Molecular identification of P2X receptors in vascular smooth muscle cells from rat anterior, posterior, and basilar arteries.
BACKGROUND
Purinergic P2X receptors in vascular smooth muscle cells (VSMCs) play an important role in physiological stimulatory responses to the extracellularly released ATP. The aim of this work was to identify molecular P2X receptor subunits in VSMCs isolated from rat anterior, posterior and basilar arteries using a number of contemporary laboratory techniques.


METHODS
P2X mediated ionic currents were recorded using amphotericin B perforated patch clamp method. Gene expression analysis was performed using RT-PCR in manually collected VSMCs. The expression of proteins was confirmed by fluorescent immunocytochemistry.


RESULTS
Under voltage clamp conditions VSMCs stimulated by application of 10 μmol/l selective P2X receptor agonist αβ-meATP, the biphasic currents consisting of rapidly rising rapidly desensitizing and slowly desensitizing components were observed in freshly isolated myocytes from all three arteries. Using RT-PCR, the expression of genes encoding only P2X1 and P2X4 receptor subunits was detected in preparations from all three arteries. The expression of corresponding P2X1 and P2X4 receptor subunit proteins was confirmed in isolated VSMCs.


CONCLUSIONS
Our work therefore identified that in major arteries of rat cerebral circulation VSMCs express only P2X1 and P2X4 receptors subunits. We can propose that these P2X receptor subunits participate in functional P2X receptor structures mediating ATP-evoked stimulatory responses in cerebral vascular myocytes in vivo.
Division of Biomedical Sciences, St. George's, University of London, London, United Kingdom; Laboratory of Molecular Pharmacology and Biophysics of Cell Signalling, Bogomoletz Institute of Physiology, Kyiv, Ukraine. Electronic address: m.harhun@gmail.com.
Maksym I
MI
Harhun
Laboratory of Molecular Pharmacology and Biophysics of Cell Signalling, Bogomoletz Institute of Physiology, Kyiv, Ukraine.
Khrystyna
K
Sukhanova
Laboratory of Molecular Pharmacology and Biophysics of Cell Signalling, Bogomoletz Institute of Physiology, Kyiv, Ukraine.
Dmitri
D
Gordienko
Laboratory of Molecular Pharmacology and Biophysics of Cell Signalling, Bogomoletz Institute of Physiology, Kyiv, Ukraine.
Yulia
Y
Dyskina
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002536:Cerebral Arteries / Q000166:cytology*; D015870:Gene Expression / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology; D051381:Rats; D058474:Receptors, Purinergic P2X1 / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4 / Q000378:metabolism*
D058911:Purinergic P2X Receptor Agonists; D058474:Receptors, Purinergic P2X1; D058848:Receptors, Purinergic P2X4; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate
ATP; Artery; Cerebral circulation; Homomeric P2X1 receptor; αβ-meATP