Fluoxetine (Prozac), a widely used antidepressant, is said to exert its medicinal effects almost exclusively by blocking the serotonin uptake systems. The present study shows that both muscle and neuronal nicotinic acetylcholine receptors are blocked, in a noncompetitive and voltage-dependent way, by f luoxetine, which also increases the rate of desensitization of the nicotinic receptors. Because these receptors are very widely distributed in the both central and peripheral nervous systems, the blocking action of f luoxetine on nicotinic receptors may play an important role in its antidepressant and other therapeutical effects. Our findings will help to understand the mode of action of f luoxetine, and they may also help to develop more specific medicinal drugs.Nicotinic acetylcholine receptors (nAcChoRs) mediate the transmission of signals across the vertebrate neuromuscular junction as well as across central and peripheral synapses (1, 2). Another widely distributed system is that in which serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter (3, 4), and it is now evident that the two systems are not completely specific. For example, dopamine activates 5HT receptors (5), and there are marked cross-interactions between serotonergic and cholinergic systems. For instance, atropine, a muscarinic antagonist, blocks both acetylcholine (AcCho) receptors and 5HT receptors in snail neurons (6); dihydro-␤-erythroidine and tubocurarine, nicotinic antagonists, block 5HT 3 receptors (7) and serotonin, as well as various serotonergic agonists and antagonists, block in a noncompetitive manner both muscle and neuronal nAcChoRs (8-10). Finally, 5HT acts as an antagonist on wild-type neuronal ␣ 7 nAcChoR, but it acts as an agonist on the ␣ 7 nAcChoR mutated in the M2 transmembrane region (11,12). Because fluoxetine (Prozac), a highly efficient inhibitor of 5HT uptake, is extensively used in the treatment of depression, eating disorders, and other diseases of the brain (13), we decided to see if, like other serotonergic agents, fluoxetine would alter the function of nAcChoRs. MATERIALS AND METHODSThe experiments were performed on voltage-clamped Xenopus oocytes expressing muscle ␣ 1 ␤ 1 ␥␦ or neuronal (␣ 2 ␤ 4 or ␣ 3 ␤ 4 ) nAcChoRs. The complementary RNA preparation, oocyte injection, and electrophysiological recordings have been described elsewhere (8, 9). Briefly, RNAs encoding mouse muscle (␣ 1 , ␤ 1 , ␥, and ␦) or rat neuronal (␣ 2 , ␣ 3 , and ␤ 4 ) nAcChoR subunits were transcribed in vitro. Equal quantities of cRNA subunits were combined to obtain ␣ 1 ␤ 1 ␥␦, ␣ 2 ␤ 4 , and ␣ 3 ␤ 4 nAcChoR subtypes. Xenopus laevis oocytes were dissected from the ovary and maintained at 16ЊC in Barth's solution containing 88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO 3 ) 2 , 0.41 mM CaCl 2 , 0.82 mM MgSO 4 , 2.4 mM NaHCO 3 , 5 mM Hepes (pH 7.4; NaOH), and 0.1 mg͞ml gentamicin sulfate. The next day each oocyte was injected with 0.5-50 ng of the corresponding cRNA mixture in a volume of 50 nl water, and about 2 days later the follicullar ...

In Xenopus oocytes expressing neuronal nicotinic acetylcholine receptors (nAcChoRs), made up of a2 and f84 subunits, acetylcholine (AcCho) elicited ionic membrane currents (AcCho currents) that were modulated by serotonergic agents. Both agonists and antagonists specific for various serotonin (5-hydroxytryptamine, 5HT) receptor subtypes interacted directly with a2,B4 nAcChoRs: 5HT, (±)-8-hydroxy-2-(di-n-propylamino)tetralin, methysergide, spiperone, and ketanserin reversibly reduced the amplitude of AcCho currents and accelerated their decay. The AcChocurrent time course decayed with two exponential functions. In the presence of 5HT, the fast time constant of current decay (Tf) was not greatly modified, but the slow time constant (Ts) was reduced. With AcCho and 5HT both at 100 ,uM, Ts was reduced from 140 s to 85 s. The order of potency for inhibition of AcCho current amplitudes was (+)-8-hydroxy-2-(di-npropylamino)tetralin > methysergide > spiperone > ketanserin > 5HT. The inhibition was voltage-dependent but the magnitude of the voltage dependence for the different blockers did not correspond to their blocking potency: e.g., the block with spiperone was stronger than with 5HT, but it was less voltage-dependent. Our results suggest that serotonergic agents block neuronal nAcChoRs in a noncompetitive manner, similar to the block of muscle nAcChoR by curare and other substances. These results show that neuronal nAcChoR channels that have been activated by their specific neurotransmitter may be modulated by nonspecific neurotransmitters and their antagonists. These effects may help to better understand brain functions as well as the mode of action of the many serotonergic agents that are used in medical practice.The nicotinic acetylcholine receptor (nAcChoR) is a ligandgated ion channel that opens after it binds the neurotransmitter acetylcholine (AcCho) and that mediates synaptic transmission between nerve and muscle as well as between neurons in the peripheral and central nervous systems. The muscle nAcChoR is a pentameric structure composed of four homologous subunits (a, /3, y or £, and 8) with a stoichiometry of a2P/Y8, whereas nAcChoRs expressed in the peripheral and central nervous systems appear to be composed of two different subunits, one a-like and the other /3-like, with a probable stoichiometry of a2133 (1, 2).The function of nAcChoRs can be modified not only by their specific neurotransmitter and related agonists and antagonists, but also by other neurotransmitters such as serotonin (5-hydroxytryptamine, 5HT) (3, 4) and substance P (5, 6).However, the mechanisms involved in the modulation of nAcChoRs by 5HT are still not well understood. In this paper, we report that neuronal nAcChoRs are affected by serotonergic agents in much the same way that 5HT acts on muscle and Torpedo nAcChoRs (4). These agents act on nAcChoRs in a noncompetitive and voltage-dependent way, probably blocking the open nAcChoR channel complex, as has been proposed for the actions of curare (7, 8), substance P (6), ni...

No abstract

Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin The structure-function relationship of muscle AcChoRs has been studied by altering their glycosylation, subunit structure, and subunit composition. These changes were found to alter the levels of receptor expression (4), receptor assembly and membrane incorporation (5-10), agonist sensitivity (11-13), and channel gating or desensitization (14)(15)(16)(17)(18)(19)

A study was made of the effects of several monoamine-uptake inhibitors on membrane currents elicited by acetylcholine (ACh-currents) generated by rat neuronal ␣2␤4 and mouse muscle nicotinic acetylcholine receptors (AChRs) expressed in Xenopus laevis oocytes. For the two types of receptors the monoamine-uptake inhibitors reduced the ACh-currents albeit to different degrees. The order of inhibitory potency was norfluoxetine Ͼ clomipramine Ͼ indatraline Ͼ fluoxetine Ͼ imipramine Ͼ zimelidine Ͼ 6-nitro-quipazine Ͼ trazodone for neuronal ␣2␤4 AChRs, and norfluoxetine Ͼ fluoxetine Ͼ imipramine Ͼ clomipramine Ͼ indatraline Ͼ zimelidine Ͼ trazodone Ͼ 6-nitro-quipazine for muscle AChRs. Thus, the most potent inhibitor was norfluoxetine, whilst the weakest ones were trazodone, 6-nitro-quipazine and zimelidine. Effects of the tricyclic antidepressant imipramine were studied in more detail. Imipramine inhibited reversibly and non-competitively the ACh-current with a similar inhibiting potency for both neuronal ␣2␤4 and muscle AChRs. The half-inhibitory concentrations of imipramine were 3.65 ± 0.30 M for neuronal ␣2␤4 and 5.57 ± 0.19 M for muscle receptors. The corresponding Hill coefficients were 0.73 and 1.2 respectively. The inhibition of imipramine was slightly voltage-dependent, with electric distances of ෂ0.10 and ෂ0.12 for neuronal ␣2␤4 and muscle AChRs respectively. Moreover, imipramine accelerated the rate of decay of AChcurrents of both muscle and neuronal AChRs. The ACh-current inhibition was stronger when oocytes, expressing neuronal ␣2␤4 or muscle receptors, were preincubated with imipramine alone than when it was applied after the ACh-current had been generated, suggesting that imipramine acts also on non-activated or closed AChRs. We conclude that monoamine-uptake inhibitors reduce ACh-currents and that imipramine regulates reversibly and noncompetitively neuronal ␣2␤4 and muscle AChRs through similar mechanisms, perhaps by interacting externally on a non-conducting state of the AChR and by blocking the open receptor-channel complex close to the vestibule of the channel. These studies may be important for understanding the regulation of AChRs as well as for understanding antidepressant-and side-effects of monoamine-uptake inhibitors. Molecular Psychiatry (2001) 6, 511-519.

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (␣ 1 ␤ 1 ␥␦, ␣ 1 ␤ 1 ␥, and ␣ 1 ␤ 1 ␦) and neuronal (␣ 2 ␤ 2 and ␣ 2 ␤ 4 ) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 M) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was ␣ 1 ␤ 1 ␥␦ > ␣ 2 ␤ 4 > ␣ 2 ␤ 2 . With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltageindependent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltagedependent, with an electrical distance of Ϸ0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptorchannel complex.Nicotinic acetylcholine receptors (AcChoRs) are members of a gene superfamily that includes GABA A , glycine, and 5HT 3 receptors (1). They are activated by the neurotransmitter acetylcholine (AcCho), and they mediate fast synaptic transmission at the neuromuscular junction and throughout the vertebrate nervous system (2-4). Additionally, AcChoRs are regulated by a wide variety of substances (5, 6), including strychnine, a selective antagonist of glycine-gated Cl Ϫ channels (7) that inhibits AcChoRs at the neuromuscular junction (8) and different types of neurons (9-13). Moreover, it appears that this inhibition depends on the subtype of nicotinic receptor involved. For instance, in bovine adrenal chromaffin cells and rat hippocampal neurons, strychnine inhibits AcChoRs competitively whereas for ␣ 4 ␤ 2 -containing AcChoRs the inhibition is noncompetitive (10, 13). Here, we report the effects of strychnine on muscle AcChoRs made up of ␣ 1 ␤ 1 ␥␦, ␣ 1 ␤ 1 ␥, or ␣ 1 ␤ 1 ␦ muscle subunits and on two subtypes of neuronal AcChoRs (␣ 2 ␤ 2 or ␣ 2 ␤ 4 ). MATERIALS AND METHODSThe methods were as previously described (14-16). In brief, cDNA clones encoding embryonic mouse muscle AcChoR subunits (␣ 1 , ␤ 1 , ␥, and ␦) or rat neuronal AcChoR subunits (␣ 2 , ␤ 2 , or ␤ 4 ) were used to make cRNAs that were suspended in RNase-free water at a concentration of 1 g͞l. Mixtures then were made with equal quantities of the requ...

No abstract

The corpus callosum (CC) is the main white matter tract involved in interhemispheric brain communication. We establish that uptake of [3H]5-hydroxytryptamine (5-HT) in CC is partially inhibited by some antidepressants. Slices of the adult rat CC had a high-affinity uptake of 5-HT. About 80% of this uptake was Na+ dependent, with a Michaelis-Menten constant, Km, of 420 +/- 80 nM and a rate of 5-HT uptake, Vmax, of 9.5 +/- 0.8 pmol/mg protein/min. The 5-HT uptake was reduced approximately 60% at pH 5 compared with that at pH 7. Fluoxetine (Prozac) inhibited only 43% of 5-HT uptake in a concentration-dependent manner, with an affinity constant, Ki, of 44.7 +/- 10.0 nM. We also studied the effects of other monoamine uptake inhibitors, all at 10 microM, and found that zimelidine, imipramine, and clomipramine inhibited 5-HT uptake in the CC by approximately 30-40%. The fluoxetine-insensitive 5-HT uptake was not altered by high concentrations of dopamine plus norepinephrine. The present data show that Na(+)-dependent 5-HT uptake occurs in the CC and optic nerve and that this uptake is partially sensitive to antidepressants and probably mediated by the serotonin transporter, which may be relevant during depression.