Purinoreceptor-mediated current in myocytes from renal resistance arteries

Abstract

Background and purpose: Ionotropic purinoreceptors (P2X) in renal vascular smooth muscle cells (RVSMCs) are involved in mediating the sympathetic control and paracrine regulation of renal blood flow (RBF). Activation of P2X receptors elevates [Ca(2+)](i) in RVSMCs triggering their contraction, leading to renal vasoconstriction and decrease of RBF. The goal of the present work was to characterize the P2X receptor-mediated ionic current (I(P2X)) and to identify the types of P2X receptors expressed in myocytes isolated from interlobar and arcuate arteries of rat kidney.

Experimental approach: The expression of P2X receptors in isolated RVSMCs was analysed by reverse transcription (RT)-PCR. I(P2X) and membrane potential were recorded using the amphotericin B-perforated patch method.

Key results: RT-PCR analysis on single RVSMCs showed the presence of genes encoding P2X1 and P2X4 receptors. Under voltage clamp conditions, the selective P2X receptor agonist alphabeta-methylene ATP (alphabeta-meATP) evoked I(P2X) similar to that induced by ATP. Under current clamp conditions, both ATP and alphabeta-meATP evoked a spike-like membrane depolarization followed by a sustained depolarization, linking P2X receptors in RVSMCs to sympathetic control of renal vascular tone. A selective antagonist of P2X1 receptors, NF279, reduced I(P2X) amplitude by approximately 65% concentration-dependently manner within the nanomolar to sub-micromolar range. The residual current was resistant to micromolar concentrations of NF279, but was inhibited by sub-millimolar to millimolar concentrations of NF279.

Conclusions and implications: Two types of functional P2X receptors, monomeric P2X1 and heteromeric P2X1/4 receptors, are expressed in RVSMCs. Our study has identified important targets for possible pharmacological intervention in the sympathetic control of renal circulation.

Figure 3

(Aa) Traces of the inward current elicited in RVSMCs by 10 µM ATP, 10 µM αβ-meATP and 10 µM UTP at holding potential −60 mV. (Ab) The peak current density for I_P2X induced by 10 µM of ATP (n= 7), 10 µM of αβ-meATP (n= 123) and 10 µM of UTP (n= 7). (Ba) Action of NF279 and NF449 on the inward current activated by 10 µM αβ-meATP. (Bb) Inhibition of I_P2X by 2 nM NF279 (n= 5) and 2 nM NF449 (n= 7). * indicates P < 0.05.

Figure 1

RT–PCR analysis of P2X receptor expression in RVSMCs from rat renal resistance arteries. (A) The purity of the preparation was verified using primers designed to amplify genes encoding the markers for certain cell types which can be found in blood vessel: SM-MHC (745 bp) for smooth muscle cells, CD34 (852 bp) for fibroblasts and endothelial cells, PGP9.5 (510 bp) for neurons, NG2 (996 bp) for pericytes. As a positive control, primers for β-actin (721 bp) were used. Primers were tested for their specificity using cDNA preparations from a mixture of rat brain and liver tissue (Aa). The RVSMCs preparation showed expression of genes encoding β-actin and SM-MHC only. (Ab). (B) Primers designed to amplify genes encoding P2X receptors sub-types: P2X1 (830 bp), P2X2 (1007 bp), P2X3 (1018), P2X4 (725 bp), P2X5 (995 bp) and P2X7 (973 bp) were tested for their specificity using cDNA preparations of rat brain and liver tissue (Ba) before using them with SMCs preparations (Bb). RVSMCs were found to express genes encoding only two types of P2X receptor proteins: P2X1 and P2X4.

Figure 2

Electrical events induced by ATP and αβ-meATP in RVSMCs. 2 s pulses of ATP (10 µM) or selective P2X receptor agonist αβ-meATP (10 µM) evoked similar inward currents (Aa and Ba correspondingly) or changes of the cell membrane potential (Ab and Bb, respectively) suggesting a crucial role of P2X receptors in mediating the response of RVSMCs to ATP.

Figure 4

(A) Kinetics of re-sensitization of P2X receptors in RVSMCs. (Aa) The 2 s pulses of 10 µM αβ-meATP were repetitively applied with different intervals, as indicated. Virtually complete recovery of the peak current density was observed within 7 min. Representative traces recorded in single cell illustrate some variability in the relative contribution of the sustained component to I_P2X (arrows). (Ab) Averaged time-course of I_P2X re-sensitization: the averaged peak current densities were normalized to the peak current density induced by the first αβ-meATP application and plotted versus duration of intervals between subsequent αβ-meATP applications (n= 6–7 cell for each point). (B) Voltage–dependence of I_P2X. (Ba) Representative current traces recorded in single RVSMC. (Bb) Current–voltage relationship for the peak I_P2X revealed inward rectification (n= 5).

Figure 5

(A) Concentration–dependence of activation of I_P2X by αβ-meATP. (Ab) Traces of I_P2X evoked by various concentrations of αβ-meATP were normalized to the peak amplitude of the maximal response evoked by 100 µM of αβ-meATP. (Ab) Averaged normalized peak amplitude of I_P2X was plotted versus corresponding concentration of αβ-meATP (n= 5–6). The concentration–dependence curve fitted to the experimental points using non-linear least square minimization algorithm revealed an EC₅₀= 1.1 ± 0.1 µM. (B) Dependence of I_P2X on extracellular Ca²⁺ concentration ([Ca²⁺]_o). (Ba) I_P2X was elicited by repetitive (with 8 min interval) applications of 10 µM αβ-meATP to RVSMC incubated in external solutions with gradually increasing [Ca²⁺]_o, as indicated. (Bb) The plot of the dependence of I_P2X on [Ca²⁺]_o revealed that increase in [Ca²⁺]_o from 1 to 5 mM reduced I_P2X by 33 ± 7% (n= 5–6).

Figure 6

(A) Concentration–dependence of inhibition of I_P2X by NF279 (Aa) Traces of I_P2X evoked by repetitive (with 8 min interval) applications of αβ-meATP following 5 min pre-incubation with gradually increasing concentrations of NF279, as indicated. Currents were normalized to the peak amplitude of I_P2X recorded in the absence of NF279. (Ab) Averaged normalized peak amplitude of I_P2X was plotted versus corresponding concentration of NF279 (n= 5–12 for each data point). The best fit of the theoretical sigmoidal curves to the experimental points using a non-linear least square minimization algorithm resulted in two distinct sigmoid curves. Thus, two components with different sensitivity to NF279 contributed to I_P2X: the high-affinity component (with IC₅₀= 6.8 ± 0.4 nM, fitted by red sigmoid) and the low-affinity component (with IC₅₀= 71.4 ± 2.8 µM, fitted by blue sigmoid). (B) Potentiation of NF279-resistant I_P2X by ivermectin. (Ba) I_P2X persisting in the presence of 2 µM NF279 was augmented by 3 µM of ivermectin, a positive P2X receptor modulator. (Bb) Summary of the data obtained in five RVSMCs. For each cell, the peak current density detected in the presence of the drug or drug combination was normalized to that in control. NF279 (2 µM) reduced the peak current density relative to its control magnitude. Subsequent incubation with 3 µM ivermectin in the presence of 2 µM NF279 partly reversed the inhibition due to NF279. * indicates P < 0.05.