Sympathoinhibitory Function of the α2A-Adrenergic Receptor Subtype
Abstract—Presynaptic α2-adrenergic receptors (α2-AR) are distributed throughout the central nervous system and are highly concentrated in the brain stem, where they contribute to neural baroreflex control of blood pressure (BP). To explore the role of the α2A-AR subtype in this function, we compared BP and plasma norepinephrine and epinephrine levels in genetically engineered mice with deleted α2A-AR gene to their wild-type controls. At baseline, the α2A-AR gene knockouts (n=11) versus controls (n=10) had higher systolic BP (123±2.5 versus 115±2.5 mm Hg, P<0.05), heart rate (730±15 versus 600±18 b/min, P<0.001), and norepinephrine (1.005±0.078 versus 0.587±0.095 ng/mL, P<0.01), respectively. When submitted to subtotal nephrectomy and given 1% saline as drinking water, both α2A-AR gene knockouts (n=14) and controls (n=14) became hypertensive, but the former required 15.6±2.5 days versus 29.3±1.4 days for the controls (P<0.001). End-point systolic BP was similar for both at 155±2.1 versus 152±5.2 mm Hg, but norepinephrine and epinephrine levels were twice as high in the knockouts at 1.386±0.283 and 0.577±0.143 versus 0.712±0.110 and 0.255±0.032 ng/mL, respectively, P<0.05 for both. We conclude that the α2A-AR subtype exerts a sympathoinhibitory effect, and its loss leads to a hypertensive, hyperadrenergic state.
The sympathetic nervous system (SNS) is one of the major pressor systems involved in the regulation of blood pressure (BP). Aberrant function of various neuroendocrine components of the SNS contributes to abnormal BP in response to environmental stimuli, such as excessive sodium intake. Indeed, a large body of literature indicates that 1 of the mechanisms by which salt-loading raises BP is via activation of the central SNS, which leads to increased sympathetic outflow.1 2 Experimental studies in animals with pharmacological probes in vivo3 4 5 and radioligand techniques in vitro6 7 have suggested that salt-induced hypertension is associated with alterations in the functional characteristics of the α2-adrenergic receptors (α2-AR) in the central nervous system (CNS). None of these methods, however, can differentiate among the α2-AR subtypes (α2A, α2B, or α2c) involved in this process.
Recently, genetically engineered mice that are deficient in each one of the α2-AR subtypes became available.8 9 In our first series of experiments with these mice, we used animals deficient for the α2B-AR gene (+/−) and α2C-AR gene knockouts (−/−) compared with their wild-type counterparts (α2B +/+ and α2C +/+, respectively) to study the role of each subtype in determining salt-sensitivity. We found that the ability to develop hypertension in response to salt-loading requires a full complement of the α2B-AR subtype gene. Indeed, the α2B +/− mice failed to raise their BP after subtotal nephrectomy and dietary salt-loading for 5 weeks, whereas their wild-type counterparts became consistently hypertensive. On the contrary, knockout mice for the α2C subtype gene had the expected hypertensive response to chronic salt-loading and behaved no differently from their wild-type counterparts in this respect.10
The present experiments were designed to explore the role of the α2A-AR subtype gene in normal BP regulation and in the hypertensive response to salt-loading by comparing α2A-AR gene knockouts (−/−) to their wild-type counterparts.
Mice with deletion of the α2A-AR subtype gene (−/−) and their wild-type controls (+/+) were used in these studies. The animals, 8 to 10 weeks old, weighed 21.8 to 30.7 g and were housed in the animal facility of our institution (Boston University School of Medicine) and given free access to food (Purina Certified Rodent Chow, 5002) and distilled water. In animals submitted to subtotal nephrectomy, drinking water was replaced with 1% saline.
Genotypes were determined by polymerase chain reaction from DNA that were isolated from the tail or spleen of the animals. To screen the α2A-AR line, MA.GF1 (CGCTCAAAGCTCCCCAAAAC), MA.GB1 (GCTTCAGGTTGTACTCGATGGC), and PGK.2 (TGAGACGTGCTACTTCCA-TTTGTC) primers were used to detect the intact α2A-AR gene (246 bp) or the interrupted α2A-AR gene (368 bp). Each 25-μL polymerase chain reaction contained 0.2 μmol/l each primer, 0.2 mmol/l each dNTP, 2 mmol/l Mg2+, 10 mmol/l Tris-HCl, pH 8.3, 50 mmol/l KCl, 0.025 U of AmpliTaq Gold (Perkin Elmer) and was incubated as follows: 95°C for 12 minutes followed by 30 cycles of 94°C for 30 seconds, 55o for 30 seconds, and 75o for 1 minute 30 seconds followed by 75o for 5 minutes. Bands were separated on 3% to 4% NuSieve agarose gels. All experiments were conducted in accordance with the guidelines for the care and use of animals approved by the Boston University Medical Center.
The first set of experiments was designed to determine whether deletion of the α2A-AR gene affects baseline BP, heart rate (HR), and sympathetic outflow in these animals. One group (n=11) of α2A-AR knockout mice (α2A-AR −/−) along with 1 group (n=10) of their wild-type counterparts (α2A-AR +/+) were used in this protocol. In both groups, control (baseline) tail-cuff systolic BP and HR measurements were obtained with a computerized tail-cuff system (BP 2000 Visitech Systems), as described elsewhere.10 The system has the ability to determine systolic BP and HR in 4 mice simultaneously and uses a photoelectric sensor to detect the cuff pressure at which blood flow to the tail is eliminated. Mice were trained for 5 consecutive days (each session consisted of 20 unrecorded measurements) to familiarize the animals to the tail-cuff apparatus. Subsequently, BP and HR measurements were recorded daily for another 5 consecutive days. Each session consisted of 20 measurements for each mouse daily and the mean BP and HR for the day were calculated.
Subsequently, tail-cuff BP was confirmed by direct measurement via arterial catheterization, as described elsewhere.11 Arterial catheterization was performed in all animals under anesthesia induced by sodium pentobarbital (50 mg/kg IP). A modified polyethylene catheter (PE-50) was introduced into the right iliac artery for direct BP recording and was tunneled subcutaneously and exteriorized at the back of the animal’s neck. Subsequently, the catheter was filled with heparin in 0.9% saline solution, sealed with heat, and attached to the animal’s nape. After surgery, the animals were returned to their cages and allowed an overnight recovery period. On the following day, the arterial catheter was connected to a BP transducer attached to a recorder (model 220S, Gould Inc) for direct BP monitoring. Direct control (baseline) BP was recorded for ≥1 hour, and blood was then drawn from the arterial line for determination of control plasma catecholamine levels.
The second set of experiments explored the role of the α2A-AR in salt-induced hypertension. One group (n=14) of α2A-AR knockout mice (α2A-AR −/−) and 1 group (n=14) of their wild-type counterparts (α2A-AR +/+) were used in these studies. Mice were submitted to subtotal nephrectomy, given 1% saline as drinking water, and handled as described elsewhere.10
Tail-cuff systolic BP and HR measurements were obtained ≥3 times a week, and mice were followed for a maximum period of 35 days or until they became hypertensive, ie, their tail-cuff systolic BP reached 150 mm Hg or an increase by ≥40 mm Hg from baseline was recorded and sustained for 3 consecutive days. The BP and HR measurements of the last 3 days were averaged, and the mean was considered as the end-point tail-cuff BP and HR for the animal. The end-point tail-cuff BP was confirmed by direct measurement via arterial catheterization at the end of the protocol, as described elsewhere.11 Direct end-point BP was recorded for ≥1 hour, and blood was then drawn from the arterial line for determination of plasma catecholamine and creatinine levels.
Determination of Plasma Catecholamine and Creatinine Levels
For assay of plasma catecholamine levels, 100 μL of blood was drawn slowly from the arterial line. EGTA (90 mg/mL), reduced glutathione (60 mg/mL) solution RPN 532 (Amersham Life Sciences), was used as anticoagulant and antioxidant, as recommended for the BioTrak Catecholamine Research Assay System TRK 995 (Amersham Life Sciences), which was used for norepinephrine and epinephrine measurement. Mouse plasma (10 to 20 μL) was diluted to 50 μL with sterile water to produce the 50 μL vol needed in the assay (with subsequent correction for dilution in calculation) and with 20 pg of each catecholamine standard to determine recovery. The assay is sensitive to ≈2 pg norepinephrine or epinephrine per tube. Plasma creatinine was measured on blood samples (500 μL) drawn into heparinized tubes from the arterial line, with a commercially available colorimetric kit from Sigma Diagnostics.
All data are presented as mean±SEM. Student’s t test for paired and unpaired data was used, as appropriate. The Mann-Whitney Rank Sum test was used for nonparametric data. Differences at P<0.05 were considered to be significant.
Figure 1⇓ presents baseline data obtained by the tail-cuff method and direct arterial catheterization in 8- to 10-week-old α2A-AR −/− mice and their wild-type counterparts. Figures 1A⇓ and 1B⇓ show that tail-cuff BP and HR were significantly higher in the α2A-AR −/− mice at 123±2.5 mm Hg and 730±15 bpm, respectively, than their wild-type (+/+) counterparts at 115±2.5 mm Hg and 600±18 bpm, respectively. Consistent with the tail-cuff measurements, direct mean arterial pressure (Figure 1C⇓) was significantly higher in the α2A-AR knockout mice than their wild-type counterparts.
Control plasma catecholamine levels in both groups are shown in Figure 2⇓. Plasma norepinephrine levels were significantly higher in the α2A-AR −/− group at 1.005±0.078 versus 0.587±0.095 ng/mL in the +/+, whereas plasma epinephrine levels were not different between the 2 groups at 0.356±0.090 versus 0.267±0.048 ng/mL, respectively.
Figure 3⇓ summarizes data obtained from subtotally nephrectomized salt-fed mice in both groups. Figure 3A⇓ shows that baseline (ie, before surgery) tail-cuff BP was higher in the α2A-AR −/− mice versus the wild-type group, with numbers similar to those of protocol 1. However, both groups became hypertensive after subtotal nephrectomy and salt-loading, which resulted in comparable end-point BP measurements (155±2.1 and 152±5.2 mm Hg, respectively). Figure 3B⇓ shows that tail-cuff HR was higher at baseline in the α2A-AR −/− mice (697±11 versus 609±12 bpm in +/+), but end-point HR was not different between the 2 groups. A significant increase in BP and HR from baseline was observed in both groups after subtotal nephrectomy and 1% saline (paired t test, P<0.001). As shown in Figure 3C⇓, direct mean arterial pressure at end point was comparable in both α2A-AR −/− and α2A-AR +/+ mice.
Figure 4⇓ shows end-point plasma catecholamine levels in both groups. Plasma norepinephrine and epinephrine levels were significantly higher in the α2A-AR −/− mice at 1.386±0.283 and 0.577±0.143 ng/mL, respectively, than their wild-type (+/+) counterparts at 0.712±0.110 and 0.255±0.032 ng/mL.
Table 1⇓ shows that no difference existed between the knockout mice (−/−) and their wild-type counterparts (+/+) in regard to body weight at baseline and end point or ratio of remnant kidney weight to body weight at end point. Mean plasma creatinine levels were comparable in both groups and indicated that the residual renal function was similar in both knockout and wild-type animals. After subtotal nephrectomy and 1% saline, however, the α2A-AR −/− mice became hypertensive faster than their wild-type counterparts (15.6±2.5 versus 29.3±1.4 days, respectively; P<0.001).
Several lines of evidence over the years have supported the notion that α2-AR in the CNS exert a sympathoinhibitory effect. This has been exploited clinically by the development of pharmacological agents with α2-agonistic properties (eg, clonidine) that suppress the central SNS and produce a sustained antihypertensive action.12 However, existing α2-agonists are nonselective for α2-AR subtypes. Genetic targeting of each one of the α2-AR gene subtypes has now produced evidence that suggests that the long-recognized sympathoinhibitory effect of central presynaptic α2-AR may be a function of the α2A-AR subtype.13 14 These receptors are abundantly distributed throughout the CNS, but highly concentrated in the brain stem,15 which is known to be the center of neural baroreflex control.16 17 Indeed, introduction of minute amounts of hypertonic saline directly into certain brain stem nuclei, such as the nucleus tractus solitarii, produces long-lasting systemic hypertensive responses18 and a hyperadrenergic state that can be explained by temporary reversal of this sympathoinhibitory effect.
The present experiments corroborate and extend these findings by demonstrating heightened sympathetic activity in α2A-AR gene knockout mice under various conditions: at baseline, 8 to 10 week old α2A-AR gene knockout mice already displayed significantly higher BPs and HRs, accompanied by about twice as high levels of circulating norepinephrine, in comparison to their genetically intact counterparts. After subtotal nephrectomy and dietary salt-loading, it took an average of ≈2 weeks for the α2A −/− mice to become hypertensive, as opposed to >4 weeks for the α2A +/+ mice. However, by end point, both groups had similar BP and HR levels, which indicated that removal of the SNS restraining effect of the α2A-AR hastened the development of salt-induced hypertension without altering the magnitude of the final response. Furthermore, at end point, both norepinephrine and epinephrine levels in the α2A −/− mice were twice as high as those of the α2A +/+. Consistent with these data is the recent finding that α2-AR agonists, which normally produce a hypotensive effect via central sympathoinhibition, were unable to produce such hypotensive effect in α2A-AR knockouts.19 This suggests that the α2A-AR is the major presynaptic receptor subtype that regulates norepinephrine release from sympathetic neurons, although a residual presynaptic α2-mediated effect was still detectable in α2A-AR knockouts, evidently contributed by one of the other subtypes.
These findings can be compared with those obtained on mice with complete or partial deletion of the other 2 α2-AR gene subtypes10 : homozygous α2C-AR gene knockout mice had normal BP at baseline, and after partial nephrectomy and dietary salt-loading, they developed hypertension to the same degree and in the same time frame as their wild-type controls, which indicated that the α2C-AR subtype has no function relevant to salt-sensitivity and/or BP elevation. On the contrary, mice deficient in the α2B-AR gene subtype, as mentioned earlier, were unable to raise their BP after subtotal nephrectomy and salt-loading. These findings indicated that the α2B-AR subtype is the one responsible for the development of salt-induced hypertension, although they did not provide information as to whether its role is central (ie, related to central SNS activation) or peripheral (ie, related to alterations in vascular tone or renal handling of sodium).
In combination, the data suggest that the central α2A-AR is the component mainly responsible for the known tonic sympathoinhibitory function of the presynaptic α2-AR of the CNS, because its loss leads to a hypertensive, hyperadrenergic state, whereas the α2B-AR is necessary for the hypertensive response to salt-loading. The pathophysiological implications of these findings are extremely important and may be directly applicable to optimizing the treatment of hypertension, chronic heart failure, anxiety disorders, and other conditions characterized by a hyperadrenergic state. Available pharmacological tools are nonselective; thus, a desirable action is inevitably accompanied by undesirable side effects (eg, the antihypertensive and bradycardic effect of clonidine cannot be separated from the drowsiness and impotence). Identification of the exact α2-AR subtype responsible for other specific α2-AR–mediated functions, such as mental alertness, sexual arousal, and other functions, may permit the development of corrective interventions or pharmacological tools designed to selectively alter 1 function without adversely affecting others.
This work was supported by the Hypertension SCOR Grant P50 HL-55001. The authors are indebted to Dr. Brian K. Kobilka from Stanford University for kindly supplying the breeder genetically engineered mice.
- Received April 6, 1999.
- Revision received April 27, 1999.
- Accepted May 5, 1999.
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