摘要:

Abstract:Recent advances in our knowledge of the blood–brain barrier (BBB) have in part been made by studying the properties and function of cerebral endothelial cells in vitro. After an era of working with a fraction, enriched in cerebral microvessels by centrifugation, the next generation of in vitro BBB model systems was introduced, when the conditions for routinely culturing the endothelial cells were established. This review summarizes the results obtained from this rapidly growing field. It can be stated with certainty that, in addition to providing a better insight into the chemical composition of cerebral endothelial cells, much has been learned from these studies about the characteristics of transport processes and cell‐to‐cell interactions during the last 12 years. With the application of new technologies, the approach offers a new means of investigation, applicable not only to biochemistry and physiology but also to the drug research, and may improve the transport of substances through the BBB. The in vitro approach has been and should remain an excellent model of the BBB to help unravel the complex molecular interactions underlying and regulating the permeability of the cerebral endoth

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09272.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The role that inositol lipids play in cellular signaling events in eukaryotic cells remains one of the most intensively investigated areas of cell biology. In this respect, phosphoinositide‐mediated signal transduction in the CNS is no exception; major advances have been made since a previous review on this subject (Fisher and Agranoff, 1987). Not only have stimulated phosphoinositide turnover and its physiological sequelae been demonstrated repeatedly in a variety of neural preparations, but, in addition, the detailed molecular mechanisms underlying these events continue to unfold. Here we review the progress that has occurred in selected aspects of this topic since 1987. In the first two sections of this article, emphasis is placed on novel functional roles for the inositol lipids and on recent insights into the molecular characteristics and regulation of three key components of the phosphoinositide signal transduction system, namely, the inositol lipid kinases, phospholipases C (PLCs), and the inositol 1,4,5‐trisphosphate[I(1,4,5)P3] receptor. The metabolic fate of I(1,4,5)P3in neural tissues, as well as its control, is also detailed. Later we focus on identification of the multiple receptor subtypes that are coupled to inositol lipid turnover and discuss possible strategies for intervention into phosphoinositide‐mediated signal transduction. Due to space limitations, an extensive evaluation of the diacylglycerol/protein kinase C (DAG/PKC) limb of the signal transduction pathway is not included (for reviews, see Nishizuka, 1988; Kanoh et al.,

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09273.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The presence of benzodiazepine binding sites in rat vas deferens was detected using [3H]Ro 5‐4864 as a radioligand. The binding of [3H]Ro 5‐4864 to the mitochondrial sites is saturable, reversible, and temperature and time dependent. The association rate constant (k1) was 8.7 ± 0.7 × 107M−1min−1, and the dissociation rate constant (k‐1) was 0.031 ± 0.003 min−1. The dissociation constant (KD) determined by saturation binding was 5.22 ± 0.56 nM. The density of binding was 4,926 ± 565 fmol/mg of protein. The Hill coefficient of binding was 0.99 ± 0.01, an indication that [3H]Ro 5‐4864 binds to a single site. The [3H]Ro 5‐4864 binding was inhibited competitively by Ro 5‐4864 and 2‐hydroxy‐5‐nitrobenzyl‐6‐thioguanosine and noncompetitively by PK 11195, nitrendipine, α,β‐methylene‐ATP, and carboxyatractyloside and was not affected by clonazepam, dicyclohexylcarbodiimide, or protoporphyrin IX. Our data indicate that [3H]Ro 5‐4864 binding sites are not identical to those labeled by PK 11195. These binding sites are modulated by the ADP/ATP mitochondrial carrier, and an interaction of dihydropyridines and [3H]Ro 5‐4864 bin

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09274.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The neuroblastoma line SK‐N‐SH consists of distinct and interconverting cell types, which include a neuroblast phenotype (SH‐SY5Y), an epithelial phenotype (SH‐EP), and an intermediate cell type (SH‐IN). In SH‐SY5Y cells, only muscarinic receptor activation produced stimulation of phosphoinositide turnover, whereas in SH‐EP cells, where muscarinic receptors are not present, the peptides bradykinin, endothelin, and angiotensin II stimulated phosphoinositide hydrolysis with EC50values of 16, 6, and 0.7 nM, respectively, and a rank order of maximal effects of brady‐kinin>endothelin>angiotensin II. Fetal calf serum at concentrations between 1 and 10% was also a potent stimulator of phosphoinositide hydrolysis in SH‐EP cells but not in SH‐SY5Y cells. In the intermediate cell clone, SH‐IN, phosphoinositide hydrolysis was stimulated not only by muscarinic receptors, but also by endothelin, bradykinin, and serum, an indication that this cell type harbors all the kinds of receptors that are differentially expressed in the other two cell types. The effects of the three peptides—bradykinin, endothelin, and angiotensin II—on phosphoinositide hydrolysis in SH‐EP cells were additive, a result suggesting that the three kinds of receptors may activate distinct transducer proteins and/or phospholipase C subtypes. Pretreatment of intact SH‐EP cells with pertussis toxin under conditions sufficient to ADP‐ribosylate 90–95% of the endogenous guanine nucleotide regulatory protein substrates did not impair the ability of any of the receptors to stimulate phosphoinositide hydrolysis in any of the cell types. In contrast, short‐term exposure to the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (1 μM) abolished the stimulation of phosphoinositide hydrolysis mediated by peptide receptors in SH‐EP cells and partially inhibited that by muscarinic receptors in SH‐SY5Y cells. Prolonged incubation of SH‐EP cells with phorbol ester resulted in a recovery of receptor responsiveness, the extent and rate of which were different for each receptor type. In contrast, there was no recovery of responsiveness for muscarinic receptors in SH‐SY5Y cells. The pattern of phorbol ester‐mediated effects depended on the cell rather than on the receptor type. In fact, muscarinic receptor responsiveness in SH‐IN, the intermediate cell type, was desensitized by and recovered from treatment with phorbol esters in a manner more similar to peptide receptors in SH‐EP than to muscarinic receptors in SH‐SY5Y. These data suggest that the transduction mechanisms by which distinct receptor types are coupled to phosphoinositide hydrolysis in the three cell phenotypes differ

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09275.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with32Piproduced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased32P incorporation into TH. At 10 Hz, the phosphorylation of Ser19and Ser40was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300 μMtetrahydrobiopterin. Nicotine (3 μM, 3 min) increased Ser19phosphorylation, vasoactive intestinal polypeptide (10 μM, 3 min) increased Ser40phosphorylation, and muscarine (100 μM, 3 min) increased TH phosphorylation primarily at Ser19and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulat

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09276.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The effect of unilateral labyrinthectomy followed by the process of vestibular compensation on the incorporation of radioactive phosphate into frog brain proteins was investigated. Phosphoproteins were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by autoradiography. The present data show that unilateral labyrinthectomy affects the incorporation of32P into various frog brain proteins. In particular, the phosphorylation of a 20‐kDa protein appeared enhanced during early stages of vestibular compensation (4–12 days). This 20‐kDa protein was shown to be immunologically related to myelin basic protein and its phosphorylation was regulated by an endogenous calcium/calmodulin‐dependent protein kinase. These data might indicate that in addition to neuronal components, components of glial origin are also involved in biochemical events that lead to functional recovery after neurona

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09277.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Antibodies raised against the synthetic peptide NH2‐QKSDDDYEDYASNKTC‐COOH (γ2 1–15 Cys), which corresponds to the N‐terminal amino acid sequence with a C‐terminal cysteine of the human γ2 subunit of the γ‐aminobutyric acidA(GABAA) receptor, were used to study the quantitative immunoprecipitation of agonist benzodiazepine binding sites from bovine brain. Anti‐γ2 1–15 Cys antibodies were found to immunoprecipitate specifically in parallel [3H]flunitrazepam‐ and [3H]muscimol‐reversible binding sites in a dose‐dependent manner. The maximum percentages of [3H]flunitrazepam binding sites immunoprecipitated from detergent extracts of bovine cerebral cortex, cerebellum, and hippocampus were 68, 77, and 83%, respectively. Immunoprecipitation studies with anti‐α1 324–341 antibodies carried out in parallel with anti‐γ2 1–15 Cys antibodies provided evidence for the promiscuity of the γ2 subunit within native GABAAreceptors. These results substantiate the association of the γ2 p

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09278.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Neuraminidase activities in oligodendroglial cells were characterized using rats of different ages. Rat oligodendroglial cells had intrinsic neuraminidase activities directed toward GM3 andN‐acetylneuramin(2–3)lactitol (NL). Developmental profiles of the neuraminidase activities toward the two substrates in oligodendroglial cells were different from each other. The neuraminidase activity toward GM3 increased rapidly with the onset of active myelination and, after 26 days of development, reached the adult level which was about 18 times higher than that in myelin. At the adult age, oligodendroglial cells had the highest neuraminidase activity toward GM3 among the individual brain cell types examined. The activity of NL‐neuraminidase showed a less remarkable developmental profile, with a peak value at 26 days. The UDP‐galactose:ceramide galactosyltransferase activity in oligodendroglial cells increased during the period of active myelination and, afterward, returned to the basal level. The enrichment and unique developmental profile in oligodendroglial cells of the neuraminidase activity toward GM3 suggest that this enzyme may play an important role in the formation and maintenance of the myelin

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09279.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The role of myelin‐associated neuraminidase in ganglioside metabolism was examined using rats of ages ranging from 17 to 97 days. The neuraminidase activity directed toward the ganglioside GM3 in the total myelin fraction was high during the period of active myelination and, thereafter, decreased rapidly to the adult level. The ganglioside composition became simpler during development with an increasing amount of GM1 and decreasing percentages of di‐and polysialogangliosides. The decrease in the proportion of GD1a was most prominent, whereas relative amounts of GD1b and GT1b increased transiently before reducing to the adult levels. The heavy myelin subfraction contained higher percentages of di‐and polysialo‐species compared to the light myelin fraction at young and adult ages. The in vitro incubation of myelin of young rats under an optimal condition for neuraminidase action produced a profile of ganglioside changes similar to that observed in in vivo development. These results strongly suggest that myelin‐associated neuraminidase may play a pivotal role in the developmental changes in the ganglioside composition of rat bra

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09280.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Cerebrospinal fluid (CSF) from 20 male patients with nonneurologic disease (age 64.5 ± 2.8 SEM) was analyzed for the presence of the serpin α1‐antichymotrypsin (α1‐ACT). A chymotrypsin‐specific chromogenic substrate (succinyl‐Ala‐Ala‐Pro‐Phe‐p‐nitroanilide) was used to examine the CSF samples. All CSF samples showed inhibitory activity ranging from 45 to 80% inhibition. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of the samples revealed the presence of a 68‐kDa protein migrating identical to authentic human plasma α1‐ACT. Complex formation was performed with iodinated bovine chymotrypsin for several representative CSF samples having the highest chymotrypsin inhibitory activity. Comparison was made with complex formation performed with commercially available authentic human plasma α1‐ACT. These studies showed the formation of complexes at 37°C, regardless of whether the sample was subsequently boiled or not. In the case of CSF, two complex bands, mass smaller than with plasma α1‐ACT, were formed at the lower temperature whereas a single higher Mrband was formed when the samples were boiled. To determine whether cleavage of the serpin occurred, these studies were repeated using human neutrophil cathepsin G as target protease. A complex of approximately 90 kDa was formed with human α1‐ACT under these same conditions. α1‐ACT has been detected in senile amyloid plaques in brains of Alzheimer's disease patients, the only plasma serine protease inhibitor localized to these structures. Another serpin, protease nexin I, is also found in these plaques, but this inhibitor does not circulate in plasma. Recently, the β‐amyloid precursor protein itself has been identified as another serine protease inhibitor, protease nexin II, known to form complexes with bovine chymotrypsin as well as with the epidermal growth factor‐binding protein. Protease nexin II does circulate in plasma; however, complexes of this inhibitor with chymotrypsin are twice as large as the complexes this protease forms with plasma α1‐ACT or with the CSF samples. Using antisera to the β‐amyloid precursor protein, other workers have detected both 125‐kDa and 105‐kDa proteins in human CSF, the 125 kDa reportedly detected by antisera against the Kunitz serine protease inhibitor‐containing domain. The studies with α1‐ACT in CSF should now allow us to evaluate this serpin and its function in CSF of pa

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb09281.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY