The release of amyloidogenic amyloid-␤ peptide (A␤) from amyloid-␤ precursor protein (APP) requires cleavage by ␤-and ␥-secretases. In contrast, ␣-secretase cleaves APP within the A␤ sequence and precludes amyloidogenesis. Regulated and unregulated ␣-secretase activities have been reported, and the fraction of cellular ␣-secretase activity regulated by protein kinase C (PKC) has been attributed to the ADAM (a disintegrin and metalloprotease) family members TACE and ADAM-10. Although unregulated ␣-secretase cleavage of APP has been shown to occur at the cell surface, we sought to identify the intracellular site of PKC-regulated ␣-secretase APP cleavage. To accomplish this, we measured levels of secreted ectodomains and C-terminal fragments of APP generated by ␣-secretase (sAPP␣) (C83) versus ␤-secretase (sAPP␤) (C99) and secreted A␤ in cultured cells treated with PKC and inhibitors of TACE/ ADAM-10. We found that PKC stimulation increased sAPP␣ but decreased sAPP␤ levels by altering the competition between ␣-versus ␤-secretase for APP within the same organelle rather than by perturbing APP trafficking. Moreover, data implicating the trans-Golgi network (TGN) as a major site for ␤-secretase activity prompted us to hypothesize that PKC-regulated ␣-secretase(s) also reside in this organelle. To test this hypothesis, we performed studies demonstrating proteolytically mature TACE intracellularly, and we also showed that regulated ␣-secretase APP cleavage occurs in the TGN using an APP mutant construct targeted specifically to the TGN. By detecting regulated ␣-secretase APP cleavage in the TGN by TACE/ADAM-10, we reveal ADAM activity in a novel location. Finally, the competition between TACE/ADAM-10 and ␤-secretase for intracellular APP cleavage may represent a novel target for the discovery of new therapeutic agents to treat Alzheimer's disease.Posttranslational processing of the amyloid-␤ precursor protein (APP) 1 is implicated in the pathology of Alzheimer's disease (AD). The amyloidogenic amyloid-␤ peptide (A␤) fragment is generated through an initial APP cleavage by ␤-secretase(s) to define the NH 2 terminus of A␤. This cleavage generates sAPP␤, a secreted ectodomain of APP, and C99, the 99-amino acid C-terminal fragment that contains the transmembrane and cytoplasmic domains of APP. C99 is further cleaved by ␥-secretase(s), releasing the A␤ peptide, which is either 40 or 42 amino acids long (i.e. A␤ 1-40 and A␤ ; reviewed in Ref. 1). Because the majority of secreted A␤ appears to be generated in the trans-Golgi network (TGN), this is likely to be a major locus for ␤-and ␥-secretase activities (2). In contrast to secreted A␤, intracellular A␤ 1-42 is generated in the endoplasmic reticulum (ER)/intermediate compartment (IC) (3-6). This suggests that both ␤-secretase and an A␤ 1-42 generating ␥-secretase are also active in the ER/IC.In addition to the TGN and ER/IC amyloidogenic processing pathways, APP can be processed by ␣-secretase, which cleaves APP at position 16 within the A␤ domain to generate sAPP␣ (the ect...

Bladder contraction is mediated by a number of intracellular biochemical reactions including glycolysis, mitochondria1 respiration, phosphocreatine kinase, and various ATPases. In order to understand how alterations in intracellular metabolism can affect bladder function (or how alterations in bladder function can affect intracellular metabolism), we must first understand and describe normal bladder metabolism. Using the rabbit as a model for bladder function, the present study compares the intracellular concentrations of a variety of important compounds between the bladder base and body.The intracellular concentrations of phosphocreatine (PC), creatine, ATP, ADP, and AMP were determined in samples of rabbit urinary bladder body and base. The values for PC and creatine were significantly lower in the bladder base than in the bladder body. Adenine nucleotide concentrations were similar in the two sections.The concentrations of NAD + NADH, NADP, CTP, CDP, CMP, GTP, GDP, GMP, and UTP, UDP, UMP were also measured. Of these compounds, the concentrations of GTP and UTP in the bladder body were nearly double those in the bladder base. The intracellular concentrations of the other tissue constituents were similar in the two bladder sections.These studies demonstrate that the bladder is not biochemically homogeneous but that the bladder base and body differ not only functionally but also metabolically.Experiments in which strips of bladder body were incubated in vitro in oxygenated Tyrode's solution containing glucose showed that the tissue concentrations of PC, ATP, GTP, and UTP decreased substantially following 30 min of incubation, with no further decrease during a subsequent 60-min incubation.