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Abstract
Particulate fractions from fresh bovine corneal endothelium exhibited high affinity, specific binding by a potent muscarinic cholinergic radioligand, [3H]QNB. Particulate fraction binding sites exhibited half maximal binding at approximately 0.3 nM [3H]QNB and reached a maximal binding capacity of 820 fmoles/mg of protein at 3 nM [3H]QNB. Muscarinic cholinergic antagonists and agonists competed with [3H]QNB when incubated concurrently with the tissue, showing relative potencies expected of these agents when binding to muscarinic cholinergic receptors. Particulate fractions prepared from cultured bovine corneal endothelium exhibited qualitatively similar [3H]QNB binding characteristics, but maximal binding capacity was only about one-fifth of its fresh-tissue counterpart. Intact cultured cells showed 3-fold more specific [3H]QNB binding than did their particulate fractions. Incubation of intact corneal endothelial cells with muscarinic cholinergic agonists such as carbachol stimulated cyclic [3H]GMP 3-fold from endogenous [3H]GTP within 1 min of incubation. The effect diminished rapidly and returned to control levels within 8 min. Carbachol stimulation of cyclic [3H]GMP was concentration-dependent, reaching half maximal stimulation at 1 microM. Atropine was a potent, competitive inhibitor of carbachol stimulation of cyclic [3H]GMP in endothelial cultures, requiring only 1 microM to completely block the carbachol response. These experiments demonstrate the existence of muscarinic cholinergic receptors in bovine corneal endothelium and their control of cyclic GMP levels in this tissue.