Guanylate cyclase occurs in two forms, one cytosolic and the other bound to the cell membrane. These two forms respond to different stimuli. The stimulus for the cytosolic form is nitric oxide formed from cytosolic nitric oxide synthase.' Consequently the histochemical demonstration of these enzymes requires procedures for retaining soluble cytosolic enzymes during the histochemical reactions.
GUANYLATE CYCLASE: HISTOCHEMISTRY
With slight modifications, the same histochemical method as has been used for demonstrating adenosine triphosphatase activity has been used by for localizing guanylate cyclase activity. Some have used such a method for studying it in bone. Generally the tissue has been perfused briefly in vivo (e.g. 5-8 min) with paraformaldehyde (or with formaldehyde-gluteraldehyde (e.g. 1 percent: 2 percent) or glutaraldehyde alone (1 percent for 5min)4. Thick slices of tissue (e.g. 40-8Opm) were subjected to the reaction medium containing either GTP or guanylyl imidodiphosphate as substrate, with lead nitrate or citrate to precipitate any liberated phosphate. This has then been converted to the coloured or black sulphide. Inhibitors of non-specific alkaline phosphatases and of phosphodiesterases were included in the r?ilwuon medium as well as manganese ions, theophylline and occasionally sodium aide; in all studies, either sucrose or glucose was included in the reaction-medium. In different studies the pH of the reaction medium varied from pH 7-42 to pH 8.5.4 various groups of workers (e.g. references "I3 I?