RESUMO
This study aimed to comprehensively investigate the in vitro metabolism of statins. The metabolism of clinically relevant concentrations of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and their metabolites were investigated using human liver microsomes (HLMs), human intestine microsomes (HIMs), liver cytosol, and recombinant cytochrome P450 enzymes. We also determined the inhibitory effects of statin acids on their pharmacological target, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. In HLMs, statin lactones were metabolized to a much higher extent than their acid forms. Atorvastatin lactone and simvastatin (lactone) showed extensive metabolism [intrinsic clearance (CLint) values of 3700 and 7400 µl/min per milligram], whereas the metabolism of the lactones of 2-hydroxyatorvastatin, 4-hydroxyatorvastatin, and pitavastatin was slower (CLint 20-840 µl/min per milligram). The acids had CLint values in the range <0.1-80 µl/min per milligram. In HIMs, only atorvastatin lactone and simvastatin (lactone) exhibited notable metabolism, with CLint values corresponding to 20% of those observed in HLMs. CYP3A4/5 and CYP2C9 were the main statin-metabolizing enzymes. The majority of the acids inhibited HMG-CoA reductase, with 50% inhibitory concentrations of 4-20 nM. The present comparison of the metabolism and pharmacodynamics of the various statins using identical methods provides a strong basis for further application, e.g., comparative systems pharmacology modeling. SIGNIFICANCE STATEMENT: The present comparison of the in vitro metabolic and pharmacodynamic properties of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin and their metabolites using unified methodology provides a strong basis for further application. Together with in vitro drug transporter and clinical data, the present findings are applicable for use in comparative systems pharmacology modeling to predict the pharmacokinetics and pharmacological effects of statins at different dosages.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Intestinos/metabolismo , Fígado/metabolismo , Microssomos/fisiologia , Biotransformação , Sistema Enzimático do Citocromo P-450/metabolismo , Citosol/metabolismo , Desenho de Fármacos/métodos , Desenvolvimento de Medicamentos/métodos , Eliminação Hepatobiliar , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/classificação , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Concentração Inibidora 50 , Taxa de Depuração Metabólica/efeitos dos fármacos , Farmacologia em RedeRESUMO
Prostasomes are nanosized microvesicles secreted by acinar epithelial cells of the prostate gland. Furthermore, they are intracellular microvesicles inside another larger vesicle, a so-called storage vesicle, equivalent to multivesicular bodies of late endosomal origin. Prostasomes are thought to play an important role in intercellular communication by direct interaction primarily between the immobile acinar cells of the prostate gland and the mobile spermatozoa. Prostasomes transfer not only membrane components but also genetic material to spermatozoa. They are rich in various transferable bioactive molecules (e.g., receptors and enzymes) that promote the fertilizing ability of spermatozoa. In this review, the pleiotropic biological effects of prostasomes that are relevant for successful fertilization will be discussed. The ability to synthesize and export prostasomes to the extracellular space is observed not only in normal prostate epithelial cells but also in malignant prostate cells. Release of prostasomes by prostate cancer cells suggests a role in malignant cell growth and proliferation. These findings may provide new therapeutic and diagnostic strategies.
Assuntos
Comunicação Celular/fisiologia , Microssomos/fisiologia , Próstata/metabolismo , Biomarcadores Tumorais/análise , Células Epiteliais/metabolismo , Humanos , Infertilidade Masculina/imunologia , Masculino , Microssomos/química , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/etiologia , Espermatozoides/fisiologiaRESUMO
The V1a receptor has emerged as an attractive target for a range of indications including Raynaud's disease and dysmenorrhoea. As part of an effort to discover a new class of orally active V1a antagonist, we optimised a highly lipophilic, metabolically unstable lead into a range of potent, selective and metabolically stable V1a antagonists. In this communication, we demonstrate the series-dependent effect of limiting the number of rotatable bonds in order to decrease Cytochrome P450-mediated metabolism. This effort culminated in the discovery of PF-184563, a novel, selective V1a antagonist with excellent in vitro and in vivo properties.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Benzodiazepinas/síntese química , Benzodiazepinas/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Dismenorreia/tratamento farmacológico , Antagonistas de Hormônios/síntese química , Antagonistas de Hormônios/farmacologia , Triazóis/síntese química , Triazóis/farmacologia , Benzodiazepinas/química , Benzodiazepinas/metabolismo , Estabilidade de Medicamentos , Feminino , Antagonistas de Hormônios/química , Antagonistas de Hormônios/metabolismo , Humanos , Microssomos/fisiologia , Estrutura Molecular , Triazóis/química , Triazóis/metabolismoRESUMO
We describe a novel series of inhibitors of the type 1 glycine transporter (GlyT1) as an approach to relieving the glutamatergic deficit that is thought to underlie schizophrenia. Synthesis and SAR follow-up of a series of octahydro-cyclopenta[c]pyrrole derivatives afforded potent in vitro inhibition of GlyT1 as well as in vivo activity in elevating CSF glycine. We also found that a 3-O(c-pentyl), 4-F substituent may serve as a surrogate for the widely used 3-trifluoromethoxy group, suggesting its application as an isostere for future medicinal chemistry studies.
Assuntos
Ciclopentanos/química , Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Pirróis/química , Animais , Linhagem Celular , Ciclopentanos/farmacologia , Cães , Proteínas da Membrana Plasmática de Transporte de Glicina/fisiologia , Humanos , Microssomos/efeitos dos fármacos , Microssomos/fisiologia , Pirróis/farmacologiaRESUMO
The sarcolemma of smooth muscle cells is composed of alternating stiff actin-binding, and flexible caveolar domains. In addition to these stable macrodomains, the plasma membrane contains dynamic glycosphingolipid- and cholesterol-enriched microdomains, which act as sorting posts for specific proteins and are involved in membrane trafficking and signal transduction. We demonstrate that these lipid rafts are neither periodically organized nor exclusively confined to the actin attachment sites or caveolar regions. Changes in the Ca(2+) concentration that are affected during smooth muscle contraction lead to important structural rearrangements within the sarcolemma, which can be attributed to members of the annexin protein family. We show that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca(2+) sensitivity, and that the extraction of annexins II and VI by detergent is prevented by elevated Ca(2+) concentrations. Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex (Babiychuk, E.B., R.J. Palstra, J. Schaller, U. Kämpfer, and A. Draeger. 1999. J. Biol. Chem. 274:35191-35195). Annexin II promotes the Ca(2+)-dependent association of lipid raft microdomains, whereas annexin V interacts with glycerophospholipid microcompartments. These interactions bring about a new configuration of membrane-bound constituents, with potentially important consequences for signaling events and Ca(2+) flux.
Assuntos
Anexina A2/fisiologia , Anexina A6/fisiologia , Cálcio/fisiologia , Membrana Celular/fisiologia , Membranas Intracelulares/fisiologia , Lipídeos de Membrana/fisiologia , Microssomos/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Fosfolipídeos/fisiologia , Membrana Celular/ultraestrutura , Colesterol/fisiologia , Colo , Glicoesfingolipídeos/fisiologia , Humanos , Membranas Intracelulares/ultraestrutura , Microssomos/ultraestrutura , Modelos Biológicos , Músculo Liso/ultraestruturaRESUMO
The membrane anchor for the molecular motor kinesin is a critical site involved in intracellular membrane trafficking. Monoclonal antibodies specific for the cytoplasmic surface of chick brain microsomes were used to define proteins involved in microtubule-dependent transport. One of four antibodies tested inhibited plus-end-directed vesicle motility by approximately 90 percent even as a monovalent Fab fragment and reduced kinesin binding to vesicles. This antibody bound to the cytoplasmic domain of kinectin, an integral membrane protein of the endoplasmic reticulum that binds to kinesin. Thus, kinectin acted as a membrane anchor protein for kinesin-driven vesicle motility.
Assuntos
Cinesinas/fisiologia , Proteínas de Membrana/fisiologia , Organelas/fisiologia , Animais , Anticorpos Monoclonais , Encéfalo/fisiologia , Embrião de Galinha , Dineínas/fisiologia , Técnicas In Vitro , Microssomos/fisiologia , Movimento/fisiologia , Ligação ProteicaRESUMO
The inositol 1,4,5-trisphosphate (IP3) receptor, a Ca(2+)-permeable channel, plays a key role in intracellular Ca2+ signaling. The effects of ATP on the IP3 receptor at the single-channel level were characterized after channel incorporation into planar lipid bilayers. ATP alone was not sufficient to open the IP3-gated channel, but addition of ATP or nonhydrolyzable ATP analogs in the presence of IP3 increased the frequency of channel openings 4.8-fold and increased the average duration of channel openings 2.5-fold; channel conductance was unchanged. High concentrations of ATP (> 4 mM) decreased channel activity most probably by competing with IP3-binding site. Allosteric modulation of IP3-induced Ca2+ release by ATP may contribute to the maintenance of cell viability during periods of energy starvation.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Canais de Cálcio , Cerebelo/fisiologia , Retículo Endoplasmático/fisiologia , Inositol 1,4,5-Trifosfato/farmacologia , Canais Iônicos/fisiologia , Microssomos/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Citoplasmáticos e Nucleares , Animais , Cálcio/metabolismo , Cães , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/efeitos dos fármacos , Cinética , Bicamadas Lipídicas , Matemática , Modelos Neurológicos , Receptores de Superfície Celular/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Triadin 1 (TRD) is an integral membrane protein that associates with the ryanodine receptor (RyR2), calsequestrin (CASQ2) and junctin to form a macromolecular Ca signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). To define the functional role of TRD, we examined the effects of adenoviral-mediated overexpression of the wild-type protein (TRD(WT)) or a TRD mutant lacking the putative CASQ2 interaction domain residues 200 to 224 (TRD(Del.200-224)) on intracellular Ca signaling in adult rat ventricular myocytes. Overexpression of TRD(WT) reduced the amplitude of I(Ca)- induced Ca transients (at 0 mV) but voltage dependency of the Ca transients was markedly widened and flattened, such that even small I(Ca) at low and high depolarizations triggered maximal Ca transients. The frequency of spontaneous Ca sparks was significantly increased in TRD(WT) myocytes, whereas the amplitude of individual sparks was reduced. Consistent with these changes in Ca release signals, SR Ca content was decreased in TRD(WT) myocytes. Periodic electrical stimulation of TRD(WT) myocytes resulted in irregular, spontaneous Ca transients and arrhythmic oscillations of the membrane potential. Expression of TRD(Del.200-224) failed to produce any of the effects of the wild-type protein. The lipid bilayer technique was used to record the activity of single RyR2 channels using microsome samples obtained from control, TRD(WT) and TRD(Del.200-224) myocytes. Elevation of TRD(WT) levels increased the open probability of RyR2 channels, whereas expression of the mutant protein did not affect RyR2 activity. We conclude that TRD enhances cardiac excitation-contraction coupling by directly stimulating the RyR2. Interaction of TRD with RyR2 may involve amino acids 200 to 224 in C-terminal domain of TRD.
Assuntos
Arritmias Cardíacas/fisiopatologia , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Transporte/fisiologia , Proteínas Musculares/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Adenoviridae/genética , Animais , Arritmias Cardíacas/genética , Cálcio/fisiologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cães , Estimulação Elétrica , Expressão Gênica , Vetores Genéticos/genética , Peptídeos e Proteínas de Sinalização Intracelular , Ativação do Canal Iônico/fisiologia , Bicamadas Lipídicas , Substâncias Macromoleculares , Masculino , Potenciais da Membrana , Proteínas de Membrana/fisiologia , Microssomos/fisiologia , Oxigenases de Função Mista/fisiologia , Modelos Cardiovasculares , Proteínas Musculares/biossíntese , Proteínas Musculares/química , Proteínas Musculares/genética , Miócitos Cardíacos/ultraestrutura , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Retículo Sarcoplasmático/metabolismo , Transdução GenéticaRESUMO
The cytochrome P450-dependent monooxygenase system catalyzes the metabolism of xenobiotics and endogenous compounds, including hormones and retinoic acid. In order to establish the role of these enzymes in embryogenesis, we have inactivated the system through the deletion of the gene for the electron donor to all microsomal P450 proteins, cytochrome P450 reductase (Cpr). Mouse embryos homozygous for this deletion died in early to middle gestation (approximately 9.5 days postcoitum [dpc]) and exhibited a number of novel phenotypes, including the severe inhibition of vasculogenesis and hematopoiesis. In addition, defects in the brain, limbs, and cell types where CPR was shown to be expressed were observed. Some of the observed abnormalities have been associated with perturbations in retinoic acid homeostasis in later embryogenesis. Consistent with this possibility, embryos at 9.5 dpc had significantly elevated levels of retinoic acid and reduced levels of retinol. Further, some of the observed phenotypes could be either reversed or exacerbated by decreasing or increasing maternal retinoic acid exposure, respectively. Detailed analysis demonstrated a close relationship between the observed phenotype and the expression of genes controlling vasculogenesis. These data demonstrate that the cytochrome P450 system plays a key role in early embryonic development; this process appears to be, at least in part, controlled by regional concentrations of retinoic acid and has profound effects on blood vessel formation.
Assuntos
Vasos Sanguíneos/embriologia , Sistema Enzimático do Citocromo P-450/fisiologia , Embrião de Mamíferos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/genética , Tretinoína/metabolismo , Animais , Vasos Sanguíneos/anormalidades , Embrião de Mamíferos/irrigação sanguínea , Morte Fetal/genética , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Homeostase/genética , Camundongos , Camundongos Mutantes , Microssomos/fisiologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Fenótipo , Receptores do Ácido Retinoico/genética , Tretinoína/farmacologia , Vitamina A/metabolismoRESUMO
Alpha-synuclein (alpha-syn), particularly in its aggregated forms, is implicated in the pathogenesis of Parkinson's disease and other related neurological disorders. However, the normal biology of alpha-syn and how it relates to the aggregation of the protein are not clearly understood. Because of the lack of the signal sequence and its predominant localization in the cytosol, alpha-syn is generally considered exclusively an intracellular protein. Contrary to this assumption, here, we show that a small percentage of newly synthesized alpha-syn is rapidly secreted from cells via unconventional, endoplasmic reticulum/Golgi-independent exocytosis. Consistent with this finding, we also demonstrate that a portion of cellular alpha-syn is present in the lumen of vesicles. Importantly, the intravesicular alpha-syn is more prone to aggregation than the cytosolic protein, and aggregated forms of alpha-syn are also secreted from cells. Furthermore, secretion of both monomeric and aggregated alpha-syn is elevated in response to proteasomal and mitochondrial dysfunction, cellular defects that are associated with Parkinson's pathogenesis. Thus, intravesicular localization and secretion are part of normal life cycle of alpha-syn and might also contribute to pathological function of this protein.
Assuntos
Córtex Cerebral/fisiologia , Exocitose/fisiologia , Neurônios/fisiologia , alfa-Sinucleína/fisiologia , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Vetores Genéticos , Humanos , Microssomos/fisiologia , Ratos , alfa-Sinucleína/genéticaRESUMO
Human placenta differs more than any other organ between species. This is the primary reason to develop models utilizing human tissue to study placental functions. There are no major ethical restrictions using human placenta for scientific studies. Also, the size of human placenta enables a great number of different parameters to be studied in one placenta. The most important cell types considering transplacental transfer, are the trophoblasts differentiating into syncytiotrophoblasts facing maternal circulation, and endothelial cells of fetal vessels. Primary trophoblasts are difficult to culture and do not grow in monolayer thus inhibiting studies on the polarized functions of transport. Several cell lines originating from trophoblasts have been developed, of which BeWo cells seem most useful for transport studies, because they grow in a tight monolayer. Placental tissue can also be retained as explant cultures, although the trophoblast viability is very restricted despite of culture conditions. Cotyledons of human placenta can be retained viable in an isolated organ perfusion. Perfused placental tissue stays viable longer than placental tissue in tissue culture. Although human placental perfusion is the most tedious experimental method to study placental functions, there are several good reasons to develop it further: transplacental transfer and molecular mechanisms of genotoxic compounds can be studied. Placental perfusion is the only experimental method that retains fully the structure of placenta for polarized transport. Furthermore, perfusion of placentas from mothers, who smoke, use illegal drugs or have a disease, allows studies on the impact of such factors on fetal exposure to genotoxic agents.
Assuntos
Troca Materno-Fetal , Mutagênicos/toxicidade , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Microssomos/efeitos dos fármacos , Microssomos/fisiologia , Modelos Biológicos , Perfusão , Placenta/anatomia & histologia , Placenta/efeitos dos fármacos , Placenta/fisiologia , Gravidez , Técnicas de Cultura de TecidosRESUMO
We have studied the plasma membrane fluidity of rat C6 glioblastoma cells and simian virus 40-transformed mouse liver cells in culture that had been rendered resistant to doxorubicin. This was done by the evaluation of fluorescence anisotropy of two probes; diphenylhexatriene was used on membrane microsomal fractions, and trimethylammonium-diphenylhexatriene was used on whole cell suspensions as a plasma membrane-specific probe since it does not enter the cells. A higher degree of membrane fluidity was exhibited with both techniques by doxorubicin-resistant glioblastoma cells as compared to the doxorubicin-sensitive strain, but in the transformed liver cells no such alteration was seen in the physical properties of their plasma membranes. A higher degree of acyl group unsaturation was noticed in the glioblastoma cells but not in the transformed liver cells upon acquisition of doxorubicin resistance. A similar simultaneous increase in acyl group unsaturation and membrane fluidity can be obtained easily by growing the sensitive cells with a medium supplemented with exogenous polyunsaturated fatty acids. This alteration does not modify the sensitivity of the cells to doxorubicin. We conclude from our work that the increase in membrane fluidity, which is frequently associated with drug resistance, is neither necessary nor sufficient for the expression of the resistance. The reason for a link between cell resistance to doxorubicin and plasma membrane fluidity remains to be found.
Assuntos
Doxorrubicina/farmacologia , Glioma/fisiopatologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/fisiologia , Resistência a Medicamentos , Ácidos Graxos Insaturados/fisiologia , Polarização de Fluorescência , Glioma/patologia , Glioma/ultraestrutura , Fluidez de Membrana , Lipídeos de Membrana/fisiologia , Microssomos/fisiologia , RatosRESUMO
Inhibitory monoclonal antibodies (MAbs) were used to determine the contribution of epitope-specific cytochrome P-450 isozymes in rat liver microsomes to hydroxylation of the esophageal carcinogen methyl-n-amylnitrosamine. These P-450-catalyzed reactions form 2-, 3-, 4-, and 5-hydroxymethyl-n-amylnitrosamine, formaldehyde (demethylation), and pentaldehyde (depentylation). With uninduced microsomes from male rats, MAb 1-68-11 inhibited 4-hydroxylation by 73% and demethylation by 46%. This indicated the major contribution of constitutive male-specific P-450 IIC11 to the metabolism. Inhibition studies with MAbs 2-66-3 and 1-91-3 indicated that P-450 IIB1 contributed 19% and IIE1 35% to demethylation. With uninduced microsomes from females, MAb 1-68-11 produced similar inhibitions to those in male rats, indicating that female-specific P-450 IIC12 (which is closely related to IIC11) also catalyzed 4-hydroxylation and demethylation. With microsomes from 3-methylcholanthrene-induced male rats, P-450 IA1 and/or IA2 were responsible for 60% of 3-hydroxylation and 40% of depentylation. With microsomes from phenobarbital-treated rats, P-450 IIB1 and IIB2 catalyzed all 6 reactions but especially 4-hydroxylation and depentylation, which were 50-75% inhibited by MAb 2-66-3. Microsomes from Aroclor-induced males behaved as if they were induced by both 3-methylcholanthrene and phenobarbital. After treatment with isoniazid (a P-450 IIE1 inducer), inhibition by MAb 1-91-3 indicated a 45% contribution of P-450 IIE1 to demethylation, and both P-450 IIE1 and IIB1 (or IIB2) appear to have been induced. A major finding with uninduced microsomes was the high specificity of MAb 1-68-11 for inhibiting 4-hydroxylation, indicating that P-450 IIC11 and IIC12 catalyzed most of this omega-1-hydroxylation. In microsomes from induced rats, the MAb inhibitions showed the role of the induced P-450 IA1 (or IA2), IIB1 (or IIB2), and IIE1 in methyl-n-amylnitrosamine hydroxylation at different positions, as well as the presence of P-450 IIC11. This study illustrates the usefulness of inhibitory MAbs for defining the contribution of individual P-450s to position-specific metabolism.
Assuntos
Carcinógenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Isoenzimas/metabolismo , Nitrosaminas/metabolismo , Animais , Anticorpos Monoclonais , Arocloros/farmacologia , Hidroxilação , Isoniazida/farmacologia , Fígado/enzimologia , Masculino , Metilcolantreno/farmacologia , Microssomos/fisiologia , Fenobarbital/farmacologia , Coelhos , Ratos , Ratos Endogâmicos , Fatores Sexuais , Especificidade por Substrato/efeitos dos fármacosRESUMO
The relative effectiveness of a variety of carbohydrates in preserving the structural and functional integrity of membranes at low water activities was studied, using Ca-transporting microsomes from muscle as a model membrane. The order of effectiveness (greatest to lowest) was: trehalose, lactose, maltose, cellobiose, sucrose, glucose, fructose, sorbitol, raffinose, myo-inositol, glycerol. At the highest concentrations of the most effective sugars tested, microsomes were obtained upon rehydration that were similar structurally and functionally to fresh membranes. The least effective carbohydrates, alcohol sugars, all appear to be fusogenic. A structural explanation for relative effectiveness of the sugars was sought, but no clear relationship was found, except that effectiveness does not appear to be related to the number of position of hydroxyl groups available for hydrogen bonding.
Assuntos
Carboidratos/fisiologia , Membranas/fisiologia , Animais , Técnica de Fratura por Congelamento , Ligação de Hidrogênio , Fusão de Membrana , Microssomos/fisiologia , Nephropidae , Oxirredução , Álcoois Açúcares , ÁguaRESUMO
When Tetrahymena pyriformis cells grown at 39 degrees C were chilled to 15 degrees C, a rapid desaturation of microsomal phospholipid-bound fatty acids was observed. A concurrent but even more rapid change in the physical properties of the microsomal lipids was detected by steady-state fluorescence polarization measurements of the probe 1,6-diphenyl-1,3,5-hexatriene in lipid multilamellar vesicles. Whereas polarization vs. temperature plots of lipids from 39 degrees C-grown cells showed discrete break points (abrupt slope changes thought to indicate altered phase separation rates) at characteristic temperatures, plots made using lipids from equivalent cells chilled to 15 degrees C for 15 or 20 min lacked such clearly defined break points. The sharp break points reappeared in plots of microsomal lipids from cells maintained at 15 degrees C for 30 min or longer, but in these curves the temperature of each break point was several degrees lower than in 39 degrees C-cell lipids and nearly the same as in cells fully acclimated to low temperature (48 h or more at 15 degrees C). Fluorescence polarization studies on mixtures of natural lipids from 39 degrees C-cells and cells shifted to 15 degrees C or on mixtures of natural and synthetic lipids revealed that each of the two break points in a polarization vs. temperature plot can respond to changes in lipid composition independently of the other. It is concluded that the expeditious desaturation of certain key fatty acids, perhaps coupled with limited retailoring of phospholipid molecular species, leads to pronounced physical changes in Tetrahymena microsomal membranes as the first step of low temperature acclimation.
Assuntos
Aclimatação , Membranas Intracelulares/fisiologia , Lipídeos de Membrana/fisiologia , Microssomos/fisiologia , Tetrahymena pyriformis/fisiologia , Animais , Temperatura Baixa , Ácidos Graxos/análise , CinéticaRESUMO
Using the fluorescent anion 8-anilino-1-naphthalenesulphonate (ANS) for determining the membrane surface potential necessitates that the intrinsic affinity constant Ki for the ANS sites be known. Two methods are presented which do not rely on a determination of Ki at high ionic strength. They are respectively applied to neutral membranes (egg phosphatidylcholine liposomes) and highly charged natural ones (horse bean microsomes and liposomes from their phospholipids). The value of Ki appears to be insensitive to the level of occupancy of the sites, the KCl concentration and the pH in large ranges. Furthermore, the classical Gouy-Chapman model seems to describe correctly the whole set of data, provided apparent mean molecular areas larger than the published crystallographic ones are admitted.
Assuntos
Membranas/fisiologia , Modelos Biológicos , Naftalenossulfonato de Anilina , Corantes Fluorescentes , Matemática , Potenciais da Membrana , Microssomos/fisiologia , Concentração Osmolar , Fenômenos Fisiológicos VegetaisRESUMO
The effect of temperature on native microsomal membrane vesicles isolated from Tetrahymena is investigated by wide angle X-ray diffraction. A 4.2 reflection, typical for lipids in the crystalline state, can be recorded in the temperature range between 0 degree C and 35 degrees C. Quantitative evaluation of this reflection reveals a broad thermotropic 'two-stage' liquid crystalline equilibrium crystalline lipid phase separation with a 'breakpoint' at approx. 18 degrees C. This 'breakpoint' coincides with the emergency of lipid-protein segregations in endomembranes of intact Tetrahymena cells as previously visualized by freeze-etch electron microscopy.
Assuntos
Membranas Intracelulares/fisiologia , Lipídeos de Membrana/fisiologia , Microssomos/fisiologia , Tetrahymena pyriformis/fisiologia , Animais , Cristalização , Membranas Intracelulares/ultraestrutura , Matemática , Conformação Molecular , Temperatura , Difração de Raios XRESUMO
1. The proportion of ethanolamine phosphoglycerides in microsomal fractions of goldfish intestine increases at low environmental temperatures. The fatty acyl composition also changes, the proportion of C22:6 and C20:4 fatty acids increasing in positions 1 and 2 and position 2 respectively. The proportion of C16:0 and C18:0 fatty acids falls in position 1 and there is an apparent switch of C18:1 and C20:1 fatty acids from position 2 to position 1. 2. The proportion of choline phosphoglycerides does not depend on the previous environmental temperature of the fish. Temperature-dependent changes in fatty acyl composition in positions 1 and 2 take place in a way similar to that described for ethanolamine phosphoglycerides, but in this case C22:6 substitution is confined to position 2. 3. Choline phosphoglycerides have been further separated into 7 different molecular species. The amounts of species 3 to 7 increase and the amount of species 2 decreases at low adaptation temperature. These changes only account for part of total change in fatty acyl composition. The remaining changes occur by chain substitution within species. 4. Present results show temperature adaptation to be highly complex, involving both quantitative and qualitative changes in different phospholipids. The possible physiological significance of these changes are discussed together with the effects these changes might have on cholesterol-phospholipid interactions.
Assuntos
Aclimatação , Cyprinidae/fisiologia , Carpa Dourada/fisiologia , Mucosa Intestinal/fisiologia , Lipídeos de Membrana/fisiologia , Fosfolipídeos/fisiologia , Adenosina Trifosfatases/metabolismo , Animais , Cromatografia em Camada Fina , Membranas/ultraestrutura , Microssomos/fisiologia , Microssomos/ultraestrutura , TemperaturaRESUMO
Fluorescence probe molecules, trans-parinaric acid and 1,6-diphenylhexatriene, were utilized to characterize the structure of plasma membranes, microsomes and mitochondria from B16 melanoma cells. High metastastic B16-F10 and low metastatic B16-F1 melanoma cell lines had markedly different membrane structures. The fluorescence polarization, fluorescence lifetime and limiting anisotropy of trans-parinaric acid were significantly lower (P less than 0.05) in all three membrane fractions of the B16-F1 cell line than in the corresponding membranes of the B16-F10 cell line. These data indicated less restriction to rotational motion in the solid lipid domains of B16-F1 cell membranes preferentially sensed by trans-parinaric acid. The limiting anisotropy of both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene was significantly lower in the outer monolayer than the inner monolayer of the plasma membrane of B16-F1 cells but not in B16-F10 cells. A breakpoint in Arrhenius plots of fluorescence near 30-34 degrees C indicated the presence of a phase separation that was assigned to the inner monolayer of the plasma membrane. However, no differences in this breakpoint temperature were noted between the B16-F1 and B16-F10 melanoma membranes. Thus, more fluid solid membrane domains and a distinct transbilayer fluidity difference were characteristic of plasma membranes from low metastatic B16-F1 melanoma cells in contrast to high metastatic B16-F10 melanoma cells.
Assuntos
Melanoma/fisiopatologia , Fluidez de Membrana , Animais , Membrana Celular/fisiologia , Corantes Fluorescentes , Membranas Intracelulares/fisiologia , Melanoma/patologia , Lipídeos de Membrana/fisiologia , Camundongos , Microssomos/fisiologia , Mitocôndrias/fisiologia , Metástase Neoplásica , Fosfolipídeos/fisiologia , Espectrometria de Fluorescência , Ácido Trinitrobenzenossulfônico/farmacologiaRESUMO
The effect of temperature on steroid C-21 hydroxylation and substrate-cytochrome P-450 binding reaction under turnover conditions (NADPH + O2 are investigated. The Arrhenius activity plot exhibited a single break, while the van 't Hoff plot of the substrate dissociation constant (Ks) exhibited four breaks between 10 and 40 degrees C which corresponded to the characteristic temperatures of the lipids' phase transitions. Unlike the case of the Ks value, the detergent Triton X-114 was without effect on the Arrhenius activity plot. This indicates that the single break in the case of the enzyme activity is distinct from but not necessarily independent of the multiple breaks inthe case of the Ks. At physiologic temperature and concentration of the substrate, the free energy (--9.5 kcal/mol) of the substrate-cytochrome binding reaction is more than sufficient to account for the apparent activation energy (6.6 kcal/mol) of the overall hydroxylation. This suggests that the substrate-cytochrome P-450 binding reaction has the potential of being a source of energy for the overall reaction.