Sandwich ELISA assay for the quantitation of palytoxin and its analogs in natural samples.

Palytoxins are potent marine biotoxins that have recently become endemic to the Mediterranean Sea, and are becoming more frequently associated with seafood. Due to their high toxicity, suitable methods to quantify palytoxins are needed. Thus, we developed an indirect sandwich ELISA for palytoxin and 42-hydroxy-palytoxin. An intralaboratory study demonstrated sensitivity (limit of detection, LOD = 1.1 ng/mL; limit of quantitation, LOQ = 2.2 ng/mL), accuracy (bias of 2.1%), repeatability (RSDr = 6% and 9% for intra- and interassay variability, respectively) and specificity: other common marine toxins (okadaic acid, domoic acid, saxitoxin, brevetoxin-3, and yessotoxin) do not cross-react in this assay. It performed well in three different matrices: observed LOQs were 11.0, 9.6, and 2.4 ng/mL for mussel extracts, algal net samples and seawater, respectively, with good accuracy and precision. The LOQ in seafood is 11 µg palytoxin/kg mussel meat, lower than that of the most common detection technique, LC-MS/MS.

Comparative cytotoxicity of gambierol versus other marine neurotoxins.

Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine organisms contaminated with those toxins results in seafood poisonings. In many cases, the lack of toxic material turns out to be an obstacle to make the toxicological investigations needed. In this study, we evaluate the cytotoxicity of several marine toxins on neuroblastoma cells, focusing on gambierol and its effect on cytosolic calcium levels. In addition, we compared the effects of this toxin with ciguatoxin, brevetoxin, and gymnocin-A, with which gambierol shares a similar ladder-like backbone, as well as with polycavernoside A analogue 5, a glycosidic macrolide toxin. For this purpose, different fluorescent dyes were used: Fura-2 to monitor variations in cytosolic calcium levels, Alamar Blue to detect cytotoxicity, and Oregon Green 514 Phalloidin to quantify and visualize modifications in the actin cytoskeleton. Data showed that, while gambierol and ciguatoxin were successful in producing a calcium influx in neuroblastoma cells, gymnocin-A was unable to modify this parameter. Nevertheless, none of the toxins induced morphological changes or alterations in the actin assembly. Although polycavernoside A analogue 5 evoked a sharp reduction of the cellular metabolism of neuroblastoma cells, gambierol scarcely reduced it, and ciguatoxin, brevetoxin, and gymnocin-A failed to produce any signs of cytotoxicity. According to this, sharing a similar polycyclic ether backbone is not enough to produce the same effects on neuroblastoma cells; therefore, more studies should be carried out with these toxins, whose effects may be being underestimated.

Assessment of the hydrolysis process for the determination of okadaic acid-group toxin ester: presence of okadaic acid 7-O-acyl-ester derivates in Spanish shellfish.

The contamination of different types of shellfish by okadaic acid (OA)-group toxin esters is an important problem that presents serious risk for human health. During previous investigations carried out in our laboratory by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS), the occurrence of a high percentage of esters in relation to the total OA equivalents has been observed in several shellfish species. The determination of these kinds of toxins using LC/MS or other chemical methods requires a hydrolysis step in order to convert the sterified compounds into the parent toxins, OA, dinophysistoxins-1 (DTX-1) and dinophysistoxins-2 (DTX-2). Most of the hydrolysis procedures are based on an alkaline hydrolysis reaction. However, despite hydrolysis being a critical step within the analysis, it has not been studied in depth up to now. The present paper reports the results obtained after evaluating the hydrolysis process of an esterified form of OA by using a standard of 7-O-acyl ester with palmitoyl as the fatty acid (palOA). Investigations were focused on checking the effectiveness of the hydrolysis for palOA using methanol as solvent standard and matrices matched standards. From the results obtained, no matrix influence on the hydrolysis process was observed and the quantity of palOA converted into OA was always above 80%. The analyses of different Spanish shellfish samples showed percentages of palOA in relation to the total OA esters ranging from 27% to 90%, depending on the shellfish specie.

Ultrastructural damage to heart tissue from repeated oral exposure to yessotoxin resolves in 3 months.

Yessotoxin (YTX), an algal toxin contaminating edible shellfish, was previously shown to induce ultrastructural changes in some cardiac muscle cells of mice after acute (1 and 2mg/kg) or daily repeated oral exposure (1 and 2mg/kg/day, for 7 days). Therefore, the temporal evolution of the ultrastructural myocardial alterations and the development of other signs of toxicity induced by a repeated daily oral administration of YTX (1mg/kg/day, for 7 days) to mice were evaluated within 3 months after the treatment. Symptoms, food consumption, body weight, gross pathology and histopathology of the main organs and tissues were observed, and plasma levels of transaminases, lactate dehydrogenase, creatinine and creatinine phosphokinase were measured. Heart, liver, kidneys and cerebellum were also analysed by transmission electron microscopy. In addition, the blood concentration of YTX was determined by a direct enzyme linked immunosorbent assay (ELISA) 24h after the last toxin administration. No mortality or other treatment-related changes, including histological or hematoclinical parameters, were recorded in mice administered with YTX. Similarly, electron microscopy did not reveal any ultrastructural alteration in the liver, kidneys, and cerebellum associated with YTX treatment. In contrast, changes in cardiac muscle cells near to the capillaries (clusters of rounded mitochondria and disorganization of myofibrils) were observed 24h after the treatment. These changes were also noted 30 days after the toxin administration, while after 90 days no differences in cardiac muscle cells between control and YTX-treated mice were observed, which indicated a recovery of the ultrastructural alterations induced by the toxin.

Effect of maitotoxin on cytosolic Ca2+ levels and membrane potential in purified rat brain synaptosomes.

In this study, the effects of the marine toxin maitotoxin on cytosolic Ca2+ levels and membrane potential in rat brain synaptosomes were evaluated. Maitotoxin (10 ng/ml) caused a remarkable increase of intrasynaptosomal Ca2+ levels monitored by the fluorescent probe fura-2. This increase was prevented by the removal of external Ca2+ ions. Tetrodotoxin, as well as the removal of extracellular Na+ ions, failed to affect maitotoxin-induced increase of intrasynaptosomal Ca2+ levels. Also the complete removal of all monovalent and divalent cations, except Ca2+ ions, from the incubation medium (0.32 M sucrose substitution), was unable to prevent the effect of maitotoxin on intrasynaptosomal Ca2+ levels. Maitotoxin (0.3-10 ng/ml), produced a dose-dependent depolarization of synaptosomal membranes, which required the presence of extracellular Ca2+ ions. The substitution of extracellular Na+ with choline or the removal of all cations from the incubation medium and their replacement with an isotonic concentration of sucrose (0.32 M), did not prevent the depolarizing effect exerted by maitotoxin. Also under these two ionic conditions, the effect of maitotoxin on membrane potential was critically dependent on the presence of 1 mM extracellular Ca2+. The depolarizing effect exerted by maitotoxin on synaptosomal membrane potential was also observed when extracellular Ca2+ ions were substituted with an equimolar concentration of Ba2+ or Sr2+ ions. In summary, these results appear to suggest that, in presence of 1 mM extracellular Ca2+ ions, maitotoxin depolarizes synaptosomal plasmamembrane by promoting the influx of extracellular Ca2+ ions. This enhanced influx of Ca2+ causes an increase of intrasynaptosomal Ca2+ levels.

Maitotoxin-induced free Ca changes in single rat hepatocytes.

We report the results using bioluminescent and fluorescent indicators to investigate maitotoxin-induced free Ca changes in single rat hepatocytes. Maitotoxin generated a steadily rising free Ca increase after a long lag period. The free Ca increase was dependent on extracellular calcium and could be antagonised by chelation of extracellular calcium or the inclusion of nickel in the superfusate. Manganese-induced quench of cytoplasmic Fura2 dextran revealed an accelerated rate of calcium entry during the final period of the lag phase, immediately prior to the free Ca increase. Imaging experiments demonstrated a markedly different part of free Ca mobilisation compared with glycogenolytic stimuli. Moreover, the use of a combination of hormonal stimuli and maitotoxin revealed that some cells could exhibit free Ca oscillations despite steadily rising intracellular free Ca level. The significance of these observations in terms of the mechanism of action of maitotoxin and the mechanism of free Ca transient generation is discussed.

Maitotoxin-induced calcium entry in human lymphocytes: modulation by yessotoxin, Ca(2+) channel blockers and kinases.

We have studied the effect of the ciguatera-related toxin maitotoxin (MTX) on the cytosolic free calcium concentration ([Ca(2+)]i) of human peripheral blood lymphocytes loaded with the fluorescent probe Fura2 and the regulation of MTX action by different drugs known to interfere in cellular Ca(2+) signalling mechanisms and by the marine phycotoxin yessotoxin (YTX). MTX produced a concentration-dependent elevation of [Ca(2+)]i in a Ca(2+)-containing medium. This effect was stimulated by pretreatment with YTX 1 microM and NiCl(2) 15 microM. The voltage-independent Ca(2+) channel antagonist 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl]-1H-imidazole hydrochloride (SKF96365) blocked the MTX-induced [Ca(2+)]i elevation, while the L-type channel blocker nifedipine had no effect. Pretreatment with NiCl(2) or nifedipine did not modify YTX-induced potentiation of MTX effect, and SKF96365-induced inhibition was reduced in the presence of YTX, which suggest different pathways to act on [Ca(2+)]i. Preincubation with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H-89) or genistein (10 microM) also had no effect on the MTX-induced [Ca(2+)]i increment. In contrast, the PKC inhibitor bisindolilmaleimide I (GF109203X 1 microM) potentiated the MTX effect, whereas phosphatidylinositol (PI) 3-kinase inhibition with wortmannin (10 nM) reduced the MTX-elicited Ca(2+) entry. In summary, MTX produced Ca(2+) influx into human lymphocytes through a SKF96365-sensitive, nifedipine-insensitive pathway. The MTX-induced [Ca(2+)]i elevation was stimulated by the marine toxin YTX through a mechanism insensitive to SKF96365, nifedipine or NiCl(2). It was also stimulated by the divalent cation Ni(2+) and PKC inhibition and was partially inhibited by PI 3-kinase inhibition.

Homologous desensitization with gonadotropin-releasing hormone (GnRH) also diminishes gonadotrope responsiveness to maitotoxin: a role for the GnRH receptor-regulated calcium ion channel in mediation of cellular desensitization.

Maitotoxin (MTX) stimulates gonadotropin release from pituitary cell cultures. The time course and efficacy of LH release in response to GnRH and to MTX are similar; both secretagogues require extracellular Ca2+ and are inhibited by the selective Ca2+ ion channel antagonist methoxyverapamil (D600). LH release in response to either GnRH or MTX is not measurably inhibited by two other chemical classes of Ca2+ ion channel inhibitors represented by nifedipine and by diltiazem. The two secretagogues are nonadditive in their action on LH release when presented at high doses and prior studies indicate that MTX has no endogenous ionophoretic activity. These observations indicate that MTX likely stimulates LH release due to activation of the GnRH receptor associated Ca2+-ion channel in the gonadotrope. We have therefore assessed the functional state of this channel during the development of homologous desensitization of the gonadotrope to GnRH by measuring the ability of MTX to stimulate LH release. Cells were desensitized with GnRH in the presence of 3 mM EGTA. Under these conditions, the cells become refractory to GnRH in the absence of gonadotropin release since the latter process, but not the former, requires extracellular Ca2+. Accordingly, this approach allows assessment of the degree of desensitization in the absence of the influence of gonadotropin depletion. Such desensitized cells are less responsive to GnRH. Desensitized pituitary cells also respond with diminished efficacy and potency to MTX three or more hours after GnRH treatment but not at an earlier time (1 h) when GnRH receptors are diminished. These data are consistent with a model in which homologous desensitization is viewed as developing in two phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study.

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).

Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator.

Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.

The effects of maitotoxin on 45Ca2+ flux and hormone release in GH3 rat pituitary cells.

Maitotoxin has been reported to activate calcium channels and stimulate calcium-dependent functions in several tissues, but a thorough investigation of 45Ca2+ fluxes is lacking. To characterize the influence of maitotoxin on 45Ca2+ flux in greater detail, we incubated dispersed GH3 pituitary tumor cells in 45Ca2+ with maitotoxin and other agents affecting calcium channels. Within 10 sec of exposure, maitotoxin induced a net calcium influx in cells at isotopic equilibrium. Calcium uptake was concentration dependent between 0.4 and 40 ng/ml maitotoxin and was inhibited by antagonists of voltage-dependent calcium channels but not by inhibitors of sodium channels. PRL and GH release from perifused GH3 cells was stimulated within 1 min by maitotoxin. We conclude that maitotoxin causes a rapid, concentration-dependent influx of calcium through presumed voltage-dependent endogenous calcium channels, culminating in enhanced hormone release. This potent toxin may provide a more precise understanding of the role of calcium in the stimulus-secretion coupling process.

Dopamine decreases 7315a tumor cell prolactin release induced by calcium mobilization.

The rat pituitary tumor 7315a secretes PRL and ACTH. Although dopamine has no effect on unstimulated PRL release from this tumor, dopamine decreases the adenylate cyclase activity in tumor cell homogenates in a manner similar to that in normal pituitary tissue. However, it was observed that under basal conditions, 7315a tumor cells have an abnormal calcium metabolism because 1) basal PRL release from tumor cells is not modified by the calcium channel blocker D-600 and is only moderately decreased by low calcium, treatments that markedly decrease normal pituitary PRL release; 2) D-600 had no effect on basal 7315a tumor calcium uptake, but blocked the increase in calcium uptake due to the calcium channel activator maitotoxin; 3) increasing the medium Ca+2 concentration above 5 mM increases 7315a PRL release, whereas this treatment decreases PRL release from normal pituitary cells. Maitotoxin and the calcium ionophore A23187 increased 7315a tumor cell PRL release in a manner similar to that in normal pituitary cells. Because dopamine blocks PRL release induced by maitotoxin, A23187, or elevated medium calcium concentration in 7315a tumor cells, the refractoriness of basal 7315a tumor cell PRL release to dopamine may be due to the abnormal calcium balance of the tumor cells under basal conditions.

Maitotoxin, a potent, general activator of phosphoinositide breakdown.

Maitotoxin (MTX), a potent marine toxin, elicits a calcium-dependent activation of cells that can be inhibited by calcium channel blockers like nifedipine. MTX also stimulates phosphoinositide breakdown in smooth muscle cells, NCB-20 cells and PC12 cells through a nifedipine-insensitive mechanism. We now report that MTX stimulates phosphoinositide breakdown in a wide variety of cells, and appears to represent the first general activator of this second messenger-generating system. MTX-induced stimulation of phosphoinositide breakdown is dependent in every cell line on the presence of extracellular calcium. In differentiated HL60 cells, in which a chemotactic peptide (fMLP) activates phosphoinositide breakdown via a pertussis toxin-sensitive mechanism, MTX-induced stimulation is not affected by pertussis toxin treatment. A phorbol ester has no effect on the response to MTX. Thus, MTX stimulates phosphoinositide breakdown through a calcium-dependent mechanism that at least in three cell lines (PC12, NCB20 and HL60) is not mediated by a pathway that involves a pertussis toxin-sensitive guanine nucleotide-binding protein.

Transient Ca2+-dependent activation of ERK1 and ERK2 in cytotoxic responses induced by maitotoxin in breast cancer cells.

Treatment of MCF-7 breast cancer cells with the marine toxin maitotoxin (MTX) induces cell death. The cytotoxic effects are clearly detectable within 2-4 h after cell treatment with 10(-10)-10(-9) M concentrations of MTX. The response was found to depend on extracellular Ca2+, inasmuch as cell death was prevented when culture dishes received MTX, following addition of EGTA. MTX caused transient phosphorylation of extracellular signal-regulated kinase isoforms 1 and 2 (ERK1 and ERK2) mitogen-activated protein kinase isoforms in MCF-7 cells, which was maximal 15 min after toxin addition to culture vessels. The effect was dependent on influx of extracellular Ca2+, as it was abolished by EGTA, and was induced by ionophores, such as A23187 and ionomycin. Our findings show that signaling pathways involving Ca2+ ions may cause activation of ERK1 and ERK2 in cell death responses.

Differential effects of maitotoxin on ATP secretion and on phosphoinositide breakdown in rat pheochromocytoma cells.

Maitotoxin (MTX) induced exocytotic secretion of ATP from PC12 rat pheochromocytoma cells. The threshold for stimulation of secretion was at concentrations of about 2 ng/ml of MTX. Maximal release occurred at 40 ng/ml. MTX-induced ATP release required the presence of calcium in the extracellular medium and could be inhibited by nifedipine, a specific blocker of voltage-dependent calcium channels. In addition to the effects on ATP secretion from PC12 cells, MTX stimulated the breakdown of phosphoinositides, as measured by the accumulation of [3H]inositol phosphates. Maximal stimulation of phosphoinositide breakdown was reached at only 0.5-1.0 ng/ml MTX. MTX at concentrations required to evoke ATP release (greater than 2 ng/ml) had lesser or no effect on phosphoinositide breakdown. Although stimulation of phosphoinositide breakdown by MTX was dependent on extracellular calcium, it was insensitive to the calcium channel blockers nifedipine, D-600 and cobalt ions. The different concentration range required to elicit these responses and the varying sensitivity to calcium channel blockers indicate that MTX-evoked secretion and MTX-stimulated phosphoinositide breakdown are independent phenomena in PC12 cells.

[Toxic complex from parrotfish]. / Le complexe toxinique des poissons perroquets.

Clinical and epidemiological observations suggested that a complex toxic molecule is involved in the parrotfish flesh (Scarus gibbus) poisoning from Gambier Islands. The fat soluble extract obtained from the muscles upon ciguatoxin preparation showed two toxic substances after fractionation by DEAE cellulose column chromatography. The major toxin is different from ciguatoxin judging by its chromatographic behaviour. The other is closely similar to (or identical with) ciguatoxin from the moray eel Gymnothorax javanicus. They were named SG1 for the new toxin and SG2 for the ciguatoxin like compound. Successive filtrations on Sephadex LH-20 of SG1 and SG2 gave respectively a lethality to mice of 0.03 microgram/g and 0.06 microgram/g. SG1, specifically occurs in the muscles of the parrotfish family (scaritoxin) while it is absent from other ciguateric fishes. According to that specificity and the lack of SG1 in S. gibbus liver and gut contents, the origin of scaritoxin is briefly discussed.

Maitotoxin increases inositol phosphates in rat anterior pituitary cells.

Maitotoxin is a potent marine poison that mobilizes calcium in most vertebrate cell types and accelerates secretion from anterior pituitary cells. It is not known whether voltage-sensitive calcium channels or other mechanisms initiate the effects of maitotoxin on anterior pituitary cells. Changes in intracellular Ca2+ levels may also be achieved by releasing internal calcium stores via inositol trisphosphate (InsP3). Indeed, maitotoxin rapidly increased inositol phosphate accumulation in a concentration-dependent manner. Calcium channel antagonists such as nifedipine and verapamil did not block this response nor did calcium-mobilizing agents (BAYk8644, A23187) mimic this effect. These data suggest that the mechanism by which maitotoxin acts at the pituitary may include the activation of an enzyme that produces the calcium-mobilizing signal InsP3.

Contraction and increase in tissue calcium content induced by maitotoxin, the most potent known marine toxin, in intestinal smooth muscle.

Maitotoxin (MTX), the most potent known marine toxin, isolated from toxic dinoflagellates and poisonous fish, caused a dose-dependent contraction of the guinea-pig isolated ileum and taenia caeci at concentrations of 100 pg to 30 ng/ml. These contractile responses to MTX (3 ng/ml) in both tissues were abolished by incubation in Ca2+-free solution and were markedly inhibited by treatment with methoxyverapamil (D600), but were not affected by tetrodotoxin and atropine. Furthermore, MTX markedly elevated tissue Ca2+ content of the taenia caeci. These results suggest that MTX activates Ca2+ channels in the smooth muscle membrane of both tissues to increase Ca2+ influx and thus induces contractions.

The mechanism of action of maitotoxin in relation to Ca2+ movements in guinea-pig and rat cardiac muscles.

Maitotoxin (MTX), the most potent marine toxin known, produced a dose-dependent positive inotropic effect on guinea-pig isolated left atria and rat ventricle strips at concentrations of 0.1 ng to 4 ng ml-1. MTX (4 ng ml-1) also exhibited a positive chronotropic effect on guinea-pig right atria. The MTX-induced inotropic effect was almost abolished by Co2+ or verapamil, but was little affected by propranolol, reserpine or tetrodotoxin. The tissue Ca content of guinea-pig left atria was increased by MTX (2-30 ng ml-1) in a dose-dependent manner, and this increase was markedly inhibited by Co2+ or verapamil. Furthermore, on the rat isolated cardiac myocytes MTX (0.01-10 ng ml-1) caused an arrhythmogenic effect which was followed by their transformation into irreversibly rounded cells. The effects of MTX on the isolated cells were inhibited by verapamil or Ca2+-free solution. These results suggest that the excitatory effects of MTX on heart muscle are caused by a direct action on the cardiac muscle membrane mainly due to an increase in Ca2+ permeability.

The mode of inotropic action of ciguatoxin on guinea-pig cardiac muscle.

1. Ciguatoxin (CTX) caused a dose-dependent increase in the contractile force of the guinea-pig isolated left atria at concentrations ranging from 0.1 to 10 ng ml-1 with the ED50 value of 0.5 ng ml-1. 2. In the atria, tetrodotoxin (5 x 10(-7) M) inhibited markedly the inotropic action of CTX. The inotropic effect of CTX at low concentrations was abolished by practolol (10(-5) M) and reserpine (2 mg kg-1 daily, for 3 days), whereas that of CTX at high concentrations was partially inhibited by both drugs. 3. In single atrial cells, CTX (3 ng ml-1) produced a marked increase in the amplitude of longitudinal contractions. 4. CTX (3 ng ml-1) caused marked prolongation in the falling phase of action potentials of atrial strips without affecting the maximum rate of rise of action potentials and membrane resting potentials. The effect of CTX on action potentials was abolished by tetrodotoxin (10(-6) M). 5. The whole-cell patch-clamp experiments on myocytes revealed that CTX (20 ng ml-1) shifted the current-voltage curve of Na inward currents by 40 mV in the negative direction. CTX caused a small sustained Na inward current even at resting membrane potentials. 6. These results suggest that the inotropic action of lower concentrations of CTX is primarily due to an indirect action via noradrenaline release, whereas that of higher concentrations is caused not only by an indirect action but also by a direct action on voltage-dependent Na channels of cardiac muscle. It is also suggested that CTX activates cardiac muscle Na channels by modifying the voltage-dependence of channel activation to increase Na inward currents, thus producing cardiotonic actions.