0013-7227/91/1283-1505$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 3 Printed in U.S.A.
NORA J. HEBERTt, JANE H. KIM, AND CHARLES S. NICOLL Department of Integratiue Biology, Cancer Research Laboratory, and Group in Endocrinology, University of California, Berkeley, California 94720
ABSTRACT. The possibility that the liver contributes to the galactopoietic effects of PRL was assessed in lactating rats in which endogenous PRL secretion was suppressed by injections of bromocriptine. Pup weight gain over a 5-day period (i.e. days 7-12 of lactation) was used as an index of lactational performance in dams. Osmotic minipumps were used to infuse different doses of ovine (o) PRL into either the external jugular vein (JV) or the hepatic portal vein of the dams at a constant rate. The latter route of delivery would directly expose the liver to a higher concentration of PRL than would the former one. Twice daily sc injections of bromocriptine (1.5 mg/kg-injection) in corn oil into the dams completely suppressed litter weight gain. Infusion of oPRL into the JV at a dose of 2.0 mg/ratday restored lactation to normal in the drug-treated mothers. Electrophoretic
S
TUDIES in our laboratory led to the proposal of a pituitary-hepatic axis that modulates the growthpromoting actions of PRL (1-3). Specifically, it was suggested that lactogenic hormones stimulate the secretion by the liver of a factor that acts in concert with those hormones to promote the growth of target organs, such as the mammary gland or pigeon crop-sac. The evidence that led to the proposal of this synlactin hypothesis was derived from several kinds of physiological studies. It was shown that slices of the liver of several vertebrate species secrete synlactin activity in vitro when the organ is removed from animals in physiological states in which PRL is known to be active (4). In addition, injections of PRL into rats (3), pigeons (2), and frogs (5) caused their liver to acquire the capacity to secrete synlactin activity. Finally, experiments that involved direct delivery of PRL to the liver in vivo via the hepatic portal vein indicated that a PRL-hepatic axis was of physiological significance in pigeons (6), rats (7), and bullfrog tadpoles (5). Received September 26, 1990. Address requests for reprints to: Dr. Charles S. Nicoll, Department of Integrative Biology, 281 LSA, University of California, Berkeley, California 94720. * This work was supported by NIH Grant DK-38879. t Trainee on USPHS Training Grant CA-09041 awarded by the NCI, DHHS.
analysis indicated that about 95% of the oPRL remained in the intact monomeric form when incubated in the infusion solvent in the minipump at 37 C for 2 days, but by 4 and 6 days of incubation the amounts of that form decreased by about 25% and 50%, respectively. Measurement of serum oPRL levels by RIA showed that they were fairly constant, and after 5 days of infusion, the final concentration was directly related to the dose infused. Continuous infusion of oPRL into the JV was equally effective at restoring pup weight gain as was infusion into the hepatic portal vein over a wide range of doses. Hence, a physiological role of the liver in the maintenance of lactation by PRL is not supported by these experiments. {Endocrinology 128: 1505-1510,1991)
Hoeffler and Frawley (8) extended the idea that a hepatic action of PRL may be of physiological importance when they reported that slices of the liver of lactating rats secreted in vitro a factor that increased casein secretion by rat mammary cells in the reverse hemolytic plaque bioassay. The factor was not secreted by liver slices from virgins, and it partially mimicked the lactogenic effects of PRL. Thus, their results suggest that the liver may be involved in milk synthesis. In view of these findings, the possible role of the liver in mediating or modulating the galactopoietic actions of PRL in vivo was investigated in our present study. Lactating rats in which endogenous PRL secretion was inhibited by treatment with bromocriptine (BC) were used to assess the effectiveness of PRL replacement therapy when the hormone was infused either directly into the hepatic portal vein (HPV) or via the external jugular vein (JV). The former route of delivery would expose the liver to higher concentrations of PRL than the latter. Thus, low doses of PRL should be more effective at restoring lactation when given via the HPV than when infused into the JV. Materials and Methods Assay for the maintenance of lactation Rats (20 days pregnant; 300-400 g) of the Long-Evans strain were purchased from Simonsen Laboratories (Gilroy, CA) and 1505
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On the Possible Role of the Liver in the Galactopoietic Action of Prolactin in the Rat*
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ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
Stability of oPRL in solvent at 37 C The following procedures were used to determine the effects of incubation of oPRL at body temperature in the infusion solvent. Two minipumps and their attached catheters were filled with the solvent containing the hormone at a concentration of 5 ng/fd. The minipumps were placed in scintillation vials containing 16 ml 0.9% NaCl, and the catheters protruded through a 3-mm diameter hole in the vial cap. The two catheters were inserted into a 1.5-ml Eppendorf vial with their tips extending to the bottom. The vial contained 0.5 ml mineral oil, which prevented evaporation of the approximately 50 n\ fluid that were pumped into the vial each day. The entire assembly was incubated at 37 C for 6 days, which is equivalent to the
duration of the incubation of the pumps in the experiments (1 day in saline at 37 C and 5 days in the animal). On days 2, 4, and 6 the approximately 100 ^1 of the solution that was transferred to the Eppendorf vial from the minipumps were removed, and 2-n\ aliquots were processed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% gel), as previously described (11). Coomassie stain was used to reveal protein bands, and a densitometer was employed to measure the relative amounts of proteins in the gel. Serial blood sampling
To evaluate the levels of oPRL achieved by constant infusion of the hormone, six male rats, weighing 250 g, were divided into two groups of three each. Males were used because we wished only to validate the infusion system, and circulating levels of endogenous PRL are low in these animals. One group was implanted with left JV catheters attached to osmotic minipumps filled with an infusion dose of 300 ng oPRL/day. The second group received the same iv dose of oPRL via the HPV. In addition, an indwelling right atrial catheter was inserted into each animal for blood collection. The method of Harms and Ojeda (12) was used for this procedure with some modification. Briefly, a 10-cm length of Silastic silicone tubing (id, 0.020 in.; od, 0.037 in.; Dow-Corning Corp., Midland, MI) was introduced into the right JV as far as the right atrium (27 mm) and secured with two ligatures and cyanoacrylate glue. The catheter was passed to the back of the neck sc and exteriorized through a horizontal incision between the ears. The catheters were filled with physiological saline and stoppered with brass contact pins (Wyle, Santa Clara, CA), and patency was monitored daily. Two days after surgery, the atrial catheters were extended with 12- to 18-in. lengths of silicone tubing, and blood samples of 0.2-0.25 ml were taken every 15 min for 4 h. Blood volume was maintained with blood from donor male rats bearing indwelling atrial catheters. Serum samples were frozen and stored at -20 C until assayed for oPRL. RIAs To verify the effectiveness of suppression of endogenous PRL secretion by BC treatment, levels of rat (r) PRL were measured in a homologous RIA system using a kit provided by the NIH. Iodination grade rPRL (NIDDK rPRL 1-5) was used as label and standard. rPRL was considered suppressed at a plasma level of 12 ng/ml or less. Circulating oPRL also was measured by homologous RIA. The antibody was raised in rabbits against oPRL (NIDDK oPRL-17) in the laboratory of Dr. George Stabenfeldt (University of California, Davis). Iodination grade oPRL (NIDDK oPRL 1-2) was used as label and standard. All serum samples were tested in a single RIA for each hormone. There was no apparent cross-reactivity between oPRL and the antibody to rPRL at concentrations of standard as high as 8 fig/m\. An equivalent lack of cross-reactivity between rPRL standard and oPRL antibody was observed. Statistical analysis All values are given as the mean ± SE, except where noted. Statistical significance was evaluated by one- and two-way analysis of variance and the Newman-Keuls multiple range test (13) or Student's t test, where appropriate.
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housed in a controlled temperature environment (22-23 C) on a 12-h light, 12-h dark cycle, with lights on at 0600 h. Water and Purina rat chow (Ralston-Purina, St. Louis, MO) were available ad libitum. The day of birth was designanted day 0 of lactation, and litters were adjusted to 10 pups each on day 2 and to 8 on day 5. The mothers were implanted with indwelling catheters attached to Alzet osmotic minipumps (model 2001, Alza, Palo Alto, CA) on the seventh day of lactation, as previously described (9). The minipumps were filled with either solvent containing varying concentrations of ovine (o) PRL (NIDDK oPRL 19) or solvent alone. The solvent consisted of 0.4 M NaHCO3, 1.6% glycerol, 0.02% sodium azide, and 250 U/ ml porcine sodium heparin (Sigma, St. Louis, MO), pH 8, and the oPRL was infused at doses of 0, 50,100, 300, 600, and 2000 Mg/day. The catheterization procedures were described in detail by Griffen et al. (10), and the anesthetic used was a cocktail consisting of ketamine HC1, xylazine, and acepromazine maleate (9). Briefly, an intrahepatic route of delivery was achieved by making a midline incision 3 cm caudal to the thorax through the skin and abdominal wall of the rat. The cecum was then exposed, and a branch of a mesenteric vein, leading to the HPV, was isolated. The catheter was then inserted and advanced approximately 2 cm into the vessel and secured with surgical silk and cyanoacrylate glue. For systemic infusion the right external JV was catheterized. A 1-cm incision was made 1-2 cm to the right of midline just rostral to the clavicle, and the JV was exposed and separated from surrounding tissues. The catheter was inserted and advanced approximately 1 cm into the vein and secured as before. The pump was placed sc on the ventral side of the animal. The pumps were immersed in 0.9% NaCl at 37 C for 24 h to ensure that solvent flow had started before they were placed in the animal. On the day of surgery, the dams received one sc injection of 2-bromo-a-ergocryptine methane sulfate (BC; 1.5 mg/kg; Sigma). Thereafter, they were injected twice daily at a dose of 3 mg/kg-day to suppress endogenous PRL secretion, except on the last day when they again received only one injection. The drug was given as an emulsion in corn oil in a volume of 80 /A/ 100 g BW. Treatment lasted 5 days, and pup weight was recorded daily at 1700 h. Pup growth over the 5-day period was used as a measure of the extent to which lactation was maintained. On day 12 all of the animals were killed, and blood was collected from the mothers for analysis of PRL by RIA. Serum samples were frozen and stored at -20 C.
Endo • 1991 Voll28«No3
ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
Results
TABLE 1. Concentration of rPRL in serum of lactating rats Serum rPRL (ng/ml)
Group
Treatment
A B C D E F
None JV-2.0 mg oPRL/day + BC injection JV-solvent + oil injection HPV-solvent + oil injection JV-solvent + BC injection HPV-solvent + BC injection
12-
163.7 ± 10.5 ± 126.8 ± 344.1 ± 6.7 ± 2.1 ±
72.6 2.2 39.7 59.0 1.5 1.0
(7)
108
JV catheterization with solvent infusion did not affect pup growth (C us. A), abdominal surgery with HPV infusion of solvent led to a 24% decrease in weight gain (C us. D, P < 0.02, by t test). Similarly, HPV infusion of solvent caused the pups nursing on BC-injected dams to lose weight, which was a significantly greater inhibition than that seen in the JV-infused BC-injected mothers (E us. F, P < 0.03, by t test). The mothers that received abdominal implants experienced a loss in weight that stabilized within 2 days of surgery. When dams received 2.0 mg oPRL/day to the JV, litter growth was fully restored (B us. E, P < 0.01). Infusion of oPRL into either the JV or the HPV produced dose-related increases in pup weight gain (Fig. 2). Although two-way analysis of variance of the data from all of the treatment groups suggested that the hepatic route of delivery was less effective at maintaining lactation (P < 0.04), there were no significant differences between HPV and JV administration at individual doses of oPRL. If the data from the HPV-catheterized animals were corrected for the effects of abdominal surgery (by increasing litter growth by 2.3 g), the pup weight gain of the HPV-infused mothers became slightly, but nonsignificantly, greater than that of animals with mothers given the hormone into the JV. A corresponding doserelated increase in serum oPRL was observed in the samples collected on day 12 of the experiment (Fig. 3). Again, jugular delivery appeared to raise oPRL to a greater extent overall {P < 0.02), but a significant difference in serum levels was seen only at the highest dose (600 Mg/day; P < 0.01). Constant infusion of oPRL at a rate of 300 Mg/day into male rats resulted in serum concentrations that varied over a wide range among the different animals (Fig. 4), and four of the six showed a gradual change in concentration over the 4-h period. One animal that received HPV infusion showed striking fluctuations in serum oPRL levels. Circulating oPRL in these males
6-j
i
o-
4
5
2 (6)
0
5
-4-
no treat -ment
JV-2 mg PRL/day + BC
I JV solv + oil
8-
c HPV
6-
i / |
£ HPV solv + oil
JV solv + BC
HPV solv + BC
FlG. 1. Effects of BC treatment, surgery, and oPRL replacement (2 mg/day) on lactational performance of dams, as assessed by pup weight gain between days 7 and 12 of lactation. The mothers received solvent (solv) or oPRL infusions into either the JV or HPV. They also received twice daily injections of either oil or BC. The number of litters per group is shown in parentheses above each column. *, P < 0.02 compared to JV-solv plus oil; • * , P < 0.03 compared to JV-solv plus BC. Differences were analyzed by Student's t test.
a 3
a
4-
50
100
600
Dose of oPRL (ng/day)
FIG. 2. Effects of constant infusion of different doses of oPRL into either the JV or HPV of PRL-suppressed rat dams on pup weight gain over a 5-day period. Each group contained six dams, except the JV-100 pig and HPV-50 ^g groups, which had five each, and the JV-50 Mg group, which had seven mothers.
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Table 1 shows the effects of the treatment on serum rPRL concentrations in the different experimental groups. In the three groups that did not receive BC injections (groups A, C, and D) blood rPRL levels were elevated, as would be expected in lactating dams. However, the mean serum rPRL concentration of group D was significantly greater (P < 0.01) than that of either group A or C. This difference may be a result of the stress of abdominal surgery and the presence of the minipumps and catheter in the peritoneal cavity. By contrast, the circulating concentration of the hormone in all of the rats that were treated with BC was severely suppressed (groups B, E, and F). The effects of suppressing endogenous PRL secretion on lactational performance are shown in Fig. 1. Between days 7 and 12 of lactation, weight gains of pups suckling untreated control mothers (A) or those given oil injections plus solvent infusion to the JV (C) were 11.1 ± 0.4 and 10.7 ± 0.5 g, respectively. Subcutaneous administration of BC to the mothers at a dose of 3 mg/kg-day suppressed growth of their suckling pups by essentially 100% (E and F us. A, P < 0.01). The data also show that there was a significant effect of surgery. Although the
1507
1508
ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
Endo • 1991 Voll28«No3
80i
I O) 60
c
20
100
200
300
400
500
600
14
Dose of oPRL (ng/day)
FIG. 3. Serum levels of oPRL after 5 days of constant infusion into either the JV or HPV of BC-treated lactating rats. All groups had six dams each, except for the following: the HPV-50 and -300 fig groups and the JV-100 ng group had five each; the JV-0 and -50 Mg/day groups had four and seven, respectively.
[ng/ml)
80-
60-
a. JV- Infusion
* * " " ' *
*
* ^
oc
-.
«....—•—•..
„...«—,—•—•
200200
100 b. HPV-lnfusion 200-1
100
time
FIG. 5. Electrophoretic analysis of oPRL that was incubated in the infusion solvent in minipumps for O, 2, 4, and 6 days at 37 C. The right column shows mol wt markers. Note the formation of an apparent dimer of about 40K mol wt and a smaller molecule of about 21K.
^-»
40-
a o
DAYS OF INCUBATION
200
(min)
FIG. 4. Serum levels of oPRL after 2 days of constant infusion at a dose of 300 jttg/rat-day into either the JV or HPV of individual male rats. Blood samples of 0.2-0.25 ml were taken every 15 min for 4 h. Each line represents the data from one animal. Note differences in scale on the ordinates of the two graphs.
after 2 days of infusion were generally higher than those in lactating females given the 300 Mg/day dose for 5 days (Fig. 3). Electrophoretic-densitometric analysis showed that the amount of oPRL that remained in the intact monomeric form (mol wt, 23K) decreased during the period of incubation in solvent at 37 C (Fig. 5). By 2, 4, and 6 days the amounts of the monomer remaining were approximately 95%, 75%, and 50%, respectively. Some of this reduction in the 23K PRL involved conversion to a form about the size of a dimer and formation of a smaller form of about 21K.
Discussion The results presented herein confirm those of other reports which showed that administration of BC inhibits
lactational performance. However, the treatment that we used was much more effective than the regimens used by others. Knight et al. (14) found that implants of BC or twice daily injections of the drug (0.4 mg/day) in mice caused only about a 30% inhibition of litter weight gain, and Fliickiger and Wagner (15) reported that BC injections inhibited lactation in rats (i.e. pup weight gain) to a lesser extent than they inhibited ovum implantation. In fact, sc administration of 10 mg/kg BC at any time during an 8-h mother-pup separation effectively inhibited suckling-induced PRL release (16). Yet, this dose reduced weight gain by only 60% during midlactation. In the present study, a 3 mg/kg-day dose given twice daily inhibited growth by 100%. The greater effectiveness of BC treatment in our experiments relative to the results of Fliickiger and colleagues (15,16) may be due primarily to differences in treatment schedules and vehicle. Fliickiger and Wagner (15) injected dams with BC at 1700 h and recorded litter weight 16 h later at 0900 h. It seems likely that a single injection of BC in rats is not maximally inhibitory, as experiments on mice have shown that twice daily injections were more effective than single injections at inhibiting lactation (14). The electrophoretic analysis showed that the monomeric (23K) form of oPRL that remained in the infusion solvent decreased during the course of incubation at 37 C. This decrease was slight during the first 2 days of incubation, but it became more rapid thereafter. Nevertheless, about 50% of the hormone remained as a monomer by the end of the experimental period. Whether the other forms that were produced (i.e. an apparent dimer, a 21K form, and presumably fragments) are bioactive and/or immunoactive was not determined. In any case, the rate of weight gain of litters of mothers infused with oPRL did not decrease with the duration of the
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0
ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
i
because it was used previously to show preferential effects of HPV vs. JV delivery of PRL and insulin. English et al. (7) recently showed that infusion of a low dose of oPRL directly to the liver of 6-week-old female rats stimulated mammary lobulo-alveolar growth; JV delivery of the same dose had no significant effect. Similarly, intrahepatic delivery of oPRL to pigeons potentiated the mitogenic effects of locally injected PRL on the crop-sac mucosa, while JV infusion of the same dose was ineffective (6). Evidence that the liver plays a role in PRL's antimetamorphic actions in amphibian tadpoles also has been reported (5). Finally, Griffen et al. (10) demonstrated that infusion of a low dose of insulin into the HPV, but not the JV, stimulated growth and elevated serum insulin-like growth factor-I concentrations in diabetic male rats. Although the other studies that involved intrahepatic infusion of oPRL (6, 7) used a pulse delivery schedule, whereas we employed a constant rate of delivery in our present study, this difference in schedule does not account for the lack of a preferential galactopoietic effect of HPV infusion. Studies in progress indicate that pulse delivery of PRL into the HPV is no more effective at restoring lactation in BC-suppressed dams than is JV delivery in pulses. Hoeffler and Frawley (8) reported that their liver lactogenic factor (LLF) increased plaque size (i.e. casein release from rat mammary cells), but not the numbers of plaques formed (i.e. cells secreting casein) in the reverse hemolytic plaque bioassay. Although PRL increased both parameters, LLF was more potent at stimulating casein secretion from cells already capable of that function. In addition, LLF was active in the absence of PRL. Frawley et al. (23) also mentioned some unpublished findings which indicate that injections of the purified LLF partially restored the lactational capacity of lactating rats given BC. Nevertheless, our present physiological studies do not support the hypothesis of hepatic mediation of PRL's galactopoietic action in vivo. Furthermore, in view of the evidence that the liver of lactating rats can secrete synlactin activity (24), and PRL injections stimulate the liver to secrete that activity (24), it seems unlikely that synlactin has a galactopoietic function in addition to its mitogenic activity (1-7, 24). However, it remains to be determined whether the liver participates in the initiation of lactation (i.e. has a lactogenic function) at the end of pregnancy. Acknowledgments The authors would like to thank Richard Lin for technical assistance in performing the RIAs and the preparation of this manuscript. We also are indebted to Dr. George Stabenfeldt of the Department of Veterinary Medical Reproduction of the University of California, Davis, for the antibody to oPRL. The purified oPRL and the reagents used in the RIAs were generous
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experiment. Thus, there is no evidence of a significant reduction in the bioactivity of the infused hormone as the experiments progressed. The experiment with male rats showed that in most cases the minipumps maintained relatively constant serum levels of oPRL in conscious animals over a period of 4 h. The one exceptional animal, which showed marked fluctuations, had a HPV catheter. The portal vasculature is the major contributor to the hepatic circulation, and the flow within it varies considerably with food intake, intestinal peristalsis, and other conditions (17,18). Thus, it is surprising that only one of the three rats with HPV catheters showed large fluctuations in its serum PRL levels. A difference in the final serum oPRL concentration in infused dams between the HPV and JV routes of delivery was seen only at the 600 Mg/day dose. The lack of a difference at the lower doses is surprising, as the liver is considered to be a major site of peptide hormone disposal, and infusion of the hormone into the HPV might be expected to activate hepatic clearance. Possibly, the hepatic clearance process is activated only at high concentrations of the hormone. The observed serum levels of oPRL at the end of the experiment can be compared with expected levels by using data in the literature. Grosvenor (19) reported that the ti/2 of oPRL in the lactating rat is 3 min, and the distribution volume (DV) in animals of the size used in our experiments is about 30 ml (20). The MCR = k x DV, where k = 0.693/t1/2 = 0.231 min"1. The expected hormone concentration, He, = infusion rate/MCR. Thus, at the highest dose given via the JV (600 /ig/rat-day), He = 417 /Lig/min • 6.93 ml min"1 = 60 ng/ml. This expected concentration is in excellent agreement with the observed value of 65 ng/ml. Grosvenor (19) also reported that the circulating ti/ 2 of oPRL was considerably greater in nonlactating females. Consequently, the higher serum oPRL concentration in male rats compared to that in the lactating females may be due to differences in MCR. Constant infusion of oPRL at a dose rate of 2.0 mg/ rat-day completely restored lactation in the BC-treated dams. In previous studies, twice daily injections of similar or higher doses of oPRL, with or without replacement therapy with other hormones, restored lactation to only about 50% of normal in hypophysectomized rats (21, 22). Thus, constant infusion of the hormone is apparently much more effective in this regard than twice daily injection. The failure of intrahepatic delivery of oPRL to have a greater restorative effect on the suppressed lactation of the BC-treated rats compared to the JV route of delivery was unexpected in view of the findings of Hoeffler and Frawley (8). It is unlikely that this lack of difference is due to technical problems with the infusion system,
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ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
gifts from the National Hormone and Pituitary Program of the NIH.
References
12. Harms PG, Ojeda SR 1974 A rapid and simple procedure for chronic cannulation of the rat jugular vein. J Appl Physiol 36:391-392 13. Zar JH 1974 Biostatistical Analysis. Prentice-Hall, Englewood Cliffs 14. Knight CH, Calvert DT, Flint DJ 1986 Inhibitory effect of bromocriptine on mammary gland development and function in lactating mice. J Endocrinol 110:263-270 15. Fliickiger E, Wagner HR 1968 2-Br-a-Ergokryptin: beeinflussing von fertilitat und laktation bei der ratte. Experientia 24:1130-1131 16. Fliickiger E, Kovacs E 1974 Inhibition by 2-Br-a-ergocryptinemesilate (CB-154) of suckling-induced prolactin depletion in lactating rats. Experientia 30:1173 17. Rappaport AM 1980 Hepatic blood flow. In: Javitt NB (ed) International Review of Physiology. University Park Press, Baltimore, pp 1-65 18. Withrington PG, Richardson PDI 1986 Hepatic hemodynamics and microcirculation. In: Thurman RG, Kauffman FC, Jungermann K (eds) Regulation of Hepatic Metabolism-Intra- and Intercellular Compartmentation. Plenum Press, New York, pp 27-53 19. Grosvenor CE 1967 Disappearance rate of exogenous prolactin from serum of female rats. Endocrinology 80:195-200 20. Nicoll CS, Swearingen KC, Mattheij JAM 1984 Relationship between depletion and release of prolactin in the lactating rat: a quantitative analysis. In: Mena F, Valverde-R CM (ed) Prolactin Secretion: A Multidisciplinary Approach. Academic Press, New York, pp 285-301 21. Bintarningsih, Lyons WR, Johnson RE, Li CH 1958 Hormonallyinduced lactation in hypophysectomized rats. Endocrinology 63:540-548 22. Cowie AT, Tindal JS 1961 The maintenance of lactation in the rat after hypophysial anterior lobectomy during pregnancy. J Endocrinol 22:403-408 23. Frawley LS, Schwabe C, Miller III HA, Betts JG, Hoeffler JP, Simpson MT 1988 Characterization and physiological role of a liver lactogenic factor. In: Hoshino K (ed) Prolactin Gene Family and Its Receptors. Elsevier (Biomedical Division), Amsterdam, pp 49-59 24. Nicoll CS, Anderson TR, Hebert NJ, Russell SM 1985 Comparative aspects of the growth-promoting actions of prolactin on its target organs: evidence for synergism with an insulin-like growth factor. In: MacLeod RM, Thorner MO, Scapagnini U (ed) Prolactin. Basic and Clinical Correlates. Liviana Press, Padua, Fidia Res Ser, vol 1:393-410
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1. Anderson TR Rodriguez J, Nicoll CS, Spencer EM 1983 The synlactin hypothesis: prolactin's mitogenic action may involve synergism with a somatomedin-like molecule. In: Spencer EM (ed) Insulin-Like Growth Factors/Somatomedins. de Gruyter, Berlin, pp 71-78 2. Anderson TR, Pitts D, Nicoll CS 1984 Prolactin's mitogenic action on the pigeon crop-sac mucosal epithelium involves direct and indirect mechanisms. Gen Comp Endocrinol 54:236-246 3. Nicoll CS, Hebert NJ, Russell SM 1985 Lactogenic hormones stimulate the liver to secrete a factor that acts synergistically with prolactin to promote growth of the pigeon crop-sac mucosal epithelium in vivo. Endocrinology 116:1449-1453 4. Delidow BC, Hebert N, Steiny S, Nicoll CS 1986 Secretion of prolactin-synergizing activity (synlactin) by the liver of ectothermic vertebrates in vitro. J Exp Zool 238:147-153 5. Delidow BC, Baldocchi RA, Nicoll CS 1988 Evidence for hepatic involvement in the regulation of amphibian development by prolactin. Gen Comp Endocrinol 70:418-424 6. Mick CW, Nicoll CS 1985 Prolactin directly stimulates the liver in vivo to secrete a factor (synlactin) which acts synergistically with the hormone. Endocrinology 116:2049-2053 7. English DE, Russell SM, Katz L, Nicoll CS 1990 Evidence for a role of the liver in the mammotrophic action of prolactin. Endocrinology 126:2252-2256 8. Hoeffler JP, Frawley LS 1987 Liver tissue produces a potent lactogen that partially mimics the actions of prolactin. Endocrinology 120:1679-1681 9. Schlechter NL, Russell SM, Greenberg S, Spencer EM, Nicoll CS 1986 A direct growth effect of growth hormone in rat hindlimb shown by arterial infusion. Am J Physiol 250:E231-E253 10. Griffen SC, Russell SM, Katz LS, Nicoll CS 1987 Insulin exerts metabolic and growth-promoting effects by a direct action on the liver in vivo: clarification of the functional significance of the portal vascular link between the beta cells of the pancreatic islets and the liver. Proc Natl Acad Sci USA 84:7300-7304 11. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685
Endo»1991 Vol 128 • No 3
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Vol. 128, No. 3 Printed in U.S.A.
NORA J. HEBERTt, JANE H. KIM, AND CHARLES S. NICOLL Department of Integratiue Biology, Cancer Research Laboratory, and Group in Endocrinology, University of California, Berkeley, California 94720
ABSTRACT. The possibility that the liver contributes to the galactopoietic effects of PRL was assessed in lactating rats in which endogenous PRL secretion was suppressed by injections of bromocriptine. Pup weight gain over a 5-day period (i.e. days 7-12 of lactation) was used as an index of lactational performance in dams. Osmotic minipumps were used to infuse different doses of ovine (o) PRL into either the external jugular vein (JV) or the hepatic portal vein of the dams at a constant rate. The latter route of delivery would directly expose the liver to a higher concentration of PRL than would the former one. Twice daily sc injections of bromocriptine (1.5 mg/kg-injection) in corn oil into the dams completely suppressed litter weight gain. Infusion of oPRL into the JV at a dose of 2.0 mg/ratday restored lactation to normal in the drug-treated mothers. Electrophoretic
S
TUDIES in our laboratory led to the proposal of a pituitary-hepatic axis that modulates the growthpromoting actions of PRL (1-3). Specifically, it was suggested that lactogenic hormones stimulate the secretion by the liver of a factor that acts in concert with those hormones to promote the growth of target organs, such as the mammary gland or pigeon crop-sac. The evidence that led to the proposal of this synlactin hypothesis was derived from several kinds of physiological studies. It was shown that slices of the liver of several vertebrate species secrete synlactin activity in vitro when the organ is removed from animals in physiological states in which PRL is known to be active (4). In addition, injections of PRL into rats (3), pigeons (2), and frogs (5) caused their liver to acquire the capacity to secrete synlactin activity. Finally, experiments that involved direct delivery of PRL to the liver in vivo via the hepatic portal vein indicated that a PRL-hepatic axis was of physiological significance in pigeons (6), rats (7), and bullfrog tadpoles (5). Received September 26, 1990. Address requests for reprints to: Dr. Charles S. Nicoll, Department of Integrative Biology, 281 LSA, University of California, Berkeley, California 94720. * This work was supported by NIH Grant DK-38879. t Trainee on USPHS Training Grant CA-09041 awarded by the NCI, DHHS.
analysis indicated that about 95% of the oPRL remained in the intact monomeric form when incubated in the infusion solvent in the minipump at 37 C for 2 days, but by 4 and 6 days of incubation the amounts of that form decreased by about 25% and 50%, respectively. Measurement of serum oPRL levels by RIA showed that they were fairly constant, and after 5 days of infusion, the final concentration was directly related to the dose infused. Continuous infusion of oPRL into the JV was equally effective at restoring pup weight gain as was infusion into the hepatic portal vein over a wide range of doses. Hence, a physiological role of the liver in the maintenance of lactation by PRL is not supported by these experiments. {Endocrinology 128: 1505-1510,1991)
Hoeffler and Frawley (8) extended the idea that a hepatic action of PRL may be of physiological importance when they reported that slices of the liver of lactating rats secreted in vitro a factor that increased casein secretion by rat mammary cells in the reverse hemolytic plaque bioassay. The factor was not secreted by liver slices from virgins, and it partially mimicked the lactogenic effects of PRL. Thus, their results suggest that the liver may be involved in milk synthesis. In view of these findings, the possible role of the liver in mediating or modulating the galactopoietic actions of PRL in vivo was investigated in our present study. Lactating rats in which endogenous PRL secretion was inhibited by treatment with bromocriptine (BC) were used to assess the effectiveness of PRL replacement therapy when the hormone was infused either directly into the hepatic portal vein (HPV) or via the external jugular vein (JV). The former route of delivery would expose the liver to higher concentrations of PRL than the latter. Thus, low doses of PRL should be more effective at restoring lactation when given via the HPV than when infused into the JV. Materials and Methods Assay for the maintenance of lactation Rats (20 days pregnant; 300-400 g) of the Long-Evans strain were purchased from Simonsen Laboratories (Gilroy, CA) and 1505
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On the Possible Role of the Liver in the Galactopoietic Action of Prolactin in the Rat*
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ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
Stability of oPRL in solvent at 37 C The following procedures were used to determine the effects of incubation of oPRL at body temperature in the infusion solvent. Two minipumps and their attached catheters were filled with the solvent containing the hormone at a concentration of 5 ng/fd. The minipumps were placed in scintillation vials containing 16 ml 0.9% NaCl, and the catheters protruded through a 3-mm diameter hole in the vial cap. The two catheters were inserted into a 1.5-ml Eppendorf vial with their tips extending to the bottom. The vial contained 0.5 ml mineral oil, which prevented evaporation of the approximately 50 n\ fluid that were pumped into the vial each day. The entire assembly was incubated at 37 C for 6 days, which is equivalent to the
duration of the incubation of the pumps in the experiments (1 day in saline at 37 C and 5 days in the animal). On days 2, 4, and 6 the approximately 100 ^1 of the solution that was transferred to the Eppendorf vial from the minipumps were removed, and 2-n\ aliquots were processed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% gel), as previously described (11). Coomassie stain was used to reveal protein bands, and a densitometer was employed to measure the relative amounts of proteins in the gel. Serial blood sampling
To evaluate the levels of oPRL achieved by constant infusion of the hormone, six male rats, weighing 250 g, were divided into two groups of three each. Males were used because we wished only to validate the infusion system, and circulating levels of endogenous PRL are low in these animals. One group was implanted with left JV catheters attached to osmotic minipumps filled with an infusion dose of 300 ng oPRL/day. The second group received the same iv dose of oPRL via the HPV. In addition, an indwelling right atrial catheter was inserted into each animal for blood collection. The method of Harms and Ojeda (12) was used for this procedure with some modification. Briefly, a 10-cm length of Silastic silicone tubing (id, 0.020 in.; od, 0.037 in.; Dow-Corning Corp., Midland, MI) was introduced into the right JV as far as the right atrium (27 mm) and secured with two ligatures and cyanoacrylate glue. The catheter was passed to the back of the neck sc and exteriorized through a horizontal incision between the ears. The catheters were filled with physiological saline and stoppered with brass contact pins (Wyle, Santa Clara, CA), and patency was monitored daily. Two days after surgery, the atrial catheters were extended with 12- to 18-in. lengths of silicone tubing, and blood samples of 0.2-0.25 ml were taken every 15 min for 4 h. Blood volume was maintained with blood from donor male rats bearing indwelling atrial catheters. Serum samples were frozen and stored at -20 C until assayed for oPRL. RIAs To verify the effectiveness of suppression of endogenous PRL secretion by BC treatment, levels of rat (r) PRL were measured in a homologous RIA system using a kit provided by the NIH. Iodination grade rPRL (NIDDK rPRL 1-5) was used as label and standard. rPRL was considered suppressed at a plasma level of 12 ng/ml or less. Circulating oPRL also was measured by homologous RIA. The antibody was raised in rabbits against oPRL (NIDDK oPRL-17) in the laboratory of Dr. George Stabenfeldt (University of California, Davis). Iodination grade oPRL (NIDDK oPRL 1-2) was used as label and standard. All serum samples were tested in a single RIA for each hormone. There was no apparent cross-reactivity between oPRL and the antibody to rPRL at concentrations of standard as high as 8 fig/m\. An equivalent lack of cross-reactivity between rPRL standard and oPRL antibody was observed. Statistical analysis All values are given as the mean ± SE, except where noted. Statistical significance was evaluated by one- and two-way analysis of variance and the Newman-Keuls multiple range test (13) or Student's t test, where appropriate.
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housed in a controlled temperature environment (22-23 C) on a 12-h light, 12-h dark cycle, with lights on at 0600 h. Water and Purina rat chow (Ralston-Purina, St. Louis, MO) were available ad libitum. The day of birth was designanted day 0 of lactation, and litters were adjusted to 10 pups each on day 2 and to 8 on day 5. The mothers were implanted with indwelling catheters attached to Alzet osmotic minipumps (model 2001, Alza, Palo Alto, CA) on the seventh day of lactation, as previously described (9). The minipumps were filled with either solvent containing varying concentrations of ovine (o) PRL (NIDDK oPRL 19) or solvent alone. The solvent consisted of 0.4 M NaHCO3, 1.6% glycerol, 0.02% sodium azide, and 250 U/ ml porcine sodium heparin (Sigma, St. Louis, MO), pH 8, and the oPRL was infused at doses of 0, 50,100, 300, 600, and 2000 Mg/day. The catheterization procedures were described in detail by Griffen et al. (10), and the anesthetic used was a cocktail consisting of ketamine HC1, xylazine, and acepromazine maleate (9). Briefly, an intrahepatic route of delivery was achieved by making a midline incision 3 cm caudal to the thorax through the skin and abdominal wall of the rat. The cecum was then exposed, and a branch of a mesenteric vein, leading to the HPV, was isolated. The catheter was then inserted and advanced approximately 2 cm into the vessel and secured with surgical silk and cyanoacrylate glue. For systemic infusion the right external JV was catheterized. A 1-cm incision was made 1-2 cm to the right of midline just rostral to the clavicle, and the JV was exposed and separated from surrounding tissues. The catheter was inserted and advanced approximately 1 cm into the vein and secured as before. The pump was placed sc on the ventral side of the animal. The pumps were immersed in 0.9% NaCl at 37 C for 24 h to ensure that solvent flow had started before they were placed in the animal. On the day of surgery, the dams received one sc injection of 2-bromo-a-ergocryptine methane sulfate (BC; 1.5 mg/kg; Sigma). Thereafter, they were injected twice daily at a dose of 3 mg/kg-day to suppress endogenous PRL secretion, except on the last day when they again received only one injection. The drug was given as an emulsion in corn oil in a volume of 80 /A/ 100 g BW. Treatment lasted 5 days, and pup weight was recorded daily at 1700 h. Pup growth over the 5-day period was used as a measure of the extent to which lactation was maintained. On day 12 all of the animals were killed, and blood was collected from the mothers for analysis of PRL by RIA. Serum samples were frozen and stored at -20 C.
Endo • 1991 Voll28«No3
ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
Results
TABLE 1. Concentration of rPRL in serum of lactating rats Serum rPRL (ng/ml)
Group
Treatment
A B C D E F
None JV-2.0 mg oPRL/day + BC injection JV-solvent + oil injection HPV-solvent + oil injection JV-solvent + BC injection HPV-solvent + BC injection
12-
163.7 ± 10.5 ± 126.8 ± 344.1 ± 6.7 ± 2.1 ±
72.6 2.2 39.7 59.0 1.5 1.0
(7)
108
JV catheterization with solvent infusion did not affect pup growth (C us. A), abdominal surgery with HPV infusion of solvent led to a 24% decrease in weight gain (C us. D, P < 0.02, by t test). Similarly, HPV infusion of solvent caused the pups nursing on BC-injected dams to lose weight, which was a significantly greater inhibition than that seen in the JV-infused BC-injected mothers (E us. F, P < 0.03, by t test). The mothers that received abdominal implants experienced a loss in weight that stabilized within 2 days of surgery. When dams received 2.0 mg oPRL/day to the JV, litter growth was fully restored (B us. E, P < 0.01). Infusion of oPRL into either the JV or the HPV produced dose-related increases in pup weight gain (Fig. 2). Although two-way analysis of variance of the data from all of the treatment groups suggested that the hepatic route of delivery was less effective at maintaining lactation (P < 0.04), there were no significant differences between HPV and JV administration at individual doses of oPRL. If the data from the HPV-catheterized animals were corrected for the effects of abdominal surgery (by increasing litter growth by 2.3 g), the pup weight gain of the HPV-infused mothers became slightly, but nonsignificantly, greater than that of animals with mothers given the hormone into the JV. A corresponding doserelated increase in serum oPRL was observed in the samples collected on day 12 of the experiment (Fig. 3). Again, jugular delivery appeared to raise oPRL to a greater extent overall {P < 0.02), but a significant difference in serum levels was seen only at the highest dose (600 Mg/day; P < 0.01). Constant infusion of oPRL at a rate of 300 Mg/day into male rats resulted in serum concentrations that varied over a wide range among the different animals (Fig. 4), and four of the six showed a gradual change in concentration over the 4-h period. One animal that received HPV infusion showed striking fluctuations in serum oPRL levels. Circulating oPRL in these males
6-j
i
o-
4
5
2 (6)
0
5
-4-
no treat -ment
JV-2 mg PRL/day + BC
I JV solv + oil
8-
c HPV
6-
i / |
£ HPV solv + oil
JV solv + BC
HPV solv + BC
FlG. 1. Effects of BC treatment, surgery, and oPRL replacement (2 mg/day) on lactational performance of dams, as assessed by pup weight gain between days 7 and 12 of lactation. The mothers received solvent (solv) or oPRL infusions into either the JV or HPV. They also received twice daily injections of either oil or BC. The number of litters per group is shown in parentheses above each column. *, P < 0.02 compared to JV-solv plus oil; • * , P < 0.03 compared to JV-solv plus BC. Differences were analyzed by Student's t test.
a 3
a
4-
50
100
600
Dose of oPRL (ng/day)
FIG. 2. Effects of constant infusion of different doses of oPRL into either the JV or HPV of PRL-suppressed rat dams on pup weight gain over a 5-day period. Each group contained six dams, except the JV-100 pig and HPV-50 ^g groups, which had five each, and the JV-50 Mg group, which had seven mothers.
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Table 1 shows the effects of the treatment on serum rPRL concentrations in the different experimental groups. In the three groups that did not receive BC injections (groups A, C, and D) blood rPRL levels were elevated, as would be expected in lactating dams. However, the mean serum rPRL concentration of group D was significantly greater (P < 0.01) than that of either group A or C. This difference may be a result of the stress of abdominal surgery and the presence of the minipumps and catheter in the peritoneal cavity. By contrast, the circulating concentration of the hormone in all of the rats that were treated with BC was severely suppressed (groups B, E, and F). The effects of suppressing endogenous PRL secretion on lactational performance are shown in Fig. 1. Between days 7 and 12 of lactation, weight gains of pups suckling untreated control mothers (A) or those given oil injections plus solvent infusion to the JV (C) were 11.1 ± 0.4 and 10.7 ± 0.5 g, respectively. Subcutaneous administration of BC to the mothers at a dose of 3 mg/kg-day suppressed growth of their suckling pups by essentially 100% (E and F us. A, P < 0.01). The data also show that there was a significant effect of surgery. Although the
1507
1508
ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
Endo • 1991 Voll28«No3
80i
I O) 60
c
20
100
200
300
400
500
600
14
Dose of oPRL (ng/day)
FIG. 3. Serum levels of oPRL after 5 days of constant infusion into either the JV or HPV of BC-treated lactating rats. All groups had six dams each, except for the following: the HPV-50 and -300 fig groups and the JV-100 ng group had five each; the JV-0 and -50 Mg/day groups had four and seven, respectively.
[ng/ml)
80-
60-
a. JV- Infusion
* * " " ' *
*
* ^
oc
-.
«....—•—•..
„...«—,—•—•
200200
100 b. HPV-lnfusion 200-1
100
time
FIG. 5. Electrophoretic analysis of oPRL that was incubated in the infusion solvent in minipumps for O, 2, 4, and 6 days at 37 C. The right column shows mol wt markers. Note the formation of an apparent dimer of about 40K mol wt and a smaller molecule of about 21K.
^-»
40-
a o
DAYS OF INCUBATION
200
(min)
FIG. 4. Serum levels of oPRL after 2 days of constant infusion at a dose of 300 jttg/rat-day into either the JV or HPV of individual male rats. Blood samples of 0.2-0.25 ml were taken every 15 min for 4 h. Each line represents the data from one animal. Note differences in scale on the ordinates of the two graphs.
after 2 days of infusion were generally higher than those in lactating females given the 300 Mg/day dose for 5 days (Fig. 3). Electrophoretic-densitometric analysis showed that the amount of oPRL that remained in the intact monomeric form (mol wt, 23K) decreased during the period of incubation in solvent at 37 C (Fig. 5). By 2, 4, and 6 days the amounts of the monomer remaining were approximately 95%, 75%, and 50%, respectively. Some of this reduction in the 23K PRL involved conversion to a form about the size of a dimer and formation of a smaller form of about 21K.
Discussion The results presented herein confirm those of other reports which showed that administration of BC inhibits
lactational performance. However, the treatment that we used was much more effective than the regimens used by others. Knight et al. (14) found that implants of BC or twice daily injections of the drug (0.4 mg/day) in mice caused only about a 30% inhibition of litter weight gain, and Fliickiger and Wagner (15) reported that BC injections inhibited lactation in rats (i.e. pup weight gain) to a lesser extent than they inhibited ovum implantation. In fact, sc administration of 10 mg/kg BC at any time during an 8-h mother-pup separation effectively inhibited suckling-induced PRL release (16). Yet, this dose reduced weight gain by only 60% during midlactation. In the present study, a 3 mg/kg-day dose given twice daily inhibited growth by 100%. The greater effectiveness of BC treatment in our experiments relative to the results of Fliickiger and colleagues (15,16) may be due primarily to differences in treatment schedules and vehicle. Fliickiger and Wagner (15) injected dams with BC at 1700 h and recorded litter weight 16 h later at 0900 h. It seems likely that a single injection of BC in rats is not maximally inhibitory, as experiments on mice have shown that twice daily injections were more effective than single injections at inhibiting lactation (14). The electrophoretic analysis showed that the monomeric (23K) form of oPRL that remained in the infusion solvent decreased during the course of incubation at 37 C. This decrease was slight during the first 2 days of incubation, but it became more rapid thereafter. Nevertheless, about 50% of the hormone remained as a monomer by the end of the experimental period. Whether the other forms that were produced (i.e. an apparent dimer, a 21K form, and presumably fragments) are bioactive and/or immunoactive was not determined. In any case, the rate of weight gain of litters of mothers infused with oPRL did not decrease with the duration of the
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0
ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
i
because it was used previously to show preferential effects of HPV vs. JV delivery of PRL and insulin. English et al. (7) recently showed that infusion of a low dose of oPRL directly to the liver of 6-week-old female rats stimulated mammary lobulo-alveolar growth; JV delivery of the same dose had no significant effect. Similarly, intrahepatic delivery of oPRL to pigeons potentiated the mitogenic effects of locally injected PRL on the crop-sac mucosa, while JV infusion of the same dose was ineffective (6). Evidence that the liver plays a role in PRL's antimetamorphic actions in amphibian tadpoles also has been reported (5). Finally, Griffen et al. (10) demonstrated that infusion of a low dose of insulin into the HPV, but not the JV, stimulated growth and elevated serum insulin-like growth factor-I concentrations in diabetic male rats. Although the other studies that involved intrahepatic infusion of oPRL (6, 7) used a pulse delivery schedule, whereas we employed a constant rate of delivery in our present study, this difference in schedule does not account for the lack of a preferential galactopoietic effect of HPV infusion. Studies in progress indicate that pulse delivery of PRL into the HPV is no more effective at restoring lactation in BC-suppressed dams than is JV delivery in pulses. Hoeffler and Frawley (8) reported that their liver lactogenic factor (LLF) increased plaque size (i.e. casein release from rat mammary cells), but not the numbers of plaques formed (i.e. cells secreting casein) in the reverse hemolytic plaque bioassay. Although PRL increased both parameters, LLF was more potent at stimulating casein secretion from cells already capable of that function. In addition, LLF was active in the absence of PRL. Frawley et al. (23) also mentioned some unpublished findings which indicate that injections of the purified LLF partially restored the lactational capacity of lactating rats given BC. Nevertheless, our present physiological studies do not support the hypothesis of hepatic mediation of PRL's galactopoietic action in vivo. Furthermore, in view of the evidence that the liver of lactating rats can secrete synlactin activity (24), and PRL injections stimulate the liver to secrete that activity (24), it seems unlikely that synlactin has a galactopoietic function in addition to its mitogenic activity (1-7, 24). However, it remains to be determined whether the liver participates in the initiation of lactation (i.e. has a lactogenic function) at the end of pregnancy. Acknowledgments The authors would like to thank Richard Lin for technical assistance in performing the RIAs and the preparation of this manuscript. We also are indebted to Dr. George Stabenfeldt of the Department of Veterinary Medical Reproduction of the University of California, Davis, for the antibody to oPRL. The purified oPRL and the reagents used in the RIAs were generous
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experiment. Thus, there is no evidence of a significant reduction in the bioactivity of the infused hormone as the experiments progressed. The experiment with male rats showed that in most cases the minipumps maintained relatively constant serum levels of oPRL in conscious animals over a period of 4 h. The one exceptional animal, which showed marked fluctuations, had a HPV catheter. The portal vasculature is the major contributor to the hepatic circulation, and the flow within it varies considerably with food intake, intestinal peristalsis, and other conditions (17,18). Thus, it is surprising that only one of the three rats with HPV catheters showed large fluctuations in its serum PRL levels. A difference in the final serum oPRL concentration in infused dams between the HPV and JV routes of delivery was seen only at the 600 Mg/day dose. The lack of a difference at the lower doses is surprising, as the liver is considered to be a major site of peptide hormone disposal, and infusion of the hormone into the HPV might be expected to activate hepatic clearance. Possibly, the hepatic clearance process is activated only at high concentrations of the hormone. The observed serum levels of oPRL at the end of the experiment can be compared with expected levels by using data in the literature. Grosvenor (19) reported that the ti/2 of oPRL in the lactating rat is 3 min, and the distribution volume (DV) in animals of the size used in our experiments is about 30 ml (20). The MCR = k x DV, where k = 0.693/t1/2 = 0.231 min"1. The expected hormone concentration, He, = infusion rate/MCR. Thus, at the highest dose given via the JV (600 /ig/rat-day), He = 417 /Lig/min • 6.93 ml min"1 = 60 ng/ml. This expected concentration is in excellent agreement with the observed value of 65 ng/ml. Grosvenor (19) also reported that the circulating ti/ 2 of oPRL was considerably greater in nonlactating females. Consequently, the higher serum oPRL concentration in male rats compared to that in the lactating females may be due to differences in MCR. Constant infusion of oPRL at a dose rate of 2.0 mg/ rat-day completely restored lactation in the BC-treated dams. In previous studies, twice daily injections of similar or higher doses of oPRL, with or without replacement therapy with other hormones, restored lactation to only about 50% of normal in hypophysectomized rats (21, 22). Thus, constant infusion of the hormone is apparently much more effective in this regard than twice daily injection. The failure of intrahepatic delivery of oPRL to have a greater restorative effect on the suppressed lactation of the BC-treated rats compared to the JV route of delivery was unexpected in view of the findings of Hoeffler and Frawley (8). It is unlikely that this lack of difference is due to technical problems with the infusion system,
1509
1510
ROLE OF LIVER IN GALACTOPOIETIC ACTION OF PRL
gifts from the National Hormone and Pituitary Program of the NIH.
References
12. Harms PG, Ojeda SR 1974 A rapid and simple procedure for chronic cannulation of the rat jugular vein. J Appl Physiol 36:391-392 13. Zar JH 1974 Biostatistical Analysis. Prentice-Hall, Englewood Cliffs 14. Knight CH, Calvert DT, Flint DJ 1986 Inhibitory effect of bromocriptine on mammary gland development and function in lactating mice. J Endocrinol 110:263-270 15. Fliickiger E, Wagner HR 1968 2-Br-a-Ergokryptin: beeinflussing von fertilitat und laktation bei der ratte. Experientia 24:1130-1131 16. Fliickiger E, Kovacs E 1974 Inhibition by 2-Br-a-ergocryptinemesilate (CB-154) of suckling-induced prolactin depletion in lactating rats. Experientia 30:1173 17. Rappaport AM 1980 Hepatic blood flow. In: Javitt NB (ed) International Review of Physiology. University Park Press, Baltimore, pp 1-65 18. Withrington PG, Richardson PDI 1986 Hepatic hemodynamics and microcirculation. In: Thurman RG, Kauffman FC, Jungermann K (eds) Regulation of Hepatic Metabolism-Intra- and Intercellular Compartmentation. Plenum Press, New York, pp 27-53 19. Grosvenor CE 1967 Disappearance rate of exogenous prolactin from serum of female rats. Endocrinology 80:195-200 20. Nicoll CS, Swearingen KC, Mattheij JAM 1984 Relationship between depletion and release of prolactin in the lactating rat: a quantitative analysis. In: Mena F, Valverde-R CM (ed) Prolactin Secretion: A Multidisciplinary Approach. Academic Press, New York, pp 285-301 21. Bintarningsih, Lyons WR, Johnson RE, Li CH 1958 Hormonallyinduced lactation in hypophysectomized rats. Endocrinology 63:540-548 22. Cowie AT, Tindal JS 1961 The maintenance of lactation in the rat after hypophysial anterior lobectomy during pregnancy. J Endocrinol 22:403-408 23. Frawley LS, Schwabe C, Miller III HA, Betts JG, Hoeffler JP, Simpson MT 1988 Characterization and physiological role of a liver lactogenic factor. In: Hoshino K (ed) Prolactin Gene Family and Its Receptors. Elsevier (Biomedical Division), Amsterdam, pp 49-59 24. Nicoll CS, Anderson TR, Hebert NJ, Russell SM 1985 Comparative aspects of the growth-promoting actions of prolactin on its target organs: evidence for synergism with an insulin-like growth factor. In: MacLeod RM, Thorner MO, Scapagnini U (ed) Prolactin. Basic and Clinical Correlates. Liviana Press, Padua, Fidia Res Ser, vol 1:393-410
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1. Anderson TR Rodriguez J, Nicoll CS, Spencer EM 1983 The synlactin hypothesis: prolactin's mitogenic action may involve synergism with a somatomedin-like molecule. In: Spencer EM (ed) Insulin-Like Growth Factors/Somatomedins. de Gruyter, Berlin, pp 71-78 2. Anderson TR, Pitts D, Nicoll CS 1984 Prolactin's mitogenic action on the pigeon crop-sac mucosal epithelium involves direct and indirect mechanisms. Gen Comp Endocrinol 54:236-246 3. Nicoll CS, Hebert NJ, Russell SM 1985 Lactogenic hormones stimulate the liver to secrete a factor that acts synergistically with prolactin to promote growth of the pigeon crop-sac mucosal epithelium in vivo. Endocrinology 116:1449-1453 4. Delidow BC, Hebert N, Steiny S, Nicoll CS 1986 Secretion of prolactin-synergizing activity (synlactin) by the liver of ectothermic vertebrates in vitro. J Exp Zool 238:147-153 5. Delidow BC, Baldocchi RA, Nicoll CS 1988 Evidence for hepatic involvement in the regulation of amphibian development by prolactin. Gen Comp Endocrinol 70:418-424 6. Mick CW, Nicoll CS 1985 Prolactin directly stimulates the liver in vivo to secrete a factor (synlactin) which acts synergistically with the hormone. Endocrinology 116:2049-2053 7. English DE, Russell SM, Katz L, Nicoll CS 1990 Evidence for a role of the liver in the mammotrophic action of prolactin. Endocrinology 126:2252-2256 8. Hoeffler JP, Frawley LS 1987 Liver tissue produces a potent lactogen that partially mimics the actions of prolactin. Endocrinology 120:1679-1681 9. Schlechter NL, Russell SM, Greenberg S, Spencer EM, Nicoll CS 1986 A direct growth effect of growth hormone in rat hindlimb shown by arterial infusion. Am J Physiol 250:E231-E253 10. Griffen SC, Russell SM, Katz LS, Nicoll CS 1987 Insulin exerts metabolic and growth-promoting effects by a direct action on the liver in vivo: clarification of the functional significance of the portal vascular link between the beta cells of the pancreatic islets and the liver. Proc Natl Acad Sci USA 84:7300-7304 11. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685
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