Increased apelin receptor gene expression in the subfornical organ of spontaneously hypertensive rats.

The vascular organ of the lamina terminalis, subfornical organ (SFO), and area postrema comprise the sensory circumventricular organs (CVO) which are central structures that lie outside the blood brain barrier and are thought to provide an interface between peripherally circulating signals and the brain through their projections to central autonomic structures. The SFO expresses mRNA for the G protein-coupled apelin receptor (APJ, gene name aplnr) and exogenous microinjection of the neuropeptide apelin (apln) to the SFO elicits a depressor effect. Here we investigated the expression and cellular distribution of aplnr, apln and the recently described ligand apela (apela) in the CVOs and investigated whether differences in the levels of expression of apelinergic gene transcripts in these regions might underlie the chronic elevated blood pressure seen in hypertension. We carried out multiplex in situ hybridization histochemistry on CVO tissue sections from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) controls. Confocal immunofluorescent images indicated strong aplnr expression, with lower levels of apln and modest apela expression, in the CVOs of both WKY rats and SHRs, in both neurons and glia. The expression level of aplnr transcripts was increased in the SFO of SHRs compared to WKY rats. Our data may highlight a potential dysfunction in the communication between CVOs and downstream signalling pathways in SHRs, which may contribute to its different phenotype/s

Vasopressin potentiates corticotropin-releasing hormone-induced insulin release from mouse pancreatic β-cells

Arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH) have both been implicated in modulating insulin secretion from pancreatic β-cells. In the present study, we investigated the insulin-secreting activities of AVP and CRH in wild-type and AVP VIb receptor knockout mice. Both neuropeptides stimulated insulin secretion from isolated mouse pancreatic islets. The response of islets to CRH was increased fourfold by concomitant incubation with a subthreshold dose of AVP that alone did not stimulate insulin secretion. Activation of the endogenously expressed M3 receptor by the cholinergic agonist carbachol also potentiated CRH-induced insulin secretion, indicating that the phenomenon may be pathway specific (i.e. Ca2+-phospholipase C) rather than agonist specific. The protein kinase C (PKC) inhibitors Ro-31-8425 and bisindolylmaleimide I attenuated the potentiating effect of AVP on CRH-stimulated insulin secretion and blocked AVP-stimulated insulin secretion. A possible interaction between the PKC and protein kinase A pathways was also investigated. The phorbol ester phorbol myristate acetate (PMA) stimulated insulin secretion, while the addition of both PMA and CRH enhanced insulin secretion over that measured with either PMA or CRH alone. Additionally, no AVP potentiation of CRH-stimulated insulin secretion was observed upon incubation in Ca2+-free Krebs–Ringer buffer. Taken together, the present study suggests a possible synergism between AVP and CRH to release insulin from pancreatic β-cells that relies at least in part on activation of the PKC signaling pathway and is dependent on extracellular Ca2+. This is the first example of a possible interplay between the AVP and CRH systems outside of the hypothalamic–pituitary–adrenal axis

Agonist-induced internalization and desensitization of the apelin receptor

Apelin acts via the G protein-coupled apelin receptor (APJ) to mediate effects on cardiovascular and fluid homeostasis. G protein-coupled receptor (GPCR) trafficking has an important role in the regulation of receptor signalling pathways and cellular functions, however in the case of APJ the mechanisms and proteins involved in apelin-induced trafficking are not well understood. We generated a stable HEK-293 cell line expressing N-terminus HA-tagged mouse (m) APJ, and used a semi-automated imaging protocol to quantitate APJ trafficking and ERK1/2 activation following stimulation with [Pyr(1)]apelin-13. The mechanisms of [Pyr(1)]apelin-13-induced internalization and desensitization were explored using dominant-negative mutant (DNM) cDNA constructs of G protein-coupled receptor kinase 2 (GRK2), β-arrestin1, EPS15 and dynamin. The di-phosphorylated ERK1/2 (ppERK1/2) response to [Pyr(1)]apelin-13 desensitized during sustained stimulation, due to upstream APJ-specific adaptive changes. Furthermore, [Pyr(1)]apelin-13 stimulation caused internalization of mAPJ via clathrin coated vesicles (CCVs) and also caused a rapid reduction in cell surface and whole cell HA-mAPJ. Our data suggest that upon continuous agonist exposure GRK2-mediated phosphorylation targets APJ to CCVs that are internalized from the cell surface in a β-arrestin1-independent, EPS15- and dynamin-dependent manner. Internalization does not appear to contribute to the desensitization of APJ-mediated ppERK1/2 activation in these cells

The effects of apelin on hypothalamic–pituitary–adrenal axis neuroendocrine function are mediated through corticotrophin-releasing factor- and vasopressin-dependent mechanisms

The apelinergic system has a widespread expression in the central nervous system (CNS) including the paraventricular nucleus, supraoptic nucleus and median eminence, and isolated cells of the anterior lobe of the pituitary. This pattern of expression in hypothalamic nuclei known to contain corticotrophin-releasing factor (CRF) and vasopressin (AVP) and to co-ordinate endocrine responses to stress has generated interest in a role for apelin in the modulation of stress, perhaps via the regulation of hormone release from the pituitary. In this study, to determine whether apelin has a central role in the regulation of CRF and AVP neurones, we investigated the effect of i.c.v. administration of pGlu-apelin-13 on neuroendocrine function in male mice pre-treated with the CRF receptor antagonist, α-helical CRF9–41, and in mice-lacking functional AVP V1b receptors (V1bR KO). Administration of pGlu-apelin-13 (1 mg/kg i.c.v.) resulted in significant increases in plasma ACTH and corticosterone (CORT), which were significantly reduced by pre-treatment with α-helical CRF9–41, indicating the involvement of a CRF-dependent mechanism. Additionally, pGlu-apelin-13-mediated increases in both plasma ACTH and CORT were significantly attenuated in V1bR KO animals when compared with wild-type controls, indicating a role for the vasopressinergic system in the regulation of the effects of apelin on neuroendocrine function. Together, these data confirm that the in vivo effects of apelin on hypothalamic–pituitary–adrenal neuroendocrine function appear to be mediated through both CRF- and AVP-dependent mechanisms

Abnormal fluid homeostasis in apelin receptor knockout mice

The apelinergic system, comprised of apelin and its G protein-coupled receptor (APJ; APLNR as given in MGI Database), is expressed within key regions of the central nervous system associated with arginine vasopressin (AVP) synthesis and release as well as in structures involved in the control of drinking behaviour, including the magnocellular neurones of the hypothalamus, circumventricular organs, and the pituitary gland. This localisation is indicative of a possible functional role in fluid homeostasis. We investigated a role for APJ in the regulation of fluid balance using mice deficient for the receptor. Male APJ wild-type and knockout (APJ−/−) mice were housed in metabolic cages to allow determination of water intake and urine volume and osmolality. When provided with free access to water, APJ−/− mice drank significantly less than wild-types, while their urine volume and osmolality did not differ. Water deprivation for 24 h significantly reduced urine volume and increased osmolality in wild-type but not in APJ−/− mice. Baseline plasma AVP concentration increased comparably in both wild-type and APJ−/− mice following dehydration; however, APJ−/− mice were unable to concentrate their urine to the same extent as wild-type mice in response to the V2 agonist desmopressin. Analysis of c-fos (Fos as given in MGI Database) mRNA expression in response to dehydration showed attenuation of expression within the subfornical organ, accentuated expression in the paraventricular nucleus, but no differences in expression in the supraoptic nucleus nor median pre-optic nucleus in APJ−/− mice compared with wild-type. These findings demonstrate a physiological role for APJ in mechanisms of water intake and fluid retention and suggest an anti-diuretic effect of apelin in vivo

Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues

Recently, the G protein-coupled receptor GPR30 has been identified as a novel oestrogen receptor (ER). The distribution of the receptor has been thus far mapped only in the rat central nervous system. This study was undertaken to map the distribution of GPR30 in the mouse brain and rodent peripheral tissues. Immunohistochemistry using an antibody against GPR30 revealed high levels of GPR30 immunoreactivity (ir) in the forebrain (e.g. cortex, hypothalamus and hippocampus), specific nuclei of the midbrain (e.g. the pontine nuclei and locus coeruleus) and the trigeminal nuclei and cerebellum Purkinje layer of the hindbrain in the adult mouse brain. In the rat and mouse periphery, GPR30-ir was detected in the anterior, intermediate and neural lobe of the pituitary, adrenal medulla, renal pelvis and ovary. In situ hybridisation histochemistry using GPR30 riboprobes, revealed intense hybridisation signal for GPR30 in the paraventricular nucleus and supraoptic nucleus (SON) of the hypothalamus, anterior and intermediate lobe of the pituitary, adrenal medulla, renal pelvis and ovary of both rat and mouse. Double immunofluorescence revealed GPR30 was present in both oxytocin and vasopressin neurones of the paraventricular nucleus and SON of the rat and mouse brain. The distribution of GPR30 is distinct from the other traditional ERs and offers an additional way in which oestrogen may mediate its effects in numerous brain regions and endocrine systems in the rodent

Expression and functional implications of the renal apelinergic system in rodents

Apelin binds to the G protein-coupled apelin receptor (APJ; gene name aplnr) to modulate diverse physiological systems including cardiovascular function, and hydromineral and metabolic balance. Recently a second endogenous ligand for APJ, named apela, has been discovered. We confirm that apela activates signal transduction pathways (ERK activation) in cells expressing the cloned rat APJ. Previous studies suggest that exogenous apela is diuretic, attributable wholly or in part to an action on renal APJ. Thus far the cellular distribution of apela in the kidney has not been reported. We have utilized in situ hybridization histochemistry to reveal strong apela labelling in the inner medulla (IM), with lower levels observed in the inner stripe of the outer medulla (ISOM), of rat and mouse kidneys. This contrasts with renal aplnr expression where the converse is apparent, with intense labelling in the ISOM (consistent with vasa recta labelling) and low-moderate hybridization in the IM, in addition to labelling of glomeruli. Apelin is found in sparsely distributed cells amongst more prevalent aplnr-labelled cells in extra-tubular regions of the medulla. This expression profile is supported by RNA-Seq data that shows that apela, but not apelin or aplnr, is highly expressed in microdissected rat kidney tubules. If endogenous tubular apela promotes diuresis in the kidney it could conceivably do this by interacting with APJ in vasculature, or via an unknown receptor in the tubules. The comparative distribution of apela, apelin and aplnr in the rodent kidney lays the foundation for future work on how the renal apelinergic system interacts

Fibrotic interstitial lung disease - palliative care needs:a World-Café qualitative study

Objectives: The importance of palliative care in those with advanced fibrotic interstitial lung diseases (F-ILD) is recognised, but the palliative care requirements of patients and caregivers affected by F-ILD regardless of disease course are not established. We set out to explore this and identify optimal solutions in meeting the needs of a F-ILD population in Ireland. Methods: Implementing a World-Café qualitative research approach, we captured insights evolving, iteratively in interactive small group discussions in response to six predefined topics on palliative care and planning for the future. Thirty-nine stakeholders participated in the World-Café including 12 patients, 13 caregivers, 9 healthcare professionals, 4 industry representatives and 1 representative of the clergy. Results: Palliative care emerged as fundamental to the care and treatment of F-ILDs, regardless of disease progression. Unmet palliative care needs were identified as psychological and social support, disease education, inclusion of caregivers and practical/legal advice for disease progression and end-of-life planning. Participants identified diagnosis as a particularly distressing time for patients and families. They called for the introduction of palliative care discussions at this early-stage alongside improvements in integrated care, specifically increasing the involvement of primary care practitioners in referrals to palliative services. Conclusion: Patients and caregivers need discussions on palliative care associated with F-ILD to be included at the point of diagnosis. This approach may address persisting inadequacies in service provision previously identified over the course of the last decade in the UK, Ireland and European F-ILD patient charters.</p