Harold P. Jones

Calmodulin-dependents stimulation of the NADPH oxidase of human neutrophils

The NADPH oxidase of human neutrophils is highly sensitive to calcium concentration and is inhibited in intact cells and cell-free preparations by various phenothiazine drugs. Addition of calmodulin to preparations of NADPH oxidase stimulates enzymatic rates from 1.4-2.5-fold. Addition of calmodulin and calcium, but not calcium alone, to NADPH oxidase preparations which have been inactivated by EDTA results in the restoration of activity. No activation is observed when membrane preparations containing latent NADPH oxidase are exposed to calcium and calmodulin. These studies suggest a role for calmodulin in the control of NADPH oxidase but that calmodulin alone is not sufficient for activation.

A comparative study of neutrophil purification and function

Several different methods are currently used by numerous laboratories for the isolation of human neutrophils. These methods include partial purification by dextran sedimentation followed by water lysis, and more complete purification procedures utilizing discontinuous density gradients coupled with dextran sedimentation and in some cases hypotonic lysis. Some investigators refrain from using certain purification schemes because certain steps or reagents used in a particular method might adversely affect the functional parameter of the neutrophil they wish to measure. In spite of these concerns there has been no systematic comparison of the functional status of neutrophils prepared by the various methodologies. In this study we have compared 4 commonly used methods of isolation with neutrophil function. The results of this study indicate that while neutrophil yield and purity were determined by isolation procedure, all cells were equivalent with regard to chemotactic performance, ability to degranulate, and ability to produce superoxide and hypochlorous acid.

Calmodulin-dependent NAD kinase of human neutrophils

NAD kinase from human neutrophils has been partially purified by sequential application of Red Agarose, ion-exchange, and gel-filtration chromatography. The enzyme has a broad pH optimum, 7.0-9.5, is strictly dependent upon the presence of Mg2+, and in the absence of calcium exhibits Km values of 0.6 and 0.9 mM for NAD and ATP, respectively. NAD kinase activity is extremely sensitive to free calcium concentration, with half-maximal activity observed at free calcium concentrations of approximately 0.4 microM. In cellular extracts calcium-dependent activation of NAD kinase increases the maximum velocity of the reaction from 2- to 5-fold while not affecting Km values for NAD and ATP. The activity of the partially purified NAD kinase is stimulated 3.5-fold by the addition of calmodulin in the presence of calcium. This stimulation is inhibited by the addition of 20 microM trifluoperazine to the incubation. These data are interpreted as implicating calmodulin in NAD kinase regulation. The total concentration of NADP + NADPH in the human neutrophil used increased 2.2-fold in response to activation by phorbol myristic acetate. Finally, neutrophil NAD kinase has a Mr, based upon gel filtration, of 169,000.

Effect of allopurinol on neutrophil superoxide production, chemotaxis, or degranulation☆

Recent studies examining the effect of allopurinol on bacterial killing by leukocytes [Tubaro et al., Biochem. Pharmac. 29, 3018 (1980); Tritsch and Neiswander, Life Sci. 32, 1359 (1983)] have been interpreted by those authors as proof that xanthine oxidase is the major superoxide producing enzyme in activated leukocytes. To test the assertion that xanthine oxidase is involved in the production of superoxide by activated human neutrophils, the xanthine oxidase content of neutrophils was measured, and the effect of allopurinol on neutrophil functions, including superoxide production, was studied. Neutrophils were found to contain a level of xanthine oxidase insufficient to account for the flux of superoxide associated with neutrophil activation. Allopurinol did not inhibit superoxide production induced by opsonized zymosan, phorbol myristic acetate, or formylmethionylleucylphenylalanine. Furthermore, neither chemotaxis nor degranulation was affected by allopurinol. Allopurinol was also found ineffective in blocking superoxide-mediated carrageenan-induced foot edema in the rat. These studies are interpreted as evidence that xanthine oxidase is not a major superoxide-generating system in activated neutrophils as has been suggested by others.

A tungsten-supplemented diet delivered by transplacental and breast-feeding routes lowers intestinal xanthine oxidase activity and affords cytoprotection in ischemia-reperfusion injury to the small intestine

Ischemia-reperfusion injury has been implicated as playing a major role in the development of necrotizing enterocolitis, a major cause of morbidity and mortality in the newborn. A tungsten-supplemented molybdenum-free diet can reduce xanthine oxidase (XO) enzyme activity in the intestine, which in turn reduces the generation of oxygen radicals after an ischemia-reperfusion insult. This study evaluated the ability of this diet to be effective by indirect means, ie, transplacental and breast-feeding routes. XO activity of the intestine was measured in three groups of CD-1 white rats: I, weanlings fed the tungsten diet or standard chow for 1 week; II, 1-day-old rat pups whose mothers were maintained on the tungsten or standard chow for 7 to 10 days prior to term; and III, rat pups at 1 and 3 weeks after birth whose lactating mothers were maintained on the tungsten or standard chow. Some animals from group III also underwent either a 30- or 60-minute episode of occlusion of the superior mesenteric artery (SMA) to evaluate the protective effects of the diet. XO activity was significantly reduced in all groups receiving the tungsten diet (P less than .0001). Blinded histopathologic studies of the entire small bowel showed significantly less villar necrosis (P less than .05) and fibrosis (P less than .0001) in the tungsten-treated group than in the controls. In the 60-minute occlusion study all tungsten-group animals survived, whereas 7 of 12 in the control group died of intestinal infarction within 24 hours (P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Activation-associated alterations in neutrophil pyridine nucleotide levels: a potential regulatory role for calcium and calmodulin

The concentration of NADP + NADPH in resting human neutrophils has been measured to be 24.0 +/- 2.7 X 10(-18) mol/cell. Upon activation with opsonized zymosan A, phorbol myristic acetate or N-formylmethionyl-leucylphenylalanine, neutrophil NADP + NADPH pools increase to 80.5, 84.0, and 54.0 X 10(-18) mol/cell, respectively. These increases in pyridine nucleotide concentration are blocked by the addition of the calcium antagonist 8-(N,N-dimethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, while calcium ionophore A23187, in the presence of calcium, will trigger the increase in the absence of other stimuli. Calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide also inhibit stimulus-induced increases in the NADP + NADPH pool. These studies are interpreted as suggesting a role for calcium and calmodulin, and possibly protein kinase C in the regulation of pyridine nucleotide concentration in the activated neutrophil.

Purification and characterization of calpain from human polymorphonuclear leukocytes.

Recent studies have demonstrated that a calcium-sensitive protease converts Ca2+/phospholipid-dependent protein kinase C to a Ca2+/phospho-lipid-independent form during the activation of human neutrophils. In this paper, the results of the purification and characterization of a calcium-dependent cytosolic protease from neutrophils is reported. Calcium-dependent protease has been purified 1062-fold from human neutrophils and behaves as a single species on native polyacrylamide gels. The protease is active in the neutral pH range with no observable activity at pH values greater than 8.0, has an absolute requirement for calcium for expression of activity with half-maximal activity observed at 12 /μM free calcium, and has an apparent molecular weight of 110,000 based on gel filtration. The protease requires the presence of dithiothreitol for activity and is inhibited by sulfhydryl inhibitors, leupeptin, and antipain but not by serine protease inhibitors, pepstatin, or orthophenanthroline. The protease is also susceptible to inactivation by autoproteolysis. Based on the similarities of this calcium-dependent protease with calpains from a variety of other mammalian tissues, the protease isolated from human neutrophils appears to be a calpain I.

Inhibition of neutrophil response by mepacrine

Clinical and experimental evidence supports neutrophil involvement in the pulmonary complications of adult respiratory distress syndrome. Preliminary evidence indicates that mepacrine salvages pulmonary function in experimental models of adult respiratory distress syndrome, possibly by inhibiting neutrophil activation [E. M. Canham et al., Am. Rev. resp. Dis. 127, 594 (1983)]. This study examines the effect of mepacrine on neutrophil responses involved in pulmonary dysfunction associated with adult respiratory distress syndrome. A comparison is made between the ability of mepacrine to inhibit a specific neutrophil response utilizing different stimuli and the ability to inhibit different neutrophil responses to a single stimulus. Neutrophils were activated by a soluble stimulus, phorbol myristic acetate, and a particulate stimulus, heat-inactivated opsonized group B streptococcus. Mepacrine inhibited superoxide production in response to both phorbol myristic acetate (IC50 = 5.3 +/- 1.2 microM) or opsonized group B streptococcus (16.1 +/- 1.7 microM). Chemotaxis in response to n-formylmethionylleucylphenylalanine was also inhibited (41.1 +/- 2.2 microM). Finally, aggregation stimulated by either streptococcus or phorbol myristic acetate was inhibited by mepacrine (73.0 +/- 9.8 microM and 77.0 +/- 19.2 microM respectively). A comparison of the IC50 values demonstrates that the inhibitory effect of mepacrine is response dependent and stimulus independent. The results of this study are consistent with the proposal that mepacrine protects against the pulmonary complications associated with adult respiratory distress syndrome by its action as an inhibitor of neutrophil function.

Calmodulin-dependency of human neutrophil phosphodiesterase.

A recent study has reported that the phosphodiesterases of human neutrophils are calmodulin-insensitive (Smolen and Geosits,Inflammation8:193–199, 1984). In the present study, two forms of human neutrophil phosphodiesterase were separated by chromatography on DEAE-52. Peak I phosphodiesterase is activated 2.3-fold by calcium and calmodulin but is not stimulated by either calcium or calmodulin alone. Calmodulin-dependent activation of the phosphodiesterase is blocked by both 20μM trifluoperazine and 20μM W-7. Peak II is not stimulated by calmodulin. These findings suggest that calmodulin may play an important role in regulating alterations in cyclic nucleotide metabolism that accompany neutrophil activation.

[50] Calcium and calmodulin in neutrophil activation

Publisher Summary The importance of calcium in neutrophil activation has been appreciated for more than 20 years. Nevertheless, the biochemical mechanisms linking elevations in cytosolic calcium to neutrophil responses are poorly understood. Approximately four years ago, it was proposed that calmodulin might play a role in the calcium-mediated activation of the neutrophil. Since that time, numerous laboratories have implicated or suggested a role for calmodulin in the various components of bacterial killing. To establish that calmodulin may play a role in the mediation of a neutrophil function, it is important to demonstrate the calcium dependency of the event. This chapter defines criteria that can be used to establish calmodulin involvement in neutrophil responses and discusses the approaches that may be utilized to satisfy these criteria. There are two basic approaches for the quantification of calmodulin in neutrophil homogenates. One of these depends upon the biological activity of the regulator while the other depends upon its immunological recognition.