Claire H. Feetham

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.

TRPV4 and KCa ion channels functionally couple as osmosensors in the paraventricular nucleus

Transient receptor potential vanilloid type 4 (TRPV4) and calcium‐activated potassium channels (KCa) mediate osmosensing in many tissues. Both TRPV4 and KCa channels are found in the paraventricular nucleus (PVN) of the hypothalamus, an area critical for sympathetic control of cardiovascular and renal function. Here, we have investigated whether TRPV4 channels functionally couple to KCa channels to mediate osmosensing in PVN parvocellular neurones and have characterized, pharmacologically, the subtype of KCa channel involved.

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

•  What is the central question of this study? Previously, we showed that Gnasxl knock‐out mice are lean and hypermetabolic, with increased sympathetic stimulation of adipose tissue. Do these mice also display elevated sympathetic cardiovascular tone? Is the brain glucagon‐like peptide‐1 system involved? •  What is the main finding and its importance? Gnasxl knock‐outs have increased blood pressure, heart rate and body temperature. Heart rate variability analysis suggests an elevated sympathetic tone. The sympatholytic reserpine had stronger effects on blood pressure, heart rate and heart rate variability in knock‐out compared with wild‐type mice. Stimulation of the glucagon‐like peptide‐1 system inhibited parasympathetic tone to a similar extent in both genotypes, with a stronger associated increase in heart rate in knock‐outs. Deficiency of Gnasxl increases sympathetic cardiovascular tone.

Benzamil sensitive ion channels contribute to volume regulation in canine chondrocytes.

Chondrocytes exist within cartilage and serve to maintain the extracellular matrix. It has been postulated that osteoarthritic (OA) chondrocytes lose the ability to regulate their volume, affecting extracellular matrix production. In previous studies, we identified expression of epithelial sodium channels (ENaC) in human chondrocytes, but their function remained unknown. Although ENaC typically has Na+ transport roles, it is also involved in the cell volume regulation of rat hepatocytes. ENaC is a member of the degenerin (Deg) family, and ENaC/Deg‐like channels have a low conductance and high sensitivity to benzamil. In this study, we investigated whether canine chondrocytes express functional ENaC/Deg‐like ion channels and, if so, what their function may be.

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.

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.

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.

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 200mM sucrose. Addition of 200mM sucrose resulted in mean cell shrinkage of 44±1% after 30mins. 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.

TRPV4 and KCa: Modelling the Perfect Couple?

The hypothalamus is responsible for maintaining body fluid osmolarity within a narrow range (∼290-300mOsm) [1]; in particular the paraventricular nucleus (PVN) is thought to have a key role in osmoregulation. Previously we have shown changes in action current frequency upon hypotonic challenge within the PVN using patch-clamp electrophysiology [2], with evidence suggesting these changes are due to the activation of the transient receptor potential vanilloid 4 (TRPV4) and calcium-activated potassium (KCa) channels.Our data supports a functional coupling between the TRPV4 and KCa channels, leading us to the hypothesis that upon hypotonic challenge TRPV4 activates KCa through influx of Ca2+, leading to an efflux of K+, as shown in other cells [3] leading to hyperpolarisation of the cell. This hyperpolarisation itself is suggested to create a positive feedback loop increasing the driving force for Ca2+ entry [4]. To investigate this hypothesis we have developed our existing NEURON model (University of Yale) [5], modelling the action of TRPV4 and KCa.In the model decreased osmolarity caused action potential (AP) frequency reduction, with a 93±3% decrease of AP frequency at 290mOsm and a half maximum of 304±0.4mOsm, dependant on starting parameters. Block of TRPV4 or KCa channels prevented this effect with AP frequency remaining at 100±0% regardless of osmolarity. To test positive feedback we simulated TRPV4 activity and measured simulated TRPV4 Ca2+ current with and without the presence of KCa activity. Breaking the positive feedback loop by block of KCa, as predicted, significantly reduced TRPV4 Ca2+ current by 640±0.1pA/s.cm2 (n=7; p<0.005).This model, together with our previous data provides further evidence for a functional coupling between TRPV4 and KCa channels within neurones in the PVN, supporting our hypothesis of a positive feedback system.