Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation.

The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA)-differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK-1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10(-6) M)-induced Ca2+ increase. In contrast, the addition of SP (10(-6) M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP-1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full-length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA-differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype.

Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens.

In order to assess the consequences of a concomitant blockade of P2X-receptors and ecto-nucleotidases, effects of 13 P2-receptor antagonists were investigated on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP) and ATP and on the removal of ATP from the incubation medium by vas deferens tissue. Increasing concentrations of all antagonists reduced and finally abolished contractions elicited by alpha,beta-MeATP (3 microM), with IC50-values ranging from 1.1 to 100 microM. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), 6-azophenyl-4-amino-5-hydroxy-naphthalene-1,3-disulphonate (NH02), 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A also progressively reduced and finally abolished contractions elicited by ATP (1 mM). 8,8'-[Carbonylbis(imino-3, 1-phenylenecarbonyl-imino)]-bis-(1,3,5-naphthalenetrisulphonate ) (NF023), suramin, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), trypan blue and reactive blue 19, in contrast, caused only partial blockade, by 34-43% maximally; reactive blue 2 and reactive red 2 had no effect; and 6,6'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-4-amino-5-hydroxy-naphtha lene-1,3-disulphonate (NH01) and Evans blue even enhanced the response to ATP. For antagonists causing full or partial inhibition, the IC50-values against ATP were close to those against alpha,beta-MeATP. All antagonists attenuated the removal of ATP, with IC25%-values ranging from 0.8 microM to >320 microM. The results confirm the frequent combination, in one antagonist molecule, of P2-receptor blockade and blockade of ecto-nucleotidases. This dual action underlies the effect of such compounds on contractions of the vas deferens elicited by ATP which, for certain substances (e.g., suramin, reactive blue 2), can be explained by a simple model in which the antagonist simultaneously blocks the degradation of ATP and a single contraction-mediating receptor (P2X1). Several observations, however, do not conform with this model, and the existence of multiple contraction-mediating receptors for ATP or multiple, pharmacologically distinct ecto-nucleotidases has to be considered.

On the suitability of adenosine 3'-phosphate 5'-phosphosulphate as a selective P2Y receptor antagonist in intact tissues.

Agonist and antagonist effects of the putative P2Y1 receptor antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC50 3.3 microM). The response was attenuated by the P2 receptor antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and reactive red 2 with apparent Kd values (0.27 and 0.29 microM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of alpha,beta-methylene ATP (alpha,beta-MeATP) in the taenia coli. Incubation with PAPS (10-100 microM) attenuated the P2Y receptor-mediated relaxation caused by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS); PAPS (100 microM) also attenuated the relaxation caused by alpha,beta-MeATP, as well as the alpha1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC50 24.7 microM), which was reduced by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; 1 microM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 microM) attenuated the P2Y receptor-mediated relaxation caused by ADPbetaS; PAPS (100 microM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 microM) attenuated the P2X receptor-mediated contraction elicited by alpha,beta-MeATP but did not alter the alpha1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

On the suitability of adenosine 3'-phosphate 5'-phosphosulphate as a selective P2Y receptor antagonist in intact tissues.

Agonist and antagonist effects of the putative P2Y1 receptor antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC50 3.3 microM). The response was attenuated by the P2 receptor antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and reactive red 2 with apparent Kd values (0.27 and 0.29 microM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of alpha,beta-methylene ATP (alpha,beta-MeATP) in the taenia coli. Incubation with PAPS (10-100 microM) attenuated the P2Y receptor-mediated relaxation caused by 5'-O-(2-thiodiphosphate) (ADPbetaS); PAPS (100 microM) also attenuated the relaxation caused by alpha,beta-MeATP, as well as the alpha1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC50 24.7 microM), which was reduced by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; 1 microM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 microM) attenuated the P2Y receptor-mediated relaxation caused by ADPbetaS; PAPS (100 microM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 microM) attenuated the P2X receptor-mediated contraction elicited by alpha,beta-MeATP but did not alter the alpha1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

P2-receptor antagonists: IV. Blockade of P2-receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2.

Effects of reactive blue 2 and twelve structurally related compounds were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP; mediated by P2X-receptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y-receptors), and the degradation of ATP by rat vas deferens tissue. All compounds, except acid blue 41 and acid blue 129 (at up to 100 microM), shifted the concentration-response curve of alpha,beta-MeATP in the rat vas deferens to the right. Most increased, but uniblue A greatly decreased, the maximum of the curve. In the case of cibacron blue 3GA and reactive blue 19, of which three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from unity. The apparent Kd values of the effective substances ranged between 0.7 and 111 microM. Most compounds increased the contraction of the rat vas deferens elicited by high K+. In the guinea-pig taenia coli, all compounds, except uniblue A and reactive blue 19 (at up to 100 microM), shifted the concentration-response curve of ADPbetaS to the right in a parallel manner. In the case of acid blue 129 and acid blue 80, of which three concentrations were tested, the slope of the Arunlakshana-Schild regression did not differ from unity. The apparent Kd values of the effective substances were between 0.7 and 69 microM. Most compounds also reduced the relaxation of the guinea-pig taenia coli elicited by noradrenaline. The removal of ATP from the medium by vas deferens tissue was decreased only by reactive blue 2, cibacron blue 3GA, uniblue A and reactive blue 19, with IC25% values between 17 and 62 microM. The structure-activity relationships for P2X- and P2Y-receptor blockade in this series are strikingly dissimilar. In reactive blue 2 and its isomers, for example, both the 1-amino-anthraquinone-2-sulphonate core and the 'side-chain' of the molecule are involved in P2X-receptor binding; P2Y-receptor affinity, in contrast, resides largely or totally in the anthraquinone core. The most promising antagonists are uniblue A which is P2X- versus P2Y-selective and acid blue 129 which is P2Y- versus P2X-selective, both with few, if any, non-P2-receptor effects at concentrations blocking the respective P2-subtype.

Characterization by antagonists of P2-receptors mediating endothelium-dependent relaxation in the rat aorta.

The receptors through which 2-methylthio ATP (MeSATP), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), UTP and ATP elicit endothelium-dependent relaxation of noradrenaline-precontracted rings of the rat aorta were characterized by means of a series of antagonists. The acetylcholine-induced relaxation and the degradation of MeSATP, UTP and ATP were also studied. The potency of the nucleotides at producing relaxation decreased in the order MeSATP (EC50 0.24 microM) > ADP beta S (0.43 microM) > UTP (1.09 microM) > ATP (3.53 microM). MeSATP, ADP beta S and UTP did not cause relaxation when the endothelium had been destroyed; high concentrations of ATP still caused some relaxation. The relaxation by MeSATP, ADP beta S and UTP became very small after treatment of the rings with NG-nitro-L-arginine methyl ester; the relaxation by ATP was less affected. Pre-exposure to MeSATP (100 microM) abolished or almost abolished the relaxation normally elicited by MeSATP and ADP beta S, did not change that elicited by UTP and slightly enhanced the relaxation elicited by ATP. Of nine compounds examined as antagonists, six attenuated selectively the effect of some or all of the nucleotides (as compared to acetylcholine): suramin, reactive blue 2, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), reactive red 2 and 5,5'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-7-amino -6-hydroxy-naphthalene-1,4-disulphonate (NH05). Decreases of maximal relaxations and slopes different from unity in Schild plots often indicated non-competitive kinetics of the antagonism. For each of the six 'selective' antagonist, the apparent Kd values against MeSATP and against ADP beta S were similar: none of the six differentiated between MeSATP and ADP beta S. Also, for each of four 'selective' antagonists, the apparent Kd values against UTP and against ATP were similar: none of the four differentiated between these two nucleotides (two antagonists did not act against UTP and ATP in the 'selective' concentration range). On the other hand, for five of the six 'selective' antagonists (the exception being NH05), the apparent Kd values against MeSATP and ADP beta S were considerably lower than those against UTP and ATP. At the highest concentrations tested against agonist-evoked relaxations, the antagonists did not alter the removal from the incubation medium, by pieces of rat aorta, of MeSATP, UTP and ATP. It is concluded that nucleotides cause endothelium-dependent relaxation of the rat aorta through two sites: a P2Y-receptor and a P2U-receptor. The receptors may be pharmacologically similar to a bovine endothelial P2Y (P2Y1) and a cloned rat P2U (P2Y2) receptor, respectively. ATP acts mainly through the P2U-receptor. Suramin, reactive blue 2, iso-PPADS, PPADS and reactive red 2 are more potent at the P2Y- than the P2U-receptor. NH05 does not discriminate between the two receptors but is the most potent P2U antagonist so far described.

Vasoconstrictor and vasodilator effects of guanine nucleotides in the rat aorta.

Although GTP, like ATP and UTP, is stored in platelet dense granules, little is known about its vascular effects. The present study was carried out in order to characterize the effects of GTP and related compounds in the rat aorta. Contractions were examined in aortic rings at resting tension. In rings with intact endothelium, GTP, GDP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) caused small contractions. In endothelium-denuded rings, the contractions were unchanged or increased and persisted after desensitization of P2X-receptors by alpha,beta-methylene ATP. Relaxations were examined in aortic rings precontracted with noradrenaline. In rings with intact endothelium, GTP (EC50 131 microM), GDP (no maximal effect obtained), GTP gamma S (EC50 6.8 microM) and guanosine (EC50 822 microM) caused prominent relaxation, whereas GDP beta S caused further contraction. In endothelium-denuded rings, the relaxant effect of GTP was greatly reduced, that of GDP and guanosine was unchanged, and that of GTP gamma S was abolished. Relaxations by GTP and GTP gamma S in endothelium-intact rings were studied in more detail. The relaxation by GTP was slightly and the relaxation by GTP gamma S greatly reduced after treatment with NG-nitro-L-arginine methyl ester. Pre-exposure to a high concentration of the P2Y-receptor agonist 2-methylthio ATP (MeSATP) did not attenuate the effects of GTP and GTP gamma S. Four compounds previously identified as antagonists at the P2Y- and P2U-receptors of rat aortic endothelium--suramin, reactive blue 2, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS) and 5,5'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-7-amino-6- hydroxynaphthalene-1,4-disulphonate (NH05)--were tested against GTP and GTP gamma S. Suramin, reactive blue 2 and iso-PPADS were much less potent against GTP and GTP gamma S than previously found against (the P2Y effect of) MeSATP. Suramin, iso-PPADS and NH05 were about as potent against GTP and GTP gamma S as previously found against (the P2U effect of) UTP and in particular ATP. It is concluded that guanine nucleotides can cause both contraction and relaxation of the rat aorta. The high concentrations of GTP and GDP required, and in the case of contraction the small size of the response, make a physiological role of the vascular effects of these nucleotides unlikely. GTP and GTP gamma S elicit endothelium-dependent relaxation through P2U-receptors. GTP in addition relaxes the aorta through smooth muscle receptors, possibly by way of its degradation product guanosine. The stable analog GTP gamma S is a relatively potent and selective agonist for the endothelial P2U-receptor.

[The role of the experiment in evaluating psychotropic substances]. / Câteva consideratii privind rolul experimentului în evaluarea substantelor psihotrope.

Designing new experimental models on animals for testing psychotropic drugs encounter important difficulties. The criteria for choosing a certain model, some pharmacokinetic aspects and few examples of models used in psychotropic drug testing are briefly presented.