Ventajas y desventajas financieras y económicas de los instrumentos financieros

In trod u ction

The previous chapters 3 and 4 have described that the TTX-S and TTX-R sodium currents o f DRG neurones are selectively lost 24 hours after infection with wt HSV 17^. The mechanism o f loss o f sodium conductance can be attributed to an event downstream o f viral DNA replication in the lytic cycle o f wt HSV 1 ? \ This event could be the synthesis o f a late protein which, by an unknown mechanism, causes the loss o f sodium currents. It is the unknown mechanism which is under scrutiny in this chapter.

One could speculate that the loss o f sodium current was directly linked to a loss o f sodium channels. Decreased sodium channel density from the surface o f DRG neurones would certainly result in a reduction o f sodium conductance. A possible mechanism for sodium channel loss from the plasma membrane o f DRG neurones is down regulation by internalisation.

There is evidence in the literature that HSV-1 infected cells had increased internalisation o f cell surface proteins. For example, the altered endocytosis o f low density lipoprotein (LDL) receptors during wt HSV 17^ infection o f arterial smooth muscle cells showed this characteristic. LDL receptors provide cells with an exogenous source o f cholesterol for making new membrane. When arterial smooth muscle cell cultures were infected with wt HSV 17^ the intracellular levels of cholesterol and cholesteryl ester increased by more than 50 fold and showed an increased binding and internalisation o f [^^^I]-LDL (Hsu et al. 1995). If, however, the arterial cell cultures were infected with a HSV-1 viral protein kinase mutant, containing a deletion in gene Us3, the increased cholesterol accumulation did not occur. This suggested a correlation between viral protein kinase activity and increased LDL internalisation and thus accumulation o f cholesterol (Hsu et al. 1995; Purves et al. 1987). It is not clear, however, whether this correlation stems from viral kinase enhanced LDL receptor internalisation or by altered activity o f kinase dependent proteins involved in cholesterol metabolism, such as, 3-hydroxy-3-methylgluaryl- coenzyme A (HMG-CoA) reductase which catalyses the rate limiting step in cholesterol synthesis, (Goldstein & Brown, 1990).

This evidence shows that certain cells infected with HSV-1 are capable o f increased internalisation o f surface membrane proteins and that this may be protein kinase dependent. More specifically o f interest here is whether sodium channels are internalised in wt HSV 17^ infected DRG neurones.

Previously, it has been shown that incubation o f neonatal rat brain cultures with veratridine (a sodium channel activator) in depolarising medium, such as 50mM KCl, for as short a period as one hour, results in a dramatic decrease in surface sodium channels (Dargent & Couraud, 1990). In these experiments the density o f sodium

channels was monitored by [ H] saxitoxin or in some cases scorpion toxin binding, where scorpion toxin was both the channel activator and probe. Experiments to investigate if the decrease in surface sodium channels was a result o f internalisation were based on the work o f Haigler et al. (1980). In these experiments, radiolabeled ligands bind to cell surface receptors (in this case sodium channels), and after a period o f incubation the cells were washed with a weak acid to remove ligand bound to the receptors on the cell surface. After the weak acid wash, any radioactivity associated with the cells would result from internalised probe bound to sodium channels. Acid resistant radioactivity was detected in neonatal rat brain cultures which had been treated with veratridine in depolarising medium, indicating that sodium channels were internalised. Synaptosomes prepared from adult rat brains or adult hippocampal slices did not behave similarly, as internalisation o f sodium channels was not observed (Dargent fl/. 1994).

The prolonged opening o f sodium channels, either by treatment with veratridine or scorpion toxin, allows sodium influx and an increase in intracellular sodium concentration. The fact that acid resistant radioactivity is not present when experiments were performed in sodium free media or in the presence o f sodium channel blocker TTX suggested that increased intracellular sodium concentration may be the trigger for sodium channel internalisation (Paillart et al. 1996). The sodium ionophore amphotei/cin B also induces sodium channel down regulation (Dargent & Couraud, 1990). The experiments in neonatal rat brain cultures provide evidence o f a mechanism for neuronal voltage-gated sodium channel internalisation, triggered by an increase in intracellular sodium concentration, resulting in a loss o f sodium channels from the cell surface and hence a loss o f sodium conductance (Dargent et al. 1994). Internalisation o f sodium channels after sodium influx was not mediated by protein kinase C, cAMP dependent protein kinase, cGMP protein kinase, tyrosine phosphorolation or intracellular calcium concentration (Dargent et al. 1995).

Sodium conductance is lost during a wt HSV 17^ infection o f DRG neurones. The experiments described in this chapter investigate the possibility that the loss o f sodium conductance observed with wt HSV 17^ infection were the result o f sodium channel internalisation. One explanation could be that during a wt HSV 17^ infection

intracellular sodium concentration rises, triggering a down regulation o f sodium channels by internalisation. The effects o f increased intracellular sodium concentration in DRG neurones has not been analysed before. The possibility that sodium channels are internalised during a wt HSV infection, and the possibility that any such internalisation is triggered by raised intracellular sodium concentration, was explored.

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