The recent demonstration of K+ channel dysfunction in fibroblasts from Alzheimer disease (AD) patients and past observations of Ca2+-mediated K+ channel modulation during memory storage suggested that AD, which is characterized by memory loss and other cognitive deficits, might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin-induced Ca2+ release, which is inositol trisphosphate-mediated, is shown here to be greatly enhanced in AD fibroblasts compared with fibroblasts from control groups. Bradykinin, another activator of phospholipase C, elicits similar enhancement of Ca2+ signaling in AD fibroblasts. By contrast, thapsigargin, an agent that releases Ca2+ by direct action on the endoplasmic reticulum, produced no differences in Ca2+ increase between AD and control fibroblasts. Depolarization-induced Ca2+ influx data previously demonstrated the absence of between-group differences of Ca2+ pumping and/or buffering. There was no correlation between the number of passages in tissue culture and the observed Ca2+ responses. Furthermore, cells of all groups were seeded and analyzed at the same densities. Radioligand binding experiments indicated that the number and affinity of bombesin receptors cannot explain the observed differences. These and previous observations suggest that the differences in bombesin and bradykinin responses in fibroblasts and perhaps other cell types are likely to be due to alteration of inositol trisphosphatemediated release of intracellular Ca2+.A number of cellular changes have been observed in fibroblasts from patients with Alzheimer disease (AD). These include abnormality of glucose and energy-related metabolism (1), defective release of a cholinergic factor (2), abnormal f8-amyloid expression and processing (3), changes in Ca2+ metabolism (30-34), and altered p-adrenergic-induced cAMP formation (4). The recent demonstration of K+ channel dysfunction in AD fibroblasts (5, 6) and past observations of Ca2+-mediated K+ channel modulation during memory storage (7) suggested that AD, which is characterized by memory loss and other cognitive deficits (8, 9), might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin (10-12), an agent that activates phospholipase C (PLC) to generate inositol 1,4,5-trisphosphate (1P3) (13)(14)(15) different for AD and control fibroblasts. f-Amyloid protein (23-25) itself, while causing the previously observed inactivation of K+ channels in AD fibroblasts, had no effect on the bombesin-elicited Ca2+ signals. These and other findings, together with measurements of bombesin receptor number, suggest that PLC/G-protein coupling and/or IP3 receptors are responsible for differences in Ca2+ responses between AD and non-AD fibroblasts. METHODSCell Lines. Human skin fibroblasts (Table 1) were purchased from the Coriell Cell Repositories (Camden, NJ). Cells were seeded and maintained as described (5). The number of passages was not significantly different between groups [AD, 10.9 ± 1.3 (mean ± SEM), n = 10; AC, 11.5 + 0.8, n = 8;...

Since memory loss is characteristic of Alzheimer disease (AD), and since K+ channels change during acquisition of memory in both molluscs and mammals, we investigated K+ channel function as a possible site of AD pathology and, therefore, as a possible diagnostic index as well. A 113-pS tetraethylammonium (TEA)-sensitive K+ channel was consistently absent from AD fibroblasts, while it was often present in young and aged control fibroblasts. A second (166-pS) K+ (28). Membrane potential was measured both prior to obtaining outside-out patches and by using the "perforated-patch" technique (28,37). Before recording, culture medium was replaced with the following solution (mM): NaCl 150, KCI 5, CaCl2 2, MgCl2 1, Hepes (NaOH) 10, pH = 7.4. Pipettes were made from Blue Tip capillary tubes (i.d. 1.1-1.2 mm) by using a BB-CH Mecanex puller and then filled with a high-K+ solution (mM): KCI 140, CaCl2 2, MgCl2 1, Hepes (NaOH) 10, pH = 7.4. Pipette resistances were -6 MQl. Records were obtained by using an Axopatch-1C amplifier (dc to 10 kHz), stored on tape (Toshiba PCM video recorder), and later transferred to a personal computer using an Axolab interface. Only recordings lasting for at least 3 min were considered for final analysis. The pClamp suite of programs was used for singlechannel data acquisition and analysis. Amplifier, interface, and software were obtained from Axon Instruments (Foster

The experiments in this paper identify multiple calcium compartments in cultured human fibroblasts and reveal abnormalities in one of these pools in cells from Alzheimer patients. In the presence of external calcium, bradykinin (BK) increased cytosolic free calcium ([Ca2+]i) about 3-fold and then [Ca2+]i rapidly declined. Omission of calcium from the media did not affect the BK-induced peak, which indicates that the peak reflects internal stores. Other compounds that also released calcium from internal stores included A23187 (a calcium ionophore), thapsigargin (Tg; an inhibitor of endoplasmic reticulum ATPase), and FCCP (an uncoupler of oxidative phosphorylation). The [Ca2+]i response to sequential addition of compounds in calcium-free media identified discrete internal calcium stores. BK depleted internal calcium pools such that subsequent stimulation with BK, FCCP or bombesin did not increase [Ca2+]i. However, A23187 or thapsigargin still elicited responses. A23187 depleted essentially all internal calcium pools. Either Tg or FCCP reduced the calcium stores that could be released by BK or A23187. Thus, cellular calcium compartments that respond to BK and A23187 partially overlap. The common pool includes Tg-and FCCP-sensitive compartments. Calcium stores were examined in cells from Alzheimer disease patients, because previous studies suggest that their calcium homeostasis is altered. A23187 addition to BK-treated cells produced a 95% greater response in cell lines from Alzheimer patients (n = 7) than in those from controls (n = 5). Thus, various calcium stores can be pharmacologically distinguished in fibroblasts and at least one of these compartments is abnormal in Alzheimer's disease.