Article

Pharmacological and molecular characterization of P2X receptors in rat pelvic ganglion neurons

British Journal of Pharmacology (impact factor: 4.41). 09/1998; 125(4):771 - 781. DOI:10.1038/sj.bjp.0702118 pp.771 - 781

ABSTRACT 1The presence and characteristics of P2X receptors on neurons of the rat major pelvic ganglia (MPG) have been studied using whole cell voltage-clamp, in situ hybridization and immunohistochemistry.2Rapid application of ATP (100 μM) to isolated rat MPG neurons induced moderately large inward currents (0.33–5.3 nA) in 39% of cells (108/277). The response to ATP occurred very rapidly, with an increase in membrane conductance, and desensitized slowly.3The concentration-response curve for ATP yielded an EC50 of 58.9 μM. The agonist profile was ATP2MeSATP=ATPS>BzATP, while ,β-MeATP, β,-MeATP, UTP and ADP were all inactive at concentrations up to 100 μM.4The response to ATP was antagonized by suramin (pA2=5.6), reactive blue-2 (IC50=0.7 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS).5Lowering the pH from 7.4 to 6.8 produced a marked potentiation (to 339% of control) of the responses to ATP (30 μM), while raising the pH to 8.0 attenuated the responses (to 20% of control). The EC50s for ATP were 28.8, 58.9 and 264 μM at pH 6.8, 7.4 and 8.0, respectively.6Co-application of ATP with Zn2+ produced a marked enhancement of the responses to ATP, with an EC50 of 9.55 μM. In the presence of Zn2+ (30 μM), the EC50 for ATP was decreased to 4.57 μM.7In situ hybridization revealed that the P2X receptor transcripts levels in rat MPG neurons are P2X2>P2X4>P2X1, P2X3, P2X5 and P2X6. The immunohistochemical staining revealed a small number of neurons with strong P2X2 immunoreactivity.8In conclusion, our results indicate that there are P2X receptors present on MPG neurons. The pharmacological characteristics of these receptors, the in situ hybridization and immunohistochemical evidence are consistent with them being of the P2X2 subtype, or heteromultimers, with P2X2 being the dominant component.British Journal of Pharmacology (1998) 125, 771–781; doi:10.1038/sj.bjp.0702118

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    Joel R Gever, Debra A Cockayne, Michael P Dillon, Geoffrey Burnstock, Anthony P D W Ford
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    ABSTRACT: Significant progress in understanding the pharmacological characteristics and physiological importance of homomeric and heteromeric P2X channels has been achieved in recent years. P2X channels, gated by ATP and most likely trimerically assembled from seven known P2X subunits, are present in a broad distribution of tissues and are thought to play an important role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. The known homomeric and heteromeric P2X channels can be distinguished from each other on the basis of pharmacological differences when expressed recombinantly in cell lines, but whether this pharmacological classification holds true in native cells and in vivo is less well-established. Nevertheless, several potent and selective P2X antagonists have been discovered in recent years and shown to be efficacious in various animal models including those for visceral organ function, chronic inflammatory and neuropathic pain, and inflammation. The recent advancement of drug candidates targeting P2X channels into human trials, confirms the medicinal exploitability of this novel target family and provides hope that safe and effective medicines for the treatment of disorders involving P2X channels may be identified in the near future.
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    Cleber A Trujillo, Arthur A Nery, Antonio Henrique B Martins, Paromita Majumder, Fernando A Gonzalez, Henning Ulrich
    [show abstract] [hide abstract]
    ABSTRACT: P2X receptors play an important role in communication between cells in the nervous system. Therefore, understanding the mechanisms of inhibition of these receptors is important for the development of new tools for drug discovery. Our objective has been to determine the pharmacological activity of the antagonist suramin, the most important antagonist of purinergic receptor function, as well as to demonstrate its noncompetitive inhibition and confirm a competitive mechanism between ATP and TNP-ATP in 1321N1 glial cells stably transfected with the recombinant rat P2X(2) receptor. A radioligand binding assay was employed to determine whether suramin, TNP-ATP, and ATP compete for the same binding site on the receptor. TNP-ATP displaced [alpha-32P]ATP, whereas suramin did not interfere with [alpha-32P]ATP-receptor binding. To determine the inhibition mechanism relevant for channel opening, currents obtained in fast kinetic whole-cell recording experiments, following stimulation of cells by ATP in the presence of suramin, were compared to those obtained by ATP in the presence of TNP-ATP. Supported by a mathematical model for receptor kinetics [Breitinger, H. G., Geetha, N., and Hess, G. P. (2001) Biochemistry 40, 8419-8429], the inhibition factors were plotted as functions of inhibitor or agonist concentrations. Analysis of the data indicated a competitive inhibition mechanism for TNP-ATP and a noncompetitive inhibition for suramin. Taken together, both data support a noncompetitive inhibition mechanism of the rat recombinant P2X(2) receptor by suramin, confirm the competitive inhibition by TNP-ATP, and allow the prediction of a model for P2X(2) receptor inhibition.
    Biochemistry 02/2006; 45(1):224-33. · 3.42 Impact Factor
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Keywords

1The presence
 
8.0 attenuated
 
agonist profile
 
desensitized slowly.3The concentration-response curve
 
dominant component.British Journal
 
immunohistochemical evidence
 
marked enhancement
 
marked potentiation
 
MPG neurons
 
P2X receptor transcripts levels
 
P2X receptors
 
P2X2 subtype
 
Pharmacology
 
rat MPG neurons
 
rat MPG neurons induced
 
receptors
 
situ hybridization
 
strong P2X2 immunoreactivity.8In conclusion
 
whole cell voltage-clamp
 
μM.7In situ hybridization
 

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