Extract of the marine alga Prymnesium patelliferum induces release of acetylcholine from cholinergic nerves in the rat bronchial smooth muscle.

An extract of the marine algal flagellate Prymnesium patelliferum enhanced the spontaneous as well as the K+ evoked (51 mM K+) release of [3H]acetylcholine and endogenous acetylcholine from autonomic cholinergic nerves of rat bronchial smooth muscle. The effects were concentration-dependent and reversible. The enhancement of the K+ evoked release by the algal extract was partly dependent on extracellular Ca2+ and was significantly suppressed by the organic Ca2+ blockers omega-conotoxin GVIA (1 microM), diltiazem (100 microM), nifedipine (100 microM) and flunarizine (100 microM). The enhancement of the spontaneous release seemed Ca2+ independent and not sensitive to the Na+ channel blocker tetrodotoxin. Sphingosine (20 microM), a protein kinase C inhibitor, strongly potentiated the enhancement of spontaneous release of [3H]acetylcholine induced by the algal extract whereas another protein kinase C inhibitor. 1-(5-quinolinesulphonyl)-2-methylpiperazine (H-7) (20 microM), was without effect. A similar potentiation as seen with sphingosine was observed with procaine (100 microM) and flunarizine (100 microM). The results indicate that the enhancement of the K+ evoked release of [3H]acetylcholine by the toxic extract of P. patelliferum was partly caused by activation of voltage-dependent Ca2+ channels. The increase in the spontaneous release of [3H]acetylcholine and endogenous acetylcholine induced by the algal extract alone may be caused by an ionophore-like property of the algal extract. This effect of the algal extract may be enhanced by compounds that facilitate the interaction of the algal toxin with the plasma membrane such as the lipophilic compounds flunarizine, procaine and sphingosine.

The effects of a purified toxic extract of Prymnesium patelliferum on transport of ions through the plasma membrane of synaptosomes.

Extract of the ichthyotoxic marine alga Prymnesium patelliferum has been shown to have several different effects on the transport of neurotransmitters across nerve membranes. It inhibits the sodium dependent uptake of L-glutamate and GABA and enhances the calcium-dependent release of acetylcholine. We have therefore investigated the effects of a purified toxic extract of P. patelliferum on some membrane properties using rat brain synaptosomes. We found that under conditions where the algal extract inhibited the uptake of L-glutamate, it increased the intracellular concentrations of Na+ and Ca2+, stimulated efflux of K+ determined as 86Rb efflux, and depolarized the synaptosomal membrane. There was no effect on Na+/K(+)-ATPase or ouabain-insensitive ATPase activities. Further, there was no leakage of the cytosolic marker LDH, indicating that the various effects of the algal extract were not due to nonspecific leakage or lysis of the synaptosomes. The rise in the cytosolic concentration of free Ca2+ induced by the algal extract was dependent on extracellular Ca2+, and was inhibited by flunarizine (1-100 microM) but not by the Ca2+ channel blockers omega-conotoxin GVIA (1 microM), diltiazem (100 microM), nifedipine (100 microM) or verapamil (100-500 microM). The increase in Na+ influx induced by the algal extract was insensitive to tetrodotoxin (3 microM) and procaine (100 microM), whereas both the Na+ influx and the membrane depolarization were inhibited by flunarizine (1-100 microM). The increase in K+ efflux was insensitive to flunarizine (5-100 microM). From these results it appears that the toxic extract of P. patelliferum increases the permeability of synaptosomes to Ca2+, Na+ and K+ and that these effects may be responsible for the plasma membrane depolarization and the disturbance of the neurotransmitter transport processes.

Toxin of the marine alga Prymnesium patelliferum enhances voltage dependent Ca(2+)-currents, elevates the cytosolic Ca(2+)-concentration and facilitates hormone release in clonal rat pituitary cells.

The marine flagellate Prymnesium patelliferum produces toxins lethal to fish. The toxin extracted from the alga has haemolytic, cytotoxic and neurotoxic effects, but the action mechanisms of the toxin are not known in detail. We have examined the toxin effects on the voltage sensitive Ca(2+)-currents, the cytosolic Ca(2+)-level ([Ca2+]i) and the prolactin release in clonal rat anterior pituitary GH4C1 cells, which possess T- and L-type Ca(2+)-channels. The trans-membrane Ca(2+)-current was recorded using whole-cell voltage clamp. After 5-15 min exposure to the algal toxin at a final concentration of 50,000-100,000 cells mL-1, the Ca(2+)-currents through both the T- and L-channels showed a 2-3-fold enhancement. The voltage sensitivity of the Ca(2+)-currents was not affected by the algal toxin, and the toxin-induced currents were inhibited by 100 microM of the Ca(2+)-channel blocker D-600. In toxin-exposed cells microfluorometric measurements based on fura-2 revealed an increase of [Ca2+]i from 100-150 to 300-500 nM. This elevation was delayed and partially inhibited by 100 microM D-600. The algal toxin induced prolactin release in a dose-dependent manner, and this effect was inhibited by the Ca(2+)-channel blocker verapamil. We therefore conclude that the toxin of P. patelliferum affects the Ca2+ homeostasis of the pituitary cells by increasing the leak through voltage sensitive Ca(2+)-channels, resulting in increased [Ca2+]i and secretion of prolactin.

Toxicity of four potentially ichthyotoxic marine phytoflagellates determined by four different test methods.

The toxicity of the marine phytoflagellates Prymnesium parvum. Prymnesium patelliferum, Chrysochromulina polylepis, and Chrysochromulina leadbeateri isolated from ichthyotoxic blooms in Norwegian coastal waters was compared using four different test methods developed for the detection of toxins produced by these species. The test methods were (1) lethality to the crustacean Artemia salina exposed to living algae, (2) hemolytic activity (lysis of human erythrocytes) by crude algal lipid extracts, and inhibition of the uptake of the neurotransmitters L-glutamate and gamma-aminobutyric acid (GABA) into (3) synaptosomes and (4) synaptic vesicles of rat brain by crude algal lipid extracts. All test methods indicated different levels of toxicity among the algal species. Prymnesium parvum, P. patelliferum, and C. polylepis were toxic as determined by all four test methods. The cultured strain of C. leadbeateri, although isolated from a toxic algal bloom, appeared nontoxic by the methods used here, and served as a negative control. The hemolytic activity of P. parvum extract was about nine times higher than that of P. patelliferum extract, whereas the activity was only two to three times higher using the other three methods. Chrysochromulina polylepis had higher toxic activity than P. patelliferum except for less inhibitory effect on synaptosomes. The inhibition of synaptosomal and vesicular neurotransmitter uptake systems by extracts of P. parvum, P. patelliferum, and C. polylepis appeared to be due to similar mechanisms of action, since similar inhibition ratios between the uptake of L-glutamate and GABA were obtained in both synaptosomes and synaptic vesicles. We suggest that P. parvum, P. patelliferum, and C. polylepis produce a set of similar toxins and that the relative amounts of each toxin vary among the three species.

Effect of toxin of Prymnesium patelliferum on neurotransmitter transport mechanisms: development of a sensitive test method.

A crude extract of the ichthyotoxic phytoflagellate Prymnesium patelliferum strongly inhibited the uptake of neurotransmitters into isolated nerve endings (synaptosomes) and synaptic vesicles. These systems were about 100 and 10 times more sensitive toward the algal toxin, respectively, than a standard hemolysis assay often used for testing the toxicity of Prymnesium spp. and other ichthyotoxic algae. Prymnesium patelliferum grown in phsophorus-deficient (-P) medium was about five times more active than when grown in phosphorus sufficient (+P) medium. The inhibition ratio between the high-affinity synaptosomal uptake of L-glutamate and gamma-aminobyturic acid (GABA) was 1/2.7 for the -P culture and 1/1.9 for the +P culture. The inhibition ratios for the low-affinity vesicular uptake of L-glutamate, GABA, and dopamine (DA) were 1/5.8/0.3 and 1/1.7/0.2, respectively. The synaptosomal transport of L-glutamate is a rapid, simple, and sensitive test method for toxicity determination of Prymnesium spp. and will be a useful tool in the further isolation and purification of the toxic principles of this and other related algae. It is suggested that the toxin interferes with ion channels or acts as an ionopore.