The OPG/RANKL/RANK pathway is a promising target for the design of therapeutic agents used in the treatment of osteoporosis. E09241 with an N-methylpyridine-chlorofuranformamide structural skeleton was previously identified to decrease bone loss and thus protect against osteoporosis in ovariectomized rats through increasing osteoprotegerin (OPG) expression. In this study, 36 derivatives of E09241 (3a) were prepared. The synthesis, up-regulation of OPG activities, SAR (structure–activity relationship), and cytotoxicity of these compounds are presented. Compounds with good up-regulating OPG activities could inhibit RANKL (the receptor activator of nuclear factor-kappa B ligand)-induced osteoclastogenesis in RAW264.7 cells. Particularly, compounds 3c and 3i1 significantly reduced NFATc1 and MMP-9 protein expression through inhibition of the NF-κB and MAPK pathways in RANKL induced RAW264.7 cells. In addition, compounds 3c and 3v significantly promoted osteoblast differentiation in MC3T3-E1 cells in osteogenic medium, and compounds 3c, 3v, and 3i1 obviously increased OPG protein expression and secretion in MC3T3-E1 cells. Furthermore, the pharmacokinetic profiles, acute toxicity, and hERG K+ channel effects of compounds 3a, 3c, 3e, 3v, and 3i1 were investigated. Taken together, these results indicate that N-methylpyridine-chlorofuranformamide analog 3i1 could serve as a promising lead for the development of new agents for treating osteoporosis.

Inhibitory GABA(A) receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk(-) cells stably expressing human recombinant GABA(A) subunits (alpha1beta1-3gamma2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl(-) buffer using a voltage/ion probe reader. The influence of different betasubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at alpha1beta1gamma2, alpha1beta2gamma2, and alpha1beta3gamma2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC(50) values of 2.4 +/- 0.2, 2.5 +/- 0.2, and 1.3 +/- 0.1 microM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC(50) values of 8.7 +/- 0.9, 9.2 +/- 0.5, and 11.7 +/- 1.2, and 43.7 +/- 6.4, 24.8 +/- 1.6, and 26.1 +/- 2.4 microM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, pentobarbital, and steroids, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one, were unaffected by the beta isoform present in the receptor complex. However, several compounds displayed beta2/3 subunit selectivity, notably loreclezole, R(-)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at beta2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at alpha1beta1gamma2. Diflunisal was the most efficacious compound, eliciting greater potentiation than loreclezole (90 +/- 14% and 109 +/- 14% at beta3 and beta2, respectively, compared with 62 +/- 6% and 56 +/- 3%), whereas niflumic acid exhibited the lowest efficacy. An additional agent, olsalazine, weakly potentiated responses at all three receptors without any selectivity. This study identifies and characterizes a variety of allosteric modulators for which betasubunits are an important determinant of efficacy and potency.

Training, assessment and accreditation of anaesthesiologists within the European Union (EU) is generally well organized and of a high standard. However, variations occur, both within and between countries, for a number of reasons, almost all of which are related to issues outside the control of the medical profession. I believe that the quality aspirations of all those involved in anaesthesiology training in the EU would, given optimal circumstances, be the same. At present, each Member State organizes its own manpower planning, training schemes, assessment and accreditation processes, awarding national certificates of completion of anaesthesiology training, which in turn lead to inclusion on the country's 'Specialist Medical Register'. This degree of national independence is to be welcomed. Such training programmes vary in intensity, duration and assessment. Some include examinations, others include the testing of general and specific skills, and others still, have less clear end-points. The crucial factor is that, once a doctor is included on the Specialist Register, and provided these doctors obtained their primary medical qualification in an EU Member State, they are legally able to apply for inclusion on the Specialist Register of another EU Member State. Ironically, other anaesthesiologists, equally well qualified and on the Specialist Register of an EU Member State, but with their primary medical qualification in a non-EU country, cannot enjoy this freedom of exchange. To many of us, this is simply bureaucratic nonsense! So while centralized 'approval' of national training schemes would be welcome, the imposition of a single centralized and international training programme would be inappropriate. As European anaesthesiologists, should we be concerned about this, and if so what should we be doing? Perhaps we should ask, what does the EU need and who should take the lead? Should it be the European Board of Anaesthesiology, under UEMS, or the proposed new confederation of ESA, European Academy of Anaesthesiology (EAA) and CENSA, or even a new body altogether? I believe that our objectives should be to raise the standards of anaesthesiology and its related disciplines, in Europe for the benefit of our patients, to improve training and assessment and to encourage scientific meetings and research. Greater uniformity of training, accreditation and assessment would facilitate additional opportunities for integration and harmonization, enhancement of professional standards, movement of professionals between countries and development of the speciality. The current methods of assessment, training and accreditation in Europe vary between and even within countries. Individual Diplomas are awarded, different criteria for specialist recognition are used, different periods of training are required and there is variable recognition of other countries' qualifications and accreditation. In addition, Europe has some unique problems related to language, variable individual practice and resources and the supply and demand for doctors. I believe that the European Academy should have a major role in the assessment of training and specialist recognition within Europe. There is great potential and opportunity for the expansion and use of the European Diploma, either as part of a national qualification process or as an internationally recognized qualification. It could be linked to hospital accreditation through the joint process, which already exists between the Academy and UEMS. The European Board, under UEMS, could and should establish criteria of Fellowship and even Membership, which would be open to all anaesthesiologists in Europe and would facilitate professional harmonization. However, the European Diploma is only testing knowledge. If we accept that, in future, competence in a trainee should be assessed as the possession of the knowledge, skills and attitudes required to undertake safe clinical practice at a level commensurate with their stage of training, then the EDA would certainly fulfil the requirement for a test of knowledge, as part of competency-based training. Clinical skills would then be tested by work place assessment and attitudes and behaviour, the commonest single cause of failure to proceed, assessed against commonly agreed standards. So how could these suggestions be taken forward? There is no doubt that the time is ripe for the Specialist Anaesthetic Section of the UEMS, to formerly establish the title of Fellowship within the European Board of Anaesthesiology. The European Board of Anaesthesiology itself already exists as a Working Group of the UEMS Section of Anaesthesiology, Reanimation and Intensive Care as explained in Simon de Lange's Editorial in the European Journal of Anaesthesiology in 2001 [1]. Many UEMS Specialist Sections operate European Boards, from which European Board 'Fellowships' can be obtained (e.g. Certificate of Fellowship of the European Board of 'Speciality'). Although these 'Fellowships' have no legal standing and do not confer a right to practise in the EU, they are a mark of quality and excellence. However, in no way do they replace the national anaesthesiology qualifications of individual countries. Indeed, there is also no restriction based upon the nationality of the applicants. Essentially colleagues from any country in the world can be registered as 'Fellows' by the European Boards, just as in the case of the North American Boards [2]. It would seem therefore, that we, as anaesthesiologists within the Specialist Section of UEMS, have the opportunity to strengthen the existing European Board and to decide upon the qualifications for the award of Board Fellowship. We urgently need to move ahead on this issue, because much of the work done within our section at present is done in a vacuum, without the central focus of Board Fellowship. Guidelines for Continuing Education and Professional Development (CEPD), anaesthetic training, advice to the new accession countries of the EU about quality and structure of anaesthetic training programmes and even hospital visiting and accreditation would all be given so much more purpose if linked to a formal Board and to the attaining of a quality standard, by becoming Fellows. It is vital that the Specialist Anaesthetic Section of UEMS, through the Board, develops into a major influence in professional standards, training and accreditation. How could Board Fellowship be developed? Firstly, we must ask what are we trying to achieve? The Board is Europe-wide, not a national organization, so its Fellows should have an international perspective on issues and the Board will need to be able to take an equally international view. Secondly, we must decide whether we think the Board should be inclusive or exclusive? Do we want to include all those involved in specialist anaesthesiology practice in Europe? The answer here I feel, is that Board Fellowship is a title of distinction and a mark of quality and therefore any applicant who fills the admission requirements should be able to become a Fellow. That said, and in the interests of inclusivity, I believe that there should be a second category of membership, open to all and from which suitable candidates could aspire to Fellowship. If this is to be achieved, several important points need to be clarified: 1. The work and structure of the Board must be seen to be relevant to EU anaesthesiology. 2. The title of Fellow must be a title of distinction and a mark of individual excellence. 3. The title of Fellow must not be devalued by awarding it to everyone; particularly those who only intend to work in Europe for a short time. 4. Board Fellowship must also be achievable and attainable by a significant number of suitably qualified anaesthesiologists. It must not be perceived as elitist and only ever available to a few anaesthesiologists who, somehow, 'have the right connections'. Various routes of admission to Fellowship of the European Board of Anaesthesiology (FEBA) might be considered, given that it should be considered as a mark of excellence, coming on top of the national qualification as a medical specialist. If applications were organized by a Nominations Committee of the Board, I firmly believe that excellence could be maintained through the following routes of admission to Fellowship, if the criteria set down were suitably robust. Possible routes of admission to Board Fellowship Examination Holders of international European qualifications in anaesthesiology, for example the European Diploma of Anaesthesiology and Intensive Care, who are, by definition, on the Specialist Register of a European country. This means that although the examination only tests knowledge, candidates will already have had their skills and attitudes assessed and accredited as part of their national qualification process. Academic 1. Senior academics in anaesthesiology departments in a European Member State, for example Professors, Readers, Senior Lecturers. 2. Academicians of the EAA. 3. Individual academic nominations to the Nominations Committee. Training and accreditation Permanent specialist members of anaesthesiology departments visited and accredited under the UEMS/EAA hospital visiting programme. Clear rules about anaesthesiologists who work for a time in an accredited hospital and then go elsewhere in the world would have to be established, and whether and for how long such people can use the title of FEBA. National nomination National specialist societies could have the opportunity to nominate individual anaesthesiologists of distinction to the Nominations Committee of the Board, for approval and award of the title. Limits could be set for individual countries based on criteria, such as total patient population, number of anaesthesiologists or a maximum number of nominations per year. How could the Board be further developed? In the interests of inclusiveness and unity of our Speciality, I believe that, if we are considering the proposals above, we should also introduce a category of Membership of the European Board (MEBA). This could be by application and open to all those on the Specialist Anaesthesiology Register of a European Member State. Members of the Board, of say 10 years of specialist anaesthesiology practice, would then be able to apply and be considered for Fellowship status, with the support of their National Anaesthesiology Society. Although, to my knowledge, this has not been considered before, I do not see that it would contravene the terms under which a UEMS Specialist Section Board operates. Furthermore, I believe it has three major advantages: 1. That all individuals involved in European anaesthesiology will feel part of a single Professional Specialist organization. 2. Board Membership and Fellowship will be seen to be attainable by everyone. 3. The distinct title of Fellow, denoting excellence will be enhanced. As often happens, our speciality has the opportunity to take a lead in what I believe is an exciting and unifying venture for anaesthesiology in a new and expanding Europe. The attainment and recognition of excellence is what we would want for our patients and here is a golden opportunity to achieve it. Peter Simpson Chairman of Examinations; European Academy of Anaesthesiology; Richmond, UK President; Royal College of Anaesthetists; London, UK

We have studied the pharmacological regulation of mitochondrial activity in a human neuroblastoma cell line. Cyclosporin A was found to directly alter mitochondrial membrane potential and to decrease mitochondrial permeability as measured using calcein. The estrogen receptor ligands tamoxifen, nafoxidine and clomiphene were identified as agents which affect mitochondrial membrane potential in a cyclosporin A-like manner. Also when mitochondrial permeability was measured using calcein, tamoxifen, nafoxidine and clomiphene were effective in inhibiting dye loss from mitochondria. Nafoxidine and cyclosporin A inhibit effects of mastoparan on SH-SY5Y mitochondria. These studies indicate that estrogen receptor ligands appear to affect mitochondria in a cyclosporin A-like manner in human neuroblastoma cells.

Selective modulators of γ-aminobutyric acid, type A (GABAA) receptors containing α4subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(−tk) cells, we stably expressed the native GABAA receptor subunit combinations α3β3γ2,α4β3γ2, and, for the first time, α4β3δ and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABAA receptor antagonists and, for α3β3γ2and α4β3γ2 receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with α4 and β3, δ subunits, compared with γ2, conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. α4β3δ responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3–7-fold more efficacious at α4β3δ compared with α4β3γ2. The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABAA receptor. For GABAA receptors containing α4 subunits, our experiments reveal that inclusion of δ instead of γ2subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of α4β3δ receptors, consistent with a central role for this subunit combination in premenstrual syndrome.

Many physiologically important activities of oligodendrocyte progenitor cells (O‐2A cells), including proliferation, migration and differentiation, are regulated by cytosolic Ca2+ signals. However, little is known concerning the mechanisms of Ca2+ signalling in this cell type. We have studied the interactions between Ca2+ entry, Ca2+ release from endoplasmic reticulum and Ca2+ regulation by mitochondria in influencing cytosolic Ca2+ responses in O‐2A cells. Methacholine (MCh; 100 μM) activated Ca2+ waves that propagated from several initiation sites along O‐2A processes. During a Ca2+ wave evoked by MCh, mitochondrial membrane potential was often either depolarized (21 % of mitochondria) or hyperpolarized (20 % of mitochondria), as measured by changes in the fluorescence of 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazole carbocyanine iodide (JC‐1). Stimulation with kainate (100 μM) evoked a slowly rising, sustained cytosolic Ca2+ elevation in O‐2A cells. This also, in some cases, resulted in either a depolarization (15 % of mitochondria) or hyperpolarization (12 % of mitochondria) of mitochondrial membrane potential. Simultaneous measurement of cytosolic (fluo‐3 AM) and mitochondrial (rhod‐2 AM) Ca2+ responses revealed that Ca2+ elevations in the cytosol evoked by either MCh or kainate were translated into long‐lasting Ca2+ elevations in subpopulations of mitochondria. In some mitochondria, Ca2+ signals appeared to activate Ca2+ release into the cytosol. Inhibition of the mitochondrial Na+‐Ca2+ exchanger by CGP‐37157 (25 μM) decreased kainate Ca2+ response amplitude and increased the rate of return of the response to basal Ca2+ levels. Thus, both ionotropic and metabotropic stimulation evoke changes in mitochondrial membrane potential and Ca2+ levels in O‐2A cells. Ca2+ uptake into some mitochondria is activated by Ca2+ entry into cells or release from stores. Mitochondrial Ca2+ release appears to play a key role in shaping kainate‐evoked Ca2+ responses.

We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP(3)R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP(3)Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP(3)Rs (InsP(3)R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP(3)R2 immunofluorescence were compared in the same cell, domains of elevated Ca2+ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP(3)R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca2+ release sites that support regenerative wave propagation include increased levels of InsP(3)R2 expression.

The mechanisms involved in Ca2+ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-D-aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca2+ concentration ([Ca2+]i) response to CCh (to 45 +/- 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 +/- 6% of the control). CCh-evoked [Ca2+]i responses were inhibited by preincubation with thapsigargin (10 microM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 microM), ryanodine (10 microM), or dantrolene (25 microM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca2+]i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 microM) evoked a much diminished increase in [Ca2+]i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 +/- 14%; ACPD, 27 +/- 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP3) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca2+ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca2+]i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP3 receptors, appear to be crucial to the mAChR-mediated [Ca2+]i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP3-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP3 receptors may not contribute significantly to NMDA-evoked elevation of [Ca2+]i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca2+ stores is proposed.

Abstract: The [Ca2+]1 of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K+ level), which both activate Ca2+ influx. The possibility that these stimuli activate Ca2+‐induced Ca2+ release was investigated using granule cells loaded with fura 2‐AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca2+]1 response to K+ or NMDA, was found to reduce the response to both agents in a concentration‐dependent manner. Preincubation with thapsigargm (10 μM) substantially reduced the plateau phase of the [Ca2+], response to K+ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μM) also reduced both the K+‐evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K+. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K+‐ and NMDA‐induced elevation of [Ca2+]1 appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca2+ pools.