Expression of adenylate cyclase subtypes II and IV in the human outflow pathway

PURPOSE. It has been demonstrated that low doses of pilocarpine and other m uscarinics substantially increase outflow facility in the isolated human ou tflow system devoid of ciliary muscle. These cholinergic-induced facility r esponses were thought possibly to be due to elevation of cAMP as a result o f the presence of adenylate cyclases II (AC-II) and IV (AC-TV). Therefore, whether these isoforms are present in outflow tissues was examined. METHODS. Human anterior segments were perfused with carbachol (10(-9)-10(-5 ) M), and outflow facility and cAMP levels in the perfusate were measured s imultaneously. Isolated trabecular meshwork (TM) were incubated with carbac hol (10(-7) M), and the subsequent changes in cAMP were measured by radioim munoassay. AC-II and AC-IV were characterized in ocular tissue with reverse transcription-polymerase chain reaction and in situ hybridization. RESULTS. Outflow facility increased, in a dose-dependent manner, by 10%, 16 %, and 27% in response to 10(-9), 10(-7); and 10(-5) M carbachol, respectiv ely. Similarly, cAMP increased by 9%, 70%, and 210% in response to 10(-9), 10(-7), and 10(-5) M carbachol, respectively. In addition, cAMP levels sign ificantly increased by 39% in isolated TM strips incubated with 10(-7) M ca rbachol. AC-II was detected in most normal tissue examined, but not in any cultured cell lines or any glaucomatous tissue. AC-IV was also widely expre ssed in most normal tissues, faintly detected in some glaucoma tissue, but not detected in most cultured cells. CONCLUSIONS. The presence of AC-II and AC-IV in outflow tissues supports th e hypothesis that cholinergics may indeed exert an effect on outflow facili ty, mediated by cAMP which is independent of muscle contraction.