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D P Lew

Publications and source records attributed to D P Lew.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1995

Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes.

Staphylococcus aureus 8325-4 has the potential to express two distinct cell wall-associated fibronectin-binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin-coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB. A double fnbAfnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non-conserved N-terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild-type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion-defective phenotype of the 8325-4 fnbAfnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325-4 to adhere to fibronectin-coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea-pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo.

Adhesins, Bacterial↗
PubMed1995

Comparison of sparfloxacin, temafloxacin, and ciprofloxacin for prophylaxis and treatment of experimental foreign-body infection by methicillin-resistant Staphylococcus aureus.

The prophylactic and therapeutic activities of three broad-spectrum fluoroquinolones were evaluated in two different experimental models of foreign-body infections caused by methicillin-resistant Staphylococcus aureus (MRSA) susceptible to quinolones. In a guinea pig model of prophylaxis, subcutaneously implanted tissue cages were infected at a > 90% rate by 10(2) CFU of MRSA in control animals. A single dose of 50 mg of ciprofloxacin per kg of body weight administered intraperitoneally 3 h before bacterial challenge was less effective than an equivalent regimen of either sparfloxacin or temafloxacin in decreasing the rate of experimental infection in tissue cages challenged with increasing inocula of MRSA. In a rat model evaluating the therapy of chronic tissue cage infection caused by MRSA, the efficacy of a 7-day high-dose (50-mg/kg twice-daily) regimen of sparfloxacin, temafloxacin, or ciprofloxacin was compared to that of vancomycin (50 mg/kg twice daily). Active levels of sparfloxacin, temfloxacin, or ciprofloxacin were continuously present in tissue cage fluid during therapy, exceeding their MBCs for MRSA by 6- to 20-fold. Either temafloxacin, sparfloxacin, or vancomycin was significantly (P < 0.01) more active than ciprofloxacin in decreasing the viable counts of MRSA in tissue cage fluids. The different activities of ciprofloxacin compared with those of the other two quinolones against chronic tissue cage infections caused by MRSA did not involve the selective emergence of quinolone-resistant mutants. Temafloxacin and ciprofloxacin, which showed the most prominent differences in their in vivo activities, however, exhibited similar bactericidal properties and pharmacokinetic parameters in the rat model. In conclusion, both temafloxacin and sparfloxacin were significantly more active than ciprofloxacin for the prophylaxis or treatment of experimental foreign-body infections caused by a quinolone-susceptible strain of MRSA.

Animals↗
PubMed1995

Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts.

We used an ex vivo canine arteriovenous shunt model, previously developed to study plasma protein adsorption and thrombogenesis on polymeric biomaterials, to define the role of host proteins in promoting adhesion of Staphylococcus aureus. Either polyethylene or polyvinyl chloride tubings were exposed to canine blood for 5, 15, or 60 min at a flow rate of 300 ml/min and then were flushed in phosphate-buffered saline (PBS), cut into 1.5-cm segments, and stored at -70 degrees C. After thawing, each segment was preincubated in 0.5% albumin in PBS to prevent nonspecific staphylococcal attachment to surfaces that were not exposed to blood. Each segment was then incubated with 4 x 10(6) CFU of [3H]thymidine-labelled S. aureus per ml for 60 min at 37 degrees C in an in vitro adhesion assay. Two site-specific mutants of S. aureus were tested: one specifically defective in adhesion to surface-bound fibronectin (FnAd-def) and the other defective in adhesion to fibrinogen (FgAD-def) [corrected]. Compared with their respective parental strains, the FgAd-def, but not the FnAd-def, mutant of S. aureus showed a strong (> 80%) decrease in attachment to ex vivo tubings. The adhesion of each strain of S. aureus onto polyethylene was consistently more than twofold higher than the adhesion onto polyvinyl chloride segments exposed to flowing blood for 5 or 15 min, but adhesion became similar to that on polyvinyl chloride after 60 min of exposure. In conclusion, the specific adhesion-defective mutants of S. aureus suggested that fibrinogen was the most active adhesion-promoting protein in a short-term blood-material interaction. The experimental approach described in this study should prove useful for screening materials thought to be resistant to protein-mediated staphylococcal adhesion and colonization.

Animals↗
PubMed1995

Quinolones and osteomyelitis: state-of-the-art.

The present review provides a critical quantitative analysis of the use of quinolones in osteomyelitis. Only papers published in peer-reviewed journals and related to the following areas were selected: experimental osteomyelitis, penetration of quinolones into human bone and clinical use in comparative and non-comparative studies. Cumulated results show clinical success rates of more than 90% in osteomyelitis caused by Enterobacteriaceae, after prolonged oral use of ciprofloxacin. However, further comparative studies using oral quinolones as single agents or in combination (versus standard parenteral therapy) are required in osteomyelitis due to S. aureus or P. aeruginosa, or in more complicated situations such as diabetic osteomyelitis or foreign body infection.

4-Quinolones↗
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↗
PubMed1994

Role of fibronectin in staphylococcal adhesion to metallic surfaces used as models of orthopaedic devices.

Staphylococcal infection of various prosthetic and internal fixation devices is a major complication associated with orthopaedic surgery. This study investigated the role of the host protein fibronectin in promoting adhesion of Staphylococcus aureus and Staphylococcus epidermidis to metallic surfaces representing materials used for orthopaedic devices. Pure human fibronectin was adsorbed in vitro onto coverslips (0.8 x 0.8 cm) of stainless steel, pure titanium, or titanium-aluminum-niobium alloy. In vitro bacterial adhesion was promoted more strongly by the metallic surfaces coated with fibronectin than by albumin-coated controls for two strains of S. aureus and one strain of S. epidermidis. Furthermore, with the fibronectin-coated coverslips, bacterial adhesion to titanium alloy was significantly greater than adhesion to stainless steel. Adhesion of the three staphylococcal strains was promoted more strongly by coverslips explanted from the subcutaneous space of guinea pigs and tested under similar conditions than by albumin-coated controls. Incubation of either in vitro fibronectin-coated or explanted metallic coverslips with anti-fibronectin antibodies produced a significant decrease in staphylococcal adhesion. These results suggest that the presence of fibronectin on the surface of implanted metallic devices is an important determinant of colonization of orthopaedic biomaterials by staphylococci.

Adsorption↗
PubMed1994

Comparative efficacies of imipenem, oxacillin and vancomycin for therapy of chronic foreign body infection due to methicillin-susceptible and -resistant Staphylococcus aureus.

The efficacies of imipenem when directed against methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) strains of Staphylococcus aureus were compared with those of oxacillin and vancomycin in a subcutaneous rat model, using chronically infected tissue cages. At three weeks after inoculation, stable chronic infections were established with average bacterial counts exceeding 10(6) cfu/mL tissue cage fluid for both strains. Intraperitoneal administration (twice a day for 7 days) of imipenem (80 mg/kg) or oxacillin (200 mg/kg) produced peak levels of 23 or 45 mg/L and through levels of < 0.1 and 5.7 mg/L, respectively. The therapeutic regimens of either imipenem (P < 0.001) or oxacillin (P < 0.02) administered for 7 days led to significant reductions in bacterial counts in the tissue cage fluids of animals chronically infected with MSSA. In contrast, imipenem was not effective against chronic MRSA tissue cage infections, despite the relatively low MIC of the infecting strain and the use of high dose (120 mg/kg) therapy. In-vitro susceptibility testings of MRSA performed before and after imipenem therapy demonstrated the emergence of a highly resistant subpopulation.

Animals↗
PubMed1994

Single-dose pharmacokinetics of oral fleroxacin in bacteremic patients.

Fleroxacin is a new broad-spectrum quinolone which can be given by the oral route. The present study was designed to assess the influence of bacteremia on the pharmacokinetics of a single oral dose of fleroxacin. Thirteen patients with proven bacteremia (one or more pairs of positive blood cultures, no hypotension) were given a single 400-mg fleroxacin dose orally on two occasions while also receiving standard antibiotic therapy. The first dose was administered 12 to 36 h after the last positive blood culture was drawn (day 1), and a second dose was administered 7 days later (day 7 +/- 2) to compare the pharmacokinetics between the acute and the convalescent phases of the disease. Following each administration of fleroxacin, serial plasma samples were collected for up to 72 h and were analyzed for unchanged drug by a reversed phase high-pressure liquid chromatography technique. There were no significant changes in the following pharmacokinetic parameters (mean standard deviation) the maximum concentration of drug in serum (6.4 +/- 1.5 versus 6.7 +/- 1.9 mg/liter), the minimum concentration of drug in serum, defined as the concentration of drug in serum at 24 h postdose (3.0 +/- 1.7 versus 2.5 +/- 1.2 mg/liter), the time to the maximum concentration of drug in serum (2.3 +/- 1.4 versus 2.0 +/- 1.2 h), and the elimination half-life (19.7 +/- 8.0 versus 17.9 +/- 6.9 h). Fleroxacin clearances were compared for each individual patient. A positive correlation (R2 = 0.787) was found between the values measured on day 1 and day 7. Oral clearance of fleroxacin (CL = CL/F, where F is bioavailability was slightly, but not significantly, reduced during the bacteremic phase (oral clearance, 43.8+/- 23.5 versus 48.5 +/- 17.5 ml/min.). When compared with previous results obtained in healthy young subjects, longer times to the maximum concentration of drug in serum and elimination half-lives and higher areas under the curve were observed. This could be due to the bacteremic state, the old age of the patients (mean, 66 years), and the low renal clearance (mean calculated creatinine clearance, 71.1 ml/min). A single oral dose of 400 mg of fleroxacin provides sufficient levels in serum to cover susceptible microorganisms for at least 24 h in bacteremic patients. Renal function appeared to be the key element that had to be taken into consideration to adapt fleroxacin dosage profiles in our patient population. Bacteremia itself appeared to amplify that phenomenon, but to a much lesser extent than renal function did.

Administration, Oral↗
PubMed1994

Teicoplanin alone or combined with rifampin compared with vancomycin for prophylaxis and treatment of experimental foreign body infection by methicillin-resistant Staphylococcus aureus.

The prophylactic and therapeutic activities of teicoplanin were evaluated in two different experimental models of foreign body infections caused by methicillin-resistant Staphylococcus aureus (MRSA). In a guinea pig model of prophylaxis, subcutaneously implanted tissue cages were infected at a > 90% rate by 10(2) CFU of MRSA in control animals. A single dose of 30 mg of teicoplanin per kg of body weight administered intraperitoneally 6 h before bacterial challenge was as effective as vancomycin in preventing experimental infection in tissue cages injected with either 10(2), 10(3), or 10(4) CFU of MRSA. In a rat model evaluating the therapy of chronic tissue cage infection caused by MRSA, the efficacy of a 7-day high-dose (30 mg/kg once daily) regimen of teicoplanin was compared with that of vancomycin (50 mg/kg twice daily). Whereas high levels of teicoplanin were found in tissue cage fluid, continuously exceeding its MBC for MRSA by 8- to 16-fold, no significant reduction in the viable counts of MRSA occurred during therapy. In contrast, either vancomycin alone or a combined regimen of high-dose teicoplanin plus rifampin (25 mg/kg twice daily) could significantly decrease the viable counts in tissue cage fluids. Whereas the bacteria recovered from tissue cage fluids during therapy showed no evidence of teicoplanin resistance, they failed to be killed even by high levels of this antimicrobial agent. The altered susceptibility of in vivo growing bacteria to teicoplanin killing might in part explain the defective activity of this antimicrobial agent when used as monotherapy against chronic S. aureus infections. These data may indicate the need for a combined regimen of teicoplanin with other agents such as rifampin to optimize the therapy of severe staphylococcal infections.

Animals↗
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

Intracellular Ca2+ and the regulation of early response gene expression in HL-60 myeloid leukemia cells.

To gain direct insight into the action of the second messenger Ca2+ on transcriptional regulation, we have developed an intact cell model in which the intracellular free Ca2+ concentration ([Ca2+]i) can be measured, set, and varied at any level within the physiological range and in which the expression of early response genes is assayed in parallel. Using promyelocytic HL-60 cells, we have observed an exquisite sensitivity to Ca2+ of c-fos, c-jun, and zif268 mRNA accumulation, since early and maximal inductions were observed at 200-300 nM [Ca2+]i. At early times (10-20 min), the [Ca2+]i dose dependence of c-fos transcription and mRNA accumulation displayed a bell shape since c-fos expression was barely modified at high (700-1,250 nM) [Ca2+]i. The threshold [Ca2+]i concentration for prolonged (60 min) c-fos mRNA accumulation was greater than 200 nM. This indicates that the quantitative effects of Ca2+ on a given gene can vary markedly as a function of both the [Ca2+]i concentration and the duration of stimulation. Strikingly, a [Ca2+]i perturbation of only 1 min was sufficient for full induction of c-fos and zif268 transcripts. This demonstrates that a transient perturbation of [Ca2+]i has long term effects on gene expression. The half-life of c-fos mRNA (16 min) was unaltered by Ca2+. Nuclear run-on analysis of the distribution of RNA polymerase II along the c-fos locus indicated that Ca2+ promotes a small increase in transcriptional initiation and a pronounced relief of a block to transcriptional elongation beyond intron 1. The extreme sensitivity to [Ca2+]i, in terms of both the length of time and the dose of [Ca2+]i required for maximal gene induction, demonstrates that Ca2+ is a major physiological regulator of early response gene expression. In addition, the results indicate that a c-fos intragenic element is the main target of Ca(2+)-regulated transcriptional activation.

Calcium↗
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↗