Muscarinic Receptor-mediated Cyclic GMP Formation Research Articles

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

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 microM (compared to 96 microM 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).

Antagonism by the diacylglycerol kinase inhibitor R59 022 of muscarinic receptor-mediated cyclic GMP formation and binding of [ 3H N-methylscopolamine

R59 022 possessed potent antimuscarinic properties in inhibiting muscarinic agonist-mediated [ 3 H] cyclic GMP response and antagonist binding in mouse neuroblastoma N1E-115 cells

Blockade of receptor-mediated cyclic GMP formation by hydroxyeicosatetraenoic acid.

Receptor-mediated cyclic GMP formation in N1E-115 murine neuroblastoma cells appears to involve oxidative metabolism of arachidonic acid. Evidence in support of this includes the blockade of this response by lipoxygenase inhibitors, e.g., eicosatetraynoic acid (ETYA) or other metabolic perturbants, e.g., methylene blue. It was recently discovered that the lipoxygenase products 15-hydroxyeicosatetraenoic (15-HETE) acid and 12-HETE, like ETYA, were inhibitors of M1 muscarinic receptor-mediated cyclic GMP formation. In the present report, the effects of monoHETEs are explored in more detail, particularly with regard to the function of the muscarinic receptor. Like 12-HETE and 15-HETE (IC50 = 13 and 11 microM, respectively), 5-HETE inhibited the cyclic GMP response to the muscarinic receptor (IC50 = 10 microM). All three of these monoHETEs were shown also to be inhibitors of the cyclic GMP responses to receptors stimulated by carbachol, histamine, thrombin, neurotensin, and bradykinin. 15-HETE was shown to inhibit the muscarinic receptor-mediated response in a complex manner (apparent noncompetitive and uncompetitive components; IC50 = 18 and 2 microM, respectively). 15-HETE did not inhibit either the M1 muscarinic receptor-stimulated release of [3H]inositol phosphates from cellular phospholipids or the M2 muscarinic receptor-mediated inhibition of hormone (prostaglandin E1)-induced AMP formation. It seemed possible that the monoHETEs could enter into biochemical pathways for arachidonate in N1E-115 cells. [3H]Arachidonate and the three [3H]-monoHETEs all rapidly labeled the membrane lipids of intact N1E-115 cells, with each [3H]eicosanoid producing a unique labeling profile. [3H]15-HETE labeling was noteworthy in that 85% of the label found in the phospholipids was in phosphatidylinositol (PI;t1/2 to steady state = 3 min). Exogenous 15-HETE inhibited the labeling of PI by [3H]arachidonate (IC50 = 28 microM) and elevated unesterified [3H]arachidonate levels. Thus, the mechanism of blockade of receptor-mediated cyclic GMP responses by monoHETEs is likely to be more complex than the simple inhibition of cytosolic mechanisms, e.g., generation of a putative second messenger by lipoxygenase, and may involve also alterations of membrane function accompanying the redistributions of esterified arachidonate.

Blockade of N1E-115 murine neuroblastoma muscarinic receptor function by agents that affect the metabolism of arachidonic acid

Inhibitors of arachidonate metabolism and perturbants of the oxidation-reduction state of the cell were employed to develop a pharmacologic profile for muscarinic receptor-mediated cyclic GMP formation in murine neuroblastoma cells (clone N1E-115). Several lipoxygenase inhibitors [eicosatetraynoic add (ETYA), nordihydroguaiaretic acid (NDGA), FPL 57231, FPL 55712, BW755c, propylgallate, and AA861] blocked the elevation of [ 3H]cyclic GMP induced by muscarinic receptor activation. The cyclooxygenase inhibitors indomethacin and ibuprofen were two orders of magnitude less potent in blocking the muscarinic receptor-mediated [ 3H]cyclic GMP response than in blocking cyclooxygenase in other systems. ETYA and NDGA did not affect the muscarinic inhibition of the prostaglandin E 1-mediated increases in [ 3H]cyclic AMP levels in N1E-115 cells. ETYA did not have a reproducible effect on the muscarinic receptor-induced release of inositol phosphates. Thus, these lipoxygenase inhibitors appeared to be selective for the effector system coupled to the low-affinity muscarinic agonist-receptor conformation, i.e. that which induces cyclic GMP formation. Other effective inhibitors of the cyclic GMP response were methylene blue, catalase, bromphenacyi bromide, retinal, dithiothreitol, quinacrine, and oxidized glutathione. The antioxidant α-tocopherol in the concentration range of 100 μM to 1 mM potentiated the receptor response. Arachidonic acid itself was an inhibitor of the muscarinic receptor-mediated cyclic GMP response ( ic 50 = 45 μM). Linoleic acid and oleic acid were less potent ( ic 50 = 130 and 190μM, respectively), and stearic acid was ineffective. When arachidonic acid was air-oxidized, its inhibitory potency was increased 10-fold. Most but not all of the spontaneously-produced oxidative metabolites, separable by reverse-phase high pressure liquid chromatography, were inhibitory to the receptor response. Enzymatically synthesized 12-hydroxyeicosatetraenoic add and 15-hydroxyeicosatetraenoic add inhibited the muscarinic receptor [ 3H]cyclic GMP response, with ic 50 values of 17 and 8 μM respectively. Catalase was effective in blocking the muscarinic cyclic GMP response ( ic 50 = 5μM) while having no effect on either the muscarinic receptor-induced inositol phosphate release or the reduction of cyclic AMP levels. Thus, the effector system for increasing cyclic GMP in these cells displays many of the expected characteristics for the involvement of a lipoxygenase or a related enzyme that oxidatively metabolizes arachidonate in order to activate the guanylate cyclase. A number of pharmacologic agents differentiate between this effector system and those to which the muscarinic receptor couples in order to effect cyclic AMP decreases or the release of inositol phosphates.

Involvement of calcium channels in short-term desensitization of muscarinic receptor-mediated cyclic GMP formation in mouse neuroblastoma cells.

Incubation of mouse neuroblastoma cells (clone N1E-115) with Mn2+ resulted in a rapid and transient increase in cyclic GMP formation. This effect appears to be due to an increase in calcium influx because it did not occur in the absence of extracellular calcium or in the presence of verapamil, a calcium transport inhibitor. In addition, Mn2+ inhibited muscarinic receptor-mediated cyclic GMP responses. The ability of Mn2+ to increase cyclic GMP levels was markedly diminished in cells desensitized to the effects of carbamoylcholine, suggesting that this densensitization involves inactivation of calcium entry.

Long-term regulation of muscarinic acetylcholine receptors on cultured nerve cells

Incubation of mouse neuroblastoma cells (clone 1LE-115) with the muscarinic agonist, carbachol, resulted in a time-dependent desensitization to muscarinic receptor-mediated cyclic GMP formation and a decrease in the number and affinity of muscarinic receptors as measured by the binding to intact cells of the muscarinic antagonist, [ 3H]quinuclidinyl benzilate (QNB). The decrease in responsiveness to cyclic GMP formation reached a maximum after a 15-minute exposure to 1 mM carbachol (short-term desensitization) whereas changes in [ 3H] QNB binding became apparent only after a one hour exposure and reached a maximum after about 12 hrs (long-term desensitization). Recovery of sensitivity after short-term desensitization was rapid, suggesting that this process may involve a conformational change in the muscarinic receptor. In contrast, recovery after long-term desensitization was prolonged and could be slowed by the inhibition of protein synthesis. These results indicate that different cellular mechanisms are involved in the short-term and long-term desensitization of muscarinic receptors.

Desensitisation of muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells

THE loss of sensitivity of a tissue to an agonist (perhaps a neurotransmitter) after prolonged exposure to this compound is called desensitisation and may be specific1 or nonspecific2. Although many different mechanisms may be involved in nonspecific desensitisation3, specific desensitisation (tachyphylaxis) has long been considered to involve the agonist–receptor complex4–7. Our interest in the interaction of certain psychotropic drugs with muscarinic acetylcholine receptors of cultured nerve cells8,9 has led us to study the desensitisation of these receptors. They mediate the formation of guanosine 3′,5′-cyclic phosphate (cyclic GMP)8–10, a response which has yielded much information about the interactions of agonists and antagonists with the muscarinic receptor8,9, although a direct coupling of this receptor and guanylate cyclase has not been demonstrated. We report here our use of this receptor effect to characterise the specific desensitisation of muscarinic acetylcholine receptors of intact mouse neuroblastoma cells (clone N1E–115).

Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells—: Ionic dependence and effects of local anesthetics

A new assay technique for measuring receptor-mediated cyclic GMP formation by cultured mouse neutroblastoma cells was used to study the role of ions in, and the effects of local anesthetics on, the function of the muscarinic receptor. The technique involved radioactively labeling intracellular stores of GTP by incubating cells with [ 3H]guanine and isolating [ 3H]cyclic GMP with a cation exchange resin (Dowex-50 +) column. High-pressure liquid chromatography of cell extracts and of eluates from the Dowex column showed that after 45 min the majority of the radioactivity in the cell extracts was [ 3H]GTP and that, for carbamylcholine-stimulated cells, greater than 90 per cent of the radioactivity in the eluates was [ 3H]cyclic GMP. In the absence of external Na + ([Na +] e , with cesium chloride as osmotic filler) or external ([Ca 2+] e ), the carbamylcholine-stimulated formation of [ 3H]cyclic GMP was about 60 and 10 per cent of control, respectively, while removal of other ions had no significant effect. There was little difference in the responses at 10 mM vs 110 mM−[Na +] e , whereas the optimal [Ca 2+], was around 5 mM. Ca 2+ increased [ 3H]cyclic GMP formation in response to carbamylcholine without affecting the apparent affinity of this agonist for the receptor. Local anesthetics were apparently competitive inhibitors of carbamylcholine with equilibrium dissociation constants ( K B ) in the range of 6–250 μM. The rank order for the apparent affinity of local anesthetics for the muscarinic receptor was tetracaine = butacaine = procaine > dibucaine = lidocaine > ethyl aminobenzoate.