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Cell cultures are often used to study physiological processes in health and disease. It is well-known that cells change their gene expression in vitro compared to in vivo, but it is rarely experimentally addressed. High glucose is a known trigger of apoptosis in proximal tubular cells (PTC). Here we used RNA-seq to detect differentially expressed genes in cultures of primary rat PTC, 3 days old, compared to cells retrieved directly from rat outer renal cortex and between PTC exposed to 15 mM glucose and control for 8 h. The expression of 6,174 genes was significantly up- or downregulated in the cultures of PTC compared to the cells in the outer renal cortex. Most altered were mitochondrial and metabolism related genes. Gene expression of proapoptotic proteins were upregulated and gene expression of antiapoptotic proteins were downregulated in PTC. Expression of transporter related genes were generally downregulated. After 8 h, high glucose had not altered the gene expression in PTC. The current study provides evidence that cells alter their gene expression in vitro compared to in vivo and suggests that short-term high glucose exposure can trigger apoptosis in PTC without changing the gene expression levels of apoptotic proteins. [ABSTRACT FROM AUTHOR]

Background: Blockers of angiotensin II type 1 receptor (AT1R) and the voltage gated calcium channel 1.2 (CaV1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT1R signaling and whether there is a reciprocal regulation of AT1R signaling by CaV1.2.Methods: To elucidate these questions, we have studied the Ca2+ signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of CaV1.2 blockers. Semi-quantitative imaging methods were used to assess the plasma membrane expression of AT1R and G-protein activation.Results: Repeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca2+ response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca2+ response. The up-regulation of the Ca2+ response to repeated 1 nM AngII doses and the down-regulation of the Ca2+ response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT1R plasma membrane expression. The Ca2+ response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the CaV1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT1R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT1R is sensitive to calcium influx.Conclusions: Our findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca2+ channel blockers as mono-therapy in hypertension. [ABSTRACT FROM AUTHOR]

Postsynaptic receptor trafficking plays an essential role in tuning neurotransmission and signal plasticity and has emerged as a potential therapeutic target in neuropsychiatrie disease. Using a novel application of fluorescence recovery after photobleaching in rat hippocampal neurons, we examined transport from the soma to dendrites of seven G-protein-coupled receptors (GPCRs) implicated in mood disorders. Most GPCRs were delivered to dendrites via lateral diffusion, but one GPCR, the serotonin IB receptor (5-HT1B), was delivered to the dendrites in secretory vesicles. Within the dendrites, 5-HT1Bwere stored in a reservoir of accessible vesicles that were recruited to preferential sites in plasma membrane, as observed with superecliptic pHluorin labeling. After membrane recruitment, 5-HT1B transport via lateral diffusion and temporal confinement to inhibitory and excitatory synapses was monitored by single particle tracking. These results suggest an alternative mechanism for control of neuronal activity via a GPCR that has been implicated in mood regulation. [ABSTRACT FROM AUTHOR]

Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na+,K+-ATPase. The analysis gave new information on how dense the D1 receptor and Na+,K+-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na+,K+-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc [ABSTRACT FROM AUTHOR]

Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+- ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (a3 isoform) in the postsynaptic region of the spine. Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines. [ABSTRACT FROM AUTHOR]

Sodium excretion is bidirectionally regulated by dopamine, acting on D1-like receptors (DIR) and angiotensin II, acting on AT1 receptors (AT1R). Since sodium excretion has to be regulated with great precision within a short frame of time, we tested the short-term effects of agonist binding on the function of the reciprocal receptor within the D1R-AT1R complex in renal proximal tubule cells. Exposure of rat renal proximal tubule cells to a D1 agonist was found to result in a rapid partial internalization of AT1R and complete abolishment of AT1R signaling. Similarly, exposure of rat proximal tubule cells and renal tissue to angiotensin II resulted in a rapid partial internalization of D1R and abolishment of D1R signaling. D1R and AT1R were, by use of coimmunoprecipitation studies and glutathione-S-transferase pull-down assays, shown to be partners in a multiprotein complex. Na+-K+-ATPase, the target for both receptors, was included in this complex, and a region in the COOH-terminal tail of DIR (residues 397-416) was found to interact with both AT1R and Na+-K+-ATPase. Results indicate that AT1R and D1R function as a unit of opposites, which should provide a highly versatile and sensitive system for short-term regulation of sodium excretion. [ABSTRACT FROM AUTHOR]

The dopaminergic and glutamatergic systems interact to initiate and organize normal behavior, a communication that may be perturbed in many neuropsychiatric diseases, including schizophrenia. We show here that NMDA, by allosterically modifying NMDA receptors, can act as a scaffold to recruit laterally diffusing dopamine D1 receptors (D1R) to neuronal spines. Using organotypic culture from rat striatum transfected with D1R fused to a fluorescent protein, we show that the majority of dendritic D1R are in lateral diffusion and that their mobility is confined by interaction with NMDA receptors. Exposure to NMDA reduces the diffusion coefficient for D1R and causes an increase in the number of D1R-positive spines. Unexpectedly, the action of NMDA in potentiating D1R recruitment was independent of calcium flow via the NMDA receptor channel. Thus, a highly energy-efficient, diffusion- trap mechanism can account for intraneuronal interaction between the glutamatergic and dopaminergic systems and for regulation of the number of D1R-positive spines. This diffusion trap system represents a molecular mechanism for brain plasticity and offers a promising target for development of antipsychotic therapy. [ABSTRACT FROM AUTHOR]

Dopamine D1 receptors have been implicated in various neurodevelopmental disorders, including attention-deficit/hyperactivity disorder. However, little is known about potential late maturational changes of the motor inhibitory and stimulatory role of these receptors. Here, we investigated the effects of a full and selective D1 receptor agonist, SKF-81297, on motor activity and expression of the plasticity-associated gene, c-fos , in the prefrontal cortex and striatum of juvenile and adolescent male rats. In general, SKF-81297 produced a biphasic effect on motor activity (locomotor and rearing activity), which consisted of an initial short inhibition followed by a long-lasting stimulation. These effects were dose- and age- dependent. The inhibitory phase was more pronounced in adolescent than in juvenile rats whereas the opposite was true for the stimulatory phase. During the initial inhibitory phase of the drug, c-fos mRNA expression was increased in the prefrontal cortex of juvenile rats but reduced in adolescent rats. There was also an increase in c-fos mRNA expression in the medial-dorsal striatum and olfactory tubercle, which was more evident in juvenile rats. In contrast, during the stimulatory phase, c-fos mRNA expression was increased in both the dorsal and ventral striatum, especially in the nucleus accumbens, as well as in the prefrontal cortex, in both age groups. The increase of c-fos mRNA in the dorsal striatum, however, was more pronounced in juvenile rats. These results indicate the presence of two distinct D1 receptor populations within the frontal-striatal circuitry, which have opposite effects on motor activity, and which have different maturational profiles. [ABSTRACT FROM AUTHOR]

Renal dopamine1 receptor (D1R) can be recruited from intracellular compartments to the plasma membrane by D1R agonists and endogenous dopamine. This study examines the role of the cytoskeleton for renal D1R recruitment. The studies were performed in LLCPK-1 cells that have the capacity to form dopamine from L-dopa. In approximately 50% of the cells treated with L-dopa the D1R was found to be translocated from intracellular compartments towards the plasma membrane. Disruption of the microtubulin network by nocodazole significantly prevented translocation. In contrast, depolymerization of actin had no effect. In control cells D1R colocalized with NBD-C6-ceramide, a trans-Golgi fluorescent marker. This colocalization was disrupted in L-dopa-treated cells. Tetanus toxin, an inhibitor of exocytosis, prevented L-dopa-induced receptor recruitment. L-Dopa treatment resulted in activation of protein kinase C (PKC). To test the functional effect of D1R recruitment, the capacity of D1R agonists to activate PKC was studied. Activation of D1R significantly translocated PKC-α from intracellular compartments to the plasma membrane. Disruption of microtubules abolished D1R-mediated – but not phorbol-ester-mediated – translocation of PKC. We conclude that renal D1R recruitment requires an intact microtubulin network and occurs via Golgi-derived vesicles. These newly recruited receptors couple to the PKC signaling pathway. [ABSTRACT FROM AUTHOR]

Examines selective up-regulation of dopamine D1 receptors in dendritic spines ny N-methyl-D-aspartate (NMDA) receptor activation. Recruitment of D1 receptors from the interior of the cell to the plasma membrane following NMDA activation; Ability of NMDA to balance between the D1 and D2 receptor signaling pathways.

The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3´, 5´-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons. Primary culture of striatal neurons were immunofluorescently labeled for DARPP-32 with Alexa-594 and for the dopamine D1 receptor (D1R) with atto-647N. Dual-color STED microscopy revealed discrete localizations of DARPP-32 and D1R in the spine structure, with clustered distributions in both head and neck. Dissected spine structures reveal that the DARPP-32 signal rarely overlapped with the D1R signal. The D1R receptor is positioned in an “aggregated” manner primarily in the spine head and to some extent in the neck, while DARPP-32 forms several neighboring small nanoclusters spanning the whole spine structure. The DARPP-32 clusters have a mean size of 52 +/- 6 nm, which is close to the resolution limit of the microscope and corresponds to the physical size of a few individual phosphoprotein immunocomplexes. Dissection of synaptic proteins using superresolution microscopy gives possibilities to reveal in better detail biologically relevant information, as compared to diffraction-limited microscopy. In this work, the dissected postsynaptic topology of the DARPP-32 phosphoprotein provides strong evidence for a compartmentalized and confined distribution in dendritic spines. The protein topology and the relatively low copy number of phosphoprotein provides a conception of DARPP-32’s possibilities to fine-tune the regulation of synaptic signaling, which should have an impact on the performance of the neuronal circuits in which it is expressed. [ABSTRACT FROM AUTHOR]

Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons.Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform) in the postsynaptic region of the spine.Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines. [ABSTRACT FROM AUTHOR]