Detection of multiple H(3) receptor affinity states utilizing [(3)H]A-349821, a novel, selective, non-imidazole histamine H(3) receptor inverse agonist radioligand

1. A-349821 is a selective histamine H(3) receptor antagonist/inverse agonist. Herein, binding of the novel non-imidazole H(3) receptor radioligand [(3)H]A-349821 to membranes expressing native or recombinant H(3) receptors from rat or human sources was characterized and compared with the binding of the agonist [(3)H]N-α-methylhistamine ([(3)H]NαMH). 2. [(3)H]A-349821 bound with high affinity and specificity to an apparent single class of saturable sites and recognized human H(3) receptors with 10-fold higher affinity compared to rat H(3) receptors. [(3)H]A-349821 detected larger populations of receptors compared to [(3)H]NαMH. 3. Displacement of [(3)H]A-349821 binding by H(3) receptor antagonists/inverse agonists was monophasic, suggesting recognition of a single binding site, while that of H(3) receptor agonists was biphasic, suggesting recognition of both high- and low-affinity H(3) receptor sites. 4. pK(i) values of high-affinity binding sites for H(3) receptor competitors utilizing [(3)H]A-349821 were highly correlated with pK(i) values obtained with [(3)H]NαMH, consistent with labelling of H(3) receptors by [(3)H]A-349821. 5. Unlike assays utilizing [(3)H]NαMH, addition of GDP had no effect on saturation parameters measured with [(3)H]A-349821, while displacement of [(3)H]A-349821 binding by the H(3) receptor agonist histamine was sensitive to GDP. 6. In conclusion, [(3)H]A-349821 labels interconvertible high- and low-affinity states of the H(3) receptor, and displays improved selectivity over imidazole-containing H(3) receptor antagonist radioligands. [(3)H]A-349821 competition studies showed significant differences in the proportions and potencies of high- and low-affinity sites across species, providing new information about the fundamental pharmacological nature of H(3) receptors

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field