Inhibition of tumor necrosis factor-alpha (TNF-alpha) production and arthritis in the rat by GW3333, a dual inhibitor of TNF-alpha-converting enzyme and matrix metalloproteinases.

Tumor necrosis factor-alpha (TNF)-converting enzyme (TACE) cleaves the precursor form of TNF, allowing the mature form to be secreted into the extracellular space. GW3333, a dual inhibitor of TACE and matrix metalloproteinases (MMPs), was compared with an anti-TNF antibody to evaluate the importance of soluble TNF and MMPs in rat models of arthritis. Oral administration of GW3333 completely blocked increases in plasma TNF after LPS for up to 12 h. In a model wherein intrapleural zymosan injection causes an increase in TNF in the pleural cavity, GW3333 completely inhibited the increase in TNF in the pleural cavity for 12 h. Under these dosing conditions, the plasma levels of unbound GW3333 were at least 50-fold above the IC(50) values for inhibition of individual MMPs in vitro. In a model wherein bacterial peptidoglycan polysaccharide polymers reactivate a local arthritis response in the ankle, a neutralizing anti-TNF antibody completely blocked the ankle swelling over the 3-day reactivation period. GW3333 administered b.i.d. over the same period also inhibited ankle swelling, with the highest dose of 80 mg/kg being slightly less active than the anti-TNF antibody. In a 21-day adjuvant arthritis model, the anti-TNF antibody did not inhibit the ankle swelling or the joint destruction, as assessed by histology or radiology. GW3333, however, showed inhibition of both ankle swelling and joint destruction. In conclusion, GW3333 is the first inhibitor with sufficient duration of action to chronically inhibit TACE and MMPs in the rat. The efficacy of GW3333 suggests that dual inhibitors of TACE and matrix metalloproteinases may prove therapeutic as antiarthritics.

Extracts of Ginkgo biloba leaves inhibit monoamine oxidase.

Extracts of Ginkgo biloba leaves produce reversible inhibition of rat brain monoamine (MAO). Both MAO-A and -B types were inhibited to a similar extent. The MAO inhibitory compound(s) were present in dried or fresh Ginkgo biloba leaves as well as in commercially available capsules of Ginkgo biloba and appear to be heat stable with relatively low molecular weight. MAO inhibition by Ginkgo biloba may be a mechanism underlying reported anti-stress and anxiolytic activities of this natural product.

Stimulation of carnitine acetyltransferase in PC12 cells by nerve growth factor: relationship to choline acetyltransferase stimulation.

The activity of carnitine acetyltransferase (acetyl-CoA:L-carnitine O-acetyltransferase) was found to be at least 50-fold higher than that of choline acetyltransferase in PC12 cells. Nerve growth factor stimulated both enzymes in a parallel manner with respect to concentration of NGF and culture time. The stimulation of both enzymes was completely inhibited by 10 microM 6-thioguanine, an inhibitor of protein kinase N. Results are discussed with reference to the hypothesis that the two enzymes may be functionally related in neuronal cells.

Effects of GTP?S and other nucleotides on phosphoinositide metabolism in crude rat brain synaptosomal preparations.

Crude nerve-ending preparations from rat brain were labeled with either [(32)P]phosphate or myo[2-(3)H]inositol in order to observe effects of guanosine 5?-[?-thio]triphosphate (GTP?S) and other nucleotides on phosphoinositides, phosphatidate and inositol phosphates. This system exhibited typical responses to muscarinic agonists, including acetylcholine-a decrease in net labeling of [(32)P]polyphosphoinositides, an increase in labeling of [(32)P]phosphatidate, and a stimulation of [(3)H]inositol phosphate formation. GTP?S and other nucleotides may not readily penetrate intact synaptosomal membranes to cause activation of phospholipase C via an interaction with G proteins, and, as might be expected, there was no indication that G-protein interaction occurred in these preparations. However, other effects were observed. GTP?S decreased net [(32)P] incorporation in phosphatidylinositol (PI) and polyphosphoinositides in a dose-dependent manner. GTP?S also caused an initial marked stimulation of [(32)P] labeling of phosphatidate, suggesting a possible inhibition in the conversion of phosphatidic acid to PI. Other nucleotides [GTP, ATP, Gpp(NH)p, GMP] produced qualitatively similar effects on phosphoinositides. Thus GTP?S and other nucleotides, at physiologically relevant concentrations, may influence phosphoinositide turnover via extracellular or other mechanisms, in addition to the proposed interaction of GTP with G-proteins within membranes.

Acetyl-L-carnitine as a precursor of acetylcholine.

Synthesis of [3H]acetylcholine from [3H]acetyl-L-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [3H] and [14C] labeled acetylcholine were produced when [3H]acetyl-L-carnitine and D-[U-14C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-L-carnitine to acetylcholine was dependent on concentration of acetyl-L-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-L-carnitine in the treatment of age-related cholinergic deficits.