Gerhard J. Molderings

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.

Direct inhibition by cannabinoids of human 5‐HT3A receptors: probable involvement of an allosteric modulatory site

Excised outside‐out patches from HEK293 cells stably transfected with the human (h) 5‐HT3A receptor cDNA were used to determine the effects of cannabinoid receptor ligands on the 5‐HT‐induced current using the patch clamp technique. In addition, binding studies with radioligands for 5‐HT3 as well as for cannabinoid CB1 and CB2 receptors were carried out. The 5‐HT‐induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): Δ9‐THC, WIN55,212‐2, anandamide, JWH‐015 and CP55940. The WIN55,212‐2‐induced inhibition was not altered by SR141716A, a CB1 receptor antagonist. WIN55,212‐3, an enantiomer of WIN55,212‐2, did not affect the 5‐HT‐induced current. WIN55,212‐2 did not change the EC50 value of 5‐HT in stimulating current, but reduced the maximum effect. The CB1 receptor ligand [3H]‐SR141716A and the CB1/CB2 receptor ligand [3H]‐CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5‐HT3A receptor cDNA, WIN55,212‐2, CP55940, anandamide and SR141716A did not affect [3H]‐GR65630 binding, but 5‐HT caused a concentration dependent‐inhibition. In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5‐HT3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5‐HT3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs.

Involvement of presynaptic imidazoline receptors in the α2-adrenoceptor-independent inhibition of noradrenaline release by imidazoline derivatives

SummaryAn involvement of imidazoline recognition sites in the modulation of transmitter release was investigated in the rabbit pulmonary artery and aorta preincubated with [3H]noradrenaline and superfused with physiological salt solution containing cocaine, corticosterone and propranolol. Electrical impulses were applied transmurally at 0.66 or 2 Hz. In the absence of further drugs, rauwolscine as well as the imidazoline derivatives BDF 6143 [4-chloro-2-(2-imidazoline-2-ylamino)-isoindoline], idazoxan and phentolamine increased the 3H overflow from the pulmonary artery, evoked by electrical stimulation at 2 Hz; the effect was due to the α2-autoreceptor blocking property of these drugs. The maximum increase in overflow obtainable with the imidazolines was considerably lower than with rauwolscine. The concentration-response curves of the imidazolines were bell-shaped. At 0.66 Hz, BDF 6143 did not facilitate, but concentration-dependently inhibited, whereas idazoxan failed to change the evoked 3H overflow. When, at the stimulation frequency of 2 Hz, presynaptic α2-adrenoceptors were blocked by rauwolscine and/or pre-exposure to phenoxybenzamine, the electrically evoked 3H overflow from the pulmonary artery and/or aorta was inhibited by the following imidazoline derivatives: the α2-adrenoceptor antagonists BDF 6143, idazoxan and phentolamine, the α1-adrenoceptor agonist with α2-blocking property cirazoline as well as the α2-adrenoceptor agonists clonidine and moxonidine. The maximum inhibition caused by BDF 6143 was greater than that due to clonidine and moxonidine; the latter two, hence, behaved as partial agonists. At the stimulation frequency of 0.66 Hz, the imidazolines exhibited a higher potency than, but a similar intrinsic activity to that at 2 Hz. Noradrenaline did not affect the evoked 3H overflow. The BDF 6143-induced inhibition of evoked 3H overflow was not modified by metitepine, atropine, theophylline, dipyridamole and indometacin, but was counteracted by the partial agonists clonidine and moxonidine.The results exclude the possibility that α1- and α2-adrenoceptors, 5-HT1 receptors, muscarine receptors, P1 purinoceptors and prostaglandin receptors are involved in the imidazoline-induced inhibition of noradrenaline release. They provide evidence indicating that the inhibitory effect is mediated by imidazoline receptors on the postganglionic sympathetic nerve terminals of the rabbit pulmonary artery and aorta.

Binding of [3H]clonidine to I1-imidazoline sites in bovine adrenal medullary membranes

SummaryImidazolines bind with high affinity not only to α-adrenoceptors but also to specific imidazoline binding sites (IBS) labelled by either [3H]clonidine or [3H]idazoxan and termed I1- and I2-IBS, respectively. Since bovine adrenal chromaffin cells lack α2-adrenoceptors, we investigated the pharmacological characteristics of [3H]clonidine binding sites in the bovine adrenal medulla. The binding of [3H]clonidine was rapid, reversible, partly specific (as defined by naphazoline 0.1 mmol/l; 55% specific binding at [3H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [3H]clonidine to bovine adrenal medullary membranes was concentration-dependently inhibited by various imidazolines, guanidines and an oxazoline derivative but not, or with negligible affinity, by rauwolscine and (−)-adrenaline. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases the single detectable site) was as follows: naphazoline >- BDF 7579 (4-chloro-2-isoindolinyl guanidine) >-clonidine>- cirazoline >_ BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)isoindoline hydrochloride) > BDF 7572 (4,7-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) > moxonidine = rilmenidine > BDF 6100 (2-(2-imidazolin-2-ylamino)-isoindoline) = idazoxan > phentolamine > aganodine = guanabenz > amiloride > histamine. This rank order is compatible with the pharmacological properties of the I1-IBS. The non-hydrolysable GTP-analogue Gpp(NH)p (5′guanylylimidodiphosphate; 100 μmol/l) inhibited specific [3H]clonidine binding by about 50%. Equilibrium [3H]clonidine binding was also significantly reduced by K+ and Mg2+ In conclusion, [3H]clonidine labels non-adrenergic high-affinity sites in plasma membranes of the bovine adrenal medulla; these sites exhibit the pharmacological properties of I1-IBS, but not of I2-IBS. Furthermore, the IBS in the adrenal medulla appear to be coupled to a G-protein.

Agmatine: clinical applications after 100 years in translation.

Agmatine (decarboxylated arginine) has been known as a natural product for over 100 years, but its biosynthesis in humans was left unexplored owing to long-standing controversy. Only recently has the demonstration of agmatine biosynthesis in mammals revived research, indicating its exceptional modulatory action at multiple molecular targets, including neurotransmitter systems, nitric oxide (NO) synthesis and polyamine metabolism, thus providing bases for broad therapeutic applications. This timely review, a concerted effort by 16 independent research groups, draws attention to the substantial preclinical and initial clinical evidence, and highlights challenges and opportunities, for the use of agmatine in treating a spectrum of complex diseases with unmet therapeutic needs, including diabetes mellitus, neurotrauma and neurodegenerative diseases, opioid addiction, mood disorders, cognitive disorders and cancer.

The pharmacological properties of the presynaptic serotonin autoreceptor in the pig brain cortex conform to the 5-HT1D receptor subtype

SummaryThe effects of serotonin receptor agonists and antagonists on the electrically (3 Hz) evoked 3H overflow were determined on pig brain cortex slices preincubated with 3H-serotonin and superfused with physiological salt solution containing indalpine (an inhibitor of serotonin uptake) plus phentolamine. The potencies of the serotonin receptor agonists and antagonists were compared with their affinities for 5-HT1A, 5-HT1B, 5-HT1c, and 5-HT1D binding sites in pig or rat tissue membranes; in addition, the potencies of the agonists were compared to their potencies in inhibiting adenylate cyclase activity in membranes of calf substantia nigra. In the superfusion experiments on pig brain cortex slices the following rank orders of potencies were obtained: agonists, serotonin > 5-methoxytryptamine = 5-carboxamidotryptamine >R U 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole) > SDZ 21009 (4(3-terbutylamino- 2-hydroxypropoxy)indol- 2-carbonic-acid-isopropylester) ≥ yohimbine ≥ cyanopindolol > 8-OHDPAT (8-hydroxy-2-(di-n-propylamino)tetralin) ≥ CGS 12066 B (7-trifluoromethyl-4(4-methyl-l-piperazinyl)-pyrrolo[1,2-a]quinoxaline); ipsapirone and urapidil were ineffective; antagonists (antagonism determined against 5methoxytryptamine as an agonist), metitepine > metergoline > mianserin. Propranolol, spiperone or mesulergine did not produce a shift of the concentration-response curve for 5-methoxytryptamine. The potencies of the serotonin receptor agonists in pig brain cortex slices were significantly correlated with their affinities for 5-HT1c and 5-HT1D binding sites in membranes of the pig choroid plexus and caudate nucleus, respectively, but not with their affinities for 5-HT1A and 5-HT1B sites in membranes of the cerebral cortex of pig and rat, respectively. The agonist potencies in decreasing 3H overflow were also significantly correlated with their potencies in inhibiting adenylate cylase activity in calf substantia nigra (i.e., a 5-HT1D receptor-mediated effect). In conclusion, the pig brain cortical 5-HT autoreceptor probably belongs to the 5-HT1D subtype. The involvement of 5-HT1c recognition sites was excluded by the low potency of mianserin as an antagonist and, in particular, by the ineffectiveness of the 5-HT1c receptor antagonist mesulergine.

N-Methyl-d-aspartate (NMDA) receptor-mediated stimulation of noradrenaline release, but not release of other neurotransmitters, in the rat brain cortex: receptor location, characterization and desensitization

SummaryRat brain cortex slices and synaptosomes (in a few experiments also hippocampal synaptosomes) preincubated with 3H-noradrenaline, 3H-5-hydroxytryptamine, 3H-choline, 3H-glutamate or 3H-γ-aminobutyric acid were used to investigate the 3H-transmitter release in response to exposure to N-methyl-d-aspartate (NMDA) and other excitatory amino acids. The slices and synaptosomes were superfused with Mg2+-free, otherwise physiologically composed salt solution.In cortical slices preincubated with 3H-noradrenaline, NMDA concentration-dependently stimulated 3H overflow, whereas no such effect occurred in slices preincubated with 3H-5-hydroxytryptamine, 3H-choline, 3H-λutamate or 3H-γ-aminobutyric acid. In cortical slices preincubated with 3H-noradrenaline, the NMDA-evoked 3H overflow was abolished by tetrodotoxin, presence of Mg2+ 1.2 mmol/l or absence of Ca2+. 2-Amino-5-phosphonovaleric acid produced a parallel shift to the right of the NMDA concentration-response curve, whereas (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate (MK-801) not only shifted the concentration-response curve to the right but also reduced the maximum effect of NMDA. Other excitatory amino acid receptor agonists also stimulated 3H overflow, yielding the following rank order of potency: NMDA > l-glutamate > l-aspartate. Kainate and, in particular, quisqualate exhibited only low potencies and/or intrinsic activities. Prolonged (25 min) exposure of 3HNA-preincubated cortical slices to a high NMDA concentration produced a short-lasting peak of 3H overflow, followed by a second phase lasting as long as the compound was present; in this phase, 3H overflow was clearly less pronounced and gradually decreased with time. The stimulatory effect of a high NMDA concentration was concentration-dependently reduced by 20 min of pre-superfusion with NMDA or l-glutamate at concentrations which by themselves produced either no or, at the most, moderate increase in 3H efflux in the two 5-min periods before application of the NMDA stimulus; in contrast, the veratridine-evoked 3H overflow was increased by pre-exposure to these NMDA concentrations. Neither in 3H-noradrenaline-preincubated synaptosomes prepared from the cortex or hippocampus nor in cortical synaptosomes preincubated with 3H-5-hy-droxytryptamine did NMDA evoke 3H overflow. The veratridine-evoked 3H overflow from 3H-noradrenaline-preincubated cortical synaptosomes was not affected by simultaneous administration of NMDA.It is concluded that NMDA selectively stimulates noradrenaline release in the rat brain cortex via NMDA receptors which appear not to be located on the noradrenergic nerve terminals. The NMDA receptor is rapidly desensitized in response to continuous application of NMDA (“tachyphylaxis”) or l-glutamate (“cross-tachyphylaxis”).

Inhibition of noradrenaline release from the sympathetic nerves of the human saphenous vein by presynaptic histamine H3 receptors.

SummaryThe human saphenous vein was used to examine whether presynaptic histamine receptors can modulate noradrenaline release and, if so, to determine their pharmacological characteristics. Strips of this blood vessel were incubated with [3H]noradrenaline and subsequently superfused with physiological salt solution containing desipramine and corticosterone. Electrically (2 Hz) evoked 3H overflow was inhibited by histamine and the H3 receptor agonist R-(−)-α-methylhistamine. Histamine-induced inhibition of electrically evoked tritium overflow was not affected by α2-adrenoceptor blockade by rauwolscine. S-(+)-α-methylhistamine (up to 10 μmol/l) as well as the histamine H1 and H2 receptor agonists 2-(2-thiazolyl)ethylamine (up to 3 μmol/l) and dimaprit (up to 30 μmol/l), respectively, were ineffective. The selective histamine H3 receptor antagonist thioperamide abolished the inhibitory effect of histamine. The histamine H2 and H1 receptor antagonists ranitidine and pheniramine, respectively, did not affect the histamine-induced inhibition of evoked tritium overflow. The present results are compatible with the suggestion that the sympathetic nerves of the human saphenous vein are endowed with inhibitory presynaptic histamine receptors of the H3 class.

Pharmacological characterization of the imidazoline receptor which mediates inhibition of noradrenaline release in the rabbit pulmonary artery

SummaryImidazoline receptors involved in modulation of noradrenaline release were characterized in the rabbit pulmonary artery preincubated with [3H]noradrenaline and superfused with physiological salt solution containing cocaine, corticosterone and propranolol. Tritium overflow was evoked by transmural electrical stimulation.The α2-adrenoceptor blocking imidazolines tolazoline, BDF 6100 [2-(2-imidazoline-2-ylamino)-isoindoline] and BDF 7572 (4,7-dichloro-derivative of BDF 6100) increased the electrically evoked 3H overflow; the concentration-response curves were bell-shaped. In contrast, two other imidazolines, i. e. moxonidine and clonidine, two guanidine derivatives structurally related to BDF 6100, i. e. aganodine and BDF 7579 [4-chloro(2-isoindolinel)-guanidine], as well as the catecholamine noradrenaline concentration-dependently inhibited the evoked 3H overflow. The concentration-response curves for moxonidine, clonidine, aganodine, BDF 7579 and noradrenaline were shifted to the right by rauwolscine. The apparent pA2 value of rauwolscine against moxonidine was 8.22, whereas those against clonidine, aganodine, BDF 7579 and noradrenaline were in the range of 6.37–6.77 and, hence, considerably lower than reported for α2-adrenoceptors. In the presence of rauwolscine an inhibitory effect was also observed with the α2-adrenoceptor blocking imidazolines tolazoline, BDF 6100, BDF 7572, and the imidazolineST 587 [2-(2-chloro-5-trifluoromethylphenylimino)-imidazoline]; the rank order of potency of all guanidines and imidazolines investigated was: aganodine > BDF 7579 > BDF 7572 > BDF 6100 > clonidine > ST 587 > moxonidine > tolazoline. Amiloride, 1-benzylimidazole and histamine were ineffective. After irreversible blockade of α-adrenoceptors by preexposure to phenoxybenzamine, evoked 3H overflow was still inhibited by aganodine, BDF 7579 and noradrenaline. Under this condition the maximal effects obtainable with the guanidines and in particular with noradrenaline were lower than in the presence of rauwolscine.These findings are compatible with our previous suggestion that imidazoline receptors mediating inhibition of noradrenaline release exist on the sympathetic nerve terminals of the rabbit pulmonary artery. Comparison of the present data with those obtained in other preparations containing imidazoline recognition sites revealed that those sites are different from the present ones. It is conceivable that the receptor characterized here represents an allosteric site of the α2-adrenoceptor or a so far undescribed α-adrenoceptor subtype since it can be activated not only by imidazolines and guanidines but also by noradrenaline and can be blocked by rauwolscine. Comparison of the properties of isoindolines substituted with either aminoimidazoline or guanidine reveals that the imidazolines (e.g. BDF 7572) possess both α2-adrenoceptor antagonistic and imidazoline receptor agonistic properties, whereas the analogous guanidines (e. g. aganodine) are imidazoline receptor agonists as well as α2-adrenoceptor agonists.

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors.

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT1B receptor ligand, and of BRL-15572, a preferential h5-HT1D receptor ligand, on the presynaptic h5-HT1B and h5-HT1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT1D heteroreceptors in the human atrium. The brain preparations, preincubated with [3H]serotonin ([3H]5-HT), and the segments of atrial appendages, preincubated with [3H]noradrenaline, were superfused with modified Krebs’ solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K+ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2μM; concentration 154 times higher than its Ki at h5-HT1D receptors) but was antagonized by SB-216641 (0.1μM; concentration 100 times higher than its Ki at h5-HT1B receptors; apparent pA2 8.45). SB-216641 (0.1μM) by itself facilitated, whereas BRL-15572 (2μM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1μM) also facilitated, and BRL-15572 (2μM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K+-evoked tritium overflow. This response was unaffected by BRL-15572 (300nM) but antagonized by SB-216641 (15nM; drug concentrations 23 and 15 times higher than their Ki at h5-HT1D and h5-HT1B receptors, respectively). Both drugs, given alone, did not modify the K+-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300nM; 23 times Ki at h5-HT1D receptors) but not by SB-216641 (30nM; 30 times Ki at h5-HT1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT1B and 5-HT1D receptors in functional studies.