Background Brain-derived neurotrophic factor (BDNF) is a neurotrophin present in the intestine where it participates in survival and growth of enteric neurons, augmentation of enteric circuits, and stimulation of intestinal peristalsis and propulsion. Previous studies largely focused on the role of neural and mucosal BDNF. The expression and release of BDNF from intestinal smooth muscle and the interaction with enteric neuropeptides has not been studied in gut. Methods The expression and secretion of BDNF from smooth muscle cultured from rabbit longitudinal intestinal muscle in response to substance P and pituitary adenylate cyclase activating peptide (PACAP) was measured by western blot and ELISA. BDNF mRNA was measured by rt-PCR. Key Results The expression of BNDF protein and mRNA was greater in smooth muscle cells from the longitudinal muscle than from circular muscle layer. PACAP and substance P increased the expression of BDNF protein and mRNA in cultured longitudinal smooth muscle cells. PACAP and substance P also stimulated the secretion of BDNF from cultured longitudinal smooth muscle cells. Chelation of intracellular calcium with BAPTA prevented substance P-induced increase in BDNF mRNA and protein expression as well as substance P-induced secretion of BDNF. Conclusions & Inferences Neuropeptides known to be present in enteric neurons innervating the longitudinal layer increase the expression of BDNF mRNA and protein in smooth muscle cells and stimulate the release of BDNF. Considering the ability of BDNF to enhance smooth muscle contraction, this autocrine loop may partially explain the characteristic hypercontractility of longitudinal muscle in inflammatory bowel disease.

Recent studies have identified AMP-activated kinase (AMPK) as a target of Ca 21/calmodulin-dependent kinase kinase (CaMKKb) and a negative regulator of myosin light-chain (MLC) kinase (MLCK). The present study examined whether a change in expression or activity of AMPK is responsible for hypercontractility of intestinal longitudinal muscle during inflammation or in response to proinflammatory cytokines. In mouse colonic longitudinal muscle cells, acetylcholine (ACh) stimulated AMPK and MLCK phosphorylation and activity and induced MLC 20 phosphorylation and muscle contraction. Blockade of CaMKKb with STO609 (7-oxo-7H-benzimidazo[2,1-a]benz [de]isoquinoline-3-carboxylic acid acetate) inhibited AMPK and MLCK phosphorylation and augmented MLCK activity, MLC 20 phosphorylation, and smooth muscle cell contraction. In muscle cells isolated from the colon of TNBS (2,4,6-trinitrobenzenesulfonic acid)-treated mice or from strips treated with interleukin-1b or tumor necrosis factor-a, nuclear factor kB was activated as indicated by an increase in p65 phosphorylation and IkBa degradation, and AMPK was phosphorylated at a cAMP-dependent protein kinase (PKA)-specific site (Ser 485 ) that is distinct from the stimulatory CaMKKb site (Thr 172 ), resulting in attenuation of ACh-stimulated AMPK activity and augmentation of MLCK activity and muscle cell contraction. Inhibition of nuclear factor-kB activity with MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal Z-LLL-CHO) or PKA activity with myristoylated PKA inhibitor 14-22 amide blocked phosphorylation of AMPK at Ser 485 and restored MLCK activity and muscle cell contraction to control levels. The results imply that PKA released from IkBa complex phosphorylated AMPK at a PKA-specific site and inhibited its activity, thereby relieving the inhibitory effect of AMPK on MLCK and increasing MLCK activity and muscle cell contraction. We conclude that hypercontractility of intestinal longitudinal muscle induced by inflammation or proinflammatory cytokines is mediated by nuclear factor kB/PKA-dependent inhibition of AMPK and activation of MLCK.

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and β nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-β-erythroidine, a α4β2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and β4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.

Thin filament associated proteins such as calponin, caldesmon, tropomyosin and smoothelin are thought to regulate acto-myosin interaction and thus, muscle contraction. However, the effect of inflammation on the expression of thin filament-associated proteins is not known. The aim of the present study is to determine the changes in the expression of calponin, caldesmon, tropomyosin, and smoothelin in colonic smooth muscle from TNBS- and DSS-induced colitis in mice. Expression of h-caldesmon, h2-calponin, α-tropomyosin and smoothelin-A was measured by qRT-PCR, and western blot. Contraction in response to acetylcholine in dispersed muscle cells was measured by scanning micrometry. mRNA and protein expression of α-actin, h2-calponin, h-caldesmon, smoothelin and α-tropomyosin in colonic muscle strips from mice with TNBS- or DSS-induced colitis was significantly increased compared to control animals. Contraction in response to acetylcholine was significantly decreased in muscle cells isolated from inflamed regions of TNBS- or DSS-treated mice compared to control mice. Our results show that increase in the expression of thin-filament associated contractile proteins, which inhibit acto-myosin interaction, could contribute to decrease in smooth muscle contraction in inflammation.

Introduction: To identify whether there is an association between maternal socioeconomic status as in living style, and preeclampsia in women from Pakistan, and if so, to what extent it increases the risk of developing preeclampsia. Methods: This is a case-control study, consisting of 74 normal pregnant and 76 already diagnosed preeclamptic Pakistani females, recruited from various hospitals in Karachi. Their socioeconomic status was assessed through the standard of living index (SLI) using the National Family Health Surveys (NFHS) criteria, which considers different living facilities as an indicator of socioeconomic status. Odds ratios of preeclampsia were calculated for low, middle and high socioeconomic status and other maternal anthropometric parameters. Results: Based on their groups, it appeared that the highest percentage of preeclamptic females (n= 65; 43.3%) and the lowest of control patients (n=14; 9.3%) lie in lower socioeconomic groups and the results were highly significantly different. Study observed that having lower socioeconomic status (P<0.001) significantly increase risk of having preeclampsia by 9% and middle socioeconomic status has no role in prevalence of preeclampsia shown by 0.0 odds ratio. In addition, odds ratio of age and PreBMI for the preeclamptic females were found as 1.37 (P<0.001) and 1.55 (P<0.001) respectively, which shows that each year increase in age, increases risk of preeclampsia by 1.37 times and 1.55 times with each increased in BMI. Conclusion: Living facilities are indications of socioeconomic status and are strong risk factors of preeclampsia