The activity of the sodium pump depends on the catalytic properties of its subunit constituents. The Na⁺,K⁺-ATPase consists of three types of subunit, α, β and λ, each of which is expressed as multiple isoforms (4 α, 4 β and at least 2 λ). We have previously shown that the α1 and 2 isoforms are expressed in uterine smooth muscle of the rat (Floyd et al. 2003). Contraction of uterine smooth muscle is dependent on the electrogenic action of the Na⁺,K⁺-ATPase, which creates cation gradients across the cell membrane to facilitate propagation of contraction and return of the membrane to resting potential. Previous molecular studies identified changes in the expression of α subunit mRNAs throughout pregnancy but little is known of the specific changes in protein expression during this period. Furthermore, there are no published reports of Na⁺,K⁺-ATPase isoform expression and cellular localisation in the uterus. The aim of this study was to determine whether there are any changes in levels of expression of Na⁺,K⁺-ATPase isoforms throughout pregnancy in the rat and to document any alterations in isoform protein expression.

Western blots were used to study the levels of α1, α2, α3 and β1, β2, β3 isoforms in tissues from non-pregnant and pregnant rats at days 10, 16 and 21 of pregnancy. We also studied the regional distribution of all isoforms in non-pregnant and term-pregnant (day 21) rats.

Non-pregnant female Wistar rats and animals at days 10, 16 and 21 of pregnancy were humanely killed by cervical dislocation under CO₂ anaesthesia. Three uterine horns per group were snap frozen in liquid nitrogen for Western blot analysis. Uterine horns from non-pregnant and 21-day pregnant rats were fixed in neutral buffered formalin before paraffin embedding and sectioning (6 µm thickness). Tissue sections were subjected to antigen retrieval in the presence of sodium dodecyl sulphate. Immunohistochemical analysis was performed after non-specific binding was blocked with 10 % normal goat serum (NGS). Total protein was extracted from snap frozen samples and run on SDS-PAGE gels. Western blotting was performed using isoform specific antibodies. Rat brain and kidney homogenates were used as positive controls for α2/α3 and α1 isoform expression respectively.

Immunohistochemistry using a pan α antibody demonstrated high Na⁺,K⁺-ATPase expression in the outer longitudinal and inner circular smooth muscle layers and the epithelial lining of the endometrium. Western blot results confirmed that the major α isoform expressed in rat uterus was α1. However, lower levels of the α2 isoform were also present. The α3 isoform was not detectable by Western blotting. The expression level of the α2 isoform appeared to diminish with progression of pregnancy whereas the α1 isoform levels were unaffected.

These results suggest that there may be a shift in the Na⁺,K⁺-ATPase isoforms during pregnancy which may affect intracellular [Na⁺]_i and hence [Ca²⁺]_i. This may explain the increase in uterine smooth muscle contraction during the latter stages of pregnancy.

The authors would like to thank the Wellcome Trust and MRC for funding.