NCB-20 Cell Research Articles

Discrimination in 5-HT(3) receptor binding in murine brain and cultured cell preparations.

One-hundred ninety-one ligands were screened at 5-HT(3) receptors in membranes from rat brain and NCB20 cells for their ability to displace the selective, high-affinity 5-HT(3) receptor antagonist, [(125)I]DAIZAC ([(125)I]( S)-5-chloro-3-iodo-2-methoxy- N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide). Thirty-seven compounds having structures related to benzamide, dibenzepine, serotonin, phenylbiguanide, or arylpiperzine were selected for more extensive displacement studies in membranes from rat and mouse brains, from two cultured cell preparations expressing heteromeric mouse-derived 5-HT(3) receptor proteins (NCB20 and NG108-15 cell lines), and from recombinant Sf9 cells expressing homomeric 5-HT(3A) receptors. [(125)I]DAIZAC bound specifically to a single site in each of the five tissue preparations with high affinity ( K(D) 0.12-0.19 nM). The densities of [(125)I]DAIZAC-labeled 5-HT(3) receptors were 7.4-7.5 fmol/mg protein in membranes from murine brain, and 38, 99, and 1588 fmol/mg protein in membranes from cultured NCB20, NG108-15, and recombinant Sf9 cells, respectively. The affinity of substituted benzamides ( n=10) was similar in all five tissue preparations. The affinity of dibenzepines ( n=17) was significantly higher in membranes from cultured cells as compared to membranes from rat and mouse brain, but similar in the two brain membrane preparations, and in each of the cultured cell membrane preparations. Serotonin-, phenylbiguanide-, and quipazine-analogs ( n=10), which typically function as 5-HT (5-hydroxytryptamine) agonists, exhibited significantly higher apparent p K(i) values in membranes from rat brain and Sf9 recombinant cells than in membranes from the three preparations expressing heteromeric mouse-derived 5-HT(3) receptor proteins ( F=7.52, P<0.001). These findings confirm that there are both species and cell-type dependent differences in binding to 5-HT(3) receptors, and that care must be taken when comparing results between experimental paradigms that utilize different sources of 5-HT(3) receptors.

Neurochemical and electrophysiological evidence for the existence of a functional γ-hydroxybutyrate system in NCB-20 neurons

Clonal neurohybridoma NCB-20 cells express a valproate-insensitive succinic semialdehyde reductase activity that transforms succinic semialdehyde into γ-hydroxybutyrate. This activity (1.14±0.16 nmol/min/mg protein) was similar to the lowest activity existing in adult rat brain. [ 3H] γ-Hydroxybutyrate labels a homogeneous population of sites on NCB-20 cell membranes ( K d =250±44.4 nM, B max=180±16.2 fmol/mg protein) that apparently represents specific γ-hydroxybutyrate binding sites characterized previously on brain cell membranes. Finally, an Na +-dependent uptake of [ 3H] γ-hydroxybutyrate was expressed in NCB-20 cells with a K m of 35+21.1 μM and a V max of 80±14.2 pmol/min/mg protein. A three-day treatment with 1 mM dibutyryl-cyclic-AMP induced a three-fold increase in the cellular succinic semialdehyde reductase activity. In parallel, a K +-evoked release of [ 3H] γ-hydroxybutyrate occurred. This release was Ca 2+ dependent and was not present in undifferentiated cells. Cyclic-AMP treatment induced a decrease of [ 3H] γ-hydroxybutyrate binding sites, which could be due to spontaneous γ-hydroxybutyrate release. Patch-clamp experiments carried out on differentiated NCB-20 cells revealed the presence of Ca 2+ conductances which were partially inhibited by 50 μM γ-hydroxybutyrate. This γ-hydroxybutyrate-induced effect was blocked by the γ-hydroxybutyrate receptor antagonist NCS-382, but not by the GABA B antagonist CGP-55 845. These results demonstrate the presence of an active γ-hydroxybutyratergic system in NCB-20 cells which possesses the ability to release γ-hydroxybutyrate. These cells express specific γ-hydroxybutyrate receptors which modulate Ca 2+ currents independently of GABA B receptors.

Significantly different fatty acid profiles in various phospholipid head groups of neuroblastoma cell lines

We studied lipid profiles of hybrid cells derived from fusion of sympathetic ganglia and neuroblastoma cells (NCB 20, F 11) and also CHO (Chinese hamster ovary) cells. The proportion of saturated to unsaturated fatty acids changed significantly in different phospholipid fractions. We observed 35.4 % of total fatty acids as saturated fatty acids in the phosphatidyl ethanolamine (PE) fraction of NCB-20 cell lines while 47.2% of total fatty acids were saturated in the phosphatidyl inositol (PI) fraction. In general, in neuroblastoma cell lines, we observed the lowest proportion of saturated fatty acids in the PE fraction while the other lipid fraction showed a much higher proportion of saturated fatty acids. On the other hand the PE fraction of a non neuronal cell line, CHO, showed 57.4% saturated fatty acids in the PE fraction. This significant difference in saturated to unsaturated fatty acid ratios in different phospholipid head groups may be linked to the different biological functions of those hybrid cells.

Neuronal death, cytoplasmic calcium and internucleosomal DNA fragmentation: evidence for DNA fragments being released from cells

Neuronal death, secondary to endogenous agents such as glutamate, may involve changes in cytoplasmic calcium. Besides its well recognized role as a second messenger mediating cellular response, calcium is necessary for the activation of endonuclease(s), resulting in DNA fragmentation and cell death. Therefore, we investigated the relationship between changes in cytoplasmic calcium, DNA fragmentation and neuronal death, using PC12 and NCB-20 cell lines. The calcium ionophore, A23187, caused a dose-dependent increase in cytoplasmic calcium, loss of cell viability, increased lactate dehydrogenase (LDH)-release, and DNA fragmentation. DNA fragments, typical of internucleosomal digestion of genomic DNA, characteristic of endonuclease(s) activation, were consistently detected in the incubating medium. Release of DNA fragments into the medium was seen with A23187 in concentrations as low as 10 nM, and within an hour of treatment. Furthermore, calcium added to preparations of PC12 nuclei also produced DNA fragmentation, although, less pronounced than when intact cells were treated with A23187. The findings indicate that A23187-induced neuronal death involves the activation of endonuclease(s). The role of cytoplasmic calcium in this process is supported by evidence that A23187 selectively mobilizes cytoplasmic calcium, and that calcium can directly activate endonuclease(s) in nuclear preparations.

Inhibition of cyclic AMP accumulation in intact NCB-20 cells as a direct result of elevation of cytosolic Ca2+.

Earlier studies established that adenylyl cyclase in NCB-20 cell plasma membranes is inhibited by concentrations of Ca2+ that are achieved in intact cells. The present studies were undertaken to prove that agents such as bradykinin and ATP, which elevate the cytosolic Ca2+ concentration ([Ca2+]i) from internal stores in NCB-20 cells, could inhibit cyclic AMP (cAMP) accumulation as a result of their mobilization of [Ca2+]i and not by other mechanisms. Both bradykinin and ATP transiently inhibited [3H]cAMP accumulation in parallel with their transient mobilization of [Ca2+]i. The [Ca2+]i rise stimulated by bradykinin could be blocked by treatment with thapsigargin; this thapsigargin treatment precluded the inhibition of cAMP accumulation mediated by bradykinin (and ATP). A rapid rise in [Ca2+]i, as elicited by bradykinin, rather than the slow rise evoked by thapsigargin was required for inhibition of [3H]cAMP accumulation. Desensitization of protein kinase C did not modify the inhibitory action of bradykinin on [3H]cAMP. Effects of Ca2+ on phosphodiesterase were also excluded in the present studies. The accumulated data are consistent with the hypothesis that hormonal mobilization of [Ca2+]i leads directly to the inhibition of cAMP accumulation in these cells and presumably in other cells that express the Ca(2+)-inhibitable form of adenylyl cyclase.

Voltage-clamp study of calcium currents during differentiation in the NCB-20 neuronal cell line.

1. Calcium currents (ICa) were studied in voltage-clamped NCB-20 cells. In undifferentiated cells, voltage steps from hyperpolarized potentials (-80/-100 mV) essentially revealed transient ICa showing characteristics classically described for "T-type" channels. In about 50% of the cells, there was a residual current at the end of the step; no ICa was elicited from a holding potential of -50 mV. 2. In contrast, 100% of the cells differentiated with dibutyryl cyclic AMP (cAMP) displayed a residual current in addition to the transient one, and depolarizing steps from a holding potential of -50 mV induced a sustained current. In these cells, Bay K 8644 elicited both a negative shift in voltage dependence and a moderate increase of the sustained component. 3. Although these changes in Ca2+ channel physiology result from chemically induced differentiation, they might not be directly related to the concomitant morphologic differentiation. 4. In undifferentiated NCB-20 cells, T-type Ca2+ currents can be elicited in relative isolation.

Ethanol Fails to Modify [3H]GR65630 Binding to 5‐HT3 Receptors in NCB‐20 Cells and in Rat Cerebral Membranes

Low concentrations of ethanol have been found to enhance the electrophysiologic effect of serotonin (5-HT) acting at 5-HT3 receptors on NCB-20 cells. To determine whether this action of ethanol reflects a change in the agonist-receptor interaction, the effect of ethanol (100 mM) on agonist and antagonist binding to 5-HT3 receptor was studied in vitro in membrane from NCB-20 cells and from cortex plus hippocampus of rat. The antagonist [3H]GR65630 was used to label 5-HT3 recognition sites. Ethanol did not change the characteristics of saturable [3H]GR65630 binding in either membrane preparation. In competition studies, the agonists 5-HT and 2-methyl-5-HT completely inhibited the binding of [3H]GR65630 to NCB-20 cell membranes, while in brain membranes the maximum displacement of specific [3H]GR65630 binding by 5-HT was approximately 30%. Ethanol decreased the affinity of the receptor for 2-methyl-5-HT, but not to 5-HT in NCB-20 cells, and had no effect on agonist binding in brain membranes. The results indicate that enhancement of 5-HT responses at 5-HT3 receptors by ethanol is not a result of changes in the equilibrium binding characteristics of the agonist.

The human σ site, which resembles that in NCB20 cells, may correspond to a low-affinity site in guinea pig brain

1,3-di(2-[5- 3H]tolyl)Guanidine ([ 3H]DTG) was found to bind to a single saturable population of binding sites in human cerebral cortex and NCB20 cells, a second low-affinity site was apparent in guinea pig brain. Displacement studies were performed to determine the pharmacology of the [ 3H]DTG binding site in these 3 membrane preparations. In human cortical tissue and NCB20 cell membranes the (+)-stereoisomers of benzomorphans displaced binding with Hill coefficients close to one, displayed similar affinity and did not give the biphasic displacement curve characteristic of guinea pig membranes. The pIC 50 of the low-affinity component of the σ binding site in guinea pig brain correlates best with the affinity of drugs for the binding site in human cortex.

Characteristics of 5‐HT3 binding sites in NG108‐15, NCB‐20 neuroblastoma cells and rat cerebral cortex using [3H]‐quipazine and [3H]‐GR65630 binding

1. The biochemical and pharmacological properties of 5-HT3 receptors in homogenates of NG108-15 and NCB-20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [3H]-quipazine and [3H]-GR65630 binding. 2. In NG108-15 and NCB-20 cell homogenates, [3H]-quipazine bound to a single class of high affinity (NG108-15: Kd = 6.2 +/- 1.1 nM, n = 4; NCB-20: Kd = 3.0 +/- 0.9 nM, n = 4; means +/- s.e.means) saturable (NG108-15: Bmax = 1340 +/- 220 fmol mg-1 protein; NCB-20: Bmax = 2300 +/- 200 fmol mg-1 protein) binding sites. In rat cortical homogenates, [3H]-quipazine bound to two populations of binding sites in the absence of the 5-hydroxytryptamine (5-HT) uptake inhibitor, paroxetine (Kd1 = 1.6 +/- 0.5 nM, Bmax1 = 75 +/- 14 fmol mg-1 protein; Kd2 = 500 +/- 300 nM, Bmax2 = 1840 +/- 1040 fmol mg-1 protein, n = 3), and to a single class of high affinity binding sites (Kd = 2.0 +/- 0.5 nM, n = 3; Bmax = 73 +/- 6 fmol mg-1 protein) in the presence of paroxetine. The high affinity (nanomolar) component probably represented 5-HT3 binding sites and the low affinity component represented 5-HT uptake sites. 3. [3H]-paroxetine bound with high affinity (Kd = 0.02 +/- 0.003 nM, n = 3) to a site in rat cortical homogenates in a saturable (Bmax = 323 +/- 45 fmol mg-1 protein, n = 3) and reversible manner. Binding to this site was potently inhibited by 5-HT uptake blockers such as paroxetine and fluoxetine (pKi s = 8.6-9.9), while 5-HT3 receptor ligands exhibited only low affinity (pK; < 7). No detectable specific [3H]-paroxetine binding was observed in NG108-15 or NCB-20 cell homogenates. 4. [3H]-quipazine binding to homogenates of NG108-15, NCB-20 cells and rat cortex (in the presence of 0.1 microM paroxetine) exhibited similar pharmacological characteristics. 5-HT3 receptor antagonists competed for [3H]-quipazine binding with high nanomolar affinities in the three preparations and the rank order of affinity was: (S)-zacopride > quarternized ICS 205-930 2 granisetron > ondansetron > ICS 205-209 (R)-zacopride > quipazine > renzapride > MDL-72222 > butanopride > metoclopramide. 5. [3H]-GR65630 labelled a site in NCB-20 cell homogenates with an affinity (Kd = 0.7 + 0.1 nms n = 4) and density (B__ = 1800 + 1000 fmol mg- protein) comparable to that observed with [3H]-quipazine. Competition studies also indicated a good correlation between the pharmacology of 5-HT3 binding sites when [3H]-GR65630 and [3H]-quipazine were used in these cells. 6. In conclusion, [3H]-quipazine labelled 5-HT3 receptor sites in homogenates of NG108-15 cells, NCB-20 cells and rat cerebral cortex. In rat cortical homogenates, [3H]-quipazine also bound to 5-HT uptake sites, which could be blocked by 0.1 microM paroxetine. The pharmacological specificity of the 5-HT3 receptor labelled by [3H]-quipazine was similar in the neuroblastoma cells and rat cortex and was substantiated in NCB-20 cells by the binding profile of the selective 5-HT3 receptor antagonist, [3H]-GR65630.

Intracellular calcium in NCB-20 cells: elevation by depolarization and ethanol but not by glutamate

Intracellular Ca 2+, measured in NCB-20 cell; Glutamate;N-methyl- d-aspartate; Fura-2 not by the excitatory amino acids l-glutamate andN-methyl- d-aspartate. These amino acids failed to raise intracellular Ca 2+ levels even in the presence of elevated extracellular Ca 2+, in the absence of extracellular Mg 2+ or Na +, under depolarizing conditions, or in differentiated cells, and induced neither 45Ca 2+ uptake nor excitotoxic cell death. NCB-20 cells thus appear to express functional voltage-gated, but notN-methyl- d-aspartate receptor-gated, Ca 2+ channels.

Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells.

A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.

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 [ 3 H]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 [ 3 H]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 ( M r 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 M r 68,000 and 90,000 polypeptides. These results indicate that the labeled bands represent constituents of at least two different PCP binding proteins. The M r 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.

Phospholipase C activity in NCB-20 cells is inhibited by protein kinase A-mediated phosphorylation of low molecular mass GTP-binding proteins.

We have previously shown that bradykinin-induced production of second messengers such as inositol trisphosphate and diacylglycerol in neurotumor cells is inhibited by raising cellular cyclic AMP levels, which in turn inhibit phospholipase C. A monoclonal antibody to phospholipase C-II immunoprecipitated the 140-kDa form of phospholipase C-II from [35S]methionine/[3H]eucine-labeled cells, but not [32P]orthophosphate-labeled phospholipase C-II, following treatment with either forskolin or dibutyryl cyclic AMP. This suggested that phospholipase C is not the target for cyclic AMP-dependent protein kinase-mediated phosphorylation. In vitro studies confirmed that phospholipase C activity was inhibited by raising cellular cAMP levels, and partial sensitivity to Bordetella pertussis toxin suggested the involvement of a GTP-binding protein which could be the target for protein kinase A. The involvement of a GTP-binding protein in coupling the bradykinin receptor to phospholipase C was further suggested by the ability of both guanosine 5'-O-(thio-triphosphate) and fluoride (NaF) to release inositol phosphates from NCB-20 cell membranes previously labeled with [3H]inositol. Both effects were blocked by pretreatment of the cells with protein kinase A activators, further suggesting a GTP-binding protein as the target for protein kinase A-mediated phosphorylation. When whole NCB-20 cell extracts were blotted onto nitrocellulose and incubated with [alpha- 32P]GTP, a major 24-kDa band plus minor bands at 22 and 20 kDa were revealed by autoradiography. A pH 3.0/6.0 soluble (basic protein) NCB-20 cell extract revealed the major 24-kDa band plus the 20-kDa band, and similar basic proteins were shown to be heavily phosphorylated following [32P]orthophosphate labeling and pretreatment with forskolin. The size and ability to bind GTP on Western blots are characteristic of the ras, rho, smg, etc. family of GTP-binding proteins recently suggested to be the much sought after GPLC (Lapetina, E.G., Lacal, J. C., Reep, B. R., and Molina y Vedia, L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3131-3134; Wang, P., Nishihata, J., Takabori, E., Yamamoto, K., Toyoshima, S., and Osawa, T. (1989) J. Biochem. (Tokyo) 105, 461-466; Nagata, K.-I., Nagao, S., and Nozawa, Y. (1989) Biochem. Biophys. Res. Commun. 160, 235-242). We propose that GPLC is uniquely sensitive to protein kinase A-mediated phosphorylation and that phosphorylation inhibits stimulus-secretion coupling in these cells.

Segregation of discrete Gsα‐mediated responses that accompany homologous or heterologous desensitization in two related somatic hybrids

1. Prostacyclin and adenosine A2 receptors activate adenylate cyclase in the neuroblastoma hybrid cell lines NG108-15 and NCB-20. Prolonged exposure of NG108-15 cells to iloprost (a stable analogue of prostacyclin) results in a subsequent reduction in the capacity for adenylate cyclase activation by iloprost, the adenosine analogue 5'-(N-ethyl)-carboxamidoadenosine (NECA) or NaF. In contrast prolonged exposure of NCB-20 cells to iloprost results only in the loss of iloprost responsiveness. 2. Iloprost pretreatment of NG108-15 cells also magnified the morphine-dependent inhibition of iloprost-stimulated adenylate cyclase activity from 36 to 48%. This change was not due to lower iloprost stimulation following desensitization, since the % inhibition of adenylate cyclase activity by morphine in control cells was constant irrespective of enzyme activity. 3. These heterologous effects observed in NG108-15 cells following iloprost pretreatment may involve changes in the GS alpha protein, since there was a reduction of about 30% in the cholera toxin-induced [32P]-ADP-ribosylation of a 45 kDa protein from cell membranes (corresponding to the extent of loss of NECA or NaF responsiveness). A similar reduction was not observed in NCB-20 cells. 4. These results indicate that iloprost pretreatment induces different forms of desensitization in NG108-15 and NCB-20 cell lines. The heterologous desensitization in the former may, like the human platelet, involve a functional loss of GS alpha from the cell membrane. Changes in the activity of GS alpha may also account for the heterologous effects on receptors that mediate inhibition of adenylate cyclase.

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

1 The characteristics of transmembrane currents evoked by 5-hydroxytryptamine (5-HT) in the neuroblastoma x Chinese hamster brain cell line NCB-20 and neuroblastoma clonal cell line N1E-115 have been studied under voltage-clamp conditions by the whole-cell recording and outside-out membrane patch modes of the patch-clamp technique. 2 In 73% of NCB-20 cells examined (n = 221), and all N1E-115 cells studied (n = 80), 5-HT (10 microM) elicited a transient inward current at negative holding potentials, this being associated with an increase in membrane conductance. In both cell lines responses to 5-HT reversed in sign at a potential of approximately -2 mV and demonstrated inward rectification. 3 The reversal potential of 5-HT-induced currents (E5-HT) recorded from either NCB-20 or N1E-115 cells was unaffected by total replacement of internal K+ by Cs+. In N1E-115 cells, reducing internal K+ concentration from 140 to 20 mM produced a positive shift in E5-HT of approximately 28 mV, whereas reducing external Na+ from 143 to 20 mM was associated with a negative shift in E5-HT of about 37 mV. A large reduction in internal Cl- concentration (from 144 to 6 mM) had little effect on E5-HT. 4 5-HT-induced currents of NCB-20 cells were unaffected by methysergide (1 microM) or ketanserin (1 microM), but were reversibly antagonized by GR38032F (0.1-1.0 nM) with an IC50 of 0.25 nM. GR 38032F (0.3 nM) reduced 5-HT-induced currents in N1E-115 cells to approximately 26% of their control value. 5 On outside-out membrane patches excised from both NCB-20 and N1E-115 cells, 5-HT induced small inward currents which could not be clearly resolved into discrete single channel events. Such responses were: (i) reversibly antagonized by GR 38032F (1 nM) (ii) reversed in sign at 0 mV, and (iii) subject to desensitization. 6 Fluctuation analysis of inward currents evoked by 5-HT (1 microM) in N1E-115 cells suggests that 5-HT gates a channel with a conductance of approximately 310fS. Such a relatively small conductance could readily explain why the response of outside-out membrane patches to 5-HT cannot at present be resolved into clear single channel events.

MRNA from NCB-20 cells encodes the N-methyl-D-aspartate/phencyclidine receptor: a Xenopus oocyte expression study.

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) receptors. We report here that Xenopus oocytes injected with NCB-20 cell poly(A)+ RNA express N-methyl-D-aspartate (NMDA)-activated channels and that these channels include the PCP receptor site. In injected oocytes, NMDA application evoked a partially desensitizing inward current that was potentiated by glycine, blocked by the competitive antagonist D-2-amino-5-phosphonovaleric acid, blocked by Mg2+ and by Zn2+, and blocked in a use-dependent manner by the PCP receptor ligands PCP and MK-801. There was little or no response to kainate or quisqualate (agonists of the other excitatory amino acid receptors), to gamma-aminobutyric acid (an inhibitory transmitter), or to glycine (an inhibitory transmitter as well as an allosteric potentiator of NMDA channels). Thus, NMDA/PCP receptors expressed from NCB-20 cell mRNA exhibit properties similar to those of the neuronal receptors. The absence of expression of other excitatory amino acid receptors in this system makes it particularly useful for study of NMDA-evoked responses without interference from responses mediated by other receptors. Moreover, NCB-20 mRNA may be an appropriate starting material for cloning the cDNA(s) encoding the NMDA/PCP-receptor complex.

The putative prostacyclin receptor antagonist (FCE-22176) is a full agonist on human platelets and NCB-20 cells

The action of FCE-22176 on prostacyclin receptors of human platelets and the NCB-20 cell line have been examined. FCE-22176 is a full agonist in both systems, mediating activation of adenylate cyclase. The concentration required for half-maximum enzyme activation was 174 nM in platelet membranes and 193 nM in homogenates of NCB-20 cells. The binding of [3H]iloprost to human platelet or NCB-20 cell membranes was inhibited by FCE-22176, and Ki values of 400 and 280 nM were obtained.

σ receptors on NCB-20 hybrid neurotumor cells labeled with (+)[ 3H]SKF 10,047 and (+)[ 3H]3-PPP

(+)[ 3H]SKF 10,047 and (+)[ 3H]3-PPP label a homogeneous population of sites in NCB-20 cell membranes that apparently represent benzomorphan specific binding sites previously reported for this cell line. Their drug specificity indicates that these sites are very similar to σ receptor binding sites labeled in brain tissues by these ligands and do not represent PCP receptors.

Prostaglandin endoperoxide analogues which are both thromboxane receptor antagonists and prostacyclin mimetics.

Two prostaglandin endoperoxide analogues, EP 035 and EP 157, behave as specific thromboxane receptor antagonists on isolated smooth muscle preparations such as rabbit aorta, dog saphenous vein and guinea-pig trachea. However, in human platelet-rich plasma (PRP) they produce an unsurmountable block of aggregation induced by a wide range of agents (ADP, platelet-activating factor, thrombin); this inhibitory profile is typical of that seen with either prostaglandin I2 (PGI2) or PGD2. EP 035 and EP 157 induce large increases in cyclic AMP levels (up to 20 times basal) in human PRP. Simultaneous exposure to PGE1 markedly reduces their effect on cyclic AMP; exposure to PGD2 is much less effective in this respect. The adenylate cyclase inhibitor SQ 22,536 opposes the inhibitory action of EP 035, EP 157, iloprost (a stable PGI2 analogue) and PGD2 on platelet aggregation. However, the xanthone derivative AH 6809 blocks the inhibitory action of PGD2 but does not affect EP 035, EP 157 and PGI2 and its structural analogues. EP 035 and EP 157 displace [3H]-iloprost binding to the PGI2 receptor on human platelet membranes. Displacing ability is ranked as follows: iloprost greater than 6a-carba PGI2 greater than EP 157 greater than EP 035 greater than EP 164 (alpha-dinor derivative of EP 157). This order of potency is the same as that found for activation of adenylate cyclase in homogenates of washed human platelets and for inhibition of aggregation in washed human platelets. The activities of EP 035 and EP 157 were studied in two other systems containing PGI2 receptor-adenylate cyclase complexes, the NCB-20 cell line and human lung tissue. In both cases stimulation of adenylate cyclase was found but maximum rates were below that achieved with iloprost. These effects of EP 035 and EP 157 could be correlated with their abilities to displace [3H]-iloprost binding. 5 These results indicate that EP 035 and EP 157 inhibit the aggregation of human platelets by acting as agonists at the PGI2 receptor linked to adenylate cyclase. They represent a class of compound with both thromboxane receptor blocking activity and prostacyclin mimetic activity.

Identification of the prostacyclin receptor by radiation inactivation.

Evidence has been obtained for a specific protein receptor for prostacyclin on cells of the NCB-20 somatic hybrid. A new stable prostacyclin analog, 5-[(E)-(1S,5S,6R,7R) - 7 - hydroxy-6-[(E) - (3S,4RS) -3-hydroxy-4-methyl-1 -octen-6-inyl]bicyclo[3.3.0]-octan-3-ylidene]pentanoic acid (Iloprost, ZK36374) activates adenylate cyclase of NCB-20 cell membranes to an extent similar to prostacyclin and with a comparable high affinity. The binding of [3H]Iloprost to NCB-20 membranes was rapid with an association rate constant (k+1) of 2.01 X 10(5) M-1 s-1 at 20 degrees C. The rate constant for the dissociation of the ligand-receptor complex (k-1) was 1.19 X 10(-3) s-1, giving a dissociation constant (k-1/k+1) of 5.9 nM. The equilibrium dissociation constant was 29.9 nM, and the membranes had a maximum binding capacity of 347 fmol mg-1 protein. Radiation inactivation has been employed to determine the molecular weights of the functional prostacyclin receptor and components of the adenylate cyclase system in the plasma membrane of the NCB-20 cells. Cell membranes were lyophilized prior to irradiation, which lead to the formation of high-molecular-weight aggregates. The aggregation was avoided, however, when membranes were prepared in an isotonic Tris-HCl buffer containing sucrose. Molecular weight values of 111,000 for the catalytic subunit of adenylate cyclase, 89,000 for the regulatory subunit, and 83,000 for the prostacyclin receptor were obtained. Loss of [3H]Iloprost binding capacity after irradiation of lyophilized membranes yielded a molecular weight value (mean +/- S.E.) for the prostacyclin receptor of 82,800 +/- 12,900 (n = 3).