PUCE (Pontificia Universidad Católica del Ecuador)

The observed differences in the biological responses of arterial cells to minimally and oxidized LDL suggests that each class of LDL induces the activation of distinct signalling pathways. Since, the involvement of cytosolic Ca^^ fluxes in cellular responses to extracellular signals is a ubiquitous feature o f cellular signalling mechanisms, studies were undertaken to investigate whether native and progressively modified LDL induce discrete patterns of cytosolic Ca^^ fluxes.

The data presented here, demonstrate that nLDL, mmLDL and oxLDL elicit cytosolic Ca^^ fluxes in bladder carcinoma T24 cells. The kinetics of the cytosolic Ca^^ fluxes were determined by the degree of oxidative modification of the LDL. Progressive oxidation of LDL promoted increases in the overall magnitude of the cytosolic Ca^^ response via increases in the peak amplitude and prolongation of elevated cytosolic Ca^^ levels. Cytosolic Ca^^ levels in cells exposed to nLDL were transiently increased, but returned to basal levels rapidly. Exposure of cells to fully oxidized LDL resulted in extension of elevated cytosolic Ca^^ such that the cytosolic Ca^^ levels did not return to basal levels. Increased cytosolic Ca^^ levels were sustained for more than 10 minutes. Sustained elevations of cytosolic Ca^^ are associated with the induction of apoptosis (Trump B.F.,

1995) and the link the ability of oxLDL to induce apoptosis with its ability to promote sustained elevation of cytosolic Ca^\

These data demonstrate that the characteristics of cytosolic Ca^^ flux are discrete for nLDL, mmLDL and oxLDL. As the LDL is progressively oxidized, the cellular response is altered resulting in increases in the peak amplitudes and the decay times. These data suggest that oxidation of LDL causes larger amounts of Ca^^ to be released and leads to suppression of the activity of pathways involved in the removal of cytosolic Ca^^.

The results of studies of intracellular Ca^^ rises in other cell types demonstrate similar kinetics of cytosolic Ca^^ transients as reported here. Exposure of endothelial cells to nLDL (O.Smg of LDL apo B/ml) results in increases in cytosolic Ca^^ transients that are maximal after approximately 16 seconds and decay to basal within 160 seconds (Allen S., 1998). In contrast to the findings reported here, these authors did not detect cytosolic Ca^^ transients in response to mmLDL (0.1 mg of LDL apo B/ml). The absence of a Ca^^ response to mmLDL (0.1 mg of LDL apo B/ml) has also been reported by Essler et al (1999). In our system, these concentrations of mmLDL were sufficient to elicit cytosolic Ca^^ transients, indeed, we observed Ca^^ responses to mmLDL at concentrations as low O.OOSmg o f LDL apo B/ml. Furthermore, the results of Mietus-Snyder et al (2000), demonstrate that induction o f the scavenger A receptor in smooth muscle cells by mmLDL is dependent upon Ca^^ flux.

In addition to the investigation of differential responses to native and minimally modified LDL, the effect of LDL concentration upon the kinetics of Ca^^ transients within each class of LDL; native, minimally or fully oxidized LDL was studied. The normal plasma LDL apo B concentration is around 0.5mg/ml, which is equivalent to ~ lp M LDL. Cells were exposed to native and oxidatively modified LDL at concentrations in the range of 0.005 to 0.5 mg of LDL apo B/ml. The characteristics of the induction of Ca^^ transients were compared between different concentrations of native, minimally modified and fully oxidized LDL to

determine whether varying the concentration of each class of LDL elicits discrete cytosolic Ca^^ responses in T24 cells. The results demonstrate that for each class of LDL; native, minimally modified or fully oxidized, the magnitude of the response is correlated with the concentration of the LDL stimulus. Further analysis of the data revealed that the increased magnitude of the response resulted from increases in the peak amplitudes and decay times. Thus increasing the concentration of LDL results in an increase in Ca^^ release and an attenuated rate of removal of Ca^^ from the cytosol. A concentration-dependent effect of LDL upon Ca^^ transients has been observed previously. Allen et al (1998) demonstrated a concentration-dependent effect of nLDL upon the magnitude of Ca^^ transients in endothelial cells. Furthermore, a positive correlation between the nLDL concentration and the peak amplitude of the Ca^^ response was identified as a mechanism for the increased magnitude o f the response. Similarly, the results reported here demonstrate concentration- dependent increases in the peak amplitude. Our data shows that attenuation of the rate of removal of cytosolic Ca^\ detected as an increase in the decay time, in concert with an elevation of the peak amplitude are responsible for increasing the magnitude of the responses as the LDL concentration is increased.

The data presented here and elsewhere demonstrate that each class and concentration of LDL elicits a unique characteristic pattern of cytosolic Ca^^ flux. This suggests the existence of a mechanism by which the concentration of LDL and the degree of oxidation can be ‘sensed’ by the cells. The sensor mechanism translates the extracellular signals into a unique characteristic pattern of Ca^^ flux. The distinct pattern of Ca^^ flux in turn, encodes a set of phenotypic instructions appropriate to the nature of the extracellular stimulus. In this way cells can elicit differential responses to native, minimally modified and fully oxidized LDL. Our data alludes to the possibility that the extent of oxidative modification of LDL is ‘sensed’ by the cell and is encoded into the decay time of the cytosolic Ca^^ elevation. An appropriate analogy is that each LDL species invokes a ‘signature response’, which results in the induction of the appropriate cellular response.

The existence of ‘signature responses’ to distinct agonists and varying concentrations of agonist has been documented previously. Ca^^ is an intracellular messenger that relays information within cells to regulate their activity. However, almost all cellular processes are regulated by calcium (figure 6.14) and in order to co-ordinate all its different functions, Ca^^ signals need to be flexible and precisely regulated. This versatility arises through the ability of Ca^^ ions to act in the contexts of space, time and amplitude.

The data demonstrate that the induction o f cytosolic Ca^^ transients by nLDL samples from different donors is variable in terms o f the rate o f onset and decay. These differences may arise from differences in the LDL profile between donor samples. The