Project description:The catalytic subunit gp91phox (Nox2) of the NADPH oxidase of mammalian phagocytes is activated by microbes and immune mediators to produce large amounts of reactive oxygen species (ROS) which participate in microbial killing. Homologs of gp91phox, the Nox and Duox enzymes, were recently described in a range of organisms, including plants, vertebrates, and invertebrates such as Drosophila melanogaster. While their enzymology and cell biology are being extensively studied in many laboratories, little is known about in vivo functions of Noxes. Here, we establish and use an inducible system for RNAi to discover functions of dNox, an ortholog of human Nox5 in Drosophila. We report here that depletion of dNox in musculature causes retention of mature eggs within ovaries, leading to female sterility. In dNox-depleted ovaries and ovaries treated with a Nox inhibitor, muscular contractions induced by the neuropeptide proctolin are markedly inhibited. This functional defect results from a requirement for dNox-for the proctolin-induced calcium flux in Drosophila ovaries. Thus, these studies demonstrate a novel biological role for Nox-generated ROS in mediating agonist-induced calcium flux and smooth muscle contraction.
Project description:Asthma is a chronic respiratory condition characterized by airway inflammation, remodeling, and hyperresponsiveness to triggers causing airway constriction. Bronchial smooth muscle plays a critical role by narrowing airways, leading to obstruction and breathing difficulties, often exacerbated by mast cell infiltration and histamine release. Whereas current treatments, including bronchodilators, corticosteroids, and biologics provide effective management for most patients, alternative therapies are needed for difficult-to-treat asthma. Recent research highlights the potential of therapeutic ketosis, achieved through dietary interventions or supplementation with exogenous ketones, to reduce airway hyperresponsiveness and inflammation. Ketone bodies, known for providing energy during carbohydrate scarcity, also influence asthma by activating cell-surface receptors and transporters. In vivo, interventions like weight loss and caloric restriction increase ketone body levels, correlating with improved asthma symptoms, reduced oxidative stress, and inflammation. These effects suggest ketone bodies, particularly β-hydroxybutyrate, may play a therapeutic role in mitigating bronchoconstriction and smooth muscle contraction in asthma. We utilize human bronchial smooth muscle cells (in vitro) and mouse precision-cut lung slices (PCLS) (ex vivo) to assess the effects of BHB on histamine-induced bronchoconstriction. Brightfield microscopy showed that BHB reduces contraction in human bronchial smooth muscle cells, an effect involving free fatty acid receptor 3 (FFAR3) activation. Light microscopy of PCLS revealed that BHB inhibits airway narrowing and cellular extrusion, demonstrating its ability to mitigate bronchoconstriction by suppressing smooth muscle contraction. These results implicate bronchial smooth muscle as a cellular target of therapeutic ketosis, an important contributor to the beneficial effects of BHB in preclinical models of asthma.
Project description:Both smooth muscle (SM) and non-muscle (NM) myosin II are expressed in hollow organs such as the bladder and uterus, but their respective roles in contraction and corresponding physiological functions remain to be determined. In this report, we assessed their roles by analyzing mice deficient of Myl9, a gene encoding the SM myosin regulatory light chain (SM RLC). We find that global Myl9-deficient bladders contracted with an apparent sustained phase, despite no initial phase. This sustained contraction was mediated by NM myosin RLC (NM RLC) phosphorylation by myosin light chain kinase (MLCK). NM myosin II was expressed abundantly in the uterus and young mice bladders, of which the force was accordingly sensitive to NM myosin inhibition. Our findings reveal distinct roles of SM RLC and NM RLC in SM contraction.
Project description:The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca(2+)-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.
Project description:The smooth muscle cell directly drives the contraction of the vascular wall and hence regulates the size of the blood vessel lumen. We review here the current understanding of the molecular mechanisms by which agonists, therapeutics, and diseases regulate contractility of the vascular smooth muscle cell and we place this within the context of whole body function. We also discuss the implications for personalized medicine and highlight specific potential target molecules that may provide opportunities for the future development of new therapeutics to regulate vascular function.
Project description:The ceca play an important role in the physiology of the gastrointestinal tract in chickens. Nevertheless, there is a gap of knowledge regarding the functionality of the ceca in poultry, especially with respect to physiological cecal smooth muscle contraction. The aim of the current study is the ex vivo characterization of cecal smooth muscle contraction in laying hens. Muscle strips of circular cecal smooth muscle from eleven hens are prepared to investigate their contraction ex vivo. Contraction is detected using an isometric force transducer, determining its frequency, height and intensity. Spontaneous contraction of the chicken cecal smooth muscle and the influence of buffers (calcium-free buffer and potassium-enriched buffer) and drugs (carbachol, nitroprusside, isoprenaline and Verapamil) affecting smooth muscle contraction at different levels are characterized. A decrease in smooth muscle contraction is observed when a calcium-free buffer is used. Carbachol causes an increase in smooth muscle contraction, whereas atropine inhibits contraction. Nitroprusside, isoprenaline and Verapamil result in a depression of smooth muscle contraction. In conclusion, the present results confirm a similar contraction behavior of cecal smooth muscles in laying hens as shown previously in other species.
Project description:Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cβ (PP1cβ) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cβ may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the β isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not result in an apparent phenotype in mice up to 6 months of age and did not affect smooth muscle contractions ex vivo In contrast, smooth muscle-specific conditional PP1cβ knockout decreased contractile force development in bladder, ileal, and aortic tissues and reduced mouse survival. Bladder smooth muscle tissue from WT mice was selectively permeabilized to remove soluble PP1cβ to measure contributions of total (α-toxin treatment) and myosin-bound (Triton X-100 treatment) phosphatase activities toward phosphorylated RLC in myofilaments. Triton X-100 reduced PP1cβ content by 60% and the rate of RLC dephosphorylation by 2-fold. These results are consistent with the selective dephosphorylation of RLC by both MYPT1-bound and -unbound PP1cβ forms in smooth muscle.
Project description:Oriented smooth muscle layers in the intestine contract rhythmically due to the action of interstitial cells of Cajal (ICC) that serve as pacemakers of the intestine. Disruption of ICC networks has been reported in various intestinal motility disorders, which limit the quality and expectancy of life. A significant challenge in intestinal smooth muscle engineering is the rapid loss of function in cultured ICC and smooth muscle cells (SMC). Here we demonstrate a novel approach to maintain the function of both ICC and SMC in vitro. Primary intestinal SMC mixtures cultured on feeder cells seeded electrospun poly(3-caprolactone) scaffolds exhibited rhythmic contractions with directionality for over 10 weeks in vitro. The simplicity of this system should allow for wide usage in research on intestinal motility disorders and tissue engineering, and may prove to be a versatile platform for generating other types of functional SMC in vitro.
Project description:BackgroundHigh-dose-rate radiotherapy has shown promising results with respect to normal tissue preservation. We developed an ex vivo model to study the physiological effects of experimental radiotherapy in the rodent esophageal smooth muscle.MethodsWe assessed the physiological parameters of the esophageal function in ex vivo preparations of the proximal, middle, and distal segments in the organ bath. High-dose-rate synchrotron irradiation was conducted using both the microbeam irradiation (MBI) technique with peak doses greater than 200 Gy and broadbeam irradiation (BBI) with doses ranging between 3.5-4 Gy.ResultsNeither MBI nor BBI affected the function of the contractile apparatus. While peak latency and maximal force change were not affected in the BBI group, and no changes were seen in the proximal esophagus segments after MBI, a significant increase in peak latency and a decrease in maximal force change was observed in the middle and distal esophageal segments.ConclusionNo severe changes in physiological parameters of esophageal contraction were determined after high-dose-rate radiotherapy in our model, but our results indicate a delayed esophageal function. From the clinical perspective, the observed increase in peak latency and decreased maximal force change may indicate delayed esophageal transit.