Novel autonomic neurotransmitters and intestinal function.

A wide variety of substances, including amines and peptides, have been detected within the complex neuronal pathways of the enteric nervous system using immunohistochemical techniques. In this article we have discussed some of the more recent data on the effects of these substances on intestinal activity. We have also commented on the many difficulties associated with ascribing neurotransmitter status to individual compounds. The technique of immunoblockade of neurogenic functional responses has been used in an attempt to identify some of the putative neurotransmitter substances. The search for selective antagonists continues.

Effects of nitric oxide (NO) and NO donors on the membrane conductance of circular smooth muscle cells of the guinea-pig proximal colon.

1. The membrane conductance changes underlying the membrane hyperpolarizations induced by nitric oxide (NO), S-nitroso-L-cysteine (NC) and sodium nitroprusside (SNP) were investigated in the circular smooth muscle cells of the guinea-pig proximal colon, by use of standard intracellular microelectrode recording techniques. 2. NO (1%), NC (2.5-25 microM) and SNP (1-1000 microM) induced membrane hyperpolarization in a concentration-dependent manner, the hyperpolarizations to NO and NC developing more rapidly than those to SNP. The slower-developing responses to SNP were mimicked by the membrane permeable analogue of guanosine 3':5' cyclic-monophosphate (cyclic GMP), 8-bromo-cyclic GMP (500 microM), and by isoprenaline (10 microM). 3. The hyperpolarizations to NC and SNP were reduced in a low Ca2+ (0.25 mM) saline and upon the addition of haemoglobin (20 microM), but were not effected by NG-nitro-L-arginine (L-NOARG) (100 microM) or omega-conotoxin GVIA (100 nM). the hyperpolarizations to SNP were also significantly reduced by methylene blue (50 microM). 4. Apamin (250 nM) depolarized the membrane potential approximately 10 mV and reduced the initial transient component of the hyperpolarization to NO (1%) and NC (25 microM), but had no effects on the hyperpolarizations to SNP and cyclic GMP. Tetraethylammonium (TEA) (5-15 mM), had little effect on the membrane responses to NO(1%), NC(2.5-25 microM), SNP(100(-1000) microM) or cyclic GMP(500 microM). However, TEA (5-15 mM) reduced the membrane hyperpolarizations to SNP (10 microM) and isoprenaline (10 microM) in a concentration-dependent manner. The hyperpolarization to isoprenaline (10 microM) remaining in the presence of 15 mM TEA was blocked by ouabain (10 microM). 5. The amplitude of electronic potentials (1 s duration) elicited during NO donor hyperpolarizations were little changed or only slightly reduced (5-25%). However, the amplitude of the electrotonic potentials elicited during maintained electrically-induced hyperpolarizations of similar amplitude were significantly increased (30-150%), suggesting that the non-linear membrane properties of the proximal colon partially mask an increase in membrane conductance elicited during the NO donor hyperpolarizations. 6. Membrane hyperpolarization in the presence of an NO donor, 8-bromo-cyclic GMP, isoprenaline, or upon application of a maintained hyperpolarizing electrical current, often evoked oscillations of the membrane potential. These oscillations were prevented by Cs+ (1 mM). 7. These results indicate that NO and NC hyperpolarize the circular muscle of the proximal colon by activating at least two TEA-resistant membrane K+ conductances, one of which is sensitive to apamin blockade. The K+ conductance increases activated by SNP or 8-bromo-cyclic GMP were little effected by apamin, perhaps suggesting a common mechanism. In contrast, the hyperpolarization to isoprenaline appears to involve the activation of TEA-sensitive Ca2(+)-activated K+ ('BK') channels, as well as a Na:K ATPase. Finally, the 'background' membrane conductance of the circular muscle cells of the proximal colon decreased upon membrane hyperpolarization to reveal oscillations of the membrane potential which may well represent 'pacemaker' or 'slow wave' activity.

Enteric nerve stimulation evokes a premature colonic migrating motor complex in mouse.

The effects of enteric nerve stimuli were investigated on spontaneously occurring colonic migrating motor complexes (CMMCs) in isolated mouse colon. Changes in circular smooth muscle tension were recorded simultaneously from the proximal, mid and distal regions of an in vitro preparation of whole mouse colon at 36 +/- 1 degrees C. The CMMCs were recorded from all preparations with a mean interval between contractions ranging from 135.2 +/- 9.3 to 163.3 +/- 22.4 s. The CMMCs migrated spontaneously from the proximal to distal colon and were abolished by tetrodotoxin (1 micromol L-1). In approximately half of all trials (57 of 103, n = 31), trains of stimuli (20 Hz, 2-5 s, 1 ms, 40-70 V) delivered to the mid or distal regions of colon, during the intervals between CMMCs, elicited a premature CMMC. However, similar trains of stimuli delivered to the proximal colon were without similar effects (33 trials, n = 13). It is suggested that in isolated whole mouse colon, CMMCs can be evoked prematurely by trains of electrical stimuli applied to the enteric nerves. The observation that nerve stimuli failed to evoke a premature CMMC from the proximal colon suggests that selective activation of functional ascending pathways may be required to initiate a premature CMMC.

Endogenous nitric oxide release modulates the direction and frequency of colonic migrating motor complexes in the isolated mouse colon.

Spontaneous colonic migrating motor complexes (CMMCs) were recorded from circular muscle at three sites along the isolated mouse colon. The interval between CMMCs was decreased from approximately 3 min in control solution, by approximately 55% in a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (L-NNA; 100 micromol L-1). This was associated with a shift in migration direction of CMMCs, such that CMMCs migrated in an oral direction. Application of the endogenous substrate for NOS, L-arginine, at a low concentration used to mimic plasma concentration (134 micromol L-1), or a high concentration (5 mmol L-1) suppressed CMMCs (for at least 15 min) which were replaced by high frequency (10-15 min-1), short duration (half width approximately 1.5 s) contractions of variable amplitudes (largest in the proximal region) until CMMCs resumed. CMMCs remained in the presence of D-arginine (134 micromol L-1 and 5 mmol L-1). Apamin (250 nmol L-1) did not alter the interval between CMMCs, however, additional nonmigrating contractions were observed between the CMMCs in the distal region. In addition to its effects on smooth muscle tone, NO, but not apamin-sensitive channels, plays an important role in suppressing the frequency of migrating contractions in the isolated mouse colon. Consideration should be given to the inclusion of L-arginine, in in vitro experiments where there may be spontaneous activity in NOS containing neurones.

Murine intestinal migrating motor complexes: longitudinal components.

Spontaneous migrating contractions have been described in the circular muscle of the isolated mouse colon and terminal ileum, however, spontaneous events equivalent to these have not been reported in the longitudinal muscle. The longitudinal muscle shortenings in the colon and ileum, which are of similar form, frequency and pharmacology to the circular muscle colonic and ileal migrating motor complexes (CMMCs and IMMCs), are recorded in the present study. The spontaneous ileal and colonic longitudinal muscle shortenings appear to be neurally organized as they are abolished by tetrodotoxin (1 micro mol L-1), hexamethonium (500 micro mol L-1) and morphine (1 micro mol L-1). Endogenously released nitric oxide slowed the frequency of spontaneous ileal and colonic longitudinal muscle shortenings and 5-hydroxytryptamine increased their frequency. Hyoscine (1 micro mol L-1) abolished longitudinal shortenings in the ileum and reduced the amplitude of longitudinal shortening by approximately 44% in the colon. Shortenings were effectively abolished by nifedipine (1 micro mol L-1). Surgical sectioning of the colon identified that each region of the colon contracted longitudinally in an independent fashion; the distal colon contracted to the greatest amplitude and lowest frequency. The longitudinal preparation is suitable to initially assess the actions of novel pharmacological agents on spontaneous, neurally coordinated, CMMCs and IMMCs in emptied isolated murine intestines.

Colonic migrating motor complexes (CMMCs) in the isolated mouse colon.

Spontaneous contractions were recorded from the circular muscle layer at three sites along the isolated mouse colon. The interval between contractions was approximately 4.5 min. The mean duration of the contractions ranged from 26 sec in the distal colon to 45 sec in the proximal colon. Contractions migrating more than half the length of the colon were termed colonic migrating motor complexes (CMMCs). Over 90% of tissues demonstrated migration predominantly in an aboral direction. Hyoscine (10(-6) M) decreased the amplitude of the CMMCs by at least 40% but had no significant effect on the interval or duration of the CMMCs. Nifedipine (10(-6) M) significantly decreased the amplitude of the CMMCs by 95% but did not alter the duration or the interval between the CMMCs. Hexamethonium (5 x 10(-4) M) and tetrodotoxin (TTX; 2 x 10(-6) M) abolished all CMMC activity. TTX increased the resting tone of the preparations. Nitro-L-arginine (10(-4) M) increased the resting tone of the preparations and significantly decreased the interval between the CMMCs by approximately 80% but had no significant effect on the duration of the CMMCs. The results suggest CMMCs migrate predominantly in an aboral direction and are neurogenic in origin. Nitric oxide may be involved in maintaining inhibition of the muscle between CMMCs.

Motility in the isolated mouse colon: migrating motor complexes, myoelectric complexes and pressure waves.

This study has used mechanical, together with pressure/volume recordings or electrophysiological recordings, to investigate the spontaneous activity in isolated preparations of mouse colon. In the former preparations, when not distended with fluid, spontaneous colonic migrating motor complexes (CMMCs) were observed using isotonic transducers. When the colons were distended with fluid, CMMCs continued at an increased frequency and in addition were associated temporally, with rises in intraluminal pressure and pulses of distally ejected fluid. 5-Hydroxytryptamine (1 micro mol L-1) or NG-nitro-l-arginine (100 micro mol L-1) increased the frequency of propulsive activity and this activity was abolished by hexamethonium (500 micro mol L-1). In a second preparation, myoelectric complexes recorded from circular muscle cells in colons using intracellular microelectrodes, were found to correlate in frequency and phase with CMMCs. The experiments indicate that CMMCs are intimately related to pressure waves in the fluid-filled viscus and the muscle membrane potential changes that have been recorded during myoelectric complexes are likely to be analogous to those occurring during fluid-filled propulsive activity.

Effects of sympathetic nerve stimulation on membrane potential in the circular muscle layer of mouse distal colon.

Little is known about the effects of sympathetic nerve stimulation on the membrane potential of colonic smooth muscle. In the distal colon of the mouse, intracellular microelectrodes were used to record the effects of lumbar colonic (LCN) and intermesenteric nerve (IMN) stimulation on circular muscle membrane potential in vitro. A two-compartment organ bath was used to selectively perfuse the colon and inferior mesenteric ganglion (IMG). In the presence of nifedipine (1-2 microM) (to the colonic compartment only), spontaneous depolarizations (myoelectric complexes, MCs) were recorded about every 4 min. MCs consisted of a prolonged burst of rapid oscillations in membrane potential (approximately 2 Hz) that were superimposed on a slow depolarization (mean amplitude 12 mV). Single electrical stimuli (600 microseconds duration) delivered to the LCN or IMN did not elicit a detectable change in the membrane potential. However, trains of stimuli (e.g., 60 pulses at 10-20 Hz) to the LCN or IMN during the intervals between MCs evoked a depolarization (with superimposed action potentials in the absence of nifedipine). Trains of stimuli delivered during the plateau phase of the MC reduced or abolished the rapid oscillations, without a further membrane depolarization. The MC period was unaffected by stimulation of the IMN or LCN. Responses were abolished by the selective perfusion of guanethidine (1 microM) to the colon, or by severing the LCN. Hexamethonium (500 microM) (to the colon) abolished MCs, induced sustained depolarization and attenuated the amplitude of the sympathetic depolarizations by 74%. In hexamethonium, sympathetic responses remained attenuated when the membrane of the circular muscle was repolarised by sodium nitroprusside (1 microM). Immunohistochemical studies of the colon revealed intense immunoreactivity for tyrosine hydroxylase in the myenteric plexus but not in the circular muscle layer. It is suggested that responses to sympathetic nerve stimulation in the circular muscle layer of the mouse colon are secondary to actions on the enteric nervous system.

Neural integrity is essential for the propagation of colonic migrating motor complexes in the mouse.

The mechanisms that underlie the propagation of contractions along the colon are uncertain. We have examined whether spontaneous colonic migrating motor complexes (CMMCs) migrate through a region of muscle paralysis, or through a region where neural transmission was disrupted in the isolated mouse colon. Mouse colon was mounted in a separately perfused three-compartment organ bath and recordings of circular muscle tension were made. Drug application was restricted to the middle compartment. Application of nifedipine (1 micromol L(-1)), an l-type calcium channel antagonist, reduced the contraction amplitude by approximately 94%, without affecting the form of contractions in the proximal and distal compartments. Moreover, CMMCs appeared to remain temporally related in all compartments. In contrast, interruption of neural transmission in the middle compartment by either tetrodotoxin (1.6 micromol L(-1)), hexamethonium (500 micromol L(-1)) or a low-calcium, high-magnesium solution abolished CMMCs in this compartment; contractions recorded in the proximal and distal compartments became slower in frequency and were no longer synchronized. The experiments suggest that there may be more than one 'pacemaker' generating spontaneous CMMCs and that CMMCs can migrate through a region where there is minimal tension generation, but not through a region where neural integrity has been compromised.

Antagonism of non-cholinergic excitatory junction potentials in the guinea-pig ileum by a substance P analogue antagonist.

In the presence of atropine electrical transmural stimulation (using repetitive volleys, e.g. 3 pulses at 50 Hz applied every 4 s) of full thickness longitudinal strips of guinea-pig ileum produced non-cholinergic excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs) in the circular muscle layer. After abolition of the IJPs with apamin, the non-cholinergic EJPs clearly showed facilitation. In the presence of apamin and the substance P analogue antagonist, [D-Arg1,D-Pro2,D-Trp7-9,Leu11]-substance P (SPA), the non-cholinergic EJPs were reduced by 60-90%; transmural stimulation now revealed an apamin-resistant IJP followed by a slow depolarization. The atropine-resistant EJPs are probably caused by the release of substance P (or a similar compound) and are likely to underlie the non-cholinergic contractions reported to occur in this tissue.

Evidence that myoelectric complexes in the isolated mouse colon may not be of myogenic origin.

The hypothesis that spontaneous depolarisations (myoelectric complexes, MCs) can occur in the absence of neuronal activity, depending on the level of the membrane potential, was systematically studied. In control Krebs' solution, MCs were recorded approximately every 5 min and were abolished by tetrodotoxin (TTX, 1.6 microM). However, TTX also induced sustained membrane depolarisation (19 mV) in the circular muscle. To test whether MCs were blocked by the depolarisation induced by TTX, graded membrane repolarisations were generated, in the continuing presence of TTX, using sodium nitroprusside (SNP, 10 nM-1 microM). Under these conditions, MC activity was not restored. The addition of SNP (1 microM) to control preparations, in normal Krebs' solution, hyperpolarised the membrane of the circular muscle cells, but did not inhibit ongoing MC activity. It is suggested that the underlying mechanisms involved in MC generation are unlikely to be dependent upon the level of membrane potential in circular smooth muscle.

Activity following colonic distension in enteric sensory fibres projecting to the inferior mesenteric ganglion in the guinea pig.

In this investigation the characteristics of the response to colonic distension of afferent fibres projecting to the inferior mesenteric ganglion (IMG) of the guinea pig were studied in vitro. Intracellular membrane potential recordings were made from neurons in the IMG. Post-synaptic potentials arising from activity in afferent fibres were recorded in these cells following distensions of a small segment of colon placed in a separate and independently perfused organ bath. These afferent fibres showed both transient and sustained responses to distension. Application of a solution containing 0.25 mM Ca2+ and 10 mM2+ Mg2+ to the colon (but not to the IMG) reduced the overall response but did not abolish the activity in these fibres. It is concluded that some afferent fibres which respond to colonic distension project directly to the IMG.

Intrinsic control of the gut.

In most mammals (except ruminants) activity in the gastrointestinal (GI) tract depends upon the condition or state of the animal, namely, fasted or fed. The fasted state is characterized by a caudally migrating, cycling motor complex, showing periods of intense contractile and secretory activity alternating with periods of quiescence. Although the mechanisms involved in the transition from the fasted to the fed state are not fully understood it seems likely that both states utilize intrinsically located neural control mechanisms and common neuronal pathways to the effector tissues. We have commented on the reported properties of the myenteric neurones and their projections to the muscle layers. The data suggests that there are both cholinergic and non-cholinergic excitatory motor neurones supplying the muscle layers. In the guinea-pig, at least, the projections of the neurones to the circular muscle layer run for relatively short distances in oral-aboral axis of the gut. The non-cholinergic excitatory transmitter substance may be Substance P or a similar tachykinin. Other excitatory nerves may well be present. There are at least two mechanisms used by non-cholinergic non-adrenergic inhibitory nerves supplying the muscle layers. In the guinea-pig ileum, there are at least two distinct projections of inhibitory motor neurones; both have aborally directed projections. The first of these is relatively short and the other long (greater than 10 mm). Individual myenteric neurones appear to contain unique and perhaps identifying groups of peptides. The functional role of many of these peptides, either within the myenteric plexus or their projections to the muscle layers, remains to be elucidated. The projections of the neurones of the submucous plexus run primarily to the mucosa. Both cholinergic and non-cholinergic secretomotor neurones appear to be present. The activation of local neural reflexes, which results in secretomotor activity, may involve submucous sensory neurones containing acetylcholine and Substance P together with cholinergic interneurones. Projections from the myenteric to the submucous plexus are likely to be involved in the coordination of intestinal movement and secretomotor activity. A simplified schematic diagram of some of the neuronal circuitry of the submucous plexus has been developed and includes the findings from immunocytochemical and electrophysiological studies.

Non-cholinergic fast and slow post-stimulus depolarization in the guinea-pig ileum.

Intracellular membrane potential recordings were made from circular muscle cells in the guinea-pig ileum in vitro at 30 degrees C in the presence of atropine (1.4 microM). Following transmural nerve stimulation the inhibitory junction potential (i.j.p.) was followed by a post-stimulus depolarization (p.s.d.). Both were abolished by tetrodotoxin. P.s.d. consisted of two phases; (i) an early fast phase (f.p.s.d.) which peaked about 1 s following the stimulus and (ii) a late slow phase (s.p.s.d.) which had a latency of 1-2 s and peaked approximately 4 s following the stimulus. Both f.p.s.d. and s.p.s.d. were observed when the membrane was hyperpolarized by anodal current to the reversal potential of the i.j.p. F.p.s.d. and s.p.s.d. were therefore unlikely to result solely from a decrease in membrane conductance to potassium ions. During f.p.s.d., hyperpolarizing electronic potentials were reduced in amplitude suggesting that f.p.s.d. was associated with an increase in membrane conductance. There were no consistent changes in the amplitude of the electronic potentials during s.p.s.d. When the membrane potential was hyperpolarized by prolonged anodal current, an additional relatively rapid depolarizing potential could be induced by either transmural stimulation or depolarizing electrotonic current pulses.

The passive membrane properties and excitatory junction potentials of the guinea pig deferens.

1. Electrotonic potentials were recorded from the superficial smooth muscle cells of the guinea-pig vas deferens using the method of Abe & Tomita (1968), in response to low-amplitude, long-duration (greater than or equal to 2 sec) pulses. 2. Averaging techniques were used to increase the signal/noise ratio, and the intracellularly recorded electrotonic potentials were corrected for extracellular voltage drop across the bath series resistance. 3. Since the length of tissue in the stimulating and recording compartments affects the time course of electrotonic potentials (see Appendix and Bywater & Redman, 1978) the passive membrane properties were measured with known amounts of tissue in these two compartments. 4. The length constant (lambda) was 0.86 mm and the membrane time constant (tau m) 270 msec. 5. Excitatory junction potentials (e.j.p.s) were recorded and averaged in response to field stimulation of intact branches of the hypogastric nerve. The mean time constant of the exponential decay phase of the e.j.p. (288 msec) was similar to the membrane time constant (tau m = 270 msec). 6. As the e.j.p.s showed little change in amplitude or time constant of decay when recorded up to several millimetres from the stimulating electrode it was assumed that the tissue was isopotential during the e.j.p., and an estimate was made of the time course of the underlying junctional current. 7. The estimated time course of the junctional current during an e.j.p. was similar to the observed time course of a spontaneous junction potential (s.e.j.p.). 8. As the time course of the junctional current during an s.e.j.p.is similar to the time course of the potential change it is likely that the factors which determine the time current underlying the s.e.j.p. also determine the time course of the e.j.p. current.

Non-cholinergic excitatory and inhibitory junction potentials in the circular smooth muscle of the guinea-pig ileum.

Intracellular micro-electrodes have been used to record evoked non-cholinergic junction potentials from the muscle layers of strips of guinea-pig ileum cut parallel to the longitudinal muscle. Transmural stimulation with single pulses, 0.6 ms duration produced inhibitory junction potentials (i.j.p.s) in the circular muscle layer. In the circular muscle layer, transmural stimulation with repetitive volleys (e.g. three pulses, 0.6 ms, 50 Hz delivered every 4 s) at low stimulus strengths (25-40 mA) produced non-cholinergic excitatory junction potentials (e.j.p.s) after an initial i.j.p. At higher stimulus strengths (greater than 40 mA) i.j.p.s occurred following each volley but were superimposed on a prolonged depolarization. Following repetitive volley stimulation every four seconds in the presence of apamin (0.25 microM), the i.j.p.s. were abolished and the non-cholinergic e.j.p.s clearly showed facilitation. At higher stimulus strengths (and at volley repetition rates of less than 0.1 Hz) volley stimulation now produced an apamin-resistant slow hyperpolarization followed by a distinct slow depolarization. Electrotonic potentials were readily recorded from the superficially located longitudinal muscle cells: in this muscle layer transmural stimulation produced small, slow changes in membrane potential. These results suggest that non-cholinergic excitatory and inhibitory nerve fibres primarily supply the circular rather than the longitudinal muscle layer in the guinea-pig ileum.

Neurogenic slow depolarizations and rapid oscillations in the membrane potential of circular muscle of mouse colon.

1. Intracellular microelectrodes have been used to record the electrical activity of smooth muscle cells of the circular layer from full length strips of mouse colon in vitro. The membrane potential was unstable and showed slow depolarizations (mean amplitude, 10.9 mV; mean frequency, 0.008 Hz; mean duration, 56.4 s). 2. A variable number (mean fifty-six) of rapid oscillations in membrane potential (mean amplitude, 10.2 mV) with a frequency of approximately 2 Hz and a duration of approximately 400 ms were superimposed on each slow depolarization. Occasionally, action potentials arose from the rapid oscillations. The action potentials, but neither the slow depolarizations nor the rapid oscillations, were abolished by 1.0 microM-nifedipine. 3. The majority of the slow depolarizations and the associated rapid oscillations migrated aborally along the colon at a velocity of between 0.5 and 1.5 mm s-1; in the distal colon the slow depolarization was often preceded by a small hyperpolarization. 4. During the rising and plateau phase of the slow depolarization the amplitude of electronic potentials was decreased. Hyperpolarization induced by passing current during the slow depolarization increased the amplitude of the rapid oscillations. 5. Transmural electrical stimulation (single pulses) in the presence of nifedipine evoked (1 mm anal to the stimulating electrodes) an inhibitory junction potential which was sometimes preceded by an excitatory junction potential. The amplitude, of the evoked inhibitory junction potential was decreased during the rising and plateau phase of the slow depolarization. 6. The slow depolarization and the rapid oscillations were abolished by hexamethonium (500 microM), morphine (1-10 microM) and tetrodotoxin (3.1 microM). Atropine (3.5 microM) abolished the rapid oscillations and reduced the amplitude of the slow depolarization. 7. Atropine (3.5 microM) and morphine (10 microM) abolished the evoked excitatory junction potential whilst tetrodotoxin (3.1 microM) abolished both the excitatory and the inhibitory junction potential. 8. It is suggested that the migrating depolarization and accompanying oscillations, which are neurogenic in origin, represent the electrical correlate in the circular muscle layer of the migrating colonic motor complex which has been associated with the propulsion of faecal pellets along the colon.

Nerve-mediated contractile and electrical activity of the guinea-pig choledocho-duodenal junction.

The intrinsic motor innervation of the guinea-pig choledocho-duodenal junction was investigated by recording the contractile and intracellular electrical activity of smooth muscle from different regions of this tissue. Electrical transmural nerve stimulation evoked phasic contractions in rings of muscle from the ampulla (0.45 s-1) and tonic contractions in rings of muscle from the choledochal sphincter. Intracellular microelectrode recordings from muscle strips from these two regions revealed that excitatory junction potentials (peak amplitude 7 mV) evoked by transmural nerve stimulation were more conspicuous in muscle strips from the choledochal sphincter, but inhibitory junction potentials (peak amplitude 13 mV) were of larger amplitude in muscle strips from the ampulla. Contractions and membrane depolarization evoked by transmural nerve stimulation were sensitive to 1.4 microM atropine and abolished by 3.1 microM tetrodotoxin. Histological studies on the choledocho-duodenal junction also revealed that the distribution of smooth muscle was non-uniform along the tissue. These results suggest that the two regions may have different functions in the motility of the choledocho-duodenal junction.

Atropine-resistant depolarization in the guinea-pig small intestine.

1. Junction potentials were recorded from the circular muscle cells of the guinea-pig ileum following transmural stimulation in the presence of atropine at 30 degrees C.2. Single stimuli produced a transient hyperpolarization, the inhibitory junction potential (i.j.p.). At high stimulus strengths the i.j.p. was followed by a post-stimulus depolarization (PSD).3. During repetitive stimulation the magnitude of the hyperpolarization decreased; however, at the end of the stimulus period the PSD was enhanced and often reached threshold for the generation of action potentials. Thus, the size of the PSD was not directly related to the degree of the preceding hyperpolarization.4. Hyperpolarization of the circular muscle cells was produced by the application of anodal current using large external electrodes. Rapid cessation of the applied current produced a transient after-depolarization which was shorter in time course than the PSD following the i.j.p. If the applied anodal current was reduced slowly (at a rate which mimicked the decrease in the hyperpolarization during repetitive nerve stimulation) no after-depolarization was observed.5. Conditioning hyperpolarization of the circular muscle cells reduced the amplitude of the i.j.p. The i.j.p. was reversed at membrane potentials greater than approximately -90 mV.6. The PSD did not appear to be due to the extracellular accumulation of potassium ions following the i.j.p. since the PSD persisted even when the i.j.p. was reversed.7. The neurotoxin apamin reversibly abolished the i.j.p. and unmasked a transient excitatory junction potential (e.j.p.) with a variable latency (350-900 ms).

Effect of substance P on non-cholinergic fast and slow post-stimulus depolarization in the guinea-pig ileum.

Membrane potentials were recorded with intracellular electrodes from the circular muscle cells of the guinea-pig ileum, in vitro, in the presence of atropine (1.4 X 10(-6) M) at 30 degrees C. Under such conditions, the preparations did not show any spontaneous activity. Exposure of the tissue to substance P (10(-7)-10(-6)M) for a short period of time (less than 5 min) caused depolarization and action potentials. At this time the membrane resistance appeared to be decreased and after transmural nerve stimulation the fast post-stimulus depolarization, which followed the inhibitory junction potential (IJP), triggered action potentials. After prolonged exposure of the tissue to substance P (greater than 10 min), the contractile activity of the preparation subsided, the membrane potential was decreased by 6.4 +/- 1.1 mV and the membrane resistance was increased; following transmural nerve stimulation, the amplitude of the IJP was increased, the fast post-stimulus depolarization was abolished and the later slow post-stimulus depolarization was enhanced and in some preparations reached threshold for action potential generation. In the latter preparations in the absence of transmural nerve stimulation, the membrane potential occasionally showed regular oscillations of approximately 10 mV in amplitude every 5-8 s. When the tissue was hyperpolarized by anodal current, after prolonged exposure to substance P, the fast post-stimulus depolarization did not return indicating that its abolition was not a secondary effect due to the depolarization of the tissue by substance P.