scholarly journals Functional expression of P2X4 receptor in capillary endothelial cells of the cochlear spiral ligament and its role in regulating the capillary diameter

2011 ◽  
Vol 301 (1) ◽  
pp. H69-H78 ◽  
Author(s):  
T. Wu ◽  
M. Dai ◽  
X. R. Shi ◽  
Z. G. Jiang ◽  
A. L. Nuttall
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Pyridoxal Phosphate ◽  
Regional Blood Flow ◽  
Reversal Potential ◽  
Lateral Wall ◽  
Disulfonic Acid ◽  
Spiral Ligament ◽  
Dependent Manner ◽  
P2x4 Receptor

The cochlear lateral wall generates the endocochlear potential (EP), which creates a driving force for the hair cell transduction current and is essential for normal hearing. Blood flow at the cochlear lateral wall is critically important for maintaining the EP. The vulnerability of the EP to hypoxia suggests that the blood flow in the cochlear lateral wall is dynamically and precisely regulated to meet the changing metabolic needs of the cochlear lateral wall. It has been reported that ATP, an important extracellular signaling molecule, plays an essential role in regulating cochlear blood flow. However, the cellular mechanism underlying ATP-induced regional blood flow changes has not been investigated. In the current study, we demonstrate that 1) the P2X4 receptor is expressed in endothelial cells (ECs) of spiral ligament (SL) capillaries. 2) ATP elicits a characteristic current through P2X4 on ECs in a dose-dependent manner (EC50 = 0.16 mM). The ATP current has a reversal potential at ∼0 mV; is inhibited by 5-(3-bromophenyl)-1,3-dihydro-2 H-benzofuro[3,2- e]-1,4-diazepin-2-one (5-BDBD), LaCl3, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), and extracellular acidosis; and is less sensitive to α,β-methyleneadenosine 5′-triphosphate (α,β-MeATP) and 2′- and 3′- O-(4-benzoyl-benzoyl) adenosine 5′-triphosphate (BzATP). 3) ATP elicits a transient increase of intracellular Ca2+ in ECs. 4) In accordance with the above in vitro findings, perilymphatic ATP (1 mM) caused dilation in SL capillaries in vivo by 11.5%. Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), a nonselective inhibitor of nitric oxide synthase, or 5-BDBD, the specific P2X4 inhibitor, significantly blocked the dilation. These findings support our hypothesis that extracellular ATP regulates cochlear lateral blood flow through P2X4 activation in ECs.

scholarly journals Toll-Like Receptor-Triggered Calcium Mobilization Protects Mice against Bacterial Infection through Extracellular ATP Release

2014 ◽  
Vol 82 (12) ◽  
pp. 5076-5085 ◽  
Author(s):  
Hua Ren ◽  
Yunfei Teng ◽  
Binghe Tan ◽  
Xiaoyu Zhang ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Immune Responses ◽  
Pyridoxal Phosphate ◽  
Atp Release ◽  
Extracellular Atp ◽  
Calcium Mobilization ◽  
Innate Immune ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Innate Immune Responses ◽  
Toll Like Receptor

ABSTRACTExtracellular ATP (eATP), released as a “danger signal” by injured or stressed cells, plays an important role in the regulation of immune responses, but the relationship between ATP release and innate immune responses is still uncertain. In this study, we demonstrated that ATP was released through Toll-like receptor (TLR)-associated signaling in bothEscherichia coli-infected mice and lipopolysaccharide (LPS)- or Pam3CSK4-treated macrophages. This ATP release could be blocked completely only byN-ethylmaleimide (NEM), not by carbenoxolone (CBX), flufenamic acid (FFA), or probenecid, suggesting the key role of exocytosis in this process. Furthermore, LPS-induced ATP release could also be reduced dramatically through suppressing calcium mobilization by use of U73122, caffeine, and thapsigargin (TG). In addition, the secretion of interleukin-1β (IL-1β) and CCL-2 was enhanced significantly by ATP, in a time- and dose-dependent manner. Meanwhile, macrophage-mediated phagocytosis of bacteria was also promoted significantly by ATP stimulation. Furthermore, extracellular ATP reduced the number of invading bacteria and protected mice from peritonitis by activating purinergic receptors. Mechanistically, phosphorylation of AKT and ERK was overtly increased by ATP in antibacterial immune responses. Accordingly, if we blocked the P2X- and P2Y-associated signaling pathway by using suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid), tetrasodium salt (PPADS), the ATP-enhanced immune response was restrained significantly. Taken together, our findings reveal an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of calcium mobilization-mediated ATP release in infectious diseases.

scholarly journals Pyridoxine Stimulates Filaggrin Production in Human Epidermal Keratinocytes

2021 ◽  
Author(s):  
Miyuki Fujishiro ◽  
Shoichi Yahagi ◽  
Shota Takemi ◽  
Takafumi Sakai ◽  
ichiro sakata
Keyword(s):  
Pyridoxal Phosphate ◽  
Seborrheic Dermatitis ◽  
Epidermal Differentiation ◽  
Disulfonic Acid ◽  
Epidermal Keratinocytes ◽  
Marker Genes ◽  
Dependent Manner ◽  
Human Epidermal Keratinocytes ◽  
Concentration Dependent Manner ◽  
Protein Levels

Abstract Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical studies have revealed that PN deficiency causes skin problems such as seborrheic dermatitis and stomatitis. However, the detailed effects of PN and its mechanism of action in epidermal function are poorly understood. In this study, we examined the effects of PN on epidermal function in normal human epidermal keratinocytes and found that PN specifically causes an increase in the expression of profilaggrin mRNA, among marker genes of terminal epidermal differentiation. In addition, PN treatment caused an increase in the production of filaggrin protein in a concentration-dependent manner. Treatment with P2x purinoceptor antagonists, namely, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate and TNP-ATP hydrate, induced an increase in the filaggrin protein levels. Moreover, we showed that elevated filaggrin production induced upon PN treatment was suppressed by ATP (known as P2x purinoceptor agonist). This study is the first to report that PN causes an increase in filaggrin transcription and production, and these results suggest that PN-induced filaggrin production may be a useful target as a daily care component in atopic dermatitis, wherein filaggrin levels are specifically reduced.

Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells

1997 ◽  
Vol 273 (1) ◽  
pp. C214-C222 ◽  
Author(s):  
V. G. Manolopoulos ◽  
T. Voets ◽  
P. E. Declercq ◽  
G. Droogmans ◽  
B. Nilius
Keyword(s):  
Endothelial Cells ◽  
Anion Channel ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Organic Osmolytes ◽  
Dependent Manner ◽  
Anion Channels ◽  
Whole Cell Patch Clamp ◽  
Osmolyte Efflux ◽  
Dose Dependent

We used a combined biochemical, pharmacological, and electrophysiological approach to study the effects of hyposmotic swelling on organic osmolyte efflux in endothelial cells (EC). In [3H]taurine-loaded monolayers of calf pulmonary artery EC (CPAEC), hyposmolality activated time- and dose-dependent effluxes of [3H]taurine. Swelling-activated [3H]taurine efflux (Jtau swell)in CPAEC was inhibited by the anion channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), fenamates, and also quinine (in a pH-dependent manner), ATP, and the phospholipase A2 inhibitor 4-bromophenacyl bromide. In contrast, Jtau swell was partly or totally insensitive to bumetanide, forskolin, phorbol 12-myristate 13-acetate, and staurosporine. Swelling also activated myo-[3H]inositol efflux that was blocked by tamoxifen, NPPB, DIDS, and niflumic acid. Moreover, the cellular content of taurine and other amino acids was significantly reduced in osmotically activated CPAEC. Finally, in whole cell patch-clamp experiments, taurine, glycine, aspartate, and glutamate exhibited significant permeability for swelling-activated anion channels. In conclusion, hyposmotic swelling activates efflux of taurine and other organic osmolytes in EC. In addition, our results suggest that anion channels may provide a pathway for swelling-activated efflux of organic osmolytes in EC.

Abstract 221: The P2x4 Receptor is Required for Neuroprotection via Ischemic Preconditioning

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Tomohiko Ozaki ◽  
Rieko Muramatsu ◽  
Toshiyuki Fujinaka ◽  
Toshiki Yoshimine ◽  
Toshihide Yamashita
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Vascular Endothelial Cells ◽  
Ischemic Tolerance ◽  
Receptor Expression ◽  
Vascular Endothelial ◽  
P2x4 Receptor ◽  
Flow Changes

Background: Ischemic preconditioning (IPC), a procedure consisting of transient ischemia and subsequent reperfusion, provides ischemic tolerance against prolonged ischemia in the brain. Although the blood flow changes mediated by IPC are primarily perceived by vascular endothelial cells, the role of these cells in ischemic tolerance has not fully clarified. In this research, we focused on the role of P2X4 receptor, which sense blood flow changes and is expressed on vascular endothelial cells. Methods: We administrated P2X4 receptor inhibitor into lateral ventricle of C57BL/6J male mice (8-10 weeks) and then conducted middle cerebral artery occlusion (MCAO). Fifteen minutes MCAO was done as IPC 48 hours before 60 minutes MCAO. To examine the necessity of P2X4 receptor expression in vascular endothelial cells, we generated a conditional knockout (CKO) mouse in which the P2X4 receptor was knocked down in VE-cadherin-positive vascular endothelial cells. To investigate molecular change by IPC, we obtained cerebrovascular endothelial cells of mice 48 hours after IPC, and real time PCR and ELISA were evaluated. To examine the molecular expression change on vascular endothelial cells by blood flow, we used in vitro culture system which generates fluid flow and real time PCR was evaluated. Inhibition of P2X4 receptor expression was conducted by P2X4 receptor siRNA transfection. Results: P2X4 receptor antagonist abolished neuroprotection via IPC. Moreover, the effect of IPC to P2X4 receptor CKO mice was smaller than control mice, the infarct volume of P2X4 receptor CKO was larger than control mice after 60 minutes MCAO (p<0.05, Control, n=4; CKO, n=6). IPC induced expression of osteopontin mRNA (p<0.05, n=5). Osteopontin administration attenuates the increase of infarct formation induced by P2X4 receptor inhibition (p<0.05, Control, n=5; Osteopontin, n=6). In vitro, shear stress upregulated expression of osteopontin mRNA (p<0.05, n=3). This upregulation was inhibited by P2X4 receptor siRNA (p<0.05, Control siRNA, n=6; P2X4 receptor siRNA, n=7). Conclusion: These results demonstrate a novel mechanism whereby vascular endothelial cells are involved in ischemic tolerance by way of the pathway about P2X4 receptor and osteopontin.

Analysis of purine- and pyrimidine-induced vascular responses in the isolated rat cerebral arteriole

2001 ◽  
Vol 280 (2) ◽  
pp. H767-H776 ◽  
Author(s):  
Tetsuyoshi Horiuchi ◽  
Hans H. Dietrich ◽  
Shinichiro Tsugane ◽  
Ralph G. Dacey
Keyword(s):  
Smooth Muscle ◽  
Pyridoxal Phosphate ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Cerebral Microvessels ◽  
Vascular Responses ◽  
Single Receptor ◽  
Low Concentrations ◽  
High Concentrations ◽  
Cerebral Arteriole

Effects of extraluminal UTP were studied and compared with vascular responses to ATP and its analogs in rat cerebral-penetrating arterioles. UTP, UDP, 2-methylthio-ATP, and α,β-methylene-ATP dilated arterioles at the lowest concentration and constricted them at high concentrations. Low concentrations of ATP dilated the vessels; high concentrations caused a biphasic response, with transient constriction followed by dilation. Endothelial impairment inhibited ATP- and UTP-mediated dilation and potentiated constriction to UTP but not to ATP. ATP- and 2-methylthio-ATP- but not UTP-mediated constrictions were inhibited by desensitization with 10−6M α,β-methylene-ATP or 3 × 10−6M pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). PPADS at 10−4M abolished the UTP-mediated constriction and induced vasodilation in a dose-dependent manner but did not affect the dilation to ATP. These results suggest that in rat cerebral microvessels 1) ATP and 2-methylthio-ATP induce transient constriction via smooth muscle P2X1receptors in the cerebral arteriole, 2) UTP stimulates two different classes of P2Yreceptors, resulting in constriction (smooth muscle P2Y4) and dilation (possibly endothelial P2Y2), and 3) ATP and UTP produce dilation by stimulation of a single receptor (P2Y2).

P2X receptors in mouse Leydig cells

2006 ◽  
Vol 290 (4) ◽  
pp. C1009-C1017 ◽  
Author(s):  
Luiz Artur Poletto Chaves ◽  
Endrigo Piva Pontelli ◽  
Wamberto Antonio Varanda
Keyword(s):  
Leydig Cells ◽  
Pyridoxal Phosphate ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Apparent Dissociation Constant ◽  
Whole Cell

ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 μM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5′- O-(3-thiotriphosphate) (ATPγS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response ( Kd) of 44, 110, and 637 μM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP ≥ ATPγS > 2-MeS-ATP > 2′,3′- O-(4-benzoylbenzoyl)-ATP (BzATP). αβ-Methylene-ATP (αβ-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (p Ka) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability ( P)Ca/ PNa = 5.32], but not to chloride ( PCl/ PNa = 0.03) or N-methyl-d-glucamine (NMDG) ( PNMDG/ PNa = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between −90 and −50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant (τ) = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X2.

Adenoviral PR39 improves blood flow and myocardial function in a pig model of chronic myocardial ischemia by enhancing collateral formation

2006 ◽  
Vol 290 (3) ◽  
pp. R494-R500 ◽  
Author(s):  
Mark J. Post ◽  
Kaori Sato ◽  
Masahiro Murakami ◽  
Jialin Bao ◽  
Daniela Tirziu ◽  
...  
Keyword(s):  
Blood Flow ◽  
Myocardial Ischemia ◽  
Regional Blood Flow ◽  
Hypoxia Inducible Factor ◽  
Receptor Expression ◽  
Pig Model ◽  
Individual Growth ◽  
Dependent Manner ◽  
Chronic Myocardial Ischemia ◽  
Collateral Formation

Angiogenic therapy with individual growth factors or “master switch” genes is being evaluated for treatment of advanced coronary artery disease. In this study, we investigated the efficacy and mechanism of PR39, a gene capable of activating VEGF and fibroblast growth factor (FGF)-2-dependent pathways. PR39 enhances hypoxia-inducible factor-1α (HIF-1α)-dependent gene expression by selectively inhibiting proteasome degradation of this transcription factor. In addition, PR39 also stimulates expression of the FGF receptors (FGFR)-1 and syndecan-4. In a pig model of chronic myocardial ischemia, we used angiography, MRI, and microsphere regional blood flow to evaluate the efficacy of intramyocardial adenoviral protein arginine-rich peptide (Ad-PR39) injections. Ad-PR39 improved collateral scores, regional perfusion, and regional function in a dose-dependent manner. Local VEGF, VEGFR-1, VEGFR-2, syndecan, and FGFR-1 levels were 16–75% upregulated after Ad-PR39 injections as assessed by real-time PCR, suggesting upregulation of VEGF and FGF pathways. PR39 is an angiogenic peptide that improves perfusion and function of ischemic myocardium, at least in part, through collateral formation. The dual mechanism, i.e., stimulation of HIF-1α and FGF receptor expression, likely accounts for the functional benefits of PR39.

scholarly journals Lactate dilates cochlear capillaries via type V fibrocyte-vessel coupling signaled by nNOS

2011 ◽  
Vol 301 (4) ◽  
pp. H1248-H1254 ◽  
Author(s):  
Min Dai ◽  
Yue Yang ◽  
Xiaorui Shi
Keyword(s):  
Blood Flow ◽  
Inner Ear ◽  
Regional Blood Flow ◽  
High Energy ◽  
No Synthase ◽  
Monocarboxylate Transporter ◽  
No Production ◽  
Spiral Ligament ◽  
Monocarboxylate Transporter 1 ◽  
Type V

Transduction of sound in the inner ear demands tight control over delivery of oxygen and glucose. However, the mechanisms underlying the control of regional blood flow are not yet fully understood. In this study, we report a novel local control mechanism that regulates cochlear blood flow to the stria vascularis, a high energy-consuming region of the inner ear. We found that extracellular lactate had a vasodilatory effect on the capillaries of the spiral ligament under both in vitro and in vivo conditions. The lactate, acting through monocarboxylate transporter 1 (MCT1), initiated neuronal nitric oxide (NO) synthase (nNOS) and catalyzed production of NO for the vasodilation. Blocking MCT1 with the MCT blocker, α-cyano-4-hydroxycinnamate (CHC), or a suppressing NO production with either the nonspecific inhibitor of NO synthase, NG-nitro-l-arginine methyl ester (l-NAME), or either of two selective nNOS inhibitors, 3-bromo-7-nitroindazole or (4S)- N-(4-amino-5[aminoethyl]aminopentyl)- N′-nitroguanidine (TFA), totally abolished the lactate-induced vasodilation. Pretreatment with the selective endothelial NO synthase inhibitor, l- N5-(1-iminoethyl)ornithine (l-NIO), eliminated the inhibition of lactate-induced vessel dilation. With immunohistochemical labeling, we found the expression of MCT1 and nNOS in capillary-coupled type V fibrocytes. The data suggest that type V fibrocytes are the source of the lactate-induced NO. Cochlear microvessel tone, regulated by lactate, is mediated by an NO-signaled coupling of fibrocytes and capillaries.

Do P2X purinergic receptors regulate skeletal muscle blood flow during exercise?

2004 ◽  
Vol 286 (2) ◽  
pp. H633-H639 ◽  
Author(s):  
John B. Buckwalter ◽  
Jessica C. Taylor ◽  
Jason J. Hamann ◽  
Philip S. Clifford
Keyword(s):  
Skeletal Muscle ◽  
Blood Flow ◽  
Femoral Artery ◽  
Purinergic Receptors ◽  
Pyridoxal Phosphate ◽  
Muscle Blood Flow ◽  
Sympathetic Nerves ◽  
P2x Receptor ◽  
Disulfonic Acid ◽  
Skeletal Muscle Blood Flow

Although there is evidence that sympathetic nerves release ATP as a neurotransmitter to produce vasoconstriction via P2X purinergic receptors, the role of these receptors in the regulation of blood flow to exercising skeletal muscle has yet to be determined. We hypothesized that there is tonic P2X receptor-mediated vasoconstriction in exercising skeletal muscle. To test this hypothesis, the effect of P2X receptor blockade on skeletal muscle blood flow was examined in six exercising mongrel dogs. P2X receptor antagonism was accomplished with pyridoxal-phosphate-6-azophenyl-2′4′-disulfonic acid (PPADs). Animals were instrumented chronically with flow probes on the external iliac arteries of both hindlimbs and a catheter in one femoral artery. PPADs (40 mg) was infused as a bolus into the femoral artery catheter during steady-state exercise at 6 miles/h. Intra-arterial infusion of PPADs increased iliac blood flow from 542 ± 55 to 677 ± 69 ml/min ( P < 0.05) and iliac vascular conductance from 5.17 ± 0.62 to 6.53 ± 0.80 ml·min–1·mmHg–1. The PPADs infusion did not affect blood flow in the contralateral iliac artery. These data support the hypothesis that P2X purinergic receptors produce vasoconstriction in exercising skeletal muscle.

Adenosine triphosphate increases the reactivity of the afferent arteriole to low concentrations of norepinephrine

2007 ◽  
Vol 293 (6) ◽  
pp. R2225-R2231 ◽  
Author(s):  
Michael Hultström ◽  
En Yin Lai ◽  
Zufu Ma ◽  
Örjan Källskog ◽  
Andreas Patzak ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Vascular Reactivity ◽  
Pyridoxal Phosphate ◽  
Cytosolic Calcium ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Afferent Arteriole ◽  
Response Curves ◽  
Atp Receptors ◽  
Low Concentrations

Adenosine triphosphate (ATP) and norepinephrine (NE) interact in the control of blood flow in the kidney. A combined effect of NE and ATP has not been previously investigated at the level of the afferent arteriole (Af). We studied the effects of ATP on the contractile response of the Af to NE. Vascular reactivity to ATP, NE, and their combination was investigated in isolated perfused Af from mice. The roles of α-adrenoceptors and P2-ATP-receptors were investigated by use of specific agonists and antagonists. Cytosolic calcium was measured using the fluorescent calcium dye fura-2. ATP in concentrations from 10−12 to 10−4 mol/l induced transient contractions. NE constricted the Af in a dose-dependent manner and induced significant contractions at > 10−7 mol/l. Treatment with ATP (10−8 and 10−6 mol/l) increased the NE response. Diameters were reduced by 20% already at 10−11 mol/l NE during ATP treatment of 10−6 mol/l. ATP increased the calcium response to NE significantly at 10−8 and 10−7mol/l NE. The P2-type ATP receptor blocker pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (10−5 mol/l) abolished the sensitization of the NE response by ATP. The α1-blocker prazosin (10−7 mol/l) inhibited the ATP effect, as did the α2-blocker yohimbine (10−7 mol/l). Neither the phenylephrine- nor clonidine-induced concentration response curves was affected by ATP in the bath solution. Costimulation with ATP enhances the response of the Af to NE. This effect is mediated by increased cytosolic calcium. The enhancing effect involves P2-type ATP receptors and both α1- and α2-adrenoceptors.

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