Effects of prostaglandins Fα on dog thyroid cyclic AMP level and function

Prostaglandins F_1α and F_2α, at high concentrations (≥28 μM) enhanced cyclic AMP accumulation in dog thyroid slices. At lower concentrations, they inhibited the cyclic AMP accumulation induced by thyrotropin (TSH), prostaglandin E₁, and cholera toxin. This effect was rapid in onset and of short duration, calcium-dependent and suppressed by methylxanthines. Prostaglandin F_α also inhibited TSH-induced secretion and activated iodine binding to proteins. These characteristics are similar to those of carbamylcholine action, except that prostaglandins F did not enhance cyclic GMP accumulation. The effect of prostaglandin F_α was not inhibited by atropine, phentolamine and adenosine deaminase and can therefore not be ascribed to an induced secretion of acetylcholine, norepinephrine or adenosine. It is suggested that prostaglandins F act by increasing influx of extracellular Ca²⁺. Arachidonic acid also inhibited the TSH-induced cyclic AMP accumulation. However this effect was specific for TSH, it was enhanced in the absence of calcium and was not inhibited by methylxanthines or by indomethacin at concentrations which completely block its conversion to prostaglandin F_α. Arachidonic acid action is sustained. This suggests that arachidonic acid inhibits thyroid adenylate cyclase at the level of its TSH receptor and that this effect is not mediated by prostaglandin F_α or any other cyclooxygenase product.     

Adenylate cyclase from guinea pig lung: further characterization and inhibitory effects of substrate analogs and cyclic nucleotides

A potent (K_i = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N⁶,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg²⁺ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg²⁺ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E₁, E₂, and F_2α, histamine, and glucagon.     

High-affinity binding sites for prostaglandin E on human lymphocytes.

Binding sites on human lymphocytes for prostaglandins were examined by incubating cells with [³H]prostaglandin (PG) A₁, E₁, E₂, F_1α, and F_2α. Specific reversible binding for [³H]PGE₁ and E₂ was found with a K_d of ~2 × 10^?9M and a B max of ~200 binding sites per cell, assuming uniform distribution. We detected no specific binding of [³H]PGA₁, F_1α, or F_2α to lymphocytes. Also, the addition of 10- to 1000-fold greater amounts of unlabeled PGA, F_1α, or F_2α did not inhibit the binding of [³H]PGE. The time course of [³H]PGE binding appeared to be bimodal with one component complete within 5 min at 37 °C and another component of binding increasing over a 40-min incubation. We feel that the rapid component of binding may represent cell surface receptors for PGE while the slower component may represent a specific uptake mechanism for PGE into the cell. Glass adherent cells had fewer binding sites than nonadherent cells. Preincubation of the cells overnight resulted in a loss of binding sites.     

Effects of epoxymethano analogues of prostaglandin endoperoxides on aggregation,on release of 5-hydroxytryptamine and on the metabolism of 3′,5′-cyclic AMP and cyclic GMP in human platelets

The effects on human platelets of two synthetic analogues of prostaglandin endoperoxides were examined in order to explore the relationship between aggregation and prostaglandin and cyclic nucleotide metabolism, and to help elucidate the role of the natural endoperoxide intermediates in regulating platelet function.Both analogues (Compound I, (15S)-hydroxy-9α,11α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid, and Compound II, (15S)-hydroxy-11α,9α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid) caused platelets to aggregate, an effect which could be inhibited by prostaglandin E₁ but not by indomethacin. Compound II produced primary, reversible aggregation at concentrations which did not induce release of 5-hydroxytryptamine. Production of thromboxane B₂ and malonyldialdehyde was monitored as an index of endogenous production of prostaglandin endoperoxides and thromboxane A₂ and were increased after incubation of human platelets with thrombin, collagen or arachidonic acid. However, neither malonydialdehyde nor thromboxane B₂ levels were significantly influenced by the endoperoxide analogues. Both analogues produced a small elevation of adenylate cyclase activity in platelet membranes and of cyclic AMP content in intact platelets, but neither had any modifying effect on the much greater stimulation of adenylate cyclase and cyclic AMP levels by prostaglandin E₁. Of all the aggregating agents tested, only arachidonic acid produced any significant increase in platelet cyclic GMP levels.These results suggest that the epoxymethano analogues of prostaglandin endoperoxides induce platelet aggregation independently of thromboxane biosynthesis and without inhibiting adenylate cyclase or lowerin platelet cyclic AMP levels. They therefore differ from better known aggregating agents such as ADP, epinephrine and collagen, which increase thromboxane A₂ production and reduce cyclic AMP levels, at least in platelets previously exposed to prostaglandin E₁.     

Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland

Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E₁ and E₂. Prostaglandin F_1α and F_2α have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE₁ is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE₁.Both PGE and PGF synthesis were monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La³⁺ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B₂ by washed platelets exposed to collagen or thrombin. In addition, La³⁺ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B₂ was not affected.La³⁺ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E₁, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La³⁺, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane.La³⁺ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E₁ and to F^?, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La³⁺ could be reversed by the addition of Ca²⁺ up to 10 mM. The stimulation of cyclic AMP production by La³⁺ may largely explain the inhibitory effect of La³⁺ upon platelet secretion and thromboxane B₂ production. These results also suggest that Ca²⁺ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

Catalytic and kinetic properties of purified high-affinity cyclic AMP phosphodiesterase from dog kidney

High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; Mg²⁺, Mn²⁺, and Co²⁺ each supported activity, but highest activity was obtained with Mg². The temperature optimum ranged from 30 to 45 °C and depended on substrate concentration; the E_a = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at > 20 μm. The K_m for cyclic AMP hydrolysis was 2 μm, and that for cyclic GMP hydrolysis was 312 μm. The K_i values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their K_m values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with K_i values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.     

Comparison of the effects of prostaglandin I2 and prostaglandin E2 stimulation of the rat kidney adenylate cyclase-cyclic AMP systems

Prostacyclin (Prostaglandin I₂) effects on the rat kidney adenylate cyclase-cyclic AMP system were examined. Prostaglandin I₂ and prostaglandin E₂, from 8 · 10^?4 to 8 · ^?7 M stimulated adenylate cyclase to a similar extent in cortex and outer medulla. In inner medulla, prostaglandin I₂ was more effective than prostaglandin E₂ at all concentrations tested. Both prostaglandin I₂ and prostaglandin E₂ were additive with antidiuretic hormone in outer and inner medulla. Prostaglandin I₂ and prostaglandin E₂ were not additive in any area of the kidney, indicating both were working by similar mechanisms. Prostaglandin I₂ stimulation of adenylate cyclase correlated with its ability to increase renal slice cyclic AMP content. Prostaglandin I₂ and prostaglandin E₂ (1.5 · 10^?4 M) elevated cyclic AMP content in cortex and outer medulla slices. In inner medulla, with Santoquin® (0.1 mM) present to suppress endogenous prostaglandin synthesis, prostaglandin I₂ and prostaglandin E₂ increased cyclic AMP content. 6-Ketoprostaglandin F_1α, the stable metabolite of prostaglandin I₂, did not increase adenylate cyclase activity or tissue cyclic AMP content. Thus, prostaglandin I₂ activates renal adenylate cyclase. This suggests that the physiological actions of prostaglandin I₂ may be mediated through the adenylate cyclase-cyclic AMP system.     

The activation by Ca2+ of platelet phospholipase A2: Effects of dibutyryl cyclic adenosine monophosphate and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate

Thrombin-induced release of arachidonic acid from human platelet phosphatidylcholine is found to be more than 90% impaired by incubation of platelets with 1 mM dibutyryl cyclic adenosine monophosphate (Bt₂ cyclic AMP) or with 0.6 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist. Incorporation of arachidonic acid into platelet phospholipids is not enhanced by Bt₂ cyclic AMP. The addition of external Ca²⁺ to thrombin-treated platelets incubated with Bt₂ cyclic AMP or TMB-8 does not counteract the observed inhibition. However, when divalent cation ionophore A23187 is employed as an activating agent, much less inhibition is produced by Bt₂ cyclic AMP or TMB-8. The inhibition which does result can be overcome by added Ca²⁺. Inhibition of arachidonic acid liberation by Bt₂ cyclic AMP, but not by TMB-8, can be overcome by high concentrations of A23187. When Mg²⁺ is substituted for Ca²⁺, ionophore-induced release of arachidonic acid from phosphatidylcholine of inhibitor-free controls is depressed and inhibition by Bt₂ cyclic AMP is slightly enhanced. The phospholipase A₂ activity of platelet lysates is increased by the presence of added Ca²⁺, however, the addition of either A23187 or Bt₂ cyclic AMP is without effect on this activity. We suggest that Bt₂ cyclic AMP may promote a compartmentalization of Ca²⁺, thereby inhibiting phospholipase A activity. The compartmentalization may be overcome by ionophore. By contrast, TMB-8 may immobilize platelet Ca²⁺ stores in situ or restrict access of Ca²⁺ to phospholipase A in a manner not susceptible to reversal by high concentrations of ionophore.     

Effects of prostaglandins on rat renal adenylate cyclase-cyclic AMP systems

The effects of prostaglandin (PG) E₁, E₂, A₁, F_1α, F_2α or D₂ on the rat renal cortical, outer medullary and inner medullary adenylate cyclase-cyclic AM systems were examined. While high concentrations (8X10⁻⁴M) of each prostaglandin stimulated adenylate cyclase activity in each area of the kidney, PGE₁ was the only prostaglandin to stimulate at 10⁻⁷M. PGA's were the only prostaglandins tested besides PGE's which stimulated adenylate cyclase at less than 10⁻⁴M. This effect of PGA's was limited to the outer medulla. PGD₂ was the least stimulatory. Observations with renal slices yielded qualitatively results. The PGE's were the most potent in each area with PGA's only stimulatory in the outer medulla. O₂ deprivation (5% O₂) lowered the slice cyclic AMP content in each area of the kidney. In the cortex and outer medulla, prostaglandin mediated increases in cyclic AMP content were either lower or absent at 5% O₂ compared to 95% O₂. However, in the inner medulla PGE stimulation was observed only at 5% O₂ and not 95% O₂. No other prostaglandins were found to increase inner medullary cyclic AMP content at 95% or 5% O₂. These results illustrate that the adenylate cyclase-cyclic AMP system responds uniquely to prostaglandins in each area of the kidney. Consideration of these results along with correlative observations suggests that inner medullary produced PGE's may act as local modulators of inner medullary adenylate cyclase.     

Prostaglandin stimulation of adenosine 3′,5′-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cycle AMP level in cultured chondrocytes were examined. Prostaglandin E₁ at 0.4 to 30 μM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D₂ and E₂ also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E₁. Prostaglandin A₁ had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E₁, D₂ or E₂ was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E₁, E₂, or D₂ on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E₁, E₂, and D₂ increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

Stimulative effect of prostaglandins on production of hexosamine-containing substances by cultured fibroblasts (2) early effect of various prostaglandins at various doses

Early effects of various prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts, which were derived from a rat carrageenin granuloma, were studied. At the stationary phase, the cells were exposed for 6 h to one of the prostaglandin A₁ (PGA₁), A₂, B₁, B₂, D₂, F_1α, F_2α, E₁, E₂ or arachidonic acid in various concentrations ranging from 0.01 to 10 μg/ml for all the stimuli and from 10 pg to 10 μg/ml for PGF_2α. The activity of the cells in incorporating ³H-glucosamine into hexosamine-containing substances (acidic) glycosaminoglycans and glycoproteins) during this period was compared with that of control cells. All the stimuli tested showed more or less stimulative effect on the synthesis of hexosamine-containing substances at their specific concentrations. PGF_2α was found to be the most potent stimulant and its stimulative effect was found significant even at the low concentration of 100 pg/ml. PGD₂, F_1α and E₂ were the next potent stimuli. Their optimum dose were around 1 μg/ml but they still had significant stimulation at the concentration of 0.01 μg/ml. Effect of PGE₂ was rather mild. Stimulation by PGA₁, A₂, B₁ and B₂ or arachidonic acid was seen at high dose, and its seemed to be non-specific. The results suggested that these prostaglandins such as PGF_2α, D₂, F_1α and E₂ play some important role on regulating the production of intercellular ground substances.     

Accumulation of adenosine 3',5'-monophosphate in rat brain slices: effects of prostaglandins.

Prostaglandin E₁ and E₂ and 15(S)-15-methyl PGE₂ methyl ester stimulate the accumulation of radioactive cyclic AMP in brain slices from Sprague-Dawley rats, labelled during a prior incubation with [¹⁴C] adenine. Prostaglandins A₁ and B₁ have marginal effects and prostaglandin F_1α has no effect. Relatively high concentrations of about 80 μM PGE₁, PGE₂ and 15(S)-15-methyl PGE₂ are required to elicit a maximal 2–5 fold increase in accumulation of cyclic AMP in slices from cerebrum, but significant increases are elicited by 3.5 μM prostaglandin. Similar increases are elicited in slices from neocortex, striatum or midbrain-thalamus-hypothalamus, while lesser increases pertain in slices from cerebellum, medulla-pons or hippocampus. The accumulation of cyclic AMP elicited by PGE₁ in slices from cerebrum was not blocked by naloxone, propranololphentolamine, tetracaine, theophylline, or by nearly equimolar concentrations of either of two prostaglandin antagonists, 7-oxa-13-prostynoic acid and the dibenzoxazepine hydrazide, SC 19220. Morphine potentiated the effects of PGE₁. The combination of 85 μM PGE₁ with either isoproterenol, norepinephrine, adenosine or veratridin did not increase the accumulation of cycli AMP significantly above those elicited by the isoproterenol, norepinephrine, adenosine or veratridine alone. The combined effect of PGE₁ and norepinephrine in the presence of a β-adrenergic antagonist, sotalol, was, however, additive. The results indicate that PGE₁ stimulates cyclic AMP formation in rat brain slices, but that it either has antagonist activity with respect to accumulations of cyclic AMP-elicited by other agents or has no detectable agonist activity when cyclases are maximally stimulated by other agents.     

Formation of adenosine 3′,5′-monophosphate from adenosine in mouse thymocytes

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3′:5′-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E₁, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E₁ and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly wihtin 1 min and was maximal by 10 to 20 min with approx. 2 and 10 μM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E₁, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [¹⁴C] adenine exhibited dramatic increases in cyclic [¹⁴C]AMP 10 min after addition of adenosine or prostaglandin E₁ which corresponded to simultaneously determined increases in total cyclic AMP. Using [¹⁴C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.     

Effect of acetaminophen on prostaglandin E2 and prostaglandin F2α synthesis in the renal inner medulla of rat

Effects of acetaminophen on the renal inner medullary production of prostaglandin E₂ and F_2α were compared with the well-known effects of aspirin on this process. Acetaminophen was found to elicit a dose-dependent inhibition of both prostaglandin E₂ and F_2α accumulation in media with a K_i of 100–200 μM. This inhibition could not be accounted for by increased accumulation of prostaglandins within slices. Acetaminophen inhibition was reversed by removal of acetaminophen during the incubation or by addition of arachidonic acid. Similar manipulations did not reverse aspirin or indomethacin-mediated inhibition of prostaglandin synthesis. Thin-layer and gas chromatographic analysis of acetaminophen following incubation with slices demonstrated that this material was identical to authentic acetaminophen. This, in addition to the lack of an effect of glutathione on inhibition, suggests that acetaminophen does not have to be metabolized to exert this inhibition. Arachidonic acid did not alter the metabolism or increase the efflux of acetaminophen. Lower levels of prostaglandin E₂ observed with 5 mM acetaminophen and 1 mM aspirin caused a corresponding decrease in cyclic AMP content. Removal of acetaminophen from the second incubation or addition of arachidonic acid caused increases in both prostaglandin E₂ and cyclic AMP. Aspirin inhibition of cyclic AMP content was not reversed by similar manipulations. In vivo inhibition of inner medullary prostaglandin E₂ and prostaglandin F_2α synthesis was observed 2 h after a 375 mg/kg, intraperitoneal injection of acetaminophen. These data suggest that acetaminophen, like aspirin, is capable of reducing tissue prostaglandin synthesis. However, the mechanisms by which these two analgesic and antipyretic agents elicit their inhibition of prostaglandin synthesis are quite different.     

Morphological Differentiation induced by Prostaglandin in Mouse Neuroblastoma Cells in Culture

PROSTAGLANDIN is known to affect concentrations of cyclic AMP in some cells¹. Dibutyryl cyclic AMP induces irreversible differentiation of mouse neuroblastoma cells in vitro², which raises the question of whether prostaglandin would mimic this effect. I report here that prostaglandins (PG)E₁ and PGE₂ induce irreversible morphological differentiation of mouse neuroblastoma cells in culture as shown by axon formation, whereas PGF₂α does not.     

Prostaglandin F2alpha and insulin stimulate phosphate uptake and (Na+, K+) ATPase activity in resting mouse fibroblast cultures.

The (Na⁺, K⁺)ATPase transport system in resting 3T3 Swiss mouse fibroblasts is rapidly activated by prostaglandin F₂α and insulin, which initiate DNA synthesis in these cells. Prostaglandin F₂α, but not insulin, promotes a rapid increase in P_i uptake which is partially coupled to the Na⁺ pump. This rapid activation of both transport systems occurs by a mechanism which does not require fluctuation of cyclic AMP levels or new protein synthesis. A subsequent protein synthesis-dependent increase in P_i uptake is stimulated by insulin and prostaglandin F₂α. These results suggest that different types of control of membrane transport occur during growth stimulation.     

Formation of guanosine 3′,5′-cyclic monophosphate by thyroliberlin in cultured rat pituitary cells a possible role in stimulation of prolactin synthesis

In a clonal strain of rat pituitary tumour cells (GH₄C₁ cells), thyroliberin stimulated prolactin secretion and synthesis: effects that could be demonstrated after 5 min and 4–5 h of treatment, respectively. Within 0.5–5 min after addition of thyroliberin, maximal increases (2–4 hold) in cellular cyclic GMP concentrations were observed, and this rise preceded or occurred simultaneously with that of cyclic AMP. After 60 min of treatment the concentrations of the cyclic nucleotides had returned to control values. Half maximal and maximal stimulation of cyclic GMP elevations were obtained with approx. 2·10⁹ and approx. 27·10^?9 thyroliberin, respectively. Aminophylline increased both cyclic GMP and cyclic AMP, and potentiated the stimulatory effects of thyroliberin on both cyclic nucleotides. The dibutyryl derivative of cyclic GMP (10^?4–10^?6 M) stimulated prolactin synthesis, but not hormone release. Prostaglandin E₂ (3·10^?7 M) stimulated cellular cyclic AMP concentrations, but did not affect cyclic GMP levels. We conclude that thyroliberin in the GH₄C₁ ccell strain stimulates cyclic GMP formation, in addition to elevate cyclic AMP concentrations. The stimulatory effect on cyclic GMP is probably not secondary to the rise in cyclic AMP concentration, since prostaglandin E₂ elevates only cyclic GMP is involved in the action of thyroliberin on prolactin, the present results suggest a role on hormone synthesis.