Sodium excretion is bidirectionally regulated by dopamine, acting on D1-like receptors (D1R) 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 ϩ -K ϩ -ATPase, the target for both receptors, was included in this complex, and a region in the COOH-terminal tail of D1R (residues 397-416) was found to interact with both AT1R and Na ϩ -K ϩ -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.AT 1 receptors; Na ϩ -K ϩ -ATPase; calcium signaling RENAL SODIUM EXCRETION IS bidirectionally regulated by angiotensin II (ANG II) and dopamine (13). Long-term dopamine exposure is known to decrease AT 1 receptors (AT1R) in renal proximal tubular cells (7). Furthermore, studies by Zeng et al. (21) have shown that long-term stimulation of AT1R results in an upregulation of D1-like receptors (D1R). This effect was not observed in spontaneously hypertensive rats, indicating that the interaction between AT1R and D1R has an impact on blood pressure regulation. Since sodium excretion must be regulated with great precision over a short period of time, it is important that control mechanisms are able to exert their effects within a short time frame. The aim of the current study has been to explore the short-term effects of ANG II exposure on D1R and the short-term effects of a D1-agonist on AT1R. Our approach has been to test the hypothesis that AT1R and D1R form a heteromeric signaling complex, where activation of either receptor may cause internalization and/or interruption of the signaling capacity of the other.The studies were performed using rat proximal tubule cells, since these cells express both AT1R and D1R in both the apical and the basolateral membrane (12,18). Previous studies from our laboratory have shown that in these cells Na ϩ -K ϩ -ATPase, the enzyme responsible for active sodium transport, is bidirectionally regulated by ANG II and dopamine (2). MATERIALS AND METHODS Cells and tissue.All studies were performed using outer cortical tissue from young (3-5 wk) male Sprague-Dawley rats. Immediately after the animals were killed, 250-m slices were taken from the outer renal cortex using a microtome. The outer 250-m region of the rat renal cortex contains Ͼ90% proxi...

BackgroundThe 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.ResultsWith 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.ConclusionsA 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.

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.

In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,KATPase in the plasma membrane.

Here we report that the 20kD C‐terminus tail of the heparan sulfate proteoglycan agrin(C20) can activate a calcium signaling pathway that acts to protect neurons from apoptosis. It is now well documented that the ubiquitous integral plasma membrane protein Na,K‐ATPase(NKA) functions both as an ion pump and a signal transducer. Previous studies have shown that ouabain, a specific ligand of the catalytic α subunit of NKA, can trigger signaling pathways that involve activation of Inositol 1,4,5‐trisphosphate receptor (IP3R). In COS7 cells expressing either the ubiquitous endogenous α1 subunit or transfected with the neuron specific α3 subunit, C20 triggers highly regular, slow calcium oscillations with a power spectrum almost identical to that observed for ouabain, and the NKAα binding sites for C20 and for ouabain are shown to partially overlap. Exposure of cells to C20 enhances the interaction between NKA and IP3R, while expression of a peptide that disrupts the interaction between NKA and IP3R significantly reduces the efficacy of agrin signaling. Slow calcium oscillations will typically activate NF‐kB, which in most systems will have an anti‐apoptotic effect. C20, in concentrations that trigger calcium oscillations, also activate NF‐kB and protects from H2O2 apoptosis in neurons. Our results indicate that C20, which in the synapse is cleaved from agrin, may act as an endogenous neuroprotector.