GASTROINTESTINAL CALMODULIN-DEPENDENT PROTEIN KINASE II

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The broad long-term objective of this project is to characterize calmodulin-dependent protein kinase II from smooth muscle cells of the proximal colon and fundus. Calmodulin-dependent protein kinase II is a multifunctional Ser/Thr protein kinase having an Oligomeric structure of 10-12 subunits. The brain enzyme is composed of the alpha and beta isoforms, while the delta and gamma isoforms are predominantly expressed in smooth muscle. Cytosolic Ca2+ levels in phasic smooth muscle oscillate, but remain elevated in tonic smooth muscle. The isoform composition of calmodulin-dependent protein kinase II modulates its response to Ca2+ oscillations. The calmodulin-dependent protein kinase II isoforms expressed in fundus and proximal colon smooth muscle are unknown. A major goal of this project is to test the hypothesis that different calmodulin-dependent protein kinase II isoforms are expressed in proximal colon and fundus smooth muscle cells. First strand cDNAs generated from purified mRNA of fundus or proximal colon smooth muscle cells will be used in PCR reactions to amplify calmodulin-dependent protein kinase II cDNAs. Isoform-specific oligonucleotides will be designed using the nucleotide sequences of the known isoforms. The enzymatic characteristics of colonic and fundic smooth muscle cell calmodulin-dependent protein kinase II will be investigated by using holoenzymes obtained by baculovirus expression [and endogenous holoenzymes purified from cultured fundus and proximal colon smooth muscle cells.] The kinetics of generation of Ca2+/calmodulin-independent activity levels will be determined using in vitro kinase assays. Calmodulin-dependent protein kinase II plays a pivotal role in transducing Ca2+ signals into physiological vents. The studies from this project will identify and elucidate the enzymatic characteristics of calmodulin-dependent protein kinase II expressed in fundus and proximal colon smooth muscle. These findings will further our understanding of the role of calmodulin-dependent protein kinase II in regulating the Ca2+- dependent behavior of tonic and phasic gastrointestinal smooth muscle cells.