The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

Loss of the PGE1 requirement for MDCK cell growth associated with a defect in cyclic AMP phosphodiesterase

Prostaglandin E₁(PGE₁), one of the components in the hormone-supplemented, serum-free medium for Madin Darby Canine Kidney (MDCK) cells (Medium K-1), is required for both long-term growth and for dome formation. Variant cells have been isolated from MDCK populations, which lack the PGE₁, requirement for long-term growth in Medium K-1. These variants will be useful in identifying the molecular events initiated by PGE₁ which are necessary for the growth response to be observed. The growth and functional properties of five independently isolated PGE₁ independent clones have been examined. Normal MDCK cells grew at an equivalent rate in Medium K-1 and in serum-supplemented medium; the growth rate was lower in Medium K-1 lacking PGE₁. In contrast, PGE₁ independent clone 1 grew at an equivalent rate in Medium K-1 minus PGE₁, and in serum-supplemented medium. When PGE₁ was added to K-1 minus PGE₁, less growth of PGE₁ independent clone 1 was observed. A similar observation was made with one other PGE₁ independent clone which was studied. A hormone deletion study indicated that PGE₁ independent clone 1 still retained growth responses to the other four supplements in Medium K-1 (insulin, transferrin, T₃, and hydrocortisone). The molecular alterations associated with loss of the PGE₁ requirement for long-term growth were examined. At confluency, all of the PGE₁ independent clones studied had higher intracellular cyclic AMP levels following PGE₁ treatment, as compared with normal MDCK cells. The increased cyclic AMP levels in the variant cells could result from a number of different types of defects, including reduced cyclic adenylic acid (cyclic AMP) efflux, an increased affinity of PGE₂ for the PGE₁ receptor, or a defect in cyclic AMP metabolism. However, in all of the variant clones studied a decreased rate of cyclic AMP degradation by cyclic AMP phosphodiesterase was observed. Thus, the increased cyclic AMP levels in the PGE₁ independent variants may result from alterations which affect cyclic AMP metabolism. The effect of PGE₁ on dome formation by the variant cells was also examined. The frequency of dome formation by PGE₁ independent clone 1 was enhanced in a dosage-dependent manner, like normal MDCK cells. This observation suggests that PGE₁ affects MDCK cell growth and dome formation by different mechanisms.     

Cyclic AMP response to prostaglandin E1 in mononuclear cells from peripheral blood and synovial fluid of patients with rheumatoid arthritis

The cyclic AMP response to prostaglandin E₁ (PGE₁) was studied in peripheral blood (PB) and synovial fluid (SF) mononuclear cells from patients with rheumatoid arthritis (RA). The PGE₁ induced accumulation of cyclic AMP was consistently (7 of 8 patients) less in cell suspensions derived from SF than in suspensions of equivalent numbers of mononuclear cells obtained simultaneously from PB. The high PB/SF cyclic AMP ratio was seen most clearly at the lowest concentration (10⁻⁶M) of PGE₁ tested. There was no correlation between the patients' therapy and cyclic AMP response to PGE₁. The high PB/SF cyclic AMP ratio was not accounted for by the presence of platelets in PB cell suspensions.     

Effects of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells

Prostaglandins of the E-series (PGE₁ and PGE₂) may be involved in disease-related, localized loss of bone. E-prostaglandins increase the cyclic AMP content of many cells; and, to determine if their effects on bone are mediated by cyclic AMP, we examined the effects of E-prostaglandins and of other agents on the cyclic AMP content of cultured bone cells. PGE₂ produced a rapid, marked and dose-related increase in the cyclic AMP content of confluent monolayers of bone cells isolated from newborn rat calvaria. At 2.8 × 10⁻⁶ M, PGE₁ and PGE₂ had approximately the same effect, while the effect of PGF_2α was much less pronounced. In the presence of theophylline, PGE₂ had a more marked effect than parathyroid hormone (PTH) and the combination of PGE₂ and PTH had a synergistic effect. The divalent, cationic, ionophore, A23187, produced an increase in cellular cyclic AMP and had an additive effect in combination with PGE₂. Synthetic salmon calcitonin (CT), which inhibits the bone resorptive effect of PGE₂, increased cellular cyclic AMP and had an additive effect in combination with PGE₂. A prostaglandin antagonist, SC-19220, partially inhibited the resorptive effect of PGE₂ and reduced its effect on cellular cyclic AMP. The calcium antagonist, D600, inhibited the bone resorptive effects of PGE₂ but had no effect on increased cellular cyclic AMP produced by PGE₂.The marked effect of PGE₂ on bone cell cyclic AMP suggests that this action is involved in the mechanism of PGE₂-related bone loss. The fact that agents with different effects on PGE₂-induced increases in cellular cyclic AMP can inhibit its resorptive actions, suggests that PGE₂-induced changes in cyclic AMP may be related less to its resorptive actions than to its inhibitory effect on bone formation.     

The ability of calcium to change cyclic AMP from a stimulator to an inhibitor to thymic lymphoblast proliferation

Calcium is a major regulator of thymic lymphoblast proliferation in vivo and in vitro. The proliferative activity of the lymphoblasts in thymic lymphocyte (thymocyte) populations in vitro is both constant and low in the presence of calcium concentrations between 0 and 1.0 mM, but higher concentrations increase proliferation by an endogenous cyclic AMP-mediated promotion of the initiation of DNA synthesis. Lower concentrations (10^?7 to 10^?5 M) of exogenous cyclic AMP (but not 5′-AMP) stimulate lymphoblast proliferation in a low-calcium (0.5 mM) medium, but higher concentrations do not. However, all exogenous cyclic AMP concentrations between 10^?7 and 10^?3 M (but again not 5′-AMP) block the stimulation of lymphoblast proliferation in a high-calcium (1.5 mM) medium. Exogenous cyclic AMP does not prevent calcium from “activating” lymphoblasts, but it reversibly blocks the reaction responsible for the initiation of DNA synthesis in these stimulated cells. Finally, cyclic AMP's inhibitory action, in contrast to its stimulatory action in low-calcium medium, is not specific for the cyclic nucleotide since a low, non-mitogenic concentration of cyclic GMP also prevents calcium from stimulating DNA synthesis and cell proliferation.     

Stimulatory effects of prostaglandin E1 on rat anterior pituitary cyclic amp and luteinizing hormone release

The effect of prostaglandin E₁ (PGE₁) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE₁ to incubation medium over a concentration range of 10^-6 to 10^-4 M produced a graded increase in pituitary cyclic AMP. At the lowest concentration (10^-6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE₁.Ten minutes after intravenous administration of 5 μg of PGE₁ to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE₁ (20 μg) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE₁ effect on LH release is mediated by the adenyl cyclase — cyclic AMP system.     

Effect of Noradrenaline and Prostaglandin E<Subscript>1</Subscript> on Adenosine 3′,5′;-Monophosphate Formation in Isolated Pericardial Fat Cells of Man

NORADRENALINE increases the intracellular concentration of adenosine 3′,5′-monophosphate (cyclic AMP)^1,2 which, in turn, enhances glycogenosis³ and lipolysis^4,5 in adipose tissue by increasing Phosphorylase and lipase activities. Prostaglandin E₁ (PGE₁) antagonizes the induced increases in Phosphorylase activity^6,7 and glycerol release in human adipose tissues^8,9 and isolated adipocytes⁷. The finding that the stimulatory effects of the cyclic AMP analogue N⁶—O² dibutyryl cyclic AMP, which mimics the hormonal effect of noradrenaline in human fat cells, are not blocked by PGE₁⁷ suggests that noradrenaline and PGE₁ alter fat cell metabolism by acting on the adenyl cyclase system¹⁰. Whether noradrenaline and PGE₁ alter concentrations of cyclic AMP in human fat cells, however, has not been reported.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production

The ability of prostaglandin I₂ (PGI₂) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE₂. However, a concentration of 15 μg/ml PGI₂ was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 μg/ml PGE₂, which produced the maximum response. It therefore appears that PGI₂ is not more effective than PGE₂ in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI₂ appeared to be able to modify the stimulation of cyclic AMP production by follicle- stimulating hormone (FSH), but whether or not PGI₂ plays any role in follicular function remains to be established.     

Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis

The steroidogenic response to ACTH and prostaglandin E₂ (PGE₂) was studied in cat adrenocortical cells dispersed with trypsin. The dose-response curves of both agents were qualitatively and quantitatively similar. Exposure to PGE₂ or ACTH in the presence of labeled steroid precursor (acetate) resulted in the accumulation of comparable levels of steroid intermediates and end-product. Submaximal or maximal concentrations of ACTH and PGE₂ given simultaneously elicited a response which was no greater than that obtained with either stimulant alone. Although calcium was required for optimal PGE₂ stimulation of steroid production, this requirement for calcium was less than the requirement with ACTH as the stimulant, but greater than with butyryl cyclic AMP. PGE₂-induced increase in the adrenal cyclic AMP was not statistically significant and was small in relation to that found with equipotent steroidogenic ACTH concentrations. The possible relationship between prostaglandins, cyclic AMP, and calcium in the action of ACTH is discussed.     

Studies on growth hormone secretion VI. Effects of dibutyryl cyclic AMP,prostaglandin E1, and indomethacin on growth and hormone secretion by rat pituitary tumor cells in culture

The growth of rat pituitary tumor cells (GH₁ line) maintained in monolayer culture was inhibited by dibutyryl cyclic AMP in a dose-related fashion. Neither PGE₁ (2.8 × 10^?5M) nor indomethacin (2.8 × 10^?6M) had any significant effect on cell proliferation. Release of GH into the culture medium was stimulated by the cyclic AMP derivative but not by PGE₁ or indomethacin. In short term experiments (15 min.) both in intact monolayers and in trypsin-treated cells incubated in suspension, PGE₁ caused a 2–10 fold increase in cyclic AMP levels. This response, however, appeared to be of short duration reaching a maximum in 10 minutes. It is suggested that, at least in this line of pituitary tumor cells, PGE₁ does not mimic the effect of cyclic AMP, for it probably cannot sustain the elevated intracellular levels of this nucleotide which seem to be necessary for growth inhibition and enhanced GH secretion.     

Metabolism and effect of prostaglandin H2 in adipose tissue

Prostaglandin H₂ (PGH₂) inhibited noradrenaline induced cyclic AMP accumulation in isolated rat fat cells in a dose-dependent manner. IC₅₀ was 10 – 25 ng/ml both in the absence and in the presence of theophylline. The degree of inhibition produced by PGH₂ increased with time of incubation. A stable PGH₂ analog did not inhibit cyclic AMP accumulation. PGH₂ was rapidly converted by isolated fat cells to PGD₂, PGE₂ and PGH_2α, but no formation of thromboxane B₂ was found either or . PGE₂ was a more potent inhibitor than PGH₂ of noradrenaline induced cyclic AMP accumulation. PGD₂ enhanced cyclic AMP accumulation in a limited concentration interval, while PGF_2α was essentially uneffective.Our results suggest that PGH₂ is an inhibitor of cyclic AMP formation in isolated rat fat cells only after conversion to PGE₂. A physiological role for PGH₂ as a modulator of lipolysis is considered unlikely.     

Effect of Carbachol and 56 mM-Potassium Chloride on the Cyclic AMP-Mediated Induction of Tyrosine Hydroxylase in Neuroblastoma Cells in Culture

Abstract: Tyrosine hydroxylase (TH) activity is increased two- to threefold in neuroblastoma cell line NBP² maintained in culture for 48 h in the presence of either the inhibitor of cyclic AMP-phosphodiesterase (PDE), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20- 1724), or the activator of adenylate cyclase, prostaglandin E₁ (PGE₁). Cyclic AMP levels are elevated 70–80% and 30–40% throughout the 48-h treatment with RO 20-1724 and PGE₁, respectively. Carbachol does not affect either basal TH activity or cyclic AMP levels in the cells. However, the cholinergic agonist delays the induction of TH elicited by either RO 20-1724 or PGE₁. This delay is prevented by atropine. The elevation in cyclic AMP levels elicited by either RO 20-1724 or PGE₁ is blocked for 1 h or 15 min. respectively, after treatment with carbachol. Cyclic AMP levels then begin to rise until they reach those levels observed in the presence of RO 20-1724 or PGE₁ alone by 12 h or 1 h of treatment, respectively. Time course studies demonstrate that this transient inhibition of the elevation of cyclic AMP is associated with a 48-h delay in the induction of TH elicited by either RO 20-1724 or PGE₁. In contrast, the induction elicited by 8-bromo cyclic AMP is unaffected by carbachol. A depolarizing concentration (56 mM) of KCl produces a 24-h delay in the induction of TH elicited by RO 20-1724, without affecting the concomitant elevation of cyclic AMP produced by the PDE inhibitor. Furthermore, 56 mM-KCl inhibits the induction of TH elicited by 8-bromo cyclic AMP. It thus appears that carbachol delays the induction of TH by transiently inhibiting the elevation of cyclic AMP, whereas potassium depolarization delays the induction of TH by inhibiting a process with a site of action that is distal to the elevation of cyclic AMP.     

Antagonism of prostaglandin-promoted pituitary cyclic amp accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid

The studies reported here confirm the previously observed potent stimulus to growth hormone (GH) secretion by prostaglandin E₁ (PGE₁). Proportional increments in GH secretion were observed following $\text{in vitro}$ addition of PGE₁ over a concentration range of 10^?7 to 10^?5 M. Growth hormone secretion could not be further stimulated by higher concentrations of prostaglandin. Prostaglandin E₁ also increased cyclic AMP concentration in the pituitary explants in a proportional fashion, which correlated closely with its potency as a growth hormone secretogogue. In order to define more precisely the mechanism by which prostaglandin acts, the effects of prostaglandin antagonist, 7-oxa-13-prostynoic acid, on GH secretion and cyclic AMP accumulation were investigated. Addition of the antagonist alone had no consistent effects on GH secretion or cyclic AMP levels in the pituitary. However, the antagonist significantly reduced the stimulation of hormone release and cyclic AMP accumulation found following addition of PGE₁. Increasing the concentration of antagonist further diminished prostaglandin stimulated hormone release and nucleotide accumulation. The antagonist failed to block the stimulatory effects of theophylline and dibutyryl cyclic AMP on GH release, indicating that the inhibition observed occurred prior to intracellular accumulation of the cyclic nucleotide. These results are consistent with the hypothesis that a prostaglandin receptor on the pituitary somatotrope is linked to the adenyl cyclase-cyclic AMP system.     

Stimulation of guanosine 3',5'-cyclic monophosphate accumulation in rat anterior pituitary gland in vitro by synthetic somatostatin

Synthetic somatostatin stimulated cyclic GMP accumulation with dose dependency (10 ng/ml – 10 μg/ml in a dose examined) in rat anterior pituitary gland in vitro. The stimulation of cyclic GMP levels in the gland was observed after 2 min incubation with somatostatin. Cyclic AMP production induced by TRH or PGE₁ was supressed by this GH release inhibiting factor, while cyclic GMP concentration in the gland was elevated. The present results seem to suggest that inhibitory effect on GH release by somatostatin in anterior pituitary gland is mediated through change in concentration of cyclic AMP and cyclic GMP in the target cells.     

Regulations of macrophage-mediated tumor cell killing by prostaglandins: Comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E₂ (PGE₂) and prostaglandin I₂ (PGI₂), the latter detected as its stable metabolite, 6-keto PGF_1α. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10⁻⁷M for PGI₂ and 3 × 10⁻⁸M for PGE₂. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE₂ and PGI₂; however, only PGE₂ had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.     

Relationship Between the Actions of Calcium Ions, Opioids and Prostaglandin E1 on the Level of Cyclic AMP in Neuroblastoma × Glioma Hybrid Cells

Abstract: Neuroblastoma × glioma hybrid cells increase their intracellular concentration of cyclic AMP in response to prostaglandin E₁ (PGE₁). This effect is inhibited by opioids. The response to PGE₁ is positively correlated with the concentration of Ca²⁺ in the incubation medium. The Ca²⁺ antagonists Co²⁺ and La³⁺, the Ca²⁺ chelator EGTA and a blocker of Ca²⁺ influx into cells, Segontin, inhibit the response to PGE₁. At low external concentrations of Ca²⁺ the response to PGE₁ is enhanced by the Ca²⁺ ionophore A23187. The effects of A23187 and Segontin point to a cytosolic site of Ca²⁺ action. Lack of Ca²⁺ reduces the level of cyclic AMP even in the absence of PGE₁ and the presence of an inhibitor of cyclic AMP phosphodiesterase. Ca²⁺ is required even for an increase in the level of cyclic AMP in cells pretreated with cholera toxin. The increases in level of cyclic AMP evoked by PGE, in a neuroblastoma and by PGE₁ or noradrenaline in a glioma cell line do not depend on Ca²⁺. The response of the hybrid cells to the opioid leucine-enkephalin appears not to rely on the presence of Ca²⁺. Even changing the intracellular concentration of Ca²⁺ by the ionophore A23187 does not alter the effect of the opioid. The analogy between opioids and lack of Ca²⁺ in the short-term (minutes) experiments mentioned holds also for long-term (hours) experiments. Cells chronically exposed to opioids or to low concentrations of Ca²⁺ display an enhanced maximal response to PGE₁.     

Endogenous synthesis of prostaglandins E2 and I2 in 3T3 fibroblasts transformed by polyoma virus: Effects on adenosine 3′:5′-monophosphate production

Prostaglandins (PG)E₁, E₂ and I₂ were produced by polyoma virus transformed (py) 3T3 fibroblasts. The levels of PGE₁, PGE₂ and 6-keto-PGF_1α (degradation product of PGI₂) were 22.7, 225 and 33.2 ng/ml medium, respectively, 72 h after medium change. The stimulatory potencies of exogenous PGE₁, PGE₂ and PGI₂ on adenosine 3′:5′-monophosphate (cyclic AMP) formation were similar. Therefore, the prostaglandin mediated increase in cyclic AMP levels observed during growth of these cells (Claesson, H.-E., Lindgren, J.Å. and Hammarström, S. (1977) Eur. J. Biochem. , 13) is largely (>80%) mediated by PGE₂ and to lesser extents by PGE₁ and PGI₂.     

The roles of calcium and cyclic AMP in the stimulatory action of parathyroid hormone on thymic lymphocyte proliferation

Both the parathyroid hormone (PTH) and the calcium ion increase the cellular content of cyclic adenosine 3′,5′-monophosphate (cyclic AMP), promote the initiation of deoxyribonucleic acid synthesis and stimulate the proliferation of rat thymocytes maintained in vitro. The ability of cyclic AMP to serve as the mediator of the mitogenic actions of both PTH and calcium is established by the fact that cyclic AMP itself stimulates cell proliferation in the absence of PTH and extracellular calcium. Neither PTH nor calcium appear to raise the cellular cyclic AMP level by increasing the nucleotide's synthesis by adenylate cyclase (formerly adenyl cyclase); PTH concentrations as high as 50 μg per ml of medium do not increase the enzyme's activity (in the presence or absence of calcium) and mitogenic calcium concentrations inhibit it. PTH also does not directly affect isolated thymocyte phosphodiesterase, but mitogenic calcium levels inhibit the enzyme's activity. Additional experiments show that it is calcium which raises the cyclic AMP level in cells treated with PTH, and some possible calcium-mediated mechanisms by which the hormone could elevate the cellular cyclic AMP levels are discussed. Thus, the mitogenic action of PTH is primarily mediated by calcium while cyclic AMP is the ultimate implementor of the hormonal action. However, calcium has a dual role and evidence is presented which indicates that besides raising the cellular cyclic AMP level, it also controls the operation of cyclic AMP's mitogenic end-reaction.     

Effect of Prostaglandins on Hepatic Cyclic Nucleotide Concentration,Carbohydrate and Lipid Metabolism

The effects of exogenous prostaglandin E₁ (PGE₁) or prostaglandin E₂ (PGE₂) were studied in the isolated perfused rat liver and in the intact canine liver in order to determine the possible physiological role of prostaglandins on hepatic carbohydrate and lipid metabolism. The data indicate that PGE₁ and PGE₂ did not stimulate cyclic AMP (cAMP) and cyclic GMP (cGMP) concentrations in intact dog liver and PGE₁ failed to stimulate cAMP or cGMP in fed or fasted perfused rat liver. PGE₁ did not promote hyperglycemia, glycogenolysis, lipolysis, or prevent epinephrine-induced hyperglycemia in the isolated perfused rat liver. Other known glycogenolytic agents including glucagon and epinephrine increased cAMP and glycogenolysis in the same perfusion system. This study does not support a physiologic role for PGE₁ on hepatic glycogenolysis or lipolysis. If PGE₁ subsequently is found to influence other metabolic parameters such as lipogenesis, gluconeogenesis, ureogenesis or amino acid transport in isolated perfused liver, such alterations would probably occur independent of changes in cyclic nucleotide activity.     

Concanavalin A and the initiation of thymic lymphoblast DNA synthesis and proliferation by a calcium-dependent increase in cyclic GMP level

Exposure of a thymic lymphocyte population (suspended in serum-free synthetic medium) to the phytomitogen concanavalin A (Con A) causes brief (within the first 8 to 12 minutes) rises in the cellular contents of cyclic AMP and cyclic GMP. However, the rise in the cyclic GMP level is calcium (extracellular)-dependent, but the cyclic AMP rise is not. These changes are followed during the next hour by the initiation of DNA synthesis by a large fraction of the lymphoblast subpopulation which, like the preceding cyclic GMP rise, is calcium-dependent. The stimulated lymphoblasts eventually progress into mitosis. Additional observations indicate that Con A operates by sensitizing lymphoblasts to calcium ions which, in turn, cause the initiation of DNA synthesis by a process mediated by cyclic GMP, but not cyclic AMP.