Both sunitinib and sorafenib are effective treatments for pheochromocytoma in a xenograft model.

Pheochromocytomas and paragangliomas are rare neuroendocrine tumors which develop from chromaffin cells of the adrenal medulla and extra-adrenal sites, leading to excess catecholamine release and hypertension. Many of the tumors are characterized by a high vascularity, suggesting the possible implementation of anti-angiogenic therapies for patients. Here, the efficacy of the tyrosine kinase inhibitors sunitinib and sorafenib was investigated in vivo and in vitro. Oral treatment with either sunitinib or sorafenib (40mg/kg/day) for 14days induced a marked reduction in the volume and weight of PC12 pheochromocytoma cell tumor xenografts in mice. Assessment of tumoral neo-angiogenesis, assessed by morphometric analysis of the vascular network after CD31 immunolabeling, showed that both sunitinib and sorafenib reduced the microvessel area (-85% and -80%, respectively) and length (-80% and -78%, respectively) in treated compared to control tumors. In addition, the number of vessel nodes was significantly lower in treated tumors (-95% and -84%, respectively). Furthermore, cleaved caspase 3 immunolabeling revealed a marked increase in the number of apoptotic cells in tumors from treated animals. Sunitinib and sorafenib could exert a direct effect on PC12 cell viability in vitro. While sunitinib induced a rapid (4h) and pronounced (5-fold) increase in caspase-3/7-dependent apoptosis, sorafenib seems to exert its cytotoxic activity through a different mechanism. Altogether, our data demonstrate that sunitinib and sorafenib have the ability to impair pheochromocytoma development by inhibiting angiogenesis and reducing tumor cell viability. These results strongly suggest that both sunitinib and sorafenib could represent valuable therapeutic tools for pheochromocytoma.

Differential expression and processing of secretogranin II in relation to the status of pheochromocytoma: implications for the production of the tumoral marker EM66.

We have previously demonstrated that measurement of tissue concentrations of the secretogranin II (SgII or SCG2 as listed in the HUGO database)-derived peptide EM66 may help to discriminate between benign and malignant pheochromocytomas and that EM66 represents a sensitive plasma marker of pheochromocytomas. Here, we investigated the gene expression and protein production of SgII in 13 normal adrenal glands, and 35 benign and 16 malignant pheochromocytomas with the goal to examine the molecular mechanisms leading to the marked variations in the expression of EM66 in tumoral chromaffin tissue. EM66 peptide levels were 16-fold higher in benign than in malignant pheochromocytomas and had an area under the receiver-operating characteristic curve of 0.95 for the distinction of benign and malignant tumors. Q-PCR experiments indicated that the SgII gene was significantly underexpressed in malignant tumors compared with benign tumors. Western blot analysis using antisera directed against SgII and SgII-derived fragments revealed lower SgII protein and SgII-processing products in malignant tumors. Western blot also showed that low p-cAMP-responsive element-binding (CREB) concentrations seemed to be associated with the malignant status. In addition, the prohormone convertase PC1 and PC2 genes and proteins were overexpressed in benign pheochromocytomas compared with malignant pheochromocytomas. Low concentrations of EM66 found in malignant tumors are associated with reduced expression and production of SgII and SgII-derived peptides that could be ascribed to a decrease in SgII gene transcription, probably linked to p-CREB down-regulation, and to lower PC levels. These findings highlight the mechanisms leading to lower concentrations of EM66 in malignant pheochromocytoma and strengthen the notion that this peptide is a suitable marker of this neuroendocrine tumor.

Clinical and dimensional characteristics of euthymic bipolar patients with or without suicidal behavior.

BACKGROUND: The clinical and dimensional features associated with suicidal behaviour in bipolar patients during euthymic states are not well characterised. METHODS: In a sample of 652 euthymic bipolar patients, we assessed clinical features with the Diagnostic Interview for Genetics Studies (DIGS) and dimensional characteristics with questionnaires measuring impulsivity/hostility and affective lability/intensity. Bipolar patients with and without suicidal behaviour were compared for these clinical and dimensional variables. RESULTS: Of the 652 subjects, 42.9% had experienced at least one suicide attempt. Lifetime history of suicidal behaviour was associated with being a woman, a history of head injury, tobacco misuse and indicators of severity of bipolar disorder including early age at onset, high number of depressive episodes, positive history of rapid cycling, alcohol misuse and social phobia. Indirect hostility and irritability were dimensional characteristics associated with suicidal behaviour in bipolar patients, whereas impulsivity and affective lability/intensity were not associated with suicidal behaviour. LIMITATIONS: This study had a retrospective design with no replication sample. CONCLUSIONS: Bipolar patients with earlier onset, mood instability (large number of depressive episodes, rapid cycling) and/or particular addictive and anxiety comorbid disorders might be at high risk of suicidal behaviour. In addition, hostility dimensions (indirect hostility and irritability), may be trait components associated with suicidal behaviour in euthymic bipolar patients.

Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibians.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide of the secretin/glucagon/vasoactive intestinal polypeptide superfamily, has been initially characterized in mammals in 1989 and, only 2 years later, its counterpart has been isolated in amphibians. A number of studies conducted in the frog Rana ridibunda have demonstrated that PACAP is widely distributed in the central nervous system (particularly in the hypothalamus and the median eminence) and in peripheral organs including the adrenal gland. The cDNAs encoding the PACAP precursor and 3 types of PACAP receptors have been cloned in amphibians and their distribution has been determined by in situ hybridization histochemistry. Ontogenetic studies have revealed that PACAP is expressed early in the brain of tadpoles, soon after hatching. In the frog Rana ridibunda, PACAP exerts a large array of biological effects in the brain, pituitary, adrenal gland, and ovary, suggesting that, in amphibians as in mammals, PACAP may act as neurotrophic factor, a neurotransmitter and a neurohormone.

Pituitary adenylate cyclase-activating polypeptide stimulates secretoneurin release and secretogranin II gene transcription in bovine adrenochromaffin cells through multiple signaling pathways and increased binding of pre-existing activator protein-1-like transcription factors.

Secretoneurin (SN) is a novel bioactive peptide that derives from the neuroendocrine protein secretogranin II (SgII) by proteolytic processing and participates in neuro-immune communication. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP-38) dose-dependently stimulates (EC(50) approximately 3 nM) SN release (up to 4-fold) and SgII gene expression (up to 60-fold) in cultured bovine adrenochromaffin cells. The effect of PACAP on both SN secretion and SgII mRNA levels is rapid and long lasting. We analyzed in this neuroendocrine cell model the transduction pathways involved in both SN secretion and SgII gene transcription in response to PACAP. The cytosolic calcium chelator BAPTA-AM and the nonselective calcium channel antagonist NiCl(2) equally inhibited both secretion of the peptide and transcription of the SgII gene, indicating a major contribution of calcium influx in PACAP-induced SN biosynthesis and release in chromaffin cells. Inhibition of protein kinase A (PKA) or C (PKC) also reduced PACAP-evoked SN release but did not alter the stimulatory effect of PACAP on SgII mRNA levels. Conversely, application of mitogen-activated protein kinase inhibitors suppressed PACAP-induced SgII gene expression. The effect of PACAP on SgII mRNA levels, like the effect of the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA), was not affected by cycloheximide, whereas the effects of the PKA stimulator forskolin or cell-depolarization by high K(+) were significantly reduced by the protein synthesis inhibitor. PACAP and TPA both increased the binding activity of the SgII cAMP response element to trans-acting factors present in chromaffin cell nuclear extracts, which are recognized by antibodies to activator protein-1-related proteins. These data indicate that SN biosynthesis is regulated by PACAP in chromaffin cells through complex signaling cascades, suggesting that SN may play a function during trans-synaptic stimulation of the adrenal medulla.

Ontogeny of pituitary adenylate cyclase-activating polypeptide (PACAP) in the frog (Rana ridibunda) tadpole brain: immunohistochemical localization and biochemical characterization.

The anatomic distribution and biochemical characteristics of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in the central nervous system of the frog, Rana ridibunda, during development. Three to four days after hatching, at stages IV-VII, PACAP-immunoreactive perikarya were detected in the dorsal thalamus within the anterior ventral area, and a few fibers were found in the medial pallium. Positive cell bodies were first observed in the hypothalamus at stages VIII-IX, at the level of the dorsal and ventral infundibular nuclei. In these regions, the number of positive perikarya increased during ontogeny. In tadpoles, during the mid- and late premetamorphosis, a more complex organization of the PACAP-immunoreactive system was found in the thalamus with the appearance, at stages IX-XII, of two additional groups of positive neurons in the ventrolateral area and posterocentral nucleus. At stages XIII-XVIII of larval development and subsequent larval stages, PACAP-immunoreactive fibers were found in the median eminence. In newly metamorphosed animals, several additional groups of positive perikarya appeared in the medial pallium, the preoptic nucleus, the torus semicircularis, the tegmentum of the mesencephalon, and the cerebellum. The immunoreactive peptide contained in the tadpole brain was characterized by high performance liquid chromatography analysis combined with radioimmunoassay quantification. At all stages investigated, the predominant form of PACAP-immunoreactive material coeluted with synthetic frog PACAP38. The occurrence of PACAP soon after hatching indicates that the peptide may exert neurotrophic activities. The existence of immunoreactive elements in several thalamic regions at mid- and late premetamorphic stages suggests that PACAP may act as a neurotransmitter, neuromodulator, or both, during ontogenesis. Finally, the presence of PACAP-immunoreactive perikarya in hypothalamic nuclei and nerve fibers in the median eminence supports the view that PACAP may play a role in the control of pituitary hormone secretion during larval development.

Molecular evolution of the growth hormone-releasing hormone/pituitary adenylate cyclase-activating polypeptide gene family. Functional implication in the regulation of growth hormone secretion.

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to the same superfamily of regulatory neuropeptides and have both been characterized on the basis of their hypophysiotropic activities. This review describes the molecular evolution of the GHRH/PACAP gene family from urochordates to mammals and presents the hypothesis that the respective roles of GHRH and PACAP in the control of GH secretion are totally inverted in phylogenetically distant groups of vertebrates. In mammals, GHRH and PACAP originate from distinct precursors whereas, in all submammalian taxa investigated so far, including birds, amphibians and fish, a single precursor encompasses a GHRH-like peptide and PACAP. In mammals, GHRH-containing neurons are confined to the infundibular and dorsomedial nuclei of the hypothalamus while PACAP-producing neurons are widely distributed in hypothalamic and extrahypothalamic areas. In fish, both GHRH- and PACAP-immunoreactive neurons are restricted to the diencephalon and directly innervate the adenohypophysis. In mammals and birds, GHRH plays a predominant role in the control of GH secretion. In amphibians, both GHRH and PACAP are potent stimulators of GH release. In fish, PACAP strongly activates GH release whereas GHRH has little or no effect on GH secretion. The GHRH/PACAP family of peptides thus provides a unique model in which to investigate the structural and functional facets of evolution.

Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide that was first isolated from ovine hypothalamic extracts on the basis of its ability to stimulate cAMP formation in anterior pituitary cells. PACAP belongs to the vasoactive intestinal polypeptide (VIP)-glucagon-growth hormone releasing factor-secretin superfamily. The sequence of PACAP has been remarkably well conserved during the evolution from protochordate to mammals, suggesting that PACAP is involved in the regulation of important biological functions. PACAP is widely distributed in the brain and peripheral organs, notably in the endocrine pancreas, gonads, and respiratory and urogenital tracts. Characterization of the PACAP precursor has revealed the existence of a PACAP-related peptide whose activity remains unknown. Two types of PACAP binding sites have been characterized. Type I binding sites exhibit a high affinity for PACAP and a much lower affinity for VIP whereas type II binding sites have similar affinity for PACAP and VIP. Molecular cloning of PACAP receptors has shown the existence of three distinct receptor subtypes, the PACAP-specific PAC1 receptor, which is coupled to several transduction systems, and the two PACAP/VIP-indifferent VPAC1 and VPAC2 receptors, which are primarily coupled to adenylyl cyclase. PAC1 receptors are particularly abundant in the brain and pituitary and adrenal glands whereas VPAC receptors are expressed mainly in the lung, liver, and testis. The wide distribution of PACAP and PACAP receptors has led to an explosion of studies aimed at determining the pharmacological effects and biological functions of the peptide. This report reviews the current knowledge concerning the multiple actions of PACAP in the central nervous system and in various peripheral organs including the endocrine glands, the airways, and the cardiovascular and immune systems, as well as the different effects of PACAP on a number of tumor cell types.

Occurrence and effect of PACAP in the human fetal adrenal gland.

In the study reported in this paper, we characterized PACAP in the human fetal adrenal gland and we investigated the effect of PACAP on steroid secretion from cultured fetal adrenal cells. The adrenal gland from 20-week-old fetuses contained substantial concentrations of PACAP-immunoreactive 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 beta-adrenoreceptor agonist isoproterenol mimicked the stimulatory effect of PACAP on both steroid secretion whereas preincubation of fetal cells with the beta-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.

The effect of the endozepine triakontatetraneuropeptide on corticosteroid secretion by the frog adrenal gland is mediated by activation of adenylyl cyclase and calcium influx through T-type calcium channels.

We have recently found that, in the frog adrenal gland, endozepines are present in chromaffin cells and we have shown that the triakontatetraneuropeptide TTN is a potent stimulator of corticosteroid secretion in vitro. In the present study, we have investigated the transduction mechanisms mediating the corticotropic effect of TTN on adrenocortical cells. Incubation of adrenal explants with graded concentrations of TTN induced a dose-dependent increase in cAMP formation, but did not affect polyphosphoinositide metabolism. Pretreatment of adrenal cells with the protein kinase A inhibitor H-89 markedly reduced the stimulatory effect of TTN on corticosterone and aldosterone secretion by perifused cells, whereas the phospholipase C inhibitor U-73122 did not affect the TTN-evoked stimulation of corticosteroid output. Incubation of adrenal cells with cholera toxin abolished the stimulatory effect of TTN on steroid secretion. Administration of a brief pulse of TTN (10(-6) M) in the vicinity of cultured adrenocortical cells induced a robust increase in the concentration of intracellular calcium ([Ca2+]i). Repeated pulses of TTN resulted in a gradual attenuation of the responses, indicating the existence of a desensitization phenomenon. Incubation of the cells with the T-type calcium channel blocker mibefradil significantly reduced the TTN-evoked [Ca2+]i increase, whereas the L-type calcium channel blocker nifedipine and the N-type calcium channel blocker omega-conotoxin GVIA had no effect. Incubation of adrenal cells with H-89 markedly reduced the stimulatory effect of TTN on [Ca2+]i. The involvement of calcium in steroid secretion induced by TTN has also been investigated. Administration of mibefradil significantly reduced the TTN-evoked stimulation of steroid production, whereas nifedipine was devoid of effect. Taken together, these data indicate that in frog adrenocortical cells, the endozepine TTN stimulates cAMP formation and calcium entry through T-type calcium channels. The effects of TTN on the adenylyl cyclase/protein kinase A pathway and calcium influx both contribute to the stimulatory action of the peptide on corticosteroid secretion.

Characterization and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites in the brain of the frog Rana ridibunda.

The biochemical characteristics and the distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites have been investigated in the brain of the frog Rana ridibunda by using [(125)I]PACAP27 as a radioligand. Membrane-binding studies revealed the existence of high-affinity receptors for frog PACAP38 and PACAP27. In contrast, the [Des-His(1)]PACAP38 analogue had a much lower affinity and vasoactive intestinal polypeptide did not produce any displacement of the binding. Autoradiographic labeling of frozen brain sections revealed that the highest concentrations of PACAP receptors were located in the olfactory bulb, pallium, striatum, habenular nuclei, ventromedial thalamic nucleus, corpus geniculatum, posterior tubercle, dorsal part of the magnocellular preoptic nucleus, tectum, and the molecular cell layer of the cerebellum. Moderate binding was observed in the septum, in most parts of the thalamus, the dorsal hypothalamic nucleus, the median eminence, the ventral nuclei of the tegmentum, the torus semicircularis, and the interpeduncular and isthmi nuclei. The present data provide the first biochemical characterization and anatomic distribution of PACAP binding sites in the brain of a nonmammalian vertebrate species. The widespread distribution of specific PACAP receptors in the frog brain suggests that the peptide does not act solely as a hypophysiotropic factor, but likely fulfills neurotransmitter functions, neuromodulator functions, or both.

Distribution, characterization, and growth hormone-releasing activity of pituitary adenylate cyclase-activating polypeptide in the European eel, Anguilla anguilla.

The complementary DNA encoding pituitary adenylate cyclase-activating polypeptide (PACAP) has been cloned from two species of teleost fishes, the Sockeye salmon and the Thai catfish, and the amino acid sequence of PACAP has been determined in another teleost, the stargazer. However, to date, the detailed distribution of PACAP immunoreactivity has never been investigated in the fish brain. In the present study, we have determined the localization of PACAP-immunoreactive neurons in the central nervous system of a primitive teleost fish, the European eel Anguilla anguilla, using an antiserum raised against PACAP27. PACAP-positive perikarya were exclusively observed in the diencephalon, i.e. in the preoptic nucleus of the hypothalamus and in the dorsal and ventral nuclei of the thalamus. PACAP-immunoreactive fibers were detected in various areas of the brain, notably in the ventral telencephalon, the diencephalon, the mesencephalon, the cerebellar valvula, and the medulla oblongata. In addition, a dense accumulation of PACAP-containing nerve terminals was found in the pars distalis of the pituitary. The PACAP-like immunoreactivity contained in the eel brain was characterized by HPLC analysis combined with RIA quantification. The major form of PACAP-immunoreactive material coeluted with mammalian PACAP38. Molecular cloning of the PACAP precursor has previously shown that in fish, PACAP and GH-releasing hormone (GHRH) originate from the same precursor. We have thus investigated the effects of PACAP and GHRH on GH secretion from eel pituitary cells in primary culture. Dose-response experiments revealed that PACAP27 and PACAP38 possessed the same efficacy, but PACAP38 was 12 times more potent than PACAP27 in stimulating GH release (ED50 = 4.3 x 10(-10) and 3.5 x 10(-9) M, respectively). In contrast, GHRH, even at a high concentration (10(-6) M), had no effect on GH release. Taken together, these data indicate that in the eel, PACAP may play a significant role in the regulation of somatotrope cells: 1) PACAP-immunoreactive neurons are exclusively located in the diencephalon and send numerous projections in the pars distalis; and 2) PACAP, but not GHRH, dose dependently stimulates GH secretion from cultured eel pituitary cells.

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.

The distribution and pharmacological properties of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were studied in the fetal human adrenal gland during the second trimester of gestation. Autoradiographic studies, using [125I]PACAP27 as a radioligand, 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 [125I]PACAP27 binding (IC50 = 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 (IC50 = 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 (ED50 = 0.07 +/- 0.02 nmol/L), as well as in cells maintained in culture for 5 days (5.4 +/- 1.8 nmol/L). In contrast, 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 medulla 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.