[Functional dissociation between apelin receptor signaling and endocytosis: implications for the effects of apelin on arterial blood pressure]. / Dissociation fonctionnelle entre la signalisation et l'endocytose du récepteur de l'apéline: implication dans les effets de l'apéline sur la pression artérielle.

Apelin is a peptide involved in the regulation of body fluid homeostasis and cardiovascular functions, that was recently isolated as the endogenous ligand for the human orphan APJ receptor, a G protein-coupled receptor which shares 31% amino-acid sequence identity with the angiotensin II type 1 receptor. The predominant molecular forms of apelin naturally occuring in vivo are apelin 36, apelin 17 (K17F) and the pyroglutamyl form of apelin 13 (pE13F). We investigated the structure-activity relationships of apelin at the rat apelin receptor, tagged at its C-terminal end with enhanced green fluorescent protein and stably expressed in CHO cells. We compared the abilities of N- and C-terminal deleted fragments of K17F (KFRRQRPRLSHKGPMPF) to bind with high affinity to the apelin receptor, to inhibit cAMP production and to induce apelin receptor internalization. The first five N-terminal and the last two C-terminal amino acids of K17F were not essential for apelin binding or cAMP response. In contrast, deletion of the arginine in position 6 drastically decreased binding and cAMP response. The full-length sequence of K17F was the most potent inducer of apelin receptor internalization because successive N-terminal amino-acid deletions progressively reduced internalization and the removal of a single amino acid, the phenylalanine in position 17 at the C-terminus of K17F abolished this process. Thus, K16P binds with high affinity to the apelin receptor and strongly inhibits cAMP production, but does not induce apelin receptor endocytosis. These data indicate that apelin receptor signaling (coupling to Gi) and endocytosis are functionally dissociated, possibly reflecting the existence of several conformational states of this receptor, stabilized by the binding of different apelin fragments to the receptor. We then investigated the consequences for biological activity of this functional dissociation by evaluating the effects of various apelin fragments, injected iv, on arterial blood pressure in normotensive Wistar Kyoto rats. We showed that apelin fragments, that did not induce receptor internalization in vitro but kept their ability to activate receptor coupling to Gi, did not decrease arterial blood pressure. Our data showed that hypotensive actions of apelin peptides correlate with the ability of those ligands to internalize. Thus, the depressor response of apelin may be controlled by apelin receptor endocytosis, which is probably required for initiation of a second wave of signal transduction. The development of biaised agonists of the apelin receptor capable of promoting only one specific signal transduction pathway may therefore offer new therapeutic avenues for the treatment of cardiovascular disorders.

Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain.

Apelin, a peptide recently isolated from bovine stomach tissue extracts, has been identified as the endogenous ligand of the human orphan APJ receptor. We established a stable Chinese hamster ovary (CHO) cell line expressing a gene encoding the rat apelin receptor fused to the enhanced green fluorescent protein, to investigate internalization and the pharmacological profile of the apelin receptor. Stimulation of this receptor by the apelin fragments K17F (Lys1-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and pE13F (pGlu5-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) resulted in a dose-dependent inhibition of forskolin-induced cAMP production and promoted its internalization. In contrast, the apelin fragments R10F (Arg8-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and G5F (Gly13-Pro-Met-Pro-Phe17) were inactive. The physiological role of apelin and its receptor was then investigated by showing for the first time in rodent brain: (i) detection of apelin neurons in the supraoptic and paraventricular nuclei by immunohistochemistry with a specific polyclonal anti-apelin K17F antibody; (ii) detection of apelin receptor mRNA in supraoptic vasopressinergic neurons by in situ hybridization and immunohistochemistry; and (iii) a decrease in vasopressin release following intracerebroventricular injection of K17F, or pE13F, but not R10F. Thus, apelin locally synthesized in the supraoptic nucleus could exert a direct inhibitory action on vasopressinergic neuron activity via the apelin receptors synthesized in these cells. Furthermore, central injection of pE13F significantly decreased water intake in dehydrated normotensive rats but did not affect blood pressure. Together, these results suggest that neuronal apelin plays an important role in the central control of body fluid homeostasis.

Morning cortisol as a risk factor for subsequent major depressive disorder in adult women.

BACKGROUND: Whether individual differences in cortisol contribute to subsequent major depressive disorder (MDD) is unknown. AIMS: To determine whether premorbid levels of salivary cortisol and dehydroepiandrosterone (DHEA) were associated with subsequent MDD and how these related to psychosocial factors known to increase the risk for MDD. METHOD: Adult women (n=116) were recruited from general practices. None was currently depressed; 83 were 'psychosocially vulnerable' to MDD, 33 were not. Salivary steroids (cortisol and DHEA at 08.00 h and 20.00 h), recent life events, current mood and social support were assessed at entry. Onset of MDD was recorded during 13 months' follow-up. RESULTS: There were no associations between salivary cortisol or DHEA and recent life events or vulnerability. Twenty-eight onsets of MDD occurred during the follow-up period. This was associated with: severe adverse life events and difficulties during the follow-up period; mean morning cortisol levels at entry; and the presence of any of three vulnerability factors. CONCLUSIONS: Individual differences in morning salivary cortisol levels may represent an independent risk factor for subsequent MDD. The origin of these differences in cortisol is not yet understood.

Immunocytochemical localization of the insulin-responsive glucose transporter 4 (Glut4) in the rat central nervous system.

We have previously reported that the insulin-responsive glucose transporter GLUT4 is strongly expressed by discrete areas of the rat brain (Leloup et al. [1996] Molec. Brain Res. 38:45-53). In the present study, a sensitive immunocytochemical technique has been used to analyze extensively the anatomical and ultrastructural localizations of GLUT4 in the rat central nervous system in order to gain insight into the physiological role of this transporter. We confirm that GLUT4 is expressed by numerous neurons of the brain and spinal cord, whereas glial cells are more scarcely labeled. In both light and electron microscopy, we observe that the immunoreactivity for GLUT4 is localized mainly in the somatodendritic portion of neurons, where some cisterns of rough endoplasmic reticulum, ribosomal rosettes, certain Golgi saccules, and some intracytoplasmic vesicles are labeled. In contrast, axons and nerve terminals are only occasionally immunostained in certain brain regions such as the neocortex and the ventricular surfaces for example. The GLUT4-immunoreactive structures appear concentrated and most prominently immunostained in motor areas, such as the sensorimotor cortex, most basal ganglia and related nuclei, the cerebellum and deep cerebellar nuclei, a number of reticular fields, motor nuclei of cranial nerves, and motor neurons of the ventral horn of the spinal cord. The labeled regions, which also include some sensory nuclei, are often those in which Vissing et al. ([1996] J. Cerebral Blood Flow Metab. 16:729-736) have shown that exercise stimulates local cerebral glucose utilization, so that GLUT4 might be involved in this effect. On the other hand, the fact that the anatomical localizations of GLUT4 reported here generally agree with the distribution of insulin- or insulin-receptor- related receptors is important since it indicates that the translocation of GLUT4 might also be regulated by insulin in the central nervous system.