James A. Roper

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.

G protein-coupled receptors in the hypothalamic paraventricular and supraoptic nuclei - serpentine gateways to neuroendocrine homeostasis

Graphical abstract Highlights ► The paraventricular and supraoptic nuclei of the hypothalamus are regulators of homeostasis. ► Over one hundred G protein-coupled receptors are expressed in each of these nuclei. ► The receptors have many functions including modulating neuropeptide synthesis and release. ► 20–30% of the receptors are ‘orphans’ whose endogenous ligand and function is unknown.

Pituitary-Adrenal Response to Acute and Repeated Mild Restraint, Forced Swim and Change in Environment Stress in Arginine Vasopressin Receptor 1b Knockout Mice

Arginine vasopressin and corticotrophin‐releasing hormone synthesised and released from the hypothalamic paraventricular nucleus are the prime mediators of the hypothalamic‐pituitary‐adrenal (HPA) axis response to stress. These neurohormones act synergistically to stimulate adrenocorticotophin (ACTH) secretion from the anterior pituitary, culminating in an increase in circulating glucocorticoids. Arginine vasopressin mediates this action at the arginine vasopressin 1b receptor (Avpr1b) located on pituitary corticotrophs. Arginine vasopressin is regarded as a minor ACTH secretagogue in rodents but evidence suggests that it has a role in mediating the neuroendocrine response to some acute and chronic stressors. To investigate the role of the Avpr1b in the HPA axis response to an acute and chronic (repeated) stress, we measured the plasma ACTH and corticosterone concentrations in three stress paradigms in both Avpr1b knockout and wild‐type mice. Single acute exposure to restraint, forced swim and change in environment stressors elevated both plasma ACTH and corticosterone concentrations in wild‐type animals. Conversely, the ACTH response to the acute stressors was significantly attenuated in Avpr1b knockout mice compared to their wild‐type counterparts. Plasma corticosterone concentrations were reduced in Avpr1b knockout mice in response to change in environment but not to mild restraint or forced swim stress. Irrespective of genotype, there was no difference in the plasma ACTH or corticosterone concentrations in response to acute and repeated stressors. The data show that a functional Avpr1b is required for an intact pituitary ACTH response to the acute and chronic stressors used in this study. Furthermore, the normal corticosterone response to repeated exposure to change in environment stress also requires the Avpr1b to drive HPA axis responsiveness.

Attenuated Stress Response to Acute Lipopolysaccharide Challenge and Ethanol Administration in Vasopressin V1b Receptor Knockout Mice

The arginine vasopressin (Avp) 1b receptor (Avpr1b) present on anterior pituitary corticotrophs is involved in the stimulation of adrenocorticotrophic hormone (ACTH) secretion, especially during times of stress. Corticotrophin‐releasing hormone (CRH) is considered the major ACTH secretagogue during acute stress whereas Avp appears to be the more dominant mediator of the hypothalamic‐pituitary‐adrenal (HPA) axis response during chronic stress situations. To investigate the role of the Avpr1b in the HPA axis response to acute stress, we measured ACTH and corticosterone (CORT) plasma levels in Avpr1b knockout (KO) mice and wild‐type controls in response to bacterial lipopolysaccharide (LPS) challenge and ethanol (EtOH) administration. Mice deficient in Avpr1b had markedly compromised plasma ACTH and CORT responses to acute (30 min) LPS, but normal ACTH and CORT response to more extended exposure (4 h) to the immune system activator. The plasma ACTH and CORT levels stimulated by intoxicating, sedative doses of EtOH (3.2 and 4 g/kg) were significantly decreased in the Avpr1b KO mice compared to wild‐type littermates. Significantly higher EtOH‐induced plasma ACTH and CORT secretion was measured in female than in male Avpr1b wild‐type mice. There were no differences in the blood alcohol levels following acute EtOH administration in Avpr1b KO or wild‐type mice of either gender. Our results clearly suggest that Avpr1b plays a significant role in the HPA axis response to acute immune stress and EtOH intoxication.

Syndecan and integrin interactomes: large complexes in small spaces

Highlights ► Syndecans are cleared from adhesion sites to allow integrin engagement. ► Literature-curated interactomes differ between syndecan-1 and syndecan-4. ► Overlap between interactomes of syndecan or integrin describe adhesion formation. ► Switching of syndecan signalling by phosphorylation.

The role of the arginine vasopressin Avp1b receptor in the acute neuroendocrine action of antidepressants

In times of stress the hypothalamic-pituitary-adrenal (HPA) axis is activated and releases two neurohormones, corticotropin-releasing hormone (Crh) and arginine vasopressin (Avp), to synergistically stimulate the secretion of adrenocorticotropin hormone (ACTH) from the anterior pituitary, culminating in a rise in circulating glucocorticoids. Avp mediates its actions at the Avp V1b receptor (Avpr1b) present on pituitary corticotropes. Dysregulation of the stress response is associated with the pathophysiology of depression and a major treatment involves increasing the availability of monamines at the synaptic cleft. Acute administration of selective serotonin reuptake inhibitors (SSRI) and tricyclic antidepressants (TCA) has previously been shown to activate the HPA axis. The present study was undertaken to evaluate the involvement of the Avpr1b in the HPA axis response to acute SC administration of an SSRI (fluoxetine 10mg/kg) and a TCA (desipramine 10mg/kg). We measured plasma ACTH and corticosterone (CORT) levels and neuropeptide mRNA expression in the hypothalamic paraventricular nucleus (PVN) of Avpr1b knockout (KO) mice and wild-type controls. Fluoxetine and desipramine administration significantly attenuated plasma ACTH and CORT levels in male and female Avpr1b KO mice when compared to their wild-type counterparts. Avp, oxytocin (Oxt) and Crh mRNA expression in the PVN did not change in fluoxetine-treated male Avpr1b KO or wild-type mice. In contrast, fluoxetine treatment increased PVN Avp mRNA levels in female Avpr1b wild type but not KO animals. PVN Oxt mRNA levels increased in fluoxetine-treated female mice of both genotypes. The data suggests that the Avpr1b is required to drive the HPA axis response to acute antidepressant treatment and provides further evidence of a sexual dichotomy in the regulation of PVN Avp/Oxt gene expression following antidepressant administration.

Role of Nociceptin/Orphanin FQ and NOP Receptors in the Response to Acute and Repeated Restraint Stress in Rats

Central nociceptin/orphanin FQ (N/OFQ)‐expressing neurones are abundantly expressed in the hypothalamus and limbic system and are implicated in the regulation of activity of the hypothalamic‐pituitary‐adrenal axis (HPA) and stress responses. We investigated the role of the endogenous N/OFQ receptor (NOP) system using the nonpeptidic NOP antagonist, JTC‐801 [N‐(4‐amino‐2‐methylquinolin‐6‐yl)‐2‐(4‐ethylphenoxy‐methyl)benzamide monohydrochloride], during the HPA axis response to acute physical/psychological stress (60 min of restraint). Although i.v. JTC‐801 (0.05 mg/kg in 100 μl) had no significant effect on restraint‐induced plasma corticosterone release at 30 or 60 min post‐injection, i.v. JTC‐801 (0.05 mg/kg in 100 μl) in quiescent rats significantly increased basal plasma corticosterone at the 30‐min time‐point compared to i.v. vehicle (1% dimethysulphoxide in sterile saline). Central injection of JTC‐801 i.c.v. was associated with increased Fos expression in the parvocellular paraventricular nucleus 90 min after infusion compared to vehicle control. These findings contrast to the effects of i.c.v. UFP‐101, a NOP antagonist that we have previously shown to have no effect on HPA activity in quiescent rats. To determine whether restraint stress was associated with compensatory changes in N/OFQ precursor (ppN/OFQ) or NOP receptor mRNAs, in a separate study, we undertook reverse transcriptase‐polymerase chain reaction and in situ hybridisation analysis of ppN/OFQ and NOP transcripts in the brains of male Sprague–Dawley rats. In support of an endogenous role for central N/OFQ in psychological stress, we found that acute restraint significantly decreased preproN/OFQ transcript expression in the hippocampus 2 h after stress compared to unstressed controls. PpN/OFQ mRNA was also reduced in the mediodorsal forebrain 4 h after stress. NOP mRNA was reduced in the hypothalamus 2 h after restraint and at 4 h in mediodorsal forebrain and hippocampus. In situ hybridisation analysis showed that acute restraint significantly decreased ppNN/OFQ in the central amygdala, with significantly increased expression in bed nucleus and reticular thalamus associated with repeated restraint. There was a strong trend for reduced NOP mRNA in the bed nucleus of acute and repeated restraint groups, although there were no other significant changes seen. Although the exact mechanisms require elucidation, the findings obtained in the present study provide evidence indicating that the endogenous N/OFQ system is involved in both acute and chronic restraint stress responses. In summary, our findings confirm the significant role of endogenous NOP receptors and tonic N/OFQ function in the response to the psychological stress of restraint.

Attenuated Stress Response to Acute Restraint and Forced Swimming Stress in Arginine Vasopressin 1b Receptor Subtype (Avpr1b) Receptor Knockout Mice and Wild-Type Mice Treated with a Novel Avpr1b Receptor Antagonist

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin‐releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic‐pituitary‐adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild‐type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress‐induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild‐type mice that had been pre‐treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint‐stressed knockout male mice, and in Avpr1b‐antagonist‐treated male wild‐type mice. By contrast, plasma CORT levels were not reduced in acutely restraint‐stressed female knockout animals, or in female wild‐type animals pre‐treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic‐pituitary‐adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors.

Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure

ABSTRACT Sustained forward migration through a fibrillar extracellular matrix requires localization of protrusive signals. Contact with fibronectin at the tip of a cell protrusion activates Rac1, and for linear migration it is necessary to dampen Rac1 activity in off-axial positions and redistribute Rac1 from non-protrusive membrane to the leading edge. Here, we identify interactions between coronin-1C (Coro1C), RCC2 and Rac1 that focus active Rac1 to a single protrusion. Coro1C mediates release of inactive Rac1 from non-protrusive membrane and is necessary for Rac1 redistribution to a protrusive tip and fibronectin-dependent Rac1 activation. The second component, RCC2, attenuates Rac1 activation outside the protrusive tip by binding to the Rac1 switch regions and competitively inhibiting GEF action, thus preventing off-axial protrusion. Depletion of Coro1C or RCC2 by RNA interference causes loss of cell polarity that results in shunting migration in 1D or 3D culture systems. Furthermore, morpholinos against Coro1C or RCC2, or mutation of any of the binding sites in the Rac1–RCC2–Coro1C complex delays the arrival of neural crest derivatives at the correct location in developing zebrafish, demonstrating the crucial role in migration guidance in vivo.

Ultrasonic Stimulation of Mouse Skin Reverses the Healing Delays in Diabetes and Aging by Activation of Rac1

Chronic skin-healing defects are one of the leading challenges to lifelong well-being, affecting 2-5% of populations. Chronic wound formation is linked to age and diabetes and frequently leads to major limb amputation. Here we identify a strategy to reverse fibroblast senescence and improve healing rates. In healthy skin, fibronectin activates Rac1 in fibroblasts, causing migration into the wound bed, and driving wound contraction. We discover that mechanical stimulation of the skin with ultrasound can overturn healing defects by activating a calcium/CamKinaseII/Tiam1/Rac1 pathway that substitutes for fibronectin-dependent signaling and promotes fibroblast migration. Treatment of diabetic and aged mice recruits fibroblasts to the wound bed and reduces healing times by 30%, restoring healing rates to those observed in young, healthy animals. Ultrasound treatment is equally effective in rescuing the healing defects of animals lacking fibronectin receptors, and can be blocked by pharmacological inhibition of the CamKinaseII pathway. Finally, we discover that the migration defects of fibroblasts from human venous leg ulcer patients can be reversed by ultrasound, demonstrating that the approach is applicable to human chronic samples. By demonstrating that this alternative Rac1 pathway can substitute for that normally operating in the skin, we identify future opportunities for management of chronic wounds.