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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
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.
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Abstract |
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), 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 decreased release of neurotransmitters from autonomic nerves.
M2 muscarinic receptors inhibit the release of neurotransmitters 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; Dammann et al., 1989; Cost and Majewski, 1991), bladder (Somogyi and de Groat, 1992; Tobin and Sjogren, 1995), and gastrointestinal tract (Goyal, 1988; Lambrecht et al., 1999; Coulson et al., 2002). The importance of M2 muscarinic receptors in regulating acetylcholine release from parasympathetic nerves has been demonstrated in the airways using agonists, such as pilocarpine, and antagonists, such as gallamine and methoctramine. Stimulating M2 muscarinic receptors 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 M2 muscarinic receptors with gallamine 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 release is speculated to be a consequence of increased function of inhibitory, neuronal M2 muscarinic receptors (Oberhauser et al., 2001). In animals, diabetes has been demonstrated to increase M2 muscarinic receptor function in the airways in vivo (Belmonte et al., 1997) and in vitro (Coulson et al., 2002) and in the gastrointestinal tract in vitro (Coulson et al., 2002). The mechanisms by which diabetes increases neuronal M2 muscarinic receptor function are unknown but insulin does reverse changes in M2 receptor function in the airways (Belmonte et al., 1997; Belmonte et al., 1998).
The aim of this study was to determine whether insulin reverses the diabetes-induced increase in neuronal M2 muscarinic receptor function and restores parasympathetic nerve function in the gastrointestinal tract.
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Materials and Methods |
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Induction of Diabetes Mellitus. Diabetes mellitus was induced by injection of 65 mg kg–1 streptozotocin into the tail vein of rats lightly anesthetized with sodium pentobarbitone (30 mg kg–1). Control rats were injected with the same volume (1 ml kg–1) of 0.1 M sodium citrate buffer (pH 4.5). Seven days after treatment, rats were given daily subcutaneous injections of insulin (0.2, 0.6, or 2 U per rat) for 7 days. The concentration of glucose in whole blood was measured in every animal immediately before each experiment by a standard glucometer (Accu-Chek Instant; Roche Diagnostics, Indianapolis, IN).
Measurement of Muscarinic Receptor Function. After 7 days, rats were killed with sodium pentobarbitone (60 mg i.p.). The ileum was removed and placed in Krebs-Henseleit solution containing propranolol (10–6 M; to block the effects of sympathetic nerve stimulation).
The ileum was rinsed with Krebs-Henseleit solution, the most distal 10 cm discarded, and the remaining ileum cut into 1- to 2-cm segments. Ileal segments were mounted longitudinally between two zigzag platinum electrodes in a 5-ml water-jacketed organ bath that contained Krebs-Henseleit solution with propranolol (10–6 M), bubbled with 5% CO2 and 95% O2, and kept at 37°C. The strips were placed under 1.0-g isometric tension and allowed to equilibrate.
During a 1-h equilibration period, each tissue was washed every 15 min with Krebs-Henseleit solution. After equilibration, a cumulative concentration-response curve to acetylcholine (10–8 M–3 x 10–4 M) or a frequency-response curve to electrical field stimulation (EFS, 1–20 Hz, 100 V, 0.2-ms pulse duration for 5 s every minute) was constructed on each preparation. The preparations were then washed every 15 min for 1 h with Krebs-Henseleit solution to remove acetylcholine from the bath and to allow tissues to reestablish baseline tension. Stable baseline responses to EFS (5 Hz) were then obtained. M2 muscarinic receptor function was tested by measuring the ability of an agonist, pilocarpine (10–10–10–3 M), to inhibit and an antagonist, methoctramine (10–9–10–4 M), to potentiate EFS-induced contraction. Pilocarpine and methoctramine were added cumulatively to the bath at 5-min intervals.
In rat ileum, the selective M2 muscarinic receptor antagonist methoctramine potentiates EFS-induced contraction due to blockade of neuronal M2 muscarinic receptors (Coulson et al., 2002). Concurrently, methoctramine reduces muscarinic agonist-induced contraction due to blockade of postjunctional M3 muscarinic receptors (Giraldo et al., 1988; Coulson et al., 2002). Thus, the effect of methoctramine on postjunctional M3 muscarinic receptors was determined by testing the ability of methoctramine to inhibit carbachol-induced contraction of smooth muscle. The tissues were precontracted with the carbachol (10–4 M) and then methoctramine (10–9–10–4 M) was added cumulatively to the bath at 5-min intervals. The effect of methoctramine on prejunctional M2 muscarinic receptors is the difference between the inhibition of carbachol-induced contraction and inhibition of EFS-induced contraction.
The ability of postjunctional M3 muscarinic receptors to respond to agonists was tested by measuring the ability of cumulative concentrations of acetylcholine (10–8–3 x 10–4 M) to contract ileum.
At the end of each experiment, the muscarinic antagonist atropine (10–4 M) was added to the organ baths. Atropine blocked EFS-induced contractions, confirming that these contractions were mediated via muscarinic receptors.
Expression and Statistical Analysis of Data. All data are expressed as means ± S.E.M. The n values equal the numbers of animals that contributed to the mean. The weights of ileal tissues from rats made diabetic with streptozotocin were significantly greater than those taken from nondiabetic rats (Table 1). This diabetes-induced increase in ileal weight has been shown by other authors to be due to an increase in both mucosal and smooth muscle mass (Nowak et al., 1990). Because smooth muscle mass is increased in streptozotocin-treated rats, the contractile responses to EFS, carbachol, and acetylcholine are expressed as the increase in grams of tension above baseline per milligram of tissue.
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The effects of pilocarpine and methoctramine on EFS-induced contraction are expressed as the ratio of contraction in the presence of drug to the contraction in the absence of drug. Because neither pilocarpine nor methoctramine altered the baseline tension, contractions to EFS in the absence and presence of drug were obtained in the same animal. Differences in the effects of pilocarpine and methoctramine between control and streptozotocin-treated rats were compared using multifactorial analysis of variance. P < 0.05 was considered statistically significant.
Drugs. Acetylcholine, atropine, carbachol, methoctramine, pilocarpine, pirenzepine, propranolol, sodium pentobarbitone, and streptozotocin were all purchased from Sigma-Aldrich (St. Louis, MO). All drugs were dissolved in Krebs' solution except streptozotocin, which was dissolved in 0.1 M sodium citrate buffer, pH 4.5. Insulin (NPH human insulin isophane suspension, Humulin N, 100 U ml–1) was purchased from Eli Lilly & Co. (Indianapolis, IN). Insulin was diluted in sterile saline so that each rat received 0.1 ml of insulin s.c. per day.
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Results |
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Diabetic rats weighed significantly less than controls and insulin treatment had no effect on animal weights in either control or diabetic rats (Table 1).
Effect of EFS. EFS of isolated ileum caused frequency-dependent contraction of gastrointestinal smooth muscle that was significantly less in diabetic rats compared with controls (Fig. 1A), indicating decreased nerve function in diabetics. In insulin-treated rats, contraction to EFS was not significantly different between control and diabetic rats (Fig. 1, B–D), showing that insulin restores nerve function in diabetics back to controls.
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Neuronal M2 Muscarinic Receptor Function. Pilocarpine inhibited EFS-induced contractions in a concentration-related manner (Fig. 2, open symbols), demonstrating functional M2 muscarinic receptors. The concentration-response curve to pilocarpine was shifted significantly to the left in diabetic rats (Fig. 2A, closed symbols), demonstrating an increase in M2 muscarinic receptor response to pilocarpine. In insulin-treated rats, there was no significant difference between pilocarpine concentration-response curves between control and diabetic rats (Fig. 2, B–D), showing that insulin restores M2 muscarinic receptor function in diabetics back to controls.
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The ability of neuronal M2 muscarinic receptors to respond to endogenous acetylcholine was measured using the selective antagonist methoctramine (see Materials and Methods). Methoctramine inhibited both carbachol- and EFS-induced contractions of gastrointestinal smooth muscle (Fig. 3) due to blockade of postjunctional M3 muscarinic receptors. However the inhibition of EFS-induced contractions was significantly less than the inhibition of carbachol-induced contractions (Fig. 3), indicating that methoctramine potentiated EFS-induced contraction. This potentiation was a result of neuronal M2 muscarinic receptor blockade.
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The potentiation of the EFS-induced response, i.e., the difference between the inhibition of EFS- and carbachol-induced contraction, was significantly greater in diabetics than controls (Fig. 3A). This confirms that diabetes increases M2 muscarinic receptor function.
In rats treated with insulin, the potentiation of the EFS-induced response by methoctramine was not significantly different between control and diabetic rats (Fig. 3, B–D), showing that insulin reversed the diabetes-induced increase in M2 muscarinic receptor function.
The inhibition of carbachol-induced contractions by methoctramine was not different between all the groups of rats (Fig. 3).
Postjunctional M3 Muscarinic Receptor Function. Acetylcholine-induced concentration-dependent contractions of gastrointestinal smooth muscle, which were not significantly different among control, diabetic, or insulin-treated control and diabetic rats (Fig. 4). This demonstrated that insulin did not alter the function of the postjunctional M3 muscarinic receptors.
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Effect of Insulin on M2 Muscarinic Receptor Function in Nondiabetic Rats. Pilocarpine inhibited contractions to EFS in all groups of rats (Fig. 2). The degree of inhibition was dependent on the concentration of insulin (Fig. 5A). In diabetic rats and nondiabetic rats treated with 0.6 or 2 U of insulin, the maximum response to pilocarpine was greater than that in control rats. This indicated that M2 muscarinic receptor function was increased in rats depleted of insulin (diabetic) or supplemented with insulin (nondiabetic treated with 0.6 or 2 U of insulin).
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Conversely, methoctramine potentiated contractions to EFS in all groups of rats (Fig. 3). The degree of potentiation was dependent on concentration of insulin (Fig. 5B). The maximum response to methoctramine was greater in diabetic rats and nondiabetic rats treated with 0.6 or 2 U of insulin versus controls, confirming that M2 muscarinic receptor function was increased in these animals.
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Discussion |
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-blocker propranolol and was blocked by the muscarinic antagonist atropine, indicating that contraction was mediated by the release of acetylcholine from parasympathetic nerves onto muscarinic receptors. In rats made diabetic by administration of the pancreatic -cell toxin streptozotocin, the contraction to EFS was significantly less than that in control rats (Fig. 1), showing that diabetes decreases parasympathetic nerve function in the gastrointestinal tract.
Decreased parasympathetic nerve function in diabetic rats was a consequence of an increase in the function of the inhibitory M2 muscarinic receptors on the nerves. This was established by measuring the ability of the muscarinic agonist pilocarpine to inhibit and the muscarinic antagonist methoctramine to potentiate EFS-induced contraction. The inhibitory effect of pilocarpine was significantly greater in diabetic rats than in controls (Fig. 2), showing that diabetes increases M2 muscarinic receptor function. This was supported by data obtained with the muscarinic antagonist methoctramine (Fig. 3). The function of postjunctional M3 muscarinic receptors was unchanged by diabetes because concentration-response curves to acetylcholine were not different between control and diabetic rats (Fig. 4). These data confirm our previous observations (Coulson et al., 2002).
Insulin reversed the diabetes-induced reduction in nerve function seen by the restoration of EFS-induced contraction in insulin-treated diabetic rats. This reversal occurred simultaneously with a restoration of normal function of the neuronal M2 muscarinic receptors demonstrated with pilocarpine and methoctramine (Figs. 2 and 3). Insulin did not change the magnitude of contractions to acetylcholine in diabetic rats (Fig. 4), showing that the function of postjunctional M3 muscarinic receptors was unaltered by insulin. Thus, increased M2 muscarinic receptor function and the consequent decrease in nerve function in the gastrointestinal tract of diabetic rats is due to decreased insulin. This reversal of diabetes-induced changes in M2 muscarinic receptor function by insulin has also been demonstrated in the airways in vitro (our unpublished data) and in vivo (Belmonte et al., 1997, 1998).
The doses of insulin used in the current study did not reverse diabetes-induced hyperglycemia, indicating that the increase in M2 muscarinic receptor function that occurs in diabetes is more likely due to insulin insufficiency rather than to elevated blood glucose. This suggests that in normal rats insulin tonically inhibits neuronal M2 muscarinic receptors, thereby increasing acetylcholine release from parasympathetic nerves. Loss of insulin removes this inhibition and increases M2 muscarinic receptor function, leading to decreased acetylcholine release.
The mechanisms by which insulin inhibits neuronal M2 muscarinic receptor function are unknown but may occur, in part, via a direct interaction between insulin receptors and muscarinic receptors. Insulin receptors have been demonstrated on cholinergic neurons in the central nervous system (Araujo et al., 1989; Holdengreber et al., 1998) and on autonomic neurons in the periphery (Reinhardt et al., 1993; Karagiannis et al., 1997) and may be present on parasympathetic neurons in the gastrointestinal tract. Binding of insulin to its tyrosine kinase-linked receptor induces a cascade of phosphorylation events, including the activation of insulin receptor substrates, phosphotidylinositol-3 kinase, and protein kinase C (Dupont and LeRoith, 2001; Lizcano and Alessi, 2002). Insulin may regulate M2 muscarinic receptor function by altering the signal transduction events that follow M2 muscarinic receptor activation (Soltoff and Toker, 1995) or possibly by phosphorylating tyrosine residues on M2 muscarinic receptors. Muscarinic receptors are G protein-coupled receptors, and there is also some evidence that insulin receptor activation phosphorylates adjacent G protein-coupled receptors (Hadcock et al., 1992; Baltensperger et al., 1996).
Another explanation for the findings that insulin inhibits neuronal M2 muscarinic receptor function is that insulin interacts with M2 muscarinic receptors to impair agonist binding. In the absence of insulin, which occurs in diabetic rats, muscarinic receptor agonist binding to M2 muscarinic receptors is increased (Belmonte et al., 1997). Treatment of diabetic rats with insulin restores binding to that seen in control rats (Belmonte et al., 1997).
The role that insulin plays in modulating neurotransmission in the gastrointestinal tract does not seem to be limited to parasympathetic nerves. Insulin has been demonstrated to increase noradrenaline release from sympathetic nerves in the guinea pig ileum (Cheng et al., 1997). This increase may be due in part to inhibition of neuronal M2 muscarinic receptors. M2 muscarinic receptors have been localized to sympathetic nerves where they limit noradrenaline release (Racke et al., 1992; Yokotani and Osumi, 1993; Lambrecht et al., 1999). Thus, inhibition of M2 muscarinic receptor function by insulin would lead to increased noradrenaline release.
The results from our study seemed to indicate that the effects of insulin on M2 muscarinic receptor function were not only dose-related but followed a bell-shaped dose-response curve (Fig. 5). M2 muscarinic receptor function was increased when insulin levels were low (diabetic rats) or when insulin levels were high (nondiabetic rats given 0.6 or 2 U of insulin). Bell-shaped responses have been described previously for insulin where the response to insulin decreases at high concentrations (De Meyts, 1994). The mechanism(s) responsible for this "nonclassical" dose-response curve are incompletely understood but may reflect receptor cross-linking, which has been proposed to occur at high insulin concentrations (De Meyts, 1994; De Meyts et al., 1994).
In conclusion, insulin seems to play an important role in modulating neurotransmission in the gastrointestinal tract. Low or high levels of insulin increase neuronal M2 muscarinic receptor function, leading to decreased acetylcholine release and smooth muscle contraction in response to parasympathetic nerve stimulation. This may be a mechanism by which autonomic nerve function is decreased in the gastrointestinal tract of diabetics, contributing to dysmotility. Increased neuronal M2 muscarinic receptor function may also occur in other organs affected by diabetes, such as the heart and bladder, contributing to heart disease and genitourinary disorders.
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Footnotes |
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ABBREVIATION: EFS, electrical field stimulation.
Address correspondence to: Dr. Fiona R. Coulson, School of Health Science, Griffith University, Gold Coast Campus, PMB 50, Gold Coast Mail Centre QLD 9726, Australia. E-mail: f.coulson{at}mailbox.gu.edu.au
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