UTP-dependent inhibition of Na+ absorption requires activation of PKC in endometrial epithelial cells

Abstract

The objective of this study was to investigate the mechanism of uridine 5'-triphosphate (UTP)-dependent inhibition of Na(+) absorption in porcine endometrial epithelial cells. Acute stimulation with UTP (5 microM) produced inhibition of sodium absorption and stimulation of chloride secretion. Experiments using basolateral membrane-permeabilized cell monolayers demonstrated a reduction in benzamil-sensitive Na(+) conductance in the apical membrane after UTP stimulation. The UTP-dependent inhibition of sodium transport could be mimicked by PMA (1 microM). Several PKC inhibitors, including GF109203X and Gö6983 (both nonselective PKC inhibitors) and rottlerin (a PKCdelta selective inhibitor), were shown to prevent the UTP-dependent decrease in benzamil-sensitive current. The PKCalpha-selective inhibitors, Gö6976 and PKC inhibitor 20-28, produced a partial inhibition of the UTP effect on benzamil-sensitive Isc. Inhibition of the benzamil-sensitive Isc by UTP was observed in the presence of BAPTA-AM (50 microM), confirming that activation of PKCs, and not increases in [Ca(2+)](i), were directly responsible for the inhibition of apical Na(+) channels and transepithelial Na(+) absorption.

Figure 1.

Effect of UTP on basal sodium transport. (A) Representative trace showing that addition of 5 μM benzamil to the apical solution blocked most of the basal Isc in monolayers maintained under serum free conditions, (n = 9, N = 4). (B) Apical addition of UTP (1 μM) caused a rapid increase in Isc followed by a slow decrease back to the basal Isc. Subsequent addition of benzamil had little inhibitory effect, but addition of NPPB (100 μM at each arrow) blocked all of the remaining Isc, (n = 15, N = 4). The scale bar applies to both Fig. 1, A and B. (C) After pretreatment with benzamil (5 μM), apical addition of UTP (5 μM) caused a rapid increase in Isc, similar to what is shown Fig. 1 B. Addition of NPPB (100 μM at each arrow) blocked all of the remaining Isc, (n = 6). Statistical analysis is provided in Fig. 6.

Figure 2.

Effects of UTP and PMA on insulin-stimulated Na⁺ transport. (A) Representative trace showing the time-dependent increase in Isc stimulated by 850 nM insulin added to the basolateral solution. Addition of 1 μM UTP to the apical solution inhibited both the insulin-stimulated and basal Isc. See results for mean ± SEM data for basal Isc, insulin-stimulated Isc and residual Isc after UTP. (B) Addition of 1 μM phorbol 12-myristate 13-acetate (PMA) produced a similar decrease in insulin-stimulated and basal current as observed with UTP. See results for mean ± SEM data for basal Isc, insulin-stimulated Isc, and residual Isc after PMA. (C) Temporal changes in [Ca²⁺]_i in response to PMA and UTP were determined using fura 2-AM as described in materials and methods. Transient elevations in [Ca²⁺]_i were observed after 1 and 5 μM UTP in Ca²⁺-containing HBSS. No elevation in [Ca²⁺]_i was detected after 1 μM PMA. This data represents the mean trace calculated using calcium measurements from 20 different cells.

Figure 3.

Current-voltage relationships for the benzamil-sensitive pathway in the apical membrane. (A) Experiments were performed using monolayers cultured in serum-free media with insulin for 3 d. Benzamil-sensitive difference currents were obtained using a voltage step protocol that ranged from −100 to 95 mV in 15-mV increments from a holding potential of 0 mV. Benzamil (5 μM) was added apically to either permeabilized control cell monolayers (top trace) or to permeabilized monolayers activated with 5 μM UTP (bottom trace). (B) Benzamil-sensitive current-voltage relationships were obtained in response to a voltage step protocol from −90 to 90 mV in 15-mV increments from a holding potential of 0 mV. Benzamil (5 μM) was added to the apical solution in the absence or presence of UTP (1 μM).

Figure 4.

Effect of UTP on Isc in the presence or absence of chloride. (A) Representative trace showing chloride dependence of the UTP (5 μM) response. Monolayers were bathed in standard porcine saline solution or chloride-free solution, replacing chloride with methane sulfonate. The peak UTP-activated current was reduced under chloride-free conditions (n = 6). (B) Bar graph showing the benzamil (5 μM) and NPPB (100 μM) -sensitive currents in the absence or presence of chloride. The NPPB-sensitive current was reduced under chloride-free conditions, whereas the benzamil-sensitive current remained the same, (n = 6 for each). Asterisk indicates significant difference between the NPPB-sensitive Isc under control and Cl⁻-free conditions.

Figure 5.

Effect of UTP on apical membrane Cl^- conductance. (A) Representative trace showing the transient change in apical membrane current after stimulation with 1 μM UTP using monolayers where the basolateral membrane was permeabilized with amphotericin B (10 μM). The basolateral membrane was bathed with KMeSO₄ saline solution, while the apical membrane was bathed with standard porcine saline solution. (B) Current-voltage relationship showing the UTP-activated current obtained in response to voltage steps from −90 to 90 mV in 15-mV increments from a holding potential of 0 mV.

Figure 6.

PKC inhibitors prevent decrease of benzamil-sensitive Isc by UTP. (A) Representative trace showing the benzamil-sensitive current in the absence or presence UTP (5 μM), and after pretreatment with both Gö6983 (10 μM) and UTP, (n = 8, 14, and 9, respectively, N = 3). (B) Bar graph illustrating the benzamil-sensitive current after benzamil only (5 μM, n = 8), after UTP (5 μM, n = 14), after PMA (0.5 μM, n = 4), after UTP in the presence of Gö6983 (10 μM, n = 9), after UTP in the presence of rottlerin (2.5 μM, n = 5), after UTP in the presence of Gö6976 (100 nM, n = 6 and 10 μM, n = 4, respectively), or after UTP in the presence of the cell permeable, myristoylated PKC inhibitor 20–28 (10 μM, n = 4). Asterisk indicates significant differences compared with the benzamil-sensitive current after UTP. The benzamil-sensitive currents following pretreatment with rottlerin and Gö6983 (a nonselective PKC inhibitor) were not significantly different from benzamil treatment alone.

Figure 7.

PKC inhibitors prevent inhibition of the apical membrane benzamil-sensitive current by UTP. (A) Current-voltage relationship comparing the benzamil-sensitive current in monolayers pretreated with GF109203X in the presence and absence of UTP (n = 4). (B) Bar graph showing the fraction of remaining current after UTP stimulation of monolayers pretreated with either GF109203X (n = 4), rottlerin (n = 5) or Gö6976 (n = 4) at −90 mV. Monolayers pretreated with GF109203X did not exhibit significant inhibition of the benzamil-sensitive current following UTP stimulation.

Figure 8.

Western blot analysis of PKC isoforms in monolayers maintained under serum free conditions. Antibodies used for these Western blots were obtained from Santa Cruz Biotechnology, Inc., and were rabbit anti–human antibodies to various PKC isoforms as indicated in materials and methods. Antibody labeling observed in whole-cell lysates were from epithelial cells maintained under serum-free conditions for 76 h.

Figure 9.

Effect of BAPTA-AM on changes in [Ca^{2 +}]_i in response to UTP. (A) Temporal changes in [Ca²⁺]_i in response to UTP were determined using fura 2-AM as described in materials and methods. Upon stimulation with 5 μM UTP in Ca²⁺-containing HBSS, control cells initially exhibited a transient elevation in [Ca²⁺]_i, followed by a gradual decline to a new sustained elevated [Ca²⁺]_i. Removing UTP from the bathing solution returned the intracellular calcium concentrations back to baseline levels. (B) BAPTA-AM reduced the basal level of intracellular calcium by 80 nM, and upon stimulation with UTP, [Ca²⁺]_i increased by only 20 nM. For this figure, measurements were obtained from 20 cells in each of three separate experiments.

Figure 10.

BAPTA-AM does not effect the UTP-mediated inhibition of sodium absorption. (A) Representative trace showing that the peak UTP-activated Isc response is diminished after pretreatment with 50 μM BAPTA-AM. (B) Representative trace showing that benzamil is not effective in reducing Isc following treatment with UTP (5 μM) in monolayers pretreated with BAPTA-AM. (C) Bar graph illustrating that pretreatment with BAPTA-AM diminished the UTP-activated current, but failed to affect inhibition of the benzamil-sensitive current by UTP, (n = 6 for each). Asterisk represents significant difference compared with cells that were not pretreated with BAPTA-AM.