Inhibition of 5-HT3 receptor-mediated ion current by divalent metal cations in NCB-20 neuroblastoma cells.

1. The effect of micromolar concentrations of divalent metal cations on ion current activated by 5-hydroxytryptamine (5-HT) was investigated in NCB-20 neuroblastoma cells by the use of the whole-cell, patch-clamp technique. 2. Ion current activated by 5-HT in these cells was mimicked by 5-HT3 receptor agonists, blocked by nanomolar concentrations of selective 5-HT3 receptor antagonists and reversed polarity at approximately 0 mV. These properties indicate that this current is carried primarily if not exclusively by the nonspecific cation channel activated by the 5-HT3 receptor. 3. The Group IIb metal cations Cd2+ and Zn2+ and the Group Ib cation Cu2+ inhibited 5-HT-activated current with inhibition increasing in a concentration-dependent manner over micromolar concentrations of the ions. The order of potency of the ions for inhibiting 5-HT-activated current was Zn2+ (IC50 = 20 microM) greater than or equal to Cu2+ (IC50 = 25 microM) greater than Cd2+ (IC50 = 75 microM) at -50 mV. The other divalent metal cations tested (Ba2+, Co2+, Mg2+, Mn2+, and Ni2+) produced little or no inhibition of 5-HT-activated current at concentrations up to 200 microM. 4. Inhibition of 5-HT-activated current by Cd2+ and Zn2+ was dependent on membrane potential with the Kd increasing e-fold per 72 and 52 mV, respectively. Inhibition by Cu2+ was much less voltage dependent with the Kd increasing e-fold per 233 mV. 5. Inhibition by all three cations decreased with increasing concentration of agonist over a range of 5-HT concentrations from 1 to 10 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 5-hydroxyindole on ethanol potentiation of 5-hydroxytryptamine (5-HT)3 receptor-activated ion current in NCB-20 neuroblastoma cells

We examined the effect of 5-hydroxyindole (5-HI) on the potentiation of 5-hydroytryptamine (5-HT)(3) receptor function by ethanol (EtOH) so as to study whether EtOH potentiates channel function through increasing activation or blocking desensitization. We measured 5-HT(3) receptor current using a whole-cell voltage clamp technique with a method of rapid drug application in NCB-20 neuroblastoma cells. The 5-HI, an agent that block receptor desensitization, increased the decay time constant (tau), not the peak of 5-HT(3) receptor-mediated currents induced by 10 microM 5-HT. EtOH did not change the peak amplitude and tau of the current induced by 10 microM 5-HT. Coapplication of EtOH and 5-HI with 5-HT caused no increase in the peak currents, but tau was further increased. Therefore, a further block in desensitization could be induced by 5-HI, despite the presence of EtOH. These results indicate that EtOH potentiates 5-HT(3) receptor function, with these effects due at least in part by increasing channel activation rather than by blocking desensitization.     

5-HT4, 5-HT6, 5-HT7; molecular pharmacology of adenylate cyclase stimulating receptors

Throughout the nervous system the receptors for the neurotransmitter serotonin form an unusually diverse set. Receptor-mediated responses to serotonin utilize a large collection of biochemical second messenger pathways, such as the stimulation or inhibition of adenylate cyclase activity, the hydrolysis of phosphoinositides, the mobilization of calcium and direct gating of ion channels. This diversity of structure and activity has been substantiated by the application of molecular cloning techniques which have now yielded at least 15 distinct molecular entities. The most recent subset of receptors for serotonin to be cloned are those that couple to the stimulation of adenylate cyclase activity. These subtypes: 5-HT₄, 5-HT₆ and 5-HT₇, although sharing a common signal transduction pathway, are remarkably divergent in their amino acid sequences, distribution in the brain and pharmacological properties. This digression from the expected relationships of receptor subtypes based on other serotonin receptors, as well as other biogenic amino receptor families, is unexpected and raises many questions about the extreme diversity of this signaling system.     

Characterization of platelet-activating factor-induced elevation of cytosolic free-calcium level in neurohybrid NCB-20 cells

The effect of platelet-activating factor on the intracellular cytosolic level of free calcium ([Ca2+]i) was studied in neurohybrid NCB-20 cells. In fura-2-loaded NCB-20 cells, platelet-activating factor induced an immediate and concentration-dependent increase in [Ca2+]i with a maximum increase of 334 +/- 27 nM above a basal value of 147 +/- 6 nM (n = 40). Platelet-activating factor-induced [Ca2+]i mobilization was inhibited by the platelet-activating factor antagonists BN 50739, WEB 2086, SRI 63-441 and BN 52021 in a dose-dependent manner with IC50 values of 12, 38, 897 and 45000 nM, respectively. The calcium-channel blockers nifedipine (10 microM) and diltiazem (10 microM) had no effect on the platelet-activating factor-induced increase in [Ca2+]i; however, extracellular Ca(2+)-depletion caused a 63.6 +/- 4.7% reduction of platelet-activating factor-induced increase in [Ca2+]i (n = 5, P less than 0.001). The remaining 36% contributed from intracellular sources was completely inhibited by 10 microM of 8-(N,N-diethylamine)octyl 3,4,5-trimethoxytenzoate hydrochloride (TMB-8). NCB-20 cells exhibited homologous desensitization to sequential addition of platelet-activating factor, but no heterologous desensitization between platelet-activating factor and bradykinin or ATP was observed. These data suggest that activation of the neuronal platelet-activating factor receptor results in an increase in [Ca2+]i primarily via a receptor-operated rather than a voltage-dependent calcium-channel and to a lesser extent from intracellular Ca2+ release. Our findings may contribute to an understanding of the mechanism of platelet-activating factor actions on neuronal cells.     

Adenosine receptors linked to adenylate cyclase activity in human neuroblastoma cells: Modulation during cell differentiation

In IMR32 neuroblastoma cells, the two adenosine receptor agonists N⁶-R-phenylisopropyl-adenosine and5'-N-ethylcarboxamidoadenosine dose-dependently stimulated membrane adenylate cyclase activity with potencies consistent with the presence of adenosine receptors of the A₂-subtype. The S enantiomer of N⁶-R-phenylisopropyladenosine induced a significantly lower stimulation of adenylate cyclase, accordingly to its lower ability to activate adenosine receptors. These effects were selectively counteracted by the adenosine receptor antagonist theophylline and, conversely, were not affected by the A₁-adenosine receptor selective blocker 8-cyclopentyl-1,3-dipropylxanthine. No adenosine receptors belonging to the A₁-subtype seem, therefore, to be present in this cell line, as also shown by the lack of inhibitory activity of_{N⁶-R-phenylisopropyladenosine} on both basal and forskolin-stimulated adenylate cyclase activity. Activation of A₂-receptors did not modify intracellular basal calcium levels, did not influence calcium influx through voltage-dependent calcium channels and did not modify calcium influx and redistribution induced by muscarinic receptor activation. Prolonged exposure of cells to either N⁶-R-phenylisopropyladenosine or5'-N-ethylcarboxamidoadenosine was associated with a small but significant degree of morphological differentiation, comparable to that induced by dibutyryl cAMP, and therefore presumably related to the prolonged increase of intracellular cAMP levels elicited by the two adenosine agonists. After cellular differentiation induced with either dibutyryl cAMP or 5-bromode-oxyuridine, a selective desensitization of A₂-receptor stimulated adenylate cyclase activity was found.

IMR32 cells are a good model for studying the characteristics of the A₂-adenosine receptors in a human neuronal tissue and its regulation during neuronal differentiation or pharmacological treatments.     

Photoaffinity labeling and binding studies reveal the existence of two types of phencyclidine receptors in the NCB-20 cell line

The mouse neuroblastoma-Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptor-like sites. We report here that polypeptide components of NCB-20 cell PCP sites were identified with the photolabile PCP derivative [3H]N-[1-(3-azidophenyl)cyclohexyl]piperidine ([3H]AZ-PCP). The pharmacological selectivity of [3H]AZ-PCP binding (under reversible conditions) was similar to that observed for [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to NCB-20 cell membranes. Inhibition of [3H]TCP binding by AZ-PCP, dexoxadrol or MK-801 was biphasic, suggesting the presence of two types of PCP sites on NCB-20 cells. Photolysis of NCB-20 cell membranes pre-equilibrated with [3H]AZ-PCP, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), revealed the presence of 5 major labeled bands (Mr 90,000, 68,000, 49,000, 40,000 and 33,000), a pattern similar to that observed for rat brain membranes. MK-801 and D-2-amino-5-phosphonovaleric acid (D-(-)-AP5) selectively inhibited the labeling of Mr 68,000 and 90,000 polypeptides. These results indicate that the labeled bands represent constituents of at least two different PCP binding proteins. The Mr 68,000 and 90,000 components appear to correspond to a high-affinity site, which comprises approximately 20% of total [3H]TCP sites in these cells, and exhibits the pharmacology expected for the PCP receptor of the N-methyl-D-aspartate (NMDA)-gated channel.     

Clozapine downregulates 5-hydroxytryptamine6 (5-HT6) and upregulates 5-HT7 receptors in HeLa cells

Clozapine is an atypical antipsychotic with high affinity for several serotonin receptors. This drug causes paradoxical downregulation of 5-hydroxytryptamine(2A) (5-HT)(2A) receptors, but its modulation of other serotonin receptors has not been studied. We examined the effects of clozapine and several other drugs on the regulation of rat 5-HT(6) and 5-HT(7) receptors individually expressed in transfected HeLa cells. Both 5-HT(6) and 5-HT(7) receptor densities (B(max)) were reduced by 5-carboxamidotryptamine, an agonist, and methiothepin, an inverse agonist. Clozapine reduced 5-HT(6) B(max). This suggests that 5-HT(6) receptors are also paradoxically downregulated by the antagonist clozapine. 5-Hydroxytryptamine(7) receptor B(max), on the other hand, was increased by clozapine. Clozapine's modulation of the 5-HT(6) and 5-HT(7) receptor levels may be important in the action of this atypical antipsychotic.     

Beta-adrenergic receptors and adenylate cyclase activity in the parotid acinar cells from acute streptozotocin-induced diabetic rats

The changes in beta-adrenergic receptors and in adenylate cyclase (AC) activity were investigated in parotid glands from rats with acute diabetic mellitus (DM) induced by a single injection of streptozotocin (STZ, 80 mg/kg). The animals were divided into three groups: control rats, DM rats, and insulin-treated DM rats. Experiments were performed 7 days after the injection of STZ. Amylase and norepinephrine (NE) contents in parotid glands were markedly decreased in DM rats in comparison with control rats. The density of beta-adrenergic receptor decreased in DM rats, but its affinity for ligand was unaffected. The effect of GTP on isoprenaline (ISO)-stimulated adenylate cyclase (AC) activity significantly decreased in DM rats, but forskolin-stimulated AC activity was unaltered. In addition, diabetes induced the blunted response of AC activity to ISO. The changes in AC activity and in amylase content induced by diabetes were restored by insulin, but those in NE content and receptor density could not. These observations indicate that diabetes decreases NE and amylase contents, receptor density, and receptor-AC coupling in parotid gland, and that these changes would occur in the earlier stage of acute STZ-induced diabetic state.     

Effect of chronic activation of 5-HT3 receptors on 5-HT3, 5-HT(1A) and 5-HT(2A) receptors functional activity and expression of key genes of the brain serotonin system

The dopamine D3 receptor gene (DRD3) is considered being one of the candidate genes contributing to the development of tardive dyskinesia (TD). In a recent meta-analysis with mixed ethnicities, only a barely positive association was found between the functional DRD3 Ser9Gly polymorphism and TD in patients with schizophrenia (OR = 1.17; 95% CI: 1.01-1.37; p = 0.041). To further evaluate the controversial association between the polymorphism and TD using only Japanese subjects, we tested the association in a case-control design. We also conducted a meta-analysis including 8 studies with 3 East Asian populations (Japanese, Chinese, and Korean). In our Japanese case-control sample (43 with TD/157 without TD), we found no association between the DRD3 Ser9Gly polymorphism in schizophrenia and TD (genotype: p = 0.92; allele: p = 1.00). Furthermore, no significant difference in the mean AIMS score among the three genotypic groups was observed in our sample. The meta-analysis comprising 1291 East Asian subjects also showed no association between the polymorphism and TD; the Mantel-Haenszel pooled OR for TD among carriers of the DRD3 Ser9Gly of the eight Asian studies was 0.94 (95% CI: 0.78-1.12). Overall, our results suggest that the DRD3 Ser9Gly polymorphism may not confer susceptibility to TD in East Asian populations. Given that the Ser9Gly variant may play a putative role in the DRD3 function, further studies on the DRD3 are warranted.     

Characterization of an adenylate cyclase system sensitive to histamine H2-receptor excitation in cells from dog gastric mucosa

A method of preparing viable cells from dog gastric mucosa is described. Cyclic AMP in these cells is elevated by histamine and 4-methyl histamine but 2-methyl histamine is only a weak agonist. The effects on cyclic AMP levels are inhibited competitively by metiamide and burimamide which give apparent K_Bvalues of 3.5×10^–7 M and 2.3×10^–6 M, respectively. These values are similar to those reported for other histamine H₂-receptor systems. The H₁-receptor antagonists, mepyramine and chlorpheniramine, have no inhibitory effect on the histamine induced elevation of cyclic AMP: promethazine inhibits the system but not by a competitive mechanism. It is concluded that the histamine stimulated adenylate cyclase system is probably located in the parietal cell component.     

Muramyl dipeptide (MDP) and 5-HT receptors. Neuroimmunomodulatory effects of MDP are probably not mediated through 5-HT4 or 5-HT1A receptors

A possible interaction of immunomodulator muramyl dipeptide (MDP) with 5-HT₄ and 5-HT_1A receptors was investigated. The activation of 5-HT₄ receptors releases acetylcholine from nerve terminals, thereby contracting the guinea-pig distal ileum. The whole ileum segments were therefore cut and placed into the bath. The preparations were contracted by 5-HT (10 nM-3.2 µM); these contractions were totally abolished in the presence of atropine (1 µM) and significantly attenuated in the presence of SDZ-205,557 (320 nM). The 5-HT evoked contractions remained unchanged in the presence of MDP (5, 50 or 500 nM). MDP (10 nM-3.2 µM) could not directly contract the preparations. In further experiments, the possible interaction of MDP with 5-HT_1A receptors was investigated. The activation of 5-HT_1A receptors inhibits the release of acetylcholine from nerve terminals, thereby decreasing the height of electrically evoked neurogenic twitches of guinea-pig ileum. The whole ileum segments were cut, placed into the bath and stimulated electrically. Selective 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) decreased the height of twitches and this effect was significantly attenuated in the presence of 5-HT antagonist metergoline (1 µM). The effect of 8-OH-DPAT remained unchanged in the presence of MDP (5, 50 or 500 nM). MDP (10 nM-3.2 µM) did not exert any direct effect on the preparations. These results suggest that MDP interacts with neither 5-HT₄ nor 5-HT_1A receptors.     

Excitability of small-diameter trigeminal ganglion neurons by 5-HT is mediated by enhancement of the tetrodotoxin-resistant sodium current due to the activation of 5-HT(4) receptors and/or by the inhibition of the transient potassium current

The aims of the present study were to investigate whether the activation of the 5-HT receptor subtypes (5-HT(4) and 5-HT(3)) acted significantly on the modification of the tetrodotoxin-resistant sodium current (I(NaR)) in small-sized rat trigeminal ganglion (TG) neurons and whether the inhibition of the transient K(+) current (I(A)) contributed to the excitability in those neurons. 5-HT applications in at concentrations ranging from 0.01-10 microM significantly increased the peak I(NaR). One micromolar 5-HT application caused the greatest increase in the peak I(NaR) amplitude accompanied by a hyperpolarizing shift in the activation curve. A similar modification of I(NaR) properties was also obtained via the application of the 5-HT(4) receptor agonist, RS 67333, in concentrations ranging from 0.001-1 microM. The largest effects of 5-HT (1 microM) and RS 67333 (0.1 microM) on the modification of I(NaR) were abolished by pretreatment with ICS 205-930 (a 5-HT(3/4) receptor antagonist, 10 microM), which showed no significant effect on the baseline I(NaR). However, ICS 205-930 application at 30 microM caused a significant decrease in the baseline I(NaR). Phenylbiguanide (a 5-HT(3) receptor agonist) did not significantly alter I(NaR) properties when applied in concentrations ranging from 1 to 100 microM. The application of 0.1 microM RS 67333 decreased the transient K(+) current (I(A)) by approximately 31%. The threshold for action potential generation was significantly lower after the application of 0.1 microM RS 67333. Furthermore, 0.1 microM RS 67333 application increased the number of action potentials and the resting membrane potential got more positive, but it decreased the duration of depolarization phase of action potential. In addition, neither the additional application of 1 microM 5-HT in the presence of 10 microM forskolin, a stimulator of adenylyl cyclase, nor the opposite applications of 5-HT and forskolin caused the enhancement of increased I(NaR), which indicates the presence of an 'occluding effect.' These results suggest that the 5-HT-induced modification of I(NaR) is mediated by the activation of 5-HT(4) receptors, involving a cAMP-dependent signaling pathway, and that the inhibition of I(A) following the application of a 5-HT(4) receptor agonist also contributes to the increased number of action potentials.     

Drug-induced changes of adenylate cyclase activity in cells from asthmatic and nonasthmatic subjects.

Monolayers of adherent mononuclear leukocytes prepared from normal subjects and asthmatic children whose bronchodilater therapy did not include sympathomimetic drugs, respond to relatively low concentrations of isoproterenol (ISO) and prostaglandin E1 with increased intracellular cyclic adenosine monophosphate (cAMP) levels. The magnitude of in vitro responses to ISO is decreased by previous contact of the cells with ISO or other, orally effective, adrenergic drugs. The desensitization is rapid, concentration- and time-dependent, and is readily reversible after removal of the desensitizing drug. The phenomenon exhibits pharmacologic specificity. Cells in which the response to restimulation with ISO was decreased exhibited full sensitivity to prostaglandin E1. No differences in these behaviors were detected in cells from normal or asthmatic subjects. The results suggest that earlier observations reporting decreased responses to beta adrenergic stimulation in asthmatics may have been due to the treatment of these patients with sympathomimetic agents and not caused by a disease-related beta adrenergic receptor dysfunction.     

5-HT activates vagal afferent cell bodies in vivo: role of 5-HT2 and 5-HT3 receptors

Occipital artery (OA) injections of 5-HT elicit pronounced reductions in heart rate and mean arterial blood pressure (MAP) in urethane-anesthetized rats by activation of vagal afferent cell bodies in the ipsilateral nodose ganglion. In contrast, internal carotid artery (ICA) and i.v. injections elicit similar cardiovascular responses by activation of peripheral vagal afferent terminals. The aim of this study was to examine the roles of 5-HT3 and 5-HT2 receptors in the 5-HT-induced activation of vagal afferent cell bodies and peripheral afferent terminals in urethane-anesthetized rats. OA, ICA and i.v. injections of 5-HT elicited dose-dependent reductions in heart rate and MAP that were virtually abolished after i.v. administration of the 5-HT3 receptor antagonists, MDL 7222 or ICS 205-930. The responses elicited by the OA injections of 5-HT were markedly diminished after i.v. injection of the 5-HT2 receptor antagonists, xylamidine or ketanserin, whereas the responses elicited by i.v. or ICA injections of 5-HT were not affected. The present findings suggest that (1) 5-HT3 and 5-HT2 receptor antagonists gain ready access to nodose ganglion cells upon i.v. administration, and (2) functional 5-HT3 and 5-HT2 receptors exist on the cell bodies of vagal afferent neurons mediating the cardiovascular responses elicited by OA injections of 5-HT. These findings also support a wealth of evidence that 5-HT3 receptors exist on the peripheral terminals of vagal afferents, and although they do not discount the possibility that 5-HT2 receptors exist on peripheral vagal afferent terminals, it appears that activation of these receptors does not have pronounced effects on 5-HT3 receptor activity on terminals that mediate the hemodynamic responses to 5-HT.     

5-Hydroxytryptamine (5-HT)-induced depolarization in isolated abdominal vagus nerves in the rat: involvement of 5-HT3 and 5-HT4 receptors.

In this study, we developed an electrophysiological technique for the in vitro measurement of isolated abdominal vagus nerve depolarization in the rat. This technique was used to compare abdominal and cervical vagus nerve depolarization values. Both 5-HT and a selective 5-HT3 agonist, 2-CH3-5HT, caused a concentration-dependent depolarization in rat isolated abdominal vagus nerves in vitro. Isolated cervical vagus nerves also showed concentration-dependent depolarization, although the isolated cervical vagus nerve depolarization was approximately 60% of that of the isolated abdominal vagus nerves at similar concentration ranges of 5-HT and 2-CH3-5HT in vitro. In isolated abdominal vagus nerves, a selective 5-HT3 antagonist, granisetron, produced a concentration-dependent decrease, but reduced the maximal response of 5-HT-induced depolarization in vitro. In isolated abdominal vagus nerves, selective 5-HT4 antagonist, SB204070, produced parallel and concentration-dependent shifts to the right on the concentration-response curves to 5-HT in vitro. These findings suggest that this electrophysiological method for evaluating isolated abdominal vagus nerve depolarization is a useful technique for the estimation of 5-HT-induced depolarization.     

Expression of 5-HT3 receptors in PC12 cells treated with NGF and 8-Br-cAMP.

1. To determine the functional development of neurons, we applied nerve growth factor (NGF) or 8-bromo-cyclic-adenosine monophosphate (8-Br-cAMP) to PC12 cells and recorded the 5-hydroxytryptamine (5-HT)-induced response by the use of a patch-clamp technique. 2. Cultured PC12 cells expressed 5-HT-sensitive receptors, which are almost absent in untreated cells, in the continuous presence of NGF or 8-Br-cAMP for a period of 10 days. 3. Activation of the receptors by 5-HT produced a transient inward current. In a K(+)-free solution, the reversal potential (E5-HT) of I5-HT was +10.3 mV, and the current-voltage (I-V) relation showed inward rectification at positive potentials. 4. The permeability ratio for monovalent cations was Na+:Li+:K+:Rb+:Cs+ = 1:1.19:0.89:0.94:0.91, indicating that a 5-HT-induced current is passing through the ligand-gated large cation channel. 5. 2-Methyl-5-HT, a specific 5-HT3 agonist, induced a similar inward current, even though the current amplitude was smaller and the activation and inactivation kinetics were slower than those of 5-HT. 6. ICS-205-930, a specific 5-HT3 antagonist, inhibited the 5-HT-induced current in a concentration-dependent manner with a noncompetitive inhibition profile. Spiperone, a 5-HT1 and 5-HT2 families antagonist, and ketanserine, 5-HT2 family antagonist, did not affect the 5-HT-induced response. 7. The time to peak (tp) as well as fast and slow time constants (tau if and tau is) decreased with increasing 5-HT concentration.(ABSTRACT TRUNCATED AT 250 WORDS)