GASTROINTESTINAL CALMODULIN-DEPENDENT PROTEIN KINASE II

该项目的广泛的长期目标是表征近端结肠和胃底平滑肌细胞的钙调蛋白依赖性蛋白激酶II。 钙调素依赖性蛋白激酶II是一种多功能的丝氨酸/苏氨酸蛋白激酶，具有10-12个亚基的寡聚体结构。 脑酶由α和β同种型组成，而δ和γ同种型主要在平滑肌中表达。在时相性平滑肌中胞浆Ca 2+水平振荡，但在紧张性平滑肌中保持升高。 钙调素依赖性蛋白激酶II的亚型组成调节其对Ca 2+振荡的反应。 钙调素依赖性蛋白激酶II亚型在胃底和近端结肠平滑肌中表达尚不清楚。 该项目的主要目标是检验不同的钙调蛋白依赖性蛋白激酶II亚型在近端结肠和胃底平滑肌细胞中表达的假设。从胃底或近端结肠平滑肌细胞的纯化mRNA产生的第一链cDNA将用于PCR反应以扩增钙调蛋白依赖性蛋白激酶II cDNA。将使用已知同种型的核苷酸序列设计同种型特异性寡核苷酸。将使用通过杆状病毒表达获得的全酶[和从培养的胃底和近端结肠平滑肌细胞纯化的内源性全酶]研究结肠和胃底平滑肌细胞钙调素依赖性蛋白激酶II的酶特性。 将使用体外激酶测定来确定Ca 2 +/钙调蛋白非依赖性活性水平的生成动力学。钙调素依赖性蛋白激酶II在将Ca 2+信号转导到生理通道中起着关键作用。 本研究将鉴定和阐明钙调素依赖性蛋白激酶II在胃底和近端结肠平滑肌中表达的酶学特征。 这些发现将进一步我们的钙调素依赖性蛋白激酶II在调节紧张性和阶段性胃肠平滑肌细胞的钙依赖行为的作用的理解。

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.