Augmentation of ouabain sensitivity of rat liver Na/K‐ATPase by in vivo adenovirus‐mediated expression of the Na/K‐ATPase α2 subunit

Abstract

These are the first experiments to study the effect of in vivo expression of the Na/K-ATPase ot2 subunit which serves as a receptor for cardiac glycosides. The ct2 subunit is not normally expressed in rat liver, so hepatocytes which lack endogenous <x2 protein are a logical first target to study the effects of a2 expression on membrane Na/K-ATPase activity. At 3 days after ot2 adenovirus vector infusion, Wistar rat livers contained ct2 DNA, ct2 mRNA, and a2 protein. Rat liver membrane ouabain binding activity and the sensitivity of Na/KATPase activity to ouabain significantly increased. Total membrane Na/K-ATPase was regulated at a constant level while expressed a2 activity represented 10% of the total active Na/KATPase sites in a2 transduced rat liver. These studies are the first to establish a paradigm in which an endogenous drug receptor is expressed to alter cellular pharmacologie sensitivity.

Downregulation of cardiac myocyte Na+-K+-ATPase by adenovirus-mediated expression of an α-subunit fragment

“…3, transfection with the ␣ 2 -vector resulted in a modest time-dependent increase in the level of intact ␣ 2 and in significant expression of two ␣ 2 -fragments of ϳ60 kDa and 34 kDa. The Northern blot analysis of total cellular RNA from neonatal myocytes and A7r5 cells transfected with the ␣ 2 -virus showed the presence of the appropriate ␣ 2 -specific message of the same size as that we showed earlier in the transfected rat livers (5). Although the detailed mechanisms involving the expression of the truncated ␣ 2fragments in cardiac myocytes and A7r5 cells remain to be determined (see DISCUSSION), the results of the above experiments on myocytes and A7r5 cells suggest that the expression of the intact ␣ 2 -subunit in the transfected cells is accompanied by its rapid degradation, with the most stable product being a 60-kDa fragment.…”

“…Finally, although the determination of the cause of the expression of the truncated ␣ 2 is not the focus of this report, it is appropriate that we briefly address this issue. The adenoviral ␣ 2 -vector used here produces a functional full-length ␣ 2 -subunit in the intact rat liver (5). If the ␣ 2 -fragments noted in the transfected myocytes and A7r5 cells are indeed due to the initial expression of the intact ␣ 2 and its subsequent fragmentation as suggested by our data, the question arises as to why the expressed full-length ␣ 2 is less stable in the cultured cells used here than in the intact liver.…”

“…Adenoviral vectors and transfections. The replication-deficient Ad5E1a,E1b,E3b-deleted ␣ 2 (H5.010CMV␣ 2 ) and the control ␤-galactosidase (H5.010CMVlacZ) adenovirus-derived expression vectors were made, amplified, and purified as described earlier (5). Cultured cells were washed with the transfection medium, which was the same as the culture medium but with 2% FBS.…”

Multiple Signal Transduction Pathways Link Na+/K+-ATPase to Growth-related Genes in Cardiac Myocytes

“…Virus was amplified in human kidney 293 cells, and the viral particles were purified from 293 cell lysates by cesium chloride gradient ultracentrifugation then desalted by dialysis (29). The concentration of recombinant adenovirus was determined based on the absorbance at 260 nm where 1 optical density unit corresponds to 10 12 particles/ml.…”

Cryopreservation of Rat Precision-cut Liver Slices is Associated with Major Metabolic Stress and Ionic Perturbations

“…After sequential differential centrifugations (120 g for 5 min; 6800 g for 15 min; 48380 g for 30 min), the final pellet was resuspended in 8% saccharose and 30 mM imidazol/HCl pH 7.4. Na + /K + -ATPase activity was determinated using slight modifications of a method previously described by measuring the rate of release of inorganic phosphate (Pi) from ATP at 37°C [20]. Briefly, the incubation mixture (500 µl) consisted of 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 25 mM KCl, 3 mM MgCl 2 , 1 mM EGTA and 5 mM NaN 3 .…”