Pharmacology

An increasing number of appetite-regulating peptides are being discovered. The list of regulators inhibiting food intake is considerably longer than that of appetite stimulators. In many cases, the peptides inhibiting food intake facilitate fear reactions, whereas the majority of Ž . the agents reducing anxiety responses stimulate appetite. Cocaine-and amphetamine-regulated transcript CART cDNA was isolated from hypothalamic libraries and CART was reported to inhibit food intake and to mediate the anorectic effects of leptin. Here, we show Ž . that the active core fragment of CART CART , 0.04-5.0 nmol injected into lateral cerebral ventricle not only inhibits food intake, 89 -103 but also causes a dose-dependent increase in anxiety-like reactions in elevated plus-maze test. Intracerebroventricular administration of Ž . CART 0.04-5.0 nmol did not inhibit water intake and did not affect spontaneous locomotor activity in the open field test ruling 82 -103 out unspecific effects of the peptide. Our results suggest that CART could be an endogenous factor in the brain mediating the effects of stress on appetite. q

The pro-opiomelanocortin-derived peptides decrease food intake possibly via MC4 receptor. In this study we compared the effects of a-melanocyte-stimulating hormone (MSH), b-MSH and g 1 -MSH (0.2, 1.0 and 5.0 mg, i.c.v.) on food intake. a-MSH and b-MSH inhibited spontaneous food intake in a dose dependent manner, whereas the g 1 -MSH did not. a-MSH and b-MSH but not g 1 -MSH (all 5.0 mg, i.c.v.) inhibited fasting-induced food intake about 50%. None of the three peptides inhibited¯uid consumption in water-deprived (24 h) rats. It is suggested that MC 3 receptor, activated selectively by g 1 -MSH, is not involved in the regulation of food intake. q

We designed and synthesized several novel cyclic MSH analogues and tested their affinities for cells expressing the MC1, MC3, MC4 and MC5 receptors.One of the substances HS028 (cyclic [AcCys11, dichloro-D-phenylalanine14, Cys18, Asp-NH222]-β-MSH11–22) showed high affinity (Ki of 0.95nM) and high (80 fold) MC4 receptor selectivity over the MC3 receptor. HS028 thus shows both higher affinity and higher selectivity for the MC4 receptor compared to the earlier first described MC4 receptor selective substance HS014.HS028 antagonised a α-MSH induced increase in cyclic AMP production in transfected cells expressing the MC3 and MC4 receptors, whereas it seemed to be a partial agonist for the MC1 and MC5 receptors.Chronic intracerebroventricularly (i.c.v.) administration of HS028 by osmotic minipumps significantly increased both food intake and body weight in a dose dependent manner without tachyphylaxis for a period of 7 days.This is the first report demonstrating that an MC4 receptor antagonist can increase food intake and body weight during chronic administration providing further evidence that the MC4 receptor is an important mediator of long term weight homeostasis.We designed and synthesized several novel cyclic MSH analogues and tested their affinities for cells expressing the MC1, MC3, MC4 and MC5 receptors.One of the substances HS028 (cyclic [AcCys11, dichloro-D-phenylalanine14, Cys18, Asp-NH222]-β-MSH11–22) showed high affinity (Ki of 0.95nM) and high (80 fold) MC4 receptor selectivity over the MC3 receptor. HS028 thus shows both higher affinity and higher selectivity for the MC4 receptor compared to the earlier first described MC4 receptor selective substance HS014.HS028 antagonised a α-MSH induced increase in cyclic AMP production in transfected cells expressing the MC3 and MC4 receptors, whereas it seemed to be a partial agonist for the MC1 and MC5 receptors.Chronic intracerebroventricularly (i.c.v.) administration of HS028 by osmotic minipumps significantly increased both food intake and body weight in a dose dependent manner without tachyphylaxis for a period of 7 days.This is the first report demonstrating that an MC4 receptor antagonist can increase food intake and body weight during chronic administration providing further evidence that the MC4 receptor is an important mediator of long term weight homeostasis.British Journal of Pharmacology (1999) 126, 27–34; doi:10.1038/sj.bjp.0702264

The DNAs encoding three melanocortin receptor subtypes (melanocortin MC 1 receptor, melanocortin MC 3 receptor and melanocortin MC 5 receptor) were expressed individually in COS (CV-1 Origin, SV40) cells to characterise their ligand binding properties. The results indicated that [125I][Nle 4, D-Phe7]a-MSH (melan~yte sti~nulating hormone) bound to a single saturable site with K o values of 85.1 _+ 8.0 pmol/1 (mean + S.E.M), 396 + 65 pmol/l and 5.05 + 1.00 nmol/l for melanocortin MC 1 receptor, melanocortin MC 3 receptor and melanocortin MC 3 receptor, respectively. The melanocortin MC 1 receptor and the melanocortin MC 5 receptor showed a similar potency order to the melanocortic peptides examined which was markedly different from the potency order of the melanocortin MC 3 receptor. The melanocortin MC 1 receptor and melanocortin MC 5 receptor had a relatively higher affinity for a-MSH than 1,-MSH and/3-MSH, whereas the melanocortin MC 3 receptor had higher affinity for desacetyl-a-MSH, y-MSH and /3-MSH compared to a-MSH. The inclusion of the endopeptidase inhibitor phosphoramidon to prevent the breakdown of ACTH-(1-39) (adrenocorticotrophic hormone) to c~-MSH, decreased ACTH-(1-39) binding affinity showing that ACTH-(1-39) had a much lower affinity for melanocortin MC 1 receptor than reported earlier.

The presence of the α " -adrenoceptor subtypes in various parts of the pig and rabbit eyes was investigated using [$H]-prazosin radioligand binding. The characterization of the subtypes was achieved by performing competition experiments with various subtype selective drugs. In the pig retina, both α "A -and α "B adrenoceptors were detected and the proportion of sites was 70 % α "A -and 30 % α " Β -adrenoceptors, respectively. In the pig iris, ciliary body and choroid, which are melanin-rich tissues, the non-specific binding of [$H]-prazosin was too high to detect any of the α " -adrenoceptor subtypes. However, in the albino rabbit iris, ciliary body and retina both α "A -and α "B -adrenoceptors were detected. The proportion of sites in the iris was 60 % α "A -and 40 % α "B -adrenoceptors, respectively. In the ciliary body and rabbit retina the proportion of sites were 70 % α "A -and 30 % α " Β -adrenoceptors. Only the α " Α -adrenoceptor subtype was detected in the rabbit choroid.

Abstract: We have demonstrated previously that [3H]idazoxan, besides being able to bind to α2-adrenergic receptors, may also bind to a nonadrenergic idazoxan-receptor site with high affinity. The idazoxan receptor is tightly bound to cellular membranes, and we have now developed a method to solubilize it from the guinea pig cerebral cortex by using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The CHAPS-solubilized receptor retains its binding properties for drugs: the membrane-bound, as well as the solubilized, idazcxan receptor shows high affinities for a number of imidazolines (cirazoline, idazoxan, tolazoline, naphazoline, tramazoline, clonidine, and oxymetazoline), some imidazoles (medetomidine, detomidine), and guanfacine. By contrast, catecholamines (adrenaline, noradrenaline, isoprenaline, and dopamine) and a number of other neurotransmitters and neuromodulators (serotonin, histamine, glutamic acid, γ-aminobutyric acid, glycine, and adenosine) show negligible affinities for the idazoxan receptor. Moreover, the idazoxan receptor shows grossly different binding properties for histamine, cimetidine, and imidazole-4-acetic acid compared to what has been described for the nonadrenergic imidazole site labeled by p-[3H]aminoclonidine, indicating that the two receptor sites are distinct. Radioligand binding data further indicate that cirazoline is an idazoxan receptor-selective drug (KD= 1 nM) showing a 50–210-fold selectivity for binding to the idazoxan receptor when compared to α2-adrenergic receptors and an about 500-fold selectivity when compared to α1-adrenergic receptors. We have also reviewed the literature for possible nonadrenergic actions of idazoxan and cirazoline, and we suggest that idazoxan receptors might be involved in the control of prolactin release from the pituitary.

We have previously reported that leptin, the product of the obese (ob) gene, may play a physiologically relevant role in the generation of estradiol/progesteroneinduced luteinizing hormone (LH) and prolactin (PRL) surges in female rats. In the present study, we examined whether the stimulatory effect of leptin on the hormonal surges is mediated through the melanocortin (MC) 4 receptor in the brain, as is leptin's effect on feeding behavior. We also explored whether the MC4 receptor participates in tonic stimulation of steroid-induced LH and PRL surges. Experiments were performed on both normally fed and 3-day starved rats, which were ovariectomized and primed with estradiol and progesterone. At 11:00 h on the day of the experiments, the normally fed rats received an intracerebroventricular administration of artificial cerebrospinal fluid (vehicle), SHU 9119 (a nonselective MC3/MC4 receptor antagonist, 1.0 nmol), or HS014 (a selective MC4 receptor antagonist, 1.0 nmol). The 3-day starved rats were given vehicle, recombinant mouse leptin (0.3 nmol), leptin (0.3 nmol) ؉ SHU9119 (1.0 nmol), or leptin (0.3 nmol) ؉ HS014 (1.0 nmol). From 11:00 to 18:00 h, blood was collected every 30 min to measure LH and PRL. The 3-day starvation completely abolished both LH and PRL surges, but leptin significantly reinstated these hormonal surges. Both SHU9119 and HS014 significantly decreased the magnitude of LH and PRL surges in normally fed rats and also significantly blocked the leptin stimulation of the hormonal surges in starved rats. These results suggest that the MC4 receptor may be the pivotal subtype of MC receptors mediating the leptin stimulation of LH and PRL surges. The data also suggest that endogenous MC(s) may tonically stimulate the hormonal surges in normally fed rats via the MC4 receptor. This is the first report describing a physiological role of a specific MC receptor in regulating the reproductive axis.

We synthesized a novel series of cyclic melanocyte stimulating hormone (MSH) analogues and tested their binding properties on cells transiently expressing the human melanocortin1 (MC1), MC3, MC4 and MC5 receptors.We discovered that compounds with 26 membered rings of [Cys4,D-Nal7,Cys11]α-MSH(4–11) displayed specific MC4 receptor selectivity. The preference order of the different MC receptor subtypes for the novel [Cys4D-Nal7Cys11]α-MSH(4–11) analogues are distinct from all other known MSH analogues, particularly as they bind the MC4 receptor with high and the MC1 receptor with low relative affinities.HS964 and HS014 have 12 and 17 fold MC4/MC3 receptor selectivity, respectively, which is much higher than for the previously described cyclic lactam and [Cys4,Cys10]α-MSH analogues SHU9119 and HS9510.HS964 is the first substance showing higher affinity for the MC5 receptor than the MC1 receptor.HS014, which was the most potent and selective MC4 receptor ligand (Ki 3.2 nM, which is ∼300 fold higher affinity than for α-MSH), was also demonstrated to antagonize α-MSH stimulation of cyclic AMP in MC4 receptor transfected cells.We found that a compound with a 29 membered ring of [Cys3,Nle10,D-Nal7,Cys11]α-MSH(3–11) (HS010) had the highest affinity for the MC3 receptor.This is the first study to describe ligands that are truly MC4 selective and a ligand having a high affinity for the MC3 receptor. The novel compounds may be of use in clarifying the physiological roles of the MC3, MC4 and MC5 receptors.We synthesized a novel series of cyclic melanocyte stimulating hormone (MSH) analogues and tested their binding properties on cells transiently expressing the human melanocortin1 (MC1), MC3, MC4 and MC5 receptors.We discovered that compounds with 26 membered rings of [Cys4,D-Nal7,Cys11]α-MSH(4–11) displayed specific MC4 receptor selectivity. The preference order of the different MC receptor subtypes for the novel [Cys4D-Nal7Cys11]α-MSH(4–11) analogues are distinct from all other known MSH analogues, particularly as they bind the MC4 receptor with high and the MC1 receptor with low relative affinities.HS964 and HS014 have 12 and 17 fold MC4/MC3 receptor selectivity, respectively, which is much higher than for the previously described cyclic lactam and [Cys4,Cys10]α-MSH analogues SHU9119 and HS9510.HS964 is the first substance showing higher affinity for the MC5 receptor than the MC1 receptor.HS014, which was the most potent and selective MC4 receptor ligand (Ki 3.2 nM, which is ∼300 fold higher affinity than for α-MSH), was also demonstrated to antagonize α-MSH stimulation of cyclic AMP in MC4 receptor transfected cells.We found that a compound with a 29 membered ring of [Cys3,Nle10,D-Nal7,Cys11]α-MSH(3–11) (HS010) had the highest affinity for the MC3 receptor.This is the first study to describe ligands that are truly MC4 selective and a ligand having a high affinity for the MC3 receptor. The novel compounds may be of use in clarifying the physiological roles of the MC3, MC4 and MC5 receptors.

A phage display system for the selection of peptides binding to heterologously expressed human melanocortin receptor 1 on the surface of insect cells has been established. It could be shown that phage particles displaying the natural ligand ␣-melanocyte-stimulating hormone bind selectively to cells expressing this receptor and that these phages exhibit biological activity on mouse B16F1 melanoma cells. Insect cells were superior to other cell lines tested and have been used to select binders from a small library, in which critical determinants (Phe 7 -Arg 8 -Trp 9 ) were kept, whereas the flanking regions where allowed to variate freely. One peptide displaying little similarity with native hormone was found that binds to the receptor also in its free form with an affinity of 7 nM. It showed a remarkable selectivity for this receptor, because it binds to the other melanocortin receptor subtypes with a maximum affinity of 21 M. This is the first time phage display has been used successfully with G-protein-coupled receptors lacking an extracellular binding domain.

We have developed an alignment-independent method for classification of G-protein coupled receptors (GPCRs) according to the principal chemical properties of their amino acid sequences. The method relies on a multivariate approach where the primary amino acid sequences are translated into vectors based on the principal physicochemical properties of the amino acids and transformation of the data into a uniform matrix by applying a modified autocross-covariance transform. The application of principal component analysis to a data set of 929 class A GPCRs showed a clear separation of the major classes of GPCRs. The application of partial least squares projection to latent structures created a highly valid model (cross-validated correlation coefficient, Q 2 ‫ס‬ 0.895) that gave unambiguous classification of the GPCRs in the training set according to their ligand binding class. The model was further validated by external prediction of 535 novel GPCRs not included in the training set. Of the latter, only 14 sequences, confined in rapidly expanding GPCR classes, were mispredicted. Moreover, 90 orphan GPCRs out of 165 were tentatively identified to GPCR ligand binding class. The alignment-independent method could be used to assess the importance of the principal chemical properties of every single amino acid in the protein sequences for their contributions in explaining GPCR family membership. It was then revealed that all amino acids in the unaligned sequences contributed to the classifications, albeit to varying extent; the most important amino acids being those that could also be determined to be conserved by using traditional alignment-based methods.

The published data for six melanocortin peptides binding to wild-type and chimeric melanocortin MC 1 /MC 3 receptors were analysed using the novel proteo-chemometrics modelling approach. The chimeric receptors and the peptides were coded using binary descriptors and used to correlate with the experimental data for affinity or selectivity for peptides binding to receptors. Correlations were achieved using partial least squares projection to latent structures (PLS) and statistically valid models were obtained. The models were further improved by adding cross-terms and applying orthogonal signal correction. The models were validated using external prediction, with half of the data being excluded from the modelling. Interpretation of the results using PLS coefficient plots revealed that the binding pocket for the melanocortins is located between the first, second, third, sixth and seventh transmembrane regions of the melanocortin receptors, in good agreement with previous three-dimensional models for the interactions of melanocortins with melanocortin receptors. Further, analysis of cross-terms between peptide descriptors indicated that the proteo-chemometrics modelling is able to distinguish between differences in the conformational space of the peptides that affect binding affinity and selectivity.

Three different nitro compounds glyceryl mononitrate (GMN), glyceryl dinitrate (GDN) and ethylene glycol dinitrate (EGDN) were tested on histamine contracted bovine mesenteric artery. GDN and EGDN caused a dose-dependent relaxation which was accompanied by an increase in the endogenous cGMP level. The ED50-value for GDN and EGDN with regard to the relaxant action was 1.06 X 10(-6) M and 4.20 X 10(-8), respectively. GMN was almost completely ineffective as a relaxing agent and did not cause any significant change in cGMP. Regression analysis revealed a significant correlation between relaxation and increase in cGMP for both GDN and EGDN. The regression model for EGDN could be significantly improved by including the squared cGMP change, indicating a non-linear relationship. With regard to GDN a hyperbolic relationship between relaxation and cGMP increase was found. A slight improvement of the regression model was found for EGDN when the change in cAMP was included. For GMN and GDN no ...

Preventive and therapeutic effects of nitrite supplementation in experimental inflammatory bowel disease, Redox Biology, http://dx.Abstract Background: Inorganic nitrate and nitrite have emerged as alternative substrates for nitric oxide (NO) generation in the gastrointestinal tract, and have shown to be protective against drug-induced gastric injury. The aim of this study was to investigate the preventive and therapeutic effects of nitrate and nitrite in a model of experimental colitis. Methods: Colitis was induced in mice by administrating dextran sulfate sodium (DSS) with concurrent administration of nitrite (1mM) or nitrate (10 mM) in the drinking water for 7 days. A therapeutic approach was also investigated by initiating nitrite treatment 3 days after DSS-induced colitis. Clinical and inflammatory markers were assessed and the colonic mucus thickness was measured in vivo. The effect of nitrite on wound healing was evaluated using colon epithelial cells. Results: Concurrent administration of DSS and nitrite (1mM) alleviated inflammation as determined by reduced disease activity index score (DAI) and increased colon length, while nitrate (10mM) only reduced the DAI-score. Nitrite also displayed therapeutic effects by ameliorating established colonic inflammation with reduced colonic expression of iNOS and improving histopathology. DSS-induced decrease in colonic mucus thickness was completely prevented by nitrite administration. In addition, goblet cell abundance was lower by DSS treatment, but was increased by addition of nitrite. Further studies using colon epithelial cells revealed an NO-dependent improvement in wound healing with nitrite administration. Work supported by The Ruth and Richard Juhlin's Foundation, Dr. P Håkanssons/Druvan Stiftelse, Karolinska Institutet Foundation (S.B.), Vinnova (CIDaT) (J.O.L.), Vetenskapsrådet -08646 (LH) and K2012-99x (MP), Ragnar Söderberg foundation (MP), Nanna Svartz foundation (MP).