Differences in the signaling pathways of α1A- and α1B-adrenoceptors are related to different endosomal targeting

Aims: To compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. Methods: Using CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). Results and Conclusions: Constitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin

Applications of fluorescence and bioluminescence resonance energy transfer to drug discovery at G protein coupled receptors

The role of G protein coupled receptors (GPCRs) in numerous physiological processes that may be disrupted or modified in disease makes them key targets for the development of new therapeutic medicines. A wide variety of resonance energy transfer (RET) techniques such as fluorescence RET and bioluminescence RET have been developed in recent years to detect protein–protein interactions in living cells. Furthermore, these techniques are now being exploited to screen for novel compounds that activate or block GPCRs and to search for new, previously undiscovered signaling pathways activated by well-known pharmacologically classified drugs. The high resolution that can be achieved with these RET methods means that they are well suited to study both intramolecular conformational changes in response to ligand binding at the receptor level and intermolecular interactions involving protein translocation in subcellular compartments resulting from external stimuli. In this review we highlight the latest advances in these technologies to illustrate general principles

The cholinergic regulation of potassium (86Rb ) permeability in sweat glands isolated from patients with cystic fibrosis

Sweat glands isolated from skin obtained from normal subjects and patients with cystic fibrosis (CF) were pre-loaded with 86Rb+ and superfused with a physiological salt solution and the rate of 86Rb+ efflux was measured as an indicator of cellular potassium permeability. Acetylcholine always evoked a permeability increase in the glands from control subjects and this response could be resolved into calcium-dependent and calcium-independent components. Sweat glands from CF patients did not show such consistent responses. In three individuals the glands were abnormally insensitive to acetylcholine but normal responsiveness was seen in a fourth case. It is proposed that CF can induce dysfunction of calcium-dependent control processes in sweat glands

Blockade of muscarinic acetylcholine receptors facilitates motivated behaviour and rescues a model of antipsychotic-induced amotivation.

Disruptions to motivated behaviour are a highly prevalent and severe symptom in a number of neuropsychiatric and neurodegenerative disorders. Current treatment options for these disorders have little or no effect upon motivational impairments. We assessed the contribution of muscarinic acetylcholine receptors to motivated behaviour in mice, as a novel pharmacological target for motivational impairments. Touchscreen progressive ratio (PR) performance was facilitated by the nonselective muscarinic receptor antagonist scopolamine as well as the more subtype-selective antagonists biperiden (M1) and tropicamide (M4). However, scopolamine and tropicamide also produced increases in non-specific activity levels, whereas biperiden did not. A series of control tests suggests the effects of the mAChR antagonists were sensitive to changes in reward value and not driven by changes in satiety, motor fatigue, appetite or perseveration. Subsequently, a sub-effective dose of biperiden was able to facilitate the effects of amphetamine upon PR performance, suggesting an ability to enhance dopaminergic function. Both biperiden and scopolamine were also able to reverse a haloperidol-induced deficit in PR performance, however only biperiden was able to rescue the deficit in effort-related choice (ERC) performance. Taken together, these data suggest that the M1 mAChR may be a novel target for the pharmacological enhancement of effort exertion and consequent rescue of motivational impairments. Conversely, M4 receptors may inadvertently modulate effort exertion through regulation of general locomotor activity levels