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The roles of interstitial cells in the regulation of motility in the rectoanal region
AuthorCobine, Caroline A.
AdvisorKeef, Kathleen D.
Biochemistry and Molecular Biology
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Interstitial cells of Cajal (ICC) have previously been described as participants in the regulation of smooth muscle motility within the gastrointestinal (GI) tract. These cells are involved in pacemaking as well as being mediators of neuromuscular transmission. The major focus of this dissertation is on the role of interstitial cells in the neural control of rectoanal motility but as ICC also serve as pacemakers in the GI tract, the mechanisms underlying spontaneous electrical and contractile activity are also addressed.Initial studies were undertaken to characterize the morphology and distribution of ICC as well as PDGFRá+ cells, a second class of interstitial cells recently suggested to participate in neuromuscular transmission within the GI tract, in the monkey and mouse rectoanal region. Both cell types were distributed throughout the rectoanal region but their morphology differed between region and between species. Intramuscular ICC in the monkey rectum and mouse IAS had a morphology similar to that previously described for ICC participating in neuromuscular transmission in other regions of the GI tract, i.e. spindle-shaped. Intramuscular ICC in the monkey IAS differed in their morphology in that these cells more closely resembled pacemaker ICC. While intramuscular PDGFRá+ cells (PDGFRá+-IM) in the mouse and monkey IAS were highly branched cells those in the monkey rectum were less branched. PDGFRá+ cell numbers were significantly greater than ICC-IM with each ICC-IM closely associated with several PDGFRá+ cells. The role of interstitial cells in inhibitory neuromuscular transmission was examined by undertaking both functional and morphological studies. Stimulation of inhibitory motor nerves gave rise to purinergic relaxation and hyperpolarization in the mouse IAS and monkey rectum but not in the monkey IAS whereas all three regions exhibited functional nitrergic responses. Examination of the relationship of ICC and PDGFRá+ cells to nNOS+ nerves also revealed differences between regions. In the monkey rectum and mouse IAS ICC-IM were closely associated with nNOS+ nerves whereas in the monkey IAS they were not. In contrast, PDGFRá+-IM were closely associated with nNOS+ nerves in all three muscles. The functional role of ICC in inhibitory neuromuscular transmission in the rectoanal region was examined by comparing inhibitory neural responses in the Kit mutant W/Wv mouse IAS (where ICC-IM are absent) to those of wild-type (WT) mice. The electrical and contractile events underlying nitrergic transmission were attenuated in these mice suggesting that ICC-IM participate in nitrergic neuromuscular transmission. In contrast, the purinergic pathway was intact. Examination of PDGFRá+-IM revealed that these cells were present in the W/Wv mouse in comparable numbers to that observed in the WT mouse IAS and that these cells were closely associated with nitrergic nerves despite the absence of ICC-IM.To provide further morphological evidence that interstitial cells participate in inhibitory neuromuscular transmission we also examined the expression of several postjunctional effector proteins involved in inhibitory neuromuscular transmission in the monkey and mouse rectoanal region Firstly, postjunctional proteins previously described to mediate nitrergic neuromuscular transmission, i.e. guanylate cyclase (GC) and cGMP-dependent protein kinase I (PKGI) were examined in ICC and PDGFRá+ cells. GC expression was observed in ICC-IM as well as in PDGFRá+ cells in all three regions. However, differences between the mouse and monkey were revealed when PKGI expression was examined. Unlike GC, PKGI expression was limited to PDGFRá+ cells in the monkey IAS and rectum. These data provide morphological evidence that PDGFRá+ cells transmit signals to adjacent SMC via a GC/PKG pathway in the monkey rectoanal region. In contrast, the absence of PKGI from interstitial cells in the mouse IAS (as well as from other GI regions examined) is less straightforward. A number of different mechanisms could account for these observations including the possibility that a PKGI-independent mechanism is involved. This issue is further discussed in chapter 6. To further investigate the role of interstitial cells in purinergic neuromuscular transmission we examined the expression of SK3, the small-conductance calcium-activated potassium channel demonstrated to underlie hyperpolarization in response to purines. SK3 was expressed in PDGFRá+-IM in the mouse IAS and monkey rectum but not in ICC-IM. In contrast, SK3 expression was absent from the monkey IAS where purinergic nerves do not contribute to inhibitory neural responses. These data provide consistent morphological evidence that PDGFRá+-IM cells underlie purinergic neuromuscular transmission. Finally some experiments were undertaken to examine the relationship of excitatory nerves to ICC in the monkey and mouse rectoanal region. The identity of the excitatory motor nerves in the IAS differed between the monkey and the mouse with excitatory transmission being predominantly sympathetic in the monkey IAS and cholinergic in the mouse IAS and monkey rectum. Immunohistochemical studies revealed that the relationships between sympathetic nerves and ICC in the monkey rectoanal region was very similar to that observed for nitrergic nerves with little association in the IAS and close association in the rectum. Since sympathetic nerves perform a neuromodulatory role in the rectum it is possible that this association is intimately related to the presence of other nerves near ICC-IM. The role of ICC in cholinergic neuromuscular transmission was addressed in the mouse IAS with functional studies comparing excitatory transmission in WT and W/Wv mice. These studies revealed that cholinergic depolarization was absent in mice lacking ICC-IM suggesting that ICC-IM mediate the electrical responses associated with cholinergic transmission in this muscle. As cholinergic contractions were intact in these mice, a cell other than ICC-IM may also participate in this neural response.In conclusion, this dissertation provides morphological and functional evidence supporting a role for interstitial cells in the neural control of motility in the rectoanal region although significant differences were found in the properties of ICC and PDGFRá+ cells between species and between regions. Consistent morphological evidence was obtained for PDGFRá+ cells as participants in purinergic transmission in muscles with functional purinergic responses. Likewise the morphology and expression patterns of PDGFRá+ cells in the monkey rectoanal region was compatible with their participation in nitrergic neuromuscular transmission. In contrast, the morphology and expression patterns of proteins in ICC-IM and in PDGFRá+ cells of the mouse were more complex. In the mouse IAS some evidence was obtained for the participation of ICC-IM and PDGFRá+ cells in nitrergic neuromuscular transmission whereas in the monkey IAS the features of ICC-IM was more in keeping with the generation of pacemaker potentials than as mediators of neuromuscular transmission.