0021-972X/91/7302-0335$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 73, No. 2 Printed in U.S.A.
Hormone-Responsive Adenylyl Cyclase in the Human Fallopian Tube* TOM TANBO, REIDAR BJ0RNERHEIM, THOMAS ABYHOLM, AND VIDAR HANSSON Departments of Obstetrics and Gynecology (T.T., T.A.) and Medicine B (R.B.), National Hospital, and the Institutes of Pathology (R.B.) and Medical Biochemistry (V.H.), University of Oslo, Oslo, Norway
ABSTRACT. Hormone-responsive adenylyl cyclase (AC) activity in biopsies from normal human Fallopian tubes was studied. Enzyme activity with a Km of 0.15 mmol/L and a maximum velocity of 13.8 pmol/mg-min in the basal condition was demonstrated. The addition of prostaglandin Ei (PCEO, PGE2, PGF2a, vasoactive intestinal polypeptide, isoproterenol, and terbutaline increased enzyme activity, with no change in the Km.
T
Maximum stimulation of AC activity was obtained with PGEi, resulting in a 2- to 8-fold increase in AC activity. The response of AC to PGEi revealed a possible topographical variation, with lowest responses to PGEi in the isthmus. No such segmental variation in AC activity and response was seen after stimulation with PGF2a, vasoactive intestinal polypeptide, or isoproterenol. (J Clin Endocrinol Metab 73: 335-340, 1991)
procedure was performed as a minilaparotomy, during which one tube was removed. At the same time a biopsy of the endometrium was taken for phase determination. The patients had given their written consent, and the study protocol had been approved by the ethical committee of the hospital. Immediately after removal, the tube was put in ice-cold Earle's medium (Gibco, Paisley, Scotland). Connective tissue and the serosal lining of the tube were quickly removed, and biopsies containing tissue from all layers of the tubal wall were taken from the isthmus, the ampullary-isthmic junction, and the ampulla. In addition, biopsies were taken from the fimbriae. The tissue samples were immediately put into liquid nitrogen for storage until further use.
HE FUNCTION of the Fallopian tube is exquisitely dependent on hormones. Estrogens, progesterone, catecholamines, prostaglandins (PGs), and vasoactive intestinal polypeptide (VIP) are known to affect myosalpingeal contractility, while estrogens and PGs of the E and F type modulate ciliary activity in the endosalpinx (1). Adrenergic and VIPergic nerves have been located in the wall of the Fallopian tube (2, 3). Furthermore, adrenergic receptors of the /32 subtype have previously been demonstrated in the rabbit oviduct (4). Agonist binding to /?-adrenergic receptors stimulates adenylyl cyclase (AC) activity. The cellular actions of VIP, glucagon, gonadotropins, and PGs of the E type are also mediated through stimulation of AC activity. The purpose of this study was to examine AC activity and response to agonists in biopsies from human Fallopian tubes and determine possible topographic differences in AC activity and response.
Chemicals, enzymes, and drugs Myokinase, Tris, ATP, cAMP, GTP, EDTA, sodium dodecyl sulfate, VIP, PGE1( PGE2) and PGF2« were obtained from Sigma Chemical Co. (St. Louis, MO). Creatine phosphate and creatine kinase were obtained from Calbiochem (La Jolla, CA). [32P]ATP and [3H]cAMP were purchased from Amersham (Aylesbury, Buckinghamshire, United Kingdom). Isoproterenol was obtained as the commercially available Isoprenalin (NAF, Oslo, Norway), and terbutaline (Bricanyl) was purchased from Draco (Lund, Sweden). hCG (Physex) was obtained from Leo (Balleriip, Denmark), and FSH (Metrodin) from Serono (Aubonne Switzerland). Glucagon was purchased from Novo (Copenhagen, Denmark).
Materials and Methods Fallopian tube biopsies Fallopian tube biopsies were obtained during sterilization procedures in women with macroscopically normal tubes. The Received December 27,1989. Address requests for reprints to: Dr. Tom Tanbo, Department of Obstetrics and Gynecology, National Hospital, Oslo, Norway. * This work was supported by grants from the Norwegian Cancer Society, Torsted's Foundation, the Nordic Insulin Foundation, Dr. Alexander Malthe's Foundation, and the Norwegian Research Council for Science and the Humanities.
Preparations of membranes Unless otherwise stated, the biopsies were weighed and homogenized in 20 vol TE buffer (10 mmol/L Tris-HCl and 1 mmol/L EDTA, pH 7.4) using an Ultra-Turrax homogenizer 335
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(Janke-Kunkel AG, Staufen, Germany) at rheostat setting 7 twice for 5 s each time. The homogenates were filtered through a nylon mesh of 70 fim, and membrane particles were prepared from the homogenates by centrifugation at 27,000 x g for 30 min at 4 C. In a previous study using the same AC assay on human myocardial preparations, no difference was found in cAMP formation after one or two centrifugations (5). The supernatant was discharged, and the pellet was resuspended to the original volume of TE buffer containing 0.1% BSA. AC assay In general, AC activity was determined in 20 fih aliquots of membrane particles in a final volume of 50 fiL containing 0.04 mmol/L GTP, 1 mmol/L EDTA, 1 mmol/L cAMP (with ~104 cpm [3H]cAMP), 20 mmol/L creatine phosphate, 0.2 mg/mL creatine kinase, 0.02 mg/mL myokinase, and 25 mmol/L TrisHC1 buffer, pH 7.4. For the MgATP and Mg2+ concentrationresponse studies, various concentrations of ATP (0.077-1.0 mmol/L) with constant specific activity (173 cpm/pmol) and various concentrations of Mg2+ (0.4-18.4 mmol/L) were used. In experiments in which ATP and Mg2+ concentrations were constant, concentrations producing optimal relative responses were used: ATP, 0.2 mmol/L (with 2 x 106 cpm [32P]ATP); and MgCl2, 3.5 mmol/L. The concentrations of hormonal agonists are indicated in the figure legends. Incubations were carried out at 32 C for 12 min. The reactions were stopped with 0.1 mL of a stop solution containing 40 mmol/L cAMP, 10 mmol/L ATP, and 1% sodium dodecyl sulfate, followed by mixing and immediate cooling to 0 C. The [32P]cAMP formed and [3H]cAMP added to monitor recovery were isolated according to the method of Salomon et al. (6) using Dowex chromatography and alumina chromatography with minor modifications, as described by Birnbaumer et al. (7). Overall recovery was 63-82%. The imidazole eluates from the alumina columns were collected in scintillation vials containing 5 mL Instagel II (Packard, Downers Grove, IL). Radioactivity was measured in a Delta 300 liquid scintillation counter, with an efficiency of 40%. Protein was determined by the method of Lowry et al. (8) using BSA as the standard, and the ATP concentrations were calculated by optical densitometry measurements at 260 nm.
Results Effects of hormonal agonists AC activity and response to varying concentrations of isoproterenol, terbutaline, VIP, PGE b PGF2«, glucagon, hCG, and FSH were examined in preparations of membrane particles from a human Fallopian tube removed in the midluteal phase. As shown in Fig. 1, isoproterenol, terbutaline, PGEi, PGF2«, and VIP caused a concentration-dependent increase in AC activity. Basal activity was 1.64 pmol/mg protein-min. Maximal stimulation was obtained with 1 x 10"4 mmol/L PGEi, resulting in an 8-fold increase in AC activity (13.32 pmol cAMP/mg
o"to concentration (M) FIG. 1. AC activity and responses to different concentrations of various hormones are shown. ISO, Isoproterenol; TERB, terbutaline. Enzyme activity is given as: relative response = stimulated/basal. Each point is the mean of triplicates ± SD.
0 TO1 TO* concentration (M) FIG. 2. AC activity in response to different concentrations of PGEi and PGE2. The biopsy was taken during a sterilization procedure in the early postpartum period. Enzyme activity is shown as the relative response.
protein-min). VIP, PGF2a, isoproterenol, and terbutaline stimulated AC to a lesser degree. In the study depicted in Fig. 2, a biopsy from the isthmus taken on the second day postpartum after a spontaneous delivery was used. In this case basal AC activity was much higher (9.0 pmol cAMP/mg protein • min, but responses to PGEi and PGE2 (1 x 10"4 mmol/ L) were lower (2-fold). Glucagon, hCG, and FSH did not increase AC activity in human Fallopian tube (results not shown).
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Effects of
Figure 3 shows the effects of increasing concentrations of Mg2+ on AC activity and responses to PGE1} VIP, and isoproterenol. The figure also demonstrates that the relative responses to the different agonists decreased slightly with increasing Mg2+ concentration. The maximal relative response was obtained at approximately 0.5 mmol/L free Mg2+. At this concentration of Mg2+, however, both basal and stimulated AC activities were low; consequently, 3.5 mmol/L Mg2+ were used in the experiments. Effects of MgATP Figure 4 depicts AC activity and responses to PGEi, VIP, and isoproterenol at different ATP concentrations.
i
0 .2 .4 concentration of ATP ImM)
8
K)
12
14
18
10 12 16 18 2 4 6 8 14 concentration of Mg+-MmM) FIG. 3. Effects of different Mg*+ concentrations on AC activity in the Fallopian tube. Fixed concentrations of isoproterenol (ISO), VIP, and PGEi (GEi) were used for stimulation of enzyme activity. The upper panel depicts cAMP formed in picomoles per mg protein/min. The lower panel shows the relative response. Each point is the mean and SD of triplicates.
6
FIG. 4. AC activity at different substrate (ATP) concentrations. Fixed concentrations of isoproterenol (ISO), VIP, and PGEi were used for stimulation of enzyme activity. The upper panel gives AC activity in picomoles of cAMP formed per mg protein/min, while the lower panel depicts the relative response. Each point represents the mean ± SD of triplicates.
The Mg2+ concentration at each ATP concentration was 1.5 mmol/L in excess of EDTA and ATP concentrations. Constant specific radioactivity was used (173 cpm/pmol), so that radioactive and nonradioactive ATP varied to the same extent. Increasing concentrations of ATP caused a dose-dependent increase in cAMP production, which approached saturation at concentrations above 0.5 mmol/L (Fig. 4). Double reciprocal plotting of the data gave linear Lineweaver-Burke plots, indicating an apparent Km of 0.15
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TANBO ET AL.
338 C25r
BASAL
JCE & M • 1991 Vol73-No2
ular, periovulatory, and luteal phases, respectively (Table 1). In all three patients the AC response to PGEi was lowest in the isthmus, with a tendency to increased responses in the lateral direction. This was not the case for AC responses to PGF2«, VIP, and isoproterenol. The most pronounced difference in the AC response to PGEi in different tubal segments was seen in the tube removed during the luteal phase (Fig. 6).
Discussion
VS(mivH)
FlG. 5. Double reciprocal plots of the ATP dose-response study depicted in Fig. 4. Kra values for basal and stimulated activities were approximately 0.15 mmol/L. Isoproterenol (ISO), VIP, and PGEi increased AC activity by increasing the Vmax, but there was no change in the apparent Km.
mmol/L and a maximum velocity (Vmax) of 13.76 pmol/ mg-min (Fig. 5). PGEi, VIP, and isoproterenol stimulated AC activity by increasing the Vmax, and no change in apparent Km was observed. Topographic distribution of AC activity and response AC activity and responses to PGEi, PGF2«, VIP, and isoproterenol were examined in membrane particle suspensions from four different regions of the tube: the fimbriae, the ampullae, the ampullary-isthmic junction, and the isthmus. Possible segmental changes in AC activity and response were examined in three different Fallopian tubes removed from patients during the follic-
Our study demonstrates agonist-responsive AC activity in human Fallopian tubes and indicates possible variation in PGEi-responsive AC activity in different tubal segments. The AC activity and response to hormonal agonists share characteristics of other somatic AC systems with regard to dependence on Mg2"1" and ATP (5, 7). The AC response to PGEi revealed considerable variation from patient to patient (2- to 8-fold). This is probably related to the endocrinological situation of the patients from whom the tissues were removed and the part of the tube that was used for the membrane particle preparations. The high basal activity and relatively low response to PGEi in preparations removed shortly after delivery may be a result of high levels of PGEs in this tissue during delivery (9). This would tend to increase basal activity and reduce hormonal response. The concentrations of PGEi and VIP required to activate AC activity in human Fallopian tube were somewhat higher than those reported in other systems (10-12). The reason for this is unknown, but it may be related to rapid degradation of the agonist, their receptors, or both. A similar high ED50 for AC activation was found for a PGEi-stimulated rat Leydig cell tumor (12).
TABLE 1. AC activity and response to stimulation with PGEi, PGF2a, VIP, and isoproterenol (Iso) in three human Fallopian tubes from different stages of the menstrual cycle AC response to hormone agonists Tube
Region
Basal activity PGEj
PGF2a
VIP
Iso
I AIJ A F
2.76 4.40 5.06 3.37
2.29 (6.31) 3.22 (14.15) 2.37 (11.98) 3.83 (12.90)
1.41 1.19 1.07 1.50
(3.90) (5.24) (5.41) (5.05)
2.00 (5.53) 1.79 (7.86) 2.21 (11.18) 2.19 (7.39)
1.29 1.03 0.84 1.47
I AIJ A F
4.30 2.17 3.36 4.52
2.12 (9.11) 3.19 (6.92) 3.27 (10.99) 3.07 (13.89)
1.03 0.96 1.16 1.32
(4.42) (2.08) (3.81) (5.95)
1.61 (6.91) 2.10 (4.55) 2.08 (6.99) 1.29 (5.84)
0.95 (4.08) 1.41 (3.06) 1.54 (5.18) 0.98 (4.41)
I AIJ A F
3.45 4.83 4.69 6.24
1.97 2.66 3.24 3.72
1.30 0.63 1.29 1.39
(4.47) (3.06) (6.05) (8.67)
0.96 1.29 1.29 0.94
1.66 (5.74) 1.02 (4.95) 1.19 (5.56) 0.83 (5.20)
(6.88) (12.83) (15.19) (23.24)
(3.32) (6.24) (6.05) (5.90)
(3.55) (4.51) (4.26) (4.97)
Tube 1 was taken in the follicular phase, tube 2 in the periovulatory phase, and tube 3 in the luteal phase. I, Isthmus; AIJ, ampullary-isthmic junction; A, ampulla; F, fimbriae. Basal activity is given in picomoles of cAMP formed per mg protein/min, while the AC response is given as the relative response, with cAMP formed in parentheses.
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HORMONE-RESPONSIVE AC IN FALLOPIAN TUBE
1
ill Isthmus
AIJ
Ampulla
Fimbriae
in • Ampulla
Fimbriae
Q)
8 Isthmus
•I
AIJ
FIG. 6. AC activity from various segments of a Fallopian tube removed in the midluteal phase. AIJ, Ampullary-isthmic junction. Stimulation with fixed concentrations of PGEi, VIP, PGF2, and isoproterenol was used. The upper panel shows cAMP formed in picomoles per mg protein -min. The points indicated are the mean ± SD of triplicates. The lower panel shows the relative response. The horizontal line in the lower panel depicts basal activity. D, PGEi; M, PGF2n; B, VIP; ^, isoproterenol; D, basal.
An interesting finding from this study was that in three different patients, the AC response to PGEi was lower in the isthmus than in the other segments of the tube. This variation in PGEi-responsive AC was agonist specific and was not observed for VIP, isoproterenol, or PGF2cv. PGEs have previously been shown to be localized primarily in the epithelium of the human Fallopian tube (13). It is, therefore, possible that the lower PGEi-responsive AC activity in the istmus simply reflects the relative content of epithelial and fibromuscular tissue in the segments of the human Fallopian tube. The low AC response to isoproterenol stimulation was unexpected, since the human Fallopian tube is shown to be heavily innervated with adrenergic nerves, especially in the isthmus (2). Using the [125I]cyanopindolol binding
339
assay technique, 0-adrenergic receptors have been demonstrated in the rabbit oviduct (4). In the myosalpinx of human Fallopian tubes, /3-adrenergic receptors have been characterized by measuring dissociation constants for adrenoceptor-antagonist complexes (14). Unpublished data on [125I]cyanopindolol binding from our laboratory have, however, demonstrated very low concentrations of /32-adrenergic receptors in biopsies from human Fallopian tubes. This observation may indicate that adrenergic activity in human Fallopian tubes is of predominantly a-adrenergic origin. VIPergic nerves and VIP activity have been demonstrated in the human Fallopian tube (3). A preferential localization to the myosalpinx in the isthmus region of the tube has been shown for adrenergic activity (15). VIP and norepinephrine have been shown to modulate muscular tension in the isthmus, thus participating in the pre- and early postovulatory tube locking mechanism (1). Since the tubal wall in the isthmus region is dominated by muscle tissue and sparse epithelium in contrast to the ampullae and fimbriae, in which a reverse relation exists, a difference in VIP- and isoproterenol-stimulated AC activity in various regions of the tube would be expected. This, however, was not observed. Whether the receptors for the various AC agonists are present in the same cell cannot be determined from the present study. This study has demonstrated AC activity in the human Fallopian tube with similar kinetic characteristics as that in other somatic cells (6, 8). The biopsies studied have contained tissue from both the muscular and epithelial layers of the tubal wall. We are currently investigating a possible difference in AC activity in the endosalpinx and myosalpinx at different phases of the menstrual cycle. References 1. Jansen RPS. Endocrine response in the Fallopian tube. Endocr Rev. 1984;5:525-51. 2. Owman C, Rosengren E, Sj^berg NO. Adrenergic innervation of the human female reproductive organs: a histochemical and chemical investigation. Obstet Gynecol. 1967;30:763-73. 3. Aim P, Alumets J, Hakansson R. Vasoactive intestinal polypeptide nerves in the female genital tract. Am J Obstet Gynecol. 1980;136:349-51. 4. Kleinstein J. Binding properties of beta adrenergic receptors in oviductal smooth muscle membranes. Gynecol Obstet Invest. 1984;17:242-6. 5. Golf S, Jahnsen T, Attramadal H, Fr^ysaker T, Hansson V. Catecholamine responsive adenylate cyclase in human myocardial preparations. Properties and optimalization of assay conditions. Scand J Lab Clin Invest. 1984;44:317-27. 6. Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974;58:541-8. 7. Birnbaumer L, Yang PC, Hunzicker-Dunn M, Bockaert J, Duran JM. Adenylyl cyclase activities in ovarian tissues. I. Homogenization and conditions of assay in Graafian follicles and corpora lutea of rabbits, rats and pigs: regulation by ATP, and some comparative properties. Endocrinology. 1976:99:163-84. 8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:26575.
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9. Husslein P, Sinzinger H. Concentration of 13,14-dihydro-15-ketoPGE2 in the maternal peripheral plasma during labor of spontaneous onset. Br J Obstet Gynaecol. 1984;91:228-30. 10. Wiik P. Homologous regulation of adenylate cyclase-coupled receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes. Regul Peptides. 1988;20:323-33. 11. Bj^ro T, ^stberg BC, Sand 0, et al. Vasoactive intestinal peptide and peptide with N-terminal histidine and C-terminal isoleucine increase prolactin secretion in cultured rat pituitary cells (GH4Ci) via a cAMP-dependent mechanism which involves transient elevation of intracellular Ca2+. Mol Cell Endocrinol. 1987:49:119-28. 12. Erichsen AA, Jahnsen T, Attramadal H, Hansson V. A transplant-
JCE & M • 1991 Vol 73 • No 2
able rat Leydig cell tumor. II. Adenylyl cyclase activation by prostaglandin Ei, isoproterenol and glucagon. J Steroid Biochem. 1984:21:545-8. 13. Nieder J, Augustin W. Prostaglandin E and F profiles in human Fallopian tubes during different phases of the menstrual cycle. Gynecol Obstet Invest. 1986:21:202-7. 14. Helm G, Owman CH, Sj^berg N-O, Walles B. Quantitative pharmacological characterization of /8-receptors and two types of areceptors mediating sypathomimetic smooth muscle response in the human Fallopian tube at various cyclic stages. Acta Physiol Scand. 1982:114:425-32. 15. Ottesen B. Vasoactive intestinal polypeptide as a neurotransmitter in the female genital tract. Am J Obstet Gynecol. 1983:147:208-24.
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Vol. 73, No. 2 Printed in U.S.A.
Hormone-Responsive Adenylyl Cyclase in the Human Fallopian Tube* TOM TANBO, REIDAR BJ0RNERHEIM, THOMAS ABYHOLM, AND VIDAR HANSSON Departments of Obstetrics and Gynecology (T.T., T.A.) and Medicine B (R.B.), National Hospital, and the Institutes of Pathology (R.B.) and Medical Biochemistry (V.H.), University of Oslo, Oslo, Norway
ABSTRACT. Hormone-responsive adenylyl cyclase (AC) activity in biopsies from normal human Fallopian tubes was studied. Enzyme activity with a Km of 0.15 mmol/L and a maximum velocity of 13.8 pmol/mg-min in the basal condition was demonstrated. The addition of prostaglandin Ei (PCEO, PGE2, PGF2a, vasoactive intestinal polypeptide, isoproterenol, and terbutaline increased enzyme activity, with no change in the Km.
T
Maximum stimulation of AC activity was obtained with PGEi, resulting in a 2- to 8-fold increase in AC activity. The response of AC to PGEi revealed a possible topographical variation, with lowest responses to PGEi in the isthmus. No such segmental variation in AC activity and response was seen after stimulation with PGF2a, vasoactive intestinal polypeptide, or isoproterenol. (J Clin Endocrinol Metab 73: 335-340, 1991)
procedure was performed as a minilaparotomy, during which one tube was removed. At the same time a biopsy of the endometrium was taken for phase determination. The patients had given their written consent, and the study protocol had been approved by the ethical committee of the hospital. Immediately after removal, the tube was put in ice-cold Earle's medium (Gibco, Paisley, Scotland). Connective tissue and the serosal lining of the tube were quickly removed, and biopsies containing tissue from all layers of the tubal wall were taken from the isthmus, the ampullary-isthmic junction, and the ampulla. In addition, biopsies were taken from the fimbriae. The tissue samples were immediately put into liquid nitrogen for storage until further use.
HE FUNCTION of the Fallopian tube is exquisitely dependent on hormones. Estrogens, progesterone, catecholamines, prostaglandins (PGs), and vasoactive intestinal polypeptide (VIP) are known to affect myosalpingeal contractility, while estrogens and PGs of the E and F type modulate ciliary activity in the endosalpinx (1). Adrenergic and VIPergic nerves have been located in the wall of the Fallopian tube (2, 3). Furthermore, adrenergic receptors of the /32 subtype have previously been demonstrated in the rabbit oviduct (4). Agonist binding to /?-adrenergic receptors stimulates adenylyl cyclase (AC) activity. The cellular actions of VIP, glucagon, gonadotropins, and PGs of the E type are also mediated through stimulation of AC activity. The purpose of this study was to examine AC activity and response to agonists in biopsies from human Fallopian tubes and determine possible topographic differences in AC activity and response.
Chemicals, enzymes, and drugs Myokinase, Tris, ATP, cAMP, GTP, EDTA, sodium dodecyl sulfate, VIP, PGE1( PGE2) and PGF2« were obtained from Sigma Chemical Co. (St. Louis, MO). Creatine phosphate and creatine kinase were obtained from Calbiochem (La Jolla, CA). [32P]ATP and [3H]cAMP were purchased from Amersham (Aylesbury, Buckinghamshire, United Kingdom). Isoproterenol was obtained as the commercially available Isoprenalin (NAF, Oslo, Norway), and terbutaline (Bricanyl) was purchased from Draco (Lund, Sweden). hCG (Physex) was obtained from Leo (Balleriip, Denmark), and FSH (Metrodin) from Serono (Aubonne Switzerland). Glucagon was purchased from Novo (Copenhagen, Denmark).
Materials and Methods Fallopian tube biopsies Fallopian tube biopsies were obtained during sterilization procedures in women with macroscopically normal tubes. The Received December 27,1989. Address requests for reprints to: Dr. Tom Tanbo, Department of Obstetrics and Gynecology, National Hospital, Oslo, Norway. * This work was supported by grants from the Norwegian Cancer Society, Torsted's Foundation, the Nordic Insulin Foundation, Dr. Alexander Malthe's Foundation, and the Norwegian Research Council for Science and the Humanities.
Preparations of membranes Unless otherwise stated, the biopsies were weighed and homogenized in 20 vol TE buffer (10 mmol/L Tris-HCl and 1 mmol/L EDTA, pH 7.4) using an Ultra-Turrax homogenizer 335
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(Janke-Kunkel AG, Staufen, Germany) at rheostat setting 7 twice for 5 s each time. The homogenates were filtered through a nylon mesh of 70 fim, and membrane particles were prepared from the homogenates by centrifugation at 27,000 x g for 30 min at 4 C. In a previous study using the same AC assay on human myocardial preparations, no difference was found in cAMP formation after one or two centrifugations (5). The supernatant was discharged, and the pellet was resuspended to the original volume of TE buffer containing 0.1% BSA. AC assay In general, AC activity was determined in 20 fih aliquots of membrane particles in a final volume of 50 fiL containing 0.04 mmol/L GTP, 1 mmol/L EDTA, 1 mmol/L cAMP (with ~104 cpm [3H]cAMP), 20 mmol/L creatine phosphate, 0.2 mg/mL creatine kinase, 0.02 mg/mL myokinase, and 25 mmol/L TrisHC1 buffer, pH 7.4. For the MgATP and Mg2+ concentrationresponse studies, various concentrations of ATP (0.077-1.0 mmol/L) with constant specific activity (173 cpm/pmol) and various concentrations of Mg2+ (0.4-18.4 mmol/L) were used. In experiments in which ATP and Mg2+ concentrations were constant, concentrations producing optimal relative responses were used: ATP, 0.2 mmol/L (with 2 x 106 cpm [32P]ATP); and MgCl2, 3.5 mmol/L. The concentrations of hormonal agonists are indicated in the figure legends. Incubations were carried out at 32 C for 12 min. The reactions were stopped with 0.1 mL of a stop solution containing 40 mmol/L cAMP, 10 mmol/L ATP, and 1% sodium dodecyl sulfate, followed by mixing and immediate cooling to 0 C. The [32P]cAMP formed and [3H]cAMP added to monitor recovery were isolated according to the method of Salomon et al. (6) using Dowex chromatography and alumina chromatography with minor modifications, as described by Birnbaumer et al. (7). Overall recovery was 63-82%. The imidazole eluates from the alumina columns were collected in scintillation vials containing 5 mL Instagel II (Packard, Downers Grove, IL). Radioactivity was measured in a Delta 300 liquid scintillation counter, with an efficiency of 40%. Protein was determined by the method of Lowry et al. (8) using BSA as the standard, and the ATP concentrations were calculated by optical densitometry measurements at 260 nm.
Results Effects of hormonal agonists AC activity and response to varying concentrations of isoproterenol, terbutaline, VIP, PGE b PGF2«, glucagon, hCG, and FSH were examined in preparations of membrane particles from a human Fallopian tube removed in the midluteal phase. As shown in Fig. 1, isoproterenol, terbutaline, PGEi, PGF2«, and VIP caused a concentration-dependent increase in AC activity. Basal activity was 1.64 pmol/mg protein-min. Maximal stimulation was obtained with 1 x 10"4 mmol/L PGEi, resulting in an 8-fold increase in AC activity (13.32 pmol cAMP/mg
o"to concentration (M) FIG. 1. AC activity and responses to different concentrations of various hormones are shown. ISO, Isoproterenol; TERB, terbutaline. Enzyme activity is given as: relative response = stimulated/basal. Each point is the mean of triplicates ± SD.
0 TO1 TO* concentration (M) FIG. 2. AC activity in response to different concentrations of PGEi and PGE2. The biopsy was taken during a sterilization procedure in the early postpartum period. Enzyme activity is shown as the relative response.
protein-min). VIP, PGF2a, isoproterenol, and terbutaline stimulated AC to a lesser degree. In the study depicted in Fig. 2, a biopsy from the isthmus taken on the second day postpartum after a spontaneous delivery was used. In this case basal AC activity was much higher (9.0 pmol cAMP/mg protein • min, but responses to PGEi and PGE2 (1 x 10"4 mmol/ L) were lower (2-fold). Glucagon, hCG, and FSH did not increase AC activity in human Fallopian tube (results not shown).
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HORMONE-RESPONSIVE AC IN FALLOPIAN TUBE
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Effects of
Figure 3 shows the effects of increasing concentrations of Mg2+ on AC activity and responses to PGE1} VIP, and isoproterenol. The figure also demonstrates that the relative responses to the different agonists decreased slightly with increasing Mg2+ concentration. The maximal relative response was obtained at approximately 0.5 mmol/L free Mg2+. At this concentration of Mg2+, however, both basal and stimulated AC activities were low; consequently, 3.5 mmol/L Mg2+ were used in the experiments. Effects of MgATP Figure 4 depicts AC activity and responses to PGEi, VIP, and isoproterenol at different ATP concentrations.
i
0 .2 .4 concentration of ATP ImM)
8
K)
12
14
18
10 12 16 18 2 4 6 8 14 concentration of Mg+-MmM) FIG. 3. Effects of different Mg*+ concentrations on AC activity in the Fallopian tube. Fixed concentrations of isoproterenol (ISO), VIP, and PGEi (GEi) were used for stimulation of enzyme activity. The upper panel depicts cAMP formed in picomoles per mg protein/min. The lower panel shows the relative response. Each point is the mean and SD of triplicates.
6
FIG. 4. AC activity at different substrate (ATP) concentrations. Fixed concentrations of isoproterenol (ISO), VIP, and PGEi were used for stimulation of enzyme activity. The upper panel gives AC activity in picomoles of cAMP formed per mg protein/min, while the lower panel depicts the relative response. Each point represents the mean ± SD of triplicates.
The Mg2+ concentration at each ATP concentration was 1.5 mmol/L in excess of EDTA and ATP concentrations. Constant specific radioactivity was used (173 cpm/pmol), so that radioactive and nonradioactive ATP varied to the same extent. Increasing concentrations of ATP caused a dose-dependent increase in cAMP production, which approached saturation at concentrations above 0.5 mmol/L (Fig. 4). Double reciprocal plotting of the data gave linear Lineweaver-Burke plots, indicating an apparent Km of 0.15
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TANBO ET AL.
338 C25r
BASAL
JCE & M • 1991 Vol73-No2
ular, periovulatory, and luteal phases, respectively (Table 1). In all three patients the AC response to PGEi was lowest in the isthmus, with a tendency to increased responses in the lateral direction. This was not the case for AC responses to PGF2«, VIP, and isoproterenol. The most pronounced difference in the AC response to PGEi in different tubal segments was seen in the tube removed during the luteal phase (Fig. 6).
Discussion
VS(mivH)
FlG. 5. Double reciprocal plots of the ATP dose-response study depicted in Fig. 4. Kra values for basal and stimulated activities were approximately 0.15 mmol/L. Isoproterenol (ISO), VIP, and PGEi increased AC activity by increasing the Vmax, but there was no change in the apparent Km.
mmol/L and a maximum velocity (Vmax) of 13.76 pmol/ mg-min (Fig. 5). PGEi, VIP, and isoproterenol stimulated AC activity by increasing the Vmax, and no change in apparent Km was observed. Topographic distribution of AC activity and response AC activity and responses to PGEi, PGF2«, VIP, and isoproterenol were examined in membrane particle suspensions from four different regions of the tube: the fimbriae, the ampullae, the ampullary-isthmic junction, and the isthmus. Possible segmental changes in AC activity and response were examined in three different Fallopian tubes removed from patients during the follic-
Our study demonstrates agonist-responsive AC activity in human Fallopian tubes and indicates possible variation in PGEi-responsive AC activity in different tubal segments. The AC activity and response to hormonal agonists share characteristics of other somatic AC systems with regard to dependence on Mg2"1" and ATP (5, 7). The AC response to PGEi revealed considerable variation from patient to patient (2- to 8-fold). This is probably related to the endocrinological situation of the patients from whom the tissues were removed and the part of the tube that was used for the membrane particle preparations. The high basal activity and relatively low response to PGEi in preparations removed shortly after delivery may be a result of high levels of PGEs in this tissue during delivery (9). This would tend to increase basal activity and reduce hormonal response. The concentrations of PGEi and VIP required to activate AC activity in human Fallopian tube were somewhat higher than those reported in other systems (10-12). The reason for this is unknown, but it may be related to rapid degradation of the agonist, their receptors, or both. A similar high ED50 for AC activation was found for a PGEi-stimulated rat Leydig cell tumor (12).
TABLE 1. AC activity and response to stimulation with PGEi, PGF2a, VIP, and isoproterenol (Iso) in three human Fallopian tubes from different stages of the menstrual cycle AC response to hormone agonists Tube
Region
Basal activity PGEj
PGF2a
VIP
Iso
I AIJ A F
2.76 4.40 5.06 3.37
2.29 (6.31) 3.22 (14.15) 2.37 (11.98) 3.83 (12.90)
1.41 1.19 1.07 1.50
(3.90) (5.24) (5.41) (5.05)
2.00 (5.53) 1.79 (7.86) 2.21 (11.18) 2.19 (7.39)
1.29 1.03 0.84 1.47
I AIJ A F
4.30 2.17 3.36 4.52
2.12 (9.11) 3.19 (6.92) 3.27 (10.99) 3.07 (13.89)
1.03 0.96 1.16 1.32
(4.42) (2.08) (3.81) (5.95)
1.61 (6.91) 2.10 (4.55) 2.08 (6.99) 1.29 (5.84)
0.95 (4.08) 1.41 (3.06) 1.54 (5.18) 0.98 (4.41)
I AIJ A F
3.45 4.83 4.69 6.24
1.97 2.66 3.24 3.72
1.30 0.63 1.29 1.39
(4.47) (3.06) (6.05) (8.67)
0.96 1.29 1.29 0.94
1.66 (5.74) 1.02 (4.95) 1.19 (5.56) 0.83 (5.20)
(6.88) (12.83) (15.19) (23.24)
(3.32) (6.24) (6.05) (5.90)
(3.55) (4.51) (4.26) (4.97)
Tube 1 was taken in the follicular phase, tube 2 in the periovulatory phase, and tube 3 in the luteal phase. I, Isthmus; AIJ, ampullary-isthmic junction; A, ampulla; F, fimbriae. Basal activity is given in picomoles of cAMP formed per mg protein/min, while the AC response is given as the relative response, with cAMP formed in parentheses.
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HORMONE-RESPONSIVE AC IN FALLOPIAN TUBE
1
ill Isthmus
AIJ
Ampulla
Fimbriae
in • Ampulla
Fimbriae
Q)
8 Isthmus
•I
AIJ
FIG. 6. AC activity from various segments of a Fallopian tube removed in the midluteal phase. AIJ, Ampullary-isthmic junction. Stimulation with fixed concentrations of PGEi, VIP, PGF2, and isoproterenol was used. The upper panel shows cAMP formed in picomoles per mg protein -min. The points indicated are the mean ± SD of triplicates. The lower panel shows the relative response. The horizontal line in the lower panel depicts basal activity. D, PGEi; M, PGF2n; B, VIP; ^, isoproterenol; D, basal.
An interesting finding from this study was that in three different patients, the AC response to PGEi was lower in the isthmus than in the other segments of the tube. This variation in PGEi-responsive AC was agonist specific and was not observed for VIP, isoproterenol, or PGF2cv. PGEs have previously been shown to be localized primarily in the epithelium of the human Fallopian tube (13). It is, therefore, possible that the lower PGEi-responsive AC activity in the istmus simply reflects the relative content of epithelial and fibromuscular tissue in the segments of the human Fallopian tube. The low AC response to isoproterenol stimulation was unexpected, since the human Fallopian tube is shown to be heavily innervated with adrenergic nerves, especially in the isthmus (2). Using the [125I]cyanopindolol binding
339
assay technique, 0-adrenergic receptors have been demonstrated in the rabbit oviduct (4). In the myosalpinx of human Fallopian tubes, /3-adrenergic receptors have been characterized by measuring dissociation constants for adrenoceptor-antagonist complexes (14). Unpublished data on [125I]cyanopindolol binding from our laboratory have, however, demonstrated very low concentrations of /32-adrenergic receptors in biopsies from human Fallopian tubes. This observation may indicate that adrenergic activity in human Fallopian tubes is of predominantly a-adrenergic origin. VIPergic nerves and VIP activity have been demonstrated in the human Fallopian tube (3). A preferential localization to the myosalpinx in the isthmus region of the tube has been shown for adrenergic activity (15). VIP and norepinephrine have been shown to modulate muscular tension in the isthmus, thus participating in the pre- and early postovulatory tube locking mechanism (1). Since the tubal wall in the isthmus region is dominated by muscle tissue and sparse epithelium in contrast to the ampullae and fimbriae, in which a reverse relation exists, a difference in VIP- and isoproterenol-stimulated AC activity in various regions of the tube would be expected. This, however, was not observed. Whether the receptors for the various AC agonists are present in the same cell cannot be determined from the present study. This study has demonstrated AC activity in the human Fallopian tube with similar kinetic characteristics as that in other somatic cells (6, 8). The biopsies studied have contained tissue from both the muscular and epithelial layers of the tubal wall. We are currently investigating a possible difference in AC activity in the endosalpinx and myosalpinx at different phases of the menstrual cycle. References 1. Jansen RPS. Endocrine response in the Fallopian tube. Endocr Rev. 1984;5:525-51. 2. Owman C, Rosengren E, Sj^berg NO. Adrenergic innervation of the human female reproductive organs: a histochemical and chemical investigation. Obstet Gynecol. 1967;30:763-73. 3. Aim P, Alumets J, Hakansson R. Vasoactive intestinal polypeptide nerves in the female genital tract. Am J Obstet Gynecol. 1980;136:349-51. 4. Kleinstein J. Binding properties of beta adrenergic receptors in oviductal smooth muscle membranes. Gynecol Obstet Invest. 1984;17:242-6. 5. Golf S, Jahnsen T, Attramadal H, Fr^ysaker T, Hansson V. Catecholamine responsive adenylate cyclase in human myocardial preparations. Properties and optimalization of assay conditions. Scand J Lab Clin Invest. 1984;44:317-27. 6. Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974;58:541-8. 7. Birnbaumer L, Yang PC, Hunzicker-Dunn M, Bockaert J, Duran JM. Adenylyl cyclase activities in ovarian tissues. I. Homogenization and conditions of assay in Graafian follicles and corpora lutea of rabbits, rats and pigs: regulation by ATP, and some comparative properties. Endocrinology. 1976:99:163-84. 8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:26575.
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9. Husslein P, Sinzinger H. Concentration of 13,14-dihydro-15-ketoPGE2 in the maternal peripheral plasma during labor of spontaneous onset. Br J Obstet Gynaecol. 1984;91:228-30. 10. Wiik P. Homologous regulation of adenylate cyclase-coupled receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes. Regul Peptides. 1988;20:323-33. 11. Bj^ro T, ^stberg BC, Sand 0, et al. Vasoactive intestinal peptide and peptide with N-terminal histidine and C-terminal isoleucine increase prolactin secretion in cultured rat pituitary cells (GH4Ci) via a cAMP-dependent mechanism which involves transient elevation of intracellular Ca2+. Mol Cell Endocrinol. 1987:49:119-28. 12. Erichsen AA, Jahnsen T, Attramadal H, Hansson V. A transplant-
JCE & M • 1991 Vol 73 • No 2
able rat Leydig cell tumor. II. Adenylyl cyclase activation by prostaglandin Ei, isoproterenol and glucagon. J Steroid Biochem. 1984:21:545-8. 13. Nieder J, Augustin W. Prostaglandin E and F profiles in human Fallopian tubes during different phases of the menstrual cycle. Gynecol Obstet Invest. 1986:21:202-7. 14. Helm G, Owman CH, Sj^berg N-O, Walles B. Quantitative pharmacological characterization of /8-receptors and two types of areceptors mediating sypathomimetic smooth muscle response in the human Fallopian tube at various cyclic stages. Acta Physiol Scand. 1982:114:425-32. 15. Ottesen B. Vasoactive intestinal polypeptide as a neurotransmitter in the female genital tract. Am J Obstet Gynecol. 1983:147:208-24.
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