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Effects of some agents on receptor- and cell volume-controlled changes in the ionic permeability of isolated guinea-pig hepatocytes

Sandford, Christine Anne; (1993) Effects of some agents on receptor- and cell volume-controlled changes in the ionic permeability of isolated guinea-pig hepatocytes. Doctoral thesis (Ph.D.), University College London (United Kingdom). Green open access

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Abstract

Sodium-linked amino acid transport causes an increase in the membrane permeability of liver cells to potassium. This effect on permeability is generally attributed to the concomitant rise in cell volume produced by the inward movement of solute. It is thought to form the basis of the subsequent regulatory volume decrease. The first part of this study examined the pharmacology and electrical characteristics of cell volume regulation in isolated guinea-pig hepatocytes using intracellular recording techniques and the whole cell variant of the patch clamp technique. In patch clamp studies, cell swelling was induced by the application of hydrostatic pressure to the shank of the patch pipette. This produced a large outward K+ current in voltage clamped cells, apparently without a rise in cytosolic free Ca2+ concentration. Spectral analysis of the current noise during this response suggested that the K+ channel involved has a unitary conductance of 7pS. In microelectrode recordings, the volume activated potassium conductance (Gv) is sometimes triggered as a consequence of electrolyte leakage from the electrode and the influx of osmotically obliged water. This experimental artefact was turned to advantage in the current study to test the ability of a variety of agents to inhibit Gv. Gv demonstrated a similar sensitivity to cetiedil to the potassium conductances that subserve the Gardos effect in red blood cells and the volume regulatory decrease described in lymphocytes subjected to hypotonic stress. Its insensitivity to oligomycin A and oxpentifylline, however, sets Gv apart from these cetiedil sensitive conductances. The second part of this study looks at various aspects of the hormonal regulation of liver cell membrane permeability. The changes in membrane conductance evoked by P2-purinoceptor activation were investigated first. Previous workers have demonstrated that Ca2+ mobilizing agonists trigger Ca2+-activated K+ and Cl- permeabilities (PK(Ca) Pcl(ca)) in the liver. In the present study, it was shown that whereas low concentrations of ATP exclusively activate the Ca2+-gated K and Cl channels, larger concentrations also induce a transient depolarizing current which flows through a separate conductive pathway that does not depend on intracellular Ca2+. The mechanisms coupling hormonal receptor stimulation to the activation of PK(Ca) Pci(ca) were addressed next. Fast oscillations in membrane conductance were observed when the hepatocytes were stimulated by submaximal concentrations of either the Ca2+ mobilizing agonist ATP or the cAMP-dependent agonist salbutamol. The cellular basis for this oscillatory activity was explored by employing the whole cell patch technique to introduce the putative second messengers cAMP and inositol 1,4,5 trisphosphate (IP3) into the cells. The results obtained suggest the release of intracellular Ca2+ by both cAMP and IP3 can be pulsatile. Finally, the potentiating effect of salbutamol on the membrane response to ATP, described previously in liver slices and cell suspensions, was re-examined at the single cell level. The dose-response relationship of single cells to ATP was found to shift to the left in the presence of salbutamol, so that near threshold concentrations of this agonist became sufficient in some cells to set in motion the series of cyclical fluctuations in cytosolic calcium, normally associated with more intense stimulation. These results are discussed in the context of Berridge's two pool model of intracellular calcium homeostasis.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Effects of some agents on receptor- and cell volume-controlled changes in the ionic permeability of isolated guinea-pig hepatocytes
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: (UMI)AAI10017717; Health and environmental sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10109568
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