General Pharmacology-the Vascular System最新文献

The antithrombotic and thrombolytic activities of Agkisacutacin (Agk), a component isolated from Agkistrodon acutus, were determined in vitro and in vivo. The models employed included Chandler's model, arterio-venous shunt model and pulmonary embolus model. The effects of Agkisacutacin on coagulation, plasma fibrinogen and platelet aggregation induced by collagen, adenosine diphosphate (ADP) and thrombin were also investigated. The results showed that Agkisacutacin can significantly inhibit thrombus formation in Chandler's and arterio-venous shunt models, and accelerate thrombolysis of pulmonary emboli in rats. The data suggested that Agkisacutacin possessed antithrombotic and thrombolytic activities. Agkisacutacin was also partial characterized.

Angiotensin converting enzyme (ACE, kininase II) is an endothelial luminal ectoenzyme expressed abundantly on the pulmonary capillary endothelium and recognized as the site for the conversion of circulating angiotensin I to II. In the present study, we have applied recently developed methodologies for assaying pulmonary capillary endothelium-bound (PCEB) ACE activity in man, to estimate the interaction of an ACE inhibitor (enalaprilat) with PCEB ACE in human subjects. Trace amounts of the specific ACE substrate, ³H-benzoyl-Phe-Ala-Pro (³H-BPAP; 40 Ci or 2 nmol), was injected as a bolus into the subclavian vein and immediately blood was withdrawn from a radial arterial catheter. Plasma concentrations of surviving substrate and product (³H-benzoyl-Phe) were estimated and BPAP utilization was calculated during a single transpulmonary passage, at baseline (T₀) and at 15 min (T₁₅) and 2 h (T₁₂₀) after intravenous administration of 1.5 g/kg enalaprilat in 12 normotensive subjects. This treatment had no significant effect on mean arterial pressure (91±6 vs. 84±7 vs. 88±6 mm Hg for T₀, T₁₅ and T₁₂₀, respectively), but significantly decreased serum and PCEB ACE activities. When normalized to predrug (T₀) activity levels, enalaprilat inhibited PCEB and serum ACE activities at T₁₅ 74±6% and 68±6%, respectively. However, 2 h after enalaprilat (T₁₂₀), PCEB ACE inhibition was maintained at 66±7%, whereas serum ACE inhibition was reduced to 46±8% (P<.01 from PCEB ACE), suggesting a preferential PCEB ACE inhibitory effect of enalaprilat.

The influence of pretreatment with 1 through 300 mg/kg ip of isoniazid (ISO) on blood pressure and heart rate responses to 0.1 mg/kg iv of hydralazine (HYD) was assessed in rats anesthetized with chloralose–urethane. HYD hypotension was significantly enhanced by ISO at doses between 3 and 300 mg/kg ip. Heart rate was not influenced by HYD in control or pretreated animals. Depressor responses to 0.2 mg/kg iv of pinacidil (PIN) were also potentiated by ISO at 100 and 300, but not at 30 mg/kg. Similarly, ISO decreased cerebral γ-aminobutyric acid (GABA) at the two highest doses; 30 mg/kg was without effect. Pretreatment of rats with ISO at 1 through 300 mg/kg failed to influence HYD-induced relaxation of aortic rings. These results were interpreted as indicating that potentiation of HYD hypotension by high doses of ISO is not specific for that vasodilator and is related to decreased cerebral GABA, as postulated previously. Lower doses could specifically potentiate the HYD-induced hypotensive effect by inhibition of semicarbazide-sensitive amine oxidase (SSAO), since both ISO and HYD are potent inhibitors of this enzyme. In support of this hypothesis, the SSAO inhibitors, benserazide (100 mg/kg ip) and mexiletine (50 mg/kg ip), were also found to enhance HYD hypotension.

We examined the role of endothelium-derived relaxing factors nitric oxide (NO), endothelium-derived hyperpolarising factor (EDHF), and prostaglandins (PGs) to P_2Y1- and P_2Y2-purinoceptor-induced vasodilation in isolated rat kidney. To do it, we analysed the renal response to ATP, 2-methylthio ATP, and UTP in rat renal vasculature under normal conditions and after the administration of: N^w-nitro-l-arginine (l-NAME), increased K⁺ concentration, indomethacin, and l-NAME and increased K⁺ together. Our results indicate that the vasodilator response to P_2Y1- and P_2Y2-purinoceptor activation in the isolated perfused kidney of rats is subserved by EDHF and NO.

The present study further examined the functional presence and the signal transduction mechanism(s) for adenosine A_2A and A_2B receptors through nitric oxide (NO) and the guanosine 3′, 5′-cyclic monophosphate (cGMP) pathway in cultured porcine coronary artery endothelial cells (PCAEC). The application of adenosine receptor agonists, NECA, CGS-21680 and CAD between 10⁻⁷ and 10⁻⁴ M, enhanced the production of NO (measured as nitrite) in a dose-dependent manner. On the basis of EC₅₀ values, these agonists showed the following order of potency: NECA>CGS-21680>CAD. This order appears to be of the A₂ adenosine receptor subtype. Similarly, the same concentrations of adenosine agonists evoked the production of cGMP in a dose-dependent manner, exhibiting a rank order that is similar to that of NO production. NO synthase inhibitor, N-nitro-l-arginine methylester (l-NAME, 10⁻⁵ M), inhibited the production of NO and cGMP, which was reversed by l-arginine (10⁻⁴ M). Selective A_2A adenosine receptor antagonists, ZM-241385 and SCH-58261, at 10⁻⁷ M, significantly inhibited the effects of CGS-21680, but only partly inhibited the effect of NECA on NO and cGMP production. Along with the earlier molecular evidence from this laboratory [Am. J. Physiol. 279 (2000) H650], the present data further support the presence of both A_2A and A_2B receptors in PCAEC. These results further support that coronary endothelial cells express functional A_2A and A_2B adenosine receptors, leading to GMP production through the NO-synthase-linked mechanism. This is the first direct evidence where an A_2B adenosine receptor has been linked to NO production in cultured endothelial cells and could play a role in coronary artery physiology and pathophysiology.

Experiments were undertaken to determine if nitric oxide (NO) plays a role in regulation of basal blood flow in the oral cavity of pentobarbital anesthetized cats and, if so, to quantify this effect using dose–response relationships. Blood flow was continuously measured from the surface of the tongue and mandibular gingiva (laser-Doppler flowmetry) and from the lingual artery (ultrasonic flowmetry). Cardiovascular parameters also were recorded. Administration of the nonselective inhibitor of nitric oxide synthase (NOS), l-NAME (0.08–20 mg/kg iv), produced a dose-related increase of blood pressure associated with decreases of blood flow at all three measurement sites. Maximal blood flow depression of 50–60% was seen 30–60 min after administration of 1.25 mg/kg of l-NAME. d-NAME (1.25 mg/kg iv) was inactive at all sites. Subsequent administration of l-arginine partially reversed effects of l-NAME in the lingual artery and tongue, but not in the gingival circulation. The neuronally selective NOS inhibitor, 7-nitroindazole (7-NI, 30 mg/kg ip), was devoid of effect on any of the measured parameters. These results suggest that endothelial (but not neuronally derived) NO plays an important role in control of basal blood flow in oral tissues of the cat.

This study sought to determine whether vascular endothelial growth factor (VEGF)-induced permeabilisation of pulmonary endothelium to macromolecules could be related to a permissive role for neutrophil-derived VEGF in neutrophil transmigration. Treatment of human pulmonary artery endothelial cell (HPAEC) monolayers with 1, 10 or 100 ng/ml VEGF for 15 min or 1, 10 ng/ml for 90 min significantly increased endothelial permeability to trypan blue-labelled albumin (TB-BSA). These increases were correlated with changes in the cellular distribution of F-actin, as visualised by rhodamine–phalloidin staining: increased stress fibre formation, cellular elongation and formation of intercellular gaps after 15 min; at 90 min, there was also evidence of microspike formation and extension of spindle processes from the cell surface. Treatment of human neutrophil suspensions with 200 nM phorbol myristyl acetate (PMA), n-formyl-methionyl leucylphenylalanine (fMLP, 10 nM), interleukin-8 (IL-8, 10 nM) (but not with leukotriene B₄ (LTB₄) 100 nM), for 30 min caused significant extracellular release of neutrophil VEGF stores. A permissive role for neutrophil-derived VEGF in facilitating migration across HPAEC monolayers was assessed in experiments using a functional blocking antihuman VEGF antibody. In the presence of this antibody (10 μg/ml), neutrophil migration in response to fMLP (10 nM), IL-8 (10 nM) or LTB₄ (100 nM) was not significantly different to that in the absence of antibody. We conclude that neutrophil-derived VEGF does not play a functional role in facilitating neutrophil migration across pulmonary vascular endothelium, despite its ability to induce cytoskeletal changes and enhance endothelial macromolecular permeability.

The change of blood pressure and heart rate after intravenous injection of Korea red ginseng (KRG) were studied in the conscious normotensive and one-kidney, one-clip Goldblatt hypertensive (1K, 1C-GBH) rats. Crude saponin (CS) of KRG (50, 100 mg/kg iv) induced a hypotensive effect and bradycardia in a dose-dependent manner in the anesthetized rats. On the other hand, CS of KRG (100 mg/kg) induced a hypotensive effect and reflex tachycardia in the conscious rats. Saponin-free fraction (SFF) of KRG did not affect them in the anesthetized normotensive rats (P>.05). The maximal hypotensive effect by CS of KRG in the conscious 1K, 1C-GBH hypertensive rats and l-nitroarginine methyl ester (l-NAME, 40 mg/kg)-treated conscious hypertensive rats was not different from that of conscious normotensive rats (Δ31.6±6.3, Δ27.5±5.8 vs. Δ26.7±4.3 mmHg, P>.05). However, pretreatment of l-NAME significantly inhibited the reflex tachycardia by CS of KRG (70.8±7.0 vs. 30.6±15.0 bpm, P<.05). Hemolysate-sensitive nitric oxide (NO) current by the CS of KRG was greater than that of the SFF of KRG (651.9±128.2 pA for CS and 164.9±92.5 pA for SFF, P<.001). These findings suggest that KRG has a hypotensive effect and its effect may be due to saponin fraction of KRG in the conscious rats. The releasing effect of NO of KRG, like NO donor, may be partly contributed to the hypotensive effect of KRG.

Previously we described ENAP-1, a 90-kDa protein that is tyrosine-phosphorylated in endothelial cells in response to bradykinin (BK) stimulation and is associated with endothelial nitric oxide synthase (eNOS). Subsequently, other investigators demonstrated that eNOS interacts with heat shock protein 90 (Hsp90) following stimulation of endothelial cells with vascular endothelial growth factor (VEGF), histamine, or fluid shear stress. Therefore, we tested the hypotheses that ENAP-1 and Hsp90 are the same protein and that BK activation of eNOS is dependent on Hsp90. Immunoblotting of immunoprecipitated Hsp90 with anti-phosphotyrosine antibody shows that Hsp90 is tyrosine-phosphorylated in response to BK stimulation of bovine aortic endothelial cells (BAECs). Coimmunoprecipitation of Hsp90 with anti-eNOS antibody reveals a Hsp90–eNOS complex in endothelial cells under basal conditions that is increased following BK stimulation. Taken together with the tyrosine phosphorylation data, these data suggest that ENAP-1 is Hsp90. BK-stimulated nitric oxide (NO) release is completely blocked by pretreatment with geldanamycin, a specific inhibitor of Hsp90, illustrating the importance of the Hsp90–eNOS interaction. In vitro binding assays with Hsp90–glutathione-S-transferase fusion proteins show direct binding of eNOS with the middle domain (residues 259–615) of Hsp90.

The aim of this work was to investigate the influence of endothelin on myocardial ischemia and reperfusion in anaesthetized dogs. Animals were submitted to left thoracotomy and 120 min of left anterior descending coronary occlusion, followed by 180 min of reperfusion. Arterial blood pressure and electrocardiogram (ECG) were recorded in order to analyze heart rate (HR)–pressure product and production of ectopic beats. Infarcted areas were identified by a macroscopic staining method and infarct size was expressed as percentage of risk zone. To inhibit the effects of endothelin in a group of animals, we administered intravenously an endothelin synthesis inhibitor (phosphoramidon) and in another group, an endothelin-1 A receptor blocker (BQ-123). Phosphoramidon decreased the HR–pressure product during reperfusion period, and both, phosphoramidon and BQ-123 decreased infarct size by 40% and the number of ventricular ectopic beats by 88% and 68%, respectively, as compared to the saline treated dogs. In conclusion, endothelin seems to play a deleterious role on the myocardium submitted to ischemia and reperfusion.