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/10⁶ 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.     

Effects of continuous oral nicotine administration on brain nicotinic receptors and responsiveness to nicotine in C57Bl/6 mice

 The route of drug delivery is an important consideration in studies that evaluate the long-term biobehavioral adaptations that occur in response to chronic drug administration. Continuous infusions (intravenous or subcutaneous) or intermittent intraperitoneal (or subcutaneous) injections are the most commonly utilized routes of chronic drug delivery in these studies. The purpose of the present study was to determine the effects of chronic oral nicotine exposure on sensitivity to nicotine and brain nicotinic cholinergic receptors in female C57Bl/6 mice. Mice were randomized to different treatment groups that received 2% saccharin, containing 0–200 μg/ml nicotine (free base). In preliminary experiments, radiotelemetry devices were implanted in the mice; consumption of the nicotine-containing drinking solution caused a significant increase in home-cage nocturnal (but not diurnal) activity and also altered circadian alterations in body temperature. Oral nicotine exposure resulted in dose-related elevations in plasma levels of cotinine, a primary nicotine metabolite. Continuous exposure (30 days) to oral nicotine (200 μg/ml) resulted in the expression of significant tolerance to the locomotor depressant and hypothermic actions of acute nicotine challenge. This tolerance was accompanied by a significant increase in brain nicotinic receptor number assessed by quantitative autoradiography using [³H]-cytisine (α4 nAChr) and [¹²⁵I]-α-bungarotoxin (α7 nAChr) as radioligands. These results suggest that chronic oral nicotine delivery to female C57Bl/6 mice results in behavioral and biochemical changes that resemble changes that occur following other routes of chronic nicotine delivery. Received: 30 January 1998 / Final version: 25 June 1998     

Both A chain and B chain of beta-bungarotoxin are functionally involved in the facilitation of spontaneous transmitter release in Xenopus nerve-muscle cultures.

In the present study, Xenopus nerve-muscle cultures were used to explore the functional roles of A chain (a phospholipase A(2) subunit) and B chain (a non-phospholipase A(2) subunit) of Bungarus multicinctus beta-bungarotoxin. It was found that beta-bungarotoxin induced an increment of the frequency of spontaneous synaptic currents (SSCs) in the nerve-muscle cultures. Modification of beta-bungarotoxin with pyridoxal-5'-phosphate or substitution of Ca(2+) with Ba(2+) in buffer abolished the phospholipase A(2) activity of beta-bungarotoxin and the facilitatory phase of SSC as well. Antibodies that were directed specifically against A chain or B chain effectively inhibited phospholipase A(2) activity, and as a consequence the SSC frequency was not greatly different from the control rate. These results suggest that both A and B chains are indispensable parts of beta-bungarotoxin for inducing the facilitation of SSC frequency with Xenopus nerve-muscle cultures.     

The effect of acute alloxan diabetes on the sensitivity of the rat skeletal neuromuscular junction to drugs

Summary Preparations from alloxan diabetic rats showed a reduced sensitivity to the neuromuscular blocking action of (+)-tubocurarine but no alteration in sensitivity to the deplolarizing neuromuscular blocking drug decamethonium. Physostigmine was less effective in augmenting twitch height in preparations from alloxan diabetic rats and such preparations had a significantly lowered total cholinesterase activity compared with control preparations. An additional observation was a reduction in the effectiveness of the pre-junctionally active agent β-bungarotoxin in producing neuromuscular blockade in physostigmine-treated preparations from alloxan diabetic rats. All the changes produced by alloxan administration were prevented by treatment with insulin.     

Antibodies in sera from patients with myasthenia gravis do not bind to nicotinic acetylcholine receptors from human brain

Nicotinic acetylcholine receptors (AChRs) from brains of chickens and rats have recently been purified and characterized (Whiting and Lindstrom, Biochemistry, 25 (1986) 2082-2093; J. Neurosci., 6 (1986) 3061-3069; Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 595-599). Using both antisera and monoclonal antibodies prepared to AChRs from rat brain, we have demonstrated the existence of a homologous AChR in human brain. Here we report that antibodies to muscle AChRs in the sera of patients with myasthenia gravis (MG) do not bind to AChRs from human brain. Similarly, there was no binding of sera from patients with Guillain-Barré, amyotrophic lateral sclerosis, multiple sclerosis, or Lambert-Eaton myasthenic syndrome. Additionally, no binding of any of these sera to the alpha-bungarotoxin (alpha-Bgt) binding protein from human brain could be detected. This data is consistent with other data using antibodies to AChRs from muscle and nerve in demonstrating that the AChR in brain is antigenically distinct from the AChR in skeletal muscle AChR, and, together with the lack of central neurological symptoms in MG, suggests that the low concentrations of anti-AChR antibodies in the cerebrospinal fluid of MG patients do not bind to AChRs in brain.     

Acetylcholine receptor concentration in the mimic musculature of the rat following denervation and reinnervation

Summary The facial musculature of the rat was denervated by cutting the facial nerve. Over a period of 41 days no facial movements were observed. Acetylcholine receptor concentrations, determined by [¹²⁵I]--bungarotoxin binding, increased sharply in the early stage of denervation (at day 10) and were still significantly higher than in the controls after 41 days.When cutting of the facial nerve was followed by immediate nerve repair (primary suture), facial movements returned on about day 16. The receptor concentrations reflected changes monitored by clinical observations. At day 10, when denervation of the facial muscles was still complete, receptor concentrations corresponded to those found in the permanently denervated muscles. At day 16 the reinnervated muscles of half the animals displayed muscle activity and had receptor concentrations identical to those found for normal (control) tissue. The other half of the animals, with no muscular activity detectable, had receptor concentrations as high as in permanently denervated tissue. After 23 days the receptor concentrations had essentially decreased to control levels and all rats had regained complete facial function.     

In vivo effects of 3-iodocytisine: Pharmacological and genetic analysis of hypothermia and evaluation of chronic treatment on nicotinic binding sites

Several cytisine derivatives have been developed in the search for more selective drugs at nicotinic acetylcholine receptors (nAChR). Binding experiments in transfected cell lines showed that the iodination of cytisine in the position 3 of the pyridone ring increased potency at α7-nAChR and to a lesser extent at the α4β2 subtypes, both of which are widely expressed in the brain. However, no in vivo studies have been published on this compound. Inhibition curves presented here using wild type, β2, and β4-null mutant mice confirm that 3-IC binds to α4β2∗, α7∗ and α3β4∗ receptors with higher affinity than cytisine (asterisk indicates the receptor may contain additional subunits, Lukas et al., 1999). Intraperitoneal injection of 3-iodocytisine (3-IC) induced considerable dose-dependent hypothermia in DBA/2J and C57BL/6J mice. This response was blocked by mecamylamine and partially inhibited by hexamethonium. β4-null mice displayed significantly less 3-IC-induced hypothermia than wild-type mice, β2-null mice were somewhat less affected than wild types, while responses of α7∗-null mice were similar to wild types. Mice treated chronically with 3-IC display a marked increase in α7∗ and α4β2∗ binding sites determined by radioligand binding in membrane preparations from cerebral cortex and hippocampus. Quantitative autoradiographic analysis of 28 brain regions of mice treated with 3-IC was consistent with the membrane binding, detecting an increase of cytisine-sensitive [¹²⁵I]epibatidine binding sites, while cytisine-resistant [¹²⁵I]epibatidine sites were unchanged. [¹²⁵I]α-Bungarotoxin binding sites also exhibited up-regulation. These results give a first evaluation of in vivo consequences of 3-IC as a potent agonist with marked effects on mice.     

Heterogeneity of antibodies directed against the α-bungarotoxin binding site on human acetylcholine receptor and severity of myasthenia gravis

A rapid and sensitive radioimmunological method is described, using decamethonium (DC), which revealed antibodies which blocked α-bungarotoxin (α-Bgt) binding to human acetylcholine receptor (AChR) in 98% of myasthenia gravis (MG) patients' sera tested. These sera had anti-AChR antibody titres by the conventional assay. The titre of blocking antibodies (1 to 110 nM) could be measured and was found to produce from 1 to 54% inhibition of α-Bgt binding. No relationship was found between these titres and anti-AChR antibody titres. MG sera were divided into 2 major groups on the basis of their blocking effects, with and without DC, but there was no correlation between these and the clinical status, as defined by Osserman's classification. However, no sera from asymptomatic or ocular MG patients had the dual capacities of blocking α-Bgt binding, directly and in the presence of DC.     

Immunological cross-reactivity between the α-bungarotoxin-binding component from rat brain and nicotinic acetylcholine receptor

An α-bungarotoxin-binding protein was partially purified from rat brain and, when complexed with [¹²⁵I]α-bungarotoxin, was shown to behave as a single radiolabelled protein that is distinct from the similarly complexed nAChR from Torpedo marmorata. The α-bungarotoxin-binding protein was used as antigen in radioimmunoassays for rabbit anti-(rat muscle nAChR) and rabbit anti-(Torpedo nAChR) antibodies, giving titres approximately 5% and 0.5%, respectively, of those obtained by using homologous antigen in the same assay.     

Differential co-localization of nicotinic acetylcholine receptor subunits with calcium-binding proteins in retinal ganglion cells

Immunohistochemistry was used to examine the co-occurrence of nicotinic acetylcholine receptor subunits with calcium-binding proteins in ganglion cells of the chick retina. The α3 subunit was rarely observed in ganglion cells containing calbindin, calretinin, or parvalbumin. On the other hand, the α8 subunit was more often co-localized with all calcium-binding proteins studied. These results may be related to the high calcium permeability of nicotinic receptors that contain the α8 subunit.