Molecular Human Reproduction, Vol. 6, No. 9, 835-842,
September 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Changes in ß2-adrenoceptor expression and in adenylyl cyclase and phosphodiesterase activity in human uterine leiomyomas
1 Division of Pharmacology, Department of Medicine and Care, 2 Division of Cell Biology, Department of Biology and Surgery and 3 Division of Obstetrics and Gynecology, Department of Health and Environment, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden
Abstract
Uterine leiomyoma is a very common benign tumour with unclear pathophysiology in adult women. In the present study we have investigated the expression level of 
2- and ß2-adrenoceptors, and the adenylyl cyclase and phosphodiesterase activity in leiomyoma tissue compared with adjacent myometrium. Our results show that the 2/ß2-adrenoceptor ratio is increased in leiomyoma, due to a significant decrease in ß2-adrenoceptor expression. These changes were not due to an increased innervation, as the tumour tissue was completely devoid of nerve fibres. Moreover, the adenylyl cyclase activity of leiomyoma membranes was found to be ~50% lower, whereas the phosphodiesterase activity was significantly increased (by ~100%). We found that stimulating an increase in intracellular cyclic AMP, by adenylyl cyclase activity through ß2-adrenoceptors (isoprenaline), by direct enzyme activation (forskolin), or by inhibition of phosphodiesterase activity (papaverine), potently blocked both protein and DNA synthesis in cultured leiomyoma smooth muscle cells. Our results imply the adrenoceptors might be involved in, or a consequence of, leiomyoma growth. The results also suggest a new interesting approach for leiomyoma pharmacotherapy.
adenylyl cyclase/adrenergic receptors/cAMP/leiomyoma/phosphodiesterase
Introduction
Uterine leiomyomata which occur in 40% of all women aged >50 years, represent the most prevalent tumours in adult women (Rosai, 1989
). These benign tumours, often incorrectly referred to as fibroids, are primarily clonal and each one arises independently from a single myometrial smooth muscle cell (Townsend et al., 1970). There is a strong correlation between leiomyoma and infertility. It has been demonstrated that the reproductive outcome after laparoscopic myomectomy for interstitial and/or subserosal myomata in infertile women with associated infertility factors, is >50% (Dubuisson et al., 2000).
Research on the pathophysiological origin of uterine leiomyoma has focussed particularly on ovarian steroids (Koutsilieris, 1992). However, no investigation has yet generated a pharmaceutical treatment able to replace surgery for leiomyomata. On the other hand, benign prostate hyperplasia (BPH) in men, which also consists of smooth muscle tissue, has been examined from a completely different perspective. When the physiological role of 1-adrenoceptors (1-AR) in prostate smooth muscle cells was studied, a reduced proliferation was obtained by inhibition of 1-AR stimulation. So in clinics today, different 1-AR antagonists are frequently used in BPH therapy (Hieble and Ruffo, 1996).
A number of investigations have shown that smooth muscle growth in different species (including humans) can, at least to some extent, be regulated by 1-, 2- and/or ß2-AR (Noveral and Grunstein, 1994; Caine and Raz, 1995; Tomlinson et al., 1995; Yu et al., 1995; Nilsson et al., 1998). Generally, - and ß-AR generate opposing effects regarding smooth muscle cell proliferation. While stimulation of -AR promotes proliferation, activation of ß-AR generates an inhibitory response. The latter effect is mediated by stimulation of adenylyl cyclase (AC) and thereby an elevation of intracellular cyclic AMP (cAMP). Leiomyoma is characterized by hyperplastic/hypertrophic smooth muscle tissue, similar to that observed in BPH (Thompson and Applemean, 1971; Hieble and Ruffo, 1996) However, it is not known whether adrenoceptors are involved in the regulation of growth of uterine leiomyomata.
The main purpose of the present study was to compare the expression of 2- and ß2-AR in uterine leiomyoma and surrounding myometrium. Moreover, the purpose was to examine whether there are any changes in the activity of enzymes that are directly involved in regulation of the cAMP level, i.e. AC and phosphodiesterase (PDE). Finally, we aimed at clarifying whether cAMP regulating agents influence the growth of uterine leiomyoma smooth muscle cells in vitro.
Materials and methods
Materials
[3H]-Rauwolscine (NET 722) and [125I]-cAMP (NEK-035) was purchased from NENTM Life Science Products (Boston, MA, USA); [3H]-dihydralprenolol ([3H]-DHA), [methyl-3H]-thymidine (TRA 120) and [35S]-methionine (AG 1594) were obtained from Amersham Pharmacia Biotech Ltd, Buckinghamshire, UK). Phentolamine (Regitina®; Ciba, Basel, Switzerland); propranolol, 3-isobutyl methyl xanthine (IBMX) and (Na)2ATP were obtained from Sigma Chemical Co (St Louis, MO, USA). The anion exchange column matrix AG® 1-X8, (Resin 200–400) mesh, was obtained from Bio-Rad Laboratories. Papaverin® was obtained from NM Pharma, Stockholm, Sweden. Medetomidine was a gift from Orion Corp./Farmos (Turku, Finland). The antibodies against -smooth muscle actin that was used to characterize cultured cells were also obtained from Sigma. The cell culture medium, Dulbecco's modified Eagle's medium (DMEM), non-essential amino acids (100x), sodium pyruvate (100 mmol/l), penicillin–streptomycin (1000 IU/ml), fetal bovine serum (FBS) and trypsin were all purchased from Gibco (Life Technologies, USA). Polyclonal antibodies against the general nerve marker protein gene product 9.5 (PGP 9.5) were purchased from Biogenesis, Sandown, NH, USA. Finally, the polyclonal antibodies against the sympathetic marker tyrosin hydroxylase (TH) were ordered from Affinity Research Ltd (Nottingham, UK).
Tissue collection
Biopsies were obtained from non-pregnant premenopausal women aged 35–55 years, undergoing hysterectomy due to symptomatic uterine leiomyomata at the Division of Obstetrics and Gynaecology, University Hospital of Linköping. The Ethical Committee of the University of Linköping approved collection and use of the biopsies for these experiments. All patients consented to the biopsies well in advance of the surgery, and they were also well informed about the experimental aim of the study. Biopsies were taken from tumours lacking any signs of necrosis, and from surrounding myometrial tissue without visible signs of leiomyoma. The uterine leiomyoma and myometrial samples were immediately placed in sterile tubes containing sterilized Ringer's solution and transported on ice to the laboratory. Each patient served as her own control in all studies. Tissue that was not used immediately was frozen with liquid nitrogen and stored at –80°C.
Membrane preparation
The biopsies were cut into small pieces and put in ice-cold 50 mmol/l Tris–HCl buffer containing 25 mmol/l MgCl (pH 7.4, from here on referred to as TM). The tissue slices were homogenized for 30 s at 20 000 rpm using an Ultra Turrax T25 (Janke & Kunkel, IKA® Labortechnik, Staufen, Germany) and subsequently centrifuged at 1100 g at 4°C for 15 min. This process was repeated and the following supernatants were centrifuged at 48 000 g for 30 min to pellet the membrane fraction. The membrane pellets were re-suspended in ice-cold TM and homogenized using a glass-Teflon® homogenizer. The protein concentration of the membrane suspension was analysed using Coomassie Protein Assay Reagent (Pierce) according to the instructions of the manufacturer. Membrane preparations not used immediately were frozen (–80°C) and stored until analysis.
Radioligand binding studies
The first experiments were designed to quantify the expression of 2- and ß2-AR in human uterine leiomyoma and surrounding myometrium using a radioligand binding technique. The samples of normal myometrial tissue were taken at some distance from the tumour to ensure that no leiomyoma tissue was included. The specimens were collected from nine pre-menopausal women, all serving as their own controls in representative saturation studies. However, in one patient the membrane preparation was just sufficient for the ß2-AR analysis. Each concentration of radioligand (0.05–4 nmol/l [3H]-DHA for AR ß2-analysis; 0.3–12 nmol/l [3H]-rauwolscine for AR 2-analysis) was run at least in triplicate. The membrane protein samples (200–400 µg, in TM) of respective tissue were incubated with increasing concentrations of [3H]-rauwolscine or [3H]-DHA for 1 h at room temperature in a final experimental volume of 500 µl. The samples were diluted with 5 ml ice-cold TM and then a rapid vacuum filtration was performed through a Whatman GF/C filter using a Brandel M48 sample harvester which terminated the radioligand incubation. Thereafter, the filters were washed three times with ice-cold TM and transferred to scintillation tubes containing Ultima GoldTM (Packard, USA) scintillation liquid. The analysis was performed using a 1217 Rackbeta liquid scintillation counter (LKB Wallac, Turku, Finland). Non-specific binding was determined using 100 µmol/l phentolamine (-AR antagonist) or 100 µmol/l propranolol (ß-AR antagonist). To evaluate the results of the radioligand studies, a non-linear regression analysis method was used (Prism GraphPad Software®, USA).
Immunohistochemistry
Specimens were collected from five hysterectomy patients. The specimens included leiomyoma tissue and healthy myometrial tissue at some distance from the tumour. A modification of the immunohistochemical technique described by Coons (Coons, 1958) was used. Briefly, specimens (5x5x10 mm) were placed in ice-cold 4% paraformaldehyde (PFA) within 5 min after completion of the hysterectomy. The specimens were fixed in cold PFA for 24 h, rinsed and cryoprotected with 10% sucrose. Frozen sections were cut at 14 µm in a Leitz 1720 Digital kryostat. The sections were mounted on chromalun gelatinated slides and incubated with polyclonal rabbit antibodies against the general nerve marker gene product PGP 9.5 (1: 2000, Biogenesis, USA) or against the sympathetic marker TH (1:50, Affinity Research Ltd, UK). The sera were diluted with phosphate buffered saline (PBS) containing 3% normal donkey serum, 3% bovine serum albumin (BSA) and 0.3% Triton X-100. After 12 h incubation with the primary serum, the sections were incubated with donkey anti-rabbit antibodies conjugated with tetramethyl rhodamine isothiocyanate (TRITC; 40 min; PGP 9.5 1:160; TH 1:20; Jackson Immunoresearch Laboratories Inc, USA). The immunostained sections were coverslipped with antifading agent and examined in a microscope (Zeiss Axiophot) in the fluorescence mode. In the controls, the primary antibodies were omitted.
Adenylyl cyclase assay
The AC-activities of leiomyoma and myometrial membranes from seven patients were determined using a radioimmunoassay (Steiner et al., 1972; Salomon et al., 1974) with [125I]-cAMP as a labelled substance and antibodies that were prepared in our own laboratory. Briefly, 10 µg membrane protein/sample was incubated together with 90 mmol/l Tris–HCl (pH 7.5), 10 mmol/l MgCl2, 2 mmol/l IBMX and 2 mmol/l ATP at 37°C for 5 min with or without forskolin, and terminated by heating the samples to 90°C for 3 min. The final incubation volume was 100 µl. Then ~50 mg of Al2O3 and 300 µl of Tris–HCl (50 mmol/l, pH 7.5) was added to each sample and mixed well before being centrifuged at 1000 g to pellet the protein-Al complex. Each radioimmunoassay was analysed using a 1282 Compugamma CS (LKB Wallac). The cAMP values were calculated from a standard curve, fitted by an unweighted linear regression method.
Phosphodiesterase assay
The PDE activities of uterine leiomyoma and myometrial membranes from six patients were determined by using [3H]-cAMP as labelled substrate according to a previously described method (Thompson and Appleman, 1971) with some modifications. Briefly, 40 µg membrane protein/sample was incubated in a final incubation volume of 500 µl together with 40 mmol/l Tris–HCl (pH 7.5), and [3H]-cAMP corresponding to 200 000 d.p.m. (3.6x10–8 mol/l) at 30°C for 30 min. The assay was terminated by heating the samples to 95°C for 2 min and thereafter put on ice. To determine the auto-hydrolysis of cAMP, two samples were incubated without membranes. To each sample including the controls without membranes, 100 µl (1 µg/µl) 5'-nucleotidase (snake venom, Ophiophagus Hannah) was added and incubated at 37°C for an additional 30 min. The incubation was terminated by heating the samples to 95°C for 2 min and thereafter put on ice. The adenosine was separated using an anion exchange column (AG® 1-X8, Resin 200–400 mesh; Bio-Rad), and analysed using liquid scintillation technique.
Establishment of leiomyoma smooth muscle cell culture
Biopsies were briefly washed in 70% EtOH, transferred to a continuous laminar flow-hood and placed on 150 mm dishes containing culture medium with the following components: DMEM, 1% non-essential amino acids, 1% sodium pyruvate, and 1% penicillin–streptomycin. Biopsies were minced into pieces of ~3 mm3 and incubated in collagenase (2 mg/ml) for 6x15 min. After each incubation period, the suspended cells were separated from the biopsies and poured into a new tube and centrifuged (1500 g for 5 min) to pellet the cell fraction. Each pellet was resuspended in the above-mentioned DMEM solution also containing 10% FBS (from here referred to as DMEM) and spread out on culture dishes. The cells were incubated for 2–3 weeks before being trypsinized and split into new culture dishes. The cells were characterized as smooth muscle cells using a monoclonal antibody against smooth muscle -actin, distinguishing smooth muscle cells from fibroblasts (Skalli et al., 1986). Incubation was carried out in an incubator at 37°C, in a humidified atmosphere (95%) with 5% CO2 and 95% air.
[3H]-Thymidine incorporation
The DNA synthesis in cultured leiomyoma smooth muscle cells was determined by measuring the incorporation of [3H]-thymidine. The cells were spliced into 24- or 96-well plates with 50x103 and 10x103 cells/well respectively, and incubated first in DMEM for 48 h, and then in starvation medium, without FBS. The cells were conditioned in this medium for 24 h before starting the proliferation studies. The leiomyoma smooth muscle cells were supplied with [3H]-thymidine and in the first experiment treated in three separate groups as follows: (i) control; (ii) forskolin (10–7 mol/l), (iii) isoprenaline (10–5 mol/l). In the second investigation, the following study was set up: (i) control; (ii) forskolin (10–6 mol/l); (iii) papaverin (10–5 mol/l); (iv) forskolin (10–6 mol/l) and papaverin (10–5 mol/l). In the third assay, the following conditions were set up: (i) control; (ii) medetomidine (10–7 mol/l) and (iii) medetomidine (10–7 mol/l) and yohimbine (10–6mol/l). In all experiments, the control conditions correspond to DMEM. After 24 h incubation the smooth muscle cells were rinsed once with PBS and subsequently treated with 5% v/v ice-cold trichloroacetic acid (TCA) for 30 min to terminate the incubation. Thereafter, the cultured smooth muscle cells were dissolved in 0.2 or 0.5 ml KOH (0.5 mol/l) for 1 h at room temperature and analysed by the above mentioned liquid scintillation technique.
[35S]-methionine incorporation
The detection of protein synthesis in cultured leiomyoma smooth muscle cells was assessed in the same way as the DNA synthesis except for the use of [35S]-methionine incorporation. Briefly, the cells were cultured on 24-well plates in DMEM for 48 h, and then 48 h in starvation medium. After starvation the cells were supplied with [35S]-methionine and thereafter the following experimental model was set up: (i) control; (ii) forskolin (10–6 mol/l); (iii) papaverin (10–6mol/l). After 24 h incubation, the smooth muscle cells were rinsed once with PBS and subsequently treated with 5% v/v ice-cold TCA for 30 min to terminate the incubation. Thereafter, the cultured smooth muscle cells were dissolved in 0.5 ml KOH (0.5 mol/l) for 1 h at room temperature and thereafter analysed using the liquid scintillation technique as described.
Statistical analysis
The results from the radioligand experiments were compared using an ordinary Wilcoxon matched-pairs signed rank test which also was used to compare the AC and PDE activities; P < 0.05 was considered to be statistically significant. A one-way analysis of variance with a Bonferroni test was used for statistical analysis of the proliferation studies; P < 0.05 was considered to be statistically significant.
Results
Adrenergic receptor expression
The binding of the antagonists [3H]-rauwolscine and [3H]-DHA to human uterine myometrial and leiomyoma membrane was a saturable process with high affinity and specificity. The non-specific binding of 3H-DHA and 3H-rauwolscine was found to be ~5 and 20% respectively. In each case, the data were best fitted to a one-site binding model. However, there was a great diversity in the expression level (Bmax) of both receptor types between different patients. In general, the ß2-AR expression was significantly down-regulated (P < 0.05) in the leiomyoma samples (Figure 1, 2a). In some patients, we could also detect an upregulation of the 2-AR expression (Figure 2b). However, when comparing all patients no significant increase in the 2-AR population was detected. The mean values of Kd and Bmax for [3H]-DHA (measuring ß2-AR expression) were 0.22 nmol/l and 38 fmol/mg for leiomyoma whereas corresponding myometrial values were 0.26 nmol/l and 63 fmol/mg respectively. The mean values from the [3H]-rauwolscine binding studies (measuring 2-AR expression) were 5.2 nmol/l and 335 fmol/mg for the leiomyoma samples whereas the myometrial values were 4.3 nmol/l and 283 fmol/mg respectively.
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Myometrial and leiomyoma innervation
In sections incubated with antibodies against PGP 9.5, normal myometrium and leiomyoma tissue differed markedly with respect to the occurrence of nerve fibres. Grossly, normal myometrium contained numerous fluorescent nerve fibres which occurred in relation to blood vessels or as interstitial bundles (Figure 3a
). In leiomyoma tissue, few, if any, PGP 9.5 immunoreactive fibres were seen (Figure 3b). Sections incubated with antibodies against TH presented fewer fluorescent nerve fibres compared to PGP 9.5-incubated sections, but principally, a similar normal/leiomyoma difference was observed. The distribution of TH-immunoreactive fibres in normal myometrium was similar to that described above (Figure 3c). In leiomyoma tissue, TH-immunoreactive fibres were absent (Figure 3d). All control sections were immunonegative.
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Adenylyl cyclase activity
The AC-activity in membrane suspensions from myometrial and leiomyoma tissue was measured, at 37°C with or without forskolin stimulation (10–5 mol/l) combined with IBMX (2 mmol/l) as described in the Materials and methods section. As can be seen in Figure 4 and 5a
, the AC-activity is lower in leiomyoma compared to myometrium and it also differs between patients. Furthermore, according to the Wilcoxon matched-pairs signed-rank test, there was a significant decrease of total AC-activity in the leiomyoma membrane compared to the myometrial control preparations (P < 0.05). After 5 min of stimulation, the mean cAMP content in leiomyoma samples was 51 ± 4.2% of corresponding myometrial samples.
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Phosphodiesterase activity
To compare the enzyme regulated hydrolysis of cAMP between leiomyoma and myometrial tissue, a PDE assay was set up including six patients. Also regarding the PDE-activity, a great variation between the patients was found. However, according to the Wilcoxon matched-pairs signed-ranks test there was a significantly increased PDE-activity in the leiomyoma membranes compared with corresponding myometrial samples (P < 0.05). In all cases except one, the activity was 100% higher or more in the leiomyoma samples compared with the myometrial controls (Figure 5b
).
[3H]-Thymidine incorporation
To determine whether the intracellular cAMP level could influence the growth of human uterine leiomyoma, an ordinary proliferation study on cultured leiomyoma smooth muscle cells using [3H]-thymidine incorporation was set up. First, compared with untreated control cells, incubation with forskolin (10–7 mol/l) or isoprenaline (10–5 mol/l) caused reductions of 73 and 50% respectively (Figure 6a). To examine to what extent the PDE-activity could influence the proliferation, cultured cells were treated with papaverin, either separately or in combination with forskolin. The results showed a notable anti-proliferative effect of papaverin alone and when combining with forskolin (10–6 mol/l), there was an additive down-regulation of [3H]-thymidine incorporation (Figure 6b). On the contrary, the opposite effect was produced by 2-AR stimulation. The 2-AR agonist, medetomidine (10–7 mol/l), resulted in a significantly increased [3H]-thymidine incorporation that was totally blocked using the 2-AR agonist, yohimbine (10–6 mol/l) (Figure 6c). All results from the [3H]-thymidine incorporation studies were found to be significant, according to analysis of variance (ANOVA) including a Bonferroni post test.
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[35S]-Methionine incorporation
To further investigate the effects of cAMP regarding leiomyoma smooth muscle cell growth, a protein assay was up set using [35S]-methionine incorporation. As regarding DNA synthesis both forskolin (10–6 mol/l) and papaverin (10–6 mol/l) had an inhibitory effect. Compared with control cells, incubation with forskolin and papaverin induced 40 and 30% reduction respectively (Figure 7
). The results from the [35S]-methionine incorporation studies were found to be significant according to ANOVA including a Bonferroni post test.
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Discussion
Uterine leiomyomata are the most prevalent indication for hysterectomy (Weber et al., 1997), which constitutes the most common surgery on adult women (Carlson et al., 1993). In the USA, 1.7 million women underwent hysterectomy from 1988 to 1990 (Wilcox et al., 1993). So far, there is no successful pharmacological treatment of these tumours but in clinical preoperative treatment, gonadotrophin-releasing hormone (GnRH) agonists are used to induce a temporary tumour-regression and thereby diminish the operative blood loss (Lumsden et al., 1987; Adamson, 1992). Several recent studies have demonstrated that adrenoceptors and AC/cAMP are involved in regulation of prostate, vascular, tubular renal and airway smooth muscle hyperplasia and/or hypertrophy (Noveral and Grunstein, 1994; Tomlinson et al., 1995; Hieble and Ruffo, 1996; Matousovic et al., 1997; Nilsson et al., 1998). In the present study we found interesting differences between uterine leiomyoma and surrounding myometrial tissue with respect to 2- and ß2-AR expression, innervation and AC/PDE-activity. In addition, our results from cultured uterine leiomyoma smooth muscle cell support the theory that the above-mentioned alterations could be involved in the regulation of tumour growth.
Firstly, the 2-AR and ß2-AR populations in uterine leiomyoma and normal myometrium were compared. Radioligand studies with [3H]-rauwolscine and [3H]-DHA demonstrated a significant down-regulation of the leiomyoma ß2-AR expression, while the 2-AR expression was to a certain extent increased in the tumour tissue of some patients. However, no significant up-regulation of the 2-AR expression could be verified when a statistical analysis was done. One explanation could be that a very high 2-AR expression was also found in the surrounding myometrium (average value 283 fmol/mg protein). In fact, when relating the myometrial 2-AR expression to the gestational stage of guinea pigs or rats, very attractive results were found. The myometrium increases mostly in mass until mid-way through pregnancy. At this stage of pregnancy, the 2-AR expression had increased to ~10-times the basal level, and after that quickly reduces to basal values (Arkinstall and Jones, 1988; Legrand et al., 1993). This coupling between a high 2-AR level and uterine smooth muscle growth and/or a high steroid hormone plasma concentration has been observed in several different species (Kovacs and Falkay, 1993). 2-AR have been shown to stimulate proliferation in cultured smooth muscle cell originating from pregnant human myometrium (Nilsson et al., 1998). However, 2-AR could also be involved in the regulation mechanism of apoptosis. Recently, a group of dimeric phosphoserine binding proteins called 14–3–3 proteins have been found to block apoptosis by differential regulation of the MAPK pathways (Xing et al., 2000). Furthermore, the third intracellular loop of the 2-AR has been found to interact with the 
isoform of 14–3–3 proteins (Prezeau et al., 1999). Shifted - and ß-AR expression levels have also been described in benign prostate hyperplasia (BPH). For example, the total occurrence of 1-AR mRNA in biopsies from BPH samples was found to be significantly higher than in non-BPH samples (Nasu et al., 1996) and the ß-AR expression in BPH was ~50% of that in non-BPH tissue (Tsujii et al., 1992).
As it is well known that both mechanical and chemical sympathectomy can elicit significant shifts between 2- and ß2-AR expressions (Bahouth, 1992; Legrand et al., 1993), it was relevant to investigate the occurrence of nerve fibres in leiomyoma and myometrial biopsies. The results showed an almost completely denervated tumour tissue, in contrast to a well-innervated surrounding myometrium (Figure 3a–d). Consequently, the shifted 2- /ß2-AR balance was not a secondary result from an abnormally dense tumour innervation. In addition, these results indicate a pure post-synaptic 2-AR population.
Moreover, as 2- and ß2-AR are coupled to Gi- and Gs-proteins respectively and, therefore, generate divergent AC responses, a comparative investigation of the AC-activity was performed. Interestingly, the total AC-activity was markedly reduced in the tumour tissue (Figure 4 and 5a). Down-regulated AC-activity in hyperplastic smooth muscle tissue has never been shown before whereas similar findings have been demonstrated in rat prostate cancer cells (Steiner et al., 1994).
Not only does the AC catalysed generation of cAMP regulate the basal cAMP level, but also the PDE controlled hydrolysis. Therefore, a comparative study of the PDE-activity was performed in uterine leiomyoma and myometrium. The analysis of the hydrolysed labelled cAMP in leiomyoma samples demonstrated PDE-activity which was ~100% higher, compared with the myometrial samples.
To determine how, and to what extent, the above-mentioned alterations in receptor expression and enzyme activity could influence cell growth, in-vitro assays were set up to measure DNA- and protein synthesis in leiomyoma smooth muscle cell. The results of these studies correspond very well with previously obtained experimental data. First, the [3H]-thymidine incorporation was effectively inhibited not only by low concentrations of forskolin but also by application of the ß-AR selective agonist, isoprenaline. Likewise, when incubating the cells with the PDE inhibitor, papaverin, DNA synthesis was significantly reduced and by combining papaverin with forskolin an additive effect was obtained. To ensure a PDE-inhibiting effect of papaverin on cultured smooth muscle cells, a radioimmunoassay was set up. The results showed a significant increase in the intracellular cAMP level (not shown). To investigate whether 2-AR stimulation generates a positive mitogenic effect in vitro, cells were incubated with the 2-AR selective agonist, medetomidine. A modest but significant increase in the [3H]-thymidine incorporation was produced when the cells were incubated with medetomidine. This effect could be totally inhibited by yohimbine, a selective 2-AR antagonist. Analogous studies on cultured smooth muscle cell, from both human and other species (and including human myometrial cells) show that an increased intracellular cAMP level inhibits proliferation (Souness et al., 1992; Noveral and Grunstein, 1994; Pan et al., 1994; Tomlinson et al., 1995; Nilsson et al., 1998).
When the protein synthesis was measured by [35S]-methionine incorporation, the results obtained were similar to the DNA studies. Both direct AC stimulation with forskolin and PDE inhibition with papaverin generated a considerably reduced incorporation compared to untreated cells (Figure 7). So far, we do not know how cAMP inhibits proliferation, but there are several possibilities. One possible way is an inhibition of the Ras-Raf-MEK-MAPK (mitogen activated protein kinase) pathway through a cAMP-activated PKA, which phosphorylates the Ras biding site of Raf-1 (Schwartz and Baron, 1999). Another tempting theory could be that cAMP stimulates apoptosis, as shown in other types of cell (Muhl et al., 1996).
In summary, this study shows that the 2/ß2-AR ratio is increased in leiomyoma compared with normal myometrium, due to a significantly decreased ß2-AR density. The changes in the 2/ß2 -AR populations were not due to an increased innervation, as the tumour tissue was completely devoid of nerve fibres. Moreover the AC-activity of leiomyoma membrane was found to be ~50% lower, compared with control tissue whereas the PDE-activity was 100% higher in leiomyoma. Finally, we found that cAMP has a potent growth inhibiting effect on cultured leiomyoma smooth muscle cell. The present study suggests that cAMP increasing agents might be a new approach for leiomyoma pharmaco-therapy. However, further research is clearly needed, particularly with regard to the effect of cAMP on myometrial cells (Nilsson et al., 1998). A potential therapy would be to combine ß2-adrenoceptor agonists with a specific phosphodiesterase inhibitor.
Acknowledgments
This study was kindly supported by grants from the Swedish Natural Science Research Council, The County Council of Östergötland, Magnus Bergvall Foundation for Medical Research, Sandoz, the Swedish Medical Research Council (K1999-14x-013046-01A; 3761). The authors would also like to express their appreciation to the staff at the division of Obstetrics and Gynecology, and to the patients who kindly provided the biopsies.
Notes
4 To whom correspondence should be addressed at: Division of Pharmacology, Department of Medicine and Care, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden. E-mail: per.adolfsson{at}far.liu.se 
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Submitted on March 27, 2000; accepted on June 21, 2000.
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