Changes In Cyclic Nucleotide Levels Research Articles

Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone.

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in 'second messengers' derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.

Local changes in fractional saturation of cGMP- and cAMP-receptors in intestinal microvilli in response to cholera toxin and heat-stable Escherichia coli toxin

Cyclic nucleotide modulation of electrolyte transport across intestinal brushborder membranes is initiated by binding of cGMP and cAMP to high-affinity receptors at the interior of the microvilli. Previously these receptors have been identified by photoaffinity-labelling techniques as regulatory domains of cGMP- and cAMP-dependent protein kinases. In the present study, the receptor concentration in isolated brushborder membrane vesicles and their fractional saturation in absorptive and secretory states of the tissue were estimated. In microvillous membrane vesicles isolated from rat small intestine in the absorptive state, about 10% of the total number of cGMP receptors (25.5 pmol/mg protein) and 40% of all cAMP receptors (28.7 pmol/mg protein) were occupied by endogenous cyclic nucleotides. Luminal exposure of the intestinal segments in vivo to heat-stable Escherichia coli toxin for 3–5 min increased the occupancy of cGMP receptors by about 5-fold without affecting receptor-bound cAMP levels. In contrast, incubation with cholera toxin for 2 h increased the fractional saturation solely of cAMP receptors by 2-fold. Addition of heat-stable E. coli toxin to cholera toxin-pretreated segments, again raising the cGMP levels by 5-fold, did not reduce the amount of receptor-bound cAMP. This finding argues against the concept that increased levels of cAMP during cholera would mimick cGMP effects on ion transport by low-affinity binding to microvillar cGMP receptors. This analysis of local changes in cyclic nucleotide levels at the microvillous level might help to explore the mechanism of action of other secretagogues or antidiarrhoeal agents and to delineate a possible compartmentation of cGMP and cAMP pools within the intestinal mucosa responding differently to external signals.

GTP-binding proteins couple cardiac muscarinic receptors to a K channel

Binding of acetylcholine (ACh) to cardiac muscarinic ACh receptors (mAChR) activates a potassium channel that slows pacemaker activity. Although the time course of this activation suggests a multi-step process with intrinsic delays of 30-100 ms, no second-messenger system has been demonstrated to link the mAChR to the channel. Changes in cyclic nucleotide levels (cyclic AMP and cyclic GMP) do not affect this K channel or its response to muscarinic agonists. Indeed, electrophysiological experiments argue against the involvement of any second messenger that diffuses through the cytoplasm. We report here that coupling of the mAChR in embryonic chick atrial cells to this inward rectifying K channel requires intracellular GTP. Furthermore, pretreatment of cells with IAP (islet-activating protein from the bacterium Bordetella pertussis) eliminates the ACh-induced inward rectification. As IAP specifically ADP-ribosylates two GTP-binding proteins, Ni and No, that can interact with mAChRs, we conclude that a guanyl nucleotide-binding protein couples ACh binding to channel activation. This represents the first demonstration that a GTP-binding protein can regulate the function of an ionic channel without acting through cyclic nucleotide second messengers.

The effects of valproate and phenytoin on the cAMP and cGMP levels in nervous tissue.

Earlier studies have demonstrated that valproic acid (VPA) and phenytoin (PHT) influence the excitability properties of crayfish axons through different mechanisms. PHT was found to antagonize the electrophysiologic effects of VPA. The purpose of the present study was to determine if the electrophysiologic effects of VPA and PHT are correlated with changes in the cellular levels of either cAMP or cGMP as these substances are known to influence membrane excitability. It was found that PHT (0.1 mM) has no effect on the levels of either cAMP or cGMP within crayfish neural tissue. VPA (4.0 mM) also has no effect on cAMP levels. However, it does significantly reduce the levels of cGMP. Pretreatment of neural tissue with PHT has been shown to eliminate the effects of VPA on membrane excitability. It was found that this pretreatment has no influence on VPA's ability to reduce cGMP levels. The effect of VPA on cGMP levels is observed in the absence of spontaneous activity. Therefore, it is concluded that the observed reduction in cGMP levels does not represent the modulation of cGMP levels that is known to accompany activity. Two experiments demonstrate that the 4-mV depolarization of membranes by VPA can not account for its effect on cGMP levels. In the first, pretreatment with PHT abolished the depolarizing effect on VPA but not its effect on cGMP. In the second, a concentration of ouabain which depolarizes crayfish neural tissue by 8-10 mV without producing spike activity had no effect on either cAMP or cGMP levels. These experiments effectively dissociate the electrophysiologic response to VPA and PHT from changes in cyclic nucleotide levels.

Cyclic nucleotides and their relationship to complement-component-C2 synthesis by human monocytes.

The time courses of changes in cyclic nucleotide levels in monocytes have been studied. Histamine and prostaglandin E2 (PGE2) produced a rapid rise in cyclic AMP (peak 15 min) levels, which returned to normal within 4h, whereas cholera toxin, NaF and phosphodiesterase inhibitors produced slow sustained rises lasting over 24h. With the exception of isobutylmethylxanthine (10 mumol X 1(-1), none of these reagents altered cyclic GMP levels. alpha 1-Adrenergic and nicotinic cholinergic receptor-ligand interactions and imidazole produced rapid and relatively short-lived falls in cyclic AMP, and rises in cyclic GMP. In contrast, prostaglandin synthetase inhibitors produced delayed but more sustained falls in cyclic AMP but no rises in cyclic GMP. Agents that increased cyclic AMP decreased complement-component-C2 production, and those that decreased cyclic AMP increased C2 production. Agents that increased cyclic GMP alone (ascorbate, nitroprusside and prostaglandin F2 alpha) did not affect C2 production. Antigen-antibody complexes that stimulate C2 synthesis produced falls in cyclic AMP and rises in cyclic GMP similar to those produced by adrenergic and cholinergic ligands. Serum-treated complexes and anaphylatoxins, which inhibited C2 production, were associated with changes in cyclic AMP similar to those produced by histamine and PGE2. These data suggest that there are two transmembrane signals involved in the regulation of C2 production by monocytes. The inhibitory signal is adenylyl cyclase activation. The stimulatory signal is not so obvious, but may be Ca2+ influx, since the time courses of changes in cyclic nucleotides produced by agents that stimulate C2 synthesis are identical, and alpha 1-adrenergic agonists cause the formation of Ca2+ channels.

Insulin regulation of sugar transport in giant muscle fibres of the barnacle.

1. Sugar transport in the giant muscle cells of Balanus nubilus is accelerated during contractile activity and exposure to porcine insulin. The characteristics of hexose-transfer regulation in the giant muscle cells have been examined by studying the transport of 3-O-methylglucose (a non-metabolized sugar) in both intact giant fibres and fibres subjected to internal solute control by internal dialysis.2. Sugar transport in barnacle muscle is mediated by a saturable process which is inhibited by both phloretin and cytochalasin B. Insulin increases the capacity of the transport system with little effect on its apparent affinity for sugar. Under the same conditions insulin increases 3-O-methylglucose-displaceable cytochalasin B binding. The effects of insulin on transport are half-maximal at 5 muM-insulin and are abolished by both insulin antibody and phloretin. The intact barnacle releases an insulin-like material in response to a rise in blood glucose levels.3. Insulin increases the cyclic GMP (cGMP) content and reduces the cyclic AMP (cAMP) content of barnacle muscle. Experiments with fibres injected with aequorin show that insulin also lowers cytosolic ionized Ca levels. The changes in cyclic nucleotide levels induced by insulin precede the effects on sugar transport and cytosolic ionized Ca. During repetitive contractile activity, cAMP, cGMP and ionized Ca levels are raised.4. Agents which raise the cAMP content of barnacle muscle normally inhibit sugar transport. Dibutyryl cAMP also inhibits transport. Alterations in cytosolic ionized Ca levels in intact fibres are without effect on sugar transport. Nevertheless, stimulation of transport by insulin is blunted when cytosolic ionized Ca is lowered by intracellular injection of the Ca-chelating agent, EGTA.5. Sugar uptake in the internally dialysed fibre is inhibited by intracellular application of cAMP. Internal application of Ca and cGMP stimulate sugar uptake in the dialysed fibre. Cyclic AMP reduces the capacity of the transport system whereas Ca and cGMP increase the capacity of the saturable transfer system. Cyclic AMP and cGMP act at kinetically independent sites. Internal ATP (2 mM) inhibits sugar uptake in the dialysed fibre by some 40%, possibly through the production of cAMP.6. External insulin stimulates sugar uptake in the dialysed fibre even when ionized Ca levels are buffered using EGTA. Stimulation by insulin requires the presence of cytosolic ATP and is potentiated by internal application of 1 mM-GTP. In the dialysed fibre stimulation of transport by insulin is greater than that brought about by Ca and cGMP.7. The stimulation of transport by insulin in the intact fibre and its inhibition by dibutyryl cAMP are abolished by intracellular injection of Gpp(NH)p. Injection of intact fibres with GTPgammaS potentiates the stimulation of transport by insulin and renders insulin-activation of transport irreversible. Injection of intact fibres with ATPgammaS leads to the irreversible inhibition of transport.8. Injection of intact fibres with cAMP phosphodiesterase lowers cAMP levels close to zero and stimulates sugar transport. Application of insulin to diesterase-injected fibres still stimulates transport in the absence of altered cytosolic cAMP.

Modification by islet-activating protein of direct and indirect inhibitory actions of adenosine on rat atrial contraction in relation to cyclic nucleotide metabolism.

Adenosine decreased the force of contraction of isolated rat left atria (direct action) and inhibited the positive inotropic action of isoproterenol (indirect action). The direct action of adenosine was not accompanied by changes in cyclic nucleotide levels, while the isoproterenol-induced increase in cyclic AMP level was reduced by the nucleoside. Reserpine pretreatment and pindolol did not affect the direct action of adenosine. Both the direct and indirect actions of adenosine were enhanced by dipyridamole and dilazep and inhibited by 1-methyl-3-isobutylxanthine and theophylline. Among adenosine derivatives, N6-phenylisopropyladenosine was most potent, and 2'-deoxyadenosine was least effective in inducing the negative inotropic action in the absence and presence of isoproterenol. In atria pretreated with islet-activating protein (IAP) both the direct and indirect actions of adenosine, and the actions of N6-phenylisopropyladenosine were markedly attenuated compared with those in nontreated atria. The isoproterenol-induced increase in cyclic AMP level was not reduced by adenosine in IAP-treated atria. These results indicate that adenosine produces a dual action on rat atria: it decreases the force of contraction directly by a mechanism independent of cyclic AMP metabolism, and inhibits the positive inotropic action of isoproterenol by reducing the drug-induced accumulation of cyclic AMP. IAP attenuates both of these inhibitory actions of adenosine on rat atria.

Cyclic nucleotides and the release of vasopressin from the rat posterior pituitary gland.

When rat posterior pituitary glands were stimulated by a high concentration of potassium, a peak of cyclic AMP and a peak of cyclic GMP were detected after 0.5 min and 1 min, respectively, whereas the rate of release of vasopressin was maximal only after 2 min. When calcium was omitted from the medium, no significant changes in cyclic nucleotide levels were found and the vasopressin release remained at the basal rate. During cold-stimulated (10 degrees C) release of vasopressin, a peak of cyclic AMP was detected after 5 min simultaneously with the maximal rate of vasopressin release. The significance of the cyclic nucleotides in the release of vasopressin is discussed.

Changes in cyclic nucleotide levels correlated with growth, division, and morphology in [formula omitted] chemostat culture

To explore the possibility that cyclic nucleotides control green algal growth and division, Chlamydomonas chemostat cultures were assayed for cyclic nucleotides. Substantial qualities of cAMP were found in cells and in extracellular millieu. Most cGMP molecules were extracellular. Slowing cell growth by slowing chemostat dilution caused reversible changes in cellular morphology and cyclic nucleotide levels. During slowed growth cAMP level increased dramatically; cGMP level decreased. New cells resulting from division were not released from original cell wall.

O-62 - Age-related changes in cyclic nucleotide levels of guinea-pig tracheal smooth muscle to various agents

O-62 - Age-related changes in cyclic nucleotide levels of guinea-pig tracheal smooth muscle to various agents

Changes in cyclic nucleotide levels and phosphodiesterase and protein kinase activities in mitogenically stimulated 3T3 fibroblasts.

Activities of cyclic-nucleotide-hydrolysing enzymes cAMP-, cCMP- and cGMP phosphodiesterase, the intracellular concentrations of cAMP, cCMP, and cGMP, and the activity of the cAMP-dependent protein kinase were studied in serum-starved 3T3 cells stimulated to proliferate by serum. Within 1 and 2 min after stimulation the activities of cAMP- and cGMP phosphodiesterase were unaffected while the concentration of cGMP was raised and that of cAMP lowered, suggesting increased synthesis of cGMP and simultaneously reduced synthesis of cAMP. 48 h after stimulation, when the cells multiplied rapidly, both the cAMP phosphodiesterase and the cCMP phosphodiesterase were reduced. Evidence was also obtained that cAMP-dependent protein kinase is important for expressing the cAMP effect in the 3T3 cells.

Changes in cyclic nucleotide levels during erythroid differentiation in Friend leukemia cells

Changes in cyclic nucleotide levels were observed in Friend leukemia cells exposed to the inducers of erythroid differentiation dimethylsulfoxide (DMSO), sodium butyrate and hypoxanthine. The most extensive changes in cAMP levels occurred during the initial 24 h growth period. In uninduced cultures, cAMP concentration increased 35-fold compared with a 7-fold increase in the presence of inducer. During the same period. cGMP levels rose about 3-fold in uninduced cells compared with a 20-fold increase in induced cells.

Cardiac effects of prostaglandins E1 and F1 alpha.

The effects of prostaglandin E1 (PGE1) and prostaglandin F1 alpha (PGF1 alpha) were studied on perfused rat hearts and isolated rat atria. Both PGE1 and PGF1 alpha produced dose-dependent increases in right atrial rate but had no effect on left atrial tension development. PGE1 (10(-4) M) increased right atrial cyclic AMP content without changing phosphorylase a activity. PGF1 alpha (10(-4) M) did not change right atrial cyclic AMP or cyclic GMP content. Both prostaglandins had no effect on left atrial cyclic nucleotide content. When infused at a rate of 1 microgram/min, PGE1 produced a time-dependent increase in cyclic AMP content in the Langendorff perfused hearts but did not alter contractile force development or phosphorylase a activity. An infusion of PGF1 alpha produced a dose-dependent increase in tension development which was secondary to a negative chronotropic effect. PGF1 alpha (1 microgram/min) did not produce any changes in cyclic nucleotide levels or phosphorylase a activity in the Langendorff perfused hearts. These results show that PGE1 can selectively increase myocardial cyclic AMP content without altering contractile force or phosphorylase activity and that PGF1 alpha does not increase rat cardiac AMP levels.

Patterns of cyclic nucleotides and related enzymes in normal and gross-virus-transformed rat thymocytes

Cyclic nucleotide (cAMP and cGMP) intracellular levels and cyclase and phosphodiesterase activities were determined in normal and Gross-virus-transformed thymocytes. Many changes in cyclic nucleotide levels and in cyclase activities were observed, but the differences are difficult to evaluate since the changes clearly depend on how the data are expressed: cell number, protein content or DNA content. However, the cAMP cGMP molar ratio was significantly lower in transformed than in normal thymocytes, thus indicating that a relative prevalence of cGMP over cAMP was present in these cells. Transformed thymocytes were sensitive to the stimulating effect of azide and theophylline, but, at variance with normal cells, did not respond to serotonin, carbamylcholine or d,l-isoproterenol. Preincubation with theophylline allowed a cAMP response to d,l-isoproterenol, but no cGMP response to serotonin or carbamylcholine was found. Alterations in the subcellular distribution of guanylate cyclase were also found in transformed thymocytes, as suggested by the relative increase in the particulate enzyme activity of these cells. Both cAMP and cGMP phosphodiesterase activities were significantly higher in transformed than in normal thymocytes. These data are in agreement with the hypothesis that cyclic nucleotide metabolism is abnormal in malignancy.

Diurnal changes in cyclic nucleotide levels in the hypothalamus of the rat.

The concentrations of cAMP and cGMP in the rat hypothalamus varied considerably over a 24-h period. The cAMP levels were found to be relatively higher in the morning and lower during the afternoon and night. Cyclic GMP levels reached highest values during the period of darkness and were lowest during the day. Deprivation of food did not have any appreciable effect on the pattern of these changes.

Changes in cyclic nucleotide levels and dimorphic transition in Candida albicans.

The relationship between the levels of cyclic nucleotides and dimorphic transition in Candida albicans was examined. The results showed that cells of this pathogenic fungus contained both cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP), the concentration of the latter being about one-tenth that of the former in stationary-phase cells of the yeast form. Our results further indicated that germ tube formation induced by incubation at 40 degrees C followed a rise in cAMP concentration in the cell with no accompanying change in cGMP content. Cysteine, which suppressed germination, also reversed the increase in intracellular cAMP concentration. Dibutyryl cAMP (1 MM) significantly promoted germination in proline medium at temperatures of 32 to 34 degrees C. These results suggested that cAMP was one of the controlling factors in the morphological transition in Candida albicans.

The time course of changes in cyclic nucleotide levels during cholinergic inhibition of positive inotropic actions of isoprenaline and theophylline in the isolated canine ventricular myocardium.

The time course of changes in cyclic AMP and cyclic GMP levels during the cholinergic inhibition of the positive inotropic actions of isoprenaline and theophylline was assessed in the isolated canine right ventricular myocardium. 1. Carbachol (3×10−6 mol/l) caused biphasic changes in force of contraction and cyclic AMP levels when administered during the positive inotropic response to isoprenaline (10−6 mol/l): a positive inotropic action superimposed on the slowly developing negative inotropic action which was preceded by a reduction of cyclic AMP levels. The positive and negative inotropic actions of, and the reduction of cyclic AMP levels induced by carbachol in the presence of isoprenaline were inhibited by atropine (3×10−7 mol/l). The cyclic GMP levels were increased after the administration of carbachol in the presence of isoprenaline in a monophasic manner and reached a maximal level 120 s after the administration; the elevation was also abolished by atropine. The half-time (T1/2) for cyclic GMP elevation after the administration of carbachol was about 50 s. 2. Carbachol (3×10−6 mol/l) administered during the positive inotropic response to theophylline (2×10−3 mol/l) abolished the inotropic action of theophylline in a monophasic manner:T1/2, 40 s; a reduction of cyclic AMP levels (T1/2, 10 s) and an elevation of cyclic GMP levels (T1/2, 20 s) preceded the changes in force of contraction caused by carbachol. 3. The present results indicate that the reduction of cyclic AMP levels occurred immediately after the administration of carbachol during cholinergic inhibition of the positive inotropic actions of isoprenaline and theophylline; the elevation of cyclic GMP levels and reduction of force of contraction occurred following the changes in cyclic AMP levels. Considering the mediator role of cyclic AMP in cardiac contraction, it appears that the reduction of cyclic AMP levels is responsible for the cholinergic inhibition of the actions of isoprenaline and theophylline in the canine ventricular myocardium.

On the mechanism of action of the phagocytosis-stimulating peptide tuftsin.

Incubation of human polymorphonuclear leukocytes (PMNL) or thioglycollate-stimulated mouse peritoneal macrophages with the phagocytosis-stimulating peptide, tuftsin (2.5 X 10(-7) M, at 37 degrees C), caused an increase of 80-90% in intracellular cGMP levels, accompanied by a decrease of 20-25% in intracellular cAMP levels. Significant changes in cyclic nucleotide levels were detectable after 4 min of incubation, were maximal at 10-20 min and persisted for at least 60 min. The concentration dependences of the stimulatory effect of tuftsin on modulation on intracellular levels of cyclic nucleotides and on phagocytosis are similar, suggesting a cause and effect relationship between the two phenomena. This notion is further supported by the finding that 8-Br-cGMP and 8-Br-cAMP elicit stimulatory and inhibitory effects on macrophage phagocytosis, respectively. Measurement of 45Ca2+ influx into PMNL and macrophages in the presence and absence of tuftsin did not reveal any change in 45Ca2+ uptake from the media. However, tuftsin did enhance release of 45Ca2+ from cells preloaded with the isotope. Results suggest that modulation of both the amount of cell-associated 45Ca2+ and the intracellular levels of cyclic nucleotides are key steps in the mechanism by which tuftsin augments phagocytosis.

The effect of serum and multiplication stimulating activity on L6 myoblast growth: The lack of correlation with cyclic nucleotide changes

The role of cAMP and cGMP in triggering proliferation of rat myoblasts was evaluated by: (1) measuring effects of mitogens on intracellular cyclic nucleotide levels, and (2) observing effects of agents which altered cyclic nucleotide levels on cell proliferation. Multiplication stimulating activity (MSA, 1 microgram/ml), a member of the somatomedin family, stimulated cell proliferation after 48 hr. It had little effect on cellular cyclic nucleotide levels, measured by radioimmunoassay. Horse serum (HS) and fetal bovine serum (FBS) stimulated cell proliferation approximately equally. Neither affected cAMP levels; FBS reduced cGMP to 33% of control values, but HS had no effect. Thus, there was no simple correlation between mitogenic action and cyclic nucleotide levels at any time from 5 min to 24 hr after addition of a purified mitogen or serum. Furthermore, agents which caused substantial changes in cyclic nucleotide levels had no effect on cell proliferation. Prostaglandin E1 (5 microM) elevated cAMP 440% without affecting cGMP levels or cell growth. A potent phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX, 0.1 mM), when added alone or in the presence of MSA, HS, or FBS, elevated cAMP 200% and cGMP 167%, but it had little effect on their mitogenic action. Lastly, a purified mitogen such as MSA, unlike serum, must be present for extended periods of time in order to stimulate cell proliferation. This makes it unlikely that a trigger mechanism functions in initiating cell division. We conclude that neither cAMP nor cGMP appear to be second messengers for the mitogenic action of MSA or serum on muscle cells.

Effects of Indomethacin on Cyclic Nucleotide Levels and Histamine Release from Rat Serosal Mast Cells

Abstract Previous studies in homogeneous and mixed cell systems on the changes in cyclic nucleotide levels that accompany the immunologic activation of mast cells for histamine release have not considered the extent to which these changes occur secondarily to the generation of the oxidative products of arachidonate. The recognition that mast cells themselves generate oxidative products of arachidonate upon immunologic activation and that exogenous prostaglandins can modulate cellular levels of 3′,5′-cyclic adenosine monophosphate (cyclic AMP) prompted the assessment of the effects of endogenous oxidation of arachidonate on changes in cyclic nucleotide levels accompanying immunologic activation. Thus, immunologic activation of purified rat serosal mast cells was carried out in the presence of a concentration of indomethacin that completely suppressed the oxidative conversion of arachidonate to the prostaglandins D2 (PGD2) and F2a (PGF2a) without having any effect on the time course and magnitude of histamine release. The immunologic activation of rat mast cells gave an early and a late monophasic rise of both cyclic AMP and 3′,5′-cyclic guanosine monophosphate (cyclic GMP). The inhibition of cyclo-oxygenase by 10 µM indomethacin abolished both monophasic rises in cyclic GMP and the second monophasic rise in cyclic AMP, but had no significant effect on the early peak of cyclic AMP that followed immunologic activation. Thus, those changes in cyclic nucleotide levels that are suppressed by indomethacin are not part of the primary activation-secretion event of mast cells and may be secondary to the generation of oxidative products of arachidonate. In contrast, the indomethacin-resistant elevations in cyclic AMP observed in the homogeneous rat mast cell system 5 to 15 sec after activation may represent an early event in the coupled activation-secretion response.