MEASUREMENT OF THE TOTAL CONCENTRATION OF FUNCTIONAL NA+,K+-PUMPS IN RUMEN EPITHELIUM

Using the technique of vanadate-facilitated [H-3]ouabain binding we have developed a simple and reliable assay for measuring the concentration of[H-3]ouabain binding sites in small fresh or frozen biopsies of rumen epithelium papillae. In bovine and ovine rumen epithelium obtained from the cranio-ventral rumen sac the concentration of [H-3]ouabain binding sites was 1.6-4.9 nmol g dry wt(-1)(n = 32) and 3.7-5.2 nmol g dry wt(-1) (n = 6), respectively. When incubated in oxygenated Krebs-Ringer bicarbonate buffer fresh biopsies of rumen epithelium maintained a high K+ and low Na+ content for at least 6 h. Na+ loading of the biopsies induced about 20-fold increase of the Na+,K+-pump activity based on measurement of ouabain suppressible net [Rb-86(+)] influx. The ouabain suppressible net influx of [Rb-86(+)] measured in Na+ loaded biopsies showed a close correlation to the [H-3]ouabain binding capacity (r = 0.80, P < 0.01) and corresponded to 47 +/- 2% (n = 9) of the theoretical maximum flux rate. The ouabain suppressible net influx of K+ and [Rb-86(+)] were linearly related (r = 0.73; P < 0.001). The net Na+ efflux was 1.21 times the net K+ influx. It is concluded that rumen epithelium has a large capacity for active Na+/K+ transport and that there is agreement between the concentration of [H-3]ouabain binding sites in the epithelium and the ouabain suppressible rate of net [Rb-86(+)] influx in Na+ loaded biopsies in spite of some uncertainty about the maximum turnover number of the Na+,K+-pump in rumen epithelium.