scholarly journals Exocytotic release of ATP and activation of P2X receptors in dissociated guinea pig stellate neurons

2006 ◽  
Vol 291 (5) ◽  
pp. C1062-C1071 ◽  
Author(s):  
John D. Tompkins ◽  
Rodney L. Parsons
Keyword(s):  
Guinea Pig ◽  
Pyridoxal Phosphate ◽  
Purinergic Receptor ◽  
P2x Receptors ◽  
Disulfonic Acid ◽  
Inward Current ◽  
Protein Receptor ◽  
Vesicular Release ◽  
Cell Recording ◽  
Inward Currents

Activation of P2X receptors by a Ca2+- and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent release of ATP was measured using patch-clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the Ca2+ ionophore ionomycin (1.5 and 3 μM). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca2+, depolarizing voltage steps (−60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of Ba2+ for Ca2+ augmented the postdepolarization frequency of ASTICs, while eliminating the larger transient current. Perfusion with an ionomycin-containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [Ca2+] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of Ca2+ with Mg2+, demonstrating a Ca2+ requirement. ASTICs were Na+ dependent, inwardly rectifying, and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (10 μM) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 μM and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 μM). The presence of botulinum neurotoxin E (BoNT/E) in the whole-cell recording electrode significantly attenuated the ionomycin-induced ASTIC activity, whereas phorbol ester treatment potentiated this activity. These results suggest that ASTICs are mediated by vesicular release of ATP and activation of P2X receptors.

Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons

2002 ◽  
Vol 283 (6) ◽  
pp. G1238-G1248 ◽  
Author(s):  
Carlos Barajas-López ◽  
Luis M. Montaño ◽  
Rosa Espinosa-Luna
Keyword(s):  
P2x Receptors ◽  
Disulfonic Acid ◽  
Pharmacological Properties ◽  
Pipette Solution ◽  
Outward Currents ◽  
C 23 ◽  
Cell Recording ◽  
Inward Currents ◽  
Inhibitory Interactions ◽  
Subtype 3

Inhibitory interactions between 5-HT subtype 3 (5-HT3) and P2X receptors were characterized using whole cell recording techniques. Currents induced by 5-HT ( I 5-HT) and ATP ( I ATP) were blocked by tropisetron (or ondansetron) and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, respectively. Currents induced by 5-HT + ATP ( I 5-HT+ATP) were only as large as the current induced by the most effective transmitter, revealing current occlusion. Occlusion was observed at membrane potentials of −60 and 0 mV (for inward currents), but it was not present at +40 mV (for outward currents). Kinetic and pharmacological properties of I 5-HT+ATP indicate that they are carried through 5-HT3 and P2X channels. Current occlusion occurred as fast as activation of I 5-HT and I ATP, was still present in the absence of Ca2+ or Mg2+, after adding staurosporine, genistein, K-252a, or N-ethylmaleimide to the pipette solution, after substituting ATP with ∝,β-methylene ATP or GTP with GTP-γ-S in the pipette, and was observed at 35°C, 23°C, and 8°C. These results are in agreement with a model that considers that 5-HT3 and P2X channels are in functional clusters and that these channels might directly inhibit each other.

scholarly journals P2X7receptors mediate deleterious renal epithelial-fibroblast cross talk

2011 ◽  
Vol 300 (1) ◽  
pp. F62-F70 ◽  
Author(s):  
Murugavel Ponnusamy ◽  
Li Ma ◽  
Rujun Gong ◽  
Maoyin Pang ◽  
Y. Eugene Chin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Pyridoxal Phosphate ◽  
Purinergic Receptor ◽  
Purinergic Signaling ◽  
Kidney Injury ◽  
Fibroblast Cell ◽  
Underlying Mechanism ◽  
Disulfonic Acid ◽  
Small Interference Rna ◽  
Renal Epithelial Cells

Peritubular fibroblasts in the kidney are the major erythropoietin-producing cells and also contribute to renal repair following acute kidney injury (AKI). Although few fibroblasts were observed in the interstitium adjacent to damaged tubular epithelium in the early phase of AKI, the underlying mechanism by which their numbers were reduced remains unknown. In this study, we tested the hypothesis that damaged renal epithelial cells directly induce renal interstitial fibroblast death by releasing intracellular ATP and activating purinergic signaling. Exposure of a cultured rat renal interstitial fibroblast cell line (NRK-49F) to necrotic renal proximal tubular cells (RPTC) lysate or supernatant induced NRK-49F cell death by apoptosis and necrosis. Depletion of ATP with apyrase or inhibition of the P2X purinergic receptor with pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid blocked the deleterious effect of necrotic RPTC supernatant. The P2X7receptor, an ATP-sensitive purinergic receptor, was not detected in cultured NRK-49F cells but was inducible by necrotic RPTC supernatant. Treatment with A438079, a highly selective P2X7receptor inhibitor, or knockdown of the P2X7receptor with small interference RNA diminished renal fibroblast death induced by necrotic RPTC supernatant. Conversely, overexpression of the P2X7receptor potentiated this response. Collectively, these findings provide strong evidence that damaged renal epithelial cells can directly induce the death of renal interstitial fibroblasts by ATP activation of the P2X7receptor.

scholarly journals ATP is involved in myocardial and vascular effects of exogenous bradykinin in ejecting guinea pig heart

1999 ◽  
Vol 277 (2) ◽  
pp. H818-H825 ◽  
Author(s):  
Peter B. Anning ◽  
Bernard D. Prendergast ◽  
Philip A. MacCarthy ◽  
Ajay M. Shah ◽  
Derek C. Buss ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Guinea Pig ◽  
Coronary Flow ◽  
Pyridoxal Phosphate ◽  
Left Ventricular ◽  
Luciferase Assay ◽  
Disulfonic Acid ◽  
Vascular Effects ◽  
Guinea Pig Heart ◽  
Nitric Oxide Release

It has recently been reported that bradykinin induces selective left ventricular (LV) relaxation in isolated guinea pig hearts via the release of nitric oxide. Exogenous bradykinin also induces vasodilation, which is only partly due to nitric oxide release. In the present study we investigated the role of adenyl purines on these bradykinin-induced effects. Isolated ejecting guinea pig hearts were studied. LV pressure was monitored by a 2-Fr micromanometer-tipped catheter. ATP concentrations were measured using a luciferin-luciferase assay. Bradykinin (1 and 100 nM) caused a progressive acceleration of LV relaxation together with a transient increase in coronary flow. These effects were inhibited by the nonselective P2 purinoceptor antagonist suramin (1 μM, n = 6) but were unaffected by the selective P2x purinoceptor antagonist pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid (1 μM, n = 6). These myocardial and vascular effects of bradykinin were associated with increased ATP levels in coronary effluent. These data suggest that the selective enhancement of LV relaxation and rise in coronary flow induced by exogenous bradykinin involve endogenous ATP and the subsequent stimulation of P2 purinoceptors.

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

2006 ◽  
Vol 291 (5) ◽  
pp. C985-C994 ◽  
Author(s):  
Kevin P. Monaghan ◽  
Sang Don Koh ◽  
Seungil Ro ◽  
Jonghun Yeom ◽  
Burton Horowitz ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Phospholipase C ◽  
Threshold Voltage ◽  
Pyridoxal Phosphate ◽  
Disulfonic Acid ◽  
Smooth Muscle Relaxation ◽  
External Application ◽  
Voltage Dependent ◽  
Inward Currents ◽  
Stimulation Of

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca2+-activated K+ channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current ( IVNSCC) and a relatively high-threshold voltage-activated (L-type) Ca2+ current ( IL). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect IVNSCC or IL in dialyzed cells. ATP (1 mM) increased IVNSCC and decreased IL in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on IVNSCC. ADP decreased IL but had no effect on IVNSCC. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on IVNSCC. ATP stimulation of IVNSCC was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y4 is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates IVNSCC and depresses IL via binding of P2Y4 receptors and stimulation of the phospholipase C/PKC pathway.

scholarly journals Cloning and characterization of a functional P2X receptor from larval bullfrog skin

2001 ◽  
Vol 281 (3) ◽  
pp. C954-C962 ◽  
Author(s):  
Philip J. Jensik ◽  
Doyle Holbird ◽  
Michael W. Collard ◽  
Thomas C. Cox
Keyword(s):  
Pyridoxal Phosphate ◽  
Rana Catesbeiana ◽  
Short Circuit ◽  
Short Circuit Current ◽  
P2x Receptors ◽  
P2x Receptor ◽  
Disulfonic Acid ◽  
Acid Protein ◽  
Sensory Reception ◽  
Tadpole Skin

ATP activates an apical-to-basolateral nonselective cation current across the skin of larval bullfrogs ( Rana catesbeiana) with similarities to currents carried by some P2X receptors. A functional P2X receptor was cloned from tadpole skin RNA that encodes a 409-amino acid protein with highest protein homology to cP2X8. RT-PCR showed that this transcript was found in skin, heart, eye, brain, and skeletal muscle of tadpoles but not in skin, brain, or heart of adults. After transcribed RNA from this clone was injected into Xenopus oocytes, application of ATP activated a transient current similar to other P2X receptors and the ATP-activated transient in short-circuit current ( I sc) across intact skin. The agonists 2-methylthio-ATP and adenosine-5′- O-(thiotriphoshate) also activated transient currents. α,β-Methylene-ATP and ADP were poor agonists of this receptor. Suramin and pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid tetrasodium (PPADS) were potent antagonists, and PPADS showed an irreversible blockade of this receptor to agonist activation. Under external Na+-free, Ca2+/Mg2+-free conditions ( N-methyl-d-glucamine replacement, 0.5 mM EGTA), ATP activated a steadily increasing inward current. Fluorescence microscopy showed that propidium was entering the cells, suggesting that a relatively large pore size was formed under zero divalent conditions. This clone has some characteristics consistent with previously described ATP-activated I sc in the tadpole skin. Because the clone is not found in adult skin, it may have some exclusive role in the tadpole such as sensory reception by the skin or triggering apoptosis at metamorphosis.

Regulation of transcervical permeability by two distinct P2 purinergic receptor mechanisms

2002 ◽  
Vol 282 (1) ◽  
pp. C75-C83 ◽  
Author(s):  
George I. Gorodeski
Keyword(s):  
Phase I ◽  
Phase Ii ◽  
Time Course ◽  
Pyridoxal Phosphate ◽  
Calcium Influx ◽  
Purinergic Receptor ◽  
Calcium Mobilization ◽  
Disulfonic Acid ◽  
Calcium Dependent ◽  
Acute Increase

Micromolar concentrations of ATP stimulate biphasic change in transepithelial conductance across CaSki cultures, an acute increase (phase I response) followed by a slower decrease ( phase II response). Phase I and phase II responses involve two distinct calcium-dependent pathways, calcium mobilization and calcium influx. To test the hypothesis that phase I and phase II responses are mediated by distinct P2 purinergic receptors, changes in permeability were uncoupled by blocking calcium mobilization with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA) or by lowering extracellular calcium, respectively. Under these conditions ATP EC50was 25 μM for phase Iresponse and 2 μM for phase II response. The respective agonist profiles were ATP > UTP > adenosine 5′- O-(3-thiotriphosphate) (ATP-γS) = N6-([6-aminohexyl]carbamoylmethyl)adenosine 5′-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-γS. Suramin blocked phase Iresponse and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2′,4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course and pharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and a saturable concentration-dependent effect (EC50of 2–3 μM). RT-PCR experiments revealed expression of mRNA for both the P2Y2and P2X4receptors. These results suggest that the ATP-induced phase I and phase IIresponses are mediated by distinct P2 purinergic receptor mechanisms.

Mechanisms of mechanotransduction by specialized low-threshold mechanoreceptors in the guinea pig rectum

2005 ◽  
Vol 289 (3) ◽  
pp. G397-G406 ◽  
Author(s):  
Vladimir P. Zagorodnyuk ◽  
Penny Lynn ◽  
Marcello Costa ◽  
Simon J. H. Brookes
Keyword(s):  
Guinea Pig ◽  
Pyridoxal Phosphate ◽  
Transient Receptor Potential ◽  
Ruthenium Red ◽  
Transient Receptor Potential Channels ◽  
Disulfonic Acid ◽  
Free Solution ◽  
Circular Smooth Muscle ◽  
Low Threshold

The guinea pig rectum, but not the colon, is innervated by a specialized class of distension-sensitive mechanoreceptors that have transduction sites corresponding to rectal intraganglionic laminar endings (rIGLEs). Rectal mechanoreceptors recorded in vitro had low threshold to circumferential stretch, adapted slowly, and could respond within 2 ms to mechanical stimulation by a piezo-electric probe. Antagonists to ionotropic N-methyl-d-aspartate (NMDA; CGS 19755, memantine) and non-NMDA (6,7-dinitroquinoxaline-2,3-dione) glutamate receptors did not affect mechanotransduction. In normal Krebs solution, the P2X purinoreceptor agonist α,β-methylene ATP reduced mechanoreceptor firing evoked by distension but simultaneously relaxed circular smooth muscle and inhibited stretch-induced contractions. Neither ATP nor α,β-methylene ATP affected mechanotransduction when transduction sites were directly compressed with von Frey hairs. The P2 purinoreceptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid did not affect stretch-induced firing but reduced the inhibitory effect of α,β-methylene ATP on stretch-induced firing. Under isometric conditions, blocking synaptic transmission in Ca2+-free solution reduced stretch-evoked firing but not when basal tension was restored to control levels. Under isotonic condition, Ca2+-free solution did not significantly affect load-evoked firing. The blockers of mechanogated and/or transient receptor potential channels, benzamil, Gd3+, SKF 96365, and ruthenium red inhibited stretch-induced firing but, in parallel, significantly reduced stretch-induced contractions. Benzamil and SKF 96365 were able to inhibit mechanotransduction when transduction sites were compressed with von Frey hairs. The results show that mechanotransduction is rapid but does not depend on fast exocytotic release of mediators. It is likely that stretch-activated ion channels on rIGLEs are involved in direct, physical mechanotransduction by rectal low-threshold mechanoreceptors.

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. C352-C362 ◽  
Author(s):  
In Deok Kong ◽  
Sang Don Koh ◽  
Kenton M. Sanders
Keyword(s):  
Guinea Pig ◽  
Pyridoxal Phosphate ◽  
Receptor Blocker ◽  
Disulfonic Acid ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Relaxation Responses ◽  
Spontaneous Transient Outward Currents

Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to −50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca2+ with equimolar Mn2+ caused STOCs to “run down.” Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude “mini-STOCs” remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca2+-activated K+ channels (SK channels). Application of ATP or 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid, a P2 receptor blocker. Similarly, pretreatment of cells with U-73122 (1 μM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP3) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P2Y receptors results in localized Ca2+ release via PLC- and IP3-dependent mechanisms. Release of Ca2+ is coupled to STOCs, which are composed of currents mediated by large-conductance Ca2+-activated K+ channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation.

scholarly journals Purinergic Signaling Modulates Survival/Proliferation of Human Dental Pulp Stem Cells

2018 ◽  
Vol 98 (2) ◽  
pp. 242-249 ◽  
Author(s):  
S. Zhang ◽  
D. Ye ◽  
L. Ma ◽  
Y. Ren ◽  
R.T. Dirksen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Purinergic Receptor ◽  
Purinergic Signaling ◽  
P2x Receptors ◽  
Dental Pulp Stem Cells ◽  
Receptor Expression ◽  
Receptor Antagonists ◽  
Inward Currents ◽  
Human Dental Pulp

Human dental pulp stem cells (hDPSCs) reside in postnatal dental pulp and exhibit the potential to differentiate into odontoblasts as well as neurons. However, the intercellular signaling niches necessary for hDPSC survival and self-renewal remain largely unknown. The objective of this study is to demonstrate the existence of intercellular purinergic signaling in hDPSCs and to assess the impact of purinergic signaling on hDPSC survival and proliferation. hDPSCs were isolated from extracted third molars and cultured in minimum essential medium. To demonstrate responsiveness to ATP application and inhibitions by purinergic receptor antagonists, whole cell patch-clamp recordings of ATP-induced currents were recorded from cultured hDPSCs. Immunofluorescence and enzymatic histochemistry staining were performed to assess purinergic receptor expression and ectonucleotidase activity in hDPSCs, respectively. To determine the effects of purinergic signaling on hDPSC, purinergic receptor antagonists and an ectonucleotidase inhibitor were applied in culture medium, and hDPSC survival and proliferation were assessed with DAPI staining and Ki67 immunofluorescence staining, respectively. We demonstrated that ATP application induced inward currents in hDPSCs. P2X and P2Y receptors are involved in the generation of ATP-induced inward currents. We also detected expression of NTPDase3 and ectonucleotidase activity in hDPSCs. We further demonstrated that purinergic receptors were tonically activated in hDPSCs and that inhibition of ectonucleotidase activity enhanced ATP-induced inward currents. Furthermore, we found that blocking P2Y and P2X receptors reduced—and inhibition of ecto-ATPase activity enhanced—the survival and proliferation of hDPSCs, while blocking P2X receptors alone affected only hDPSC proliferation. Autocrine/paracrine purinergic signaling is essential for hDPSC survival and proliferation. These results reveal potential targets to manipulate hDPSCs to promote tooth/dental pulp repair and regeneration.

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