The cannabinoid receptor agonist CP-55,940 and ethyl arachidonate interfere with [(3)H]batrachotoxinin A 20 alpha-benzoate binding to sodium channels and inhibit sodium channel function

Recent investigations in our laboratory showed that voltage-gated sodium channels (VGSCs) in brain are sensitive to inhibition by various synthetic cannabinoids and endocannabinoids. The present experiments examined the effects of the cannabinoid-1 (CB1) receptor agonist CP-55,940 and ethyl arachidonate on [(3)H]batrachotoxinin A 20 alpha-benzoate ([(3)H]BTX-B]) binding and VGSC-dependent depolarization of the nerve membrane in synaptoneurosomes isolated from mouse whole brain. CP-55,940 acted as a full inhibitor of [(3)H]BTX-B binding and its IC(50) was established at 22.3 microM. At its maximum effect concentration, ethyl arachidonate achieved partial (approximately 70%) inhibition and was less effective than CP-55,940 as an inhibitor of binding (IC(50)=262.7 microM). The potent CB1 receptor antagonist AM251 (2 microM) had no significant effect on the displacement of [(3)H]BTX-B by either compound (P>0.05). Scatchard analyses showed that CP-55,940 and ethyl arachidonate reduce the binding of [(3)H]BTX-B by lowering its B(max) but ethyl arachidonate also increased the K(d) of radioligand binding. In kinetic experiments, CP-55,940 and ethyl arachidonate were found to boost the dissociation of [(3)H]BTX-B from VGSCs to rates that exceed the maximum velocity achievable by veratridine, indicating they operate as allosteric inhibitors of [(3)H]BTX-B binding. Neither compound was effective at changing the initial rate of association of [(3)H]BTX-B with sodium channels. CP-55,940 and ethyl arachidonate inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes with IC(50)s of 3.2 and 50.1 microM respectively. These inhibitory effects were again not influenced by 2 microM AM251. Our data demonstrate that the potent cannabinoid receptor agonist CP-55,940 and the ethyl ester of arachidonic acid have the ability to associate with VGSCs and inhibit their function independently of effects on CB1 receptors. Binding data comparisons using mouse brain preparations indicate CP-55,940 is approximately 10,000 times more potent as a CB1 receptor ligand than a sodium channel ligand while ethyl arachidonate shows a much smaller differential. Ethyl arachidonate has been shown previously to be the principal metabolite of ethanol in the brains of intoxicated individuals and effects of this ester on VGSCs and CB1 receptors may contribute to the depressant effects of alcohol.     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.     

Binding of [3H]batrachotoxinin A-20-alpha-benzoate to a high affinity site associated with house fly head membranes

1. [3H]Batrachotoxinin A-20-alpha-benzoate (BTX-B), a radioligand that labels the alkaloid activator recognition site of the voltage-sensitive sodium channel, was bound specifically to high affinity, saturable sites in a subcellular preparation from house fly (Musca domestica L.) heads that was shown previously to contain binding sites for other sodium channel-directed ligands. 2. Specific binding of [3H]BTX-B was observed in the presence of 140 mM sodium or potassium and was inhibited by choline ion. 3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom stimulated the specific binding of [3H]BTX-B four-fold, increasing the proportion of specific binding of 10 nM [3H]BTX-B from less than 15% to 40%. Equilibrium dissociation studies in the presence of scorpion venom gave an equilibrium dissociation constant (KD) for [3H]BTX-B of 80 nM and a maximal binding capacity (Bmax) of 1.5 pmol/mg protein. 4. Parallel experiments in the absence of venom gave a KD value of 140 nM and a Bmax of 1.3 pmol/mg protein, indicating that scorpion venom stimulated [3H]BTX-B binding by increasing the affinity of this site approximately two-fold. 5. The specific binding of [3H]BTX-B was inhibited by the sodium channel activators aconitine and batrachotoxin and, to a lesser extent, by the anticonvulsant diphenylhydantoin. However, several other sodium channel-directed neurotoxins known to exert allosteric effects on the binding of [3H]BTX-B to mammalian brain preparations did not affect the binding of [3H]BTX-B to house fly head membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

20(S)-protopanaxadiol and the ginsenoside Rh2 inhibit Na+ channel-activated depolarization and Na+ channel-dependent amino acid neurotransmitter release in synaptic fractions isolated from mammalian brain

The ginsenoside Rh(2) and its aglycone 20(S)-protopanaxadiol are known to inhibit the binding of [(3)H]batrachotoxinin 20alpha-benzoate to site 2 on voltage-gated sodium channels and electrophysiological investigations conducted by others have shown that ginsenosides cause voltage-dependent inhibition of reconstituted forms of the sodium channel. Here we describe the actions of Rh(2) and 20(S)-protopanaxadiol on sodium channel function and release of neurotransmitters resulting from activation of native sodium channels in synaptic preparations isolated from whole mouse brain. Rh(2) and 20(S)-protopanaxadiol inhibited veratridine-dependent (tetrodotoxin-suppressible) depolarization of synaptoneurosomes as determined using the rhodamine 6G method although 20(S)-protopanaxadiol was more potent as an inhibitor than Rh(2). Veratridine- (sodium channel-) dependent release of the neurotransmitters L-glutamate and GABA was almost fully inhibited by 20(S)-protopanaxadiol, however, less complete inhibition was observed with Rh(2). At its maximum inhibitory concentration, Rh(2) also produced release of l-glutamate and GABA from synaptosomes, in contrast to 20(S)-protopanaxadiol. We conclude that low to moderate micromolar concentrations of Rh(2) and 20(S)-protopanaxadiol inhibit sodium channel function and sodium channel-activated release of neurotransmitters. Apparently the ginsenoside Rh(2) cannot achieve complete inhibition of sodium channel-activated transmitter release because at high concentrations it also stimulates release.     

The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors.

It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.     

Binding of [3H]batrachotoxinin A-20-α-benzoate to a high affinity site associated with house fly head membranes

1. [³H]Batrachotoxinin A-20-α-benzoate (BTX-B), a radioligand that labels the alkaloid activator recognition site of the voltage-sensitive sodium channel, was bound specifically to high affinity, saturable sites in a subcellular preparation from house fly (Musca domestica L.) heads that was shown previously to contain binding sites for other sodium channel-directed ligands.2. Specific binding of [³H]BTX-B was observed in the presence of 140 mM sodium or potassium and was inhibited by choline ion.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom stimulated the specific binding of [³H]BTX-B four-fold, increasing the proportion of specific binding of 10 nM [³H]BTX-B from less than 15% to 40%. Equilibrium dissociation studies in the presence of scorpion venom gave an equilibrium dissociation constant (K_D) for [³H]BTX-B of 80 nM and a maximal binding capacity (B_max) of 1.5 pmol/mg protein.4. Parallel experiments in the absence of venom gave a K_D value of 140 nM and a B_max of 1.3 pmol/mg protein, indicating that scorpion venom stimulated [³H]BTX-B binding by increasing the affinity of this site approximately two-fold.5. The specific binding of [³H]BTX-B was inhibited by the sodium channel activators aconitine and batrachotoxin and, to a lesser extent, by the anticonvulsant diphenylhydantoin. However, several other sodium channel-directed neurotoxins known to exert allosteric effects on the binding of [³H]BTX-B to mammalian brain preparations did not affect the binding of [³H]BTX-B to house fly head membranes.6. These studies provide evidence for a high affinity binding site in house fly head membrane preparations that exhibits properties expected of the activator recognition site of the voltage-sensitive sodium channel but does not respond to several compounds known to modify allosterically the binding of [³H]BTX-B to sodium channels in mammalian brain.     

Hormonal interactions with benzodiazepine binding sites in vitro

Prostaglandin A1 and hormones like corticosteroids and DL-Thyroxin (T4) inhibit binding of [3H]RO 5-4864 and [3H] Clonazepam to their respective binding sites with inhibition constants in the low micromolar range. The corticosteroid Cortisone inhibits [3H] RO 5-4864, but not [3H] Clonazepam binding in a competitive manner with an inhibition constant of 4.3 +/- 0.7 microM, Prostaglandin A1 inhibits [3H] Clonazepam, but not [3H] RO 5-4864 binding in a competitive manner with an inhibition constant of 6 +/- 1.2 microM and DL-Thyroxin (T4) inhibits both [3H] RO 5-4864 and [3H] Clonazepam binding with inhibition constants of 12.1 +/- 2.2 and 1.6 +/- 0.4 microM respectively. While the inhibition of [3H] RO 5-4864 binding by DL-Thyroxin (T4) is competitive, the inhibition of [3H] Clonazepam binding is of the mixed type as indicated by Scatchard Plot.     

Effects of saturated long-chain N-acylethanolamines on voltage-dependent Ca2+ fluxes in rabbit T-tubule membranes

The effects of saturated long-chain (C: 16-22) N-acylethanolamines and a series of saturated fatty acids with the same length of carbon chains were investigated on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with (45)Ca(2+) and membrane potentials were generated by establishing potassium gradients across the vesicle using the ionophore valinomycin. Arachidonoylethanolamide and docosaenoylethanolamide but not palmitoylethanolamide and stearoylethanolamide (all 10 microM) caused a significant inhibition of depolarization-induced (45)Ca(2+) fluxes and specific binding of [(3)H]Isradipine to transverse tubule membranes. On the other hand, saturated fatty acids including palmitic, stearic, arachidic, and docosanoic acids (all 10 microM) were ineffective in functional and radioligand binding experiments. Additional experiments using endocannabinoid metabolites suggested that whereas ethanolamine and arachidic acids were ineffective, arachidonoylethanolamide inhibited Ca(2+) effluxes and specific binding of [(3)H]Isradipine. Further studies indicated that only those fatty acids containing ethanolamine as a head group and having a chain length of more than 18 carbons were effective in inhibiting depolarization-induced Ca(2+) effluxes and specific binding of [(3)H]Isradipine. In conclusion, results indicate that depending on the chain length and the head group of fatty acid, N-acylethanolamines have differential effects on the function of voltage-dependent Ca(2+) channels and on the specific binding of [(3)H]Isradipine in skeletal muscle membranes.     

A new class of calcium entry blockers defined by 1,3-diphosphonates. Interactions of SR-7037 (belfosdil) with receptors for calcium channel ligands

Tetrabutyl-2(2-phenoxyethyl)-1,3-propylidene diphosphonate (SR-7037) completely displaced dihydropyridine [( 3H]PN200-110), phenylalkylamine [( 3H]D888), and benzothiazepine [( 3H]diltiazem) ligands from brain L-type calcium channels. Half-maximal inhibition of [3H]PN200-110 binding occurred at 19 nM with a Hill coefficient of 0.96. SR-7037 primarily decreased the affinity for [3H]PN200-110 with a small, but significantly, effect on the maximal binding capacity. Kinetic studies showed that this was due to an increased radioligand dissociation rate from 0.04 min-1 to 0.43 min-1 in the presence of the diphosphonate. Displacement of [3H]D888 by SR-7037 was biphasic with respective IC50 of 44 and 8400 nM. Likewise, unlabeled (-)-D888 identified two sites with IC50 values of 0.9 and 27 nM. Both SR-7037 (1000 nM) and D888 (200 nM) accelerated radioligand dissociation about 2-fold. [3H]Diltiazem binding was inhibited by SR-7037 with an IC50 value of 29 nM. The inhibition of dihydropyridine binding by SR-7037 is enhanced by most divalent cations at millimolar concentrations with the following potency: Mn2+ greater than Mg2+ greater than Ca2+ greater than Co2+. Barium has the opposite effect. The half-maximal effect of calcium occurred at 6 microM free ion. Specific binding of [3H]D888 was antagonized in the presence of 1 mM CaCl2. It is concluded that SR-7037 has allosteric interactions with the dihydropyridine receptor of the L-type calcium channel. The differential effect of Ca2+ on the potency of D888 and diltiazem relative to that of SR-7037 indicates that the three drugs may bind to nonequivalent sites. These results support specific calcium channel inhibition, possibly at a novel site, as the primary mechanism of the diphosphonate's pharmacological actions.     

Effects of isoflurane on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with alcohols

The effects of racemic (+/-) and (+)- and (-)-stereoisomers of isoflurane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. In the concentration range 0.5 to 2 mM, (+/-)-isoflurane inhibited (45)Ca(2+) fluxes and functionally modulated the effects of the Ca(2+) channel antagonist nifedipine (1-10 microM). Isoflurane-induced inhibition of (45)Ca(2+) fluxes was not significantly affected by pretreatment with either pertussis toxin (5 microg/ml) or phorbol 12-myristate 13-acetate (50 nM). Further experiments indicated that there were no significant differences between (+)- and (-)-stereoisomers of isoflurane with respect to the extent of inhibition of (45)Ca(2+) fluxes. Radioligand binding studies indicated that racemic and (+)- and (-)-isoflurane were equally effective in displacing the specific binding of [(3)H]PN 200-110 to transverse tubule membranes. There were no apparent differences between the effects of (+)- and (-)-isoflurane on the characteristics of [(3)H]PN 200-110 binding. Although the concentrations of isoflurane for the inhibitions of (45)Ca(2+) fluxes and radioligand bindings were similar, the concentrations of n-alcohols required for the inhibition of (45)Ca(2+) fluxes were lower than those for the displacement of radioligand. Comparison of the data for the displacement of [(3)H]PN 200-110 binding and the inhibition of (45)Ca(2+) fluxes by isoflurane and by n-alcohols suggested that both isoflurane and n-alcohols may have more than a single binding site. In conclusion, results indicate that isoflurane, independent of intracellular Ca(2+) levels, nonstereospecifically inhibits the function of voltage-dependent Ca(2+) channels and this effect is mediated through multiple binding sites.     

Procaine isothiocyanate: An irreversible inhibitor of the specific binding of [3H]batrachotoxinin-A benzoate to sodium channels

[³H]Batrachotoxinin-A benzoate ([³H]BTX-B) binds with high affinity to sites on voltage sensitive sodium channels in synaptoneurosomes from guinea pig cerebral cortex. Local anesthetics competitively antagonize the binding of [³H]BTX-B. An irreversible local anesthetic, procaine isothiocyanate (PRIT) and a tritiated derivative ([³H]PRIT) have been prepared. PRIT inhibits the binding of [³H]BTX-B in a noncompetitive, irreversible manner (apparent K_i=13 M) whereas the parent compound, procaine, inhibits in a competitive, reversible manner (K_i=40 M). The dissociation rate of [³H]BTX-B from sites on the sodium channel is greatly accelerated in a concentration dependent manner in the presence of PRIT. A 50% increase in the dissociation rate of [³H]BTX-B is achieved in the presence of 0.98 M PRIT. [³H]PRIT binds irreversibly to three proteins in synaptoneurosomes with apparent molecular weights of 20, 42, and 68 kDa. Protection studies with procaine and other local anesthetics suggest that only the 68 kDa species was related to local anesthetic binding.     

Influence of formamidines on batrachotoxin in a 20α-benzoate binding to neural membranes from pyrethroid susceptible and resistant tobacco budworm moths Heliothis virescens

1. Amitraz stimulated [³H]batrachotoxin in A 20-α-benzoate ([³H]BTX-B) binding to neural membranes from pyrethroid susceptible (S) and resistant (R) tobacco budworm moths, but N′-(2,4-xylyl)-N-methylformamidine (SN 49844) stimulated binding only with S moths.2. Chlordimeform stimulated [³H]BTX-B binding only with R moths, and N-(4-chloro-o-tolyl)-N-methylformamidine (demethylchlordimeform) yielded no significant stimulation with either strain.3. A mixture of amitraz and deltamethrin, a pyrethroid that previously had been shown to enhance [³H]BTX-B binding with tobacco budworm moths, also gave significant stimulation of radioligand binding with S moths.4. When membranes were prepared from S moths at various intervals following topical application of amitraz, deltamethrin, or a mixture of amitraz and deltamethrin, biphasic stimulation of [³H]BTX-B binding was observed, with maximum enhancement occurring at 2 and 6 hr.5. These results provided a basis for suggesting that a formamidine binding site is located on or closely associated with the sodium channel protein.6. Whether this site is the same as the pyrethroid/DDT binding domain remains to be demonstrated; however, similarities in responses elicited by amitraz and deltamethrin alone and in combination indicate that some relationship may exist.     

Interactions of δ-Conotoxins with Alkaloid Neurotoxins Reveal Differences Between the Silent and Effective Binding Sites on Voltage-Sensitive Sodium Channels

Abstract: The δ-conotoxin-TxVIA from Conus textile (δTxVIA) is a mollusk-specific conotoxin that slows sodium channel inactivation exclusively in mollusk neuronal membranes but reveals high-affinity binding to both mollusk (effective binding) and rat brain (silent binding) neuronal membranes, despite not having any toxic effect in vertebrates in vivo and in vitro. Using binding studies with radioactive δTxVIA we demonstrate that a different mollusk-specific conotoxin, δ-conotoxin-GmVIA from the venom of Conus gloriamaris , possesses "silent" and effective binding properties in rat brain and mollusk sodium channels, respectively. Binding studies and electrophysiological tests with both vertebrate muscle and insect neuronal preparations have indicated that the silent binding sites of δTxVIA are highly conserved in a wide range of distinct vertebrate and insect sodium channels. Direct probing of receptor site 2 by a tritiated derivative of batrachotoxin ([³H]BTX-B) revealed that [³H]BTX-B binding in mollusk sodium channels is of high affinity with no addition of enhancing ligands, unlike [³H]BTX-B binding in rat brain. In contrast to the negative allosteric modulation of δTxVIA binding by veratridine, δTxVIA is not able to affect the binding of [³H]BTX-B in mollusk neuronal membranes but reduces [³H]BTX-B binding in rat brain in the presence of α-scorpion toxins. The latter finding indicates the existence of a pharmacological distinction between the silent and effective binding sites of δTxVIA and points out possible functionally important structural differences between molluscan and rat brain sodium channels.     

Aporphine metho salts as neuronal nicotinic acetylcholine receptor blockers

(S)-Aporphine metho salts with the 1,2,9,10 oxygenation pattern displaced radioligands from recombinant human alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChR) at low micromolar concentrations. The affinity of the nonphenolic glaucine methiodide (4) (vs [(3)H]cytisine) was the lowest at alpha4beta2 nAChR (K(i)=10 microM), and predicentrine methiodide (2) and xanthoplanine iodide (3), with free hydroxyl groups at C-2 or C-9, respectively, had the highest affinity at these receptors (K(i) approximately 1 microM), while the affinity of the diphenolic boldine methiodide (1) was intermediate between these values. At homomeric alpha7 nAChR, xanthoplanine had the highest affinity (K(i)=10 microM) vs [(125)I]alpha-bungarotoxin while the other three compounds displaced the radioligand with K(i) values between 15 and 21 microM. At 100 microM, all four compounds inhibited the responses of these receptors to EC(50) concentrations of ACh. The effects of xanthoplanine iodide (3) were studied in more detail. Xanthoplanine fully inhibited the EC(50) ACh responses of both alpha7 and alpha4beta2 nACh receptors with estimated IC(50) values of 9+/-3 microM (alpha7) and 5+/-0.8 microM (alpha4beta2).     

Specific inhibition of [3H] saxitoxin binding to skeletal muscle sodium channels by geographutoxin II, a polypeptide channel blocker

Geographutoxin II (GTX II), a peptide toxin isolated from Conus geographus, inhibited [3H]saxitoxin binding to receptor sites associated with voltage-sensitive Na channels in rat skeletal muscle homogenates and rabbit T-tubular membranes with K0.5 values of 60 nM for homogenates and 35 nM for T-tubular membranes in close agreement with concentrations that block muscle contraction. Scatchard analysis of [3H]saxitoxin binding to T-tubular membranes gave values of KD = 9.3 nM and Bmax = 300 fmol/mg of protein and revealed a primarily competitive mode of inhibition of saxitoxin binding by GTX II. The calculated KD values for GTX II were 24 nM for T-tubules and 35 nM for homogenates, respectively. In rat brain synaptosomes, GTX II caused a similar inhibitory effect on [3H]saxitoxin binding at substantially higher concentrations (K0.5 = 2 microM). In contrast, binding of [3H]batrachotoxin A 20-alpha-benzoate and 125I-labeled scorpion toxin to receptor sites associated with Na channels in synaptosomes was not affected by GTX II at concentrations up to 10 microM. Furthermore, [3H]saxitoxin binding to membranes of rat superior cervical ganglion was only blocked 10% by GTX II at 10 microM. These results indicate that GTX II interacts competitively with saxitoxin in binding at neurotoxin receptor site 1 on the sodium channel in a highly tissue-specific manner. GTX II is the first polypeptide ligand for this receptor site and the first to discriminate between this site on nerve and adult muscle sodium channels.     

WIN 17317-3, a new high-affinity probe for voltage-gated sodium channels.

The iminodihydroquinoline WIN 17317-3 was previously shown to inhibit selectively the voltage-gated potassium channels, K(v)1.3 and K(v)1.4 [Hill, R. J., et al. (1995) Mol. Pharmacol. 48, 98-104; Nguyen, A., et al. (1996) Mol. Pharmacol. 50, 1672-1679]. Since these channels are found in brain, radiolabeled WIN 17317-3 was synthesized to probe neuronal K(v)1 channels. In rat brain synaptic membranes, [(3)H]WIN 17317-3 binds reversibly and saturably to a single class of high-affinity sites (K(d) 2.2 +/- 0.3 nM; B(max) 5.4 +/- 0.2 pmol/mg of protein). However, the interaction of [(3)H]WIN 17317-3 with brain membranes is not sensitive to any of several well-characterized potassium channel ligands. Rather, binding is modulated by numerous structurally unrelated sodium channel effectors (e.g., channel toxins, local anesthetics, antiarrhythmics, and cardiotonics). The potency and rank order of effectiveness of these agents in affecting [(3)H]WIN 17317-3 binding is consistent with their known abilities to modify sodium channel activity. Autoradiograms of rat brain sections indicate that the distribution of [(3)H]WIN 17317-3 binding sites is in excellent agreement with that of sodium channels. Furthermore, WIN 17317-3 inhibits sodium currents in CHO cells stably transfected with the rat brain IIA sodium channel with high affinity (K(i) 9 nM), as well as agonist-stimulated (22)Na uptake in this cell line. WIN 17317-3 interacts similarly with skeletal muscle sodium channels but is a weaker inhibitor of the cardiac sodium channel. Together, these results demonstrate that WIN 17317-3 is a new, high-affinity, subtype-selective ligand for sodium channels and is a potent blocker of brain IIA sodium channels.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Effects of the sulfhydryl reagentN-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes

1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of [3H]batrachotoxinin A-20-α-benzoate and [3h]saxitoxin to receptor sites associated with sodium channels in trout brain synaptoneurosomes

1. [³H]Batrachotoxinin A-20-α-benzoate ([³H]BTX-b) and [³H]saxitoxin ([³H]STX), radioligands that bind to distinct sites on the voltage-sensitive sodium channel, were bound specifically to saturable sites in rainbow trout (Oncorhynchus mykiss) brain synaptoneurosomes.2. Specific [³H]BTX-B binding was temperature dependent with highest levels of specific [³H]BTX-B binding observed at 7°C. Specific binding was inversely correlated with assay temperature at temperatures above 7°C.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom (ScV) stimulated specific [³H]BTX-B binding at 27°C, but not at 7°C. The dihydropyrazole insecticide RH 3421 inhibited specific [³H]BTX-B binding at 7°C but had no effect on specific binding at 27°C. The sodium channel activators veratridine and aconitine and the local anesthetic dibucaine inhibited specific [³H]BTX-B binding at both 7°C and 27°C.4. Displacement experiments in the presence of ScV at 27°C gave an equilibrium dissociation constant (K_d) for [³H]BTX-B of 710 nM and a maximal binding capacity (B_max) of 11.3 pmol/mg protein. Kinetic experiments established the rates of association (1.17 × 10⁵min⁻¹ nM⁻¹) and dissociation (0.0514min⁻¹) of the ligand-receptor complex.5. The binding of [³H]STX reached apparent saturation at 7.5 nM. Scatchard analysis of the saturation data indicated a K_d of 3.8nM and a B_max of 1.9 pmol/mg protein.6. These studies provide evidence for high affinity, saturable binding sites for [³H]BTX-B and [³H]STX in trout brain preparations. Whereas certain neurotoxins modified the specific binding of [³H]BTX-B in trout brain synaptoneurosomes in a predictable fashion, other compounds known to affect specific [³H]BTX-B binding in mammalian brain preparations had no effect on specific [³H]BTX-B binding in the trout.