Epithelial ion transport and luminal fluid composition in the vaginal cul-de-sac of the Australian brushtail possum Trichosurus vulpecula.

Abstract

The brushtail possum (Trichosurus vulpecula) is an introduced pest in New Zealand and there is interest in developing alternatives to existing toxin-based controls. The vaginal cul-de-sac is a structure of unknown function that is unique to marsupials. During the follicular stage of oestrous, the epithelium lining the cul-de-sac proliferates and a mucus-rich luminal fluid is secreted. Following ovulation, the epithelium regresses and the fluid disappears. Identifying the epithelial transport processes mediating the cyclic changes in cul-de-sac fluid may assist with the development of a possum-specific contraceptive. The Ussing short-circuit (Isc) technique and measurements of transcript and protein expression of the epithelial Na+ channel (ENaC) were used to determine if variations in electrogenic Na+ transport are associated with cul-de-sac fluid disappearance. Spontaneous Isc (<20 µA cm-2 during anoestrus, 60-80 µA cm-2 in cycling animals) was inhibited by serosal ouabain. Mucosal amiloride (10 µmol L-1), an inhibitor of ENaC, had little effect on follicular Isc but reduced luteal Isc by ≈35%. This amiloride-sensitive Isc was dependent on mucosal Na+ and the IC50-amiloride (0.95 µmol L-1) was consistent with ENaC-mediated Na+ absorption. αENaC mRNA expression was low in anoestrus, but increased to similar levels in the follicular and luteal phases. However, in follicular animals αENaC immunoreactivity in epithelial cells was evident throughout the cytoplasm, but was restricted to the apical pole of cells from luteal animals. These data suggest that increased Na+ absorption contributes to fluid absorption during the luteal phase and is regulated by insertion of ENaC into the apical membrane of epithelial cells. In the follicular stage, 79±6% of the amiloride-insensitive Isc was inhibited by the anion channel blocker 5-Nitro-2-(3-phenylpropylamino)-benzoic-acid (NPPB, 100 µmol L-1 mucosal). This NPPB sensitive current (INPPB) was reduced when both Cl- and HCO3- were substituted from the serosal solution, suggesting that HCO3- and Cl- secretion contribute to the formation of cul-de-sac fluid. In Cl--free conditions, the carbonic anhydrase (CA) blocker acetazolamide (1 mmol L-1 mucosal and serosal) inhibited 31±6% of the Isc, whereas in HCO3-/CO2-free conditions the Na+/K+/2Cl- cotransporter (NKCC1) blocker bumetanide (10 µmol L-1 serosal) inhibited 28±6% of Isc. Collectively, these data indicate that HCO3- and Cl- secretion can occur in the follicular phase, and are facilitated by CA and NKCC1, respectively. CA mediated HCO3- secretion may involve an apical Na+/H+ exchanger (NHE) as the INPPB of cul-de-sac tissue was dependent on mucosal Na+, and mucosal 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 200 µmol L-1) an NHE inhibitor, blocked 35.8±5% of the spontaneous Isc. RT-PCR results indicate that NHE2 may be expressed. Cul-de-sac fluid from follicular animals had low Cl- activity (7.7±9.4 mmol L-1) and [Na+] (25.3±17.2 mmol L-1), while [HCO3-] (93.3±83.4 mmol L-1) and [Ca2+] (75±28.8 mmol L-1) were high. However, there was no correlation between the [Ca2+] and [HCO3-] in this fluid. Two of 10 samples measured contained low (<5 mmol L-1) [CO32-] and no CO32- was detected in the remaining 8 samples suggesting that CaCO3 precipitation is limited. As interactions between Ca2+ and HCO3- are pH dependent, the absence of CaCO3 precipitation could be a consequence of H+ secretion by apical NHEs. This study demonstrates that cyclic changes in the expression or activity of epithelial ion transport proteins in the cul-de-sac could account for differences in luminal fluid volume and composition during the oestrous cycle. Furthermore, it is proposed that modifying the volume of cul-de-sac fluid, by inducing the translocation of ENaC into the apical membrane in the follicular stage, or modifying the properties of fluid by inhibiting the NHEs involved in H+ secretion, could produce a contraceptive effect.