Antenatal betamethasone increases vascular reactivity to endothelin-1 by upregulation of CD38/cADPR signaling.

Antenatal steroid administration is associated with hypertension in adult life; however, the mechanisms underlying this phenomenon are unclear. The aim of this study was to further characterize the effects of antenatal glucocorticoid exposure on the endothelin (ET-1) system, specifically to ascertain the role of the cyclic adenosine diphosphate ribose (cADPR)/ryanodine receptor pathway in the increased sensitivity to ET-1 observed in the offspring exposed to antenatal glucocorticoids. Pregnant sheep were randomly treated with betamethasone (Beta; 0.17 mg/kg) or vehicle at 80 and 81 days of gestation. In adults, we studied endothelium-denuded arterial segments of the brachial arteries. ET-1-induced vasoconstriction was significantly higher in the arteries from Beta sheep (F=3.5, P<0.05). Inhibition of ADP-ribosyl cyclase with 2-2'-dihydroxy-azobenzene significantly decreased the ET-1-induced contraction in Beta but not in vehicle-treated sheep. Nicotinamide attenuated ET-1 contraction in both, but it was significantly more pronounced in the Beta-treated sheep. No significant differences were observed following KCl-induced (6.25-75 mM) contraction. Nicotinamide (10 mM) significantly attenuated the KCl-induced vasoconstriction in both groups. In KCl (62.5 mM)-constricted arteries, the effect of nicotinamide (NIC) was significantly greater in the vehicle-treated sheep (50% relaxation v. 40% relaxation; t=2.2, P<0.05). In contrast, the sodium nitroprusside (SNP) relaxation was not statistically different. An additive effect was observed when NIC and SNP were used in combination and it was also more pronounced in vehicle-treated sheep. We conclude that the increased response to ET-1 is mediated by activation of the CD38/cADPR signaling pathway. Further studies are required to identify the effectors downstream from cADPR affected by exposure to antenatal steroids.

Connexin 43 ontogeny in fetal sheep adrenal glands.

We examined fetal sheep adrenal glands from 99 to 130 days of gestational age (dGA) to see how connexin 43 (Cx43), the major if not the only adult adrenal gap junction protein, changes expression as the adrenal cortex emerges from the well-documented refractory period to participate in labor and delivery. Immunocytochemical technique and Western blot were used to examine changes in the quantity and quality of Cx43. In addition, adrenal glands of ACTH infused fetuses or of fetuses from dexamethasone injected ewes underwent image analysis quantification after Cx43 immunostaining. Finally, fetal adrenal glands, from fetuses splanchnic nerve sectioned (SPLX) at 125 dGA, were examined for the pattern of Cx43 immunostaining at 131 days of gestation. From 100 to 130 dGA, the amount of Cx43 in cells of the adrenal cortex increased steadily while the pattern of immunoreactivity changed from predominantly cytoplasmic to membrane bound. At 100-103 dGA, ACTH infusion increased the size of the cortex, but decreased the expression of Cx43 per unit area while dexamethasone had no effect on either parameter. Lastly, the expression of Cx43 as a membrane bound protein was not delayed or reversed by SPLX. We conclude that the described changes in Cx43 are most intriguing given their temporal relationship to the described preparturient increases in ACTH and cortisol in peripheral fetal plasma as term approaches and deserve further investigation.

Developmental and regional expression patterns of Type I Nitric Oxide Synthase mRNA and protein in fetal sheep brain during the last third of gestation.

Type I NOS (nNOS) catalytic activity represents the activity of full-size protein and truncated protein variants originated from many different spliced mRNA variants. Splice mRNA variants are thought to be important in determining the differential organ and subcellular expression of Type I NOS. The present study was directed to increase our understanding of the developmental regulation of Type I NOS in fetal brain. In four discrete areas of the fetal brain, we measured steady-state mRNA levels and catalytic activity and protein mass in the soluble and particulate fractions. Under general anesthesia, we collected sensory-motor cortex, striatum, hippocampus and cerebellum from sheep fetuses at 105, 115, 125 and 135 days gestation (32 fetuses). NOS protein in the soluble and particulate fractions was characterized using Western blot (molecular weight) and arginine to citrulline conversion (enzymatic activity). At the mRNA level, steady state levels were determined using probes directed against exon 2 and exon 21/22 by ribonuclease protection assay (RPA). Our data show that NOS catalytic activity is regulated in a region, subcellular and temporal manner. NOS activity was higher in the soluble fraction in all brain regions and significantly higher levels were found in the soluble fraction of striatum and particulate fraction of hippocampus (P<0.05 by ANOVA). Western blot analysis revealed three distinct molecular weight bands for Type I NOS (155, 144 and 136 kDa). The bands were present in all brain regions and in both cellular compartments with the 155 kDa band being the most abundant molecular form. Truncated protein variants accounted for 25% and 15% of total Type I NOS protein in the soluble fraction and particulate fraction respectively. RPA analysis showed a differential regulation of mRNA variants with exon 2 frame deletions in striatum and hippocampus. A coordinated increase with advancing gestational age of catalytic activity, the full-length protein, the protein variants and steady state mRNA levels was observed in cortex and striatum as demonstrated by higher levels at 125 and 135 days gestation (P<0.05 by ANOVA). NOS enzymatic activity was Ca(2+) and calmodulin dependent. However, in the particulate fraction 20% of the NOS activity was resistant to calmodulin inhibition. In summary, fetal brain Type I NOS mRNA variants are differentially regulated according to brain regions. Our data suggests that exon 2 deleted mRNA variants have low translation efficiency as indicated by the lack of parallel expression of truncated Type I NOS protein variants.

Physiologic role of nitric oxide in the maintenance of uterine quiescence in nonpregnant and pregnant sheep.

OBJECTIVE: This study evaluated the role of nitric oxide in the maintenance of uterine quiescence in nonpregnant and pregnant ewes. STUDY DESIGN: Sixteen ovariectomized nonpregnant and 10 pregnant (115 days' gestation) chronically instrumented ewes were studied. Uterine contractility was assessed by electromyography and intrauterine pressure recordings. Nitric oxide synthase inhibition was induced with nitro-L -arginine methyl ester or aminoguanidine (4. 5 mg/kg per hour) given during estrogen replacement with 17beta-estradiol (100 microg/d) or in late gestation. In the pregnant group we evaluated the ability of nitric oxide synthase inhibition to alter the responsiveness to oxytocin-induced uterine contractility. Blood pressure and common internal iliac artery blood flow were assessed to confirm nitric oxide synthase inhibition. In addition, the effects of the nitric oxide donor nitroglycerin and the cyclooxygenase inhibitor indomethacin were studied in nonpregnant sheep. The effect of nitric oxide in vitro on myometrial spontaneous and induced contractions was also studied. RESULTS: In nonpregnant estrogen-replaced sheep, nitric oxide synthase inhibition and nitroglycerin administration did not alter uterine contractility, despite significant changes in blood pressure. In contrast, indomethacin decreased electromyographic results to 70% of baseline after 1 hour and 47% after 2 hours. In pregnant ewes nitric oxide synthase inhibition failed to alter uterine contractility in response to oxytocin. These findings are in contrast to results of the in vitro study in which nitric oxide was shown to relax sheep myometrium. CONCLUSION: The absence of significant effects of nitric oxide synthase inhibition and nitric oxide donors on uterine contractility in vivo suggests that nitric oxide does not play a physiologic role in the regulation of uterine contractility in nonpregnant or pregnant ewes.

Functional and molecular characterization of nitric oxide synthase in the endometrium and myometrium of pregnant sheep during the last third of gestation.

OBJECTIVE: This study was undertaken to characterize the biochemical and expression profiles of the nitric oxide synthase isoforms present in the sheep uterus during late gestation. STUDY DESIGN: Myometrium and endometrium were obtained from 28 time-mated pregnant sheep that were under halothane general anesthesia. Tissues were kept frozen at -80 degrees C until they were homogenized for the measurement of (1) nitric oxide synthase activity according to the carbon 14-labeled arginine-citrulline conversion assay, (2) nitric oxide synthase protein mass according to Western blot analysis, and (3) nitric oxide synthase messenger ribonucleic acid according to the ribonuclease protection assay. The nitric oxide synthase activity assay included 8 parallel treatments for biochemical characterization, in particular with the arginase inhibitors ornithine and (+)-S-2-amino-5-iodoacetamidopentanoic acid. RESULTS: The biochemical characterization of nitric oxide synthase indicated that the predominant form of nitric oxide synthase in endometrium and myometrium (80%-90%) was calcium-calmodulin dependent. In endometrium 50% of reduced nicotinamide adenine dinucleotide-dependent arginine metabolism was accounted for by the presence of alternative arginine metabolic pathways. Expressions of type 1 and type 3 nitric oxide synthase were demonstrated in endometrium and myometrium by Western blot and ribonuclease protection assay. Although no significant decrease in nitric oxide synthase activity or protein mass was observed, a significant decrease in myometrial type 1 nitric oxide synthase messenger ribonucleic acid occurred in sheep not in labor at 140 days' gestation (P <.05 by analysis of variance; term is 144 +/- 5 days). CONCLUSION: In the gravid sheep uterus the predominant nitric oxide synthase isoforms are type 1 in myometrium and type 3 in endometrium. Despite a decrease in type 1 nitric oxide synthase messenger ribonucleic acid, enzymatic activity and type 1 nitric oxide synthase protein mass do not decrease before parturition.

Nonpregnant sheep uterine type I and type III nitric oxide synthase expression is differentially regulated by estrogen.

The aim of this study was to characterize the effect of estrogen on the expression of neuronal and endothelial isoforms of nitric oxide (NO) synthase (NOS) in myometrium, endometrium, and caruncle (nonglandular endometrium) in nonpregnant sheep. Twenty sheep were castrated during synchronized estrus (Days 14-16) and 4 days after surgery treated i.v. through the jugular with 100 microg/day of estradiol-17beta for 5 (n = 6) or 8 (n = 6) days or with vehicle (n = 8). Nitric oxide synthase mRNA was measured by ribonuclease protection assay, and NOS protein mass was measured by Western immunoblotting. Data were analyzed by ANOVA and Tukey's test. The three distinct uterine compartments studied contained the mRNA and protein for the neuronal (type I NOS) and the endothelial (type III NOS) isoforms of NOS. However, no inducible NOS was detected. Estrogen exhibited a differential effect on NOS expression in a tissue compartment- and NOS isoform-specific manner. In myometrium and caruncles, but not in endometrium, type I NOS mRNA and protein mass increased significantly (p < 0.05) after 5 or 8 days of estrogen. In contrast, type III NOS increased significantly in myometrium only after 8 days, whereas in endometrium and caruncles the increase was significant in the 5-day treatment group (p < 0.05). We conclude that the expression of type I NOS and type III NOS in the uterus are differentially regulated by estrogen. This differential regulation suggests that the NO produced within the uterus serves more than one physiological role. In myometrium it may be a uterorelaxant and regulate glucose utilization, and in endometrium and myometrium it may regulate blood flow.

Estrogen increases nitric oxide synthase activity in the uterus of nonpregnant sheep.

OBJECTIVE: The aim of this study was to characterize nitric oxide synthase activity in endometrium and myometrium of nonpregnant sheep and to determine whether estrogen administration affects uterine nitric oxide synthase activity. STUDY DESIGN: Nonpregnant sheep were castrated during synchronized estrus and 4 days after surgery were treated with 100 micrograms/day of 17 beta-estradiol for 3 days. Nitric oxide synthase activity was measured by the citrulline conversion assay. RESULTS: Citrulline generation found in soluble and particulate fractions had all the characteristics of nitric oxide synthase, namely, it was strictly dependent on reduced nicotinamide adenine dinucleotide phosphate and enhanced by flavin nucleotides and tetrahydrobiopterin. Estrogen administration significantly increased Ca(++)-dependent nitric oxide synthase activity in myometrium but not in endometrium. The effect of estrogen was more pronounced in the membrane-associated enzyme activity (approximately fivefold). Estrogen treatment increased myometrial nitric oxide synthase activity from 9.0 +/- 2.4 to 20.0 +/- 3.7 pmol/mg of protein per 30 minutes in the soluble fraction and from 12.0 +/- 5.1 to 62.0 +/- 13.1 pmol/mg of protein per 30 minutes in the particulate fraction (mean +/- SEM, p < 0.05 by t test). The increase in nitric oxide synthase activity was not mediated by an increase in tetrahydrobiopterin availability, as shown to be the case in macrophages. CONCLUSION: These data show that in the nonpregnant sheep uterus > 90% of the nitric oxide synthase activity found in myometrium is Ca++ dependent and is up-regulated by estrogen in a tissue-specific manner.

Maternal and fetal cardiovascular effects and placental transfer of the oxytocin antagonist atosiban in late-gestation pregnant sheep.

OBJECTIVES: Atosiban is a synthetic oxytocin antagonist that is currently undergoing dose-ranging clinical trials. To date, no data are available on the cardiovascular effects of combined oxytocin and vasopressin blockade during late pregnancy. Our aims were (1) to determine the effects of atosiban infusion on the maternal and fetal cardiovascular system and on uterine blood flow and (2) to determine the maternal pharmacokinetics and the rate of placental transfer of atosiban. STUDY DESIGN: Five chronically catheterized pregnant sheep were treated with a 2-hour infusion of atosiban (300 micrograms.min-1) at 116 to 126 days' gestation. Maternal and fetal blood pressure and heart rate and uterine blood flow were measured before and during the infusion. Maternal and fetal arterial blood samples were obtained throughout the experiment for measurement of plasma atosiban levels and blood gas values. RESULTS: No significant change in maternal cardiovascular parameters or uterine blood flow were observed. Similarly, no changes in fetal blood pressure and arterial blood gases were present during the infusion of the atosiban. Maternal plasma levels of atosiban reached a maximum of 585.2 +/- 82.2 (ng/ml mean +/- SD) at the end of the infusion and decreased biexponentially with a mean t1/2 alpha of 17 minutes and a mean t1/2 beta of 2.2 hours. Fetal plasma levels of atosiban were at or below the detection limit. CONCLUSION: Atosiban does not significantly affect maternal or fetal cardiovascular parameters when it is administered for 2 hours in late-pregnant sheep. Although significant levels were measured in maternal blood, negligible transfer to the fetus occurred.

Role of the sympathetic innervation on uterine electromyographic activity in nonpregnant ovariectomized sheep under estrogen supplementation.

The aims of the present study were to characterize the sympathetic innervation of the nonpregnant sheep uterus, to determine the catecholamine content in myometrium (MYO) and endometrium, and to study the effects of chemical sympathectomy (CHSPX) on uterine catecholamine content and on uterine electromyographic (EMG) activity recorded from the MYO and mesometrium (MESO) in the nonpregnant ovariectomized sheep. After synchronization of estrus, 9 nonpregnant sheep were anesthetized with halothane, ovariectomized, and fitted with vascular catheters and EMG electrodes. Estradiol-17 beta was administered intravascularly at a rate of 50 micrograms/24 h for 10 days. CHSPX was induced with 6-hydroxy dopamine (20 mg/kg). Uterine tissues were obtained for determination of catecholamine content by HPLC and for immunocytochemical staining using an antibody against tyrosine hydroxylase (TH). In nonpregnant ovariectomized sheep, TH immunostaining was present in nerve fibers located in endometrium and MYO. In all layers of the uterus, catecholamine fibers were found in the proximity of blood vessels as well as in defined regions of the parenchyma. Throughout the uterus, norepinephrine content and TH immunostaining were dramatically decreased after CHSPX. CHSPX decreased uterine short EMG event activity in both MYO and MESO. Contracture-type activity was not affected in MYO and was increased in MESO. We conclude that sympathetic innervation modulates the MYO and MESO EMG activity in nonpregnant ovariectomized sheep under estradiol supplementation, and that the removal of the sympathetic innervation induces a decrease in the spontaneous activity.

Lack of effect of maternal cocaine administration on myometrial electromyogram and maternal plasma oxytocin concentrations in pregnant sheep at 124-146 days' gestational age.

Although cocaine abuse during human pregnancy is associated with an increased incidence of preterm labor, there are few reports on the effects of cocaine on myometrial activity during pregnancy in experimental animals. Cocaine (0.5, 1, or 2 mg/kg) or vehicle was randomly administered intravenously to 15 pregnant ewes between 124-146 days' gestation (term is 147 days). Neither cocaine nor vehicle administration altered total myometrial electromyographic activity from pre-dose levels 1 or 6 hours after administration. Maternal arterial plasma oxytocin did not change during the study. Using a positive control, we confirmed observations of other investigators that administration of 2 mg/kg cocaine significantly increases maternal arterial blood pressure. The results indicate that cocaine does not stimulate myometrial contractility significantly in late pregnancy in sheep.

Further characterization of the electromyographic activity of the myometrium and mesometrium in nonpregnant sheep under estrogen supplementation.

The effects of estrogen administration on uterine contractility varies with animal species. In nonpregnant ovariectomized sheep, estrogen administration has been reported either to inhibit, inhibit then stimulate, or only stimulate uterine contractility. The aim of the present study was to determine the effects of prolonged estrogen administration in the electromyographic (EMG) activity recorded from the myometrium and mesometrium in nonpregnant ovariectomized sheep after estrous synchronization by inserting vaginal progesterone sponges 14 days before surgery. Surgery was performed on four ewes under halothane anesthesia. Bilateral oophorectomy was performed, and stainless steel EMG electrodes were sewn to the mesometrium and myometrium in both left and right horns of the uterus. Blood samples were taken at 1000 h from the uterine vein for 13,14-dihydro-15-keto-prostaglandin F2 alpha determination, and from the femoral artery for estradiol determination. Starting on Day 7 after surgery, estradiol 17 beta (50 micrograms/24 h) was infused continuously into the jugular vein. Estrogen administration had a different effect on the EMG activity recorded from myometrium and mesometrium. The myometrial response to estrogen was an increase in the frequency of short EMG events from 19.0 +/- 8.7 to 57.0 +/- 5.0 (p less than 0.05) for events less than 60 sec, and from 2.70 +/- 0.83 to 10.30 +/- 1.36 (p less than 0.05) for events lasting greater than 60 sec but less than less than 180 sec. In contrast, there was no stimulatory effect of estrogen on mesometrial EMG for both types of short events less than 60 sec, and greater than 60 but less than less than 180 sec.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphasic effect of a prostacyclin methyl ester analog on uterine contractility in the chronically catheterized pregnant rhesus monkey in late gestation.

We determined the in vivo effects of a prostacyclin methyl ester analog (PGI2) administration on myometrial activity in twelve chronically- catheterized pregnant rhesus monkeys during the last third of pregnancy under three different states of myometrial contractility: postsurgical contractions, spontaneous contractures and oxytocin induced contractions. Prostacyclin (200 micrograms) was administered to four monkeys 2-3 h after surgery, to eight monkeys having only contractures, and to four monkeys having oxytocin induced contractions. Vehicle administration was performed in six animals having contractures and to four having oxytocin induced contractions. In all three experimental paradigms PGI2 administration elicited a biphasic response in uterine contractility. An initial increase in the 5 min average intrauterine pressure (IUP) value from 2.3 +/- 1.29 to 5.8 +/- 2.29; 3.6 +/- 2.38 to 7.6 +/- 3.59; and 2.3 +/- 0.65 to 8.6 +/- 0.72 (SD); was observed in the post-surgical, spontaneous contracture and oxytocin induced contraction groups respectively (mmHg, P less than 0.05). This increase was followed by a fall in the 5 min average IUP from 2.3 +/- 1.29 to 0.6 +/- 0.49; 3.6 +/- 2.38 to 1.3 +/- 0.86; and 2.3 +/- 0.65 to 0.1 +/- 0.11 in the post-surgical, spontaneous contracture and oxytocin induced contraction groups respectively (mmHg, P less than 0.05). A fall in mean arterial blood pressure from 89 +/- 9.0 to 55 +/- 10.9 (mmHg, P less than 0.05) and a compensatory tachycardia from 108 +/- 18.2 to 164 +/- 56.2 (beats.min-1, P less than 0.05) was observed by 10 min after PGI2 administration.(ABSTRACT TRUNCATED AT 250 WORDS)