Política de desarrollo rural territorial y el plan nacional de desarrollo.

This thesis examined the capacity o f a neuronal cell line to buffer calcium, the cross-talk between different receptors and the resultant effect on the calcium responses to two agonists, and the effects on calcium signalling o f expression of the protein annexin VI.

The calcium buffering capacity of NlE-115 cells, a neuronal cell line was assessed through the use of a caged-calcium compound, NP-EGTA. Photo­ destruction o f NP-EGTA led to a small release o f calcium, distributed uniformly throughout the cytoplasm of the cells. The calcium release was small enough that no calcium buffers within the cell would saturate giving misleading results, as may occur at the periphery of a cell when the calcium load applied to a cell is via an influx o f calcium across the plasma membrane. The result obtained indicated a very high buffering capacity in these cells. This may reflect the technique used to assess the buffering capacity in these cells, or may simply reflect a high calcium buffering capacity in these cells.

The ability of a cell to control any calcium changes that occur is vital. A concentration of free calcium in the cytoplasm that remains high for significant periods o f time can be pathological for cells. Since calcium is such an important second messenger within cells, tight control over intracellular concentration must be maintained through controlling the magnitude or duration o f calcium changes, or both. The remainder of this thesis examined calcium signalling in A431 cells, in two aspects. Firstly the interplay between agonists on the calcium responses by cells was examined, by comparing both the magnitude o f calcium changes observed following application o f two agonists, and also the pattern of the

calcium change observed. Secondly, the role of the calcium sensitive phospholipid binding protein, annexin VI, in calcium signalling \vas examined.

Cells are not isolated units, that are only stimulated by one agonist at a time, and the interplay between agonists and both their specific receptor and other receptors is crucial. In this thesis, the cross-talk between BK and EGF on A431 cells was examined. The response of the cells to EGF follov^ng prior stimulation with BK was compared to the response with no prior stimulation. Pre-treatment with BK shifted the dose response curve to EGF to the left, but also depressed the peak response. It is likely that the EGF-R has been affected through some downstream effect of BK binding to the BK-R.

When cells were stimulated with EGF followed by BK, the response to BK was increased. Pre-treatment with EGF, which increased the magnitude o f the response to BK, led to potentiation of the BK-R or a downstream component.

Expression of annexin VI in A431 cells is known to reduce growth rates and induce contact-inhibition. The role of annexin VI in the calcium signalling elicited by stimulation o f the cells with EGF was examined, as well as a comparison o f the changes in cytosolic calcium concentration observed following photodestruction of NP-EGTA with parental cells, lacking in annexin VI. The results of this thesis show that annexin VI is affecting the response o f cells to EGF by inhibiting calcium influx across the plasma membrane - the secondary calcium response to EGF. It may be doing so by buffering local calcium levels at the membrane, leading to the inhibition o f hyperpolarisation, but the comparison of calcium release between parental cells and annexin VT^ cells indicated no difference in the ability to buffer calcium. Other experiments comparing the response o f the cell types to BK indicated an effect on calcium signalling being

located to the EGF-R. It would seem more likely that annexin VI is exerting its effect at the level o f the EGF-R than by buffering calcium.

Calcium is an important, near ubiquitous second messenger in cells. Anything that affects the magnitude, pattern or duration o f calcium changes in a cell will have profound effects on the response of the cell to a stimulus. This thesis examined three factors that could affect the calcium signals within a cell - the ability of a cell to buffer calcium changes, thus affecting the magnitude o f a calcium change observed for a given calcium load; the cross-talk between different receptors, which affects both the magnitude and pattern of response to an agonist; and the effect o f specific protein expression, which again modulates both the size and style of response seen in cells expressing the protein in comparison with cells which are lacking in the protein. It is evident from this study that a huge amount of variation in the responses o f cells can be elicited by altering any or all o f these factors.