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Biomedical subjects

S Nigam

Publications and source records attributed to S Nigam.

At least 73 records · Page 4Linked to original sources
PubMed1992

Enhancement by staurosporine of platelet-activating factor formation in N-formyl peptide-challenged human neutrophils is mediated by intracellular platelet-activating factor binding sites.

Staurosporine potentiates the formation of platelet-activating factor (PAF) and causes a sustained elevation of intracellular Ca2+ ([Ca2+]i). WEB 2086, a specific PAF-receptor antagonist, inhibits both potentiation of PAF formation and elevation of [Ca2+]i by 78% and 65%, respectively. Moreover, the PAF produced by FMLP and/or Staurosporine was completely retained in the cell. This suggests that the effect of staurosporine in FMLP-stimulated neutrophils may be mediated by the action of endogenously produced PAF, which in turn leads to an increase in [Ca2+]i and PAF formation. We conclude that PAF is the major product of human neutrophils which reacts via specific intracellular PAF binding sites to stimulate the phospholipase A2, and its synthesis is under control of a staurosporine-sensitive protein kinase.

Alkaloids↗
PubMed1992

Arachidonic acid release and platelet-activating factor formation by staurosporine in human neutrophils challenged with n-formyl peptide.

Staurosporine, a putative protein kinase C (PKC) inhibitor, increased the release of [14C]arachidonic acid dose dependently between 100 nM and 1000 nM in human neutrophils challenged with 100 nM N-formyl-methionine-leucine-phenylalanine (FMLP). Staurosporine also increased the formation of leukotriene B4 (LTB4) and platelet-activating factor (PAF) in a dose-dependent manner. In addition, exogenously added lyso-PAF further augmented [3H]PAF formation in staurosporine-pretreated human neutrophils stimulated by FMLP, thus suggesting an activation of acetyl-CoA: lyso-PAF acetyltransferase by staurosporine. The potentiation of [14C]arachidonic acid release and [3H]PAF formation by staurosporine was further enhanced in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA), which pinpoints a mechanism other than the modulation of PKC in this process, inasmuch as staurosporine antagonizes PMA-induced O2- production and [3H]PAF formation. Additional studies with other putative PKC inhibitors also revealed the potentiating effects of 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7, 20 microM) and sphingosine (2.5 microM) on FMLP-induced [14C]arachidonic acid release and [3H]PAF formation. We therefore conjecture that staurosporine-sensitive protein kinases including PKC are not involved in the activation of phospholipase A2 and acetyl-CoA:lyso-PAF acetyltransferase.

Alkaloids↗
PubMed1992

Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-trisphosphate, diacylglycerol and intracellular calcium.

Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 microM concentration. The IP3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3-1.0 microM, did not affect the initial mobilization of FMLP-induced intracellular Ca2+ (Ca2+i), although a sustained elevation of cytosolic Ca2+ level was observed within 5 min. This effect could not be suppressed, even by 1 microM phorbol-myristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (less than or equal to 100 nM) were unable to affect FMLP-induced IP3 production, DG accumulation and Ca2+i, the PMA-inhibited initial Ca2+i signal and IP3 formation triggered by FMLP were almost completely restored. At higher concentrations (greater than or equal to 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-bisphosphate (PIP2) breakdown, thus causing accumulation of IP3 and DG and an elevation of C2+i level. Whereas IP3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.

Alkaloids↗
PubMed1992

Clinical significance of prostacyclin and thromboxane in cancer of the female breast and genital tract.

Studies investigating the role of thromboxane A2 and prostacyclin in cancer of the female breast and genital tract are reviewed. Whereas thromboxane A2 was found to promote the tumour growth and metastasis, prostacyclin exerted a protective effect in maintaining vascular and platelet homeostasis. Thus, monitoring of prostacyclin and thromboxane levels in plasma and urine of cancer patients may be essential for the evaluation of tumour growth and metastasis. Of all modulators of thromboxane and prostacyclin biosynthesis, nafazatrom was found to exhibit promising results for the treatment of advanced breast cancer, although its use in the routine therapy is questionable at this stage.

Breast Neoplasms↗
PubMed1992

Loss of cytosolic 15-lipoxygenase activity in A23187-stimulated human leukocytes: involvement of a translocation process?

We focused our study on the subcellular redistribution of 15-lipoxygenase in human leukocytes challenged with A23187 and arachidonic acid (AA). We found that in cytosolic fractions of stimulated cells the 15-lipoxygenase (15-LO) activity, measured as 15-HETE, was 65% less than in controls. However, no activity was found in cell membranes. This effect was complete within 2 min of incubation and was correlated in a dose dependent manner to exogenously added AA. No significant difference in cytosolic distribution of 15-LO activity was observed when cells were stimulated in presence of various concentrations of Ca++. Immunoblot analysis showed that the loss of cytosolic 15-LO activity registered after challenging with A23187 was associated with a concomitant loss of the enzyme content in the cytosol, suggesting the possibility of a translocation process. Neither the 15-LO activity nor the enzyme was, however, found in the cell membrane under our present experimental conditions. But, addition of protease inhibitors showed a slight increase of 15-LO activity in the membrane fraction. Despite the small effect, this may indicate a translocation of 15-LO following challenge of human leukocytes with A23187.

Adult↗
PubMed1991

Isolation of a novel arachidonic acid metabolite 3-hydroxy-5,8,11,14-eicosatetraenoic acid (3-HETE) from the yeast Dipodascopsis uninucleata UOFs-Y128.

When arachidonic acid (AA) is added to the yeast Dipodascopsis uninucleata UOFS Y128, one of the major metabolites isolated and purified with the help of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) is 3-hydroxy-5,8,11,14-eicosatetraenoic acid (3-HETE). The structure of this new AA metabolite was elucidated mainly by electron impact (EI) mass spectrometry (MS). Strikingly, the formation of this new metabolite was found to be inhibited by aspirin.

Arachidonic Acid↗
PubMed1991

Hepoxilins modulate second messenger systems in the human neutrophil.

In this chapter, we will review recent findings which implicate the hepoxilins as modulators of second messenger systems in the human neutrophil. We have shown that the hepoxilins affect calcium homeostasis in the cell and that they stimulate the release of arachidonic acid and diradylglycerol but not inositol phosphate indicating a mode of action for these 12-lipoxygenase metabolites that is independent of phospholipase C activation. In fact lipid analyses indicate that the phospholipid affected by the hepoxilins is phosphatidyl choline, and that this phospholipid is hydrolyzed by a phospholipase D. These findings indicate that the hepoxilins, which are formed by the platelet as well as the neutrophil, may affect neutrophil activation through a potential cell-cell interaction in the circulation or at pathologic sites to initiate or potentiate the inflammatory process.

8,11,14-Eicosatrienoic Acid↗
PubMed1991

Nasal challenge studies with bradykinin: influence upon mediator generation.

Previous studies have shown that nasal allergen provocation leads to dose-dependent increases of inflammatory mediators, e.g. histamine, kinins, LTC4 and PGD2 in nasal lavages. To investigate further the interaction of these mediators, a titration study with intranasal bradykinin (Bk) application (maximal dose 100 nmol/nostril) and consecutive lavage were performed in eight grass-pollen-allergic patients out of season, and five controls. The nasal lavages were analysed for albumin, N-alpha-tosyl-L-arginine methyl ester (TAME) esterase activity, histamine, 9 alpha,11 beta-PGF2, and LTC4. The clinical reactions were measured with a subjective symptom score. A dose-dependent elevation of albumin was found which was significantly higher in patients with allergic and non-allergic rhinitis compared with normal volunteers. TAME-esterase activity also increased in relation to the dosage of Bk given without significant difference between the various groups. No influence on histamine, LTC4 and 9 alpha,11 beta-PGF2, release (PGD2 metabolite) was seen. Short-lasting clinical symptoms like irritation, sneezing, and obstruction were noticed after the two highest Bk dosages (10 and 100 nmol). We conclude that intranasally applied Bk induces a dose-dependent plasma leakage into the nasal cavity, which is significantly higher in patients with seasonal allergic rhinitis out of season compared to normals. Bk does not seem to affect the mast cell since histamine, LTC4 and 9 alpha,11 beta-PGF2 levels do not alter. The ability to induce relevant symptoms of rhinitis provides strong support for the hypothesis that kinins may be important mediators of inflammatory disorders of the upper airways.

Adult↗
PubMed1991

Increased concentrations of eicosanoids and platelet-activating factor in menstrual blood from women with primary dysmenorrhea.

Prostanoids, leukotrienes and platelet-activating factor were measured by radioimmunoassay in menstrual blood of seven women with primary dysmenorrhea and five healthy controls. The eicosanoids and PAF concentrations in dysmenorrheic patients were significantly higher than those found in healthy women (P less than 0.005 for PGF2 alpha, 11-dehydro-TXB2, 2,3-dinor-TXB2 and LTC4/D4; P less than 0.001 for PAE; P less than 0.05 for PGE2 and 2,3-dinor-6-keto-PGF1 alpha). Whereas no relationship could be found between the concentrations of PGF2 alpha, PGE2, 11-dehydro- and 2,3-dinor-TXB2 and severity of primary dysmenorrhea, a close correlation between LTC4/D4 and PAF and severity of the disease was observed, particularly in patients who responded poorly to therapy with prostaglandin synthetase inhibitors. We conclude that the hyperstimulation of myometrial activity is not caused by selective stimulation of one metabolic pathway of arachidonic acid, but rather by an overall stimulation of phospholipid metabolism. The assessment of prostanoids, leukotrienes and PAF in menstrual blood many be useful as a direct index of primary dysmenorrhea, and the development of their antagonists may have therapeutic implications in improved treatment of the disease.

Adult↗
PubMed1990

Receptor-mediated action of hepoxilin A3 releases diacylglycerol and arachidonic acid from human neutrophils.

We have previously shown that hepoxilin A3 increases the intracellular concentration of Ca+2 in human neutrophils. Herein we address the initial events of hepoxilin action on the neutrophil which precede the rise in intracellular calcium. We show that hepoxilin A3 at 10-1000 nM concentrations releases from [1-14C]-arachidonic acid labeled neutrophils diacylglycerol and unesterified arachidonic acid in a time and concentration dependent fashion. The release of arachidonic acid and diacyglycerol are receptor-mediated events which are blocked by pertussis toxin. This data shows that hepoxilin A3 stimulates phospholipases C and A2 in the cell which may be involved in the rise in cytosolic calcium. Thus, hepoxilins may represent a hitherto unrecognised class of cellular mediators.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Lipoxin A4 and lipoxin B4 stimulate the release but not the oxygenation of arachidonic acid in human neutrophils: dissociation between lipid remodeling and adhesion.

The profiles of actions of lipoxin A4 (LXA4) and lipoxin B4 (LXB4), two lipoxygenase-derived eicosanoids, were examined with human neutrophils. At nanomolar concentrations, LXA4 and LXB4 each stimulated the release of [1-14C]arachidonic acid from esterified sources in neutrophils. Lipoxin-induced release of [1-14C]arachidonic acid was both dose- and time-dependent and was comparable to that induced by the chemotactic peptide f-met-leu-phe. Time-course studies revealed that lipoxin A4 and lipoxin B4 each induced a biphasic release of [1-14C]arachidonic acid, which was evident within seconds (5-15 sec) in its initial phase and minutes (greater than 30 sec) in the second phase. In contrast, the all-trans isomers of LXA4 and LXB4 did not provoke [1-14C]AA release. Lipoxin-induced release of arachidonic acid was inhibited by prior treatment of the cells with pertussis toxin but not by its beta-oligomers, suggesting the involvement of guaninine nucleotide-binding regulatory proteins in this event. Dual radiolabeling of neutrophil phospholipid classes with [1-14C]arachidonic acid and [3H]palmitic acid showed that phosphatidylcholine was a major source of lipoxin-induced release of [1-14C]arachidonic acid. They also demonstrated that lipoxins rapidly stimulate both formation of phosphatidic acid as well as phospholipid remodeling. Although both LXA4 and LXB4 (10(-8)-10(-6) M) stimulated the release of [1-14C]arachidonic acid, neither compound evoked its oxygenation by either the 5- or 15-lipoxygenase pathways (including the formation of LTB4, 20-COOH-LTB4, 5-HETE, or 15-HETE). LXA4 and LXB4 (10(-7) M) each stimulated the elevation of cytosolic Ca2+ as monitored with Fura 2-loaded cells, albeit to a lesser extent than equimolar concentrations of FMLP. Neither lipoxin altered the binding of [3H]LTB4 to its receptor on neutrophils. In addition, they did not stimulate aggregation or induce adhesion of neutrophils to human endothelial cells. Results indicate that both LXA4 and LXB4 stimulate the rapid remodeling of neutrophil phospholipids to release arachidonic acid without provoking either aggregation or the formation of lipoxygenase-derived products within a similar temporal and dose range. Together they indicate that LXA4 and LXB4 display selective actions with human neutrophils and suggest that these eicosanoids possess unique profiles of action which may regulate neutrophil function during inflammation.

Arachidonic Acids↗
PubMed1989

5-Hydroperoxyeicosatetraenoic acid (5-HPETE) enhances the synthesis of 1-O-alkyl-2-sn-acetyl-glycero-3-phosphocholine (PAF) in fMet-Leu-Phe-stimulated HL-60 cells: key role of diacylglycerol (DAG) in activation of protein kinase C (PKC).

We investigated the effect of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) on the PAF formation in fMet-Leu-Phe-stimulated HL-60 cells. 5-HPETE was found to enhance the PAF synthesis in fmlp-stimulated cells without causing additional mobilization of intracellular calcium. However, a significant increase in diacylglycerol (DAG) levels due to 5-HPETE was observed, which in turn activated the protein kinase C (PKC). Obviously, PKC is responsible for the activation of phospholipase A2 and the release of lyso-PAF and AA from complex lipid stores. Further, the dose-dependent increase in DAG production in absence of simultaneous increase in total inositol phosphates is indicative of an additional source for DAG besides PIP2.

Calcium↗
PubMed1988

Arachidonic acid metabolites and their circadian rhythm in patients with allergic bronchial asthma.

The aim of this study was to investigate circadian variation in concentrations of arachidonic acid (AA) metabolites in relation to the circadian pattern in bronchial patency. Blood samples were obtained at 4-hr intervals from 2000 of 1 day until 1400 of the next from 12 diurnally active asthmatic and six diurnally active non-asthmatic patients. Bloods were analyzed for the prostanoids thromboxane A2 (measured as stable metabolite 6-keto-PGF1a), PGE2 and PGF2a. Airways patency was assessed by self-measurement of peak expiratory flow (PEF). In asthmatics, circadian variation was detected in PEF as well as PGE2 and TXB2. The circadian trough of the PEF rhythm closely coincided with the circadian peak of the PGE2 and TXB2 rhythms. In the controls, the PEF was not circadian rhythmic. Of the AA metabolites only 6-keto-PGF1a exhibited 24-hr bioperiodicity in the controls. The controls exhibited a significantly higher circadian mean of PEF (P less than 0.001), while the asthmatics had a lower 24-hr average PGE2 but greater mean TXB2/PGE2 ratio. The obstructive effect caused by the overall 24-hr deficiency of PGE2 in asthmatics is possibly amplified by the increased of TXB2 during the early morning hours. This dissociation of the temporal patterns in TXB2 and PGE2 levels over the 24 hr is discussed as a characteristic finding for asthmatics.

Adult↗
PubMed1987

Inhibition of lipoxygenase products by retinoids in human blood cells.

Arotinoids, third generation retinoids, inhibited significantly 12-lipoxygenase in platelets and 5-lipoxygenase in human neutrophils. The doses required for inhibition of 12-lipoxygenase in platelets were as low as 1 pM and of 5-lipoxygenase in neutrophils as low as 100 pM. No cytotoxicity was observed in neutrophils at concentrations used, as assayed by exclusion of trypan blue dye. In contrast to various studies reported in the literature, arotinoids used inhibited ionophore-induced arachidonic acid (AA) release in platelets and neutrophils of humans. The doses required for 50% inhibition of AA release were between 10 pM and 75 nM for platelets and between 1 and 50 nM for neutrophils. The data suggest that arotinoids are excellent dual inhibitors of cyclooxygenase and lipoxygenase pathways of AA metabolism. The mechanism for inhibition of eicosanoids lies apparently in the inhibition of AA deacylation from phospholipid membrane. It cannot, however, be ruled out that the accumulation of AA results from arotinoid-induced stimulation of reacylation of AA into the phospholipid membrane.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

Assessment of the pathogenetic role of physical exercise in renal stone formation.

The effects of moderate physical exercise (performed on a bicycle ergometer to 70-75% of maximum oxygen consumption) without fluid replenishment on urinary chemistries and crystallization of kidney stone-forming substances were compared to those of rest in six normal subjects. Moderate physical exercise significantly decreased urinary pH [from 6.35 +/- 0.32 (+/-SD) to 5.79 +/- 0.33; P less than 0.05] and citrate [from 121.1 +/- 63.5 to 88.2 +/- 44.4 mg/6-h period from initiation of physical exercise; P less than 0.05 (630 +/- 331 to 459 +/- 231 mumol/6 h)], owing to induced metabolic acidosis. The total renal excretion of stone-forming constituents decreased [for example, calcium from 31.2 +/- 15.8 to 21.4 +/- 6.5 mg/6 h (0.8 +/- 0.4 to 0.5 +/- 0.2 mmol/6 h), phosphorus from 155 +/- 42 to 127 +/- 27 mg/6 h (5.01 +/- 1.4 to 4.1 +/- 0.9 mmol/6 h), and uric acid from 172 +/- 60 to 117 +/- 13 mg/6 h (1.0 +/- 0.4 to 0.7 +/- 0.1 mmol/6 h), each P less than 0.05], probably due to extracellular volume contraction (from sweating) and enhanced renal tubular reabsorption. However, the urinary concentration of stone-forming constituents significantly increased during and after moderate exercise because of the fall in urinary volume from 847 +/- 312 to 290 +/- 36 ml/6 h (P less than 0.01). Thus, urinary calcium oxalate saturation increased significantly from 2.62- to 6.68-fold saturation (P less than 0.01). The urinary undissociated uric acid concentration significantly rose [from 31.6 +/- 24.8 to 125.7 +/- 60.3 mg/L (0.19 +/- 0.15 to 0.76 +/- 0.36 mmol/L; P less than 0.01)], due to higher total uric acid concentration and reduced urinary pH. The saturation of calcium phosphate (brushite) did not change significantly, because the rise in urinary calcium concentration was compensated for by reduced phosphate dissociation (from lower urinary pH). The propensity for spontaneous precipitation of calcium oxalate was greater after exercise, as less soluble oxalate was required to elicit nucleation of calcium oxalate [58.0 +/- 21.2 to 49.0 +/- 16.4 mg/L (644 +/- 236 to 544 +/- 182 mumol/L); P less than 0.05]. The results suggest that moderate physical exercise, without increased fluid intake to compensate for excessive sweating, may cause the crystallization of uric acid and calcium oxalate in urine and may enhance the risk of the formation of renal stones composed of these salts.

Adult↗