Takeshi Y. Hiyama

Nax channel involved in CNS sodium-level sensing

Mammals feel thirsty or an appetite for salt when the correct balance between water and sodium in the body fluid has been disrupted, but little is known about the mechanism in the brain that controls salt homeostasis. It has been postulated that the existence of both an osmoreceptor and a specific sodium receptor is essential if the experimental data are to be encompassed. Several candidate osmoreceptors have been identified, and here we show that the Nax channel in the circumventricular organs (CVO) is a probable candidate for the specific sodium receptor.

The Subfornical Organ is the Primary Locus of Sodium-Level Sensing by Nax Sodium Channels for the Control of Salt-Intake Behavior

Dehydration causes an increase in the sodium (Na) concentration and osmolarity of body fluid. For Na homeostasis of the body, controls of Na and water intake and excretion are of prime importance. However, the system for sensing the Na level within the brain that is responsible for the control of Na- and water-intake behavior remains to be elucidated. We reported previously that the Nax channel is preferentially expressed in the circumventricular organs (CVOs) in the brain and that Nax knock-out mice ingest saline in excess under dehydrated conditions. Subsequently, we demonstrated that Nax is a Na-level-sensitive Na channel. Here we show that the subfornical organ (SFO) is the principal site for the control of salt-intake behavior, where the Nax channel is the Na-level sensor. Infusion of a hypertonic Na solution into the cerebral ventricle induced extensive water intake and aversion to saline in wild-type animals but not in the knock-out mice. Importantly, the aversion to salt was not induced by the infusion of a hyperosmotic mannitol solution with physiological Na concentration in either genotype of mice. When Nax cDNA was introduced into the brain of the knock-out mice with an adenoviral expression vector, only animals that received a transduction of the Nax gene into the SFO among the CVOs recovered salt-avoiding behavior under dehydrated conditions. These results clearly show that the SFO is the center of the control of salt-intake behavior in the brain, where the Na-level-sensitive Nax channel is involved in sensing the physiological increase in the Na level of body fluids.

Autoimmunity to the Sodium-Level Sensor in the Brain Causes Essential Hypernatremia

Na(x) is the sodium-level sensor of body fluids in the brain involved in sodium homeostasis. Na(x)-knockout mice do not stop ingesting salt even when dehydrated. Here we report a case with clinical features of essential hypernatremia without demonstrable hypothalamic structural lesions, who was diagnosed as a paraneoplastic neurologic disorder. The patient had autoantibodies directed against Na(x), along with a ganglioneuroma composed of Schwann-like cells robustly expressing Na(x). The removal of the tumor did not reduce the autoantibody levels or relieve the symptoms. Intravenous injection of the immunoglobulin fraction of the patients serum into mice induced abnormalities in water/salt intake and diuresis, which led to hypernatremia. In the brains of these mice, cell death was observed along with focal deposits of complement C3 and inflammatory infiltrates in circumventricular organs where Na(x) is specifically expressed. Our findings thus provide new insights into the pathogenesis of hypernatremia relevant to the sodium-level-sensing mechanism in humans.

Nax sodium channel is expressed in non-myelinating Schwann cells and alveolar type II cells in mice

Na(x) is an extracellular sodium-level-sensitive sodium channel expressed in the circumventricular organs in the brain, essential loci for the sodium-level homeostasis in body fluids. Here, we examined the localization of Na(x) throughout the visceral organs at the cellular level. In visceral organs including lung, heart, intestine, bladder, kidney and tongue, a subset of Schwann cells within the peripheral nerve trunks were highly positive for Na(x). An electron microscopic study indicated that these Na(x)-positive cells were non-myelinating Schwann cells. In the lung, Na(x)-positive signals were also observed in the alveolar type II cells, which actively absorb sodium and water to aid gas exchange through the alveolar surface. It was thus suggested that the Na(x) sodium channel is involved in controlling the local extracellular sodium level through sodium absorption activity.

Osmosensitivity of Transient Receptor Potential Vanilloid 1 Is Synergistically Enhanced by Distinct Activating Stimuli Such as Temperature and Protons

In animals, body-fluid osmolality is continuously monitored to keep it within a narrow range around a set point (∼300 mOsm/kg). Transient receptor potential vanilloid 1 (TRPV1), a cation channel, has been implicated in body-fluid homeostasis in vivo based on studies with the TRPV1-knockout mouse. However, the response of TRPV1 to hypertonic stimuli has not been demonstrated with heterologous expression systems so far, despite intense efforts by several groups. Thus, the molecular entity of the hypertonic sensor in vivo still remains controversial. Here we found that the full-length form of TRPV1 is sensitive to an osmotic increase exclusively at around body temperature using HEK293 cells stably expressing rat TRPV1. At an ambient temperature of 24°C, a slight increase in the intracellular calcium concentration ([Ca2+]i) was rarely observed in response to hypertonic stimuli. However, the magnitude of the osmosensitive response markedly increased with temperature, peaking at around 36°C. Importantly, the response at 36°C showed a robust increase over a hypertonic range, but a small decrease over a hypotonic range. A TRPV1 antagonist, capsazepine, and a nonspecific TRP channel inhibitor, ruthenium red, completely blocked the increase in [Ca2+]i. These results endorse the view that the full-length form of TRPV1 is able to function as a sensor of hypertonic stimuli in vivo. Furthermore, we found that protons and capsaicin likewise synergistically potentiated the response of TRPV1 to hypertonic stimuli. Of note, HgCl2, which blocks aquaporins and inhibits cell-volume changes, significantly reduced the osmosensitive response. Our findings thus indicate that TRPV1 integrates multiple different types of activating stimuli, and that TRPV1 is sensitive to hypertonic stimuli under physiologically relevant conditions.

Distinct neural mechanisms for the control of thirst and salt appetite in the subfornical organ

Body fluid conditions are continuously monitored in the brain to regulate thirst and salt-appetite sensations. Angiotensin II drives both thirst and salt appetite; however, the neural mechanisms underlying selective water- and/or salt-intake behaviors remain unknown. Using optogenetics, we show that thirst and salt appetite are driven by distinct groups of angiotensin II receptor type 1a-positive excitatory neurons in the subfornical organ. Neurons projecting to the organum vasculosum lamina terminalis control water intake, while those projecting to the ventral part of the bed nucleus of the stria terminalis control salt intake. Thirst-driving neurons are suppressed under sodium-depleted conditions through cholecystokinin-mediated activation of GABAergic neurons. In contrast, the salt appetite-driving neurons were suppressed under dehydrated conditions through activation of another population of GABAergic neurons by Nax signals. These distinct mechanisms in the subfornical organ may underlie the selective intakes of water and/or salt and may contribute to body fluid homeostasis.

The Nax Channel What It Is and What It Does

Na x , which is preferentially expressed in the glial cells of sensory circumventricular organs in the brain, is a sodium channel that is poorly homologous to voltage-gated sodium channels. We previously reported that Na x is a sodium concentration ([Na+])-sensitive, but not a voltage-sensitive channel that is critically involved in body-fluid homeostasis. Nax-knockout mice do not stop ingesting salt even when dehydrated and transiently develop hypernatremia. [Na+] in body fluids is strictly controlled at 135 to 145 mM in mammals. Although the set point must be within this range, Na x was shown to have a threshold value of ~150 mM for extracellular [Na+] ([Na+]o) for activation in vitro. Therefore, the [Na+]o dependency of Na x in vivo is presumably modified by an as yet unidentified mechanism. We recently demonstrated that the [Na+]o dependency of Na x in the subfornical organ was adjusted to the physiological range by endothelin-3. Pharmacological experiments revealed that endothelin receptor B signaling was involved in this modulation of Na x gating through protein kinase C and ERK1/2 activation. In addition, we identified a case of essential hypernatremia caused by autoimmunity to Na x . Occurrence of a ganglioneuroma composed of Schwann-like cells that robustly expressed Na x was likely to induce the autoimmune response in this patient. An intravenous injection of the immunoglobulin fraction of the patient’s serum, which contained anti-Na x antibodies, into mice reproduced the patient’s symptoms. This review provides an overview of the physiological functions of Na x by summarizing our recent studies.Nax, which is preferentially expressed in the glial cells of sensory circumventricular organs in the brain, is a sodium channel that is poorly homologous to voltage-gated sodium channels. We previously reported that Nax is a sodium concentration ([Na+])-sensitive, but not a voltage-sensitive channel that is critically involved in body-fluid homeostasis. Nax-knockout mice do not stop ingesting salt even when dehydrated and transiently develop hypernatremia. [Na+] in body fluids is strictly controlled at 135 to 145 mM in mammals. Although the set point must be within this range, Nax was shown to have a threshold value of ~150 mM for extracellular [Na+] ([Na+]o) for activation in vitro. Therefore, the [Na+]o dependency of Nax in vivo is presumably modified by an as yet unidentified mechanism. We recently demonstrated that the [Na+]o dependency of Nax in the subfornical organ was adjusted to the physiological range by endothelin-3. Pharmacological experiments revealed that endothelin receptor B signaling was ...

Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution

The LacZ gene, which encodes Escherichia coli β-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.

Nax-deficient mice show normal vasopressin response to dehydration

In dehydrated animals, the antidiuretic hormone vasopressin (VP) is released from the nerve terminals of magnocellular neurons of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) into the systemic circulation at the posterior pituitary. Increases in sodium (Na+)-level and osmolality in body fluids upon dehydration are reportedly sensed by a Na+-sensor and/or an osmosensor, respectively. However, it is still unknown whether both are involved in the regulation of production and/or release of VP. Na(x) is the cerebral Na+-level sensor and Na(x)-knockout mice do not stop ingesting salt even when dehydrated. Here we examined VP production/release in Na(x)-knockout mice, and found that they are normal in the VP response to dehydration or intraperitoneal-administration with hypertonic saline. In situ hybridization using an intron-specific probe showed that VP gene expression in the SON did not differ from wild-type mice when dehydrated. Also, there was no significant difference in the activity of subfornical organ neurons projecting to the SON between the two genotypes when stimulated by water deprivation. Furthermore, Na(x)-knockout mice showed a normal response in urine excretion to dehydration. All these results indicate that the information of Na+-level increase detected by Na(x) does not contribute to the control of VP production/release.

Involvement of Nax sodium channel in peripheral nerve regeneration via lactate signaling

Nax, a sodium concentration‐sensitive sodium channel, is expressed in non‐myelinating Schwann cells of the adult peripheral nervous system, but the pathophysiological role remains unclear. We found that functional recovery of the hind paw responses from the sciatic nerve transection was delayed in Nax knockout ( Nax−/− ) mice. Histological analyses showed a decrease in the number of regenerated myelinated axons in Nax−/− sciatic nerves. The delay in the recovery in Nax−/− mice was improved by lactate and inhibited by a monocarboxylate transporter inhibitor. In vitro experiments using cultured Schwann cells showed that lactate release was enhanced by endothelin (ET)‐1 and blocked by an ET receptor type B antagonist. Here, it is conceivable that Nax was activated by ET‐1. The amount of lactate release by ET‐1 was lower in Nax−/− mice than in wild‐type mice. These results indicated that Nax is functionally coupled to ET for lactate release via ET receptor type B and is involved in peripheral nerve regeneration.