Ivar von Kügelgen

G protein-coupled receptor P2Y5 and its ligand LPA are involved in maintenance of human hair growth

Hypotrichosis simplex is a group of nonsyndromic human alopecias. We mapped an autosomal recessive form of this disorder to chromosome 13q14.11–13q21.33, and identified homozygous truncating mutations in P2RY5, which encodes an orphan G protein–coupled receptor. Furthermore, we identified oleoyl-L-α-lysophosphatidic acid (LPA), a bioactive lipid, as a ligand for P2Y5 in reporter gene and radioligand binding experiments. Homology and studies of signaling transduction pathways suggest that P2Y5 is a member of a subgroup of LPA receptors, which also includes LPA4 and LPA5. Our study is the first to implicate a G protein–coupled receptor as essential for and specific to the maintenance of human hair growth. This finding may provide opportunities for new therapeutic approaches to the treatment of hair loss in humans.

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells—so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine–A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Inhibition by nucleotides acting at presynaptic P2-receptors of sympathetic neuro-effector transmission in the mouse isolated vas deferens

SummaryEffects of nucleotides and nucleosides on smooth muscle tension and the release of previously stored [3H]-noradrenaline were studied in the mouse isolated vas deferens. The tissue was stimulated twice by 20 electrical field pulses delivered at 2 Hz (S1, S2).α, \-Methylene-ATP, ATPγS, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine (up to 100 μmol/1) and uridine (up to 1 mmol/1). The electrically evoked overflow of tritium was reduced by the drugs in the following order of potency: ATPγS > ATP = adenosine > UTP; α,\-meth-ylene-ATP (up to 10 µmol/l) and uridine (up to 1 mmol/1) did not significantly change the evoked overflow. 8-(p-Sulphophenyl)theophylline did not alter the contractile responses to the nucleotides; it prevented the overflow-inhibiting effect of adenosine and reduced that of UTP; the overflow-inhibiting effects of ATP and ATPγS were not significantly attenuated. After prolonged exposure to α,β-methylene-ATP, all contractile nucleotide effects were abolished; in contrast, the depression by adenosine and the nucleotides of the evoked overflow of tritium persisted. None of the effects was changed by indometacin, yohimbine or reactive blue 2.It is concluded that ATP, ATPγS, α,\-methylene-ATP and UTP produce contraction of the vas deferens by activation of P2x-receptors. Moreover, the nucleotides inhibit per se the release of [3H]-noradrenaline (and presumably the co-transmitter mixture of noradrenaline and ATP); the effect of ATP is not, or only to a small extent, due to breakdown to adenosine. The presynaptic site of action of the purine nucleotides is a P2-receptor which differs from the P2X-receptor and may be a reactive blue 2-resistant “P2y-like” receptor.

Evidence for two separate vasoconstriction-mediating nucleotide receptors, both distinct from the P2X-receptor, in rabbit basilar artery: a receptor for pyrimidine nucleotides and a receptor for purine nucleotides

SummaryUridine 5′-triphosphate- (UTP-) and adenosine 5′-triphosphate-(ATP) induced vasoconstriction was studied in the rabbit basilar artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure.Serotonin, histamine and noradrenaline caused concentration-dependent vasoconstriction, with potency decreasing in that order. Of the nucleotides tested, UTP, UDP, UMP, CTP, ATP, ADP, adenosine 5′-O-(3-thio)triphosphate (ATPγS), and β,γ-imido adenosine 5′-triphosphate (AMP-PNP) elicited concentration-dependent vasoconstriction, whereas AMP, 2-methylthio-ATP, α, β-methylene-ATP and β,γ-methylene-ATP up to 10−3 mol/l caused no or only a very small increase in perfusion pressure. The order of potency of the pyrimidine nucleotides was: UTP = UDP ≫ UMP = CTP; that of the purine nucleotides was: ATPγS > AMP-PNP > ATP > ADP > 2-methylthio-ATP = α, β-methylene-ATP = β,γ-methylene-ATP. The vasoconstrictor effects of UTP and ATP were not or only to a minor degree influenced by: phentolamine; a mixture of atropine, diphenhydramine and methysergide; indometacin; nordihydroguaiaretic acid; denervation by 6-hydroxydopamine; or mechanical removal of endothelium. Prolonged exposure to α,β-methylene-ATP elicited only a very small vasoconstriction and did not change the constrictor effects of UTP or ATP. Prolonged exposure to ATPγS elicited marked vasoconstriction; subsequently, responses to ATP were reduced whereas those to UTP were, if anything, slightly enhanced. Reactive blue 2 reduced neither the UTP- nor the ATP-induced vasoconstriction. ATP 10−3 mol/l elicited marked additional vasoconstriction after precontraction with UTP 10−3 mol/l, whereas UTP elicited only a very small additional vasoconstriction when its concentration was doubled from 10−3 to 2 × 10−3 mol/l.It is concluded that, in the rabbit basilar artery, the vasoconstrictor response to UTP is mediated by a pyrimidine nucleotide receptor which is distinct from the P2-purinoceptor, and that the vasoconstrictor response to ATP is mediated by a P2-receptor which is distinct from the known P2-subtypes.

Release of noradrenaline and ATP by electrical stimulation and nicotine in guinea-pig vas deferens

SummaryEffects of electrical stimulation and nicotine on ATP and tritium outflow and smooth muscle tension were studied in the guinea-pig isolated vas deferens preincubated with [3H]-noradrenaline. ATP was measured using the luciferase technique.Electrical stimulation caused biphasic contractions and an acceleration of ATP and tritium outflow. The contraction amplitude and the overflow of ATP increased markedly, whereas the overflow of tritium increased only slightly with the frequency of stimulation (1–10 Hz; constant number of 60 pulses). The contraction amplitude did not increase with an increase in pulse number (20–540 pulses; constant frequency of 5 Hz), whereas the overflow of ATP increased slightly, and that of tritium markedly. Nicotine caused monophasic, transient contractions and, again, an acceleration of ATP and tritium outflow. Contractions, ATP and tritium overflow increased with the concentration of nicotine (56–320 μmol/l) in an approximately parallel manner. The influence of some drugs on responses to electrical stimulation (60 pulses, 5 Hz) and nicotine (180 μmol/l) was investigated. Tetrodotoxin blocked all effects of electrical stimulation but did not change those of nicotine. The reverse was true for hexamethonium. Neither electrical stimulation nor nicotine caused contraction or an increase in ATP outflow after pretreatment with 6-hydroxydopamine. The main effects of prazosin 0.3 μmol/l were to reduce electrically evoked contractions (above all second phase) as well as nicotine-evoked contractions and the nicotine-evoked overflow of ATP (the latter by about 81 %). Prazosin also tended to diminish the electrically evoked overflow of ATP. α,ß-Methylene-ATP 10 μmol/l elicited a transient contraction and ATP overflow on its own. The main change in the subsequent state of desensitization was a decrease of the first phase of electrically evoked contractions. The main effects of prazosin combined with desensitization by α,ß-methylene-ATP were marked decreases of electrically evoked contractions (by 94%), the electrically evoked overflow ATP (by 66%), nicotine-evoked contractions (by 97%) and the nicotinee-voked overflow of ATP (by 70%).It is concluded that both electrical stimulation and nicotine release noradrenaline and ATP in guinea-pig vas deferens. Only part of the evoked overflow of ATP (about 32%) is neural in origin. Another part probably originates from smooth muscle cells where it is released by neurogenic noradrenaline acting at α1-adrenoceptors. Corelease leads to cotransmission: electrically as well as nicotine-evoked contractions consist of adrenergic and purinergic components. Varying types of stimulation release cotransmitter mixtures of varying composition. Electrical stimulation at high frequency (for example 10 Hz) and with low pulse numbers (for example 20 pulses) seems to release the cotransmitters at a relatively high ATP/noradrenaline ratio. Activation of prejunctional nicotine receptors seems to release the cotransmitters at a relatively low ATP/noradrenaline ratio.

Evidence for P2-purinoceptor-mediated inhibition of noradrenaline release in rat brain cortex

1 Some postganglionic sympathetic axons possess P2Y‐like P2‐purinoceptors which, when activated, decrease the release of noradrenaline. We examined the question of whether such receptors also occur at the noradrenergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [3H]‐noradrenaline, then superfused with medium containing desipramine (1 μm) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz. 2 The selective adenosine A1‐receptor agonist, N6‐cyclopentyl‐adenosine (CPA; 0.03‐3 μm) as well as the non‐subtype‐selective agonist 5′‐N‐ethylcarboxamido‐adenosine (NECA; 0.3‐3 μm) reduced the evoked overflow of tritium, whereas the adenosine A2a‐receptor agonist, 2‐p‐(2‐carbonylethyl)‐phenethylamino‐5′‐N‐ethylcarboxamido‐adenosine (CGS‐21680; 0.003–30 μm) and the adenosine A3‐receptor agonist N6‐2‐(4‐aminophenyl)ethyl‐adenosine (APNEA; 0.03‐3 μm) caused no change. Of the nucleotides tested, ATP (30–300 μm), adenosine‐5′‐0‐(3‐thiotriphosphate) (ATP7S; 30–300 μm), adenosine‐5′‐0‐(2‐thiodiphosphate) (ADPγS; 30–300 μm), P1, P4‐di(adenosine‐5−)‐tetraphosphate (Ap4A; 30–300 μm) and the preferential P2Y‐purinoceptor agonist, 2‐methylthio‐ATP (300 μm) decreased the evoked overflow of tritium. The P2X‐purinoceptor agonist, α,β‐methylene‐ATP (3–300 μm) caused no change. 3 The A1‐selective antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 10 nm) attenuated the effects of the nucleosides CPA (apparent pKB value 9.8) and NECA as well as of the nucleotides ATP (apparent pKB 9.3), ATP7S (apparent pKB 9.2) and ADPβS (apparent pKB 8.7). CGS‐21680 and APNEA were ineffective also in the presence of DPCPX. The A2‐selective antagonist 1,3‐dipropyl‐8‐(3,4‐dimethoxystyryl)‐7‐methylxanthine (KF‐17837) reduced the effects of CPA, NECA and ATPγS only when given at a concentration of 300 nm but not at 10 nm. 4 The P2‐purinoceptor antagonists, suramin (300 μm), reactive blue 2 (30 μm) and cibacron blue 3GA (30 μm) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentration‐response curve of ATPγS to the right (apparent pKB values 3.7 and 5.0, respectively). Reactive blue 2 also attenuated the effect of ATPγS, and cibacron blue 3GA attenuated the effect of ATP, but in these cases the agonist concentration‐response curves were not shifted to the right. There was no antagonistic effect of suramin against ATP and ADPβS. 5 The results indicate that rat cerebrocortical noradrenergic axons possess, in addition to the known adenosine A1‐receptor, a separate purinoceptor for nucleotides (P2) which, in contrast to the A1‐receptor, is blocked by suramin, reactive blue 2 and cibacron blue 3GA. Nucleotides such as ATP and ATPγS activate both receptors. Inconsistencies in antagonist effects against nucleotides are probably due to this activation of two receptors. The presynaptic P2‐purinoceptor is P2Y‐like, as it is in the peripheral sympathetic nervous system.

Interaction of adenine nucleotides, UTP and suramin in mouse vas deferens: suramin-sensitive and suramin-insensitive components in the contractile effect of ATP

SummaryEffects of various nucleotides, nucleosides and noradrenaline on smooth muscle tension were studied in the isolated mouse vas deferens. α,β-Methylene-ATP, ATPγS, noradrenaline, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine or uridine (up to 100 μmol/l). Prolonged incubation with α,β-methylene-ATP (concentration increased stepwise from 0 to 15 μmol/l) selectively reduced contractions induced by ATP and UTP but not those induced by noradrenaline, and there was cross-tachyphylaxis between ATP and UTP. Suramin (10–300 μmol/l) did not alter the response to noradrenaline but shifted the concentration-response curves for α,β-methylene-ATP, ATPγS, UTP and lower concentrations of ATP (0.1–1 μol/l) to the right. The pA2-values of suramin were 5.2 against α,β-methylene-ATP, 4.8 against ATPyS, 5.1 against UTP and 5.4 against lower concentrations of ATP. The effects of higher concentrations of ATP were largely resistant to suramin. The results indicate that the mouse vas deferens possesses contraction-mediating smooth muscle P2X-receptors. UTP also acts at this receptor, and there is no evidence for a separate UTP receptor. The selective inhibition of nucleotide- but not noradrenaline-induced contractions by suramin confirms the view that suramin is a selective P2-antagonist. The resistance against suramin of part of the effect of ATP suggests that ATP activates a suramin-insensitive site in addition to the P2X-receptor.

Molecular pharmacology, physiology, and structure of the P2Y receptors.

The P2Y receptors are a widely expressed group of eight nucleotide-activated G protein-coupled receptors (GPCRs). The P2Y(1)(ADP), P2Y(2)(ATP/UTP), P2Y(4)(UTP), P2Y(6)(UDP), and P2Y(11)(ATP) receptors activate G(q) and therefore robustly promote inositol lipid signaling responses. The P2Y(12)(ADP), P2Y(13)(ADP), and P2Y(14)(UDP/UDP-glucose) receptors activate G(i) leading to inhibition of adenylyl cyclase and to Gβγ-mediated activation of a range of effector proteins including phosphoinositide 3-kinase-γ, inward rectifying K(+) (GIRK) channels, phospholipase C-β2 and -β3, and G protein-receptor kinases 2 and 3. A broad range of physiological responses occur downstream of activation of these receptors ranging from Cl(-) secretion by epithelia to aggregation of platelets to neurotransmission. Useful structural models of the P2Y receptors have evolved from extensive genetic analyses coupled with molecular modeling based on three-dimensional structures obtained for rhodopsin and several other GPCRs. Selective ligands have been synthesized for most of the P2Y receptors with the most prominent successes attained with highly selective agonist and antagonist molecules for the ADP-activated P2Y(1) and P2Y(12) receptors. The widely prescribed drug, clopidogrel, which results in irreversible blockade of the platelet P2Y(12) receptor, is the most important therapeutic agent that targets a P2Y receptor.

Stable adenine nucleotides inhibit [3H]-noradrenaline release in rabbit brain cortex slices by direct action at presynaptic adenosine A1-receptors.

SummaryEffects of adenosine and nucleotides on the release of previously stored [3H]-noradrenaline were studied in rabbit brain cortex slices. The slices were stimulated twice, in most experiments by 6 electrical field pulses delivered at 100 Hz.Adenosine and the nucleotides AMP, ADP, ATP, AMPS, ADPβS, ATPyS, β,γ-imido-ATP and β,γ-methyl-ene-ATP all reduced the evoked overflow of tritiated compounds. For purines for which concentration-response curves were determined, the order of potency was adenosine > ATP ≈ ATPyS β,γ-imido-ATP ≈ ADP > β,γ-methylene-ATP. AMP 30 Etmol/l and AMPS 30 μmol/l were approximately equieffective with 30 μmol/l of adenosine and ATPγS, and ADPβS, 30 μmol/l was approximately equieffective with 30 μmol/l of ADP. α,β-Methylene-ADP, 2-methylthio-ATP, UTP and GTPγS did not change the evoked overflow of tritium. α,β-Methylene-ATP caused an increase; however, the increase was small and became significant only after 59 min of exposure to α,β-methylene-ATP or when the slices were stimulated by 30 pulses, 10 H2. Neither adenosine deaminase (100 U/l) nor the blocker of 5′-nucleotidase, α,β-methylene-ADP (10 μmol/l), attenuated the inhibition caused by ATP, ATPyS and β,γ-methylene-ATP, despite the fact that adenosine deaminase abolished the effect of adenosine. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nmol/l) shifted the concentration-response curves of adenosine, ATPyS, β,γ-imido-ATP and β,γ-methylene-ATP to the right by very similar degrees. 8(p-Sulphophenyl)-theophylline (30 and 300 μmol/l) also markedly antagonized the inhibition produced by ATPγS. α,β-Methylene-ATP (10 and 30 μmol/l) and suramin (100 gmol/l) did not modify the effects of adenosine, ATPγS and β,γ-methylene-ATP.It is concluded that nucleotides themselves can inhibit the release of noradrenaline in the rabbit brain cortex. The nucleotides and adenosine seem to act at the same site, i.e., the A1 subtype of the P1-purinoceptor. The results support the notion that metabolically stable, phosphate chain-modified nucleotides such as ATPγS, β,γ-imido-ATP and β,γ-methylene-ATP can be potent P1 agonists. No evidence was found for presynaptic P2X-, P2Y- or P3-purinoceptors.

Prejunctional modulation of noradrenaline release in mouse and rat vas deferens: contribution of P1- and P2-purinoceptors.

1 Prejunctional purinoceptors modulating the release of noradrenaline were compared in mouse and rat vas deferens. Tissue slices were preincubated with [3H]‐noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 60 pulses, 1 Hz. 2 In mouse vas deferens, 2‐chloroadenosine (IC50 0.24 μm), β,γ‐methylene‐ATP (IC50 3.8 μm), α,β‐methylene‐ATP (IC50 2.9 μm) and 2‐methylthio‐ATP (only 30 μm tested) reduced the evoked overflow of tritium. 8‐Cyclopentyl‐1,3‐dipropylxanthine (DPCPX), 10 nm, antagonized the effect of 2‐chloroadenosine (apparent pKB 10.2) as well as of β,γ‐methylene‐ATP (apparent pKB 9.6) and α,β‐methylene‐ATP. Suramin, 300 μm, attenuated the effect of 2‐chloroadenosine at best very slightly, antagonized the effect of β,γ‐methylene‐ATP (apparent pKB 4.5) and, when combined with DPCPX 10 nm, caused a further marked shift to the right of the concentration‐response curve of β,γ‐methylene‐ATP beyond the shift produced by DPCPX alone. 3 In rat vas deferens, 2‐chloroadenosine (IC50 0.20 μm), β,γ‐methylene‐ATP (IC50 4.8 μm); α,β‐methylene‐ATP (IC50 3.0 μm) and 2‐methylthio‐ATP (only 30 μm tested) also reduced the evoked overflow of tritium. DPCPX, 10 nm, antagonized the effect of 2‐chloroadenosine (apparent pKB 9.7) as well as of β,γ‐methylene‐ATP (apparent pKB 9.6) and α,β‐methylene‐ATP. Suramin, 300 μm, did not change the effect of 2‐chloroadenosine, attenuated the effect of β,γ‐methylene‐ATP at best very slightly and, when combined with DPCPX, caused at best a very small shift to the right of the concentration‐response curve of β,γ‐methylene‐ATP beyond the shift produced by DPCPX alone. 4 It is concluded that prejunctional purinoceptor mechanisms in mouse and rat vas deferens are similar. In either species, both nucleosides such as adenosine and nucleotides such as β,γ‐methylene‐ATP activate a common release‐inhibiting receptor which is a P1‐ or, more specifically, A1‐purinoceptor. There seems to be no need to postulate the existence of a novel prejunctional P3‐purinoceptor. Moreover, the sympathetic terminal axons possess an additional P2‐purinoceptor in both species which is activated by some nucleotides such as β,γ‐methylene‐ATP and 2‐methylthio‐ATP, although the activation of the P2‐purinoceptor by β,γ‐methylene‐ATP is difficult to demonstrate in the rat.