Antagonists of calcium fluxes and calmodulin block activation of the p21-activated protein kinases in neutrophils.
Neutrophils stimulated with fMLP or a variety of other chemoattractants that bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecular masses of approximately 63 and 69 kDa (gamma- and alpha-Pak). Previous studies have shown that products of phosphatidylinositol 3-kinase and tyrosine kinases are required for the activation of Paks. We now report that a variety of structurally distinct compounds which interrupt different stages in calcium/calmodulin (CaM) signaling block activation of the 63- and 69-kDa Paks in fMLP-stimulated neutrophils. These antagonists included selective inhibitors of phospholipase C (1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), the intracellular Ca(2+) channel (8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate), CaM (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), and CaM-activated protein kinases (N-[2-(N-(chlorocinnamyl)-N:-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide). This inhibition was dose-dependent with IC(50) values very similar to those that interrupt CaM-dependent reactions in vitro. In contrast, less active analogues of these compounds (1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(4-aminobutyl)-1-naphthalenesulfonamide; promethazine; 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine]) did not affect activation of Paks in these cells. CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), but not their less-active analogues (N-(6-aminohexyl)-1-naphthalenesulfonamide; promethazine), were also found to block activation of the small GTPases Ras and Rac in stimulated neutrophils along with the extracellular signal-regulated kinases. These data strongly suggest that the Ca(2+)/CaM complex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases.
Lian
J P
JP
Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
Crossley
L
L
Zhan
Q
Q
Huang
R
R
Coffer
P
P
Toker
A
A
Robinson
D
D
Badwey
J A
JA
D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D007424:Intracellular Fluid / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D002147:Calmodulin; D004791:Enzyme Inhibitors; D013449:Sulfonamides; C040184:W 12; D009240:N-Formylmethionine Leucyl-Phenylalanine; C017967:W 7; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D020744:rac GTP-Binding Proteins; D018631:ras Proteins; D002118:Calcium