Abstract

Polymorphonuclear leukocytes, PMNs, incubated in a chemoattractant undergo a time-dependent decrease in responsiveness to the chemoattractant; i.e. they desensitize or adapt. We have examined the role of ligand-induced changes at early steps in signal transduction for adaptation of PMNs to chemoattractants. The chemoattractant stimulation of a pertussis toxin-sensitive GTPase activity on PMN membranes was used as an assay of signal transduction. We find a decreased basal GTPase activity and a decrease in the ability of N-formylnorleucylleucylphenylalanine (FN-LLP) to stimulate this activity on membranes prepared from PMNs incubated with the chemotactic peptide FNLLP. The basal GTPase activity is decreased by up to 70% and the peptide-stimulated GTPase activity by up to 95% on membranes from PMNs incubated for 20 min at 37 degrees C in 10(-7) M FNLLP. The decrease in peptide-stimulated GTPase activity cannot be accounted for by the decreased number of FNLLP receptors on the membranes. Rather, receptors that remain available for binding stimulate the GTPase activity with a decreased efficiency. The ligand-induced change in GTPase activity is not stimulus specific. GTPase activity stimulated by both C5a and LTB4 was decreased on membranes from PMNs incubated in FNLLP. The decrease in chemoattractant-stimulated GTPase activity is partially reversed if cells are subsequently incubated at 37 degrees C in the absence of peptide prior to membrane preparation. We detected no quantitative or qualitative change in either pertussis toxin substrates or immunoreactive G proteins when membranes from control and FNLLP-treated cells were compared.

Highlights

  • Polymorphonuclear leukocytes, PMNs, incubated in a chemoattractant undergo a time-dependent decrease in responsiveness to the chemoattractant; i.e. they desensitize or adapt

  • Crude membrane fractions from PMNs have a basal GTPase activity in the absence of added ligand.This activity was decreased by more than 50% on membranes prepared from PMNs treated with pertussis toxin

  • This basal pertussis toxin-sensitive activity could be due to endogenous GTPase activity of the Gproteins

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Summary

MATERIALS ANDMETHODS

Reagents-Phosphocreatine and creatine phosphokinase were obtained from Boehringer Mannheim. At the end of the experiment, the dishes were washed for 15 min with three exchanges of 4 "C 0.9% NaCl. Crude Membrane Fraction-Cells pelleted from suspension or scraped from dishes were resuspended in 50 mM Tris-HC1, 10 mM MgClz,1mM phenylmethylsulfonyl fluoride, 50 mM @-glycerolphosphate, and 2.5 mM EGTA, pH 7.4, a t 1X lo' cells/ml and were broken by nitrogen cavitation (350psi for 15min, 4 "C)T. his cell homogenate was spun at 100 X g for 9 min to remove unbroken cells and nuclei. 27,000 X g for 15 min a t 4 "C.The pellet was resuspended to 0.5-1 mg/ml protein in 10 mM potassium phosphate buffer containing 0.25 M sucrose, 2 mM EGTA, and 10 mMMgC12, pH 7.2, aliquoted, and frozen a t -70 'C. Free phosphate detected (in the absence of membranes) was between 1-2% of total counts added. England Nuclear 46 Ci/mmol) to membrane fractions was determined by incubating 25 pg ofmembrane protein with 1X lo-' M [3H]FNLLP inthe presence or absence of 1 X. Release of 32Pifrom [ T - ~ ~ P ] G TwPas measured a t various timesin the presence (solid) and absence (open) of M FNLLP

RESULTS
GTPase activity
Chemoattractant Effects on PMNMembrane GTPase activity
DISCUSSION
Chemoattractant Effects on PMN Membrane GTaPcatisveity
Full Text
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