In response to ingestion of nutrients enteroendocrine L cells secrete a potent incretin hormone, glucagon‐like peptide‐1 (GLP‐1) to enhance glucose‐dependent release of insulin and regulate glucose metabolism. Therapies related to GLP‐1 (e.g., exendin‐4) are currently approved for type 2 diabetes. The GLP‐1 receptor (GLP‐1R) is expressed in cells of the gastrointestinal tract (enteric neurons, enterocytes) and elsewhere (pancreatic b cells, heart, and vascular smooth muscle). In pancreatic b cells, GLP‐1R are coupled to stimulatory G protein, Gs, generation of cAMP and activation of cAMP‐dependent protein kinase. Although a decrease in the motility of stomach and colon are commonly associated with diabetes, the expression and function of GLP‐1R in gastrointestinal smooth muscle are not known. Aim To test the hypothesis that GLP‐1 regulates smooth muscle function by acting on GLP‐1R expressed on smooth muscle. Methods Intestine and colon were removed from mice, and smooth muscle cells isolated by collagenase digestion, and cultured in DMEM‐10. Expression of GLP‐1R mRNA was measured by RT‐PCR using specific primers and the expression of GLP‐1R protein was measured by western blot using GLP‐1R specific antibody (sc‐66911 and 1:200 dilution). The effect of GLP‐1 on basal activity and on acetylcholine (ACh)‐induced contraction was measured in muscle strips and intact colon in organ bath experiments. Results Amplification of GLP‐1R mRNA was obtained in mRNA isolated from mucosa derived from colon and intestine and also in mRNA obtained from tissue preparations devoid of mucosa suggesting expression of GLP‐1R mRNA in mucosal as well as non‐mucosal cells (enteric neurons or muscle cells) of the colon and intestine. The specificity of GLP‐1R primers was corroborated using mRNA from a pancreatic b cell line (MIN‐6) shown to express GLP‐1R in our previous studies. A similar pattern of GLP‐1R protein expression was obtained with western blot. To identify the GLP‐1R expression specifically in muscle cells devoid of other cells such as enteric neurons, expression was examined in cultured smooth muscle. Expression of GLP‐1R mRNA was confirmed in these pure smooth muscle cell cultures of colon and intestine. The role of GLP‐1R in the regulation of smooth muscle function was analyzed in organ bath studies. The addition of GLP‐1 (1 nM‐1 mM) caused dose‐dependent relaxation of basal tone in colonic muscle strips and relaxation of acetylcholine (ACh, 1 mM)‐induced phasic activity and tonic contractions in both muscle strips and intact colon. The effect of GLP‐1 on ACh‐induced contraction suggests a role of smooth muscle Gs‐coupled GLP‐1R in mediating relaxation. Conclusion Colonic smooth muscle cells express GLP‐1R and GLP‐1 inhibits both basal and acetylcholine‐induced contraction. Studies are underway to identify the mechanism of action of GLP‐1 to inhibit colonic muscle function. Support or Funding Information Supported by grants DK15564, DK28300 and DK34153.
Short-chain fatty acids (SCFAs) accelerate colonic transit. This study examined whether this action was mediated by activation of the peristaltic reflex. SCFAs (acetate, butyrate, or propionate) were applied to the central compartment of a three-compartment flat-sheet preparation of the rat middle to distal colon. The release of serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and CGRP was measured in all three compartments. Ascending contraction and descending relaxation were measured in the orad and caudad compartments. The addition of SCFAs at physiological to supraphysiological concentrations (0.5-100 mM) to the central compartment elicited concentration-dependent ascending contraction and descending relaxation (EC50 approximately 5 mM). At this concentration, SCFAs induced an 8- to 11-fold increase in 5-HT release and a 2- to 3-fold increase in CGRP release in the central compartment only. They had no effect on BDNF release. CGRP release was inhibited by a 5-HT4 but not a 5-HT3 receptor antagonist. Ascending contraction and descending relaxation were also inhibited by 5-HT4 and by CGRP receptor antagonists added to the central compartment. 5-HT and CGRP release, as well as ascending contraction and descending relaxation induced by mechanical stimulation of the mucosa (2-8 strokes), were significantly augmented by 1 mM acetate. Acetate (1 mM) also doubled propulsive velocity in isolated whole segments of the guinea pig colon. In conclusion, chemical stimulation of the mucosa by SCFAs triggers a peristaltic reflex mediated by the release of 5-HT from mucosal cells and activation of 5-HT4 receptors on sensory CGRP-containing nerve terminals. This SCFA-induced peristaltic pathway augments the peristaltic reflex elicited by mechanical stimulation of the mucosa.
In gastrointestinal (GI) smooth muscle, inhibitory transmitters (e.g., vasoactive intestinal peptide (VIP)) that are coupled to activation of G s protein mediate relaxation via generation of cAMP and activation of cAMP‐dependent protein kinase (PKA). Cyclic AMP levels are regulated by the activities of adenylyl cyclase (AC) and phosphodiesterases (PDEs). Previous studies in gastrointestinal muscle showed selective expression of ACV/VI and cAMP‐specific PDE4D5. Diabetes‐associated GI motility disorders are common and are correlated with the decrease in neuronal nitric oxide synthase expression and activity, and density of interstitial cells of Cajal. Changes in the smooth muscle AC/cAMP/PKA pathway leading to decrease in relaxation could also contribute to decreased motility. However, diabetes‐induced changes in the expression and activities of ACV/VI and PDE4D5 in the smooth muscle are not known. Aim To test the hypothesis that downregulation of ACV/VI and upregulation of PDE4D5 expression in smooth muscle contribute to decreased relaxation and motility in diabetes. Methods Gastric and colonic smooth muscle from control and diabetic (db/db and ob/ob) mice were used to measure the expression of ACV/VI and PDE4D5, cAMP levels, and muscle relaxation. Expression of ACV/VI and PDE4D5 was measured by western blot. cAMP levels in response to forskolin, VIP or isoproterenol were measured by radioimmunoassay. Muscle relaxation in response to forskolin, VIP or isoproterenol was measured in muscle strips (organ bath studies) and isolated muscle cells (scanning micrometry). Gastric emptying and colonic pellet propulsion were measured as indices of gastrointestinal motility. Results Smooth muscle isolated from the stomach and the colon of db/db and ob/ob mice showed a decrease in ACV/VI expression and an increase in PDE4D5 expression compared to the stomach and colon of control mice. Similar changes in the expression of ACV/VI and PDE4D5 were obtained in control muscle treated with 30 mM glucose for 48 h. Forskolin‐induced cAMP levels, measured in the presence or absence of 3‐isobutyl‐1‐methylxanthine, were significantly reduced in gastric and colonic smooth muscle of db/db and ob/ob mice compared to control. A similar decrease in cAMP levels in diabetic mice was obtained with Gs‐coupled receptor agonists, VIP, and isoproterenol. Forskolin‐induced cAMP levels were also decreased in control muscle treated with 30 mM glucose. Consistent with the decrease in cAMP levels, muscle relaxation in response to forskolin, VIP or isoproterenol was decreased in gastric and colonic smooth muscle of db/db and ob/ob mice compared to control. Both gastric emptying and the velocity of propulsion of fecal pellets in colonic segments were decreased in diabetic mice suggesting a decrease in gastrointestinal motility in diabetes. Conclusion In diabetic stomach and colon, downregulation of ACV/VI expression and upregulation of PDE4D5 expression in smooth muscle causes a decrease in cAMP levels and muscle relaxation leading to a decrease in gastrointestinal motility. Support or Funding Information R01DK28300
Gastrointestinal peristalsis is significantly dependent on the enteric nervous system. Constipation due to reduced peristalsis is a major side-effect of morphine, which limits the chronic usefulness of this excellent pain reliever in man. The ionic basis for the inhibition of enteric neuron excitability by morphine is not well characterized as previous studies have mainly utilized microelectrode recordings from whole mount myenteric plexus preparations in guinea pigs. Here we have developed a Swiss-Webster mouse myenteric neuron culture and examined their electrophysiological properties by patch-clamp techniques and determined the mechanism for morphine-induced decrease in neuronal excitability. Isolated neurons in culture were confirmed by immunostaining with pan-neuronal marker, β-III tubulin and two populations were identified by calbindin and calretinin staining. Distinct neuronal populations were further identified based on the presence and absence of an afterhyperpolarization (AHP). Cells with AHP expressed greater density of sodium currents. Morphine (3 µM) significantly reduced the amplitude of the action potential, increased the threshold for spike generation but did not alter the resting membrane potential. The decrease in excitability resulted from inhibition of sodium currents. In the presence of morphine, the steady-state voltage dependence of Na channels was shifted to the left with almost 50% of channels unavailable for activation from hyperpolarized potentials. During prolonged exposure to morphine (two hours), action potentials recovered, indicative of the development of tolerance in single enteric neurons. These results demonstrate the feasibility of isolating mouse myenteric neurons and establish sodium channel inhibition as a mechanism for morphine-induced decrease in neuronal excitability.
Abnormalities in extrinsic and intrinsic innervation and in the distribution of interstitial cells of Cajal are known to contribute to altered gut motility in diabetes. Smooth muscle dysfunction in relation to diabetes has not been fully explored. Aim. To test the hypothesis that upregulation of RhoA/Rho kinase pathway and downregulation of cGMP/PKG pathway contribute to increased muscle tone in diabetes. Methods. Colonic smooth muscle cells from type 1 (NOD) or type 2 diabetes (db/db) mice were used to measure i) RhoA and PDE5 expression; 2) ACh‐induced Rho kinase activity and muscle contraction; 3) NO‐induced PDE5 activity, cGMP levels and muscle relaxation. Colonic segments were used to measure peristaltic reflex and pellet propulsion. Results. Smooth muscle cells isolated from the colon of NOD and db/db mice showed an increase in RhoA and PDE5 expression, Rho kinase and PDE5 activity, and muscle contraction, and a decrease in cGMP levels and muscle relaxation relative to control mice. Consistent with an increase in RhoA and PDE5 expression, orad contraction was increased by 20%, caudad relaxation decreased by 60%, and pellet propulsion decreased by 35% in db/db mice relative to control. Discussion. In diabetic colon upregulation of the RhoA/Rho kinase pathway and downregulation of the cGMP/PKG pathway causes an increase in muscle contraction and decrease in relaxation leading to a decrease in pellet propulsion.
