Enhanced osmotic responsiveness in angiotensin AT1a receptor deficient mice: evidence for a role for AT1b receptors

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1 Exp Physiol 90.5 pp Experimental Physiology Enhanced osmotic responsiveness in angiotensin AT1a receptor deficient mice: evidence for a role for AT1b receptors Yanfang Chen, Hao Chen and Mariana Morris Department of Pharmacology and Toxicology, Wright State University School of Medicine, Dayton, OH 45450, USA Experiments were performed to study the role of angiotensin (Ang) AT1a and AT1b receptor subtypes in osmotic regulation of blood pressure using gene deletion and pharmacological methods. The cardiovascular effects of hypertonic saline (HS) or vasopressin (VP) delivered via vascular catheters were measured in Ang AT1a gene deletion (AT1a / ) and control (AT1a+/+) mice. Blood pressure (BP) and heart rate (HR) were recorded in conscious mice using direct carotid catheters. Plasma osmolality and VP concentration were also measured. The major finding was that deletion of AT1a receptors resulted in enhanced BP response to osmotic stimulation. This was seen after acute HS injection (20 µl, 20% NaCl). The peak percentage change in mean arterial pressure (MAP) was 15.4 ± 1.9% versus 28.1 ± 2.4% (AT1a+/+versus AT1a /, respectively). Losartan (AT1 antagonist), but not PD (AT2 antagonist), inhibited the HS-induced MAP response, specifically in AT1a / mice. Plasma osmolality and VP concentration were elevated after HS injection with no differences noted between groups. Vascular injection of VP (5 ng g 1 ) increased BP and HR, with similar MAP response between groups. Evidence shows that removal of Ang AT1a receptors results in a significant enhancement in the pressor response to acute osmotic stimulation. Studies of AT1 receptor blockade indicate that complementary Ang AT1b receptors, but not AT2 receptors, may be involved in the osmotic response. (Received 13 April 2005; accepted after revision 2 June 2005; first published online 8 June 2005) Corresponding author Yanfang Chen: Department of Pharmacology and Taxicology, 3640 Colonel Glenn Highway, Wright State University School of Medicine, Dayton, OH , USA. yanfang.chen@wright.edu The brain angiotensin (Ang) system is important in regulating blood pressure (BP) and volume homeostasis (Phillips, 1987; Fitzsimons, 1998; Baltatu et al. 2001; Diz et al. 2002). Ang II in the central nervous system (CNS) acts mainly through AT1 receptors which are expressed in rodents as the highly complementary, AT1a and AT1b subtypes (Lenkei et al. 1998; De Gasparo et al. 2000). Differentiation of the AT1 subtypes requires molecular methods because of similarity at the genomic and proteomic levels. Using PCR and in situ hybridization methods, results have shown a similar pattern of expression in brainstem and hypothalamic regions with high levels in regions that receive afferent information on pressure and osmotic statue (Chen & Morris, 2001; Chen et al. 2003, 2004b). Both AT1a and AT1b receptor subtypes are present in subfornical organ (SFO), anterior ventricular forebrain and the dorsal vagal complex/nucleus tractus solitarii (DVC/NTS). Osmotic stimulation in mice produced effects on AT1a and AT1b expression, which were dependent on the mode of administration. When 2% NaCl was given as the sole drinking fluid, plasma osmolality and brain AT1a mrna were increased (Chen et al. 2003). When NaCl was given via the diet, plasma osmolality was unchanged while hypothalamic AT1a mrna was decreased (Chen et al. 2004b). Evidence for activation of AT1b receptors was seen in the brainstem under conditions of dehydration and in the absence of AT1a receptors (Rocha et al. 2005). Questions as to the physiological function of the AT1a and AT11b receptors have relied on molecular genetic manipulations because the receptors cannot be differentiated with pharmacological agents. Overexpression of AT1a receptors in brain resulted in enhanced cardiovascular sensitivity to central Ang II stimulation (Lazartigues et al. 2002). In contrast, deletion of the AT1a receptor produced animals with low BP, increased water DOI: /expphysiol

2 740 Y. Chen and others Exp Physiol 90.5 pp intake and abnormal kidney function (Ito et al. 1995; Oliverio et al. 2000b). Davisson et al. (2000) reported that CNS AT1b receptors are important in drinking behaviour as the drinking response to central administration of Ang II was absent in AT1b-deficient mice. There is also evidence that Ang AT1b receptors are important in BP control (Oliverio et al. 1997; Zhou et al. 2003). AT1b receptors are present in arterioles and neuroendocrine tissues as determined using PCR with enzyme restriction digest (Zhou et al. 2003, 2005). In fact, Ang AT1b appears to the dominant form in the vasculature (Zhou et al. 2003, 2005). Chronic blockade of AT1 with losartan reduced BP in AT1a / mice (Oliverio et al. 1997) and BP was lower in the combined AT1a/AT1b knockout mice as compared to animals with the single AT1a gene deletion (Oliverio et al. 1998). Data also showed that AT1a / mice are more sensitive to the effects of dehydration and salt loading (Oliverio et al. 2000a) intermsof BP and CNS activation (Morris et al. 2001). This along with other data suggests that both Ang AT1a and AT1b signalling participate in the osmotic control of blood pressure. The objective of the present study was to further characterize the role of AT1b receptors in osmotic regulation of BP by conducting studies in Ang AT1a / mice. We examined the effect of a vascular osmotic challenge on BP, heart rate (HR) and plasma VP concentration, as well as the effect of acute VP injection on BP and HR. Methods Animals Adult male AT1a / and AT1a+/+ mice (27 35 g) were used for these studies. Animals were generated from a breeding colony maintained at Wright State University. The original founders were provided by Dr Thomas Coffman (Duke University, Durham, NC, USA) (Ito et al. 1995). The genotype was determined by PCR analysis of genomic DNA isolated from tail tissue according to previously reported methods (Ito et al. 1995). Mice were housed singly at 22 C and 100 Lux, under a 12-h light 12-h dark cycle with access to water and standard mouse chow (0.4% NaCl, Harlan Teklad, Madison, WI, USA) ad libitum.theinvestigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no , revised 1996). Measurement of blood pressure and heart rate Mean arterial pressure (MAP) and HR were measured in mice using direct carotid arterial catheters. Detailed methods for catheter implantation have been previously described (Li et al. 1999; Joaquim et al. 2004). Briefly, mice were anaesthetized with a ketamine xylazine mixture (70:6mgkg 1, i.m.) and surgery was conducted using a dissection microscope. A microrenathane catheter (o.d., mm; Braintree Scientific, Inc., Braintree, MA, USA) was inserted into the carotid artery. The catheter was covered with a spring and attached to a swivel at the top of the cage. The catheter was connected through a flowthrough BP transducer to a syringe pump. Heparinized saline (80 U ml 1 ) was continuously infused (25 µlh 1 ) in order to maintain catheter patency. The transducer was calibrated and connected to a computerized data acquisition system (BIOPAC System Inc. Goleta, CA, USA). Mice were allowed to recover for at least 7 days before any experiments were started. In considering that ingestion of water evokes pressor response and inhibits the release of VP, water and food were removed from the cages before experiments. Experimental protocols Experiment 1 was designed to investigate the effect of injection of hypertonic (HS) or isotonic saline (IS) into the carotid arterial catheter on BP and HR in AT1a / and+/+ mice. The procedures were: baseline BP recording (10 min); HS injection (20 µl, 20% NaCl) or IS injection (20 µl, 0.9% NaCl); and BP recording (10 min). Experiment 2 was designed to investigate the effect of AT1 receptor blockade on HS-induced changes in BP and HR in AT1a / and+/+ mice. The procedures were: baseline BP recording (10 min);hs injection (20 µl, 20% NaCl) within 20 s; BP recording (10 min); losartan injection (20 mg kg 1,20µl) or sham injection (IS, 20 µl); 30 min interval; BP basal recording (10 min); HS injection (20 µl, 20% NaCl) within 20 s; and BP recording (10 min). Experiment 3 was designed to investigate the effect of AT2receptor blockade (PD123319, 15 mg kg 1,20µl) on HS-induced changes in BP and HR in AT1a / and+/+ mice. The procedures were similar to those described for Experiment 2. Experiment 4 was designed to investigate the effect of HS injection on plasma osmolality and plasma VP concentration in AT1a / and+/+ mice. Blood samples (200 µl) were collected through the carotid arterial catheter into pre-cooled 1.5-ml tubes, before and immediately after (within 1 min) HS injection. The time point for the blood collection was determined on the basis of previous experiments (Chen et al. 2004a). Osmolality was measurement using 50 µl of the 200 µl collected blood sample. The remaining blood sample was centrifuged immediately and plasma was stored at 80 C for radioimmunoassay (RIA). Experiment 5 was designed to investigate the effect of VP (5 ng g 1 ) injected into the carotid arterial catheter on BP and HR in AT1a / and+/+ mice. The procedures were:

3 Exp Physiol 90.5 pp AT1b receptors in osmonsensitivity 741 Table 1. Effect of intracarotid HS on plasma osmolality and VP Osmolality VP Groups Treatment (mosm/kg) (pg ml 1 ) AT1a+/+ Con ± ± 0.8 AT1a / Con ± ± 0.7 AT1a+/+ HS ± ± 1.4 AT1a / HS ± ± 0.7 Values are mean ± S.E.M, P < 0.01, HS vs. Con, n = 5 per group. baseline BP recording (10 min); VP injection or control injection (0.9% NaCl); and BP recording (10 min). Plasma osmolality was measured using freezing-point osmometry (Advanced Instruments Inc., Norwood, MA, USA). Plasma VP concentration was measured by RIA (Morris et al. 1999). Intra- and interassay variations were monitored to establish the continued reliability of the RIA procedure and antiserum. All samples from within an experiment were measured in the same assay to reduce variation. The data are presented as means ± s.e.m. Comparisons between groups, treatments and time course were made in the acute osmotic stimulation study using a two or threeway ANOVA for analysis. One-way ANOVA was used for comparisons in the VP injection study. If a statistically significant effect was found, a Newman Keuls test was performed to detect the difference between the groups. P < 0.05 was used as a criterion for significance. Table 2. Effect of losartan on intra-carotid HS-induced changes in MAP in AT1a+/+ and AT1a / mice MAP (mmhg) Groups Treatment HS 1 min HS 5 min HS 10 min AT1a+/+ Con 19.0 ± ± ± 2.5 AT1a / Con 23.6 ± ± ± 2.7 AT1a+/+ Losartan 10.7 ± ± ± 2.1 AT1a / Losartan 18.3 ± ± ± 0.7 Values are mean ± S.E.M. Three-way ANOVA shows group effect (F [1,65] = 5.4, P = 0.02), treatment effect (F [1,65] = 50.3, P < 0.001) and time effect (F [2,65] = 15.8, P < 0.001). There are no individual differences. Basal MAP and HR were ± 4.3 mmhg and ± 17.3 beats min 1 in AT1a+/+ mice, and 86.3 ± 4.6 mmhg and ± 27.6 beats min 1 in AT1a / mice, respectively. n = 6 7 per group ± 1.8% (P = 0.052), respectively. There were no significant differences in HR (Figs 2B and 3B). Effect of pharmacological blockade of AT1 and AT2 receptors on basal MAP and HR In order to determine whether Ang AT1b or AT2 receptors were involved in the control of basal BP and HR, we tested the effect of AT1 and AT2 receptor antagonists, losartan and PD123319, respectively. Results showed that the AT1 antagonist losartan did not alter basal MAP in AT1a+/+ Results Effect of acute intracarotid HS injection on plasma osmolality and VP HS caused an increase in plasma osmolality with no difference noted between the groups. The elevated systemic osmolality induced a 2- to 4-fold increase in plasma VP concentration with similar responses between groups (Table 1). Effect of acute intracarotid HS injection on MAP and HR AT1a / mice had a lower basal MAP than AT1a+/+ mice, with no difference in HR (Fig. 1). IS produced no effect on MAP or HR in either AT1a+/+ or AT1a / mice (data not shown). Acute intracarotid HS injection produced a rapid increase in MAP in both AT1a+/+ and AT1a / mice with a return to baseline within min. HS induced a greater increase in MAP in AT1a / as compared to in AT1a+/+ mice (Tables 2 and 3, group effect, P = 0.02). The percentage increases of MAP 1, 5 and 10 min after HS injection were (AT1a / versus AT1a+/+, Fig. 2A): ± 2.4% versus ± 1.9% (P = 0.003), ± 3.0% versus ± 1.5% (P = 0.04) and ± 3.9% versus Figure 1. MAP (A) and HR (B) ofat1a+/+ and AT1a / mice under basal condition and after losartan treatment (20 mg kg 1 ) Two-way ANOVA, followed by Newman Keuls post hoc test, was used for analysis. AT1a / mice had a lower basal MAP (F [1,22] = 85.2, P < 0.001). Losartan treatment did not alter the basal MAP (F [1,22] = 2.83, p = 0.11). There were no significant differences in HR in baseline values or after treatment in both groups. n = 6 7 per group. Values are mean ± S.E.M. P < 0.01 AT1a / versus AT1a+/+, with or without losartan.

4 742 Y. Chen and others Exp Physiol 90.5 pp Table 3. Effect of PD on intra-carotid HS induced changes in MAP in AT1a+/+ and AT1a / mice MAP (mmhg) Groups Treatment HS 1 min HS 5 min HS 10 min AT1a+/+ Con 17.2 ± ± ± 4.9 AT1a / Con 22.4 ± ± ± 2.6 AT1a+/+ PD ± ± ± 2.2 AT1a / PD ± ± ± 0.8 Values are mean ± S.E.M. Three-way ANOVA shows group effect (F [1,48] = 6.1, P = 0.02), treatment effect (F [1,48] = 0.09, P > 0.05) and time effect (F [2,48] = 12.0, P < 0.001). There are no individual differences. Basal MAP and HR were ± 4.6 mmhg and ± 24.7 beats min 1 in AT1a+/+ mice, and 81.5 ± 2.2 mmhg and ± 10.2 beats min 1 in AT1a / mice, respectively. n = 5 6 per group. or AT1a / mice (Fig. 1A). Likewise the AT2 antagonist, PD treatment did not alter basal MAP in AT1a+/+ or AT1a / mice (Fig. 3A). Losartan and PD produced no effect on basal HR (Figs 1B and 3B). Effect of pharmacological blockade of AT1 and AT2 receptors on the cardiovascular response to HS In order to determine whether Ang AT1b or AT2 receptors were involved in the HS response, we tested the effect of AT1 and AT2 receptor antagonists, losartan and PD123319, respectively. To determine whether the second HS treatment may augment the BP response, we set saline injection for treatment controls in experiments 2 and 3. Results showed that the second injection of HS induced similar BP response as compared to the first HS injection in saline controls in AT1a+/+ and AT1a / mice (data not shown). Results showed that losartan blocked the higher MAP response in AT1a / mice, but not in AT1a+/+ mice (Table 2 and Fig. 2A). Losartan produced no effect on HR after osmotic stimulation (Fig. 2B). The AT2 antagonist, PD did not inhibit the MAP response to osmotic stimulation (Table 3, Fig. 4A). The AT2 antagonist did not affect HR after HS injection (Fig. 4B). Effect of acute intracarotid VP on MAP and HR In order to determine whether AT1a+/+ and AT1a / mice have a different pressor response to VP, we tested the effect of acute VP injection. Results showed that VP (5 ng g 1 ) induced an increase in MAP and HR in both groups with a maximum response seen in 1 min. There were no difference in maximum MAP and HR response to VP between AT1a+/+ and AT1a / mice (Table 4). Figure 2. Effect of systemic osmotic stimulation on change of MAP (A) and HR (B) inat1a+/+ and AT1a / mice with and without losartan pretreatment Three-way ANOVA, followed by Newman Keuls post hoc test, was used for analysis. For MAP there was a significant effect of group (F [1,65] = 23.6, P < ), time (F [2,65] = 10.1, P = 0.002) and treatment (F [1,65] = 70.5, P = ) as well as an interaction between group and treatment (F [1,65] = 6.7, P = 0.012). There were no significant changes in HR. n = 6 7 per group. Values are mean ± S.E.M. P < 0.01, P < 0.05, AT1a / versus AT1a+/+; ##P < 0.01, #P < 0.05, AT1a / versus AT1a / plus losartan. Figure 3. MAP and HR of AT1a+/+ and AT1a / mice under basal condition and after treatment with the Ang AT2 antagonist PD Two-way ANOVA, followed by Newman Keuls post hoc test, was used for analysis. AT1a / mice had a lower basal MAP (F [1,16] = 153.9, P < 0.001). PD treatment did not alter the basal MAP (F [1,16] = 2.7, P = 0.12) or HR in AT1a+/+ and AT1a / mice. n = 5 per group. Values are mean ± S.E.M. P < 0.01 AT1a / versus AT1a+/+, with or without PD

5 Exp Physiol 90.5 pp AT1b receptors in osmonsensitivity 743 Discussion Neuronal osmoreceptors are capable of transducing changes in external osmotic pressure into electrical signals that activate CNS areas involved in the control of BP and water homeostasis. Sensitive structures in the CNS include the rostral anterior ventricle, median preoptic (MnPO), SFO and the DVC/NTS (Carlson et al. 1997; Toney et al. 2003). Anatomical studies by us (Chen & Morris, 2001; Chen et al. 2003, 2004b) and others (Kakar et al. 1992; Burson et al. 1994) have shown that both AT1a and AT1b receptors are present in mouse brain, specifically in regions responsive to osmotic and pressor stimulation. Physiological studies using receptor knockout strains suggest that both AT1a and AT1b are involved in the control of BP and water homeostasis (Oliverio et al. 1997; 2000a,b;Zhuet al. 1998; Ruan et al. 1999; Davisson et al. 2000; Zhou et al. 2003). This was suggested by the lower basal BP in AT1a / mice and the depressor response to losartan (Ito et al. 1995; Oliverio et al. 1997). There is also evidence that the AT1 receptors are involved in central osmotic pathways (Chen et al. 2003). The response to dehydration is enhanced in the AT1a / mice, seen as increased c-fos and VP mrna expression in the paraventricular region (Morris et al. 2001). There is also an increased BP response to dietary salt in the AT1a / strain (Oliverio et al. 2000a). Finally, recent data show that there is enhanced cellular osmosensitivity as seen in isolated supraoptic neurones using patch-clamp electrophysiological recording (Skalska et al. 2005). To extend our studies of the role of AT1a receptors in osmosensitivity, we tested the effect of vascular injection of HS in AT1a / and+/+ mice. Using carotid arterial catheters, the objective was to directly and quickly stimulate brain osmoreceptors with similar increases in osmolality. The fact that the NaCl was injected into the carotid artery and that the pressure response occurs rapidly suggests that the pathway may be via the CNS. Results showed that HS produced a greater pressor response in AT1a / mice, indicating enhanced osmosensitivity. This supports other studies which showed enhanced responsiveness to more chronic osmotic stimuli (Oliverio et al. 2000a; Morris et al. 2001; Rocha et al. 2005). It is interesting that a model in which the renin angiotensin system is over- rather then under-expressed in brain, the mren2 transgenic rat, also showed an enhanced pressor response to osmotic stimuli (Nishioka et al. 1999). This result suggests a relationship between enhanced central angiotensinergic drive and central osmotic responses. However, from that study it is not clear whether the AT1a or AT1b receptor signals mediated the hypersensitivity. The difference may be related to the different route in which the osmotic stimulation was applied. To evaluate whether AT1b or AT2 receptors are involved in the osmotic cascade, losartan (AT1 antagonist) or Table 4. Effect of intra-carotid vasopressin injection on MAP and HR Groups n MAP (mmhg) MAP (%) HR (bpm) HR (%) AT1a+/ ± ± ± ± 4.6 AT1a / ± ± ± ± 6.1 Values are mean ± S.E.M. One-way ANOVA shows group effect in MAP (absolute change, F [1,8] = 0.05, P = 0.82; percentage change, F [1,8] = 2.2, P = 0.18) and in HR (absolute change, F [1,8] = 0.47, P = 0.51; percentage change, F [1,8] = 0.27, P = 0.62). Basal MAP and HR were ± 2.1 mmhg and ± 46.9 beats min 1 in AT1a+/+ mice, and 81.0 ± 3.7 mmhg and ± 21.8 beats min 1 in AT1a / mice, respectively. PD (AT2 receptor antagonist) were used to block the AT1b (remaining AT1 receptors) or AT2 receptors in AT1a / mice. The results showed that acute blockade of AT1 or AT2 receptors had no effect on basal BP or HR in AT1a+/+ or AT1a / mice. A previous study had shown that chronic losartan treatment lowered BP in this strain (Oliverio et al. 1997). There is the possibility that losartan may have two different mechanisms (sites) of action in the two studies. It is possible that short-term use of losartan in the present study blocks AT1 receptors in the periphery, whereas losartan may act at both central Figure 4. Effect of systemic osmotic stimulation on change of MAP (A) and HR (B) inat1a+/+ and AT1a / mice, with and without PD pretreatment Three-way ANOVA, followed by Newman Keuls post hoc test, was used for analysis. For MAP there was a significant effect of group (F [1,48] = 15.0, P = ) and time (F [2,48] = 12.6, P < ), but not treatment (F [1,48] = 1.4, P = 0.24). There were no significant changes in HR. n = 5 6 per group. Values are mean ± S.E.M. P < 0.01, P < 0.05, AT1a+/+ versus AT1a /.

6 744 Y. Chen and others Exp Physiol 90.5 pp and peripheral receptors when used for a longer duration in the study of Oliverio et al. (1997). Another major finding was that the enhanced HS pressor response in AT1a / mice was prevented by losartan, an effect which was not observed in the receptor-intact group. This may mean that the effect of HS in AT1a+/+ mice involves the central receptors and that in the AT1a / mice both central receptors and those accessed by circulating Ang II are involved. For example, there are AT1b receptors in SFO (Chen & Morris, 2001; Chen et al. 2003). This suggests that Ang AT1b receptors might be involved because losartan acts to inhibit the remaining AT1 signal (AT1b). A previous study had shown that AT1b mrna is upregulated in the brainstem of AT1a / animals and in mice consuming a high-salt diet (Chen et al. 2002, 2004b). These data together suggest that the AT1b receptors are activated when the AT1a receptors are deleted, and this alteration in AT1b receptors may be responsible for the enhanced osmotic sensitivity. In contrast, PD pretreatment did not antagonize the osmotic pressor responses in AT1a / and AT1a+/+ mice, suggesting that AT2 receptors did not play a role in the osmotic pressor response. There are several reports in rats that demonstrated the role for AT1 receptors in mediating osmotic responses (Chen & Toney, 2001; Zhang & Leenen, 2001). Losartan microinjected into bilateral PVN is reported to have no effect on resting renal sympathetic nerve activity (RSNA) but reduces RSNA response to HS (Chen & Toney, 2001). Peripheral administration of losartan can prevent the BP response induced by chronic central osmotic stimulation (Zhang & Leenen, 2001). A cellular osmosensitivity study in isolated supraoptic neurones using path-clamp electrophysiological recording provides direct evidence of the hyper-osmosensitivity in AT1a / mice (Skalska et al. 2005). The findings in this study deserve further exploration of its mechanisms. Using antisense oligonucleotides for the AT1 receptor suggested that the AT1 receptors in PVN mediated the salt-sensitive hypertension in mren-2 transgenic rats (Li et al. 1996). Recently, we have developed RNA interference (RNAi) methods for silencing AT1a and AT1b receptors. Our study using adenovirus-mediated small hairpin RNA (shrna) for site-specific silencing down AT1a receptors in mouse brain provides a new tool for this purpose (Chen et al. 2005). The data being collected by researchers using this new technique will help to further disclose the functions of these two receptor subtypes. VP, a neuropeptide secreted from the neurohypophysis, is important in regulation of water balance and BP (Reghunandanan et al. 1991; Michelini & Morris, 1999; Oliverio et al. 2000a). Osmotic stimulation induces VP release and the pathway may involve central Ang receptors (Qadri et al. 1993; Bourque et al. 1994; Chen et al. 2004a). To determine whether VP plays a role in the altered pressor response, we measured HS-induced VP release in AT1a / and AT1 a+/+ mice. Data showed that the plasma VP levels were similar in both groups under basal and osmotically stimulated conditions. This suggests that VP does not mediate the enhanced BP response in AT1a / mice and indicates that AT1a receptors are not necessary to maintain osmotic control of VP release. In previous studies we showed a reduced plasma VP response to dehydration in AT1a / mice in association with an enhanced central neurosecretory response (Morris et al. 1999; 2001). However, dehydration is a stimulus that produces both increased osmolality and hypovolaemia, which is different from the effect of acute HS injection. The interpretation of the plasma VP data must be tempered by results from our laboratory which show that the time course for neurosecretion in mice is extremely rapid (Chen et al. 2004a). The plasma VP response to systemic HS was almost instantaneous with a return to baseline levels within 1 min. This makes it difficult to say with certainty that there are not early changes in VP, not detected because of the experimental protocol. To determine whether altered response to VP accounts for the altered osmotic sensitivity, we measured the VP-induced pressor response in AT1a / and+/+ mice. The results showed that the VP-induced BP and HR responses were similar in AT1a / and AT1a+/+ mice. This observation did not support the possibility that the sensitivity of plasma VP mediates the osmotic hypersensitivity in AT1a / mice. 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