Adenylate cyclase and acetylcholine release regulated by separate serotonin receptors of somatic cell hybrids.

Abstract

Serotonin activates adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of NCB-20 neuroblastoma-brain hybrid cells with an activation constant of 530 nM, but has little or no effect on cellular cyclic AMP or cyclic GMP content of (4,5). Serotonin also induces contractions in ileal smooth muscle (6). However, central neuronal responses to serotonin in mammals are predominantly inhibitory and are mediated through either pre-or postsynaptic receptors, which can be distinguished by the differences in their responses to D-lysergic acid diethylamide (LSD) (5, 7). In molluscan neurons, as many as six kinds of response to serotonin have been detected (8).Serotonin-dependent activation of adenylate cyclase is preserved after electrolytic lesion of the raphe nuclei, which greatly reduces the number of serotonergic neurons innervating the colliculus. This suggests that collicular serotonin receptors that mediate activation of adenylate cyclase are postsynaptic receptors (2).In this report, effects mediated by two species of serotonin receptor in NCB-20 neuroblastoma-brain hybrid cells are described. One receptor is coupled to the activation of adenylate cyclase, and the other mediates cell depolarization and acetylcholine release. The receptors are distinguished on the basis of functional response, specificity, and desensitization.MATERIALS AND METHODS Cells. The N1E-1 15 adrenergic neuroblastoma cell line was cloned from C-1300 mouse neuroblastoma (9). The NG108-15 neuroblastoma-glioma hybrid cell line (unpublished results) was derived by Sendai virus-induced fusion of C-1300 mouse neuroblastoma clone N18TG2 resistant to 6-thioguanine (10) with rat glioma clone C6BU-1, resistant to 5-bromodeoxyuridine (11). The NCB-20 neuroblastoma-fetal Chinese hamster brain hybrid cell line (12) Adenylate Cyclase Assay. Cells were cultured as described (13). Confluent cells were detached from flasks by gentle tapping and were washed three times, each with 10 ml of Dulbecco's phosphate-buffered saline (without Ca2+ or Mg2+ ions) adjusted to 340 mOsm with NaCl. Cells were recovered by centrifugation (150 X g for 5 min) after each wash, and after the third wash were suspended in 25 mM Tris-HCl, pH 7.4/290 mM sucrose (2 ml/75-cm2 flask) and homogenized at 4°C with 50 strokes of a Dounce homogenizer. Homogenates were frozen and stored in a liquid N2 freezer. Each 100-Al reaction mixture contained 50 mM Tris-HCl (pH 7.4); 87 mM sucrose; 20 mM creatine phosphate, disodium salt (Sigma); 10 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisenment" in accordance with 18 U. S. C'. §1734 solely to indicate this fact.

The properties of 5‐HT3 receptors in clonal cell lines studied by patch‐clamp techniques

“…5-HT has previously been shown to stimulate the formation of cyclic AMP in the latter system (Berry- Kravis & Dawson, 1983) and also to evoke a membrane depolarization, the two effects occurring via pharmacologically distinct subtypes of 5-HT receptor (MacDermot et al, 1979). The present work demonstrates that the electrical response in cells is, like that of the NIE-115 clone, mediated by 5-HT3 receptors.…”

“…Firstly, the enhancement of adenylate cyclase activity shows no decline in the continued presence of 5-HT, whereas the depolarization and inward current elicited by 5-HT desensitize rapidly (MacDermot et al, 1979). Secondly, both D-lysergic acid diethylamide and methysergide act as partial agonists at '5-HT1-like' receptors in NCB-20 cells, but neither compound affects the electrical response to 5-HT (MacDermot et al, 1979 and present study).…”

“…Cells of the clonal lines NCB-20 (Minna et al, 1975) and N1E-1 15 (Amano et al, 1972) were grown essentially as described by MacDermot et al (1979) and Khimi et al (1976) respectively. Briefly, NCB-20 cells were cultured in 25 cm2 'Nunclon' tissue culture flasks (Gibco) containing 10ml of Dulbecco's modified Eagle medium (DMEM) supplemented with 5% (vol/vol) foetal calf serum (FCS), hypoxanthine (13.6mg l-), aminopterin (0.178 mgl-), thymidine (3.88mgl-'), streptomycin (50mgl 1) and penicillin (5 x I04iu 1 -).…”

THE BINDING OF [3H]‐PROSTACYCLIN TO MEMBRANES OF A NEURONAL SOMATIC HYBRID

“…This cell line (Minna, Yavelow & Coon, 1975) is derived from a mouse neuroblastoma and brain of foetal Chinese hamster. It expresses many functions of highly differentiated nerve cells including acetylcholine synthesis, electrical excitability and competence to form stable, physiologically active synapses with myotubes in co-culture (MacDermot, Higashida, Wilson, Matsuzawa, Minna & Nirenberg, 1979). This system provides a convenient source of neuronal cells that are free from contamination with vascular, erthropoietic and other prostacyclin-sensitive tissues.…”

Neuropeptide Y in neuroblastoma × glioma hybrid cells

“…The NG108-15 cell line possesses many neuronal properties including eIectricaI excitability [lt2] mediated by activation of specific sodium charmels f3] and the ability to form synapses in vitro [ 1, 2,4]. These cells synthesize the neurotransmitter acetylcholine which is rekeased in response to stimulation [5].…”