A decline in myometrial nitric oxide synthase expression is associated with labor and delivery.

The mechanisms that maintain relative uterine quiescence during pregnancy remain largely unknown. A possible role for nitric oxide has recently emerged, however, the expression of nitric oxide synthase within human myometrium at midgestation, a time when the uterus is normally quiescent, has not been investigated. The purpose of this study was to identify cell types in human myometrium that contain inducible nitric oxide synthase (iNOS), and to examine changes in its expression during pregnancy and labor. We found that iNOS is expressed in smooth muscle cells of pregnant myometrium. Expression of iNOS was highest in myometrium of preterm not-in-labor patients. At term, iNOS expression fell by 75%, and was barely detectable in preterm in-labor or term in-labor specimens. There was no staining in the myocytes of nonpregnant myometrium. Western blotting also revealed a similar pattern of changes in iNOS expression. In summary, iNOS expression in the myocytes of human myometrium is increased greatly during pregnancy, and declines towards term or with labor. Significantly, preterm inlabor patients also had a large decline in iNOS expression. These data suggest that changes in myometrial iNOS expression may participate in the regulation of uterine activity during human pregnancy.

Rabbit uterine oxytocin receptors and in vitro contractile response: abrupt changes at term and the role of eicosanoids.

We examined the relation between increased uterine oxytocin receptor concentration and increased in vivo sensitivity of the rabbit uterus to oxytocin at the end of gestation. We determined oxytocin receptor concentrations in myometrium and decidua on different days near term of gestation and postpartum. We also examined the in vitro contractile response to oxytocin on days 30 and 5 days postpartum, when the uterus is unresponsive in vivo, and on day 31 (term), when the uterus is exquisitely sensitive to this hormone in vivo. In addition, we tested the role of endogenous eicosanoids and decidual oxytocin receptors in the myometrial contractile response to oxytocin by examining the contractile response in the presence of the cyclooxygenase/lipoxygenase inhibitor sodium meclofenamate or in muscle strips from which the decidua had been removed by scraping. The concentration of specific binding sites for [3H]oxytocin in myometrial and also decidual membrane preparations was determined. We demonstrate that contractile sensitivity to oxytocin increases at least 4-fold between days 30 and 31 (term) of gestation, and this is accompanied by a nearly 10-fold increase in the concentration of oxytocin-binding sites in both decidua and myometrium. The lesser sensitivity to oxytocin on day 30 was, however, only apparent in the presence of meclofenamate, which suggests that endogenous eicosanoids contribute to the preterm response to oxytocin measured in vitro. The maximal response to oxytocin (integrated area) increased 2-fold between day 30 and term. Thus, an increase in both sensitivity and maximal response to oxytocin could be demonstrated at term in vitro. Five days after parturition, maximal response and uterine sensitivity measured in the presence of meclofenamate had returned to those of the preterm uterus, and the concentration of oxytocin-binding sites had declined. In contrast, sensitivity and maximal response to the cholinergic agonist carbamylcholine declined between day 30 and term. These results support a highly regulated physiological role for oxytocin in parturition which depends primarily on changes in receptor concentration.

Restoration of estrogen-dependent progesterone receptor expression in a uterine myocyte cell line.

Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.

Prostaglandins modulate hormonal effects on rabbit myometrial alpha 1-adrenergic responses.

Estrogen increases the alpha 1-adrenergic contractile sensitivity of the rabbit uterus. Since estrogen treatment increases prostaglandin (PG) production by the perfused rabbit uterus, and PGs contribute to the alpha 1-adrenergic contractile response, we postulated that estrogen's effects on PG production or response may play a role in the increased alpha 1-adrenergic sensitivity induced by estrogen. We studied the effects of the eicosanoid synthesis inhibitor meclofenamate (60 microM) on the response to epinephrine (10(-9)-10(-5) M) of uterine strips from ovariectomized, mature, and estrogen-treated rabbits in terms of both contractile response and PGE2 and PGF2 alpha production. We also measured the contractile response to PGE2 and PGF2 alpha (both 10(-10)-10(-5) M) and KCl (70 mM) of uterine strips from these groups. We found that in the ovariectomized rabbits, meclofenamate decreased PG production, but did not alter the alpha 1-adrenergic sensitivity. In the mature rabbit uterus, meclofenamate decreased both PGE2 and PGF2 alpha production and reduced the alpha 1-adrenergic sensitivity. In the estrogen-treated rabbit uterus, meclofenamate decreased PGF2 alpha, but not PGE2, production and did not alter the alpha 1-adrenergic sensitivity. Finally, meclofenamate reduced the contractile response to KCl in all three groups, and exposure to PGE2 increased the contractile response to KCl in both the mature and estrogen-treated rabbits. We conclude that PGs play a role in the increase in the alpha 1-adrenergic sensitivity of the uterus in mature rabbits, and that this may be the result of an estrogen-mediated alteration in the postreceptor effects of PGs.

Rabbit myometrial oxytocin and alpha 2-adrenergic receptors are increased by estrogen but are differentially regulated by progesterone.

Both myometrial oxytocin and alpha 2-adrenergic receptors are induced by estrogen. To compare the regulation of these two receptor populations by progesterone, we measured myometrial receptor concentration in ovariectomized steroid-treated and in pregnant rabbits. To control for the effects of estrogen withdrawal, we used concomitant rather than sequential presentation of estrogen and progesterone in ovariectomized rabbits. Estradiol increased both myometrial oxytocin and alpha 2-adrenergic receptor concentrations in ovariectomized rabbits after 8 days of treatment. Simultaneous progesterone administration during the last 4 days of estradiol treatment reversed the induction of oxytocin, but not alpha 2-adrenergic, receptors. Similarly, administration of the antiprogestin RU 38486 to pregnant rabbits on day 27 of gestation resulted in premature delivery and evoked an increase in myometrial oxytocin receptor concentration mimicking that observed at term (day 31). However, RU 38486 did not significantly affect alpha 2-adrenergic receptor concentration. Our data provide further support for involvement of oxytocin receptors in parturition, but do not indicate a comparable function for myometrial alpha 2-adrenergic receptors.

Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells.

Previous work has suggested that nerve growth factor treatment of PC12 cells induces neuronal nitric oxide synthase, and possibly also endothelial nitric oxide synthase (NOS) and inducible NOS. To further analyze this process we exposed rat pheochromocytoma (PC12) cells to increasing concentrations of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), nerve growth factor (NGF), and vascular endothelial cell growth factor (VEGF). Changes in NOS expression were then analyzed by Western blotting, using antisera generated against the three isoforms of NOS. Our results demonstrate that neuronal NOS was induced by growth factors that promote both differentiation (bFGF, NGF) and proliferation (EGF). nNOS levels were unaffected by VEGF treatment. In contrast, the levels of endothelial and inducible NOS were undetectable in these same cells, suggesting that different clonal lines of PC12 cells have different isoform complements.

Superior cavopulmonary anastomosis suppresses the activity and expression of pulmonary angiotensin-converting enzyme.

BACKGROUND: Superior cavopulmonary anastomosis is widely used for palliation of various forms of univentricular heart defects. However, clinically significant pulmonary arteriovenous malformations develop in 15% to 25% of patients after surgery. OBJECTIVE: To assess altered regulation of pulmonary vascular tone caused by superior cavopulmonary anastomosis in an ovine model. METHODS: Lambs, aged 35 to 45 days, underwent an end-to-end anastomosis of the superior vena cava to the right pulmonary artery. In age-matched controls, a sham operation was performed. Arteriovenous malformations were detectable by contrast echocardiography by 8 weeks after surgery. Animals (n = 24) were studied at various time points after the operations. Expression of angiotensin-converting enzyme messenger RNA, protein levels, and enzyme activity were measured in lung homogenates. Levels of angiotensin II were measured by enzyme-linked immunosorbent assay. RESULTS: Expression of angiotensin-converting enzyme messenger RNA and protein was significantly reduced at 1 to 5 weeks after superior cavopulmonary anastomosis. Angiotensin-converting enzyme activity in the right lung of animals subjected to superior cavopulmonary anastomosis was reduced 86% +/- 1% (standard deviation) compared with control values at 1 week (P =.003) and 77% +/- 8.5% at 2 weeks (P <.001) after surgery. This correlated with a 59% +/- 3.5% (P =.007) reduction in angiotensin II levels up to 5 weeks after cavopulmonary anastomosis. By 15 weeks after the operations, angiotensin II levels were equivalent to control levels (P =.19). CONCLUSIONS: Superior cavopulmonary anastomosis causes an early reversible reduction in activity and expression of angiotensin-converting enzyme, resulting in decreased circulating levels of the vasoconstrictor angiotensin II. These results suggest that the ability of the pulmonary endothelium to regulate vascular tone is inhibited after superior cavopulmonary anastomosis. Dilation of the affected vasculature induced by cavopulmonary anastomosis may contribute to the disordered vascular remodeling observed in this setting.

Sanguinarine: a positive inotropic alkaloid which inhibits cardiac Na+,K+-ATPase.

In isolated, isometrically contracting left guinea pig atria, sanguinarine, a benzophenanthridine alkaloid from the papaveracea Sanguinaria canadensis, produced a concentration-dependent positive inotropic effect. Between 2.3 x 10(-6) M and 6.5 x 10(-5) M, sanguinarine increased contractility by 108% which was comparable to the maximal inotropic effect of ouabain. Within the same concentration range, sanguinarine caused inhibition of Na+,K+-ATPase isolated from guinea pig myocardium. 100% inhibition of Na+,K+,ATPase activity occurred at 1 x 10(-4) M sanguinarine. The I50 for enzyme inhibition and the ED50 for the inotropic action of sanguinarine were the same (6-6.5 x 10(-6) M) indicating that both effects may be causally related.

Myometrial characteristics of the Syrian hamster uterine smooth muscle cell line, SHM.

We describe several characteristics of a novel smooth muscle cell line, SHM (Syrian hamster myometrium) derived from a primary uterine leiomyosarcoma which was induced by chronic estrogen plus androgen treatment of a female Syrian (golden) hamster. To determine the usefulness of the SHM cell line as a model for understanding myometrial function and its regulation, we have examined the morphologic and immunocytochemical properties of these cells, and the ability of uterotonic agonists to activate transmembrane signaling via phosphoinositide hydrolysis. The SHM cells exhibited a spindle-shape, smooth musclelike morphology when subconfluent, and a more compact, stellate shape at confluence. Like primary myocytes, SHM cells expressed the intermediate filament desmin and the contractile protein alpha smooth muscle actin, but not the epithelial antigen cytokeratin. Norepinephrine and bradykinin, which stimulate contraction and inositol polyphosphate production in the uterus, also stimulated inositol polyphosphate production in SHM cells. The maximal phosphoinositide signaling responses were lower in SHM cells compared with primary hamster uterine myocytes. We conclude that the SHM cell line exhibits primary uterine myocyte characteristics, and may therefore be a useful system for examining the mechanisms through which myometrial functions are regulated.

Myometrial characteristics of the syrian hamster uterine smooth muscle cell line, SHM.

We describe several characteristics of a novel smooth muscle cell line, SHM (Syrian hamster myometrium) derived from a primary uterine leiomyosarcoma which was induced by chronic estrogen plus androgen treatment of a female Syrian (golden) hamster. To determine the usefulness of the SHM cell line as a model for understanding myometrial function and its regulation, we have examined the morphologic and immunocytochemical properties of these cells, and the ability of uterotonic agonists to activate transmembrane signaling via phosphoinositide hydrolysis. The SHM cells exhibited a spindle-shape, smooth musclelike morphology when subconfluent, and a more compact, stellate shape at confluence. Like primary myocytes, SHM cells expressed the intermediate filament desmin and the contractile protein alpha smooth muscle actin, but not the epithelial antigen cytokeratin. Norepinephrine and bradykinin, which stimulate contraction and inositol polyphosphate production in the uterus, also stimulated inositol polyphosphate production in SHM cells. The maximal phosphoinositide signaling responses were lower in SHM cells compared with primary hamster uterine myocytes. We conclude that the SHM cell line exhibits primary uterine myocyte characteristics, and may therefore be a useful system for examining the mechanisms through which myometrial functions are regulated.

Myocardial cholinergic signaling changes with age.

We examined the linkage of cholinergic receptors to the phosphoinositide signaling pathway to elucidate one facet of the autonomic response mechanism in fetal and adult sheep. Cholinergic stimulation with carbachol increases the production of 3H-inositol mono-, bis-, and trisphosphates in a time- and concentration-dependent manner in both fetal and adult myocardium. However, the maximal stimulation of inositol polyphosphates above basal activity was much greater in fetal (120 +/- 11%) than in adult (20 +/- 7%) myocardium (mean +/- SEM). Saturation binding analysis of myocardial muscarinic receptors using 3H-N-methylscopolamine revealed significantly higher receptor concentration in fetal (240 +/- 25 fmol/mg protein) than in adult (78 +/- 15 fmol/mg protein) myocardium (mean +/- SEM). Binding competition studies revealed a pattern of selectivity-atropine less than 4-diphenylacetoxy-N-methylpiperidine methiodide less than pirenzepine less than or equal to (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride less than or equal to 11-2[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one 116-compatible with the presence of muscarinic receptor (MR)2, MR3, and/or MR5 subtypes. Receptor subtype determination by Northern blot analysis revealed mRNA specific for the MR2 subtype in both fetal and adult myocardium, although expression was greater in fetal heart. We conclude that decreases in MR2 subtype protein and mRNA levels parallel the age-related decrease in carbachol-stimulated PLC activity. Our studies demonstrate differences between fetal and adult myocardium in the concentration of muscarinic cholinergic receptors and their linkage to a putative calcium mobilizing signaling pathway and suggest that this pathway may play a different role in the fetus than in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of uterine smooth muscle function during gestation.

The uterus is unique among smooth muscular organs in that, during pregnancy, it undergoes profound, largely reversible, changes orchestrated by the ovarian hormones. These changes facilitate uterine adaptation to the stretch induced by the growing fetus such that a state of myometrial contractile quiescence can be maintained. This quiescent state usually is maintained until fetal development is sufficient for extrauterine life, at which point unknown mechanisms precipitate conversion to a highly contractile state. Throughout pregnancy, signaling mechanisms for myometrial contractility are altered--first to promote quiescence and then again to promote contractions. The mechanisms responsible for these changes are only partially understood. This review attempts to summarize salient features of many of the changes in uterine contractile signaling and the current state of ongoing investigations of their mechanisms. We have also highlighted some newer information and concepts from nonuterine tissues, which we believe may provide insight into the control of uterine smooth muscle function. Some detail has been omitted, and can be found in the many excellent reviews cited. We hope that this discussion may stimulate the interests of other investigators. The diverse areas of inquiry offer hope that this decade will lead to a fuller understanding of myometrial function and the development of vastly improved approaches for the control of preterm labor.

Estrogen increases adrenergic- but not cholinergic-mediated production of inositol phosphates in rabbit uterus.

alpha 1-Adrenergic and muscarinic cholinergic stimuli activate uterine contraction. Estrogen increases adrenergic but not cholinergic sensitivity of rabbit myometrium independent of its effects on adrenoceptor concentration. Since both alpha 1-adrenergic and muscarinic receptors are coupled to phosphatidylinositol hydrolysis, we tested the hypothesis that estrogen increases adrenergic- but not cholinergic-mediated inositol triphosphate production. We found that maximal production of inositol phosphates stimulated by norepinephrine was increased approximately 3-fold following estrogen treatment. Cholinergic-stimulated production was not increased by estrogen treatment. These results demonstrate that the effect of estrogen to enhance uterine adrenergic sensitivity is associated with an increased post-receptor response. The nature of the selectivity of estrogen for adrenergic versus cholinergic response remains obscure, but the results suggest the presence of parallel pathways for receptor activation of a common post-receptor response.

The concentration of estrogen receptors in rabbit uterine myocytes decreases in culture.

OBJECTIVE: The purpose of this study was to determine whether the concentration of estrogen receptors in cultured myocytes is preserved after dispersion. STUDY DESIGN: Primary myocytes were prepared from rabbit myometrium by collagenase dispersion after removing the endometrium and were isolated with Percoll density gradients. The cells were assayed for estrogen receptor concentration at intervals after dispersion by means of a whole-cell binding assay. Unpaired t test was used for comparisons. RESULTS: The concentration of estrogen receptors on the first day after dispersion was 12,058 +/- 1096 sites per cell (mean +/- SEM) and decreased to 4389 +/- 1223 site per cell within 9 to 14 days after dispersion (63% decline, p < 0.001). A similar decrease was observed when 2 nmol/L estradiol was present in the medium. CONCLUSION: The concentration of estrogen receptors in isolated rabbit uterine myocytes decreases after dispersion. This may partly explain the difficulty of demonstrating in vitro estrogen effects on myocytes, which are well established in vivo.

Rabbit myometrial adrenergic sensitivity is increased by estrogen but is independent of changes in alpha adrenoceptor concentration.

The ability of estrogen to increase the alpha-1 adrenergic sensitivity of rabbit uterine smooth muscle was compared with its effects on the concentration of alpha adrenoceptor subtypes in myometrium. Compared to ovariectomized controls, estrogen treatment was found to increase the adrenergic contractile sensitivity of rabbit uterus determined in vitro. Estrogen treatment increased uterine alpha-2 adrenoceptor concentration nearly 5-fold. Mature rabbits (endogenous estrogen) had the same uterine sensitivity and alpha-2 adrenoceptor concentration as estrogen-treated rabbits. Alpha-1 receptor concentration was increased only in the estrogen-treated group, and was accompanied by increased maximal contractile response. Thus, the increase in adrenergic sensitivity after estrogen was correlated closely with an increased concentration of alpha-2 adrenoceptors. However, enhanced adrenergic sensitivity persisted after alpha-1 and alpha-2 receptor concentration returned to pre-estrogen levels in rabbits pretreated with estrogen before ovariectomy. Studies utilizing subtype-selective competitive antagonists verified that contractile response is mediated primarily by alpha-1 adrenoceptors, with no apparent influence of alpha-2 adrenoceptors. Finally, we found that adrenergic sensitivity is altered by temperature and divalent cation concentration, but these effects do not prevent the expression of the regulatory action of estradiol. We conclude that estrogen increases the alpha-1 adrenergic sensitivity of rabbit uterus without changes in alpha-1 receptors, and thus may act on postreceptor response events.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of prostaglandin E1 receptors in cavernous tissue of men, monkeys and dogs.

Prostaglandin E1 (PGE1) has been shown to relax the muscles of the corpora cavernosa and inhibit spontaneous activity, and clinical trials have proved its safety and effectiveness when given intracavernously to induce erection. Through use of a specific PGE1 receptor binding assay, we undertook this study to quantify its receptor density and measure binding affinity. The cavernous tissue of normal and impotent men as well as that of monkeys and dogs was studied in an attempt to understand their different responses to the intracavernous injection of PGE1. Our results showed a lower receptor density in impotent men than in normal men and monkeys and a complete absence of receptors in dogs. These findings correlated well with the clinical response to intracavernous injection of PGE1.

Nitric oxide synthase activity in the pregnant uterus decreases at parturition.

The mechanisms that mediate changes in uterine activity from a quiescent state during pregnancy to active labor at parturition are unknown. Nitric oxide (NO), a potent mediator of smooth muscle relaxation, and its presence in the uterus is the subject of this report. Nitric oxide synthase (NOS) activity was demonstrated in nerves, blood vessels and decidua of gravid rat uterus by the NADPH-diaphorase staining method. Uterine tissue fixed during labor demonstrated markedly less NOS. Quantitation of NOS activity in subcellular fractions of pregnant and laboring uterus revealed its presence in both the cytosolic and the membranous compartments of uterine homogenates. In both cellular subfractions the enzyme activity decreased significantly from pregnancy to term. We conclude NOS is present in multiple structures within the uterus. Its presence in two cellular compartments suggests more than one form of NOS may be present in the uterus. Reduction in NOS activity at parturition suggests NO may contribute to the maintenance of uterine contractile quiescence during gestation.

Hormonal regulation myometrial adrenergic responses: the receptor and beyond.

The contractile response of the uterus is modified by sex steroids. In rabbit uterus, oestrogen promotes alpha-adrenergically-mediated contraction, whilst progesterone treatment results in beta-adrenergic relaxation. Examination of the mechanisms responsible for these changing adrenergic responses with sex steroids reveals multiple sites of regulation. Oestrogen increases alpha 1-receptor concentration and the linkage of the receptor to phospholipase C. In addition to this direct effect to promote contraction, oestrogen also uncouples the beta-receptor from adenylate cyclase. Progesterone, conversely, promotes relaxation through beta-receptors by uncoupling alpha 2-receptors from inhibition of adenylate cyclase. Thus sex steroids can regulate specific agonist responses at and beyond the receptor.

Progesterone prevents linkage of rabbit myometrial alpha 2-adrenergic receptors to inhibition of adenylate cyclase.

The uterine response to adrenergic stimulation is determined by the hormonal milieu. This response is particularly well characterized in the rabbit. In this species, as in humans, the response of the uterus to sympathetic stimulation is alpha-adrenergically mediated contraction with elevated circulating estrogen. However, with progesterone predominance, similar stimulation inhibits uterine contractions, a response mediated by beta-adrenergic receptors acting through their second message, cyclic adenosine monophosphate. We studied the mechanisms by which sex steroids regulate myometrial adrenergic responses. In this study, we questioned whether part of the effect of sex steroids could be explained by an alteration of the coupling of the alpha 2-adrenergic receptor to the inhibition of adenylate cyclase. We found that in the progesterone-treated rabbit, although alpha 2-receptors are present, they are not linked to inhibition of cyclic adenosine monophosphate synthesis. The net synthesis of cyclic adenosine monophosphage in response to endogenous catecholamines is determined by their activation of beta-adrenergic receptors to increase and alpha 2-receptors to decrease cyclic adenosine monophosphate formation. Thus the uncoupling of alpha 2-receptors contributes to increased intracellular cyclic adenosine monophosphate in myometrium of progesterone-treated animals consistent with the reported predominance of beta-adrenergic contractile responses in this setting.