Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.
In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism; D002923:Cilia / Q000187:drug effects / Q000201:enzymology*; D004533:Egtazic Acid / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010247:Paramecium / Q000187:drug effects / Q000201:enzymology*
D002147:Calmodulin; D004533:Egtazic Acid; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D008274:Magnesium; D002118:Calcium