Long-term blockade by toxin F of nicotinic synaptic potentials in cultured sympathetic neurons
Abstract
The effects of a recently identified blocker of neuronal nicotinic transmission, toxin F, were studied in cultured sympathetic neurons. Single principal neurons, dissociated from superior cervical ganglia of newborn rats, were grown on cardiac myocytes in microculture. The toxin blocked nicotinic synaptic potentials in these cultures but had no effect on muscarinic interactions. When toxin F was applied by addition to the perfusion medium, the concentration required for blocking most of the nicotinic potential was 40 nM, and the recovery from blockade was slow (t1/2 = 95 ± 64min). When the toxin was briefly applied by pressure ejection from a pipette, the concentration in the pipette necessary for blockade was 21 μM, and 30–60% of the response recovered within a few minutes while the remainder recovered slowly (t1/2of the remainder= 105 ± 82min).
One possible explanation for the difference in recovery time is that toxin F binds initially with low affinity to the nicotinic receptor but with time the toxin-receptor complex converts to a high affinity state. The presence of dihydro-β-erythroidine during perfusion of toxin F prevented the long-lasting blockade by the toxin, suggesting that toxin F and dihydro-β-erythroidine act through a common binding site. The specificity, potency, and slow reversibility of the effects of toxin F make it useful as a probe for studying neuronal nicotinic receptors of cultured sympathetic neurons.
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Pharmacology of Neuronal Nicotinic Acetylcholine Receptor Subtypes
1997, Advances in PharmacologyThis chapter begins with the properties of receptors expressed in the oocyte. Considerable data exist on the properties of neuronal nicotinic receptors in cells isolated from both rat and chick peripheral ganglia or specific brain nuclei. The remainder of the review considers the problem of associating specific properties with known combinations of receptor subunits. Although genes can be included in gene families on the basis of their similarities to other members of the family, the most compelling test of relatedness is function. The Xenopus oocyte has provided a convenient and powerful means of assessing the function of proteins thought to be subunits of ligand-gated ion channels. Injection of RNA transcribed from cDNA clones encoding the appropriate receptor subunits or injection of the cDNA in which the coding sequence is placed downstream of a promoter, such as SV40 (simian virus) or CMV (cytomegalovirus), results in the appearance of functional ligand-gated ion channels on the surface of the oocyte. Oocytes do not express an endogenous functional nicotinic receptor, although it has been reported that they express receptor coding sequences. Oocytes do, however, express a calcium-gated chloride channel. This chapter discusses the rat and chick neuronal nicotinic receptors, properties of the receptors in vivo—pre- and post- synaptic and receptors, presynaptic nicotinic receptors in striatum, hippocampus, and interpeduncular nucleus, somatic nicotinic responses from the central nervous system, chick ciliary ganglion binding of I-labeled neuronal bungarotoxin, and the two classes of nicotinic binding sites in rat superior cervical ganglion. Some useful pharmacological tools allow the limited identification of subunits in native receptors. Block by α-bungarotoxin identifies α7, α8, or α9 subunits; activation of a receptor bycytisine indicates a α7 or β4 subunit; and neuronal bungarotoxin block identifies a β2 subunit. Neuronal nicotinic receptors are highly permeable to calcium, unlike muscle nicotinic receptors.
Fast synaptic transmission in sympathetic ganglia is mediated by acetylcholine, acting on nicotinic receptors, yet muscarinic receptors are also present and are involved in the production of slow postsynaptic potentials. In order further to elucidate the role of muscarinic receptors in ganglionic transmission their distribution in the rat superior cervical sympathetic ganglion was investigated autoradiographically by use of the tritiated irreversible muscarinic ligand propylbenzilylcholine mustard. It was observed that this agent blocked the carbachol-evoked hydrolysis of inositol phospholipids in the ganglion and that this response to carbachol is itself inhibitable by selective muscarinic antagonists with a potency sequence which indicates involvement primarily of M1 receptors. Light microscope autoradiography showed that labelling inhibitable by atropine and by the M1-selective muscarinic antagonist pirenzepine was essentially confined to the margins of neuronal somata and regions of dendritic arborization, which include synaptic contacts. Quantitative electron microscope autoradiography showed that binding of the radioligand, of which approximately 70% was inhibitable by atropine and 68% by pirenzepine, was associated predominantly with surface membranes of neuronal somata, dendrites, other neurites (including axons and uncharacterized dendrites) and nerve terminal profiles, in the approximate ratios 95:85:52:45. Of the inhibitable binding over neuronal membranes in the ganglion little more than 3% was found to be synaptically located, and this involved para- or peri-synaptic regions of nerve terminal contacts rather than the specialized synaptic zone. About 5% of the inhibitable binding over neuronal membranes involved non-synaptic surfaces of nerve terminals and preterminal axon segments; almost 70% was distributed over non-synaptic surfaces of neuronal somata and dendrites, and about 21 % upon other neurites. Binding sites were found not to be more highly concentrated at or adjacent to synapses than over other regions of neuronal surface membranes. About 50%, possibly more, of the binding on non-synaptic surfaces of nerve endings, and about 7% of binding upon dendritic membranes, was of non-M1, possibly M2 type, inhibitable by atropine but not by pirenzepine. Non-synaptic neuro-neuronal appositions, which involve dendrites and somata and often lie adjacent to synapses, showed rather more than twice the binding expected for each membrane individually; and neuronal membrane exposed to basal lamina lining ganglionic tissue spaces showed high levels of binding. Little inhibitable binding was seen over membranes of satellite and Schwann cells, or over cytoplasmic territories or ganglionic interstitial tissue. A model was constructed of the distribution of label, which showed that the observed results for total binding could be approximately matched by assuming the following relative densities of ligand binding sites: interstitial tissue space and supporting cells 1, soma cytoplasm 3, cytoplasm of dendrites, neurites and nerve terminals 4.5, surfaces of mesodermal elements 15, surfaces of neurites and nerve endings including sites of synapse 45, surfaces of dendrites 90, surfaces of neuronal somata 120, non-synaptic neuro-neuronal appositions 180.
It is concluded that functional muscarinic receptors in this sympathetic ganglion, predominantly of the M1 type linked with slow depolarizations, but including some non-M, receptors, are widely distributed over non-synaptic surfaces of the neuronal somata and dendrites and are not concentrated at synapses. Presynaptic autoreceptors are also present, of which half or more are of non-Ma possibly M2, type which might be inhibitory. The presence of M4 receptors is not excluded. The observed distribution suggests that muscarinic receptors in the ganglion are appropriately located to be capable of exploiting acetylcholine spilling over from sites of release from preganglionic nerve endings, for which there is evidence in vivo, thereby assisting the co-ordination or reciprocal regulation of recruitment and activity in ganglionic neuronal populations with similar and, or, with opposing functions.
[3H]Noradrenaline release was studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Acetylcholine elicited a concentration- and time-dependent increase in3H outflow which was half-maximal at about 300 μM and within 5 s. The overflow induced by 10 s exposure to 300 μM acetylcholine was reduced by the nicotinic antagonist hexamethonium, but increased by the muscarinic antagonist atropine. Cd2+ (300 μM) prevented the overflow evoked by electrical field stimulation, but reduced acetylcholine-induced overflow by less than 50%. Removal of extracellular Ca2+ abolished stimulation-evoked tritium overflow, irrespective of the stimulus. The selective α2-adrenoceptor agonist UK 14,304 inhibited acetylcholine-evoked overflow to a significantly smaller extent (≈25% maximal inhibition) than electrically induced overflow (≥45% maximal inhibition). These inhibitory effects were antagonized by the α2-adrenoceptor antagonist yohimbine. Noradrenaline (0.1 μM) reduced acetylcholine-evoked overflow to the same extent as did UK 14,304 (0.1 μM). UK 14,304 had no effect when3H overflow was evoked by acetylcholine in the presence of 300 μM Cd2+. Currents through nicotinic acetylcholine receptors and voltage-activated Ca2+ currents were studied with the whole-cell variant of the patch-clamp technique. UK 14,304 reduced nicotinic acetylcholine receptor currents and voltage-activated Ca2+ currents with similar potency and efficacy. Yohimbine, however, antagonized only the inhibition of voltage-activated Ca2+ currents, but not the effects of UK 14,304 on nicotinic receptor currents. Furthermore, yohimbine per se reduced currents through nicotinic receptors. Noradrenaline (10 μM) inhibited voltage-dependent Ca2+ currents just as did UK 14,304 (10 μM), but failed to reduce currents through nicotinic acetylcholine receptor channels. Cd2+ (300 μM) abolished voltage-activated Ca2+ currents and reduced nicotinic acetylcholine receptor currents by 65%.
These results indicate that acetylcholine evokes noradrenaline release from rat sympathetic neurons by activation of nicotinic receptors and restricts this release via muscarinic receptors. The acetylcholine-induced transmitter release is based on two mechanisms, one involving and the other one bypassing voltage-dependent Ca2+ channels. α2-Adrenoceptor activation reduces voltage-activated Ca2+ currents and affects exclusively the component of acetylcholine-induced release which involves voltage-dependent Ca2+ channels. These results support the hypothesis that voltage-activated Ca2+ channels are the sole site of autoinhibitory α2-adrenergic effects on transmitter release from rat sympathetic neurons. The inhibitory effects of α2-adrenoceptor agonists and antagonists on currents through nicotinic acetylcholine receptors are not mediated by an α2-adrenoceptor.
Mechanism of long-lasting block of ganglion nicotinic receptors by mono-ammonium compounds with long aliphatic chain
1994, Journal of the Autonomic Nervous SystemThe effect of long-chain mono-ammonium compounds (long-chain MACs), t-butyldecylammonium (IEM-1078), 2,2,6,6-tetramethyldecylpiperidine (IEM-1559), and diisopropyldecylammonium (IEM-1194), on nicotinic acetylcholine receptors (nAChRs) was studied in sympathetic ganglion neurons using the patch clamp method. Long-chain MACs (1–10 μM) strongly inhibited acetylcholine (ACh)-induced current (ACh-current); the block persisted for hours after washing the drugs out. Short-chain MACs had a much weaker and completely reversible blocking effect. Suppression of ACh-current by MACs was dose- and voltage-dependent; it was absent at low ACh doses or at potentials ≥ 60 mV and increased with higher ACh doses or hyperpolarization. The second of two ACh-currents induced by paired application of ACh was inhibited by long-chain MACs more strongly than the first. This use-dependent block also persisted for hours after washing the drugs out. Additional inhibition of the second ACh-current was reduced by lengthening the time interval between ACh applications in the pair. Time constants of the recovery of the second ACh-current in the presence and after washing out of long-chain MACs were similar, ranging from 45 to 140 s at −50 mV for different long-chain MACs, and decreased with de- or hyperpolarization. The use-dependent block produced by long-chain MACs could be prevented by another long-chain MAC with a small ammonium head (IEM-1195, 75–100 μM) or trimethaphan (30 μM), a competitive antagonist of ACh in ganglia. Neither the short-chain MAC (IEM-1405, 100 μM) nor ACh (100 μM) could exert this protective effect. Long-chain MACs did not exert any use-, dose- or voltage-dependent suppression of ACh-current when applied intracellularly. Single-channel conductance was not affected by IEM-1194 (3–10 μM). We suggest that inhibition of ACh-current by long-chain MACs is accounted for by (i) a long-lasting, apparently irreversible, binding of the drug near the channel of nAChR via its long alipathic chain and (ii) a slow reversible block of the nAChR channel with the MAC's ammonium head.
MRNAs encoding muscarinic and substance p receptors in cultured sympathetic neurons are differentially regulated by LIF or CNTF
1994, Developmental BiologyLeukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have previously been shown to regulate neuronal choice of neurotransmitter. In this present study, these factors were shown to specifically and differentially regulate levels of both muscarinic (subtypes m1, m2, m3, m4, and m5) and substance P receptor (SPR) mRNAs in sympathetic neurons of the rat superior cervical ganglion (SCG) using solution hybridization/RNase protection analysis. In vivo, neonatal rat SCG expressed predominantly m2 (10.31 ± 0.43 pg mRNA/μg total RNA) and some ml (1.54 ± 0.84 pg/μg) muscarinic receptor mRNA, which increased developmentally to adult levels (m2 mRNA levels being 60% higher than those in neonates). By contrast, m3, m4, and m5 subtype mRNAs were much less abundant at all time points measured. A similar developmental regulation was found in dissociated SCG neurons in vitro. After 16 days in culture, m2 mRNA increased 334% to 15.76 ± 0.68 pg/μg, while m1 mRNA changed little (2.03 ± 1.00 pg/μg). However, LIF or CNTF treatment (5 ng/ml, 14 days) in sister cultures completely blocked this developmental increase. Further, LIF treatment blocked the normal muscarinic receptor-mediated increase in intracellular calcium (fura-2 imaging), indicating a functional change in receptor phenotype. By contrast, levels of SPR mRNA, which were low in untreated cultures (0.037 ± 0.025 pg SPR mRNA/μg total RNA), were elevated by LIF or CNTF treatment, to 0.866 ± 0.034 pg/μg and 0.662 ± 0.148 pg/μg, respectively. These observations indicate that muscarinic and SPR receptor expression are differentially regulated by the same factors in SCG neurons and that neuronal choice of receptor phenotype may be, at least in part, specifically regulated by cytokines/growth factors in the cellularmilieu.
Leukemia inhibitory factor and ciliary neurotrophic factor regulate expression of muscarinic receptors in cultured sympathetic neurons
1993, Developmental BiologyRegulation of muscarinic receptor expression was studied in cultured sympathetic neurons of the neonatal rat superior cervical ganglion (SCG). Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF), both previously shown to regulate neurotransmitter development in cultured SCG neurons (Yamamori et al., 1989; Saadat et al., 1989), were examined for effects on receptor expression. Exposure of SCG neurons to LIF or CNTF (5 ng/ml) prevented the normal developmental increase in muscarinic receptors as measured by whole cell binding of N -methyl[3H]scopolamine. Reduction in receptor binding was detected within 2 to 4 days of treatment, with a 65-80% reduction after 16 days. Scatchard analysis demonstrated a reduction in total receptor number (Bmax) with no significant change in receptor affinity (Kd). Concentrations of 1 ng/ml of either factor reduced receptor expression with near-maximal effectiveness at doses of 10 ng/ml. The decrease in muscarinic receptors was not blocked by atropine, indicating that it was not agonist induced. Treatment with LIF or CNTF did not affect the survival of cultured neurons. Further, effects on receptor expression were reversible after discontinuance of treatment. Finally, treatment with either factor increased overall protein synthesis, indicating the integrity of cellular metabolism of cultures and hence the specificity of the decrease in muscarinic receptor number. LIF and CNTF thus regulate receptor as well as neurotransmitter development and could therefore play a role during synaptogenesis in the developing nervous system.