M3 muscarinic receptors on murine HSDM1C1 cells: Further functional,regulatory, and receptor binding studies

In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M₃-selective radioligand, [³H]4-DAMP, to cell homogenates was characterized. Carbachol (EC₅₀=9 M), (+)muscarine (EC₅₀=4.5 M) and cis-dioxolane (EC₅=0.72 M) were full agonists which stimulated PI turnover by 13.3±1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM–10 M muscarine during the last 24h of [³H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cisdioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. pertussis toxin (5–2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 M) PI turnover. The half-maximal effect of TPA was produced at 1.8±0.3 nM. [³H]4-DAMP binding to cell homogenates was of high affinity (K_d=0.19±0.04 nM) and moderate capacity (B_max=201±22 fmol/mg protein). The pharmacological specificity (4-DAMP>p-FHHSiD>dicyclomine>pirenzepine>methoctramine>AFDX-116 >gallamine) resembled that for [³H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M₃ receptors on HSDM1C1 cells. These receptors have been shown to be influenced by pertussis toxin, an active phorbol ester and to exhibit desensitization.     

Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ET_A and ET_B receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of ¹²⁵I-ET-1 (nonselective bivalent radioligand), ¹²⁵I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K _D of 71 pM and a B _max of 120 fmol mg^–1. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K _D and 8.0 fmol mg^–1 of B _max, whereas 10 nM sarafotoxin S6c (ET_B agonist) exerted little change in these binding parameters (K _D, 72 pM; B _max, 110 fmol mg^–1).3. Competition binding studies with a fixed amount (3.8 pM) of ¹²⁵I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ET_B agonist), and BQ-788 (ET_B antagonist) competitively inhibited ¹²⁵I-ET-1 binding with K _is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for ¹²⁵I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of ¹²⁵I-IRL 1620 (ET_B radioligand), IRL1620 bound to a single population of the ET_B receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. ¹²⁵I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K _is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ET_A antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for ¹²⁵I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ET_A and the ET_B receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ET_A and ET_B receptors with a functional binding capability for ET receptor-ligands, the ET_B receptor does not independently recognize ET-1 without the aid of the ET_A receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ET_A–ET_B receptor heterodimer.     

The serotonin 5-HT1D receptor: A progress review

Most of the known neurotransmitters interact with more than one type of receptor. Some of them even dispose of receptor subtypes to exert their actions. Serotonin, far from being an exception to that, possesses at least 3 classes of receptors, which have all been reported to be heterogeneous, although convincing data only exist for the 5-HT₁ class. This name has been proposed in 1979, two years before the introduction of A and B in the nomenclature to account for the observed heterogeneity of these cites. The 5-HT_1C receptor subtype was first described in 1984 and the last member of the family, named 5-HT_1D, was characterized in 1987. The pharmacological profiles, the signal transducing systems and the anatomical localizations, both at the regional and cellular levels, of all these subtypes have been investigated and possible functions have been proposed for each of them. Moreover, last and most definitive demonstration of the subtype individuality, the gene or complementary DNA coding for the 5-HT_1A and 5-HT_1C (and 5-HT₂) receptors have been cloned and sequenced. Such data are still missing for 5-HT_1D (and 5-HT_1B) receptors, but will certainly be provided in the next few years. However and waiting for this decisive clue, the characterization of the 5-HT_1D subtype leaves no doubt concerning its significance as a functional 5-HT receptor. This review will concentrate on the characteristics of this subtype of 5-HT receptor.Abbreviations 5-CT 5-carboxamidotryptamine - 5-MeOT 5-methoxy-tryptamine - 5-MeODMT N,N-dimethyl-5-methoxytryptamine - 8-OH-DPAT 8-hydroxy-2[di-n-propylamino]tetralin - CYP cyanopindolol - DHE dihydroergotamine - DOI 2,5-dimethoxy-4-iodophenylisopropylamine - DP-5-CT N,N-dipropyl 5-carboxamidotryptamine - ICPY 2-iodo-cyanopindolol - mCPP m-chloro-phenyl-piperazine - TFMPP m-trifluoro-methyl-phenyl-piperazine - E_MAX Maximal effect - EC₅₀ Half maximal effective concentration - K_D Dissociation constant - K_B Antagonist dissociation constant     

[3H]Adenine is a suitable radioligand for the labeling of G protein-coupled adenine receptors but shows high affinity to bacterial contaminations in buffer solutions

[³H]Adenine has previously been used to label the newly discovered G protein-coupled murine adenine receptors. Recent reports have questioned the suitability of [³H]adenine for adenine receptor binding studies because of curious results, e.g. high specific binding even in the absence of mammalian protein. In this study, we showed that specific [³H]adenine binding to various mammalian membrane preparations increased linearly with protein concentration. Furthermore, we found that Tris-buffer solutions typically used for radioligand binding studies (50 mM, pH 7.4) that have not been freshly prepared but stored at 4°C for some time may contain bacterial contaminations that exhibit high affinity binding for [³H]adenine. Specific binding is abolished by heating the contaminated buffer or filtering it through 0.2-μm filters. Three different, aerobic, gram-negative bacteria were isolated from a contaminated buffer solution and identified as Achromobacter xylosoxidans, A. denitrificans, and Acinetobacter lwoffii. A. xylosoxidans, a common bacterium that can cause nosocomial infections, showed a particularly high affinity for [³H]adenine in the low nanomolar range. Structure–activity relationships revealed that hypoxanthine also bound with high affinity to A. xylosoxidans, whereas other nucleobases (uracil, xanthine) and nucleosides (adenosine, uridine) did not. The nature of the labeled site in bacteria is not known, but preliminary results indicate that it may be a high-affinity purine transporter. We conclude that [³H]adenine is a well-suitable radioligand for adenine receptor binding studies but that bacterial contamination of the employed buffer solutions must be avoided. Anke C. Schiedel and Heiko Meyer contributed equally to this work.     

Endothelin-1 Binding to Endothelin Receptors in the Rat Anterior Pituitary Gland: Interaction in the Recognition of Endothelin-1 Between ETA and ETB Receptors

1. ¹²⁵I-Endothelin (ET)-1 binding to the rat anterior pituitary gland was saturable and single, with a K _d of 71 pM and a B _max of 120 fmol/mg.2. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, the binding parameters were 8.3 pM and 8.0 fmol/mg, whereas 10 nM sarafotoxin S6c (ET_Bagonist) exerted little change in these binding parameters (K _d,72pM;B _max, 110 fmol/mg).3. ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620, and BQ-788 competitively inhibited ¹²⁵I-ET-1 binding, only when 1.0 M BQ-123 was present in the incubation buffer.4. Thus, the ET_B receptor is capable of binding ET-1 when the ET_A receptor is being occupied by BQ-123. A collaboration mechanism between the ET_A and the ET_B receptor may function in the recognition of ET-1, a typical bivalent ligand.     

Pharmacological modulation of NMDA receptor activity and the advent of negative and positive allosteric modulators

The NMDA receptor (NMDAR) family of l-glutamate receptors are well known to have diverse roles in CNS function as well as in various neuropathological and psychiatric conditions. Until recently, the types of agents available to pharmacologically regulate NMDAR function have been quite limited in terms of mechanism of action and subtype selectivity. This has changed significantly in the past two years. The purpose of this review is to summarize the many drug classes now available for modulating NMDAR activity. Previously, this included competitive antagonists at the l-glutamate and glycine binding sites, high and low affinity channel blockers, and GluN2B-selective N-terminal domain binding site antagonists. More recently, we and others have identified new classes of NMDAR agents that are either positive or negative allosteric modulators (PAMs and NAMs, respectively). These compounds include the pan potentiator UBP646, the GluN2A-selective potentiator/GluN2C and GluN2D inhibitor UBP512, the GluN2D-selective potentiator UBP551, the GluN2C/GluN2D-selective potentiator CIQ as well as the new NMDAR-NAMs such as the pan-inhibitor UBP618, the GluN2C/GluN2D-selective inhibitor QZN46 and the GluN2A inhibitors UBP608 and TCN201. These new agents do not bind within the l-glutamate or glycine binding sites, the ion channel pore or the N-terminal regulatory domain. Collectively, these new allosteric modulators appear to be acting at multiple novel sites on the NMDAR complex. Importantly, these agents display improved subtype-selectivity and as NMDAR PAMs and NAMs, they represent a new generation of potential NMDAR therapeutics.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

The third intracellular loop of D1 and D5 dopaminergic receptors dictates their subtype-specific PKC-induced sensitization and desensitization in a receptor conformation-dependent manner

We previously showed that phorbol-12-myristate-13-acetate (PMA) mediates a robust PKC-dependent sensitization and desensitization of the highly homologous human Gs protein and adenylyl cyclase (AC)-linked D1 (hD1R) and D5 (hD5R) dopaminergic receptors, respectively. Here, we demonstrate using forskolin-mediated AC stimulation that PMA-mediated hD1R sensitization and hD5R desensitization is not associated with changes in AC activity. We next employed a series of chimeric hD1R and hD5R to delineate the underlying structural determinants dictating the subtype-specific regulation of human D1-like receptors by PMA. We first used chimeric receptors in which the whole terminal region (TR) spanning from the extracellular face of transmembrane domain 6 to the end of cytoplasmic tail (CT) or CT alone were exchanged between hD1R and hD5R. CT and TR swaps lead to chimeric hD1R and hD5R retaining PMA-induced sensitization and desensitization of wild type parent receptors. In striking contrast, hD1R sensitization and hD5R desensitization mediated by PMA are correspondingly switched to PMA-induced receptor desensitization and sensitization following the IL3 swap between hD1R and hD5R. Cell treatment with the PKC blocker, Gö6983, inhibits PMA-induced regulation of these chimeric receptors in a similar fashion to wild type receptors. Further studies with chimeras constructed by exchanging IL3 and TR show that PMA-induced regulation of these chimeras remains fully switched relative to their respective wild type parent receptor. Interestingly, results obtained with the exchange of IL3 and TR also reveal that the D1-like subtype-specific regulation by PMA, while fully dictated by IL3, can be modulated in a receptor conformation-dependent manner. Overall, our results strongly suggest that IL3 is the critical determinant underlying the subtype-specific regulation of human D1-like receptor responsiveness by PKC.