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

Abstract

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 14CO 2 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.