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The luminal fluid microenvironment of the uterus is important for sperm capacitation and embryo development. In an attempt to understand the possible role of Na(+)/H(+) exchangers (NHEs) in uterine function, the mRNAs of different NHE isoforms as well as their subcellular localization (apical versus basolateral) and functional activity were investigated in mouse endometrial epithelial cells using reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and intracellular pH (pH(i)) measurement techniques. The presence of NHE1, NHE2, and NHE4, but not NHE3 mRNAs were revealed by RT-PCR. Immunostaining showed that NHE1, NHE2, and NHE4 were present in both apical and basolateral membranes. The pH(i) recovery from intracellular acidification was Na(+)-dependent; however, the rate of pH(i) recovery depending on basolateral Na(+) was 12.4 times faster than that depending on apical Na(+). The Na(+)-dependent rate of pH(i) recovery was also inhibited by amiloride, indicating H(+) extrusion through NHEs; however, the amiloride sensitivity of the apical membrane was less than that of the basolateral membrane, suggesting the involvement of different types of NHEs in the two membranes. The results indicate that the basolaterally located NHE1, NHE2, and NHE4, in addition to participating in the homeostatic control of intracellular pH, may play a role in H(+) extrusion in order to achieve transepithelial HCO(3)(-) secretion. The apically located NHEs may be involved in mediating Na(+) absorption as alternatives of or complementary to epithelial Na(+) channels.

The present study investigated the involvement of Na+-HCO3- cotransporter in mediating cAMP-stimulated HCO3- secretion across the cultured mouse endometrial epithelium using the short-circuit current (I(SC)) technique and intracellular pH measurement. Forskolin stimulated a rise in the I(SC), 55.6% and 52.1% of which could be reduced by the removal of extracellular Cl- or by eliminating the contribution of Cl- secretion by bumetanide, an inhibitor of Na+-K+-2Cl- cotransporter, respectively. More than 80% reduction in the forskolin-induced I(SC) was obtained when both Cl- and HCO3- in the bath were removed or in HCO3--free solution with bumetanide, indicating that the I(SC) depended on both Cl- and HCO3-. The presence of the Na+ channel-blocker amiloride in the apical solution did not reduce the forskolin-induced I(SC); however, the I(SC) could be abolished by removing Na+ from the bathing solution, suggesting that the Cl-- and HCO3--dependent I(SC) was also dependent on basolateral Na+. The forskolin-stimulated I(SC) could be reduced 43.6% by removal of HCO3- and 47.9% by a Na+-HCO3--cotransporter inhibitor, dihydrogen-4,4'-didsothiocyanostilbene-2,2'-disulfonic acid (H2DIDS). The inhibitory effect of H2DIDS was observed in Cl--free solution, but not when HCO3- was removed, thus confirming its effect on HCO3--dependent transport. Intracellular pH measurements demonstrated that the recovery from cellular acidification depended on the presence of both basolateral Na+ and HCO3-, further indicating the involvement of Na+-HCO3- cotransporter. Reverse transcription-polymerase chain reaction experiments confirmed the expression of Na+-HCO3- cotransporter in the mouse endometrium. The results suggest that basolaterally located Na+-HCO3- cotransporter is involved in mediating cAMP-stimulated HCO3- secretion across the mouse endometrial epithelium.

Previous studies have demonstrated that Cl- secretion by the mouse endometrial epithelium is under neurohormonal influence. The present study characterized the Cl- conductance activated by a number of agonists in the mouse endometrial epithelial cells using the whole-cell voltage-clamp technique. Adrenaline (1 microM), prostaglandin (PG) E2 (5-10 microM), and PGF2alpha (100 microM) activated a whole-cell current that exhibited a linear I-V relationship as well as time- and voltage-independent characteristics. However, the current magnitude varied with different agonists. The agonist-activated current could be mimicked by an adenylate cyclase activator, forskolin (10 microM), and suppressed by an adenylate cyclase inhibitor, MDL12330A, suggesting the involvement of cAMP. Current characteristics remained the same after cation replacement, leaving Cl- as the major permeant ion species in the solutions. The reversal potential of the agonist-induced current was close to the equilibrium potential of Cl- in the presence of a Cl- gradient, indicating the activation of Cl- conductance. The agonist-induced current was inhibited by the Cl- channel blocker diphenylamine 2,2'-dicarboxylic acid (DPC), but not by the Cl- channel blocker 4,4'-diisothiocyanatostibene-2, 2'-disulfonic acid (DIDS). The anion selectivity sequence of the current was NO3-Br-Cl-I-. The observed electrophysiological properties of the agonist-induced Cl- conductance were consistent with those reported for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel expressed in many epithelia. The expression of CFTR in the mouse endometrial cells was also demonstrated by Western blot analysis. It appears that neurohormonal regulation of the uterine fluid in the mouse endometrium converges on the cAMP-activated Cl- channel, presumably CFTR.

The uterine fluid composition is largely determined by the absorptive and secretory activities of the endometrial epithelium. The present study explored the cellular mechanisms involved in adrenaline-stimulated anion secretion across the cultured mouse endometrial epithelium using the short-circuit current (ISC) technique in conjunction with transporter inhibitors and channel blockers. Cultured endometrial epithelial monolayers responded to basolateral application of adrenaline with an increase in ISC, which was attributable to both Cl- and HCO3- secretion. When extracellular Cl- or HCO3- was removed, the adrenaline-induced response, as measured by the total charge transfer per unit area, was reduced to 53% and 46%, respectively. When both Cl- and HCO3- were absent from the bathing solutions, the adrenaline-induced response was reduced to only 2% of the response when both ions were present, indicating substantial contribution of Cl- and HCO3- secretion to the adrenaline-stimulated response. Cellular mechanisms, e.g., transporters and ion channels, involved in Cl- or HCO3- secretion were investigated separately. Cl- secretion was found to depend on the activities of basolaterally located Na+-K+-ATPase, Na+-K+-2Cl- cotransporter, and K+ channels, while evidence suggested that HCO3- secretion depends substantially on basolaterally situated Na+-HCO3- cotransporter and Na+-H+ exchanger. Similar to what was seen for Cl- exit, a large portion of HCO3- appeared to exit apically through anion channels. The results indicate that the uterine fluid composition in the mouse may be regulated by adrenaline through stimulation of both Cl- and HCO3- secretion and may be fine-tuned through an elaborate operation of different cellular mechanisms.