Intranasal Losartan Decreases Perivascular Beta Amyloid, Inflammation, and the Decline of Neurogenesis in Hypertensive Rats.

The contribution of the local angiotensin receptor system to neuroinflammation, impaired neurogenesis, and amyloid beta (Aß) accumulation in Alzheimer's disease (AD) and in hypertension is consistent with the remarkable neuroprotection provided by angiotensin receptor blockers (ARBs) independent of their blood pressure-lowering effect. Considering the causal relationship between hypertension and AD and that targeting cerebrovascular pathology with ARBs does not necessarily require their systemic effects, we tested intranasal losartan in the rat model of chronic hypertension (spontaneously hypertensive stroke-prone rats, SHRSP). Intranasal losartan at a subdepressor dose decreased mortality, neuroinflammation, and perivascular content of Aß by enhancing key players in its metabolism and clearance, including insulin-degrading enzyme, neprilysin, and transthyretin. Furthermore, this treatment improved neurologic deficits and increased brain IL-10 concentration, hippocampal cell survival, neurogenesis, and choroid plexus cell proliferation in SHRSP. Losartan (1 µM) also reduced LDH release from cultured astroglial cells in response to toxic glutamate concentrations. This effect was completely blunted by IL-10 antibodies. These findings suggest that intranasal ARB treatment is a neuroprotective, neurogenesis-inducing, and Aß-decreasing strategy for the treatment of hypertensive stroke and cerebral amyloid angiopathy acting at least partly through the IL-10 pathway.

Age-dependent astroglial vulnerability to hypoxia and glutamate: the role for erythropoietin.

Extracellular accumulation of toxic concentrations of glutamate (Glu) is a hallmark of many neurodegenerative diseases, often accompanied by hypoxia and impaired metabolism of this neuromediator. To address the question whether the multifunctional neuroprotective action of erythropoietin (EPO) extends to the regulation of extracellular Glu-level and is age-related, young and culture-aged rat astroglial primary cells (APC) were simultaneously treated with 1mM Glu and/or human recombinant EPO under normoxic and hypoxic conditions (NC and HC). EPO increased the Glu uptake by astrocytes under both NC and especially upon HC in culture-aged APC (by 60%). Moreover, treatment with EPO up-regulated the activity of glutamine synthetase (GS), the expression of glutamate-aspartate transporter (GLAST) and the level of EPO mRNA. EPO alleviated the Glu- and hypoxia-induced LDH release from astrocytes. These protective EPO effects were concentration-dependent and they were strongly intensified with age in culture. More than a 4-fold increase in apoptosis and a 2-fold decrease in GS enzyme activity was observed in APC transfected with EPO receptor (EPOR)-siRNA. Our in vivo data show decreased expression of EPO and a strong increase of EPOR in brain homogenates of APP/PS1 mice and their wild type controls during aging. Comparison of APP/PS1 and age-matched WT control mice revealed a stronger expression of EPOR but a weaker one of EPO in the Alzheimer's disease (AD) model mice. Here we show for the first time the direct correlation between the extent of differentiation (age) of astrocytes and the efficacy of EPO in balancing extracellular glutamate clearance and metabolism in an in-vitro model of hypoxia and Glu-induced astroglial injury. The clinical relevance of EPO and EPOR as markers of brain cells vulnerability during aging and neurodegeneration is evidenced by remarkable changes in their expression levels in a transgenic model of AD and their WT controls.

Therapeutic efficacy of intranasally delivered mesenchymal stem cells in a rat model of Parkinson disease.

Safe and effective cell delivery remains one of the main challenges in cell-based therapy of neurodegenerative disorders. Graft survival, sufficient enrichment of therapeutic cells in the brain, and avoidance of their distribution throughout the peripheral organs are greatly influenced by the method of delivery. Here we demonstrate for the first time noninvasive intranasal (IN) delivery of mesenchymal stem cells (MSCs) to the brains of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. IN application (INA) of MSCs resulted in the appearance of cells in the olfactory bulb, cortex, hippocampus, striatum, cerebellum, brainstem, and spinal cord. Out of 1 × 106 MSCs applied intranasally, 24% survived for at least 4.5 months in the brains of 6-OHDA rats as assessed by quantification of enhanced green fluorescent protein (EGFP) DNA. Quantification of proliferating cell nuclear antigen-positive EGFP-MSCs showed that 3% of applied MSCs were proliferative 4.5 months after application. INA of MSCs increased the tyrosine hydroxylase level in the lesioned ipsilateral striatum and substantia nigra, and completely eliminated the 6-OHDA-induced increase in terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine, 5'-triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining of these areas. INA of EGFP-labeled MSCs prevented any decrease in the dopamine level in the lesioned hemisphere, whereas the lesioned side of the control animals revealed significantly lower levels of dopamine 4.5 months after 6-OHDA treatment. Behavioral analyses revealed significant and substantial improvement of motor function of the Parkinsonian forepaw to up to 68% of the normal value 40-110 days after INA of 1 × 106 cells. MSC-INA decreased the concentrations of inflammatory cytokines-interleukin-1ß (IL-1ß), IL-2, -6, -12, tumor necrosis factor (TNF), interferon-γ (IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF)-in the lesioned side to their levels in the intact hemisphere. IN administration provides a highly promising noninvasive alternative to the traumatic surgical procedure of transplantation and allows targeted delivery of cells to the brain with the option of chronic application.

Intranasal delivery of cells to the brain.

The safety and efficacy of cell-based therapies for neurodegenerative diseases depends on the mode of cell administration. We hypothesized that intranasally administered cells could bypass the blood-brain barrier by migrating from the nasal mucosa through the cribriform plate along the olfactory neural pathway into the brain and cerebrospinal fluid (CSF). This would minimize or eliminate the distribution of cellular grafts to peripheral organs and will help to dispense with neurosurgical cell implantation. Here we demonstrate transnasal delivery of cells to the brain following intranasal application of fluorescently labeled rat mesenchymal stem cells (MSC) or human glioma cells to naive mice and rats. After cells crossed the cribriform plate, two migration routes were identified: (1) migration into the olfactory bulb and to other parts of the brain; (2) entry into the CSF with movement along the surface of the cortex followed by entrance into the brain parenchyma. The delivery of cells was enhanced by hyaluronidase treatment applied intranasally 30 min prior to the application of cells. Intranasal delivery provides a new non-invasive method for cell delivery to the CNS.

Lentiviral transfection of ependymal primary cultures facilitates the characterisation of kinocilia-specific promoters.

Ependymal primary cultures (EPCs) are an established model for studying ependymal cell biochemistry and the biology of kinocilia-bearing cells. However, the difficulty in causing them to express transgenes at high efficiency has been an important drawback of the system. Indeed plasmid-based transfection attempts remain at an efficiency below 1% and fail to elicit reporter gene expression, namely green fluorescent protein (GFP) synthesis, in any of the kinocilia-bearing cells of the cultures. Human immunodeficiency virus pseudotyped with the vesicular stomatitis virus envelope glycoprotein (HIV/VSV-G) and encoding GFP under the control of the ubiquitously recognised promoter of elongation factor 1 alpha (EF1alpha) also does not cause transgene expression in the kinocilia-bearing cells of an EPC when applied at multiplicities of infection (MOIs) of up to 40 and destroys the culture when the MOI is increased further. In contrast, HIV/VSV-G encoding GFP under the control of a promoter specifically active in kinocilia-bearing cells leads to transgene expression in up to 79% of the kinociliated cells of an EPC when applied at an MOI of 20. This has permitted the initial characterisation of the promoter for the gene specifically transcribed in kinocilia-bearing cells, wdr16. The results have identified two regions of 100 nucleotides length each, which are critical for promoter activity and contain putative binding sites for the transcription factors Foxd1, Sox17 and Spz1. It appears that wdr16 is controlled by a bidirectional promoter also responsible for regulating the syntaxin 8 gene.

Expression of pyruvate carboxylase in cultured oligodendroglial, microglial and ependymal cells.

The mitochondrial enzyme, pyruvate carboxylase (PC; EC 6.4.1.1) is considered to play a significant role in the intermediary metabolism of neural tissue. PC-catalyzed carboxylation of pyruvate to oxaloacetate is a major anaplerotic reaction in brain. Anaplerosis is essential for homeostasis of the members of the tricarboxylic acid (TCA) cycle. Several biochemical pathways rely on withdrawing TCA cycle members. Prominent among these are biosynthesis of fatty acids and of non-essential amino acids such as aspartate, asparagine, glutamate and glutamine, gluconeogenesis, glycogen synthesis, and regeneration of NADPH. The expression of PC in brain has already been described and assigned to astrocytes. Since pyruvate carboxylase deficiency is associated with malformations of the brain, e.g., inadequate development of the corpus callosum and the lack of myelination, one can hypothesize that PC may be expressed also in glial cells other than astrocytes. Therefore, the expression of PC was investigated in cultured oligodendroglial, microglial, and ependymal cells. As assessed by RT-PCR, all these cultures contain PC mRNA. This mRNA is generated in a transcription process that is regulated by the "distal class" of promoters of the PC gene. The expression of PC among cultured glial cells was studied with a rabbit antiserum by immunoblotting and immunocytochemistry. The results indicate that PC is not only expressed in cultured astroglial cells but also in cultured oligodendrocytes, microglial cells, and ependymocytes. It appears that the intermediary metabolism of these cells includes the anaplerotic action of PC as well as possibly also functions of the enzyme in biosynthetic pathways and the provision of NADPH for defense against reactive oxygen species.

Expression of the brain and muscle isoforms of glycogen phosphorylase in rat heart.

Heart glycogen represents a store of glucosyl residues which are mobilized by the catalysis of glycogen phosphorylase (GP) and are mainly destined to serve as substrates for the generation of ATP. The brain isoform of GP (GP BB) was studied in rat heart in comparison with the muscle isoform (GP MM) to find functional analogies to the brain. Western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed that at the protein level, but not at the mRNA level, the content of GP BB is similar in heart and brain. In contrast, GP MM is more abundant in the heart than in the brain. Immunocytochemically GP BB was colocalized with GP MM in cardiomyocytes. GP MM was also detected in interstitial cells identified as fibroblasts. The physiological role of co-expression of GP BB and GP MM in cardiomyocytes and in brain astrocytes is discussed in a comparative way.

Renal expression of the brain and muscle isoforms of glycogen phosphorylase in different cell types.

Kidney contains glycogen. Glycogen is degraded by glycogen phosphorylase (GP). This enzyme comes in three isoforms, one of which, the brain isozyme (GP BB), is known to occur in kidney. Its pattern of distribution in rat kidney was studied in comparison to that of the muscle isoform (GP MM) with the aim to see if for GP BB and GP MM there were functional similarities in brain and kidney. In immunoblotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments, both isozymes and their respective mRNAs were found in kidney homogenates. GP BB was immunocytochemically detected in collecting ducts which were identified by the marker protein aquaporin-2. GP MM was localized exclusively in interstitial cells of cortex and outer medulla. These cells were identified as fibroblasts by their expression of 5'-ectonucleotidase (cortex) or by their morphology (outer medulla). The physiological role of both isozymes is discussed in respect to local demands of energy and of proteoglycan building blocks.

Expression of 3-hydroxyisobutyrate dehydrogenase in cultured neural cells.

The branched-chain amino acids (BCAAs)--isoleucine, leucine, and valine--belong to the limited group of substances transported through the blood-brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched-chain alpha-keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate-limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched-chain amino acids. Unlike the acyl-CoA derivates in the irreversible part of valine catabolism, 3-hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine-derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3-hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity-purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT-PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double-labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy.

Thrombin causes the enrichment of rat brain primary cultures with ependymal cells via protease-activated receptor 1.

Ependymal cell culture models from rat have been developed over the last 20 years to facilitate biochemical studies on this least-studied glial cell type. The cell culture protocol calls for the presence of thrombin, which is essential for obtaining a high proportion of multiciliated ependymal cells. The serine protease appears to act via protease-activated receptor 1 to prevent the apoptosis of ependymal precursors and enhance their proliferation without affecting contaminating cells. Unciliated precursors differentiate into polyciliated ependymocytes by passing through a stage of monociliation. The message for protease-activated receptor (PAR) 1 is initially abundant in the cultures, but its level declines as the cells differentiate. Besides PAR 1, signalling through PAR 2 also promotes ciliation in rat brain primary cultures, albeit to a lesser degree than the thrombin receptor. Thrombin and other proteases may be involved in the regulation of ventricular wall development. This action would be mediated mainly by PAR1.

Angiotensin receptor type 1 blockade in astroglia decreases hypoxia-induced cell damage and TNF alpha release.

The present study investigated the role of angiotensin receptors (AT-R) in the survival and inflammatory response of astroglia upon hypoxic injury. Exposure of rat astroglial primary cultures (APC) to hypoxic conditions (HC) led to decreased viability of the cells and to a 3.5-fold increase in TNF-alpha release. AT-R type1 (AT1-R) antagonist losartan and its metabolite EXP3174 decrease the LDH release (by 36 +/- 9%; 45 +/- 6%) from APC under HC. Losartan diminished TNF-alpha release (by 40 +/- 15%) and the number of TUNEL-cells by 204 +/- 38% under HC, alone and together with angiotensin II (ATII), while EXP3174 was dependent on ATII for its effect on TNF-alpha. The AT2-R antagonist, PD123.319, did not influence the release of LDH and TNF-alpha under normoxic (NC) and HC. These data suggest that AT1-R may decrease the susceptibility of astrocytes to hypoxic injury and their propensity to release TNF-alpha. AT1-R antagonists may therefore be of therapeutic value during hypoxia-associated neurodegeneration.

Biosynthesis of Wdr16, a marker protein for kinocilia-bearing cells, starts at the time of kinocilia formation in rat, and wdr16 gene knockdown causes hydrocephalus in zebrafish.

The rat ortholog of the WD40 repeat protein Wdr16 is abundantly expressed in testis and cultured ependymal cells. Low levels are found in lung and brain, respectively, while it is absent from kinocilia-free tissues. In testis and ependymal primary cultures, Wdr16 messenger RNA appears concomitantly with the messages for sperm-associated antigen 6, a kinocilia marker, and for hydin, a protein linked to ciliary function and hydrocephalus. In testis, ependyma and respiratory epithelium, the Wdr16 protein is up-regulated together with kinocilia formation. The wdr16 gene is restricted to genera in possession of kinocilia, and it is strongly conserved during evolution. The human and zebrafish proteins are identical in 62% of their aligned amino acids. On the message level, the zebrafish Wdr16 ortholog was found exclusively in kinocilia-bearing tissues by in situ hybridisation. Gene knockdown in zebrafish embryos by antisense morpholino injection resulted in severe hydrocephalus formation with unaltered ependymal morphology or ciliary beat. Wdr16 can be considered a differentiation marker of kinocilia-bearing cells. In the brain, it appears to be functionally related to water homeostasis or osmoregulation.

Distribution of secretory pathway Ca2+ ATPase (SPCA1) in neuronal and glial cell cultures.

1. Secretory pathway Ca(2+) ATPase type 1 (SPCA1) is a newly recognized Ca(2+)/Mn(2+)-transporting pump localized in membranes of the Golgi apparatus. 2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues. 3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains. 4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells. 5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically. 6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, alpha-tubulin or MBP. 7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells. 8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.

Immunocytochemical localization of 3-methylcrotonyl-CoA carboxylase in cultured ependymal, microglial and oligodendroglial cells.

To evaluate the ability of ependymal, microglial and oligodendroglial cells to degrade leucine, the presence of 3-methylcrotonyl-CoA carboxylase (MCC) was investigated in cultures of these cells. MCC is a biotin-containing heterodimeric enzyme that is specific for the irreversible part of the leucine catabolic pathway. It has been reported previously that in cell culture MCC is expressed in astrocytes and a subpopulation of neurones. In the present study ependymal, microglial and oligodendroglial cell cultures, derived from the brains of newborn rats, were examined for the expression of MCC by RT-PCR, western blotting and immunocytochemistry. The results of RT-PCR and western blotting showed the presence of mRNA as well as protein of both subunits of MCC in ependymal, microglial and oligodendroglial cell cultures. Immunocytochemical investigation of the cellular and subcellular distribution of MCC demonstrated a mitochondrial location of MCC in all neuroglial cell types investigated. The ubiquitous expression of MCC in glial cells demonstrates the ability of the cells to engage in the catabolism of leucine transported into the brain, mainly for the generation of energy.

Natriuretic peptides, but not nitric oxide donors, elevate levels of cytosolic guanosine 3',5'-cyclic monophosphate in ependymal cells ex vivo.

Atrial natriuretic peptide-(1-28) (ANP), brain natriuretic peptide-(1-32) (BNP) and C-Type natriuretic polypeptide (CNP) occur in the brain, are concentrated in the anteroventral area of the third cerebral ventricle and participate in the regulation of body fluid homeostasis. The ventricles of the mammalian brain are lined by a continuous monolayered epithelium of polyciliated ependymal cells. In the adult rat, the ependymocytes continue to express the intermediate filament vimentin, but do not contain glial fibrillary acidic protein. Ependymal functions are poorly understood, but may extend to osmoregulation and volume sensing. Ependymal cells possess receptors for the natriuretic peptides, and in cell culture respond to them with an increase in their cyclic GMP content. In this study, a cyclic GMP-specific antibody was employed together with an ex vivo brain slice system to assess the ependymal response to ANP, BNP and CNP under close to life-like conditions. While ANP in concentrations of 0.1 nM and 1 nM had no effect, at concentrations of 10nM and 100 nM it increased ependymal cyclic GMP levels in a concentration-dependent manner. The other natriuretic peptides BNP, and CNP, also increased the cyclic GMP content of ependymocytes, while nitric oxide (NO) donors had no effect. However, in contrast to the natriuretic peptides, the NO donors elevated the level of cyclic GMP in the brain parenchyma below the ependymal layer.

Glycogen metabolism in rat ependymal primary cultures: regulation by serotonin.

Ependymal primary cultures are a model for studying ependymal energy metabolism. Intracellular glycogen is built up in the cultures dependent on culture age and the presence of glucose and glutamate. This energy store is mobilized upon glucose withdrawal, stimulation with isoproterenol, forskolin or serotonin and after uncoupling of oxidative phosphorylation from ATP production. Serotonin regulates ependymal glycogen metabolism predominantly via 5-HT receptor (5-HTR) 7, which elicits an increase in the level of ependymal cyclic AMP. Although the most abundant mRNAs for serotonin receptors are those of 5-HTR 2B and 5-HTR 3A, ependymal cells in primary culture do not respond to serotonin with an increase in their concentration of cytosolic calcium ions. The mRNAs of 5-HTRs 1A, 6, 1B, 5B, 7, 1/2C and 5A are also detectable in order of decreasing abundance. The mRNAs for 5-HTRs 1D, 1F, 3B and 4 are absent from the cultured cells. The ability of serotonin to mobilize ependymal glycogen depends on the culture age and the time allowed for glycogen buildup. During glycogen buildup time, glutamate is consumed by the cells. An increased ability of 5-HT to mobilize ependymal glycogen stores is noticed after the depletion of glutamate from the glycogen buildup medium. In ependymal primary cultures, cilia are colocalized with glycogen phosphorylase isozyme BB, while the MM isoform is not expressed. It is known from the literature that an increase in the concentration of cytosolic cAMP in ependymal cells leads to a decrease in ciliary beat frequency. Therefore, the present data point towards a function for ependymal glycogen other than supplying energy for the movement of cilia.

Uptake and metabolism of serotonin by ependymal primary cultures.

Serotonin uptake and metabolism was studied in ependymal primary cultures. Serotonin uptake was facilitated by two different systems, one of which was the neuronal serotonin transporter SERT, exhibiting a Vmax value of 3.8+/-0.1 pmol x min(-1) x (mg protein)(-1) and an apparent Michaelis-Menten constant of 0.41+/-0.03 microM. The main product of metabolism was 5-hydroxyindole acetic acid, which resulted from the action of monoamine oxidase A. This enzyme showed a maximal rate of 0.85+/-0.02 nmol x min(-1) x (mg protein)(-1) and a Michaelis-Menten constant of 78+/-5 microM. Ependymal cells were able to dispose of extracellular serotonin with initial rates of approximately 600 pmol x min(-1) x (mg protein)(-1) and of 4 pmol x min(-1) x (mg protein)(-1) when challenged with 500 microM and 1 microM extracellular serotonin, respectively. Ependymal cells are concluded to facilitate the "sink" action of the CSF by removing waste compounds upon passing of the fluid from the parenchymal extracellular space into the ventricular system.

Regulation by insulin and insulin-like growth factor of 2-deoxyglucose uptake in primary ependymal cell cultures.

Ependymal cells have been reported to express the facilitative glucose carriers GLUT1, GLUT2, and GLUT4, as well as glucokinase. They are therefore speculated to be part of the cerebral glucose sensing system and may also respond to insulin with alterations in their glucose uptake rate. A cell culture model was employed to study the functional status of ependymal insulin-regulated glucose uptake in vitro. Insulin increased the uptake of the model substrate 2-deoxyglucose (2-DG) dependent on the insulin concentration. This was due to a near doubling of the maximal 2-DG uptake rate. Insulin-like growth factor (IGF-1) was at least 10 times more potent than insulin in stimulating the rate of ependymal 2-DG uptake, suggesting that IGF-1, rather than insulin, is the physiological agonist regulating glucose transport in ependymal cells. The predominant glucose transporter in ependymal cell cultures was found to be GLUT1, which is apparently regulated by IGF-1 in ependymal cells.

Atrial natriuretic peptides elevate cyclic GMP levels in primary cultures of rat ependymal cells.

The aim of this study was to examine the effect of atrial natriuretic peptides on primary cultures of ependymal cells, as measured by changes in intracellular levels of cyclic GMP. Incubation of ependymal cells with rat atrial natriuretic peptide-(1-28) (rANP) elicited a 30-fold increase in ependymal cGMP content within 1 min and more than a 100-fold increase within 10 min to a plateau value of approximately 30 pmol/mg protein. The C-type natriuretic peptide (CNP) elicited a similar increase in cGMP levels; however the maximal effect was observed within 1 min and the levels subsequently dropped by 90% to a low plateau within 10 min. A comparison of the concentration-response curves for rANP, human ANP-(1-28) (hANP) and CNP showed that rANP, hANP and CNP had similar effects, with regards to elevation of cGMP levels at high concentrations, but with differing EC50 values. These results demonstrate the presence of a heterogenous population of functional ANP receptors i n cultured ependymalcells suggesting that ANP may regulate specific ependymal cell activity.