| The gastrointestinal (GI) tract requires a myriad of coordinated contractile patterns to efficiently digest food, reabsorb water and electrolytes, and to propagate fecal matter in the aboral direction. These coordinated contractions are controlled by internal and external influences on smooth muscle activity, including pace setting by interstitial cells of Cajal, and regulatory inputs from the enteric and autonomic nervous systems. Contraction in colonic muscle is associated with an increase in the level of cytosolic Ca2+ [Ca2+]i. The source of Ca2+ that leads to contraction is controversial as to whether or not release of Ca 2+ from the intracellular stores is required. Current literature suggests the functional role of the sarcoplasmic reticulum (SR) in some smooth muscle is to provide a negative feedback mechanism on contractions and it is not a source of contractile Ca2+.;Previously, in colonic myocytes loaded with a fluorescent Ca2+ dye we have identified spontaneous localized releases of Ca 2+ from the SR that did not lead to cell shorting or contraction, but instead led to the activation of Ca2+-activated K+ channels on the plasma membrane causing hyperpolarization. These events are similar to what has been identified in other smooth muscles, called "sparks." Due to the differences in SR Ca2+ release channels responsible for these Ca2+ transients in colonic myocytes the events that we observed were termed "puffs.";This dissertation has characterized mechanisms that modulate the activity of Ca2+ puffs in colonic myocytes. We observed that the neuropeptides, VIP and PACAP, increased Ca2+ transients and STOC frequency and amplitude; and this mechanism may contribute to the post-junctional responses including membrane hyperpolarization and inhibition of contraction. We also characterized a novel mechanism that modulates the coupling strength between localized Ca2+ transients and the large conductance Ca 2+-activated K+ (BK) channels. This appears to be a negative feedback mechanism to control the excitability of colonic muscles. This dissertation also investigated the possibility that post-translation modifications on the accessory beta1 subunit, namely glycosylation, affect the characteristics of BK channels. The regulation of Ca2+ transients and Ca2+-activated K+ channels reveal what may be novel targets disrupted in pathophysiological disorders. |
