Simultaneous measurement of tyrosine hydroxylase activity and phosphorylation in bovine adrenal chromaffin cells

A method for simultaneous measurement of tyrosine hydroxylase (TH) activation and phosphorylation in permeabilised and intact bovine adrenal chromaffin cells (BACCs) was established. Permeabilised cells were stimulated with cyclic AMP (1-10 μM) in the presence of [³²P]ATP and L-[carboxyl- ¹⁴C]tyrosine. Intact BACCs were preincubated with ³²P(i) for 3 h and stimulated with forskolin (1-5 μM) in the presence of L-[carboxyl- ¹⁴C]tyrosine. On stimulation each well was covered with a sealed 'chimney' fitted with a small plastic cup containing 300 μl of 1.0 M NaOH that trapped the ¹⁴CO₂ released. TH activity was determined by measuring ¹⁴C radioactivity. TH phosphorylation was measured in the same cells by separating the solubilized proteins on SDS PAGE followed by autoradiography and/or HPLC analysis. It was found that H89, a protein kinase A inhibitor, significantly blocked both TH phosphorylation and activation in response to cyclic AMP in permeabilised cells. However, in intact cells, H89 was effective only in respect to forskolin-stimulated TH activity and did not block the forskolin-stimulated TH phosphorylation of Ser-40. The reason(s) for this lack of correlation between TH activation and phosphorylation is presently not understood.