Cultivation modes affect the morphology, biochemical composition, and antioxidant and anti-inflammatory properties of the green microalga Neochloris oleoabundans.

Microalgae are considered promising sustainable sources of natural bioactive compounds to be used in biotechnological sectors. In recent years, attention is increasingly given to the search of microalgae-derived compounds with antioxidant and anti-inflammatory properties for nutraceutical or pharmacological issues. In this context, attention is usually focused on the composition and bioactivity of algae or their extracts, while less interest is driven to their biological features, for example, those related to morphology and cultivation conditions. In addition, specific studies on the antioxidant and anti-inflammatory properties of microalgae mainly concern Chlorella or Spirulina. The present work was focused on the characterization of the Chlorophyta Neochloris oleoabundans under two combinations of cultivation modes: autotrophy and glucose-induced mixotrophy, each followed by starvation. Biomass for morphological and biochemical characterization, as well as for extract preparation, was harvested at the end of each cultivation phase. Analyses indicated a different content of the most important classes of bioactive compounds with antioxidant/anti-inflammatory properties (lipids, exo-polysaccharides, pigments, total phenolics, and proteins). In particular, the most promising condition able to prompt the production of antioxidant algal biomass with anti-inflammatory properties was the mixotrophic one. Under mixotrophy, beside an elevated algal biomass production, a strong photosynthetic metabolism with high appression of thylakoid membranes and characteristics of high photo-protection from oxidative damage was observed and linked to the overproduction of exo-polysaccharides and lipids rather than pigments. Overall, mixotrophy appears a good choice to produce natural bioactive extracts, potentially well tolerated by human metabolism and environmentally sustainable.

A3 adenosine receptor: pharmacology and role in disease.

The study of the A(3) adenosine receptor (A(3)AR) represents a rapidly growing and intense area of research in the adenosine field. The present chapter will provide an overview of the expression patterns, molecular pharmacology and functional role of this A(3)AR subtype under pathophysiological conditions. Through studies utilizing selective A(3)AR agonists and antagonists, or A(3)AR knockout mice, it is now clear that this receptor plays a critical role in the modulation of ischemic diseases as well as in inflammatory and autoimmune pathologies. Therefore, the potential therapeutic use of agonists and antagonists will also be described. The discussion will principally address the use of such compounds in the treatment of brain and heart ischemia, asthma, sepsis and glaucoma. The final part concentrates on the molecular basis of A(3)ARs in autoimmune diseases such as rheumatoid arthritis, and includes a description of clinical trials with the selective agonist CF101. Based on this chapter, it is evident that continued research to discover agonists and antagonists for the A(3)AR subtype is warranted.

Adenosine receptors and cancer.

The A(1), A(2A), A(2B) and A(3) G-protein-coupled cell surface adenosine receptors (ARs) are found to be upregulated in various tumor cells. Activation of the receptors by specific ligands, agonists or antagonists, modulates tumor growth via a range of signaling pathways. The A(1)AR was found to play a role in preventing the development of glioblastomas. This antitumor effect of the A(1)AR is mediated via tumor-associated microglial cells. Activation of the A(2A)AR results in inhibition of the immune response to tumors via suppression of T regulatory cell function and inhibition of natural killer cell cytotoxicity and tumor-specific CD4+/CD8+ activity. Therefore, it is suggested that pharmacological inhibition of A(2A)AR activation by specific antagonists may enhance immunotherapeutics in cancer therapy. Activation of the A(2B)AR plays a role in the development of tumors via upregulation of the expression levels of angiogenic factors in microvascular endothelial cells. In contrast, it was evident that activation of A(2B)AR results in inhibition of ERK1/2 phosphorylation and MAP kinase activity, which are involved in tumor cell growth signals. Finally, A(3)AR was found to be highly expressed in tumor cells and tissues while low expression levels were noted in normal cells or adjacent tissue. Receptor expression in the tumor tissues was directly correlated to disease severity. The high receptor expression in the tumors was attributed to overexpression of NF-kappaB, known to act as an A(3)AR transcription factor. Interestingly, high A(3)AR expression levels were found in peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing animals and cancer patients, reflecting receptor status in the tumors. A(3)AR agonists were found to induce tumor growth inhibition, both in vitro and in vivo, via modulation of the Wnt and the NF-kappaB signaling pathways. Taken together, A(3)ARs that are abundantly expressed in tumor cells may be targeted by specific A(3)AR agonists, leading to tumor growth inhibition. The unique characteristics of these A(3)AR agonists make them attractive as drug candidates.

Pharmacological characterization of P2X1 and P2X3 purinergic receptors in bovine chondrocytes.

OBJECTIVE: The aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE(2)) release. METHODS: Saturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X(1) and P2X(3) purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE(2) release) the potency of purinergic agonists and antagonists was evaluated. RESULTS: Bovine chondrocytes expressed P2X(1) and P2X(3) purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5'-triphosphate (ATP) and alpha,beta-methyleneATP were able to increase NO and PGE(2) release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists. CONCLUSIONS: These data demonstrate for the first time the presence of functional P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE(2) release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption.

Dose and time effects of caffeine intake on human platelet adenosine A(2A) receptors : functional and biochemical aspects.

BACKGROUND-We determined whether repeated caffeine administration at different dosages and for different periods of time (400 or 600 mg/d for 1 week or 400 mg/d for 2 weeks) upregulates human platelet adenosine A(2A) receptors and is accompanied by increases in cAMP accumulation and decreases in aggregation and calcium levels after stimulation of A(2A) receptors by the selective agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HE-NECA). METHODS AND RESULTS-Platelets were obtained from peripheral venous blood of 45 healthy human volunteers at the end of 2 weeks of caffeine abstinence and at 12, 60, and 108 hours after the last dose of caffeine. The lowest dose of caffeine, when given for only 7 days, had no effect. Increasing the total dose, either by giving 400 mg/d for 14 days or giving 600 mg/d, resulted in binding assays performed with the adenosine A(2A) receptor radioligand [(3)H]SCH 58261 [5-amino-7(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1, 5-c]pyrimidine], in the upregulation of A(2A) receptors. Moreover, the potency of HE-NECA to produce antiaggregatory effects, a rise in cAMP accumulation, and a decrease in calcium levels was significantly increased. CONCLUSIONS-Chronic caffeine intake can lead to upregulation of adenosine A(2A) receptors, which is accompanied by sensitization, in a time- and dose-dependent manner, to the actions of the agonist HE-NECA.

Platelet alpha2-adrenoceptor alterations in patients with essential hypertension are normalized after treatment with doxazosin but not propranolol.

OBJECTIVE: Marked alterations have been demonstrated to occur in the platelet alpha2-adrenoceptors of patients with essential hypertension. The purpose of this study was to determine whether antihypertensive treatment with alpha-adrenergic blocker doxazosin or beta-adrenergic blocker propranolol can affect the affinity and the density of platelet alpha2-adrenoceptors in such patients. SUBJECTS AND METHODS: In two groups of 22 previously untreated, essential hypertensive patients, the mean affinity (Kd) and density (B(max)) of platelet alpha2-adrenoceptors were studied by [3H]-UK 14304 binding assays; the first assays were performed before any medication was begun, the second were performed after treatment for up to 13 weeks with doxazosin or propranolol. A third group of 22 healthy normotensive volunteers matched by age, sex and body mass index was used as control. RESULTS: Blood pressure did not differ significantly in the two hypertensive groups, and treatment with the two drugs resulted in closely similar, normal blood pressure levels. Kd and B(max) values were significantly higher in the two hypertensive groups than in controls. After treatment with propranolol the binding parameters did not change significantly, whereas after treatment with doxazosin Kd and B(max) returned to normotensive values. CONCLUSIONS: In previously untreated, essential hypertensive patients platelet alpha2-adrenoceptors have a lower affinity but a higher density than in normotensive subjects. Despite similar effects on blood pressure, the treatment with the alpha-adrenergic blocker doxazosin is followed by restoration of normal findings in the binding assays of platelet alpha2-adrenoceptors whereas the treatment with the beta-adrenergic blocker propranolol does not alter the Kd and B(max) values.

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: influence of the chain at the N(8) pyrazole nitrogen.

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.

Synthesis, molecular modeling, and opioid receptor affinity of 9, 10-diazatricyclo[4.2.1.1(2,5)]decanes and 2,7-diazatricyclo[4.4.0. 0(3,8)]decanes structurally related to 3,8-diazabicyclo[3.2. 1]octanes.

Various lines of evidence, including molecular modeling studies, imply that the endoethylenic bridge of 3,8-diazabicyclo[3.2. 1]octanes (DBO, 1) plays an essential role in modulating affinity toward mu opioid receptors. This hypothesis, together with the remarkable analgesic properties observed for N(3) propionyl, N(8) arylpropenyl derivatives (2) and of the reverted isomers (3), has prompted us to insert an additional endoethylenic bridge on the piperazine moiety in order to identify derivatives with increased potency toward this receptor class. In the present report, we describe the synthesis of the novel compounds 9,10-diazatricyclo[4.2. 1.1(2,5)]decane (4) and 2,7-diazatricyclo[4.4.0.0(3,8)]decane (5), as well as the representative derivatives functionalized at the two nitrogen atoms by propionyl and arylpropenyl groups (6a-e, 7a-d). Opioid receptor binding assays revealed that, among the compounds tested, the N-propionyl-N-cinnamyl derivatives 6a and 7a exhibited the highest mu-receptor affinity, and remarkably, compound 7a displayed in vivo (mice) an analgesic potency 6-fold that of morphine.

Fluorosulfonyl- and bis-(beta-chloroethyl)amino-phenylamino functionalized pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives: irreversible antagonists at the human A3 adenosine receptor and molecular modeling studies.

A series of pyrazolotriazolopyrimidines was previously reported to be highly potent and selective human A(3) adenosine receptor antagonists (Baraldi et al. J. Med. Chem. 2000, 43, 4768-4780). A derivative having a methyl group at the N(8) pyrazole combined with a 4-methoxyphenylcarbamoyl moiety at N(5) position, displayed a K(i) value at the hA(3) receptor of 0.2 nM. We now describe chemically reactive derivatives which act as irreversible inhibitors of this receptor. Electrophilic groups, specifically sulfonyl fluoride and nitrogen mustard (bis-(beta-chloroethyl)amino) moieties, have been incorporated at the 4-position of the aryl urea group. Membranes containing the recombinant hA(3) receptor were preincubated with the compounds and washed exhaustively. The loss of ability to bind radioligand following this treatment indicated irreversible binding. The most potent compound in irreversibly binding to the receptor was 14, which contained a sulfonyl fluoride moiety and a propyl group at the N(8) pyrazole nitrogen. The bis-(beta-chloroethyl)amino derivatives displayed a much smaller degree of irreversible binding than the sulfonyl fluoride derivatives. A computer-generated model of the human A(3) receptor was built and analyzed to help interpret these results. The model of the A(3) transmembrane region was derived using primary sequence comparison, secondary structure predictions, and three-dimensional homology building, using the recently published crystal structure of rhodopsin as a template. According to our model, sulfonyl fluoride derivatives could dock within the hypothetical TM binding domain, adopting two different energetically favorable conformations. We have identified two amino acids, Ser247 and Cys251, both in TM6, as potential nucleophilic partners of the irreversible binding to the receptor.

Further studies on nociceptin-related peptides: discovery of a new chemical template with antagonist activity on the nociceptin receptor.

Three series of nociceptin (NC)-related peptides were synthesized and their abilities (i) to bind to the NC sites expressed in mouse forebrain membranes, (ii) to inhibit the electrically evoked contraction of the mouse vas deferens, and (iii) to inhibit forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human recombinant NC receptor (CHONCR) were investigated. The compounds of the first series (a series) have an ordinary Xaa1-Gly2 bond, those of the second series (b series) have a Xaa1psi(CH2-NH)Gly2 pseudopeptide bond, and those of the third series (c series) have a peptoid (Nxaa1-Gly2) structure. The affinity values measured in the binding assay and in the two functional assays with the compounds of the three series showed high levels of correlation. Thus, (I) the compounds of the a series in which Phe1 was substituted with Tyr, Cha, or Leu acted as potent NC receptor agonists; (II) the b series compounds behaved as NC receptor antagonists in the mouse vas deferens and as full agonists in CHO(NCR) cells with different potencies depending on the first amino acid residue, [Phe1psi(CH2-NH)Gly2]NC(1-17)NH2 and [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 being the most potent compounds; (III) the compounds of the third series were all inactive both as agonists and as antagonists with the exception of [Nphe1]NC(1-17)NH2 and [Nphe1]NC(1-13)NH2, which behaved as NC receptor antagonists both in the isolated tissue and in CHO(NCR) cells (pKB 6.1-6.4). In conclusion, this study demonstrates that chemical requirements for NC receptor agonists are different from those of antagonists. Moreover, modifications of the steric orientation of the aromatic residue Phe1 in the NC sequence as obtained with the pseudopeptide bond between Phe1 and Gly2 or with the displacement of the benzyl side chain by one atom, as in Nphe1, lead respectively to reduction or elimination of efficacy. Indeed, in contrast to [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 which has been reported to exhibit agonist activity in several assays involving either central or recombinant NC receptors, [Nphe1]NC(1-13)NH2 antagonizes the effect of NC at human recombinant NC receptors and in the mouse tail withdrawal assay.

Changes in hippocampal and cortical B1 bradykinin receptor biological activity in two experimental models of epilepsy.

An increased response to the activation of receptors mediating excitatory effects may be involved in some forms of epilepsy. In this study, it has been tested whether B1 bradykinin receptors (which mediate excitatory effects in the peripheral nervous system and have little constitutional expression in the central nervous system) may be proposed in this role. Two experimental models of epilepsy (kindling and kainate) have been employed, and glutamate outflow experiments have been performed in hippocampal and cortical slices taken from control, kindled and kainate-treated rats. The endogenous B1 receptor agonist Lys-des-Arg9-bradykinin (10(-7) M) did not affect electrically-evoked glutamate overflow in control animals, but concentration-dependently increased it in kindled rats (maximal effect +40 to + 50%) and, to a lesser extent (+20%), in kainate-treated rats. These effects were fully prevented by the selective B1 receptor antagonist R-715 (10(-6) M), but not by the selective B2 receptor antagonist Hoe 140 (10(-6) M). The observed changes in B1 bradykinin receptor biological activity may play a role in epileptic hyperexcitability.

The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea-pig ventricular myocytes.

1. We used electrophysiological and binding techniques to determine the effects of oxygen free radicals (OFRs) generated by dihydroxyfumaric acid (DHF, 5 mM) on calcium current and dihydropyridine binding sites in guinea-pig isolated ventricular myocytes. 2. Binding of [3H]-PN200-110 to isolated ventricular myocytes revealed one population of binding sites with a KD of 0.11 +/- 0.01 nM and Bmax of 139.1 +/- 6.9 fmol mg-1 protein (n = 24). After 15 min of exposure to DHF, the density, but not the affinity of [3H]-PN200-110 binding sites was significantly (P < 0.01) reduced to 35% of the control value (Bmax = 49.4 +/- 3.7 fmol mg-1 protein, KD = 0.11 +/- 0.01 nM, n = 15). In the presence of superoxide dismutase (SOD) and catalase (CAT) the reduction in [3H]-PN200-110 binding sites was almost completely prevented (Bmax = 120.5 +/- 7.4 in control, n = 4 and 98.8 +/- 7.4 fmol mg-1 protein in DHF plus SOD and CAT, n = 4). KD values were not modified (0.08 +/- 0.01 in control and 0.09 +/- 0.01 nM in DHF plus SOD and CAT). 3. The time-course of the reduction of [3H]-PN200-110 binding sites by OFRs was paralleled by the decrease in L-type calcium current (Ica,L) measured in patch-clamped guinea-pig ventricular myocytes either in the absence or in the presence of EGTA in the patch pipette. In the former conditions OFRs induced the appearance of calcium-dependent alterations, i.e. the transient inward current, within 10 min. After 30 min of incubation with DHF, [3H]-PN200-110 binding sites were reduced to 25% of the control value. 4. In myocytes incubated with the antilipoperoxidant agent, butylated hydroxytoluene (BHT, 50 microM), the decrease in [3H]-PN200-110 binding sites caused by DHF was partially prevented (Bmax values after 30 min exposure to DHF were 55.5 +/- 1.9 and 23.7 +/- 5.9 fmol mg-1 protein in the presence and in the absence of BHT respectively, P < 0.05). BHT did not affect the decrease in [3H]-PN200-110 binding sites during the first 15 min of exposure to DHF, but was able to prevent completely the further decrease occurring during the following 15 min of incubation with OFRs. 5. Our results demonstrate that the OFR-induced decrease in calcium current is associated with a reduction in DHP binding sites. The decrease in calcium current and in calcium channels may be implicated in the mechanical dysfunction associated with oxidative stress.

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding.

1. The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg-1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments. 3. Thermodynamic data indicate that [3H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A-adenosine receptors. 4. It is concluded that in human lymphocyte membranes [3H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A-receptor. The presence of A2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.

[3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes.

1. The present study describes the direct labelling of A2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[l,5-c]pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.34 nM and 75 fmol mg(-1) protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [3H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments. 3. Thermodynamic data indicated that [3H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A2A adenosine receptors. 4. It was concluded that in human neutrophil membranes, [3H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A2A adenosine receptors of other tissues. The receptors labelled by [3H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

Pharmacological and biochemical characterization of purified A2a adenosine receptors in human platelet membranes by [3H]-CGS 21680 binding.

1. The binding properties of human platelet A2a adenosine receptors, assayed with the A2a-selective agonist, [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine ([3H]-CGS 21680), are masked by a non-receptorial component, the adenotin site. In order to separate A2a receptors from adenotin sites, human platelet membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulphonate (CHAPS). The soluble platelet extract was precipitated with polyethylene glycol (PEG) and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. 2. The present paper describes the binding characteristics of the selective A2a agonist, [3H]-CGS 21680, to this purified platelet membrane preparation. In addition, receptor affinity and potency of several adenosine agonists and antagonists were determined in binding and adenylyl cyclase studies. 3. Saturation experiments revealed a single class of binding site with Kd and Bmax values of 285 nM and 2.07 pmol mg-1 of protein respectively. Adenosine receptor ligands competed for the binding of 50 nM [3H]-CGS 21680 to purified protein, showing a rank order of potency consistent with that typically found for interactions with the A2a adenosine receptors. In the adenylyl cyclase assay the compounds examined exhibited a rank order of potency very close to that observed in binding experiments. 4. Thermodynamic data indicated that [3H]-CGS 21680 binding to the purified receptor is totally entropy-driven in agreement with results obtained in rat striatal A2a adenosine receptors. 5. It is concluded that in the purified platelet membranes there is a CGS 21680 binding site showing the characteristic properties of the A2a receptor. This makes it possible to use this compound for reliable radioligand binding studies on the A2a adenosine receptor of human platelets.

Human vascular kinin receptors of the B2 type characterized by radioligand binding.

1. The human umbilical vein responds to bradykinin (BK) with contractions that are mediated by B2 receptors. In the present study, the corresponding vascular smooth muscle B2 binding sites have been investigated. 2. [3H]-BK, a full agonist labelled ligand, was used to demonstrate a single binding site giving a Kd value of 0.51+/-0.02 nM and a Bmax of 24+/-1 fmol mg(-1) protein. Scatchard plots were linear (r=0.98) in the 0.05-5 nM range of concentrations. Non-specific binding was found to be 30% of total binding. 3. Competition binding curves gave the following order of potency for various B2 receptor agonists: BK-[Hyp3]-BK > or = Lys-BK >> [Aib7]-BK >>> [desArg9]-BK, which is typical of B2 receptors. There was no binding to B1 receptors since the selective B1 receptor ligand, Lys-[desArg9]BK was inactive up to 10 microM (n=4). 4. Characterization of the binding site with antagonists, performed with three chemically distinct series of peptide and non-peptide compounds, revealed a high affinity of Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK) (Ki 0.17 nM; n=4) which was more potent that FR 173657 ([(E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinol inyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl] acrylamide]) (Ki 1.94 nM; n=4), D-Arg-[Hyp3,D-Phe7,Leu8]-BK (Ki 256 nM; n=4) and Win 64338 (phosphonium, [[4[[2[[bis(cyclohexylamino)methylene]amino]-3-(2-naphthalenyl)-1-oxopro pyl]amino]phenyl]methyl]tributyl, chloride, monohydrochloride) (Ki 1,450 nM; n=4). 5. The present study describes and characterises B2 receptor binding sites in the vascular smooth muscle of the human umbilical vein. The binding assay appears to be suitable for studying new agonists or antagonists designed to activate or block the B2 receptor class that mediate the majority of the physiopathological effects of kinins in man.

Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells.

1. The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line. 2. The A3 subtype was found by means of RT-PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with K(D) of 1.9+/-0.2 nM and B(max) of 1.3+/-0.1 pmol mg(-1) of protein. 3. The pharmacological profile of [3H]-MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype. 4. Thermodynamic data indicated that [3H]-MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy- and enthalpy-driven, according with that found in cells expressing the recombinant human A3 subtype. 5. In functional assays the high affinity A3 agonists Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and stimulate Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx. 6. The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]-DPCPX binding with a K(D) of 0.9+/-0.1 nM and B(max) of 42+/-3 fmol mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a K(D) of 2.5+/-0.3 nM and a B(max) of 1.4+/-0.2 pmol mg(-1) of protein. 7. In conclusion, by means of the first antagonist radioligand [3H]-MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells.

Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line.

1. The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. 2. Adenosine receptors were detected by RT - PCR experiments. A1 receptors were characterized using [3H]-DPCPX binding with a KD of 1.9+/-0.2 nM and Bmax of 23+/-7 fmol x mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a KD of 5.1+/-0.2 nM and a Bmax of 220+/-7 fmol x mg(-1) of protein. A3 receptors were studied with the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 3.3+/-0.7 nM and Bmax of 291+/-50 fmol x mg(-1) of protein. 3. The pharmacological profile of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the different adenosine receptor subtype. 4. Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy- and enthalpy-driven. 5. In functional assays the high affinity A2A agonists HE-NECA, CGS 21680 and A2A - A2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A3 agonists Cl-IB-MECA, IB-MECA and NECA were able to stimulate Ca2+ mobilization. In conclusion, all these data indicate, for the first time, that adenosine receptors with a pharmacological and biochemical profile typical of the A1, A2A, A2B and A3 receptor subtype are present on A375 melanoma cell line.

Binding thermodynamics at the human neuronal nicotine receptor.

The thermodynamic parameters deltaGo (standard free energy), deltaHo (standard enthalpy) and deltaSo (standard entropy) of the binding equilibrium of eleven ligands (six agonists and five antagonists) to the neuronal nicotinic receptor were determined by affinity measurements carried out on human thalamus membranes at six different temperatures (0, 10, 20, 25, 30, 35 degrees) and deltaG vs. T plot analysis. Affinity constants were obtained by saturation experiments for [3H]-cytisine, a ganglionic nicotinic agonist, or its displacement in inhibition assays for the other compounds. The deltaG vs T plots appeared to be reasonably linear in the full temperature range for most of the compounds investigated (equilibrium heat capacity change,deltaCo(p) approximately 0), with the exception of the three agonists cytisine, nicotine and methylcarbachol (deltaCo(p) of the order of -720 / -1610 J mol(-1) K(-1)). Thermodynamic parameters were in the range -53.3 < or =deltaHo < or = -28.9 kJ mol(-1) and -41 < or = deltaSo < or = 69 J mol(-1) K(-1) for agonists, and 8.7 < or = deltaHo < or = 68.2 kJ mol(-1) and 99 < or = deltaSo < or = 311 J mol(-1) K(-1) for antagonists, indicating that agonistic binding was both enthalpy- and entropy-driven, while antagonistic binding was totally entropy-driven. Agonists and antagonists were, therefore, thermodynamically discriminated. Experimental results were discussed with particular regard to the following points: 1) reasons why membrane receptors displayed unusually low values of deltaCo(p); 2) possible reasons for the phenomenon of thermodynamic discrimination between agonists and antagonists particularly in connection with ligand-gated ion channel receptors; and 3) the origin of the recurrent phenomenon of enthalpy-entropy compensation which has been observed for neuronal nicotinic receptor ligands as well as for all membrane receptors studied thus far.

Early changes in adenosine A1 receptors in cerebral cortex slices submitted to in vitro ischemia.

The effects of brain ischemia on the maximum binding capacity (Bmax) and affinity (Kd) of A1 receptors were studied in the rat cerebral cortex, with an in vitro approach. The results were correlated with changes in 3H-adenosine release, studied under identical experimental conditions. Fifteen minutes of in vitro 'ischemia' (hypoxic, glucose-free medium) induced a significant increase in both Bmax (2398+/-132 fmol/mg protein, 151% of the control, P < 0.05) and in Kd (2.43+/-0.12 nM, 161% of the control, P < 0.01). At the same time, an increase in tritium efflux from [3H]-adenosine labeled cerebral cortex slices to 324% of the control was observed. A trend toward normalization was evident 5-15 min after 'reoxygenation' (restoring normal medium), but the binding parameters were still altered after 60 min (Bmax 2110+/-82 fmol/mg protein, Kd 2.26+/-0.14 nM, P < 0.01 vs the corresponding control) as was adenosine release (196% of the control). These findings suggest that the increased availability of adenosine and its receptors may be a defense mechanism against ischemic injury, while the reduced affinity of A1 receptors, possibly due to desensitization, may be a sign of ischemia-induced cellular damage.