Cholinergic receptors in renal medullary collecting duct cells

Intrarenal administration of cholinergic agents produces diuresis. However, neither cholinergic innervation or specific cholinergic receptors have been shown to be present in the kidney. Recently, we have demonstrated that carbachol, a cholinergic agent, stimulates phosphoinositide hydrolysis in the inner medullary collecting duct (IMCD) cells. The effect was blocked by atropine (a cholinergic antagonist), suggesting that phosphoinositide hydrolysis occurs through the interaction of carbachol with specific cholinergic receptors in these cells. Therefore, we examined the cholinergic receptors in IMCD cells by measurement of radioligand binding of a cholinergic receptor antagonist, I-quinuclidinyl (phenyl-4-3H)benzilate([3H]QNB). The IMCD cells were prepared from rabbit kidneys by incubating the inner medullary slices with collagenase and treating the isolated cells with hypotonic solution to lyse cells other than IMCD cells. Binding of [3H]QNB to IMCD cells was measured at 37 degrees C for 60 min in the absence (total binding) and the presence (nonspecific binding) of 100 microM atropine (a muscarinic receptor antagonist). The specific binding (the difference between total and nonspecific binding) of [3H]QNB to IMCD cells was saturable with a Bmax (maximum binding sites) of 27.5 fmol/mg of protein and Kd (dissociation constant) of 0.27 nM. Atropine, but not hexamethonium (a nicotinic antagonist), was able to displace [3H]QNB from IMCD cells with a Ki of 0.1 microM. It is, therefore, concluded that specific high affinity muscarinic receptors are present in IMCD cells. These receptors may play a role in producing the pharmacologic actions of cholinergic agents on the kidney.