II. Phorbol ester binding to a human lymphoblastoid B-cell line, LA350, stimulates 32P incorporation into selected phospholipids and immunoglobulin secretion

Autor: Howard M. Rosenblatt, William T. Shearer, Karyl S. Barron, Frank M. Orson, Ellen B. Gilliam
Rok vydání: 1988
Předmět:
Zdroj: Cellular Immunology. 111:316-331
ISSN: 0008-8749
DOI: 10.1016/0008-8749(88)90096-2
Popis: Anti-mu antibody binds to surface IgM on LA350, a transformed human B-cell line, and causes the immediate (5 min) hydrolysis of phosphatidylinositol (PI) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol followed by a subsequent (48-72 hr) increase in immunoglobulin M (IgM) production. Phorbol myristate acetate (PMA) in a dose-dependent fashion inhibited completely the anti-mu-stimulated hydrolysis of PI and its resynthesis (PI cycle) from phosphatidic acid (PA) (P less than 0.001). Phorbol dibutyrate (PD), but not the inactive methyl ester derivative of PMA (PMA-ME), inhibited the anti-mu stimulation of the PI cycle (P less than 0.001). Conversely, PMA and PD, but not PMA-ME, stimulated in a dose-dependent fashion the metabolic events consistent with an activation of a putative phosphatidylcholine (PC) cycle. For example, at 10(-8) M PMA there was a 300% increase in the acute (1 hr) incorporation of [3H]choline into PC (P less than 0.001), a 680% increase in the acute (1 hr) incorporation of 32P into PC (P less than 0.001), but no net synthesis of PC as measured by the lack of PMA-stimulated incorporation of 32P into PC in LA350 prelabeled for 24 hr. Also in cells labeled to equilibrium with [3H]choline and in pulse-chase experiments we established that PMA produces a rapid incorporation of choline phosphate into PC and a rapid breakdown of PC, yielding choline metabolites released as choline itself into external medium surrounding the cell. Binding studies with [3H]PD demonstrated a dissociation constant of 20 mM and 5.3 x 10(5) total binding sites per cell. PMA was as effective as cold PD in inhibiting [3H]PD binding (P less than 0.001), but PMA-ME was ineffective. PMA and PD, but not PMA-ME, produced a similar dose-dependent (maximal at 10(-8) M) increase (300%) in immunoglobulin production as measured by either an ELISA assay or a reverse hemolytic plaque assay (P less than 0.001). Thus, activation of either the PI or the PC cycle results in significant enhancement in immunoglobulin production in LA350. Although PMA turns off the PI cycle, it turns on the PC cycle. A common mechanism to explain these findings might be the activation of protein kinase C, indirect via diacylglycerol release in the PI cycle stimulation by anti-mu and direct in the PC cycle stimulation by PMA by virtue of direct binding to protein kinase C.
Databáze: OpenAIRE