Functional, endogenously expressed corticotropin‐releasing factor receptor type 1 (CRF1) and CRF1receptor mRNA expression in human neuroblastoma SH‐SY5Y cells

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Abstract
Abstract—Corticotropin‐releasing factor (CRF) is a hypothalamic 41‐amino acid peptide which stimulates corticotropin (ACTH) release from the anterior pituitary and is also involved in the body response to stress. CRF, receptors represent a potential target for novel antidepressant/anxiolytic drugs. The aim of the present study was to search for a human cell line expressing native, functional CRF, receptors as a starting material for screening purposes. We identified CRF, receptors functionally coupled to cAMP formation in human neuroblastoma SH‐SY5Y cells. CRF induced concentration‐dependent increases in cAMP accumulation in SH‐SY5Y cells (maximal increase 6.9 ± 0.9 fold over basal values,n= 14). This effect was mimicked by related peptides with similar potencies: (mean pEC50value) human/rat CRF (8.63), rat urocortin (9.32), sauvagine (8.97), urotensin I (8.93), ovine CRF (8.81). The efficacies of these agonists were nearly the same, with the exception of ovine CRF which was slightly less efficacious (75% the Emaxof CRF). The responses to CRF were competitively antagonised by the following peptide fragments (mean pKBvalue): α‐helical‐CRF (9–41) (7.54), [D‐Phe12, Nle21,38, CαMeLeu37]CRF (12–41) (8.36) and [D‐Tyr12]astressin (9.49) and by the selective, non‐peptidic CRF, receptor antagonists, CP‐154,526 (7.76) and antalarmin (9.19). Estimation of receptor density by [125I]Tyr0‐ovine CRF saturation binding yielded a modest number of binding sites (Bmax12 fmol/mg protein, KD0.2 nM). Analysis of mRNA by reverse transcription‐polymerase chain reaction clearly revealed the presence of mRNA for CRF1receptors in SH‐SY5Y cells. A slight signal for CRF2receptor mRNA was also observed. We conclude that neuroblastoma SH‐SY5Y cells are endowed with native CRF1receptors positively coupled to cAMP formation. They therefore constitute a useful functional model for the search of CRF1selective compounds with potential anxiolytic/antidepressant activity.