Effect of Prolactin on Sodium and Potassium Concentrations in Mammary Alveolar Tissue IAN R. FALCONER* AND JOHN M. ROWE Department of Biochemistry and Nutrition, University of Neiv England, Armidale, Australia 2351 ABSTRACT. A series of investigations is described in which changes in mammary tissue content of monovalent ions are measured following treatment with prolactin. These effects of prolactin are compared with the effects of ouabain and prolactin together. In vitro experiments were carried out using slices of lactating mammary gland from rabbits, held in continuously gassed (95% O2:5% CO2) Krebs bicarbonate buffer for 1 h in the presence or absence of prolactin (43.5 nM [lju,g/ml], ouabain (0.1 mM) or both. In the presence of prolactin [Na+] decreased and [K+] increased in the whole slices, and also when calculated as intracellular ion concentrations. Ouabain had the reverse effect; ouabain and prolactin together also had the reverse effect to prolactin alone. The tissue slices were sensitive to prolactin concentrations as low as 2.2 nM (50 ng/ml) in the incubation medium. The decreased [Na+] in the tissue slices showed a significant dose/response
I
relationship up to a prolactin concentration of 6.5 nM, above which no further decreases were observed. In vivo studies utilized rabbits in mid-pseudopregnancy, which were injected intraductally with prolactin (0.435 nmol [10 fig] per duct), ouabain (20 nmol per duct) or both. At 7.5 h later [I4C-]lactose was administered iv to provide measurements of extracellular volume, and at 8 h the rabbits were killed. Similarly to the in vitro experiment, prolactin caused a decrease in whole tissue and calculated intracellular [Na+] and an increase in [K+] compared to control glands. These effects were reversed by treatment with ouabain plus prolactin. It is concluded that prolactin activates the extrusion of Na+ from, and the entry of K+ into, mammary cells both in lactating and pre-lactating tissue. This effect can be reversed by ouabain. (Endocrinology 101: 181, 1977)
NTRACELLULAR changes associated with the stimulation by prolactin of mammary alveolar tissue have been shown to be preceded by an initial binding of the hormone to a surface receptor (1,2). However, unlike the majority of polypeptide hormones which activate or deactivate membrane-bound adenylate cyclase, no conclusive evidence exists to relate prolactin with a mechanism of this type (3,4). A survey of the comparative endocrinology of prolactin shows that it is implicated in the survival and adaptation of many marine species to variations in the osmotic environment (5). The importance of the pituitary in adaptive osmoregulation in fish was demonstrated by Epstein et at. (6) where hypophysectomy reduced the activity of the Na+/K+-ATPase located in the gill filaments of Fundulus heteroclitus. This in-
volement of prolactin in osmoregulation and ionic balance extends throughout the vertebrates to man (7) where studies by Horrobin et al. (8) have shown that ovine prolactin causes reduced renal excretion of sodium, potassium and water. The demonstrated action of prolactin on cation permeability and transport in gill and renal tissue of fish may therefore extend to the mammary alveolar cells, which supply cations to the milk. Considerable quantities of cations are secreted in milk, and the proportions vary during lactation (9-11). The present study was therefore undertaken to examine the effect of prolactin and ouabain on whole tissue and intracellular [Na + ], [K+] and [Cl] in vitro using lactating mammary gland slices, and in vivo by intraductal injections to midpseudopregnant rabbits.
Received November 15, 1976. Supported by the Nuffield Foundation, the NH & MRC of Australia and by the University of New England. *To whom reprint requests should be addressed.
Materials and Methods Hormones Prolactin (NIH P-S9), potency 30.3 IU/mg, growth hormone (NIH GH-S11), potency 0.56 IU/
181
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FALCONER AND ROWE
182
mg, were gifts from the Endocrinology Study Section, National Institutes of Health, Bethesda, Maryland. Bromocryptin (CB154) was a gift from Prof. E. Flukiges and Dr. H. Friedle, Sandoz, Basle; ouabain was obtained from Sigma, St. Louis, Mo. Animals Adult female rabbits were used either as virgin animals for induction of pseudopregnancy, or during the 2nd to 3rd week of lactation as a source of lactating tissue for slice incubations. In vitro studies Lactating rabbits 2-3 weeks post-partum were killed by cervical dislocation. Prior to killing bromocryptin [2-Br-a-Ergocryptine-mesilate (CB154)] was administered daily for 3 days at 1 mg/kg sc to inhibit prolactin secretion (12). Mammary gland tissue was removed immediately after killing and known weights of tissue were retained for determination of Na+, K+, Cl" and water content by the procedure described below. For experiments 500 mg quantities of tissue slices (approx. 2 x 5 x 0.7 mm thick, cut by a Mickle tissue chopper) were prepared, and transferred into conical flasks containing 25 ml Krebsbicarbonate buffer (with 4 ml rabbit serum/1), pH 7.4. Prolactin, growth hormone, bovine serum albumin (Commonwealth Serum Labs., Melbourne, Australia) and ouabain were dissolved in Krebs buffer and added to the appropriate flask together with 0.1 fxCi [14C]lactose, to provide measurement of the extracellular volume. After 1 h of incubation at 37 C with
continuous gassing with 5% CO2: 95% O2, a sample of the medium was taken for [I4C]lactose measurement and the incubation terminated by decanting the medium. In vivo studies Human chorionic gonadotrophin (100 IU in 0.5 ml sterile 0.9% saline) was administered iv to mature virgin estrous rabbits to induce pseudopregnancy. Intraductal injections of prolactin, ouabain or both at the 12th to 14th day of pseudopregnancy were carried out under anaesthesia, with tlie prolactin and ouabain dissolved in a solution of [Na + ], [K+] and [Cl"] similar to colostrum (13) containing Dextran Blue 2000 (1 mg/ml) in order to locate the injected glands at the time of removal. Each duct received 0.435 nmol (10 fig) prolactin, or 20 nmol ouabain or both. Several ducts were injected in each gland with 200 fx\ of solution and gently massaged to enhance distribution. Seven and one-half hours after the intraductal injections, 5 fxCi of [14C]lactose was injected through the marginal ear vein to provide measurement of the extracellular volume and the rabbit killed 30 min later by cervical dislocation. Blood samples were immediately collected into heparinized vessels and later centrifuged at 1000 x g for 15 min, and the plasma samples were retained for [14Cllactose measurement. Alveolar tissue associated with the injected duct systems was removed and samples taken for water content determinations and Na+, K+, Cl~ and [14C]lactose analysis.
TABLE 1. Monovalent ion concentration, water content and extracellular space in slices of lactating alveolar tissue from rabbits after incubation with increasing amounts of prolactin, growth hormone (43.5 x 10~9M [l.O/ttg/ml]) and albumin (1.0ju,g/ml)
Na +
K+
ci-
Water content ml/kg wet tissue; mean ± SEM
105.5 ± 3.4 100.5 ± 4.4 94.5 ± 3.3* 97.2 ± 2.1* 93.2 ± 3.4* 99.5 ± 3.9 103.2 ± 7.7
43.8 ± 3.1 48.0 ± 3.9 49.1 ± 2.0 47.6 ± 2.4 51.9 ± 4.8* 49.1 ± 4.2 47.2 ± 3.2
78.5 ± 3.8 81.0 + 7.2 67.8 + 9.3 74.8 + 3.0 77.3 ±3.1 73.5 ± 4.7 74.3 ± 2.4
748 ± 26 784 + 17 748 ± 13 760 ± 9 750 ± 15 759 + 21 737 ± 13
Monoval jnt ion concentration minol/kg wet tissue ; mean ± SEM Treatment
Calculated intracellular ion concentration - mmo 1/1 intracellular water; mean ± SEM
ci-
ratio
Extract llular space ml/kg we I IISMIL mean :t SEM
87.2 ± 9.8 84.3 + 16.9 64.2 + 21.4 76.5 ± 7.1 86.0 + 7.5 71.0 ± 11.2 76.2 ± 6.4
1.37 1.08 1.05 1.10 0.93* 1.07 1.13
336:t 3 4 357:t 18 3 1 5 :t 37 331 :t 27 327:t 20 344 :t 19 365:t 3 0
Na + /K + K
N
Prolactin concentration (M x 10") 0 2.2 6.5
17.4 43.5 Growth hormone Albumin
139.0 115.8 114.2 116.8 109.2 121.5 137.3
t t t t t ± +
8.2 10.2 7.5 4.9 8.0 9.3 20.7
101.2 it 107.7 it 109.0 ii 106.2 it 117.5 it 113.8:t 121.3 :t
7.4 9.1 4.7 11.3 9.9* 9.9 8.6
Two experiments, each treatment with 3 replicates (n = 6). Significance of difference between control and treated slices by analysis of variance * P < 0.05.
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PROLACTIN AND MONOVALENT CATIONS
183
Analytical procedure All tissue samples were digested in 4 ml of 10% acetic acid for 10 h at 100 C followed by centrifugation for 1 h at 1000 x g. Samples from the supernatant were taken for Na+ and K+ analysis using an atomic absorption spectrometer (AA100, Techtron Pty. Ltd., Melbourne, Australia) and Cl~ determined by titration (14). [14C]Lactose was counted in a scintillation fluid comprising toluene and Triton X100 (2:1) containing POPOP (0.1 g/1) and PPO (4 g/1).
110
= -16-5X*10496 = 0-47
IS ioo UJ
Results Effect of in vitro incubation of mammary tissue with or without prolactin, growth hormone or albumin The water content, and Na+ and Cl~ concentrations of the mammary gland slices increased and [K+] decreased following a 1 h incubation in Krebs' buffer compared to fresh tissue concentrations (Table 2). The increased water uptake was apparently due to the absence of a sufficient concentration of macromolecules to prevent water uptake in the slices by colloid osmotic pressure (15). The difference between the ionic content of fresh and incubated slices is due largely to the equilibrium of the ions present in the incubation medium with those in the milk space which has a lower [Na+] and [Cl~] and higher [K+]. Further ionic changes may be attributed to the lack of ionic pumps situated on the apical surface of alveolar cells allowing Na+, K+ and Cl~ to diffuse into the cell according to their electrochemical gradients (15). Prolactin in the range of 2.2-43.5 nM (50-1,000 ng/ml) markedly affected the whole tissue and calculated intracellular [Na+] and [K+] (Table 1). The whole tissue [Na+] decreased by 5 mmol/kg wet tissue at a prolactin concentration of 2.2 nM, with a further decrease of 6 mmol/kg wet tissue at 6.5 nM prolactin. A regression analysis showed the decrease in the Na+ content to be significantly correlated with increasing prolactin concentration up to 6.5 nM (Fig. 1). The whole tissue and calcu-
z
90
< cc UJ
z o o
80
0 PROLACTIN
A
8
CONCENTRATION (nM)
FIG. 1. Relationship between prolactin concentration in the incubation medium and the Na+ concentration of mammary alveolar tissue slices from a lactating rabbit after 1 h at 37 C in Krebs-bicarbonate buffer.
lated intracellular [K+] increased with increasing prolactin concentrations up to 43.5 nM. No significant change was observed in the total tissue and calculated intracellular [Cl~] concentration over the range of prolactin concentrations. A similar response to prolactin at 2.2 nM was seen with growth hormone at 43.5 nM in the incubation medium (Table 1). However, the effect of growth hormone was not significant. Albumin (1/Ag/ml) had no significant effect on either the whole tissue slice or calculated intracellular [Na + ], [K+] or [Cl"] (Table 1). Effects of in vitro incubation of mammary tissue with ouabain and prolactin, or ouabain alone Incubated slices of lactating mammary glands showed a Na+ increase of 22 and a
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FALCONER AND ROWE
184
TABLE 2. Monovalent ion concentration, water content and extracellular space in slices of lactating mammary alveolar tissue from rabbits after incubation in the presence and absence of prolactin (43.5 x 10~9 M [1.0/ig/ml]) and/or ouabain (0.1 HIM) Extracellular space ml/kg wet tissue; mean ± SEM
Na +
K+
ci-
mean ± SEM
Na+
K+
ci-
54.6 ± 4.0 107.3 ± 2.2 98.6 ± 1.9*
74.3 + 4.0 46.8 ± 1.3 50.5 ± 1.6t
54.1 ± 1.4 80.9 ± 1.8 77.0 ± 2.1
717 ± 4.6 764 ± 12.9 773 + 11.4
— 137.2 ± 6.3 109.2 ± 5.2*
— 126.1 ± 3.6 132.5 ± 4.5*
—
—
—
82.2 ± 5.8 69.1 ± 6.6
1.09 0.82f
412 ± 14.9 411 ± 10.7
129.4 ± 3.8f 133.1 ±3.1f
19.8 ± 0.9f 21.0 ± l.Of
79.8 + 2.4 80.3 ± 2.3
775 ± 11.8 773 ± 11.6
192.1 ± 9.9t 204.7 + 8.9f
45.8 ± 2.41 78.0 ± 6.9 50.4 ± 2.6f 79.4 ± 6.9
4.19f 4.06f
393 ± 14.8 404 + 13.7
Treatment Tissue before incubation Control Prolactin Prolactin + Ouabain Ouabain
Calculated intracellular ion concentration - mmol/1 intracellular water; meai ± SEM
Water content ml/kg wet tissue;
Monovalent ion concentration mmol/kg wet tissue: mean ± SEM
ratio
Five experiments, each treatment with 5 replicates (n = 25). Significance of difference between control and treated slices by analysis of variance * P < 0.05, f P < 0.01.
K+ decrease of 27 mmol/kg wet tissue in re- crease of 10 mmol/kg wet tissue (Table 3). sponse to a combination of ouabain (0.1 mM) The calculated intracellular [Na+] decrease and prolactin (43.5 nM) in the medium and [K+] increase were 31 and 28 mmol/1 (Table 2). The calculated intracellular [Na+] intracellular water, respectively (Table 3). increase and [K+] decrease were 55, and 80 In both experiments, prolactin showed no mmol/1 intracellular water, respectively. significant effect on the [Cl~] content. The Cl~ of the tissue slices did not change Mammary glands which received intrasignificantly (Table 2). ductal injections of ouabain (20 nmol/duct) The effect of ouabain alone on the mono- and prolactin (0.435 nmol/duct) showed a valent ion content of mammary gland slices [Na+] increase of 12 and [K+] decrease of 6 closely resembled that of ouabain plus pro- mmol/kg wet tissue (Table 3). The calculated lactin (Table 2). intracellular [Na+] increase and [K+] decrease were 41 and 20 mmol/1 intracellular Effects in vivo of prolactin and/or ouabain water, respectively (Table 3). The increased administration to mammary glands of extracellular water content of the ouabainpseudopregnant rabbits treated glands (for which there is no obvious Mammary glands of mid-pseudopregnant explanation) effectively masked changes in rabbits which received intraductal in- intracellular ion concentration due to this jections of prolactin (0.435 nmol/duct) treatment (Table 3). In general there was showed a [Na+] decrease of 11 and [K+] in- no obvious effect of either prolactin or TABLE 3. Monovalent ion concentration and extracellular space in mammary alveolar tissue from mid-pseudopregnant rabbits after intraductal injections of prolactin (0.435 nmol [10 fig]) and/or ouabain (20 nmol), or neither, in a solution of ionic composition resembling colostrum Monovalent ion concentration mmol/kg wet tissue; mean ± SEM Treatment
Na +
K+
Not injected Control injection Prolactin Prolactin + Ouabain Ouabain
92.4 ± 2.7 96.2 + 3.0 85.2 + 2.4 f 107.7 + 4.5f 104.4 ± 1.9*
36.1 + 2.3 33.2 ± 1.6 42.9 ± 1.8* 26.9 ± 1.5* 30.1 + 1.3
ci76.7 ± 3.5 75.2 + 3.0 74.3 + 2.2 • 79.0 + 2.9 75.1 + 3.5
Calculated intracellular ion concentration — mmol/1 intracellular water; mean + SEM Na+ 87.5 ± 95.2 + 64.0 + 136.0 + 122.9 +
K+ 8.8 10.4 7.6* 15.6* 7.0*
107.2 ± 7.4 102.8 + 5.4 130.8 + 5.9f 83.1 + 5.3* 100.4 + 4.6
ci94.7 it 88.1 it 88.6 it 102.9 :t 83.7 it
11.1 10.8 7.6 10.0 13.2
Na+/K+ ratio
Extracellular space ml/kg wet tissue; mean ± SEM
0.82 0.92 0.49 f 1.64 f 1.22*
428 + 39 443 + 25 432 + 20 449 ± 32 469 + 28
Four experiments, each treatment with 5 replicates (n = 20). Significance of difference between control and treated glands by analysis of variance * P < 0.05, f P < 0.01.
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PROLACTIN AND MONOVALENT CATIONS
185
the hypothesis that prolactin controls the transport of Na+ across the basal membranes of the mammary alveolar cell, and thus the intracellular Na+ content and Na+:K+ ratio Discussion of the cells. Our studies provide no inFrom these results we conclude that in formation on the existence of "tight" or vitro and in vivo prolactin has a significant "leaky" cell junctions in mammary alveoli influence upon the Na+ and K+ content (and as described by Taylor et al. (11). Since prolactin apparently activates the therefore Na+/K+ ratio) of mammary alveoouabain-sensitive Na+/K+ pump, this offers lar tissue. The action of prolactin upon the + + intracellular [Na ] and [K ] is in opposition an effective method + by+ which relatively to that of ouabain. This would suggest that large changes in Na /K ratio within the prolactin modulates these changes in intra- cell may occur. Such an effect could act cellular ions by way of the Na+/K+-ATPase as an intracellular "messenger" for prolacsince ouabain is a specific inhibitor of this tin, since variations in intracellular Na/K enzyme in mammary as well as other tis- ratio have been shown to have marked efsues (15). The presence of both the Na+/ fects on a number of key biosynthetic sysK+-ATPase (16) and the prolactin receptors tems. Evidence of this role of intracellu(1) in the basal region of the plasma mem- lar monovalent cations is provided by Lubin brane of alveolar cells is consistent with this (18), who demonstrated that the rate of protein synthesis was regulated by the intrasuggestion. cellular potassium concentration in EsThe conclusions drawn from the data of cherichia coli B and Bacillus subtilis. The this paper are in agreement with our earlier same author, using a tissue culture of aniexperiments in which prolactin was shown 22 + mal cells incubated in Amphotericin B to decrease the content of Na in in(which produces graded leaks of small ions), cubated slices of lactating mammary glands demonstrated a depression in the rates of (17). The inclusion of ouabain in the earlier protein and DNA synthesis which paralexperiments also blocked the action of 22 + leled the intracellular potassium loss caused prolactin and raised the content of Na by Amphotericin B. Quastel and Kaplan in the slices, thus again indicating a pos+ + (19), studying lymphocyte activation by sible role of the Na /K -ATPase in the acphytohaemagglutinin, found that the stimution of prolactin. lated synthesis of DNA, RNA and protein Evidence for changes in sodium transport was inhibited to almost the same extent by into the milk in lactation which respond to low concentrations of ouabain. This inprolactin, has been provided by Gachev (9) hibition could be prevented by increasing and Taylor et at. (11), who showed that in the K+ content of the culture medium. They late lactation in the rabbit there is a fall in concluded that activation of the Na+/K+ milk lactose and K+ concentrations, and ATPase system was involved with the an increase in Na+ and Cl~ concentra- process of lymphocyte stimulation, and was tions. These changes could be reversed by critically dependent upon increased intragiving prolactin. Since the ionic composition cellular potassium. Cellular growth has also of normal milk (at any rate for Na+ and K+) been found to be directly related to the level reflects the intracellular ionic composition, of activity of the Na+/K+-ATPase in mouse it is likely that the monovalent cation con- lymphoblasts (20). centration in milk is significantly regulated by ion transport at the basal membrane of References the cell. Thus, both the in vitro and in vivo studies reported here, and the in vivo studies 1. Birkinshaw, M., and I. R. Falconer, J Enclocrinol 55: 323, 1972. of milk composition quoted above, support ouabain on the extracellular space or water content of mammary tissue in vivo or in vitro.
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186
FALCONER AND ROWE
2. Turkington, R. W., Biochem Biophys Res Commun 41: 1362, 1970. 3. Turkington, R. W., In Wolstenholme, G. E. W., and J. Knights (eds.), Lactogenic Hormones, Churchill-Livingstone, Edinburgh and London, 1972, p. 111. 4. Sapag-Hagar, M., and R. L. Greenbaum, Eur J Biochem 47: 303, 1974. 5. Bern, H. A., and C. S. Nicoll, Recent Prog Horm Res 24: 681, 1968. 6. Epstein, F. H., A. I. Katz, and G. W. Pickford, Science 156: 1245, 1967. 7. Bern, H. A., Am Zool 15: 937, 1975. 8. Horrobin, D. F., I. U. Lloyd, A. Lipton, P. G. Burstyn, M. Durkin, and K. L. Muiruri, Lancet 2: 352, 1971. 9. Gachev, M., Zh Obshch Biol 24: 382, 1963. 10. Linzell, J. L., and M. Peaker, Physiol Rev 51: 564, 1971.
Endo • 1977 Vol 101 • No 1
11. Taylor, J. C., M. Peaker, and J. L. Linzell,7 Endocrinol 65: 26P, 1975. 12. Hart, I. C . J Endocrinol 57: 179, 1973. 13. Coates, M. E., M. E. Gregory, and S. B. Thompson, BrJ Nutr 18: 583, 1964. 14. Cotlove, E., V. Tranthan, and R. L. Bowman, 7 Lab Clin Med 50: 358, 1958. 15. Vreeswijk, J. H. A., J. J. H. H. M. de Pont, and S. C. Bonting, Pflugers Arch 356: 347, 1975. 16. Kinura, T.JJap Obstet Gynaecol Soc 21: 301,1967. 17. Falconer, I. R., and J. M. Rowe, Nature 256: 327, 1975. 18. Lubin, M., Nature 213: 451, 1967. 19. Quastel, M. R., and J. G. Kaplan, Exp Cell Res 62: 407, 1970. 20. Shank, B. B., and N. E. Smith, J Cell Physiol 87: 377, 1976.
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I
relationship up to a prolactin concentration of 6.5 nM, above which no further decreases were observed. In vivo studies utilized rabbits in mid-pseudopregnancy, which were injected intraductally with prolactin (0.435 nmol [10 fig] per duct), ouabain (20 nmol per duct) or both. At 7.5 h later [I4C-]lactose was administered iv to provide measurements of extracellular volume, and at 8 h the rabbits were killed. Similarly to the in vitro experiment, prolactin caused a decrease in whole tissue and calculated intracellular [Na+] and an increase in [K+] compared to control glands. These effects were reversed by treatment with ouabain plus prolactin. It is concluded that prolactin activates the extrusion of Na+ from, and the entry of K+ into, mammary cells both in lactating and pre-lactating tissue. This effect can be reversed by ouabain. (Endocrinology 101: 181, 1977)
NTRACELLULAR changes associated with the stimulation by prolactin of mammary alveolar tissue have been shown to be preceded by an initial binding of the hormone to a surface receptor (1,2). However, unlike the majority of polypeptide hormones which activate or deactivate membrane-bound adenylate cyclase, no conclusive evidence exists to relate prolactin with a mechanism of this type (3,4). A survey of the comparative endocrinology of prolactin shows that it is implicated in the survival and adaptation of many marine species to variations in the osmotic environment (5). The importance of the pituitary in adaptive osmoregulation in fish was demonstrated by Epstein et at. (6) where hypophysectomy reduced the activity of the Na+/K+-ATPase located in the gill filaments of Fundulus heteroclitus. This in-
volement of prolactin in osmoregulation and ionic balance extends throughout the vertebrates to man (7) where studies by Horrobin et al. (8) have shown that ovine prolactin causes reduced renal excretion of sodium, potassium and water. The demonstrated action of prolactin on cation permeability and transport in gill and renal tissue of fish may therefore extend to the mammary alveolar cells, which supply cations to the milk. Considerable quantities of cations are secreted in milk, and the proportions vary during lactation (9-11). The present study was therefore undertaken to examine the effect of prolactin and ouabain on whole tissue and intracellular [Na + ], [K+] and [Cl] in vitro using lactating mammary gland slices, and in vivo by intraductal injections to midpseudopregnant rabbits.
Received November 15, 1976. Supported by the Nuffield Foundation, the NH & MRC of Australia and by the University of New England. *To whom reprint requests should be addressed.
Materials and Methods Hormones Prolactin (NIH P-S9), potency 30.3 IU/mg, growth hormone (NIH GH-S11), potency 0.56 IU/
181
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Endo • 1977 Vol 101 • No 1
FALCONER AND ROWE
182
mg, were gifts from the Endocrinology Study Section, National Institutes of Health, Bethesda, Maryland. Bromocryptin (CB154) was a gift from Prof. E. Flukiges and Dr. H. Friedle, Sandoz, Basle; ouabain was obtained from Sigma, St. Louis, Mo. Animals Adult female rabbits were used either as virgin animals for induction of pseudopregnancy, or during the 2nd to 3rd week of lactation as a source of lactating tissue for slice incubations. In vitro studies Lactating rabbits 2-3 weeks post-partum were killed by cervical dislocation. Prior to killing bromocryptin [2-Br-a-Ergocryptine-mesilate (CB154)] was administered daily for 3 days at 1 mg/kg sc to inhibit prolactin secretion (12). Mammary gland tissue was removed immediately after killing and known weights of tissue were retained for determination of Na+, K+, Cl" and water content by the procedure described below. For experiments 500 mg quantities of tissue slices (approx. 2 x 5 x 0.7 mm thick, cut by a Mickle tissue chopper) were prepared, and transferred into conical flasks containing 25 ml Krebsbicarbonate buffer (with 4 ml rabbit serum/1), pH 7.4. Prolactin, growth hormone, bovine serum albumin (Commonwealth Serum Labs., Melbourne, Australia) and ouabain were dissolved in Krebs buffer and added to the appropriate flask together with 0.1 fxCi [14C]lactose, to provide measurement of the extracellular volume. After 1 h of incubation at 37 C with
continuous gassing with 5% CO2: 95% O2, a sample of the medium was taken for [I4C]lactose measurement and the incubation terminated by decanting the medium. In vivo studies Human chorionic gonadotrophin (100 IU in 0.5 ml sterile 0.9% saline) was administered iv to mature virgin estrous rabbits to induce pseudopregnancy. Intraductal injections of prolactin, ouabain or both at the 12th to 14th day of pseudopregnancy were carried out under anaesthesia, with tlie prolactin and ouabain dissolved in a solution of [Na + ], [K+] and [Cl"] similar to colostrum (13) containing Dextran Blue 2000 (1 mg/ml) in order to locate the injected glands at the time of removal. Each duct received 0.435 nmol (10 fig) prolactin, or 20 nmol ouabain or both. Several ducts were injected in each gland with 200 fx\ of solution and gently massaged to enhance distribution. Seven and one-half hours after the intraductal injections, 5 fxCi of [14C]lactose was injected through the marginal ear vein to provide measurement of the extracellular volume and the rabbit killed 30 min later by cervical dislocation. Blood samples were immediately collected into heparinized vessels and later centrifuged at 1000 x g for 15 min, and the plasma samples were retained for [14Cllactose measurement. Alveolar tissue associated with the injected duct systems was removed and samples taken for water content determinations and Na+, K+, Cl~ and [14C]lactose analysis.
TABLE 1. Monovalent ion concentration, water content and extracellular space in slices of lactating alveolar tissue from rabbits after incubation with increasing amounts of prolactin, growth hormone (43.5 x 10~9M [l.O/ttg/ml]) and albumin (1.0ju,g/ml)
Na +
K+
ci-
Water content ml/kg wet tissue; mean ± SEM
105.5 ± 3.4 100.5 ± 4.4 94.5 ± 3.3* 97.2 ± 2.1* 93.2 ± 3.4* 99.5 ± 3.9 103.2 ± 7.7
43.8 ± 3.1 48.0 ± 3.9 49.1 ± 2.0 47.6 ± 2.4 51.9 ± 4.8* 49.1 ± 4.2 47.2 ± 3.2
78.5 ± 3.8 81.0 + 7.2 67.8 + 9.3 74.8 + 3.0 77.3 ±3.1 73.5 ± 4.7 74.3 ± 2.4
748 ± 26 784 + 17 748 ± 13 760 ± 9 750 ± 15 759 + 21 737 ± 13
Monoval jnt ion concentration minol/kg wet tissue ; mean ± SEM Treatment
Calculated intracellular ion concentration - mmo 1/1 intracellular water; mean ± SEM
ci-
ratio
Extract llular space ml/kg we I IISMIL mean :t SEM
87.2 ± 9.8 84.3 + 16.9 64.2 + 21.4 76.5 ± 7.1 86.0 + 7.5 71.0 ± 11.2 76.2 ± 6.4
1.37 1.08 1.05 1.10 0.93* 1.07 1.13
336:t 3 4 357:t 18 3 1 5 :t 37 331 :t 27 327:t 20 344 :t 19 365:t 3 0
Na + /K + K
N
Prolactin concentration (M x 10") 0 2.2 6.5
17.4 43.5 Growth hormone Albumin
139.0 115.8 114.2 116.8 109.2 121.5 137.3
t t t t t ± +
8.2 10.2 7.5 4.9 8.0 9.3 20.7
101.2 it 107.7 it 109.0 ii 106.2 it 117.5 it 113.8:t 121.3 :t
7.4 9.1 4.7 11.3 9.9* 9.9 8.6
Two experiments, each treatment with 3 replicates (n = 6). Significance of difference between control and treated slices by analysis of variance * P < 0.05.
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PROLACTIN AND MONOVALENT CATIONS
183
Analytical procedure All tissue samples were digested in 4 ml of 10% acetic acid for 10 h at 100 C followed by centrifugation for 1 h at 1000 x g. Samples from the supernatant were taken for Na+ and K+ analysis using an atomic absorption spectrometer (AA100, Techtron Pty. Ltd., Melbourne, Australia) and Cl~ determined by titration (14). [14C]Lactose was counted in a scintillation fluid comprising toluene and Triton X100 (2:1) containing POPOP (0.1 g/1) and PPO (4 g/1).
110
= -16-5X*10496 = 0-47
IS ioo UJ
Results Effect of in vitro incubation of mammary tissue with or without prolactin, growth hormone or albumin The water content, and Na+ and Cl~ concentrations of the mammary gland slices increased and [K+] decreased following a 1 h incubation in Krebs' buffer compared to fresh tissue concentrations (Table 2). The increased water uptake was apparently due to the absence of a sufficient concentration of macromolecules to prevent water uptake in the slices by colloid osmotic pressure (15). The difference between the ionic content of fresh and incubated slices is due largely to the equilibrium of the ions present in the incubation medium with those in the milk space which has a lower [Na+] and [Cl~] and higher [K+]. Further ionic changes may be attributed to the lack of ionic pumps situated on the apical surface of alveolar cells allowing Na+, K+ and Cl~ to diffuse into the cell according to their electrochemical gradients (15). Prolactin in the range of 2.2-43.5 nM (50-1,000 ng/ml) markedly affected the whole tissue and calculated intracellular [Na+] and [K+] (Table 1). The whole tissue [Na+] decreased by 5 mmol/kg wet tissue at a prolactin concentration of 2.2 nM, with a further decrease of 6 mmol/kg wet tissue at 6.5 nM prolactin. A regression analysis showed the decrease in the Na+ content to be significantly correlated with increasing prolactin concentration up to 6.5 nM (Fig. 1). The whole tissue and calcu-
z
90
< cc UJ
z o o
80
0 PROLACTIN
A
8
CONCENTRATION (nM)
FIG. 1. Relationship between prolactin concentration in the incubation medium and the Na+ concentration of mammary alveolar tissue slices from a lactating rabbit after 1 h at 37 C in Krebs-bicarbonate buffer.
lated intracellular [K+] increased with increasing prolactin concentrations up to 43.5 nM. No significant change was observed in the total tissue and calculated intracellular [Cl~] concentration over the range of prolactin concentrations. A similar response to prolactin at 2.2 nM was seen with growth hormone at 43.5 nM in the incubation medium (Table 1). However, the effect of growth hormone was not significant. Albumin (1/Ag/ml) had no significant effect on either the whole tissue slice or calculated intracellular [Na + ], [K+] or [Cl"] (Table 1). Effects of in vitro incubation of mammary tissue with ouabain and prolactin, or ouabain alone Incubated slices of lactating mammary glands showed a Na+ increase of 22 and a
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Endo • 1977 Vol 101 • No 1
FALCONER AND ROWE
184
TABLE 2. Monovalent ion concentration, water content and extracellular space in slices of lactating mammary alveolar tissue from rabbits after incubation in the presence and absence of prolactin (43.5 x 10~9 M [1.0/ig/ml]) and/or ouabain (0.1 HIM) Extracellular space ml/kg wet tissue; mean ± SEM
Na +
K+
ci-
mean ± SEM
Na+
K+
ci-
54.6 ± 4.0 107.3 ± 2.2 98.6 ± 1.9*
74.3 + 4.0 46.8 ± 1.3 50.5 ± 1.6t
54.1 ± 1.4 80.9 ± 1.8 77.0 ± 2.1
717 ± 4.6 764 ± 12.9 773 + 11.4
— 137.2 ± 6.3 109.2 ± 5.2*
— 126.1 ± 3.6 132.5 ± 4.5*
—
—
—
82.2 ± 5.8 69.1 ± 6.6
1.09 0.82f
412 ± 14.9 411 ± 10.7
129.4 ± 3.8f 133.1 ±3.1f
19.8 ± 0.9f 21.0 ± l.Of
79.8 + 2.4 80.3 ± 2.3
775 ± 11.8 773 ± 11.6
192.1 ± 9.9t 204.7 + 8.9f
45.8 ± 2.41 78.0 ± 6.9 50.4 ± 2.6f 79.4 ± 6.9
4.19f 4.06f
393 ± 14.8 404 + 13.7
Treatment Tissue before incubation Control Prolactin Prolactin + Ouabain Ouabain
Calculated intracellular ion concentration - mmol/1 intracellular water; meai ± SEM
Water content ml/kg wet tissue;
Monovalent ion concentration mmol/kg wet tissue: mean ± SEM
ratio
Five experiments, each treatment with 5 replicates (n = 25). Significance of difference between control and treated slices by analysis of variance * P < 0.05, f P < 0.01.
K+ decrease of 27 mmol/kg wet tissue in re- crease of 10 mmol/kg wet tissue (Table 3). sponse to a combination of ouabain (0.1 mM) The calculated intracellular [Na+] decrease and prolactin (43.5 nM) in the medium and [K+] increase were 31 and 28 mmol/1 (Table 2). The calculated intracellular [Na+] intracellular water, respectively (Table 3). increase and [K+] decrease were 55, and 80 In both experiments, prolactin showed no mmol/1 intracellular water, respectively. significant effect on the [Cl~] content. The Cl~ of the tissue slices did not change Mammary glands which received intrasignificantly (Table 2). ductal injections of ouabain (20 nmol/duct) The effect of ouabain alone on the mono- and prolactin (0.435 nmol/duct) showed a valent ion content of mammary gland slices [Na+] increase of 12 and [K+] decrease of 6 closely resembled that of ouabain plus pro- mmol/kg wet tissue (Table 3). The calculated lactin (Table 2). intracellular [Na+] increase and [K+] decrease were 41 and 20 mmol/1 intracellular Effects in vivo of prolactin and/or ouabain water, respectively (Table 3). The increased administration to mammary glands of extracellular water content of the ouabainpseudopregnant rabbits treated glands (for which there is no obvious Mammary glands of mid-pseudopregnant explanation) effectively masked changes in rabbits which received intraductal in- intracellular ion concentration due to this jections of prolactin (0.435 nmol/duct) treatment (Table 3). In general there was showed a [Na+] decrease of 11 and [K+] in- no obvious effect of either prolactin or TABLE 3. Monovalent ion concentration and extracellular space in mammary alveolar tissue from mid-pseudopregnant rabbits after intraductal injections of prolactin (0.435 nmol [10 fig]) and/or ouabain (20 nmol), or neither, in a solution of ionic composition resembling colostrum Monovalent ion concentration mmol/kg wet tissue; mean ± SEM Treatment
Na +
K+
Not injected Control injection Prolactin Prolactin + Ouabain Ouabain
92.4 ± 2.7 96.2 + 3.0 85.2 + 2.4 f 107.7 + 4.5f 104.4 ± 1.9*
36.1 + 2.3 33.2 ± 1.6 42.9 ± 1.8* 26.9 ± 1.5* 30.1 + 1.3
ci76.7 ± 3.5 75.2 + 3.0 74.3 + 2.2 • 79.0 + 2.9 75.1 + 3.5
Calculated intracellular ion concentration — mmol/1 intracellular water; mean + SEM Na+ 87.5 ± 95.2 + 64.0 + 136.0 + 122.9 +
K+ 8.8 10.4 7.6* 15.6* 7.0*
107.2 ± 7.4 102.8 + 5.4 130.8 + 5.9f 83.1 + 5.3* 100.4 + 4.6
ci94.7 it 88.1 it 88.6 it 102.9 :t 83.7 it
11.1 10.8 7.6 10.0 13.2
Na+/K+ ratio
Extracellular space ml/kg wet tissue; mean ± SEM
0.82 0.92 0.49 f 1.64 f 1.22*
428 + 39 443 + 25 432 + 20 449 ± 32 469 + 28
Four experiments, each treatment with 5 replicates (n = 20). Significance of difference between control and treated glands by analysis of variance * P < 0.05, f P < 0.01.
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PROLACTIN AND MONOVALENT CATIONS
185
the hypothesis that prolactin controls the transport of Na+ across the basal membranes of the mammary alveolar cell, and thus the intracellular Na+ content and Na+:K+ ratio Discussion of the cells. Our studies provide no inFrom these results we conclude that in formation on the existence of "tight" or vitro and in vivo prolactin has a significant "leaky" cell junctions in mammary alveoli influence upon the Na+ and K+ content (and as described by Taylor et al. (11). Since prolactin apparently activates the therefore Na+/K+ ratio) of mammary alveoouabain-sensitive Na+/K+ pump, this offers lar tissue. The action of prolactin upon the + + intracellular [Na ] and [K ] is in opposition an effective method + by+ which relatively to that of ouabain. This would suggest that large changes in Na /K ratio within the prolactin modulates these changes in intra- cell may occur. Such an effect could act cellular ions by way of the Na+/K+-ATPase as an intracellular "messenger" for prolacsince ouabain is a specific inhibitor of this tin, since variations in intracellular Na/K enzyme in mammary as well as other tis- ratio have been shown to have marked efsues (15). The presence of both the Na+/ fects on a number of key biosynthetic sysK+-ATPase (16) and the prolactin receptors tems. Evidence of this role of intracellu(1) in the basal region of the plasma mem- lar monovalent cations is provided by Lubin brane of alveolar cells is consistent with this (18), who demonstrated that the rate of protein synthesis was regulated by the intrasuggestion. cellular potassium concentration in EsThe conclusions drawn from the data of cherichia coli B and Bacillus subtilis. The this paper are in agreement with our earlier same author, using a tissue culture of aniexperiments in which prolactin was shown 22 + mal cells incubated in Amphotericin B to decrease the content of Na in in(which produces graded leaks of small ions), cubated slices of lactating mammary glands demonstrated a depression in the rates of (17). The inclusion of ouabain in the earlier protein and DNA synthesis which paralexperiments also blocked the action of 22 + leled the intracellular potassium loss caused prolactin and raised the content of Na by Amphotericin B. Quastel and Kaplan in the slices, thus again indicating a pos+ + (19), studying lymphocyte activation by sible role of the Na /K -ATPase in the acphytohaemagglutinin, found that the stimution of prolactin. lated synthesis of DNA, RNA and protein Evidence for changes in sodium transport was inhibited to almost the same extent by into the milk in lactation which respond to low concentrations of ouabain. This inprolactin, has been provided by Gachev (9) hibition could be prevented by increasing and Taylor et at. (11), who showed that in the K+ content of the culture medium. They late lactation in the rabbit there is a fall in concluded that activation of the Na+/K+ milk lactose and K+ concentrations, and ATPase system was involved with the an increase in Na+ and Cl~ concentra- process of lymphocyte stimulation, and was tions. These changes could be reversed by critically dependent upon increased intragiving prolactin. Since the ionic composition cellular potassium. Cellular growth has also of normal milk (at any rate for Na+ and K+) been found to be directly related to the level reflects the intracellular ionic composition, of activity of the Na+/K+-ATPase in mouse it is likely that the monovalent cation con- lymphoblasts (20). centration in milk is significantly regulated by ion transport at the basal membrane of References the cell. Thus, both the in vitro and in vivo studies reported here, and the in vivo studies 1. Birkinshaw, M., and I. R. Falconer, J Enclocrinol 55: 323, 1972. of milk composition quoted above, support ouabain on the extracellular space or water content of mammary tissue in vivo or in vitro.
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186
FALCONER AND ROWE
2. Turkington, R. W., Biochem Biophys Res Commun 41: 1362, 1970. 3. Turkington, R. W., In Wolstenholme, G. E. W., and J. Knights (eds.), Lactogenic Hormones, Churchill-Livingstone, Edinburgh and London, 1972, p. 111. 4. Sapag-Hagar, M., and R. L. Greenbaum, Eur J Biochem 47: 303, 1974. 5. Bern, H. A., and C. S. Nicoll, Recent Prog Horm Res 24: 681, 1968. 6. Epstein, F. H., A. I. Katz, and G. W. Pickford, Science 156: 1245, 1967. 7. Bern, H. A., Am Zool 15: 937, 1975. 8. Horrobin, D. F., I. U. Lloyd, A. Lipton, P. G. Burstyn, M. Durkin, and K. L. Muiruri, Lancet 2: 352, 1971. 9. Gachev, M., Zh Obshch Biol 24: 382, 1963. 10. Linzell, J. L., and M. Peaker, Physiol Rev 51: 564, 1971.
Endo • 1977 Vol 101 • No 1
11. Taylor, J. C., M. Peaker, and J. L. Linzell,7 Endocrinol 65: 26P, 1975. 12. Hart, I. C . J Endocrinol 57: 179, 1973. 13. Coates, M. E., M. E. Gregory, and S. B. Thompson, BrJ Nutr 18: 583, 1964. 14. Cotlove, E., V. Tranthan, and R. L. Bowman, 7 Lab Clin Med 50: 358, 1958. 15. Vreeswijk, J. H. A., J. J. H. H. M. de Pont, and S. C. Bonting, Pflugers Arch 356: 347, 1975. 16. Kinura, T.JJap Obstet Gynaecol Soc 21: 301,1967. 17. Falconer, I. R., and J. M. Rowe, Nature 256: 327, 1975. 18. Lubin, M., Nature 213: 451, 1967. 19. Quastel, M. R., and J. G. Kaplan, Exp Cell Res 62: 407, 1970. 20. Shank, B. B., and N. E. Smith, J Cell Physiol 87: 377, 1976.
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