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 ∼63 and 69 kDa (γ- and α-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-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), the intracellular Ca2+ 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 IC50 values very similar to those that interrupt CaM-dependent reactions in vitro. In contrast, less active analogues of these compounds (1-[6-((17β-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 Ca2+/CaM complex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases.

Activation signals from bacterial stimuli set into motion a series of events that alter the abbreviated lifespan of neutrophils. These studies show that the bacterial chemoattractant, formyl-Met-Leu-Phe (fMLP), promotes the phosphorylation/inactivation of the FOXO subfamily of forkhead transcription factors (FKHR, FKHR-L1, and AFX) through the phosphatidylinositol-3-kinase/Akt (protein kinase B) and the RAS mitogen-activated protein kinase pathways. Furthermore, fMLP stimulation causes the inducible expression of the prosurvival Bcl-2 family member Mcl-1, which then binds to a complex containing FKHR. These studies show that fMLP-stimulated neutrophils coordinate the regulation of FOXO transcription factors and the survival factor Mcl-1, a mechanism that may allow neutrophils to alter their survival.

OBJECTIVEPreemptive administration of analgesic medication is more effective than medication given after the onset of the painful stimulus. The efficacy of preoperative or preemptive pain relief after thoracolumbosacral spine surgery has not been well studied. The present study was a double-blind, placebo-controlled randomized trial of preemptive analgesia with a single-shot epidural injection in adult patients undergoing spine surgery.METHODSNinety-nine adult patients undergoing thoracolumbosacral operations via a posterior approach were randomized to receive a single shot of either epidural placebo (group 1), hydromorphone alone (group 2), or bupivacaine with hydromorphone (group 3) before surgery at the preoperative holding area. The primary outcome was the presence of opioid sparing and rescue time—defined as the time interval from when a patient was extubated to the time pain medication was first demanded during the postoperative period. Secondary outcomes include length of stay at the postanesthesia care unit (PACU), pain score at the PACU, opioid dose, and hospital length of stay.RESULTSOf the 99 patients, 32 were randomized to the epidural placebo group, 33 to the hydromorphone-alone group, and 34 to the bupivacaine with hydromorphone group. No significant difference was seen across the demographics and surgical complexities for all 3 groups. Compared to the control group, opioid sparing was significantly higher in group 2 (57.6% vs 15.6%, p = 0.0007) and group 3 (52.9% vs 15.6%, p = 0.0045) in the first demand of intravenous hydromorphone as a supplemental analgesic medication. Compared to placebo, the rescue time was significantly higher in group 2 (187 minutes vs 51.5 minutes, p = 0.0014) and group 3 (204.5 minutes vs 51. minutes, p = 0.0045). There were no significant differences in secondary outcomes.CONCLUSIONSThe authors’ study demonstrated that preemptive analgesia in thoracolumbosacral surgeries can significantly reduce analgesia requirements in the immediate postoperative period as evidenced by reduced request for opioid medication in both analgesia study groups who received a preoperative analgesic epidural. Nonetheless, the lack of differences in pain score and opioid dose at the PACU brings into question the role of preemptive epidural opioids in spine surgery patients. Further work is necessary to investigate the long-term effectiveness of preemptive epidural opioids and their role in pain reduction and patient satisfaction.Clinical trial registration no.: NCT02968862 (clinicaltrials.gov)

Stimulation of neutrophils with the chemoattractant fMet-Leu-Phe (fMLP) triggers phosphorylation/inactivation of the K K-and L L-isoforms of glycogen synthase kinase 3 (GSK-3) with phosphorylation of the K K-isoform predominating. These reactions were monitored with a phosphospecific antibody that only recognized the K K-or L L-isoforms of GSK-3 when these proteins were phosphorylated on serine residues 21 and 9, respectively. Inhibitor studies indicated that phosphorylation of GSK-3K K may be catalyzed by the combined action of p90-RSK and Akt and may represent a new strategy by which G proteincoupled receptors inactivate GSK-3. Inactivation of GSK-3 may be one of the mechanisms that delay apoptosis in fMLPstimulated neutrophils. ß