RESUMEN
The action of platelet activating factor (PAF) on subcellular distribution and activity of protein kinase C (PKC) isoforms in rabbit platelets was analyzed. The results showed an increase of PKC alpha in membrane fraction, concomitantly with a decrease in cytosolic fraction after 5 min PAF treatment, indicating that a translocation of PKC alpha occurred. In addition, PKC zeta was redistributed in a "reverse" form, from the membrane to cytosolic fraction after PAF treatment. PAF induced an increase of PKC alpha activity, whereas a decrease rather than increase in PKC zeta was observed by using immunoprecipitation assays. In addition, some results indicated that PI3 kinase activation was not involved in PAF-induced PKC zeta translocation as occur in several cells and with other agonists. These actions were time- and concentration-dependent, and were inhibited by the treatment with a PAF antagonist. No translocation was observed when the platelets were incubated with lysoPAF, a PAF related compound. The redistribution of PKC isoforms take place through the activation of high specificity PAF binding sites. The pretreatment of the rabbit platelets with staurosporine, a putative inhibitor of PKC, completely blocked the PAF-evoked aggregation without affecting to PAF-evoked shape change and serotonin release. All together, these data could suggest that the specific translocation of PKC isoforms play an important role in the activation of rabbit platelets.
Asunto(s)
Plaquetas/enzimología , Isoenzimas/sangre , Factor de Activación Plaquetaria/farmacología , Proteína Quinasa C/sangre , Animales , Plaquetas/citología , Plaquetas/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Factor de Activación Plaquetaria/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Conejos , Serotonina/sangreRESUMEN
In this study we report that protein kinase C zeta (PKC zeta), one of the atypical isoforms of the PKC family located predominantly in cytosol, is redistributed by C2-ceramide treatment in isolated hepatocytes. PKC zeta increased in membrane and nuclear fractions after 30 min of treatment with C2-ceramide in a dose- and time-dependent manner. The action of C2-ceramide was inhibited by wortmannin and LY 294002, indicating that C2-ceramide-induced PKC zeta increase in both nucleus and membrane fractions is mediated by phosphatidylinositol 3-kinase (PI3-kinase) activation. In addition, a significant translocation of PI3-kinase to the nucleus was observed after C2-ceramide treatment.
Asunto(s)
Núcleo Celular/metabolismo , Hepatocitos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Androstadienos/farmacología , Animales , Membrana Celular/metabolismo , Separación Celular , Cromonas/farmacología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Hepatocitos/química , Hepatocitos/efectos de los fármacos , Isoenzimas/metabolismo , Masculino , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Ratas Wistar , Esfingosina/antagonistas & inhibidores , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , WortmaninaRESUMEN
Sphingosylphosphorylcholine (SPC) induces a rapid increase of intracellular Ca2+ concentration in isolated synaptosomes. This effect is dose-dependent and is also dependent on extracellular Ca2+. Sphingosine (SPH) has a smaller effect and treatment with psychosine (PSY) is ineffective, which suggests that phosphorylation of the 1-carbon of SPH is required for the SPC to act as a Ca2+ release agonist in synaptosomes. Experiments performed in the presence of heparin or ryanodine indicate that SPC-elicited Ca2+ release is not mediated by IP3 or ryanodine receptors. Finally, our results show that the effect of SPC on Ca2+ concentration is nimodipine-sensitive, suggesting that SPC possibly activates a specific sphingolipid-gated Ca2+ channel in synaptosomes.
Asunto(s)
Calcio/metabolismo , Fosforilcolina/farmacología , Esfingolípidos/farmacología , Esfingosina/farmacología , Sinaptosomas/metabolismo , Animales , Anticoagulantes/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Heparina/farmacología , Nimodipina/farmacología , Fosforilcolina/análogos & derivados , Psicosina/farmacología , Ratas , Rianodina/farmacología , Esfingosina/análogos & derivados , Sinaptosomas/efectos de los fármacosRESUMEN
When isolated rat liver nuclei were treated with platelet-activating factor (PAF), a rapid increase in the mass of diacylglycerol (DAG) occurred. This effect was dose- and time-dependent. The maximum effect was observed after 1 min of 10(-7) M PAF treatment. A concomitant decrease of polyphosphoinositides and phosphatidic acid (PA) levels was observed. PAF-induced DAG accumulation was inhibited by the treatment with WEB 2086 or PCA-4248, specific PAF-receptor antagonists. This result may suggest that PAF exerts its action in the nucleus through specific nuclear PAF binding sites. The findings described herein are due to the activation of phospholipase C, as the results from experiments using U73122, a phospholipase C inhibitor, indicate. These are the first data on the action of
Asunto(s)
Núcleo Celular/efectos de los fármacos , Diglicéridos/biosíntesis , Hepatocitos/metabolismo , Fosfatidilinositoles/metabolismo , Factor de Activación Plaquetaria/farmacología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Adenosina Trifosfato/metabolismo , Animales , Núcleo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Hidrólisis , Masculino , Glicoproteínas de Membrana Plaquetaria/metabolismo , Ratas , Ratas Wistar , Factores de Tiempo , Fosfolipasas de Tipo C/metabolismoRESUMEN
In this study we have analyzed the distribution of protein kinase C isoforms in cytosol, membrane, and nucleus in HL60 cells. Furthermore, we have studied the redistribution of these isoforms after cyclic AMP treatment. Protein kinase C localization and cyclic AMP-induced translocation was demonstrated by Western blot analysis. Cytosol, membrane and nucleus in HL60 cells expressed the abundance of protein kinase C alpha, betaI, betaII, delta, lambda, and zeta isoforms. After cyclic AMP treatment, the amount of protein kinase C betaI and zeta increased only in the nucleus, while protein kinase C delta increased in the three fractions tested. These effects were dependent on the cyclic AMP concentration and duration of action. Our results suggest the existence of cross-talk between the cyclic AMP system and protein kinase C in HL60 cells. Taking into account the processes regulated by protein kinase C, these findings also suggest that cyclic AMP plays a regulatory role in various cellular responses in HL60 cells, such as differentiation and gene expression. The increase observed in PKC delta was due to cyclic AMP-dependent protein kinase C activation, and the synthesis of enzyme was probably activated by the nucleotide.
Asunto(s)
Núcleo Celular/enzimología , AMP Cíclico/farmacología , Citosol/enzimología , Membranas Intracelulares/enzimología , Proteína Quinasa C/metabolismo , Transporte Biológico , Compartimento Celular , Células HL-60 , Humanos , Isoenzimas/metabolismo , Transducción de SeñalRESUMEN
The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat brain slices. PAF induced a time- and concentration-dependent increase in tyrosine phosphorylation of a doublet of approximately 125 kDa. These proteins were identified by immunoprecipitation as p125(FAK) and p130(Cas), using monoclonal antibodies. This effect was mediated by PAF receptors, as shown by its inhibition by the action of a PAF antagonist. The tyrosine phosphorylation evoked by PAF was dependent, at least in part, on external calcium. The involvement of protein kinase C was demonstrated by the synergistic effect of TPA on PAF-stimulated tyrosine phosphorylation. The finding that PAF stimulates tyrosine phosphorylation of both focal adhesion protein p125(FAK) and p130(Cas) suggests that PAF might modulate the integrin mediated signal transduction in the brain.
Asunto(s)
Encéfalo/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Fosfoproteínas/metabolismo , Factor de Activación Plaquetaria/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas , Animales , Encéfalo/metabolismo , Proteína Sustrato Asociada a CrK , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Técnicas In Vitro , Fosforilación , Pruebas de Precipitina , Ratas , Proteína p130 Similar a la del Retinoblastoma , Estimulación QuímicaRESUMEN
Sphingosylphosphorylcholine (SPC) caused a rapid increase of Ca2+ concentration in isolated brain nuclei. This effect was prevented by nimodipine, an inhibitor of L-type Ca2+ channels, and by thapsigargin, an inhibitor of Ca(2+)-ATPase. Neither heparin nor U73122 modified this effect, suggesting that phospholipase C activation and inositol 1,4,5-trisphosphate (IP3) production are not involved. Results also indicated that SPC-induced increase in Ca2+ concentration is not protein kinase C-dependent.
Asunto(s)
Encéfalo/metabolismo , Calcio/metabolismo , Núcleo Celular/metabolismo , Fosforilcolina/análogos & derivados , Esfingosina/análogos & derivados , Animales , Encéfalo/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Núcleo Celular/efectos de los fármacos , Estrenos/farmacología , Heparina/farmacología , Técnicas In Vitro , Cinética , Nimodipina/farmacología , Fosforilcolina/farmacología , Pirrolidinonas/farmacología , Ratas , Esfingosina/farmacología , Tapsigargina/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The sphingolipids, sphingosine (SPH), sphingosylphosphorylcholine (SPC) and psycosine induce a rapid and transient rise in nuclear free Ca2+ concentration in a dose dependent manner. To determine whether these sphingolipids act by a IP3-dependent pathway, we tested the increase of Ca2+ in the presence of heparin, an antagonist of IP3 receptor or U70122, an inhibitor of phospholipase C. Results indicate that the effect of both SPH and SPC, but not that of psychosine, is partially mediated by IP3 production. The sphingolipid-induced Ca2+ mobilization was unaffected by the inhibition of protein kinase C, but was totally abolished in the presence of nimodipine, a L-type Ca2+ channel inhibitor. The results could indicate the existence of a sphingosine-gated Ca2+-permeable channel in liver nuclei.
Asunto(s)
Calcio/metabolismo , Núcleo Celular/metabolismo , Hígado/metabolismo , Esfingolípidos/farmacología , Animales , Colorantes Fluorescentes , Fura-2 , Hígado/ultraestructura , Masculino , Ratas , Ratas WistarRESUMEN
Arachidonic acid treatment in isolated liver nuclei resulted in a rapid and transient increase of Ca2+ concentration in the nucleoplasm which was monitored with the Ca(2+)-sensitive dye fura-2 dextran. This effect was associated with a decrease of Ca2+ concentration in the nuclear envelope as measured with fura-2 AM. Our results indicate that arachidonic acid causes a Ca2+ release from the nuclear envelope to the nucleoplasm similar to that evoked by inositol trisphosphate (IP3). The arachidonic acid-induced Ca2+ mobilization in the nucleus was not due to the metabolites of arachidonic acid. Experiments performed in the presence of ATP and Ca2+ indicate that arachidonic acid-induced Ca2+ mobilization in the nucleus takes place in a non ATP-dependent way. Taken together, these results suggest that arachidonic acid may contribute to the regulation of nuclear Ca2+ mobilization.
Asunto(s)
Ácido Araquidónico/farmacología , Calcio/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Ácido Araquidónico/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Fura-2 , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/farmacología , Transporte Iónico/efectos de los fármacos , Masculino , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/metabolismo , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
The action of endothelin-1 (ET-1) on phosphoinositide metabolism was studied in rat synaptosomes. ET-1 caused an early and transitory decrease of 32P incorporation into phosphoinositides, concomitantly with an increase into phosphatidic acid (PA). This effect was time-dependent and was not found in the absence of exogenous calcium. Furthermore, ET-1 caused an increase in the generation of inositol phosphates and diacylglycerol (DAG). In addition, the peptide provoked a translocation of protein kinase C from the cytosol to membrane.
Asunto(s)
Endotelina-1/farmacología , Fosfatidilinositoles/metabolismo , Proteína Quinasa C/metabolismo , Sinaptosomas/metabolismo , Animales , Calcio/farmacología , Membrana Celular/metabolismo , Corteza Cerebral , Citoplasma/metabolismo , Diglicéridos/metabolismo , Fosfatos de Inositol/metabolismo , Fosfatos/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolípidos/metabolismo , RatasRESUMEN
We have suggested that substance P, in cerebral cortex, causes a phosphatidylinositol (PI) breakdown by a dual mechanism suggesting the involvement of either phospholipase A2 or phospholipase C. We have presently characterized further these effects. Substance P (65 pM) provoked an increase in lysoPI concomitant with a decrease in PI level. This finding confirms the involvement of phospholipase A2 activation. To study the involvement of phospholipase C in the action of higher doses (0.65 microM) of the peptide, we used pulse-chase experiments (where phospholipid depletion was monitored) and short-term 32P-labeled slices (where phospholipid synthesis was studied). Substance P evoked an acceleration of both hydrolysis and resynthesis of PI as early as 15 s. A prolonged exposure (30 min) resulted in stimulation of PI hydrolysis without subsequent resynthesis. The peptide did not cause any effect on inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. These alterations in PI metabolism take place simultaneously with a generation of diacylglycerol which showed two maxima at both indicated times.
Asunto(s)
Encéfalo/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Sustancia P/farmacología , Animales , Encéfalo/metabolismo , Hidrólisis , Técnicas In Vitro , Masculino , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacosRESUMEN
In the present study, experiments were conducted to determine the effect of platelet-activating factor (PAF) on (Na+,K+)-ATPase in rat cerebral cortex. PAF, but not lysoPAF, inhibited (Na+,K+)ATPase activity, in a dose- and time-dependent manner, 10(-7) to 10(6) M being the most effective dose. These effects were abolished in the presence of PCA-4248, a PAF antagonist, indicating that the PAF effect may be mediated by its specific membrane receptors. Omission of external calcium caused an increase in the basal activity and abolished the PAF effect on (Na+,K+)ATPase. The present study demonstrates that PAF inhibits (Na+,K+)ATPase activity in the cerebral cortex and suggests that PAF released during certain pathological conditions, such as ischemia, may act on ATPase. This could be one possible mechanism of PAF action that needs further attention.
Asunto(s)
Encéfalo/enzimología , Factor de Activación Plaquetaria/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Encéfalo/efectos de los fármacos , Calcio/fisiología , Dihidropiridinas/farmacología , Ácido Egtácico/farmacología , Técnicas In Vitro , Masculino , Factor de Activación Plaquetaria/análogos & derivados , Factor de Activación Plaquetaria/antagonistas & inhibidores , Ratas , Ratas WistarRESUMEN
In rat brain microvessels, tetrahydroaminoacridine (THA) caused a significant inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities, in a dose-dependent manner. THA resulted to be a more potent inhibitor of BuChE than AChE. Lineweaver-Burk plots showed that Km (app) and V were altered by THA, indicating mixed competitive/non-competitive inhibition. The results of the present study also established that the three molecular forms of BuChE (G1, G2 and G4), recently described to be present in brain microvessels, are inhibited after THA treatment.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Tacrina/farmacología , Acetilcolinesterasa/efectos de los fármacos , Animales , Butirilcolinesterasa/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Microcirculación/enzimología , Ratas , Factores de TiempoRESUMEN
The effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on phosphoinositide metabolism in cerebral microvessels were examined. Treatment of microvessels with TPA evoked a dose-dependent increase in the 32P-orthophosphate incorporation into polyphosphoinositides, phosphatidic acid (PA) and phosphatidylcholine (PC). The effect on PC was found only after a lag period. Experiments with membranes isolated from microvessels indicated a TPA-induced activation of phosphoinositide kinases. Evidence that this effect was mediated by protein kinase C (PKC) activation was provided by the reversal of the effect in the presence of staurosporine. Concomitantly with these observations, an increase of diacylglycerol (DAG) production was evoked without formation of inositol phosphates. Therefore, we suggest that TPA also stimulates PC metabolism. These results support a regulatory role for PKC in phospholipid signaling pathways in the blood-brain barrier (BBB).
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/irrigación sanguínea , Fosfatidilcolinas/metabolismo , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol) , Acetato de Tetradecanoilforbol/farmacología , Animales , Diglicéridos/metabolismo , Técnicas In Vitro , Masculino , Microcirculación/efectos de los fármacos , Microcirculación/metabolismo , Fosforilación , Fosfotransferasas/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Ratas EndogámicasRESUMEN
(Na+ + K+)ATPase activity in cerebral cortex was modulated by insulin action depending on the Mg2+ concentration. Thus, in homogenates in the presence of 1-3 mM Mg2+, insulin stimulated the enzyme, whereas in the presence of 4-6 mM Mg2+ inhibition was observed. Exposure of synaptosomal membranes to the soluble fraction resulted in inhibition of ATPase activity in a dose-dependent manner. The inhibitory effect of insulin was regulated by a cytoplasmic factor in a dose-dependent manner. Similar variations to those obtained with a crude synaptosomal fraction were obtained by using a partially purified ATPase. These results indicated the importance of soluble factors in the modulation of ATPase by insulin and add more evidence in support for a role of insulin as a neuromodulator.
Asunto(s)
Corteza Cerebral/enzimología , Citoplasma/fisiología , Insulina/farmacología , Magnesio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Sinaptosomas/enzimología , Animales , Relación Dosis-Respuesta a Droga , Cinética , Masculino , Ratones , Sinaptosomas/efectos de los fármacosRESUMEN
The action of platelet-activating factor (PAF) on phosphoinositide hydrolysis was studied in rat brain slices. PAF produced a significant increase of 32P incorporation into phosphoinositides and phosphatidic acid (PA), in a dose- and time-dependent manner. Concomitantly, an increase of inositol phosphates and diacylglycerol (DAG) production was observed. Both inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were detected as early as 5 s and they returned immediately to basal levels; concomitantly, formation of inositol monophosphate (IP) was detected. These findings demonstrated that PAF causes a rapid hydrolysis of polyphosphoinositides in cerebral cortex by a phospholipase C-dependent mechanism followed by subsequent resynthesis.
Asunto(s)
Corteza Cerebral/enzimología , Fosfatidilinositoles/metabolismo , Factor de Activación Plaquetaria/farmacología , Fosfolipasas de Tipo C/metabolismo , Animales , Diglicéridos/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Técnicas In Vitro , Fosfatos de Fosfatidilinositol , Ratas , Fosfolipasas de Tipo C/efectos de los fármacosRESUMEN
Vanadate, over a concentration range from 0.1 to 0.5 mM, stimulated the incorporation of (32P)-orthophosphate into PI and PA in brain microvessels. At concentrations higher than 0.5 mM, the stimulatory effect of vanadate decreased. Concommitantly, an enhanced DAG production was observed, indicating that vanadate stimulated PI turnover. All these effects were evident at all the times tested. Experiments performed in the presence of pertussis toxin (IAP) indicated that a IAP-sensitive G-protein does not mediate the vanadate stimulated PI effect in brain microvessels.
Asunto(s)
Encéfalo/irrigación sanguínea , Toxina del Pertussis , Fosfatos/metabolismo , Fosfatidilinositoles/metabolismo , Vanadatos/farmacología , Factores de Virulencia de Bordetella/farmacología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Diglicéridos/metabolismo , Relación Dosis-Respuesta a Droga , Hidrólisis , Masculino , Microcirculación/metabolismo , Ácidos Fosfatidicos/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The effect of insulin on phosphoinositide metabolism in the cerebral cortex was examined using 32P as precursor. A maximal increase was detected as early as 15 s; phospholipid labeling declined after this initial peak but then increased to another maximum at 30 min. The levels of these phospholipids were unchanged at the earliest time examined, but at 30 min insulin caused an increase in the content of all phospholipids tested. In pulse-chase experiments, insulin stimulated depletion of 32P-labeled phosphoinositides only at 15 s. On the other hand, insulin treatment caused a biphasic diacyglycerol (DAG) production. We conclude that in cerebral cortex, insulin has a dual mechanism of action on phosphoinositide metabolism. First, insulin causes a rapid but transient hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism, followed by subsequent resynthesis; thereafter, insulin increases de novo phospholipid synthesis.
Asunto(s)
Encéfalo/metabolismo , Insulina/farmacología , Fosfatidilinositoles/metabolismo , Animales , Encéfalo/efectos de los fármacos , Diglicéridos/metabolismo , Masculino , Fosfolípidos/metabolismo , Ratas , Ratas Endogámicas , Fosfolipasas de Tipo C/metabolismoRESUMEN
The treatment of cerebral cortex slices with substance P caused alterations in the phospholipid levels. A significant loss of phosphatidylinositol in a dose-dependent manner was observed. In contrast, the levels of the major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were enhanced by the peptide. The effect of substance P on the fatty acid composition of phospholipids was also studied. The most relevant event was the decrease in the content of both stearic and arachidonic acids of phosphatidylinositol. This decrease was more evident at the lowest substance P concentration tested (65 pM). These results are consistent with the phosphatidylinositol breakdown caused by substance P in some tissues. Furthermore, our data indicate that this breakdown is selective depending on the peptide dose. Thus, in the presence of very low doses of substance P (65 pM) a preferential degradation of 1-acyl(predominantly stearoyl)-2-arachidonoylglycerophosphoinositol molecular species occurs, whereas high doses of the peptide (0.65 microM) induce a generalized hydrolysis of phosphatidylinositol without showing any preference towards molecular species rich in arachidonic acid. Hence we describe for the first time a dual, dose-dependent mechanism for phosphatidylinositol hydrolysis by substance P, suggesting the possibility that either phospholipase A2 or phospholipase C activation is involved.
Asunto(s)
Corteza Cerebral/metabolismo , Fosfatidilinositoles/metabolismo , Sustancia P/farmacología , Animales , Ácidos Grasos/metabolismo , Hidrólisis , Técnicas In Vitro , Masculino , Ratas , Ratas EndogámicasRESUMEN
The effects of insulin on brain alkaline phosphatase activity have been examined. Insulin inhibited the activity of alkaline phosphatase on brain microvessels in in vitro experiments. The inhibition observed was of the non-competitive type. These observations indicate that the hormone is able to induce neurochemical modifications revealed in this case as changes in the phosphate transfer enzymes in brain microvessels.