Molecular and Cellular Endocrinology 4 (1976) 131-137 0 North-Holland Publishing Company
RAPID INTERACTION EXPLANTS James A. RILLEMA
OF PROLACTIN
WITH MOUSE MAMMARY GLAND
and Larry D. ANDERSON
Department of Physiology, Michigan 48201, U.S.A.
Wayne State University, School of Medicine, Detroit,
Received 8 September 1975; accepted 28 October 1975
Exposure of mouse mammary gland explants to prolactin at O”C, for periods as brief as 10 seconds, caused a stimulation of labeled uridine incorporation into RNA during a subsequent incubation for 4 h at 37°C. Furthermore, a 2-h wash of the prolactinexposed explants in media at 0°C did not attenuate the hormonal effect. A similar exposure of explants to insulin, followed by a 2-h wash at O”C, caused the abolition of the insulin stimulation of labeled uridine incorporation into RNA. These results suggest that there is a rapid and relatively stable interaction of prolactin with the mammary gland, while the interaction of insulin with this tissue would appear to be less stable. Keywords:
prolactin; insulin; mammary gland; RNA synthesis.
Prolactin and insulin have a number of rapid actions on regulating the metabolism of the mammary gland (Turkington et al., 1973; Topper and Oka, 1974). Included among these is the stimulation of labeled uridine into RNA by both of these hormones. The effect of insulin on uridine metabolism occurs within minutes following exposure of mammary gland explants to this hormone (Rillema, 1975a). In contrast, the prolactin stimulation of uridine metabolism is only demonstrable 34 h following the addition of prolactin to medium bathing mammary gland explants (Rillema, 1973, 1975b). The present studies were designed to study the nature plants
of the early interaction of insulin and prolactin with mammary of mice. Specifically, experiments were carried out to determine
hormonal explants
effects which
MATERIALS Porcine
on labeled
are exposed
uridine to prolactin
incorporation or insulin
gland exwhether
into RNA are demonstrable
in
at 0°C for brief periods.
AND METHODS insulin
(Lot PJ 5682)
was a gift from the Eli Lilly Company. 131
Ovine pro-
132
J.A. Rillema, L.D. Anderson
lactin (NIHP-S-9) was a gift from the National Institutes of Arthritis and Metabolic Diseases. Other hormones and chemicals were purchased from the following sources: Hydroco~isone from Chas. Pfizer and Co.; Nystatin from E.R. Squibb and Sons kc.; Medium 199, ~njciIlin and streptomyc~~ from ~i~~~~i~~~g~~a~ Associates Inc.; ]5-3Hj uridine (27.8 Cifmmole) from the New England Nuclear Corp. Midpregnant (lo-14 day) Swiss-Webster mice were purchased from Spartan Research Animals Inc., Haslett, MI. In these studies mammary gland explants were prepared and incubated by procedures described in a detailed manner in an earlier publication (Rillema, 1973). Briefly, 3-5 mg explants were initially prepared. In each incubation vessel 3 explants were then placed on siliconized lens paper floating on 2 ml Medium 199. When the effects of brief exposures to prolactin were to be studied, the explants were preincubated for 2 days at 37°C with medium containing 2.5 yg/ml insulin and 2.5 pg/ ml hydrocortisone. The vessels containing the explants were then placed on ice. After cooling, the explants were immersed for 10 s or I min in media at 0°C containing insulin, hydrocortisone and 0, 50 or 250 ngfml prolactin. The explants were then blotted on filter paper and placed in 25 m1 ice-cold Medium 199 containing insulin plus hydrocortisone, but no prolactin. The explants were placed in fresh, icecold 25ml volumes of Medium 199 after 5, 10, 15, 30 and 60 min. After 2 h at O”C, the explants were again placed on siliconized iens paper floating on Medium 199 containing insulin, hydro~ortisone, and, in certain cases, 50 ng/ml prolactin. When the rate of 13H] uridine incorporation into RNA was to be studied, the tissues were then incubated at 37°C for a 4-h period. [3H] Uridine (0.5 &i/ml) was added to the media 30 min prior to termination of the incubations. The rate of [3H] uridine incorporation into RNA was then determined by methods employed eariier (Rillema, 1973). When the rate of [3H] leucine incorporation into casein was to be measured; the tissues were in~nbated at 37OC for a 10-h period following the washes at 0°C. [3H] Leucine (1 &i/ml) was added to the media 2 h prior to termination of the incubations. The amount of (3H]leucine incorporated into a caseinrich protein fraction was then determined by methods previously reported (Rillema, 1973). Experiments similar to those described above were also carried out with insulin. Modifications in the experimenta protocol were as follows. Explants were prepared and preincubated for 1 day in Medium 199 containing no hormones. The explants were then immersed for 1 nun in media at 0°C containing no or 2.5 Erg/ml insulin. Then, following the washes in 25ml volumes of Medium 199 at O”C, one set of explants, not previously exposed to insulin, was immersed for I min in the media at O’C containing 2.5 +&ml insulin. All explants were then blotted on filter paper and placed on siliconized lens paper floating on Mediu~?~ 199 containing 0.5 #X/ml [3H] uridine and, in certain cases, 2.5 pg/ml insulin. Incubation was then carried out at 37°C for 1 h and the amount of [“H] uridine incorporated into RNA was subsequently determined. Statistical differences were determined by an analysis of variance.
133
Prolactin and the mammary gland
RESULTS Table 1 shows the results of experiments in which the effect of prolactin on labeled uridine incorporation into RNA was tested in explants exposed for brief periods of time to prolactin. In preliminary experiments 50 rig/ml prolactin was established as the minimal hormonal concentration with which consistent effects on uridine incorporation into RNA were observed during a 4-h incubation period at 37°C. Exposure of explants for 1 min or 10 s at 0°C to this concentration of prolactin produced effects comparable to that observed when prolactin was present for the entire 4-h incubation period at 37°C (table 1). Further, the fact that the explants were washed for 2 h at 0°C in large volumes of Medium 199 prior to incubation at 37”C, suggests that the interaction of prolactin with mammary tissue probably involves a high affinity association. Table 2 shows the effect of brief exposures to prolactin on labeled leucine incorporation into casein. In contrast to the effects observed on uridine incorporation, brief exposure of explants to prolactin at O’C did not cause a stimulatory effect on leucine incorporation into casein. This was observed even when explants were exposed to prolactin concentrations as high as 2.5 pg/ml. It must be noted, however, that the effect of prolactin on casein synthesis only becomes demonstrable after a 8-10 h incubation period at 37”C, while the uridine incorporation effect becomes apparent much earlier, i.e. after a 3-4 h incubation period at 37°C. Experiments similar to those described above were carried out with regard to the insulin stimulation of labeled uridine incorporation into RNA. Table 3 contains the results of these studies. In contrast to prolactin, exposure of explants to insulin for
Table 1 13H] Uridine incorporation into RNA in mammary gland explants exposed to prolactin for 1 min or 10 s at 0°C. Explants were preincubated for 2 days with medium containing insulin plus hydrocortisone. The tissues were then cooled to 0°C after which certain of the tissues were exposed to prolactin at 0°C for 1 min or 10 s. All tissues were then blotted on filter paper and washed in 25ml volumes of media at 0°C; the tissues were placed in fresh media after 5, 10, 13, 30 and 60 min. After 2 h at O”C, the tissues were incubated at 37’C for a 4-h period with prolactin added to certain of the flasks. 0.5 pCi/ml [3H] uridine was added to the flasks 30 min prior to termination of the incubations. Prolactin exposure at 0°C
Prolactin added at 37°C
[ 3H] Uridine incorporation into RNA (dpm/pg RNA)
_ _ 1 min, 250 ng/ml 1 min, 50 ng/ml 10 s, 50 ng/ml
50 ng/ml -
179 275 283 252 282
-
+ + f r f
12 * 16 15 15 15
* Numbers in the table are means + standard errors of explants from 7 flasks.
J.A. Rillema, L.D. Anderson
134
Table 2 13H]Leucine incorporation into casein in mammary gland explants exposed to prolactin for 1 min at 0°C. Experimental protocol was the same as that described in table 1 with the following modifications. The final incubation period at 37°C was for a 10-h period and 1 pCi/ml [3H] leucine was present in the media for 2 h prior to termination of the incubations. Prolactin
exposure
at 0°C
_ _
added
at 37°C
_
in the table are means
+ standard
[ 3H] Leucine incorporation into casein (DPM/mg wet wt) 795 1421 895 811
50 ng/ml ._ _
50 ng/ml 2.5 pg/ml * Numbers
Prolactin
error of explants
+ * + f
33 * 75 55 52
from 7 flasks.
1 min at 0°C followed by a 2-h wash period at O”C, caused no change in the rate of labeled uridine incorporation into RNA. This occurred despite the facts that (a) a high concentration of insulin (2.5 pg/ml) was used, and (b) the insulin effect occurs more rapidly than the prolactin effect. One set of tissues was also immersed in the insulin media at 0°C immediately prior to the I-h incubation period at 37’C. The fact that an effect of insulin on labeled uridine incorporation was observed in these tissues demonstrates that an adequate amount of insulin was present in the tissues exposed to insulin prior to the 2-h wash period to cause metabolic effects.
Table 3 [ 3H] Uridine incorporation into RNA in mouse mammary gland explants exposed to insulin for I min at 0°C. Explants were preincubated for 1 day with medium containing no hormones. The tissues were then cooled to 0°C after which certain of the tissues were exposed to insulin at 0°C for 1 min. The tissues were then blotted on filter paper and washed in 25-ml volumes of media at 0°C; the tissues were placed in fresh media after 5, 10,15, 30 and 60 min. After 2 h at 0°C certain tissues (+) were exposed to insulin at O’C for 1 min and then blotted. All tissues were then incubated at 37’C for 1 h in media which contained 0.5 &i/ml [3H] uridine, and in certain cases insulin. 1-min insulin exposure
Insulin added at 37°C
[ 3H] Uridine incorporation into RNA (dpm/pg RNA)
195 319 214 272
Before 2-h 0°C wash
After 2-h 0°C wash
-
_
_
_
_ _
2.5 pg/ml _ _
+ _ * Numbers
+
in the table are means + S.E.M. of explants
from 7 flasks.
i 11 * ?- 24 i- 15 f 16
Prolactin and the mammary gland
13.5
DISCUSSION The above experiments lead us to conclude that prolactin interacts with mammary gland tissues rapidly and with a high affinity. The rapid interaction with prolactin is supported by the experiment in which a 10-s exposure of explants at 0°C to a relatively low concentration of prolactin (SO ng/ml) was found to be sufficient to produce a stimulation of uridine incorporation into RNA when these tissues were subsequently incubated for a 4-h period at 37’C. The high affinity of mammary tissues for prolactin is suggested by the fact that extensive washing of tissues exposed to prolactin for brief periods at 0°C did not attenuate the prolactin response. Kostyo and Schmidt (1961) have reported that growth hormone in muscle tissue behaves in a similar manner. Several laboratories have recently studied the binding properties of prolactin in homogenates of mammary glands from rabbits (Shiu and Friesen, 1973a,b), rats (Kelly et al., 1974) and mice (O’Keefe and Cuatrecasas, 1974). In all these species, high affinity binding sites for prolactin have been discovered; dissociation constants of l-10 X 10e9 M have been reported in most cases. The high affinity of prolactin for mammary tissues, which is suggested by the experiments in this report, is compatible with the idea that the binding sites studied by other laboratories may be the physiological receptors for prolactin. Further supporting this thesis is the observation of Shiu and Friesen (1974a) that prolactin does not dissociate from its binding site in rabbit mammary tissues when the temperature is reduced to below 4°C. Thus, the data in this report, in conjunction with the observations of other laboratories, make tenable the idea that the rapid interaction of prolactin in mammary tissues may be with its physiological receptor site. An alternative possibility, which cannot be discarded, is’ that prolactin may initially interact with mammary tissues in a nonspecific manner and, during the incubation period at 37”C, the prolactin may become available for its receptor sites. In any case, it is clear that metabolically active amounts of prolactin accumulate rapidly in mammary gland explants. Moreover, since effects of prolactin on uridine incorporation were observed after 4 h at 37’C following the brief exposure to the hormone at O”C, a sustained exposure of mammary tissues to prolactin is apparently not required in order that the metabolic effects of this hormone may become manifest. Furthermore, periodical measurements of the plasma levels of prolactin may not necessarily reflect the physiological role of this hormone on regulating the metabolism of the mammary gland or perhaps other tissues. Only occasional surges of prolactin release may be required for maximal biological effects of this hormone (Tyson et al., 1972a,b). The fact that tissues exposed for brief periods to prolactin did not stimulate leutine incorporation into casein may be a result of the extended incubation period required before enhanced casein synthesis becomes apparent. It is possible that the prolactin taken up during the brief exposure period at 0°C is in some manner inactivated during the 10-b incubation period at 37°C: the stimulation of casein synthe-
136
J.A. Riilema, L.D. Anderson
sis may, therefore, require a more frequent or perhaps a continuous delivery of the hormone to the mammary gland. The interaction of insulin with mammal tissue appears to be quite different from that of prolactin, despite the finding that the affinities of both these hormones for their respective binding’sites in this tissue are quite similar (Turkington et al., 1973; O’Keefe and Cuatrecases, 1974). An insulin effect on uridine incorporation was not apparent in tissues exposed to a high concentration of insulin (2.5 @g/ml) for I min at 0°C followed by a 2-h wash period at 0°C. That an adequate amount of insulin to produce a response was present in these tissues following the brief exposure to the insulin solution was demonstrated by the fact that an insulin response was observed in tissues immersed in the insulin-containing media immediately prior to the incubation period at 37°C. Several explanations can be offered concerning these observations. First, it is possible that the insulin binding sites are not available at O*C and that the response observed in the tissues exposed to insulin immediately prior to the 37°C incubation may be due to the insulin which acccumulated in extracellular water during the brief exposure period. Second, it is also possible that the insulin binding sites may become occupied at O’C but when the tissues are warmed to 37’C, the insulin may be released by the tissue. It is of interest that insulin is also washed out of fat and muscle tissues at temperatures higher than 0°C (Stadie et al., 1949; Crofford, 1968). Finaly, it may also be that the small amount of insulin remaining in the tissues following the 2-h wash at 0°C may be rapidly inactivated when the tissues are warmed to 37°C. In any event, it seems reasonable to conclude that biologically active quantities of insulin are rapidly taken up by mammary tissues at 0°C but that enough insulin is removed from the tissue by a 2-h wash in media at 0°C such that metabolic effects are no longer apparent during a subsequent incubation period at 37°C.
ACKNOWLEDGEMENT This investigation was supported by NIH grant number HD-06571 National Institutes of Child Health and Human Development, U.S.A.
from the
REFERENCES Crofford, O.B. (1968) J. Biol. Chem. 243,362. Kelly, P.A., Bradley, C., Shiu, R.P.C., Meites, J. and Friesen, H.G. (1974) Proc. Sot. Exp. Biol. Med. 146,816. Kostyo, J.L. and Schmidt, J.E. (1961) Am. J. Physiol. 299,675. O’Keefe, E. and Cuatrecasas, P. (1974) Biochim. Biophys. Acta 343,64. Rillema, J.A. (1973) Endocrinology 92, 1673. Rillema, J.A. (1975a) Am. J. Physiol. 228,1531. Rillema, J.A. (1975b) Endocrinology 96,1307.
Prolactin and the mammary gland
137
Shiu, R.P.C. and Friesen, H.G. (1974a) Biochem. J. 140,301. Shiu, R.P.C. and Friesen, H.G. (1974b) J. Biol. Chem. 249, 7902. Stadie, W.C., Haugaard, N., March, J.B. and Hills, A.G. (1949) Am. J. Med. Sci. 218,275. Topper, Y.J. and Oka, T. (1974) In: Lactation, Vol. 1, Eds.: B.L. Larson and V.R. Smith (Academic Press, New York) p. 327. Turkington, R.W., Majumder, G.C., Kadohama, N., MacIndoe, J.M. and Frantz, W.L. (1973) Rec. Prog. Horm. Res. 29,417. Tyson, J.E., Friesen, H.G. and Anderson, M.S. (1972a) Science 177,897. Tyson, J.E., Hwang, P., Guyda, H. and Friesen, H.G. (1972b) Am. J. Obstet. Gynecol. 113,14.
RAPID INTERACTION EXPLANTS James A. RILLEMA
OF PROLACTIN
WITH MOUSE MAMMARY GLAND
and Larry D. ANDERSON
Department of Physiology, Michigan 48201, U.S.A.
Wayne State University, School of Medicine, Detroit,
Received 8 September 1975; accepted 28 October 1975
Exposure of mouse mammary gland explants to prolactin at O”C, for periods as brief as 10 seconds, caused a stimulation of labeled uridine incorporation into RNA during a subsequent incubation for 4 h at 37°C. Furthermore, a 2-h wash of the prolactinexposed explants in media at 0°C did not attenuate the hormonal effect. A similar exposure of explants to insulin, followed by a 2-h wash at O”C, caused the abolition of the insulin stimulation of labeled uridine incorporation into RNA. These results suggest that there is a rapid and relatively stable interaction of prolactin with the mammary gland, while the interaction of insulin with this tissue would appear to be less stable. Keywords:
prolactin; insulin; mammary gland; RNA synthesis.
Prolactin and insulin have a number of rapid actions on regulating the metabolism of the mammary gland (Turkington et al., 1973; Topper and Oka, 1974). Included among these is the stimulation of labeled uridine into RNA by both of these hormones. The effect of insulin on uridine metabolism occurs within minutes following exposure of mammary gland explants to this hormone (Rillema, 1975a). In contrast, the prolactin stimulation of uridine metabolism is only demonstrable 34 h following the addition of prolactin to medium bathing mammary gland explants (Rillema, 1973, 1975b). The present studies were designed to study the nature plants
of the early interaction of insulin and prolactin with mammary of mice. Specifically, experiments were carried out to determine
hormonal explants
effects which
MATERIALS Porcine
on labeled
are exposed
uridine to prolactin
incorporation or insulin
gland exwhether
into RNA are demonstrable
in
at 0°C for brief periods.
AND METHODS insulin
(Lot PJ 5682)
was a gift from the Eli Lilly Company. 131
Ovine pro-
132
J.A. Rillema, L.D. Anderson
lactin (NIHP-S-9) was a gift from the National Institutes of Arthritis and Metabolic Diseases. Other hormones and chemicals were purchased from the following sources: Hydroco~isone from Chas. Pfizer and Co.; Nystatin from E.R. Squibb and Sons kc.; Medium 199, ~njciIlin and streptomyc~~ from ~i~~~~i~~~g~~a~ Associates Inc.; ]5-3Hj uridine (27.8 Cifmmole) from the New England Nuclear Corp. Midpregnant (lo-14 day) Swiss-Webster mice were purchased from Spartan Research Animals Inc., Haslett, MI. In these studies mammary gland explants were prepared and incubated by procedures described in a detailed manner in an earlier publication (Rillema, 1973). Briefly, 3-5 mg explants were initially prepared. In each incubation vessel 3 explants were then placed on siliconized lens paper floating on 2 ml Medium 199. When the effects of brief exposures to prolactin were to be studied, the explants were preincubated for 2 days at 37°C with medium containing 2.5 yg/ml insulin and 2.5 pg/ ml hydrocortisone. The vessels containing the explants were then placed on ice. After cooling, the explants were immersed for 10 s or I min in media at 0°C containing insulin, hydrocortisone and 0, 50 or 250 ngfml prolactin. The explants were then blotted on filter paper and placed in 25 m1 ice-cold Medium 199 containing insulin plus hydrocortisone, but no prolactin. The explants were placed in fresh, icecold 25ml volumes of Medium 199 after 5, 10, 15, 30 and 60 min. After 2 h at O”C, the explants were again placed on siliconized iens paper floating on Medium 199 containing insulin, hydro~ortisone, and, in certain cases, 50 ng/ml prolactin. When the rate of 13H] uridine incorporation into RNA was to be studied, the tissues were then incubated at 37°C for a 4-h period. [3H] Uridine (0.5 &i/ml) was added to the media 30 min prior to termination of the incubations. The rate of [3H] uridine incorporation into RNA was then determined by methods employed eariier (Rillema, 1973). When the rate of [3H] leucine incorporation into casein was to be measured; the tissues were in~nbated at 37OC for a 10-h period following the washes at 0°C. [3H] Leucine (1 &i/ml) was added to the media 2 h prior to termination of the incubations. The amount of (3H]leucine incorporated into a caseinrich protein fraction was then determined by methods previously reported (Rillema, 1973). Experiments similar to those described above were also carried out with insulin. Modifications in the experimenta protocol were as follows. Explants were prepared and preincubated for 1 day in Medium 199 containing no hormones. The explants were then immersed for 1 nun in media at 0°C containing no or 2.5 Erg/ml insulin. Then, following the washes in 25ml volumes of Medium 199 at O”C, one set of explants, not previously exposed to insulin, was immersed for I min in the media at O’C containing 2.5 +&ml insulin. All explants were then blotted on filter paper and placed on siliconized lens paper floating on Mediu~?~ 199 containing 0.5 #X/ml [3H] uridine and, in certain cases, 2.5 pg/ml insulin. Incubation was then carried out at 37°C for 1 h and the amount of [“H] uridine incorporated into RNA was subsequently determined. Statistical differences were determined by an analysis of variance.
133
Prolactin and the mammary gland
RESULTS Table 1 shows the results of experiments in which the effect of prolactin on labeled uridine incorporation into RNA was tested in explants exposed for brief periods of time to prolactin. In preliminary experiments 50 rig/ml prolactin was established as the minimal hormonal concentration with which consistent effects on uridine incorporation into RNA were observed during a 4-h incubation period at 37°C. Exposure of explants for 1 min or 10 s at 0°C to this concentration of prolactin produced effects comparable to that observed when prolactin was present for the entire 4-h incubation period at 37°C (table 1). Further, the fact that the explants were washed for 2 h at 0°C in large volumes of Medium 199 prior to incubation at 37”C, suggests that the interaction of prolactin with mammary tissue probably involves a high affinity association. Table 2 shows the effect of brief exposures to prolactin on labeled leucine incorporation into casein. In contrast to the effects observed on uridine incorporation, brief exposure of explants to prolactin at O’C did not cause a stimulatory effect on leucine incorporation into casein. This was observed even when explants were exposed to prolactin concentrations as high as 2.5 pg/ml. It must be noted, however, that the effect of prolactin on casein synthesis only becomes demonstrable after a 8-10 h incubation period at 37”C, while the uridine incorporation effect becomes apparent much earlier, i.e. after a 3-4 h incubation period at 37°C. Experiments similar to those described above were carried out with regard to the insulin stimulation of labeled uridine incorporation into RNA. Table 3 contains the results of these studies. In contrast to prolactin, exposure of explants to insulin for
Table 1 13H] Uridine incorporation into RNA in mammary gland explants exposed to prolactin for 1 min or 10 s at 0°C. Explants were preincubated for 2 days with medium containing insulin plus hydrocortisone. The tissues were then cooled to 0°C after which certain of the tissues were exposed to prolactin at 0°C for 1 min or 10 s. All tissues were then blotted on filter paper and washed in 25ml volumes of media at 0°C; the tissues were placed in fresh media after 5, 10, 13, 30 and 60 min. After 2 h at O”C, the tissues were incubated at 37’C for a 4-h period with prolactin added to certain of the flasks. 0.5 pCi/ml [3H] uridine was added to the flasks 30 min prior to termination of the incubations. Prolactin exposure at 0°C
Prolactin added at 37°C
[ 3H] Uridine incorporation into RNA (dpm/pg RNA)
_ _ 1 min, 250 ng/ml 1 min, 50 ng/ml 10 s, 50 ng/ml
50 ng/ml -
179 275 283 252 282
-
+ + f r f
12 * 16 15 15 15
* Numbers in the table are means + standard errors of explants from 7 flasks.
J.A. Rillema, L.D. Anderson
134
Table 2 13H]Leucine incorporation into casein in mammary gland explants exposed to prolactin for 1 min at 0°C. Experimental protocol was the same as that described in table 1 with the following modifications. The final incubation period at 37°C was for a 10-h period and 1 pCi/ml [3H] leucine was present in the media for 2 h prior to termination of the incubations. Prolactin
exposure
at 0°C
_ _
added
at 37°C
_
in the table are means
+ standard
[ 3H] Leucine incorporation into casein (DPM/mg wet wt) 795 1421 895 811
50 ng/ml ._ _
50 ng/ml 2.5 pg/ml * Numbers
Prolactin
error of explants
+ * + f
33 * 75 55 52
from 7 flasks.
1 min at 0°C followed by a 2-h wash period at O”C, caused no change in the rate of labeled uridine incorporation into RNA. This occurred despite the facts that (a) a high concentration of insulin (2.5 pg/ml) was used, and (b) the insulin effect occurs more rapidly than the prolactin effect. One set of tissues was also immersed in the insulin media at 0°C immediately prior to the I-h incubation period at 37’C. The fact that an effect of insulin on labeled uridine incorporation was observed in these tissues demonstrates that an adequate amount of insulin was present in the tissues exposed to insulin prior to the 2-h wash period to cause metabolic effects.
Table 3 [ 3H] Uridine incorporation into RNA in mouse mammary gland explants exposed to insulin for I min at 0°C. Explants were preincubated for 1 day with medium containing no hormones. The tissues were then cooled to 0°C after which certain of the tissues were exposed to insulin at 0°C for 1 min. The tissues were then blotted on filter paper and washed in 25-ml volumes of media at 0°C; the tissues were placed in fresh media after 5, 10,15, 30 and 60 min. After 2 h at 0°C certain tissues (+) were exposed to insulin at O’C for 1 min and then blotted. All tissues were then incubated at 37’C for 1 h in media which contained 0.5 &i/ml [3H] uridine, and in certain cases insulin. 1-min insulin exposure
Insulin added at 37°C
[ 3H] Uridine incorporation into RNA (dpm/pg RNA)
195 319 214 272
Before 2-h 0°C wash
After 2-h 0°C wash
-
_
_
_
_ _
2.5 pg/ml _ _
+ _ * Numbers
+
in the table are means + S.E.M. of explants
from 7 flasks.
i 11 * ?- 24 i- 15 f 16
Prolactin and the mammary gland
13.5
DISCUSSION The above experiments lead us to conclude that prolactin interacts with mammary gland tissues rapidly and with a high affinity. The rapid interaction with prolactin is supported by the experiment in which a 10-s exposure of explants at 0°C to a relatively low concentration of prolactin (SO ng/ml) was found to be sufficient to produce a stimulation of uridine incorporation into RNA when these tissues were subsequently incubated for a 4-h period at 37’C. The high affinity of mammary tissues for prolactin is suggested by the fact that extensive washing of tissues exposed to prolactin for brief periods at 0°C did not attenuate the prolactin response. Kostyo and Schmidt (1961) have reported that growth hormone in muscle tissue behaves in a similar manner. Several laboratories have recently studied the binding properties of prolactin in homogenates of mammary glands from rabbits (Shiu and Friesen, 1973a,b), rats (Kelly et al., 1974) and mice (O’Keefe and Cuatrecasas, 1974). In all these species, high affinity binding sites for prolactin have been discovered; dissociation constants of l-10 X 10e9 M have been reported in most cases. The high affinity of prolactin for mammary tissues, which is suggested by the experiments in this report, is compatible with the idea that the binding sites studied by other laboratories may be the physiological receptors for prolactin. Further supporting this thesis is the observation of Shiu and Friesen (1974a) that prolactin does not dissociate from its binding site in rabbit mammary tissues when the temperature is reduced to below 4°C. Thus, the data in this report, in conjunction with the observations of other laboratories, make tenable the idea that the rapid interaction of prolactin in mammary tissues may be with its physiological receptor site. An alternative possibility, which cannot be discarded, is’ that prolactin may initially interact with mammary tissues in a nonspecific manner and, during the incubation period at 37”C, the prolactin may become available for its receptor sites. In any case, it is clear that metabolically active amounts of prolactin accumulate rapidly in mammary gland explants. Moreover, since effects of prolactin on uridine incorporation were observed after 4 h at 37’C following the brief exposure to the hormone at O”C, a sustained exposure of mammary tissues to prolactin is apparently not required in order that the metabolic effects of this hormone may become manifest. Furthermore, periodical measurements of the plasma levels of prolactin may not necessarily reflect the physiological role of this hormone on regulating the metabolism of the mammary gland or perhaps other tissues. Only occasional surges of prolactin release may be required for maximal biological effects of this hormone (Tyson et al., 1972a,b). The fact that tissues exposed for brief periods to prolactin did not stimulate leutine incorporation into casein may be a result of the extended incubation period required before enhanced casein synthesis becomes apparent. It is possible that the prolactin taken up during the brief exposure period at 0°C is in some manner inactivated during the 10-b incubation period at 37°C: the stimulation of casein synthe-
136
J.A. Riilema, L.D. Anderson
sis may, therefore, require a more frequent or perhaps a continuous delivery of the hormone to the mammary gland. The interaction of insulin with mammal tissue appears to be quite different from that of prolactin, despite the finding that the affinities of both these hormones for their respective binding’sites in this tissue are quite similar (Turkington et al., 1973; O’Keefe and Cuatrecases, 1974). An insulin effect on uridine incorporation was not apparent in tissues exposed to a high concentration of insulin (2.5 @g/ml) for I min at 0°C followed by a 2-h wash period at 0°C. That an adequate amount of insulin to produce a response was present in these tissues following the brief exposure to the insulin solution was demonstrated by the fact that an insulin response was observed in tissues immersed in the insulin-containing media immediately prior to the incubation period at 37°C. Several explanations can be offered concerning these observations. First, it is possible that the insulin binding sites are not available at O*C and that the response observed in the tissues exposed to insulin immediately prior to the 37°C incubation may be due to the insulin which acccumulated in extracellular water during the brief exposure period. Second, it is also possible that the insulin binding sites may become occupied at O’C but when the tissues are warmed to 37’C, the insulin may be released by the tissue. It is of interest that insulin is also washed out of fat and muscle tissues at temperatures higher than 0°C (Stadie et al., 1949; Crofford, 1968). Finaly, it may also be that the small amount of insulin remaining in the tissues following the 2-h wash at 0°C may be rapidly inactivated when the tissues are warmed to 37°C. In any event, it seems reasonable to conclude that biologically active quantities of insulin are rapidly taken up by mammary tissues at 0°C but that enough insulin is removed from the tissue by a 2-h wash in media at 0°C such that metabolic effects are no longer apparent during a subsequent incubation period at 37°C.
ACKNOWLEDGEMENT This investigation was supported by NIH grant number HD-06571 National Institutes of Child Health and Human Development, U.S.A.
from the
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Shiu, R.P.C. and Friesen, H.G. (1974a) Biochem. J. 140,301. Shiu, R.P.C. and Friesen, H.G. (1974b) J. Biol. Chem. 249, 7902. Stadie, W.C., Haugaard, N., March, J.B. and Hills, A.G. (1949) Am. J. Med. Sci. 218,275. Topper, Y.J. and Oka, T. (1974) In: Lactation, Vol. 1, Eds.: B.L. Larson and V.R. Smith (Academic Press, New York) p. 327. Turkington, R.W., Majumder, G.C., Kadohama, N., MacIndoe, J.M. and Frantz, W.L. (1973) Rec. Prog. Horm. Res. 29,417. Tyson, J.E., Friesen, H.G. and Anderson, M.S. (1972a) Science 177,897. Tyson, J.E., Hwang, P., Guyda, H. and Friesen, H.G. (1972b) Am. J. Obstet. Gynecol. 113,14.
