The Molecular and Pharmacological Properties of Muscarinic Cholinergic Receptors Expressed by Rat Sweat Glands Are Unaltered by Denervation
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The Journal of Neuroscience, December 1991, 77(12): 37633771 The Molecular and Pharmacological Properties of Muscarinic Cholinergic Receptors Expressed by Rat Sweat Glands Are Unaltered by Denervation Michael P. Grant,‘-* Story C. Landis,* and Ruth E. Siegel’ Departments of ‘Pharmacology and *Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106 Previous studies have indicated that denervation of adult Muscarinic cholinergic receptors are a family of closely related rodent sweat glands results in the loss of secretory respon- molecular subtypes that possess distinct functional properties siveness to muscarinic agonists. To elucidate the molecular (reviewed by Nathanson, 1987; Bonner, 1989; Hulme et al., basis of this loss, we have characterized the muscarinic 1990). The heterogeneity of this receptor class was initially re- cholinergic receptor present in adult rat sweat glands and vealed in pharmacological studies. Based on differing affinities examined the effects of cholinergic denervation on its prop- for the antagonist pirenzepine in competition experiments with erties and expression. When homogenates of gland-rich tis- [N-methyl-3H]-scopolamine (t3H]NMS), muscarinic receptors sue from adult animals were assayed with [KmethyL3H]- were divided into three broad pharmacological classes (desig- scopolamine, a high-affinity muscarinic antagonist, the con- nated by “M”): a high-affinity or M, site found predominantly centration of muscarinic receptors was 301 fmollmg protein in brain (K, < 10 nM) and intermediate- and low-affinity sites and the affinity was 13 1 pm. Autoradiographic analysis dem- located predominantly in peripheral autonomic targets (K;s = onstrated that ligand binding sites were detectable only on 300 and 800-1000 nM, respectively), which have been collec- glands. In competition studies with well-characterized mus- tively designated as M, (Hammer et al., 1980). Parotid and carinic agents, the receptor exhibited typical muscarinic lacrimal glands possess the intermediate-affinity site, while heart pharmacology. Further investigation with the selective mus- contains the low-affinity site (Hammer et al., 1980). The char- carinic antagonists 4-diphenylacetoxy-Kmethylpiperidine acterization of these classes was further refined following the methiodide, pirenzepine, and AF DX-116 revealed that the development of two additional compounds; 4diphenylacetoxy- sweat gland receptor belongs to the M, glandular pharma- N-methylpiperidine methiodide (4-DAMP) possesses high af- cological subtype. In situ hybridization histochemistry with finity for most exocrine gland receptors and AF DX-116 pos- receptor subtype-specific oligonucleotide probes indicated sesses high affinity for the heart receptor (Barlow and Shepherd, that rat sweat glands express the m3 molecular receptor 1986; Hammer et al., 1986). Based on these studies, the M, subtype. Seven days after sciatic nerve transection, when receptors have been subclassified as M, cardiac and M, glandular denervated glands were compared to those on the contra- (Barlow and Shepherd, 1986; Hammer et al., 1986). lateral unoperated side, there was no significant difference Subsequent molecular cloning studies have demonstrated that either in the concentration or affinity of muscarinic binding in both humans and rats muscarinic receptors comprise a family sites or in receptor density or distribution. Furthermore, the of five related genes (designated by “m”), ml-m5 (Kubo et al., molecular subtype and the level of its expression were un- 1986a,b; Bonner et al., 1987, 1988; Peralta et al., 1987a,b). The changed. Thus, it appears that muscarinic binding sites and mRNAs that correspond to these proteins have a heterogeneous m3 receptor mRNA are present in denervated sweat glands distribution in peripheral autonomic targets and CNS (Peralta that are unresponsive to muscarinic stimulation. These re- et al., 1987a; Buckley et al., 1988; Maeda et al., 1988). For each sults suggest that the regulation of responsiveness occurs molecular subtype, competition studies performed with cell lines at a point distal to the expression of muscarinic receptors. transfected with cDNAs encoding a single molecular subtype have yielded distinct pharmacological profiles (Peralta et al., 1987a; Akiba et al., 1988; Bonner et al., 1988; Buckley et al., 1989); for example, the ml receptor possesses high affinity for Received Mar. 29, 1991; revised June 19, 1991; accepted June 27, 1991. pirenzepine and low affinity for AF DX-116, while the m2 re- We thank Drs. Marshall Snavely and James Unnerstall for their assistance in ceptor exhibits high affinity for AF DX- 116 and low affinity for developing the binding assay protocol and Dr. George Dubyak for helpful dis- pirenzepine and 4-DAMP. These two molecular subtypes, ml cussions. We also thank Dr. Steven Jones for his assistance with data analysis and and m2, are believed to correspond to the M, and M, heart review of the manuscript. This work was supported by NIH Grant NS 23678 and NIDDK Grant P30 27651 to S.C.L. and a grant from Mathers Foundation to pharmacological subtypes. Analysis of cell expression systems R.E.S. M.P.G. was supported by NIH Grant GM07382-14. has also suggested that particular molecular subtypes are pref- Correspondence should be addressed to Michael P. Grant, Department of Neu- rosciences, Case Western Reserve University, School of Medicine, 10900 Euclid erentially coupled to specific intracellular effecters. The m 1, m3, Avenue, Cleveland, OH 44106-4975. and m5 subtypes interact with phospholipase C and thus acti- Copyright 0 1991 Society for Neuroscience 0270-6474/91/l 13763-09$05.00/O vate the phosphoinositide (PI) pathway, whereas the m2 and 3764 Grant et al. - Denervation and Muscarinic Receptors m4 receptors are coupled to inhibition of adenyl cyclase (Ash- cholinergic innervation plays a central role in the maintenance kenaziet al., 1987; Fukudaet al., 1988; Joneset al., 1988; Peralta of secretory function at a site distinct from the receptor. et al., 1988). Although the diversity of this receptor family is Preliminary reports of these studies have appeared previously now well established, relatively little is known about the specific (Grant and Landis, 1988, 1989). distributions of the different molecular subtypes in peripheral tissues and the relationship between particular molecular sub- Materials and Methods types and pharmacological properties in vivo. Materials. Carbamylcholine, atropine sulfate, chloral hydrate, pilocar- Choline@ denervation of peripheral targets produces di- pine, oxotremorine sesquifumarate, dithiothreitol (DTT), acetic anhy- verse effects on muscarinic receptor expression. Denervation of dride, triethanolamine, dextran sulfate, bovine serum albumin, ssDNA, and yeast tRNA were obtained from Sigma (St. Louis, MO). Pirenzepine cat iris by extirpation of the ciliary ganglion produces no change and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were in the concentration or affinity of muscarinic receptors (Sachs purchased from Research Biochemical Inc. (Natick, MA). AF DX- 116 et al., 1979). In contrast, parasympathectomy of rat urinary was the kind gift of Boehringer Ingelheim (Ridgefield, CT). Terminal bladder produces an increase in the concentration of muscarinic deoxynucleotidyl transferase was obtained corn Bethesda Research Labs. receptors; a portion of this increase, however, may result from [N-methyl-3H]-scopolamine (85 Wmmol), %-deoxyadenosine 5’-[(Y- thioltriuhosohate (1320 Wmmol), and Formula 963 were from New the compensatory hypertrophy that accompanies bladder de- E&a&l Nuclear (Boston, MA). I%otographic emulsion NTB-3, D- 19 nervation (Nilvebrant et al., 1986). Finally, denervation of rat developer, and fixer were from Kodak (Rochester, NY). All other re- parotid gland produces a decrease in the number of muscarinic agents were obtained from Fisher and were reagent grade or better. receptors (Talamo et al., 1979). Since muscarinic receptors are Animals. Litters and adult Sprague-Dawley rats were obtained from Zivic-Miller (Zelienople, PA). The sweat glands of adult rats were de- heterogeneous and the several subtypes may be regulated dif- nervated by cutting the sciatic nerve. Rats were deeply anesthetized with ferently, the varying responses to denervation could reflect dif- chloral hydrate, the lateral aspect of the upper thigh was shaved, and ferences in the molecular subtype expressed by target cells. In the surgical area was swabbed with ethanol. A 2-3 cm longitudinal none of the studies to date, however, has the expression of incision was made over the head of the humerus. The sciatic nerve was specific molecular subtypes been examined. Therefore, it re- visualized by reflecting the inferior aspect of the gluteus maximus su- periorly and reaching under the biceps femoris. A 1.5 cm segment of mains unclear whether the observed changes in the level of sciatic nerve was removed, and the wound was closed with wound clips. receptor expression, or lack thereof, are related to the specific Secretion assay. Sweat secretion was assayed by making a silicone target cell types or, alternatively, whether they are a function of elastic (Kerr Co., Romulus, MI) mold of the plantar surface of the rat the molecular subtype(s) expressed. footpad as described previously (Kennedy et al., 1984; Stevens and Rat eccrine sweat glands provide an interesting model system Landis, 1987). Animals were injected subcutaneously with 3 mg&g of pilocarpine, and the impression