PROCEDIMIENTOS PARA LAS MEDICIONES FOTOMÉTRICAS EN ILUMINACIÓN

4.5.2.1. Complex II+III

Loss of complex II+III activity was observed in liver, astrocytes and neurones following OHPA treatment. In isolated liver mitochondria, loss of complex II+III was dose dependent, and was observed within 15 minutes of being incubated with OHPA, suggesting that inhibition is a result of either OHPA or 3-oxo-4- pentenoate directly interacting with the enzyme. Loss of complex II+III activity was shown not to be due to either OHPA or 3 -oxo-4-pentenoate interfering with the assay. Despite HBDH activity being similar in astrocytes and neurones, loss of complex II+III activity was greater in neurones, compared to astrocytes. In astrocytes, a 21% loss in complex II+III activity was observed after 18 hours, while activity was unaffected after 4 or 8 hours of treatment. Complex II+III activity was inhibited by 41% in neurones treated with OHPA for 4 hours, with a 68% loss o f activity observed after 18 hours. Loss of complex II+III was still observed when expressed against CS activity suggesting that the loss of activity

was not due to just a possible decrease in mitochondrial number (see below; section 4.5.2.2).

The greater susceptibility of neuronal complex II+III may in part be due to the depletion of mitochondrial GSH. However cellular GSH status cannot fully explain the differential effect of OHPA on complex II+III in astrocytes and neurones, as inhibition is still observed in astrocytes despite mitochondrial GSH being maintained, and cytosolic GSH concentration increased. It is not known whether the loss of activity is due to inhibition of either complex II and III, or both o f them. Alternatively, co-enzyme Qiomay be affected by OHPA treatment, as the complex II+III assay requires endogenous Qio(King, 1967). The complexes need to be assayed separately to see if either is affected by OHPA. Cellular levels o f Qio also need to be determined.

The inhibition of complex II+III by OHPA or 3 -oxo-4-pentenoate in liver, astrocytes, and neurones limits its use for investigating the function of mitochondrial GSH. OHPA would be unsuitable for investigating the role of mitochondrial GSH in protecting the ETC, as it will be difficult to distinguish whether any effects that are observed are due to depletion of mitochondrial GSH, or the direct interaction of the drug with the ETC. However, the drug may be useful in liver and neurones for investigating GSH transport in to mitochondria.

4.5.2.2. Citrate synthase

Loss o f CS was observed in neurones treated with OHPA for both 4 and 18 hours, while no loss in activity was observed in either astrocytes or liver. The 25% loss of CS activity after 4 hours is probably due to inhibition of the enzyme rather than a decrease in neuronal mitochondrial number, as the activities of both complexes I and IV were unaffected by OHPA treatment. Incubation of neuronal homogenates with OHPA during the CS assay had no effect on activity, indicating that loss in activity was not due to OHPA interfering with the assay. This is endorsed by the observation that neither liver nor astrocyte CS activity was affected by OHPA exposure. A 37% loss of CS activity was observed following OHPA treatment for 18 hours. It is difficult to ascertain whether this loss in activity is due to a decrease

in mitochondrial number, or inhibition of the enzyme, since complexes I to IV were all inhibited at this time point. Neuronal homogenates were assayed for monoamine oxidase, an enzyme located on the outer mitochondrial membrane (Ragan et al., 1987), following OHPA treatment in order to determine whether a decrease in mitochondrial number occurs. However, enzyme activity could not be accurately determined due to the poor sensitivity of the assay. Monoamine oxidase activity in purified rat brain mitochondria has been reported to be 2.0 ± 0.2 nmol/min/mg protein (Heales et al., 1995). Monoamine oxidase activity is likely to be considerably lower in neuronal homogenates, with detection of the oxidation of the substrate (benzylamine) below the sensitivity of the spectrophotometer. Unfortunately, other mitochondrial enzymes that could be measured, such as aconitase and a-ketoglutarate dehydrogenase, are also susceptible to oxidative stress (Gardner et al., 1994; Chinopoulos et al., 1999; Park et al., 1999). Measurement of the amount of mitochondrial DNA present in OHPA treated neurones may be a method to determine if mitochondria are indeed lost following OHPA treatment.

4.S.2.3. Complex I

A 36% loss of complex I activity was observed in neurones treated with OHPA for 18 hours. The 36% loss of complex I activity was concomitant with a 42% depletion in cellular GSH concentration. The depletion of GSH could result in complex I becoming more susceptible to the effects o f OHPA treatment. However, if OHPA treatment does result in loss of mitochondrial number (see above; 4.5.2.2), no inhibition of complex I was observed when expressed against CS activity, suggesting that the loss of activity maybe due to loss of mitochondria. No loss of complex I activity was observed in liver mitochondria depleted of GSH by OHPA. This could support the hypothesis that loss of complex I activity in neurones is due to a reduction in the number of mitochondria. Alternatively, complex I may have been unaffected in liver mitochondria because of the short incubation period (15 minutes).

4.S.2.4. Complex IV

Treatment o f neurones with 0.5 mM OHPA for 18 hours resulted in a 37% loss of enzyme activity, while liver incubated with 1 mM OHPA for 15 minutes resulted in a 40% loss of activity. The depletion of mitochondrial GSH may leave complex IV more susceptible to inhibition by free radicals generated by mitochondria (Wullner et al., 1999; Paradies et al., 1999), or the direct action of OHPA or 3- oxo-4-pentenoate.

In the original paper that reported the depletion of isolated liver mitochondrial GSH by OHPA, Shan et al (1993) showed that 0.75 mM OHPA resulted in a 30% decrease in mitochondrial oxygen consumption. However they did not indicate whether this was significant or not. The inhibition of complexes II+III and IV shown here could be contributing towards the possible decrease in oxygen consumption.