Brain Research. 551) ( 1991) 129- 132 © 1991 Elsevier Science Publishers B.V. (X)06-8993/91/$03.50 A I)ONIS 0(X1689939124672 D BRES 24672 129 Characterization of muscarinic receptor subtype of rat eccrine sweat gland by autoradiography Nora E. Torres, Paula J. Zollman and Phillip A. Low Department of Neurology, Mayo Foundation, Rochester, MN 55905 (U. S.A.) (Accepted 19 February 1991) Key words: Autoradiography; Muscarinic" Sweat Gland; Eccrine: Rat Thc muscarinic cholinergic receptor of rat eccrine sweat gland was characterized using quantitative autoradiography and [3H]QNB as radioligand. The distribution of radioligand was maximal in the secretory coil. Autoradiographic competition binding studies were performed using selective antagonists to M~ (pirenzepine), M, (AF-DX 116), and M~ (4-DAMP) and the classical nonselective antagonist atropine, pKi for pirenzepine, AF-DX 116, 4-DAMP, and atropine was 6.58, 5.47, 8.511, and 8.66 respectively indicating that the eccrine sweat gland muscarinic receptor was predominantly M~. The eccrine sweat gland is innervated by postganglio- nic sympathetic sudomotor fibers and pharmacologically is cholinergic muscarinic in humans and several species including the rat and monkey 7'~~'16. Knowledge of the receptor subtype is important since the response of the sweat gland to direct stimulation provides an index of postganglionic sympathetic axonal integrity ~. However, the muscarinic receptor subtype is largely unknown. Pirenzepine (PZ) binds to M~ receptors with high affinity and selectivity 5"'~. M 2 (previously M 2 'cardiac') and Ms (previously M 2 "glandular') receptors have low affinity binding to PZ 4'ss'. M 2 and M 3 can be differentiated by differential affinity to AF-DX 116 (11-[[2-(diethylamino)- methyl]- l -piperidinyl]acetyl-5,11-dihydro-6H-pyrido[2,3-b]- [1,4]benzodiazepine-6-one) and 4-DAMP (4-diphenyla- cetoxy-N-methylpiperidine methbromide). M z receptors have a high affinity toward AF-DX 116; M 3 receptors have low affinity binding to AF-DX 1166, and high affinity binding to 4-DAMP ~'13. We performed autoradiographic competition binding studies of the displacement of [3H]QNB (quinuclidinyl- benzilate) with the selective muscarinic antagonists: pirenzepine, AF-DX 116 and 4-DAMP and the classical nonselective antagonist atropine. Our studies were done on male, 250-300 g, Sprague- Dawley rats that were anesthetized with intraperitoneal pcntobarbital (511 mg/kg). Salivary glands and distal foot pads (regions B and C according to Kennedy et al. ~) were dissected. All the studies on sweat glands were done on foot pads B and C because they have the highest number of sweat glands. The tissue was rapidly frozen using isopentane cooled in liquid nitrogen. Cryostat sections (10 um for autoradiography; 30 lgm for preliminary biochemical studies) were thaw-mounted on glass slides coated with gelatin/chrome-alum. The sections were stored overnight at 4 °C in a desiccator with drierite and the next day were stored at -20 °C until the day of the incubation (never more than 7 days). Before incubation, the sections were allowed to dry at room temperature. As the eccrine sweat glands of rat foot pads were too minute for direct liquid scintillation counting, preliminary biochemical studies to identify optimal conditions for labeling the receptors with [3H]QNB were done using another peripheral exocrine tissue, the submaxillary salivary gland which is known to have a high density of muscarinic receptor binding sites 4"~. The slides contain- ing tissue sections were incubated in 1 nM [3H]QNB with and without atropine for 120 min at 25 °C. The slides were then transferred to PBS buffer at 4 °C for different times in order to determine the wash time that resulted in the highest specific to nonspecific labeling ratio. At 20 min the specific to nonspecific binding ratio was 7:1 and at 60 min, 20:1 with a slow loss of specific binding. A 60 min wash time (2 × 30 min) was adopted for all subsequent experiments. In order to identify conditions under which binding was at equilibrium, the secticms were incubated in 1 nM [~H]QNB with and without atropine for different times at 25 °C. For this experiment a wash time of 20 min was selected. While nonspecific binding remained constant ('orrewondence: P.A l,ow, Department of Neurology, Mayo Foundation, Rochester. MN 55905. U.S.A.