Biomedical subjects

J L Bueb

Publications and source records attributed to J L Bueb.

At least 19 recordsLinked to original sources

PubMed2001

Role of G(i)-proteins in norepinephrine-mediated vasoconstriction in rat tail artery smooth muscle.

We showed, in rat de-endothelialised tail artery, that pertussis toxin (PTX) (1 microg/mL, 2 hr) attenuated norepinephrine (NE)-induced vasoconstriction without modifying intracellular calcium concentration [Ca2+](i) mobilisation. We suggested the existence of two NE-induced intracellular pathways: a first, which would be insensitive to PTX and lead to [Ca2+](i) mobilisation, and a second sensitive to PTX and involved in the [Ca2+](i) sensitivity of NE-induced contraction. The aim of this study was to demonstrate the existence of the second intracellular pathway. PTX-sensitive G(i/o)-proteins in rat tail artery SMC membrane were identified by immunoblot and ADP-ribosylation. [(32)P]ADP-ribosylation of alpha(i/o)-subunits was demonstrated in situ by perfusing rat de-endothelialised tail artery segments with PTX (1 microg/mL, 2 hr), which suggested that G(i/o)-protein inactivation was involved in the reduction by PTX of the [Ca2+](i) sensitivity of NE-induced contraction. Coupling between G(i/o)-proteins and NE receptors was confirmed by the NE-induced increase in G(i/o)-specific GTPase activity (24.1 +/- 1.9 vs 8.8 +/- 0.4 pmol P(i)/mg protein at 5 min; P < 0.05 vs basal). [(3)H]Prazosin-binding data showed the presence of a heterogeneous alpha(1)-AR population in rat tail artery smooth muscle cells. We demonstrated the in vitro coupling between alpha(1A)-AR subtype and alpha(i)-subunits. In conclusion, we identified, in rat de-endothelialised tail artery, a PTX-sensitive G(i/o)-protein-modulated pathway that is coupled to NE receptors via alpha(1A)-AR. We suggest that NE stimulates two alpha(1)-AR-mediated intracellular pathways: a first, which is mediated by a G(q)-protein and leads to [Ca2+](i) mobilisation and contraction, and a second, which is mediated by a G(i)-protein and is involved in the amplification of the [Ca2+](i) sensitivity of NE-induced tension.

Analysis of Variance↗

PubMed2001

Receptor-independent effects of natural cannabinoids in rat peritoneal mast cells in vitro.

Cannabinoids can activate CB(1) and CB(2) receptors. Since a CB(2) mRNA has been described in rat peritoneal mast cells (RPMC), we investigated a series of cannabinoids and derivatives for their capacity to stimulate RPMC. Effects of natural cannabinoids Delta(9)-tetrahydrocannabinol (Delta(9)-THC), Delta(8)-THC, endocannabinoids (anandamide, palmitoylethanolamide) and related compounds (N-decanoyl-, N-lauroyl-, N-myristoyl-, N-stearoyl- and N-oleoyl-ethanolamines; N-palmitoyl derivatives (-butylamine, -cyclohexylamine, -isopropylamine); and N-palmitoyl, O-palmitoylethanolamine), and synthetic cannabinoids including WIN 55,212-2, SR141716A and SR144528 were assessed for their capacity to induce histamine release or prime RPMC stimulated by compound 48/80. Only Delta(9)-THC and Delta(8)-THC could induce non-lytic, energy- and concentration-dependent histamine releases from RPMC (respective EC(50) values: 23.5+/-1.2; 53.4+/-20.6 microM, and maxima: 71.2+/-5.5; 55.7+/-2.7% of the total RPMC histamine content). These were not blocked by CB(1) (SR141716A) or CB(2) (SR144528) antagonists, but reduced by pertussis toxin (100 ng/ml). Endocannabinoids and analogues did neither induce histamine secretion, nor prime secretion induced by compound 48/80 (0.2 microg/ml). Delta(9)-THC and Delta(8)-THC induced in vitro histamine secretion from RPMC through CB receptor-independent interactions, partly involving G(i/o) protein activation.

Amides↗

PubMed2001

Lack of involvement of pertussis toxin-sensitive G-proteins in norepinephrine-induced vasoconstriction of rat aorta smooth muscle.

Several studies have shown that stimulation of pertussis toxin (PTX)-sensitive G-proteins amplified alpha-adrenoceptor (alpha-AR) agonist-induced vasoconstriction in small muscular and resistance arteries. The aim of this study was to assess the potential involvement of PTX-sensitive G-proteins in norepinephrine (NE)-induced constriction in a large diameter artery, the rat aorta. PTX (1 microg/mL, 2 hr; 3 microg/mL, 4 hr) did not modify concentration-response curves to NE in endothelium-denuded aortic rings. However, several lines of evidence suggested that aortic smooth muscle cells (SMC) had a PTX-sensitive G-protein pathway. [alpha-(32)P]ADP-ribosylation of G(i/o)-proteins by PTX (3 microg/mL, 4 hr) was demonstrated in situ in the intact aorta without endothelium. alpha(i/o) subunits were identified in vitro by both immunoblotting and ADP-ribosylation experiments in rat aorta SMC membranes. The measurement of G(i/o)-specific GTPase activity evidenced an effective coupling between NE receptors and G(i/o)-proteins, as NE induced an increase in basal G(i/o)-specific GTPase activity (20.7 +/- 2.8 vs 7.2 +/- 2.2 pmol P(i)/mg protein at 5 min; P < 0.05 vs basal). Co-immunoprecipitation revealed the in vitro coupling between alpha(1D)-ARs and G(i)-protein in rat aorta SMC membranes. In conclusion, we identified a PTX-sensitive G(i/o)-protein pathway in rat endothelium-denuded aorta. We showed an effective coupling between NE receptors and G(i)-proteins via alpha(1D)-ARs. Since PTX has no effect on NE-induced vasoconstriction, the PTX-sensitive G(i)-protein pathway does not play a predominant role in NE-induced responses in rat aorta SMC in contrast to small diameter muscular and resistance arteries.

Adrenergic alpha-Agonists↗

PubMed2000

Pertussis toxin-sensitive G(i)-proteins and intracellular calcium sensitivity of vasoconstriction in the intact rat tail artery.

1. We studied the involvement of pertussis toxin (PTX)-sensitive G-proteins in the sensitivity of arterial constriction to intracellular calcium ([Ca(2+)](i)) mobilization. 2. Vasoconstriction was measured in vitro in perfused, de-endothelialized rat tail arteries loaded with the calcium-sensitive dye, fura-2 and treated or not with PTX (30 - 1000 ng ml(-1)). Arteries were stimulated with noradrenaline (NA, 0.1 - 100 microM) or KCl (15 - 120 mM). 3. KCl elicited a smaller vasoconstrictor response (E(max)=94+/-8 mmHg) than NA (E(max)=198+/-9 mmHg) although [Ca(2+)](i) mobilization was similar (E(max)=123+/-8 and 135+/-7 nM for KCl and NA, respectively). PTX (1000 ng ml(-1)) had no effect on [Ca(2+)](i) mobilization but lowered NA- (but not KCl-) induced vasoconstriction (E(max)=118+/-7 mmHg). 4. G(i/o)-proteins were revealed by immunoblotting with anti-G(i alpha) and anti-G(o alpha) antibodies in membranes prepared from de-endothelialized tail arteries. [alpha(32)P]-ADP-ribosylation of G-proteins by PTX (1000 ng ml(-1)) was demonstrated in the intact rat tail artery (pixels in the absence of PTX: 3150, presence: 25053). 5. In conclusion, we suggest that smooth muscle cells possess a PTX-sensitive G(i)-protein-mediated intracellular pathway which amplifies [Ca(2+)](i) sensitivity of contraction in the presence of agonists such as NA.

Animals↗

PubMed1999

Release of O2- by human umbilical cord blood-derived eosinophils: role of intra- and extracellular calcium.

The aim of our study was to investigate the physiologic mechanisms involved in eosinophil activation as an essential prerequisite to disrupting the biochemical cascade that triggers inflammation, thereby attenuating the effect of this activation or, ideally, preventing it from occurring. We have, therefore, examined the nature of the fMLP- and PAF-induced [Ca2+]i rise and the relationship between the [Ca2+]i rise and O2- production in human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5. These cells responded to fMLP or PAF (1 microM each) with an increase in [Ca2+]i (217.3 +/- 22.1 and 197.8 +/- 22.1 nM respectively) which was associated with production of O2- (40.2 +/- 8.2 and 35.2 +/- 7.6 pmol/min/10(6) cells respectively). The role of Ca2+ in the induced respiratory burst was studied by changing the availability of Ca2+ in the intra- and extracellular compartments. Removal or chelation of extracellular Ca2+ induced a reduction of both the fMLP and PAF-induced [Ca2+]i rise and O2- production. Chelation of intracellular Ca2+ induced a concentration-dependent inhibition of fMLP- and PAF-induced [Ca2+]i rise and caused a decrease in O2- production. SK&F 96365 had a stimulatory effect on PAF-induced [Ca2+]i rise and on fMLP-induced O2- production, this phenomenon was not observed with extracellular Ca2+ removal or chelation. Furthermore, Ni2+ exhibited an inhibition of both fMLP and PAF-induced [Ca2+]i rise and O2- production. Finally, both fMLP and PAF induced an increase in divalent cation influx that was further augmented by thapsigargin. Our results indicate that fMLP and PAF dependent O2- production in human eosinophils require intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation.

Calcium↗

PubMed1998

Effect of SK&F 96365 on extracellular Ca2+ -dependent O2- production in neutrophil-like HL-60 cells.

Store-operated Ca2+ entry is referred to a capacitative current activated by Ca2+ -stores depletion in various non-excitable cells. Neutrophil-like HL-60 cells responded to N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLP) by an early O2- production preceded by a [Ca2+]i rise. Cell stimulation in the absence of extracellular Ca2+ resulted in a major reduction of [Ca2+]i rise and O2- production. A purported inhibitor of store-operated Ca2+ entry, SK&F 96365 (1-(beta-(3-(4-methoxy-phenyl)propoxyl)-4-methoxy-phenetyl)- 1H-imidazole hydrochloride), inhibited extracellular Ca2+ -dependent [Ca2+]i rise by 30% but did not alter O2- production. In conclusion, SK&F 96365 did not modify extracellular Ca2+ -dependent O2- production, despite a significant but limited reduction in fMLP-activated membrane Ca2+ fluxes which can be ascribed to store-operated Ca2+ entry. Furthermore, Ca2+ influx is necessary for a full induction and maintenance of the biological response.

Calcium↗

PubMed1998

The M2 muscarinic receptor antagonist methoctramine activates mast cells via pertussis toxin-sensitive G proteins.

Methoctramine, a selective M2 muscarinic cholinergic receptor antagonist, has been reported to activate phosphoinositide breakdown at high concentrations. Its polyamine structure suggests a putative activation of guanine nucleotide-binding proteins (G proteins). Incubation of methoctramine with rat peritoneal mast cells resulted in a dose-dependent noncytotoxic histamine release, with an EC50 of 20 microM and a maximum effect at 1 mM. Atropine, pirenzepine and HHSiD neither inhibited methoctramine-induced histamine release nor stimulated histamine release. Histamine release and inositol phosphates generation induced by methoctramine were both inhibited by pertussis toxin pretreatment. Benzalkonium chloride, a selective inhibitor of histamine secretion induced by basic secretagogues, inhibited the secretory response to methoctramine. [p-Glu5, D-Trp7,9,l0]-SPs5-11 (GPAnt-2), a well-characterized antagonist of G proteins, blocked the methoctramine-induced histamine release when the antagonist was allowed to reach its intracellular target by streptolysin O-permeabilization. The response to methoctramine was prevented by the hydrolysis of sialic acid residues of the cell surface by neuraminidase. The response of mast cells was restored by permeabilization of the plasma membrane. These results demonstrate that methoctramine, following its entry into the cell and the involvement of pertussis toxin-sensitive G proteins, activates phosphoinositide hydrolysis leading to mast cell exocytosis.

Animals↗

PubMed1997

Human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5 respond to fMLP with [Ca2+]i variation and O2- production.

In the presence of interleukin-3 and interleukin-5, eosinophil precursors from human umbilical cord blood mononuclear cells were regularly differentiated into mature eosinophil-like cells expressing normal morphology and cyanide-resistant peroxidase. O2- production and [Ca2+]i rise were measured in these in vitro differentiated eosinophils after fMLP stimulation; with dihydrorhodamine-123 and fura-2, respectively. Umbilical cord blood-derived eosinophils responded to fMLP (0.01 nM to 3 microM) with a concentration-dependent production of O2- (EC50 = 63.1 +/- 17.2 nM; Emax = 71.0 +/- 6.2 pmol/min/10(6) cells). O2- production was correlated with an fMLP concentration-dependent increase in [Ca2+]i (EC50 = 32.5 +/- 14.9 nM; Emax = 200.0 +/- 23.9 nM). These results indicate that human umbilical cord blood-derived eosinophils demonstrate functional characteristics similar to adult human peripheral blood eosinophils after activation by fMLP. Therefore, the large numbers of eosinophils (2-3 x 10(6)/ml cord blood) which can be obtained by culture of human cord blood mononuclear cells may serve as a useful model for future studies which will provide insight into the pathogenesis of diseases associated with eosinophils.

Adult↗

PubMed1996

Endothelin-1 does not modulate O2.release and [Ca(2+)]i variations in resting or differentiated HL-60 cells.

Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O2.) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01-100 nM) was not able to modulate O2. generation stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, EC50 = 4.24 +/- 1.63 nM in the absence and 3.16 +/- 1.95 nM in the presence of ET-1). Neither did ET-1 (0.01-100 nM) promote the mobilization of intracellular calcium ions or modulate fMLP-induced [Ca(2+)]i increase in this model of human neutrophils. Phosphoramidon, a neutral endopeptidase inhibitor, was not able to reveal any biological (O2.) or biochemical ([Ca(2+)]i response to ET-1 in the absence or in the presence of fMLP in these cells. These results indicate that DMSO-differentiated neutrophil-like HL-60 cells are not sensitive to ET-1 in terms of O2. generation or [Ca(2+)]i variations.

Calcium↗

PubMed1995

A double-labelling fluorescent assay for concomitant measurements of [Ca2+]i and O2. production in human macrophages.

To measure intracellular free Ca2+ concentration ([Ca2+]i) and superoxide (O2) production in human alveolar macrophages, we used the fluorescent Ca2+ indicator fura-2 and the O2-sensitive dye dihydrorhodamine-123, which becomes fluorescent in its oxidized form, rhodamine-123. We describe a new double-dye technique whereby the kinetics of both [Ca2+]i levels and O2. production can be monitored simultaneously. This technique was developed in the dimethylsulfoxide-differentiated monocytic-like U-937 cell line (not equal to U-937), validated by comparison with single dye measurements and applied to human alveolar macrophages. The chemotactic peptide N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine induced in both cell types a similar transient elevation in [Ca2+]i, followed within seconds by a sustained increase in O2 production, which was however 4-fold weaker in not equal to U-937 cells. These results indicate that O2 production is an early event following the stimulation of human alveolar macrophages. This new double-dye technique may be relevant to other O2 ion-producing cells and could help to define more precisely the kinetics of the events leading to this biological response.

Calcium↗

PubMed1992

Natural polyamines stimulate G-proteins.

The natural polyamines spermine and spermidine, the biosynthetic precursor putrescine and their analogues cadaverine and tyramine stimulate the GTPase activity of purified GTP-binding proteins (Go/Gi) from calf brain reconstituted into phospholipid vesicles. The order of potency was spermine greater than spermidine greater than putrescine = cadaverine greater than tyramine. The physiological relevance of this observation was assessed, showing the same order of potency of polyamines in the stimulation of peritoneal and tracheal rat mast cells. The activation of rat mast cells by polyamines was inhibited by benzalkonium chloride or by a 2 h pretreatment of the cells with pertussis toxin. The increase in inositol phosphates evoked by polyamines was also inhibited by pertussis toxin. Therefore we propose that intracellular polyamines might control the basal level of second messengers and modulate extracellular signals transduced through G-protein-coupled receptors.

Animals↗

PubMed1991

Evidence for the interaction of mast cell-degranulating peptide with pertussis toxin-sensitive G proteins in mast cells.

K(+)-channel blocker properties have been reported for mast cell-degranulating peptide (MCD) in the central nervous system, but its action mechanism in mast cells remains unknown. We studied the effect of MCD on the membrane potential of rat peritoneal mast cells using the fluorescent probe bis-oxonol. Unexpectedly, MCD induced a decrease in bis-oxonol fluorescence, in a rapid and then a slower phase, suggesting hyperpolarization of mast cells. Other K(+)-channel blockers, tetraethylammonium and 4-aminopyridine, did not significantly modify the bis-oxonol fluorescence and did not alter the effect of MCD. The late phase of bis-oxonol fluorescence decrease was inhibited by ouabain and by potassium deprivation, whereas histamine release was not affected. The first phase of putative hyperpolarization induced by MCD coincided with histamine release and with the generation of inositol polyphosphates. Prior treatment of the cells with pertussis toxin inhibited these effects of MCD. MCD stimulated the GTPase activity of purified G proteins (G0/Gi) in a concentration-dependent manner. These results indicate that the effect of MCD on mast cells is unrelated to K+ channels but that it is relevant to the activation of pertussis toxin-sensitive G proteins leading to the activation of phospholipase C. A direct interaction of MCD with G proteins is proposed, which, unlike mastoparan, does not require positive cooperativity.

Amino Acid Sequence↗

PubMed1991

G-proteins as targets for non-immunological histamine releasers.

The molecular mechanism of action of several non-immunological histamine releasers has been investigated using pertussis toxin which interfers, via ADP-ribosylation, with some G-proteins. Pertussis toxin (100 ng/ml) inhibited histamine release induced by compound 48/80, substance P, mastoparan, peptide 401, bradykinin and spermine showing that a G-protein sensitive to pertussis toxin was involved in the non-immunological histamine release. All these compounds directly activate purified G-proteins. The sensitivity to pertussis toxin of this direct stimulatory effect was demonstrated for compound 48/80, mastoparan and substance P. Altogether these results suggest that a direct activation of G-protein might be the molecular mechanism of action of histamine secretagogues acting through a pertussis toxin sensitive G-protein and in this way mimic agonist-ligand receptor interaction.

Animals↗

PubMed1991

Molecular basis for cellular effects of naturally occurring polyamines.

The naturally occurring polyamines, putrescine, spermidine and spermine, and the analogue cadaverine, induce a dose-dependent histamine release from rat peritoneal mast cells. Spermine was the most active among these polycationic metabolites, followed by spermidine and putrescine. The histamine release was inhibited by a 2 h pretreatment of the cells with pertussis toxin (100 ng/ml), demonstrating the involvement of a pertussis toxin-sensitive GTP-binding regulatory protein during the exocytotic process. Experiments performed with purified Go/Gi proteins reconstituted into phospholipid vesicles showed a direct stimulation of GTPase activity by the polyamines. This direct stimulation of G proteins and the consequent activation of the coupled effectors may represent a new mechanism of action for natural polyamines controlling receptor-dependent processes.

Animals↗

PubMed1990

A pertussis toxin-sensitive G protein is required to induce histamine release from rat peritoneal mast cells by bradykinin.

Bradykinin, kallidin (Lys-bradykinin) and [Thi 5,8, D-Phe7]-bradykinin, a functional B2 antagonist, induce histamine release from rat peritoneal mast cells. The histamine release is dependent upon added calcium when mast cells are placed in calcium-free medium 30 min before being triggered with the kinins. Histamine release was dose-dependently inhibited by pertussis toxin (1-100 ng/ml) and by benzalkonium chloride (0.1-3 micrograms/ml). The efficiency of ionophore A23187 on histamine release was affected neither by pertussis toxin nor by benzalkonium chloride. The parallel response of rat peritoneal mast cells to kinins and to substance P suggest that these peptides have the same mechanisms of action i.e. activation of a pertussis toxin-sensitive G protein and of phospholipase C defining a peptidergic triggering pathway of mast cells.

Animals↗

PubMed1990

G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides.

The neuropeptide substance P, the venom peptide mastoparan and the synthetic polyamine compound 48/80 activate rat peritoneal mast cells, leading to rapid histamine release by exocytosis. Although these effects are inhibited by pertussis toxin and involve a transient increase in IP3, no selective membrane receptors have been identified. However, it has recently been shown that these compounds activate G proteins in vitro. Here Yves Landry and colleagues discuss the proposal that direct activation of G protein is the physiological mechanism of action of substance P on rat peritoneal mast cells, this mechanism being mimicked by mastoparan and 48/80, and possibly by other cationic amphiphilic peptides such as kinins. These compounds might be of help in defining the interaction between membrane receptors and G proteins.

Amino Acid Sequence↗

PubMed1990

[Neuropeptides and inflammation: presumed mechanisms in neurogenic inflammation].

Neuropeptides, among which substance P, VIP (Vasoactive intestinal peptide), somatostatin, neurotensin, dynorphin and enkephalins, are able to modulate inflammatory processes. Increasing interest is now devoted to these peptides in different inflammatory diseases, concerning skin, lung and joins. The effect of substance P can be dependent on its C-terminal moiety implicating by this way an interaction with specific neurokinin receptors or can be dependent on its N-terminal moiety which does not involve a specific membrane receptor. Such diversity of the action mechanisms of peptides should influence the evolution of the anti-inflammatory therapeutic.

Arthritis↗

PubMed1990

Activation of Gi-like proteins, a receptor-independent effect of kinins in mast cells.

The peptide hormones bradykinin and kallidin (Lys-bradykinin), as well as their analogues [des-Arg9]-bradykinin, a selective B1 agonist, [des-Arg9,Leu8]-bradykinin, a selective B1 antagonist, and [Thi5,8,D-Phe7]-bradykinin and D-Arg0-[Hyp3,D-Phe7]-bradykinin, two selective B2 antagonists, induced rapid histamine release from purified rat peritoneal mast cells. In contrast, the N-terminal fragment bradykinin-(1-5) was inactive. These peptides also activate the GTPase activity of GTP-binding proteins (G proteins) (Go/Gi) purified from calf brain, with an order of potency identical to that observed on mast cells, [Thi5,8,D-Phe7]-bradykinin much greater than kallidin greater than bradykinin greater than D-Arg0-[Hyp3,D-Phe7]-bradykinin greater than [des-Arg9]-bradykinin greater than [des-Arg9,Leu8]-bradykinin greater than bradykinin-(1-5). This correlation suggested that G proteins are the targets of kinins in mast cells. Accordingly, the concomitant increase in inositol trisphosphates and release of histamine elicited by kinins were inhibited by pertussis toxin pretreatment of mast cells. The inhibitory effect of benzalkonium chloride showed that the G proteins involved belong to the Gi type. GTPase activity was measured in the supernatant of homogenized mast cells but not in the membranous fraction. This activity was stimulated by kinins and by the venom peptide mastoparan. The potency of peptides was similar to that observed with purified bovine G proteins. Sodium dodecyl sulfate-gel electrophoresis of mast cell supernatant revealed pertussis toxin-induced ADP-ribosylation of two proteins, in the Mr 41,000 and 40,000 range, i.e., similar to purified alpha-subunits of Gi1 and Gi2 or Gi3 subtypes. The data support the proposal that bradykinin and analogues act like mastoparan, substance P, and compound 48/80, interacting first with sialic acid residues of the cell surface and then with Gi-like proteins, inducing phospholipase C activation and intracellular calcium mobilization.

Adenosine Diphosphate Ribose↗