Endocrine Abstracts

Endokinin B (EKB) is a recently identified tachykinin whose role is not yet fully understood. EKB displays equivalent affinity for the three tachykinin receptors as substance P (SP) which it is often found co-expressed with. SP has been implicated in a variety of neuroendocrinological responses including stress regulation, and affective and anxiety-related behaviours. An important aspect, therefore, is to dissect a distinct role for EKB from that of SP. Here, we have attempted to elucidate whether EKB and SP stimulate divergent signalling pathways in an astrocytoma cell line U251MG, which has high endogenous levels of the tachykinin NK1 receptor. We have investigated stimulation of PKCβII-dependent ERK1/2 signalling using western blotting and confocal microscopy following tachykinin receptor activation. Both SP and EKB phosphorylated (activated) PKCβII through the NK1 receptor with subsequent phosphorylation of ERK1/2 occurring via c-Raf. ERK1/2 was maximally phosphorylated after 2.5 min with SP and after 5 min with EKB; the minimum dose of agonist required for ERK1/2 phosphorylation was 10 nM SP and 100 nM EKB. ERK1/2-dependent p90RSK phosphorylation was also detected. Confocal fluorescence microscopy demonstrated ERK1/2 to migrate to the nucleus after challenge with both SP and EKB. We conclude that EKB stimulates similar PKCβII-ERK1/2 signalling to that of SP following NK1 receptor activation, both being blocked by an NK1 antagonist. However phosphorylation as a result of EKB challenge was not as immediate or robust as SP and a 10-fold greater dose of EKB was required. Migration of phosphorylated ERK1/2 to the nucleus suggests that both SP and EKB can initiate gene transcription. Antagonists developed for attenuating SP/EKB mediated transcription through PKCβII-ERK1/2 signalling could be of use to target events associated with stress and behavioural responses.