The cytochemical localization of adenylate cyclase: fact or artifact?

In a study of the location of adenylate cyclase activity in rat pancreas with the method of Reik et al. (Science 168:382, 1970), as modified by Howell and Whitfield (J Histochem Cytochem 20:873, 1972) it was found that (a) unspecific staining occurs in rat pancreatic tissue fragments incubated in the Reik-Howell medium in the absence of substrate; (b) addition of adenylyl-imidodiphosphate (AMP-PNP) as substrate, either alone or together with stimulants of rat pancreas adenylate cyclase (secretin. NaF), does not result in increased precipitation; (c) cytochemical incubation of isolated rat pancreatic acinar cells and of rat liver and kidney fragments does not lead to substrate-specific precipitation. In subsequent chemical studies we have found that cyclic adenosine monophosphate (AMP) formation from [alpha32P]AMP-PNP in the presence of rat pancreatic particulate matter is very low in the Reik-Howell medium without lead ions, but is stimulated by addition of lead nitrate (4 mM). Whereas heat-treatment of the particulate matter abolishes all cyclic AMP formation in the absence of lead ions, it actually increases cyclic AMP production in the presence of 4 mM lead nitrate. This indicates that the cyclic AMP formation in the complet Reik-Howell medium occurs by a nonenzymatic mechanism. In addition, this medium shows a tendency to become turbid, particularly when calcium ions are added to the medium, suggesting a possible explanation for the apparently specific cytochemical detection observed by other authors. A revised cytochemical medium, with barium replacing lead and with a pH of 8.9 (optimal for adenylate cyclase with AMP-PNP substrate), leaves rat pancreatic adenylate cyclase activity intact and hormone sensitive, while it is still able to precipitate imidodiphosphate. However, cytochemical incubation of isolated rat pancreatic acinar cells in this revised medium in the presence of AMP-PNP and secretin does not yield an electron-dense precipitate, showing that the enzyme activity is to low to produce sufficient imidodiphosphate. These findings throw further doubt on the validity of the cytochemical detection of adenylate cyclase, reported by other investigators, notwithstanding the alleged positive results.