Differential effects of physostigmine and organophosphates on nicotinic receptors in neuronal cells of different species.

The effects of the carbamate physostigmine and of the organophosphates (OPs) parathion, paraoxon and phenyl saligenin cyclic phosphate (PSP) were examined on different subtypes of neuronal nicotinic acetylcholine receptors (nAChR). Stimulation with 1 mM ACh induced transient nicotinic inward currents in mouse N1E-115 and human SH-SY5Y neuroblastoma and in locust thoracic ganglion cells. All four acetylcholinesterase (AChE) inhibitors reduced the nicotinic currents in a concentration-dependent manner. Parathion is about 50 times more potent in blocking nAChR, compared to its active AChE inhibiting metabolite paraoxon. The relative blocking potency of the different AChE inhibitors was the same in all cell types, and followed the order parathion > physostigmine > PSP > paraoxon. In N1E-115 cells the IC50 values of block amounted to 2 microM, 30 microM, 39 microM and 96 microM for parathion, physostigmine, PSP and paraoxon, respectively. In all cell types, the nicotinic currents were equally blocked by parathion. Human nAChR in SH-SY5Y cells appeared more sensitive to block by physostigmine, PSP and paraoxon, while these AChE inhibitors similarly inhibited nicotinic currents in insect cells and in mouse neuroblastoma cells. The observation that the concentration-dependence of block is different from that of AChE inhibition, indicates a distinct interaction of AChE inhibitors with nAChR. Only in locust cells physostigmine induced a non-desensitizing inward current, that appeared to originate from nAChR activation. Occasionally, the OPs were able to activate slow ionic currents in mouse, but not in human and locust cells. As the OP-induced agonistic activity in mouse cells was not associated with the blocking action, the target site appeared to be distinct from nAChR. These results show that AChE inhibitors block nAChR with different potencies, dependent on the compound and the receptor subtype, and may activate distinct ion currents in neuronal cells of different species origin.

Physostigmine and acetylcholine differentially activate nicotinic receptor subpopulations in Locusta migratoria neurons.

The effects of acetylcholine (ACh) and physostigmine (PHY) on thoracic ganglion neurons of Locusta migratoria were investigated using whole-cell and cell-attached voltage clamp. ACh activated whole-cell currents with variable amplitudes, time course and ion channel block between cells, suggesting differential expression of nicotinic acetylcholine receptor (nAChR) subtypes. This was supported by selective block of the peak of the currents by the alpha7-specific alpha-conotoxin ImI. PHY at 100 microM evoked smaller whole-cell currents with variable amplitudes and marginal desensitization. The PHY/ACh amplitude ratio varied between cells, and was positively related to the time constant of decay of the ACh response. EC50 values for the peak amplitude of the ACh- and PHY-induced currents were 50 microM and 3 microM, respectively. Both agonists activated nAChR, indicated by equal voltage-dependence and reversal potentials and the same pharmacological properties of ACh and PHY responses. In addition, PHY and ACh induced ion channel block. Co-application and cross-desensitization experiments showed that ACh and PHY activate the same nAChR subpopulations. Both agonists activated nicotinic single channels with three conductance levels, which were equal for ACh and PHY, indicating activation of the same nAChR subtypes by both agonists. However, for all levels PHY displayed a lower open probability than ACh. Taken together, different whole-cell responses appear to originate from differential activation, desensitization and ion channel block by ACh and PHY of distinct nAChR populations.

Differential muscarinic receptor binding of acetylcholinesterase inhibitors in rat brain, human brain and Chinese hamster ovary cells expressing human receptors.

Displacement of muscarinic radioligands by the cholinesterase inhibitors parathion, paraoxon, physostigmine and phenyl saligenin cyclic phosphate was examined in rat cortex and brain stem, human cortex and brain stem, and in Chinese hamster ovary (CHO) cells expressing human M2 or M4 muscarinic acetylcholine receptors. None of the cholinesterase inhibitors tested significantly affected binding of the antagonist [3H]quinuclinidyl benzilate. However, the agonist [3H]oxotremorine-methiodide (3H]oxo-M) was displaced by all compounds tested in a differential manner. Parathion only marginally displaced [H]oxo-M binding with pKi values < 5 in all tissue or cell types. In rat brain paraoxon, physostigmine and phenyl saligenin cyclic phosphate displaced [3H]oxo-M with pKi values of 7.5, 7.0 and 6.1, respectively. The cholinesterase inhibitors displaced [3H]oxo-M in human brain at 15- to 250-fold higher concentrations, that is with pKi values of 6.3, 4.6 and 4.2, respectively. Maximal displacement of [3H]oxo-M varied between 25% and 95%, depending on the species and the compound. Human receptors in brain and in CHO cells were equally sensitive to displacement of [3H]oxo-M by parathion, physostigmine and phenyl saligenin cyclic phosphate. However, paraoxon displaced [3H]oxo-M at > or = 35-fold lower concentrations from human receptors in brain than in CHO cells. In conclusion, the data show that cholinesterase inhibitors interfere with agonist binding to muscarinic acetylcholine receptors. The species-selectivity of the displacement appears to result from differences between rat and human muscarinic acetylcholine receptors. In addition, for paraoxon marked differences exist between the sensitivity of human muscarinic acetylcholine receptors in brain tissue and of those expressed in clonal CHO cells.

In vitro approaches to species and receptor diversity in cellular neurotoxicology.

Effects of selective and non-selective neurotoxic compounds on membrane currents mediated by nicotinic acetylcholine receptors (nAChR), natively expressed in cultured cells and artificially expressed in Xenopus oocytes, have been investigated in vitro using voltage clamp techniques. Mammalian neuronal nAChR in cultured mouse N1E-115 cells, muscle type nAChR in cultured mouse BC3H1 cells and insect neuronal nAChR in dissociated locust thoracic ganglion neurons show interspecies differences in sensitivity to neurotoxic compounds. The nitromethylene heterocyclic insecticide WL145004 and physostigmine selectively agonize the insect type nAChR. The mouse neuronal and muscle types of nAChR are much less sensitive to and are partially inhibited by WL 145004. Intraspecies differences have been investigated for the effects of Pb(2+) on subtypes of nAChR expressed in Xenopus oocytes. The nature of the effect of the heavy metal Pb(2+) depends on the combination of mammalian neuronal a and beta nAChR subunits. Ion currents mediated by alpha4beta2 and alpha3beta4 nAChR are inhibited and those mediated by alpha3beta2 nAChR are potentiated by Pb(2+). Distinct sensitivities of subtypes of mammalian neuronal nAChR to agonists and antagonists are employed to characterize native nAChR in N 1E-115 cells. Implications of receptor diversity for neurotoxicology are discussed.

Can human polyclonal immunoglobulin raise the threshold for convulsions in rats?

In the present preliminary study we explored the possibility of an anticonvulsant effect of normal human immunoglobulin in an animal epilepsy model based on direct cortical stimulation in freely moving rats. After human immunoglobulin administration a significant and prolonged elevation of the threshold for convulsions was measured in 12% (6/49) of the total group of outbred Wistar rats. In the subgroup of more than seven months old Wistar rats this was 67% (6/9). When a threshold increasing effect of immunoglobulin occurred, it was detectable within 0.5-1 hour after administration, reached its maximum after approximately two hours and continued for at least 40 hours.