During conditions of moderate sodium excess, the dopaminergic system sits at the fulcrum of homeostatic control of water and electrolyte balance and blood pressure (1, 2). Dopamine promotes natriuresis by inhibiting sodium chloride reabsorption in specific segments of the nephron. Dopamine exerts its action on dopamine receptors, which belong to the family of G protein-coupled receptors (GPCRs). The signal transduction that follows ligand occupation of a GPCR is tightly regulated to limit the specificity and extent of cellular response. GPCR-mediated signal transduction is rapidly dampened via receptor desensitization or the waning of the responsiveness of the receptor to agonist with time. Desensitization involves receptor phosphorylation and is carried out by either GPCR kinases (GRKs) or second messenger-activated kinases such as protein kinase A and protein kinase C. Homologous desensitization involves GRKs that selectively phosphorylate only agonist-activated receptors, whereas heterologous desensitization is carried out by second messenger-dependent kinases that indiscriminately phosphorylate agonist-activated receptors and those that have not been exposed to the agonist (7).The GRKs are serine/threonine protein kinases comprising seven isoforms that are grouped into three subfamilies. GRK1 and GRK7 belong to the rhodopsin kinase subfamily and are expressed exclusively in the retina (8 -10). GRK2 and GRK3 phosphorylate the ␤-adrenergic receptor and belong to the ␤-adrenergic receptor kinase subfamily (11), and GRK4, GRK5, and GRK6 belong to the GRK4 subfamily. GRK4 is highly enriched in the testis and, to a lesser degree, in the kidneys (12, 13). Four splice variants of human GRK4 result from the alternative splicing of exons 2 and 15 (11). GRK4-␣ is considered the full-length version, whereas GRK4-␤, -␥, and -␦ are shortened versions of . The coding region of the GRK4 gene, whose 4p16.3 locus has been linked to essential hypertension (15, 16), contains several single nucleotide polymorphisms, including R65L, A142V, and A486V, which have been linked to * This work was supported, in whole or in part, by National Institutes of Health Grants HL023081, HL074940, DK039308, HL092196, and HL068686.

Dopamine cellular signaling, via the D 1 receptor (D 1 R), involves both protein kinase A (PKA) and protein kinase C (PKC), but the PKC isoform involved has not been determined. Therefore, we tested the hypothesis that the D 1 R-mediated inhibition of NADPH oxidase activity involves crosstalk between PKA and specific PKC isoform(s). In HEK-293 cells heterologously expressing human D 1 R (HEK-hD 1 ), fenoldopam, a D 1 R agonist, and phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited oxidase activity in a time-and concentration-dependent manner. The D 1 R-mediated inhibition of oxidase activity (68.1±3.6%) was attenuated by two different PKA inhibitors, H89 (10 µmol/L) (88±8.1%) and Rp-cAMP (10 µmol/L) (97.7±6.7%), and two different PKC inhibitors, bisindolylmaleimide I (1 µmol/L) (94±6%) and staurosporine (10 nmol/ L) (93±8%), which by themselves, had no effect (n=4-8/group). The inhibitory effect of PMA (1 µmol/L) on oxidase activity (73±3.2%) was blocked by H89 (100±7.8%) (n=5-6/group). The PMA-mediated inhibition of NADPH oxidase activity was accompanied by an increase in PKCθ S676 , an effect that was also blocked by H89. Fenoldopam (1 µmol/L) also increased PKCθ S676 in HEK-hD 1 and human renal proximal tubule (RPT) cells. Knockdown of PKCθ with siRNA in RPT cells prevented the inhibitory effect of fenoldopam on NADPH oxidase activity. Our studies demonstrate for the first time that cross-talk between PKA and PKCθ plays an important role in the D 1 R-mediated negative regulation of NADPH oxidase activity in human kidney cells.

Dopamine-mediated regulation of Na+-K+-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na+-K+-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na+-K+-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na+-K+-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na+-K+-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na+-K+-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na+-K+-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis.

D1‐like dopamine receptors inhibit NADPH oxidase activity via stimulation of protein kinase A (PKA) and inhibition of phospholipase D. We tested the hypothesis that a cross‐talk between PKA and protein kinase C (PKC) is involved in the regulation of NADPH oxidase activity in HEK‐293 cells heterologously expressing human D1 dopamine receptor (hD1R) (n=6/group). The D1‐like receptor agonist, fenoldopam (Fen., 1 μM), decreased NADPH oxidase activity (63±5.0% vs. vehicle=101.6±6.2%) that was completely blocked by the D1‐like receptor antagonist, Sch23390 (5μM) (84±6.2%), which, by itself, had no effect. The PKA inhibitor, H89 (10 μM), by itself, had no effect on NADPH oxidase activity but prevented (85.6±5.0%) the inhibitory effect of fenoldopam (P<0.01, vs. others, ANOVA Newman‐Keuls test). The D1R‐mediated decrease in oxidase activity (65±3.0%) was also completely reversed by PKC inhibitors, e.g., bisinolylmaleimide I (1 μM)(94±6.0%) and staurosporine (10 nM) (93.4±7.8%), which, by themselves, had no effect (P<0.001). Moreover, the PKC activator, phobol‐12‐myristate‐13‐acetate (PMA,1 μM), inhibited oxidase activity (72.7±8.1%), that was accompanied by increased PKCθS647 phosphorylation, effects that were also abolished by H89 (103±7.4%), which, by itself, had no effect (P<0.05). We conclude that D1R inhibition of NADPH oxidase activity is dependent on both PKA and PKC (PKCθS647) signaling. Our studies demonstrate for the first time that a cross‐talk between PKA and PKC signaling pathways plays an important role in the D1R negative regulation of NADPH oxidase activity in human kidney cells.