Na-K pump site density and ouabain binding affinity in cultured chick heart cells

Journal Article · Sun Nov 01 04:00:00 EST 1987 · Am. J. Physiol.; (United States)

OSTI ID:5520770

Lobaugh, L A; Lieberman, M

The possible existence of multiple (/sup 3/H)ouabain binding sites and the relationship between ouabain binding and Na-K pump inhibition in cardiac muscle were studied using cultured embryonic chick heart cells. (/sup 3/H)ouabain bound to a single class of sites in 0.5 mM K (0.5 Ko) with an association rate constant (k+1) of 3.4 X 10(4) M-1.s-1 and a dissociation rate constant (k-1) of 0.0095 s. Maximal specific (/sup 3/H)ouabain binding RT to myocyte-enriched cultures is 11.7 pmol/mg protein and Kd is 0.43 microM in 0.5 Ko, whereas Kd,apparent is 6.6 microM in 5.4 Ko. The number of binding sites per myocyte was calculated by correcting for the contribution of fibroblasts in myocyte-enriched cultures using data from homogeneous fibroblast cultures (RT = 3.3 pmol/mg protein; Kd = 0.19 microM in 0.5 Ko). Equivalence of (/sup 3/H)ouabain binding sites and Na-K pumps was implied by agreement between maximal specific binding of (/sup 3/H)ouabain and /sup 125/I-labeled monoclonal antibody directed against Na+-K+-ATPase (approximately 2 X 10(6) sites/cell). However, (/sup 3/H)ouabain binding occurred at lower concentrations than inhibition of ouabain-sensitive /sup 42/K uptake in 0.5 Ko. Further studies in both 0.5 K and 5.4 Ko showed that ouabain caused cell Na content Nai to increase over the same range of concentrations that binding occurred, implying that increased Nai may stimulate unbound Na-K pumps and prevent a proportional decrease in /sup 42/K uptake rate. The results show that Na-K pump inhibition occurs as a functional consequence of specific ouabain binding and indicate that the Na-K pump is the cardiac glycoside receptor in cultured heart cells.

• Research Organization: Duke Univ. Medical Center, Durham, NC
• OSTI ID: 5520770
• Journal Information: Am. J. Physiol.; (United States), Journal Name: Am. J. Physiol.; (United States) Vol. 253; ISSN AJPHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
AFFINITY
ALKALI METAL ISOTOPES
ALKALI METALS
ANIMALS
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIRDS
BODY
CARBOHYDRATES
CARDIAC GLYCOSIDES
CARDIOTONICS
CARDIOVASCULAR AGENTS
CARDIOVASCULAR SYSTEM
CELL CULTURES
CHICKENS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ELEMENTS
ENZYMES
FOWL
GLYCOSIDES
HEART
HOURS LIVING RADIOISOTOPES
HYDROLASES
INHIBITION
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
METALS
MUSCLES
MYOCARDIUM
NUCLEI
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
OUABAIN
PHOSPHOHYDROLASES
POTASSIUM
POTASSIUM 42
POTASSIUM ISOTOPES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
SODIUM
STROPHANTHINS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES