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Apelin receptors (ApelinRs) are expressed along an increasing cortico-medullary gradient in collecting ducts (CDs). We showed here that iv injection of apelin 17 (K17F) in lactating rats characterized by increases in both synthesis and release of arginine vasopressin (AVP) increased diuresis concomitantly with a significant decrease in urine osmolality and no change in Na(+) and K(+) excretion. Under these conditions, we also observed a significant decrease in apical aquaporin-2 immunolabeling in CD, with a cortico-medullary gradient, suggesting that K17F-induced diuresis could be linked to a direct action of apelin on CD. We then examined the potential cross talk between V1a AVP receptor (V1a-R), V2 AVP receptor (V2-R) and ApelinR signaling pathways in outer medullary CD (OMCD) and inner medullary CD microdissected rat CD. In OMCD, expressing the 3 receptors, K17F inhibited cAMP production and Ca(2+) influx induced by 1-desamino-8-D-arginine vasopressin a V2-R agonist. Similar effects were observed in inner medullary CD expressing only V2-R and ApelinR. In contrast, in OMCD, K17F increased by 51% the Ca(2+) influx induced by the stimulation of V1a-R by AVP in the presence of the V2-R antagonist SR121463B, possibly enhancing the physiological antagonist effect of V1a-R on V2-R. Thus, the diuretic effect of apelin is not only due to a central effect by inhibiting AVP release in the blood circulation as previously shown but also to a direct action of apelin on CD, by counteracting the antidiuretic effect of AVP occurring via V2-R.

Apelin is a bioactive peptide identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ in 1998. The present data show that apelin modulates the activity of magnocellular and parvocellular oxytocin (OXY) neurons in the lactating rat. A combination of in situ hybridization and immunohistochemistry demonstrated the presence of apelin receptor mRNA in hypothalamic OXY neurons. Double immunofluorescence labeling then revealed the colocalization of apelin with OXY in about 20% of the hypothalamic OXY-positive neurons. Intracerebroventricular apelin administration inhibited the activity of magnocellular and parvocellular OXY neurons, as shown by measuring the c-fos expression in OXY neurons or by direct electrophysiological measurements of the electrical activity of these neurons. This effect was correlated with a decrease in the amount of milk ejected. Thus, apelin inhibits the activity of OXY neurons through a direct action on apelin receptors expressed by these neurons in an autocrine and paracrine manner. In conclusion, these findings highlight the inhibitory role of apelin as an autocrine/paracrine peptide acting on OXY neurons during breastfeeding.

Cortical thick ascending limb (CTAL) naturally expresses the angiotensin II (AngII) receptor type 1A (AT(1)-R), the bradykinin (BK) receptor type 2 (B(2)-R), and the insulin receptor. This segment is made of a single morphologically distinct cell type. AngII and BK are involved in same transduction pathways but differ markedly in their physiological actions on Na(+) transport. Besides, the insulin signaling intersects with those of AngII and BK at multiple levels and especially by stimulation on Na(+) reabsorption. Thus, the CTAL is a biologically suitable model to study the cross talk between G protein-coupled receptors or G protein-coupled receptors and receptor tyrosine kinase. In this work, the cross talks between AngII, BK, and insulin signaling are studied in rat CTAL by measuring changes in [Ca(2+)](i). We show that BK exerts negative modulatory effects on AngII-induced [Ca(2+)](i) responses dependent on tyrosine kinase and MAPK pathways. Moreover, in the presence of BK, AngII-induced Na(+) transport is suppressed. These effects suggest an interaction between AT(1)-R and B(2)-R. We show a positive interaction between the insulin receptor and the AT(1)-R through a protein kinase A-dependent mechanism that involves MAPK cascade, leading to the stimulation of the Ca(2+) influx induced by AngII. The presence of such interactions brings additional arguments for a complex and fine regulation of CTAL functions and puts forward the potentially beneficial effect of BK across this segment, in case of hyperinsulinemia or insulin resistance, by its negative feedback on AngII actions.

The cortical thick ascending limb (CTAL) coexpresses angiotensin (Ang) II/Ang III receptor type 1A (AT(1A)-R) and bradykinin (BK) receptor type 2 (B2-R). In several cell types, these two receptors share the same signaling pathways, although their physiological functions are often opposite. In CTAL, little is known about the intracellular transduction events leading to the final physiological response induced by these two peptides. We investigated and compared in this segment the action of Ang II/III and BK on intracellular calcium concentration ([Ca2+]i) response and metabolic CO2 production, an index of Na+ transport, by using inhibitors of protein kinase C (bisindolylmaleimide), Src tyrosine kinase (herbimycin A and PP2), and MAPK/ERK (PD98059 and UO126). Ang II/III and BK (10(-7) mol/liter) released Ca2+ from the same intracellular pools but activated different Ca2+ entry pathways. Ang II/III- or BK-induced [Ca2+]i increases were similarly potentiated by bisindolylmaleimide. Herbimycin A and PP2 decreased similarly the [Ca2+]i responses induced by Ang II/III and BK. In contrast, PD98059 and UO126 affected the effects of BK to a larger extent than those of Ang II/III. Especially, the Ca2+ influx induced by BK was more strongly inhibited than that induced by Ang II/III in the presence of both compounds. The Na+ transport was inhibited by BK and stimulated by Ang II/III. The inhibitory action of BK on Na+ transport was blocked by UO126, whereas the stimulatory response of Ang II/III was potentiated by UO126 but blocked by bisindolylmaleimide. These data suggest that the inhibitory effect of BK on Na+ transport seems to be directly mediated by an increase in Ca2+ influx dependent on MAPK/ERK pathway activation. In contrast, the stimulatory effect of Ang II/III on Na+ transport is more complex and involves PKC and MAPK/ERK pathways.

Apelin, a neuropeptide recently identified as the endogenous ligand for the G protein-coupled receptor APJ, is highly concentrated in brain structures involved in the control of body fluid homeostasis including the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. To clarify the implication of apelin in the regulation of water balance, we sought to determine whether apelin colocalized with arginine vasopressin (AVP) in the rat SON and PVN. We also investigated the effects of water deprivation on the levels of apelin within these two nuclei by comparison with those of AVP. Using dual immunolabeling confocal microscopy, we found that a large proportion of apelin-immunoreactive neurons colocalized AVP within both the SON and PVN, but that the two peptides were segregated within distinct subcellular compartments inside these cells. Both the number and labeling intensity of magnocellular apelin-immunoreactive cells increased significantly after 24- or 48-h dehydration, whereas the number and labeling density of AVP-immunoreactive neurons significantly decreased. The dehydration-induced increase in apelin immunoreactivity was markedly diminished by central injection of a selective vasopressin-1 receptor antagonist. Conversely, the effect of dehydration was mimicked by a 16-min intracerebroventricular infusion of AVP, again in a vasopressin-1 receptor antagonist-reversible manner. These results provide additional evidence for the involvement of the neuropeptide apelin in the control of body fluid homeostasis. They further suggest that the dehydration-induced release of AVP from magnocellular hypothalamic neurons may be responsible for the observed increase in immunoreactive apelin levels within the same neurons and thus that the release of one peptide may block that of another peptide synthesized in the same cells.

In this study, a sequential analysis of pathways involved in the regulation of GH secretagogue receptor subtype 1a (GHSR-1a) signaling has been undertaken to characterize the process of rapid desensitization that is observed after ghrelin binding. This process was evaluated by studying the binding of [(125)I]ghrelin, measurement of intracellular calcium mobilization, and confocal microscopy. The results indicate that GHSR-1a is mainly localized at the plasma membrane under unstimulated conditions and rapidly desensitizes after stimulation. The agonist-dependent desensitization is not mediated by protein kinase C because phorbol ester, phorbol-12-myristate-13-acetate, failed to block the ghrelin-induced calcium response. The ghrelin/GHSR-1a complex progressively disappears from the plasma membrane after 20 min exposure to ghrelin and accumulates in the perinuclear region after 60 min. Colocalization of the internalized GHSR-1a with the early endosome marker (EEA1) after 20 min exposure to ghrelin suggests that endocytosis occurs via clathrin-coated pits, which is consistent with the lack of internalization of this receptor observed after potassium depletion. Different from other G protein-coupled receptors, GHSR-1a showed slow recycling. Surface binding slowly recovered after agonist treatment and returned to control levels within 360 min. Furthermore, inhibition of vacuolar H(+)-ATPases prevented recycling of the receptor, suggesting that the nondissociation of the ligand/receptor complex is responsible for this effect. The GHSR-1a internalization may explain the characteristic physiological responses mediated by this receptor.

Recent studies have pointed out the differential role of angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in cardiac hypertrophy and fibrosis during pathological cardiac growth. Because senescence is characterized by an important cardiovascular remodeling, we examined the age-related expression of cardiac Ang II receptors in rats. AT1 and AT2 receptor subtype messenger RNA (mRNA) levels were quantitated by RT-PCR. In parallel, specific Ang II densities were determined in competition binding experiments using specific antagonists. AT1a and AT1b mRNA levels were markedly up-regulated (5.6-fold) in the left ventricle of 24-month-old rats compared with 3-month-old rats, but not in the right ventricle. In contrast, AT2 gene expression was increased in both ventricles of senescent rats (4.2- and 2.8-fold in the left and right ventricles, respectively). Similarly, AT1 and AT2 gene expression was increased 2.3- and 2-fold, respectively, in freshly isolated cardiomyocytes from aged rats. Furthermore, AT1 and AT2 specific binding was increased in the aged left ventricular myocardium. Even though the mechanistic pathway of this up-regulation of Ang II receptor subtype gene expression might be intrinsic to developmental gene reprogramming, the up-regulation of AT1 mRNA accumulation in the left ventricle during aging could also be secondary to age-related hemodynamic changes, whereas increased AT2 gene expression in both ventricles may depend upon hormonal and humoral factors.