Lack of Rapid Desensitization of Ca²⁺ Responses in Transfected CHO Cells Expressing the Rat Neurotensin Receptor Despite Agonist‐Induced Internalization

Abstract

Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization of the neurotensin receptor at the second messenger level. Stimulation with nanomolar concentrations of neurotensin elicited rapid rises in the cytosolic calcium concentration ([Ca2+]i), which remained elevated throughout the peptide application. A significant response was already detected with neurotensin concentrations as low as 0.01 nM. This high efficiency of neurotensin in mediating this calcium response contrasts with the nanomolar affinity of the peptide for its receptor measured in binding experiments. Evidence indicated that the initial elevation of the [Ca2+]i resulted from release of Ca2+ from intracellular stores, whereas the sustained response involved an influx of extracellular origin. Return to the basal level was only reached after extensive washing of the peptide or its displacement with the neurotensin receptor antagonist SR48692. After washing, further stimulations were still able to mediate an increase in the [Ca2+]i, indicating an apparent absence of rapid desensitization of the intracellular signaling pathway that mediates calcium mobilization. In contrast with this absence of response desensitization, the neurotensin receptors were found to internalize after stimulation with the peptide. This internalization was maximal after 30 min and accounted for ∼70% of the number of neurotensin binding sites located at the cell surface. These results indicate that despite the functional properties of the rat neurotensin receptor present in Chinese hamster ovary cells after transfection, the intracellular signaling pathway triggered by stimulation with neurotensin seems to be resistant to desensitization. This might be related to the high efficiency of the intracellular signaling pathway coupled to the neurotensin receptor observed in these cells. A possible absence of desensitization of the neurotensin receptor itself is also discussed.

Role of Calcium in Neurotensin-Evoked Enhancement in Firing in Mesencephalic Dopamine Neurons

“…Considering the coupling of this receptor to PLC and IP 3 production (Bozou et al, 1989;Tanaka et al, 1990;Hermans et al, 1992), the hypothesis that its activation causes intracellular Ca 2ϩ elevation in DAergic neurons is reasonable but was untested. Calcium mobilization by NT has been demonstrated previously in other cell types (Sato et al, 1991;Hermans et al, 1995;Schaeffer et al, 1995;Borges et al, 1996;Trudeau, 2000). The present experiments provide the first detailed characterization of NT-evoked intracellular Ca 2ϩ increase in DAergic neurons.…”

Europium-Labeled Epidermal Growth Factor and Neurotensin: Novel Probes for Receptor-Binding Studies

“…The observation that Ca 2ϩ mobilization can be obtained at low NT concentrations may be related to the fact that only a small fraction of the high-affinity NT receptors is required to fully activate Ca 2ϩ mobilization as previously reported (18). On the other hand, it is possible that the same efficacy of NT and Eu-NT on Ca 2ϩ mobilization relates to the amplification process taking place in the endoplasmic reticulum (18). Decline of the ligand-receptor affinity due to peptide modification has been reported in other cases where DTPA labeling of bioactive peptides has been described (19).…”

“…In agreement with the impaired binding capacity, intracellular Ca 2ϩ mobilization by Eu-NT was even less efficient compared with native NT. The observation that Ca 2ϩ mobilization can be obtained at low NT concentrations may be related to the fact that only a small fraction of the high-affinity NT receptors is required to fully activate Ca 2ϩ mobilization as previously reported (18). On the other hand, it is possible that the same efficacy of NT and Eu-NT on Ca 2ϩ mobilization relates to the amplification process taking place in the endoplasmic reticulum (18).…”

Evidence for the dual coupling of the rat neurotensin receptor with pertussis toxin‐sensitive and insensitive G‐proteins

“…CHO‐NTR cells respond to NT stimulation with a large release of Ca 2+ from the endoplasmic reticulum followed by sustained entry of Ca 2+ from the extracellular space [19]. This response is triggered by InsP 3 as shown by its sensitivity to micro‐injected heparin [22].…”

“…Using transfected Chinese hamster ovary (CHO) cells expressing NTS1 (CHO‐NTR), we have previously reported on the NT‐induced accumulation of inositol phosphates (InsP) [10], production of arachidonic acid [18], mobilization of intracellular calcium [19] and increase in [ 35 S]GTPγS binding [20]. On the basis of further biochemical analysis, we now provide evidence that these functional responses are dependent on the dual coupling of the receptor with both PTx‐sensitive and insensitive G‐proteins.…”