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a The Johns Hopkins Medical Institutions, Departments of Anesthesiology/Critical Care Medicine and Environmental Health Sciences, Baltimore, Maryland 21205
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In rats, sensitivity to isoproterenol is reduced on the last day of gestation (Day 21) compared with mid gestation (Day 14) [1]. Effects at any of the three components of the ß-adrenergic receptor-Gs-adenylyl cyclase pathway could produce alterations in sensitivity to isoproterenol. Pregnancy-related changes in the coupling [2] and expression [3, 4] of ß-adrenergic receptors and Gs [5, 6] have been reported, but potential alterations to the third component of the pathway, adenylyl cyclase protein, have not been evaluated directly. We questioned whether adenylyl cyclase activity and protein levels increase during pregnancy, and whether rapid decreases in adenylyl cyclase at the end of gestation coincide with the onset of labor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonpregnant (virgin adult, 200–225 g) and pregnant Sprague-Dawley rats were obtained from Charles River (Boston, MA). For adenylyl cyclase activity assays using fresh uterine tissue, day of gestation was timed to within three days, so that mid gestation was defined as Days 12–14 and late gestation was defined as Days 18–20. Delivery occurred on Day 22. For [3H]forskolin binding assay, we obtained exactly timed-dated pregnant rats (Day 14, Day 20, Day 21, or Day 22). Investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals. Rats were anesthetized briefly with inhaled halothane (1%) until loss of spontaneous movement and were then killed by decapitation. Intrapartum (Day 22 of gestation) rats were killed after delivery of at least one pup but before delivery of all fetuses. Uterine horns were removed quickly. Mesentery and fat were trimmed, and uterine horns were incised longitudinally along the mesenteric border. Fetuses, placentas, and implantation sites were discarded, and the endometrium was removed by scraping the luminal surface with a razor blade. Uterine horns were minced into fragments (about 1 mm3) that were placed in cold buffer solution (see below).
Membrane Preparation
For measurement of adenylyl cyclase activity, minced tissue fragments were placed in cold buffer containing Tris HCl (50 mM), MgCl2 (10 mM), EDTA (2 mM), and dithiothreitol (0.5 mM), pH 7.4. Plasma membrane fractions were produced from minced tissue fragments by homogenization with a Tissuemizer homogenizer (Tekmar Co., Cincinnati, OH) at 4°C. The homogenate was centrifuged at 1000 x g for 10 min at 4°C to remove nondisrupted tissue, and the supernatant was centrifuged at 48 000 x g for 30 min. The resulting membrane pellet was resuspended in cold buffer (50 mM Tris HCl, 10 mM MgCl2) at a protein concentration of 1–2 mg/ml. Because frozen and thawed membranes tended to manifest global reductions in adenylyl cyclase activity, for these studies adenylyl cyclase activity was measured immediately in freshly prepared membranes.
For [3H]forskolin binding assays, membrane fractions were prepared in a manner designed to maximize specific binding. The cold buffer consisted of Tris HCl (50 mM), MgCl2 (10 mM), leupeptin (0.01 mg/ml), and aprotinin (0.01 mg/ml), pH 7.5. The procedure was similar to the protocol used to prepare membranes for adenylyl cyclase activity assay, except that after homogenization with a Tissuemizer homogenizer, tissue fragments underwent further homogenization with 30 strokes of a Potter-Elvehjem glass homogenizer, using a motor-driven Teflon (DuPont, Wilmington, DE) pestle at 4°C. After centrifugation, membrane pellets were suspended in cold buffer at a concentration of 3–6 mg/ml. [3H]Forskolin binding assays were performed immediately, and membranes from different gestational states were assayed in parallel within the same experiment. Different gestational states were compared in a single assay on the same day.
Measurement of Adenylyl Cyclase Activity
Adenylyl cyclase activity was measured in plasma membrane fractions in triplicate samples by incubating membrane fractions in a solution (0.1 ml final volume, 30°C) containing Na-HEPES (50 mM, pH 8.0), NaCl (50 mM), MgCl2 (7 mM), EGTA (0.4 mM), BSA (0.25 mg/ml), [
-32P]ATP (0.1 mM, 0.1–0.2 mCi/mmol), cAMP (1 mM), creatine phosphate (5 mM), and creatine phosphokinase (50 U/ml). The reaction was initiated by the addition of membrane protein (0.03–0.05 mg) and was terminated after 10 min by the addition of buffer (0.1 ml) containing SDS (2%), HEPES (50 mM, pH 7.5), ATP (2 mM), and cAMP (0.5 mM), with 40 nM [3H]cAMP (25 Ci/mmol) (to allow subsequent calculation of column recoveries), and by boiling for 3 min. Increases in adenylyl cyclase activity in response to isoproterenol were determined in the presence of GTP (10-5 M), and assays containing NaF (10 mM) also contained AlCl3 (10 µM). Newly synthesized [32P]cAMP was separated from the precursor [32P]ATP by sequential column chromatography over Dowex and alumina [7]. Adenylyl cyclase activity was expressed as picomoles of cAMP/10 min per milligram protein. Protein concentrations were determined with the method of Lowry et al. [8], using BSA as the standard.
[3H]Forskolin Binding Assay
Membranes were incubated in a total volume of 0.125 ml containing Tris HCl (50 mM), MgCl2 (10 mM), leupeptin (0.01 mg/ml), and aprotinin (0.01 mg/ml), pH 7.5, with [3H]forskolin. Incubations with [3H]forskolin were conducted in the presence or absence of unlabeled forskolin (10 µM) to determine nonspecific binding. Cytochalasin B (40 µM) was included to prevent nonspecific binding to the glucose transporter [9]. Gpp(NH)p (100 µM) and NaF (10 mM) were included to maximize affinity of adenylyl cyclase molecules for forskolin. Preliminary studies were undertaken to determine optimal duration of incubation (0.5–3 h) and membrane protein content (0.05–0.5 mg/tube). Simultaneous controls, without membrane protein, were included. Reactions were terminated by vacuum filtration through GF/C glass fiber filters (Brandell, Gaithersburg, MD) with a cell harvester, followed by 5 washes with cold buffer lacking leupeptin and aprotinin. Incorporation of 3H was determined by scintillation counting of filters. Specific binding (total binding minus nonspecific binding) was calculated after subtraction of counts obtained in control (without membranes) filters. Initial assays determined saturating concentrations of [3H]forskolin (25–150 nM). For these assays, uteri from 4–6 nonpregnant or pregnant rats were pooled. In each experiment, maximum specific binding was determined, and responses at other concentrations were expressed as a percentage of maximum. For studies of gestation-related changes in [3H]forskolin binding, the uterus of a single rat of each gestational state was harvested for each assay. For these studies, to minimize the total number of uteri required, the maximum number of forskolin binding sites was estimated by using a single, saturating concentration of [3H]forskolin.
Statistical Analysis
We used one-way ANOVA and the Student-Neuman-Keuls Method for comparisons of adenylyl cyclase activity between separate assays. For other comparisons within a single assay, i.e., [3H]forskolin concentration-response and gestational comparisons of [3H]forskolin binding, we used ANOVA for repeated measures. When the null hypothesis was rejected, Dunnett's test was used to compare results from pregnant rats with controls (nonpregnant). Results are expressed as means ± SE. We used P < 0.05 to indicate statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Basal adenylyl cyclase activity in myometrial membranes from nonpregnant rats was 26.9 ± 2.1 pmol cAMP/mg protein per 10 min (n = 10) and differed significantly depending on gestational state (P < 0.0001, Fig. 1). During pregnancy, basal adenylyl cyclase activity increased to 60.2 ± 3.5 (n = 6, P < 0.05) and to 124.7 ± 4.1 (n = 3, P < 0.05) pmol cAMP/mg protein per 10 min at mid (Days 12–14) and late (Days 18–20) gestation, respectively, when compared with the nonpregnant state. During labor (Day 22), basal adenylyl cyclase activity (75.3 ± 2.0 pmol cAMP/mg protein per 10 min, n = 3) decreased significantly (P < 0.05) when compared with basal activity during late gestation.
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Similar gestation-related changes in adenylyl cyclase activity occurred during stimulation of G proteins with GTP (10-5 M; Fig. 2, P < 0.0001); ß-adrenergic receptors with GTP (10-5 M) and isoproterenol (10-4 M; Fig. 3, P < 0.0001); or adenylyl cyclase with MnCl2 (20 mM), (Fig. 4, P < 0.001). GTP-stimulated adenylyl cyclase activity increased from 53.6 ± 5.8 pmol cAMP/mg protein per 10 min (n = 10) in myometrial membranes from nonpregnant rats to 90.9 ± 5.4 pmol cAMP/mg protein per 10 min (n = 6, P < 0.05) and to 225.0 ± 7.5 pmol cAMP/mg protein per 10 min (n = 3, P < 0.05) at mid and late gestation, respectively. GTP-stimulated adenylyl cyclase activity was significantly lower in myometrial membranes from intrapartum rats (140.0 ± 3.0 pmol cAMP/mg protein per 10 min, n = 3, P < 0.05) when compared with late gestation. For isoproterenol stimulation, adenylyl cyclase activity increased from 57.3 + 6.4 pmol cAMP/mg protein per 10 min (n = 10) in myometrial membranes from nonpregnant rats to 100.6 ± 5.5 pmol cAMP/mg protein per 10 min (n = 6, P < 0.05) and to 255.0 ± 7.5 pmol cAMP/mg protein per 10 min (n = 3, P < 0.05) at mid and late gestation, respectively. A significant decrease in isoproterenol-stimulated adenylyl cyclase activity, to 155.7 ± 3.7 pmol cAMP/mg protein per 10 min (n = 3, P < 0.05), occurred intrapartum. During direct stimulation with MnCl2, adenylyl cyclase activity was 289.0 ± 34.8 pmol cAMP/mg protein per 10 min (n = 10) in myometrial membranes from nonpregnant rats and did not differ significantly at mid gestation (272.6 ± 21.2 pmol cAMP/mg protein per 10 min, n = 6). MnCl2-stimulated adenylyl cyclase activity increased significantly at late gestation (647.0 ± 21.2 pmol cAMP/mg protein per 10 min, n = 3, P < 0.05) and then decreased significantly intrapartum (417 ± 25.7 pmol cAMP/mg protein per 10 min, n = 3, P < 0.05).
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[3H]Forskolin Binding in Nonpregnant, Pregnant (Day 14, Day 20, Day 21), and Intrapartum (Day 22) Rats
In preliminary studies, specific binding did not increase further for incubations of greater than 45 min (specific binding = 68–78%) or for protein content greater than 0.15 mg (specific binding = 62–65%). We therefore selected an incubation duration of 1 h and a protein content of 0.15–0.3 mg for subsequent studies. For both nonpregnant and pregnant rats, [3H]forskolin produced concentration-dependent increases in specific binding that reached a plateau (n = 4–5, P = 0.01). Specific binding did not differ significantly (P > 0.05) for [3H]forskolin concentrations of 75, 100, 125, or 150 nM. We therefore selected 100 nM [3H]forskolin to estimate maximum forskolin binding sites (Bmax) in further experiments involving rats of different gestational states.
Maximum [3H]forskolin binding showed gestation-related changes (P < 0.001, Fig. 5). In each experiment, Bmax increased from nonpregnant rats (83.0 ± 6.5 fmol/mg protein, n = 5) to rats of gestational Day 14 (106.9 ± 15.5 fmol/mg protein, n = 4, P < 0.05) and to rats of gestational Day 20 (162.2 ± 12.6 fmol/mg protein, n = 5, P < 0.05). At gestational Day 21, all rats manifested an abrupt decrease in Bmax (89.1 ± 13.8 fmol/mg protein, n = 4) to levels similar to those of nonpregnant or intrapartum rats (Bmax = 62.5 ± 24.5 fmol/mg protein, n = 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we evaluated changes in adenylyl cyclase activity throughout pregnancy and during labor. Basal (Fig. 1) and isoproterenol-stimulated (Fig. 3) adenylyl cyclase activity increased during pregnancy and decreased during labor. This pattern was also apparent during stimulation of components of the pathway distal to the ß-adrenergic receptor (Figs. 2 and 4), suggesting that effects of pregnancy were not limited to the receptor. The significant increases in adenylyl cyclase activity at late gestation during stimulation with MnCl2 (Fig. 4), which directly activates the adenylyl cyclase enzyme, indicated an effect of pregnancy on the adenylyl cyclase enzyme. The abrupt decrease in MnCl2-stimulated adenylyl cyclase activity in myometrium from intrapartum rats suggested that decreased activity of this enzyme may facilitate labor.
Alterations in the first two components of the ß-adrenergic receptor-Gs-adenylyl cyclase pathway in rat myometrium have been described. Using saturation radioligand binding studies, Maltier et al. [3] observed an approximate 50% decrease in ß2-adrenergic binding sites from gestational Day 18 to Day 21. These decreases appeared to be mediated, in part, by reductions in the synthesis of ß2-adrenergic receptors, since concentrations of ß2-adrenergic receptor mRNA were reduced at the end of gestation [1]. Furthermore, a greater proportion of ß-adrenergic receptors were reported to be in the low-affinity state, uncoupled from the G protein, on the day of gestation in the rat. Uncoupling of ß-adrenergic receptors, in conjunction with decreased density, resulted in decreased isoproterenol-stimulated adenylyl cyclase activity [2], as in our study (Fig. 3). Gestation-related changes in G proteins have also been described. Expression of Gs protein, measured by ADP ribosylation or immunoblotting, increased from mid to late gestation in rats and was accompanied by increased NaF- or cholera toxin-stimulated adenylyl cyclase activity [10, 11]. In our study, increases in GTP-stimulated adenylyl cyclase activity during pregnancy (Fig. 2) may have resulted from increased expression of the G protein.
Pregnancy affects human myometrial ß-adrenergic receptors and G proteins in a similar manner. Isoproterenol-stimulated adenylyl cyclase activity decreased in myometrial membranes from women at term (39–40 wk), compared to those at preterm (32–35 wk) gestation [12], although another study was unable to duplicate these findings [13]. As in the rat, these findings resulted, in part, from decreased concentrations of ß-adrenergic receptors [14]. Changes distal to the ß-adrenergic receptor, evidenced by increased expression of myometrial Gs during pregnancy [5], with down-regulation of G protein expression and 5'-guanylyl-imidodiphosphate-stimulated adenylyl cyclase activity during labor [6], were also similar to those in the rat.
Effects of pregnancy on the third component of the pathway, the adenylyl cyclase enzyme, have been explored only to a limited extent. In previous studies in rat myometrium, changes in the adenylyl cyclase enzyme were evaluated by measuring forskolin-stimulated cAMP accumulation or adenylyl cyclase activity. Tanfin and Harbon [11] showed increased forskolin-stimulated adenylyl cyclase activity and cAMP generation in myometrium from late-gestation (Days 19–21), compared with mid-gestation (Day 12), rats. These changes presumably resulted from increased function and/or quantity of adenylyl cyclase protein at late gestation, but effects were similar at Day 19 and Day 21, and intrapartum changes were not reported. Similar to our findings of decreased MnCl2-stimulated adenylyl cyclase in intrapartum rats (Fig. 4), two studies reported decreased forskolin-stimulated cAMP generation [2] or adenylyl cyclase activity [15] on the day of delivery (Day 22) compared with Day 15 of gestation, but the authors could not determine the precise timing of these changes because responses on Days 16–21 were not measured. Suzuki et al. [16], in an attempt to clarify the time course of these changes, observed greater forskolin-stimulated adenylyl cyclase activity on gestational Days 17 and 21, when compared with that in nonpregnant rats. Although responses tended to be lower on Day 21 compared with Day 17, the authors did not determine whether these responses differed significantly.
In the present study, we directly evaluated effects of pregnancy on the adenylyl cyclase enzyme by measuring changes in total adenylyl cyclase protein and by stimulating adenylyl cyclase activity with MnCl2, throughout the course of pregnancy and labor. We selected MnCl2 to produce maximal direct stimulation of the adenylyl cyclase enzyme [17], rather than forskolin, because maximum forskolin stimulation may require G protein activation [18]. The parallel increases and decreases in adenylyl cyclase quantity and activity from the nonpregnant state to late gestation and then labor (Figs. 4 and 5) provided evidence that changes in enzyme quantity were largely responsible for the observed changes in activity, but did not eliminate the possibility that enzyme function was also altered. Although one previous study in rat myometrium showed decreased maximum [3H]forskolin binding on gestational Day 22, compared with Day 14, the functional significance of these findings was uncertain because the study did not include measurements of MnCl2-stimulated adenylyl cyclase activity or [3H]forskolin binding on other gestational days [15].
Changes in myometrial adenylyl cyclase activity and protein are likely to have important implications in determining uterine tone. In cardiac myocytes, increased adenylyl cyclase protein content, by overexpression of adenylyl cyclase isoform VI, proportionally increased production of cAMP, independent of ß-adrenergic receptor or G protein expression [19]. If myometrial cells respond in a similar fashion, then increased adenylyl cyclase protein at gestational Days 14 and 20 (Fig. 5) would promote uterine quiescence during pregnancy by enhancing conversion of intracellular ATP to cAMP, which mediates uterine relaxation. Interestingly, the abrupt and profound decrease in enzyme quantity occurred on the day before delivery (Fig. 5). On Day 21 of gestation, with the return of adenylyl cyclase protein to levels similar to those of nonpregnant animals, loss of uterine quiescence would occur, thus facilitating the onset of labor on Day 22.
The total quantity of myometrial adenylyl cyclase protein depends on the quantities of isoforms of this enzyme. Using Northern blotting, Suzuki et al. [16] identified mRNA for five isoforms, and Mhaouty-Kodja et al. [15] identified mRNA for seven isoforms in fresh pregnant rat uterus. Using the more sensitive technique of reverse transcription-polymerase chain reaction, we identified mRNA for eight isoforms (II–IX) in fresh pregnant rat uterus and localized these isoforms to the myometrium cells by documenting their presence in cultured myometrial cells [20]. Our observed changes in total adenylyl cyclase protein (Fig. 5) may reflect changes in production of the isoforms. Suzuki et al. [16] demonstrated increased quantities of mRNA for the five isoforms on gestational Day 17, compared with those in the nonpregnant state, and decreases on gestational Day 21, but other investigators [15] were unable to detect differences from Days 14 to Day 22, perhaps because of the limited sensitivity of the technique. An alternative explanation for the change in total adenylyl cyclase protein quantity is a change in the rate of protein degradation, but this possibility has not yet been explored.
Mechanisms that control gestation-related changes in the quantity of adenylyl cyclase have not yet been elucidated. Sex steroids regulate expression and coupling of rat myometrial ß-adrenergic receptors [2, 3, 21] and Gs [10]. Whether sex steroids regulate adenylyl cyclase expression in a similar fashion is uncertain, since exposure of cultured rabbit myometrial cells to estradiol and progesterone enhanced isoproterenol-stimulated, but not basal or NaF-stimulated, adenylyl cyclase activity [22].
In conclusion, this study provides the first direct evidence of changes in myometrial adenylyl cyclase protein from the nonpregnant state to pregnancy (mid and late gestation) and during labor. Changes in adenylyl cyclase activity during direct stimulation of the enzyme with MnCl2 provide further evidence of the functional importance of these changes in protein quantities. The increased quantity and activity of total adenylyl cyclase during late gestation appear to be important in facilitating uterine quiescence during pregnancy. The sudden, profound decreases in quantity and activity of the enzyme on the last day of pregnancy would result in loss of uterine quiescence and may therefore be important in the mechanism of parturition.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH R29 HD34782. 
2 Correspondence: Karen S. Lindeman, The Johns Hopkins Hospital, 600 North Wolfe Street, Meyer 297, Baltimore, MD 21287-7294. FAX: 410 955 8978; klindema{at}jhmi.edu
Accepted: December 13, 1999.
Received: January 6, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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s in pregnancy. Endocrinology 1993; 132:2484–2490.[Abstract]s in human myometrium in term and preterm labor: a mechanism for parturition. J Clin Endocrinol Metab 1994; 79:1835–1839.[Abstract]This article has been cited by other articles:
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  J. A. Mitchell, T. C. Ting, S. Wong, B.F. Mitchell, and S. J. Lye Parathyroid Hormone-Related Protein Treatment of Pregnant Rats Delays the Increase in Connexin 43 and Oxytocin Receptor Expression in the Myometrium Biol Reprod, August 1, 2003; 69(2): 556 - 562. [Abstract] [Full Text] [PDF]
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 R. Landau, V. Dishy, A. J.J. Wood, C. M. Stein, and R. M. Smiley Disproportionate Decrease in {alpha}- Compared With {beta}-Adrenergic Sensitivity in the Dorsal Hand Vein in Pregnancy Favors Vasodilation Circulation, August 27, 2002; 106(9): 1116 - 1120. [Abstract] [Full Text] [PDF]
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 J. F. Telfer, H. Itoh, A. J. Thomson, J. E. Norman, K. Nakao, J. S. Campa, L. Poston, R. M. Tribe, and R. R. Magness Activity and Expression of Soluble and Particulate Guanylate Cyclases in Myometrium from Nonpregnant and Pregnant Women: Down-Regulation of Soluble Guanylate Cyclase at Term J. Clin. Endocrinol. Metab., December 1, 2001; 86(12): 5934 - 5943. [Abstract] [Full Text] [PDF]
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