Functional expression of NaV1.7 channels in freshly dispersed mouse bronchial smooth muscle cells.

Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at ∼21°C. Stepping from -100 mV to -20 mV evoked inward currents of mean amplitude -275 pA. These inactivated (tau = 1.1 ms) and were abolished when external Na+ was substituted with N-Methyl-d-glucamine. In current-voltage protocols, current peaked at -10 mV and reversed between +20 and +30 mV. The V1/2s of activation and inactivation were -25 and -86 mV, respectively. The current was highly sensitive to tetrodotoxin (IC50 = 1.5 nM) and the NaV1.7 subtype-selective blocker, PF-05089771 (IC50 = 8.6 nM), consistent with NaV1.7 as the underlying pore-forming α subunit. Two NaV1.7-selective antibodies caused membrane-delineated staining of isolated SMC, as did a nonselective pan-NaV antibody. RT-PCR, performed on groups of ∼15 isolated SMCs, revealed transcripts for NaV1.7 in 7/8 samples. Veratridine (30 µM), a nonselective NaV channel activator, reduced peak current evoked by depolarization but induced a sustained current of 40 pA. Both effects were reversed by tetrodotoxin (100 nM). In tension experiments, veratridine (10 µM) induced contractions that were entirely blocked by atropine (1 µM). However, in the presence of atropine, veratridine was able to modulate the pattern of activity induced by a combination of U-46619 (a thromboxane A2 mimetic) and PGE2 (prostaglandin E2), by eliminating bursts in favor of sustained phasic contractions. These effects were readily reversed to control-like activity by tetrodotoxin (100 nM). In conclusion, mouse bronchial SMCs functionally express NaV1.7 channels that are capable of modulating contractile activity, at least under experimental conditions.

Fast voltage-dependent sodium (NaV ) currents are functionally expressed in mouse corpus cavernosum smooth muscle cells.

BACKGROUND AND PURPOSE: Corpus cavernosum smooth muscle (CCSM) exhibits phasic contractions that are coordinated by ion channels. Mouse models are commonly used to study erectile dysfunction, but there are few published electrophysiological studies of mouse CCSM. We describe the voltage-dependent sodium (NaV ) currents in mouse CCSM and investigate their function. EXPERIMENTAL APPROACH: We used electrophysiological, pharmacological and immunocytochemical methods to study the NaV currents in isolated CCSM cells from C57BL/6 mice. Tension measurements were carried out using crural sections of the corpus cavernosum in whole tissue. KEY RESULTS: Fast, voltage-dependent, sodium currents in mouse CCSM were induced by depolarising steps. Steady-state activation and inactivation curves revealed a window current between -60 and -30 mV. Two populations of NaV currents, 'TTX-sensitive' and 'TTX-insensitive', were identified. TTX-sensitive currents showed 48% block with the NaV channel subtype-specific blockers ICA-121431 (NaV 1.1-1.3), PF-05089771 (NaV 1.7) and 4,9-anhydro-TTX (NaV 1.6). TTX-insensitive currents were resistant to blockade by A803467, specific for NaV 1.8 channels. Immunocytochemistry confirmed expression of NaV 1.5 and NaV 1.4 in freshly dispersed CCSM cells. Veratridine, a NaV channel activator, reduced time-dependent inactivation of NaV currents and increased duration of evoked action potentials. Veratridine induced phasic contractions in CCSM strips, reversible with TTX and nifedipine but not KB-R7943. CONCLUSION AND IMPLICATIONS: There are fast, voltage-dependent, sodium currents in mouse CCSM. Stimulation of these currents increased contractility of CCSM in vitro, suggesting an involvement in detumescence and potentially providing a clinically relevant target in erectile dysfunction. Further work will be necessary to define its role.

Veratridine: A Janus-Faced Modulator of Voltage-Gated Sodium Ion Channels.

Voltage-gated sodium ion channels (NaVs) are integral to both neuronal and muscular signaling and are a primary target for a number of proteinaceous and small molecule toxins. Included among these neurotoxins is veratridine (VTD), a C-nor-D homosteroidal alkaloid from the seeds of members of the Veratrum genus. VTD binds to NaV within the pore region, causing a hyperpolarizing shift in the activation threshold in addition to reducing peak current. We have characterized the activity of VTD against heterologously expressed rat NaV1.4 and have demonstrated that VTD acts on the channel as either an agonist or antagonist depending on the nature of the electrophysiological stimulation protocol. Structure-activity studies with VTD and VTD derivatives against NaV mutants show that the functional duality of VTD can be decoupled. These findings suggest that the dichotomous activity of VTD may derive from two distinct, use-dependent binding orientations of the toxin.

Development of a high-throughput fluorescent no-wash sodium influx assay.

Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495-575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1-1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1-1.7 activator veratridine (EC50 10-29 µM) or the NaV1.1-1.8 inhibitor tetracaine (IC50's 6-66 µM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.

Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR.

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Nav1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Nav1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Nav1.4. Here, we determine the NMR structure of a selectively 13C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By 13C NMR, we obtain NMR structural constraints as 13C chemical shifts and the 1H-2H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the 1H-2H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel NavRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Nav1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.

Xanthurenic Acid Formation from 3-Hydroxykynurenine in the Mammalian Brain: Neurochemical Characterization and Physiological Effects.

Xanthurenic acid (XA), formed from 3-hydroxykynurenine (3-HK) in the kynurenine pathway of tryptophan degradation, may modulate glutamatergic neurotransmission by inhibiting the vesicular glutamate transporter and/or activating Group II metabotropic glutamate receptors. Here we examined the molecular and cellular mechanisms by which 3-HK controls the neosynthesis of XA in rat, mouse and human brain, and compared the physiological actions of 3-HK and XA in the rat brain. In tissue homogenates, XA formation from 3-HK was observed in all three species and traced to a major role of kynurenine aminotransferase II (KAT II). Transamination of 3-HK to XA was also demonstrated using human recombinant KAT II. Neosynthesis of XA was significantly increased in the quinolinate-lesioned rat striatum, indicating a non-neuronal localization of the process. Studies using rat cortical slices revealed that newly produced XA is rapidly released into the extracellular compartment, and that XA biosynthesis can be manipulated experimentally in the same way as the production of kynurenic acid from kynurenine (omission of Na+ or glucose, depolarizing conditions, or addition of 2-oxoacids). The synthesis of XA from 3-HK was confirmed in vivo by striatal microdialysis. In slices from the rat hippocampus, both 3-HK and XA reduced the slopes of dentate gyrus field EPSPs. The effect of 3-HK was reduced in the presence of the KAT inhibitor aminooxyacetic acid. Finally, both 3-HK and XA reduced the power of gamma-oscillatory activity recorded from the hippocampal CA3 region. Endogenous XA, newly formed from 3-HK, may therefore play a physiological role in attentional and cognitive processes.

Isolation of 6-deoxytetrodotoxin from the pufferfish, Takifugu pardalis, and a comparison of the effects of the C-6 and C-11 hydroxy groups of tetrodotoxin on its activity.

Identification of new tetrodotoxin (TTX, 1) analogues would be significant in the elucidation of its biosynthetic pathway and a study of its structure-activity relationships. In this study, a new TTX analogue, 6-deoxyTTX (2), was isolated from the ovary of the pufferfish, Takifugu pardalis, and the structure was determined using spectroscopic methods. Compound 2 was also identified in other marine animals, Nassarius snail and blue-ringed octopuses, using LC-MS. Furthermore, we investigated the voltage-gated sodium channel blocking activity of 2 by examination of the inhibitory activities to cytotoxicity induced by ouabain and veratridine in mouse neuroblastoma cells (Neuro-2a). The activities were then compared with those of 1, 11-deoxyTTX (3), and 6,11-dideoxyTTX (4). The EC50 value for 2 was estimated to be 6.5±2.2 nM, approximately 3-fold larger than that of 1 (2.1±0.6 nM) and approximately 20-fold smaller than that of 3. These results suggested that contribution of the C-6 hydroxy group to the activity is less than that of the C-11 hydroxy group.

Selectivity problems with drugs acting on cardiac Na⁺ and Ca²âº channels.

With the increase of our knowledge on cardioactive agents it comes more and more clear that practically none of the currently used compounds shows absolute selectivity to one or another ion channel type. This is particularly true for Na(+) and Ca(2+) channel modulators, which are widely applied in the clinical practice and biomedical research. The best example might be probably the marine guanidine poison tetrodotoxin, which has long been considered as a selective Na(+) channel blocker, while recently it turned out to effectively inhibit cardiac Ca(2+) currents as well. In the present study the cross actions observed between the effects of various blockers of Na(+) channels (such as toxin inhibitors, class I antiarrhythmics and local anesthetics) and Ca(2+) channels (like phenylalkylamines, dihydropyridine compounds, diltiazem and mibefradil) are overviewed in light of the known details of the respective channel structures. Similarly, activators of Na(+) channels, including veratridine and batrachotoxin, are also compared. The binding of tetrodotoxin and saxitoxin to Cav1.2 and Nav1.5 channel proteins is presented by construction of theoretical models to reveal common structures in their pore forming regions to explain cross reactions. Since these four domain channels can be traced back to a common ancestor, a close similarity in their structure can well be demonstrated. Thus, the poor selectivity of agents acting on cardiac Na(+) and Ca(2+) channels is a consequence of evolution. As a conclusion, since the limited selectivity is an intrinsic property of drug receptors, it has to be taken into account when designing new cardioactive compounds for either medical therapy or experimental research in the future.

Solution NMR analysis of the binding mechanism of DIVS6 model peptides of voltage-gated sodium channels and the lipid soluble alkaloid veratridine.

Voltage-gated sodium channels (VGSCs) are responsible for generating action potentials in nervous systems. Veratridine (VTD), a lipid soluble alkaloid isolated from sabadilla lily seed, is believed to bind to segment 6 of VGSCs and act as a partial agonist. However, high resolution structural interaction mechanism between VGSCs and VTD is difficult to elucidate because of the large size and membrane localization of VGSCs. Here, the authors designed model peptides corresponding to domain IV segment 6 (DIVS6) of rat skeletal muscle Na(v)1.4 and analyzed the complex of the model peptides and VTD by solution NMR analysis to obtain structural information of the interaction. The model peptides successfully formed an α-helices, which is the suspected native conformation of DIVS6, in aqueous 2,2,2-trifluoroethanol, a membrane-mimicking solvent. The VTD binding residues of the model peptide were identified using the NMR titration experiments with VTD, including a newly discovered VTD binding residue Leu14 (µ1-L1580 in Na(v)1.4), which has not been reported by point mutation studies. Mapping of VTD binding residues on the model peptide revealed the hydrophobic interaction surface. NMR titration experiments with a non-toxic analog of VTD, veracevine, also indicated that the steroidal backbone of VTD interacts with the hydrophobic interaction surface of DIVS6 and that the 3-acyl group of VTD possibly causes neurotoxicity by interacting with domain I segment 6 and/or domain IV segment 4.

Investigating the in vitro metabolism of veratridine: characterization of metabolites and involved cytochrome P450 isoforms.

Veratridine is a lipid-soluble alkaloid extracted from Veratrum officinale and other species of the family Liliaceae. Veratridine prevents inactivation of Na(+) channel via binding the receptor site 2, causes influx of sodium ion and depolarization and induces apoptosis of neuronal cells. In the present study, we investigated the metabolism of veratridine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of veratridine in rat liver microsomes. The metabolites were separated and assayed by liquid-chromatography-electrospray ionization-ion trap tandem mass spectrometry (LC-ESI-QIT-MSn), and further identified by their mass spectra and chromatographic behaviors. Result showed that four CYP isoforms (CYP1A, CYP2B, CYP2E1, CYP3A) were involved in the metabolism of veratridine in vitro and seven metabolites of veratridine were detected incubating with rat liver microsomes. Some of the metabolites were presumed to be potential mediates of neurotoxicity via protein binging. Further research in vivo needs to link the metabolism of veratridine to its toxicity.

Sodium channel activators: model of binding inside the pore and a possible mechanism of action.

Sodium channel activators, batrachotoxin and veratridine, cause sodium channels to activate easier and stay open longer than normal channels. Traditionally, this was explained by an allosteric mechanism. However, increasing evidence suggests that activators can bind inside the pore. Here, we model the open sodium channel with activators and propose a novel mechanism of their action. The activator-bound channel retains a hydrophilic pathway for ions between the ligand and conserved asparagine in segment S6 of repeat II. One end of the activator approaches the selectivity filter, decreasing the channel conductance and selectivity. The opposite end reaches the gate stabilizing it in the open state.

Use of two detection methods to discriminate ciguatoxins from brevetoxins: application to great barracuda from Florida Keys.

In Florida (USA), numerous cases of human ciguatera fish poisoning, as well as neurotoxic shellfish poisoning following consumption of local seafood products, have been reported. By using in parallel, the sodium channel receptor binding assay (RBA), and the ouabain/veratridine-dependent cytotoxicity assay (N2A assay), we established criteria to identify, detect, and quantify ciguatoxins in fish extracts, with a brevetoxin as internal standard. Results showed that the Caribbean ciguatoxin C-CTX-1 exhibited an 8-fold higher potency in the RBA than brevetoxins and, a 440 and 2300-fold higher potency in the N2A assay than PbTx-1 and PbTx-3, respectively. Moreover, a sensitivity comparison between assays revealed that the N2A assay was more sensitive (12-fold) for ciguatoxin analysis, whereas the RBA was more sensitive (3-24-fold) for brevetoxins analysis. Based on the relative potency between toxins and the opposite sensitivity of both assays we have used the RBA and the N2A assay to screen great barracuda (Sphyraena barracuda) collected from the Florida Keys for ciguatoxins and brevetoxins. Fish extract analysis showed a sodium channel-dependent activity consistent with the presence of ciguatoxins, and not brevetoxins. Among 40 barracudas analyzed, 60% contained ciguatoxin levels in their liver measurable by the N2A assay with the most toxic fish containing 2.1ppb C-CTX-1 equivalents.

Veratridine modifies the TTX-resistant Na+ channels in rat vagal afferent neurons.

A number of neurotoxins from venoms of invertebrates and plants are ligands for voltage-gated Na+ channels and are useful tools for studying Na+ channel function and structure. Using whole-cell recordings from vagal afferent nodose neurons, we studied neurotoxins that target Na+ channels. We asked whether Ts3 (an alpha-scorpion toxin) and/or veratridine (a lipid-soluble toxin), could modify the TTX-resistant Na+ current generated by vagal afferent nodose neurons. Nodose TTX-resistant current was not affected by Ts3, whereas Ts3 slowed inactivation of the current generated by TTX-sensitive current component. We found that veratridine inhibited the TTX-resistant Na+ currents on rat nodose neurons. Interestingly, veratridine-modified Na+ channels developed a persistent current that accounted for the large tail current observed. We propose that veratridine modifies TTX-resistant Na+ channels through a mechanism distinct from its actions on other voltage-gated Na+ channels.

delta-Atracotoxins from australian funnel-web spiders compete with scorpion alpha-toxin binding but differentially modulate alkaloid toxin activation of voltage-gated sodium channels.

delta-Atracotoxins from the venom of Australian funnel-web spiders are a unique group of peptide toxins that slow sodium current inactivation in a manner similar to scorpion alpha-toxins. To analyze their interaction with known sodium channel neurotoxin receptor sites, we studied their effect on [3H]batrachotoxin and 125I-Lqh II (where Lqh is alpha-toxin II from the venom of the scorpion Leiurus quinquestriatus hebraeus) binding and on alkaloid toxin-stimulated 22Na+ uptake in rat brain synaptosomes. delta-Atracotoxins significantly increased [3H]batrachotoxin binding yet decreased maximal batrachotoxin-activated 22Na+ uptake by 70-80%, the latter in marked contrast to the effect of scorpion alpha-toxins. Unlike the inhibition of batrachotoxin-activated 22Na+ uptake, delta-atracotoxins increased veratridine-stimulated 22Na+ uptake by converting veratridine from a partial to a full agonist, analogous to scorpion alpha-toxins. Hence, delta-atracotoxins are able to differentiate between the open state of the sodium channel stabilized by batrachotoxin and veratridine and suggest a distinct sub-conductance state stabilized by delta-atracotoxins. Despite these actions, low concentrations of delta-atracotoxins completely inhibited the binding of the scorpion alpha-toxin, 125I-Lqh II, indicating that they bind to similar, or partially overlapping, receptor sites. The apparent uncoupling between the increase in binding but inhibition of the effect of batrachotoxin induced by delta-atracotoxins suggests that the binding and action of certain alkaloid toxins may represent at least two distinguishable steps. These results further contribute to the understanding of the complex dynamic interactions between neurotoxin receptor site areas related to sodium channel gating.

Effects of veratridine on sodium currents and fluxes.

Veratridine causes Na+ channels to stay open during a sustained membrane depolarization by abolishing inactivation. The consequential Na+ influx, either by itself or by causing a maintained depolarization, leads to many secondary effects such as increasing pump activity, Ca2+ influx, and in turn exocytosis. If the membrane is voltage clamped in the presence of the alkaloid, a lasting depolarizing impulse induces, following the "normal" transient current, another much more slowly developing Na+ current that reaches a constant level after a few seconds. Repolarization then is followed by an inward tail current that slowly subsides. Development of these slow currents is enhanced by additional treatment with agents that inhibit inactivation. Most of these phenomena can be satisfactorily explained by assuming that Na+ channels must open before veratridine binds to them, and that the slow current changes reflect the kinetics of binding and unbinding. It is unclear, however, where the alkaloid stays when it is not bound. Although the effect sets in promptly, once this pool is filled, access to it from outside must be impeded since in most preparations veratridine can only partially be washed out. Cooling acts as if the available concentration is reduced, but this reversible "reduction" takes much longer to develop than the cold-induced changes in kinetics. Several authors assume that the binding site, site 2, is accessed from the lipid phase of the membrane. Considerations of this kind are often based on experiments with batrachotoxin, the widely used site-2 ligand which has a much higher affinity and acts as a full agonist in contrast to the partial agonist veratridine. Batrachotoxin thus lends itself to binding studies using radiolabeled derivatives. Such experiments may eventually lead to the characterization of neurotoxin site 2; the first promising steps have been taken. Modern techniques of molecular biology will almost certainly be successful, and one hopes for point-mutated channels with distinctly different reactions also to veratridine. A considerable amount of research is still required to clarify the structural basis for the numerous allosteric interactions with other sites, the mechanism of the very large potential shift of activation, the reduced single-channel conductance and selectivity, and the chemical nature of the different affinities of the site-2 toxins. Note Added in Proof. A report on point mutations with effects on neurotoxin site 2 (see Sect. 8) has just appeared: Wang S-Y, Wang GK (1988) Point mutations in segment I-S6 render voltage-gated Na+ channels resistant to batrachotoxin. Proc Natl Acad USA 95:2653-2658. In microliter muscle Na+ channels expressed in mammalian cells, mutation Asn434Lys leads to complete, Asn434Ala to partial insensitivity to 5 mM batrachotoxin. (Asn434 corresponds to Asn419 of Trainer et al. 1996). The mutant channel displays almost normal current kinetics and in the presence of veratridine little, if any, slow tail current. However, veratridine inhibits peak Na+ currents in the mutant which may point to a complex structure of site 2.

Competition for binding between veratridine and KIFMK: an open channel blocking peptide of the RIIA sodium channel.

Veratridine, an alkaloid isolated from the rhizome of V. album, binds and slows the inactivation of the brain sodium channels. The synthetic pentapeptide KIFMK causes a voltage- and use-dependent open-channel block of the RIIA (rat brain type IIA) sodium channel (Eaholtz, Scheuer & Catterall, 1994). Our studies on the RIIA sodium channel expressed in CHO cells reveal that the fraction of veratridine modified sodium channels decreases linearly with increasing KIFMK concentration. However, the time constant for dissociation of veratridine from the channel remains unchanged in the presence of a high concentration of KIFMK, as opposed to that in the presence of QX314 where the dissociation appears to be more complex. These data are consistent with mutually exclusive binding of the open channel blocking peptide and veratridine to the brain sodium channel.

Ibogaine selectively inhibits nicotinic receptor-mediated catecholamine release.

The effects of ibogaine, a putative anti-addictive drug, on stimulated catecholamine release were examined in cultured chromaffin cells to clarify its mechanism(s) of action. Low concentrations of ibogaine (1-10 microM) had a selective inhibitory action on nicotinic receptor-mediated catecholamine release, while higher concentrations (100 microM) inhibited additional modes of stimulated catecholamine release. These results suggest a selective inhibitory action of ibogaine at the nicotinic acetylcholine receptor, possibly at the receptor ion channel site.

Characterization of [3H]brevetoxin binding to voltage-dependent sodium channels in adrenal medullary cells.

We have previously reported that in bovine adrenal chromaffin cells Ptychodiscus brevis toxin-3 (PbTx-3) does not alter the veratridine-induced 22Na influx when given alone, but increases the influx of 22Na when co-applied with either alpha- or beta-scorpion venom (Wada et al. 1992). In the present study, we characterized [3H]PbTx-3 binding in bovine adrenal chromaffin cells. [3H]PbTx-3 binding was saturable, reversible and of high-affinity with an equilibrium dissociation constant (Kd) of 32.0 +/- 4.9 nmol/l and a maximum binding capacity (Bmax) of 6.2 +/- 1.2 pmol/4 x 10(6) cells (4.5 +/- 0.9 pmol/mg cell protein). A Hill plot revealed the lack of cooperative interaction among the binding sites. Unlabelled PbTx-3 inhibited [3H]PbTx-3 binding with an IC50 of 31 nmol/l. However, tetrodotoxin, veratridine, alpha- and beta-scorpion venom, or veratridine in combination with either alpha- or beta-scorpion venom did not alter [3H]PbTx-3 binding. All these results suggest that PbTx-3 binds to a site (site 5) distinct from the previously known four toxin binding sites, which does not gate voltage-dependent Na channels by itself, but is specifically involved in the allosteric modulation of Na channels in adrenal medullary cells.

Relation between veratridine reaction dynamics and macroscopic Na current in single cardiac cells.

Veratridine modification of Na current was examined in single dissociated ventricular myocytes from late-fetal rats. Extracellularly applied veratridine reduced peak Na current and induced a noninactivating current during the depolarizing pulse and an inward tail current that decayed exponentially (tau = 226 ms) after repolarization. The effect was quantitated as tail current amplitude, Itail (measured 10 ms after repolarization), relative to the maximum amplitude induced by a combination of 100 microM veratridine and 1 microM BDF 9145 (which removes inactivation) in the same cell. Saturation curves for Itail were predicted on the assumption of reversible veratridine binding to open Na channels during the pulse with reaction rate constants determined previously in the same type of cell at single Na channels comodified with BDF 9145. Experimental relationships between veratridine concentration and Itail confirmed those predicted by showing (a) half-maximum effect near 60 microM veratridine and no saturation up to 300 microM in cells with normally inactivating Na channels, and (b) half-maximum effect near 3.5 microM and saturation at 30 microM in cells treated with BDF 9145. Due to its known suppressive effect on single channel conductance, veratridine induced a progressive, but partial reduction of noninactivating Na current during the 50-ms depolarizations in the presence of BDF 9145, the kinetics of which were consistent with veratridine association kinetics in showing a decrease in time constant from 57 to 22 and 11 ms, when veratridine concentration was raised from 3 to 10 and 30 microM, respectively. As predicted for a dissociation process, the tail current time constant was insensitive to veratridine concentration in the range from 1 to 300 microM. In conclusion, we have shown that macroscopic Na current of a veratridine-treated cardiomyocyte can be quantitatively predicted on the assumption of a direct relationship between veratridine binding dynamics and Na current and as such can be successfully used to analyze molecular properties of the veratridine receptor site at the cardiac Na channel.

Inhibitory effect of okadaic acid on carbachol-evoked secretion of catecholamines in cultured bovine adrenal medullary cells.

We examined the effect of okadaic acid on catecholamine secretion caused by carbachol in cultured bovine adrenal medullary cells. Treatment of cells with 100 nM okadaic acid for 3-24 hr produced an inhibition of catecholamine secretion stimulated by carbachol. The half-maximal and maximal inhibition of secretion was observed at 40 nM and 300 nM okadaic acid for 24 hr, respectively. Okadaic acid also inhibited veratridine- and high K(+)-induced secretion but not ionomycin-induced secretion. Okadaic acid strongly suppressed 45Ca2+ influx and slightly inhibited 22Na+ influx in carbachol-stimulated cells. These results suggest that okadaic acid inhibits carbachol-evoked secretion of catecholamines mainly by suppression of Ca2+ influx in adrenal medullary cells.