Insights into the redox cycle of human quinone reductase 2

Abstract

NRH:quinone oxidoreductase 2 (QR2) is a cytosolic enzyme that catalyzes the reduction of quinones, such as menadione and co-enzymes Q. With the aim of understanding better the mechanisms of action of QR2, we approached this enzyme catalysis via electron paramagnetic resonance (EPR) measurements of the by-products of the QR2 redox cycle. The variation in the production of oxidative species such as H₂O₂, and subsequent hydroxyl radical generation, was measured during the course of QR2 activity under aerobic conditions and using pure human enzyme. The effects on the activity of the following were compared: (i) synthetic (N-benzyldihydronicotinamide, BNAH) or natural (nicotinamide riboside, NRH) co-substrates; (ii) synthetic (menadione) or natural (co-enzyme Q0, Q2) substrates; (iii) QR2 modulators and inhibitors (melatonin, resveratrol and S29434); (iv) a pro-drug activated via a redox cycle [CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide]. The results were also compared with those obtained with human QR1. The production of hydroxyl radicals is: (i) observed whatever the substrate/co-substrate used; ii) quenched by adding catalase; (iii) not observed with the specific QR2 inhibitor S29434; (iv) observed with the pro-drug CB1954. While QR2 produced free radicals with this pro-drug, QR1 gave no EPR signal showing the strong reducing capacity of QR2. In conclusion, EPR analysis of QR2 enzyme activity through free radical production enables modulators and effective inhibitors to be distinguished.     

Insights into the redox cycle of human quinone reductase 2

NRH:quinone oxidoreductase 2 (QR2) is a cytosolic enzyme that catalyzes the reduction of quinones, such as menadione and co-enzymes Q. With the aim of understanding better the mechanisms of action of QR2, we approached this enzyme catalysis via electron paramagnetic resonance (EPR) measurements of the by-products of the QR2 redox cycle. The variation in the production of oxidative species such as H(2)O(2), and subsequent hydroxyl radical generation, was measured during the course of QR2 activity under aerobic conditions and using pure human enzyme. The effects on the activity of the following were compared: (i) synthetic (N-benzyldihydronicotinamide, BNAH) or natural (nicotinamide riboside, NRH) co-substrates; (ii) synthetic (menadione) or natural (co-enzyme Q0, Q2) substrates; (iii) QR2 modulators and inhibitors (melatonin, resveratrol and S29434); (iv) a pro-drug activated via a redox cycle [CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide]. The results were also compared with those obtained with human QR1. The production of hydroxyl radicals is: (i) observed whatever the substrate/co-substrate used; ii) quenched by adding catalase; (iii) not observed with the specific QR2 inhibitor S29434; (iv) observed with the pro-drug CB1954. While QR2 produced free radicals with this pro-drug, QR1 gave no EPR signal showing the strong reducing capacity of QR2. In conclusion, EPR analysis of QR2 enzyme activity through free radical production enables modulators and effective inhibitors to be distinguished.     

Design,synthesis, and biological evaluation of resveratrol analogues as aromatase and quinone reductase 2 inhibitors for chemoprevention of cancer

A series of new resveratrol analogues were designed and synthesized and their inhibitory activities against aromatase were evaluated. The crystal structure of human aromatase (PDB 3eqm) was used to rationalize the mechanism of action of the aromatase inhibitor 32 (IC₅₀ 0.59 μM) through docking, molecular mechanics energy minimization, and computer graphics molecular modeling, and the information was utilized to design several very potent inhibitors, including compounds 82 (IC₅₀ 70 nM) and 84 (IC₅₀ 36 nM). The aromatase inhibitory activities of these compounds are much more potent than that for the lead compound resveratrol, which has an IC₅₀ of 80 μM. In addition to aromatase inhibitory activity, compounds 32 and 44 also displayed potent QR2 inhibitory activity (IC₅₀ 1.7 μM and 0.27 μM, respectively) and the high-resolution X-ray structures of QR2 in complex with these two compounds provide insight into their mechanism of QR2 inhibition. The aromatase and quinone reductase inhibitors resulting from these studies have potential value in the treatment and prevention of cancer.     

Optimization of thiazole analogues of resveratrol for induction of NAD(P)H:quinone reductase 1 (QR1)

NAD(P)H:quinone reductase 1 (QR1) belongs to a class of enzymes called cytoprotective enzymes. It exhibits its cancer protective activity mainly by inhibiting the formation of intracellular semiquinone radicals, and by generating α-tocopherolhydroquinone, which acts as a free radical scavenger. It is therefore believed that QR1 inducers can act as cancer chemopreventive agents. Resveratrol (1) is a naturally occurring stilbene derivative that requires a concentration of 21 μM to double QR1 activity (CD = 21 μM). The stilbene double bond of resveratrol was replaced with a thiadiazole ring and the phenols were eliminated to provide a more potent and selective derivative 2 (CD = 2.1 μM). Optimizing the substitution pattern of the two phenyl rings and the central heterocyclic linker led to a highly potent and selective QR1 inducer 9o with a CD value of 0.087 μM.     

A family of antimicrobial and immunomodulatory peptides related to the frenatins from skin secretions of the Orinoco lime frog Sphaenorhynchus lacteus (Hylidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus (Hylidae, Hylinae) revealed the presence of three structurally related host-defense peptides with limited sequence similarity to frenatin 2 from Litoria infrafrenata (Hylidae, Pelodryadinae) and frenatin 2D from Discoglossus sardus (Alytidae). Frenatin 2.1S (GLVGTLLGHIGKAILG.NH₂) and frenatin 2.2S (GLVGTLLGHIGKAILS.NH₂) are C-terminally α-amidated but frenatin 2.3S (GLVGTLLGHIGKAILG) is not. Frenatin 2.1S and 2.2S show potent bactericidal activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MIC ≤16 μM) but are less active against a range of Gram-negative bacteria. Frenatin 2.1S (LC₅₀ = 80 ± 6 μM) and 2.2S (LC₅₀ = 75 ± 5 μM) are cytotoxic against non-small cell lung adenocarcinoma A549 cells but are less hemolytic against human erythrocytes (LC₅₀ = 167 ± 8 μM for frenatin 2.1S and 169 ± 7 μM for 2.2S). Weak antimicrobial and cytotoxic potencies of frenatin 2.3S demonstrate the importance of C-terminal α-amidation for activity. Frenatin 2.1S and 2.2S significantly (P < 0.05) increased production of proinflammatory cytokines IL-1β and IL-23 by lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and frenatin 2.1S also enhanced production of TNF-α. Effects on IL-6 production were not significant. Frenatin 2.2S significantly downregulated production of the anti-inflammatory cytokine IL-10 by LPS-stimulated cells. The data support speculation that frenatins act on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms. They may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.     

Thieno[2,3-b]pyridines—A new class of multidrug resistance (MDR) modulators

To identify new potent multidrug resistance modulators, we have synthesized a series of novel thieno[2,3-b]pyridines and furo[2,3-b]pyridines, and examined their stucture–activity relationships. All synthesized compounds were tested to determine BCRP1, P-gp, and MRP1 inhibitor activity, and most potent MDR modulators were also screened for their toxicity, cytotoxicity and Ca²⁺ channel antagonist activity. Among these compounds, thieno[2,3-b]pyridine (6r) was found to exhibit a potent P-gp inhibitory action with EC₅₀ = 0.3 ± 0.2 μM, MRP1 inhibitory action with EC₅₀ = 1.1 ± 0.1 μM and BCRP1 inhibitory action with EC₅₀ = 0.2 ± 0.05 μM and may represent suitable candidate for further pharmacological studies.     

DNA integrity of fresh and frozen canine epididymal spermatozoa

The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1 mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3 ± 3.6) and samples frozen with (4.2 ± 3.8) or without (3.6 ± 3.7) melatonin. Sperm motility was significantly (p < 0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p < 0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

Design,synthesis, and biological characterization of novel PEG-linked dimeric modulators for CXCR4

CXCR4 dimerization has been widely demonstrated both biologically and structurally. This paper mainly focused on the development of structure-based dimeric ligands that target CXCL12–CXCR4 interaction and signaling. This study presents the design and synthesis of a series of [PEG]_n linked dimeric ligands of CXCR4 based on the knowledge of the homodimeric crystal structure of CXCR4 and our well established platform of chemistry and bioassays for CXCR4. These new ligands include [PEG]_n linked homodimeric or heterodimeric peptides consisting of either two DV3-derived moieties (where DV3 is an all-d-amino acid analog of N-terminal modules of 1–10 (V3) residues of vMIP-II) or hybrids of DV3 moieties and CXCL12_1–8. Among a total of 24 peptide ligands, four antagonists and three agonists showed good CXCR4 binding affinity, with IC₅₀ values of <50 nM and <800 nM, respectively. Chemotaxis and calcium mobilization assays with SUP-T1 cells further identified two promising lead modulators of CXCR4: ligand 4, a [PEG₃]₂ linked homodimeric DV3, was an effective CXCR4 antagonist (IC₅₀ = 22 nM); and ligand 21, a [PEG₃]₂ linked heterodimeric DV3–CXCL12_1–8, was an effective CXCR4 agonist (IC₅₀ = 407 nM). These dimeric CXCR4 modulators represent new molecular probes and therapeutics that effectively modulate CXCL12–CXCR4 interaction and function.     

New melatonin (MT1/MT2) ligands: Design and synthesis of (8,9-dihydro-7H-furo[3,2-f]chromen-1-yl) derivatives

Herein we describe the synthesis of novel tricyclic analogues issued from the rigidification of the methoxy group of the benzofuranic analogue of melatonin as MT₁ and MT₂ ligands. Most of the synthesized compounds displayed high binding affinities at MT₁ and MT₂ receptors subtypes. Compound 6b (MT₁, K_i = 0.07 nM; MT₂, K_i = 0.08 nM) exhibited with the vinyl 6c and allyl 6d the most interesting derivatives of this series. Functional activity of these compounds showed full agonist activity with EC₅₀ in the nanomolar range. Compounds 6a (EC₅₀ = 0.8 nM and E_max = 98%) and 6b (EC₅₀ = 0.2 nM and E_max = 121%) exhibited good pharmacological profiles.     

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background: Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods: TXS and VEGF metabolite levels were measured in NSCLC serum samples (n = 46) by EIA. TXB₂ levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results: Serum TXB₂ levels were correlated with VEGF (p < 0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p < 0.0001) and microvessel density (CD-31; p < 0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p < 0.0001) and female patients (p < 0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p < 0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion: TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.     

Experimentally induced stress,oxidative load and changes in immunity in a tropical wild bird,Perdicula asiatica: involvement of melatonin and glucocorticoid receptors

Throughout the year, birds encounter various environmental challenges such as extreme temperatures, rainfall and shortage of food. Here we report on the effect of stress on the general immunity of wild birds as measured by several assays including melatonin, an anti-stress hormone. We selected Perdicula asiatica, a wild tropical bird, and exposed them to experimental stressors such as water deprivation, food deprivation and immobilization, i.e., stressors that they would encounter in a natural environment. We measured the oxidative load in the spleen in terms of superoxide dismutase (SOD), catalase activity and thiobarbituric acid-reactive substance (TBARS) levels. The immune status was judged by total leukocyte count (TLC), heterophil/lymphocyte ratios (H/L) and percent stimulation ratio of splenocytes (%SR). The peripheral levels of melatonin and corticosterone were also determined and correlated with the expression of melatonin (Mel_1a/Mel_1b) and glucocorticoid receptors. Our results showed a significant (p < 0.05) decrease in splenic SOD and catalase activity, while a significant (p < 0.05) increase in TBARS and a corticosterone level was observed. Stressful conditions also decreased the immune status as reflected by the low values of H/L ratios, TLC and %SR. In contrast, melatonin pretreatment significantly (p < 0.05) reduced the oxidative stress and improved the immune parameters when compared to untreated control birds. This suggests that melatonin prevents/alleviates oxidative damage and suppresses the immune status induced by stressful conditions via its membrane receptor expression (Mel_1a and Mel_1b) in P. asiatica.     

Oral administration of melatonin modulates the expression of tumor necrosis factor-α (TNF-α) gene in irradiated rat cervical spinal cord

AimWe aimed to determine the changes in TNF-α expression and Malondialdehyde (MDA) level in a short time after irradiation. Furthermore, we evaluated the effect of melatonin on the modulation of TNF-α gene expression.BackgroundThe radio-sensitivity of the cervical spinal cord limits the dose of radiation which can be delivered to tumors in the neck region. There is increasing evidence that TNF-α has a role in the development of the acute phase of spinal cord injury.Materials/MethodsFour groups of rats were investigated. Group 1 (vehicle treatment) served as the control. Group 2 (radiation) was treated with the vehicle, and 30 min later, the rats were exposed to radiation. Group 3 (radiation + melatonin) was given an oral administration of melatonin (100 mg/kg body weight) and 30 min later exposed to radiation in the same manner as in group 2. Group 4 (melatonin-only) was also given an oral administration of melatonin (100 mg/kg body weight). 5 mg/kg of melatonin was administered daily to rats in groups 3 and 4, and the vehicle was administered daily to rats in groups 1 and 2.ResultsThree weeks after irradiation, TNF-α gene up-regulated almost 5 fold in the irradiated group compared to the normal group. TNF-α gene expression in the melatonin pretreatment group, compared to the radiation group, was significantly down-regulated 3 weeks after irradiation (p < 0.05). MDA levels increased after irradiation and then significantly decreased under melatonin treatment.ConclusionWe suggest that inhibition of TNF-α expression by oral administration of melatonin may be a therapeutic option for preventing radiation-induced spinal cord injury.     

Rumen microbial responses in fermentation characteristics and production of CLA and methane to linoleic acid in associated with malate or fumarate

An in vitro incubation in batch was conducted to investigate the effect of propionate precursor (malate or fumarate) on fermentation characteristics, and production of CLA and methane by rumen microbes when incubated with linoleic acid (C_18:2). Sixty milligrams of C_18:2 alone (LA), 60 mg C_18:2 with 24 mM malic acid (M-LA), or 60 mg C_18:2 with 24 mM fumaric acid (F-LA) was added to 150 ml culture solution consisting of 75 ml strained rumen fluid and 75 ml McDougall's artificial saliva. Culture solution for incubation was also made without malate, fumarate, and C_18:2 (control). Two grams of feed consisting of 1.4 g concentrate and 0.6 g ground alfalfa (DM basis) was also added to the culture solution of each treatment. An in vitro incubation in batch was made anaerobically in a shaking incubator for up to 12 h at 39 °C.The pH of the culture solution was increased (P<0.0001) in M-LA or F-LA treatments from 3 h to 12 h compared with the control and LA treatments. At 12 h incubation, the concentration of total VFA in the culture solution was higher (P<0.01) in M-LA and F-LA than in control and LA treatments. Concentration of C₃ by M-LA and F-LA was increased at 3 h (P<0.01), 6 h (P<0.01) and 12 h (P<0.01) compared with control and LA. However, no difference in C₃ concentration was observed between control and LA, or between M-LA and F-LA. Accumulated total gas produced for up to 12 h incubation was increased (P<0.01) by M-LA or F-LA compared with the control. Accumulated total methane produced for up to 12 h incubation, however, was greatly reduced (P<0.01) by all the supplements compared with control, and its production from M-LA or F-LA was smaller than the LA. The M-LA or F-LA also increased (P<0.05–<0.001) the concentrations of cis9, trans11-CLA for all incubation times and trans10, cis12-CLA at 1 h (P<0.01), 3 h (P<0.05), and 12 h (P<0.05) incubation times compared with LA.It can be concluded that malate and fumarate, as propionate precursors, act as alternative electron sinks and may compete with CH₄ generation and bio-hydrogenation of C_18:2 in the utilization of metabolic H₂. The highest CLA concentration at the early incubation stage (1 h) was accompanied by reduced propionate proportion. Linoleic acid is also considered one of the potential alternatives to suppress CH₄ generation.     

Factores de riesgo de mortalidad en pacientes mayores de 65 años hospitalizados por COVID-19

Background and objectiveCOVID-19 is a disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has caused a global pandemic that we are currently suffering from.Objectiveto identify factors associated with the death of patients aged 65 years or older hospitalized for COVID-19.Materials and methodsRetrospective cohort study. We included patients aged 65 years or older who were hospitalized for COVID-19 and dead o discharged between March 5 and 25, 2020. We used univariable and multivariable logistic regression methods to explore the risk factors associated with in-hospital death.Results277 patients were included in this study. The bivariate analysis showed significant differences (p < 0.05) between survivors and non survivors: age, increased dependence and comorbidity, history of ischemic heart disease, renal failure and non-hematological neoplasms, heart failure during admission, leukocytosis, elevated creatinine, PCR, GOT and troponin Ic values, lymphopenia, and decreased blood pH and SatO2. Multivariate logistic regression revealed that age ≥ 65 years (OR: 4.23 (95% CI: 1.43-12.52; p = 0.009), lymphopenia < 1000/μL (OR: 2.36 (95% CI: 1.07-5.20; p = 0.033), creatinine > 1.2 mg/dL (OR: 3.08 (95% CI: 1.37-6.92; p = 0.006), SatO₂ < 90% (OR: 2.29 (95% CI: 1.01-5.21; p = 0.049) and troponin Ic > 11 ng/mL (OR: 2.32 (95% CI: 1.04-5.16; p = 0.040) were independently associated with higher hospital mortality.ConclusionsOlder age, lymphopenia, SatO₂ < 90%, elevated creatinine and troponin Ic values were independently associated with higher mortality in hospitalized patients with COVID-19, these factors could help clinicians to identify patients with poor prognosis.     

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part II

Spirocyclopropane- and spiroazetidine-substituted tetracycles 13D–E and 16A are described as orally active MK2 inhibitors. The spiroazetidine derivatives are potent MK2 inhibitors with IC₅₀ <3 nM and inhibit the release of TNFα (IC₅₀<0.3 μM) from hPBMCs and hsp27 phosphorylation in anisomycin stimulated THP-1 cells. The spirocyclopropane analogues are less potent against MK2 (IC₅₀ = 0.05–0.23 μM), less potent in cells (IC₅₀ <1.1 μM), but show good oral absorption. Compound 13E (100 mg/kg po; bid) showed oral activity in rAIA and mCIA, with significant reduction of swelling and histological score.     

Esculentin-2CHa: A host-defense peptide with differential cytotoxicity against bacteria,erythrocytes and tumor cells

The host-defense peptide, esculentin-2CHa (GFSSIFRGVA¹⁰KFASKGLGK D²⁰LAKLGVDLVA³⁰ CKISKQC) shows potent (MIC ≤ 6 μM) growth inhibitory activity against clinical isolates of multidrug-resistant strains of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia and differential cytotoxic activity against human erythrocytes (LC₅₀ = 150 μM) and human non-small cell lung adenocarcinoma A549 cells (LC₅₀ = 10 μM). Esculentin-2CHa significantly (P < 0.01) stimulates the release of the anti-inflammatory cytokine IL-10 by mouse lymphoid cells and elevates its production after stimulation with concanavalin A and significantly (P < 0.05) stimulates TNF-α production by peritoneal macrophages. Effects on IL-6 and IL-1β production were not significant. Removal of the hydrophobic N-terminal hexapeptide (GFSSIF) from esculentin-2CHa results in abolition of growth inhibitory activity against S. aureus and cytotoxic activity against erythrocytes and A549 cells as well as a marked (≥16-fold) reduction in potency against A. baumannii and S. maltophilia. The primary structure of esculentin-2 has been poorly conserved between frog species but evolutionary pressure has acted to maintain the hydrophobic character of this N-terminal hexapeptide sequence. Removal of the cyclic C-terminal domain (CKISKQC) and replacement of the Cys³¹ and Cys³⁷ residues by serine resulted in appreciable decreases in cytotoxicity against all microorganisms and against mammalian cells. The more cationic [D20K, D27K] analog showed a modest increase in potency against all microorganisms (up to 4-fold) but a marked increase in cytotoxicity against erythrocytes (LC₅₀ = 11 μM) and A549 cells (LC₅₀ = 3 μM).     

Differential dopamine receptor subtype regulation of adenylyl cyclases in lipid rafts in human embryonic kidney and renal proximal tubule cells

Dopamine D₁-like receptors (D₁R and D₅R) stimulate adenylyl cyclase (AC) activity, whereas the D₂-like receptors (D₂, D₃ and D₄) inhibit AC activity. D₁R, but not the D₅R, has been reported to regulate AC activity in lipid rafts (LRs). We tested the hypothesis that D₁R and D₅R differentially regulate AC activity in LRs using human embryonic kidney (HEK) 293 cells heterologously expressing human D₁ or D₅ receptor (HEK-hD₁R or HEK-hD₅R) and human renal proximal tubule (hRPT) cells that endogenously express D₁R and D₅R. Of the AC isoforms expressed in HEK and hRPT cells (AC3, AC5, AC6, AC7, and AC9), AC5/6 was distributed to a greater extent in LRs than non-LRs in HEK-hD₁R (84.5 ± 2.3% of total), HEK-hD₅R (68.9 ± 3.1% of total), and hRPT cells (66.6 ± 2.2% of total) (P < 0.05, n = 4/group). In HEK-hD₁R cells, the D₁-like receptor agonist fenoldopam (1μM/15 min) increased AC5/6 protein (+ 17.2 ± 3.9% of control) in LRs but decreased it in non-LRs (− 47.3 ± 5.3% of control) (P < 0.05, vs. control, n = 4/group). By contrast, in HEK-hD₅R cells, fenoldopam increased AC5/6 protein in non-LRs (+ 67.1±5.3% of control, P < 0.006, vs. control, n = 4) but had no effect in LRs. In hRPT cells, fenoldopam increased AC5/6 in LRs but had little effect in non-LRs. Disruption of LRs with methyl-β-cyclodextrin decreased basal AC activity in HEK-D₁R (− 94.5 ± 2.0% of control) and HEK-D₅R cells (− 87.1 ± 4.6% of control) but increased it in hRPT cells (6.8 ± 0.5-fold). AC6 activity was stimulated to a greater extent by D₁R than D₅R, in agreement with the greater colocalization of AC5/6 with D₁R than D₅R in LRs. We conclude that LRs are essential not only for the proper membrane distribution and maintenance of AC5/6 activity but also for the regulation of D₁R- and D₅R-mediated AC signaling.