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Am J Physiol Heart Circ Physiol 290: H1251-H1258, 2006. First published November 4, 2005; doi:10.1152/ajpheart.00203.2005
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In vivo evidence for endothelin-1-mediated attenuation of {alpha}1-adrenergic stimulation

Gerard D’Angelo,1,2 Jennifer S. Pollock,1,2,3 and David M. Pollock1,2,3,4

1Vascular Biology Center, Departments of 2Physiology, 3Pharmacology, and 4Surgery, Medical College of Georgia, Augusta, Georgia

Submitted 2 March 2005 ; accepted in final form 31 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experiments were designed to determine the influence of endothelin A (ETA) receptors on the pressor response to acute environmental stress in Dahl salt-resistant (DR) and Dahl-sensitive (DS) rats. Mean arterial pressure (MAP) was chronically monitored by telemetry before and after treatment with the selective ETA receptor antagonist ABT-627. Rats were restrained and subjected to pulsatile air jet stress (3 min). In untreated animals, the total pressor response (area under the curve) to acute stress was not different between DR vs. DS rats (8.1 ± 1.7 vs. 15.6 ± 2.6 mmHg x 3 min, P = 0.10). Conversely, treatment with ABT-627 potentiated the total pressor response only in DR rats (36.3 ± 6.2 vs. 22.6 ± 5.9 mmHg x 3 min, DR vs. DS, P < 0.05). Treatment with ABT-627 allowed greater responses in anesthetized DR rats to exogenous phenylephrine (1–4 µg/kg) during ganglionic blockade (P < 0.05) and produced a significant increase in plasma norepinephrine at baseline and during stress in conscious DR rats compared with untreated animals (P < 0.05). ETA receptor blockade had no effect on these responses in DS rats. Our results suggest that endothelin-1 can inhibit {alpha}-adrenergic-mediated effects in DR, but not DS rats, consistent with the hypothesis that ETA receptor activation functions to reduce sympathetic nerve activity and responses in vascular smooth muscle to sympathetic stimulation.

vascular smooth muscle; salt-sensitive hypertension; sympathetic nervous system

ENDOTHELIN-1 (ET-1) is a 21-amino acid peptide produced in numerous cell types, including the endothelium, vascular smooth muscle, renal tubular epithelium, cardiac myocytes, and neurons (20). In the vasculature, ET-1 functions as a potent vasoconstrictor and mitogen both in vivo and in vitro (9, 12, 34). Responses to ET-1 are mediated by two receptor subtypes, endothelin A (ETA) and B (ETB) (1, 23). The ETA receptor is found primarily on vascular smooth muscle, where its activation causes a sustained vasoconstriction and resultant increase in mean arterial pressure (MAP). Conversely, ETB receptors have been identified in the vascular endothelium (24, 27); coupled to an increase in nitric oxide (NO) (29) and prostacyclin (6), ETB receptor activation is thus responsible for the transient decrease in MAP observed immediately after the infusion of exogenous ET-1 (6, 16). ET-1 is generally thought to be responsible for elevating MAP principally in experimental models of salt-sensitive hypertension, given the effect of selective ETA or mixed ETA/ETB receptor antagonists to lower MAP after the induction of hypertension by a high-salt diet (2, 4, 8, 15).

Sudden spikes in arterial pressure, such as those caused by acute environmental stress, are believed to play a role in the onset of acute cardiovascular events, as well as the development of chronic cardiovascular disease. A causal relationship between ET-1 and the pressor response to stress has been implicated in clinical studies that have demonstrated a rapid increase in plasma ET-1 during the response to acute physical and mental stress (10, 19, 28). We have recently found, however, that the integrated pressor response to acute environmental stress is blunted in ETB receptor-deficient rats when placed on a high-salt diet, an experimental model of endothelin-dependent hypertension (3). Moreover, this effect is reversed by ETA receptor blockade. Together, these findings suggest that ET-1 attenuates the sympathetically mediated increase in MAP.

In the present study, the role of ETA receptors in the pressor response to acute stress was evaluated in Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats. Normotensive DS rats are more sensitive to the pressor effects of exogenous ET-1 compared with DR rats (14). Moreover, ETA receptor blockade reduces arterial pressure in DS rats made hypertensive by a high-salt diet. Thus the aim of this study was to test the hypothesis that ETA receptor blockade would cause greater enhancement of the pressor response to acute environmental stress in DS rats. Experiments in cultured cells have demonstrated that ET-1 can desensitize {alpha}-adrenergic receptors and thereby downregulate {alpha}-adrenergic signaling pathways (11, 30). Thus one potential mechanism by which ET-1 can reduce the pressor response to stress is by lowering the sensitivity of vascular smooth muscle to the vasoconstrictor effect of sympathetic activation through heterologous desensitization. Experiments were therefore designed to also test the hypothesis that ETA receptor blockade will heighten vascular smooth muscle responsiveness to {alpha}1-adrenergic stimulation.

Environmental stress was applied by subjecting DS and DR rats to air jet stress, a well-established method to evoke a rapid increase in sympathetic nerve activity (7, 17, 18). Cardiovascular responses to acute air jet stress were examined in animals that were continuously monitored by telemetry. The extent to which this response is modulated by ET-1 acting on ETA receptors was determined by monitoring the pressor response in untreated animals and in those given the selective ETA receptor antagonist ABT-627. To determine the effect of ETA receptor blockade on vascular smooth muscle responsiveness, we measured the pressor response to exogenous phenylephrine in anesthetized animals.


   METHODS
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 METHODS
RESULTS
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Animal model. All experiments used 9- to 12-wk-old male DR and DS rats (Harlan Laboratories; Indianapolis, IN) fed standard rat chow containing 0.8% NaCl and tap water ad libitum. Rats were housed in the animal care facility at the Medical College of Georgia, which is approved by the American Association for the Accreditation of Laboratory Animal Care. All protocols have been approved by the Institutional Animal Care and Use Committee.

Telemetry. Telemetry transmitters (Data Sciences,) were implanted according to the manufacturer’s specifications. Rats were anesthetized intraperitoneally with ketamine (50 mg/kg)-xylazine (10 mg/kg). The abdominal aorta was then exposed by a midline incision and briefly occluded. The transmitter catheter was inserted into a hole made by a 21-gauge needle just proximal to the iliac bifurcation and secured in place with tissue glue (Vetbond). The transmitter body was attached to the abdominal wall along the incision line with 4-O proline suture as the incision is closed. The skin was closed with staples that were removed 7 days after the incision had healed. Rats were returned to individual housing for data collection and allowed 8–10 days to recover from surgery before being placed on dietary protocols and subjected to stress. Animals were housed in a room separate from that used for studying the stress response. The individual rat cages were placed on top of the telemetry receivers, and MAP and heart rate (HR) were continuously (i.e., 10-s sampling periods at regularly scheduled 10-min intervals) recorded throughout the study using the Dataquest ART Acquisition program.

Air jet stress. DR and DS rats (n = 7 for each strain) were subjected to two sessions of acute air jet stress spaced 1 wk apart. Before each stress session, animals from each strain were divided into two groups, control and those treated with the selective ETA receptor antagonist ABT-627 (33). Treated animals were given ABT-627 at a concentration to deliver a dose of 5 mg·kg–1·day–1 for 3 days in their drinking water before the stress session. For those animals receiving ABT-627 before the first stress session, drug treatment was discontinued immediately after the stress session. ABT-627 has a plasma elimination half-life of 6.2 h (31) and thus was fully cleared before the second stress session. After the first session, treatment was reversed such that the previously untreated rats were given ABT-627 for 3 days before the second stress session.

On the day they were subjected to air jet stress, rats were quietly brought to a sound-proofed room. Immediately after the telemetry recording software was started, the room was vacated. The animals were then allowed to acclimate to their surroundings for 15–30 min in their cages, that is, until which time they ceased exploring the new environment and their pressures stabilized. Animals were then placed in tubular Plexiglas restrainers with sufficient aeration, and MAP and HR were continuously monitored by telemetry for at least 15 min before initiating air jet stress. When necessary, animals were monitored for up to an additional 10 min to allow the animals to adapt to being restrained, such that 3–5 min of stable MAP and HR recordings were obtained before exposure to air jet stress. Stress consisted of pulses (2-s duration delivered every 10 s for 3 min) of compressed air (15 lb/in2) aimed at the forehead from a 1/8-in. opening at the front of the tube. After the 3-min stress period, MAP and HR were monitored for 20 additional minutes while the animals were still in the restrainer, and post-air jet values were obtained. At the end of this poststress period, animals were returned to their cages and brought back to their holding room.

Whole animal pressor responses. DR and DS animals were either left untreated (water alone) (n = 6 for each strain) or administered the highly selective ETA receptor antagonist A-127722 (10 mg·kg–1·day–1; n = 5 for each strain) (21) in their drinking water for three days. A-127722 is 1:1 racemic mixture, of which the (+)-enantiomer ABT-627 is the biologically active form; conversely, the (–)-enantiomer is inert (21). Animals were anesthetized with thiobutabarbital (Inactin; 65 mg/kg ip), and the right jugular vein and femoral artery were isolated and cannulated with PE-50 for drug infusion and monitoring of MAP, respectively. After a 30-min equilibration period, phenylephrine (0.5, 1, 2, 4, 8, 16, and 24 µg/kg), an {alpha}1-adrenergic-specific agonist, was administered in randomized order. MAP was allowed to return to baseline between each dose. To eliminate endogenous sympathetic vasomotor tone and baroceptor-reflex-mediated responses, animals were given chlorisondamine (5 mg·ml–1·kg–1 iv). Effective blockade was confirmed by the absence of reflex bradycardia after constrictor administration. For each experiment, the responses to phenylephrine were tested in both the absence and presence of chlorisondamine; responses in the presence of ganglionic blockade were always tested second, following a 30-min reequilibration period after restoration of MAP in response to the last phenylephrine dose. All measurements were recorded using a PowerLab data-acquisition system.

Plasma catecholamine concentration. DR (n = 13) and DS (n = 18) rats were anesthetized intraperitoneally with ketamine (50 mg/kg)-xylazine (10 mg/kg), and catheters (Braintree Scientific, Braintree, MA) were inserted into the jugular vein. Catheters were routed subcutaneously and exteriorized at the back of the neck; catheters were filled with heparin (1,000 U/ml). For 2 days after the surgery, animals were placed into the restrainers for at least 15 min, consistent with the duration used for the acute stress protocol, and catheters were flushed to maintain patency. On days 3 and 4 postsurgery, blood (500 µl) was drawn from restrained animals to determine baseline (unstressed) plasma catecholamine levels. Blood was replaced with sterile saline (0.9% NaCl), and catheters were refilled with heparin. On day 5 postsurgery, animals were subjected to the acute stress protocol, and blood (500 µl) was drawn over the 30- to 60-s interval of air jet stress. As before, blood was replaced with sterile saline, and catheters were refilled with heparin. Catheter patency was maintained for the next 4 days, after which time the animals were placed on ABT-627 (5 mg·kg–1·day–1 for 3 days in the drinking water). Blood (500 µl) was drawn on each of the first 2 days of treatment with ABT-627, and on the third day of treatment, animals were again subjected to the acute-stress protocol. Alternatively, a group of animals was given ABT-627 immediately after the second baseline blood draw, i.e., day 4 postsurgery, and subjected to air jet stress after 3 days of treatment; thus these animals were stressed only once. Baseline catecholamine levels during treatment with ABT-627 in these animals were similar to those animals that had previously been subjected to air jet stress, and so the baseline values during treatment with ABT-627 were pooled. Samples were centrifuged at 2,000 g for 10 min at 4°C, and plasma was removed, aliquoted, and stored at –80°C until analyses could be performed. Plasma concentrations of epinephrine (Epi) and norepinephrine (NE) were determined by radioimmunoassay (BI-CAT-RIA, ALPCO Diagnostics, Windham, NH).

Plasma ET-1 concentration. DR and DS (n = 5 for each strain) rats treated with or without ABT-627 for 3 days were anesthetized with pentobarbital sodium (65 mg/kg ip), and a terminal arterial blood sample was obtained. Samples were centrifuged at 2,000 g for 10 min at 4°C, and plasma was removed, aliquoted, and stored at –80°C until analyses could be performed. Plasma ET-1 concentration was determined by ELISA (QuantiGlo; R&D Systems, Minneapolis, MN).

Statistical analysis. Data are expressed as means ± SE. All baseline MAP and HR values are reported as 24-h means. Total pressor response refers to the change in MAP during the 3 min of air jet stress and was determined by the equation {sum}[(P – PBase) x 0.067], where P refers to each MAP data point recorded during the delivery of air jet stress, PBase is the average pressure during the 3 min just before the onset of the air pulses, and 0.067 is the 4-s data collection interval. Data are expressed as the area under the curve (AUC; mmHg x min). Responses to phenylephrine in anesthetized animals are reported as the peak change in MAP from the baseline MAP. Baseline catecholamine values represent the average values obtained over the 2 days before subjecting the animals to air jet stress. Statistical analysis of the total pressor response was made by two-way ANOVA, followed by Newman-Keuls test for multiple comparisons. Phenylephrine dose-response curves were analyzed two-way ANOVA with repeated measures. Differences are considered significant at P < 0.05.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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Baseline cardiovascular parameters. Baseline (24-h) MAP was slightly but significantly higher in DS rats, whereas HR was not different (Fig. 1). Treatment with ABT-627 lowered baseline MAP in DS rats such that MAP was no longer different from that in DR animals (Fig. 1A). The change in MAP was accompanied by a significant increase in HR in DR but not in DS animals (Fig. 1B).


Figure 1
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  		Fig. 1. Baseline mean arterial pressure (MAP) (A) and heart rate (HR) (B) in Dahl salt-resistant and Dahl salt-sensitive rats. Animals were either untreated or given the endothelin A (ETA) receptor antagonist ABT-627 (5 mg·kg–1·day–1) for 3 days. Data represent average values over the 24-h period before being subjected to acute air jet stress. *P < 0.05.

 
Plasma ET-1 concentrations were not different in untreated DR and DS rats (Fig. 2). ABT-627 produced significant increases in both DR and DS rats; the ABT-627-mediated increase was significantly greater in DR animals.


Figure 2
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  		Fig. 2. Effect of ETA receptor antagonist on plasma endothelin-1 (ET-1) in Dahl salt-resistant and Dahl salt-sensitive rats. Animals were either untreated or given ABT-627 (5 mg·kg–1·day–1) for 3 days. *P < 0.05

 
Cardiovascular responses to stress. Cardiovascular responses to acute air jet stress in DR and DS rats are shown in Fig. 3, A and B, respectively. Typically, there was an initial rapid rise in MAP at the start of air jet stress, followed by oscillatory changes that were coincident with the delivery of subsequent air pulses. The initial response in the absence of ABT-627 was smaller in DS vs. DR rats although this difference was not significant (24 ± 5 vs. 33 ± 4 mmHg, DS vs. DR, P = 0.07). During the last minute of air jet stress, MAP was comparable to the pre-air jet baseline for both strains (Fig. 3, A and B). Comparison of the total pressor response, calculated as the area under the curve (AUC), indicated that the integrated response was not statistically elevated in DS vs. DR animals (P = 0.10) (Fig. 4). Both DR and DS rats displayed an abrupt bradycardic response at the start of air jet stress; HR then returned toward the pre-air jet value during the last minute of the stress period (data not shown).


Figure 3
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  		Fig. 3. Effect of ETA receptor antagonist on pressor response to acute environmental stress (A, B) and on blood pressure recovery after the termination of air jet stress (C, D) in Dahl salt-resistant (A, C) and Dahl salt-sensitive (B, D) rats. Animals were either untreated or given ABT-627 (5 mg·kg–1·day–1) for 3 days. Animals were restrained and subjected to pulsatile air jet stress. MAP was continuously monitored by telemetry. Dashed lines in C and D represent average MAP during 3 min before the start of air jet stress.

 

Figure 4
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  		Fig. 4. Summary of integrated pressor response (area under the curve; AUC) to acute air jet stress (A) and integrated MAP during the 20-min poststress period (B) in Dahl salt-resistant and Dahl salt-sensitive rats (n = 7 for each strain). Animals were either untreated or given the ETA receptor antagonist ABT-627 (5 mg·kg–1·day–1) for 3 days. AUC was calculated as the sum of the MAP data points during or after air jet stress minus the average MAP obtained over the 3 min before the start of air jet stress. *P < 0.05

 
To determine the extent the ETA receptor is involved in the pressor response to acute environmental stress, animals were given the selective ETA receptor antagonist ABT-627 for 3 days and then were subjected to the acute stress protocol. ABT-627 did not alter the height of the initial response for either strain. In DR rats treated with ABT-627, the increase in MAP during the last minute of air jet stress relative to the pre-air jet baseline was significantly higher than in those DR animals not given the compound (12 ± 3 vs. 2 ± 1 mmHg, treated vs. untreated, P < 0.05) (Fig. 3A). Consequently, comparison of the total pressor response indicated that ABT-627 caused an increase in DR rats (P < 0.05 vs. untreated) (Fig. 4A). Conversely, in DS animals treated with ABT-627, there was no difference in the relative increase in MAP during the last minute of air jet stress (7 ± 2 vs. 4 ± 3 mmHg, treated vs. untreated) (Fig. 3B) nor was there any significant enhancement of the total pressor response (Fig. 4A). Throughout the stress response, HR was essentially not different in DR and DS rats given the antagonist compared with their respective untreated controls (data not shown).

MAP and HR were monitored for 20 min after the stress period to assess the extent of recovery (Fig. 3, C and D). In all animals, MAP returned quickly to or below the pre-air jet baseline. In untreated and treated DR rats, average MAP during the last 5 min of the recovery period was 6 ± 2 (P < 0.05 vs. average MAP 3-min before air jet stress) and 2 ± 2 mmHg below, respectively, the pre-air jet baseline (Fig. 3C). Corresponding values for DS animals were 10 ± 3 and 4 ± 1 mmHg (P < 0.05 for each vs. average MAP 3 min before air jet stress), respectively (Fig. 3D). Comparison of the integrated MAP during the 20-min poststress period revealed a significant decrease in DS but not DR rats (Fig. 4B). Similarly, HR fell below pre-air jet baseline for all animals during the poststress period (data not shown). During the last 5 min poststress, average values for HR were not different between DR and DS rats or between untreated controls and those treated with the ETA receptor antagonist for each strain.

Whole animal pressor response to exogenous phenylephrine. To determine whether the increase in the stress-mediated pressor response in DR rats treated with an ETA receptor antagonist was due to augmented {alpha}-adrenergic responsiveness, we examined whole animal pressor responses to exogenous phenylephrine in anesthetized animals. Chlorisondamine produced comparable decreases in MAP in DR and DS rats, both in untreated animals (–30 ± 2 vs. –34 ± 2 mmHg, DR vs. DS) and in those given the ETA receptor antagonist (–32 ± 2 vs. –31 ± 4 mmHg, DR vs. DS). In the absence of chlorisondamine, responses to lower concentrations (0.5–2 µg/kg) of phenylephrine were not affected by treatment with A-127722 in DR animals (Fig. 5A); however, the maximum response to phenylephrine (24 µg/kg) was reduced ~12% ({Delta}MAP = 61 ± 3 vs. 69 ± 2 mmHg, treated vs. untreated, P < 0.05) (data not shown). Conversely, in the presence of chlorisondamine, phenylephrine responses at the lower dose range (0.5–2 µg/kg; P < 0.05) were augmented by ~22–25% in DR rats given the ETA receptor antagonist compared with controls (Fig. 5B), whereas the maximum response was unaltered (data not shown). Figure 6 shows the responses to the low dose range (0.5–4 µg/kg) of phenylephrine in DS rats. Treatment with A-127722 exerted no effect on phenylephrine responses either in the absence (Fig. 6A) or presence (Fig. 6B) of ganglionic blockade; similarly, pressor responses to the higher dose range (8–24 µg/kg) were not different between untreated and treated animals (data not shown).


Figure 5
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  		Fig. 5. Effect of ETA receptor antagonist on whole animal pressor response to exogenous phenylephrine (PE) in anesthetized Dahl salt-resistant rats in the absence (A) and presence (B) of autonomic ganglionic blockade using chlorisondamine (5 mg·kg–1·ml–1). Animals were either untreated or given A-127722 (10 mg·kg–1·day–1) for 3 days. *P < 0.05 vs. respective untreated response.

 

Figure 6
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  		Fig. 6. Effect of ETA receptor antagonist on whole animal pressor response to exogenous PE in anesthetized Dahl salt-sensitive rats in the absence (A) and presence (B) of autonomic ganglionic blockade using chlorisondamine (5 mg·kg–1·ml–1). Animals were either untreated or given A-127722 (10 mg·kg–1·day–1) for 3 days.

 
Effect of ETA receptor blockade on baseline and stressed catecholamine levels. To determine whether ETA receptor blockade augments the pressor response to stress by causing an increase in plasma catecholamine levels, DR and DS rats were fitted with venous catheters, and blood was drawn in unstressed and stressed animals. ABT-627 had no effect on the baseline plasma Epi concentration in DR rats but significantly reduced the stress-induced increase in plasma Epi (Fig. 7A). Conversely, ETA receptor blockade produced a significant increase in plasma NE concentration both at baseline and during air jet stress in DR rats (Fig. 7B). In DS rats, ABT-627 had no effect on baseline or stress-mediated increases in the plasma Epi (Fig. 8A) or NE concentration (Fig. 8B).


Figure 7
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  		Fig. 7. Effect of ETA receptor antagonist on plasma concentration of epinephrine (Epi) (A) and norepinephrine (NE) (B) at baseline and during air jet stress Dahl salt-resistant rats. Animals were either untreated or given ABT-627 (5 mg·kg–1·day–1) for 3 days. *P < 0.05.

 

Figure 8
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  		Fig. 8. Effect of ETA receptor antagonist on plasma concentration of Epi (A) and NE (B) at baseline and during air jet stress Dahl salt-sensitive rats. Animals were either untreated or given ABT-627 (5 mg·kg–1·day–1) for 3 days.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
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We have previously found that conditions reflective of an increase in tissue ET-1 activity suppress the pressor response to acute environmental stress and that this is reversed by ETA receptor antagonism (3). Experiments were performed in DS and DR rats in light of a previous report that DS rats exhibit increased vascular reactivity to ET-1 (14). Contrary to our hypothesis that ABT-627 would augment the response to acute stress in DS rats, we have found that ETA receptor blockade potentiates the pressor response in DR but not in DS animals. Consistent with this result, exogenous phenylephrine produced larger increases in MAP in ganglion blocked DR but not DS rats treated with an ETA receptor antagonist, suggesting that enhanced vascular smooth muscle responsiveness accounts at least partially for the effect of ETA receptor blockade on the response to acute stress. Moreover, ABT-627 produced a significant increase in baseline plasma NE concentration; plasma NE concentration was also greater in treated DR rats during air jet stress compared with DS animals. The enhanced pressor response in DR rats was coupled with a larger increase in plasma ET-1 concentration produced by administration of the ETA receptor antagonist. Finally, treatment with ABT-627 tended to attenuate blood pressure recovery during the poststress period in both strains.

Previously, we found that the enhanced pressor response after administration of an ETA receptor antagonist occurred in a genetic model of ET-1-dependent hypertension (3). In the present study, however, normotensive DS rats did not demonstrate any change in pressor responses, both to acute stress and to {alpha}1-adrenergic stimulation with ETA receptor blockade. Moreover, baseline HR was invariant, despite a significant decrease in MAP after treatment with ABT-627. Similarly, the pressor response to acute stress was comparable in normotensive and hypertensive DS rats (3). Together, these results indicate an inherent inability of the DS rat to adapt to alterations in ET-1 activity.

The experiments described herein address the pressor response to acute stress, which by virtue of the time frame studied is presumed to be mediated by the sympathetic nervous system, given that sympathetic nerve activity is rapidly increased in response to air jet stress (7, 18). Our results suggest that heightened ET-1 activity may actually blunt the pressor response because the response was enhanced under conditions of ETA receptor blockade. It therefore stands to reason that modulating catecholamine release and/or sensitivity of the target tissue may represent potential mechanisms by which ET-1 can affect sympathetic control of blood pressure.

To address the possibility that ET-1 may exert an effect postsynaptically, we examined the whole animal pressor response to exogenous phenylephrine in anesthetized animals. Experiments were performed in the presence of ganglionic blockade to prevent buffering of the pressor response by the baroceptor reflex, thereby allowing us to assess vascular responses directly. We observed that ETA receptor blockade augmented the phenylephrine-mediated response in DR but not DS rats, suggesting that ET-1 can depress {alpha}1-adrenergic-mediated vasoconstriction in these animals. Because this occurred in the dose range that elicited increases in MAP comparable to those seen during the stress response, it supports the contention that increased vascular smooth muscle responsiveness may contribute to the enhanced stress response seen with ETA receptor blockade. The argument that ET-1 can downregulate {alpha}1-adrenergic receptor activity is supported by previous studies in cultured rat-1 fibroblasts, where ET-1 markedly increased basal phosphorylation of {alpha}1-adrenergic receptors, an index of receptor desensitization, and attenuated the norepinephrine-induced rise in cytosolic calcium (11, 30). Moreover, ETA receptor blockade reversed the effect on {alpha}1-adrenergic receptor phosphorylation, indicating that the desensitizing effects are mediated through this pathway (11).

The presence of ET-1 receptors has been demonstrated on postganglionic sympathetic neurons (5), allowing for the possibility that ET-1 may also exert presynaptic regulation. This notion is supported by our finding that ABT-627 caused a significant rise in baseline plasma NE concentration in DR animals. Together with the observed increase in baseline HR in DR rats treated with ABT-627, these data suggest that basal sympathetic nerve activity is elevated by ETA receptor blockade in DR animals. We also found that ETA receptor antagonism yielded greater plasma NE concentration in DR rats during stress, consistent with results from several laboratories suggesting that ET-1 suppresses stimulated catecholamine release (25, 26, 32). The absolute increase in plasma NE above the respective baselines was not different in treated vs. untreated DR animals, that is, there was a parallel rise in plasma NE during stress. These data therefore suggest that a larger absolute change in plasma NE cannot account for the heightened total pressor response to stress in these animals. One possible explanation is that the corelease of other neurotransmitters may also be modulated by ET-1. Specifically, Hoang et al. (13) found that ET-1 inhibited nerve stimulation-induced neuropeptide Y release from the perfused mesenteric arterial bed. This effect, however, was sensitive to ETB but not ETA receptor blockade.

As noted in our previous study (3), an important caveat of the experimental paradigm is the use of two stressors, restraint followed by air jet stress. In the present study, we have consistently found that within each strain, restraint causes parallel increases in both MAP and HR relative to that level noted just before restraint in untreated animals and those given ABT-627. This suggests that the degree of sympathetic activation caused by placing the animals into the restrainers was comparable and that any elevation in sympathetic activity cannot explain the enhanced pressor response to air jet stress caused by ETA receptor blockade in DR rats. In our earlier study, we also found that increases in HR caused by restraint alone, which presumably reflect elevated sympathetic nerve activity, did not track with altered pressor responses to air jet stress produced by high-salt diet or a high-salt diet plus ETA receptor antagonism. Moreover, alteration of the pressor response occurred independently of changes in baseline MAP. Together, these data suggest that the effect of ETA receptor blockade on the pressor response to air jet stress can be differentiated from the effects of restraint alone.

The larger increase in plasma ET-1 concentration noted in the present study with ABT-627 in DR vs. DS rats suggests there is a greater inhibition of ET-1 binding to ETA receptors in the salt-resistant animals. Reasons for this difference are not necessarily straightforward. It is unlikely that tissue levels of ET-1 are lower in prehypertensive DS rats because both preproET-1 mRNA (14) and ET-1 protein levels (2) have been shown to be comparable in vascular segments from DR and DS rats. Because ETB receptors function to maintain the circulating ET-1 concentration at a low level, one possibility is that there is an upregulation of ETB receptors or a decrease in the ratio of ETA:ETB receptors in DS rats. Accordingly, this would account for the relative insensitivity of the acute pressor response to ETA receptor blockade in DS rats. Precedence for this explanation comes from our previous work demonstrating an apparent decrease in the ETA:ETB receptor ratio in the kidney in DOCA-salt hypertensive rats (22). Alternatively, it is conceivable that the binding affinity of ET-1 to the ETA receptor in normotensive DS rats is higher, thus requiring higher concentrations of antagonist to inhibit the binding of ET-1 to its receptor. To our knowledge, however, there is currently no evidence to support this idea. To address the possibility that the ETA:ETB receptor ratio is lower in DS rats, future studies will test the hypothesis that ETB receptor blockade produces greater increases in baseline MAP and plasma ET-1 in DS vs. DR rats.

In summary, we found that ETA receptor blockade significantly enhanced the pressor response to air jet stress in DR, but not DS rats. ETA receptor blockade also augmented the pressor response to exogenous phenylephrine during ganglionic blockade and caused a significant increase in plasma NE at baseline and during stress in DR but not DS rats. Finally, the larger increase in the plasma ET-1 concentration caused by ABT-627 suggests that inhibition of ET-1 binding is greater in DR vs. DS animals. Together, these data suggest that ET-1 modulates sympathetically mediated responses, such that inhibition of ETA-mediated effects reveals increased vascular smooth muscle responsiveness to {alpha}-adrenergic stimulation and removal of sympathoinhibition by ET-1. In the context of our hypothesis, these data indicate that the modulation of ETA-dependent responses is impaired in prehypertensive DS rats.


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 GRANTS
REFERENCES
 
This study was supported by grants from the National Heart, Lung, and Blood Institute (D. M. Pollock, HL-64776; J. S. Pollock, HL-69999) and from the American Heart Association (G. D’Angelo, Scientist Development Grant 0530361N; D. M. Pollock, Established Investigator 0340443N; J. S. Pollock, Established Investigator 0440073N).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the excellent technical assistance of Hiram Ocasio, Andrea Chancey, and Amy Dukes.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. D’Angelo, Vascular Biology Center, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta, GA 30912-2500 (e-mail: gdangelo{at}mail.mcg.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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