Kinetic Effects of Terbium Ions on Muscarinic Acetylcholine Receptors of Murine Neuroblastoma Cells

Preincubation of murine neuroblastoma cells (clone N1E-115) with terbium chloride resulted in a significant potentiation of carbachol-mediated increase in cyclic GMP formation. This effect was accompanied by a shift of the peak response from 30 s to 120 s and a 6-fold decrease in carbachol concentration producing half-maximal responses, in addition to a significant increase in the Hill coefficient. Terbium ions also caused a significant decrease in the affinity and an increase in the maximum binding of [3H]quinuclidinyl benzilate to muscarinic receptors, the change in affinity being mainly due to a decrease in the association rate. Preincubation of cells with 1 mM carbachol for 4 h (the desensitized state of the muscarinic receptor) resulted in a decrease in the ability of terbium to alter [3H]quinuclidinyl benzilate binding. The effects of terbium reported here might be due to its affecting muscarinic receptor-effector coupling, which is considered to be lost upon receptor desensitization.     

Effect of Some Calcium Antagonists on Muscarinic Receptor-Mediated Cyclic GMP Formation

Several calcium antagonists were screened for their effect on muscarinic acetylcholine receptor-mediated cyclic GMP formation in murine neuroblastoma cells (clone N1E-115). Mn2+, Ni2+, and verapamil rapidly antagonized the response noncompetitively, with the following order of potency: verapamil greater than Mn2+ greater than Ni2+. The effects of Mn2+ and Ni2+, but not those of verapamil, were largely reversed by increasing extracellular calcium concentration. Additional effects of these agents included displacement of [3H]quinuclidinyl benzilate binding by verapamil and elevation of cyclic GMP levels by Mn2+ and Ni2+ in the absence of agonists. These results are in support of the hypothesis that receptor-mediated cyclic GMP formation by these cells is dependent upon entry of calcium into the cell and demonstrate the complexity of the effects of calcium antagonists.     

Role of Intercellular and Intracellular Communication by Nitric Oxide in Coupling of Muscarinic Receptors to Activation of Guanylate Cyclase in Neuronal Cells

Abstract: Muscarinic receptor-mediated cyclic GMP formation and release of nitric oxide (NO) (or a precursor thereof) were compared in mouse neuroblastoma N1E-115 cells. [³H]Cyclic GMP was assayed in cells prelabeled with [³H]guanine. Release of NO upon the addition of muscarinic agonists to unlabeled neuroblastoma cells (NO donor cells) was quantitated indirectly by its ability to increase the [³H]cyclic GMP level in labeled cells whose muscarinic receptors were inactivated by irreversible alkylation (NO detector cells). Carbachol increased NO release in a concentration-dependent manner, with half-maximal stimulation at 173 μ M (compared to 96 μ M for direct activation of cyclic GMP formation). The maximal effect of carbachol in stimulating release of NO when measured indirectly was lower than that in elevating [³H]cyclic GMP directly in donor cells. Hemoglobin was more effective in blocking the actions of released NO than in attenuating direct stimulation of [³H]cyclic GMP synthesis. There was a good correlation between the ability of a series of muscarinic agonists to release NO or to activate [³H]cyclic GMP formation directly, and the potency of pirenzepine in inhibiting the two responses. Furthermore, there was a similar magnitude of desensitization of both responses by prolonged receptor activation or stimulation of protein kinase C. NO release was also regulated in relation to the cellular growth phase. A model is proposed in which a fraction of NO generated upon receptor activation does not diffuse extracellularly and stimulates cyclic GMP synthesis within the same cell where it is formed (locally acting NO). The remainder of NO that is extruded extracellularly might travel to neighboring cells (neurotransmitter NO) or might be taken back into the cells of origin (homing NO).     

Regulation of neuronal nitric oxide synthase by histone,protamine, and myelin basic protein

We examined the effects of endogenous basic proteins rich in the amino acidL-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca²⁺ gradient by a Ca²⁺ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influex of extracellular Ca²⁺ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca²⁺ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism ofL-arginine intoL-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.     

Regulation of Muscarinic Receptor-Mediated Cyclic GMP Synthesis by Cultured Mouse Neuroblastoma Cells

Mouse neuroblastoma clone N1E-115 has muscarinic acetylcholine receptors that mediate cyclic GMP synthesis. This receptor-mediated response is not significantly higher than background until the cells have been maintained in the stationary phase for at least 1 week. The basis of the influence of time in culture on the cyclic GMP response was investigated. The relative amount of cyclic GMP synthesized by intact cells was measured by radioactively labeling the GTP pool with [³H]guanine, incubating cells with agonists, and then chromatographically isolating [³H]cyclic GMP. Carbamylcholine-, ionophore X-537A-, and sodium azide-induced cyclic GMP formation increased with time in culture to a maximum of 13-, 9-, and 2.5-fold above basal, respectively. There was no change in the number or the apparent affinity of the muscarinic receptors as measured by [³H]quinuclidinyl benzylate ([³H]QNB) binding. In addition, there was no change in the apparent affinity of the receptors for agonist as measured by the ability of carbamylcholine to displace the specific binding of [³H]QNB. Guanylate cyclase activity per milligram protein and per cell in-creased six- and sevenfold, respectively, from day 0 to day 22. However, this increase in guanylate cyclase appeared to precede the marked increase in sensitivity of the cells to agonists. These data suggest that, in addition to guanylate cyclase and muscarinic receptors, there is another factor which is responsible for the development of this muscarinic receptor-mediated response.     

Temperature Dependence of Muscarinic Acetylcholine Receptor Activation, Desensitization, and Resensitization

Abstract: The effects of temperature on muscarinic acetylcholine receptor activation, desensitization, and resensitization were studied with the use of intact mouse neuroblastoma cells (clone N1E-115), which have muscarinic receptors that mediate cyclic GMP synthesis. Below 15-20°C, activation or desensitization of muscarinic receptors by carbamylcholine and recovery from desensitization (caused by carbamylcholine at 37°C) did not occur. Above these temperatures, the apparent rates of receptor-mediated cyclic GMP synthesis, desensitization, and recovery of sensitivity increased as the incubation temperature was increased. Arrhenius plots of the data yielded activation energies of 25, 14, and 23 kcal.mol⁻¹ for activation, desensitization, and resensitization, respectively. These data suggest that a certain degree of membrane phospholipid fluidity is required for these processes to occur.     

Regulation of M1 Muscarinic Receptor-Mediated Signaling in Intact Cells by Exogenous,but Not Endogenously Produced,Nitric Oxide

Regulation of nitric oxide (NO) formation is critical to ensure maintenance of appropriate cellular concentrations of this labile, signaling molecule. This study investigated the role exogenous and endogenously produced NO have in feeding back to regulate NO synthesis in intact cells. Two NO donors inhibited activation of neuronal NO synthase (nNOS) in response to the muscarinic receptor agonist carbachol in Chinese hamster ovary (CHO) cells stably transfected with the M₁ muscarinic receptor and nNOS. The presence of the NO scavenger [2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide · potassium salt] (C-PTIO) potentiated carbachol-induced activation of nNOS in transfected CHO cells. C-PTIO also potentiated nNOS activity in response to the Ca²⁺ ionophore ionomycin. In contrast, the NO scavenger oxyhemoglobin depressed carbachol- and ionomycin-induced NO formation. These discrepant results suggest that it is unlikely that endogenously produced NO induces feed back inhibition at the level of nNOS activation itself. Exogenous sources of NO inhibited carbachol-induced inositol phosphates formation. However, endogenously produced NO did not appear to feed back to regulate phosphoinositide hydrolysis as there was no difference in [³H]inositol phosphates formation between cells that do or do not express nNOS. There was also no change in carbachol-induced [³H]inositol phosphates formation in the presence or absence of a NOS inhibitor or the NO scavenger C-PTIO. A decrease in the carbachol-mediated transient Ca²⁺ peak was observed in cells that express nNOS as compared to cells lacking the enzyme, suggesting that endogenous NO might inhibit receptor mediated Ca²⁺ signaling. This conclusion, however, was not supported by the lack of ability of a NOS inhibitor to modulate carbachol-induced Ca²⁺ elevations. Taken together, these results highlight differences in the regulation of the nNOS activation cascade by endogenous vs. exogenous sources of NO.     

Arachidonic Acid Inhibition of Muscarinic Receptor-Mediated Nitric Oxide Production Occurs at the Level of Calcium Mobilization in Chinese Hamster Ovary Cells

Strong evidence supports that nitric oxide (NO) alters cell signaling pathways involving arachidonic acid (AA). Little is known, however, about the reciprocal modulation of nitrergic pathways by AA. The effects of exogenous AA on signal transduction of M₁ muscarinic acetylcholine receptors were investigated in a model system of stably transfected Chinese hamster ovary cells. AA concentration-dependently inhibited the effects of carbachol in producing NO (IC₅₀ = 191 M) but did not alter inositol phosphate production or M₁ receptor binding. AA inhibited both carbachol-induced transient and sustained increase in intracellular calcium concentration ([Ca²⁺]_i; IC₅₀ = 11 and 12 M, respectively). Furthermore, AA-induced increase in [Ca²⁺]_i cross-desensitizes with thapsigargin, but AA does not inhibit Ca²⁺-ATPase activity. These data support the concept that AA concentration-dependently inhibits receptor-mediated NO production at the level of calcium mobilization.     

Prolonged inhibition of brain nitric oxide synthase by short-term systemic administration of nitro-l-arginine methyl ester

We studied the dose-response characteristics and the temporal profile of inhibition of brain nitric oxide (NO) synthase (NOS) elicited by i.v. administration of the NOS inhibitor nitro-l-arginine methyl ester (L-NAME). L-NAME was administered i.v. in awake rats equipped with a venous cannula. L-NAME was injected in cumulative doses of 5, 10, 20 and 40 mg/kg and rats were sacrificed 30 min after the last dose. NOS catalytic activity was assayed in forebrain cytosol as the conversion of [³H]l-arginine into [³H]l-citrulline. L-NAME attenuated brain NOS activity in a dose-dependent manner but enzyme activity could not be inhibited by more than 50%. After a single 20 mg/kg injection of L-NAME the inhibition of brain NOS activity was time dependent and reached a stable level at 2 hrs (52% of vehicle). Inhibition after a single injection was still present at 96 hrs, albeit to a lower magnitude. We conclude that intravenous administration of L-NAME in rats at concentrations commonly used in physiological experiments leads to a dose and time-dependent but partial inhibition of brain NOS catalytic activity. The finding that the inhibition persists for several days after a single administration is consistent with the hypothesis that nitro-L-arginine, the active principle of L-NAME, binds to NOS irreversibly.     

Endogenous expression of nNOS protein in several neuronal cell lines

Several neuronal cell lines were screened for endogenous expression of neuronal nitric oxide synthase (nNOS) protein using Western blot analysis. Detectable levels of the nNOS protein were evident in the SK-N-SH, SH-SY5Y, and N1E-115 neuroblastoma cell lines, as well as the NG108-15 neuroblastoma x glioma hybrid. Only trace amounts were visible in Neuro2A human neuroblastoma cells. The presence of endogenously expressed nNOS in these cells may allow for the study of the interaction between nNOS and the endogenous receptor systems expressed in the same cells.