Paracrine Regulation of Renal Function by Dopamine

Sources of L-DOPA

As previously mentioned, the major source of renal dopamine production is the renal tubular decarboxylation of L-DOPA taken up by renal tubule cells from the glomerular filtrate and circulation. Plasma L-DOPA, which is about 2 μM, reflects catecholamine turnover from sympathetically innervated tissues, such as skeletal muscle, heart, adrenal gland, and gut. There are non-neuronal sources of plasma L-DOPA, because patients with pure autonomic failure have normal plasma levels of L-DOPA. Indeed, L-DOPA is produced by non-neuronal cells that express TH in mesenteric organs (gastrointestinal tract, spleen, and pancreas ). Plasma L-DOPA sulfate levels increase after meals, possibly related to the amount of DOPA in the food. Inhibition of the decarboxylation of L-DOPA during a protein meal doubles its plasma levels, indicating that normally a protein meal-induced increase in plasma L-DOPA is immediately decarboxylated. Tyrosinase activation, decreased degradation of L-DOPA, and renal demethylation of 3-O-methyl DOPA may be additional sources of urinary dopamine.

Renal Tubular Uptake of L-DOPA

As already indicated, the rate-limiting step in the synthesis of dopamine by the kidney is the uptake of L-DOPA by renal tubule cells from the circulation and glomerular filtrate. The conversion of L-DOPA to dopamine occurs mainly in the proximal tubule, since AADC activity is highest in this nephron segment, although AADC is also present in more distal nephron segments. L-DOPA is taken up by the renal tubules via organic cation transporters located at both apical and basolateral membranes. These include sodium-dependent amino acid transporters, L-amino acid transporter-2 (LAT-2), and related to b0,+ amino acid transporter (rBAT), sodium-dependent L-amino acid transporters, ASCT2 and B0AT1, and as yet unidentified transporters. rBAT is predominantly expressed at the apical membrane, while LAT-2 is expressed at both apical and basolateral membranes. In normotensive rats, LAT-2 and rBAT may account for more than 50% of the L-DOPA uptake in renal proximal tubules, while ASCT2 and B0AT1 may account for the rest. In contrast in SHRs, 50% of L-DOPA uptake occurs via LAT-1, 25% via LAT-2, and 25% via ASCT2 and B0AT1. Aging is associated with a decreased tubular uptake of L-DOPA resulting in a decrease in urinary dopamine production, the mechanism of which has not been determined.

Aromatic Amino Acid Decarboxylase (AADC) Activity

Renal AADC activity is dependent on L-DOPA concentration and not on its Km. However, the increase in urinary dopamine in sodium-replete states may be caused, in part, by increased AADC activity. Patients with AADC deficiency have normal or increased levels of urinary dopamine that may be explained, in part, via tyramine hydroxylation by renal CYP2D6. Hypertonic saline can increase the expression of AADC, that is mediated, in part, by TonEBP.

Dopamine Transporters

The accumulation of monoamines from the cytoplasm to storage organelles and their secretion are dependent on vesicular monoamine transporters 1 and 2 (VMAT-1, VMAT-2). VMAT-2 is expressed in neurons and certain neuroendocrine cells, while VMAT-1 is expressed in endocrine and rat renal proximal tubule cells. VMAT-1 may participate in the dopaminergic regulation of renal ion transport, because renal tubular VMAT-1 mRNA and protein expression are increased by a high sodium diet. However, dopamine synthesized in renal tubules is not stored, but is transported both to the basolateral membrane and into the tubular lumen, where it acts on its receptors locally. Dopamine is secreted into the basolateral space via organic acid cationic transporters, while dopamine probably diffuses into the lumen where it is concentrated, reaching high nanomolar to low micromolar concentrations (see above), as a result of water reabsorption and can stimulate its receptors in more distal nephron segments.

Dopamine Metabolism

Dopamine that is synthesized in the renal tubule cells is probably not converted to norepinephrine or epinephrine. Dopamine β-hydroxylase (DBH) mRNA is not expressed in bovine or rat kidneys. However, DBH is expressed in adrenergic nerves supplying the kidney, and inhibition of DBH increases renal dopamine concentration three-fold. In tissues expressing phenylethanolamine- N -methyl transferase (PNMT) (e.g., adrenal medulla, heart, and some areas of the brain) norepinephrine is converted to epinephrine ( Figure 19.1 ). PNMT is also not expressed in human renal proximal tubules, but is expressed in glomeruli, renal distal tubules, and endothelial cells. Non-specific NMT, which is expressed in non-neuronal tissues including the kidney, enables the synthesis of epinephrine even after denervation. More recent studies have also shown that TH and DBH are expressed in mesangial and renal proximal tubule cells in culture, in a pig renal tubular cell line (LLCPK) with proximal tubule and distal tubule characteristics, in a canine distal tubular cell line, MDCK, and mouse inner medullary collecting duct cells. Because TH is not expressed in renal proximal tubules, the presence of TH in LLCPK could be indicative of its distal tubular characteristics. Although dopamine may have access to non-specific NMT, the increase in urinary dopamine with salt loading is not accompanied by an increase in urinary norepinephrine or epinephrine.

In renal tubules, dopamine is degraded both by deamination via monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetic acid (DOPAC), and by methylation via COMT to 3-methoxytyramine. Newly formed dopamine is metabolized predominantly by MAO-A rather than MAO-B, especially in the proximal tubule. In contrast, dopamine is metabolized by COMT in more distal nephron segments. A novel flavin adenine dinucleotide-dependent amine oxidase named renalase or MAO-C has been identified in the kidney, but its role in the intrarenal metabolism of renal dopamine is unknown. Acute inhibition of COMT activity may be more important than MAO in the short-term, while the converse is true for chronic inhibition. The inhibition of dopamine degrading enzymes can increase its concentration. However, the increase in urinary dopamine with high salt intake is probably not related to any decrease in dopamine degradation.

Dietary Influence Including Salt Intake

Feeding increases urinary dopamine that have been related to the amount of sodium in the diet. Most studies have shown that a low sodium intake is associated with low urinary dopamine, while the converse occurs with a high sodium diet. This could be due to an increased spillover of L-DOPA into the arterial blood, as ingestion of food/protein which contains sodium causes an increase in circulating L-DOPA. However, plasma L-DOPA levels may not be increased by a high sodium diet. Even if circulating L-DOPA is not increased by salt intake, the increase in urinary dopamine with salt loading is associated with an increase in urinary excretion of L-DOPA. Therefore, the increase in urinary excretion of dopamine appears to be secondary to an increase in the uptake of L-DOPA by renal proximal tubules, presumably from the glomerular filtrate and circulation. There may also be a mechanism by which L-DOPA is delivered to the kidney independent of circulating L-DOPA (e.g., via the adrenal gland).

Chloride may be more important than sodium in the regulation of renal dopamine production, because an increase in the intake of chloride without sodium increases urinary dopamine, while sodium bicarbonate does not. An increase in the intake of phosphate or calcium also increases renal dopamine production. Blockade of N-type, but not L-type, calcium channels can decrease urinary dopamine excretion.

An important factor in the increase in urinary dopamine caused by sodium loading is the preferential egress of dopamine into the renal tubular lumen, rather than into the interstitium. In LLC-PK1 cells dopamine egresses at the apical surface by a non-saturable process, while its egress at the basolateral surface is saturable, with the latter 5–7-fold higher than the former. The egress of dopamine in LLC-PK1 is probably not related to VMAT-1, because the egress of dopamine is not inhibited by dopamine transporter inhibitors. However, chronic sodium loading increases the expression of the VMAT-1 in the renal proximal tubule, which may contribute to the modification of the polarity of dopamine secretion during sodium loading. Inhibition of sodium hydrogen exchanger (NHE) activity decreases efflux of dopamine into the peritubular space, which presumably favors luminal outflow. Thus, dopamine, by inhibiting proximal tubular luminal NHE activity, may facilitate its own egress into the tubular lumen. The preferential luminal efflux and tubular fluid reabsorption result in high nanomolar to low micromolar concentrations of dopamine in the tubular lumen, concentrations that approach the EC ₅₀ of dopamine and D ₁ -like receptor agonists to stimulate adenylyl cyclase and phospholipase C activity. This mechanism, and the fact that dopamine can also be synthesized in more distal nephron segments, albeit to a lower extent relative to the proximal tubule, enables dopamine to stimulate its receptors in more distal nephron segments.

There may not always be a relationship among dietary sodium, urinary dopamine, and sodium excretion. Mühlbauer and colleagues have claimed that food intake rather than sodium is responsible for the increase in urinary dopamine. However, all the acute saline loading studies were performed in rats in which food, but not water, was withheld for 24 hours. Differences in the amount and duration of the sodium load in the food, as well as differences in the strains of rats studied (see below) are possible explanations. In humans, increasing sodium intake from 20 to >200 mmol/day increases renal dopamine production that peaks by the second day, followed by a gradual decline to 50% of the peak value by the 5th day. In rats, sodium chloride loading maximally increases dopamine excretion on the first few days, with the excretion decreasing close to control levels at 1 week, only to gradually increase again from 2 to 4 weeks. Renal dopamine concentrations are not higher in non-salt loaded rats than in rats loaded with salt for 6 weeks. There may also be an interaction between sodium and other substances in food, e.g., protein. Ingestion of food with very low sodium content (<0.02% sodium) is not associated with an increase in sodium excretion (unpublished data).

The renal production of dopamine is strain-dependent in rodents. Urine dopamine is lower in Sprague-Dawley than in Dahl salt-sensitive or -resistant rats or in WKY and SHRs. A strain of WKY rat increases urinary dopamine after 24 hours of salt loading at 4 weeks, but not at 12 weeks of age. Uninephrectomy increases urinary dopamine in Wistar-Han from Harlan, but not from Charles-Rivers or WKY rats from Harlan. The increase in urinary dopamine with chronic sodium chloride loading is less in the salt-sensitive C57BL/6 than the salt-resistant SJL mice from Jackson Laboratory.

The renal production of dopamine is also age-dependent. Renal dopamine synthesis increases with development. However, in contrast to the stimulatory effect of sodium intake on dopamine excretion in normal adult humans, in pre-term infants sodium supplementation prevents, while sodium restriction increases, urinary dopamine ; the significance of this in terms of sodium balance remains to be determined. The stimulatory effect of increased dietary sodium on renal dopamine production and ability to excrete salt load are diminished with aging. Urinary dopamine excretion is also decreased in patients with chronic renal failure, but due to loss of nephron number rather than an inability to synthesize dopamine.

Gender may influence the urinary dopaminergic response to a sodium load. For example, sodium loading has been reported to increase urinary dopamine excretion in Chinese females, but not Chinese males. Ethnic differences in the relationship between urine dopamine and sodium have been reported. The blunted increase in urinary dopamine in response to acute volume expansion in blacks may be caused by reduced decarboxylation of L-DOPA.

Patients with type 1 or type 2 diabetes have decreased basal urinary dopamine, as well as a deficient response to high sodium chloride diet or infusion. Decreased dopamine production in human adults and children with type 2 diabetes may affect the ability to excrete a sodium load. Rats with type 1 diabetes also have decreased renal dopamine production.

Patients with salt-sensitive, low renin or non-modulating hypertension, and some with normal renin hypertension have a blunted increase in urinary dopamine in response to salt loading, despite higher rates of L-DOPA excretion. Protein intake or high dietary sodium intake may not increase urinary dopamine in essential hypertensive patients or even normotensive subjects with a family history of hypertension. However, salt-sensitive and normal renin essential hypertensives have normal basal urinary dopamine. Mild exercise has been reported to increase renal dopamine production in Stage 1 hypertensive subjects. Borderline and young hypertensives have higher basal urinary dopamine than control subjects or stable hypertensive patients, suggesting that the renal dopaminergic system may act as an early defense against hypertension that fails during its progression.

There are also abnormalities in renal dopamine production in animal models of genetic hypertension. Renal dopamine production may be decreased in adult spontaneously hypertensive and Dahl salt-sensitive rats relative to their normotensive controls, the WKY and Dahl salt-resistant rat, respectively. However, urinary free dopamine and renal tissue dopamine are actually increased in young SHRs in comparison with WKY rats, but the difference disappears at age 16 weeks. In the young Dahl salt-sensitive rat, urinary dopamine is normal and increases with salt intake, as in Dahl salt-resistant rats, although kidney levels of dopamine are lower in Dahl salt-sensitive than in salt-resistant rats on a high salt diet. In adult Dahl salt-sensitive rats, the urinary excretion of dopamine is not increased or may even be decreased in response to acute volume expansion or salt loading. Mice deficient of D ₂ dopamine receptors have salt-sensitive hypertension and decreased renal dopamine production.

Dopamine Receptor Subtypes

Dopamine receptors probably evolved from a common ancient ancestor over 750 million years ago, before the divergence of vertebrates and invertebrates. Dopamine receptors belong to the α group of the rhodopsin-like family of GPCRs, characterized by seven transmembrane domains. Based on their ability to stimulate or inhibit adenylyl cyclase, dopamine receptors have been classified into two families ( Table 19.1 ): D ₁ -like receptors stimulate adenylyl cyclases, while D ₂ -like receptors inhibit adenylyl cyclases. There are two D ₁ -like receptors in eutherian mammals, D _1A and D _1B , also known as D ₁ and D ₅ in humans; archosaurs have four D ₁ -like receptors, D _1A, D _1B , D _1C , and D _1D ; all other jawed vertebrates have three D1-like receptors, D _1A , D _1B , and D _1C . The D ₂ -like receptors include D ₂ (D ₂ R), D ₃ (D ₃ R), and D ₄ (D ₄ R). The rank order affinity of dopamine to its receptors is: D ₃ R≥D ₄ R>D ₅ R≥D ₂ R>D ₁ R.

Based on site-directed mutagenesis and protein modeling of some GPCRs, including D ₂ R, the binding of their respective agonist has been suggested to occur at the hydrophobic transmembrane domains, i.e., aspartate residues in transmembrane domains II and III, two serine residues in transmembrane V, and a phenylalanine residue in transmembrane VI. Dopamine receptors have not been crystallized, but studies of the crystallized β2-adrenergic receptor suggest that agonist binding occurs at the extracellular ends of helices III, IV, V, and VII, with the cytosolic ends mediating G-protein activation. Nuclear magnetic resonance spectroscopy has revealed three distinct conformations of the β ₂ AR: unliganded receptor or neutral antagonist, inverse agonist, and agonist. The binding pocket of the A _2A adenosine receptor is slightly different, with the binding pocket closer to helices VI and VII, suggesting GPCR specificity.

Dopamine Receptor Signaling

Receptor activation, at least for rhodopsin, occurs by a disruption of the Arg135/Glu134 ionic bond in transmembrane VI resulting in the formation of a new Arg135/Tyr223 interaction in transmembrane V261. This causes the GPCR to function as a guanine nucleotide exchange factor promoting the exchange of GDP with GTP on the Gα-subunit, resulting in the dissociation and release of the Gα-subunit from the Gβγ-subunit. The dissociated G-protein-subunits then initiate the activation or inhibition of downstream effectors. Hydrolysis of GTP by the intrinsic GTPase activity of Gα promotes the reassociation of the G-protein-subunits. RGS (regulators of G-protein signaling) acting as a GTPase-activating protein (GAP), stimulate the hydrolysis of GTP and, therefore, the reassociation of the G-protein-subunits. RGS act as a GAP for GαS, Gαi and Gαq. While a primary function of RGS is to suppress G-protein signaling via GAP activity, RGS have non GAP actions such as direct antagonism of Gα and binding of Gβ/γ. Specific RGS have been reported to regulate dopamine receptors. For example, RGS7 with Gβ5 can regulate dopamine receptor signaling and RGS9-2, but not RGS4, inhibits D ₂ R internalization, while RGS19 inhibits D ₂ R signaling. RGS4 may be involved in D ₁ R signaling. In contrast, RGS2 may be regulated by both D ₁ R and D ₂ R.

In some instances, agonist specific ligand binding does not cause dissociation of the protein subunits, but rather a conformational change. Heterotrimeric G-proteins can also interact with proteins other than GPCRs to regulate physiological processes. These include activators of G-protein signaling (AGS) which promote guanine nucleotide exchange independent of GPCRs and activate or inhibit adenylyl cyclase or phospholipase C. Dopamine receptors have been reported to decrease AGS1 expression. In addition, GPCR signaling can be independent of G-protein-subunits. For example, the inotropic effect of angiotensin II with occupation of AT _1a R in cardiomyocytes occurs via a Gαq/PKC-independent, but GRK6/β-arrestin 2-dependent, mechanism.

Both D ₁ -like receptors, D ₁ R and D ₅ R couple to Gα _S and Gαq. There are also differences in G-protein linkage between D ₁ R and D ₅ R. For example, the D ₁ R, but not D ₅ R, also couples to Go and Gα _Olf , which is expressed in renal tubules and macula densa. In contrast, D ₅ R, but not D ₁ R, couples to Gz and Gα _12/13 . Gα _12/13 proteins may have functions different from those ascribed to GPCR activation, including cell movement, actin cytoskeleton, and protein phosphatase 2A activity. Because the recycling of D ₁ R is partly under the control of protein phosphatase 2A or a protein phosphatase 2-like enzyme, this may be a mechanism for D ₅ R and D ₁ R cross-talk.

The linkage of G-protein-subunits to the specific D ₁ -like receptor may be tissue-specific. This apparently discordant linkage of D ₁ -like receptors to G-protein-subunits can be explained by recent observations that a specific ligand acting on the same receptor recruits specific signaling proteins that elicit a specific phenotype. For example, most D ₁ -like receptor agonists stimulate both adenylyl cyclase and phospholipase C. However, there are exceptions; SKF83822 stimulates adenylyl cyclase but not phospholipase C, whereas SKF83959 stimulates phospholipase C but actually inhibits adenylyl cyclase.

Adenylyl Cyclase and Protein Kinase A (PKA)

As indicated above, D ₁ -like receptors are linked to stimulation of adenylyl cyclase which leads to the formation of cAMP. There are at least 10 isoforms of adenylyl cyclase (adenylyl cyclases I-IX and adenylyl cyclase short). In the neostriatum, the D ₁ R is linked to adenylyl cyclase V. However, adenylyl cyclase V is not expressed in the rat renal proximal tubules (but may be expressed in humans, unpublished observations). In kidney tubule cells, D ₁ R is linked to adenylyl cyclase VI, while D ₅ R is linked to adenylyl cyclase IV. cAMP binds to the regulatory subunits of PKA resulting in the activation (disinhibition) of its catalytic subunits. There are two PKA isoforms, but D ₁ -like receptors, specifically D ₁ R, may utilize PKA II rather than PKA I. There are several PKA substrates, among which are dopamine and cAMP-regulated 32 kDa phosphoprotein (DARPP-32) which inhibits protein phosphatase 1. In contrast to the stimulatory effect of the D ₁ R/PKA pathway on DARPP-32, the D ₁ R/phospholipase C pathway inhibits DARPP-32. The roles of PKA, DARPP-32, and protein-phosphatases in renal sodium transport are discussed below.

Phospholipase C

Phospholipase C catalyzes the formation of diacylglycerol and inositol phosphates. D ₁ -like receptors are linked to phospholipase C, via Gαq, independent of adenylyl cyclase, a phenomenon that was initially described in renal cortical tubules. There are several isoforms of phospholipase C; the D ₁ R directly stimulates phospholipase Cβ1 in renal cortical but not in medullary membranes, and indirectly stimulates phospholipase Cγ via PKA and PKC, in fibroblasts.

In neuroblastoma cells, the D ₁ R is not linked to G _q and in neurons, D ₁ R-mediated stimulation of phospholipase C may require the presence of D ₂ R while D ₅ R, by itself, can increase phospholipase C-mediated calcium mobilization that is inhibited by D ₂ R. However, in a pituitary adenoma rat cell line (GH4C1) transfected with the D ₅ R, the D ₅ R activation actually decreases inositol phosphate production. The report that D ₁ -like receptor-stimulated phospholipase C persists in D ₁ ^−/− mice has been taken to indicate that there is a D ₁ -like receptor other than D ₁ R that can stimulate phospholipase C. This is indeed the case, as the D ₅ R is also linked to phospholipase C activation in neural tissue (hippocampus, cortex, and striatum).

Phospholipase D

Phospholipase D (PLD) hydrolyzes phospholipids, such as phosphatidylcholine, to form phosphatidic acid and the free polar head group of the phospholipid substrate. Phosphatidic acid can be cleaved by phosphatidic acid phosphohydrolase to produce diacylglycerol; both are important second messengers in the “late” response of cells to certain stimuli. The two mammalian isoforms of PLD (PLD1 and PLD2) have ~50% identity, and are distributed widely in mammalian tissues and cells. They are believed to play an important role in the regulation of cell function and cell fate by a variety of extracellular signals. D ₅ R inhibits PLD2 activity in renal proximal tubule cells, while both D ₁ R and D ₅ R can inhibit phospholipase D activity in vascular smooth muscle cells. These actions lead to inhibition of the production of reactive oxygen species (ROS). The D ₃ R heterologously expressed in HEK293 cells has been reported to activate PLD, via Rho and independent of Gi/Go. The relationship between PLD and endogenously expressed D ₃ R remains to be determined.

Protein Kinase C

Protein kinase C (PKC) isoforms are classified into three groups: (1) conventional PKCs (cPKC-α, -βI, -βII, and -γ); (2) novel PKCs (nPKC-δ, -ε, -θ, and -η/G); and (3) atypical PKCs (aPKC-λ(ι) and -ξ) and PKCμ, also called PKD which consists of PKD1, PKD2, and PKD3. In renal tubule cells, D ₁ -like receptors can stimulate PKCθ and PKCξ, and inhibit PKCδ. The D ₁ R increases the phosphorylation of PKCθ ^S676 in human embryonic kidney (HEK293) cells. D ₁ -like receptors can also translocate specific PKC isoforms. Thus, D ₁ -like receptors translocate conventional (α, β) and novel (ε) PKCs from renal tubule cytosol into membranes, and the novel PKC-δ from membranes to cytosol. Some studies have shown that the stimulation of PKC by dopamine may be secondary to PKA activation.

While PKA and PKC mediate the actions of D ₁ R, both can also regulate D ₁ R signaling. PKC activation can produce differential D ₁ R and D ₅ R signaling. PKC can increase the membrane expression of D ₁ R in vascular smooth muscle cells. In HEK293 cells, novel PKC isoforms stimulate D ₁ R signaling, but inhibit D ₅ R signaling. However, in HEK293T cells, PKCα, β1, γ, δ, and ε constitutively phosphorylate and dampen D ₁ R signaling. Ethanol enhances D ₁ R signaling by inhibiting PKCγ and PKCδ. PKA and PKC have been reported to phosphorylate D ₁ R, modulating the rate of agonist-induced D ₁ R desensitization and intracellular trafficking. RanBP9 and RanBP10 interact with both PKCδ/γ to increase the phosphorylation and desensitization of D ₁ R in neural and renal proximal tubule cells. RanBP9 and RanBP10 may function as scaffolding proteins to regulate the spatial and temporal organization of D ₁ R and PKC. In renal proximal tubule cells, NHERF-1 is necessary for the D ₁ R-mediated stimulation of cAMP accumulation and PKC activity.

D ₂ -Like Receptors

The D ₂ -like receptors couple to G-proteins Gα _i and G _o , and inhibit adenylyl cyclases.

There are two isoforms of D ₂ R; postsynaptic D ₂ R effects are mediated by the long isoform, D _2L R, while the presynaptic D ₂ R effects are mediated by the short isoform, D _2S R. The D _2S R is localized at the plasma membrane, while the D _2L R is localized in the perinuclear region in several cell lines heterologously expressing D _2S R and D _2L R. However, in renal proximal tubule cells that express the D _2L R rather than the D _2S R, the D _2L R is expressed mainly at the plasma membrane. The particular G-protein-subunit that interacts specifically with either D ₂ R isoform is not clear. The D ₂ R can couple to the same extent to Gαi (Gαi2 and Gαi3) and Gz, but Gαo may be the major G-protein that is activated by D _2S R and D _2L R ; neither D _2S R nor D _2L R subtype is linked to Gq11 or Gα _12/13 . A splice variant of Gαi2, sGα _i2 , is important in the intracellular localization of D ₂ R, and its dissociation from D ₂ R following D ₂ R agonist stimulation increases cell surface D ₂ R expression.

The D ₄ R, like the D ₂ R, couples to Gαi (Gα _i2 and Gα _i3 ), Gαo, Gz, and Gαt2. Different numbers of 16 amino acid repeats in the third cytoplasmic loop cause several human D ₄ R isoforms (e.g., D4-2, D4-4, and D4-7). The role of these D ₄ R isoforms in cell function remains to be determined. However, the D ₄ R long (at least one 7 to 10 repeat) has been reported to be associated with higher diastolic and systolic blood pressure.

The D ₃ R linkage to Gα _i is not robust, in contrast to that observed for D ₂ R and D ₄ R. In rat renal proximal tubule cells the D ₃ R stimulation of phospholipase C is via Gα _i3 . The signaling of D ₃ R, like the D ₂ R and D ₄ R, may be mediated by Gαo. The utilization of G-proteins in D ₂ R and D ₃ R-mediated stimulation of extracellular signal-regulated kinase differs, D ₂ R utilizes Gα _i while D ₃ R utilizes Gα/ο and the βγ-subunit of G _i . The D ₃ R also couples to Gz and, in the presence of pertussis toxin, the D ₃ R can also couple to Gαs. The D ₃ R can also couple to Gq11. The linkage of the D ₃ R to other effectors, such as inhibition of K ⁺ and Ca ²⁺ , may be more sensitive than its weak linkage to G-proteins. The D ₃ R is also insensitive to GTP, relative to the other dopamine receptor subtypes. There could be seven distinct alternatively spliced D ₃ R variants. The full-length D ₃ R and a shorter receptor isoform, the D ₃ S, bind to dopamine. The five other alternatively spliced D ₃ R variants do not bind dopamine, but one of them, D ₃ Rnf, regulates receptor dimerization.

Adenylyl Cyclase

Although D ₂ -like receptors inhibit adenylyl cyclase activity, in many cell lines heterologously expressing the D ₃ R, its linkage to Gα _i is not robust. However, the D ₃ R robustly inhibits adenylyl cyclase isoform V, but because this adenylyl cyclase isoform is not expressed in the rat renal proximal tubule, it is expressed in human renal proximal tubule cells (unpublished data). D ₃ R action in this rat nephron segment is not due to Gα _i signaling. Because D ₃ R can couple to Gαs in the presence of pertussis toxin, the D ₃ R could increase adenylyl cyclase activity in this nephron segment in the rat. This has not been directly tested, but D ₁ R and D ₃ R dimerization increases the ability of dopamine to stimulate adenylyl cyclase activity. In contrast, the co-expression of D ₂ R with D ₃ R increases the ability of D ₂ -like agonists to inhibit adenylyl cyclase activity. This becomes a conundrum in cells expressing these dopamine receptor subtypes, but at least in renal proximal tubules there is no evidence of D ₁ R and D ₂ R or D ₂ R and D ₃ R heterodimerization (unpublished data). However, in the kidney and vascular smooth muscle D ₁ R and D ₃ R do interact, and this interaction probably predominates over the other possible D ₁ -like and D ₂ -like interactions. While D ₂ R, by itself, stimulates Na ⁺ K ⁺ -ATPase activity, simultaneous stimulation of D ₁ -like and other D ₂ -like receptors inhibits Na ⁺ K ⁺ -ATPase activity. Furthermore, D _2L R and D _4.4 R can potentiate PKC-stimulated adenylyl cyclase II activity through the release of β/γ-subunits, and D _2L R can sensitize adenylyl cyclase VI. Although these adenylyl cyclase isoforms are expressed in renal proximal tubule cells, this interaction probably does not occur in renal proximal tubule cells because quinpirole, a D ₂ -like receptor agonist with preference for the D ₃ R and D ₄ R over the D ₂ R, does not stimulate adenylyl cyclase activity (unpublished studies). As stated above, there are species differences because adenylyl cyclase V is expressed in the human (unpublished data) but not in the rat renal proximal tubule.

Phospholipase C

In neural cells, D ₂ R, via Gβ/γ, stimulates phospholipase C and specifically phospholipase Cβ4 in the intermediate lobe of the pituitary gland _. D ₁ R and D ₂ R can interact to stimulate phospholipase C in neural cells, but as stated above there is no evidence for D ₂ R and D ₃ R interaction in renal proximal tubule cells. D ₂ R and D ₅ R can also heterodimerize to stimulate phospholipase C in neural tissue and HEK-293 cells. The D _2S R can also stimulate phospholipase D, but the latter enzyme is inhibited by D ₅ R. These effects need not be counter-regulatory, because as previously mentioned the D _2S R is presynaptic, while the D ₅ R inhibition of phospholipase D is postsynaptic in renal proximal tubule cells (in particular). In addition, presynaptic inhibition of adrenergic neurotransmitters by D _2S R facilitates the inhibitory effects of dopamine on ion transport.

Dopamine Regulation of Ion Channels

D ₁ -like receptors inhibit voltage-gated K ⁺ channels and G-protein-regulated inward rectifying channels, but stimulate L-type Ca ²⁺ channels. D ₁ -like receptors inhibit G-protein-dependent inward rectifier K-like channels in medial prefrontal cortex via PKC. In contrast, D ₂ -like receptors stimulate voltage-gated K ⁺ channels and G-protein-regulated inward rectifying channels, but inhibit L-type Ca ²⁺ channels. Both D ₁ -like and D ₂ -like receptors inhibit N-, P-, and Q-type Ca ²⁺ channels and transient sodium channels, but stimulate persistent P sodium channels. We have reported that the D ₃ R-mediated vasorelaxation of rat mesenteric arteries is due to stimulation of small- and/or large-Ca ²⁺ -activated K ⁺ channels. In human coronary vascular smooth muscle cells, D ₅ R stimulates Ca ²⁺ -activated big K ⁺ channel, via cAMP-mediated stimulation of PKG.

Dopamine Regulation of Mitogen-Activated Protein (MAP) Kinase and other Kinases

D ₁ -like receptors may activate MAP kinases, including p38MAP kinase, and c-jun amino-terminal kinase. In the brain, D ₁ -like receptors can also activate ERK, but ERK1/2 is actually inhibited by D ₅ R but not by D ₁ R in human renal proximal tubule cells. D ₂ -like receptors can also activate MAP kinase via Gβ/γ. D ₂ -like receptor-mediated stimulation of Na ⁺ K ⁺ -ATPase in the kidney is linked to the p44/42 MAP kinase pathway. Aberrant D ₁ R activation of ERK1/2/MAP kinase has been reported in D ₁ R supersensitivity. However, in the kidney, MAP kinase reduces D ₁ R affinity and G-protein coupling.

Dopamine inhibits DNA synthesis of cultured human mesangial cells. In rat vascular smooth muscle cells, D ₁ R or D ₃ R does not affect vascular smooth muscle cell proliferation, but inhibits the proliferative effect of insulin and norepinephrine. D ₂ R, via inhibition of MAP kinase, inhibits angiotensin II-induced hypertrophy of cultured neonatal rat ventricular myocytes. However, dopamine increases the phosphorylation of and activates p44/42 MAP kinases in rabbit renal proximal tubule cells in primary culture. In human renal proximal tubule cells and Chinese hamster ovary cells, the D ₃ R promotes mitogenesis or cell proliferation through the activation of MAP kinases. The variable effects of dopamine on MAP kinase in smooth muscle and epithelial cells may be related to tissue- or cell-specificity.

DARPP-32

As stated above, DARPP-32 was identified initially as a major target for dopamine-activated adenylyl cyclase in striatum. Since the mid-1980s, DARPP-32 has been acknowledged as a crucial mediator of the biochemical, electrophysiological, transcriptional, and behavioral effects of dopamine. The state of DARPP-32 phosphorylation has been shown to provide a mechanism for integrating information arriving at dopaminoceptive neurons, in multiple brain regions, via a variety of neurotransmitters, neuromodulators, neuropeptides, and steroid hormones. Activation of PKA or PKG stimulates DARPP-32 phosphorylation at Thr, and thereby converts DARPP-32 into a potent inhibitor of protein phosphatase-1 (PP1). Protein phosphatase-2B (PP2B) is the most effective protein phosphatase in dephosphorylating DARPP-32 at Thr. Thus, DARPP-32 acts as an amplifier of PKA and PKG-mediated signaling when it is phosphorylated on Thr, which converts it into an inhibitor of PP1. The role of DARPP-32 in signaling has turned out to be very complex. Under basal conditions, DARPP-32 is phosphorylated at Thr and inhibits PKA. Thus, DARPP-32 has the unique property of acting either as an inhibitor of PP1 or an inhibitor of PKA. However, under hyperdopaminergic conditions, the phosphorylated state of Thr is reduced, allowing increased phosphorylation at Thr. This positive feedback loop acts as a switch to potentiate dopaminergic signaling. Cdk5, a cyclin-dependent kinase family member, also phosphorylates DARPP-32 at Thr. Furthermore, protein phosphatase 2A dephosphorylates DARPP-32 both at Thr and Thr, and the state of phosphorylation of DARPP-32 at Thr depends on the phosphorylation state of two serine residues, Ser and Ser. DARPP-32 has been demonstrated with biochemical, immunohistochemical, and in situ hybridization techniques to have an anatomical distribution similar to that of dopaminoceptive cells possessing D ₁ Rs in the central nervous system. DARPP-32 is also expressed in several peripheral tissues, including brown fat cells, parathyroid, retina, and renal tissue. The ability of D ₁ R to stimulate protein phosphatase 2A may serve as a negative feedback in the D ₁ R and DARPP-32 relationship, and prevent the inhibitory effect of DARPP-32 on PKA.

Regulation of Dopamine Receptor Function