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Endothelins activate Ca(2+)-gated K(+) channels via endothelin B receptors in CD-1 mouse erythrocytes
A Rivera1, M A Rotter, C Brugnara
1Department of Laboratory Medicine Bader 7, The Children's Hospital, Boston, Massachusetts 02115, USA. rivera_a@a1.tch.harvard.edu
Insights
Endothelins activate calcium-activated potassium channels (Gardos channels) in mouse red blood cells via ET(B) receptors. This mechanism involves protein kinase C and influences cell dehydration, a factor in sickle cell disease.
Area of Science:
- Physiology
- Molecular Biology
- Pharmacology
Background:
- Cell dehydration in sickle cell disease is linked to calcium-activated potassium channels.
- The Gardos channel in mouse erythrocytes is a key target for understanding these processes.
Purpose of the Study:
- To investigate the role of endothelins in regulating erythrocyte Gardos channels.
- To elucidate the specific receptors and signaling pathways involved in endothelin-mediated Gardos channel activation.
Main Methods:
- Utilized patch-clamp electrophysiology to measure Gardos channel activity (Vmax, K0.5).
- Employed radioligand binding assays to characterize endothelin receptor interactions.
- Investigated receptor subtype involvement using specific agonists and antagonists (IRL 1620, BQ-788).
- Assessed the role of protein kinase C using calphostin C.
Main Results:
- Endothelin-1 (ET-1) significantly increased Gardos channel Vmax and decreased its Ca(2+) affinity.
- ET-1 and ET-3 demonstrated dose-dependent activation of the Gardos channel.
- High-affinity binding sites for ET-1 were identified on erythrocytes.
- The endothelin B (ET(B)) receptor was implicated in channel activation, as shown by agonist/antagonist studies.
- Calphostin C inhibited ET-1-induced Gardos channel activation and protein kinase C activity.
Conclusions:
- Endothelins regulate erythrocyte Gardos channels through ET(B) receptors.
- A calphostin-sensitive pathway, likely involving protein kinase C, mediates this regulation.
- Findings provide insights into the molecular mechanisms underlying cell dehydration in sickle cell disease.
Abstract:
Cell dehydration mediated by Ca(2+)-activated K(+) channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca(2+)-activated K(+) channel (Gardos channel) with maximal velocity (V(max)) of 0.154 +/- 0.02 mmol. l cells(-1). min(-1) and an affinity constant (K(0.5)) for Ca(2+) of 286 +/- 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their V(max) by 88 +/- 9% (n = 8) and decreased their K(0.5) for Ca(2+) to 139 +/- 63 nM (P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC(50) of 75 +/- 20 nM for ET-1 and 374 +/- 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 +/- 51 and 785 +/- 143 nM and with capacity of binding sites of 298 +/- 38 and 1,568 +/- 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ET(B)) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ET(B) receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ET(B) receptors and a calphostin-sensitive mechanism.