Neurally released ATP mediates endothelium-dependent hyperpolarization in the circular smooth muscle cells of chicken anterior mesenteric artery

Abstract

The object of the present study was to clarify the neurotransmitter(s) controlling membrane responses to electrical field stimulation (EFS) in the circular smooth muscle cells of first-order branches of chicken anterior mesenteric artery.EFS (five pulses at 20 Hz, 1 ms) evoked a hyperpolarization of amplitude--21.6+/-1.2 mV, total duration 21.8+/-1.2 s and latency 641.7+/-81.9 ms. The response was tetrodotoxin-sensitive and nonadrenergic noncholinergic (NANC) in nature. The NANC response was blocked by the nonspecific purinergic antagonist, suramin, indicating that the response is mediated by the neurotransmitter adenosine 5'-triphosphate (ATP). Either desensitization or blockade of P2Y receptor with its putative agonist 2-methylthioATP (1 microM for 30 min) or with its antagonist cibacron blue F3GA (10 microM), respectively, abolished the purinergic hyperpolarization. PPADS at concentrations up to 100 microM had no effect on the EFS-induced response, indicating that this response is mediated through P2Y, but not P2X, receptor. In addition, the response was completely abolished by two specific P2Y1 receptor antagonists, namely, MRS 2179 (300 nM) and A3P5PS (10 microM). Removal of the endothelium abolished the purinergic hyperpolarization, which was converted, in some preparations, to a small depolarization, indicating that the hyperpolarizing response is endothelium-dependent. The present study suggests that in first-order branches of chicken anterior mesenteric artery, ATP released from perivascular nerves may diffuse to the endothelium-activating P2Y1 receptor to induce release of an inhibitory substance that mediates hyperpolarization in the circular smooth muscle.

Figure 1

Hyperpolarization produced by single and multiple nerve stimulation at 33°C bath temperature. EFS-evoked hyperpolarization produce by a single pulse (a) or 2 (b), 5 (c) or 10 (d) pulses at 20 Hz. Membrane potential was −62 mV.

Figure 2

Effect of endothelium denudation on nerve stimulation using a train of five pulses. (A) Typical recordings showings EFS-evoked hyperpolarization in artery with endothelium. (B) Typical recordings showing EFS-evoked hyperpolarization in artery without endothelium. Membrane potential for (A, Ba, Bb) were −63, −62 and −63 mV, respectively.

Figure 3

Effects of suramin on the EFS (using a train of five pulses)-evoked hyperpolarization. (a) Typical recording showing the effect of suramin (500 μM; n=5) on the EFS-evoked hyperpolarization. (b) Summary graph showing concentration-dependent inhibition of suramin on the amplitude of EFS-evoked hyperpolarization. (c) Typical recording showing the effect of PPADS (100 μM; n=5) on the EFS-evoked hyperpolarization. Membrane potentials for (a, c) were −60 and −59 mV, respectively.

Figure 4

Effects of CBF3GA on the EFS (using a train of five pulses)-evoked hyperpolarization. (a) Typical recording showing the effect of CBF3GA (100 μM; n=6) on the EFS-evoked hyperpolarization. (b) Summary graph showing concentration-dependent inhibition of CBF3GA on the amplitude of EFS-evoked hyperpolarization. Membrane potential for (a) was −62 mV.

Figure 5

Effect of desensitization of P2Y receptor by its agonist, 2-MeSATP, on the EFS (using a train of five pulses)-evoked hyperpolarization. (A) Typical recordings showings EFS-evoked hyperpolarization. (B) Typical recording showing the effect of P2Y receptor desensitization by its agonist 2-MeSATP that EFS-evoked hyperpolarization was converted to a small depolarization (1 μM; n=3). Membrane potential for (A, Ba, Bb) were −62, −60 and −61 mV, respectively.

Figure 6

Effects of P2Y1 receptor antagonists on the EFS (using a train of five pulses)-evoked hyperpolarization and effect of P2X receptor antagonist on depolarization. (a) Typical recording showing the effect of MRS 2179 (300 nM; n=5) on the EFS-evoked hyperpolarization. (b) Typical recording showing the effects of A3P5PS (10 μM; n=7) on the EFS-evoked hyperpolarization, and effect of PPADS (100 μM; n=3) on the A3P5PS-induced depolarization. Membrane potentials for (a, b) were −60 and −62, respectively.

Figure 7

Effect of exogenously applied ATP on membrane potential and muscular tone of chicken mesenteric artery. Summary graph showing concentration-dependent membrane responses of ATP (n=6) and summary graph showing concentration-dependent relaxation of ATP (n=7).