Methionine-enkephalin in a procine endometrial cell line and its responsiveness to potassium depolarization

Life Sciences, Vol. Printed in the USA

51, pp.

1981-1990

Pergamon

Press

METHIONINE-ENKEPHALIN IN A PORCINE ENDOMETRIAL CELL LINE AND ITS RESPONSIVENESS TO POTASSIUM DEPOLARIZATION

Wan-I Li~ Department of Physiology and Pharmacology, College of Veterinary Medicine The University of Georgia, Athens, GA 30602 (Received

in final

form October

9, 1992)

Summary Immunoreactive methionine-enkephalin (ir-MENK) has been identified in the porcine uterine fluid and endometrium. Previously, we have established a porcine endometrial cell line of epithelial origin (PE-1) by transfecting primary endometrial cells with temperature sensitive SV40 DNA. The current study was conducted to identify and characterize irMENK present in PE-1 cells, and to investigate the effect of KCI depolarization on the kinetics of ir-MENK secretion. PE-1 cells were cultured at 33C until confluency was reached (33C cells), after which they were incubated at 40C for 2 days (40C cells). Ir-MENK in PE-1 cells was analyzed by Sephadex G-15 gel filtration and reverse phase (RP)HPLC. Analysis of 40C cell extract by Sephadex G-15 and RP-HPLC indicated that the major portion of ir-MENK present in PE-1 cells was eluted at a position similar to that of synthetic MENK. The effect of temperature on ir-MENK synthesis in PE-1 cells was examined by measuring ir-MENK content in 33C and 40C cells over a 14-day culture period. Compared to 33C cells, 40C cells maintained higher and steadier levels of ir-MENK, suggesting that synthesis of ir-MENK is temperature sensitive. KCI stimulated ir-MENK secretion at all concentrations tested (5-60 mM for 60 min), with 30 mM being the optimal concentration. Temporal analysis of ir-MENK secretion showed that incubation for 60 min with 30 mM KCI allowed maximal secretion. Secretion of ir-MENK from PE-1 cells resulted in depletion of ir-MENK in cell content. These results demonstrate that PE-1 cells contain ir-MENK which is biochemically similar to synthetic MENK, PE-1 cells synthesize ir-MENK in a temperature sensitive manner, and these cells secrete irMENK upon KCI stimulation. Evidence has been provided that the uterus synthesizes and secretes endogenous opioid peptides (EOP). ~-Endorphin has been demonstrated in the endometrium of women (1) and pigs (2), and in the uterine secretions of women, cows (3), and pigs (4). Methionine-enkephalin (MENK) has been detected in uterine fluids of cows, women (1), pigs (5), and rabbits (6), as well as in a rabbit endometrial cell line (HREH9) (6). Identification of mRNA for pro-opiomelanocortin (7), proenkephalin (8), and prodynorphin (9) in the uterine tissue further suggests that de novo synthesis of EOP occurs within the uterus. 1 Address all correspondence and requests for reprints to: Dr. Wan-I Li, Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602.

Copyright

0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights

reserved.

1982

I r - M E N K and P o r c i n e E n d o m e t r i a l

Cell L i n e

Vol.

51, No. 25,

1992

The biological significance of uterine EOP remains to be elucidated. Investigation into the hormonal regulation of EOP synthesis and secretion in the endometrium is essential in understanding their physiological role(s). We have previously established a porcine endometrial cell line (PE-1) by transfecting primary endometrial cells with temperature sensitive-SV40 (ts-SV40) DNA (10). This cell line exhibits temperature sensitivity in cell morphology, cell growth rate, and protein synthesis rate. At the permissive temperature (33C), PE-1 cells grow rapidly, doubling in number by approximately 48 hr. The cells exhibit spindle-shape morphology, a feature typical of cells being transformed. When PE-1 cells are incubated at the nonpermissive temperature (40C), they grow slowly and become rounded in shape. At 40C, their metabolic rate increass, as indicated by higher protein synthesis. The aim of the current study was to (a) identify immunoreactive MENK (ir-MENK) in PE-1 cells, (b) biochemically characterize ir-MENK, (c) examine if ir-MENK synthesis in PE-1 cells is temperature sensitive, and (d) investigate the kinetics of ir-MENK secretion from PE-1 cells. Materials and Methods Growth of PE-1 Cells: The PE-1 cell line, established by transforming primary porcine endometrial cells with ts-SV40, has been previously characterized (10). The cells were cultured in (z-Modified Minimum Essential Medium ((z-MEM) supplemented with 4% fetal bovine serum (FBS) (GIBCO-BRL, Grand Island, NY), streptomycin (10 pg/ml), penicillin (10 U/ml), insulin (5 I~g/ml), and 1% dextrose, and this was termed (zMEM-4. The o~-MEM-4 was changed twice weekly. Cells were grown at 33C (33C cells) in 75 cm 2 culture flasks until confluency was apparent, and then transferred to a 40C (40C cells) environment and incubated for an additional 2 days before being used for experiments. Cell Collection and Extraction: In the identification and biochemical characterization of ir-MENK in PE-1 cells, the medium was decanted and 40C cells were isolated by trypsin treatment (1.5 ml, 0.5 mg/ml; Sigma, St. Louis, MO). Cell suspensions were collected in 50-ml conical tubes and pelleted by centrifugation at 1000 x g (Beckman T J-6, Beckman Instruments, Palo Alto, CA) for 10 rain at 4C. Cell pellets were extracted with 0.1 N acetic acid, homogenized using a Polytron homogenizer (Brinkmann, Inc., Westbury, NY) at a speed setting of 3 for three 5-sec bursts, and centrifuged at 30,000 x g (Beckman J2-21) for 30 min at 4C. Acetic acid was chosen because it extracts the highest tissue level of ir-MENK, as compared to HCI (0.1 N) or phosphate buffer saline-EDTA (0.1 M) (6). The supernatant was lyophilized using a Labconco benchtop lyophilizer (Labconco Corp., Kansas City, MO) and reconstituted with deionized water (d.w.). Reconstituted cell extracts were centrifuged at 10,000 x g (Beckman J2-21) for 20 min at 4C, and filtered through 0.80 and 0.22 pm filters. The resulting filtrates were used for chromatographic characterization of ir-MENK. Chromatographic analysis: Ir-MENK present in the reconstituted extract of 40C cells was chromatographically characterized as previously reported (5). Reconstituted extract was applied to a Sephadex G-15 column (58 x 1.6 cm) to determine the molecular weight (tool wt) of ir-MENK. The Sephadex G-15 column was precalibrated with blue dextran (void volume, Vo), MENK (mol wt = 0.58 KD, Kav = 1.07), MENKArg-Phe (0.88 KD, Kav= 0.68), MENK-Lys (0.73 KD, Kav= 0.98), and Des-Tyr 1-MENK (0.41 KD, Kav= 1.75) (Peninsula Laboratories Inc., Belmont, CA). These peptides were eluted from the column with 0.1 N acetic acid containing 0.1% BSA and 0.01% sodium azide. Fractions (2 ml) were collected, oxidized overnight with 1% H202, lyophilized, reconstituted in appropriate volumes of radioimmunoassay (RIA) buffer (11), and used for the measurement of ir-MENK by RIA.

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51, No.

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1992

Ir-MENK

and P o r c i n e

Endometrial

Cell L i n e

1983

To further characterize the ir-MENK identified in the gel filtration profiles, hydrophobicity of ir-MENK was compared to that of synthetic MENK-related peptides. Fractions of ir-MENK coinciding with synthetic MENK were pooled, lyophilized, reconstituted with d.w., filtered, and applied to a Waters i~Bondapak C18 column (0.39 x 30 cm) for reverse phase-high performance liquid chromatography (RP-HPLC) analysis. The HPLC system included two M510 pumps, a U6K sample injector, a M481 absorbance detector, an automated gradient controller, M740 data modules (Waters Associate, Milford, MA), and a FRAC-100 fraction collector (Pharmacia LKB, Inc., Piscataway, NJ). The column was equilibrated with 0.1% trifluoroacetic acid and washed with HPLC grade H20 for 5 min. A linear gradient was initiated with a rate of 2% acetonitrile/min for 30 min, followed by a linear gradient of 4%/min for 5 min. The flow rate was 2 ml/min. Fractions (1 ml) were collected, lyophilized, reconstituted with d.w., oxidized overnight with 1% H202, after which RIA buffer was added and fractions assayed for MENK by RIA. Each extract sample was analyzed in triplicate by gel filtration and RP-HPLC. The recovery rates of ir-MENK from gel filtration and RP-HPLC were 84.6 + 5.3% and 80.1 + 8.1%, respectively. Loss of ir-MENK in the column chromatographies may have been partly due to the fact that some fractions contained ir-MENK below the sensitivity limit of MENK RIA used (4-8 pg).

Temperature sensitivity in ir-MENK synthesis: To determine whether the synthesis of ir-MENK in PE-1 cells is temperature sensitive, the content of ir-MENK in 33C cells and 40C cells was measured by modification of a previously described method (12). After removal of medium, PE-1 cells were dissociated from the flask. Cell suspensions were pooled, thoroughly mixed, and distributed in equal volumes into 72 culture flasks (75 cm2). Flasks were randomly assigned into three groups, and incubated at either 33C or 40C, or shifted to 33C after 7 days of incubation at 40C. Cells from two flasks of each group were collected and extracted as aforementioned. Culture medium in the remaining culture flasks was replaced daily with (~-MEM-4. Secretion of ir-MENK by PE-1 cells: The effects of KCI depolarization on the secretion of ir-MENK by PE-1 cells was investigated. The first experiment was designed to determine the optimal concentration of KCI needed for the stimulation of ir-MENK release. Sterile KCI solution was prepared by dissolving KCI powder in 0.9% NaCI saline. Ultrafiltered KCI solution was added to 40C cells to achieve final KCI concentrations of 5, 15, 30, or 60 mM, and cells were incubated at 40C for a period of 60 min. Culture medium was then collected, centrifuged to remove cellular debris, ultrafiltered with PM10 membrane, and lyophilized. The 40C cells were isolated by trypsin treatment, centrifuged, extracted with 0.1 N acetic acid, homogenized, and lyophilized. The lyophilized medium or cell extract was reconstituted with d.w., oxidized overnight with H20 2, and used for measurement of ir-MENK levels. The control group was incubated for the same time period, except that only ultrafiltered saline was added. After the optimal KCI concentration was determined, the next experiment undertaken was to study the temporal kinetics of ir-MENK release from PE-1 cells utilizing the optimal KCI concentration. PE-1 cells were grown under the same conditions as for the first experiment. KCI was added to flasks to achieve a final concentration of 30 mM. Cell culture medium was collected after 15, 30, 45, 60, 90, or 120 min of incubation in the presence of KCI. After incubation, cells and medium were collected and processed as described for the first experiment and used for the measurement of ir-MENK levels. Cells incubated with only saline for the same time intervals were used as control. Experiments were conducted twice in triplicate.

1984

Ir-MENK and Porcine Endometrial Cell Line

240 i

~

A. Sephadex G-15

Vol.

51, No. 25, 1992

MENK

200 160

~ 120 8o

0

.

I

I

22

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26

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13 17 21 25 29 33 37 41 45 49 53 Fraction #

Fig. 1. (A) Sephadex G-15 chromatographic profile of ir-MENK in 40C cells. Arrows indicate the fractions (2 ml each) in which blue dextran (Vo) or synthetic MENK eluted. The major peak, which accounted for 74.7% of the total ir-MENK applied to the column, was eluted at a position similar to that of synthetic MENK. (B) Fractions of ir-MENK co-eluting with synthetic MENK in Sephadex G-15 gel filtration were pooled and fractionated by RP-HPLC. A chromatographic profile of RP-HPLC revealed that a major portion (81.5%) of the total ir-MENK applied possessed a hydrophobicity similar to synthetic MENK. Arrows indicate fractions (1 ml each) in which Vo or synthetic MENK eluted. MENK RIA: The RIA for MENK was performed using rabbit anti-MENK sulfoxide #25. This antiserum has been shown to be highly specific to MENK and other MENKrelated peptides (13). On a mass basis, it cross-reacts with MENK-Lys, MENK-ArgPhe, and MENK-Arg-Gly-Leu at 53%, 45% and 42%, respectively. The sensitivity of the assay was 4-8 pg/tube. Since the antiserum detects the oxidized form of MENK, aliquots of samples were oxidized with 1% H20 2 prior to the assay. The inter-and intra-assay coefficients of variation, as determined by measuring PE-1 cell extract, were 13.6% and 7.2%, respectively. RIA procedures were those described by Kumar et al. (14). This MENK RIA system has been used for the identification of ir-MENK in the porcine uterus (5), and rabbit uterus, in viv0 and in vitro (6).

Vol.

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1992

I r - M E N K and P o r c i n e E n d o m e t r i a l

Cell L i n e

1985

Statistical analysis: Results were analyzed by least squares analysis of variance using the General Linear Models procedure of the Statistical Analysis System (15). Data from KCI stimulation on ir-MENK release were expressed as a percentage of the control group (100%) and then analyzed by ANOVA. Additional multiple comparisons were performed using the Student-Newman-Keuls test to detect the depolarization effects of KCI on the release of ir-MENK into cell culture medium and on the cell content of ir-MENK. Results from the temporal kinetics of KCl on PE-1 cells were expressed as a percent change of control. Data were analyzed by T-test to assess the effect of 30 mM KCl on the temporal release of ir-MENK. A difference of p < 0.05 was considered statistically significant. Results

Chromatographic analysis of ir-MENK in PE-1 cells: Sephadex G-15 profiles of PE-1 cell extract displayed 2 peaks of ir-MENK. The major peak, which accounted for 74.7% of the total ir-MENK applied to the column, eluted at a position similar to that of synthetic MENK. A minor peak coinciding with Vo was also observed (Fig. 1A). Fractions of ir-MENK co-eluting with synthetic MENK were pooled and analyzed by RP-HPLC, which revealed a major portion (81.5%) of the ir-MENK from the Sephadex G-15 fractions also possessed a hydrophobicity similar to synthetic MEN K (Fig. 1B). 100 -~ N

==

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Days Fig. 2. Concentration of ir-MENK in 40C cells was higher and steadier than that of 33C cells, suggesting that synthesis of ir-MENK in PE-1 cells is also temperature-sensitive. A decline in ir-MENK concentration was observed for 40C cells when shifted back to 33C.

Synthesis of ir-MENK in PE-1 cells: The synthesis of ir-MENK in PE-1 cells cultured at the permissive and nonpermissive temperatures was examined. Over a period of 14 days, 40C cells had a higher rate of ir-MENK synthesis per million cells than 33C cells, and maintained a steadier levels of ir-MENK (Fig. 2). KCI Depolarization Effect: KCI depolarization of PE-1 cells produced a general stepwise increase in the release of ir-MENK into culture medium as cells were incubated with increasing concentrations of KCI, from 5 to 30 mM for 60 rain. It is found that the optimal concentration of KCI required to stimulate ir-MENK release from PE-1 cells was 30 mM. Further increase of KCI (60 mM) caused a reduction of ir-MENK release,

1986

I r - M E N K and P o r c i n e E n d o m e t r i a l

Cell L i n e

500450400350300250-

C

Vol.

51, No.

25, 1992

C

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t..

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KC! Concentration (mM) Fig. 3. Secretion of ir-MENK in response to KCI depolarization. KCI solution was added to 40C cells to achieve final concentrations of 5, 15, 30, or 60 mM, while cell cultures devoid of KCI served as controls. Cells were incubated for 60 min, after which the medium was collected, processed, and measured for ir-MENK. Data were expressed as the mean + SEM for 6 flasks in each group. Different letters on top of each bar represent significant difference at p < 0.05 level. 110-

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Vol.

51, No.

25,

1992

I r - M E N K and P o r c i n e E n d o m e t r i a l

Cell Line

1987

as compared with the optimal concentration (Fig. 3). The majority (>90%) of PE-1 cells were viable at the end of KCl-treatment, as was confirmed by the trypan blue exclusion method. The cells revealed a decreasing trend in the ir-MENK content with increasing KCl concentration, but a significant decrease in cell content was observed only in cells treated with 30 (72.4 + 6.7%) and 60 mM KCI (70.5 + 8.2%) (Fig. 4). 600[ ] Control 500. [ ] KCI * u m t... 400iiiiiiiiiiiiii iiiiiii!iiiiiiii~i iiiiii!i!iiiiii!i 300. ,

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Time (rain) Fig. 5. Secretion of ir-MENK from PE-1 cells depolarized with 30 mM in KCI over different time intervals. KCl solution was added to 40C cells to achieve a final concentration of 30 mM, while cell cultures devoid of KCl served as controls. Cells were incubated for 15, 30, 45, 60, 90, or 120 min, after which the medium was collected, processed, and measured for ir-MENK. Data are expressed as the mean + SEM for 6 flasks in each group. Significant effects of 30 mM KCl over time (p < 0.05) vs. control are indicated by asterisks (*). Using 30 mM KCl to examine the temporal kinetics of ir-MENK release revealed that incubation of PE-1 cells for 15 to 60 min resulted in a step-wise elevation of ir-MENK release. Compared to controls, ir-MENK release was significantly increased during 30 min, 45 min, and 60 min. Incubation of PE-1 cells for longer times did not further increase ir-MENK release (Fig. 5). With increasing incubation time, the cell content of ir-MENK showed a decreasing trend in the ir-MENK content. A significant decrease in cell content was observed in cells incubated for 60 min, 90 min, and 120 min, as compared to controls (Fig. 6). Discussion In this study, we have used Sephadex G-15 gel filtration chromatography combined with RP-HPLC and MENK RIA to identify the presence of ir-MENK in the medium and cell extract of PE-1 cells. The majority (74.7 %) of ir-MENK in PE-1 cell extracts eluted at a position similar to that of synthetic MENK. RP-HPLC analysis further revealed that a major portion (81.5%) of ir-MENK from the Sephadex G-15 fractions also possessed a hydrophobicity identical to synthetic MENK. This is in agreement with the biochemical characteristics of ir-MENK identified in porcine endometrial tissues (5), suggesting that the synthesis and post-translational processing of proenkephalin in PE-1 cells are similar to ~ viv0 porcine endometrial tissues. A parallel observation has also shown that the majority of ir-MENK present in the rabbit uterus and a ts-

I r - M E N K a n d P o r c i n e E n d o m e t r i a l Cell L i n e

1988

Vol.

51, No. 25, 1992

SV40-transformed rabbit endometrial cell line (HRE-H9) possessed a mol wt and hydrophobicity similar to those of synthetic MENK (6).

[] 110100-

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Time (min) Fig. 6. Content of ir-MENK in PE-1 cells depolarized with 30 mM in KCl over different time intervals. KCl solution was added to 40C cells to achieve final concentration of 30 mM, while cell cultures devoid of KCl served as controls. Cells were incubated for 15, 30, 45, 60, 90, or 120 min, after which the cells were collected, extracted, and measured for ir-MENK. Data are expressed as the mean + SEM for 6 flasks in each group. Significant effects of 30 mM KCl over time (p < 0.05) vs. control are indicated by asterisks (*). In all three experimental groups, there was a decline in the content of cellular ir-MENK over a 14-day incubation period, possibility due to the end-product feedback inhibition of ir-MENK synthesis. Compared with 33C cells, 40C cells were able to maintain higher and steadier levels of ir-MENK. This observation suggests that at the nonpermissive temperature (40C), PE-1 cells have a higher metabolic rate than cells grown at the permissive temperature (33C). Several cell lines established by ts-SV40 transformation possess temperature sensitivity in their metabolic functions. The synthesis of l~-endorphin in a rabbit HRE-H9 cell line was higher at 40C (12). Similarly, synthesis of keratin in ts-SV40-transformed human epidermal cell (16), and production of o~-fetoprotein, albumin, and transferrin in transformed liver cells (17) were significantly increased at 40C. On the other hand, in a ts-SV40-transformed rat granulosa cell line, gene expression of insulin-like growth factor I was slightly higher at 330 (18). Expression of ornithine decarboxylase activity was higher at 33C in a human endometrial stromal cell line transfected by ts-SV40 DNA (19). Upon depolarization by KCl, PE-1 cells secreted ir-MENK in a dose responsive manner, indicating that ir-MENK present in culture medium was a secretory product rather than a substance which may have leaked through disrupted membranes of dead cells. The response to KCl stimulation is also indicative that organelles involved in secretory actions are functional in PE-1 cells. The optimal concentration of KCl used in this study (30 raM) was higher than KCl concentration used for the induction of

Vol.

51, No.

25,

1992

I r - M E N K and P o r c i n e E n d o m e t r i a l

Cell Line

1989

ir-MENK secretion from rabbit HRE-H9 cells (15 raM) (6), but lower than concentrations used for testing in vitro viability of neural tissues (56-60 mM) (20, 21). Although ir-MENK secretion was significantly augmented at KCI concentration as low as 5 mM, cell content of ir-MENK was not significantly reduced until higher doses were used (30-60 mM). This finding, combined with the fact that ir-MENK concentration is approximately 100-fold higher in the cell content (56.4 pg / mg protein) than in the medium (0.79 pg / mg protein), suggests that only a minor portion of ir-MENK is secreted in response to KCI stimulation. Depolarization with 60 mM KCI for 60 min resulted in an increase of ir-MENK secretion which was less than that with 15 and 30 mM. Interestingly, the total cell content of ir-MENK in the 60 mM group was the lowest among all the doses tested. It is likely that long time incubation of 40C cells with high KCI concentration may hinder the ir-MENK synthesizing processes and/or cause some cell death. Another possibility is that incubation with high KCI concentrations increases metabolism of MENK. As a consequence, in the high-dose group (60 mM) there may be less ir-MENK in the 40C cells available for secretion. A similar effect was shown in rabbit HRE-H9 cells treated with 30-60 mM KCI for 3-6 hr. In the temporal study, ir-MENK was significantly secreted beginning 30 min after stimulation, while prolonged incubation (90-120 min) did not induce further secretion. However, cell content of ir-MENK was inversely proportional to the incubation period, suggesting that synthesis of ir-MENK in PE-1 cells may be disturbed by long term, high salt conditions. Presently, it is not clear what physiological role(s) MENK plays in the porcine uterus. EOP have been implicated to modulate immune responses (see 22, 23 for review). Both ~-endorphin and MENK have been identified in the porcine uterus and uterine secretion (2, 4, 5). An involvement of EOP in modulating the local immune response of the uterine environment is possible and worth studying. Cell lines transformed by temperature-sensitive SV40 have been used for studies of the human placenta (24), rat ovary (18), and the macrophage (25), and other tissues such as the liver (see 26 for review). It appears that the PE-1 cell line is an ideal model for the study of the mechanisms of action of endometrial EOP production and release. In addition, its secretory products under different hormonal conditions may be used for functional analysis, such as in vitro lymphocyte activity testing. AcknowledQments The authors thank Dr. M.S.A. Kumar for providing MENK sulfoxide antiserum, and Dr. D.C. Ferguson for the use of HPLC instruments.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

T. WAHLSTROM, T. LAATIKAINEN, K. SALMINEN, and J. LEPPALUOTO, Life Sci. 3__66987-990 (1985) W.I. LI, H. WU, and C.L. CHEN, Biol. Reprod. 46740-746 (1992) F. PETRAGLIA, F. FACCHINETTI, K. M'FUTA, M. RUSPA, J.J. BONAVERA, F. GANDOLFI, and A.R. GENAZZANI, Fertil. Steril. 46247-251 (1986) W.I. LI, C.L. CHEN, P.J. HANSEN, and F.W. BAZER, Endocrinology 12111111115 (1987) W.I. LI, L.C. SUNG, and F.W. BAZER, Endocrinology 12821-26 (1991) W.I. LI, H. WU, and A.M. KUMAR, Biol. Reprod. 4_5_691-697 (1991) D.F. JIN, K.E. MUFFLY, W.C. OKULICZ, and D.L. KILPATRICK, Endocrinology 1221466-1471 (1988)

1990

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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Cell L i n e

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51, No. 25,

1992

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