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M Quik

Publications and source records attributed to M Quik.

At least 37 records · Page 2Linked to original sources
PubMed2000

Differential alterations in nicotinic receptor alpha6 and beta3 subunit messenger RNAs in monkey substantia nigra after nigrostriatal degeneration.

Our previous studies showed that alpha4, alpha6, alpha7, beta2, beta3 and beta4 nicotinic acetylcholine receptor messenger RNAs are present in monkey substantia nigra, with a particularly intense and localized labelling of the alpha6 and beta3 subunit messenger RNAs to this brain region. Because loss of nigrostriatal neurons is a central feature of Parkinson's disease and evidence suggests that nicotinic agonists potentiate antiparkinsonian effects of L-dopa, experiments were done to determine whether nicotinic receptor subunit messenger RNAs and binding sites were altered in the basal ganglia after nigrostriatal degeneration. Squirrel monkeys (Saimiri sciureus) were rendered parkinsonian by systemic injection of the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine. Behavioral studies showed that this treatment decreased baseline motor activity to 36+/-11% of control. One month after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, caudate and putamen dopamine levels were reduced to 51+/-15% and 43+/-6% of control, respectively, while the number of tyrosine hydroxylase-positive neurons in the substantia nigra was 75+/-6% of control. Despite the reduction in nigral cell number after nigrostriatal degeneration, there were no changes in alpha4, alpha7, beta2 and beta4 messenger RNA levels in the substantia nigra. In contrast, alpha6 mRNA levels were significantly increased (143+/-10%) and the beta3 transcript decreased (62+/-6%) in the substantia nigra after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Declines were also observed in [125I]epibatidine binding in both the caudate-putamen and substantia nigra, with no change in alpha7 receptor binding. These results may suggest a dissociation in the regulation of receptor messenger RNA and binding sites, and/or that there are differential alterations in the different receptor subtypes measured using [125I]epibatidine. The changes in the two nicotinic receptor subunit messenger RNAs, alpha6 and beta3, which exhibit a selective localization to the substantia nigra, may indicate that nicotinic receptors containing these subunits are altered after nigrostriatal degeneration.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

Investigating levodopa-induced dyskinesias in the parkinsonian primate.

Research into the cause of dyskinesias arising from levodopa treatment has been vexingly limited, partly due to the lack of an inexpensive and widely available animal model. Rodents do not develop levodopa-induced dyskinesias in a clinically recognizable form. However, nonhuman primates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism readily develop levodopa-induced dyskinesias that are virtually indistinguishable from those seen in patients with Parkinson's disease. We have developed and validated a five-point Global Primate Dyskinesia Rating Scale to accurately measure these dyskinesias. Monkeys with MPTP-induced parkinsonism were then investigated to evaluate the relationship between dyskinesias, parkinsonism and severity of the nigrostriatal lesion. All parkinsonian animals were responsive to levodopa, and developed dyskinesias within 2-3 days of levodopa administration. Monkeys treated with only a single injection of MPTP also developed dyskinesias, even though they were not parkinsonian. It would appear that there is a different threshold of striatal dopamine depletion for parkinsonism and dyskinesias in the monkey. Finally, three hypotheses, put forward to explain the genesis of dyskinesias, are reviewed, and various experimental approaches suggested for each.

Animals↗
PubMed2000

Nitric oxide is involved in acetylcholinesterase inhibitor-induced myopathy in rats.

Excess activation of muscle nicotinic acetylcholine receptors due to genetic mutations, as seen in slow channel congenital myasthenic syndrome, or acetylcholinesterase (AChE) inhibition results in muscle cell degeneration. Our recent work showed that nitric oxide synthase (NOS) inhibitors prevent nicotine-induced muscle cell death in culture. In the present study, we examined the effects of NOS inhibition on nicotinic receptor-mediated myopathy in vivo. Rats injected with the AChE inhibitor paraoxon demonstrate a 90-fold increase in the number of dying muscle cells compared with control as evidenced histologically by centralized nuclei and the presence of degenerating profiles. Coadministration of the nonspecific NOS inhibitor nitro-L-arginine methyl ester or the neuronal NOS-specific inhibitor 7-nitroindazole dramatically reduced the presence of such degenerating profiles to approximately 20% of that seen with paraoxon alone. These results show that inhibition of NOS, as well as neuronal NOS, significantly reduces AChE inhibitor-induced muscle cell degeneration, suggesting that increased nitric oxide production mediates such myopathy.

Animals↗
PubMed1997

Modulation of alpha7 nicotinic receptor-mediated calcium influx by nicotinic agonists.

Our previous work had demonstrated stable expression of rat alpha7 alpha-bungarotoxin (alpha-BGT) binding sites by GH4C1 rat pituitary cells, a clonal line that does not endogenously express nicotinic receptors. The stably expressed alpha7 sites had similar binding affinities, pharmacological profiles, kinetic properties, and molecular size as rat brain alpha-BGT receptors, suggesting that they represent a good model system for studying receptor function. The present data show that nicotinic receptor agonists increase intracellular calcium levels ([Ca2+]i), as assessed using Fura-2, in alpha7/GH4C1 cells in a dose-dependent manner with EC50 values that correlate well with the affinity of these ligands for alpha7/GH4C, alpha-BGT receptors. Nicotinic receptor antagonists inhibited agonist-induced increases in [Ca2+]i, with IC50 values in the nanomolar to micromolar range. The nicotinic agonist-induced increase in [Ca2+]i required extracellular calcium and did not occur in the presence of CdCl2, suggesting that agonist-induced increases in [Ca2+]i are due to an influx of extracellular calcium through voltage-gated calcium channels. Preexposure of the alpha7/GH4C1 cells to 8-bromo cAMP resulted in an enhanced [Ca2+]i in response to agonist, suggesting that phosphorylation by adenylate cyclase may regulate receptor responsiveness. Interestingly, short-term preexposure (40-60 sec) of the cells to subthreshold concentrations of nicotinic agonist-enhanced receptor-stimulated calcium influx (up to 55%) while activating agonist concentrations completely blocked receptor-mediated responses. Long-term exposure of alpha7/GH4C1 cells to K+ resulted in about a 2-fold increase in alpha-BGT receptors and in agonist-evoked calcium influx. The sensitivity of these up-regulated receptors were modulated by subthreshold and activating concentrations of agonist in a manner similar to control receptors. The present results, demonstrating a biphasic regulation of alpha7 receptor-mediated calcium influx by nicotinic agonists, suggest that these receptors may play an important role in neuronal function under control and depolarizing conditions.

Animals↗
PubMed1997

Involvement of nitric oxide in nicotinic receptor-mediated myopathy.

Previous studies have shown that nicotinic cholinergic agonists induce muscle cell degeneration. Although an involvement of calcium is well documented, subsequent intracellular steps have not been identified. The present experiments test whether nitric oxide (NO) may play such a role. Both the irreversible nitric oxide synthase inhibitor L-5N-iminoethyl ornithine and L-nitroarginine methyl ester, a reversible inhibitor, protected the muscle cells from the myopathic effects of nicotine. These results may suggest that nicotinic receptor stimulation produces an increase in NO that results in muscle cell degeneration. In line with this interpretation, exposure of the muscle cultures to the NO donor sodium nitroprusside resulted in a dose-dependent decline in myotube branch points. Neither L-5N-iminoethyl ornithine nor nitroprusside altered the binding of the nicotinic receptor agonist 125I-alpha-bungarotoxin to muscle cells in culture, which indicates that the effect of these agents was not mediated through an interaction at the nicotinic receptor recognition site. The results with agents that inhibit guanylate cyclase or modify extracellular levels of cGMP suggest an involvement of this cyclic nucleotide in the nicotinic receptor-mediated myopathy. To conclude, the present results suggest that nicotinic receptor activation causes skeletal muscle degeneration through an increase in NO production and a possible involvement of cGMP.

Animals↗
PubMed1996

Characterization of nicotinic receptors in immortalized hippocampal neurons.

Nicotinic acetylcholine receptors, particularly nicotinic alpha-bungarotoxin (alpha-BGT) receptors, are present in relatively high concentrations in rat hippocampus. Because of the difficulties encountered in studying receptors using primary cells in culture, especially for biochemical work, we investigated the possibility of using an immortalized cell line from embryonic rat hippocampus (H19-7). RNase protection assays show that alpha 4, alpha 7 and beta 2 neuronal nicotinic receptor subunit mRNAs are present in differentiated but not undifferentiated H19-7 cells, while alpha 2, alpha 3, alpha 5 and beta 3 subunit mRNAs were not detectable under either condition. In line with these results, the present data demonstrate that the H19-7 cells express cell surface nicotinic alpha-BGT binding sites, which were maximal after seven days of differentiation in culture. The receptors were saturable, of high affinity (Kd = 1.30 nM and Bmax = 11.70 fmol/10(5) cells) and had a pharmacological profile similar to that observed for CNS alpha-BGT receptors. On the other hand, although alpha 4 and beta 2 neuronal nicotinic subunit mRNAs were present in differentiated H19-7 cells, no [3H]cytisine binding was observed. Because immortalized cell lines have the advantage that they provide a limitless supply of cells as compared to primary cell cultures, but yet are not malignant in origin, the present results may suggest that the H19-7 immortalized hippocampal cell line represent a useful CNS model system for examining alpha-BGT nicotinic receptors.

Alkaloids↗
PubMed1996

Similarity between rat brain nicotinic alpha-bungarotoxin receptors and stably expressed alpha-bungarotoxin binding sites.

The present results demonstrate stable expression of alpha-bungarotoxin (alpha-BGT) binding sites by cells of the GH4C1 rat pituitary clonal line. Wild-type GH4C1 cells do not express alpha-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor alpha2, alpha3, alpha4, alpha5, alpha7, beta2, or beta3 subunits. However, GH4C1 cells stably transfected with rat nicotinic receptor alpha7 cDNA (alpha7/GH4C1 cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of teh predicted size. The alpha7/GH4C1 cells also express saturable, high-affinity binding sites for 125I-labeled alpha-BGT, with a KD of 0.4 nM and Bmax of 3.2 fmol/10(6) intact cells. 125I-alpha-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or alpha7/GH4C1 cells. Furthermore, KD and Ki values for 125I-alpha-BGT binding sites on intact alpha7/GH4C1 cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the alpha-BGT binding sites expressed in alpha7/GH4C1 cells was similar to that of the native brain alpha-BGT receptor. Chronic exposure of alpha7/GH4C1 cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of alpha-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of alpha-BGT binding sites in transfected alpha7/GH4C1 cells resemble those for brain nicotinic alpha-BGT receptors. If the heterologously expressed alpha-BGT binding sites in the present study are composed solely of alpha7 subunits, the results could suggest that the rat brain alpha-BGT receptor has a similar homooligomeric structure. Alternatively, if alpha-BGT binding sites exist as heterooligomers of alpha7 plus some other previously identified or novel subunit(s), the data would indicate that the alpha7 subunits play a major role in determining properties of the alpha-BGT receptor.

3T3 Cells↗
PubMed1995

Production of polyclonal antisera that recognize and distinguish between the extracellular domains of neuronal nicotinic acetylcholine receptor subunits.

Ligand-gated ion channels are oligomeric transmembrane proteins that usually contain more than one kind of monomer. The variety of monomers available to participate in oligomer formation and the apparent latitude in acceptable monomer combinations allows considerable diversity. Mechanisms for identifying the monomers comprising specific receptors are needed. We have generated affinity-purified polyclonal antisera that recognize the extracellular domain of nine neuronal nicotinic acetylcholine receptor (nAChR) subunits and distinguish between them. We prepared these antisera by immunizing rabbits with bacterially expressed recombinant protein representing the N-terminal extracellular domain of each neuronal nAChR subunit followed by affinity purification of antibodies against synthetic peptides corresponding to residues 68-81 of the alpha 1 subunit. We demonstrate subunit specificity of each affinity-purified antisera by western blots of the bacterially expressed protein and immunoblot against peptide. We further used these antibodies to demonstrate expression of neuronal nAChR subunits on the surface of transiently transfected simian kidney (COS-7) cells.

Amino Acid Sequence↗
PubMed1994

alpha-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). Interestingly, both the nicotine and cytisine induced increases in H69 cell number were blocked by alpha-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 alpha-bungarotoxin receptor. This idea is further supported by experiments which show (1) that H69 cells possess high affinity alpha-bungarotoxin sites (Kd = 25 nM, Bmax = 10.4 fmol/10(6) cells) with the characteristics of a nicotinic alpha-bungarotoxin receptor and (2) that the potencies of nicotinic receptor ligands in the alpha-bungarotoxin binding assay were similar to those observed in the functional studies. Northern analysis showed that mRNA for alpha 7, a putative nicotinic alpha-bungarotoxin binding subunit, and for alpha 5 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 alpha-bungarotoxin receptor population. These results suggest that the alpha-bungarotoxin site may be involved in modulating proliferative responses in neuroendocrine derived SCLC cells.

Alkaloids↗
PubMed1994

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

The snake toxin alpha-bungarotoxin distinguishes between neuronal nicotinic receptor subtypes. In chick ciliary ganglion neurons, activation of alpha-bungarotoxin-sensitive nicotinic receptors has been proposed to produce elevations in intracellular calcium levels. In the present study we show that prolonged treatment with alpha-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 alpha-bungarotoxin. These results suggest that nicotinic alpha-bungarotoxin receptors are not primarily involved in nicotine-mediated increases in intracellular calcium in bovine adrenal medullary cells.

Adrenal Medulla↗
PubMed1994

The pharmacology of the nicotinic antagonist, chlorisondamine, investigated in rat brain and autonomic ganglion.

1. A single administration of the ganglion blocker, chlorisondamine (10 mg kg-1, s.c.) is known to produce a quasi-irreversible blockade of the central actions of nicotine in the rat. The mechanism of this persistent action is not known. It is also unclear whether chlorisondamine can block neuronal responses to excitatory amino acids and whether chronic blockade of nicotinic responses also occurs in the periphery. 2. Acute administration of chlorisondamine (10 mg kg-1, s.c.) to rats resulted in a blockade of central nicotinic effects (ataxia and prostration) when tested 1 to 14 days later, but caused no detectable cell death in tissue sections sampled throughout the rostrocaudal extent of the brain which were stained in order to reveal neuronal degeneration. 3. Long-term blockade of central nicotinic effects by chlorisondamine was not associated with significant alterations in the density (Bmax) of high-affinity [3H]-nicotine binding to forebrain cryostat-cut sections. 4. In cultured dissociated mesencephalic cells of the foetal rat, chlorisondamine and mecamylamine inhibited [3H]-dopamine release evoked by N-methyl-D-aspartate (NMDA, 10(-4) M), but only at high concentrations (IC50 approx. 600 and 70 microM, respectively). A high concentration of chlorisondamine (10(-3) M) had no effect on responses to quisqualate (10(-5) M) and only slightly reduced responses to kainate (10(-4) M). Mecamylamine (10(-3) M) was ineffective against both agonists. 5. In adult rat hippocampal slices, chlorisondamine depressed NMDA receptor-mediated synaptically-evoked field potentials, but again only at high concentrations (10(-4)-10(-3) M). Synaptic responses that were mediated by non-NMDA excitatory amino acid receptors were less affected. 6. In rat isolated superior cervical ganglion, electrically-evoked synaptic transmission was reduced 1 h after acute in vivo administration of chlorisondamine (0.1 mg kg-1, s.c.). However, in vivo administration of a higher dose (10 mg kg-1, s.c.) did not significantly affect ganglionic transmission when tested two weeks later, despite the continued presence of central nicotinic blockade.7. These results indicate that the persistent CNS nicotinic blockade by chlorisondamine is not accompanied by changes in nicotinic [3H]-nicotine binding site density or by neuronal degeneration in the brain; that at doses sufficient to produce nicotinic receptor blockade, chlorisondamine acts in a pharmacologically selective manner; and that chronic central blockade is not accompanied by long-term peripheral ganglionic blockade.

Amino Acids↗
PubMed1993

Modulation of nicotinic acetylcholine receptor function in bovine adrenal chromaffin cells by thymopentin.

Thymopentin is a five amino acid peptide which corresponds to amino acids 32-36 of the thymic polypeptide thymopoietin. Previous work had shown that TP-5 could modulate responsiveness at the muscle-type nicotinic receptor. The present studies show that neuronal nicotinic receptor function, measured as radiolabelled noradrenaline release from bovine adrenal medullary cells, was attenuated by thymopentin. The pentapeptide exhibited specificity for nicotinic receptor evoked release, since thymopentin did not affect potassium-stimulated secretion of [3H]noradrenaline. It appears, therefore, that thymopentin antagonizes catecholamine release by specifically modulating nicotinic receptor responsiveness.

Acetylcholine↗