Suppression of an amyloid beta peptide-mediated calcium channel response by a secreted beta-amyloid precursor protein.

Secreted isoforms of the beta-amyloid precursor protein potently enhance neuronal survival in cell cultures exposed to toxic amyloid beta peptide. Lowering of intracellular calcium levels to offset the increases in intraneuronal calcium caused by amyloid beta peptide is thought to underly this neuroprotection. Because we have shown previously that an amyloid beta peptide-mediated potentiation of calcium channel currents may contribute to this cytosolic calcium overload, the present study examined the effects of a secreted beta-amyloid precursor protein on the calcium channel response to amyloid beta peptide. When compared with untreated cultured rat hippocampal neurons, cells that underwent a 24 h preincubation with beta-amyloid precursor protein 751 displayed decreases in the relative size of the calcium channel response to amyloid beta peptide. A membrane-permeable analog of cyclic GMP, a second messenger believed to be involved in the calcium regulation process mediated by beta-amyloid precursor proteins, also attenuated the modulatory calcium channel response. Co-application of beta-amyloid precursor protein 751 with amyloid beta peptide did not alter calcium channel response to amyloid beta peptide. Taken together, these findings suggest that secreted beta-amyloid precursor proteins can suppress a calcium channel response to amyloid beta peptide that is potentially injurious to the cell, and as such, may define a neuroprotective mechanism that is specific for amyloid beta toxicity.

Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons.

Neurotoxicity induced by overstimulation of N-methyl-D-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1beta-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

Neuronal membrane conductance activated by amyloid beta peptide: importance of peptide conformation.

Whole-cell voltage-clamp recording and circular dichroism (CD) spectroscopy were used to assess the importance of amyloid beta peptide (A beta) conformation for eliciting homeostatically disruptive membrane conductances in embryonic rat hippocampal neurons. A beta that assumed a random coil conformation when freshly dissolved in water did not alter cell resting ('leak') membrane conductances. In contrast, after several days incubation ('aging'), the same peptide samples became capable of activating a large, rapid onset and potentially toxic increase in leak membrane conductance that coincided temporally with a transition in peptide conformation from random coil to beta-sheet. Interestingly, this membrane activity was not mimicked with chemically equivalent A beta s that immediately adopted a beta-sheet conformation in water ('pre-aged'). These findings suggest that, under conditions that allow for a gradual transition of random coil A beta to beta-sheet structures, peptide conformation may be an important determinant of the toxic consequences of A beta-mediated membrane conductances.

Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro.

Amyloid beta peptide (A beta), the major protein constituent of senile plaques in patients with Alzheimer's disease, is believed to facilitate the progressive neurodegeneration that occurs in the latter stages of this disease. Early attempts to characterize the structure-activity relationship of A beta toxicity in vitro were compromised by the inability to reproducibly elicit A beta-dependent toxicity across different lots of chemically equivalent peptides. In this study we used CD spectroscopy to demonstrate that A beta secondary structure is an important determinant of A beta toxicity. Solubilized A beta was maximally toxic when the peptide adopted a beta-sheet conformation. Three of the four A beta lots tested had a random coil conformation and were weakly toxic or inactive, whereas the single A beta lot exhibiting toxic activity at low peptide concentrations had significant beta-sheet structure. Incubation of the weakly toxic A beta lots in aqueous stock solutions for several days before use induced a time-dependent conformational transition from random coil to beta-sheet and increased A beta toxicity in three different toxicity assays. Furthermore, the secondary structure of preincubated A beta was dependent upon peptide concentration and pH, so that beta-sheet structures were attenuated when peptide solutions were diluted or buffered at neutral and basic pH. Our data could explain some of the variable toxic activity that has been associated with A beta in the past and provide additional support for the hypothesis that A beta can have a causal role in the molecular neuropathology of Alzheimer's disease.

beta-Amyloid peptide in vitro toxicity: lot-to-lot variability.

beta A4 peptide (beta AP) accumulates in amyloid plaques of Alzheimer's disease and may contribute to neuronal degeneration. Conflicting observations have been reported regarding the direct in vitro and in vivo neurotoxicity of beta AP. We have assessed in vitro beta AP toxicity in high density primary rat hippocampal cultures and found marked lot-to-lot differences in the neurotoxic properties of beta AP. One lot of beta AP from a commercial supplier resulted in significant direct neurotoxicity at 10 microM, while 2 other lots from the same supplier were essentially nontoxic. Three additional lots of beta AP from unrelated sources were also nontoxic at 10 microM. Initial biochemical characterization has not yet revealed any marked differences among the various lots of beta AP. Low levels of endotoxin (ca., 1 EU/ml) were detected in several beta AP preparations but did not correlate with neurotoxicity. Our observation that lot-to-lot variability of beta AP occurred even under identical in vitro culture conditions may account for part of the present controversy in this area.

Mechanism of impaired responses of cerebral arterioles during diabetes mellitus.

The goal of this study was to determine the mechanism of impaired responses of cerebral arterioles during diabetes mellitus. To induce diabetes, rats were injected with streptozotocin. Rats were characterized as diabetic by a blood glucose of greater than 300 mg/dl. Diameter of pial arterioles was measured with intravital microscopy in nondiabetic and diabetic rats during superfusion with acetylcholine (ACh), ADP, the thromboxane (Tx) analogue U-46619, and nitroglycerin. ACh increased pial arteriolar diameter in nondiabetic rats and did not alter diameter in diameter in diabetic rats. ADP increased pial arteriolar diameter in nondiabetic rats and produced minimal changes in diameter of arterioles in diabetic rats. Tx analogue U-46619 produced similar constriction of cerebral arterioles in nondiabetic and diabetic rats. In addition, nitroglycerin produced similar dilatation of cerebral arterioles in nondiabetic and diabetic rats, suggesting that impaired dilatation of cerebral arterioles in diabetic rats was not related to nonspecific impairment of vasodilatation. Next, we examined the possibility that impaired responses of cerebral arterioles in diabetic rats in response to ACh and ADP may be related to production of a cyclooxygenase constrictor substance. Indomethacin and the TxA2-prostaglandin (PG) H2 receptor antagonist SQ 29548 restored dilator responses to ACh and ADP in diabetic rats toward that observed in nondiabetic rats. Indomethacin and SQ 29548 did not alter responses in nondiabetic rats. Thus diabetes mellitus impairs endothelium-dependent responses of cerebral arterioles. The mechanism of impaired responses of cerebral arterioles during diabetes mellitus appears to be related to the production of a cyclooxygenase constrictor substance and presumably related to stimulation of the TxA2-PGH2 receptor.

Substance P modulates single-channel properties of neuronal nicotinic acetylcholine receptors.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.

Serotonin enhances the excitatory acetylcholine response in the RB cell cluster of Aplysia californica.

The RB cells, an identified cluster of serotonergic neurons in the abdominal ganglion of Aplysia californica, are excited by ACh. We have found that serotonin (5-HT) enhances the ACh responses recorded from the RB cells. The locus and specificity of the serotonergic interaction with the ACh response were analyzed using the conventional two-electrode voltage-clamp technique. We compared the current responses evoked in RB cells by constant-current iontophoretic pulses of ACh in control solutions and in solutions containing various concentrations of 5-HT. Serotonin consistently enhances the ACh evoked response in a dose-dependent manner while simultaneously directly activating the RB cells by turning on a small steady-state inward current. The dose-response characteristics for the 5-HT-mediated steady-state current differ, however, from those of the 5-HT-induced ACh response facilitation. The ACh response enhancement appears to be an effect of 5-HT upon RB cell ACh receptors since the enhancement is present under conditions that eliminate presynaptic input, and 5-HT is still capable of enhancing the iontophoretically evoked response when the AChE-resistant agonist carbachol is substituted for ACh. We tested other neurons in the Aplysia CNS exhibiting ACh evoked responses qualitatively similar to the RB cell ACh responses; 5-HT does not enhance the iontophoretically evoked ACh responses of these cells. Also, although the RB cells are responsive to dopamine and histamine, these putative neurotransmitters do not enhance the ACh response.

Evaluation of antibiotic efficacy using electron microscopy: morphological effects of guanylureido cephalosporin, chlorobenzoylureido cephalosporin, BL-P1654, and carbenicillin on Pseudomonas aeruginosa.

The response of Pseudomonas aeruginosa to carbenicillin, BL-P1654, and two cephalosporins (112384 and 112883) was evaluated by minimal inhibitory concentration determinations, [(14)C]leucine uptake studies, morphological studies of colonial growth, and mouse intraperitoneal inoculations. Spheroplast formation and bacterial lysis were not the early response; instead, cell division was inhibited and long filaments were formed. Spheroplast formation and bacterial lysis were not observed in the first 7 h of incubation in minimal inhibitory concentrations of antibiotic.

Evaluation of antibiotic efficacy using electron microscopy: morphological effects of guanylureido cephalosporin, BL-P1654, and carbenicillin on Escherichia coli.

Early response of Escherichia coli to minimal inhibitory concentrations of 112883, BL-P1654, and carbenicillin was determined by [(14)C]leucine uptake and scanning electron microscopy morphology studies. [(14)C]leucine uptake was inhibited later by carbenicillin than by BL-P1654 or 112883. Cellular swelling at 30 min, septal region swelling, and lysis were the progressive morphological changes with BL-P1654 and 112883. Carbenicillin-treated cells showed septal region swelling, beaded chains, and lysis.