Pacemaker shift in the gastric antrum of guinea-pigs produced by excitatory vagal stimulation involves intramuscular interstitial cells

doi:10.1113/jphysiol.2002.018614

Pacemaker shift in the gastric antrum of guinea-pigs produced by excitatory vagal stimulation involves intramuscular interstitial cells

G. D. S. Hirst, E. J. Dickens and F. R. Edwards

Department of Zoology, University of Melbourne, Victoria 3010, Australia

ABSTRACT

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Abstract
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Methods
Results
Discussion
References

Intracellular recordings were made from isolated bundles of the circular muscle layer of guinea-pig gastric antrum and the responses produced by stimulating intrinsic nerve fibres were examined. After abolishing the effects of stimulating inhibitory nerve terminals with apamin and L-nitroarginine (NOLA), transmural nerve stimulation often evoked a small amplitude excitatory junction potential (EJP) and invariably evoked a regenerative potential. Neurally evoked regenerative potentials had similar properties to those evoked in the same bundle by direct stimulation. EJPs and neurally evoked regenerative potentials were abolished by hyoscine suggesting that both resulted from the release of acetylcholine and activation of muscarinic receptors. Neurally evoked regenerative potentials, but not EJPs, were abolished by membrane hyperpolarization, caffeine and chloride channel blockers. In the intact antrum, excitatory vagal nerve stimulation increased the frequency of slow waves. Simultaneous intracellular recordings of pacemaker potentials from myenteric interstitial cells (ICC_MY) and slow waves showed that the onset of each pacemaker potential normally preceded the onset of each slow wave but vagal stimulation caused the onset of each slow wave to precede each pacemaker potential. Together the observations suggest that during vagal stimulation there is a change in the origin of pacemaker activity with slow waves being initiated by intramuscular interstitial cells (ICC_IM) rather than by ICC_MY.

(Received 7 February 2002; accepted after revision 30 March 2002)
Corresponding author G. D. S. Hirst: Department of Zoology, University of Melbourne, Victoria 3010, Australia. Email: d.hirst{at}zoology.unimelb.edu.au

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Most regions of the gastrointestinal tract generate slow waves. These are initiated by a network of interstitial cells of Cajal which most frequently lie near the myenteric plexus (ICC_MY; Sanders, 1996). Thus tissues taken from mutant mice which lack the myenteric network of interstitial cells (Ward et al. 1994) or from tissues in which the development of ICC_MY has been impaired (Ward et al. 1997, 1999; Ordog et al. 1999) fail to generate slow waves. Intracellular recordings made from ICC_MY of either the guinea-pig or the mouse gastric antrum show they generate large amplitude, long lasting pacemaker potentials (Dickens et al. 1999; Hirst & Edwards, 2001; Hirst et al. 2002a). Pacemaker potentials spread passively to the circular muscle layer where they trigger the secondary regenerative component of the slow wave (Ohba et al. 1975; Dickens et al. 1999; Hirst et al. 2002a). In the gastric antrum a distinct set of interstitial cells of Cajal lie in the circular muscle layer. Rather than forming a linked network of cells, like the pacemaking interstitial cells of the myenteric region, individual intramuscular interstitial cells of Cajal (ICC_IM) are distributed amongst the smooth muscle cells making up the circular layer (Burns et al. 1997). The secondary component of the slow wave appears to be initiated by this second population of intramuscular interstitial cells of Cajal as it is absent in tissues which lack ICC_IM (Dickens et al. 2001; Hirst et al. 2002a). When single bundles of circular muscle, which contain ICC_IM and smooth muscle cells but lack ICC_MY, are stimulated directly they generate the secondary regenerative component of the slow wave (Suzuki & Hirst, 1999; Edwards et al. 1999; van Helden et al. 2000). Secondary regenerative potentials result from Ca²⁺ release from intracellular stores (Suzuki & Hirst, 1999; van Helden et al. 2000), followed by the activation of anion-selective channels (Hirst et al. 2002b). Slow waves are absent in tissues devoid of type-1 inositol 1,4,5-trisphosphate (IP₃) receptors (Suzuki et al. 2000) and are abolished by 2-aminoethoxydiphenyl borate (2APB) (Hirst & Edwards, 2001), an inhibitor of IP₃ induced Ca²⁺ release (Maruyama et al. 1997), suggesting that internal Ca²⁺ release during each slow wave involves the formation of IP₃.

As well as generating the secondary component of the slow wave, ICC_IM have been suggested to be intermediaries in the pathway by which neuronal information modifies contractile activity in the gastrointestinal tract. Thus, the responses to both inhibitory and excitatory nerve stimulation are greatly attenuated in tissues devoid of ICC_IM (Burns et al. 1996; Ward et al. 2000). However the nature of the functional connection between intrinsic nerve terminals and ICC_IM has not been investigated. The initial experiments, described in this report, examined the process of excitatory neurotransmission in isolated bundles of circular muscle with the effects of inhibitory nerve stimulation being blocked by apamin and L-nitroarginine (NOLA) (Dickens et al. 2000). The experimental observations support the view that ICC_IM, rather than smooth muscle cells, are the major target for excitatory nerve terminals. The subsequent experiments examined the responses produced by stimulating excitatory vagal projections to the intact gastric antrum. Short trains of high frequency excitatory nerve stimulation evoked premature slow waves; longer trains of lower frequency stimulation increased the frequency of occurrence of slow waves. Simultaneous recordings of pacemaker potentials and slow waves showed that the site of initiation of rhythmical activity switched from ICC_MY to the circular muscle layer. The results are discussed in relation to the idea that during excitatory nerve stimulation the dominant pacemaker switches from ICC_MY to ICC_IM.

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

The procedures described were approved by the animal experimentation ethics committee at the University of Melbourne. Guinea-pigs of either sex were stunned, exsanguinated and the stomach removed. Three different preparations were used. In the first set of experiments, single bundles of circular muscle were isolated as described previously (Suzuki & Hirst, 1999). Briefly, the antral region was isolated and immersed in oxygenated physiological saline, composition (mM): NaCl, 120; NaHCO₃, 25; NaH₂PO₄, 1.0; KCl, 5; MgCl₂, 2; CaCl₂, 2.5 and glucose, 11; bubbled with 95 % O₂-5 % CO₂. After removing the mucosa, and then the longitudinal muscle layer, single bundles of circular muscle (diameter 150-200 µm, length 400-800 µm) were dissected free and pinned in a recording chamber. Preparations were impaled with two independently mounted sharp electrodes, one was used to record membrane potential changes and the other to inject current so changing the membrane potential of the preparations. A pair of platinum stimulating electrodes were positioned, one on either side of the preparation, to allow intramuscular nerve terminals to be stimulated. In the second set of experiments, the stomach along with the vagal nerve trunk innervating the dorsal side of the stomach were removed (Dickens et al. 2000). The antrum was isolated, taking care to maintain the integrity of the vagal trunk, and immersed in oxygenated physiological saline. The mucosa was dissected away and the preparations were pinned out in a recording chamber that had a base consisting of a microscope coverslip coated with Sylgard silicone resin (Dow Corning Corp, Midland, MI, USA). Under a dissecting microscope a region of antrum was immobilized with fine pins and the serosa removed. The vagus was then drawn into a suction electrode for stimulation. Preparations were viewed with an inverted compound microscope and intracellular recordings were made from the circular layer using sharp microelectrodes. When simultaneous recordings were made from different types of cells, the same preparations were impaled using two independent microelectrodes (Dickens et al. 1999). The electrodes were placed within 100 µm of each other and within the boundaries of the same circular muscle bundle. In the third set of experiments, preparations of antral longitudinal muscle were prepared in the same way as has been described previously (Hirst & Edwards, 2001). These preparations were pinned out in a recording chamber with the serosal surface downwards. A pair of platinum stimulating electrodes, one above and one below the sheet of longitudinal muscle, were used to stimulate the network of ICC_MY which remained attached to the longitudinal muscle layer (Hirst & Edwards, 2001). ICC_MY were impaled with sharp microelectrodes. In each set of experiments microelectrodes (90-150 M $Omega$ ) were filled with 0.5 M KCl. Membrane potential changes and membrane currents were amplified using an Axoclamp-2B amplifier (Axon Instruments, Foster City, CA, USA), low pass filtered (cut-off frequency, 100 Hz) digitized and stored on computer for later analysis. During each experiment preparations were constantly superfused with physiological saline solution warmed to 37 °C; unless stated otherwise, nifedipine (1 µM) was added to the physiological saline to reduce the amplitudes of the contractions associated with each excitatory response. In most of the experiments involving nerve stimulation the effects of stimulating inhibitory nerves were abolished by adding apamin (0.1 µM) and NOLA (1-10 µM) to the physiological saline (see Dickens et al. 2000). All data are expressed as means ± standard error of the mean (S.E.M.). Student's t test was used to determine whether data sets differed, with P < 0.05 taken to indicate a significant difference.

2-Aminoethoxydiphenyl borate (2APB) and L-nitroarginine (NOLA) (obtained from Calbiochem, San Diego, CA, USA), 4,4-diisothiocyano-2,2-stilbene disulphonic acid (DIDS), anthracene-9-carboxylic acid (9-AC), caffeine, tetrodotoxin, apamin, and nifedipine (obtained from Sigma) were used in these experiments.

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Responses evoked by transmural stimulation of isolated bundles of circular muscle

Intracellular recordings were made from electrically short bundles of circular muscle dissected from the antral region. When preparations were impaled with two electrodes, one being used to pass current and the other to record membrane potential changes, a current pulse invariably evoked an electrotonic potential (Suzuki & Hirst, 1999). The time course of onset, or offset, of each electrotonic potential was adequately described by a single exponential with time constants in the range 110-410 ms (195 ± 20 ms, n = 19). Preparations had input resistances in the range 1.5-8.2 M $Omega$ (3.5 ± 0.4 M $Omega$ , n = 19) and resting potentials in the range -59 to -73 mV (-63.2 ± 0.9 mV, n = 19). Depolarizing current pulses, or the break of hyperpolarizing pulses, evoked regenerative potentials with properties similar to those described previously (Suzuki & Hirst, 1999). In this set of experiments regenerative potentials had peak amplitudes in the range 20.0-48.1 mV (31.8 ± 1.5 mV, n = 19). A brief stimulating pulse, 0.1-0.2 ms, delivered to the pair of stimulating electrodes, evoked an inhibitory junction potential (IJP; Fig. 1A). This was followed after a variable delay by a response that resembled the regenerative potential evoked by direct stimulation in that preparation (Fig. 1C). The properties of IJPs have not been examined further other than to note that they were reversed in polarity when the membrane potential was hyperpolarized by some 40-50 mV and were abolished by adding apamin (0.1 µM) and NOLA (1 µM) to the physiological saline (Fig. 1B). In six of the 19 preparations examined, adding apamin and NOLA to the physiological saline revealed an excitatory junction potential (EJP) which when evoked by a single stimulus had a peak amplitude of some 1.0-2.5 mV (1.6 ± 0.2 mV, n = 6). EJPs were detected in a higher proportion of preparations when trains of stimuli were applied but they could not be characterized in control solutions as they coincided with the onset of the regenerative potential.

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Figure 1. Regenerative responses evoked by transmural stimulation in isolated bundles of antral circular muscle, effects of apamin, NOLA and hyoscine
A single transmural stimulus, pulse width 0.1 ms (vertical line), initiated an IJP followed, after a latency of some 6 s, by a regenerative potential (A). After adding apamin (0.1 µM) and NOLA (1 µM) the same stimulus initiated only a regenerative response (B). The resting potential was -62 mV throughout. Neurally evoked regenerative potentials, but not those initiated by direct stimulation, were abolished by hyoscine. A regenerative potential evoked by a train of five stimuli (pulse width 0.1 ms, frequency 5 Hz, indicated by filled bar) was followed some 30 s later by one evoked by direct stimulation (C). After adding hyoscine (0.1 µM) the response to transmural stimulation was abolished but the response to direct stimulation persisted (D). The resting potential of this segment of tissue was -65 mV throughout. The voltage and current calibration bars apply to all traces, the upper time calibration bar applies to the upper two traces, the lower time calibration bar to the two lower traces.

All the responses to transmural nerve stimulation, detected after blocking the effects of inhibitory nerve stimulation with apamin and NOLA, were rapidly abolished by hyoscine (0.1 µM; n = 4), suggesting that they resulted from the release of ACh, followed by the activation of muscarinic receptors (Fig. 1D). Hyoscine had no effect on regenerative responses evoked by direct stimulation of the muscle bundles (Fig. 1D). In all subsequent experiments tissues were bathed in physiological saline containing apamin (0.1 µM) and NOLA (1 µM).

Properties of neurally evoked regenerative potentials recorded from bundles of circular muscle

Single transmural stimuli either initiated regenerative potentials which started at variable delays after the stimulus or caused an increase in the discharge of membrane noise (Fig. 2A). Increasing the number of stimuli shortened the latency before the onset of each regenerative potential and decreased the latency fluctuation (Fig. 2B and C). In most preparations a regenerative response of minimum latency, 0.98 ± 0.11 s (n = 19), was initiated by three impulses (Fig. 2). In a few preparations, up to five impulses were required (see Fig. 3A). This pattern of behaviour was very similar to that detected when isolated bundles of circular muscle were stimulated directly with current pulses of increasing intensity (see Suzuki & Hirst, 1999). The mean amplitude of the responses evoked by a train of three impulses at 5 Hz was 31.1 ± 1.6 mV (n = 19), a value not significantly different from that of the regenerative potential evoked by direct stimulation.

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Figure 2. Effect of increasing number of stimuli on latency of neurally evoked regenerative potentials
A shows four successive responses evoked by single transmural stimuli. Note that on three occasions a regenerative potential was initiated but on one occasion a slow depolarization, associated with an increase in membrane noise, was detected. B shows four successive responses evoked by pairs of transmural stimuli, delivered at 5 Hz, and each pair of stimuli initiated a regenerative response. Increasing the number of stimuli in each train to three, delivered at 5 Hz (C), also regularly initiated a regenerative potential and the latency fluctuations were reduced. The resting potential was -64 mV throughout. The time and voltage calibration bars apply to all traces.

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Figure 3. Interaction between neurally evoked and directly initiated regenerative potentials
A shows a regenerative potential initiated by a train of five stimuli, delivered at 5 Hz. When this train of stimuli was preceded by a regenerative potential, initiated by direct stimulation, the response to nerve stimulation was much reduced (B). Similarly, C shows a regenerative potential initiated by direct stimulation. When this response was preceded by a regenerative potential, initiated by a train of five stimuli, delivered at 5 Hz, the response to direct stimulation was much reduced (D). The resting potential of this preparation was -68 mV throughout. Neurally initiated regenerative responses appeared not to be triggered by a depolarizing action of neurally released ACh. When a regenerative potential was initiated by nerve stimulation (E) it was not preceded by a substantial depolarization. When brief (500 ms) depolarizing currents were applied a threshold depolarization of ~6 mV was required to trigger a regenerative potential. When this depolarization failed to trigger a regenerative potential it triggered an increase in membrane noise (F). The resting potential of this preparation was -64 mV throughout. The time, voltage and current calibration bars apply to all traces.

One of the characteristics of regenerative potentials is that they are followed by a partial refractory period which lasts for several seconds (Suzuki & Hirst, 1999). To test whether neurally evoked regenerative responses and those initiated by direct stimulation reflected activation of a common pathway, the interaction between neurally evoked and directly evoked responses was examined. An experiment is shown in Fig. 3A-D. It can be seen that a train of five impulses initiated a regenerative response (Fig. 3A) but if this train of stimuli was preceded by a directly evoked regenerative response, the train of stimuli only caused an increased discharge of membrane noise (Fig. 3B). Conversely a pulse of depolarizing current, applied from the second intracellular electrode, readily initiated a regenerative potential (Fig. 3C) but this was abolished if it was preceded by a neurally evoked response (Fig. 3D). Further support for the idea that responses to neurally released ACh and direct stimulation were similar came by determining power spectral density curves of the membrane noise which occurred during the falling phase of both responses (see Edwards et al. 1999). When this was done the spectral density curves obtained from the two responses, initiated by direct or neural stimulation, were indistinguishable (data not shown).

Regenerative potentials can be initiated by brief periods of membrane depolarization (Suzuki & Hirst, 1999). The possibility was considered that neuronally released ACh might cause a depolarization that was not readily detected because of the ongoing discharge of membrane noise. This idea was tested by determining the amplitude of membrane depolarization required to initiate a regenerative potential and comparing it with the depolarization readily detected during neurally evoked regenerative potentials. An experiment is shown in Fig. 3E and F. It can be seen that three impulses triggered a slow depolarization that led into the upstroke of the regenerative response (Fig. 3E). When the threshold for initiation of a regenerative potential was determined directly by passing a depolarizing current pulse for 500 ms through the second intracellular electrode, it can be seen that a depolarization of some 6 mV failed to trigger a regenerative response on one occasion and on the other triggered a regenerative response which started after a latency of about 2 s (Fig. 3F). The threshold depolarization, determined in this way in 15 of the preparations studied, was 8.9 ± 1.6 mV. This value differs significantly from the amplitude of EJPs evoked by single stimuli, suggesting that the depolarization produced by an EJP is in itself insufficient to trigger a regenerative response and that regenerative responses are triggered by some other action of neurally released ACh.

Regenerative responses, initiated by direct muscle stimulation are reduced in amplitude by 2APB (Hirst et al. 2002b). The effect of 2APB (40 µM) was examined on neurally and directly evoked regenerative potentials (Fig. 4A-D). In control solutions, neurally evoked regenerative responses had a mean peak amplitude of 27.7 ± 1.7 mV, whilst those evoked by direct stimulation had a mean peak amplitude of 26.7 ± 1.5 mV (Fig. 4A and B). The application of 2APB was associated with a period of membrane depolarization which lasted for some 10-15 min, after this time the membrane potential returned to its control value. When responses were evoked by direct stimulation and by trains of neural stimuli, both responses were reduced in amplitude. In 2APB the directly evoked regenerative potential had a mean amplitude of 1.3 ± 0.4 mV and the neurally evoked response had a mean amplitude of 0.8 ± 0.5 mV (n = 4). In two of these experiments the responses to nerve stimulation were completely abolished but in the other two it was found that the trains of neural stimuli had evoked a 2APB resistant EJP; the EJPs had peak amplitudes of 2.1 and 1.1 mV (see Fig. 4C).

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Figure 4. Effect of 2APB and DIDS on neurally evoked and directly initiated regenerative potentials
A-D show the effect of 2APB (40 µM) on neurally evoked and directly initiated regenerative potentials. A regenerative potential initiated by nerve stimulation (A) was reduced in amplitude by adding 2APB to the physiological saline and allowed an EJP to be detected (B). Similarly a regenerative potential initiated by direct stimulation (C) was also reduced in amplitude by 2APB (D). The resting potential was -66 mV when each set of recordings was made. E-H show the effect of DIDS (40 µM) on neurally evoked and directly initiated regenerative potentials. Regenerative potentials initiated by nerve stimulation (E) and direct stimulation (G) were reduced in amplitude by DIDS (F and H). The resting potential was -67 mV in E and G and -69 mV in F and H. The time, voltage and current calibration bars apply to all traces.

Regenerative responses, initiated by transmural nerve stimulation and by direct stimulation were also reduced in amplitude by DIDS (100 µM; Fig. 4E-H). In control solution neurally evoked regenerative responses had peak amplitudes of 25.4 ± 3.1 mV (Fig. 4E), whilst those evoked by direct stimulation had peak amplitudes of 24.6 ± 2.6 mV (Fig. 4G). In DIDS the values were 1.1 ± 0.7 and 1.7 ± 0.1 mV, respectively (n = 3; Fig. 4G and H). This effect of DIDS was readily reversed by washing with drug-free physiological saline. The observations suggest that regenerative potentials, triggered by nerve stimulation or by direct stimulation, involve the formation of IP₃ and result from a similar increase in conductance, presumably one involving anion selective channels (Hirst et al. 2002b).

Comparison between neurally evoked regenerative potentials and EJPs recorded from bundles of circular muscle

It has been pointed out that on a few occasions it was possible to detect an EJP, followed after a delay by a regenerative potential when a single transmural impulse was applied and that increasing the number of stimuli shortened the latency before the onset of a regenerative potential. It became apparent that EJPs could be detected in a higher proportion of preparations when more stimuli were applied and that EJPs had properties different to those of regenerative potentials. This section will compare the properties of the two responses. Both clearly resulted from the neural release of ACh since both were abolished by hyoscine.

Membrane hyperpolarization decreased the amplitudes of neurally evoked regenerative potentials and eventually abolished them (Fig. 5A). With moderate hyperpolarization, 10-15 mV, increasing the number of transmural stimuli allowed a regenerative potential to be initiated but this was also abolished by further membrane hyperpolarization. In contrast the EJP, which at resting membrane potential was barely distinguishable from the discharge of membrane noise, or from the onset of the neurally evoked regenerative potential (Fig. 5A), persisted. In 10 of the 14 preparations examined in this way, hyperpolarization revealed an EJP provided that three or more stimuli were applied. Even profound membrane hyperpolarization, >30 mV, failed to increase the amplitude of EJPs in any of the preparations examined.

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Figure 5. Comparison between the properties of EJPs and neurally evoked regenerative potentials
A shows the effect of membrane potential on the amplitudes of neurally evoked EJPs and regenerative potentials. As the membrane was progressively hyperpolarized, the amplitude of the regenerative potential was reduced until at negative potentials it was not detected. In contrast, although the amplitudes of EJPs slightly decreased with membrane hyperpolarization, an EJP was detected at all potentials. After the break of the hyperpolarizing pulses, a regenerative potential was initiated. B and C compare and contrast the effect of a prior regenerative potential on the ability of nerve stimulation to initiate an EJP and a regenerative potential. Note that the EJP, which was difficult to detect when not preceded by a regenerative potential (B), persisted during the refractory period which followed a regenerative potential (C). The resting potential was -73 mV throughout. The time, voltage and current calibration bars apply to all traces.

It has been pointed out that during the refractory period which follows a regenerative potential, transmural stimulation fails to trigger a regenerative potential (Fig. 3). In contrast during the refractory period, which follows each regenerative potential, transmural stimulation continued to evoke an EJP (Fig. 5B and C).

Characteristically regenerative potentials are rapidly reduced in amplitude by moderate concentrations of caffeine (Suzuki & Hirst, 1999). In the present series of experiments caffeine (1 mM) reduced the amplitude of regenerative responses initiated by both transmural nerve stimulation and direct muscle stimulation (Fig. 6A and B). In control solution neurally evoked regenerative responses had a mean peak amplitude of 26.4 ± 2.6 mV; those evoked by direct stimulation had a mean peak amplitude of 28.4 ± 1.9 mV. In caffeine the values were 1.3 ± 0.6 and 1.0 ± 0.6 mV, respectively (n = 6). In four of the experiments, the trains of neural stimuli were found to evoke an EJP at hyperpolarized potentials; these persisted in caffeine. The peak amplitudes of the caffeine resistant EJPs, recorded at resting membrane potential, were 3.7, 2.0, 4.7 and 0.9 mV. All effects of caffeine were rapidly reversed by washing with drug-free physiological saline. Similarly regenerative potentials are reduced in amplitude by 9-AC (Hirst et al. 2002b). In these experiments 9-AC (500 µM) reduced the amplitude of regenerative responses initiated by both transmural nerve and direct stimulation (Fig. 6C and D). In control solution neurally evoked regenerative responses had a mean peak amplitude of 31.4 ± 3.2 mV; those evoked by direct stimulation had a mean peak amplitude of 31.9 ± 3.6 mV. In 9-AC the values were 4.2 ± 1.5 and 7.1 ± 3.6 mV, respectively (n = 4). In three of the experiments, the trains of neural stimuli continued to evoke an EJP in the presence of 9-AC, these had peak amplitudes of 2.8, 3.1 and 4.1 mV when measured at the resting potential. From all the experiments where neurally evoked regenerative potentials were abolished by caffeine, 2APB or 9-AC, EJPs, determined at the resting potential, had a mean peak amplitude of 3.3 ± 0.4 mV (n = 9), a value significantly different to that of the depolarization required to directly initiate regenerative responses using 500 ms depolarizing pulses of current, i.e. 8.9 ± 1.6 mV (n = 15).

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Figure 6. Effect of caffeine and 9-AC on neurally evoked and directly initiated regenerative potentials
A shows successive traces where the response to nerve stimulation was recorded at resting potential and during a period of membrane hyperpolarization. At resting potential, -65 mV, a train of stimuli initiated a regenerative potential; hyperpolarization revealed an EJP. Regenerative potentials, triggered by nerve stimulation or by the break of membrane hyperpolarization, were reduced in amplitude by caffeine (1 mM; B) but the EJP persisted, resting membrane potential -68 mV. C shows responses triggered in the same way in a different preparation. Again regenerative potentials, triggered by nerve stimulation or by the break of membrane hyperpolarization, were reduced in amplitude by 9-AC (500 µM; D) but the EJP persisted. The time, voltage and current calibration bars apply to all traces.

Effect of vagal stimulation on slow waves and pacemaker potentials

The previous sets of experiments have shown that cholinergic nerve stimulation evokes a regenerative potential in isolated bundles of the circular layer of gastric antrum, apparently because neurally released ACh directly stimulates ICC_IM. The role of this pathway in the neural control of antral motility was examined by recording slow waves from intact segments of antrum and determining the effects of vagal stimulation on the discharge of slow waves. As has been shown in dog stomach (Vogalis & Sanders, 1990), it was found that brief trains of high frequency vagal stimulation, four to eight impulses at 10 Hz, delivered in the interval between slow waves, evoked premature slow waves in the guinea-pig antrum. Although premature slow waves had a mean amplitude of 30.2 ± 1.2 mV, which was not significantly different from that of spontaneous slow waves, 31.6 ± 1.9 mV (n = 6), the shape of premature slow waves differed from that of spontaneous slow waves in that they lacked an inflection on the rising phase (Fig. 7D). Long trains of vagal stimuli, delivered at 1-5 Hz for 50 s, increased the frequency of slow waves. With increasing stimulus frequency a corresponding increase in the magnitude of each response was observed. With 2 Hz stimulation, the membrane potential between slow waves depolarized by 8.3 ± 1.0 mV and the frequency of slow waves rose from 4.0 ± 0.3 to 4.4 ± 0.3 min^-1 during stimulation, Student's paired t test indicated that this change was statistically significant (n = 6). Both types of responses, that is those produced by brief trains of high frequency vagal stimulation and the increase in frequency produced by low frequency vagal stimulation, persisted after blocking L-type Ca²⁺ channels with nifedipine (1 µM) and were abolished by hyoscine (1 µM).

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Figure 7. Effect of a brief train of vagal stimuli on the generation of pacemaker potentials and slow waves in the gastric antrum
The upper two panels show pacemaker potentials (A) and slow waves (B) recorded simultaneously. A brief train of stimuli, eight at 10 Hz, applied to the vagus evoked a premature pacemaker potential (A) and a premature slow wave (B). The time courses of pacemaker potentials and slow waves recorded prior to stimulation (C) and those triggered by the burst of vagal stimuli (D) are displayed in the bottom section of the figure on a faster time scale. It can be seen that prior to stimulation the upstroke of the pacemaker potential (continuous trace) preceded that of the slow wave (dashed trace) (C). In contrast, the start of the premature slow wave (dashed trace) preceded the start of the premature pacemaker potential (continuous trace) (D). The voltage calibration bar applies to all traces, the upper time calibration bar applies to the upper two traces, the lower bar applies to the expansions.

The possibility that vagal stimulation evoked premature slow waves by triggering premature regenerative potentials in the circular muscle layer was considered. Simultaneous intracellular recordings were made from ICC_MY and from cells in the circular muscle layer (n = 4). All recordings were made in the presence of nifedipine (1 µM), this reduced muscle movements and allowed recordings to be made from the two cell types for several minutes. Recordings from ICC_MY were identified on electrophysiological grounds, with the pacemaker potentials having amplitudes in excess of 40 mV and maximum rates of rise in excess of 0.1 V s^-1 (Dickens et al. 1999; Hirst & Edwards, 2001). In each preparation, prior to stimulation, much of the rising phase of the pacemaker potential preceded that of the initial component of the slow wave (Fig. 7C). A brief train of vagal stimuli, eight at 10 Hz, evoked a premature slow wave and a premature pacemaker potential (Fig. 7A and B) but the start of the premature slow wave lacked an initial component and preceded the start of the pacemaker potential (Fig. 7D). With longer trains of low frequency vagal stimuli, 1-5 Hz for 50 s, the frequency of occurrence of pacemaker potentials and slow waves increased synchronously and the duration of pacemaker potentials was shortened (Fig. 8A and B). Again when the recordings were overlaid and inspected at higher scan speed, prior to stimulation, much of the rising phase of the pacemaker potential preceded that of the initial component of the slow wave (Fig. 8C) but during vagal stimulation the start of each slow wave again preceded that of the associated pacemaker potential (Fig. 8D and E).

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Figure 8. Effect of low frequency vagal stimulation on the generation of pacemaker potentials and slow waves in the gastric antrum
The upper two panels show pacemaker potentials (A) and slow waves (B) recorded simultaneously. A long train of stimuli, 50 s at 2 Hz, applied to the vagus increased the rate of occurrence of pacemaker potentials (A) and slow waves (B). The time courses of pacemaker potentials and slow waves recorded prior to stimulation (C) and during vagal stimuli (D and E) are displayed in the bottom section of the figure on a faster time scale. It can be seen that prior to stimulation the upstroke of the pacemaker potential (continuous trace) preceded that of the slow wave (dashed trace) (C). In contrast, the start of each slow wave recorded during the period of vagal stimulation (dashed trace) preceded the start of the associated pacemaker potential (continuous trace) (D and E). The voltage calibration bar applies to all traces, the upper time calibration bar applies to the upper two traces, the lower bar applies to the expansions.

Electrical excitability of pacemaker cells

The previous set of experiments have indicated that vagal stimulation causes a shift of the dominant pacemaker from ICC_MY to the circular layer with pacemaker potentials occurring at an increased frequency (Fig. 7 and Fig. 8). These observations could be explained if premature regenerative potentials, initiated in the circular muscle layer, triggered premature pacemaker potentials in ICC_MY. For this to be the case, as well as being coupled to the circular layer (Dickens et al. 1999), the network of ICC_MY would have to be electrically excitable. This point was tested directly by recording from a network of ICC_MY and determining if a pacemaker potential could be evoked by electrical stimulation. Recordings were made from the network of ICC_MY which remains attached to the longitudinal muscle layer (Hirst & Edwards, 2001); the network was stimulated directly using a pair of stimulating electrodes. The effects of nerve stimulation were abolished by hyoscine (1 µM) and tetrodotoxin (1 µM) and direct muscle stimulation by nifedipine (1 µM).

Pacemaker potentials were recorded from ICC_MY. In the absence of stimulation, pacemaker potentials occurred at a mean frequency of 3.4 ± 0.3 min^-1 (n = 6). When a stimulating pulse, 10-20 ms, was applied at varying intervals after the end of a pacemaker potential a premature pacemaker potential could be initiated. Trains of impulses, delivered at a higher frequency than the naturally occurring pacemaker frequency, generated a repeated discharge of pacemaker potentials (Fig. 9). As with the recordings of pacemaker potentials made from intact antral preparations during periods of vagal stimulation (Fig. 8), evoked pacemaker potentials were briefer in duration than those recorded during normal pacemaker activity (Fig. 9). Presumably this reflects the relationship between pacemaker duration and the preceding interval detected during normal pacemaker activity, with the paced potentials occurring after short intervals with shorter durations (Hirst & Edwards, 2001). When the conduction velocity of pacemaker potentials through the network of ICC_MY was estimated by measuring the separation between the stimulating electrodes and the impaled cells, along with the latency of an evoked pacemaker potential, it was found to be 5.4 ± 0.7 mm s^-1 (n = 6). These experiments indicate that a voltage-dependent ion channel must be present in the membranes of ICC_MY and that this channel can be activated by membrane depolarization (for further discussion see Hirst & Edwards, 2001).

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Figure 9. Effect of electrical stimulation on discharge of pacemaker potentials by ICC_IM
A shows the effect of stimulating a network of ICC_MY, attached to the longitudinal muscle layer, with a train of three impulses delivered at 0.1 Hz. B shows the amplitude of the stimulating pulses, pulse width 15 ms. It can be seen that each stimulus evokes a premature pacemaker potential and that when their frequency of occurrence was increased, their duration was reduced. The effects of concurrently stimulating intrinsic nerves was abolished by TTX (1 µM) and hyoscine (1 µM), the effects of direct muscle stimulation were abolished by nifedipine (1 µM). The resting membrane potential was -67 mV.

DISCUSSION

Top
Abstract
Introduction
Methods
Results
Discussion
References

These experiments have shown that cholinergic nerve stimulation evokes two distinct types of responses in the circular muscle layer of guinea-pig antrum. One type of response resembles the regenerative response which is initiated in this tissue by ICC_IM following direct stimulation. The other type of response, an EJP, may result from activation of muscarinic receptors present on smooth muscle cells. In intact tissues excitatory vagal stimulation evokes a premature slow wave which originates in the circular layer.

Neurally evoked regenerative potentials, like those evoked by direct stimulation, were abolished by 2APB (Fig. 4, see Hirst et al. 2002b), by the chloride channel blockers DIDS and 9-AC (Fig. 4 and Fig. 6, see Hirst et al. 2002b) and by caffeine (Fig. 6, see Suzuki & Hirst, 1999). During the refractory period, which follows each regenerative potential, nerve stimulation failed to evoke a regenerative response (Fig. 3, see Suzuki & Hirst, 1999). The simplest explanation for these findings is that neurally released ACh stimulates muscarinic receptors located on ICC_IM which in turn activate the same pathway as that activated by membrane depolarization (Fig. 1). It has been suggested that voltage activation of regenerative potentials involves the formation of IP₃, followed by the release of calcium ions from internal stores (Suzuki & Hirst, 1999; van Helden et al. 2000). One possibility is that in this tissue neurally released ACh activates sets of muscarinic receptors which are also linked to phospholipase C, as has been demonstrated in many tissues (Berridge, 1993). This idea is supported by the finding that the responses to excitatory nerve stimulation and slow waves are absent in mutant mice which lack IP₃ type 1 receptors (Suzuki et al. 2000) and both responses are abolished by 2APB (Fig. 4) which blocks IP₃-induced Ca²⁺ release (Maruyama et al. 1997; see however Prakriya & Lewis, 2001). Similarly it has been shown that membrane hyperpolarization reduces agonist-induced formation of IP₃ in some smooth muscles (Itoh et al. 1992; Ganitkevich & Isenberg, 1993); in preparations of circular muscle, membrane hyperpolarization reduced and then blocked the neural activation of regenerative potentials (Fig. 5 and Fig. 6).

The second type of response detected, an EJP, also resulted from activation of muscarinic receptors. EJPs were distinguishable from neuronally initiated regenerative potentials; they persisted in the presence of caffeine, 2APB, the chloride channel blockers DIDS and 9-AC and during the refractory period which follows a regenerative potential. EJPs also persisted when the membrane potential of the isolated bundles was hyperpolarized. However unlike most excitatory synaptic potentials their amplitudes were not increased by membrane hyperpolarization (Fig. 5). These observations suggest that EJPs result from the activation of muscarinic receptors on smooth muscle cells. Such receptors have been shown to be linked to cation selective channels which show rectification at negative potentials (Benham et al. 1985; Inoue & Isenberg, 1990a). Moreover such channels are linked by a G-protein but do not appear to require the formation of IP₃ for activation (Inoue & Isenberg, 1990b). It is to be stressed, however, that the receptors linked to cation selective channels have not previously been shown to be functionally innervated in the gastrointestinal tract. In the longitudinal muscle of the small intestine, where cholinergic nerve terminals directly innervate smooth muscle cells (Klemm, 1995), the responses to nerve stimulation differ from those attributable to receptors linked to cation selective channels. Those activated by neuronally released ACh are associated with a substantial increase in the internal concentration of calcium ions (Cousins et al. 1993, 1995) whereas such an increase could not be readily detected following the muscarinic activation of cation selective channels (Pacaud & Bolton, 1991). Which pathway is involved in the longitudinal muscle layer of the gastric antrum is not known, however, it is clear that excitatory nerve stimulation does not involve ICC_IM, since although these cells are present in both layers of the gastric fundus (Burns et al. 1997), they are absent in the longitudinal layer of the gastric antrum (Hirst & Edwards, 2001, 2002a). Whatever the case the contribution made by EJPs to neuronal responses detected in the circular layer of the antrum is small, being of insufficient amplitude to trigger regenerative responses (Fig. 3).

The demonstration of a functional connection between intrinsic excitatory nerves and ICC_IM provides an explanation for how excitatory nerve stimulation evokes premature slow waves (Vogalis & Sanders, 1990). Neurally released ACh triggers a regenerative potential in the circular muscle layer (Fig. 1). In intact tissue this is manifest as a premature slow wave which precedes the normally occurring pacemaker potential generated by ICC_MY (Fig. 7 and Fig. 8). The finding that the rate of pacemaker potentials is consequently increased is consistent with the finding that the myenteric layer of interstitial cells is electrically coupled to the circular layer (Dickens et al. 1999) and that the network of ICC_MY can be paced electrically (Fig. 9). Pacing of slow waves in intact tissues has been described previously (Publicover & Sanders, 1986) and presumably this has a similar basis. These observations also suggest, that as with inhibitory neuronal projections in stomach (Dickens et al. 2000), few, if any, excitatory neuronal projections form functional contacts with the pacemaker network of myenteric interstitial cells, i.e. ICC_MY.

All of the observations made in this report, on the nature of functional neuronal connections within the wall of the stomach, are consistent with the suggestion that ICC_IM are essential intermediaries to the transfer of excitatory neuronal information to gastric muscle (Ward et al. 2000). Thus the dominant response, triggered by excitatory nerve stimulation in the circular layer, resembles the secondary regenerative component of the slow wave which is generated by interstitial cells lying in the circular layer (Dickens et al. 2001; Hirst et al. 2002). Moreover regenerative responses, which are the dominant responses produced by neural stimulation involve the activation of anion selective channels. Although such channels have been detected in several smooth muscle cells (Large, 1984; van Helden, 1988; Large & Wang, 1996; Greenwood & Large, 1999) and interstitial-like cells in other tissues (Sergeant et al. 2001) they have not been detected in intestinal smooth muscle. The finding that the contribution of cation selective channels to responses produced by neurally released ACh in the antrum is small, suggests that smooth muscle cells receive little functional innervation and that ACh activates receptors located near its points of release (Hirst et al. 1996). Presumably such points of release are located in close apposition to ICC_IM (Ward et al. 2000).

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

BENHAM, C. D., BOLTON, T. B. & LANG, R. J. (1985). Acetylcholine activates an inward current in single mammalian smooth muscle cells. Nature 328, 275-278

BERRIDGE, M. J. (1993). Inositol trisphosphate and calcium signaling. Nature 361, 315-325 [Medline]

BURNS, A. J., HERBERT, T. M., WARD, S. M. & SANDERS, K. M. (1997). Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit. Cell and Tissue Research 290, 11-20 [Medline]

BURNS, A. J., LOMAX, A. E., TORIHASHI, S., SANDERS, K. M. & WARD, S. M. (1996). Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proceedings of the National Academy of Sciences of the USA 93, 12008-12013 [Abstract]

COUSINS, H. M., EDWARDS, F. R. & HIRST, G. D. S. (1995). Neuronally released and applied acetylcholine on the longitudinal muscle of the guinea-pig ileum. Neuroscience 65, 193-207 [Medline]

COUSINS, H. M., EDWARDS, F. R., HIRST, G. D. S. & WENDT, I. R. (1993). Cholinergic neuromuscular transmission in the longitudinal muscle of the guinea-pig ileum. Journal of Physiology 471, 61-86 [Abstract]

DICKENS, E. J., EDWARDS, F. R. & HIRST, G. D. S. (2000). Vagal inhibitory projections to rhythmically active cells in the antral region of guinea-pig stomach. American Journal of Physiology - Gastrointestinal and Liver Physiology 279, G388-399 [Medline]

DICKENS, E. J., EDWARDS, F. R. & HIRST, G. D. S. (2001). Selective knockout of intramuscular interstitial cells reveals their role in the generation of slow waves in mouse stomach. Journal of Physiology 531, 827-833 [Abstract/Full Text]

DICKENS, E. J., HIRST, G. D. S. & TOMITA, T. (1999). Identification of rhythmically active cells in guinea-pig stomach. Journal of Physiology 514, 515-531 [Abstract/Full Text]

EDWARDS, F. R., HIRST, G. D. S. & SUZUKI, H. (1999). Unitary nature of regenerative potentials recorded from circular smooth muscle of guinea-pig antrum. Journal of Physiology 519, 235-250 [Abstract/Full Text]

GANITKEVICH, V. YA. & ISENBERG, G. (1993). membrane potential modulates inositol 1, 4, 5-trisphophate-mediated Ca²⁺ transients in guinea-pig coronary myocytes. Journal of Physiology 470, 35-44 [Abstract]

GREENWOOD, I. A. & LARGE, W. A. (1999). Modulation of the decay of Ca²⁺-activated Cl^- currents in rabbit portal vein smooth muscle cells by external anions. Journal of Physiology 516, 365-376 [Abstract/Full Text]

HIRST, G. D. S., BECKETT, E. A. H., SANDERS, K. M. & WARD, S. M. (2002a). Regional variation in contribution of ICC_MY and ICC_IM to generation of slow waves in mouse gastric antrum. Journal of Physiology 540, 1003-1012 [Abstract/Full Text]

HIRST, G. D. S., BRAMICH, N. J., TERAMOTO, N., SUZUKI, H. & EDWARDS, F. R. (2002b). Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca²⁺]_i and Cl^- channels. Journal of Physiology 540, 907-919 [Abstract/Full Text]

HIRST, G. D. S., CHOATE, J. K., COUSINS, H. M., EDWARDS, F. R. & KLEMM, M. F. (1996). Transmission by post-ganglionic axons of the autonomic nervous system: the importance of the specialized neuroeffector junction. Neuroscience I73, 7-23

HIRST, G. D. S. & EDWARDS, F. R. (2001). Generation of slow waves in the antral region of guinea-pig stomach - a stochastic process. Journal of Physiology 535, 165-180 [Abstract/Full Text]

INOUE, R. & ISENBERG, G. (1990a). Effect of membrane potential on acetylcholine induced inward current in guinea-pig ileum. Journal of Physiology 424, 57-71 [Abstract]

INOUE, R. & ISENBERG, G. (1990b). Acetylcholine activates non selective cation channels in guinea pig ileum through a G protein. American Journal of Physiology 258, C1173-1178 [Medline]

ITOH, M., SEKI, N., SUZUKI, H., ITO, S., KAJIKURA J. & KURIYAMA, H. (1992). Membrane hyperpolarization inhibits agonist-induced synthesis of inositol 1,4,5-trisphosphate in rabbit mesenteric artery. Journal of Physiology 451, 307-328 [Abstract]

KLEMM, M. F. (1995). Neuromuscular junctions made by nerve fibres supplying the longitudinal muscle of the guinea-pig. Journal of the Autonomic Nervous System 55, 155-164 [Medline]

LARGE, W. A. (1984). The effect of chloride removal on the responses of the isolated rat anococcygeus muscle to $alpha$ ₁-adrenoceptor stimulation. Journal of Physiology 352, 17-29 [Abstract]

LARGE, W. A. & WANG, Q. (1996). Charactersitics and physiological role of the Ca²⁺ -activated Cl^- conductance in smooth muscle. American Journal of Physiology 271, C435-454 [Medline]

MARUYAMA, T., KANAJI, T., NAKADE, S., KANNO, T. & MIKOSHIBA, K. (1997). 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P₃-induced Ca²⁺ release. Journal of Biochemistry 122, 498-505 [Medline]

OHBA, M., SAKAMOTO, Y. & TOMITA, T. (1975). The slow wave in the circular muscle of the guinea-pig stomach. Journal of Physiology 253, 505-516 [Abstract]

ORDOG, T., WARD, S. M. & SANDERS, K. M. (1999). Interstitial cells of Cajal generate electrical slow waves in the murine stomach. Journal of Physiology 518, 257-269 [Abstract/Full Text]

PACAUD, P. & BOLTON, T. B. (1991). Relation between muscarinic receptor cationic current and intestinal calcium in guinea-pig jejunal smooth muscle. Journal of Physiology 441, 477-499 [Abstract]

PRAKRIYA, M. & LEWIS, R. S. (2001). Potentiation and inhibition of Ca²⁺ release-activated Ca²⁺ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP₃ receptors. Journal of Physiology 536, 3-19 [Abstract/Full Text]

PUBLICOVER, N. G. & SANDERS, K. M. (1986). Effects of frequency on the wave form of propagated slow waves in canine gastric antral muscle. Journal of Physiology 371, 179-189 [Abstract]

SANDERS, K. M. (1996). A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111, 492-515 [Medline]

SERGEANT, G. P., HOLLYWOOD, M. A., MCCLOSKEY, K. D., THORNBURY, K.D. & MCHALE, N. G. (2001). Specialised pacemaking cells in the rabbit urethra. Journal of Physiology 526, 359-366 [Abstract/Full Text]

SUZUKI, H. & HIRST, G. D. S. (1999). Regenerative potentials evoked in circular smooth muscle of the antral region of guinea-pig stomach. Journal of Physiology 517, 563-573 [Abstract/Full Text]

SUZUKI, H., TAKANO, H., YAMAMOTO, Y., KOMURO, T., SAITO, M., KATO, K. & MIKOSHIBA, K. (2000). Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor. Journal of Physiology 525, 105-111 [Abstract/Full Text]

VAN HELDEN, D. F. (1988). Electrophysiology of neuromuscular transmission in guinea-pig mesenteric veins. Journal of Physiology 401, 469-488 [Abstract]

VAN HELDEN, D. F., IMTIAZ, M. S., NURGALIYEVA, K., VON DER WEID, P. & DOSEN, P. J. (2000). Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea-pig gastric pylorus. Journal of Physiology 524, 245-265 [Abstract/Full Text]

VOGALIS, F. & SANDERS, K. M. (1990). Excitatory and inhibitory neural regulation of canine pyloric smooth muscle. American Journal of Physiology 259, G125-133 [Medline]

WARD, S. M., BECKETT, E. A., WANG, X., BAKER, F., KHOYI, M. & SANDERS, K. M. (2000). Interstitial cells of Cajal mediate cholinergic neurotransmission from enteric motor neurons. Journal of Neuroscience 20, 1393-1403 [Abstract/Full Text]

WARD, S. M., BRENNAN, M. F., JACKSON, V. M. & SANDERS, K. M. (1999). Role of PI3-kinase in the development of interstitial cells and pacemaking in murine gastrointestinal smooth muscle. Journal of Physiology 516, 835-846 [Abstract/Full Text]

WARD, S. M., BURNS, A. J., TORIHASHI, S. & SANDERS, K. M. (1994). Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. Journal of Physiology 480, 91-97 [Abstract]

WARD, S. M., HARNEY, S. C., BAYGUINOV, J. R., MCLAREN, G. J. & SANDERS, K. M. (1997). Development of electrical rhythmicity in the murine gastrointestinal tract is specifically encoded in the tunica muscularis. Journal of Physiology 505, 241-258 [Abstract]

Acknowledgements

This project was supported by a grant from the Australian NH&MRC. We are grateful to Dr Narelle Bramich for her critical reading of the manuscript.

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BENHAM, C. D., BOLTON, T. B. & LANG, R. J. (1985). Acetylcholine activates an inward current in single mammalian smooth muscle cells. Nature 328, 275-278
BERRIDGE, M. J. (1993). Inositol trisphosphate and calcium signaling. Nature 361, 315-325	[Medline]
BURNS, A. J., HERBERT, T. M., WARD, S. M. & SANDERS, K. M. (1997). Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit. Cell and Tissue Research 290, 11-20	[Medline]
BURNS, A. J., LOMAX, A. E., TORIHASHI, S., SANDERS, K. M. & WARD, S. M. (1996). Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proceedings of the National Academy of Sciences of the USA 93, 12008-12013	[Abstract]
COUSINS, H. M., EDWARDS, F. R. & HIRST, G. D. S. (1995). Neuronally released and applied acetylcholine on the longitudinal muscle of the guinea-pig ileum. Neuroscience 65, 193-207	[Medline]
COUSINS, H. M., EDWARDS, F. R., HIRST, G. D. S. & WENDT, I. R. (1993). Cholinergic neuromuscular transmission in the longitudinal muscle of the guinea-pig ileum. Journal of Physiology 471, 61-86	[Abstract]
DICKENS, E. J., EDWARDS, F. R. & HIRST, G. D. S. (2000). Vagal inhibitory projections to rhythmically active cells in the antral region of guinea-pig stomach. American Journal of Physiology - Gastrointestinal and Liver Physiology 279, G388-399	[Medline]
DICKENS, E. J., EDWARDS, F. R. & HIRST, G. D. S. (2001). Selective knockout of intramuscular interstitial cells reveals their role in the generation of slow waves in mouse stomach. Journal of Physiology 531, 827-833	[Abstract/Full Text]
DICKENS, E. J., HIRST, G. D. S. & TOMITA, T. (1999). Identification of rhythmically active cells in guinea-pig stomach. Journal of Physiology 514, 515-531	[Abstract/Full Text]
EDWARDS, F. R., HIRST, G. D. S. & SUZUKI, H. (1999). Unitary nature of regenerative potentials recorded from circular smooth muscle of guinea-pig antrum. Journal of Physiology 519, 235-250	[Abstract/Full Text]
GANITKEVICH, V. YA. & ISENBERG, G. (1993). membrane potential modulates inositol 1, 4, 5-trisphophate-mediated Ca²⁺ transients in guinea-pig coronary myocytes. Journal of Physiology 470, 35-44	[Abstract]
GREENWOOD, I. A. & LARGE, W. A. (1999). Modulation of the decay of Ca²⁺-activated Cl^- currents in rabbit portal vein smooth muscle cells by external anions. Journal of Physiology 516, 365-376	[Abstract/Full Text]
HIRST, G. D. S., BECKETT, E. A. H., SANDERS, K. M. & WARD, S. M. (2002a). Regional variation in contribution of ICC_MY and ICC_IM to generation of slow waves in mouse gastric antrum. Journal of Physiology 540, 1003-1012	[Abstract/Full Text]
HIRST, G. D. S., BRAMICH, N. J., TERAMOTO, N., SUZUKI, H. & EDWARDS, F. R. (2002b). Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca²⁺]_i and Cl^- channels. Journal of Physiology 540, 907-919	[Abstract/Full Text]
HIRST, G. D. S., CHOATE, J. K., COUSINS, H. M., EDWARDS, F. R. & KLEMM, M. F. (1996). Transmission by post-ganglionic axons of the autonomic nervous system: the importance of the specialized neuroeffector junction. Neuroscience I73, 7-23
HIRST, G. D. S. & EDWARDS, F. R. (2001). Generation of slow waves in the antral region of guinea-pig stomach - a stochastic process. Journal of Physiology 535, 165-180	[Abstract/Full Text]
INOUE, R. & ISENBERG, G. (1990a). Effect of membrane potential on acetylcholine induced inward current in guinea-pig ileum. Journal of Physiology 424, 57-71	[Abstract]
INOUE, R. & ISENBERG, G. (1990b). Acetylcholine activates non selective cation channels in guinea pig ileum through a G protein. American Journal of Physiology 258, C1173-1178	[Medline]
ITOH, M., SEKI, N., SUZUKI, H., ITO, S., KAJIKURA J. & KURIYAMA, H. (1992). Membrane hyperpolarization inhibits agonist-induced synthesis of inositol 1,4,5-trisphosphate in rabbit mesenteric artery. Journal of Physiology 451, 307-328	[Abstract]
KLEMM, M. F. (1995). Neuromuscular junctions made by nerve fibres supplying the longitudinal muscle of the guinea-pig. Journal of the Autonomic Nervous System 55, 155-164	[Medline]
LARGE, W. A. (1984). The effect of chloride removal on the responses of the isolated rat anococcygeus muscle to $alpha$ ₁-adrenoceptor stimulation. Journal of Physiology 352, 17-29	[Abstract]
LARGE, W. A. & WANG, Q. (1996). Charactersitics and physiological role of the Ca²⁺ -activated Cl^- conductance in smooth muscle. American Journal of Physiology 271, C435-454	[Medline]
MARUYAMA, T., KANAJI, T., NAKADE, S., KANNO, T. & MIKOSHIBA, K. (1997). 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P₃-induced Ca²⁺ release. Journal of Biochemistry 122, 498-505	[Medline]
OHBA, M., SAKAMOTO, Y. & TOMITA, T. (1975). The slow wave in the circular muscle of the guinea-pig stomach. Journal of Physiology 253, 505-516	[Abstract]
ORDOG, T., WARD, S. M. & SANDERS, K. M. (1999). Interstitial cells of Cajal generate electrical slow waves in the murine stomach. Journal of Physiology 518, 257-269	[Abstract/Full Text]
PACAUD, P. & BOLTON, T. B. (1991). Relation between muscarinic receptor cationic current and intestinal calcium in guinea-pig jejunal smooth muscle. Journal of Physiology 441, 477-499	[Abstract]
PRAKRIYA, M. & LEWIS, R. S. (2001). Potentiation and inhibition of Ca²⁺ release-activated Ca²⁺ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP₃ receptors. Journal of Physiology 536, 3-19	[Abstract/Full Text]
PUBLICOVER, N. G. & SANDERS, K. M. (1986). Effects of frequency on the wave form of propagated slow waves in canine gastric antral muscle. Journal of Physiology 371, 179-189	[Abstract]
SANDERS, K. M. (1996). A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111, 492-515	[Medline]
SERGEANT, G. P., HOLLYWOOD, M. A., MCCLOSKEY, K. D., THORNBURY, K.D. & MCHALE, N. G. (2001). Specialised pacemaking cells in the rabbit urethra. Journal of Physiology 526, 359-366	[Abstract/Full Text]
SUZUKI, H. & HIRST, G. D. S. (1999). Regenerative potentials evoked in circular smooth muscle of the antral region of guinea-pig stomach. Journal of Physiology 517, 563-573	[Abstract/Full Text]
SUZUKI, H., TAKANO, H., YAMAMOTO, Y., KOMURO, T., SAITO, M., KATO, K. & MIKOSHIBA, K. (2000). Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor. Journal of Physiology 525, 105-111	[Abstract/Full Text]
VAN HELDEN, D. F. (1988). Electrophysiology of neuromuscular transmission in guinea-pig mesenteric veins. Journal of Physiology 401, 469-488	[Abstract]
VAN HELDEN, D. F., IMTIAZ, M. S., NURGALIYEVA, K., VON DER WEID, P. & DOSEN, P. J. (2000). Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea-pig gastric pylorus. Journal of Physiology 524, 245-265	[Abstract/Full Text]
VOGALIS, F. & SANDERS, K. M. (1990). Excitatory and inhibitory neural regulation of canine pyloric smooth muscle. American Journal of Physiology 259, G125-133	[Medline]
WARD, S. M., BECKETT, E. A., WANG, X., BAKER, F., KHOYI, M. & SANDERS, K. M. (2000). Interstitial cells of Cajal mediate cholinergic neurotransmission from enteric motor neurons. Journal of Neuroscience 20, 1393-1403	[Abstract/Full Text]
WARD, S. M., BRENNAN, M. F., JACKSON, V. M. & SANDERS, K. M. (1999). Role of PI3-kinase in the development of interstitial cells and pacemaking in murine gastrointestinal smooth muscle. Journal of Physiology 516, 835-846	[Abstract/Full Text]
WARD, S. M., BURNS, A. J., TORIHASHI, S. & SANDERS, K. M. (1994). Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. Journal of Physiology 480, 91-97	[Abstract]
WARD, S. M., HARNEY, S. C., BAYGUINOV, J. R., MCLAREN, G. J. & SANDERS, K. M. (1997). Development of electrical rhythmicity in the murine gastrointestinal tract is specifically encoded in the tunica muscularis. Journal of Physiology 505, 241-258	[Abstract]

				S. K. Sarna Are interstitial cells of Cajal plurifunction cells in the gut? Am J Physiol Gastrointest Liver Physiol, February 1, 2008; 294(2): G372 - G390. [Abstract] [Full Text] [PDF]

				Y. Takeda, S. D. Koh, K. M. Sanders, and S. M. Ward Differential expression of ionic conductances in interstitial cells of Cajal in the murine gastric antrum J. Physiol., February 1, 2008; 586(3): 859 - 873. [Abstract] [Full Text] [PDF]

				E. E. Daniel, A. E. Yazbi, M. Mannarino, G. Galante, G. Boddy, J. Livergant, and T. E. Oskouei Do gap junctions play a role in nerve transmissions as well as pacing in mouse intestine? Am J Physiol Gastrointest Liver Physiol, March 1, 2007; 292(3): G734 - G745. [Abstract] [Full Text] [PDF]

				K. M. Sanders and S. M. Ward Kit mutants and gastrointestinal physiology J. Physiol., January 1, 2007; 578(1): 33 - 42. [Abstract] [Full Text] [PDF]

				H. Chen, D. Redelman, S. Ro, S. M. Ward, T. Ordog, and K. M. Sanders Selective labeling and isolation of functional classes of interstitial cells of Cajal of human and murine small intestine Am J Physiol Cell Physiol, January 1, 2007; 292(1): C497 - C507. [Abstract] [Full Text] [PDF]

				K. M. Sanders and S. M. Ward Interstitial cells of Cajal: a new perspective on smooth muscle function J. Physiol., November 1, 2006; 576(3): 721 - 726. [Abstract] [Full Text] [PDF]

				G. D. S. Hirst and F. R. Edwards Electrical events underlying organized myogenic contractions of the guinea pig stomach J. Physiol., November 1, 2006; 576(3): 659 - 665. [Abstract] [Full Text] [PDF]

				S. M. Ward and K. M. Sanders Involvement of intramuscular interstitial cells of Cajal in neuroeffector transmission in the gastrointestinal tract J. Physiol., November 1, 2006; 576(3): 675 - 682. [Abstract] [Full Text] [PDF]

				A. S. Forrest, T. Ordog, and K. M. Sanders Neural regulation of slow-wave frequency in the murine gastric antrum Am J Physiol Gastrointest Liver Physiol, March 1, 2006; 290(3): G486 - G495. [Abstract] [Full Text] [PDF]

				G. D. S. Hirst, A. P. Garcia-Londono, and F. R. Edwards Propagation of slow waves in the guinea-pig gastric antrum J. Physiol., February 15, 2006; 571(1): 165 - 177. [Abstract] [Full Text] [PDF]

				H. Hashitani, A. P. Garcia-Londono, G. D. S. Hirst, and F. R. Edwards Atypical slow waves generated in gastric corpus provide dominant pacemaker activity in guinea pig stomach J. Physiol., December 1, 2005; 569(2): 459 - 465. [Abstract] [Full Text] [PDF]

				K.-J. Won, K. M. Sanders, and S. M. Ward Interstitial cells of Cajal mediate mechanosensitive responses in the stomach PNAS, October 11, 2005; 102(41): 14913 - 14918. [Abstract] [Full Text] [PDF]

				G. Song, G David, S Hirst, K. M Sanders, and S. M Ward Regional variation in ICC distribution, pacemaking activity and neural responses in the longitudinal muscle of the murine stomach J. Physiol., April 15, 2005; 564(2): 523 - 540. [Abstract] [Full Text] [PDF]

				F. R Edwards and G. D. S Hirst An electrical description of the generation of slow waves in the antrum of the guinea-pig J. Physiol., April 1, 2005; 564(1): 213 - 232. [Abstract] [Full Text] [PDF]