Geneeskundige Stichting Koningin Elisabeth ... - GSKE - FMRE

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β-endorphine (β-end) and β-lipotropin (β-LPH). The aim of the present project is to study the
effects of different stress types (processive vs. systemic and acute vs. chronic stressors) on the
POMC-processing in the chicken.
The goal of the present study was to develop a competitive PCR assay to measure changes
in the expression of pro-opiomelanocortin (POMC) mRNA in myelomonocytic HD11 cells after
Salmonella typhimurium lipopolysaccharide (LPS) stimulation. HD11 cells are chicken myelomonocytic
cells (macrophages) transformed by the v-myc-encoding MC29 virus. Pro-opiomelanocortin
mRNA could be detected in HD11 cells by reverse transcriptase – polymerase chain reaction
(RT-PCR). To our knowledge, this is the first observation of a POMC RNA transcript in avian
macrophages. Based on this observation, the effect of stimulation with bacterial LPS on the
POMC expression in HD11 cells was investigated by means of a newly developed competitive
PCR assay. For this purpose, the HD11 cells received a one-hour LPS challenge with LPS concentrations
ranging from 0 to 100 ng/ml. A PCR MIMIC (consisting of a heterologous DNA
fragment flanked by templates for the gene-specific primers) was used as an internal control in
the competitive PCR. A ten-fold dilution series of the MIMIC was co-amplified with a constant
amount of experimental cDNA. While HD11 POMC mRNA expression showed an increase of
one order of magnitude following treatment with 100 ng/ml LPS as compared to untreated controls,
no significant differences could be observed after treatment with 50 and 10 ng/ml LPS.
Quantitative measurement of POMC mRNA levels is a first step towards a better understanding
of the physiological role of non-hypophysial POMC-derived peptides in the response to immune
stress in birds.
PACAP and the PACAP Type I receptor in chicken brain
To map in detail the brain areas in which pituitary adenylate cyclase-activating polypepide
(PACAP) may play a significant role in birds, the distribution of PACAP and PACAP type I
receptor (PAC1-R) mRNA was examined throughout the entire chicken brain by using in situ
hybridization histochemistry. Widespread distribution of both PACAP and its receptor mRNA
were found. The telencephalic areas where the most intense signals for PACAP mRNA were
found included the hyperstriatum accessorium, the hippocampus, and the archistriatum. In the
diencephalon, a group of neurons that highly expressed PACAP mRNA was observed from the
anterior medial hypothalamic nucleus to the inferior hypothalamic nucleus. Moderate expression
was found in the paraventricular nucleus and the preoptic region. A second large group
of neurons containing PACAP message was found within the nucleus dorsolateralis anterior thalami
and extended caudally to the area around the nucleus ovoidalis and the nucleus paramedianus
internus thalami. Furthermore, expression of PACAP message was observed within the
bed nucleus of the pallial commissure, nucleus spiriformis medialis, optic tectum, cerebellar cortex,
olfactory bulbs, and several nuclei within the brainstem (dorsal vagal and parabrachial
complex, reticular formation). The highest expression of PAC1-R mRNA was found in the dorsal
telencephalon, olfactory bulbs, lateral septum, optc tectum, cerebellum, and throughout the
hypothalamus and the thalamus. The presence of PACAP and PAC1-R mRNA in a variety of
brain areas in birds suggests that PACAP mediates several physiologically important processes
in addition to regulating activity of the pituitary gland.