Nutrition Coverage in Medical Licensing Examinations in Germany: An Analysis of Six Nationwide Exams.

The state of nutrition education in medicine is inadequate, with nutrition-related topics being poorly integrated into lectures. Most medical students receive only a few contact hours of nutrition instruction during their entire time at medical school. Identifying potential barriers that may explain the paucity of nutritional knowledge in medical students is thus of paramount importance. The extent of nutrition coverage in the second part of Germany's nationwide medical licensing exam is currently unknown. We addressed this issue and assessed nutrition content, as well as students' scores, in this pivotal test prior to their graduation. We performed a post hoc analysis of six nationwide medical licensing examinations (2018-2020) undertaken by 29,849 medical students and screened 1920 multiple-choice questions for nutrition-related content. Nutrition-related questions accounted for a minority of the questions (2.1%, n = 40/1920). A considerable number of the questions (n = 19) included only a single nutrition-related answer option that was frequently incorrect and served as a distractor. About 0.5% of questions were entirely nutrition related. Despite undeniable barriers, the inclusion of additional nutrition-related examination questions could serve as an incentive to engage students and medical schools in enhancing medical nutrition education. The recently published competence-oriented learning objective catalog in Germany could play a pivotal role in this context, leading to better recognition of nutrition-related topics in medical education.

Pharmacokinetics and brain σ1 receptor occupancy of MR309, a selective σ1 receptor antagonist.

AIMS: Preclinical studies of MR309, a selective sigma-1 receptor (σ1R) antagonist, support a potential role in treating neuropathic pain. We report 2 studies that provide insight into the pharmacokinetics (PK) and brain σ1R binding of MR309. METHODS: Steady-state PK of MR309 (400 mg once daily and 200 mg twice-daily [BID] for 10 days; EudraCT 2015-001818-99 [PK study]) and the relationship between MR309 plasma exposure and brain σ1R occupancy (EudraCT 2017-000670-11 [positron emission tomography study]) were investigated in healthy volunteers. Positron emission tomography using the σ1R ligand [11 C]SA4503 was conducted at baseline, and 2 and 8 hours after a single dose of MR309 (200-800 mg). The relationship between brain σ1R occupancy and MR309 exposure was explored using data-driven model fitting. RESULTS: MR309 was well tolerated, brain σ1R occupancy ranged between 30.5 and 74.9% following single-dose MR309 (n = 7). MR309 BID provided a plasma PK profile with less fluctuation than once daily dosing (n = 16). MR309 200 mg BID yielded average steady state plasma concentrations between 2000 and 4000 ng/mL in the PK study, which corresponded to an estimated brain σ1R occupancy of 59-74%. CONCLUSION: MR309 200 mg BID dose was below the 75% σ1R occupancy threshold expected to elicit maximal antinociceptive effect as observed in neuropathic pain models. Further investigations of MR309 for neuropathic pain will require higher brain σ1R occupancy, and establish the optimal dose by elucidating the clinical impact of a broad range of brain σ1R occupancy across different neuropathic pain indications.

Mu opioid receptor knockout mouse: Phenotypes with implications on restless legs syndrome.

Restless legs syndrome (RLS) is characterized by an irresistible need to move the legs while sitting or lying at night with insomnia as a frequent consequence. Human RLS has been associated with abnormalities in the endogenous opioid system, the dopaminergic system, the iron regulatory system, anemia, and inflammatory and auto-immune disorders. Our previous work indicates that mice lacking all three subtypes of opioid receptors have a phenotype similar to that of human RLS. To study the roles of each opioid receptor subtype in RLS, we first used mu opioid receptor knockout (MOR KO) mice based on our earlier studies using postmortem brain and cell culture. The KO mice showed decreased hemoglobin, hematocrit, and red blood cells (RBCs), with an appearance of microcytic RBCs indicating anemia. Together with decreased serum iron and transferrin, but increased ferritin levels, the anemia is similar to that seen with chronic inflammation in humans. A decreased serum iron level was also observed in the wildtype mice treated with an MOR antagonist. Iron was increased in the liver and spleen of the KO mice. Normal circadian variations in the dopaminergic and serotoninergic systems were absent in the KO mice. The KO mice showed hyperactivity and increased thermal sensitivity in wakefulness primarily during what would normally be the sleep phase similar to that seen in human RLS. Deficits in endogenous opioid system transmission could predispose to anemia of inflammation and loss of circadian variations in dopaminergic or serotonergic systems, thereby contributing to an RLS-like phenotype.

Analgesic and Respiratory Depressant Effects of R-dihydroetorphine: A Pharmacokinetic-Pharmacodynamic Analysis in Healthy Male Volunteers.

BACKGROUND: There is an ongoing need for potent opioids with less adverse effects than commonly used opioids. R-dihydroetorphine is a full opioid receptor agonist with relatively high affinity at the µ-, δ- and κ-opioid receptors and low affinity at the nociception/orphanin FQ receptor. The authors quantified its antinociceptive and respiratory effects in healthy volunteers. The authors hypothesized that given its receptor profile, R-dihydroetorphine will exhibit an apparent plateau in respiratory depression, but not in antinociception. METHODS: The authors performed a population pharmacokinetic-pharmacodynamic study (Eudract registration No. 2009-010880-17). Four intravenous R-dihydroetorphine doses were studied: 12.5, 75, 125, and 150 ng/kg (infused more than 10 min) in 4 of 4, 6 of 6, 6 of 6, and 4 of 4 male subjects in pain and respiratory studies, respectively. The authors measured isohypercapnic ventilation, pain threshold, and tolerance responses to electrical noxious stimulation and arterial blood samples for pharmacokinetic analysis. RESULTS: R-dihydroetorphine displayed a dose-dependent increase in peak plasma concentrations at the end of the infusion. Concentration-effect relationships differed significantly between endpoints. R-dihydroetorphine produced respiratory depression best described by a sigmoid EMAX-model. A 50% reduction in ventilation in between baseline and minimum ventilation was observed at an R-dihydroetorphine concentration of 17 ± 4 pg/ml (median ± standard error of the estimate). The maximum reduction in ventilation observed was at 33% of baseline. In contrast, over the dose range studied, R-dihydroetorphine produced dose-dependent analgesia best described by a linear model. A 50% increase in stimulus intensity was observed at 34 ± 11 pg/ml. CONCLUSIONS: Over the dose range studied, R-dihydroetorphine exhibited a plateau in respiratory depression, but not in analgesia. Whether these experimental advantages extrapolate to the clinical setting and whether analgesia has no plateau at higher concentrations than investigated requires further studies.

A randomized pilot study to investigate the effect of opioids on immunomarkers using gene expression profiling during surgery.

Endogenous opioid peptides and exogenous opioids modulate immune function, and animal and human studies have shown that some have a depressant immunomodulatory effect. This is potentially of high clinical significance, eg, in cancer patients and surgery. The primary objective of this pilot study was to evaluate the effect of morphine and oxycodone on immune pathways associated with immunosuppression in gynecological laparotomy patients. Gene expression was analyzed in CD4, CD8, and natural killer (NK) cells using the 3' Affymetrix microarray. Patients were randomized to receive morphine, oxycodone, or nonopioid "control" analgesia during and after surgery. Genes demonstrating differential expression were those with a ≥±2-fold difference and P-value ≤0.05 after analysis of variance. Cytometric bead array and NK cell degranulation assay were used to investigate changes in serum cytokine concentration and in NK cell cytotoxicity, respectively. Forty patients had satisfactory RNA which was hybridized to gene chips. Genes were identified (Partek Genomics Suite 6.6) at baseline, 2, 6, and 24 hours and were either ≥2-fold upregulated or downregulated from baseline. At 2 hours, a large number of genes were downregulated with morphine but not with control analgesia or oxycodone. Statistically significant increases in IL-6 concentrations were induced by morphine only; NK cell activity was suppressed with morphine, but maintained with oxycodone and epidural analgesia. Gene expression profiles suggest that at 2 hours, post incision morphine appeared to be immunosuppressive as compared to oxycodone and nonopioid control analgesia.

Hyperactivity, dopaminergic abnormalities, iron deficiency and anemia in an in vivo opioid receptors knockout mouse: Implications for the restless legs syndrome.

Previous studies have uncovered a potential role of the opioid system in iron hemostasis and dopamine metabolism. Abnormalities in both of these systems have been noted in human RLS. Autopsy studies of human RLS have shown an endogenous opioid deficiency in the thalamus. Opioids, particularly prolonged-release oxycodone/naloxone, have been approved in Europe to be a second-line therapy for severe restless legs syndrome (RLS). To study the role of opioid receptors in the pathogenesis of RLS, we used a triple knockout (KO) mouse strain that lack mu, delta, and kappa opioid receptors and explored the behavioral and biochemical parameters relevant to RLS. The triple KO mice showed hyperactivity and a trend of increased probability of waking during the rest period (day) akin to that in human RLS (night). Surprisingly, triple KO mice also exhibit decreased serum iron concentration, evidence of anemia, a significant dysfunction in dopamine metabolism akin to that noted in human RLS, as well as an increased latency in response to thermal stimuli. To our knowledge, this is the first study to demonstrate that the endogenous opioid system may play a role in iron metabolism and subsequently in the pathogenesis of anemia. It is also the first study showing that opioid receptors are involved in the production of motor restlessness with a circadian predominance. Our findings support the role of endogenous opioids in the pathogenesis of RLS, and the triple KO mice can be used to understand the relationship between iron deficiency, anemia, dopaminergic dysfunction, and RLS.

Assessment of Transdermal Buprenorphine Patches for the Treatment of Chronic Pain in a UK Observational Study.

BACKGROUND: Opioids provide effective analgesia for moderate-to-severe, chronic pain. Transdermal buprenorphine (TDB) is available in the UK as weekly, lower-dose (5-20 µg/h) patches and twice-weekly, higher dose (35-70 µg/h) patches. This prospective, observational, multicenter study of patients with various chronic pain conditions assessed the safety, perceptions, and discontinuation of treatment with TDB in a real-world, non-interventional setting (ClinicalTrials.gov study ID: NCT01225861). METHODS: Patients aged ≥18 years who were already receiving or initiating treatment with TDB were recruited in the UK during routine clinical visits and were followed for 6 visits or 9 months (whichever came first). Self-reported treatment adherence, patient satisfaction, and safety data were collected at each study visit. RESULTS: Of 465 patients, 272 were already receiving 7-day TDB at the study start (TDB experienced), 146 were TDB naïve, and 47 were prescribed twice-weekly TDB. Most patients were female (72.9 %) and overweight/obese (body mass index ≥25: 75.3 %). The median age was 67 years, and the mean duration of pain was 11.1 years. Arthritis/other musculoskeletal disorders (39.6 %) were the most common causes of pain. Mild adverse events were commonly reported. Skin irritations, which were most frequent in 7-day TDB-experienced patients (45.6 %), rarely resulted in treatment discontinuation (8.8 %). Nearly all patients used TDB in accordance with treatment recommendations. Most patients reported that TDB was 'effective'/'very effective' at relieving pain and were 'satisfied'/'very satisfied' with TDB therapy. CONCLUSION: In everyday clinical practice, TDB was well tolerated and patients were satisfied with their therapy. Self-reported adherence to TDB was very high, and adverse events rarely resulted in treatment discontinuation. Opportunities were identified to limit common adverse events associated with TDB.

Prolonged-release oxycodone-naloxone for treatment of severe pain in patients with Parkinson's disease (PANDA): a double-blind, randomised, placebo-controlled trial.

BACKGROUND: Pain is a common non-motor symptom of Parkinson's disease. We investigated the analgesic efficacy of prolonged-release oxycodone-naloxone (OXN PR) in patients with Parkinson's disease and chronic, severe pain. METHODS: We did this phase 2 study in 47 secondary care centres in the Czech Republic, Germany, Hungary, Poland, Romania, Spain, and the UK. We enrolled patients with Hoehn and Yahr Stage II-IV Parkinson's disease, at least one type of severe pain, and an average 24-h pain score of at least 6 (assessed on an 11-point rating scale from 0=no pain to 10=pain as bad as you can imagine). Participants were randomly assigned (1:1) with a validated automated system (block size four) to either oral OXN PR or placebo for 16 weeks (starting dose oxycodone 5 mg, naloxone 2·5 mg, twice daily). Patients and investigators were masked to treatment assignment. The primary endpoint was average 24-h pain score at 16 weeks in the full analysis population. This study is registered with EudraCT (2011-002901-31) and ClinicalTrials.gov (NCT01439100). FINDINGS: We enrolled 202 patients; 93 were assigned to OXN PR and 109 to placebo; the full analysis population consisted of 88 patients versus 106 patients. Least squares mean average 24-h pain score at 16 weeks in the full analysis population was 5·0 (95% CI 4·5 to 5·5) in the OXN PR group versus 5·6 (5·1 to 6·0) in the placebo group (difference -0·6, 95% CI -1·3 to 0·0; p=0·058). Similar proportions of patients in each group had adverse events (60/92 [65%] vs 76/109 [70%]), treatment-related adverse events (52/92 [57%] vs 62/109 [57%]), and serious adverse events (5/92 [5%] vs 7/109 [6%]). Treatment-related nausea was more common in the OXN PR group than in the placebo group (16/92 [17%] vs 10/109 [9%]), as was treatment-related constipation (16/92 [17%] vs 6/109 [6%]). INTERPRETATION: The primary endpoint, based on the full analysis population at week 16, was not significant. Nonetheless, the results of this study highlight the potential efficacy of OXN PR for patients with Parkinson's disease-related pain and might warrant further research on OXN PR in this setting. FUNDING: Mundipharma Research.

Prolonged release oxycodone-naloxone for treatment of severe restless legs syndrome after failure of previous treatment: a double-blind, randomised, placebo-controlled trial with an open-label extension.

BACKGROUND: Opioids are a potential new treatment for severe restless legs syndrome. We investigated the efficacy and safety of a fixed-dose combination of prolonged release oxycodone-naloxone for patients with severe restless legs syndrome inadequately controlled by previous, mainly dopaminergic, treatment. METHODS: This multicentre study consisted of a 12-week randomised, double-blind, placebo-controlled trial and 40-week open-label extension phase done at 55 sites in Austria, Germany, Spain, and Sweden. Patients had symptoms for at least 6 months and an International RLS Study Group severity rating scale sum score of at least 15; patients with severe chronic obstructive pulmonary disease or a history of sleep apnoea syndrome were excluded. Patients were randomly assigned (1:1) to either study drug or matched placebo with a validated interactive response technology system in block sizes of four. Study drug was oxycodone 5·0 mg, naloxone 2·5 mg, twice per day, which was up-titrated according to investigator's opinion to a maximum of oxycodone 40 mg, naloxone 20 mg, twice per day; in the extension, all patients started on oxycodone 5·0 mg, naloxone 2·5 mg, twice per day, which was up-titrated to a maximum of oxycodone 40 mg, naloxone 20 mg, twice per day. The primary outcome was mean change in severity of symptoms according to the International RLS Study Group severity rating scale sum score at 12 weeks. This study is registered with ClinicalTrials.gov (number NCT01112644) and with EudraCT (number 2009-011107-23). FINDINGS: We screened 495 patients, of whom 306 were randomly assigned and 276 included in the primary analysis (132 to prolonged release oxycodone-naloxone vs 144 to placebo). 197 patients participated in the open-label extension. Mean International RLS Study Group rating scale sum score at randomisation was 31·6 (SD 4·5); mean change after 12 weeks was -16·5 (SD 11·3) in the prolonged release oxycodone-naloxone group and -9·4 (SD 10·9) in the placebo group (mean difference between groups at 12 weeks 8·15, 95% CI 5·46-10·85; p<0·0001). After the extension phase, mean sum score was 9·7 (SD 7·8). Treatment-related adverse events occurred in 109 of 150 (73%) patients in the prolonged release oxycodone-naloxone group and 66 of 154 (43%) in the placebo group during the double-blind phase; during the extension phase, 112 of 197 (57%) had treatment-related adverse events. Five of 306 (2%) patients had serious treatment-related adverse events when taking prolonged release oxycodone-naloxone (vomiting with concurrent duodenal ulcer, constipation, subileus, ileus, acute flank pain). INTERPRETATION: Prolonged release oxycodone-naloxone was efficacious for short-term treatment of patients with severe restless legs syndrome inadequately controlled with previous treatment and the safety profile was as expected. Our study also provides evidence of open-label long-term efficacy of this treatment. Opioids can be used to treat patients with severe restless legs syndrome who have had no benefit with first-line drugs. FUNDING: Mundipharma Research.

Live cell imaging of G protein-coupled receptors.

Live cell imaging experiments with G protein-coupled receptors (GPCRs) tagged with fluorescent fusion proteins were originally performed to study trafficking and subcellular location of these important drug targets. In the past decade, however, substantial progress came from improved imaging methods and from the cloning of novel fluorescent fusion proteins. Today, these methods allow to visualize not only GPCR interactions but also, e.g., receptor activation, trafficking between subcellular compartments, and to measure transport kinetics. Here, we summarize recent progress in live cell imaging of GPCRs using a confocal laser scanning microscope.

A double-blind, placebo-controlled study on the effect of buprenorphine and fentanyl on descending pain modulation: a human experimental study.

OBJECTIVES: The descending pain inhibitory system is impaired in chronic pain and it is important to know how analgesics interact with this system. The aim of this human experimental pain, double-blind, randomized, placebo-controlled, 3 way cross-over study was to investigate the effect of 2 different opioids on descending pain inhibition using conditioning pain modulation (CPM) as a screening tool. METHODS: Twenty-two healthy male volunteers were randomized to 72 hours of treatment with transdermal patches of fentanyl (25 µg/h), buprenorphine (20 µg/h), or placebo. The CPM was induced by immersing the hand into cold (3.0 ± 0.3°C) water and the evoked pain was continuously rated on a visual analogue scale (VAS). The test stimulus [pressure pain tolerance threshold (PPTol)] was applied to the contra-lateral arm. The CPM test was performed at baseline, 24, 48, and 72 hours after application of the patches. RESULTS: The opioid treatments did not significantly (F=2.249; P=0.07) modulate the PPTol over the treatment period compared with placebo. The CPM-evoked PPTol increases (percentage increase from what was obtained at the baseline before patch application) were significantly enhanced by buprenorphine (P=0.004) and fentanyl (P=0.005) compared with placebo, with no differences between the 2 active drugs. Fentanyl significantly attenuated the time to cold water-evoked VAS peak compared with placebo (P=0.005), and the same trend was observed for buprenorphine (P=0.06). The VAS pain intensity was not affected. DISCUSSION: The opioids buprenorphine and fentanyl significantly potentiate the effect of descending pain inhibition in healthy volunteers.

Localisation of endothelin B receptor variants to plasma membrane microdomains and its effects on downstream signalling.

The endothelin B (ET(B)) receptor can undergo a proteolytic cleavage resulting in an unglycosylated N-terminally truncated receptor. We investigated whether ET(B) receptor processing affects caveolar localisation and mitogenic signalling. Distinct subcellular localisations of ET(B) receptor constructs and epidermal growth factor (EGF) receptor ligands were analysed performing detergent-free caveolae preparations and total internal reflection fluorescence microscopy. ET(B) receptor-induced transactivation of the EGF receptor and its downstream signalling was investigated performing shedding assays and ERK1/2 phosphorylation analyses. In COS7 cells, the N-terminally truncated but not the full-length or glycosylation-deficient ET(B) receptor localised to caveolae. In caveolae-free HEK293 cells, only ET(B) receptor constructs fused to caveolin-2 localised to membrane microdomains. A caveolar accumulation of the ET(B) receptor disfavoured EGF receptor ligand shedding. Nonetheless, the activation of ERK1/2 was efficient and long-lasting. In HEK293 cells, the shedding activity was also impaired by N-terminal truncation. The subsequent ERK1/2 phosphorylation was long-lasting only for the full-length ET(B) receptor. We conclude that the ET(B) receptor localisation might depend on the presence of caveolae within the cell investigated. The data further suggest that caveolar enrichment of ET(B) receptors does not facilitate the release of EGF receptor ligands. However, independent of their localisation, ET(B) receptors are able to induce an ERK1/2 phosphorylation.

Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB.

The molecular basis for recognition of peptide ligands endothelin-1, -2 and -3 in endothelin receptors is poorly understood. Especially the origin of ligand selectivity for ET(A) or ET(B) is not clearly resolved. We derived sequence-structure-function relationships of peptides and receptors from mutational data and homology modeling. Our major findings are the dissection of peptide ligands into four epitopes and the delineation of four complementary structural portions on receptor side explaining ligand recognition in both endothelin receptor subtypes. In addition, structural determinants for ligand selectivity could be described. As a result, we could improve the selectivity of BQ3020 about 10-fold by a single amino acid substitution, validating our hypothesis for ligand selectivity caused by different entrances to the receptors' transmembrane binding sites. A narrow tunnel shape in ET(A) is restrictive for a selected group of peptide ligands' N-termini, whereas a broad funnel-shaped entrance in ET(B) accepts a variety of different shapes and properties of ligands.

Endothelin potentiates TRPV1 via ETA receptor-mediated activation of protein kinase C.

BACKGROUND: Endothelin-1 (ET-1) both stimulates nociceptors and sensitizes them to noxious stimuli, an effect probably mediated by the ETA receptor (ETAR) expressed in sensory neurons. The cellular mechanisms of this ET-1-mediated effect are only poorly understood. TRPV1, the heat-, pH- and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released in response to noxious stimuli and during inflammation, is a potential target for the action of ET-1. RESULTS: We studied the effects of ET-1 on TRPV1 in sensory neurons from the dorsal root ganglion (DRG) and in HEK293 cells coexpressing TRPV1 and the ETAR. Specific 125I-ET-1 binding sites (817 +/- 92 fmol/mg) were detected in membrane preparations of DRG with an ETAR/ETBR ratio of 60:40. In an immunofluorescence analysis, coexpression of TRPV1 and the ETAR was found in a subpopulation of primary sensory neurons. ET-1 strongly potentiated capsaicin-induced TRPV1 currents in some neurons, and in HEK293 cells co-expressing TRPV1 and the ETAR. Weaker potentiation was observed in HEK293 cells coexpressing TRPV1 and the ETBR. ETAR activation also increased responses to low pH and heat. In HEK293 cells, strong potentiation of TRPV1 like that induced by ET-1 via the ETAR could be induced by PKC activation, but not with activators of the adenylyl cyclase or the PKA pathway. Furthermore, inhibition of PKC with bisindolylmaleimide X (BIM X) or mutation of the PKC phosphorylation site S800 completely prevented ETAR-mediated potentiation. CONCLUSION: We conclude that ET-1 potentiates TRPV1 by a PKC-dependent mechanism and that this could play a major role in the algogenic and hyperalgesic effects of ET-1 described in previous studies.

Essential role of ubiquitin-specific protease 8 for receptor tyrosine kinase stability and endocytic trafficking in vivo.

Posttranslational modification by ubiquitin controls multiple cellular functions and is counteracted by the activities of deubiquitinating enzymes. UBPy (USP8) is a growth-regulated ubiquitin isopeptidase that interacts with the HRS-STAM complex. Using Cre-loxP-mediated gene targeting in mice, we show that lack of UBPy results in embryonic lethality, whereas its conditional inactivation in adults causes fatal liver failure. The defect is accompanied by a strong reduction or absence of several growth factor receptor tyrosine kinases (RTKs), like epidermal growth factor receptor, hepatocyte growth factor receptor (c-met), and ERBB3. UBPy-deficient cells exhibit aberrantly enlarged early endosomes colocalizing with enhanced ubiquitination and have reduced levels of HRS and STAM2. Congruently immortalized cells gradually stop proliferation upon induced deletion of UBPy. These results unveil a central and nonredundant role of UBPy in growth regulation, endosomal sorting, and the control of RTKs in vivo.

Functional expression of mammalian receptors and membrane channels in different cells.

In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.

Mu-opioid receptor activation modulates transient receptor potential vanilloid 1 (TRPV1) currents in sensory neurons in a model of inflammatory pain.

Current therapy for inflammatory pain includes the peripheral application of opioid receptor agonists. Activation of opioid receptors modulates voltage-gated ion channels, but it is unclear whether opioids can also influence ligand-gated ion channels [e.g., the transient receptor potential vanilloid type 1 (TRPV1)]. TRPV1 channels are involved in the development of thermal hypersensitivity associated with tissue inflammation. In this study, we investigated mu-opioid receptor and TRPV1 expression in primary afferent neurons in the dorsal root ganglion (DRG) in complete Freund's adjuvant (CFA)-induced paw inflammation. In addition, the present study examined whether the activity of TRPV1 in DRG neurons can be inhibited by mu-opioid receptor (mu-receptor) ligands and whether this inhibition is increased after CFA inflammation. Immunohistochemistry demonstrated colocalization of TRPV1 and mu-receptors in DRG neurons. CFA-induced inflammation increased significantly the number of TRPV1- and mu-receptor-positive DRG neurons, as well as TRPV1 binding sites. In whole-cell patch clamp studies, opioids significantly decreased capsaicin-induced TRPV1 currents in a naloxone- and pertussis toxinsensitive manner. The inhibitory effect of morphine on TRPV1 was abolished by forskolin and 8-bromo-cAMP. During inflammation, an increase in TRPV1 is apparently rivaled by an increase of mu-receptors. However, in single dissociated DRG neurons, the inhibitory effects of morphine are not different between animals with and without CFA inflammation. In in vivo experiments, we found that locally applied morphine reduced capsaicin-induced thermal allodynia. In summary, our results indicate that mu-receptor activation can inhibit the activity of TRPV1 via G(i/o) proteins and the cAMP pathway. These observations demonstrate an important new mechanism underlying the analgesic efficacy of peripherally acting mu-receptor ligands in inflammatory pain.

Mass-spectrometric identification of a novel angiotensin peptide in human plasma.

OBJECTIVE: Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients. METHODS AND RESULTS: Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an angiotensin octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Ala1 instead of Asp1. Des[Asp1]-[Ala1]-Ang II, in the following named Angiotensin A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp1. Ang A has the same affinity to the AT1 receptor as Ang II, but a higher affinity to the AT2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD 123319, suggesting a lower intrinsic activity at the AT1 receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A/Ang II was higher in end-stage renal failure patients. CONCLUSIONS: Ang A is a novel human strong vasoconstrictive angiotensin-derived peptide, most likely generated by enzymatic transformation through mononuclear leukocyte-derived aspartate decarboxylase. Plasma Ang A concentration is increased in end-stage renal failure. Because of its stronger agonism at the AT2 receptor, Ang A may modulate the harmful effects of Ang II.

Endothelin A and endothelin B receptors differ in their ability to stimulate ERK1/2 activation.

Endothelin-1 (ET-1) acts on two different G protein-coupled receptors, namely the endothelin A (ET(A)) and the endothelin B (ET(B)) receptors. Both receptor subtypes show differences in their tissue expression and signal transduction. In the present study, we compared the ability of ET(A) and ET(B) receptors to stimulate extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, we analyzed the role of the extracellular N terminus for ERK1/2 activation, because the ET(B) receptor undergoes an agonist-dependent N-terminal proteolysis. ET-1 stimulation of HEK293 cells stably expressing the ET(A) receptor induced a monophasic, but sustained ERK1/2 activation, whereas the ET(B) receptor showed a biphasic ERK1/2 activation. A truncated mutant ET(B) receptor, lacking the proteolytically cleaved N terminus (delta2-64 ET(B)) revealed only a monophasic and transient ERK1/2 activation. Treatment of HEK293 delta2-64 ET(B) cell clones with ET-1 and a synthetic NT27-64 peptide, corresponding to the N-terminally cleaved fragment of the ET(B) receptor and ET-1, did not restore the biphasic activation of ERK1/2. A chimeric ET(B) receptor in which the N terminus was replaced by the N terminus of the ET(A) receptor elicited biphasic ERK1/2 activation. The presented data suggest that an intact N terminus of the ET(B) receptor is necessary for the second phase of ERK1/2 activation. However, it appears that the length of the N terminus rather than a specific sequence motif is required for biphasic ERK1/2 activation.

Endothelin-1 potentiates capsaicin-induced TRPV1 currents via the endothelin A receptor.

Endothelin-1 (ET-1) both stimulates nociceptors and sensitizes them to painful stimuli. The cellular mechanisms of the ET-1-mediated effects are only poorly understood. TRPV1, the heat-, proton-, and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released by painful stimuli and during inflammation, is a potential target for the action of ET-1. In immunocytochemistry of rat lumbar dorsal root ganglion using TRPV1- and ET(A) receptor-specific antibodies, both proteins were found to be co-expressed in small sensory neurons. To provide evidence that ET-1 can modulate TRPV1 activity via the ET(A) receptor, we used HEK 293 cells transiently co-expressing a fusion protein of TRPV1 and the yellow fluorescent protein (TRPV1-YFP) and the ET(A) receptor. In whole-cell patch clamp recordings of HEK293 cells co-expressing TRPV1-YFP and the ET(A) receptor, capsaicin (10 nM) elicited small currents, which were markedly potentiated when capsaicin (10 nM) and ET-1 (100 nM) were applied simultaneously. The data indicate that ET-1 potentiates TRPV1 activity via the ET(A) receptor and that this process is likely to play a crucial role in the pain-producing and pain-potentiating effects of ET-1. Thus, ET(A) receptor antagonists may be of importance in painful states with increased circulating ET-1 levels, as found in cancer and in chronic inflammation.