Propionic acid stimulates superoxide generation in human neutrophils

Short-chain carboxylic acids are the metabolic by-products of pathogenic anaerobic bacteria and are found at sites of infection in millimolar quantities. We previously reported that propionic acid, one of the short-chain carboxylic acids, induces an increase in intracellular Ca2+ ([Ca2+]i) in human neutrophils. Here we investigate the effect of propionic acid on superoxide generation in human neutrophils. Propionic acid (10 mm) induced inositol 1,4, 5-trisphosphate (IP3) formation and a rapidly transient increase in [Ca2+]i, but not superoxide generation, whereas 1 microm formylmethionyl-leucyl-phenylalanine (fMLP), a widely used neutrophil-stimulating bacterial peptide, stimulated not only IP3 formation and Ca2+ mobilization but also superoxide generation. The IP3 level induced by propionic acid was slightly lower than that induced by fMLP. The transient increase in [Ca2+]i induced by propionic acid immediately returned to the basal level, whereas a sustained increase in [Ca2+]i, which was higher than the basal level, following a transient increase in [Ca2+]i was induced by fMLP. The peak level induced by propionic acid was lower than that with fMLP. In the absence of extracellular Ca2+, thapsigargin, a potent inhibitor of endoplasmic reticulum Ca2+-ATPase, induced an increase in [Ca2+]i even after propionic acid stimulation, but not after fMLP. The Ca2+ ionophore A23187 and thapsigargin induced superoxide generation by themselves. Propionic acid enhanced the superoxide generating effect of A23187 and thapsigargin. These results suggest that Ca2+ mobilization induced by propionic acid is much weaker than that with fMLP, and propionic acid is able to generate superoxide in the presence of a Ca2+ ionophore and a Ca2+ influx activator.     

Effect of the calcium ionophore A23187 on superoxide generation in phorbol ester stimulated human neutrophils

The calcium ionophore, A23187, and the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), interacted synergistically to elicit an accelerated superoxide production response in human neutrophils. The lag period preceding PMA-induced superoxide generation was decreased in a dose-dependent manner by A23187 at a concentration range from 1.0 X 10(-8) to 1.0 X 10(-5) M. Superoxide production rate, however, was subject to biphasic effects. While the rate was potentiated in a dose-dependent manner at A23187 concentrations below 1.0 X 10(-6) M, inhibitory influences became manifest at higher concentrations. Total superoxide production was subject to inhibitory effects, characterized by a mean inhibitory dose of 1.3 X 10(-6) M. The synergistic interaction of A23187 with PMA is consistent with a role for protein kinase C in neutrophil activation. Inhibition at high A23187 concentrations appeared to result from the effects of elevated intracellular Ca2+ levels on either NADPH oxidase itself, or some step in the transduction process linking protein kinase C to the oxidase complex.     

Novel priming compounds of cystathionine metabolites on superoxide generation in human neutrophils

Human peripheral blood polymorphonuclear leukocytes were preincubated with cystathionine and cystathionine metabolites found in the urine of patients with cystathioninuria. Among the cystathionine metabolites, cystathionine ketimine and N-acetyl-S-(3-oxo-3-carboxy-n-propyl) cysteine (NAc-OCPC) significantly enhanced the N-formylmethionylleucylphenylalanine (fMLP)-induced superoxide generation, but cystathionine, NAc-cystathionine, and cyclothionine did not enhance the superoxide generation. Cystathionine ketimine and NAc-OCPC also enhanced superoxide generation induced by opsonized zymosan (OZ) but not that induced by arachidonic acid (AA) and phorbol 12-myristate 13-acetate (PMA). Superoxide generation induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein, an inhibitor of tyrosine kinase, and was enhanced by 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C. Cystathionine ketimine and NAc-OCPC markedly also increased phosphorylation of 45-kDa protein in human neutrophils and the phosphorylation depended on the concentrations of cystathionine ketimine and NAc-OCPC. The phosphorylation of 45-kDa protein induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein and herbimycin A, inhibitors of tyrosine kinase, but was not inhibited by H-7 and staurosporine, inhibitors of protein kinase C. Cystathionine metabolites and l-cystathionine sulfoxides were separated into two diastereoisomers, CS-I and CS-II. CS-I enhanced the superoxide generation induced by AA and PMA but not that induced by fMLP and OZ. In contrast, CS-II enhanced the superoxide generation induced by fMLP and OZ, but not that induced by AA and PMA.     

Influence of various antibiotics on superoxide generation by normal human neutrophils

Among the several killing mechanisms displayed by human neutrophils, the oxidative system is the most efficient. We have studied the influence of various antibiotics on the generation of superoxide by isolated human polymorphonuclear leukocytes (PMNL) stimulated by phorbol-myristate acetate. Among the antibiotics tested, only coumermycin significantly inhibited superoxide generation; this effect was dose-related, it depended on extracellular calcium concentration and was potentiated by sub-inhibitory concentrations of calcium channel-blocking agents. Coumermycin inhibited the influx of calcium produced by the ionophore A23187 as well as directed chemotaxis in agar and the intracellular killing of a highly susceptible strain of S. aureus. These inhibitory effects required at least 15 min of preincubation of the PMNL. Coumermycin, at clinically achievable serum concentrations, significantly impaired several PMNL functions. The mechanism could be a specific or a non-specific interaction with calcium-channels.     

Effect of degranulation on superoxide dismutase activity in human neutrophils

Resting neutrophils possess cytosolic cyanide-sensitive (CNs) Superoxide dismutase (SOD) and cyanide-insensitive (CNi) SOD, located in an undefined organelle of the 27,000 g sedimentable fraction of its homogenate. Stimulated neutrophils generate large amounts of Superoxide anion, part of which is released in the extracellular medium and contributes to changes that occur in inflammatory foci. Our purpose was to assess whether or not the neutrophil upon stimulation secreted either or both CNs and CNi SOD activity, because the process could protect against the release of Superoxide anion. Human neutrophils stimulated in vitro with phorbol myristate acetate released 32.6% and 53% of their content in myeloperoxidase (an azurophilic granule marker) and vitamin B₁₂ binding activity, respectively. The CNi SOD was not secreted at all, whereas 16% and 23% of CNs SOD were released by resting and stimulated neutrophils, respectively. In contrast, lactate dehydrogenase, a cytosolic marker, was released by both resting and stimulated cells (∼-9%). These results suggest that CNi SOD is not located in the granules but in another organelle that does not degranulate upon stimulation and consequently does not protect against Superoxide anion formed by neutrophils in the extracellular medium. In contrast, CNs SOD is slightly but significantly released (P <.02) and may be protective. Neutrophils from two patients with chronic granulomatous disease behaved similarly to control neutrophils but their content of both types of SOD was higher than that of the controls.     

Circadian periodicity of urinary volume, creatinine and 5-hydroxyindole acetic acid excretion in healthy Indians

The circadian periodicity of urinary output, creatinine (Cr) and 5-hydroxyindole acetic acid (5-HIAA) excretion was studied under near-tropical conditions in 130 healthy volunteers (65 men and 65 women, 16-75 years of age) with a diurnal activity from about 06:30 to about 22:00 and nocturnal rest. These volunteers were divided into 4 groups, 16-30, 31-45, 46-60 and 61-75 years of age, comprising 20, 20, 15 and 10 participants of each gender, respectively. A marked circadian rhythm was recorded for urine volume, Cr and 5-HIAA excretion in healthy Indians of different ages. The acrophase tended to be delayed in the older age group. The relative amplitude decreased with advancing age, notably in women. Overall, men produced a larger urine volume as compared to women. Excretions of Cr and 5-HIAA in healthy Indian volunteers of different ages may be influenced by diet, societal relations, climate and/or geographic location. The contribution of such factors in metabolism and degradation warrants further study.     

Influence of carvedilol on superoxide generation and enzyme release from stimulated human neutrophils

Activation of neutrophils induces generation of reactive oxygen species and release of granule enzymes, which not only participate in the bactericidal mechanisms of these cells, but also in possible tissue damage. We studied the effect of carvedilol (CARV) [0.1-100 micromol/l], an antihypertensive and cardiovascular drug with antioxidative properties, on superoxide generation (SO) and myeloperoxidase (MPO) release from isolated human neutrophils stimulated with fMLP, a specific receptor activator, or with PMA, a receptor bypassing stimulus. Unstimulated cells showed neither SO formation nor MPO release after preincubation with drug. CARV decreased fMLP and PMA stimulated MPO release and SO generation dose dependently. The inhibitory effect of CARV may attributed to non-specific action since its effect was not influenced by the type of stimulation. It might inhibit SO generation as well as MPO release either by membrane-operating stimulus (fMLP) or membrane bypassing activator (PMA).     

A time-course study on superoxide generation and protein kinase C activation in human neutrophils

The time course of superoxide generation and membrane association of protein kinase C was studied in human neutrophils stimulated by PMA, FMLP, ionomycin and A23187. The initiation of superoxide generation in PMA; ionomycin- and A23187-stimulated neutrophils was characterized by a lag period of at least 30 s in contrast to a lag period of 10-15 s in FMLP-stimulated cells. The time course of membrane association of protein kinase C in PMA-stimulated neutrophils was highly dependent upon the PMA concentration used for stimulation. However, membrane association of protein kinase C preceded superoxide generation in cells stimulated by 10-300 ng/ml PMA. FMLP, ionomycin and A23187 induced membrane association of protein kinase C in a few seconds and always before superoxide generation. It is concluded that membrane association of protein kinase C in PMA-, FMLP-, ionomycin- and A23187-stimulated neutrophils precedes superoxide generation, and thereby may be part of the mechanism initiating NADPH-oxidase activity. A simple correlation between the two parameters could not be proven, indicating that also other activation mechanisms are decisive in the activation of NADPH-oxidase.     

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils: Relationship to calcium mobilization,aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to ‘stage’ events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10^?7 M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 μg/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up ⁴⁵Ca²⁺ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O^?₂ (10–15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O^?₂ generation and aggregation (30–35 s).     

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils. Relationship to calcium mobilization, aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to 'stage' events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10(-7) M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 microgram/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up 45Ca2+ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O2-. (10-15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O2-. generation and aggregation (30-35 s).     

Effect of acetylsalicylic acid on urinary excretion of prostaglandin E in stroke patients

In 12 of 76 stroke patients complicated by the syndrome of inappropriate secretion of antidiuretic hormone (SIADH), a significant increase in urinary prostaglandin E (PGE) (p less than 0.005), and a significant positive relationship between the plasma arginine vasopressin (AVR) level and urinary PGE excretion were observed (r = 0.72, p less than 0.05). The experimental results are consistent with the view that renal PGE acts as a modulator of ADH. Nowadays acetylsalicylic acid (ASA), an inhibitor of prostaglandin biosynthesis, is widely used in ischemic stroke, it was felt necessary to study the effect of this drug on urinary PGE excretion. Therefore various daily doses of ASA were given orally for 3 days to patients with ischemic stroke. PGE values in 24-hour urine samples were measured every day for 3 days before administration of the drug and for 3 days during ASA administration. In 10 patients who took 75 mg of ASA, the decrease in urinary PGE excretion was not statistically significant. On the other hand when ASA was administered 300 mg once in 19 patients or 300 mg 4 times in 11 cases, urinary PGE excretion decreased significantly (p less than 0.05 and p less than 0.05 respectively). In another group of 8 patients who were observed before, during and after the ASA administration, a daily oral dose of 300 mg for 3 days caused a significant decrease in urinary PGE excretion during these 3 days (p less than 0.05). The urinary PGE excretion returned to the control level within 3 days after cessation of the ASA administration.     

Acute creatine ingestion in human: consequences on serum creatine and creatinine concentrations

The aim of the study was to explore the effect of an acute dose of creatine (Cr) ingestion on serum Cr and serum creatinine (Crn) concentrations. Sixteen healthy subjects ingested a single dose of Cr (20 g) followed by the measurement of serum Cr and Crn concentration for 3 h up to a maximum of 6 h (n=6). In response to Cr ingestion a large rise in serum Cr concentration was observed (by 50 folds) occurring approximately 2 1/2h after the ingestion (peak value of 2.17 +/- 0.66 mmol x l(-1)). We also found a moderate but significant rise in serum Crn concentration averaging 13 % after 3 h (peak value at 99.5 +/- 10.5 micromol x l(-1)). A dose response curve obtained in two case studies, in whom different doses of Cr were ingested (0, 2.5, 5, 10, 15, 20 g and 0, 10, 20, 30 g), showed that serum Cr concentration as well as the peak time increased linearly with Cr ingestion. In addition, acute Crn ingestion (5 g) resulted in a substantial increase in serum Crn concentration (by 10 folds) but led to a minor rise in serum Cr concentration (by 2 folds). These results suggest that when acute doses of Cr are ingested in humans, the degree of conversion of exogenous Cr to Crn in the stomach and the gut can be considered as negligible following the first 6 h of ingestion. However, further studies are required to explore the prolonged effect of Cr on Crn metabolism.     

A study in twins of the urinary excretion of vanilmandelic acid, 17-keto-steroids, 17-hydroxycorticosteroids, and creatinine]

The 24-hour urinary excretion rates of creatinine, vanilmandelic acid (VMA), 17-hydroxycorticosteroids, 17-ketosteroids were analysed in 30 female twin pairs. For each of these variables, a comparative study based on the breakdown of total variance into interpair and intrapair variance was undertaken, first on identical (MZ) and fraternal (DZ) twins and then on twins who live together or separately. The comparison of test results shows a strong likeness in the MZ group and in the living together group with respect to the urinary excretion of 17-OH. The variability of VMA excretion can be explained by genetic factors while the variability of 17-ketosteroids and creatinine can be explained with reference to environmental factors.     

Effect of calcium ion on fatty acid-induced generation of superoxide in guinea pig neutrophils

When phospholipases of plasma membranes are activated by certain stimuli, unsaturated fatty acids are liberated. Because unsaturated fatty acids enhance the transmembrane movement of calcium ions, the fatty acids released may modulate intracellular calcium homeostasis in various cells, including neutrophils. To determine the physiological function of these unsaturated fatty acids, we studied the effects of various fatty acids on superoxide generation and on changes in intracellular calcium contents of guinea pig neutrophils. Some unsaturated fatty acids, arachidonate and linoleate, stimulated the rate of superoxide generation concomitant with the increase in the amount of intracellular calcium. In contrast, the saturated fatty acid, myristate, stimulated the generation of superoxide without affecting the content of intracellular calcium. The stimulating actions of arachidonate and myristate were increased dramatically by the presence of a low concentration (1 microM) of extracellular calcium ion. The rate of superoxide generation in fatty acid-treated neutrophils was inhibited by chlorpromazine, an inhibitor of such calcium-binding proteins as C-kinase. These and other observations suggest that liberated unsaturated fatty acids increase the amount of intracellular calcium and enhance C-kinase activity also that the increased activity of the enzyme is involved in the chain of events leading to the stimulation of superoxide generation in fatty acid-treated neutrophils.