Bovine Coronary Artery Research Articles

Identification of Epoxyeicosatrienoic Acids as Endothelium-Derived Hyperpolarizing Factors

Endothelial cells release several compounds, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF), that mediate the vascular effects of vasoactive hormones. The identity of EDHF remains unknown. Since arachidonic acid causes endothelium-dependent relaxations of coronary arteries through its metabolism to epoxyeicosatrienoic acids (EETs) by cytochrome P450, we wondered if the EETs represent EDHFs. Precontracted bovine coronary arteries relaxed in an endothelium-dependent manner to methacholine. The cytochrome P450 inhibitors, SKF 525A and miconazole, significantly attenuated these relaxations. They were also inhibited by tetraethylammonium (TEA),an inhibitor of Ca2+-activated K+ channels, and by high [K+]0 (20 mmol/L). Methacholine also caused hyperpolarization of coronary smooth muscle (-27 +/- 3.9 versus -40 +/- 5.1 mV), which was completely blocked by SKF 525A and miconazole. In vessels prelabeled with [3H] arachidonic acid, methacholine stimulated the release of 6-ketoprostaglandin F1alpha, 12-HETE, and the EETs. Arachidonic acid relaxed precontracted coronary arteries, which were also blocked by TEA, charybdotoxin, another Ca2+-activated K+ channel inhibitor, and high [K+]0. 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET relaxed precontracted coronary vessels (EC50, 1 X 10(-6) mol/L). The four regioisomers were equally active. TEA, charybdotoxin, and high [K+]0 attenuated the EET relaxations. 11,12-EET hyperpolarized coronary smooth muscle cells from -37 +/- 0.2 to -59 +/- 0.3 mV. In the cell-attached mode of patch clamp, both 14,15-EET and 11,12-EET increased the open-state probability of a Ca2+-activated K+ channel in coronary smooth muscle cells. This effect was blocked by TEA and charybdotoxin. These data support the hypothesis that the EETs are EDHFs.

Calcium signalling and autacoid production in endothelial cells are modulated by changes in tyrosine kinase and phosphatase activity.

The vascular endothelium is the source of a number of vasodilator and vasoconstrictor autacoids and is thus a key regulator of vascular homeostasis. We studied the effects of altering the balance between protein tyrosine kinase and phosphatase activity on Ca2+ signalling and phosphotyrosine levels in cultured human endothelial cells, as well as on autacoid production in native endothelial cells. In isolated segments of rabbit aorta and carotid artery, as well as in bovine coronary arteries, the tyrosine phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate initiated endothelium-dependent relaxations which could be attributed to the release of nitric oxide and the endothelium-derived hyperpolarizing factor. In cultured endothelial cells incubation with PAO resulted in a time-dependent accumulation in 6-keto prostaglandin F1 alpha, the stable metabolite of prostacyclin, as well as in an increase in the intracellular concentration of free Ca2+ ([Ca2+]i). Inhibition of tyrosine kinases attenuated both the PAO-induced relaxation and the increase in endothelial [Ca2+]i. Western blot analysis of endothelial cells treated with the tyrosine phosphatase inhibitors revealed a time-dependent increase in the tyrosine phosphorylation of a series of bands in both the Triton X-100-soluble and Triton X-100-insoluble (cytoskeletal) fractions. These observations suggest that alterations in cellular levels of phosphotyrosine may have profound effects on vascular homeostasis by modulating Ca2+ signalling and autacoid production in endothelial cells.

The synergistic effect of elastase and hydrogen peroxide on vascular endothelial cell injury is due to the production of hydroxylradical in the endothelial cells.

Protease inhibitors such as aprotinin and urinastatin inhibited vascular endothelial cell injury induced by PMA-stimulated leukocytes, although their inhibitors did not suppress the production of active oxygen species released from leukocytes. On the other hand, in the presence of pancreas elastase (10 micrograms/ml), hydrogen peroxide (50 microM) caused severe injury of endothelial cells isolated from the bovine carotid artery (% specific 51Cr release, % SR = 42.9 +/- 3.3%), although the % SR elicited by elastase or hydrogen peroxide alone, respectively, was below 1%. Elastase and hydrogen peroxide acted synergistically on the injury of endothelial cells from the bovine carotid artery similarly to that in the endothelial cells isolated from the bovine coronary artery and human umbilical vein. Furthermore, elastase derived from both pancreas and leukocyte induced this synergistic action on endothelial cell injury. To clarify the mechanism of vascular endothelial cell injury induced by the combination of elastase and hydrogen peroxide, we examined the effects of various radical scavengers and protease inhibitors. Deferoxamine mesylate completely inhibited the endothelial cell injury, while protease inhibitors such as antitrypsin and macroglobulin had a protective effect. Pretreatment of endothelial cells with deferoxamine mesylate also protected against this cytotoxicity. These findings suggested that the synergistic effect of elastase and hydrogen peroxide on the endothelial cell injury is due to the production of hydroxylradical in the endothelium and that this synergistic action might be partially involved in the endothelial cell injury induced by activated leukocytes.

Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells

Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [ 14C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F 1α, PGE 2, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11- hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12( S)-, 15( S)-, and 11( R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11( R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF 1α and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF 1α exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.

The effect of edothelium on the response to propofol on bovine coronary artery rings

In this study, the effect of propofol on isolated bovine coronary artery tone was studied in artery rings precontracted with PGF2a. Propofol, in concentrations of 10-6-10-5 M did not change vascular smooth muscle tone, but at high concentrations (10-4-10-1 M) produced relaxation in rings with intact endothelium. In rings denuded of endothelium or treated with methylene blue, propofol produced relaxation at 10-3-10-1 M concentrations, but there was a significant decrease in relaxation compared to endothelium intact rings. In the presence of a calcium agonist (Bay K 8644; 10-5 M), propofol produced constriction in rings denuded of endothelium. These results suggest that high concentrations of propofol may have vasorelaxant effect on bovine coronary artery and that these effects may be due to actions on the endothelium and mediated by calcium channels.

Endothelium-mediated vasodilation during ACE inhibition.

ACE inhibitors are superior to other vasodilators in the treatment of congestive heart failure and may be advantageous in patients with myocardial infarction and hypertension. The mechanisms mediating these beneficial effects are not clear. The present article discusses the mechanisms leading to augmented release of endothelium-derived nitric oxide during ACE inhibition. Acute potentiation of bradykinin (Bk)-induced vasodilation was studied in rings of bovine and human coronary arteries mounted in organ chambers for recording of isometric force. The ACE inhibitors captopril, enalaprilat, fosinoprilat, lisinopril, or ramiprilat alone did not affect vascular tone in isolated coronary tone in isolated coronary artery preparations with intact endothelium. However, in the presence of exogenous Bk, kallidin, or one of the slowly degradable Bk2-receptor agonists D-Arg(Hyp3)-Bk or [Hyp3-Tyr(Me)8]-Bk they elicited potent concentration-dependent relaxations. Relaxations in response to lisinopril were not observed in the presence of other vasodilators. They were prevented by mechanical removal of the endothelium, inhibition of nitric oxide synthase or Bk2-receptor blockade. The data indicate that ACE inhibitors potentiate the effects of Bk on endothelial cells by a local mechanism, probably independent of the degradation of bradykinin. The chronic effects of ACE inhibitors on endothelial function were compared with those of selective angiotensin(AT)1-receptor blockade in cyclosporin A (CsA) treated rats. Chronic AT blockade alone does not affect endothelium-dependent relaxation and increases contractions to ATII in the rot aorta. Combination of CsA with either an ACE-Inhibitor or an AT2 receptor antagonist prevented the endothelial dysfunction in the rat arta observed after CsA alone.(ABSTRACT TRUNCATED AT 250 WORDS)

772-4 Intravascular Heating Facilitates Local Heparin Delivery and Attenuates Washout During Balloon Angioplasty with Hydrogel-Coated Balloons

Although previous studies have demonstrated that heparin can be delivered to the arterial wall during balloon angioplasty with hydrogel-coated balloons, drug washout over the first hour is rapid. The purpose of this study was to determine whether vascular heating during local heparin delivery with hydrogel-coated, radiofrequency-powered thermal angioplasty balloons would affect immediate drug deposition and the subsequent persistence of heparin within the arterial wall. To evaluate the effect ofheat on heparin deposition, fluorescein-labeled heparin was delivered in vitro to 13 bovine coronary arteries either at 50°C (n = 3), at 60°C (n = 3) or without heat (n = 7). Heparin deposition was graded on a 1–3 scale as extending into the inner 1/3, the middle 1/3 or outer 1/3 of the arterial wall. To assess the effect of heat on the intramural persistence of heparin , 3 H-heparin was delivered in vivo to 13 porcine peripheral artery pairs. The balloon:artery ratio was 1.2: 1 as guided by intravascular ultrasound. One artery in each pair was heated during heparin delivery to either 50°C (n = 11) or 80°C (n = 2). The contralateral control vessel was not heated. Arteries were harvested at one hour for scintillation counting. In both the in vivo and in vitro studies, balloon inflations were at 6 atm for 2.5 minutes. In all heated vessels thermal energy was applied for 90 secs. Supplemental heating during local drug delivery significantly increased both the immediate deposition and the persistence of intramurally delivered heparin. In vitro, the heparin deposition score was 2.3 ± 0.5 for heated arteries and 1.3 ± 0.8 for controls (p = < 0.03). In vivo, more heparin was present at one hour in the heated arteries than in the control arteries in 11 of 13 vessel pairs (heated vessels, 58 ± 46 units; non-heated controls, 43 ± 42 units, p = 0.02). Vascular heating at the time of local heparin delivery with hydrogei-coated balloons increases both the immediate delivery and the persistence of heparin in the arterial wall.

Heat-induced changes in the mechanical behavior of passive coronary arteries.

We performed in vitro pressure-diameter and axial force-length experiments on nondiseased, passive bovine coronary arteries subjected to bath temperatures from 21 to 80 degrees C for 90 s to 4 hr. Over the strain ranges studied, we found that: (a) vessel behavior remained the same over 20 min of testing at 21 to 55 degrees C, (b) vessels stiffened multiaxially after 5 min of exposure to 60 degrees C and continued to stiffen over 20 min of testing, (c) dramatic multiaxial vessel stiffening and shrinkage occurred after 90 s of exposure to 70 and 80 degrees C, and (d) heat-induced changes at 70 degrees C depended on the intraluminal pressure during heating. Thus, passive bovine coronary arteries exhibit a complex thermomechanical behavior that depends on the temperature, duration of thermal exposure, and the mechanical loads applied during heating.

Potentiation of PDGF-induced growth responses in coronary artery smooth muscle cells by thromboxane.

Mitogenic effects of TXA2 in vascular smooth muscle cells are discussed to be dependent on the age of the donor organism. The present study investigates the contribution of TXA2 on PDGF-induced proliferation of bovine coronary artery smooth muscle cells (BCA-SMC) isolated from adult animals. Radioligand binding studies revealed high affinity TXA2 binding sites (Kd = 1.6 nM) in these cells. TXA2-mimetics alone showed no proliferative effect in BCA-SMC, assessed by [3H]thymidine incorporation. However, PDGF-stimulated proliferation was potentiated two-fold receptor-dependently by TXA2-mimetics. Thus, vasoconstrictory eicosanoids released from activated platelets might aggravate proliferation of vascular smooth muscle cells at sites of vessel injury in the adult organism.

P1-127 - Bradykinm-induced endothelium-dependent biphasic relaxation of bovine coronary artery in vitro

P1-127 - Bradykinm-induced endothelium-dependent biphasic relaxation of bovine coronary artery in vitro

Antagonistic affinity profile of imperialine on different subtypes of muscarinic receptors

In order to identify the receptor subtype responsible for acetylcholine (ACh)-induced relaxation of bovine coronary artery, we determined the affinity of six subtype-selective muscarinic antagonists and compared them with affinity estimates obtained for bovine left atria. At low concentrations, ACh potently relaxed circular strips of coronary artery with endothelium (EC50 0.15 μM), but contracted them at higher agonist concentrations with potencies that depended on the presence or absence of endothelium: EC50 1.8 μM (without endothelium); 4.6 JμM (with endothelium). The pA2 values obtained for antagonism of relaxant responses to ACh were: pirenzepine (M1-selective) 7.38 ± 0.12; AF-DX 116 (11-[2-(diethylamino-methyl)-1-piperidinylacetyl]-5, 11-dihydro-6H-pyrido(2,3-b)l,4-benzodiazepine-6-one; M2-selective) 5.79 ± 0.09; and 4-diphenylacetoxy-N-methylpiperidine-methobromide (4-DAMP; M3/M1-selective) 9.07 ± 0.12. The corresponding Schild slopes were 0.98 ± 0.07 for pirenzepine, 1.17 ± 0.09 for AF-DX 116 and 1.01 ± 0.04 for 4-DAMP. For the following three antagonists, pKg values were determined at two different antagonist concentrations: dicyclomine (M1-selective) 7.49 ± 0.10, cyclohexylphenyl-(2-piperidinoethyl)-silanol (CPPS; M3-selective) 8.0 ± 0.10, and parafluoro-hexahydrosila-difenidol (pFHHSiD; M3-selective) 7.87 ± 0.10. For comparison, the antagonism of methacholine-induced negative inotropy in left atria was determined for three antagonists, yielding the following pA2 values: pirenzepine 5.98 ± 0.14; AF-DX 116 6.81 ± 0.14 and 4-DAMP 7.99 ± 0.14. The slopes of the corresponding Schild plots were 1.05 ± 0.10,1.14 ± 0.12 and 1.08 ± 0.08, respectively. The results indicate that ACh relaxes precontracted circular strips of bovine coronary arteries with intact endothelium by activation of muscarinic receptors most closely corresponding to the M3 subtype.

Vasoreactivity and structure of human coronary arteries irradiated by excimer laser.

In this study the effects of excimer laser (308 nm) angioplasty on arteries have been studied. We report histology and vasoreactivity properties of human atherosclerotic and normal coronary arteries and bovine coronary arteries after exposure to pulsed excimer laser irradiation. Segments of isolated and pharmacologically active human coronary arteries were obtained within 5-8 hr postmortem. Segments of bovine arteries were obtained from fresh hearts. Side branches were ligated and vessels perfused with Krebs-Ringer's-bicarbonate solution in a perfusion apparatus to maintain their viability. Excimer laser irradiation was performed using a fused silica fiber advanced through the artery within a polyethylene cannula to assist with steerability and to protect against perforation. The fiber delivered a total of 40-60 J at its tip at 100 mJ/pulse at 15 pulses/sec. A total of 5 human atherosclerotic, 2 human normal, and 8 bovine arteries were used. Ablation of plaque was accomplished with remarkable ease and little resistance. Histologic studies showed clear-cut wedges inscribed by the catheter along the arterial wall without charring or thermal effects of coagulation necrosis. Vasoreactivity was assessed by measuring flow changes during perfusion before and after excimer angioplasty using a transient challenge with the vasoconstrictor serotonin. All excimer-irradiated arteries showed no increase in vasoreactivity during the challenge and showed preservation of relaxation properties following the challenge. In conclusion, coronary artery angioplasty by excimer laser appears to allow for effective plaque ablation with simultaneous preservation of structure and pharmacological properties of arteries.

NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium.

In this study we examined the intracellular sources of superoxide anion (O2-.) in cultured bovine coronary endothelium, employing lucigenin (250 microM)-elicited chemiluminescence (CL). In the homogenate from these cells, 100 microM NADPH increased O2-. by 81% from 8.9 +/- 1.5 to 16.0 +/- 1.5 x 10(5) cpm/mg protein (P < 0.01, n = 8). In the presence of 100 microM NADH, however, CL increased by 458% from 8.9 +/- 1.6 to 49.6 +/- 12.0 x 10(5) cpm/mg protein (P < 0.01, n = 8). Scavengers of O2-., superoxide dismutase (100 micrograms/ml), or 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (Tiron, 10 mM) inhibited NADH-mediated CL by 70 and 83%, respectively. Neither hypoxanthine (100 microM) nor antimycin (10 microM)+succinate (5 mM) had any significant effect on basal CL levels, thereby excluding xanthine oxidase and mitochondria, respectively, as a detectable sources of O2-. generation. The presence of NAD+ (100 microM) and lactate (1 mM) increased CL by 88% (n = 8, P < 0.01). In the intact cells, basal production of CL was increased by 205% (P < 0.01) by 5 mM lactate, but not by 5 mM pyruvate, and CL was inhibited by 10 mM Tiron, suggesting the reduction of cytosolic NAD by lactate dehydrogenase stimulates O2-. production. Diphenyliodonium at 1 and 10 microM inhibited both NADH-mediated CL in homogenate and lactate-mediated CL in intact endothelium by 50 and 33%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the new A2 adenosine receptor antagonist 8FB-PTP, an 8 substituted pyrazolo-triazolo-pyrimidine, on in vitro functional models.

1. We have characterized the in vitro pharmacological profile of putative A2 adenosine antagonists, two non-xanthine compounds, 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (8FB-PTP) and 5-amino-9-chloro-2-(2-furyl 1,2,4-triazolo [1,5-c] quinazoline (CGS 15943), and the xanthine derivative (E)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropyl- xanthine (KF 17837). 2. In binding studies on bovine brain, 8FB-PTP was the most potent (Ki = 0.074 nM) and selective (28 fold) drug on A2 receptors, whereas CGS 15943 and KF 17837 exhibited affinity in the low and high nanomolar range, respectively, and showed little selectivity. 3. In functional studies, 8FB-PTP antagonized 5'-N-ethyl-carboxamidoadenosine (NECA)-induced vasorelaxation of bovine coronary artery (pA2 = 7.98) and NECA-induced inhibition of rabbit platelet aggregation (pA2 = 8.20). CGS 15943 showed weak activity in the platelet aggregation model (pA2 = 7.43) and failed to antagonize NECA-induced vasodilatation. KF 17837 was ineffective in both models up to micromolar concentrations. 4. Antagonism of A1-mediated responses was tested versus 2-chloro-N6-cyclopentyladenosine (CCPA) in rat atria. 8FB-PTP and CGS 15943 also antagonized competitively the negative chronotropic response induced by CCPA. Conversely, KF 17837 was unable to reverse A1-mediated responses. 5. 8FB-PTP is a potent and competitive antagonist of responses mediated by A2 adenosine receptors. The data provided a basis to reduce, by further chemical modifications, the affinity at A1 receptor and therefore enhance A2 receptor selectivity.

Expression and subcellular distribution of basic fibroblast growth factor are regulated during migration of endothelial cells.

Migration of endothelial cells is involved in normal and pathological angiogenesis and in re-endothelialization after vascular injury or rupture of atherosclerotic plaques. Several types of endothelial cells are known to synthesize basic fibroblast growth factor (bFGF); in some of these, migration is increased by exogenous bFGF and inhibited by anti-bFGF antibodies. Using immunocytochemical techniques and RNase protection analysis, we studied endothelial cells from bovine coronary arteries and veins as well as from adrenal microvessels. We found that bFGF mRNA and peptide were present in confluent endothelial cells and were upregulated during migration stimulated by removal of some cells from the monolayer. During migration, extracellular matrix stores of bFGF were depleted, and bFGF immunoreactivity began to accumulate in the cytoplasm of endothelial cells between 2 and 6 hours. After migration had begun, but before the initiation of DNA synthesis, bFGF immunoreactivity increased in the nuclei and nucleoli. Exogenous bFGF stimulated endothelial migration, and antibodies to bFGF markedly inhibited migration, suggesting that an intracrine function of nuclear bFGF is not sufficient for cell migration. In all three types of endothelial cells studied, bFGF was identified as an endogenous regulator, but not as the sole regulator, or migration. Moreover, bFGF expression and subcellular localization were found to be regulated during endothelial cell migration.

S-nitrosothiol modulation of tolerance to glyceryl trinitrate in bovine isolated coronary artery

The aim of the study was to ascertain whether induction of tolerance to glyceryl trinitrate (GTN) was modified by nitrosothiols. Vasodilator responses to GTN and to two nitrosothiols, namely S-nitroso- N-acetylpenicillamine (SNAP) and S-nitroso- N-acetylcaptopril (SNOCAP) were measured in bovine coronary artery rings constricted with the thromboxane mimetic agent U46619. Pre-exposure of vessels to SNAP (10 μM) for 1 h resulted in cross-tolerance to GTN comparable with tolerance induced by GTN (1.0 μM). Tolerance was increased after pre-exposure to both agents. SNAP alone in lower concentrations (0.01, 0.1 and 1.0 μM) did not produce cross tolerance to GTN, but in combination with GTN (1.0 μM) caused a small but significant decrease in tolerance to GTN. This attenuating effect of SNAP on GTN tolerance was reproduced by SNOCAP (0.01 μM). We conclude that the nitrosothiols exert a biphasic effect on induction of tolerance to GTN.

Tumor Necrosis Factor Inhibits Stimulated but Not Basal Release of Nitric Oxide

Tumor necrosis factor alpha (TNF alpha) increases nitric oxide (NO) synthase in vascular endothelium, but it inhibits endothelium-dependent relaxation (EDR) of vascular smooth muscle. We tested whether TNF alpha inhibits the response to, or release of, NO in bovine pulmonary artery (BPA) using the technique of perfusion-superfusion bioassay and ozone chemiluminescence. Effluent from the perfused BPA with endothelium (donor)-relaxed endothelium-rubbed bovine coronary artery (BCA) (detector). Moreover, effluent from the donor stimulated with acetylcholine (ACh) or bradykinin (BK) (0.001 to 100 nmol) relaxed the detector. Direct application of these agonists to the detector failed to produce relaxation. Basal and agonist-stimulated effluent from the donor treated with L-NG-monomethylarginine (LNMMA) (100 microM) suppressed effluent-mediated relaxation of the detector. ACh and BK released LNMMA-inhibitable nitrite and nitrate from the BPA. Thus, the effluent contained NO. Exposure of the donor to TNF alpha (1.25 micrograms/ml) for 60 min did not affect basal release of NO, but it attenuated bioassayable and chemiluminescence-detectable NO release by ACh and BK. The inhibition of NO release was directly related to the magnitude of inhibition of EDR by ACh and BK. Thus, TNF alpha selectively inhibits receptor-mediated release of NO without affecting basal release of NO. This effect differs from that of L-arginine-based inhibitors of NO and represents a unique physiologic mechanism of regulation of NO in the endothelium.

Effects of selective A1 and A2 adenosine receptor agonists on cardiovascular tissues

We investigated the negative chronotropic and vasodilating properties of new selective A1 and A2 adenosine agonists such as 2-chloro-N6-cyclopentyladenosine (CCPA) and 2-hexynyl-5'-N-ethyl-carboxamidoadenosine (2-hexynyl-NECA) as compared with reference adenosine analogues. The potency of these compounds on heart rate was assessed in the rat atrial preparation and their activity on the vascular tone was determined in both rat aorta and bovine coronary artery. CCPA was found to be the most potent A1 agonist of those currently available in producing negative chronotropic effects (EC50 = 8.2 nM). The A1 antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) blocked CCPA activity in a dose-dependent manner. There was also a significant correlation between its biological effect and the affinity for A1 receptors as measured in the rat brain by [3H]-N6-cyclohexyladenosine (3[H]-CHA) binding. The A2 selective agonist 2-hexynyl-NECA showed vasodilating properties comparable with those observed with the reference compounds, CGS 21680 and NECA. EC50 values were 596 and 569 nM in rat aorta and bovine coronary artery, respectively. Moreover, the rank order of potency was similar in the two vascular districts examined, suggesting that the rat aorta is a useful model for studying the effects of adenosine derivatives on vascular tone. In addition, the potency of the compounds in inducing vasodilation was found to be correlated with their affinity for A2 receptors as measured in the rat striatum by 3[H]-CGS 21680 binding. These data further support that A1 receptors are involved in depressing cardiac activity and A2 receptors in inducing vasorelaxation.

Role of PGI2 and epoxyeicosatrienoic acids in relaxation of bovine coronary arteries to arachidonic acid.

Metabolites of arachidonic acid regulate several physiological processes, including vascular tone. The purpose of this study was to determine which metabolites of arachidonic acid are produced by bovine coronary arteries and which may regulate coronary vascular tone. Arachidonic acid induced a concentration-related, endothelium-dependent relaxation [one-half maximum effective concentration (EC50) of 2 x 10(-7) M and a maximal relaxation of 91 +/- 2% at 10(-5) M] of bovine coronary arteries that were contracted with U-46619, a thromboxane mimetic. The concentration of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), a metabolite of prostaglandin I2 (PGI2), increased from 82 +/- 6 to 328 +/- 24 pg/ml with arachidonic acid (10(-5) M). Treatment with the cyclooxygenase inhibitor indomethacin attenuated arachidonic acid-induced relaxations by approximately 50% and blocked the synthesis of 6-keto-PGF1 alpha. PGI2 caused a concentration-related relaxation (EC50 of 10(-8) M and a maximal relaxation of 125 +/- 11% at 10(-7) M). BW755C, a cyclooxygenase and lipoxygenase inhibitor, inhibited arachidonic acid-induced relaxation to the same extent as indomethacin. When vessels were treated with both indomethacin and BW755C, the inhibition of relaxation was the same as either inhibitor alone. SKF 525a, a cytochrome P-450 inhibitor, reduced arachidonic acid-induced relaxation by approximately 50%. When SKF 525a was given in combination with indomethacin, the relaxation by arachidonic acid was almost completely inhibited. SKF 525a inhibited the synthesis of epoxyeicosatrienoic acids (EETs).(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of Basic Fibroblast Growth Factor in Bovine Endothelial Cells: Immunohistochemical and Biochemical Studies

To gain insight into the mechanisms of synthesis, storage, and release of basic fibroblast growth factor (bFGF), we studied the immunohistochemical localization of bFGF in bovine coronary artery, coronary sinus, and adrenal capillary endothelial cells grown in culture. Light and electron microscopic immunohistochemical studies were performed using the ABC immunoperoxidase method on p-formaldehyde-fixed cells. Five different anti-bFGF antibodies gave similar results in all cell types. In subconfluent cells, immunoreactivity was noted in the nuclear chromatin, nucleoli, cytosol, cytoplasmic vesicles (some of which appeared to fuse with the plasma membrane), and extracellular matrix. No reaction was found in endoplasmic reticulum or Golgi zones. Confluent cells demonstrated less immunoreactivity in the nuclei and cytosol but more in the extracellular matrix. Some cells of senescent morphology showed only cytoplasmic staining; however, no cells were found with only nuclear staining. Biochemical studies showed that three forms of bFGF (18, 24, and 26 kDa) were present in endothelial cells and varied with different culture conditions. Protection analysis indicated that bFGF mRNA is less abundant in postconfluent cells than in subconfluent cells. These data suggest that subconfluent cells synthesize bFGF and transport it into the nucleus and exocytotic vesicles, while confluent cells synthesize little bFGF but store it in extracellular matrix, cytoplasmic vesicles, and nuclei.