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PubMed1980

Central respiratory and circulatory depression caused by intravascular saxitoxin.

1 In cats anaesthetized with pentobarbitone and vagotomized, observations were made on the phrenic nerve action potential and the diaphragm electromyogram (EMG) at constant end-tidal Pco(2). Arterial blood pressure was stabilized by intravenous infusions of noradrenaline.2 Intravenous administration of saxitoxin (STX) initially abolished respiratory activity in the EMG and caused a slowing of oscillation in the central phrenic neurogram. Additional STX produced apneustic phrenic discharges followed by a progressive loss of nerve action potentials.3 The inspiratory centre in the medulla oblongata was stimulated electrically to evoke a sustained phrenic nerve discharge. STX, given intravenously, resulted in the elimination of spontaneous nerve activity without interfering with the evoked response.4 The cephalic intravascular infusion of STX into a carotid or vertebral artery depressed spontaneous respiratory activity while sparing EMG activity evoked by electrical stimulation of the intact phrenic nerve.5 Spontaneous respiratory discharge in the phrenic nerve was eliminated by smaller doses of STX administered intra-arterially than were required intravenously. In addition, onset of and recovery from neural silence occurred faster following intra-arterial injection of STX.6 Depressant effects on arterial blood pressure coincided with those on respiration when STX was given intra-arterially.7 An electrophysiological assay on frog sartorius muscle was used to measure STX in the cerebrospinal fluid. Levels of STX detected were proportional to amounts of the toxin infused intra-arterially.8 It is concluded that STX exchanges rapidly between blood and brain to bring about central depression and this adds to its peripheral paralytic actions.

Animals↗
PubMed1974

Complementary lowering of the behavioural and physiological thermoregulatory set-points by tetrodotoxin and saxitoxin in the cat.

1. Injections of tetrodotoxin or saxitoxin (25 ng in 0.10 ml.) into a lateral cerebral ventricle caused deep body temperature of cats to fall approximately the same amount (2 degrees C) whether the animals were resting in the cold (4 degrees C) or were responding to escape heat.2. Continuous exposure to heat either prevented the hypothermic response or enhanced the level of tachypnoea required to lower body temperature. Tetrodotoxin also caused hypothermia when an animal was lever pressing to obtain heat in the cold environment.3. These results provide evidence that agents which alter the set-point for physiological thermoregulatory activity produce a complementary shift in the behavioural set-point as well.

Animals↗
PubMed1976

A new method for labelling saxitoxin and its binding to non-myelinated fibres of the rabbit vagus, lobster walking leg, and garfish olfactory nerves.

1. A new method of labelling saxitoxin (STX) is described, based on transfer of tritium from tritiated water to the toxin. 2. The radiochemical purity of the labelled toxin has been directly determined, rather than being based on indirect biochemical means, as in previous experiments with Wilzbach-labelled STX and TTX. 3. The specific activity of the labelled toxin, 66 d..m.f-mole-1, corresponds with one tritium atom per molecule STX, an improvement of about 300-fold over other means of labelling TTX and STX. 4. The binding of this toxin to rabbit, lobster and garfish olfactory nerve fibres has been re-examined. 5. The density of sodium channels calculated on the basis of the binding of the toxin is about four to six-times the values previously reported.

Animals↗
PubMed1979

Characteristics of saxitoxin binding to the sodium channel of sarcolemma isolated from rat skeletal muscle.

1. The characteristics of saxitoxin (STX) binding to the mammalian Na channel have been studied in purified sarcolemma isolated from rat skeletal muscle. 2. STX binds specifically to isolated sarcolemma with a Kd of 1.43 x 10(-9) M and Bmax of 7-8 p-mole STX bound/mg membrane protein at 0 degrees C in the presence of 140 mM-NaCl. In rat muscle homogenate under the same conditions the corresponding values are Kd = 1.53 x 10(-9) M and Bmax = 0.15-0.20 p-mole/mg protein (18-20 p-mole/g wet wt.). Membrane purification produced a fortyfold increase in STX binding site concentration per milligram protein. Calculated binding site density in isolated sarcolemma was about 30 sites/micron 2 of membrane surface. 3. Denervation (10-14 days) results in a 43% reduction in the density of high-affinity STX binding sites in purified sarcolemma, but the Kd for this class of sites is not changed. 4. In sarcolemma, the apparent Kd for STX binding is dependent on temperature pH and ionic strength. The Q10 for Kd between 0 and 40 degrees C is 1.3. Protonation of a group having a pK of 6.0 markedly raises Kd without affecting Bmax. Apparent Kd increases eightfold when ionic strength is raised from 20 to 600 mM. 5. Dissociation and association rate constants for STX binding are temperature dependent with Q10 of 2.6 and 1.9 respectively between 0 and 20 degrees C. 6. STX binding is competitively inhibited by monovalent and divalent cations under conditions of constant total ionic strength. An affinity sequence of Tl+ greater than Li+ greater than Na+ greater than K+ greater than Rb+ greater than Cs+ is seen for the monovalent cation-binding site. 7. The STX binding site is relatively stable to heat and to enzymic degradation. A specific modifier of carboxyl residues inactivates subsequent STX binding. This process can be prevented by the presence of STX during the reaction. 8. Characteristics of the STX binding site in isolated sarcolemma are compared to those reported for other isolated excitable membranes and for studies of whole muscle and muscle homogenate. Sarcolemma provides a potential source of enriched Na channels for further purification efforts in a mammalian system.

Animals↗
PubMed1980

Saxitoxin binding to sodium channels of rat skeletal muscles.

1. The saturable binding of exchange-labelled tritiated saxitoxin (STX) to the extensor digitorum longus (e.d.l.), diaphragm and soleus muscles of adult rats was studied. By measuring STX uptake to small pieces of muscle, the dissociation constant (KD) and binding capacity could be determined for individual muscles. 2. The affinity for STX is very similar in all three muscles, with a KD of 4.3 +/- 0.3, 5.1 +/- 0.5 and 4.9 +/- 0.5 nM (mean +/- S.E. of mean) at 4 degrees C for e.d.l., diaphragm and soleus respectively. The maximum binding capacity, which varies between different muscles, is 52.6 +/- 2.5, 40.3 +/- 4.9 and 23.8 +/- 1.1 f-mole.mg wet wt.-1 respectively. 3. The affinity of e.d.l. for tetrodotoxin (TTX), measured by inhibition of STX binding, is 12.1 +/- 1.4 nM at 4 degrees C. Raising the temperature to 37 degrees C increases the KD for STX to 6.8 +/- 0.8 nM and the KD for TTX to 47.5 +/- 4.5 nM. 4. STX binding is pH dependent; protons compete with STX for the binding site as if there were a titratable acidic group with a pK of 5.5. 5. The binding capacity of the diaphragm is not uniform along the length of the muscle fibres. Binding at the ends of the fibres is only 78% of that in the central region. 6. Denervation of e.d.l. for 7 days causes no change in the affinity for STX. There is a slight reduction in the binding capacity from 54 +/- 5 to 43 +/- 3 f-mole.mg wet wt.-1. There is no change in the diameter of the muscle fibres.

Animals↗
PubMed1988

Neural regulation of [3H]saxitoxin binding site numbers in rat neonatal muscle.

1. Neural regulation of the density of sodium (Na+) channels in rat muscle was studied by measuring specific binding of tritiated saxitoxin ([3H]STX) to muscles from rat hindlimbs during normal development and in rats in which neuromuscular function was interrupted by sciatic nerve section or neuromuscular blockade with botulinum toxin (BoTX). 2. The normal developmental increase in [3H]STX binding site numbers followed a simple exponential with a time constant of 12 days. The most rapid incorporation of channels coincided with the onset of accelerated muscle growth and increased neuromuscular activity at 2 weeks of age. 3. Elimination of neuromuscular activity retarded muscle growth and inhibited the normal incorporation of Na+ channels into neonatal muscle. Muscles denervation was more effective than BoTX paralysis: denervation at 2 weeks of age prevented the normal 3-fold increase in the binding site density between 2 and 3 weeks of age while age-matched BoTX-treated muscles incorporated an average of 66% of the normal Na+ channel incorporation. 4. Denervation and BoTX treatment were equally effective in reducing the numbers of [3H]STX binding sites in adult muscle. A reduction of 30% in binding sites brought the numbers to levels which corresponded with levels normally seen in muscles at 3 weeks of neonatal development. 5. It was concluded that the neural influence on incorporation of Na+ channels into membranes of neonatal muscle is, at least in part, mediated by neuromuscular activity.

Animals↗
PubMed2003

Saxitoxin blocks L-type ICa.

Saxitoxin (STX) and tetrodotoxin (TTX) are frequently used to selectively block sodium channels. In this study, we provide evidence that commercial STX also inhibits L-type Ca2+ currents (I(Ca,L)) in adult mouse ventricular myocytes (VMs) and tsA-201 cells that were transiently cotransfected with three calcium channel subunits. We measured inhibition of sodium currents (INa) in mouse VMs, of I(Ca,L) in mouse VM and tsA-201 cells, and intracellular calcium concentration ([Ca2+]i) transients in single mouse VMs. STX or TTX was abruptly applied before the test voltage pulse using a rapid solution switcher device. STX (10 microM; Calbiochem) and TTX (60 microM; Sigma-Aldrich) completely blocked INa in mouse VMs. However, STX at 10 microM also reduced I(Ca,L) in mouse VM by 39% (P < 0.0001; n = 14), whereas TTX at 60 microM had no effect on I(Ca,L). STX (10 microM; Calbiochem) reduced the amplitude of the [Ca2+]i transients in mouse VMs by 36% (P < 0.0001; n = 10). In contrast, TTX (60 microM; Sigma-Aldrich) only reduced the amplitude of the [Ca2+]i transients by 9% (P = 0.003; n = 5). STX (10 microM) obtained from Sigma-Aldrich showed a similar inhibitory effect on I(Ca,L) (33%) (P < 0.0001; n = 5) in mouse VMs. STX (Calbiochem) inhibited the calcium currents of tsA-201 cells in a dose-dependent manner. This inhibition was voltage-independent. The current-voltage relationship of calcium currents in tsA-201 cells was not altered by STX. These results indicate that STX partially blocks L-type Ca2+ channels and thus provide further evidence that its effects are not specific for Na+ channels.

Animals↗
PubMed2003

Effects of saxitoxin (STX) and veratridine on bacterial Na+ -K+ fluxes: a prokaryote-based STX bioassay.

Saxitoxin (STX) is a potent natural sodium channel blocker and represents a significant health concern worldwide. We describe here the antagonistic effects of STX and veratridine (VTD), an Na+ channel activator, on three gram-negative bacteria and their application to an STX bioassay. STX reduced the total cellular levels of both Na+ and K+, as measured by flame photometry, whereas VTD increased the cellular concentrations relative to control ion fluxes in the cyanobacterium Cylindrospermopsis raciborskii AWT205. Endogenous STX production in toxic cyanobacterial strains of C. raciborskii and Anabaena circinalis prevented cell lysis induced by VTD stress. Microscopic cell counts showed that non-STX producing cyanobacteria displayed complete cell lysis and trichome fragmentation 5 to 8 h after addition of VTD and vanadate (VAN), an inhibitor of sodium pumps. The addition of STX, or its analogue neoSTX, prior to treatment with VTD plus VAN prevented complete lysis in non-STX-producing cyanobacteria. VTD also affected cyanobacterial metabolism, and the presence of exogenous STX in the sample also ameliorated this decrease in metabolic activity, as measured by the cellular conversion of tetrazolium into formazan. Reduced primary metabolism was also recorded as a decrease in the light emissions of Vibrio fischeri exposed to VTD. Addition of STX prior to VTD resulted in a rapid and dose-dependent response to the presence of the channel blocker, with samples exhibiting resistance to the VTD effect. Our findings demonstrate that STX and VTD influence bacterial Na+ and K+ fluxes in opposite ways, and these principles can be applied to the development of a prokaryote-based STX bioassay.

Bacteriolysis↗
PubMed2004

Identification of an Na(+)-dependent transporter associated with saxitoxin-producing strains of the cyanobacterium Anabaena circinalis.

Blooms of the freshwater cyanobacterium Anabaena circinalis are recognized as an important health risk worldwide due to the production of a range of toxins such as saxitoxin (STX) and its derivatives. In this study we used HIP1 octameric-palindrome repeated-sequence PCR to compare the genomic structure of phylogenetically similar Australian isolates of A. circinalis. STX-producing and nontoxic cyanobacterial strains showed different HIP1 (highly iterated octameric palindrome 1) DNA patterns, and characteristic interrepeat amplicons for each group were identified. Suppression subtractive hybridization (SSH) was performed using HIP1 PCR-generated libraries to further identify toxic-strain-specific genes. An STX-producing strain and a nontoxic strain of A. circinalis were chosen as testers in two distinct experiments. The two categories of SSH putative tester-specific sequences were characterized by different families of encoded proteins that may be representative of the differences in metabolism between STX-producing and nontoxic A. circinalis strains. DNA-microarray hybridization and genomic screening revealed a toxic-strain-specific HIP1 fragment coding for a putative Na(+)-dependent transporter. Analysis of this gene demonstrated analogy to the mrpF gene of Bacillus subtilis, whose encoded protein is involved in Na(+)-specific pH homeostasis. The application of this gene as a molecular probe in laboratory and environmental screening for STX-producing A. circinalis strains was demonstrated. The possible role of this putative Na(+)-dependent transporter in the toxic cyanobacterial phenotype is also discussed, in light of recent physiological studies of STX-producing cyanobacteria.

Amino Acid Sequence↗
PubMed1986

Effects of saxitoxin analogues and ligand competition on sodium currents of squid axons.

Saxitoxin (STX) and several STX analogues from dinoflagellates (genus Protogonyaulax) block sodium conductance in squid giant axons with variable potencies. Toxins, analyzed under voltage clamp, are 21-sulfosaxitoxin, 21-sulfosaxitoxin 11 alpha-hydroxysulfate, 21-sulfosaxitoxin 11 beta-hydroxysulfate, (B1, C1, C2, respectively) and gonyautoxins 2 and 3. The potency sequence for the toxins examined is STX greater than gonyautoxin 3 greater than B1 greater than C2 greater than gonyautoxin 2 much greater than C1. Guanidine, when substituted for sodium in external seawater, reduced the potency of STX to block inward current but did not affect tetrodotoxin activity. Methylguanidine also reduced the ability of STX to block outward sodium current. Inhibitory constants for guanidine and methylguanidine were 116 and 187 mM, respectively. Competition can be explained by binding at or near the toxin binding site but not by surface potential alteration.

Amphibian Proteins↗
PubMed1985

Saxitoxin binding and "fast" sodium channel inhibition in sheep heart plasma membrane.

We compared specific [3H]saxitoxin (STX) binding to isolated sheep ventricular sarcolemmal vesicles with inhibition of maximal action potential upstroke velocity (V max) by STX and tetrodotoxin (TTX) in sheep trabeculae carneae. In sarcolemmal vesicles purified 30 to 40 times over cardiac homogenate, STX binding at 0 degrees C in Na-free solution exhibited both high-affinity sites (KD = 0.22 +/- 0.05 nM, n = 85 +/- 13 fmol/mg protein) and low-affinity sites (KD = 11 +/- 4 nM, n = 360 +/- 42). The STX-inhibition constant for V max in Tyrode solution at 37 degrees C was 280 nM. TTX was approximately 10% as effective as STX in displacing bound [3H]STX and inhibiting V max. Allowing for different experimental conditions during [3H]STX binding and V max measurements, we suggest that the low-affinity sites are physiologically relevant "fast" Na+ channels of myocardial cells. Combining morphometric data for plasmalemmal area of mammalian cardiac myocytes with n for low-affinity sites, we estimate 3.6-7.6 fast Na+ channels/micron2 plasmalemma.

Action Potentials↗
PubMed1987

Saxitoxin differentiates between two types of Na+-dependent potentials in the Retzius cell of hirudinid leeches.

The effects of tetrodotoxin (TTX) and saxitoxin (STX) on the action potentials recorded in Ca2+-free solution in the absence of Ca2+ and K+ currents were investigated in the Retzius cell of three hirudinid leech species (Hirudo medicinalis, Macrobdella decora and Poecilobdella granulosa) and in the glossiphoniid leech Haementeria ghilianii. In the four leech species, stimulation of the Retzius cell in the presence of 25 mmol l-1 tetraethylammonium chloride (TEA), 3 mmol l-1 4-aminopyridine (4-AP) and 2mmol l-1 Mn2+ evoked prolonged action potentials consisting of an initial fast-rising spike followed by a plateau lasting several hundreds of milliseconds. The amplitude and duration of both components of action potentials recorded under these conditions were dependent on [Na+]o. In the hirudinid leeches the initial spike was unaffected by TTX and STX whereas the plateau was selectively blocked by micromolar concentration of STX. In Haementeria both the initial spike and the subsequent plateau were sensitive to nanomolar concentrations of STX and TTX with an estimated ED50 of approximately 20 nmol l-1 for inhibition of Vmax of the fast spike. The results suggest that there are two types of ionic currents mediating the two distinct components of Na+-dependent action potentials in the Retzius cell: (1) a fast-inactivating one, presumably underlying the normal spike which is TTX- and STX-resistant in hirudinid leeches but sensitive to both agents in Haementeria and (2) a low-threshold, prolonged current which underlies the plateau recorded from these cells in the absence of Ca2+ and K+ currents and which is selectively blocked by STX in hirudinid leeches but sensitive to both STX and TTX in Haementeria. It is likely that the variable kinetic and pharmacological properties that characterize the various Na+ potentials in these identified homologous neurones may be of functional significance and result from differences in the molecular structure of their Na+ channels.

4-Aminopyridine↗
PubMed1976

The local anesthetic activity of saxitoxin alone and with vasoconstrictor and local anesthetic agents.

STX (saxitoxin), alone and with various vasoconstrictor and local anesthetic agents, was evaluated for its ability to produce topical anesthesia on the rabbit cornea, peripheral nerve block in the rat, and epidural anesthesia in the dog. High frequency and long duration of block can be attained if sufficiently high concentrations of STX are used, although latency is long and the doses used may produce systemic toxicity. Frequency of satisfactory blocks and mean duration of block can be increased and systemic toxicity reduced if STX is administered with a vasoconstrictor agent. Conventional local anesthetic agents also enhance the nerve blocking activity of STX. When appropriate concentrations of STX, vasoconstrictor and local anesthetic agents are used, systemic toxic effects are not manifested and the blocks produced exhibit the rapid onset and high frequency of block characteristic of the local anesthetic agent and the remarkably long duration of STX.

Anesthesia, Epidural↗
PubMed1986

The sodium channel from rat brain. Role of the beta 1 and beta 2 subunits in saxitoxin binding.

Procedures are described for the selective removal of the beta 1 or the beta 2 subunits from the detergent-solubilized channel from rat brain, and the functional integrity of the resulting protein complex is examined. Treatment of the channel with 1.0 M MgCl2 followed by sedimentation through sucrose gradients results in complete separation of beta 1 from the alpha-beta 2 complex and complete loss of [3H]saxitoxin (STX) binding activity. At intermediate MgCl2 concentrations, the loss of beta 1 and the loss of [3H]STX binding activity are closely correlated. Tetrodotoxin (TTX) quantitatively stabilizes the solubilized complex against both the loss of beta 1 and the loss of [3H]STX binding activity. This indicates that association of the alpha and beta 1 subunits is required to maintain the STX/TTX binding site in a conformation with high affinity for STX and TTX in the detergent-solubilized state. Treatment of the solubilized sodium channel with dithiothreitol in the presence of TTX causes specific release of the beta 2 subunit, without significantly removing beta 1. There is little or no correlation between the amount of beta 2 in the sodium channel complex and the ability of the preparation to bind [3H]STX. We conclude from these studies that the presence of beta 1, but not beta 2, is required for the integrity of the STX/TTX binding site of the solubilized and purified rat brain sodium channel.

Animals↗
PubMed1986

3H-batrachotoxinin-A benzoate binding to voltage-sensitive sodium channels: inhibition by the channel blockers tetrodotoxin and saxitoxin.

The sodium channel blockers tetrodotoxin (TTX) and saxitoxin (STX) and the channel activator batrachotoxin (BTX) produce their effects by binding to separate and distinct sites on the channel protein. The fact that TTX- and STX-modified sodium channels are blocked to sodium flux has precluded drawing any direct conclusions regarding the effect of TTX/STX on BTX binding based on electrophysiological or 22Na flux measurements. Nevertheless, these sites have been presumed to be non-interacting. In this study, 3H-batrachotoxinin-A benzoate (BTX-B), a tritiated congener of BTX, has been used to provide a direct assessment of these binding interactions. Equilibrium specific binding of 3H-BTX-B to sodium channels in vesicular preparations of mouse brain in the presence of scorpion toxin was measured using a filtration assay procedure. At 25 degrees C both TTX and STX inhibit 3H-BTX-B binding in a concentration-dependent and noncompetitive manner. This inhibition is markedly temperature-dependent, being negligible at 37 degrees C and maximal at 18 degrees C, the lowest temperature investigated. Scatchard analysis of BTX-B binding isotherms at 25 degrees C in the presence and absence of 1 microM TTX revealed that inhibition is due to a 3-fold decrease in the affinity of BTX-B binding with no change in the number of binding sites (Bmax). The concentration dependence for TTX inhibition of both specific 3H-STX and 3H-BTX-B binding is identical, suggesting that inhibition of 3H-BTX-B binding is due to a direct effect of TTX/STX binding at their specific sodium channel site. The channel blockers did not alter the binding of scorpion toxin under these assay conditions, nor did BTX-B affect the binding of 3H-STX.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Isolation of membranes enriched in "tetrodotoxin-insensitive" saxitoxin-binding sites from mammalian ventricle. Receptor solubilization.

Purification and characterization of Na+ channel protein from mammalian ventricular myocytes has heretofore been complicated by the low concentration of Na+ channels and by the finding that mammalian ventricles contain both tetrodotoxin (TTX)-sensitive channels (TSC), with high affinity for saxitoxin (STX), and TTX-insensitive channels (TIC), with low affinity for STX. Most (perhaps all) of the sodium current for myocardial cell action potentials is carried by TIC. Most, if not all, of the cardiac TSC reside in nerves innervating the heart. To isolate TIC in sufficient quantity for further study, we prepared t-tubular sarcolemmal vesicles from large (sheep) hearts with techniques designated to minimize contamination from nerve plasmalemma. Discontinuous sucrose density gradient centrifugation of these membranes produced membrane vesicles, some of which contained no detectable TSC (range 94-100% TIC, or 0-6% TSC), at a concentration of 200-1500 fmol total sites/mg protein, with yields of 4.0-25.0 mg protein/100 g starting material (ventricle). TTX-insensitive STX-binding sites were solubilized from the membranes by 1% digitonin (and with less stability by Triton X-100). The equilibrium binding constant and dissociation rate coefficient for STX binding to the digitonin-solubilized sites were similar to those of the binding sites for the unsolubilized membranes. Unlabeled TTX competed with [3H]STX for the site with 14 times less affinity than did unlabeled STX. Digitonin-solubilized sites had a half-life for STX binding of about 24 h. Binding could be further stabilized by addition of Mg2+ or Ca2+ and exogenous phospholipid.

Animals↗
PubMed1989

Development of saxitoxin-sensitive and insensitive sodium channels in cultured neonatal rat astrocytes.

Voltage-sensitive Na channels were studied in cultures of neonatal rat cortical astrocytes. These channels were present at all times in culture as determined by tracer 22Na+ influx in the presence of batrachotoxin (BTX) and sea anemone polypeptide toxin (AxTx). The affinity of saxitoxin (STX) binding and sensitivity to STX inhibition of sodium influx were utilized to characterize these channels. Up to 7 d in culture, high-affinity 3H-STX binding (Kd of 0.2 nM at 4 degrees C) was very low, and 22Na+ influx was inhibited only by high concentrations (Ki = 170 nM) of STX. From 7 to 14 d, total specific binding of STX increased to a maximum of over 2 pmol/mg protein and remained constant for 28 d. By 14 d, inhibition of 22Na+ influx by STX was clearly biphasic, indicating the presence of 2 populations of channels with Ki's of 0.2 nM and 150 nM. At 14 d in culture, binding of 3H-STX to astrocyte membranes revealed the presence of 2 specific sites. During this second week, increasing numbers of high-affinity STX binding sites and increasing sensitivity to the inhibition of BTX + AxTx-stimulated 22Na+ influx by STX coincided with the change in morphology of primitive flat polygonal cells to highly branched stellate forms characteristic of mature astrocytes in vivo. Changes in culture conditions modified the time course of the onset of high STX affinity binding. Twenty-four hours after changing to serum-free G5 medium, there was both an 8-fold increase in STX binding sites and a change to a stellate shape in all cells. The results suggest that although low-affinity STX Na channels are always present in astrocytes, after 7 d in culture a different population of channels appears with the high affinity for STX characteristic of adult neuronal sodium channels. This spontaneous process is greatly accelerated by changing to a chemically defined medium.

Animals↗
PubMedDate not supplied

Indirect enzyme-linked immunosorbent assay for saxitoxin in shellfish.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of saxitoxin (STX). Antibodies against STX were demonstrated in rabbits 5 weeks after immunizing with STX-bovine serum albumin (STX-HCHO-BSA). In the ELISA, STX-HCHO-BSA or polylysine-STX was coated onto the microtiter plate, followed by incubation with standard toxin and anti-STX antibody. The amount of antibody bound to the solid phase was determined by incubation with goat anti-rabbit IgG peroxidase conjugate and a reaction with chromogenic substrate. Competitive indirect ELISA revealed that the anti-serum did not cross-react with either carbamoyl-neo-STX-sulfate or tetrodotoxin. The antibodies for STX cross-reacted with decarbamoyl-STX and neo-STX about 56% and 16% as much as they did with STX, respectively. The lower detection limits for STX, decarbamoyl-STX, and neo-STX in this system were about 25, 45, and 156 pg per assay, respectively. When STX added to clams or mussels was assayed, the detection limit for STX was about 50-100 ppb, and recoveries were in the range of 86.8-107%.

Animals↗