Date of Award
Doctor of Philosophy (PhD)
Electrical and Computer Engineering
Dr. Jan D. Huizinga
Knowledge of the origin and characteristics of the intestinal pacemaker activity, and the characteristics of intercellular communication throughout the musculature is instrumental for the understanding of the mechanisms through which gastrointestinal (GI) motility is regulated. This thesis makes a significant contribution to provide electrophysiological and morphological evidence supporting the hypothesis that interstitial cells of Cajal (ICCs) are the Gl pacemaker cells. The pacemaker activity of the GI tract triggers the slow-wave.type action potentials (slow waves) which are coherent with the phasic contractions for facilitating peristaltic movement. Origins of the slow waves at different portions of the GJ tract always coincide with the locations of the ICCs. The objectives of this study were to identify the cellular origin of the pacemaker activity and to thoroughly investigate the mechanism of intercellular communication in the canine colon using electrophysiological and microscopic techniques. The cellular origin of the pacemaker activity was further examined by studying simultaneously the ontogenesis of the pacemaker activity and the ICCs in the neonatal mouse small intestine.
The cellular origin of the pacemaker activity was studied by employing the photodynamic property of methylene blue (Chapter 3). We previously demonstrated that the submuscular ICCs of the canine colon selectively accumulate methylene blue. In this undertaken study, we further illustrated that incubation with 50 μM methylene blue and subsequent intense illumination resulted in abolition of the pacemaker activity. Following methylene blue incubation, intense illumination first changed the mitochondrial conformation in the ICCs from very condensed to orthodox, and progressively imposed more severe damages, such as swollen and ruptured mitochondria, loss of cytoplasmic contrast and detail, and rupture of the plasma membrane. No damage was seen in smooth muscle cells and nerves. The correlation between selective lesioning of ICCs and loss of the pacemaker activity strongly supports that ICCs play an essential role in the generation of the pacemaker activity.
The regulatory mechanism of the pacemaker frequency was investigated with a focus on the effects of cyclopiazonic acid (CPA), a specific inhibitor of the endoplasmic reticulum (ER) Ca²⁺-pump (Chapter 4). CPA dose dependently decreased the pacemaker frequency. Similarly, chelating cytosolic Ca²⁺ with BAPTA also decreased the pacemaker frequency. The pacemaker frequency was also decreased by neomycin (inhibiting inositol 1,4,5-triphosphate (IP₃) synthesis) and caffeine (inhibiting the IP₃-sensitive Ca²⁺ channels in the ER membrane). Electron microscopy showed that the smooth ER forms an extensive network of subsurface cisternae which is closely associated with large areas of the cytoplasmic face of the plasma membrane. These structures were the most extensive in the ICCs, slightly less in branching smooth muscle cells and far less in circular muscle cells. Based on the electrophysiological and morphological observations, we hypothesize that the Ca²⁺ refilling cycle of the IP3-sensitive calcium stores associated with the plasma membrane, determines the frequency of the pacemaker activity generated by the submuscular ICC-smooth-muscle network of the canine colon.
The ontogenesis of the pacemaker activity and the ICCs in the small intestine of neonatal mice was studied to further substantiate the pacemaker role of ICCs (Chapter 5). The pacemaker component of the slow waves was fingerprinted by its resistance to L-type Ca²⁺-channel blockers and sensitivity to cyclopiazonic acid, CPA, (Chapter 4). All isolated musculature of the neonatal mouse small intestine (newborn, unfed-7 days old) spontaneously generated action potentials. The presence of the pacemaker component in different age groups was examined by verapamil, a L-type Ca²⁺ channel blocker, and CPA. In conclusion, electrophysiological and morphological evidences were obtained to demonstrate that both the pacemaker activity and the, ICC network were immature at birth but fully developed in 2 days old neonatal mice.
Communication between the longitudinal and the circular muscle layers are essential for producing co-ordinated motility in the musculature. Through electrophysiological measurements with microelectrodes, the study of neurobiotin spread using confocal microscopy and the investigation of the cellular structure at the electron-microscopic level, the cellular mechanisms of communication between the two muscle layers was studied. We positively demonstrated the existence of low-resistance pathways. We also provided evidence that the ICCs associated with the myenteric plexus facilitated electrotonic coupling between the two muscle layers across the myenteric plexus of the canine colon (Chapter 7).
In the longitudinal muscle layer, no positive evidence for electrical coupling between smooth muscle cells has yet been presented. We thoroughly examined the properties of electrical coupling in the longitudinal muscle layer of the canine colon (Chapter 8). The properties of electrical coupling between longitudinal muscle cells were compared with that between the circular muscle cells. Three electrical coupling parameters were measured: (i) the input resistance, (ii) the space constant (determined by the method developed with a double-electrode technique (Chapter 6)), and (iii) the phase relationship of simultaneously recorded electrical activities. Furthermore, a detailed electron microscopic investigation revealed the absence of gap junctions in the longitudinal muscle layer; whereas, numerous close apposition contacts were observed. These observations put forward the hypothesis that the pathways for electrical coupling between longitudinal muscle cells are consituted by close apposition contacts.
Communication between circular muscle (CM) lamellae is necessary to generate propulsive phasic contractions for facilitating peristalsis along the longitudinal axis of the GI tract. The submuscular ICCs are extensively coupled to the underlying branching smooth muscle (bSM) cells forming an ICC-bSM network covering the entire submucosal surface of the canine colon. There is another ICC network located in the myenteric plexus. The roles of the submuscular ICC-bSM network, the myenteric ICC network and the longitudinal muscle layer in mediating communication across the CM lamellae were studied by simultaneous recordings with surface electrodes using different types of muscle strip preparations (Chapter 9). Electrophysiological evidence demonstrated that, within the pure circular musculature, circular muscle cells were electrically coupled along a CM lamella, oriented circumferentially around the lumen, but electrically insulated across CM lamellae. The submuscular ICC-bSM network, but not the longitudinal muscle nor the myenteric plexus, was shown to be essential for mediating communication between CM lamellae such that co-ordinated motility can be exhibited with neig,hbouring CM lamellae through excitation-contraction coupling.
In summary, employing a number of electrophysiological and microscopic techniques, this dissertation presents novel evidence (i) to substantiate the pacemaker role of ICCs in the GI tract; (ii) to put forward a hypothesis that the pacemaker-frequency regulatory mechanism is synchronized with the ER Ca²⁺ refilling cycle; and, (iii) on the heterogeneity of mechanisms through which intercellular communication occurs along the radial, circumferential and longitudinal axes of the intestinal musculature.
Liu, Louis Wing Cheong, "Pacemaker Activity and Intercellular Communication in the Intestinal Musculature" (1995). Open Access Dissertations and Theses. Paper 2237.