A common binding site for primary prostanoids in vascular smooth muscles: a definitive discrimination of the binding for thromboxane A2/prostaglandin H2 receptor agonist from its antagonist

Differences in binding characteristics between agonists and antagonists for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were examined in rat cultured vascular smooth muscle cells (VSMC). Scatchard analysis indicated the existence of two binding sites for the TXA2/PGH2 agonist, whereas a single class of recognition sites for the receptor antagonists were observed with approximately the same maximum binding capacity (Bmax) as a high-affinity binding site of the agonist. Weak binding inhibition by approx. 100 nM of primary prostanoids (PGE1, PGF2 alpha and PGD2) was detected only with the TXA2/PGH2 agonist, and not with the antagonist. Primary prostanoids as well as TXA2/PGH2 agonists (U46619 and STA2) suppressed the [3H]PGF2 alpha and [3H]PGE1 binding with almost the same potency, whereas TXA2/PGH2 antagonists (S-145, SQ29,548 and ONO3708) did not. The Bmax value of the binding sites was roughly identical in PGF2 alpha, PGE1 and a low-affinity binding site of U46619. These results suggest the existence of two binding sites for TXA2/PGH2 in VSMC, i.e., a high-affinity binding site corresponding to that of the TXA2/PGH2 antagonists and a low-affinity binding site in common with primary prostanoids.     

3H]prostaglandin F2 alpha membrane binding reexamined

Tritiated prostaglandin F2 alpha ([3H]PGF2 alpha) binding to bovine corpora luteal membranes has been reexamined from the viewpoint of eventual PGF2 alpha receptor purification. Several modifications of the literature on PGF2 alpha binding allow for a more stabilized [3H]PGF2 alpha PGF2 alpha receptor complex which should then facilitate the PGF2 alpha receptor purification. Of particular importance were: identification of protease inhibitors which protect [3H]PGF2 alpha binding and protease inhibitors which are detrimental to subsequent [3H]PGF2 alpha binding; the finding that EGTA treatment of tissue homogenates greatly protects subsequent [3H]PGF2 alpha binding; the observation that Mn(+)+ substitutes for Ca(+)+ and, in fact, among the divalent cations Mn(+)+ greater than Mg(+)+ greater than Ca(+)+ in facilitating [3H]PGF2 alpha binding where as Cd(+)+, Cu(+)+ and Zn(+)+ either have no effect or are detrimental to this binding; the lack of effect of ATP, GTP, GDP and cAMP or of kinase and phosphatase inhibitors and activators to alter binding of [3H]PGF2 alpha to isolated membranes; and the ease with which the [3H]PGF2 alpha-PGF2 alpha receptor complex can be removed from the membrane in spite of the receptor being an integral membrane protein. A new simple technique for separating protein bound [3H]PGF2 alpha (PGF2 alpha receptor-[3H]PGF2 alpha complexes) from free [3H]PGF2 alpha by use of hydroxyapatite (HAP) is introduced. This HAP method is of particular use in solubilized membrane preparations (but can also be used during PG radioimmunoassays to separate free PG from antibody bound PG). These changes were required to facilitate subsequent chromatographic steps leading to identification and purification of the PGF2 alpha receptor. (ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells

We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.     

High-affinity prostaglandin E receptors attenuate adenylyl cyclase activity in isolated bovine myometrial membrane

Purified bovine myometrial plasma membranes were used to characterize prostaglandin (PG) E2 binding. Two binding sites were found: a high-affinity site with a dissociation constant (KD) of 0.27 +/- 0.08 nM and maximum binding (Bmax) of 102.46 +/- 8.6 fmol/mg membrane protein, and a lower affinity site with a KD = 6.13 +/- 0.50 nM and Bmax = 467.93 +/- 51.63 fmol/mg membrane protein. Membrane characterization demonstrated that [3H]PGE2 binding was localized in the plasma membrane. In binding competition experiments, unlabelled PGE1 displaced [3H]PGE2 from its receptor at the same concentrations as did PGE2. Neither PGF2 alpha nor PGD2 effectively competed for [3H]PGE2 binding. Adenylyl cyclase activity was inhibited at concentrations of PGE2 that occupy the high-affinity receptor. These data demonstrate that two receptor sites, or states of binding within a single receptor, are present for PGE2 in purified myometrial membranes. PGE2 inhibition of adenylyl cyclase activity support the view that cAMP has a physiological role in the regulation of myometrial contractility by PGE2.     

Phorbol ester stimulates cyclooxygenase-2 expression and prostanoid production in cardiac myocytes

Phorbol-12-myristate- 13-acetate (PMA) has been shown to induce hypertrophy of cardiac myocytes. The prostaglandin endoperoxide H synthase isoform 2 (cyclooxygenase-2, COX-2) has been associated with enhanced growth and/or proliferation of several types of cells. Thus we studied whether PMA induces COX-2 and prostanoid products PGE(2) and PGF(2alpha) in neonatal ventricular myocytes and whether endogenous COX-2 products participate in their growth. In addition, we examined whether PMA affects interleukin-1beta (IL-1beta) stimulation of COX-2 and PGE(2) production. PMA (0.1 micromol/l) stimulated growth, as indicated by a 1.6-fold increase in [(3)H]leucine incorporation. PMA increased COX-2 protein levels 2. 8-fold, PGE(2) 3.7-fold, and PGF(2alpha) 2.9-fold. Inhibition of either p38 kinase or protein kinase C (PKC) prevented PMA-stimulated COX-2. Inhibition of COX-2 with either indomethacin or NS-398 had no effect on PMA-stimulated [(3)H]leucine incorporation. Exogenous administration of PGF(2alpha), but not PGE(2), stimulated protein synthesis. Treatment with IL-1beta (5 ng/ml) increased COX-2 protein levels 42-fold, whereas cotreatment with IL-1beta and PMA stimulated COX-2 protein only 32-fold. IL-1beta did not affect control or PMA-stimulated protein synthesis. These findings indicate that: 1) PMA, acting through PKC and p38 kinase, enhances COX-2 expression, but chronic treatment with PMA partially inhibits IL-1beta stimulation of COX-2; and 2) exogenous PGF(2alpha) is involved in neonatal ventricular myocyte growth but endogenous COX-2 products are not.     

Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells

Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum-free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10(-7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.     

Prostaglandin E1 binds to Z protein of rat liver

Z protein or fatty-acid-binding protein is abundant in the cytosol of many cell types including liver cells. It is considered to play an important role in intracellular transport and metabolism of long-chain fatty acids and other organic anions. We studied the role of Z protein in the metabolism of prostaglandin E1 (PGE1). Binding of tritiated prostaglandin E1 to this fatty-acid-binding protein (Z protein) purified from rat liver was determined. The binding of [3H]prostaglandin E1 to Z protein is rapid, saturable and reversible. Scatchard analysis of [3H]PGE1 binding to Z protein showed a single class of binding sites with a dissociation constant (Kd) of 37 nM. The binding capacity is 110 nmol/mg Z protein. Optimal [3H]PGE1 binding occurred at pH 7.4. The presence of 3 mM MgCl2 stimulated the prostaglandin E1 binding to Z protein. Competition experiments show that the binding of this autacoid to Z protein is highly specific. It could not be displaced by other prostaglandins (PGA1, PGA2, PGE2, PGB1, PGI2, PGD2, PGF2 alpha, and 6-keto PGF1 alpha). Z protein might be involved in the metabolism of prostaglandins in the cytosol.     

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.     

Effects of prostaglandin E2 and F2 alpha on cytoplasmic pH in a clonal osteoblast-like cell line, MOB 3-4.

Prostaglandin E2 (PGE2, 5 ng/ml to 5 micrograms/ml) induced a dose-dependent increase in cAMP accumulation, inositol phosphates (IPs) accumulation, and cytoplasmic free Ca2+ ([Ca2+]i) in a clonal osteoblast-like cell line, MOB 3-4. In contrast, prostaglandin F2 alpha (PGF2 alpha, 5 ng/ml to 5 micrograms/ml) stimulated increases in IPs accumulation and [Ca2+]i without stimulating an increase in cAMP accumulation. Both PGE2 (greater than 0.5 micrograms/ml) and PGF2 alpha (greater than or equal to 5 micrograms/ml) increased cytoplasmic pH (pHi) from approximately 7.15 to 7.35 in BCECF-loaded cells. A tumor promotor, phorbol 12-myristate 13-acetate (PMA, 0.1-100 nM) also increased pHi without effect on phosphoinositide hydrolysis. Both PGE2-(5 micrograms/ml) and PMA- (100 nM) induced cytoplasmic alkalinization was inhibited by removal of extracellular Na+, or by pretreatment of the cells with amiloride (0.5 mM), an inhibitor of Na+/H+ exchange, or H-7 (100 microM), a nonspecific inhibitor of protein kinase C. Thus, MOB 3-4 cells appeared to possess PGE2 receptors and PGF2 alpha receptors: the former are coupled to adenylate cyclase and phospholipase C, and the latter are predominantly coupled to phospholipase C. Also the cells appeared to possess an amiloride-sensitive Na+/H+ exchange activity, which increases pHi in response to PGE2 and PGF2 alpha, as well as to PMA. Long-term (48 hr) exposure of the cells to PGE2 at a high concentration (5 micrograms/ml), but not to PGF2 alpha and PMA, decreased DNA synthesis in the serum-deficient medium. Thus, cytoplasmic alkalinization appeared insufficient for cell replication. At least in MOB 3-4 cells, the inhibitory effect of PGE2 on DNA synthesis may be due to the cAMP messenger system.     

Prostaglandin moieties that determine receptor binding specificity in the bovine corpus luteum.

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.     

Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets.

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.     

Astrocytes possess prostaglandin F2 alpha receptors coupled to phospholipase C.

We examined the effect of prostaglandin (PG) F2 alpha on phosphoinositide (PI) hydrolysis in rat cultured astrocytes. PGF2 alpha stimulated the formation of [3H]inositol phosphates in [3H]inositol-labeled astrocytes with the ED50 value of 23 nM, whereas PGD2 and PGE2 were much less effective than PGF2 alpha. Transformation of astrocytes was accompanied by an increase in the stimulatory response of PGF2 alpha. Pretreatment of the astrocytes with pertussis toxin and cholera toxin did not affect the PGF2 alpha-evoked PI hydrolysis. In the digitonin-permeabilized astrocytes, PGF2 alpha significantly enhanced the GTP gamma S-evoked PI hydrolysis in the presence of Ca2+. These results indicate that rat cultured astrocytes possess PGF2 alpha receptors coupled to phospholipase C.     

Inhibition of (3H) prostaglandin F2alpha binding to its receptors by progesterone.

The specific binding of [3H] prostaglandin (PG) F2alpha to bovine corpus luteum cell membranes was inhibited by progesterone. Progesterone inhibition of binding was dependent on membrane protein and independent of [3H] PGF2alpha concentrations in the medium. The lower inhibition of binding at high protein concentrations can be overcome by increasing the amounts of progesterone added. Progesterone inhibition of binding appears to be due to a decrease in the receptor number rather than a decrease in the receptor affinities. The kinetic properties (association and dissociation rates) of the remaining receptors were unchanged. The inhibition of [3H] PGF2alpha binding was observed by preincubating the membranes with progesterone or by adding at the beginning but not during incubation. The concentrations of progesterone that inhibited binding by about 50% do occur in bovine corpora lutea of estrous cycle and pregnancy.     

Endothelin-1 stimulates phospholipid hydrolysis and prostaglandin F2α production in primary human decidua cell cultures

The regulation of prostaglandin (PG) production by the uterine decidua may be an important mechanism controlling the onset and maintenance of human parturition. The present in vitro study has evaluated the potential for endothelin-1 (ET-1) to activate cell signalling and PGE2 alpha production in human primary decidua cell cultures. ET-1 stimulated a dose-dependent increase in inositol phospholipid hydrolysis and PG precursor release as evidenced by respective increases in [3H] inositol monophosphate accumulation and [14C] arachidonate release from radiolabelled decidua cells. PGF2 alpha production was increased in some but not all cell preparations in response to ET-1 alone. Pretreatment of decidua cells with interleukin-1 beta (IL-1 beta) enhanced PGF2 alpha production but not arachidonate release in response to ET-1. These in vitro observations support a possible role for ET-1 in the regulation of decidual PG production during parturition.     

Gene expression of arachidonate cyclooxygenase pathway leading to the delayed synthesis of prostaglandins E2 and F2alpha in response to phorbol 12-myristate 13-acetate and action of these prostanoids during life cycle of adipocytes

Several types of prostaglandin (PG)s are synthesized in adipocytes and involved differently in the control of adipogenesis. To elucidate how the PG synthesis is regulated at different stages in the life cycle of adipocytes, we examined the gene expression of arachidonate cyclooxygenase (COX) pathway leading to the delayed synthesis of PGE2 and PGF2alpha and their roles in adipogenesis after exposure of cultured cells to phorbol 12-myristate 13-acetate (PMA), which is a useful system for monitoring mitogen-induced changes. While the expression of COX-1 remained constitutive, mRNA and protein levels of COX-2 were up-regulated by treatment with PMA. Preadipocytes exhibited higher gene expression of cytosolic phospholipase A2alpha (cPLA2alpha) and PGF synthase. In contrast, three isoforms of PGE synthase are expressed constitutively during all phases. The delayed synthesis of PGE2 and PGF2alpha following the stimulation for 24 with a mixture of PMA and calcium ionophore A23187 was the highest in preadipocytes, reflecting the increased expression levels of cPLA2alpha and COX-2. Cultured cells treated with PMA during the differentiation phase and then exposed to the maturation medium, or cells treated with PMA in the maturation medium after the differentiation phase showed the suppression of adipogenesis in adipocytes. The attenuating effect of PMA was additionally enhanced when the cell were treated along with A32187 during the differentiation phase, suggesting the involvement of endogenous PGs. The cells at the stages of the differentiation and maturation phases were highly sensitive to exogenous PGE2 and PGF2alpha, respectively, resulting in the marked suppression of the stored fats in adipocytes. Taken together, these results provided the evidence for the distinct gene expression of isoformic enzymes in the COX pathway leading to the synthesis of PGE2 and PGF2alpha and the specific action of these prostanoids at different cycle stages of adipocytes.     

Requirement for prostaglandin F2 alpha in 17 beta-estradiol stimulation of DNA synthesis in rabbit endometrial cultures

We have hypothesized that two of the endogenously synthesized endometrial prostaglandins (PGs), prostaglandin F2 alpha (PGF2 alpha), and prostaglandin E1 (PGE1), play a regulatory role in growth control of the rabbit endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used to examine the possible role of these PGs in the mechanism of action of 17 beta-estradiol on DNA synthesis. Towards this end, binding, second messenger and DNA synthesis experiments were performed. 17 beta-estradiol stimulation resulted in a time dependent (optimal: approximately 6 h) and 17 beta-estradiol concentration dependent (optimal: approximately 10(-7) M 17 beta-estradiol in phenol red-containing medium) increase in [3H]PGF2 alpha binding. Scatchard type analysis of the binding data revealed an increase in receptor number while the receptor affinity for [3H]PGF2 alpha remained the same as in the control treated cultures. This 17 beta-estradiol stimulated increase in PGF2 alpha receptor allowed a suboptimal concentration of PGF2 alpha (10(-9) M) to increase intracellular levels of inositol polyphosphates, while by itself this concentration of PGF2 alpha caused no significant change in intracellular inositol polyphosphate levels. 17 beta-estradiol, alone among the several studied steroid hormones, could increase [3H]PGF2 alpha binding. Proliferation studies revealed that, in these primary cultures of rabbit endometrium, 17 beta-estradiol could increase DNA synthesis but not in the presence of indomethacin, unless PGF2 alpha was added to the medium at a concentration (10(-10) M) near or above what is normally accumulated in the medium by these cultures. In the absence of 17 beta-estradiol stimulation, addition of these same low concentrations of PGF2 alpha had no effect on DNA synthesis. Apparently, through its effect on the PGF2 alpha receptor, 17 beta-estradiol enhances the PGF2 alpha stimulated DNA synthesis response approximately 100 fold. The DNA synthesis induced by 17 beta-estradiol can be inhibited by PGE1, as can PGF2 alpha-induced DNA synthesis. We propose that 17 beta-estradiol may be mediating its mitogenic effect through an alteration of the prostaglandin agonist:antagonist control of proliferation in rabbit endometrial cultures. In addition we suggest that, if 17 beta-estradiol acts to increase PGF2 alpha, receptors as part of its mode of action, this may be of importance in other tissues possessing both prostaglandin and 17 beta-estradiol receptors.     

Activity of phospholipase C in ovine luteal tissue in response to PGF2 alpha, PGE2 and luteinizing hormone.

Four ewes were utilized to determine the effects of prostaglandin (PG) F2 alpha, PGE2 and luteinizing hormone (LH) on activity of phospholipase C (PLC) in ovine luteal tissue. Corpora lutea were collected on d 10 post-estrus and six slices from one corpus luteum from each ewe were pre-incubated with [3H]-inositol prior to incubation with one of 6 treatments. Treatments were 1) control, 2) PGF2 alpha (100 ng/ml), 3) PGE2 (10 ng/ml), 4) LH (10 ng/ml), 5) PGF2 alpha + PGE2 and 6) PGF2 alpha + LH. Phospholipase C was determined indirectly by measuring the accumulation of [3H]-inositol mono-, bis- and tris-phosphates (IP, IP2, IP3). Effects of PGF2 alpha (0 vs. PGF2 alpha) and luteotropic treatment (0 vs. PGE2 vs. LH) and their interactions were determined by analysis of variance. There was a significant main effect of PGF2 alpha (P less than 0.01) as concentrations of IP, IP2, IP3 and total [3H]-inositol phosphates were greater in tissue slices treated with PGF2 alpha, regardless of luteotropic treatment. Within groups receiving no PGF2 alpha (1,3,4), no effect of luteotropic treatment was observed. Within groups receiving PGF2 alpha (2,5,6), LH caused a significant (P less than .05) increase in the accumulation of total [3H]-inositol phosphates. Thus, PGF2 alpha can stimulate the activity of PLC in ovine luteal tissue and LH can potentiate this effect.     

Solubilization and Characterization of Prostaglandin E2 Binding Protein from Porcine Cerebral Cortex

The specific binding protein for prostaglandin (PG) E2 was solubilized in an active form from the crude mitochondrial (P2) fraction of porcine cerebral cortex. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 4 degree C for 30 min, the PGE2 binding to the supernatant fraction (103,000 g, 60 min) was determined by the polyethylene glycol method. The maximum yield (approximately 30% of the binding activity to the P2 fraction) was obtained with 10 mM CHAPS. The specific [3H]PGE2 binding to the solubilized fraction was time-dependent and the equilibrium was reached at around 60 min at 37 degrees C. By dilution of the reaction mixture, the binding site-[3H]PGE2 complex formed after 5-min incubation slowly dissociated, whereas that formed after 60-min incubation did not dissociate to a significant extent. The binding was highly specific for PGE2 and inhibited by unlabeled PGs in the following order: PGE2 greater than PGE1 much greater than PGF2 alpha greater than PGE2 methyl ester greater than PGA2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGD2. Scatchard analyses of the solubilized fraction suggested the presence of high- and low-affinity sites. Heat treatment and preincubation with trypsin or proteinase K markedly reduced the binding. The binding activity was eluted in a single peak both from gel filtration and from ion-exchange columns using HPLC. These results suggest that a specific protein solubilized may be responsible for the binding site.     

The existence of distinct classes of prostaglandin E2 receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-E1.

We have reported previously that PGE2 evoked an increase in intracellular calcium level ([Ca2+]i) in mouse osteoblastic cells (1). Here, we investigated the effects of PGE1 and PGF2 alpha on cAMP production and [Ca2+]i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but had no effect on [Ca2+]i, whereas PGF2 alpha evoked only [Ca2+]i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+]i. From the Scatchard plot analysis of PGE2 it was confirmed that there were two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein). As the increase in [Ca2+]i was caused by PGF2 alpha and PGE2, but not by PGE1, we investigated the displacement of [3H]-PGF2 alpha binding. The displacement capacity of unlabeled PGE2 was about 110 of that of PGF2 alpha, while that of PGE1 was very low even at 500-fold excess. These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems.     

Protein kinase C modulates effects of prostanoids on cyclic adenosine monophosphate in guinea pig chief cells

In the course of examining the role of protein kinase C in signal transduction in dispersed chief cells from guinea pig stomach, we observed that phorbol esters inhibit prostaglandin (PG)-stimulated increases in cyclic adenosine monophosphate (cAMP). Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, decreased maximal levels of PGE2-stimulated cAMP by 40%. This dose-dependent effect was observed within 30 sec and was maximal by 1 min of incubation at 37 degrees C. Phorbols that do not activate protein kinase C did not have this effect. Adding H7, a protein kinase C inhibitor, abolished the inhibitory effects of PMA, indicating that these effects are not caused by activation of cyclic nucleotide phosphodiesterases. PMA did not alter the increase in cellular cAMP caused by cholera toxin, forskolin, secretin, or vasoactive intestinal peptide. Hence the site of these prostanoid-specific actions of protein kinase C does not appear to be stimulatory or inhibitory guanine nucleotide binding proteins or the catalytic component of the adenylyl cyclase system. In dispersed chief cells, activation of protein kinase C may inhibit prostanoid-induced stimulation of the adenylyl cyclase system by a direct effect on prostaglandin receptors.