Insulin Regulates Neuronal M 2 Muscarinic Receptor Function in the Ileum of Diabetic Rats Fiona R. Coulson, David B. Jacoby, and Allison D. Fryer Division of Physiology, Department of Environmental Health Sciences, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland Received September 4, 2003; accepted November 4, 2003 ABSTRACT Acetylcholine release from cholinergic nerves in the gastroin- testinal tract is limited by neuronal M 2 muscarinic receptors. In diabetic animals, M 2 muscarinic receptor function in the ileum is increased, leading to decreased acetylcholine release and smooth muscle contraction in response to nerve stimulation. The mechanisms responsible for increased M 2 muscarinic re- ceptor function are unknown but may contribute to the gastro- intestinal dysmotility that occurs frequently in diabetics. In this study, we investigated whether insulin modulates M 2 musca- rinic receptor function in the gastrointestinal tract of diabetic rats. M 2 muscarinic receptor function was tested by measuring the ability of an agonist, pilocarpine, to inhibit and an antago- nist, methoctramine, to potentiate electrical field stimulation (EFS)-induced contraction of ileum in vitro. Insulin administra- tion (0.2, 0.6, and 2 U s.c. daily for 7 days) reversed the diabetes-induced increase in M 2 muscarinic receptor function and restored normal contractions to EFS. Insulin had no effect on the function of postjunctional M 3 muscarinic receptors, de- termined by measuring contractile responses to acetylcholine. These data suggest that insulin tonically inhibits neuronal M 2 muscarinic receptors. Thus, loss of insulin removes this inhibi- tion and increases M 2 muscarinic receptor function leading to decreased acetylcholine release and contraction to EFS. In nondiabetic rats, there was a trend that higher insulin doses (0.6 and 2 U) increased M 2 muscarinic receptor function, sug- gesting a bell-shaped concentration-response relationship for insulin. In conclusion, lack of insulin or excess insulin increases M 2 muscarinic receptor function in rat ileum. This mechanism may contribute to decreased acetylcholine release in the gas- trointestinal tract of diabetics, resulting in dysmotility. Decreased function of the autonomic nerves is a serious complication of diabetes and contributes to heart disease (Aronson, 2001), genitourinary disorders (Dunsmuir and Holmes, 1996) and gastrointestinal dysmotility (Horowitz and Fraser, 1994; Bittinger et al., 1999) in diabetic patients. Loss of autonomic nerve function has long been considered to be due to irreversible nerve damage. Although this may be a consequence of chronic diabetes, other mechanisms may also contribute to decreased autonomic function such as de- creased release of neurotransmitters from autonomic nerves. M 2 muscarinic receptors inhibit the release of neurotrans- mitters from autonomic nerves. These inhibitory, neuronal receptors are present on parasympathetic and sympathetic nerves throughout the autonomic nervous system, supplying the lungs (Gallagher et al., 1975; Fryer and Maclagan, 1984; Mak and Barnes, 1990), heart (Hancock et al., 1987; Dam- mann et al., 1989; Cost and Majewski, 1991), bladder (Somo- gyi and de Groat, 1992; Tobin and Sjogren, 1995), and gas- trointestinal tract (Goyal, 1988; Lambrecht et al., 1999; Coulson et al., 2002). The importance of M 2 muscarinic re- ceptors in regulating acetylcholine release from parasympa- thetic nerves has been demonstrated in the airways using agonists, such as pilocarpine, and antagonists, such as gal- lamine and methoctramine. Stimulating M 2 muscarinic re- ceptors with pilocarpine inhibits acetylcholine release from parasympathetic nerves and decreases bronchoconstriction in response to vagal nerve stimulation by more than 80% (Fryer and Maclagan, 1984; Minette and Barnes, 1988; Baker et al., 1992). Blocking M 2 muscarinic receptors with gal- lamine or methoctramine enhances acetylcholine release and increases bronchoconstriction in response to vagal nerve stimulation 5–10-fold (Fryer and Maclagan, 1984; Minette and Barnes, 1988; Kilbinger et al., 1991; Patel et al., 1995). In diabetes, the release of acetylcholine from the heart (Oberhauser et al., 2001) and the corpus cavernosum (Blanco et al., 1990) has been shown to be reduced and worsen with the duration of diabetes. This reduction in acetylcholine re- lease is speculated to be a consequence of increased function of inhibitory, neuronal M 2 muscarinic receptors (Oberhauser This work was funded by the National Institutes of Health Grants HL- 55543 (to A.D.F.), HL-54659 (to D.B.J.), HL-61013 (D.B.J.), HL-10342 (to A.D.F.) and by a grant from the American Heart Association (to A.D.F.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.057570. ABBREVIATION: EFS, electrical field stimulation. 0022-3565/04/3082-760 –766$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 308, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 57570/1125591 JPET 308:760–766, 2004 Printed in U.S.A. 760 at ASPET Journals on July 19, 2018 jpet.aspetjournals.org Downloaded from