Glutamate, by activating N-methyl-D-aspartate (NMDA) receptors, alters the balance between dopamine D1 and D2 receptor signaling, but the mechanism responsible for this effect has not been known. We report here, using immunocytochemistry of primary cultures of rat neostriatal neurons, that activation of NMDA receptors recruits D1 receptors from the interior of the cell to the plasma membrane while having no effect on the distribution of D2 receptors. The D1 receptors were concentrated in spines as shown by colocalization with phalloidin-labeled actin filaments. The effect of NMDA on D1 receptors was abolished by incubation of cells in calcium-free medium and was mimicked by the calcium ionophore ionomycin. Recruitment of D1 receptors from the interior of the cell to the membrane was confirmed by subcellular fractionation. The recruited D1 receptors were functional as demonstrated by an increase in dopamine-sensitive adenylyl cyclase activity in membranes derived from cells that had been pretreated with NMDA. These results provide evidence for regulated recruitment of a G protein-coupled receptor in neurons, provide a cell biological basis for the effect of NMDA on dopamine signaling, and reconcile the conflicting hyperdopaminergic and hypoglutamatergic hypotheses of schizophrenia. Increasing evidence indicates that interactions between different types of neurotransmitter receptors represent a major mechanism of neuronal plasticity. A variety of physiological and pharmacological stimuli alter the efficacy of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic transmission (1, 2). These changes in signaling have been accounted for in large part by changes in the distribution of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors between spines and the interior of the cell (3, 4). Although signaling in the dopaminergic pathway also shows a great deal of plasticity, little information is available about its cellular basis. In the present study, we examined the possibility that an alteration in the distribution of dopamine receptors might contribute to the ability of N-methyl-D-aspartate (NMDA) to alter the balance between D1 and D2 receptor signaling pathways. Materials and MethodsCell Culture. Cultures of striatal neurons were prepared from 18-to 19-day rat embryos. The cells were plated on poly-D-lysinecoated glass coverslips and cultured in Eagle's MEM plus F-12 (1:1). The cultures were grown in the presence of 5% FBS, penicillin, or streptomycin (100 mg͞ml). To suppress the growth of glial cells, FBS was equilibrated with N2 (1%) 2 days after plating, and cytosine arabinoside (5 M) was added for 48 h to the culture medium on day 5. Cells were maintained in culture for 2-3 weeks before experiments; at that time, virtually all cells from embryonic rat striatum seemed to be mature neurons. They resembled typical medium spiny neurons and stained positively for neuronal markers (5).Immunostaining. Cells were fixed for 20 min in ice-cold 2% paraformaldehyde, permeabilized with 0....

Short-term regulation of sodium metabolism is dependent on the modulation of the activity of sodium transporters by first and second messengers. In understanding diseases associated with sodium retention, it is necessary to identify the coupling between these messengers. We have examined whether dopamine, an important first messenger in tubular cells, activates and translocates various protein kinase C (PKC) isoforms. We used a proximal tubular-like cell line, LLCPK-1 cells, in which dopamine was found to inhibit Na(+)-K(+)-ATPase in a PKC-dependent manner. Translocation of PKC isoforms was studied with both subcellular fractionation and confocal microscopy. Both techniques revealed a dopamine-induced translocation from cytosol to plasma membrane of PKC-alpha and -epsilon, but not of PKC-delta, -gamma, and -zeta. The process of subcellular fractionation resulted in partial translocation of PKC-epsilon. This artifact was eliminated in confocal studies. Confocal imaging permitted detection of translocation within 20 s. Translocation was abolished by a phospholipase C inhibitor and by an antagonist against the dopamine 1 subtype (D(1)) but not the 2 subtype of receptor (D(2)). In conclusion, this study visualizes in renal epithelial cells a very rapid activation of the PKC-alpha and -epsilon isoforms by the D(1) receptor subtype.

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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-C(6)-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-alpha 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.