Functional Role Of Non-selective Cation Channels On Colonic Excitability In Physiological And Pathological Conditions
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Aims:Integrity of smooth muscle mechanisms is essential for maintaining colonic excitability. This excitability is regulated in part, by the activity of specific ion channels that can affect the resting membrane potential. The experiments performed in this thesis investigated multiple aspects of colonic excitability in various species (human, monkey and mouse) including activity of specific ion channels, their molecular candidates and the regulation of these ion channels in physiological and pathological conditions. Since the resting membrane potential is generally more positive than <italic>E</italic><sub>k</sub> in gastrointestinal smooth muscle, firstly we hypothesized that non-selective cation channels were involved in setting the resting membrane potential. Secondly we examined intracellular signaling mechanisms of non-selective cation channels that contribute to membrane excitability. Since smooth muscle excitability can be changed in pathological conditions, we also examined the effects of histamine to mimic the acute effects of inflammation in two different species (monkey and mouse). Finally we investigated the functional role of non-selective cation channels in an animal model of colitis. Results:Patch clamp techniques, conventional microelectrode recordings, isometric force measurements and molecular studies were employed to examine colonic excitability. Firstly, we found that basally activated non-selective cation channels contribute to the resting membrane potential in human and monkey colonic smooth muscle cells. Basal influx of Na<super>+</super> and Ca<super>2+</super> ions through these channels were responsible for generating relatively positive membrane potentials thus increasing the excitability of colonic smooth muscle cells. Among Transient Receptor Potential subfamily members, molecular candidates for basally activated non-selective cation channels were different in human and monkey colonic smooth muscle cells. Initial plans to examine phospholipase C (PLC)-dependent regulation of basally activated non-selective cation channels were changed on discovery of unexpected effects of a PLC-activator on colonic contractility. To understand the decreased contractility by this drug, we tested the effects of this PLC-activator on various ionic conductances. Both the PLC activator and its inactive analog decreased colonic contractile amplitude, suppressed delayed rectifier K<super>+</super> and L-type Ca<super>2+</super> currents and also transiently increased intracellular Ca<super>2+</super>. These multiple PLC-independent effects suggest that extreme caution must be employed when using these drugs to decipher PLC-related pathways.Since several reports have suggested a possible link between Rho-kinase and ion channel activity, we investigated the involvement of Rho-kinase in colonic excitability at both the tissue and cellular level. Nerve-evoked and carbachol-induced contractions were significantly decreased independent of neuronal influences suggesting that the Rho-kinase inhibitor acts directly on smooth muscle. In addition, carbachol-induced depolarization was significantly reduced by Rho-kinase inhibitors suggesting that Rho-kinase may be involved in regulating agonist stimulated non-selective cation channels. Patch-clamp experiments revealed that Rho-kinase inhibition did not affect basally activated non-selective cation currents but GTPγS-activated currents were inhibited by a Rho-kinase inhibitor. Thus Rho-kinase may play a role in regulation of receptor-activated non-selective cation channels in colonic smooth muscle.In order to examine the functional role of non-selective cation channels in pathological conditions, we examined the effects of the inflammatory mediator, histamine, on colonic excitability in monkey and murine colonic smooth muscle tissue and cells. In monkey colonic tissue, histamine induced significant depolarization and contraction. A similar effect was observed with application of either H1 or H4 receptor agonists. A 5pS non-selective cation channel was activated by histamine in monkey colonic smooth muscle cells. In contrast, in murine colonic tissue, histamine caused hyperpolarization and decreased contractility. This hyperpolarization was prevented by pretreatment with the ATP-sensitive K<super>+</super> channel blocker, glibenclamide, suggesting activation of these channels through the H2/G<sub>s</sub>/cAMP/PKA pathway. Patch clamp experiments also revealed that glibenclamide inhibited an ATP-sensitive K<super>+</super> current activated by histamine. These different contractile responses were proposed to be due to differential expression of specific histamine receptors in the colons from these two species. Finally, inflammation induced by treatment with dextran sulphate sodium in mice caused a significant shift in the resting membrane potential of inflamed colonic smooth muscle to more negative values. Furthermore, carbachol-induced depolarization and contractile amplitude were significantly attenuated in the inflamed smooth muscle suggesting that there may be changes in expression of specific Transient Receptor Potential channels in smooth muscle cells during inflammation. Conclusions: The studies in this dissertation addressed for the first time 1) the contribution of non-selective cation channels to the resting membrane potential, 2) potential molecular candidates for non-selective cation channels and 3) contribution of non-selective cation channels to colonic excitability in physiological and inflammatory conditions.