A great deal of data has been amassed suggesting that cationic peptides are able to translocate into eucaryotic cells in a temperature-independent manner. Although such peptides are widely used to promote the intracellular delivery of bioactive molecules, the mechanism by which this cell-penetrating activity occurs still remains unclear. Here, we present an in vitro study of the cellular uptake of peptides, originally deriving from protegrin (the SynB peptide vectors), that have also been shown to enhance the transport of drugs across the blood-brain barrier. In parallel, we have examined the internalization process of two lipid-interacting peptides, SynB5 and pAntp-(43-58), the latter corresponding to the translocating segment of the Antennapedia homeodomain. We report a quantitative study of the time-and dose-dependence of internalization and demonstrate that these peptides accumulate inside vesicular structures. Furthermore, we have examined the role of endocytotic pathways in this process using a variety of metabolic and endocytosis inhibitors. We show that the internalization of these peptides is a temperatureand energy-dependent process and that endosomal transport is a key component of the mechanism. Altogether, our results suggest that SynB and pAntp-(43-58) peptides penetrate into cells by an adsorptive-mediated endocytosis process rather than temperature-independent translocation.

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We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18‐α‐glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7‐dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO4 and 4‐hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte‐astrocyte coupling but no astrocyte‐neuron or neuron‐neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA‐treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO4/GA. Elevations of neuronal intracellular calcium levels ([Ca2+]i) induced by FeSO4 were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca2+ and the L‐type Ca2+ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO4 and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate‐ and FeSO4‐induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.

Cholinergic pathways serve important functions in learning and memory processes, and deficits in cholinergic transmission occur in Alzheimer disease (AD). A Cholinergic pathways projecting from basal forebrain regions to hippocampus and neocortex are believed to play important roles in learning and memory processes (1). In Alzheimer disease (AD) cholinergic systems are disrupted, and neurons ultimately die in several connected brain regions including hippocampus and basal forebrain (2). However, it is unclear whether deficits in cholinergic signaling occur prior to neuronal loss. Acetylcholine binding to muscarinic cholinergic receptors initiates the heterotrimeric G protein cycle, which commences with the exchange of GTP for GDP on a-subunits and the subsequent dissociation of fry subunits. The activated, GTP-bound form of the a-subunit stimulates (or inhibits) its effector, then undergoes inactivation by intrinsic GTPase activity, which converts GTP to GDP by hydrolytic cleavage of the y phosphate bond. Cholinergic agonist stimulation of M1, M3, and M5 receptors activates G proteins of the pertussis toxin-insensitive Gq/11 family. Gq/11 subunits stimulate phospholipase C, which catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate, resulting in the liberation of diacylgylcerol and inositol triphosphate (IP3). Diacylgylcerol activates protein kinase C, and IP3 induces the release of Ca2+ from endoplasmic reticulum (3).

Alzheimer's disease is a progressive neurodegenerative disorder that affects primarily learning and memory functions. There is significant neuronal loss and impairment of metabolic functioning in the temporal lobe, an area believed to be crucial for learning and memory tasks. Aggregated deposits of amyloid beta-peptide may have a causative role in the development and progression of AD. We review the cellular actions of A beta and how they can contribute to the cytotoxicity observed in AD. A beta causes plasma membrane lipid peroxidation, impairment of ion-motive ATPases, glutamate uptake, uncoupling of a G-protein linked receptor, and generation of reactive oxygen species. These effects contribute to the loss of intracellular calcium homeostasis reported in cultured neurons. Many cell types other than neurons show alterations in the Alzheimer's brain. The effects of A beta on these cell types is also reviewed.