RESUMEN
Intracellular recordings were made from either sheets or isolated bundles of the circular muscle layer of guinea-pig proximal colon and the responses evoked by stimulating inhibitory nerve fibres were analysed. Inhibitory junction potentials (IJPs), evoked by single stimuli, had two components which could be separated on their pharmacological and temporal characteristics and their voltage sensitivities. The initial component, which was abolished by apamin and reduced in amplitude by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), had a brief time course: its amplitude was changed when the external concentration of potassium ions ([K+](o)) was changed. The second component of the IJP had a slower onset than the first component, was abolished by l-nitroarginine (NOLA) and oxadiazolo quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase: its amplitude was little affected by changing [K+](o) and was increased when the membrane potential of the circular layer was hyperpolarized. The observations suggest that the initial component of the IJP results from the release of ATP which triggers an increase in membrane conductance to K+ and that the second component results from the release of nitric oxide which suppresses a background inward current.
Asunto(s)
Colon/fisiología , Inhibición Neural/fisiología , Animales , Apamina/farmacología , Colon/efectos de los fármacos , Femenino , Cobayas , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Inhibición Neural/efectos de los fármacosRESUMEN
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.
Asunto(s)
Motilidad Gastrointestinal/fisiología , Intestinos/fisiología , Contracción Muscular/fisiología , Músculo Liso/fisiología , Complejo Mioeléctrico Migratorio/fisiología , Anestésicos Locales/farmacología , Animales , Depuradores de Radicales Libres/farmacología , Bloqueadores Ganglionares/farmacología , Hexametonio/farmacología , Intestinos/efectos de los fármacos , Ratones , Morfina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Complejo Mioeléctrico Migratorio/efectos de los fármacos , Nifedipino/farmacología , Óxido Nítrico/metabolismo , Técnicas de Cultivo de Órganos , Serotonina/farmacología , Tetrodotoxina/farmacología , Vasodilatadores/farmacologíaRESUMEN
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.
Asunto(s)
Colon/fisiología , Motilidad Gastrointestinal/fisiología , Complejo Mioeléctrico Migratorio/fisiología , Animales , Colon/efectos de los fármacos , Electrofisiología , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Masculino , Ratones , Microelectrodos , Músculo Liso/fisiología , Complejo Mioeléctrico Migratorio/efectos de los fármacos , Técnicas de Cultivo de Órganos , Estimulación Física , PresiónRESUMEN
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.
Asunto(s)
Colon/fisiología , Sistema Nervioso Entérico/fisiología , Motilidad Gastrointestinal/fisiología , Complejo Mioeléctrico Migratorio/fisiología , Anestésicos Locales/farmacología , Animales , Colon/efectos de los fármacos , Estimulación Eléctrica , Sistema Nervioso Entérico/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Ratones , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Complejo Mioeléctrico Migratorio/efectos de los fármacos , Técnicas de Cultivo de Órganos , Tetrodotoxina/farmacologíaRESUMEN
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.
Asunto(s)
Colon/fisiología , Complejo Mioeléctrico Migratorio/fisiología , Transmisión Sináptica/fisiología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Colon/efectos de los fármacos , Técnicas In Vitro , Ratones , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Complejo Mioeléctrico Migratorio/efectos de los fármacos , Nifedipino/farmacología , Transmisión Sináptica/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
1. Intracellular microelectrodes were used to record spontaneous and evoked inhibitory junction potentials (IJP) from the circular muscle layer of the mid-distal region of the mouse isolated colon in the presence of nifedipine (1 micromol/L) and hyoscine (1 micromol/L). 2. The length of the tissue preparation (> 1 cm) or the presence of the mucosa had no effect on the frequency of spontaneous IJP. 3. Hexamethonium (500 micromol/L) reduced the frequency of spontaneous IJP to approximately 70% of the control frequency, whereas D-tubocurarine (280 micromol/L) reduced the frequency to approximately 17% of control. Apamin (250 nmol/L) abolished all spontaneous IJP activity. 4. The greater inhibition of spontaneous IJP in the presence of D-tubocurarine compared with hexamethonium is discussed as a possible 'apamin-like' effect. 5. Although electrically evoked IJP (single pulse at 15 V, 0.6 msec) were not significantly affected by hexamethonium, D-tubocurarine and apamin reduced the amplitude of evoked IJP to approximately 65 and 50% of control, respectively. 6. These results suggest that the properties of spontaneous IJP cannot be inferred by a study of evoked IJP alone.
Asunto(s)
Colon/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Antagonistas Nicotínicos/farmacología , Animales , Colon/fisiología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Femenino , Hexametonio/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Mutantes , Inhibición Neural/fisiología , Neuronas/fisiología , Receptores Nicotínicos/fisiología , Tubocurarina/farmacologíaRESUMEN
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.
Asunto(s)
Colon/fisiología , Motilidad Gastrointestinal/fisiología , Óxido Nítrico/metabolismo , Animales , Apamina/farmacología , Arginina/farmacología , Colon/inervación , Electrofisiología , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/fisiología , Inhibidores Enzimáticos/farmacología , Motilidad Gastrointestinal/efectos de los fármacos , Técnicas In Vitro , Ratones , Ratones Mutantes , Neuronas Motoras/fisiología , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/farmacologíaRESUMEN
The effects of 5-hydroxytryptamine (5-HT) and related drugs on colonic migrating motor complexes (CMMCs) were evaluated in isolated colons from the heterozygotes of pie-bald lethal mice. 5-HT produced a dose-related increase in the frequency of CMMCs without any change in the amplitude or duration of the CMMC contractions themselves. The 5-HT(2) agonist, alpha-methyl 5-HT, (100 nM-1 microM) increased the frequency of CMMCs whilst the 5-HT(3) agonist, 2-methyl 5-HT, did so at 10 microM. The 5-HT(4) agonist, 5-methoxy dimethyl tryptamine oxalate did not alter the frequency of CMMCs in the concentration range 1 nM-10 microM. The 5-HT(3) receptor antagonist, ondansetron, increased the interval between CMMCs in the concentration range 100 nM-1 microM, whilst the 5-HT(1) receptor antagonist, methiothepin, the 5-HT(2) receptor antagonist, cyproheptadine and the 5-HT(4) receptor antagonist, SDZ 205 557, had no significant effects on the interval between CMMCs in the concentration range 1 nM-10 microM. The effects of 5-HT did not appear to be altered by the presence of ondansetron (1 microM) or cyproheptadine (1 microM). However, in the presence of ondansetron (1 microM), the further addition of cyproheptadine (1 microM) effectively abolished CMMCs. Furthermore, in the combined presence of these antagonists the effects of 5-HT were severely diminished. It is suggested that the frequency of CMMCs may be under the influence of endogenously released 5-HT in this preparation
Asunto(s)
Colon/inervación , Motilidad Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/fisiología , Neuronas Motoras/efectos de los fármacos , Agonistas de Receptores de Serotonina/farmacología , Serotonina/análogos & derivados , Ácido 4-Aminobenzoico/farmacología , Animales , Ciproheptadina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Técnicas In Vitro , Masculino , Metiotepina/farmacología , Metoxidimetiltriptaminas/farmacología , Ratones , Ratones Mutantes , Neuronas Motoras/fisiología , Ondansetrón/farmacología , Serotonina/farmacología , Antagonistas de la Serotonina/farmacología , para-AminobenzoatosRESUMEN
1. Movements of the gastrointestinal tract are required for the digestion of food and the expulsion of waste products. 2. The present paper will discuss the nature of electrical rhythms underlying some intestinal motility patterns. 3. The rhythms are generated by pacemakers with cycle rates appropriate to controlling individual contractions, motor patternings or switching between different motor programmes. 4. Electrical rhythms are discussed with periods of the order of seconds, minutes and hours. 5. Particular discussion is centred on rhythms recorded from the small and large intestine of the mouse.
Asunto(s)
Ritmo Circadiano , Intestinos/fisiología , Peristaltismo/fisiología , Animales , Electrofisiología , Ratones , Contracción Muscular/fisiología , Factores de TiempoRESUMEN
Intracellular electrophysiological techniques were used to record the spontaneous myoelectric activity in the circular muscle layer of an in vitro preparation of whole mouse colon. In 34 out of 58 preparations, spontaneous depolarisations (myoelectric complexes, MCs) were recorded cyclically, about every 4 min. In these preparations, apamin (250 nM) and NG-nitro-L-arginine (NOLA, 100 microM) depolarised the membrane potential between MCs by 8 mV or 13 mV, respectively. Tetrodotoxin (1.6 microM) abolished MCs and also induced depolarisation (17 mV). In the remaining 24 preparations, MCs were not recorded and the membrane potential was significantly depolarised compared to the membrane potential between MCs. NOLA (100 microM), apamin (250 nM) and tetrodotoxin (1.6 microM) were without significant effect on membrane potential. It is suggested that in preparations that exhibit MC cycling, membrane potential between MCs is maintained in a state of tonic inhibition, predominantly mediated by nitrergic mechanisms generated via spontaneously active inhibitory neurons. Apamin-sensitive channels may also be involved in the inhibition.
Asunto(s)
Colon/inervación , Transmisión Sináptica/efectos de los fármacos , Animales , Apamina/farmacología , Colon/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Óxido Nítrico Sintasa/antagonistas & inhibidores , Tetrodotoxina/farmacologíaRESUMEN
Intracellular microelectrodes were used to record spontaneous and evoked inhibitory junction potentials (IJPs) from the circular muscle layer of an in vitro preparation of whole mouse colon. Membrane potential recordings were made from cells of the mid to distal region of colon at 36 +/- 1 degrees C in a modified Krebs' solution that contained atropine (1 microM) and nifedipine (1-2 microM). Spontaneously occurring hyperpolarisations of irregular amplitude and frequency (range: up to 20 mV and 2 Hz) were recorded that were resistant to N(G)-nitro-L-arginine (NOLA, 100 microM), but were abolished by tetrodotoxin (TTX, 1.6 microM) or apamin (250 nM). These were considered to be spontaneous IJPs as a consequence of activity in inhibitory motor neurons. Single electrical stimuli (0.6 ms, 15 V), elicited a fast IJP, whose time course could be superimposed on spontaneous IJPs of similar amplitude. The amplitude of evoked IJPs was not depressed by NOLA (100 microM). However, in NOLA (100 microM), further addition of apamin (250 nM) significantly depressed the amplitudes of the evoked IJPs by 44%. NOLA- and apamin-resistant evoked IJPs were abolished by TTX (1.6 microM). It is suggested, that in the circular muscle layer of mouse colon, NO does not mediate the fast hyperpolarisations associated with spontaneous or evoked IJPs. Apamin abolished spontaneous IJPs, but electrical stimuli evoked an IJP with apamin-sensitive and resistant components both of which were non-nitrergic in origin.
Asunto(s)
Colon/fisiología , Músculo Liso/fisiología , Inhibición Neural/fisiología , Animales , Apamina/farmacología , Colon/efectos de los fármacos , Colon/inervación , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Neuronas/fisiología , Nitroarginina/farmacología , Tetrodotoxina/farmacologíaRESUMEN
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.
Asunto(s)
Colon/fisiología , Complejo Mioeléctrico Migratorio/fisiología , Animales , Colon/citología , Colon/efectos de los fármacos , Femenino , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Mutantes , Complejo Mioeléctrico Migratorio/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
1. The effects of noradrenaline and isoprenaline on the Ca2+i-insensitive, voltage-activated K+ current in smooth muscle cells from the circular muscle layer of the guinea pig proximal colon were investigated by using standard whole-cell patch-clamp techniques at room temperature (22-24 degrees C). 2. The Ca2+-activated K+ current was eliminated by bathing cells in tetraethylammonium (TEA;2-5 mM) and a Ca2+-entry blocker (Cd2+, 0.1 mM) or nifedipine, 2-10 microM) and by internally perfusing cells with 3 mM EGTA. 3. Two Ca2+i-insensitive, voltage-activated K+ currents were recorded at potentials positive to -50 mV: (a) a transient K+ current (IKto) that was blocked by 4-aminopyridine (5 mM) and (b) a delayed rectifier-type K+ current (IKdel) that was blocked by TEA (>10 mM). 4. Both noradrenaline (10-50 microM) and isoprenaline (5-50 microM) reduced the amplitudes of IKto and IKdel irreversibly after a slow onset (2-5 min). This reduction was mimicked by forskolin (50-100 microM) and by 8 bromo-c-AMP (500 microM). 5. The voltage of half-maximal availability (V0.5) of IKto (-74.6+/-2.3 mV) was unaffected by isoprenaline (10 microM) (-76.7+/-3.6 mV, n=4), but the background "leak" current (Ileak) was increased from -48+/-9 to -70+/-20 pA. 6. Our data suggest that stimulation of beta-adrenoceptors in the circular muscle layer of the guinea pig proximal colon inhibits voltage-activated Ca2+i-insensitive K+ currents.
Asunto(s)
Agonistas Adrenérgicos/farmacología , Músculo Liso/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Colforsina/farmacología , Colon/citología , Colon/efectos de los fármacos , Cobayas , Técnicas In Vitro , Isoproterenol/farmacología , Potenciales de la Membrana/efectos de los fármacos , Músculo Liso/citología , Técnicas de Placa-ClampRESUMEN
Intracellular microelectrodes were used to record electrically evoked inhibitory junction potentials (IJPs) and electrotonic potentials during spontaneous cyclical depolarisations (myoelectric complexes, MCs) in the circular muscle layer of mouse colon in vitro. In the presence of nifedipine (1-2 microM) and atropine (1 microM), MCs were recorded every 264 +/- 18 s. Between MCs, single electrical stimuli (15 V, 0.6 ms, every 8 s) elicited IJPs whose amplitudes remained constant. In comparison, during the depolarising phase of MCs, the mean IJP amplitude was reduced by 61 +/- 7%, while during the late plateau and early repolarising phase of MCs, IJP amplitude was increased (up to 20%). NG-nitro-L-arginine (NOLA, 100 microM) abolished the repolarisation phase between MCs, so that the circular muscle remained depolarised and the amplitude of MCs was reduced by 73 +/- 6%. However, the amplitude of evoked IJPs was unaffected, as was the decrease in their amplitude during the depolarising phase of the residual MCs. In the presence of NOLA (100 microM), the further addition of apamin (250 nM) reduced the amplitude of evoked IJPs by approximately half. However, the amplitudes of NOLA- and apamin-resistant IJPs were also attenuated by 82 +/- 5% during the depolarising phase of residual MCs (amplitude: 1.9 +/- 1 mV). However, during this phase, the amplitude of an electrotonic potential (evoked by extracellular current application) was not attenuated. Addition of hexamethonium (500 microM), or tetrodotoxin (TTX) (1.6 microM) to solutions containing NOLA and apamin were without effect on membrane potential, but the residual MCs and the cyclical attenuation in IJP amplitude were abolished. During the intervals between MCs, membrane potential is maintained under tonic inhibition, via spontaneous release of inhibitory neurotransmitter(s), predominantly through nitrergic mechanisms. The cyclical attenuation in the amplitude of the non-nitrergic IJP does not arise from cyclical postjunctional changes in membrane resistance or potential. Moreover, the generation of the depolarising phase of MCs involves the simultaneous suppression of both nitrergic and non-nitrergic inhibitory neurotransmission. It is suggested that MCs arise from presynaptic suppression of ongoing inhibitory neurotransmitter release.
Asunto(s)
Colon/inervación , Complejo Mioeléctrico Migratorio/fisiología , Inhibición Neural/fisiología , Animales , Apamina/farmacología , Colon/fisiología , Resistencia a Medicamentos , Estimulación Eléctrica , Electrofisiología , Inhibidores Enzimáticos/farmacología , Potenciales Evocados/efectos de los fármacos , Hexametonio/farmacología , Ratones , Ratones Endogámicos , Músculo Liso/fisiología , Complejo Mioeléctrico Migratorio/efectos de los fármacos , Óxido Nítrico/agonistas , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Nitroprusiato/farmacología , Tetrodotoxina/farmacologíaRESUMEN
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.
Asunto(s)
Colon/inervación , Colon/fisiología , Músculo Liso/fisiología , Sistema Nervioso Simpático/fisiología , Animales , Estimulación Eléctrica/métodos , Femenino , Ganglios Simpáticos/fisiología , Bloqueadores Ganglionares/farmacología , Hexametonio/farmacología , Inmunohistoquímica , Masculino , Potenciales de la Membrana/fisiología , Mesenterio/inervación , Ratones , Ratones Mutantes , Complejo Mioeléctrico Migratorio/efectos de los fármacos , Complejo Mioeléctrico Migratorio/fisiología , Nitroprusiato/farmacologíaRESUMEN
Recent electrophysiological studies of the properties of intestinal reflexes and the neurons that mediate them indicate that the intrinsic sensory neurons may transmit to second order neurons via either fast (30-50 ms duration) or slow (10-60 s duration) excitatory synaptic potentials or both. Which of these possible modes of transmission is involved in the initiation of motility reflexes has not been determined and it is not clear and what the consequences of the different forms of synaptic transmission would be for the properties of the reflex pathways. In the present study, this question has been addressed by the use off a suite of computer programs, Plexus, which was written to simulate the activity of the neurons of the enteric nervous system during intestinal reflexes. The programs construct a simulated enteric nerve circuit based on anatomical and physiological data about the number, functions and interconnections of neurons involved in the control of motility. The membrane potentials of neurons are calculated individually from physiological data about the reversal potentials and membrane conductances for Na+, K+ and Cl-. Synaptic potentials are simulated by changes in specific conductances based on physiological data. The results of each simulation are monitored by recording the membrane potentials of up to 16 separate defined neurons and by recording the summed activity of whole classes of neurons as a function of time and location in the stimulated network. The present series of experiments simulated the behaviour of a network consisting of 18,898 sensory neurons and 3708 ascending interneurons after 75% of the sensory neurons lying in the anal 10 mm of a 30 mm long segment of small intestine were stimulated once. The results were compared with electrophysiological data recorded from myenteric neurons during ascending reflexes evoked either by distension or mechanical stimulation of the mucosa. When transmission from sensory neurons to ascending interneurons was via fast excitatory synaptic potentials, the latencies and durations of the simulated responses were too brief to match the electrophysiologically recorded responses. When transmission from sensory neurons was via slow excitatory synaptic potentials, the latencies were very similar to those recorded physiologically, but the durations of the stimulated responses were much longer than seen in physiological experiments. The latencies and durations of simulated and physiologically recorded responses matched only when the firing of ascending interneurons was limited to the beginning of a slow excitatory synaptic (in this study by limiting the duration of the decrease in K+ conductance). The simulation provided several physiologically testable predictions, indicating that Plexus is an important tool for the investigation of the properties and behaviour of the enteric nervous system.
Asunto(s)
Simulación por Computador , Intestinos/inervación , Modelos Neurológicos , Neuronas Aferentes/fisiología , Reflejo/fisiología , Humanos , Vías Nerviosas/fisiología , Periodo Refractario Electrofisiológico , Factores de TiempoRESUMEN
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.
Asunto(s)
Colon/fisiología , Complejo Mioeléctrico Migratorio/fisiología , Nifedipino/farmacología , Animales , Colon/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hexametonio/farmacología , Técnicas In Vitro , Ratones , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Complejo Mioeléctrico Migratorio/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Escopolamina/farmacología , Tetrodotoxina/farmacologíaRESUMEN
The nature of the electrically- or stretch-evoked nonadrenergic, noncholinergic (NANC) inhibitory junction potentials (IJPs) in circular smooth muscle cells of the guinea-pig proximal and distal colon were investigated using standard intracellular microelectrode recording techniques. We have confirmed that the NANC IJP, recorded in the presence of hyoscine (1 microM) and nifedipine (1 microM), can be divided into two components with apamin (250 nM), a blocker of the small conductance Ca2(+)-activated K+ channels. Both the apamin-sensitive and the apamin-resistant components of the IJP were blocked by tetrodotoxin (1.6 microM) or by lowering the external Ca2+ concentration (to 0.25 mM). The apamin-sensitive IJP was also blocked by omega-conotoxin GVIA (100 nM), a blocker of 'N-type' Ca2+ channels. The apamin-resistant IJP and rebound post-stimulus depolarization (PSD) were reduced upon exposure to either NG-L-arginine (NOLA), an inhibitor of nitric oxide synthase (NOS), or the nitric oxide (NO) scavenger, haemoglobin. The effects of NOLA were partially reversed in the presence of excess L-arginine, a substrate for NOS, suggesting that NO, or a related NO-donor compound, is likely to be the apamin-resistant inhibitory transmitter. Blockade of either the apamin-sensitive or apamin-resistant IJP was associated with membrane depolarization and a decrease in the membrane conductance in the absence of nerve stimulation. In the proximal colon, the apamin-resistant IJP and PSD could both be demonstrated to arise from an increase in the membrane conductance after subtraction of a non-linear background conductance. The hyperpolarization upon repetitive NANC nerve stimulation was mimicked by the NO donor, S-nitroso-L-cysteine (2.5-25 microM), which evoked a transient apamin-sensitive, but omega-conotoxin GVIA resistant, component followed by a slower apamin-resistant component. These results suggest that neurally-released NO has a number of actions in the guinea-pig colon, causing apamin-resistant hyperpolarization and depolarization, as well as directly opening apamin-sensitive K+ channels.
Asunto(s)
Apamina/farmacología , Arginina/farmacología , Colon/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Animales , Estimulación Eléctrica , Femenino , Cobayas , MasculinoRESUMEN
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.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Colon/metabolismo , Músculo Liso/metabolismo , Óxido Nítrico/farmacología , Animales , Calcio/fisiología , Colon/efectos de los fármacos , Colon/ultraestructura , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Cobayas , Hemoglobinas/farmacología , Técnicas In Vitro , Magnesio/fisiología , Potenciales de la Membrana/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/ultraestructura , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Nitroprusiato/farmacología , Técnicas de Placa-Clamp , Penicilamina/análogos & derivados , Penicilamina/antagonistas & inhibidores , Penicilamina/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , S-Nitroso-N-Acetilpenicilamina , Vasodilatadores/antagonistas & inhibidores , Vasodilatadores/farmacologíaRESUMEN
BACKGROUND/AIMS: Little is known about the mechanisms controlling colonic migrating electrical activity. This study investigates the neural processes involved in the generation of migrating myoelectric complexes in the isolated mouse colon. METHODS: Intracellular electrophysiological recordings were obtained from the circular muscle layer of the mouse colon in vitro in the presence of 2 mumol/L nifedipine. RESULTS: Complexes occurred approximately every 3 minutes and consisted of 1 mumol/L hyoscine-sensitive rapid oscillations (approximately 2 Hz) superimposed on a slow depolarization (approximately 17 mV); the latter was often preceded by a precomplex hyperpolarization (approximately 7 mV) that was reduced by 250 nmol/L apamin. Five hundred micromolars of hexamethonium or 2 mumol/L of tetrodotoxin abolished the complexes and depolarized the muscle by 8.7 +/- 1.3 mV (n = 9) or 12.1 +/- 1.4 mV (n = 5), respectively. Carbachol (50 nmol/L to 5 mumol/L) produced dose-dependent depolarizations but without rapid oscillations. The nitric oxide synthase inhibitor NG-nitro-L-arginine (100 mumol/L) depolarized the tissue by 17.2 +/- 1.6 mV (n = 8) but had no effect on the rapid oscillations. In the presence of 2 mumol/L tetrodotoxin, 5 mumol/L sodium nitroprusside produced a sustained hyperpolarization (15.5 +/- 2.0 mV; n = 5) but did not restore complexes. CONCLUSIONS: In the isolated mouse colon, the membrane potential between complexes is maintained by the release of inhibitory neurotransmitters (including nitric oxide), and the formation of complexes involves disinhibition and the simultaneous activation of cholinergic motor nerves.