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He Y, Sun Z, He X, Mi Y. AFM is used to study the biophysics of hypertension-induced tachyarrhythmia. Microsc Res Tech 2023; 86:1099-1107. [PMID: 37422907 DOI: 10.1002/jemt.24365] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Revised: 05/15/2023] [Accepted: 05/19/2023] [Indexed: 07/11/2023]
Abstract
Patients with long-lasting hypertension often suffer from atrial or ventricular arrhythmias. Evidence suggests that mechanical stimulation can change the refractory period and dispersion of the ventricular myocyte action potential through stretch-activated ion channels (SACs) and influence cellular calcium transients, thus increasing susceptibility to ventricular arrhythmias. However, the specific pathogenesis of hypertension-induced arrhythmias is unknown. In this study, through clinical data, we found that a short-term increase in blood pressure leads to a rise in tachyarrhythmias in patients with clinical hypertension. We investigated the mechanism of this phenomenon using a combined imaging system(AC) of atomic force microscopy (AFM) and laser scanning confocal microscopy. After mechanical distraction to stimulate ventricular myocytes isolated from Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), we synchronously monitored cardiomyocyte stiffness and intracellular calcium changes. This method can reasonably simulate cardiomyocytes' mechanics and ion changes when blood pressure rises rapidly. Our results indicated that the stiffness value of cardiomyocytes in SHR was significantly more extensive than that of normal controls, and cardiomyocytes were more sensitive to mechanical stress; In addition, intracellular calcium increased rapidly and briefly in rats with spontaneous hypertension. After intervention with streptomycin, a SAC blocker, ventricular myocytes are significantly less sensitive to mechanical stimuli. Thus, SAC is involved in developing and maintaining ventricular arrhythmias induced by hypertension. The increased stiffness of ventricular myocytes caused by hypertension leads to hypersensitivity of cellular calcium flow to mechanical stimuli is one of the mechanisms that cause arrhythmias. The AC system is a new research method to study the mechanical properties of cardiomyocytes. This study provides new techniques and ideas for developing new anti-arrhythmic drugs. HIGHLIGHT: The mechanism of hypertension-induced tachyarrhythmia is not precise. Through this study, it is found that the biophysical properties of myocardial abnormalities, the myocardium is excessively sensitive to mechanical stimulation, and the calcium flow appears to transient explosive changes, leading to tachyarrhythmia.
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Affiliation(s)
- Yin He
- Emergency Department, Beijing Anzhen Hospital, Capital Medical University, Beijing, People's Republic of China
| | - Zhifu Sun
- Otolaryngology head and neck surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing, People's Republic of China
| | - Xiaonan He
- Emergency Department, Beijing Anzhen Hospital, Capital Medical University, Beijing, People's Republic of China
| | - Yuhong Mi
- Emergency Department, Beijing Anzhen Hospital, Capital Medical University, Beijing, People's Republic of China
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Klemm L, Seydewitz R, Siebert T, Böl M. Three-dimensional multi-field modelling of gastric arrhythmias and their effects on antral contractions. Comput Biol Med 2023; 153:106488. [PMID: 36592609 DOI: 10.1016/j.compbiomed.2022.106488] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2022] [Revised: 12/19/2022] [Accepted: 12/25/2022] [Indexed: 12/31/2022]
Abstract
The contraction activation of smooth muscle in the stomach wall (SW) is coordinated by slow electrical waves. The interstitial cells of Cajal (ICC), specialised pacemaker cells, initiate and propagate these slow waves. By establishing an electrically coupled network, each ICC adjusts its intrinsic pacing frequency to a single dominant frequency, to be a key aspect in modelling the electrophysiology of gastric tissue. In terms of modelling, additional fields associated with electrical activation, such as voltage-dependent calcium influx and the resulting deformation, have hardly been considered so far. Here we present a three-dimensional model of the electro-chemomechanical activation of gastric smooth muscle contractions. To reduce computational costs, an adaptive multi-scale discretisation strategy for the temporal resolution of the electric field is used. The model incorporates a biophysically based model of gastric ICC pacemaker activity that aims to simulate stable entrainment and physiological conduction velocities of the electrical slow waves. Together with the simulation of concomitant gastric contractions and the inclusion of a mechanical feedback mechanism, the model is used to study dysrhythmias of gastric slow waves induced by abnormal stretching of the antral SW. The model is able to predict the formation of stretch-induced gastric arrhythmias, such as the emergence of an ectopic pacemaker in the gastric antrum. The results show that the ectopic event is accompanied by smooth muscle contraction and, although it disrupts the normal propagation pattern of gastric slow electrical waves, it can also catalyse the process of handling indigestible materials that might otherwise injure the gastric SW.
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Affiliation(s)
- Lisa Klemm
- Institute of Mechanics and Adaptronics, Technische Universität Braunschweig, Braunschweig D-38106, Germany
| | - Robert Seydewitz
- Institute of Mechanics and Adaptronics, Technische Universität Braunschweig, Braunschweig D-38106, Germany
| | - Tobias Siebert
- Institute of Sport and Motion Science, University of Stuttgart, Stuttgart D-70569, Germany
| | - Markus Böl
- Institute of Mechanics and Adaptronics, Technische Universität Braunschweig, Braunschweig D-38106, Germany.
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3
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PKC-Mediated Orai1 Channel Phosphorylation Modulates Ca2+ Signaling in HeLa Cells. Cells 2022; 11:cells11132037. [PMID: 35805121 PMCID: PMC9266177 DOI: 10.3390/cells11132037] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Revised: 05/15/2022] [Accepted: 05/27/2022] [Indexed: 12/04/2022] Open
Abstract
The overexpression of the Orai1 channel inhibits SOCE when using the Ca2+ readdition protocol. However, we found that HeLa cells overexpressing the Orai1 channel displayed enhanced Ca2+ entry and a limited ER depletion in response to the combination of ATP and thapsigargin (TG) in the presence of external Ca2+. As these effects require the combination of an agonist and TG, we decided to study whether the phosphorylation of Orai1 S27/S30 residues had any role using two different mutants: Orai1-S27/30A (O1-AA, phosphorylation-resistant) and Orai1-S27/30D (O1-DD, phosphomimetic). Both O1-wt and O1-AA supported enhanced Ca2+ entry, but this was not the case with O1-E106A (dead-pore mutant), O1-DD, and O1-AA-E106A, while O1-wt, O1-E106A, and O1-DD inhibited the ATP and TG-induced reduction of ER [Ca2+], suggesting that the phosphorylation of O1 S27/30 interferes with the IP3R activity. O1-wt and O1-DD displayed an increased interaction with IP3R in response to ATP and TG; however, the O1-AA channel decreased this interaction. The expression of mCherry-O1-AA increased the frequency of ATP-induced sinusoidal [Ca2+]i oscillations, while mCherry-O1-wt and mCherry-O1-DD decreased this frequency. These data suggest that the combination of ATP and TG stimulates Ca2+ entry, and the phosphorylation of Orai1 S27/30 residues by PKC reduces IP3R-mediated Ca2+ release.
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Joshi V, Strege PR, Farrugia G, Beyder A. Mechanotransduction in gastrointestinal smooth muscle cells: role of mechanosensitive ion channels. Am J Physiol Gastrointest Liver Physiol 2021; 320:G897-G906. [PMID: 33729004 PMCID: PMC8202201 DOI: 10.1152/ajpgi.00481.2020] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Mechanosensation, the ability to properly sense mechanical stimuli and transduce them into physiologic responses, is an essential determinant of gastrointestinal (GI) function. Abnormalities in this process result in highly prevalent GI functional and motility disorders. In the GI tract, several cell types sense mechanical forces and transduce them into electrical signals, which elicit specific cellular responses. Some mechanosensitive cells like sensory neurons act as specialized mechanosensitive cells that detect forces and transduce signals into tissue-level physiological reactions. Nonspecialized mechanosensitive cells like smooth muscle cells (SMCs) adjust their function in response to forces. Mechanosensitive cells use various mechanoreceptors and mechanotransducers. Mechanoreceptors detect and convert force into electrical and biochemical signals, and mechanotransducers amplify and direct mechanoreceptor responses. Mechanoreceptors and mechanotransducers include ion channels, specialized cytoskeletal proteins, cell junction molecules, and G protein-coupled receptors. SMCs are particularly important due to their role as final effectors for motor function. Myogenic reflex-the ability of smooth muscle to contract in response to stretch rapidly-is a critical smooth muscle function. Such rapid mechanotransduction responses rely on mechano-gated and mechanosensitive ion channels, which alter their ion pores' opening in response to force, allowing fast electrical and Ca2+ responses. Although GI SMCs express a variety of such ion channels, their identities remain unknown. Recent advancements in electrophysiological, genetic, in vivo imaging, and multi-omic technologies broaden our understanding of how SMC mechano-gated and mechanosensitive ion channels regulate GI functions. This review discusses GI SMC mechanosensitivity's current developments with a particular emphasis on mechano-gated and mechanosensitive ion channels.
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Affiliation(s)
- Vikram Joshi
- 1Division of Gastroenterology & Hepatology, Enteric NeuroScience Program (ENSP), Mayo Clinic, Rochester, Minnesota
| | - Peter R. Strege
- 1Division of Gastroenterology & Hepatology, Enteric NeuroScience Program (ENSP), Mayo Clinic, Rochester, Minnesota
| | - Gianrico Farrugia
- 1Division of Gastroenterology & Hepatology, Enteric NeuroScience Program (ENSP), Mayo Clinic, Rochester, Minnesota,2Department of Physiology & Biomedical Engineering, Mayo Clinic, Rochester, Minnesota
| | - Arthur Beyder
- 1Division of Gastroenterology & Hepatology, Enteric NeuroScience Program (ENSP), Mayo Clinic, Rochester, Minnesota,2Department of Physiology & Biomedical Engineering, Mayo Clinic, Rochester, Minnesota
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Klemm L, Seydewitz R, Borsdorf M, Siebert T, Böl M. On a coupled electro-chemomechanical model of gastric smooth muscle contraction. Acta Biomater 2020; 109:163-181. [PMID: 32294551 DOI: 10.1016/j.actbio.2020.04.007] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2019] [Revised: 03/31/2020] [Accepted: 04/02/2020] [Indexed: 12/13/2022]
Abstract
The stomach is a central organ in the gastrointestinal tract that performs a variety of functions, in which the spatio-temporal organisation of active smooth muscle contraction in the stomach wall (SW) is highly regulated. In the present study, a three-dimensional model of the gastric smooth muscle contraction is presented, including the mechanical contribution of the mucosal and muscular layer of the SW. Layer-specific and direction-dependent model parameters for the active and passive stress-stretch characteristics of the SW were determined experimentally using porcine smooth muscle strips. The electrical activation of the smooth muscle cells (SMC) due to the pacemaker activity of the interstitial cells of Cajal (ICC) is modelled by using FitzHugh-Nagumo-type equations, which simulate the typical ICC and SMC slow wave behaviour. The calcium dynamic in the SMC depends on the SMC membrane potential via a gaussian function, while the chemo-mechanical coupling in the SMC is modelled via an extended Hai-Murphy model. This cascade is coupled with an additional mechano-electrical feedback-mechanism, taking into account the mechanical response of the ICC and SMC due to stretch of the SW. In this way the relaxation responses of the fundus to accommodate incoming food, as well as the typical peristaltic contraction waves in the antrum for mixing and transport of the chyme, have been well replicated in simulations performed at the whole organ level. STATEMENT OF SIGNIFICANCE: In this article, a novel three-dimensional electro-chemomechanical model of the gastric smooth muscle contraction is presented. The propagating waves of electrical membrane potential in the network ofinterstitial cells of Cajal (ICC) and smooth muscle cells (SMC) lead to a global pattern of change in the calciumdynamics inside the SMC. Taking additionally into account the mechanical response of the ICC and SMC due to stretch of the stomach wall, also referred to as mechanical feedback-mechanism, the result is a complex spatio-temporal regulation of the active contraction and relaxation of the gastric smooth muscle tissue. Being a firstapproach, in future view such a three-dimensional model can give an insight into the complexload transferring system of the stomach wall, as well as into the electro-chemomechanicalcoupling process underlying smooth muscle contraction in health and disease.
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Affiliation(s)
- Lisa Klemm
- Institute of Solid Mechanics, Technische Universität Braunschweig, Braunschweig D-38106, Germany
| | - Robert Seydewitz
- Institute of Solid Mechanics, Technische Universität Braunschweig, Braunschweig D-38106, Germany
| | - Mischa Borsdorf
- Institute of Sport and Motion Science, University of Stuttgart, Stuttgart D-70569, Germany
| | - Tobias Siebert
- Institute of Sport and Motion Science, University of Stuttgart, Stuttgart D-70569, Germany
| | - Markus Böl
- Institute of Solid Mechanics, Technische Universität Braunschweig, Braunschweig D-38106, Germany.
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6
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Sarco-Endoplasmic Reticulum Calcium Release Model Based on Changes in the Luminal Calcium Content. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2020; 1131:337-370. [DOI: 10.1007/978-3-030-12457-1_14] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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7
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Role of BK Ca in Stretch-Induced Relaxation of Colonic Smooth Muscle. BIOMED RESEARCH INTERNATIONAL 2016; 2016:9497041. [PMID: 28018918 PMCID: PMC5149602 DOI: 10.1155/2016/9497041] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/22/2016] [Revised: 09/30/2016] [Accepted: 10/23/2016] [Indexed: 12/15/2022]
Abstract
Stretch-induced relaxation has not been clearly identified in gastrointestinal tract. The present study is to explore the role of large conductance calcium-activated potassium channels (BKCa) in stretch-induced relaxation of colon. The expression and currents of BKCa were detected and the basal muscle tone and contraction amplitude of colonic smooth muscle strips were measured. The expression of BKCa in colon is higher than other GI segments (P < 0.05). The density of BKCa currents was very high in colonic smooth muscle cells (SMCs). BKCa in rat colonic SMCs were sensitive to stretch. The relaxation response of colonic SM strips to stretch was attenuated by charybdotoxin (ChTX), a nonspecific BKCa blocker (P < 0.05). After blocking enteric nervous activities by tetrodotoxin (TTX), the stretch-induced relaxation did not change (P > 0.05). Still, ChTX and iberiotoxin (IbTX, a specific BKCa blocker) attenuated the relaxation of the colonic muscle strips enduring stretch (P < 0.05). These results suggest stretch-activation of BKCa in SMCs was involved in the stretch-induced relaxation of colon. Our study highlights the role of mechanosensitive ion channels in SMCs in colon motility regulation and their physiological and pathophysiological significance is worth further study.
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8
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Kinetics on Demand Is a Simple Mathematical Solution that Fits Recorded Caffeine-Induced Luminal SR Ca2+ Changes in Smooth Muscle Cells. PLoS One 2015; 10:e0138195. [PMID: 26390403 PMCID: PMC4577101 DOI: 10.1371/journal.pone.0138195] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2015] [Accepted: 08/27/2015] [Indexed: 12/02/2022] Open
Abstract
The process of Ca2+ release from sarcoplasmic reticulum (SR) comprises 4 phases in smooth muscle cells. Phase 1 is characterized by a large increase of the intracellular Ca2+ concentration ([Ca2+]i) with a minimal reduction of the free luminal SR [Ca2+] ([Ca2+]FSR). Importantly, active SR Ca2+ ATPases (SERCA pumps) are necessary for phase 1 to occur. This situation cannot be explained by the standard kinetics that involves a fixed amount of luminal Ca2+ binding sites. A new mathematical model was developed that assumes an increasing SR Ca2+ buffering capacity in response to an increase of the luminal SR [Ca2+] that is called Kinetics-on-Demand (KonD) model. This approach can explain both phase 1 and the refractory period associated with a recovered [Ca2+]FSR. Additionally, our data suggest that active SERCA pumps are a requisite for KonD to be functional; otherwise luminal SR Ca2+ binding proteins switch to standard kinetics. The importance of KonD Ca2+ binding properties is twofold: a more efficient Ca2+ release process and that [Ca2+]FSR and Ca2+-bound to SR proteins ([Ca2+]BSR) can be regulated separately allowing for Ca2+ release to occur (provided by Ca2+-bound to luminal Ca2+ binding proteins) without an initial reduction of the [Ca2+]FSR.
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9
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Kurth F, Franco-Obregón A, Casarosa M, Küster SK, Wuertz-Kozak K, Dittrich PS. Transient receptor potential vanilloid 2-mediated shear-stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix. FASEB J 2015. [PMID: 26207028 DOI: 10.1096/fj.15-275396] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The developmental sensitivity of skeletal muscle to mechanical forces is unparalleled in other tissues. Calcium entry via reputedly mechanosensitive transient receptor potential (TRP) channel classes has been shown to play an essential role in both the early proliferative stage and subsequent differentiation of skeletal muscle myoblasts, particularly TRP canonical (TRPC) 1 and TRP vanilloid (TRPV) 2. Here we show that C2C12 murine myoblasts respond to fluid flow-induced shear stress with increments in cytosolic calcium that are largely initiated by the mechanosensitive opening of TRPV2 channels. Response to fluid flow was augmented by growth in low extracellular serum concentration (5 vs. 20% fetal bovine serum) by greater than 9-fold and at 18 h in culture, coincident with the greatest TRPV2 channel expression under identical conditions (P < 0.02). Fluid flow responses were also enhanced by substrate functionalization with laminin, rather than with fibronectin, agreeing with previous findings that the gating of TRPV2 is facilitated by laminin. Fluid flow-induced calcium increments were blocked by ruthenium red (27%) and SKF-96365 (38%), whereas they were unaltered by 2-aminoethoxydiphenyl borate, further corroborating that TRPV2 channels play a predominant role in fluid flow mechanosensitivity over that of TRPC1 and TRP melastatin (TRPM) 7.
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Affiliation(s)
- Felix Kurth
- *Department of Biosystems and Science Engineering and Institute for Biomechanics, Eidgenössische Technische Hochschule Zürich, Switzerland; Department of Surgery, Yong Loo Lin School of Medicine, and Department of Physiology, National University of Singapore, Singapore; and National University Hospital Sports Centre, Singapore
| | - Alfredo Franco-Obregón
- *Department of Biosystems and Science Engineering and Institute for Biomechanics, Eidgenössische Technische Hochschule Zürich, Switzerland; Department of Surgery, Yong Loo Lin School of Medicine, and Department of Physiology, National University of Singapore, Singapore; and National University Hospital Sports Centre, Singapore
| | - Marco Casarosa
- *Department of Biosystems and Science Engineering and Institute for Biomechanics, Eidgenössische Technische Hochschule Zürich, Switzerland; Department of Surgery, Yong Loo Lin School of Medicine, and Department of Physiology, National University of Singapore, Singapore; and National University Hospital Sports Centre, Singapore
| | - Simon K Küster
- *Department of Biosystems and Science Engineering and Institute for Biomechanics, Eidgenössische Technische Hochschule Zürich, Switzerland; Department of Surgery, Yong Loo Lin School of Medicine, and Department of Physiology, National University of Singapore, Singapore; and National University Hospital Sports Centre, Singapore
| | - Karin Wuertz-Kozak
- *Department of Biosystems and Science Engineering and Institute for Biomechanics, Eidgenössische Technische Hochschule Zürich, Switzerland; Department of Surgery, Yong Loo Lin School of Medicine, and Department of Physiology, National University of Singapore, Singapore; and National University Hospital Sports Centre, Singapore
| | - Petra S Dittrich
- *Department of Biosystems and Science Engineering and Institute for Biomechanics, Eidgenössische Technische Hochschule Zürich, Switzerland; Department of Surgery, Yong Loo Lin School of Medicine, and Department of Physiology, National University of Singapore, Singapore; and National University Hospital Sports Centre, Singapore
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Lopez-Ayon GM, Liu HY, Xing S, Maria OM, LeDue JM, Bourque H, Grutter P, Komarova SV. Local membrane deformation and micro-injury lead to qualitatively different responses in osteoblasts. F1000Res 2014; 3:162. [PMID: 25254108 PMCID: PMC4168753 DOI: 10.12688/f1000research.4448.1] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 07/11/2014] [Indexed: 11/24/2022] Open
Abstract
Micro-damage of bone tissue is known to regulate bone turnover. However, it is unknown if individual bone cells can differentiate between membrane deformation and micro-injury. We generated osteoblasts from mouse bone marrow or bone morphogenetic protein 2-transfected C2C12 cells. Single cells were mechanically stimulated by indentation with the atomic force microscopy probe with variable force load either resulting in membrane deformation only, or leading to membrane penetration and micro-injury. Changes in the cytosolic free calcium concentration ([Ca (2+)] i) in fluo4-AM loaded cells were analyzed. When deformation only was induced, it resulted in an immediate elevation of [Ca (2+)] i which was localized to the probe periphery. Multiple consecutive local Ca (2+) responses were induced by sequential application of low level forces, with characteristic recovery time of ~2 s. The duration of [Ca (2+)] i elevations was directly proportional to the tip-cell contact time. In contrast, cell micro-injury resulted in transient global elevations of [Ca (2+)] i, the magnitude of which was independent of the tip-cell contact time. Sequential micro-injury of the same cell did not induce Ca (2+) response within 30 s of the first stimulation. Both local and global Ca (2+)elevations were blocked in Ca (2+)-free media or in the presence of stretch-activated channel blocker Gd (3+). In addition, amount of Ca (2+) released during global responses was significantly reduced in the presence of PLC inhibitor Et-18-OCH 3. Thus, we found qualitative differences in calcium responses to mechanical forces inducing only membrane deformation or deformation leading to micro-injury.
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Affiliation(s)
- G Monserratt Lopez-Ayon
- Center for the Physics of Materials and the Department of Physics, McGill University, 3600 University, Montreal, Quebec, H3A 2T8, Canada
| | - Heng-Yen Liu
- Faculty of Dentistry, McGill University, 3640 University, Montreal, Quebec, H3A 0C7, Canada ; Shriners Hospital for Children - Canada, 1529 Cedar Ave, Montreal, Quebec, H3G IA6, Canada
| | - Shu Xing
- Center for the Physics of Materials and the Department of Physics, McGill University, 3600 University, Montreal, Quebec, H3A 2T8, Canada ; Faculty of Dentistry, McGill University, 3640 University, Montreal, Quebec, H3A 0C7, Canada
| | - Osama M Maria
- Faculty of Dentistry, McGill University, 3640 University, Montreal, Quebec, H3A 0C7, Canada
| | - Jeffrey M LeDue
- Center for the Physics of Materials and the Department of Physics, McGill University, 3600 University, Montreal, Quebec, H3A 2T8, Canada
| | - Helene Bourque
- Center for the Physics of Materials and the Department of Physics, McGill University, 3600 University, Montreal, Quebec, H3A 2T8, Canada
| | - Peter Grutter
- Center for the Physics of Materials and the Department of Physics, McGill University, 3600 University, Montreal, Quebec, H3A 2T8, Canada
| | - Svetlana V Komarova
- Faculty of Dentistry, McGill University, 3640 University, Montreal, Quebec, H3A 0C7, Canada ; Shriners Hospital for Children - Canada, 1529 Cedar Ave, Montreal, Quebec, H3G IA6, Canada
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Guerrero-Hernández A, Ávila G, Rueda A. Ryanodine receptors as leak channels. Eur J Pharmacol 2013; 739:26-38. [PMID: 24291096 DOI: 10.1016/j.ejphar.2013.11.016] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2013] [Accepted: 11/21/2013] [Indexed: 01/18/2023]
Abstract
Ryanodine receptors are Ca(2+) release channels of internal stores. This review focuses on those situations and conditions that transform RyRs from a finely regulated ion channel to an unregulated Ca(2+) leak channel and the pathological consequences of this alteration. In skeletal muscle, mutations in either CaV1.1 channel or RyR1 results in a leaky behavior of the latter. In heart cells, RyR2 functions normally as a Ca(2+) leak channel during diastole within certain limits, the enhancement of this activity leads to arrhythmogenic situations that are tackled with different pharmacological strategies. In smooth muscle, RyRs are involved more in reducing excitability than in stimulating contraction so the leak activity of RyRs in the form of Ca(2+) sparks, locally activates Ca(2+)-dependent potassium channels to reduce excitability. In neurons the enhanced activity of RyRs is associated with the development of different neurodegenerative disorders such as Alzheimer and Huntington diseases. It appears then that the activity of RyRs as leak channels can have both physiological and pathological consequences depending on the cell type and the metabolic condition.
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Affiliation(s)
| | | | - Angélica Rueda
- Departamento de Bioquímica, Cinvestav, Mexico city, México
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12
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Wenger KH, El-Awady AR, Messer RLW, Sharawy MM, White G, Lapp CA. Pneumatic pressure bioreactor for cyclic hydrostatic stress application: mechanobiology effects on periodontal ligament cells. J Appl Physiol (1985) 2011; 111:1072-9. [PMID: 21757574 DOI: 10.1152/japplphysiol.01175.2010] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
A bioreactor system was developed to provide high-amplitude cyclic hydrostatic compressive stress (cHSC) using compressed air mixed commercially as needed to create partial pressures of oxygen and carbon dioxide appropriate for the cells under investigation. Operating pressures as high as 300 psi are achievable in this system at cyclic speeds of up to 0.2 Hz. In this study, ligamentous fibroblasts from human periodontal ligaments (n = 6) were compressed on two consecutive days at 150 psi for 3 h each day, and the mRNA for families of extracellular matrix protein and protease isoforms was evaluated by real-time PCR array. Several integrins were significantly upregulated, most notably alpha-3 (6.4-fold), as was SPG7 (12.1-fold). Among the collagens, Col8a1 was highly upregulated at 53.5-fold, with Col6a1, Col6a2, and Col7a1 also significantly upregulated 4.4- to 8.5-fold. MMP-1 was the most affected at 122.9-fold upregulation. MMP-14 likewise increased 17.8-fold with slight reductions for the gelatinases and a significant increase of TIMP-2 at 5.8-fold. The development of this bioreactor system and its utility in characterizing periodontal ligament fibroblast mechanobiology in intermediate-term testing hold promise for better simulating the conditions of the musculoskeletal system and the large cyclic compressive stresses joints may experience in gait, exertion, and mastication.
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Affiliation(s)
- Karl H Wenger
- Department of Orthopaedic Surgery, Georgia Health Sciences University, Augusta, GA 30912, USA.
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Shklyar TF, Safronov AP, Toropova OA, Pollack GH, Blyakhman FA. Mechanoelectric potentials in synthetic hydrogels: Possible relation to cytoskeleton. Biophysics (Nagoya-shi) 2011. [DOI: 10.1134/s0006350910060084] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
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15
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Abstract
The sarcoplasmic reticulum (SR) of smooth muscles presents many intriguing facets and questions concerning its roles, especially as these change with development, disease, and modulation of physiological activity. The SR's function was originally perceived to be synthetic and then that of a Ca store for the contractile proteins, acting as a Ca amplification mechanism as it does in striated muscles. Gradually, as investigators have struggled to find a convincing role for Ca-induced Ca release in many smooth muscles, a role in controlling excitability has emerged. This is the Ca spark/spontaneous transient outward current coupling mechanism which reduces excitability and limits contraction. Release of SR Ca occurs in response to inositol 1,4,5-trisphosphate, Ca, and nicotinic acid adenine dinucleotide phosphate, and depletion of SR Ca can initiate Ca entry, the mechanism of which is being investigated but seems to involve Stim and Orai as found in nonexcitable cells. The contribution of the elemental Ca signals from the SR, sparks and puffs, to global Ca signals, i.e., Ca waves and oscillations, is becoming clearer but is far from established. The dynamics of SR Ca release and uptake mechanisms are reviewed along with the control of luminal Ca. We review the growing list of the SR's functions that still includes Ca storage, contraction, and relaxation but has been expanded to encompass Ca homeostasis, generating local and global Ca signals, and contributing to cellular microdomains and signaling in other organelles, including mitochondria, lysosomes, and the nucleus. For an integrated approach, a review of aspects of the SR in health and disease and during development and aging are also included. While the sheer versatility of smooth muscle makes it foolish to have a "one model fits all" approach to this subject, we have tried to synthesize conclusions wherever possible.
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Affiliation(s)
- Susan Wray
- Department of Physiology, School of Biomedical Sciences, University of Liverpool, Liverpool, Merseyside L69 3BX, United Kingdom.
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16
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17
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Kitsiopoulou E, Hatziefthimiou AA, Gourgoulianis KI, Molyvdas PA. Resting tension affects eNOS activity in a calcium-dependent way in airways. Mediators Inflamm 2007; 2007:24174. [PMID: 17515950 PMCID: PMC1868075 DOI: 10.1155/2007/24174] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2006] [Accepted: 02/05/2007] [Indexed: 11/18/2022] Open
Abstract
The alteration of resting tension (RT) from 0.5 g to 2.5 g increased significantly airway smooth muscle contractions induced by acetylcholine (ACh) in rabbit trachea. The decrease in extracellular calcium concentration [Ca2+]o from 2 mM to 0.2 mM reduced ACh-induced contractions only at 2.5 g RT with no effect at 0.5 g RT. The nonselective inhibitor of nitric oxide synthase (NOS), NG-nitro-L-arginine methyl ester (L-NAME) increased ACh-induced contractions at
2.5 g RT. The inhibitor of inducible NOS, S-methylsothiourea or neuronal
NOS, 7-nitroindazole had no effect. At 2.5 g RT, the reduction of [Ca2+]o from 2 mM to 0.2 mM abolished the effect of L-NAME on ACh-induced contractions. The NO precursor L-arginine or the tyrosine kinase inhibitors erbstatin A and genistein had no effect on ACh-induced contractions obtained at 2.5 g RT. Our results suggest that in airways, RT affects ACh-induced contractions by modulating the activity of epithelial NOS in a calcium-dependent, tyrosine-phosphorylation-independent way.
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Affiliation(s)
- Eudoxia Kitsiopoulou
- Department of Physiology, Medical School, University of Thessaly, Papakiriazi 22, 41222 Larissa, Greece
| | - Apostolia A. Hatziefthimiou
- Department of Physiology, Medical School, University of Thessaly, Papakiriazi 22, 41222 Larissa, Greece
- *Apostolia A. Hatziefthimiou:
| | | | - Paschalis-Adam Molyvdas
- Department of Physiology, Medical School, University of Thessaly, Papakiriazi 22, 41222 Larissa, Greece
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18
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Abstract
Bladder dysfunction is a common complication of diabetic autonomic neuropathy; however, its cause remains uncertain. We have recently identified a novel IgG autoantibody (Ab) in patients with type 1 diabetes that acts as an agonist at the dihydropyridine (DHP) site of L-type voltage-gated calcium channels (VGCC), disrupting neuronal regulation of visceral smooth muscle. In the present study, passive transfer to mice of IgG from patients with type 1 diabetes was used to investigate the role of anti-VGCC Abs in mediating diabetic bladder dysfunction. Injection of mice with diabetic immunoglobulin (IgG) with anti-VGCC activity induced features of an overactive bladder, including phasic detrusor contractions and a loss of bladder wall compliance. The bladder overactivity is mimicked by the DHP agonist Bay K8644, reversed by the DHP antagonist nicardipine, but is insensitive to the motor nerve blocker tetrodotoxin, indicating that the anti-VGCC Ab acts at the level of the bladder detrusor itself. This study reports the first evidence of Ab-mediated bladder dysfunction in type 1 diabetes, which may be part of a wider spectrum of smooth muscle and cardiac abnormalities.
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Affiliation(s)
- E-C Wan
- Department of Immunology, Allergy & Arthritis, Flinders Medical Centre, Flinders University, Bedford Park, SA, Australia
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19
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Morita H, Honda A, Inoue R, Ito Y, Abe K, Nelson MT, Brayden JE. Membrane Stretch-Induced Activation of a TRPM4-Like Nonselective Cation Channel in Cerebral Artery Myocytes. J Pharmacol Sci 2007; 103:417-26. [PMID: 17420615 DOI: 10.1254/jphs.fp0061332] [Citation(s) in RCA: 96] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022] Open
Abstract
Stretch-activated cation channels (SACs) have been observed in many types of smooth muscle cells. However, the molecular identity and activation mechanisms of SACs remain poorly understood. We report that TRPM4-like cation channels are activated by membrane stretch in rat cerebral artery myocytes (CAMs). Negative pressure (> or =20 mmHg, cell-attached mode) activated single channels (approximately 20 pS) in isolated CAMs. These channels were permeable to Na(+) and Cs(+) and inhibited by Gd(3+) (30 microM) and DIDS (100 microM). The effect of negative pressure was abolished by membrane excision, but subsequent application of Ca(2+) (>100 nM) to the intracellular side of the membrane restored single channel activity that was indistinguishable from SACs. Caffeine (5 mM), which depletes SR Ca(2+)-stores, first activated and then abolished SACs. Tetracaine (100 microM), a ryanodine receptor antagonist, inhibited SACs. Overexpression of hTRPM4B in HEK293 cells resulted in the appearance of cation channels that were activated by both negative pressure and Ca(2+) and which had very similar biophysical and pharmacological properties as compared with SACs in CAMs. These studies indicate that TRPM4-like channels in CAMs can be activated by membrane stretch, possibly through ryanodine receptor activation, and this may contribute to the depolarization and concomitant vasoconstriction of intact cerebral arteries following mechanical stimulation.
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MESH Headings
- 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology
- Animals
- Boron Compounds/pharmacology
- Calcium Channels/genetics
- Calcium Channels/physiology
- Cell Line
- Cell Membrane/physiology
- Cells, Cultured
- Cerebral Arteries/cytology
- Cerebral Arteries/metabolism
- Cerebral Arteries/physiology
- Female
- Gadolinium/pharmacology
- Gene Expression/drug effects
- Humans
- Male
- Membrane Potentials/drug effects
- Membrane Potentials/physiology
- Myocytes, Smooth Muscle/cytology
- Myocytes, Smooth Muscle/metabolism
- Myocytes, Smooth Muscle/physiology
- Patch-Clamp Techniques
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Rats
- Rats, Sprague-Dawley
- Reverse Transcriptase Polymerase Chain Reaction
- Stress, Mechanical
- TRPC Cation Channels/genetics
- TRPC Cation Channels/physiology
- TRPM Cation Channels/genetics
- TRPM Cation Channels/physiology
- TRPV Cation Channels/genetics
- TRPV Cation Channels/physiology
- Transient Receptor Potential Channels/genetics
- Transient Receptor Potential Channels/physiology
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Affiliation(s)
- Hiromitsu Morita
- Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA.
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20
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Wang JHC, Thampatty BP. An introductory review of cell mechanobiology. Biomech Model Mechanobiol 2006; 5:1-16. [PMID: 16489478 DOI: 10.1007/s10237-005-0012-z] [Citation(s) in RCA: 361] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2005] [Accepted: 12/08/2005] [Indexed: 11/30/2022]
Abstract
Mechanical loads induce changes in the structure, composition, and function of living tissues. Cells in tissues are responsible for these changes, which cause physiological or pathological alterations in the extracellular matrix (ECM). This article provides an introductory review of the mechanobiology of load-sensitive cells in vivo, which include fibroblasts, chondrocytes, osteoblasts, endothelial cells, and smooth muscle cells. Many studies have shown that mechanical loads affect diverse cellular functions, such as cell proliferation, ECM gene and protein expression, and the production of soluble factors. Major cellular components involved in the mechanotransduction mechanisms include the cytoskeleton, integrins, G proteins, receptor tyrosine kinases, mitogen-activated protein kinases, and stretch-activated ion channels. Future research in the area of cell mechanobiology will require novel experimental and theoretical methodologies to determine the type and magnitude of the forces experienced at the cellular and sub-cellular levels and to identify the force sensors/receptors that initiate the cascade of cellular and molecular events.
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Affiliation(s)
- J H-C Wang
- MechanoBiology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, 210 Lothrop St. BST, E1640, Pittsburgh, PA 15213, USA.
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21
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Thorneloe KS, Nelson MT. Ion channels in smooth muscle: regulators of intracellular calcium and contractility. Can J Physiol Pharmacol 2005; 83:215-42. [PMID: 15870837 DOI: 10.1139/y05-016] [Citation(s) in RCA: 134] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Smooth muscle (SM) is essential to all aspects of human physiology and, therefore, key to the maintenance of life. Ion channels expressed within SM cells regulate the membrane potential, intracellular Ca2+ concentration, and contractility of SM. Excitatory ion channels function to depolarize the membrane potential. These include nonselective cation channels that allow Na+ and Ca2+ to permeate into SM cells. The nonselective cation channel family includes tonically active channels (Icat), as well as channels activated by agonists, pressure-stretch, and intracellular Ca2+ store depletion. Cl--selective channels, activated by intracellular Ca2+ or stretch, also mediate SM depolarization. Plasma membrane depolarization in SM activates voltage-dependent Ca2+ channels that demonstrate a high Ca2+ selectivity and provide influx of contractile Ca2+. Ca2+ is also released from SM intracellular Ca2+ stores of the sarcoplasmic reticulum (SR) through ryanodine and inositol trisphosphate receptor Ca2+ channels. This is part of a negative feedback mechanism limiting contraction that occurs by the Ca2+-dependent activation of large-conductance K+ channels, which hyper polarize the plasma membrane. Unlike the well-defined contractile role of SR-released Ca2+ in skeletal and cardiac muscle, the literature suggests that in SM Ca2+ released from the SR functions to limit contractility. Depolarization-activated K+ chan nels, ATP-sensitive K+ channels, and inward rectifier K+ channels also hyperpolarize SM, favouring relaxation. The expression pattern, density, and biophysical properties of ion channels vary among SM types and are key determinants of electrical activity, contractility, and SM function.
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Affiliation(s)
- Kevin S Thorneloe
- Department of Pharmacology, College of Medicine, University of Vermont, Burlington 05405, USA.
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22
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Charras GT, Williams BA, Sims SM, Horton MA. Estimating the sensitivity of mechanosensitive ion channels to membrane strain and tension. Biophys J 2005; 87:2870-84. [PMID: 15454477 PMCID: PMC1304704 DOI: 10.1529/biophysj.104.040436] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Bone adapts to its environment by a process in which osteoblasts and osteocytes sense applied mechanical strain. One possible pathway for the detection of strain involves mechanosensitive channels and we sought to determine their sensitivity to membrane strain and tension. We used a combination of experimental and computational modeling techniques to gain new insights into cell mechanics and the regulation of mechanosensitive channels. Using patch-clamp electrophysiology combined with video microscopy, we recorded simultaneously the evolution of membrane extensions into the micropipette, applied pressure, and membrane currents. Nonselective mechanosensitive cation channels with a conductance of 15 pS were observed. Bleb aspiration into the micropipette was simulated using finite element models incorporating the cytoplasm, the actin cortex, the plasma membrane, cellular stiffening in response to strain, and adhesion between the membrane and the micropipette. Using this model, we examine the relative importance of the different cellular components in resisting suction into the pipette and estimate the membrane strains and tensions needed to open mechanosensitive channels. Radial membrane strains of 800% and tensions of 5 10(-4) N.m(-1) were needed to open 50% of mechanosensitive channels. We discuss the relevance of these results in the understanding of cellular reactions to mechanical strain and bone physiology.
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Affiliation(s)
- Guillaume T Charras
- Bone and Mineral Centre, Department of Medicine, University College London, London, United Kingdom.
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23
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Zou H, Lifshitz LM, Tuft RA, Fogarty KE, Singer JJ. Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs—fundamental calcium events. Cell Calcium 2004; 35:523-33. [PMID: 15110142 DOI: 10.1016/j.ceca.2004.01.019] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2003] [Accepted: 01/25/2004] [Indexed: 11/29/2022]
Abstract
Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca(2+) permeable ion channel using Ca(2+) indicators like fluo-3. These Single Channel Ca(2+) Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca(2+) sparks and Ca(2+) puffs caused by Ca(2+) release from intracellular stores (due to the opening of ryanodine receptors and IP(3) receptors, respectively). In contrast to intracellular Ca(2+) release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca(2+) handling in the vicinity of a channel with a known Ca(2+) influx, to obtain the Ca(2+) current passing through plasma membrane cation channels in near physiological solutions, to localize Ca(2+) permeable ion channels on the plasma membrane, and to estimate the Ca(2+) currents underlying those elementary events where the Ca(2+) currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca(2+) channels, and stretch-activated channels. For the L-type Ca(2+) channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca(2+) currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.
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Affiliation(s)
- Hui Zou
- Department of Physiology and Biomedical Imaging Group, University of Massachusetts Medical School, Worcester, MA 01655, USA.
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24
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Abstract
Mechanosensitive ion channels are the primary transducers that convert mechanical force into an electrical or chemical signal in hearing, touch, and other mechanical senses. Unlike vision, olfaction, and some types of taste, which all use similar kinds of primary heterotrimeric GTP-binding protein-coupled receptors, mechanosensation relies on diverse types of transducer molecules. Unrelated types of channels can be used for the perception of various mechanical stimuli, not only in distant groups of organisms, but also in separate locations of the same organism. The extreme sensitivity of the transduction mechanism in the auditory system, which relies on an elaborate structure of rigid cilia, filamentous links, and molecular motors to focus force on transduction channels, contrasts with that of the bacterial channel MscL, which is opened by high lateral tension in the membrane and fulfills a safety-valve rather than a sensory function. The spatial scales of conformational movement and force in these two systems are described, and are shown to be consistent with a general physical description of mechanical channel gating. We outline the characteristics of several types of mechanosensitive channels and the functional contexts in which they participate in signaling and cellular regulation in sensory and nonsensory cells.
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Affiliation(s)
- Sergei Sukharev
- Department of Biology, University of Maryland, College Park, MD 20742, USA.
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25
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Abstract
Background—
During head-up tilt (HUT), peripheral vasoconstriction occurs. This response requires appropriate communication between the sympathetic nerve terminal and vascular smooth muscle cell in the neurovascular space. Both of these cell types require extracellular calcium ([Ca
2+
]
o
) for proper activation and function. We hypothesize that [Ca
2+
]
o
rises with tilt and in the process contributes to vasoconstriction.
Methods and Results—
We used microdialysis techniques in the lower-limb skeletal muscle to measure [Ca
2+
]
o
changes in this space with HUT. [Ca
2+
]
o
was measured in 10 healthy subjects during HUT. We found a 62% increase in the dialysate [Ca
2+
] (0.223±0.018 to 0.353±0.028 mmol/L) with HUT.
Conclusions—
This result implies a significant increase in [Ca
2+
]
o
in the neurovascular space during HUT. This represents the first report of such in situ [Ca
2+
]
o
measurements in humans. This rise in [Ca
2+
]
o
may provide a mechanism for proper cell-cell interaction, helping to promote peripheral vasoconstriction during HUT. How this [Ca
2+
]
o
transient affects the nerve terminal, vascular smooth muscle cells, or both remains to be determined.
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Affiliation(s)
- Soraya Samii
- Division of Cardiology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pa 17033, USA
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26
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Wagner MB, Kumar R, Joyner RW, Wang Y. Induced automaticity in isolated rat atrial cells by incorporation of a stretch-activated conductance. Pflugers Arch 2004; 447:819-29. [PMID: 14727114 DOI: 10.1007/s00424-003-1208-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2003] [Accepted: 09/30/2003] [Indexed: 10/26/2022]
Abstract
Stretch of the atrium and sympathetic activity have been implicated as substrates for atrial fibrillation. We investigate how a model of stretch in combination with sympathetic stimulation can induce automaticity in atrial cells. We adapted our coupling clamp circuit so that a model ionic current that represents stretch-activated channels (SACs) was injected into an isolated rat atrial cell in real time. This current was calculated as ISAC= GSAC (Vm-ESAC), where GSAC and ESAC are the conductance and reversal potential of SACs and Vm is the cell's membrane potential. Repetitive automaticity was induced by a sufficiently large GSAC and this critical value of GSAC was decreased by exposure to isoproterenol. The critical value of GSAC decreased from 0.63+/-0.05 nS (mean+/-SE) in control to 0.40+/-0.07 nS in isoproterenol (P<0.05). Additionally, after exposure to isoproterenol, automaticity continued after GSAC was no longer applied and was accompanied by delayed after-depolarizations. In three cells, repetitive automaticity could not be induced at any value of GSAC. Exposure to 10 nM isoproterenol converted these cells to cells with repetitive automaticity in response to GSAC. We conclude that automaticity can be induced in isolated rat atrial cells by application of a model of SACs. Exposure to isoproterenol enhances this effect.
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Affiliation(s)
- Mary B Wagner
- Todd Franklin Cardiac Research Laboratory, The Sibley Heart Center, Department of Pediatrics, Emory University, 2040 Ridgewood Drive, Atlanta, GA 30322, USA
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27
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Zhang Y, Paterson WG. Role of sarcoplasmic reticulum in control of membrane potential and nitrergic response in opossum lower esophageal sphincter. Br J Pharmacol 2003; 140:1097-107. [PMID: 14530211 PMCID: PMC1574123 DOI: 10.1038/sj.bjp.0705537] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
1. We previously demonstrated that a balance of Ca2+-activated Cl- current (ICl(Ca)) and K+ current activity sets the resting membrane potential of opossum lower esophageal sphincter (LES) circular smooth muscle at approximately -41 mV, which leads to continuous spike-like action potentials and the generation of basal tone. Ionic mechanisms underlying this basal ICl(Ca) activity and its nitrergic regulation remain unclear. Recent studies suggest that spontaneous Ca2+ release from sarcoplasmic reticulum (SR) and myosin light chain kinase (MLCK) play important roles. The current study investigated this possibility. Conventional intracellular recordings were performed on circular smooth muscle of opossum LES. Nerve responses were evoked by electrical square wave pulses of 0.5 ms duration at 20 Hz. 2. In the presence of nifedipine (1 microm), substance P (1 microm), atropine (3 microm) and guanethidine (3 microm), intracellular recordings demonstrated a resting membrane potential (MP) of -38.1+/-0.7 mV (n=25) with spontaneous membrane potential fluctuations (MPfs) of 1-3 mV. Four pulses of nerve stimulation induced slow inhibitory junction potentials (sIJPs) with an amplitude of 6.1+/-0.3 mV and a half-amplitude duration of 1926+/-147 ms (n=25). 3. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific guanylyl cyclase inhibitor, abolished sIJPs, but had no effects on MPfs. Caffeine, a ryanodine receptor agonist, hyperpolarized MP and abolished sIJPs and MPfs. Ryanodine (20 microm) inhibited the sIJP and induced biphasic effects on MP, an initial small hyperpolarization followed by a large depolarization. sIJPs and MPfs were also inhibited by cyclopiazonic acid, an SR Ca2+ ATPase inhibitor. Specific ICl(Ca) and MLCK inhibitors hyperpolarized the MP and inhibited MPfs and sIJPs. 4. These data suggest that (1). spontaneous release of Ca2+ from the SR activates ICl(Ca), which in turn contributes to resting membrane potential; (2). MLCK is involved in activation of ICl(Ca); (3). inhibition of ICl(Ca) is likely to underlie sIJPs induced by nitrergic innervation.
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Affiliation(s)
- Yong Zhang
- Gastrointestinal Disease Research Unit and Departments of Medicine, Biology and Physiology, Queen's University, Kingston, Ontario, Canada
| | - William G Paterson
- Gastrointestinal Disease Research Unit and Departments of Medicine, Biology and Physiology, Queen's University, Kingston, Ontario, Canada
- Author for correspondence:
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28
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Wu SN, Lin PH, Hsieh KS, Liu YC, Chiang HT. Behavior of nonselective cation channels and large-conductance Ca2+-activated K+ channels induced by dynamic changes in membrane stretch in cultured smooth muscle cells of human coronary artery. J Cardiovasc Electrophysiol 2003; 14:44-51. [PMID: 12625609 DOI: 10.1046/j.1540-8167.2003.02040.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
INTRODUCTION The effects of membrane stretch on ion channels were investigated in cultured smooth muscle cells of human coronary artery. METHODS AND RESULTS In the cell-attached configuration, membrane stretch with negative pressure induced two types of stretch-activated (SA) ion channels: a nonselective cation channel and a large-conductance Ca2+-activated K+ (BK(Ca)) channel. The single-channel conductances of SA cation and BK(Ca) channels were 26 and 203 pS, respectively. To elucidate the mechanism of activation of these SA channels and to minimize mechanical disruption, a sinusoidal change in pipette pressure was applied to the on-cell membrane patch. During dynamic changes in pipette pressure, increases in SA cation channel activity was found to coincide with increases in BK(Ca) channel activity. In the continued presence of cyclic stretch, the activity of SA cation channels gradually diminished. However, after termination of cyclic stretch, BK(Ca) channel activity was greatly enhanced, but the activity of SA cation channels disappeared. CONCLUSION This study is the first to demonstrate that the behavior of SA cation and BK(Ca) channels in coronary smooth muscle cells is differentially susceptible to dynamic changes in membrane tension.
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Affiliation(s)
- Sheng-Nan Wu
- Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung City, Taiwan, ROC.
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29
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Lyford GL, Strege PR, Shepard A, Ou Y, Ermilov L, Miller SM, Gibbons SJ, Rae JL, Szurszewski JH, Farrugia G. alpha(1C) (Ca(V)1.2) L-type calcium channel mediates mechanosensitive calcium regulation. Am J Physiol Cell Physiol 2002; 283:C1001-8. [PMID: 12176756 DOI: 10.1152/ajpcell.00140.2002] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the alpha(1C) L-type calcium channel subunit (Ca(V)1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a beta(2) calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac alpha(1C) splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the alpha(1C) COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming alpha(1C)-subunit.
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Affiliation(s)
- Greg L Lyford
- Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 55905, USA
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30
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Li L, Jin NG, Piao L, Hong MY, Jin ZY, Li Y, Xu WX. Hyposmotic membrane stretch potentiated muscarinic receptor agonist-induced depolarization of membrane potential in guinea-pig gastric myocytes. World J Gastroenterol 2002; 8:724-7. [PMID: 12174386 PMCID: PMC4656328 DOI: 10.3748/wjg.v8.i4.724] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the relationship between hyposmotic membrane stretch and muscarinic receptor agonist-induced depolarization of membrane potential in antral gastric circular myocytes of guinea-pig.
METHODS: Using whole cell patch-clamp technique recorded membrane potential and current in single gastric myocytes isolated by collagenase.
RESULTS: Hyposmotic membrane stretch hyperpolarized membrane potential from -60.0 mV ± 1.0 mV to -67.9 mV ± 1.0 mV. TEA (10 mmol/L), a nonselective potassium channel blocker significantly inhibited hyposmotic membrane stretch-induced hyperpolarization. After KCl in the pipette and NaCl in the external solution were replaced by CsCl to block the potassium current, hyposmotic membrane stretch depolarized the membrane potential from -60.0 mV ± 1.0 mV to -44.8 mV ± 2.3 mV (P < 0.05), and atropine (1 μmol/L) inhibited the depolarization of the membrane potential. Muscarinic receptor agonist Carbachol depolarized membrane potential from -60.0 mV ± 1.0 mV to -50.3 mV ± 0.3 mV (P < 0.05) and hyposmotic membrane stretch potentiated the depolarization. Carbachol induced muscarinic current (Icch) was greatly increased by hyposmotic membrane stretch.
CONCLUSION: Hyposmotic membrane stretch potentiated muscarinic receptor agonist-induced depolarization of membrane potential, which is related to hyposmotic membrane stretch-induced increase of muscarinic current.
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Affiliation(s)
- Lin Li
- Department of Physioloy, Yanbian University College of Medicine, Juzi 121, Yanji 133000, Jilin Province, China.
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31
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Charras GT, Horton MA. Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation. Biophys J 2002; 82:2970-81. [PMID: 12023220 PMCID: PMC1302085 DOI: 10.1016/s0006-3495(02)75638-5] [Citation(s) in RCA: 187] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The skeleton adapts to its mechanical usage, although at the cellular level, the distribution and magnitude of strains generated and their detection are ill-understood. The magnitude and nature of the strains to which cells respond were investigated using an atomic force microscope (AFM) as a microindentor. A confocal microscope linked to the setup enabled analysis of cellular responses. Two different cell response pathways were identified: one, consequent upon contact, depended on activation of stretch-activated ion channels; the second, following stress relaxation, required an intact microtubular cytoskeleton. The cellular responses could be modulated by selectively disrupting cytoskeletal components thought to be involved in the transduction of mechanical stimuli. The F-actin cytoskeleton was not required for responses to mechanical strain, whereas the microtubular and vimentin networks were. Treatments that reduced membrane tension, or its transmission, selectively reduced contact reactions. Immunostaining of the cell cytoskeleton was used to interpret the results of the cytoskeletal disruption studies. We provide an estimate of the cellular strain magnitude needed to elicit intracellular calcium responses and propose a model that links single cell responses to whole bone adaptation. This technique may help to understand adaptation to mechanical usage in other organs.
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Affiliation(s)
- Guillaume T Charras
- The Bone and Mineral Center, The Rayne Institute, Department of Medicine, University College, London WC1E 6JJ, United Kingdom
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32
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Abstract
Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor-mediated Ca(2+) release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to approximately 120% of slack length (DeltaL = 20) evoked Ca(2+) release from intracellular stores in the form of single Ca(2+) sparks and propagated Ca(2+) waves. Ca(2+) release was not due to calcium-induced calcium release, as release was observed in Ca(2+)-free extracellular solution and when free Ca(2+) ions in the cytosol were strongly buffered to prevent increases in [Ca(2+)](i). Stretch-induced calcium release (SICR) was not affected by inhibition of InsP(3)R-mediated Ca(2+) release, but was completely blocked by ryanodine. Release occurred in the absence of previously reported stretch-activated currents; however, SICR evoked calcium-activated chloride currents in the form of transient inward currents, suggesting a regulatory mechanism for the generation of spontaneous currents in smooth muscle. SICR was also observed in individual myocytes during stretch of intact urinary bladder smooth muscle segments. Thus, longitudinal stretch of smooth muscle cells induces Ca(2+) release through gating of RYR. SICR may be an important component of the physiological response to increases in luminal pressure in smooth muscle tissues.
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Affiliation(s)
- Guangju Ji
- Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
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33
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Zou H, Lifshitz LM, Tuft RA, Fogarty KE, Singer JJ. Visualization of Ca2+ entry through single stretch-activated cation channels. Proc Natl Acad Sci U S A 2002; 99:6404-9. [PMID: 11983921 PMCID: PMC122961 DOI: 10.1073/pnas.092654999] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Stretch-activated channels (SACs) have been found in smooth muscle and are thought to be involved in myogenic responses. Although SACs have been shown to be Ca(2+) permeable when Ca(2+) is the only charge carrier, it has not been clearly demonstrated that significant Ca(2+) passes through SACs in physiological solutions. By imaging at high temporal and spatial resolution the single-channel Ca(2+) fluorescence transient (SCCaFT) arising from Ca(2+) entry through a single SAC opening, we provide direct evidence that significant Ca(2+) can indeed pass through SACs and increase the local [Ca(2+)]. Results were obtained under conditions where the only source of Ca(2+) was the physiological salt solution in the patch pipette containing 2 mM Ca(2+). Single smooth muscle cells were loaded with fluo-3 acetoxymethyl ester, and the fluorescence was recorded by using a wide-field digital imaging microscope while SAC currents were simultaneously recorded from cell-attached patches. Fluorescence increases at the cell-attached patch were clearly visualized before the simultaneous global Ca(2+) increase that occurred because of Ca(2+) influx through voltage-gated Ca(2+) channels when the membrane was depolarized by inward SAC current. From measurements of total fluorescence ("signal mass") we determined that about 18% of the SAC current is carried by Ca(2+) at membrane potentials more negative than the resting level. This would translate into at least a 0.35-pA unitary Ca(2+) current at the resting potential. Such Ca(2+) currents passing through SACs are sufficient to activate large-conductance Ca(2+)-activated K(+) channels and, as shown previously, to trigger Ca(2+) release from intracellular stores.
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Affiliation(s)
- Hui Zou
- Department of Physiology and Biomedical Imaging Group, University of Massachusetts Medical School, Worcester, MA 01655, USA.
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Hill MA, Zou H, Potocnik SJ, Meininger GA, Davis MJ. Invited review: arteriolar smooth muscle mechanotransduction: Ca(2+) signaling pathways underlying myogenic reactivity. J Appl Physiol (1985) 2001; 91:973-83. [PMID: 11457816 DOI: 10.1152/jappl.2001.91.2.973] [Citation(s) in RCA: 204] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The smooth muscle of arterioles responds to an increase in intraluminal pressure with vasoconstriction and with vasodilation when pressure is decreased. Such myogenic vasoconstriction provides a level of basal tone that enables arterioles to appropriately adjust diameter in response to neurohumoral stimuli. Key in this process of mechanotransduction is the role of changes in intracellular Ca(2+). However, it is becoming clear that considerable complexity exists in the spatiotemporal characteristics of the Ca(2+) signal and that changes in intracellular Ca(2+) may play roles other than direct effects on the contractile process via activation of myosin light-chain phosphorylation. The involvement of Ca(2+) may extend to modulation of ion channels and release of Ca(2+) from the sarcoplasmic reticulum, alterations in Ca(2+) sensitivity, and coupling between cells within the vessel wall. The purpose of this brief review is to summarize the current literature relating to Ca(2+) and the arteriolar myogenic response. Consideration is given to coupling of Ca(2+) changes to the mechanical stimuli, sources of Ca(2+), involvement of ion channels, and spatiotemporal aspects of intracellular Ca(2+) signaling.
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Affiliation(s)
- M A Hill
- Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia.
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35
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Abstract
The rat middle colon spontaneously generates regularly occurring giant contractions (GCs) in vitro. We investigated the neurohumoral and intracellular regulation of these contractions in a standard muscle bath. cGMP content was measured in strips and single smooth muscle cells. The circular muscle strips generated spontaneous GCs. Their amplitude and frequency were significantly increased by tetrodotoxin (TTX), omega-conotoxin, N(omega)-nitro-L-arginine (L-NNA), and the dopamine D(1) receptor antagonist Sch-23390. The GCs were unaffected by hexamethonium, atropine, and antagonists of serotonergic (5-HT(1--4)), histaminergic (H(1--2)), and tachykininergic (NK(1--2)) receptors but enhanced by NK(3) receptor antagonism. The guanylate cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ) also enhanced GCs to the same extent as TTX and L-NNA, and each of the three agents prevented the effects of the others. GCs were abolished by electrical field stimulation, S-nitroso-N-acetyl-penicillamine, and 8-bromo-cGMP. BAY-K-8644 and apamin enhanced the GCs, but they were abolished by D-600. Basal cGMP content in strips was decreased by TTX, L-NNA, or ODQ, but these treatments had no effect on cGMP content of enzymatically dissociated single smooth muscle cells. We conclude that spontaneous contractions in the rat colonic muscle strips are not generated by cholinergic, serotonergic, or histaminergic input. Constitutive release of nitric oxide from enteric neurons sustains cGMP synthesis in the colonic smooth muscle to suppress spontaneous in vitro GCs.
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Affiliation(s)
- A Gonzalez
- Department of Surgery, Medical College of Wisconsin, Milwaukee 53266, USA
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36
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Abstract
The simplest cell-like structure, the lipid bilayer vesicle, can respond to mechanical deformation by elastic membrane dilation/thinning and curvature changes. When a protein is inserted in the lipid bilayer, an energetic cost may arise because of hydrophobic mismatch between the protein and bilayer. Localized changes in bilayer thickness and curvature may compensate for this mismatch. The peptides alamethicin and gramicidin and the bacterial membrane protein MscL form mechanically gated (MG) channels when inserted in lipid bilayers. Their mechanosensitivity may arise because channel opening is associated with a change in the protein's membrane-occupied area, its hydrophobic mismatch with the bilayer, excluded water volume, or a combination of these effects. As a consequence, bilayer dilation/thinning or changes in local membrane curvature may shift the equilibrium between channel conformations. Recent evidence indicates that MG channels in specific animal cell types (e.g., Xenopus oocytes) are also gated directly by bilayer tension. However, animal cells lack the rigid cell wall that protects bacteria and plants cells from excessive expansion of their bilayer. Instead, a cortical cytoskeleton (CSK) provides a structural framework that allows the animal cell to maintain a stable excess membrane area (i.e., for its volume occupied by a sphere) in the form of membrane folds, ruffles, and microvilli. This excess membrane provides an immediate membrane reserve that may protect the bilayer from sudden changes in bilayer tension. Contractile elements within the CSK may locally slacken or tighten bilayer tension to regulate mechanosensitivity, whereas membrane blebbing and tight seal patch formation, by using up membrane reserves, may increase membrane mechanosensitivity. In specific cases, extracellular and/or CSK proteins (i.e., tethers) may transmit mechanical forces to the process (e.g., hair cell MG channels, MS intracellular Ca(2+) release, and transmitter release) without increasing tension in the lipid bilayer.
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Affiliation(s)
- O P Hamill
- Physiology and Biophysics, University Of Texas Medical Branch, Galveston, Texas 77555, USA.
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Daniel EE, Kwan CY, Janssen L. Pharmacological techniques for the in vitro study of intestinal smooth muscles. J Pharmacol Toxicol Methods 2001; 45:141-58. [PMID: 11687381 DOI: 10.1016/s1056-8719(01)00131-9] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
MESH Headings
- Animals
- Dose-Response Relationship, Drug
- Electric Stimulation
- Humans
- In Vitro Techniques
- Intestine, Large/drug effects
- Intestine, Large/innervation
- Intestine, Large/physiology
- Intestine, Small/drug effects
- Intestine, Small/innervation
- Intestine, Small/physiology
- Muscle Contraction/drug effects
- Muscle Contraction/physiology
- Muscle, Smooth/drug effects
- Muscle, Smooth/innervation
- Muscle, Smooth/physiology
- Xenobiotics/pharmacology
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Affiliation(s)
- E E Daniel
- Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada
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