The long-acting β2-adrenoceptor agonist olodaterol attenuates pulmonary inflammation

Background and Purpose

β₂-adrenoceptor agonists are widely used in the management of obstructive airway diseases. Besides their bronchodilatory effect, several studies suggest inhibitory effects on various aspects of inflammation. The aim of our study was to determine the efficacy of the long-acting β₂-adrenoceptor agonist olodaterol to inhibit pulmonary inflammation and to elucidate mechanism(s) underlying its anti-inflammatory actions.

Experimental Approach

Olodaterol was tested in murine and guinea pig models of cigarette smoke- and LPS-induced lung inflammation. Furthermore, effects of olodaterol on the LPS-induced pro-inflammatory mediator release from human parenchymal explants, CD11b adhesion molecule expression on human granulocytes TNF-α release from human whole blood and on the IL-8-induced migration of human peripheral blood neutrophils were investigated.

Key Results

Olodaterol dose-dependently attenuated cell influx and pro-inflammatory mediator release in murine and guinea pig models of pulmonary inflammation. These anti-inflammatory effects were observed at doses relevant to their bronchodilatory efficacy. Mechanistically, olodaterol attenuated pro-inflammatory mediator release from human parenchymal explants and whole blood and reduced expression of CD11b adhesion molecules on granulocytes, but without direct effects on IL-8-induced neutrophil transwell migration.

Conclusions and Implications

This is the first evidence for the anti-inflammatory efficacy of a β₂-adrenoceptor agonist in models of lung inflammation induced by cigarette smoke. The long-acting β₂-adrenoceptor agonist olodaterol attenuated pulmonary inflammation through mechanisms that are separate from direct inhibition of bronchoconstriction. Furthermore, the in vivo data suggest that the anti-inflammatory properties of olodaterol are maintained after repeated dosing for 4 days.Tables of Links

Therapeutic targets of triple-negative breast cancer: a review

Breast cancer (BC) is the second most common cause of cancer deaths. Triple-negative breast cancer (TNBC) does not show immunohistochemical expression of oestrogen receptors, progesterone receptors or HER2. At present, no suitable treatment option is available for patients with TNBC. This dearth of effective conventional therapies for the treatment of advanced stage breast cancer has provoked the development of novel strategies for the management of patients with TNBC. This review presents recent information associated with different therapeutic options for the treatment of TNBC focusing on promising targets such as the Notch signalling, Wnt/β-catenin and Hedgehog pathways, in addition to EGFR, PARP1, mTOR, TGF-β and angiogenesis inhibitors.Tables of Links

IL–1β and IL–18: inflammatory markers or mediators of hypertension?

Chronic inflammation in the kidneys and vascular wall is a major contributor to hypertension. However, the stimuli and cellular mechanisms responsible for such inflammatory responses remain poorly defined. Inflammasomes are crucial initiators of sterile inflammation in other diseases such as rheumatoid arthritis and gout. These pattern recognition receptors detect host-derived danger-associated molecular patterns (DAMPs), such as microcrystals and reactive oxygen species, and respond by inducing activation of caspase-1. Caspase-1 then processes the cytokines pro-IL-1β and pro-IL-18 into their active forms thus triggering inflammation. While IL-1β and IL-18 are known to be elevated in hypertensive patients, no studies have examined whether this occurs downstream of inflammasome activation or whether inhibition of inflammasome and/or IL-1β/IL-18 signalling prevents hypertension. In this review, we will discuss some known actions of IL-1β and IL-18 on leukocyte and vessel wall function that could potentially underlie a prohypertensive role for these cytokines. We will describe the major classes of inflammasome-activating DAMPs and present evidence that at least some of these are elevated in the setting of hypertension. Finally, we will provide information on drugs that are currently used to inhibit inflammasome/IL-1β/IL-18 signalling and how these might ultimately be used as therapeutic agents for the clinical management of hypertension.Tables of Links

MD-2 as the target of a novel small molecule,L6H21, in the attenuation of LPS-induced inflammatory response and sepsis

Background and Purpose

Myeloid differentiation 2 (MD-2) recognizes LPS, which is required for TLR4 activation, and represents an attractive therapeutic target for severe inflammatory disorders. We previously found that a chalcone derivative, L6H21, could inhibit LPS-induced overexpression of TNF-α and IL-6 in macrophages. Here, we performed a series of biochemical experiments to investigate whether L6H21 specifically targets MD-2 and inhibits the interaction and signalling transduction of LPS-TLR4/MD-2.

Experimental Approach

The binding affinity of L6H21 to MD-2 protein was analysed using computer docking, surface plasmon resonance analysis, elisa, fluorescence measurements and flow cytometric analysis. The effects of L6H21 on MAPK and NF-κB signalling were determined using EMSA, fluorescence staining, Western blotting and immunoprecipitation. The anti-inflammatory effects of L6H21 were confirmed using elisa and RT-qPCR in vitro. The anti-inflammatory effects of L6H21 were also evaluated in septic C57BL/6 mice.

Key Results

Compound L6H21 inserted into the hydrophobic region of the MD-2 pocket, forming hydrogen bonds with Arg⁹⁰ and Tyr¹⁰² in the MD-2 pocket. In vitro, L6H21 subsequently suppressed MAPK phosphorylation, NF-κB activation and cytokine expression in macrophages stimulated by LPS. In vivo, L6H21 pretreatment improved survival, prevented lung injury, decreased serum and hepatic cytokine levels in mice subjected to LPS. In addition, mice with MD-2 gene knockout were universally protected from the effects of LPS-induced septic shock.

Conclusions and Implications

Overall, this work demonstrated that the new chalcone derivative, L6H21, is a potential candidate for the treatment of sepsis. More importantly, the data confirmed that MD-2 is an important therapeutic target for inflammatory disorders.Tables of Links

Ten things you should know about protein kinases: IUPHAR Review 14

Many human malignancies are associated with aberrant regulation of protein or lipid kinases due to mutations, chromosomal rearrangements and/or gene amplification. Protein and lipid kinases represent an important target class for treating human disorders. This review focus on ‘the 10 things you should know about protein kinases and their inhibitors'', including a short introduction on the history of protein kinases and their inhibitors and ending with a perspective on kinase drug discovery. Although the ‘10 things’ have been, to a certain extent, chosen arbitrarily, they cover in a comprehensive way the past and present efforts in kinase drug discovery and summarize the status quo of the current kinase inhibitors as well as knowledge about kinase structure and binding modes. Besides describing the potentials of protein kinase inhibitors as drugs, this review also focus on their limitations, particularly on how to circumvent emerging resistance against kinase inhibitors in oncological indications.

Tables of Links

Ketamine and phencyclidine: the good,the bad and the unexpected

The history of ketamine and phencyclidine from their development as potential clinical anaesthetics through drugs of abuse and animal models of schizophrenia to potential rapidly acting antidepressants is reviewed. The discovery in 1983 of the NMDA receptor antagonist property of ketamine and phencyclidine was a key step to understanding their pharmacology, including their psychotomimetic effects in man. This review describes the historical context and the course of that discovery and its expansion into other hallucinatory drugs. The relevance of these findings to modern hypotheses of schizophrenia and the implications for drug discovery are reviewed. The findings of the rapidly acting antidepressant effects of ketamine in man are discussed in relation to other glutamatergic mechanisms.Tables of Links

Identification of matrine as a promising novel drug forhepatic steatosis and glucose intolerance with HSP72 as an upstream target

Background and Purpose

Matrine is a small molecule drug used in humans for the treatment of chronic viral infections and tumours in the liver with little adverse effects. The present study investigated its therapeutic efficacy for insulin resistance and hepatic steatosis in high-fat-fed mice.

Experimental Approach

C57BL/J6 mice were fed a chow or high-fat diet for 10 weeks and then treated with matrine or metformin for 4 weeks. The effects on lipid metabolism and glucose tolerance were evaluated.

Key Results

Our results first showed that matrine reduced glucose intolerance and plasma insulin level, hepatic triglyceride content and adiposity in high-fat-fed mice without affecting caloric intake. This reduction in hepatosteatosis was attributed to suppressed lipid synthesis and increased fatty acid oxidation. In contrast to metformin, matrine neither suppressed mitochondrial respiration nor activated AMPK in the liver. A computational docking simulation revealed HSP90, a negative regulator of HSP72, as a potential binding target of matrine. Consistent with the simulation results, matrine, but not metformin, increased the hepatic protein level of HSP72 and this effect was inversely correlated with both liver triglyceride level and glucose intolerance.

Conclusions and Implications

Taken together, these results indicate that matrine may be used for the treatment of type 2 diabetes and hepatic steatosis, and the molecular action of this hepatoprotective drug involves the activation of HSP72 in the liver.Tables of Links

A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases

The universal second messenger cAMP is generated upon stimulation of G_s protein-coupled receptors, such as the β₂-adreneoceptor, and leads to the activation of PKA, the major cAMP effector protein. PKA oscillates between an on and off state and thereby regulates a plethora of distinct biological responses. The broad activation pattern of PKA and its contribution to several distinct cellular functions lead to the introduction of the concept of compartmentalization of cAMP. A-kinase anchoring proteins (AKAPs) are of central importance due to their unique ability to directly and/or indirectly interact with proteins that either determine the cellular content of cAMP, such as β₂-adrenoceptors, ACs and PDEs, or are regulated by cAMP such as the exchange protein directly activated by cAMP. We report on lessons learned from neurons indicating that maintenance of cAMP compartmentalization by AKAP5 is linked to neurotransmission, learning and memory. Disturbance of cAMP compartments seem to be linked to neurodegenerative disease including Alzheimer''s disease. We translate this knowledge to compartmentalized cAMP signalling in the lung. Next to AKAP5, we focus here on AKAP12 and Ezrin (AKAP78). These topics will be highlighted in the context of the development of novel pharmacological interventions to tackle AKAP-dependent compartmentalization.Tables of Links

Gaddum Memorial Lecture 2014: receptors as an evolving concept: from switches to biased microprocessors

This review is based on the JR Vane Medal Lecture presented at the BPS Winter Meeting in December 2014 by T. Kenakin. A recording of the lecture is included as supporting information and can also be viewed online here: https://www.youtube.com/watch?v=xrP81AQ8l-8. Pharmacological models used to describe drug agonism and antagonism have evolved over the past 20 years from a parsimonious model describing single active and inactive receptor states to models of multiconformational receptor systems modified by ligand conformational selection. These latter models describe the observed, presently underexploited, pharmacological mechanism of ligand-directed biased signalling. Biased signals can be quantified with transduction coefficients (ΔΔLog(τ/K_A) values), a scale grounded in the Black/Leff operational model; this enables the optimization of biased profiles through medicinal chemistry. The past decades have also brought the availability of new technologies to measure multiple functional effects mediated by seven transmembrane receptors. These have confirmed that drugs can have many efficacies, which may be collaterally linked, that is there is no linear sequence of activities required. In addition, new functional screening assays have introduced increasing numbers of allosteric ligands into drug discovery. These molecules are permissive (they do not necessarily preclude endogenous signalling in vivo); therefore, they may allow better fine tuning of pathological physiology. The permissive quality of allosteric ligands can also change the quality of endogenous signalling efficacy (‘induced bias’) as well as the quantity of signal; in this regard, indices related to ΔΔLog(τ/K_A) values (namely ΔLog(αβ) values) can be used to quantify these effects for optimization in the drug discovery process. All of these added scales of drug activity will, hopefully, allow better targeting of candidate molecules towards therapies.Tables of Links

PDE7 inhibitor TC3.6 ameliorates symptomatology in a model of primary progressive multiple sclerosis

Background and Purpose

cAMP plays an important role in the transduction of signalling pathways involved in neuroprotection and immune regulation. Control of the levels of this nucleotide by inhibition of cAMP-specific PDEs such as PDE7 may affect the pathological processes of neuroinflammatory diseases like multiple sclerosis (MS). In the present study, we evaluated the therapeutic potential of the selective PDE7 inhibitor, TC3.6, in a model of primary progressive multiple sclerosis (PPMS), a rare and severe variant of MS.

Experimental Approach

Theiler’s murine encephalomyelitis virus-induced demyelinated disease (TMEV-IDD) is one of the models used to validate the therapeutic efficacy of new drugs in MS. As recent studies have analysed the effect of PDE7 inhibitors in the EAE model of MS, here the TMEV-IDD model was used to test their efficacy in a progressive variant of MS. Mice were subjected to two protocols of TC3.6 administration: on the pre-symptomatic phase and once the disease was established.

Key Results

Treatment with TC3.6 ameliorated the disease course and improved motor deficits of infected mice. This was associated with down-regulation of microglial activation and reduced cellular infiltrates. Decreased expression of pro-inflammatory mediators such as COX-2 and the cytokines, IL-1β, TNF-α, IFN-γ and IL-6 in the spinal cord of TMEV-infected mice was also observed after TC3.6 administration.

Conclusion

These findings support the importance of PDE7 inhibitors, and specifically TC3.6, as a novel class of agents with therapeutic potential for PPMS. Preclinical studies are needed to determine whether their effects translate into durable clinical benefits.Tables of Links

Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del-CFTR by a CDK-independent mechanism

Background and Purpose

The most common mutation in cystic fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CF transmembrane conductance regulator (CFTR) protein. Because CF is an orphan disease, therapeutic strategies aimed at improving mutant CFTR functions are needed to target the root cause of CF.

Experimental Approach

Human CF airway epithelial cells were treated with roscovitine 100 μM for 2 h before CFTR maturation, expression and activity were examined. The mechanism of action of roscovitine was explored by recording the effect of depleting endoplasmic reticulum (ER) Ca²⁺ on the F508del-CFTR/calnexin interaction and by measuring proteasome activity.

Key Results

Of the cyclin-dependent kinase (CDK) inhibitors investigated, roscovitine was found to restore the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. Neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, corrected F508del-CFTR trafficking demonstrating that the correcting effect of roscovitine was independent of CDK inhibition. Competition studies with inhibitors of the ER quality control (ERQC) indicated that roscovitine acts on the calnexin pathway and on the degradation machinery. Roscovitine was shown (i) to partially inhibit the interaction between F508del-CFTR and calnexin by depleting ER Ca²⁺ and (ii) to directly inhibit the proteasome activity in a Ca²⁺-independent manner.

Conclusions and Implications

Roscovitine is able to correct the defective function of F508del-CFTR by preventing the ability of the ERQC to interact with and degrade F508del-CFTR via two synergistic but CDK-independent mechanisms. Roscovitine has potential as a pharmacological therapy for CF.Table of Links

Pleiotropic,heart rate-independent cardioprotection by ivabradine

Background and Purpose

In pigs, ivabradine reduces infarct size even when given only at reperfusion and in the absence of heart rate reduction. The mechanism of this non-heart rate-related cardioprotection is unknown. Hence, in the present study we assessed the pleiotropic action of ivabradine in more detail.

Experimental Approach

Anaesthetized mice were pretreated with ivabradine (1.7 mg·kg⁻¹ i.v.) or placebo (control) before a cycle of coronary occlusion/reperfusion (30/120 min ± left atrial pacing). Infarct size was determined. Isolated ventricular cardiomyocytes were exposed to simulated ischaemia/reperfusion (60/5 min) in the absence and presence of ivabradine, viability was then quantified and intra- and extracellular reactive oxygen species (ROS) formation was detected. Mitochondria were isolated from mouse hearts and exposed to simulated ischaemia/reperfusion (6/3 min) in glutamate/malate- and ADP-containing buffer in the absence and presence of ivabradine respectively. Mitochondrial respiration, extramitochondrial ROS, mitochondrial ATP production and calcium retention capacity (CRC) were assessed.

Key Results

Ivabradine decreased infarct size even with atrial pacing. Cardiomyocyte viability after simulated ischaemia/reperfusion was better preserved with ivabradin, the accumulation of intra- and extracellular ROS decreased in parallel. Mitochondrial complex I respiration was not different without/with ivabradine, but ivabradine significantly inhibited the accumulation of extramitochondrial ROS, increased mitochondrial ATP production and increased CRC.

Conclusion and Implications

Ivabradine reduces infarct size independently of a reduction in heart rate and improves ventricular cardiomyocyte viability, possibly by reducing mitochondrial ROS formation, increasing ATP production and CRC.Tables of Links

Effects of chronic treatment with the new ultra-long-acting β2-adrenoceptor agonist indacaterol alone or in combination with the β1-adrenoceptor blocker metoprolol on cardiac remodelling

Background and Purpose

The ability of a chronic treatment with indacaterol, a new ultra-long-acting β₂-adrenoceptor agonist, to reverse cardiac remodelling and its effects in combination with metoprolol, a selective β₁-adrenoceptor antagonist, were investigated on myocardial infarction in a rat model of heart failure (HF).

Experimental Approach

We investigated the effects of indacaterol and metoprolol, administered alone or in combination, on myocardial histology, β-adrenoceptor-mediated pathways, markers of remodelling and haemodynamic parameters in a rat model of HF. Five groups of rats were assessed: sham-operated rats; HF rats; HF + indacaterol 0.3 mg·kg⁻¹·day⁻¹; HF + metoprolol 100 mg·kg⁻¹·day⁻¹; HF + metoprolol + indacaterol. All pharmacological treatments continued for 15 weeks.

Key Results

Treatment with either indacaterol or metoprolol significantly reduced the infarct size in HF rats. However, the combination of indacaterol and metoprolol reduced the infarct size even further, reduced both BP and heart rate, reversed the decrease in ejection fraction, normalized left ventricular systolic and diastolic internal diameters, normalized the decreased β₁ adrenoceptor mRNA expression as well as cardiac cAMP levels and reduced cardiac GPCR kinase 2 expression, compared with the untreated HF group.

Conclusion and Implications

The results of our study demonstrated an additive interaction between indacaterol and metoprolol in normalizing and reversing cardiac remodelling in our experimental model of HF. The translation of these findings to clinical practice might be of interest, as this combination of drugs could be safer and more effective in patients suffering from HF and COPD.Tables of Links

Identification and optimization of 2-aminobenzimidazole derivatives as novel inhibitors of TRPC4 and TRPC5 channels

Background and Purpose

Transient receptor potential canonical (TRPC) channels play important roles in a broad array of physiological functions and are involved in various diseases. However, due to a lack of potent subtype-specific inhibitors the exact roles of TRPC channels in physiological and pathophysiological conditions have not been elucidated.

Experimental Approach

Using fluorescence membrane potential and Ca²⁺ assays and electrophysiological recordings, we characterized new 2-aminobenzimidazole-based small molecule inhibitors of TRPC4 and TRPC5 channels identified from cell-based fluorescence high-throughput screening.

Key Results

The original compound, M084, was a potent inhibitor of both TRPC4 and TRPC5, but was also a weak inhibitor of TRPC3. Structural modifications of the lead compound resulted in the identification of analogues with improved potency and selectivity for TRPC4 and TRPC5 channels. The aminobenzimidazole derivatives rapidly inhibited the TRPC4- and TRPC5-mediated currents when applied from the extracellular side and this inhibition was independent of the mode of activation of these channels. The compounds effectively blocked the plateau potential mediated by TRPC4-containing channels in mouse lateral septal neurons, but did not affect the activity of heterologously expressed TRPA1, TRPM8, TRPV1 or TRPV3 channels or that of the native voltage-gated Na⁺, K⁺ and Ca²⁺ channels in dissociated neurons.

Conclusions and Implications

The TRPC4/C5-selective inhibitors developed here represent novel and useful pharmaceutical tools for investigation of physiological and pathophysiological functions of TRPC4/C5 channels.Tables of Links

A non-erythropoietic peptide derivative of erythropoietin decreases susceptibility to diet-induced insulin resistance in mice

Background and Purpose

The haematopoietic activity of erythropoietin (EPO) is mediated by the classic EPO receptor (EpoR) homodimer, whereas tissue-protective effects are mediated by a heterocomplex between EpoR and the β-common receptor (βcR). Here, we investigated the effects of a novel, selective ligand of this heterocomplex – pyroglutamate helix B surface peptide (pHBSP) – in mice fed a diet enriched in sugars and saturated fats.

Experimental Approach

Male C57BL/6J mice were fed a high-fat high-sucrose diet (HFHS) for 22 weeks. pHBSP (30 μg·kg⁻¹ s.c.) was administered for the last 11 weeks. Biochemical assays, histopathological and immunohistochemical examinations and Western blotting were performed on serum and target organs (liver, kidney and skeletal muscle).

Key Results

Mice fed with HFHS diet exhibited insulin resistance, hyperlipidaemia, hepatic lipid accumulation and kidney dysfunction. In gastrocnemius muscle, HFHS impaired the insulin signalling pathway and reduced membrane translocation of glucose transporter type 4 and glycogen content. Treatment with pHBSP ameliorated renal function, reduced hepatic lipid deposition, and normalized serum glucose and lipid profiles. These effects were associated with an improvement in insulin sensitivity and glucose uptake in skeletal muscle. Diet-induced overproduction of the myokines IL-6 and fibroblast growth factor-21 were attenuated by pHBSP and, most importantly, pHBSP markedly enhanced mitochondrial biogenesis in skeletal muscle.

Conclusions and Implications

Chronic treatment of mice with an EPO derivative, devoid of haematopoietic effects, improved metabolic abnormalities induced by a high-fat high-sucrose diet, by affecting several levels of the insulin signalling and inflammatory cascades within skeletal muscle, while enhancing mitochondrial biogenesis.Table of Links

Analgesic tolerance to morphine is regulated by PPARγ

Background and Purpose

Opioid drugs are potent analgesics. However, their chronic use leads to the rapid development of tolerance to their analgesic effects and subsequent increase of significant side effects, including drug dependence and addiction. Here, we investigated the role of PPARγ in the development of analgesic tolerance to morphine in mice.

Experimental Approach

We monitored analgesia on alternate days using the tail immersion test.

Key Results

Daily administration of morphine (30 mg·kg⁻¹, bid) resulted in the rapid development of tolerance to thermal analgesia. Co-administration of pioglitazone (10 and 30 mg·kg⁻¹, bid) significantly attenuated the development and expression of tolerance. However, pretreatment with GW-9662 (5 mg·kg⁻¹, bid), a selective PPARγ antagonist, completely abolished this effect. Injection of GW-9662 and a lower dose of morphine (15 mg·kg⁻¹, bid) accelerated the development of tolerance to its antinociceptive effect. Subsequently, we found that conditional neuronal PPARγ knockout (KO) mice develop a more rapid and pronounced tolerance to morphine antinociception compared with wild-type (WT) controls. Moreover, in PPARγ KO mice, pioglitazone was no longer able to prevent the development of morphine tolerance.

Conclusions and Implications

Overall, our results demonstrate that PPARγ plays a tonic role in the modulation of morphine tolerance, and its pharmacological activation may help to reduce its development. These findings provide new information about the role of neuronal PPARγ and suggest that combining PPARγ agonists with opioid analgesics may reduce the development of tolerance and possibly attenuate the potential for opioid abuse.Table of Links

Endothelin-1 contributes to endothelial dysfunction and enhanced vasoconstriction through augmented superoxide production in penile arteries from insulin-resistant obese rats: role of ETA and ETB receptors

Background and Purpose

We assessed whether endothelin-1 (ET-1) inhibits NO and contributes to endothelial dysfunction in penile arteries in a model of insulin resistance-associated erectile dysfunction (ED).

Experimental Approach

Vascular function was assessed in penile arteries, from obese (OZR) and lean (LZR) Zucker rats, mounted in microvascular myographs. Changes in basal and stimulated levels of superoxide (O₂⁻) were detected by lucigenin-enhanced chemiluminescence and ET receptor expression was determined by immunohistochemistry.

Key Results

ET-1 stimulated acute O₂⁻ production that was blunted by tempol and the NADPH oxidase inhibitor, apocynin, but markedly enhanced in obese animals. ET-1 inhibited the vasorelaxant effects of ACh and of the NO donor S-nitroso-N-acetyl-DL-penicillamine in arteries from both LZR and OZR. Selective ET_A (BQ123) or ET_B receptor (BQ788) antagonists reduced both basal and ET-1-stimulated superoxide generation and reversed ET-1-induced inhibition of NO-mediated relaxations in OZR, while only BQ-123 antagonized ET-1 actions in LZR. ET-1-induced vasoconstriction was markedly enhanced by NO synthase blockade and reduced by endothelium removal and apocynin. In endothelium-denuded penile arteries, apocynin blunted augmented ET-1-induced contractions in OZR. Both ET_A and ET_B receptors were expressed in smooth muscle and the endothelial layer and up-regulated in arteries from OZR.

Conclusions and Implications

ET-1 stimulates ET_A-mediated NADPH oxidase-dependent ROS generation, which inhibits endothelial NO bioavailability and contributes to ET-1-induced contraction in healthy penile arteries. Enhanced vascular expression of ET_B receptors contributes to augmented ROS production, endothelial dysfunction and increased vasoconstriction in erectile tissue from insulin-resistant obese rats. Hence, antagonism of ET_B receptors might improve the ED associated with insulin-resistant states.Tables of Links

Salvinorin A analogues PR-37 and PR-38 attenuate compound 48/80-induced itch responses in mice

Background and Purpose

The opioid system plays a crucial role in several physiological processes in the CNS and in the periphery. It has also been shown that selective opioid receptor agonists exert potent inhibitory action on pruritus and pain. In this study we examined whether two analogues of Salvinorin A, PR-37 and PR-38, exhibit antipruritic properties in mice.

Experimental Approach

To examine the antiscratch effect of PR-37 and PR-38 we used a mouse model of compound 48/80-induced pruritus. In order to elucidate the mechanism of action of tested compounds, specific antagonists of opioid and cannabinoid receptors were used. The effect of PR-37 on the CNS was assessed by measuring motor parameters and exploratory behaviours in mice.

Key Results

PR-37 and PR-38, jnjected s.c., significantly reduced the number of compound 48/80-induced scratching behaviours in mice in a dose- and time-dependent manner. PR-38 was also active when orally administered. The antiscratch activity of PR-37 was blocked by the selective κ opioid receptor antagonist, nor-binaltorphimine, and that of PR-38 by the selective μ opioid receptor antagonist, β-funaltrexamine.

Conclusion and Implications

In conclusion, a novel framework for the development of new antipruritic drugs derived from salvinorin A has been validated.Table of Links

α1-Adrenoceptor activation of PKC-ε causes heterologous desensitization of thromboxane receptors in the aorta of spontaneously hypertensive rats

Background and Purpose

In the aorta of adult spontaneously hypertensive (SHR), but not in that of normotensive Wistar-Kyoto (WKY), rats, previous exposure to phenylephrine inhibits subsequent contractions to PGE₂. The present experiments were designed to examine the mechanism(s) underlying this inhibition.

Experimental Approach

Isometric tension was measured in isolated rings of SHR and WKY aortae. Gene expression and protein presence were measured by quantitative real-time PCR and Western blotting respectively.

Key Results

In aorta of 18 weeks SHR, but not age-matched WKY, pre-exposure to phenylephrine inhibited subsequent contractions to PGE₂ that were mediated by thromboxane prostanoid (TP) receptors. This inhibition was not observed in preparations of pre-hypertensive 5-week-old SHR, and was significantly larger in those of 36- than 18-week-old SHR. Pre-exposure to the PKC activator, phorbol 12,13-dibutyrate, also inhibited subsequent contractions to PGE₂ in SHR aortae. The selective inhibitor of PKC-ε, ε-V1-2, abolished the desensitization caused by pre-exposure to phenylephrine. Two molecular PKC bands were detected and their relative intensities differed in 36-week-old WKY and SHR vascular smooth muscle. The mRNA expressions of PKC-α, PKC-ε, PK-N2 and PKC-ζ and of G protein-coupled kinase (GRK)-2, GRK4 and β-arrestin2 were higher in SHR than WKY aortae.

Conclusions and Implications

These experiments suggest that in the SHR but not the WKY aorta, α₁-adrenoceptor activation desensitizes TP receptors through activation of PKC-ε. This heterologous desensitization is a consequence of the chronic exposure to high arterial pressure.Tables of Links

Pharmacologically distinct phenotypes of α1B-adrenoceptors: variation in binding and functional affinities for antagonists

Background and Purpose

The pharmacological properties of particular receptors have recently been suggested to vary under different conditions. We compared the pharmacological properties of the α_1B-adrenoceptor subtype in various tissue preparations and under various conditions.

Experimental Approach

[³H]-prazosin binding to α_1B-adrenoceptors in rat liver (segments, dispersed hepatocytes and homogenates) was assessed and the pharmacological profiles were compared with the functional and binding profiles in rat carotid artery and recombinant α_1B-adrenoceptors.

Key Results

In association and saturation-binding experiments with rat liver, binding affinity for [³H]-prazosin varied significantly between preparations (K_D value approximately ten times higher in segments than in homogenates). The binding profile for various drugs in liver segments also deviated from the representative α_1B-adrenoceptor profile observed in liver homogenates and recombinant receptors. L-765,314 and ALS-77, selective antagonists of α_1B-adrenoceptors, showed high binding and antagonist affinities in liver homogenates and recombinant α_1B-adrenoceptors. However, binding affinities for both ligands in the segments of rat liver and carotid artery were 10 times lower, and the antagonist potencies in α_1B-adrenoceptor-mediated contractions of carotid artery were more than 100 times lower than the representative α_1B-adrenoceptor profile.

Conclusions and Implications

In contrast to the consistent profile of recombinant α_1B-adrenoceptors, the pharmacological profile of native α_1B-adrenoceptors of rat liver and carotid artery varied markedly under various receptor environments, showing significantly different binding properties between intact tissues and homogenates, and dissociation between functional and binding affinities. In addition to conventional ‘subtype’ characterization, ‘phenotype’ pharmacology must be considered in native receptor evaluations in vivo and in future pharmacotherapy.Table of Links