Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.
Ciliary muscle is a smooth muscle characterized by a rapid response to muscarinic receptor stimulation and sustained contraction. Although it is evident that these contractions are Ca2+-dependent, detailed molecular mechanisms are still unknown. In order to elucidate the role of Ser/Thr protein phosphatase 2A (PP2A) in ciliary muscle contraction, we examined the effects of okadaic acid and other PP2A inhibitors on contractions induced by carbachol (CCh) and ionomycin in bovine ciliary muscle strips (BCM). Okadaic acid inhibited ionomycin-induced contraction, while it did not cause significant changes in CCh-induced contraction. Fostriecin showed similar inhibitory effects on the contraction of BCM. On the other hand, rubratoxin A inhibited both ionomycin- and CCh-induced contractions. These results indicated that PP2A was involved at least in ionomycin-induced Ca2+-dependent contraction, and that BCM had a unique regulatory mechanism in CCh-induced contraction.
Type 2 diabetic men commonly experience erectile dysfunction for which phosphodiesterase-5 (PDE5) inhibitors like sildenafil (Viagra) are often recommended. By preventing degradation of cyclic guanosine monophosphate (cGMP) in vascular smooth muscle, these inhibitors also enhance arterial vasorelaxant effects of nitric oxide donors (which stimulate cGMP synthesis). In the present work, we confirmed this enhancing effect after co-administration of sildenafil with nitroprusside to freshly-isolated rat tail arterial tissues. However, in the same tissues we also observed that sildenafil does not enhance but rather attenuates vasorelaxant effects of three commonly-used antidiabetic drugs, i.e. the biguanide metformin and the thiazolidinediones pioglitazone and rosiglitazone. Indeed, sildenafil completely blocked vasorelaxant effects of low concentrations of these drugs. In addition, we found that this same novel anti-vasorelaxant interaction of sildenafil with these agents was abolished by either 1) omitting extracellular glucose or 2) inhibiting specific smooth muscle glycolytic pathways; pathways known to preferentially utilize extracellular glucose to fuel certain adenosine triphosphate (ATP)-dependent ion transporters: e.g. ATP-sensitive K channels, sarcoplasmic reticulum Ca-ATPase, plasma membrane Ca-ATPase and Na/K-ATPase. Accordingly, we suspect that altered activity of one or more of these ion transporters mediates the observed attenuating (anti-vasorelaxant) interaction of sildenafil with the antidiabetic drugs. The present results are relevant because hypertension is so common and difficult to control in Type 2 diabetes. The present data suggest that sildenafil might interfere with the known antihypertensive potential of metformin and the thiazolidinediones. However, they do not suggest that it will interact with them to cause life-threatening episodes of severe hypotension, as can occur when it is co-administered with nitrates.
Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of β-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and β-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications.
Aim: Inflammatory bowel disease is characterized by the presence of gastrointestinal motility disturbances; however alterations in the gastric myoelectrical activity have not been characterized. In this study we have recorded the gastric myoelectrical activity in patients with ulcerative colitis (UC) and Crohn’s disease (CD) during their clinical remission. Materials and Methods: Gastric activity was assessed using electrogastrography (EGG) in patients with UC (n = 60), CD (n = 40) and healthy controls (n = 40). In each case, their response to water load test, as well as the dominant frequency (DF), dominant power (DP) and the power ratio (PR) of the electrical activity were recorded. Results: In healthy controls, the resting DF was 2.57 ± 1.05 cycles per minute (cpm), which decreased after water ingestion (2.34 ± 0.99 cpm; P = 0.001). Compared to healthy controls, patients with UC had low resting DF (bradygastria) (2.57 ± 1.05 vs. 1.86 ± 1.28 cpm; P = 0.01). The change in DF after water ingestion was insignificant in patients with UC and CD. Post-water ingestion, healthy controls exhibited an increase in the DP as compared to the resting state, (7.1 [2.93, 102.56] vs. 15.94 [3.92, 133.41] μV 2; P = 0.02). Patients with UC (1.26 [0.14, 9.83] vs. 3.27 [0.61, 42.12] μV2) and CD (2.54 [0.44, 47.06] vs. 15.8 [0.1, 126.68] μV2) also showed a significant increase in the DP post-water ingestion. Conclusions: Patients with ulcerative colitis have altered resting gastric myoelectrical activity during the remission phase of the disease.
Uridine triphosphate (UTP) can be released from damaged cells to cause vasoconstriction. Although UTP is known to act through P2Y receptors and PLC activation in vascular smooth muscle, the role of PKC in generating the response is somewhat unclear. Here we have used Tat-linked membrane permeable peptide inhibitors of PKC to assess the general role of PKC and also of specific isoforms of PKC in the UTP induced contraction of rat mesenteric artery. We examined the effect of PKC inhibition on UTP induced contraction, increased cytoplasmic Ca2+ and reduction of K+ currents and found that PKC inhibition caused a relatively small attenuation of contraction but had little effect on changes in cytoplasmic Ca2+. UTP attenuation of both voltage-gated (Kv) and ATP-dependent (KATP) K+ currents was abolished when intracellular Ca2+ was decreased from 100 to 20 nM. PKC inhibition reduced slightly the ability of UTP to attenuate Kv currents but had no effect on KATP current inhibition. In conclusion, both UTP induced contraction of mesenteric artery and the inhibition of Kv and KATP currents of mesenteric artery smooth muscle cells by UTP are relatively independent of PKC activation; furthermore, the inhibition of both Kv and KATP currents requires intracellular Ca2+.
Studies that evaluate the mechanisms for increased airway responsiveness are very sparse, although there are reports of exercise-induced bronchospasm. Therefore, we have evaluated the tracheal reactivity and the rate of lipid peroxidation after different intensities of swimming exercise in rats. Thus, male Wistar rats (age 8 weeks; 250–300 g) underwent a forced swimming exercise for 1h whilst carrying attached loads of 3, 4, 5, 6 and 8% of their body weight (groups G3, G4, G5, G6 and G8, respectively; n=5 each). Immediately after the test, the trachea of each rat was removed and suspended in an organ bath to evaluate contractile and relaxant responses. The rate of lipid peroxidation was estimated by measuring malondialdehyde levels. According to a one-way ANOVA, all trained groups showed a significant decrease in the relaxation induced by aminophylline (10–12–10–1 M) (pD2=3.1, 3.2, 3.3, 3.3 and 3.2, respectively for G3, G4, G5, G6 and G8) compared to the control group (pD2=4.6) and the Emax values of G5, G6, G8 groups were reduced by 94.2, 88.0 and 77.0%, respectively. Additionally, all trained groups showed a significant increase in contraction induced by carbachol (10–9–10 –3 M) (pD2=6.0, 6.5, 6.5, 7.2 and 7.3, respectively for G3, G4, G5, G6 and G8) compared to the control group (pD2=5.7). Lipid peroxidation levels of G3, G4 and G5 were similar in both the trachea and lung, however G6 and G8 presented an increased peroxidation in the trachea. In conclusion, a single bout of swimming exercise acutely altered tracheal responsiveness in an intensity-related manner and the elevation in lipid peroxidation indicates a degree of oxidative stress involvement.
Two-photon microscopy (2PM) can enable high-resolution deep imaging of thick tissue by exciting a fluorescent dye and protein at anastomotic sites in the mouse small intestine in vivo. We performed gut surgery and transplanted neural stem cells (NSC) from the embryonic central nervous system after marking them with the fluorescent cell linker, PKH26. We found that neurons differentiated from transplanted NSC (PKH [+]) and newborn enteric neurons differentiated from mobilized (host) NSC (YFP [+]) could be localized within the granulation tissue of anastomoses. A 5-HT4-receptor agonist, mosapride citrate (MOS), significantly increased the number of PKH (+) and YFP (+) neurons by 2.5-fold (P<0.005). The distribution patterns of PKH (+) neurons were similar to those of YFP (+) neurons. On the other hand, the 5-HT4-receptor antagonist, SB-207266 abolished these effects of MOS. These results indicate that neurogenesis from transplanted NSC is facilitated by activation of 5-HT4-receptors. Thus, a combination of drug administration and cell transplantation could be more beneficial than exclusive cell transplantation in treating Hirschsprung’s disease and related disorders including post rectal cancer surgery. The underlying mechanisms for its action were explored using immunohistochemistry of the longitudinal mouse ileum and rat rectal preparations including an anastomosis. MOS significantly increased the number of new neurons, but not when co-administered with either of a protein tyrosine kinase receptor, c-RET two inhibitors. The c-RET signaling pathway contributes to enteric neurogenesis facilitated by MOS. In the future, we would perform functional studies of new neurons over the thick granulation tissue at anastomoses, using in vivo imaging with 2PM and double transgenic mice expressing a calcium indicator such as GCaMP6 and channelrhodopsin.