Molecular characterization of alpha 2-adrenergic receptors regulating intestinal electrolyte transport

Norepinephrine (NE) is an important neuromodulator of active Na+ and Cl- transport by the small intestine; however, the cellular targets and the adrenergic receptor (AR) subtype mediating its effects on ion transport have not been clearly defined. NE inhibited short-circuit current in submucosal-mucosal sheets of porcine distal jejunum under basal conditions and after electrical transmural stimulation of intrinsic neurons. A membrane fraction (P2) prepared from the submucosa of porcine jejunum was enriched in specific [3H]saxitoxin binding sites, relative to other submucosal fractions. This fraction contained homogeneous and high affinity sites binding the alpha 2-AR antagonist [3H]yohimbine (Kd = 0.39 +/- 0.03 nM). A prazosin versus oxymetazoline Ki ratio of 218 was obtained for the submucosal AR binding site, suggesting that it represents a neuronal alpha 2A-AR. A cell membrane fraction prepared from the mucosa exhibited specific and saturable high affinity binding of the muscarinic cholinergic antagonist [3H] quinuclidinyl benzilate (Kd = 77 +/- 9 pM) but displayed minimal specific binding of [3H]saxitoxin or [3H]yohimbine. A [32P]cDNA probe derived from the human alpha 2-C10 gene encoding the alpha 2A-AR hybridized to a 3.8-kilobase message that was prevalent in poly(A)+ RNA isolated from the jejuno-ileal submucosa and was also detected in porcine cerebral cortex and kidney; no message was detected in RNA isolated from the jejunal mucosa. These results suggest that NE modulates active ion transport in the small intestine through interactions with a submucosal alpha 2A-AR probably associated with enteric neurons.