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.     

The effect of prostaglandins on the cyclic AMP content of limb mesenchymal cells

We have been investigating the hypothesis that prostaglandins including prostaglandin E2 (PGE2) produced during the critical condensation phase of limb chondrogenesis are involved in the regulation of cartilage differentiation by acting as local modulators of cyclic AMP (cAMP) accumulation. The purpose of the present study was to determine directly whether PGE2 and other prostanoids which had previously been shown to stimulate in vitro chondrogenic differentiation do indeed elevate the cAMP content of limb mesenchymal cells, and to determine whether the ability of various prostanoids to increase cAMP production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis. We have found that PGE2 does indeed elicit a striking elevation in the cAMP content of subridge mesenchymal cells, indicating that the cells possess adenylate cyclase-coupled receptors for this molecule. The effect of PGE2 on cAMP accumulation is potentiated by a phosphodiesterase inhibitor, thus paralleling the potentiating effect phosphodiesterase inhibitors have on PGE2-stimulated in vitro chondrogenesis. The effect of PGE2 on cAMP content is dose-dependent with a 3-fold increase seen at 10(-8)M, which is the lowest concentration at which PGE2 effectively stimulates chondrogenesis. PGE1, which is just as effective as PGE2 in stimulating chondrogenesis, is just as effective as PGE2 in stimulating cAMP accumulation. PGA1, which is a much less effective stimulator of chondrogenesis than PGE2 or PGE1, is less than half as potent as these molecules in elevating cAMP levels. PGF1 alpha, 6-keto PGF1 alpha, and thromboxane B2, which have little or no effect on chondrogenesis, have little or no effect on cAMP content.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of cholera toxin on cyclic AMP accumulation and bone resorption in cultured mouse calvaria

We have utilized the adenylate cyclase stimulator, cholera toxin, as a tool to test the role of cyclic AMP as a mediator of the effects on bone resorption by the calcium-regulating hormones, parathyroid hormone (PTH) and calcitonin. The effects on bone resorption were studied in an organ culture system using calvarial bones from newborn mice. Cyclic AMP response was assayed in calvarial bone explants and isolated osteoblasts from neonatal mouse calvaria. Cholera toxin caused a dose-dependent cAMP response in calvarial bones, seen at and above approx. 1-3 ng/ml and calculated half-maximal stimulation (EC50) at 18 ng/ml. The stimulatory effect of cholera toxin could be potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 0.2 mmol/l). Cyclic AMP accumulation in the bones was maximal after 4-6 h, and thereafter declined. However, activation of the adenylate cyclase was irreversible and the total amount (bone + medium) of cAMP produced, in the presence of IBMX (0.2 mmol/l), increased with time, for at least 48 h. In osteoblast-like cells cholera toxin (1 microgram/ml) stimulated the cellular levels of cAMP with a peak after 60-120 min, which could be potentiated with IBMX. The total cAMP accumulation indicated an irreversible response. In short-term bone organ cultures (at most, 24 h) cholera toxin, at and above 3 ng/ml, inhibited the stimulatory effect of PTH (10 nmol/l) on 45Ca release from prelabelled calvarial bones. The inhibitory effect of cholera toxin (0.1 microgram/ml) on 45Ca release was significant after 6 h and the calculated IC50 value at 24 h was 11.2 ng/ml. Cholera toxin (0.1 microgram/ml) also inhibited PTH-stimulated (10 nmol/l) release of Ca2+, inorganic phosphate (Pi), beta-glucuronidase, beta-N-acetylglucosaminidase and degradation of organic matrix (release of 3H from [3H]proline-labelled bones) in 24 h cultures. 45Ca release from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxyvitamin D3 (0.1 mumol/l) was also inhibited by cholera toxin (0.3 microgram/ml) in 24-h cultures. The inhibitory effect of cholera toxin on bone resorption was transient, and in long-term cultures (120 h) cholera toxin caused a dose-dependent, delayed stimulation of mineral mobilization (Ca2+, 45Ca, Pi), degradation of matrix and release of the lysosomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of dibutyryl cyclic AMP and cytochalasin B on cultured human glioma cells.

Cultured human glioma cells (138 MG) exposed to dibutyryl cyclic AMP (dbc-AMP; 0.1--5 mM) attained an arborized shape with thin processes extending from a rounded cell body. Cytochalasin B (CB; 1--1 muM) induced similar morphological changes. The processes in both dbc-AMP and CB treated cells were formed by retraction of the cell margin. Colchicine (1muM) completely and liver treated phalloidin (0.1 mg/ml) partially inhibited the morphological alterations induced by dbc-AMP and CB. Dbc-AMP was found to arrest cell movement, cell division and uptake of 2-deoxy-D-glucose. CB has the same effects but was more potent. The effects of dbc-AMP and CB could be due to interference with a common cellular structure, e.g. microfilaments.     

Effects of dibutyryl cyclic AMP and prostaglandin E1 on cultured human glioma cells

Dibutyryl cyclic AMP (db-cAMP) and prostaglandin E₁ (PGE₁) induced morphological alterations in cultured human glioma cells (138 MG). Cells in serum-free medium, treated with db-cAMP (1 mM) or PGE₁ (10μg/ml), within 1–3 h showed multiple thin processes resembling those of normal glial cells. These processes increased in size during a 24 h incubation. In serum-containing medium the appearance of cells with multiple processes was delayed. The induced morphological alterations were reversible upon exchange with fresh serum-containing but not with serum-deprived medium. Actinomycin D (5 μg/ml) did not prevent the changes induced by PGE₁ or db-cAMP. Inhibition of protein synthesis with cycloheximide (10 μg/ml) did not arrest the initial (1–3 h) changes in morphology but blocked further growth of the processes on prolonged incubation. Vinblastine sulphate (0.1 μg/ml) completely inhibited the alterations induced by PGE₁ or db-cAMP.     

Effects of prostaglandins F alpha on dog thyroid cyclic AMP level and function

Prostaglandins F1 alpha and F2 alpha, at high concentrations (greater than or equal to 28 microM) 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 alpha also inhibited TSH-induced secretion and activated iodide 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 alpha 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 alpha. 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 alpha or any other cyclooxygenase product.     

Effects of cyclic AMP on the kinetics of serum protein synthesis in cultured mouse hepatoma cells

The mouse hepatoma cell line, Hepa, was cultured in the presence of either 1 mM N⁶, O^2′-dibutyryl cyclic AMP (Bt₂cAMP), 0.5 mM 3-isobutyl-1-methylxanthine, or 1 μg/ml cholera toxin. The synthesis and secretion of albumin, α-fetoprotein, and transferrin were elevated above controls by 24 h reaching two- to fourfold stimulations within 72 h. These effects were reversible and were specific for the serum proteins. The stimulation of serum protein synthesis was accompanied by a decrease in the rate of cell proliferation. Protein synthetic parameters were analyzed in Hepa cells 72 h after exposure to N⁶,O^2′-dibutyryl cyclic AMP. The cellular rate of albumin synthesis was increased fourfold and the relative rate of albumin synthesis was increased approximately threefold. N⁶,O^2′-Dibutyryl cyclic AMP did not affect the size distribution of either total Hepa polyribosomes or of albumin-synthesizing polyribosomes. The elongation rate on total mRNA and on albumin mRNA was decreased by approximately 40%. These results indicate that the rate of initiation of total Hepa mRNA and of albumin mRNA also decreased by 40%. The nonspecific nature of the N⁶,O^2′-dibutyryl cyclic AMP effect on Hepa protein synthetic parameters must be due to an alteration in the level of a common substrate, perhaps ATP. The specific threefold increase in the relative rate of albumin synthesis with no alteration in polyribosome sizes requires a threefold increase in the relative amount of functional albumin mRNA in Hepa cells. This prediction was confirmed by cell-free translation of Hepa polyribosomes.     

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.     

Synthesis of prostaglandins and cyclic AMP by cultured embryonic palate mesenchyme at various population densities

During embryonic development, facial and palate mesenchymal cells exhibit differential growth rates. Normal palatal growth is regulated in part by hormones and growth factors. Because hormonal responsiveness of some cells correlates with their cell density, we have investigated the relationship between embryonic palate mesenchymal cell population density and their ability to synthesize prostaglandins (PGs) and cyclic AMP. Primary cultures of palate mesenchymal cells exhibited typical lag, log, and stationary phases of growth with a doubling time of 32-34 hrs. The ability of cells to produce PGE2 in response to a calcium ionophore (A23187), an activator of phospholipase A2 (melittin), arachidonic acid, or serum was maximal during the period of early exponential growth. Prostaglandin F2 alpha synthesis in response to A23187 or arachidonic acid showed a similar transient increase also corresponding temporally to the period of early exponential growth. The ability to synthesize PGF2 alpha in response to melittin, however, failed to diminish after early exponential growth. The pattern of cAMP synthesis in response to isoproterenol and PGE1 was different from that seen for induced prostaglandin synthesis. A transient increase in sensitivity to isoproterenol and PGE1 was seen that corresponded temporally to the period of late exponential growth just prior to attainment of confluency. Decreased sensitivity to stimulation of either prostaglandin or cAMP production as the cells became confluent was shown to be a density-dependent phenomenon; confluent cultures that were subcultured to reestablish logarithmic growth exhibited density-dependent hormonal responses identical to those seen in primary cultures. The ability of palate mesenchymal cells to synthesize both prostaglandins and cAMP, thought to be critical for proper palatal development, might thus be related to local differential craniofacial growth rates.     

Dibutyryl cyclic AMP decreases proliferative activity and the cholesteryl ester content in cultured cells of atherosclerotic human aorta

Dibutyryl cyclic AMP (DB cAMP) decreases by 2-5-fold the uptake of labeled thymidine into the intimal cells isolated from atherosclerotic human aorta. This effect depends on the DB cAMP concentration and the duration of its action. 0.1–1.0 mM concentration of DB cAMP reduces the level of cholesteryl esters in cells taken from atherosclerotic lesions by 1.5-2-fold. It is assumed that cAMP derivatives, and compounds which increase its intracellular content may impede the development of such atherosclerotic manifestations as smooth muscle cell proliferation and lipoidosis.     

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.     

Rapid axonal transport in vitro. Effects of derivatives of cyclic AMP and other agents acting on the cyclic AMP system

The effects of agents known to affect the cyclic AMP (cAMP) system in nervous tissue have been studied on the rapid axonal transport in vitro of [³H]leucine-labeled proteins in the frog sciatic nerve. The transport was inhibited by 3 different cAMP analogues; dibutyryl cAMP (1 mM), zeatin (0.5 mM), and zeatin riboside (0.5 mM), whereas another N⁶-substituted adenine derivative, N⁶-, isopentyl-adenine (DMA) (0.5 mM), and also dibutyryl cyclic GMP (1 mM), lacked effects. Two inhibitors of cAMP phosphodiesterases, papaverine and RO 207222, increased the level of cAMP in the nerve and arrested the transport. Papaverine was very potent and caused a reversible transport block at 0.05 mM. Adenosine (3 mM) increased the cAMP content about 16 times, much more than any of the other drugs tested, but only inhibited the transport by about 50%. Veratridine, a depolarizing agent, irreversibly blocked the transport at a low concentration (0.01 mM), which did not change the cAMP level. Transport inhibitory effects by another depolarizing substance, ouabain, and tricyclic psychotropic agent, chlorpromazine, have been described earlier. Ouabain (0.1 mM), in contrast to chlorpromazine (0.1 mM), caused a small increase in the cAMP content. The present results do not suggest the existence of a close relationship between rapid axonal transport and the cAMP system. Transport inhibitory effects due to disturbed energy metabolism will be discussed.     

Effect of hormones on cyclic AMP levels in cultured human cells.

Cultured cells derived from human adipose tissue grew more slowly and had significantly higher basal levels of cyclic AMP than cultured fibroblasts. Cyclic AMP levels in cultured adipose tissue cells were unaffected by epinephrine and were elevated 15-fold by prostaglandin E₁ while fibroblast cyclic AMP levels were elevated 27-fold by epinephrine and 95-fold by prostaglandin E₁. These results support the postulate that the cultured adipose tissue cell is a distinct cell type which may represent an adipocyte or preadipocyte in culture.     

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.     

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.