mHypoA-2/10 cells express β₂-AR and β₃-AR. In (A) reads per kilobase of transcript per million reads mapped values for α₁-, α₂- and β-AR are shown as the mean ± S.D. RNAs from six distinct cell pools were used. In (B), aequorin expressing cells were stimulated with the indicated ligand by automatic injection at time point 5 second. In (C) saturation-binding experiments with total membrane fractions (20 µg) are shown. ¹²⁵I-CYP (3.125–400 pM) was either used alone (total binding) or together with 10 µM propranolol (unspecific binding). Receptor-bound ¹²⁵I-CYP was calculated by subtracting unspecific from total binding. Results of 3 independent experiments performed in triplicates are expressed as the mean ± S.D. Data were fitted using a nonlinear fit (one-site specific binding). In (D to E) data using cells stably expressing the CREB-dependent luciferase reporter are shown. In (D) serum-starved cells were stimulated or not for 4 hours at 37°C with either NE (10 µM), ISO (10 µM), or Sal (100 nM). Cells were then lysed, luciferase activity determined, and x-fold over basal calculated. Data of 5 independent experiments performed in quadruplicates are expressed as the mean ± S.D. In (E), cells were preincubated for 30 minutes with 10 µM of the PKC inhibitors BIM-X or (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), the PKA inhibitor KT-5720, or the exchange factor directly activated by cAMP inhibitor ESI-09. In (F), serum-starved cells were stimulated for 4 hours at 37°C with either NE (10 µM) alone or together with increasing concentrations of ICI118551, SR59230A, or phentholamine. Cells were then lysed, luciferase activity determined, and data obtained with NE alone set to 100%. All other signals were calculated as percentage. Data of 6 independent experiments performed in triplicates were expressed as the mean ± S.D. and analyzed by a nonlinear fit (two-site fit logIC₅₀).

Competition binding with membranes from mHypoA-2/10 cells at distinct temperatures. Competition-binding experiments with total membrane fractions (20 µg) and ¹²⁵I-CYP (75 pM) performed for 1 hour at 36°C (blue symbols) or at 38°C (red symbols) are shown. In (A) increasing concentrations of NE and in (B–D) of Sal were used to detect specific binding. The total ¹²⁵I-CYP binding in the absence of any agonist was set to 100%, and all other values calculated as percentage. In (C) and (D), membranes were preincubated or not with 10 µM of Gpp(NH)p. Data of 3 (A and B) or 4 (C and D) independent experiments performed in triplicates were expressed as the mean ± S.D and fitted using a nonlinear fit (one-site logIC₅₀ in A or two-site fit logIC₅₀ in B to D). Parameters of the binding curves are given in Table 1.

NE- or Sal-induced cAMP accumulation is enhanced at 36°C. cAMP accumulation was determined for 1 hour with 1 mM IBMX alone or with NE, FSK (both 10 µM), or Sal (100 nM) in A to D. In (E) increasing concentrations of NE were used. Blue bars present data obtained at 36°C, red bars data at 38°C, and black bars at 37°C. In (A and E) data are presented as the ratio cAMP/cAMP + ATP, in (B) as x-fold over basal in (C) data obtained at 38°C and in (D) at 37°C were set to 100%. Data of 7 (A–C), 3 (D), or 4 (E) independent experiments performed in triplicates were expressed as the mean ± S.D. Data shown in (A–C) are the same. Two-way ANOVA followed by Ṧidák’s post hoc test was used to determine statistical differences. In (E), data were fitted using a nonlinear fit (log_agonist versus response variable slope; four parameters)

NE-induced cytosolic cAMP accumulation and efflux. In (A), cytosolic cAMP accumulation for 10, 20, 40, or 60 minutes with 1 mM IBMX alone or with 10 µM NE was detected at 37°C. In (B), cAMP levels in the supernatants of the same samples are shown. In (C), cAMP levels in the supernatants of cells stimulated with 1 mM IBMX and 10 µM NE are shown in the presence or not of 100 µM indomethacin. In (D), data from (A) and (B) were compiled. Data of 4 independent experiments performed in triplicates were expressed as the mean ± S.D. One-way ANOVA followed by Tukey’s post hoc test was used in (A, B, and D) and unpaired t test in (C) to determine statistical differences.

NE-induced cytosolic cAMP accumulation and efflux are temperature-sensitive. Cytosolic cAMP accumulation (A) and efflux (B) induced by 10 µM NE in the presence of 1 mM IBMX was measured for 20 and 60 minutes at 36 or 38°C. In (C), rEC-cAMP at 20 or 60 minutes were calculated. In (D), data from (A) and (B) were compiled. Blue bars present data obtained at 36°C, red bars data at 38°C. Data of 4 independent experiments performed in triplicates were expressed as the mean ± S.D. Two-way ANOVA followed by Ṧidák’s post hoc test (A–C) or Tukey’s post hoc test (D) was used to determine statistical differences.

NE-induced ³H-cAMP degradation is enhanced at 38°C. In (A), ³H-cAMP degradation in homogenates from unstimulated cells was measured for 10, 20, 40, or 60 minutes with or without 1 mM IBMX at 37°C. As a control (input), the same amount of ³H-cAMP was incubated without homogenates and also purified. No statistical differences between homogenates with IBMX and the input control were observed (data not shown). In (B and C), ³H-cAMP degradation in homogenates of cells treated with 10 µM NE for 30 minutes at 37°C without IBMX was measured for 20 minutes at 36 or 38°C. Blue bars present data obtained at 36°C, red bars data at 38°C. In (B), raw data are shown as ³H-cAMP in decays per minute. In (C), ³H-cAMP degradation was calculated as percentage of the corresponding input control. 7 independent experiments performed in triplicates were expressed as the mean ± S.D. Two-way ANOVA followed by Ṧidák’s post hoc test was used in (B) and two-tailed t test in (C) to determine statistical differences.

Basal and NE-induced thyreoliberin promoter activity is increased at 36°C. Thyreoliberin promoter activity was monitored using mHypoA-2/10 cells stably expressing a luciferase promoter construct encoding the rat thyreoliberin promoter. Basal and NE (10 µM)-induced promoter activity was measured for 1, 2, 3, and 4 hours at 36°C (blue bars) or 38°C (red bars). In (A), basal reporter activity is given in RLU. In (B), x-fold over basal values of NE-stimulated cells are provided. Data of 4 independent experiments performed in triplicates were expressed as the mean ± S.D. Two-way ANOVA followed by Ṧidák’s post hoc test was used to determine statistical differences.

BAY60-6583 but not BK-induced CREB activation is enhanced at 36°C. In (A, B, and D), data using cells stably expressing the CREB-dependent luciferase reporter are shown. In (C), ligand-induced cytosolic cAMP accumulation is given as x-fold of basal after stimulation of cells with BAY60-6583 or BK (both 1 µM) at 37°C for 30 minutes. Data of 3 independent experiments performed in triplicates are compiled as the mean ± SD and two-tailed t test performed to determine statistical differences. In (A), CREB reporter–expressing mHypoA-2/10 cells were stimulated for 4 hours at 37°C with BAY60-6583 or BK (both 1 µM). Data of 3 independent experiments performed in triplicates are compiled as the mean ± SD and two-tailed t test performed todetermine statistical differences. In (C), cells were preincubated with the PKC inhibitors BIM-X or Gö6983 (both 10 µM) and the stimulated with BK (1 µM) for 4 hours at 37°C. Data of 3 independent experiments performed in triplicates are compiled as the mean ± SD, and one-way ANOVA followed by Ṧidák’s post hoc test used to determine statistical differences. In (D), cells were either incubated for 4 hours at 36 or 38°C and then for additional 4 hours stimulated with BAY60-6583 or BK (both 1 µM). Bars in red indicate data obtained at 38°C, bars in blue at 36°C. Data obtained at 38°C were set to 100%. Two-tailed t test was performed to determine statistical differences.

NE- or Sal-induced cAMP accumulation is enhanced at 36°C in various cell lines. cAMP accumulation was measured after stimulation of cells with NE (10 µM) at 37°C (A) or at 36 and 38°C with NE (10 µM) or Sal (100 nM) in (B–E). In (A), data of 5 independent experiments performed in quadruplicates are compiled as the mean ± SD. In (B), mHypoA-2/12 cells, in (C), H1299 cells, in (D), primary HBSMCs, and in (E), data of HaCaT cells are shown. Bars in red indicate data obtained at 38°C, bars in blue at 36°C. Data obtained at 38°C were set to 100%. Two-way ANOVA followed by Ṧidak‘s post hoc test was used to determine statistical differences.