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K H Krause

Publications and source records attributed to K H Krause.

At least 55 records · Page 3Linked to original sources
PubMed1996

Highly supralinear feedback inhibition of Ca2+ uptake by the Ca2+ load of intracellular stores.

Net Ca2+ uptake into intracellular Ca2+ stores of homogenized cells is transient, even when the extravesicular Ca2+ concentration is kept constant. To study the mechanism underlying the phenomenon, we have investigated 45Ca2+ uptake by HL-60 cell homogenates. The initial rate of Ca2+ uptake as well as the final amount of stored Ca2+ were a function of the extravesicular Ca2+ concentration. However, Ca2+ uptake stopped independently of the extravesicular Ca2+ concentration after approximately 10 min. Studies using Ca2+-ATPase inhibitors demonstrated that the transient nature of the net uptake was not due to Ca2+ efflux. Monovalent cation ionophores did not influence the Ca2+ uptake curves, excluding a relevant involvement of pH and membrane potential. Together with the observation of a continued Ca2+ uptake in the presence of the intralumenal Ca2+ chelator oxalate, these results strongly suggest a feedback inhibition of Ca2+ uptake by the Ca2+ load of intracellular stores. The concentration-inhibition relationship between the Ca2+ load and the rate of Ca2+ uptake was highly supralinear (slope factor >/= 4). IC50 and maximum of the dose-inhibition curve, but not the slope factor were a function of the extravesicular free Ca2+ concentration. A series of three logistic equations derived from our data allowed an appropriate description of the behavior of Ca2+ uptake. Our results suggest, in addition to its well known activation by cytosolic Ca2+ concentration, a highly supralinear feedback inhibition of Ca2+ uptake by the Ca2+ load of intracellular stores. The steepness of the feedback inhibition might have a profound effect on spatial and temporal behavior of the Ca2+ signal.

Biological Transport↗
PubMed1996

Organization of Ca2+ stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor.

In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA)-type Ca(2+)-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca(2+)-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca(2+)-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca(2+)-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.

Calcium↗
PubMed1996

Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx.

The widely distributed and highly conserved Ca(2+)-binding protein calreticulin has been suggested to play a role as a Ca2+ storage protein of intracellular Ca+ stores. To test this hypothesis, we have generated a mouse L fibroblast cell line stably transfected with a calreticulin expression vector. The calreticulin content of the overexpressers was increased by 1.6 +/- 0.2-fold compared with mock-transfected cells. The total cellular Ca2+ content of calreticulin-overexpressing and control cells, as assessed by equilibrium 45Ca+2 uptake, was 141 +/- 8 and 67 +/- 6 pmol of Ca2+/10(6) cells, respectively (i.e. a 2.1 +/- 0.2-fold increase in the Ca2+ content of calreticulin-overexpressing cells). Over 80% of the increased Ca2+ content was found within thapsigargin-sensitive Ca2+ stores. The pattern of calreticulin distribution, revealed by immunofluorescence microscopy, showed an endoplasmic reticulum-like pattern and was identical in overexpressers and control cells. In overexpressers, cytosolic free [Ca2+] elevations due to Ca2+ release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast, thapsigargin-induced Ca2+ and Mn2+ influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active involvement of calreticulin in the regulation of store-operated Ca2+ influx.

Adenosine Triphosphate↗
PubMed1996

Proton currents in human eosinophils.

The killing of metazoan parasites by eosinophils involves the activation of a respiratory burst oxidase. To investigate whether human eosinophils possess an H+ conductance that might participate in the extrusion of H+ generated by the respiratory burst, we employed the whole cell patch-clamp technique under conditions designed to isolate putative H+ currents. We observed a slow activation of outward currents by depolarizing voltage steps. The reversal potential (Erev) of the currents was a function of the H+ gradient, demonstrating that the current was carried by H+. The H+ conductance was activated by cytosolic acidification and reversibly blocked by divalent and trivalent cations. During large prolonged depolarizing voltage steps, the current activation was followed by a decrease in current. This was due to cytosolic H+ depletion, as evidenced by 1) a change in Erev and 2) a cytosolic alkalinization. We also observed a rundown of the current, possibly due to the loss of a cytosolic factor necessary for H+ current activity. An elevated pipette Ca2+ concentration (1 microM) activated the H+ conductance, suggesting that the cytosolic Ca2+ concentration is involved in the physiological regulation of H+ currents. The Ca(2+)-activated currents had properties similar to the currents observed at low Ca2+ concentrations (Erev, high-affinity block by Zn2+, kinetics of tail currents, kinetics of rundown). The Ca2+ effect might be mediated by phospholipase A2, inasmuch as 1) the currents were also activated by arachidonic acid, 2) the Ca2+ effect and the arachidonic acid effect were not additive, and 3) the Ca2+ effect, but not the arachidonic acid effect, was blocked by a phospholipase A2 inhibitor. Taken together, our results demonstrate that human eosinophils have large H+ currents that are activated by physiological intracellular signals. The electrophysiological properties of the H+ currents and their regulation strongly suggest that they participate in H+ extrusion during the respiratory burst.

Calcium↗
PubMed1996

Presumed bilateral cytomegalovirus-induced optic neuropathy in an immunocompetent person. A case report.

Ocular manifestations of an acquired cytomegalovirus (CMV) infection are often associated with the patient being in an immunocompromised state. In the vast majority of cases, the affliction appears as a necrotic retinitis; an isolated optic nerve lesion is seen less frequently. To date, only two cases of CMV retinitis and one case of bilateral acute retinal necrosis have been reported in healthy individuals. We describe a 32-year-old healthy woman who developed bilateral papillitis. Tests were positive for acute CMV infection but negative for an immunocompromised state. We changed the antiviral therapy from acyclovir to foscarnet in association with corticosteroids, and resolution of the papillitis and recovery of visual acuity were obtained.

Adult↗
PubMed1995

[Characterization of Ca2+ influx in human neutrophils using the patch clamp technique].

In neutrophils, Ca2+ influx across the plasma membrane occurs in response to a variety of agonists. It plays an important role in the regulation of neutrophil function, in particular exocytosis and release of inflammatory mediators. Using the combination of patch clamp and microfluorimetry techniques, we have found the following characteristics of neutrophil Ca2+ influx: (a) neutrophils do not possess voltage-dependent Ca2+ channels; (b) Ca2+ influx is activated by depletion of intracellular Ca2+ stores, either by Ins(1,4,5)P3, or by Ca(2+)-ATPase inhibitors; (c) Ca2+ influx is not activated by [Ca2+]i elevations and not associated with the activation of non-selective cation channels; (d) Ca2+ influx is associated with the activation of a small, voltage-independent current through a highly Ca(2+)-selective conductance. The current was only seen upon depletion of intracellular Ca2+ stores and showed a pharmacological profile similar to the fluorimetrically detected Ca2+ influx.

Calcium↗
PubMed1995

Intracellular pattern of cytosolic Ca2+ changes during adhesion and multiple phagocytosis in human neutrophils. Dynamics of intracellular Ca2+ stores.

The subcellular pattern of cytosolic free Ca2+ ([Ca2+]i) changes in human polymorphonuclear neutrophils (PMNs) was studied using imaging of fura-2 fluorescence (time resolution 12.5 ratios/s) to determine whether PMNs could obtain directional information from the [Ca2+]i signal. [Ca2+]i changes were observed during initial adherence, the subsequent chemotactic movement, and the phagocytosis of opsonized yeast particles. Initial adherence was followed by a rapid increase in [Ca2+]i (from 90 +/- 10 to 290 +/- 40 nmol/L in 6.5 +/- 2.5 seconds; +/- SEM, n = 10), apparently homogeneously distributed over the entire cytoplasm, which preceded the spreading of the PMNs. [Ca2+]i increases after the contact of the PMNs with yeast particles were of lower mean amplitude; [Ca2+]i increased simultaneously throughout the cytosol. In the absence of extracellular Ca2+, multiple phagocytotic events could proceed normally without a mandatory [Ca2+]i transient. In PMNs polarized on phagocytosis, gradients in [Ca2+]i could be observed. [Ca2+]i was more elevated in the periphagosomal area than in the remaining parts. Taken together, these data show that [Ca2+]i waves do not provide the neutrophil with directional information during chemotaxis and phagocytosis. Sustained small inhomogeneity of [Ca2+]i levels are consistent with a proposed redistribution of releasable Ca2+ stores on phagocytosis.

Adult↗
PubMed1995

Highly cooperative Ca2+ elevations in response to Ins(1,4,5)P3 microperfusion through a patch-clamp pipette.

To study the initial kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations with a high time resolution and to avoid the problem of cell-to-cell heterogeneity, we have used the combined patch-clamp/microfluorimetry technique. The mathematical description of the microperfusion of Ins(1,4,5)P3 and the subsequent Ca2+ release consists of a monoexponential decay (cytosolic Ins(1,4,5)P3 concentration) and a Hill equation (Ins(1,4,5)P3 dose-response curve). Two additional Hill equations and an integration were necessary to include a putative dependence of Ins(1,4,5)P3-induced Ca2+ release on [Ca2+]i. Best-fitting analysis assuming [Ca2+]i-independent Ca2+ release yielded Hill coefficients between 4 and 12. The high cooperativity was also observed with the poorly metabolizable analog Ins(2,4,5)P3 and was independent of extracellular [Ca2+]. Best-fitting analysis including a positive [Ca2+]i feedback suggested a cooperativity on the level of Ins(1,4,5)P3-induced channel opening (n = 2) and an enhancement of Ins(1,4,5)P3-induced Ca2+ release by [Ca2+]i. In summary, the onset kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations in single HL-60 granulocytes showed a very high cooperativity, presumably because of a cooperativity on the level of channel opening and a positive Ca2+ feedback, but not because of Ca2+ influx or Ins(1,4,5)P3 metabolism. This high cooperativity, acting in concert with negative feedback mechanisms, might play an important role in the fine-tuning of the cellular Ca2+ signal.

Calcium↗
PubMed1994

Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils.

Subcellular gradients of cytosolic free Ca2+ concentration, [Ca2+]i, are thought to be critical for the localization of functional responses within a cell. A potential but previously unexplored mechanism for the generation of gradients of [Ca2+]i is the accumulation of Ca2+ stores at the site of Ca2+ action. The distribution of the Ca2+ store markers Ca(2+)-dependent adenosine triphosphatase and calreticulin was investigated in resting and phagocytosing human neutrophils. Both proteins showed an evenly distributed fine granular pattern in nonphagocytosing cells, but became markedly concentrated in the filamentous actin-rich cytoplasmic area around the ingested particle during phagocytosis. This redistribution began at early stages of phagocytosis and did not depend on an increase in [Ca2+]i. Thus, accumulation of Ca2+ stores in a restricted area of the cell may contribute to the generation of localized increases in [Ca2+]i.

Actins↗
PubMed1994

Rapid heparin-sensitive Ca2+ release following Ca(2+)-ATPase inhibition in intact HL-60 granulocytes. Evidence for Ins(1,4,5)P3-dependent Ca2+ cycling across the membrane of Ca2+ stores.

In many cell types, emptying of intracellular Ca2+ stores after application of inhibitors of the intracellular Ca(2+)-ATPase (e.g. thapsigargin) is astonishingly rapid. It was the aim of this study to elucidate the underlying mechanism. We first compared thapsigargin-induced emptying of intracellular Ca2+ stores in intact and homogenized HL-60 granulocytes. Thapsigargin-induced Ca2+ release was rapid in intact cells (33.9 +/- 4.9% of store content/min), but it was slow in permeabilized or homogenized cells (7.7 +/- 3.9 and 12 +/- 3.8% of store content/min respectively). To study whether the Ins(1,4,5)P3 receptor might be involved in thapsigargin-induced Ca2+ release, we tested the effect of heparin, a competitive Ins(1,4,5)P3 antagonist. In homogenized and permeabilized preparations, heparin did not interfere with thapsigargin-induced Ca2+ release. In contrast, when introduced into intact cells by an endocytosis/osmotic-shock procedure, heparin, but not the inactive de-N-sulphated heparin, decreased the rate of Ca2+ release by approx. 70%. Heparin inhibited Ca2+ release in response to the Ins(1,4,5)P3-generating receptor agonist N-formylmethionyl-leucyl-phenylalanine (f-MLP) (50 nM) and to thapsigargin (50 nM) at comparable concentrations. Heparin inhibition was competitive for f-MLP-induced, but not for thapsigargin-induced, Ca2+ release. In permeabilized cells, the addition of low Ins(1,4,5)P3 concentrations before thapsigargin increased the rate of thapsigargin-induced Ca2+ release 4-fold. Taken together, our results suggest that the rapid Ca(2+)-ATPase-inhibitor-induced Ca2+ release is due to a partial activation of the Ins(1,4,5)P3 receptor in resting cells. This implies Ca2+ cycling across the membrane of Ins(1,4,5)P3-sensitive Ca2+ stores in resting cells.

Calcium↗
PubMed1994

Characterization of receptor-mediated and store-regulated Ca2+ influx in human neutrophils.

1. It is not known to what extent the emptying of intracellular Ca2+ stores participates in the mediation of chemoattractant-induced Ca2+ influx in human neutrophils. To study this question, we compared the properties of bivalent-cation influx in response to the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenyl-alanine (f-MLP) and to the microsomal Ca(2+)-ATPase inhibitor thapsigargin. 2. The influx pathway activated by f-MLP and thapsigargin had identical properties of permeation. Mn2+ influx became saturated at around 1 mM extracellular Mn2+, whereas Ca2+ influx did not become saturated up to concentrations of 10 mM. 3. The influx of the two bivalent cations, Mn2+ and Ca2+, was activated to a similar extent and with identical kinetics of activation. 4. The Mn2+ influx activated by f-MLP and thapsigargin was blocked, with identical dose-inhibition curves, by four imidazole analogues. 5. The same relationship between the emptying of Ca2+ stores and bivalent-cation influx was observed for f-MLP and thapsigargin, with a half-maximal activation of the influx at 40% emptying of intracellular stores. 6. In conclusion, neutrophils possess a single type of Ca(2+)-influx pathway that is activated by receptor agonists and by store depletion. Receptor agonists activate this influx pathway to a large extent, if not completely, through the depletion of intracellular Ca2+ stores.

Biological Transport↗
PubMed1993

The regulation of store-dependent Ca2+ influx in HL-60 granulocytes involves GTP-sensitive elements.

In granulocytes, emptying of intracellular Ca2+ stores activates Ca2+ influx across the plasma membrane. To study the putative role of GTP-binding proteins in this process, we have introduced non-hydrolyzable guanosine phosphate analogues into the cytosol of non-permeabilized HL-60 granulocytes using an endocytosis-hypoosmotic shock procedure. At the cytosolic concentrations obtained (100-500 microM), neither guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) nor guanosine 5'-3-O-(thio)diphosphate (GDP beta S) affected basal [Ca2+]i. Ca2+ release in response to the receptor agonist fMet-Leu-Phe, the Ca(2+)-ATPase inhibitor thapsigargin, or the Ca2+ ionophore ionomycin was also unaffected by GTP gamma S or GDP beta S. In contrast, the activation of the Ca2+ influx pathway by fMet-Leu-Phe or by thapsigargin was blocked by GTP gamma S but not by GDP beta S. The GTP gamma S effect was mimicked by NaF. The GTP gamma S and NaF effects were independent of protein kinase C activation and actin polymerization. Our results demonstrate that a GTP-sensitive element is involved in the signaling between intracellular Ca2+ stores and plasma membrane Ca2+ channels. The identical effects of GTP gamma S and NaF suggest that the GTP-sensitive element is a heterotrimeric G-protein.

Biological Transport↗
PubMed1993

Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C.

Stimulation of human neutrophils by the receptor agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) results in a respiratory burst, catalysed by an NADPH oxidase. Concomitantly, phospholipase D (PLD) is activated. To investigate the role of protein kinase C (PKC) in these neutrophil responses, we have compared the effects of staurosporine and a structural analogue of staurosporine (cgp41251), that reflects a higher selectivity towards PKC [Meyer, Regenass, Fabbro, Alteri, Rösel, Müller, Caravatti and Matter (1989) Int. J. Cancer 43, 851-856]. Both staurosporine and cgp41251 dose-dependently inhibited the production of superoxide induced by phorbol 12-myristate 13-acetate (PMA). Both compounds also caused inhibition of the fMLP-induced respiratory burst, but with a lower efficacy during the initiation phase of this response. This latter observation cannot be taken as evidence against PKC involvement in the activation of the respiratory burst, because pretreatment of neutrophils with ionomycin before PMA stimulation also results in a lower efficacy of inhibition. Activation of PLD by fMLP was enhanced in the presence of staurosporine, but not in the presence of cgp41251. Enhancement of PLD activation was also observed in the presence of H-89, an inhibitor of cyclic-AMP-dependent protein kinase (PKA). Both staurosporine and H-89 reversed the dibutyryl-cyclic-AMP-induced inhibition of PLD activation, whereas cgp41251 was without effect. These results indicate that the potentiating effect of staurosporine on PLD activation induced by fMLP does not reflect a feedback inhibition by PKC activation, but instead a feedback inhibition by PKC activation. Taken together, our results indicate that in human neutrophils: (i) PKC activity is not essential for fMLP-induced activation of PLD; (ii) PKC activity does play an essential role in the activation of the respiratory burst by fMLP, other than mediating or modulating PLD activation; (iii) there exists a negative-feedback mechanism on fMLP-induced PLD activation by concomitant activation of PKA.

Alkaloids↗
PubMed1993

Ca2+ and Mg2+ regulation of inositol 1,4,5-triphosphate binding in myeloid cells.

Inhibition of inositol 1,4,5-triphosphate (Ins(1,4,5)P3) binding by Ca2+ and Mg2+ remains a controversial issue: the Ca2+ effect may also be explained by Ins(1,4,5)P3 generation due to a Ca(2+)-sensitive phospholipase C, and the Mg2+ effect by Ins(1,4,5)P3 degradation. In this study, we have, therefore, investigated the effects of Ca2+ and Mg2+ on Ins(1,4,5)P3 binding to membranes of the myeloid cell line HL-60 and assayed in parallel phospholipase C activity and Ins(1,4,5)P3 degradation. The assays for Ins(1,4,5)P3 binding, Ins(1,4,5)P3 generation, and Ins(1,4,5)P3 degradation were performed under identical experimental conditions. Ca2+ significantly and reversibly decreased Ins(1,4,5)P3 binding at submicromolar concentrations. Ca2+ stimulated Ins(1,4,5)P3 generation only at concentrations above 1 microM. At 1 microM [Ca2+], Ins(1,4,5)P3 binding was inhibited by 46 +/- 6%, but no Ins(1,4,5)P3 generation was observed. [Mg2+] between 0 and 1 mM slightly stimulated, while higher concentrations inhibited, Ins(1,4,5)P3 binding. Mg2+ did not activate phospholipase C. Neither Ca2+ nor Mg2+ induced relevant Ins(1,4,5)P3 degradation under the conditions of the binding assay. The effects of Ca2+ and Mg2+ on Ins(1,4,5)P3 binding were conserved after solubilization of HL-60 membranes. However, only Mg2+, but not Ca2+, inhibited Ins(1,4,5)P3 binding to the receptor that had been partially purified by heparin affinity chromatography. The Ca2+ sensitivity of Ins(1,4,5)P3 binding to the partially purified receptor could be reconstituted by addition of the flow-through of the heparin column. No Ca(2+)-dependent Ins(1,4,5)P3 generation was observed in the reconstituted system. Thus, we conclude that both Ca2+ and Mg2+ can inhibit Ins(1,4,5)P3 binding independently of phospholipase C activation and Ins(1,4,5)P3 breakdown. The Ca2+ effect depends upon factors that can be separated biochemically from the Ins(1,4,5)P3 receptor. Mg2+ acts either directly on the Ins(1,4,5)P3 receptor or on a closely associated target.

Calcium↗