| BackgroundCell volume constancy is challenged by any alteration of intracellular and/or extracellular osmolarity. For the living cells, because of the metabolism and the transportation of solute into or out of the cells, the cells always face changes of osmolarity. Because most cell membranes are highly permeable to water, which follows a hydrostatic and osmotic pressure gradient. Water may permeat by diffusion or through water channel. Thus, the cell volume will change. To avoid or counteract untoward alterations of their volume, cells have developed a great number of cell volume regulatory mechanism, which primarily serve to dissipate osmotic gradient across the cell membraneThe regulation of cell volume is an essential function coupled to a variety of physiological process, such as cell proliferation, differentiation and migration. Under physiological condition, cells respond with swelling or shrinkage to osmotic perturbation, but swollen or shrunken cells thereafter exhibit volume regulation. Under pathophysiological condition, cells often undergo a persistent swelling or shrinkage without showing volume regulation. So volume regulation is an important physiological function of the cells.In the face of anisotonic conditions, most mammalian cells can regulate, in a non-steady-state manner, their volume after transient osmotic shrinkage or swelling,this was called regulatory volume decrease (RVD) and regulatory volume increase (RVI). RVD means that cell volume will go back to the normal state after the cell swelling, and RVI means the cell can regulate their volume to normal state after the cell shrinkage.Our aim is concentrated in the mechanism of RVD. It has been understood that most cells accomplish their regulatory volume decrease by release of K+ and Cl" through the activation of K"1" channels and /or anion channels.In human epithelial Intestine 407 cells, there already have some reports about the RVD time course, the Ca2+ dependence of RVD and Cl" channel in RVD by Okada's laboratory, but the identity of the K+ channel responsible for RVD is still unknown Specific AimsThe aim of this thesis is to study the mechanism of RVD systematically, especially the identity of the K+ channels activated in RVD by effectively combination of molecular biology and electrophysiological methods. Specifically it can be divided into several parts. The first part is to record the whole cell current when cells are exposed to hypotonic solution to see the channels activated during RVD. Then to isolate the hypotonicity activated Cl" current and study the electrophysiological and pharmacological characteristics of VSOR. The third part is using RT-PCR to study the expression of different Ca2+-activated K+ channels' mRNA in Intestine 407 cells. The fourth part is to isolate the K+ whole cell current activated by hypotonic stress and to test its Ca2+ dependency and sensitivity to Ca -activated, intermediate conductance K+ channel blocker clotrimazole(CLT) and using single channel recording (cell-attached) to make sure that the same channel can be activated by hypotonic stress at single channel level. After that, the characteristics of ionomycin-induced Ca2+-activated K+ current in Intestine 407 cells and their sensitivity to known K+ channel blockers and different specific Ca2+-activated K+channel blockers were tested. The last part is using electro-measuring system to measure the cell volume and study the time course of the RVD and test the effect of different K+ and Cl" channel blockers on RVD course. MethodsThe technical methods used in this paper consist of the following items. The first is to culture the human epithelial cell line intestine 407 cells. Then to check the expression of different types of K+ channels in intestine 407 cells by using RT-PCR method. The third is using patch-clamp whole cell recording to record hypotonicity activated K+ and Cl" current and ionomycin-induced current. And using patch-clamp single channel recordings (inside-out and cell-attached mode) to study the single...
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