Interferon-tau modulates phorbol ester-induced production of prostaglandin and expression of cyclooxygenase-2 and phospholipase-A(2) from bovine endometrial cells

Antiluteolytic actions of bovine interferon-tau (bIFN-tau) require suppression of prostaglandin F(2 alpha) (PGF(2 alpha)) production. Our objective was to test whether bIFN-tau could block PGF(2 alpha) production and synthesis of phospholipase A(2) (PLA(2)) and cyclooxygenase-2 (COX-2) enzymes induced by a protein kinase C (PKC) stimulator (phorbol 12,13 dibutyrate; PDBu). Bovine endometrial epithelial (BEND) cells were treated with PDBu in the presence or absence of bIFN-tau. Medium samples were analyzed for concentrations of PGF(2 alpha), whole-cell extracts were analyzed for abundance of PLA(2) and COX-2 by immunoblotting, and RNA extracts were examined for steady-state levels of COX-2 mRNA by Northern blotting. The PDBu stimulated production of PGF(2 alpha) between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA(2) by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN-tau suppressed PGF(2 alpha) production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA(2) proteins. Added after a 3-h stimulation with PDBu alone, bIFN-tau suppressed PGF(2 alpha) production after 1 h. Bovine IFN-tau inhibited intracellular mechanisms responsible for PGF(2 alpha) production in BEND cells, and this could be through both cytosolic and nuclear actions.     

Nicergoline stimulates protein kinase C mediated alpha-secretase processing of the amyloid precursor protein in cultured human neuroblastoma SH-SY5Y cells.

We investigated the ability of the antidementia agents, nicergoline, aniracetam and hydergine to stimulate PKC mediated alpha-secretase amyloid precursor protein (APP) processing in cultured human neuroblastoma SH-SY5Y cells. Western immunoblotting of cell conditioned media using the Mabs 22C11 and 6E10 revealed the presence of 2 bands with molecular mass of 90 and 120 kDa, corresponding to possible alternatively glycosylated forms of secreted APP (APPs). Short-term (30 min and 2 h) treatment of cells with nicergoline gave an increased intensity of both bands, compared to non-treated cells. Maximal nicergoline effects, of the order of 150-200% over basal APPs release, were seen at concentrations between 1 and 10 microM. Under the same condition, 1 microM PdBu, used as a positive control, gave 500-1000% increases of basal APPs release. In contrast, aniracetam and hydergine, did not show any effect on APPs secretion. 2 h treatment with nicergoline had no effect on cellular full-length APP levels, as determined by immunoblotting of cell extracts with 22C11 and CT15 antibodies. Immunoblotting with PKC isoform specific antibodies of soluble and membrane fractions prepared from 2 h treated cells, showed that nicergoline (50 microM) and PdBu (1 microM) both induced translocation of PKC alpha, gamma and epsilon, but not PKC beta. The involvement of PKC in mediating nicergoline stimulated APPs release was also studied using specific inhibitors. 1 microM calphostin C, a broad range PKC inhibitor, significantly reduced both PdBu (1 microM) and nicergoline (10 microM) induced APPs release. In contrast, Go6976 (1 microM), a selective PKC alpha and beta1 inhibitor, as well as the cAMP-dependent protein kinase inhibitor, H89 (1 microM) were without effect. These results indicate that nicergoline can modulate alpha-secretase APP processing by a PKC dependent mechanism that is likely to involve the gamma and epsilon isoforms of this enzyme.     

Oxytocin stimulates prostaglandin F2alpha secretion from porcine endometrial cells through activation of calcium-dependent protein kinase C

The mechanism for oxytocin's (OT) stimulation of PGF2alpha secretion from porcine endometrium is not clear, but is thought to involve mobilization of intracellular Ca2+ and subsequent activation of protein kinase C (PKC). This study determined: (1) if mobilization of inositol trisphosphate-sensitive Ca2+ by thapsigargin or activation of PKC by phorbol 12-myristate 13-acetate (PMA) could stimulate PGF2alpha release from luminal epithelial, glandular epithelial and stromal cells of porcine endometrium and (2) if inhibitors of various PKC isotypes could attenuate the ability of OT, thapsigargin and PMA to stimulate PGF2alpha secretion from these cells. Thapsigargin and PMA each stimulated (P < 0.01) PGF2alpha secretion from all three endometrial cell types examined. However, the effects of thapsigargin and PMA were synergistic (P < 0.05) only in stromal cells. Three protein kinase C inhibitors (i.e. G?6976, G?6983 and Ro-31-8220) differentially attenuated (P < 0.05) the ability of OT, thapsigargin and PMA to stimulate PGF2alpha release. These results are consistent with the hypothesis that OT mobilizes Ca2+ to activate a Ca2+-dependent PKC pathway to promote PGF2alpha secretion from porcine endometrial cells. The differing pattern of response to isotype-specific inhibitors of PKC among cell types suggests that distinct PKC isoforms are differentially expressed in luminal epithelial, glandular epithelial and stromal cells.     

Platelet aggregation induced by alpha 2-adrenoceptor and protein kinase C activation. A novel synergism.

Adrenaline or UK 14304 (a specific alpha 2-adrenoceptor agonist) and phorbol ester (phorbol 12,13-dibutyrate; PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol; DiC8) synergistically induced platelet aggregation and ATP secretion. The effect on aggregation was more pronounced than the effect on secretion, and it was observed in aspirinized, platelet-rich plasma or suspensions of washed aspirinized platelets containing ADP scavengers. No prior shape change was found. In the presence of adrenaline, DiC8 induced reversible aggregation and PdBu evoked irreversible aggregation that correlated with the different kinetics of DiC8- and PdBu-induced protein kinase C activation. Adrenaline and UK 14304 did not induce or enhance phosphorylation induced by DiC8 or PdBu of myosin light chain (20 kDa), the substrate of protein kinase C (47 kDa), or a 38 kDa protein. Immunoprecipitation studies using a Gcommon alpha antiserum or a Gi alpha antiserum showed that Gi alpha is not phosphorylated after exposure of platelets to PdBu or PdBu plus adrenaline. Adrenaline, PdBu or adrenaline plus PdBu did not cause stimulation of phospholipase C as reflected in production of [32P]phosphatidic acid. Adrenaline caused a small increase of Ca2+ in the platelet cytosol of platelets loaded with Indo-1; this effect was also observed in the absence of extracellular Ca2+. However, under conditions of maximal aggregation induced by adrenaline plus PdBu, no increase of cytosolic Ca2+ was observed. Platelet aggregation induced by PdBu plus adrenaline was not inhibited by a high intracellular concentration of the calcium chelator Quin-2. These experiments indicate that alpha 2-adrenoceptor agonists, known to interact with Gi, and protein kinase C activators synergistically induced platelet aggregation through a novel mechanism. The synergism occurs distally to Gi protein activation and protein kinase C-dependent protein phosphorylation and does not involve phospholipase C activation or Ca2+ mobilization.     

Regulation of amyloid precursor protein (APP) secretion by protein kinase calpha in human ntera 2 neurons (NT2N)

Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic APP products. Protein kinase C (PKC) has been shown to regulate APPs secretion, and PKCalpha and PKCepsilon have been implicated in APPs secretion in fibroblasts. This study examined the PKC isoform involved in regulated APPs secretion in human NT2N neurons and in CHO cells stably expressing APP(695). Inhibition of PMA-induced APPs secretion with the PKC inhibitors Calphostin C and GF109203X demonstrated that PKC is involved in PMA-regulated APPs secretion in NT2N cells. The specific PKC isoforms present in NT2N and CHO695 cells were identified, and PKCalpha and PKCepsilon were found to translocate from cytosol to membranes in NT2N and CHO695 cells. Translocation of PKC to the membrane allows for activation of the enzyme, as well as for positioning of the enzyme close to its substrate. Long-term PMA treatment led to complete downregulation of PKCalpha in NT2N cells and to downregulation of PKCalpha and PKCepsilon in CHO695 cells. PKCalpha downregulation in the NT2N cells resulted in loss of PMA-regulated APPs secretion and a substantial reduction in constitutive APPs secretion. Downregulation of PKCalpha and PKCepsilon in CHO695 cells resulted in loss of PMA-regulated APPs secretion; however, constitutive APPs secretion was unaffected. These findings suggest that PKCalpha is involved in PMA-regulated APPs secretion in NT2N cells and PKCalpha and/or PKCepsilon is involved in PMA-regulated APPs secretion in CHO695 cells.     

Oxytocin stimulates prostaglandin F(2alpha) secretion from porcine endometrial cells through activation of calcium-dependent protein kinase C*

The mechanism for oxytocin's (OT) stimulation of PGF(2alpha) secretion from porcine endometrium is not clear, but is thought to involve mobilization of intracellular Ca(2+) and subsequent activation of protein kinase C (PKC). This study determined: (1) if mobilization of inositol trisphosphate-sensitive Ca(2+) by thapsigargin or activation of PKC by phorbol 12-myristate 13-acetate (PMA) could stimulate PGF(2alpha) release from luminal epithelial, glandular epithelial and stromal cells of porcine endometrium and (2) if inhibitors of various PKC isotypes could attenuate the ability of OT, thapsigargin and PMA to stimulate PGF(2alpha) secretion from these cells. Thapsigargin and PMA each stimulated (P < 0.01) PGF(2alpha) secretion from all three endometrial cell types examined. However, the effects of thapsigargin and PMA were synergistic (P < 0.05) only in stromal cells. Three protein kinase C inhibitors (i.e. G?6976, G?6983 and Ro-31-8220) differentially attenuated (P < 0.05) the ability of OT, thapsigargin and PMA to stimulate PGF(2alpha) release. These results are consistent with the hypothesis that OT mobilizes Ca(2+) to activate a Ca(2+)-dependent PKC pathway to promote PGF(2alpha) secretion from porcine endometrial cells. The differing pattern of response to isotype-specific inhibitors of PKC among cell types suggests that distinct PKC isoforms are differentially expressed in luminal epithelial, glandular epithelial and stromal cells.     

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein is associated with bovine luteal oxytocin exocytosis

The ruminant corpus luteum, in addition to producing progesterone, synthesizes and secretes oxytocin (OT) during the estrous cycle. Secretion of oxytocin occurs by exocytosis of membrane-encapsulated granules of this hormone. Exocytosis of oxytocin involves transport of granules through a cytoskeletal matrix including an actin cortex closely associated with the plasma membrane (PM). Actin filaments crosslinked by various proteins give rise to the structural integrity of the cortex. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by protein kinase C (PKC), crosslinks actin filaments and anchors the actin network to the inner leaflet of the PM. There is evidence that the intact actin cortex may serve as a barrier, precluding fusion of transport vesicles with the PM. In some secretory cells, phosphorylation of MARCKS has resulted in its translocation from the PM to the cytoplasm with an associated disassembly of the actin cortex. Prostaglandin F(2alpha) (PGF(2alpha)) stimulation of the bovine corpus luteum during the midluteal phase of the estrous cycle activates PKC, which is associated with an increase in OT secretion in vivo and in vitro. Data are presented demonstrating that stimulation of bovine luteal cells with PGF(2alpha) on Day 8 of the cycle promotes rapid phosphorylation of MARCKS protein and causes its translocation from the PM to the cytoplasm and concomitant, enhanced exocytosis of OT. These data are consistent with the premise that MARCKS plays a role in the exocytotic process.     

Down-regulation of oxytocin-induced cyclooxygenase-2 and prostaglandin F synthase expression by interferon-tau in bovine endometrial cells

Oxytocin (OT) is responsible for the episodic release of luteolytic prostaglandin (PG) F2alpha from the uterus in ruminants. The attenuation of OT-stimulated uterine PGF2alpha secretion by interferon-tau (IFN-tau) is essential for prevention of luteolysis during pregnancy in cows. To better understand the mechanisms involved, the effect of recombinant bovine IFN-tau (rbIFN-tau) on OT-induced PG production and cyclooxygenase-2 (COX-2) and PGF synthase (PGFS) expression in cultured endometrial epithelial cells was investigated. Cells were obtained from cows at Days 1-3 of the estrous cycle and cultured to confluence in RPMI medium supplemented with 5% steroid-free fetal calf serum. The cells were then incubated in the presence or absence of either 100 ng/ml OT or OT+100 ng/ml rbIFN-tau for 3, 6, 12, and 24 h. OT significantly increased PGF2alpha and PGE2 secretion at all time points (p < 0.01), while rbIFN-tau inhibited the OT-induced PG production and reduced OT receptor binding in a time-dependent manner. OT increased the steady-state level of COX-2 mRNA, measured by Northern blot, which was maximal at 3 h (9-fold increase) and then decreased with time (p < 0.01). OT also caused an increase in COX-2 protein, which peaked at 12 h (11-fold increase), as measured by Western blot. Addition of rbIFN-tau suppressed the induction of COX-2 mRNA (89%, p < 0.01) and COX-2 protein (50%, p < 0.01) by OT. OT also increased PGFS mRNA, and this stimulation was attenuated by rbIFN-tau (p < 0.01). To ensure that the decrease in COX-2 was not solely due to down-regulation of the OT receptor, cells were stimulated with a phorbol ester (phorbol 12-myristate 13-acetate; PMA) in the presence and absence of rbIFN-tau. The results showed that rbIFN-tau also decreased PMA-stimulated PG production and COX-2 protein. It can be concluded that rbIFN-tau inhibition of OT-stimulated PG production is due to down-regulation of OT receptor, COX-2, and PGFS.     

Angiotensin II stimulates hypertrophic growth of cultured neonatal rat ventricular myocytes: roles of PKC and PGF2alpha

Angiotensin II (Ang II) has been shown to regulate growth in smooth muscle cells. Protein kinase C (PKC), which mediates Ang II action, has been implicated in myocardial cell hypertrophy. Acute pressure overload in the left ventricles has been demonstrated to produce prostaglandin F2 alpha (PGF2alpha) release. Therefore, we used cultured neonatal rat ventricular myocytes to study Ang II, PKC and PGF2alpha and their relationship to hypertrophy. The amount of PGF2alpha produced was determined by radioimmunoassay, Ang II-induced hypertrophy and PGF2alpha release. Pretreatment with 10(-6) M of PKC inhibitor, 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7), blocked Ang II-induced hypertrophy and PGF2alpha release. In neonatal rat ventricular myocytes that were treated with either Ang II or PKC activator (Phorbol 12, 13, dibutyrate; PDBu), PKC enzyme assay showed PKC was translocated from the cytosol to the membrane which indicates activation. This suggests that PKC mediates, in part, Ang II-induced PGF2alpha release and hypertrophy. In summary, Ang II activates PKC, which causes PGF2alpha release and hypertrophy, and this PGF2alpha release and hypertrophy can be overcome by pretreatment with PKC inhibitor.     

Spatiotemporal interactions of myristoylated alanine-rich C kinase substrate (MARCKS) protein with the actin cytoskeleton and exocytosis of oxytocin upon prostaglandin F2alpha stimulation of bovine luteal cells

In the bovine corpus luteum (CL) phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in response to prostaglandin F2alpha (PGF2alpha) is correlated with the secretion of oxytocin. The present study was conducted to 1) examine the intracellular translocation characteristics of wild-type and mutant forms of a green fluorescent protein (GFP)-conjugated MARCKS (MARCKS-GFP) after PGF2alpha treatment and 2) evaluate PGF2alpha-induced temporal changes in MARCKS-GFP and actin cortex associated with exocytosis of oxytocin. In experiment 1, cells of the bovine CL were cultured on coverslips overnight. Then, wild-type and mutant MARCKS-GFP constructs were transfected separately into cells and expression was detected through fluorescence microscopy. Forty-eight hours after transfection, cells were treated with vehicle, PGF2alpha (56 nM), or a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA], 1 microM). Treatment of cells expressing wild-type MARCKS-GFP with PGF2alpha and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min. Phosphorylation mutant MARCKS-GFP (m3) protein was localized on the plasma membrane, and treatments did not cause its translocation to the cytoplasm. Myristoylation mutant MARCKS-GFP (G2A) was observed solely in the cytoplasm, and no changes were detected in the intracellular location of this mutant MARCKS after treatment. In experiment 2, luteal cells were transfected with one of the three MARCKS-GFP constructs. Cells were then fixed and probed sequentially for oxytocin and filamentous actin. Results revealed that only wild-type MARCKS-GFP transfected large luteal cells contained advanced signs of exocytosis (peripheral movement of oxytocin vesicles; shorter actin filaments) with translocation of MARCKS-GFP from membrane to cytoplasm in response to PGF2alpha treatment. These data demonstrate that phosphorylation of membrane-bound MARCKS protein is requisite for exocytosis of oxytocin to occur in bovine large luteal cells.     

Specific translocation of protein kinase Calpha to the plasma membrane requires both Ca2+ and PIP2 recognition by its C2 domain

The C2 domain of protein kinase Calpha (PKCalpha) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCalpha and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCalpha Ca2+-binding loops (CBLs) and PIP2 binding site (beta-strands 3-4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCalpha C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on beta-strands 3-4, which bind to plasma membrane PIP2.     

Effects of prostaglandins E2 and F2alpha (PGE2; PGF2alpha), trilostane,mifepristone, palmitic acid (PA), indomethacin (INDO), ethamoxytriphetol (MER-25), PGE2 + PA,or PGF2alpha + PA on PGE2, PGF2alpha,and progesterone secretion by bovine corpora lutea of mid-pregnancy in vitro

The effects of PGE2, PGF2alpha, trilostane, RU-486, PA, INDO, MER-25, PGE2, or PGF2alpha + PA on secretion of progesterone, PGE2, or PGF2alpha by bovine corpora lutea (CL) of mid-pregnancy in vitro for 4 and 8 hr was examined. Secretion of PGE2 and PGF2alpha increased with time in culture (P < or = 0.05). PGE2 and PGE2 + PA increased (P < or = 0.05) secretion of progesterone at 4 and 8 h, progesterone secretion was increased (P < or = 0.05) at 4 h; but not at 8 h (P > or = 0.05) by trilostane, mifepristone, PGF2alpha and PGF2alpha + PA, and was decreased at 8 h by PGF2alpha and PGF2alpha + PA. Indomethacin decreased (P < or = 0.05) secretion of PGE2, PGF2alpha, and progesterone at 4 and 8 h. Trilostane, PA, PGF2alpha, RU-486 and PGF2alpha + PA increased (P < or = 0.05) PGE2 at 4 h only. Palmitic acid decreased (P < or = 0.05) PGF2alpha at 4 h, while trilostane, RU-486, or MER-25 did not affect (P < or = 0.05) PGE2 of PGF2alpha secretion. It is concluded that PGE2 of luteal tissue origin is the luteotropin at mid-pregnancy in cows. Also, it is suggested that PA may alter progesterone secretion by affecting the inter conversion of PGE2 and PGF2alpha.     

Parathyroid hormone-mediated regulation of Na+-K+-ATPase requires ERK-dependent translocation of protein kinase Calpha

Parathyroid hormone (PTH) inhibits Na+-K+-ATPase activity by serine phosphorylation of the alpha1 subunit through protein kinase C (PKC)- and extracellular signal-regulated kinase (ERK)-dependent pathways. Based on previous studies we postulated that PTH regulates sodium pump activity through isoform-specific PKC-dependent activation of ERK. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH stimulated membrane translocation of PKCalpha by 102 +/- 16% and PKCbetaI by 41 +/- 7% but had no effect on PKCbetaII and PKCzeta. Both PKCalpha and PKCbetaI phosphorylated the Na+-K+-ATPase alpha1 subunit in vitro. PTH increased the activity of PKCalpha but not PKCbetaI. Coimmunoprecipitation assays demonstrated that treatment with PTH enhanced the association between Na+-K+-ATPase alpha1 subunit and PKCalpha, whereas the association between Na+-K+-ATPase alpha1 subunit and PKCbetaI remained unchanged. A PKCalpha inhibitory peptide blocked PTH-stimulated serine phosphorylation of the Na+-K+-ATPase alpha1 subunit and inhibition of Na+-K+-ATPase activity. Pharmacologic inhibition of MEK-1 blocked PTH-stimulated translocation of PKCalpha, whereas transfection of constitutively active MEK-1 cDNA induced translocation of PKCalpha and increased phosphorylation of the Na+-K+-ATPase alpha1 subunit. In contrast, PTH-stimulated ERK activation was not inhibited by pretreatment with the PKCalpha inhibitory peptide. Inhibition of PKCalpha expression by siRNA did not inhibit PTH-mediated ERK activation but significantly reduced PTH-mediated phosphorylation of the Na+-K+-ATPase alpha1 subunit. Pharmacologic inhibition of phosphoinositide 3-kinase blocked PTH-stimulated ERK activation, translocation of PKCalpha, and phosphorylation of the Na+-K+-ATPase alpha1 subunit. We conclude that PTH stimulates Na+-K+-ATPase phosphorylation and decreases the activity of Na+-K+-ATPase by ERK-dependent activation of PKCalpha.     

Effect of PGF2alpha, indomethacin, tamoxifen, or estradiol-17beta on incidence of abortion, progesterone, and pregnancy-specific protein B (PSPB) secretion in 88- to 90-day pregnant sheep

One objective of this experiment was to evaluate our hypotheses that estradiol-17beta regulates secretion of pregnancy specific protein B (PSPB) and that secretion of progesterone during pregnancy is regulated by a prostanoid by examining the effects of prostaglandin F2alpha (PGF2alpha), a luteolyic agent; indomethacin, a prostanoid synthesis inhibitor; tamoxifen, an estrogen receptor antagonist; estradiol 17-beta; and interaction of these factors on the incidence of abortion and progesterone and PSPB secretion. Another objective was to determine if there is a luteal source of PSPB. Weights of corpora lutea were decreased (P < or = 0.05) by PGF2alpha, indomethacin, PGF2alpha + tamoxifen, PGF2alpha + indomethacin, and PGF2alpha + estradiol-17beta but not (P > or = 0.05) by tamoxifen or estradiol-17beta alone. No ewe treated with PGF2alpha alone aborted (P > or = 0.05). Forty percent of ewes treated with PGF2alpha + estradiol-17beta aborted (P < or = 0.05), but ewes were not aborted by any other treatment within the 72-h sampling period. Profiles of progesterone in jugular venous blood differed (P < or = 0.05) among control, indomethacin-, tamoxifen-, and PGF2alpha + indomethacin-treated ewes. Progesterone in jugular venous blood of control ewes decreased (P < or = 0.05) by 24 h, followed by a quadratic increase (P < or = 0.05) from 24 to 62 h. Progesterone in jugular venous blood of indomethacin-, PGF2alpha-, PGF2alpha- + tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + estradiol-17beta-, and tamoxifen-treated ewes was reduced (P < or = 0.05) by 18 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Progesterone in vena cava and in uterine venous blood was reduced (P < or = 0.05) at 72 h in PGF2alpha-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + tamoxifen-, and PGF2alpha + estradiol-17beta-treated ewes. Weights of placentomes did not differ among treatment groups (P > or = 0.05). Profiles of PSPB in inferior vena cava blood differed (P < or = 0.05) among control, estradiol-17beta-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, and PGF2alpha + tamoxifen-treated 88- to 90-day pregnant ewes. Concentrations of PSPB in inferior vena cava blood were increased (P < or = 0.05) in indomethacin-, estradiol-17beta-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated 88- to 90-day pregnant ewes within 6 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Concentrations of PSPB in uterine venous blood of indomethacin-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated ewes were greater (P < or = 0.05) at 72 h than at 0 h. PSPB in ovarian venous blood did not differ (P > or = 0.05) adjacent or opposite to the ovary with the corpus luteum. It is concluded from these data that estrogen regulates placental secretion of PSPB and that a prostanoid, presumably prostaglandin E, regulates placental secretion of progesterone during 88-90 days of gestation in sheep and that there is no luteal source of PSPB.     

Polyunsaturated fatty acids and bovine interferon-tau modify phorbol ester-induced secretion of prostaglandin F2 alpha and expression of prostaglandin endoperoxide synthase-2 and phospholipase-A2 in bovine endometrial cells

Embryonic mortality in cattle may occur because of inadequate inhibition of uterine secretion of prostaglandin (PG) F2alpha mediated by bovine interferon-tau (bIFN-tau). The objectives of the present study were to determine whether polyunsaturated fatty acids inhibit secretion of PGF2alpha from bovine endometrial cells induced by stimulating protein kinase C with phorbol 12,13 dibutyrate (PDBu) and to investigate possible mechanisms of action. Confluent cells were exposed for 24 h to 100 microM of linoleic, arachidonic (AA; C20:4, n-6), linolenic (LNA; C18:3, n-3), eicosapentaenoic (EPA; C20:5, n-3), or docosahexaenoic (DHA; C22:6, n-3) acid. After incubation, cells were washed and stimulated with PDBu. The EPA, DHA, and LNA attenuated secretion of PGF2alpha in response to PDBu. The EPA and DHA were more potent inhibitors than LNA. The EPA inhibited secretion of PGF2alpha at 6.25 microM. Secretion of PGF2alpha in response to PDBu decreased with increasing incubation time with EPA. Both bIFN-tau and EPA inhibited secretion of PGF2alpha, and their inhibitory effects were additive. The bIFN-tau, but not EPA, reduced the abundance of PG endoperoxide synthase-2 (PGHS-2) mRNA. Incubation with 100 microM EPA, DHA, or AA for 24 h followed by treatment with PDBu did not affect concentrations of PGHS-2 and phospholipase A2 proteins. The EPA and DHA inhibit secretion of PGF2alpha through a mechanism different from that of bIFN-tau. The effect of EPA on PGF2alpha secretion may be caused by competition with AA for PGHS-2 activity or reduction of PGHS-2 activity. The use of EPA and DHA to inhibit uterine secretion of PGF2alpha and to improve embryonic survival in cattle warrants further investigation.     

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids.

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2alpha (PGF2alpha) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 microM [Ca2+]e, both PGF2alpha and U46619 (10(-5) M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 microM [Ca2+]e, PGF2alpha and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx--PKC activity--contraction relationship showed a 45Ca2+-influx threshold of approximately 7 micromol x kg(-1) x min(-1) for maximal PKC activation by PGF2alpha and U46619. 45Ca2+ influx > 10 micromol x kg(-1) x min(-1) was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed alpha-, delta-, epsilon-, and zeta-PKC in porcine coronary artery. In unstimulated tissues, alpha- and epsilon-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of epsilon-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of alpha-PKC was observed at 600 microM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent epsilon-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent alpha-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.