Long-term hypoxia alters calcium regulation in near-term ovine myometrium.

Previous studies showed that long-term hypoxia (LTH) during pregnancy alters myometrial contractility. The present study was designed to test the hypothesis that LTH during pregnancy suppresses myometrial contractility in sheep by affecting the calcium signaling cascade. Pregnant sheep were maintained at high altitude (3820 m) from Day 30 to Day 139 of gestation, when the animals were killed for collection of myometrial tissue. Tissue was also collected from age-matched, normoxic controls. Circular and longitudinal layers were separated, and strips from each layer were mounted in a muscle bath. After pretreatment with 10(-8) M oxytocin, the strips were exposed to increasing half- or quarter-log doses of nifedipine (L-type calcium-channel blocker), ruthenium red, ryanodine (blockers of inositol 1,4,5-trisphosphate-insensitive calcium stores), or 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; phospholipase C inhibitor). Area under the contraction curve was analyzed, and pD(2) (log of concentration yielding 50% of maximum response) values and maximum relaxation responses were calculated. The maximum relaxation response to nifedipine was increased in both longitudinal (P < 0.01) and circular (P < 0.05) myometrial layers from LTH compared to control tissue, whereas no difference was observed in response to ruthenium red or ryanodine. The maximum relaxation response to NCDC was lower in the LTH circular layer (P < 0.05). Together, these data are indicative of an increase in the dependence of ovine uterine smooth muscle on extracellular calcium influx through the L-type, voltage-gated calcium channels following LTH. This appears to occur not through an increase in L-type calcium channels but, rather, through a possible decline in importance of the oxytocin-induced, phospholipase C-mediated pathway, resulting in a greater proportion of extracellular calcium contributing to contraction. Layer-dependent differences also exist between the circular and longitudinal myometrium in response to phospholipase C inhibition.

Long-term hypoxia changes myometrial responsiveness and oxytocin receptors in the pregnant ewe: differential effects on longitudinal versus circular smooth muscle.

Previous studies from our laboratory demonstrated that long-term hypoxia (LTH) altered in vitro contractile responses to oxytocin in full-thickness myometrial strips from pregnant sheep. The present study was designed to determine, first, if the reduced contractile response to oxytocin following LTH is the result of combined effects on longitudinal and circular smooth muscle or if the effect is specific to a single muscle layer and, second, if the reduced contractile response to oxytocin following LTH is caused by changes in oxytocin-receptor protein. Pregnant ewes were maintained at high altitude (3820 m) from Day 30 to Days 137-142 of gestation, when the ewes were killed for collection of myometrial tissue. Tissue was also collected from age-matched, normoxic controls. Longitudinal and circular layers were separated, length-tension curves generated to determine optimal resting tension, and all strips exposed to increasing half-log doses of oxytocin ranging from 10-12 to 10-6.5 M. The expression of oxytocin-receptor protein was measured using Western blot analysis. We found that LTH did not affect KCl-induced contraction of either smooth muscle layer, whereas the sensitivity of both myometrial layers to oxytocin was altered. A decreased maximum contractile response of the circular layer to oxytocin was also observed. Additionally, LTH decreased expression of oxytocin-receptor protein in the circular layer and increased levels in the longitudinal layer. Results from the present study indicate that LTH alters contractile responses and oxytocin-receptor protein expression in a layer-specific manner in the pregnant sheep myometrium.

Inhibition of renal arachidonic acid omega-hydroxylase activity with ABT reduces blood pressure in the SHR.

The mechanism-based cytochrome P-450 (CYP) inhibitor 1-aminobenzotriazole (ABT) was characterized as an inhibitor of renal arachidonic acid metabolism and administered to spontaneously hypertensive rats (SHRs) to determine the effect of reduced eicosanoid production on mean arterial pressure (MAP). A single intraperitoneal dose of ABT to Sprague-Dawley rats caused a dose-dependent loss of renal CYP content, arachidonic acid metabolism, and CYP4A protein. In the cortex and outer medulla, ABT showed a high degree of selectivity for the CYP4A enzymes, reflected by the potent inhibition of 19- and 20-hydroxyeicosatetraenoic acid (19- and 20-HETE) formation. A 50 mg/kg dose of ABT reduced cortical 20-HETE formation to 16.1 +/- 0.82% of control and outer medullary 20-HETE formation to 23.8 +/- 0.45% of control. In contrast, there was no inhibition of renal epoxygenase activity at this dose. Renal CYP content, arachidonic acid omega- and (omega-1)-hydroxylase activity, and CYP4A protein levels gradually return to control levels by 72 h after a single dose of ABT. Cortical 20-HETE formation recovered from 17.9 +/- 3.15% of control at 6 h to 84.8 +/- 4.67% of control at 72 h after ABT administration. A single injection of ABT to 7-wk-old SHRs caused an acute reduction in MAP, which remained suppressed for at least 12 h. The effect was maximal within 4 h and averaged 17-23 mmHg during the 4- to 12-h period after administration. 20-HETE formation was inhibited 85% in the cortex and 70-80% in the outer medulla during the period when MAP was reduced. A structurally related ABT analog 1-hydroxybenzotriazole had no effect on blood pressure or renal arachidonic acid metabolism. These results identify ABT as a selective inhibitor of renal CYP4A activity and provide further support for a role for 20-HETE in the regulation of blood pressure.

Pharmacokinetic and fetal cardiovascular effects of enalaprilat administration to maternal rhesus macaques.

OBJECTIVE: The purpose of this study was to determine the extent of placental transfer of the angiotensin-converting enzyme inhibitor enalaprilat and the effects on maternal and fetal cardiovascular parameters. STUDY DESIGN: Between gestational days 122 and 126 (term 167 days) five rhesus macaques underwent surgery for implantation of maternal and fetal vascular catheters. At least 4 days after surgery maternal and fetal blood pressures and heart rates were recorded for 1 hour. This was followed by a 5-minute maternal venous infusion of saline solution vehicle and recording for an additional hour. Enalaprilat was then infused over 5 minutes through the maternal femoral artery at doses of 0.05, 0.1, or 0.2 mg/kg. Maternal and fetal arterial blood samples were collected for determination of blood gas status and plasma enalaprilat concentrations. RESULTS: Enalaprilat rapidly crossed the placenta, and fetal values for areas under the concentration time curve were 50% to 65% of maternal values across dose groups. Drug was retained in the fetal plasma approximately threefold to fourfold longer than in maternal plasma. Maternal heart rate, blood pressure, arterial Po2 and pH were unchanged after enalaprilat infusion, as were fetal heart rate and blood gases. In contrast, fetal arterial pressure decreased significantly (19% to 23%, p < 0.01) after maternal treatment with 0.1 and 0.2 mg/kg and remained depressed throughout the 6-hour study interval. At 0.05 mg/kg fetal arterial pressure was decreased by 13% from baseline; differences were not significantly different (p > 0.05). CONCLUSIONS: Results from this study indicate that enalaprilat rapidly crosses the primate placenta with a single intravenous administration to the mother, resulting in significant and prolonged reduction of fetal arterial pressure. Because maternal cardiovascular parameters were unaffected, enalaprilat appears to have a direct effect on fetal arterial pressure.

Myometrial contractile responsiveness to oxytocin after dexamethasone suppression of circadian uterine activity in pregnant rhesus macaques during late gestation.

OBJECTIVE: This study was designed to determine if dexamethasone alters myometrial responsiveness to oxytocin or oxytocin secretion. STUDY DESIGN: Studies were conducted in rhesus macaques (n = 6), between 144 and 148 days' gestation (term 167 days). The first study was conducted at 9 AM and repeated 36 hours later at 9 PM. At 9 AM the following morning a continuous maternal dexamethasone infusion (15 micrograms/kg/hr given intravenously) was initiated, and the study was repeated at 9 PM, 60 hours later. Four doses of oxytocin (500, 1000, 2000, and 4000 pg/kg/min) were administered as 1-minute pulses every 5 minutes for 30 minutes. The number of contractions per pulse (contraction/pulse ratio) was used to determine differences in myometrial responsiveness. RESULTS: Before dexamethasone infusion there was a circadian rhythm in uterine activity with peak contractile events between 8 and 10 PM (p < 0.01), whereas during infusion the rhythm was ablated. At oxytocin dose 1, the 9 AM contraction/pulse ratio (0.3 +/- 0.1) was lower than that for 9 PM (0.6 +/- 0.2) and for 60 hours later (0.6 +/- 0.1) (mean +/- SE, p < 0.05). Similar results were observed at dose 2, whereas no differences in the contraction/pulse ratio were noted at dose 3. Basal plasma oxytocin concentrations were unaffected by dexamethasone treatment, whereas plasma estradiol and cortisol concentrations were reduced compared with control values (p < 0.01). CONCLUSIONS: (1) There is a differential sensitivity to oxytocin between morning and evening and (2) the dexamethasone-induced loss of the uterine contractile rhythm is not the result of a loss of myometrial sensitivity to oxytocin or to a suppression of plasma oxytocin concentrations.

Circadian myometrial and endocrine rhythms in the pregnant rhesus macaque: effects of constant light and timed melatonin infusion.

Six chronically catheterized rhesus macaques maintained on a 12-hour-light/dark cycle (lights on from 7 AM to 7 PM) showed a nocturnal uterine activity rhythm with peak contractile events between 9 and 11 PM (p less than 0.05). In blood samples collected at 3-hour intervals over a 24-hour period, we determined that plasma melatonin and progesterone concentrations were elevated at night whereas estradiol, estrone, and cortisol reached peak concentrations in the early morning (p less than 0.05). Lights were then left on for the remainder of the study. After 12 days in constant light, daily rhythms in uterine activity and plasma steroid levels were relatively unchanged, whereas melatonin concentrations were suppressed. Animals then received a timed infusion of melatonin (0.2 mg/kg/hr each day from 7 PM to 6 AM daily until delivery). The nocturnal uterine activity rhythm and the rhythms in plasma steroid concentrations were maintained. We conclude that the 24-hour patterns in maternal uterine activity and plasma steroid hormone levels are circadian rhythms generated by an endogenous biologic clock and do not appear to be driven by the pattern of melatonin in circulation.