Dopamine Receptors on Adrenal Chromaffin Cells Modulate Calcium Uptake and Catecholamine Release

The presence of dopamine-containing cells in sympathetic ganglia, i.e., small, intensely fluorescent cells, has been known for some time. However, the role of dopamine as a peripheral neurotransmitter and its mechanism of action are not well understood. Previous studies have demonstrated the presence of D2 dopamine receptors on the surface of bovine adrenal chromaffin cells using radioligand binding methods and dopamine receptor inhibition of catecholamine release from perfused adrenal glands. In the present study, we provide evidence confirming a role of dopamine receptors as inhibitory modulators of adrenal catecholamine release from bovine chromaffin cell cultures and further show that the mechanism of modulation involves inhibition of stimulated calcium uptake. Apomorphine gave a dose-dependent inhibition (IC50 = 1 microM) of 45Ca2+ uptake stimulated by either nicotine (10 microM) or membrane depolarization with an elevated K+ level (60 mM). This inhibition was reversed by a series of specific (including stereospecific) dopamine receptor antagonists: haloperidol, spiperone, sulpiride, and (+)-butaclamol, but not (-)-butaclamol. In addition, the calcium channel agonist Bay K 8644 was used to stimulate uptake of 45Ca2+ into chromaffin cells, and this uptake was also inhibited by the dopamine receptor agonist apomorphine. The combined results suggest that dopamine receptors on adrenal chromaffin cells alter Ca2+ channel conductance, which, in turn, modulates catecholamine release.     

Pharmacological and immunological identification of native alpha7 nicotinic receptors: evidence for homomeric and heteromeric alpha7 receptors

Controversy surrounds the expression of alpha7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, alpha7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [(125)I]alpha-bungarotoxin (alphaBGT) binding reaches equilibrium within 4 h and is saturable with a K(d) value of 4.2 nM. Using homologous competition experiments, the K(i) for binding of alphaBGT was 1.9 nM. These data are consistent with the expression of homomeric alpha7 nAChRs. Methyllycaconatine (MLA), which binds alpha7 nAChRs with high affinity, inhibits [(125)I]alphaBGT binding in a concentration-dependent manner with a K(i) of 30.6 nM; this value is approximately 10 fold higher than the reported affinity of MLA for alpha7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate alpha7 nAChRs, as has been previous demonstrated for bovine adrenal alpha3beta4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes alpha7 nAChR protein, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes alpha3 and alpha5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. Together, these results support the expression of alpha7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric alpha7 nAChRs.     

Effects of Antimitotic Agents on Secretion and Detergent Extractibility of Adrenal Nicotinic Acetylcholine Receptors

1. Evidence exists that associations of adrenal nicotinic acetylcholine receptors (nAChRs) with the cytoskeleton play an important role in signal transduction pathways by maintaining these receptors in a functional state. These studies were designed to explore this possibility and elucidate the mechanism by which antimitotic agents inhibit activation of adrenal nAChRs.2. Functional studies demonstrated that vincristine, tubulozole, podophyllotoxin, and demecolcine inhibited nAChR-stimulated catecholamine release noncompetitively and in a concentration-dependent manner, with IC₅₀ values of 3 (1–10), 5 (2–10), 8 (4–15), and 19 (9–39) M, respectively.3. Detergent extraction experiments indicated that approximately 36% of adrenal nAChRs were associated with the detergent-insoluble cytoskeletal fraction. When chromaffin cells were first treated with antimitotic agents and then detergent solubilized, a significant reduction occurred in the population of adrenal nAChRs associated with the detergent-insoluble cytoskeleton.4. These studies support an association of adrenal nAChRs with microtubules and suggest that the mechanism by which the antimitotic drugs interfere with the signal transduction pathway is by inducing dissociation of nAChRs from the microtubular network.     

Regulation of Phenylethanolamine N-Methyltransferase Gene Expression by Imidazoline Receptors in Adrenal Chromaffin Cells

Abstract: As adrenal medullary chromaffin cells express imidazoline binding sites in the absence of α₂-adrenergic receptors, these cells provide an ideal system in which to determine whether imidazolines can influence catecholamine gene expression through nonadrenergic receptors. This study evaluates the ability of clonidine and related drugs to regulate expression of the gene for the epinephrine-synthesizing enzyme phenylethanolamine N -methyltransferase (PNMT) in the rat adrenal gland and in bovine adrenal chromaffin cell cultures. In vivo, PNMT and tyrosine hydroxylase (TH) mRNA levels increase in rat adrenal medulla after a single injection of clonidine. Clonidine also dose-dependently stimulates PNMT mRNA expression in vitro in primary cultures of bovine chromaffin cells, with a threshold dose of 0.1 μ M . Other putative imidazoline receptor agonists, including cimetidine, rilmenidine, and imidazole-4-acetic acid, likewise enhance PNMT mRNA production showing relative potencies that correlate with their binding affinities at chromaffin cell I₁-imidazoline binding sites. The effects of clonidine on PNMT mRNA appear to be distinct from and additive with those exerted by nicotine. Moreover, neither nicotinic antagonists nor calcium channel blockers, which attenuate nicotine's influence on PNMT mRNA production, diminish clonidine's effects on PNMT mRNA. Although 100 μ M clonidine diminishes nicotine-stimulated release of epinephrine and norepinephrine in chromaffin cells, this effect appears unrelated to stimulation of imidazoline receptor subtypes. This is the first report to link imidazoline receptors to neurotransmitter gene expression.     

Serotonin Modulates Nicotinic Responses of Adrenal Chromaffin Cells

Abstract: 5-Hydroxytryptamine (5-HT) specifically and reversibly inhibits nicotine-induced currents and catecholamine release in bovine adrenal chromaffin cells in culture. Pharmacological analysis indicates that the inhibition is not mediated by known 5-HT receptor subtypes. The inhibition is noncompetitive over a range of nicotine concentrations between 1 and 100 μM. Preincubation with either 5-HT or substance P significantly protects the response from nicotine-induced desensitization. It is concluded that 5-HT inhibits nicotinic acetylcholine receptors on bovine adrenal chromaffin cells, probably by binding to a noncompetitive site on the receptor itself. Because both blood and the chromaffin cells contain 5-HT, the inhibition provides an opportunity for negative control of catecholamine secretion from the adrenals.     

Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-β-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide–Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.     

The Relationship Between Arachidonic Acid Release and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

Increased arachidonic acid release occurred during activation of catecholamine secretion from cultured bovine adrenal medullary chromaffin cells. The nicotinic agonist 1,1-dimethyl-4- phenylpiperazinium (DMPP) caused an increased release of preincubated [3H]arachidonic acid over a time course which corresponded to the stimulation of catecholamine secretion. Like catecholamine secretion, the DMPP-induced [3H]arachidonic acid release was calcium-dependent and was blocked by the nicotinic antagonist mecamylamine. Depolarization by elevated K+, which induced catecholamine secretion, also stimulated arachidonic acid release. Because arachidonic acid release from cells probably results from phospholipase A2 activity, our findings indicate that phospholipase A2 may be activated in chromaffin cells during secretion.     

Amino acids outside of the loops that define the agonist binding site are important for ligand binding to insect nicotinic acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the α subunits and another three loops (D, E, and F) of the non-α subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-α subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens , a target-site mutation (Y151S) was found within two α subunits (Nlα1 and Nlα3). In Drosophila S2 cells and Xenopus oocytes, Nlα1 can form functional receptors with rat β2 subunit. However, the same thing was not observed in Nlα3. In the present paper, by exchanging the corresponding regions between Nlα1 and Nlα3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding.     

Two Distinct ATP Receptors Activate Calcium Entry and Internal Calcium Release in Bovine Chromaffin Cells

Abstract: ATP, an established neurotransmitter, causes elevation of cytosolic Ca²⁺ and catecholamine secretion when applied to chromaffin cells in the intact adrenal gland. The ATP-induced rise in Ca²⁺ is due both to release from internal stores and to entry across the plasma membrane. The latter source of Ca²⁺ causes secretion; the primary role of Ca²⁺ released from internal stores remains undetermined. In this article, we have studied the nucleotide specificity for activating the two types of Ca²⁺ increases. The agonist potency order for the increase in fluorescence from fura-2-loaded chromaffin cells due to release of Ca²⁺ from internal stores is ATP = UTP > ADP > 2-methylthio-ATP, α,β-methylene ATP, identifying the receptor as a P_2U purinoceptor. The potency order for secretion is 2-methylthio-ATP > ATP > α,β-methylene ATP, ADP, UTP, placing the receptor in the P_2Y subtype. Thus, two distinct receptors are responsible for Ca²⁺ release and secretion. Agonists were more effective in the absence of extracellular Mg²⁺, suggesting that ATP uncomplexed with divalent cations binds preferentially to both receptors. The low response of both receptors to ADP distinguishes them from the ATP receptor on these cells that inhibits voltage-dependent Ca²⁺ current and secretion.     

Regulation of catecholamine release in human adrenal chromaffin cells by β-adrenoceptors

The adrenal gland plays a fundamental role in the response to a variety of stress situations. After a stress condition, adrenal medullary chromaffin cells release, by exocytosis, high quantities of catecholamine (epinephrine, EP; norepinephrine, NE), especially EP. Once in the blood stream, catecholamines reach different target organs, and induce their biological actions through the activation of different adrenoceptors. Adrenal gland cells may also be activated by catecholamines, through hormonal, paracrine and/or autocrine system. The presence of functional adrenoceptors on human adrenal medulla and their involvement on catecholamines secretion was not previously evaluated. In the present study we investigated the role of β(1)-, β(2)- and β(3)-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture. We observed that the β-adrenoceptor agonist (isoproterenol) and β(2)-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β(2)-adrenoceptor antagonist (ICI 118,551; 100 nM) and β(3)-adrenoceptor antagonist (SR 59230A; 100 nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β(1)-adrenoceptor antagonist (atenolol; 100 nM) did not change the isoproterenol- neither the nicotine-evoked catecholamine release from human adrenal chromaffin cells. Moreover, our results show that the protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) are intracellular mechanisms involved in the catecholamine release evoked by salbutamol. In conclusion, our data suggest that the activation of β(2)- and β(3)-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.     

Functional expression of the α7 and α4-containing nicotinic acetylcholine receptors on the neonatal rat carotid body

The carotid bodies (CBs) are chemosensory organs that respond to hypoxemia with transmitter neurosecretion, leading to a respiratory reflex response. It has been proposed that acetylcholine is a key regulator of transmitter release through activation of presynaptic nicotinic acetylcholine receptors (nAChRs). In the present work, we studied the identity of such nAChRs and their contribution to catecholamine release from CBs. Neonatal rat CBs were placed in a recording chamber for electrochemical recordings or disassociated for voltage-clamp studies on isolated cells. Fast nicotine superfusion increases catecholamine release from intact CBs. This response was diminished reversibly by the non-selective nAChR blocker hexamethonium, by the selective α7 blocker α-bungarotoxin and by the α4-containing nAChR blocker erysodine. In isolated CB cells the nAChR agonists nicotine, acetylcholine and cytisine all evoke inward currents with similar potencies. The nicotine-evoked current was fully blocked by mecamylamine and partially inhibited by α-bungarotoxin or erysodine. However, the combination of both α-bungarotoxin an erysodine failed to suppress this response. Immunodetection studies confirm the presence of α7 and α4 subunits in isolated dopaminergic CB cells. Our results show that activation of α7 and/or α4-containing nAChR subtypes have the ability to regulate catecholamine release from intact CB due to activation of fast inward currents expressed in chemoreceptor cells. Therefore, our results suggest that both nAChR subtypes contribute to the cholinergic nicotinic regulation of catecholamine signaling in the carotid body system.     

Roles of Nicotinic and Muscarinic Receptors in Calcium Signaling and Transmitter Release

Activation of acetylcholine receptors (AChR) triggers catecholamine release from adrenal chromaffin cells and release of neurotransmitters in neuron-to-neuron and neuromuscular junctions, including those on smooth muscle cells. Calcium ions play the role of the main intracellular messenger, which mediates these processes. In our study, we explored the properties of Ca²⁺ signaling triggered by activation of AChR by analyzing the characteristics of Ca²⁺ transients induced by selective activation of nicotinic (nAChR) and muscarinic (mAChR) cholinoreceptors using Fura-2 fluorescent measurements in experiments on rat chromaffin cells. Two populations of chromaffin cells, which in a different manner responded to AChR stimulation, were classified. We found that the mean frequency of quantum release induced by ACh is considerably higher than that during hyperpotassium cell depolarization. Comparative analysis of single secretory events showed that, in the case of stimulation by ACh, single secretory spikes demonstrate faster kinetic characteristics than those induced by depolarization. Statistical analysis of the integral magnitude (area) of single secretory spikes evoked by both types of stimulation showed no significant difference despite amplitude and kinetic dissimilarities between such secretory events. Mathematical modeling of the dynamics of the exocytotic processes led to the conclusion that the reason for the specific kinetic characteristics of single secretory responses may be different diameters of the secretory pores formed during fusion of secretory vesicles with the plasma membrane.     

Functional co-expression of two insect nicotinic receptor subunits (Nlα3 and Nlα8) reveals the effects of a resistance-associated mutation (Nlα3Y151S) on neonicotinoid insecticides

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. Previously, we have identified a nAChR point mutation (Y151S) associated with insecticide resistance in the brown planthopper Nilaparvata lugens . Although this mutation has been identified in two different N. lugens nAChR subunits (Nlα1 and Nlα3) because of difficulties in heterologous expression of Nlα3; its influence on agonist potency has been examined only in Nlα1-containing nAChRs. Here we describe the cloning of a novel nAChR subunit from N. lugens (Nlα8), together with evidence for its co-assembly with Nlα3 in native and recombinant nAChRs. This has, for the first time, enabled the functional effects of the Nlα3^Y151S mutation to be examined. The Nlα3^Y151S mutation has little effect on agonist potency of acetylcholine but has a dramatic effect on neonicotinoid insecticides (reducing I _max values and increasing EC₅₀ values). The apparent affinity of neonicotinoids was higher and the effect of the Y151S mutation on neonicotinoid agonist potency was more profound in Nlα3-containing, rather than Nlα1-containing nAChR. We conclude that Nlα3- and Nlα1-containing nAChRs may be representative of two distinct insect nAChR populations.     

Tetrapeptides on N- and C-Terminal Regions of Mastoparan Inhibit Catecholamine Release from Chromaffin Cells by Blocking Nicotinic Acetylcholine Receptor

Mastoparan (MP), a tetradecapeptide in wasp venom, has been reported to evoke catecholamine release, but also reported to inhibit secretory response upon nicotinic stimulation in adrenal chromaffin cells. To elucidate the inhibitory mechanism of MP, we examined the effect of two MP fragments (INLK-NH2 and KKIL-NH2) on catecholamine release in bovine adrenal chromaffin cells. These MP fragments inhibited catecholamine release induced by nicotinic stimulation in a noncompetitive manner. These fragments did not affect catecholamine release evoked by high [K+] or by other secretagogues, neither caused catecholamine release by themselves. Replacement by hydrophobic and basic amino acids of the MP fragments enhanced the inhibitory effects on ACh-evoked catecholamine release. Among 23 analogs of the MP fragments, (Nle)3-R-NH2 showed the most potent inhibition with IC50 = 541 microM. These results suggested that the MP fragments selectively inhibit the secretory response to nicotinic stimulation by attacking nAChR on the site(s) made up of hydrophobic and acidic amino acids but other than ACh-binding sites. This mechanism may explain the inhibitory action of MP on nicotine-evoked catecholamine release.     

Regulation of Nicotinic Receptor Subtypes Following Chronic Nicotinic Agonist Exposure in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick α4β2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the K _i values for binding inhibition and EC₅₀ values for agonist-induced α4β2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing α3, α5, β2, and β4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.     

Dopaminergic Inhibition of Catecholamine Secretion from Chromaffin Cells: Evidence that Inhibition Is Mediated by D4 and D5 Dopamine Receptors

Abstract: Previous studies have suggested that activation of D2-like dopamine receptors inhibits catecholamine secretion from adrenal chromaffin cells. The purpose of this study was to determine whether the activation of D1-like receptors on chromaffin cells affects either catecholamine release from the cells or the inhibition of secretion by D2-like dopamine receptors. Both D1- and D2-selective agonists inhibited secretion elicited by dimethylphenylpiperazinium (DMPP), veratridine, and high K⁺ levels. The D1-selective agonists 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (Cl-APB) and SKF-38393 inhibited DMPP-stimulated catecholamine secretion in a concentration-dependent manner; 50% inhibition was obtained with ～10 µM Cl-APB and ～100 µM SKF-38393. Of the D2-selective agonists, bromocriptine was a more potent inhibitor of DMPP-stimulated catecholamine release than was quinpirole. The inhibition of secretion caused by Cl-APB or SKF-38393 was additive with the inhibition caused by bromocriptine. Pertussis toxin treatment (50 ng/ml, 18 h) attenuated the inhibitory effect of D2-selective, but not D1-selective, dopamine agonists. In addition, forskolin-stimulated adenylyl cyclase activity was inhibited by D2-selective, but not D1-selective, agonists. Neither D1- nor D2-selective agonists stimulated adenylyl cyclase activity in the cells, although cyclase activity was stimulated by forskolin, carbachol, and vasoactive intestinal peptide. DMPP-stimulated Ca²⁺ uptake was inhibited by both D1- and D2-selective dopamine agonists. PCR analysis was used to determine which of the dopamine receptor subtypes within the D1-like and D2-like subfamilies was responsible for the observed inhibition. PCR analysis indicated that mRNA for only D4 and D5 dopamine receptor subtypes was present in chromaffin cells. These combined data suggest that D1- and D2-selective agonists inhibit Ca²⁺ uptake and catecholamine secretion by activating D4 and D5 dopamine receptors on chromaffin cells.     

Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites

In isolated bovine adrenal chromaffin cells, beta-endorphin, dynorphin, and levorphanol caused a dose-dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 microM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD = 0.59 nM). beta-Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5-I2Tyr1]-beta-endorphin, [3,5-I2Tyr1]-dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with beta-endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh-evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 microM, respectively). Inhibition of CA release was also obtained with naloxone (ID50 = 14) microM) and naltrexone (ID50 greater than 10(-4) M), two classical antagonists of opiate receptors, and this effect was additive to that of beta-endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.     

Differential Effects of Hypoxia on Ligand Binding Properties of Nicotinic and Muscarinic Acetylcholine Receptors on Cultured Bovine Adrenal Chromaffin Cells

Abstract: Hypoxia is known to disturb neuronal signal transmission at the synapse. Presynaptically, hypoxia is reported to suppress the release of neurotransmitters, but its postsynaptic effects, especially on the function of neurotransmitter receptors, have not yet been elucidated. To clarify the postsynaptic effects, we used cultured bovine adrenal chromaffin cells as a model of postsynaptic neurons and examined specific binding of l -[³H]nicotine (an agonist for nicotinic acetylcholine receptors: nAChRs) and ²²Na⁺ flux under control and hypoxic conditions. Experiments were performed in media preequilibrated with a gas mixture of either 21% O₂/79% N₂ (control) or 100% N₂ (hypoxia). Scatchard analysis of the specific binding to the cells revealed that the K_D under hypoxic conditions was twice as large as that under control conditions, whereas the B _max was unchanged. When the specific [³H]nicotine binding was kinetically analyzed, the association constant ( k ₁) but not the dissociation constant ( k ₋₁) was decreased to 40% of the control value by hypoxia. When the binding assay was performed using the membrane fraction, these changes were not observed. Nicotine-evoked ²²Na⁺ flux into the cells was suppressed by hypoxia. In contrast, specific [³H]quinuclidinyl benzilate binding to the intact cells was unaffected by hypoxia. These results demonstrate that hypoxia specifically suppresses the function of nAChRs (and hence, neuronal signal transmission through nAChRs), primarily by acting intracellularly.