00l3-7227/91/1292-0889$03.00/0 Endocrinology Copyrights 1991 by The Endocrine Society
Vol. 129, No. 2 Printed in U.S.A.
Effect of Prostaglandin F2a on Cytosolic Free Calcium Ion Concentrations in Rat Luteal Cells* MARIE R. RODWAY, KENNETH G. BAIMBRIDGE, BASIL HO YUEN, AND PETER C. K. LEUNG Departments of Obstetrics and Gynecology (M.R.R., B.H. Y., P.C.K.L.) and Physiology (K.G.B.), University of British Columbia, Vancouver, British Columbia, Canada
the concentration of PGF2(t. Perifusion with low calcium buffer reduced, then eliminated, the [Ca2+]i response to PGF2(Y. Perifusion of cells with PGF2o resulted in a single transient [Ca2+]i response, similar to that after short term exposure to PGF2n. Many (67%) of the cells that responded to PGF2(, also responded to GnRH. No additive increase in [Ca2+]i was seen when PGF2n and GnRH were administered together. The source of the calcium appears to be intracellular stores that are shared by GnRH and PGF2n. (Endocrinology 129: 889-895, 1991)
ABSTRACT. Changes in cytosolic free calcium concentrations ([Ca2+]i) in response to prostaglandin F2o (PGF2o) were measured in single rat luteal cells, using the calcium-sensitive fluorescent dye fura-2. A total of 112 cells were studied in 20 experiments. The average resting [Ca2+]i was 113 ± 6.4 nM. In 59 cells (53%), there was a 4.6 ± 0.2-fold increase in [Ca2+]i within 29.3 ± 1.0 sec of PGF2n administration, and the cells recovered by 78.0 ± 4.5 sec. The magnitude of the increase in [Ca"+]i in response to PGF2(, was not changed by an increase in
P
on [Ca2+]i in individual rat luteal cells and the relationship between PGF2«- and GnRH-induced increases in [Ca2+]i was examined.
ROSTAGLANDIN F2tv (PGF2(V) has effects on the function of the corpus luteum in the rat (1-4). PGF2« is synthesized in the rat ovary (5), and receptors for PGF2l( have been found in rat ovaries (6, 7). Although the second messenger responsible for the effect of PGF 2a on luteal function has not been conclusively established (8-12), PGF2,v does induce the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] in rat granulosa and luteal cells (4, 12, 13). The products of PtdIns(4,5)P2 breakdown, diacylglycerol and inositol 1,4,5-trisphosphate [Ins(l,4,5)P3], are second messengers, which activate protein kinase-C and induce increases in cytosolic free calcium ion ([Ca2+]i) concentrations, respectively (14, 15). Numerous studies have used calcium ionophores (16, 17), 45Ca (18), calcium channel blockers (16,18), calmodulin inhibitors (8, 18), and inhibitors of intracellular calcium release (8) to investigate the importance of calcium to modulation of the effects of PGF2a . However, the precise role of intracellular calcium ions in the action of PGF2(V is still largely unknown. In these experiments we have investigated the effect of PGF2« on [Ca2+]i in individual rat luteal cells. In addition, the effect of GnRH
Materials and Methods Luteal cells were prepared as described previously (19). Immature (23-25 days old) female Sprague-Dawley rats were injected with 25 IU PMSG (Ayerst, Rouses Point, IL), then with 25 IU hCG (Sigma, St. Louis, MO) 48 h later. Five days after hCG injection, rats were killed by cervical dislocation, and the ovaries were removed. Enzymatic dispersion was carried out for 1 h at 37 C in a shaking water bath, using collagenase (type 1) and DNase (type 1) with 0.2% BSA in HEPES buffer at pH 7.2. The resulting supernatant was filtered through a 210-Mm gauge mesh, then centrifuged at 1000 x g for 10 min. The cells were washed twice with McCoy's 5A medium (Gibco, Grand Island, NY) containing transferrin, insulin, and cortisol (all from Sigma). The cells were counted, plated at a density of 0.2 million cells/2-ml well in multiwell culture plates with glass coverslips, and incubated at 37 C in a humidified atmosphere of 5% CO2 in air for 4 days. For intracellular calcium measurements, the cells were preloaded with 5 fiM fura-2-acetoxyl-ester (fura-2AM, Molecular Probes, Inc., Eugene, OR) in 1% dimethylsulfoxide for 1.5 h before use, as described previously (20-22). Coverslips were loaded onto a laminar flow-through chamber (volume, 350 IJL). A water-tight seal was achieved using silicone seal. This chamber was inserted into a stainless steel holder, which was mounted onto the stage of a Zeiss Jenalumar microscope equipped for epifluorescense (Zeiss, New York, NY). The light source was a 200-watt mercury arc lamp powered by a DC
Received February 2,1991. Address requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Room 2H30, Grace Hospital, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. * This work was supported by grants from the Medical Research Council of Canada (to K.G.B. and P.C.K.L.).
889
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EFFECT OF PGF2(V ON [Ca 2+ ]i
890
power supply. The light was first passed through one of three differential interference filters (350, 365, or 380 nm; bandwidth, 10 nm), which was mounted in a turret rotated by a computercontrolled stepping motor. The light was then passed through a 410-nm dichroic mirror and a X100 apochromat oil immersion lens with a numerical aperture of 1.4 and an adjustable diaphragm to reduce the light intensity. A field diaphragm in the light path before the dichroic mirror was used to reduce the area of illumination to the size of a single cell. All fluorescent light passed back through the dichroic mirror and a 450-nm band pass filter to reduce background fluorescence. The emitted fluorescence was deflected to either the eyepieces or a camera port, in which was mounted a photomultiplier tube. The photomultiplier tube converted the fluorescence into DC voltage. The voltage was converted to a digital form, and measurements of fluorescence ratios, corrected for background, were obtained over a 0.5-sec period, every 5 sec. Calcium concentration was calculated (20) from the fluorescence intensity ratio (350 nm/ 380 nm) by the formula: [Ca2+]i = Kd X (R - Rmin/Rmax - R) X /? (mean fluorescence at 380 nm in minimum calcium/mean fluorescence at 380 nm in maxium calcium). Calibration of fura-2 in rat luteal cells produced the following values: Rmjn = 0.36, Rmax = 1.5, and /5 = 1.43. Perifusion with Earle's Balanced Salt Solution (EBSS), EBSS with no added calcium (designated low calcium EBSS), or EBSS containing calcium channel blockers was performed at a rate of 4 ml/min. The stainless steel holder was maintained at 36 C. Hormones were administered in 500-^1 aliquots and washed through with the perifusion medium, except where otherwise indicated. Numbers are reported as the mean ± SEM.
Endo • 1991 Vol 129 • No 2
800 600 400 200
10
FIG. 1. The effect of PGF2n on [Ca2+]i in a single rat luteal cell. Cells were prepared as described in Materials and Methods. PGF2« was administered in a concentration of 10~6 M in 500 ^1 EBSS at the times indicated by arrows. Similar results were seen in 59 individual cells in 20 experiments.
600
400 •
O 200
Results
20
Response to PGF2a PGF2(V caused a change in [Ca2+]i in 59 (53%) of 112 cells tested in 20 experiments. The average resting calcium concentration was 113 ± 6.4 nM, the average fold response was 4.6 ± 0.2, the average time to respond was 29.3 ± 1 . 0 sec, and the average time to return to the resting calcium concentration was 78 ± 4.5 sec (Fig. 1). In some experiments the interval between PGF2fV administration was reduced in an attempt to determine the minimum interval necessary for PGF 2a to produce a response of similar magnitude. In 3 separate experiments, the responses to PGF2« were similar in magnitude if the interval was greater than 7 min. If the interval was less, the magnitude of response decreased (Fig. 2).
20
Time (min)
30
50
Time (min) FIG. 2. The effect of a decrease in the time between administrations of agonist on alterations in [Ca2+]i in a single rat luteal cell. PGF2,, (10~6 M in 500 nl) was administered at the times indicated by arrows. The spacing of arrows corresponds to time intervals of 7.2, 6.5, 6.0, 3.7, 3.7, 2.3, and 1.0 min. Three experiments produced similiar results.
Concentration-response relationship and minimum effective concentration The minimum effective concentration of PGF 2a in luteal cells ranged from 10~"8-10~5 M (the minimum effective concentration in two cells was 10"5 M, in six was 10~6 M, in five was 10~7 M, and in three was 10~8 M). The magnitude of the increase in [Ca2+]i in response to increasing concentrations of PGF 2a did not change in six luteal cells studied (Fig. 3).
0 •— 0
10
Time (min) FIG. 3. An investigation of the dose response to PGF2n in a single rat luteal cell. PGF2O was administered in concentrations from 10"9-l0~s M in 500 p\ EBSS at the times indicated by arrows. Five cells tested produced similar results.
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EFFECT OF PGF2a ON [Ca2+]i
891
Low calcium perifusion experiments Perifusion of luteal cells with low calcium EBSS (11 cells in 8 experiments) caused a reduction, then an elimination, of the increase in [Ca2+]i in response to PGF2((. In 8 of the 11 cells the normal response to PGF2tv was recovered after reperifusion with EBSS containing 1.8 raM calcium. The time to elimination of the response to PGF2(V in low calcium EBSS varied from 11-24 min (Fig. 4). Resting [Ca2+]i fell significantly (by t test, P < 0.01) during perifusion with low calcium EBSS.
600 " 400 . O 200 -
10
Time (min)
Response to GnRH in cells responsive to PGF2a Thirty cells in which PGF2« induced an increase in [Ca'2+]i were subsequently treated with GnRH. Twenty (67%) also responded to GnRH. The average fold response was 4.5 ± 0.4, the average time to response was 28.5 ± 2.3 sec, and the average time to recovery was 72 ± 7.2 sec; these values were similar to those found for PGF2
Vol. 129, No. 2 Printed in U.S.A.
Effect of Prostaglandin F2a on Cytosolic Free Calcium Ion Concentrations in Rat Luteal Cells* MARIE R. RODWAY, KENNETH G. BAIMBRIDGE, BASIL HO YUEN, AND PETER C. K. LEUNG Departments of Obstetrics and Gynecology (M.R.R., B.H. Y., P.C.K.L.) and Physiology (K.G.B.), University of British Columbia, Vancouver, British Columbia, Canada
the concentration of PGF2(t. Perifusion with low calcium buffer reduced, then eliminated, the [Ca2+]i response to PGF2(Y. Perifusion of cells with PGF2o resulted in a single transient [Ca2+]i response, similar to that after short term exposure to PGF2n. Many (67%) of the cells that responded to PGF2(, also responded to GnRH. No additive increase in [Ca2+]i was seen when PGF2n and GnRH were administered together. The source of the calcium appears to be intracellular stores that are shared by GnRH and PGF2n. (Endocrinology 129: 889-895, 1991)
ABSTRACT. Changes in cytosolic free calcium concentrations ([Ca2+]i) in response to prostaglandin F2o (PGF2o) were measured in single rat luteal cells, using the calcium-sensitive fluorescent dye fura-2. A total of 112 cells were studied in 20 experiments. The average resting [Ca2+]i was 113 ± 6.4 nM. In 59 cells (53%), there was a 4.6 ± 0.2-fold increase in [Ca2+]i within 29.3 ± 1.0 sec of PGF2n administration, and the cells recovered by 78.0 ± 4.5 sec. The magnitude of the increase in [Ca"+]i in response to PGF2(, was not changed by an increase in
P
on [Ca2+]i in individual rat luteal cells and the relationship between PGF2«- and GnRH-induced increases in [Ca2+]i was examined.
ROSTAGLANDIN F2tv (PGF2(V) has effects on the function of the corpus luteum in the rat (1-4). PGF2« is synthesized in the rat ovary (5), and receptors for PGF2l( have been found in rat ovaries (6, 7). Although the second messenger responsible for the effect of PGF 2a on luteal function has not been conclusively established (8-12), PGF2,v does induce the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] in rat granulosa and luteal cells (4, 12, 13). The products of PtdIns(4,5)P2 breakdown, diacylglycerol and inositol 1,4,5-trisphosphate [Ins(l,4,5)P3], are second messengers, which activate protein kinase-C and induce increases in cytosolic free calcium ion ([Ca2+]i) concentrations, respectively (14, 15). Numerous studies have used calcium ionophores (16, 17), 45Ca (18), calcium channel blockers (16,18), calmodulin inhibitors (8, 18), and inhibitors of intracellular calcium release (8) to investigate the importance of calcium to modulation of the effects of PGF2a . However, the precise role of intracellular calcium ions in the action of PGF2(V is still largely unknown. In these experiments we have investigated the effect of PGF2« on [Ca2+]i in individual rat luteal cells. In addition, the effect of GnRH
Materials and Methods Luteal cells were prepared as described previously (19). Immature (23-25 days old) female Sprague-Dawley rats were injected with 25 IU PMSG (Ayerst, Rouses Point, IL), then with 25 IU hCG (Sigma, St. Louis, MO) 48 h later. Five days after hCG injection, rats were killed by cervical dislocation, and the ovaries were removed. Enzymatic dispersion was carried out for 1 h at 37 C in a shaking water bath, using collagenase (type 1) and DNase (type 1) with 0.2% BSA in HEPES buffer at pH 7.2. The resulting supernatant was filtered through a 210-Mm gauge mesh, then centrifuged at 1000 x g for 10 min. The cells were washed twice with McCoy's 5A medium (Gibco, Grand Island, NY) containing transferrin, insulin, and cortisol (all from Sigma). The cells were counted, plated at a density of 0.2 million cells/2-ml well in multiwell culture plates with glass coverslips, and incubated at 37 C in a humidified atmosphere of 5% CO2 in air for 4 days. For intracellular calcium measurements, the cells were preloaded with 5 fiM fura-2-acetoxyl-ester (fura-2AM, Molecular Probes, Inc., Eugene, OR) in 1% dimethylsulfoxide for 1.5 h before use, as described previously (20-22). Coverslips were loaded onto a laminar flow-through chamber (volume, 350 IJL). A water-tight seal was achieved using silicone seal. This chamber was inserted into a stainless steel holder, which was mounted onto the stage of a Zeiss Jenalumar microscope equipped for epifluorescense (Zeiss, New York, NY). The light source was a 200-watt mercury arc lamp powered by a DC
Received February 2,1991. Address requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Room 2H30, Grace Hospital, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. * This work was supported by grants from the Medical Research Council of Canada (to K.G.B. and P.C.K.L.).
889
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 13:48 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF PGF2(V ON [Ca 2+ ]i
890
power supply. The light was first passed through one of three differential interference filters (350, 365, or 380 nm; bandwidth, 10 nm), which was mounted in a turret rotated by a computercontrolled stepping motor. The light was then passed through a 410-nm dichroic mirror and a X100 apochromat oil immersion lens with a numerical aperture of 1.4 and an adjustable diaphragm to reduce the light intensity. A field diaphragm in the light path before the dichroic mirror was used to reduce the area of illumination to the size of a single cell. All fluorescent light passed back through the dichroic mirror and a 450-nm band pass filter to reduce background fluorescence. The emitted fluorescence was deflected to either the eyepieces or a camera port, in which was mounted a photomultiplier tube. The photomultiplier tube converted the fluorescence into DC voltage. The voltage was converted to a digital form, and measurements of fluorescence ratios, corrected for background, were obtained over a 0.5-sec period, every 5 sec. Calcium concentration was calculated (20) from the fluorescence intensity ratio (350 nm/ 380 nm) by the formula: [Ca2+]i = Kd X (R - Rmin/Rmax - R) X /? (mean fluorescence at 380 nm in minimum calcium/mean fluorescence at 380 nm in maxium calcium). Calibration of fura-2 in rat luteal cells produced the following values: Rmjn = 0.36, Rmax = 1.5, and /5 = 1.43. Perifusion with Earle's Balanced Salt Solution (EBSS), EBSS with no added calcium (designated low calcium EBSS), or EBSS containing calcium channel blockers was performed at a rate of 4 ml/min. The stainless steel holder was maintained at 36 C. Hormones were administered in 500-^1 aliquots and washed through with the perifusion medium, except where otherwise indicated. Numbers are reported as the mean ± SEM.
Endo • 1991 Vol 129 • No 2
800 600 400 200
10
FIG. 1. The effect of PGF2n on [Ca2+]i in a single rat luteal cell. Cells were prepared as described in Materials and Methods. PGF2« was administered in a concentration of 10~6 M in 500 ^1 EBSS at the times indicated by arrows. Similar results were seen in 59 individual cells in 20 experiments.
600
400 •
O 200
Results
20
Response to PGF2a PGF2(V caused a change in [Ca2+]i in 59 (53%) of 112 cells tested in 20 experiments. The average resting calcium concentration was 113 ± 6.4 nM, the average fold response was 4.6 ± 0.2, the average time to respond was 29.3 ± 1 . 0 sec, and the average time to return to the resting calcium concentration was 78 ± 4.5 sec (Fig. 1). In some experiments the interval between PGF2fV administration was reduced in an attempt to determine the minimum interval necessary for PGF 2a to produce a response of similar magnitude. In 3 separate experiments, the responses to PGF2« were similar in magnitude if the interval was greater than 7 min. If the interval was less, the magnitude of response decreased (Fig. 2).
20
Time (min)
30
50
Time (min) FIG. 2. The effect of a decrease in the time between administrations of agonist on alterations in [Ca2+]i in a single rat luteal cell. PGF2,, (10~6 M in 500 nl) was administered at the times indicated by arrows. The spacing of arrows corresponds to time intervals of 7.2, 6.5, 6.0, 3.7, 3.7, 2.3, and 1.0 min. Three experiments produced similiar results.
Concentration-response relationship and minimum effective concentration The minimum effective concentration of PGF 2a in luteal cells ranged from 10~"8-10~5 M (the minimum effective concentration in two cells was 10"5 M, in six was 10~6 M, in five was 10~7 M, and in three was 10~8 M). The magnitude of the increase in [Ca2+]i in response to increasing concentrations of PGF 2a did not change in six luteal cells studied (Fig. 3).
0 •— 0
10
Time (min) FIG. 3. An investigation of the dose response to PGF2n in a single rat luteal cell. PGF2O was administered in concentrations from 10"9-l0~s M in 500 p\ EBSS at the times indicated by arrows. Five cells tested produced similar results.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 13:48 For personal use only. No other uses without permission. . All rights reserved.
EFFECT OF PGF2a ON [Ca2+]i
891
Low calcium perifusion experiments Perifusion of luteal cells with low calcium EBSS (11 cells in 8 experiments) caused a reduction, then an elimination, of the increase in [Ca2+]i in response to PGF2((. In 8 of the 11 cells the normal response to PGF2tv was recovered after reperifusion with EBSS containing 1.8 raM calcium. The time to elimination of the response to PGF2(V in low calcium EBSS varied from 11-24 min (Fig. 4). Resting [Ca2+]i fell significantly (by t test, P < 0.01) during perifusion with low calcium EBSS.
600 " 400 . O 200 -
10
Time (min)
Response to GnRH in cells responsive to PGF2a Thirty cells in which PGF2« induced an increase in [Ca'2+]i were subsequently treated with GnRH. Twenty (67%) also responded to GnRH. The average fold response was 4.5 ± 0.4, the average time to response was 28.5 ± 2.3 sec, and the average time to recovery was 72 ± 7.2 sec; these values were similar to those found for PGF2
