In this study three major subtypes of nicotinic acetylcholine receptors were characterized pharmacologically using the calcium influx through the ion channel as a robust functional assay system. Human alpha3beta4 receptors and alpha4beta2 receptors were cloned and stably expressed in HEK293 cells. [(125)I]epibatidine saturation binding yielded a B(max) of 4420+/-840 fmol/mg protein for the alpha4beta2 receptor ( n=4) and 518+/-15 fmol/mg protein for the alpha3beta4 receptor ( n=4). As a source for muscle type of nicotinic receptor, the TE671 cell line was used which expresses endogenously the human fetal alpha1beta1gammadelta subtype of nicotinic receptor. Stimulation of these nicotinic receptor subtypes in the different cell lines led to calcium transients that peaked 5-10 s after agonist application and declined thereafter. Eleven agonists were tested in this study and their efficacy and potency at the three nicotinic receptor subtypes were determined (epibatidine, ABT594, anatoxin, ABT418, nicotine, DMPP, cytisine, ABT089, choline, GTS21, AAR17779). This pharmacological characterization of agonist-induced elevation of intracellular free Ca(2+) revealed a distinct rank order of agonist potency for each receptor subtype. Epibatidine showed at all three subtypes the highest potency and was a full agonist. The agonist-elicited response could be blocked by co-incubation of different antagonists from which mecamylamine did not display a strong subtype specificity. These data illustrate that the assessment of calcium transients upon receptor stimulation is a powerful tool for rapid examination of the functional properties of nicotinic receptors.

Objectives The aim of this study was to deliver insights from multiple stakeholders into actual and future collaboration for health technology assessment (HTA) in general and in oncology in particular. Methods Eighteen semi-structured interviews were conducted with experts from European HTA bodies (HTAbs), former board members of the European Network for Health Technology Assessment (EUnetHTA), and representatives from the pharmaceutical industry, a regulatory agency, academia, and patient organizations. The stakeholders were asked about their support of the EUnetHTA’s intent, about the general strengths and challenges of the EUnetHTA and its Joint Action 3 (JA 3), the strengths and challenges of the clinically oriented HTA collaboration in oncology during JA 3 across the technology life cycle, about future challenges to HTA in oncology with consequences for collaboration, and about collaboration in the economic domains of HTA. The transcribed interviews were analyzed qualitatively. Results The participants perceived the intention and work quality of the EUnetHTA as positive. The experts described methodological, procedural, and capacity challenges in early dialogues (EDs) and rapid relative effectiveness assessments (REAs) meant to analyze clinical effectiveness in oncology. The majority attached increasing importance to collaboration in the future to cope with the uncertainty of HTA. Several stakeholders also proposed the incorporation of joint postlaunch evidence generation (PLEG) activities. Some gave sporadic suggestions for voluntary nonclinical collaboration as well. Conclusion Stakeholders’ continued readiness to discuss the remaining challenges to and sufficient resources for implementing HTA regulation, as well as further cooperative expansion along the technology life cycle, are necessary for improved HTA collaboration in Europe.