Expression of adenylyl cyclase isoforms in neutrophils.
In the present study, we have identified the expression of adenylyl cyclase (AC) isoforms in rat neutrophils according to the mRNA analysis and the distinct mode of regulation of isoform activity. Agarose gel electrophoresis of reverse transcription-polymerase chain reaction (RT-PCR)-amplified products resulted in a single band of the expected size for each product with nucleotide sequences corresponding to AC1 to AC9. AC1 was abundant, while AC2, 6 and 9 were of moderate expression among the AC isoforms in neutrophils based on the quantitative real-time RT-PCR analysis. Exposure of neutrophils to Ca(2+) ionophore A23187, isoproterenol and forskolin stimulated cellular cyclic AMP accumulation. EDTA and the calmodulin (CaM) antagonist, trifluoperazine, prevented the A23187-induced response. Pretreatment with pertussis toxin (PTX) inhibited the alpha(2)-adrenergic agonist, UK14304-induced cellular cyclic AMP elevation. In addition, UK14304 augmented the cyclic AMP elevation when cells were stimulated by isoproterenol. Phorbol 12-myristate 13-acetate (PMA) attenuated the augmentation response of UK14304 and isoproterenol. Treatment of the membrane preparations from rat neutrophils with Ca(2+)/CaM, forskolin, isoproterenol, GTPgammaS or Gbetagamma all increased cyclic AMP production. The addition of protein kinase C (PKC) catalytic fragment and Gbetagamma augmented the Ca(2+)/CaM- and isoproterenol-stimulated AC activity, respectively. However, forskolin and the activated protein kinase A (PKA) attenuated the GTPgammaS- and isoproterenol-stimulated AC activity, respectively. KT5720, a PKA inhibitor, reversed the inhibition by PKA. Taken together, these data suggest the presence of four groups of AC isoforms in rat neutrophils.
Chang
Ling-Chu
LC
Department of Education and Research, Taichung Veterans General Hospital, 160, Chung Kang Road, Sec. 3, 407, Taichung, Taiwan, ROC.
Wang
Chung-Jieh
CJ
Lin
Yi-Lee
YL
Wang
Jih-Pyang
JP
D000262:Adenylyl Cyclases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007527:Isoenzymes; D012333:RNA, Messenger; D000262:Adenylyl Cyclases