Membrane Of Choroid Plexus Research Articles

In vitro investigation of the effect of proinflammatory cytokines on mouse choroid plexus membrane transporters Ncbe and NKCC1

Intraventricular hemorrhage is a potentially life-threatening condition. Approximately 20% of patients develop posthemorrhagic hydrocephalus with increased ventricular volume and intracranial pressure. Hydrocephalus develops partially due to increased secretion of cerebrospinal fluid by the choroid plexus. During hemorrhage a multitude of factors are released into the cerebrospinal fluid. Many of these have been implicated in the hypersecretion. In this study, we have investigated the isolated effect of inflammatory components, on the abundance of two membrane transporters involved in cerebrospinal fluid secretion by the choroid plexus: the Na+-dependent Cl−/HCO3− exchanger, Ncbe, and the Na+, K+, 2Cl− cotransporter, NKCC1. We have established a primary choroid plexus epithelial cell culture from 1 to 7 days old mouse pups. Seven days after seeding, the cells formed a monolayer. The cells were treated with either tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), or interleukin 6 (IL-6) to mimic inflammation. The data show that treatment with TNFα, and IL-1β only transiently increased NKCC1 abundance whereas the effect on Ncbe abundance was a transient decrease. IL-6 however significantly increased NKCC1 (242%), the phosphorylated NKCC1 (147%), as well as pSPAK (406%) abundance, but had no effect on Ncbe. This study suggests that the inflammatory pathway involved in hypersecretion primarily is mediated by activation of basolateral receptors in the choroid plexus, mainly facilitated by IL-6. This study highlights the complexity of the pathophysiological circumstances occurring during intraventricular hemorrhage.

Membrane transporters control cerebrospinal fluid formation independently of conventional osmosis to modulate intracranial pressure

BackgroundDisturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the details underlying the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP.MethodsExperimental rats were employed for in vivo determinations of CSF secretion rates, ICP, blood pressure and ex vivo excised choroid plexus for morphological analysis and quantification of expression and activity of various transport proteins. CSF and blood extractions from rats, pigs, and humans were employed for osmolality determinations and a mathematical model employed to determine a contribution from potential local gradients at the surface of choroid plexus.ResultsWe demonstrate that CSF secretion can occur independently of conventional osmosis and that local osmotic gradients do not suffice to support CSF secretion. Instead, the CSF secretion across the luminal membrane of choroid plexus relies approximately equally on the Na+/K+/2Cl− cotransporter NKCC1, the Na+/HCO3− cotransporter NBCe2, and the Na+/K+-ATPase, but not on the Na+/H+ exchanger NHE1. We demonstrate that pharmacological modulation of CSF secretion directly affects the ICP.ConclusionsCSF secretion appears to not rely on conventional osmosis, but rather occur by a concerted effort of different choroidal transporters, possibly via a molecular mode of water transport inherent in the proteins themselves. Therapeutic modulation of the rate of CSF secretion may be employed as a strategy to modulate ICP. These insights identify new promising therapeutic targets against brain pathologies associated with elevated ICP.

CrossTalk opposing view: NKCC1 in the luminal membrane of choroid plexus is outwardly directed under basal conditions and contributes directly to cerebrospinal fluid secretion

CrossTalk opposing view: NKCC1 in the luminal membrane of choroid plexus is outwardly directed under basal conditions and contributes directly to cerebrospinal fluid secretion

Cotransporter-mediated water transport underlying cerebrospinal fluid formation

Cerebrospinal fluid (CSF) production occurs at a rate of 500 ml per day in the adult human. Conventional osmotic forces do not suffice to support such production rate and the molecular mechanisms underlying this fluid production remain elusive. Using ex vivo choroid plexus live imaging and isotope flux in combination with in vivo CSF production determination in mice, we identify a key component in the CSF production machinery. The Na+/K+/2Cl− cotransporter (NKCC1) expressed in the luminal membrane of choroid plexus contributes approximately half of the CSF production, via its unusual outward transport direction and its unique ability to directly couple water transport to ion translocation. We thereby establish the concept of cotransport of water as a missing link in the search for molecular pathways sustaining CSF production and redefine the current model of this pivotal physiological process. Our results provide a rational pharmacological target for pathologies involving disturbed brain fluid dynamics.

Expression of Aquaporins in the Rat Choroid Plexus

Background: Aquaporins (AQPs) are water channels that play a key role in water transport in many epithelia. Recent studies have shown that AQP1, located in the apical membrane of choroid plexus cells, has an important role in cerebrospinal fluid (CSF) production. However, the mechanism of water transport through the basolateral membrane of choroid plexus (CP) cells is yet to be determined. Objectives: In this study, the expression and localization of AQP1, AQP4 and AQP5 in the rat lateral ventricle CP were investigated by immunocytochemistry, western analysis and flowcytometry. Materials and Methods: Choroidal epithelial cells of the lateral ventricle in Wistar rats were isolated and grown in Dulbecco's Modified Eagle's medium <b>(</b>DMEM) supplement, which was refreshed every two days. After five days, the expression patterns of AQP1, AQP4 and AQP5 were determined by immunoblotting, immuncytochemistry and flowcytometry. Results: The immunocytochemistry data revealed the expression of AQP1 and AQP4 in the membrane and the cytoplasmof AP cells, respectively. Through western analysis, the AQP1 antibody detected two bands of approximately 27 and 32 kDa. A single peptide of 29 kDa was recognized by the AQP4 antibody. Quantitative flowcytometry revealed CP cells exhibiting a high level of AQP1 and AQP4 proteins (95.39% and 92.21%, respectively). According to immunocytochemistry, AQP5 is weakly expressed in the cytoplasm of CP cells. Anti-AQP5 antibody recognized a pale band of approximately28 kDa by western analysis. Conclusions: These observations suggested that AQP4 may have an important role in CSF secretion; however, expression of AQP4 or AQP5 was not detected in the cell membrane. Thus, how water crosses the basolateral membrane of CP cells remains to be determined.

A Role for the Proton-coupled Folate Transporter (PCFT-SLC46A1) in Folate Receptor-mediated Endocytosis

Recently, this laboratory identified a proton-coupled folate transporter (PCFT), with optimal activity at low pH. PCFT is critical to intestinal folate absorption and transport into the central nervous system because there are loss-of-function mutations in this gene in the autosomal recessive disorder, hereditary folate malabsorption. The current study addresses the role PCFT might play in another transport pathway, folate receptor (FR)-mediated endocytosis. FRalpha cDNA was transfected into novel PCFT(+) and PCFT(-) HeLa sublines. FRalpha was shown to bind and trap folates in vesicles but with minimal export into the cytosol in PCFT(-) cells. Cotransfection of FRalpha and PCFT resulted in enhanced folate transport into cytosol as compared with transfection of FRalpha alone. Probenecid did not inhibit folate binding to FR, but inhibited PCFT-mediated transport at endosomal pH, and blocked FRalpha-mediated transport into the cytosol. FRalpha and PCFT co-localized to the endosomal compartment. These observations (i) indicate that PCFT plays a role in FRalpha-mediated endocytosis by serving as a route of export of folates from acidified endosomes and (ii) provide a functional role for PCFT in tissues in which it is expressed, such as the choroid plexus, where the extracellular milieu is at neutral pH.

Expression and localization of Na-driven Cl-HCO 3− exchanger (SLC4A8) in rodent CNS

The Na +-driven Cl-HCO 3 exchanger (NDCBE or SLC4A8) is a member of the so lute carrier 4 (SLC4) family of HCO 3 − transporters, which includes products of 10 genes with similar sequences. Most SLC4 members play important roles in regulating intracellular pH (pH i). Physiological studies suggest that NDCBE is a major pH i regulator in at least hippocampal (HC) pyramidal neurons. We generated a polyclonal rabbit antibody directed against the first 18 residues of the cytoplasmic N terminus (Nt) of human NDCBE. By Western blotting, the antibody distinguishes NDCBE—as a purified Nt peptide or a full-length transporter (expressed in Xenopus oocytes)—from other Na +-coupled HCO 3 − transporters. By Western blotting, the antiserum recognizes an ∼135-kDa band in several brain regions of adult mice: the cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and HC. In CX, PNGase F treatment reduces the molecular weight to ∼116 kDa. By immunocytochemistry, affinity-purified (AP) NDCBE antibody stains the plasma membrane of neuron cell bodies and processes of rat HC neurons in primary culture as well as freshly dissociated mouse HC neurons. The AP antibody does not detect substantial NDCBE levels in freshly dissociated HC astrocytes, or astrocytes in HC or CB sections. By immunohistochemistry, the AP antibody recognizes high levels of NDCBE in neurons of CX, HC (including pyramidal neurons in Cornu Ammonis (CA)1-3 and dentate gyrus), substantial nigra, medulla, cerebellum (especially Purkinje and granular cells), and the basolateral membrane of fetal choroid plexus. Thus, NDCBE is in a position to contribute substantially to pH i regulation in multiple CNS neurons.

Thyroid Transcription Factor-1 Facilitates Cerebrospinal Fluid Formation by Regulating Aquaporin-1 Synthesis in the Brain

In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5'-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation.

Functional Role of the C Terminus of Human Organic Anion Transporter hOAT1

Functional Role of the C Terminus of Human Organic Anion Transporter hOAT1

Expression of human organic anion transporters in the choroid plexus and their interactions with neurotransmitter metabolites.

The purpose of the present study was to elucidate the expression of human organic anion transporter 1 (hOAT1) and hOAT3 in the choroid plexus of the human brain and their interactions with neurotransmitter metabolites using stable cell lines. Immunohistochemical analysis revealed that hOAT1 and hOAT3 are expressed in the cytoplasmic membrane and cytoplasm of human choroid plexus. Neurotransmitter metabolites, namely, 5-methoxyindole-3-acetic acid (5-MI-3-AA), homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HI-3-AA), N-acetyl-5-hydroxytryptamine (NA-5-HTT), melatonin, 5-methoxytryptamine (5-MTT), 3,4-dihidroxymandelic acid (DHMA), 5-hydroxytryptophol, and 5-methoxytryptophol (5-MTP), but not methanephrine (MN), normethanephrine (NMN), and 3-methyltyramine (3-MT), at 2 mM, inhibited para-aminohippuric acid uptake mediated by hOAT1. On the other hand, melatonin, 5-MI-3-AA, NA-5-HTT, 5-MTT, 5-MTP, HVA, 5-HI-3-AA, VMA, DOPAC, 5-hydroxytryptophol, and MN, but not 3-MT, DHMA, and NMN, at 2 mM, inhibited estrone sulfate uptake mediated by hOAT3. Differences in the IC(50) values between hOAT1 and hOAT3 were observed for DHMA, DOPAC, HVA, 5-HI-3-AA, melatonin, 5-MI-3-AA, 5-MTP, 5-MTT, and VMA. HOAT1 and hOAT3 mediated the transport of VMA but not HVA and melatonin. These results suggest that hOAT1 and hOAT3 are involved in the efflux of various neurotransmitter metabolites from the cerebrospinal fluid to the blood across the choroid plexus.

Mechanism of Organic Anion Transport across the Apical Membrane of Choroid Plexus

The mechanism and membrane localization of choroid plexus (CP) organic anion transport were determined in apical (or brush border) membrane vesicles isolated from bovine choroid plexus and in intact CP tissue from cow and rat. Brush border membrane vesicles were enriched in Na(+),K(+)-ATPase (20-fold; an apical marker in CP) and demonstrated specific, sodium-coupled transport of proline, glucose, and glutarate. Vesicular uptake of the anionic herbicide 2, 4-dichlorophenoxyacetic acid (2,4-D) was markedly stimulated by an inward sodium gradient but only in the presence of glutarate, indicating the presence of apical dicarboxylate/organic anion exchange. Consistent with this interpretation, an imposed outward glutarate gradient stimulated 2,4-D uptake in the absence of sodium. Under both conditions, uptake was dramatically slowed and overshoot was abolished by probenecid. Likewise, apical accumulation of 2,4-D by intact bovine choroid plexus tissue in vitro was stimulated by external glutarate in the presence of sodium. Glutarate stimulation was abolished by 5 mM LiCl. Identical findings were obtained using rat CP tissue, which showed both sodium/glutarate-stimulated 2,4-D (tissue/medium (T/M) approximately 8) and p-aminohippurate (T/M = 2) transport. Finally, since the renal exchanger (rROAT1) has been cloned in rat kidney, a rROAT1-green fluorescent protein construct was used to analyze exchanger distribution directly in transiently transfected rat CP. As predicted by the functional studies, the fluorescently tagged transporter was seen in apical but not basolateral membranes of the CP.

Choline uptake across the ventricular membrane of neonate rat choroid plexus.

The uptake of [3H]choline from the cerebrospinal fluid (CSF) side of the rat neonatal choroid plexus was characterized in primary cultures of the choroidal epithelium grown on solid supports. Cell-to-medium concentration ratios were approximately 5 at 1 min and as high as 70 at 30 min. Apical choline uptake was facilitated; the Km was approximately 50 microM. Several organic cations (e.g., hemicholinium-3 and N1-methylnicotinamide) inhibited uptake. The reduction or removal of external Na+ or the addition of 5 mM LiCl had no effect on uptake. However, increasing external K+ concentration from 3 to 30 mM depolarized ventricular membrane potential (-70 to -15 mV) and reduced uptake to 45% of that for the control. Treatment with 1 mM ouabain or 2 mM BaCl2 reduced uptake 45%, and intracellular acidification reduced uptake to approximately 90% of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled to Na+ influx but is sensitive to plasma membrane electrical potential.

Identification of endothelin receptor subtypes in sheep choroid plexus.

Endothelin (ET) and its G-protein-coupled receptors are distributed in a wide variety of tissues, including the brain. In this study, we have identified and characterized the endothelin receptor subtypes in sheep choroid plexus. Competitive binding experiments using [125I]ET-1 and the receptor subtype-selective ligands, ET-1, ET-3, BQ-123, Sarafotoxin 6c, and [Ala1,3,11,15] ET-1 demonstrated the presence of both ETA and ETB receptor subtypes in the ratio of 30:70. In addition, a small fraction of the total binding sites exhibited affinities for ET-1 in the subpicomolar range. Chemical crosslinking of [125I]ET-1 with bis(sulfosuccinimidyl)-suberate (BS3) to choroid plexus membranes revealed the presence of two bands, with apparent molecular masses of 89 and 45 kDa, corresponding to the ETA receptor, and three bands, with apparent molecular masses of 75, 58, and 33 kDa, corresponding to the ETB receptor. Of considerable interest was the finding that dimers of the [125I]ET-1-occupied ETA receptor could be identified by crosslinking, as could apparent dimers and tetramers of [125I]ET-1, but only when bound to receptor. In addition to mapping the distribution of ET receptors in sheep choroid plexus, our results strongly suggest that ET-1 binding to the ETA receptor leads to dimer formation.

The choroid plexus epithelium is the site of the organic anion transport protein in the brain.

The mRNA for organic anion transport protein (oatp) was previously shown to be present in abundance in liver and kidney, and in small amounts in brain. Data obtained from experiments with reverse transcriptase-PCR techniques and in situ hybridization analysis showed that the oatp mRNA is present within the brain, localized to the choroid plexus. A sequence-specific antibody to the oatp polypeptide demonstrated the presence of the expected polypeptide with a molecular weight of 80,000 plus an immunoreactive species with a higher molecular weight in preparations of choroid plexus membranes. Examination of the choroid plexus by fluorescence confocal microscopy revealed that immunoreactive oatp polypeptide is localized to the apical surface of the choroid plexus epithelial cells, which contacts the cerebrospinal fluid. This localization of oatp is consistent with previous experiments showing vectorial transport of organic anions between the choroid plexus and the cerebrospinal fluid.

The fodrin-ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2.

A unique feature of the choroid plexus as a single-layer epithelium is its localization of Na+K(+)-ATPase at its apical (lumenal) surface. In contrast, a band 3 (AE1)-related anion exchanger protein has been localized to the basolateral surface of the choroid plexus. Both Na+K(+)-ATPase and AE1 in other tissues have been shown to bind via ankyrin to the spectrin-actin-based membrane cytoskeleton. Since linkage of integral membrane proteins to the membrane cytoskeleton is important for their restriction to specialized domains of the cell surface, we investigated the polarity of the choroid plexus membrane cytoskeleton. We developed isoform-specific antibodies to confirm the identity of choroid plexus band 3-related polypeptide as AE2. We demonstrated that ankyrin, fodrin/spectrin, actin, myosin, and alpha-actinin are predominantly apical in choroid plexus and preferentially colocalize with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2. Colchicine administration did not alter the polarity of apical cytoskeletal and transport proteins or basolateral AE2 in choroid plexus, suggesting that biosynthetic targeting of these proteins is not microtubule dependent. In choroid plexus papilloma, Na+K(+)-ATPase and AE2 were decreased in amount and failed to preserve their polarized distributions.

Characterization of prolactin receptor in human brain and choroid plexus

We have studied the binding of 125I-labeled human prolactin (PRL) to membranes from various regions of the human brain (hypothalamus, cerebral cortex, cerebellum and choroid plexus) derived from autopsy specimens. Among the various regions studied, the choroid plexus of both male and female subjects showed the highest specific binding and a clearly detectable specific binding was also observed in the hypothalamus of both sexes, whereas it was very low in other brain regions. No significant sex differences in PRL binding to various brain regions were observed except for the hypothalamus where a higher binding was seen in female subjects. The binding did not vary with the age of the subjects. Moreover, the cause of death and the time elapsed from death to autopsy in this study did not affect the binding significantly. The binding of 125I-labeled human PRL to hypothalamus and choroid plexus membranes from female specimens was inhibited in a dose-dependent manner by both unlabeled human and ovine PRL and by human growth hormone (GH), but not by other polypeptide hormones. Scatchard analysis of the binding revealed the presence of saturable binding sites with low capacity and high affinity for human PRL ligand. These results provide strong preliminary evidence for the presence of PRL binding sites in the human brain.

Membrane peptidases in the peripheral nervous system of the pig: Their localization by immunohistochemistry at light and electron microscopic levels

The presence and cellular localization of five membrane peptidases has been investigated in peripheral nerves, including those of the autonomic nervous system, in the pig. Endopeptidase-24.11 (“enkephalinase”) peptidyl dipeptidase A, aminopeptidase N, aminopeptidase W and dipeptidyl peptidase IV were studied by both enzymic assays of membranes prepared from samples of nerve and by immunoperoxidase histochemistry at light and in two cases, endopeptidase-24.11 and aminopeptidase W, at electron microscopic levels. All five peptidases could be quantified by enzymic assay, though the activities were about 1% of those in renal microvilli and less than those of choroid plexus membranes. Endopeptidase-24.11 was associated with Schwann cell membranes in all types of nerve examined, including major nerves containing predominantly myelinated fibres as well as autonomic nerves, such as the vagus and splenic nerves and the sympathetic chain, staining being observed in membranes associated with myelinated and unmyelinated fibres. The Schwann cell location of endopeptidase-24.11 was confirmed by correlation with immunostaining for glial fibrillary acidic protein and by electron microscopy. This peptidase is known to have a wide repertoire of susceptible substrates among neuropeptides which was here shown to include vasoactive intestinal polypeptide ( K m 268μM, k cat 568 min −1), one of a number of neuropeptides present in peripheral nerve fibres. Three of the peptidases, peptidyl dipeptidase A, aminopeptidase N and dipeptidyl peptidase IV, were associated with microvessels of peripheral nerves. Aminopeptidase N was also observed in connective tissue elements, including the perineurium. Aminopeptidase W was unique among the five peptidases in having a neuronal localization. This was observed in unmyelinated and myelinated nerves and was supported by comparison with the pattern of staining observed for neurofilament protein and by electron microscopic immunoperoxidase staining. This observation was unexpected since aminopeptidase W has not been detected as a neuronal marker in the brain. Some possible roles for the membrane peptidases in peripheral nerves are discussed.

The hydrolysis of brain and atrial natriuretic peptides by porcine choroid plexus is attributable to endopeptidase-24.11

The hydrolysis of the porcine 26-residue brain natriuretic peptide (BNP-26) and its counterpart human 28-residue atrial natriuretic peptide (alpha-hANP) by pig membrane preparations and purified membrane peptidases was studied. When the two peptides were incubated with choroid plexus membranes, the products being analysed by h.p.l.c., alpha-hANP was degraded twice as fast as BNP. The h.p.l.c. profiles of alpha-hANP hydrolysis, in short incubations with choroid plexus membranes, yielded alpha hANP' as the main product, this having been previously shown to be the result of hydrolysis at the Cys7-Phe8 bond. In short incubations this cleavage was inhibited 84% by 1 microM-phosphoramidon, a specific inhibitor of endopeptidase-24.11. BNP-26 was hydrolysed by choroid plexus membranes, kidney microvillar membranes and purified endopeptidase-24.11 in a manner that yielded identical h.p.l.c. profiles. In the presence of phosphoramidon, hydrolysis by the choroid plexus membranes was 94% inhibited. Captopril had no effect and, indeed, no hydrolysis of BNP-26 by peptidyl dipeptidase A (angiotensin-converting enzyme) was observed even after prolonged incubation with the purified enzyme. The stepwise hydrolysis of BNP-26 by endopeptidase-24.11 was investigated by sequencing the peptides produced during incubation. The initial product resulted from hydrolysis at Ser14-Leu15, thereby opening the ring. This product (BNP') was short-lived; further degradation involved hydrolysis at Ile12-Gly13, Arg8-Leu9, Gly17-Leu18, Val22-Leu23, Arg11-Ile12 and Cys4-Phe5. Thus endopeptidase-24.11 is the principal enzyme in renal microvillar and choroid plexus membranes hydrolysing BNP-26 and alpha-hANP.

Membrane peptidases in the pig choroid plexus and on other cell surfaces in contact with the cerebrospinal fluid.

A comprehensive survey of 11 peptidases, all of which are markers for renal microvillar membranes, has been made in membrane fractions prepared from pig choroid plexus. Two fractionation schemes were explored, both depending on a MgCl2-precipitation step, the preferred one having advantages in speed and yield of the activities. The specific activities of the peptidases in the choroid-plexus membranes were, with the exception of carboxypeptidase M, lower than in renal microvillar membranes: those of aminopeptidase N, peptidyl dipeptidase A ('angiotensin-converting enzyme') and gamma-glutamyltransferase were 3-5-fold lower, those of aminopeptidase A and endopeptidase-24.11 were 12-15 fold lower, and those of dipeptidyl peptidase IV and aminopeptidase W were 50-70-fold lower. Carboxypeptidase M had a similar activity in both membranes. Alkaline phosphatase and (Na+ + K+)-activated ATPase were more active in the choroid-plexus membranes. No activity for microsomal dipeptidase, aminopeptidase P and carboxypeptidase P could be detected. Six of the peptidases and (Na+ + K+)-activated ATPase were also studied by immunoperoxidase histochemistry at light- and electron-microscopic levels. Endopeptidase-24.11 and (Na+ + K+)-activated ATPase were uniquely located on the brush border, and the other two peptidases appeared to be much more abundant on the endothelial lining of microvessels. Dipeptidyl peptidase IV and aminopeptidase W were also detected in microvasculature. Pial membranes associated with the brain and spinal cord also stained positively for endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A. The immunohistochemical studies indicated the subcellular fractionation did not discriminate between membranes derived from epithelial cells (i.e. microvilli) and those from endothelial cells. The possible significance of these studies in relation to neuropeptide metabolism and the control of cerebrospinal fluid production is discussed.

5-HT1C receptor-mediated stimulation of inositol phosphate production in pig choroid plexus. A pharmacological characterization.

1) 5-HT (5-hydroxytryptamine, serotonin) induces inositol phosphate production in a pig choroid plexus preparation. This effect has been pharmacologically characterized and the data compared to those obtained from radioligand binding studies performed with [3H]mesulergine to 5-HT1C sites in pig choroid plexus membranes. 2) The rank order of potency of agonists stimulating inositol phosphate production was: alpha-methyl-5-HT greater than 1-methyl-5-HT greater than DOI greater than bufotenine = SKF 83566 = 5-HT greater than 5-MeO-DMT greater than 5-MeOT = RU 24969 greater than SCH 23390 greater than 5-CT. 8-OH-DPAT was virtually devoid of activity at 100 mumol/l. 3) The increase in inositol phosphate production induced by 5-HT and other agonists was surmountably antagonised by mesulergine, ketanserin and spiperone with pKB values of 8.7, 6.7 and 5.3, respectively. 4) The rank order of potency of antagonists was: metergoline greater than mesulergine greater than LY 53857 greater than ritanserin greater than methiothepin greater than mianserin greater than cyproheptadine greater than pirenperone greater than cinanserin greater than ketanserin greater than spiperone. The following antagonists were virtually devoid of activity at 100 mumol/l; pindolol, 21-009 and yohimbine. 5) The results obtained both with agonists and antagonists strongly support the view that 5-HT1C receptors mediate agonist induced production of inositol phosphates in pig choroid plexus.(ABSTRACT TRUNCATED AT 250 WORDS)