Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca²⁺]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca²⁺]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca²⁺ probe. Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca²⁺]i in both small and large luteal cells, having an effect at concentrations as low as 5μM; and was maximally effective at 50μM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca²⁺]i, with NDGA exerting a greater stimulation of [Ca²⁺i than indomethacin. Prostaglandin F_2α (PGF_2α) as well as prostaglandin E₂ (PGE₂) increased [Ca²⁺ in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca²⁺]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF_2α and PGE₂) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca²⁺]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.     

Epidermal growth factor (EGF), tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium and stimulate arachidonic acid release and PGE2/6-keto PGF1 alpha production in cultured porcine thyroid cells

Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium ([Ca2+]i) and stimulate arachidonic acid release and production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in cultured porcine thyroid cells. Addition of EGF, TPA or A23187 to the cells loaded with fura-2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i, which is the earliest event after mitogen stimulation. This [Ca2+]i response occurs immediately, reaching a maximum within several seconds. EGF, TPA and A23187 stimulate arachidonic acid release and PGE2 and 6-keto PGF1 alpha production; the maximum effects are obtained after 2-4 h incubation. EGF, TPA and A23187 increase [Ca2+]i and then stimulate arachidonic acid release and PG production.     

Developmental sensitivity of the bovine corpus luteum to prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1): is ET-1 a mediator of the luteolytic actions of PGF2alpha or a tonic inhibitor of progesterone secretion?

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.     

Stimulatory and inhibitory effects of prostaglandins on the gonadotropin-sensitive adenylate cyclase in the monkey corpus luteum

Detailed analysis of the action of prostaglandins (PGs) on the corpus luteum in primate species is very limited. In this study we examined the response of the adenylate cyclase system to PGs in homogenates prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. The conversion of [alpha 32p] ATP to [32p] cyclic AMP (cAMP) was assessed in the absence (control activity; 50 microM GTP) and presence of various concentrations of seven PGs and arachidonic acid, either alone or in combination with 250 nM hCG. Cyclic AMP production increased up to three-fold in the presence of PGD2, PGE2, PGI2 or PGF2 alpha; however PGA2, PGB2, 13, 14-dihydro-15-keto PGE2 and arachidonic acid alone did not alter cAMP levels. In dose-response studies, adenylate cyclase was 10 and 100-fold more sensitive to PGD2 (Vmax at 1 X 10(-5) M) than to PGE2 or to PGI2 and PGF2 alpha, respectively. Activity in the presence of hCG plus either PGD2, PGE2, PGI2 or PGF2 alpha did not differ from that for hCG (or the PG) alone. In contrast, addition of PGA2 or arachidonate inhibited (p less than 0.05) hCG-stimulated cAMP production by 50 and 100 percent. We conclude that the gonadotropin-sensitive adenylate cyclase of the macaque corpus luteum is also modulated by several PGs. These factors may either mimic (e.g., PGD2, PGE2, PGI2) or suppress (PGA2) gonadotropin-stimulated cAMP production and possibly cAMP-mediated events in luteal cells.     

Anti-apoptotic roles of prostaglandin E2 and F2alpha in bovine luteal steroidogenic cells

Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8-12 of the cycle) were treated for 24 h with PGE2 (0.001-1 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.     

Sensitivity of bovine corpora lutea to prostaglandin F2alpha is dependent on progesterone, oxytocin, and prostaglandins.

Prostaglandin (PG) F2alpha that is released from the uterus is essential for spontaneous luteolysis in cattle. Although PGF2alpha and its analogues are extensively used to synchronize the estrous cycle by inducing luteolysis, corpora lutea (CL) at the early stage of the estrous cycle are resistant to the luteolytic effect of PGF2alpha. We examined the sensitivity of bovine CL to PGF2alpha treatment in vitro and determined whether the changes in the response of CL to PGF2alpha are dependent on progesterone (P4), oxytocin (OT), and PGs produced locally. Bovine luteal cells from early (Days 4-5 of the estrous cycle) and mid-cycle CL (Days 8-12 of the estrous cycle) were preexposed for 12 h to a P4 antagonist (onapristone: OP; 10(-4) M), an OT antagonist (atosiban: AT; 10(-6) M), or indomethacin (INDO; 10(-4) M) before stimulation with PGF2alpha. Although OP reduced P4 secretion (p < 0.001) only in early CL, it reduced OT secretion in the cells of both phases examined (p < 0.001). OP also reduced PGF2alpha and PGE2 secretion (p < 0.01) from early CL. However, it stimulated PGF2alpha secretion in mid-cycle luteal cells (p < 0.001). AT reduced P4 secretion in early and mid-cycle CL (p < 0.05). Moreover, PGF2alpha secretion was inhibited (p < 0.05) by AT in early CL. The OT secretion and the intracellular level of free Ca2+ ([Ca2+]i) were measured as indicators of CL sensitivity to PGF2alpha. PGF2alpha had no influence on OT secretion, although [Ca2+]i increased (p < 0.05) in the early CL. However, the effect of PGF2alpha was augmented (p < 0.01) in cells after pretreatment with OP, AT, and INDO in comparison with the controls. In mid-cycle luteal cells, PGF2alpha induced 2-fold increases in OT secretion and [Ca2+]i. However, in contrast to results in early CL, these increases were magnified only by preexposure of the cells to AT (p < 0.05). These results indicate that luteal P4, OT, and PGs are components of an autocrine/paracrine positive feedback cascade in bovine early to mid-cycle CL and may be responsible for the resistance of the early bovine CL to the exogenous PGF2alpha action.     

Inhibitors of arachidonic acid metabolism eliminate the increase in cytosolic free calcium induced by the mitogen concanavalin A in rat thymocytes

Using inhibitors of arachidonic acid (AA) metabolism, the possible involvement of AA products in the generation of [Ca2+]i and the pHi rise induced by the mitogen concanavalin A (Con A) in rat thymocytes has been studied. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) and the phospholipase A2 inhibitor bromophenacyl bromide (10 microM) eliminated the [Ca2+]i signal induced by Con A; the cyclooxygenase blocker indomethacin also inhibited it. However, neither NDGA nor indomethacin suppressed the pHi rise stimulated by Con A. Exogenous AA induced an increase in [Ca2+]i but not in the pHi. These results indicate that AA metabolites, probably of the lipoxygenase pathway, take part in the generation of the [Ca2+]i response to the mitogen. In contrast, they appear not to be involved in the pHi rise evoked by Con A.     

Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.     

PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells

The hypotheses that PKCepsilon is necessary for: 1) PGF2alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKCepsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKCepsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.     

Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells.

Prostaglandins (PGs) are autocrine or paracrine hormones that may interact with circulating hormones such as parathyroid hormone (PTH) in bone. We examined the interaction of the PGs, PGF2 alpha, PGE2, and 6-keto-PGF1 alpha with PTH to enhance the rapid, initial transient rise in free cytosolic calcium ([Ca2+]i) and cAMP levels stimulated by PTH. Pretreatment of UMR-106, MC3T3-E1, and neonatal rat calvarial osteoblast-like cells by PGs resulted in an enhancement of the early transient rise in [Ca2+]i stimulated by PTH. PGF2 alpha was approximately 100 times more potent than PGE2. PGE2 itself was more potent than 6-keto-PGF1 alpha in enhancing PTH-stimulated rise in [Ca2+]i. Near-maximal augmentation was achieved at PGF2 alpha doses of 10 nM and PGE2 of 1 microM. The degree of augmentation in [Ca2+]i by PGF2 alpha was independent of preincubation time. PGF2 alpha pretreatment did not alter the EC50 for the PTH-induced [Ca2+]i increase but only the extent of rise in [Ca2+]i at each dose of PTH. The augmented increase in [Ca2+]i was mostly due to enhanced PTH-mediated release of Ca2+ from intracellular stores. PGF2 alpha did not stimulate an increase in PTH receptor number as assessed by [125I]-PTH-related peptide binding. PG pretreatment partially reversed PTH inhibition of cell proliferation, suggesting that an increase in [Ca2+]i may play a role in tempering the anti-proliferative effect of PTH mediated by cAMP. These studies suggest a new mode by which PGs can affect cellular activity.     

Involvement of lipoxygenase products of arachidonic acid metabolism in bovine luteal function

A series of experiments was conducted to determine the effects of lipoxygenase products of arachidonic acid (AA) metabolism on the function of the bovine corpus luteum (CL). In the first experiment, reaction products of soybean lipoxidase-AA were added to dispersed bovine luteal cells in increasing concentrations. These lipoxygenase products resulted in a dose-related reduction in the biosynthesis of progesterone and 6-keto-prostaglandin (PG)F1 alpha, while the synthesis of PGF2 alpha was unaffected. In a second experiment, the addition of 5-hydroxyeicosatetraenoic acid (5-HETE), a specific lipoxygenase product, again resulted in a reduction in progesterone and 6-keto-PGF1 alpha, with no change in PGF2 alpha synthesis. Extremely high endogenous concentrations of 5-HETE were measured in luteal tissues (36 +/- 17 to 46 +/- 13 ng/10(6) cells) in a third experiment. In the fourth experiment, an inhibitor of the lipoxygenase pathways, nordihydroguaiaretic acid (NDGA) infused into the uterine lumen twice daily on Days 14-18 of the estrous cycle delayed luteolysis and resulted in lengthened estrous cycles (27.2 +/- 0.3 vs 21.5 +/- 1.0 days for controls, p less than 0.05). Thus, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism delays luteolysis, possibly by removing the preferential inhibition of PGF1 alpha biosynthesis caused by 5-HETE and other products of the lipoxygenase system. Collectively, these results suggest that products of the lipoxygenase pathway are involved in luteolysis in normal heifers.     

Influence of nitric oxide and noradrenaline on prostaglandin F(2)(alpha)-induced oxytocin secretion and intracellular calcium mobilization in cultured bovine luteal cells

Although prostaglandin (PG) F(2alpha) released from the uterus has been shown to cause regression of the bovine corpus luteum (CL), the neuroendocrine, paracrine, and autocrine mechanisms regulating luteolysis and PGF(2alpha) action in the CL are not fully understood. A number of substances produced locally in the CL may be involved in maintaining the equilibrium between luteal development and its regression. The present study was carried out to determine whether noradrenaline (NA) and nitric oxide (NO) regulate the sensitivity of the bovine CL to PGF(2alpha) in vitro and modulate a positive feedback cascade between PGF(2alpha) and luteal oxytocin (OT) in cows. Bovine luteal cells (Days 8-12 of the estrous cycle) cultured in glass tubes were pre-exposed to NA (10(-5) M) or an NO donor (S-nitroso-N:-acetylpenicillamine [S-NAP]; 10(-4) M) before stimulation with PGF(2alpha) (10(-6) M). Noradrenaline significantly stimulated the release of progesterone (P(4)), OT, PGF(2alpha), and PGE(2) (P: < 0.01); however, S-NAP inhibited P(4) and OT secretion (P: < 0.05). Oxytocin secretion and the intracellular level of free Ca(2+) ([Ca(2+)](i)) were measured as indicators of CL sensitivity to PGF(2alpha). Prostaglandin F(2alpha) increased both the amount of OT secretion and [Ca(2+)](i) by approximately two times the amount before (both P: < 0.05). The S-NAP amplified the effect of PGF(2alpha) on [Ca(2+)](i) and OT secretion (both P: < 0.001), whereas NA diminished the stimulatory effects of PGF(2alpha) on [Ca(2+)](i) (P: < 0.05). Moreover, PGF(2alpha) did not exert any additionally effects on OT secretion in NA-pretreated cells. The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL function. While NA stimulates P(4) and OT secretion, NO may inhibit it in bovine CL. Both NA and NO are likely to stimulate the synthesis of luteal PGs and to modulate the action of PGF(2alpha). Noradrenaline may be the factor that is responsible for the limited action of PGF(2alpha) on CL and may be involved in the protection of the CL against premature luteolysis. In contrast, NO augments PGF(2alpha) action on CL and it may be involved in the course of luteolysis.     

Prostaglandin E1 and F2alpha specific binding in bovine corpora lutea: comparison with luteolytic effects.

Preliminary characterization indicated the presence of separate prostaglandin (PG)E1 and (PG)F2alpha binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 x 10(-9)M and 1.1 x 10(-8)M for PGE1 and PGF2alpha, respectively. Competition of several natural prostaglandins for the PGE1 and PGF2alpha bovine luteal specific binding sites indicates specificity for the 9-keto or 9alpha-hydroxyl moiety, respectively. Differences in relative ability to inhibit 3H-PG binding were found due to sensitivity to the absence or presence of the 5, 6-cis-double bond as well. Bovine luteal function was affected following treatment of heifers with 25 mg PGF2alpha as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contract, treatment with 25 mg PGE1 resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE1 treatment compared to pretreatment values. In so far as data obtained in vitro on PGF2alpha relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF2alpha induced luteolysis in the bovine, PGF2alpha relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE1 relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Effects of selective protein kinase c isozymes in prostaglandin2alpha-induced Ca2+ signaling and luteinizing hormone-induced progesterone accumulation in the mid-phase bovine corpus luteum

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.     

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.     

Tumor necrosis factor-alpha stimulates prostaglandin F2alpha secretion by bovine luteal cells via activation of mitogen-activated protein kinase and phospholipase A2 pathways

It has been well demonstrated that tumor necrosis factor-alpha (TNFalpha) stimulates prostaglandin (PG) F2alpha secretion by bovine corpus luteum (CL) in vitro. The objective of the present study was to clarify the intracellular signaling pathway of TNFalpha to stimulate PGF2alpha production in cultured bovine luteal cells. Bovine luteal cells that were obtained from mid- (days 8-12 after ovulation) CL were incubated with TNFalpha (0.6 nM) and/or various compounds as follows: U-73122 (an inhibitor of phospholipase [PL] C), ACA (an inhibitor of PL-A2), H-89 (an inhibitor of protein kinase [PK] A), calphostin C (an inhibitor of PK-C), L-NAME/L-NORG (inhibitors of nitric oxide synthase), and PD98059 (an inhibitor of mitogen-activated protein kinase [MAPK] kinase). Although U-73122 (0. 1-10 microM), H-89 (0.1-10 microM), calphostin C (0.01-1 microM) and L-NAME/L-NORG (1-100 microM) did not affect TNFalpha-induced PGF2alpha secretion by the cultured cells, ACA (1-100 microM) and PD98059 (0.1-100 microM) inhibited TNFalpha-stimulated PGF2alpha secretion by the cells in a dose-dependent fashion (P < 0.05 or lower). These findings suggest that TNFalpha activates the MAPK and PL-A2 pathways in bovine luteal cells to stimulate PGF2alpha secretion.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.     

Lipoxygenase inhibitors suppress intracellular calcium rise induced by ionomycin in rat thymocytes

The lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and 15S-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE) have been found to suppress the rise in free cytoplasmic Ca2+ concentration [( Ca2+]i) induced by the Ca2+ ionophores ionomycin and A23187 in rat thymocytes. Bromophenacyl bromide (BPB), a phospholipase A2 (PLA2) inhibitor, produced a much weaker inhibitory effect, and indomethacin, a cyclo-oxygenase inhibitor, practically did not influence the [Ca2+]i response to ionomycin. These findings implicate the involvement of LO product(s) in the [Ca2+]i rise triggered by the Ca2+ ionophores. The contribution of the NDGA-sensitive component to the ionomycin-induced [Ca2+]i rise was significant in the ionomycin concentration range of 0.1 nM to 0.1 microM whereas at higher doses of the ionophore it gradually diminished. By contrast, the [Ca2+]i rise induced by exogenous arachidonic acid (AA) or melittin, a PLA2 activator, was not suppressed but potentiated by NDGA. Ionomycin and exogenous AA also elicited opposite changes in thymocyte cytoplasmic pH (pHi): the former elevated the pHi while the latter induced a pronounced acidification of the cytoplasm. This difference in the pHi responses may account for the different sensitivity of ionomycin- and AA-elicited [Ca2+]i signal to LO inhibitors.     

Effect of leukotriene B4, prostaglandin E2 and arachidonic acid on cytosolic-free calcium in human neutrophils

Changes in cytosolic free calcium [Ca2+]i and release of beta-glucuronidase in response to leukotriene B4 (LTB4) were measured in intact neutrophils loaded with the fluorescent Ca2+ indicator, quin 2. LTB4 (10(-10) M or higher) caused a rapid rise in [Ca2+]i due to influx from the extracellular medium and release from intracellular pools as well as enzyme release. PGE2 (3 microM) did not alter [Ca2+]i whereas arachidonic acid (10 microM) raised [Ca2+]i. Pretreatment of cells with the chemotactic peptide FMLP inhibited the subsequent rise of [Ca2+]i induced by LTB4. Since chemotactic peptides activate the lipoxygenase pathway of arachidonic acid metabolism, it may be speculated that endogenous LTB4 generation is involved in neutrophil activation.     

Control of steroidogenesis in small and large bovine luteal cells

Evidence was cited to show that: (1) prostacyclin (PGI2) plays a luteotrophic role in the bovine corpus luteum and that products of the lipoxygenase pathway of arachidonic acid metabolism, especially 5-hydroxyeicosatetraenoic acid play luteolytic roles; (2) oxytocin of luteal cell origin plays a role in development, and possibly in regression, of the bovine corpus luteum; and (3) luteal cells arise from two sources; the characteristic small luteal cells at all stages of the oestrous cycle and pregnancy are of theca cell origin; the large cells are of granulosa cell origin early in the cycle, but a population of theca-derived large cells appears later in the cycle. Results of in vitro studies with total dispersed cells and essentially pure preparations of large and small luteal cells indicate that: (1) the recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system is involved in progesterone synthesis in the bovine corpus luteum; (2) activation of protein kinase C is stimulatory to progesterone synthesis in the small luteal cells; (3) activation of protein kinase C has no effect on progesterone synthesis in the large luteal cells; and (4) protein kinase C exerts its luteotrophic effect in total cell preparations, in part at least, by stimulating the production of prostacyclin. The protein kinase C system may cause down regulation of LH receptors in the large cells.