The selectivity of ��-adrenoceptor agonists at human ��1-, ��2- and ��3-adrenoceptors

Background and purpose:

There are two important properties of receptor–ligand interactions: affinity (the ability of the ligand to bind to the receptor) and efficacy (the ability of the receptor–ligand complex to induce a response). Ligands are classified as agonists or antagonists depending on whether or not they have efficacy. In theory, it is possible to develop selective agonists based on selective affinity, selective intrinsic efficacy or both. This study examined the affinity and intrinsic efficacy of 31 β-adrenoceptor agonists at the three human β-adrenoceptors to determine whether the current agonists are subtype selective because of affinity or intrinsic efficacy.

Experimental approach:

Stable clonal CHO-K1 cell lines, transfected with either the human β₁, β₂ or β₃-adrenoceptor, were used, and whole-cell [³H]-CGP 12177 radioligand binding and [³H]-cAMP accumulation were measured.

Key results:

Several agonists were found to be highly subtype selective because of selective affinity (e.g. salmeterol and formoterol, for the β₂-adrenoceptor over the β₁ or β₃), while others (e.g. isoprenaline) had little affinity–selectivity. However, the intrinsic efficacy of salmeterol, formoterol and isoprenaline was similar across all three receptor subtypes. Other ligands (e.g. denopamine for β₁; clenbuterol, AZ 40140d, salbutamol for β₂) were found to have subtype-selective intrinsic efficacy. Several ligands appeared to activate two agonist conformations of the β₁- and β₃-adrenoceptors.

Conclusions and implications:

There are agonists with subtype selectivity based upon both selective affinity and selective intrinsic efficacy. Therefore, there is scope to develop better selective agonists based upon both selective affinity and selective intrinsic efficacy.This article is commented on by Kenakin, pp. 1045–1047 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00764.x     

DNR declaration �� emergency medical system nurses�� opinions

Advance directive and other declarations of will made by patients in a case of mental illness still raise ethical and legal issues. In Poland there is no legal regulation, neither research about code of conduct in situation of do not attempt resuscitation. There are also not enough studies regarding Healthcare workers?? opinion about DNAR declaration (Do Not Attempt Resuscitation). The study is aimed at finding out emergency medical system nurses opinion on the subject of enforcing do not attempt resuscitation in situation of circulatory and respiratory arrest. Methods: The research was conducted by means of the diagnostic survey method applying a self ?? constructed questionnaire. The study was carried out among 82 (100%) nurses, from September to December 2011. Obtained information were analyzed statistically, Chi-square of independence with assumed p ?? 0.05. level of significance was used for statistical analysis. Results: The study of the respondents?? opinion shows that 67% Healthcare employees think that DNAR declaration should be obligatory in Poland. Contrary opinion has 7.3% of respondents. In their opinion the decision to refrain from resuscitating should be made by attending physician ? 46.3% and medical board ? 29.3%. Information enclosed within DNAR declaration, in most of respondents?? opinion ? 59.5%, should be only passed on in written form. Conclusions: Majority of respondents agree that patients have a right to refrain from resuscitating as a self ? determination act. Respondents concur the introduction of DNAR declaration in Polish Healthcare system. In respondents?? opinion that decision should be required in written form and an attending physician should decide about its implementation, what violates the existing rule. The execution of living will declaration raises ethical issues. Additionally, it also appears as public/social problem. The last stage of incurable disease is given as justifiable circumstances of DNAR.     

��4��2 Nicotinic acetylcholine receptors, willing if able

Li and Steinbach apply nonstationary noise analysis to the whole-cell current responses of low sensitivity α4β2 nAChR stably-expressed in HEK cells. These receptors represent one of the most important nAChR subtypes in brain, and also one of the most difficult to study in native tissues. They found the activating properties of the full agonists ACh and nicotine to be similar with regard to P_open and single channel conductance, whereas the weak α4β2 partial agonist cytisine caused channels to open with low probability but increased single channel conductance. When optimally stimulated by either of the full agonists, approximately 80% of the available receptors opened at the peak of the response. However, comparisons of whole-cell current to estimates of total cell surface receptors, indicated that only about 7% of the total receptor population can be activated. These observations provide important and intriguing new pieces of the brain nicotine receptor puzzle.     

Antibody-Mediated ��Universal�� Osteoclast Targeting Platform using Calcitonin as a Model Drug

Purpose

To generate and characterize a specific monoclonal antibody (mAb) against recombinant human RANK receptor and to develop an antiresorptive strategy using this mAb as an osteoclast-targeting platform that selectively targets osteoclast cells whilst delivering an attached (i.e. chemically conjugated) active drug cargo.

Methods

Using hybridoma technology, we generated a specific monoclonal antibody (mAb) against recombinant human RANK receptor and characterized by SDS PAGE, ELISA, Western Blot and immunocytochemistry, then synthesized osteoclast-targeting bioconjugates of salmon calcitonin (sCT) using this antibody by generating thiol groups on mAb using 2-Iminothiolane and subsequently reacting them with sCT-PEG-MAL synthesised from sCT and NHS-PEG-MAL. To test the efficacy of the conjugate in vitro, osteoclasts were generated from precursor RAW 264.7 cells by dosing with the cytokines macrophage-colony-stimulating factor (M-CSF), and RANK Ligand (RANKL) and TRAP activity assay, Resorption Pit Assay, TRAP staining were performed. Cytotoxicity of the mAb-sCT conjugate was also evaluated in RAW 264.7 cells; sCT bioactivity and CTR binding potential were evaluated by in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells.

Results

Generation of antibody against human RANK receptor was confirmed by SDS PAGE, ELISA and Western Blot. Immunocytochemistry confirmed the osteoclast targeting potential of the antibody. Successful conjugation of the antibody with sCT was confirmed by SDS PAGE and ELISA.Multinucleated osteoclast formation was confirmed by staining for tartrate-resistant acid phosphatase (TRAP). Conjugate functionality was confirmed by TRAP activity and Resorption Pit assay, showing the inhibitory effect on osteoclast differentiation. cAMP assay confirmed the retention of calcitonin bioactivity after conjugation.

Conclusions

Our strategy offers the potential for a ??universal?? osteoclast-targeting platform??one that targets the RANK receptor on osteoclast cells by simply altering the conjugated cargo in order to affect the specific regulation of osteoclast cells.     

The ��apparent clearance�� of free phenytoin in elderly vs. younger adults

AIMS

To test the hypothesis that the ‘apparent clearance’ of free phenytoin is reduced in elderly patients.

METHODS

Two separate studies were conducted comparing free phenytoin ‘apparent clearance’ in elderly vs. younger adults. The first study was a retrospective analysis of free phenytoin concentrations measured at Christchurch Hospital from 1997 to 2006. In the second study free phenytoin concentrations were measured prospectively in ambulatory subjects who were taking phenytoin regularly.

RESULTS

In the retrospective study (n = 29), free phenytoin ‘apparent clearance’ was 0.27 ± 0.04 l kg⁻¹ day⁻¹ (95% CI 0.19, 0.34) in the elderly cohort vs. 0.37 ± 0.06 l kg⁻¹ day⁻¹ (95% CI 0.22, 0.52) in younger adults, but the difference was not statistically significant. In the prospective study, free phenytoin ‘apparent clearance’ showed a non-significant trend to being reduced in the elderly patients (0.12 ± 0.02 l kg⁻¹ day⁻¹, 95% CI 0.07, 0.17) compared with the younger cohort (0.18 ± 0.07 l kg⁻¹ day⁻¹, 95% CI 0.09, 0.26) in those not taking interacting drugs (n = 21).

CONCLUSIONS

This research does not prove the hypothesis that the ‘apparent clearance’ of free phenytoin is reduced in the elderly. However, the trends found in these two studies are supported by trends in the same direction in other published studies, suggesting an age effect.     

Activation of PPAR�� Attenuates IFN�� and IL-1��-induced Cell Proliferation in Astrocytes: Involvement of IL-6 Independent Pathway

The present study demonstrates the effect of fibrates, agonists of PPARα on cytokines-induced proliferation in primary cultured astrocytes. Alone or combination treatment with cytokines, such as IL-1β (10 ng/ml), IFNγ (10 ng/ml), and TNF-α (10 ng/ml) cause a significant increase of cell proliferation in a time-dependent manner. Treatment of astrocytes with bezafibrate and fenofibrate (0, 5, and 10 µM) reduced the IFNγ and IL-1β-induced cell proliferation in a dose-dependent manner. To address the involvement of IL-6 on the IFNγ and IL-1β-induced cell proliferation, released IL-6 level was measured. IFNγ and IL-1β cause an increase of released IL-6 protein level in a time-dependent manner. Furthermore, pretreatment with IL-6 antibody (0, 0.1, 1, 2.5, and 5 ng/ml) dose-dependently inhibited the IFNγ and IL-1β-induced cell proliferation. However, bezafibrate and fenofibrate did not affect increased mRNA and protein levels of IL-6 in IFNγ and IL-1β-stimulated astrocytes. Taken together, these results clearly suggest that activation of PPARα attenuates the IFNγ and IL-1β-induced cell proliferation through IL-6 independent pathway.     

Tuning in to the ��right�� calcium channel regulation in experimental models of diabetes

Elucidation of cellular and molecular mechanisms underlying vascular disease is of fundamental importance to the development of pharmacological agents to target these pathways. Pinho et al. in this issue of the BJP provide highly compelling evidence that the δ isoform of phosphatidyl inositol 3-kinase (PI3K δ) was upregulated and accounted for the increase in L-type, voltage-gated, Ca channel current in aortic vascular smooth muscle (VSM) cells of a mouse model of type 1 diabetes. There are several key issues of broad fundamental significance to this work. Firstly, what is the ‘right’ answer about calcium channel regulation in diabetes? Conflicting reports of increased and decreased Ca channel current may be due to specificity of the vascular bed and species. Then, the time course of diabetic vasculopathy may influence the expression of contractile versus proliferative phenotypes of VSM. Also the metabolic characterization of diabetes may enlighten or confound any study of diabetic vascular disease. These issues need attention to move forward work in this area.

LINKED ARTICLE

This article is a commentary on Pinho et al., pp. 1458–1471 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2010.00955.x     

Quercetin Inhibits ��3��4 Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Quercetin mainly exists in the skin of colored fruits and vegetables as one of flavonoids. Recent studies show that quercetin, like other flavonoids, has diverse pharmacological actions. However, relatively little is known about quercetin effects in the regulations of ligand-gated ion channels. In the previous reports, we have shown that quercetin regulates subsets of homomeric ligand-gated ion channels such as glycine, 5-HT_3A and α7 nicotinic acetylcholine receptors. In the present study, we examined quercetin effects on heteromeric neuronal α3β4 nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal α3 and β4 subunits. Treatment with acetylcholine elicited an inward peak current (I_ACh) in oocytes expressing α3β4 nicotinic acetylcholine receptor. Co-treatment with quercetin and acetylcholine inhibited I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptors. The inhibition of I_ACh by quercetin was reversible and concentration-dependent. The half-inhibitory concentration (IC₅₀) of quercetin was 14.9±0.8 µM in oocytes expressing α3β4 nicotinic acetylcholine receptor. The inhibition of I_ACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate α3β4 nicotinic acetylcholine receptor and this regulation might be one of the pharmacological actions of quercetin in nervous systems.     

Low Doses of 17��-Estradiol and 17��-Estradiol Facilitate, Whereas Higher Doses of Estrone and 17��- and 17��-Estradiol Impair, Contextual Fear Conditioning in Adult Female Rats

Estrogens are known to exert significant structural and functional effects in the hippocampus of adult rodents. In particular, 17β-estradiol can improve, impair, or have no effect on hippocampus-dependent learning and memory depending on dose and time of administration. The effects of other forms of estrogen, such as estrone and 17α-estradiol, on hippocampus-dependent learning have not been as thoroughly investigated. Therefore, the purpose of this study was to investigate the effects of 17β-estradiol, estrone, and 17α-estradiol at three different doses on two different tasks: hippocampus-dependent contextual fear conditioning and hippocampus-independent cued fear conditioning. Adult ovariectomized female rats were injected with one of the estrogens at one of the three doses 30 mins before conditioning to assess the rapid effects of these estrogens on acquisition. Twenty-four hours later memory for the context was examined and 1 h later memory for the cue (tone) was assessed. Levels of synaptophysin were examined in the dorsal hippocampus of rats to identify a potential synaptic correlate of hormonal effects on contextual fear conditioning. Low 17β-estradiol and 17α-estradiol enhanced, whereas high 17β-estradiol and 17α-estradiol impaired, contextual fear conditioning. Only the middle dose of estrone severely impaired contextual fear conditioning. Estrogens did not alter performance in the hippocampus-independent cued task. Synaptophysin expression was increased by estrone (at a middle and high dose) and 17β-estradiol (at a middle dose) in the CA3 region of the hippocampus and was not correlated with cognition. The results of this study indicate that estradiol can positively or negatively influence hippocampus-dependent learning and memory, whereas estrone impairs hippocampus-dependent learning and memory in a dose-dependent manner. These results have important therapeutic implications, as estrone, a main component of a widely used hormone replacement therapy, was shown to have either a negative effect or no effect on learning and memory. It may be possible to use 17α-estradiol and lower doses of estrogens as potential alternatives in hormone replacement therapies.     

Physicians�� perception of CPOE implementation

Objective To identify perceptions held by physicians of the benefits of computerized physician order entry (CPOE) and factors influencing its successful implementation in the context of the increased presence of a clinical pharmacist on ward. Setting A 2000-bed University Hospital. Method A cross-section opinion survey was conducted of all permanent physicians of the hospital to determine their perception on the benefits, or otherwise, of CPOE. Questionnaires, built upon the analysis of 10 preliminary semi-structured interviews with physicians, were sent to physicians by electronic and paper mail. It comprised three sections with a 4 level Likert scale: general perception of CPOE benefits (items 1.1?C1.8); opinion on the introduction of the CPOE system in the hospital (item 2); opinion on the presence of a pharmacist on ward (item 3). A fourth section recorded the respondent??s profile. Main outcome measures Level of agreement on the items describing the general perception of CPOE benefits; opinion on the introduction of a CPOE system in the hospital; and opinion on the pharmacist??s presence on ward. A Principal Component Analysis (PCA) was conducted on sections one and two. Analysis of this PCA representation in terms of the respondents?? profile was performed. Results One hundred and one physicians (18%) participated in the survey. Most (83%) physicians favoured the implementation of a CPOE (item 2). Among the advantages of CPOE, the greatest agreement concerned items related to safety and regulatory issues (from 80 to 76% agreement). Other items related to management issues were perceived as less tangible benefits (from 50 to 67% agreement). The increased presence of a pharmacist on the ward was supported by 94% of physicians. The PCA representation using profile items produced a 2-factor solution, accounting for 68% of the variance, with former experience of collaboration with a pharmacist (P = 0.002) and senior physician status (P = 0.013) positively influencing the perception of the CPOE. Conclusion Endorsement by senior physicians and the presence of a clinical pharmacist on ward promote a positive attitude towards CPOE and facilitate its implementation.     

The neuronal nicotinic ��4��2 receptor has a high maximal probability of being open

Background and purpose:

A fundamental property of transmitter-gated ion channels is the probability a channel will be open (P_open) when stimulated by a concentration of agonist that elicits a maximal response. This value is critical for interpreting steady-state concentration–response relationships in terms of channel activation, and for understanding the actions of drugs that potentiate responses. We used analysis of non-stationary noise to estimate the maximal probability the nicotinic α4β2 receptor is open.

Experimental approach:

HEK293 cells stably transfected to express human α4β2 nicotinic receptors were studied using whole-cell voltage clamp. Nicotinic agonists (acetylcholine, nicotine, cytisine and 5-iodo A-85380) were applied, and the relationship between variance of the elicited whole-cell current and mean current was analysed.

Key results:

The variance did not increase linearly with the mean current. For acetylcholine and nicotine the relationship between variance and mean indicates that the maximal P_open is greater than 0.8. The number of agonist-activatable channels was estimated to be about 1000 per cell. The mean single channel conductance at −60 mV was indistinguishable when currents were elicited by acetylcholine (18 pS), nicotine (17 pS) or 5-iodo A-85380 (17 pS), whereas the value for cytisine was larger (24 pS).

Conclusions and implications:

The neuronal nicotinic α4β2 receptor has a maximal probability of being open that is greater than 0.8. This conclusion applies to the receptor containing three α4 and two β2 subunits (the low-sensitivity stoichiometry), but may not apply to the receptor containing two α4 and three β2 subunits (the high-sensitivity stoichiometry).This article is commented on by Papke, pp. 1903–1905 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00868.x     

N ��-Methylated phenylhistamines exhibit affinity to the hH4R��a pharmacological and molecular modelling study

Histamine H(1)-receptor agonists and antagonists exhibit affinity to the human histamine H(4)-receptor (hH(4)R). However, the pharmacological profiles between hH(1)R and hH(4)R exhibit similarities and differences. Since suprahistaprodifen and trifluoromethylphenylhistamine show significant affinity to hH(4)R, the aim of this study was to analyse a large number of new phenylhistamines, histaprodifens and phenoprodifens at hH(4)R to extend the pharmacological profile of these compound classes at hH(4)R. The hH(4)R-RGS19 fusion protein was co-expressed with G(αi2) and G(β1γ2) in Sf9 insect cells, and [(3)H]histamine competition binding as well as GTPase assays were performed. Based on adequate crystal structures, homology models of hH(4)R were generated. Molecular modelling studies, including molecular dynamics and prediction of Gibbs energy of ligand binding, were performed in order to explain the pharmacological data at hH(4)R on molecular level. The exchange of the phenyl moiety of phenylhistamines into the diphenylpropyl moiety of histaprodifens acts, in contrast to hH(1)R, as partial agonism-inverse agonism switch at hH(4)R. Based on our studies, some phenylhistamine derivatives with significantly higher affinity at hH(4)R than at hH(1)R were identified. The molecular dynamic simulations revealed two different conformations for the highly conserved Trp(6.48), suggested to be involved in receptor activation. Furthermore, the predicted Gibbs energy of ligand binding for six selected phenylhistamines was in very good agreement with the experimentally determined affinities. We identified phenylhistamine derivatives with higher affinity at hH(4)R than at hH(1)R. Besides, we have identified partial agonism-inverse agonism switch between phenylhistamines and histaprodifens at hH(4)R. These results are very important to understand selectivity between hH(1)R and hH(4)R and to design new potent H(1)R and/or H(4)R receptor ligands.     

Troglitazone induces cytotoxicity in part by promoting the degradation of peroxisome proliferator-activated receptor �� co-activator-1�� protein

BACKGROUND AND PURPOSE

Troglitazone (Tro), rosiglitazone (Rosi) and pioglitazone (Pio) are anti-diabetic thiazolidinediones that function as ligands for peroxisome proliferator-activated receptor γ (PPARγ); however, Tro has been withdrawn from the market due to liver toxicity issues. Mitochondrial dysfunction induced by Tro has been suggested to be an important mechanism behind its cytotoxicity. Constitutively active nuclear hormone receptors, oestrogen-related receptor α and γ are thought to regulate mitochondrial mass and oxidative phosphorylation together with their co-activators PPARγ co-activator-1α and -1β (PGC-1α and PGC-1β). Hence, in this study, we investigated whether Tro affects the expression and activity levels of these regulators.

EXPERIMENTAL APPROACH

Cellular viability was measured by an ATP-based assay. Mitochondrial mass and reactive oxygen species (ROS) were quantified by two different fluorogenic probes. Apoptosis was measured by an Annexin-V-based kit. Gene expression at the levels of mRNA and protein was measured by quantitative RT-PCR and Western analysis. Over-expression of PGC-1α was mediated by an adenovirus.

KEY RESULTS

Tro, but not Rosi or Pio, selectively stimulated PGC-1α protein degradation. As a result, Tro reduced mitochondrial mass, and superoxide dismutases 1 and 2 expressions, but induced ROS to initiate apoptosis. Using a ubiquitin–proteasome inhibitor MG132, it was established that blocking PGC-1α degradation partially suppressed the reduction of mitochondrial mass. Importantly, over-expressing PGC-1α partially restored the Tro-suppressed mitochondrial mass and attenuated the cytotoxic effects of Tro.

CONCLUSIONS AND IMPLICATIONS

Collectively, these results suggest that PGC-1α degradation is an important mechanism behind the cytotoxic effects of Tro in the liver.