Objective: The aim of this study was to evaluate the possible role of prostacyclin (PGI2) in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). Methods: Measurement of mRNA and protein levels of PGH synthase (PGHS)-1, PGI2 synthase and the PGI2 receptor, in the thoracic aorta was performed in SHR aged 5, 10, 20, and 40 weeks old and in age-matched normotensive Wistar–Kyoto (WKY) rats with a competitive polymerase chain reaction method and immunoblotting. Aortic production of 6-keto-PGF1α, the main metabolite of PGI2, was also measured. Results: Compared with age-matched WKY rats, PGHS-1 mRNA and protein levels in the thoracic aorta of SHR increased with age, reaching three- and twofold higher than WKY rats at 40 weeks old, respectively. PGI2 synthase mRNA and protein levels in SHR were significantly higher than in WKY rats at 20 and 40 weeks old. In contrast, PGI2 receptor mRNA levels in SHR were consistently lower than in WKY rats at all ages. Conclusions: These results provide evidence that hypertension elicits alterations in levels of arachidonic acid metabolites, including PGH2 and PGI2. They also suggest that the decreased expression of PGI2 receptor mRNA in prehypertensive SHR could be one of the causes of hypertension in SHR.
Time for primary review 25 days.
Arachidonic acid metabolites, including prostaglandins (PG) produced in vessel walls, are known to serve important roles in vessel function such as regulation of vessel tone [1–3], platelet adhesion and lipid metabolism [5–8]. Strain differences in PG production in vessel wall derived from spontaneously hypertensive rats (SHR) and Wistar–Kyoto rats (WKY) have been reported [6, 7, 9–12], and also altered PG synthesis has been linked with aging [6, 7, 9–14]. We reported previously that PGH2 may be an endothelium-derived contracting factor by comparing acetylcholine-induced contraction in SHR and WKY aorta [14–16]. From previous observations, activated synthesis of PGs, including PGH2 and prostacyclin (PGI2), might be involved in the pathogenesis of hypertension in SHR.
PGI2, the main metabolite of arachidonic acid, has potential effects such as vasodilation, antiplatelet-aggregation, antimitogenicity for vessel smooth muscle cells and modulation of cholesterol turnover [5, 12, 17–19]. Thus, PGI2 is important for the regulation of vessel function. However, no studies have been conducted to elucidate the molecular biological mechanisms of PGI2 synthesis, i.e., mRNA and protein expression, in physiological or pathophysiological conditions. Recently, in addition to the nucleotide sequence of PGH synthase-1 (PGHS-1) (known as the rate-limiting step of PGI2 synthesis ) those of rat PGI2 synthase (unpublished data, registered at the National Center for Biotechnology Information in 1996, accession number U53855) and PGI2 receptor have been reported, and we evaluated levels of expression of their mRNAs and proteins by a competitive polymerase chain reaction (PCR) method and immunoblotting, respectively.
In the present study to identify the possible role of PGI2 in the onset and development of hypertension, we measured temporal regulation of PGHS-1, PGI2 synthase and PGI2 receptor gene and protein expressions in the thoracic aorta of SHR.
2.1 Animal model
This investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985). Rats were maintained under a 12 h light/12 h dark cycle at 23°C and were fed standard laboratory chow and water ad libitum. Measurements were performed in male SHR (the Okamoto strain) at the age of 5, 10, 20, 40 weeks and in age-matched WKY (Chubu Kagaku Shizai, Nagoya, Japan) (n=8 for each age). Systolic blood pressure was measured by the tail-cuff method (BP98A; Softron, Tokyo, Japan). The rats were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally). After perfusion with saline containing 1000 U/l heparin to avoid platelet aggregation, the thoracic aorta was quickly excised and cleaned of adherent connective tissue. Part of each aorta was immediately used for measurement of 6-keto-PGF1α, while the remainder was frozen in liquid nitrogen for RNA and protein extraction and stored at −80°C until extraction.
2.2 Production of 6-keto-PGF1α in the aortic wall
PGI2 production in the aortic wall was evaluated by measuring 6-keto-PGF1α, the main metabolite of PGI2, with a sensitive chemiluminescence enzyme immunoassay kit (Assay Designs, Ann Arbor, MI, USA). The aorta was cut into pieces about 10 mm square (100 mg), rinsed in cold saline and incubated in 1 ml of Tris–HCl buffer (pH 7.5, 37°C) containing 0.05 mmol/l arachidonic acid . After 45 min incubation, the concentration of 6-keto-PGF1α in the solution was measured.
2.3 RNA preparation
Total cellular RNA was extracted using a total RNA extraction kit (Isogen, Nippon Gene, Tokyo, Japan). The frozen thoracic aortas were ground individually into fine powder in a stainless-steel pod and suspended in lysis buffer. After 15 min, the buffer was sonicated for 30 s (Biomic model 7250B, Seiko, Tokyo, Japan). After centrifugation for 3 min at 12 000 g, the supernatant was used. Extraction was performed according to the manufacturer's protocol. The concentration and purity were measured three times for each sample in a spectrophotometer (LKB Ultra Spec, Amco, Tokyo, Japan) with absorbance at 260 nm to 280 nm. Only samples in which the ratio of absorbance at 260 nm to 280 nm was >1.7 were used.
2.4 Reverse transcription and competitive PCR
The relative expression of each mRNA in SHR and WKY was evaluated by a reverse transcription PCR method with appropriate internal controls [24–26]. We constructed a heterologous mutant of endogenous cDNA by PCR and transcribed by SP6 RNA polymerase to make mimic RNA competitors (cRNA) for PGHS-1, PGI2 synthase, and PGI2 receptor with a commercial kit (competitive RNA construction kit, Takara, Tokyo, Japan), as illustrated in Fig. 1. Briefly, as an example of PGI2 synthase, primers for mimic RNA was designed as follows; Forward: 5′--TGGTGTGGGATCTGCGTACAGTACGGTCATCATCTGACAC-3′ and 5′-CATTATCGCTACGCATTACTCCTCCACTCCATACA-3′. (The boxed sequence corresponds to SP6 RNA polymerase, underlined sequences correspond to primer for PGI2 synthase shown in Table 1, and simple sequences correspond to primers for a DNA template. Sequences of the marked portion are similar in PGHS-1 and PGI2 receptor). At first, DNA competitors were constructed with a DNA template of known sequence (λDNA: nucleotides 29 760–30 000). To make RNA competitors, these DNA competitors were used as a template for transcription by the SP6 RNA polymerase according to the transcription protocol. The size of endogenous DNA and mimic DNA are as follows: PGHS-1, 151 basepairs (bp) and 130 bp; PGI2 synthase, 297 bp and 270 bp; and PGI2 receptor, 222 bp and 200 bp, respectively. Total RNA (5 μg) from each aorta and cRNA were reverse transcribed using a first-strand cDNA synthesis kit (Ready-to-Go, You-Prime First-Strand beads, Pharmacia, La Jolla, CA, USA), in which Moloney Murine Leukemia Virus reverse transcriptase catalyzes first-strand cDNA synthesis. After quantification, serial dilution was used as an internal standard for competitive PCR. RNA aliquots (2 μl) were mixed with 2 μl of cRNA at different concentrations and reverse transcribed with 0.5 μg of oligo(dT)12–18 primer (Invitrogen, San Diego, CA, USA). Aliquots of cDNA and mimic DNA were amplified in a DNA thermal cycler (TP3000, Takara) with 0.25 μmol/l each of forward and reverse primers, and 0.5 U of Taq DNA polymerase (Takara) in 50 μl of 10 mmol/l Tris–HCl (pH 8.3), 50 mmol/l KCl, 2 mmol/l MgCl2, and 0.2 mmol/l of each deoxy-NTP. PCR was performed for 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min with a final 10 min extension period. Specific primers for PGHS-1, PGI2 synthase and PGI2 receptor were designed according to previous reports [20, 22]as shown in Table 1. The reaction products (10 μl) were subjected to electrophoresis on a 3% agarose gel, which was stained with ethidium bromide and photographed on a UV transilluminator. Signal intensity was quantified by densitometric analysis (Imagemaster, Pharmacia, Tokyo, Japan). The intensity ratios between cDNA and internal standard were plotted as a function of known amounts of the competitive template. The intra- and interassay variabilities were 10.7% and 12.7%, respectively. The amount of each mRNA was expressed as attomole/ng RNA for PGHS-1 and PGI2 receptor and attomole/pg RNA for PGI2 synthase.
The numbers on the right correspond to the nucleotide sequences in each cDNA.
The expression levels of PGHS-1 and PGI2 synthase protein were evaluated by immunoblotting as previously described . Briefly, electrophoresis was performed by using 10% sodium dodecyl sulfate-polyacrylamide gel. The proteins obtained from rat aortas were transferred to polyvinyl difluoride membrane (Immobilon-P, Millipore, Bedford, MA, USA). Membranes were blocked in skim milk, washed three times with TRIS-buffered saline with 0.2% Tween-20 (TBST), and incubated with specific monoclonal antibodies (diluted 1:500 in TBST) for 1 h at room temperature. Membranes were washed three times with TBST, incubated with antirabbit immunoglobulin antibody (1:5000), washed again three times with TBST, and developed with enhanced chemiluminescence reagent (ECL Western Blotting Analysis System, Amersham, Tokyo, Japan). Signal intensity of each protein was quantified and corrected by that of α-actin. The relative intensity was evaluated for protein expression. The specific monoclonal antibodies for PGHS-1, PGI2 synthase and α-actin were purchased from Cayman Chemical (La Jolla, CA, USA).
2.6 Statistical analysis
Data are expressed as means±SEM. For statistical analyses, repeated measure analysis of variance (ANOVA) were performed to compare systolic blood pressure between two strains. Statistical significance was determined by one-way or two-way ANOVA combined with a Student's t-test with Bonferroni correction. A level of p<0.05 was accepted as statistically significant.
3.1 Systolic blood pressure
The changes in systolic blood pressure of SHR and WKY in each age group are shown in Table 2. In both strains, systolic blood pressure increased with age. In SHR, systolic blood pressure was significantly higher than in WKY except at 5 weeks old.
|Body weight (g)|
|Systolic blood pressure (mmHg)|
|Production of 6-keto-PGF1α (ng/100 mg tissue)|
|Body weight (g)|
|Systolic blood pressure (mmHg)|
|Production of 6-keto-PGF1α (ng/100 mg tissue)|
Data are means±SEM, ap<0.05, bp<0.01 vs. age-matched controls (n=8 in each group).
3.2 Production of 6-keto-PGF1α in the aorta
Temporal changes of 6-keto-PGF1α levels in aorta are shown in Table 2. In both strains, the production of 6-keto-PGF1α increased with age. In SHR, the production increased by 12-fold at the age of 40 weeks, whereas in age-matched WKY the increase was sevenfold (p<0.01).
3.3 Alteration of PGHS-1, PGI2 synthase and PGI2 receptor mRNA expression
The changes in expression of each mRNA in the thoracic aorta of SHR and WKY are shown in Fig. 2. In WKY, no significant changes were observed in PGHS-1 mRNA levels. In SHR, however, level of this mRNA increased after 20 weeks, and a threefold increase was seen at the age of 40 weeks compared with 5 weeks (p<0.01). The expression of PGI2 synthase in both strains was low until 10 weeks, and increased markedly after 20 weeks in both strains. At 40 weeks, PGI2 synthase gene expression increased in SHR and WKY by about 18- and 12-fold compared with the value at 5 weeks, respectively (p<0.01). In SHR, the expression was significantly higher than in WKY at 40 weeks (p<0.05). In contrast, the level of PGI2 receptor gene expression decreased gradually with age in both strains. In SHR, the expression was suppressed at all ages compared with WKY (p<0.05).
3.4 Analysis of PGHS-1 and PGI2 synthase protein expression
The changes in expression of PGHS-1 and PGI2 synthase protein in the thoracic aorta of SHR and WKY are shown in Fig. 3. In WKY, no significant changes were observed in PGHS-1 synthase protein levels. In SHR, however, level of this protein increased after 20 weeks, and a twofold increase was seen at the age of 40 weeks compared with 5 weeks (p<0.05). PGI2 synthase protein level increased with age in both strains. Marked increase of this protein was observed in SHR after 20 weeks and the level of expression was twofold higher than in WKY at 20 weeks. However, no difference was observed at 40 weeks between the two strains.
4.1 Animal model and alterations of blood pressure
SHR of the Okamoto strain is the most widely used animal model of human essential hypertension [28, 29]. Considerable effort has focused on several factors for blood pressure regulation including the renin–angiotensin system [30–32], natriuresis , endothelin synthesis , nitric oxide synthesis [35, 36], lipid metabolism [6–8]as well as PGs. However, the etiology of hypertension remains obscure. The synthesis of PGs, including PGH2 and PGI2, is activated with age and this activation might serve important roles in the development of hypertension. In the present study to identify the possible role of PGI2 in the pathogenesis of hypertension, we selected rats at 5, 10, 20 and 40 weeks old to represent prehypertensive, developmental, established and chronic stages of hypertension, respectively, as previously described in SHR . In both normotensive WKY and SHR, the systolic blood pressure increased with age. In SHR, systolic blood pressure increased after 10 weeks and remained higher than in WKY. These results are consistent with those of previous studies [14, 30, 37], and revealed that the ages which we observed in the present study were good representatives of each stage of hypertension.
4.2 PGHS-1 gene and protein expression
In SHR, the levels of PGHS-1 mRNA and protein increased with age and resulted in a threefold and twofold increase at 40 weeks compared with 5 weeks, respectively, whereas no significant change was observed in WKY. In PGHS-1, gene and protein expression increased parallel to aging in SHR. In contrast, aging did not affect the expression in WKY. Similarly, Ge et al. reported that PGHS-1 expression, which was evaluated by both reverse transcriptase PCR and Western blotting, was twofold higher in aorta derived from SHR than WKY at 35 weeks old . Excessive expression of PGHS-1 supports the hypothesis that hypertension elicits an alteration in arachidonic acid metabolites levels, resulting in enhanced synthesis of PGH2 in the arterial endothelium. Furthermore, these and previous findings [14–16]suggested that PGH2 is a possible candidate for endothelium-derived contracting factor in acetylcholine-induced contraction in SHR.
4.3 PGI2 synthase gene and protein expression
The expression of PGI2 synthase mRNA increased after 20 weeks in both strains. In SHR, there was an 18-fold increase at 40 weeks old compared with 5 weeks old and the level was significantly higher than that in WKY at 40 weeks old (p<0.05). Furthermore, the enhanced expression of PGI2 synthase protein were confirmed by immunoblotting. The protein levels increased after 20 weeks in both strains and the level in SHR reached twofold higher than that in WKY at the age of 20 weeks (p<0.01). However, the protein level was similar between the two strains at 40 weeks.
Systolic blood pressure gradually became elevated with age, whereas the level of PGI2 synthase expression at the developmental stage remained almost equivalent to that at the prehypertensive stage. This suggests that PGI2 synthase expression is not activated initially at the development of hypertension to regulate vascular tone, but is gradually activated after the establishment of hypertension. Time course of changes in PGI2 synthase gene expression was similar to that of 6-keto-PGF1α production in the aortic wall. These results are consistent with previous observations of increases in the production of PGI2 and capacity of its release with age [23, 39]. In SHR, PGI2 synthase protein expression reached a plateau after 20 weeks and the increase did not become parallel to 6-keto-PGF1α production. We speculate that a part of the increase in PGI2 production may be compensated by activation of PGI2 synthase at the chronic hypertensive stage. This result suggests that the posttranslational regulation underlies PGI2 production in the aorta of SHR. In this respect, further examination will be required to elucidate the role of PGI2 in SHR, especially at more chronic hypertensive stages than 40 weeks of age.
4.4 PGI2 receptor gene expression
PGI2 receptor gene expression decreased with age in both strains. Notably, in SHR, the expression was suppressed at the prehypertensive stage compared with WKY and was consistently lower than in WKY at all ages. Since previous studies have shown no differences in PGI2 receptor number between 6-week-old SHR and age-matched WKY , the decrease in PGI2 receptor induced by suppressed gene expression with age may cause overproduction of PGI2. It has been reported that PGI2-induced relaxation in the vasculature of SHR and WKY decreased with age, and the relaxation was greater in WKY than in SHR [23, 38, 41]. Our findings may provide an explanation for this phenomenon that overproduction of PGI2 and decreased number of PGI2 receptor caused a diminished response of aorta to PGI2 with age. In addition, increased synthesis of PGI2 may also exacerbate hypertension by paradoxically constricting vessels as suggested by Rapoport and Williams . They proposed that overproduction of PGI2 may elicit the thromboxane A2/PGH2 receptor-independent and -dependent components of acetylcholine-induced contraction in the rat aorta.
Recently, Yukawa et al. found two subtypes of thromboxane A2 receptor (endothelial type and placental type), of which signal transduction pathways are different, and observed characteristic differences in cultured cells overexpressing each type of receptor . In the vessel wall, the endothelial type receptor is dominant and activates PGI2 production by prolonged stimulation of thromboxane A2 produced in endothelium and platelets. In fact, higher concentrations of thromboxane A2 are observed in serum or urine in several hypertensive diseases [44–46]. Thus, PGI2 production may be stimulated to a greater extent via the thromboxane A2 and PGH2 pathway, and this hypothesis is consistent with our findings.
We observed an increase in PGHS-1 mRNA and protein expression with age and marked elevation of PGI2 synthase mRNA and protein expression after establishment of hypertension in SHR. These results provide evidence that hypertension elicits an alteration in arachidonic acid metabolites levels including PGH2 and PGI2. In contrast, PGI2 receptor mRNA expression was suppressed before the development of hypertension compared with WKY. Alterations in expression of genes related to PGI2 might be partly involved in the pathogenesis of hypertension in SHR.
We thank Dr. Keiji Naruse for helpful discussion and excellent technical assistance.