Translocon Subunits of the COP9 Signalosome Complex Are a Central Hub for Regulating Multiple Photoresponsive Processes and Autophagic Flux in Magnaporthe oryzae.

Photodependent processes, including circadian rhythm, autophagy, ubiquitination, neddylation/deneddylation, and metabolite biosynthesis, profoundly influence microbial pathogenesis. Although a photomorphogenesis signalosome (COP9/CSN) has been identified, the mechanism by which this large complex contributes to the pathophysiological processes in filamentous fungi remains unclear. Here, we identified eight CSN complex subunits in the rice blast fungus Magnaporthe oryzae and functionally characterized the translocon subunits containing a nuclear export or localization signal (NES/NLS). Targeted gene replacement of these CSN subunits, including MoCSN3, MoCSN5, MoCSN6, MoCSN7, and MoCSN12, attenuated vegetative growth and conidiation and rendered the deletion strains nonpathogenic. MoCSN7 deletion significantly suppressed arachidonic acid catabolism, and compromised cell wall integrity in M. oryzae. Surprisingly, we also discovered that MoCSN subunits, particularly MoCsn7, are required for the cAMP-dependent regulation of autophagic flux. Therefore, MoCSN significantly contributes to morphological, physiological, and pathogenic differentiation in M. oryzae by fostering cross-talk between multiple pathways.

Pharmacological characterization of the zebrafish Hrh2a histamine H2 receptor.

The zebrafish, Danio rerio, is a widely adopted in vivo model that conserves organs such as the liver, kidney, stomach, and brain, being, therefore, suitable for studying human diseases, drug discovery and toxicology. The brain aminergic systems are also conserved and the histamine H1, H2 and H3 receptors were previously cloned and identified in the zebrafish brain. Genome studies identified another putative H2 receptor (Hrh2) with ∼50% sequence identity with H2 receptor orthologs. In this study, we recombinantly expressed both zebrafish H2 receptor paralogs (hrh2a and hrh2b) and compared their pharmacology with the human H2 receptor ortholog. Our results showed that both zebrafish receptors conserve all the class A GPCR motifs. However, in contrast with the Hrh2a paralog, the Hrh2b does not possess all the amino acid residues shown to participate in histamine binding. The zebrafish Hrh2a receptor displays high affinity for [3H]-tiotidine with a binding profile for H2 receptor ligands similar to that of the human H2 receptor. The zebrafish Hrh2a receptor couples to GαS and Gαq/11 proteins, resulting in cAMP accumulation and activation of several reporter genes linked to the Gαq/11 pathway. Additionally, this receptor shows high constitutive activity, with histamine potency in the low nanomolar range for cAMP accumulation and the micromolar range for the activation of the NFAT response element. Moreover, dimaprit and amthamine seem to preferentially activate GαS over Gαq/11 proteins via the zebrafish Hrh2a receptor. These results can contribute to clarifying the functional roles of the H2 receptor in zebrafish.

Functional Activity of Enantiomeric Oximes and Diastereomeric Amines and Cyano Substituents at C9 in 3-Hydroxy-N-phenethyl-5-phenylmorphans.

The synthesis of stereochemically pure oximes, amines, saturated and unsaturated cyanomethyl compounds, and methylaminomethyl compounds at the C9 position in 3-hydroxy-N-phenethyl-5-phenylmorphans provided µ-opioid receptor (MOR) agonists with varied efficacy and potency. One of the most interesting compounds, (2-((1S,5R,9R)-5-(3-hydroxyphenyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-9-yl)acetonitrile), was found to be a potent partial MOR agonist (EC50 = 2.5 nM, %Emax = 89.6%), as determined in the forskolin-induced cAMP accumulation assay. Others ranged in potency and efficacy at the MOR, from nanomolar potency with a C9 cyanomethyl compound (EC50 = 0.85 nM) to its totally inactive diastereomer, and three compounds exhibited weak MOR antagonist activity (the primary amine 3, the secondary amine 8, and the cyanomethyl compound 41). Many of the compounds were fully efficacious; their efficacy and potency were affected by both the stereochemistry of the molecule and the specific C9 substituent. Most of the MOR agonists were selective in their receptor interactions, and only a few had δ-opioid receptor (DOR) or κ-opioid receptor (KOR) agonist activity. Only one compound, a C9-methylaminomethyl-substituted phenylmorphan, was moderately potent and fully efficacious as a KOR agonist (KOR EC50 = 18 nM (% Emax = 103%)).

Potent MOR Agonists from 2'-Hydroxy-5,9-dimethyl-N-phenethyl Substituted-6,7-benzomorphans and from C8-Hydroxy, Methylene and Methyl Derivatives of N-Phenethylnormetazocine.

(-)-5,9-Dimethyl-6,7-benzomorphan (normetazocine) derivatives with a para-OH or ortho-F substituent in the aromatic ring of the N-phenethyl moiety were synthesized and found to have subnanomolar potency at MOR, and both were fully efficacious in vitro. These new compounds, (1R,5R,9R)-6,11-dimethyl-3-(2-fluorophenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol and (1R,5R,9R)-6,11-dimethyl-3-(4-hydroxyphenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol, were more potent than the unsubstituted compound N-phenethylnormetazocine and about 30 or 40 times more potent than morphine, respectively. A variety of substituents in the ortho, meta, or para position in the aromatic ring of the N-phenethyl moiety were synthesized, 25 of these compounds, and found to have varying effects on potency and efficacy as determined by the forskolin-induced cAMP accumulation assay. The N-phenethyl moiety was also modified by increasing chain length to form a N-phenylpropyl side chain with and without a para-nitro moiety, and by an N-cinnamyl side chain. Also, an indole ethylamine normetazocine was synthesized to replace the N-phenethylamine side chain in normetazocine. The phenylpropylamine, propenylamine (cinnamyl) and the para-nitropropylamine had little or no MOR potency. The indole-ethylamine on the normetazocine nucleus, however, had moderate potency (MOR EC50 = 12 nM), and was fully efficacious (%Emax = 102%) in the cAMP assay. Retention of the N-phenethyl moiety and the addition of alkyl and alkenyl moieties on C8 in (-)-N-phenethylnormetazocine gave a C8-methylene derivative that had subnanomolar potency at MOR and a C8-methyl analog that had nanomolar potency. Five C8-substituted compounds were synthesized.

A MOR Antagonist with High Potency and Antagonist Efficacy among Diastereomeric C9-Alkyl-Substituted N-Phenethyl-5-(3-hydroxy)phenylmorphans.

The 5-(3-hydroxy)phenylmorphan structural class of compounds are unlike the classical morphinans, 4,5-epoxymorphinans, and 6,7-benzomorphans, in that they have an equatorially oriented aromatic ring rather than the axial orientation of that ring found in the classical opioids. This modified and simplified opioid-like structure has been shown to retain antinociceptive activity, depending on its stereochemistry and substituents, and some of them have been found to be much more potent than morphine. A simple C9-hydroxy-5-(3-hydroxy)phenylmorphan enantiomer was found to be about 500 times more potent than morphine in vivo. We have previously examined C9-alkenyl and hydroxyalkyl substituents in the N-phenethyl-5-(3-hydroxy)phenylmorphan class of compounds. Comparable C9-alkyl (methyl through butyl) substituents, with their sets of diastereomers, have not been explored. All these compounds have now been synthesized to determine the effect chain-length and stereochemistry at the C9 position in the molecule might have on their interaction with opioid receptors. We now report the synthesis and in vitro activity of 16 compounds, the C9-methyl, ethyl, propyl, and butyl diastereomers, using the inhibition of forskolin-induced cAMP accumulation assay. Several potent (sub-nanomolar and nanomolar) MOR compounds were found to be selective agonists with varying efficacy. Of greatest interest, a selective MOR antagonist was discovered; it did not display any DOR or KOR agonist activity in vitro, was three times more potent than naltrexone, and was found to antagonize the EC90 of fentanyl at MOR to a greater extent than naltrexone.

Discovery of a Potent Highly Biased MOR Partial Agonist among Diastereomeric C9-Hydroxyalkyl-5-phenylmorphans.

All possible diastereomeric C9-hydroxymethyl-, hydroxyethyl-, and hydroxypropyl-substituted 5-phenylmorphans were synthesized to explore the three-dimensional space around the C9 substituent in our search for potent MOR partial agonists. These compounds were designed to lessen the lipophilicity observed with their C9-alkenyl substituted relatives. Many of the 12 diastereomers that were obtained were found to have nanomolar or subnanomolar potency in the forskolin-induced cAMP accumulation assay. Almost all these potent compounds were fully efficacious, and three of those chosen for in vivo evaluation, 15, 21, and 36, were all extremely G-protein biased; none of the three compounds recruited beta-arrestin2. Only one of the 12 diastereomers, 21 (3-((1S,5R,9R)-9-(2-hydroxyethyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), was a MOR partial agonist with good, but not full, efficacy (Emax = 85%) and subnanomolar potency (EC50 = 0.91 nM) in the cAMP assay. It did not have any KOR agonist activity. This compound was unlike morphine in that it had a limited ventilatory effect in vivo. The activity of 21 could be related to one or more of three well-known theories that attempt to predict a dissociation of the desired analgesia from the undesirable opioid-like side-effects associated with clinically used opioids. In accordance with the theories, 21 was a potent MOR partial agonist, it was highly G-protein biased and did not attract beta-arrestin2, and it was found to have both MOR and DOR agonist activity. All the other diastereomers that were synthesized were either much less potent than 21 or had either too little or too much efficacy for our purposes. It was also noted that a C9-methoxymethyl compound with 1R,5S,9R stereochemistry (41) was more potent than the comparable C9-hydroxymethyl compound 11 (EC50 = 0.65 nM for 41 vs. 2.05 nM for 11). Both 41 and 11 were fully efficacious.

Comparative neuropharmacology of structurally distinct non-fentanyl opioids that are appearing on recreational drug markets worldwide.

BACKGROUND: The emergence of novel synthetic opioids (NSOs) is contributing to the opioid overdose crisis. While fentanyl analogs have historically dominated the NSO market, a shift towards non-fentanyl compounds is now occurring. METHODS: Here, we examined the neuropharmacology of structurally distinct non-fentanyl NSOs, including U-47700, isotonitazene, brorphine, and N-desethyl isotonitazene, as compared to morphine and fentanyl. Compounds were tested in vitro using opioid receptor binding assays in rat brain tissue and by monitoring forskolin-stimulated cAMP accumulation in cells expressing the human mu-opioid receptor (MOR). Compounds were administered subcutaneously to male Sprague-Dawley rats, and hot plate antinociception, catalepsy score, and body temperature changes were measured. RESULTS: Receptor binding results revealed high MOR selectivity for all compounds, with MOR affinities comparable to those of morphine and fentanyl (i.e., nM). All drugs acted as full-efficacy MOR agonists in the cyclic AMP assay, but nitazene analogs had greater functional potencies (i.e., pM) compared to the other drugs (i.e., nM). When administered to rats, all compounds induced opioid-like antinociception, catalepsy, and body temperature changes, but nitazenes were the most potent. Similar to fentanyl, the nitazenes had faster onset and decline of in vivo effects when compared to morphine. In vivo potencies to induce antinociception and catalepsy (i.e., ED50s) correlated with in vitro functional potencies (i.e., EC50s) but not binding affinities (i.e., Kis) at MOR. CONCLUSIONS: Collectively, our findings indicate that non-fentanyl NSOs pose grave danger to those individuals who use opioids. Continued vigilance is needed to identify and characterize synthetic opioids as they emerge in clandestine drug markets.

Bias-force guided simulations combined with experimental validations towards GPR17 modulators identification.

Glioblastoma Multiforme (GBM) is known to be by far the most aggressive brain tumor to affect adults. The median survival rate of GBM patient's is < 15 months, while the GBM cells aggressively develop resistance to chemo- and radiotherapy with their self-renewal capacity which suggests the pressing need to develop novel preventative measures. We have recently proved that GPR17 -an orphan G protein-coupled receptor- is highly expressed on the GBM cell surface and it has a vital role to play in the disease progression. Despite the progress made on GBM downregulation, there still remain difficulties in developing a promising modulator for GPR17, till date. Here, we have performed robust virtual screening combined with biased-force pulling molecular dynamic (MD) simulations to predict high-affinity GPR17 modulators followed by experimental validation. Initially, the database containing 1379 FDA-approved drugs were screened against the orthosteric binding pocket of the GPR17. The external bias-potentials were then applied to the screened hits during the MD simulations which enabled to predict a spectrum of rupture peak force values that were used to select four approved drugs -ZINC000003792417 (Sacubitril), ZINC000014210457 (Victrelis), ZINC000001536109 (Pralatrexate) and ZINC000003925861 (Vorapaxar)- as top hits. The hits selected turns out to demonstrate unique dissociation pathways, interaction pattern, and change in polar network over time. Subsequently the selected hits with GPR17 were measured by inhibiting the forskolin-stimulated cAMP accumulation in GBM cell lines, LN229 and SNB19. The ex vivo validations shows that Sacubitril drug can act as a full agonist, while Vorapaxar functions as a partial agonist for GPR17. The pEC50 of Sacubitril was identified as 4.841 and 4.661 for LN229 and SNB19, respectively. Small interference of the RNA (siRNA)- silenced the GPR17 to further validate the targeted binding of Sacubitril with GPR17. In the current investigation, we have identified new repurposable GPR17 specific drugs which are likely to increase the opportunity to treat orphan deadly diseases.

Humulane-Type Macrocyclic Sesquiterpenoids From the Endophytic Fungus Penicillium sp. of Carica papaya.

Three new humulane-type sesquiterpenoids, penirolide A (1), penirolide B (2), and 10-acetyl-phomanoxide (3), together with three known compounds aurasperone A (4), pughiinin A (5), and cyclo(l-Leu-l-Phe) (6) were isolated from the endophytic fungus Penicillium sp. derived from the leaves of Carica papaya L. Their structures including their absolute configurations were determined based on the analysis of NMR and HRESIMS spectra, NMR chemical shifts, and ECD calculations. Compounds 2, 3, 5, and 6 significantly inhibited glucagon-induced hepatic glucose production, with EC50 values of 33.3, 36.1, 18.8, and 32.1 µM, respectively. Further study revealed that compounds 2, 3, 5, and 6 inhibited hepatic glucose production by suppression of glucagon-induced cAMP accumulation.

The Intriguing Effects of Substituents in the N-Phenethyl Moiety of Norhydromorphone: A Bifunctional Opioid from a Set of "Tail Wags Dog" Experiments.

(-)-N-Phenethyl analogs of optically pure N-norhydromorphone were synthesized and pharmacologically evaluated in several in vitro assays (opioid receptor binding, stimulation of [35S]GTPγS binding, forskolin-induced cAMP accumulation assay, and MOR-mediated ß-arrestin recruitment assays). "Body" and "tail" interactions with opioid receptors (a subset of Portoghese's message-address theory) were used for molecular modeling and simulations, where the "address" can be considered the "body" of the hydromorphone molecule and the "message" delivered by the substituent (tail) on the aromatic ring of the N-phenethyl moiety. One compound, N-p-chloro-phenethynorhydromorphone ((7aR,12bS)-3-(4-chlorophenethyl)-9-hydroxy-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one, 2i), was found to have nanomolar binding affinity at MOR and DOR. It was a potent partial agonist at MOR and a full potent agonist at DOR with a δ/µ potency ratio of 1.2 in the ([35S]GTPγS) assay. Bifunctional opioids that interact with MOR and DOR, the latter as agonists or antagonists, have been reported to have fewer side-effects than MOR agonists. The p-chlorophenethyl compound 2i was evaluated for its effect on respiration in both mice and squirrel monkeys. Compound 2i did not depress respiration (using normal air) in mice or squirrel monkeys. However, under conditions of hypercapnia (using air mixed with 5% CO2), respiration was depressed in squirrel monkeys.

The endomorphin-1/2 and dynorphin-B peptides display biased agonism at the mu opioid receptor.

BACKGROUND: Opioid agonist activation at the mu opioid receptor (MOR) can lead to a wide variety of physiological responses. Many opioid agonists share the ability to selectively and preferentially activate specific signaling pathways, a term called biased agonism. Biased opioid ligands can theoretically induce specific physiological responses and might enable the generation of drugs with improved side effect profiles. METHODS: Dynorphins, enkephalins, and endomorphins are endogenous opioid agonist peptides that may possess distinct bias profiles; biased agonism of endogenous peptides could explain the selective roles of these ligands in vivo. Our purpose in the present study was to investigate biased signaling and potential underlying molecular mechanisms of bias using 35S-GTPγS and cAMP assays, specifically focusing on the role of adenylyl cyclases (ACs) and regulators of G-protein signaling proteins (RGSs) in CHO, N2a, and SH-SY5Y cell lines, all expressing the human MOR. RESULTS: We found that endomorphin-1/2 preferentially activated cAMP signaling, while dynorphin-B preferentially activated 35S-GTPγS signaling in most cell lines. Experiments carried out in the presence of an isoform selective RGS-4 inhibitor, and siRNA knockdown of AC6 in N2a cells did not significantly affect the bias properties of endomorphins, suggesting that these proteins may not play a role in endomorphin bias. CONCLUSION: We found that endomorphin-1/2 and dynorphin-B displayed contrasting bias profiles at the MOR, and ruled out potential AC6 and RGS4 mechanisms in this bias. This identified signaling bias could be involved in specifying endogenous peptide roles in vivo, where these peptides have low selectivity between opioid receptor family members.

Mesenchymal stem cells from bone marrow attenuated the chronic morphine-induced cAMP accumulation in vitro.

Even though opioid tolerance is both a common and a major challenge in medicine, treatment with opioids is currently the primary method used to treat acute and chronic pain. The cAMP accumulation induced by chronic morphine is regarded as one of the molecular mechanisms leading to its tolerance and dependence characteristics. In the present study, we differentiated SH-SY5Y cells into neuron-like cells by retinoic acid (RA), pretreated these cells with morphine, and tested their cAMP levels under different conditions, including co-culture with bone marrow-derived human mesenchymal stem cells from bone marrow (hMSCs-BM) at various hMSCs-BM/SH-SY5Y ratios (1:5, 1:25, and 1:125), by direct cell-to-cell contact or without cell-to-cell contact, and by conditioned medium (CM) from hMSCs-BM. We found that chronic treatment with 10 µM morphine led to cAMP upregulation in those RA-differentiated SH-SY5Y cells while the morphine induced-cAMP accumulation was significantly attenuated by co-culturing with hMSCs-BM by direct cell-to-cell contact at a lower cell ratio (1:25) and a higher cell ratio (1:5). However, at neither the low or higher cell ratios could hMSCs-BM inhibit morphine-induced cAMP accumulation in RA-differentiated SH-SY5Y cells without cell-to-cell contact. In summary, hMSCs-BM can successfully inhibit morphine-induced cAMP up-regulation in RA-differentiated SH-SY5Y cells by cell-to-cell contact at a higher ratio, suggesting that hMSCs-BM may serve as valuable therapeutics to minimize the risk of drug abuse and addiction in the treatment of morphine tolerance and dependence.

Pharmacological evaluation of new constituents of "Spice": synthetic cannabinoids based on indole, indazole, benzimidazole and carbazole scaffolds.

PURPOSE: In the present study we characterized a series of synthetic cannabinoids containing various heterocyclic scaffolds that had been identified as constituents of "Spice", a preparation sold on the illicit drug market. All compounds were further investigated as potential ligands of the orphan receptors GPR18 and GPR55 that interact with some cannabinoids. METHODS: The compounds were studied in radioligand binding assays to determine their affinity for human cannabinoid CB1 and CB2 receptors expressed in CHO cells, and in cAMP accumulation assays to study their functionality. RESULTS: Structure-activity relationships were analyzed. The most potent CB1 receptor agonist of the present series MDMB-FUBINACA (12) (Ki = 98.5 pM) was docked into the human CB1 receptor structure, and a plausible binding mode was identified showing high similarity with that of the co-crystallized THC derivatives. MDMB-CHMCZCA (41) displayed a unique profile acting as a full agonist at the CB1 receptor subtype, but blocking the CB2 receptor completely. Only a few weakly potent antagonists of GPR18 and GPR55 were identified, and thus all compounds showed high CB receptor selectivity, mostly interacting with both subtypes, CB1 and CB2. CONCLUSIONS: These results will be useful to assess the compounds' toxicological risks and to guide legislation. Further studies on 41 are warranted.

Synthesis and Structure-Activity Relationships of LP1 Derivatives: N-Methyl-N-phenylethylamino Analogues as Novel MOR Agonists.

The opioid pharmacological profile of cis-(-)-N-normetazocine derivatives is deeply affected by the nature of their N-substituents. Here, our efforts were focused on the synthesis and pharmacological evaluation of novel derivatives of the lead LP1, a multitarget opioid analgesic compound featuring an N-phenylpropanamido substituent. LP1 derivatives 5a-d and 6a-d were characterized by flexible groups at the N-substituent that allow them to reposition themselves relative to cis-(-)-N-normetazocine nucleus, thus producing different pharmacological profiles at the mu, delta and kappa opioid receptors (MOR, DOR and KOR) in in vitro and in vivo assays. Among the series, compound 5c, with the best in vitro and in vivo profile, resulted a MOR agonist which displays a KiMOR of 6.1 nM in a competitive binding assay, and an IC50 value of 11.5 nM and an Imax of 72% in measurement of cAMP accumulation in HEK293 cells stably expressing MOR, with a slight lower efficacy than LP1. Moreover, in a mouse model of acute thermal nociception, compound 5c, intraperitoneally administered, exhibits naloxone-reversed antinociceptive properties with an ED50 of 4.33 mg/kg. These results expand our understanding of the importance of N-substituent structural variations in the opioid receptor profile of cis-(-)-N-normetazocine derivatives and identify a new MOR agonist useful for the development of novel opioid analgesics for pain treatment.

2-OMe-lysophosphatidylcholine analogues are GPR119 ligands and activate insulin secretion from ßTC-3 pancreatic cells: Evaluation of structure-dependent biological activity.

GPR119 receptor has been proposed as a metabolic regulator playing a pivotal role in the modulation of glucose homeostasis in type 2 diabetes. GPR119 was identified on pancreatic ß cells and its ligands have the ability to enhance glucose-stimulated insulin secretion (GSIS). Lysophosphatidylcholine (LPC) was shown to potentiate GSIS and our present studies indicate that 2-methoxy-lysophosphatidylcholine (2-OMe-LPC) analogues, unable to undergo 1→2 acyl migration, stimulate GSIS from murine ßTC-3 pancreatic cells even more efficiently. Moreover, biological assays in engineered Tango™ GPR119-bla U2OS cells were carried out to ascertain the agonist activity of 2-OMe-LPC at GPR119. 2-OMe-LPC possessing in sn-1 position the residues of myristic, palmitic, stearic and oleic acid were also evaluated as factors regulating [Ca2+]i mobilization and cAMP levels. Extension of these studies to R- and S-enantiomers of 14:0 2-OMe-LPC revealed that the overall impact on GSIS does not depend on chirality, however, the intracellular calcium mobilization data show that the R enantiomer is significantly more active than S one. Taking into account differences in chemical structure between various native LPCs and their 2-methoxy counterparts the possible binding mode of 2-OMe-LPC to the GPR119 receptor was determined using molecular modeling approach.

Glucose-Dependent Insulinotropic Peptide Prevents Serum Deprivation-Induced Apoptosis in Human Bone Marrow-Derived Mesenchymal Stem Cells and Osteoblastic Cells.

Human bone marrow-derived mesenchymal stem cells (hBMSC) are able to differentiate into cells of connective tissue lineages, including bone and cartilage. They are therefore considered as a promising tool for the treatment of bone degenerative diseases. One of the major issues in regenerative cell therapy is the biosafety of fetal bovine serum used for cell culture. Therefore, the development of a culture medium devoid of serum but preserving hBMSC viability will be of clinical value. The glucose-dependent insulinotropic peptide (GIP) has an anti-apoptotic action in insulin-producing cells. Interestingly, GIP also exerts beneficial effects on bone turnover by acting on osteoblasts and osteoclasts. We therefore evaluated the ability of GIP to prevent cell death in osteoblastic cells cultured in serum-free conditions. In hBMSC and SaOS-2 cells, activation of the GIP receptor increased intracellular cAMP levels. Serum deprivation induced apoptosis in SaOS-2 and hBMSC that was reduced by 30 and 50 %, respectively, in the presence of GIP. The protective effect of GIP involves activation of the adenylate cyclase pathway and inhibition of caspases 3/7 activation. These findings demonstrate that GIP exerts a protective action against apoptosis in hBMSC and suggest a novel approach to preserve viability of hBMSC cultured in the absence of serum.

Analysis of functional selectivity through G protein-dependent and -independent signaling pathways at the adrenergic α(2C) receptor.

Although G protein-coupled receptors (GPCRs) are traditionally categorized as Gs-, Gq-, or Gi/o-coupled, their signaling is regulated by multiple mechanisms. GPCRs can couple to several effector pathways, having the capacity to interact not only with more than one G protein subtype but also with alternative signaling or effector proteins such as arrestins. Moreover, GPCR ligands can have different efficacies for activating these signaling pathways, a characteristic referred to as biased agonism or functional selectivity. In this work our aim was to detect differences in the ability of various agonists acting at the α2C type of adrenergic receptors (α2C-ARs) to modulate cAMP accumulation, cytoplasmic Ca(2+) release, ß-arrestin recruitment and receptor internalization. A detailed comparative pharmacological characterization of G protein-dependent and -independent signaling pathways was carried out using adrenergic agonists (norepinephrine, phenylephrine, brimonidine, BHT-920, oxymetazoline, clonidine, moxonidine, guanabenz) and antagonists (MK912, yohimbine). As initial analysis of agonist Emax and EC50 values suggested possible functional selectivity, ligand bias was quantified by applying the relative activity scale and was compared to that of the endogenous agonist norepinephrine. Values significantly different from 0 between pathways indicated an agonist that promoted different level of activation of diverse effector pathways most likely due to the stabilization of a subtly different receptor conformation from that induced by norepinephrine. Our results showed that a series of agonists acting at the α2C-AR displayed different degree of functional selectivity (bias factors ranging from 1.6 to 36.7) through four signaling pathways. As signaling via these pathways seems to have distinct functional and physiological outcomes, studying all these stages of receptor activation could have further implications for the development of more selective therapeutics with improved efficacy and/or fewer side effects.