Saponin Effects of Prolactin-Like Stimulation of Ornithine Decarboxylase Activity in Mouse Mammary Gland Explants P. B. Koduri and J. A. Rillema Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan, U.S.A.
Saponin, a naturally occurring plant glycoside, was found to elicit a prolactin-like stimulation of ornithine decarboxylase (ODC) activity in mouse mammary gland explants. A dose-response activation of ODC was observed with saponin at concentrations between 2 and 10(j.g/ ml. At concentrations of 10 and 15 ug/ml, saponin effected a response similar to that of PRL; when tested in concert, PRL and saponin caused a nonadditive response. The timecourse of the saponin and PRL effects on ODC activation were not different; a maximum response occurred 2 - 4 hours after addition of saponin. The saponin and PRL responses were abolished by antibiotics (puromycin and cyclohexamide) that inhibit protein synthesis, but not by actinomycin D which inhibits RNA synthesis. Finally, saponin, by itself, did not affect the rate of milk product formation, but at higher concentrations (above 0.5 ug/ml) impaired the PRL stimulation of lipid and casein synthesis. Key words Saponin - Prolactin - Mouse Mammary Glands - Ornithine Decarboxylase - Casein - Lipids
Introduction Saponins are naturally occurring glycosides, predominately of plant origin, that are well-known for their foaming and hemolytic activities. Saponin is commonly used to permeabilize cell membranes for introducing certain molecules into cells that are not normally transported intracellularly (Wassler, Jonasson, Perrson and Frier 1987). This effect seems to be due chiefly to its property to form complexes with cholesterol. During the course of studies designed to determine the optimal concentrations of saponin needed for permeabilization of mammary cells, the observations included in this report evolved. These observations may provide some insight into the mechanism by which prolactin expresses its effects on target cells. Materials and Methods Midpregnant (10-14 days of pregnancy) Swiss Webster mice were used in all experiments; they were purchased from Horm. metab. Res. 24 (1992) 562-564 © Georg Thieme Verlag Stuttgart • New York
Harlan Laboratories, Inc. (Indianapolis, IN). Ovine PRL was a gift from NIAMDD. Other materials were obtained from the following sources: Cortisol from Charles Pfizer and Co. (New York, NY); Hank's balanced salt solution and medium 199-Earle's salts, from Gibco Laboratories (Grand Island, NY); bovine insulin, penicillin, and streptomycin from Eli Lilly Co. (Indianapolis, IN); actinomycin D, puromycin and cyclohexamide from Sigma Chem. Co. (St. Louis, MO); [l-14C]-omithine (52.5mCi/mmol), hyamine hydroxide, [4,5-3H]-leucine (50.0 Ci/ mmol) and [14C]-acetate (1.9mCi/mmol) from New England Nuclear Corp.; saponin from Sigma Chem. Co. (St. Louis, MO). Mice were killed by cervical dislocation, and the caudal pair of mammary glands was removed and placed in medium 199 Earle's salts. Explants (3-5 mg each) were then prepared as described in detail earlier (Rillema 1973). The glands were cut into pieces weighing about 3 mg each and placed on siliconized lens paper floating on medium 199. When the effect of saponin and/or PRL on ODC activity was to be determined explants were placed on siliconized lens paper floating on 2 ml medium 199 Earle's salts containing 1 ug/ml insulin and 10-7 M Cortisol. Explants were randomly distributed among treatment combinations. All incubations were carried out in polypropylene vials maintained at 37 °C in an atmosphere of 95 % O2, 5 % CO2. After a 1 day culture, the tissues were preincubated with saponin for 1 hour and PRL (1 ug/ml) was added subsequently. Incubation was continued for an additional 3 hours. In order to study the time-course for saponin stimulation of ODC activity, tissues were incubated with saponin (10 ug/ml) for times from 0-10 hr. To study the effects of metabolic inhibitors, tissues were incubated with the antibiotics, saponin and/or PRL for 4 hours. The tissues were next homogenized in 50 mM Tris buffer (pH 7.4) and ODC activity was determined using a modification (Rillema 1976) of the methods described by Janne and Williams-Ashman (1971). ODC activity was expressed as DPM/l0 mg wet tissue weight. When the effects of saponin on PRL stimulation of casein synthesis were to be determined, explants were cultured for 30 hours with Media 199 containing insulin (1 ug/ml), Cortisol 10-7 M and saponin. PRL (1 ug/ml) was then added to the medium and incubations were continued for 16 hours. [3H]-leucine (0.5uCi/ml) was added to the media 2 hours prior to termination of the incubations. The rate of [3H]-leucine incorporation into a casein rich protein fraction was used as an index of the rate of casein synthesis. Methods used to isolate and quantitate the labelled macromolecules were as described earlier (Rillema 1973). Results were expressed as DPM [3H]-leucine incorporated /mg wet tissue weight. When the effects of saponin on the PRL stimulation of lipid synthesis were to be determined, the explants were first cultured for 36 hours with medium 199 containing insulin (1 ug/ml) and Cortisol (10-7M). PRL (1 ug/ml) and saponin (0-10 ug/ml) were then added to the culture medium and incubations continued for 18 hours. In the final 2 hours of incubation, tissues were pulse labeled with [14C]acetate. The tissues were then weighed and homogenized in 2 ml dH20 and the lipids were extracted by the method of Bligh and Dyer (1959). RadioReceived: 12 Dec 1991
Accepted: 25 March 1992
Downloaded by: Universite Laval. Copyrighted material.
Summary
Horm. metab. Res. 24 (1992)
Fig. 1 Effect of saponin on prolactin stimulation of ODC activity. Explants were cultured for 24 hr in the presence of 1 u,g/ml insulin plus 10-7 M Cortisol. Explants were then incubated with saponin (0-15|ig/ml) for 1 hr. PRL (1 ug/ml) was added to certain vials and incubation was continued for 3 hr. ODC activity was determined as described in the text. Numbers represent the mean ±SE of 6 observations. 'Significantly greater than control (p
Saponin, a naturally occurring plant glycoside, was found to elicit a prolactin-like stimulation of ornithine decarboxylase (ODC) activity in mouse mammary gland explants. A dose-response activation of ODC was observed with saponin at concentrations between 2 and 10(j.g/ ml. At concentrations of 10 and 15 ug/ml, saponin effected a response similar to that of PRL; when tested in concert, PRL and saponin caused a nonadditive response. The timecourse of the saponin and PRL effects on ODC activation were not different; a maximum response occurred 2 - 4 hours after addition of saponin. The saponin and PRL responses were abolished by antibiotics (puromycin and cyclohexamide) that inhibit protein synthesis, but not by actinomycin D which inhibits RNA synthesis. Finally, saponin, by itself, did not affect the rate of milk product formation, but at higher concentrations (above 0.5 ug/ml) impaired the PRL stimulation of lipid and casein synthesis. Key words Saponin - Prolactin - Mouse Mammary Glands - Ornithine Decarboxylase - Casein - Lipids
Introduction Saponins are naturally occurring glycosides, predominately of plant origin, that are well-known for their foaming and hemolytic activities. Saponin is commonly used to permeabilize cell membranes for introducing certain molecules into cells that are not normally transported intracellularly (Wassler, Jonasson, Perrson and Frier 1987). This effect seems to be due chiefly to its property to form complexes with cholesterol. During the course of studies designed to determine the optimal concentrations of saponin needed for permeabilization of mammary cells, the observations included in this report evolved. These observations may provide some insight into the mechanism by which prolactin expresses its effects on target cells. Materials and Methods Midpregnant (10-14 days of pregnancy) Swiss Webster mice were used in all experiments; they were purchased from Horm. metab. Res. 24 (1992) 562-564 © Georg Thieme Verlag Stuttgart • New York
Harlan Laboratories, Inc. (Indianapolis, IN). Ovine PRL was a gift from NIAMDD. Other materials were obtained from the following sources: Cortisol from Charles Pfizer and Co. (New York, NY); Hank's balanced salt solution and medium 199-Earle's salts, from Gibco Laboratories (Grand Island, NY); bovine insulin, penicillin, and streptomycin from Eli Lilly Co. (Indianapolis, IN); actinomycin D, puromycin and cyclohexamide from Sigma Chem. Co. (St. Louis, MO); [l-14C]-omithine (52.5mCi/mmol), hyamine hydroxide, [4,5-3H]-leucine (50.0 Ci/ mmol) and [14C]-acetate (1.9mCi/mmol) from New England Nuclear Corp.; saponin from Sigma Chem. Co. (St. Louis, MO). Mice were killed by cervical dislocation, and the caudal pair of mammary glands was removed and placed in medium 199 Earle's salts. Explants (3-5 mg each) were then prepared as described in detail earlier (Rillema 1973). The glands were cut into pieces weighing about 3 mg each and placed on siliconized lens paper floating on medium 199. When the effect of saponin and/or PRL on ODC activity was to be determined explants were placed on siliconized lens paper floating on 2 ml medium 199 Earle's salts containing 1 ug/ml insulin and 10-7 M Cortisol. Explants were randomly distributed among treatment combinations. All incubations were carried out in polypropylene vials maintained at 37 °C in an atmosphere of 95 % O2, 5 % CO2. After a 1 day culture, the tissues were preincubated with saponin for 1 hour and PRL (1 ug/ml) was added subsequently. Incubation was continued for an additional 3 hours. In order to study the time-course for saponin stimulation of ODC activity, tissues were incubated with saponin (10 ug/ml) for times from 0-10 hr. To study the effects of metabolic inhibitors, tissues were incubated with the antibiotics, saponin and/or PRL for 4 hours. The tissues were next homogenized in 50 mM Tris buffer (pH 7.4) and ODC activity was determined using a modification (Rillema 1976) of the methods described by Janne and Williams-Ashman (1971). ODC activity was expressed as DPM/l0 mg wet tissue weight. When the effects of saponin on PRL stimulation of casein synthesis were to be determined, explants were cultured for 30 hours with Media 199 containing insulin (1 ug/ml), Cortisol 10-7 M and saponin. PRL (1 ug/ml) was then added to the medium and incubations were continued for 16 hours. [3H]-leucine (0.5uCi/ml) was added to the media 2 hours prior to termination of the incubations. The rate of [3H]-leucine incorporation into a casein rich protein fraction was used as an index of the rate of casein synthesis. Methods used to isolate and quantitate the labelled macromolecules were as described earlier (Rillema 1973). Results were expressed as DPM [3H]-leucine incorporated /mg wet tissue weight. When the effects of saponin on the PRL stimulation of lipid synthesis were to be determined, the explants were first cultured for 36 hours with medium 199 containing insulin (1 ug/ml) and Cortisol (10-7M). PRL (1 ug/ml) and saponin (0-10 ug/ml) were then added to the culture medium and incubations continued for 18 hours. In the final 2 hours of incubation, tissues were pulse labeled with [14C]acetate. The tissues were then weighed and homogenized in 2 ml dH20 and the lipids were extracted by the method of Bligh and Dyer (1959). RadioReceived: 12 Dec 1991
Accepted: 25 March 1992
Downloaded by: Universite Laval. Copyrighted material.
Summary
Horm. metab. Res. 24 (1992)
Fig. 1 Effect of saponin on prolactin stimulation of ODC activity. Explants were cultured for 24 hr in the presence of 1 u,g/ml insulin plus 10-7 M Cortisol. Explants were then incubated with saponin (0-15|ig/ml) for 1 hr. PRL (1 ug/ml) was added to certain vials and incubation was continued for 3 hr. ODC activity was determined as described in the text. Numbers represent the mean ±SE of 6 observations. 'Significantly greater than control (p
