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Received 23 March 1998; accepted after revision 8 September 1998.
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ABSTRACT |
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INTRODUCTION |
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Inhibitory junction potentials (IJPs) evoked by stimulation of non-adrenergic, non-cholinergic (NANC) nerves in many cases cause relaxation of gastrointestinal smooth muscle by decreasing the influx of Ca2+ via voltage-operated calcium channels (VOCCs). Knowledge of mechanisms underlying the generation of IJPs helps to identify not only the types of ionic channels which mediate the hyperpolarizing responses, but also the neurotransmitters involved. Currently, a number of candidates have been documented as NANC inhibitory transmitters using electrophysiological techniques (for a review see Bennett, 1997). These include adenosine 5'-triphosphate (ATP; for reviews see Burnstock, 1972, 1990), nitric oxide (NO; for reviews see Rand, 1992; Sanders & Ward, 1992; Rand & Li, 1995) and the neuropeptides, pituitary adenylyl cyclase-activating peptide (PACAP; Schworer et al. 1992; Kishi et al. 1996) and vasoactive intestinal peptide (VIP; Furness & Costa, 1987).
The contribution of NANC neurotransmitters to IJP generation and to regulation of gastrointestinal smooth muscle contractility appears to be tissue dependent (Costa et al. 1986; for a review see Bennett, 1997). This is based on varying effects of agents such as apamin and NO inhibitors on NANC IJPs in different tissues. For instance, in the guinea-pig internal anal sphincter, NANC IJPs appeared to be biphasic (Rae & Muir, 1996), comprising a fast phase (i.e. rapid in onset) followed by a slower phase, which prolonged the duration of the IJP. Apamin was claimed to inhibit the fast phase of the IJP whereas inhibition of NOS with L-NAME abolished the slow phase without affecting the first phase (Rae & Muir, 1996). In the guinea-pig taenia coli similar biphasic IJPs have been reported with the first phase again blocked by apamin although NOS inhibition failed to affect either phase of the IJP (Bridgewater et al. 1995). By contrast, NOS inhibition is thought to affect both phases of the NANC IJP in the canine ileocolonic sphincter including the apamin-sensitive phase (Ward et al. 1992). Therefore, complex mechanisms appear to underlie NANC inhibition of gastrointestinal smooth muscle, which can be characterized pharmacologically and appear to be attributed to the release and interaction of multiple neurotransmitters.
In the rat the anococcygeus - an example of a smooth muscle innervated by NANC nerves generally regarded as being nitrergic (Gillespie et al. 1989; Li & Rand, 1989; Hobbs & Gibson, 1990; Liu et al. 1991) - NANC relaxations have been demonstrated not to be associated with significant changes in membrane potential or conductance (Creed et al. 1975; Creed & Gillespie, 1977). Thus, NANC relaxations in this preparation were most likely to be due to non-ionic or metabotropic mechanisms. However, we have shown that NANC relaxations in the rat anococcygeus were blocked by the L-type VOCC inhibitor, nifedipine (Selemidis & Cocks, 1997). Although the study did not involve measurement of membrane potential, it provided indirect evidence that NANC relaxations in rat anococcygeus, like other NANC-innervated preparations, involved hyperpolarization. This finding prompted the aim of the present study, which was to examine the effect of NANC nerve stimulation on membrane potential in the rat anococcygeus using conventional intracellular recording techniques. We show for the first time that NANC nerve stimulation evokes apamin- and NO-sensitive IJPs in the rat anococcygeus indicating that the inhibitory innervation of this tissue is similar to other NANC-innervated preparations from the gastrointestinal tract.
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METHODS |
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Tissue preparation
Male Sprague-Dawley rats (
300 g) were killed by CO2 asphyxiation. Silk sutures were tied on either end of the anococcygeus muscles in situ and the tissues removed from the animal. Tissues were then pinned with fine-gauge steel wires longitudinally between two parallel platinum electrodes on a Sylgard-base organ chamber and superfused constantly (5 ml min-1; Minipuls 3-Gilson) with pre-warmed (35°C) carbogenated (95 % O2, 5 % CO2) Krebs solution of the following composition (mM: Na+, 143·1; K+, 5·9; Ca2+, 2·5; Mg2+, 1·2; Cl-, 127·8; HCO3-, 25·0; SO42-, 1·2; H2PO4-, 1·2; and glucose, 11·0).
Electrophysiological apparatus
Capillary glass microelectrodes (borosilicate glass capillaries, GC 120F10, Clark Electromedical Instruments) were made using a microelectrode puller (Model P-87; Sutter Instrument Co.) and 'backfilled' with KCl (0·5 M) and had resistances in the range 80-150 M
. A fine Ag-AgCl-coated electrode connected to a headstage (HS-2; Axon Instruments) was placed in the microelectrode to transmit changes in electrical events occurring in the smooth muscle to the recording devices. These responses were recorded against a second 'reference' Ag-AgCl electrode placed in the bath medium. Evoked electrical events were amplified (Axonprobe-1A multipurpose microelectrode amplifier; Axon Instruments), digitized (sampling rate, 1024 points per record) with an analog-to-digital converter (DMA interface, model TL-1; Axon Instruments), and visualized on a storage oscilloscope. Permanent registrations of evoked events were achieved on a Sony digital audio tape (DAT) and SCAN analysis program on a PC for the determination of latency, peak amplitude, time to peak, amplitude at 4 s and duration of responses.
Experimental protocol
After a 45 min equilibration period in normal Krebs solution, smooth muscle cells were impaled with the aid of a Zeiss microelectrode manipulator and microscope (Stemi SV-6; Zeiss optics) to establish the resting membrane potential. All preparations were then exposed to Krebs solution containing guanethidine (30 µM), atropine (1 µM) and propranolol (1 µM) (GAP Krebs) to induce NANC conditions. Tissues were exposed to GAP Krebs for the remainder of the experiment. This treatment caused membrane potential depolarization from a resting level that ranged from between -60 and -70 mV to between -20 and -30 mV. The depolarization in response to GAP Krebs was a result of activation of 
-adrenoceptors by released noradrenaline from adrenergic nerves because of guanethidine's indirect sympathomimetic activity. Resting membrane potentials before and after guanethidine treatment were consistent with previous electrophysiological studies in the rat anococcygeus muscle (Creed et al. 1975). Also, like that observed by Creed et al. (1975), IJPs to electrical field stimulation were very small (1·5 mV) under these conditions. Therefore, after 30-60 min exposure to GAP Krebs, the -adrenoceptor antagonist phentolamine was titrated at increments of 0·01 µM into the Krebs solution to reduce the -adrenoceptor-dependent depolarization caused by guanethidine and to stabilize the impalements. The maximum concentration of phentolamine used was 0·05 µM. Control responses to electrical field stimulation (EFS; 1, 2 or 5 pulses at 5 Hz delivered in square wave pulses via a trigger, 0·5 ms duration, 60-80 V; Applegarth trigger and stimulator, Oxford, UK) of NANC inhibitory nerves were obtained only when a steady membrane potential between -40 and -50 mV was achieved. Duplicate control responses from each cell were obtained and averaged to determine the initial sensitivity of the cells. Following the control responses, tissues were either untreated, which served as time control, or treated separately with tetrodotoxin (1 µM), apamin (0·1 or 1 µM), charybdotoxin (0·01 or 0·1 µM), iberiotoxin (0·1 µM), glibenclamide (10 µM), N G-nitro-L-arginine (L-NOARG; 100 µM), N G-nitro-D-arginine (D-NOARG; 100 µM), 1-H-oxodiazol-[1,2,4]-[4,3-a] quinoxaline-1-one (ODQ; 10 µM) or dimethyl sulphoxide (DMSO; 0·1 %) added to the perfusing solution. Some tissues exposed to apamin (0·1 µM) were subsequently treated with L-NOARG (100 µM). In all treatment groups an exposure period of 30 min was allowed before responses to EFS were recorded. Also, in time control experiments, a similar 30 min period of no drug treatment was allowed before EFS was repeated. Therefore, in a single cell the effect of drug treatment could be determined with a single impalement of 45-60 min.
Drugs
Guanethidine sulphate, L-NOARG, D-NOARG, phentolamine hydrochloride and L-propranolol hydrochloride were from Sigma; apamin, charybdotoxin, glyburide (glibenclamide), iberiotoxin, ODQ and tetrodotoxin were from Sapphire Bioscience (Alexandria, NSW, Australia); atropine sulphate was from Research Biochemicals International (USA). Stock solutions (10 mM) of guanethidine, atropine, propranolol, phentolamine, apamin (0·1 mM) and tetrodotoxin (1 mM) were prepared in distilled water. L-NOARG (100 mM) and D-NOARG (100 mM) were prepared in NaHCO3 (1 M). Charybdotoxin (10 µM) and iberiotoxin (10 µM) were prepared by dissolution in a buffer solution of the following composition: 0·1 % BSA, 100 mM NaCl, 10 mM Tris and 1 mM EDTA; pH 7·5. ODQ (10 mM) and glibenclamide (10 mM) were prepared in DMSO (100 %). Further dilutions of all stock solutions were in distilled water.
Data presentation and statistical analysis
Since the time course of the IJPs was biphasic, two amplitude responses were measured. The first was the maximum amplitude (peak) of the IJP and the second, the amplitude of the response at 4 s (4 s amplitude), both measured in millivolts (mV) from the pre-stimulus resting membrane potential. Because the initial fast phase lasted between 1 and 3 s, an index of the slow phase was the 4 s amplitude. At this time any contribution of the first phase would be minimal. Time to peak (s) was recorded from the first stimulus artefact. The first control responses (including peak amplitude, time to peak and 4 s amplitude) of each treatment group were compared with each other with a one-way analysis of variance (ANOVA) using the Tukey-Kramer method for multiple comparisons. Similarly, the responses following drug treatments were compared with the time control responses using an ANOVA (Tukey- Kramer). The first control responses and those obtained 30 min later (i.e. time control group) were compared using Student's paired t test. In all cases, significance was accepted at the P < 0·05 level.
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RESULTS |
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Responses to EFS of NANC nerves
In the absence of drugs, the resting membrane potential of rat anococcygeus smooth muscle cells ranged between -60 and -70 mV. When NANC conditions were imposed with GAP Krebs and phentolamine, the membrane potential depolarized to between -40 and -50 mV. Under these conditions EFS (1, 2 or 5 pulses at 5 Hz, 0·5 ms duration, 60-80 V) evoked pulse-dependent IJPs in the smooth muscle. The NANC IJP evoked by a single stimulus had a rapid onset and reached a peak of 3·9 ± 0·5 mV (n = 6) in 0·80 ± 0·04 s (n = 6). The time course of the NANC IJP was biphasic, comprising a fast-to-peak phase (fast phase) followed by a second, slower phase (slow phase) which delayed repolarization (Fig. 1). This was more evident when two and five pulses were delivered at 5 Hz (Fig. 1). The duration of NANC IJPs was in the range 10-20 s. In time control experiments (conducted 30 min after first control responses) it was observed that the peak amplitude (1 pulse: 2·2 ± 0·2 mV; n = 6) and 4 s amplitude (1 pulse: 0·6 ± 0·1 mV; n = 6) were slightly, but significantly reduced (Student's paired t test; P < 0·05) when compared with the first control IJPs (peak amplitude for 1 pulse: 3·9 ± 0·5 mV; n = 6) (4 s amplitude for 1 pulse: 1·1 ± 0·2 mV; n = 6) (Fig. 1; see also Table 1 for 5 pulses, 5 Hz responses). However, time had no significant effect on the time to peak (1 pulse: 0·74 ± 0·03 s, n = 6; Table 1) or duration of the IJP. The first control responses to EFS of peak amplitude, time to peak and 4 s amplitude within each treatment group were not significantly different from one another (P > 0·05; ANOVA, n = 6).
Tetrodotoxin (1 µM) nearly abolished both phases of the NANC IJP suggesting that the IJP was neuronal in origin (Fig. 2). In some cases, a small depolarization to EFS was observed which most probably represented a stimulus artefact (Fig. 2).
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Superimposed digitized recordings of membrane potential showing the 1st control (control) and sequential, time control (30 min later) inhibitory junction potentials (IJPs) following electrical field stimulation (1, 2 and 5 pulses (P) at 5 Hz every 45-60 s, 0·5 ms duration, 60-80 V) of NANC inhibitory nerves in rat isolated anococcygeus muscle. A downward deflection signifies hyperpolarization whereas transient spikes indicate stimulus artefacts for each pulse. The amplitude of the IJPs in time controls was slightly reduced compared with the 1st control IJPs; however, the duration and time to peak remained unaffected. Horizontal dotted line indicates the pre-stimulus resting membrane potential (-40 to -50 mV). Vertical (mV) and horizontal (time scale; s) bars apply to all traces. All recordings shown in this panel are from a single cell and are representative of 6 cells from 6 separate animals. | ||
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Superimposed digitized recordings of membrane potential showing the 1st control (control) inhibitory junction potentials (IJPs) and sequential IJPs in the presence of tetrodotoxin (TTX; 1 µM) following electrical field stimulation (1, 2 and 5 pulses (P) at 5 Hz every 45-60 s, 0·5 ms duration, 60-80 V) of NANC inhibitory nerves in rat isolated anococcygeus muscle. A downward deflection signifies hyperpolarization whereas transient spikes indicate stimulus artefacts for each pulse. TTX almost abolished the IJPs but in some cases (as shown here) it revealed a small depolarization followed by a small hyperpolarization. Horizontal dotted line indicates the pre-stimulus resting membrane potential (-40 mV). Vertical (mV) and horizontal (time scale; s) bars apply to all traces. All recordings shown in this panel are from a single cell and are representative of 6 cells from 6 separate animals. | ||
Effect of K+ channel inhibitors on EFS-evoked IJPs
In the presence of apamin (0·1 µM), an inhibitor of small-conductance Ca2+-activated K+ channels, the peak of the NANC IJP (1 pulse: 1·4 ± 0·4 mV; n = 6) was significantly (P < 0·05) smaller than the time control (Fig. 3; Table 1). Furthermore, the IJP was slower in onset and time to peak (1 pulse: 1·95 ± 0·02 s; n = 6; P < 0·01) than the time control (Table 1). However, the 4 s amplitude (1 pulse: 0·7 ± 0·4 mV; n = 6) was not significantly different from the time control (Table 1). Increasing the concentration of apamin to 1 µM had no further effect on the NANC IJP (n = 2; data not shown). Thus, it is likely that a different mechanism underlies the apamin-resistant slow phase of the NANC IJP.
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Superimposed digitized recordings of membrane potential showing the 1st control (control) inhibitory junction potentials (IJPs) and sequential IJPs in the presence of apamin (0·1 µM) following electrical field stimulation (1, 2 and 5 pulses (P) at 5 Hz every 45-60 s, 0·5 ms duration, 60-80 V) of NANC inhibitory nerves in rat isolated anococcygeus muscle. A downward deflection signifies hyperpolarization. Apamin inhibited the fast phase of the IJP by reducing the peak amplitude and by increasing the time to peak. Horizontal dotted line indicates the pre-stimulus resting membrane potential (-48 to -52 mV). Vertical (mV) and horizontal (time scale; s) bars apply to all traces. All recordings shown in this panel are from a single cell and are representative of 6 cells from 6 separate animals. | ||
The peak (1 pulse: 2·9 ± 0·9 mV, n = 6), time to peak (1 pulse: 0·76 ± 0·05 s, n = 6), and the 4 s amplitude (1 pulse: 0·7 ± 0·2 mV, n = 6) were unaffected by the large-conductance Ca2+-activated K+ channel inhibitor, charybdotoxin (0·01 µM; Table 1). Also, IJPs remained unaffected even in the presence of a higher concentration of charybdotoxin (0·1 µM; n = 2; data not shown). In addition, the more selective inhibitor of large-conductance Ca2+-activated K+ channels, iberiotoxin (0·1 µM; n = 2), and the inhibitor of ATP-sensitive K+ channels, glibenclamide (10 µM; n = 2), both failed to affect either phase of the IJP (data not shown).
Effect of N G-nitro-L-arginine and ODQ on NANC IJPs
In the presence of L-NOARG (100 µM) the peak amplitude (1 pulse: 1·9 ± 0·5 mV, n = 6) and time to peak (1 pulse: 0·8 ± 0·06 s, n = 6) were both not significantly different from time controls suggesting that L-NOARG had no effect on the fast, apamin-sensitive phase of the IJP (Fig. 4; Table 1). However, the 4 s amplitude (1 pulse: L-NOARG; 0·2 ± 0·09 mV; n = 6) was significantly (P < 0·05) reduced by L-NOARG (Fig. 4; Table 1) and the duration markedly reduced to between 1 and 3 s. D-NOARG (100 µM) had no effect on NANC IJPs (n = 2; data not shown). Pre-treatment with a combination of L-NOARG (100 µM) and apamin (0·1 µM) almost abolished both phases of the NANC IJP (Fig. 5). The small depolarization to EFS following L-NOARG plus apamin probably represents a stimulation artefact.
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Superimposed digitized recordings of membrane potential showing 1st control (control) inhibitory junction potentials (IJPs) and sequential IJPs in the presence of L-NOARG (100 µM) following electrical field stimulation (1, 2 and 5 pulses (P) at 5 Hz every 45-60 s, 0·5 ms duration, 60-80 V) of NANC inhibitory nerves in rat isolated anococcygeus muscle. Downward deflection signifies hyperpolarization whereas transient spikes indicate stimulus artefacts for each pulse. Note the time to peak of the IJP was unaffected by L-NOARG treatment but the duration reduced to between 1 and 3 s, therefore, L-NOARG had no effect on the fast phase (compared with time controls in Fig. 1) but abolished the second phase. Horizontal dotted line indicates the pre-stimulus resting membrane potential (-40 to -50 mV). Vertical (mV) and horizontal (time scale; s) bars apply to all traces. All recordings shown in this panel are from a single cell and are representative of 6 cells from 6 separate animals. | ||
Table 1. Characterization of NANC IJPs
| 1st control responses | |||||
| Experimental groups: | No treatment | Apamin | L-NOARG | CBTX | ODQ |
| Peak (mV) | 9·38 ± 0·94 | 8·56 ± 0·95 | 7·55 ± 0·96 | 8·41 ± 1·12 | 9·76 ± 1·01 |
| Time to peak (s) | 1·26 ± 0·02 | 1·29 ± 0·03 | 1·29 ± 0·06 | 1·26 ± 0·06 | 1·30 ± 0·04 |
| 4 s amplitude (mV) | 5·10 ± 0·76 | 4·99 ± 1·22 | 3·79 ± 0·40 | 4·92 ± 0·49 | 4·78 ± 1·30 |
| 2nd responses after treatment | |||||
| Time control | Apamin | L-NOARG | CBTX | ODQ | |
| Peak (mV) | 6·17 ± 0·57  | 3·46 ± 0·60 * | 5·16 ± 0·68 | 5·95 ± 1·00 | 7·21 ± 0·80 |
| Time to peak (s) | 1·24 ± 0·02 | 4·24 ± 0·90 * | 1·24 ± 0·05 | 1·31 ± 0·05 | 1·29 ± 0·03 |
| 4 s amplitude (mV) | 2·98 ± 0·39 | 3·11 ± 0·53 | 0·27 ± 0·23 * | 2·64 ± 0·66 | 1·29 ± 0·69 * |
Significant difference from 1st control response. Responses are means ± The soluble guanylate cyclase selective inhibitor ODQ (10 µM) produced a similar pattern of inhibition of the NANC IJP as L-NOARG (i.e. selectively inhibited the slow phase; Fig. 6). Whilst the duration of the response was reduced to 1-3 s by ODQ, the peak and time to peak were not significantly different from the time control (Table 1). DMSO (0·1 %), at the concentration used to prepare ODQ (10 µM), had no effect on either phase of the NANC IJP (data not shown; n = 2).
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View larger version [in this window] [in a new window] |
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Superimposed digitized recordings of membrane potential showing 1st control (control) inhibitory junction potentials (IJPs) and sequential IJPs in the presence of a combination of apamin (0·1 µM) and L-NOARG (100 µM) following electrical field stimulation (1, 2 and 5 pulses (P) at 5 Hz every 45-60 s, 0·5 ms duration, 60-80 V) of NANC inhibitory nerves in rat isolated anococcygeus muscle. A downward deflection signifies hyperpolarization whereas transient spikes indicate stimulus artefacts for each pulse. The combined treatment of apamin and L-NOARG nearly abolished the IJP and in some cases revealed a small depolarization which is most likely to be a stimulus artefact. Horizontal dotted line indicates the pre-stimulus resting membrane potential (-40 to -45 mV). Vertical (mV) and horizontal (time scale; s) bars apply to all traces. All recordings shown in this panel are from a single cell and are representative of 6 cells from 6 separate animals. | ||
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View larger version [in this window] [in a new window] |
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Superimposed digitized recordings of membrane potential showing the 1st control (control) inhibitory junction potentials (IJPs) and sequential IJPs in the presence of ODQ (10 µM) following electrical field stimulation (1, 2 and 5 pulses (P) at 5 Hz every 45-60 s, 0·5 ms duration, 60-80 V) of NANC inhibitory nerves in rat isolated anococcygeus muscle. A downward deflection signifies hyperpolarization whereas transient spikes indicate stimulus artefacts for each pulse. Like L-NOARG (Fig. 4), ODQ inhibited the second, slow phase of the IJP because the time to peak was unaffected and the duration reduced to between 1 and 3 s. Horizontal dotted line indicates the pre-stimulus resting membrane potential (-45 mV). Vertical (mV) and horizontal (time scale; s) bars apply to all traces. All recordings shown in this panel are from a single cell and are representative of 6 cells from 6 separate animals. | ||
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DISCUSSION |
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The present study demonstrates unequivocal NANC nerve-evoked biphasic IJPs in smooth muscle of the rat isolated anococcygeus. These NANC IJPs were pulse dependent and tetrodotoxin sensitive indicating that they were neuronal in origin. Furthermore, the biphasic nature of the NANC IJPs suggested that they involved more than one mechanism or inhibitory transmitter. The first phase of the NANC IJP was apamin sensitive, fast in onset and time to peak, whereas the second was slower compared with the first phase and was almost abolished by L-NOARG and ODQ. Thus, small-conductance Ca2+-activated K+ channels and a NO-sensitive, cGMP-dependent mechanism both appeared to contribute to the generation of biphasic NANC IJPs in the rat anococcygeus.
Our previous finding that NANC-mediated relaxation in the rat anococcygeus was blocked by the L-type VOCC inhibitor nifedipine (Selemidis & Cocks, 1997) and the findings of the present study suggest that hyperpolarization underlies relaxation to NANC nerve stimulation in this preparation. Previous electrophysiological reports, however, concluded that NANC relaxations in the rat anococcygeus were not associated with hyperpolarization (Creed et al. 1975; Creed & Gillespie, 1977). A possible explanation for the discrepancy between our and these earlier findings may be related to the choice of recording conditions. Thus, Creed et al. (1975) showed that the adrenergic neurone blocker guanethidine, used to eliminate adrenergic responses, caused membrane depolarization from a resting level of -60 mV to -20 mV. Such conditions of near-maximum depolarization may have affected the ability of hyperpolarizing neurotransmitters to activate K+ channels, the majority of which are regulated by Ca2+ and or voltage (Rudy, 1988; Latorre et al. 1989). Like Creed et al. (1975) we also found that it was difficult to record IJPs in highly depolarized assay conditions (S. Selemidis & T. Cocks, unpublished observations). However, when we controlled the degree of depolarization to guanethidine with the -adrenoceptor antagonist phentolamine, at concentrations that do not affect K+ channels (McPherson & Angus, 1989), the resting membrane potential in the smooth muscle was in the range -40 to -50 mV. Then, under these conditions, the K+ channel-dependent mechanisms appeared to be more permissive to activation by hyperpolarizing neurotransmitters and as such, IJPs were generated.
This study revealed an apamin-, L-NOARG- and ODQ-sensitive biphasic IJP. By contrast, in separate functional studies the relaxation to NANC inhibitory nerve stimulation in the rat anococcygeus was unaffected by apamin but was blocked by nifedipine and virtually abolished with the subsequent addition of L-NOARG plus oxyhaemoglobin (Selemidis & Cocks, 1997). Similar biphasic IJPs blocked by apamin (fast phase) and L-NOARG or L-NAME (slow phase) have been reported for the guinea-pig internal anal sphincter (Rae & Muir, 1996), mouse gastric fundus (Mashimo et al. 1996) and guinea-pig circular muscle of the colon (Maggi & Giuliani, 1993). Whilst Rae & Muir (1996) claimed that apamin also blocked the accompanying relaxation to NANC nerve stimulation, the lack of group data for any functional measurements combined with conflicting results from single experiments (see Figs 1 and 3 in Rae & Muir, 1996) make it difficult to assess this claim. Regardless, from their data, apamin appeared to have had little if any effect on the NANC relaxation as we reported in the rat anococcygeus (Selemidis & Cocks, 1997). A similar apparent dissociation between the effects of apamin on the electrical and mechanical responses to NANC nerve stimulation also occurs in the mouse gastric fundus. As stated above, Mashimo et al. 1996, found that the fast-phase component of the biphasic IJP was blocked by apamin, whereas in functional studies, we found that the relaxation to NANC inhibitory nerve stimulation was completely unaffected by apamin (S. Selemidis & T. Cocks, unpublished observations). Therefore, as in the rat anococcygeus, apamin-sensitive IJPs in the guinea-pig internal anal sphincter and mouse gastric fundus do not appear to contribute directly to smooth muscle relaxation. By contrast, in the guinea-pig taenia coli (Maas, 1981; Selemidis et al. 1997) and circular muscle of the guinea-pig colon (Maggi & Giuliani, 1993) the apamin-sensitive IJPs contribute directly to NANC relaxations.
One possible role for the fast apamin-sensitive phase of the IJP in the rat anococcygeus is that originally alluded to by Rae & Muir (1996) in the guinea-pig internal anal sphincter. They hypothesized that the initial fast component of the composite IJP acts to prime the tissue to respond with relaxation to the second slower component of the IJP. Another possibility is that the fast-phase IJP in these tissues is conducted, perhaps by Ca2+ waves which are known to travel between cells (Young et al. 1996), to other remote smooth muscle cells to increase their threshold for activation. Whatever the roles, if any, our results agree with previous findings that apamin-sensitive NANC IJPs in the same gastrointestinal preparations do not appear to contribute directly to relaxation.
Whilst the findings of the present study and our previous study (Selemidis & Cocks, 1997) point against a role for the apamin-sensitive IJP in mediating relaxation, the NO-induced IJP appears to directly contribute to relaxation. In our previous paper (Selemidis & Cocks, 1997), the nifedipine-sensitive component of the NANC relaxation was also partly L-NOARG and oxyhaemoglobin sensitive. This suggests that the NO-induced IJP observed here causes sufficient membrane hyperpolarization to evoke closure of nifedipine-sensitive, L-type VOCCs and relaxation. The remaining nifedipine-resistant relaxation observed in our previous study (Selemidis & Cocks, 1997), which was abolished by L-NOARG and oxyhaemoglobin, may have also been due to the NO-induced IJP causing relaxation independently of the closure of L-type VOCCs. For example, in bovine (Drummond & Cocks, 1996) and canine (Yamagishi et al. 1992) coronary arteries the ATP-sensitive potassium (KATP) channel opener levcromakalim caused membrane hyperpolarization but the relaxation was not due to the closure of VOCCs but to a reduction in the level of inositol trisphosphate (IP3). Therefore, a similar effect of the NO-induced IJP may underlie the NO-dependent, nifedipine-resistant component of the NANC relaxation (Selemidis & Cocks, 1997). It is also possible that this remaining NO-dependent response is mediated by a mechanism independent of changes in membrane potential.
The mechanisms and pharmacological profiles of NANC IJPs provide important information as to the identity of the neurotransmitters involved. In the rat anococcygeus, NANC IJPs were biphasic in time course with each phase characterized by its selective sensitivity to apamin (fast phase) or L-NOARG and ODQ (slow phase). Thus, two distinct neurotransmitters appear to mediate the IJPs: NO and an as yet to be identified apamin-sensitive substance. Similar biphasic IJPs have been reported in other NANC-innervated tissues (for a review see Bennett, 1997) such as the guinea-pig internal anal sphincter (Rae & Muir, 1996), circular muscles of the ileum (He & Goyal, 1993) and colon (Maggi & Giuliani, 1993) and guinea-pig taenia coli (Bridgewater et al. 1995). In these tissues, ATP has been suggested as the transmitter evoking the fast, apamin-sensitive phase. By analogy, ATP may represent the unknown transmitter evoking the fast phase in the rat anococcygeus. An objection to this proposal, however, is that exogenously applied ATP has been found to cause depolarization in the rat anococcygeus (Byrne & Large, 1984). A possible explanation for the inability to record hyperpolarization in response to exogenously applied ATP is that the concentration of ATP reached in the neuroeffector junction is low (i.e. inactivated by junctional ectoATPases) and as such ATP fails to activate junctional purinoceptors (possibly P2Y). The more accessible depolarization-mediating purinoceptors (most likely P2X) located outside the neuroeffector junction would be preferentially activated by ATP and result in an overall depolarization. Until specific P2X and P2Y purinoceptor antagonists are developed such interactions between intra-and extra-junctional purinoceptors and the role of ATP will remain speculative. However, conventional but non-selective ATP antagonists such as suramin, Reactive Blue 2 and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) may be useful in identifying the non-NO first phase of the IJP.
Like ATP, pituitary adenylyl cyclase-activating peptide (PACAP), which has been localized immunohistochemically in and shown to be released from enteric nerves, mimics NANC inhibitory nerve stimulation in several gut smooth muscle preparations when applied exogenously (Jin et al. 1994). Moreover, and also like ATP, the inhibitory effects of PACAP are apamin sensitive (McConalogue et al. 1995). Thus, as for ATP, the use of PACAP antagonists (e.g. PACAP6-38; see Jin et al. 1994) would also be important in assessing any possible role for this peptide as a candidate mediating the apamin-sensitive fast phase of the IJP in the rat anococcygeus.
A NO-dependent mechanism appeared to contribute to the NANC IJP in the rat anococcygeus since the potent L-arginine analogue inhibitor of NOS, L-NOARG, but not its stereoisomer, D-NOARG, selectively inhibited the slow phase of the biphasic IJP. NO has been shown to evoke smooth muscle hyperpolarization in many tissues via cGMP-dependent (Robertson et al. 1993) and cGMP-independent (Bolotina et al. 1994) mechanisms. Either mechanism ultimately involves activation of K+ channels. In the present study, the selective guanylate cyclase inhibitor, ODQ, inhibited the slow phase of the IJP to a similar degree as L-NOARG suggesting that a cGMP-dependent K+ channel mechanism underlies the NO-mediated hyperpolarization. Although activation of apamin- and charybdotoxin-sensitive K+ channels by NO has been shown in several tissues (Ward et al. 1992; Bolotina et al. 1994; Yamakage et al. 1996), in the rat anococcygeus similar K+ channels were unlikely to have been activated by NO since the NO-dependent slow component of the IJP was unaffected by both apamin and charybdotoxin. In addition, iberiotoxin, which is a more selective inhibitor of large-conductance Ca2+-activated K+ channels than charybodotoxin and glibenclamide, an inhibitor of ATP-sensitive K+ channels, also failed to affect the NANC IJPs. It is possible, however, that K+ channels were not involved at all; instead, the hyperpolarization occurred as a result of closure of either non-selective cation channels or closure of chloride channels.
In conclusion, NANC nerve stimulation of the rat anococcygeus results in the generation of smooth muscle IJPs, which, as for other gut smooth muscle preparations innervated by similar NANC inhibitory nerves, are biphasic in time course. These NANC IJPs are characterized by two pharmacologically distinct phases; the first is fast and apamin sensitive, whereas the second is slow and apamin, charybdotoxin and glibenclamide resistant but blocked by L-NOARG. Therefore, we propose that NO and a non-NO transmitter which may represent the putative factor, nerve-derived hyperpolarizing factor (NDHF; Selemidis & Cocks, 1997) both contribute to smooth muscle NANC IJPs in the rat anococcygeus.
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This work was funded by the National Health and Medical Research Council (NHMRC) of Australia.
Corresponding author
T. M. Cocks: Department of Pharmacology, Triradiate Building, University of Melbourne, Parkville, Victoria 3052, Australia.
Email: t.cocks{at}pharmacology.unimelb.edu.au
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