The role of prostaglandins in hypertension. I. The release of prostaglandins by aorta strips of renal, DOCA-salt, and spontaneously hypertensive rats

The release of prostaglandin-like (PG-like) material by aorta strips of normotensive and hypertensive rats has been studied in vitro. When incubated in an oxygenated Krebs solution kept at 37 degrees C, aorta strips removed from 8- and 12-week-old spontaneously hypertensive (SH) rats generate 1.2-2.5 times more PG-like material than aorta strips from age-matched normotensive Wistar (NW) rats. The overproduction of PG-like material by aorta strips of SH rats did not precede the development of hypertension in SH rats. Aorta strips derived from renal and DOCA-salt hypertensive rats produced 1.5-3 times more PG-like material than aorta strips from NW rats. The production of PG-like material by aorta strips of renal and DOCA-salt hypertensive rats was largely reduced when hypertension was interrupted in these animals, thus suggesting that the alteration taking place in the arteries of hypertensive rats (namely increased production of PGs) during the development of hypertension was reversible. The production of PG-like material by aorta strips of hypertensive rats was inhibited by indomethacin. Analysis of the PG-like material by bioassays and thin-layer chromatography suggests the presence of PGE2 and PGE1. The possible involvement of these PGs in the pathogenesis of hypertension in rats is discussed.     

In vitro production of prostaglandins by isolated aorta strips of normotensive and hypertensive rats.

When suspended in oxygenated Krebs solution at 37 degrees C, strips derived from thoracic aortae of spontaneously hypertensive rats maintain their initial intrinsic tone and release prostaglandin-like material in the suspending medium, while similar preparations from normal Wistar rats relax progressively and produce significantly smaller amounts of prostaglandins. Indomethacin, a potent antagonist of prostaglandin synthesis, has two major effects: it favors the relaxation of both strips of hypertensive rats and of normal rats; and it inhibits the accumulation of prostaglandin-like material in the suspending medium, as evaluated with a specific and sensitive biological assay (rat stomach strip or chick rectum). Carotid and femoral arteries taken from the same animals show similar differences as the aorta strips, with regard to the production of prostaglandin-like material. The generation of prostaglandin is markedly decreased by the absence of O2, while it is unaffected by the absence of the extracellular Ca2+. It is proposed that the absence of relaxation of aorta strips taken from hypertensive, compared to normal rats, is due to increased intramural synthesis and release of prostaglandins.     

Enhanced release of a ‘prostacyclin-like’ substance from aortic strips of spontaneously hypertensive rats

The release of an endogenous ‘prostacyclin-like’ substance from aortic strips of 8 male Wistar rats of the New Zealand genetically hypertensive strain (GH) was compared with that of 8 weight, age and sex matched normotensive Wistar control rats. The amount of ‘prostacyclin-like’ substance released by the aortic strips into tris buffer, under the influence of mechanical stimulation, was measured by its ability to inhibit human platelet aggregation as compared to the inhibitory effect of standard prostacyclin sodium salt. It was shown that generation of this substance increased with incubation time and that a significantly greater amount was produced by GH rats.     

Prostacyclin-induced contraction of isolated aortic strips from normal and spontaneously hypertensive rats (SHR)

Prostacyclin (PGl₂) (500-5,000 ng/ml) produced a concentration-dependent increase in contractile tension of isolated thoracic aortic strips (AS) from normotensive (WKY) and spontaneously hypertensive rats (SHR). No significant differences were noted between this response to PGl₂ in these two groups. Lower concentrations of PGl₂ (10 pg/ml — 100 ng/ml) caused neither contraction nor relaxation of agonist-contracted tissue. PGl₂ (500-5,000 ng/ml) did not relax KCl or methoxamine contracted AS. In concentrations above 100 ng/ml, PGl₂ caused a further increase in tension in KCl-depolarized preparations. The constrictor effect of PGl₂ on AS was attenuated by verapamil pretreatment or removal of extracellular Ca⁺⁺ from the physiological buffer. This inhibitory effect of Ca⁺⁺ deficiency on the PGl₂ response was significantly greater in AS from SHR compared to WKY tissue. The stable metabolite of PGl₂, 6-keto PGF_1a, caused a weak constrictor effect (40% of KCl reference contraction) over the concentration range 1,000–5,000 ng/ml. Contraction induced by PGl₂ was not prevented by pretreatment with antagonists of adrenergic, histamine, serotonin or cholinergic receptors. The contraction response of the rat AS to PGl₂ is similar to that reported for porcine coronary artery and rabbit aortic tissues in vitro.     

Effects of hydrogen-rich water and ascorbic acid treatment on spontaneously hypertensive rats

Hydrogen-rich water (HW) has been suggested to possess antioxidant properties of value in treatments of lifestyle diseases and for prevention of latent pathologies. To date, the potential benefits of HW against the deleterious effects of excessive salt intake and hypertension have not been investigated. Here, we first examined the effects of HW or HW supplemented with 0.1% ascorbic acid (HWA) on spontaneously hypertensive rats (SHR) that had been fed a normal diet. In comparison to control rats given distilled water (DW), we found that HW did not significantly influence systolic blood pressure (SBP) or diastolic blood pressure (DBP) in SHR; however, the increase in SBP and DBP were inhibited in the HWA group. Next, four groups of SHR were given DW, 0.1% ascorbic acid-added DW (DWA), HW, or HWA in combination with a 4% NaCl-added diet. SHR fed the 4% NaCl-added diet showed increased hypertension; HWA treatment resulted in a significant reduction in blood pressure. The HWA group tended to have lower plasma angiotensin II levels than the DW group. In addition, urinary volumes and urinary sodium levels were significantly lower in the HWA group than the DW group. Urinary isoprostane, an oxidative stress marker, was also significantly lower in the HWA group, suggesting that the inhibitory effect of HWA on blood pressure elevation was caused by a reduction in oxidative stress. These findings suggest a synergistic interaction between HW and ascorbic acid, and also suggest that HWA ingestion has potential for prevention of hypertension.     

Effects of superovulation, arachidonic acid or endometrium on release of prostaglandins and synthesis of proteins by bovine trophoblast

This study was conducted in vitro to examine factors that may regulate prostaglandin release by bovine trophoblast and endometrial slices. Trophoblastic tissues and endometrial slices were recovered from superovulating and normally-ovulating cattle on day 16 or 20 of pregnancy and incubated for 24 h. Release of PGF2 alpha and 13,14-dihydro-15-keto-PGF2 alpha (PGFM), and incorporation of [14C]-leucine into proteins were quantified and expressed per microgram DNA, which gives a measure of cellular activity. Activity of trophoblastic tissue for synthesizing protein was decreased (P less than .05) and for releasing PGFM was increased (P less than .05) on day 20 compared to day 16 of pregnancy. Neither superovulation nor day of pregnancy altered trophoblastic activity for releasing PGF2 alpha. Superovulation increased (P less than .05) endometrial release of PGF2 alpha. Endometrial release of PGF2 alpha was less (P less than .05) on day 20 than on day 16 of pregnancy. When arachidonic acid (0, 100, 200 or 400 micrograms) was added at the start of incubation, trophoblastic release of PGF2 alpha changed (P less than .05) quadratically with dose of arachidonic acid. When arachidonic acid was added 8 h after the start of incubation, trophoblastic release of PGF2 alpha increased linearly (P less than .01) with dose of arachidonic acid. Adding arachidonic acid to incubation medium did not affect trophoblastic or endometrial protein synthesis. Endometrial slices suppressed (P less than .05) trophoblastic protein synthesis and release of PGF2 alpha. Apparently, endometrium can modulate trophoblastic release of prostaglandins and synthesis of proteins in vitro, and trophoblastic tissue from superovulated cattle 16 or 20 days pregnant can be used to study trophoblastic synthesis of prostaglandins and proteins.     

Effects of superovulation, arachidonic acid or endometrium on release of prostaglandins and synthesis of proteins by bovine trophoblast

This study was conducted in vitro to examine factors that may regulate prostaglandin release by bovine trophoblast and endometrial slices. Trophoblastic tissues and endometrial slices were recovered from superovulating and normally-ovulating cattle on day 16 or 20 of pregnancy and incubated for 24 h. Release of PGF_2^α and 13,14-dihydro-15-keto-PGF_2^α (PGMF), and incorporation of [¹⁴C]-leucine into proteins were quantified and expressed per μg DNA, which gives a measure of cellular activity. Activity of trophoblastic tissue for synthesizing protein was decreased (P<.05) and for releasing PGMF was increased (P<.05) on day 20 compared to day 16 of pregnancy. Neither supercovulation nor day of pregnancy altered trophoblastic activity for releasing PGF_2^α. Supercovulation increased (P<.05) endometrial release of PGF_2^α. Endometrial release of PGF_2^α was less (P<.05) on day 20 than on day 16 of pregnancy. When arachidonic acid (0, 100, 200 or 400 μg) was added at the start of incubation, trophoblastic release of PGF_2^α changed (P<.05) quadratically with dose of arachidonic acid. When arachidonic acid was added 8 h after the start of incubation, triphoblastic release of PGF_2^α increased linearly (P<.01) with dose of arachidonic acid. Adding arachidonic acid to incubation medium did not affect trophoblastic or endometrial protein synthesis. Endometrial slices suppressed (P<.05) trophoblastic protein synthesis and release of PGF_2^α. Apparently, endometrium can modulate trophoblastic release of prostaglandins and synthesis of proteins in vitro, and trophoblastic tissue from supercovulated cattle 16 or 20 days pregnant can be used to study trophoblastic synthesis of prostaglandins and proteins.     

Effect of various prostaglandins on the release of arachidonic acid from cultured fibroblasts

Effect of various prostaglandins on the release of arachidonic acid from [¹⁴C]arachidonic acid labeled fibroblasts was studied. Prostaglandin(PG) F_2α was found to enhance the release of radioactive arachidonic acid from the cells. The stimulatory effect was dose dependent, and was greater than that of bradykinin. The active compounds can be ranked in potency for the release of arachidonic acid from the pre-labeled cells per cent of control: PGF_2α(200.1%)>PGF_1α (141.8%)>PGD₂ (137.1%)>thromboxane B₂ (113.7%)>PGE₂ (109.4%). On the other hand, PGI₂ showed a strong inhibitory effect on the arachidonic acid release from the pre-labeled cells (the value was only 69% of the control), while 6-ketoPGF_1α, an end metabolite of PGI₂, had no effect.     

Puberty and ovulatory release of gonadotropins in spontaneously hypertensive rats

The age at vaginal opening, estrous cyclicity, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) on the day of proestrus, and number of ova and ovarian weight as measured on the day of estrus in spontaneously hypertensive (SH) and genetically matched normotensive Wistar Kyoto (WKY) female rats were compared. In SH rats, there was a significant delay in the vaginal opening, but the regular 4-day estrous cycle followed afterwards. No significant changes were observed in the afternoon increase in serum LH, FSH and PRL on the day of proestrus in SH and WKY rats, although the basal levels of LH and PRL in the morning (11:00 h) were lower in SH rats than in WKY rats. The mean number of ova in SH rats was also less than in WKY rats, whereas the ovarian relative weight was similar in both species of rats. It can be said that SH rats undergo certain, but not critical, endocrine and/or neuroendocrine changes related to reproduction.     

Effects of arachidonic acid and prostaglandins on lung function in the intact dog

The effects of the prostaglandin (PG) precursor, arachidonic acid (AA), and the primary PG's, PGF2alpha, and PGD2, on lung function were compared in 39 intact-chest, paralyzed, artificially ventilated dogs. Intravenous AA decreased dynamic compliance (Cdyn) and functional residual capacity and increased airway resistance (Rl) and transpulmonary pressure at end-passive expiration. The decrease in Cdyn correlated closely with a rise in pulmonary arterial pressure (Ppa). Indomethacin abolished airway and vascular responses to AA, but did not attenuate responses to the PG's. The effects of AA, PGD2, and PGF2alpha on lung function and Ppa were similar, whereas PGE2 had little effect. Vagotomy attentuated the RL increase in response to AA, PGE2alpha, and PGD2 and the Cdyn decrease in response to the PG's. The effects of the PG's on compliance were greater than those produced by mechanically increasing pulmonary venous pressure. The present studies suggest that the PG precursor is rapidly converted to agents that have marked effect on both pulmonary vessels and airways, particularly peripheral airways, in the dog.     

Endothelial-dependent relaxant effects of vaso-active intestinal polypeptide and arachidonic acid in rat aortic strips

The relaxant actions of vaso-active intestinal polypeptide (VIP), acetylcholine (ACh), histamine and papaverine have been compared using circular muscle strips of rat aorta contracted with noradrenaline (NA). Arachidonic acid (AA) in a low dose (6.7 × 10 − 7M) also relaxed the aorta. The relaxant actions of all these substances except papaverine were abolished by removal of the endothelial cells. Higher doses of AA (6.7–13.4 × 10–6M) contracted aortic strips in the absence of NA but the con tractile effect “faded” while AA was still present in the bathing fluid. De-endothelialisation abolished this “fade” portion of the response leaving a sustained contracture. Indomethacin inhibited the contractile effect of AA revealing a weak inhibitory effect. However, it did not affect the relaxations induced by VIP, ACh, histamine or papaverine. ETYA abolished the relaxant actions of all these substances except papaverine. The results are consistent with the hypothesis that VIP, ACh and histamine relax the rat aorta via an endothelial-dependent mechanism which may involve the synthesis of a lipoxygenase product.     

Endothelial-dependent relaxant effects of vaso-active intestinal polypeptide and arachidonic acid in rat aortic strips

The relaxant actions of vaso-active intestinal polypeptide (VIP), acetylcholine (ACh), histamine and papaverine have been compared using circular muscle strips of rat aorta contracted with noradrenaline (NA). Arachidonic acid (AA) in a low dose (6.7 X 10(-7M) also relaxed the aorta. The relaxant actions of all these substances except papaverine were abolished by removal of the endothelial cells. Higher doses of AA (6.7-13.4 X 10(-6M) contracted aortic strips in the absence of NA but the con tractile effect "faded" while AA was still present in the bathing fluid. De-endothelialisation abolished this "fade" portion of the response leaving a sustained contracture. Indomethacin inhibited the contractile effect of AA revealing a weak inhibitory effect. However, it did not affect the relaxations induced by VIP, ACh, histamine or papaverine. ETYA abolished the relaxant actions of all these substances except papaverine. The results are consistent with the hypothesis that VIP, ACh and histamine relax the rat aorta via an endothelial-dependent mechanism which may involve the synthesis of a lipoxygenase product.     

Effects of prostaglandins and arachidonic acid on baboon cerebral and mesenteric arteries

Effects of prostaglandins (PGs) E1, E2, F2 alpha and I2 in a wide range of concentration were examined in mesenteric and cerebral arteries isolated from mature baboons. PGs E1, E2 and F2 alpha at low concentrations (10(-10) to 10(-7) M) elicited relaxation in helically cut strips of cerebral arteries precontracted with phenylephrine. In contrast, the PGs did not cause relaxation in the mesenteric artery. PGI2 (10(-9) to 10(-6) M) produced marked relaxation in both arteries. The EC25 for PGI2 in the mesenteric artery was significantly lower than that in the cerebral artery. During baseline conditions, cerebral arteries contracted in response to high concentrations (greater than 10(-7) M) of PGs E1, E2 and F2 alpha. In mesenteric arteries, a large contraction was induced by PGs F2 alpha and E2 but not by PGE1. Arachidonic acid (10(-6) M) produced an aspirin-inhibitable relaxation in both arteries to a similar extent, so that the vasodilator PG(s) formed in the two different arterial walls appear to exert a similar relaxant action. Thus, the baboon mesenteric artery was more sensitive to PGI2 for the relaxant effect than was the cerebral artery, while PGs F2 alpha, E1 and E2 caused only a contraction in the mesenteric artery but both relaxation and contraction in the cerebral artery.     

Effects of prostaglandins and arachidonic acid on baboon cerebral and mesenteric arteries

Effects of prostaglandins (PGs) E₁, E₂, F_2α and I₂ in a wide range of concentration were examined in mesenteric and cerebral arteries isolated from mature baboons. PGs E₁, E₂ and F_2α at low concentrations (10⁻¹⁰ to 10⁻⁷ M) elicited relaxation in helically cut strips of cerebral arteries precontracted with phenylephrine. In contrast, the PGs did not cause relaxation in the mesentric artery. PGI₂ (10⁻⁹ to 10⁻⁶ M) produced marked relaxation in both arteries. The EC₂₅ for PGI₂ in the mesenteric artery was significantly lower than that in the cerebral artery. During baseline conditions, cerebral arteries contracted in response to high concentrations (greater than 10⁻⁷ M) of PGs E₁, E₂ and F_2α. In mesentric arteries, a large contraction was induced by PGs F_2α and E₂ but not by PGE₁. Arachidonic acid (10⁻⁶ M) produced an aspirin-inhibitable relaxation in both arteries to a similar extent, so that the vasodilator PG(s) formed in the two different arterial walls appear to exert a similar relaxant action. Thus, the baboon mesenteric artery was more sensitive to PGI₂ for the relaxant effect than was the cerebral artery, while PGs F_2α, E₁ and E₂ caused only a contraction in the mesenteric artery but both relaxation and contraction in the cerebral artery.     

Changes of beta-adrenoceptors in the aortic muscle cells of spontaneously hypertensive rats

The characteristics of [125I]monoiodocyanopindolol (ICYP) binding to beta-adrenoceptors of cultured aortic smooth muscle cells derived from 4-week-old spontaneously hypertensive rats (SHR) and the Wistar-Kyoto normotensive rats (WKY) were examined. During optimization of the binding assays, we found that the specific binding of ICYP by intact cells was masked by a high level of nonspecific ICYP accumulation in intact cells presumably owing to the lipophilic nature of ICYP. Optimal specific ICYP binding requires that the cells be gently lysed with hypotonic dilution followed by a freeze-and-thaw cycle. Under most experimental conditions tested, the total number of ICYP binding sites in WKY aortic muscle cells was considerably and consistently smaller than that in SHR cells. There was no difference in the Kd values for ICYP binding to SHR and WKY cells. However, when ICYP binding was carried out using crude membrane fractions with well-defined plasma membrane content isolated from aortic muscle strips of adult rats, we found no difference in the number of beta-adrenoceptor sites between SHR and WKY. Morphological evidence indicated that cultured SHR aortic muscle cells contained a greater proportion of larger cells with multinuclear features. These results suggest that an increase in the number of beta-adrenoceptor density per cell in SHR may be associated with cellular hypertrophy of aortic smooth muscle cells. We conclude that under cultured conditions, a higher incidence of polyploid smooth muscle cells in the SHR as compared with WKY was expressed earlier than under in vivo conditions. Therefore, the interpretation of results obtained from cultured cell studies in relation to under in vivo conditions should be exercised with caution.     

Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus

The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.