Serum Prolidase Activity as a Biomarker for Choroid Plexus Calcification.

The choroid plexus (CP) performs multiple functions such as secretion and reabsorption. CP also acts as the blood-cerebrospinal fluid barrier. Prolidase plays an important role in collagen metabolism by degrading imidodipeptides, in which proline or hydroxyproline residue is located at the C-terminal end. Serum prolidase activity (SPA) may reflect the degree of fibrosis and inflammation. Choroid plexus calcification (CPC) is considered as the physiological calcification of the brain, and CPC is diagnosed by the presence of calcification in the anatomical region on computed tomography (CT). Here, CPC and non-calcified CP were defined by Hounsfield Units (HU) values of > 150 and < 50, respectively. We aimed to measure SPA in subjects with CPC and those with non-calcified CP. This study included 89 subjects who were admitted to the neurology clinic and underwent CT: 44 subjects with CPC and 45 subjects with non-calcified CP. The neurological examination of all subjects was normal; namely, the subjects with CPC were asymptomatic. The SPA level was significantly higher in the CPC group than that in the non-calcified CP group (p < 0.002), and there was a significant positive correlation between vitamin D and SPA levels in the CPC group. In contrast, the vitamin D and parathyroid hormone levels were higher in the CPC group, but the difference was not statically significant (p > 0.05). These findings indicate that SPA is a biomarker for CPC that may be predictive of future brain disease.

Fibronectin changes in eosinophilic meningitis with blood-CSF barrier disruption.

Fibronectin, which is present at relatively low levels in healthy central nervous systems (CNS), shows increased levels in meningitis. In this study, fibronectin processing was correlated with the increased permeability of the blood-cerebrospinal fluid (CSF) barrier as well as with the formation of eosinophil infiltrates in angiostrongyliasis meningitis. The immunohistochemistry results show matrix metalloproteinase-9 (MMP-9) is localized in the choroid plexus epithelium. Coimmunoprecipitation demonstrated fibronectin strongly binds MMP-9. Furthermore, treatment with the MMP-9 inhibitor GM6001 significantly inhibited fibronectin processing, reduced the blood-CSF barrier permeability, and decreased the eosinophil counts. The decreased fibronectin processing in CSF implies decreased cellular invasion of the subarachnoid space across the blood-CSF barrier. Therefore, increased fibronectin processing may be associated with barrier disruption and participate in the extravasation and migration of eosinophils into the CNS during experimental parasitic infection.

Antenatal betamethasone exposure is associated with lower ANG-(1-7) and increased ACE in the CSF of adult sheep.

Antenatal betamethasone (BM) therapy accelerates lung development in preterm infants but may induce early programming events with long-term cardiovascular consequences. To elucidate these events, we developed a model of programming whereby pregnant ewes are administered BM (2 doses of 0.17 mg/kg) or vehicle at the 80th day of gestation and offspring are delivered at term. BM-exposed (BMX) offspring develop elevated blood pressure; decreased baroreflex sensitivity; and alterations in the circulating, renal, and brain renin-angiotensin systems (RAS) by 6 mo of age. We compared components of the choroid plexus fourth ventricle (ChP4) and cerebral spinal fluid (CSF) RAS between control and BMX male offspring at 6 mo of age. In the choroid plexus, high-molecular-weight renin protein and ANG I-intact angiotensinogen were unchanged between BMX and control animals. Angiotensin-converting enzyme 2 (ACE2) activity was threefold higher than either neprilysin (NEP) or angiotensin 1-converting enzyme (ACE) in control and BMX animals. Moreover, all three enzymes were equally enriched by approximately 2.5-fold in ChP4 brush-border membrane preparations. CSF ANG-(1-7) levels were significantly lower in BMX animals (351.8 ± 76.8 vs. 77.5 ± 29.7 fmol/mg; P < 0.05) and ACE activity was significantly higher (6.6 ± 0.5 vs. 8.9 ± 0.5 fmol·min(-1)·ml(-1); P < 0.05), whereas ACE2 and NEP activities were below measurable limits. A thiol-sensitive peptidase contributed to the majority of ANG-(1-7) metabolism in the CSF, with higher activity in BMX animals. We conclude that in utero BM exposure alters CSF but not ChP RAS components, resulting in lower ANG-(1-7) levels in exposed animals.

ATP7A gene addition to the choroid plexus results in long-term rescue of the lethal copper transport defect in a Menkes disease mouse model.

Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment (daily subcutaneous copper injections) is not entirely effective in the majority of affected individuals. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age. We documented that mo-br mice on C57BL/6 background were not rescued by peripheral copper administration, and used this model to evaluate brain-directed therapies. Neonatal mo-br mice received lateral ventricle injections of either adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A) complementary DNA (cDNA), copper chloride, or both. AAV5-rsATP7A showed selective transduction of choroid plexus epithelia and AAV5-rsATP7A plus copper combination treatment rescued mo-br mice; 86% survived to weaning (21 days), median survival increased to 43 days, 37% lived beyond 100 days, and 22% survived to the study end point (300 days). This synergistic treatment effect correlated with increased brain copper levels, enhanced activity of dopamine-ß-hydroxylase, a copper-dependent enzyme, and correction of brain pathology. Our findings provide the first definitive evidence that gene therapy may have clinical utility in the treatment of Menkes disease.

[3beta-Hydroxysteroid dehydrogenase in ependimocytes of brain lateral ventricle lining and vascular plexus villi in rats of different age].

Using the histochemical method, the activity of 3beta-hydroxysteroid dehydrogenase (HSDH) was studied in the brain of laboratory male albino rats of different age groups: 5-6 days (n = 6), 45-50 days (n = 12), and 6 months (n = 15). The quantitative assessment of reaction intensity was performed with the cytospectrophotometer. The results obtained indicate that the ependimocytes lining the brain lateral ventricles and covering the villi of their vascular plexuses are characterized by the presence of HSDH activity typical to that of steroid-producing cells. In this regard ependimocytes may be attributed to the cells that can produce neurosteroids. It was established that HSDH activity in ependimocytes was minimal in the early postnatal period and considerably increased by the prepuberty period, remaining at this level in adult animals.

Beta-adrenergic-sensitive adenylate cyclase in secretory cells of choroid plexus.

Biochemical evidence supporting the sympathetic control of cerebrospinal fluid production has been obtained through identification of a specific beta-adrenergic-sensitive adenylate cyclase in the choroid plexus. The enzyme, which is localized in the secretory epithelium, is activated by low concentrations of isoproterenol and norepinephrine and appears separate from beta-adrenergic-sensitive adenylate cyclase present in cerebral blood vessels.

Potential pathophysiological role of D-amino acid oxidase in schizophrenia: immunohistochemical and in situ hybridization study of the expression in human and rat brain.

D-Amino acid oxidase (DAO) is a peroxisomal flavoenzyme that catalyzes oxidative deamination of a wide range of D-amino acids. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) type glutamate receptor. The gene for DAO was reported to be associated with schizophrenia. Since DAO is expected to be one of the key enzymes in the regulation of NMDA neurotransmission, the modulation of the enzyme activity is expected to be therapeutical for neuronal disorders. In search of the pathophysiological role of DAO, we analyzed the distribution of DAO mRNA and protein in the rat and human brain. In rat, the distribution of DAO mRNA was newly detected in choroid plexus (CP) epithelial cells in addition to glial cells of pons, medulla oblongata, and especially Bergmann glia of cerebellum. Moreover, to investigate how DAO expression level is altered in schizophrenia, we performed immunohistochemistry in the human brain. In agreement with the results in the rat brain, the immunoreactivity for DAO was detected in glial cells of rhombencephalon and in CP. Furthermore, higher level of DAO expression was observed in schizophrenic CP epithelial cells than that in non-schizophrenic cases. These results suggest that an increase in DAO expression in parts of the brain is involved in aberrant D-amino acid metabolism. In particular, gene expression of DAO in CP suggests that DAO may regulate D-amino acid concentration by modulating the cerebrospinal fluid and may be regarded as a potential therapeutic target for schizophrenia.

Drug metabolizing enzyme expression in rat choroid plexus: effects of in vivo xenobiotics treatment.

The presence of drug metabolizing enzymes in extrahepatic tissues such as the choroid plexus (CP) suggests that the CP, like the blood-brain barrier, affords a metabolic protection to the brain against xenobiotics. The CP, which is the principal site of formation of the cerebrospinal fluid (CSF), controls the exchange of many endogenous compounds and exogenous molecules between brain tissue and CSF. We present the changes in mRNA expression and enzymatic activities of UDP-glucuronosyltransferase, UGT1A6 isoform and NADPH-cytochrome P450 reductase, after in vitro treatment with xenobiotic molecules known to act in the liver as inducers or inhibitors of these drug metabolizing enzymes. Five study groups of male Sprague-Dawley rats were treated separately with 3-methylcholantrene (3-MC), phenobarbital (PB), dexamethasone (DEX), cyclosporine (CsA) or paraquat (PQ). Choroidal 1-naphthol glucuronidation activities were significantly induced by 3-MC and PQ administration (354 +/- 85 and 257 +/- 49 vs. 115 +/- 24 nmol/h per mg protein, in control group), whereas the other molecules were without effect. Accordingly, UGT1A6 mRNA expression, measured by RT-PCR, was 2.3-fold higher after 3-MC treatment and 2.1-fold higher after PQ administration. By contrast, reductase activities and mRNA expression remained unchanged in the isolated choroids plexus in these experimental conditions. We present for the first time evidences that the choroids plexus express transcripts for both UGT1A6 and NADPH-cytochrome P450 reductase, and their mRNA expression can be differently regulated by exogenous factors. These results emphasize that xenobiotics could modulate the biotransformation of exogenous and/or endogenous compounds in the choroids plexus, and underline the role of UGTs in the maintenance of brain homeostasis.

Low levels of Na, K-ATPase and carbonic anhydrase II during choroid plexus development suggest limited involvement in early CSF secretion.

UNLABELLED: In the adult, cerebrospinal fluid (CSF) is produced by the actions of numerous transporters and enzymes creating ion gradients that drive the entry of water into the ventricles via the aquaporin-1 water channels (AQP1). It is not known when in development CSF secretion starts but, in the rat, it has been postulated to occur around the time of birth. However, recent evidence suggests that the secretion may start much earlier, as soon as the lateral choroid plexuses first appear (around E14). PURPOSE: To investigate the developmental profiles of two major enzymes responsible for CSF secretion in the adult, Na, K-ATPase (NKA) and carbonic anhydrase II (CAII). METHODS: The developmental profiles of both enzymes were investigated using immunohistochemistry and Western Blot analysis in tissue from embryonic day (E) 15, 18, postnatal day (P) 0, 9 and adult rats. RESULT: Western Blot analysis showed low levels of NKA at E15 followed by a progressive increase with age. Immunohistochemistry confirmed the presence of NKA on the apical membrane of the lateral ventricular choroid plexus epithelium from E15 onwards. Western Blot analysis of CAII was complicated by its presence in blood, but the amount of protein increased with age. Immunohistochemically, CAII appeared in the lateral ventricular choroid plexus between P0 and P9. CONCLUSIONS: The low levels of NKA and CAII during early choroid plexus development indicate that other mechanisms, such as the previously described specific protein transfer across epithelial cells, may be involved in early CSF secretion and movement of water into the cerebral ventricles.

Corticosteroids, 11beta-hydroxysteroid dehydrogenase isozymes and the rabbit choroid plexus.

The epithelial cells of the choroid plexus (CP) are responsible for cerebrospinal fluid (CSF) secretion into the ventricles of the brain. The balance between CSF production and drainage, in part, facilitates a normal intracranial pressure. The secretion of Na(+) and anions by the CP creates an osmotic gradient driving water into the ventricles. This is opposite to classical Na(+) transporting tissues, such as the kidney, where Na(+) and water reabsorption is mediated by 11beta-hydroxysteroid dehydrogenase type 2 that protects the mineralocorticoid receptor by abrogating active cortisol to inactive cortisone. In the human ocular ciliary epithelium, Na(+) and water secretion is dependent on a novel mediator of ciliary epithelial Na(+) transport, 11beta-HSD type 1 (11beta-HSD1), that generates intraocular cortisol. In a mechanism analogous to that of the embryologically related ocular ciliary epithelium, we propose that autocrine regulation of intracranial cortisol is dependent on 11beta-HSD1 expression in the CP epithelial cells. By conducting immunolocalisation studies on brains from New Zealand White Albino rabbits, we defined the expression of 11beta-HSD1 in the secretory CP epithelial cells. Enzyme assays performed on intact rabbit CP whole tissue explants confirmed predominant 11beta-HSD1 activity, generating cortisol that was inhibited by glycyrrhetinic acid (an 11beta-HSD inhibitor). Using the real time-polymerase chain reaction, rabbit CP tissue was found to express levels of 11beta-HSD1, glucocorticoid receptor alpha and serum and glucocorticoid-regulated kinase 1 mRNA comparable to that expressed in rabbit ocular ciliary body, thereby highlighting the similarity between these two tissues. Furthermore, an enzyme-linked immunosorbent assay of rabbit CSF revealed a median cortisol concentration of 1.7 nmol/l (range 1.4-4.3 nmol/l, n = 9). Our data have identified a functional 11beta-HSD1 within the CP, mediating intracranial cortisol bioavailability. Expression of 11beta-HSD1 may be fundamental in the regulation of CSF secretion and the local generation of cortisol may represent a pathophysiological mechanism underlying cortisol-dependent neuroendocrine diseases.

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase.

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.

Macromolecules involved in production and metabolism of beta-amyloid at the brain barriers.

One of the notable features of Alzheimer's disease (AD) is the overabundance of beta-amyloid peptides in brain fluids, leading to the formation and deposition of insoluble amyloid plaques. Previous work in this lab demonstrates that the normal choroid plexus, a primary component of the blood-cerebrospinal fluid barrier, has the capacity to remove beta-amyloid from the cerebrospinal fluid, potentially preventing the formation of beta-amyloid plaques. The purpose of this work was to determine whether the choroid plexus and/or the brain capillaries, a primary component of the blood-brain barrier, possessed the capacity to produce or degrade beta-amyloid peptides. Using quantitative real-time RT-PCR, immunodetection and enzyme activity assays, we demonstrated the presence in brain barriers of several key enzymes involved in beta-amyloid production, namely, amyloid precursor protein and beta-secretase, and in beta-amyloid metabolism and alternate processing, such as insulin degrading enzyme, endothelin-converting enzyme-1, neprilysin and alpha-secretase. Furthermore, beta-amyloid presence, in the absence of its application in culture media, was detected in an immortalized choroidal epithelial cell line, known as Z310 cells. The ability of the choroid plexus to produce and degrade beta-amyloid, in addition to its transport function, suggests a vital role of this tissue in maintaining beta-amyloid homeostasis. Disruption of this homeostasis due to aging, injury or toxicant exposure may contribute to accumulation of beta-amyloid peptides in the brain fluids, leading to AD.

Increased matrix-metalloproteinase-2 and matrix-metalloproteinase-9 expression in the brain of dystrophic mdx mouse.

Brain edema and severe alterations of the glial and endothelial cells have recently been demonstrated in the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy, and an increase in microvessel density in patients affected by Duchenne muscular dystrophy has also been shown. In order to further elucidate the mechanisms underlying the angiogenetic processes occurring in Duchenne muscular dystrophy, in this study we analyzed matrix-metalloproteinase-2 and -9 expression in the brain of 20-month-old mdx and control mice by means of immunohistochemistry, in situ hybridization, immunoblotting and gelatin zymography. Moreover, we studied vascular endothelial growth factor expression by means of Western blot and immunohistochemistry, and by dual immunofluorescence using anti-vascular endothelial growth factor and anti matrix-metalloproteinase-2 and-9 antibodies. Ultrastructural features of the brain choroidal plexuses were evaluated by electron microscopy. Spatial relationships between endothelium and astrocyte processes were studied by confocal laser microscopy, using an anti-CD31 antibody as a marker of endothelial cells, and anti-glial fibrillary acidic protein (GFAP) as a marker of glial cells. The results demonstrate that high expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 protein content occurs in mdx brain and in choroidal plexuses where, by in situ hybridization, matrix-metalloproteinase-2 and matrix-metalloproteinase-9 mRNA was localized in the epithelial cells. Moreover, matrix-metalloproteinase-2 mRNA was found in both mdx perivascular astrocytes and blood vessels, while matrix-metalloproteinase-9 mRNA was localized in mdx vessels. Through zymography, increased expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in mdx brain compared with the controls. These enhanced matrix-metalloproteinase levels in mdx mice were found to be associated with increased vascular endothelial growth factor expression, as determined by immunoblotting and immunocytochemistry and with ultrastructural alterations of the mdx choroidal epithelial cells and brain vessels, as previously reported [Nico B, Frigeri A, Nicchia GP, Corsi P, Ribatti D, Quondamatteo F, Herken R, Girolamo F, Marzullo A, Svelto M, Roncali L (2003) Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice. Glia 42:235-251]. Indeed, in the mdx epithelial cells of the plexuses, the apical microvilli were located on the lateral membranes, whereas in the controls they were uniformly distributed over the free ventricular surface. Moreover, by dual immunofluorescence, a colocalization of vascular endothelial growth factor and matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in the ependymal and epithelial cells of plexuses in mdx mice and, under confocal laser microscopy, mdx CD-31 positive vessels were enveloped by less GFAP-positive astrocyte processes than the controls. Overall, these data point to a specific pathogenetic role of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 in neurological dysfunctions associated with Duchenne muscular dystrophy.

Cell death, caspase activation, and HMGB1 release of porcine choroid plexus epithelial cells during Streptococcus suis infection in vitro.

The choroid plexus epithelium constitutes the structural basis of the blood-cerebrospinal fluid barrier. We previously demonstrated that Streptococcus suis (S. suis), a relevant cause of bacterial meningitis in pigs and humans, affects porcine choroid plexus epithelial cell (PCPEC) barrier function and integrity. We now characterized PCPEC cell death and investigated whether apoptosis or necrosis is responsible for the cytotoxicity after infection with different S. suis isolates. We found S. suis strain-dependent histone associated DNA-fragments quantified by ELISA. This response could partially be inhibited by cylcoheximide, cytochalasin D, dexamethasone, herbimycin A, but most effectively by the pan-caspase inhibitor zVAD-fmk. We further detected caspase-3 and -9 activation after infection with all tested S. suis isolates that could also be blocked by zVAD-fmk. However, we found a significantly stronger caspase activity with the protein kinase inhibitor staurosporine. All tested S. suis isolates induced loss of cell viability in PCPEC as shown with the Live/Dead assay, but strain dependent lactate dehydrogenase-release. Both parameters could not be influenced by zVAD-fmk. Immunostaining showed release of high-mobility group box 1 (HMGB1) protein from the nucleus, indicative of necrosis. Transmission electron microscopy showed cell swelling, cytoplasmic vacuolization, loss of membrane integrity, nuclear fermentation but no nuclear condensation, indices for a primarily necrotic cell morphology. Taken together, our findings indicate that S. suis causes cell death in PCPEC by different mechanisms. Although apoptosis may be involved in the process of PCPEC cell death, necrosis seems to be the predominant mechanism. Through inflammation in the choroid plexus during bacterial meningitis, the blood-cerebrospinal fluid barrier function will be compromised.

The alkaline phosphatase in human plexus chorioideus.

The content of alkaline phosphatase isozymes in various brain regions was determined by monoclonal immunocatalytic assays. The levels of the isozymes in human brain tissues were low compared with those in other human tissues, liver, kidney, bone, intestine and placenta. Plexus chorioideus in the brain, however, was found to express significant amounts of alkaline phosphatase activity. The purified isozyme from human plexus chorioideus demonstrated a single 70 kDa protein band on SDS-polyacrylamide gel which coincides with that of tissue-unspecific alkaline phosphatase from human liver. The isozyme expressed in the plexus was confirmed to be the tissue-unspecific alkaline phosphatase isozyme with regard to its reactivity with monoclonal antibodies specific for liver alkaline phosphatase, heat stability, and the inhibition by amino acids. This finding adds new dimensions to the functional role this isozyme may play.

Polarization of the Na+, K(+)-ATPase in epithelia derived from the neuroepithelium.

The neuroepithelium generates a fascinating group of epithelia. One of their intriguing properties is how they polarize the distribution of the Na+, K(+)-ATPase. Typically, this ion pump is concentrated in the basolateral membrane, but it is concentrated in the apical membranes of the retinal pigment epithelium and the epithelium of the choroid plexus. A comparison of their development with that of systemic epithelia yields insights into how cells polarize the distribution of this and other membrane proteins. The polarization of the Na+, K(+)-ATPase depends upon the interplay between different sorting signals and different types of polarity mechanisms. These include intracellular targeting signals that direct the delivery of newly synthesized proteins, and maintenance signals that stabilize proteins in the proper membrane domain. Conflicting signals appear to be arranged in a hierarchy that can be rearranged as cells respond to certain environmental stimuli. Part of this response is mediated by changes in the distribution and composition of the cortical cytoskeleton.

Chronic ACE inhibition by quinapril modulates central vasopressinergic system.

OBJECTIVE: The role of the brain as a target for angiotensin converting enzyme (ACE) inhibitors in the treatment of heart failure and hypertension is unclear. To test the hypothesis that ACE inhibitors may modulate other central neuropeptide systems such as the central vasopressin system, we studied the effects of chronic treatment with the ACE inhibitor, quinapril, on ACE activity and on central vasopressin content in specific brain areas in rats. METHODS: 22 rats were chronically treated with quinapril (6 mg.kg-1 BW per gavage daily for 6 weeks; untreated controls, n = 14). ACE density in various brain regions was assessed by in vitro autoradiography using the specific ACE inhibitor, 125I-351A. Vasopressin content was determined in 19 brain areas (micropunch technique) known to be involved in cardiovascular regulation. RESULTS: Following chronic quinapril treatment ACE was significantly decreased in the thalamus (-38%), hypothalamus (-37%), hypophysis (-35%), cerebellum (-36%) choroid plexus (-20%), and locus coeruleus (-35%). Additionally, a marked reduction in serum ACE activity (-97%) was observed. Plasma levels of vasopressin were significantly decreased after quinapril treatment (0.97[s.e.m. 0.11] vs. 1.63[0.24] pg.ml-1 in controls, P < 0.05). Vasopressin content was significantly reduced in 9 of 19 specific brain areas. Regarding the hypothalamic vasopressin-producing nuclei, vasopressin was decreased in the paraventricular (292[197] vs. 2379[585] pg.mg-1 crotein in controls; P < 0.001) and supraoptic nuclei (13618[1979] vs. 24525[3894] pg.mg-1 protein; P < 0.05), but not in the suprachiasmatic nucleus. Vasopressin content was significantly reduced in brain areas connected by vasopressinergic fibres originating in the hypothalamic paraventricular nucleus: namely central gray, subcommissural organ, organum vasculosum laminae terminalis, dorsal raphe nucleus, and locus coerules. Vasopressin content was also significantly reduced in the median eminence (5887[1834] vs. 28321[4969] pg.mg-1 protein, P < 0.001), where the hormone is mainly concentrated in the hypothalamo-hypophysial tract. CONCLUSIONS: Autoradiographic studies in vitro indicate that orally administered quinapril suppresses central ACE activity after chronic treatment. ACE inhibition by quinapril strongly influences vasopressin content in important brain areas which are involved in central cardiovascular regulation. Therefore, central modulatory effects of ACE inhibitors may also contribute to overall therapeutic efficacy.

Enzyme histochemistry of the choroid plexus in old rats.

The influence of ageing on the metabolic profile of lateral ventricle choroid plexus epithelial cells from young (3-month-old) and aged (26-month-old) male Wistar rats was studied using enzyme histochemical techniques. The following enzymatic activities related to energy transduction were examined: lactate-(LDH) and succinate- (SDH) dehydrogenases; NADH2-tetrazolium reductase (NADHD) and alpha-glycerophosphate-dehydrogenase (GPDH). The intensity of enzymatic staining within single choroid plexus epithelial cells from young and old animals was assessed microphotometrically. In the choroid plexus epithelial cells of young rats NADHD was the enzymatic activity more heavily stained; cell levels of LHD and GPDH were approximately the same and SDH reactivity was less intense. In old age LDH was reduced by 9.3%, SDH was reduced by 26.1%, NADHD was reduced by 8.6% and GPDH was reduced by 3.6%. The possibility that impaired energy transduction mechanisms at the level of choroid plexus epithelium in old age may influence functional activity of the choroid plexus is discussed.

Differential distribution of nicotinamide adenine dinucleotide phosphate-diaphorase and neural nitric oxide synthase in the rat choroid plexus. A histochemical and immunocytochemical study.

This study used NADPH diaphorase (NADPHd) histochemistry and neuronal nitric oxide synthase immunocytochemistry to examine the localization of nitric oxide synthase in the choroid plexus of the lateral ventricles and the fourth ventricle of rat brain. That the NADPHd reaction product in choroid plexus was specific to nitric oxide synthase was evaluated: (i) by comparison to immunocytochemical labelling for nitric oxide synthase; and (ii) by comparing NADPHd histochemical staining in choroid plexus and brain (rich in nitric oxide synthase-positive and NADPHd-positive neurons) in the presence or absence of iodonium diphenyl or dichlorophenolindophenol, two potent albeit non-selective inhibitors of nitric oxide synthase activity. In brain, NADPHd histochemistry homogeneously stained neuronal cell bodies, axons and dendrites, while it produced particulate cytoplasmic staining of all epithelial cells in the choroid plexuses of the lateral and fourth ventricles. Within the choroid plexus of the lateral ventricles, NADPHd-positive nerve fibres were also observed around blood vessels and coursing among the epithelial cells. The distribution of immunoreactivity for nitric oxide synthase in brain and in nerve fibres in the choroid plexuses of the lateral ventricles resembled the distribution of histochemical labelling for NADPHd. Choroid plexus epithelial cells were, however, devoid of nitric oxide synthase immunoreactivity. Consistent with this, iodonium diphenyl and dichlorophenolindophenol (0.1 mM) inhibited NADPHd histochemical staining in brain neurons and in choroid plexus nerve fibres, but not in choroid plexus epithelial cells. These results demonstrate that the choroid plexus of the lateral ventricles in rat brain is innervated by nitric oxide synthase-positive nerve fibres. These nitric oxide synthase-positive nerve fibres may have an important role in the regulation of cerebrospinal fluid balance. Although choroid plexus epithelial cells contain an enzyme with NADPHd activity, this enzyme is not nitric oxide synthase.

Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus.

Na+,K+-ATPase plays a central role in the mechanism of cerebrospinal fluid secretion by the choroid plexus. We have used an antiserum to the 100 KD catalytic polypeptide of the enzyme purified from mouse brain (30) to localize the catalytic unit in mouse choroid plexus at the light and electron microscopic levels. Pre-embedding immunostaining with the peroxidase-conjugated second antibody technique showed that microvillar borders facing the ventricle were intensely reactive. In contrast, basal and lateral plasma membrane surfaces were devoid of activity. Identical localization was obtained with a post-embedding procedure in which protein A-gold was used to stain immunoreactive sites on thin sections of Lowicryl-embedded tissue. For comparison, immunogold staining was shown to be restricted to basolateral membranes of kidney medullary ascending thick limbs. The apical localization of Na+,K+-ATPase in choroid plexus is in striking contrast to the almost exclusive basolateral localization seen in other ion-transporting tissues. The immunocytochemical data are completely consistent with physiological data on choroidal epithelial transport and with light microscopic autoradiographic localization of [3H]-ouabain binding sites.