Inhibition by tetrodotoxin

In vivo microdialysis is a technique that allows sampling of extracellular levels of neurotransmitters in discrete regions of the brain. The extracellular neurotransmitter levels provide an indication of the net activity of a particular set of neurons, including release, synthesis, and uptake, from conscious unanesthetized animals. For example, in vivo microdialysis studies have shown that extracellular 5-HT levels measured under appropriate conditions are dependent on the concentration of Ca2+ or K+ in the perfusion fluid, is inhibited by tetrodotoxin, and is predominately neuronal in origin (45). In addition, specific neural processes can be measured after the local application of agents through the microdialysis probe, such as release after application of hypertonic KC1, rate of synthesis after synthesis inhibitors, or the local effects of drugs (46). This technique has made it possible to more accurately quantitate and characterize the... 

Na and Ca Influx. PTX caused a concentration-dependent increase in Na and Ca influxes into PC12 cells at concentrations of 10" to 10" M and 10" to 10" M, respectively. The PTX-induced Ca influx was markedly inhibited by Co but not by verapamil or nifedepine, whereas the PTX-induc Na influx was not affected by tetrodotoxin. 

Xie, X. M., Dale, T. J., Trezise, D. J. Voltage-dependent inhibition of tetrodotoxin-resistant Na+ currents in rat sensory neurons by lamotrigine. Soc. Neurosci. Abstr. 1996, 22, 33.3. 

Thaliporphine (0.1-100 pM), a potent vasoconstrictor, was observed to produce contractions in the isolated guinea-pig ileum in a concentration-dependent manner. These contractions were not affected by pretreatment of the ileum with tetrodotoxin, phentolamine, prazosin, propranolol, naloxone, atropine, diphenhydramine, methysergide, indomethacin or staurosporine. However, the contraction was markedly inhibited by nifedipine and verapamil, suggesting that thaliporphine produces intestinal smooth muscle contraction by a direct effect on muscle mediated by an increased Ca+2 influx through voltage-dependent Ca+2 channels [327]. 

Finally, 2-AG has been found to induce contractions in the longitudinal smooth muscle from the guinea-pig distal colon in vitro in a tetrodotoxin-sensitive manner. This response was mimicked by anandamide, but not by the cannabinoid receptor agonist WIN 55,212-2 or the vanilloid receptor agonist AM404 and was not inhibited by antagonists of cannabinoid or vanilloid receptors (Kojima et al. 

Figure 6.3 Reduction by tetrodotoxin (TTX, lOjlmoFl) and (+)-kavain (400Hmol/l) of ouabain-induced (200(lmoFl) Na influx into rat cerebrocortical synaptosomes. From top to bottom Control, (+)-kavain-treated (lower thick-bne trace), and TTX-treated (lower thin-line trace). Inset Increase in [Na /min of control (C), TTX and (+)-kavain (K). Traces represent mean + S.E., = 6, P < 0.001. Reprinted from Gleitz etal. (1995) (+)-Kavain inhibits the verattidine-activated voltage-dependent Na chatmels in synaptosomes prepared from rat cerebral cortex. Neuropharmacology, 34, 1133—1138, 1995, with permission from Elsevier Science.

Figure 4. The inhibition of veratridine-dependent transmitter release by tetrodotoxin. Reproduced with permission Ref. 2. Copyright 1985, Society of Chemical Industry.

Several studies employing oocytes of the clawed frog, Xenopus laevis, for the in vitro translation of sodium channel encoding mRNAs (53-55) suggest that this experimental system may be particularly useful toward this end. The biophysical properties of sodium channels expressed in oocytes following injection of rat brain mRNA were similar to those of sodium channels in their native membrane environment, and were specifically inhibited by the sodium channel blockers tetrodotoxin and saxitoxin (i5.). Sodium channels encoded by mRNAs from rat skeletal muscle and eel electroplax have also been expressed in Xenopus oocytes (56-57). To date the expression of insect sodium channels in the Xenopus oocyte has not been reported, but the utility of this system for the translation and expression of insect acetylcholine receptor mRNA has recently been demonstrated (58). Successful application of this methodology to the expression of insect mRNAs encoding functional sodium channels offers a novel method to test some of the hypotheses for the molecular basis of the kdr mechanism. 

Phenytoin also affects calcium uptake in synaptosomes. Similar to Mn, it inhibits the action of both veratridine and K, but like tetrodotoxin it is much more effective against veratridine (Figure 2). Stimulated Ca uptake produced at all concentrations of K between 15 and 64 mM is inhibited 20% by 0.1 mM phenytoin, and this is unaffected by tetrodotoxin. Examination of the dose-related effect of phenytoin on stimulated Ca uptake produced by 23 and 64 mM K reveals identical proportional inhibition of both. Thus, inhibition of K-induced Ca uptake by phenytoin is independent of K concentration. In contrast, there appears to be a competitive interaction between veratridine and phenytoin. Phenytoin has a much greater inhibitory effect, proportionally, on Ca uptake stimulated by low concentrations of veratridine than that produced by higher concentrations. For example, 35 yM phenytoin caused 50% inhibition of Ca uptake produced by 5 yM veratridine, but 225 yM phenytoin is required for 50% inhibition of the Ca uptake produced by 100 yM veratridine. 

Thus, phenytoin appears to inhibit K-induced and veratridine-induced calcium uptake by different mechanisms. Present data demonstrate that phenytoin has an action similar, but not identical, to that of tetrodotoxin, as well as an action like, but also not identical, to that of Mn. Other investigators have reported that phenytoin blocks Na channels in lobster nerve and squid giant axon (11-13) and this is not a Ca-dependent process. The present data demonstrate that phenytoin is able to block K-stimulated Ca uptake, and this is unaffected by tetrodotoxin, indicating that this effect is independent of Na. We think that all of the data together suggest that phenytoin can inhibit both Na and Ca conductances in nervous tissue and that these are separate and independent processes. The present results also indicate that Na conductance may be more sensitive to the inhibitory action of phenytoin, but more research will be necessary to prove this contention. 

The cause of min.e.p.p. is the opening of the same channels as in the case of e.p.p. This has been checked in experiments with tetrodotoxin, a poison which selectively blocks only the sodium channels and makes the spike impossible both in the nerve and muscle fibres. After tetrodotoxin s inhibition of the sodium spike, the min.e.p.p. induced by the electrotonic depolarization of the nerve endings are not changed (Fig. 3A). The local depolarization induced by microapplication of ACh also is not influenced by tetrodotoxin. 

Batrachotoxin is extremely potent in antagonizing axonal transport 198). It would appear that the basis of the effect of batrachotoxin on axonal transport is dependent on interactions with sodium channels and the resultant influx of sodium ions. The effect is blocked by tetrodotoxin. In the mollusc, Aplysia californica, it has been proposed that the inhibition of axonal transport by batrachotoxin is not due to interactions with sodium channels 169), but this interpretation has been questioned 105). Blockade of axonal transport by batrachotoxin reduced uptake and transport of nerve growth factor at distal terminals 41), increased activity of certain muscle lysosomal enzymes 40), and altered uptake of calcium in muscle sarcoplasmic reticulum 264, 265). Batrachotoxin inhibits saltatory movements in neuroblastoma cells 105). This inhibitory effect is blocked by tetrodotoxin. 

Batrachotoxin is an extremely potent cardiotonic agent 129,132,133, 156,183,233). Its actions lead ultimately to arrhythmias and cardiac arrest. The basis for the action of batrachotoxin in cardiac preparations is linked to activation of sodium channels and can be antagonized by tetrodotoxin. Unlike the cardiotonic cardiac glycosides, batrachotoxin has little effect on Na -K -ATPase, causing only a slight inhibition of the enzyme at a concentration (60 pM) much higher than that usually employed for depolarization of cells (79). 

---