL XOD to generate reactive oxygen species. In addition, perfusion of pancreatic acinar cells with 20 mU/mL XOD in the absence of extracellular calcium led to a transient increase in [Ca(2+)](i,) that blocked the initiation of the Ca(2+) signals in response to 20 pM CCK-8. Similarly, XOD was also able to block acetylcholine evoked Ca(2+) spikes. However, reactive oxygen species had no effect either on Ca(2+) extrusion or on re-uptake into intracellular stores, but CCK-8-evoked Ca(2+) entry was reduced by XOD. In conclusion, our results show that XOD-evoked reactive oxygen species generation leads to a reduction either of Ca(2+) mobilization, following stimulation of pancreatic acinar cells with the Ca(2+)-mobilizing agonists CCK-8 and acetylcholine, and Ca(2+) influx evoked by CCK-8 depletion of intracellular stores. The possible XOD inhibitory mechanism on Ca(2+) mobilization by agonists is discussed.     

The mechanism for the contraction induced by leukotriene C4 in guinea-pig taenia coli

The mechanism underlying the LTC(4)-induced contraction of guinea-pig taenia coli was determined using the simultaneous measurements of [Ca(2+)](i) and force in whole muscle preparations. Additional experiments were performed in receptor coupled permeabilized preparation. For comparison purposes, the contraction which was induced by a typical G-protein mediated agonist, carbachol was also characterized. LTC(4) induced a contraction in the guinea-pig taenia coli in a concentration-dependent manner. The maximal response was obtained at 100 nM and the EC(50) value was 5.4+/-1.9 nM. Both LTC(4) and carbachol induced increases in [Ca(2+)](i) and force. The maximum force induced by 100 nM LTC(4) was significantly smaller than that induced by 10 microM carbachol, although an increase in [Ca(2+)](i) produced by both agonists was similar. In the permeabilized preparations, carbachol, but not LTC(4), induced an additional force development at a fixed Ca(2+) concentration. LTC(4) induced no increase in [Ca(2+)](i) and force in the Ca(2+)-free solution, while carbachol induced transient increases in both [Ca(2+)](i) and force in a Ca(2+)-free solution. Both diltiazem and SK&F 96365 significantly inhibited the LTC(4)- and carbachol-induced increases in [Ca(2+)](i) and force in normal PSS. The inhibitory pattern of [Ca(2+)](i) by these drugs was also similar. We thus conclude that LTC(4) induces the contraction of the guinea-pig taenia coli mainly through Ca(2+) influx via both the diltiazem-sensitive and SK&F 96365-sensitive Ca(2+) channels, without affecting either the Ca(2+)-sensitivity or the intracellular Ca(2+) release. These results indicated that the mechanism underlying the LTC(4)-induced contraction differs greatly from that for conventional G-protein mediated agonists, such as carbachol.     

Lindane increases in vitro respiratory burst activity and intracellular calcium levels in rainbow trout (Oncorhynchus mykiss) head kidney phagocytes

Phagocytic cells are the main actors of the fish immune system. They secrete reactive oxygen species (ROS) involved in their bactericidal activity. The effects of lindane on ROS production in rainbow trout phagocytes are contradictory. Here, we study the effects of high concentrations of lindane on ROS production (by chemiluminescence) and on intracellular calcium levels ([Ca(2+)](i)) (by spectrofluorimetry) in trout phagocytes. In these cells, lindane from 2.5 to 10 μM, increases ROS production and has no effect on [Ca(2+)](i). From 25 to 200 μM, lindane leads to a rise in ROS production (maximal value measured: 41152+/-6253 RLU for 100 μM lindane) associated with an increase in [Ca(2+)](i) (+3149+/-96 nM for 100 μM lindane) and with cytotoxicity which appears 2 min after addition of 100 μM lindane (25.4+/-3.75%; P<0.05). In the absence of extracellular calcium, ROS production of lindane-treated cells remains significantly higher than in controls (maximal value measured: 1899+/-254 RLU for 25 μM lindane), a significant decrease in [Ca(2+)](i) is observed in cells treated with 5 or 10 μM lindane (-54+/-35 nM for 10 μM lindane), and an increase in [Ca(2+)](i) in cells treated with 100 μM lindane (330+/-33 nM). The rise in [Ca(2+)](i) induced by lindane is inhibited when cells are preincubated with thapsigargin (Thaps). We conclude that lindane induces an increase in [Ca(2+)](i)50 μM) alter Ca(2+) homeostasis in the absence of extracellular Ca(2+), confirming that lindane can affect other intracellular stores of Ca(2+). At low concentrations (<25 μM), lindane stimulates ROS production by Ca(2+)-independant mechanisms without inducing cytotoxicity. From 25 μM, lindane increases [Ca(2+)](i) and maximal cytotoxicity appears from 100 μM lindane. Lindane toxicity in fish phagocytes may be associated with high [Ca(2+)](i) and high ROS production. Thus, ROS are beneficial in protection of the organism but when ROS are produced in excess, they can be toxic for cells and tissues.     

CCKA receptor antagonism inhibits mechanisms underlying CCK-8-stimulated insulin release in isolated rat islets.

The influence of the cholecystokinin (CCK)A receptor antagonist, L-364,718, on beta-cell activation was examined in isolated perifused prelabelled rat islets. Insulin secretion and 3H efflux from myo-[2-3H]inositol-prelabelled islets (reflecting phosphoinositide hydrolysis) stimulated by CCK-8 (100 nM) were both inhibited by L-364,718, partially at 1 nM and totally at 10 nM. 45Ca2+ efflux from prelabelled islets was markedly stimulated by CCK-8. This stimulation was inhibited equally by 1 and 10 nM L-364,718. CCK-8 stimulated the 86Rb+ efflux (reflecting K+ movements) from prelabelled islets, which probably reflects an indirect effect of CCK-8 due to opening of Ca(2+)-activated K+ channels. This 86Rb+ efflux was inhibited by L-364,718 at 10 nM but not affected by L-364,718 at 1 nM. It is concluded that insulin secretion, phosphoinositide hydrolysis, Ca2+ and K+ movements stimulated by CCK-8 in isolated islets are all events mediated by CCKA receptors. The L-364,718-induced inhibition of phosphoinositide hydrolysis was most closely correlated to the inhibition of insulin secretion. This suggests that induction of cellular events activated through stimulation of phosphoinositide hydrolysis is a major mechanism underlying CCK-8-stimulated insulin secretion.     

High sensitivity bioassay of paralytic (PSP) and amnesic (ASP) algal toxins based on the fluorimetric detection of [Ca(2+)](i) in rat cortical primary cultures.

A high sensitivity bioassay able to recognise small amounts of paralytic and amnesic toxins in algal acetic extracts is described. The method is based on the measure of intracellular [Ca(2+)](i) in primary cultures of rat cortical neurones preloaded with Fura-2 and submitted to electrical field stimulation. Under normal conditions the basal [Ca(2+)](i) level was about 50-100 nM and was nearly doubled during the peaks induced by trains of electrical pulses at 10 Hz for 10 s. Saxitoxin (STX) 3.5 nM and tetrodotoxin (TTX) 24 nM halved the peaks height without affecting basal [Ca(2+)](i). Conversely, domoic acid increased the basal [Ca(2+)](i) (EC(50)=3. 7 microM) and decreased the calcium peaks (EC(50)=7.3 microM). CNQX (a competitive antagonist of AMPA/KA receptors) at 10 microM shifted to the right by a factor of 3 the concentration-response curves of domoic acid. The extracts of non-toxic algae were well tolerated by up to 10 microg protein/ml, whereas extracts of Alexandrium lusitanicum at 1-4 microg protein/ml reduced [Ca(2+)](i) peaks and increased basal calcium levels. This toxic effect of A. lusitanicum was unexpected since parallel HPLC analysis showed only the presence of gonyautoxins, known to act like saxitoxin. Therefore, the bioassay on rat cortical neurones revealed a complex composition of the toxins present in A. lusitanicum. The relevance of fluorimetric detection of [Ca(2+)](i) in primary neuronal cultures in the evaluation of algal risk is stressed.     

Triethyltin increases cytosolic Ca(2+) levels in human osteoblasts

In human osteosarcoma MG63 cells, effect of triethyltin, an environmental toxicant, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2. Triethyltin (1-50 μM) caused a rapid and sustained plateau rise of [Ca(2+)](i) in a concentration-dependent manner (EC(50)=10 μM). Triethyltin-induced [Ca(2+)](i) rise was prevented by 50% by removal of extracellular Ca(2+) but was not altered by voltage-gated Ca(2+) channel blockers. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of triethyltin on [Ca(2+)](i) was attenuated by 60%; also, pretreatment with triethyltin abolished thapsigargin-induced [Ca(2+)](i) increase. Depletion of mitochondrial Ca(2+) with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 μM) did not affect triethyltin-induced Ca(2+) release. U73122, an inhibitor of phoispholipase C, abolished ATP (but not triethyltin)-induced [Ca(2+)](i) rise. A low concentration (1 μM) of triethyltin failed to alter ATP and bradykinin-induced [Ca(2+)](i) rises. These findings suggest that triethyltin rapidly increases [Ca(2+)](i) in osteoblasts by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release via as yet unidentified mechanism(s).