Objectives: The stoichiometric binding model (Frelin C, Guedin D. Cardiovasc Res 1994;28:1613-1622) implies that most endothelin in tissues is bound onto receptors rather than in a free state. The objective of this study was to assay receptor bound endothelins in normal rat tissues.
Methods: We first defined acidic conditions that promoted a mild and reversible denaturation of ETA and ETB receptors and that allowed dissociation of bound [125I]endothelin-1. The action of an acid wash on [125I]endothelin-1 binding to cell or tissue homogenates was then investigated.
Results: Acid washing of homogenates prepared from rat brain capillary endothelial cells that express prepro endothelin-1 mRNAs unmasked receptor sites. Acid washing of cardiac, lung, brain or liver homogenates did not increase [125I]endothelin-1 binding. An acid wash of kidney homogenates increased 2.2 fold [125I]endothelin-1 binding. Experiments using BQ-123 further indicated that the acid treatment of renal homogenates mainly unmasked ETA receptors. Masked renal ET receptors were mainly localized in the medulla. Treatment of rats with phosphoramidon decreased the density of masked ET receptors in kidney homogenates.
Conclusion: As much as 50% of endothelin receptors in renal tissues are masked by endogenous endothelins. Most cardiac receptors are free of bound endothelins. These suggest that endothelins act as local rather than systemic mediators.