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P D Lew

Publications and source records attributed to P D Lew.

At least 37 records · Page 2Linked to original sources
PubMed1987

Control of cytosolic free calcium by intracellular organelles in bovine adrenal glomerulosa cells. Effects of sodium and inositol 1,4,5-trisphosphate.

The regulation of cytosolic free Ca2+ concentration ([Ca2+]c) by intracellular organelles was studied in permeabilized bovine adrenal glomerulosa cells. Two compartments, with distinct characteristics, were able to pump Ca2+. A first pool, sensitive to ruthenium red and presumably mitochondrial, required respiratory chain substrates to maintain [Ca2+]c around 700 nM. Ca2+ efflux from this compartment was activated by Na+ (ED50 = 5 mM). Inositol 1,4,5-trisphosphate (IP3) had no effect on this pool. A second nonmitochondrial pool required ATP to lower [Ca2+]c to about 200 nM and released Ca2+ transiently upon addition of IP3. When the two systems were allowed to work simultaneously, the nonmitochondrial pool regulated [Ca2+]c and IP3 released Ca2+ in a concentration-dependent manner (EC50 = 0.6 microM). Under these conditions the mitochondria seemed Ca2+ depleted. Upon repeated stimulations with IP3, a marked attenuation of the response was observed. This phenomenon was due to Ca2+ sequestration by a nonmitochondrial IP3-insensitive pool. Neither dantrolene (200 microM) nor 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (10 microM) were able to abolish IP3-induced Ca2+ release, though both compounds efficiently inhibited aldosterone production in intact cells stimulated with angiotensin II (10 nM) or K+ (12 mM). These results suggest that in permeabilized adrenal glomerulosa cells: the nonmitochondrial pool is responsible for buffering [Ca2+]c and for releasing Ca2+ in response to IP3; at resting [Ca2+]c levels, the mitochondria appear Ca2+ depleted; when [Ca2+]c rises above their set point, the mitochondria accumulate Ca2+ as a function of [Na+]c; 4) the mitochondria are not involved in the desensitization mechanism of the response to IP3.

Adenosine Triphosphate↗
PubMed1987

Role of cytosolic free calcium and phospholipase C in leukotriene-B4-stimulated secretion in human neutrophils. Comparison with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine.

Various leukotriene analogues were tested for their capacity to raise the cytosolic free calcium concentration, [Ca2+]i, and to stimulate exocytosis in human neutrophils. Their order of potency for both parameters was LTB4 greater than the stereochemical isomer of LTB4, (5S, 12S)-LTB4 much much greater than the sulphidopeptides LTD4, LTC4. The correlation between [Ca2+]i and secretion indicates that an increase of [Ca2+]i above a threshold level of about 300 nM is necessary for stimulating secretion with LTB4. This threshold is about an order of magnitude higher than that required for the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). The increase in [Ca2+]i elicited by LTB4 was unaffected by increasing cellular cAMP, while secretion was completely inhibited. These results indicate, that similar to fMet-Leu-Phe, leukotrienes generate other signals in addition to [Ca2+]i elevations. Contrary to previous claims, leukotrienes stimulate polyphosphoinositide hydrolysis, as indicated by the increase in [3H]inositol trisphosphate, InsP3, observed upon stimulation of myo[3H]inositol-labelled neutrophils with LTB4 or (5S, 12S)-LTB4. The two InsP3 isomers [Ins(1,4,5)P3 and Ins(1,3,4P3] were separated by high-pressure liquid chromatographed and, as reported for other cell types, the formation of Ins(1,4,5)P3 precedes that of Ins(1,3,4)P3. Maximal stimulatory doses of LTB4 or (5S, 12S)-LTB4 produce about 50% the amount of InsP3 generated by equimolar concentrations of fMet-Leu-Phe. The present observations suggest that, though the transmembrane signalling systems activated by LTB4 and fMet-Leu-Phe are the same, the different efficacy of these two agonists at stimulating neutrophil functions is due, at least in part, to a different degree of activation of phospholipase C.

Calcium↗
PubMed1987

Cell surface expression of fMet-Leu-Phe receptors on human neutrophils. Correlation to changes in the cytosolic free Ca2+ level and action of phorbol myristate acetate.

We have studied how cytosolic free Ca2+ ([Ca2+]i) changes and phorbol myristate acetate (PMA) exposure affects ligand-independent cell surface expression of fMet-Leu-Phe receptors on human neutrophils. Mere incubation primed neutrophils to double their binding of fMet-Leu-Phe. This spontaneous increase of peptide binding was unaffected by changes in the extracellular calcium concentration. However, depression of the [Ca2+]i totally abolished the increased binding of fMet-Leu-Phe. Scatchard-Plot analysis revealed that the observed increase of peptide binding was due to an increased number of receptors. Normalization of the [Ca2+]i in cells where it was initially depressed resulted in a slow but progressive increase in fMet-Leu-Phe binding. The rate of receptor recruitment could be enhanced by rapidly increasing the [Ca2+]i by addition of ionomycin. Addition of PMA to cells with near maximal receptor expression led to a marked reduction of fMet-Leu-Phe binding without affecting [Ca2+]i. These observations suggest the existence of a dual regulatory mechanism for up- and down-regulation of fMet-Leu-Phe receptors on the cell surface of human neutrophils.

Aminoquinolines↗
PubMed1987

Subcellular distribution of Ca2+ pumping sites in human neutrophils.

The distribution of nonmitochondrial Ca2+ pumping sites and the site of action of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) were studied in subcellular fractions of human neutrophils. In homogenates, two different Ca2+ pools could be observed: a mitochondrial Ca2+ pool and a nonmitochondrial, ATP-dependent, Ins 1,4,5-P3-responsive Ca2+ pool. When the homogenate was separated into microsomes, primary granules, and secondary granules, the nonmitochondrial Ca2+ pumping and the Ins 1,4,5-P3-induced Ca2+ release occurred only in the microsomal fraction. In a gradient developed to separate different microsomal organelles, maximal Ca2+ pumping activity occurred in fractions of low densities. Correlations between Ca2+ uptake and organelle markers were negative for the endoplasmic reticulum (r = -0.49) and positive for plasma membrane (r = 0.47), Golgi (r = 0.62), and endosomes (r = 0.96). Because the Ca2+ pumping organelles in these fractions were insensitive to micromolar vanadate and digitonin treatment, they are unlikely to be plasma membrane vesicles. We conclude first that microsomal fractions of human neutrophils contain organelles that lower the ambient free Ca2+ concentration and respond to Ins 1,4,5-P3. Second, granules are not involved in intracellular Ca2+ regulation in neutrophils. Third, nonendoplasmic reticulum organelles, such as endosomes, Golgi elements, or yet undefined specialized structures, play a major role in intracellular Ca2+ homeostasis in human neutrophils.

Adenosine Triphosphate↗
PubMed1986

The role of cytosolic free calcium in the generation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate in HL-60 cells. Differential effects of chemotactic peptide receptor stimulation at distinct Ca2+ levels.

The generation of the two inositol trisphosphate (IP3) isomers, 1,4,5-IP3 and 1,3,4-IP3, and its relation to changes in the cytosolic free calcium concentration, [Ca2+]i, in response to the chemotactic peptide fMet-Leu-Phe was studied in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Stimulation by fMet-Leu-Phe within seconds transiently elevates 1,4,5-IP3 to peak values averaging 8-fold basal levels, and leads to a concomitant rise in [Ca2+]i and to degranulation. These responses are followed by a slower and more sustained rise in 1,3,4-IP3. Alterations in [Ca2+]i modulate differentially the generation of the two IP3 isomers. At [Ca2+]i lower than 30 nM, no IP3 is generated upon fMet-Leu-Phe stimulation. Working at normal resting [Ca2+]i, but preventing the fMet-Leu-Phe induced transient rise in [Ca2+]i (by prior depletion of intracellular Ca2+ stores and working in calcium-free medium) the fMet-Leu-Phe stimulation of 1,3,4-IP3 levels is attenuated, whereas the response of 1,4,5-IP3 is not significantly altered. Maintained elevation of [Ca2+]i to micromolar levels with the Ca2+ ionophore ionomycin generates enhanced 1,3,4-IP3 levels in the absence of fMet-Leu-Phe, whereas the fMet-Leu-Phe stimulation of 1,4,5-IP3 generation is markedly inhibited. Pertussis toxin selectively abolishes the fMet-Leu-Phe-induced IP3 production, whereas ionomycin stimulation of 1,3,4-IP3 generation is unaffected. These findings indicate that in intact cells: receptor-triggered phosphatidylinositol bisphosphate phosphodiesterase activation has a minimal Ca2+ requirement, but does not depend on a previous or concomitant rise in [Ca2+]i; Ca2+ elevations above micromolar levels decrease the fMet-Leu-Phe-induced generation of 1,4,5-IP3; and 1,3,4-IP3 generation is not directly linked to receptor activation and appears to result both from increased [Ca2+]i and 1,4,5-IP3 levels.

Calcium↗
PubMed1986

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent.

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.

Adenosine Triphosphate↗
PubMed1986

[Acute Neisseria subflava endocarditis].

A 50-year-old woman who developed acute endocarditis on a mitral Starr valve due to Neisseria subflava is reported. This microorganism, which belongs to the normal flora of the upper respiratory tract, is a very rare cause of endocarditis since only 9 cases have been described. The patient developed various complications including right hemiplegia. Cure was obtained by medical therapy alone due to the high susceptibility of the bacteria to a third generation cephalosporin (moxalactam). This is the third time a neurological complication of this kind is reported during N. subflava endocarditis. A high frequency of neurological and systemic embolic complications appears to be an important feature of endocarditis due to N. subflava and other saprophytic Neisseria.

Echocardiography↗
PubMed1986

Chronic Q fever endocarditis with massive splenomegaly in childhood.

Two children with congenital heart disease developed persistent fever, anemia, and hepatosplenomegaly. Both were shown to have intracardiac vegetations and evidence of infection with Coxiella burnetti. Thus, the same clinical manifestations of Q fever may develop in both children and adults.

Adolescent↗
PubMed1986

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils.

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.

Calcium↗
PubMed1986

Effects of vasopressin and its analogs on rat aortic smooth muscle and renal medullary tubular cells: characterization of receptor subtypes.

The response to activation of specific receptors by arginine-vasopressin (AVP) has been studied in two cell models. In rat aortic smooth muscle cells in monolayer culture, the response of cytosolic free calcium ([Ca2+]i) upon addition of various agonists or antagonists was examined using the fluorescent calcium probe Quin 2. Activation of the vasopressin (V1) subtype of receptor resulted in a transient rise of [Ca2+]i, which could be prevented by a selective V1 antagonist but not by the calcium channel blocker, nifedipine and that was only slightly reduced in the absence of calcium in the medium. Release of 6-keto-prostaglandin F1 alpha (PGF1 alpha), a stable metabolite of prostacyclin, was also observed in response to AVP. We conclude that one of the primary intracellular events following activation of V1 receptors on smooth muscle cells is a rise of [Ca2+]i released from intracellular stores, which mediates smooth muscle contraction and prostacyclin production. In superfused rat renal medullary tubular cells, the study of the release of cyclic AMP (cAMP) and prostaglandin E2 (PGE2) upon stimulation by AVP and various analogs (agonists or antagonists) permitted demonstration of the presence of two subtypes of receptors. The V2 type appeared to be linked to adenylate cyclase and was responsible for cAMP release, mediating the hydroosmotic effect of AVP, whereas the V1 type was related to calcium, mediating the prostaglandin production.

Animals↗
PubMed1986

Calcium mobilization by inositol 1,4,5-trisphosphate during activation of islet, pituitary, and myeloid cells.

We have investigated whether the initial rise in cytosolic free Ca2+, observed when cells are activated by certain receptor ligands, is mediated by inositol 1,4,5-trisphosphate (IP3). The chemotactic peptide formyl-methionyl-leucyl-phenylalanine increased cytosol Ca2+ (as measured with the Quin-2 technique) in human neutrophils, even in the absence of extracellular Ca2+, thus demonstrating Ca2+ mobilization. Similar findings were obtained with thyrotropin-releasing hormone in GH3 cells (pituitary cell line) and with carbamylcholine in RINm5F cells, an insulin-secreting line. IP3 levels were raised by the three agonists in their respective target cells within 5 s. The action of IP3 was assessed in digitonin-permeabilized cells. Such cell preparations display two steady-state levels, to which ambient free Ca2+ is lowered: one approximately 0.7 microM, maintained by mitochondria, another at 0.1-0.2 microM. The latter was ATP-dependent and was shown to be due to Ca2+ pumping by the endoplasmic reticulum. IP3 mobilized Ca2+ from the ATP-dependent pool, but not from mitochondria. The effect of IP3 is transient, which could be due to rapid degradation of the compound. However, when IP3 degradation by the permeabilized cells was measured, the half-time of degradation was short in RINm5F cells and GH3 cells, but much longer in neutrophils. IP3 degradation could thus not be the only explanation for the transient nature of the evoked Ca2+ mobilization. It is concluded that the early rise in cytosol Ca2+ following receptor-mediated cell activation is induced by IP3, thus attesting to the messenger function of this phospholipid breakdown product.

Adenosine Triphosphate↗
PubMed1986

Effect of the synthetic inhibitor tosylamino-phenylethyl-chloromethylketone on chemotactic peptide receptor activation and superoxide production in human neutrophils.

It was previously shown that inhibitors such as tosylamido-phenylethyl-chloromethylketone (TPCK) inhibit superoxide production by human neutrophils. These studies suggested that a chymotrypsin-like protease inhibited by TPCK was involved in the activation of the neutrophils oxidative system. In this study, we attempted to define the step in cellular activation and/or cell function inhibited by TPCK. TPCK 10(-5) M did not inhibit the following early events thought to be involved in the activation of oxidase. 1) f-met-leu-phe-induced activation of phospholipase C assessed by the production of inositol-tris-phosphate (IP3), 2) f-met-leu-phe-induced membrane potential changes, 3) f-met-leu-phe-induced increase in free cytosolic calcium, and 4) phorbol-myristate acetate-induced protein phosphorylation in 32P labeled neutrophils. We also showed that TPCK 10(-5) M inhibited bactericidal activity of neutrophils on Staphylococcus aureus, whereas it did not inhibit the ingestion rate of endotoxin-coated Oil red O particles. We conclude that 1) TPCK at the concentration of 10(-5) M inhibits superoxide production but not ingestion of Oil red O particles and 2) TPCK inhibits superoxide production at a step distal from calcium mobilization and protein phosphorylation. Radiolabeled TPCK may therefore be a useful tool to study, whether a protease is involved in the activation of the oxidative system distal to second messenger generation.

Adult↗
PubMed1986

Characterization of fMet-Leu-Phe receptor-mediated Ca2+ influx across the plasma membrane of human neutrophils.

N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe) stimulation of human neutrophils leads to a rapid increase of the cytosolic free Ca2+ concentration, [Ca2+]i, which is significantly reduced by removal of extracellular calcium. In the present study we show that fMet-Leu-Phe-induced [Ca2+]i increases are, in part, mediated by an increase of the plasma membrane permeability to Ca2+. This conclusion is based on the following evidence. In the presence of extracellular calcium, addition of La3+ reduced the fMet-Leu-Phe-induced [Ca2+]i increase to approximately the same level as that observed in the absence of extracellular calcium. A net increase of the plasma membrane permeability for Mn2+ could be observed after fMet-Leu-Phe stimulation, as revealed by intracellular quenching of the quin2 signal. The influx of Mn2+, like that of Ca2+, was inhibited by La3+ and was more pronounced in the absence of extracellular Ca2+, suggesting competition for the same pathway. Temporal dissociation of intracellular Ca2+ release from stores and Ca2+ influx from the medium could be demonstrated by readdition of calcium to cells stimulated in the absence of this cation. This second [Ca2+]i increase could be abolished either by giving the specific chemotactic peptide receptor antagonist, BOC-Met-Leu-Phe, or Co2+. We could also show that the fMet-Leu-Phe-dependent Ca2+ influx was not due to the activation of voltage-dependent calcium channels since depolarization either by K+ or gramicidin D did not affect the resting [Ca2+]i, nor did it affect a subsequent [Ca2+]i increase induced by fMet-Leu-Phe. Furthermore, nifedipine and verapamil, at concentrations known to block classical voltage-dependent calcium channels, had no significant effects on the Ca2+ influx induced by fMet-Leu-Phe. We suggest that fMet-Leu-Phe promotes influx of Ca2+ ions across the plasma membrane of human neutrophils by opening of receptor-dependent calcium channels.

Adult↗
PubMed1986

Effects of atrial natriuretic peptide on the stimulation by angiotensin II of various target cells.

The effects of the synthetic (1-24) atrial natriuretic peptide (atriopeptin III) on angiotensin II-induced changes in cytosolic free calcium and biological responses have been examined in three cell types: adrenal glomerulosa cells, vascular smooth muscle cells and renal cortical tubular cells. Atriopeptin III did not affect the transient changes in cytosolic free calcium induced by angiotensin II in adrenal glomerulosa and vascular smooth muscle cells, nor did it alter resting cytosolic free calcium levels. In contrast with its known inhibitory effects on angiotensin II-induced aldosterone production and vasoconstriction, atriopeptin III did not prevent either angiotensin II-stimulated prostaglandin 6-keto-F1 alpha or prostaglandin E2 (PGE2) synthesis in cultured vascular smooth muscle cells and dispersed renal cortical tubular cells, respectively. These data show that atrial natriuretic peptide is not an endogenous calcium-blocking agent, and indicate that the atrial peptide is not a general inhibitor of all angiotensin II-induced biological responses.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Cytosolic free calcium levels in monolayers of cultured rat aortic smooth muscle cells. Effects of angiotensin II and vasopressin.

A rise in cytosolic free calcium ([Ca2+]i) is thought to be the principal mediator in vascular smooth muscle contraction. Quantitative changes of [Ca2+]i in response to two vasoconstrictor peptide hormones, angiotensin II and vasopressin, were directly measured in monolayers of adherent cultured rat aortic smooth muscle cells loaded with the fluorescent calcium indicator Quin 2. Angiotensin II induced rapid, concentration-dependent rises in [Ca2+]i from 1.53 +/- 0.27 X 10(-7) (n = 16) up to 1.2 X 10(-6) M, with ED50 of 0.45 X 10(-9) M, an effect which was blocked by the antagonist analogue [Sar1, Ala8]angiotensin II. Vasopressin also elicited transient rises in [Ca2+]i to peak levels of about 8 X 10(-7) M, with ED50 of 1.05 X 10(-9) M, and this response was completely abolished by a vasopressor antagonist. In calcium-free medium, basal [Ca2+]i levels fell to 0.92 +/- 0.24 X 10(-7) M (n = 4), and both hormones were still able to raise [Ca2+]i, although to a lesser extent. Readdition of extracellular calcium following the [Ca2+]i transient induced a second, slower [Ca2+]i rise. In calcium-containing medium, lanthanum ion (2 X 10(-5) M) reduced peptide-evoked [Ca2+]i rises to the values observed in calcium-free medium. Stimulation with each peptide completely desensitized the smooth muscle cells to a subsequent identical challenge, with little crosstachyphylaxis. Potassium ion (50 mM) only minimally affected [Ca2+]i levels. The calcium channel blocker nifedipine (10(-6) M) did not prevent the [Ca2+]i rises induced by angiotensin II, vasopressin, or potassium. These findings indicate that the two physiologically important vasoconstrictor hormones angiotensin II and vasopressin rapidly raise [Ca2+]i in cultured vascular smooth muscle cells, in part by mobilizing calcium from intracellular pools and in part through activation of receptor-operated calcium channels.

Aminoquinolines↗
PubMed1985

Cytosolic free calcium and intracellular calcium stores in neutrophils from hypertensive subjects.

Alterations in intracellular calcium have been implicated in the pathogenesis of essential hypertension. To see whether this is a generalized phenomenon we assessed cytosolic free calcium and intracellular calcium stores in neutrophils from normo- and hypertensive subjects, by trapping the fluorescent calcium indicator quin2 in intact cells. Ten patients with untreated essential hypertension were compared with 10 age- and sex-matched normotensive subjects. The levels of cytosolic free calcium and intracellular calcium stores releasable by the calcium ionophore ionomycin did not differ. No significant relationship was found between blood pressure and the calcium parameters in all 20 subjects studied. The results indicate that essential hypertension is not associated with a membrane defect in calcium handling of all human cell systems, leading to generalized increases in resting values of cytosolic free calcium. Neutrophils do not appear to be a good model for intracellular calcium handling in vascular smooth muscle.

Adult↗