Correspondence to: Scott M. O'Grady, Departments of Physiology and Animal Science, 495 Animal Science/Veterinary Medicine Building, University of Minnesota, St. Paul, Minnesota 55108. Fax: 612-625-2743; E-mail:ograd001{at}tc.umn.edu.
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Abstract |
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The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na+ transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na+ transport two- to threefold by increasing Na+-K+ ATPase transport activity and basolateral membrane K+ conductance without increasing the apical membrane amiloride-sensitive Na+ conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na+ absorption with a further increase in Na+-K+ ATPase transport activity and an increase in apical membrane amiloride-sensitive Na+ conductance. The effect of insulin on the Na+-K+ ATPase was the result of an increase in Vmax for extracellular K+ and intracellular Na+, and an increase in affinity of the pump for Na+. Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of -1 and
-2 isoforms, but not the
-3 isoform of Na+-K+ ATPase, which did not change in the presence of insulin. Insulin-stimulated Na+ transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)3], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na+ transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na+-K+ ATPase activation.
Key Words: insulin-like growth factor I, epithelial ion transport, membrane transport, ouabain, amiloride
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INTRODUCTION |
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The transport-related activity of the surface and glandular endometrial epithelium plays an important role in regulation of uterine lumen electrolyte composition, pH, and fluid volume, providing a suitable environment for fertilization and implantation of the developing embryo (, cAMP, and gastrin-releasing peptide (
The first demonstration of an effect of insulin on transepithelial Na transport was reported by -2 isoform is selectively inserted into the plasma membrane (
-1 and
-2 isoforms and an increase in Vmax associated with the
-2 isoform of the pump. In hepatocytes, skeletal muscle cells, and 3T3-L1 adipocytes, increases in Na+-K+ ATPase transport activity appear to be due to elevations in intracellular [Na+] resulting from activation of either Na+-H+ exchange (
Specific binding sites for insulin and IGF-I have been identified in normal endometrium and endometrial cancer cells (
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MATERIALS AND METHODS |
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Materials
Insulin, IGF-I, ouabain, indomethacin, nonessential amino acids, and high purity grade salts were purchased from Sigma Chemical Co. Hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)3], wortmannin, okadaic acid, and PD-98059 (2'-amino-3'-methoxyflavone) were purchased from Biomol Research Laboratories. Amiloride, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and calyculin A were purchased from Research Biochemicals International, and benzamil from Molecular Probes. Dulbecco's modified Eagle's medium (DMEM), Dulbecco's phosphate buffer saline (DPBS), fetal bovine serum (FBS), collagenase (type 1), kanamycin, penicillin-streptomycin, and fungizone were purchased from GIBCO BRL.
Cell Isolation and Culture
Porcine uterine tissues were collected from 45-mo-old Yorkshire-Pietrain cross pigs purchased from stock herds maintained by the University of Minnesota College of Agriculture. Uterine tissues from adult, precycling animals were used to minimize variability in electrolyte transport properties at different stages of the estrus cycle. Uteri were obtained from pigs that were killed at the University of Minnesota Meat Sciences Laboratory by the captive bolt euthanasia procedure approved by the University Animal Care Committee and supervised by a USDA certified veterinarian. Uterine tissue was placed in ice cold porcine Ringer solution containing (mM): 130 NaCl, 6 KCl, 3 CaCl2, 0.7 MgCl2, 20 NaHCO3, 0.3 NaH2PO4, 1.3 Na2HPO4, gassed with 95% O2/5% CO2, pH 7.4. After removal of the serosal muscle layer, the endometrial tissue was minced into small pieces (1 mm3) and washed twice with Ca2+- and Mg2+-free DPBS. The tissue fragments were then subjected to collagenase digestion and the epithelial glands were isolated as described previously (
Measurement of Basal Electrical Parameters
Transepithelial resistance of the cell monolayers was measured using the EVOM epithelial voltohmmeter coupled to Ag/AgCl "chopstick" electrodes (World Precision Instruments). After 10 d, the confluent culture inserts were mounted in Ussing Chambers, bathed on both sides with standard porcine Ringer solution maintained at 37°C, and bubbled with 95% O2 /5% CO2. Transepithelial potential difference, tissue conductance, and short circuit current (Isc) were measured with the use of voltage-clamp circuitry from JWT Engineering. The data from the voltage clamp experiments was digitized, stored, and analyzed using Workbench data acquisition software (Kent Scientific Corp.), and recorded with a Compaq pentium microcomputer. All cells were pretreated with indomethacin (10 µM) added to both apical and basolateral solutions at least 10 min before the beginning of the experiment.
Measurement of Pump Current
Pump current was measured using amphotericin B (10 µM) to permeabilize the apical membrane of monolayers mounted in Ussing chambers. To determine the [K+] dependence of the pump, monolayers were bathed on both sides with NaMeSO4 Ringer solution containing (mM): 120 NaMeSO4, 30 mannitol, 3 calcium gluconate, 1 MgSO4, 20 NaHCO3, 0.3 NaH2PO4, 1.3 Na2HPO4, gassed with 95% O2/5% CO2, pH 7.4. Increasing intracellular K+ concentration was accomplished by replacement of NaMeSO4 Ringer solution with an equivalent volume of KMeSO4 Ringer solution. KMeSO4 Ringer solution contained (mM): 120 KMeSO4, 30 mannitol, 5 NaCl, 3 calcium gluconate, 1 MgSO4, 20 KHCO3, 0.3 KH2PO4, 1.3 K2HPO4, gassed with 95% O2/5% CO2, pH 7.4. To determine the [Na+] dependence of the pump, monolayers were bathed on both sides with N-methyl-D-glucamine (NMDG)MeSO4 Ringer solution containing (mM): 130 NMDGMeSO4, 30 mannitol, 3 calcium gluconate, 1 MgSO4, 10 KHCO3, 0.3 KH2PO4, 1.3 K2HPO4, gassed with 95% O2/5% CO2, pH 7.4, and a NaMeSO4 Ringer solution containing different concentrations of Na+ was used to replace NMDGMeSO4 Ringer solution. The Na+ and K+ dependence of pump current was determined using the Hill equation: Ip = Imax [S]n/([S]n + K0.5), and its linear expression: log (Ip/Imax - Ip) = n log[S] - log K0.5, where Ip is the pump current stimulated by an increase in intracellular [Na+] or extracellular [K+], Imax is the maximal pump current, S is an intracellular [Na+] or extracellular [K+], K0.5 is the apparent dissociation constant, and n is the Hill coefficient. The kinetic parameters were determined by nonlinear regression or by linear regression analysis (PrismTM 2.0; GraphPad Software).
Measurement of Membrane Permeability
Currentvoltage relationships were determined using amphotericin Bpermeabilized monolayers mounted in Ussing chambers. The intracellular compartment was bathed with KMeSO4 Ringer solution and amphotericin B (10 µM) was added to permeabilize the membrane. The extracellular compartment was bathed with standard porcine Ringer solution or NaMeSO4 Ringer solution. An epithelial voltage clamp (World Precision Instruments) in combination with an LM-12 A-D interface (Dagan Corp.) were used to voltage clamp the monolayers and record the data. The voltage step commands and the resultant currents were generated using pCLAMP software (Axon Instruments). Currentvoltage (IV) relationships were obtained by a series of voltage step commands described in the figure legends. The compound-sensitive components were obtained by subtracting the currents before and after addition of the compound. The Na+:K+ selectivity ratio (PNa/PK) was calculated from reversal potential (Erev) measurements using the Goldman-Hodgkin-Katz equation {Erev = RT/zF ln (PNa [Na+])/(PK [K+])}.
Ouabain Binding
Epithelial cells (3 x 105) were subcultured onto 6.5-mm transparent permeable membranes containing 10% FBS in DMEM. After 34 d, the cells were placed in serum-free Phenol-Redfree DMEM for 48 h, followed by insulin treatment (850 nM) for 24 h. Specific [3H] ouabain binding was performed at 37°C in humidified incubator of 5% CO2 in air, using a procedure modified from
Immunocytochemistry
Epithelial cells were allowed to grow on permeable membrane filters in DMEM supplemented with 10% FBS for 57 d, followed by serum-free Phenol Redfree DMEM for 2 d. Insulin (850 nM) was then added to the serum-free media for 2 d. The monolayers were then washed twice in DPBS, pH 7.4, permeabilized with 0.3% Triton X-100 in DPBS at room temperature for 10 min, and then fixed in methanol at -20°C for 10 min. After fixation, filters were cut from their supports. The cells were then washed with DPBS and incubated with DPBS containing 1% bovine serum albumin and 10% normal goat serum to block nonspecific binding at room temperature for 1 h. Then the cells were incubated overnight with isoform-specific mouse monoclonal antibody against the -1 and polyclonal antibodies to
-2 and
-3 isoforms of the rat Na+-K+ ATPase, 1:200 (Upstate Biotechnology Inc.) at 4°C. After washing, the cells were incubated with indocarbocyanine (cy3)-labeled goat antirabbit antibody, 1:400 (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After the final wash, the filters were placed on a glass slide. The cells and filters were embedded in fluoromount (Gallard Schlessinger) and examined by confocal microscopy, using a MRC1024 laser confocal microscope (Bio-Rad Laboratories) equipped with krypton-argon lasers.
Western Blot Analysis
Cell monolayers, as prepared for immunocytochemistry, were solubilized with lysis buffer (50 mM Tris-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 µg/ml aprotinin, and 1 mM NaF, pH 7.4) at 37°C for 30 min and homogenized. A protein assay was performed using a BCA Protein Assay Kit by Pierce. Proteins were separated by PAGE (8%). Electroblotting was done using Immobilon-P (Millipore Corp.). The electroblot assembly was placed into the electroblotting apparatus (Trans-Blot Cell; Bio-Rad Laboratories) and blotting was performed at 16 V overnight on ice. After the blots were removed, they were washed twice, and then blocked in freshly prepared 1x TBS-tween containing 3% nonfat dry milk (MLK) for 30 min at 2025°C with constant agitation. After washing, blots were reacted overnight in primary antibody, 15 or 100 ml freshly prepared 1x TBS-tween containing 3% MLK with appropriate dilution of the primary antibody (antirat -1 monoclonal antibody and rabbit-antirat
-2 polyclonal antibody from Upstate Biotechnology Inc.). The next day, blots were washed and reacted with secondary antibody, either goat antirabbit, alkaline phosphataselabeled or goat antimouse, alkaline phosphataselabeled. Secondary antibody was diluted 1:3,000 (33 µl into 100 ml) in 1x TBS-tween containing 3% MLK and reacted for ~2 h. After washing, alkaline phosphatase color reagent was added to 100-ml 1x alkaline phosphatase color development buffer at room temperature. Blots were incubated in development buffer until bands were clearly developed.
Statistics
All values are presented as means ± SEM, n is the number of monolayers, and N is the number of animals in each experiment. The differences between control and treatment means were analyzed using a t test for paired or unpaired means, where appropriate. A value of P < 0.05 was considered statistically significant. The EC50 values for insulin and IGF-I and the IC50 values for benzamil and amiloride were determined using a four parameter logistic function to fit the data (PrismTM 2.0).
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RESULTS |
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Long-Term Effects of Insulin and IGF-I on Electrolyte Transport
The basal electrical properties of cultured epithelial cells used in this study have been previously described (
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Short-Term Effect of Insulin on Isc
Addition of 850 nM insulin to the basolateral solution of monolayers produced an increase in Isc within 5 min that reached a maximal plateau response of 36 ± 2 µA (n = 11, N = 5) in 3045 min, as illustrated in Figure 2 A. The maximal Isc response was sustained for as long as 3 h. Addition of 65 nM IGF-I to the basolateral solution produced the same response as insulin with a maximal plateau response of 36 ± 3 µA (n = 6, N = 3). Subsequent addition of 10 µM benzamil to the apical solution inhibited both the basal and insulin-stimulated Isc. Concentrationresponse curves for benzamil and amiloride are shown in Figure 2 B with IC50 values of 25 nM for benzamil and 194 nM for amiloride. Pretreatment with 10 µM benzamil completely inhibited the insulin-stimulated Isc (data not shown). In addition, pretreatment with 50 µM HNMPA-(AM)3, a specific inhibitor of insulin receptor tyrosine kinase activity, for 30 min inhibited the insulin-stimulated increase in Isc (7 ± 2 µA, n = 6, N = 4), as illustrated in Figure 3 A. The concentrationresponse relationships for insulin and IGF-I presented in Figure 3 B demonstrated that threshold concentrations were ~0.85 nM for insulin and 0.13 nM for IGF-I. The EC50 values were 12 nM for insulin and 2.5 nM for IGF-I.
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Na+ and K+ Dependence of the Na+-K+ ATPase
To determine whether the increase in Isc produced by insulin was the result of stimulation of the Na+-K+ ATPase, experiments were performed using amphotericin Bpermeabilized monolayers mounted in Ussing chambers bathed on both sides with either NaMeSO4 Ringer solution containing 5 mM BaCl2 or NMDGMeSO4 Ringer solution containing 10 mM KHCO3. BaCl2 was used to inhibit basolateral K+ channels and limit the contribution of K+ recycling to pump activation. The increase in extracellular [K+] was accomplished by replacement of NaMeSO4 solution with an equal volume of KMeSO4 Ringer solution containing different concentrations of K+. To determine the Na+ dependence of the pump, NaMeSO4 Ringer solution with 10 mM KHCO3 and different concentrations of Na+ was used to replace NMDGMeSO4 Ringer solution.
Figure 4 A shows the increase in pump current (Ip) produced by increasing K+ concentrations in the basolateral solution of insulin-treated monolayers. The stimulated Ip was completely inhibited by 10 µM ouabain added to the basolateral solution (data not shown). When Ip was plotted as a function of basolateral [K+], it revealed that insulin treatment increased Imax from 20 ± 2 µA (n = 11, N = 5) to 63 ± 6 µA (n = 9, N = 5), with an increase in the K0.5 value from 1.8 ± 0.2 to 2.9 ± 0.2 mM. Stimulation of pump current was also dependent on intracellular [Na+], as shown in Figure 5 A. The relationship of Ip and [Na+] revealed that insulin treatment increased Imax from 18 ± 1 µA (n = 8, N = 4) to 42 ± 5 µA (n = 10, N = 4) with a significant decrease in the K0.5 value from 39 ± 2 to 24 ± 2 mM. Hill coefficients were 2.1 ± 0.3 for K+ and 1.2 ± 0.1 for Na+ under insulin treatment, which was not significantly different from corresponding control values (1.9 ± 0.2 for K+ and 1.3 ± 0.1 for Na+), as illustrated in Figure 4 B and 5 B.
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Ouabain-sensitive Current Across the Basolateral Membrane
The acute (1520 min) effects of insulin on the currentvoltage relationship of the Na+-K+ ATPase is shown in Figure 6. The experiment was performed using amphotericin Bpermeabilized monolayers, as described in the previous section. The apical surface of the epithelium was bathed with KMeSO4 Ringer solution supplemented with 5 mM NaCl, and amphotericin B was used to permeabilize the apical membrane. Standard Ringer solution was used to bathe the basolateral surface of the epithelium. The IV relationship of the pump was obtained using a voltage step protocol ranging from -90 to +90 mV (15-mV steps) at a holding potential of 0 mV. The difference currents were obtained by subtracting the current before and 10 min after basolateral addition of 100 µM ouabain. The ouabain-sensitive current after pretreatment with 850 nM insulin for 15 min was approximately twofold greater in magnitude, with no change in conductance when compared with current responses under basal conditions.
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Effect of Insulin on Basolateral Membrane K+ Permeability
The effect of insulin on basolateral K+ permeability is shown in Figure 7. The apical surface of the epithelium was permeabilized with amphotericin B and bathed with KMeSO4 Ringer solution without NaCl, while the basolateral surface was bathed with standard Ringer solution. The insulin-sensitive current obtained after basolateral addition of 850 nM insulin for 15 min exhibited slight outward rectification with a mean reversal potential of -53 ± 3 mV (n = 9, N = 4). Replacement of standard Ringer solution with KCl Ringer solution shifted the reversal potential toward zero (3 ± 2, n = 6, N = 4). Acute insulin (850 nM) treatment (1530 min) had no effect on apical membrane conductance.
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Effect of Insulin on Benzamil-sensitive Current
To characterize the properties of the benzamil/amiloride-sensitive Na+ channel located in the apical membrane, experiments were performed with amphotericin Bpermeabilized monolayers, as previously mentioned. The monolayers were bathed on the apical side with NaMeSO4 Ringer solution and on the basolateral side with KMeSO4 Ringer solution containing amphotericin B. The benzamil-sensitive current was obtained before and 2 min after apical addition of 5 µM benzamil. The currentvoltage relationship of benzamil-sensitive current is shown in Figure 8. Treatment with 850 nM insulin for 4 d showed a mean reversal potential of 65 ± 4 mV (n = 7, N = 4), which was not significantly different from control cells in serum-free media (72 ± 4 mV, n = 5, N = 3). The basal benzamil-sensitive conductance was 0.17 ± 0.05 mS. Stimulation with insulin significantly increased the conductance to 0.37 ± 0.05 mS. Interestingly, a 30-min treatment with 850 nM insulin produced no significant change in reversal potential (69 ± 8 mV, n = 6, N = 3) or conductance (0.18 ± 0.05 mS) compared with control monolayers. The Na+ to K+ selectivity ratio of the benzamil-sensitive current in the presence of insulin was calculated to be 11.6:1.
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Mechanism of Insulin Action
Previous studies using a rat skeletal muscle cell line demonstrated that the activation of Na+-K+ ATPase by insulin may involve the phosphatidylinositol 3-kinase (PI-3 kinase) signaling pathway (
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Additional experiments were performed to determine the effect of phosphatase inhibitors on insulin-stimulated pump current, as illustrated in Figure 10. The Na-K ATPase was stimulated by basolateral addition of 5 mM KMeSO4 Ringer solution before and after treatment with insulin for 30 min. Addition of 850 nM insulin to the basolateral solution had no effects on basal current (-5 ± 1 µA, n = 25, N = 9). A subsequent addition of 5 mM KMeSO4 Ringer solution produced a rapid increase in current that was completely inhibited by 100 µM ouabain, as shown in Figure 10 A. Compared with the control monolayers, insulin stimulated an increase in pump current from 18 ± 2 µA (n = 12, N = 5) to 32 ± 1 µA (n = 7, N = 4). Pretreatment with 100 nM okadaic acid for 30 min before addition of insulin abolished the insulin-stimulated pump current and decreased the basal pump current to 14 ± 2 µA (n = 6, N = 4), as shown in Figure 10 B.
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Short- and Long-term Effects of Insulin on Conductance and Pump Current
Figure 11 compares the apical Na+ conductance (Figure 8) and the pump current stimulated by 30-min and 4-d treatments with insulin. Administration of insulin for 30 min significantly increased K+-stimulated pump current with no effect on Na+ conductance. However, treatment with insulin for 4 d significantly increased both Na+ conductance and K+-stimulated pump current, suggesting that long-term treatment with insulin produced an increase in Na+ permeability of the apical membrane in addition to further activation of Na+-K+ ATPase.
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Identification of Na-K-ATPase Subunit Isoforms
The presence of -1,
-2, and
-3 isoforms of the Na+-K+ ATPase were determined by immunofluorescence and Western blotting. Figure 12 shows the summation of immunofluorescence images (13) obtained at 3-µm steps as the microscope was focused from the filter towards the apical membrane of glandular epithelial cell monolayers. The cell monolayers were labeled using a monoclonal antibody to
-1 and polyclonal antibodies to
-2 and
-3 isoforms of fusion proteins of the rat Na+-K+ ATPase. The cell monolayers were labeled with antibodies to the
-1 and
-2 isoforms, but not the
-3 isoform of the Na+-K+ ATPase. The labeling pattern of both
-1 and
-2 isoforms was intensely localized to the basolateral membrane. The cells grown in serum-free media in the absence and presence of 850 nM insulin exhibited the same pattern of labeling for both
-1 and
-2 isoforms. The control monolayers incubated with preimmune serum gave images identical to that observed with the
-3 isoform antibody (data not shown).
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A representative Western blot is also presented in Figure 12 and confirms the expression of -1 and
-2 isoforms of the Na+-K+ ATPase. The monoclonal antiNa+-K+ ATPase
-1 antibody labeled an ~95-kD protein and the polyclonal antiNa+-K+ ATPase antibody labeled a protein of ~100 kD, consistent with
-1 and
-2 isoforms of the Na+-K+ ATPase.
[3H] Ouabain Binding
To determine whether insulin increased transport activity of the pump as a result of an increase in Na+-K+ ATPase concentration in the basolateral membrane, specific [3H] ouabain binding was performed with the cell monolayers cultured in serum-free media in the absence or presence of 850 nM insulin. Figure 13 shows the specific binding of [3H] ouabain to endometrial epithelial cells as a function of ouabain concentration. Analysis of [3H] ouabain binding revealed a single class of binding sites with total receptor concentration (Bmax) of 13.9 ± 2.4 pmol/mg protein and Kd of 252.8 ± 90.5 nM for insulin-treated cells (n = 5). The Bmax and Kd of insulin-treated cells did not significantly differ from those of control cells (Bmax = 11.4 ± 3.9 pmol/mg protein and Kd = 237.0 ± 173.4 nM).
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DISCUSSION |
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Early studies of toad urinary bladder demonstrated that insulin stimulates transepithelial Na+ transport by activation of the Na+-K+ ATPase without increasing apical membrane Na+ permeability (
Unlike the results obtained using A6 cells, we find in the present study that the acute (within 30 min after stimulation) increase in Na+ transport after treatment with insulin or IGF-I is not the result of an increase in apical Na+ conductance. Insulin and IGF-I increase Na+ absorption by stimulating Na+-K+ ATPase activity and by increasing basolateral membrane K+ permeability. Stimulation of Na+-K+ ATPase activity involves increases in both Vmax and Na+ affinity, and a small decrease in K+ affinity. Previous studies of Na+-K+ ATPase stimulation by insulin in skeletal muscle indicated that increases in Vmax may be the result of insertion of pumps into the membrane (-1 and
-2 isoforms of the Na+-K+ ATPase are present in the basolateral membrane. Previous studies with adipocytes showed that insulin stimulation produced decreases in K0.5 for Na+ of both the
-1 and
-2 isoforms of the pump (
-2 isoform was remarkably similar to pump current measurements reported in this study for endometrial epithelial cells (from 39 to 24 mM). Estimates of Vmax based on a theoretical fit of the 86Rb+ uptake data in adipocytes also suggested a twofold increase in Vmax, but this prediction was not experimentally confirmed. It was concluded from the studies of
-1 isoform was greater than that of the
-2 isoform in its contribution to basal pump activity in the absence of insulin. However, treatment with insulin produced selective activation of the
-2 isoform so that
-2 fractional activity was dominant under insulin-stimulated conditions. Stimulation of the
-2 isoform by insulin has also been reported in brain (
-2 isoform could account for both the increase in Na+ affinity and Vmax observed after insulin stimulation reported in this study for endometrial epithelial cells. However, it is worth noting that in renal epithelia, where only the
-1 isoform is present, insulin also produces a marked stimulation of the Na+-K+ ATPase.
Although insulin did not produce an acute increase in apical Na+ conductance, we found that longer-term treatment (4 d) with insulin resulted in a greater than twofold increase in apical Na+ conductance and an additional increase in pump current. One possible explanation for the enhanced Na+ absorption following longer term exposure to insulin may be related to the growth-stimulating effects of insulin on cultured cells. Although we did not examine this possibility directly, it is worth noting that no significant change in total protein content was observed in monolayers treated with insulin for 4 d compared with control monolayers under serum-free conditions. In addition, primary endometrial epithelial cells exhibit density-dependent arrest, making it less likely that the cell population would increase twofold after achieving confluence. Another possible explanation could involve regulation of Na+ channel expression at the transcriptional level that could result in an increase in apical membrane Na+ conductance. Regulation at this level would likely follow a time course on the order of hours or perhaps days that could be consistent with the longer-term actions of insulin on Na+ conductance. At present, evidence in support of this idea is not available, but insulin is known to stimulate DNA replication and transcription of other proteins involved in cell cycle regulation.
An interesting observation from this study, relating to the Na+ dependence of the Na+-K+ ATPase, is that the degree of cooperativity for Na+ ion binding is less than that previously reported for the Na+-K+ ATPase in adipocytes (
Experiments using amphotericin B to permeabilize the apical membrane demonstrated the presence of an insulin-activated, outwardly rectifying conductance in the basolateral membrane. Replacement of standard porcine Ringer solution with high KCl Ringer solution shifted the reversal potential to near 0 mV, suggesting K+ as the current-carrying ion. Insulin-dependent increases in basolateral K+ conductance presumably contributes to the electrical driving force for apical Na+ uptake and could serve to offset decreases in K+ recycling that would occur in the face of sustained hyperpolarization of the basolateral membrane.
It is generally accepted that the first step in the insulin/IGF-I signaling cascade involves receptor binding followed by stimulation of receptor-mediated tyrosine kinase activity. Previous studies in A6 cells demonstrated that insulin-stimulated Na+ transport was inhibited by tyrosine kinase inhibitors ( subunit through activation of PP-1, and PI-3 kinase appears to be involved in an earlier step in the signaling cascade. In addition, recent studies in A6 cells demonstrated that PI-3 kinase inhibitors blocked insulin-stimulated Na+ transport and insulin-stimulated PI-3 kinase activity (
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Insulin and IGF-I have been previously shown to stimulate endometrial epithelial cell proliferation (
Submitted: 26 May 1999
Revised: 23 August 1999
Accepted: 24 August 1999
1used in this paper: IGF-I, insulin-like growth factor I; Isc, short circuit current; IV, currentvoltage; MAP, mitogen-activated protein; NMDG, N-methyl-D-glucamine; PI-3 kinase, phosphatidylinositol 3-kinase
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