Neurotransmitter-Stimulated Ion Transport Across Cultured Bovine Mammary Epithelial Cell Monolayers1

J. Dairy Sci. 84:2622–2631  American Dairy Science Association, 2001.

Neurotransmitter-Stimulated Ion Transport Across Cultured Bovine Mammary Epithelial Cell Monolayers1 C. R. Schmidt,* R. W. Carlin,* J. M. Sargeant,† and B. D. Schultz* *Department of Anatomy and Physiology and †Food Animal Health & Management Center Kansas State University, Manhattan, KS 66506

ABSTRACT Bovine mammary epithelial (BME-UV) and myoepithelial (BMM-UV) cell lines were acquired with the goal of developing an in vitro model of mammary epithelia for the study of ion transport. The bovine mammary cell lines were successfully cultured on commercially available permeable supports, and results suggest that mammary epithelial cells, but not myoepithelial cells, form tight junctions necessary to perform a barrier function. Electrogenic ion transport was not observed in basal conditions. Acute exposure to norepinephrine or forskolin caused prototypic increases in short circuit current accompanied by a reduction in transmural resistance indicative of anion secretion through a conductive pathway. Bumetanide and N-(4-methyphenylsulfonyl)-N′-(4-trifluoro- methylphenyl)urea, inhibitors of Na+/K+/Cl− cotransport and cystic fibrosis transmembrane conductance anion channels, respectively, reduced forskolin-stimulated ion transport. Amiloride, an inhibitor of epithelial sodium channels, had no effect on basal or forskolin-stimulated ion transport. However, naturally occurring and synthetic corticosteroids induced the expression of amiloride sensitive current indicative of sodium absorption. Chronic exposure to increased apical ionic strength and/or reduced carbohydrate concentration were associated with reduced transepithelial resistance although forskolin-stimulated ion transport was unaffected. These results demonstrate that neurotransmitters and steroid hormones act directly on bovine mammary epithelial cells to acutely and chronically modulate the volume and composition of their secretions. The in vitro system that we describe can be further exploited to characterize

Received March 27, 2001. Accepted July 18, 2001. Corresponding author: B. D. Schultz; e-mail: bschultz@vet. ksu.edu. 1 Contribution no. 01-382-J from the Kansas Agricultural Experiment Station.

cellular and molecular mechanisms associated with mammary function in health and disease. (Key words: bovine, mammary, epithelia, ion transport) Abbreviation key: CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; DASU02 = N-(4-methyphenylsulfonyl)-N′-(4-trifluoro- methylphenyl)urea; Isc = short-circuit current; MEM = modified Eagle mix, PDte = transepithelial potential difference; Rte = transepithelial resistance; TBM = typical bovine medium. INTRODUCTION Milk sales are reported to have the second highest impact of any single commodity on the farm economy in the United States. Nearly $20 billion in sales per year account for approximately 12% of total farm cash receipts. The desire to increase production efficiency has directed many agricultural resources and much research effort toward a better understanding of the relationships of genetics, animal nutrition, housing, and pregnancy to milk production. However, the mammary epithelium is ultimately responsible for milk production, and the cow provides an appropriate microenvironment for this activity. The optimum environment for epithelial function (e.g., presence of hormones, neurotransmitters, salts, carbohydrates, etc.) and the mechanisms controlling epithelial secretion of the various milk components remain to be fully elucidated. Mechanisms of ion transport across mammary epithelium are poorly understood, and the molecular identity of most membrane-bound ion transport proteins remains to be determined. Comprehensive reviews (Shennan, 1998; Neville, 1999; Shennan and Peaker, 2000) detailing what is known of mammary epithelial function and the gaps in our knowledge have recently been published. Both the ouabain-inhibitable Na+/K+ ATPase and the bumetanide-sensitive Na+/K+/Cl− cotransporter are reportedly present in the basolateral membrane of mammary epithelia (Shennan and

2622

2623

MAMMARY EPITHELIAL ION TRANSPORT

Peaker, 2000). A K+ conductance (Blatchford and Peaker, 1988; Smith et al., 1990; Shennan, 1992; Stelwagen et al., 1999; Silanikove et al., 2000) and/or a nonselective cation conductance (Blatchford and Peaker, 1988; Stelwagen et al., 1999) has been documented in lipid membranes isolated from milk although the channels in these cell-free vesicles remain unidentified and appear to be unregulated. Likewise, an apical Cl− channel has also been postulated. Currently, we report the development of an in vitro system to study bovine mammary epithelial ion transport. A transformed bovine mammary cell line, BMEUV (Zavizion et al., 1996a) that was previously reported to synthesize αs1-casein and α-lactalbumin was obtained. Conditions were identified and optimized to promote the development of a resistive epithelial monolayer. The monolayer responds to corticosteroids and to adrenergic stimulation with changes in ion transport. The results suggest that BME-UV cells provide an excellent model for the study of mammary epithelial transport mechanisms. MATERIALS AND METHODS Bovine Mammary Epithelial Cells Bovine mammary epithelial cells (BME-UV) and bovine mammary myoepithelial cells (BMM-UV) were generously provided by the University of Vermont, Department of Animal and Food Sciences. The cells have previously been described and suitable growth conditions for culture on an impermeant substrate have been reported (Zavizion et al., 1996a,b). Experiments were conducted on cells from the first 20 passages after receipt. Monolayer Culture Cells were initially cultured in a medium similar to that previously described (Table 1, typical bovine medium, TBM; Zavizion et al., 1996a). Stock cell cultures were grown in 75-cm2 tissue culture treated, vented flasks (cat. #430641, Corning, Inc., Corning, NY) at 37°C in 5% CO2. Cells were subcultured once every 3 to 5 d when flasks were ∼70% confluent. Passage of cells in the flasks was performed by aspirating spent medium from the flask, rinsing with PBS for cell culture (composition in mM; 140, NaCl; 2, KCl; 1.5, KH2PO4; 15, Na2HPO4) and then incubating the cells in 1.5 ml of dissociation solution (PBS for cell culture containing 2.5 g of trypsin/L and 2.65 mM disodium EDTA) for 2 min. The dissociation solution was removed, and the cells were allowed to incubate at 37°C for 5 to 6 min. Once the cells had detached from the flask, cells were suspended in 6 ml of TBM and the suspension was

pipetted 5 to 10× to facilitate disruption of cell aggregates. Three drops of the suspension were seeded onto a 75-cm2 flask containing 10 ml of TBM. Concomitantly, tissue culture treated permeable polyester supports (Costar Snapwell #3801, Fisher Scientific, Pittsburgh, PA) were seeded with 3 to 4 drops of the initial cell suspension. Media in flasks and on Snapwells was changed daily to provide optimal conditions for monolayer development and cell proliferation. The basolateral side of cell monolayers in Snapwell chambers was always exposed to TBM as were all cells grown in flasks. The apical aspect of cell monolayers was exposed to TBM or a medium of defined salt composition as indicated (Table 2). During initial studies, both the apical and the basolateral aspects of the epithelial monolayers were exposed to TBM. Subsequently, media of defined salt composition were developed to test whether the cells were sensitive to changes in apical cation and carbohydrate concentrations. Listed in Table 1 are the components for the base solution for all alternate media. Salt and carbohydrate concentrations for alternate media are listed in Table 2. Prior to use, all media were supplemented with 1% insulin-transferrin-sodium selenite media supplement solution and 1% penicillin-streptomycin. Each medium was pH adjusted to 7.3, filter sterilized using 0.22-µm filters, and stored in opaque bottles. Na+, K+, and Cl− concentrations were adjusted by substituting NaCl, KCl, NaHCO3, and KHCO3 to achieve the indicated concentrations as measured with ion-specific electrodes (K+, Orion Ion Plus; Na+ and Cl−, Accumet, Fisher Scientific) interfaced with dual-channel ion meters (Accumet AR25, Fisher Scientific). HCO3− concentration was consistent in all media. Lactose and mannitol concentrations were adjusted to maintain consistent osmolality in all media. Experiments were conducted to test for effects of chronic exposure to selected steroid hormones that are thought to affect milk production. For these experiments, dexamethasone (100 nM), cortisol (500 nM), or prednisolone (1 µM) were included in the TBM. Epithelial monolayers were exposed to the selected steroid hormones for 7 to 12 d prior to evaluation of ion transport. Electrophysiology Bovine mammary epithelial cell monolayers were evaluated for transepithelial potential difference (PDte), transepithelial electrical resistance (Rte), basal short-circuit current (Isc; an indicator of net ion transport), and changes in Isc stimulated by selected physiological and pharmacological agents using a modified Ussing chamber (model DCV9, Navicyte, San Diego, Journal of Dairy Science Vol. 84, No. 12, 2001

2624

SCHMIDT ET AL. Table 1. Typical bovine and base media composition.1,2 Typical bovine medium (TBM)

Component Dulbecco’s Modified eagle media (DMEM)3 Ham F-12 nutrient mixture (F-12)3 RPMI-1640 (RPMI)3 NCTC 135 (NCTC)3 Fetal bovine serum; heat inactivated (FBS) Newborn calf serum (NCS) Iron-supplemented bovine calf serum (FeBCS) Lactalbumin hydrosylate L-Ascorbic acid MEM Amino acid solution 50X (MEM AA) MEM Nonessential amino acid solution 100X (MEM nonAA) RPMI 1640 amino acid solution 50X (RPMI AA) MEM vitamins solutions 100X (MEM Vit) RPMI 1640 vitamins solution 100X (RPMI Vit) Lactose CaCl2 Ca(NO3)2 CuSO4 Fe(NO3)3 FeSO4 MgCl2 MgSO4 Na2HPO4 ZnSO4 Lipoic acid Hypoxanthine Glucose Linoleic acid Putrescine 2HCl Choline chloride Sodium pyruvate

21.25% 21.25% 25.5% 17 % 10 % 3% 2% 1 mg/ml 10 µg/ml

Base solution for ion substitution studies (media #2–#18)

17 % 10 % 3% 2% 1 mg/ml 10 µg/ml 0.85% 0.425% 0.85% 0.85% 0.425%

3 mM 0.445 mM 0.108 mM 2.12 × 10−6 5.25 × 10−5 6.33 × 10−4 0.127 mM 0.276 mM 1.8 mM 6.34 × 10−4 2.15 × 10−4 0.006 mM 10.3 mM 6.05 × 10−5 2.12 × 10−4 0.018 mM 0.212 mM

mM mM mM

mM mM mM mM

1 Before use, all media were supplemented with 1% insulin-transferrin-sodium selenite and 1% penicillinstreptomycin. 2 Each medium was pH adjusted to 7.3, filter sterilized with 0.22-µm filters, and stored in opaque bottles. 3 Reconstituted per manufacturers instructions.

Table 2. Ionic and carbohydrate composition of defined media.1 Medium #

Na+ mEq

K+ mEq

Cl− mEq

Lact. mM

Man. mM

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

67 66 90 152 62 59 60 50 71 59 84 162 59 59 57 55 71

92 46 46 48 29 63 143 33 141 48 50 52 31 68 155 37 158

141 92 139 203 82 114 197 71 188 98 130 190 86 124 190 93 191

3 240 160 0 280 200 40 284 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 240 160 0 280 200 40 284 0

1 Base solution composition is listed in Table 1. Concentration of Na+, K+, and Cl− were determined with ion selective electrodes. Lactose (Lact.) and mannitol (Man.) concentrations were based upon the amount added to the media during preparation.

Journal of Dairy Science Vol. 84, No. 12, 2001

CA) as previously described (Sedlacek et al., in press). For electrical measurements, cell monolayers were bathed in Ringer solution (composition in mM; 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, and 1.2 MgCl2) maintained at 39°C and continuously bubbled with 5% CO2: 95% O2. After recording PDte the monolayers were clamped to zero mV and Isc measured continuously with a voltage clamp apparatus (model 558C, University of Iowa, Department of Bioengineering, Iowa City, IA). Periodically, a 1-mV pulse was applied across the monolayer and the resultant change in current was used to calculate monolayer resistance by employing Ohm’s law. Data were digitally acquired at 1 Hz with a Macintosh computer (Apple Computer, Cuppertino, CA) using an MP100A-CE interface and Aqknowledge software (version 3.2.6, BIOPAC Systems, Santa Barbara, CA). Trichome Staining Cells on Snapwell inserts were fixed in 10% buffered neutral formalin (Fisher Scientific) overnight at 4°C

2625

MAMMARY EPITHELIAL ION TRANSPORT

followed by three 15-min rinses in PBS for cytochemistry (in mM: 5, KH2PO4; 15, K2HPO4; 150, NaCl; pH 7.2 to 7.4) and stored in PBS for cytochemistry + 0.02% sodium azide. Epithelial cell monolayers were dehydrated in increasing concentrations of ethanol to absolute, followed by 100% glass-distilled acetone (Electron Microscopy Sciences, Fort Washington, PA) and embedded in Epon/Araldite (Electron Microscopy Sciences). Embedded tissues were cut into 0.6- to 0.8-µm semithin sections (Ultracut E ultramicrotome, Reichert-Jung, Austria) using a diamond knife (Diatome, Fort Washington, PA). Sections were heat-fixed on glass slides and stained as previously described (Kurotaki, 1972). Chemical Sources Forskolin (Coleus forskohlii) was purchased from Calbiochem (La Jolla, CA). Dulbecco’s modified Eagle media, Ham F-12 nutrient mixture (F-12), fetal bovine serum, penicillin (10 units/ml) and streptomycin (10 µg/ml) premix, and trypsin plus EDTA were purchased from GibcoBRL (Rockville, MD). Fe-Fortified bovine calf serum and newborn calf serum were purchased from Hyclone (Logan, UT). Amiloride, bumetanide, glucose, insulin-transferrin-sodium selenite media supplement, lactalbumin hydrosylate, lactose, L-ascorbic acid, mannitol, modified Eagle medium (MEM) amino acid premix (MEM AA), MEM nonessential AA premix (MEM nonAA), MEM vitamin premix (MEM Vit), NCTC 135, norepinephrine, pyruvate, RPMI 1640, RPMI AA, and RPMI Vit were purchased from Sigma (St. Louis, MO). All other chemicals were of highest grade available and purchased from reputable sources. Stock Solutions for Ussing Chamber Experiments Stock solutions were prepared as follows: forskolin, 10 mM in ethanol; norepinephrine, 10 mM in 1 mM ascorbic acid; amiloride, 10 mM in H2O; bumetanide, 20 mM in ethanol. N-(4-methyphenylsulfonyl)-N′-(4trifluoro- methylphenyl)urea (DASU-02), 100 mM in dimethyl sulfoxide. Forskolin, DASU-02, and bumetanide were stored at −20°C. Amiloride was stored at 4°C. All other modulators were freshly dissolved on the day of the experiment.

Figure 1. Bovine mammary epithelial cells (BME-UV) form a confluent monolayer and exhibit a pseudostratified structure when cultured on a permeable support. Trichrome stain, 0.8-µM semithin section. Bar = 10 µm.

appropriate, multivariate comparisons were conducted with ANOVA PROC GLM (SAS Institute, Inc., Cary, NC). The probability of a type 1 error of less than 5% was considered statistically significant. RESULTS Bovine Mammary Epithelial Cells Form Continuous Monolayers Bovine mammary epithelial cells were initially cultured under conditions similar to those previously described (Zavizion et al., 1996a). When grown in tissue culture flasks, mammary cells attached, proliferated, and dispersed to reach ∼70% confluency within 3 to 5 d after subculture. If allowed to grow to confluency in the culture flask, BME-UV cells exhibited a cobblestone pattern typical of epithelial cells as previously described (Zavizion et al., 1996a). When grown on permeable supports and assessed by light microscopy, cells exhibited a pseudostratified cuboidal morphology (Figure 1). Cells formed a continuous monolayer in which all cells appeared to have contact with the permeable support. Nuclei containing nucleoli were observed at various levels throughout the monolayer. The cells have previously been reported to exhibit microvilli and desmosomes (Zavizion et al., 1996a). Taken together, these results suggest that BME-UV cells form an epithelial monolayer that acts as a barrier between dissimilar fluid compartments.

Statistical Analysis

Bovine Mammary Epithelial Cells Form an Electrically Tight Monolayer that Actively Transports Ions

Where appropriate, numerical data from Ussing chamber experiments are presented as the arithmetical mean and standard error of the mean using culture well as the experimental unit. The Student’s t-test (Excel, Microsoft Corporation, Redman, WA) was employed to assess the likelihood of population differences. Where

Electrophysiological techniques were employed to provide a stringent test of epithelial function. Initially, experiments were performed to test whether BME-UV or BMM-UV cells formed resistive monolayers when grown on permeable supports. Cells of both types were seeded at equal density, grown on Snapwell supports Journal of Dairy Science Vol. 84, No. 12, 2001

2626

SCHMIDT ET AL.

for 14 to 16 d and evaluated for Rte. BME-UV cell monolayers displayed significantly higher Rte (52 ± 10 Ω cm2; n = 9) compared with BMM-UV cell monolayers (10 ± 2 Ω cm2; n = 5), which were indistinguishable from filters without cells. (8 ± 1 Ω cm2; n = 3; Figure 2A). Although BME-UV cells developed into a resistive monolayer, PDte remained near zero (−0.07 ± 0.14 mV). These results suggest that BME-UV cells form tight junctions necessary to perform a barrier function, but that active electrogenic ion transport cannot be observed in basal conditions. Forskolin, an activator of the cAMP cascade, caused a prototypical response that was characterized by a rapid increase in Isc followed by a sustained plateau. Persistant elevations in ion transport were routinely observed for periods in excess of 10 min (Figure 2B). Occasionally, the initial response included a transient peak in Isc (e.g., see Figure 5B below). The increase in Isc was coupled with a reduction in Rte (Figure 2C) suggesting that a conductive transcellular pathway was activated by exposure to forskolin. Myoepithelial cells (BMM-UV) did not respond to forskolin (not shown). The hypothesis that an apical medium with a higher K+:Na+ ratio would affect electrophysiological parameters was tested. BME-UV cell monolayers were grown with either TBM (Table 1; 142 mEq Na+, 6.6 mEq K+) or medium #2 (Table 2, 68 mEq Na+, 80 mEq K+) on the apical side, and TBM on the basolateral side. Exper-

Figure 3. Time- and culture-dependent development of electrophysiological properties by bovine mammary epithelial (BME-UV) cell monolayers. (A) Rte increases in all monolayers over the time period evaluated regardless of media composition (P ≤ 0.05). Medium #2 was associated with greater Rte at all time points tested (P ≤ 0.003). (B) The basal PDte was not significantly different from zero in either media at any time point (P > 0.3). (C) Acute exposure to forskolin induced an increase in Isc at all time points tested. Responses were consistently greater in medium #2 at every time point tested (P ≤ 0.05). No time-dependent differences were detected. (D) Forskolininduced change in Rte increases in all monolayers over the time period evaluated regardless of media composition (P < 0.05). Medium #2 was associated with greater ∆Rte at all time points tested (P ≤ 0.05). Compositions of typical bovine medium and medium #2 are provided in Tables 1 and 2.

Figure 2. Bovine mammary epithelial (BME-UV) cells exhibit characteristics commonly associated with secretory epithelial cells when cultured on permeable supports. (A) BME-UV cell monolayers (BME) exhibit higher electrical resistance than mammary myoepithelial cell monolayers (BMM), which are not different from culture supports with no cells (Filter). Traces showing BME-UV Isc (B) and Rte (C) are shown. Periodic Isc deflections result from a 1-mV pulse that was used to calculate Rte. In basal conditions, Isc and Rte were stable. Arrows indicate the addition of forskolin, an activator of adenylyl cyclase, to the tissue chambers. Forskolin caused an increase in Isc and a corresponding decrease in Rte. Results are typical of nine experiments. Journal of Dairy Science Vol. 84, No. 12, 2001

iments were conducted 7 to 14 d postseeding in modified Ussing chambers to test for the developmental time course of Rte, PDte, and forskolin-stimulated ∆Isc and ∆Rte (Figure 3). Rte increased over the duration of the experiment regardless of the apical medium that was employed (Figure 3A; P ≤ 0.05; n = six or seven paired observations at each time point). However, media with an increased K+:Na+ ratio (i.e., medium #2) was associated with a significantly greater Rte at all time points tested (P ≤ 0.003). Rte approached a plateau of approxi-

MAMMARY EPITHELIAL ION TRANSPORT

mately 80 Ω cm2 after 14 d in culture. In either medium, PDte was not significantly different from zero regardless time point (Figure 3B). Forskolin-induced elevations in Isc did not change over the duration of the experiment although monolayers exposed to medium #2 provided consistently greater responses at all time points tested (Figure 3C; P ≤ 0.05). Monolayers cultured in medium #2 responded with a greater forskolin-induced ∆Rte (P ≤ 0.05; Figure 3D), a result that might be interpreted as a greater density of responsive ion channels. However, circuit analysis cannot confirm this conclusion from the available results. The results in Figure 3 indicate that BME-UV cells are fully capable of ion transport shortly after subculture, but that epithelial cell association develops over time. An apical medium with a high K+:Na+ ratio promotes greater epithelial resistance and responsiveness to pharmacological stimuli. Experiments were conducted to test the response of mammary epithelial monolayers to a physiological agonist, norepinephrine. Invariably, norepinephrine exposure was associated with an acute increase in Isc (Figure 4) similar to that previously observed with forskolin. Responses to norepinephrine were observed only when the basolateral aspect of the monolayer was exposed, indicating the adrenergic receptor location. Bumetanide, an inhibitor of Na+/K+/Cl− cotransporter, reduced both forskolin and norepinephrine stimulated current (Figure 4) when applied to the basolateral face of the epithelial monolayer. A significantly smaller effect of bumetanide was observed during apical exposure that might reflect modest paracellular permeability to this compound. DASU-02, a chloride channel blocker (Schultz et al., 1997; Schultz et al., 1999; O’Donnell et al., 2000), also reduced both norepinephrine (Figure 4B) and forskolin (not shown) stimulated Isc. These results indicate that bovine mammary epithelial cells express ion transport mechanisms that can be acutely modulated by neurotransmitters. The results strongly suggest that the stimulated ion transport is Cl− secretion because it is partially inhibited by both bumetanide and DASU-02. Glucocorticoids that are either elevated during mastitis (cortisol) or used in the treatment of mastitis (dexamethasone, prednisolone) were evaluated for direct effects on mammary epithelial ion transport. Epithelial exposure to both cortisol (Figure 5B) and dexamenthasone (Figure 5C) were associated with an elevated basal Isc and reduced Rte (Figure 5D). The increased basal current and reduced resistance are consistent with the expression of a conductive ion permeation pathway. Amiloride was employed to test the hypothesis that corticosteroids induced the expression of the epithelial Na+ channel (ENaC). The Isc was reduced and Rte was increased by 10 µM amiloride, effects that are consis-

2627

tent with inhibition of ENaC. Furthermore, amiloride was effective only from the apical membrane (Figure 5C) providing additional evidence for the selected targeting of ion transport mechanisms in BME-UV cell monolayers. Exposure to prednisolone was likewise associated with an elevated basal current that was sensitive to amiloride (not shown). The response to forskolin was unchanged by chronic corticosteroid exposure. Further studies were conducted to test for effects of apical ion concentration and Na+:K+ ratio on Rte and forskolin-stimulated ∆Isc. Lactose and mannitol were used to maintain osmolality and test for the possibility that milk lactose concentration modulates epithelial ion transport. As depicted in Figure 6A, mannitol was associated with statistically greater Rte than lactose regardless of Na+:K+ ratio or ionic strength of the apical medium. Perhaps more striking was the significant reduction in Rte that accompanied increased ionic strength in the presence of either lactose or mannitol. Because osmolality could not be maintained without

Figure 4. Bovine mammary epithelial cells respond to different pharmacological and physiological stimuli. (A, B) Acute exposure to norepinephrine resulted in an increase transepithelial current. Acute exposure to bumetanide (basolateral) and DASU-02 led to a reduction in norepinephrine stimulated current. Results are typical of 11, 4, 4, and 3 observations for effects of norepinephrine, basolateral bumetanide, apical bumetanide, and DASU-02, respectively. (C) Forskolinstimualted responses were significantly greater than responses to norepinephrine (n = 11; P < 0.03). Responses after basolateral additions of bumetanide were statistically greater than the responses after apical additions of bumetanide following either forskolin or norepinephrine (n = 4, each; P ≤ 0.02). Abbreviations are as follows: Amil, 10 µM amiloride; Norep, 10 µM norepinephrine; Bum, 20 µM bumetanide; For, 2 µM forskolin; AP, apical; BL, basolateral. DASU02 was used at a concentration of 100 µM. Journal of Dairy Science Vol. 84, No. 12, 2001

2628

SCHMIDT ET AL.

Figure 5. Bovine mammary cell monolayers respond to chronic glucocorticoid exposure with expression of amiloride-sensitive ion transport. (A) Control, no response to amiloride and a typical forskolin response. Chronic exposure to 100 nM dexamethasone (B) or 500 nM cortisol (C) induced an amiloride-sensitive basal Isc. (D) Chronic exposure to dexamethasone was associated with reduced Rte in both typical bovine medium and medium #2 (n = 5; P < 0.02). Abbreviations are as follows: Amil, 10 µM amiloride; For, 2 µM forskolin; AP, apical; BL, basolateral; Dex, dexamethasone; TBM, typical bovine medium.

reciprocally changing carbohydrate (lactose or mannitol) and ionic concentration, we are unable to determine whether the reductions in Rte are caused by an increase in ionic strength or by a decrease in carbohydrate concentration. BME-UV cell monolayers were relatively insensitive to changes in Na+:K+ ratio. At high ionic strength, resistance was low regardless of whether the Na+:K+ ratio was 1:3 or 2:1. At lower ionic strength that included increased carbohydrate concentration, Rte was equally unaffected by changes in Na+:K+ ratio. These results suggest that changes in the ionic strength of milk might contribute to changes in the integrity of the mammary epithelium. Forskolin-stimulated ion transport was not affected by changes in apical ion or carbohydrate concentration (Figure 6B). These results again suggest that the acutely modulated ion transport activity of BME-UV cells is regulated by mechanisms distinctly different than epithelial resistance. In summary, the results show that BME-UV cells form an epithelial monolayer that acutely responds to adrenergic stimulation with increases in anion secretion. Over the first 2 weeks in culture, monolayer integJournal of Dairy Science Vol. 84, No. 12, 2001

Figure 6. Permeability of bovine mammary epithelial (BME-UV) monolayers is modulated by apical cation and carbohydrate concentration. Osmolality was held constant, while Na+, K+, lactose, and mannitol concentrations were varied. (A) Media containing mannitol was associated with higher Rte than media containing lactose (P ≤ 0.0001). Media containing increased concentrations of either Na+ or K+ (while the other was held constant and carbohydrate concentration decreased to maintain osmolality) were associated with reduced Rte (P ≤ 0.0001). Although significant interactions between carbohydrate and cation concentration were observed, identical trends were observed in each carbohydrate. (B) No significant effects of carbohydrate or cation concentration were observed on forskolin-stimulated ∆Isc. The two right bars in each panel represent responses in typical bovine medium and medium #2, respectively. Ion-specific electrodes were employed to measure the cation concentration of each media after the addition of serum. Lactose and mannitol concentrations are based upon the amount added during media preparation. Abbreviations are as follows: Lac, lactose; Man, mannitol. Concentrations are reported in mEq or mM.

MAMMARY EPITHELIAL ION TRANSPORT

rity, as assessed by changes in Rte, and Isc increase and can be modulated by chronic glucocorticoid exposure. Composition of the apical solution (i.e., the milk compartment) significantly alters epithelial integrity. We believe that these results are the first to show direct effects of neurotransmitters and corticosteriods on bovine mammary epithelial cells with resultant changes in epithelial ion transport. DISCUSSION Mechanisms of ion transport that contribute to milk composition or volume are poorly understood. Ion transport studies have been conducted within the teat of the cow (Blatchford and Peaker, 1988); however, very few studies are present that have looked at ion transport across bovine mammary epithelium in a defined environment. The cell culture system herein described provides a means of testing and characterizing epithelial mechanisms of ion transport that will lead to a better understanding of bovine mammary function. While there are numerous reports regarding the culture of bovine mammary epithelial cells, few have focused on developing an electrically tight polarized monolayer and none has been employed to study ion transport. Both Smits et al. (1996), and Delabarre et al. (1997), reported that primary epithelial cell cultures could develop a resistance of up to 2000 Ω cm2 during a 1- to 2-wk culture period. Each of these systems is well suited for their respective applications (leukocyte diapedesis and protein secretion, respectively), but have drawbacks that might limit their usefulness for the study of ion transport. In each case, the authors employed relatively complex media containing numerous hormones and equally complex substrates to produce the epithelial monolayers. The cultures contained multiple cell types; one by design and the other because it is a primary culture. Unfortunately, one cannot be certain that observed effects on epithelial function in such a system are due to direct interactions with epithelial cells; nonepithelial cells may modulate epithelial function by paracrine mechanisms. Thus, there is precedent for culturing bovine mammary epithelial cell monolayers that are capable of forming an electrically tight barrier and of secreting milk proteins; however, such systems may not be well suited for studies focusing on mechanisms of ion transport mediated exclusively by epithelial cells. At least two clonal bovine mammary epithelial cell lines have been described, MAC-T (Huynh et al., 1991) and BME-UV (Zavizion et al., 1996a). These cell lines have been reported to synthesize and/or secrete milk proteins (α-lactalbumin, αs1-casein, and/or β-casein) and the BME-UV cells exhibit morphological character-

2629

istics of epithelia including microvilli, desmosomes, and the presence of cytokeratins. These cell lines appear to express many attributes typically associated with mammary epithelium; however, reports have not been forthcoming regarding their ability to form resistive monolayers or to actively transport ions. We obtained the clonal BME-UV cell line and have shown that resistive monolayers are formed when cells were cultured on commercially available substrates. Culture conditions were similar to those previously described for this cell line although neither prolactin nor cortisol were initially included. The results showed that these conditions are sufficient for the formation of a resistive epithelial monolayer. Microscopy was employed to demonstrate that the resistance was not an artifact of cells forming multilayer structures, but that the cells displayed an epithelial morphology consistent with previous descriptions. Transepithelial resistance increased during the duration of the experiment indicating that the cells could mature to this level of epithelial differentiation. In contrast, closely paired myoepithelial cells (BMM-UV) did not exhibit transepithelial resistance when cultured in identical conditions. Thus, the transformed BME-UV cell line responds to the need for a model in which ion transport across a mammary epithelium can be studied in a well-defined environment free of nonepithelial cell types (Shennan, 1998). Mechanisms of ion transport across mammary epithelium are poorly understood. Shennan and Peaker (2000) provided a review of what is known regarding epithelial ion transport and the gaps in our knowledge. It should first be noted that the mammary epithelium effectively maintains substantial concentration gradients for monovalent ions. These gradients must be generated by active epithelial transport and maintained by tight junctions. Second, the presence of a lumen positive transepithelial potential suggests that active mechanisms provide for either cation secretion or anion absorption. Third, the reported distribution of Na+ and K+ across the apical membrane is near their equilibrium potential, suggesting the presence of highly permeant pathways for these ions. Fourth, Cl− is distributed across the apical membrane well away from its equilibrium potential. Thus, the Cl− electrochemical potential could readily be harnessed to acutely increase the volume of secretion across the epithelium. Numerous mechanisms are required to develop these ion gradients. However, the identity of only one of the components is recognized with any degree of certainty, the ouabain-inhibitable Na+/K+-ATPase. We, too, observed that oubain inhibited forskolin-stimulated ion transport across the BME-UV monolayers (not shown). This effect was manifested only from the basolateral aspect of the monolayers supporting the expected location of Journal of Dairy Science Vol. 84, No. 12, 2001

2630

SCHMIDT ET AL.

this membrane ion transporter. Shennan and Peaker (2000) also suggest that the Cl− loading step at the basolateral membrane is likely a Na+/K+/Cl− cotransporter that is inhibited by loop diuretics (e.g., bumetanide, furosemide). However, they indicate that the cotransporter would not theoretically function with its commonly accepted stoichiometry of 1:1:2 in the ionic conditions reported for the bovine mammary gland (∆G > 0). This inconsistency suggests that either a relatively unique cotransporter with a different stoichiometry is present in mammary epithelium (Russell, 1983), or the distribution of ions is different than has been reported. Our results indicate that the bumetanide inhibitable cotransporter is present in the basolateral membrane of the epithelial cells although the stoichiometry remains to be determined. Little is known regarding conductive pathways in the apical membrane of polarized mammary epithelial cells. It has been suggested that the apical membrane is unable to discriminate between Na+ and K+ (Blatchford and Peaker, 1988; Shennan and Peaker, 2000). However, an amiloride-sensitive current was expressed by the BME-UV cells in situations in which natural and synthetic corticosteroids were employed to mimic the physiological or clinical situation. The elevated basal current and reduced resistance as a result of chronic exposure to corticosteroids are consistent with ENaC expression. These results suggest an additional mechanism by which stress, by increasing cortisol levels and the subsequent expression of ENaC, can reduce the volume of milk output. A K+ conductance has been documented in membranes isolated from milk (Smith et al., 1990; Shennan, 1992; Stelwagen et al., 1999; Silanikove et al., 2000), although the channels in these cell-free vesicles are unidentified and appear to be unregulated. The molecular identities of nonselective or K+-selective apical conductances remain to be determined. Both forskolin and norepinephrine acutely stimulated an increase in short-circuit current that we interpret as anion secretion based upon its insensitivity to amiloride, and sensitivity to both bumetanide and DASU-02. The identity of the apical Cl− conductance involved in this transport is unknown, although inhibition by DASU-02 suggests that the cystic fibrosis (CF) anion channel participates in this response (Schultz et al., 1997, 1999; O’Donnell et al., 2000). Previously published reports suggested that the CF transmembrane conductance regulator (CFTR) is not likely a major contributor to the overall anion conductance across human mammary epithelium as evidenced by normal milk composition in CF mothers (Welch et al., 1981; Shiffman et al., 1989; Michel and Mueller, 1994). One caveat with this conclusion, however, is that it is based on CF patients with mild forms of the disease and Journal of Dairy Science Vol. 84, No. 12, 2001

thus may represent patients who exhibit some CFTR channel activity. Regardless, the reported distribution of ions (Shennan and Peaker, 2000) suggests that the anion conductive pathway is tightly regulated. The molecular identity and regulatory mechanisms associated with this channel are particularly important since our results suggest that the composition or volume of epithelial secretions can be acutely modulated by anion channel activity. Adrenergic stimulation of milk secretion is not without precedent. Blum et al. (1989) reported that adrenalin, noradrenalin, and dopamine reduced milk removal from cows. However, these effects were mitigated by αadrenergic receptor blockade. Futhermore, β-adrenergic stimulation (either with a β-agonist or a nonselective agonist in the presence of an α-antagonist) was associated with increased milk yield, increased peak flow rate and decreased time to peak flow. Our results provide a cellular mechanism that can account for these observations. Additional studies are required to further elucidate the pathway responsible for norepinephrinestimulated ion transport. The effects of changing apical ion concentrations on the epithelium have important implications. Electrical conductivity, an indicator of total ion concentration, has been employed as an early indicator of mastitis (Ziv et al., 1998; Hillerton and Semmens, 1999) and changes in the ionic composition of milk during mastitis have been well established (i.e., the concentration of Na+ increases dramatically and the concentration of K+ declines, resulting in an increase in the Na+:K+ ratio and a net increase in cation concentration; (VandeputteVan Messom and Burvenich, 1993; Hoeben et al., 1999). Our results suggest that the activity of epithelial cells can be modulated to affect changes in the ionic composition of milk and that increased cation concentrations contribute to a reduction in epithelial barrier integrity. This hypothesis challenges current dogma, which suggests that a loss of epithelial barrier function must precede changes in milk ionic composition. The bovine mammary epithelial cell system that we have developed will provide an excellent model to test for effects of lipopolysaccharide, cytokines, and other proinflamatory agents on epithelial secretion and changes in epithelial resistance. CONCLUSIONS When cultured on permeable supports, the BME-UV cell line responds to a pressing need for a model in which epithelial function can be studied in a well-defined environment free of nonepithelial cell types (Shennan, 1998). The cell line has previously been shown to synthesize milk proteins (α-lactabumin and αs1-casein;

MAMMARY EPITHELIAL ION TRANSPORT

Zavizion et al., 1996a). The current results extend the utility of this bovine mammary epithelial cell line by demonstrating that it forms electrically resistive monolayers, that activity of the monolayers is acutely modulated by physiological neurotransmitters, that activity of the monolayers is chronically modulated by naturally occurring and synthetic hormones, and that the monolayers are affected by the composition of the apical medium as would be expected in vivo. These attributes indicate that the BME-UV cell line and the conditions that we have identified provide an excellent model to study cellular and epithelial mechanisms of ion transport associated with milk secretion. Further exploitation of this system will allow for insights regarding mammary development, lactation, involution, and pathology. ACKNOWLEDGMENTS The authors extend their thanks to Jeff White for cell acquisition, to Roger Sedlacek and Rebecca Quesnell for technical assistance, and to Ginger Biesenthal, Pam Say, and Bonnie Thompson for accounting and clerical support. Special thanks are in order for John Tomich and Kathy Mitchell for their assistance in nurturing the academic and scientific development of C. R. Schmidt. This research was supported by the Kansas State University College of Veterinary Medicine Dean’s Research Fund (BDS), by the Kansas State University Agricultural Experiment Station (Animal Health and Disease Program of Research, Section 1433; BDS and JMS), and a Cancer Research Award to CRS from the Kansas State University Center for Basic Cancer Research (CRS). Contribution no. 01-382-J from the Kansas Agricultural Experiment Station. REFERENCES Blatchford, D. R., and M. Peaker. 1988. Effect of ionic composition of milk on transepithelial potential in the goat mammary gland. J. Physiol. (Lond.) 402:533–541. Blum, J. W., D. Schams, and R. Bruckmaier. 1989. Catecholamines, oxytocin and milk removal in dairy cows. J. Dairy Res. 56:167–77. Delabarre, S., C. Claudon, and F. Laurent. 1997. Influence of several extracellular matrix components in primary cultures of bovine mammary epithelial cells. Tissue Cell 29:99–106. Hillerton, J. E., and J. E. Semmens. 1999. Comparison of treatment of mastitis by oxytocin or antibiotics following detection according to changes in milk electrical conductivity prior to visible signs. J. Dairy Sci. 82:93–98. Hoeben, D., C. Burvenich, P. J. Eppard, and D. L. Hard. 1999. Effect of recombinant bovine somatotropin on milk production and composition of cows with Streptococcus uberis mastitis. J. Dairy Sci. 82:1671–1683.

2631

Huynh, H. T., G. Robitaille, and J. D. Turner. 1991. Establishment of bovine mammary epithelial cells (MAC-T): An in vitro model for bovine lactation. Exp. Cell Res. 197:191–199. Kurotaki, M. 1972. [Differential staining of epon-resin-embedded tissue for light microscopy observation (including review of the literature on general technics of semi-thin sectioning)]. Kaibogaku Zasshi 47:237–250. Michel, S. H., and D. H. Mueller. 1994. Impact of lactation on women with cystic fibrosis and their infants: a review of five cases [see comments]. J. Am. Diet. Assoc. 94:159–165. Neville, M. C. 1999. Physiology of lactation. Clin. Perinatol. 26:251– 279, v. O’Donnell, E. K., R. L. Sedlacek, A. K. Singh, and B. D. Schultz. 2000. Inhibition of enterotoxin-induced porcine colonic secretion by diarylsulfonylureas in vitro. Am. J. Physiol. Gastrointest. Liver Physiol. 279:G1104–G1112. Russell, J. M. 1983. Cation-coupled chloride influx in squid axon. Role of potassium and stoichiometry of the transport process. J. Gen. Physiol. 81:909–925. Schultz, B. D., A. K. Singh, D. C. Devor, and R. J. Bridges. 1999. Pharmacology of CFTR chloride channel activity. Physiol. Rev. 79:S109–S144. Schultz, B. D., A. Takahashi, C. Liu, R. A. Frizzell, and M. Howard. 1997. FLAG epitope positioned in an external loop preserves normal biophysical properties of CFTR. Am. J. Physiol. 273:C2080–C2089. Sedlacek, R. L., R. W. Carlin, A. K. Singh, and B. D. Schultz. 2001. Culture of epithelial cells from porcine vas deferens for ion transport studies. Am. J. Physiol.: (In press) Shennan, D. B. 1992. K+ and Cl− transport by mammary secretory cell apical membrane vesicles isolated from milk. J. Dairy Res. 59:339–348. Shennan, D. B. 1998. Mammary gland membrane transport systems. J. Mammary Gland Biol. Neoplasia 3:247–258. Shennan, D. B., and M. Peaker. 2000. Transport of milk constituents by the mammary gland. Physiol. Rev. 80:925–951. Shiffman, M. L., T. W. Seale, M. Flux, O. R. Rennert, and P. T. Swender. 1989. Breast-milk composition in women with cystic fibrosis: Report of two cases and a review of the literature. Am. J. Clin. Nutr. 49:612–617. Silanikove, N., A. Shamay, D. Shinder, and A. Moran. 2000. Stress down regulates milk yield in cows by plasmin induced beta-casein product that blocks K+ channels on the apical membranes. Life Sci. 67:2201–2212. Smith, K. M., S. A. McNeillie, and D. B. Shennan. 1990. K+ (Rb+) transport by a mammary secretory cell apical membrane fraction isolated from goats’ milk. Exp. Physiol. 75:349–358. Smits, E., E. Cifrian, A. J. Guidry, P. Rainard, C. Burvenich, and M. J. Paape. 1996. Cell culture system for studying bovine neutrophil diapedesis. J. Dairy Sci. 79:1353–1360. Stelwagen, K., V. C. Farr, and H. A. McFadden. 1999. Alteration of the sodium to potassium ratio in milk and the effect on milk secretion in goats. J. Dairy Sci. 82:52–59. Vandeputte-Van Messom, G. V., and C. Burvenich. 1993. Effect of somatotropin on changes in milk production and composition during coliform mastitis in periparturient cows. J. Dairy Sci. 76:3727–3741. Welch, M. J., D. L. Phelps, and A. B. Osher. 1981. Breast-feeding by a mother with cystic fibrosis. Pediatrics 67:664–666. Zavizion, B., M. van Duffelen, W. Schaeffer, and I. Politis. 1996a. Establishment and characterization of a bovine mammary epithelial cell line with unique properties. In Vitro Cell. Dev. Biol. Anim. 32:138–148. Zavizion, B., M. van Duffelen, W. Schaeffer, and I. Politis. 1996b. Establishment and characterization of a bovine mammary myoepithelial cell line. In Vitro Cell. Dev. Biol. Anim. 32:149–158. Ziv, G., M. Shem-Tov, and F. Ascher. 1998. Combined effect of ampicillin, colistin and dexamethasone administered intramuscularly to dairy cows on the clinico-pathological course of E. coli-endotoxin mastitis. Vet. Res. 29:89–98.

Journal of Dairy Science Vol. 84, No. 12, 2001