Α-bungarotoxin Receptors Research Articles

Behavioural and pharmacological profiles of zebrafish administrated pyrrolidinyl benzodioxanes and prolinol aryl ethers with high affinity for heteromeric nicotinic acetylcholine receptors.

Prolinol aryl ethers and their rigidified analogues pyrrolidinyl benzodioxanes have a high affinity for mammalian α4β2 nicotinic acetylcholine receptors (nAChRs). Electrophysiological studies have shown that the former are full agonists and the latter partial agonists or antagonists of human α4β2 receptors, but their in vivo effects are unknown. As α4β2 nAChRs play an important role in the cognition and the rewarding effects of nicotine, we tested the effects of two full agonists and one antagonist on spatial learning, memory and attention in zebrafish using a T-maze task and virtual object recognition test (VORT). The effect of a partial agonist in reducing nicotine-induced conditioned place preference (CPP) was also investigated. In comparison with the vehicle alone, the full agonists MCL-11 and MCL-28 induced a significant cognitive enhancement as measured by the reduced running time in the T-maze and increased attention as measured by the increased discrimination index in the VORT. MCL-11 was 882 times more potent than nicotine. The two compounds were characterised by an inverted U-shaped dose-response curve, and their effects were blocked by the co-administration of the antagonist MCL-117, which alone had no effect. The partial agonist MCL-54 induced CPP and had an inverted U-shaped dose-response curve similar to that of nicotine but blocked the reinforcing effect of co-administered nicotine. Binding studies showed that all of the compounds have a higher affinity for heteromeric [3H]-epibatidine receptors than [125I]-αBungarotoxin receptors. MCL-11 was the most selective of heteromeric receptors. These behavioural studies indicate that full agonist prolinol aryl ethers are very active in increasing spatial learning, memory and attention in zebrafish. The benzodioxane partial agonist MCL-54 reduced nicotine-induced CPP, and the benzodioxane antagonist MCL-117 blocked all agonist-induced activities.

The Role of Palmitoylation in Functional Expression of Nicotinic α7 Receptors

Neuronal alpha-bungarotoxin receptors (BgtRs) are nicotinic receptors that require as yet unidentified post-translational modifications to achieve functional expression. In this study, we examined the role of protein palmitoylation in BgtR expression. BgtR alpha7 subunits are highly palmitoylated in neurons from brain and other cells capable of BgtR expression, such as pheochromocytoma 12 (PC12) cells. In PC12 cells, alpha7 subunits are palmitoylated with a stoichiometry of approximately one palmitate per subunit, and inhibition of palmitoylation blocks BgtR expression. In cells incapable of BgtR expression, such as human embryonic kidney cells, alpha7 subunits are not significantly palmitoylated. However, in these same cells, chimeric subunits with the N-terminal half of alpha7 fused to the C-terminal half of serotonin-3A receptor (alpha7/5-HT3A) subunits form functional BgtRs that are palmitoylated to an extent similar to that of BgtRalpha7 subunits in PC12 cells. Palmitoylation of PC12 and alpha7/5-HT3A BgtRs occurred during assembly in the endoplasmic reticulum (ER). In conclusion, our data indicate a function for protein palmitoylation in which palmitoylation of assembling alpha7 subunits in the ER has a role in the formation of functional BgtRs.

β3 subunit is present in different nicotinic receptor subtypes in chick retina

Although the neuronal nicotinic β3 subunit was cloned several years ago, it has only recently been shown to form heteromeric channels when associated with other nicotinic subunits, and very little information is available concerning its assembly in the native nicotinic receptors of the nervous system. Using subunit-specific antibodies and immunoprecipitation experiments, we have identified the retina as being the chick central nervous system (CNS) area that expresses the highest level of the β3 subunit. Sequential immunopurification experiments showed that there are at least two populations of β3-containing receptors in chick retina: in one, the β3 subunit is associated with the α6 and β4 subunits; in the other more heterogeneous population, the β3 subunit is associated with the α2, α3, α4, β2 and β4 subunits. Both of these receptor populations bind [ 3H]epibatidine and a number of nicotinic receptor agonists with high affinity (nM) and nicotinic receptor antagonists with a lower affinity (μM). The greatest pharmacological difference between the two populations is the affinity for the α-conotoxin MII, which inhibits binding to α6-containing receptors and not that to β3-containing receptors. We also searched for the presence of the β3 subunit associated with the α-bungarotoxin binding subunits α7 and/or α8 in retina and chick brain. Immunoprecipitation studies using anti-β3 antibodies did not detect any specific α-bungarotoxin labeled receptors, thus, indicating that the β3 subunit is not present in the α-bungarotoxin receptors of these areas.

Agonist activation and α-bungarotoxin inhibition of wild type and mutant α7 nicotinic acetylcholine receptors

The properties of wild type and mutant rat nicotinic α7 receptors expressed in Xenopus oocytes were investigated using electrophysiology and site-directed mutagenesis. When compared at individual agonist concentrations, neither the normalised nicotinic, nor acetylcholine, responses of the wild type receptors were significantly different from the corresponding responses obtained from a first extracellular domain mutant, phenylalanine 189tyrosine ( P>0.05). The dissociation constants ( K D) of the wild type (4.7 nM) and Phe 189Tyr mutant (5.2 nM) receptors for α-bungarotoxin were estimated by an electrophysiological approach. The similarity of the results suggests that the mutation did not lead to a widespread disruption of structure–function relationships, although a slight change in nicotine sensitivity may have occurred. In contrast, the mutations (Tyr 190Gln, first extracellular domain), (Glu 261Ala, M2 region) severely compromised receptor function. An additional mutation was made in a negatively charged motif of the second extracellular domain which is conserved in homomeric nicotinic receptors. This mutation, Asp 268Ala, also caused a loss of function. Thus the structure–function relationships in nicotinic α7 receptors have parallels with heteromeric nicotinic receptors, but there may also be some marked differences.

4-Oxystilbene compounds are selective antagonists for neuronal nicotinic α-bungarotoxin receptors

4-Oxystilbene compounds are selective antagonists for neuronal nicotinic α-bungarotoxin receptors

Neuronal α-Bungarotoxin Receptors Differ Structurally from Other Nicotinic Acetylcholine Receptors

We have characterized the alpha-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of alpha7-containing receptors that bind alpha-Bgt with high affinity (Kd = 94 pM). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and alpha-Bgt block approximately 90% of the whole-cell current. The binding of nicotinic agonists to cell-surface BgtRs was highly cooperative with four different agonists showing Hill coefficients in the range of 2.3-2.4. A similar agonist binding cooperativity was observed for BgtR homomers formed from chimeric alpha7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for alpha7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and alpha7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and alpha7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.

Nicotinic receptor distribution in the human thalamus: autoradiographical localization of [ 3H]nicotine and [ 125I] α-bungarotoxin binding

The thalamus plays a major role in relaying and transforming information that is relayed to the cortex and in turn modulates cortical outputs. The reticular nucleus projects to the other thalamic nuclei, modulating and integrating their activity. The distribution of high affinity nicotine and α-bungarotoxin ( αBTX) receptors in the human thalamus has been investigated by radioligand autoradiography in post mortem human tissue. [ 3H]nicotine binding in the human thalamus was high in most thalamic nuclei, especially in the lateral dorsal, the medial geniculate, lateral geniculate and anterior nuclei. The distribution of [ 125I] αBTX binding was quite distinct from [ 3H]nicotine binding. [ 125I] αBTX binding was generally lower (<0.26–11.62 fmol/mg protein compared with 6.68–36.17 fmol/mg protein for nicotine binding) and concentrated in the reticular nucleus, with discrete groups of cells displaying higher binding in the latter. These results indicate differences between the distribution of nicotinic receptors in humans and those previously reported in mice and monkeys. Changes in high affinity nicotine and αBTX receptors in the thalamus may contribute to symptoms observed in neuropathological conditions associated with disorders of perception and movement such as Dementia with Lewy Bodies, Alzheimer's Disease and Schizophrenia.

Linkage of strain-specific nicotinic receptor α 7 subunit restriction fragment length polymorphisms with levels of α-bungarotoxin binding in brain

Inbred mouse strains have been shown to differ in their levels of brain α-bungarotoxin binding. These differences in α-bungarotoxin receptors have been shown to correlate with an animal's sensitivity to nicotine-induced seizures. Recent studies have shown that the α 7 nicotinic acetylcholine receptor subunit is the major α-bungarotoxin binding site in rodent brain. In this report, we examined whether mouse strains that differ in levels of α-bungarotoxin binding and sensitivity to nicotine-induced convulsions also differ for the α 7 subunit. A full-length murine α 7 cDNA was cloned and sequenced and found to be identical to that of a mouse α 7 cDNA recently reported. Subsequently, a comparison of α 7 cDNA sequences and RNA species was performed between two strains (C3H/2 and DBA/2) that differ in levels of brain α-bungarotoxin binding and sensitivity to nicotine-induced seizures. The only difference observed was a single nucleotide difference in the open reading frame of α 7 that does not affect the primary amino acid sequence. Inbred strains were also surveyed for restriction fragment length polymorphisms at the α 7 locus. Strain-specific polymorphisms were identified, and F 2 and backcross animals from a classic genetic cross between C3H/2 and DBA/2 mice were compared for the inheritance of α 7 genotype and α-bungarotoxin receptor levels. A significant association between genotype and receptor levels was observed in both the F 2 and backcross generations. These results indicate that α 7 genotype is an important determinant of α-bungarotoxin receptor levels.

Ciliary neurotrophic factor regulates nicotinic acetylcholine receptors on human neuroblastoma cells

We have investigated the effects of several neurokine/cytokine family members on the level of α-bungarotoxin-binding to neuronal nicotinic acetylcholine receptors. Exposure of human neuroblastoma cells (SH-SY5Y and IMR-32) to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor or oncostatin-M resulted in a 30–40% decline in α-bungarotoxin receptors on the cells with no decrease seen in either muscarinic acetylcholine receptors or in L-type Ca 2+ channels. The level of nicotinic receptor was not affected by the related cytokine, interleukin-6. Treatment of IMR-32 cells with 40 pM CNTF produced a half-maximal decrease of α-bungarotoxin binding which compared well with the affinity estimated from binding of 125I-CNTF ( K i ≈ 40 pM) and the concentration causing c- fos activation in SH-SY5Y cells, as detected by nuclear run-on assays (60–120 pM). Previous results have indicated that the differentiating agents, phorbol esters and retinoic acid, also decrease nicotinic receptor numbers. Here the effects of CNTF, which did not induce neural differentiation, were enhanced by differentiation with 12- O-tetradecanoylphorbol 13-acetate (10 nM) and prevented by retinoic acid (10 μM). Therefore, the response of neuroblastoma cells to cytokines may be under developmental control. These cells offer a system to examine cytokine responses and signal transduction mechanisms during neural development.

Regulation of nicotinic acetylcholine receptors on human neuroblastoma cells during differentiation

Neuronal nicotinic acetylcholine receptors are expressed on a variety of cells in the nervous system where they play key roles in synaptic transmission and information transfer. Little is known, however, about the molecular mechanisms that control their expression, distribution, and function during nervous system development. We have investigated the control of expression during differentiation of one class of acetylcholine receptors that bind α-bungarotoxin of human neuroblastoma cells. We report that induction of differentiation of SH-SY5Y, SK-n-SH or IMR-32 cells by the phorbol ester 12- O-tetradecanoyl phorbol 13-myristate (10 nM, TPA) or by retinoic acid resulted in as much as a 70% decline in α-bungarotoxin receptors on the cells. The response to the phorbol ester was blocked by the protein kinase C inhibitors staurosporine and bisindolylmaleimide. The decrease in receptors induced by 10 μM retinoic acid was not affected by either agent. However, responses to lower (10 nM) concentrations of retinoic acid were blocked by staurosporine but not bisindolylmaleimide, suggesting a dual mechanism of action for retinoic acid in regulating acetylcholine receptors. It appears that acetylcholine receptors on neuroblastoma cells are regulated during differentiation by both protein kinase C-dependent and -independent mechanisms.

Native Nicotinic Acetylcholine Receptors in Human Imr32 Neuroblastoma Cells: Functional, Immunological and Pharmacological Properties

IMR32 cells express two classes of surface nicotinic receptors: those labelled with high affinity by [125I]neuronal toxin, and those labelled by [125I]alpha-bungarotoxin. Whole-cell patch-clamp recordings indicate that both classes of receptor are able to elicit inward currents that are totally blocked by d-tubocurarine but only partially blocked by alpha-bungarotoxin. In IMR32 cells, nicotine induces an increase in the intracellular level of free Ca2+. This increase, which is also completely blocked by d-tubocurarine and only partially blocked by alpha-bungarotoxin and Cd2+, is due to extracellular calcium influx through both the nicotinic receptors and the voltage-activated Ca2+ channels. By using subunit-specific polyclonal antibodies, we have demonstrated that the alpha-bungarotoxin receptors contain the alpha 7 subunit, but none of the other subunits whose transcripts are present in IMR32 cells. The pharmacological profile of these human alpha 7-containing alpha-bungarotoxin receptors is similar to that observed in the native chick alpha 7 receptor, but there are also some species-specific differences.

The characterization of α-bungarotoxin receptors on the surface of parasympathetic neurons in the frog heart

Receptors for α-bungarotoxin are found on the surface of parasympathetic neurons in the frog cardiac ganglion by light microscopic autoradiography. Competition studies suggest that these receptors are cholinergic and indicate that they are also recognized by neuronal bungarotoxin (κ-bungarotoxin). These receptors are outnumbered by those recognized exclusively by neuronal bungarotoxin. Unlike neuronal bungarotoxin receptors, α-bungarotoxin receptors are not concentrated at synaptic sites. Fluorescence techniques fail to find evidence for clusters of α-bungarotoxin receptors anywhere on the neuronal surface. The possible function of these receptors, which apparently do not play a role in fast synaptic transmission, is discussed.

α-Bungarotoxin blocks the nicotinic receptor mediated increase in cell number in a neuroendocrine cell line

Exposure of H69 small cell lung carcinoma cells to nicotinic agonists resulted in a significant increase (up to 100%) in cell number after 6 to 12 days. The effect of nicotine (10 −8 M to 10 −4 M) was both dose and time dependent as was that of another nicotinic agonist cytisine (10 −6 M to 10 −4 M). Interstingly, both the nicotine and cytisine induced increases in H69 cell number were blocked by α-bungarotoxin, as well as d-tubocurarine a nicotinic blocker which appears to interact with most nicotinic receptors. These results suggest that the nicotine induced increase in cell number is mediated through an interaction at the nicotinic α-bungarotoxin receptor. This idea is further supported by experiments which show (1) that H69 cells possess high affinity α-bungarotoxin sites ( K d = 25 nM, B max = 10.4 fmol/10 6 cells) with the characteristics of a nicotinic α-bungarotoxin receptor and (2) that the potencies of nicotinic receptor ligands in the α-bungarotoxin binding assay were similar to those observed in the functional studies. Northern analysis showed that mRNA for α7, a putative nicotinic α-bungarotoxin binding subunit, and for α5 were present in H69 cells. The present data provide further evidence that nicotine increases cell number in small cell lung carcinoma and are the first to show that this effect is mediated through an interaction at the nicotinic α-bungarotoxin receptor population. These results suggest that the α-bungarotoxin site may be involved in modulating proliferative responses in neuroendocrine derived SCLC cells.

Nicotine-induced intracellular calcium changes are not antagonized by α-bungarotoxin in adrenal medullary cells

The snake toxin α-bungarotoxin distinguishes between neuronal nicotinic receptor subtypes. In chick ciliary ganglion neurons, activation of α-bungarotoxin-sensitive nicotinic receptors has been proposed to produce elevations in intracellular calcium levels. In the present study we show that prolonged treatment with α-bungarotoxin did not affect the nicotine-evoked calcium response in suspended chromaffin cells. On the other hand, the classical nicotinic receptor blocker d-tubocurarine potently blocked nicotinic receptor mediated effects. The degree of inhibition of the nicotinic response observed with d-tubocurarine was not modified by prior treatment with α-bungarotoxin. These results suggest that nicotinic α-bungarotoxin receptors are not primarily involved in nicotine-mediated increases in intracellular calcium in bovine adrenal medullary cells.

A role for the nicotinic α-bungarotoxin receptor in neurite outgrowth in PC12 cells

The addition of nicotine decreased neuritic outgrowth in PC12 cells in culture. This effect occurs as early as one day after addition of nicotine to the culture medium in a concentration-dependent manner. The nicotine-induced decline in neunte outgrowth was prevented by d-tubocurarine (10 − M) indicating that the effect was mediated through a nicotinic receptor. α-Bungarotoxin (10 −8 M) was also able to inhibit the nicotine-induced decrease in process formation in a dose-dependent manner. The concentrations of α-bungarotoxin required to affect process outgrowth correlated with those required to inhibit radiolabelled α-bungarotoxin binding. α-Bungarotoxin had no effect on [ 3H]noradrenaline release, a functional response mediated through the α-bungarotoxin-insensitive neuronal nicotinic acetylcholine receptor, suggesting that α-bungarotoxin specifically interacts with the neuronal α-bungarotoxin receptor. The present results suggest a functional role for the neuronal nicotinic α-bungarotoxin receptor in neurite outgrowth.

Phorbol esters and K + up-regulate α-bungarotoxin binding sites in cultured chromaffin cells through a related mechanism

Nicotinic α-bungarotoxin (α-BGT) receptors in cultured chromaffin cells are up-regulated in response to long term (days) exposure to nicotinic antagonists, elevated K +, and activators of protein kinase C (PKC), such as the phorbol ester, 4β-phorbol 12-myristate 13-acetate (PMA). The present experiments were done to determine whether their was any interaction in the ability of PMA and K + to up-regulate the α-BGT receptors. Chromaffin cells were treated for 3 days with both 100 nM PMA and 20 mM K −, concentrations which produce maximal responses on their own. The increase in α-BGT binding after the combined treatment was the same as that seen with K + alone suggesting that K + inhibited the PMA-mediated effect. The K +-induced increase in toxin binding was partially prevented by polymyxin B, an agent which completely inhibited the PMA induced increase. The time courses of the increases in binding induced by both K + and PMA were similar in that the most marked increases in binding were observed at the later time points. The PMA-induced up-regulation was partially inhibited by an activator of adenylate cyclase, a result similar to that previously seen with K +. The present studies suggest that the up-regulation of α-BGT receptors induced by K + shares similarities with that induced by phorbol esters. The observations that K + inhibited the PMA induced increase and that a PKC inhibitor partially blocked the K + response suggest that the K + and PMA mediated induction of the α-BGT sites may be linked with the effects of K + preceding those of PMA.

A functional α-bungarotoxin receptor is present in chick cerebellum: Purification and characterization

It has recently been demonstrated that α-bungarotoxin receptors, which behave as functional nicotinic receptors, are present in chick CNS. In this paper, we report the purification and characterization of a functional α-bungarotoxin receptor from chick cerebellum, a nervous tissue in which a clear inhibition of induced nicotine effects has been reported in vivo. This receptor contains at least three subunits of apparent mol. wt 52,000, 57,000 and 67,000. The use of monoclonal antibodies specific for the α7 subunit demonstrated that 75% of the molecules present in our purified preparation belong to the α7 subtype and that this antibody labels the 57,000 band in western blot, thus indicating that this is the toxin binding subunit. Reconstruction experiments in planar lipid bilayers show that this α-bungarotoxin receptor forms a cation selective channel whose opening is blocked by d-tubocurarine. Binding experiments on immobilized receptors over an α-bungarotoxin-Sepharose affinity column show that the ligand binding subunit is presen t in vivo in two copies per receptor. Immunological, pharmacological and functional experiments show that this purified receptor is very similar, but not identical, to the previously characterized chick optic lobe receptor, thus indicating the heterogeneity of these α-bungarotoxin receptors in the CNS.

A human skeletal muscle cell line obtained from an adult donor

A cell line (RCMH) in permanent culture was established from surgically removed adult normal human skeletal muscle by exposure to conditioned media obtained from thyroid cells. Cells proliferated indefinitely but displayed density inhibition of growth while maintaining some differentiated marker. Under certain incubation conditions, cells fused into myotube-like structures, with a concomitant increase in muscle specific proteins, such as human myoglobin, skeletal muscle myosin, desmin and dystrophin, as identified using immunocytochemical procedures. In addition, RCMH cells displayed high affinity receptors for α-bungarotoxin ( B max = 0.7 pmol/mg protein, K d = 1.5 nM) and dihydropyridines ( B max = 0.3 pmol/mg protein, K d = 0.5 nM for [ 3H]PN200-110); these values are comparable to those reported for muscle cells in primary culture. Patch-clamp studies showed the presence of 42 pS carbachol gated channels and of 5 pS calcium channels (current carried by barium); chloride and potassium channels were also seen. This new cell line appears to be a convenient model system to study skeletal muscle function.

Thymopoietin, a thymic polypeptide, potently interacts at muscle and neuronal nicotinic alpha-bungarotoxin receptors.

Current studies suggest that several distinct populations of nicotinic acetylcholine (ACh) receptors exist. One of these is the muscle-type nicotinic receptors with which neuromuscular nicotinic receptor ligands and the snake toxin alpha-bungarotoxin interact. alpha-Bungarotoxin potently binds to these nicotinic receptors and blocks their function, two characteristics that have made the alpha-toxin a very useful probe for the characterization of these sites. In neuronal tissues, several populations of nicotinic receptors have been identified which, although they share a nicotinic pharmacology, have unique characteristics. The alpha-bungarotoxin-insensitive neuronal nicotinic receptors, which may be involved in mediating neuronal excitability, bind nicotinic agonists with high affinity but do not interact with alpha-bungarotoxin. Subtypes of these alpha-toxin-insensitive receptors appear to exist, as evidenced by findings that some are inhibited by neuronal bungarotoxin whereas others are not. In addition to the alpha-bungarotoxin-insensitive sites, alpha-bungarotoxin-sensitive neuronal nicotinic receptors are also present in neuronal tissues. These latter receptors bind alpha-bungarotoxin with high affinity and nicotinic agonists with an affinity in the microM range. The function of the nicotinic alpha-bungarotoxin receptors are as yet uncertain. Thymopoietin, a polypeptide linked to immune function, appears to interact specifically with nicotinic receptor populations that bind alpha-bungarotoxin. Thus, in muscle tissue where alpha-bungarotoxin both binds to the receptor and blocks activity, thymopoietin also potently binds to the receptor and inhibits nicotinic receptors-mediated function. In neuronal tissues, thymopoietin interacts only with the nicotinic alpha-bungarotoxin site and not the alpha-bungarotoxin-insensitive neuronal nicotinic receptor population. These observations that thymopoietin potently and specifically interacts with nicotinic alpha-bungarotoxin-sensitive receptors in neuronal and muscle tissue, together with findings that thymopoietin is an endogenously occurring agent, could suggest that this immune-related polypeptide represents a ligand for the alpha-bungarotoxin receptors. The function of thymopoietin at the alpha-bungarotoxin receptor is as yet uncertain; however, a potential trophic, as well as other roles are suggested.

Light-dark variation in response to chronic nicotine treatment and the density of hypothalamic α-bungarotoxin receptors

Chronic nicotine administration increased locomotor activity during the light, but not the dark, in rats maintained on a 12:12-hr light/dark cycle, but the period and peak of the circadian rhythm (CR) were not affected. In Experiment I, 24 male rats were implanted with battery-operated telemeters and locomotor activity was continuously measured for 10 days before and 10 days after the implantation of osmotic mini-pumps which delivered 0, 0.5, 3.0 or 10 mg/kg/day of (±)-nicotine tartrate. Nicotine increased locomotor activity during the light in a dose-dependent manner. Tolerance to the stimulant effects of nicotine during the light occurred in 5–6 days. To determine if the stimulant properties of nicotine were associated with light as opposed to disruption by the environmental stimuli normally present during the day in our animal facility, a second experiment was conducted in which rats were treated with saline or 10 mg/kg/day (±)-nicotine di(+)hydrate tartrate and maintained on a reversed light/dark cycle. Again nicotine increased activity during the light (21:00–09:00) but not dark (09:00–21:00). In a third experiment, the density of α-bungarotoxin binding sites was found to be significantly decreased when animals were sacrificed at 06:00 in comparison with animals sacrificed at 10:00 and 14:00