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PubMed1998

Quantitative assessment of [Ca2+]i levels in rat skeletal muscle in vivo.

Intracellular free Ca2+ concentration ([Ca2+]i) plays an essential role in physiological regulatory processes and common pathological conditions. Better understanding of these phenomena is still hampered by problems encountered in the quantitative assessment of [Ca2+]i changes, especially in blood-perfused organs. This study demonstrates that the ratiometric fluorescence technique can be adapted for quantitative in vivo [Ca2+]i determinations. The rat spinotrapezius muscle was topically loaded with indo 1-AM and imaged by a cooled digital camera. Ratio images were calculated in small regions (100 micrometers x 100 micrometers) practically devoid of large vessels in the resting state, after 30 min of ischemia, 20 min of reperfusion, or ionomycin or manganate treatments. When we assumed an average [Ca2+]i of 100 nM in the resting blood-perfused muscle, ischemia increased [Ca2+]i to approximately 200 nM. During reperfusion [Ca2+]i decreased to approximately 140 nM. Ionomycin induced an increase in [Ca2+]i to well above 750 nM. Manganate reduced Ca2+-dependent fluorescence to virtually zero. Our main conclusion is that changes in [Ca2+]i can be monitored and quantitatively determined in vivo.

Animals↗
PubMed1994

Activation of G proteins may inhibit or stimulate surfactant secretion in rat alveolar type II cells.

To investigate how G proteins regulate surfactant secretion, we subjected rat alveolar type II cells to conditions known to activate or to inactivate G proteins. AlF-4, which activates G proteins, inhibited secretion in intact cells. Guanosine-5'-O-(3-thiotriphosphate), which activates G proteins in permeabilized cells, stimulated secretion at basal cytosolic [Ca2+], but inhibited secretion at higher [Ca2+]. In contrast, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), which inactivates G proteins, stimulated secretion at each [Ca2+] tested. Because treatment with GDP beta S stimulated secretion at basal cytosolic [Ca2+], surfactant secretion appears to be subject to G protein-regulated tonic inhibition. Pertussis toxin (PTX) inhibited terbutaline- and ionomycin-stimulated secretion in intact cells, but did not inhibit secretion stimulated by either forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate. Inhibition by PTX of terbutaline-stimulated, but not 8-bromoadenosine 3',5'-cyclic monophosphate- or forskolin-stimulated secretion, suggests that PTX-sensitive G proteins regulate beta-adrenergic-stimulated surfactant secretion proximal to second messenger generation. Inhibition of ionomycin-stimulated secretion, however, suggests that PTX-sensitive G proteins may also regulate non-receptor-mediated secretory events.

Aluminum Chloride↗
PubMed1998

Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability.

Cytosolic Ca2+ concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+ induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 microM) produced higher [Ca2+]i levels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]i response in RPMVECs, we investigated whether reduced activation of store-operated Ca2+ entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+ followed by readdition media with 2 mM extracellular Ca2+ increased RPMVEC [Ca2+]i to a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]i in RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 microM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]i promotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]i signaling pathways or dominant Ca(2+)-independent mechanisms from controlling cellular gap formation and permeability.

Animals↗
PubMed2004

Regulation and identity of intracellular calcium stores involved in membrane cross talk in the early distal tubule of the frog kidney.

The early distal tubule (EDT) of the frog nephron, similar to the thick ascending limb in mammals, mediates the transepithelial absorption of NaCl. The continued absorption of NaCl in the face of varying Na(+) load is maintained by coordination of the activity of ion-transporting proteins in the apical and basolateral membranes, so-called pump-leak coupling. Previous studies identified intracellular Ca(2+), originating from an intracellular Ca(2+) store, as playing a key role in pump-leak coupling in the EDT (Cooper GJ, Fowler MR, and Hunter M. Pflügers Arch 442: 243-247, 2001). The purpose of the experiments described in this paper was to identify the intracellular Ca(2+) storage pools in the renal diluting segment. Store Ca(2+) movements were monitored by the fluorescence of mag-fura 2 in permeabilized segments of frog EDTs. The presence of both ATP and Ca(2+) was required to maintain store Ca(2+) content. Removal of either of these substrates resulted in a passive leak of Ca(2+) from the stores. The uptake of Ca(2+) into the store was sensitive to the SERCA inhibitor 2,5-di(tert-butyl) hydroquinone, whereas Ca(2+) release from the store was stimulated by IP(3) but not cADPR. Store Ca(2+) was insensitive to the mitochondrial ATP synthase inhibitor oligomycin, and, under conditions that energized Deltapsi(m), the complex 1 inhibitor rotenone and the protonophore FCCP. Ionomycin was able to mobilize store Ca(2+) following exposure to IP(3). These results suggest that the endoplasmic reticulum is a dominant Ca(2+) store in the frog EDT. A second pool, sensitive to ionomycin but not IP(3), may overlap with the IP(3)-sensitve pool. The data also rule out any contribution by mitochondria to EDT Ca(2+) cycling.

Animals↗
PubMed1991

Inhibitory effect of angiotensin II on renal tubular transport.

This study was designed to examine the intracellular mechanism of inhibitory action of high concentration of angiotensin II (ANG II) on proximal tubular transport in rat kidneys by microperfusion methods. Perfusion of ANG II (10(-6) M) to peritubular capillaries caused a reduction of both fluid and HCO3- transport (Jv and JHCO3-, respectively) by 33 and 26%, respectively. These inhibitory effects were blocked by the ANG II-receptor antagonist [Sar1, Ile8]ANG II (10(-5) M). Similar degrees of inhibition on Jv and JHCO3- were observed when ionomycin (10(-7) and 10(-6) M), a Ca2+ ionophore, was added to capillary perfusate. Moreover, there was no additive effect when both ANG II and ionomycin were perfused together through capillaries, suggesting that both agents work via the same mechanism, presumably by increasing cytosolic Ca2+ concentration ([Ca2+]i). Inhibitory effects of ANG II on proximal tubular transport were still observed in a Ca2(+)-free perfusate containing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, indicating that these effects do not require influx of Ca2+ from extracellular medium. Furthermore, the observation that TMB-8, an agent that prevents intracellular Ca2+ mobilization, completely eliminated the effect of ANG II strongly suggests that intracellular Ca2+ rather than Ca2+ influx mediates effects of ANG II on proximal tubular transport. Direct measurement of [Ca2+]i by use of fura-2 in isolated proximal tubular cells showed slight but statistically significant increases in [Ca2+]i. Taken together, these observations support the idea that intracellular Ca2+ serves as a second messenger in the inhibitory effect of high concentrations of ANG II on Jv and JHCO3- in proximal tubule of kidney.

Angiotensin II↗
PubMed1992

Injury-induced changes in cytosolic Ca2+ in individual rabbit proximal tubule cells.

Cell injury was studied in cultured rabbit proximal tubular epithelial cells using digital-imaging fluorescent microscopy to relate changes in cytosolic Ca2+ ([Ca2+]i) to bleb formation and cell death. Fura-2-loaded cells were treated in normal (1.37 mM) and low (less than 5 microM) extracellular Ca2+ ([Ca2+]e) with 1) inhibitors of glycolysis (iodoacetate) and/or mitochondrial oxidation (KCN), 2) thiol-modifying reagents (N-ethylmaleimide, p-chloromercuribenzene, and p-chloromercuribenzene sulfonate), and 3) Ca2+ ionophore (ionomycin). All three types of injury produced both [Ca2+]e-independent and [Ca2+]e-dependent increases in [Ca2+]i. KCN + iodoacetate +/- [Ca2+]e did not produce blebbing or death within 60-90 min. Thiol modifiers and ionomycin produced blebbing, which correlated with sustained threefold or greater elevations of [Ca2+]i and loss of viability only after [Ca2+]i had risen severalfold. Blebbing and cell death could be prevented or delayed by treatment in low [Ca2+]e. Trypsin (x0.5) caused a transient (less than 5 min) elevation in [Ca2+]i as well as increases in intracellular Ca2+ pools.

Animals↗
PubMed1993

Nucleotide receptors regulate membrane ion transport in renal epithelial cells.

Regulation of plasma membrane ion transport by endogenous purinergic receptors was assessed in a distal renal (A6) cell line. Nucleotide analogues stimulated Na-K-Cl cotransport activity with relative potencies of ATP > UTP > ATP gamma S > 2-methylthio-ATP = alpha,beta-methylene ATP. Activation of nucleotide receptors with extracellular ATP and nucleotide analogues increased intracellular calcium concentration ([Ca2+]i) primarily by release of intracellular calcium stores, with relative potency of agonists similar to that seen for stimulation of Na-K-Cl cotransport. Neither the change in [Ca2+]i nor the stimulation of cotransport was abolished by the adenosine receptor antagonist 8-(4-[N-(2-aminoethyl)carbamoylmethoxy]-phenyl)-1,3-dipropylxanthi ne (XAC). In contrast to the adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine, nucleotide analogues had no discernible effect on cytosolic adenosine 3',5'-cyclic monophosphate levels or adenylyl cyclase activity. To address possible mechanisms for stimulation of Na-K-Cl cotransport by the nucleotide receptor, 125I efflux and patch-clamp studies were used to measure chloride secretion. ATP and ionomycin markedly enhanced 125I efflux and whole cell currents, consistent with activation of chloride conductance pathways. Diphenylamine-2-carboxylate, a chloride channel blocker, eliminated the effects of ionomycin, forskolin, adenosine, and ATP on Na-K-Cl cotransport. This study demonstrates that nucleotide receptors in this model of renal epithelium initiate distinct regulation of Na-K-Cl cotransport. Nucleotide receptors may effect their responses through primary activation of membrane chloride channels.

Biological Transport↗
PubMed2004

Effect of renal cell carcinomas on the development of type 1 T-cell responses.

PURPOSE: We reported that in renal cell carcinoma patients with active disease, T-cell reactions to the tumor-associated antigens MAGE-6 and EphA2 are highly skewed toward TH2-type cytokine responses [interleukin (IL) 5]. Herein, we determined whether tumor-derived products, including gangliosides isolated from renal cell carcinoma patients, participate in the down-regulation of type 1 T-cell responses. EXPERIMENTAL DESIGN: T cells from healthy volunteers or renal cell carcinoma patients were cultured in the presence and absence of supernatants derived from renal cell carcinoma explants or with gangliosides isolated from those tumor supernatants. T cells were stimulated or not with either autologous dendritic cells pulsed with superantigen (Staphylococcus enterotoxin B) or with phorbol 12-myristate 13-acetate and ionomycin and then were assessed for type 1 or type 2 responses (cytokine production and gene expression) and apoptosis. RESULTS: Tumor supernatants efficiently inhibited the TH1-type responses [interferon (IFN) gamma] of T cells stimulated with either S. enterotoxin B or phorbol 12-myristate 13-acetate and ionomycin but had no inhibitory effect on activated T-cell production of type 2 cytokines (IL-4, IL-5, and IL-10). Likewise, IFN-gamma mRNA and protein production were inhibited when T cells were cocultured with either renal cell carcinoma supernatant-derived gangliosides or a commercial source of purified GD1a. It was also determined that gangliosides impair type 1 responses by inducing apoptosis of activated T cells. CONCLUSIONS: We propose that renal cell carcinoma-derived tumor products such as gangliosides can induce a type 2 bias in antitumor immunity by initiating apoptosis in the IFN-gamma-producing type 1 effector cells. This represents a relevant mechanism by which renal cell carcinoma can inhibit protective antitumor immunity.

CD4 Lymphocyte Count↗
PubMed1999

Whole-blood flow-cytometric analysis of induction of adhesion molecules expression on eosinophils stimulated by phorbol-12-myristate-13-acetate/ionomycin.

BACKGROUND: Activation of eosinophils is closely associated with the pathology of allergic inflammatory disease, especially bronchial asthma. We recently investigated the activation of eosinophils by applying whole blood to a flow cytometer. We measured here beta1 and beta2 integrin on eosinophils stimulated by phorbol-12-myristate-13-acetate (PMA)/ionomycin to evaluate eosinophil activation in vitro using whole blood. METHODS: Heparinized whole blood was diluted with the same volume of RPMI 1640, then cells were incubated in the presence or absence of PMA and ionomycin for 45 min at 37 degrees C. After hemolyzation with lysing solution, flow-cytometric findings for CR3, LFA1-alpha, LFA1-beta and VLA-4 expression on eosinophils were examined. RESULTS: Mean fluorescent intensity (MFI) of CR3 and LFA1-beta stimulated by PMA and ionomycin was significantly higher than that of the unstimulated control. MFI of LFA1-alpha showed no significant difference from the unstimulated control. On the other hand, MFI of VLA-4 tended to decrease. CONCLUSIONS: Our method to distinguish eosinophils from various cell groups in whole blood is simple and time-saving, similar to conditions in vivo and may allow intensive investigation of eosinophils in clinical laboratories as well as in research laboratories. We are currently investigating the influence of different kinds of stimulations, regulation factors or agents on eosinophils using this method.

Carcinogens↗
PubMed2006

Stimulation of erythrocyte phosphatidylserine exposure by paclitaxel.

Side effects of cytostatic treatment include development of anemia resulting from either decreased generation or accelerated clearance of circulating erythrocytes. Recent experiments revealed a novel kind of stress-induced erythrocyte death, i.e. eryptosis, which is characterized by enhanced cytosolic Ca(2+) levels, increased ceramide formation and exposure of phosphatidylserine at the cell surface. The present study explored whether cytostatic treatment with paclitaxel (Taxol) triggers eryptosis. Blood was drawn from cancer patients before and after infusion of 175 mg/m2 Taxol. The treatment significantly decreased the hematocrit and significantly increased the percentage of annexin-V-binding erythrocytes in vivo (by 37%). In vitro incubation of human erythrocytes with 10 microM paclitaxel again significantly increased annexin-V-binding (by 129%) and augmented the increase of annexin-V-binding following cellular stress. The enhanced phosphatidylserine exposure was not dependent on caspase-activity but paralleled by erythrocyte shrinkage, increase of cytosolic Ca(2+) activity, ceramide formation and activation of calpain. Phosphatidylserine exposure was similarly induced by docetaxel but not by carboplatin or doxorubicin. Moreover, eryptosis was triggered by the Ca(2+) ionophore ionomycin (10 microM). In mice, ionomycin-treated eryptotic erythrocytes were rapidly cleared from circulating blood and sequestrated into the spleen. In conclusion, our data strongly suggest that paclitaxel-induced anemia is at least partially due to induction of eryptosis.

Animals↗
PubMed2006

Upregulation of arginase by H2O2 impairs endothelium-dependent nitric oxide-mediated dilation of coronary arterioles.

OBJECTIVE: Overproduction of reactive oxygen species such as hydrogen peroxide (H2O2) has been implicated in various cardiovascular diseases. However, mechanism(s) underlying coronary vascular dysfunction induced by H2O2 is unclear. We studied the effect of H2O2 on dilation of coronary arterioles to endothelium-dependent and endothelium-independent agonists. METHODS AND RESULTS: Porcine coronary arterioles were isolated and pressurized without flow for in vitro study. All vessels developed basal tone and dilated dose-dependently to activators of nitric oxide (NO) synthase (adenosine and ionomycin), cyclooxygenase (arachidonic acid), and cytochrome P450 monooxygenase (bradykinin). Intraluminal incubation of vessels with H2O2 (100 micromol/L, 60 minutes) did not alter basal tone but inhibited vasodilations to adenosine and ionomycin in a manner similar as that by NO synthase inhibitor L-NAME. H2O2 affected neither endothelium-dependent responses to arachidonic acid and bradykinin nor endothelium-independent dilation to sodium nitroprusside. The inhibited adenosine response was not reversed by removal of H2O2 but was restored by excess L-arginine. Inhibition of L-arginine consuming enzyme arginase by alpha-difluoromethylornithine or N(omega)-hydroxy-nor-L-arginine also restored vasodilation. Administering deferoxamine, an inhibitor of hydroxyl radical production, prevented the H2O2-induced impairment of vasodilation to adenosine. Western blot and reverse-transcription polymerase chain reaction results indicated that arginase I was upregulated after treating vessels with H2O2. CONCLUSIONS: H2O2 specifically impairs endothelium-dependent NO-mediated dilation of coronary microvessels by reducing L-arginine availability through upregulation of arginase. The formation of hydroxyl radicals from H2O2 may contribute to this process.

Animals↗
PubMed1989

Collagen-stimulated human platelet aggregation is mediated by endogenous calcium-activated neutral protease.

To clarify the physiological role of calcium-activated neutral protease (CANP) in human platelets, we loaded the platelets with a Ca2+ -sensitive fluorescent dye, fura-2, and measured the degree of aggregation, cytosolic calcium ion concentration [( Ca2+]i), and proteolysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At physiological concentration of Ca2+ (1 mM) in the incubation medium, [Ca2+]i was below 0.5 microM and platelet aggregation was not shown. Ionomycin (0.15 microM) or collagen (50 micrograms/ml), but not ADP (10 microM), sharply enhanced the [Ca2+]i to near 1 microM and caused the aggregation. A calcium entry blocker, verapamil, completely abolished both the [Ca2+]i rise and the aggregation. NCO-700, a membrane permeable inhibitor against cysteine proteases (including CANP), dose-dependently blocked the aggregation but did not change the [Ca2+]i transient. SDS-PAGE revealed that filamin, talin, and 70 kDa protein were specifically degraded when platelets were aggregated by ionomycin or collagen and that the proteolysis was not observed when the aggregation was blocked by verapamil or NCO-700. These data provided evidence that Ca2+ entry exceeding 0.5 microM is essential, but not sufficient per se, and that activation of cysteine protease, most likely CANP, is involved in the platelet aggregation by collagen or calcium ionophore.

Blood Platelets↗
PubMed1996

Intracellular Ca2+, intercellular electrical coupling, and mechanical activity in ischemic rabbit papillary muscle. Effects of preconditioning and metabolic blockade.

During myocardial ischemia, electrical uncoupling and contracture herald irreversible damage. In the present study, we tested the hypothesis that an increase of intracellular Ca2+ is an important factor initiating these events. Therefore, we simultaneously determined tissue resistance, mechanical activity, pH(0), and intracellular Ca2+ (with the fluorescent indicator indo 1, Molecular Probes, Inc) in arterially perfused rabbit papillary muscles. Sustained ischemia was induced in three experimental groups: (1) control, (2) preparations preconditioned with two 5-minute periods of ischemia followed by reperfusion, and (3) preparations pretreated with 1 mmol/L iodoacetate to block anaerobic metabolism and minimize acidification during ischemia. In a fourth experimental group, intracellular Ca2+ was increased under nonischemic conditions by perfusing with 0.1 mmol/L ionomycin and 0.1 mumol/L gramicidin. Ca2+ transients and contractions rapidly disappeared after the induction of ischemia. In the control group, diastolic Ca2+ began to rise after 12.6 +/- 1.3 minutes of ischemia; uncoupling, after 14.5 +/- 1.2 minutes of ischemia; and contracture, after 12.6 +/- 1.5 minutes of ischemia (mean +/- SEM). Preconditioning significantly postponed Ca2+ rise, uncoupling, and contracture (21.5 +/- 4.0, 24.0 +/- 4.1, and 23.0 +/- 5.3 minutes of ischemia, respectively). Pretreatment with iodoacetate significantly advanced these events (1.9 +/- 0.7, 3.6 +/- 0.9, and 1.9 +/- 0.2 minutes of ischemia, respectively). In all groups, the onset of uncoupling always followed the start of Ca2+ rise, whereas the start of contracture was not different from the rise in Ca2+. Perfusion with ionomycin and gramicidin permitted estimation of a threshold [Ca2+] for electrical uncoupling of 685 +/- 85 nmol/L. In conclusion, the rise in intracellular Ca2+ is the main trigger for cellular uncoupling during ischemia. Contracture is closely associated with the increase of intracellular Ca2+ during ischemia.

Animals↗
PubMed1998

LDLs impair vasomotor function of the coronary microcirculation: role of superoxide anions.

Oxidized LDLs (Ox-LDLs) inhibit endothelium-dependent dilation of isolated conduit arteries in a manner comparable to the impairment demonstrated in atherosclerotic vessels. However, it is not known whether the microvessels, which do not develop atherosclerotic lesions, are susceptible to Ox-LDL. Since endothelial release of NO plays an important role in vasodilation and since its dysfunction associated with atherosclerosis has been shown to extend into the coronary microcirculation, we hypothesized that Ox-LDLs impair endothelium-dependent vasodilation of coronary arterioles by reducing the synthesis and/or release of NO. To test this hypothesis, porcine subepicardial vessels (50 to 100 microm) were isolated, cannulated, and pressurized to 60 cm H2O without flow for in vitro study. Isolated vessels developed basal tone and dilated in a dose-dependent manner to the endothelium-dependent vasodilators serotonin, ATP, and ionomycin. These vasodilatory responses were inhibited by the NO synthase inhibitor NG-monomethyl-L-arginine and were subsequently reversed by extraluminal administration of the NO precursor L-arginine (3 mmol/L), suggesting the involvement of NO in these vasomotor responses. Intraluminal incubation of the vessels with native LDL (N-LDL) or Ox-LDL (1 mg protein/mL) significantly attenuated dilations to serotonin, ATP, and ionomycin. Ox-LDL produced more severe inhibition than did N-LDL, and the inhibitory effect was comparable to that of NG-monomethyl-L-arginine. The inhibitory effects of N-LDL and Ox-LDL were reversed by exogenous L-arginine (3 mmol/L) and were prevented by sodium dihydroxybenzene disulfonate (Tiron), a cell-permeable superoxide scavenger. In contrast, administration of the cell-impermeable superoxide scavenger superoxide dismutase prevented the inhibitory effect of N-LDL but not of Ox-LDL. In addition, the inhibitory effects of LDL were not restored by D-arginine or by removal of intraluminal LDL. Neither N-LDL nor Ox-LDL altered endothelium-independent vasodilation to sodium nitroprusside. These results indicate that coronary arterioles are susceptible to LDLs that specifically impair endothelium-dependent vasodilation by reducing NO synthesis. It is suggested that the initiation of superoxide anion production and the subsequent L-arginine deficiency may be responsible for the detrimental effect of LDL.

Animals↗
PubMed1997

Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation.

Endothelial cell (EC) gap formation and barrier function are subject to dual regulation by (1) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal adhesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125FAK (FAK), represent target regulatory sites involved in thrombin-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key effector enzyme involved in thrombin-induced EC barrier dysfunction. Although vinculin and talin were phosphorylated in situ under basal conditions in 32P-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting showed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not further increased after thrombin. In contrast, both thrombin and the thrombin receptor-activating peptide (TRAP) produced an increase in FAK phosphotyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunction in a myosin light chain kinase-independent manner, was used to increase intracellular Ca2+ and evaluate the Ca2+ sensitivity of this observation. In contrast to thrombin, ionomycin effected a dramatic decrease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting distinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphorylation of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability models that do not depend on an increase in myosin 20-kD regulatory light chain phosphorylation.

Animals↗
PubMed1993

Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.

The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, 20% with secretory vesicles and the rest with plasma membranes. Stimulation with nanomolar concentrations of FMLP resulted in the translocation of Mac-1 from secretory vesicles to the plasma membrane, and only minimal translocation from specific granules and gelatinase granules. Stimulation with PMA or Ionomycin resulted in full translocation of Mac-1 from secretory vesicles and gelatinase granules to the plasma membrane, and partial translocation of Mac-1 from specific granules. These findings were corroborated by flow cytometry, which demonstrated a 6-10-fold increase in the surface membrane content of Mac-1 in response to stimulation with FMLP, granulocyte-macrophage colony stimulating factor, IL-8, leukotriene B4, platelet-activating factor, TNF-alpha, and zymosan-activated serum, and a 25-fold increase in response to Ionomycin. Thus, secretory vesicles constitute the most important reservoir of Mac-1 that is incorporated into the plasma membrane during stimulation with inflammatory mediators.

Cell Compartmentation↗
PubMed1996

Human blood monocytes, but not alveolar macrophages, reveal increased CD11b/CD18 expression and adhesion properties upon receptor-dependent activation.

The beta 2 integrin receptor CD11b/CD18 mediates adhesion to the endothelial lining as well as to extracellular matrix components. The present study was undertaken to investigate peripheral human blood monocytes (BMs) and alveolar macrophages (AMs) with respect to quantitative levels of CD11b/CD18 and adhesion properties in relation to the state of activation. BMs and AMs were recruited from healthy subjects. Quantitative analysis of the surface expression of CD11b/CD18 by flow cytometric technique and adhesion properties to albumin-coated surfaces were performed both on resting and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated cells. Receptor independent stimuli (phorbol-12-myristate-13-acetate (PMA) and ionomycin) were used in additional experiments. Intracellular stored CD11b/CD18 was evaluated by flow cytometry and immunofluorescence microscopy. The surface expression of CD11b/CD18 on resting BMs increased fivefold (p < 0.01) upon fMLP activation. On resting AMs, the surface expression of CD11b/CD18 was significantly higher (p < 0.01) compared to resting BMs but did not increase further upon activation with fMLP, PMA or ionomycin. In contrast to BMs, no evidence for an additional intracellular pool of CD11b/CD18 was found in AMs. The adherence of resting BMs did not significantly differ from the adherence of resting AMs. After fMLP activation, the adherence of BMs, but not AMs, increased significantly (p < 0.05). Our results indicate that in vivo differentiation of human blood monocytes into alveolar macrophages implies reduced responsiveness to fMLP in terms of CD11b/CD18 upregulation and adhesion properties, and that the lack of upregulation of CD11b/CD18 on alveolar macrophages presumably depends on the absence of an additional intracellular pool.

Adolescent↗
PubMed2001

Endothelial [Ca2+]i is an integrating signal for the vascular tone in rat aortae.

BACKGROUND: Although various endothelium-dependent relaxing factors (endothelial autacoids) are released upon the elevation of endothelial cytosolic free Ca2+ concentration (EC [Ca2+]i), the quantitative relationship between EC [Ca2+]i and vascular tone remains to be established. Moreover, whether the basal release of endothelial autacoids is modulated by basal EC [Ca2+]i is still unclear. We assessed these issues by using a novel method that allows simultaneous recording of EC [Ca2+]i and vascular displacement in dissected rat aortic segments. RESULTS: Receptor-dependent (acetylcholine) or independent (ionomycin) agonists caused immediate EC [Ca2+]i elevation followed by vasorelaxation in preparations pre-contracted with phenylephrine. Low doses of agonists induced small EC [Ca2+]i elevations (about 100 nmol/L) and concomitant half-maximal vasorelaxation. At high doses, agonists elevated EC [Ca2+]i to micromol/L range with little additional vasodilatation. When EC [Ca2+]i was plotted against the vasorelaxation, the curves were almost identical for both acetylcholine and ionomycin treatments, in the presence or absence of various endothelial autacoid inhibitors. Calcium-free solution reduced basal EC [Ca2+]i and induced a drastic vasoconstriction. Endothelial autacoid inhibitors reduced EC [Ca2+]i changes and abolished both agonist-induced vasodilatation and calcium-free solution-induced vessel contraction. When the EC [Ca2+]i was completely chelated by 40 micromol/L BAPTA, the acetylcholine-evoked vasorelaxation could be abolished as well. However, when the EC [Ca2+]i was partially chelated by 20 micromol/L BAPTA, the acetylcholine-evoked vasorelaxation was almost unaffected. CONCLUSIONS: These results indicate that vascular tone is modulated by subtle changes of EC [Ca2+]i level, which seems to serve as an integrating signal in both basal and stimulated states.

Animals↗