Mechanisms of Regulation of Neurotensin Receptors

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Abstract

Since its discovery in 1973, the neuropeptide neurotensin has been demonstrated to be involved in the control of a broad variety of physiological activities in both the central nervous system and in the periphery. Pharmacological studies have shown that the biological effects elicited by neurotensin result from its specific binding to cell membrane neurotensin receptors that have been characterized in various tissue and in cell preparations. In addition, it is now well documented that most of these responses are subject to rapid desensitization. Such desensitization results in transient responses to sustained peptide applications, or to tachyphylaxis during successive stimulations in the same conditions. More recently, desensitization of neurotensin signalling was investigated at the cellular and molecular levels. In cultured cells, regulation at the second messenger level, receptor internalization, and receptor down-regulation processes have been reported. These are proposed to play a critical role in the control of cell responsiveness to neurotensin. This review aims to compile recent data on the different biochemical processes involved in the regulation of the neurotensin receptor and to discuss the physiological consequences of this regulation in vivo.

Introduction

Neurotensin is a tridecapeptide (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) that was first isolated from bovine hypothalamus during the purification of substance P (Carraway and Leeman, 1973). Its name was chosen on the basis of its neuronal localization and the vasodilatation that is observed after its local administration. It is now well established that neurotensin is present in both the CNS, where it plays the role of neurotransmitter or neuromodulator, and in some peripheral tissues, mainly in the gastrointestinal tract, where it may be considered as a hormone. In the CNS, both anatomical and pharmacological data suggested a close relationship between neurotensin and the dopaminergic systems, essentially in the nigrostriatal and mesolimbic systems Nemeroff 1986, Kitabgi 1989. Decreased locomotor activity and hypothermia are some of the physiological responses observed after central administration of neurotensin and that are thought to be directly related to the modulatory effect of neurotensin on the dopaminergic systems. Neurotensin has been shown to control the synthesis and the release of dopamine, as well as the sensitivity of some dopamine receptors. As a consequence, both inhibition and enhancement of dopaminergic transmission are mediated by neurotensin, depending on the brain area. For a more complete review of the biological effects of neurotensin in the CNS and in the peripheral tissues, see Kitabgi and Nemeroff (1992) and Kitabgi et al. (1985).

Most of the effects observed after administration of neurotensin result from the specific interaction of the peptide with cell surface neurotensin receptors. The first binding experiments conducted with neurotensin suggested the existence of at least two neurotensin binding sites in homogenates prepared from various mammalian tissues, although only one high-affinity neurotensin receptor was clearly identified and characterized (Vincent, 1992). The high-affinity neurotensin receptor has been cloned from both rat and human tissues Tanaka et al. 1990, Vita et al. 1993; the low-affinity binding site was thought to correspond to a neurotensin acceptor site, devoid of any biological function. More recently, however, there have been further proposals of the existence of multiple neurotensin receptor subtypes Dubuc et al. 1994, Labbé-Jullié et al. 1994, Le et al. 1996, and there have now been three reports of the molecular cloning of a second neurotensin receptor, from human, rat, and mouse tissues, respectively Vita et al. 1997, Chalon et al. 1996, Mazella et al. 1996. The sequence similarity between this second receptor and the high-affinity neurotensin receptor was 64% in rat. From a pharmacological point of view, this second receptor binds neurotensin with a lower affinity and is recognized by levocabastine; therefore, this site clearly corresponds to what was previously called the neurotensin acceptor site. The two cloned neurotensin receptors differ by their tissue distributions and their ontogenic profiles. In situ hybridization studies revealed that the low-affinity neurotensin receptor was mainly expressed in the hippocampus, in the piriform cortex, and also in the cerebellar cortex, the latter being devoid of high-affinity neurotensin receptors. On the contrary, the low-affinity receptor is not detected in the substantia nigra and the ventral tegmental area, two regions associated with central dopaminergic activity, in which the highest levels of the high-affinity neurotensin receptor have been reported. The high-affinity neurotensin receptor was characterized in tissues, essentially rat and murine brain, as well as in cell cultures, primary cultures of neurons, and cell lines (see Section 1.3). Both neurotensin receptors have sequence homology with the family of G-protein-coupled receptors with seven transmembrane spanning domains connected by intracellular and extracellular loops (Tanaka et al., 1990). Accordingly, the binding of the agonist neurotensin to the high-affinity receptor was found to be modulated by guanine nucleotides (Mills et al., 1988). Surprisingly, guanine nucleotide modulation of agonist binding has not been reported for the low-affinity receptor, so functional coupling of this second neurotensin receptor with intracellular G-proteins is not yet clearly established, despite its seven transmembrane spanning domain structure. Neurotensin stimulation of Xenopus oocytes injected with the mRNA encoding the low-affinity receptor induced cell depolarization, although the mechanism of this effect is unclear. Given the lack of evidence for a functional role of the low-affinity receptor, the rest of this review will focus on the properties and modulation of the high-affinity neurotensin receptor.

The biochemical properties of the high-affinity neurotensin receptor are summarized in Table 1. Until 1993, only peptide analogues of neurotensin with weak agonist or antagonist properties were available for the study of the neurotensin receptor. The receptor, therefore, was characterized in binding experiments using tritiated or iodinated neurotensin, which binds with an affinity in the nanomolar or subnanomolar range Kitabgi et al. 1977, Lazarus et al. 1977. Binding studies conducted with neurotensin fragments or with modified peptides indicated that all of the binding affinity of neurotensin resides in its carboxyl-terminal end, between amino acids 8 and 13, whereas its amino-terminal end does not bind to receptor. With a few exceptions, a similar structure-activity relationship was observed in multiple biological assays, demonstrating that these effects were receptor mediated. Recently, nonpeptide neurotensin receptor antagonists have been developed Gully et al. 1993, Gully et al. 1997. These compounds (SR 48692 and SR 142948A) constitute precious tools, not only for the in vitro study of the neurotensin receptor, but also for the understanding of the importance of the neurotensin transmission observed in vivo (Gully et al., 1995).

The first evidence for an intracellular response to neurotensin was obtained in electrophysiological studies (Okuma and Osumi, 1982). Membrane currents induced by neurotensin are proposed to result from biochemical cascades leading to the production of intracellular second messengers Wu et al. 1995, Wu and Wang 1995. It is now well established that the major intracellular effector activated by neurotensin is phospholipase C (PLC), which is responsible for the hydrolysis of phosphatidyl inositol 4,5-bisphosphate to generate two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. The production of inositol phosphates induced by neurotensin, initially measured in rat brain slices (Goedert et al., 1984), was also demonstrated in the majority of primary and cell line cultures in which neurotensin receptors are expressed (cultured neurons, N1E115 cells, HT29 cells, NG108-15 cells, bovine chromaffin cells) Snider et al. 1986, Imaizumi et al. 1989, Amar et al. 1986, Amar et al. 1987, Bommer and Herz 1989, Weiss et al. 1988. In cultured neurons and in cell lines, this inositol phosphate production was further correlated with an increase in intracellular calcium concentration Snider et al. 1986, Imaizumi et al. 1989, Sato et al. 1991, Bozou et al. 1989a, Woll and Rozengurt 1989. Paradoxically, despite the activation of PLC, there have been no reports of neurotensin-induced diacylglycerol production and protein kinase C (PKC) activation (Bozou et al., 1989b). Accordingly, there was no evidence for involvement of PKC in the depolarization of dopaminergic neurons by neurotensin (Wu and Wang, 1995). On the contrary, a possible involvement of PKC was proposed in the neurotensin-induced potentiation of dopamine release from the rat retina (Okada et al., 1992) and in the neurotensin activation of mitogen-activated protein kinase in transfected Chinese hamster ovary (CHO) cells (Poinot Chazel et al., 1996). Accordingly, the neurotensin-induced dopamine release from mesencephalic cell cultures was found to be decreased after down-regulation of PKC activity by phorbol ester treatment (Brouard et al., 1994). The inability of PKC activators or inhibitors to affect the neurotensin-induced inhibition of some midbrain dopamine neurons contrasts with the effect of cyclic AMP (cAMP) modulating agents on the neurotensin response measured in the same conditions (Shi and Bunney, 1992). Both positive and negative modulation of adenylyl cyclase activity by neurotensin were reported in some cultured cells Bozou et al. 1986, Yamada et al. 1993c, but such responses to neurotensin are not the rule, since no modulation of cAMP levels by neurotensin was observed in rat brain slices (Goedert et al., 1984) or in Cl.16E cells (derived from HT29 cells) (Augeron et al., 1992). In HT29 cells, an intracellular calcium increase was found to potentiate the cAMP production induced by forskolin (Warhurst et al., 1994). Finally, a potential role for neurotensin-induced cAMP production and protein kinase A activation was suggested by the heterologous phosphorylation and desensitization of dopamine D2 receptor in the rat brain, supporting evidence for a coupling of the neurotensin receptor with the G-protein Gs (Kalivas, 1993). In N1E115 cells, neurotensin initially was found to produce an accumulation of cyclic GMP (cGMP) (Gilbert and Richelson, 1984). However, this effect was shown to be a consequence of the increase in intracellular calcium level, which activates a biochemical cascade, resulting in the intracellular production of nitric oxide Marsault and Frelin 1992, Yamada et al. 1992a, McKinney et al. 1990. In a similar fashion, neurotensin-induced opening of calcium channels observed in various systems Memo et al. 1986, Kullak et al. 1987 was also proposed to be related to the phosphoinositide-calcium release signalling cascade Memo et al. 1986, Hermans et al. 1995. Interestingly, Slusher et al. (1994) recently reported the neurotensin-induced production of cGMP in the olfactory bulb. The functional coupling of the neurotensin receptor was also measured at the G-protein level, as it was found that neurotensin induced an increase in the binding of radiolabelled hydrolysis-resistant guanine nucleotide to G-protein in homogenates of transfected CHO cells expressing the rat neurotensin receptor (Hermans et al., 1997b). The different second messenger pathways associated with the neurotensin receptor stimulation are summarized in Table 2.

Since its discovery in 1973, many studies of the biological effects of neurotensin have shown that the responses to neurotensin were particularly subject to rapid desensitization. Desensitization is characterized by transient responses to sustained peptide applications, lack of response to repeated neurotensin applications, and by a shift to the right and a depression of the maximum response of the neurotensin concentration-response curve. Indeed, desensitization was observed in the first report on the actions of neurotensin, where the systemic hypotensive effect of neurotensin exhibited acute tachyphylaxis, and an absence of response to subsequent applications of the peptide for more than 60 min after the first agonist dose (Carraway and Leeman, 1973). However, responses recovered several hours later. Similar results on the neurotensin-induced hypotension in normal rats were obtained using neurotensin analogues (Di Paola and Richelson, 1990). Desensitization of the cell responses to neurotensin was also reported in numerous electrophysiological studies of neurons from rat cortex (Audinat et al., 1989), guinea pig inferior mesenteric ganglion (Stapelfeldt and Szurszewski, 1989), basal forebrain cholinergic neurons (Farkas et al., 1994), oocytes injected with brain mRNA Augeron et al. 1992, Warhurst et al. 1994, and the ileal mucosa preparation (Kachur et al., 1982). In many systems, neurotensin was found to induce the release of various neurotransmitters, including dopamine, γ-aminobutyric acid, and acetylcholine, and all of these responses were found to show rapid desensitization Nakamoto et al. 1987, Faggin and Cubbedu 1990. In mesencephalic cell cultures, the neurotensin-induced dopamine release was suppressed by both short- and long-term treatments with phorbol ester. Similar modulations of responses by phorbol esters have been described for other receptors, and probably are related to a PKC-dependent desensitization process (Brouard et al., 1994). In peripheral tissues, desensitization to neurotensin also seems to be the rule, especially in studies concerning the effects of neurotensin in the gastrointestinal tract. For example, neurotensin dose dependently modulated motility of rat proximal colon; a 20-min exposure to 5 nM neurotensin shifted the neurotensin concentration-response curve to the right and depressed the maximum response. Concentrations above 10 nM induced an almost complete loss of tissue response, although extensive washing of the tissue resulted in a time-dependent recovery of the response (Mulè et al., 1995). Other reports of desensitization of neurotensin responses include modulation of ion transport in the porcine distal jejunum (Brown and Treder, 1989), contraction of smooth muscle of the isolated rat uterus (Lebrun et al., 1988), and of the rat fundus (Huidobro-Toro and Kullak, 1985). In the latter tissue, cross-desensitization was observed between neurotensin and its active analogues. In addition, a correlation has been reported between the efficacy of the fragments or analogues of neurotensin to induce the biological effect and their ability to cause tachyphylaxis. Table 3 gives some selected examples of desensitization of neurotensin responses. As indicated in Table 3, the rate of recovery of response differs greatly between studies. Some authors report a maximal recovery after only a few minutes of washout Kachur et al. 1982, Gibson et al. 1984, whereas others did not show any recovery of response even after prolonged (up to 3 hr) washout (Lebrun et al., 1988).

Despite the many reports of rapidly desensitizing neurotensin responses, it is intriguing that there have been some studies of neurotensin responses in which desensitization was not observed. Thus, Okuma and Osumi (1982) showed that the release of noradrenaline induced by neurotensin from rat hypothalamic slices was not reduced during successive stimulations (Okuma and Osumi, 1982). Katsoulis and Conlon (1988) showed that the contraction of longitudinal muscle strips from the guinea pig gastric corpus was long lasting, indicating absence of desensitization. More recently, in electrophysiological studies of dopaminergic neurons of the ventral tegmental area, Seutin et al. (1989) reported that the activation by neurotensin was long lasting and exhibited little tachyphylaxis; prolonged infusion of peptide produced a sustained effect, and successive infusions gave similar activation. Accordingly, absence of tachyphylaxis was also reported in substantia nigra dopaminergic neurons (Pinnock, 1985). In bovine chromaffin cells, desensitization of the neurotensin-induced opiate and catecholamine release was found to be negligible during application for at least 2 hr (Bommer and Herz, 1989). Finally, intracerebroventricular injection of neurotensin was found to induce an increase in blood pressure, without any sign of tachyphylaxis (Sumners et al., 1982). The reason for the absence of tachyphylaxis in these instances is not apparent; possibilities include the existence of further (desensitization-resistant) neurotensin receptor subtypes or tissue-dependent differences in mechanisms inducing neurotensin receptor desensitization.

Section snippets

Regulation of the neurotensin receptor

Despite many reports of desensitization of neurotensin responses, there is only limited information on the molecular and cellular mechanisms underlying desensitization. Differences in desensitization onset and offset rates may reflect multiple molecular or cellular processes. However, in addition to homologous desensitization to neurotensin, heterologous desensitization of responses was also reported between neurotensin and other neurotransmitters, including substance P and acetylcholine (the

Conclusions

The neurotensin receptor undergoes rapid desensitization and internalization after stimulation with the agonist in a variety of cellular and tissue systems. The desensitization process, which is proposed to precede the internalization of the cell surface receptor, results in transient responses to sustained peptide applications. The molecular mechanisms involved in this desensitization remain unknown. However, by analogy with other G-protein-coupled receptors, the desensitization at the

Acknowledgements

The authors wish to thank the Belgian Queen Elisabeth Medical Foundation, the Fund for Medical Scientific Research (Belgium) (FRSM 3-458090 and 3-452991), and the Ministry of Scientific Policy (Belgium) (ARC 89/95-135). E. Hermans is Senior Research Assistant of the National Fund for Scientific Research (FNRS). We also thank Cécile Hermans for the revision of the manuscript.

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