Schonhoff Christopher M, Gillin Henry, Webster Cynthia R L, Anwer M Sawkat
Department of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, MA 01536, USA.
Hepatology. 2008 Apr;47(4):1309-16. doi: 10.1002/hep.22162.
Cyclic adenosine monophosphate (cAMP) stimulates translocation of Na(+)-taurocholate (TC) cotransporting polypeptide (Ntcp) and multidrug resistant associated protein 2 (Mrp2) to the plasma membrane. Because cAMP activates phosphoinositide-3-kinase (PI3K) and protein kinase C (PKC) activation is PI3K-dependent, the aim of the current study was to determine whether cAMP activates conventional and novel PKCs in hepatocytes and whether such activation plays a role in cAMP-stimulated Ntcp and Mrp2 translocation. The effect of cAMP on PKCs, TC uptake, and Ntcp and Mrp2 translocation was studied in isolated rat hepatocytes using a cell-permeable cAMP analog, CPT-cAMP. The activity of PKCs was assessed from membrane translocation of individual PKCs, and phospho-specific antibodies were used to determine PKCdelta phosphorylation. TC uptake was determined from time-dependent uptake of (14)C-TC, and a cell surface biotinylation method was used to determine Ntcp and Mrp2 translocation. CPT-cAMP stimulated nPKCdelta but not cPKCalpha or nPKCepsilon, and induced PI3K-dependent phosphorylation of nPKCdelta at Thr(505). Rottlerin, an inhibitor of nPKCdelta, inhibited cAMP-induced nPKCdelta translocation, TC uptake, and Ntcp and Mrp2 translocation. Bistratene A, an activator of nPKCdelta, stimulated nPKCdelta translocation, TC uptake, and Ntcp and Mrp2 translocation. The effects of cAMP and bistratene A on TC uptake and Ntcp and Mrp2 translocation were not additive.
These results suggest that cAMP stimulates Ntcp and Mrp2 translocation, at least in part, by activating nPKCdelta via PI3K-dependent phosphorylation at Thr(505).
环磷酸腺苷（cAMP）可刺激牛磺胆酸钠（TC）共转运多肽（Ntcp）和多药耐药相关蛋白2（Mrp2）向质膜的转位。由于cAMP可激活磷酸肌醇-3-激酶（PI3K），且蛋白激酶C（PKC）的激活依赖于PI3K，因此本研究的目的是确定cAMP是否能激活肝细胞中的传统型和新型PKC，以及这种激活是否在cAMP刺激的Ntcp和Mrp2转位中发挥作用。使用细胞可渗透的cAMP类似物CPT-cAMP，在分离的大鼠肝细胞中研究了cAMP对PKC、TC摄取以及Ntcp和Mrp2转位的影响。通过单个PKC的膜转位评估PKC的活性，并使用磷酸特异性抗体确定PKCδ的磷酸化。通过（14）C-TC的时间依赖性摄取来确定TC摄取，并使用细胞表面生物素化方法来确定Ntcp和Mrp2转位。CPT-cAMP刺激新型PKCδ，但不刺激传统型PKCα或新型PKCε，并诱导PI3K依赖性的新型PKCδ在苏氨酸（505）处的磷酸化。新型PKCδ抑制剂罗特列林抑制了cAMP诱导的新型PKCδ转位、TC摄取以及Ntcp和Mrp2转位。新型PKCδ激活剂双萜烯A刺激了新型PKCδ转位、TC摄取以及Ntcp和Mrp2转位。cAMP和双萜烯A对TC摄取以及Ntcp和Mrp2转位的影响并非相加性的。
这些结果表明，cAMP至少部分地通过PI3K依赖性磷酸化苏氨酸（505）激活新型PKCδ来刺激Ntcp和Mrp2转位。
