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Prostaglandin E2 induces it's own secretion
Poster Presentations Monday 21 July
EFA & Eicosanoids 1997 - Edinburgh
P53
P54
The Involvement Of Calcium In Prostaglandin Production By The Guinea-pig Placenta. H. Aitken & NL Poyser, Department of Pharmacology, University ~)1 Edinburgh Medical School, 1 George Square, Edinburgh. EH8 9JZ. 11K.
Prostaglandin E 2 Induces It's Own Secretion. C. Weems ~, Y. Weems ~, M. Lammogliaz and R. Randet z. Animal Science Dept., ~University of Hawaii, Honolulu HI 96822; 2Texas A&M University, Overton TX 75864, U.S.A.
Prostaglandin (PG) synthesis by the guinea-pig placenta is mediated by cyclooxygenase (Cox-2). The outputs of PGF:~, and 6-keto-PGF,~, but not of POE:, from the guinea-pig placenta in culture significantly (P < 0.05) increased 7- and 3-fold, respectively, between days 22 and 29 of pregnancy. The calcium requirement for ttlis increased PG production has been studied. Day 29 guinea-pig placenta was cultured for 24 h and PG output lug/100 tug tissue/h) was measured by radioimmunoassay. Results (mean _+ sere. n = 5) were analysed by the paired "t" test. Lack of extracellular calcium (by using calcium-depleted medium) had no effect on PGF_,~ and PGE_~ outputs, but significantly (P < 0.05) decreased 6-keto-PGF,~ output from 0.74 _+ 0.06 to 0.51 -+ 0.06. EGTA (2 raM; a calcium chelator) and TMB-8 (30 /aM: an intracellular calcium antagonis0 significantly (P < 0.05) reduced PGF_,~ output ii-om 2.00 ± 0.32 to 0.52 +- 0.05 and 0,74 _+ 0.11, respectively, and 6-ketoPGF~,~ output from 1.14 _+0.20 to 0.66 ± 0.08 and 0.40 _+ 0,1)4, respectively. TMB-8 also significantly (P < 0.05) reduced PGE, output ti-om 0.56 -+ (). 11 to 0.27 ± 0.06. Two calmodulin antag~mists, namely trifluoperazine (TFP; 100 and 200 ~M) mid W-7 (150 alld 300 p.M) hod no inhibilory eflect on PG production, except TFP (100 ~M) which significantly (P < 0.05) reduced 6keto-PGF,= output from 1,14 -+ 0.20 to 0.44 +_0.08. These results indicate that the production of PGF~, 6-keto-PGF,~ and, to a lesser extent, PGE, by the guinea-pig placenta is dependent upon intracellular calcium. The production of 6-keto-PGF~ is also dependent upon extracellular calcimn. Calmodolin does not appear to be involved in the action of c',dciunL Although the outputs of PGF,.~ and PGE_~are apparently not dependent up~m extracellulax calcium two calcium channel blockers, namely nifedipine (1 ~M) and verapamil (1 ~M), significantly (P < I).05) reduced PGF_~ output ti-nm 2.12 -+0.16 to 1.05 -+0.03 and 0.99 _+0.04, respectively, and PGE_, output from 0.61 + 0.04 to 0.36 ± 0.05 and 0.40 _+ 0.03, respectively. NiIEdipine (1 ~M) also significantly (P < 0.05) reduced 6-keto-PGF~,~ output fi'om 0.74 _+0.03 to 0.60 -+ 0.02. The mechanism by which nitedipine ~md verapamit reduces PG output is not apparent and requires further study.
The Placenta secretes half the progesterone at day 90 of pregnancy in sheep (Y.S. Weems, et at., Prostaglandins 43:203, 1992). Placental secretion of progesterone and prostaglandin (PG) E2 (PGE2) in vitro 7 days after ovariectomy increases (4 a' Intl. Congr. Essential Fatty Acids and Eicosanoids, 1997). The objective was to determine whether PGE2 stimulates bovine placenr,fl secretion of progesterone in vitro. Day 200 (n=9) or 270 (n=9) Brahman placenta slices were collected by caesarean section, treatments randomized within each animal, minced and incubated in 5 ml of M199 containing 20~tg/ml 25-OH-Cholesterol, 25raM Hopes Buffer and Earle's salts, 0.1% BSA, penicillin (100 IU/ml) and streptomycin sulfate (0.2 mg/ml) in vitro for 4 or 8 hr at 39° C, pH 7.2 and under 95:5% air and COz, respectively, in the presence or absence of prostaglandin synthesis inhibitors. Treatments were: M199, indnmcthacin (INDO; 2 0 ~ I ) , meclofenamic acid (MECLO; 20~tM), PGEz (0, 1, 10 or 100 og/ml) PGEz +INDO, PGE2 + MECLO, or Pregnancy Specific Protein B (PSPB; 10 ~tg/ml). Progesterone, estradiol-17~, PGFzt~, PGE and PSPB were quantified by RIA. Data were analyzed by a factmial ANOVA- INDO and MECLO reduced (P<0.05) PGFzt~ and PGE, respectively, by 61 and 94 and 43 and 95%. No treatment affected secretion of progesterone, estradiol-17~ or PSPB (P>0.05). PGE2 increased (P<0.05) secretion of PGE by 270 day placenta by 3 fold even in the presence of INDO or MECLO, but not by 200 day placenta (P>0.05). PSPB increased (P<0.0.~ secretion of PGE by 200 day placenta. It is concluded that PGF~ does not affect placental secretion of progesterone and that INDO and MECLO inhibition of PG synthesis is not entirely through inhibition of cyclooxygenase (COX) 1 and 2 but may he downstream at the level of specific PG isomerases, through another unidentified COX, or through a nonenzymatic PG synthesis pathway.
227
P55
P56
Ovariectomy Affects Placental Secretion of Prostaglandins, Steroids and Proteins in Ewes. C. Weems l, Y. Wcems l, P. Bridges t, G. Sasser2, B. LeaMaster t, and D. Vincent. ~Animal Science Dept., University of Hawaii, Honolulu, HI 96822; ZUniverslty of Idaho, Moscow, ID 83843, USA.
Estradiol-I7fl, Indomethacin or Tamoxifen Affect Placental Function of Ewes. Y. Wecms l, P. Bridges x, G. Sasser2, B. Le~Iaster 1, D. Vincent t, and C. Wcems t. Animal Science Dept., tUniversity of Hawaii, Honolulu HI 96822; ZUniverslty of ldaho, Moscow, ID 83843, USA.
A model for regulation of urine placental secretion of progesterone (P4), when sheep are not a b o r t e d with p r o s t a g l a n d i n Fzvt (PGFzo0, has been proposed 0Neems, Y.S. et al., P r o s t a g l a u d i n s 43:203, 1992; 46:277, 1993; 48:377, 1994) whereby estradiol-17fl regulates placental secretion of p r e g n a n c y specific protein B (PSPB); PSPB regulates placental secretion of p r o s t a g l a n d i n E (PGE) and P G E regulates placental secretion of P4 at 90 days of p r e g n a n c y in sheep. The objective was to determine the effects of ovariectomy on ovine placental secretion of P4, PGE, estradiol-17fl, PSPB and PGF2o:. Ninety day p r e g n a n t ewes were ovariectomized (OVX; N=5) or l a p a r o t o m i z e d only (n=6) and a j u g u l a r venous catheter and a vena cava catheter were installed via the saphenous vein with the catheter tip 5 cm cranial to the j u n c t u r e of the ovarian vein and vena cava. J u g u l a r and vena cava blood w e r e collected at 0 and every 6 hr t h r o u g h 0600 hr on day 7 for analysis of P4, PSPB, PGE, estradiol-17fl or PGF2~ by RIA. Placentome slices were collected on day 7 and i n c u b a t e d for 4 hr at 3 9 ° C in M-199 containing Hepes Buffer, E a r l e ' s Salts, 25-OH cholesterol, penicillin, s t r e p t o m y c i n sulfate and 0.1% BSA. Media were analyzed for P4, PSPB, estradiol-17[~, P G E and PGF2~ by RIA. Placentome slices from OVX 90 day p r e g n a n t ewes in vitro secreted 3 fold more P4 (P_<0.05), two fold more estradioI-17fl, 2 fold m o r e PSPB (P_<0.05) but did not affect PGFz~ when c o m p a r e d to o v a r y intact controls (P_>0.05). These d a t a are consistent with the hypothesis that estradiol-17IJ regulates ovine placental secretion of PSPB, PSPB regulates placental secretion of P G E and P G E regulates placental secretion of P4 in sheep.
Prustaglandin F2c~ (PGF2ct) is luteolytic but does not abort intact or ovariectomized 90-100 day pl'egnant ewes (Weems, Y.S. et aL, Prostaglandins 43:203, 1992). Pregnancy specific protein B (PSPB), estradiol-1711 and prostaglandin E (PGE) appear to protect placental steroidogenesis from P G F ~ (Weems, Y.S. et at., Prostaglandins, 46:277, 1993; 48:377, 1994). The objective of this experiment was to determine the effects of estradiol-17fi, indomethaein or tamoxifen on placental function of 90 day pregnant ewes. Ninety day pregnant ewes were fitted with a jugular venous and vena cava catheter and received: vehicle, PGF2~ (8 rag/58 kg/BW) i.m. at 0 hr, estradiol-171~ (500/~g) i.m. every 6 hr or PGF2ct at 0 hr and estradiol-171~, indomethacin (100 mg) or tamoxifeu (40 ml0 Lm. every 6 hr. Five ewes were in each group. Jugular venous and inferior veda cava blood were collected every 6 hr from 0-60 hr, every 20 vain from 62-66 hr and at 72 hr for analysis for PGF2~, PGE, estradiul-171~, progesterone or PSPB by RIA. Profiles of PGF:ct, progesterone, PSPB and estradiol-17fl were analyzed by a split-plot ANOVA for repeated measures. Profiles of PGFzct (P<0.05) or estradiol171I (P<0.05) in vena cava plasma of PGF2~ + estradiol-17fl differed (P<0.05) from the otber treatment groups but did not differ among the other 5 treatment groups (P>0.05). Profiles of progesterone in jugular plasma in the indomethacin group were lower (P_<0.05) than controls. Estradiol-17fl alone (P<0.05) or tamoxifen (P<0.05) prevents the decline in PSPB wlfich was elevated (P_<0.05) at 6 hr in all groups. Abortions occurred only in the PGFz~ + estradiol-17fl group (40%). These data are consistent with our hypothesis of regulation of placental function, however, when both exogenous PGF2ct and estradlol-17fl are given, the placenta secretes both PGF2~ and estradiol-171~ which is detrimental to pregnancy.
EFA & Eicosanoids 1997 - Edinburgh
P53
P54
The Involvement Of Calcium In Prostaglandin Production By The Guinea-pig Placenta. H. Aitken & NL Poyser, Department of Pharmacology, University ~)1 Edinburgh Medical School, 1 George Square, Edinburgh. EH8 9JZ. 11K.
Prostaglandin E 2 Induces It's Own Secretion. C. Weems ~, Y. Weems ~, M. Lammogliaz and R. Randet z. Animal Science Dept., ~University of Hawaii, Honolulu HI 96822; 2Texas A&M University, Overton TX 75864, U.S.A.
Prostaglandin (PG) synthesis by the guinea-pig placenta is mediated by cyclooxygenase (Cox-2). The outputs of PGF:~, and 6-keto-PGF,~, but not of POE:, from the guinea-pig placenta in culture significantly (P < 0.05) increased 7- and 3-fold, respectively, between days 22 and 29 of pregnancy. The calcium requirement for ttlis increased PG production has been studied. Day 29 guinea-pig placenta was cultured for 24 h and PG output lug/100 tug tissue/h) was measured by radioimmunoassay. Results (mean _+ sere. n = 5) were analysed by the paired "t" test. Lack of extracellular calcium (by using calcium-depleted medium) had no effect on PGF_,~ and PGE_~ outputs, but significantly (P < 0.05) decreased 6-keto-PGF,~ output from 0.74 _+ 0.06 to 0.51 -+ 0.06. EGTA (2 raM; a calcium chelator) and TMB-8 (30 /aM: an intracellular calcium antagonis0 significantly (P < 0.05) reduced PGF_,~ output ii-om 2.00 ± 0.32 to 0.52 +- 0.05 and 0,74 _+ 0.11, respectively, and 6-ketoPGF~,~ output from 1.14 _+0.20 to 0.66 ± 0.08 and 0.40 _+ 0,1)4, respectively. TMB-8 also significantly (P < 0.05) reduced PGE, output ti-om 0.56 -+ (). 11 to 0.27 ± 0.06. Two calmodulin antag~mists, namely trifluoperazine (TFP; 100 and 200 ~M) mid W-7 (150 alld 300 p.M) hod no inhibilory eflect on PG production, except TFP (100 ~M) which significantly (P < 0.05) reduced 6keto-PGF,= output from 1,14 -+ 0.20 to 0.44 +_0.08. These results indicate that the production of PGF~, 6-keto-PGF,~ and, to a lesser extent, PGE, by the guinea-pig placenta is dependent upon intracellular calcium. The production of 6-keto-PGF~ is also dependent upon extracellular calcimn. Calmodolin does not appear to be involved in the action of c',dciunL Although the outputs of PGF,.~ and PGE_~are apparently not dependent up~m extracellulax calcium two calcium channel blockers, namely nifedipine (1 ~M) and verapamil (1 ~M), significantly (P < I).05) reduced PGF_~ output ti-nm 2.12 -+0.16 to 1.05 -+0.03 and 0.99 _+0.04, respectively, and PGE_, output from 0.61 + 0.04 to 0.36 ± 0.05 and 0.40 _+ 0.03, respectively. NiIEdipine (1 ~M) also significantly (P < 0.05) reduced 6-keto-PGF~,~ output fi'om 0.74 _+0.03 to 0.60 -+ 0.02. The mechanism by which nitedipine ~md verapamit reduces PG output is not apparent and requires further study.
The Placenta secretes half the progesterone at day 90 of pregnancy in sheep (Y.S. Weems, et at., Prostaglandins 43:203, 1992). Placental secretion of progesterone and prostaglandin (PG) E2 (PGE2) in vitro 7 days after ovariectomy increases (4 a' Intl. Congr. Essential Fatty Acids and Eicosanoids, 1997). The objective was to determine whether PGE2 stimulates bovine placenr,fl secretion of progesterone in vitro. Day 200 (n=9) or 270 (n=9) Brahman placenta slices were collected by caesarean section, treatments randomized within each animal, minced and incubated in 5 ml of M199 containing 20~tg/ml 25-OH-Cholesterol, 25raM Hopes Buffer and Earle's salts, 0.1% BSA, penicillin (100 IU/ml) and streptomycin sulfate (0.2 mg/ml) in vitro for 4 or 8 hr at 39° C, pH 7.2 and under 95:5% air and COz, respectively, in the presence or absence of prostaglandin synthesis inhibitors. Treatments were: M199, indnmcthacin (INDO; 2 0 ~ I ) , meclofenamic acid (MECLO; 20~tM), PGEz (0, 1, 10 or 100 og/ml) PGEz +INDO, PGE2 + MECLO, or Pregnancy Specific Protein B (PSPB; 10 ~tg/ml). Progesterone, estradiol-17~, PGFzt~, PGE and PSPB were quantified by RIA. Data were analyzed by a factmial ANOVA- INDO and MECLO reduced (P<0.05) PGFzt~ and PGE, respectively, by 61 and 94 and 43 and 95%. No treatment affected secretion of progesterone, estradiol-17~ or PSPB (P>0.05). PGE2 increased (P<0.05) secretion of PGE by 270 day placenta by 3 fold even in the presence of INDO or MECLO, but not by 200 day placenta (P>0.05). PSPB increased (P<0.0.~ secretion of PGE by 200 day placenta. It is concluded that PGF~ does not affect placental secretion of progesterone and that INDO and MECLO inhibition of PG synthesis is not entirely through inhibition of cyclooxygenase (COX) 1 and 2 but may he downstream at the level of specific PG isomerases, through another unidentified COX, or through a nonenzymatic PG synthesis pathway.
227
P55
P56
Ovariectomy Affects Placental Secretion of Prostaglandins, Steroids and Proteins in Ewes. C. Weems l, Y. Wcems l, P. Bridges t, G. Sasser2, B. LeaMaster t, and D. Vincent. ~Animal Science Dept., University of Hawaii, Honolulu, HI 96822; ZUniverslty of Idaho, Moscow, ID 83843, USA.
Estradiol-I7fl, Indomethacin or Tamoxifen Affect Placental Function of Ewes. Y. Wecms l, P. Bridges x, G. Sasser2, B. Le~Iaster 1, D. Vincent t, and C. Wcems t. Animal Science Dept., tUniversity of Hawaii, Honolulu HI 96822; ZUniverslty of ldaho, Moscow, ID 83843, USA.
A model for regulation of urine placental secretion of progesterone (P4), when sheep are not a b o r t e d with p r o s t a g l a n d i n Fzvt (PGFzo0, has been proposed 0Neems, Y.S. et al., P r o s t a g l a u d i n s 43:203, 1992; 46:277, 1993; 48:377, 1994) whereby estradiol-17fl regulates placental secretion of p r e g n a n c y specific protein B (PSPB); PSPB regulates placental secretion of p r o s t a g l a n d i n E (PGE) and P G E regulates placental secretion of P4 at 90 days of p r e g n a n c y in sheep. The objective was to determine the effects of ovariectomy on ovine placental secretion of P4, PGE, estradiol-17fl, PSPB and PGF2o:. Ninety day p r e g n a n t ewes were ovariectomized (OVX; N=5) or l a p a r o t o m i z e d only (n=6) and a j u g u l a r venous catheter and a vena cava catheter were installed via the saphenous vein with the catheter tip 5 cm cranial to the j u n c t u r e of the ovarian vein and vena cava. J u g u l a r and vena cava blood w e r e collected at 0 and every 6 hr t h r o u g h 0600 hr on day 7 for analysis of P4, PSPB, PGE, estradiol-17fl or PGF2~ by RIA. Placentome slices were collected on day 7 and i n c u b a t e d for 4 hr at 3 9 ° C in M-199 containing Hepes Buffer, E a r l e ' s Salts, 25-OH cholesterol, penicillin, s t r e p t o m y c i n sulfate and 0.1% BSA. Media were analyzed for P4, PSPB, estradiol-17[~, P G E and PGF2~ by RIA. Placentome slices from OVX 90 day p r e g n a n t ewes in vitro secreted 3 fold more P4 (P_<0.05), two fold more estradioI-17fl, 2 fold m o r e PSPB (P_<0.05) but did not affect PGFz~ when c o m p a r e d to o v a r y intact controls (P_>0.05). These d a t a are consistent with the hypothesis that estradiol-17IJ regulates ovine placental secretion of PSPB, PSPB regulates placental secretion of P G E and P G E regulates placental secretion of P4 in sheep.
Prustaglandin F2c~ (PGF2ct) is luteolytic but does not abort intact or ovariectomized 90-100 day pl'egnant ewes (Weems, Y.S. et aL, Prostaglandins 43:203, 1992). Pregnancy specific protein B (PSPB), estradiol-1711 and prostaglandin E (PGE) appear to protect placental steroidogenesis from P G F ~ (Weems, Y.S. et at., Prostaglandins, 46:277, 1993; 48:377, 1994). The objective of this experiment was to determine the effects of estradiol-17fi, indomethaein or tamoxifen on placental function of 90 day pregnant ewes. Ninety day pregnant ewes were fitted with a jugular venous and vena cava catheter and received: vehicle, PGF2~ (8 rag/58 kg/BW) i.m. at 0 hr, estradiol-171~ (500/~g) i.m. every 6 hr or PGF2ct at 0 hr and estradiol-171~, indomethacin (100 mg) or tamoxifeu (40 ml0 Lm. every 6 hr. Five ewes were in each group. Jugular venous and inferior veda cava blood were collected every 6 hr from 0-60 hr, every 20 vain from 62-66 hr and at 72 hr for analysis for PGF2~, PGE, estradiul-171~, progesterone or PSPB by RIA. Profiles of PGF:ct, progesterone, PSPB and estradiol-17fl were analyzed by a split-plot ANOVA for repeated measures. Profiles of PGFzct (P<0.05) or estradiol171I (P<0.05) in vena cava plasma of PGF2~ + estradiol-17fl differed (P<0.05) from the otber treatment groups but did not differ among the other 5 treatment groups (P>0.05). Profiles of progesterone in jugular plasma in the indomethacin group were lower (P_<0.05) than controls. Estradiol-17fl alone (P<0.05) or tamoxifen (P<0.05) prevents the decline in PSPB wlfich was elevated (P_<0.05) at 6 hr in all groups. Abortions occurred only in the PGFz~ + estradiol-17fl group (40%). These data are consistent with our hypothesis of regulation of placental function, however, when both exogenous PGF2ct and estradlol-17fl are given, the placenta secretes both PGF2~ and estradiol-171~ which is detrimental to pregnancy.