Abstract
Abstract: :
Purpose:To investigate mRNA expression of the neurotrophins (NTs) nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4) and their receptors (NTRs) TrkA, TrkB, TrkC and p75 in adult rat conjunctiva and to deterrmine the effects of NTs on p42/44 mitogen-activated protein kinase (MAPK) activity. Methods:Total RNA was isolated from conjunctiva and brain (positive control) and used for complementary DNA (cDNA) synthesis by reverse transcription. The cDNA was then amplified by PCR using specific primers for NTs and NTRs. To evaluate the effects of NTs on the activation of MAPK, pieces of conjunctiva were removed, placed in keratinocyte basal medium, and incubated with NTs and cholinergic agonist carbachol (positive control) for varying concentrations or times. After stimulation, conjunctival pieces were sonicated and centrifuged. Proteins in the supernatant were analyzed by Western blot using antibodies specific to phosphorylated (activated) p42/44-MAPK or total p42-MAPK. Immunoreactive bands were quantified, and data were expressed as fold increase over basal. Results:PCR products of the expected sizes for the NTs NGF, BDNF, NT3, NT4 and for the NTRs TrkA, TrkB, TrkC and p75 were detected in the conjunctiva and brain (positive control). No bands were obtained when PCR was performed in the absence of cDNA. NGF, BDNF, and NT3 (all at 100ng/ml) activated MAPK by 2.8, 3.9, and 2.3 fold respectively (n=4). The positive control carbachol (10-4 M) increased MAPK activity 2 fold above basal. Conclusions:We conclude that NT and NTR mRNA are present and that the NTs NGF, BDNF, and NT3 activate MAPK in conjunctival tissue.
Keywords: conjunctiva • growth factors/growth factor receptors • gene/expression