A model with original conditions and materials to test potential inhibitors of cyclooxygenase-2 versus cyclooxygenase-1 in isolated human blood cells.

The inflammatory process can be influenced by an enzyme known as cyclooxygenase, which exists in two isoforms: COX-1, the constitutive form, and COX-2, which is only synthesized in the case of inflammation and has negative side effects. To decrease these undesirable effects, current research seeks to discover compounds that selectively inhibit COX-2 versus COX-1. When added to the samples, arachidonic acid activates the synthesis of COX-2. This explains why the two isoforms proceed in different ways. As for COX-1, human lymphocytes and monocytes were activated with 50 microM of calcium ionophore for 1 h at 37 degrees C with 50 microM of arachidonic acid, and the quantity of thromboxane B(2) (TxB(2)) was measured by radioimmuno-assay (RIA). As for COX-2, cells were first incubated with acetylsalicylic acid to block COX-1, then stimulation of COX-2 was performed using 8 mcg/ml lipopolysaccharide (LPS) for 3 h at 37 degrees C with 50 microM arachidonic acid. The IC(50) values for reference compounds were reproducible, taking into consideration the passage through the plasmatic membrane and the transduction signal, while avoiding the problems of binding with plasma proteins and using identical cells, unlike other models.

Investigation of the inhibitory effects of HA-1077 and Y-32885 on the translocation of PKCbetaI, PKCbetaII and PKCzeta in human neutrophils.

To transmit the information inside the cell, one possibility is the action of an enzyme called kinase that phosphorylates other proteins. To study these enzymes, chemical compound synthesis was needed to know the function and the mechanism of activation. The major difficulty is creating a specific molecule for one kinase. In this study, we test the action of Rho-kinase inhibitors (HA-1077 and Y-32885) on protein kinase C (PKC) in the respiratory burst in the human polymorphonuclear neutrophils. We have shown that these compounds could inhibit the anion superoxide production. To prove their action on PKC, we have shown a decrease of binding of a specific ligand (phorbol-12,13-dibutyrate) with each inhibitor. During its activation, PKC was translocated from the cytoplasm to the plasmic membrane. We have also shown an inhibition of this translocation, proving an inhibition of PKC by HA-1077 and Y-32885.

Investigation of the inhibitory effects of chelerythrine chloride on the translocation of the protein kinase C betaI, betaII, zeta in human neutrophils.

The protein kinase C (PKC) is a serine/threonine kinase, consisting of different isoforms, implicated in numerous processes of signal transduction. To understand this enzyme well, different pharmacological tools were developed. To activate PKC specifically, phorbol esters were previously used but recent research has shown that these compounds are able to stimulate other proteins. Our model is the respiratory burst in the polymorphonuclear neutrophils. A decrease in the inflammatory process was measured using chelerythrine chloride. Action on PKC was proved by a binding study and by showing the absence of translocation of this enzyme from the cytoplasm to the plasmic membrane during stimulation.