22.21 Death of Cerebellar Granule Neurons Induced
by Piperine is Distinct from that Induced by Low
Potassium Medium
In this study, piperine-induced cell death in cultured cerebellar granule neurons was
compared with that induced by K+ withdrawal. Exposures to piperine resulted in
concentration-related neuronal death. In contrast to low K+-induced granule neuronal death, piperine-induced death was unaffected by inhibitors of protein synthesis and endonuclease activity. It is well-known that death of cerebellar granule
neurons induced by K+ withdrawal displays the requisite morphological and biochemical characteristics of apoptosis including the dependence on de novo protein
synthesis and endonuclease activation (Unchern et al. 1998). Therefore, our data
suggest an involvement of non-apoptotic mechanism in piperine-induced granule
neuronal death. It was notable that a decrease in cellular MTT reduction preceded
the onset of neuronal death induced by both piperine and low K+ exposures. This
indicated that compromised mitochondrial function may be, at least partly,
responsible for the observed neuronal death. It was evident that neurons rapidly lost
their mitochondrial membrane potential, energy charge, and subsequently the
ability to metabolize MTT before proceeding to necrosis. Due to its high
lipophilicity and unsaturated double bonds, it is conceivable that piperine may
directly interact with neuronal membranes and initiate lipid peroxidation. This
speculation is in accordance with an observation that piperine (25–100 µM)
increased lipid peroxidation in isolated epithelial cells of rat jejunum (Johri et al.
1992). The consequences of piperine-induced lipid peroxidation are the disruption
of neuronal membrane integrity and the liberation of cytotoxic reactive oxygen
species (ROS). ROS have been implicated as mediators of both glutamate-induced
cell death (Gunasekar et al. 1995) and low K+ -induce apoptosis of cerebellar
granule neurons (Schulz et al. 1996). However, we did not observe any protective
effects of lipophilic free radical scavengers against low K+ -induced granule neuronal death. This ineffectiveness may be due to the fact that major intracellular sites
of oxygen free radical generation in apoptosis include mitochondria, endoplasmic
reticulum, and perhaps nuclear membranes (Korsmeyer et al. 1995); whereas the
free radical scavengers used in our study exert their effects mainly on cell membranes. Schulz et al. (1996) observed that vitamin E (>2 mM) partially protected
cerebellar granule neurons against apoptosis induced by 24 h K+ withdrawal
(Schulz et al. 1996). At very high concentrations, the protective effect of vitamin E
may be related to its stabilizing effect on the cell membrane rather than its
antioxidant or free radical scavenging property (Diplock 1982).
22 Piperine: Sources, Properties, Applications …
775
by Piperine is Distinct from that Induced by Low
Potassium Medium
In this study, piperine-induced cell death in cultured cerebellar granule neurons was
compared with that induced by K+ withdrawal. Exposures to piperine resulted in
concentration-related neuronal death. In contrast to low K+-induced granule neuronal death, piperine-induced death was unaffected by inhibitors of protein synthesis and endonuclease activity. It is well-known that death of cerebellar granule
neurons induced by K+ withdrawal displays the requisite morphological and biochemical characteristics of apoptosis including the dependence on de novo protein
synthesis and endonuclease activation (Unchern et al. 1998). Therefore, our data
suggest an involvement of non-apoptotic mechanism in piperine-induced granule
neuronal death. It was notable that a decrease in cellular MTT reduction preceded
the onset of neuronal death induced by both piperine and low K+ exposures. This
indicated that compromised mitochondrial function may be, at least partly,
responsible for the observed neuronal death. It was evident that neurons rapidly lost
their mitochondrial membrane potential, energy charge, and subsequently the
ability to metabolize MTT before proceeding to necrosis. Due to its high
lipophilicity and unsaturated double bonds, it is conceivable that piperine may
directly interact with neuronal membranes and initiate lipid peroxidation. This
speculation is in accordance with an observation that piperine (25–100 µM)
increased lipid peroxidation in isolated epithelial cells of rat jejunum (Johri et al.
1992). The consequences of piperine-induced lipid peroxidation are the disruption
of neuronal membrane integrity and the liberation of cytotoxic reactive oxygen
species (ROS). ROS have been implicated as mediators of both glutamate-induced
cell death (Gunasekar et al. 1995) and low K+ -induce apoptosis of cerebellar
granule neurons (Schulz et al. 1996). However, we did not observe any protective
effects of lipophilic free radical scavengers against low K+ -induced granule neuronal death. This ineffectiveness may be due to the fact that major intracellular sites
of oxygen free radical generation in apoptosis include mitochondria, endoplasmic
reticulum, and perhaps nuclear membranes (Korsmeyer et al. 1995); whereas the
free radical scavengers used in our study exert their effects mainly on cell membranes. Schulz et al. (1996) observed that vitamin E (>2 mM) partially protected
cerebellar granule neurons against apoptosis induced by 24 h K+ withdrawal
(Schulz et al. 1996). At very high concentrations, the protective effect of vitamin E
may be related to its stabilizing effect on the cell membrane rather than its
antioxidant or free radical scavenging property (Diplock 1982).
22 Piperine: Sources, Properties, Applications …
775