TRPM7 Is Involved in Volume Regulation in Salivary Glands

2017 ◽  
Vol 96 (9) ◽  
pp. 1044-1050 ◽  
Author(s):  
J.M. Kim ◽  
S. Choi ◽  
K. Park
Keyword(s):  
Critical Role ◽  
Regulatory Volume Decrease ◽  
Specific Inhibitor ◽  
Acinar Cells ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Current Profile ◽  
Patch Clamp Technique ◽  
Extracellular Medium ◽  
Intracellular Ca

Under hypotonic conditions, the regulatory volume decrease (RVD) is essential to maintain physiological homeostasis and functions in diverse biological systems. Intracellular Ca2+ has been reported as an important mediator of this response, but the underlying Ca2+ mechanism responsible for RVD is still controversial. Here we investigate the role of Ca2+ in the RVD response using live-cell imaging, microspectrofluorimetry, and a patch-clamp technique. A typical RVD was observed in submandibular gland acinar cells after swelling in a hypotonic solution, whereas intracellular Ca2+ chelation completely inhibited the RVD response. The incidence and magnitude of the Ca2+ transient were proportional to the degree of hypotonicity of the extracellular medium, and there was a close relationship between intracellular Ca2+ concentration and the volumetric changes of the cells. Notably, this response was mediated by Ca2+-induced Ca2+ release, which is triggered by Ca2+ influx via stretch-activated TRPM7 channels. Furthermore, we detected the generation of Cl− currents in the swelling acinar cells upon hypotonic stress, and the current profile matched that of the Ca2+-activated Cl− currents. A specific inhibitor of Cl− currents also inhibited the RVD response. In conclusion, an intracellular Ca2+ increase in response to osmotically induced cell swelling plays a critical role in RVD in salivary gland acinar cells.

Dihydropyridine-sensitive cell volume regulation in proximal tubule: the calcium window

1990 ◽  
Vol 259 (6) ◽  
pp. F950-F960 ◽  
Author(s):  
N. A. McCarty ◽  
R. G. O'Neil
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Cell Swelling ◽  
Channel Blockers ◽  
Ca Channel ◽  
Hypotonic Solution ◽  
Dependent Manner ◽  
Intracellular Ca

The mechanism underlying the activation of hypotonic cell volume regulation was studied in rabbit proximal straight tubule (PST). When isolated non-perfused tubules were exposed to hypotonic solution, cells swelled rapidly and then underwent a regulatory volume decrease (RVD). The extent of regulation after swelling was highly dependent on extracellular Ca concentration ([Ca2+]o), with a half-maximal inhibition (K1/2) for [Ca2+]o of approximately 100 microM. RVD was blocked by the Ca-channel blockers verapamil, lanthanum, and the dihydropyridines (DHP) nifedipine and nitrendipine, implicating voltage-activated Ca channels in the RVD response. Using the fura-2 fluorescence-ratio technique, we observed that cell swelling caused a sustained rise in intracellular Ca ([Ca2+]i) only when [Ca2+]o was normal (1 mM) but not when [Ca2+]o was low (1-10 microM). Furthermore, external Ca was required early on during swelling to induce RVD. If RVD was initially blocked by reducing [Ca2+]o or by addition of verapamil during hypotonic swelling, volume regulation could only be restored by subsequently inducing Ca entry within the first 1 min or less of exposure to hypotonic solution. These data indicate a "calcium window" of less than 1 min, during which RVD is sensitive to Ca, and that part of the Ca-dependent mechanism responsible for achieving RVD undergoes inactivation after swelling. It is concluded that RVD in rabbit PST is modulated by Ca via a DHP-sensitive mechanism in a time-dependent manner.

Potassium conductance activated during regulatory volume decrease by mudpuppy red blood cells

1996 ◽  
Vol 270 (4) ◽  
pp. R801-R810
Author(s):  
L. J. Bergeron ◽  
A. J. Stever ◽  
D. B. Light
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Enhanced Recovery ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
K Channel ◽  
Ionophore A23187 ◽  
Patch Clamp Technique ◽  
Extracellular Medium ◽  
Volume Decrease

The cellular basis of regulatory volume decrease (RVD) by mudpuppy (Necturus maculosus) red blood cells (RBCs) was examined. Volume regulation was inhibited by replacing extracellular Na+ with K+. In contrast, addition of gramicidin (5 microM) to the extracellular medium enhanced RVD. The K(+)-channel blocker quinine (1 mM) also inhibited RVD, and this inhibition was reversed by gramicidin (5 microM). In addition, a 0 Ca(2+)-EGTA Ringer blocked RVD, whereas the Ca2+ ionophore A23187 ( microM) enhanced recovery of cell volume. The stretch-activated ion channel antagonist gadolinium (10 microM) inhibited RVD, and this effect was reversed by A23187 (2 microM). Furthermore, the calmodulin inhibitors pimozide (10 microM) and N-(6-aminohexyl)-5-chloro-1-napthalene-sulfonamide (0.1 mM) blocked RVD, and this inhibition was reversed with gramicidin (5 microM). Consistent with these findings, a K(+)-selective membrane conductance was activated by exposing RBCs to a 0.5x Ringer solution (observed with the whole cell patch clamp technique). This conductance was inhibited by quinine (1 mM), gadolinium (10 microM), and pimozide (10 microM). These results indicate that cell swelling activates a K+ conductance by a Ca(2+)-calmodulin-dependent mechanism and that this channel mediates K+ loss during RVD.

Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells

1995 ◽  
Vol 73 (7-8) ◽  
pp. 535-544 ◽  
Author(s):  
C. Lo ◽  
J. Ferrier ◽  
H. C. Tenenbaum ◽  
C. A. G. McCulloch
Keyword(s):  
Cell Volume ◽  
Intracellular Ph ◽  
Binding Proteins ◽  
Kinase Inhibitor ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Osteosarcoma Cells ◽  
Extracellular Medium ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of ~30% in suspended (i.e., round) cells and ~60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with B APTA–AM and by depletion of Cl−, indicating the role of calcium-regulated K+ and Cl− efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (~0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na+-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl−-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl− that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.Key words: cell volume, pH, osteoblast, G proteins, actin.

Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated K+ channels

2000 ◽  
Vol 279 (5) ◽  
pp. R1647-R1658 ◽  
Author(s):  
C. Duranton ◽  
E. Mikulovic ◽  
M. Tauc ◽  
M. Avella ◽  
P. Poujeol
Keyword(s):  
Potassium Channels ◽  
Primary Cultures ◽  
Dicentrarchus Labrax ◽  
Sea Bass ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Ionic Substitution ◽  
Gill Cells

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl− channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K+ channel of 123 ± 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K+ over Na+ and Ca2+. The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 μM), and gadolinium (500 μM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K+ channel activity is consistent with the hypothesis that stretch-activated K+ channels could mediate an increase in K+ conductance during cell swelling.

scholarly journals Flavonoid Myricetin ModulatesGABAAReceptor Activity through Activation ofCa2+Channels and CaMK-II Pathway

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Xiao Hu Zhang ◽  
Ze Gang Ma ◽  
Dewi Kenneth Rowlands ◽  
Yu Lin Gou ◽  
Kin Lam Fok ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Binding Site ◽  
Primary Culture ◽  
Brain Slice ◽  
Specific Inhibitor ◽  
Patch Clamp Technique ◽  
Intracellular Ca ◽  
Gabaergic Activity ◽  
Brain Slice Preparation ◽  
Dependent Mechanism

The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated byGABAAreceptor. This effect of myricetin was not blocked by theGABAAreceptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca2+/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca2+imaging studies found that myricetin could increase Ca2+current and intracellular Ca2+concentration, respectively, via T- and L-type Ca2+channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhancesGABAAreceptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists ofGABAAreceptor.

Mechanisms of Ca2+-stimulated fluid secretion by porcine bronchial submucosal gland serous acinar cells

2010 ◽  
Vol 298 (2) ◽  
pp. L210-L231 ◽  
Author(s):  
Robert J. Lee ◽  
J. Kevin Foskett
Keyword(s):  
Basolateral Membrane ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
Cell Swelling ◽  
Solute Content ◽  
Intracellular Ca ◽  
Shrinkage And Swelling ◽  
Serous Cell ◽  
Rat Parotid ◽  
Necessary And Sufficient

The serous acini of airway submucosal glands are important for fluid secretion in the lung. Serous cells are also sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. However, the mechanisms of serous cell fluid secretion remain poorly defined. In this study, serous acinar cells were isolated from porcine bronchi and studied using optical techniques previously used to examine fluid secretion in rat parotid and murine nasal acinar cells. When stimulated with the cholinergic agonist carbachol, porcine serous cells shrank by ∼20% (observed via DIC microscopy) after a profound elevation of intracellular [Ca2+] ([Ca2+]i; measured by simultaneous fura 2 fluorescence imaging). Upon removal of agonist and relaxation of [Ca2+]i to resting levels, cells swelled back to resting volume. Similar results were observed during stimulation with histamine and ATP, and elevation of [Ca2+]i was found to be necessary and sufficient to activate shrinkage. Cell volume changes were associated with changes in [Cl−]i (measured using SPQ fluorescence), suggesting that shrinkage and swelling are caused by loss and gain of intracellular solute content, respectively, likely reflecting changes in the secretory state of the cells. Shrinkage was inhibited by niflumic acid but not by GlyH-101, suggesting Ca2+-activated secretion is mediated by alternative non-CFTR Cl− channels, possibly including Ano1 (TMEM16A), expressed on the apical membrane of porcine serous cells. Optimal cell swelling/solute uptake required activity of the Na+K+2Cl− cotransporter and Na+/H+ exchanger, both of which are expressed on the basolateral membrane of serous acini and likely contribute to sustaining transepithelial secretion.

scholarly journals Effect of osmotic stress on the expression of TRPV4 and BKCa channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes

2014 ◽  
Vol 306 (11) ◽  
pp. C1050-C1057 ◽  
Author(s):  
Ismail M. Hdud ◽  
Ali Mobasheri ◽  
Paul T. Loughna
Keyword(s):  
Osmotic Stress ◽  
P38 Mapk ◽  
Transient Receptor Potential ◽  
Articular Chondrocytes ◽  
Regulatory Volume Decrease ◽  
Specific Inhibitor ◽  
Cell Swelling ◽  
Transient Receptor Potential Vanilloid ◽  
Channel Expression ◽  
Trpv4 Channel

The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and large-conductance Ca2+-activated K+ (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process. Isolated equine articular chondrocytes were subjected to anisosmotic conditions, and TRPV4 and BKCa channel expression and ERK1/2 and p38 MAPK protein phosphorylation were investigated using Western blotting. Results indicate that the TRPV4 channel contributes to the early stages of hypo-osmotic stress, while the BKCa channel is involved in responding to elevated intracellular Ca2+ and mediating regulatory volume decrease. ERK1/2 is phosphorylated by hypo-osmotic stress ( P < 0.001), and p38 MAPK is phosphorylated by hyperosmotic stress ( P < 0.001). In addition, this study demonstrates the importance of endogenous ERK1/2 phosphorylation in TRPV4 channel expression, where blocking ERK1/2 by a specific inhibitor (PD98059) prevented increased levels of the TRPV4 channel in cells exposed to hypo-osmotic stress and decreased TRPV4 channel expression to below control levels in iso-osmotic conditions ( P < 0.001).

scholarly journals Interaction between bradykinin and natriuretic peptides via RGS protein activation in HEK-293 cells

2012 ◽  
Vol 303 (12) ◽  
pp. C1260-C1268 ◽  
Author(s):  
Marina Dobrivojević ◽  
Aleksandra Sinđić ◽  
Bayram Edemir ◽  
Stefanie Kalweit ◽  
Wolf-Georg Forssmann ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Natriuretic Peptide ◽  
Natriuretic Peptides ◽  
Specific Inhibitor ◽  
Rgs Proteins ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Intracellular Ca

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential ( Vm) and intracellular Ca2+ using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B2R) and Ca2+-dependent Cl− channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 μM). The BK-induced Ca2+ signal was blocked by the B2R inhibitor HOE 140. [Des-Arg9]-bradykinin, an activator of B1R, had no effect on intracellular Ca2+. NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K+ channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca2+ signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 μM) completely inhibited the BK-induced Ca2+ signaling. UTP, another activator of the PLC-mediated Ca2+ signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B2R signaling pathway, NP may also positively interact in pathological conditions caused by BK.

scholarly journals Ca2+- and Volume-sensitive Chloride Currents Are Differentially Regulated by Agonists and Store-operated Ca2+ Entry

2005 ◽  
Vol 125 (2) ◽  
pp. 197-211 ◽  
Author(s):  
Alexander Zholos ◽  
Benjamin Beck ◽  
Vadym Sydorenko ◽  
Loïc Lemonnier ◽  
Pascal Bordat ◽  
...  
Keyword(s):  
Imaging Techniques ◽  
Regulatory Volume Decrease ◽  
Human Keratinocytes ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Cytoplasmic Calcium ◽  
Intact Cells ◽  
Chloride Currents ◽  
Pipette Solution ◽  
Transient Elevation

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca2+-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca2+-activated (ICl(Ca)) and volume-regulated (ICl, swell) chloride currents. NPPB and DIDS more efficiently inhibited ICl(Ca) and ICl, swell, respectively. Cell swelling caused by hypotonic solution invariably activated ICl, swell while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling–induced ICl, swell, while its inactive analogue U-73343 had no effect. ICl(Ca) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, ICl, swell could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced ICl, swell in a nonadditive manner, suggesting their convergence on a common pathway. ICl, swell and ICl(Ca) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated ICl(Ca), but abolished ICl, swell, thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that ICl, swell can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a “selection” switch between closely localized channels.

scholarly journals Oxidative stress affects responsiveness to hypotonicity of renal cells

2014 ◽  
Vol 87 (2) ◽  
Author(s):  
Rossana Morabito ◽  
Giuseppa La Spada ◽  
Silvia Dossena ◽  
Angela Marino
Keyword(s):  
Oxidative Stress ◽  
Cell Volume ◽  
Critical Role ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Isotonic Solution ◽  
Renal Cells ◽  
Cell Shrinkage ◽  
Hypotonic Stress ◽  
Volume Decrease

Oxidative stress plays a critical role in the pathophysiology of several kidney diseases and is the consequence of alterations like ischemic events. The regulatory volume decrease (RVD) is an homeostatic response essential to many cells, including renal cells, to counteract changes in the osmolarity of the external medium. The aim of the present work is to verify whether oxidative stress affects RVD in a model of renal cells (human embryonic kidney cells, HEK 293 Phoenix). To accomplish this aim, the experimental procedure consisted in: i) cell culture preparation and treatment with 200 μM H<sub>2</sub>O<sub>2</sub>; and ii) measurement of cell volume changes in isotonic conditions or following hypotonic stress. H<sub>2</sub>O<sub>2</sub> added to the extracellular isotonic solution induced a significant reduction in cell volume, and added to the extracellular hypotonic solution dramatically impaired the expected osmotic cell swelling. Pre-incubation of cells in an extracellular isotonic solution containing H<sub>2</sub>O<sub>2</sub> prevented cell from swelling after hypotonic stress application. In conclusion, H<sub>2</sub>O<sub>2</sub> leads to cell shrinkage in isotonic conditions, inhibits the hypotonicity-induced cell swelling and consequently prevents RVD, hypothetically due to an activation of transport pathways determining ion loss and, in turn, water efflux. Cell shrinkage in isotonic conditions is a hallmark of apoptosis and is known as the apoptotic volume decrease.

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