Inositol Phosphate Accumulation Research Articles

Identification of a potential modification site in human stromal cell‐derived factor‐1

Selective modification of proteins is an important tool to study their function. However, it is still challenging to identify the best position to avoid a loss of activity. By using a 6-nitroveratryl (Nvoc)-modification approach, we facilitate the identification of a potential modification site as Nvoc can be removed in situ by UV irradiation and accordingly allows directly the comparison of the biological activity of the modified and the unmodified protein derived from the same precursor. As a test system, we used stromal cell-derived factor-1 (SDF-1), which is involved in a wide range of physiological functions, mainly hematopoiesis and embryonic organ development. This chemokine is a potential candidate in regenerative medicine because of its capability to attract stem cells to distinct localizations. First, we synthesized the wildtype and the Nvoc-modified C-terminal segments SDF-1(50-68) and studied their secondary structure formation by circular dichroism spectroscopy. By using the intein-mediated purification with a affinity chitin binding tag system, we then expressed the peptide thioester M-[A(49)]-SDF-1(1-49)-MESNA recombinantly, in which the valine at position 49 was replaced by a more suitable alanine residue to allow improved cleavage and ligation. After ligation and refolding, the biological activity was proven in a cell-based inositol phosphate accumulation assay prior and after Nvoc removal, which showed that neither the alanine 49 nor the attached Nvoc group impair the activity of the analog. The study shows that lysine 56 is a potential site to introduce labels site-specifically in SDF-1.

The Constitutive Activity of the Human Muscarinic M3 Receptor Unmasks Differences in the Pharmacology of Anticholinergics

An activator protein 1-driven luciferase reporter assay was developed to monitor the activation of the human muscarinic M3 receptor (hM3-R) and evaluate functional potencies of different anticholinergics in Chinese hamster ovary cells. This assay proved to be superior to previously used functional assays [i.e., inositol phosphate accumulation (J Pharmacol Exp Ther 330:660-668, 2009)], thanks to the longer incubation times that allow reaching of pseudoequilibrium for ligands with slower dissociation kinetics, the long-acting muscarinic antagonists. Interestingly, within this system the hM3-R efficiently signaled in an agonist-independent manner. All the antagonists tested were able to inhibit the hM3-R constitutive activity in a concentration-dependent fashion, behaving as full inverse agonists. Curiously, significant differences in potency as antagonists (against carbachol) and inverse agonists were seen for some compounds (N-methyl scopolamine and tiotropium). Given the potential for inverse agonists to cause receptor up-regulation, the effect of chronic exposure to anticholinergics on the expression levels of hM3-R was also tested. Again, significant differences were seen, with some ligands (e.g., tiotropium) producing less than half of the receptor up-regulation caused by other anticholinergics. This study shows that anticholinergics can exhibit differential behaviors, which depend on the pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation. In addition, differences in the potential of anticholinergics to induce hM3-R up-regulation might have clinical relevance, because many are on the market or in clinical trials as chronic treatment for chronic obstructive pulmonary disease, for example.

Novel effects mediated by bradykinin and pharmacological characterization of bradykinin B2 receptor antagonism in human synovial fibroblasts

Bradykinin (BK) and B2 receptors have been implicated in the pathophysiology of osteoarthritis (OA), and synovitis is one of its hallmarks. Here, the selective B2 receptor antagonists MEN16132 and icatibant have been pharmacologically characterized in human synovial cells. Radioligand and functional studies (inositol phosphate (IP) accumulation, interleukin (IL)-6 and IL-8 release) were performed in cultured synoviocytes. [3H]-BK saturation studies indicated receptor density (Bmax) and K(d) values of 121,550 sites per cell and 1.14 nM respectively. In synoviocytes, MEN16132 (pK(I) 8.9) was threefold more potent than icatibant (pK(I) 8.4). Both antagonists showed competitive antagonism in the BK-induced IP assay (control EC50 0.45 nM), with pK(B) values of 9.9 (MEN16132) and 8.1 (icatibant). 24h incubation with BK induced IL-6 (EC50 216 nM) and IL-8 (EC50 53 nM) release. Both MEN16132 (IL-6: pIC50 8.1; IL-8: pIC50 8.4) and icatibant (IL-6: pIC50 6.6; IL-8: pIC50 6.7) completely prevented this BK-induced release. Indomethacin did not affect the basal or the IL-6/IL-8 release induced by BK, whereas nordihydroguaiaretic acid decreased the basal release, although BK still increased IL-6 and IL-8 production. BK-induced IL-8 release was attenuated by inhibitors of phospholipase C (U73122), p38 (SB203580), JNK (SP600125), ERK 1/2 (PD98059) MAPKs, phosphoinositide 3-kinase (LY294002), NF-kappaB (BAY-117085) and by the glucocorticoid dexamethasone. Bradykinin via B2 receptors can participate in inflammatory events in synovitis. MEN16132 is a highly potent B2 receptor antagonist capable of blocking pro-inflammatory responses to BK evoked in human synoviocytes.

Dual effect of the novel peptide antagonist K‐14585 on proteinase‐activated receptor‐2‐mediated signalling

Here we have examined the effects of the novel peptide antagonist N-[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indol-5-yl]aminocarbonyl]-glycinyl-L-lysinyl-L-phenylalanyl-N-benzhydrylamide (K-14585) on proteinase-activated receptor (PAR)(2)-mediated intracellular signalling events. Using NCTC2544 cells expressing PAR(2), we assessed the effects of K-14585 on PAR(2)-mediated [(3)H] inositol phosphate accumulation, MAP kinase activation, p65 NFkappaB phosphorylation and DNA binding and IL-8 production. Pretreatment with K-14585 (5 microM) inhibited [(3)H] inositol phosphate levels stimulated by PAR(2)-activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) in PAR(2)-expressing NCTC2544 cells. K-14585 pretreatment did not influence PAR(2)-mediated extracellular regulated kinase activation but inhibited p38 MAP kinase phosphorylation. At a higher concentration (30 microM), K-14585 alone stimulated p38 MAP kinase activation. These effects were replicated in EAhy926 cells, endogenously expressing PAR(2), but not in parental or PAR(4)-expressing NCTC2544 cells, suggesting these effects were PAR(2)-dependent. SLIGKV-mediated stimulation of p38 MAP kinase phosphorylation was substantially reduced by the G(q/11) inhibitor YM-254890, without affecting K-14585-mediated phosphorylation. Pretreatment with K-14585 inhibited PAR(2)-mediated p65 NFkappaB phosphorylation and NFkappaB-DNA binding. K-14585 (30 microM) alone stimulated comparable NFkappaB reporter activity to SLIGKV-OH. K-14585 inhibited SLIGKV-stimulated IL-8 production, but given alone increased IL-8. While SLIGKV-induced IL-8 formation was reduced by both SB203580 and YM-254890, the response to K-14585 was sensitive to SB203580 but not YM-254890. These data reveal that K-14585 has a duality of action functioning both as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling. The data also suggest two modes of p38 MAP kinase activation emanating from PAR(2), one G(q/11)-dependent and the other G(q/11)-independent.

Elevated Expression of GPR34 in Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma and Its Association with Increased Cell Growth, Erk Activation, and AP-1 and CRE-Mediated Transcription.

Abstract 3927Poster Board III-863In previous studies we characterized the t(X;14)(p11.4;q32) translocation in a patient with MALT lymphoma and found that GPR34, an orphan G-protein coupled receptor (GPCR), was highly expressed due to its juxtaposition to the IGHSA2 switch region. As part of a larger MALT gene expression-profiling project, we have now acquired gene expression analysis on the patient carrying the t(X;14)(p11;q32) translocation and have confirmed overexpression of GPR34. We then measured GPR34 mRNA expression in a panel of MALT lymphomas (n=17) and found that GPR34 was expressed at levels higher than that seen in normal B cells (mean, 11.3 fold; median, 5.5; range, 1.4-64 fold). When analyzed separately, 70% (12/17) had an expression level greater than 3-fold over normal B cells. Of note, in a gastric MALT lymphoma specimen, we found a 64 fold increase in GPR34 mRNA expression. FISH studies performed on this specimen showed an extra intact GPR34 signal but no translocation involving IGH or GPR34, suggesting that other mechanisms, including gene dosage effect, can upregulate GPR34. Elevated expression of GPR34 mRNA was also detected in other histologic types of NHL, but not to the extent seen in MALT lymphoma. Taken together, these data suggest that GPR34 is commonly overexpressed in MALT lymphoma and that deregulation of GPR34 expression can occur independent of a t(X;14)(p11.4;q32) translocation.The receptor encoded by GPR34 is most similar to the PY2 receptor subfamily of GPCR and GPR34 mRNA transcripts are abundant in mast cells while lower levels were detected in other immune cells including B cells. Signals from GPR34 have been briefly described and the results to date suggest that overexpression of GPR34 results in an accumulation of inositol phosphates. To further characterize the impact of GPR34 overexpression on cell signaling, HeLa cells were transduced with a retroviral expression plasmid (pBMN-GFP) that expresses GPR34 and GFP. GFP expressing cells were isolated and overexpression of GPR34 mRNA was confirmed by PCR and GPR34 protein expression was detected by flow cytometry. When normalized to the isotype control, pBMN-GPR34 cells expressed 17-fold more GPR34 on their cell surface compared to the pBMN-vector control cells. To determine which signaling pathways were affected by GPR34 overexpression, pBMN-GPR34 or pBMN-vector control cells were transfected with an AP-1, CRE, NF-κB, E2F, SRE, NFAT, or ISRE- luciferase reporter plasmid. Upon normalization with renilla, pBMN-GPR34 expressing cells had increased luciferase activity (n=3) driven by AP-1 (5.35-fold), CRE (4.7), NF-κB (2.8-fold), and E2F (2.13) when compared to pBMN-vector control cells. ISRE, NFAT, and SRE mediated luciferase expression was similar in the GPR34 and control cells. AP-1 and CRE have been implicated in a large variety of cellular processes, including transformation, and both AP-1 and CRE activity is induced upon activation of MAP kinases. To determine if MAPK activity was also upregulated in GPR34 expressing cells, we analyzed the phosphorylation status of Erk1/2 in pBMN-GPR34 cells by western blot and found that Erk1/2 was constitutively phosphorylated in GRP34 expressing cells (1.8 fold increase) compared to vector control cells. Increased phosphorylation of PKC-α/β was also detected in pBMN-GPR34 cells (3.5 fold increase compared to control cells). To determine the biologic impact of GRP34 overexpression on cell growth, the proliferation rates of pBMN-control and pBMN-GPR34 cells were compared and it was found that proliferation of GPR34 expressing cells was 2.2 times higher than that seen in control cells. Because the MAPK kinase pathway was found to be active in the pBMN-GPR34 cells, we tested the effect of the MEK inhibitor PD98059 on proliferation and saw a dose dependent decrease in proliferation of GPR34 expressing cells. These results suggest that GPR34-mediated proliferation is Erk-dependent.In summary, these data suggest that deregulation of GPR34 is commonly found in MALT lymphoma and that overexpression of GPR34 results in activation of Erk1/2, phosphorylation of PKC, and results is AP-1 and CRE mediated transcription. Additionally, our data suggest that overexpression of GPR34 results in increased cell growth that is MAPK-dependent. Taken together, this studies indicate that overexpression of a GPCR, GPR34, may be a novel mechanism by which MALT, lymphoma, and potentially other subtypes of NHL, develop. Disclosures:No relevant conflicts of interest to declare.

Muscarinic cholinoceptor activation by pilocarpine triggers apoptosis in human skin fibroblast cells

The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChRs) on apoptosis in human skin fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with pilocarpine in the presence or absence of specific antagonists. Pilocarpine stimulates apoptosis, total inositol phosphates (InsP) accumulation and nitric oxide synthase (NOS) activity. All these effects were inhibited by atropine, mustard hydrochloride (4-DAMP) and pirenzepine, indicating that M(1) and M(3) mAChRs are implicated in pilocarpine action. Pilocarpine apoptotic action is accompanied by caspase-3 and JNK activation. The intracellular pathway leading to pilocarpine-induced biological effects involved phospholipase C, calcium/calmodulin and extracellular calcium as U-73122, W-7, verapamil, BAPTA and BAPTA-AM blocked pilocarpine effects. L-NMMA, a NOS inhibitor, had no effect, indicating that the enzyme does not participate in the apoptosis phenomenon. These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on the apoptotic human skin fibroblast process.

The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

Identification and Analysis of Two Splice Variants of Human G2A Generated by Alternative Splicing

G2A is a G protein-coupled receptor that can be induced by various stressors. G2A is reported to have proton-sensing activity that mediates intracellular inositol phosphate (IP) accumulation with decreasing pH. We previously showed that G2A is also activated by some oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid (9-HODE). In this study, we identified a novel alternative splice variant of G2A (G2A-b) that has a partially different N terminus compared with the G2A originally reported (G2A-a). The two splice variants of G2A show similar tissue distributions, but G2A-b is expressed more abundantly. There was no difference between the two variants in 9-HODE-induced cellular responses, such as intracellular calcium mobilization and GDP/GTP exchange of Galpha protein, and in proton-sensitive IP accumulation. However, G2A-b showed a higher basal activity in terms of IP accumulation. Mutagenesis study revealed that the difference in the basal activity is attributable to the K7 residue that exists only in G2A-a. We further demonstrated that an R42A mutation largely impaired both the basal and proton-sensing activities, but did not affect the 9-HODE-induced intracellular calcium increase. Taken together, we found an additional novel G2A variant (G2A-b) that is the major transcript with functional response to ligand stimulation as well as G2A-a, and succeeded in discriminating proton-sensing and oxidized fatty acid-sensing activities of G2A.

Nelotanserin, a Novel Selective Human 5-Hydroxytryptamine2A Inverse Agonist for the Treatment of Insomnia

5-Hydroxytryptamine (5-HT)(2A) receptor inverse agonists are promising therapeutic agents for the treatment of sleep maintenance insomnias. Among these agents is nelotanserin, a potent, selective 5-HT(2A) inverse agonist. Both radioligand binding and functional inositol phosphate accumulation assays suggest that nelotanserin has low nanomolar potency on the 5-HT(2A) receptor with at least 30- and 5000-fold selectivity compared with 5-HT(2C) and 5-HT(2B) receptors, respectively. Nelotanserin dosed orally prevented (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 5-HT(2A) agonist)-induced hypolocomotion, increased sleep consolidation, and increased total nonrapid eye movement sleep time and deep sleep, the latter marked by increases in electroencephalogram (EEG) delta power. These effects on rat sleep were maintained after repeated subchronic dosing. In healthy human volunteers, nelotanserin was rapidly absorbed after oral administration and achieved maximum concentrations 1 h later. EEG effects occurred within 2 to 4 h after dosing, and were consistent with vigilance-lowering. A dose response of nelotanserin was assessed in a postnap insomnia model in healthy subjects. All doses (up to 40 mg) of nelotanserin significantly improved measures of sleep consolidation, including decreases in the number of stage shifts, number of awakenings after sleep onset, microarousal index, and number of sleep bouts, concomitant with increases in sleep bout duration. Nelotanserin did not affect total sleep time, or sleep onset latency. Furthermore, subjective pharmacodynamic effects observed the morning after dosing were minimal and had no functional consequences on psychomotor skills or memory. These studies point to an efficacy and safety profile for nelotanserin that might be ideally suited for the treatment of sleep maintenance insomnias.

Activating autoantibodies against the calcium-sensing receptor detected in two patients with autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type 1 (APS1) is an autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene. Hypoparathyroidism occurs in 80% of patients with APS1 and has been suggested to result from an autoimmune reaction against the calcium-sensing receptor (CaSR) in parathyroid cells. Anti-CaSR binding antibodies have previously been detected in patients with APS1. The aim of this study was to determine whether anti-CaSR antibodies present in APS1 patients could modulate the response of the CaSR to stimulation by Ca(2+). The results indicated that two of the 14 APS1 patients included in the study had anti-CaSR antibodies that stimulated the receptor. These antibodies were detected by their ability to increase both Ca(2+)-dependent extracellular signal-regulated kinase phosphorylation and inositol phosphate accumulation in human embryonic kidney 293 cells expressing the CaSR. An important implication of the present results is that although the majority of APS1 patients do not have CaSR-stimulating antibodies, there may be a small but substantial minority of patients in whom the hypoparathyroid state is the result of functional suppression of the parathyroid glands rather than their irreversible destruction.

The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+‐binding protein 2 modulates receptor function

Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

Effect of the Calcimimetic R-568 [3-(2-Chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine] on Correcting Inactivating Mutations in the Human Calcium-Sensing Receptor

Over 257 mutations in the human calcium-sensing receptor (hCaSR) gene have been reported. Heterozygous inactivating mutations can result in familial hypocalciuric hypercalcemia (FHH), whereas homozygous inactivating mutations can cause life-threatening neonatal severe hyperparathyroidism (NSHPT). Activating mutations in the hCaSR can result in hypercalciuria and hypocalcemia. A recent publication on the successful treatment of a patient suffering from FHH with the hCaSR positive allosteric modulator cinacalcet prompted our interest in exploring the molecular pharmacology of calcimimetics to correct signaling defects associated with inactivating hCaSR mutations. We prepared 11 mutant hCaSRs, previously identified in patients suffering from NSHPT or FHH, and tested their ability to couple to inositol phosphate accumulation and intracellular calcium mobilization in transiently transfected human embryonic kidney 293 and Chines hamster ovary cells using the calcimimetic R-568 [3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine]. We found that extracellular Ca(2+) was significantly less potent on the mutated receptors compared with wild-type hCaSR. However, R-568 was able to enhance the potency of extracellular Ca(2+) toward the mutant hCaSRs. Furthermore, R-568 increased the maximal agonist response on several of the mutant CaSRs. We applied a novel operational model of allosteric modulation/agonism that provided a common mechanism to account for the behavior of the wild-type and mutant hCaSRs. The data provide evidence for the potential use of calcimimetics to treat diseases such as FHH and NSHPT where severe hypercalcemia can be life-threatening.

Characterization of P2Y receptor subtypes functionally expressed on neonatal rat cardiac myofibroblasts

Little is known about P2Y receptors in cardiac fibroblasts, which represent the predominant cell type in the heart and differentiate into myofibroblasts under certain conditions. Therefore, we have characterized the phenotype of the cells and the different P2Y receptors at the expression and functional levels in neonatal rat non-cardiomyocytes. Non-cardiomyocyte phenotype was determined by confocal microscopy by using discoidin domain receptor 2, alpha-actin and desmin antibodies. P2Y receptor expression was investigated by reverse transcription-polymerase chain reaction and immunocytochemistry, and receptor function by cAMP and inositol phosphate (IP) accumulation induced by adenine or uracil nucleotides in the presence or absence of selective antagonists of P2Y(1) (MRS 2179, 2-deoxy-N(6)-methyl adenosine 3',5'-diphosphate diammonium salt), P2Y(6) (MRS 2578) and P2Y(11) (NF 157, 8,8'-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-fluoro-3,1-phenylene)carbonylimino]]bis-1,3,5-naphthalene trisulphonic acid hexasodium salt) receptors. G(i/o) and G(q/11) pathways were evaluated by using Pertussis toxin and YM-254890 respectively. The cells (>95%) were alpha-actin and discoidin domain receptor 2-positive and desmin-negative. P2Y(1), P2Y(2), P2Y(4), P2Y(6) were detected by reverse transcription-polymerase chain reaction and immunocytochemistry, and P2Y(11)-like receptors at protein level. All di- or tri-phosphate nucleotides stimulated IP production in an YM-254890-sensitive manner. AMP, ADPbetaS, ATP and ATPgammaS increased cAMP accumulation, whereas UDP and UTP inhibited cAMP response, which was abolished by Pertussis toxin. MRS 2179 and NF 157 inhibited ADPbetaS-induced IP production. MRS 2578 blocked UDP- and UTP-mediated IP responses. P2Y(1)-, P2Y(2)-, P2Y(4)-, P2Y(6)-, P2Y(11)-like receptors were co-expressed and induced function through G(q/11) protein coupling in myofibroblasts. Furthermore, P2Y(2) and P2Y(4) receptor subtypes were also coupled to G(i/o). The G(s) response to adenine nucleotides suggests a possible expression of a new P2Y receptor subtype.

APD791, 3-Methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide, a Novel 5-Hydroxytryptamine 2A Receptor Antagonist: Pharmacological Profile, Pharmacokinetics, Platelet Activity and Vascular Biology

We have evaluated the receptor pharmacology, antiplatelet activity, and vascular pharmacology of APD791 [3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide] a novel 5-hydroxytryptamine 2A (5-HT(2A)) receptor antagonist. APD791 displayed high-affinity binding to membranes (K(i) = 4.9 nM) and functional inverse agonism of inositol phosphate accumulation (IC(50) = 5.2 nM) in human embryonic kidney cells stably expressing the human 5-HT(2A) receptor. In competition binding assays, APD791 was greater than 2000-fold selective for the 5-HT(2A) receptor versus 5-HT(2C) and 5-HT(2B) receptors, and was inactive when tested against a wide panel of other G-protein-coupled receptors. APD791 inhibited 5-HT-mediated amplification of ADP-stimulated human and dog platelet aggregation (IC(50) = 8.7 and 23.1 nM, respectively). Similar potency was observed for inhibition of 5-HT-stimulated DNA synthesis in rabbit aortic smooth muscle cells (IC(50) = 13 nM) and 5-HT-mediated vasoconstriction in rabbit aortic rings. Oral administration of APD791 to dogs resulted in acute (1-h) and subchronic (10-day) inhibition of 5-HT-mediated amplification of collagen-stimulated platelet aggregation in whole blood. Two active metabolites, APD791-M1 and APD791-M2, were generated upon incubation of APD791 with human liver microsomes and were also indentified in dogs after oral administration of APD791. The affinity and selectivity profiles of both metabolites were similar to APD791. These results demonstrate that APD791 is an orally available, high-affinity 5-HT(2A) receptor antagonist with potent activity on platelets and vascular smooth muscle.

Metabolite Profiling of Two Novel Low Phytic Acid (lpa) Soybean Mutants

A GC-based approach was applied to compare the metabolite profiles of two low phytic acid (lpa) soybean mutants and their respective wild-types. The lpa mutants (Gm-lpa-TW75-1 and Gm-lpa-ZC-2) were grown together with the wild-types (Taiwan 75 and Zhechun no. 3) in three and four field trials, respectively. HPLC analysis revealed a phytic acid reduction of -53% for Gm-lpa-TW75-1 and of -46% for Gm-lpa-ZC-2. For Gm-lpa-TW75-1, no accumulation of lower inositol phosphates was observed, whereas Gm-lpa-ZC-2 exhibited significantly increased contents of the lower inositol phosphates InsP(3), InsP(4), and InsP(5) compared to the corresponding wild-type. The metabolite profiling revealed that compared to the wild-types, 40% (Gm-lpa-TW75-1) and 21% (Gm-lpa-ZC-2) of the detected peaks were statistically significantly different in the lpa mutants grown at one field trial. However, the majority of these differences were shown to be related to environmental impact and natural variability rather than to the mutation event. Identification of consistent metabolic changes in the lpa mutants revealed decreased contents of myo-inositol, galactinol, raffinose, stachyose, and the galactosyl cyclitols galactopinitol A, galactopinitol B, and fagopyritol B1 compared to the wild-type. These consistently pronounced changes in Gm-lpa-TW75-1 confirmed the suggested mutation target. Consideration of the metabolic changes observed for Gm-lpa-ZC-2 (accumulation of lower inositol phosphates and increased myo-inositol contents) indicated a mutation event affecting the latter biosynthetic steps leading to phytic acid. The study demonstrated the applicability of metabolite profiling for the detection of changes in the metabolite phenotype induced by mutation breeding and its power in assisting in the elucidation of mutation events.

A novel GalR2-specific peptide agonist

The galanin peptide family and its three receptors have with compelling evidence been implicated in several high-order physiological disorders. The co-localization with other neuromodulators and the distinct up-regulation during and after pathological disturbances has drawn attention to this neuropeptide family. In the current study we present data on receptor binding and functional response for a novel galanin receptor type 2 (GalR2) selective chimeric peptide, M1145 [(RG)(2)-N-galanin(2-13)-VL-(P)(3)-(AL)(2)-A-amide]. The M1145 peptide shows more than 90-fold higher affinity for GalR2 over GalR1 and a 76-fold higher affinity over GalR3. Furthermore, the peptide yields an agonistic effect in vitro, seen as an increase in inositol phosphate (IP) accumulation, both in the absence or the presence of galanin. The peptide design with a N-terminal extension of galanin(2-13), prevails new insights in the assembly of novel subtype specific ligands for the galanin receptor family and opens new possibilities to apply the galanin system as a putative drug target.

Regulation of Oxytocin Receptor Responsiveness by G Protein-Coupled Receptor Kinase 6 in Human Myometrial Smooth Muscle

Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.

Phospholipase C isozymes as effectors of Ras superfamily GTPases

The physiological effects of many extracellular stimuli are initiated through receptor-promoted activation of phospholipase C and inositol lipid signaling pathways. The historical view that phospholipase C-promoted signaling primarily occurs through activation of heterotrimeric G proteins or tyrosine kinases has expanded in recent years with the realization that at least three different mammalian phospholipase C isozymes are directly activated by members of the Ras superfamily of GTPases. Thus, Ras, Rap, Rac, and Rho GTPases all specifically regulate certain phospholipase C isozymes, and insight into the physiological significance of these signaling responses is beginning to accrue. High resolution three-dimensional structures of phospholipase C isozymes also are beginning to shed light on their mechanism of activation.

Assessment of the contents of phytic acid and divalent cations in low phytic acid ( lpa) mutants of rice and soybean

The objective of the study was to investigate the influence of mutational breeding on the contents of nutritionally relevant minerals in low phytic acid ( lpa) mutants compared to their wild-types. Three lpa rice ( Os-lpa-XQZ-1, Os-lpa-XS110-1, Os-lpa-XS110-2) and two lpa soybean mutants ( Gm-lpa-TW75-1, Gm-lpa-ZC-2) were analyzed regarding their contents of phytic acid, lower inositol phosphates and the minerals calcium, iron and zinc. Additionally, cadmium was included into the spectrum of divalent cations analyzed. The phytic acid reduction in lpa rice was consistently more pronounced in Os-lpa-XS110-1 than in Os-lpa-XQZ-1 and Os-lpa-XS110-2. However, only for Os-lpa-XQZ-1, levels of calcium, iron and zinc were found to be consistently increased, whereas the cadmium level was shown to be predominantly decreased compared to the wild-type. Investigation of the two lpa soybean mutants, characterized by absence ( Gm-lpa-TW75-1) and accumulation ( Gm-lpa-ZC-2) of lower inositol phosphates, respectively, revealed no consistently decreased or increased contents of calcium, iron, zinc and cadmium. The data do not change the view that lpa mutations do not result in systematic increases or decreases of mineral contents in these crops. However, on the basis of the molar ratios of phytate/minerals in lpa rice and soybean mutants which are considered as predictive parameters, an improved bioavailability of minerals in the lpa materials can be expected.

The effect of oxytocin on progesterone secretion, phosphoinositide hydrolysis and intracellular mobilisation of Ca 2+ in porcine luteal cells

Oxytocin (OT) is involved in the regulation of steroid secretion by the corpus luteum (CL) in pigs, but OT signal transduction in the porcine CL has not been identified. In this study, the effects of OT on in vitro progesterone (P4) secretion, phosphoinositide (PI) hydrolysis and intracellular mobilisation of Ca2+ ([Ca2+]i) were investigated in porcine luteal cells during the early (days 3-5), mid(days 8-10) and late luteal phases (days 12-14) of the oestrous cycle. Basal concentrations of P4 and accumulation of inositol phosphates (IPs) were higher (P < 0.05) on days 3-5 and 8-10 of the oestrous cycle than on days 12-14. Basal [Ca2+]i mobilisation did not differ among studied periods of the oestrous cycle. Oxytocin (10(-7) M) enhanced P4 secretion and PI hydrolysis (P < 0.05) by luteal cells harvested on days 8-10 of the oestrous cycle. Moreover, OT started to increase mobilisation of [Ca2+]i at the 15th (days 3-5 and 8-10) or 30th second (days 12-14) in porcine luteal cells. It was concluded that in pigs OT acts as a regulator of steroidogenesis, stimulating P4 secretion in mature CL. This OT action may be mediated by changes in PI hydrolysis and [Ca2+]i mobilisation.