Identification of the 11,14,15- and 11,12, 15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta.

A number of endothelium-derived relaxing factors have been identified including nitric oxide, prostacyclin, and the epoxyeicosatrienoic acids. Previous work showed that in rabbit aortic endothelial cells, arachidonic acid was metabolized by a lipoxygenase to vasodilatory eicosanoids. The identity was determined by the present study. Aortic homogenates were incubated in the presence of [U-14C]arachidonic acid, [U-14C]arachidonic acid plus 15-lipoxygenase (soybean lipoxidase), or [U-14C]15-hydroxyeicosatetraenoic acid (15-HPETE) and analyzed by reverse phase high pressure liquid chromatography (RP-HPLC). Under both experimental conditions, there was a radioactive metabolite that migrated at 17.5-18.5 min on RP-HPLC. When the metabolite was isolated from aortic homogenates, it relaxed precontracted aortas in a concentration-dependent manner. Gas chromatography/mass spectrometry (GC/MS) of the derivatized metabolite indicated the presence of two products; 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 11,14,15-THETA. A variety of chemical modifications of the metabolite supported these structures and confirmed the presence of a carboxyl group, double bonds, and hydroxyl groups. With the combination of 15-lipoxygenase, arachidonic acid, and aortic homogenate, an additional major radioactive peak was observed. This fraction was analyzed by GC/MS. The mass spectrum was consistent with this peak, containing both the 11-hydroxy-14, 15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-H-11,12-EETA. The hydroxyepoxyeicosatrienoic acid (HEETA) fraction also relaxed precontracted rabbit aorta. Microsomes derived from rabbit aortas also synthesized 11,12,15- and 11,14,15-THETAs from 15-HPETE, and pretreatment with the cyctochrome P450 inhibitor, miconazole, blocked the formation of these products. The present studies suggest that arachidonic acid is metabolized by 15-lipoxygenase to 15-HPETE, which undergoes an enzymatic rearrangement to 11-H-14,15-EETA and 15-H-11,12-EETA. Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-THETA, which relaxed rabbit aorta. Thus, the 15-series THETAs join prostacyclin, nitric oxide, and epoxyeicosatrienoic acids as new members of the family of endothelium-derived relaxing factors.

Methacholine-induced contraction of rabbit pulmonary artery: role of platelet-endothelial transcellular thromboxane synthesis.

Arachidonic acid- and methacholine-induced contractions of rabbit pulmonary arteries are mediated by thromboxane (TX) A2. Although removal of the endothelium abolishes the contractions, endothelial cells isolated from pulmonary arteries do not synthesize TXA2. Further studies described here showed that the expression of TX synthase was evident in platelets and intact pulmonary artery but not in endothelial cells. These studies examined the role of platelet TXA2 production in the vasoconstrictor response to methacholine. Endothelial cells were incubated with platelets in the presence or absence of methacholine. Methacholine caused an increase in TXB2 production. Pretreatment of endothelial cells with aspirin (100 micromol/L) before the addition of platelets did not impair the ability of methacholine to increase TXB2 synthesis. Conversely, if platelets were pretreated with aspirin, methacholine failed to stimulate TXB2. Using endothelial cells with their cellular lipids labeled with [3H]arachidonic acid, methacholine did not stimulate the production of [3H]TXB2. When the endothelial cells were incubated with methacholine and control platelets, [3H]TXB2 was detected. If aspirin-treated platelets were incubated with endothelial cells, methacholine did not increase the production of [3H]TXB2. However, pretreatment of the endothelial cells with aspirin did not affect the ability of methacholine to induce [3H]TXB2 release. This suggests that methacholine stimulated the endothelial cell to release arachidonic acid, which was transferred to the platelets and metabolized to TXA2. To test whether this cell-cell interaction is necessary for methacholine-induced contractions, rabbits were administered aspirin (20 mg/kg) for 2 days. On day 4, methacholine-induced contractions of pulmonary arteries were depressed in aspirin-treated compared with control subjects. Control arteries synthesized 6-keto-prostaglandin F1alpha and TXB2. Aspirin treatment inhibited both pulmonary artery and platelet TXB2 production but had no effect on vessel 6-keto-prostaglandin F1alpha. These studies implicate platelets as a vascular source of TXA2 and indicate that both endothelial cells and platelets may be required for methacholine-induced TXA2 synthesis and vasoconstriction.

Vascular smooth muscle thromboxane A2 receptors mediate arachidonic acid-induced sudden death in rabbits.

We recently identified a subgroup of rabbits (called nonresponders) that were deficient in vascular thromboxane A2 receptors. Thromboxane A2-mediated platelet aggregation was not different between responders and nonresponders. In the present study, we utilized these nonresponders as a model to study the relative contribution of the platelet and vascular thromboxane A2 receptors to the observed hemodynamic responses associated with arachidonic acid-induced sudden death. Mean arterial pressure was slightly but not significantly lower in the nonresponders compared with the responders. However, nonresponders were protected from arachidonic acid-induced sudden death. While 100% of the responders died at the 2.0 mg dose of arachidonic acid, only 27% of nonresponders died at this same dose. Administration of the thromboxane A2 mimetic U46619 (5 micrograms/kg IV) decreased blood pressure by 41 +/- 6 mm Hg in responders but had no effect in the nonresponders. The affinity and density of thromboxane A2 receptors in cultured aortic vascular smooth muscle cells obtained from both responders and nonresponders were assessed using radioligand binding. The Kd values were not different (4.4 +/- 1.0 versus 2.4 +/- 0.6 nmol/L, responder versus nonresponder). However, there was a significant decrease in the density of receptors from vascular smooth muscle cells of nonresponders (Bmax = 397 +/- 59 versus 157 +/- 59 fmol/10(6) cells, responder versus nonresponder, P < .01). U46619 produced a concentration-dependent increase in [3H]-thymidine incorporation into responder vascular smooth muscle cells but had no effect in the nonresponder cells. Using an anti-thromboxane A2 receptor antibody, we compared the amount of receptor expressed in aortic tissue obtained from responders and nonresponders. Consistent with the results observed with [3H]-thymidine uptake and radioligand binding assays, the expression of thromboxane A2 receptor protein was decreased in nonresponder compared with responder vascular tissue. Platelet thromboxane A2 receptor expression was not different. These studies demonstrate that the vascular smooth muscle cells of nonresponder rabbits are deficient in the thromboxane A2 receptor. Furthermore, the reduction in arachidonic acid-induced sudden death in nonresponders indicates that the vascular smooth muscle thromboxane A2 receptor mediates this effect.

Contribution of arachidonic acid metabolites to reduced norepinephrine-induced contractions in hypercholesterolemic rabbit aortas.

Because alterations in the aortic metabolism of arachidonic acid and in vascular responsiveness occur in hypercholesterolemic rabbits, we hypothesized that an arachidonic acid metabolite may contribute to the regulation of vascular tone. Aortic contractions to norepinephrine were investigated in rabbits fed either standard chow or chow containing 2% cholesterol. In normal rabbits, norepinephrine (10(-6) M) elicited a 126 +/- 2% contraction compared with a 95 +/- 2% contraction in cholesterol-fed rabbits. The factor mediating the depressed response was endothelium-dependent because removal of the endothelium blocked the decrease in norepinephrine-induced contractions observed in the cholesterol-fed rabbits. The endothelium-derived factor was not nitric oxide, because blockade of nitric oxide synthase with nitro-L-arginine did not abolish the decreased response in the cholesterol-fed rabbits. Pretreatment of aortas with a cyclooxygenase inhibitor, indomethacin (10(-5) M) caused a slight decrease in the norepinephrine-induced contractions, suggesting that the factor could be a vasoconstrictor cyclooxygenase metabolite or a vasodilatory lipoxygenase or cytochrome P450 epoxygenase metabolite. Pretreatment with the thromboxane A2/prostaglandin H2-receptor antagonist, SQ 29458, had no effect on norepinephrine-induced contractions. Whereas the lipoxygenase inhibitor, nordihydroguaiaretic acid (5 x 10(-5) M), caused a slight increase in the contractions to norepinephrine in cholesterol-fed rabbits compared with normal rabbits, the cytochrome P450 epoxygenase inhibitor, metyrapone (10(-4) M), produced a greater enhancement of norepinephrine-induced contractions in cholesterol-fed rabbits but had no effect on responses in the normal rabbits. Characterization of [3H]arachidonic acid metabolism in cholesterol-fed aortic tissue indicated that norepinephrine stimulated the synthesis of both lipoxygenase and epoxygenase metabolites in an endothelium-dependent manner. This study demonstrated that (a) an endothelium-derived metabolite of arachidonic acid regulates vascular tone, (b) this metabolite appears to be a lipoxygenase or cytochrome P450 product or both, and (c) the activity or synthesis of the factor is enhanced by hypercholesterolemia.

Vasorelaxation by an endothelium-derived metabolite of arachidonic acid.

Arachidonic acid elicited relaxation responses in normal rabbit aorta precontracted with norepinephrine. The relaxation response was enhanced by the cyclooxygenase inhibitor indomethacin and inhibited by lipoxygenase inhibitors, including nordihydroguaiaretic acid and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. The cytochrome P-450 epoxygenase inhibitor metyrapone had no effect on arachidonic acid-induced relaxations. The present study hypothesized that a lipoxygenase metabolite of arachidonic acid mediated the response. Incubation of rabbit aorta with [14C]arachidonic acid resulted in the synthesis of a previously unidentified 14C-labeled metabolite and was called the unknown factor. Production of the unknown factor was not inhibited by indomethacin and decreased by lipoxygenase inhibitors. Production of the unknown factor and arachidonic acid-induced relaxations were dependent on an intact endothelium, indicating that the cellular source of the unknown relaxant factor was the endothelial cell. This was confirmed by demonstrating the ability of cultured rabbit aortic endothelial cells to produce the unknown factor from [14C]arachidonic acid. Feeding rabbits a 2% cholesterol diet for 2 wk induced hypercholesterolemia without causing atherosclerosis. In the cholesterol-fed rabbits, indomethacin enhanced arachidonic acid-induced relaxations in norepinephrine-precontracted aortas (maximal relaxation 49.0 +/- 2.5 vs. 35.5 +/- 1.7%, cholesterol-fed vs. normal) and increased production of the unknown factor compared with normal rabbits. The partially purified unknown factor elicited an approximately 26% inhibition of the vasoconstrictor response to norepinephrine in intact rabbit aorta. Further purification of the unknown factor by reverse-phase high-pressure liquid chromatography system resulted in isolation of a radioactive product that relaxed precontracted rabbit aorta. Therefore these data suggest that in normal and hypercholesterolemic rabbit aorta the endothelium produces an unknown metabolite of arachidonic acid that causes vasorelaxation and may regulate vascular tone.

Reduced pulmonary artery vasoconstriction in methacholine in cholesterol-fed rabbits.

Alterations in vascular tone are well documented in hypercholesterolemia, yet little is known about the role of dietary cholesterol in endothelium-dependent contractions of pulmonary arteries. Methacholine and arachidonic acid cause endothelium-dependent contractions in normal rabbit pulmonary artery that are mediated by thromboxane A2. We tested the effect of these agonists on pulmonary arteries from rabbits fed standard rabbit chow or chow supplemented with 2% cholesterol for 2 weeks. Arachidonic acid-induced contractions did not differ in the groups. However, methacholine-induced contractions were significantly depressed in cholesterol-fed rabbits. Vascular thromboxane A2 production was similar in normal and cholesterol-fed rabbits. Pretreatment with the nitric oxide synthase inhibitor nitro-L-arginine had no effect on contractions observed with methacholine in normal rabbits but enhanced methacholine-induced contractions in cholesterol-fed rabbits. In norepinephrine-precontracted vessels, methacholine caused a small relaxation response in normal rabbits. In contrast, in cholesterol-fed rabbits, methacholine produced enhanced relaxations, suggesting that cholesterol feeding augments relaxations and decreases contractions by increasing nitric oxide. However, nitric oxide synthase activity in pulmonary arteries from cholesterol-fed and normal rabbits was not different between the two groups. In an additional experiment, the calcium-dependent potassium channel blocker charybdotoxin had little effect on methacholine-induced contractions in cholesterol-fed rabbits. In summary, the present study demonstrates that hypercholesterolemia alters pulmonary artery vascular contractions and relaxations to methacholine. This effect is not mediated by a decreased production of thromboxane A2 or by an increased production of nitric oxide. Although the mechanisms mediating the altered vascular responses is still unknown, the results from this study clearly indicate that the regulation of vascular tone is different in normal and hypercholesterolemic vessels.

Role of endothelium-derived metabolites of arachidonic acid in enhanced pulmonary artery contractions in female rabbits.

Previous studies reported sex differences in production of endothelium-derived substances and suggested that these compounds may be involved in regulation of vascular tone under both normal and pathological conditions. The present study was designed to compare the effects of endothelium-dependent contractions in pulmonary artery vessels obtained from male and female rabbits. Rings of intrapulmonary arteries were suspended under isometric tension in oxygenated Krebs' buffer. In male rabbit pulmonary artery, arachidonic acid and methacholine elicited endothelium-dependent, concentration-related contractions (maximal contraction, 79 +/- 4% and 54 +/- 4% of the KCl contractions, respectively). In contrast, endothelium-dependent arachidonic acid- and methacholine-induced contractions were greater in female pulmonary arteries (maximal response, 113 +/- 7% and 101 +/- 6% of the KCl contractions, respectively). There was no difference in KCl-induced contractions in female and male pulmonary arteries (1.2 +/- 0.1 versus 1.3 +/- 0.1 g, respectively). In male rabbits, the vasoconstrictor responses to arachidonic acid and methacholine were inhibited by the cyclooxygenase inhibitor indomethacin. We have previously identified thromboxane A2 as the endothelium-dependent contracting factor in male rabbits. However, indomethacin only partially inhibited arachidonic acid-induced contractions in female pulmonary arteries (maximal inhibition, 46% of the control response) suggesting that a noncyclooxygenase metabolite of arachidonic acid mediates contraction in female rabbits. Likewise, indomethacin only partially inhibited methacholine-induced contractions of female pulmonary arteries. The combined cyclooxygenase/lipoxygenase inhibitor BW 755C and the lipoxygenase inhibitor nordihydroguaiaretic acid completely blocked arachidonic acid-induced contractions in females. Furthermore, both basal and stimulated production of thromboxane B2, as measured by radioimmunoassay, were similar in female and male pulmonary arteries. When segments of pulmonary arteries obtained from female and male rabbits were incubated with 14C-arachidonic acid and the extracted metabolites were resolved by reverse-phase high-performance liquid chromatography, there was an enhanced production of metabolites in females. Pretreatment with indomethacin attenuated metabolism of all products in the males but enhanced production of some compounds in vessels from the females. These observations suggest that the enhanced vasoconstrictor response to arachidonic acid in female pulmonary arteries is due to a lipoxygenase metabolite of arachidonic acid.

Decrease in vascular TxA2 receptors in a subgroup of rabbits unresponsive to a TxA2 mimetic.

In rabbit pulmonary artery, endothelium-dependent contractions to arachidonic acid and methacholine are mediated by thromboxane (Tx) A2. The TxA2 mimetics U-46619 and (1S-[1 alpha,2 beta(5Z),3 alpha(1E,3R*),4 alpha])-7-[3- [3-hydroxy-4-(4'-iodophenoxy)-1-butenyl]-7-oxabicyclo(2.2.1)hep tan-2-yl]- 5-heptenoic acid (I-BOP), norepinephrine, and endothelin produced endothelium-independent contractions. Arachidonic acid, methacholine, U-46619, and I-BOP failed to produce contractions in a subgroup of rabbits (25%). Nonresponder arteries contracted similarly to norepinephrine and endothelin as responder arteries. The affinity (Kd) and density (Bmax) of TxA2 receptors in crude pulmonary artery membranes were assessed via equilibrium binding studies using 125I-BOP. There was no difference in Kd between the two groups (0.49 +/- 0.17 vs. 0.32 +/- 0.14 nM, responder vs. nonresponder). There was a significant decrease in Bmax (123 +/- 21 vs. 28 +/- 11 fmol/mg protein, responder vs. nonresponder; P < 0.01) of receptors in the nonresponders. Nonresponder aortas also did not contract to U-46619 and exhibited a decrease in TxA2 receptors, indicating that the difference is not specific for the pulmonary artery. Nonresponder platelets aggregated to U-46619, suggesting that the platelet receptor is not altered. TxA2-receptor expression may be regulated in vivo. Nonresponder rabbits may provide a useful model for studying these receptors in vascular disease.

Endothelium-dependent contractions in rabbit pulmonary artery are mediated by thromboxane A2.

This study was designed to characterize the endothelium-dependent contracting factor (EDCF) released by arachidonic acid (AA) and methacholine (MeCH) in the rabbit pulmonary artery. AA and MeCH contract the rabbit pulmonary artery; however, the effects of both are blocked by denuding the vessels and by administration of indomethacin (a cyclooxygenase inhibitor), dazoxiben (a thromboxane [TX] synthase inhibitor), and SQ29548 (a TXA2/prostaglandin [PG] H2 receptor antagonist). When segments of rabbit pulmonary artery were incubated with [14C]AA and the [14C] metabolites were resolved by reverse-phase high-performance liquid chromatography (HPLC), radioactive products were observed that comigrated with 6-keto-PGF1 alpha and TXB2, the stable metabolites of prostacyclin and TXA2. The TXB2 radioactive peak was rechromatographed on normal-phase HPLC and again migrated with TXB2. Finally, the structures of derivatized [14C]6-keto-PGF1 alpha and [14C]TXB2 peaks were confirmed by gas chromatography/mass spectrometry. The synthesis of [14C]6-keto-PGF1 alpha and [14C]TXB2 was inhibited by removal of the endothelium and by indomethacin. Dazoxiben inhibited the synthesis of [14C]TXB2 but not [14C]6-keto-PGF1 alpha. Using specific radioimmunoassays, AA and MeCH stimulated 6-keto-PGF1 alpha and TXB2 release. Indomethacin blocked the production of both 6-keto-PGF1 alpha and TXB2, whereas dazoxiben only blocked TXB2. In a superfusion/bioassay system, AA stimulated an endothelium-intact donor vessel to release a labile substance that contracted an indomethacin-treated endothelium-denuded recipient vessel. The EDCF released by AA had an approximate half-life of 30 seconds. Cultured rabbit pulmonary arterial endothelial cells synthesized 6-keto-PGF1 alpha but not TXB2. Immunohistochemical studies indicated the presence of cyclooxygenase, but not TX synthase, in pulmonary artery endothelial cells. TXA2 appears to be the EDCF released by AA and MeCH in rabbit pulmonary artery; however, TXA2 is not produced by endothelial cells but may arise from cells that adhere to the luminal surfaces, such as platelets or macrophages.

Arachidonic acid- and acetylcholine-induced relaxations of rabbit aorta.

The present study investigated the role of arachidonic acid and acetylcholine in mediating endothelium-dependent relaxations of rabbit aorta. Isolated thoracic aortic rings were precontracted with a submaximal concentration of norepinephrine, and the effect of various agents on arachidonic acid- and acetylcholine-induced relaxations was examined. Arachidonic acid elicited a concentration-related relaxation that was potentiated by the cyclooxygenase inhibitor indomethacin. Treatment with the lipoxygenase inhibitor nordihydroguaiaretic acid completely blocked but the cytochrome P450 inhibitor metyrapone had no effect on arachidonic acid-induced relaxation. NG-Monomethyl-L-arginine and nitro-L-arginine, compounds that inhibit the nitric oxide-like endothelium-derived relaxing factor, had little or no effect on arachidonic acid-induced relaxations. In contrast, nordihydroguaiaretic acid, metyrapone, NG-monomethyl-L-arginine, and nitro-L-arginine all attenuated the relaxation to acetylcholine; however, indomethacin had no effect on acetylcholine-induced relaxations. Arachidonic acid and acetylcholine had no effect on denuded rabbit aorta. Incubation of rabbit aorta with [14C]arachidonic acid resulted in the synthesis of major radioactive metabolites that comigrated with the prostaglandins and hydroxyeicosatetraenoic acids. Indomethacin selectively inhibited prostaglandin formation, nordihydroguaiaretic acid attenuated both prostaglandins and hydroxyeicosatetraenoic acids, and metyrapone blocked the epoxyeicosatrienoic acids. Additionally, acetylcholine elicited a twofold increase in tissue cyclic guanosine monophosphate content in contrast to a 59% reduction in cyclic guanosine monophosphate content observed with arachidonic acid. Therefore, these data suggest that in rabbit aorta, arachidonic acid-induced relaxations are mediated by an endothelium-dependent factor (or factors) that differs from the factor (or factors) released by acetylcholine. These results support the existence of multiple endothelium-derived relaxing factors.

Enhanced synthesis of epoxyeicosatrienoic acids by cholesterol-fed rabbit aorta.

Arachidonic acid metabolism via cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase was investigated in thoracic aortic tissue obtained from rabbits fed either standard rabbit chow or chow containing 2% cholesterol. Aortic strips were incubated with [14C]arachidonic acid and A23187. Metabolites from extracted media were resolved by high-pressure liquid chromatography (HPLC). Normal and cholesterol-fed rabbit aortas synthesized prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs). The major cyclooxygenase products were 6-keto-PGF1 alpha and PGE2. Basal aortic 6-keto-PGF1 alpha production was slightly reduced in cholesterol-fed compared with normal rabbits. 12(S)- and 15(S)-HETE were the major aortic lipoxygenase products from both normal and cholesterol-fed rabbits. The structures were confirmed by gas chromatography-mass spectrometry (GC-MS). Only cholesterol-fed rabbit aortas metabolized arachidonic acid via cytochrome P-450 epoxygenase to the epoxyeicosatrienoic acids (EETs). 14,15-, 11,12-, 8,9-, and 5,6-EET were identified based on comigration on HPLC with known 14C-labeled standards and typical mass spectra. Incubation of normal aorta with 14,15-EET decreased the basal synthesis of 6-keto-PGF1 alpha. The other EETs were without effect. The four EET regioisomers relaxed the norepinephrine-precontracted normal and cholesterol-fed rabbit aorta. The relaxation response to 14,15-EET was greater in aortas from cholesterol-fed rabbits. These studies demonstrate that hypercholesterolemia, before the development of atherosclerosis, alters arachidonic acid metabolism via both the cyclooxygenase and epoxygenase pathways.

Method of removal of aortic endothelium affects arachidonic acid metabolism and vascular reactivity.

To assess endothelium-dependent responses of blood vessels in vitro, endothelial cells are removed by a variety of mechanical means. We sought to determine if the method of removal of the endothelium affected arachidonic acid metabolism and vascular reactivity of isolated strips of rabbit aorta. Thoracic aorta of New Zealand White rabbits were excised and sectioned into strips with a sharp razor blade. The luminal surface of the vessel was then gently stroked (denuded-1) or forcefully rubbed (denuded-2) with a moist cotton swab. Vessels were then either fixed in 3% glutaraldehyde and processed for electron microscopy, incubated with [14C]arachidonic acid and 20 microM A23187 for determination of arachidonic acid metabolism, incubated with 20 microM A23187 for measurement of endogenous release of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and 12-hydroxyeicosatetraenoic acid (12-HETE) by specific radioimmunoassays, or suspended in an organ chamber filled with Krebs bicarbonate solution for vascular reactivity experiments. Electron micrographs showed that denuded-1 vessels lacked an endothelial cell layer and had slight degeneration of the smooth muscle cells. Additionally, these vessels had a diminished capacity to produce 6-keto-PGF1 alpha as compared to control vessels (214 +/- 25 vs. 360 +/- 36 pg/mg of tissue, P less than 0.05). Denuded-2 vessels contained severe degeneration and rupture of smooth muscle cells in addition to the loss of the endothelial cell layer. While the 6-keto-PGF1 alpha concentration (168 +/- 23 pg/mg) was less in denuded-2 vessels, HPLC indicated that the production of [14C]12-HETE was markedly increased in these vessels as compared to control or denuded-1 vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Augmented vasoconstrictor responses to serotonin precede development of atherosclerosis in aorta of WHHL rabbit.

Watanabe heritable hyperlipidemic (WHHL) rabbits have elevated concentrations of plasma cholesterol and develop progressive atherosclerosis. The present investigation was undertaken to evaluate the vascular responses to vasoactive compounds of aorta from WHHL rabbits and normal New Zealand White (NZW) rabbits at 1 and 6 months of age. Rings of distal thoracic aorta were suspended under isometric tension in oxygenated Krebs buffer. Developed tension was measured in response to graded concentrations of agonists. Maximal responses to KCl (40 mM) were the same in aortas from the 1-month-old and 6-month-old WHHL and NZW rabbits. Aortas from 1-month-old animals were more sensitive to serotonin than aortas from 6-month-old animals. Aortas from WHHL rabbits exhibited an increased maximal response to serotonin when compared with NZW controls. In contrast, the constrictor responses to norepinephrine were reduced in WHHL rabbits compared with NZW rabbits at both age groups. Methacholine decreased tension development in serotonin-contracted vessels. This relaxation was greatest in aortas from NZW rabbits. In 1-month-old NZW rabbits fed a high cholesterol diet, the constrictor responses to serotonin and the relaxation responses to methacholine did not differ from NZW rabbits ingesting a normal diet. However, the responses to norepinephrine were markedly attenuated in the hypercholesterolemic NZW rabbits. Microscopic evaluation of the aortas revealed occasional adherent leukocytes and irregularities in the vascular endothelium in 1-month-old WHHL animals. These changes were greater in aortas from 6-month-old WHHL animals, with more adherent leukocytes, adherent platelets, and severe irregularities in the endothelial surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Eicosapentaenoic acid alters vascular reactivity and platelet adhesion in Watanabe heritable hyperlipidemic rabbits.

Feeding a diet rich in eicosapentaenoic acid (EPA) to Watanabe heritable hyperlipidemic (WHHL) rabbits resulted in an attenuated aortic contractile response to the vasoconstrictor agent serotonin when compared to responses from WHHL rabbits fed normal rabbit chow. In contrast, only the maximal contractile response to norepinephrine was reduced in EPA-fed rabbit aortas. Additionally, methacholine-induced relaxations were potentiated in aortas obtained from the EPA-fed rabbits. When platelets obtained from EPA-fed rabbits were incubated with arachidonic acid, there was a reduced ability of the platelets to adhere to albumin-coated discs in comparison to control rabbit platelets. These data indicate a potentially beneficial effect of EPA in atherosclerotic WHHL rabbits.

Characterization of arachidonic acid metabolism in Watanabe heritable hyperlipidemic (WHHL) and New Zealand white (NZW) rabbit aortas.

WHHL rabbits develop progressive atherosclerosis. There are no visible signs of the disease at 1 month, however, by 12 months, the formation of aortic plaques is extensive. This study characterized arachidonic acid (AA) metabolism in 1 and 12 month old WHHL and NZW rabbit aortas. Vessels incubated with 14C-AA and A23187 metabolized AA to a number of oxygenated products as identified by high pressure liquid chromatography. The major AA metabolites produced by WHHL and NZW aortas were 6-keto PGF1 alpha, PGE2, 12- and 15-hydroxyeicosatetraenoic acids (HETEs). The structures of the HETEs were confirmed by gas chromatography-mass spectrometry. Indomethacin blocked the synthesis of prostaglandins (PGs) but not HETEs whereas ETYA, NDGA or removal of the endothelium attenuated the production of both PGs and HETEs. Measurement of 6-keto PGF1 alpha, 12- and 15-HETE by specific radioimmunoassays indicated that as the rabbits aged and as atherosclerosis progressed, aortas lost the ability to synthesize 6-keto PGF1 alpha and 15-HETE. Prior to the development of atherosclerosis, 1 month old WHHL aortas produced 70% less 15-HETE than did NZW aortas. Atherosclerotic aortas from 12 month old WHHLs synthesized 60% less 6-keto PGF1 alpha during stimulation with AA or A23187 than did 12 month old NZW aortas. We conclude that the development and expression of atherosclerosis in WHHL rabbits impairs the ability of aortas to metabolize AA to both PGs and HETEs.

Angiotensin II contributes to blood pressure maintenance in conscious rats treated with yohimbine.

Yohimbine (1 mg/kg s.c.) produced significant and persistent increases in plasma renin concentration and plasma norepinephrine concentration in conscious rats, but mean arterial pressure (MAP) and heart rate were unchanged. The subsequent i.v. infusion of the angiotensin II receptor antagonist saralasin (100 micrograms/kg per min) caused a significant decrease (-17%) in MAP. We conclude that yohimbine-induced renin release, and the resultant rise in plasma angiotensin II concentration, prevents the decrease in MAP which would result from the blockade of vascular alpha-adrenoceptors by yohimbine.

The pharmacokinetic properties of yohimbine in the conscious rat.

We used high performance liquid chromatography with fluorescence detection to measure the concentration of yohimbine in serum and brain of conscious Sprague-Dawley rats at various times after the i.v. injection of 1 mg/kg of yohimbine. The serum concentration-time profile of yohimbine was biphasic with a rapid distribution phase (t1/2 alpha = 0.048 h) followed by a very slow elimination phase t1/2 beta = 16.3 h). The clearance of yohimbine was 11 ml/h.kg-1, and the volume of distribution was 259 ml/kg. Increasing doses (0.3, 1 and 3 mg/kg, i.v.) of yohimbine produced non-linear increases in serum yohimbine concentration. Yohimbine entered the brain rapidly (5,000 ng/g at 5 min after 1 mg/kg, i.v.) and disappeared from brain with a t1/2 beta of 7.7 h. In contrast to serum yohimbine concentration, increasing doses of yohimbine (0.3, 1 and 3 mg/kg) produced linear increases in brain yohimbine concentration, a phenomenon which is consistent with concentration-dependent binding of yohimbine to plasma proteins. The rapid entry of yohimbine into the brain, the slow rate of elimination of yohimbine from serum and brain and the linear relationship of brain yohimbine concentration as a function of dose should be taken into consideration whenever yohimbine is to be used as a probe of alpha 2-adrenoceptor function in vivo.

The mechanism of yohimbine-induced renin release in the conscious rat.

These studies were designed to determine the role of the central nervous system, the sympathetic nervous system, the adrenal glands and the renal sympathetic nerves in yohimbine-induced renin release in conscious rats. Yohimbine (0.3-10 mg/kg, s.c.) caused time- and dose-related increases in plasma renin activity (PRA) and concentration (PRC) which were accompanied by time- and dose-related elevations of plasma norepinephrine (NE) and epinephrine (Epi) concentrations. Significant positive correlations were found between the increases in PRA and the increases in plasma NE and Epi concentrations caused by yohimbine, and propranolol (1.5 mg/kg, s.c.) blocked 90% of yohimbine (3 mg/kg, s.c.)-induced renin release. Over the entire spectrum of doses of yohimbine, the increases in PRA and plasma NE and Epi concentrations were positively correlated with the decreases in mean arterial pressure (MAP), but the y-intercept was positive in every case and the 1 mg/kg dose of yohimbine consistently increased PRA independent of any change in MAP. Complete renal denervation, as evidenced by a greater than 90% reduction in renal NE content, did not alter the increase in PRA caused by yohimbine (3 mg/kg, s.c.). An increase in circulating plasma catecholamine concentrations appeared to mediate yohimbine-induced renin release since propranolol prevented the rise in PRA caused by yohimbine in renal denervated rats. Prior adrenalectomy (Adx) also failed to prevent the rise in PRA produced by yohimbine (3 mg/kg, s.c.), but a combination of Adx and renal denervation caused a significant impairment of yohimbine-induced renin release. However, neither Adx alone nor the combination of Adx and renal denervation affected the increase in plasma NE concentration caused by yohimbine. Complete transection of the spinal cord at C8 caused a drastic reduction in plasma catecholamine concentrations but did not change basal PRC. Yohimbine (3 mg/kg, s.c.) did not increase PRC or plasma catecholamine concentrations after spinal transection. Based on these results, we conclude that 1) the stimulation of renin release by yohimbine is a secondary neurohormonal consequence of the generalized increase in sympathetic activity caused by yohimbine, 2) the sympathoadrenal activation produced by yohimbine results from an action in the brain which is amplified by the simultaneous blockade of prejunctional alpha 2-adrenoceptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Yohimbine induces sympathetically mediated renin release in the conscious rat.

The preferential alpha 2-adrenergic antagonist yohimbine (4 mg/kg s.c.) caused a time-related increase in serum renin activity and heart rate in conscious Sprague-Dawley rats. Although mean arterial pressure was not decreased significantly over the 2-h period, heart rate was elevated significantly at 15 and 30 min post-injection. In contrast, serum renin activity remained elevated for up to 2 h with a 9-fold and 9.7-fold increase occurring at 30 and 60 min post-injection, respectively. Yohimbine (0.3, 1, 3 and 10 mg/kg s.c.) elicited a dose-related increase in serum renin activity and heart rate (30 min post-injection). The 1 mg/kg dose of yohimbine did not alter blood pressure whereas the 3 mg/kg dose caused a variable decrease in mean arterial pressure. The highest dose of yohimbine (10 mg/kg) significantly lowered blood pressure. The beta-adrenergic receptor antagonist propranolol (1.5 mg/kg s.c.), blocked the renin release and tachycardia caused by yohimbine (1 and 3 mg/kg s.c.), and the ganglionic blocking agent chlorisondamine partially inhibited the renin release elicited by 3 mg/kg (s.c.) of yohimbine. The prostaglandin synthetase inhibitors indomethacin (5 mg/kg s.c.) and meclofenamate (5 mg/kg s.c.) impaired the ability of yohimbine (3 mg/kg) to elevate SRA but did not alter the hemodynamic effects of yohimbine. Thus, the increase in renin release caused by yohimbine appears to be mediated by the sympathetic nervous system. Because the smaller doses of yohimbine increase renin release in the absence of a decrease in mean arterial pressure, it is unlikely that yohimbine stimulates renin release by baroreflex-mediated activation of the renal sympathetic nerves.(ABSTRACT TRUNCATED AT 250 WORDS)