Mapacalcine specifically blocks hypoxia-induced calcium influx in rat hepatocytes.

Post ischaemic cell calcium invasion has been described as one of the main causes of graft failure. Protective effects of calcium antagonists have been investigated but are not convincing and their mechanisms of action remain unclear. In this work we tested the protective effect of a new calcium inhibitor described to block a calcium current insensitive to all known calcium blockers. Specific mapacalcine receptors were first characterized on rat hepatocytes membranes using the 125I-labeled mapacalcine. 45Ca fluxes were then measured on cultured hepatocytes submitted (or not) to an hypoxic period. The action of mapacalcine was investigated on the ischaemia-induced calcium influx. We demonstrate here that: (a) there are specific receptors for mapacalcine in rat hepatocytes; (b) Mapacalcine is able to specifically block ischaemia-induced calcium influx with an IC50 of 0.3 micro m and does not significantly interact with the basal calcium flux. Our work demonstrates that the mapacalcine receptor is a cellular structure directly involved in the phenomenon of postischaemic cell invasion by calcium. Specific block of ischaemia-induced Ca2+ influx by mapacalcine suggests that the development of a panel of pharmacological drugs acting on this receptor could lead to the discovery of therapeutic agents able to protect cells against one of the events responsible for organ failure after transplantation or simply after an ischaemic period. Moreover, identification of the cellular protein which binds mapacalcine may become an important step in the research of mechanisms involved in postischaemic cell invasion by calcium.     

Mapacalcine Protects Mouse Neurons against Hypoxia by Blocking Cell Calcium Overload

Stroke is one of a major cause of death and adult disability. Despite intense researches, treatment for stroke remains reduced to fibrinolysis, a technique useful for less than 10% of patients. Finding molecules able to treat or at least to decrease the deleterious consequences of stroke is an urgent need. Here, we showed that mapacalcine, a homodimeric peptide purified from the marine sponge Cliona vastifica, is able to protect mouse cortical neurons against hypoxia. We have also identified a subtype of L-type calcium channel as a target for mapacalcine and we showed that the channel has to be open for mapacalcine binding. The two main L-type subunits at the brain level are CaV1.3 and CaV1.2 subunits but mapacalcine was unable to block these calcium channels.Mapacalcine did not interfere with N-, P/Q- and R-type calcium channels. The protective effect was studied by measuring internal calcium level variation triggered by Oxygen Glucose Deprivation protocol, which mimics stroke, or glutamate stimulation. We showed that NMDA/AMPA receptors are not involved in the mapacalcine protection. The protective effect was confirmed by measuring the cell survival rate after Oxygen Glucose Deprivation condition. Our data indicate that mapacalcine is a promising molecule for stroke treatment.     

Production,in Pichia pastoris,of a recombinant monomeric mapacalcine,a protein with anti-ischemic properties

Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.     

Asymmetric localization of Notch2 on the microvillous surface in choroid plexus epithelial cells

Notch family molecules are transmembrane receptors that play various roles in contact-dependent cell–cell interactions in a wide range of organs. In the brain, Notch2, but not the other members of Notch, is expressed in the choroid plexus at an exceptionally high level. We immunohistochemically examined the cellular and subcellular localization of Notch2 protein in the choroid plexus using confocal and electron microscopy. Unexpectedly, Notch2 was asymmetrically localized on the microvillous surface of epithelial cells in the choroid plexus of both postnatal and adult rats. This localization pattern of Notch2 suggests its novel and unknown role independent of contact with adjacent cells in the choroid plexus. In organotypic cultures of the choroid plexus, the addition of anti-Notch2 antibody resulted in deformation of microvilli in epithelial cells, which suggests a role of Notch2 in the maintenance of the microvillous structure in choroid plexus epithelial cells.     

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.     

Immunochemical and biochemical distinction of subtypes of atrial natriuretic peptide receptor

The presence of subtypes of atrial natriuretic peptide (ANP) receptor has been demonstrated by examining the immunological features and ligand specificities of the receptors in various bovine tissues. The antibody probe used was the antiserum raised against the bovine lung ANP receptor, and the tissues examined for the possible presence of different types of ANP receptor were the lung, kidney, adrenal cortex, ovary, choroid plexus, and vascular tissues. When incubated with Triton extracts of these tissues, the antiserum strongly cross-reacted with the ovary, kidney, and choroid plexus receptors as well as the homologous lung receptor (type I). The adrenal and vascular receptors were recognized only weakly, however, suggesting the presence of distinct ANP receptors (type II). In support of this immunochemical subtyping, type I and type II receptors showed a marked difference in their ability to bind the ANP analog atriopeptin I (ANP5-25): type I receptors in the lung exhibited a moderate affinity for atriopeptin I with a KD of 10(-9) M; however, type II receptors in adrenal and artery showed only a weak affinity for the analog with a KD of 10(-6) M. Structural analysis of affinity-labeled ANP receptors by SDS-PAGE indicated that type I and type II receptors have similar disulfide-linked dimeric structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin, bradykinin and endothelin signalling in a sheep choroid plexus cell line

The secretion of cerebrospinal fluid by the epithelial cells of choroid plexus is regulated by membrane receptors coupled to adenylyl cyclases or to phospholipase C. These intracellular signalling pathways as their interactions were investigated in a sheep choroid plexus cell line. Endothelin-1, bradykinin and serotonin induced a transient dose-dependent increase in intracellular calcium. EC 50 were 10(-8) M for endothelin-1, 10(-8) M for bradykinin and 10(-6) M for serotonin. Maximal increase in intracellular calcium was comparable for bradykinin and serotonin, but was 3 to 5 fold larger for endothelin-1. Successive stimulations with endothelin-1, serotonin or bradykinin elicited calcium increases similar to single stimulations reflecting absence of heterologous desensitization between these receptors. Forskolin-induced cAMP accumulation was potentiated by bradykinin, but not by serotonin and endothelin-1. This potentiation resulted from an increase in cAMP production rather than to an inhibition of cAMP hydrolysis. These data suggest that serotonin, endothelin-1 and bradykinin each use specific signalling pathways in the sheep choroid plexus cells.     

Uniform cell-autonomous tumorigenesis of the choroid plexus by papovavirus large T antigens.

The simian virus 40 (SV40) large tumor antigen (T antigen) under its natural regulatory elements induces choroid plexus papillomas in transgenic mice. Because these tumors develop focally after several months, it has been suggested that secondary cellular alterations are required to induce a tumor in this tissue. In contrast to SV40, the related lymphotropic papovavirus early region induces rapid nonfocal choroid plexus neoplasia in transgenic mice. Here, using hybrid gene constructs, we showed that T antigen from either virus in in fact sufficient to induce these tumors. Their abilities to induce proliferative abnormalities in other tissues, such as kidney and thymus, were also indistinguishable. Differences in the rate of choroid plexus tumorigenesis reflected differences in the control regions of the two viruses, rather than differences in T antigen per se. Under SV40 regulation, expression was limited to a fraction of the choroid plexus cells prior to the formation of focal tumors. When SV40 T antigen was placed under lymphotropic papovavirus control, in contrast, expression was generally uniform in the choroid plexus and rapid expansion of the tissue ensued. We found a direct relationship between T-antigen expression, morphological transformation, and proliferation of the choroid plexus epithelial cells. Analysis of mosaic transgenic mice indicated further that T antigen exerts its mitogenic effect cell autonomously. These studies form the foundation for elucidating the role of various T-antigen subactivities in tumorigenesis.     

Insulin-like growth factor II messenger ribonucleic acids are synthesized in the choroid plexus of the rat brain

Previous studies demonstrating the presence of immunoreactive insulin-like growth factors (IGFs) and their receptors in the brain suggest a role of the IGFs in the central nervous system. IGF-II has been implicated as the predominant IGF in brain of mature animals based on studies of immunoreactive peptide and of IGF-II mRNAs. To obtain information about the sites of synthesis of IGF-II in adult rat brain, a 32P-labeled 31 base long synthetic oligodeoxyribonucleotide complementary in sequence to trailer peptide coding sequences in rat IGF-II mRNA (IGF-II 31 mer) was hybridized with coronal sections of fixed rat brain. The IGF-II 31 mer showed specific hybridization with the choroid plexus throughout rat brain, whereas in other brain regions, structures or cells, hybridization was not discernibly above background. These findings suggest that the choroid plexus is a primary site of synthesis of IGF-II, a probable source of IGF-II in cerebrospinal fluid, and a potential source of IGF-II for actions on target cells within the adult rat brain.     

THE ELECTRON MICROSCOPY OF THE CHOROID PLEXUS

1. The choroid plexus of the rat has been studied in detail by electron microscopy. Samples from the frog, rabbit, and cat have also been examined without noting significant differences. 2. The surface of the ependymal epithelium is covered by pedicels of variable size. There is reason for thinking of these structures as labile. They may actually pinch off and contribute to the secretory product. In any case, the surface area is vastly increased by their presence. Polypoid border seems an apt term to apply to this type of surface. 3. There is also a great expansion of the basal surface of ependymal cells. In the vicinity of cell junctions this surface is deeply infolded, and continuous with elaborate interdigitations of the lateral intercellular surfaces. Analogous infolding of the basal cell surface is known to exist in other epithelia also noted for their water transport (kidney tubules, salivary gland, and ciliary body). 4. Pretreatment of rats with diamox, an agent known to block cerebro-spinal fluid production, did not produce an important morphological change in the features of the ependyma, or any other part of the choroid plexus. 5. Capillaries of the choroid plexus have a very attenuated endothelium. This is seen to be fenestrated. It is thought this probably represents the condition in life, and is not simply a fixation artefact. 6. Pial cells tend to interpose sheets of cytoplasm between the capillaries and ependyma. The sheets are not continuous, however, and so would not constitute a serious diffusion barrier. These cells belong to the reticuloendothelial system, and undergo shape changes, and probably increase in number, when the system is stimulated by the repeated injection of trypan blue.     

Binding of a pure 125I-monoiodoleptin analog to mouse tissues: a developmental study

The preparation of a pure 125I-labeled monoiododerivative of mouse leptin is described. This radiolabeled analog has been used to characterize and localize central and peripheral leptin binding sites (Ob-R) of the mouse at different stages of its development. The affinity values found in membrane homogenates of various mouse tissues are similar and range between 0.1 and 0.3 nM, indicating that all the Ob-R isoforms have a similar affinity. Leptin binding sites are highly expressed at the membrane level in lung, intestine, kidney, liver, and skin and to a lesser degree in stomach, heart, and spleen. Brain, thymus, and pancreas homogenates are devoid of any specific binding. The distribution of mouse Ob-R has also been explored by autoradiography and dipping techniques on whole mouse sections. In lung, leptin binding sites are located at the pulmonary parenchyma and at the bronchiolar epithelial level. Binding sites are expressed all along the digestive tract from the tongue to the rectum (esophagus, stomach, intestine, colon, and rectum). In muscular visceral structures (stomach, intestine, and bladder) the binding is mainly present in the lamina propria. During development, leptin receptors are early expressed in the liver, kidney, and bone. In the lung, the Ob-R level increased gradually from birth to adulthood where the expression is maximal. By contrast, leptin receptors located in the medulla of the kidney remain remarkably constant all along the development. A broad signal is present in cartilage and bone particularly in vertebrae, limb, and ribs. Interestingly, leptin receptors are barely detectable in the mouse brain except in the choroid plexus and leptomeninges, whereas in the rat brain leptin binding sites are located in the thalamus, the piriform cortex, the cerebellum (at the granular and molecular cell layer), and the pineal gland.     

Regional Distribution and Relative Abundance of Serotonin2c Receptors in Human Brain: Effect of Suicide

Abnormalities in serotonin receptor subtypes have been observed in the postmortem brain of suicide victims. We examined the regional distribution of serotonin (5HT)(2C) receptor mRNA in several areas of the human brain and also compared its protein and mRNA expression in the prefrontal cortex (PFC), hippocampus, and choroid plexus between suicide victims and normal control subjects. 5HT(2C) receptors were found to be distributed in several areas of the human brain (in order of abundance): highly concentrated and richest in choroid plexus; hypothalamus; nucleus accumbens; with the lowest abundance in PFC and cerebellum. Comparison of 5HT(2C) receptors between suicide victims and control subjects showed higher protein levels in the PFC but not the hippocampus or choroid plexus of suicide victims. However, there were no significant differences in mRNA levels between suicide victims and control subjects in these brain areas. These results suggest that 5HT(2C) receptors are richly distributed throughout the brain with the highest level in the choroid plexus and that abnormalities in protein expression of 5HT(2C) receptors in the PFC may be associated with suicide.     

Sonic hedgehog is required for vascular outgrowth in the hindbrain choroid plexus

Critical to the exchange and metabolic functions served by tissues like brain choroid plexi and lung is the coherent development of an epithelial sheet of large surface area in tight apposition to an extensive vascular bed. Here, we present functional experiments in the mouse demonstrating that Sonic hedgehog (Shh) produced by hindbrain choroid plexus epithelium induces the extensive vascular outgrowths and vascular surface area fundamental to choroid plexus functions, but does not induce the more specialized endothelial cell features of fenestrations and bore size. Our findings indicate that these Shh-dependent vascular elaborations occur even in the presence of Vegf and other established angiogenic factors, suggesting either that the levels of these factors are inadequate in the absence of Shh or that a different set of factors may be more essential to choroid plexus outgrowth. Transducing the Shh signal is a perivascular cell—the pericyte—rather than the more integral vascular endothelial cell itself. Moreover, our findings suggest that hindbrain choroid plexus endothelial cells, as compared to other vascular endothelial cells, are more dependent upon pericytes for instruction. Thus, in addition to Shh acting on the progenitor pool for choroid plexus epithelial cells, as previously shown, it also acts on choroid plexus pericytes, and together serves the important role of coordinating the development of two disparate yet functionally dependent structures—the choroid plexus vasculature and its ensheathing epithelium.     

Distribution of transferrin synthesis in brain and other tissues in the rat

Levels of transferrin mRNA were measured by hybridization to transferrin cDNA in extracts from various areas of rat brain and other tissues. The highest concentrations of transferrin mRNA were found in the liver and the choroid plexus of the lateral and third ventricles. Lower concentrations were observed in the medulla and thalamus, choroid plexus of the fourth ventricle, cortex, hypothalamus, cerebellum, pituitary, testis, placenta, stomach, spleen, kidney, muscle, and heart. Yolk sac, small intestine, and adrenal glands did not contain detectable transferrin mRNA levels. The size of transferrin mRNA was the same in liver, brain, and testis. Upon incubation of choroid plexus pieces with [14C]leucine in vitro, about 4% of the radioactive protein secreted into the medium was found to be transferrin. Together with previous data (Dickson, P.W., Howlett, G.J., and Schreiber, G. (1985) J. Biol. Chem. 260, 8214-8219; Dickson, P.W., Aldred, A.R., Marley, P.D., Bannister, D., and Schreiber (1986) J. Biol. Chem. 261, 3475-3478) the obtained data suggest that the choroid plexus plays a role in maintenance of homeostasis in the microenvironment of the central nervous system by synthesizing and secreting plasma proteins.     

Vasopressin V1a receptor signaling in a rat choroid plexus cell line

A new cell line was derived from primary culture of rat choroid plexus (RCP) by immortalization with the TSOri minus adenovirus. The selected clone expressed vasopressin V1a receptors at a density of 64,000 sites per cell, and a K(d) of 7.2 nM. Addition of vasopressin to the RCP cells induced a transient calcium peak comparable to V1a receptor signalling in different expression systems. This [Ca(2+)](i) increase was dose-dependent with an EC(50) of 22 nM vasopressin. Similar [Ca(2+)](i) increase was elicited by addition of serotonin, angiotensin II, endothelin-1, and bradykinin. Heterologous desensitization of V1a receptor was observed in RCP cells exposed to the phorbol ester PMA or following stimulation of other receptors coupled to the phosphoinositide pathway. Positive immunolabelling with Factor VIII, Flt1 and CD 34 antibodies suggests that this new RCP cell line originated from endothelial cells of rat choroid plexus.     

High Activities of Glutamine Transaminase K (Dichlorovinylcysteine β-Lyase) and ω-Amidase in the Choroid Plexus of Rat Brain

Abstract: Certain halogenated hydrocarbons, e.g., dichlo-roacetylene, are nephrotoxic to experimental animals and neurotoxic to humans; cysteine-S-conjugate β-lyases may play a role in the nephrotoxicity. We now show that with dichlorovinylcysteine as substrate the only detectable cysteine-S-conjugate β-lyase in rat brain homogenates is identical to glutamine transaminase K. The predominant (mitochondrial) form of glutamine transaminase K in rat brain was shown to be immunologically distinct from the predominant (cytosolic) form of the enzyme in rat kidney. Glutamine transaminase K and ω-amidase (constituents of the glutaminase II pathway) activities were shown to be widespread throughout the rat brain. However, the highest specific activities of these enzymes were found in the choroid plexus. The high activity of glutamine transaminase K in choroid plexus was also demonstrated by means of an immunohistochemical staining procedure. Glutamine transaminase K has a broad specificity toward amino acid and α-keto acid substrates. The ω-amidase also has a broad specificity; presumably, however, the natural substrates are α-ketoglutaramate and α-ketosuccinamate, the α-keto acid analogues of glutamine and aspara-gine, respectively. The high activities of both glutamine transaminase K and ω-amidase in the choroid plexus suggest that the two enzymes are linked metabolically and perhaps are coordinately expressed in that organ. The data suggest that the natural substrate of glutamine transaminase K in rat brain is indeed glutamine and that the metabolism of glutamine through the glutaminase II pathway (i.e., l -glutamine and α-keto acid α-ketoglutarate and l -amino acid + ammonia) is an important function of the choroid plexus. Moreover, the present findings also suggest that any explanation of the neurotoxicity of halogenated xenobiotics must take into account the role of glutamine transaminase K and its presence in the choroid plexus.     

A novel role for the choroid plexus in BMP-mediated inhibition of differentiation of cerebellar neural progenitors

Cerebellar granule cells, the most abundant neurons in the mammalian brain, arise in the rhombic lip located at the roof of the brain's fourth ventricle. Bordering the rhombic lip is the choroid plexus, a non-neuronal structure, composed of blood vessels enveloped by epithelial cells. Here, we show a striking decrease in neural differentiation of rhombic lip-derived cells, which failed to extend neuritic processes and attenuate Math1 promoter activity, when co-cultured with choroid plexus cells. Moreover, a blocking antibody against BMP7, a morphogenetic protein expressed in the choroid plexus, blocked the inhibitory effect of the choroid plexus, whereas purified BMP7 mimicked this effect, demonstrating causal involvement of BMP. On the other hand, the BMP antagonist NBL1 promoted neurogenesis in rhombic lip cultures from Math1 null mice displaying arrested differentiation. Our data indicate that besides its secretory and barrier functions, the choroid plexus has a novel role in attenuating the differentiation of adjacent neural progenitors.     

H2 Histamine Receptors on the Epithelial Cells of Choroid Plexus

A major site of cerebrospinal fluid production in vertebrates is the choroid plexus. The epithelial cells of the choroid plexus accumulate intracellular cyclic AMP in response to several effectors, including histamine. Since histamine is known to regulate fluid secretion in the stomach via H2 histamine receptors, we asked whether H2 receptors might also be present on epithelial cells of bovine choroid plexus. Using agonists and antagonists of histamine, we show that an agonist and antagonist pair specific for the H2 subtype were clearly more effective than an H1 agonist and antagonist pair in mimicking or inhibiting histamine stimulation of cellular cyclic AMP. Analysis by Schild plot allowed assignment of an apparent dissociation constant to the H2 antagonist metiamide which was 34-fold lower than that of its H1 counterpart, diphenhydramine. These results indicate that epithelial cells of the choroid plexus possess H2 histamine receptors.