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 effect of cyclic AMP and prostaglandins on the fibrinolytic activity of mouse neuroblastoma cells.

The C1300 mouse neuroblastoma cell line was found to produce plasminogen activator which is secreted into the growth medium. The intra- and extracellular activities of this enzyme were markedly increased (up to 14 fold) by treatment with cyclic AMP agents. Prostaglandins E₁ and E₂ and butyric acid were the most efficient inducers followed by propionic acid and dibutyryl cyclic AMP. Theophylline was found to be ineffective. The highest enzyme activities were found in cells exposed simultaneously to prostaglandin E₁ and dibutyryl cyclic AMP.     

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 X 10(-6) M, PGE1 and PGE2 had approximately the same effect, while the effect of PGF2alpha 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.     

Effect of epidermal growth factor on synthesis of prostaglandins and cyclic AMP by embryonic palate mesenchymal cells

The influence of epidermal growth factor (EGF) on the ability of murine embryonic palate mesenchymal (MEPM) cells to be stimulated to synthesize cAMP and prostaglandins was investigated. Preincubation of MEPM cells with EGF enhanced, in a dose-dependent fashion, (1) the responsiveness of MEPM cells to prostaglandin E1-induced elevation of intracellular levels of cAMP, and (2) the responsiveness of cells to calcium ionophore (A23187) and melittin-induced synthesis of prostaglandins E2 and F2 alpha. Hormonal responsiveness of MEPM cells to EGF, prostaglandins and cAMP has been implicated as being involved in controlling various aspects of normal oro-facial development. We show here that EGF can potentiate hormonal responsiveness of these cells and thus allows consideration of EGF as a factor which may modulate hormonally regulated craniofacial growth and differentiation.     

The effect of collagen on the cyclic AMP content of embryonic somites.

Previous studies have demonstrated that collagen substrates stimulate in vitro somite chondrogenesis, and that agents that elevate intracellular cyclic AMP levels in hibit the ability of somites to respond to the inductive influence of collagen. In the present investigation, radiommunoassay was utilized to compare the cyclic AMP content of somite explants cultured on purified Type I collagen substrates with control explants cultured on Millipore filters. During the period of culture, the cyclic AMP content of collagen-treated explants is significantly lower than the cyclic AMP content of control explants. The cyclic AMP content of collagen-treated explants is 66% of control values as early as one hour following the initiation of culture, and the cyclic AMP content of collagen-treated explants remains lower than controls throughout the 3-day cultured period. The greatest difference in the cyclic AMP content of collagen-treated and control explants is observed at the seventeenth hour of culture, at which time the cyclic AMP content of collagen-treated explants is 56% of controls. These results combined with previous studies provides support for the hypothesis that collagen elicits a reduction in the cyclic AMP content of embroyic somites and that this reduction is necessary to trigger chondrogenic differentiation.     

Effects of prostaglandins on cyclic AMP levels in isolated cells from developing chick limbs

Effects of prostaglandins (PGs) on accumulation of cyclic AMP (cAMP) in the presence of a phosphodiesterase inhibitor were investigated in cells isolated from avian limb buds at various stages of development. Cells were responsive to PGE2 at the earliest stage investigated (stage 20-21) which was well in advance of specific cytodifferentiation of limb tissues. At three later stages (24-25; 26-28; 30-32), the responsiveness of cells isolated from the developing skeletal anlagen of the limb progressively increased coincident with the differentiation and maturation of the cartilage phenotype. Cells isolated from stage 26-28 cartilage rods were responsive also to prostacyclin (PGI2); however, the response produced was only about 50% of the response to an equivalent concentration of PGE2. Cells were not responsive to either PGF2 alpha or 6-keto PGF1 alpha, at concentrations of 30-33 micrograms/ml demonstrating a degree of specificity for PGE2 and PGI2. In the absence of the phosphodiesterase inhibitor, PGE2 increased cAMP accumulation two-fold over the controls and produced a concentration-dependent response between 0.3-30 micrograms/ml. The results demonstrate that PGs are capable of modulating cAMP levels of undifferentiated limb mesenchymal cells as well as embryonic cartilage cells and suggest a role for these compounds in limb chondrogenesis.     

Regulation of cyclic AMP metabolism in rabbit cortical collecting tubule cells by prostaglandins

Prostaglandin E1 (PGE1) at 1 nM inhibits arginine-vasopressin (AVP)-induced water reabsorption in the rabbit cortical collecting tubule (RCCT), while 100 nM PGE1, by itself, stimulates water reabsorption (Grantham, J. J., and Orloff, J. (1968) J. Clin. Invest. 47, 1154-1161). To investigate the basis for these two responses, we measured the effects of prostaglandins on cAMP metabolism in purified RCCT cells. In freshly isolated cells, PGE2, PGE1, and 16,16-dimethyl-PGE2 acting at high concentrations (0.1-10 microM) stimulated cAMP accumulation; however, one PGE2 analog, sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), failed to stimulate cAMP accumulation or to antagonize PGE2-induced cAMP formation; PGD2, PGF2 alpha, and a PGI2 analog, carbacyclin (6-carbaprostaglandin I2), also failed to stimulate cAMP synthesis. These results suggest that there is a PGE-specific stimulatory receptor in RCCT cells which mediates activation of adenylate cyclase. Occupancy of this receptor would be anticipated to cause water reabsorption by the collecting tubule. At lower concentrations (0.1-100 nM) PGE2, PGE1, 16,16-dimethyl-PGE2, and, in addition, sulprostone inhibited AVP-induced cAMP accumulation by fresh RCCT cells in the presence of cAMP phosphodiesterase inhibitors. Pertussis toxin pretreatment of RCCT cells blocked the ability of both PGE2 and sulprostone to inhibit AVP-induced cAMP accumulation. In membranes prepared from RCCT cells, sulprostone prevented stimulation of adenylate cyclase by AVP. These results suggest that E-series prostaglandins (including sulprostone) can act through an inhibitory PGE receptor(s) coupled to the inhibitory guanine nucleotide regulatory protein, Gi, to block AVP-induced cAMP synthesis by RCCT cells. Occupancy of this receptor would be expected to cause inhibition of AVP-induced water reabsorption in the intact tubule. Curiously, after RCCT cells were cultured for 5-7 days, PGE2 no longer inhibited AVP-induced cAMP accumulation, but PGE2 by itself could still stimulate cAMP accumulation. In contrast to PGE2, epinephrine acting via an alpha 2-adrenergic, Gi-linked mechanism did block AVP-induced cAMP formation by cultured RCCT cells. This implies that some component of the inhibitory PGE response other than Gi is lost when RCCT cells are cultured.     

Inhibition of chondrogenesis by retinoic acid in limb mesenchymal cells in vitro: Effects on PGE2 and cyclic AMP concentrations

Effects of retinoic acid (RA) on prostaglandin E₂ (PGE₂) and cyclic AMP (cAMP) concentrations were investigated in high density, micromass cultures of mesenchymal cells derived from chick limb buds. Exposure of cells during the initial 24 h of culture to RA concentrations between 0.05–1.0 μg/ml inhibited chondrogenesis in a dose-dependent manner with 1.0 μg/ml totally inhibiting cartilage formation. Concentrations of PGE₂ and cAMP increased during the prechondrogenic period in control cells in a closely related way and remained elevated throughout the six-day period examined. Addition of RA (0.05 and 0.5 μg/ml) did not significantly alter cAMP concentrations at any time point, but significantly elevated PGE₂ levels relative to control cells in six-day cultures in a concentration-dependent manner. Addition of dibutyryl cAMP enhanced chondrogenesis in control cells between days 3 and 4, but failed to alter the inhibitory effect of RA on chondrogenesis. The results indicate that while PGE₂ and cAMP are important signals in cartilage differentiation, the inhibitory effects of RA on this process are mediated through some other mechanism.     

The effect of cyclic AMP on the maturation and degranulation of mast cells.

In short-term tissue cultures dibutyryl cAMP inhibits the cortisone-induced degranulation of mast cells. The effect of methylxanthine was found to be similar but developed at a slower rate. The increase of cAMP level also affected the maturation of mast cells, as the ratio of cells of mixed granulation increased, compared to the alcian blue- and safranin-present model inhibited degranulation.     

The effect of prostaglandins on in vitro limb cartilage differentiation

A variety of studies indicate that a key event in limb chondrogenic differentiation is a cellular condensation process during which an intimate cell-cell interaction occurs that triggers cartilage differentiation by elevating cAMP levels. It has recently been demonstrated that when limb mesenchymal cells are subjected to high density monolayer culture under conditions conducive to chondrogenesis, they synthesize several prostaglandins, including PGE2 and prostacyclin, which are important local modulators of cAMP formation in a number of cells and tissues. In the present study, we demonstrate that exogenous PGE2 stimulates the in vitro chondrogenic differentiation of the subridge mesoderm of the embryonic chick limb bud. The stimulatory effect of PGE2 is greatly potentiated by the phosphodiesterase inhibitor, theophylline, suggesting its influence on chondrogenesis is mediated by its ability to increase cAMP levels. The stimulatory effect of PGE2 is dose-dependent and can be detected at a concentration as low as 10(-8)M. PGE1 is just as effective as PGE2 in stimulating in vitro chondrogenesis, whereas PGA1 and PGF1 alpha are less than half as effective. Thromboxane B2 has no effect on chondrogenesis. On the basis of our results, the possibility that endogenous prostaglandins might regulate limb cartilage differentiation by acting as local regulators of cAMP content is discussed.     

Interactions between parathyroid hormone and prostaglandins on renal cortical cyclic AMP

Effects of parathyroid hormone (PTH) and several prostaglandins (PGs) on cyclic AMP (cAMP) metabolism were studied and compared in isolated renal cortical tubules from male hamsters. Both production and intracellular degradation of cAMP were increased by PTH and each of the PGs tested (PGE₂, PGE₁, PGI₂). Production of cAMP was increased to similar levels by maximal concentrations of PTH and each PG, however, degradation of cAMP was significantly higher in response to PTH than with any of the PGs. This difference in intracellular degradation of cAMP was responsible for the much higher concentrations of cAMP in renal cortical tubules exposed to PGs (PGE₁, PGE₂, PGI₂) than to PTH. Submaximal amounts of each PG produced additive increases in cAMP concentrations in the presence of maximal amounts of PTH. Additivity of the combined responses was lost, however, as the PGs concentrations reached their maximas. The results suggest that renal PGs (PGE₂ and PGI₂) may modulate the effects of PTH on cAMP concentrations in renal cortical tubules.     

Interactions between parathyroid hormone and prostaglandins on renal cortical cyclic AMP

Effects of parathyroid hormone (PTH) and several prostaglandins (PGs) on cyclic AMP (cAMP) metabolism were studied and compared in isolated renal cortical tubules from male hamsters. Both production and intracellular degradation of cAMP were increased by PTH and each of the PGs tested (PGE2, PGE1, PGI2). Production of cAMP was increased to similar levels by maximal concentrations of PTH and each PG, however, degradation of cAMP was significantly higher in response to PTH than with any of the PGs. This difference in intracellular degradation of cAMP was responsible for the much higher concentrations of cAMP in renal cortical tubules exposed to PGs (PGE1, PGE2, PGI2) than to PTH. Submaximal amounts of each PG produced additive increases in cAMP concentrations in the presence of maximal amounts of PTH. Additivity of the combined responses was lost, however, as the PGs concentrations reached their maxima. The results suggest that renal PGs (PGE2 and PGI2) may modulate the effects of PTH on cAMP concentrations in renal cortical tubules.     

Modulatory effect of cyclic AMP on calcium fluxes in FRTL-5 cells

The aim of the present study was to investigate the effect of cAMP on calcium fluxes in Fura 2 loaded thyroid FRTL-5 cells. Preincubating the cells with the phosphodiesterase inhibitor Ro-201724 decreased the ATP-stimulated entry of calcium, while having no effect on the release of sequestered calcium. Pretreatment with forskolin decreased both the release of sequestered calcium and the entry of calcium in response to ATP. We then incubated the cells with phenylisopropyl adenosine (PIA), a P₂₁-receptor agonist earlier shown to decrease cAMP in FRTL-5 cells. Although we did not observe a decrease in cellular cAMP after PIA, the ATP-evoked calcium response was enhanced. Forskolin decreased calcium entry induced by thapsigargin a Ca^2?-ATPase inhibitor, but forskolin had no effect on the thapsigargin-evoked release of sequestered calcium. Addition of calcium to cells stimulated with ATP in a calcium-free buffered resulted in a rapid influx of calcium. This response in [Ca²⁺]_i was decreased in cells pretreated with forskolin. In cells stimulated with thapsigargin, the increase in [Ca²⁺]_i after addition of calcium was inhibited in part by forskolin and enhanced by PIA. The results suggest that cAMP may regulate calcium fluxes in FRTL-5 cells Furthermore, PIA increased agonist-induced calcium entry through a presently unknown mechanism. © 1993 Wiley-Liss, Inc.     

The effect of calcium and cyclic AMP on amylase release in digitonin-permeabilized parotid gland cells

Rat parotid cells were permeabilized with digitonin to examine their secretory dynamics. Cells were isolated by a modification of the method previously described by Hootman [1985). J. Biol. Chem. 260, 4186-4194) in which alpha-chymotrypsin was included. The final preparation consisted of approx. 40-60% single cells. The cells were 85-90% viable by trypan blue exclusion and secreted amylase when stimulated with isoproterenol. Digitonin (2 or 5 microM) was sufficient for permeabilization while 2 microM digitonin was somewhat more effective in maintaining cell integrity as indicated by lactate dehydrogenase release. Digitonin had minimal effects on intracellular granules in the whole cell and was, thus, relatively selective. The response of digitonin-permeabilized cells to calcium (without secretagogues) in the incubation medium was monitored by amylase release. For a wide range of applied free calcium concentrations (1 X 10(-7) M to 10(-4) M) a statistically significant increase in amylase secretion was observed. Control cells did not release amylase to a similar extent without secretagogue. Cyclic AMP (50 microM) significantly enhanced amylase secretion from digitonin-treated cells at all concentrations of free calcium tested. Neither calcium nor cyclic AMP alone was sufficient to stimulate maximal amylase release. Our results provide direct evidence for a model in which calcium and cyclic AMP work on separate pathways as interacting regulators of exocytosis.     

Release of cyclic AMP from macrophages by stimulation with prostaglandins.

The effect of various prostaglandins (PG)on the generation of cyclic AMP in rat peritoneal macrophages has been studied in vitro. PGE1 produced a rapid intracellular accumulation of cyclic AMP which was followed by its release into the extracellular space. More cyclic AMP was released with prostaglandins of the E-type than with A- and F-types. It is suggested that release of cyclic AMP from macrophages may participate in the modulation of leukocyte function.     

The dose dependent effect of cyclic AMP on ribonucleotide reductase in mitogen stimulated mononuclear cells

Cyclic adenosine monophosphate arrests proliferating T lymphocytes in the G1 phase of the cell cycle. Here we demonstrate that exogenous and endogenous elevations in cyclic AMP concentration result in diminished mitogen stimulation, cell cycle arrest, and decreased ribonucleotide reductase messenger RNA concentrations in peripheral blood mononuclear cells. At lower concentrations (less than 1mM) of dibutyryl cyclic AMP that do not generate cell cycle arrest there is inhibition of ribonucleotide reductase activity without decreased messenger RNA concentration for the M2 subunit of ribonucleotide reductase. However, at higher concentrations of dibutyryl cyclic AMP there is G1 cell cycle arrest and reduced M2 specific messenger RNA concentration. Thus, cyclic AMP inhibition of lymphocyte activation may occur by different mechanisms that are dose dependent.     

Rat osteogenic sarcoma cells: Effects of some prostaglandins, their metabolites and analogues on cyclic AMP production

Cyclic AMP production by freshly isolated cells, from a ³²P-induced transplantable rat osteogenic sarcoma, was stimulated by PGE₁, PGE₂ and to a less extent by PGF_2α and PGA₂. In the case of PGE₂, the cyclic AMP content of cells was miximal within 5 min. The 13, 14-dihydro derivatives of PGE₁, PGE₂ and PGF_2α had approximately 40% of the activity of the parent prostaglandin whilst, in every case, the metabolites (15-keto and 13,14-dihydro-15-keto) had very little activity. Two prostaglandin endoperoxide analogues (U44069 and U46619) had only 10% of the activity of an equimolar dose of PGE₂. The data presented in this paper demonstrates similarities between the responses of these cells and cells derived from bony tissue in terms of the ability of prostaglandins to stimulate bone resorption in tissue culture.