In vitro action of PG F2 alpha on progesterone and cAMP synthesis in small bovine luteal cells

The action of prostaglandin F2 alpha (PG F2 alpha) on incubated small bovine luteal cells in the presence or in the absence of bovine luteinizing hormone (LH) or dibutyryl cyclic adenosine monophosphate (db cAMP) was investigated. In the absence of LH and db cAMP, PG F2 alpha stimulated progesterone synthesis at concentrations of 10 ng/ml and 100 ng/ml but had no effects at concentrations below 1 ng/ml. PG F2 alpha partially inhibited the LH or db cAMP stimulated progesterone synthesis. This inhibition was maximal for PG F2 alpha concentrations around 100 pg/ml whereas distinctly higher or lower concentrations were without effect. At the concentration of 100 pg/ml, PG F2 alpha partially inhibited the LH induced cAMP accumulation. These results demonstrate an "in vitro" action of PG F2 alpha on bovine luteal cells. They indicate that the luteolytic action of PG F2 alpha in the bovine species could involve, as already suggested for the rat, both an inhibition of the LH induced synthesis of cAMP and an inhibition of the action of cAMP.     

Prostaglandin production in cultured gastric mucosal cells: role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE2, PGI2 and thromboxaneA2 (TXA2) in amounts of 316 +/- 18, 100 +/- 7 and 30 +/- 5 pg per 10(5) cells in 10 min, respectively, in response to 10 microM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1-5 mM) did not alter the amount of subsequent AA-induced PGE2 production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE2 synthesis, while it increased intracellular cAMP accumulation. Our studies suggest AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, AA-induced PG production is limited in these cells, and it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

Prostaglandin production in cultured gastric mucosal cells: Role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE₂, PGI₂ and thromboxaneA₂ (TXA₂) in amounts of 316±18, 100±7 and 30±5 pg per 10⁵ cells in 10 min, respectively, in response to 10μM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1–5mM) did not alter the amount of subsequent AA-induced PGE₂ production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE₂ synthesis, while it increased intracellular cAMP accumulation. Our studies suggest (1) AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, (2) AA-induced PG production is limited in these cells, and (3) it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

Mediation of macrophage collagenase production by 3'-5' cyclic adenosine monophosphate

The production of collagenase by lipopolysaccharide-(LPS) activated guinea pig macrophages is mediated by prostaglandins (PG) of the E series. After stimulation of guinea pig macrophages with LPS, extracellular PGE levels and cellular cAMP levels are elevated. Indomethacin inhibits not only PG synthesis, but also cAMP and collagenase production in LPS-stimulated macrophage cultures. In these indomethacin-inhibited cultures containing LPS, dibutyryl (dB) cAMP, or cholera toxin can restore macrophage collagenase production but not PG synthesis. Moreover, dBcAMP and cholera toxin enhance collagenase production in LPS-activated cultures. Initial activation of the macrophages by an agent such as LPS is a prerequisite for synthesis of collagenase, since in the absence of LPS, dBcAMP or cholera toxin alone are ineffective stimuli. These findings clearly demonstrate a role for PG-induced elevations of cAMP in the production of collagenase by LPS-activated macrophages.     

Regulation of proteoglycan and hyaluronan synthesis by elevated level of intracellular cyclic adenosine monophosphate in peritubular cells from immature rat testis

The effects of an increase in intracellular cAMP concentration on proteoglycan (PG) synthesis by peritubular (PT) cells from immature rat testis were investigated. In the presence of dBcAMP for 72 h, the [³H]-hexosamine incorporation in secreted PG and in cellassociated PG was reduced, whereas [³⁵S]-sulfate radioactivity was enhanced in secreted PG and not affected in cell-associated PG. Cholera toxin and IBMX, known to generate high intracellular cAMP levels, induced similar changes. Cyclic AMP did not alter PG protein moiety synthesis but enhanced PG turnover. Cholera toxin and dBcAMP profoundly modified PG characteristics: (1) Apparent molecular weight of PG was increased. (2) This was due to an increase in glycosaminoglycans (heparan sulfate (HS) and chondroitin sulfate (CS)) length. (3) The number of glycosaminoglycan chains was presumably reduced. (4) Heparan sulfate and chondroitin sulfate chains of medium and cell layer-associated PG appeared oversulfated. (5) The pattern of cell layer associated PG was modified with a decrease in HSPG and a correlative increase in CSPG. Cholera toxin and dBcAMP also dramatically stimulated hyaluronan synthesis by possible phosphorylation induced activation of hyaluronan synthase(s).     

Tumor necrosis factor stimulates prostaglandin production and cyclic AMP levels in rat cultured mesangial cells

Human recombinant tumor necrosis factor-alpha (TNF) was found to stimulate the production of prostaglandins (PG) by cultured rat mesangial cells. This effect was demonstrable from 6 h, was dose dependent and affected the synthesis of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. It required both RNA and protein synthesis but was not associated with a modification of cell proliferation. TNF also stimulated adenosine 3'-5' cyclic monophosphate (cAMP) levels in the mesangial cell culture medium. Indomethacin suppressed the effect of TNF on PGs but only reduced that on cAMP, indicating that PG production partly mediates the increase in cAMP. These findings demonstrate that mesangial cells can be a target for TNF and that the mechanism of TNF action includes stimulation of both PG production and cAMP levels.     

Formation of cyclic adenosine monophosphate (cAMP) in the preovulatory rabbit follicle: role of prostaglandins and steroids

The preovulatory increase in follicular prostaglandins (PG) stimulated by luteinizing hormone (LH) is dependent upon 3'-5'-cyclic adenosine monophosphate (cAMP) and is essential for ovulation. It has been proposed that follicular PG stimulate a second rise in cAMP, independent of LH. This study examined the temporal relationships among PGE2, PGF2 alpha 6-keto-PGF1 alpha, estradiol-17 beta, progesterone, testosterone, androstenedione and the biphasic increases of cAMP in follicles of rabbits. Does received indomethacin (IN, 20 mg/kg, i.v.; n = 30) or phosphate buffer (C; n = 30), 0.5 h before 50 ug of LH. At laparotomy at 0, 0.5, 1, 2, 4 or 8 h after LH, blood was collected from each ovarian vein and two follicles per ovary were aspirated of fluid and excised. Plasma and follicular tissue and fluid were assayed for PG and steroids. Tissue and fluid were assayed for cAMP. In C does, cAMP (pmol/follicle) in tissue increased from 11.3 at 0 h to 14.2 at 0.5 h, decreased at 1 h (5.4) and increased linearly through 8 h to 14.5. In IN-treated does, cAMP remained high from 0.5 (13.2) to 2 h (16.3), decreased at 4 h (7.9) then increased again by 8 h (15.5). Indomethacin decreased all PG in follicular tissue but 6-keto-PGF1 alpha rose after 2 h, whereas PGE2 and PGF2 alpha did not. Estradiol-17 beta, progesterone, and androstenedione did not vary with treatment; testosterone was increased (P less than .05) by IN. PGE2 or PGF2 alpha may terminate the first phase of cAMP production, rather than initiate the second phase.     

The Bombyx mori sex pheromone biosynthetic pathway is not mediated by cAMP

In most moths, sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN). How the extracellular PBAN signal is turned into a biological response has been the focus of numerous studies. In the classical scheme of signal transduction, activated G proteins relay the extracellular signal to downstream effector molecules such as calcium channels and adenylyl cyclase. The role of calcium in PBAN signaling has been clearly demonstrated, but the possible involvement of cAMP is not as straightforward. While cAMP has been shown to be necessary for PBAN signaling in most heliothine species, there has been no definitive demonstration of its role in Bombyx mori. To address this question, we used degenerate RT-PCR to clone two Gs subunits, designated P50Gs1 and P50Gs2, from B. mori pheromone gland (PG) cDNAs. The two Gs proteins were expressed in all tissues examined and were not up-regulated in accordance with adult eclosion. Even though two bands corresponding to the approximate molecular weights of P50Gs1 and P50Gs2 were detected in PG homogenates, the Gs antagonist, NF449, had no effect on sex pheromone production. Furthermore, no changes in the intracellular cAMP levels were detected following PBAN stimulation.     

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.     

The role of various prostaglandins on the correlation between permeability to albumin and cAMP levels in the isolated mesentery.

Prostaglandins (PG) have been shown to raise the level of cyclic AMP (cAMP) in various tissues, and to increase permeability. Whether both events are linked, is at present a matter of speculation. We have investigated the effects of PGE1, E2, A1, A2, F1alpha and F2alpha on an isolated rat mesentery placed in a diffusion cell (surface area : 2 sq.cm). The PGs (5 microgram/ml) increased the passage of (I 125) - Albumin across the mesentery. In other experiments, diks of rat mesentery (surface area : 2 sq.cm) have been incubated in assay tubes, and cAMP levels measured by a binding protein assay. We have observed an excellent correlation between increases in permeability and cAMP levels (r=0.961). In order of increasing potency on both parameters, the PGs may be classified as follows : PGF, PGA and PGE. In the rat mesentery, under the influence of prostaglandins, increases in permeability and in cAMP levels are apparently connected.     

The role of various prostaglandins on the correlation between permeability to albumin and cAMP levels in the isolated mesentery

Prostaglandins (PG) have been shown to raise the level of cyclic AMP (cAMP) in various tissues, and to increase permeability. Whether both events are linked, is at present a matter of speculation. We have investigated the effects of PGE1, E2, A1, A2, F1α and F2α on an isolated rat mesentery placed in a diffusion cell (surface area : 2 sq.cm). The PGs (5 μg/ml) increased the passage of (I 125)-Albumin across the mesentery.In other experiments, diks of rat mesentery (surface area : 2 sq.cm) have been incubated in assay tubes, and cAMP levels measured by a binding protein assay. We have observed an excellent correlation between increases in permeability and cAMP levels (r=0.961). In order of increasing potency on both parameters, the PGs may be classified as follows : PGF, PGA and PGE.In the rat mesentery, under the influence of prostaglandins, increases in permeability and in cAMP levels are apparently connected.     

The role of cyclic nucleotides and prostaglandins in heart function.

A short review of the role of cyclic nucleotides and prostaglandins (PGs) in normal and pathological functions of the heart is given. Possible interrelationships of these two regulatory systems have been studied by using spontaneously beating rat atria preparations. Addition of noradrenaline (NA) to the incubate (1 . 10(-6) M) caused an increase in amplitude and frequency which was preceded and parallelled by an elevation of the tissue cAMP level. A transient increase in cGMP and PGE values was also seen. Propranolol (5 . 10(-6) M) abolished the increase in amplitude and frequency as well as in cAMP and PGE concentrations. Indomethacin (1 . 10(-5) M) inhibited the formation of PGE. The increase in cGMP was blocked by phenoxybenzamine. Interchange between beta- and alpha-receptors according as the temperature is lowered has been described earlier. Hypothermia (20 degrees C) had a positive inotropic effect on the atria and increased the tissue cAMP concentration. Loading of the atria caused an increase in cAMP without any effects on cGMP or PGs. Slight hypoxia did not change the cAMP or PG levels, but elevated the cGMP values. Arrhythmias induced by hypo- or hyperpotassemia did not modify the biochemical parameters measured. PGF2alpha (1. 10(-5) M) normalized the atrial rhythm and increased the amplitude without changing cyclic nucleotide or PG levels. PGE1 (1 . 10(-4) M) increased the amplitude of normorhythmic atria and the tissue concentration of cAMP. PGE2 was the only PG tested which stimulated the heart adenylate cyclase in vitro. There seems to be close but complicated relationships between cyclic nucleotides and PGs in the heart.     

Prostaglandins and cyclic nucleotides in the dynamic development of an experimental poisoning syndrome caused by Yersinia pestis lipopolysaccharide

This work deals with the influence of Y. pestis lipopolysaccharide (LPS), introduced intraperitoneally in a dose of 2 LD50, on the content of prostaglandins (PG), such as PGE, PGF2 alpha and 6-keto-PGF1 alpha, thromboxane, cAMP and cGMP in the liver, lungs and blood plasma of guinea pigs in the process of the development of experimental intoxication. The content of thromboxane in blood plasma increased 2.4-fold in 2 hours after intoxication and remained elevated for as long as 5 hours. Other parameters of blood plasma remained unchanged. The data obtained in this investigation indicate that thromboxane, known as a regulator of thrombogenesis, may induce early disturbances in microcirculation. A change in the content of PG was shown to occur in pulmonary tissue 2 and 5 hours after the beginning of intoxication. The content of PG in liver tissue was found to occur at a later period of the toxic action. The concentration of cyclic nucleotides (CN) in the tissues under study sharply increased even at the initial stage of the development of shock in guinea pigs. The effect of LPS on the metabolism of PG and CN, revealed in this investigation, resembles the effect produced by the thermostable fraction of "mouse" toxin.     

Stimulation of cAMP formation by prostaglandin D2 in primary cultures of bovine adrenal medullary cells: identification of the responsive population as fibroblasts

In primary cultures of bovine adrenal medulla, chromaffin cells responded to prostaglandin (PG) E2 by stimulating phosphoinositide metabolism (Yokohama et al. (1988) J. Biol. Chem. 263, 1119-1122). In contrast, nonchromaffin cells were found to respond to PGD2 by elevating their intracellular cAMP level. The formation of cAMP was detected at as low as 0.1 nM PGD2 and increased more than 100-fold over the basal level at 0.1 microM, and the response was specific for PGD2 (greater than PGE1 greater than PGE2 greater than PGF2 alpha = PGI2). The magnitude of cAMP formation and its specificity to PGD2 were retained throughout a 40-day culture period. Based on the inhibitory effect of cis-4-hydroxy-L-proline, an inhibitor of collagen synthesis, on cAMP formation, morphology, and immunoreactivity of cells to anti-collagen type I antiserum, the responsive cells were identified as fibroblasts. These results taken together demonstrate that the adrenal medulla is composed of chromaffin and nonchromaffin cells, which respond to PGE2 and PGD2, respectively, by two different signal transduction pathways. The cAMP formation by PGD2 was also observed in fibroblasts from bovine embryonic trachea among cell lines tested, suggesting that some populations of fibroblasts responsive to PGD2 exist in various tissues and may discriminate the signal from that of PGE1 or PGE2.     

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.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.     

The embryo influences adenylate cyclase activity and hormonal response in rabbit myometrium during early pregnancy

The effect of pseudopregnancy (PSPG; days: 0 (estrus), 1, 6.5, 9 and 15) and pregnancy (PG; days: 6.5, 9 and 15) on adenylate cyclase (AC) activity was verified in rabbit myometrium. During PSPG, there was a time related decline in basal activity from 71 +/- 16.2 (D 0) to 13.1 +/- 1.6 (PSPG-D9) pmoles cAMP formed/mg prot-min. Stimulation of the enzyme by GTP, Isoproterenol (ISO), Prostaglandin E2 (PGE2) or Sodium Fluoride (NaF) followed a similar pattern. AC activity was compared in myometrial tissues of pregnant animals (PG) separated into embryonic (ES) and interembryonic (IES) sites. On days 6.5 and 9, AC activity measured in tissues from PG rabbits (ES and IES) was always higher than that found in tissues from PSPG animals on corresponding days. On day 6.5, AC activity was slightly higher (p less than 0.01) in ES than in IES. This was confirmed on day 9 where basal as well as GTP, ISO and PGE2 stimulated activities were higher in ES than in IES (p less than 0.001). Dose response to ISO, expressed as % of GTP, were similar on D0, 1, 6.5 and 15 of PSPG. However, on day 9, there was a striking diminution in response reflected by a lower stimulation at suboptimal dose (0.1 microM; p less than 0.05) from 115 +/- 2 on day 0 to 104 +/- 4 on day 9. These results suggest that protein content which is increased during pseudopregnancy could be responsible for the decline of AC activity. However, results obtained on day 9 and 15 suggest that other factors are involved. Dose responses to ISO during PG showed an alteration in response on days 6.5 and 9 at ES. It was reflected by a higher stimulation at suboptimal (0.1 microM) and optimal doses (100 microM). These results suggest that myometrial AC activity could be regulated by the presence of the embryo.     

Role of proteoglycans on testosterone synthesis by purified Leydig cells from immature and mature rats.

In order to characterize an involvement of proteoglycans (PG) in the regulation of Leydig cell function, we have examined the effects of para-nitrophenyl-beta-D-xyloside (PNPX), a specific inhibitor of PG synthesis and para-nitrophenyl-beta-D-galactoside (PNPG), an inefficient structural analogue, on testosterone production by purified Leydig cells from immature and mature rats, in the presence or not of various concentrations of hCG during 24 h. Whatever the age, the addition of PNPX induces a decrease of [35S] and [3H] incorporations into cell layer associated-PG; these latter being less numerous (-50 and -25%, respectively in immature and mature rat), and less sulfated (-40%) when compared to control Leydig cells. In immature Leydig cells, the inhibition of PG synthesis decreases both the basal and weakly stimulable-hCG or -(Bu)2cAMP or -LH testosterone synthesis. In mature Leydig cells, the PG inhibition has no effect on testosterone production both in the absence of hCG and in the presence of weak amounts of hCG but increases it in the presence of subsaturating hCG concentrations. Whatever the age, the inhibition of PG synthesis is ineffective in the presence of saturating amounts of either hCG or (Bu)2cAMP. These effects are maintained in the presence of MIX, PMA, but are not observed in the presence of 22R-hydroxycholesterol. Therefore, our results suggest that in rat Leydig cells, the inhibition of PG synthesis affects the signal transduction at a step distal to cyclic AMP and more precisely, the cholesterol supply to the mitochondria by acting on its cellular distribution (free and esterified cholesterol).     

Influence of prostaglandins and of cyclic nucleotides on the metabolism of 25-hydroxyvitamin D3 in primary chick kidney cell culture

Prostaglandins (PG) are likely to be involved in a number of regulatory mechanisms in the kidney, which may be mediated by cyclic nucleotides. The present study describes the effects of prostaglandins and cyclic nucleotides on the hydroxylases of 25-hydroxycholecalciferol (25(OH)D₃) in a primary chick kidney cell culture. 3–30 nM PG E₂ produced significant increases in the 25(OH)D₃-1-hydroxylase associated with decreases in the 25(OH)D₃-24-hydroxylase at 6 hours but not at 1 hour. PG F_2α, in concentrations between 0.3 nM and 3 μM affected the hydroxylases in a similar manner. A significant increase of 1-hydroxylase activity was observed with 0.1 mM cyclic AMP (cAMP) or dibutyryl cyclic AMP (dbcAMP) in 6 hours, but again no effect on either hydroxylase was observed when the incubation time was reduced to 1 hour. These results suggest that PG E₂ and PG F_2α might be involved in the regulation of renal 25(OH)D₃ metabolism, and that the effects on the 25(OH)D₃-hydroxylases might be mediated by cAMP.