RESUMO
In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.
Assuntos
Micropartículas Derivadas de Células/efeitos dos fármacos , GMP Cíclico/análogos & derivados , Selectina-P/biossíntese , Agregação Plaquetária/efeitos dos fármacos , Receptores de Trombina/fisiologia , Tionucleotídeos/farmacologia , Trombina/farmacologia , Micropartículas Derivadas de Células/fisiologia , Células Cultivadas , AMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Diclororribofuranosilbenzimidazol/análogos & derivados , Diclororribofuranosilbenzimidazol/farmacologia , Humanos , Fatores de TempoRESUMO
Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced.
Assuntos
Trifosfato de Adenosina/sangue , Plaquetas/metabolismo , Glioxilatos/sangue , Glioxilatos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Adenina/sangue , Difosfato de Adenosina/sangue , Difosfato de Adenosina/farmacologia , Adenilil Ciclases/sangue , Plaquetas/efeitos dos fármacos , Radioisótopos de Carbono/sangue , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Cinética , Ácido Láctico/sangue , Masculino , TrombinaRESUMO
Resveratrol is a polyphenol that among other sources occurs in grapes and for this reason, red wines also contain considerable amounts of this compound. Interactions of resveratrol with pure molecular species of phosphatidylcholine (PC), phosphatidylethanoloamine (PE) and phosphatidylserine (PS) were studied with the Langmuir technique on monolayers and with differential scanning calorimetry on unilamellar liposomes. Resveratrol caused a modest increase in the mean molecular area (MMA) of dipalmitoyl-PC (DPCC) and PE (DPPE) monolayers, but profoundly increased the MMA of dipalmitoyl-PS (DPPS). The resveratrol-induced increase in MMA was enlarged in PS species containing stearolyl and oleoyl acyls suggesting that increase in the acyl chain length and unsaturation enhanced the resveratrol-phospholipid interaction. In liposomes resveratrol lowered T(m) (main transition temperature) and increased T(C1/2) (transition interval) in DPPE, DPPS and 1-palmitoyl-2-oleoyl-PS (POPS), suggesting that resveratrol causes an increase in the anisotropy of these liposomes. In DPPE and POPS liposomes resveratrol caused a lowering of ΔH, further substantiating a pure enhancement of anisotropy for these molecular species. However, resveratrol caused a marked increase of ΔH in DPPS liposomes, indicating that, in addition to increase anisotropy, DPPS and resveratrol attracted each other specifically. This study has clearly shown interactions between resveratrol and glycerophospholipids on a molecular level and that these interactions are influenced by the acyl chain length, degree of unsaturation and head group of the lipids.
Assuntos
Glicerofosfolipídeos/química , Lipídeos de Membrana/química , Estilbenos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Varredura Diferencial de Calorimetria , Lipossomos/química , Fosfatidilcolinas/química , Fosfatidilserinas/química , Resveratrol , Temperatura de TransiçãoRESUMO
Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH-as observed by liposome content leakage and circular dichroism experiments-and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET.
Assuntos
Lactalbumina/química , Ácidos Oleicos/química , Fosfolipídeos/química , Polímeros/química , Lipossomas Unilamelares/química , Animais , Bovinos , Dicroísmo Circular , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Ácido Oleico/química , Dobramento de ProteínaRESUMO
Adrenaline is not a true platelet agonist, but enhances aggregation, dense granule secretion, and phospholipase C induced by other agonists. In the present work we investigated the effect of adrenaline on other platelet responses. It strongly potentiated ADP-induced shape change in platelet-rich plasma, particularly when aggregation was prevented by EDTA. The degree of potentiation increased with increasing concentrations of ADP. Thrombin-induced α-granule secretion, measured by the release of fibrinogen in gel-filtered platelets, was also potentiated by adrenaline at thrombin concentrations above 0.05 U/ml. In contrast, adrenaline had little effect on thrombin-induced secretion of ß-acetyl-hexosaminidase and potentiated very little liberation of arachidonate at high thrombin concentrations. When autocrine stimulation was inhibited by the removal of secreted ADP by creatine phosphate/creatine phosphate kinase and specific blocking of the thromboxane A(2) and fibrinogen receptors, the potentiation of thrombin-induced ADP + ATP secretion by adrenaline was reduced and this reduction was mostly due to the blocking of the thromboxane A(2) receptor. Protein tyrosine phosphorylation by both thrombin and collagen was reduced by adrenaline, and inhibitors of autocrine stimulation counteracted this reduction.
Assuntos
Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Epinefrina , Agregação Plaquetária/efeitos dos fármacos , Trombina/farmacologia , Difosfato de Adenosina/metabolismo , Plaquetas/citologia , Plaquetas/metabolismo , Forma Celular , Células Cultivadas , Cromatografia em Camada Fina , Colágeno/metabolismo , Sinergismo Farmacológico , Ácido Edético/farmacologia , Ácidos Eicosanoicos/metabolismo , Eletroforese em Gel de Poliacrilamida , Epinefrina/farmacologia , Fibrinogênio/metabolismo , Hexosaminidases/metabolismo , Humanos , Fosforilação , Plasma Rico em Plaquetas/citologia , Plasma Rico em Plaquetas/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismoRESUMO
We and others have previously demonstrated that nitric oxide (NO)-induced inhibition of platelet shape change is important in regulating platelet adhesion and aggregation, and therapeutic intervention of this pathway is clinically relevant for secondary prevention of stroke with dipyridamole. In the present study, we investigated whether dipyridamole affected the shape change of aspirinated platelets. Platelet shape change was inhibited using both authentic NO and sodium nitroprusside, as monitored by light scattering and mean platelet volume measurements. Dipyridamole synergized with NO, even at supra-therapeutic levels, to inhibit thrombin-induced shape change and further potentiated cAMP dependent protein kinase (PKA) mediated phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser157, even without altered levels of platelet cAMP. The effect of dipyridamole on NO-inhibited shape change depended on cGMP synthesis as evaluated by inhibition of soluble guanylyl cyclase. Measured increases in cGMP levels by dipyridamole and NO was assessed by mathematical modeling and found to be consistent with inhibition of phosphodiesterase 5 (PDE5). The model could explain the unexpected efficiency of dipyridamole in inhibiting PDE5 at the measured cGMP levels, by the majority of cGMP being bound to cGMP-dependent protein kinase (PKG). Still, selective activators of PKG failed to extend NO-mediated inhibition of the thrombin-induced platelet shape change, suggesting that PKG was not responsible for the inhibitory effect of NO and dipyridamole on shape change. The effects of dipyridamole were independent of the prostanoid and ADP pathways. Thus, the effect of dipyridamole on NO-mediated inhibition of platelet shape change may be an important and additional beneficial therapeutic effect of dipyridamole, which we suggest, is acting though localized amplification of the NO/cGMP/Phosphodiesterase3/cAMP/PKA-pathway. Probably, the efficiency of dipyridamole could be amplified clinically with NO donors.
Assuntos
Plaquetas/efeitos dos fármacos , Dipiridamol/farmacologia , Óxido Nítrico/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Trombina/farmacologia , Plaquetas/fisiologia , Moléculas de Adesão Celular/metabolismo , Forma Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Sinergismo Farmacológico , Humanos , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Óxido Nítrico/metabolismo , Nitroprussiato/metabolismo , Nitroprussiato/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Acidente Vascular Cerebral/prevenção & controle , Trombina/metabolismoRESUMO
We have earlier investigated the interaction of the antipsychotic drugs chlorpromazine(CPZ) and olanzapine(OLP) with glycerophospholipid monolayers. These experiments were carried out at high and low temperatures and showed that OLP had a more pronounced effect on the packing of the phospholipid (PL) monolayers than CPZ. At pH 7.36, where OLP consists of one positive and one neutral species. In the present work we have studied the interaction of the drugs with monolayers of PLs by the Langmuir technique at pH 6.00 and 10.00 at 37°C. The PLs were palmitoylphosphatidyl-choline(DPPC), 1-stearoyl-2-arachinodonoylphoshatidylcholine(SAPC),dipalmitoylphosphatidyl-serine(DPPS) and 1-palmitoyl-2-oleoylphosphatidylserine(POPS). OLP has a pKa around 7.4, with one neutral and one positive species at pH 6.00 and pH 10.00, respectively. CPZ has pKa value around 9.4, and is positively charged at pH 6.00 and neutral at pH 10.00. Our studies revealed that the surface area of DPPC with CPZ in the subphase did not change at pH 6.00. In contrast, OLP increased the mean molecular area(MMA) of DPPC at pH 6.00, while CPZ caused distinct increase in MMA on the monolayer packing of all the other PLs, including monolayers of DPPC at pH 10.00. OLP, increased MMA of all PLs at both pHs. Further, OLP increased MMA of DPPC (pH 10.00), SAPC (pH 10.00), DPPS (pH 6.00) and POPS (pH 6.00) at 30mN/m, the expected MMA of biological membranes. CPZ had the more pronounced effect at lift-off and gave an effect of the monolayers with negatively charged head groups in accordance our earlier experiments. However, CPZ affected the packing of the SAPC monolayer both at pH 6.00 and 10.00, and DPPC at pH 10.00. Both these PLs have neutral choline head group. Our results suggest that both drugs intercalate in the PL monolayers, and that the intercalation might involve electrostatic interaction with the head groups or hydrophobic interaction with the acyl chains of the PLs, or both. Probably the drugs intercalate to different extents depending on charge of both the drugs and the PL head groups. Our investigation may suggest that the interaction of CPZ and OLP with membrane PLs could be linked to both the psychotropic and the side effects.
Assuntos
Glicerofosfolipídeos/química , Psicotrópicos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Benzodiazepinas/química , Clorpromazina/química , Concentração de Íons de Hidrogênio , Olanzapina , Fosfatidilcolinas/química , Fosfatidilserinas/química , TemperaturaRESUMO
Patients respond differently to psychotropic drugs, and this is currently a controversial theme among psychiatrists. The effects of 16 psychotropics on cell membrane parameters have been reported. These drugs belong to three major groups used in therapeutic psychiatry: antipsychotics, antidepressants, and anxiolytic/hypnotics. Human platelets, lacking dopamine (D(2)) receptors (proposed targets of most psychotropics), have been used as a cell model. Here we discuss the effects of these drugs on three metabolic phenomena and also results from Langmuir experiments. Diazepam, in contrast to the remaining drugs, had negligible effects on metabolic phenomena and had no effects in Langmuir experiments. Psychotropic drugs may work through intercalation in membrane phospholipids. It is possible that the fluidity of membranes, rich in essential fatty acids, the content being influenced by diet, could be a contributing factor to the action of psychotropics. This might in turn explain the observed major differences in therapeutic response among patients.
RESUMO
Intercalation of drugs in the platelet membrane affects phospholipid-requiring enzymatic processes according to the drugs' intercalation capability. We investigated effects of Promethazine, Citalopram, Ziprasidone, Risperidone, and Diazepam on phospholipase A(2) (PLA(2)) and polyphosphoinositide (PPI) metabolism in thrombin-stimulated human platelets. We also examined effects of the drugs on monolayers of glycerophospholipids using the Langmuir technique. Diazepam did not influence PLA( 2 ) activity, had no effects on PPI cycle, and caused no change in mean molecular area of phospholipid monolayers. The remaining psychotropic drugs affected these parameters in different ways and levels of potency suggesting that they act by being intercalated between the molecules of adjacent membrane phospholipids, thus causing changes in substrate availability for phospholipid-hydrolyzing enzymes (PLA(2) and Phospholipase C). We show that several psychotropic drugs can also have other cellular effects than receptor antagonism. These effects may be implicated in the psychotropic effects of the drugs and/or their side effects.
RESUMO
Polyethylene glycol (PEG), a high-molecular-weight colloid present in new organ preservation solutions, protects against cold ischemia injuries leading to better graft function of transplanted organs. This protective effect cannot be totally explained by immuno-camouflaging property or signaling-pathway modifications. Therefore, we sought for an alternative mechanism dependent on membrane fluidity. Using the Langmuir-Pockles technique, we show here that PEGs interacted with lipid monolayers of defined composition or constituted by a renal cell lipid extract. High-molecular-weight PEGs stabilized the lipid monolayer at low surface pressure. Paradoxically, at high surface pressure, PEGs destabilized the monolayers. Hypothermia reduced the destabilization of saturated monolayer whereas unsaturated monolayer remained unaffected. Modification of ionic strength and pH induced a stronger stabilizing effect of PEG 35,000 Da which could explain its reported higher effectiveness on cold-induced injuries during organ transplantation. This study sheds a new light on PEG protective effects during organ preservation different from all classical hypotheses.
RESUMO
OBJECTIVE: Local anesthetics are the most commonly used drugs in dentistry, with a wide range of effects, including antimicrobial activity. High antimicrobial effects have recently been reported on oral microbes from articaine hydrochloride, revealed by the minimum inhibitory concentration and minimal bactericidal concentration. Additionally, articaine has recently been used as an alkaline component in endodontic materials with a proposed antibacterial activity. However, the detailed mechanisms of action have not been discussed. MATERIAL AND METHODS: We determined the Langmuir surface pressure/molecular area isotherms of prokaryotic lipid monolayers, as well as the phospholipid phase transitions, by employing differential scanning calorimetry on unilamellar prokaryotic liposomes (bilayers). RESULTS: Articaine hydrochloride was found to interact with the prokaryotic membrane lipids in both monolayers and bilayers. An increase of the phospholipid molecular area of acidic glycerophospholipids as well as a decrease in phase transition temperature and enthalpy were found with increasing articaine hydrochloride concentration. The thermodynamic changes by adding articaine hydrochloride to prokaryotic membrane lipids are potentially related to the effects observed from antimicrobial peptides resulting from membrane insertion, aggregate composition, pore formation, and lysis. CONCLUSION: Interaction of articaine hydrochloride with prokaryotic membrane lipids is indicated. Hence, further research is necessary to gain insight into where these compounds exert their effects at the molecular level.
Assuntos
Antibacterianos/química , Carticaína/química , Membrana Celular/química , Lipídeos de Membrana/química , Fosfolipídeos/química , Anestesia Dentária , Anestésicos Locais/química , Varredura Diferencial de Calorimetria , Relação Dose-Resposta a Droga , Lipossomos , Transição de Fase , Células Procarióticas , Temperatura de TransiçãoRESUMO
OBJECTIVE: To compare the effects of chlorpromazine (CPZ), prochlorperazine (PCP), trifluoperazine (TFP), clozapine (CLO), haloperidol (HPD), quetiapine (QTP), pimozide (PMZ), and olanzapine (OLP) as well as the tricyclic antidepressants amitriptyline AMI, imipramine IMI, and nortriptyline NTP on thrombin-induced liberation of arachidonic acid AA in platelets. METHODS: This work was carried out at the Department of Biomedicine, University of Bergen, Norway in 2006-2007. Human platelets pre labelled with [3H] arachidonate were incubated with thrombin in the absence and presence of the drugs, and the amount of free [3H] arachidonate liberated was determined. Myosin light chain (MLC) phosphorylation was determined in [32P] phosphate-labelled platelets after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The effects of the drugs on the molecular area and surface pressure of phospholipid monolayers were determined in the Langmuir apparatus. RESULTS: All drugs reduced arachidonate liberation with the ranking order of increasing potency: OLPAssuntos
Antidepressivos Tricíclicos/efeitos adversos
, Antipsicóticos/efeitos adversos
, Ácido Araquidônico/biossíntese
, Plaquetas/efeitos dos fármacos
, Células Cultivadas
, Fosfolipases A2 do Grupo IV/metabolismo
, Humanos
, Lipídeos de Membrana/metabolismo
, Cadeias Leves de Miosina/metabolismo
, Fosforilação
, Trombina/metabolismo
RESUMO
Incubation of platelets with increasing concentrations of thrombin produced large amounts of phosphatidic acid (PA) and distinct changes in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2), prominent metabolites in the polyphosphoinositide (PPI) cycle. The relation between normalized PA and PIP or PIP2 levels in such thrombin-treated platelets from 22 normal donors gave a very similar pattern, suggesting tight control of the metabolites in the polyphosphoinositide (PPI) cycle. Prochlorperazine (PCP), trifluoperazine (TFP), haloperidol (HPD), quetiapine (QTP), pimozide (PMZ) and clozapine (CLO) interfered with this tight coupling produced by treating platelets with increasing thrombin concentrations. All drugs decreased the formation of PA at a given thrombin concentration, a decrease that varied greatly among platelets from different donors. This made it difficult to treat the PIP/PA and PIP2/PA relationships with ordinary, descriptive statistics. The data were therefore subjected to regression analysis using polynomials of second or first degree and gave the interference ranking order: PCP>TFP>>PMZ = HPD>CLO>QTP. All six drugs increased the mean molecular area of monolayers of dipalmitoyl phosphatidylserine on pure water at 37 degrees C by 20-50%, while they had little effect on monolayers of dipalmitoyl phosphatidylcholine. These results suggest that the drugs are membrane-active and may intercalate in biomembranes containing negatively charged phospholipids. Since human platelets do not contain D2 receptors, the interference with the tight coupling of PPI cycle metabolites was not receptor-mediated. We suggest that the drugs are intercalated in the plasma membrane and alter the relative, spatial positioning of phospholipid-consuming enzymes and thereby alter the velocities of the enzyme-catalyzed reactions. Such intercalation could be part of the side effects of the drugs and may explain their psychotropic action(s).
Assuntos
Antipsicóticos/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Membrana Celular , Clorpromazina/farmacologia , Fosfatos de Fosfatidilinositol/metabolismo , Psicotrópicos/farmacologia , Plaquetas/citologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Clozapina/farmacologia , Dibenzotiazepinas/farmacologia , Haloperidol/farmacologia , Humanos , Estrutura Molecular , Pimozida/farmacologia , Proclorperazina/farmacologia , Psicotrópicos/metabolismo , Fumarato de Quetiapina , Transdução de Sinais/fisiologia , Trombina/metabolismo , Trifluoperazina/farmacologiaRESUMO
The typical antipsychotics chlorpromazine (CPZ) and trifluoperazine (TFP) increase the mean molecular area (mma) of acidic, but not neutral, glycerophospholipids in monolayers at pH 7.36 measured by the Langmuir technique. The atypical antipsychotic olanzapine (OLP(1)) is structurally similar to TFP. We have therefore studied the effects of OLP on glycerophospholipid monolayers and in comparison with CPZ. Olanzapine (10 microM, in subphase, pH 7.36) influenced the isotherms (surface pressure versus mma) in monolayers of the neutral dipalmitoyl phosphatidylcholine (DPPC) and the acidic dipalmitoyl phosphatidylserine (DPPS) or 1-palmitoyl-2-oleoylphosphatidylserine (POPS) in the increasing order of mma: DPPSAssuntos
Benzodiazepinas/farmacologia
, Glicerofosfolipídeos/química
, Membranas Artificiais
, Psicotrópicos/farmacologia
, 1,2-Dipalmitoilfosfatidilcolina/química
, Concentração de Íons de Hidrogênio
, Olanzapina
, Fosfatidilserinas/química
, Promazina/química
, Promazina/farmacologia
, Propriedades de Superfície/efeitos dos fármacos
, Temperatura
RESUMO
Offshore oil production releases large amounts of lipophilic compounds in produced water into the ocean. In 2004, 143 million m(3) produced water, containing approximately 13 tons of long-chain (>C(4)) alkylphenols (AP), was discharged from installations in the Norwegian sector of the North Sea. Long-chain APs are known to cause endocrine disruption in a number of species. However, relatively little is known about their long-term effects in the marine environment. In the present study, Atlantic cod (Gadus morhua) were exposed (0.02 to 80 mg AP/kg) to a mixture (1:1:1:1) of APs (4-tert-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol and 4-n-heptylphenol) or 17 beta-estradiol (5 mg E2/kg) for 5 weeks and the effect on the fatty acid profile and cholesterol content in the membrane lipids from the liver and the brain was studied. We also determined the interaction between different para-substituted APs and glycerophospholipids (native phospholipids extracted from cod liver and brain) and model phosphatidylcholine (PC 16:0/22:6 n-3) in monolayers with the Langmuir-Blodget technique. The study demonstrated that APs and E2 alter the fatty acid profile in the polar lipids (PL) from the liver to contain more saturated fatty acids (SFA) and less n-3 polyunsaturated fatty acids (n-3 PUFA) compared with control. In the brain of the exposed groups a similar effect was demonstrated, although with higher saturation of the fatty acids found in the neutral lipids (mainly cholesterol ester), but not in the polar lipids. The AP and E2 exposure also gave a decline in the cholesterol levels in the brain. The in vitro studies showed that APs increased the mean molecular areas of the PLs in the monolayers at concentrations down to 5 microM, most likely due to intercalation of the APs between PL molecules. The increase in molecular area increased with the length of the alkyl side chain.
Assuntos
Química Encefálica/efeitos dos fármacos , Colesterol/análise , Ácidos Graxos/análise , Gadus morhua/metabolismo , Fígado/efeitos dos fármacos , Fenóis/toxicidade , Animais , Estradiol/farmacologia , Feminino , Fígado/química , Fluidez de Membrana/efeitos dos fármacos , Lipídeos de Membrana/análiseRESUMO
We have studied the interaction of trifluoperazine (TFP) with monolayers of various glycerophospholipids at 37 degrees C. TFP (1-10 microM) had little effect on surface pressure/molecular area isotherms in monolayers (on pure water) of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine but greatly increased the mean molecular area (mma) of dipalmitoylphosphatidylserine; the increment was greatest between 0 and 1 microM, and a further increase to 10 microM TFP gave only a slight increase in mma. With phosphatidylserine (PS)-containing stearoyl and varying acyls in the sn-1 and -2 positions, respectively, TFP increased the mma in a manner that depended on the number of double bonds and chain length. In mixtures of DPPC with two of these PS species the TFP-induced mma of the monolayers (on buffer, pH 7.4) increased linearly with the proportion of PS. Both PS and TFP have ionizable groups, and the TFP-induced mma increase had optima at pH 5.0 and 7.0. We conclude that the TFP-PS interaction is mainly, but not entirely, driven by electrostatic interactions between the TFP cation and PS headgroup anion, with an insertion of the phenothiazine moiety among the acyls in the monolayer that depends on the packing of the acyls.
Assuntos
Glicerofosfolipídeos/química , Fosfatidilserinas/química , Trifluoperazina/química , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Lipídeos/química , Lipídeos de Membrana/química , Modelos Químicos , Modelos Moleculares , Modelos Estatísticos , Fosfolipídeos/química , Pressão , Propriedades de Superfície , TemperaturaRESUMO
The molecular classes of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) from the basal ganglia, cerebellum, cortex, erythrocytes and blood platelets of female rats were separated by an isocratic HPLC method using a silica column and ultraviolet detection. Each glycerophospholipid class were thereafter derivatized to dimethylphosphatidic acid (PA) molecular species, separated by reverse phase HPLC and detected by an evaporative laser scatter to quantify the different glycerophospholipid species. The distribution of molecular species in each class of the glycerophospholipids in the three brain areas was very similar with a predominance of the 18:0/22:6 species and very little of the 18:0/20:4 species. In contrast, the 18:0/20:4 species predominated in the blood cells which had a very low proportion of 18:0/22:6. These results are discussed on the background that platelets have been extensively used as a model for neurons and our previous physicochemical observation that phenothiazines appear to interact specifically with the 18:0/22:6 species of PS.
Assuntos
Plaquetas/química , Encéfalo/citologia , Glicerofosfolipídeos/análise , Modelos Biológicos , Neurônios/química , Animais , Gânglios da Base/química , Cerebelo/química , Córtex Cerebral/química , Cromatografia Líquida de Alta Pressão , Feminino , Glicerofosfolipídeos/classificação , Fosfatidilcolinas/análise , Fosfatidiletanolaminas/análise , Fosfatidilserinas/análise , Ratos , Ratos Sprague-DawleyRESUMO
Vasostatin-I (CgA1-76) is a naturally occurring and biologically active N-terminal peptide derived from chromogranin A (CgA), produced and secreted at high concentrations by neuroendocrine tissues and also from a range of neuroendocrine tumors. This study aims to examine the hypothesis that in the absence of classical protein receptors CgA1-76 may, like its two derived peptides CgA1-40 and CgA47-66, perturb the lipid microenvironment of other membrane receptors, as a basis for the largely inhibitory activities of these CgA peptides. The nature of the interactions between phospholipids and vasostatin-derived fragments was studied in the Langmuir film balance apparatus at 37 degrees C. The synthetic peptides CgA1-40 and CgA47-66 and a recombinant fragment (VS-I) containing vasostatin-I (Ser-Thr-Ala-CgA1-78) were compared for their effects on monolayers of phosphatidylcholine and phosphatidylethanolamine from pig brain and defined species of phosphatidylserine. Marked differences in surface pressure-area isotherms and phase-transition plateaus were apparent with the three classes of phospholipids on VS-I, CgA1-40 and CgA47-66 in physiological buffer or pure water. The results indicate that VS-I and CgA47-66 at 5-10 nM concentrations may engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions, VS-I in particular enhancing the fluidity of saturated species of phosphatidylserine.
Assuntos
Cromograninas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Animais , Encéfalo/metabolismo , Cromogranina A , Cromograninas/farmacologia , Interações Medicamentosas , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/farmacologia , Fosfolipídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Suínos , TemperaturaRESUMO
The polyunsaturated fatty acid docosahexaenoic acid (DHA, 22:6, n-3) is found at a level of about 50% in the phospholipids of neuronal tissue membranes and appears to be crucial to human health. Dipalmitoyl phosphatidylcholine (DPPC, 16:0/16:0 PC) and the DHA containing 1-stearoyl-2-docosahexenoyl phosphatidylserine (SDPS) were used to make DPPC (60%)/SDPS (40%) bilayers with and without 10 mol% chlorpromazine (CPZ), a cationic, amphiphilic phenothiazine. Resonances that are present in 13C NMR spectrum of the DPPC (60%)/SDPS (40%) sample and that disappear in presence of 10% CPZ most probably are due to the special interface environment, e.g. the hydrophobic mismatch, at the interface of DPPC and SDPS microdomains in the DPPC/SDPS bilayer. In itself the appearance of resonances at novel chemical shift values is a clear demonstration of a unique chemical environment in the DPPC (60%)/SDPS (40%) bilayer. The findings of the study presented here suggest CPZ bound to the phosphate of SDPS will slow down and partially inhibit such a DHA acyl chain movement in the DPPC/SDPS bilayer. This would affect the area occupied by a SDPS molecule (in the bilayer) and probably the thickness of the bilayer where SDPS molecules reside as well. It is quite likely that such CPZ caused changes can affect the function of proteins embedded in the bilayer.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Clorpromazina/química , Ácidos Docosa-Hexaenoicos/química , Bicamadas Lipídicas/química , Microdomínios da Membrana/química , Fosfatidilserinas/química , Isótopos de Carbono , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
INTRODUCTION: In order to investigate if decompression sickness involves platelet activation an animal model was evaluated. MATERIALS AND METHODS: Twenty-four thiopentone-midazolam-fentanyl-anaesthetized pigs in four groups received 5-min infusions of adenosine diphosphate (25 mg/kg) or platelet activating factor (0.4 microg/kg). Groups 1 and 2 (adenosine diphosphate, n=6 and platelet activating factor, n=6) were studied for 30 min and then sacrificed. Groups 3 and 4 (adenosine diphosphate, n=6 and platelet activating factor, n=6) were sacrificed immediately afterwards to study short-term changes. Haemodynamics, platelet counts and post mortem lung platelet aggregates were registered. Groups 1 and 2 also had indium platelet labelling, lung scintigraphy and platelet accumulation index calculations performed. RESULTS: Adenosine diphosphate induced immediate and more profound transient shocks. Platelet and leukocyte count decreases and occurrences of post mortem lung platelet aggregates were significantly more profound in the 5-min adenosine diphosphate group (Group 3) than in the platelet activating factor group (Group 4). With platelet labelling there were positive platelet accumulation index trends in the 30-min adenosine diphosphate group (Group 1). Adenosine diphosphate also produced platelet aggregation in platelet-rich porcine plasma. Only adenosine diphosphate (an intermediate platelet agonist) showed signs of platelet activation when considering all platelet parameters. The model should be further evaluated with different bolus doses of adenosine diphosphate, but may be used to evaluate if gas bubbles introduced into the circulation (as with decompression sickness), or possibly if clinical drugs, might produce platelet activation in vivo.