Ptychodiscus Brevis Research Articles

Dexmedetomidine and clonidine inhibit the function of NaV1.7 independent of α2-adrenoceptor in adrenal chromaffin cells

Besides being administered systemically for sedation and analgesia, α(2)-agonists such as dexmedetomidine and clonidine have been administered with intrathecal, epidural, or perineural injections, leading to an antinociceptive effect at the spinal cord or peripheral nerve level. However, the mechanism for this remains unclear. In the present study, we examined whether dexmedetomidine and clonidine could inhibit the function of tetrodotoxin-sensitive Na(+) channels, which play important roles in the generation of pain. Cultured bovine adrenal chromaffin cells expressing the tetrodotoxin-sensitive Na(v)1.7 Na(+) channel isoform were incubated in KRP buffer containing 2μCi (22)NaCl for 5min without or with dexmedetomidine or clonidine in the absence or presence of veratridine, α-scorpion venom, β-scorpion venom, Ptychodiscus brevis toxin-3 or ouabain. Cells were then washed and counted radioactively. Dexmedetomidine and clonidine reduced veratridine-induced (22)Na(+) influx via Na(v)1.7 in a concentration-dependent manner (EC(50)=50μM and 530μM), even in the presence of ouabain, an inhibitor of Na(+), K(+)-ATPase. Dexmedetomidine and clonidine shifted the concentration-response curve of veratridine for (22)Na(+) influx downward without altering the EC(50) of veratridine. Atipamezole and yohimbine, α(2)-antagonists, did not prevent the inhibition of veratridine-induced (22)Na(+) influx by dexmedetomidine. Dexmedetomidine and clonidine combined with lidocaine induced more inhibition of veratridine-induced (22)Na(+) influx than each drug did individually. Atipamezole and yohimbine did not prevent the lidocaine-enhancing effect of dexmedetomidine and clonidine. Dexmedetomidine and clonidine inhibit the function of Na(v)1.7 independent of α(2)-adrenoceptor. These results may lead to a deeper understanding of the peripheral antinociceptive effects of α (2)-agonists.

GABAergic system for Ptychodiscus brevis toxin-induced depression of synaptic transmission elicited in isolated spinal cord from neonatal rats

The involvement of inhibitory transmitters for Ptychodiscus brevis toxin (PbTx)-induced depression of spinal synaptic transmission in neonatal rats was investigated. Stimulation of a dorsal root evoked monosynaptic reflex (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root. The PbTx depressed the reflexes in a concentration-dependent manner and this depression was blocked by GABAA antagonist, bicuculline (1 M). GABA also produced depression of the reflexes in a concentration-dependent manner. Simultaneous application of sub maximal concentrations of PbTx (28 M) and GABA (30 M) enhanced the depression (>75%). In contrast, PbTx alone (28 M) depressed the MSR and the PSR by 33 and 47%, respectively, and GABA (30 M) alone depressed the reflexes by 30%. The N-methyl- -aspartate receptor antagonist, -2-amino-5-phosphono-pentanoic acid (10 M), blocked the PbTx-induced depression of MSR and also the enhancement of GABA response by PbTx. A glycine receptor antagonist, strychnine (1 M), failed to block the depression by the toxin up to 28 M; however, the depression was attenuated significantly at 84 M of the toxin. The results indicate that PbTx depressed the spinal reflexes via GABAA receptors. Furthermore, the potentiation of GABAergic action by PbTx requires the N-methyl-D-aspartate-dependent mechanism.

Red tide toxin produces in vitro depolarization of human airway smooth muscle

Background. Brevetoxin (PbTx), taken from the earlier species name Ptychodiscus brevis, is a red algae toxin. It has been associated with clinically observed bronchoconstriction in nonasthmatics. In asthmatics, similar exposures may produce severe transient effects, sometimes requiring emergency treatment, thus suggesting that asthmatics are more sensitive to this toxin. As such, we have investigated potential mechanisms in vitro. Methods. Membrane potentials of in vitro airway smooth muscle (ASM) preparations were measured with a microelectrode before, during, and after the exposure to PbTx (0.01–1.2 μg/mL) in strip preparations (SPs) and cultured ASM reaggregate preparations. The latter preparation results in the disruption of normal peripheral nervous ASM associations through enzymatic dissociation of cells. Results. We observed an increased level of depolarization in asthmatic preparations at the same level of exposure. Exposure to PbTx produced concentration-dependent depolarization in both nonasthmatic and asthmatic in vitro SPs. In the former, responses did not occur in the presence of the blocking agents such as atropine or tetrodotoxin (TTX). In asthmatic SPs, atropine and TTX produced little effect, whereas verapamil blocked the PbTx-induced depolarization. The toxin was without effect in nonasthmatic cultured cells, whereas acetylcholine produced depolarization that was blocked in the presence of atropine, but not TTX or verapamil. In contrast, the toxin produced significant depolarization in cultured asthmatic ASM cells, which were unaffected by either atropine or TTX but were blocked by verapamil. Conclusions. We propose that PbTx directly affects asthmatic ASM whereas the effect is neurally mediated in nonasthmatics.

Red tide phenomenon leading to panic attack and mass casualty among children in coastal Kerala

Red tide phenomenon is an event that occurs with varying intensity almost every year.(1) This occurs along the coastal areas. This is frequently described along the Florida coast and Gulf of Mexico.(2) The timing of the phenomenon is difficult to predict. This occurs almost every month along the coast of Florida. The waste products and decaying matter along the seashore get washed into the sea especially during rains. These nutrients washed from the coastal areas into the sea water are consumed by the unicellular phytoplankton algae, Cochlodeneum Krikoids, which forms the lower most organism in the food chain.(3) The right salinity, temperature and nutrients are thought to nourish these organisms. These may proliferate seasonally and reach enormous amounts thereby discoloring the water surface; hence this phenomenon is also referred to as ’Dinoflagellate boom’. Most species produce red pigments and so this phenomenon is often referred to as red tide phenomenon. Some produce reddish brown, yellow or green pigments as well.(3) The phenomenon though referred to as red tide phenomenon, it has nothing to do with tidal activity. These algae are usually harmless, but some of them may produce very potent toxins. With massive proliferation, they may deplete the seawater of oxygen leading to death of fishes in water. Usually fishes that dwell in deep waters are affected. Following widespread death of fishes, there may be release of noxious gases. Inhalation of these gases by human beings can cause clinical manifestations such as syncopial attacks, headache, drowsiness, abdominal pain, nausea, vomiting and panic attacks.(4) However, it is interesting to note that the panicky situation is rarely associated with significant morbidity or mortality.(5) The algae are also consumed by shell fishes which when consumed by human beings can cause gastrointestinal manifestations. Hence human beings are advised to abstain from consuming certain harmful shellfishes during the phenomenon. During red tides, the dinoflagellate, Ptychodiscus Brevis is known to elaborate several neurotoxins such as saxitoxin that inhibit the sodium potassium pump and nerve conduction. Filter feeding molluscs concentrate these toxins and ingestion of these shellfishes may lead to poisoning. Previously, there was a similar event in the same locality with significant morbidity and a few mortality (unpublished).

Chronic lithium treatment up-regulates cell surface Na V1.7 sodium channels via inhibition of glycogen synthase kinase-3 in adrenal chromaffin cells: Enhancement of Na + influx, Ca 2+ influx and catecholamine secretion after lithium withdrawal

In cultured bovine adrenal chromaffin cells expressing Na V1.7 isoform of voltage-dependent Na + channels, we have previously reported that lithium chloride (LiCl) inhibits function of Na + channels independent of glycogen synthase kinase-3 (GSK-3) (Yanagita et al., 2007). Here, we further examined the effects of chronic lithium treatment on Na + channels. LiCl treatment (1–30 mM, ≥12 h) increased cell surface [ 3H]saxitoxin ([ 3H]STX) binding by ∼32% without altering the affinity of [ 3H]STX binding. This increase was prevented by cycloheximide and actinomycin D. SB216763 and SB415286 (GSK-3 inhibitors) also increased cell surface [ 3H]STX binding by ∼31%. Simultaneous treatment with LiCl and SB216763 or SB415286 did not produce an increased effect on [ 3H]STX binding compared with either treatment alone. LiCl increased Na + channel α-subunit mRNA level by 32% at 24 h. LiCl accelerated α-subunit gene transcription by 35% without altering α-subunit mRNA stability. In LiCl-treated cells, LiCl inhibited veratridine-induced 22Na + influx as in untreated cells. However, washout of LiCl after chronic treatment enhanced veratridine-induced 22Na + influx, 45Ca 2+ influx and catecholamine secretion by ∼30%. Washout of LiCl after 24 h treatment shifted concentration–response curve of veratridine upon 22Na + influx upward, without altering its EC 50 value. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na + influx by two-fold in untreated and LiCl-treated cells. Whole-cell patch-clamp analysis indicated that I– V curve and steady-state inactivation/activation curves were comparable between untreated and LiCl-treated cells. Thus, GSK-3 inhibition by LiCl up-regulated cell surface Na V1.7 via acceleration of α-subunit gene transcription, enhancing veratridine-induced Na + influx, Ca 2+ influx and catecholamine secretion.

An efficient synthesis of O,O- Di Propyl (E)-2-[1-methyl 2-oxopropylidene]phosphorohydrazidothiolate (E) Oxime and Its Analogues: A Potential Marine Toxin

An efficient method for the synthesis of Ptychodiscus brevis toxin O,O- di n-propyl (E)-2-[1-methyl 2-oxopropylidene]phosphorohydrazidothiolate (E) oxime (TG-1) and its analogues has been developed using thermally stable and recyclable silica gel and Na2SO4 as a condensing agent and water scavenger, respectively. The compounds were evaluated against fish Rasbora daniconius by determining the LC50 and LC90 values. The results of biological evaluation showed that these compounds have high degree of toxicity.

Lysophosphatidic acid‐LPA1 receptor–Rho–Rho kinase‐induced up‐regulation of Nav1.7 sodium channel mRNA and protein in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion

In cultured bovine adrenal chromaffin cells, chronic (> or = 24 h) treatment with lysophosphatidic acid (LPA) augmented veratridine-induced 22Na+ influx via Na(v)1.7 by approximately 22% (EC(50) = 1 nmol/L), without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated channel. LPA enhanced veratridine (but not nicotine)-induced 45Ca2+ influx via voltage-dependent calcium channel and catecholamine secretion. LPA shifted concentration-response curve of veratridine for 22Na+ influx upward, without altering the EC(50) of veratridine. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na+ influx by twofold in non-treated and LPA-treated cells. Whole-cell patch-clamp analysis showed that peak Na+ current amplitude was greater by 39% in LPA (100 nmol/L for 36 h)-treated cells; however, I-V curve and steady-state inactivation/activation curves were comparable between non-treated and LPA-treated cells. LPA treatment (> or = 24 h) increased cell surface [3H]saxitoxin binding by approximately 28%, without altering the K(d) value; the increase was prevented by cycloheximide, actinomycin D, or Ki16425, dioctylglycerol pyrophosphate 8:0 (two inhibitors of LPA(1) and LPA3 receptors), or botulinum toxin C3 (Rho inhibitor), Y27632 (Rho kinase inhibitor), consistent with LPA(1) receptor expression in adrenal chromaffin cells. LPA raised Nav1.7 mRNA level by approximately 37%. Thus, LPA-LPA(1) receptor-Rho/Rho kinase pathway up-regulated cell surface Nav1.7 and Nav1.7 mRNA levels, enhancing veratridine-induced Ca2+ influx and catecholamine secretion.

Lithium inhibits function of voltage-dependent sodium channels and catecholamine secretion independent of glycogen synthase kinase-3 in adrenal chromaffin cells

Lithium has been proven to be effective in the therapy of bipolar disorder, but its mechanism of pharmacological action is not clearly defined. We examined the effects of lithium on voltage-dependent Na + channels, nicotinic acetylcholine receptors, and voltage-dependent Ca 2+ channels, as well as catecholamine secretion in cultured bovine adrenal chromaffin cells. Lithium chloride (LiCl) reduced veratridine-induced 22Na + influx in a concentration-dependent manner, even in the presence of ouabain, an inhibitor of Na +, K +-ATPase. Glycogen synthase kinase-3 (GSK-3) inhibitors (SB216763, SB415286 or the GSK-3 inhibitor IX) did not affect veratridine-induced 22Na + influx, as well as inhibitory effect of LiCl on veratridine-induced 22Na + influx. Enhancement of veratridine (site 2 toxin)-induced 22Na + influx caused by α-scorpion venom (site 3 toxin), β-scorpion venom (site 4 toxin), or Ptychodiscus brevis toxin-3 (site 5 toxin), still occurred in the presence of LiCl in the same manner as in the control cells. LiCl also reduced veratridine-induced 45Ca 2+ influx and catecholamine secretion. In contrast, LiCl (≦30 mM) had no effect on nicotine-induced 22Na + influx, 45Ca 2+ influx and catecholamine secretion, as well as on high K +-induced 45Ca 2+ influx and catecholamine secretion. Chronic treatment with LiCl at 100 mM (but not at ≦30 mM) significantly reduced cell viability in a time-dependent manner. These results suggest that lithium selectively inhibits Na + influx thorough Na + channels and subsequent Ca 2+ influx and catecholamine secretion, independent of GSK-3 inhibition.

Trichlorocyanuric Acid: An Efficient Reagent for One‐Pot Synthesis of Ptychodiscus brevis (PB‐1) Toxin and Its Derivatives.

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Trichlorocyanuric Acid: An Efficient Reagent for One-Pot Synthesis ofPtychodiscus Brevis(PB-1) Toxin and its Derivatives

Trichlorocyanuric acid is an efficient regent for simple and one-pot synthesis of dialkyl, N-cycloctylphosphoraamidates (PB-1) and its various derivatives in excellent yields. The procedure is operationally simple and involves the reaction of various dialkylphosphites with trichloroisocyanuric acid in acetonitrile and subsequent treatment with amines in presence KF-celite to give PB-1 and derivatives.

Ptychodiscus brevis toxin-induced depression of spinal reflexes involves 5-HT via 5-HT 3 receptors modulated by NMDA receptor

The involvement of 5-hydroxytryptaminergic (5-HT) system for the Ptychodiscus brevis toxin (PbTx)-induced depression of spinal reflexes was evaluated. The reflex potentials were recorded at ventral root by stimulating the corresponding dorsal root in neonatal rat spinal cord in vitro. Superfusion of PbTx (2.8–84 μM) depressed the monosynaptic (MSR) and polysynaptic (PSR) reflexes in a concentration-dependent manner. The depression of the reflexes was maximal with 84 μM of the toxin. Ondansetron (0.1 μM), a 5-HT 3 receptor antagonist, blocked the PbTx-induced depression of MSR and PSR. Spiperone (a 5-HT 2A antagonist) or ketanserin (5-HT 2A/2C antagonist and also at 5-HT 1B/1D) failed to block the PbTx-induced depression of the reflexes. The 5-HT concentration of the cords was increased by four-fold after exposure to PbTx (28 μM) and the increase was not seen in the cords pretreated with dl-2 amino-5-phosphonovaleric acid (APV, a NMDA receptor antagonist). Superfusion of 5-HT or phenylbiguanide (PBG, a 5-HT 3 receptor agonist) also produced depression of the spinal reflexes in a concentration-dependent manner. The 5-HT-induced depression of reflexes was blocked by ondansetron but not by spiperone. The results demonstrate that the PbTx-induced depression of spinal reflexes involves 5-hydroxytryptamine via 5-HT 3 receptors modulated by NMDA receptor.

Ptychodiscus brevis toxin enhances the frequency-dependent depression of the monosynaptic reflex in neonatal rat spinal cord in vitro

The involvement of frequency-dependent depression (FDD) of synaptic transmission for the depressant action of the Ptychodiscus brevis toxin (PbTx) was investigated in neonatal rat spinal cord in vitro. The stimulation of a dorsal root by train of pulses (five stimuli) at different frequencies evoked potentials in the ventral root (monosynaptic reflex, MSR). Amplitude of the fifth response as percent of first response at 0.1, 0.2, 0.5, 1.0 and 2.0 Hz were 90, 80, 75, 70 and 50%, respectively. In Mg 2+-free medium, PbTx depressed the MSR and also enhanced the FDD in a concentration-dependent manner. Further, the PbTx-induced depression can well be correlated with the enhancement of FDD ( r=0.98). In the presence of Mg 2+ (1.3 mM), the FDD was greater than that in the absence of Mg 2+. But in the presence of Mg 2+ PbTx did not alter FDD, eventhough there was 25% depression at 28 μM (significantly lesser than in Mg 2+-free medium). The results indicate that the Mg 2+-sensitive component of PbTx-induced depression of MSR is mediated via the neuronal systems involving FDD.

Involvement of the GABAergic system for Ptychodiscus brevis toxin-induced depression of synaptic transmission elicited in isolated spinal cord from neonatal rats

The involvement of inhibitory transmitters for Ptychodiscus brevis toxin (PbTx)-induced depression of spinal synaptic transmission in neonatal rats was investigated. Stimulation of a dorsal root evoked monosynaptic reflex (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root. The PbTx depressed the reflexes in a concentration-dependent manner and the depression was blocked by GABA A antagonist, bicuculline (1 μM). GABA also produced depression of the reflexes in a concentration-dependent manner. Simultaneous application of submaximal concentrations of PbTx (28 μM) and GABA (30 μM) enhanced the depression (>75%). In contrast, PbTx alone (28 μM) depressed the MSR and the PSR by 33 and 47%, respectively, and GABA (30 μM) alone depressed the reflexes by 30%. The N-methyl- d-aspartate receptor antagonist, dl-2-amino-5-phosphono-pentanoic acid (10 μM), blocked the PbTx-induced depression of MSR and also the enhancement of GABA response by PbTx. A glycine receptor antagonist, strychnine (1 μM), failed to block the depression by the toxin up to 28 μM; however, the depression was attenuated significantly at 84 μM of the toxin. The results indicate that PbTx depressed the spinal reflexes via GABA A receptors. Furthermore, the potentiation of GABAergic action by PbTx requires the N-methyl- d-aspartate dependent mechanism.

Differential effects of bupivacaine enantiomers, ropivacaine and lidocaine on up-regulation of cell surface voltage-dependent sodium channels in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, (±)-bupivacaine inhibited veratridine-induced 22Na + influx (IC 50 6.8 μM). The IC 50 of (+)-bupivacaine (2.8 μM) was 6.2-, 7.4-, and 17.1-fold lower than those of (−)-bupivacaine (17.3 μM), (−)-ropivacaine (20.6 μM), and lidocaine (47.8 μM). Chronic (i.e. 3-h) treatment of cells with (±)-bupivacaine increased cell surface [ 3H]saxitoxin ([ 3H]STX) binding capacity by 48% (EC 50 of 233 μM; t 1/2=7.4 h), without changing the K d value. Treatment for 24 h with either (+)- or (−)-bupivacaine, or (−)-ropivacaine elevated [ 3H]STX binding, whereas 24-h treatment with lidocaine had no effect. The rise of [ 3H]STX binding by (±)-bupivacaine was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of cell surface vesicular exit from the trans-Golgi network; however, (±)-bupivacaine did not increase Na + channel α- and β 1-subunit mRNA levels. In cells subjected to (±)-bupivacaine treatment (1 mM for 24 h) followed by 3-h washout, veratridine-induced 22Na + influx was enhanced, even when measured in the presence of ouabain, an inhibitor of Na +,K +-ATPase. Ptychodiscus brevis toxin-3 potentiated veratridine-induced 22Na + influx by 2.3-fold in the (±)-bupivacaine-treated cells, as in non-treated cells. These results suggest that lipophilic bupivacaine enantiomers or (−)-ropivacaine acutely inhibit Na + channel gating, whereas its chronic treatment up-regulates cell surface expression of Na + channels via translational and externalization events.

Involvement of N-methyl- D-aspartate receptors for the Ptychodiscus brevis toxin-induced depression of monosynaptic and polysynaptic reflexes in neonatal rat spinal cord in vitro

The effects of Ptychodiscus brevis toxin (PbTx) on the Ia-α motoneuron synaptic transmission in neonatal rat spinal cord in vitro was examined. The stimulation of a dorsal root evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root in Mg 2+-free medium. Superfusion with PbTx (2.8–84 μM) depressed the MSR and the PSR in a concentration-dependent manner. At 2.8 μM of PbTx, the depression of MSR and PSR was 24±8.3% and 37±9.7%, respectively. The maximal depression was seen at 84 μM of the toxin (78% for MSR and 96% for PSR). The concentration of toxin required to produce 50% depression was 28.3±6.4 μM for MSR and 5.5±1.1 μM for PSR. The PbTx (28 μM) did not alter the magnitude of the dorsal root or the ventral root potentials. Addition of MgSO 4 (1.3 mM) or DL-2-amino-5-phosphonovaleric acid (APV; 10 μM) to the physiological solution abolished the PSR totally and decreased the MSR by about 30%. In both the conditions, the PbTx-induced depression of the MSR was attenuated significantly. The PbTx-induced depression was blocked completely in the presence of APV+6-cyano-7-nitroquinoxaline-2,3-dione (0.1 μM). NMDA (1 μM) by itself did not alter the magnitude of MSR or PSR but enhanced the PbTx-induced depression (28 μM) of PSR significantly. 7-Chlorokynurenic acid (3 μM; glycine B antagonist) did not block the PbTx-induced depression of MSR. D-serine (glycine B agonist) did not reverse the PbTx-induced depression of reflexes although it reversed the 7-chlorokynurenic acid-induced depression of PSR. The results indicate that the PbTx depressed the spinal reflexes without altering the magnitude of dorsal root or ventral root activity. The depression of the PSR involved NMDA receptors while that of the MSR involved NMDA and non-NMDA receptors. The PbTx actions did not involve the glycine B site of the NMDA receptor.

Relative potency of synthetic analogs of Ptychodiscus brevis toxin in depressing synaptic transmission evoked in neonatal rat spinal cord in vitro

Effects of Ptychodiscus brevis toxin (PbTx) analogs on the spinal synaptic transmission in neonatal rats in vitro were evaluated. PbTx 1/PbTx 2 had aromatic groups and PbTx 3/PbTx 4 had aliphatic groups. All the analogs depressed monosynaptic reflex (MSR) and polysynaptic reflex (PSR) in a concentration-dependent manner. The maximal depression of MSR (75% from initial) and PSR (96%) was at 84 μM for PbTx 1. Concentration to produce 25% inhibition from initial (IC25) by PbTx 1 for MSR and PSR was ⩽2.8 μM. The maximal depression of MSR (80%) was at 96 μM and PSR (100%) was at 32 μM by PbTx 2. IC25 for MSR and PSR were 5.5 μM and <3.2 μM, respectively. PbTx 3 decreased MSR by 25% maximally (=IC25) at 36 μM. The depression of PSR fluctuated and was maximal (75%) at 108 μM and IC25 was 6.2 μM. PbTx 4 depressed MSR and PSR at the maximum of 35% at 32 μM and IC25 for MSR was 8.3 μM and for PSR was 35 μM. Rank order of potency of toxins for depressing MSR was PbTx 1>PbTx 2≫PbTx 4>PbTx 3; and for PSR it was PbTx 2>PbTx 1>PbTx 3≫PbTx 4. Results indicate that the toxins having aromatic groups exhibited greater neurotoxicity.

Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels.

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Short- and long-term differential effects of neuroprotective drug NS-7 on voltage-dependent sodium channels in adrenal chromaffin cells.

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited veratridine-induced (22)Na(+) influx via voltage-dependent Na(+) channels (IC(50)=11.4 microM). The inhibition by NS-7 occurred in the presence of ouabain, an inhibitor of Na(+),K(+) ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS-7. NS-7 attenuated veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels (IC(50)=20.0 microM) and catecholamine secretion (IC(50)=25.8 microM). Chronic (>/=12 h) treatment of cells with NS-7 increased cell surface [(3)H]-STX binding by 86% (EC(50)=10.5 microM; t(1/2)=27 h), but did not alter the K(D) value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, but was not associated with increased levels of Na(+) channel alpha- and beta(1)-subunit mRNAs. In cells subjected to chronic NS-7 treatment, (22)Na(+) influx caused by veratridine (site 2 toxin), alpha-scorpion venom (site 3 toxin) or beta-scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced (22)Na(+) influx remained depressed even at higher concentration of veratridine; however, either alpha- or beta-scorpion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratridine-induced (22)Na(+) influx as in nontreated cells. These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na(+) channels, thereby reducing Ca(2+) channel gating and catecholamine secretion. Chronic treatment with NS-7 up-regulates cell surface Na(+) channels via translational and externalization events, but persistently inhibits Na(+) channel gating without impairing the cooperative interaction between the functional domains of Na(+) channels.

MASS SPECTRAL STUDIES ON SYNTHETIC ANALOGUES OF ORGANOPHOSPHORUS TOXIN ISOLATED FROM PTYCHODISCUS BREVIS

A series of structurally related organophosphorus oximes analogous to O,O-dipropyl-(E)-2-(1-methyl-2-oxopropylidene)phosphorahydrazidothioate-(E)-oxime, isolated from Ptychodiscus brevis, have been synthesised and subjected to electron impact (EI) mass spectral studies. These studies, though aimed at total identification of these compounds, resulted in certain interesting observations and hence are being reported. In order to confirm the observations under electron impact and to support the mechanism of fragmentation we have also performed tandem mass spectrometry experiments in some cases.

UPTAKE, TISSUE DISTRIBUTION, AND EXCRETION OF BREVETOXIN 3 ADMINISTERED TO RATS BY INTRATRACHEAL INSTILLATION

Brevetoxins are cyclic polyether neurotoxins produced by the marine dinoflagellate Ptychodiscus brevis. Blooms of P. brevis (red tides) are toxic to fish, marine mammals, and humans. Humans exposed to seaspray aerosols containing brevetoxins may experience respiratory tract irritation. Because a major route of human exposure to brevetoxins is via the respiratory tract, the objective of this study was to examine the toxicokinetics of brevetoxin 3 (PbTx-3) administered to the lung by intratracheal instillation. Twenty-one male F344/Crl BR rats, 12 wk of age, were administered 3H-PbTx-3 (1 muCi, 6.6 mug PbTx-3/kg) by intratracheal instillation. Groups of 3 rats were sacrificed at 0.5, 3, 6, 24, 48, and 96 h after exposure, and tissues were collected. Three additional rats were placed in glass metabolism cages for collection of urine and feces over a 7-d period. PbTx-3-associated activity was cleared rapidly from the lung and distributed throughout the body, chiefly to the carcass, intestines, and liver. Blood, brain, and fat contained the lowest percentages of the administered dose. Although a majority of the PbTx-3 was cleared rapidly from lung, liver, and kidneys, approximately 20% of the initial concentration present in each organ was retained for 7 d. Concentrations of PbTx-3 in brain and fat were low, but remained relatively constant over time. Approximately twice as much PbTx-3-associated activity was excreted in feces than in urine, with the majority of excretion occurring within 48 h after instillation. The results of this study indicate that over 80% of the PbTx-3 is rapidly absorbed from the lung to the blood and distributed to all tissues. The tissues containing the greatest amount of PbTx-3-associated activity reflect the compound's site of deposition, storage compartment, and major route of metabolism and excretion. These results illustrate that brevetoxin exposure by the respiratory route results in systemic distribution of brevetoxin and suggest that the initial respiratory irritation and bronchoconstriction may only be a part of the overall toxicological consequences associated with brevetoxin inhalation.