Gambogenic acid induces apoptosis via upregulation of Noxa in oral squamous cell carcinoma.

Gambogenic acid (GNA), a bioactive compound derived from the resin of Garcinia hanburyi, has demonstrated significant antitumor properties. However, its mechanisms of action in oral squamous cell carcinoma (OSCC) remain largely unclear. This study aimed to elucidate the apoptotic effects of GNA on OSCC cell lines CAL-27 and SCC-15. Our results indicated that GNA induced apoptosis by upregulating the pro-apoptotic protein Noxa. Mechanistic investigations revealed that GNA treatment led to the generation of reactive oxygen species (ROS), which activated endoplasmic reticulum (ER) stress, culminating in cell apoptosis. Inhibition of ROS production and ER stress pathways significantly mitigated GNA-induced Noxa upregulation and subsequent apoptosis. Furthermore, in vivo studies using a murine xenograft model demonstrated that GNA administration effectively inhibited the growth of CAL-27 tumors. Collectively, these findings underscore GNA's potential as a therapeutic agent for the treatment of OSCC.

Asymmetric total synthesis of polycyclic xanthenes and discovery of a WalK activator active against MRSA.

The development of new antibiotics continues to pose challenges, particularly considering the growing threat of multidrug-resistant Staphylococcus aureus. Structurally diverse natural products provide a promising source of antibiotics. Herein, we outline a concise approach for the collective asymmetric total synthesis of polycyclic xanthene myrtucommulone D and five related congeners. The strategy involves rapid assembly of the challenging benzopyrano[2,3-a]xanthene core, highly diastereoselective establishment of three contiguous stereocenters through a retro-hemiketalization/double Michael cascade reaction, and a Mitsunobu-mediated chiral resolution approach with high optical purity and broad substrate scope. Quantum mechanical calculations provide insight into stereoselective construction mechanism of the three contiguous stereocenters. Additionally, this work leads to the discovery of an antibacterial agent against both drug-sensitive and drug-resistant S. aureus. This compound operates through a unique mechanism that promotes bacterial autolysis by activating the two-component sensory histidine kinase WalK. Our research holds potential for future antibacterial drug development.

Discovery of Di(het)arylmethane and Dibenzoxanthene Derivatives as Potential Anticancer Agents.

A family of bifunctional dihetarylmethanes and dibenzoxanthenes is assembled via a reaction of acetals containing a 2-chloroacetamide moiety with phenols and related oxygen-containing heterocycles. These compounds demonstrated selective antitumor activity associated with the induction of cell apoptosis and inhibition of the process of glycolysis. In particular, bis(heteroaryl)methane containing two 4-hydroxy-6-methyl-2H-pyran-2-one moieties combine excellent in vitro antitumor efficacy with an IC50 of 1.7 µM in HuTu-80 human duodenal adenocarcinoma models with a high selectivity index of 73. Overall, this work highlights the therapeutic potential of dimeric compounds assembled from functionalized acetals and builds a starting point for the development of a new family of anticancer agents.

Ultra-pH-sensitive nanoparticle of gambogenic acid for tumor targeting therapy via anti-vascular strategy plus immunotherapy.

Although the combination of anti-vascular strategy plus immunotherapy has emerged as the optimal first-line treatment of hepatocellular carcinoma, lack of tumor targeting leads to low antitumor efficacy and serious side effect. Here, we report an ultra-pH-sensitive nanoparticle of gambogenic acid (GNA) encapsulated by poly(ethylene glycol)-poly(2-azepane ethyl methacrylate) (PEG-PAEMA) for tumor-targeting combined therapy of anti-vascular strategy plus immunotherapy. PEG-PAEMA-GNA nanoparticle was quite stable at pH 7.4 for 30 d. In contrast, it exerted size shrinkage, charge reversal and the release of GNA at pH 6.7 within 24 h. Moreover, PEG-PAEMA-GNA significantly enhanced the anti-vascular activity, membrane-disruptive capability and pro-apoptosis when pH changed from 7.4 to 6.7. Western blot analysis exhibits that PEG-PAEMA and its GNA nanoparticle facilitated the phosphorylation of STING protein. In vivo assays show that PEG-PAEMA-GNA not only displayed much higher tumor inhibition of 92 % than 37 % of free GNA, but also inhibited tumor vasculature, promoted the maturation of dendritic cells and recruited more cytotoxic t-lymphocytes for sufficient anti-vascular therapy and immunotherapy. All these results demonstrate that PEG-PAEMA-GNA displayed tumor-targeting combined treatment of anti-vascular therapy and immunotherapy. This study offers a simple and novel method for the combination of anti-vascular therapy and immunotherapy with high selectivity towards tumor.

Tetrahydroxanthene-1,3(2H)-diones and Oxidized Hexadiene Derivatives from Uvaria leptopoda and Their Biological Activities.

The first phytochemical investigation of the twig extract of Uvaria leptopoda resulted in the isolation and identification of three new tetrahydroxanthene-1,3(2H)-diones, uvarialeptones A-C, two new oxidized hexadiene derivatives, uvarialeptols A and B, together with ten known compounds. Their structures were elucidated by spectroscopic techniques and mass spectrometry. Uvarialeptones A and B were unprecedented tetrahydroxanthene-1,3(2H)-dione dimers which exhibited a cyclobutane ring via [2 + 2] cycloaddition from uvarialeptone C and 9a-O-methyloxymitrone, respectively. The structure of uvarialeptone A was confirmed by X-ray diffraction analysis using Mo Kα radiation. Compound 3 inhibited NO production at an IC50 value of 6.7 ± 0.1 µM.

Dihydroxanthene-based monoamine oxidase A-activated photosensitizers for photodynamic/photothermal therapy of tumors.

Small molecule photosensitizers for combined in vivo tailored cancer diagnostics and photodynamic/photothermal therapy are desperately needed. Monoamine oxidase A (MAO-A)-activated therapeutic and diagnostic compounds provide great selectivity because MAO-A can be employed as a biomarker for associated Tumors. In order to screen photosensitizers with photodynamic therapeutic potential, we have created a range of near-infrared fluorescent molecules in this work by combining dihydroxanthene parent with various heterocyclic fluorescent dyes. The NIR fluorescent diagnostic probe, DHMQ, was created by combining the screened fluorescent dye matrices with the propylamino group, which is the recognition moiety of MAO-A, based on the oxidative deamination mechanism of the enzyme. This probe has a low toxicity level and can identify MAO-A precisely. It has the ability to use fluorescence imaging on mice and cells to track MAO-A activity in real-time. It has strong phototoxicity and can produce singlet oxygen when exposed to laser light. The temperature used in photothermal imaging can get up to 50 °C, which can harm tumor cells permanently and have a positive phototherapeutic impact on tumors grown from SH-SY5Y xenograft mice. The concept of using MAO-A effectively in diseases is expanded by the MAO-A-activated diagnostic-integrated photosensitizers, which offer a new platform for in vivo cancer diagnostics and targeted anticancer treatment.

Arginine and sodium fluoride affect the microbial composition and reduce biofilm metabolism and enamel mineral loss in an oral microcosm model.

OBJECTIVE: To assess the effects of arginine, with or without sodium fluoride (NaF; 1,450 ppm), on saliva-derived microcosm biofilms and enamel demineralization. METHODS: Saliva-derived biofilms were grown on bovine enamel blocks in 0.2 % sucrose-containing modified McBain medium, according to six experimental groups: control (McBain 0.2 %); 2.5 % arginine; 8 % arginine; NaF; 2.5 % arginine with NaF; and 8 % arginine with NaF. After 5 days of growth, biofilm viability was assessed by colony-forming units counting, laser scanning confocal microscopy was used to determine biofilm vitality and extracellular polysaccharide (EPS) production, while biofilm metabolism was evaluated using the resazurin assay and lactic acid quantification. Demineralization was evaluated by measuring pH in the culture medium and calcium release. Data were analyzed by Kruskal-Wallis' and Dunn's tests (p < 0.05). RESULTS: 8 % arginine with NaF showed the strongest reduction in total streptococci and total microorganism counts, with no significant difference compared to arginine without NaF. Neither 2.5 % arginine alone nor NaF alone significantly reduced microbial counts compared to the control, although in combination, a reduction in all microbial groups was observed. Similar trends were found for biofilm vitality and EPS, and calcium released to the growth medium. CONCLUSIONS: 8 % Arginine, with or without NaF, exhibited the strongest antimicrobial activity and reduced enamel calcium loss. Also, NaF enhanced the effects of 2.5 % arginine, yielding similar results to 8 % arginine for most parameters analyzed. CLINICAL SIGNIFICANCE: The results provided further evidence on how arginine, with or without NaF, affects oral microcosm biofilms and enamel mineral loss.

Ionic Liquids Immobilized Synthesis of New Xanthenes Derivatives and their Antiproliferative, Molecular Docking, and Morphological Studies.

BACKGROUND: Xanthenes and benzoxanthenesare are highly valuable compounds in organic chemistry and medicinal chemistry. Xanthene derivatives were found to have many applications in medicinal chemistry. OBJECTIVE: This work aims to explore the synthesis of xanthene derivatives with various substituents and find the possibility of their uses as anticancer agents. METHODS: The basic starting compound through this work was the 2,3-dihydro-1H-xanthen-1-one (3), which was synthesized from the reaction of cyclohexan-1,3-dione and 2-hydroxybenzaldehyde. Compound 3 was used to synthesize new thiophene, pyrimidine, isoxazole, and thiazole derivatives based on the xanthenes nucleus. Fused xanthene derivatives were obtained through further heterocyclization reactions. Multicomponent reactions expressed in this work were carried out in the presence of solvent catalyzed by Et3N and in solvent-free ionic liquid immobilized catalyst. RESULTS: Cytotoxicity for the newly synthesized compounds toward cancer cell lines was measured, and the results revealed that many compounds exhibited high inhibitions. CONCLUSION: The antiproliferative activity of the synthesized compounds was studied on six selected cancer cell lines. The nature of the heterocyclic ring and the variations of substituted groups showed a high effect through the inhibitions of the tested compound.

Design, synthesis, and biological evaluation of gambogenic acid derivatives: Unraveling their anti-cancer effects by inducing pyroptosis.

Gambogenic acid (GNA), a caged xanthone derived from Garcinia hanburyi, exhibits a wide range of anti-cancer properties. The caged skeleton of GNA serves as the fundamental pharmacophore responsible for its antitumor effects. However, limited exploration has focused on the structural modifications of GNA. This study endeavors to diversify the structure of GNA and enhance its anti-cancer efficacy. Sulfoximines, recognized as pivotal motifs in medicinal chemistry due to their outstanding properties, have featured in several anti-cancer drugs undergoing clinical trials. Accordingly, a series of 33 GNA derivatives combined with sulfoximines were synthesized and evaluated for their anti-cancer effects against MIAPaCa2, MDA-MB-231, and A549 cells in vitro. The activity screening led to the identification of compound 12k, which exhibited the most potent anti-cancer effect. Mechanistic studies revealed that 12k primarily induced pyroptosis in MIAPaCa2 and MDA-MB-231 cells by activating the caspase-3/gasdermin E (GSDME) pathway. These findings suggested that 12k is a promising drug candidate in cancer therapy and highlighted the potential of sulfoximines as a valuable functional group in drug discovery.

Unveiling Gambogenic Acid as a Promising Antitumor Compound: A Review.

Gambogenic acid is a derivative of gambogic acid, a polyprenylated xanthone isolated from Garcinia hanburyi. Compared with the more widely studied gambogic acid, gambogenic acid has demonstrated advantages such as a more potent antitumor effect and less systemic toxicity than gambogic acid according to early investigations. Therefore, the present review summarizes the effectiveness and mechanisms of gambogenic acid in different cancers and highlights the mechanisms of action. In addition, drug delivery systems to improve the bioavailability of gambogenic acid and its pharmacokinetic profile are included. Gambogenic acid has been applied to treat a wide range of cancers, such as lung, liver, colorectal, breast, gastric, bladder, and prostate cancers. Gambogenic acid exerts its antitumor effects as a novel class of enhancer of zeste homolog 2 inhibitors. It prevents cancer cell proliferation by inducing apoptosis, ferroptosis, and necroptosis and controlling the cell cycle as well as autophagy. Gambogenic acid also hinders tumor cell invasion and metastasis by downregulating metastasis-related proteins. Moreover, gambogenic acid increases the sensitivity of cancer cells to chemotherapy and has shown effects on multidrug resistance in malignancy. This review adds insights for the prevention and treatment of cancers using gambogenic acid.

Quantifying metabolic activity of Ascaris suum L3 using resazurin reduction.

BACKGROUND: Helminth infections are an important public health problem in humans and have an even greater impact on domestic animal and livestock welfare. Current readouts for anthelmintic drug screening assays are stage development, migration, or motility that can be subjective, laborious, and low in throughput. The aim of this study was to apply and optimize a fluorometric technique using resazurin for evaluating changes in the metabolic activity of Ascaris suum third-stage larvae (L3), a parasite of high economic relevance in swine. METHODS: Ascaris suum L3 were mechanically hatched from 6- to 8-week embryonated and sucrose-gradient-enriched eggs. Resazurin dye and A. suum L3 were titrated in 96-well microtiter plates, and resazurin reduction activity was assessed by fluorometry after 24 h of incubation. Fluorescence microscopy was used to localize the resazurin reduction site within the larvae. Finally, we exposed A. suum L3 to various stress conditions including heat, methanol, and anthelmintics, and investigated their impact on larval metabolism through resazurin reduction activity. RESULTS: We show that the non-fluorescent dye resazurin is reduced inside vital A. suum L3 to fluorescent resorufin and released into the culture media. Optimal assay parameters are 100-1000 L3 per well, a resazurin concentration of 7.5 µg/ml, and incubation at 37 °C/5% CO2 for 24 h. An intact L2 sheath around the L3 of A. suum completely prevents the uptake of resazurin, while in unsheathed L3, the most intense fluorescence signal is observed along the larval midgut. L3 exposed to methanol or heat show a gradually decreased resazurin reduction activity. In addition, 24 h exposure to ivermectin at 0.625 µM, mebendazole at 5 µM, and thiabendazole from 10 to 100 µM significantly decreased larval metabolic activity by 55%, 73%, and 70% to 89%, respectively. CONCLUSIONS: Together, our results show that both metabolic stressors and anthelmintic drugs significantly and reproducibly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy-to-use method to evaluate metabolic activity of A. suum L3 in vitro.

Anti-tumor Effect of Gambogenic Acid and Its Effect on CYP2C and CYP3A after Oral Administration.

Gambogenic acid (GNA), which has a broad spectrum of anti-tumor activity, is considered as a potential anticancer ingredient. In this study, we examined the anti-tumor effect and the effect of GNA on CYP and pregnane X receptor (PXR). In anti-tumor experiments, an A549 cells tumor-bearing nude mice model was established. Tumor weights and volumes were measured. Inhibition ratio (IR) was calculated. In a pharmacokinetic study, after intragastrical administration of GNA in rats, a cocktail method was adopted to evaluate the activities of CYP2C6, 2C11 and 3A1; RT-quantitative PCR (RT-qPCR) and Western blot (WB) assays were applied to evaluate the mRNA and protein expression levels, respectively. Compared with injection, oral administration also can inhibit tumor growth. Moreover, GNA increased the activities of CYP2C11 and CYP3A1 in the high-dose group as well as the mRNA and protein expression levels. The mRNA and protein expression levels of PXR were also slightly induced. Our study suggested that, oral administration of GNA was effective in inhibiting tumor growth in mice and could induced the activities of CYP2C and CYP3A in rats.

The Design, Synthesis, and Evaluation of Novel 9-Arylxanthenedione-Based Allosteric Modulators for the δ-Opioid Receptor.

Chronic pain and depression are both widely prevalent comorbid medical conditions. While efficient, µ-opioid receptor-based medications are associated with life-threatening side effects, including respiratory depression, dependence, and addiction. The δ-opioid receptor is a promising alternative biological target for chronic pain and depression due to its significantly reduced on-target side effects compared to the µ-opioid receptor. A previous study identified two δ-opioid receptor positive allosteric modulators. Herein, we report the design of five series of compounds targeting previously unexplored regions of the originally described SAR. Analogs were assessed for their ability to potentiate the agonist response of Leu-enkephalin. Of the 30 analogs, compound 6g displayed trends toward enhancing the ERK1/2 phosphorylation signaling compared to cAMP inhibition, while compound 11c exhibited a trend in shifting the signaling bias toward cAMP inhibition. Both 6g and 11c emerged as promising tool compounds toward the design of prospective therapeutics requiring specific downstream signaling attributes.

Penicixanthene E, a new xanthene isolated from a mangrove-derived fungus Penicillium sp.

One new xanthene derivative, named penicixanthene E (1), together with one known compound 2, was isolated from the EtOAc extract of the endophytic fungus Penicillium sp. GXIMD 03101, which was identified from the mangrove Acanthus ilicifolius L. collected in the South China Sea. The structure of 1 was elucidated by 1D and 2D NMR spectral interpretation and HREISMS data. The absolute configurations of C-9 and C-11 in 1 were proposed based on electronic circular dichroism (ECD), but the configuration at C-3 in 1 was unassigned. Compound 1 represents a xanthene derivative that was first reported, in which carbon-carbon double bond has been reduced. The cytotoxic activities of all compounds were evaluated, the result showed that compound 1 has weak activity against pancreatic cancer SW1990.

BioMOF-Mn: An Antimicrobial Agent and an Efficient Nanocatalyst for Domino One-Pot Preparation of Xanthene Derivatives.

In this paper, a new Mn-based metal-organic framework [UoB-6] was obtained via a one-step ultrasonic irradiation method with the ligand (H2bdda: 4,4'-(1,4-phenylenebis(azaneylylidene))bis(methaneylylidene))dibenzoic acid. The structural integrity of the synthesized BioMOF-Mn was corroborated by FT-IR, EDX, ICP, XRD, TEM, DLS, FESEM, and BET-BJH analyses. The aerobic oxidative domino reaction of benzyl alcohols or aldehydes with dimedone derivatives was performed in the presence of the UoB-6 catalyst to produce xanthene derivatives in good yields. Hot filtration and Hg poisoning tests proved the heterogeneous nature of the catalyst. Novel synthesized xanthene-based bis-aldehydes were introduced as potent HDAC1 inhibitors according to molecular docking calculations. Finally, the inhibitory activities of Mn-MOF nanoparticles were evaluated on Escherichia coli and Candida albicans. The MIC, MBC, and MFC values were determined from 2048 to 4096 µg·mL-1 according to antimicrobial susceptibility testing methods. The inhibitory effects of antimicrobial agents can be exacerbated when loaded on BioMOFs.

Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon.

Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin.

Discovery of potent antiproliferative agents from selected oxygen heterocycles as EGFR tyrosine kinase inhibitors from the U.S. National Cancer Institute database by in silico screening and bioactivity evaluation.

A similarity search was conducted on the U.S. Enhanced National Cancer Institute Database Browser 2.2 to find structures related to 1,5-dihydroxy-9H-xanthen-9-one, a previously established EGFR-TK inhibitor. Compounds were virtually screened and selected for bioactivity testing revealed 5 candidates, mostly displayed stronger antiproliferative activities than erlotinib with IC50 values between 0.95 and 17.71 µM against overexpressed EGFR-TK cancer cell lines: A431 and HeLa. NSC107228 displayed the strongest antiproliferative effects with IC50 values of 2.84 and 0.95 µM against A431 and HeLa cancer cell lines, respectively. Three compounds, NSC81111, NSC381467 and NSC114126 inhibited EGFR-TK with IC50 values between 0.15 and 30.18 nM. NSC81111 was the best inhibitor with IC50 = 0.15 nM. Molecular docking analysis of the 3 compounds predicted hydrogen bonding and hydrophobic interactions with key residues were important for the bioactivities observed. Furthermore, calculations of the physicochemical properties suggest the compounds are drug-like and are potentially active orally.

Impact of the ACE2 activator xanthenone on tacrolimus nephrotoxicity: Modulation of uric acid/ERK/p38 MAPK and Nrf2/SOD3/GCLC signaling pathways.

AIMS: The calcineurin inhibitor tacrolimus is an effective and widely used immunosuppressant after organ transplantation to reduce graft rejection. However, its nephrotoxic effect could compel the patients to treatment discontinuation. The beneficial effects of angiotensin-converting enzyme 2 (ACE2) on the kidney and other organs have been investigated in several studies, but its role in tacrolimus nephrotoxicity still needs to be elucidated. Our study was designed to investigate effects of the ACE2 activator xanthenone on tacrolimus-induced renal injury. MATERIALS AND METHODS: Male Wistar rats were administered xanthenone (2 mg/kg) concurrently with tacrolimus (1 mg/kg) for 3 weeks, then blood and kidney tissue samples were collected for biochemical and molecular investigations. KEY FINDINGS: Co-administration of xanthenone significantly improved renal functions in tacrolimus-treated rats, where serum creatinine, urea, and uric acid levels were close to those of the normal control. Besides, xanthenone reduced renal angiotensin (ANG) II content, while elevated ANG (1-7). Relative protein expressions of p-ERK/ERK and p-p38 MAPK/p38 MAPK inflammatory signals were downregulated upon xanthenone administration with tacrolimus. In addition, xanthenone reinforced antioxidant defense against tacrolimus by enhancing protein expression of the transcription factor Nrf2 with subsequently increased mRNA expressions of the antioxidants SOD3 and GCLC. SIGNIFICANCE: These protective effects of xanthenone could be attributed to ANG II degradation to ANG (1-7) by ACE2 activation resulting in regulated inflammatory and oxidative responses in the kidney. Therefore, administration of xanthenone along with tacrolimus could be a promising therapeutic strategy to reduce the adverse effects and increase the tolerability to tacrolimus immunosuppressive therapy.

Facile and divergent optimization of chromazonarol enabled the identification of simplified drimane meroterpenoids as novel pharmaceutical leads.

The diversity of drimane hydroquinones was significantly expanded by the facile construction of (+)-chromazonarol relevant natural products, isomers, and analogues for the discovery of new pharmaceutical leads. The structure-activity relationship of (+)-chromazonarol relevant (non)-natural products was delineated via the synergistic interaction of the programmable synthesis and bioactivity-guided screening. The first divergent derivatization of (+)-chromazonarol demonstrated that the phenolic hydroxyl group is one inviolable requirement for antifungal effect. Pinpoint modification of (+)-yahazunol manifested the position of hydroxyl group was crucial for both antifungal and antitumor activities. (+)-Albaconol, (+)-neoalbaconol, and two (+)-yahazunol isomers (24 and 25) proved to be the novel pharmaceutical leads. The probable macromolecular targets were estimated to deliver new information about the biological potentials resident in (+)-yahazunol relevant products. This work also featured the first synthesis of (+)-albaconol and (+)-neoalbaconol, the first biological exploration of (+)-dictyvaric acid and improved preparation of (+)-8-epi-puupehedione and a promising pelorol analogue.

Graded heterogeneity of metabotropic signaling underlies a continuum of cell-intrinsic temporal responses in unipolar brush cells.

Many neuron types consist of populations with continuously varying molecular properties. Here, we show a continuum of postsynaptic molecular properties in three types of neurons and assess the functional correlates in cerebellar unipolar brush cells (UBCs). While UBCs are generally thought to form discrete functional subtypes, with mossy fiber (MF) activation increasing firing in ON-UBCs and suppressing firing in OFF-UBCs, recent work also points to a heterogeneity of response profiles. Indeed, we find a continuum of response profiles that reflect the graded and inversely correlated expression of excitatory mGluR1 and inhibitory mGluR2/3 pathways. MFs coactivate mGluR2/3 and mGluR1 in many UBCs, leading to sequential inhibition-excitation because mGluR2/3-currents are faster. Additionally, we show that DAG-kinase controls mGluR1 response duration, and that graded DAG kinase levels correlate with systematic variation of response duration over two orders of magnitude. These results demonstrate that continuous variations in metabotropic signaling can generate a stable cell-autonomous basis for temporal integration and learning over multiple time scales.