Regulation of Human Sickle Erythrocyte Hydration Status by Endothelin-1 Is Mediated by Casein Kinase II.

Abstract 3020

Poster Board II-996

In Sickle Cell Disease, K⁺ efflux and osmotically induced water loss leads to erythrocyte dehydration, an important step in the formation of hemoglobin S polymers and irreversible sickle cells. We have previously reported that activation of sickle erythrocyte endothelin-1 (ET-1) receptors is partially responsible for dense irreversible sickle cell formation thru mechanisms that are still undefined. Casein Kinase II (CK2), a serine/threonine kinase that phosphorylates acidic proteins, has been shown to regulate calmodulin activity and cytoskeletal proteins. Thus, we posit that CK2 may regulate cellular hydration status. CK2 activity is modulated by apigenin, emodin, heparin, and ornithine decarboxylase. Our results indicate that CK2 activity in erythrocyte membranes increases in the presence of ET-1 (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 10⁶ cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 10⁶ cell). Our data also indicate that 20 μM apigenin blocks ET-1 induced CK2 activity in sickle erythrocytes (2.8 ± 0.1 to 2.7 ± 0.4 nmol/min/mL * 10⁶ cell). We then examined the role of CK2 activation in cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), another specific CK2 inhibitor, and measured the changes in erythrocyte density by the phthalate oil method. We observed that inhibition of CK2 led to the inhibition of deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D₅₀= 1.106 to 1.100 g/mL) or TBB (D₅₀ =1.097 g/mL). Apigenin also inhibited the volume-stimulated K⁺ efflux in a dose-dependent manner (IC₅₀ = 18 ± 1 μM). These results strongly implicate CK2 as an intermediate of the ET-1 stimulated cellular volume regulatory systems in erythrocytes and suggest that regulation of CK2 activity may lead to reduction of hemoglobin S polymerization in vivo. Supported by NIH R01-HL09632 to AR.