Elsevier

Autonomic Neuroscience

Volume 165, Issue 2, 7 December 2011, Pages 178-190
Autonomic Neuroscience

Neostigmine-induced contraction and nitric oxide-induced relaxation of isolated ileum from STZ diabetic guinea pigs

https://doi.org/10.1016/j.autneu.2011.07.009Get rights and content

Abstract

Both delayed gastrointestinal transit and autonomic neuropathy have been documented in patients with diabetes mellitus. The mechanism of neostigmine, an agent that mimics release of acetylcholine from autonomic neurons by prokinetic agents, to contract smooth muscle, despite dysfunctional enteric neural pathways, was determined using isolated ilea from STZ-treated and control guinea pigs. Both bethanechol- and neostigmine-induced contractions were stronger in diabetic ileum. Bethanechol-induced contractions of control but not diabetic ileum were increased by low dose scopolamine suggesting reduced activation of presynaptic muscarinic autoreceptors in diabetic ileum. The muscarinic receptor antagonist 4-DAMP strongly, but the nicotinic receptor antagonist hexamethonium only weakly, reduced neostigmine-induced contractions of control and diabetic ilea. The amount of acetylcholine, inferred from tissue choline content, was increased in diabetic ileum. Nicotinic neural and noncholinergic postjunctional smooth muscle receptors contributed more strongly to neostigmine-induced contractions in diabetic than control ileum. Relaxation of diabetic ileum by exogenous nitric oxide generated from sodium nitroprusside was comparable to control ileum, but smooth muscle relaxation by l-arginine using neuronal nitric oxide synthase to generate nitric oxide was weaker in diabetic ileum with evidence for a role for inducible nitric oxide synthase. Despite autonomic neuropathy, neostigmine strongly contracted ileum from diabetic animals but by a different mechanism including stronger activation of postjunctional muscarinic receptors, greater synaptic acetylcholine, stronger activation of noncholinergic excitatory pathways, and weaker activation of inhibitory pathways. A selective medication targeting a specific neural pathway may more effectively treat disordered gastrointestinal transit in patients with diabetes mellitus.

Introduction

Small intestinal contractions mix luminal contents to promote digestion of nutrients by digestive enzymes and to expose nutrients to the apical surface of enterocytes for absorption and propel luminal contents towards the large intestine. Small intestinal motility can modulate the rate of gastric emptying. Arrival of nutrients to the distal small intestine delayed gastric emptying by the ileal brake (Spiller et al., 1984, Holgate and Read, 1985, Brown et al., 1992, Maljaars et al., 2008). In turn, gastric distension by a meal enhanced the flow of luminal contents from the ileum into the cecum (Kerlin and Phillips, 1983). This bidirectional coordination between stomach and ileum is mediated by vagal autonomic reflexes and gastrointestinal (GI) hormones (Maljaars et al., 2008).

Gastrointestinal motor abnormalities in patients with diabetes mellitus (DM) are often attributed to autonomic neuropathy (Scarpello and Sladen, 1978, Feldman and Schiller, 1983, Yang et al., 1984, Vinik et al., 2003). Extrinsic autonomic reflexes controlling GI function were impaired in patients with DM. Gastric acid secretion in response to sham feeding, a vagal reflex, was reduced in patients with DM (Feldman et al., 1979). Increased colonic motility in response to gastric distension, the parasympathetic gastrocolonic reflex, was absent in patients with DM reporting severe constipation (Battle et al., 1980, Battle et al., 1983). Diabetic diarrhea has been associated with autonomic neuropathy (Ellenberg, 1964, McNally et al., 1969, Whalen et al., 1969, Scarpello et al., 1976) with predominantly reports of slowed intestinal transit (Hodges et al., 1947, Whalen et al., 1969, Scarpello et al., 1976, Iber et al., 1993), but occasionally reports of accelerated intestinal transit (Muri, 1953, Vinnik et al., 1962). Hence, inappropriate postprandial intestinal motility may be the consequence of inadequate activation of extrinsic parasympathetic reflex pathways by disordered gastric motility (Samsom et al., 1995, Samsom et al., 1996, Samsom et al., 1998, Troncon et al., 1998, Rosztoczy et al., 2004, Kumar et al., 2008) or by abnormal gastric emptying (Maggs et al., 2008, Sarosiek et al., 2010), and may result in inappropriate gastric emptying through an ineffective ileal brake reflex as observed in streptozotocin (STZ) rats (Martin et al., 2004).

Some agents used to treat slowed GI transit in patients increase release of endogenous acetylcholine (Ach) from autonomic neurons (Rayner and Horowitz, 2005, Park and Camilleri, 2006, Hasler, 2007, Patrick and Epstein, 2008, Sanger and Alpers, 2008). However, in patients with dysfunctional enteric motor pathways due to neuropathy, agents that modulate the activity of enteric neural pathways may not effectively normalize GI transit (Gershon, 2004). Pathology of enteric neurons has been documented in some DM patients with diarrhea (Bennett et al., 1956, Vinnik et al., 1962, Ellenberg, 1964, Whalen et al., 1969, Yoshida et al., 1988) or gastric dysfunction (He et al., 2001, Pasricha et al., 2008, Harberson et al., 2010, Grover et al., 2011) suggesting altered activity of intrinsic enteric pathways. Since, Ach released from both extrinsic and intrinsic autonomic neurons can activate both enteric excitatory and inhibitory pathways to generate smooth muscle pressure gradients required for normal GI transit, we hypothesized that despite documented autonomic neuropathy of the vagus nerve (Robertson and Sima, 1980, Kniel et al., 1986, Regalia et al., 2002) and enteric neurons (Chandrasekharan and Srinivasan, 2007) as well as smooth muscle myopathy (Ordog, 2008) in animal models of DM, nonselective cholinergic activation of enteric excitatory and inhibitory pathways by Ach using neostigmine, an inhibitor of acetylcholinesterase (AchE), would normalize intestinal motility. Contributions of excitatory and inhibitory enteric neural pathways to neostigmine-induced contractions were evaluated and compared by assessing neuronal Ach content, direct smooth muscle contraction and relaxation to cholinergic agonists and nitric oxide (NO), and participation of enteric nicotinic and muscarinic receptors using isolated ileum from control and diabetic animals.

In animal models of DM, GI dysfunction may result from either a low concentration of effective plasma insulin, abnormal plasma c-peptide concentration, or elevated plasma glucose. This study uses a STZ-induced model of type 1 DM in guinea pigs to capitalize on the wealth of information on ileal enteric neurochemical coding and motor circuits published for guinea pig (Furness and Costa, 1987, Costa et al., 1996, Brookes, 2001, Bornstein et al., 2004). Similar to rodent models, insulin and c-peptide were very low in STZ guinea pigs (Junod et al., 1969, Howell et al., 1971, Lundquist et al., 1975, Gorray et al., 1986). However, hyperglycemia and ketosis were milder in STZ guinea pigs (Peterssen et al., 1970, Elliott and Pogson, 1977, Johnson, 1978, Schlosser et al., 1984, Schlosser et al., 1987, Gorray et al., 1986, Hootman et al., 1998) as compared to STZ rats (Junod et al., 1969, Hawkins et al., 1986). Hence, to focus on the contribution of insulin deficiency to autonomic neuropathy and motor dysfunction, the effect of neostigmine on ileal motility was determined in STZ-treated guinea pigs.

Section snippets

Induction of diabetes by streptozotocin

Diabetes was induced in male Hartley guinea pigs (n = 11, 200–300 g, Charles River Laboratories, Portage, MI) by a single intraperitoneal (ip) injection of STZ (pancreatic beta cell toxin, 50 mg/mL, 280 mg/kg, Sigma, St. Louis, MO) dissolved in citrate buffer (0.05 M, pH 4.5) (Schlosser et al., 1984). Control animals (n = 11) were given a single ip injection of citrate buffer. Animals were euthanized by CO2 narcosis 5–6 weeks after injection. Urine was tested for glucose using uristix (Siemens

Animal model

At the time of the experiment 5–6 weeks after STZ injection, diabetic animals weighed less than control animals [g: control (n = 11), 570 ± 17; diabetic (n = 11), 309 ± 19*; *p < 0.05]. Diabetic animals had a higher kidney weight to body weight ratio [g/g × 10 3: control (n = 11), 3.75 ± 0.07; diabetic (n = 11), 6.02 ± 0.31*; *p < 0.05]. At the time of sacrifice, glucose was detected in the urine of most diabetic (8/10) but no control (0/11) guinea pigs.

Basal motility

Ilea from control and diabetic animals had similar frequencies

Discussion

In these studies, neostigmine, an AchE inhibitor that increases endogenous Ach levels, was used to mimic the general mechanism of action of many prokinetic drugs. In the presence of neostigmine, sufficient endogenous Ach accumulated at synaptic junctions to activate cholinergic receptors producing strong contraction of ilea. Despite neuropathy of autonomic neurons (LePard, 2005), neostigmine-induced contractions of diabetic ileum were stronger than control ileum (Figs. 2 and 3). Many mechanisms

Acknowledgments

This study was supported by R21 NS039768 and R15 NS047106 to KJL and by ORSP at Midwestern University.

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