An electrophysiological study of excitatory purinergic neuromuscular transmission in longitudinal smooth muscle of chicken anterior mesenteric artery

Abstract

1. The object of the present study was to clarify the neurotransmitters controlling membrane responses to electrical field stimulation (EFS) in the longitudinal smooth muscle cells of the chicken anterior mesenteric artery. 2. EFS (5 pulses at 20 Hz) evoked a depolarization of amplitude 19.7+/-2.1 mV, total duration 29.6+/-3.1 s and latency 413.0+/-67.8 ms. This depolarization was tetrodotoxin (TTX)-sensitive and its amplitude was partially decreased by atropine (0.5 microM); however, its duration was shortened by further addition of prazosin (10 microM). 3. Atropine/prazosin-resistant component was blocked by the nonspecific purinergic antagonist, suramin, in a dose-dependent manner, indicating that this component is mediated by the neurotransmitter adenosine 5'-triphosphate (ATP). 4. Neither desensitization nor blocking of P2X receptor with its putative receptor agonist alpha,beta-methylene ATP (alpha,beta-MeATP, 1 microM) and its antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic (PPADS, up to 50 microM), had significant effect on the purinergic depolarization. In contrast, either desensitization or blocking of P2Y receptor with its putative agonist 2-methylthioATP (2-MeSATP, 1 microM) and its antagonist Cibacron blue F3GA (CBF3GA, 10 microM) abolished the purinergic depolarization, indicating that this response is mediated through P2Y but not P2X receptor. 5. The purinergic depolarization was inhibited by pertussis toxin (PTX, 600 ng ml(-1)). Furthermore, it was significantly inhibited by a phospholipase C (PLC) inhibitor, U-73122 (10 microM), indicating that the receptors involved in mediating the purinergic depolarization are linked to a PTX-sensitive G-protein, which is involved in a PLC-mediated signaling pathway. 6. Data of the present study suggest that the EFS-induced excitatory membrane response occurring in the longitudinal smooth muscle of the chicken anterior mesenteric artery is mainly purinergic in nature and is mediated via P2Y purinoceptors.

Figure 1

Depolarization produced by single and multiple nerve stimulation at 35 and 29°C bath temperature. (a) Depolarization produced by a single pulse (1 ms) (left panel), repetitive nerve stimulation by single pulses at 0.25 Hz resulted in facilitation and/or summation (middle panel) and 5 pulse at 20 Hz evoked depolarization with an initial spike (left panel) at 35°C bath temperature. (b) Depolarization produced by a single pulse (1 ms) (left panel) and 5 pulse at 20 Hz evoked depolarization without an initial spike (left panel) at 29°C bath temperature. Membrane potential values for (a) and (b) were −38 and −36 mV, respectively.

Figure 2

Effects of atropine and prazosin on the EFS-evoked depolarization. (a) Typical recordings showing the effects of atropine (0.5 μM) alone; n=7, and in combination with prazosin (10 μM); n=12, on the amplitude and duration of the depolarization evoked by EFS (1 ms, 5 pulses and 20 Hz). (b, c) Summary graphs showing the effects of atropine alone or combined with prazosin on the amplitude (b) and duration (c) of EFS-evoked depolarization. Membrane potential for (a) was −35 mV.

Figure 3

Effect of suramin on the EFS-evoked depolarization. (a) Typical recordings showing the effect of suramin (500 μM); n=6. (b) Summary graph showing concentration-dependent inhibition of suramin on the amplitude of the EFS-evoked depolarization. Membrane potential for (a) was −37 mV.

Figure 4

Effect of CBF3GA on the EFS-evoked depolarization. (a) Typical recordings showing the effect of CBF3GA (100 μM); n=8, on the EFS-evoked depolarization. Note that EFS-evoked depolarization was converted to a small IJP. (b) Summary graph showing concentration-dependent inhibition of CBF3GA on the amplitude of EFS-evoked depolarization. Membrane potential for (a) was −34 mV.

Figure 5

Effect of ATP on membrane potential. (a) Typical recordings showing the effect of exogenous ATP (100 μM); n=6, on the resting membrane potential under control conditions (top) and after suramin (500 μM); n=3. (b) Summary graph showing the inhibitory effect of suramin (500 μM) on the depolarizing effect of ATP. Membrane potential for (a) was −39 mV.

Figure 6

Effect of desensitization of P2 receptors by their agonists on the EFS-evoked depolarization. (a) Typical recordings showing the effect of P2Y receptor agonist; 2-MeSATP (1μM); n=4, note that EFS-evoked depolarization was converted to a small IJP (left panel) and P2X receptor agonist; α,β-MeATP (1μM); n=4 (right panel) desensitization on EFS-evoked depolarization. (b) Summary graph showing the inhibitory effect of 2-MeSATP; and the noneffect of α,β-MeATP on the recorded EFS-evoked depolarization. Membrane potential for (a) was −36 and −34 mV for the left and right panel, respectively.

Figure 7

Effect of pertussis toxin (PTX) and the phospholipase C inhibitor U-73122 on the EFS-evoked depolarization. (a) Histogram showing the sensitivity of the EFS-evoked depolarization to PTX (600 ng ml⁻¹); n=5. (b) Histogram showing the inhibitory effect of U-73122 on the recorded EFS-evoked depolarization; n=5.