Bombesin and bradykinin increase inositol phosphates and cytosolic free Ca 2+, and stimulate DNA synthesis in human endometrial stromal cells T. Endo, H. Fukue, M. Kanaya, M. Mizunuma, M. Fujii, H. Yamamoto, S. Tanaka and M. Hashimoto Department of Gynecology and Obstetrics, Sapporo Medical College, Chuoku, Sapporo, 060 Japan REVISED MANUSCRIPT RECEIVED
16
April
1991
ABSTRACT
The present studies were carried out to investigate the effect of several growth factors on human endometrial stromal cells. In human endometrial stromal cells, bombesin and bradykinin provoked an increase in intracellular free Ca2+ and in labelled inositol phosphates when pre\x=req-\ incubated with [3H]myoinositol. Some or possibly all of the initial increase in intracellular free Ca2+ represented a mobilization of Ca2+ from intracellular stores and the second phase of the response depended on Ca2+ influx from the extracellular medium. [3H]Thymidine was added to human cultured endo-
INTRODUCTION
The endometrium undergoes rapid growth and differ¬ entiation during the menstrual cycle, in preparation for embryo implantation. It is thought that oestradiol and progesterone induce the decidual transformation of the stromal cells. Although much is known about the effects of steroid hormones on endometrial physi¬ ology, relatively little information is available about the role of compounds acting on endometrial plasma membrane receptors. Proliferation continues throughout the entire menstrual cycle in human endometrial stromal cells (Ferenczy, Bertrand & Gelfand, 1979) and it has been reported that prostaglandin F2a (PGF2a) induces DNA synthesis and increases the number of cells and inositol phosphates (IPs) in primary cultures of rabbit endothelial endometrial cells (Orlicky, Lieberman & Gershenson, 1986a; Orlicky, Lieberman, Williama &
Gershenson, 1986e).
The present study was undertaken to examine the effects of bombesin, bradykinin, epidermal growth fac¬ tor (EGF), PGF2a, vasopressin and platelet-derived
metrial stromal cells with bombesin, bradykinin, epidermal growth factor (EGF), prostaglandin F2\g=a\, vasopressin and platelet-derived growth factor. Bombesin, bradykinin and EGF stimulated the incorporation of [3H]thymidine into DNA in quiescent
cells. In
conclusion, bombesin and bradykinin are growth factors which activate phospholipase C in
human endometrial stromal cells, while EGF stimulates DNA synthesis without the activation of phospholipase C.
Journal of Endocrinology (1991) 131, 313\p=n-\318
growth factor (PDGF), all of which are known to be growth factors for other cells, on DNA synthesis and the accumulation of IPs, and on intracellular Ca2+ levels in human endometrial stromal cells.
MATERIALS AND METHODS
Chemicals
Myo-2-[3H(N)]inositol (15-8Ci/mmol) and methyl[3H]thymidine (82-2 Ci/mmol) were obtained from
New England Nuclear Corporation Boston, MA, U.S.A. PGF2a was a gift from Ono Pharmaceuticals, Osaka, Japan. The commercial sources of other chemicals were as follows: Eagle's Minimal Essential medium (MEM), Nissui, Tokyo, Japan; fetal calf serum (FCS), Gibco, Grand Island, NY, U.S.A.; type-1 collagenase, Sigma, St Louis, MO, U.S.A.; type-1 dispase, Godo Shusei, Tokyo, Japan; fura 2/penta
acetoxymethyl ester, Dojin Chemicals, Kumamoto, Japan; bombesin, Peptide Institute, Osaka, Japan; arginine vasopressin (367 units/mg) and bradykinin,
Boehringer Mannheim Yamanouchi, EGF and PDGF, Sigma.
Tokyo, Japan;
Cell culture Human endometrium was obtained in the periovulatory phase from women who were not receiving any medication, whose menstrual cycles were regular and who were not suffering from any endocrinological disorder when they were undergoing hysterectomy for uterine fibroma or intraepithelial neoplasia. Consent was obtained from them preoperatively for the use of the endometrial tissues. The stage of the cycle was determined on the basis of their menstrual history and basal body temperature. Endometrium tissue samples were collected by curettage from the uterus and were minced and digested with collagenase (500 protease units/ml) and dispase (100 IU/ml) to isolate cells. The digestion was performed at 37 °C for 15 to 30 min. This incubation time is sufficiently short to prevent dispersal of glandular cells, aiding in their separation from single stromal cells. The digested sample was filtered through two layers of gauze to remove undigested endometrium. After this treatment the dispersed cells were separated into stromal cell elements and glandu¬ lar cell elements by discontinuous density gradient centrifugation. Namely, 20%, 40%, 60% and 80% Percoli (Sigma) layers were discontinuously stratified up. The endometrial cell elements treated as described above were then put on the top of the layers and cen¬ trifuged at 100 g for 30 min. Stromal cell elements were harvested between the 20% and 40% Percoli layer as described previously (Tanaka, Mizunuma, Kanaya et al. 1991). Aliquots of cell suspension (2 105 cells/ml) in Eagle's MEM with 10% FCS (v/v) were plated onto 90 mm culture dishes (Nunc, Roskilde, Denmark) and cultured at 37 °C under an atmosphere of 5% C02 in air. The culture medium was replaced every 2 days.
Analysis of inositol phosphates When the cells
were
confluent the medium
was
changed to Eagle's MEM supplemented with 5% FCS and 5 pCi myo-2-[3H]inositol/ml and the cells were incubated for a further 2 days. The labelled cells were washed twice with Hepes-buffered saline (145 mmol NaCl/1, 5 mmol KC1/1,1 mmol NaH2P04/l, 1 mmol CaCyi, 0-5 mmol MgCl2/l, 5 mmol glucose/1, 10 mmol Hepes-NaOH/1; pH 7-4) (HBS) containing 01% (w/v) bovine serum albumin, and they are then harvested in the saline using a rubber scraper. Incu¬ bation was started by adding 1 ml Li-HBS (5 105 cells/ml), a buffer in which part (10 mmol/1) of the NaCl in the HBS had been replaced with 10 mmol
LiCl/1 with or without bombesin (40, 400 nmol/1), bradykinin (40, 400 nmol/1), EGF (1, 10 ng/ml), PGF2a (01, 1 pmol/ml), vasopressin (01, 1 mIU/ml) or PDGF (10, 100 ng/ml). The reaction was termi¬ nated at 5, 15 or 30 min at 37 °C by adding 0-75 ml chloroform/methanol (1 :2, v/v). The mixture was mixed thoroughly and then divided into two phases by adding 0-25 ml each of chloroform and water. The upper phase containing [3H]IPs was removed
and diluted with 4 ml water. The radiolabelled IPs in the aqueous phase were determined by anion exchange chromatography (Sasaki & Hasegawa-
Sasaki, 1985).
Determination of intracellular free (a2
Cells
were harvested with a rubber scraper when they confluent. They were then incubated in Eagle's MEM containing 1-5 pmol fura 2/penta acetoxymethyl ester/1 at 37 °C for 30 min. After being loaded, cells were washed three times with HBS and resuspended (5xl05 cells/ml) in the saline. Changes in fura 2 fluorescence were recorded at 37 °C (HasegawaSasaki, Lutz & Sasaki, 1988) using a Hitachi 650-10s fluorimeter (Tokyo, Japan). Excitation and emission wavelengths were 340 and 500 nm with 2 and 10 nm slits respectively. Extracellular Ca2+ was depleted by suspending fura 2-loaded human endometrial stromal cells in Ca2+- and Mg2+-free Hepes-buffered saline containing 1 mmol EGTA/1. The intracellular free Ca2+ concentration ([Ca2+]) was calculated from the ratio of fura 2 fluorescence intensities at two excitation wavelengths (340 and 360 nm) (Grynkiewicz, Poenie & Tsien, 1985). were
Effects of various chemicals on DNA
synthesis
Cells were plated at a density of 1 105/cm2 in 96-well multidishes (Corning, Corning, NY, U.S.A.) in medium containing 10% (w/v) FCS. When the cells were confluent, the medium was changed to 1 ml medium containing 5% FCS. Methyl-[3H]thymidine (2pCi) was then added to each well with bombesin (400 nmol/1), bradykinin (400 nmol/1), EGF (10 ng/
ml), PGF2a (1 pmol/1), vasopressin (1 mIU/ml) or PDGF (100 ng/ml). The cells were labelled for 24 h and [3H]thymidine incorporation in human endo¬ metrial stromal cells harvester.
was
determined with
a
cell
Statistical evaluation
Results
data
were
are expressed as means ± s.e.m.. The analysed statistically by /-test. Data considered statistically significant if < 005.
were
RESULTS
1. Effects of bombesin, bradykinin, epidermal growth factor (EGF), prostaglandin F2o (PGF2a), vasopressin and platelet-derived growth factor (PDGF) on the accumulation of inositol monophosphate, inositol bisphosphate and inositol trisphosphate (IPs) in human endometrial stromal cells which were incubated for 15 min with 10 mmol LiCl in the absence (control) or presence of bombesin, bradykinin, table
Increase in IPs and |Ca2 |i in human endometrial stromal cells in response to bombesin, bradykinin, EGF, PGF2a, vasopressin and PDGF '
Figure 1 shows the time course of the stimulatory effect of bombesin on inositol monophosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3). The basal level of the IPs (IP, IP2 and IP3) remained relatively unchanged in each incubation period. A significant (P
16
April
1991
ABSTRACT
The present studies were carried out to investigate the effect of several growth factors on human endometrial stromal cells. In human endometrial stromal cells, bombesin and bradykinin provoked an increase in intracellular free Ca2+ and in labelled inositol phosphates when pre\x=req-\ incubated with [3H]myoinositol. Some or possibly all of the initial increase in intracellular free Ca2+ represented a mobilization of Ca2+ from intracellular stores and the second phase of the response depended on Ca2+ influx from the extracellular medium. [3H]Thymidine was added to human cultured endo-
INTRODUCTION
The endometrium undergoes rapid growth and differ¬ entiation during the menstrual cycle, in preparation for embryo implantation. It is thought that oestradiol and progesterone induce the decidual transformation of the stromal cells. Although much is known about the effects of steroid hormones on endometrial physi¬ ology, relatively little information is available about the role of compounds acting on endometrial plasma membrane receptors. Proliferation continues throughout the entire menstrual cycle in human endometrial stromal cells (Ferenczy, Bertrand & Gelfand, 1979) and it has been reported that prostaglandin F2a (PGF2a) induces DNA synthesis and increases the number of cells and inositol phosphates (IPs) in primary cultures of rabbit endothelial endometrial cells (Orlicky, Lieberman & Gershenson, 1986a; Orlicky, Lieberman, Williama &
Gershenson, 1986e).
The present study was undertaken to examine the effects of bombesin, bradykinin, epidermal growth fac¬ tor (EGF), PGF2a, vasopressin and platelet-derived
metrial stromal cells with bombesin, bradykinin, epidermal growth factor (EGF), prostaglandin F2\g=a\, vasopressin and platelet-derived growth factor. Bombesin, bradykinin and EGF stimulated the incorporation of [3H]thymidine into DNA in quiescent
cells. In
conclusion, bombesin and bradykinin are growth factors which activate phospholipase C in
human endometrial stromal cells, while EGF stimulates DNA synthesis without the activation of phospholipase C.
Journal of Endocrinology (1991) 131, 313\p=n-\318
growth factor (PDGF), all of which are known to be growth factors for other cells, on DNA synthesis and the accumulation of IPs, and on intracellular Ca2+ levels in human endometrial stromal cells.
MATERIALS AND METHODS
Chemicals
Myo-2-[3H(N)]inositol (15-8Ci/mmol) and methyl[3H]thymidine (82-2 Ci/mmol) were obtained from
New England Nuclear Corporation Boston, MA, U.S.A. PGF2a was a gift from Ono Pharmaceuticals, Osaka, Japan. The commercial sources of other chemicals were as follows: Eagle's Minimal Essential medium (MEM), Nissui, Tokyo, Japan; fetal calf serum (FCS), Gibco, Grand Island, NY, U.S.A.; type-1 collagenase, Sigma, St Louis, MO, U.S.A.; type-1 dispase, Godo Shusei, Tokyo, Japan; fura 2/penta
acetoxymethyl ester, Dojin Chemicals, Kumamoto, Japan; bombesin, Peptide Institute, Osaka, Japan; arginine vasopressin (367 units/mg) and bradykinin,
Boehringer Mannheim Yamanouchi, EGF and PDGF, Sigma.
Tokyo, Japan;
Cell culture Human endometrium was obtained in the periovulatory phase from women who were not receiving any medication, whose menstrual cycles were regular and who were not suffering from any endocrinological disorder when they were undergoing hysterectomy for uterine fibroma or intraepithelial neoplasia. Consent was obtained from them preoperatively for the use of the endometrial tissues. The stage of the cycle was determined on the basis of their menstrual history and basal body temperature. Endometrium tissue samples were collected by curettage from the uterus and were minced and digested with collagenase (500 protease units/ml) and dispase (100 IU/ml) to isolate cells. The digestion was performed at 37 °C for 15 to 30 min. This incubation time is sufficiently short to prevent dispersal of glandular cells, aiding in their separation from single stromal cells. The digested sample was filtered through two layers of gauze to remove undigested endometrium. After this treatment the dispersed cells were separated into stromal cell elements and glandu¬ lar cell elements by discontinuous density gradient centrifugation. Namely, 20%, 40%, 60% and 80% Percoli (Sigma) layers were discontinuously stratified up. The endometrial cell elements treated as described above were then put on the top of the layers and cen¬ trifuged at 100 g for 30 min. Stromal cell elements were harvested between the 20% and 40% Percoli layer as described previously (Tanaka, Mizunuma, Kanaya et al. 1991). Aliquots of cell suspension (2 105 cells/ml) in Eagle's MEM with 10% FCS (v/v) were plated onto 90 mm culture dishes (Nunc, Roskilde, Denmark) and cultured at 37 °C under an atmosphere of 5% C02 in air. The culture medium was replaced every 2 days.
Analysis of inositol phosphates When the cells
were
confluent the medium
was
changed to Eagle's MEM supplemented with 5% FCS and 5 pCi myo-2-[3H]inositol/ml and the cells were incubated for a further 2 days. The labelled cells were washed twice with Hepes-buffered saline (145 mmol NaCl/1, 5 mmol KC1/1,1 mmol NaH2P04/l, 1 mmol CaCyi, 0-5 mmol MgCl2/l, 5 mmol glucose/1, 10 mmol Hepes-NaOH/1; pH 7-4) (HBS) containing 01% (w/v) bovine serum albumin, and they are then harvested in the saline using a rubber scraper. Incu¬ bation was started by adding 1 ml Li-HBS (5 105 cells/ml), a buffer in which part (10 mmol/1) of the NaCl in the HBS had been replaced with 10 mmol
LiCl/1 with or without bombesin (40, 400 nmol/1), bradykinin (40, 400 nmol/1), EGF (1, 10 ng/ml), PGF2a (01, 1 pmol/ml), vasopressin (01, 1 mIU/ml) or PDGF (10, 100 ng/ml). The reaction was termi¬ nated at 5, 15 or 30 min at 37 °C by adding 0-75 ml chloroform/methanol (1 :2, v/v). The mixture was mixed thoroughly and then divided into two phases by adding 0-25 ml each of chloroform and water. The upper phase containing [3H]IPs was removed
and diluted with 4 ml water. The radiolabelled IPs in the aqueous phase were determined by anion exchange chromatography (Sasaki & Hasegawa-
Sasaki, 1985).
Determination of intracellular free (a2
Cells
were harvested with a rubber scraper when they confluent. They were then incubated in Eagle's MEM containing 1-5 pmol fura 2/penta acetoxymethyl ester/1 at 37 °C for 30 min. After being loaded, cells were washed three times with HBS and resuspended (5xl05 cells/ml) in the saline. Changes in fura 2 fluorescence were recorded at 37 °C (HasegawaSasaki, Lutz & Sasaki, 1988) using a Hitachi 650-10s fluorimeter (Tokyo, Japan). Excitation and emission wavelengths were 340 and 500 nm with 2 and 10 nm slits respectively. Extracellular Ca2+ was depleted by suspending fura 2-loaded human endometrial stromal cells in Ca2+- and Mg2+-free Hepes-buffered saline containing 1 mmol EGTA/1. The intracellular free Ca2+ concentration ([Ca2+]) was calculated from the ratio of fura 2 fluorescence intensities at two excitation wavelengths (340 and 360 nm) (Grynkiewicz, Poenie & Tsien, 1985). were
Effects of various chemicals on DNA
synthesis
Cells were plated at a density of 1 105/cm2 in 96-well multidishes (Corning, Corning, NY, U.S.A.) in medium containing 10% (w/v) FCS. When the cells were confluent, the medium was changed to 1 ml medium containing 5% FCS. Methyl-[3H]thymidine (2pCi) was then added to each well with bombesin (400 nmol/1), bradykinin (400 nmol/1), EGF (10 ng/
ml), PGF2a (1 pmol/1), vasopressin (1 mIU/ml) or PDGF (100 ng/ml). The cells were labelled for 24 h and [3H]thymidine incorporation in human endo¬ metrial stromal cells harvester.
was
determined with
a
cell
Statistical evaluation
Results
data
were
are expressed as means ± s.e.m.. The analysed statistically by /-test. Data considered statistically significant if < 005.
were
RESULTS
1. Effects of bombesin, bradykinin, epidermal growth factor (EGF), prostaglandin F2o (PGF2a), vasopressin and platelet-derived growth factor (PDGF) on the accumulation of inositol monophosphate, inositol bisphosphate and inositol trisphosphate (IPs) in human endometrial stromal cells which were incubated for 15 min with 10 mmol LiCl in the absence (control) or presence of bombesin, bradykinin, table
Increase in IPs and |Ca2 |i in human endometrial stromal cells in response to bombesin, bradykinin, EGF, PGF2a, vasopressin and PDGF '
Figure 1 shows the time course of the stimulatory effect of bombesin on inositol monophosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3). The basal level of the IPs (IP, IP2 and IP3) remained relatively unchanged in each incubation period. A significant (P
