Erythrocytes may contain a ouabain-insensitive K+-ATPase which plays a role in internal K+ balance

Abstract

Erythrocytes are useful in evaluating K + transport pathways involved in internal K + balance. Several forms of H + ,K + -ATPase have been described in nephron segments active in K + transport. Furthermore, the activity of a ouabain-insensitive isoform of H + ,K + -ATPase expressed in collecting duct cells may be modulated by acid-base status. Various assays were performed to determine if a ouabain-insensitive K + -ATPase is present in rat erythrocytes and, if so, whether it plays a role in internal K + balance. Kinetic studies demonstrated that maximal stimulation of enzyme activity was achieved with 2.5 mM K + at pH 7.4. Subsequent experiments were performed on erythrocyte membranes collected from animals submitted to varying degrees of K + homeostasis: control rats, K + -depleted rats, K + -loaded rats, and rats rendered hyperkalemic due to acute renal failure. As observed in the collecting duct cell studies, there was a significant decrease in the activity of ouabain-insensitive K + -ATPase in the erythrocytes of both K + -loaded and metabolically alkalotic K + -depleted rats. However, this enzyme activity in erythrocyte membranes of rats with metabolic acidosis-related hyperkalemia was similar to that of control animals. This finding may be interpreted as resulting from two potentially modulating factors: the stimulating effect that metabolic acidosis has on K + -ATPase and the counteracting effect that hyperkalemia and uremia have on metabolic acidosis. In summary, we present evidence of a ouabain-insensitive K + -ATPase in erythrocytes, whose activity is modulated by acid-base status and K + levels.

Study of Erythrocyte ATPases in Infants Evaluated during the Recovery Phase of Severe Dehydration Caused by Diarrhea

“…As previously mentioned, ouabain-insensitive K + -ATPase has been shown to play a role in internal potassium balance in rat erythrocytes [5]. The authors of that study found that K + -ATPase presented a kinetic response to variations in the potassium concentration of the incubation solution.…”

“…Subsequently, 0.1 ml of purified erythrocyte membranes were incubated in duplicate in 0.4 ml of medium for 1 h at 37°C as previously described [5, 12]. The incubation medium contained: 100 m M NaCl, 3 m M MgCl 2 , 136 m M Tris-base, 5 m M NaN 3 , 0.5 m M EGTA and 5 m M ATP (Sigma, St. Louis, Mo., USA), as well as trace amounts of γ[ 32 P]ATP and 0.02% deoxycholate (pH 7.4).…”

“…Total ATPase was measured in the presence of 2.5 m M KCl, and basal ATPase was determined in a K + -free incubation medium. To assure that no P-type cation-transporting ATPases contaminated the assay, 15 m M sodium vanadate (Sigma) was added to a K + -free incubation medium [5,12,13,14]. …”

“…In a recent study involving rat erythrocytes, we described an ouabain-insensitive K + -ATPase whose activity plays a role in internal potassium balance and is modulated by changes in acid-base status [5]. This enzyme is probably one of the H + -K + -ATPase isoforms that has previously been described in other tissues such as the gastric mucosa and mammalian nephron [6, 7].…”