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Identification of a sodium channel toxin from a Caribbean benthic dinoflagellate
Abstracts
308
than previously believed. In addition, the findings suggest that the PPZA-assay can be developed into a rapid and relatively simple method for detecting PSPase inhibitors in crude cell extracts produced from a variety of sources. This work was funded Administration.
in part
by a grant
(NA47FD0429)
from
the National
Oceanic
Effects on$sheries and human health linked to a toxic estuarine dinofagellate. J. M. Burkholder Jr (North Carolina State University, Raleigh, NC 27965-7612, U.S.A.).
and
Atmospheric
and H. B. Glasgow
The estuarine dinoflagellate PJiesteria piscicida gen. & sp. nov. (Steidinger et al.) has been implicated as the causative agent of c. 50% of the major fish kills (IO’ to lo9 fish) in the Albemarle-Pamlico Estuarine System, NC. Pjiesteriu piscicidu is stimulated by fresh fish secreta, and it was lethal to all 19 species of fish bioassayed in culture. This dinoflagellate produces exotoxin(s) that create both neurotoxic water and aerosols. Culture filtrate induces formation of open ulcerative sores, hemorrhaging, and death of finfish and shellfish. Human exposure to culture aerosols has been linked to narcosis, respiratory distress, nausea, eye irritation (hours to days); other autonomic nervous system dysfunction [high localized perspiring, erratic heart beat (weeks)]; and central nervous system dysfunction [sudden rages, other erratic behavior (hoursdays); reversible cognitive impairment, short-term memory loss (weeks)]. Elevated hepatic enzyme levels and high P excretion suggested hepatic and renal dysfunction (weeks); easy infection and low counts of several T-cell types indicated immune system suppression (months to years). Pfiesteria piscicida is euryhaline and eurythermal, and is stimulated by organic P enrichment, Pfiesteria-like dinoflagellates have been tracked to fish kill sites in eutrophic estuaries from Delaware Bay through the Gulf Coast. Our data point to a critical need to characterize their chronic effects on human health as well as fish recruitment, disease resistance, and survival. Identification of a sodium channel toxin from a Caribbean benthic dinofiagellate. G. Escalona de Motta,’ A. L. Rivera Rentas,’ J. A. Mercado,’ T. Tostesor? and I. Gonzalez* (‘University of Puerto Rico, Institute of Neurobiology and Department of Biology, San Juan, PR 00901, U.S.A.; and *Department of Marine Sciences, Mayagiiez, PR 00680, U.S.A.). Ostreopsis lenticularis cells treated with acetone before methanol extraction showed a ten-fold increase in mouse toxicity and an HPLC chromatogram with a major peak in the 5 min region termed by us the ostreotoxin-3 (OTx-3) peak. Intracellular recording experiments demonstrated that OTx-3-enriched extracts depolarized frog skeletal muscle and decreased the amplitude of endplate potentials without affecting the nerve impulse. This effect was partially blocked by 10 PM TTx and was totally blocked by 5 PM p-Conotoxin GIIIA QJ-CTx). Using the whole-cell configuration of the patch clamp technique we identified two pharmacologically different populations of voltage-dependent sodium channels in chick embryo neurons: TTx-sensitive channels that produced nearly 80% of the current and a minor fraction of p-CTx-sensitive channels that produced approximately 20%. These sodium currents were affected by OTx-3 extracts. The most toxic one, 223-4B, shifted the voltage dependence of both current activation and inactivation but had its major effect on the inactivation curve. There it caused a shift of 30 mV to the negative of the VW*,*without any change in the curve steepness. In the presence of p-CTx GIIIA, the remaining sodium current was not altered by the OTX-3 extract. Thus, the myotoxic actions of 0. lenticularis extracts may be explained by the presence of a sodium channel toxin that recognizes a precise subclass of voltage-gated, p-CTx GIIIA-sensitive sodium channels. Alkaloid neurotoxins from Caribbean sponges Rivera-Rentas.’ R. Rosa.’ A. D. Rodriguez,* Institute of Neurobiology, Departmeits of -‘Universidad Central de1 Caribe School of U.S.A.).
of the genus Agelas: effect on neuronal membrane channels. A. L. W. Silva3 and G. Escalona de Motta’ (University of Puerto Rico ‘Biology and *Chemistry, San Juan: PR 00961, U.S.A.: and Medicine, Department of Pharmacology, Bayambn, PR 00958,
Compounds of the C,, N, family found in tropical Agehs sponges have been shown to be active on muscle and nerve receptors. Clathrodin (CLA) and dibromosceptrin (DBS) are two of these compounds which differ in structure, CLA being a monomer and DBS a dimer, and in bromide substitutions which range from none (CLA) to four (DBS). Voltage clamp experiments on frog muscle showed that both agents altered voltage-dependent ionic currents, CLA causing an early potentiation followed by total inhibition and DBS exerting an immediate current blockade. Their effects on ionic channels were examined using CLA and DBS in radioligand binding studies which showed that DBS inhibited nearly 90% of ‘H-saxitoxin (STx) binding to rat brain synaptosomes while CLA inhibited only 30%. As these results suggest an action on sodium channels, we studied the effect of CLA and DBS on membrane sodium currents of sympathetic neurons using the whole-ceil patch clamp technique. Although low CLA concentrations produced a potentiation, both agents decreased peak current amplitudes by an average of 27% for CLA and 40% for DBS. Neither altered the voltage dependence of current activation
308
than previously believed. In addition, the findings suggest that the PPZA-assay can be developed into a rapid and relatively simple method for detecting PSPase inhibitors in crude cell extracts produced from a variety of sources. This work was funded Administration.
in part
by a grant
(NA47FD0429)
from
the National
Oceanic
Effects on$sheries and human health linked to a toxic estuarine dinofagellate. J. M. Burkholder Jr (North Carolina State University, Raleigh, NC 27965-7612, U.S.A.).
and
Atmospheric
and H. B. Glasgow
The estuarine dinoflagellate PJiesteria piscicida gen. & sp. nov. (Steidinger et al.) has been implicated as the causative agent of c. 50% of the major fish kills (IO’ to lo9 fish) in the Albemarle-Pamlico Estuarine System, NC. Pjiesteriu piscicidu is stimulated by fresh fish secreta, and it was lethal to all 19 species of fish bioassayed in culture. This dinoflagellate produces exotoxin(s) that create both neurotoxic water and aerosols. Culture filtrate induces formation of open ulcerative sores, hemorrhaging, and death of finfish and shellfish. Human exposure to culture aerosols has been linked to narcosis, respiratory distress, nausea, eye irritation (hours to days); other autonomic nervous system dysfunction [high localized perspiring, erratic heart beat (weeks)]; and central nervous system dysfunction [sudden rages, other erratic behavior (hoursdays); reversible cognitive impairment, short-term memory loss (weeks)]. Elevated hepatic enzyme levels and high P excretion suggested hepatic and renal dysfunction (weeks); easy infection and low counts of several T-cell types indicated immune system suppression (months to years). Pfiesteria piscicida is euryhaline and eurythermal, and is stimulated by organic P enrichment, Pfiesteria-like dinoflagellates have been tracked to fish kill sites in eutrophic estuaries from Delaware Bay through the Gulf Coast. Our data point to a critical need to characterize their chronic effects on human health as well as fish recruitment, disease resistance, and survival. Identification of a sodium channel toxin from a Caribbean benthic dinofiagellate. G. Escalona de Motta,’ A. L. Rivera Rentas,’ J. A. Mercado,’ T. Tostesor? and I. Gonzalez* (‘University of Puerto Rico, Institute of Neurobiology and Department of Biology, San Juan, PR 00901, U.S.A.; and *Department of Marine Sciences, Mayagiiez, PR 00680, U.S.A.). Ostreopsis lenticularis cells treated with acetone before methanol extraction showed a ten-fold increase in mouse toxicity and an HPLC chromatogram with a major peak in the 5 min region termed by us the ostreotoxin-3 (OTx-3) peak. Intracellular recording experiments demonstrated that OTx-3-enriched extracts depolarized frog skeletal muscle and decreased the amplitude of endplate potentials without affecting the nerve impulse. This effect was partially blocked by 10 PM TTx and was totally blocked by 5 PM p-Conotoxin GIIIA QJ-CTx). Using the whole-cell configuration of the patch clamp technique we identified two pharmacologically different populations of voltage-dependent sodium channels in chick embryo neurons: TTx-sensitive channels that produced nearly 80% of the current and a minor fraction of p-CTx-sensitive channels that produced approximately 20%. These sodium currents were affected by OTx-3 extracts. The most toxic one, 223-4B, shifted the voltage dependence of both current activation and inactivation but had its major effect on the inactivation curve. There it caused a shift of 30 mV to the negative of the VW*,*without any change in the curve steepness. In the presence of p-CTx GIIIA, the remaining sodium current was not altered by the OTX-3 extract. Thus, the myotoxic actions of 0. lenticularis extracts may be explained by the presence of a sodium channel toxin that recognizes a precise subclass of voltage-gated, p-CTx GIIIA-sensitive sodium channels. Alkaloid neurotoxins from Caribbean sponges Rivera-Rentas.’ R. Rosa.’ A. D. Rodriguez,* Institute of Neurobiology, Departmeits of -‘Universidad Central de1 Caribe School of U.S.A.).
of the genus Agelas: effect on neuronal membrane channels. A. L. W. Silva3 and G. Escalona de Motta’ (University of Puerto Rico ‘Biology and *Chemistry, San Juan: PR 00961, U.S.A.: and Medicine, Department of Pharmacology, Bayambn, PR 00958,
Compounds of the C,, N, family found in tropical Agehs sponges have been shown to be active on muscle and nerve receptors. Clathrodin (CLA) and dibromosceptrin (DBS) are two of these compounds which differ in structure, CLA being a monomer and DBS a dimer, and in bromide substitutions which range from none (CLA) to four (DBS). Voltage clamp experiments on frog muscle showed that both agents altered voltage-dependent ionic currents, CLA causing an early potentiation followed by total inhibition and DBS exerting an immediate current blockade. Their effects on ionic channels were examined using CLA and DBS in radioligand binding studies which showed that DBS inhibited nearly 90% of ‘H-saxitoxin (STx) binding to rat brain synaptosomes while CLA inhibited only 30%. As these results suggest an action on sodium channels, we studied the effect of CLA and DBS on membrane sodium currents of sympathetic neurons using the whole-ceil patch clamp technique. Although low CLA concentrations produced a potentiation, both agents decreased peak current amplitudes by an average of 27% for CLA and 40% for DBS. Neither altered the voltage dependence of current activation