Pharmacology of neuronal nicotinic acetylcholine receptor subtypes.

The search for the physiological function of nicotinic receptors on neurons in the brain began with their discovery. It was initially assumed that, as in ganglia and at the neuromuscular junction, nicotinic receptors would gate fast synaptic transmission in the brain. The best functional evidence now, however, points to a role in modifying the release of other transmitters. This does not preclude a postsynaptic role in transmission for nicotinic receptors in the brain, but attempts to locate such a synapse have not been successful. If fast nicotinic synapses are present in the brain, they are probably low in number and may be masked by other more prevalent synapses (such as glutamatergic) so identification will not be easy. The extent of diversity of nicotinic receptors is substantial. At the molecular level this is reflected in the number of different genes that encode receptor subunits and the multiple possible combinations of subunits that function in expression systems. From the cellular level there is a broad diversity of properties of native receptors in neurons. Some useful pharmacological tools allow the limited identification of subunits in native receptors. For example, block by alpha-bungarotoxin identifies alpha 7, alpha 8, or alpha 9 subunits; activation of a receptor by cytisine indicates an alpha 7 or beta 4 subunit; and neuronal bungarotoxin block identifies a beta 2 subunit. Despite the clues to identity gained by careful use of these agents, we have not been able to identify all the components of any native receptor based on pharmacological properties assessed from expression studies. When both pharmacological and biophysical properties of a receptor are taken into consideration, none of the combinations tested in oocytes mimics native receptors exactly. The reason for this discrepancy has been debated at length; it is possible that oocytes do not faithfully manufacture neuronal nicotinic receptors. For example, they may not correctly modify the protein after translation or they may allow a combination of subunits that do not occur in vivo. Another possibility is that correct combinations of subunits have not yet been tested in oocytes. Data from immunoprecipitation experiments suggest that many receptors contain three or more different subunits. Results from further experiments injecting combinations of three or more subunits into oocytes may be enlightening. The diversity of receptors may allow targeting of subtypes to specific locations. Nicotinic receptors are located presynaptically, preterminally, and on the cell soma. The function of the nicotinic receptors located on innervating axons is presumably to modify the release of other neurotransmitters. It is an attractive hypothesis that nicotinic receptors might be involved in modifying the weight of central synapses; however, in none of the regions where this phenomenon has been described is there any evidence for axoaxonal contacts. The presynaptic receptors described so far are pharmacologically unique; therefore, if there are different subtypes of nicotinic receptors modifying the release of different transmitters, they may provide a means of exogenously modifying the release of a particular transmitter with drugs. There are still many basic unanswered questions about nicotinic receptors in the brain. What are the compositions of native nicotinic receptors? What is their purpose on neurons? Although there is clearly a role presynaptically, what is the function of those located on the soma? Neuronal nicotinic receptors are highly permeable to calcium, unlike muscle nicotinic receptors, and this may have important implications for roles in synaptic plasticity and development. Finally, why is there such diversity? (ABSTRACT TRANCATED)

Pharmacological profiles of the GABA and acetylcholine receptors from the nematode, Ascaris suum.

The pharmacological profiles of the acetylcholine and GABA receptors of Ascaris muscle have been investigated. Acetylcholine excites the muscle through activation of a nicotinic receptor which resembles the mammalian ganglionic receptor. DMPP is a potent agonist and alpha-bungarotoxin is a weak antagonist. GABA inhibits the muscle through activation of a chloride-linked receptor which in terms of agonist profile resembles the mammalian GABA-A receptor. However, bicuculline is inactive and picrotoxin is a very weak antagonist. Avermectin acts as a non-competitive antagonist at this GABA receptor with an IC-50 value in the low microM range.

Alpha3, beta2, and beta4 form heterotrimeric neuronal nicotinic acetylcholine receptors in Xenopus oocytes.

One of the problems faced when using heterologous expression systems to study receptors is that the pharmacological and physiological properties of expressed receptors often differ from those of native receptors. In the case of neuronal nicotinic receptors, one or two subunit cDNAs are sufficient for expression of functional receptors in Xenopus oocytes. However, the stoichiometries of nicotinic receptors in neurons are not known and expression patterns of mRNA coding for different nicotinic receptor subunits often overlap. Consequently, one explanation for the discrepancy between properties of native versus heterologously expressed nicotinic receptors is that more than two types of subunit are necessary for correctly functioning receptors. The Xenopus oocyte expression system was used to test the hypothesis that more than two types of subunit can coassemble; specifically, can two different beta subunits assemble with an alpha subunit forming a receptor with unique pharmacological properties? We expressed combinations of cDNA coding for alpha3, beta2, and beta4 subunits. Beta2 and beta4, in pairwise combination with alpha3, are differentially sensitive to cytisine and neuronal bungarotoxin (nBTX). Alpha3beta4 receptors are activated by cytisine and are not blocked by low concentrations of nBTX; acetylcholine-evoked currents through alpha3beta2 receptors are blocked by both cytisine and low concentrations of nBTX. Coinjection of cDNA coding for alpha3, beta2, and beta4 into oocytes resulted in receptors that were activated by cytisine and blocked by nBTX, thus demonstrating inclusion of both beta2 and beta4 subunits in functional receptors.

The pharmacology of cholinoceptors on the somatic muscle cells of the parasitic nematode Ascaris suum.

1. Acetylcholine (ACh) elicited depolarization and an increase in input conductance of the somatic muscle cells of the parasitic nematode Ascaris suum. 2. The relative potency of nicotinic and muscarinic agents was studied in this preparation. The order of potency of these compounds was metahydroxy-phenylpropyltrimethylammonium (HPPT) greater than 1.1 dimethyl-4-phenylpiperazinium (DMPP) greater than ACh greater than carbachol greater than nicotine greater than tetramethylammonium (TMA+) greater than muscarone greater than furtrethonium greater than arecoline. Decamethonium was also a weak agonist. McN-A-343 elicited a very weak depolarization at concentrations above 1 mmol l-1. Bethanechol and methacholine were without effect up to 1 mmol l-1. Pilocarpine and muscarine elicited a slight hyperpolarization of up to 3 mV with a threshold for the response of around 500 mumol l-1. Oxotremorine (1 mmol l-1) was without effect. 3. The nitromethylene insecticide 2(nitromethylene)tetrahydro 1,3-thiazine (NMTHT), an agonist at insect nicotinic receptors, was without effect on Ascaris muscle cells up to 1 mmol l-1. 4. Mecamylamine and benzoquinonium were the most potent antagonists of the acetylcholine response. The order of potency of the other antagonists was tetraphenylphosphonium (TPP) greater than quinacrine greater than pancuronium, curare greater than trimethaphan greater than atropine greater than chlorisondamine, decamethonium greater than hexamethonium greater than dihydro-beta-erythroidine. 5. The agonist profile of the Ascaris muscle cell ACh receptor clearly indicates that it is nicotinic. The potency of ganglionic and neuromuscular nicotinic receptor antagonists in Ascaris does not enable a further subclassification of this nicotinic receptor. The Ascaris nicotinic receptor seems to possess some of the pharmacological properties of each type of vertebrate nicotinic receptor. The pharmacology of the Ascaris nicotinic receptor is discussed in relation to that of nicotinic receptors in other invertebrate preparations and in vertebrate preparations.

Calcium flux through predominantly independent purinergic ATP and nicotinic acetylcholine receptors.

Ligand-gated nicotinic acetylcholine receptors (nAChRs) and purinergic ATP receptors are often expressed in the same peripheral and central neurons, and ATP and acetylcholine (ACh) are stored together in some synaptic vesicles. Evidence has suggested that nAChRs and ATP receptors are not independent and that some agonists strongly cross-activate and desensitize both receptor types. Rat sympathetic neurons and nAChRs expressed in Xenopus oocytes were studied to determine the significance of the interactions caused by the two agonist types. Current amplitudes included with separate or combined applications of ATP and nicotine are > 90% additive and independent. Half of all neurons tested responded to either ATP or nicotine but not to both, indicating differences in the expression of the two receptors. In neurons that expressed both receptors types, the nAChRs were inhibited by the activity-dependent open-channel blocker chlorisondamine. If the purinergic and nicotinic receptors were significantly dependent and coactivated, then blocking the ion channels opened by a nicotinic agonist should diminish the current activated by a purinergic agonist. That result was not seen; rather, complete open-channel block of nAChRS with chlorisondamine did not significantly alter the amplitude or kinetics of ATP-induced currents in the same neurons. Finally, when cloned nAChR subunits were expressed in oocytes, ATP activated only very small currents compared with the current activated by Ach. For the 13 different nAChR subunit combinations that were studied, ATP (50-500 microM) activated a current that ranged from 0 to 4% of the size of the current activated by 100 microM ACh. In summary, we find that there is little cross reactivity, and nAChRs and purinergic ATP receptors are predominantly independent, acting with separable physiological characteristics. Therefore the quantitative Ca2+ flux could be separately determined for nAChRs and ATP receptors. The fraction of total current that is carried by Ca2+ was quantitatively determined by simultaneously measuring the whole cell current and the associated change in intracellular Ca2+ with fura-2. For sympathetic neurons bathed in 2.5 mM Ca2+ at a holding potential of -50 mV, Ca2+ carries 4.8 +/- 0.3% (mean +/- SE) of the inward current through neuronal nAChRs and 6.5 +/- 0.1% of the current through purinergic ATP receptors. In conclusion, activity-dependent Ca2+ influx through predominately independent populations of nAChRS and ATP neurons can produce different intracellular signals at purinergic and cholinergic synapses.