Na/K-ATPase assay in the intact guinea pig liver submitted to in situ perfusion.
We describe an assay for the enzyme Na/K-ATPase in intact guinea pig livers perfused through the portal vein with modified Hank's solution. The model uses the measurement of non-radioactive rubidium ion incorporation by liver cells, both in the absence and in the presence of the specific Na/K-ATPase inhibitor ouabain, followed by a rinsing procedure with cold saline. The concentration of Rb+ in acid-digested liver lobes was measured by atomic emission spectrometry and Na/K pump activity was calculated by the difference between the incorporation of Rb+ in the absence and in the presence of ouabain. The optimal conditions for Rb+ incorporation were: perfusion flow rate, 3 ml/min per liver; perfusion time at 37 degrees C, 60 min; rinsing time with cold saline, 5-10 min; and concentration of ouabain, 3 mM. The calculated ouabain IC(50) was 100 microM. The major advantage of this model is the possibility of testing experimental drugs affecting this enzyme in conditions close to those in the intact organ.
Dos Santos
Maria da Conceição B
Mda C
Department of Cell Biology, Biology Institute, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil.
Burth
Patricia
P
Younes-Ibrahim
Maurício
M
Gonçalves
Cassiano F
CF
Santelli
Ricardo Erthal
RE
Oliveira
Eliane Pádua
EP
de Castro Faria
Mauro Velho
MV
D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D007552:Isotonic Solutions; D008099:Liver / Q000098:blood supply / Q000201:enzymology*; D010042:Ouabain / Q000494:pharmacology; D012039:Regional Blood Flow; D012413:Rubidium / Q000032:analysis / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D013997:Time Factors
D004791:Enzyme Inhibitors; C072670:Hanks Balanced Salt Solution; D007552:Isotonic Solutions; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium