Immunohistochemical localization of cyclic AMP-responsive element binding protein (CREB)-binding protein in the pig retina during postnatal development / 대한해부학회지

This study evaluated the cellular localization of cyclic AMP-responsive element binding protein-binding protein (CBP) expression in pig retinas during postnatal development. Immunohistochemistry and Western blot analysis were performed on retinal tissue from 2-day-old, 5-week-old, and 6-month-old pigs. Western blot analysis detected the expression of CBP in the retinas of 2-day-old piglets and showed that it was significantly decreased in the retinas of 5-week-old and 6-month-old pigs. Immunohistochemically, CBP was intensely immunostained in protein kinase C alpha (PKCalpha)-positive-bipolar cells, glutamine synthetase-positive Muller cells, and in ganglion cells in 2-day-old piglets. CBP was detected weakly in the inner plexiform, outer nuclear, and rod and cone layers. CBP immunoreactivity in the ganglion cell layer was decreased in the retinas of 5-week-old and 6-month-old pigs, while clear CBP expression detected in the neurite of PKCalpha-positive bipolar cells in the inner nuclear layer. In addition, CBP immunoreactivity in Muller cells and glial fibrillary acidic protein-positive glial processes was particularly noteworthy in pig retinas, but not in rat retinas. The results indicate that CBP is expressed differentially in the retinal neurons and glial cells according to growth and animal species, and may play an important role in homeostasis in Muller cells, neurite extention in bipolar cells, and signal transduction in photoreceptor cells in the porcine retina.

Effects of sleep deprivation on learning and memory and pCREB level in hippocampus of mice / 中华行为医学与脑科学杂志

Objective To observe the effects of sleep deprivation(SD)on learning and memory and phos-phorylated cyclic AMP responsive element binding protein(pCREB) expression in hippocampus of mice,and to explore the mechanism of cognitive change after SD. Methods Twenty female C57BL/6J mice were randomly divided into sleep deprivation group(SD, n = 10) and normal cage control group (CC,n = 10). Touch method was used to establish the sleep deprivation model. 30 days after SD,all the animals were subjected for Morris Water Maze (MWM) to test mean escape latency and percentage of time spent in the target quadrant. pCREB level in hippocampus was tested with Western blot. Results The mean escape latency in SD group in the second and third day of MWM was (29.31 ±4.93) s and (25.33 ±5.06)s, respectively, and was longer than that in CC group ((26.05 ±5.96)s and (19.35 ±7. 85)s,respectively). Mice in SD group spent less time in the target quadrant than that in CC group((23.61 ±9.86)% and (37.46 ±7. 51)%,.respectively, P<0.05). Results of Western blot for pCREB revealed that the pCREB level in hippocampus in sleep deprivation group was significantly lower than that in control group(0.71 ±0.03 and 0.82 ±0.06, respectively, P<0.01) . Conclusion The impairment of spatial learning and memory ability in sleep deprivation animals may be associated with the reduction of pCREB in hippocampus.

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.