Effect of G Protein-Coupled Receptor Kinase 2 on the Sensitivity of M4 Muscarinic Acetylcholine Receptors to Agonist-Induced Internalization and Desensitization in NG108-15 Cells

NG108-15 cells express predominantly the M4 subtype of the muscarinic receptor for acetylcholine. Stimulation of these receptors by the agonist carbachol causes an inhibition of cellular adenylyl cyclase and a consequent fall in the intracellular cyclic AMP concentration. Pretreatment of the cells with carbachol caused both internalization and desensitization of the M4 receptor. Overexpression of G protein-coupled receptor kinase (GRK) 2 caused an increase in the rate constant for receptor endocytosis (from 0.06 to 0.18 min(-1)) and a decrease in the EC50 for carbachol stimulation of internalization (from 15 to 3 microM). Overexpression of a dominant negative form of GRK2 had more modest effects, reducing the rate constant for endocytosis (from 0.11 to 0.07 min(-1)) and increasing the EC50 for carbachol stimulation of internalization (from 8 to 17 microM). Neither GRK2 nor dominant negative GRK2 overexpression had any effect on the rate constant for receptor recycling following agonist removal. The time course and extent of receptor desensitization in control cells were identical to the corresponding values for receptor internalization, and the rate and extent of desensitization were again increased by GRK2 overexpression. Exposure of the cells to hyperosmolar sucrose (0.6 M) almost completely blocked agonist-induced receptor internalization in both control and GRK2-overexpressing cells. Sucrose treatment also blocked agonist-induced desensitization. We conclude that the internalization and desensitization of the M4 muscarinic receptor in NG108-15 cells can be modulated in response to changes in GRK2 activity and also that internalization plays a key role in desensitization.     

Additive Induction of Egr-1 (zif/268) mRNA Expression in Neuroblastoma × Glioma Hybrid NG108-15 Cells via Cholinergic Muscarinic, α2-Adrenergic, and Bradykinin Receptors

Abstract: Administration of carbachol, noradrenaline, and bradykinin induced Egr-1 mRNA expression within 1 h in mouse neuroblastoma × rat gliomahybrid NG108–15 cells. With specific receptor antagonists, the Egr-1 inductions by carbachol and noradrenaline were shown to be mediated via cholinergic muscarinic and α₂-adrenergic receptors, respectively. At their saturation levels for Egr-1 induction, the two agonists had additive effects when added together, but no prolongation of the effect on Egr-1 induction was observed. Addition of carbachol or noradrenaline 6 h after primary stimulation with carbachol or noradrenaline did not result in secondary Egr-1 induction, probably because of receptor desensitization. On the other hand, bradykinin consistently had an additive effect on Egr-1 induction, irrespective of the time of its addition, suggesting that the signal pathways for Egr-1 induction by carbachol or noradrenaline and by bradykinin are different. Treatment of cells with pertussis toxin or cholera toxin strongly inhibited Egr-1 induction by carbachol or noradrenaline but only partially inhibited the induction by bradykinin. Thus, the signals transduced in NG108–15 cells by different neurotransmitter receptors appear to have different effects on Egr-1 induction, depending on the times of stimulation and the combinations of receptors stimulated.     

Expression and Agonist-Induced Down-Regulation of mRNAs of m2- and m3-Muscarinic Acetylcholine Receptors in Cultured Cerebellar Granule Cells

The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization, mRNA species for m2- and m3-mAChRs but not m1- and m4-mAChRs were detected in these cells. The expression of mRNAs of both m2- and m3-mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 microM carbachol led to differential down-regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 microM) and pirenzepine (10 microM), prevented carbachol-induced m3-mAChR mRNA down-regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up-regulation of m3-mAChR mRNA. Thus, the mRNA species for both m2- and m3-mAChR subtypes are differentially expressed in culture and down-regulated by agonist stimulation. The loss of these mRNA species may play a role in the down-regulation of mAChR binding sites that occurs after desensitization.     

G Protein Modulation of ω-Conotoxin Binding Sites in Neuroblastoma X Glioma NG 108-15 Hybrid Cells

Electrophysiological evidence shows that voltage-dependent calcium channel (VDCC) activity can be regulated by a large number of neurotransmitters. In particular, guanine nucleotide binding regulatory protein (G protein)-mediated inhibitory modulation of the channel activity has been deduced from evidence that GTP analogues and purified G proteins are able to mimic this effect. The G proteins involved are pertussis toxin (PTx) sensitive. The purpose of the present study was to investigate, using biochemical techniques, whether G protein activation modulates the recognition site for omega-conotoxin GVIA (CgTx), a peptide neurotoxin that selectively labels a population of high-threshold VDCC. Undifferentiated and differentiated (1 mM dibutyryl cyclic AMP, 4 days) NG 108-15 cells were used. In both crude cellular extracts specific binding of 125I-CgTx was characterized. Differentiation induced a sixfold increase in the number of binding sites and doubled the KD value. The in vitro addition of guanylylimidodiphosphate (GMP-PNP; a nonhydrolyzable analogue of GTP) to extracts prepared from differentiated cells reduced the 125I-CgTx binding by 48%. This effect, observed in undifferentiated cells as well, was also caused by other triphosphate guanine nucleotides, such as GTP, but not by guanosine 5'-O-(2-thiodiphosphate) or adenine nucleotides. Treatment of the cells with PTx prevented the GMP-PNP effect. Moreover, the results obtained after preincubation with specific antisera raised against the alpha subunits of Gi1-2 and Go suggest that Go is the G protein responsible for the observed effect.     

Muscarinic Acetylcholine Receptors in Neuroblastoma Cells: Lack of Effect of Veratrum Alkaloids on Receptor Number

The effect of compounds that activate sodium channels on the number of muscarinic acetylcholine receptors in neuroblastoma NIE 115 cells has been investigated. The cells were used in electrically unexcitable ("control" cells) and excitable ("differentiated" cells) states. Although receptor assays using a single concentration of the radioligand [3H]scopolamine methyl chloride indicated a loss of receptors after a 6-h incubation of cells with veratrine, no true loss of receptors was seen with any of the compounds tested (veratridine, veratrine, aconitine) when full saturation analyses were performed in either control or differentiated cells. The apparent receptor loss seen with veratrine was due to a muscarinic receptor-active component of veratrine (not veratridine) occluded by the cells and released into the binding assays upon cell breakage. Veratridine and aconitine have a very low affinity for muscarinic acetylcholine receptors, and the binding of carbamoylcholine to the receptors is unaffected by tetrodotoxin, so that there is no evidence in this system for interaction between muscarinic receptors and sodium channels.     

Intracellular Calcium Pools in Neuroblastoma × Glioma Hybrid NG108–15 Cells

The intracellular nonmitochondrial calcium pools of saponin-permeabilized NG108-15 cells were characterized using inositol 1,4,5-trisphosphate (IP3) and GTP. IP3 or GTP alone induced release of 47 and 68%, respectively, of the calcium that was releasable by A23187. GTP induced release of a further 24% of the calcium after IP3 treatment, whereas IP3 induced release of a further 11% of the calcium after GTP treatment. Guanosine 5'-O-(3-thio)triphosphate had little effect on IP3-induced calcium release but completely inhibited GTP-induced calcium release. In contrast, heparin inhibited the action of IP3 but not that of GTP. The results imply the existence of at least three nonmitochondrial pools: (a) 31% is releasable by IP3 and GTP, (b) 11% is releasable by IP3 alone, and (c) 24% is releasable by GTP alone. GTP enhanced calcium uptake in the presence of oxalate with an EC50 of 0.6 microM and stimulated calcium release in the absence of oxalate with an EC50 of 0.32 microM. The similar EC50 values for these dual effects of GTP on calcium movement suggest that GTP exerts its dual action by the same mechanism.     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

Chronic Activation of Inhibitory δ-Opioid Receptors Cross-Regulates the Stimulatory Adenylate Cyclase-Coupled Prostaglandin E1 Receptor System in Neuroblastoma × Glioma (NG108-15) Hybrid Cells

Abstract: The present article investigates chronic opioid regulation of the stimulatory adenylate cyclase-coupled prostaglandin E₁ (PGE₁) receptor system in neuroblastoma × glioma (NG108-15) hybrid cells. Persistent activation of δ-opioid receptors by morphine (10 µmol/L; 3 days) substantially down-regulates the number of PGE₁ binding sites by ～30%, without affecting their affinity. Radioligand binding studies performed in the presence of GTPγS (100 µmol/L) further revealed that the remaining PGE₁ binding sites are still capable of interacting functionally with their associated stimulatory G proteins, G_s. On the postreceptor level, neither changes in the abundance nor in the intrinsic activity of the α subunit of G_s (G_sα) were found during the state of opioid dependence, as has been verified by western blot and S49 cyc^? reconstitution experiments, respectively. Evaluation of the functional interaction between PGE₁ receptors and G_s by means of receptor-stimulated, cholera toxin-catalyzed ADP-ribosylation of G_sα revealed a significant increase in the ability of PGE₁ receptors to activate G_sα (3.3-fold increase in EC₅₀; p < 0.05) in cells chronically exposed to morphine. This effect was completely blocked by coincubation of the cells together with the opiate antagonist naloxone (100 µmol/L; 3 days), whereas precipitation of morphine withdrawal by naloxone (100 µmol/L) had no further effect on sensitization in PGE₁ receptor/G_s coupling. These findings provide evidence that the stimulatory adenylate cyclase-coupled PGE₁ receptor system represents a potential target of chronic δ-opioid receptor activation in NG108-15 hybrid cells. They further suggest that sensitization in stimulatory signal transduction plays a critical role in the generation of opioid dependence.     

Muscarinic Acetylcholine Receptors in Torpedo Electric Organ: Effect of Guanine Nucleotides

Abstract: The effect of guanine nucleotides on the binding properties of presynaptic muscarinic receptors has been studied in a membrane preparation from the electric organ of Torpedo marmorata by measuring the competitive displacement of the radiolabelled antagonist, [³H]quinuclidinyl benzilate, by nonradioactive muscarinic ligands. The binding of the antagonists, atropine, scopolamine and pirenzepine was to a single class of sites [slope factors (pseudo Hill coefficients) close to 1] and was unaffected by 0.1 m M GTP. The binding of the N -methylated antagonists, N -methylatropine and N -methyl-scopolamine was more complex (slope factors <1) but also insensitive ( N- methylatropine) to 0.1 m M GTP. Agonist binding was complex and could be resolved into two binding sites with relatively high and low affinities. The proportion of high-affinity sites varied with the nature of the agonist (15–80%). Agonist binding was depressed by 0.1 m M GTP, and the order of sensitivity was oxotremorine-M > carbamoylcholine > muscarine > acetylcholine > arecoline > oxotremorine. The binding of pilocarpine, a partial agonist, was unaffected by GTP. With carbamoylcholine as a test ligand the GTP effect on agonist binding was half-maximal at 12 μM. GDP and guanylylimidodiphosphate produced comparable inhibition of carbamoylcholine binding, but GMP and cyclic GMP were ineffective, as were various adenine nucleotides. Analysis of agonist binding in terms of a two-site model indicates that the predominant effect of guanine nucleotides is to reduce the number of sites of higher affinity.     

Heterogeneity of Nucleotide Receptors in NG108-15 Neuroblastoma and C6 Glioma Cells for Mediating Phosphoinositide Turnover

Abstract: We have compared the characteristics of receptors for nucleotide analogues and the involvement of phospholipase C (PLC) in the effector mechanism in NG108-15 neuroblastoma and C6 glioma cells. The relative potency of these analogues to stimulate inositol phosphate (IP) formation is UTP > UDP ? 2-methylthio-ATP (2-MeSATP), GTP > ATP, CTP > ADP > UMP in NG108-15 cells and ATP > UTP > ADP > GTP > UDP ? 2Me-SATP, CTP, UMP in C6 glioma cells. α,β-Methylene-ATP, β,γ-methylene-ATP, AMP, and adenosine had little or no effect in both types of cells. The EC₅₀ values were 3 and 106 µM for UTP in NG108-15 and C6 glioma cells, respectively. The EC₅₀ value for ATP in C6 glioma cells was 43 µM. 2-MeSATP was threefold more potent than ATP in NG108-15 cells but had little effect in C6 glioma cells at 1 mM. In NCB-20 cells, a similar rank order of potency to that found in NG108-15 cells, i.e., UTP ? GTP > ATP > CTP, was observed. In both NG108-15 and C6 glioma cells, preincubation with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated IP production. ATP and UTP displayed no additivity in terms of IP formation at maximally effective concentrations. In contrast, endothelin-1, bradykinin, and NaF interacted in an additive manner with either nucleotide in stimulating PI hydrolysis. Pretreatment with pertussis toxin did not affect ATP-, UTP-, and GTP-stimulated IP generation in these cells, indicating that nucleotide receptors coupled to PLC by a pertussis toxin-resistant G protein in both cell types. Short-term treatment of the cells with protein kinase C (PKC) activators [phorbol 12-myristate 13-acetate (PMA) and octylindolactam V] produced a dose-dependent inhibition of ATP- and UTP-induced IP formation with a greater extent and higher susceptibility in C6 glioma cells than in NG108-15 cells. Furthermore, a 24-h exposure of the cells to PMA resulted in an obvious attenuation of nucleotide-induced IP formation in C6 glioma cells but failed to change the response in NG108-15 cells. These results suggest that distinct nucleotide receptors that respond to ATP and UTP with different selectivity exist in NG108-15 and C6 glioma cells. These heterogeneous nucleotide receptors coupled to PLC undergo discriminative modulation by PKC. NG108-15 and NCB-20 neuroblastoma are two cell lines that showed the highest specificity to extracellular UTP rather than ATP among the nucleotide receptors so far studied in various cells, suggesting the presence of a pyrimidine receptor in these cells.     

Possible Existence of Two Subsets of Platelet-Activating Factor Receptor to Mediate Polyphosphoinositide Breakdown and Calcium Influx in Neuroblastoma × Glioma Hybrid NG 108–15 Cells

Platelet-activating factor (PAF) initiated polyphosphoinositide (polyPI) breakdown and a rise of intracellular calcium concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG 108-15 cells. The accumulation of [3H]inositol trisphosphate and [3H]inositol bisphosphate was evident within 15 s after PAF stimulation, peaked at 1 min, and then gradually decayed. The increase in [3H]inositol monophosphate level was observed at 30 s, plateaued in 5 min, and was sustained up to 10 min in the presence of 10 mM LiCl. On the other hand, the rise of [Ca2+]i evoked by PAF reached a peak within 8-12 s and returned to basal levels within 1 min as measured in fura 2-loaded cells. When cells were suspended in Ca(2+)-depleted medium, the PAF-induced [Ca2+]i rise was reduced by 80%, indicating that the increase of [Ca2+]i was predominantly due to the Ca2+ influx from an extracellular source. Both PAF-induced accumulation of 3H-labeled inositol phosphates and [Ca2+]i elevation were concentration dependent with EC50 values of approximately 1 x 10(-10) and 5 x 10(-8) M, respectively. The PAF analogs 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-O-methyl-rac-glycerol-3-phosphocholine were much poorer agonists at eliciting the same responses in these cells. Pretreatment of cells with pertussis toxin caused a substantial inhibition of PAF-induced accumulation of 3H-inositol phosphates. In contrast, the rise in [Ca2+]i was not significantly affected by toxin treatment at the same concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Phospholipases on Chronic Opiate Action in Neuroblastoma × Glioma NG108-15 Hybrid Cells

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone. The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospholipids with various lipases. Treatment with phospholipase C from Clostridium welchii produced an enzyme concentration-dependent decrease of prostaglandin E1-stimulated adenylate cyclase activity in control or etorphine-treated (1 microM for 4 h) hybrid cells. In addition, incubation of hybrid cells with phospholipase C concentrations of greater than or equal to 0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment. This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations. The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C from Bacillus cereus and phospholipase D. Hydrolysis of the acyl side chains of phospholipids with phospholipase A2 did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin. Because the guanylylimidodiphosphate- and NaF-sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains.     

Choline Uptake by the Neuroblastoma X Glioma Hybrid, NG108-15

The neuroblastoma X glioma hybrid clone NG108-15 is able to release acetylcholine upon depolarization and form cholinergic neuromuscular synapses in culture. Normal functioning of cholinergic synapses is thought to be dependent on the ability of a neuron to take up extracellular choline, since neurons are unable to synthesize choline de novo. For these two reasons it became important to characterize the choline uptake system of NG108-15 cells. The uptake system appears to bear little if any resemblance to the Na⁺-dependent high-affinity choline uptake system normally associated with cholinergic neurons. Although the cells appear to possess both high- and low- affinity choline uptake systems, neither system is dependent on Na⁺ and uptake actually is increased about 60% by the substitution of sucrose for NaCl. Acetylcholine synthesis also is not dependent on Na⁺, since sucrose, substituted for NaCl, also stimulates acetylcholine synthesis. Changes in the concentrations of the other ions in the uptake medium have little effect on uptake, with the exception that elevated Ca²⁺ or Mg²⁺ reverses the stimulation of choline uptake produced by substitution of sucrose for NaCl. Choline uptake is inhibited by hemicholinium-3, but only at high concentrations of the drug (IC₅₀= 30–80 μm ). The metabolic poisons cyanide and iodoacetate inhibit uptake by only 30-40%. Growth of the cells in N⁶,O^2′ dibutyryladenosine-3′,5′-cyclic monoposphate, which promotes functional and morphological differentiation of the cells, decreased slightly the total amount of choline taken up but had no additional effect on the uptake system. Thus, it appears that NG108-15 cells are capable of forming functional cholinergic synapses with muscle cells even though the neuroblastoma does not possess the high-affinity choline uptake system normally associated with cholinergic neurons.     

Effect of Heat Shock on Intracellular Calcium Mobilization in Neuroblastoma × Glioma Hybrid Cells

Abstract: The effect of heat shock on agonist-stimulated intracellular Ca²⁺ mobilization and the expression of heat shock protein 72 (hsp72) in neuroblastoma × glioma hybrid cells (NG 108–15 cells) were examined. Hsp72 was expressed at 6 h after heat shock (42.5°C, 2 h), reached a maximum at 12 h, and decreased thereafter. Bradykinin-induced [Ca²⁺], rise was attenuated to 28% of control by heat shock at 2 h after heat shock, and reversion to the control level was seen 12 h later. When the cells were treated with quercetin or antisense oligodeoxyribonucleotide against hsp72 cDNA, the synthesis of hsp72 was not induced by heat shock, whereas bradykinin-induced [Ca²⁺]_i rise was abolished and the [Ca²⁺]_i rise was not restored. Recovery from this stressed condition was evident when cells were stimulated by the Ca²⁺-ATPase inhibitor thapsigargin, even in the presence of either quercetin or antisense oligodeoxyribonucleotide. Inositol 1,4,5-trisphosphate (IP₃) production was not altered by heat shock at 12 h after heat shock, whereas IP₃ receptor binding activity was reduced to 45.3%. In the presence of quercetin or antisense oligodeoxyribonucleotide, IP₃ receptor binding activity decreased and reached 27.2% of the control 12 h after heat shock. Our working thesis is that heat shock transiently suppresses the IPs-mediated intracellular Ca²⁺ signal transduction system and that hsp72 is involved in the recovery of bradykinin-induced [Ca²⁺]_i rise.     

Involvement of Receptor Cycling and Receptor Reserve in Resensitization of Muscarinic Responses in SH-SY5Y Human Neuroblastoma Cells

Abstract: Preexposure of SH-SY5Y cells to the muscarinic agonist carbachol caused a rapid desensitization of subsequent carbachol-stimulated intracellular Ca²⁺ responses and a slower decrease in the number of receptors at the plasma membrane. Desensitization (to 30% of the control response) was maximal after 1 min of exposure to agonist, whereas the number of cell surface receptors reached a minimum (33% of control) only after 5 min. Following agonist washout, the recovery of response was complete within 12 min, whereas the recovery of surface receptor number reached a plateau at 65% of control after 30 min. Treatment with inhibitors of endocytosis (concanavalin A) or recycling (nigericin) did not affect rapid desensitization but did decrease resensitization, suggesting that receptor cycling is involved in resensitization. Experiments with the irreversible antagonist propylbenzilylcholine mustard demonstrated that the receptor reserve for the Ca²⁺ response to 1 m M carbachol is ∼50%. Removal of this receptor reserve led to a decrease in the rate of resensitization. We propose that the existence of a receptor reserve might explain the poor correlation between functional response and surface receptor number, and that one of its roles might be to permit rapid resensitization after a significant agonist-induced decrease in surface receptor number. The purpose of receptor cycling might be to allow dephosphorylation (and reactivation) of receptors that have become phosphorylated (and inactivated) in response to agonist stimulation, because the protein phosphatase inhibitor calyculin A significantly reduced resensitization.     

Messenger RNA Levels and Binding Sites of Muscarinic Acetylcholine Receptors in Gastrointestinal Muscle Layers from Healthy Dairy Cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M₁to M₅ in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [³H]-QNB (1-quinuclidinyl-[phenyl-4-³H]-benzilate) binding by M antagonists [atropine (M_{1 ? 5}), pirenzepine (M₁), methoctramine (M₂), 4-DAMP (M₃), and tropicamide (M₄)] was used to identify receptors at the functional level. Maximal binding (B_max) was determined through saturation binding with atropine as a competitor. The mRNA levels of M₁, M₂, M₃, and M₅ represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M₄ was undetectable. The mRNA levels of M₂ and of M₃ in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [³H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [³H]-QNB binding. The [³H]-QNB binding was dose-dependent and saturable. B_max in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B_max and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.     

Chronic Activation of Muscarinic and Metabotropic Glutamate Receptors Down-Regulates Type I Inositol 1,4,5-Trisphosphate Receptor Expression in Cerebellar Granule Cells

Abstract: The ability of receptors coupled to phosphoinositide turnover to evoke accumulation of inositol 1,4,5-trisphosphate (InsP₃) over extended incubation periods, and consequently to affect the level of InsP₃ receptor expression, was studied in cultured cerebellar granule cells. The cholinergic agonist carbachol (CCh; 1 m M ) evoked a biphasic accumulation of InsP₃, a rapid three- to fourfold peak increase over control levels at ∼10 s, decreasing within 1 min to a long-lasting plateau elevation. Using an antibody against the type I InsP₃ receptor, it was demonstrated that >50% down-regulation of type I InsP₃ receptor expression in cerebellar granule cells occurred within 1 h of incubation with 1 m M CCh. Over 24 h, 1 m M CCh caused an ∼85% decrease in type I InsP₃ receptor levels, and significant decreases in immunoreactivity were evident at much lower concentrations of CCh. Direct assessment of total InsP₃ receptor expression using a radioligand binding method also detected down-regulation, but to an apparently lesser extent. 1-Aminocyclopentane-1 S ,3 R -dicarboxylic acid (200 µ M ), an agonist of metabotropic glutamate receptors, evoked a marked decrease in type I InsP₃ receptors after 24 h of incubation. These findings demonstrate that a functional consequence of maintained InsP₃ production in cerebellar granule cells is the down-regulation of InsP₃ receptor expression and that this down-regulation may be a common mechanism of action of phosphoinositide-linked receptors during prolonged stimulation.     

AT1 Angiotensin Receptors Mobilize Intracellular Calcium in a Subclone of NG108–15 Neuroblastoma Cells

The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG 108-15 cells. The subclone, C1, was prepared by fluorescence-activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration-dependent manner (1 nM-100 microM), yielding EC50 values of 437 +/- 80 nM (n = 4; slope = 1.6 +/- 0.3), 57 +/- 8 nM (n = 12; slope = 1.5 +/- 0.3), and 36 +/- 5 nM (n = 7; slope = 1.4 +/- 0.3), respectively. AIII was significantly more potent than AII (p less than 0.05). In contrast, Des-Phe8-AII, AII-hexapeptide (AII 3-8), and p-NH2-Phe6-AII (1-10 microM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108-15 cells were totally inhibited by the AT1 receptor-selective nonpeptide antagonist, DUP-753 (0.3-1 microM), the AT2-selective antagonists, EXP-655 and CGP42112A (1-10 microM), failed to block the effects of AII. DUP-753 (0.3-100 nM) produced dextral shifts of the AII-induced concentration-response curves and yielded an estimated affinity constant (pA2) of 8.5 +/- 0.2 (n = 16) using single-point analysis involving different concentrations of DUP-753. These data compared well with those obtained for the inhibition of AII-induced aortic contractions by DUP-753 (pA2 = 8.5) reported previously by others.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurochemical Characteristics of a Novel Dorsal Root Ganglion X Neuroblastoma Hybrid Cell Line, F-11

We investigated the properties of the novel dorsal root ganglion (DRG) hybrid cell line F-11 to see how closely these cells resembled normal DRG cells. Under normal growth conditions, F-11 cells appeared to contain several short neurite-like processes. However, these cells could also be grown under conditions in which they showed a much more extensive neuronal morphology, exhibiting many long neurites. Several differentiated features of DRG cells were present on F-11 cells. These included the presence of delta-opioid receptors, receptors for prostaglandins and bradykinin, and dihydropyridine-sensitive calcium channels. F-11 cells also synthesized and released a substance P-like compound, as determined by immunoreactivity. Both the number of bradykinin receptors and the voltage-sensitive calcium influx increased on cell differentiation. Opioid agonists (delta-specificity) were found to decrease cyclic AMP levels in F-11 cells in a naloxone- and pertussis toxin-reversible fashion. Bradykinin stimulated the synthesis of inositol-1,4-bisphosphate and inositol-1,4,5-trisphosphate. Ca2+ channel agonists stimulated voltage-sensitive Ca2+ influx in a dose-dependent, stereospecific manner, whereas Ca2+ channel antagonists inhibited Ca2+ influx. F-11 cells should, therefore, prove useful as models for authentic DRG neurons.     

Stimulation of Phospholipase D Activity in Human Neuroblastoma (LA-N-2) Cells by Activation of Muscarinic Acetylcholine Receptors or by Phorbol Esters: Relationship to Phosphoinositide Turnover

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.