[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.

Angiotensin III: a central regulator of vasopressin release and blood pressure.

Among the main bioactive peptides of the brain renin-angiotensin system, angiotensin (Ang) II and AngIII exhibit the same affinity for type 1 and type 2 AngII receptors. Both peptides, injected intracerebroventricularly, cause similar increases in vasopressin release and blood pressure. Because AngII is converted in vivo to AngIII, the identity of the true effector is unknown. This review summarizes new insights into the predominant role of brain AngIII in the control of vasopressin release and blood pressure and underlines the fact that brain aminopeptidase A, the enzyme forming central AngIII, could constitute a putative central therapeutic target for the treatment of hypertension.

Identification of endocrine cell populations expressing the AT1B subtype of angiotensin II receptors in the anterior pituitary.

Angiotensin II (Ang II) participates in the regulation of anterior pituitary hormone secretion by acting either directly on the anterior pituitary or indirectly on the hypothalamus. When applied directly on pituitary cells, Ang II increases both ACTH and PRL secretion and has also been reported to affect GH secretion. Three distinct subtypes of Ang II receptors (AT1A, AT1B, and AT2) have been identified; they are unequally distributed and differently regulated in various tissues. We have previously demonstrated that only AT1A receptors are present in the hypothalamus while anterior pituitary cells express predominantly the AT1B subtype. Using in situ hybridization in combination with immunohistochemistry, the aim of the present study was to identify the phenotype of the endocrine cell expressing AT1B receptor messenger RNA (mRNA) in the anterior pituitary of adult male Sprague-Dawley rats. Expression of AT1B receptor mRNA was present in 33.9 +/- 1.0% of anterior pituitary cells. AT1B mRNA is predominantly expressed by lactotropes (78.2 +/- 2.1% of AT1B mRNA-expressing cells) and to a lower degree by corticotropes (18.3 +/- 2.1%) and is not detectable in somatotropes, mammosomatotropes, gonadotropes, or thyrotropes. These results indicate that in adult male rats, Ang II, which has been shown to be synthesized in gonadotropes, can directly stimulate PRL and ACTH release from lactotropes and corticotropes through activation of AT1B receptors. As only 53.8 +/- 2.7% of lactotropes and 23.6 +/- 2.8% of corticotropes expressed AT1B mRNA, our findings suggest a functional heterogeneity of both cell types regarding their sensitivity to Ang II.

Desensitization of type 1 angiotensin II receptor subtypes in the rat kidney.

Differences involving serine residues in the sequence of the carboxyl-terminal tail of type 1 angiotensin II (Ang II) receptor subtypes AT(1A) and AT(1B) suggest differences in desensitization ability. We examined the Ang II-induced homologous desensitization patterns of both receptor subtypes in freshly isolated renal structures: glomerulus (Glom), afferent arteriole, and cortical thick ascending limb (CTAL), whose content in each subtype mRNA is different, by measuring variations in intracellular calcium concentration. A preexposure to a maximal dose of Ang II, followed by a second application of the same concentration, induced: 1) a complete desensitization in Glom, where AT(1A) and AT(1B) mRNAs were expressed in similar proportions, and 2) no or partial desensitization in afferent arteriole and CTAL, where AT(1A) mRNA was predominant. In the absence of nephron structure containing only AT(1B) mRNA, we studied rat anterior pituitary cells that exhibit high content in this subtype and observed that desensitization was not complete. In Glom, CTAL, and pituitary cells, desensitization proceeded in a dose-dependent manner. In Glom and CTAL, desensitization occurred via a PKC-independent mechanism. These results suggest that desensitization does not depend on the nature of Ang II receptor subtype but either on the proportion of each subtype in a given cell and/or on cell specific type. This could allow adaptive biological responses to Ang II appropriate to the specific function of a given cell type.

Cardiac senescence is associated with enhanced expression of angiotensin II receptor subtypes.

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.

Type 1 angiotensin II receptor subtypes in kidney of normal and salt-sensitive hypertensive rats.

We studied the localization and regulation of the two type 1 angiotensin II receptor subtypes AT(1A) and AT(1B) in different renal zones of the rat kidney by a reverse transcription-polymerase chain reaction amplification method. The yield of the reaction was quantified with an internal standard that was a 63-bp deleted mutant cRNA of the AT(1A) receptor. In kidneys of male Sprague-Dawley rats (n=4), the levels of AT(1A) and AT(1B) receptor mRNAs were highest in the inner stripe of the outer medulla, lowest in the inner medulla, and intermediate in the cortex and outer stripe of the outer medulla. Results (mean+/-SE) expressed in 10(5) molecules per microgram total RNA were for cortex outer stripe, inner stripe, and inner medulla, respectively, 171 +/- 15, 152 +/- 27, 322 +/- 10, and 73 +/- 3 for AT(1A), and 35 +/- 9, 26 +/- 1, 71 +/- 10, and 53 +/- 11 for AT(1B). In sabra rats sensitive (n=6) or resistant (n=6) to salt-induced hypertension and maintained on a normal salt diet, the percentage and level of each receptor subtype mRNA in cortex and outer stripe were similar in the two strains and comparable to those observed in Sprague-Dawley rats. However, AT(1A) of the inner stripe was significantly decreased in salt-resistant compared with salt-sensitive rats (166 +/- 28 and 318 +/- 58 10(5) molecules per microgram total RNA, respectively). These modifications were organ specific because no difference in the level of the receptor mRNAs was observed in the liver of the two Sabra rat strains, whereas a twofold increase in AT(1A) mRNA level but not in AT(1B) mRNA level was apparent in adrenal and in one renal zone, the inner stripe of the outer medulla, of hypertension-prone Sabra rats.

Tissular expression and regulation of type 1 angiotensin II receptor subtypes by quantitative reverse transcriptase-polymerase chain reaction analysis.

Recent studies have revealed that angiotensin II (Ang II) interacts with two pharmacologically different types of seven-transmembrane domain receptors, hence named Ang II type 1 and type 2 (AT1 and AT2) receptors. cDNAs for the AT1 receptor have been cloned, and the existence of two receptor subtypes, AT1A and AT1B, has been revealed in rat and mouse. This study presents a new approach for the specific quantification of AT1A and AT1B receptor mRNAs by reverse transcription and polymerase chain reaction amplification in the presence of an AT1 receptor mutant cRNA as internal standard. Absolute quantities of mRNA are then determined by extrapolation using the standard curve generated with the internal standard. Moreover, addition of this internal standard to each tube controls for both reverse transcription and polymerase chain reaction amplification in each sample. In male Wistar rats, the highest absolute AT1A receptor mRNA levels were found in liver and kidney and those for AT1B receptor mRNA in the pituitary. Expressed as a percentage of total AT1A+AT1B receptor mRNA content, AT1A receptor mRNA content was 100% in liver, 85% in lung, 73% in kidney, 65% in aorta, 48% in adrenals, and 15% in the hypophysis. Since this approach can determine absolute AT1A and AT1B receptor mRNA quantities in different organs, it allows the study of the regulation of their expression under different pathophysiological conditions. After sodium depletion, known to induce hyperactivity of the renin-angiotensin system, adrenal AT1A and AT1B receptor mRNA levels were increased by 60% and 110%, respectively. In contrast, in renovascular hypertension (two-kidney, one clip), also associated with elevated circulating plasma renin activity, adrenal AT1B receptor mRNA levels decreased by 50%, whereas there was no change in those of AT1A. Therefore, the differential distribution and regulation of these two receptor subtypes suggest that each of them might be involved in the mediation of different biological effects of Ang II.

Regulation of angiotensin II receptor subtypes by dexamethasone in rat mesangial cells.

The objective of this study was to examine the role of dexamethasone on the expression of angiotensin II (Ang II) receptors in cultured rat mesangial cells. Dexamethasone caused concentration- and time-dependent decreases in 125I-[Sar1,Ala8]Ang II binding that were prevented by glucocorticoid receptor inhibition with mifepristone. A lag time of 24 hours and a dexamethasone concentration of at least 10 nmol/L were necessary for this effect to occur. Dexamethasone-induced reduction of 125I-[Sar1,Ala8]Ang II binding resulted from decreased Ang II type 1 (AT1) receptor density. No change in the apparent dissociation constant was observed. Dexamethasone also markedly inhibited Ang II-dependent inositol phosphate accumulation. Both reverse transcription-polymerase chain reaction and Northern blot analysis using specific short probes from the 3' noncoding region of the cDNA demonstrated the presence of AT1A and AT1B receptor mRNAs in rat mesangial cells, with a slight predominance of AT1B. Therefore, we studied the effect of dexamethasone on the expression of these two subtypes in rat mesangial cells. Dexamethasone produced a time-dependent decrease of AT1B receptor mRNA that was apparent after 6 hours of incubation, whereas AT1A receptor mRNA did not change. Mifepristone also suppressed the dexamethasone-induced decrease in AT1B receptor mRNA. In conclusion, glucocorticoids diminish Ang II receptor density at the mesangial cell surface through a mechanism that implies successive interaction with the glucocorticoid receptor and specific reduction in AT1B receptor mRNA expression. This differential regulation of both AT1 receptor subtypes might allow glucocorticoids to exert adjusted effects in their various target tissues.

Tissue-specific regulation of type 1 angiotensin II receptor mRNA levels in the rat.

Most of the biological effects of the renin-angiotensin system are mediated by the binding of angiotensin II (Ang II) to the type 1 Ang II (AT1) receptor, the predominant receptor subtype present after fetal life. To study tissue-specific regulation of the expression of the AT1 receptor in the rat, we altered activity of the renin-angiotensin system by feeding rats a low (0.07% NaCl), normal (0.3% NaCl), or high (7.5% NaCl) salt chow for 14 days; infusing Ang II (200 ng/kg per minute IP) or vehicle for 7 days; and administering an angiotensin-converting enzyme inhibitor (captopril, 100 mg/dL in the drinking water) or vehicle for 7 days. Renin, angiotensinogen, and total AT1 receptor mRNA levels were measured by slot-blot hybridization with cRNA probes, and AT1 receptor subtypes (A and B) were measured by reverse transcription-polymerase chain reaction in the presence of a cRNA internal standard. Plasma renin concentration and renal renin, renal and hepatic angiotensinogen, and hepatic AT1 receptor mRNA levels were all inversely related to salt intake; in contrast, renal AT1 receptor mRNA levels were significantly lower in rats fed low salt, a difference that was exclusively due to a decrease in the AT1A subtype. This difference did not appear to be mediated by a change in the circulating levels of Ang II, because Ang II infusion reduced plasma renin concentration and renal renin mRNA with no effect on either angiotensinogen or AT1 receptor mRNA levels in kidney or liver, renal Ang II receptor density (determined by in situ autoradiography) decreased, presumably via a posttranscriptional mechanism. Similarly, inhibition of Ang II generation with captopril increased plasma renin concentration and renal renin mRNA levels without altering renal or hepatic angiotensinogen mRNA or renal AT1 receptor mRNA levels. Thus, AT1 receptor gene expression is regulated in a tissue-specific manner that is distinct from other components of systemic and local renin-angiotensin systems and that appears to be mediated by a mechanism other than through changes in the circulating levels of Ang II.

Detection of the tripeptide Tyr-Gly-Gly, a putative enkephalin metabolite in brain, using a sensitive radioimmunoassay.

A sensitive and specific radioimmunoassay has been developed for YGG. The tripeptide was previously derivatised with p-benzoquinone to prepare the immunogen and the 125I tracer as well as in samples submitted to the RIA. The sensitivity is about 1 nM as compared with 8000 nM for underivatised YGG. Measurable amounts of endogenous YGG immunoreactivity, co-eluting in HPLC with authentic YGG, were detected in mouse striatal extracts.

Distribution of angiotensin II type-2 receptor (AT2) mRNA expression in the adult rat brain.

Radioactively labeled cRNA probes were used for in situ hybridization histochemistry to establish a detailed map of the sites of expression of the recently cloned angiotensin II, type 2 (AT2) receptor mRNA in the adult rat brain. The distribution of the AT2 receptor mRNA was consistent with that of the AT2 binding sites, which were previously established by autoradiographic binding studies. Thus, high AT2 receptor mRNA expression was observed in the lateral septum, in several thalamic nuclei, in the subthalamic nucleus, in the locus coeruleus, and in the inferior olive. Due to the superior resolution and sensitivity of in situ hybridization, AT2 receptor expression was localized at the cellular level, and some additional brain nuclei expressing AT2 receptor mRNA have been identified. These include the red nucleus, the pedunculopontine tegmental nucleus, the bed nucleus of the supraoptic decussation, the paragenual nucleus, and numerous brainstem nuclei. Several brain nuclei, such as the motor hypoglossal nucleus and the cerebellar nuclei, where AT2 receptor binding had previously been identified in young animals only, showed a high expression of the AT2 receptor mRNA in the adult rat. No correlation was found between the expression of the AT2 and the type 1 (AT1) receptor mRNAs. A combination of the in situ hybridization and glial fibrillary acidic protein (GFAP) immunohistochemistry shows that the AT2 receptor in the lateral septum showed that the AT2 receptor was not detected in GFAP immunoreactive astroglial cells, therefore indicating that AT2 is neuronal rather than glial in this brain region.

Ontogeny of angiotensin II type 2 receptor mRNA expression in fetal and neonatal rat brain.

Recent studies have provided evidence for a specific role of the angiotensin II type 2 receptor (AT2) in in vitro neuron differentiation, and in AT2 knock-out mice that display central neurological anomalies. The role of AT2 in brain development is currently unknown. By using radiolabeled cRNA probes for in situ hybridization histochemistry, we determined the ontogenic development of AT2 mRNA in fetal and neonatal rat brain, from 11 days of gestation (E11) to 28 days postnatal (P28). Brain AT2 mRNA is first detected in the lateral hypothalamic neuroepithelium at E13. AT2 mRNA is detected beginning at E15 in the subthalamic and hypoglossus nuclei; at E17 in the pedunculopontine nucleus, cerebellum, motor facial nucleus, and the inferior olivary complex; at E19 in the thalamus, bed nucleus of the supraoptic decussation, interstitial nucleus of Cajal, nuclei of the lateral lemniscus, locus coeruleus, and supragenual nucleus; and at E21 in the lateral septal and medial amygdaloid nuclei, medial geniculate body, and the superior colliculus. The substantia nigra and many telencephalic and medullary nuclei express AT2 mRNA only after birth. Certain structures express AT2 mRNA strongly but transiently during embryonic life, such as the differentiating lateral hypothalamic area at E13, the superior olivary complex at E19 and E21, and the red nucleus at E15 and E17. In conclusion, during brain development, expression of AT2 mRNA appears early at E13, is strongly but transiently expressed in certain structures, and is high and persists until brain maturity in nuclei involved in motor functions and sensory integration. Our results support a dual role of AT2 during brain development in early maturation and differentiation, but also in modulation of established functions during perinatal and adult life.

Ontogeny of angiotensin II type 1 receptor mRNAs in fetal and neonatal rat brain.

Studies have demonstrated a specific function of the angiotensin II (Ang II) type 1 receptor (AT(1)) in regulation of adult central cardiovascular, fluid, and pituitary hormone release and a predominant role of the renin-angiotensin system in fetal and neonatal cardiovascular homeostasis. The pattern of brain AT(1) mRNA expression during fetal and neonatal development is currently unknown. We used radiolabeled cRNA probes for in situ hybridization histochemistry to determine the ontogenic development of the two AT(1) subtypes (AT(1a) and AT(1b)) mRNA in rat brain, from 11 days of gestation (E11) to 28 days after birth (P28). No AT(1b) mRNA was detected in the developing brain, whereas AT(1a) mRNA was first detected at E19. The age at which AT(1a) mRNA is first detected varied among different brain areas and expression predominates in areas involved in fluid homeostasis, pituitary hormone release, and cardiovascular regulation, where it persists until P28. AT(1a) mRNA expression is present from E19 onward in the median preoptic nucleus, the vascular organ of the lamina terminalis, the paraventricular nucleus, the periaqueductal gray, the nucleus raphe pallidus, the motor facial nucleus, and very weakly in the nucleus of the solitary tract and the ambiguous nucleus, and at E21 in the subfornical organ, the anterior olfactory nucleus and the piriform cortex. AT(1a) mRNA expression is present after birth in many regions, including the preoptic and lateral hypothalamic areas, the area postrema and medullary reticular nuclei. In conclusion, during brain development, expression of AT(1a) mRNA, appears in late gestation at E19, predominantly in forebrain areas involved in fluid homeostasis and cardiovascular regulation. In contrast, AT(1a) mRNA expression is absent or present only in very small amounts until after birth in many medullary nuclei, known to play an important role in cardiovascular modulation. Our results suggest that, in perinatal life, AT(1a) is involved in fluid and perhaps cardiovascular homeostasis and that the role of Ang II in modulating medullary cardiovascular centers matures later in postnatal life.

Differential inhibition of aminopeptidase A and aminopeptidase N by new beta-amino thiols.

Aminopeptidase A (APA) is a highly selective peptidase, which cleaves the N-terminal Glu or Asp residues of biologically active peptides, and has therefore been proposed to be involved in angiotensin II and CCK8 metabolism. Highly potent and selective APA inhibitors are consequently required to study the physiological regulation of these two peptides. Using, as a model, Glu-thiol (4-amino-5-mercaptopentanoic acid), which was the first efficient APA inhibitor described but is however equipotent on APA (0.14 microM) and aminopeptidase N (APN) (0.12 microM), several beta-amino thiol inhibitors have been synthesized. In these molecules, the length of the side chain was varied and the carboxylate group of Glu-thiol was replaced by other negatively charged groups, such as phosphonate, sulfonate, hydroxamate, and thiol. The inhibitory potency of one of these compounds, 22h (S)-3-amino-4-mercaptobutanesulfonate, was found to be nearly 100-fold better for APA than for APN, with an affinity (0.29 microM) almost equivalent to that of Glu-thiol. Hence, this compound is the first selective APA inhibitor reported, and as such, it should be an interesting probe to explore the physiological involvement of APA in the metabolism of neuropeptides like angiotensin II and CCK8.

Investigation of the active site of aminopeptidase A using a series of new thiol-containing inhibitors.

Aminopeptidase A (APA) and aminopeptidase N (APN) are two metallopeptidases which have been suggested to be involved in the enzymatic cascade of the renin-angiotensin system. APA liberates angotensin III from angiotensin II by releasing the N-terminal aspartate, and APN participates in the inactivation of angiotensin III. As the role of angiotensin III in the regulation of blood pressure in the central nervous system and at the periphery is controversial, it was of interest to develop selective and efficient inhibitors of APA. Starting from Glu-thiol(1), which was the first efficient APA inhibitor described, but however is equipotent on APA (Ki = 0.14 microM) and APN (Ki = 0.12 microM), beta-amino thiols bearing various carboxyalkyl chains have been synthesized and their inhibitory potencies measured on both purified enzymes. Compounds containing a carboxylated aromatic ring inhibited APA and APN with Ki values in the micromolar range but were slightly more active on APA. Conversely, inhibitors containing a cyclohexyl ring were more efficient on APN. Various modifications of the structure of Glu-thiol decreased inhibitory activity on both enzymes but increased the selectivity for APA, and compound 9d ((S)-4-amino-6-mercaptohexanoic acid) was 23 times more potent on APA (Ki = 2.0 microM) than on APN (Ki = 45 microM).

Investigation of subsite preferences in aminopeptidase A (EC 3.4.11.7) led to the design of the first highly potent and selective inhibitors of this enzyme.

The study of the physiological roles of the membrane-bound zinc-aminopeptidase A (glutamyl aminopeptidase, EC 3.4.11.7) needs the design of efficient and selective inhibitors of this enzyme. An acute exploration of aminopeptidase A active site was performed by a combinatorial approach using (3-amino-2-mercapto-acyl)dipeptides able to fit its S(1), S(1)', and S(2)' subsites. This analysis confirmed that the S(1) subsite is optimally blocked by a glutamate or isosteric residues and demonstrated that the S(1)' subsite is hydrophobic whereas the S(2)' subsite recognizes preferentially negatively charged residues derived from aspartic acid. The optimization of these structural parameters led to the synthesis of nanomolar and subnanomolar inhibitors of aminopeptidase A such as H(3)N(+)CH(CH(2)CH(2)SO(3)(-))CH(SH)CO-Ile-(3-COOH)Pro that exhibits a K(i) of 0.87 nM. The best compounds were synthesized by a stereochemically controlled route. These first described highly potent inhibitors could allow studies about the role of physiological substrates of APA such as angiotensin II and cholecystokinin CCK(8) in the central nervous system.

Distribution of apelin-synthesizing neurons in the adult rat brain.

The peptide apelin originating from a larger precursor preproapelin molecule has been recently isolated and identified as the endogenous ligand of the human orphan G protein-coupled receptor, APJ (putative receptor protein related to the angiotensin receptor AT(1)). We have shown recently that apelin and apelin receptor mRNA are expressed in brain and that the centrally injected apelin fragment K17F (Lys(1)-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe(17)) decreased vasopressin release and altered drinking behavior. Using a specific polyclonal antiserum against K17F for immunohistochemistry, the aim of the present study was to establish the precise topographical distribution of apelin immunoreactivity in colchicine-treated adult rat brain. Immunoreactivity was essentially detected in neuronal cell bodies and fibers throughout the entire neuroaxis in different densities. Cells bodies have been visualized in the preoptic region, the hypothalamic supraoptic and paraventricular nuclei and in the highest density, in the arcuate nucleus. Apelin immunoreactive cell bodies were also seen in the pons and the medulla oblongata. Apelin nerve fibers appear more widely distributed than neuronal apelin cell bodies. The hypothalamus represented, by far, the major site of apelin-positive nerve fibers which were found in the suprachiasmatic, periventricular, dorsomedial, ventromedial nuclei and in the retrochiasmatic area, with the highest density in the internal layer of the median eminence. Fibers were also found innervating other circumventricular organs such as the vascular organ of the lamina terminalis, the subfornical and the subcommissural organs and the area postrema. Apelin was also detected in the septum and the amygdala and in high density in the paraventricular thalamic nucleus, the periaqueductal central gray matter and dorsal raphe nucleus, the parabrachial and Barrington nuclei in the pons and in the nucleus of the solitary tract, lateral reticular, prepositus hypoglossal and spinal trigeminal nuclei. The topographical distribution of apelinergic neurons in the brain suggests multiple roles for apelin especially in the central control of ingestive behaviors, pituitary hormone release and circadian rhythms.

Distribution of angiotensin type-1 receptor messenger RNA expression in the adult rat brain.

Angiotensin II and angiotensin III in the brain exert their various effects by acting on two pharmacologically well-defined receptors, the type-1 (AT1) and the type-2 (AT2) receptors. Receptor binding autoradiography has revealed the dominant presence of AT1 in brain nuclei involved in cardiovascular, body fluid and neuroendocrine control. The cloning of the AT1 complementary DNA has revealed the existence of two receptor subtypes in rodents, AT1A and AT1B. Using specific riboprobes for in situ hybridization, we have previously shown that the AT1A messenger RNA is predominantly expressed in the rat forebrain; in contrast the AT1B subtype predominates in the anterior pituitary. Using a similar technical approach, the aim of the present study was to establish the precise anatomical localization of cells synthetising the AT1A receptor in the adult rat brain. High AT1A messenger RNA expression was found in the vascular organ of the lamina terminalis, the median preoptic nucleus, the subfornical organ, the hypothalamic periventricular nucleus, the parvocellular parts of the paraventricular nucleus, the nucleus of the solitary tract and the area postrema, in agreement with previous autoradiographic studies, describing a high density of AT1 binding sites in these nuclei. In addition, AT1A messenger RNA expression was detected in several brain areas, where no AT1 binding was reported previously. Thus, we identify strong expression of AT1A messenger RNA expression in scattered cells of the lateral parts of the preoptic region, the lateral hypothalamus and several brainstem nuclei. In none of these structures was the AT1B messenger RNA detectable at the microscopic level. In conclusion, it is suggested that angiotensins may exert their central effects on body fluid and cardiovascular homeostasis mainly via the AT1A receptor subtype.

Aminopeptidase A: distribution in rat brain nuclei and increased activity in spontaneously hypertensive rats.

Aminopeptidase A is a membrane-bound zinc metalloprotease which cleaves angiotensin II into angiotensin III. Using a new specific aminopeptidase A inhibitor, EC33, we evaluated its enzymatic activity in several microdissected brain nuclei involved in the control of cardiovascular functions and in the pituitary. We compared this distribution with that of the angiotensin I-converting enzyme which converts angiotensin I to angiotensin II. Aminopeptidase A activity was heterogenously distributed with a 150-fold difference between the lowest and the highest levels. The pituitary and the circumventricular organs were the richest source of enzyme, followed by the median eminence, the arcuate nucleus, the area postrema, the choroid plexus and the supraotic and paraventricular nuclei. We did not find any close parallel between aminopeptidase A and angiotensin I-converting enzyme distributions. We examined both enzymatic activities in brain nuclei of spontaneously hypertensive rats. Aminopeptidase A activity was higher in the spontaneously hypertensive rats than in age-matched Wistar Kyoto control rats. The difference was up to 2.5-fold in several brain nuclei involved in the blood pressure regulation; in contrast, no differences in angiotensin I-converting enzyme activity were found in the same regions. The close correspondence between the distribution of aminopeptidase A activity and angiotensin receptors and nerve terminals in the brain associated with the observation that aminopeptidase A activity was overactivated in the spontaneously hypertensive rats suggest that this enzyme may contribute, at least in part, to the regulation of cardiovascular functions by its ability to convert angiotensin II to angiotensin III.

A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.