Bone remodelling induced by physical stress is prostaglandin E2 mediated

In vitro cultured bone cells were found to be responsive to hormones and physical forces. A simple device has been developed which enables the direct application of physical forces to tissue culture dishes to which cells are firmly attached. The physical forces created a deformation of the dish. It was found that prostaglandin E₂ synthesis underwent a rapid increase, reaching a maximum after 20 min and then declined. Concurrent with the increase in prostaglandin E₂ was an increase in cyclic AMP production, having a maximum around 15 min. The increase in cyclic AMP was blocked by indomethacin, the prostaglandin E₂ synthesis inhibitor, indicating the dependence of cyclic AMP production on the de novo synthesis of prostaglandin E₂. Prostaglandin E₂ added to cells mimicked the effect of physical forces on the production of cyclic AMP. The increase in cyclic AMP resulted from an early rise in adenyl cyclase activity (within 5 min) and a later (10 min) increase in phosphodiesterase activity. The same physical forces also stimulatedthe incorporation of thymidine into DNA after 24 h. On addition of prostaglandin E₂ the increase in DNA synthesis was also mimicked. Pretreatment of the cells with indomethacin abolished the effect of physical forces on DNA synthesis.The results suggest a stimulus receptor mechanism for physical forces which, like hormonal effectors, are mediated by prostaglandins and stimulate cyclic AMP and DNA synthesis.We believe that physical forces stimulate bone remodelling through such a stimulus receptor system, mediated by prostaglandins.     

Some regulatory properties of glycogen phosphorylase from Streptococcus salivarius

Purified (200-fold) glycogen phosphorylase (EC 2.4.1.1) of Streptococcus salivarius was activated by AMP and NaF when assayed both in the direction of synthesis and in the direction of phosphorolysis. Activation by NaF + AMP was greater than the sum of their individual effects. In the direction of synthesis, the K_m for AMP was 0.25 mm and was decreased to 0.125 mm in the presence of NaF. The K_m for NaF was 0.49 m and was decreased to 0.40 m in the presence of AMP. Glycogen phosphorolysis was similarly affected by AMP and NaF, except that above a concentration of 2 mm AMP was inhibitory. The effects of AMP and NaF were reversible since preincubation with these compounds, followed by dialysis, restored activity almost to the control values although some inhibition of enzyme activity was noted with the samples preincubated with NaF. The presence of both NaF and AMP had no effect on the K_m values for glucose-1-P and glycogen in the direction of synthesis, but increased the V of the enzyme.When assayed in the absence of AMP and NaF in the direction of synthesis, the enzyme was slightly inhibited by glucose and glucose-6-P, and activated by P-enolpyruvate and ADP-glucose. In the presence of AMP and NaF, the enzyme was inhibited by glucose, glucose-6-P and ADP-glucose, but was activated by P-enolpyruvate. Fructose-1,6-P₂ had no effect on the enzyme. The enzyme was further activated in the absence of AMP and NaF by adenosine, ATP, GMP, cyclic AMP and ADP, and was slightly inhibited by GTP and GDP. In the presence of AMP and NaF, however, these compounds, with the exception of adenosine, either did not show any effect or were slightly inhibitory. Adenosine was slightly stimulatory with NaF + AMP, but not with AMP alone. In the direction of phosphorolysis, the enzyme was inhibited by glucose and ADP-glucose, and activated by P-enolpyruvate, fructose-1,6-P₂ and ATP, both in the presence and absence of AMP + NaF.     

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.     

Kinetics of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab (Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with P_i or arsenate as substrates. The effects of AMP, UDPG, G-6-P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to P_i changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6-P is a competitive inhibitor of G-1-P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to P_i. Arsenate is a competitive inhibitor with respect to P_i. The K_m for AMP increases in the presence of UDPG, and decreases with increasing concentrations of P_i or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for P_i, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Repression of arginase and agmatine amidinohydrolase by urea in the lichen Evernia prunastri

Two forms of arginase (EC 3.5.3.1) have been found in Evernia prunastri: (1) a light-arginase (M_r, 180 000) induced by l-arginine—urea causes repression which is reversed by cyclic AMP; (2) a constitutive heavy-arginase (M_r, 330 000) which is not affected by cyclic AMP. Agmatine amidinohydrolase (EC 3.5.3.11) is also repressed by urea but this effect is carried out at catabolite concentrations higher than those required to prevent the synthesis of the light-arginase. This repression is also relieved by cyclic AMP.     

Regulation of ovarian steroidogenesis: The disparity between 125I-labelled choriogonadotropin binding,cyclic adenosine 3′,5′-monophosphate formation and progesterone synthesis in the rat ovary

The binding of ¹²⁵I-labeled human choriogonadotropin, formation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP), and synthesis of progesterone were examined in ovarian cells from immature rats. Collagenase dispersed ovarian cells were found to respond specifically to lutropin-like activity. The equilibrium dissociation constant (K_d) for the binding of ¹²⁵I-labelled choriogonadotropin was 1.7 · 10^1?10 M. Progesterone synthesis was increased at least 40-fold and cyclic AMP formation 10-fold in response to maximum hormonal stimulation. The concentration of choriogonadotropin which stimulated progesterone synthesis maximally in Eagle's minimum essential medium ?0.1% gelatin (2 ng/ml), resulted in minimal (less than 30% of maximum) increases in cyclic AMP accumulation and hormone bindind. Similarly, binding of choriogonadotropin was not saturated at a hormone concentration (50 ng/ml) that stimulated maximal cyclic AMP formation. These results are consistent with the existence of receptor reserve in the ovarian cell. A marked shift in the dose vs. response relationship for progesterone synthesis occurred when fetal calf serum was used to supplemen Eagle's minimum essential medium, however. Under these experimental conditions, progesterone synthesis reached a maximum at a hormone concentration of the same order of magnitude as did cyclic AMP formation. It is concluded that the degree of spare receptor effect observed may depend not only on an absolute amount of excess receptor, but also on the readiness of the system to respond in a given fashion.     

Decidual adenylate cyclase and prostaglandin production in vitro

Human decidua contains an active adenylate cyclase, and a number of studies indicate that adenylate cyclase is functionally linked to increased in vitro prostaglandin synthesis. Increased decidual prostaglandin synthesis is associated with parturition, and therefore activation of adenylate cyclase may be involved in the control of human parturition. In this study, third trimester human decidual cells were preincubated for no more than 24 h prior to stimulation with a number of reagents which increase cellular cyclic AMP levels. Forskolin rapidly increased intracellular and extracellular cyclic AMP levels, but there was no increase in prostaglandin E₂ biosynthesis during incubations ranging from 5 min up to 24 h. Dibutyryl cyclic AMP or 8-bromo-cyclic AMP were also without effect on PGE₂ production, which suggests that the adenylate cyclase was not linked to the mechanisms regulating prostaglandin production. Cholera toxin increased basal cyclic AMP and PGE₂ synthesis, and was without effect on IL-1β-stimulated PGE₂ levels. PGE₂ synthesis was increased by 24 h culture with IL-1β in all the cell preparations, indicating that the cells were biologically active, and that the lack of effect of changes in cyclic AMP synthesis on PGE₂ levels could not be attributed to a defect in the prostaglandin synthetic pathway. Our findings did not agree with earlier work which showed that changes in cyclic AMP were correlated with changes in PGE₂ production by human decidual cells. It is clear that in the previous studies the decidual cells were preincubated for 4–7 days prior to stimulation, in contrast with 24 h in our investigation. We suggest that the functional link between cyclic AMP and PGE₂ synthesis reported previously may develop during culture, and not be a part of normal decidual cell function, but further studies are needed to test this hypothesis.     

The effect of 7-oxa-13 prostynoic acid on the mechanism of action of bradykinin in human synovial fibroblasts

Bradykinin, a potent inflammatory mediator, induces an increment in intracellular cyclic AMP concentrations of human synovial fibroblasts and evokes the synthesis and release of ³H-arachidonic acid and ³H-E prostaglandins from these cells pre-labeled in their phospholipids. Fetal calf serum in the media also stimulates the synthesis and release of these labeled lipids from pre-labeled human synovial fibroblasts and potentiates the bradykinin-induced cyclic AMP response. The PGE₁ analogue, 7-oxa-13 prostynoic acid, completely abrogates both the bradkinin-induced cyclic AMP response and the bradykinin- and fetal calf serum-evoked release of labeled E-prostaglandins from pre-labeled cells. In serum-free media, the prostaglandin antagonist stimulated the release of ³H-arachidonic acid from pre-labeled human synovial fibroblasts and did not inhibit the bradykinin-induced release of this lipid.     

Oxygen-18 studies of the mechanism of pyrophosphoryl group transfer catalyzed by phosphoribosylpyrophosphate synthetase.

The formation of phosphoribosylpyrophosphate (PRPP) and adenosine 5′-monophosphate (AMP) from ribose 5-phosphate and adenosine 5′-triphosphate, catalyzed by purified PRPP synthetase from Salmonella typhimurium, was conducted in ¹⁸O-enriched water. The products were isolated, and inorganic phosphate was isolated from AMP and the pyrophosphoryl moiety of PRPP. Oxygen-18 was incorporated into PRPP but not into AMP. These results indicate that PRPP synthesis proceeds with scission of a βPO bond of adenosine 5′-triphosphate. Oxygen-18 enters PRPP by prior exchange of H₂¹⁸O into ribose 5-phosphate; the rate of this exchange was measured by combined gas chromatography-mass spectrometry of the trimethylsilyl derivative of ribose 5-phosphate.     

Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro

Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10(-4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development.     

Alteration of hormone-stimulated cyclic AMP synthesis in guinea pig peritoneal macrophages.

The decrease of PGE-stimulated cyclic AMP synthesis due to pretreatment of intact cells with PGE (hormone-specific desensitization) was shown to be a rapid process in macrophages. Desensitization was found to be extensive after 5-min treatment of macrophages with PGE₂ and almost complete after 20 min. Furthermore, incubation of intact macrophages with colchicine caused a two- to sixfold increase in the rate of PGE₁-stimulated cyclic AMP synthesis in intact macrophages. Colchicine alone did not alter cyclic AMP levels. The enhancing effect of colchicine is related to its ability to disrupt microtubules. Vinblastine, another microtubule-disrupting agent, caused similar enhancement of PGE-stimulated cyclic AMP synthesis; no enhancement was found when lumicolchicine was used. Hormonestimulated cyclic AMP synthesis by colchicine-treated macrophages was also measured after cell homogenization. The enhancement of hormone sensitivity by colchicine was found to be lost upon homogenization. These findings suggest that colchicine acts at the interior of the cell to reversibly affect adenylate cyclase.     

Vasopressin and angiotensin II stimulate ureogenesis through increased mitochondrial citrulline production.

Vasopressin, angiotensin II, glucagon and epinephrine (through a cAMP-independent, alpha₁adrenergic mechanism), stimulate ureogenesis in isolated rat hepatocytes. Mitochondria, isolated from hepatocytes which were previously treated with these hormones, displayed an enhanced rate of citrulline synthesis in the presence of NH₄Cl as the nitrogen source. When mitochondria were incubated with glutamine as the nitrogen source, only those mitochondria isolated from hepatocytes previously treated with epinephrine or glucagon displayed an enhanced capacity to synthesize citrulline.When cells were incubated in the absence of extracellular calcium, the effects of vasopressin and angiotensin II on urea synthesis were abolished, whereas those of epinephrine and glucagon were only diminished. Mitochondria isolated from cells incubated under these conditions, showed that the effect of all these hormones on citrulline synthesis could still be observed. However, the effects of glucagon and epinephrine plus propranolol were larger than those of angiotensin II or vasopressin.Phosphatidylinositol labeling was significantly increased by epinephrine, vasopressin and angiotensin II both in the absence or presence of calcium. Cyclic AMP levels were significantly increased by glucagon or epinephrine but not by vasopressin or angiotensin II. The effect of epinephrine on cyclic AMP levels was blocked by propranolol both in the absence or presence of calcium.     

Cycloheximide: An adrenergic agent

Cycloheximide, a widely used inhibitor of protein synthesis, stimulates glycogenolysis, gluconeogenesis and ureogenesis in isolated rat hepatocytes. The effects of cycloheximide were compared to those of norepinephrine. Both agents, cycloheximide and norepinephrine, produced slight increases in the levels of cyclic AMP (30% increases) which were blocked by propranolol. Interestingly, it was found that the metabolic actions of norepinephrine and cycloheximide (stimulation of glycogenolysis, gluconeogenesis and ureogenesis) were only slightly diminished by the β adrenergic antagonist propranolol but abolished by the selective α₁ adrenergic antagonist prazosin. The ability of cycloheximide to inhibit protein synthesis was not affected by either prazosin or propranolol. It is concluded that the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by cycloheximide in rat hepatocytes, is an effect of the antibiotic independent of its ability to inhibit protein synthesis and that is mediated through activation of α₁ adrenoceptors. The adrenergic activity of cycloheximide should be considered when this drug is used as an inhibitor of protein synthesis.     

Dissociation of the regulation of fatty acid synthesis and cyclic AMP levels in cultured glial and neuronal cells.

Abstract— Cultured C-6 glia and neuroblastoma were utilized to investigate the relation of rates of fatty acid synthesis (from ³H₂O) to levels of cyclic AMP under conditions of short-term and long-term regulation. The data demonstrate a consistent dissociation of alterations in rates of fatty acid synthesis and levels of cyclic AMP. Thus, marked alterations in the rate of fatty acid synthesis occurred when serum or albumin-bound palmitic acid was present in the culture medium, but there were no accompanying alterations in levels of cyclic AMP. Similarly, when high intracellular and/or extracellular levels of cyclic AMP were induced by exposure of the cells to dibutyryl cyclic AMP or isoproterenol, no change in the rate of fatty acid synthesis occurred. Although the data raise serious doubt about an important role for cyclic AMP in the regulation of fatty acid synthesis, they do not rule out such a role. The findings do indicate that any such role must involve alterations in compartmentalization, metabolism or binding of the mononucleotide within the cell.     

Evidence that endogenous cyclic AMP does not modulate serum sulfation factor action on embryonic chicken cartilage

The role of cartilage cyclic AMP as a mediator or modulator of serum sulfation factor (SSF) action on embryonic chicken cartilage was assessed. Media with concentrations of rat serum (7.5%) sufficient to maximally stimulate chondromucoprotein synthesis as measured by ³⁵SO₄ incorporation did not change cartilage cyclic AMP levels. Theophylline (2.5mM) doubled cyclic AMP in cartilage incubated in media but had no effect on ³⁵SO₄ incorporation. In media containing 5% rat serum, theophylline at 0.5, 1.5 and 2.5mM caused a similar and significant rise in tissue cyclic AMP but only 2.5mM inhibited SSF stimulated ³⁵SO₄ incorporation. The data indicate that cartilage cyclic AMP neither mediates nor modulates SSF action on cartilage chondromucoprotein synthesis.     

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3′,5′-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I₂, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B₄ synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B₄ itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B₄ is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B₄ synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B₄ can limit their own activity in neutrophils.     

Evidence for the conformation about the C(5′)-O(5′) bond of AMP complexed to AMP kinase: Substrate properties of a vinyl phosphonate analog of AMP

A vinyl phosphonate analog of adenosine 5′-phosphate (AMP) was synthesized in which the CH₂OP system of AMP is replaced by CHCHP. The V_max values of this analog relative to AMP were 0.7% with rabbit muscle AMP aminohydrolase, 13.4% with rabbit muscle AMP kinase, and 6.6% with pig muscle AMP kinase. The vinyl analog of ADP produced by the kinases was a substrate of rabbit muscle pyruvate kinase. These results, together with substrate specificity properties at the AMP sites of the enzymes indicate that the C(4′)-C(5′)-O(5′)-P system of AMP is of trans character during conversion of AMP to ADP by pig or rabbit AMP kinase.     

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.     

Prostaglandins and cyclic AMP stimulate creatine kinase synthesis but not fusion in cultured embryonic chick muscle cells

Cyclic AMP levels have been measured in cultures derived from 12-day-old chick embryonic muscle. A rise in concentration was found after the onset of myoblast fusion. Cells cultured at a medium Ca²⁺ concentration of 0.1 μM did not fuse and exhibited only a small rise in cyclic AMP concentration during culture. Addition of 1.4 mM Ca²⁺ to these cells after 50 h in culture caused rapid, synchronous fusion with a concomitant rise in cyclic AMP levels. Indomethacin, an inhibitor of prostaglandin synthesis, did not inhibit fusion, but inhibited the rise in cyclic AMP concentration. Indomethacin-treated cultures exhibited lower creatine kinase levels, though no change in the ratio of the three isoenzymes was observed. Addition of prostaglandins E₁ and E₂ to indomethacin-treated cultures overcame this inhibition. We propose that prostaglandin synthesis is a consequence of the stimulation of myoblast fusion and that via cyclic AMP it stimulates protein synthesis.