Abstract 632: Flow-induced TRPV4 Activation Mediates Calcium Influx In The Thick Ascending Limb.

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Pablo D Cabral ◽  
Jeffrey L Garvin
Keyword(s):  
Blood Pressure ◽  
Intracellular Calcium ◽  
Calcium Influx ◽  
Ruthenium Red ◽  
Sodium Balance ◽  
Transient Receptor Potential Vanilloid ◽  
No Production ◽  
Thick Ascending Limb ◽  
Fura 2 ◽  
Luminal Flow

Background: The thick ascending limb is critical in the regulation of sodium balance and therefore blood pressure. We previously showed that nitric oxide (NO), which inhibits sodium chloride transport and therefore blood pressure is regulated by luminal flow in this segment. Transient receptor potential vanilloid 4 (TRPV4), a member of the TRPV family of cation channels, is expressed in the thick ascending limb. This calcium permeable channel can be activated by luminal flow in other cell types. Therefore, we hypothesized that in the thick ascending limb luminal flow induces TRPV4 activation thereby increasing calcium influx and NO production. METHODS: We used the TRPV4 antagonists Ruthenium red and RN 1734 and the TRPV4 agonist GSK1016790A and measured intracellular calcium and NO production in the absence and presence of luminal flow in isolated and perfused thick ascending limbs from Sprague Dawley rats. NO production was measured using the NO-sensitive dye DAF FM da. Intracellular calcium was measured using the ratiometric calcium sensitive dye Fura-2 AM. Results: Increasing luminal flow from 0 to 20 nL/min stimulated NO production from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min ( p <0.05, n =5). Increasing luminal flow in the presence of the TRPV4 antagonist Ruthenium red (15 μM) eliminated NO production (from 18 ± 5 to 16 ± 9 AU/min; n = 4). Flow-induced NO production was also prevented by the selective TRPV4 antagonist RN 1734 (10 μM) (from 11 ± 7 to 9 ± 2 AU/min; n = 4). Increasing luminal flow increased Fura 2 ratio units by 113 ± 20 %. However, in the presence of the TRPV4 antagonist Ruthenium red (15 μM) Fura 2 ratio units increased only by 55 ± 12 % in response to luminal flow ( p <0.04, n =6). In the absence of luminal flow, activating TRPV4 using the TRPV4 selective agonist GSK1016790A (1 ηM) increased Fura 2 ratio units by 204 ± 43 %. Conclusion: From these data we conclude that flow-induced TRPV4 activation mediates calcium influx and NO production in the thick ascending limb.

scholarly journals TRPV4 activation mediates flow-induced nitric oxide production in the rat thick ascending limb

2014 ◽  
Vol 307 (6) ◽  
pp. F666-F672 ◽  
Author(s):  
Pablo D. Cabral ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Ruthenium Red ◽  
Transient Receptor Potential Vanilloid ◽  
No Production ◽  
Thick Ascending Limb ◽  
Different Types ◽  
Luminal Flow ◽  
Transient Receptor

Nitric oxide (NO) regulates renal function. Luminal flow stimulates NO production in the thick ascending limb (TAL). Transient receptor potential vanilloid 4 (TRPV4) is a mechano-sensitive channel activated by luminal flow in different types of cells. We hypothesized that TRPV4 mediates flow-induced NO production in the rat TAL. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. Increasing luminal flow from 0 to 20 nl/min stimulated NO from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min ( n = 5; P < 0.05). The TRPV4 antagonists, ruthenium red (15 μmol/l) and RN 1734 (10 μmol/l), blocked flow-induced NO production. Also, luminal flow did not increase NO production in the absence of extracellular calcium. We also studied the effect of luminal flow on NO production in TALs transduced with a TRPV4shRNA. In nontransduced TALs luminal flow increased NO production by 47 ± 17 AU/min ( P < 0.05; n = 5). Similar to nontransduced TALs, luminal flow increased NO production by 39 ± 11 AU/min ( P < 0.03; n = 5) in TALs transduced with a control negative sequence-shRNA while in TRPV4shRNA-transduced TALs, luminal flow did not increase NO production (Δ10 ± 15 AU/min; n = 5). We then tested the effect of two different TRPV4 agonists on NO production in the absence of luminal flow. 4α-Phorbol 12,13-didecanoate (1 μmol/l) enhanced NO production by 60 ± 11 AU/min ( P < 0.002; n = 7) and GSK1016790A (10 ηmol/l) increased NO production by 52 ± 15 AU/min ( P < 0.03; n = 5). GSK1016790A (10 ηmol/l) did not stimulate NO production in TRPV4shRNA-transduced TALs. We conclude that activation of TRPV4 channels mediates flow-induced NO production in the rat TAL.

scholarly journals Luminal flow induces eNOS activation and translocation in the rat thick ascending limb

2004 ◽  
Vol 287 (2) ◽  
pp. F274-F280 ◽  
Author(s):  
Pablo A. Ortiz ◽  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Apical Membrane ◽  
Renal Medulla ◽  
Cytochalasin D ◽  
No Synthase ◽  
No Production ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Luminal Flow ◽  
Nos Inhibitor

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) acts as an autacoid to inhibit NaCl absorption in the thick ascending limb of the loop of Henle (THAL). In the vasculature, shear stress activates eNOS. We hypothesized that increasing luminal flow activates eNOS and enhances NO production in the THAL. We measured NO production by isolated, perfused THALs using a NO-sensitive microelectrode. Increasing luminal flow from 0 to 20 nl/min increased NO production by 43.1 ± 4.1 pA/mm of tubule ( n = 10, P < 0.05), and this response was blunted (92%) by the NOS inhibitor l-ωnitro-methylarginine ( P < 0.05). We studied the effect of flow on eNOS subcellular localization. In the absence of flow, eNOS was diffusely localized throughout the cell (basolateral = 33 ± 4%; middle = 27 ± 3%; apical = 40 ± 4% of total eNOS). Increasing luminal flow induced eNOS translocation to the apical membrane, as evidenced by a 60% increase in eNOS immunoreactivity in the apical membrane (from 40 ± 4 to 65 ± 2%; n = 6; P < 0.05). Disrupting the actin cytoskeleton with cytochalasin D (10 μM) reduced flow-induced NO production by 62% (from 37.1 ± 3.4 to 14.0 ± 2.4 pA/mm tubule, n = 7, P < 0.04) and blocked flow-induced eNOS translocation. Flow also increased the amount of phosphorylated eNOS (Ser1179) at the apical membrane (from 25 ± 2 to 56 ± 2%; P < 0.05). We conclude that increasing luminal flow induces eNOS activation and translocation to the apical membrane in THALs. These are the first data showing that flow regulates eNOS in epithelial cells. This may be an important mechanism for regulation of NO levels in the renal medulla.

scholarly journals Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores

2011 ◽  
Vol 301 (3) ◽  
pp. C679-C686 ◽  
Author(s):  
Peter Sobolewski ◽  
Judith Kandel ◽  
Alexandra L. Klinger ◽  
David M. Eckmann
Keyword(s):  
Endothelial Cells ◽  
Intracellular Calcium ◽  
Calcium Release ◽  
Transient Receptor Potential ◽  
Calcium Influx ◽  
Calcium Transient ◽  
Epifluorescence Microscopy ◽  
Transient Receptor Potential Vanilloid ◽  
Calcium Stores ◽  
Air Bubble

Gas embolism is a serious complication of decompression events and clinical procedures, but the mechanism of resulting injury remains unclear. Previous work has demonstrated that contact between air microbubbles and endothelial cells causes a rapid intracellular calcium transient and can lead to cell death. Here we examined the mechanism responsible for the calcium rise. Single air microbubbles (50–150 μm), trapped at the tip of a micropipette, were micromanipulated into contact with individual human umbilical vein endothelial cells (HUVECs) loaded with Fluo-4 (a fluorescent calcium indicator). Changes in intracellular calcium were then recorded via epifluorescence microscopy. First, we confirmed that HUVECs rapidly respond to air bubble contact with a calcium transient. Next, we examined the involvement of extracellular calcium influx by conducting experiments in low calcium buffer, which markedly attenuated the response, or by pretreating cells with stretch-activated channel blockers (gadolinium chloride or ruthenium red), which abolished the response. Finally, we tested the role of intracellular calcium release by pretreating cells with an inositol 1,4,5-trisphosphate (IP3) receptor blocker (xestospongin C) or phospholipase C inhibitor (neomycin sulfate), which eliminated the response in 64% and 67% of cases, respectively. Collectively, our results lead us to conclude that air bubble contact with endothelial cells causes an influx of calcium through a stretch-activated channel, such as a transient receptor potential vanilloid family member, triggering the release of calcium from intracellular stores via the IP3 pathway.

scholarly journals Shear stress increases nitric oxide production in thick ascending limbs

2010 ◽  
Vol 299 (5) ◽  
pp. F1185-F1192 ◽  
Author(s):  
Pablo D. Cabral ◽  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Shear Stress ◽  
Primary Cultures ◽  
Physiological Saline ◽  
Shear Stresses ◽  
No Production ◽  
Thick Ascending Limb ◽  
Luminal Flow ◽  
Nos Inhibitor ◽  
Stimulation Of

We showed that luminal flow stimulates nitric oxide (NO) production in thick ascending limbs. Ion delivery, stretch, pressure, and shear stress all increase when flow is enhanced. We hypothesized that shear stress stimulates NO in thick ascending limbs, whereas stretch, pressure, and ion delivery do not. We measured NO in isolated, perfused rat thick ascending limbs using the NO-sensitive dye DAF FM-DA. NO production rose from 21 ± 7 to 58 ± 12 AU/min ( P < 0.02; n = 7) when we increased luminal flow from 0 to 20 nl/min, but dropped to 16 ± 8 AU/min ( P < 0.02; n = 7) 10 min after flow was stopped. Flow did not increase NO in tubules from mice lacking NO synthase 3 (NOS 3). Flow stimulated NO production by the same extent in tubules perfused with ion-free solution and physiological saline (20 ± 7 vs. 24 ± 6 AU/min; n = 7). Increasing stretch while reducing shear stress and pressure lowered NO generation from 42 ± 9 to 17 ± 6 AU/min ( P < 0.03; n = 6). In the absence of shear stress, increasing pressure and stretch had no effect on NO production (2 ± 8 vs. 8 ± 8 AU/min; n = 6). Similar results were obtained in the presence of tempol (100 μmol/l), a O2− scavenger. Primary cultures of thick ascending limb cells subjected to shear stresses of 0.02 and 0.55 dyne/cm2 produced NO at rates of 55 ± 10 and 315 ± 93 AU/s, respectively ( P < 0.002; n = 7). Pretreatment with the NOS inhibitor l-NAME (5 mmol/l) blocked the shear stress-induced increase in NO production. We concluded that shear stress rather than pressure, stretch, or ion delivery mediates flow-induced stimulation of NO by NOS 3 in thick ascending limbs.

scholarly journals Standing calcium gradients in olfactory receptor neurons can be abolished by amiloride or ruthenium red.

1993 ◽  
Vol 102 (5) ◽  
pp. 817-831 ◽  
Author(s):  
F W Lischka ◽  
D Schild
Keyword(s):  
Spatial Distribution ◽  
Intracellular Calcium ◽  
Olfactory Receptor ◽  
Ruthenium Red ◽  
Olfactory Receptor Neurons ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Fura 2 ◽  
Receptor Neurons ◽  
In Cells

Digital imaging and the patch clamp technique were used to investigate the intracellular calcium concentration in olfactory receptor neurons using the Ca2+ indicator dyes fura-2 and fura-2/AM. The spatial distribution of Cai2+ as well as its modification by the drugs Amiloride and Ruthenium Red were studied. Resting calcium concentrations in cells loaded with fura-2/AM were between 10 and 200 nM. In cells that were loaded with the pentapotassium salt of fura-2 through the patch pipette, calcium concentrations were in the same range if ATP was added to the pipette solution. Otherwise, Ca2+ reached concentrations of approximately 500 nM. Most of the observed cells showed a standing gradient of calcium, the calcium concentrations in the distal dendritic end of the cell being higher than in the soma. In some cells, the gradient was markedly reduced or abolished by adding either Amiloride or Ruthenium Red to the bath solution. In a few cells, neither drug had any effect upon the gradient. It is suggested that the inhomogenous spatial distribution of intracellular calcium in olfactory cells of Xenopus laevis is brought about by an influx of calcium ions through two different calcium permeable conductances in the peripheral compartments of the cells. The fact that only either Ruthenium Red or Amiloride abolished the standing calcium gradient further suggested that the two conductances blocked were presumably not coexpressed in the same cells.

scholarly journals Luminal flow regulates NO and O2−along the nephron

2011 ◽  
Vol 300 (5) ◽  
pp. F1047-F1053 ◽  
Author(s):  
Pablo D. Cabral ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Shear Stress ◽  
Primary Cilia ◽  
Collecting Duct ◽  
Bicarbonate Transport ◽  
Nitrite Accumulation ◽  
No Production ◽  
Urinary Flow ◽  
Thick Ascending Limb ◽  
Luminal Flow

Urinary flow is not constant but in fact highly variable, altering the mechanical forces (shear stress, stretch, and pressure) exerted on the epithelial cells of the nephron as well as solute delivery. Nitric oxide (NO) and superoxide (O2−) play important roles in various processes within the kidney. Reductions in NO and increases in O2−lead to abnormal NaCl and water absorption and hypertension. In the last few years, luminal flow has been shown to be a regulator of NO and O2−production along the nephron. Increases in luminal flow enhance fluid, Na, and bicarbonate transport in the proximal tubule. However, we know of no reports directly addressing flow regulation of NO and O2−in this segment. In the thick ascending limb, flow-stimulated NO and O2−formation has been extensively studied. Luminal flow stimulates NO production by nitric oxide synthase type 3 and its translocation to the apical membrane in medullary thick ascending limbs. These effects are mediated by flow-induced shear stress. In contrast, flow-induced stretch and NaCl delivery stimulate O2−production by NADPH oxidase in this segment. The interaction between flow-induced NO and O2−is complex and involves more than one simply scavenging the other. Flow-induced NO prevents flow from increasing O2−production via cGMP-dependent protein kinase in thick ascending limbs. In macula densa cells, shear stress increases NO production and this requires that the primary cilia be intact. The role of luminal flow in NO and O2−production in the distal tubule is not known. In cultured inner medullary collecting duct cells, shear stress enhances nitrite accumulation, a measure of NO production. Although much progress has been made on this subject in the last few years, there are still many unanswered questions.

scholarly journals Functional Expression of Piezo1 in Dorsal Root Ganglion (DRG) Neurons

2020 ◽  
Vol 21 (11) ◽  
pp. 3834
Author(s):  
Jueun Roh ◽  
Sung-Min Hwang ◽  
Sun-Ho Lee ◽  
Kihwan Lee ◽  
Yong Ho Kim ◽  
...  
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Transient Receptor Potential ◽  
Channel Blocker ◽  
Calcium Influx ◽  
Ruthenium Red ◽  
Transient Receptor Potential Vanilloid ◽  
Drg Neurons ◽  
Physiological Functions ◽  
Virus Serotype

Piezo channels are mechanosensitive ion channels. Piezo1 is primarily expressed in nonsensory tissues, whereas Piezo2 is predominantly found in sensory tissues, including dorsal root ganglion (DRG) neurons. However, a recent study demonstrated the intracellular calcium response to Yoda1, a selective Piezo1 agonist, in trigeminal ganglion (TG) neurons. Herein, we investigate the expression of Piezo1 mRNA and protein in mouse and human DRG neurons and the activation of Piezo1 via calcium influx by Yoda1. Yoda1 induces inward currents mainly in small- (<25 μm) and medium-sized (25–35 μm) mouse DRG neurons. The Yoda1-induced Ca2+ response is inhibited by cationic channel blocker, ruthenium red and cationic mechanosensitive channel blocker, GsMTx4. To confirm the specific inhibition of Piezo1, we performed an adeno-associated virus serotype 2/5 (AAV2/5)-mediated delivery of short hairpin RNA (shRNA) into mouse DRG neurons. AAV2/5 transfection downregulates piezo1 mRNA expression and reduces Ca2+ response by Yoda1. Piezo1 also shows physiological functions with transient receptor potential vanilloid 1 (TRPV1) in the same DRG neurons and is regulated by the activation of TRPV1 in mouse DRG sensory neurons. Overall, we found that Piezo1 has physiological functions in DRG neurons and that TRPV1 activation inhibits an inward current induced by Yoda1.

scholarly journals ATP mediates flow-induced NO production in thick ascending limbs

2012 ◽  
Vol 303 (2) ◽  
pp. F194-F200 ◽  
Author(s):  
Pablo D. Cabral ◽  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Basolateral Membrane ◽  
Atp Release ◽  
P2y Receptors ◽  
P2x Receptor ◽  
No Production ◽  
Thick Ascending Limb ◽  
Rate Of Increase ◽  
Basolateral Side ◽  
Luminal Flow ◽  
Reduced Flow

Mechanical stimulation caused by increasing flow induces nucleotide release from many cells. Luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in thick ascending limbs. However, the factors that mediate flow-induced NO production are unknown. We hypothesized that luminal flow stimulates thick ascending limb NO production via ATP. We measured NO in isolated, perfused rat thick ascending limbs using the fluorescent dye DAF FM. The rate of increase in dye fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nl/min stimulated NO production from 17 ± 16 to 130 ± 37 arbitrary units (AU)/min ( P < 0.02). Increasing flow from 0 to 20 nl/min raised ATP release from 4 ± 1 to 21 ± 6 AU/min ( P < 0.04). Hexokinase (10 U/ml) plus glucose, which consumes ATP, completely prevented the measured increase in ATP. Luminal flow did not increase NO production in the presence of luminal and basolateral hexokinase (10 U/ml). When flow was increased with the ATPase apyrase in both luminal and basolateral solutions (5 U/ml), NO levels did not change significantly. The P2 receptor antagonist suramin (300 μmol/l) reduced flow-induced NO production by 83 ± 25% ( P < 0.03) when added to both and basolateral sides. Luminal hexokinase decreased flow-induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min ( P < 0.02). Basolateral hexokinase also reduced flow-induced NO production. The P2X receptor-selective antagonist NF023 (200 μmol/l) prevented flow-induced NO production when added to the basolateral side but not the luminal side. We conclude that ATP mediates flow-induced NO production in the thick ascending limb likely via activation of P2Y receptors in the luminal and P2X receptors in the basolateral membrane.

Ultra-Rapid Freezing of Isolated Skeletal Muscle Fibers

1977 ◽  
Vol 35 ◽  
pp. 576-577
Author(s):  
Joachim R. Sommer ◽  
Nancy R. Wallace
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Granular Material ◽  
Nuclear Envelope ◽  
Intracellular Calcium ◽  
Ruthenium Red ◽  
Threshold Concentration ◽  
Rapid Freezing ◽  
Frog Skeletal Muscle ◽  
Electrical Isolation

After Howell (1) had shown that ruthenium red treatment of fixed frog skeletal muscle caused collapse of the intermediate cisternae of the sarcoplasmic reticulum (SR), forming a pentalaminate structure by obi iterating the SR lumen, we demonstrated that the phenomenon involves the entire SR including the nuclear envelope and that it also occurs after treatment with other cations, including calcium (2,3,4).From these observations we have formulated a hypothesis which states that intracellular calcium taken up by the SR at the end of contraction causes the M rete to collapse at a certain threshold concentration as the first step in a subsequent centrifugal zippering of the free SR toward the junctional SR (JSR). This would cause a) bulk transport of SR contents, such as calcium and granular material (4) into the JSR and, b) electrical isolation of the free SR from the JSR.

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