Objective
Our objective was to determine whether the pregnancy and high altitude long-term hypoxia-mediated changes in uterine artery contractility were regulated by K ATP and L-type Ca 2+ channel activities.
Study Design
Uterine arteries were isolated from nonpregnant and near-term pregnant ewes that had been maintained at sea level (∼300 m) or exposed to high altitude (3801 m) for 110 days. Isometric tension was measured in a tissue bath.
Results
Pregnancy increased diazoxide, but not verapamil-induced relaxations. Long-term hypoxia attenuated diazoxide-induced relaxations in near-term pregnant uterine arteries, but enhanced verapamil-induced relaxations in nonpregnant uterine arteries. Diazoxide decreased the maximal response (E max ) of phenylephrine-induced contractions in near-term pregnant uterin arteries but not nonpregnant uterine arteries in normoxic sheep. In contrast, diazoxide had no effect on phenylephrine-induced E max in near-term pregnant uterine arteries but decreased it in nonpregnant uterine arteries in long-term hypoxia animals. Verapamil decreased the E max and pD 2 (-logEC 50 ) of phenylephrine-induced contractions in both nonpregnant uterine arteries and near-term pregnant uterine arteries in normoxic and long-term hypoxia animals, except nonpregnant uterine arteries of normoxic animals in which verapamil showed no effect on the pD 2 .
Conclusion
The results suggest that pregnancy selectively increases K ATP , but not L-type Ca 2+ channel activity. Long-term hypoxia decreases the K ATP channel activity, which may contribute to the enhanced uterine vascular myogenic tone observed in pregnant sheep at high altitude hypoxia.
Vascular smooth muscle cells express a diverse array of ion channels that play an important role in the function of vessels in both health and disease. Potassium (K + ) channels are the dominant ion channels expressed in the plasma membrane of arterial smooth muscle cells and contribute mainly to the regulation of smooth muscle tone. Activation of K + channels in vascular smooth muscle leads to decreased vascular tone, with an increase in blood flow and a decrease in blood pressure. Inhibition of K + channel activity leads to vasoconstriction. In vascular smooth muscle cells, 4 types of K + channels (K V , K Ca , K ATP , and K IR channels) have been identified to regulate the membrane potential, which in turn controls the activity of L-type Ca 2+ channels and vascular tone. K + channels may be involved in the actions of a variety of vasodilators and vasoconstrictors, and their activities and functions may be altered in patho- and/or physiologic conditions.
Pregnancy is associated with a significant decrease in uterine vascular tone, with a striking increase in uterine blood flow. The adaptation of uterine vascular tone to pregnancy is complex, and the mechanisms that contribute to the profound changes during pregnancy are poorly understood. Potassium channels may be the targets and the key mediators responsible for these pregnant-mediated alterations. Indeed, previous studies have demonstrated that Ca 2+ -activated K channel (K Ca ) in sheep and ATP-sensitive K channel (K ATP ) in guinea pigs play important roles in the regulation of uterine blood flow during pregnancy. Previous studies also have indicated that enhanced K + channel activity contributed to the vascular changes associated with normal pregnancy, eg, the fall in systemic and local vascular resistances, and the attenuated pressor responses to several vasoconstrictors.
Chronic hypoxia during the course of pregnancy is one of the most common insults to the maternal cardiovascular system and fetal development, and is thought to be associated with increased risk of preeclampsia and fetal intrauterine growth restriction (IUGR). However, in our high-altitude sheep animal model, chronic hypoxia alters maternal and fetal cardiovascular systems without significant IUGR, suggesting a compensatory adaptation to high-altitude hypoxia in sheep, which are similar to those observed in well-adapted human populations such as Tibetans and Andeans. Previous studies have demonstrated that long-term hypoxia (LTH) has profound effects on maternal uterine artery contractility. However, the mechanisms underlying LTH-mediated uterine contractility are poorly understood. Among the 4 types of K + channels, the K ATP channel has been demonstrated to be involved in the hypoxia-mediated contractility. Although the role of K ATP channels in hypoxia-mediated contractility is well established at other tissues, to our knowledge, no studies have examined the relative roles of K ATP channels in high-altitude LTH-mediated uterine artery contractility during pregnancy. Given that LTH enhances ovine uterine artery myogenic contractions during pregnancy, we tested the hypothesis that the LTH-associated enhanced uterine artery vascular tone is secondary to altered K ATP channel function during pregnancy. In addition, we also determined the L-type Ca 2+ channel activity to test whether LTH alters this channel activity in uterine artery smooth muscle cells during pregnancy. To test our hypothesis, we used sheep as the animal model. The sheep were divided into four groups: normoxic nonpregnant sheep, normoxic pregnant sheep, LTH nonpregnant sheep, and LTH pregnant sheep. The effects of K ATP channel and L-type Ca 2+ channel on uterine vascular contractility were determined among those 4 groups of animals.
Materials and Methods
Tissue preparation
As previously described, nonpregnant and time-dated pregnant sheep were obtained from the Nebeker Ranch in Lancaster, CA (altitude: ∼300 m; arterial PaO 2 : 102 ± 2 Torr). For chronic hypoxic treatment, nonpregnant and pregnant (30 days of gestation) animals were transported to the Barcroft Laboratory, White Mountain Research Station, Bishop, CA (altitude, 3801 m; maternal PaO 2 , 60 ± 2 Torr) and maintained there for ∼110 days. Animals then were transported to the laboratory at Loma Linda University. Shortly after arrival, we placed a tracheal catheter in the ewe, through which N 2 flowed at a rate to maintain PaO 2 at ∼60 Torr, and this was maintained until the time of the experimental study. Ewes were anesthetized with thiamylal (10 mg/kg) administered via the external left jugular vein. The animals then were intubated and anesthesia was maintained with 1.5% to 2.0% halothane in oxygen throughout surgery. An incision in the abdomen was made and the uterus exposed. The uterine arteries were isolated and removed without stretching and were placed in a modified Krebs’ solution (pH 7.4) of the following composition (in mM): 115.21 NaCl, 4.7 KCl, 1.80 CaCl 2 , 1.16 MgSO 4 , 1.18 KH 2 PO 4 , 22.14 NaHCO 3 , 0.03 EDTA, and 7.88 dextrose, oxygenated with a mixture of 95% O 2 -5% CO 2 . After removal of the tissues, animals were killed with an overdose of the proprietary euthanasia solution, Euthasol (pentobarbital sodium 100 mg/kg and phenytoin sodium 10 mg/kg; Virbac, Fort Worth, TX). All procedures and experimental protocols were approved by the Loma Linda University Institutional Animal Care and Use Committee and adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals .
Contraction studies
The fourth generation branches (∼0.8 mm in external diameter) of main uterine arteries were separated from the surrounding tissue, and cut into 2-mm ring segments. The small branches of uterine arteries were chosen, because they are much closer in characteristics to arterioles and play a substantial role in vascular resistance. Isometric tension was measured in the Krebs solution in a tissue bath at 37°C, as described previously. Briefly, each ring was equilibrated for 60 minutes and then gradually stretched to the optimal resting tension, as determined by the tension that developed in response to 120 mM KCl added at each stretch level. Tissues then were stimulated with cumulative additions of phenylephrine in approximate one-half log increments to generate a concentration-response curve, and contractile tensions were recorded with an online computer. After phenylephrine was washed away, tissues were relaxed to the baseline and were recovered at the resting tension for 30 minutes. The second concentration-response curves of phenylephrine-induced contractions then were repeated in the absence or presence of a K ATP channel blocker (glibenclamide, 10 μM; Research Biochemical International, Natick, MA), or K ATP channel opener (diazoxide, 10 μM; Research Biochemical International) or a L-type Ca 2+ channel blocker (verapamil, 10 μM; Research Biochemical International) for 20 minutes. For relaxation studies, the tissues were precontracted with submaximal concentrations of phenylephrine, followed by diazoxide, and verapamil, respectively, added in a cumulative manner. EC 50 values for the agonist in each experiment were taken as the molar concentration at which the contraction-response curve intersected 50% of the maximum response, and were expressed as pD 2 (−logEC 50 ) values.
Simultaneous measurement of [Ca 2+ ] i and tension
Simultaneous recordings of contraction and [Ca 2+ ] i (fura-2 signal R f340/f380 ) in the same tissue were conducted as described previously. Briefly, the arterial ring was attached to an isometric force transducer in a 5-mL tissue bath mounted on a CAF-110 intracellular Ca 2+ analyzer (model CAF-110, Jasco; Tokyo, Japan). The tissue was equilibrated in Krebs buffer under a resting tension of 0.5 g for 40 minutes. The ring was then loaded with 5 μM fura 2-AM for 3 hours in the presence of 0.02% Cremophor EL at room temperature (25°C). After loading, the tissue was washed with Krebs solution at 37°C for 30 minutes to allow for hydrolysis of fura-2 ester groups by endogenous esterase. Contractile tension and fura-2 fluorescence were measured simultaneously at 37°C in the same tissue. The tissue was illuminated alternatively (125 Hz) at excitation wavelengths of 340 and 380 nm, respectively, by means of 2 monochromators in the light path of a 75-watt xenon lamp. Fluorescence emission from the tissue was measured at 510 nm by a photomultiplier tube. The fluorescence intensity at each excitation wavelength (F 340 and F 380 , respectively) and the ratio of these 2 fluorescence values (R f340/380 ) were recorded with a time constant of 250 ms and stored with the force signal on a computer.
Data analysis
Concentration-response curves were analyzed by computer-assisted nonlinear regression to fit the data using GraphPad Prism (GraphPad Software, San Diego, CA). Results were expressed as means ± standard error of mean (SEM) obtained from the number (n) of experimental animals given. Differences were evaluated for statistical significance ( P < .05) by 2-way analysis of variance (ANOVA), followed by the Newman-Keuls post hoc test.
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