Previous studies have focused on gastric dysmotility and delayed emptying in diabetes mellitus. There is little information about the effects of hyperglycemia on colonic motility and secretion. 5-HT was reported to mediate contractile activity by activating receptors on both enteric neurons and smooth muscle cells. The aim of this study was to investigate and compare the effects of 5-HT on circular contractile activity coordinated with secretion in streptozocin-induced diabetic and non-diabetic rats. Sonomicrometry and voltage clamping techniques were used to measure motility and secretion simultaneously in isolated whole thickness colonic sheets. Male Sprague Dawley rats were injected with streptozocin (65 mg/kg body weight) in 0.1 M sodium citrate buffer, into the tail veins. Glucose levels of 300-400 mg/dl and above were achieved. The control rats were injected with the same volumes of vehicle (0.1 M sodium citrate buffer). Animals were sacrificed 10-12 days following the induction of hyperglycemia. Flat sheets of colon were mounted serosal side up in Ussing chambers. 1 mm piezocrystals were glued to the serosal surface 4-5 mm apart to measure circular contractions as decrease in inter-crystal distances (ICD). Voltage-clamping the tissues at 0 mV was used ix to measure short circuit current (Isc), indicative of chloride secretion. In diabetic rats 50 gM 5-HT caused mean amplitude of contractions of 174 ± 26 gm (n=4), which was significantly reduced as compared to the response in non-diabetic rats of 970 + 243 gm (n=4; p<0.05). The secretory response in diabetic rats paralleled the reduction in ICD (diabetic: 23 +1 gA/cm2, controls: 57 + 18 gA/cm2). Neural blockade with 0.1 gM TTX revealed a decreased myogenic contractile activity in diabetic rats. The mean amplitude of contractions after TTX in diabetic rats was 162 ± 45 gm verses controls of 612 ± 86 gm. These results suggest that the reduction of the 5-HT contractile response in diabetic rats may be a composite of direct effects on the smooth muscle as well as indirect effects on the neurons. / Department of Physiology and Health Science

Indiana University-Purdue University Indianapolis (IUPUI) / Intestinal smooth muscle development involves complex transcriptional regulation leading to cell differentiation of the circular, longitudinal and muscularis mucosae layers. Differentiated intestinal smooth muscle cells express high levels of smooth muscle-specific contractile and regulatory proteins, including telokin. Telokin is regulatory protein that is highly expressed in visceral smooth muscle. Analysis of cis-elements required for transcriptional regulation of the telokin promoter by using hypoxanthine-guanine phosphoribosyltransferase (Hprt)-targeted reporter transgenes revealed that a 10 base pair large CC(AT)₆GG ciselement, called CArG box is required for promoter activity in all tissues. We also determined that an additional 100 base pair region is necessary for transgene activity in intestinal smooth muscle cells. To examine how transcriptional regulation of intestinal smooth muscle may be altered under pathological conditions we examined the effects of diabetes on colonic smooth muscle. Approximately 76% of diabetic patients develop gastrointestinal (GI) symptoms such as constipation due to intestinal dysmotility. Mice were treated with low-dose streptozotocin to induce a type 1 diabetes-like hyperglycemia. CT scans revealed decreased overall GI tract motility after 7 weeks of hyperglycemia. Acute (1 week) and chronic (7 weeks) diabetic mice also had decreased potassium chloride (KCl)-induced colon smooth muscle contractility. We hypothesized that decreased smooth muscle contractility at least in part, was due to alteration of contractile protein gene expression. However, diabetic mice showed no changes in mRNA or protein levels of smooth muscle contractile proteins. We determined that the decreased colonic contractility was associated with an attenuated intracellular calcium increase, as measured by ratio-metric imaging of Fura-2 fluorescence in isolated colonic smooth muscle strips. This attenuated calcium increase resulted in decreased myosin light chain phosphorylation, thus explaining the decreased contractility of the colon. Chronic diabetes was also associated with increased basal calcium levels. Western blotting and quantitative real time polymerase chain reaction (qRT-PCR) analysis revealed significant changes in calcium handling proteins in chronic diabetes that were not seen in the acute state.These changes most likely reflect compensatory mechanisms activated by the initial impaired calcium response. Overall my results suggest that type 1 diabetes in mice leads to decreased colon motility in part due to altered calcium handling without altering contractile protein expression.