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 acid (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 E1-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, bromphenacyl bromide, retinal, dithiothreitol, quinacrine, and oxidized glutathione. The antioxidant alpha-tocopherol in the concentration range of 100 microM to 1 mM potentiated the receptor response. Arachidonic acid itself was an inhibitor of the muscarinic receptor-mediated cyclic GMP response (IC50 = 45 microM). Linoleic acid and oleic acid were less potent (IC50 = 130 and 190 microM, 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 acid and 15-hydroxyeicosatetraenoic acid inhibited the muscarinic receptor [3H]cyclic GMP response, with IC50 values of 17 and 8 microM respectively. Catalase was effective in blocking the muscarinic cyclic GMP response (IC50 = 5 microM) 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 may 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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Selective loss with subculturing of the low-affinity agonist site mediating cyclic GMP formation

Cells of the murine neuroblastoma clone N1E-115 possess muscarinic receptors that influence the intracellular level of cyclic nucleotides. The stimulation of [3H]cyclic GMP levels occurs only with intact cells and has an EC50 near the "low-affinity" agonist equilibrium dissociation constant (KL) determined by radioligand binding assays. The inhibition of prostaglandin E1-stimulated [3H]cyclic AMP formation has an EC50 close to the value for the "high-affinity" agonist equilibrium dissociation constant (KH). During sequential subculturing in medium supplemented with newborn bovine serum, the inhibition of [3H]cyclic AMP was maintained, but the [3H]cyclic GMP response declined dramatically, and after 7 subculturings it was essentially absent. The time course for [3H]cyclic GMP formation in a late subculture with an 88% loss of the response was identical with the time course in early subcultures. A normal [3H]cyclic GMP response to bradykinin and histamine was demonstrated to be present in cells that had lost the [3H]cyclic GMP response to carbachol. The EC50 and KD values for the two muscarinic responses and binding sites increased 3- to 4-fold after several subculturings. A 90% loss of low-affinity binding sites was closely correlated with a similar loss of the [3H]cyclic GMP response. High-affinity binding sites did not decline significantly in concentration until the 11th subculture, where the total number of muscarinic sites was only 6% of the earliest subculture. In all subcultures, however, the ability of the muscarinic receptor to decrease [3H]cyclic AMP levels was maintained. These data, which show that the subculturing of N1E-115 cells in medium supplemented with newborn calf serum results in a selective loss of one muscarinic function, strongly support the hypothesis that these cells contain two separate muscarinic receptor-effector systems. One receptor subtype or conformation has a low affinity for the agonist and mediates cyclic GMP formation. The other receptor subtype or conformation has a higher affinity for the agonist and mediates an inhibition of prostaglandin E1-stimulated cyclic AMP formation.     

Characterization of autonomic receptors in the rat sublingual gland by biochemical and radioligand assays

Summary The autonomic receptors of the rat sublingual gland were characterized by radioligand binding and by specific functional responses involving the release of K⁺ and the generation of cyclic AMP in vitro. Both muscarinic cholinergic and alpha₂-adrenergic receptors were present in moderately high density in the sublingual gland, as judged by the binding of the specific radioligands ³H-QNB and ³H-clonidine (B _max in pmol/g tissue=18.4±2.4 and 9.9±1.3, respectively). By contrast, although alpha₁ and beta-adrenergic receptors were detected, they were not present in large numbers. The B _max (pmol/g tissue) for the binding of ³H-prazosin and ³H-DHA were, respectively, 3.2±0.6 and 3.6±0.4. Stimulation of muscarinic cholinergic receptors with carbamylcholine (2×10^–5 M) caused a net release of K⁺ from sublingual slices incubated in an enriched, oxygenated Krebs Ringer bicarbonate medium of 35±7% after 10 min of incubation. Norepinephrine, phenylephrine, clonidine and isoproterenol did not induced K⁺ release from the slice preparation, but actually reduced the basal or unstimulated release of K⁺. As in the submandibular and parotid glands, the release of K⁺ from sublingual slices had two components, a passive efflux and an active uptake which depended on the activation of an ouabain-sensitive Na⁺, K⁺ ATPase. The release of K⁺ was also dependent on the presence of Ca²⁺ (2.7 mM) in the incubation medium. Stimulation of beta-adrenergic receptors with isoproterenol (10^–5 M) caused a 9-fold increase in the content of glandular cyclic AMP (from 4.0±0.6 to 36.0±2 pmol/mg/10 min). A similar increase was observed with norepinephrine (10^–4 M) in the presence of phentolamine (10^–4 M). Clonidine at concentrations of 10^–5 and 10^–4 M reduced the cyclic AMP content to below basal levels. The rat sublingual gland has functional cholinergic receptors and can release K⁺ in vitro in the presence of cholinergic agents. In contrast to the other major salivary glands, it has a low density of alpha₁-adrenergic receptors and fails to release K⁺ upon stimulation with alpha₁-agonists. The functional significance of a low density of beta-adrenergic receptors is still unclear, although there is a definite glandular cyclic AMP response upon stimulation with isoproterenol. Abundant alpha₂-adrenergic receptors in the sublingual gland are apparently negatively coupled to adenylate cyclase.     

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 ([Ca²⁺]_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 [Ca²⁺], was around 5 mM. Ca²⁺ 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.     

Inhibitory effects of mepacrine and eicosatetraynoic acid on cyclic GMP elevations caused by calcium and hormonal factors in rat ductus deferens

Summary Stimulation of cyclic GMP formation by hormones and neurotransmitters is restricted to intact cells and requires the presence of Ca²⁺ and, as shown for some tissues, of oxygen. Since peroxidized free fatty acids and oxygen radicals can stimulate soluble guanylate cyclase preparations, the effects of agents that interfere with the release or peroxidation of arachidonic acid were studied on Ca²⁺-and hormone-stimulated cyclic GMP levels in rat ductus deferens.When isolated tissue was preincubated for 30 min in Ca²⁺-free Krebs-Ringer-bicarbonate buffer, addition of Ca²⁺ (2 mM) for 3 min increased cyclic GMP levels about 3-fold. Noradrenaline and acetylcholine (0.1 mM each) added with Ca²⁺ caused 6- and 8-fold increases, respectively. Sodium nitroprusside (0.1 mM), independently of the presence of Ca²⁺, caused an 8-fold increase. Preincubation with 0.1 mM mepacrine, an inhibitor of phospholipase A₂, abolished the effects of Ca²⁺ and noradrenaline and reduced the effect of acetylcholine whereas the response to sodium nitroprusside was unchanged. Contractile responses were not affected by mepacrine. Preincubation with the arachidonic acid analogue, eicosatetraynoic acid (0.1 mM), which inhibits arachidonate peroxidation, reduced cyclic GMP responses to Ca²⁺, acetylcholine, noradrenaline and sodium nitroprusside without significant effect on contractile responses. Whereas the cyclooxygenase inhibitor, indomethacin (0.1 mM), did not affect cyclic GMP responses, nordihydroguaiaretic acid (0.1 m), an antioxidant and lipoxigenase inhibitor, reduced cyclic GMP responses to all of the above stimulants but also reduced contractile responses of the tissue.These findings support the concept that the release of arachidonic acid and possibly other unsaturated fatty acids by Ca²⁺-stimulated phospholipases and the subsequent formation of peroxidized fatty acids, which are presumably degraded under formation of an active oxygen species, are involved in hormone-induced stimulation of cyclic GMP formation.Abbreviations PGG₂ Prostaglandin G₂ - PGH₂ prostaglandin H₂ - ETYA eicosatetraynoic acid - HPETE hydroperoxyeicosatetraenoic acid - EDTA ethylenediamine tetraacetic acid Data are taken from a dissertation to be submitted by C.S. to the Pharmazeutische Fakultät, Universität Heidelberg, in partial fulfillment of the requirements of the Dr. rer. nat. degree. Preliminary reports were presented (Schultz et al., 1979a; Spies et al., 1979)     

Effect of the antitumor drug caracemide on the neurochemistry of murine neuroblastoma cells (clone N1E-115)

Because the antitumor drug caracemide causes neuropsychiatric effects in patients, we investigated its effects on the neurochemistry of cultured neuroblastoma cells (murine clone N1E-115). The drug caused a transient elevation in the level of [3H]cyclic GMP that was not blocked by receptor antagonists or by desensitization of histamine or muscarinic receptors. The EC50 for the response to caracemide was 635 microM. Preincubation of cells with caracemide led to the inhibition of muscarinic receptor-mediated [3H]cyclic GMP formation with an IC50 of 450 microM. Caracemide inhibited basal guanylate cyclase activity in homogenates noncompetitively with a Ki value of 162 microM. The drug also inhibited sodium nitroprusside-stimulated guanylate cyclase in homogenates. Caracemide did not inhibit basal adenylate cyclase activity in either intact cells or homogenates, but inhibited adenylate cyclase activated by prostaglandin E1 (PGE1) or forskolin. The muscarinic receptor-mediated reduction of PGE1-stimulated [3H]cyclic AMP formation was not affected. The Ki for the inhibition of PGE1-activated adenylate cyclase in homogenates was 110 microM. Caracemide was a competitive inhibitor of acetylcholinesterase with a Ki value of 8 microM. The drug did not inhibit, but slightly stimulated, monoamine oxidase activity in N1E-115 cells. The results indicate that caracemide can affect several neurochemical systems in neural cells in culture in a way that correlates with its neuropsychiatric effects. The N1E-115 clone thus appears to be useful for evaluating some of the molecular pharmacological effects of drugs interacting with the nervous system.     

The effect of l-dopa and propranolol on human CSF cyclic nucleotides

Human CSF cyclic AMP and cyclic GMP have been measured as possible indicators of activity of central neurotransmitter-sensitive adenylate or guanylate cyclase. In an attempt to help to identify the specific neurotransmitter systems of origin of human CSF cyclic AMP and GMP, we studied Parkinson patients with and without l-dopa therapy and schizophrenic patients before and after propranolol therapy. No effect of l-dopa or propranolol was found on CSF cyclic nucleotides. However, Parkinson patients had a 40–50% reduction of CSF cyclic AMP and a 80–90% reduction of CSF cyclic GMP compared with the schizophrenic patients. Implications of this finding are discussed.     

Exogenous GTP Increases Cyclic GMP and Inhibits Thrombin-Induced Aggregation of Washed Human Platelets: Comparison with ATP,Adenosine and Guanosine

Abstract: The effects of exogenous guanosine 5′-triphosphate (GTP), guanosine, adenosine 5′-triphosphate (ATP) and adenosine on platelet aggregation, serotonin secretion and cyclic nucleotide accumulation were studied using thrombin-stimulated washed human platelets. GTP (10 μM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion. The inhibition of aggregation was accompanied by an increase in platelet cyclic GMP. GTP did not affect cyclic AMP concentration. Adenosine (1 μM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion, and increased cyclic AMP. ATP at high concentrations (100 μM-1 mM) inhibited aggregation and serotonin secretion, and 1 mM ATP increased cyclic AMP. Guanosine was relatively ineffective in preventing aggregation and serotonin secretion and did not affect cyclic GMP. The rank order of inhibition of thrombin-induced aggregation of washed human platelets was adenosine > GTP > ATP > guanosine. In conclusion, exogenous GTP inhibits thrombin-induced aggregation and serotonin secretion of washed human platelets by increasing cyclic GMP. The results raise the possibility of a cell membrane site of action for GTP in platelets which mediates the activation of soluble guanylate cyclase suggesting that GTP may have a local antithrombotic effect also in vivo.     

Atriopeptin II-induced relaxation of rabbit aorta is potentiated by M&B 22,948 but not blocked by haemoglobin.

We examined the effects of haemoglobin (which inhibits the vascular responses to stimulation of soluble guanylate cyclase) and of M&B 22,948 (which selectively inhibits cyclic GMP phosphodiesterase) on the relaxation induced in rabbit aorta by the atrial natriuretic peptide, atriopeptin II (which stimulates particulate guanylate cyclase). Pretreatment with M&B 22,948 (100 microM) produced a 2.3 fold potentiation of atriopeptin II-induced relaxation of endothelium-denuded rings of rabbit aorta. Pretreatment with haemoglobin (10 microM) had no effect on the relaxation or the 10.9 fold increase in cyclic GMP content induced by atriopeptin II in endothelium-denuded rings of rabbit aorta. The potentiation by M&B 22,948 suggests a causal role for cyclic GMP in mediating atriopeptin II-induced vasodilatation of rabbit aorta. The inability of haemoglobin to block the atriopeptin II-induced rise in cyclic GMP suggests that it does not block stimulation of particulate guanylate cyclase. Thus, it is unlikely that a ferrous haem-containing receptor site is involved in the activation of the particulate form of guanylate cyclase as it is with soluble guanylate cyclase.     

Exogenous GTP increases cyclic GMP and inhibits thrombin-induced aggregation of washed human platelets: comparison with ATP, adenosine and guanosine.

The effects of exogenous guanosine 5'-triphosphate (GTP), guanosine, adenosine 5'-triphosphate (ATP) and adenosine on platelet aggregation, serotonin secretion and cyclic nucleotide accumulation were studied using thrombin-stimulated washed human platelets. GTP (10 microM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion. The inhibition of aggregation was accompanied by an increase in platelet cyclic GMP. GTP did not affect cyclic AMP concentration. Adenosine (1 microM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion, and increased cyclic AMP. ATP at high concentrations (100 microM-1 mM) inhibited aggregation and serotonin secretion, and 1 mM ATP increased cyclic AMP. Guanosine was relatively ineffective in preventing aggregation and serotonin secretion and did not affect cyclic GMP. The rank order of inhibition of thrombin-induced aggregation of washed human platelets was adenosine > GTP > ATP > guanosine. In conclusion, exogenous GTP inhibits thrombin-induced aggregation and serotonin secretion of washed human platelets by increasing cyclic GMP. The results raise the possibility of a cell membrane site of action for GTP in platelets which mediates the activation of soluble guanylate cyclase suggesting that GTP may have a local antithrombotic effect also in vivo.     

Cyclic GMP stimulation by vasopressin in LLC-PK1 kidney epithelial cells is l-arginine-dependent

Summary The l-arginine antagonist N^G-monomethyl-l-arginine has been shown to inhibit nitric oxide formation from l-arginine in endothelial cells. In the present study N^G-monomethyl-l-arginine was used to assess the role of l-arginine for cyclic GMP stimulation by vasopressin in a kidney epithelial cell line (LLC-PK₁). Preincubation of cells with 1 mol/l, 10 mol/l and 100 mol/l N^G-monomethyl-l-arginine decreased cyclic GMP stimulation at 1 mol/l vasopressin by 25%, 71% and 90%, respectively. This inhibition by N^G-monomethyl-l-arginine was markedly reduced by l-arginine (2 mmol/1) but not d-arginine (2 mmol/1). Cyclic GMP stimulation by the calcium ionophore A23187 was also inhibited by N^G-monomethyl-l-arginine and enantioselectively restored by l-arginine. However, N^G-monomethyl-l-arginine did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. These results suggest that, in kidney epithelial cells, vasopressin induces nitric oxide formation from l-arginine leading to activation of soluble guanylate cyclase. It is concluded that nitric oxide formation from l-arginine is not only responsible for endothelium-dependent relaxation but may be a more general pathway with regulatory function for intracellular guanylate cyclase activity.Send offprint requests to K. Schror at the above address     

Cyclic nucleotides and contractility in human and sheep umbilical arteries

Strips of human umbilical artery (HUA) and sheep umbilical artery (SUA) were freeze-clamped at selected times during drug-induced contraction or relaxation. Tissue concentrations of cyclic AMP and cyclic GMP were measured. 5-Hydrpxytryptamine (10 μM) contracted HUA and SUA, but had no detectable effect on cyclic GMP; cyclic AMP rose in SUA (but not in HUA) after contractions had begun. Histamine (4 gmM) elevated cyclic GMP in HUA with a time course that lagged behind contraction. Prostaglandin E₁ (PGE₁), PGE₂ and PGF_2α (2.8 μM with 0.1 % v/v ethanol) contracted HUA and decreased cyclic GMP. Nitroglycerin (3 μM) relaxed KCl-contracted HUA; this relaxation was preceded by an initial 29-fold elevation of cyclic GMP. Our data shows no consistent correlation of cyclic nucleotide changes with contraction of unbilical arteries and suggest that such changes are not essential for contraction of vascular smooth muscle. In contrast, the temporal relationship between cyclic GMP elevation and relaxation induced by nitroglycerin suggests a possible role for cyclic GMP.     

Stimulation of adenosine 3′,5′-monophosphate formation in guinea-pig cerebral cortical slices in a calcium free medium

Summary In guinea-pig cerebral cortical slices cyclic AMP concentrations increase during incubation with histamine+noradrenaline. After 10 min of incubation the levels of cyclic AMP start to decline. When calcium ions are omitted from the incubation medium, cyclic AMP levels do increase to a greater extent under the same conditions and do not drop during 30 min incubation. In the presence of calcium ions cyclic AMP synthesis can not be elicited by noradrenaline alone. In calcium-free Krebs-Ringer solution a pronounced effect of noradrenaline on cyclic AMP levels is observed. This effect of noradrenaline is shown to be mediated by a classical -type receptor. 5-Hydroxytryptamine, prostaglandin E ₁ and dopamine do not significantly enhance cyclic AMP formation in guinea-pig brain slices in either the presence in, or the absence of calcium ions from the incubation medium. Under depolarizing conditions of incubation the stimulatory effect of ouabain or 125 mM K⁺ is blocked in a calcium-free medium, while with the depolarizing agent veratridine no significant reduction of cyclic AMP formed during incubation in a calcium-free medium is obtained.     

Regulation of cyclic AMP levels by phorbol esters in bovine adrenal medullary cells

Cyclic AMP responses to phorbol esters were studied in cultured bovine adrenal medullary cells. Phorbol esters that activate protein kinase C (PKC: phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate) increased cellular cyclic AMP levels by up to 100% over 5 min, and this was maintained for up to 3 h. The effect was mimicked by 1,2-dioctanoyl-sn-glycerol but not by inactive phorbol esters. The effect of active phorbol esters was concentration dependent over the range 50–500 nM, and was abolished by the PKC inhibitor, Ro 31–8220 (10μM). The response was enhanced by 3-isobutyl-1-methylxanthine (1 mM) and by forskolin (0.3 μM), was enhanced following pertussis toxin pretreatment (100 ng/ml, 7.5 h) and was unaffected by removing extracellular Ca²⁺. The phorbol ester cyclic AMP response was additive with that to K⁺ depolarisation, and synergised with those to prostaglandin E₁ and dimaprit. The results indicate PKC activation increases cyclic AMP formation in bovine adrenal medullary cells, probably by a direct action on adenylate cyclase or G_s.     

Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115): cyclic GMP formation is mediated by a low affinity agonist-receptor conformation and cyclic AMP reduction is mediated by a high affinity agonist-receptor conformation

Murine neuroblastoma cells (clone N1E-115) possess two subtypes of the muscarinic receptor each of which separately mediates a cyclic nucleotide response. The formation of cyclic GMP is postulated to involve a low affinity agonist-receptor conformation, whereas the reduction of prostaglandin E1-stimulated cyclic AMP formation appears to involve a high affinity conformation. Further evidence supporting this hypothesis was obtained in experiments measuring the equilibrium dissociation constants for the full agonist carbachol by the method of partial receptor inactivation. Quinuclidinyl benzilate (QNB) was employed to occlude muscarinic receptors; measurements with [3H] QNB ensured that the amount of QNB appearing in the assay after washout had only a minimal effect on the determination of the equilibrium dissociation constants. Carbachol mediated cyclic GMP formation with an equilibrium dissociation constant (KD) of 325 microM and cyclic AMP reductions with a KD value of 13 microM. These KD values are similar to but somewhat higher than those determined by direct binding at 15 degrees, and they are strong evidence in support of the view that a low affinity conformation mediates cyclic GMP formation, whereas a high affinity conformation mediates cyclic AMP reductions.     

Pharmacological characterisation of 5-HT receptors positively coupled to adenylyl cyclase in the rat hippocampus

The pharmacological properties of 5-hydroxytryptamine (5-HT) receptors positively coupled to adenylyl cyclase in the rat hippocampus were investigated using selective agonists and antagonists. 5-HT (0.008–125 μM) stimulated cyclic AMP formation in homogenates of rat hippocampus in a concentration-dependent manner. The maximal increase in cyclic AMP formation occurred at 1 μM (141 ± 6%) and the half-maximal effect (EC₅₀) at 50 ± 22 nM. Cyclic AMP accumulation induced by 1 μM 5-HT was partly inhibited by the selective 5-HT_1A receptor antagonist WAY 100,635 (1 μM), the selective 5-HT₄ receptor antagonist SB 203,186 (1 μM), and the 5-HT_2A/C/ 5-HT₇ receptor antagonist mesulergine (25 μM). WAY 100,635, SB 203,186 and mesulergine inhibited the effect of 5-HT (1 μM) by 47%, 33% and 49%, respectively. The combination of WAY 100,635 (1 μM) with SB 203,186 (1 μM) or mesulergine (25 μM) resulted in stronger inhibition than with each antagonist alone, and the combination of all three antagonists produced almost total blockade (95%) of 5-HT-induced cyclic AMP accumulation. 5-Carboxamidotryptamine (5-CT; 0.008–125 μM), a 5-HT₁/5-HT₇ receptor agonist, and SDZ 216–454 (0.008– 125 μM), a selective 5-HT₄ receptor agonist, concentration-dependently stimulated cyclic AMP formation, but the maximal effect of each agonist was smaller than that of 5-HT alone. SDZ 216–454 (5 μM) and 5-CT (5 μM) in combination stimulated cyclic AMP formation in an additive manner. 8-OH-PIPAT and 8-OH-DPAT, two selective 5-HT_1A agonists, produced a small but significant increase in cyclic AMP formation at concentrations above 0.04 μM and 10 μM, respectively. These findings suggest that at least three 5-HT receptor subtypes, i.e. 5-HT_1A, 5-HT₇ and 5-HT₄ receptors, are involved in mediating 5-HT-induced cyclic AMP formation in rat hippocampus. Received: 28 October 1998 / Accepted: 16 March 1999     

Activation of coronary arterial guanylate cyclase by nitric oxide,nitroprusside, and nitrosoguanidine—Inhibition by calcium,lanthanum, and other cations,enhancement by thiols

Although reports that certain vasodilate s activate soluble guanylate cyclase, especially in the presence of thiols, and elevate cyclic GMP levels in smooth muscle suggest that cyclic GMP is involved in vascular smooth muscle relaxation, earlier reports that Ca²⁺ activates guanylate cyclase and that Ca²⁺ -dependent contractile agents elevate cyclic GMP levels are seemingly at odds with this hypothesis. The objective of this study was to examine the effects of Ca²⁺ related cations, and thiols on bovine coronary arterial soluble guanylate cyclase. Guanylate cyclase activity was detected in the presence of Mg²⁺ or Mn²⁺ but not of other cations. Basal activity was greater in the presence of Mn²⁺ than of Mg²⁺. Activity of guanylate cyclase stimulated by nitroprusside, nitric oxide, or nitrosoguanidine, however, was greater with Mg²⁺, although the requirement of activated enzyme for Mn²⁺ was reduced about 10-fold. Ca²⁺ markedly inhibited guanylate cyclase activation in the presence of Mg²⁺ but not of Mn²⁺. La²⁺ inhibited enzyme activation in the presence of Mg²⁺ or Mn²⁺. Neither Ca²⁺ nor La³⁺ altered basal enzymatic activity. Results that were qualitatively similar to those indicated above were observed with partially purified, heme-free, coronary arterial soluble guanylate cyclase. Nitric oxide and nitroso compounds activated partially purified enzyme, and thiols enhanced enzyme activation by nitroprusside and nitrosoguanidine without appreciably altering basal activity. Irreversible sulfhydryl binding agents such as ethacrynic acid and gold inhibited both basal and activated guanylate cyclase. These results suggest that changes in intracellular concentrations of free Ca²⁺ and sulfhydryl groups could influence the rate of formation of cyclic GMP by vasodilators and that this, in turn, could alter smooth muscle tone.     

Inhibition of pacemaker currents by nitric oxide via activation of ATP-sensitive K+ channels in cultured interstitial cells of Cajal from the mouse small intestine

We investigated the role of nitric oxide (NO) in pacemaker activity and signal mechanisms in cultured interstitial cells of Cajal (ICC) of the mouse small intestine using whole cell patch-clamp techniques at 30°C. ICC generated pacemaker potential in the current clamp mode and pacemaker currents at a holding potential of –70 mV. (±)-S-nitroso-N-acetylpenicillamine (SNAP; a NO donor) produced membrane hyperpolarization and inhibited the amplitude and frequency of the pacemaker currents, and increased resting currents in the outward direction. These effects were blocked by the use of glibenclamide (an ATP-sensitive K⁺ channel blocker), but not by the use of 5-hydroxydecanoic acid (a mitochondrial ATP-sensitive K⁺ channel blocker). Pretreatment with ODQ (a guanylate cyclase inhibitor) almost blocked the NO-induced effects. The use of cell-permeable 8-bromo-cyclic GMP also mimicked the action of SNAP. However, the use of KT-5823 (a protein kinase G inhibitor) did not block the NO-induced effects. Spontaneous [Ca²⁺]_i oscillations in ICC were inhibited by the treatment of SNAP, as seen in recordings of intracellular Ca²⁺ ([Ca²⁺]_i). These results suggest that NO inhibits pacemaker activity by the activation of ATP-sensitive K⁺ channels via a cyclic GMP dependent mechanism in ICC, and the activation of ATP-sensitive K⁺ channels mediates the inhibition of spontaneous [Ca²⁺]_i oscillations.     

Endothelium-derived relaxant factor inhibits platelet activation

Summary Experiments were designed to investigate whether platelet activation is modulated by endothelium-derived relaxant factor (EDRF) which has been shown to induce vascular smooth muscle relaxation by direct stimulation of soluble guanylate cyclase. EDRF was released from cultured bovine endothelial cells, grown on microcarrier beads, by stimulation with thimerosal in the presence of indomethacin. EDRF had no effect on the intracellular free calcium concentration (Ca_i ²⁺, measured with the fluorescent indicator indo-1) of resting washed human platelets but significantly attenuated the thrombin-induced rise of Ca_i ²⁺ from 896 ± 99 (SEM) to 509 ± 48 nmol/l. EDRF significantly increased platelet cyclic GMP levels from 0.25 ± 0.04 to 2.5 ± 0.4 pmol/10⁸ platelets and reduced the thrombin-induced aggregation to 23 ± 3% of control. EDRF had no effect on Caiz+, cyclic GMP or aggregation after a 3 min storage interval, but superoxide dismutase (shown to increase stability of the labile factor) significantly augmented the EDRF effects on Ca_i ²⁺. The antiaggregatory potency of EDRF was completely abolished in the presence of hemoglobin. The results characterize EDRF as a potent cyclic GMP-dependent antiaggregatory factor which may act synergistically in vivo with the cyclic AMP-dependent inhibitory effect of prostacyclin. Send offprint requests to R. Busse     

Evidence for cocaine and methylecgonidine stimulation of M(2) muscarinic receptors in cultured human embryonic lung cells

1. Muscarinic cholinoceptor stimulation leads to an increase in guanylyl cyclase activity and to a decrease in adenylyl cyclase activity. This study examined the effects of cocaine and methylecgonidine (MEG) on muscarinic receptors by measurement of cyclic GMP and cyclic AMP content in cultured human embryonic lung (HEL299) cells which specifically express M(2) muscarinic receptors. 2. A concentration-dependent increase in cyclic GMP production was observed in HEL299 cells incubated with carbachol, cocaine, or MEG for 24 h. The increase in cyclic GMP content was 3.6 fold for 1 microM carbachol (P < 0.01), 3.1 fold for 1 microM cocaine (P < 0.01), and 7.8 fold for 1 microM MEG (P < 0.001), respectively. This increase in cyclic GMP content was significantly attenuated or abolished by the muscarinic receptor antagonist atropine or the M(2) blocker methoctramine. 3. In contrast, cocaine, MEG, and carbachol produced a significant inhibition of cyclic AMP production in HEL299 cells. Compared to the control, HEL299 cells treated with 1 microM cocaine decreased cyclic AMP production by 30%. MEG and carbachol at 1 microM decreased cyclic AMP production by 37 and 38%, respectively. Atropine or methoctramine at 1 or 10 microM significantly attenuated or abolished the cocaine-induced decrease in cyclic AMP production. However, the antagonists alone had neither an effect on cyclic GMP nor cyclic AMP production. Pretreatment of HEL299 cells with pertussis toxin prevented the cocaine-induced reduction of cyclic AMP production. 4. Western blot analysis showed that HEL299 cells specifically express M(2) muscarinic receptors without detectable M(1) and M(3). Incubation of HEL299 cells with cocaine, carbachol, and atropine did not alter the expression of M(2) protein levels. However, the inducible isoform of nitric oxide synthase (iNOS) was induced in the presence of cocaine or carbachol and this induction was significantly attenuated after addition of atropine or methoctramine. 5. The present data show that cocaine and MEG significantly affect cyclic GMP and cyclic AMP production in cultured HEL299 cells. Our results also show that these effects result from the drug-induced stimulation of M(2) muscarinic receptors accompanied with no alterations of receptor expression. However, the induction of iNOS by cocaine may result in the increase in cyclic GMP production.