Endothelial vasodilator production by uterine and systemic arteries. VIII. Estrogen and progesterone effects on cPLA2, COX-1, and PGIS protein expression.

During ovine pregnancy, when both estrogen and progesterone are elevated, prostacyclin (PGI2) production by uterine arteries and the key enzymes for PGI2 production, phospholipase A2 (cPLA2), cyclooxygenase 1 (COX-1), and prostacyclin synthetase (PGIS), are increased. This study was conducted to determine whether exogenous estradiol-17beta (E2beta) with or without progesterone (P4) treatment would increase cPLA2, COX-1, and PGIS protein expression in ovine uterine, mammary, and systemic (renal, mental, and coronary) arteries. Nonpregnant ovariectomized sheep received vehicle (n = 10), P(4) (0.9-g controlled internal drug release vaginal implants; n = 13), E2beta (5 microg/kg bolus followed by 6 microg x kg(-1) x day(-1); n = 10), or P4 + E2beta (n = 12). Arteries were procured on Day 10, and cPLA2, COX-1, and PGIS protein were measured by Western immunoblot analysis in endothelial isolated proteins and vascular smooth muscle (VSM). The levels of cPLA2 was increased in uterine artery endothelium in ewes treated with P4 + E2beta but was not altered by any steroid treatment in renal, coronary, mammary, or omental artery endothelium or in VSM of any evaluated artery. Similarly, COX-1 was increased in uterine artery endothelium with P4 + E2beta but was not significantly altered by treatment in other endothelium or VSM. E2beta treatment increased PGIS protein in uterine and renal artery endothelium but did not alter PGIS in other endothelial tissue. P4 increased PGIS expression in the uterine, mammary, omental, and renal artery VSM, and E2beta increased PGIS expression in the uterine and omental artery VSM. Both E2beta and P4 treatments differentially alter protein expression of the key enzymes involved in PGI2 production in different artery types and may play an important role in the control of blood flow redistribution during hormone replacement therapy.     

Regulation of types I and III NOS in ovine uterine arteries by daily and acute estrogen exposure

Nitric oxide contributes to estrogen-mediated uterine vasodilation; however, the nitric oxide synthases (NOS) involved and their location within uterine arteries are incompletely documented. We investigated the effects of repetitive daily and acute estradiol-17beta (E(2)beta) exposure on uterine hemodynamics and NOS abundance and localization in uterine arteries from nonpregnant ovariectomized ewes receiving daily intravenous E(2)beta (1 microg/kg, n = 5) or no E(2)beta (n = 7) for 5 days to determine NOS abundance, cGMP contents, and NOS immunohistochemistry. Daily E(2)beta increased basal and E(2)beta-mediated rises in uterine blood flow (UBF) 36 and 43% (<0.01), respectively, calcium-dependent NOS activity 150% (P < 0.02) in endothelium-intact and -denuded ( approximately 40% of total NOS) arteries, and cGMP contents 39% (P < 0.05). Endothelial (eNOS) was detected in luminal endothelium, whereas neuronal NOS (nNOS) protein was only in the media. A second group of ewes received E(2)beta (1 microg/kg iv) for 4 days and acute intravenous E(2)beta (n = 8) or vehicle (n = 4) on day 5. UBF rose 5.5-fold (P < 0.001) 115 min after E(2)beta, at which time only endothelium-derived calcium-dependent NOS activity increased 30 +/- 13% (P < 0.05). Daily E(2)beta enhances basal and E(2)beta-mediated increases in UBF, which parallel increases in endothelium-derived eNOS and smooth muscle-derived nNOS. Acute E(2)beta, however, selectively increases endothelium-derived eNOS.     

Global protein expression profiling underlines reciprocal regulation of caveolin 1 and endothelial nitric oxide synthase expression in ovariectomized sheep uterine artery by estrogen/progesterone replacement therapy

Ovariectomized (OVX) ewes were assigned to receive vehicle, progesterone (P4, 0.9-g controlled internal drug release vaginal implants), estradiol-17beta (E2, 5 microg/kg bolus + 6 microg kg(-1) day(-1)), or P4 + E2 for 10 days (n = 3/group). Uterine artery endothelial proteins were mechanically isolated on Day 10. The samples were used for protein expression profiling by the Ciphergen Proteinchip system and immunoblotting analysis of endothelial nitric oxide synthase (NOS3, also termed eNOS) and caveolin 1. Uterine artery rings were cut and analyzed by immunohistochemistry to localize NOS3 and caveolin 1 expression. With the use of the IMAC3 protein chip with loading as little as 2 microg protein/sample, many protein peaks could be detected. Compared to vehicle controls, a approximately 133.1-kDa protein was identified to be upregulated by 2- to 4-fold in OVX ewes receiving E2, P4, and their combination, whereas a approximately 22.6-kDa protein was downregulated by 2- to 4-fold in OVX ewes receiving E2 and E2/P4, but not P4 treatments. Western blot analysis revealed that E2, P4, and their combination all increased NOS3 protein, whereas E2 and its combination with P4, but not P4 alone, downregulated caveolin 1 expression. Immunohistochemical analysis revealed that NOS3 was mainly localized in the endothelium and upregulated by E2, whereas caveolin 1 was localized in both endothelium and smooth muscle and downregulated by E2. Thus, our data demonstrate that uterine artery endothelial NOS3 and caveolin 1 are regulated reciprocally by estrogen replacement therapy. In keeping with the facts that E2, but not P4, causes uterine vasodilatation and that E2 and P4 increase NOS3 expression, but only E2 decrease caveolin 1 expression, our current study suggests that both increased NOS3 expression and decreased caveolin 1 expression are needed to facilitate estrogen-induced uterine vasodilatation.     

Development and use of an ovarian synchronization model to study the effects of endogenous estrogen and nitric oxide on uterine blood flow during ovarian cycles in sheep

The objective of the current study was to develop an ovine animal model for consistent study of uterine blood flow (UBF) changes during synchronized ovarian cycles regardless of season. Sheep were surgically bilaterally instrumented with uterine artery blood flow transducers and 5-7 days later implanted with a vaginal progesterone (P(4))-controlled internal drug-releasing device (CIDR; 0.3 g) for 7 days. On Day 6 of P(4), sheep were given two prostaglandin F(2 alpha) injections (7.5 mg i.m. 4 h apart). At CIDR removal, Experimental Day 0, zero (n = 9), 500 IU (n = 8), or 1000 IU (n = 7) eCG was injected i.m.; UBF was monitored continuously for 55-75 h. Jugular blood was sampled every 8 h to evaluate levels of P(4), estradiol-17 beta (E(2)beta) and luteinizing hormone (LH). The inhibitor of nitric oxide synthase, L-nitro-arginine methyl ester (L-NAME) was infused in a stepwise fashion unilaterally into one uterine artery at 48-50 h after 500 IU eCG and the effects on UBF were examined (n = 7). The zero-eCG group gradually increased UBF from a baseline of 17.4 +/- 3.9 to 80.5 +/- 1.1 ml/min. The 500-IU-eCG group increased UBF between 10 and 15 h from a baseline of 11 +/- 3.3 to 83.3 +/- 1.0 ml/min, whereas UBF for the 1000-IU-eCG group was higher (100.1 +/- 1.7 ml/min) than that seen in either of the other groups. Plasma P(4) fell to baseline within 8 h of CIDR removal, while E(2)beta rose gradually in association with elevations in UBF. LH surges occurred between 32 and 56 h after CIDR removal and the LH surge occurred earlier in the 1000-IU-eCG group than the other two groups (P < 0.01). L-NAME infusion dose dependently reduced maximum levels of UBF ipsilaterally by 54.6% +/- 6.2%, but contralaterally only by 27.4% +/- 8.5%. Regardless of season, either dose of eCG will result in analogous UBF responses. During the follicular phase, elevations in UBF are in part locally controlled by the de novo production of nitric oxide.     

Calcium-activated potassium channels and nitric oxide coregulate estrogen-induced vasodilation

Nitric oxide synthase (NOS) contributes to estradiol-17beta (E(2)beta)-induced uterine vasodilation, but additional mechanisms are involved, and the cellular pathways remain unclear. We determined if 1) uterine artery myocytes express potassium channels, 2) E(2)beta activates these channels, and 3) channel blockade plus NOS inhibition alters E(2)beta-induced uterine vasodilation. Studies of cell-attached patches identified a 107 +/- 7 pS calcium-dependent potassium channel (BK(Ca)) in uterine artery myocytes that rapidly increased single-channel open probability 70-fold (P < 0.05) after exposure to 100 nM E(2)beta through an apparent cGMP-dependent mechanism. In ovariectomized nonpregnant ewes (n = 11) with uterine artery flow probes and catheters, local BK(Ca) blockade with tetraethylammonium (TEA; 0.05-0.6 mM) dose dependently inhibited E(2)beta-induced uterine vasodilation (n = 37, R = 0.77, P < 0.0001), with maximum inhibition averaging 67 +/- 11%. Mean arterial pressure (MAP) and E(2)beta-induced increases (P 相似文献   

Effect of uterine blood flow occlusion on shear stress-mediated nitric oxide production and endothelial nitric oxide synthase expression during ovine pregnancy

During normal pregnancy, uterine blood flow (UBF) is increased in association with elevations of endothelial nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression. Shear stress increases endothelial-derived NO production to reduce vasomotor tone. We hypothesized that decreasing in vivo UBF, and thus shear stress, will decrease NO and/or eNOS levels. In this experiment, one of the main uterine arteries of chronically instrumented late pregnant sheep (125 +/- 1 days' gestation [mean +/- SEM]; n = 15) was occluded for 24 h. Cardiovascular parameters (systemic and uterine arterial pressure, heart rate [HR], and ipsilateral and contralateral UBF) and NO(2)/NO(3) (NO(x)) levels were evaluated. Although UBF measured using Transonic flow probes was reduced unilaterally 41.5% +/- 2.1%, uterine perfusion pressure only fell 12.2% +/- 4.5%. Systemic arterial blood pressure and HR were unaltered. Using radioactive microspheres, ipsilateral UBF was reduced approximately 28% during occlusion. The redistribution of UBF to other reproductive tissues suggests that collateral circulation develops in response to occlusion. Systemic arterial and uterine venous NO(x) levels were reduced 22.1% +/- 6.7% and 22.6% +/- 7.6%, respectively, during occlusion. Treatment with microspheres produced an unexpected initial ( approximately 2.5 h) increase in systemic arterial and uterine venous NO(x) levels by 116% +/- 30% and 97% +/- 49%, respectively. Despite a decline in NO(x) levels after 6 h, no significant differences versus preocclusion NO(x) levels were detected by 24 h of occlusion in this experimental group. In contrast, NO(x), UBF, and uterine perfusion pressure levels unexpectedly failed to return to baseline values following release of occlusion. No differences in uterine artery eNOS expression were demonstrated by Western analysis from occlusion. Thus, our data suggest that shear stress may mediate in vivo vasomotor tone via production of NO(x).     

Endothelial vasodilator production by uterine and systemic arteries. VI. Ovarian and pregnancy effects on eNOS and NO(x)

Normal pregnancy and the follicular phase of the ovarian cycle are both estrogen-dominated physiological states that are characterized by elevations in uterine blood flow and endothelial nitric oxide synthase (eNOS) protein expression in the uterine artery (UA) endothelium. It is unknown if elevations in mRNA level account for the changes in protein or eNOS activity. We tested the hypothesis that pregnancy and the follicular phase are associated with increases in eNOS mRNA and the consequent elevated expression of eNOS protein results in increased circulating nitric oxide (NO) levels. UA were obtained from pregnant (PREG; n = 8; 110-130 days gestation; term = 145 +/- 3 days), nonpregnant luteal (LUT; n = 6), nonpregnant follicular (FOL; n = 6), and nonpregnant ovariectomized (OVEX; n = 6) sheep. Circulating NO levels were analyzed as total NO(2)-NO(3) (NO(x)). Western analysis performed on UA endothelial-isolated proteins demonstrated that eNOS protein levels were OVEX = LUT < or = FOL < PREG (P < 0.05), whereas eNOS mRNA expression (RT-PCR) in UA endothelial cells obtained by limited collagenase digestion was OVEX < LUT < FOL < PREG (P < 0.05). Pregnancy dramatically elevated eNOS protein (4.1- to 6.9-fold) and mRNA (2.4- to 6.9-fold) over LUT controls (P < 0.01). Circulating NO(x) levels were not altered by ovariectomy or the ovarian cycle but were elevated from 4.4 +/- 1.1 microM in LUT to 12 +/- 4, 22 +/- 3, and 41 +/- 3 microM at 110, 120, and 130 days gestation (P < 0.01). Systemic NO(x) levels in singleton (12.5 +/- 1.6 microM) were less (P < 0.01) than in multiple (twin 27.6 +/- 6.5 microM; triplet = 46 +/- 10 microM) pregnancies. Therefore, the follicular phase and, to a much greater extent, pregnancy are associated with elevations in UA endothelium-derived eNOS expression, although significant increases in systemic NO(x) levels were only observed in the PREG group (multiple > singleton). Thus, although UA endothelial increases in eNOS protein and mRNA levels are associated with high estrogen states, increases in local UA NO production may require additional eNOS protein activation to play its important role in the maintenance of uterine blood flow in pregnancy.     

Endothelial vasodilator production by uterine and systemic arteries. IV. Cyclooxygenase isoform expression during the ovarian cycle and pregnancy in sheep

Uterine artery endothelial production of the potent vasodilator, prostacyclin, is greater in pregnant versus nonpregnant sheep and in whole uterine artery from intact versus ovariectomized ewes. We hypothesized that uterine artery cyclooxygenase (COX)-1 and/or COX-2 expression would be elevated during pregnancy (high estrogen and progesterone) and the follicular phase of the ovarian cycle (high estrogen/low progesterone) as compared to that in luteal phase (low estrogen/high progesterone) or in ovariectomized (low estrogen and progesterone) ewes. Uterine and systemic (omental) arteries were obtained from nonpregnant luteal-phase (LUT; n = 10), follicular-phase (FOL; n = 11), and ovariectomized (OVEX; n = 10) sheep, as well as from pregnant sheep (110-130 days gestation; term = 145 +/- 3 days; n = 12). Endothelial and vascular smooth muscle (VSM) COX-1 protein levels and uterine artery endothelial cell COX-1 mRNA levels were compared. Using immunohistochemistry and Western analysis, the primary location of COX-1 protein was the endothelium; that is, we observed 2.2-fold higher COX-1 protein levels in intact versus endothelium-denuded uterine artery and a 6.1-fold higher expression in the endothelium versus VSM (P < 0.05). COX-2 protein expression was not detectable in either uterine artery endothelium or VSM. COX-1 protein levels were observed to be higher (1.5-fold those of LUT) in uterine artery endothelium from FOL versus either OVEX or LUT nonpregnant ewes (P < 0.05), with substantially higher COX-1 levels seen in pregnancy (4.8-fold those of LUT). Increases in uterine artery endothelial COX-1 protein were highly correlated to increases in the level of COX-1 mRNA (r(2) = 0.66; P < 0.01) for all treatment groups (n = 6-8 per group), suggesting that increased COX-1 protein levels are regulated at the level of increased COX-1 mRNA. No change in COX-1 expression was observed between groups in a systemic (omental) artery. In conclusion, COX-1 expression is specifically up-regulated in the uterine artery endothelium during high uterine blood flow states such as the follicular phase and, in particular, pregnancy.     

Effect of estradiol-17beta on PGF and total protein content in bovine uterine flushings and peripheral plasma concentration of 13, 14-dihydro-15-keto-PGF(2alpha)

Two experiments were conducted to examine the effect of estradiol-17beta (E(2)-17beta) on content of immunoreactive prostagladin F(2)alpha (PGF, ng) and total protein (TUP, mg) in uterine flushings, as well as concentrations of 13, 14-dihydro-15-keto-PGF(2)alpha (PGFM) in plasma (Pg/ml). In experiment 1, Holstein heifers were utilized in a single reversal trial in which either E(2)-17beta (3 mg in 2 ml saline/ethanol 50:50; n=5) or vehicle alone (n=6) were given intravenously on day 14 or 15 of the estrous cycle (Period 1) following an induced estrus (day of estrus = day 0). Treatment (Trt) groups were reversed in Period 2 (Day 14 or 15 of the second estrous cycle). Jugular venous plasma was obtained before treatment (Oh), and at 5, 6, and 9h posttreatment (PT). Uterine flushings were collected nonsurgically in vivo , per cervix, via Foley catheter at 6h PT (20 ml of .9% saline per uterine horn). E(2)-17beta did not significantly alter (E(2)-17beta vs vehicle; x(-) +/- S.E.M.) PGF (1674 +/- .11 +/- 338.39 vs 1889.91 +/- 400.24 ng; P> .10) or TUP (33.25 +/- 2.57 vs 39.16 +/- 3.04 mg; P > .10). However, E(2)-17beta increased (P < .05) plasma PGFM (E(2)-17beta vs vehicle) after treatment (0h, 113.2 vs 163.8; 5h, 312.5 vs 203.9; 6h, 324.5 vs 198.0; 9h, 323.2 vs 246.8, pg/ml). In experiment 2, crossbred beef cattle received comparable treatments of either E(2)-17beta (n=5) or vehicle (n=5) on day 14 or 15 postestrus. Jugular venous plasma was obtained at 0h PT, and at 6h PT. Uterine flushings (1.9% saline, 20 ml per uterine horn) and peripheral plasma were collected at slaughter. Estradiol-17beta increased PGF (30.07 +/- 5.94 vs 8.46 +/- 2.01 ng; P> <.05) in uterine flushings as well as PGFM in plasma (E(2)-17beta : 55.82 +/- 19.13 pg/ml, at 0h and 89.31 +/- 14.02 pg/ml, at 6h, vs saline: 103.46 +/- 50.73 pg/ml, at 0h and 17.78 +/- 14.22, at 6h). Estradiol-17beta stimulated uterine production and release of PGF and protein as measured in flushings (experiment 2) as well as plasma PGFM responses (experiments 1 and 2). Uterine and/or cervical stimulation of experiment 1 may have masked uterine response to E(2)-17beta.     

Endothelial vasodilator production by uterine and systemic arteries. V. Effects of ovariectomy, the ovarian cycle, and pregnancy on prostacyclin synthase expression

Prostacyclin (PGI(2)) is a potent vasodilator, the level of which is increased during pregnancy, and is the main eicosanoid of which production is elevated in the endothelium and vascular smooth muscle (VSM) of both uterine and omental (systemic) arteries. We tested the hypothesis that during physiologic states that have high uterine blood flow, such as pregnancy and the follicular phase of the ovarian cycle (versus luteal phase and ovariectomized ewes), there is an increased level of prostacyclin synthase (PGIS) expression in ovine uterine and omental artery endothelium and VSM. To investigate this, the cellular localization and PGIS protein expression level in uterine and systemic arteries was examined by immunohistochemistry as well as by Western immunoblot analysis of endothelial-isolated protein and denuded vessels (VSM). Whole uterine, but not omental (systemic), arteries from the pregnant ewes showed an increase (P < 0.001) in PGIS expression. Further localization of PGIS protein by immunohistochemistry and quantification by Western analysis showed PGIS to be somewhat higher in the uterine artery VSM (69 +/- 7%) than endothelium (31 +/- 7%). PGIS protein levels in uterine and omental artery endothelial isolated protein were not altered by ovariectomy or the ovarian cycle, although they were both significantly elevated by pregnancy. Uterine and omental artery VSM PGIS expression levels also were not altered by ovariectomy or the ovarian cycle, whereas PGIS expression, in uterine but not omental artery VSM showed a significant elevation during pregnancy. Thus, the rise in PGI(2) production by uterine arteries observed in ovine pregnancy is paralleled by an elevation in PGIS expression in both endothelium and VSM, whereas those seen in omental arteries is associated with increases in endothelial PGIS.     

The influence of estradiol and progesterone on the concentrations of uterine oxytocin receptors and plasma PGFM in response to oxytocin in ovariectomized gilts

Peripubertal gilts (n = 25) were treated with corn oil (CO) or ovarian steroids, one month following an ovariectomy. The first day of treatment was assigned as the first day of the experiment. The gilts received: Group (Gr) I (n = 4)--CO (2 mL x day(-1) from 1st to 12th day), Gr II (n = 4) and Gr III (n = 4)--progesterone (P4; 10 to 100 mg x day(-1) from 1st to 12th day), Gr IV (n = 5)--estradiol benzoate (EB; 400 microg x day(-1) from 1st to 3rd day), Gr V (n = 4) and Gr VI (n = 4)--EB + P4 (EB 400 microg x day(-1) from 1st to 3rd day, 20 microg x day(-1) at 6th and 9th day, 50 microg at 12th day plus P4 10 to 100 mg from 4th to 15th day). All gilts were injected with oxytocin (OT; 20 IU; i.v.) on the following days of the experiment: 13th (Gr I and Gr II), 15th (Gr III and Gr IV), 16th (Gr V) and 18th (Gr VI). Concentrations of the PGF2alpha metabolite--PGFM were determined in blood samples, collected from 30 min before to 120 min after OT injection. Baseline PGFM concentrations (30 min before OT) differed among treatment groups and were the highest in Gr V and Gr VI (P < 0.01 vs. other groups). The magnitude of the PGFM response to OT increased only in four of the five gilts of Gr IV and in three of the four gilts of Gr VI, and it was higher (P = 0.009) in Gr VI than in Gr IV. In the remaining groups, PGFM concentrations did not increase above the baseline in response to OT. The day after OT injection, oxytocin receptors (OTR) were found in the uterine tissues of all animals studied. The lowest OTR concentrations were in Gr I--75.5 +/- 11.2 fmol x mg protein(-1) and the highest in Gr IV--712.9 +/- 86.7 fmol x mg protein(-1); (P < 0.05 vs. other groups). The values of K of OTR differed among groups (P < 0.001) and ranged from 1.62 +/- 0.44 nM in Gr I to 12. 08 +/- 1.9 nM in Gr VI. A positive correlation (r = 0.54; P < 0.01) between plasma E2 and uterine OTR concentrations was observed. In conclusion, E2 and P4 are involved in both PGF2 synthesis/secretion and OTR formation, however, full PGF response to OT does not develop before puberty. Estrogens are evident stimulators of uterine OTR synthesis ingilts.     

Effects of estradiol and progesterone on the reproductive tract and on uterine sex steroid receptors in female lambs

The effects of estradiol-17 beta (E(2)) and progesterone (P) on the reproductive tract and on uterine estrogen receptors and P receptors were studied in 2-mo-old female lambs (n = 11). On Days 0, 1 and 2, E(2) (1 ug/kg, Group E, n = 4), P (0.3 mg/kg, Group P, n = 4) or corn oil (control) vehicle (Group C, n = 3) were administered, and in Day 3 all lambs were slaughtered. Group E (n = 12) had E(2) serum concentrations (mean +/- SEM) of 43.8 +/- 2.2 pmol L , similar to that of the follicular phase; while P concentrations in Group P (n = 12) were similar (2.8 +/- 0.18 nmol L ) to those of the luteal phase of the ewe estrous cycle. The E(2) treatment increased the reproductive tract weight, while P treatment increased only the uterine weight. Both E(2) and P receptors from upper and middle uterine zones (including the myometrium, endometrium and caruncles) were determined by binding assays with tritiated hormones, dextran-charcoal separation and inverse Scatchard analysis. Both the E(2) and P treatments decreased E(2) and P receptor concentrations in upper and middle zones, although the upper zone had higher receptor concentrations than the middle zone (P < 0.01). E(2) receptor concentrations in the upper zone (mean +/- SEM, fmol mg prot) were 1236 +/- 34, 667 +/- 80 and 444 +/- 103 for Groups C, P and E, respectively. The P receptor concentrations were 2434 +/- 135, 1273 +/- 102 and 1536 +/- 213 for the same groups. The high uterine P receptor concentrations allowed P action without prior estrogen priming of female lambs. The present results suggest that E(2) and P might down-regulate their own and each other's receptors during development. The biological responses induced by E(2) and P, as measured by the reproductive tract weight, demonstrated that at an early stage of development uterine receptors are physiologically active.     

Endothelium-derived nitric oxide synthase protein expression in ovine placental arteries

During the third trimester, fetoplacental and uterine blood flows increase dramatically to meet the high metabolic demands of the growing fetus. We hypothesized that the expression of endothelial nitric oxide synthase (eNOS) in fetoplacental artery endothelium and the concentrations of nitric oxide (NO) and cyclic GMP (cGMP) in amniotic fluid (AF) are increased during the third trimester of ovine gestation. Placental arteries and AF were collected from ewes at 110, 120, 130, and 142 days of gestation (n = 24; mean +/- SEM term = 145 +/- 3 days). Expression of eNOS protein was measured in intact and denuded placental arteries and in endothelium-derived protein by Western analysis and confirmed by immunohistochemistry. Concentrations of NO (nitrates plus nitrites) and cGMP were determined in AF. Placental artery eNOS protein expression was localized to the endothelium, where it was markedly greater than in vascular smooth muscle. Placental artery endothelium-derived eNOS expression and AF cGMP concentrations were similar at 110 and 120 days of gestation; however, both peaked at 130 days at levels two- to threefold above baseline (P < 0.05) before returning to baseline at 142 days of pregnancy. The AF NO (nitrates plus nitrites) levels, however, increased progressively between 120 days of gestation and term (P < 0.05). We concluded that endothelium-derived placental artery eNOS levels, AF NO (nitrates plus nitrites), and AF cGMP were markedly increased during the third trimester, thus supporting a role for NO-mediated elevations in cGMP in the control of fetoplacental blood flow.     

Glucose uptake during centrally induced stress is insulin independent and enhanced by adrenergic blockade.

Glucose utilization increases markedly in the normal dog during stress induced by the intracerebroventricular (ICV) injection of carbachol. To determine the extent to which insulin, glucagon, and selective (alpha/beta)-adrenergic activation mediate the increment in glucose metabolic clearance rate (MCR) and glucose production (R(a)), we used five groups of normal mongrel dogs: 1) pancreatic clamp (PC; n = 7) with peripheral somatostatin (0.8 microg x kg(-1) x min(-1)) and intraportal replacement of insulin (1,482 +/- 84 pmol x kg(-1) x min(-1)) and glucagon (0.65 ng x kg(-1) x min(-1)) infusions; 2) PC plus combined alpha (phentolamine)- and beta (propranolol)-blockade (7 and 5 microg x kg(-1) x min(-1), respectively; alpha+beta; n = 5); 3) PC plus alpha-blockade (alpha; n = 6); 4) PC plus beta-blockade (beta; n = 5); and 5) a carbachol control group without PC (Con; n = 10). During ICV carbachol stress (0-120 min), catecholamines, ACTH, and cortisol increased in all groups. Baseline insulin and glucagon levels were maintained in all groups except Con, where glucagon rose 33%, and alpha, where insulin increased slightly but significantly. Stress increased (P < 0.05) plasma glucose in Con, PC, and alpha but decreased it in beta and alpha+beta. The MCR increment was greater (P < 0.05) in beta and alpha+beta than in Con, PC, and alpha. R(a) increased (P < 0.05) in all groups but was attenuated in alpha+beta. Stress-induced lipolysis was abolished in beta (P < 0.05). The marked rise in lactate in Con, PC, and alpha was abolished in alpha+beta and beta. We conclude that the stress-induced increase in MCR is largely independent of changes in insulin, markedly augmented by beta-blockade, and related, at least in part, to inhibition of lipolysis and glycogenolysis, and that R(a) is augmented by glucagon and alpha- and beta-catecholamine effects.     

Uterine prostaglandin and blood flow responses to estradiol-17 beta in cyclic cattle

Normal cyclic dairy cattle (n = 7) underwent a midventral laparotomy on day 17 of the estrous cycle and were fitted, ipsilateral to the CL, with: an electromagnetic flow transducer around the uterine artery (UA; n = 5); catheters within the ovarian vein (OV; n = 7) via a uterine branch of the ovarian vein, uterine branch of the ovarian artery (UBOA; n = 5) and facial artery (FA; n = 7). On day 18, blood samples were collected at 30 min intervals for 1 h prior to injection of estradiol-17 beta (E2; 3 mg) and 12 h post-E2. Uterine blood flow (UBF) was monitored continuously and plasma samples analyzed for PGF2 alpha and PGFM. Exact locations of catheters in reproductive tracts were verified post-slaughter. Data were analyzed by method of least squares analysis of variance. Uterine blood flow (ml/min) increased above pre-E2 flow rates within 30 min post-E2 injection, peaked between 2.5 to 3.5 h and declined between 4 to 8.5 h. A small secondary rise in UBF occurred between 9 and 12 h. Regression analysis for concentrations (pg/ml) of PGF2 alpha and PGFM in the OV (i.e., [OV]-[FA]) demonstrate a similar response as PGFM concentration in the FA in that all increased at approximately 3 h, peaked between 5 and 7 h and returned to near baseline levels by 9 to 10 h post-E2. Facial artery PGFM concentrations were positively correlated with uterine production of PGF2 alpha (r = .66) and PGFM (r = .30), whereas FA PGF2 alpha concentrations were not. In three of five cows, a difference in PGF2 alpha was detected between UBOA and FA (UBOA greater than FA); supportive of a local countercurrent exchange between the uterine venous drainage and the ovarian artery.     

Uterine artery blood flow and renal sympathetic nerve activity during exercise in rabbit pregnancy

The uterine artery blood flow (UtBF) and renal sympathetic nerve activity (SNA) responses to treadmill exercise were evaluated in 12 nonpregnant (NP) and 17 term pregnant (P) rabbits. UtBF was monitored continuously with a Transonic flowprobe. Rabbits underwent three exercise trials (5-min duration) that varied in absolute workload. The rise in renal SNA with exercise was intensity related. Pregnancy did not affect the average steady-state renal SNA response expressed relative to maximum activity (P 24 +/- 1% vs. NP 23 +/- 2% of maximum smoke-elicited activity) and increased the average renal SNA response expressed relative to resting activity (P +155 +/- 19% vs. NP +84 +/- 23% from rest, P = 0.03) At rest, UtBF (P 13 +/- 3 vs. NP 1.9 +/- 0.3 ml/min) and uterine artery conductance (UtC; P 22 +/- 5 vs. NP 2.8 +/- 0.5 ml. min-1.mmHg-1 x 10-2) were elevated in the P rabbits. The average exercise-related decreases in UtBF (P -16 +/- 4% vs. NP -48 +/- 4%) and UtC (P -27 +/- 4% vs. NP -54 +/- 4%) were attenuated in the P rabbits. Pregnancy does not impair the ability to raise renal SNA but attenuates the uterine artery constrictor response to moderate to heavy dynamic exercise in rabbits. Under normal conditions, the pregnant uterine circulatory bed may be relatively protected from exercise-related redistribution of blood flow.     

Immunosuppressive activity of uterine luminal protein from steroid-treated ovariectomized heifers

Uterine luminal protein (ULP) collected from ovariectomized steroid-treated crossbred heifers was tested for immunosuppressive activity in vitro. Heifers were allotted to treatment groups and for 16 d received daily injections of the following steroids or vehicle: Control (C, corn oil only, n=10); estradiol-17beta (E(2), 1.1 mug/kg body wt, n=10); progesterone (P(4), 2.2 mg/kg body wt, n=10); and E(2)+P(4) (1.1 mug + 2.2 mg/kg body wt, n=9). On Day 17, uterine flushings were collected, concentrated and quantitated for total ULP. ULP was tested for suppression of lymphocyte blastogenesis. For each experiment, 5 x 10(5) bovine lymphocytes were incubated with 0.4 mug of phytohemagglutinin (PHA) and ULP (25 to 400 mug ULP/ml) using standard culture conditions. At 48 h, 0.5 muCi of (3H) thymidine was added to cultures with cells harvested at 60 +/- 1 h by automation. Incorporated thymidine was measured by scintillation chromatography. Mean total ULP values for C-, E(2)-, P(4)- and E(2)+P(4)-treated groups were 4.7, 8.4, 13.6, and 25.5 mg, respectively (E(2)+P(4)>C and E(2), P<0.05). ULP from all treatment groups suppressed (P<0.0001) lymphocyte blastogenesis (thymidine incorporation) to PHA; however, suppression was greater (P<0.0001) for ULP from E(2)- and P(4)-than C-treated heifers at 100 and 200 mug ULP/ml. In conclusion, E(2) and P(4) injections enhanced immunosuppressive activity of ULP secretions.     

Effect of PGE2 on uterine contractility and tone in mares

A technique for transvaginal, ultrasound-guided intrauterine injection was developed. After preliminary study using different approaches, the procedure was successful in 24 of 25 (96%) mares, based on detecting fluid in the uterine lumen during and after the injection. The technique was used to study the effect of PGE2, reportedly produced by the embryonic vesicle, on uterine contractility on Day 12 (Day 0 = ovulation). Uterine contractility was scored (1 = minimal, 4 = maximal) every 10 min for 1 h and every 30 min for the next hour by a continuous 1-min ultrasound examination of a longitudinal section of the uterine body without knowledge of group. In Experiment 1, the main effect of group (1-mL vehicle, n = 6; 0.25 microgram PGE2, n = 7) tended to be significant (P < 0.09), and the effect of time was significant (P < 0.008). The mean score was higher for the PGE2 group (2.0 +/- 0.1) than for the vehicle group (1.7 +/- 0.1). An increase in contractility occurred between 0 and 5 min in the vehicle group (P < 0.0004) and between 0 and 10 min in the PGE2 group (P < 0.04). In Experiment 2, there was a tendency (P < 0.08) for effect of group (control without injection, n = 6; 1-mL vehicle, n = 6; 0.025 microgram PGE2, n = 6). The PGE2 group (2.0 +/- 0.1) was different from the vehicle group (1.6 +/- 0.1) and the control group (1.6 +/- 0.1). An increase in contractility occurred between 0 and 20 min in the PGE2 group, and the changes were not significant in the other groups. However, scores were higher in the PGE2 group before treatment, and there were no significant effects when data were converted to percentage changes. The results for an effect of intrauterine treatment of PGE2 on uterine contractility are considered uncertain because of the transient increase in contractility from vehicle injections in Experiment 1 and the higher score in the PGE2 group before treatment, with no significant differences in percentages in Experiment 2. Indirectly, however, an effect of PGE2 was suggested by a shorter (P < 0.05) period of detectability of intrauterine fluid in the PGE2 groups (21 +/- 31 min) than in the vehicle groups (50 +/- 42 min). The shorter period was attributable to greater dispersion of the fluid as a result of increased contractility. In Experiment 3, PGE2 (10 mg, n = 5) and vehicle (4 mL, n = 5) were given intravenously. In addition to uterine contractility, uterine tone was scored (1 = minimal, 4 = maximal) by transrectal digital compression. The main effect of group was significant (P < 0.03) for uterine contractility score, which increased between 0 and 20 min after PGE2 injection. The time effect and interaction were highly significant (P < 0.0001) for uterine tone score, and tone increased in the PGE2 group between 0 and 20 min after injection. The results indicated that PGE2 should be considered as a potential stimulator of both uterine contractions and uterine tone during the time of embryo mobility in mares.     

Maturation enhances fluid shear-induced activation of eNOS in perfused ovine carotid arteries

The present study tests the hypothesis that age-dependent increases in endothelial vasodilator capacity are due to maturational increases in endothelial nitric oxide (NO) synthesis and release. Intact 4-cm carotid artery segments taken from term fetal lambs and nonpregnant adult sheep were perfused by using a closed system that enabled independent control of flow and inflow pressure and facilitated complete recovery of all NO released. Fluid shear stress induced a graded release of NO (in nmol NO x min x cm(-2) of luminal surface area) that was significantly greater in adult (890 +/- 140) than in fetal (300 +/- 40) carotid arteries at corresponding values of shear stress (5.9 +/- 0.3 dyn/cm2) but was independent of inflow pressure in both age groups. These age-related differences in NO release were not attributable to corresponding differences in endothelial NO synthase (eNOS) abundance, as eNOS protein levels (in ng of eNOS/cm2 of luminal surface area) were similar in adult (14 +/- 2) and fetal (12 +/- 1) arteries. Adult (80 +/- 15) and fetal (89 +/- 32) levels of eNOS mRNA (in 10(6) copies/cm2 of luminal surface area) were also similar. However, when NO release was normalized relative to the associated mass of eNOS protein to estimate eNOS-specific activity in situ, this value (in nmol NO x microg of eNOS(-1) x min(-1)) was significantly greater in adult (177 +/- 44) than in fetal (97 +/- 36) arteries when the endothelium was maximally activated by A-23187. Similarly, the slope of the relation between fluid shear stress and estimated eNOS-specific activity (in nmol NO x microg of eNOS(-1) x min(-1) per dyn/cm2) was also significantly greater in adult (6.8 +/- 0.1) than in fetal (2.9 +/- 0.1) arteries, which suggests that eNOS may be more sensitive to or more efficiently coupled to activating stimuli in adult compared with fetal arteries. We conclude that maturational increases in endothelial vasodilator capacity are attributable to age-dependent increases in NO release secondary to elevated eNOS-specific activity and involve more efficient coupling between endothelial activation and enhancement of eNOS activity in adult compared with fetal arteries.