[Receptor independent activation of G proteins].
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Amino Acid Sequence↗
Biomedical subjects
R Seifert
Publications and source records attributed to R Seifert.
Cationic-amphiphilic arpromidine-derived guanidines and a histamine trifluoromethyl-toluidide derivative may activate pertussis toxin-sensitive G-proteins by a receptor-independent mechanism.
Formyl peptides activate superoxide anion (O2-) formation in human neutrophils and in HL-60 cells via pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins), and histamine (HA) mediates inhibition of O2- formation via H2-receptors. We have studied the effects of lipophilic arpromidine-derived guanidines, which are potent, full H2-receptor agonists in the guinea pig atrium, on O2- formation and on activation of G-proteins in HL-60 membranes and on purified G-proteins. We have also studied the effects of a HA trifluoromethyl-toluidide derivative (HTMT), a cationic-amphiphilic HA derivative which activates O2- formation in HL-60 cells through a mechanism which is independent of known HA receptor subtypes, on G-protein activation. Guanidines, at concentrations, up to 30 mumol/l inhibited and, at concentrations above 30 mumol/l, enhanced formyl peptide-induce O2- formation in neutrophils. In HL-60 cells, guanidines per se activated O2- formation. The stimulatory effects of guanidines on O2- formation were not inhibited by H1- or H2-receptor antagonists. In HL-60 membranes, guanidines and HTMT, activated high-affinity GTPase in a PTX-sensitive manner. These substances also increased GTP hydrolysis effected by transducin and Gi/G(o)-proteins. Our data suggest that lipophilic guanidines and HTMT may act as receptor-independent activators of PTX-sensitive G-proteins, resulting in stimulation of O2- formation.
Biotransformation↗
Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels.
Sensory transduction in vertebrate photoreceptors and olfactory sensory neurons is mediated by cyclic nucleotide-gated (CNG) channels that conduct mono- and divalent cations. Ca2+ entering the cell through CNG channels intimately controls signaling pathways by regulating several key enzymes. Cloned CNG channels from photoreceptors and olfactory sensory neurons profoundly differ in their relative Ca2+ permeability, their blockage by external divalent cations, and the fraction of current carried by Ca2+. In particular, CNG channels from cone photoreceptors conduct significantly more Ca2+ than those from rod photoreceptors. Furthermore, the current through the olfactory CNG channel is entirely carried by Ca2+ at approximately 3 mM extracellular Ca2+. These results suggest that a major function of CNG channels is to provide a pathway for Ca2+ entry.
Animals↗
A 240 kDa protein represents the complete beta subunit of the cyclic nucleotide-gated channel from rod photoreceptor.
The cyclic nucleotide-gated channel from rod photoreceptors is composed of two distinct subunits (alpha and beta). The properties of the alpha subunit, which can form functional channels by itself, are modified by coexpression with a homologous polypeptide, designated the beta subunit. However, the alpha subunit from rod photoreceptor membranes copurifies with a 240 kDa protein that is significantly larger than this putative beta subunit. We now demonstrate by peptide sequencing and by cloning and functional expression of cDNA that the 240 kDa protein represents the complete beta subunit with an unusual bipartite structure. The N-terminal part is essentially identical to a glutamic acid-rich protein (GARP), whereas the C-terminal part is highly homologous to the previously cloned human "beta subunit." Expression of the complete beta subunit in HEK 293 cells results in a polypeptide with the same apparent molecular weight as the 240 kDa protein of the native rod channel. Coexpression of the alpha subunit with the full-length beta subunit yields hetero-oligomeric channels with properties characteristic of the native channel.
Amino Acid Sequence↗
[Quantitative EEG, basic disorders and smoking in etiopathogenetically different groups of paranoid-hallucinatory psychoses--an exploratory study].
We performed an exploratory study of quantitative EEG in aetiopathogenetically different paranoid-hallucinatory psychoses divided into the following groups: a) patients with familial psychoses (n = 12), b) patients with neuropsychological deficits (n = 16), c) patients with alcohol and drug abuse (n = 22) and d) patients with so-called sporadic psychoses (n = 12). We found a significant reduction of relative alpha power in the group with neuropsychological deficits. In the group with familial psychosis there was a significant reduction of absolute delta power and a significant increase of relative beta power and dominant beta frequency, especially for the frontal leads. Patients with drug abuse showed a reduction of absolute beta power and an increase of absolute and relative theta power. The group with sporadic psychosis showed a significant slowing of the dominant beta frequency and a significant increase of the absolute power of fast alpha rhythms. The group with sporadic psychoses showed lowered scores for the paranoid-hallucinatory basic symptom factor. The group with neuropsychological deficits showed the most visceral-somatoform basic symptoms, the highest nicotine consumption, increased dyskinesias and more perinatal complications. This group also showed the highest level of neuroleptic and antiparkinson medication. All in all, the group with neuropsychological deficits showed a complex interaction of somatic-exogenic and medical-iatrogenic factors. Furthermore, there was a significant positive correlation between paranoid-hallucinatory basic symptoms and nicotine abuse and high frequency beta waves.
Adult↗
Direct and indirect receptor-independent G-protein activation by cationic-amphiphilic substances. Studies with mast cells, HL-60 human leukemic cells and purified G-proteins.
Studies from several laboratories have revealed that structurally diverse substances including the wasp venom, mastoparan (MP), activate purified regulatory heterotrimeric guanine nucleotide-binding proteins (G-proteins) in a receptor-independent manner, presumably by mimicking the effects of heptahelical receptors. Mast cells and differentiated HL-60 human leukemic cells are useful model systems for the analysis of receptor-independent G-protein activation. We compared the effects of 2-phenylhistamines which are cationic-amphiphilic, too, and of MP on G-protein activation in dibutyryl cAMP-differentiated HL-60 cells and in the rat basophilic leukemia cell line, RBL 2H3. In HL-60 cells, 2-phenylhistamines show stimulatory effects which resemble those of formyl peptide receptor agonists but which cannot be attributed to agonism at classical receptors. 2-phenylhistamines do not, however, activate RBL 2H3 cells and various other myeloid cell types, pointing to cell type-specificity of receptor-independent G-protein activation. In HL-60 cells, MP shows effects on G-protein activation which differ substantially from those of formyl peptides. In RBL 2H3 membranes, MP shows similar effects on G-protein activation as in HL-60 membranes. We develop a model according to which receptor-independent G-protein activation can be subdivided into direct and indirect receptor-independent G-protein activation. In case of the former mechanism, substances like 2-phenylhistamines interact with G-protein alpha-subunits and in case of the latter mechanism, substances like MP interact with nucleoside diphosphate kinase which catalyzes the formation of GTP. This newly formed GTP is then transferred to, and cleaved by, G-protein alpha-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The class III antiarrhythmic drug amiodarone directly activates pertussis toxin-sensitive G proteins.
The class III antiarrhythmic drugs amiodarone and bretylium tosylate are cationic/amphiphilic, and various substances with these physico-chemical properties are known to directly activate heterotrimeric regulatory G proteins. We asked the question of whether class III antiarrhythmic drugs are also direct G protein activators, using HL-60 leukemic cells and purified bovine brain G proteins as model systems. In HL-60 cell membranes, aminodarone increased high affinity GTP hydrolysis with an EC50 of 7.5 microM. The stimulatory effect of amiodarone on GTP hydrolysis was inhibited by pertussis toxin. Amiodarone stimulated binding of guanosine-5'-O-(3-thio)triphosphate to, and incorporation of GTP azidoanilide into, Gi protein alpha subunits in HL-60 membranes. The drug increased the cytosolic Ca2+ concentration in HL-60 cells in the presence but not in the absence of extracellular Ca2+. Amiodarone-induced increases in the cytosolic Ca2+ concentration were reduced by pertussis toxin and by a blocker of non-selective cation channels, SK&F 96365. Amiodarone activated the GTPase of reconstituted Gi/G(o) proteins and G12 with EC50 values of 20 microM and 50 microM, respectively. Bretylium tosylate did not increase GTP hydrolysis in HL-60 membranes or with Gi/G(o) proteins. Our data suggest that amiodarone but not bretylium tosylate is a direct activator of Gi and G(o) proteins and that amiodarone activates nonselective cation channels in HL-60 cells via Gi proteins and independently of Ca2+ mobilization from intracellular stores. Future studies will have to test the hypothesis that direct G protein activation by amiodarone contributes to its toxic and/or therapeutic effects.
Amiodarone↗
Differential activation of dibutyryl cAMP-differentiated HL-60 human leukemia cells by chemoattractants.
Dibutyryl cAMP-differentiated HL-60 human leukemia cells possess receptors for the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), C5a and leukotriene B4 (LTB4). We compared the effects of these chemoattractants in HL-60 membranes and in intact HL-60 cells. fMLP, C5a and LTB4 stimulated GTP hydrolysis and guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) binding in HL-60 membranes with similar effectiveness and in a pertussis toxin (PTX)-sensitive manner. They also stimulated photolabeling of the alpha-subunits of the guanine nucleotide-binding proteins (G-proteins), Gi2 and Gi3 with similar effectiveness. Chloride salts of monovalent cations differentially enhanced and inhibited chemoattractant-induced GTP hydrolyses. C5a was less effective than fMLP in enhancing cholera toxin-catalysed ADP-ribosylation of Gi alpha 2 and Gi alpha 3, and LTB4 was ineffective. fMLP was more effective than C5a and LTB4 in stimulating Ca2+ influx in HL-60 cells. C5a- and LTB4-induced rises in cytosolic Ca2+ concentration ([Ca2+]i) were PTX-sensitive, whereas the effect of fMLP was partially PTX-insensitive. LTB4-induced rises in [Ca2+]i were more sensitive towards homologous desensitization than those induced by C5a, and the effect of fMLP was resistant in this regard. C5a was considerably less effective than fMLP in activating superoxide anion formation and azurophilic granule release, and LTB4 was ineffective. Our data suggest that fMLP, C5a and LTB4 effectively activate the G-proteins, Gi2 and Gi3, in HL-60 cells and that fMLP may additionally activate PTX-insensitive G-proteins. fMLP, C5a and LTB4 are full, partial and incomplete secretagogues, respectively, and these differences may be due to differences in homologous receptor desensitization and qualitative Gi-protein activation.
Bucladesine↗
Primary structure and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes and antennae.
Cyclic nucleotide-gated (CNG) ion channels serve as downstream targets of signalling pathways in vertebrate photoreceptors and olfactory sensory neurons. Whether CNG channels subserve similar functions in invertebrate photoreception and olfaction is unknown. We have cloned genomic DNA and cDNA encoding a cGMP-gated channel from Drosophila. The gene contains at least seven exons. Heterologous expression of cloned cDNA in both Xenopus oocytes and HEK 293 cells gives rise to functional ion channels. The Drosophila CNG channel is approximately 50-fold more sensitive to cGMP than to cAMP. The voltage dependence of blockage by divalent cations is different compared with the CNG channel of rod photoreceptors, and the Ca2+ permeability is much larger. The channel mRNA is expressed in antennae and the visual system of Drosophila. It is proposed that CNG channels are involved in transduction cascades of both invertebrate photoreceptors and olfactory sensillae.
Amino Acid Sequence↗
Evidence that inhibition of phorbol ester-induced superoxide anion formation by cyclosporin A in phagocytes is not mediated by direct inhibition of protein kinase C.
Cyclosporin A (CsA) has been reported to inhibit phorbol myristate acetate (PMA)-induced superoxide anion (O2-) formation in human neutrophils and murine macrophages. We found that CsA inhibited O2- formation in HL-60 cells induced by PMA (30 nM) and phorbol dibutyrate (200 nM) with a half-maximal effect at 1 and 0.75 microM, respectively. One possible target of CsA action is protein kinase C (PKC) [EC 2.7.1.37] since phorbol esters activate this kinase. However, CsA did not inhibit PMA-mediated reduction of histamine-induced rises in cytosolic Ca2+ concentration in, and PMA-induced differentiation of, HL-60 cells and platelet aggregation. CsA did not reduce the activity of various recombinant c-PKC isoenzymes (alpha, beta 1 and gamma), n-PKC isoenzymes (delta and epsilon), an a-PKC isoenzyme (zeta) nor of PKC purified from rat brain in vitro. These data show that CsA inhibits phorbol ester-induced O2- formation in HL-60 cells but not other phorbol ester-mediated events and that inhibition by CsA of O2- formation cannot readily be attributed to direct PKC inhibition. We also show that CsA does not change the activity of nucleoside diphosphate kinase [EC 2.7.4.6] in HL-60 membranes nor the latter's physical properties.
Animals↗
Maitotoxin activates cation channels distinct from the receptor-activated non-selective cation channels of HL-60 cells.
We investigated whether maitotoxin activates non-selective cation channels, as was recently proposed [Soergel, Yasumoto, Daly and Gusovsky (1992) Mol. Pharmacol. 41, 487-493]. Stimulation of dibutyryl cyclic AMP-differentiated HL-60 cells with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 0.1 microM), the Ca(2+)-ATPase inhibitor thapsigargin (0.1 microM) or maitotoxin (25 ng/ml) resulted in an increase in cytoplasmic free calcium concentration ([Ca2+]i). Unlike fMLP and thapsigargin, maitotoxin produced no increase in [Ca2+]i in the absence of extracellular Ca2+. The increase in [Ca2+]i induced by fMLP was blocked by pretreatment with pertussis toxin (100 ng/ml for 24 h) but not that induced by maitotoxin. Similarly, the increase in [Ca2+]i produced by fMLP but not that produced by maitotoxin was inhibited by pretreatment with phorbol myristate acetate (100 ng/ml). Both fMLP- and maitotoxin-induced increases in [Ca2+]i were blocked by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl)-1H-imid azole hydrochloride (SKF 96365) in a concentration-dependent manner. However, the maitotoxin-induced increase in [Ca2+]i was more sensitive to inhibition by SKF 96365 than the fMLP-induced increase. fMLP-induced increases in [Ca2+]i were blocked by cations with Gd3+ being more effective than Cd2+, whereas for maitotoxin Cd2+ was more effective than Gd3+. Both fMLP and thapsigargin stimulated quenching of Fura-2 fluorescence in the presence of extracellular Mn2+, whereas maitotoxin produced no Mn2+ quenching. Taken together these results suggest that maitotoxin does not stimulate the nonselective cation channel activated by fMLP, but instead activates Ca2+ influx by a different mechanism.
Bucladesine↗
Lipophilic beta-adrenoceptor antagonists and local anesthetics are effective direct activators of G-proteins.
We studied the effects of various beta-adrenoceptor (beta AR) antagonists and local anesthetics (LAs), i.e. substances possessing one basic and one lipophilic domain each, on activation of regulatory heterotrimeric guanine nucleotide-binding proteins (G-proteins). In membranes of differentiated HL-60 cells, propranolol activated high-affinity GTP hydrolysis with a half-maximal effect at 0.19 mM and a maximum at 1 mM. There was a close correlation between the log Q values (logarithm of the octanol: water partition coefficient) of beta AR antagonists and the logarithm of their effectiveness at activating GTPase (EC 3.6.1.-) in HL-60 membranes. The lipophilic LA, tetracaine, was also an effective activator of GTPase in HL-60 membranes, whereas more hydrophilic LAs were less stimulatory (bupivacaine and lidocaine) or even inhibitory (procaine). Propranolol and tetracaine also stimulated binding of guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) to HL-60 membranes, but their stimulatory effects on GTP[gamma S] binding were smaller than on GTP hydrolysis. The stimulatory effects of propranolol and tetracaine on GTPase and GTP[gamma S] binding were inhibited by pertussis toxin. Propranolol and tetracaine effectively activated GTP hydrolysis of a reconstituted mixture of bovine brain Gi/Go-proteins, but the concentrations of substances needed for GTPase activation were higher than in HL-60 membranes. Procaine showed stimulatory effects on the GTPase of Gi/Go-proteins. Our data show that beta AR antagonists and LAs activate pertussis toxin-sensitive G-proteins, presumably through interaction with the C-terminus of their alpha-subunits. Apparently, the lipophilic domain of beta AR antagonists and LAs is more important for G-protein activation than the basic domain. We discuss the possibility that activation of nucleoside diphosphate kinase by beta AR antagonists and LAs contributes to their stimulatory effects on GTP hydrolysis in HL-60 membranes.
Adrenergic beta-Antagonists↗
N-protein kinase C isoenzymes may be involved in the regulation of various neutrophil functions.
The role of protein kinase C (PKC) isoenzymes in the regulation of cell functions is largely unknown. We studied the effects of 2-(1H-indol-3-yl)-3- [1-(3-dimethylaminopropyl)-1H-indol-3-yl]-maleinimide (Gö 6850), a selective inhibitor of c- and n-PKC isoenzymes, and 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2 ,3- a]pyrrolo[3, 4-c]-carbazole (Gö 6976), an inhibitor of c-PKC isoenzymes, on various human neutrophil functions. Gö 6850 inhibited 4 beta-phorbol 12-myristate 13-acetate (PMA)-, 1,2-dicaprylyl- glycerol- and chemotactic peptide-induced superoxide anion formation with half-maximal effects at 100 nM, 240 nM and 850 nM, respectively. Gö 6850 reverted PMA-mediated inhibition of chemotactic peptide-induced rises in cytosolic Ca2+ concentration with a half-maximal effect at 480 nM. Gö 6850 (1 microM) inhibited PMA-induced lysozyme release by 55%. Gö 6976 had no effect on the parameters studied. Our data suggest the following: (1) n- Rather than c-PKC isoenzymes are involved in the regulation of various human neutrophil functions. (2) Different n-PKC isoenzymes may mediate activation of NADPH oxidase by various stimuli. (3) Different n-PKC isoenzymes may be involved in the mediation of the effects of PMA on various cell functions.
Carbazoles↗
Some taste substances are direct activators of G-proteins.
Amphiphilic substances may stimulate cellular events through direct activation of G-proteins. The present experiments indicate that several amphiphilic sweeteners and the bitter tastant, quinine, activate transducin and Gi/Go-proteins. Concentrations of taste substances required to activate G-proteins in vitro correlated with those used to elicit taste. These data support the hypothesis that amphiphilic taste substances may elicit taste through direct activation of G-proteins.
Animals↗
Human neutrophils and HL-60 cells do not possess alpha 2-adrenoceptors.
Human neutrophils have been reported to possess both alpha 2- and beta 2-adrenoceptors. While activation of beta 2-adrenoceptors is known to inhibit N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2-) production, the functional role of alpha 2-adrenoceptors is not known. We studied the effects of a range of structurally unrelated alpha 2-adrenoceptor agonists on fMLP-induced O2- production and UTP-induced increases in cytosolic free calcium concentration ([Ca2+]i) in human neutrophils. No effect of alpha 2-adrenoceptor agonists was seen on either fMLP-induced O2- production or UTP-induced increases in [Ca2+]i. alpha 2-Adrenoceptor agonists by themselves had no effect on either O2- production or [Ca2+]i. We then studied a model for neutrophils, differentiated HL-60 cells and human erythroleukaemia (HEL) cells, a cell line known to possess alpha 2-adrenoceptors. While the alpha 2-adrenoceptor agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304) and 5-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]azepin- dihydrochloride increased the [Ca2+]i in HEL cells, they had no effect by themselves on either [Ca2+]i or UTP-induced increases in [Ca2+]i in differentiated HL-60 cells. Activation of high-affinity GTPase by UK 14304 was seen in membranes from HEL cells but not in membranes from differentiated HL-60 cells. Similarly, a selective alpha 2-adrenoceptor antagonist, [3H]2-(2-methoxy-1,4-benzodioxan-2yl)-2 imidazoline, bound specifically and saturably to membranes from HEL cells, but not to membranes from HL-60 promyelocytes or differentiated HL-60 cells. Taken together, these data suggest that neither HL-60 promyelocytes nor differentiated HL-60 cells possess alpha 2-adrenoceptors, and that the lack of functional responses to alpha 2-adrenoceptor agonists in human neutrophils is due to the absence of alpha 2-adrenoceptors.
Adrenergic alpha-Agonists↗
Cross-linking of CD18 in human neutrophils induces an increase of intracellular free Ca2+, exocytosis of azurophilic granules, quantitative up-regulation of CD18, shedding of L-selectin, and actin polymerization.
Polymorphonuclear leukocytes (PMNs) exert most of their physiological functions while adherent to surfaces rather than in suspension. PMN adhesion is largely dependent on the function of the beta 2 integrins, CD11a,b,c/CD18. We mimicked engagement of beta 2 integrins by antibody cross-linking of CD18 on isolated human PMNs using both intact monoclonal antibody and F(ab')2 fragments. Within seconds of CD18 cross-linking, we observed a significant, transient rise of intracellular free Ca2+ concentration by 200-300 nM, which was largely due to Ca2+ mobilization from intracellular stores. The Ca2+ signal was blocked after pretreatment with phorbol myristate acetate, an activator of protein kinase C, but not with herbimycin A, a potent inhibitor of tyrosine kinases. In addition to the rise of intracellular free Ca2+ concentration, CD18 cross-linking induced exocytosis of azurophilic granules (release of 26% of total PMN elastase), which was significantly inhibited by herbimycin A. Moreover, 2.2-fold up-regulation of CD18 antigen and significant down-regulation of surface expression of the granulocyte adhesion molecule L-selectin were induced. Granulocyte F-actin content as measured by nitrobenzoxadiazole-phallacidin increased significantly 1 min after CD18 cross-linking. By contrast, CD18 cross-linking by soluble antibodies did not induce superoxide production, but PMNs bound to immobilized monoclonal antibodies against CD18 released significant amounts of superoxide. Initial signaling through beta 2 integrins does not appear to be mediated by a phospholipase C isoform activated through tyrosine phosphorylation, because the Ca2+ signal was not altered by herbimycin A. However, more complex cellular responses including exocytosis were found to require tyrosine phosphorylation. We show that engagement of beta 2 integrins provides an important stimulatory signal to PMNs inducing degranulation, modulation of L-selectin, and cytoskeletal changes.
Actins↗
Histamine H1-receptors in HL-60 monocytes are coupled to Gi-proteins and pertussis toxin-insensitive G-proteins and mediate activation of Ca2+ influx without concomitant Ca2+ mobilization from intracellular stores.
The results of binding studies suggest the presence of histamine H1-receptors in human monocytes, but it is not known whether these receptors are functionally active. This prompted us to study the effects of histamine (HA) on cytosolic Ca2+ concentration ([Ca2+]i) and superoxide anion (O2-) formation in HL-60 cells differentiated towards monocytes with 1 alpha,25-dihydroxycholecalciferol. In HL-60 monocytes, HA increased [Ca2+]i with a half-maximal effect at 8 microM and a maximum at 30-100 microM. Pertussis toxin (PTX) partially inhibited the stimulatory effects of HA on [Ca2+]i. Betahistine, a weak partial H1-receptor agonist, also increased [Ca2+]i, whereas H2- and H3-receptor agonists were ineffective. H1- but not H2- and H3-receptor antagonists inhibited HA-induced rises in [Ca2+]i. HA-induced rises in [Ca2+]i were desensitized in a homologous manner and were also inhibited by the activator of protein kinase C, 4 beta-phorbol 12-myristate 13-acetate. Various protein kinase C inhibitors did not interfere with homologous desensitization. The stimulatory effects of HA on [Ca2+]i were completely dependent on the presence of extracellular Ca2+ and were inhibited by the blocker of non-selective cation (NSC) channels, 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1 H-imidazole hydrochloride (SK & F 96365). HA was much less effective than the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), to induce rises in [Ca2+]i. Unlike fMLP, HA did not activate O2- formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate Ribose↗
In vitro studies on the existence of endothelial precursor cells in the subectodermal avascular region of quail wing buds.
In vitro studies were performed to investigate the angiogenic capacity of different parts of the avian limb bud. Small pieces of wing mesenchyme of the vascularized core or of the avascular subectodermal region were obtained from quail embryos at stages 18 to 25, and were cultured. The identification of the avascular wing mesenchyme was made possible after injection of India ink via the vitelline vein or by bleeding control during in vivo dissection. Tissue cultures were treated with the QH-1 antibody or/and the endothelial cell marker DiI-Ac-LDL. Endothelial cells were found in cultures of the mesenchymal core and in those of the avascular subectodermal wing mesenchyme. Moreover, their appearance was independent of the stage of the donor embryo. Although there were no vessels, the subectodermal wing mesenchyme was able to produce endothelial cells that proliferated and differentiated under in vitro conditions. Thus, endothelial precursor cells probably existed within the avascular wing mesenchyme. These cells might be identical with the QH-1-positive isolated cells that have been described in immunohistochemical studies of this region; they may contribute to the growing capillary plexus of the limb bud.
Animals↗