Mitochondrial pharmacology turns its sights on the Ca2+ uniporter

Opening paragraph…To use a well-known phrase, mitochondria are the ‘power house of the cell’ and produce ATP, the energetic currency of life.1 Although this is a vital role, mitochondria also have many other crucial roles including cell death pathways such as collapse of the mitochondrial membrane potential by opening the mitochondrial permeability transition pore (mPTP). These roles are profoundly affected by the entry of calcium into the mitochondria. Mitochondrial calcium increases the likelihood of mPTP opening, regulation of metabolic rate by activation of metabolic enzymes (dehydrogenases), reactive oxygen species production, signalling (Ca-calmodulin) and influencing the pattern of global calcium oscillations in response to receptor stimulation

Opioids and cancer survival: are we looking in the wrong place?

There is a controversial narrative in the anaesthetic literature suggesting that anaesthetic technique (including opioids) may be detrimental to survival after tumour resection. The initial observations were retrospective. Several prospective studies are ongoing; one in breast cancer has reported no adverse outcome. The evidence for an effect of opioids stems from three pieces of information: (1) opioids depress the immune system, (2) opioids potentially promote angiogenesis, and (3) opioids potentially support tumour growth. Although the evidence for (2)/(3) is unclear, combinations of these effects are beneficial to tumours and potentially promote metastatic reseeding. Accepted wisdom suggests that opioid effects are driven by opioid receptor activation but the presence of opioid receptors on immune cells for example is unlikely. Immune cells, vascular endothelium and a range of tumour cells express Toll-like receptor 4 (TLR4) receptors (for Gram-negative bacterial wall components), and there is growing evidence for opioids interacting with this alternative receptor; and for some there is paradoxical naloxone sensitivity. Is the focus on opioid receptors and cancer the wrong target? TLR4 receptor activation produces immune activation, stimulates angiogenesis, and supports tumour survival. We know that some opioids are more immune suppressive than others (there is no such comparative information for angiogenesis and tumour survival); this may correlate with TLR4 activation. If there are clusters of opioids that have more opioid than TLR4 profiles and vice versa, this may influence outcome. If this is the case, then evidence-based advice could be given for perioperative use in the oncology–anaesthesia setting.</p

Hot Topics in Opioid Pharmacology: Mixed and Biased Opioids.

Analgesic design and evaluation has been driven by the desire to create high affinity, high selectivity MOP (mu;µ) agonists. Such ligands are the mainstay of current clinical practice and include morphine and fentanyl. Advances in this sphere have come from designing pharmacokinetic advantage; rapid metabolism for remifentanil. These produce analgesia but also the adverse effect profile that currently defines this drug class; namely ventilatory depression, tolerance and abuse liability. The MOP receptor is part of a family and there are significant functional interactions between other members of the family (DOP;delta;δ, KOP;kappa;κ and NOP;nociceptin/orphanin FQ). Experimentally MOP agonism and DOP antagonism produced antinociception (animals) with no tolerance and low doses of MOP and NOP ligands synergize to antinociceptive advantage. In this latter context lack of effect of NOP agonists on ventilation is an additional advantage. Recent development has been to move towards low selectivity multifunctional ‘mixed ligands’ (e.g., Cebranopadol) or ligand mixtures (e.g., Targinact). Moreover, the observation that β-arrestin coupling underlies the side effect profile for MOP ligands (from knockout animal studies) led to the discovery of biased (to G-protein and away from β-arrestin) MOP ligands (e.g., oliceridine). There is sufficient excitement in the opioid field to suggest that opioid analgesics without significant side effects may be on the horizon and the ‘opioid holy grail’ might be in reach

Evidence for Nociceptin/Orphanin FQ (NOP) but not µ (MOP), δ (DOP) or κ (KOP) opioid receptor mRNA in whole human blood

Background While it is well known that opioids depress the immune system, the site(s) of action for this depression is highly controversial. Immune modulation could occur directly at the immune cell or centrally via the hypothalamic-pituitary-adrenal axis. In a number of studies using individual enriched immune cell populations we have failed to detect classical µ (MOP), δ (DOP) and κ (KOP) receptors. The non-classical nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is expressed on all cells examined thus far. Our hypothesis was that immune cells do not express classical opioid receptors and that using whole blood would definitively answer this question. Methods Whole blood (containing all immune cell types) was incubated with opioids (morphine and fentanyl) commonly encountered in anaesthesia and with agents mimicking sepsis [lipopolysaccharide (LPS) and peptidoglycan G (PepG)]. Opioid receptor mRNA expression was assessed by endpoint polymerase chain reaction (PCR) with gel visualisation and quantitative PCR. Results Classical MOP, DOP, and KOP receptors were not detected in any of the samples tested either at rest or when challenged with opioids, LPS or PepG. Commercial primers for DOP did not perform well in quantitative PCR, so the absence of expression was confirmed using a traditional gel-based approach. NOP receptors were detected in all samples; expression was unaffected by opioids and reduced by LPS/PepG combinations. Conclusions Classical opioid receptors are not expressed on circulating immune cells

Cannabinoid receptor expression in the bladder is altered in detrusor overactivity

Introduction: Immunohistochemical (IHC) evidence shows that cannabinoid receptors (CB) are expressed in human bladders and cannabinoid agonists are known to inhibit detrusor contractility. However, the mechanism for this inhibition remains unknown. In addition, the role of CB in detrusor overactivity (DO) is under-investigated. The aim of this study was to compare CB expression in normal and DO human bladders and to further characterise these receptors. Methods: Polymer chain reaction (PCR) was used to detect differences in CB transcripts in bladder samples. Differences in CB protein expression was assessed by IHC. Immunofluorescence (IF) was used to evaluate co-localisation of CB with nerve fibres. Receptor density and binding affinity were measured using the cannabinoid radioligand [3H]-CP-55,940. Results: There were higher levels of CB1 transcripts in the urothelium of patients with DO and lower levels in the detrusor, compared with normal bladders. Radioligand binding revealed CB density of 421 ± 104 fmol/mg protein in normal human bladders. IHC confirmed these findings at the protein level. IF staining demonstrated co-localisation of CB1 with choline acetyltransferase-(ChAT)-positive nerves in the detrusor and co-localisation with PGP9.5 in both urothelium and detrusor. CB2 was co-localised with both ChAT and PGP9.5 in the urothelium and the detrusor. Conclusions: Cannabinoid receptor expression is reduced in the detrusor of patients with DO, which may play a role in the pathophysiology of the disease. Co-localisation of CB receptors with cholinergic nerves may suggest that CB1, being localised on pre- and postsynaptic terminals, could influence neurotransmitter release. Our findings suggest the potential role of cannabinoid agonists in overactive bladder pharmacotherapy

Effects of cannabinoid receptor activation by CP55,940 on normal bladder function and irritation-induced bladder overactivity in non-awake anaesthetised rats.

INTRODUCTION AND HYPOTHESIS: This study was designed to evaluate the effects of CP55,940 on normal bladder function in vivo and examine whether it suppresses urinary frequency induced by nociceptive stimuli in the bladder. Cannabinoid receptor (CBR) activity may be involved in the regulation of bladder function. However, the role of CBR subtypes in micturition has yet to be established. CP55,940 is a synthetic analogue of tetrahydrocannabidiol, which is a psychoactive ingredient of the Cannabis plant. METHODS: Cystometry under urethane anaesthesia was performed to evaluate the effect of intravesical delivery of CP55,940 with or without administration of CB1 antagonist AM251 or CB2 antagonist AM630 on bladder function in female rats. The effects of CP55,940 were also examined in rats with urinary irritation induced by intravesical infusion of acetic acid. RESULTS: Infusion of CP55,940 significantly (p < 0.05) increased micturition interval (MI) and bladder capacity (BC) by 52 % and decreased maximal voiding pressure (MP) by 25 %. Pretreatment with AM251 or AM630 before CP55,940 administration prevented CP55,940-induced increases in MI, BC and reduced MP. Acetic acid induced urinary frequency as evidenced by a reduction in MI and was suppressed by CP55,940. CONCLUSIONS: CP55,940 decreases bladder activity and urinary frequency induced by nociceptive stimuli, probably by suppression of bladder afferent activity. Effects of CP55,940 were abolished by both CBR antagonists. This data implicates a role for the endocannabinoid system in bladder mechanoafferent function in rats. In addition, our results show that CP55,940 reverses urinary frequency exemplified in an overactive bladder model, suggesting it could be an effective treatment for patients with lower urinary tract symptoms

Exploring LPS-induced sepsis in rats and mice as a model to study potential protective effects of the nociceptin/orphanin FQ system

The nociceptin receptor (NOP) and its ligand nociceptin/orphanin FQ (N/OFQ) have been shown to exert a modulatory effect on immune cells during sepsis. We evaluated the suitability of an experimental lipopolysaccharide (LPS)-induced sepsis model for studying changes in the nociceptin system. C57BL/6 mice BALB/c mice and Wistar rats were inoculated with different doses of LPS with or without a nociceptin receptor antagonist (UFP-101 or SB-612111). In C57BL/6 mice LPS 0.85 mg/kg injection produced no septic response, whereas 1.2mg/kg produced a profound response within 5h. In BALB/c mice, LPS 4 mg/kg produced no response, whereas 7 mg/kg resulted in a profound response within 24h. In Wistar rats LPS 15 mg/kg caused no septic response in 6/10 animals, whereas 25mg/kg resulted in marked lethargy before 24h. Splenic interleukin-1β mRNA in BALB/c mice, and serum TNF-α concentrations in Wistar rats increased after LPS injection in a dose-dependent manner, but were undetectable in control animals, indicating that LPS had stimulated an inflammatory reaction. IL-1β and TNF-α concentrations in LPS-treated animals were unaffected by administration of a NOP antagonist. Similarly NOP antagonists had no effect on survival or expression of mRNA for NOP or ppN/OFQ (the N/OFQ precursor) in a variety of tissues. In these animal models, the dose-response curve for LPS was too steep to allow use in survival studies and no changes in the N/OFQ system occurred within 24h. We conclude that LPS-inoculation in rodents is an unsuitable model for studying possible changes in the NOP-N/OFQ system in sepsis

The Nociceptin/Orphanin FQ System Is Modulated in Patients Admitted to ICU with Sepsis and after Cardiopulmonary Bypass

BACKGROUND AND OBJECTIVES: Nociceptin/Orphanin FQ (N/OFQ) is a non-classical endogenous opioid peptide that modulates immune function in vitro. Its importance in inflammation and human sepsis is unknown. The objectives of this study were to determine the relationship between N/OFQ, transcripts for its precursor (pre-pro-N/OFQ [ppNOC]) and receptor (NOP), inflammatory markers and clinical outcomes in patients undergoing cardiopulmonary bypass and with sepsis. METHODS: A prospective observational cohort study of 82 patients admitted to Intensive Care (ICU) with sepsis and 40 patients undergoing cardiac surgery under cardiopulmonary bypass (as a model of systemic inflammation). Sixty three healthy volunteers, matched by age and sex to the patients with sepsis were also studied. Clinical and laboratory details were recorded. Polymorph ppNOC and NOP receptor mRNA were determined using quantitative PCR. Plasma N/OFQ was determined using ELISA and cytokines (TNF- α, IL-8, IL-10) measured using radioimmunoassay. Data from patients undergoing cardiac surgery were recorded before, 3 and 24 hours after cardiopulmonary bypass. ICU patients with sepsis were assessed on Days 1 and 2 of ICU admission, and after clinical recovery. MAIN RESULTS: Plasma N/OFQ concentrations increased (p<0.0001) on Days 1 and 2 of ICU admission with sepsis compared to matched recovery samples. Polymorph ppNOC (p= 0.019) and NOP mRNA (p<0.0001) decreased compared to healthy volunteers. TNF-α, IL-8 and IL-10 concentrations increased on Day 1 compared to matched recovery samples and volunteers (p<0.0001). Similar changes (increased plasma N/OFQ, [p=0.0058], decreased ppNOC [p<0.0001], increased IL-8 and IL-10 concentrations [both p<0.0001]) occurred after cardiac surgery but these were comparatively lower and of shorter duration. CONCLUSIONS: The N/OFQ system is modulated in ICU patients with sepsis with similar but reduced changes after cardiac surgery under cardiopulmonary bypass. Further studies are required to clarify the role of the N/OFQ system in inflammation and sepsis, and the mechanisms involved

Pharmacological Characterization of μ-Opioid Receptor Agonists with Biased G Protein or β-Arrestin Signaling, and Computational Study of Conformational Changes during Receptor Activation

In recent years, G protein vs. β-arrestin biased agonism at opioid receptors has been proposed as an opportunity to produce antinociception with reduced adverse effects. However, at present this approach is highly debated, a reason why more information about biased ligands is required. While the practical relevance of bias in the case of µ-opioid receptors (MOP) still needs to be validated, it remains important to understand the basis of this bias of MOP (and other GPCRs). Recently, we reported two cyclopeptides with high affinity for MOP, the G protein biased Dmt-c[d-Lys-Phe-pCF3-Phe-Asp]NH2 (F-81), and the β-arrestin 2 biased Dmt-c[d-Lys-Phe-Asp]NH2 (C-33), as determined by calcium mobilization assay and bioluminescence resonance energy transfer-based assay. The biased character of F-81 and C-33 has been further analyzed in the [35S]GTPγS binding assay in human MOP-expressing cells, and the PathHunter enzyme complementation assay, used to measure β-arrestin 2 recruitment. To investigate the structural features of peptide-MOP complexes, we performed conformational analysis by NMR spectroscopy, molecular docking, and molecular dynamics simulation. These studies predicted that the two ligands form alternative complexes with MOP, engaging specific ligand–receptor contacts. This would induce different displays of the cytosolic side of the seven-helices bundle, in particular by stabilizing different angulations of helix 6, that could favor intracellular coupling to either G protein or β-arrestin

In vitro and in vivo characterisation of the bifunctional MOP/DOP ligand UFP-505.

Background and purpose: Targeting more than one opioid receptor type simultaneously may have analgesic advantages in reduced side effect profile. We have evaluated the mixed MOP (μ, mu) agonist/DOP (δ, delta) antagonist UFP‐505 in vitro and in vivo. Experimental approach: We measured receptor density and function in single MOP, DOP and MOP/DOP double expression systems. GTPγ35S binding, cAMP formation and arrestin recruitment were measured. Antinociceptive activity was measured in vivo using tail withdrawal and paw pressure tests following acute and chronic treatment. In some experiments we have harvested tissue to measure receptor. Key Results: UFP‐505 bound to MOP and DOP; at MOP this binding resulted in full agonist activity and at DOP there was low efficacy partial agonism. At MOP but not DOP UFP‐505 treatment led to arrestin recruitment. Unlike morphine, UFP‐505 treatment internalized MOP and there was evidence for DOP internalization. Similar data were obtained in a MOP/DOP double expression system. In rats, acute UFP‐505 or morphine treatment was antinociceptive following i.t. administration. In tissues harvested from these experiments there was a reduction in MOP and DOP receptor density for UFP‐505 but not morphine (in agreement with in vitro data). Both Morphine and UFP‐505 induced significant tolerance. Conclusions and Implications:In this study we have shown that UFP‐505 behaves as a full agonist at the MOP receptor with variable activity at DOP. This bifunctional compound produces antinociception in rats via i.t. administration. In this paradigm dual targeting provides no advantages in terms of tolerance liability