Characterization of high affinity neurotensin receptor NTR1 in HL-60 cells and its down regulation during granulocytic differentiation.

Abstract

We investigated responses to neurotensin in human promyelocytic leukaemia HL-60 cells.

Neurotensin increased the cytosolic calcium concentration ([Ca2+]i) in a concentration-dependent manner and also produced inositol 1,4,5-trisphosphate (InsP3).

Among the tested neurotensin analogues, neurotensin 8–13, neuromedin-N, and xenopsin also increased [Ca2+]i, whereas neurotensin 1–11 and neurotensin 1–8 did not elicit detectable responses.

SR48692, an antagonist of NTR1 neurotensin receptors, blocked the neurotensin-induced [Ca2+]i increase, whereas levocabastine, which is known as an NTR2 neurotensin receptor antagonist, did not attenuate the neurotensin-evoked effect.

The expression of NTR1 neurotensin receptors was confirmed by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT–PCR).

During 1.25% dimethylsulfoxide (DMSO)-triggered granulocytic differentiation of HL-60 cells, the neurotensin-induced [Ca2+]i rise became gradually smaller and completely disappeared 4 days after treatment with DMSO. The mRNA level for neurotensin receptors was also decreased after differentiation.

The results show that HL-60 cells express NTR1 neurotensin receptors and suggest that granulocytic differentiation involves transcriptional regulation of the receptors resulting in down-regulation of the neurotensin-induced signalling.