Sui, Guiping; (1999) The electrophysiological properties of freshly isolated and cultured human and guinea-pig detrusor smooth muscle cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Uncontrolled contraction of the detrusor is a major factor in bladder instability, but the cellular mechanisms remain unknown and clinical treatment still leaves much to be desired. The objectives of this study were to characterise the electrophysiological properties of freshly isolated adult and cultured detrusor smooth muscle cells from human biopsy samples and guinea-pig bladder in order to develop a model for study of the causes of bladder instability; and to facilitate the development of functional materials suitable for bladder implants. Single detrusor myocytes were obtained from either adult guinea-pig or human urinary bladders by enzymatic dissociation after removal of the mucosa and epithelium. A suitable culture medium was developed which consisted of D-valine substituted minimal essential medium, supplemented with 10% foetal bovine serum. Cultures were maintained in a humidified incubator at 37C with 5% CO2, grown to confluence and passaged up to three times. Electrophysiological properties of freshly isolated, primary cultured and passaged cells were investigated using patch-type electrodes. Cultured detrusor smooth muscle cells presented a "hills and valleys" appearance after growing to confluence; smooth muscle specific a-actin staining was positive. The electrophysiological properties of the freshly isolated cells were retained in the cultured cells, but with some modifications. Specific membrane resistance was decreased, but membrane capacitance was larger; the resting membrane potential and action potential threshold were shifted to more depolarised values; action potential upstroke velocity was slower, attributed to smaller inward currents and action potential duration was longer. Similar results were obtained for both guinea-pig and human detrusor smooth muscle cells. Inward and steady-state outward currents were maintained in cultured cells, the current density was lower in primary cultures but outward currents increased again during subsequent passages. Activation and inactivation curves of the inward currents were shifted to more positive potentials after culture. The spontaneous transient outward current, which was recorded in freshly isolated cells, was not recorded in cultured cells. Both inward and outward currents were further characterised in guinea- pig cells. This revealed the existence of a T-type Ca2+ current in addition to the L- type currents found in freshly isolated cells, but in cultured cells only the L-type Ca2+ current was found. In freshly-isolated cells K+ currents showed transient and steady-state components; the transient current and part of the steady-state current were Ca2+- dependent, the transient current was blocked by iberiotoxin but not apamin. The results show that myocytes are less excitable in culture, this could be due to the reduced density and altered steady-state kinetics of Ca2+ currents, as well as a proportionately smaller reduction of K+ currents.

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