Angiotensin II interacts with prostaglandin F2alpha and endothelin-1 as a local luteolytic factor in the bovine corpus luteum in vitro

Recent findings suggest that the ovarian renin-angiotensin system may regulate ovarian function through the paracrine/autocrine actions of angiotensin II (Ang II). In this study, we have examined and characterized the local effects of Ang II as a luteolytic factor and its interaction with prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1) in the bovine corpus luteum (CL) of the mid-luteal phase, by using an in vitro microdialysis system (MDS). Ang II was detected in the MDS perfusate (4 pg/ml), and infusion of PGF2alpha (10(-6) M) for 2 h increased the Ang II release by 50-100% during the following experimental period, in addition to its stimulation of ET-1 release. Two 2-h infusions of Ang II (10(-7)-10(-5) M) separated by a 2-h interval induced a dose- and time-dependent decrease of progesterone (P4) release by 41-66%. When the luteal explants were pre-perfused with PGF2alpha (10(-6) M) for 2 h, two consecutive perfusions of Ang II (10(-6) M) at a 2-h interval rapidly reduced the P4 release (by 50%). This reduction occurred 6 h earlier than those of infusions of PGF2alpha or Ang II alone. The simultaneous infusion of either 1) Ang II (10(-6) M) with PGF2alpha (10(-6) M), 2) ET-1 (10(-7) M) with PGF2alpha, or 3) Ang II + ET-1 with PGF2alpha (10(-6) M) for 2 h also induced a rapid and pronounced (60%) decrease in P4 release. Perfusion with the Ang II antagonist blocked the P4-suppressing activity of Ang II alone or PGF2alpha + Ang II infusion. Ang II stimulated the release of ET-1 and oxytocin during infusion but inhibited them after infusion. These results show that Ang II is released in the bovine midcycle CL in vitro, and this peptide, either alone or together with PGF2alpha, can suppress the release of P4. As PGF2alpha directly stimulated Ang II release, Ang II may influence the critical period for starting the cascade of functional luteolysis in vivo and might lead to structural luteolysis with ET-1 as a major vasoconstrictor. The overall results suggest that Ang II may have an important role at luteolysis in the bovine CL.     

Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5x10(-7) M) stimulated PMN (6x10(6) cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8x10(-6) M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2x10(-5) and 2x10(-4) M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5x10(-6) M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8x10(-6) M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2x10(-6) to 10(-5) M), but N-hexanoyl SPN was less active (from 2x10(-5) to 2x10(-4) M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.     

Sodium nitroprusside relaxes goat coronary artery through activation of calcium-dependent K+ channels

In the present investigation we have examined the hypothesis that calcium-dependent K+ channels (K(Ca)) are involved in the sodium nitroprusside (SNP)-induced vasodilatation of goat coronary artery. SNP (10(-9)-3 x 10(-6) M), added cumulatively, relaxed K+ (30 mM)-contracted coronary artery ring segments in a concentration-dependent manner with an EC50 of 1.32 x 10(-7) M (95% CL, 0.93-1.86 x 10(-7) M; n = 21). K(Ca) blocker, tetraethyl ammonium (1 mM) caused a rightward shift in the concentration-response curve of SNP with a corresponding increase in EC50 (1.62 x 10(-6) M; 95% CL, 0.44-6.02 x 10(-6) M, n = 4) of nitro vasodilator. Lowering of extra cellular Ca2+ in the physiological saline solution to 1/4 of normal selectively attenuated the vasorelaxant response of SNP, thereby causing an increase in its EC50 (2.4 x 10(-6) M; 95% CL, 1.23-4.68 x 10(-6) M, n = 4). Exposure of the tissues to high K+ (80 mM) solution, a protocol adopted to reduce the K+ gradient across the cell membrane, markedly inhibited the coronary artery relaxations induced by SNP (EC50, 2.54 x 10(-6) M; 95% CL, 1.31-4.91 x 10(-6) M, n = 4), when compared with tissues contracted with low K+ (30 mM) solution (EC50 7.9 x 10(-8); 95% CL, 4.4 x 10(-8)-1.44 x 10(-7) M, n = 6). The results suggested that a major component of SNP-induced relaxation of goat coronary artery was mediated by K(Ca) channels.     

Role of intraluteal prostaglandin F(2alpha), progesterone and oxytocin in basal and pulsatile progesterone release from developing bovine corpus luteum

The present study examined the role of intra-luteal prostaglandin (PG) F(2alpha), progesterone (P4) and oxytocin (OT) on the corpus luteum function by using specific hormone antagonists. Luteal cells from the developing CL (days 5-7 of the estrous cycle) were exposed to P4 antagonist (onapristone, OP, 10(-4)M), OT antagonist (atosiban, AT; 10(-6)M) or indomethacin (INDO; 10(-4)M), for 12h and then stimulated with PGF(2alpha) (10(-8)M) for 4h. Pre-treatment of the cells with OP, AT or INDO resulted in an increase in P4 secretion in response to PGF(2alpha). To examine the temporal effects of P4, OT and PGs on P4 secretion, dispersed luteal cells were pre-exposed to OP, AT or INDO for 1, 2, 4, 6 or 12h. Prostaglandin F(2alpha) stimulated P4 secretion (P<0.05) after 2h of pre-exposition. In the microdyalisis study, the spontaneous release of P4 from developing CL tissue was of pulsatile nature with irregular peaks at 1-2h intervals. Treatment with OP increased the number of P4 peaks (P<0.05), whereas AT and INDO significantly reduced the number of P4 peaks detected (P<0.05). Interestingly, INDO completely blocked the pulsatile nature in the release of P4, but it secretion remained stable throughout the experimental period. These results demonstrate that luteal PGF(2alpha), OT, and P4 are components of an autocrine/paracrine intra-ovarian regulatory system responsible for the episodic (pulsatile) release of P4 from the bovine CL during the early luteal phase.     

Steroid production in vitro by granulosa, theca, and luteal cells from goat ovaries

Basal progesterone (P4) production by isolated goat ovarian cells in vitro was in the order corpus luteum (CL) greater than granulosa (G) greater than theca (TH), while estradiol (E2) production was in the order TH greater than G greater than CL. In G cells, various concentrations (0.01 to 100 micrograms/ml) of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) increased P4 and E2 secretion. Testosterone (T, 10(-9) to 10(-5) M) produced dose-dependent increases in P4 and E2 secretion. Testosterone and LH together had an additive effect on E2 secretion. The combined effect of the lower (less than 10(-6) M) concentrations of T and LH on P4 production was marginally higher than either agent alone, but the increase was statistically insignificant; at higher concentrations of T (10(-6) and 10(-5) M) in combination with LH, P4 secretion was similar to that with LH alone, but was significantly (p less than 0.01 and less than 0.001, respectively) less compared to that with T alone. Follicle-stimulating hormone and T together produced a synergistic effect on E2 and an additive effect on P4 production. In TH cells, a dose-dependent increase in P4 and E2 production was observed with LH and hCG, but the effect of FSH was not significant. Testosterone produced a dose-dependent increase in P4 and E2 secretion. Testosterone and LH together induced higher steroid production than either agent alone. However, the increase was not statistically significant compared to T alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and immunological properties of related small ruminant trophoblast interferons

The objectives of this study were 1) to evaluate the biochemical and immunological properties of caprine interferon tau (cIFNtau), 2) to determine if intrauterine injection of recombinant ovine interferon tau (roIFNtau) extends CL life span in goats, and 3) to evaluate potential side effects of intramuscular (i.m.) administration roIFNtau. Caprine IFNtau was purified, and its effects on lymphocyte proliferation were evaluated. Incorporation of 3H-thymidine into newly synthesized DNA was suppressed (P<0.05) by cIFNtau. Spanish goats were also fitted with bilateral uterine catheters at Day 7 or 8 postestrus. The goats received twice-daily intrauterine injections of 100 microg roIFNtau (n = 4) or caprine serum proteins (n = 4) from Days 14 to 18 postestrus. Intrauterine injection of roIFNtau extended CL life span compared with that of control goats (26.4 +/- 1.7 vs 17.8 +/- 1.9 d, respectively; P<0.01). Potential side effects of intramuscular injections of roIFNtau were also evaluated. Goats received 0, 1, 2 or 4 mg of roIFNt on Days 10, 13, 16 or 19 of the estrous cycle. Treatment of goats with roIFN resulted in hyperthermia (P<0.01), with rectal temperatures of 40.5 degrees C recorded after 4 h and returning to normal (38.5 degrees C) after 24 h. Concomitant with the increase in rectal temperatures was a decrease (P<0.01) in plasma progesterone concentrations. Therefore, the tau interferons of goats and sheep have similar biological properties and roIFNtau has side effects associated with other classes of interferons.     

The role of cyclooxygenase inhibition in the effect of alpha-melanocyte-stimulating hormone on reactive oxygen species production by rat peritoneal neutrophils

The effect of alpha-MSH on reactive oxygen species (ROS) production by rat peritoneal neutrophils and the effect of cyclooxygenase (COX) inhibition were investigated using the chemiluminescence (CL) technique. Cells were obtained by peritoneal lavage 4h after administration of oyster glycogen to rats and were stimulated with lipopolysaccharide (LPS) from Salmonella enderitidis and phorbol 12-myristate 13-acetate (PMA). The increasing concentrations of alpha-MSH (10(-12)-10(-6) M) were added to stimulated cells alone or along with the COX inhibitors indomethacin, ketorolac or nimesulide (10(-8)-10(-5) M). Luminol and lucigenin CL levels were significantly increased in cells stimulated with LPS and PMA compared to unstimulated ones. alpha-MSH significantly reduced lucigenin CL values and this effect was completely reversed in the presence of indomethacin (10(-8) and 10(-7) M). In conclusion, alpha-MSH inhibits the production of superoxide radicals by activated rat peritoneal neutrophils and COX contributes to this effect.     

Methylation in bovine luteal cells as a regulator of luteinizing hormone action

Experiments were conducted to determine if methylation is a part of the mechanism by which luteinizing hormone (LH) and epinephrine stimulate progesterone production by dispersed bovine luteal cells. Corpora lutea (CL) were collected from 24 Holstein heifers on Day 10 of the estrous cycle and dispersed with collagenase. Net progesterone accumulation, representing total progesterone synthesized by 10(6) cells during a 2-h incubation was determined. Cells from 7 CL were treated with 0 and 5 ng LH, in the presence and absence of methylation inhibitor, S-adenosyl-homocysteine (SAH, 1 mM). LH-stimulated progesterone production was inhibited (P less than 0.05) in the presence of SAH(209 +/- 19 vs. 119 +/- 7 ng/10(6) cells). In the absence of LH, progesterone production was unaffected (87 +/- 22 vs. 68 +/- 28) by SAH. Cells from 4 CL were treated with 10 micrograms epinephrine or 10 micrograms isoproterenol with and without SAH. Both epinephrine and isoproterenol-stimulated progesterone production was inhibited (P less than 0.05) by the presence of SAH (204 +/- 24 vs. 125 +/- 18 and 198 +/- 15 vs. 130 +/- 8). Progesterone production by cells from 4 CL was unaffected by the presence of SAH when treated with Medium 199 (M199) (75 +/- 32), 10 micrograms cholera toxin, which directly stimulates adenylate cyclase on the cytoplasmic side of plasma membranes (168 +/- 19), or 3 mM dibutyryl cAMP (210 +/- 40).(ABSTRACT TRUNCATED AT 250 WORDS)     

Different actions of noradrenaline and nitric oxide on the output of prostaglandins and progesterone in cultured bovine luteal cells

The effects of noradrenaline (NA) and nitric oxide (NO) on prostaglandins (PGs) and progesterone (P4) secretion during the development of the bovine corpus luteum (CL) were investigated. Bovine luteal cells of early and mid-cycle CL were cultured for 20 to 24 h in medium containing 10% calf serum, washed, and treated with NA or nitrergic agents for an additional 16 h in a serum-free medium. NA (10(-5) M) stimulated P4 from early and mid-cycle CL by 238% and 154% (P < 0.01), respectively. Moreover, although NA induced a twofold increase in PGE2 secretion (P < 0.01) in both examined periods, the effect of NA on PGF2alpha secretion was approximately 1.5 times higher (P < 0.05) in early than in mid-cycle CL. Two NO synthase inhibitors, L-NAME and L-NOARG (both 10(-4) M), stimulated P4 secretion only in mid-luteal cells (P < 0.01), although they did not affect the cells from early CL. Although a NO donor, S-NAP (10(-4) M) inhibited P4 secretion from mid-cycle luteal cells (P < 0.05), it strongly stimulated PGE2 in both examined phases (P < 0.001). On the other hand, the output of PGF2alpha was stimulated by S-NAP only in the cells of the mid-cycle CL (P < 0.01). The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL functions. Whereas NA may play a supporting role in luteal development, NO may participate in the functional regression of the bovine CL by inhibiting steroidogenesis.     

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.     

4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5'-tetraphosphate (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5',5'-P(1),P(5)-pentaphosphate and dAp(4)dA from the appropriate substrates, p(4)A and dATP, respectively. Formation of Ap(3)A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p(4)A and diadenosine 5',5',-P(1),P(4)-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.     

Action of exogenous potassium and calcium ions on in vitro metacyclogenesis in Trypanosoma cruzi.

The cations Ca2+ and K+ and the anions Cl-, HCO3-, and PO4- were studied for their contribution to metacyclic trypomastigote formation of Trypanosoma cruzi in starvation media consisting of phosphate-buffered saline (PBS) + 10 mM proline + 10 mM sodium acetate as well as one of the following salts: 0.035% NaHCO3 (PBSNPA), 0.035% K2CO3 (PBSKPA) or 0.035% K2HPO4 (PBSPPA). Isolates CL and DM28c were activated to transform with 5% CO2 and the percent metacyclogenesis determined after incubation for 96 h in PBS starvation media. Maximal metacyclogenesis was found with CaCl2 and KCl. In the presence of K+, the percent transformation was highest with the phosphate salt, followed by the carbonate and the chloride salts. Cells incubated in PBSNPA and the cationic ionophores A23187 (5 x 10(-6) M), lasalocid (5 x 10(-6) M), and valinomycin (10(-8) M) do not survive; addition of 2 mM CaCl2 or 17 mM KCl to DM28c cells, reversed the lethal action of the ionophores permitting differentiation into metacyclic forms. The addition of CaCl2 to CL cells incubated in ionophores abrogated the lethal effect of the ionophores but transformation was significantly different than in control preparations. Adding KCl to ionophore incubated cells resulted in normal levels of transformation except in the case of valinomycin. DM28c and CL cells incubated in PBSKPA show significantly greater metacyclogenesis in the presence of 5 mM EGTA. These results indicate that exogenous concentrations of several cations and anions significantly influence T. cruzi metacyclogenesis and that the degree of response by the parasite to free ion levels may be strain dependent.     

Secretion and gene expression of metalloproteinases and gene expression of their inhibitors in porcine corpora lutea at different stages of the luteal phase

We hypothesize that spontaneous regression of corpora lutea (CL) involves short-lasting restructure of luteal tissue with an activation of matrix metalloproteinases (MMPs) and their respective inhibitors (tissue inhibitors of metalloproteinase, TIMPs). This was tested by determining the gene expression of MMP-1, MMP-2, and MMP-9 and respective TIMP-1 and TIMP-2 in luteal tissue from sows at the early, midluteal, and late luteal phase (Days 6-8, Days 9-11, and Days 13-15 of estrous cycle). Gene expression of the three MMPs was low in early, slightly higher in midluteal, and significantly elevated (P < 0.05) in regressing CL. An inverse pattern was found for gene expression of TIMP-1 and TIMP-2. Under culture conditions, the release of MMPs was determined from steroidogenic large luteal cells (LLC). LLC harvested from regressing CL released significantly (P < 0.05) more active MMPs than cells obtained from CL at the early luteal phase. As luteolysis can be induced by prostaglandin F(2alpha) (PGF(2alpha)) and tumor necrosis factor alpha (TNF), we studied their effects on LLC under culture conditions. Treatment of cells with PGF(2alpha) or TNF (10(-7) M or 3 x 10(-9) M, respectively) induced a significantly higher release of MMPs, and gene expression was also significantly stimulated in comparison to that in untreated LLC. The gene expression of TIMPs remained unaffected by either treatment. It is concluded that at the beginning of luteolysis, MMPs are expressed and released in high amounts and that this is essential for the structural regression of the CL.     

Determination of agmatine in biological samples by capillary electrophoresis with chemiluminescence detection

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 x 10(-4)M luminol added to the CE running buffer and 5.0 x 10(-4)M NaBrO in 100 mM NaCO3-NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 x 10(-6)M (S/N=3). The precision (R.S.D.) on peak height (at 1 x 10(-5)M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at approximately 1950 ng/g wet tissue.     

Progestin implants can rescue demi-embryo pregnancies in goats: a case study

Survival after transfer of demi-embryos (i.e., half-embryos produced by embryo splitting) to recipients usually is lower than survival after transfer of intact embryos. Reduced survival after demi-embryo transfer could be due to loss of viability after splitting, failure of a viable demi-embryo to prevent corpus luteum (CL) regression in the recipient female, or a combination of factors. From a retrospective analysis of pregnancy and embryo survival rates after demi-embryo transfer in sheep and goats, we report the rescue of caprine demi-embryo pregnancies in which CL regression occurred at the end of diestrus despite the presence of a viable conceptus in the uterus with progestin implants. Day 5 or 6 morulae and blastocysts were flushed from superovulated ewes and does and split into demi-embryos of approximately equal halves. Demi-embryos were either transferred fresh to synchronized recipients of the homologous species or frozen in liquid nitrogen. Approximately half of the recipient does and ewes were treated with norgestomet implants on Day 10 of the embryo transfer cycle and again 2 wk later. Serum collected on Day 25 from recipients with implants was assayed for progesterone to determine if a CL of pregnancy had been maintained. Pregnancy was diagnosed by ultrasonography on Day 35 of gestation. Corpus luteum regression occurred despite the presence of a viable conceptus in the uterus in 6 of 55 progestin-treated caprine demi-embryo recipients and in 0 of 66 ovine demi-embryo recipients. Five of the caprine pregnancies were maintained to term with norgestomet implants and produced 5 live kids. The sixth fetus, which was carried by a progestin implant-treated 8-mo-old doeling, died at approximately 50 d of gestation. These results suggest that, at least in goats, some demi-embryos may provide inadequate signaling for maternal recognition of pregnancy, and such pregnancies can be rescued with progestin treatment to the doe.     

Protective activity of propofol,Diprivan and intralipid against active oxygen species.

We separately studied the antioxidant properties of propofol (PPF), Diprivan (the commercial form of PPF) and intralipid (IL) (the vehicle solution of PPF in Diprivan) on active oxygen species produced by phorbol myristate acetate (10(-6) M)-stimulated human polymorphonuclear leukocytes (PMN: 5 x 10(5) cells/assay), human endothelial cells (5 x 10(5) cells/assay) or cell-free systems (NaOCl or H2O2/peroxidase systems), using luminol (10(-4) M)-enhanced chemiluminescence (CL). We also studied the protective effects of Diprivan on endothelial cells submitted to an oxidant stress induced by H2O2/MPO system: cytotoxicity was assessed by the release of preincorporated 51Cr. Propofol inhibited the CL produced by stimulated PMN in a dose dependent manner (until 5 x 10(-5) M, a clinically relevant concentration), while Diprivan and IL were not dose-dependent inhibitors. The CL produced by endothelial cells was dose-dependently inhibited by Diprivan and PPF, and weakly by IL (not dose-dependent). In cell free systems, dose-dependent inhibitions were obtained for the three products with a lower effect for IL. Diprivan efficaciously protected endothelial cells submitted to an oxidant stress, while IL was ineffective. By HPLC, we demonstrated that PPF was not incorporated into the cells. The drug thus acted by scavenging the active oxygen species released in the extracellular medium. IL acted in the same manner, but was a less powerful antioxidant.     

Inhibition by angiotensin converting enzyme inhibitors of endothelin secretion from cultured human endothelial cells.

We conducted a study to determine whether angiotensin converting enzyme inhibitors (ACEIs) inhibit endothelin secretion from cultured human endothelial cells. Confluent umbilical vein endothelial cells were incubated in multi-well plates with culture medium containing either captopril (10(-6), 10(-5), 10(-4) M) or enalaprilat (10(-7), 10(-6), 10(-5) M) for 6 hours. Immunoreactive endothelin in the medium was measured by radioimmunoassay. Calf serum (CS) stimulated endothelin release in a concentration-dependent manner, and both ACEIs inhibited 5% CS-stimulated endothelin release in a concentration-dependent manner. To explore the mechanisms of ACEI-induced suppression of endothelin release, the effects of angiotensin II (10(-8), 10(-7), 10(-6) M), angiotensin converting enzyme (0.1, 1, 10 mU/ml), bradykinin (10(-8), 10(-7), 10(-6) M), and sodium nitroprusside (10(-6), 10(-5), 10(-4) M) on endothelin release were also examined. Although angiotensin II and angiotensin converting enzyme had no significant effect on endothelin release, concentration-dependent suppression occurred with bradykinin and sodium nitroprusside. These results indicate that ACEIs inhibit the stimulated release of endothelin from human endothelial cells, and provide indirect evidence that ACEI-induced ET suppression may be mediated via potentiation of autacoid formation from the cells.