Molecular analyses of NaK-ATPase abundance and a-subunit isoform distribution were performed to determine whether pump expression varies at different points through the thickness of the circular layer of colonic smooth muscle. The mRNA and polypeptides of Na,K-ATPase al and 13 subunits were twice as abundant in the submucosal region of the circular layer, which has previously been shown to generate large pump potentials. Sodium pump activity directly correlated with the relative abundance of the al polypeptide. These data show that sodium pump expression varies in electrically dissimilar regions of the circular layer.Recent studies have suggested that the gradient in resting potential across the circular muscle layer of the canine colon is due to a gradient in the contribution of the Na,K-ATPase to the resting potential (1). Drugs or ionic conditions that inhibit the Na,K-ATPase abolished the resting-membrane potential gradient, such that all cells were polarized to approximately -40 mV (1). Several differences in cells through the circular layer might be responsible for the gradient in the contribution of the sodium pump to membrane potential. There may be differences in (i) pump expression in submucosal vs. myenteric muscle cells, (ii) membrane conductance in the two regions, (iii) concentration of pump substrates such as intracellular Na', or (iv) Na' influx pathways such as differential expression of electrically silent transporters (e.g, Na+/H' exchange).The Na,K-ATPase couples the hydrolysis of ATP to the transport of three Na' ions out of the cell and two K+ ions into the cell for each pump cycle (2,3). This stoichiometry is the basis for its electrogenicity. Na,K-ATPase consists of a catalytically active a subunit (Mr = 112,000) and a glycosylated 13 subunit (protein component, Mr = 35,000). Several laboratories have identified cDNAs encoding three isoforms of the a subunit in rat brain [referred to as al, a2 (or a+), and a3] (4-7). Na,K-ATPase isozymes, which are composed of one a subunit (al, a2, or a3) and one , 1 subunit, have different affinities for sodium (8,9). Thus distribution of the isozymes in the circular muscle layer should be determined, because differential expression of these isoforms of the pump may affect the magnitude of pump current and, therefore, the contribution of the sodium pump to resting potential. Experiments in the present study demonstrate the following: (i) at the RNA level, Na,K-ATPase al isoform and 13 subunit mRNAs are at least twice as abundant in submucosal circular muscle as in myenteric circular muscle whereas the a2 isoform is equally distributed throughout the circular muscle layer, (ii) at the protein level, Na,K-ATPase al protein is more abundant in submucosal circular muscle than in myenteric circular muscle whereas the a2 isoform is equally distributed, and (iii) there is approximately twice as much sodium pump activity in submucosal circular muscle as in myenteric circular muscle.
MATERIALS AND METHODSTissue Preparation. Mongrel dogs of either sex wer...