Factors produced by activated macrophages reduce accumulation of Alzheimer's beta-amyloid protein in vascular smooth muscle cells.

Smooth muscle cells (SMCs) isolated from amyloid-angiopathy affected brain vessels accumulate intracellularly amyloid-beta peptide (A beta). Now we demonstrate that accumulation of A beta in SMCs can be reduced by factors secreted by macrophages - IL-1alpha, IL-6, TNF-alpha, TGF-beta1 or PGE2 - probably by stimulating the non-amyloidogenic processing of A beta precursor protein (PP). It is suggested that brain macrophages may regulate A betaPP/A beta metabolism under physiological conditions and prevent beta-amyloidosis. The disturbance of this regulatory function of brain macrophages may result in excessive production and accumulation of A beta.

Promotion of synthetic amyloid beta-peptide fibrillization by cell culture media and cessation of fibrillization by serum.

Dulbecco's modified Eagle's medium promotes aggregation and fibrillization of the synthetic amyloid beta 1-40 and beta 1-42 peptides more than RPMI and OPTI media. Fibrillization in all of these media is faster than in phosphate-buffered saline and Tris buffer. Normal and heat-inactivated fetal bovine and human serum abolish amyloid fibril formation in buffers and cell culture media. Fibrillar amyloid formed during 2-day-long incubation in cell culture media and buffers is defibrillized by 1-day-long treatment with human and bovine serum. This study indicates that amyloid beta fibrillogenesis in cell culture should be studied in serum-free media or in media with a low concentration of serum.

Binding of calmodulin to the neuronal cytoskeletal protein kinase type II cooperatively stimulates autophosphorylation.

The kinetics of autophosphorylation of the cytoskeletal form of the neuronal calmodulin-dependent protein kinase type II were studied as a function of calmodulin binding under the same conditions. Whereas calmodulin binding was noncooperative with respect to calmodulin concentration (Hill coefficient = 1), the activation of autophosphorylation and the phosphorylation of exogenous substrates showed marked positive cooperativity (Hill coefficient greater than or equal to 1.6). Reduction of the active calmodulin concentration by the addition of the calmodulin antagonist trifluoperazine confirmed the cooperative nature of enzyme activation, because autophosphorylation was more sensitive to the drug than was binding at high concentrations of calmodulin. At intracellular levels of calmodulin the binding and activation of autophosphorylation were cooperative functions of magnesium and calcium concentration. The calmodulin-dependent cooperative activation seems to be a unique feature of the cytoskeletal, but not the soluble, form of the protein kinase and may result from the supramolecular organization of the cytoskeletal enzyme. These observations suggest that interactions among the subunits of the oligomeric cytoskeletal calmodulin-dependent protein kinase regulate enzyme activation, enhancing the sensitivity of the enzyme to small changes in the intracellular calcium levels that may be particularly relevant to signaling at the synapse.

Competitive binding assay for drugs that interfere with calmodulin function.

A direct 125I-calmodulin-binding system utilizing a particulate cytoskeletal preparation of the neuronal calmodulin-dependent protein kinase type II provides a simple, rapid filtration-based method for assessing anti-calmodulin drug activity. The binding assay avoids potential artifacts due to direct drug effects on the standard phosphodiesterase enzyme assay while obtaining comparable IC50 values for a spectrum of drugs. It also provides a tool to probe mechanisms of action of putative anticalmodulin agents which may help elucidate selective pharmacologic modifications of pathophysiological processes.

Specific binding of solubilized adenylate cyclase to the erythrocyte cytoskeleton.

Concepts and criteria that have been developed for the study of the molecular organization of membrane-associated proteins are employed here to investigate the interaction of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with other membrane components. Detergent-solubilized adenylate cyclase can be shown to bind to erythrocyte-derived Triton X-100 shells containing cytoskeletal elements. This binding appears to be saturable with respect to adenylate cyclase concentration, and it is enhanced by the presence of divalent cations. Preactivation of the enzyme with 5'-guanylyl imidodiphosphate and isoproterenol, or with NaF, is a prerequisite for effective binding. Two exceptions to this general observation are noted: rat brain adenylate cyclase, which binds without prestimulation, and rat testicular cytosolic adenylate cyclase, which fails to bind under any of the conditions tried. The binding sites of the Triton X-100 shells are inactivated or released by treatment with various concentrations of trypsin or KCl. Moreover, exposure of the Triton X-100 shells to increasing temperatures results in a progressive loss of the adenylate cyclase binding capacity. On the basis of these and other findings, it is suggested that the adenylate cyclase complex possesses two principal domains that allow it to interact with both cytoskeletal elements and the lipid bilayer. The specific modulation of these interactions may be involved in the hormonal regulation of adenylate cyclase activity.

Activation of pigeon erythrocyte adenylate cyclase by cholera toxin. Partial purification of an essential macromolecular factor from horse erythrocyte cytosol.

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43,000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13,000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD+, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.

Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.

Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.

Properties of the interaction of fluoride- and guanylyl-5'-imidodiphosphate-regulatory proteins with adenylate cyclase.

The mechanism of activation of adenylate cyclase by guanylyl-5'-imidodiphosphate [Gpp(NH)p] and NaF has been investigated by studying the reconstitution of Gpp(NH)p and NaF sensitivity of an enzyme rendered insensitive to these agents by differential detergent extraction of a particulate brain enzyme. Such reconstitution can be achieved by the addition of macromolecular regulatory factors from membranes of various tissues. Trypsin digestion and thermal inactivation provide evidence for the existence of two distinct regulatory functions, one capable of restoring the Gpp(NH)p response and another the NaF response. The regulatory protein(s) seem to interact with their respective activators in an easily reversible, divalent cation-independent reaction. This appears to be followed by a high-affinity interaction between the catalytic and regulatory components of adenylate cyclase in a slow, temperature-dependent, divalent cation-dependent process tha produces the persistently activated state of the enzyme. The enzyme activation can be reversed by methods that separate catalytic from regulatory components and the resulting enzyme activity can be restimulated by the reconstitution technique.