Investigating the effects of D-alanine on pituitary intracellular signalling: a comparison with D-serine

Thesis

The D-amino acid, D-alanine, is a co-agonist of the N-methyl-D-Aspartate receptor (NMDAR), and an agonist of the glycine receptor (GlyR). In the rat, D-alanine is highly concentrated in the pituitary gland, where it is localized to adrenocorticotropic hormone (ACTH) containing cells. This suggests that D-alanine may influence the release of ACTH, and ultimately the secretion of the stress hormone, corticosterone. Previous studies have shown that ACTH secretion from pituitary cells involves CREB and ERK1/2 signalling. We have, therefore, investigated the influence of D-alanine on the levels of these signalling proteins (western blot) and ACTH (ELISA) in cultures of the AtT-20 mouse pituitary tumour cell-line. For comparison, the effect of D-serine, a co-agonist of central NMDARs, but not GlyRs, was also examined.

AtT-20 cells were incubated with D-alanine or D-serine, CRH, dexamethasone (DEX) and MK-801 (NMDAR antagonist) in different combinations. Data was expressed as ratios of total/phosphorylated protein, where tCREB is CREB/β-actin and pERK1/2 is pERK/ERK, and subjected to one-way ANOVA statistics.

Compared to controls, the levels of tCREB and ACTH expressed by AtT20 cells were significantly greater when incubated with 500 µM of D-alanine (+35%, p=0.011 and +12%, p=0.028 respectively) or D-serine (+29%, p= 0.028 and +13%, p=0.043 respectively) for 24 hours. The level of tCREB and ACTH in AtT20 cells was significantly lower when incubated with D-alanine and DEX for 24 hours (-30%, p= 0.019 and -44%, p=0.030 respectively), but significantly greater for pERK/ERK and ACTH in cells treated with D-serine and DEX for 24 hours compared to controls (+38%, p = 0.039 and +16%, p=0.038 respectively).

The current data show that D-alanine and D-serine differentially modulate stress hormone-mediated negative feedback signalling in AtT-20 cells. Based on our findings, the changes suggest that D-alanine might augment the inhibition of ACTH, whereas Dserine may attenuate negative feedback, and release ACTH. The elevation of both ERK (with DEX and D-serine), and CREB (with both D-amino acids alone) implies NMDAR activation. The combination of DEX and D-alanine, suggests the possible involvement of GlyRs.

Authors: Naidoo, K

• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: D-alanine; D-serine; CREB; ACTH; ERK
• Subjects: neurosciences