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.

ATP stimulates chemically sensitive and sensitizes mechanically sensitive afferents

2002 ◽  
Vol 283 (6) ◽  
pp. H2636-H2643 ◽  
Author(s):  
Jianhua Li ◽  
Lawrence I. Sinoway
Keyword(s):  
Blood Pressure ◽  
Receptor Antagonist ◽  
Pyridoxal Phosphate ◽  
Pressor Response ◽  
P2x Receptors ◽  
Triceps Surae ◽  
P2x Receptor ◽  
Disulfonic Acid ◽  
Muscle Stretch ◽  
Arterial Blood

We examined whether ATP stimulation of P2X purinoceptors would raise blood pressure in decerebrate cats. Femoral arterial injection of the P2X receptor agonist α,β-methylene ATP into the blood supply of the triceps surae muscle induced a dose-dependent increase in arterial blood pressure. The maximal increase in mean arterial pressure (MAP) evoked by 0.1, 0.2, and 0.5 mM α,β-methylene ATP (0.5 ml/min injection rate) was 6.2 ± 2.5, 22.5 ± 4.4, and 35.2 ± 3.9 mmHg, respectively. The P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (2 mM ia) attenuated the increase in MAP elicited by intra-arterial α,β-methylene ATP (0.5 mM), whereas the P2Y receptor antagonist reactive blue 2 (2 mM ia) did not affect the MAP response to α,β-methylene ATP. In a second group of experiments, we tested the hypothesis that ATP acting through P2X receptors would sensitize muscle afferents and, thereby, augment the blood pressure response to muscle stretch. Two kilograms of muscle stretch evoked a 26.5 ± 4.3 mmHg increase in MAP. This MAP response was enhanced when 2 mM ATP or 0.1 mM α,β-methylene ATP (0.5 ml/min) was arterially infused 10 min before muscle stretch. Furthermore, this effect of ATP on the pressor response to stretch was attenuated by 2 mM pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid ( P < 0.05) but not by the P1 purinoceptor antagonist 8-( p-sulfophenyl)-theophylline (2 mM). These data indicate that activation of ATP-sensitive P2X receptors evokes a skeletal muscle afferent-mediated pressor response and that ATP at relatively low doses enhances the muscle pressor response to stretch via engagement of P2X receptors.

scholarly journals Purinergic 2X receptors play a role in evoking the exercise pressor reflex in rats with peripheral artery insufficiency

2014 ◽  
Vol 306 (3) ◽  
pp. H396-H404 ◽  
Author(s):  
Audrey J. Stone ◽  
Katsuya Yamauchi ◽  
Marc P. Kaufman
Keyword(s):  
Pyridoxal Phosphate ◽  
Pressor Response ◽  
P2x Receptors ◽  
P2x Receptor ◽  
Disulfonic Acid ◽  
Femoral Arteries ◽  
Exercise Pressor Reflex ◽  
Pressor Responses ◽  
Before And After ◽  
Pressor Reflex

Purinergic 2X (P2X) receptors on the endings of thin fiber afferents have been shown to play a role in evoking the exercise pressor reflex in cats. In this study, we attempted to extend this finding to decerebrated, unanesthetized rats whose femoral arteries were either freely perfused or were ligated 72 h before the start of the experiment. We first established that our dose of pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 10 mg/kg), a P2X receptor antagonist, attenuated the pressor response to α,β-methylene ATP (10 μg/kg), a P2X receptor agonist. We then compared the exercise pressor reflex before and after infusing PPADS into the arterial supply of the hindlimb muscles that were statically contracted. In rats with freely perfused femoral arteries, the peak pressor responses to contraction were not significantly attenuated by PPADS (before PPADS: 19 ± 2 mmHg, 13 min after PPADS: 17 ± 2 mmHg, and 25 min after PPADS: 17 ± 3 mmHg). Likewise, the cardioaccelerator and renal sympathetic nerve responses were not significantly attenuated. In contrast, we found that in rats whose femoral arteries were ligated PPADS significantly attenuated the peak pressor responses to contraction (before PPADS: 37 ± 5 mmHg, 13 min after PPADS: 27 ± 6 mmHg, and 25 min after PPADS: 25 ± 5 mmHg; P < 0.05). Heart rate was not significantly attenuated, but renal SNA was at certain time points over the 30-s contraction period. We conclude that P2X receptors play a substantial role in evoking the exercise pressor reflex in rats whose femoral arteries were ligated but play only a minimal role in evoking the reflex in rats whose femoral arteries were freely perfused.

scholarly journals Spinal P2X receptor modulates muscle pressor reflex via glutamate

2009 ◽  
Vol 106 (3) ◽  
pp. 865-870 ◽  
Author(s):  
Jianhua Li ◽  
Jian Lu ◽  
Zhaohui Gao ◽  
Satoshi Koba ◽  
Jihong Xing ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Dorsal Horn ◽  
Pyridoxal Phosphate ◽  
Pressor Response ◽  
Cardiovascular Responses ◽  
P2x Receptors ◽  
P2x Receptor ◽  
Disulfonic Acid ◽  
Pressor Reflex ◽  
Stimulation Of

Static contraction of skeletal muscle evokes reflex increases in blood pressure and heart rate. Previous studies showed that P2X receptors located at the dorsal horn of the spinal cord play a role in modulating the muscle pressor reflex. P2X stimulation can alter release of the excitatory amino acid, glutamate (Glu). In this report, we tested the hypothesis that stimulation of P2X receptors enhances the concentrations of Glu ([Glu]) in the dorsal horn, and that blocking P2X receptors attenuates contraction-induced Glu increases and the resultant reflex pressor response. Contraction was elicited by electrical stimulation of the L7 and S1 ventral roots of 14 cats. Glu samples were collected from microdialysis probes inserted in the L7 level of the dorsal horn of the spinal cord, and dialysate [Glu] was determined using the HPLC method. First, microdialyzing α,β-methylene ATP (0.4 mM) into the dorsal horn significantly increased [Glu]. In addition, contraction elevated [Glu] from baseline of 536 ± 53 to 1,179 ± 192 nM ( P < 0.05 vs. baseline), and mean arterial pressure by 39 ± 8 mmHg in the control experiment. Microdialyzing the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (10 mM) into the dorsal horn attenuated the contraction induced-Glu increase (610 ± 128 to 759 ± 147 nM; P > 0.05) and pressor response (16 ± 3 mmHg, P < 0.05 vs. control). Our findings demonstrate that P2X modulates the cardiovascular responses to static muscle contraction by affecting the release of Glu in the dorsal horn of the spinal cord.

Pharmacological modulation of ion transport across wild-type and ΔF508 CFTR-expressing human bronchial epithelia

2000 ◽  
Vol 279 (2) ◽  
pp. C461-C479 ◽  
Author(s):  
Daniel C. Devor ◽  
Robert J. Bridges ◽  
Joseph M. Pilewski
Keyword(s):  
Cystic Fibrosis ◽  
Ion Transport ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Bronchial Epithelium ◽  
Disulfonic Acid ◽  
Wild Type ◽  
Pharmacological Agents ◽  
Transport Defect

Forskolin, UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen (Methoxsalen; 8-MOP), and genistein were evaluated for their effects on ion transport across primary cultures of human bronchial epithelium (HBE) expressing wild-type (wt HBE) and ΔF508 (ΔF-HBE) cystic fibrosis transmembrane conductance regulator. In wt HBE, the baseline short-circuit current ( I sc) averaged 27.0 ± 0.6 μA/cm2 ( n = 350). Amiloride reduced this I sc by 13.5 ± 0.5 μA/cm2 ( n = 317). In ΔF-HBE, baseline I sc was 33.8 ± 1.2 μA/cm2 ( n = 200), and amiloride reduced this by 29.6 ± 1.5 μA/cm2 ( n = 116), demonstrating the characteristic hyperabsorption of Na+ associated with cystic fibrosis (CF). In wt HBE, subsequent to amiloride, forskolin induced a sustained, bumetanide-sensitive I sc(Δ I sc = 8.4 ± 0.8 μA/cm2; n = 119). Addition of acetazolamide, 5-( N-ethyl- N-isopropyl)-amiloride, and serosal 4,4′-dinitrostilben-2,2′-disulfonic acid further reduced I sc, suggesting forskolin also stimulates HCO3 − secretion. This was confirmed by ion substitution studies. The forskolin-induced I scwas inhibited by 293B, Ba2+, clofilium, and quinine, whereas charybdotoxin was without effect. In ΔF-HBE the forskolin I sc response was reduced to 1.2 ± 0.3 μA/cm2 ( n = 30). In wt HBE, mucosal UTP induced a transient increase in I sc (Δ I sc = 15.5 ± 1.1 μA/cm2; n = 44) followed by a sustained plateau, whereas in ΔF-HBE the increase in I sc was reduced to 5.8 ± 0.7 μA/cm2 ( n = 13). In wt HBE, 1-EBIO, NS004, 8-MOP, and genistein increased I sc by 11.6 ± 0.9 ( n = 20), 10.8 ± 1.7 ( n = 18), 10.0 ± 1.6 ( n = 5), and 7.9 ± 0.8 μA/cm2( n = 17), respectively. In ΔF-HBE, 1-EBIO, NS004, and 8-MOP failed to stimulate Cl− secretion. However, addition of NS004 subsequent to forskolin induced a sustained Cl−secretory response (2.1 ± 0.3 μA/cm2, n = 21). In ΔF-HBE, genistein alone stimulated Cl− secretion (2.5 ± 0.5 μA/cm2, n = 11). After incubation of ΔF-HBE at 26°C for 24 h, the responses to 1-EBIO, NS004, and genistein were all potentiated. 1-EBIO and genistein increased Na+ absorption across ΔF-HBE, whereas NS004 and 8-MOP had no effect. Finally, Ca2+-, but not cAMP-mediated agonists, stimulated K+ secretion across both wt HBE and ΔF-HBE in a glibenclamide-dependent fashion. Our results demonstrate that pharmacological agents directed at both basolateral K+ and apical Cl− conductances directly modulate Cl−secretion across HBE, indicating they may be useful in ameliorating the ion transport defect associated with CF.

Role of CFTR in chloride secretion across human tracheal epithelium

1995 ◽  
Vol 269 (5) ◽  
pp. L561-L566 ◽  
Author(s):  
B. Q. Shen ◽  
R. J. Mrsny ◽  
W. E. Finkbeiner ◽  
J. H. Widdicombe
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Culture Conditions ◽  
Tracheal Epithelium ◽  
Disulfonic Acid ◽  
Acetoxymethyl Ester ◽  
Cl Channels

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.

Effects of ammonium ion and ammonia on function and morphology of in vitro frog gastric mucosa

1993 ◽  
Vol 265 (2) ◽  
pp. G277-G288 ◽  
Author(s):  
A. Yanaka ◽  
H. Muto ◽  
S. Ito ◽  
W. Silen
Keyword(s):  
Gastric Mucosa ◽  
Electrical Resistance ◽  
Rana Catesbeiana ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ammonium Ion ◽  
Gastric Epithelial Cells ◽  
Sudden Drop ◽  
Oxynticopeptic Cells

The effects of ammonium ion (NH+4) and ammonia (NH3) on function and morphology of gastric epithelial cells were studied in intact sheets of in vitro frog (Rana catesbeiana) gastric mucosa. Luminal 115 mM NH4Cl at luminal pH 8.0 (calculated [NH3] 2.7 mM), but not at 5.0 (calculated [NH3] 3 microM) induced 1) an increase in intracellular pH (pHi) in oxynticopeptic cells (OPC) and decreases in transmucosal potential difference (PD) and electrical resistance (R) in resting tissues, 2) a decrease in histamine-stimulated H+ secretion and an increase in H+ backdiffusion after removal of luminal NH4Cl, and 3) augmented acidification of OPC during luminal acidification. Serosal 30 mM NH4Cl at serosal pH 7.2 (calculated [NH3] 0.47 mM) induced 1) an increase in pHi in OPC and inhibition of the alkalinization of OPC after removal of ambient Cl-, 2) a decrease in PD associated with the increase in R and decrease in short-circuit current, effects attenuated by serosal 15 mM K+, accentuated by 0.2 mM Ba2+, and abolished by removal of ambient Cl-, 3) a sudden drop of PD in resting, but not in stimulated tissues, effects prevented by high serosal pH (7.8), serosal HCO3-, or removal of luminal Cl-, 4) a decrease in histamine-stimulated H+ secretion and an increase in H+ backdiffusion after removal of NH4Cl, and 5) augmented acidification of OPC during luminal acidification. These results suggest that 1) luminal NH3, but not NH+4, increases backdiffusion of H+ from the lumen to the mucosa, 2) serosal NH3 and/or NH+4 induces depolarization of OPC and decreases electrogenic Cl- transport, thereby attenuating the activity of the basolateral Cl(-)-HCO3- exchanger in OPC, and 3) both of these effects contribute to the augmented acidification of OPC during exposure to high luminal [H+].

Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors

1999 ◽  
Vol 277 (4) ◽  
pp. F552-F559 ◽  
Author(s):  
David E. McCoy ◽  
Amanda L. Taylor ◽  
Brian A. Kudlow ◽  
Katherine Karlson ◽  
Margaret J. Slattery ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Extracellular Nucleotides ◽  
P2x Receptor ◽  
Apical Plasma Membrane ◽  
Receptor Subtypes ◽  
Nacl Transport

Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na+ absorption [measured via Na+ short-circuit current[Formula: see text])] and stimulated Cl− secretion [measured via Cl−short-circuit current ([Formula: see text])]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate [Formula: see text] and[Formula: see text]. By RT-PCR, two P2X receptor channels (P2X3, P2X4) and two P2Y G protein-coupled receptors (P2Y1, P2Y2) were identified. Functional localization of P2 purinoceptors suggest that [Formula: see text] is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas[Formula: see text] is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit[Formula: see text] across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate [Formula: see text]through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

cAMP-dependent sulfate secretion by the rabbit distal colon: a comparison with electrogenic chloride secretion

1997 ◽  
Vol 273 (1) ◽  
pp. C148-C160 ◽  
Author(s):  
R. W. Freel ◽  
M. Hatch ◽  
N. D. Vaziri
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Disulfonic Acid ◽  
Cl Secretion ◽  
Cyclic Monophosphate ◽  
Anion Conductance ◽  
Flux Measurements

The ability of a Cl-secreting epithelium to support net secretion of an anion other than a halide was investigated with 35SO4 flux measurements across the isolated, short-circuited rabbit distal colon. In most experiments, 36Cl fluxes were simultaneously measured to validate the secretory capacity of the tissues. Serosal addition of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 0.5 mM) stimulated a sustained net secretion of SO4 (about -3.0 nmol.cm-2.h-1 from a 0.20 mM solution) via an increase in the serosal-to-mucosal unidirectional flux, whereas Ca ionophore A-23187 (1 microM, serosal) produced a more transient stimulation of SO4 and Cl secretion. Net adenosine 3',5'-cyclic monophosphate (cAMP)-dependent SO4 and Cl secretion were strongly voltage sensitive, principally through the potential dependence of the serosal-to-mucosal fluxes, indicating an electrogenic transport process. Symmetrical replacement of either Na, K, or Cl inhibited cAMP-dependent SO4 secretion, whereas HCO3-free buffers had no effect on SO4 secretion. Serosal bumetanide (50 microM) or furosemide (100 microM) reduced DBcAMP-stimulated SO4 and Cl secretion, whereas serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (50 microM) blocked DBcAMP-induced SO4 secretion while enhancing net Cl secretion and short-circuit current. Mucosal 5-nitro-2-(3-phenylpropylamino)benzoic acid partially inhibited SO4 secretion and completely inhibited Cl secretion. It is concluded that secretagogue-stimulated SO4 secretion, like Cl secretion, may be an electrogenic process mediated by diffusive efflux through an apical anion conductance. Cellular accumulation of SO4 across the basolateral membrane appears to be achieved by a mechanism that is distinct from that employed by Cl.

Action of adenosine on chloride active transport of isolated frog cornea

1979 ◽  
Vol 237 (2) ◽  
pp. F121-F127
Author(s):  
B. S. Spinowitz ◽  
J. A. Zadunaisky
Keyword(s):  
Rana Catesbeiana ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Adenyl Cyclase ◽  
Naturally Occurring ◽  
Chloride Pump ◽  
Net Flux ◽  
The One ◽  
Sustained Increase

Addition of adenosine (10–7 to 10–4 M) to the tear side of isolated corneas (Rana catesbeiana) produced a rapid, sustained increase in short-circuit current, potential difference, and radioisotopic chloride net flux. The increased net chloride flux accounted for the increased short-circuit current. Adenosine, a known activator of adenyl cyclase in other tissues, exerted its effects on chloride transport through a receptor different from the one described for epinephrine and prostaglandins in the corneal epithelium. Propranolol inhibited the epinephrine response but not the adenosine effect. Dipolyphloretin phosphate inhibited prostaglandin responses but did not affect the adenosine stimulation of chloride transport. Adenine and/or ribose, parts of the adenosine molecule, had no stimulatory effect, but 5'-AMP had a partial effect.The activation of the chloride pump with DBcAMP blocked the response to adenosine. Adenosine interacted with the effects of theophylline. Adenosine, a naturally occurring molecule, stimulated chloride transport by activation of adenyl cyclase through a separate membrane receptor in the corneal eqithelium.

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