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Endocrinology Vol. 139, No. 1 81-88
Copyright © 1998 by The Endocrine Society

ARTICLES

Potentiation of Natriuretic Peptide Action by the ß-Adrenergic Blocker Carvedilol in Hypertensive Rats: A New Antihypertensive Mechanism1

Takanobu Yoshimoto, Mitsuhide Naruse, Akiyo Tanabe, Kiyoko Naruse, Toshirou Seki, Toshihiro Imaki, Takamura Muraki, Yuzuru Matsuda and Hiroshi Demura

Department of Medicine, Institute of Clinical Endocrinology, and the Department of Pharmacology (T.M.), Tokyo Women’s Medical College, Tokyo 162; and the Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd. (Y.M.), Tokyo 194, Japan

Address all correspondence and requests for reprints to: Dr. Takanobu Yoshimoto, Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women’s Medical College, 8–1 Kawadacho, Shinjuku-ku, Tokyo 162, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment with a ß-adrenergic blocker (ß-blocker) in hypertension is associated with increased plasma atrial natriuretic peptide (ANP) levels despite a decrease in cardiac overload. The mechanism and pathophysiological significance of the phenomenon remain unclear. To clarify the role of the ANP system in the antihypertensive effects of the ß-blocker, we investigated the effects of carvedilol (30 mg/kg·day, orally, for 4 weeks) on the ANP system in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm). Plasma ANP levels showed a significant increase despite a significant decrease in blood pressure and heart rate in the carvedilol group. Although ANP messenger RNA levels in the heart did not change, messenger RNA levels of the natriuretic peptide-C (NP-C) receptor as a clearance receptor showed a significant decrease in both the aorta and lung in the carvedilol group. NP-C receptor densities were also significantly decreased in the lung in this group. The biological half-life of exogenous ANP in circulating blood was prolonged in the carvedilol group compared with that in the control group. Administration of the ANP receptor antagonist, HS-142–1, resulted in a greater increase in systolic blood pressure in the carvedilol group than in the control group. In addition, both basal and ANP-stimulated cGMP contents in the aorta were significantly higher in the carvedilol group. These results suggest that carvedilol potentiates the hypotensive action of ANP by increasing plasma ANP levels and enhancing the vascular response to ANP. These effects were closely related to the down-regulation of the NP-C receptor. The newly found mechanism seems to account for a sizable portion of the antihypertensive effects of carvedilol and could be of potential importance in the treatment of cardiovascular disease with ß-blockers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AS SYMPATHETIC activation is a common pathological feature in patients with essential hypertension (for review, see Ref.1), ß-adrenergic blockers (ß-blocker), as a sympathetic antagonist, are now classified as one first line medicine for the treatment of hypertension (2, 3). Although reduced cardiac output, blocking of presynaptic ß2-adrenergic receptor, inhibition of renin secretion, an action on the central nervous system, and stimulation of vasodilator PGs have been discussed as possible antihypertensive mechanisms of ß-blockers, no conclusive view concerning the mechanism has been reached (for review, see Ref.4).

Although ß-blockers are known to provide secondary cardioprotection after an acute myocardial infarction (for review, see Ref.5), whether they have primary cardioprotective effects remains unproved. Recently, however, Packer et al. (6) reported that treatment with carvedilol, a nonselective ß-blocker, lessens the morbidity and mortality rate in patients with chronic heart failure. The cardioprotective effects of carvedilol in the previously contraindicated condition of heart failure stimulated renewed interest concerning the mechanism of this newly developed ß-blocker in cardiovascular diseases.

Evidence has accumulated to support the importance of the natriuretic peptide (NP) system in the regulation of cardiovascular homeostasis (for review, see Refs. 7 and 8). Plasma atrial natriuretic peptide (ANP) levels increase in patients with hypertension and decrease to normal levels after long term treatment with antihypertensive agents, mainly through a reduction of cardiac afterload (9, 10, 11, 12, 13). By contrast, treatment with a ß-blocker during hypertension has been reported to increase plasma ANP levels despite the significant decrease in blood pressure (14, 15, 16, 17, 18). As ANP is a cardiac hormone with potent vasodilatative and natriuretic actions (7), the increase in plasma ANP levels may contribute to the cardiovascular effects of the ß-blocker (17, 18). However, the mechanism responsible for the increase in plasma ANP levels and the pathophysiological significance of the increased ANP remain to be elucidated.

The aim of the present study was to examine the role of the ANP system in the antihypertensive effects of carvedilol. We investigated the effects of carvedilol on plasma ANP levels, ANP messenger RNA (mRNA) expression in the heart, ANP receptor mRNA levels and density, pharmacokinetics of ANP, and ANP-induced cGMP production of the aorta in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm). In addition, the biological significance of the endogenous ANP in carvedilol-treated rats was ascertained by a specific antagonist for a biologically active ANP receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Carvedilol (19) was provided by Daiichi Seiyaku Co. (Tokyo, Japan). The specific NP-A/NP-B receptor (8) antagonist, HS-142–1 (20), was provided by Kyowa Hakko Kogyo Co. (Tokyo, Japan). The ANP analog, des[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)-NH2 (C-ANF) was purchased from Peninsula Laboratories (Belmont, CA). C-ANF (21) is a synthetic ring-deleted analog of ANP that binds specifically to the NP-C receptor (21, 22) with high affinity, but not to the NP-A and NP-B receptors due to a quite low affinity.

Animals and treatments
Animal studies were approved by the committee on animal care and use of Tokyo Women’s Medical College under the NIH Guide for the Care and Use of Laboratory Animals. Male SHR-SP/Izm rats (23) were obtained from the Disease Model Cooperative Research Association (Kyoto, Japan) at 12 weeks of age. These rats were randomly allocated into two groups: the control group and the group treated with carvedilol (30 mg/kg·day, orally) for 4 weeks (carvedilol group). Carvedilol suspended in 0.5% methylcellulose solution was orally administered by a stomach tube once in the morning. The control group was given 0.5% methylcellulose solution as vehicle. All experiments were performed after this 4 weeks of treatment. All rats were kept in temperature- and humidity-controlled rooms that were illuminated from 0800–2000 h. They were fed ad libitum a standard chow (MF, Oriental Yeast Co., Chiba, Japan).

Blood pressure measurements
Systolic blood pressure and heart rate were determined at 12 weeks of age before treatment and 1, 2, and 4 weeks after the administration of carvedilol by the tail cuff method (Manometer-Tachometer, model KN-210–1, Natsume Instruments, Tokyo, Japan) in conscious semirestrained rats after warming. Ten measurements were made per session, and the average value was used.

RNA extraction
Rats were killed by decapitation. The aorta, kidney, lung, atrium, and left ventricle with interventricular septum (LV) were quickly excised, frozen on dry ice, and stored at -80 C. Total RNA was extracted from these tissues by the acid guanidinium-thiocyanate-phenol-chloroform method (24).

Preparation of complementary RNA (cRNA) probe and sense RNA standard
Antisense cRNA probes for the NP-A receptor, NP-C receptor, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were generated by methods described previously (25, 26). The sense RNA standard of each NP receptor was synthesized using the opposite side of the promoter for antisense probes from each linearized vector as a template (25, 26).

The complementary DNA fragment of rat ANP (rANP) corresponding to 244–475 bp of rANP complementary DNA sequence (27) was subcloned into multicloning sites between the T3 and T7 promoters of plasmid Bluescript II SK- (Stratagene, La Jolla, CA). The purified plasmids were linearized by restriction enzymes, BamHI (Boehringer Mannheim, Mannheim, Germany). The antisense cRNA probe of rat ANP was synthesized in the presence of T7 RNA polymerase (Stratagene), 100 µCi [32P]UTP (800 Ci/mmol; DuPont-New England Nuclear Research Products, Boston, MA), and three other unlabeled nucleotides with RNA transcription kits (Stratagene).

Ribonuclease (RNase) protection analysis
The RNase protection assay for the quantification of the NP-A and NP-C receptor mRNA levels was performed as described previously (25, 26). Briefly, a 20-µg sample of RNA or synthesized sense RNA standard was hybridized with 1 x 105 cpm NP-A or NP-C receptor probe in 30 µl 40 mM PIPES (Nakarai Tesque Co., Kyoto, Japan), pH 6.4, containing 80% formamide, 0.4 M NaCl, and 1 mM Na2EDTA. For RNA hybridization of the sample, the GAPDH probe (1 x 105 cpm) was also added to the hybridization mixture. The mixture was heated at 85 C for 8 min and incubated at 55 C for 12 h. Nonannealing nucleic acids were digested with RNase A (Boehringer Mannheim) and T1 (Sigma Chemical Co., St. Louis, MO) at final concentrations of 40 µg/ml and 2 µg/ml, respectively, in 10 mM Tris-HCl buffer, pH 8.0, containing 300 mM NaCl and 5 mM EDTA at 37 C for 40 min. The protected fragments were electrophoresed on 5% polyacrylamide gel containing 7 M urea.

Autoradiography for NP-A, NP-C receptor mRNAs, and GAPDH mRNA was performed as described previously (25, 26). The integrated optical density of the radioactive bands for NP-A and NP-C receptors was determined densitometrically with a Computing Densitometer (model ACD-25DX, ATTO, Tokyo, Japan). Each NP receptor mRNA level was quantified from the standard curve obtained with known amounts of synthesized sense RNA and was expressed as attomoles (amol) of mRNA per 20 µg total RNA, as described previously (25, 26). The results for each NP receptor were normalized to those for GAPDH in each sample.

The ANP mRNA was also semiquantitatively analyzed by RNase protection analysis according to the same principle as that described for the NP receptor (25, 26). Five micrograms of atrial or LV RNA sample were hybridized with 1 x 105 cpm ANP probe as described above. Semiquantification of ANP mRNA in the atrium and LV was performed by densitometric analysis after autoradiography. The results of ANP mRNA were normalized to those of GAPDH in each sample.

ANP binding assay for determination of NP-C receptor density
Membrane preparation and competitive binding assay were performed according to the methods described by Schiffrin et al. (28) with some modifications. Briefly, after decapitation, lung was quickly excised, frozen on dry ice, and stored at -80 C. Lung was homogenized with ice-cold 0.25 M sucrose solution in a Polytron homogenizer (Kinematica, Luzern, Switzerland; setting 8, twice for 10 sec each time). The homogenate was centrifuged at 1500 x g at 4 C for 10 min; then the supernatant was filtered through 50-µm nylon mesh and centrifuged at 100,000 x g for 30 min at 4 C. The pellet was resuspended in a 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM MgCl2. The protein concentration was measured using a protein assay kit (Bio-Rad Laboratories, Richmond, CA). BSA was then added at a concentration of 0.2%. The composition of the assay buffer was 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM MgCl2, 0.5 mM phenylmethylsulfonylfluoride (Sigma), 0.1% bacitracin (Sigma), 1 µM aprotinin (Wako Pure Chemical Industries, Osaka, Japan), and 0.2% BSA. [125I]{alpha}-rANP (74 tetrabecquerels/mmol) was purchased from Amersham (Aylesbury, UK).

In competitive binding assay, the membrane fraction from the control group (30 µg protein) in 0.25 ml assay buffer was incubated in duplicate for 90 min at 25 C with 50 pM [125I]{alpha}-rANP (74 tetrabecquerels/mmol) and increasing concentrations of unlabeled {alpha}-rANP (Peptide Institute, Osaka, Japan) or C-ANF. Separation of bound and free radioactivity was achieved by rapid filtration through polyethylenimine-treated Whatman GF/C filters (Whatman, Clifton, NJ). The filters were washed twice with 2.5 ml 50 mM Tris-HCl buffer (pH 7.4) and then counted in an Aloka {gamma}-counter with 75% efficiency. Binding remaining in the presence of 1 µM unlabeled {alpha}-rANP was considered nonspecific binding. The homologous inhibition curve (the inhibition curve by {alpha}-rANP) and the inhibition curve by C-ANF were used for Scatchard analysis of ANP-binding sites and estimation of the NP-C receptor ratio, respectively.

The binding assay for the NP-C receptor in lung tissue was performed according to the methods described by Kishimoto et al. (29) with some modifications. The specific activity of [125I]{alpha}-rANP was adjusted with unlabeled {alpha}-rANP to 1000–1100 mCi/µmol. The membrane fraction was incubated with a saturable concentration (0.14 nM) of the adjusted [125I]{alpha}-rANP in the presence or absence of an excess amount of unlabeled C-ANF (1 µM) under the same conditions described in the competitive binding assay. The concentration of [125I]{alpha}-rANP (0.14 nM) was saturable in both the carvedilol and control groups. Separation of bound and free radioactivities was performed in the same manner as described above. To determine the specific binding to the NP-C receptor, the [125I]{alpha}-rANP binding in the presence of excess C-ANF was subtracted from the total binding.

Pharmacokinetic study
Pharmacokinetic study for exogenously administered ANP in carvedilol-treated and control SHR-SP/Izm was performed according to the methods described by Yokota et al. (30) with some modifications. Rats were anesthetized by ip injection of sodium pentobarbital (50 mg/kg), and the trachea, right carotid artery, and left jugular vein were catheterized. A bolus iv injection of 2.0 µg {alpha}-human ANP ({alpha}-hANP; Peptide Institute, Osaka Japan) dissolved in 0.1 ml saline was given. Then, 0.3-ml blood samples were periodically (0.5, 1, 1.5, 2, 3, 5, 8, 15, and 25 min after injection) withdrawn via the catheter in the artery and replaced by an equal volume of saline via the catheter in the vein. The concentration of plasma {alpha}-hANP was determined by commercially available immunoradiometric assay kits (Shionogi Co., Osaka, Japan) as described previously (31). As the 125I-labeled monoclonal antibody raised against Gly10-Arg-Met-Asp-Arg14 of {alpha}-hANP was used in the assay kits, the cross-reactivity with {alpha}-rANP was less than 1%. The half-lives of {alpha}-hANP in the fast and slow phases were calculated from the two-compartment open model by nonlinear least square regression analysis (32).

Effects of NP receptor antagonist, HS-142–1, on systolic blood pressure
The role of endogenous ANP in SHR-SP/Izm from both the carvedilol and control groups was investigated by ip bolus injection of HS-142–1 (20) (8 mg/kg BW) as described previously (33). Systolic blood pressure was recorded by the tail cuff method before and 30, 60, and 120 min after the administration of HS-142–1 with the rats under conscious semi-restrained conditions. The results were expressed as the average change from the baseline blood pressure in each rat after administration.

Aortic cyclic nucleotide contents in response to ANP
Cyclic nucleotide contents in aortic tissue in response to ANP were measured by the methods described by Schiffrin et al. (34) with some modifications. Briefly, aorta was quickly excised from the control and carvedilol groups after decapitation. The descending portion of thoracic aorta was divided into two segments (~20–30 mg each), and one was quickly frozen in liquid nitrogen and stored at -80 C for measurements of basal cGMP and cAMP contents. The other was suspended in Krebs-Ringer phosphate buffer, pH 7.4, at 37 C under bubbling with a mixture of 95% O2 and 5% CO2. The buffer consisted of the following composition: 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 11.1 mM glucose, 100 U/ml penicillin, and 100 µg/ml streptomycin. Then, 10 min before stimulation by ANP, isobutylmethylxanthine (Sigma) was added to the buffer at a concentration of 0.2 mM. Next, the tissues were exposed to 10-8 M {alpha}-rANP for an additional 10 min. The reaction was stopped by immersion of tissues in liquid nitrogen, and they were stored at -80 C.

The frozen tissues were homogenized with 2 ml ice-cold 6% perchloric acid and then centrifuged at 1500 x g at 4 C for 15 min. The pellet was resuspended with 0.5 N NaOH, and protein concentrations were determined using a protein assay kit (Bio-Rad Laboratories). The supernatant was neutralized with 0.2 ml 60% KOH and briefly centrifuged, and the final supernatant was collected and stored at -20 C for cGMP and cAMP measurements. The cGMP and cAMP contents of the tissue extracts were determined by commercially available RIA kits after a succinylation step (Yamasa Shoyu Co., Choshi, Japan) and were normalized to the amount of protein in the pellet. The antibodies against cGMP and cAMP used in the RIA kits showed less than 0.1% cross-reactivity with cAMP and cGMP, respectively.

Hormone assay methods
Trunk blood was collected into chilled test tubes with Na2EDTA plus aprotinin (500 IU/ml). Plasma samples obtained by centrifugation at 4 C were kept at -20 C until assayed. Plasma ANP concentrations were determined by commercially available RIA kits specific for {alpha}-rANP (Peninsula) after extraction on a Sep-Pak C18 cartridge (Waters, Milford, CA) and elution with 60% acetonitrile containing 0.2% ammonium acetate (pH 4.0) as described previously (35). PRA was determined by commercially available RIA kits (Incstar Co., Stillwater, MN).

Statistical analysis
Values were expressed as the mean ± SEM of multiple experiments. Differences between the two groups were analyzed by the Mann-Whitney U test. Time-dependent changes in systolic blood pressure and heart rate after the administration of carvedilol and HS-142–1 were analyzed by one-way ANOVA followed by Dunn’s test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The systolic blood pressure and heart rate showed a significant decrease after 1 week through 4 weeks of treatment with carvedilol (Fig. 1Go and Table 1Go). After 4 weeks of treatment, plasma ANP concentrations were significantly elevated in the carvedilol group compared with those in the control group, whereas there was no significant difference in PRA between the groups (Table 1Go).



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  		Figure 1. Changes in heart rate (a) and systolic blood pressure (b) before and after treatment with carvedilol (closed circle) and in the controls (open circle). Values are the mean ± SEM of six rats in each group. *, P < 0.05 vs. the control group; {dagger}, P < 0.05 vs. before treatment; §, P < 0.05 vs. 1 week after treatment.

 

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  		Table 1. Effects of 4-week treatment of carvedilol on systolic blood pressure, heart rate, plasma ANP levels, and plasma renin activity

 
ANP mRNA levels in the heart were semiquantified by RNase protection analysis. The protected bands of ANP mRNA showed single bands at the expected size (Fig. 2aGo). There was no significant difference in ANP mRNA levels in the atrium and ventricle between the carvedilol group (n = 4) and the control group (n = 4).



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  		Figure 2. Typical autoradiogram of PAGE of ANP (a), NP-A receptor (b), and NP-C receptor (c) mRNAs by RNase protection assay. Cont., Control group; Carved., carvedilol group; LV, left ventricle; NP-AR, NP-A receptor; NP-CR, NP-C receptor.

 
NP receptor mRNA levels in the aorta, kidney, and lung were semiquantified by RNase protection analysis. The protected bands of NP-A, NP-C receptor, and GAPDH mRNA showed single bands of the expected sizes (Fig. 2Go, b and c). There was no significant difference in NP-A receptor mRNA levels in the aorta and kidney between the carvedilol (n = 4) and control (n = 4) groups. NP-C receptor mRNA levels showed a significant (P < 0.05) decrease in both the aorta (3.4 ± 0.3 amol/20 µg RNA) and lung (75.7 ± 2.8 amol/20 µg RNA) of the carvedilol group compared with those inthe control group (aorta, 6.1 ± 0.2 amol/20 µg RNA; lung, 119 ± 8 amol/20 µg RNA; Fig. 3Go, a and b). There was no significant change in NP-C receptor mRNA levels in the kidney between the two groups (n = 4).



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  		Figure 3. Effects of carvedilol on NP-C receptor mRNA levels in the aorta (a) and lung (b) of SHR-SP/Izm rats. Values are the mean ± SEM. The number of experiments is shown in parentheses. *, P < 0.05 vs. the control group.

 
The inhibition curve of the competitive binding assay with [125I]{alpha}-rANP in the lung tissue of control SHR-SP/Izm was shown in Figure 4aGo. Scatchard analysis revealed a single class, high affinity binding site with a Kd of 201 ± 9 pM (n = 6) and a maximal binding capacity of 250 ± 13 fmol/mg protein (n = 6; Fig. 4bGo). An excess amount of C-ANF displaced 40 ± 3% (n = 6) of the specific [125I]{alpha}-rANP binding (Fig. 4aGo), indicating that approximately 40% of the NP receptors were classified as NP-C receptor in the lung tissue of SHR-SP/Izm rats. The NP-C receptor densities in the lung of the carvedilol group (40 ± 4.2 fmol/mg protein; n = 6) were significantly (P < 0.05) decreased compared with those in the control group (79 ± 9.3 fmol/mg protein; n = 6).



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  		Figure 4. Characterization of ANP receptor in the lung of control SHR-SP/Izm. a, Competitive inhibition curve of [125I]{alpha}-rANP binding by {alpha}-rANP (open circle) and c-ANF (closed circle). b, Scatchard transformation of ANP-specific binding. Results are expressed as the mean value of six different membrane preparations.

 
The disappearance curves of the exogenously administered {alpha}-hANP in the carvedilol and control groups are shown in Fig. 5Go. {alpha}-hANP disappeared from the circulation rapidly in the fast phase and then more slowly in the slow phase, as reported previously (30, 36). The half-life of {alpha}-hANP in the fast phase in the carvedilol group (0.8 ± 0.1 min; n = 5) was comparable with that in the control group (0.9 ± 0.1 min; n = 4). By contrast, the half-life in the slow phase was significantly (P < 0.05) longer in the carvedilol group (7.7 ± 0.8 min; n = 5) than that in the control group (4.9 ± 0.5 min; n = 4).



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  		Figure 5. The disappearance curves of {alpha}-hANP from rat circulating blood after an iv bolus injection (2.0 µg) in the carvedilol (closed circle; n = 5) and control (open circle; n = 4) groups. Values are the mean ± SEM.

 
In both groups, the biological activity of endogenous ANP was investigated in vivo using a NP receptor antagonist, HS-142–1 (Fig. 6Go). Systolic blood pressure increased significantly (P < 0.05) in both groups after 30 min of HS-142–1 administration, and the effects were eliminated 60 min after treatment. However, the extent of the increase in blood pressure was significantly (P < 0.05) greater in the carvedilol group (20 ± 3 mm Hg; n = 5) than in the control group (7 ± 2 mm Hg; n = 5).



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  		Figure 6. Effects of the NP-A/NP-B receptor antagonist HS-142–1 on systolic blood pressure in the control (n = 5) and carvedilol (n = 5) groups. Each circle indicates the change from the baseline systolic blood pressure in each rat after HS-142–1 administration. Values are the mean ± SEM. *, P < 0.05 vs. baseline value; {dagger}, P < 0.05 vs. the control group.

 
To explore whether the vascular response to ANP is altered by the decrease in the NP-C receptor gene expression of the aorta, we determined cGMP contents of the aorta in response to ANP in both the control and carvedilol groups. Basal cGMP and cAMP contents were also determined. The basal cGMP content in the aorta was significantly (P < 0.05) elevated in the carvedilol group (0.81 ± 0.1 pmol/mg protein; n = 5) compared with that in the control group (0.44 ± 0.04 pmol/mg protein; n = 5), whereas the basal cAMP content did not show any difference between the two groups [control, 4.6 ± 0.7 pmol/mg protein (n = 5); carvedilol, 5.0 ± 1.0 pmol/mg protein (n = 5)]. The ANP (10-8 M)-stimulated cGMP contents of the aorta were significantly (P < 0.05) higher in the carvedilol group (12.6 ± 2.4 pmol/mg protein) than in the control group (4.8 ± 0.6 pmol/mg protein). The incremental changes in cGMP contents of the aorta are documented in Fig. 7Go.



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  		Figure 7. Incremental changes in ANP (10-8 M)-stimulated cGMP contents of the aortic tissue in the control and carvedilol groups. Values are the mean ± SEM. The number of experiments is shown in parentheses. *, P < 0.05 vs. the control group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In agreement with previous reports (14, 15, 16, 17, 18), plasma ANP levels were significantly elevated in the carvedilol-treated SHR-SP/Izm rats. Plasma ANP levels are primarily determined by ANP synthesis and secretion from the heart (7). However, there was no significant change in the ANP mRNA levels in either the atrium or left ventricle of the carvedilol group, indicating that increases in plasma ANP levels are not attributable to changes in ANP synthesis. The present findings contrast with previous observations of other kinds of ß-blockers, in which ANP secretion was increased, possibly due to stimulation by reactive {alpha}1-adrenergic activation (37, 38) and/or by atrial distention due to the decrease in cardiac output (14, 17). The difference may be attributable at least in part to the weak {alpha}1-blocking potency (19) and to a lack of significant effect of carvedilol on cardiac output (19).

Another factor that affects plasma ANP levels is the clearance of the peptide from the circulation through the receptors. We previously reported that the NP-A receptor is up-regulated and NP-C receptor is down-regulated at the level of mRNA expression in the aorta of SHR-SP/Izm rats (25, 26). In the present study, NP-C receptor mRNA levels in the aorta decreased in the carvedilol group, whereas NP-A receptor mRNA levels in the aorta showed no significant change. In addition, we determined the NP-C receptor mRNA levels in the kidney and lung, where the NP-C receptor has been shown to be abundantly expressed (21, 39, 40). NP-C receptor mRNA levels in the lung also showed a significant decrease in the carvedilol group, although there was no significant change in the kidney. The binding density of the NP-C receptor in lung was significantly decreased in the carvedilol group compared with that in the control group. As the pulmonary circulation receives nearly the entire cardiac output and the lung has been shown to play a major role in the clearance of ANP (40, 41, 42), decreased NP-C receptors in the lung at both its mRNA and protein levels could cause a decrease in ANP clearance from the circulation and prolong the half-life of ANP.

In support of this hypothesis, we found that the half-life of plasma ANP, estimated by administering synthetic ANP in the carvedilol group, was about 2-fold greater than that in the control group. Taken together, these results suggest that the increase in plasma ANP levels in the carvedilol group is attributable not to the changes in ANP synthesis in the heart but to the decreased clearance of the peptide through the NP-C receptor and the prolongation of the half-life.

The changes in vascular NP receptor mRNA levels caused by carvedilol demonstrated in the present study are in a striking contrast to our previous findings with different types of antihypertensive agents in SHR-SP/Izm rats (25, 43, 44). The calcium channel blocker manidipine did not affect the vascular NP-A and NP-C receptor mRNA levels despite its potent antihypertensive effects (25, 43). The angiotensin type 1 receptor antagonist TCV-116 significantly increased the down-regulated NP-C receptor mRNA levels, whereas it did not show any significant effect on NP-A receptor mRNA levels (25, 44). Therefore, the decrease in vascular NP-C receptor mRNA levels is a phenomenon specific to carvedilol rather than a common phenomenon associated with blood pressure lowering by antihypertensive agents.

The biological significance of the increased plasma ANP levels in the blood pressure decrease caused by carvedilol was investigated by HS-142–1, a specific antagonist for both NP-A and NP-B receptors (20). Acute bolus administration of HS-142–1 significantly increased the systolic blood pressure in both control and carvedilol groups after 30 min of administration, and the effects were eliminated after 60 min. Although the duration of the effect to increase blood pressure by HS-142–1 was very limited, it could be attributed to its short acting nature in vivo as described previously (33, 45). The blood pressure increase caused by HS-142–1 was, however, much greater in the carvedilol group than in the control group. The results indicate that the activity of the endogenous NP system is potentiated by treatment with carvedilol. Various mechanisms have been suggested for the antihypertensive effect of ß-blocker: reduced cardiac output, action on the central nervous system, suppression of renin secretion, and production of vasodilator prostanoids (4). In addition, carvedilol has direct vasodilatory actions via {alpha}-blocking and calcium channel-blocking effects (46). All of these may contribute to the antihypertensive effect of carvedilol. In the present study, carvedilol decrease systolic blood pressure about 40 mm Hg compared with that in the control group. As the net increments in blood pressure caused by HS-142–1 between the two groups was about 13 mm Hg, the present results suggest that the potentiation of the NP system accounts for approximately one third of the antihypertensive effect of carvedilol, and that the mechanism shown in the present study may be one of the major antihypertensive mechanisms of carvedilol.

In addition, the biological significance of the down-regulated vascular NP-C receptor was investigated in an ex vivo experiment with an aortic strip. Both basal contents and ANP-stimulated responses of cGMP were exaggerated in the aorta of the carvedilol group. Interestingly, the cGMP response to ANP was reported to be exaggerated in bovine cultured endothelial cells stimulated by high NaCl (47) and in rat cultured vascular smooth muscle cells stimulated by Ang II (48). Under these conditions, the NP-C receptor was down-regulated, whereas the NP-A receptor did not change. Locally decreased ANP clearance is expected to increase the availability of ANP for the NP-A receptor, which may result in an enhancement of the cGMP response to ANP. Katafuchi et al. (47), however, demonstrated that masking the NP-C receptor with C-ANF, a biologically inactive specific ligand for the NP-C receptor, did not exaggerate the cGMP response to ANP in bovine cultured endothelial cells. Further studies are required to elucidate the mechanism for the increased sensitivity of the aorta to ANP.

The mechanism for the down-regulation of the NP-C receptor remains unknown. cAMP (29), cGMP (49, 50), and angiotensin II (25) have been reported to down-regulate the NP-C receptor in vascular cells. However, there was no significant difference in cAMP contents in the aorta or in PRA between the carvedilol and control groups, thus excluding their possible involvement in the down-regulation of NP-C receptor. By contrast, basal cGMP contents in the aorta and the response to ANP were greater in the carvedilol group than in the control group. Down-regulated NP-C receptors may potentiate ANP action and increase cGMP production, which, in turn, further down-regulates the receptors. In addition, besides the ß-blocking action, carvedilol has multiple unique actions, including those as antioxidant (for review, see Ref.51), {alpha}-blocking agent (46), and calcium channel blocker (46), which may be involved in the down-regulation of the NP-C receptor. We, however, previously reported that the calcium channel blocker did not affect vascular NP-C receptor mRNA levels in SHR-SP/Izm (25). Further studies are required to explore its actions as a {alpha}-blocking agent or an antioxidant of the NP-C receptor.

It has been well established that nitric oxide increases cGMP formation via activation of soluble guanylate cyclase. As several antioxidants were reported to activate nitric oxide action through its protection from superoxide anion (52, 53), and carvedilol has a potent antioxidant action (51), carvedilol may increase cGMP production through potentiation of nitric oxide action. Further studies are required, however, to explore this issue.

In conclusion, we have demonstrated that the ß-blocker carvedilol potentiates the hypotensive action of ANP by way of increasing plasma ANP levels and enhancing the vascular response to ANP. These effects are closely related to the specific down-regulation of the NP-C receptor. This new mechanism seems to account for a sizable part of the antihypertensive effects of carvedilol. Recombinant ANP has recently been established as one of the most powerful therapeutic agents for heart failure (54, 55). In addition, carvedilol has been shown to be effective in reducing long term morbidity and mortality rates of patients with chronic heart failure (6). Therefore, the ANP-potentiating action of carvedilol demonstrated in the present study is definitely of importance in the treatment of cardiovascular diseases, including systemic hypertension.


   Acknowledgments
 
We thank Daiichi Seiyaku Co. for supplying carvedilol.


   Footnotes
 
1 This work was supported in part by research grants from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists; research grants from the Japanese Ministry of Education, Science, and Culture, the Kidney Foundation (Tokyo, Japan) for Research of the Kidney and Nitric Oxide, and the Smoking Research Foundation (Tokyo, Japan); a grant-in-aid from the Tokyo Hypertension Conference; and a research grant from Tanabe Biomedical Conference (Tokyo, Japan). Back

Received June 10, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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