Elevated blood pressure, heart rate and body temperature in mice lacking the XLαs protein of the Gnas locus is due to increased sympathetic tone.

Imbalances of energy homeostasis are often associated with cardiovascular complications. Previous work has shown that Gnasxl-deficient mice have a lean and hypermetabolic phenotype, with increased sympathetic stimulation of adipose tissue. The Gnasxl transcript from the imprinted Gnas locus encodes the trimeric G-protein subunit XLαs, which is expressed in brain regions that regulate energy homeostasis and sympathetic nervous system (SNS) activity. To determine whether Gnasxl knock-out (KO) mice display additional SNS-related phenotypes, we have now investigated the cardiovascular system. The Gnasxl KO mice were ∼20 mmHg hypertensive in comparison to wild-type (WT) littermates (P ≤ 0.05) and hypersensitive to the sympatholytic drug reserpine. Using telemetry, we detected an increased waking heart rate in conscious KOs (630 ± 10 versus 584 ± 12 beats min(-1), KO versus WT, P ≤ 0.05). Body temperature was also elevated (38.1 ± 0.3 versus 36.9 ± 0.4°C, KO versus WT, P ≤ 0.05). To investigate autonomic nervous system influences, we used heart rate variability analyses. We empirically defined frequency power bands using atropine and reserpine and verified high-frequency (HF) power and low-frequency (LF) LF/HF power ratio to be indicators of parasympathetic and sympathetic activity, respectively. The LF/HF power ratio was greater in KOs and more sensitive to reserpine than in WTs, consistent with elevated SNS activity. In contrast, atropine and exendin-4, a centrally acting agonist of the glucagon-like peptide-1 receptor, which influences cardiovascular physiology and metabolism, reduced HF power equally in both genotypes. This was associated with a greater increase in heart rate in KOs. Mild stress had a blunted effect on the LF/HF ratio in KOs consistent with elevated basal sympathetic activity. We conclude that XLαs is required for the inhibition of sympathetic outflow towards cardiovascular and metabolically relevant tissues.

Temperature modulates PVN pre-sympathetic neurones via transient receptor potential ion channels.

The paraventricular nucleus (PVN) of the hypothalamus plays a vital role in maintaining homeostasis and modulates cardiovascular function via autonomic pre-sympathetic neurones. We have previously shown that coupling between transient receptor potential cation channel subfamily V Member 4 (Trpv4) and small-conductance calcium-activated potassium channels (SK) in the PVN facilitate osmosensing, but since TRP channels are also thermosensitive, in this report we investigated the temperature sensitivity of these neurones. Methods: TRP channel mRNA was quantified from mouse PVN with RT-PCR and thermosensitivity of Trpv4-like PVN neuronal ion channels characterised with cell-attached patch-clamp electrophysiology. Following recovery of temperature-sensitive single-channel kinetic schema, we constructed a predictive stochastic mathematical model of these neurones and validated this with electrophysiological recordings of action current frequency. Results: 7 thermosensitive TRP channel genes were found in PVN punches. Trpv4 was the most abundant of these and was identified at the single channel level on PVN neurones. We investigated the thermosensitivity of these Trpv4-like channels; open probability (Po) markedly decreased when temperature was decreased, mediated by a decrease in mean open dwell times. Our neuronal model predicted that PVN spontaneous action current frequency (ACf) would increase as temperature is decreased and in our electrophysiological experiments, we found that ACf from PVN neurones was significantly higher at lower temperatures. The broad-spectrum channel blocker gadolinium (100 µM), was used to block the warm-activated, Ca2+-permeable Trpv4 channels. In the presence of gadolinium (100 µM), the temperature effect was largely retained. Using econazole (10 µM), a blocker of Trpm2, we found there were significant increases in overall ACf and the temperature effect was inhibited. Conclusion: Trpv4, the abundantly transcribed thermosensitive TRP channel gene in the PVN appears to contribute to intrinsic thermosensitive properties of PVN neurones. At physiological temperatures (37°C), we observed relatively low ACf primarily due to the activity of Trpm2 channels, whereas at room temperature, where most of the previous characterisation of PVN neuronal activity has been performed, ACf is much higher, and appears to be predominately due to reduced Trpv4 activity. This work gives insight into the fundamental mechanisms by which the body decodes temperature signals and maintains homeostasis.

A rare human variant that disrupts GPR10 signalling causes weight gain in mice.

Disruption of brain-expressed G protein-coupled receptor-10 (GPR10) causes obesity in animals. Here, we identify multiple rare variants in GPR10 in people with severe obesity and in normal weight controls. These variants impair ligand binding and G protein-dependent signalling in cells. Transgenic mice harbouring a loss of function GPR10 variant found in an individual with obesity, gain excessive weight due to decreased energy expenditure rather than increased food intake. This evidence supports a role for GPR10 in human energy homeostasis. Therapeutic targeting of GPR10 may represent an effective weight-loss strategy.

Cell volume regulation in chondrocytes.

Chondrocytes are the cells within cartilage which produce and maintain the extracellular matrix. Volume regulation in these cells is vital to their function and occurs in several different physiological and pathological contexts. Firstly, chondrocytes exist within an environment of changing osmolarity and compressive loads. Secondly, in osteoarthritic joint failure, cartilage water content changes and there is a notable increase in chondrocyte apoptosis. Thirdly, endochondral ossification requires chondrocyte swelling in association with hypertrophy. Regulatory volume decrease (RVD) and regulatory volume increase (RVI) have both been observed in articular chondrocytes and this review focuses on the mechanisms identified to account for these. There has been evidence so far to suggest TRPV4 is central to RVD; however other elements of the pathway have not yet been identified. Unlike RVD, RVI appears less robust in articular chondrocytes and there have been fewer mechanistic studies; the primary focus being on the Na(+)-K(+)-2Cl(-) co-transporter. The clinical significance of chondrocyte volume regulation remains unproven. Importantly however, transcript abundances of several ion channels implicated in volume control are changed in chondrocytes from osteoarthritic cartilage. A critical question is whether disturbances of volume regulation mechanisms lead to, result from or are simply coincidental to cartilage damage.

The cytokine GDF15 signals through a population of brainstem cholecystokinin neurons to mediate anorectic signalling.

The cytokine, GDF15, is produced in pathological states which cause cellular stress, including cancer. When over expressed, it causes dramatic weight reduction, suggesting a role in disease-related anorexia. Here, we demonstrate that the GDF15 receptor, GFRAL, is located in a subset of cholecystokinin neurons which span the area postrema and the nucleus of the tractus solitarius of the mouse. GDF15 activates GFRALAP/NTS neurons and supports conditioned taste and place aversions, while the anorexia it causes can be blocked by a monoclonal antibody directed at GFRAL or by disrupting CCK neuronal signalling. The cancer-therapeutic drug, cisplatin, induces the release of GDF15 and activates GFRALAP/NTS neurons, as well as causing significant reductions in food intake and body weight in mice. These metabolic effects of cisplatin are abolished by pre-treatment with the GFRAL monoclonal antibody. Our results suggest that GFRAL neutralising antibodies or antagonists may provide a co-treatment opportunity for patients undergoing chemotherapy.

Ion Channels in the Paraventricular Hypothalamic Nucleus (PVN); Emerging Diversity and Functional Roles.

The paraventricular nucleus of the hypothalamus (PVN) is critical for the regulation of homeostatic function. Although also important for endocrine regulation, it has been referred to as the "autonomic master controller." The emerging consensus is that the PVN is a multifunctional nucleus, with autonomic roles including (but not limited to) coordination of cardiovascular, thermoregulatory, metabolic, circadian and stress responses. However, the cellular mechanisms underlying these multifunctional roles remain poorly understood. Neurones from the PVN project to and can alter the function of sympathetic control regions in the medulla and spinal cord. Dysfunction of sympathetic pre-autonomic neurones (typically hyperactivity) is linked to several diseases including hypertension and heart failure and targeting this region with specific pharmacological or biological agents is a promising area of medical research. However, to facilitate future medical exploitation of the PVN, more detailed models of its neuronal control are required; populated by a greater compliment of constituent ion channels. Whilst the cytoarchitecture, projections and neurotransmitters present in the PVN are reasonably well documented, there have been fewer studies on the expression and interplay of ion channels. In this review we bring together an up to date analysis of PVN ion channel studies and discuss how these channels may interact to control, in particular, the activity of the sympathetic system.

PACAP Neurons in the Ventromedial Hypothalamic Nucleus Are Glucose Inhibited and Their Selective Activation Induces Hyperglycaemia.

Background: Glucose-sensing neurons are located in several parts of the brain, but are concentrated in the ventromedial nucleus of the hypothalamus (VMH). The importance of these VMH neurons in glucose homeostasis is well-established, however, little is known about their individual identity. In the present study, we identified a distinct glucose-sensing population in the VMH and explored its place in the glucose-regulatory network. Methods: Using patch-clamp electrophysiology on Pacap-cre::EYFP cells, we explored the glucose-sensing ability of the pituitary adenylate cyclase-activating peptide (PACAP) neurons both inside and outside the VMH. We also mapped the efferent projections of these neurons using anterograde and retrograde tracing techniques. Finally, to test the functionality of PACAPVMH in vivo, we used DREADD technology and measured systemic responses. Results: We demonstrate that PACAP neurons inside (PACAPVMH), but not outside the VMH are intrinsically glucose inhibited (GI). Anatomical tracing techniques show that PACAPVMH neurons project to several areas that can influence autonomic output. In vivo, chemogenetic stimulation of these neurons inhibits insulin secretion leading to reduced glucose tolerance, implicating their role in systemic glucose regulation. Conclusion: These findings are important as they identify, for the first time, a specific VMH neuronal population involved in glucose homeostasis. Identifying the different glucose-sensing populations in the VMH will help piece together the different arms of glucose regulation providing vital information regarding central responses to glucose metabolic disorders including hypoglycaemia.

The depressor response to intracerebroventricular hypotonic saline is sensitive to TRPV4 antagonist RN1734.

Several reports have shown that the periventricular region of the brain, including the paraventricular nucleus (PVN), is critical to sensing and responding to changes in plasma osmolality. Further studies also implicate the transient receptor potential ion channel, type V4 (TRPV4) channel in this homeostatic behavior. In previous work we have shown that TRPV4 ion channels couple to calcium-activated potassium channels in the PVN to decrease action potential firing frequency in response to hypotonicity. In the present study we investigated whether, similarly, intracerebroventricular (ICV) application of hypotonic solutions modulated cardiovascular parameters, and if so whether this was sensitive to a TRPV4 channel inhibitor. We found that ICV injection of 270 mOsmol artificial cerebrospinal fluid (ACSF) decreased mean blood pressure, but not heart rate, compared to naïve mice or mice injected with 300 mOsmol ACSF. This effect was abolished by treatment with the TRPV4 inhibitor RN1734. These data suggest that periventricular targets within the brain are capable of generating depressor action in response to TRPV4 ion channel activation. Potentially, in the future, the TRPV4 channel, or the TRPV4-KCa coupling mechanism, may serve as a therapeutic target for treatment of cardiovascular disease.

An in vitro model of skeletal muscle volume regulation.

INTRODUCTION: Hypertonic media causes cells to shrink due to water loss through aquaporin channels. After acute shrinkage, cells either regulate their volume or, alternatively, undergo a number of metabolic changes which ultimately lead to cell death. In many cell types, hypertonic shrinkage is followed by apoptosis. Due to the complex 3D morphology of skeletal muscle and the difficulty in obtaining isolated human tissue, we have begun skeletal muscle volume regulation studies using the human skeletal muscle cell line TE671RD. In this study we investigated whether hypertonic challenge of the human skeletal muscle cell line TE671RD triggered cell death or evoked a cell volume recovery response. METHODS: The cellular volume of TE671RD cells was calculated from the 2D surface area. Cell death was assessed by both the trypan blue live/dead assay and the TUNEL assay. RESULTS: Medium osmolality was increased by addition of up to 200 mM sucrose. Addition of 200 mM sucrose resulted in mean cell shrinkage of 44±1% after 30 mins. At later time points (2 and 4 hrs) two separate cell subpopulations with differing mean cell volume became apparent. The first subpopulation (15±2% of the total cell number) continued to shrink whereas the second subpopulation had an increased cell volume. Cell death was observed in a small proportion of cells (approximately 6-8%). CONCLUSION: We have established that a substantial proportion of TE671RD cells respond to hypertonic challenge with RVI, but that these cells are resistant to hypertonicity triggered cell death.

NK1-receptor-expressing paraventricular nucleus neurones modulate daily variation in heart rate and stress-induced changes in heart rate variability.

The paraventricular nucleus of the hypothalamus (PVN) is an established center of cardiovascular control, receiving projections from other nuclei of the hypothalamus such as the dorsomedial hypothalamus and the suprachiasmatic nucleus. The PVN contains a population of "pre-autonomic neurones" which project to the intermediolateralis of the spinal cord and increase sympathetic activity, blood pressure, and heart rate. These spinally projecting neurones express a number of membrane receptors including GABA and substance P NK1 receptors. Activation of NK1-expressing neurones increases heart rate, blood pressure, and sympathetic activity. However, their role in the pattern of overall cardiovascular control remains unknown. In this work, we use specific saporin lesion of NK1-expressing PVN rat neurones with SSP-SAP and telemetrically measure resting heart rate and heart rate variability (HRV) parameters in response to mild psychological stress. The HRV parameter "low frequency/high frequency ratio" is often used as an indicator of sympathetic activity and is significantly increased with psychological stress in control rats (0.84 ± 0.14 to 2.02 ± 0.15; P < 0.001; n = 3). We find the stress-induced increase in this parameter to be blunted in the SSP-SAP-lesioned rats (0.83 ± 0.09 to 0.93 ± 0.21; P > 0.05; n = 3). We also find a shift in daily variation of heart rate rhythm and conclude that NK1-expressing PVN neurones are involved with coupling of the cardiovascular system to daily heart rate variation and the sympathetic response to psychological stress.