Evidence for the interaction of endophilin a3 with endogenous Kca2.3 channels in PC12 cells.

BACKGROUND/AIMS: Small-conductance calcium-activated (SK) channels play an important role by controlling the after-hyperpolarization of excitable cells. The level of expression and density of these channels is an essential factor for controlling different cellular functions. Several studies showed a co-localization of K(Ca)2.3 channels and Endophilin A3 in different tissues. Endophilin A3 belongs to a family of BAR- and SH3 domain containing proteins that bind to dynamin and are involved in the process of vesicle scission in clathrin-mediated endocytosis. METHODS: Using the yeast two-hybrid system and the GST pull down assay we demonstrated that Endophilin A3 interacts with the N-terminal part of K(Ca)2.3 channels. In addition, we studied the impact of this interaction on channel activity by patch clamp measurements in PC12 cells expressing endogenous K(Ca)2.3 channels. K(Ca)2.3 currents were activated by using pipette solutions containing 1 µM free Ca(2+). RESULTS: Whole-cell measurements of PC12 cells transfected with Endophilin A3 showed a reduction of KCa2.3 specific Cs(+) currents indicating that the interaction of Endophilin A3 with K(Ca)2.3 channels also occurs in mammalian cells and that this interaction has functional consequences for current flowing through K(Ca)2.3 channels. Since K(Ca)2.3 specific currents could be increased in PC12 cells transfected with Endophilin A3 with DC-EBIO (30 µM), a known SK-channel activator, these data also implicate that Endophilin A3 did not significantly remove K(Ca)2.3 channels from the membrane but changed the sensitivity of the channels to Ca(2+) which could be overcome by DC-EBIO. CONCLUSION: This interaction seems to be important for the function of K(Ca)2.3 channels and might therefore play a significant role in situations where channel activation is pivotal for cellular function.

Modulation of sodium transport in alveolar epithelial cells by estradiol and progesterone.

The effects of estradiol (E2) and progesterone (P) on alveolar epithelial Na+ transport were studied in isolated alveolar epithelial cells from 18- to 19-d GA rat fetuses, grown to confluence in serum-free media supplemented with E2 (0-1 µM) and P (0-2.8 µM). Short-circuit currents (ISC) were measured, showing an increase by E2 and P in a dose-dependent manner. The Na,K-ATPase subunits -α1 and -ß1 were detected by Western blotting, but total expression was not significantly altered. Furthermore, all three epithelial Na+ channel (ENaC) subunits -α, -ß, and -γ were detected, with trends toward a higher expression in the presence of E2 and P. Real-time PCR revealed an increase of α- and ß-ENaC expression but no alteration of γ-ENaC. In addition, the mRNA expression of cystic fibrosis transmembrane conductance regulator (CFTR) and Na,K-ATPase-ß1 subunit were elevated in the presence of E2 and P. Single-channel patch clamp analysis demonstrated putative highly selective and nonselective cation channels in the analyzed cells, with a higher percentage of responsive patches under the influence of E2 and P. We conclude that E2 and P increased Na+ transport in alveolar epithelial cells by enhancing the expression and activity of ENaC and Na,K-ATPase.

AQP4 expression in striatal primary cultures is regulated by dopamine--implications for proliferation of astrocytes.

Proliferation of astrocytes plays an essential role during ontogeny and in the adult brain, where it occurs following trauma and in inflammation and neurodegenerative diseases as well as in normal, healthy mammals. The cellular mechanisms underlying glial proliferation remain poorly understood. As dopamine is known to modulate proliferation in different cell populations, we investigated the effects of dopamine on the proliferation of striatal astrocytes in vitro. We found that dopamine reduced proliferation. As proliferation involves, among other things, a change in cell volume, which normally comes with water movement across the membrane, water channels might represent a molecular target of the dopamine effect. Therefore we studied the effect of dopamine on aquaporin 4 (AQP4) expression, the main aquaporin subtype expressed in glial cells, and observed a down-regulation of the AQP4-M23 isoform. This down-regulation was the cause of the dopamine-induced decrease in proliferation as knockdown of AQP4 using siRNA techniques mimicked the effects of dopamine on proliferation. Furthermore, stimulation of glial proliferation by basic fibroblast growth factor was also abolished by knocking down AQP4. In addition, blocking of AQP4 with 10 mum tetraethylammonium inhibited osmotically induced cell swelling and stimulation of glial cell proliferation by basic fibroblast growth factor. These results demonstrate a clear-cut involvement of AQP4 in the regulation of proliferation and implicate that modulation of AQP4 could be used therapeutically in the treatment of neurodegenerative diseases as well as in the regulation of reactive astrogliosis by preventing or reducing the glia scar formation, thus improving regeneration following ischemia or other trauma.

Functional alterations of the nigrostriatal dopamine system in estrogen receptor-alpha knockout (ERKO) mice.

Estrogen represents an important factor for the development and function of the nigrostriatal dopamine system. Estrogen also controls sex-specific differentiation and activity of the nigrostriatal dopaminergic system. We used an estrogen receptor-alpha knockout (-/-) model (ERKO) to study the influence of this particular receptor subtype on the regulation of functional characteristics of the male and female nigrostriatal dopamine system. On the striatal level, we found a sex-specific regulation of dopamine D1 receptors (D1) and dopamine receptor-interacting protein 78 (Drip78). In female (-/-) mice D1 receptor expression levels were increased compared to wild type (wt) animals, whereas in male (-/-) mice Drip78 mRNA levels were decreased compared to wt. In the midbrain, expression of tyrosine hydroxylase (TH) and brain-derived neurotrophic factor (BDNF) was reduced in (-/-) mice of both sexes. Glial cell line-derived neurotrophic factor (GDNF) expression was not affected. These data demonstrate that the integrity of estrogen receptor-alpha (ERalpha) signalling is necessary for the regulation of gene expression of proteins known to be important for the function of the nigrostriatal system at the postsynaptic striatal and presynaptic midbrain level.

Regulation of glutamate transporter GLAST and GLT-1 expression in astrocytes by estrogen.

Estrogen influences neuronal development and a broad spectrum of neural functions. In addition, several lines of evidence suggest a role as neuroprotective factor for estrogen in the CNS. Neuroprotection can result from direct estrogen-neuron interactions or be mediated indirectly involving the regulation of physiological properties of nonneuronal cells, such as astrocytes and microglia. Increased l-glutamate levels are associated with neurotoxic and neurodegenerative processes in the brain. Thus, the removal of l-glutamate from the extracellular space by astrocytes through the astroglial glutamate transporters GLT-1 and GLAST appears essential for maintaining a homeostatic milieu for neighboring neurons. We have therefore studied the influence of 17beta-estradiol on l-glutamate metabolism in cultured astrocytes from the neonate mouse midbrain using quantitative RT-PCR and Western blotting for both transporters as well as functional l-glutamate uptake studies. The administration of estrogen significantly increased the expression of GLT-1 and GLAST on the mRNA and protein level. Likewise, specific l-glutamate uptake by astrocytes was elevated after estrogen exposure and mimicked by dbcAMP stimulation. Induction of transporter expression and l-glutamate uptake were sensitive to ICI 182,780 treatment suggesting estrogen action through nuclear estrogen receptors. These findings indicate that estrogen can prevent l-glutamate-related cell death by decreasing extracellular l-glutamate levels through an increased l-glutamate uptake capacity by astrocytes.

Cell type-specificity of nonclassical estrogen signaling in the developing midbrain.

Estrogens have widespread biological functions in the CNS involving the coordination of developmental processes, the regulation of cell physiology, and the control of neuroendocrine systems. In the midbrain, estrogens promote the survival, maturation, and function of neurons and, in particular, of dopamine cells. Aside from classical signaling through nuclear estrogen receptors, we have provided evidence that cellular transmission of estrogen effects in the midbrain comprises a complex intracellular signaling scenario. The major conclusion drawn from our studies is that estrogens interact with yet unidentified membrane receptor complexes which stimulate the phospholipase C and induce the formation of inosite-tri-phosphate (IP(3)). This causes a rapid and transitory rise in intracellular free calcium. The modulation of calcium homeostasis is the primary nonclassical physiological response to estrogens in all cell types. Surprisingly, a different secondary downstream signaling cascade seems to be activated in each estrogen-responsive cell population, i.e. phosphatidylinositol-3 kinase (PI3-kinase) in GABAergic and cAMP/ protein kinase A (PKA) in dopaminergic neurons, mitogen-activated protein kinase (MAP-kinase) in astrocytes. The precise biological role of estrogens for the different cell types is still fragmentary. We assume that estrogens positively influence intracellular signaling mechanisms which are important for cell differentiation and survival. It remains to be elucidated what determines the cell type-specificity of these estrogen responses.

Regulation of gene expression in the developing midbrain by estrogen: implication of classical and nonclassical steroid signaling.

Estrogen plays an important role during midbrain development. This is indicated by the presence of nuclear estrogen receptors and the transient expression of the estrogen-forming enzyme aromatase. A number of recent studies have shown that estrogen promotes the differentiation and survival, as well as physiological performance, of midbrain dopaminergic cells. In addition, we have reported that both ways of cellular estrogen signaling (classical and nonclassical) as well as interactions with nonneuronal target cells are involved in the transmission of intra- and intercellular estrogen effects in this brain region. This study provides additional evidence that (i) estrogen is capable of regulating gene expression in cultured embryonic neurons and astrocytes differently and (ii) both signaling mechanisms, i.e., classically through nuclear receptors and nonclassically through the stimulation of membrane-estrogen receptors, which are coupled to distinct intracellular signal transduction cascades, contribute diversely to gene regulation. These data reveal a high degree of complexity of estrogen action at the genomic level in the developing brain. Further studies are warranted to unravel the exact contribution of the differently regulated genes for developmental estrogen action.

Developmental expression of progesterone receptor isoforms in the mouse midbrain.

Progesterone participates in the regulation of developmental processes in the brain and controls the function of distinct neural circuits. We have studied the expression of progesterone receptor (PR) isoforms in the developing and adult male and female mouse ventral midbrain. Transcripts of both receptor isoforms (PR-A and B) were detectable pre- and postnatally but regulated differentially during ontogeny. Immunoblotting revealed that only the full-length form (PR-B) is transcribed transiently into protein, whereas the truncated PR-A isoform is not detectable as protein. Although the precise function of progesterone in the developing CNS is not fully understood, our data implicate a potential role for PR signaling for the developing nigrostriatal system.

6-Hydroxydopamine leads to T2 hyperintensity, decreased claudin-3 immunoreactivity and altered aquaporin 4 expression in the striatum.

The neurotoxin 6-hydroxydopamine (6-OHDA) is frequently used in animal models to mimic Parkinson's disease. Imaging studies describe hyperintense signalling in regions close to the site of the 6-OHDA injection in T2-weighted (T2w) magnetic resonance imaging (MRI). The nature of this hyperintense signal remains elusive and still is matter of discussion. Here we demonstrate hyperintense signalling in T2w MRI and decreased apparent diffusion coefficient (ADC) values following intraventricular injection of 6-OHDA. Moreover, we show decreased GFAP immunoreactivity in brain regions corresponding to the region revealing the hyperintense signalling, probably indicating a loss of astrocytes due to a toxic effect of 6-OHDA. In the striatum, where no hyperintense signalling in MRI was observed following intraventricular 6-OHDA injection, immunohistochemical and molecular analyses revealed an altered expression of the water channel aquaporin 4 and the emergence of vasogenic edema, indicated by an increased perivascular space. Moreover, a significant decrease of claudin-3 immunoreactivity was observed, implying alterations in the blood brain barrier. These findings indicate that intraventricular injection of 6-OHDA results (1) in effects close to the ventricles that can be detected as hyperintense signalling in T2w MRI accompanied by reduced ADC values and (2) in effects on brain regions not adjacent to the ventricles, where a disturbance of water homeostasis occurs. We clearly demonstrate that 6-OHDA leads to brain edema that in turn may affect the overall results of experiments (e.g. behavioral alterations). Therefore, when using 6-OHDA in Parkinson's models effects that are not mediated by degeneration of catecholaminergic neurons have to be considered.

Dopamine regulates the expression of the glutamate transporter GLT1 but not GLAST in developing striatal astrocytes.

Dopamine and L: -glutamate are important signals which guide the development of functional neural circuits within the striatal complex. Disequilibrium of these neurotransmitter systems is believed to be etiological for the genesis of neurological and psychiatric diseases. Since dopamine plays a crucial role for the early transmitter-regulated differentiation of striatal GABAergic neurons, we emphasized that dopaminergic transmission may also be involved in the fine tuning of intra-striatal glutamate action. In this study, we report that dopamine decreases the expression of the glutamate transporter GLT1 but not GLAST in striatal astrocytes by measuring gene and protein expression. Using glutamate-uptake approaches, we demonstrate an increase in glutamate clearance of externally added glutamate in dopamine-treated cultures compared to controls. Our findings imply that dopamine regulates the availability of L: -glutamate in the developing striatum. It is also suggested that the application of dopaminergic drugs can interfere with ontogenetic processes within the striatal complex.

BDNF-dependent stimulation of dopamine D5 receptor expression in developing striatal astrocytes involves PI3-kinase signaling.

It is well known that brain-derived neurotrophic factor (BDNF) and the early nigrostriatal dopaminergic input are implicated in the regulation of developmental processes in the neostriatum. There is growing evidence that interactions between these developmental signals rather than singular actions are critical for cellular differentiation and compartmentation of the striatum. In the present report, our goal is to identify striatal target cells for BDNF and dopamine. Using primary neuronal and astroglial cell cultures, we have demonstrated that BDNF selectively regulates D(5) but not D(1) receptor expression in astrocytes. This effect was not observed in neurons. Pharmacological approaches indicated that BDNF effects on dopamine D(5) receptor expression were mediated at the intracellular level by an activation of the PI3- but not MAP-kinase cascade. FACS analysis and confocal laser microscopy revealed that the newly synthesized D(5) receptors were integrated into the plasma membrane of astrocytes. Our findings clearly show that developing striatal astrocytes are targets for BDNF. Furthermore, BDNF appears to regulate the dopamine responsiveness of astrocytes. This implicates that functional interactions between BDNF, dopamine, and astrocytes are necessary to warrant proper differentiation of the striatal anlage.

Ontogenetic expression of estrogen and progesterone receptors in the mouse lung.

In the mammalian lung, estrogen and progesterone seem to be import for the morphological and functional maturation. Despite this, only sparse information is available on the onset and ontogenic expression of estrogen receptors (ER) and progesterone receptors (PR) in the perinatal lung. The expression patterns of the two known nuclear isoforms ER-alpha and ER-beta and the PR were analyzed in the pre- and postnatal lungs of BALB/c mice. Steroid receptor mRNA expression of all three receptors were highest in the prenatal lung and declined thereafter to significantly lower levels in the postnatal and adult lung. The ontogenetic pattern of ER and PR expression supports the view that both gonadal steroids are pivotal for prenatal lung maturation and development.