UTP activates small-conductance Ca2+-activated K+ channels in murine detrusor PDGFRα+ cells

Abstract

Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction is likely due to activation of inward currents in smooth muscle cells, and prolonged relaxation may be due to activation of small-conductance Ca(2+)-activated K(+) (SK) channels via P2Y1 receptors expressed by detrusor PDGF receptor (PDGFR)α(+) cells. We investigated whether other subtypes of P2Y receptors are involved in the activation of SK channels in PDGFRα(+) cells of detrusor muscles. Quantitative analysis of transcripts revealed that P2ry2, P2ry4, and P2ry14 are expressed in PDGFRα(+) cells of P2ry1-deficient/enhanced green fluorescent protein (P2ry1(-/-)/eGFP) mice at similar levels as in wild-type mice. UTP, a P2Y2/P2Y4 agonist, activated large outward currents in detrusor PDGFRα(+) cells. SK channel blockers and an inhibitor of phospholipase C completely abolished currents activated by UTP. In contrast, UTP activated nonselective cation currents in smooth muscle cells. Under current-clamp (current = 0), UTP induced significant hyperpolarization of PDGFRα(+) cells. MRS2500, a selective P2Y1 antagonist, did not affect UTP-activated outward currents in PDGFRα(+) cells from wild-type mice, and activation of outward currents by UTP was retained in P2ry1(-/-)/eGFP mice. As a negative control, we tested the effect of MRS2693, a selective P2Y6 agonist. This compound did not activate outward currents in PDGFRα(+) cells, and currents activated by UTP were unaffected by MRS2578, a selective P2Y6 antagonist. The nonselective P2Y receptor blocker suramin inhibited UTP-activated outward currents in PDGFRα(+) cells. Our data demonstrate that P2Y2 and/or P2Y4 receptors function, in addition to P2Y1 receptors, in activating SK currents in PDGFRα(+) cells and possibly in mediating purinergic relaxation responses in detrusor muscles.

Keywords: detrusor relaxation; interstitial cells; potassium channels; purinergic receptors.

Fig. 1.

End-point gels and quantitative analysis of P2ry transcripts in unsorted cells and sorted detrusor PDGF receptor (PDGFR)α⁺ cells from P2ry1-deficient/enhanced green fluorescent protein (P2ry₁^−/−/eGFP) mice. A: all P2rys except P2ry₁ were detected in PDGFRα⁺ cells purified by FACS or in unsorted cells dispersed from detrusor muscles of P2ry₁^−/−/eGFP mice. B: summary graph showing that P2ry2, P2ry4, and P2ry14 were enriched in detrusor PDGFRα⁺ cells from P2ry₁^−/−/eGFP mice compared with unsorted cells. P2ry12 and P2ry13 were minimally expressed and thus not displayed. Expression of all transcripts was normalized to Gapdh. qPCR, quantitative PCR. ***P < 0.001.

Fig. 2.

Effects of UTP on the membrane currents and membrane potential in PDGFRα⁺ cells. A: cells were held at −40 mV. UTP (10 μM) activated outward currents in PDGFRα⁺ cells, and these responses were reproducible in response to multiple applications. B: expanded timescales at points designated as a and b in A showing current responses to ramp potentials before (a) and after (b) addition of UTP. C: UTP (10 μM) hyperpolarized PDGFRα⁺ cells under current-clamp mode [current (I) = 0] and activated outward currents in the same cells under voltage-clamp mode [V-C; holding potential (HP) = −40 mV].

Fig. 3.

Effects of UTP on membrane currents in smooth muscle cells (SMCs). A: UTP (10 μM) and ATP (100 μM) induced inward currents in SMCs dialyzed with a K⁺-rich solution at a HP of −60 mV. B: expanded timescales from control conditios (a) and in the presence of UTP (b) in A. C: UTP activated inward currents in SMCs dialyzed with Cs⁺-rich pipette solution (HP = −40 mV). D: expanded timescales from before (a) and after UTP (b) in C. Horizontal dotted lines denote 0 pA. Arrowheads denote the applications of ramp potentials (protocol shown in bottom traces in A and C).

Fig. 4.

Effect of small-conductance Ca²⁺-activated K⁺ (SK) channel blockers on the outward currents activated by UTP in PDGFRα⁺ cells. A and C: cells were held at −40 mV. UTP (10 μM) activated outward currents, and these responses were inhibited by apamin (0.3 μM; A) or UCL1684 (1 μM; C). B and D: expanded timescaless at points denoted by a and b in A and C showing the outward currents activated by UTP (B,a and D,a); these currents were blocked by apamin (B,b) or UCL1684 (D,b). Horizontal dotted lines in all A–D denote 0 pA. Arrowheads denote the application of ramp potentials (see protocols shown in bottom traces in B and D).

Fig. 5.

Effect of a phospholipase C (PLC) inhibitor on outward currents activated by UTP in PDGFRα⁺ cells. A: cells were held at −40 mV. UTP induced outward currents, and these responses were blocked by U73122 (1 μM). B: expanded timescales from points denoted by a–c in A showing current responses to ramp potentials under control conditions (a), after the addition of UTP (b), and after the addition of U73122 and UTP (c). C: cells were held at −40 mV. UTP (10 μM) activated outward currents before and after the addition of U73343 (1 μM), an inactive analog of U73122. D: expanded timescales from points denoted by a--c in C. The bottom traces in B and D depict the ramp depolarization protocols. Horizontal dotted lines in A–D denote 0 pA. Arrowheads denote the application of ramp potentials.

Fig. 6.

Effects of P2Y₁ antagonist and deletion on outward currents activated by UTP in PDGFRα⁺ cells. A: UTP (10 μM) activated outward currents in PDGFRα⁺ cells from wild-type mice before and after the addition of MRS2500 (1 μM; HP = −40 mV). B: expanded timescales showing responses to ramp potentials during only MRS2500 (a) and with MRS2500 and UTP (b) from the points denoted in A. C: UTP (10 μM) also activated outward currents at −40 mV in PDGFRα⁺ cells from P2ry₁^−/−/eGFP mice. D: current traces at expanded timescales during ramp potentials in control conditions (a) and after the addition of UTP (b) from points denoted in C. Horizontal dotted lines in A–D denote 0 pA. Arrowheads denote the applications of ramp potentials.

Fig. 7.

Effects of P2Y6 agonist and antagonist and nonselective P2Y receptor inhibitor on PDGFRα⁺ cells. A: UTP induced outward currents in the presence of MRS2578 (a selective P2Y6 antagonist). B: expanded timescales showing responses to ramp potentials before (a; pretreatment with MRS2578) and after the addition of UTP (b) from points denoted in A. C: MRS2693 (a selective P2Y6 agonist) activated only small-amplitude outward currents at a HP of −40 mV in PDGFRα⁺ cells. D: expanded timescales of ramp depolarization from control conditions (a), with MRS2693 (b), and with UTP (c) in C. The current-voltage curves in D show that the minor response to MRS2693 was not a voltage-independent SK-like current, as activated by UTP. E: suramin (a nonselective P2Y antagonist) inhibited UTP-activated outward currents at a HP of −40 mV in PDGFRα⁺ cells. F: expanded timescales of ramp depolarization from control conditions (a), with UTP (b), and UTP in the presence of suramin (c) in E. Horizontal dotted lines in A–F denote 0 pA. Arrowheads denote applications of ramp potentials.