batrachotoxinin-a-20-alpha-benzoate and Neuroblastoma

cis-9,10-Octadecenoamide (cOA) accumulates in cerebrospinal fluid during sleep deprivation and induces sleep in animals, but its cellular actions are poorly characterized. In earlier studies, like a variety of anesthetics, cOA modulated gamma-aminobutyric acidA receptors and inhibited transmitter release/burst firing in cultured neurones or synaptoneurosomes.. Here, radioligand binding ([3H]batrachotoxinin A 20-alpha-benzoate and mouse central nervous system synaptoneurosomes) and voltage clamp (whole cell recording from cultured NIE115 murine neuroblastoma) confirmed an interaction with neuronal voltage-gated sodium channels (VGSC).. cOA stereoselectively inhibited specific binding of toxin to VGSC (inhibitor concentration that displaces 50% of specifically bound radioligand, 39.5 microm). cOA increased (4x) the Kd of toxin binding without affecting its binding maximum. Rate of dissociation of radioligand was increased without altering association kinetics, suggesting an allosteric effect (indirect competition at site 2 on VGSC). cOA blocked tetrodotoxin-sensitive sodium currents (maximal effect and affinity were significantly greater at depolarized potentials; P < 0.01). Between 3.2 and 64 microm, the block was concentration-dependent and saturable, but cOA did not alter the V50 for activation curves or the measured reversal potential (P > 0.05). Inactivation curves were significantly shifted in the hyperpolarizing direction by cOA (maximum, -15.4 +/- 0.9 mV at 32 microm). cOA (10 microm) slowed recovery from inactivation, with tau increasing from 3.7 +/- 0.4 ms to 6.4 +/- 0.5 ms (P < 0.001). cOA did not produce frequency-dependent facilitation of block (up to 10 Hz).. These effects (and the capacity of oleamide to modulate gamma-aminobutyric acidA receptors in earlier studies) are strikingly similar to those of a variety of anesthetics. Oleamide may represent an endogenous ligand for depressant drug sites in mammalian brain.

Topics: Animals; Batrachotoxins; Brain; Cells, Cultured; Hypnotics and Sedatives; Male; Mice; Neuroblastoma; Oleic Acids; Patch-Clamp Techniques; Receptors, GABA; Sodium Channels

Measurement of neurotoxin binding in rat brain membranes and neurotoxin-activated 22Na+ influx in neuroblastoma cells were used to define the site and mechanism of action of pyrethroids and DDT on sodium channels. A highly potent pyrethroid, RU 39568, alone enhanced the binding of [3H]batrachotoxinin A 20-alpha-benzoate up to 30 times. This effect was amplified by the action of neurotoxins such as sea anemone toxins and brevetoxin acting at different sites of the sodium channel protein in brain membranes. The ability of various pyrethroids and DDT to enhance batrachotoxin binding was related to their capacity to activate tetrodotoxin sensitive 22Na+ uptake. These results point to an allosteric mechanism of pyrethroids and DDT action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels. Pyrethroids do not block [3H]tetrodotoxin binding, 125I-Anemonia sulcata toxin 2 binding, 125I-Tityus serrulatus toxin gamma binding at neurotoxin receptor sites 1, 3 and 4 respectively. Pyrethroids appear to act at a new neurotoxin receptor site on the sodium channel. The distribution of pyrethroid binding sites in rat brain was determined by quantitative autoradiographic procedures using the property of pyrethroids to reveal binding sites for [3H]batrachotoxinin A 20-alpha-benzoate.

Topics: Animals; Batrachotoxins; Binding Sites; Binding, Competitive; Brain; DDT; Mice; Neuroblastoma; Neurotoxins; Pyrethrins; Rats; Receptors, Neurotransmitter; Sodium Channels; Tumor Cells, Cultured