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

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. Keywords: Neurotensin, cytosolic Ca2+, phospholipase C, human hemopoietic cells, differentiation Introduction Neurotensin, the tridecapeptide ELYENKPRRPYIL, has been studied as a modulator in neuronal and non-neuronal systems (Kitabgi et al., 1985; Vincent, 1995). Neurotensin reveals its modulatory effect in almost every region of the nervous system including the cerebral cortex, terminals of striatal nucleus, hypothalamus, midbrain, cerebellar purkinje cells, retina, and spinal cord (Shi & Bunney, 1992a). In the central nervous system neurotensin is known to modulate the signals of dopaminergic neurons by regulating D2 receptors. Therefore, neurotensin displays activities similar to antipsychotics in clinical trials involving schizophrenia, Parkinson's disease, and Alzheimer's disease (Nemeroff et al., 1992). It has been also reported that neurotensin potentiates the barbiturate- and ethanol-induced sedation and that it induces antinociception, catalepsy, and hypothermia. For non-neuronal systems, major roles of neurotensin have been demonstrated in the gastrointestinal and cardiovascular system. Neurotensin is localized at the intestinal mucosa (endocrine N-cells) where it is thought to be involved in intestinal contraction (Buhner & Ehrlein, 1989). Intravenous injection of neurotensin changes the concentration of insulin, glucagon, and pituitary hormones including thyroid releasing hormone (Kitabgi et al., 1985; Schimpff et al., 1995). It also induces vasodilatation and affects vascular permeability by way of vascular, portal vein, and smooth muscle contraction (Bachelard et al., 1992). The neurotensin effect on the cardiovascular system somewhat depends on the species, because neurotensin causes hypotension in dogs but hypertension in guinea pigs. It has been reported that endothelial cells of the umbilical vein also express the neurotensin receptor which is involved in the increase of intracellular Ca2+ (Schaeffer et al., 1995) and the production of prostacyclin (Schaeffer et al., 1997). However, it is not yet known what role neurotensin plays in hemopoietic or immune cells. Neurotensin evokes two major actions at the cellular level. It causes inositol 1,4,5-trisphosphate (InsP3) production and increase in the cytosolic calcium concentration ([Ca2+]i) through the activation of phospholipase C (PLC) (Goedert et al., 1984; Bozou et al., 1989; Hermans et al., 1992). In another signalling pathway, neurotensin modulates the intracellular cyclic nucleotide level including cyclic GMP (Gilbert & Richelson, 1984; Amar et al., 1985) and cyclic AMP (Bozou et al., 1986; Yamada et al., 1994). HL-60 cells have served as a good model in which to study signal transduction of various pharmacological receptors before and after differentiation of the hemopoietic cells (Klinker et al., 1996). HL-60 cells can be forced to terminally differentiate into neutrophil-like cells by dibutyryl cyclic AMP, DMSO, or retinoic acid treatment and into adherent macrophage-like cells by 1, 25-dihydroxy vitamin D3 and phorbol esters (Klinker et al., 1996; Collins et al., 1978). Here we report the expression of PLC-coupled neurotensin receptors in HL-60 human promyelocytic leukaemia cells and the down-regulation of neurotensin-induced signalling during granulocytic differentiation into neutrophil-like cells.