It is believed that the endometrial epithelium, the mucosal lining of the uterus, has both absorptive and secretory activities which may be important for the formation of a uterine fluid environment suitable for sperm transport and embryo implantation. Measurements of luminal Na+ and K+ concentrations in a number of species, including humans (Casslen & Nilsson, 1984), rats (Nilsson & Ljung, 1985; Nordenvall, Ulmsten & Ungerstedt, 1989) and sows (Iritani, Sato & Nishikawa, 1974), have indicated that Na+ may be absorbed and K+ secreted by the endometrial epithelium during various reproductive events. Electrophysiological studies on primary cultures of human endometrial epithelial cells (Matthews, McEwan, Redfern, Thomas & Hirst, 1992, 1993a; Matthews, Thomas, Redfern & Hirst, 1993b) and the intact endometrial epithelium from immature pigs (Vetter & O'Grady, 1996) has provided direct evidence for regulated Na+ absorption and K+ secretion by the endometrial epithelium. However, less is known for the regulation of endometrial anion secretion, although short circuit current measurements on intact rat uteri have indicated that electrogenic transfer of Cl− and HCO3− into the luminal fluid occurs (Kyriakides & Levin, 1973), and X-ray microanalyses of anions in uterine secretions in the rat have demonstrated an increase in Cl− concentration during blastocyst implantation (Nilsson & Ljung, 1985).
Recently, a primary culture of mouse endometrial epithelial cells grown on permeable supports has been established and shown to have a basal short circuit current (ISC) predominantly mediated by Na+ absorption (Chan et al. 1997a). It has also been demonstrated that the cultured epithelium responds to a number of agonists with increases in the ISC which can be predominantly attributable to Cl− secretion (Chan et al. 1997a,b). In the present study the regulation of anion secretion by adrenoceptors in the mouse endometrial epithelium has been investigated further. The results suggest that anion secretion across the mouse endometrium epithelium can be regulated by β-adrenoceptors and involves a cAMP-dependent mechanism.
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The present study has demonstrated stimulation of anion secretion across the mouse endometrial epithelium by adrenoceptor agonists. Previous studies on isolated human endometrial epithelial cells have shown that a number of neurohormonal agents, including adrenaline, stimulate electrogenic ion transport (Matthews et al. 1992, 1993a,b). However, the ion species mediating the response has not been fully investigated. The present study shows that the response of the mouse endometrial epithelium to adrenaline is mainly mediated by anion secretion. The supporting evidence comes from the experiments in which the ISC was greatly reduced by replacement of Cl− in the bathing solutions and the adrenaline-activated ISC was blocked by various Cl− channel blockers applied to the apical membrane. It should be noted that replacement of both HCO3− and Cl− produced a further reduction in the adrenaline-stimulated ISC response. One possible explanation is that HCO3− secretion could be stimulated concurrently, but clarification of this point requires further studies. The possibility that Na+ absorption is involved in the adrenaline-stimulated ISC was excluded by the observation that addition of amiloride at a concentration known to block apical Na+ channels did not inhibit the adrenaline-stimulated ISC. Replacement of apical Na+ did not diminish the adrenaline-stimulated ISC either, which also indicated that Na+ absorption was not involved. The reduction in the adrenaline-stimulated ISC following bilateral replacement of Na+ observed in the present study may result from inhibition of certain basolaterally located Na+-dependent transporters. A likely candidate would be the Na+-K+-2Cl− cotransporter which has been demonstrated to play an important role in active Cl− secretion in many secreting epithelia, including the airways (review by Welsh, 1987). Another candidate could be the Na+–HCO3− cotransporter, which has been demonstrated to be responsible for substantial HCO3− secretion across the pancreatic duct of the guinea-pig (Ishiguro, Steward, Wilson & Case, 1996). These possibilities are consistent with our contention that adrenaline stimulates anion secretion across the mouse endometrial epithelium.
The present study has also demonstrated that stimulation of anion secretion by adrenaline across the mouse endometrial epithelium is mainly mediated by β-adrenoceptors. Several lines of evidence support this contention. First, agonists with higher specificity for β-adrenoceptors are more effective in activating the ISC (isoprenaline > adrenaline > noradrenaline), while the β-adrenoceptor agonist methoxamine is without effect. Adrenaline could produce further stimulation of ISC if the initial stimulant was noradrenaline, but not if it was isoprenaline. The fact that the effects of adrenaline and isoprenaline on ISC were not additive suggests that the action of adrenaline is similar to that of isoprenaline, which acts on β-adrenoceptors. While the effect of adrenaline following noradrenaline on the ISC was additive, the combined effect was similar to that produced by adrenaline alone. In contrast, no additive effect could be seen if adrenaline was added prior to noradrenaline, suggesting that their effects are likely to be mediated by the same pathway, which involves β-adrenoceptors. This notion is further supported by the studies using adrenoceptor antagonists. Propranolol, a β-adrenoceptor antagonist, was found to be more potent than the β-adrenoceptor antagonist phentolamine in blocking the agonist-induced ISC, demonstrating a predominant involvement of β-adrenoceptors.
As further support for a role for adrenoceptors in mediating the adrenaline response, the involvement of cAMP has also been demonstrated by mimicking the effect of adrenaline on the ISC by an adenylate cyclase activator, forskolin. The effects of forskolin and adrenaline on the ISC are similar in that they both induced a slow and sustained ISC compared with the previously observed rapid and transient current elicited by Ca2+-mobilizing agents such as ionomycin and ATP (Chan et al. 1997b). In addition, both forskolin- and adrenaline-induced ISC could be inhibited by various Cl− channel blockers to a similar extent. Together with the observed inhibition of the adrenaline-stimulated ISC by pretreatment with the adenylate cyclase inhibitor MDL, these results suggest a role for cAMP in the action of adrenaline and add further support to the contention that β-adrenoceptors are involved in the regulation of anion secretion in the mouse endometrium. It is interesting to note that a mean reduction of 20% in these currents could be induced by DIDS, a blocker known to inhibit the Ca2+-activated Cl− channel but not the cAMP-activated Cl− channel in various epithelia (Fuller & Benos, 1992). The adrenaline-induced response observed in the mouse endometrial epithelium may involve cross-talk between cAMP and Ca2+ signalling pathways.
Previous studies on intact rat uteri have yielded similar results, demonstrating that the effect of adrenaline on rat endometrial bioelectrical activity in vivo and in vitro was mediated by a β-adrenoceptor through cAMP and Ca2+-sensitive pathways (Levin & Phillips, 1983; Levin & Sebkhi, 1989). In contrast to the present finding that adrenaline stimulates mainly Cl− secretion in the mouse, the adrenaline-stimulated electrogenic ion secretion in the rat has been attributed to HCO3− secretion only (Levin & Scargill, 1987). Further investigation of the interaction between Cl− and HCO3− ions in the adrenaline-stimulated ISC response is required to understand fully the ionic mechanism(s) underlying the electrogenic ion transport process in the endometrium.
Regulation of ion transport across the endometrium by neurohormonal agents has also been observed in humans (Matthews et al. 1992, 1993a) and pigs (Vetter & O'Grady, 1996), but the responses appear to be different from those observed in the present study. The adrenaline-stimulated ISC observed in primary cultures of human endometrial glandular epithelial cells, which was evident even in Na+-free solution, was transient in nature in contrast to the sustained response observed in the present study. In addition, no ISC response to forskolin was observed in the human cultures, which excludes the possibility that a cAMP-dependent mechanism mediates the effect of adrenaline in the human endometrial cells. Stimulation of Na+ absorption and K+ secretion rather than anion secretion by PGF2α was observed in intact endometrium from pigs (Vetter & O'Grady, 1996), but activation of both anion secretion and Na+ absorption by PGE2 was observed in primary cultures of porcine glandular endometrium (Deachapunya & O'Grady, 1996). The different responses observed in the porcine endometrium have been attributed to the possibility that glandular cells in culture express a combination of luminal cell and glandular cell transport phenotypes and that receptors present in culture may not be present in vivo. The isolation and culture methods (McCormack & Glasser, 1980) that we have adapted for the mouse endometrium should yield predominantly luminal epithelial cells, although we cannot exclude possible contamination by glandular epithelial cells. At the present, it is difficult to determine whether the different responses of the endometrium to cAMP-evoking agents observed in different species was due to species specificity, or to the culture conditions, i.e. luminal vs. glandular epithelial cells, or intact vs. cultured cells. Clarification of this issue will require further investigation. It should also be noted that the present results were obtained from endometrial cells from reproductively immature mice. This may also contribute to the difference between the present results and those obtained from humans.
It would be of interest to investigate whether cAMP-dependent Cl− secretion is mediated by the cystic fibrosis transmembrane conductance regulator (CFTR), which has itself been shown to be a cAMP-regulated Cl− channel (Bear et al. 1992) and is expressed differently in the uterine epithelium of humans (Tizzano, Chitayat & Buchwald, 1993) and rodents (Trezise et al. 1993). Although the present study indicates that cAMP-dependent Cl− secretion across the mouse endometrial epithelium is sensitive to glibenclamide and DPC, both of which have been shown to have potent effect on CFTR (Fuller & Benos, 1992; Sheppard & Welsh, 1992), further experiments using the patch clamp technique are required to identify the Cl− channels involved. The role of the cAMP-stimulated endometrial Cl− secretion in cystic fibrosis and infertility in CF women also remains to be elucidated.
In conclusion, the present study has demonstrated that anion secretion across the mouse endometrium could be regulated via a β-adrenoceptor and involve a cAMP-dependent mechanism. Regulated anion secretion may constitute the physiological basis for the observed high pH and HCO3− content in the rabbit uterus (Vishwakarma, 1962) and the increased Cl− concentration during implantation in the rat (Nilsson & Ljung, 1985).