Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters

C. D. Smith, R. J. Uhing, R. Snyderman

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110 Scopus citations

Abstract

Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment with the PMNs with pertussis toxin (PT) or 4-β-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTPγ[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 μM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTPγS binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTPγS alone at low concentrations of Ca2+ (0.1-1 μM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTPγS, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTPγS. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol-ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.

Original languageEnglish (US)
Pages (from-to)6121-6127
Number of pages7
JournalJournal of Biological Chemistry
Volume262
Issue number13
StatePublished - 1987

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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