Mechanism of Agonist-Induced Down-Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.     

Subcellular Compartmentation of Opioid Receptors: Modulation by Enkephalin and Alkaloids

Abstract: A subclone of NG108–15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes β-glucuronidase, galactosyltransferase, 5′-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [³H]-D-Ala²D-Leu⁵-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [³H]diprenorphine, chased with various unlabeled opiates, and the distribution of ³H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [³H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells.     

Binding of Aminoalkylindoles to Noncannabinoid Binding Sites in NG108-15 Cells

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma × glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharamcological characteristics of [³H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes.2. Although the K _D of specifid [³H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the B _max was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [³H]WIN 55212-2 binding.However, IC₅₀ values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the K _i values for CP55,940 were1.2nM in brain and >5000nM in NG108-15 membranes. Moreover, sodium and GTP--S decreased [³H]WIN 55212-2 binding in brain but not in NG108-15membranes.3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindolebinding sites are not coupled to G proteins.     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

Opiate binding to membrane preparations of neuroblastoma x glioma hybrid cells NG108-15: effects of ions and nucleotides.

Interaction of a number of opiate agonists with the opiate receptors in NG108-15 cell membranes is influenced by ions, as well as certain nucleotides. Steady state binding of [³H]leu-enkephalin is increased by Mg⁺⁺ and decreased by Na⁺, GMP-P(NH)P, GTP, GDP, ITP and IMP-P(NH)P. Half-maximal inhibition produced by GMP-P(NH)P occurred at 4.6 μM. The dissociation of [³H]leu- and [³H]met-enkephalin, as well as [³H]etorphine, from these opiate receptors was also shown to be altered by both ions and nucleotides.     

Down regulation of enkephalin (δ) receptors Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue, d-Ala²-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to δ-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the α₂-adrenergic ligand, [³H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of δ-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

Muscarinic acetylcholine receptor regulation by accelerated rate of receptor loss

Sustained activation of muscarinic acetylcholine receptors on neuron-like NG108-15 hybrid cells reduces the number of [³H]quinuclidinyl benzilate binding sites per cell as much as 88%. The response occurs at concentrations of agonists commensurate with those needed to occupy receptors and inhibit adenylate cyclase. Decreases in steady-state receptor levels persist as long as activator remains present. Withdrawal of activator results in a slow increase in receptor levels that is blocked by cycloheximide. Activation shortens receptor half-life by a factor of nearly 4, indicating that regulation occurs at the level of receptor breakdown.     

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.     

Down-Regulation of Angiotensin II Receptor Subtypes and Desensitization of Cyclic GMP Production in Neuroblastoma N1E-115 Cells

Abstract: Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either ¹²⁵I-AngII or the high-affinity antagonist ¹²⁵I-[Sarc¹,Ile⁸]-AngII. This apparent down-regulation was dose related with an ED₅₀ of 1 nM, and maximal decreases of ～90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37°C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT₁ receptor subtype during the first 5 min, followed by a decline in both AT₁ and AT₂ receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc¹, Thr⁸]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface Angll receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells. These results suggest that differentiated N1E-115 cells are an excellent cell line in which to examine the factors regulating the expression of AngII receptor subtypes in the nervous system.     

The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK–AMPK–dependent mechanism

The role of muscarinic acetylcholine receptors (mAChRs) in regulating glucose uptake in L6 skeletal muscle cells was investigated. [³H]-2-Deoxyglucose uptake was increased in differentiated L6 cells by insulin, acetylcholine, oxotremorine-M and carbachol. mAChR-mediated glucose uptake was inhibited by the AMPK inhibitor Compound C. Whole cell radioligand binding using [³H]-N-methyl scopolamine chloride identified mAChRs in differentiated but not undifferentiated L6 cells and M₃ mAChR mRNA was detected only in differentiated cells. M₃ mAChRs are Gq-coupled, and cholinergic stimulation by the mAChR agonists acetylcholine, oxotremorine-M and carbachol increased Ca²⁺ in differentiated but not undifferentiated L6 cells. This was due to muscarinic but not nicotinic activation as responses were antagonised by the muscarinic antagonist atropine but not the nicotinic antagonist tubocurarine. Western blotting showed that both carbachol and the AMPK activator AICAR increased phosphorylation of the AMPKα subunit at Thr172, with responses to carbachol blocked by Compound C and the CaMKK inhibitor STO609 but not by the PI3K inhibitor wortmannin. AICAR-stimulated AMPK phosphorylation was not sensitive to STO-609, confirming that this compound inhibits CaMKK but not the classical AMPK kinase LKB1. The TAK1 inhibitor (5Z)-7-oxozeaenol and the G_i inhibitor pertussis toxin both failed to block AMPK phosphorylation in response to carbachol. Using CHO-K1 cells stably expressing each of the mAChR subtypes (M₁–M₄), it was determined that only the M₁ and M₃ mAChRs phosphorylate AMPK, confirming a G_q-dependent mechanism. This study demonstrates that activation of M₃ mAChRs in L6 skeletal muscle cells stimulates glucose uptake via a CaMKK–AMPK-dependent mechanism, independent of the insulin-stimulated pathway.     

Src homology 2 (SH2) domain containing protein tyrosine phosphatase-1 (SHP-1) dephosphorylates VEGF Receptor-2 and attenuates endothelial DNA synthesis, but not migration*

Background

Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Moreover, abrogation of β-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.

Results

Herein we report that agonist activation of M₁ mAChRs produces a sustained β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M₂ mAChRs does result in stable co-localization between the M₂ mAChR and β-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M₁ and M₂ mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M₁ and M₂ mAChRs, with the effect substantially higher on the M₂ mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M₂ mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2^{K18R, K107R, K108R, K207R, K296R}, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-arrestin 2^{K18R, K107R, K108R, K207R, K296R} increased the agonist-promoted down-regulation of the M₁ mAChR but did not result in a stable co-localiztion of the receptor with this β-arrestin lysine mutant.

Conclusion

These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M₁ and M₂ mAChRs.     

Effects of carbachol on rat Sertoli cell proliferation and muscarinic acetylcholine receptors regulation: an in vitro study

The aim of the present work was to study the effect of muscarinic agonist on cell proliferation and muscarinic acetylcholine receptors (mAChRs) regulation in rat Sertoli cells. Primary cultures of Sertoli cells were obtained from 8-day and 15-day old male Wistar rats. In proliferation assays, [methyl-3H]thymidine incorporation in Sertoli cells from 8-day and 15-day old rats reached a plateau after 60 min of carbachol incubation and decreased after 120 min of agonist incubation. Binding studies with [N-Methyl-3H]scopolamine ([3H]NMS) indicated a rapid loss of cell surface mAChRs when Sertoli cells from 15-day old rats were incubated with carbachol at 35 degrees C for 2 min. This effect was temperature-dependent. When the incubation of the cells was prolonged at 35 degrees C or at 4 degrees C, after the agonist had been washed away, 94% of mAChRs were present in the cell surface after 120 min incubation at 35 degrees C. At 4 degrees C, however, a low percentage of mAChRs was detected in the cell surface. In the presence of cycloheximide, the recycling of mAChRs to the cell surface was not changed, suggesting that the appearance of mAChRs on cell surface was not dependent on de novo receptor synthesis. In conclusion, our studies indicate that the activation of mAChRs may play a role in rat Sertoli cell proliferation. These receptors may be under regulation (internalization and recycling) when cells are exposed to muscarinic cholinergic agonist.     

A novel analogue of clonidine with opiate-receptor agonist activity

A new analogue of the α₂-adrenergic receptor ligand clonidine, N-(4-hydroxphenacetyl)-4-aminoclonidine, was synthesized. The analogue possesses opiate-receptor agonist activity in addition to α-adrenergic partial agonist activity. The analogue elicits inhibition of adenylate cyclase of NG108-15 neuroblastoma × glioma hybrid cells; most of the inhibition is reversed by the opiate-receptor antagonist naloxone. The analogue also inhibits the binding of [³H]D-Ala²-Met⁵-enkephalinamide and [³H]dihydromorphine to rat brain opiate receptors. The structure of the analogue suggests common elements in the ligand binding sites of α- and opiate receptors and may lead to a new class of opiate analgesics.     

Rabies virus decreases agonist binding to opiate receptors of mouse neuroblastoma-rat glioma hybrid cells 108-CC-15

An acute and a persistent infection with Rabies virus (HEP Flury) was established in the CNS-derived cell line 108-CC-15 (NG 108-15). These cells possess specific membrane receptors to many hormones and neurotransmitters including opiate receptors. In both cases we found an increase in the dissociation constant (K_D) for the agonist ³H -etorphine as estimated by Scatchard plot analysis. However, in both cases there was no change in the number of opiate receptors per cell compared to uninfected cells. These studies complete our previous published observations of the impairment of receptor functions in rabies virus infected cells (1).     

Specific bindings of [3H](+)PN200-110 and [125I]ω-conotoxin to crude membranes from differentiated NG108-15 cells

The characteristics of the specific bindings of [³H](+)PN200-110 (PN: L-type Ca channel antagonist) and [¹²⁵I]-conotoxin G VI A (-CgTX: neuronal L-or N-type Ca channel antagonist) to crude membranes from undifferentiated neuroblastoma x glioma hybrid NG108-15 (NG108-15) cells and differentiated cells induced with dibutyryl cAMP (Bt₂cAMP) were examined, because we have already observed that the magnitude and rate of KCL-stimulated⁴⁵Ca uptake by NG108-15 cells increased progressively during differentiation of the cells induced with Bt₂cAMP (unpublished results). The specific binding of [³H](+)PN to these crude membranes was saturable at various concentrations of 2.5–5.0 nM [³H](+)PN. Scatchard analysis showed that the specific binding of [³H](+)PN at equilibrium was significantly increased after differentiation of the NG108-15 cells with Bt₂cAMP, but that the apparent Kd value for the specific binding of [³H](+)PN was not influenced by treatment with Bt₂cAMP. The specific binding of [³H](+)PN to crude membranes from Bt₂cAMP-treated NG108-15 cells was inhibited by a calcium agonist and antagonists, the order of their inhibitory potencies being (+)PN>nitrendipine>(–)PNBay K 8644diltiazem = verapamil. Thus, PNs showed significant stereoselective inhibition of the specific binding of [³H(+)PN. On the other hand, [¹²⁵I]-CgTX at concentrations of 0.075–0.6 nM showed scarcely any specific binding to these crude membranes, although at 0.6 nM it showed specific binding to crude membranes from rat brain in the same experimental conditions. These results suggest that the increase in magnitude or rate of KCl-stimulated⁴⁵Ca uptake during differentiation of NG108-15 cells is partially due to quantitative alteration of voltage-sensitive Ca channels in the cells, and that there are scarcely any specific binding sites for [¹²⁵I]-CgTX on Bt₂cAMP-treated or untreated NG108-15 cells.     

Identification and characterization of the beta-adrenergic receptor on neuroblastoma × glioma hybrid NG108-15 cells

1. Using [3H]DHA and unlabeled L-alprenolol, a substantial amount of over 64% specific binding of beta-adrenergic receptor has been identified on the neuroblastoma x glioma hybrid NG108-15 cell, which has been proven to display numerous functional characteristics of intact neurons. 2. Beta-adrenergic receptor binding on intact NG108-15 cells does not change significantly upon morphological differentiation, induced by 1 mM dibutyryl cyclic AMP (dBcAMP). 3. The [3H]DHA binding on intact NG108-15 cells is rapid, saturable, and reversible, having a t1/2 of 1.0 min for association and 3.5 min for dissociation. 4. The affinity constant (Kd) and maximum binding capacity (Bmax) for binding of [3H]DHA to beta-adrenergic receptors on NG108-15 cells have been estimated by Scatchard plot analysis to be 2.5 and 0.23 nM, respectively. Further analysis indicates a single class of receptors for [3HDHA binding on NG108-15 cells. 5. Studies on kinetic properties have revealed on-rate (K + 1) and off-rate (K - 1) constants of 0.7 X 10(-9) M min-1 and 0.19 min-1, respectively. Further, the IC50 value and inhibition constant (Ki) for unlabeled L-alprenolol to inhibit [3HDHA binding on NG108-15 cells have been estimated to be 10(-5) and 8.9 X 10(-6) M, respectively. 6. The rank-order potency of catecholamine agonists, (-)ISO greater than (+)ISO greater than EPI greater than NE, reveals the presence of type 2 receptor for the beta-adrenergic binding on both differentiated and undifferentiated NG108-15 cells. 7. The present study indicates that the clonal neuroblastoma x glioma hybrid NG108-15 cell line possesses substantial amounts of beta-adrenergic receptors with characteristics similar to those on neuronal cells.     

Independent and Coordinate Regulation of β1- and β2-Adrenergic Receptors in Rat C6 Glioma Cells

Abstract

Rat C6 glioma cells have both β₁- and β₂-adrenergic receptors in ～ 7:3 ratio. When the cells were exposed to the β-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [³H] CGP-12177 to intact cells. Using the β₂-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both β₁ and β₂ receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of ～ 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [¹²⁵I] iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous β₁-and β₂-adrenergic receptors in the same cell line.     

Effects of pertussis toxin-catalyzed ADP-ribosylation on interactions of transducin and the inhibitory GTP-binding protein of adenylate cyclase with guanyl nucleotides

Release of bound [3H]Gpp(NH)p from NG108-15 cell membranes was induced by carbamylcholine, enkephalinamide, and norepinephrine, all of which inhibit adenylate cyclase. Release was blocked by antagonist, was greater with multiple agonists than with one, and required guanyl nucleotides. With membranes from pertussis toxin-treated cells, both total [3H] Gpp(NH)p binding and agonist-induced [3H]Gpp(NH)p release was decreased. ADP-ribosylation by toxin of transducin, the retinal GTP-binding protein which is similar in structure and function to that in cyclase, decreased [3H]Gpp(NH)p binding. Thus, the inability to demonstrate agonist-induced [3H]Gpp(NH)p release from toxin-treated NG108-15 membranes may result in part from absence of bound [3H]Gpp(NH)p.     

Multiple binding states of muscarinic acetylcholine receptors in membranes from neuroblastoma X glioma hybrid cells

Membranes of neuron-like NG108-15 hybrid cells bind [³H]quinuclidinyl benzilate (QNB) with high affinity and specificity. Greater than 90% of total [³H]QNB binding is to sites having the pharmacological specificity of muscarinic acetylcholine receptors. Three significant features characterize the interaction of ligands with these sites: (1) Specific binding of [³H]QNB at equilibrium follows a simple adsorption isotherm with an apparent K_D of 1 × 10^?10 M; (2) Rates of [³H]QNB association and dissociation are biphasic and, as the binding reaction proceeds, the fraction of readily dissociable [³H]QNB decreases; (3) Competition against [³H]QNB for specific binding sites by antagonists gives a slope of 1 when analyzed on Hill plots, but competition for binding sites by agonists gives a slope of less than 1. A simple two-step model for activation is proposed to account for these features.     

Endogenous GM1 Ganglioside of the Plasma Membrane Promotes Neuritogenesis by Two Mechanisms

The influence of GM1 on the neuritogenic phase of neuronal differentiation has been highlighted in recent reports showing upregulation of this ganglioside in the plasma and nuclear membranes concomitant with axonogenesis. These changes are accompanied by alterations in Ca²⁺ flux which constitute an essential component of the signaling mechanism for axon outgrowth. This study examines 2 distinct mechanisms of induced neurite outgrowth involving plasma membrane GM1, as expressed in 3 neuroblastoma cell lines. Growth of Neuro-2a and NG108-15 cells in the presence of neuraminidase (N'ase), an enzyme that increases the cell surface content of GM1, caused prolific outgrowth of neurites which, in the case of Neuro-2a, could be blocked by the B subunit of cholera toxin (Ctx B) which binds specifically to GM1; however, the latter agent applied to NG108-15 cells proved neuritogenic and potentiated the effect of N'ase. With N18 cells, the combination was also neuritogenic as was Ctx B alone, whereas N'ase by itself had no effect. Neurite outgrowth correlated with influx of extracellular Ca²⁺, determined with fura-2. Treatment of NG108-15 and N18 cells with Ctx B alone caused modest but persistent elevation of intracellular Ca²⁺ while a more pronounced increase occurred with the combination Ctx B + N'ase. Treatment with N'ase alone also caused modest but prolonged elevation of intracellular Ca²⁺ in NG108-15 and Neuro-2a but not N18; in the case of Neuro-2a this effect was blocked by Ctx B. Neuro-2a and N18 thus possess 2 distinctly different mechanisms for neuritogenesis based on Ca²⁺ modulation by plasma membrane GM1, while NG108-15 cells show both capabilities. The neurites stimulated by N'ase + Ctx B treatment of N18 cells were shown to have axonal character, as previously demonstrated for NG108-15 cells stimulated in this manner and for Neuro-2a cells stimulated by N'ase alone.