Proc. NatI. Acad. Sc. USA
Vol. 76, No. 1, pp. 536-540, January 1979 Physiological Sciences
Mouse mammary epithelial cells on floating collagen gels: Transepithelial ion transport and effects of prolactin (active Na+ transport/primary cell culture/midpregnant cells/cell differentiation/milk ions)
CHESTER A. BISBEE*, TERRY E. MACHENt, AND HOWARD A. BERN* *Department of Zoology and Cancer Research Laboratory and tDepartment of Physiology-Anatomy, University of California, Berkeley, California 94720
Contributed by Howard A. Bern, October 12, 1978
ABSTRACT Epithelial cells dissociated from midpregnant BALB/c mouse mammary glands were cultured for as long as 20 days as confluent monolayers on floating collagen gels. Detached gels bearing ihonolayers were placed in lucite Ussing chambers for measurement of transepithelial potential difference (PD), short-circuit current (I¢), resistance (R), and unidirectional fluxes of Na+ and Cl- during short-circuit current conditions (PD = 0). With Hanks' solution bathing both sides of cultures maintained with insulin and cortisol, PD = -12.8 mV (serosal side ground), IC = 24.6 &A/cm2, and R = 507 flcm2. Net absorption of Na+ equaled Ic, and there was no net Cltransport. PD and ISc were reduced 50% by mucosal addition of 10 &M amiloride and to zero by metabolic inhibition with nitrogen gas or by serosal addition of 0.1 mM ouabain. In similar cultures supplemented with prolactin, PD and ISC increased to -15.8 mV and 48.0 ,uA/cm2, respectively, and R decreased to 374 Qlcm2. Inhibitor effects were similar to those seen in prolactin-free cultures. Prolactin exposure resulted in a 3-fold increase in net absorption of Na+. Na+ absorption was not equivalent to ISC, and there was little Cl- absorption; therefore, prolactin induced active transport of other, as yet unidentified, ions. These effects of prolactin require at least 3 days to occur and cannot be attributed to the known contamination with neurohyophysial hormones. The prolactin-induced increase in Na+ absorption arallels its Na+-retaining ability in lower vertebrates and could be part of the mechanism that keeps milk Na+ concentration low in intact glands.
structural differentiation, secretory polarization of organelles, and appropriate biochemical responses to prolactin (12, 13). These features are similar to those seen in vivo and are markedly different from those of primary cultures grown on attached collagen gels or plastic substrates (13). The access of media to the basal side and the capacity for cell movement on floating gels appear to be the bases of these differences. Because these cultures resemble the in vivo situation so closely and respond to prolactin, they seemed to provide an ideal system to study transport and its hormonal control in the mammary gland. We have concerned ourselves with the basic electrophysiological and ion transport characteristics of midpregnant mouse mammary epithelial cell cultures, as well as the effects of the lactogenic and osmoregulatory hormone prolactin. The effect of prolactin on Na+ transport is reminiscent of its Na+-retaining ability in lower vertebrates (1, 14) and may be the cause of low Na+ concentrations in milk. This system promises to provide useful data on the mechanisms and basic control processes in the mammary gland. It may also provide a basis to evaluate the theory that prolactin and other hormones control normal and pathological cell function via changes in ion transport (1420).
Secretion of milk is a significant osmoregulatory stress for lactating females (1). During milk production, mammary epithelial cells transport large volumes of isotonic fluid (2). The ionic content of this fluid is determined at least partially by active ion transport. The mechanisms and control of these ion transport processes have been studied in vio (2-6), but there are limits to the experiments that can be performed on such an anatomically and physiologically complex gland. Therefore, we were interested in adapting the techniques of cell culture to this tissue so that we might use classic Ussing-type chambers and electrophysiological and ion flux determinations to study isolated mammary epithelia maintained as cell sheets. Use of cultured epithelial cell sheets for studies relevant to transport was pioneered by Leighton et al. (7) and by Misfeldt, Hamamoto, and Pitelka (8) on the MDCK kidney cell line. Recent work has centered on hormonal control of Na+ fluxes in this system (9) and modification of the culturing techniques for application to other cell lines (10) and to toad bladder cells (11). The major differences between the previous culture systems used and the mammary system to be described herein are that these are primary cell cultures and not cell lines and that cells are plated and maintained on collagen gels that are subsequently released from petri dishes to float freely in the medium. Cultures of midpregnant cells maintained on detached and floating gels have been shown to have extensive ultra-
MATERIALS AND METHODS Methods of dissociation and preparation of cells for culture as well as manufacture of collagen gels have been described elsewhere (13, 21). Briefly, we dissociated tissue by mincing with scalpel blades and incubating at 37°C for 2 hr with 1.0 mg of type III collagenase (Worthington Biochemical, 108 units per mg) per ml of Hanks' basic salt solution (GIBCO). One gram of tissue was added to 10 ml of this solution. The cell suspension obtained from 10- to 17-day pregnant BALB/c Crgl mice was then plated onto polymerized rat tail collagen membranes in 35-mm petri dishes at an approximate density of 4 X 105 cells per cm2 surface area. Cells adhered to the collagen gel matrix within 2 days. All cultures were maintained in a humidified incubator at 37°C in Waymouth's medium (GIBCO) supplemented with 10% calf serum (Berkeley Biological Laboratories, Berkeley, CA). Each dissociation provided two groups of cultures. One had medium supplemented with insulin at 10 ,g/ml [bovine pancreatic (Sigma), 24.9 international units per mg] and cortisol at 5 ,ug/ml (Sigma), and the medium of the other group contained these two hormones plus prolactin at 10 jig/ml (National Institutes of Health PS-11, 26.4 international units per mg). These media were established at the time of cell plating and were maintained throughout the culture period. All culture media contained 100 units of penicillin per ml and 100 jig of streptomycin per ml (both Sigma) to retard bacterial
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current; R, transepithelial resistance; Jnet, net flux; Jms, mucosal-to-
growth.
Abbreviations: PD, transepithelial potential difference; IS,, short-circuit
this fact.
serosal flux; Jsm, serosal-to-mucosal flux.
536
Physiological Sciences:
At 2-5 days after dissociation, the collagen matrix containing adhered cells was detached from the plastic petri dish and allowed to float freely in the medium. Once released, the epithelial cell sheets contract and become composed of differentiated columnar cells. These contracted sheets were used for electrophysiological and isotope flux experiments 3-15 days after release. Cultures used before 3 days showed variable stability of transport properties and often no transport at all. Presumably this time period is required for cultures to achieve complete confluency and to form functional tight junctions. A lucite Ussing chamber in which edge damage was minimized by the presence of a slight bevel and high-vacuum silicone grease (Dow Corning) around the aperture allowed us to seal the tissue without crushing the edge. Cultures were bathed on both mucosal and serosal surfaces by Hanks' solution (pH 7.2) and gassed with 95% air/5% CO2. The temperature of the cultures and their bathing solutions was maintained at 37°C by a water jacket around the chamber. Nitex nylon monofilament cloth, 150-,tm mesh (Tobler, Ernst, and Trabe), was used as a rigid support to transfer cultures from petri dish to chamber. We measured transepithelial potential difference (PD) by connecting apical and basal sides of the cultures to a high impedance electrometer (D. Lee, Sunnyvale, CA) via calomel reference electrodes (Corning) and 151 mM NaCl/4% agar bridges. PD is reported as that of the mucosal-side solution with respect to the serosal-side solution. No PD was found across the Nitex cloth or blank collagen gels alone. We used a voltage and current clamping device (D. Lee, Sunnyvale, CA) to measure transepithelial resistance (R) by passing 10-20,tA of current through Ag/AgCl electrodes imbedded in 151 mM NaCl/4% agar and measuring the resulting PD response. Short-circuit current (IS) was measured as the amount of current required to clamp the PD to 0 mV with Hanks' solution on both sides of the tissue. The resistance of solutions, Nitex cloth, and blank collagen gels was accounted for in both I. and R measurements. Permanent records of PD and current were made with a Linear Instruments model 385 dual channel chart recorder. We have measured unidirectional transepithelial ionic fluxes by using 22Na+ and 36C1- (New England Nuclear) on paired cultures. Pairing was accomplished by measuring mucosalto-serosal (Jms) and serosal-to-mucosal (Jsm) fluxes on cultures that were selected to have nearly identical ISC. In these experiments, 10,uCi of 22Na+ or 10,uCi of 36Ch- (1 Ci = 3.7 X 1010 becquerels) to 10 ml of solution on the appropriate side and 10 min was allowed for equilibration before sampling 1 ml of the opposite, "cold," side as the zero time sample. Three subsequent samples were taken at 10-min intervals. Each flux reported here was the average of these three flux periods. 22Na+ was counted with a Beckman gamma 4000 counter and 36Cl- with a Packard model 3320 Tri-Carb scintillation spectrometer. Scintiverse (Fisher) was used as the scintillating solution; 10 ml was added to 1 ml of isotope-containing Hanks' solution. We used the F test to check for equivalence of variance and the Student's t test (or the appropriate modification of this test for unequal variances) to determine statistically significant differences between the two hormone-supplemented groups in both electrophysiological and flux experiments (22). For the purpose of this paper, we consider a value of P < 0.05 to be significant. The effects of common inhibitors and stimulants of transport processes were also tested. Ouabain, acetazolamide, synthetic arginine vasopressin (385 international units per mg), and synthetic oxytocin (19 international units per mg) were obtained was kindly provided by Val R. Wagner (Hoechst-Roussel Pharmaceuticals). Amiloride and ethacrynic acid were from Merck, Sharp and Dohme Research
from Sigma. Furosemide
Proc. Natl. Acad. Sci. USA 76 (1979)
Bisbee et al.
537
Laboratory. All were added from stock solutions in microliter amounts to the 10 ml of either apical or basal solution. All other chemicals used were reagent grade. RESULTS Table 1 shows the electrophysiological properties of midpregmouse mammary epithelial cell cultures maintained in media containing either insulin and cortisol or insulin, cortisol, and prolactin. These cultures developed significant PD (serosal side ground), R, and IC when bathed on both sides by identical Hanks' solutions. Addition of prolactin to the incubation medium caused PD and Is, to increase and R to decrease. The prolactin-induced activation of electrogenic pumps could be the sole cause of this decrease in R. We tested this possibility by plotting Ic vs. R for cultures maintained in the two hormonal states. If Isc and R changes were due to the same mechanism, then the plot should be linear and show a strong correlation, with high R cultures having low Isc and vice versa. Fig. 1 shows such a plot for all cultures used to generate the data in Table 1. There is no correlation' between R and Isc for insulin and cortisol cultures, because the linear regression line has a slope of essentially zero and a correlation coefficient of -0.079. There is a slight correlation between low R and high Isc for insulin, cortisol, and prolactin cultures, but the correlation coefficient is only -0.218. We measured isotope fluxes during Isc conditions to determine the active transport processes that contribute to this ISC. Na+ and Cl- were studied first because of their importance in the aqueous phase of milk (5, 6) and because prolactin is known to affect Na+ movements in other vertebrate tissues (1, 14, 15). Table 2 shows the results of these experiments. In cultures maintained with only insulin and cortisol, there was net Na+ absorption, and this absorption was nearly identical to I.. In cultures maintained with insulin, cortisol, and prolactin, all unidirectional fluxes increased, although the effects on jCMS, jcl, and JN, were not significant at the 5% level. The most striking effects were on JNa, which increased more than 3-fold. This was due primarily to an increase in JNa. Also, it should be noted that in the insulin, cortisol, and prolactin cultures, jTNa
nant
(3.55 ,ueq/cm2-hr) was now much larger than Isc (1.94 Aeq/ cm2-hr). With regard to chloride transport, Table 2 shows that there was no significant JClt in insulin and cortisol cultures. There appeared to be a small, but statistically different from zero, JO in insulin, cortisol, and prolactin cultures, but this JC' was not statistically different from that of insulin and cortisol cultures. We also attempted to answer two additional questions regarding these observed prolactin effects: Were they short-term Table 1. Electrophysiological data from BALB/c midpregnant mouse mammary epithelial cell cultures maintained on floating collagen gels Medium supplements Insulin and cortisol Insulin, cortisol, and prolactin
IsO
n
PD,* mV
gA/cm2
R, Q-cm2
39
-12.8 + 1.0
24.6 i 1.8
507 ± 35
49
-15.8 + 0.8 P < 0.02
48.0 i 2.5 P < 0.001
374 ± 20 P < 0.01
Cultures were maintained in Waymouth's medium supplemented with 10% calf serum and either insulin (10 ug/ml) and cortisol (5 ,ug/ml) or insulin, cortisol, and prolactin (10 Ag/ml). n refers to the number of different preparations tested. + refers to SEM. * Serosa ground.
Proc. Nati. Acad. Sci. USA 76 (1979)
Physiological Sciences: Bisbee et al.
538 90r
0
0 0
0
0
0
00
E
0
0 0
0
0
u
30F * .
400
800 R Q cm2
FIG. 1. ISC vs. R in both culture groups. Insulin and cortisol cultures (0): y = -0.004X + 26.7 (solid line) and correlation coefficient (r) =
-0.079; insulin, cortisol, and prolactin cultures (0): y = -0.028X = -0.218.
+ 58.3 (broken line) and r
or long-term? and Were they due to the known contamination of prolactin preparations with arginine vasopressin or oxytocin (23-25)? Arginine vasopressin or oxytocin was added to the mucosal, serosal, or both bathing solutions at 1 milliunit per ml, a concentration at least 50 times larger than the known contamination in prolactin at 10 ,g/ml in which the cultures were maintained (23-25). Neither hormone had any effect on electrical variables for up to 45 min. Moreover, cultures maintained with insulin, cortisol, and vasopressin exhibited no difference in electrophysiological properties from those in insulin and cortisol alone. When prolactin at 100 ,tg/ml was added to either or both sides of insulin and cortisol cultures mounted in chambers, no effect on electrical properties was observed for up to 45 min. Lastly, we tested some well-known. inhibitors of active transport. Bubbling both sides of cultures from either treatment group with 95% N2/5% CO2 instead of 95% air/5% CO2 caused PD and Isc to decrease nearly to zero. Adding 100 ,uM ouabain to the serosal, but not the mucosal, side of cultures from both groups also caused PD and ISC to decrease to zero within 30 min. Amiloride added to the mucosal, but not the serosal, side of cultures from both groups caused a rapid (within 30 sec) 50% reduction of PD and I,. All of these treatments caused R to increase to 108-286% of the original value. Furosemide, ethacrynic acid, thiocyanate, and acetazolamide were added individually to both groups of membranes at 0.1-1 mM. There were no effects of any of these inhibitors when added to the serosal or mucosal solutions of any cultures.
DISCUSSION Characterization of ion transport in mammary cell culture Organ culture of mammary glands provides a method for studying direct effects of hormones on tissues. However, the physiological and anatomical complexity of these tissues makes explants and intact glands unsuitable for detailed study of hormonal effects on ion transport. Because it supplies a flat epithelial cell sheet whose growth and experimental conditions can be regulated closely, cell culture is a promising alternative. More detailed analysis of both active and passive ionic transport pathways can be accomplished by manipulation of solutions, employment of isotopic tracers, and use of metabolic and transport inhibitors that are toxic to whole animals. Unfortunately, it is difficult to maintain differentiated cell populations, even in the presence of lactogenic hormones, when cells are cultured on plastic substrates or attached collagen gels (12, 13). These cells show little secretory specialization or polarization of organelles (13). On the other hand, cells cultured on floating collagen gels in the presence of insulin and cortisol are columnar rather than squamous and show secretory differentiation (13). In addition, these cells respond to prolactin in a fashion similar to that of intact glands by showing numerous casein granules and by accumulating and secreting casein (12, 13). Such a culture system has proven to be useful herein for the study of hormone-controlled transepithelial ion transport in the mammary gland. In insulin and cortisol cultures, there were significant PD, IC, and R. ISC was equal to jNt, which was 0.95 jLeq/cm2.hr (Table 2). There was a small statistically insignificant JC1 (0.18 gzeq/cm2-hr) that was in accord with the lack of effect of Cl- transport inhibitors on these cultures. Ouabain, an inhibitor of Na+,K+-ATPase, and N2, a metabolic inhibitor, both reduced PD and ISC to zero. Amiloride, the well-known inhibitor of mucosal sodium entry (26, 27), decreased PD and ISC by 50%. The experiments with ouabain and N2 lead to the conclusion that the maintenance of active Na+ absorption is dependent upon oxidative metabolism and a functional Na+,K+-ATPase at the serosal surface. The amiloride effects indicate, somewhat surprisingly, that only a portion of the Na+ current is sensitive to this inhibitor.
Prolactin effects on ion transport in mammary cell culture Addition of prolactin caused increases in PD and Ic and a decrease in R (Table 1). These effects are due solely to long-term incubation with prolactin. Prolactin also affected the fluxes of Na+ and Cl-. jNa, jCl and jC1 all increased, but these changes were not statistically significant at the 5% level (Table 2). Jnet also increased and appeared to be different from zero, but was not statistically different from that observed in insulin and cortisol cultures. The largest changes were the 2.5-fold increase in jN" and the greater than 3-fold increase in JN, to a value of 3.55 ,teq/cm2.hr. Since there was only a concomitant doubling
Table 2. Unidirectional and net fluxes of Na+ and C1- during I., conditions Insulin and cortisol
Jmg 2.19 + 0.44
(n 5) Insulin, cortisol, and 5.26 1 0.88 (n =6) prolactin
Jnet
ISc
1.30 + 0.24 (n =5)
Jsm 1.12 : 0.11 (n =5)
0.18 + 0.31 (n 5)
1.00 : 0.21 (n =10)
2.10 k 0.33 (n 6)
1.44 + 0.20 (n =6)
0.66 A: 0.29 (n = 6)
2.09 i 0.18 (n 12)
Jsm 1.24 + 0.16 (n 5)
Jnet 0.95 ± 0.48 (n =5)
Isc
mg
0.94 + 0.07 (n 10)
1.70 + 0.23 (n =6)
3.55 + 0.82 (n =6)
1.94 + 0.21 (n =12)
P < 0.001 NS NS NS All fluxes and IC are expressed in jueq/cm2-hr. We obtained Iac values by averaging the values from cultures used for measurement of unidirectional fluxes, and we obtained net fluxes by subtracting Jam from Jma in cultures with similar ISc values. NS = P > 0.05. ± refers to SEM.
P < 0.02
NS
P < 0.05
P < 0.001
Physiological Sciences:
Bisbee et al.
in I., (to 1.94 jieq/cm2.hr), there must also have been a substantial residual active absorption of some anion or secretion of pome cation. If the above-mentioned, prolactin-induced Jcnt is real, this residual active transport would amount to 0.87 Aeq/cm2-hr, and, if it is not real, the residual current was 1.53
,ueq/cm2.hr.
As with insulin and cortisol cultures, prolactin cultures are dependent upon metabolic energy and serosal Na+,K+-ATPase for active transport as indicated by the abolition of PD and I.,
with N2 or serosal ouabain. Amiloride also caused the PD and ISC to decrease by 50%, indicating that much of the active Na+ absorption is generated by mechanisms that do not have an amiloride-sensitive mucosal entry step. The lack of effect of C1inhibitors on prolactin cultures supports the flux data indicating a lack of Jcut. There was no correlation between Isc and R in insulin and cortisol cultures; and in prolactin cultures, there was only a slight correlation between high I., and low R (Fig. 1). This indicates that some of the prolactin-induced decrease in tissue R may be due to the induction of active, electrogenic transport processes, but that most of the change in R must be due to changes in other conductive pathways. Comparison of cultures to in vivo mammary gland Lactating goat mammary gland develops a PD of about -10 mV, serosal side ground (28), whereas the midpregnant cultures maintained in media supplemented with insulin, cortisol, and prolactin exhibit PDs averaging -15.8 mV. This similarity is remarkable considering that apical membranes of cultures were bathed by Hanks', a solution high in Na+, whereas in viwo lactating mammary glands have apical membranes bathed by milk, a low Na+/high K+ solution (2, 6). If, as now seems likely, in vivo mammary glands are capable of reabsorbing Na+, the slightly more negative potential of the cultures may simply reflect a greater availability of Na+ for absorption from the high Na+ concentration at the apical membrane in the cultures. Both nonlactating glands (28) and cultures without prolactin have smaller PDs. In the cultures, prolactin caused a 3-fold increase in net active absorption of Na+. Increased Na+ reabsorption may account for the low Na+ concentration observed in milk in vivo (2, 6). Unfortunately, it is impossible to compare these measurements of JNa to the in vivo situation, because isotope fluxes of Na+ have only been measured in the blood-to-milk direction (5). Movement in this direction would not necessarily be expected to change when Na+ reabsorption, a milk-to-blood movement, is increased. Increasing the levels of plasma membrane Na+,K+-ATPase is one way in which prolactin might induce increased Na+ absorption in these cultures. There is some indication from studies on tissue fragments that this may be the case for intact mammary glands. Enzyme assays in guinea pig mammary glands have shown that resting glands (low prolactin levels expected) have about one-half the activity of lactating glands (high prolactin levels expected) on a per cell basis as determined by DNA content (29). It has been claimed that prolactin causes increased cell K+ concentration and decreased cell Na+ concentration in pseudopregnant and lactating rabbits (3, 4). We note, though, that in these studies intracellular Na+ concentrations were well above 100 mM and similar to intracellular K+ concentrations; this situation is markedly different from that in most cells (30) and from that observed in guinea pig mammary glands in two earlier studies (31, 32). Histological studies have shown that Na+,K+-ATPase is located almost exclusively on the basolateral membranes of mammary epithelial cells (33). This would be consistent with
Proc. Natl. Acad. Sci. USA 76 (1979)
539
the inhibition by ouabain after its addition to the basal side of these cultures. However, the cytological location of this enzyme has come into doubt recently (34). In addition, there is still a question about the exact correlation between Na+,K+-ATPase activity and transepithelial Na+ transport (35, 36). This culture system may provide a means for determining the effects of prolactin on Na+,K+-ATPase activity of the mammary epithelial cells and what relationship this has to transepithelial Na+ transport, without the complications inherent in whole glands or tissue slices of heterogeneous cell populations. The case for prolactin induction of active Cl- transport in cultures or in vivo is unclear. In the absence of prolactin, there was no active Cl- transport in cultures. The value for Jcet in prolactin-treated cultures is significantly different from zero, but it was not significantly different from the Jct of 0.18 ,ueq/ cm2.hr in prolactin-free cultures. Inhibitors of active chloride transport have no effect on the cultures and have little if any effect on intracellular Cl- concentrations in the guinea pig mammary gland (31, 32). Therefore, the bulk of the evidence indicates there is little net Cl- transport in the mammary gland. There are three possibilities that would account for the residual I., in prolactin cultures that are consistent with in vvo data. HCO- reabsorption (or the equivalent H+ secretion) is one possibility. Milk has low HCO3 concentrations and a pH of 6.8 (6). Secretion of K+ is a second possibility, because milk has high K+ concentrations (2). Because it is believed that K+ is passively distributed across the apical membrane of mammary epithelial cells (2), K+ pumped into the cell by the serosal membrane Na+,K+-ATPase might leak across the mucosal membrane and contribute to transepithelial transport of K+. Lastly, it is possible that Ca2+ is actively secreted, because it is concentrated in milk (2, 6) and because its movement is not influenced by altering the chemical gradient across the gland (37). Finally, with reference to the effects of prolactin on R, we have found that this change is not due solely to induction of active, electrogenic transport processes. In vivo, parturition or prolactin administration decreases the permeability of rabbit mammary glands to sucrose to one-fourth (5). This has been interpreted as a decrease in permeability of tight junctions, because sucrose is expected to move by paracellular rather than transcellular routes. Such an effect might cause an increase in R. Studies of junctional ridge ultrastructure are not conclusive, but at parturition freeze-fracture replicas tend to have ridges of more constant width and orientation (38), which is more characteristic of tissues with high-resistance tight junctions (39). In this one aspect, the correlation between the culture system and in vivo data does not seem to hold. However, it is possible that in vivo prolactin induces both an increase in cell membrane ion conductances and also a decrease in shunt permeability to sucrose, thereby causing an overall decrease in R. One of the advantages of this culture system will be separation of the paracellular and transcellular components of the transepithelial R with detailed study of transepithelial electrolyte and nonelectrolyte fluxes. The effects of prolactin on Na+ transport in these cultures parallels those effects postulated in other vertebrates (1, 14, 15). Changes in both passive and active Na+ transport may be the underlying and most basic effect of prolactin (14, 15). The present culture system provides a convenient model in which to study the detailed effects of hormonal actions and interactions on transport in the mammary gland because of its structural simplicity and its physiological resemblance to intact glands. Physiological and pathological states of this tissue may result at least in part from hormone-mediated changes in
transport, and the culture system may provide insight into these
changes. We gratefully acknowledge Drs. Dorothy R. Pitelka and Joanne T. Emerman for encouraging our applications of their culture methods. We thank Susan T. Hamamoto for teaching us the necessary cell culture techniques. This investigation was supported by National Cancer Institute Grants CA-05388 and CA-09041. 1.
2. 3. 4. 5. 6. 7. 8. 9.
10. 11.
12.
13. 14. 15. 16.
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Nicoll, C. S. & Bern, H. A. (1972) in Lactogenic Hormones, eds. Wolstenholme, G. E. W. & Knight, J. (Churchill Livingstone, Edinburgh, Scotland), pp. 299-324. Linzell, J. L. & Peaker, M. (1971) Physiol. Rev. 51,564-597. Falconer, I. R., Forsyth, J. A., Wilson, B. M. & Dils, R. (1978) Biochem. J. 172,509-516. Falconer, I. R. & Rowe, J. M. (1977) J. Endocrinol. 101, 181186. Linzell, J. L., Peaker, M. & Taylor, J. C. (1975) J. Physiol. (London) 253,547-563. Peaker, M. (1978) in Lactation, ed. Larson, B. L. (Academic, New York), Vol. 4, pp. 437-462. Leighton, J., Estes, L. W., Mansukhami, S. & Brada, Z. (1970) Cancer 26, 1022-1028. Misfeldt, D. S., Hamamoto, S. T. & Pitelka, D. R. (1976) Proc. Natl. Acad. Sci. USA 73, 1212-1216. Simmons, N. L. (1978) J. Physiol. (London) 276, 28P-29P (abstr.). Cereijido, M., Robbins, E. S., Dolan, W. J., Rotunno, C. A. & Sabatini, D. D. (1978) J. Cell Biol. 77, 853-880. Johnson, J. P., Sahib, M. K., Steele, R., Wade, J., Preston, A. S., Lawson, N. & Handler, J. S. (1978) Nephrology: Proceedings, International Congress of Nephrology, Seventh, Montreal (Karger, White Plains, NY), p. Z2 (abstr.). Emerman, J. T., Enami, J., Pitelka, D. R. & Nandi, S. (1977) Proc. Natl. Acad. Sc. USA 74,4466-4470. Emerman, J. T. & Pitelka, D. R. (1977) In Vitro 13,316-328. Hirano, T. (1977) Gunma Symp. Endocrinol. 14,45-59. Bern, H. A. (1975) Am. Zool. 15,937-948. Carrasco, L. & Smith, A. E. (1976) Nature (London) 264, 807-809.
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Vol. 76, No. 1, pp. 536-540, January 1979 Physiological Sciences
Mouse mammary epithelial cells on floating collagen gels: Transepithelial ion transport and effects of prolactin (active Na+ transport/primary cell culture/midpregnant cells/cell differentiation/milk ions)
CHESTER A. BISBEE*, TERRY E. MACHENt, AND HOWARD A. BERN* *Department of Zoology and Cancer Research Laboratory and tDepartment of Physiology-Anatomy, University of California, Berkeley, California 94720
Contributed by Howard A. Bern, October 12, 1978
ABSTRACT Epithelial cells dissociated from midpregnant BALB/c mouse mammary glands were cultured for as long as 20 days as confluent monolayers on floating collagen gels. Detached gels bearing ihonolayers were placed in lucite Ussing chambers for measurement of transepithelial potential difference (PD), short-circuit current (I¢), resistance (R), and unidirectional fluxes of Na+ and Cl- during short-circuit current conditions (PD = 0). With Hanks' solution bathing both sides of cultures maintained with insulin and cortisol, PD = -12.8 mV (serosal side ground), IC = 24.6 &A/cm2, and R = 507 flcm2. Net absorption of Na+ equaled Ic, and there was no net Cltransport. PD and ISc were reduced 50% by mucosal addition of 10 &M amiloride and to zero by metabolic inhibition with nitrogen gas or by serosal addition of 0.1 mM ouabain. In similar cultures supplemented with prolactin, PD and ISC increased to -15.8 mV and 48.0 ,uA/cm2, respectively, and R decreased to 374 Qlcm2. Inhibitor effects were similar to those seen in prolactin-free cultures. Prolactin exposure resulted in a 3-fold increase in net absorption of Na+. Na+ absorption was not equivalent to ISC, and there was little Cl- absorption; therefore, prolactin induced active transport of other, as yet unidentified, ions. These effects of prolactin require at least 3 days to occur and cannot be attributed to the known contamination with neurohyophysial hormones. The prolactin-induced increase in Na+ absorption arallels its Na+-retaining ability in lower vertebrates and could be part of the mechanism that keeps milk Na+ concentration low in intact glands.
structural differentiation, secretory polarization of organelles, and appropriate biochemical responses to prolactin (12, 13). These features are similar to those seen in vivo and are markedly different from those of primary cultures grown on attached collagen gels or plastic substrates (13). The access of media to the basal side and the capacity for cell movement on floating gels appear to be the bases of these differences. Because these cultures resemble the in vivo situation so closely and respond to prolactin, they seemed to provide an ideal system to study transport and its hormonal control in the mammary gland. We have concerned ourselves with the basic electrophysiological and ion transport characteristics of midpregnant mouse mammary epithelial cell cultures, as well as the effects of the lactogenic and osmoregulatory hormone prolactin. The effect of prolactin on Na+ transport is reminiscent of its Na+-retaining ability in lower vertebrates (1, 14) and may be the cause of low Na+ concentrations in milk. This system promises to provide useful data on the mechanisms and basic control processes in the mammary gland. It may also provide a basis to evaluate the theory that prolactin and other hormones control normal and pathological cell function via changes in ion transport (1420).
Secretion of milk is a significant osmoregulatory stress for lactating females (1). During milk production, mammary epithelial cells transport large volumes of isotonic fluid (2). The ionic content of this fluid is determined at least partially by active ion transport. The mechanisms and control of these ion transport processes have been studied in vio (2-6), but there are limits to the experiments that can be performed on such an anatomically and physiologically complex gland. Therefore, we were interested in adapting the techniques of cell culture to this tissue so that we might use classic Ussing-type chambers and electrophysiological and ion flux determinations to study isolated mammary epithelia maintained as cell sheets. Use of cultured epithelial cell sheets for studies relevant to transport was pioneered by Leighton et al. (7) and by Misfeldt, Hamamoto, and Pitelka (8) on the MDCK kidney cell line. Recent work has centered on hormonal control of Na+ fluxes in this system (9) and modification of the culturing techniques for application to other cell lines (10) and to toad bladder cells (11). The major differences between the previous culture systems used and the mammary system to be described herein are that these are primary cell cultures and not cell lines and that cells are plated and maintained on collagen gels that are subsequently released from petri dishes to float freely in the medium. Cultures of midpregnant cells maintained on detached and floating gels have been shown to have extensive ultra-
MATERIALS AND METHODS Methods of dissociation and preparation of cells for culture as well as manufacture of collagen gels have been described elsewhere (13, 21). Briefly, we dissociated tissue by mincing with scalpel blades and incubating at 37°C for 2 hr with 1.0 mg of type III collagenase (Worthington Biochemical, 108 units per mg) per ml of Hanks' basic salt solution (GIBCO). One gram of tissue was added to 10 ml of this solution. The cell suspension obtained from 10- to 17-day pregnant BALB/c Crgl mice was then plated onto polymerized rat tail collagen membranes in 35-mm petri dishes at an approximate density of 4 X 105 cells per cm2 surface area. Cells adhered to the collagen gel matrix within 2 days. All cultures were maintained in a humidified incubator at 37°C in Waymouth's medium (GIBCO) supplemented with 10% calf serum (Berkeley Biological Laboratories, Berkeley, CA). Each dissociation provided two groups of cultures. One had medium supplemented with insulin at 10 ,g/ml [bovine pancreatic (Sigma), 24.9 international units per mg] and cortisol at 5 ,ug/ml (Sigma), and the medium of the other group contained these two hormones plus prolactin at 10 jig/ml (National Institutes of Health PS-11, 26.4 international units per mg). These media were established at the time of cell plating and were maintained throughout the culture period. All culture media contained 100 units of penicillin per ml and 100 jig of streptomycin per ml (both Sigma) to retard bacterial
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current; R, transepithelial resistance; Jnet, net flux; Jms, mucosal-to-
growth.
Abbreviations: PD, transepithelial potential difference; IS,, short-circuit
this fact.
serosal flux; Jsm, serosal-to-mucosal flux.
536
Physiological Sciences:
At 2-5 days after dissociation, the collagen matrix containing adhered cells was detached from the plastic petri dish and allowed to float freely in the medium. Once released, the epithelial cell sheets contract and become composed of differentiated columnar cells. These contracted sheets were used for electrophysiological and isotope flux experiments 3-15 days after release. Cultures used before 3 days showed variable stability of transport properties and often no transport at all. Presumably this time period is required for cultures to achieve complete confluency and to form functional tight junctions. A lucite Ussing chamber in which edge damage was minimized by the presence of a slight bevel and high-vacuum silicone grease (Dow Corning) around the aperture allowed us to seal the tissue without crushing the edge. Cultures were bathed on both mucosal and serosal surfaces by Hanks' solution (pH 7.2) and gassed with 95% air/5% CO2. The temperature of the cultures and their bathing solutions was maintained at 37°C by a water jacket around the chamber. Nitex nylon monofilament cloth, 150-,tm mesh (Tobler, Ernst, and Trabe), was used as a rigid support to transfer cultures from petri dish to chamber. We measured transepithelial potential difference (PD) by connecting apical and basal sides of the cultures to a high impedance electrometer (D. Lee, Sunnyvale, CA) via calomel reference electrodes (Corning) and 151 mM NaCl/4% agar bridges. PD is reported as that of the mucosal-side solution with respect to the serosal-side solution. No PD was found across the Nitex cloth or blank collagen gels alone. We used a voltage and current clamping device (D. Lee, Sunnyvale, CA) to measure transepithelial resistance (R) by passing 10-20,tA of current through Ag/AgCl electrodes imbedded in 151 mM NaCl/4% agar and measuring the resulting PD response. Short-circuit current (IS) was measured as the amount of current required to clamp the PD to 0 mV with Hanks' solution on both sides of the tissue. The resistance of solutions, Nitex cloth, and blank collagen gels was accounted for in both I. and R measurements. Permanent records of PD and current were made with a Linear Instruments model 385 dual channel chart recorder. We have measured unidirectional transepithelial ionic fluxes by using 22Na+ and 36C1- (New England Nuclear) on paired cultures. Pairing was accomplished by measuring mucosalto-serosal (Jms) and serosal-to-mucosal (Jsm) fluxes on cultures that were selected to have nearly identical ISC. In these experiments, 10,uCi of 22Na+ or 10,uCi of 36Ch- (1 Ci = 3.7 X 1010 becquerels) to 10 ml of solution on the appropriate side and 10 min was allowed for equilibration before sampling 1 ml of the opposite, "cold," side as the zero time sample. Three subsequent samples were taken at 10-min intervals. Each flux reported here was the average of these three flux periods. 22Na+ was counted with a Beckman gamma 4000 counter and 36Cl- with a Packard model 3320 Tri-Carb scintillation spectrometer. Scintiverse (Fisher) was used as the scintillating solution; 10 ml was added to 1 ml of isotope-containing Hanks' solution. We used the F test to check for equivalence of variance and the Student's t test (or the appropriate modification of this test for unequal variances) to determine statistically significant differences between the two hormone-supplemented groups in both electrophysiological and flux experiments (22). For the purpose of this paper, we consider a value of P < 0.05 to be significant. The effects of common inhibitors and stimulants of transport processes were also tested. Ouabain, acetazolamide, synthetic arginine vasopressin (385 international units per mg), and synthetic oxytocin (19 international units per mg) were obtained was kindly provided by Val R. Wagner (Hoechst-Roussel Pharmaceuticals). Amiloride and ethacrynic acid were from Merck, Sharp and Dohme Research
from Sigma. Furosemide
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Bisbee et al.
537
Laboratory. All were added from stock solutions in microliter amounts to the 10 ml of either apical or basal solution. All other chemicals used were reagent grade. RESULTS Table 1 shows the electrophysiological properties of midpregmouse mammary epithelial cell cultures maintained in media containing either insulin and cortisol or insulin, cortisol, and prolactin. These cultures developed significant PD (serosal side ground), R, and IC when bathed on both sides by identical Hanks' solutions. Addition of prolactin to the incubation medium caused PD and Is, to increase and R to decrease. The prolactin-induced activation of electrogenic pumps could be the sole cause of this decrease in R. We tested this possibility by plotting Ic vs. R for cultures maintained in the two hormonal states. If Isc and R changes were due to the same mechanism, then the plot should be linear and show a strong correlation, with high R cultures having low Isc and vice versa. Fig. 1 shows such a plot for all cultures used to generate the data in Table 1. There is no correlation' between R and Isc for insulin and cortisol cultures, because the linear regression line has a slope of essentially zero and a correlation coefficient of -0.079. There is a slight correlation between low R and high Isc for insulin, cortisol, and prolactin cultures, but the correlation coefficient is only -0.218. We measured isotope fluxes during Isc conditions to determine the active transport processes that contribute to this ISC. Na+ and Cl- were studied first because of their importance in the aqueous phase of milk (5, 6) and because prolactin is known to affect Na+ movements in other vertebrate tissues (1, 14, 15). Table 2 shows the results of these experiments. In cultures maintained with only insulin and cortisol, there was net Na+ absorption, and this absorption was nearly identical to I.. In cultures maintained with insulin, cortisol, and prolactin, all unidirectional fluxes increased, although the effects on jCMS, jcl, and JN, were not significant at the 5% level. The most striking effects were on JNa, which increased more than 3-fold. This was due primarily to an increase in JNa. Also, it should be noted that in the insulin, cortisol, and prolactin cultures, jTNa
nant
(3.55 ,ueq/cm2-hr) was now much larger than Isc (1.94 Aeq/ cm2-hr). With regard to chloride transport, Table 2 shows that there was no significant JClt in insulin and cortisol cultures. There appeared to be a small, but statistically different from zero, JO in insulin, cortisol, and prolactin cultures, but this JC' was not statistically different from that of insulin and cortisol cultures. We also attempted to answer two additional questions regarding these observed prolactin effects: Were they short-term Table 1. Electrophysiological data from BALB/c midpregnant mouse mammary epithelial cell cultures maintained on floating collagen gels Medium supplements Insulin and cortisol Insulin, cortisol, and prolactin
IsO
n
PD,* mV
gA/cm2
R, Q-cm2
39
-12.8 + 1.0
24.6 i 1.8
507 ± 35
49
-15.8 + 0.8 P < 0.02
48.0 i 2.5 P < 0.001
374 ± 20 P < 0.01
Cultures were maintained in Waymouth's medium supplemented with 10% calf serum and either insulin (10 ug/ml) and cortisol (5 ,ug/ml) or insulin, cortisol, and prolactin (10 Ag/ml). n refers to the number of different preparations tested. + refers to SEM. * Serosa ground.
Proc. Nati. Acad. Sci. USA 76 (1979)
Physiological Sciences: Bisbee et al.
538 90r
0
0 0
0
0
0
00
E
0
0 0
0
0
u
30F * .
400
800 R Q cm2
FIG. 1. ISC vs. R in both culture groups. Insulin and cortisol cultures (0): y = -0.004X + 26.7 (solid line) and correlation coefficient (r) =
-0.079; insulin, cortisol, and prolactin cultures (0): y = -0.028X = -0.218.
+ 58.3 (broken line) and r
or long-term? and Were they due to the known contamination of prolactin preparations with arginine vasopressin or oxytocin (23-25)? Arginine vasopressin or oxytocin was added to the mucosal, serosal, or both bathing solutions at 1 milliunit per ml, a concentration at least 50 times larger than the known contamination in prolactin at 10 ,g/ml in which the cultures were maintained (23-25). Neither hormone had any effect on electrical variables for up to 45 min. Moreover, cultures maintained with insulin, cortisol, and vasopressin exhibited no difference in electrophysiological properties from those in insulin and cortisol alone. When prolactin at 100 ,tg/ml was added to either or both sides of insulin and cortisol cultures mounted in chambers, no effect on electrical properties was observed for up to 45 min. Lastly, we tested some well-known. inhibitors of active transport. Bubbling both sides of cultures from either treatment group with 95% N2/5% CO2 instead of 95% air/5% CO2 caused PD and Isc to decrease nearly to zero. Adding 100 ,uM ouabain to the serosal, but not the mucosal, side of cultures from both groups also caused PD and ISC to decrease to zero within 30 min. Amiloride added to the mucosal, but not the serosal, side of cultures from both groups caused a rapid (within 30 sec) 50% reduction of PD and I,. All of these treatments caused R to increase to 108-286% of the original value. Furosemide, ethacrynic acid, thiocyanate, and acetazolamide were added individually to both groups of membranes at 0.1-1 mM. There were no effects of any of these inhibitors when added to the serosal or mucosal solutions of any cultures.
DISCUSSION Characterization of ion transport in mammary cell culture Organ culture of mammary glands provides a method for studying direct effects of hormones on tissues. However, the physiological and anatomical complexity of these tissues makes explants and intact glands unsuitable for detailed study of hormonal effects on ion transport. Because it supplies a flat epithelial cell sheet whose growth and experimental conditions can be regulated closely, cell culture is a promising alternative. More detailed analysis of both active and passive ionic transport pathways can be accomplished by manipulation of solutions, employment of isotopic tracers, and use of metabolic and transport inhibitors that are toxic to whole animals. Unfortunately, it is difficult to maintain differentiated cell populations, even in the presence of lactogenic hormones, when cells are cultured on plastic substrates or attached collagen gels (12, 13). These cells show little secretory specialization or polarization of organelles (13). On the other hand, cells cultured on floating collagen gels in the presence of insulin and cortisol are columnar rather than squamous and show secretory differentiation (13). In addition, these cells respond to prolactin in a fashion similar to that of intact glands by showing numerous casein granules and by accumulating and secreting casein (12, 13). Such a culture system has proven to be useful herein for the study of hormone-controlled transepithelial ion transport in the mammary gland. In insulin and cortisol cultures, there were significant PD, IC, and R. ISC was equal to jNt, which was 0.95 jLeq/cm2.hr (Table 2). There was a small statistically insignificant JC1 (0.18 gzeq/cm2-hr) that was in accord with the lack of effect of Cl- transport inhibitors on these cultures. Ouabain, an inhibitor of Na+,K+-ATPase, and N2, a metabolic inhibitor, both reduced PD and ISC to zero. Amiloride, the well-known inhibitor of mucosal sodium entry (26, 27), decreased PD and ISC by 50%. The experiments with ouabain and N2 lead to the conclusion that the maintenance of active Na+ absorption is dependent upon oxidative metabolism and a functional Na+,K+-ATPase at the serosal surface. The amiloride effects indicate, somewhat surprisingly, that only a portion of the Na+ current is sensitive to this inhibitor.
Prolactin effects on ion transport in mammary cell culture Addition of prolactin caused increases in PD and Ic and a decrease in R (Table 1). These effects are due solely to long-term incubation with prolactin. Prolactin also affected the fluxes of Na+ and Cl-. jNa, jCl and jC1 all increased, but these changes were not statistically significant at the 5% level (Table 2). Jnet also increased and appeared to be different from zero, but was not statistically different from that observed in insulin and cortisol cultures. The largest changes were the 2.5-fold increase in jN" and the greater than 3-fold increase in JN, to a value of 3.55 ,teq/cm2.hr. Since there was only a concomitant doubling
Table 2. Unidirectional and net fluxes of Na+ and C1- during I., conditions Insulin and cortisol
Jmg 2.19 + 0.44
(n 5) Insulin, cortisol, and 5.26 1 0.88 (n =6) prolactin
Jnet
ISc
1.30 + 0.24 (n =5)
Jsm 1.12 : 0.11 (n =5)
0.18 + 0.31 (n 5)
1.00 : 0.21 (n =10)
2.10 k 0.33 (n 6)
1.44 + 0.20 (n =6)
0.66 A: 0.29 (n = 6)
2.09 i 0.18 (n 12)
Jsm 1.24 + 0.16 (n 5)
Jnet 0.95 ± 0.48 (n =5)
Isc
mg
0.94 + 0.07 (n 10)
1.70 + 0.23 (n =6)
3.55 + 0.82 (n =6)
1.94 + 0.21 (n =12)
P < 0.001 NS NS NS All fluxes and IC are expressed in jueq/cm2-hr. We obtained Iac values by averaging the values from cultures used for measurement of unidirectional fluxes, and we obtained net fluxes by subtracting Jam from Jma in cultures with similar ISc values. NS = P > 0.05. ± refers to SEM.
P < 0.02
NS
P < 0.05
P < 0.001
Physiological Sciences:
Bisbee et al.
in I., (to 1.94 jieq/cm2.hr), there must also have been a substantial residual active absorption of some anion or secretion of pome cation. If the above-mentioned, prolactin-induced Jcnt is real, this residual active transport would amount to 0.87 Aeq/cm2-hr, and, if it is not real, the residual current was 1.53
,ueq/cm2.hr.
As with insulin and cortisol cultures, prolactin cultures are dependent upon metabolic energy and serosal Na+,K+-ATPase for active transport as indicated by the abolition of PD and I.,
with N2 or serosal ouabain. Amiloride also caused the PD and ISC to decrease by 50%, indicating that much of the active Na+ absorption is generated by mechanisms that do not have an amiloride-sensitive mucosal entry step. The lack of effect of C1inhibitors on prolactin cultures supports the flux data indicating a lack of Jcut. There was no correlation between Isc and R in insulin and cortisol cultures; and in prolactin cultures, there was only a slight correlation between high I., and low R (Fig. 1). This indicates that some of the prolactin-induced decrease in tissue R may be due to the induction of active, electrogenic transport processes, but that most of the change in R must be due to changes in other conductive pathways. Comparison of cultures to in vivo mammary gland Lactating goat mammary gland develops a PD of about -10 mV, serosal side ground (28), whereas the midpregnant cultures maintained in media supplemented with insulin, cortisol, and prolactin exhibit PDs averaging -15.8 mV. This similarity is remarkable considering that apical membranes of cultures were bathed by Hanks', a solution high in Na+, whereas in viwo lactating mammary glands have apical membranes bathed by milk, a low Na+/high K+ solution (2, 6). If, as now seems likely, in vivo mammary glands are capable of reabsorbing Na+, the slightly more negative potential of the cultures may simply reflect a greater availability of Na+ for absorption from the high Na+ concentration at the apical membrane in the cultures. Both nonlactating glands (28) and cultures without prolactin have smaller PDs. In the cultures, prolactin caused a 3-fold increase in net active absorption of Na+. Increased Na+ reabsorption may account for the low Na+ concentration observed in milk in vivo (2, 6). Unfortunately, it is impossible to compare these measurements of JNa to the in vivo situation, because isotope fluxes of Na+ have only been measured in the blood-to-milk direction (5). Movement in this direction would not necessarily be expected to change when Na+ reabsorption, a milk-to-blood movement, is increased. Increasing the levels of plasma membrane Na+,K+-ATPase is one way in which prolactin might induce increased Na+ absorption in these cultures. There is some indication from studies on tissue fragments that this may be the case for intact mammary glands. Enzyme assays in guinea pig mammary glands have shown that resting glands (low prolactin levels expected) have about one-half the activity of lactating glands (high prolactin levels expected) on a per cell basis as determined by DNA content (29). It has been claimed that prolactin causes increased cell K+ concentration and decreased cell Na+ concentration in pseudopregnant and lactating rabbits (3, 4). We note, though, that in these studies intracellular Na+ concentrations were well above 100 mM and similar to intracellular K+ concentrations; this situation is markedly different from that in most cells (30) and from that observed in guinea pig mammary glands in two earlier studies (31, 32). Histological studies have shown that Na+,K+-ATPase is located almost exclusively on the basolateral membranes of mammary epithelial cells (33). This would be consistent with
Proc. Natl. Acad. Sci. USA 76 (1979)
539
the inhibition by ouabain after its addition to the basal side of these cultures. However, the cytological location of this enzyme has come into doubt recently (34). In addition, there is still a question about the exact correlation between Na+,K+-ATPase activity and transepithelial Na+ transport (35, 36). This culture system may provide a means for determining the effects of prolactin on Na+,K+-ATPase activity of the mammary epithelial cells and what relationship this has to transepithelial Na+ transport, without the complications inherent in whole glands or tissue slices of heterogeneous cell populations. The case for prolactin induction of active Cl- transport in cultures or in vivo is unclear. In the absence of prolactin, there was no active Cl- transport in cultures. The value for Jcet in prolactin-treated cultures is significantly different from zero, but it was not significantly different from the Jct of 0.18 ,ueq/ cm2.hr in prolactin-free cultures. Inhibitors of active chloride transport have no effect on the cultures and have little if any effect on intracellular Cl- concentrations in the guinea pig mammary gland (31, 32). Therefore, the bulk of the evidence indicates there is little net Cl- transport in the mammary gland. There are three possibilities that would account for the residual I., in prolactin cultures that are consistent with in vvo data. HCO- reabsorption (or the equivalent H+ secretion) is one possibility. Milk has low HCO3 concentrations and a pH of 6.8 (6). Secretion of K+ is a second possibility, because milk has high K+ concentrations (2). Because it is believed that K+ is passively distributed across the apical membrane of mammary epithelial cells (2), K+ pumped into the cell by the serosal membrane Na+,K+-ATPase might leak across the mucosal membrane and contribute to transepithelial transport of K+. Lastly, it is possible that Ca2+ is actively secreted, because it is concentrated in milk (2, 6) and because its movement is not influenced by altering the chemical gradient across the gland (37). Finally, with reference to the effects of prolactin on R, we have found that this change is not due solely to induction of active, electrogenic transport processes. In vivo, parturition or prolactin administration decreases the permeability of rabbit mammary glands to sucrose to one-fourth (5). This has been interpreted as a decrease in permeability of tight junctions, because sucrose is expected to move by paracellular rather than transcellular routes. Such an effect might cause an increase in R. Studies of junctional ridge ultrastructure are not conclusive, but at parturition freeze-fracture replicas tend to have ridges of more constant width and orientation (38), which is more characteristic of tissues with high-resistance tight junctions (39). In this one aspect, the correlation between the culture system and in vivo data does not seem to hold. However, it is possible that in vivo prolactin induces both an increase in cell membrane ion conductances and also a decrease in shunt permeability to sucrose, thereby causing an overall decrease in R. One of the advantages of this culture system will be separation of the paracellular and transcellular components of the transepithelial R with detailed study of transepithelial electrolyte and nonelectrolyte fluxes. The effects of prolactin on Na+ transport in these cultures parallels those effects postulated in other vertebrates (1, 14, 15). Changes in both passive and active Na+ transport may be the underlying and most basic effect of prolactin (14, 15). The present culture system provides a convenient model in which to study the detailed effects of hormonal actions and interactions on transport in the mammary gland because of its structural simplicity and its physiological resemblance to intact glands. Physiological and pathological states of this tissue may result at least in part from hormone-mediated changes in
transport, and the culture system may provide insight into these
changes. We gratefully acknowledge Drs. Dorothy R. Pitelka and Joanne T. Emerman for encouraging our applications of their culture methods. We thank Susan T. Hamamoto for teaching us the necessary cell culture techniques. This investigation was supported by National Cancer Institute Grants CA-05388 and CA-09041. 1.
2. 3. 4. 5. 6. 7. 8. 9.
10. 11.
12.
13. 14. 15. 16.
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