Interstitial cells of Cajal and slow wave generation in canine colonic circular muscle.
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Animals↗
Biomedical subjects
J D Huizinga
Publications and source records attributed to J D Huizinga.
Inhibitory innervation of colonic smooth muscle cells and interstitial cells of Cajal.
The effect of neural inhibition on the electrical activities of circular and longitudinal colonic smooth muscle was investigated. In addition, a comparative study was carried out between circular muscle preparations with and without the "submucosal" and "myenteric plexus" network of interstitial cells of Cajal (ICC) to study innervation of the "submucosal" ICC and to investigate whether or not the ICC network is an essential intermediary system for inhibitory innervation of smooth muscle cells. Electrical stimulation of intrinsic nerves in the presence of atropine caused inhibitory junction potentials (ijps) throughout the circular and longitudinal muscle layers. The ijp amplitude depended on the membrane potential and not on the location of the muscle cells with respect to the ICC network. Neurally mediated inhibition of the colon resulted in a reduction in amplitude and duration of slow wave type action potentials in circular and abolishment of spike-like action potentials in longitudinal smooth muscle, both resulting in a reduction of contractile activity. With respect to mediation by ICC, the study shows (i) "submucosal" ICC receive direct inhibitory innervation and (ii) circular smooth muscle cells can be directly innervated by inhibitory nerves without ICC as necessary intermediaries. The reversal potential of the ijp in colonic smooth muscle was observed to be approximately -76 mV, close to the estimated potassium equilibrium potential, suggesting that the nerve-mediated hyperpolarization is caused by increased potassium conductance.
Animals↗
Innervation of interstitial cells of Cajal by vasoactive intestinal polypeptide containing nerves in canine colon.
The hypothesis was tested, through structural and functional studies, that interstitial cells of Cajal receive and can respond to direct innervation from nerves containing the vasoactive intestinal polypeptide neuromediator. The submucosal network of interstitial cells of Cajal has been postulated to provide pacemaking activity for the circular muscle and to be involved in neurotransmission from nonadrenergic, noncholinergic nerves for which vasoactive intestinal polypeptide is a putative mediator. The distribution of vasoactive intestinal polypeptide and substance P immunoreactive material in nerve profiles of the enteric nervous system of the canine colon was examined. In addition, electrophysiological studies were done on the interstitial cells bordering the submucosal side of the circular muscle layer after they were electrically isolated using heptanol. The vasoactive intestinal polypeptide immunoreactivity, located exclusively in nerve large granular vesicles, was found throughout the enteric nervous system (myenteric plexus, submucous plexus, and circular muscle--submucosa interface). The highest proportion (38% compared with 22-24%) of profiles of large granular vesicles with vasoactive intestinal polypeptide immunoreactivity was found in nerve profiles of the circular muscle--submucosa interface. In contrast, substance P immunoreactivity was found in nerve profiles of myenteric plexus (33% of large granular vesicles were positive) but not associated with submucosal interstitial cell nerve network. The vasoactive intestinal polypeptide hyperpolarized interstitial cells by 9 mV when electrically isolated by 1 mM heptanol and markedly reduced (about 50%) their input membrane resistance. We conclude that the distribution of vasoactive intestinal polypeptide immunoreactivity and its action are consistent with a postulated role of the interstitial cells as a major site of neurally mediated inhibition of colonic pacemaker activity.
Animals↗
Structural characterization of interstitial cells of Cajal in myenteric plexus and muscle layers of canine colon.
We have carried out a detailed ultrastructural study of the interstitial cells near the myenteric plexus of the canine colon and defined the structural characteristics which distinguish them from other resident non-neural cells. We have also examined the interconnections of these interstitial cells with nerves, the longitudinal muscle, and the circular muscle. In addition, we sought connections between interstitial cells of the myenteric plexus and those described earlier at the inner border of the circular muscle in proximal and distal colon. The interstitial cells of the myenteric plexus were structurally distinctive, and made gap junctions with one another and occasionally with smooth muscle. There seemed to be two subsets of these interstitial cells, one associated with the longitudinal muscle and the other with the circular muscle. Cells of both subsets were often close (less than or equal to 20 nm) to nerve profiles. The interstitial cells near the longitudinal muscle layer penetrated slightly into the muscle layer, but those near the circular muscle did not and neither set contacted the other. Moreover, interstitial cells of Cajal located near the myenteric plexus were never observed to contact those at the inner border of circular muscle. The interstitial cells of Cajal at the canine colon myenteric plexus are structurally organized to provide independent pacemaking activities for the longitudinal and adjacent circular muscle. Their dense innervation suggests that they mediate neural modulation of intestinal pacemaker activities. Moreover, they lack direct contacts with the interstitial cell network at the inner border of circular muscle, which is essential for the primary pacemaking activity of circular muscle. The structural organization of interstitial cells in canine colon is consistent with their proposed role in pacemaking activity of the two muscle layers.
Animals↗
Ionic and cellular basis for slow-wave-type and spike-like action potentials.
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Action Potentials↗
Introduction to ionic basis of electrical behaviour. Origin and characteristics of gastrointestinal pacemakers.
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Animals↗
Quinidine and quinine effects on the slow wave activity of colonic circular muscle.
The slow wave plateau phase has an important role in the regulation of contractile activity in the canine colon. Quinidine (EC50 approximately 5 microM) and quinine (EC50 approximately 13 microM) inhibited in a concentration dependent manner the plateau phase. Quinidine and quinine decreased the plateau amplitude, and increased the plateau potential; whereas, they did not affect the upstroke amplitude, and the average rate of rise of the slow waves. Their specific effect on the slow wave plateau suggests that the plateau phase depolarization is mediated by a quinidine- and quinine-sensitive inward current. Quinidine and quinine will be useful experimental tools to further characterize the ionic conductances responsible for the plateau depolarization.
Animals↗
Pacemaker activity in the proximal lower oesophageal sphincter of the dog.
1. The electrical and mechanical activities of different regions of the canine lower oesophageal sphincter were measured using the single sucrose gap technique. 2. Spontaneous electrical activity was found in the region 0-6 mm oral to the squamocolumnar border. 3. The electrical activity consisted of bursts of spikes superimposed on slow waves. The slow-wave frequency ranged from 0.6 to 5 min-1 in different muscle strips. 4. The slow wave-spike complex and associated contraction were insensitive to tetrodotoxin and atropine. 5. In the pacemaker region, electrical stimulation of intrinsic nerves evoked excitatory junction potentials (atropine sensitive), inhibitory junction potentials (non-adrenergic) and post-stimulus excitation. 6. Increase in the frequency of the slow waves was obtained by muscarinic receptor stimulation (carbachol 10(-7) M) and 10 mM-KCl. 7. The distal lower oesophageal sphincter exhibited a high basal tension but did not show spontaneous electrical activity and stimulation of intrinsic nerves revealed only non-cholinergic, non-adrenergic inhibition. 8. The electrical slow-wave activity observed in the proximal sphincter may constitute the control mechanism for the phasic nature of the contractile activity seen during both the postprandial period and phase III of the interdigestive migrating myoelectric complex. 9. The neural cholinergic activity present in the proximal lower oesophageal sphincter suggests the possibility of neural modulation of the myogenic control activity.
Action Potentials↗
Role of the sodium pump in pacemaker generation in dog colonic smooth muscle.
1. The role of the Na+ pump in the generation of slow wave activity in circular muscle of the dog colon was investigated using a partitioned 'Abe-Tomita' type chamber for voltage control. 2. Blockade of the Na+ pump by omission of extracellular K+, by ouabain, or the combination of 0 mM-Na+ and ouabain, depolarized the membrane up to approximately -40 mV and abolished the slow wave activity. Repolarization back to the control membrane potential by hyperpolarizing current restored the slow wave activity. 3. Slow waves continued to be present in 0 Na+, Li+ HEPES solution. 4. The depolarization induced by the procedures to block Na+ pump activity was associated with an increase in input membrane resistance. 5. Voltage-current relationships show the presence of an inward rectification. 6. Reduction of temperature depolarized the membrane, and decreased the slow wave frequency and amplitude. The slow wave amplitude was restored by repolarization of the membrane. 7. Brief depolarizing pulses evoked premature slow waves. Brief hyperpolarizing pulses terminated the slow waves. 8. We conclude that abolition of slow wave activity by Na+ pump blockade is a direct effect of membrane depolarization and that the Na+ pump is not responsible for the generation of the slow wave. 9. Our results are consistent with the hypothesis that pacemaker activity in smooth muscle is a consequence of membrane conductance changes which are metabolically dependent.
Animals↗
Ionic basis of pacemaker generation in dog colonic smooth muscle.
1. The ionic basis of the slow waves in the circular muscle of the dog colon, in particular the ionic conductances involved in their initiation, were investigated by measuring intracellular electrical activity in the Abe-Tomita-type chamber for voltage control. 2. The depolarization that initiates the slow wave activity could be evoked by an increase in inward current and/or by a block of outward current. According to previous work, inward current could be carried by Na+, Cl-, and Ca2+ ions; K+ ions would carry outward current. 3. The Na+ channel blocker tetrodotoxin (5 x 10(-7) M) did not affect the slow wave amplitude nor its rate of rise. After omission of Na+, by replacing Na+ with N-methyl-D-glucamine, large slow waves continued to develop although some changes in slow wave characteristics occurred. 4. Replacement of 91% of the Cl- by isethionate decreased the slow wave frequency and increased the slow wave amplitude. However, NaCl substitution by sucrose increased the slow wave frequency and decreased the slow wave amplitude. 5. Slow wave activity continued to develop after blockade of Ca2+ influx by D600 (10(-6) M) or CoCl2 (1-3 mM). D600 and Co2+ did not affect the membrane potential but reduced the slow wave amplitude and abolished the plateau potential. Slow waves were abolished after omission of extracellular Ca2+ (plus 1 mM-EGTA). This suggests that Ca2+ influx is probably not necessary but extracellular presence of Ca2+ ions is indispensible for the slow wave generation. 6. The combination of 0 Na+, Li+ HEPES solution, by replacing Na+ with Li+, plus D600 depolarized the cells (up to approximately -40 mV) and abolished slow wave activity. This effect was voltage dependent since repolarization caused slow waves to return. 7. Abolition of the slow wave activity was also obtained by current-induced depolarization to approximately -40 mV. However, during high-K+-induced depolarization (to approximately -40 mV) high amplitude (16 mV) slow waves were still present, slowing that the voltage dependence of the slow waves was shifted positively. This effect probably occurs due to modification by extracellular K+ of a voltage-dependent K+ conductance, which would suggest that a K+ conductance is involved in slow wave generation. 8. In conclusion, slow waves are generated by cyclic membrane conductance changes, which are dependent on the presence of extracellular Ca2+ ions and on the membrane potential. Our data are consistent with the hypothesis that slow waves are initiated by the blockade of a K+ conductance.
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Stimulus-dependent pacemaker activity in the distal canine lower esophageal sphincter.
Electrical and mechanical properties of the distal canine lower esophageal sphincter were studied in vitro to investigate possible means of inducing pacemaker activity. Both direct excitation and block of potassium conductance were investigated. The acetylcholine analog, carbachol, induced tissue depolarization and increase in tone but no electrical slow waves. Tetraethylammonium (TEA) chloride induced depolarization and evoked continuous spiking activity and increase in tone. BaCl did not depolarize the tissue but low amplitude spiking activity developed and increased tone. The putative potassium channel blocker, aminacrine at 2 X 10(-4) M, induced electrical slow wave activity in the distal lower esophageal sphincter, with or without superimposed spikes, accompanied by phasic contractile activity. This activity closely resembled the spontaneous pacemaker activity observed previously in the proximal lower esophageal sphincter. The aminacrine-induced activity was abolished by calcium influx blockers. Aminacrine, but not TEA or BaCl, abolished the nonadrenergic nerve-mediated inhibitory junction potentials. In conclusion, block of inhibitory innervation, and induction of electrical slow waves as a control mechanism for phasic contractile activity, seems to require blockade of an aminacrine- but not TEA-sensitive potassium conductance.
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Pacemaker activity recorded in interstitial cells of Cajal of the gastrointestinal tract.
The hypothesis was tested that interstitial cells of Cajal can generate slow wave activity. Intracellular recordings were performed only in the most superficial cells at the submucosal surface of the canine colonic circular muscle layer. An omnipresent and characteristic slow wave activity was present in all cells with a mean amplitude of 37 +/- 3 mV, a frequency of 4.6 +/- 0.1 counts/min (cpm), and a duration of 5.6 +/- 0.5 s; the average resting membrane potential was -70 +/- 1 mV. To determine the type of cell from which these recordings were obtained, methylene blue was injected by microiontophoresis. The strips were immediately fixed while the microelectrode was kept in the cell. A small segment of the tissue containing this cell was then processed for electron microscopy and serially sectioned. Electron-microscopic evidence showed that the microelectrode tip was positioned in an interstitial cell of Cajal (ICC): 1) several sections were observed with round cytoplasmic lesions of decreasing diameter followed by sections from the same cell without the lesion and 2) electron-dense material was observed in these sections due to the injected methylene blue. These cells were identified as part of the ICC network present at the muscle-submucosa interface of the circular muscle and were positively identified as ICC by the presence of cell processes. This is the first report giving direct evidence for the occurrence of electrical slow waves in ICC. It is essential support for the hypothesis that ICC are the actual pacemaker cells of the gut musculature.
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Different mechanisms of contraction generation in circular muscle of canine colon.
Smooth muscle cells from the circular muscle layer of the dog colon showed a mechanical threshold of -44 mV. No gradient in mechanical threshold was measured between the cells from the submucosal and myenteric plexus surface. The threshold was passed during the upstroke and the plateau phase of the spontaneous slow-wave activity from cells at the submucosal surface and by spike potentials occurring mainly in cells at the myenteric plexus surface and sporadically in cells at the submucosal surface. Carbachol-induced specific changes in electrical and mechanical activities that were inhibited by calcium influx blockade are as follows: 1) increase in slow-wave duration; 2) decrease in plateau potential; 3) enhancement of spiking activity; and 4) increase in contractility. This indicates that calcium influx is significantly increased in the presence of carbachol in cells at both surfaces of the circular muscle layer. The increase in calcium influx could be the result of a direct action by carbachol on the calcium conductance and/or could be mediated by a decrease in outward current. The latter is suggested by the carbachol-induced membrane depolarization associated with an increase in the input resistance, which were both methoxyverapamil insensitive. The results show that an excitatory stimulus can generate contraction of the circular muscle through different electrophysiological activities. In addition, the patterns of spontaneous electrical activity and the different responses to carbachol stimulation provide further information about the heterogeneous nature of the electrical activities within the colonic circular muscle layer.
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Altered smooth muscle contraction and sodium pump activity in the inflamed rat intestine.
We examined changes in membrane function underlying the increased contractility of jejunal longitudinal muscle to carbachol in rats infected 6 days previously with Trichinella spiralis. Muscarinic receptor characteristics were examined in particulate fractions using [N-methyl-3H]scopolamine (NMS). There was a significant reduction in the total number of binding sites on muscle from infected rats, but the affinity for NMS was unchanged. Similarly, in competition studies, the binding of carbachol to high or low affinity sites was not significantly different in tissue from control or infected rats. However, we observed an 89% suppression of the activity of K+ -stimulated ouabain-sensitive p-nitrophenylphosphatase (pNPPase), an enzyme marker for the Na+ -K+ pump, in plasma membranes from infected compared with control rats. Similar results were obtained in 86Rb uptake studies. In contractility studies, evidence for the electrogenicity of the Na+ -K+ pump was obtained by demonstrating that pump activation by K+, Rb+, or Cs+ was associated with tissue relaxation with a rank order of potency that was identical to that for stimulation of pNPPase activity by these ions. Conversely, pump inhibition by vanadate increased tone and abolished phasic contractions in muscle from control or infected rats. This was accompanied by an increased response to carbachol in muscle from control but not infected rats. In addition, pump inhibition by removing extracellular K increased tone in control tissue but decreased tone in muscle from T. spiralis-infected rats, presumably because of preexisting pump suppression. These results are consistent with the hypothesis that suppression of electrogenic Na-pump activity contributes to the increased contractility of jejunal muscle in rats infected with T. spiralis.
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Interstitial cells of Cajal in the canine colon: a special communication network at the inner border of the circular muscle.
The ultrastructure of the region shown to be essential for pacemaking activity of the circular muscle of the canine colon was studied. This region, at the inner border of the circular muscle, consists of a network of several layers of interstitial cells of Cajal type III. These are interconnected to one another and to the adjacent circular muscle cells by numerous gap junctions. Elsewhere in circular muscle, gap junctions are rare and small. In addition, interstitial cells are in close (often less than 20 nm) contact with nerve varicosities containing large granular vesicles or sometimes small granular vesicles. The morphology of interstitial cells resembles that of others of type III. It is suggested that this arrangement of interstitial cells, circular smooth muscles, and nerves allows for a tightly coupled network of membrane oscillators to be subject to neural modulation.
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Heterogeneity in spontaneous and tetraethylammonium induced intracellular electrical activity in colonic circular muscle.
Marked differences were observed in the intracellular electrical activities (spontaneous and TEA-induced) comparing the submucosal and myenteric plexus surfaces of the circular muscle of the dog colon. Distinct characteristics of the cells at the myenteric plexus surface were: a less (10 mV) polarized membrane, a lower amplitude slow wave, and the occurrence of burst type spiking activity. However, slow waves with a high upstroke amplitude (approximately 2.5 times higher than the plateau) were observed in 40% of the preparations. This high upstroke amplitude was dependent on the occurrence of a regenerative membrane potential change (a spike) during the slow wave propagation into the myenteric plexus surface. Such a spike was mediated by Ca2+-influx and could be evoked or enhanced by electrical pulses or by blocking a TEA-sensitive potassium conductance. In the presence of TEA, spikes occurred in bursts. Both slow waves and spiking activities generated contraction. In conclusion, at least two types of cells exist in the circular muscle layer with marked differences in electrophysiological properties. Slow waves are generated at the submucosal surface, passively propagated to the outermost circular muscle where they induce regenerative membrane potential changes.
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Electrical correlate of circumferential contractions in human colonic circular muscle.
The role of myogenic electrical activity in the coordination of circumferential contraction of the human colon circular muscle was investigated. Five suction electrodes were placed (5-7 mm apart) on isolated rings of human colon and simultaneously electrical and motor activities were measured. In normal Krebs solution, the slow waves were not synchronised in most preparations studied. The electrical activities at the different recording sites were different with respect to slow wave frequency and amplitude, and amount of spiking activity. This resulted in irregular contractile activity. Cholinergic stimulation resulted in the development of a specific pattern of electrical activity: periodic slow wave activity with superimposed spiking activity which was synchronised over the length of the segment studied. This synchronised electrical activity resulted in regular phasic contractions at the frequency of the bursts of electrical activity (approximately 1/min). The response to carbachol was mediated by muscarinic receptors since it was blocked by atropine. The periodic activity in the continuous presence of carbachol was not the result of periodic input of neural activity as it occurred in the presence of TTX. Intrinsic properties of the muscle cells were responsible for the carbachol induced pattern of activity. The present study presents evidence that the electrical correlate of circumferential contractions is different in man compared with the most commonly studied animal models. It is a specific, stimulus induced pattern of myogenic activity. Its characteristics closely resemble those of a particular pattern of in vivo recorded activity referred to as the 'long spike bursts'.
Action Potentials↗