Department of Pharmacology, Jichi Medical School, Minamikawachi, Tochigi, 329-0498, Japan
Submitted 2 December 2002 ; accepted in final form 25 February 2003
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ABSTRACT |
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Trp; calcium channel; vasopressin; mechanosensitive channel
TRPV4 is abundantly detected in the kidney and lung by Northern blot analysis. When cerebral circumventricular organs are involved, in situ hybridization reveals the localization in detail (6). The localization of TRPV4 in the central nervous system suggests involvement of TRPV4 in regulation of water ingestion or osmolality of the body. To elucidate the osmosensing role of TRPV4, we studied mice lacking this gene and examined its possible functions in the sensation of osmolality.
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METHODS |
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Animals. All experiments were performed in accordance with national guidelines for the care and use of research animals. Experiments were performed with mice fed ad libitum and with free access to tap water. Mice were maintained in a balance cage (Sugiyama-Gen, Tokyo, Japan) capable of accurately measuring daily food and water consumption and excretion of urine. Na+ and K+ were measured by flame photometry (IL943; Instrument Lab). Osmolality was measured with an osmometer (One-Ten osmometer; Fiske). All other chemicals were determined with kits designed for colorimetric measurements.
Mandatory ingestion of water (20 ml/kg) or 2% NaCl (20 ml/kg) was performed manually through a tube within a period of 5 min. Arginine vasopressin (AVP) was measured from the serum of three mice (500 µl) or the serum of one mouse (100 µl) after intraperitoneal injection of propylene glycol as an osmotic substrate; mice were decapitated and AVP was measured by radioimmunoassay (Mitsubishi Yuka, Tokyo, Japan). For dialysis, the brain was sliced (2 mm thick) obliquely in the frontal cerebrum and hypothalamus. Bath solution contained (in mM) 127 NaCl, 1.5 KCl, 1.24 KH2PO4, 1.3 MgSO4, 26 NaHCO3, 2.4 CaCl2, and 10 glucose equilibrated with 95% O2-5% CO2 (pH 7.4, 300 mosmol/kgH2O). Osmolality was varied by addition of glycerol to 320, 340, and 360 mosmol/kgH2O. Each solution was perfused for 10 min. The perfusate was collected and freeze-dried (50°C) as a sample for AVP measurement (12).
Detection of RNA. Total RNA from the renal cortex was isolated by using a column (RNeasy; Qiagen). Reverse transcriptase (RT)-PCR was performed according to the manufacturer's protocol (RNA-PCR; Takara, Osaka, Japan) with 0.3 µg of RNA as a template. The amplification conditions consisted of incubation at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, for a total of 29 cycles.
Antibody, protein extraction, Western blot analysis, and histological analysis. Antibody was raised against the COOH-terminal peptide (CDGHQQGYAPK) in a solution using keyhole limpet hemocyanin (KLH) as a conjugate. The antigen of 1 mg/ml of distilled water with 1 ml of Freund's adjuvant was intramuscularly injected into a New Zealand White rabbit, followed by a biweekly booster injection of the same dose of the antigen in Freund's incomplete adjuvant. The titer of the serum used was >10,000 times higher than the control. To obtain polyclonal anti-TRPV4 antibodies, the IgG fraction was purified with a protein G column (HiTrap; Amersham Pharmacia, Tokyo, Japan) and further affinity-purified with a kit (Prot On; MPS). Western blot analysis with a blocking test by excess of antigen was performed to evaluate specificity (19). Renal extracts were made in (in mM) 300 sucrose, 25 imidazole, 1 EGTA, and 5 EDTA with a cocktail of protease inhibitors by homogenization. Histological staining of the brain was performed and detected by fluorescent isothiocyanate-labeled anti-rabbit IgG (Dako, Kyoto, Japan).
Statistics. The data were analyzed with Student's t-test or one-way analysis of variance (ANOVA), and the significance was calculated with Scheffé's analysis. P < 0.05 was considered statistically significant.
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RESULTS |
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Electrolyte concentrations were determined for the normal diet averaged over 1 wk (Table 1). Blood volumes of 0.20.8 ml could be sampled from one mouse. No significant alterations in electrolyte concentration were found with blood chemical analysis. The urinary Na+ concentration was low in the TRPV4/ mouse, but the total amount of Na+ excreted into the urine did not change significantly. Blood pressure and water balance were not altered between the two groups (Table 2).
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Localization of TRPV4 in osmosensing area. TRPV4 protein was detected in the renal cortical extract in TRPV4+/+ but not in TRPV4/ mice. An excess of antigen was coincubated with the antibody, resulting in the absence of the band (Fig. 2). In the central nervous system, TRPV4 is expressed in cells around the ventricle, whereas neurosensory cells are responsive to systemic or cerebrospinal fluid (CSF) osmotic pressure (21). Figure 2 shows a histologically positive signal of TRPV4 protein. The frontal part of the circumventricular area, especially the lateral portion, was positively stained. Choroid plexus of the third ventricle was also positively stained, whereas TRPV4 was not detected in other parts (cerebrum to cerebellum).
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Osmotic response in TRPV4/ mice. Using a water or 2% NaCl ingestion test, we measured serum and urine osmolality. In the water loading test (20 ml/kg at 20°C within 5 min), no significant change in urinary Na+ concentration, K+ concentration (data not shown), or osmolality was observed between the two groups (Fig. 3). However, urinary osmolality was higher at 2 h after ingestion of the 2% NaCl solution (20 ml/kg at 20°C within 5 min) in TRPV4 / mice compared with TRPV4+/+ mice. During the test, urinary Na+ concentration was increased at 1 h and then decreased, but there was no difference between the two groups. Thus unknown osmotic substrate(s) induced this change. Although abnormality of the osmotic regulation was suggested as a reason for this change, it was not determined until measurement of AVP.
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Serum AVP concentration in TRPV4/ mice. The level of serum AVP was measured by radioimmunoassay. To determine exact values, we diluted control samples and measured them repeatedly. We found that internal error for the measurement was within 0.15 pg/ml with 0.2 ml of serum. The amount of serum from each mouse was around 0.2 ml, whereas 0.5 ml of serum was required for this assay. We therefore used sera of three mice for one sample in the basal condition. To induce AVP secretion, we next measured serum osmolality in various stressful conditions, including salt overload and water deprivation for 2 days. We injected propylene glycol into the peritoneal cavity of a conscious mouse to increase serum osmolality (plus water deprivation) and sampled the whole blood within 10 s. Serum osmolality was stable under these conditions (410 ± 8 mosmol/kgH2O). Values of AVP in both conditions were compared (Fig. 4). A significant rise in AVP concentration in the TRPV4/ mouse was observed under the latter stimulated condition (n = 11 vs. 13; P < 0.01, t-test), whereas a significant difference was not obtained under the steady-state control condition.
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AVP excretion in vitro by brain slice. Because serum hyperosmolality accompanied water deprivation in vivo, the direct effect of hyperosmolality on AVP secretion through TRPV4 remained obscure. To elucidate the response of AVP to osmolality, we measured the amount of secreted AVP released from sliced brain in vitro during a stepwise increase in perfusate osmolality. Secretion of AVP during 10 min was collected, and it was shown to be increased by hyperosmotic bath solution. The rate of increment rather than the set point was exaggerated in TRPV4/ mice (Fig. 5; n = 5 in each, ANOVA).
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DISCUSSION |
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We next examined various conditions that cause Na+ metabolism to fluctuate in the body, such as salt overload or restriction. However, no significant differences were observed in TRPV4/ mice (data not shown). Short-term salt overload or restriction may induce an altered response of osmolality in urine. Traits in TRPV4/ mice are suggestive of the syndrome of inappropriate secretion of AVP (SIADH). SIADH can develop when the set point of AVP release is lower than normal (280300 mosmol/kgH2O). Addition of hypertonic saline (rather than water restriction) is a useful test to detect subtle differences in the regulation of osmolality (10). In our experiment, abnormality of AVP response in TRPV4/ mice was obviously a response to brief hypertonic saline loading. The results of this test and the higher level of AVP under a condition of hypertonic dehydration (Fig. 4) support the finding that the TRPV4/ mice exhibited an exaggerated AVP response.
However, hyponatremia was not observed in TRPV4/ mice. AVP concentrations in the steady-state condition were not high in TRPV4/ mice compared with TRPV4+/+ mice, suggesting that the set point to release AVP was not altered in TRPV4/ mice. However, hyponatremia may not be apparent in AVP transgenic mice even when the AVP level is 5 (22) or 100 (4) times higher than normal serum osmolality. Furthermore, constant infusion of AVP does not substantiate hyponatremia in rats (11). Therefore, the absence of hyponatremia or normal AVP level alone did not necessarily indicate an abnormality in the set point. We therefore examined AVP release in vitro. The results strongly suggested that the response rather than the set point was altered in TRPV4/ mice.
The mechanism of altered response of AVP to hypertonicity remains unclear. The choroid plexus is considered to be the sensor of hydrostatic or osmotic CSF (21). To our knowledge, neurological events that follow injection of hypotonic solution directly into CSF have not been examined. Therefore, the neuron network of the downregulation of AVP is not yet mapped. A rise in AVP concentration in TRPV4/ mice suggests that TRPV4 in choroid plexus cells and paraventricular cells plays a negative role in sensing hyperosmolality to release AVP. TRPV4-possessing neurons may connect the AVP-secreting network. As to the anatomic connection of TRPV4 with AVP secretion, at least two pathways are possible, directly or indirectly connecting to AVP-secreting cells (Fig. 6). Basically, it is shown in HEK-293 and endothelial cells that TRPV4 is temperature sensitive and thereby constitutively active in the range of 3440°C (25). If hyperosmolality (320 mosmol/kgH2O) inhibited the activity of the channel and reduced intracellular calcium in TRPV4-expressing cells, TRPV4 may also play an inhibitory role in AVP secretion. TRPV4 thereby connects to the network of AVP secretion directly (connection A; Fig. 6). TRPV4 may be constitutively active in the isosmotic condition to regulate the tuning of AVP secretion by hyperosmolality. TRPV4 thereby connects to the hyperosmolality-sensitive mechanism (connection B; Fig. 6) rather than directly to the secretion of AVP. Interestingly, the baroregulatory system provides a similar influence on the AVP-osmolality curve, which acute hypovolemia shifts without changing the resetting point in vivo (14). However, the baroregulatory system generally involves neurological afferents that arise in pressure-sensitive receptors in the heart, aorta, or carotid sinus. Thus the brain slice model in the present study did not reproduce the reaction of acute hypovolemia. Nevertheless, it is possible that TRPV4 plays a terminal role in transmitting the inhibitory baroregulatory system through connection B. Regulation of TRPV4 other than by osmolality, such as by protein kinase C, was recently reported (24), which suggests a further possibility for the mechanism.
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The mechanism underlying TRPV4 mirrors that of a well-known mechanism whereby the stretch-inhibitable or shrinkage-activated channel responds to hyperosmolality, transmitting a signal that enhances the secretion of AVP (1). It is assumed that AVP secretion reflects a balance of inhibitory and stimulatory inputs (1, 14). Investigation of the connection of TRPV4-positive neurons will clarify the network.
The presence of TRPV4 was also indicated in the lamina terminalis and subfornical organ (6), but we did not find a positive staining of these nuclei. Both are regarded as osmoreceptive sensory organs related to drinking behavior (8, 16). However, we did not find any difference in water intake during the salt restriction or overloading treatments.
In conclusion, TRPV4 is a swell-activated Ca2+-permeable cation channel detected in the osmosensing area in the brain. TRPV4/ mice do not exhibit abnormalities in Na+ metabolism but show an abnormal response of AVP. Thus central TRPV4 may play an inhibitory role in AVP secretion.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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