Alkaloid Veratridine Research Articles

The role of sodium in somatostatin secretion: evidence for the involvement of Na+ channels in the release mechanism.

The influence of Na+ upon the secretion of somatostatin from the isolated perfused canine pancreas was studied. The Na+ channel-opening alkaloid veratridine (10 microM) was found to cause a biphasic increase in somatostatin output as a normal Ca++ concentration of 1.3 mM. The response to veratridine was inhibited 70% by the addition of 1 microM tetrodotoxin (TTX) and was totally abolished in the absence of extracellular Ca++. TTX (1 microM) reversibly inhibited (by 30%) the glucose-induced somatostatin secretion. During Ca++ depletion, the inhibitory effect of TTX was eliminated. Partial replacement of extracellular Na+ by choline (40 mM Na+ and 100 mM choline) caused a 3- to 4-fold increase in somatostatin secretion. This finding and the fact that the somatostatin response to Na+ withdrawal required the presence of extracellular Ca++ suggest that the stimulatory effect of a decrease in extracellular Na+ concentration is mediated, at least in part, by a Na+-Ca++ countertransport mechanism. The results indicate that the Na+-mediated somatostatin release is dependent upon the extracellular Ca++ concentration. It is unlikely that Na+ per se or via redistribution of the intracellularly bound Ca++ can stimulate somatostatin secretion.

Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions

Neuroblastoma-glioma hybrid cells (NG108-15) in suspension accumulate the permeant lipophilic cation [(3)H]tetraphenylphosphonium (TPP(+)) against a concentration gradient. The steady-state level of TPP(+) accumulation is about twice as great in physiological media of low K(+) concentration (i.e., 5 mM K(+)/135 mM Na(+)) than in a medium of high K(+) concentration (i.e., 121 mM K(+)/13.5 mM Na(+)). The latter manipulation depolarizes the NG108-15 plasma membrane and indicates that the resting membrane potential (DeltaPsi) is due primarily to a K(+) diffusion gradient (K(in) (+) --> K(out) (+)). TPP(+) accumulation is time and temperature dependent, achieving a steady state in 15-20 min at 37 degrees C, and is a linear function of cell number and TPP(+) concentration (i.e., the concentration gradient is constant). The difference in TPP(+) accumulation in low and high K(+) media under various conditions has been used to calculate mean (+/-SD) DeltaPsi values of -56 +/- 3, -63 +/- 4, and -66 +/- 5 mV at 26, 33, and 37 degrees C, respectively. Importantly, these values are virtually identical to those obtained by direct electrophysiological measurements made under the same conditions. TPP(+) accumulation is abolished by the protonophore carbonylcyanide-m-chlorophenylhydrazone, whereas the neurotoxic alkaloid veratridine diminishes uptake to the same level as that observed in high K(+) media. In addition, the effect of veratridine is dependent upon the presence of external Na(+) and is blocked by tetrodotoxin. The steady-state level of TPP(+) accumulation is enhanced by monensin, indicating that this ionophore induces hyperpolarization under appropriate conditions. Finally, ouabain has essentially no effect on the steady-state level of TPP(+) accumulation in short-term experiments, suggesting that Na(+),K(+)-ATPase activity makes little contribution to the resting potential in these cells. Because many of these observations are corroborated by intracellular recording techniques, it is concluded that TPP(+) distribution measurements can provide a biochemical method for determining membrane potentials in populations of cultured neuronal cells.

Abnormal axonal discharges in R2 Neurones ofAplysia californica

1. The neurone R2 ofAplysia is normally silent. The present paper describes an abnormal electrophysiological behaviour observed in 32 R2 neurones fromAplysia californica, which displayed long spiking discharges following synaptic or direct stimulations. 2. The discharge was studied with two intra-somatic microelectrodes and up to 3 extra-axonal electrodes located along the right pleurovisceral connective. 3. Both delay measurements and changes in the shape of the extra-axonal spikes showed that the discharge originated in an axonal area, located from 5 to 14 mm from the soma, where frequent blockade of conduction occurred. 4. Intracellular recordings were performed in this axonal area and in the soma. The axonal spike was followed by a depolarizing afterpotential (DAP) which could be summed resulting in a long axonal depolarization, with associated spiking, lasting up to 4 min. 5. The axonal long potential wave persisted when the soma was hyperpolarized or when the axon was sectioned near the soma. It was strongly attenuated by 10−5 M TTX but not affected by 10 mM cobalt. 6. A similar behaviour was observed in normal R2 neurones treated by low concentrations of the alkaloid veratridine (10−7 M): long discharges originating in the axon, blockade of the spike propagation, DAP's and long depolarizations. The DAP's of veratridinized R2 axons were reversibly blocked by 10−5 M TTX. 7. The analogies with the veratridine induced behaviour suggest that the abnormal long discharges result from the alteration of the sodium conductance in the proximal part of the axon of the cell.

The functional significance of sodium channels in pancreatic beta‐cell membranes

1. The existence and functional significance of Na channels in pancreatic beta-cell membranes were investigated by studying the effects of the plant alkaloid veratridine on the temporal release of insulin from perfused isolated rat islets of Langerhans.2. 100 muM veratridine evoked a sustained threefold increase in insulin release which was almost completely inhibited by 3 muM tetrodotoxin (TTX). This action of TTX was rapidly reversible.3. The simultaneous presence of 100 muM propranolol, 100 muM phenoxy-benzamine and 10 muM atropine did not alter the magnitude of the response to 100 muM veratridine, indicating that the action of veratridine on the beta-cells was direct and was not mediated via the release of neurotrans-mitters from nerve endings within the islets.4. (45)Ca uptake by isolated islets in static incubation was increased almost threefold by 100 muM veratridine. This increase was completely inhibited by the simultaneous presence of 3 muM TTX.5. Replacement of Na(o) by choline caused a transient fourfold increase in insulin release which was associated with an increase in the uptake of (45)Ca from the extracellular space of similar magnitude. Subsequent exposure of islets to 100 muM veratridine still evoked some insulin release but this only achieved 32% of that secreted by islets exposed to veratridine in medium of normal [Na](o).6. The addition of 2.5 mM CoCl(2) to the medium caused a 62.5% inhibition of veratridine-mediated insulin release.7. In Ca-free medium supplemented with 1 mM EGTA, 100 muM veratridine evoked insulin release of equal magnitude and of similar temporal relationship to that obtained in the presence of normal [Ca](o).8. A twofold increase in insulin release that occurred in the 15 min period immediately following exposure to 1 mM ouabain was completely independent of [Ca](o). Subsequent ouabain-evoked release became increasingly dependent on [Ca](o).9. Tetrodotoxin (3 muM) inhibited the first phase of insulin release evoked by 16.7 mMd-glucose by 37% and the second phase by 20%.10. Both Na and Ca appear capable of entering through Na channels opened in the beta-cell membrane by veratridine. The increase in [Na](i), resulting from the veratridine mediated increase in P(Na+), causes depolarization of the beta-cell membrane with a consequent opening of voltage-sensitive, Co(2+)-blockable channels for additional Ca entry. An increase in [Na](i) also increases [Ca](i) by altering the equilibria of intracellular Ca-sequestering mechanisms. The small but significant reduction of glucose-mediated insulin release by TTX indicates that glucose has a rather weak action on the Na channel and a more pronounced effect on the voltage-dependent Co(2+)-blockable Ca channel.

Purification of a toxic protein from scorpion venom which activates the action potential Na+ ionophore.

Venom of the scorpion Leiurus quinquestriatus acts cooperatively with the alkaloids veratridine, aconitine, and batrachotoxin in activating the action potential Na+ ionophore. A small (Mr = 6700), basic (pI approximately 9.8), toxic polypeptide purified approximately 80-fold from this venom by ion exchange chromatography appears homogeneous by gel electrophoresis and isoelectric focusing and, like whole venom, acts cooperatively with the alkaloids veratridine, aconitine, and batrachotoxin to activate the action potential Na+ ionophore. The action of the scorpion toxin is slowly reversible. Concentration-response curves suggest interaction with a single class of sites with KD - 1.3 to 2.4 nM. The scorpion toxin is a poor activator of the Na+ ionophore when tested alone. However, treatment of cells sequentially with scorpion toxin followed by veratridine activates as well as treatment with both simultaneously suggesting that scorpion toxin binds in the absence of veratridine but does not activate the Na+ ionophore unless veratridine is present. In contrast, scorpion toxin causes 3- to 20-fold decreases in apparent KD for aconitine, veratridine, and batrachotoxin. The effect of the toxin is inhibited competitively by divalent cations and noncompetitively by tetrodotoxin (KI - 4 nM).

Interactions of neurotoxins with the action potential NA + ionophore.

Four neurotoxins that activate the action potential Na+ionophore of electrically excitable neuroblastoma cells interact with two distinct classes of sites, one specific for the alkaloids veratridine, batrachotoxin, and aconitine, and the second specific for scorpion toxin. Positive heterotropic cooperativity is observed between toxins bound at these two classes of sites. Tetrodotoxin, a specific inhibitor of the action potential Na+ current, inhibits activation by each of these toxins in a noncompetitive manner (KI=4-8 nM). These results suggest the existence of three functionally separable components of the action potential Na+ionophore: two regulatory components which bind activating neurotoxins and interact allosterically in controlling the activity of a third ion-transport component, which binds tetrodotoxin. The dissociation constant for scorpion toxin binding is increased 10-fold by depolarization of the cells with K+, suggesting that the scorpion toxin binding site is located on a voltage-sensitive regulatory component of the ionophore.

Cooperative activation of action potential Na+ ionophore by neurotoxins.

Four neurotoxins that activate the action potential Na+ ionophore of electrically excitable neuroblastoma cells interact with two distinct classes of sites, one specific for the alkaloids veratridine, batrachotoxin, and aconitine, and the second specific for scorpion toxin. Positive heterotropic cooperativity is observed between toxins bound at these two classes of sites. Tetrodotoxin is a noncompetitive inhibitor of activation by each of these toxins (KI = 4-8 nM). These results suggest the existence of three functionally separable components of the action potential Na+ ionophore: two regulatroy components, which bind activating neurotoxins and interact allosterically in controlling the activity of a third ion-transport component, which binds tetrodotoxin.