The CDP-choline cycle involves three sequential enzymatic reactions. In the first, catalyzed by choline kinase, a monophosphate is transferred from ATP to the hydroxyl oxygen of the choline, yielding phosphocholine. In the second, catalyzed by CTP : phosphocholine cytidylyltransferase (CT), cytidine-5′-monophosphate (CMP) is transferred from CTP to the phosphorus of phosphocholine, yielding cytidine-5′-diphosphocholine (also known as CDP-choline or citicoline). As discussed below, much of the CTP the human brain uses for this reaction derives from circulating uridine.33 In the third and last reaction, catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT), the phosphocholine of CDP-choline is bonded to the hydroxyl group on the 3-carbon of diacylglycerol (DAG), yielding the PC. DAG molecules containing a polyunsaturated fatty acid (PUFA) at the 2-position are preferentially utilized for this reaction.34 All three PC precursors must be obtained by brain entirely or in large part from the circulation, and because the PC-synthesizing enzymes that act on all three have low affinities for these substrates, blood levels of all three can affect the overall rate of PC synthesis.4,7
Thus, choline administration increases brain phosphocholine levels in rats35 and humans,36 because choline kinase's Km value for choline (2.6 mM)37 is much higher than usual brain choline levels (30–60 µM).38–40 Most commonly, the second CT-catalyzed reaction most influences the overall rate of PC synthesis, either because not all of the CT enzyme is fully activated by being attached to a cellular membrane41 or because local CTP concentrations are insufficient to saturate the CT. Thus, when brain CTP levels are increased by giving animals uridine,42 CTP's circulating precursor in human blood,33 PC synthesis is accelerated.42 The activity of CPT and the extent to which this enzyme is saturated with DAG can also control the overall rate of PC synthesis43: In PC-12 cells, nerve growth factor increased DAG levels fivefold, CPT activity by 70%, and the incorporation of choline into PC twofold. If rodents are given a standard diet supplemented with choline and uridine (as its monophosphate, UMP) and, also, by gavage, DHA, brain PC synthesis rapidly increases7,42 and absolute levels of PC per cell (i.e., deoxyribonucleic acid [DNA]) or per milligram of protein rise substantially (e.g., by 30% or more after several weeks of daily treatment7 (Table 1).
Sources of plasma and brain uridine
Few data are available as to whether foods other than milk contain significant quantities of free uridine or uridine-containing nucleotides, or whether consumption of any naturally occurring food, by adults, can substantially increase plasma uridine levels. What is known is that pyrimidines as well as purines are constituents of nucleic acids, i.e., ribonucleic acid (RNA), which contains uridine and cytidine, and DNA, which contains cytidine. Since RNA and DNA are components of all cells, any food consumed by humans that contains cells (e.g., meats, poultry, fish, vegetables, fruits, etc.) is, at least theoretically, a good source of nucleic acids and perhaps also of plasma pyrimidines. Evidence from in vitro studies suggests that, following enzymatic breakdown of dietary nucleic acids, pyrimidine compounds are taken up into the blood from the intestine, although no in vivo study has demonstrated, in adults, an actual increase in plasma uridine levels after eating an RNA- or DNA-containing food. The nucleic acids in foods or in breast milk have been shown in in vitro studies to be degraded to yield purine and pyrimidine nucleotides; nucleosides; and free bases.53,54 In vitro, RNA is digested by ribonucleases to yield uridine nucleotides, and these can be further hydrolyzed to uridine by phosphatases in the intestinal mucosa.55
Uridine is present as such in breast milk and also as constituents of RNA, nucleotides (5′-UMP), and nucleotide adducts (uridine-5′-diphosphate [UDP]-glucose, UDP-galactose).56,57 The total available uridine contents of pooled milk samples from 100 European women determined by a method that simulated in vivo digestion56 (i.e., by enzymatically degrading nucleic acids, nucleotides, and nucleotide adducts) were 32, 48, and 47 µM, respectively, for mothers of 2- to 10-day-old, 1-month-old, and 3-month-old babies. Available cytidine contents in the same samples were 86, 102, and 96 µM.56 Synthetic infant formulas are also routinely fortified with uridine and cytidine monophosphates.
Uridine is transported across the intestinal mucosal epithelium as such58,59 or as uracil, the free base. In rat small intestine, cytidine derived from RNA or DNA is partly deaminated to uridine.53 In humans, this deamination in intestinal mucosa and liver is probably much greater than in rats, since exogenously administered cytidine is almost undetectable as such in human plasma.33
The transport of pyrimidine nucleosides and bases across the small intestine is mediated by the sodium-dependent concentrative nucleoside transporters CNT1 and CNT2.60 The kinetic properties of this uptake have not yet been determined. Following intestinal absorption, uridine and uracil are transferred via the portal vein to the liver. In rats, the liver is probably the major organ modulating plasma uridine concentrations: more than 90% of the uridine that enters the liver via the portal vein is metabolized in a single pass61; moreover, uridine's concentration in hepatic venous plasma (1.32 ± 0.45 µM) is slightly higher than in portal (1.03 ± 0.3 µM) or arterial (1.06 ± 0.2 µM) blood, indicating that some of the uridine in the hepatic venous blood derived from de novo hepatic synthesis.
Uridine and cytidine are transported across cellular membranes in all tissues,62 including the brain, via two families of transport proteins: the Na+-independent, low-affinity, equilibrative transporters (ENT1 and ENT2; SLC29 family) and the Na+-dependent, high-affinity, concentrative transporters (CNT1, CNT2, and CNT3; SLC28 family). The two ENT proteins exhibit Km values for both uridine and cytidine in the high micromolar range (100–800 µM)63; thus, they probably mediate BBB pyrimidine uptake only when plasma levels have been elevated experimentally. In contrast, CNT2, which transports both uridine and purines like adenosine, probably mediates BBB uridine transport under physiological conditions: its Km values for uridine (and adenosine) are in the low micromolar range (9–40 µM), whereas plasma uridine levels are subsaturating, i.e., 0.9–3.9 µM in rats, 3.1–4.9 µM in humans, and around 6.5 µM in gerbils. Pyrimidines also may be taken up into brain via the choroid plexus epithelium; however, because the surface area of the BBB is so much greater (i.e., in humans 21.6 m2 versus 0.021 m2), it is clear the BBB is the major locus of uridine uptake.
Uridine and cytidine are phosphorylated to their respective nucleotides by various kinases. Thus, uridine-cytidine kinase (ATP : uridine 5′-phosphotransferase, Enzyme Commission [EC] no. 188.8.131.52) converts to UMP64,65; UMP is then converted to UDP by UMP-CMP kinase (ATP : CMP phosphotransferase, EC 184.108.40.206)66–68 and to UTP by nucleoside diphosphate kinases (nucleoside triphosphate: nucleoside diphosphate phosphotransferase, EC 220.127.116.11).65 Interconversions of uridine and cytidine and of their respective nucleotides also occur in mammalian cells. Cytidine and CMP can be deaminated to uridine and UMP,69,70 while UTP is aminated to CTP by CTP synthase (UTP : ammonia ligase [ADP-forming], E.C. 18.104.22.168).70
All of the above enzymes are unsaturated with their respective nucleoside or nucleotide substrates in brain and other tissues. For example, the Km values for uridine of uridine-cytidine kinase prepared from various tissues varied between 33 and 270 µM21,64,65 and prepared from recombinant mouse brain enzyme was 40 µM.71 Brain uridine and cytidine levels are about 22–46 pmol/mg wet weight42,72 and 6–43 pmol/mg wet weight,42 respectively. Hence, the syntheses of UTP and CTP, and the subsequent syntheses of brain PC and PE via the Kennedy pathway, depend on the availability of their pyrimidine substrates. Indeed, an increase in the supply of uridine or cytidine to neuronal cells, in vitro46,73,74 or in vivo,42 enhanced the phosphorylation of uridine and cytidine, elevating the levels of UTP, CTP, and CDP-choline.
Brain levels of particular uridine-containing compounds following uridine administration were examined in gerbils given a single dose of UMP (1 mmol/kg) 42 by gavage and killed between 5 minutes and 8 hours thereafter. Thirty minutes after gavage, plasma uridine levels were increased from 6.6 ± 0.58 to 32.7 ± 1.85 µM (P < 0.001) and brain uridine levels from 22.6 ± 2.9 to 89.1 ± 8.82 pmol/mg tissue (P < 0.001). UMP also significantly increased plasma and brain cytidine levels. However, both basally and following UMP administration, these levels were much lower than those of uridine, rising from 1.2 µM to 1.9 µM in plasma and from 5 pmol/mg tissue to 12 pmol/mg tissue in brain 30–60 minutes after gavage. (In human subjects receiving oral cytidine as CDP-choline, plasma cytidine levels did not rise detectably at all).33 Brain UTP, CTP, and CDP-choline were all elevated in gerbils 15 minutes after UMP administration (from 254 ± 31.9 to 417 ± 50.2 [P < 0.05]; 56.8 ± 1.8 to 71.7 ± 1.8 [P < 0.001]; and 11.3 ± 0.5 to 16.4 ± 1 [P < 0.001] pmol/mg tissue, respectively), returning to basal levels after 20 and 50 minutes. The smallest UMP dose that significantly increased brain CDP-choline was 0.5 mmol/kg. These results show that oral UMP, a uridine source, enhances the synthesis of CDP-choline, the immediate precursor of PC, in gerbil brain, but the increases in nucleotides or CDP-choline are short-lived and disappear long before increases in brain phosphatides become detectable. How, then, does repeated daily intake of supplemental uridine (as UMP in the test diet) ultimately raise brain PC? Probably, in part, via uridine's other mechanism of action, discussed below: activation of P2Y receptors, which then elicit longer-term downstream effects.
Sources of plasma and brain choline
Choline is present in plasma as the free base,75,76 as a constituent of phospholipids (including PC, SM, lyso-PC, choline-containing plasmalogens, and platelet-activating factor), and as PC's water-soluble metabolites (principally phosphocholine and glycerophosphocholine).77 Free choline is also found in other biologic fluids78 and concentrated within erythrocytes through the action of an uptake molecule that is unsaturated (Km value, 5–10 µM at normal plasma choline concentrations).
Plasma choline derives from three main sources: dietary choline, consumed as the free base or as a constituent of phospholipids; endogenous synthesis, principally in liver; and liberation from the membrane phosphatides of all mammalian cells. Choline is present within many foods78 (see http://www.nal.usda.gov/fnic/foodcomp/Data/Choline/Choline.html) and also in breast milk and infant formulas,79 principally as the free molecule or as phosphatides, and its plasma levels can rapidly increase several-fold after ingestion of choline-rich foods. Thus, consumption by humans of a five-egg omelet (containing about 1.3 g of choline) increased these levels from 9.8 µM to 36.6 µM within 4 hours. Prolonged fasting reduced human plasma choline levels from 9.5 µM to 7.8 µM after 7 days. Similarly, removal of all choline-containing foods from the diet for 17–19 days gradually lowered plasma choline, from 10.6 µM to 8.4 µM in humans80 and from 12.1 µM to 6.3 µM in rats, indicating plasma choline can be partially but not fully sustained by release from endogenous stores.
Dietary PC is deacylated within the gut to form lyso-PC. About half of this product is further degraded to free choline within the gut or liver. The remainder is reacylated to regenerate PC,81 which is then absorbed into the lymphatic circulation.82 Much of the dietary choline that reaches the liver via the portal circulation is destroyed by oxidation to betaine, ultimately providing methyl groups that can be used to regenerate S-adenosylmethionine (SAM) from homocysteine. The rest passes into the systemic circulation.
In 1998, the Food and Nutrition Board of the U.S. Institute of Medicine established a dietary reference intake (DRI) for choline.80,83 Since the Food and Nutrition Board did not believe existing scientific evidence allowed calculation of a Recommended Daily Allowance (RDA) for choline, it instead set an Adequate (daily) Intake (AI) level and an Upper (daily) Limit (UL) that should not be exceeded. The main criteria for determining the AI and UL were, respectively, the amount of choline needed to prevent liver damage, and the choline intake associated with choline's most sensitive adverse effect, i.e., hypotension.83 It should be noted that subsequent studies have shown the enzymes, described below, that synthesize and metabolize choline can be affected by common genetic polymorphisms that cause important person-to-person variations in dietary choline needs. Further details about dietary reference intakes and the choline content of various foods are available on the official websites of the Institute of Medicine (http://www.nap.edu/catalog/6015.html#toc) and the US Department of Agriculture (http://www.nal.usda.gov/fnic/foodcomp/Data/Choline/Choline.html).
Endogenous choline is synthesized, principally in liver but also to a small extent within brain,84–86 by the sequential addition of three methyl groups to the amine nitrogen of PE; this forms PC, which can then be hydrolyzed to liberate the choline. The methylation reactions are catalyzed by two phosphatidylethanolamine-N-methyltransferase (PEMT) enzymes: PEMT1 (EC: 22.214.171.124), which converts PE to its monomethyl derivative, and phosphatidyl-N-methylethanolamine-N-methyltransferase (PEMT2; EC: 126.96.36.199), which adds the second and third methyl groups (a single enzyme may catalyze all three methylations in liver). Both enzymes utilize SAM as the methyl donor; their Km values for SAM are 2–4 × 10−6 M and 20–110 × 10−6 M, respectively,84 while brain SAM concentrations are 10–17 µg/g wet weight (50–85 µM, assuming about 50% of the brain mass is aqueous). Hence, PEMT1 is probably fully saturated with SAM, while PEMT2 is not. PEMT activity has been identified in brain homogenates,86 particularly in synaptosomes,84 suggesting nerve terminals can synthesize choline. PE itself is formed in liver, kidney, or brain from free ethanolamine via the CDP-ethanolamine cycle (Kennedy cycle) or from the decarboxylation of PS. PS is produced, in nerve terminals87 and elsewhere, by “base exchange,” in which a serine molecule substitutes for the ethanolamine in PE or for the choline in PC.
The biosynthesis of PC, and thus of endogenous choline, by the methylation of hepatic PE is diminished in animals given inadequate amounts of vitamins required for methyl group production, i.e., B6, B12, and folate. This relationship provides a basis for administering supplemental quantities of these vitamins to subjects receiving uridine, DHA, and choline to promote membrane phosphatide formation.
Free choline is liberated from PC by the phospholipase enzymes. Phospholipase D directly cleaves the choline/phosphate bond to generate choline and phosphatidic acid. Phospholipase A2 acts on the bond connecting a fatty acid to the hydroxyl group on PC's number-2 carbon to yield that fatty acid (often arachidonic acid [AA] or DHA) and lyso-PC; the lyso-PC is then further metabolized to choline by a phosphodiesterase, or to glycerophosphocholine, then cleaved to choline by a phosphatase. Phospholipase C acts on the bond connecting the phosphate and the hydroxyl group on PC's number-3 carbon to yield DAG and phosphocholine; the phosphocholine can then be metabolized to free choline by a phosphatase.
It is estimated, on average, about 15% of the free choline that enters the human bloodstream derives from endogenous synthesis, the rest coming principally from dietary sources.88 Acute or chronic liver disease or deficiencies in methionine, folic acid, or vitamin B12 intake can thus lower plasma choline levels by impairing hepatic PC synthesis.
Cellular membranes contain most of the choline in the body, principally as PC and SM. They also contain, of course, the phosphatides PS, PE, and phosphatidylinositol (PI) as well as specific proteins, cholesterol, and various minor lipids. The quantities of choline present in brain as PC (2–2.5 mmol/g) or SM (0.25 mmol/g) are orders of magnitude greater than those of free choline (30–60 µM).
PC is highly heterogeneous, actually representing a family of compounds with differing fatty acid compositions and, consequently, differing chemical and physical properties. The fatty acid in the C-1 position of PC tends most often to be saturated (e.g., stearic or palmitic acid), while that in position C-2 is more likely to be monounsaturated (oleic acid) or polyunsaturated (e.g., the omega-3 fatty acids DHA [22:6] and eicosapentaenoic acid [EPA][20:5] or the omega-6 fatty acid AA [20:4]). Newly synthesized phosphatide molecules contain relatively larger quantities of PUFAs than the phosphatide molecules present at steady state.89 This reflects either faster turnover of PUFA-containing phosphatides, or their rapid deacylation followed by reacylation with more-saturated fatty acid species, or both. Membranes of retinal and brain cells are especially rich in PUFAs, particularly DHA (which comprises about 20% of the total fatty acids in retinal phospholipids and about 7% of those in brain phospholipids, respectively). As described below, administration of supplemental DHA accelerates PC synthesis and increases brain levels of PC and other phosphatides.
Dietary choline or choline secreted into the gut can be broken down by intestinal bacteria to form trimethylamine and related amine products. This process is responsible for the fishy odor sometimes detected in people taking large doses of choline supplements.
Because choline is, by virtue of its quaternary nitrogen atom, highly polar, it had generally been assumed that plasma choline was unavailable to the brain. Furthermore, since brain cells were also thought to be incapable of synthesizing choline de novo, the ability of cholinergic neurons to maintain the intracellular choline concentrations needed for ACh synthesis was usually attributed either to an extraordinarily effective reuptake mechanism for reutilizing choline formed from the hydrolysis of ACh or to the uptake into brain of circulating PC or lyso-PC. Since the poor affinity of choline acetyltransferase, the enzyme that catalyzes choline's conversion to ACh, for choline made it likely that intracellular choline concentrations would control brain ACh synthesis, it was broadly conjectured that choline's high-affinity uptake from the synaptic cleft controlled the rate of brain ACh synthesis.
It is no longer held that brain choline levels are sustained solely by circulating phosphatides or by the high-affinity uptake of free choline from synapses, or that variations in high-affinity uptake are responsible for observed variations in brain choline levels. Choline molecules (but not those of PC or lyso-PC) do readily cross the BBB,90,91 and brain cells do indeed synthesize choline de novo.84 Physiological variations do occur in choline levels within brain neurons; however, these result principally from changes in plasma choline concentrations after eating choline-rich foods76 or from choline's metabolism.
Free choline molecules in brain derive from four known sources: uptake from the plasma, liberation from the PC in brain membranes, high-affinity uptake from the synaptic cleft after ACh released from a cholinergic terminal has been hydrolyzed, and, probably to a minor extent, the breakdown of newly synthesized PC formed from the methylation of PE.
The brain can obtain circulating choline via two routes. Small amounts pass from the blood to the cerebrospinal fluid through the action of a specific transport protein, organic cation transporter 2, present in cells lining the choroid plexus.92 However, orders of magnitude more choline pass bidirectionally91 between the blood and the brain's extracellular fluid by facilitated diffusion. This process is catalyzed by a different transport protein, localized within endothelial cells that line the brain's capillaries.91–93 Its action is independent of sodium and can be blocked by hemicholinium-3.
This transport protein (RBE4) exhibits a relatively low Km value for choline (estimated variously as 39–42 µM or 20 µM90 or 220–450 µM91,93,94). These differences in affinities might reflect the different methods used for their measurement, but in any case, the protein would still be unsaturated at physiological plasma choline concentrations and its net activity still affected by variations in these concentrations.
Choline can pass in either direction, based on the gradient between its blood and brain levels.95 When plasma choline levels are elevated (e.g., to 50 µM in the rat) by consumption of a choline-rich meal, choline tends to enter the brain, but when plasma choline levels are low, choline's flux is in the opposite direction. It has been estimated the plasma choline concentration in rats required in order for the net choline flux to be from blood to brain is about 15 µM; below this concentration, net choline flux is presumably from brain to blood.95 Once the circulating choline has entered the brain's extracellular fluid, it can be taken up into all cells by a low-affinity transport protein (Km value, 30–100 µM) or into cholinergic nerve terminals by a high-affinity uptake protein (Km, 0.1–10 µM). The high-affinity process – unlike the passage of choline across the BBB – is energy and sodium dependent.
The choline in membrane PC can be liberated through the actions of the phospholipase enzymes, described above. In brain, the activation of each phospholipase is tightly regulated and, in general, initiated by the interaction of a neurotransmitter or other biologic signal with a receptor coupled to a G protein. For example, the phospholipase C enzymes (which act on PC to yield DAG and phosphocholine, or on PI) and phospholipase D (which acts on PC to yield phosphatidic acid and choline) are all activated when ACh attaches to M1 or M3 muscarinic receptors.
The release of choline from PC can be enhanced, and the reincorporation of choline into PC diminished, by sustained neuronal depolarization.96 This process, termed “autocannibalism,” occurs when some of the choline is diverted for the synthesis of Ach.11,97 Autocannibalism may, by decreasing the quantities of phosphatide molecules and thus of neuronal membranes, underlie the particular vulnerability of cholinergic neurons in certain diseases. It can be blocked by providing the brain with supplemental choline.
ACh released into synapses is very rapidly hydrolyzed to free choline and acetate by the acetylcholinesterases (EC 188.8.131.52; AChE) within the cholinergic synapse. Most of the free choline liberated by the hydrolysis of ACh is taken back up into its nerve terminal of origin by a high-affinity choline transporter and either reacetylated to form ACh or phosphorylated for ultimate conversion to membrane PC.
Plasma and brain DHA and EPA
The omega-3 PUFAs DHA (22:6n−3) and EPA (20:5n−3) and the omega-6 PUFA AA (22:4n−6) are long-chain derivatives of α-linolenic acid (ALA; 18:3n−3) and linoleic acid (LA; 18:2n−6), respectively. ALA and LA are essential dietary constituents for vertebrates, since these animals cannot synthesize them or their polyunsaturated products de novo. Although DHA and EPA as well as AA can be produced in humans through the elongation and desaturation of ALA and LA, respectively, the conversion of ALA to EPA or DHA is slow, since about 75% of available ALA is shunted to β-oxidation. Furthermore, the commercial oils that provide dietary ALA, like safflower, sunflower, and corn oils, also contain very high proportions of LA, thus yielding disproportionately large amounts of AA, which then suppresses the delta-6 desaturase enzyme that would convert LA to AA. Thus, additional EPA and DHA must be obtained from the diet, particularly from high-fat fish or foods fortified with deodorized omega-3 rich oils. No authoritative body has defined a requirement for DHA98; intakes as great as 3 g per day or even more have been used to lower plasma triglyceride levels in diabetes mellitus.
The uptakes of circulating PUFAs into the brain and brain cells involve both simple diffusion (also termed “flip-flop”)99 and protein-mediated transport.100,101 DHA, EPA, and AA are then transported from the brain's extracellular fluid into cells, activated to their corresponding coenzyme A (CoA) species (e.g., docosahexaenoyl-CoA, eicosapentaenoyl-CoA, or arachidonoyl-CoA), and acylated to the sn-2 position of DAG to form PUFA-rich DAG species102 for incorporation into phosphatides. DHA is acylated by a specific acyl-CoA synthetase, Acsl6,103 which exhibits a low affinity for this substrate (Km value, 26 µM)104 relative to usual brain DHA levels (1.3–1.5 µM).105 Hence, treatments that raise blood DHA levels rapidly increase its uptake into and retention by brain cells.
EPA can be acylated to DAG by the Acyl-CoA synthetase106 or it can be converted to DHA by brain astrocytes,107 allowing its effects on brain phosphatides and synaptic proteins, described below, to be mediated by DHA itself. Exogenously administered AA, like DHA, is preferentially incorporated into brain phosphatides108 as well as into other lipids, e.g., the plasmalogens. AA shares some neurochemical effects with DHA, for example, the ability to activate syntaxin-3,44 and also has other important functions, e.g., as the precursor of prostaglandins. However, unlike DHA, AA administered orally to laboratory rodents without uridine and choline apparently does not promote synaptic membrane synthesis45 or dendritic spine47 formation.
AA is widespread throughout the brain and is particularly abundant in PI and PC, and DHA is concentrated within synaptic regions of gray matter109–111 and is especially abundant in PE and PS.109–111 In contrast, EPA is found only in trace amounts in brain phosphatides, mostly in PI. No significant differences have been described between the proportions of ingested omega-3 and of omega-6 PUFAs that enter the blood, or between the rates at which radioactively labeled circulating DHA and AA are incorporated into brain phospholipids.109–112
P2Y receptors as mediators of uridine effects
How does exogenous uridine – a precursor for the cytidine compounds utilized in the syntheses of PC and other cellular lipids – increase levels of cellular proteins, specifically of various pre-and post-synaptic neuronal proteins? Most likely, by a second mechanism in which uridine and its phosphorylated products act as ligands for P2Y receptors that then can activate protein synthesis and normal neuronal differentiation.
Extracellular nucleotides can serve as ligands for a variety of ionotropic P2X and metabotropic P2Y receptors. While P2X receptors recognize adenine nucleotides, P2Y receptors can recognize both adenine and uridine nucleotides. Members of the P2Y family, G-protein-coupled receptors, are widely distributed throughout the body, including within the brain.113 To date, eight P2Y receptors of human origin (P2Y1, 2, 4, 6, 11, 12, 13, and 14) have been cloned and characterized.113
P2Y receptors that recognize adenine but not uridine nucleotides, i.e., the P2Y1, P2Y11, P2Y12, and P2Y13 subtypes, exist principally outside the brain. P2Y2 receptors, in contrast, are abundant in brain and are activated by UTP or ATP; P2Y4 receptors are activated by UTP, and P2Y6 receptors by UDP. Their activation, through coupling to phospholipase C, increases intracellular concentrations of DAG, IP3, and calcium.114
That uridine nucleotides affect neurite outgrowth as well as neuronal differentiation and function by stimulating P2Y receptors115 has been demonstrated mainly using in vitro assay systems.46,116 UTP increases neurite outgrowth by nerve-growth-factor-stimulated PC-12 cells46 and the expression of neurofilament proteins and synaptic proteins (e.g., PSD-95); these effects are blocked by P2Y receptor antagonists or by apyrase, a drug that degrades extracellular nucleotides.46 Such P2Y-receptor-mediated actions could argue for the possible utility of P2Y agonists in treating Alzheimer's disease, especially since P2Y2 receptors are known to be selectively deficient in the parietal cortex of Alzheimer's disease brains.117