Glutamine utilization by rat neutrophils: presence of phosphate-dependent glutaminase

Abstract

The capacity of rat neutrophils to utilize glutamine was investigated by 1) determination of oxygen consumption in the presence of glucose or glutamine, 2) measurement of maximal activity of phosphate-dependent glutaminase, 3) Northern blot, Western blot, and immunocytochemical detection of glutaminase, and 4) measurement of glutamine utilization and also production of ammonia, glutamate, aspartate, alanine, and lactate and decarboxylation of [U-14C]glutamine in cells incubated for 1 h. The rate of respiration by isolated neutrophils in the absence of added substrate was 5.0 nmol ⋅ min−1 ⋅ 107cells−1. Maximal activity of phosphate-dependent glutaminase was 56 nmol ⋅ min−1 ⋅ mg protein−1 in freshly obtained neutrophils; the Michaelis-Menten constant was 3.5 mM for glutamine. This enzyme activity was inhibited by 2 mM glutamate, 2 mM oxoglutarate, and 2 mM NH4Cl. The presence of glutaminase protein (65 kDa) was confirmed by Western blot and immunocytochemical detection and the presence of the mRNA (6.0 kb) by Northern blot analysis. Glutamine was utilized by neutrophils incubated for 1 h at a rate of 12.8 nmol ⋅ min−1 ⋅ mg protein−1 when the amino acid was added to the medium at 2 mM, which is three to four times higher than the physiological concentration. In the presence of 0.5 mM glutamine, the amino acid was utilized at a rate of 2.9 nmol ⋅ min−1 ⋅ mg protein−1. The addition of 0.5 mM glutamate to the incubation medium caused a marked reduction (by 70%) in glutamine utilization by neutrophils. Glucose was utilized at 7.7 nmol ⋅ min−1 ⋅ mg protein−1 when cells were incubated in 5 mM glucose. The conversion of [U-14C]glutamine to14CO2was very low: <1% was totally oxidized. The formation of ammonia was ∼27% of glutamine utilization, and the conversion of glutamine to glutamate, aspartate, alanine, and lactate accounted for ∼84.6% of the total amino acid utilized by neutrophils. In this study, evidence is presented that, in addition to lymphocytes and macrophages, neutrophils also utilize glutamine.

Polyunsaturated Fatty Acids, Ulcerative Colitis and Cancer Prevention

“…Different adhesion molecules participate in these interactions, including selectins, integrins and intercellular adhesion molecules (ICAM) (Rang & Dale 1995). Neutrophils constitute 60% of circulating leukocytes and act as the first line of cellular defence, and they may participate in both reactions as a nonspecific defence and as specific antigen reactions (Curi et al 1997). Monocytes represent approximately 3-6% of circulating leukocytes in human blood, and they migrate to different tissues where they differentiate into macrophages in response to different stimuli.…”

UCP2, a metabolic sensor coupling glucose oxidation to mitochondrial metabolism?

“…Interestingly, glutamine is also a potent enhancer of UCP2 translation (19). Glutamine is known to be an essential oxidative fuel in immune cells (43,44). In these cells, whereas glucose is essentially converted to lactate, glutamine provides metabolic intermediates such as alphaketoglutarate for the TCA cycle.…”

Glutamine and glutamate as vital metabolites

“…It is now widely accepted that glutamine is utilized at high rates by isolated cells of the immune system such as lymphocytes, mac-rophages and neutrophils (42)(43)(44). Although the activity of the first enzyme responsible for the metabolism of glutamine, phosphatedependent glutaminase, is high in these cells, the rate of oxidation is low.…”