Statistical Analysis Each and every experiment was repeated no less than in two independent transfections and the information are shown as imply ? SD. The statistical variations were tested using employing one-way ANOVA followed by Bonferonni test, p < 0.05 being considered significantly different. The Kd values for ?2C-AR and ?2B-AR at 37?C and at 30?C were calculated using Graph Pad Software and nonlinear regression for best fit to a one-site binding model. 3. Results 3.1. The effects of low-temperature on the ?2C-AR plasma membrane levels Previously it has been shown that the functional TH-302 kinase inhibitor responses to ?2C-AR stimulation are enhanced at low-temperature and that the receptor accumulates intracellularly at 37?C . However, the mechanisms underlying the particular receptor trafficking remain poorly characterized. To fill this gap, in the present study the plasma membrane ?2C-AR levels in transfected cell lines were determined by radioligand binding in intact cells. As different ?2C-AR localization were noted on in fibroblasts and neuro-endocrine cells , the effects of low-temperature were evaluated in a variety of cell lines . Exposure to 30?C significantly enhanced the ?2C-AR plasma membrane levels in all cell lines with fibroblast phenotype in time-dependent manner .
In six such cell lines, a significant improve in cell surface PD0332991 selleck receptor levels was observed soon after 6 hours, however the maximal impact was observed after 18 h exposure at 30?C . In contrast, exposure to low-temperature had no effect on the receptor levels within the neuro-endocrine cell line, PC12 .
The largest enhance of ?2C-AR plasma membrane levels at 30?C was discovered in HEK293T cells , and this cell line was chosen to further study the mechanisms involved in the regulation of receptor trafficking by low-temperature. Subsequent, the temperature ranges stimulating the ?2C-AR trafficking for the plasma membrane had been determined. Since long-term exposure at temperatures lower than 25?C induces irreversible changes in the cytoskeletal structures , the present study was limited to study the effects of temperatures above 28?C. The maximal raise inside the cell surface receptor levels was located at 30?C . As exposure to low-temperatures within the range of 28?32?C is regularly used to enhance the plasma membrane expression of misfolded proteins , the effects of low-temperature were also assessed on the closest ?2C-AR homologue, ?2B-AR. Though these two receptors share more than 80% homology, exposure to low-temperature had no impact on the ?2B-AR plasma membrane levels, . In contrast, substantial augmentation of the ?2C-AR cell surface levels was discovered in cells exposed to 30?C . Comparable results had been obtained within the purified isolated plasma membrane fraction . These increases cannot be explained by changes of your affinity with the ligand for the receptor, since equivalent Kd values have been calculated at 37?C and 30?C by the two various solutions .