PAC 1 Receptor Activation by PACAP-38 Mediates Ca 2؉ Release from a cAMP-dependent Pool in Human Fetal Adrenal Gland Chromaffin Cells* Downloaded from

International audiencePrevious studies have shown that human fetal adre-nal gland from 17-to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaf-fin cells. The aim of the present study was to identify PACAP receptor isoforms and to determine whether PACAP can affect intracellular calcium concentration ([Ca 2؉ ] i) and catecholamine secretion. Using primary cultures and specific stimulation of chromaffin cells, we demonstrate that PACAP-38 induced an increase in [Ca 2؉ ] i that was blocked by PACAP (6-38), was independent of external Ca 2؉ , and originated from thapsi-gargin-insensitive internal stores. The PACAP-triggered Ca 2؉ increase was not affected by inhibition of PLC␤ (preincubation with U-73122) or by pretreatment of cells with Xestospongin C, indicating that the inosi-tol 1,4,5-triphosphate-sensitive stores were not mobilized. However, forskolin (FSK), which raises cytosolic cAMP, induced an increase in Ca 2؉ similar to that recorded with PACAP-38. Blockage of PKA by H-89 or (R p)-cAMPS suppressed both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was also abolished by emptying the caffeine/ryanodine-sensitive Ca 2؉ stores. Furthermore, treatment of cells with or-thovanadate (100 M) impaired Ca 2؉ reloading of PACAP-sensitive stores indicating that PACAP-38 can mobilize Ca 2؉ from secretory vesicles. Moreover, PACAP induced catecholamine secretion by chromaf-fin cells. It is concluded that PACAP-38, through the PAC 1 receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion , through nonclassical, recently described, ryano-dine/caffeine-sensitive pools, involving a cAMP-and PKA-dependent phosphorylation mechanism. Pituitary adenylate cyclase-activating polypeptide is a 38-residue ␣-amidated neuropeptide (PACAP-38) 1 originally isolated from the ovine hypothalamus for its ability to stimulate cAMP formation in rat anterior pituitary cells. Processing of PACAP-38 can generate a 27-amino acid amidated peptide (PACAP-27) that exhibits 68% sequence identity with vasoac-tive intestinal polypeptide (VIP), thus identifying PACAP as a member of the VIP/secretin/glucagon superfamily of regulatory peptides (1, 2). The effects of PACAP are mediated through interaction with two types of high affinity receptors: type I receptors are selectively activated by PACAP, whereas type II receptors bind PACAP and VIP with similar affinity (3). Three isoforms of PACAP receptors have now been cloned and designated as PACAP-specific receptor I (PAC 1-R) (4, 5) and VIP/PACAP mutual receptors 1 and 2 (VPAC 1-R and VPAC 2-R) (6, 7). Both PAC 1-R (type 1 receptors) and VPAC 1-R/VPAC 2-R (type 2 receptors) belong to the seven-transmembrane domain, G-protein coupled receptor family, and are all positively coupled to adenylyl cyclase (2). Eight isoforms of PAC 1-R, resulting from alternative splicing, have been characterized to date. These variants display differential signal transduction properties with regard to adenylyl cyclase and phospholipase C (PLC) stimulation (1, 2). In addition to these classical signaling pathways , PACAP has been found to stimulate a Ca 2ϩ-calmodulin nitric oxide synthase (8) and mitogen-activated protein kinase activity (9). These various transduction mechanisms are involved in the neurotrophic activities exerted by PACAP (i.e. inhibition of apoptosis and stimulation of neurite outgrowth) during development (9-11). PACAP and its receptors are actively expressed in the adre-nal medulla (12-14). In particular, we have previously demonstrated the occurrence of PACAP-38 (15) and PACAP binding sites (16) in chromaffin cells from 16-to 20-week-old fetal human adrenal glands. Activation of these receptors by PACAP-38 causes stimulation of cAMP production and induces a modest increase in inositol 1,4,5-triphosphate (IP 3) formation (16), suggesting a role for the neuropeptide in the developin

Récepteurs de l'angiotensine II et développement de la surrénale humaine

La surrénale foetale humaine est morphologiquement et fonctionnellement différente de la glande adulte. Son cortex se compose d'une large zone frontale sécrétant du DHEA/S entourée des cellules du néocortex. Des cellules chromaffines d'origine neurale pénètrent le cortex f¶tal tôt en gestation pour migrer vers le centre de la glande. Après la naissance, la zone f¶tale dégénère, les cellules chromaffines se rassemblent pour former la médullosurrénale et le cortex achève sa différenciation pour donner les 3 zones adultes. Les changements importants dans l'organisation de la glande au cours de la gestation et dans la période postnatale suggèrent l'implication de plusieurs facteurs dans l'ontogenèse de la surrénale au cours du développement. Le but de cette étude était de caractériser les sites de liaison de l'Angiotensine II dans la surrénale f¶tale humaine au cours du second trimestre de gestation et de déterminer le rôle de l'hormone au cours du développement de la surrénale."--Résumé abrégé par UMI

Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays

Abstract: Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase and β-lactamase as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a propriety luciferase termed “NanoLuc” derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity-relationships, we screened a collection of 42,460 PubChem compounds at 10µM using purified enzyme preparations. We then compared hit rates and chemotypes of confirmed actives for each enzyme. The hit rates ranged from <0.1% for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap ranging from 40-70%, while unrelated luciferases such as firefly luciferases, GLuc, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based-enzyme stabilization can lead to rises in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these enzymes

Identification of a Novel Secretogranin II-Derived Peptide (SgII 187-252 ) in Adult and Fetal Human Adrenal Glands Using Antibodies Raised against the Human Recombinant Peptide*

International audienceMolecular cloning of secretogranin II (SgII) in phylogenetically distant species has recently revealed the existence of a highly conserved 66-amino acid peptide flanked by preserved pairs of basic residues. This observation suggested that this peptide, named EM66, which had not been described to date, could be an important processing product of SgII. The aim of the present study was to investigate the possible occurrence of EM66 in the human adrenal gland. The EM66 peptide was generated in Escherichia coli, which was programmed to make a fusion protein containing the human EM66 sequence. The affinity-purified fusion protein was used to raise poly-clonal antibodies in rabbits. The free EM66 peptide was obtained by cleavage of the fusion protein followed by high performance liquid chromatography purification. Immunohistochemical analysis using the EM66 antibodies revealed intense labeling of adrenochromaffin cells in the adult adrenal medulla and the fetal adrenal gland. A sensitive and specific RIA was developed and applied to the detection of EM66-like immunoreactivity in extracts of adult adrenal medulla and whole fetal adrenal gland after high performance liquid chro-matographic analysis. A major immunoreactive species exhibiting the same retention time as recombinant EM66 was detected in both adult and fetal adrenal extracts. Taken together, these data demonstrate that posttranslational processing of SgII actually generates EM66 in the adrenal gland. The strong conservation of the amino acid sequence of EM66 in the vertebrate phylum and the occurrence of the mature peptide in both fetal and adult chromaffin cells suggest that EM66 could play an important physiological role in the human adrenal gland. (J Clin Endocrinol Metab 83: 2944-2951, 1998

Identification of a Novel Secretogranin II-Derived Peptide (SgII 187–252 ) in Adult and Fetal Human Adrenal Glands Using Antibodies Raised against the Human Recombinant Peptide*

International audienceMolecular cloning of secretogranin II (SgII) in phylogenetically distant species has recently revealed the existence of a highly conserved 66-amino acid peptide flanked by preserved pairs of basic residues. This observation suggested that this peptide, named EM66, which had not been described to date, could be an important processing product of SgII. The aim of the present study was to investigate the possible occurrence of EM66 in the human adrenal gland. The EM66 peptide was generated in Escherichia coli, which was programmed to make a fusion protein containing the human EM66 sequence. The affinity-purified fusion protein was used to raise poly-clonal antibodies in rabbits. The free EM66 peptide was obtained by cleavage of the fusion protein followed by high performance liquid chromatography purification. Immunohistochemical analysis using the EM66 antibodies revealed intense labeling of adrenochromaffin cells in the adult adrenal medulla and the fetal adrenal gland. A sensitive and specific RIA was developed and applied to the detection of EM66-like immunoreactivity in extracts of adult adrenal medulla and whole fetal adrenal gland after high performance liquid chro-matographic analysis. A major immunoreactive species exhibiting the same retention time as recombinant EM66 was detected in both adult and fetal adrenal extracts. Taken together, these data demonstrate that posttranslational processing of SgII actually generates EM66 in the adrenal gland. The strong conservation of the amino acid sequence of EM66 in the vertebrate phylum and the occurrence of the mature peptide in both fetal and adult chromaffin cells suggest that EM66 could play an important physiological role in the human adrena

Identification of a Novel Secretogranin II-Derived Peptide (SgII 187-252 ) in Adult and Fetal Human Adrenal Glands Using Antibodies Raised against the Human Recombinant Peptide*

International audienceMolecular cloning of secretogranin II (SgII) in phylogenetically distant species has recently revealed the existence of a highly conserved 66-amino acid peptide flanked by preserved pairs of basic residues. This observation suggested that this peptide, named EM66, which had not been described to date, could be an important processing product of SgII. The aim of the present study was to investigate the possible occurrence of EM66 in the human adrenal gland. The EM66 peptide was generated in Escherichia coli, which was programmed to make a fusion protein containing the human EM66 sequence. The affinity-purified fusion protein was used to raise poly-clonal antibodies in rabbits. The free EM66 peptide was obtained by cleavage of the fusion protein followed by high performance liquid chromatography purification. Immunohistochemical analysis using the EM66 antibodies revealed intense labeling of adrenochromaffin cells in the adult adrenal medulla and the fetal adrenal gland. A sensitive and specific RIA was developed and applied to the detection of EM66-like immunoreactivity in extracts of adult adrenal medulla and whole fetal adrenal gland after high performance liquid chro-matographic analysis. A major immunoreactive species exhibiting the same retention time as recombinant EM66 was detected in both adult and fetal adrenal extracts. Taken together, these data demonstrate that posttranslational processing of SgII actually generates EM66 in the adrenal gland. The strong conservation of the amino acid sequence of EM66 in the vertebrate phylum and the occurrence of the mature peptide in both fetal and adult chromaffin cells suggest that EM66 could play an important physiological role in the human adrenal gland. (J Clin Endocrinol Metab 83: 2944-2951, 1998

Identification of a Novel Secretogranin II-Derived Peptide (SgII 187–252 ) in Adult and Fetal Human Adrenal Glands Using Antibodies Raised against the Human Recombinant Peptide 1

International audienceMolecular cloning of secretogranin II (SgII) in phylogenetically distant species has recently revealed the existence of a highly conserved 66-amino acid peptide flanked by preserved pairs of basic residues. This observation suggested that this peptide, named EM66, which had not been described to date, could be an important processing product of SgII. The aim of the present study was to investigate the possible occurrence of EM66 in the human adrenal gland. The EM66 peptide was generated in Escherichia coli, which was programmed to make a fusion protein containing the human EM66 sequence. The affinity-purified fusion protein was used to raise polyclonal antibodies in rabbits. The free EM66 peptide was obtained by cleavage of the fusion protein followed by high performance liquid chromatography purification. Immunohistochemical analysis using the EM66 antibodies revealed intense labeling of adrenochromaffin cells in the adult adrenal medulla and the fetal adrenal gland. A sensitive and specific RIA was developed and applied to the detection of EM66-like immunoreactivity in extracts of adult adrenal medulla and whole fetal adrenal gland after high performance liquid chromatographic analysis. A major immunoreactive species exhibiting the same retention time as recombinant EM66 was detected in both adult and fetal adrenal extracts. Taken together, these data demonstrate that posttranslational processing of SgII actually generates EM66 in the adrenal gland. The strong conservation of the amino acid sequence of EM66 in the vertebrate phylum and the occurrence of the mature peptide in both fetal and adult chromaffin cells suggest that EM66 could play an important physiological role in the human adrenal gland

Localization, Characterization, and Second Messenger Coupling of Pituitary Adenylate Cyclase-Activating Polypeptide Receptors in the Fetal Human Adrenal Gland during the Second Trimester of Gestation*

International audienceThe distribution and pharmacological properties of pituitary ad-enylate cyclase-activating polypeptide (PACAP) receptors were studied in the fetal human adrenal gland during the second trimester of gestation. Autoradiographic studies, using [ 125 I]PACAP27 as a ra-dioligand, revealed that PACAP-binding sites are exclusively located on chromaffin cells of adrenals from fetuses 14 –20 weeks old. Biochemical characterization of binding revealed the occurrence of a single class of PACAP-binding sites with a dissociation constant value of 0.32– 0.74 nmol/L and a binding capacity of 0.30 – 0.81 pmol/mg wet tissue. PACAP27 and PACAP38 were equipotent in competing for [ 125 I]PACAP27 binding (IC 50 0.28 – 0.64 nmol/L and 0.15– 0.81 nmol/L, respectively), and the Hill coefficients were close to 1. In contrast, vasoactive intestinal polypeptide was much less efficient in displacing the tracer (IC 50 4 –362 nmol/L), and the Hill coefficients were less than 0.6. PACAP38 induced a dose-dependent increase in cAMP production in fetal human adrenal cell suspension (ED 50 0.07 0.02 nmol/L), as well as in cells maintained in culture for 5 days (5.4 1.8 nmol/L). In constrast, PACAP38 induced a modest increase in inositol phosphate formation. These data indicate that type I PACAP receptors are present in the early stages of the human me-dulla organization during the process of migration of chromaffin cells from the periphery to the central part of the gland. The present results suggest that PACAP could be involved in the regulation of the human adrenochromaffin cells during ontogenesis. (J Clin Endocrinol Metab 83: 1299 –1305, 1998

Occurrence and Effect of PACAP in the Human Fetal Adrenal Gland

International audienceIn the study reported in this paper, we characterized PACAP in the human fetal adrenal gland and we investigated the effect of PACAP on ste-roid secretion from cultured fetal adrenal cells. The adrenal gland from 20-week-old fetuses contained substantial concentrations of PACAP-immunore-active material (88.6 ng/g wet tissue). HPLC analysis of adrenal extracts revealed the presence of both PACAP27 and PACAP38, the latter being the predominant form. Incubation of cultured fetal adrenal cells with PACAP38 (10-7 M) significantly increased cortisol and DHEAS secretion. Administration of the ␤-adrenoreceptor agonist isoproterenol mimicked the stimulatory effect of PACAP on both steroid secretion whereas preincubation of fetal cells with the ␤-adrenoreceptor antagonist propranolol suppressed the steroidogenic effect of PACAP. These data, together with the observation that PACAP receptors are exclusively located on chromaffin cells, suggest that, in the fetal human adrenal gland, the effect of PACAP on steroid secretion is mediated via the local release of catecholamines