Excitatory purinergic neurotransmission in smooth muscle of guinea-pig [corrected] taenia caeci

Abstract

Non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmission has been an area of intense interest in gut motor physiology, whereas excitatory NANC neurotransmission has received less attention. In order to further explore excitatory NANC neurotransmission, we performed conventional intracellular recordings from guinea-pig taenia caeci smooth muscle. Tissue was perfused with oxygenated Krebs solution at 35 degrees C and nerve responses evoked by either oral or aboral nerve stimulation (NS) (4 square wave pulses, 0.3 ms duration, 20 Hz). Electrical activity was characterized by slow waves upon which one to three action potentials were superimposed. Oral NS evoked an inhibitory junction potential (IJP) at either the valley or peak of the slow wave. Application of nifedipine (1 microM) abolished slow waves and action potentials, but membrane potential flunctuations (1-3 mV) and IJPs remained unaffected. Concomitant application of apamin (300 nM), a small-conductance Ca(2+)-activated K(+) channel blocker, converted the IJP to an EJP that was followed by slow IJP. Further administration of N(G)-nitro-l-arginine methyl ester (l-NAME, 200 microM), a nitric oxide synthase inhibitor, abolished the slow IJP without affecting the EJP, implying that the slow IJP is due to nitrergic innervation. The EJP was abolished by tetrodotoxin (1 microM), but was not significantly affected by atropine (3 microM) and guanethidine (3 microM) or hexamethonium (500 microM). Substance P (SP, 1 microM) desensitization caused slight attenuation of the EJP, but the EJP was abolished by desensitization with alpha,beta-methylene ATP (50 microM), a P2 purinoceptor agonist that is more potent than ATP at the P2X receptor subtype, suramin (100 microM), a non-selective P2 purinoceptor antagonist, and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 100 microM) , a selective P2X purinoceptor antagonist. In contrast, the EJP was unaffected by MRS-2179 (2 microM), a selective P2Y(1) receptor antagonist. Aboral NS evoked an apamin- and l-NAME-sensitive IJP, but virtually no NANC EJP. These data suggest the presence of polarized excitatory purinergic neurotransmission in guinea-pig taenia caeci, which appears to be mediated by P2X purinoceptors, most likely the P2X(1) subtype.

Figure 1. Anatomy of taenia caecum and intracellular recording configuration

A, photograph of guinea-pig caecum. The taenia caecum is clearly visible as a distinct thickening of longitudinal muscle, with the aboral end being the portion closest to the ascending colon. The framed area represents the portion dissected free for intracellular recording. B, intracellular recording set up. Two pairs of silver wires were placed at the oral and aboral ends of the tissue 5 mm far from each other. Intracellular recording was made in the middle of two pairs of stimulating electrodes. In all figures except Fig. 9, recordings depict results obtained with oral NS.

Figure 2. Properties of electrical activity recorded in smooth muscle of guinea-pig taenia caeci

A, raw recordings of spontaneous action potentials. B, original recordings from panel A at expanded time scale. Ba and b, IJP evoked by 1 pulse of oral NS at either valley or peak, respectively, of slow wave. Bc, an IJP induced by 4 pulses. Arrows indicated nerve stimulation. Smooth muscle of taenia caeci was characterized by slow waves on which 1–3 spontaneous action potentials were superimposed.

Figure 3. Pharmacological isolation of an EJP induced by oral NS resistant to atropine and guanethidine in the presence of nifedipine

Aa, 1 pulse NS. Bb, 4 pulse NS. The stronger (4 pulse) NS produced a larger fast IJP, followed by a slow IJP and postinhibitory EJP. Bai and ii, junction potentials induced by NS of 1 and 4 pulses 5 min after application of apamin. Bbi and ii, junction potentials superimposed before (panel A) and after apamin. Cai and ii, junction potentials 10 min after atropine and guanethidine in the presence of nifedipine and apamin. Cbi and ii, overlapped junction potentials from panel Ba and panel Ca. Application of apamin converted the IJP to an EJP followed by a slow IJP, which was not affected by concomitant application of atropine and guanethidine.

Figure 4. Effects of SP desensitization on the NANC EJP in the presence of nifedipine, apamin, atropine and guanethidine

A, raw recordings of resting membrane potential. Application of SP produced significant membrane depolarization. However, continuous exposure to SP for up to 30 min resulted in slow recovery of resting membrane potential. Ba, control. Bb and Ca, 2 and 4 min after SP. iii and iv in panel Bb and Ca, junction potentials induced by oral NS of 1 and 4 pulses before, 2 and 20 min after administration of SP, respectively. The EJP amplitude was significantly inhibited and resting membrane potential was depolarized by application of SP for 2 min. However, the EJP amplitude slowly recovered to baseline despite continuous exposure to SP, suggesting that the EJP is resistant to SP desensitization.

Figure 5. Inability of L-NAME to suppress EJP

Aa and b, junction potentials induced by oral NS of 1 and 4 pulses in the presence of nifedipine, apamin, atropine, guanethidine and SP. B, 10 min after application of l-NAME. l-NAME abolished slow IJP and left EJP intact. C, superimposed junctional potentials before and after l-NAME.

Figure 6. Effects of α,β-methylene ATP and TTX on EJP

A, original recordings of resting membrane potential for 40 min α,β-methylene ATP produced significant membrane depolarization in the presence of nifedipine, apamin, atropine, guanethidine and SP. This effect reached a maximum in 5–10 min and stayed stable until wash out. The membrane potential gradually returned to the baseline 30 min after washing. Resting membrane potential was not affected by application of TTX. Ba and b, EJPs before and 10 min after α,β-methylene ATP. Biii, superimposed EJPs. Ca and b, EJPs before and 10 min after α,β-methylene ATP. Ciii, superimposed EJPs. The EJP induced by oral NS was markedly inhibited by application of α,β-methylene ATP and fully recovered 30 min after washing out. Subsequent application of TTX abolished the EJP.

Figure 7. Inhibitory effects of suramin, a non-selective P2 purinoceptor antagonist, and PPADS, a selective P2X purinoceptor antagonist, on EJP induced by oral NS

A and C, raw recordings of resting membrane potentials. Ba–c and Da–c, the EJP induced by 4 pulses or oral NS at expanded time scale before, during and after application of suramin and PPADS, respectively. Bd and Dd, superimposed EJPs. Resting membrane potential was not affected by suramin, but was significantly depolarized by PPADS. Suramin reached maximal inhibitory effects on the EJP in 15 min, while maximal PPADS effect occurred within 10 min. Effects of suramin and PPADS reversed within 15 min after washing out.

Figure 8. The purinergic EJP induced by oral NS was not significantly affected by MRS-2179, a specific P2Y₁ antagonist

A, raw recording of membrane electrical activity for up to 20 min after application of MRS-2179. Ba–c, expanded time scale depiction of EJPs from panel A in control, 10 min and 20 min after MRS-2179. Bd, superimposed EJPs.

Figure 9. An example of junction potentials recorded in the same cell in the presence of atropine, guanethidine and SP, as induced by either oral (panel i) or aboral (panel ii) NS

A, junction potentials produced by oral nerve stimulation in control (a), and after cumulative application of apamin (b), l-NAME (c) and suramin (d). B, overlay of junction potentials corresponding to panel A. Note that the purinergic EJP is polarized, whereas the NANC IJP is not.