Ultracytochemical localization of adenylate cyclase activity in rat thymocytes

Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5′-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO4 as an activator, 5 mM theophylline as an inhibitor of 3′,5′-AMP-phosphodiesterase and 2 mM lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10−4 M adrenalin in the presence of 5′-guanylylimido-diphosphate (GMP-PNP) as well as with 10−2 M NaF. In the cells incubated in a medium devoid of theophylline and containing 5′-AMP instead of AMP-PNP, 5′-nucleotidase activity was observed in the same cell structures as AC activity. Hydrolysis of 5′-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5′-AMP in all cell structures. No staining was observed with 2 mM β-glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5′-AMP or p-nitrophenyl phosphate, but not β-glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 30 nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.