Cytosolic free calcium, a key second messenger in cellular activation.
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Calcium↗
Biomedical subjects
P D Lew
Publications and source records attributed to P D Lew.
Chemotactic peptide activation of human neutrophils and HL-60 cells. Pertussis toxin reveals correlation between inositol trisphosphate generation, calcium ion transients, and cellular activation.
The mechanism of neutrophil activation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) has been studied by pretreatment of human neutrophils with pertussis toxin. Upon stimulation with FMLP, the cytosolic-free calcium concentration, [Ca2+]i, is increased both by stimulation of calcium influx and mobilization of cellular calcium. We have measured [Ca2+]i as well as the generation of the phospholipid breakdown product inositol trisphosphate (IP3), which is thought to mediate Ca2+ mobilization. As the phosphoinositide pool in human neutrophils is difficult to prelabel with [3H]myoinositol, experiments were also carried out in the cultured human promyelocytic leukemia cell line HL-60 after differentiation with dimethylsulfoxide. Pertussis toxin pretreatment of both cell types inhibited FMLP stimulated membrane depolarization, exocytosis, and superoxide production in a dose-dependent manner. This toxin effect was selective for the receptor agonist, since stimulation of these parameters by two substances bypassing the transduction mechanism, the calcium ionophore ionomycin and the phorbolester phorbol myristate acetate, were unaffected. Rises in [Ca2+]i, as well as generation of IP3 in response to FMLP, were inhibited in parallel; for the inhibition of functional responses, slightly lower toxin concentrations were required. The attentuation of the [Ca2+]i rise was more marked in the absence of extracellular calcium, i.e., when the rise is due only to calcium mobilization. The results provide evidence that phospholipase C stimulation by FMLP resulting in IP3 generation is involved in the signal transduction mechanism. Coupling of FMLP receptor occupancy to phospholipase C activation is sensitive to pertussis toxin, suggesting the involvement of a GTP binding protein (N protein), which has been shown to be a pertussis toxin substrate. The parallel changes in [Ca2+]i and IP3 further support the hypothesis that IP3 is the calcium-mobilizing mediator in FMLP-activated cells.
Calcium↗
Intracellular calcium handling in cystic fibrosis: normal cytosolic calcium and intracellular calcium stores in neutrophils.
We used quin2, a high affinity fluorescent calcium indicator to measure free cytosolic Ca++ in neutrophils isolated from fresh heparinized blood of 13 cystic fibrosis (CF) patients (age range 6-32 yr) and 11 healthy individuals (age range 25-41 yr) as controls. The mean value of free cytosolic Ca++ for CF patients was 137 +/- 19 nM (mean +/- SD) and 141 +/- 23 nM for controls (p = NS). Calcium stores released after exposure of the cells to ionomycin in Ca++-free medium were also similar in neutrophils of CF patients (51 +/- 34 pmol/10(6) neutrophils) and in the controls (50 +/- 15 pmol/10(6) neutrophils). Stimulation of the neutrophils with the chemotactic peptide f-mlp (10(-7) M) led to similar rises in free cytosolic Ca++ with mean peak levels of 718 +/- 230 nM in CF patients and 687 +/- 321 nM in another population of 10 healthy adults. In our group of CF patients free cytosolic Ca++, intracellular Ca++ stores, and the kinetics of intracellular Ca++ transients induced by f-mlp are similar to those of normals. These findings do not support the hypothesis of a generalized defect in calcium handling at the cellular level in CF.
Adolescent↗
Ca2+ homeostasis in permeabilized human neutrophils. Characterization of Ca2+-sequestering pools and the action of inositol 1,4,5-triphosphate.
The regulation of Ca2+ transport by intracellular compartments was studied in digitonin-permeabilized human neutrophils, using a Ca2+-selective electrode. When incubated in a medium containing ATP and respiratory substrates, the cells lowered within 6 min the ambient [Ca2+] to a steady state of around 0.2 microM. A vesicular ATP-dependent and vanadate-sensitive non-mitochondrial pool maintained this low [Ca2+] level. In the absence of ATP, a higher Ca2+ steady state of 0.6 microM was seen, exhibiting the characteristics of a mitochondrial Ca2+ "set point." Both pools were shown to act in concert to restore the previous ambient [Ca2+] following its elevation. Thus, the mitochondria participate with the other pool(s) in decreasing [Ca2+] to the submicromolar range whereas only the nonmitochondrial pool(s) lowers [Ca2+] to the basal level. The action of inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a few cell types was studied. IP3 released (detectable within 2 s) Ca2+ accumulated in the ATP-dependent pool(s) but had no effect on the mitochondria. The response was transient and resulted in desensitization toward subsequent IP3 additions. Under experimental conditions in which the ATP-dependent Ca2+ influx was blocked, the addition of IP3 resulted in a very large Ca2+ release from nonmitochondrial pool. The results strongly suggest that IP3 is a second messenger mediating intracellular Ca2+ mobilization in human neutrophils. Furthermore, the nonmitochondrial pool appears to have independent influx and efflux pathways for Ca2+ transport, a Ca2+ ATPase (the influx component) and an IP3-sensitive efflux component activated during Ca2+ mobilization.
Adenosine Triphosphate↗
Correlation between cytosolic free Ca2+ and aldosterone production in bovine adrenal glomerulosa cells. Evidence for a difference in the mode of action of angiotensin II and potassium.
Quantitative changes in cytosolic free calcium [( Ca2+]i), membrane potential, and aldosterone production in response to angiotensin II and extracellular potassium were measured in intact bovine adrenal glomerulosa cells loaded with the fluorescent calcium indicator quin 2. Angiotensin II (10(-9) M) induced a rapid rise in [Ca2+]i from 124 +/- 26 nM to 204 +/- 63 nM (n = 7), which was followed by steroid production, as measured in dynamic studies with superfused adrenal cells, and by slower changes in membrane potential, as assessed with the fluorescent probe 3,3'-dipropylthiadicarbocyanine. Both [Ca2+]i rises and functional response were blocked by the antagonist analogue [Sar1,Ala8]angiotensin II in a dose-dependent manner. Potassium (3-10 mM) provoked dose-dependent increases in [Ca2+]i, with ED50 of 6.5 mM, associated with rapid changes in membrane potential, a response superimposable upon the dose-related aldosterone production induced by potassium in static incubations of quin 2-loaded glomerulosa cells (ED50 = 6.8 mM). Verapamil (2 X 10(-5) M) and nifedipine (10(-7)-10(-6) M) decreased resting [Ca2+]i and blocked entirely the rise in [Ca2+]i induced by potassium, but did not suppress the [Ca2+]i rises induced by angiotensin II. These findings indicate that two important physiological regulators of aldosterone secretion, extracellular potassium, by the opening of voltage-dependent calcium channels, and angiotensin II, by a receptor-mediated mechanism, induce rapid rises in cytosolic free calcium, which precede, and presumably trigger the steroidogenic response.
Adrenal Cortex↗
Early recognition of potentially fatal necrotizing fasciitis. The use of frozen-section biopsy.
Necrotizing fasciitis is a rare, rapidly progressing soft-tissue infection. Early clinical recognition is difficult, and there is often a fatal delay in appropriate treatment. We evaluated the use of frozen-section biopsies for the rapid diagnosis of this disease in its early stages. Nineteen cases of necrotizing fasciitis were studied over a 13-year period. In all eight cases in which frozen-section biopsy was performed within zero to four days (average, 21 hours) after the onset of symptoms, histologic study confirmed the diagnosis, and surgical d ebridement was immediately performed. Only one patient died. In 11 patients, the diagnosis was made on clinical grounds, and surgical therapy was delayed for 1 to 15 days (average, 6) after the onset of symptoms because of failure to recognize the disorder immediately. Eight patients died (P less than 0.02). Our study indicates that rapid performance of frozen-section soft-tissue biopsy early in the evolution of a suspect lesion may provide a definitive and life-saving diagnosis.
Adult↗
Effect of leukotriene B4, prostaglandin E2 and arachidonic acid on cytosolic-free calcium in human neutrophils.
Changes in cytosolic free calcium [Ca2+]i and release of beta-glucuronidase in response to leukotriene B4 (LTB4) were measured in intact neutrophils loaded with the fluorescent Ca2+ indicator, quin 2. LTB4 (10(-10) M or higher) caused a rapid rise in [Ca2+]i due to influx from the extracellular medium and release from intracellular pools as well as enzyme release. PGE2 (3 microM) did not alter [Ca2+]i whereas arachidonic acid (10 microM) raised [Ca2+]i. Pretreatment of cells with the chemotactic peptide FMLP inhibited the subsequent rise of [Ca2+]i induced by LTB4. Since chemotactic peptides activate the lipoxygenase pathway of arachidonic acid metabolism, it may be speculated that endogenous LTB4 generation is involved in neutrophil activation.
Aminoquinolines↗
Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils.
The intracellularly trapped fluorescent calcium indicator, quin 2, was used not only to monitor changes in cytosolic-free calcium, [Ca2+]i, but also to assess the role of [Ca2+]i in neutrophil function. To increase cytosolic calcium buffering, human neutrophils were loaded with various quin 2 concentrations, and [Ca2+]i transients, granule content release as well as superoxide [O2-] production were measured in response to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) and the calcium ionophore ionomycin. Receptor-mediated cell activation induced by fMLP caused a rapid rise in [Ca2+]i. The extent of [Ca2+]i rise and granule release were inversely correlated with the intracellular concentration of quin 2, [quin 2]i. These effects of [quin 2]i were more pronounced in the absence of extracellular Ca2+. The initial rate and extent of fMLP-induced O2- production were also inhibited by [quin 2]i. The rates of increase of [Ca2+]i and granule release elicited by ionomycin were also inversely correlated with [quin 2]i in Ca2+-containing medium. As the effects of ionomycin, in contrast to those of fMLP, are sustained, the final increase in [Ca2+]i and granule release were not affected by [quin 2]i. A further reduction of fMLP effects was seen when intracellular calcium stores were depleted by incubating the cells in Ca2+-free medium with ionomycin. The specificity of quin 2 effects on cellular calcium were confirmed by loading the cells with Anis/AM, a structural analog of quin 2 with low affinity for calcium which did not inhibit granule release. In addition, functional responses to phorbol myristate acetate (PMA), which stimulates neutrophils without raising [Ca2+]i, were not affected by [quin 2]i. The findings indicate that rises in [Ca2+]i control the rate and extent of granule exocytosis and O2-generation in human neutrophils exposed to the chemotactic peptide fMLP.
Aminoquinolines↗
Comparative superoxide-generating system of granulocytes from blood and peritoneal exudates.
Blood polymorphonuclear leukocytes (PMN), upon interaction with specific chemotactic stimuli, leave the blood stream and migrate to tissues. At such a location, and upon contact with invading microorganisms, they generate superoxide (O2-) as a part of the respiratory burst of phagocytosis. We have compared the O2(-)-generating system of guinea pig peritoneal exudate PMN with that of peripheral blood PMN from the same species or of human origin. The rate of O2- production by casein-induced guinea pig exudate cells in response to a particulate stimulus (opsonized zymosan) was significantly decreased when compared with peripheral blood PMN. Furthermore, the activation time of the O2(-)-generating system was shorter in exudate than in peripheral blood PMN. Important differences in sensitivity to a metabolic inhibitor were found: (i) N-ethylmaleimide increased the activation time of the O2(-)-generating system only in guinea pig exudate cells and not in blood cells; (ii) when N-ethylmaleimide was added after complete activation of the PMN, the O2- production rate was inhibited in guinea pig as well as human blood PMN, but not in guinea pig exudate PMN. We conclude that exudation markedly alters one of the most important antibacterial mechanisms of PMN, the superoxide-generating system.
Animals↗
Adenosine triphosphate-dependent calcium pump in the plasma membrane of guinea pig and human neutrophils.
Changes in cytosolic free Ca may function as a second messenger in neutrophils. Since the plasma membrane seems to be a major regulator of intracellular Ca in many cells, we characterized an energy-dependent Ca transport system in plasma membrane-enriched fractions ("podosomes") from phorbol myristate acetate-stimulated guinea pig and human neutrophils. The active Ca transport system in guinea pig podosomes exhibited a high affinity for Ca (Michaelis constant [Km]Ca 280 +/- 120 nM) and a maximum velocity of 0.83 nmol Ca/mg protein per min. Uptake showed an absolute requirement for Mg ATP (Km ATP 67 microM), whereas other trinucleotides were inactive. Ca uptake was optimal at pH 7, was azide insensitive and temperature dependent. Vanadium, an inhibitor of the Ca/Mg ATPase of heart sarcolemma, inhibited Ca pump activity by 50% at 1 microM. Ca transport was not affected in a NaCl-containing medium, an observation arguing against the presence of a Na/Ca exchange system. Calmodulin at 0.5-10 micrograms/ml stimulated the Ca pumping activity of EGTA-washed podosomes. Calmodulin depletion decreased the affinity of the Ca pump for Ca (Km Ca 2.07 microM) and its readdition restored it (Km Ca 0.55 microM). ATP-dependent Ca transport by podosomes and phagocytic vesicles was inactivated by exposure to trypsin or to the nonpenetrating sulfhydryl reagent rho-chloromercuribenzene sulfonate. Human podosomes had a Ca uptake system with properties similar to those of the guinea pig. These findings demonstrate the presence of a Ca pump in the neutrophil plasma membrane, which is active at physiological concentrations of free cytosolic Ca. By changing Ca concentrations at the cell periphery, this pump could control various motile functions of the neutrophil, such as locomotion or degranulation.
Adenosine Triphosphate↗
Phorbol myristate acetate stimulates ATP-dependent calcium transport by the plasma membrane of neutrophils.
We studied the effect of phorbol myristate acetate (PMA) on the plasma membrane ATP-dependent calcium pump in neutrophils. Plasma membrane-enriched fractions ("podosomes") from PMA-stimulated guinea pig neutrophils exhibited a twofold stimulation of ATP-dependent calcium transport when compared with control podosomes. The stimulatory effect was rapid (beginning less than 2 min after exposure to PMA) and reached maximal values within 5 min. PMA increased the maximum velocity but not the affinity of the calcium pump for Ca++. Pump activation was not preceded by a rise in cytosolic free calcium concentration [Ca++]i, as assessed by the intracellularly trapped fluorescent calcium indicator Quin 2, but instead slightly lowered [Ca++]i and prevented the rise in [Ca++]i normally induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine. These results suggest that the calcium pump in the plasma membrane of neutrophils may be stimulated by calcium-independent pathways, and that this activation could be one of the earliest events mediating some of the effects of phorbol esters.
Adenosine Triphosphate↗
Pathogenesis of foreign body infection. Evidence for a local granulocyte defect.
Implanted foreign bodies are highly susceptible to pyogenic infections and represent a major problem in modern medicine. In an effort to understand the pathogenesis of these infections, we studied the phagocytic function in the vicinity of a foreign body by using a recently developed guinea pig model of Teflon tissue cages subcutaneously implanted (Zimmerli, W., F.A. Waldvogel, P. Vaudaux, and U.E. Nydegger, 1982, J. Infect. Dis., 146:487-497). Polymorphonuclear leukocytes (PMN) purified from tissue cage fluid had poor bactericidal activity against a catalase-positive microorganism. When compared with blood or exudate PMN, they exhibited a significant reduction in their ability to generate superoxide in response to a particulate or a soluble stimulus (72 and 57%, respectively, P less than 0.001). Not only their total contents in myeloperoxidase, beta-glucuronidase, lysozyme, and B12 binding protein were significantly reduced (by 62, 21, 47, and 63%, respectively, P less than 0.01), but also their capability for further secretion of residual B12 binding protein upon stimulation. Ingestion rates of endotoxin-coated opsonized oil particles were reduced by 25% (P less than 0.05). In an effort to reproduce these abnormalities in vitro, fresh peritoneal exudate PMN were incubated with Teflon fibers in the presence of plasma. Interaction of PMN with the fibers led to significant increases in hexose monophosphate shunt activity and exocytosis of secondary granules (P less than 0.01). PMN eluted after such interaction showed defective bactericidal activity, oxidative metabolism, and granular enzyme content similar to those observed in tissue cage PMN. The local injection of fresh blood PMN into tissue cages at the time of, or 3 h after, inoculation with 100 microorganisms (Staphylococcus aureus Wood 46) reduced the infection rate from 50 to 56 cages to 1 of 21 (P less than 0.001) and 3 of 8 cages (P less than 0.001), respectively. These results suggest that the in vivo as well as in vitro interaction of PMN with a nonphagocytosable foreign body induces a complex PMN defect, which may be partly responsible for the high susceptibility to infection of foreign bodies.
Animals↗
Cytosolic free calcium and intracellular calcium stores are normal in neutrophils from patients with essential hypertension.
Cytosolic free calcium and intracellular calcium stores were measured in neutrophils by trapping intracellularly the fluorescent calcium indicator Quin-2. Ten patients with untreated essential hypertension were compared to 10- age and sex-matched normotensive subjects. Intracellular calcium concentration and calcium stores did not differ in the two groups. No significant relationship was found between blood pressure and the calcium parameters. These results suggest that essential hypertension is not associated with a generalized cellular alteration in calcium handling.
Aminoquinolines↗
Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease.
Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.
Calcium↗
Intra-abdominal Citrobacter infections: association with biliary or upper gastrointestinal source.
The clinical records of 46 patients with intra-abdominal and/or bacteremic infections caused by Citrobacter organisms were reviewed to determine whether the isolation of Citrobacter organisms from these sites could be used to determine its portal of entry. The isolation of Citrobacter organisms from intra-abdominal or pleural fluid cultures was associated with a biliary tract or upper gastrointestinal source in 36 of 41 patients. The biliary tract or small bowel was likewise the portal of entry for 8 to 13 patients with Citrobacter septicemia. Thus, the isolation of these organisms from the blood, pleural space, or abdominal cavity strongly suggests that the upper gastrointestinal (or biliary) tract is the site of significant pathologic disease. Of the antibiotics tested, moxalactam, cefotaxime, and amikacin were most effective against Citrobacter.
Adolescent↗
An ultrastructural study of the macrophages of the carrageenan-induced granuloma in the rat lung.
The intralobular injection of 0.17 ml of 2 per cent. carrageenan into the rat lung induced an inflammatory granulomatous response. This inflammation was characterized in the early stages by immediate polymorphonuclear (PMN) leucocyte infiltration into the alveoli. Within 2 days the PMN's began to disappear and were replaced by carrageenan-containing macrophages. Alveolar macrophages actively phagocytised the injected carrageenan and were the dominant cell type in he alveoli for the length of this study (365 days). These low-turnover alveolar macrophages, which with light microscopy stained pinkish-red metachromatically with toludine blue due to their carrageenan content, underwent changes in vacuole morphology as well as changes in size and shape. Throughout the course of this inflammation, these macrophages had carrageenan-containing vacuoles which could be seen undergoing fusion to form larger vacuoles which at times constituted half the size of the entire macrophage cytoplasm. The carrageenan in the vacuoles was an amorphous flocculent appearing material for the initial 14 days but changes to a more fine filamentous form for the remainder of the study. This was probably due to its partial digestion by lysosomal enzymes. The general composition of the cytoplasm remained fairly constant during the 365 days. Pinocytotic vesicles and free ribosomes were abundant and the many mitochondria were small and rounded. Smooth endoplasmic reticulum lysosomes, and Golgi apparati, although not prominent, were present but the most striking feature of the cytoplasm was the presence of numerous lamellar bodies (phagocytosised surfactant). Some of the macrophages increased greatly in overall size (five to seven times) compared to their initial size. A few isolated macrophages could be seen degenerating but no general necrosis was seen. Except for one isolated case, no epithelioid cells were observed in this carrageenan inflammation. Fibrosis, if present at all, was very localised and was only evident at day 340 post-injection. This fibrosis generally involved one or two carrageenan-containing macrophages encapsulated by a few collagen fibres. No widespread fibrosis was ever observed in this study which confirmed earlier histological and biochemical investigations.
Animals↗
In vitro efficacy of several antibiotics against intracellular S. aureus in chronic granulomatous disease.
We report the case of a one-year-old boy with chronic granulomatous disease (CGD), characterized by a defect in the polymorphonuclear leukocytes (PMN) which ingest, but do not kill catalase-positive bacteria such as S. aureus. This well-known observation led us to study the ability of clindamycin, rifampin, co-trimoxazole (TMP-SMX), methicillin and gentamicin to kill intracellular S. aureus in CGD and in normal PMN. Clindamycin killed S. aureus more effectively when these bacteria were intracellular than in the absence of PMN; rifampin was equally active on the same microorganisms in presence and absence of PMN, whereas TMP-SMX, methicillin and gentamicin were less effective in killing intracellular S. aureus. Similar results were obtained in CGD and in normal PMN. These results suggest that clindamycin and rifampin would be a reasonable choice in the treatment of staphylococcal infections in patients with CGD.
Anti-Bacterial Agents↗
Deficient phagocytosis secondary to breakdown of opsonic factors in infected exudates.
Pleural empyema, a clinical entity characterized by the simultaneous presence of large number of PMNLs and viable bacteria, is a biological paradox which has not been fully explained yet. Our preliminary studies suggest that receptor and bactericidal functions of PMNL isolated from purulent exudates, can be close to normal in this condition. Supernatants of these empyemas however have been shown to be low in heat labile opsonic activity and complement hemolytic activity. These observations have been extended by the demonstration of breakdown of IgG, C3 and factor B in infected pleural effusions as opposed to pleural fluids obtained under other conditions. The breakdown of Ig and C3 seems to be enzymatic and to occur, at least for C3, even in the absence of Ca and Mg ions: thus, direct cleavage of C3, possibly by PMNL enzymes, has to be postulated to explain these results. Present work in our laboratory is trying to explore this possibility.
Abscess↗