Natriuretic Peptide Receptors in Human Artery and Vein and Rabbit Vein Graft
Abstract Natriuretic peptides elicit their biological effects by elevation of cGMP through activation of two biologically active receptors: natriuretic peptide A receptor, which shows high affinity to atrial and brain natriuretic peptides, and natriuretic peptide B receptor, which is specific to C-type natriuretic peptide. To elucidate the implications of the natriuretic peptide system in arteries and veins, we examined the cGMP production in response to atrial and C-type natriuretic peptides and gene expressions of biologically active natriuretic peptide receptors in human gastroepiploic artery, internal mammary artery, and saphenous vein. Atrial natriuretic peptide augmented cGMP production more potently by one order of magnitude in arteries than in veins. C-type natriuretic peptide stimulated cGMP production weakly and equally in these vessels. Analyzed by reverse transcription–polymerase chain reaction, gene expression of natriuretic peptide A receptor was four times more abundant in arteries than in veins. Gene expression of natriuretic peptide B receptor was approximately the same between these vessels. We also studied the responsiveness to atrial and C-type natriuretic peptide in rabbit jugular vein grafted into carotid artery. In arterialized vein grafts 4 weeks after operation, the effects of atrial and C-type natriuretic peptides on cGMP production did not change from those in jugular veins. In conclusion, atrial natriuretic peptide stimulates cGMP production more potently in arteries than in veins due to the preferential expression of natriuretic peptide A receptor in arteries. These observations support the distinct roles of natriuretic peptides in cardiovascular homeostasis.
The natriuretic peptide family consists of at least three distinct peptides: ANP, BNP, and CNP. We and others have demonstrated that ANP and BNP are circulating hormones secreted mainly from the atrium and the ventricle, respectively.1 Although CNP was originally considered to serve as a neuropeptide,2 we recently reported that CNP is produced by vascular endothelial cells3 and that CNP is detected in human circulation.4 Thus, CNP can be considered to be a novel endothelium-derived relaxing peptide.
Natriuretic peptides elicit their pharmacological actions via activation of two subtypes of the biologically active receptor, NPR-A and NPR-B, which are the particulate guanylate cyclase itself, to elevate intracellular cGMP.5 We and others have demonstrated that ANP and, to a lesser extent, BNP potently activate NPR-A, whereas CNP selectively activates NPR-B.6 7 Several studies, including ours, demonstrated that CNP infused into healthy humans has weaker vasodepressor and natriuretic effects than ANP or BNP, suggesting a difference of expression of natriuretic peptide receptors in humans and distinct roles of these natriuretic peptides in cardiovascular homeostasis.8 9 10
In the present study, to elucidate the possible differential implications of the natriuretic peptide system in human artery and vein, we examined the cGMP production in response to ANP and CNP and gene expressions of biologically active natriuretic peptide receptors in human GEA, IMA, and SV. In addition, using a rabbit model of arterialized vein graft, we further studied the alteration of the responsiveness to ANP and CNP in the jugular vein grafted into the arterial circulation by the process of arterialization.
Preparation of Human Tissues
Segments of GEA, IMA, and SV were obtained from 13 patients with ischemic heart disease undergoing coronary artery bypass surgery (11 men, 47 to 70 years old; average, 61.1 years). Segments of GEA were also obtained from 2 patients with gastric cancer undergoing total gastrectomy (2 men, 66 and 70 years old, respectively). For cGMP production assay, the segments of the vessels were stored in ice-cold phosphate-buffered saline and used within 6 hours after sampling. For RNA preparation, the segments of vessels were immediately frozen by liquid nitrogen and stored at −70°C until RNA preparation. Informed consent was obtained from each patient. This study was approved by the ethical committee on human research of Kyoto University.
Sixteen male Japanese White rabbits weighing 2.5 to 3.0 kg were used. Rabbits were maintained under a 12-hour light schedule and were fed regular rabbit chow diet. All animals used in this study received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Surgical Technique of Rabbit Vein Graft Operation
The operative procedures were performed under aseptic conditions. Anesthesia was achieved by administration of 25 mg/kg pentobarbital sodium IV and 50 mg lidocaine hydrochloride SC. The right carotid artery and jugular vein were exposed through a vertical neck incision. After the animals were given 100 U/kg heparin sodium IV, a 15- to 20-mm segment of the artery was isolated with vascular forceps and removed. A 15- to 20-mm segment of the jugular vein was reversed and interposed in the carotid artery in an end-to-end fashion. Anastomoses were created with 10-0 nylon continuous suture at ×20 magnification. After 4 weeks of observation, the vein graft, left carotid artery, and left jugular vein were harvested from each rabbit, and cGMP production in response to ANP and CNP in these three vessels was examined. For histological examination, animals were killed by an overdose of pentobarbital sodium, and neck vessels were harvested after fixation in situ by perfusion of 10% phosphate-buffered formalin into the ascending aorta through a median sternotomy. Harvested arteries were subjected to usual histological examination, and vascular wall thickness, defined as the distance between external elastic lamina and endothelium, was measured in at least five sections from each vessel. Of 16 grafts, 14 were patent after 4 weeks of observation (patency rate, 0.875); the occluded grafts were excluded from the study.
α-Human ANP and human CNP were purchased from the Peptide Institute.
Determination of cGMP Production
The effects of ANP and CNP on cGMP production in each vessel were examined as we previously described.7 Each of the vessel segments was cleaned of surrounding tissue and was cut longitudinally on ice. Endothelial cells were denuded by gentle scraping with the blade of scalpel. Then each vessel segment was cut into fragments of approximately the same size and was subjected to cGMP production assay for either ANP or CNP and for vehicle, each in duplicate. Examined concentrations of the peptides were 10 nmol/L, 100 nmol/L, and 1 μmol/L in humans and 1 nmol/L, 10 nmol/L, 100 nmol/L, and 1 μmol/L in rabbits. Each fragment was preincubated for 30 minutes at 37°C in 250 μL Dulbecco’s modified Eagle’s medium containing 0.5% fetal calf serum and 0.5 mmol/L isobutylmethylxanthine. ANP or CNP was added to the medium and incubated for another 30 minutes at 37°C. After the incubation, trichloroacetic acid was added to a final concentration of 6% and the tissue fragments were mechanically homogenized in the medium by a Teflon homogenizer. After the trichloroacetic acid was removed by extraction three times with water-saturated ether, the amount of cGMP in the sample was determined by radioimmunoassay after succinylation as described elsewhere.11 The production of cGMP was expressed on a molar basis and was normalized by wet weight of the tissue fragments.
Characterization of Gene Expressions of Biologically Active Natriuretic Peptide Receptors
Gene expressions of NPR-A and NPR-B were characterized by the RT-PCR method. Total RNA was extracted by the guanidinium thiocyanate–CsCl method from GEA, IMA, and SV pooled from at least three patients. The specific primers for PCR were synthesized with an Applied Biosystems 381A DNA Synthesizer according to the nucleotide sequence of each receptor in humans (NPR-A: sense, 5′-GGGGATGTAGAAATGAAGGGC-3′ and antisense, 5′-TCATGGTAGAAGCAAGGCATACAGG-3′; NPR-B: sense, 5′-TGACCAGCTGAGGCTACGCA-3′ and antisense, 5′-CTACAACTTCCATATAAGGT-3′).5 12 cDNA was synthesized from 1 μg of total RNA by oligo (dT) priming with Molony murine leukemia virus reverse transcriptase (Life Technologies Inc) and was subjected to PCR using Taq DNA polymerase (Takara Shuzo). After 30 cycles of PCR, aliquots of the PCR products were size-fractionated by agarose gel electrophoresis, and then gels were subjected to ethidium bromide staining and Southern blot analysis. For Southern blot analysis, 32P-labeled synthetic oligomer was used as a probe. The nucleotide sequences of the probes were 5′-GAGCTTACAGGCTGAGCCAA-3′ for NPR-A and 5′-GGTGGTAGAGGAGACATGGAT-3′ for NPR-B. As an internal control, Southern blotting of RT-PCR product for G3PDH was also done by specific primers and oligoprobe (sense, 5′-TCAAGGCTGAGAACGGGAAGC-3′; antisense, 5′-CTTCACCACCTTCTTGATGTC-3′; oligoprobe, 5′-CTCATGACCACAGTCCAT-3′).13 Every blot was quantified by NIH Image 1.52 software after being scanned by an HP ScanJet 3c flatbed scanner (Hewlett-Packard) and was presented in arbitrary units such that 1 unit equaled the intensity of blot for G3PDH in each vessel.
To confirm the quantification by RT-PCR, we also performed PCR for NPR-A, NPR-B, and G3PDH using 0.25, 0.5, and 2 times the amount of cDNA and examined whether the intensity of blots increased according to the amount of cDNA.
All values were expressed as mean±SEM. When two mean values between different groups were compared, a two-sided unpaired Student’s t test was performed. When mean values between more than three groups were compared, factorial ANOVA followed by Fisher’s protected least significant difference was performed. Statistical significance was defined as a value of P<.05.
Effects of ANP and CNP on cGMP Production in Human GEA, IMA, and SV
Fig 1⇓ shows the effects of ANP and CNP on cGMP production in GEA, IMA, and SV segments. Basal cGMP production (vehicle) in GEA (n=3), IMA (n=6), and SV (n=7) was 22.6±6.1, 8.2±1.8, and 4.9±1.7 fmol/mg wet tissue, respectively (GEA versus IMA, P=.004; GEA versus SV, P=.0007; IMA versus SV, P=.32). In all three vessels, ANP and CNP exhibited significant augmentation of cGMP production. ANP stimulated cGMP production more potently in GEA and IMA than in SV. ANP at a concentration of 1 μmol/L augmented cGMP production by 23.3-fold in GEA and 27.4-fold in IMA but only 4.2-fold in SV. In contrast, the effect of CNP on cGMP production did not differ significantly between GEA, IMA, and SV at any concentration. CNP 1 μmol/L elevated cGMP production by 3.0-fold in GEA, 2.7-fold in IMA, and 3.2-fold in SV. Therefore, the effect of ANP was more potent by at least one order of magnitude in GEA and IMA than in SV. The effect of CNP on cGMP production was almost equipotent in GEA, IMA, and SV and was almost equivalent to the effect of ANP in SV.
Gene Expressions of Biologically Active Natriuretic Peptide Receptors in GEA, IMA, and SV
Fig 2A⇓ shows the results of Southern blot analysis on PCR-amplified products of the transcripts for two subtypes of the biologically active natriuretic peptide receptor in GEA (n=3, pooled), IMA (n=3, pooled), and SV (n=4, pooled). As shown in the figure, specific signal for NPR-A gene transcript in GEA or IMA was about four times more intense than that in SV. In contrast, the intensity of the specific signal for NPR-B gene transcript was approximately the same in GEA, IMA, and SV. Fig 2B⇓ shows the results of Southern blot analysis for NPR-A, NPR-B, and G3PDH gene transcripts using 0.25, 0.5, 1, and 2 times the amount of cDNA from GEA used in the analysis shown in Fig 2A⇓. The intensities of blots for PCR-amplified products of NPR-A, NPR-B, and G3PDH transcripts increased in an almost linear relation to the amounts of cDNA used (Fig 2B⇓).
Effects of ANP and CNP on cGMP Production in Rabbit Carotid Artery, Jugular Vein, and Arterialized Vein Graft
As shown in Fig 3⇓, in the arterialized vein grafts 4 weeks after operation, wall thickness measured in histological sections was 116±20 μm (n=5). This was significantly greater than wall thickness of intact jugular veins (21±1 μm, n=5, P<.0001) and comparable to that of intact carotid arteries (108±6 μm, n=5, P=.71).
The effects of ANP and CNP on cGMP production in rabbit vessels are shown in Fig 4⇓. Basal production of cGMP was 36.6±3.7 fmol/mg wet tissue in carotid arteries (n=15), 16.2±2.5 fmol/mg wet tissue in jugular veins (n=9), and 13.8±2.0 fmol/mg wet tissue in arterialized vein grafts (n=9) (carotid artery versus jugular vein, P=.0001; carotid artery versus vein graft, P<.0001; jugular vein versus vein graft, P=.64). ANP stimulated cGMP production more potently in carotid arteries than in jugular veins, but the difference of responsiveness to ANP between the artery and the vein was smaller than that in humans. ANP at a concentration of 1 μmol/L augmented cGMP production by 12.6-fold in carotid arteries but 6.9-fold in jugular veins. In arterialized vein grafts, ANP at a concentration of 1 μmol/L caused only a 3.6-fold increase of cGMP production. The effect of CNP on cGMP production did not differ significantly between carotid arteries, jugular veins, and arterialized vein grafts at any concentration. CNP 1 μmol/L elevated cGMP production by 6.0-fold in carotid arteries, 4.9-fold in jugular veins, and 5.0-fold in arterialized vein grafts.
The present study demonstrates that in humans, ANP augments cGMP production more potently in GEA and IMA than in SV, and CNP exerts weak stimulatory action on cGMP production similarly in GEA, IMA, and SV. In rabbit vessels, similar effects of ANP and CNP on cGMP production were observed; the stimulatory effect of ANP on cGMP production was more potent in carotid arteries than in jugular veins; and the effect of CNP was weak and almost the same in carotid arteries and in jugular veins. Most of the biological activities of natriuretic peptides are thought to be mediated by intracellular elevation of cGMP through the activation of the particulate guanylate cyclases (NPR-A and NPR-B).14 Therefore, our results suggest that ANP can elicit its vasodilating effect preferentially in arteries compared with veins. Actually, Hughes et al15 reported that ANP produced concentration-dependent relaxation in isolated human arteries, including pulmonary, brachial, uterine, and saphenous arteries, but not in saphenous veins. Wei et al16 reported that ANP but not CNP caused vasorelaxation in isolated canine renal arteries. Our results in human and rabbit arteries and veins were consistent with those results. However, Wei et al16 also reported that CNP but not ANP was a relaxing factor of isolated canine renal, saphenous, and femoral veins. We did not observe the difference in the responsiveness to CNP and ANP of human saphenous and rabbit jugular veins. The differences in the response to ANP and CNP in the veins between our results and theirs might be due to the difference in the vessels examined or the species.
Augmentation of cGMP production in response to ANP and CNP was considered to reflect the expression of biologically active natriuretic peptide receptors in the vessel. As we have reported previously, in cultured PC12 cells, which express only NPR-A, ANP and BNP at concentrations >10 nmol/L significantly increased cGMP production and 1 μmol/L of ANP caused a 20-fold increase of cGMP generation, whereas CNP exerted almost no effect even at a concentration of 1 μmol/L.7 In contrast, in cultured rat aortic smooth muscle cells with a synthetic phenotype, which express predominantly NPR-B, CNP at concentrations >100 nmol/L significantly increased cGMP production, whereas ANP and BNP slightly increased cGMP production at a concentration of 1 μmol/L.7 From these results, it is obvious that cGMP production in response to ANP reflects the amount of NPR-A and that cGMP production in response to CNP reflects the amount of NPR-B. Therefore, the differential effects of ANP and CNP on cGMP production in human and rabbit arteries and veins suggest that NPR-B is expressed almost equally in arteries and veins in low quantity, whereas NPR-A is expressed as much as NPR-B in veins but is expressed more abundantly by one or two orders of magnitude in arteries. Indeed, the results of Southern blot analysis demonstrated that NPR-B mRNA expression was about the same in human IMA, GEA, and SV, whereas the gene expression of NPR-A was more abundant in GEA and IMA than in SV.
Another important finding in the present study is that the effects of ANP and CNP on cGMP production in the rabbit jugular veins did not change when the vein was incorporated into the arterial circulation. Although the wall thicknesses of the arterialized vein grafts were comparable to those of carotid arteries, basal cGMP production of the arterialized vein grafts was significantly lower than that of carotid arteries and almost the same as that of jugular veins. From these results, it is suggested that there are some differences in characteristics between VSMCs in intact arteries and VSMCs in neointima occurring in veins. Otherwise, it is possible that 4 weeks of observation was too short for VSMCs to alter their phenotype from the “venous” to the “arterial” type.
In the present study, the concentrations of ANP and CNP necessary to augment cGMP production in isolated vessels exceeded the levels of ANP and CNP detected in human circulation under physiological and pathophysiological conditions (at most 300 pmol/L and 50 pmol/L, respectively), as we and others previously reported.1 4 17 But we have demonstrated that the elevated plasma cGMP level of stroke-prone spontaneously hypertensive rats with a plasma ANP level of 300 pmol/L was significantly reduced by the intravenous administration of our monoclonal antibody against ANP.18 Furthermore, in our recent observations, when the CNP gene was overexpressed by adenovirus-mediated gene transfer in cultured VSMCs that expressed predominantly NPR-B, the VSMCs exhibited enhanced cGMP production and a significantly lower growth rate (unpublished observations). In this system, the CNP concentration in the culture medium was about 100 pmol/L. Exogenously administered CNP at that concentration exerts no effects on cGMP production in cultured VSMCs.19 20 Therefore, a difference exists in the effective concentration of exogenously administered natriuretic peptides and endogenously produced natriuretic peptides. It is thus possible that GEA and IMA produce more cGMP than SV in vivo in response to circulating ANP and BNP at the enhanced level.
The stimulating effect of exogenous CNP on cGMP production was not as prominent as that of ANP and was almost equal in GEA, IMA, and SV. As we and others have demonstrated, CNP is produced by vascular endothelial cells and is suggested to be a local regulator of vascular tone and growth rather than a circulating hormone.3 21 We also reported that the endothelial production of CNP is markedly augmented by several growth factors and cytokines.3 22 Therefore, the possibility still remains that CNP at the enhanced level activates NPR-B in an autocrine and paracrine manner in atherosclerotic vessels.
In conclusion, the present study demonstrated that ANP stimulates cGMP production more potently in IMA and GEA than SV in humans and in the carotid artery more than the jugular vein and the arterialized vein graft in rabbits. This is due to the preferential expression of NPR-A in these arteries. Circulating ANP thus can elicit more prominent vasodilating and antiproliferative effects in arteries than in veins. CNP is considered to exert less effect than ANP on either arteries or veins. These observations support the distinct roles of natriuretic peptides in cardiovascular homeostasis: ANP as a circulating hormone and CNP as a local regulator of vascular tone and remodeling.
Selected Abbreviations and Acronyms
|ANP||=||atrial natriuretic peptide|
|BNP||=||brain natriuretic peptide|
|CNP||=||C-type natriuretic peptide|
|IMA||=||internal mammary artery|
|NPR-A||=||natriuretic peptide A receptor|
|NPR-B||=||natriuretic peptide B receptor|
|RT-PCR||=||reverse transcription–polymerase chain reaction|
|VSMC||=||vascular smooth muscle cell|
This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture. We thank Dr Ario Yamazato of Kyoto Takeda Hospital, Dr Jun-ichi Soneda of Takeda General Hospital, Dr Toshifumi Takeuchi of Mitsubishi Kyoto Hospital, and Dr Teiji Oda of Otowa Hospital for their assistance in tissue samplings.
↵1 The first two authors contributed equally to this study.
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