Binding of [3H]dihydroalprenolol and [3H]quinuclidinyl benzilate to intact cells of cultured corneal epithelium.

Abstract

In intact cultured rabbit corneal epithelial cells we have identified [3H]dihydroalprenolol ( [3H]DHA) and [3H]quinuclidinyl benzilate ( [3H]QNB) binding activities which meet criteria for beta-adrenergic and muscarinic cholinergic receptors. For saturable, propranolol-sensitive [3H]DHA binding, Bmax = 0.374 +/- 0.063 fmol/microgram protein; KDHA = 12.5 +/- 2.4 nM from Scatchard analysis. For saturable, atropine-sensitive [3H]QNB binding, Bmax = 0.403 +/- 0.053 fmol/microgram protein; KQNB = 15.4 +/- 0.7 nM. The order of potency of unlabeled adrenergic agonists in competition for [3H]DHA sites was isoproterenol greater than epinephrine greater than norepinephrine. For unlabeled cholinergic agonists competing for [3H]QNB sites, the order was oxotremorine greater than acetylcholine greater than or equal to carbamylcholine. Acetylcholine did not inhibit [3H]DHA binding, nor did isoproterenol or choline inhibit [3H]QNB binding. Effectiveness of drugs in stimulating cAMP or cGMP accumulation closely paralleled efficacy in competition for [3H]DHA or [3H]QNB sites. Results confirm the presence in intact cultured corneal epithelial cells of beta-adrenergic receptors (demonstrated by others in corneal membrane suspensions), identify in intact cells muscarinic cholinergic receptors (not previously detected in broken cell preparations), and supply evidence for receptor-mediated regulation of cyclic nucleotide levels in these cells, further supporting our hypothesis of bidirectional influence by cAMP-mediated beta-adrenergic and cGMP-mediated cholinergic "first messengers" on proliferation during healing of corneal epithelial defects.