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: L-Arginine (L-arg), widely known as a substrate for endogenous nitric oxide synthesis, can improve endothelial function associated with the vasculature, inhibit platelet aggregation, and alter the activity of vascular smooth muscle cells. P-selectin is a membrane component of the platelet alpha-granule and the endothelial cell-specific Wiebel-Palade body that plays a central role in mediating interactions between platelets and both leukocytes and the endothelium. The experiment was designed to evaluate the effect of novel microspheres with L-arg targeting P-selectin on the formation of deep vein thrombosis and repair of vascular wall in a rat model. Thrombosis of the inferior vena cava was induced by applying a piece of filter paper (5 mm × 10 mm) saturated with 10% FeCl3 solution for 5 min. Targeted microspheres with L-arg, targeted microspheres with water, and saline were injected into the tail veins of the rats after 30 min of applying the filter paper saturated with 10% FeCl3 solution. The dry weight and length of the thrombus isolated from the inferior vena cava were significantly decreased in the group with L-arg in microsphere after 24 h. No significant differences in prothrombin time, activated partial thromboplastin time, thrombin time, and fibrinogen among the groups were indicated. Images revealed that apoptosis in the vascular wall was less in the group injected with targeted microspheres with L-arg than in the other two groups at 1 and 8 d postsurgery. Meanwhile, cell proliferation was considerably excessive in the group injected with L-arg wrapped in targeted microspheres. Therefore, these novel microspheres could decrease the formation of thrombus in the early stages and in the subsequent periods of thrombosis. The microspheres can also enhance the vitality of impaired endothelial cells and reduce cell apoptosis.

Objective: Venous function is underappreciated in its role in blood pressure determination, a physiologic parameter normally ascribed to changes in arterial function. Significant evidence points to the hormone endothelin-1 (ET-1) as being important to venous contributions to blood pressure. We hypothesized that the artery and vein should similarly depend on the signaling pathways stimulated by ET-1, specifically phospholipase C (PLC) activation. This produces two functional arms of signaling: diacylglycerol (DAG; protein kinase C [PKC] activation) and inositol trisphosphate (IP3) production (intracellular calcium release)., Methods: The model was the male Sprague-Dawley rat. Isolated tissue baths were used to measure isometric contraction. Western blot and immunocytochemical analyses measured the magnitude of expression and site of expression, respectively, of IP3 receptors in smooth muscle/tissue. Pharmacologic methods were used to modify PLC activity and signaling elements downstream of PLC (IP3 receptors, PKC)., Results: ET-1-induced contraction was PLC dependent in both tissues as the PLC inhibitor U-73122 significantly reduced contraction in aorta (86% ± 4% of control; P < .05) and vena cava (49% ± 11% of control; P < .05). However, ET-1-induced contraction was not significantly inhibited by the IP3 receptor inhibitor 2-aminoethoxydiphenylborane (100 μM) in vena cava (82% ± 8% of control; P = .23) but was in the aorta (55% ± 4% of control; P < .05). All three IP3 receptor isoforms were located in venous smooth muscle. IP3 receptors were functional in both tissues as the novel membrane-permeable IP3 analogue (Bt-IP3; 10 μM) contracted aorta and vena cava. Similarly, whereas the PKC inhibitor chelerythrine (10 μM) attenuated ET-1-induced contraction in vena cava and aorta (5% ± 2% and 50% ± 5% of control, respectively; P < .05), only the vena cava contracted to the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol., Conclusions: These findings suggest that ET-1 activates PLC in aorta and vena cava, but vena cava contraction to ET-1 may be largely IP3 independent. Rather, DAG—not IP3—may contribute to contraction to ET-1 in vena cava, in part by activation of PKC. These studies outline a fundamental difference between venous and arterial smooth muscle and further reinforce a heterogeneity of vascular smooth muscle function that could be taken advantage of for therapeutic development., (Copyright © 2015 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

The Na(+)/Ca(2+) exchanger (NCX) is a bi-directional regulator of cytosolic Ca(2+), causing Ca(2+) efflux in forward-mode and Ca(2+) influx in reverse-mode. We hypothesized that reverse-mode NCX is a means of Ca(2+) entry in rat aorta (RA) and vena cava (RVC). NCX protein in RA and RVC was confirmed by immunoprecipitation. To assess NCX function, isometric contraction and intracellular Ca(2+) was measured in RA and RVC rings in response to low extracellular Na(+), endothelin-1 (ET-1), and KCl, in the presence or absence of the NCX antagonist KB-R7943. In RVC, low extracellular Na(+) caused vasoconstriction and an increase in intracellular Ca(2+) that was attenuated by 10μM KB-R7943. KB-R7943 (10 μM) attenuated maximal contraction to ET-1 in RVC (53 ± 9% of control), but not RA (91±1% of control). KB-R7943 (10 μM) reduced the maximal contraction to KCl in RA (48 ± 5%) and nearly abolished it in RVC (9 ± 2%), suggesting that voltage-dependent Ca(2+) influx may be inhibited by KB-R7943 as well. However, the L-type Ca(2+) channel inhibitor nifedipine (1 μM) did not alter ET-1-induced contraction. Our findings suggest that reverse-mode NCX is an important mechanism of Ca(2+) influx in RVC but not RA, especially during ET-1-induced contraction. Also, the effects of KB-R7943 on ET-1-induced contraction of RA and RVC are predominantly mediated by reverse-mode NCX inhibition and not due to off-target inhibition of Ca(2+) channels., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: Arterial pressure induced vein graft injury can result in endothelial loss, accelerated atherosclerosis and vein graft failure. Inflammation, including complement activation, is assumed to play a pivotal role herein. Here, we analyzed the effects of C1-esterase inhibitor (C1inh) on early vein graft remodeling., Methods: Human saphenous vein graft segments (n=8) were perfused in vitro with autologous blood either supplemented or not with purified human C1inh at arterial pressure for 6h. The vein segments and perfusion blood were analyzed for cell damage and complement activation. In addition, the effect of purified C1inh on vein graft remodeling was analyzed in vivo in atherosclerotic C57Bl6/ApoE3 Leiden mice, wherein donor caval veins were interpositioned in the common carotid artery., Results: Application of C1inh in the in vitro perfusion model resulted in significantly higher blood levels and significantly more depositions of C1inh in the vein wall. This coincided with a significant reduction in endothelial loss and deposition of C3d and C4d in the vein wall, especially in the circular layer, compared to vein segments perfused without supplemented C1inh. Administration of purified C1inh significantly inhibited vein graft intimal thickening in vivo in atherosclerotic C57Bl6/ApoE3 Leiden mice, wherein donor caval veins were interpositioned in the common carotid artery., Conclusion: C1inh significantly protects against early vein graft remodeling, including loss of endothelium and intimal thickening. These data suggest that it may be worth considering its use in patients undergoing coronary artery bypass grafting., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Studies by electromagnetic flowmetry in acute experiments on cats under conditions of the open thoracic cage and artificial ventilation of the lungs showed that 64% of venous return via the vena cava posterior was realized at the expense of the splanchnic and 36% due to the musculocutaneous vessels (abdominal basin of the caudal vein). Epinephrine (20 μg/kg) increased the contribution of the splanchnic venous blood flow to the increase in the blood flow in the vena cava posterior and reduced the contribution of the musculocutaneous veins throughout the entire duration of systemic reactions: 84% of the blood flow increase in the vena cava posterior was due to the splanchnic and just 16% due to the musculocutaneous blood flow. Norepinephrine (10 μg/kg) resulted in a phase-wise involvement of the studied compartments in blood flow increase in the vena cava posterior. During the initial period of systemic reactions (coinciding with the maximum systemic BP rise) the contribution of the musculocutaneous compartment was 13% higher, while later (by the time of the maximum elevation of venous blood flow in the studied compartments) the contribution of splanchnic veins predominated constituting 89% of venous blood flow in the vena cava posterior. These results indicate that venous blood flow increase in the splanchnic vessels largely determined the formation of changes in the vena cava posterior blood flow in response to catecholamines.

Objective: Use of autologous vein grafts for surgical revascularisation is limited by vein graft failure. Topical high-density lipoprotein (HDL) administration on the adventitial side of vein grafts was evaluated as a new therapeutic modality to improve vein graft patency and function., Methods: Caval veins of C57BL/6 apo E(-/-) mice were grafted to the right carotid arteries of recipient 3 month-old C57BL/6 TIE2-LacZ/apo E(-/-) mice. HDL (200 μg/ml; 50 μl) in 20% pluronic F-127 gel was applied on the adventitial side of vein grafts., Results: Topical HDL application reduced intimal area by 55% (p < 0.001) at day 28 compared to control mice. Blood flow quantified by micro magnetic resonance imaging at day 28 was 2.8-fold (p < 0.0001) higher in grafts of topical HDL treated mice than in control mice. Topical HDL potently reduced intimal inflammation and resulted in enhanced endothelial regeneration as evidenced by a 1.9-fold (p < 0.05) increase in the number of CD31 positive endothelial cells. HDL potently enhanced migration and adhesion of endothelial colony-forming cells (ECFCs) in vitro, and these effects were dependent on signaling via scavenger receptor-BI, extracellular signal-regulated kinases, and NO, and on increased β1 integrin expression. Correspondingly, the number of CD31 β-galactosidase double positive cells, reflecting incorporated circulating progenitor cells, was 3.9-fold (p < 0.01) higher in grafts of HDL treated mice than in control grafts., Conclusions: Topical HDL administration on the adventitial side of vein grafts attenuates vein graft atherosclerosis via increased incorporation of circulating progenitor cells in the endothelium, enhanced endothelial regeneration, and reduced intimal inflammation., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

We showed here that energy metabolism in thoracic rabbit aorta and posterior vena cava is disturbed two weeks following the induction of alloxan-induced diabetes and monoiodacetate intoxication. In the vessels studied, the alterations are manifested in the decrease in the intensity of glucose uptake, oxygen consumption, lactic acid production, and ATP resynthesis. Simultaneously, the ATP content is significantly reduced. The possible significance of these disorders in the development of Monckeberg's arteriosclerosis is discussed.

Physical exercise evokes an extensive circulatory redistribution. However, the influence of exercise upon the effects of sympathomimetic agonists in veins was not well studied. Thus, the present study aimed to determine whether a single bout of exercise modifies the effects of sympathomimetic agonists in veins and whether this exercise-induced modification may be altered by exercise training. The results have shown that the training did not change the responsiveness of the rat portal vein, but exposure of trained animals to a single bout of exercise enhanced the phenylephrine Rmax in these preparations. Such exercise-induced modifications of vascular response were territory-specific since similar modifications of response to phenylephrine were not observed in vena cava. Moreover, this exercise-induced augmentation of phenylephrine Rmax in the portal vein of trained rats was prevented by endothelium removal or in the presence of N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME), indomethacin, BQ-123 or BQ-788. In conclusion, these data indicate that the training adapted the rat portal vein to respond vigorously to sympathetic stimuli even when the animal is exposed to this exercise. This increased response to sympathetic stimuli appears to involve an enhancement of the vasocontractile influence of endothelin that supplants the modulation exerted by nitric oxide (NO) and vasodilator prostanoids.

Endothelin receptors (ET(A) and ET(B)) mediate responses to ET-1. ET(B) receptor function seems to differ between a similarly sized arterial and venous pair, the rat vena cava (RVC) and rat thoracic aorta (RA). ET(B) receptors mediate RVC contraction directly, but it is unclear whether ET(B) receptors mediate contraction in RA. Because of these apparent differences in ET(B) receptor-mediated vascular contraction, we hypothesize that relaxant ET(B)-receptor mechanisms in RVC would be different from those in RA. RA and RVC rings were isolated from rats for measurement of isometric contraction. When contracted with prostaglandin F-2alpha (PGF-2alpha) (20 microM), the ET(B) receptor agonist sarafotoxin-6c (S6c) (100 nM) significantly relaxed RA and RVC. N(omega)-Nitro-L-arginine (LNNA) (100 microM) or endothelial denudation abolished relaxation to S6c in RA. By contrast, S6c-induced relaxation of RVC was attenuated but not abolished by LNNA and endothelial denudation. RVC (PGF-2alpha-contracted) relaxed to low concentrations of ET-1, whereas under the same conditions RA responded with contraction. ET-1-induced relaxation in RA was observed only with ET(A) receptor blockade. Vessels from dopamine-beta-hydroxylase-ET(B) transgenic rats, which lack functional ET(B) receptors in the vasculature, were also used. RVC (PGF-2alpha-contracted) from these rats did not relax to ET-1. Thus, although both RA and RVC possess endothelial relaxant ET(B) receptors, RA and RVC differ in that relaxant ET(B) receptors may also be present in smooth muscle of RVC. Moreover, the mechanisms of endothelial cell ET(B) receptor-mediated relaxation in RA and RVC are not the same.

Background: Bone marrow-derived cells are recruited into the thrombus during resolution. This study explored whether mobilization of bone marrow cells with recombinant human granulocyte colony-stimulating factor (rhG-CSF) could enhance the resolution of venous thrombi and the accumulation of macrophages in thrombi and explored the effect of rhG-CSF on cysteine-cysteine chemokine receptor 2 (CCR2) expression., Methods: The Sprague-Dawley adult rats were randomly divided into four groups: control, sham-operated, thrombus, and treatment groups. Thrombi were induced in the thrombus and treatment group, which received a subcutaneous injection of rhG-CSF once daily for 6 days postoperatively. The thrombus, sham-operated, and control groups received equal volumes of 0.9% saline. The mononuclear cells in peripheral blood were analyzed by an automated hematology analyzer and counted under microscope. The cell marker CD68 was used to determine the number of macrophages in thrombi tissue sections. Levels of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP1alpha) in the peripheral blood were detected by enzyme-linked immunosorbent assay. Real-time reverse transcriptase-polymerase chain reaction and Western blot were used to analyze, respectively, the expression of CCR2 messenger RNA in the peripheral blood and CCR2 protein of THP-1 monocyte., Results: At postoperative days 3 (P < .05) and 7 (P < .01), mononuclear cells significantly increased in treatment group (2.1 +/- 0.3, 4.4 +/- 0.3 x 10(6)/L) vs the thrombus group (1.7 +/- 0.2, 1.3 +/- 0.4 x 10(6)/L). The organization and recanalization of thrombi in treatment group progressed more quickly compared with the thrombus group (P < .01). The macrophage number of the thrombus in the treatment group (338 +/- 26 cells/15 high-power fields) increased significantly vs the thrombus group (125 +/- 11 cells/15 high-power fields, P < .01). No statistical difference was observed between the thrombus and treatment group in the MCP-1 and MIP-1alpha level in peripheral blood. Expressions of the CCR2 gene in the peripheral blood of the treatment group significantly increased compared with the thrombus group (P < .05). Recombinant human G-CSF induced higher expression of CCR2 protein of human monocytic cell line THP-1., Conclusions: Bone marrow mobilization enhanced the resolution and recanalization of venous thrombi. This process was associated with increased macrophage accumulation in thrombi, which might be the result of higher CCR2 expression of monocytes., Clinical Relevance: The classic treatment of venous thrombi is anticoagulation. Anticoagulant therapy and thrombolysis both have limited effects on existing thrombi and have a small but significant risk of severe hemorrhage. In clinical practice, we lack specific treatment for patients with venous thrombosis combined with brain hemorrhage or a gastrointestinal activated ulcer, which are contraindicated for anticoagulation and thrombolytic therapy. Enhancing the resolution of venous thrombi would contribute to its therapy. Bone marrow-derived cells are recruited into the thrombus during resolution. Many of these cells express a macrophage phenotype and may represent a population of plastic stem cells that orchestrate thrombus recanalization. Recombinant human granulocyte colony stimulating factor (rhG-CSF) can mobilize monocytic lineage cells into peripheral blood and may contribute to this cell in the thrombi. If rhG-CSF enhances the resolution of venous thrombi and recanalization, it might be used to treat patients with venous thrombi, especially those who have contraindication for anticoagulation and thrombolytic therapy.

Arterial remodeling occurs in response to mechanical and neurohumoral stimuli. We hypothesized that veins, which are not exposed to higher pressures in hypertension, would demonstrate less active remodeling than arteries. We assessed remodeling with two standard measures of arterial remodeling: vessel morphometry and the expression/function of matrix metalloproteinases (MMPs). Thoracic aorta and vena cava from sham normotensive and DOCA-salt hypertensive rats (110 +/- 4 and 188 +/- 8 mmHg systolic blood pressure, respectively) were used. Wall thickness was increased in DOCA-salt vs. sham aorta (301 +/- 23 vs. 218 +/- 14 mum, P < 0.05), as was medial area, but neither measure was altered in the vena cava. The aorta and vena cava expressed the gelatinases MMP-2, MMP-9, transmembrane proteinase MT1-MMP, and tissue inhibitor of metalloproteinase-2 (TIMP-2). Immunohistochemically, MMP-2 localized to smooth muscle in the aorta and densely in endothelium/smooth muscle of the vena cava. Western and zymographic analyses verified that MMP-2 was active in all vessels and less active in the vena cava than aorta. In hypertension, MMP-2 expression and activity in the aorta were increased (59.1 +/- 3.7 and 74.5 +/- 6.1 units in sham and DOCA, respectively, P < 0.05); similar elevations were not observed in the vena cava. MMP-9 was weakly expressed in all vessels. MT1-MMP was expressed by the aorta and vena cava and elevated in the vena cava from DOCA-salt rats. TIMP-2 expression was significantly increased in the aorta of DOCA rats compared with sham but was barely detectable in the vena cava of sham or DOCA-salt hypertensive rats. These findings suggest that large veins may not undergo vascular remodeling in DOCA-salt hypertension.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. Since the role of TAFI in thrombus formation is still controversial in mice, our present study was designed to evaluate mice deficient in TAFI (TAFI(-/-)) on FeCl(3)-induced vena cava and carotid artery thrombosis. Parallel studies were carried out in wild-type mice using a potato carboxypeptidase inhibitor (PCI), a selective inhibitor of activated TAFI (TAFIa). Significant reduction in thrombus formation was observed in TAFI(-/-) mice (n = 8, P < 0.05 compared to wild-type littermates) but not in heterozygous (TAFI(+/-)) mice in 3.5% FeCl(3)-induced vena cava thrombosis. A similar effect was observed following treatment with 5 mg/kg bolus plus 5 mg/kg/h PCI in the same venous thrombosis model in C57BL/6 mice (n = 8, P < 0.01 compared to vehicle). No compositional difference was observed for the venous thrombi in TAFI(-/-) and wild-type littermates with or without PCI treatment using histological assessment. In contrast, neither TAFI deficiency nor treatment with PCI showed antithrombotic efficacy in the 3.5% FeCl(3)-induced carotid artery thrombosis model. In a tail transection bleeding time model, both TAFI deficiency and PCI treatment increased bleeding time up to 4.5 and 3.5 times, respectively, over controls (P < 0.05, n = 8). Similar ex vivo fibrinolytic activities were demonstrated for both TAFI deficiency and PCI treatment as enhanced lysis of thrombin-induced plasma clots and lysis of whole blood clot in a thrombelastograph. These data provide direct evidence for the role of TAFIa in vena cava thrombosis without the addition of exogenous thrombolytic in mice. The strong ex vivo fibrinolytic activity of TAFI deficiency or TAFIa inhibition by PCI provides a biomarker of TAFIa inhibition that tracks in vivo antithrombotic efficacy.

In hypertension, blood vessels exhibit increased reactive oxygen species production that may alter vascular tone. We previously observed that H2O2 contracted rat thoracic vena cava under resting tone and aorta contracted with KCl. In arteries but not veins, H2O2-induced contraction required extracellular Ca2+ influx. Because of this difference in Ca2+ utilization, we hypothesized that signaling pathways mediating H2O2-induced contraction in vena cava under resting tone differed from those mediating H2O2-induced contraction in aorta contracted with KCl. Inhibitors of cyclooxygenase (COX) 1 and 2 (indomethacin, 10 microM), thromboxane A2 (TXA2) receptors [ICI185282 (2RS,4RS,5SR-4-o-hydroxyphenyl-2-trifluoromethyl-1,3-dioxan-5-yl heptenoic acid), 10 microM], p38 mitogen-activated protein kinase (MAPK) [SB203580 (4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine), 10 microM], extracellular signal-regulated kinase (Erk) [PD98059 (2'-amino-3'-methoxyflavone), 10 microM], src [PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, 10 microM], and rho kinase [Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride), 10 microM], significantly reduced H2O2-induced contraction in vena cava under resting tone and aorta after KCl (30 mM) contraction. In contrast, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, 20 microM] did not reduce aortic or venous H2O2-induced contraction. p38 MAPK, Erk MAPK, and src inhibition did not reduce aortic or venous contraction to the TXA2 receptor agonist U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF(2alpha), 1 microM), whereas rho kinase inhibition significantly reduced aortic and venous contraction to U46619, and PI3-K inhibition reduced venous contraction to U46619. Our data suggest that, in rat thoracic aorta and vena cava, a COX-derived metabolite is one important mediator of H2O2 contraction, possibly via rho kinase activation, and that H2O2-induced contraction via p38 and Erk MAPK probably occurs independently of TXA2 receptor activation.

Hydrogen peroxide causes vascular contraction and relaxation and contributes to the pathogenesis of hypertension. We hypothesized that the contractile state of blood vessels governs whether H2O2 causes contraction or relaxation. Hydrogen peroxide (1 micromol/L to 1 mmol/L) concentration-dependently contracted thoracic aorta and vena cava from sham normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The maximal contraction to H2O2 was 3 times greater in DOCA aorta compared with sham aorta but unchanged in DOCA vena cava compared with sham vena cava. In prostaglandin F2alpha (20 micromol/L)-contracted aorta and vena cava from sham and DOCA rats, H2O2 (1 micromol/L to 1 mmol/L) induced a concentration-dependent relaxation that was impaired in DOCA aorta but not DOCA vena cava. In contrast, in KCl (30 mmol/L)-contracted vessels, maximal H2O2-induced contraction was enhanced 15-fold in sham aorta and 5-fold in DOCA aorta but only 2-fold in sham vena cava. Tetraethylammonium (10 mmol/L), BAY K 8644 (100 nmol/L), and ouabain (1 mmol/L) all enhanced maximal aortic H2O2-induced contraction, whereas only ouabain enhanced venous H2O2-induced contraction. The removal of extracellular Ca2+ reduced H2O2-induced contraction in KCl-contracted aorta, whereas maximal venous H2O2-induced contraction (under basal conditions) was unchanged. Our data suggest that differences in arterial and venous K+ channel activity and extracellular Ca2+ influx are responsible for differences in arterial and venous contraction to H2O2. In DOCA-salt hypertension, arterial but not venous contraction to H2O2 is enhanced, and relaxation to H2O2 is reduced.

Cyclooxygenase 2 (Cox-2), an enzyme involved in prostaglandin production, is a key player in the development of pathologic changes, such as colorectal cancer, arteriosclerosis and thrombosis. In this study, we investigated the effects of estrogens, selective estrogen receptor modulators (SERMs), pure antiestrogens and phytoestrogens on the tissue-specific expression of Cox-2 in the uterus and the v. cava of ovariectomized female rats. Cox-2 expression could be detected in the uterine epithelium and in the endothelium of the v. cava. Cox-2 expression was time-dependently stimulated after administration of 17beta estradiol (E2) in the uterus. In the v. cava, E2 treatment resulted in a stimulated expression of the progesterone receptor (PR), a gene known to be regulated by E2, whereas Cox-2 was simultaneously down-regulated. Administration of the pure antiestrogen faslodex (Fas) had no effect on Cox-2 expression. In contrast, administration of tamoxifen (Tam) resulted in a decrease of Cox-2 expression in the v. cava but does not stimulate Cox-2 expression in the uterus. Interestingly, the same expression pattern of Cox-2 could be detected after dose-dependent administration of genistein (Gen). Here, down-regulation of Cox-2 could already be detected after administration of merely 0.5 mg/(kgBW) Gen, a dose where no effects on uterine weight were observed. In summary, our results demonstrate a reverse tissue-specific regulation of Cox-2 expression by estrogens in the v. cava and uterus indicating the existence of complex molecular mechanisms which have to be characterized in future studies. Remarkably, Tam and the phytoestrogen Gen, both share the ability to decrease the expression of Cox-2 in the v. cava without effecting its uterine expression. These observations may be of great importance with respect to potential beneficial or adverse effects of estrogens, SERMs and phytoestrogens on the cardiovascular tissue.

The effects of black cohosh extracts (Rhizoma Cimicifugae racemosae) on primary estrogen target organs, like mammary gland and endometrium are better described then those on other estrogen-sensitive systems e.g. the vasculature. We therefore treated ovariectomized DA/Han rats for 17 days with an isopropanolic Cimicifuga racemosa rhizoma extract (iCR) alone and in combination with the pure antiestrogen fulvestrant. As control groups vehicle, estradiol, fulvestrant, and estradiol fulvestrant cotreatment were used. Effects of all substances were investigated by vena cava and uterine gene expression analysis using real-time-PCR. Uterus wet weight was increased after estradiol treatment compared to the negative controls but none of the other treatments including the treatment with iCR had a uterotrophic effect. While estradiol-induced changes in uterine gene expression were mainly analogous to those detectable in shorter term experiments, iCR showed no or slightly antiestrogenic effects on gene expression in the uterus. This is mirrored in the vena cava where iCR had a very minor impact on the expression of the genes analyzed. While C. racemosa is effectively used for treatment of peri- and post-menopausal symptoms for a long time its mechanism of action remains unresolved. Contrary to earlier suggestions C. racemosa does not seem to act as an estrogen agonist, but possibly as a weak antiestrogen.

The in-vivo inhibitory effects of the ethanol extract of Radix Ophiopogon japonicus (ROJ-ext) on venous thrombosis were studied in mouse and rat models and in-vitro endothelial cell-protective and anti-adhesive activities were observed in ECV304 cells injured by sodium dithionite and HL-60 adhesion to ECV304 cells injured by TNF-alpha. The in-vivo results showed that ROJ-ext significantly inhibited venous thrombosis induced by tight ligation of the inferior vena cava for 6 h in mice and for 24 h in rats by once oral administration at doses of 12.5 and 25 mg/kg. Meanwhile, ROJ-ext had no obvious effect on some coagulation parameters, which was different from warfarin, which remarkably prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) in rats at the same time. Histological analysis under light microscope and scanning electron microscope (SEM) of inferior vena cava indicated that ROJ-ext could protect endothelial cells from anoxic injury and alleviate inflammatory changes in the vein wall. On the other hand, the in-vitro studies approved that ROJ-ext significantly enhanced viability of ECV304 cells injured by sodium dithionite at the concentrations of 0.1, 1.0 and 10 mug/ml when given before and after the anoxic induction. Meanwhile, ROJ-ext remarkably inhibited adhesion of HL-60 cells to ECV304 cells injured by rh TNF-alpha at above concentrations in a dose-dependent manner. The findings of this study showed that ethanol extract of Radix Ophiopogon japonicus (ROJ-ext) inhibited venous thrombosis, which linked with its endothelial cell-protective and anti-adhesive activities. This lends scientific support to the therapeutic use of the plant for thrombotic diseases.

This study examined the nitric oxide (NO) control of the vascular smooth muscle of the ventral abdominal vein and vena cava of the toad, Bufo marinus, by using anatomical and physiological approaches. Nicotinamide adenine di-nucleotide phosphate-diaphorase histochemistry and immunohistochemistry using endothelial nitric oxide synthase (NOS) and neural NOS antibodies produced no evidence for endothelial NOS in the veins, but, neural NOS-immunoreactive perivascular nerves were present. Acetylcholine (10(-5) M) caused a vasodilation in both veins that was endothelium-independent, and which was blocked by the soluble guanylyl cyclase inhibitor, ODQ (10(-5) M). The NOS inhibitors, L-NNA (10(-4) M) and L-NAME (10(-4) M), did not significantly reduce the vasodilatory effect of acetylcholine in the veins; this suggested that the vasodilation was not due to NO. However, in the presence of phenoxybenzamine (10(-7)-10(-8) M), L-NNA significantly reduced the vasodilatory effect of acetylcholine in the veins. This unusual response is due to phenoxybenzamine partially inactivating the muscarinic receptor pool in the veins. In addition, the neural NOS inhibitor, vinyl-L-NIO (10(-5) M), significantly reduced the acetylcholine-mediated vasodilation in the presence of phenoxybenzamine. The results show that in toad veins, nitrergic nerves rather than an endothelial NO system are involved in NO-mediated vasodilation.

Vein graft failure within the first month after bypass surgery is largely because of thrombosis. However, systemic study of thrombus formation in vein grafts is still lacking, and few effective techniques are available to prevent this event. Herein, we analyzed the kinetics of thrombosis and tested the effectiveness of locally applied aspirin on prevention of the disease in a mouse model. En face analysis of vein grafts revealed that 67+/-12% and 54+/-17% of the surface areas were covered by microthrombi at 1 and 3 days, respectively. Thrombus generation was also identified by labeling of platelets and fibrin, which occurred in 35 grafts examined at 1 and 3 days and 1, 2, 4, and 8 weeks. In a fifth of grafts, the thrombus occluded the vessel lumen by > or =1/4. Furthermore, a significant loss of endothelial cells was evidenced by beta-gal staining for vein grafts in transgenic mice expressing LacZ gene controlled by TIE2-endothelial specific gene promoter. Following thrombosis, neointimal lesions were significantly increased by 4-fold 2 weeks after the operation. When vein grafts were treated locally with aspirin in pluronic gel-127, the thrombus area was significantly reduced (P<0.005) at 1, 4, and 8 weeks. Interestingly, neointimal lesions were markedly reduced in the local, but not oral, aspirin-treated group at 4 and 8 weeks by 50% to 70% (P<0.005). The mechanism of reduced lesions by locally applied aspirin involved the protection of vein graft endothelium. Thus, we provide strong evidence that thrombus formation occurs before the development of neointimal lesions in vein grafts and that local aspirin treatment successfully reduces vein graft arteriosclerosis through endothelial protection, resulting in reduction of thrombosis.

This study tested the hypothesis that depressor responses caused by tempol are not associated with reductions in vascular O2- levels in urethane-anesthetized deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We compared the effects of intravenous (IV) administration of tempol, apocynin, superoxide dismutase-polyethylene glycol (PEG-SOD), and SOD on mean arterial blood pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). In DOCA-salt rats, tempol (30 to 300 micromol/kg) dose-dependently decreased RSNA, MAP, and HR. Tempol (300 micromol/kg) decreased MAP from 140+/-5 to 83+/-4 mm Hg (P<0.05). HR decreased from 435+/-15 to 390+/-12 bpm (P<0.05). RSNA was reduced by 54%+/-6% from baseline. However, in the same rats, tempol did not reduce dihydroethidium-induced fluorescent signals in the aorta and vena cava. Apocynin (200 micromol/kg) did not lower MAP (142+/-5 mm Hg versus 140+/-6 mm Hg) or HR (428+/-15 bpm versus 420+/-13 bpm) and apocynin did not potentiate depressor responses caused by tempol. PEG-SOD (10 000 U/kg, bolus or 5000 U/kg bolus followed by a 30-minutes infusion of 500 U/kg/min) or SOD (25 000 U/kg, bolus or 10 000 U/kg bolus followed by a 30-minutes infusion of 1000 U/kg per minute) did not alter MAP or HR. It is concluded that depressor responses and decreases in HR and RSNA caused by acute tempol treatment are caused by direct sympathetic nerve activity inhibition that is not accompanied by SOD-mimetic action in the aorta or vena cava.

Introduction: Our previous studies have shown that when a segment of rat aorta was placed upstream and in series to a rat cremasteric isolated arteriole, endotoxin (ET) exposure produced significant vasodilatation. Without the aorta, no loss of tone was noted, indicating that a precursor, as of yet unidentified, was washed downstream, thereby inducing vasodilation. Prior treatment of the donor of either the aorta or the arteriole with pyrrolidine dithiocarbamate (PDTC), a potent NF-kappaB inhibitor, prevented the loss of tone. This suggests a role for NF-kappaB in the signaling pathway. The aim of this study was to determine if a large vein was also capable of releasing a similar factor in response to ET., Materials and Methods: This project followed the same experimental design except that the upstream aortic segment was replaced by a segment of vena cava. Male Sprague-Dawley rats were given an intraperitoneal (ip) injection of PDTC (100 mg/kg) or a sham injection of saline. First-order cremasteric arterioles were isolated, cannulated, and pressurized. A segment of inferior vena cava was then placed in series with the microvascular preparation. Arterioles were allowed to equilibrate and achieve spontaneous myogenic tone in a bath of warm physiological buffer over 1 h (t = 0). Internal vessel diameters were measured with video calipers and the response to ET or continued infusion of buffer was measured over 2 h (t = 120). The control group (n = 8) received a sham injection and the vessels were exposed to buffer only. The ET group (n = 7) was exposed to ET only. The PDTC group (n = 5) received PDTC only. The PDTC/ET group (n = 6) received PDTC and was exposed to ET., Results: After equilibration, spontaneous tone (measured as a percentage of maximal diameter) was similar in the four groups (t = 0). After 2 h (t = 120), the ET group had significantly less tone (30.1 +/- 3.6%; P < 0.05) than the control (44.8 +/- 2.6%), the PDTC group (43.0 +/- 1.3%), and the PDTC/ET group (49.4 +/- 3.0%)., Conclusions: These results show that the vena cava is capable of releasing a factor leading to vasodilation in response to ET in a manner similar to the aorta. Produced by the veins, it will affect venous capacitance as well as contribute to the total amount in the plasma pool affecting the tone of the resistant arterioles. Thus, it appears that these large conducting vessels, regardless of origin, play a role in the deleterious effects during septic events.

Nitroglycerin (glyceryl trinitrate, GTN) relaxes blood vessels primarily via activation of the soluble guanylyl cyclase (sGC)/cGMP/cGMP-dependent protein kinase (cGK-I) pathway. Although the precise mechanism of sGC activation by GTN in the vascular wall is unknown, the mediatory role of nitric oxide (NO) has been postulated. We tested the GTN/NO hypothesis in different types of isolated rat and rabbit blood vessels using two novel approaches: (1) EPR spin trapping using colloid Fe(DETC)2 and (2) analysis of cGK-I-dependent phosphorylation of the vasodilator-stimulated phosphoprotein at Ser239 (P-VASP). For comparison, another organic nitrate, isosorbide dinitrate (ISDN), and endothelium-dependent vasodilator, calcium ionophore A23187, were tested. We found a marked discrepancy between GTN's strong vasoactivity (vasodilation and augmentation of P-VASP) and its poor NO donor properties. In aortas precontracted with phenylephrine, GTN, ISDN, and A23187 induced nearly full relaxations (>80%) and doubling of vascular P-VASP content at concentrations of 100 nmol/L, 100 micromol/L, and 1 micromol/L, respectively. GTN applied in vasorelaxant concentrations (10 to 1000 nmol/L) did not significantly increase the basal vascular NO production, in contrast to ISDN and A23187. The absence of GTN-derived NO was confirmed in rabbit vena cava and renal artery. A significant increase in vascular NO formation was observed only at suprapharmacological GTN concentrations (>10 micromol/L). The concentration dependency of NO formation from GTN was comparable to that of ISDN, although the latter exhibits 100-folds lower vasorelaxant potency. We conclude that GTN activates the sGC/cGMP/cGK-I pathway and induces vasorelaxation without intermediacy of the free radical NO. The full text of this article is available online at http://www.circresaha.org.

Deoxycorticosterone acetate (DOCA)-salt hypertension is characterized by low renin/angiotensin but increased arterial superoxide levels. We have recently reported that the arterial endothelin-1 (ET-1) level is increased, resulting in NADPH oxidase activation and superoxide generation. However, the effect of ET-1 on venous superoxide production and its relation to venoconstriction are unknown. The present study tested the hypotheses that ET-1 stimulates venous NADPH oxidase and superoxide via its ET(A) receptors, resulting in enhanced venoconstriction in DOCA-salt hypertensive rats. Treatment with ET-1 (0.01 to 1 nmol/L), but not the selective ET(B) receptor agonist sarafotoxin s6c, of vena cavas of normal rats concentration-dependently increased superoxide levels, an effect that was abolished by the selective ET(A) receptor antagonist ABT-627. Although the ET-1 level was not increased in the vena cava and plasma, both venous NADPH oxidase activity and superoxide levels were significantly higher in DOCA-salt compared with sham rats. Moreover, ET-1 treatment (10(-9) mol/L, 10 minutes) of isolated vena cavas further elevated superoxide levels in DOCA-salt rats only but not sham rats, an effect that was abrogated by the superoxide scavenger tempol. Similarly, ET-1-induced contractions of isolated vena cavas of DOCA-salt but not sham rats were significantly inhibited by tempol. The NADPH oxidase inhibitor apocynin significantly reduced superoxide levels in vena cavas of DOCA-salt rats and in ET-1-treated vena cavas of normal rats. Finally, in vivo ET(A) receptor blockade by ABT-627 significantly lowered venous superoxide levels and blood pressure in DOCA-salt but not sham rats. These results suggest that superoxide contributes to ET-1-induced venoconstriction through an elevated venous NADPH oxidase activity in mineralocorticoid hypertension.

In anesthetized cats, nitroglycerin increased blood flow in the superior vena cava and decreased the flow in the inferior vena cava and total venous return. Simultaneous changes in right atrial pressure could be either positive or negative. The shifts in the superior vena cava flow and right atrial pressure preceded the corresponding alterations in the inferior vena cava flow and venous return.

The type and magnitude of changes in blood flow in the anterior (cranial) and posterior (caudal) caval veins and shifts in the mean right atrial pressure induced by catecholamines (epinephrine and norepinephrine) were studied in acute experiments on cats. It was found that irrespective of right atrial pressure shifts, the increase in the blood flow in the anterior vena cava was more pronounced than in the posterior vena cava and was determined by blood redistribution due to more pronounced increase in vascular resistance in the abdominal aorta basin.

The venom of Lonomia obliqua caterpillar may induce a hemorrhagic syndrome in humans, and blood incoagulability by afibrinogenemia when intravenously injected in laboratory animals. The possible antithrombotic and thrombolytic activities of L. obliqua caterpillar bristle extract (LOCBE) were evaluated in this study. The minimal intravenous dose of the extract necessary to induce afibrinogenemia and anticoagulation was 3.0 and 10.0 microg protein/kg body weight for rabbits and rats, respectively. In rabbits, this dose induced total blood incoagulability for at least 10 h and did not reduce the weight of preformed venous thrombi, in contrast to streptokinase (30,000 IU/kg). In rats, pretreatment with 5.0 and 10.0 microg/kg LOCBE prevented the formation of thrombi induced by venous stasis or by injury to the venous endothelium. The dose of 5.0 microg/kg LOCBE did not modify blood coagulation assay parameters but increased bleeding time and decreased plasma factor XIII concentration. When the extract was administered to rats at the dose of 10.0 microg/kg, the blood was totally incoagulable for 6 h. These data show that LOCBE was effective in preventing experimental venous thrombosis in rats, justifying further studies using purified fractions of the extract to clarify the mechanisms of this effect.

1. Arachidonic acid (0.01-1 microM) induced relaxation of precontracted rings of rabbit saphenous vein, which was counteracted by contraction at concentrations higher than 1 microM. Concentrations higher than 1 microM were required to induce dose-dependent contraction of vena cava and thoracic aorta from the same animals. 2. Pretreatment with a TP receptor antagonist (GR32191B or SQ29548, 3 microM) potentiated the relaxant effect in the saphenous vein, revealed a vasorelaxant component in the vena cava response and did not affect the response of the aorta. 3. Removal of the endothelium from the venous rings, caused a 10 fold rightward shift in the concentration-relaxation curves to arachidonic acid. Whether or not the endothelium was present, the arachidonic acid-induced relaxations were prevented by indomethacin (10 microM) pretreatment. 4. In the saphenous vein, PGE2 was respectively a 50 and 100 fold more potent relaxant prostaglandin than PGI2 and PGD2. Pretreatment with the EP4 receptor antagonist, AH23848B, shifted the concentration-relaxation curves of this tissue to arachidonic acid in a dose-dependent manner. 5. In the presence of 1 microM arachidonic acid, venous rings produced 8-10 fold more PGE2 than did aorta whereas 6keto-PGF1alpha and TXB2 productions remained comparable. 6. Intact rings of saphenous vein relaxed in response to A23187. Pretreatment with L-NAME (100 microM) or indomethacin (10 microM) reduced this response by 50% whereas concomitant pretreatment totally suppressed it. After endothelium removal, the remaining relaxing response to A23187 was prevented by indomethacin but not affected by L-NAME. 7. We conclude that stimulation of the cyclo-oxygenase pathway by arachidonic acid induced endothelium-dependent, PGE2/EP4 mediated relaxation of the rabbit saphenous vein. This process might participate in the A23187-induced relaxation of the saphenous vein and account for a relaxing component in the response of the vena cava to arachidonic acid. It was not observed in thoracic aorta because of the lack of a vasodilatory receptor and/or the poorer ability of this tissue than veins to produce PGE2.

In rat vena cava and aorta preincubated with [3H]noradrenaline the involvement of imidazoline receptors in modulation of [3H]noradrenaline release from sympathetic nerves was investigated. In the vena cava, the guanidine 1,3-di(2-tolyl)guanidine (DTG) inhibited the electrically evoked [3H]noradrenaline release; the inhibitory effect was more pronounced in the presence than in the absence of the alpha 2-adrenoceptor antagonist rauwolscine. The concentration-response curves of BDF 6143 [4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline], and idazoxan for their facilitatory effect on electrically evoked [3H]noradrenaline release was bell-shaped; in the presence of rauwolscine, BDF 6143 inhibited the evoked [3H]noradrenaline release, whereas idazoxan did not. After blockade of alpha 2-autoreceptors by rauwolscine, the electrically evoked [3H]noradrenaline release from vena cava was inhibited not only by DTG and BDF 6143 but also by aganodine, clonidine and cirazoline; the rank order of potency of most of the drugs was similar to that found at the presynaptic imidazoline receptors in the rabbit aorta and pulmonary artery as well as in human atrial appendages. In the presence of rauwolscine, clonidine-induced inhibition of electrically evoked [3H]noradrenaline release was counteracted by 1 microM of the selective CB1 receptor antagonist SR141716A (N-[piperidin-1-yl]-5-[4-chlorophenyl]-1-[2,4-dichlorophenyl] -4-methyl-1H-pyrazole-3-carboxamide). In the aorta, BDF 6143 and cirazoline did not modify [3H]noradrenaline release in the absence of alpha 2-adrenoceptor blockade; in the presence of rauwolscine, the electrically evoked [3H]noradrenaline release from aorta was inhibited by BDF 6143, cirazoline, aganodine and clonidine with a rank order of potency similar to that in the vena cava. SR141716A 1 microM antagonized the inhibitory effect of BDF 6143 and clonidine (in the presence of rauwolscine). In conclusion, noradrenaline release in rat vena cava and aorta is inhibited via presynaptic imidazoline receptors which appear to be related to those previously characterized in rabbit and human cardiovascular tissue.

In the present study the formation of thrombin-antithrombin-complexes (TAT) during incubation of thrombin (0.89, 4.5, 8.9 nmol/l) and antithrombin (4.6 micromol/l) on the surface of cultured human EC, derived from different parts of the circulation, and on the surface of human vessel segments was studied. In the absence of EC TAT increased over time reaching a maximum at 60 sec; 10 sec (8.9 nmol/l thrombin): 6.35+/-0.72 nmol/l, 60 sec: 10.49+/-1.04 nmol/l. In the presence of exogenous heparin (0.1 IU/ml) maximum TAT levels were already reached after 10 sec (10.75+/-0.97); cultured EC and EC on vessel segments did not show such heparin effects. Incubation of EC with heparin resulted in an EC-surface localized heparin activity only when very high doses (3.0 IU/ml) of the drug were used. When thrombin was incubated on the EC surface in the presence of AT the efficiency of the thrombomodulin(TM)-protein C(PC)-system was markedly reduced, while in the presence of exogenous heparin (0.5 IU/l) the activity of this pathway was nearly abolished. Our results demonstrate that 1) human EC do not exert heparin-like accelerating effects on TAT formation, 2) an EC localized heparin activity is only generated when EC are incubated with amounts clearly exceeding therapeutical doses, and 3) an acceleration of TAT formation at the EC surface by heparin causes a switching off of the TM-PC-system.

It has been suggested that at least the majority of mammalian presynaptic alpha2-autoreceptors belong to the genetic alpha2A/D-adrenoceptor subtype. The aim of the present study was to re-examine the alpha2-autoreceptors in tissues in which previous assignments conflicted with this alpha2A/D rule: in the rat vena cava and rat heart atria, where the autoreceptors were classified as alpha2B or similar to, but not identical with, alpha2D, and in the human kidney, where they were classified as alpha2C. Also re-examined were the autoreceptors in the guinea-pig urethra, where they were suggested to be alpha2A, in agreement with the rule, but in contrast to indications that the alpha2A/D-adrenoceptor of the guinea pig possesses alpha2D pharmacological properties. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically under autoinhibition-free or almost autoinhibition-free conditions. The Kd values of up to 14 antagonists (including the partial agonist oxymetazoline) against the release-inhibiting effect of the alpha2 agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. UK 14,304 reduced the evoked overflow of tritium with an EC50 between 6.3 and 13.2 nM. All antagonists (except prazosin in one case) shifted the concentration-inhibition curve of UK 14,304 to the right. Comparison of the Kd values thus obtained with Kd values at known alpha2 subtypes indicated that the autoreceptors in the rat vena cava, rat atria and the guinea-pig urethra were alpha2D and those in the human kidney alpha2A. For example, the pKd values of the antagonists in the rat vena cava, in rat atria and in the guinea-pig urethra were closely correlated with pKd values at the prototypic alpha2D radioligand binding sites in the bovine pineal gland (r = 0.96, P < 0.001; r = 0.92, P < 0.01; and r = 0.95; P < 0.001) and with the pKd values at the alpha2D-autoreceptors of guinea-pig atria (r = 0.99, P < 0.001; r = 0.95, P < 0.001; and r = 0.98, P < 0.001). The pKd values at the autoreceptors in rat vena cava, rat atria and guinea-pig urethra were not significantly or more loosely correlated with pKd values at alpha2A, alpha2B and alpha2C binding sites and alpha2A-autoreceptors. On the other hand, the pKd values of the antagonists in the human kidney were closely correlated with pKd values at the prototypic alpha2A radioligand binding sites in HT29 cells (r = 0.95; P < 0.001) and with pKd values at the alpha2A-autoreceptors of the pig brain cortex (r = 0.97; P < 0.001), but were not significantly or more loosely correlated with pKd values at alpha2B, alpha2C and alpha2D binding sites and alpha2D-autoreceptors. In contrast to previous suggestions, the autoreceptors in rat vena cava and atria are alpha2D, those in the human kidney alpha2A, and those in the guinea-pig urethra equally alpha2D. All, therefore, conform to the rule that alpha2-autoreceptors belong at least predominantly to the genetic alpha2A/D subtype of the alpha2-adrenoceptor. The apparent paradox of an alpha2A-autoreceptor in the urethra of the guinea pig, a species in which the genetic alpha2A/D-adrenoceptor otherwise has alpha2D pharmacological properties, is removed.

Blood and tissue O2 levels are major determinants of short-term autoregulatory adjustments in vascular smooth muscle cell (SMC) tension and may effect long-term alterations in SMC catecholamine responsiveness. We examined the hypothesis that prolonged hypoxia altered gene expression of alpha 1-adrenoceptors. After exposure of cultured aortic (in vitro) SMC to 3% O2 for 8 h, alpha 1B mRNA increased to 523% (P = 0.02) of control cells (21% O2) and to 205% (P = 0.04) in in situ organ-cultured aortic SMC. In vivo hypoxic hypoxia (10% inspired O2) similarly increased aortic SMC alpha 1B mRNA 180% (P = 0.02). In contrast, alpha 1D, alpha-actin and beta-actin mRNA levels were not changed in aortic SMC by low O2 in the in vitro, in situ, or in vivo models. Unlike aortic SMC, vena caval SMC alpha 1B mRNA expression did not change with low-O2 exposure in vitro or in vivo, nor did alpha 1D, alpha-actin or beta-actin mRNA. Aortic SMC alpha 1B transcription rate increased 360% (P = 0.02), whereas alpha 1D, alpha-actin, and beta-actin transcription was unchanged. Neither alpha 1B nor alpha 1D mRNA stability was altered by low-O2 exposure. Total alpha 1-adrenoceptor density ([3H]prazosin binding) increased 12% (P = 0.04) after 24 h of 3% O2. This was associated with a 200% increase (P < 0.01) in the chloroethylclonidine (CEC)-sensitive alpha 1-adrenoceptor population and no change in CEC-insensitive alpha 1-adrenoceptor density. Exposure of aortic SMC to 24 h of 3% O2 increased the maximum response of norepinephrine-evoked elevations in intracellular Ca2+ as measured using fura 2. Low O2 did not change responses to another G protein-coupled receptor, angiotensin II. These data suggest that reduced O2, during prolonged hypoxemia or tissue ischemia, may selectively increase expression of functionally coupled alpha 1B-adrenoceptors in arterial blood vessels.

Arginine (0.8g/kg, ip) or a vehicle was administered to rats and the levels of arginine and a large number of related amino compounds++ were measured in plasma, heart, aorta, vena cava, pancreas and bronchi at specified time intervals. Arginine levels (nmol/ml) increased in the plasma from 237 to 3172 at 15 min, 1236 at 30 min and 509 at 120 min. Peak concentrations (nmol/g) of arginine are reached in the tissues at 15 or 30 minutes with control and postinjection values of 500 and 1769 in the heart, 314 and 1563 in the aorta, 575 and 2976 in the vena cava, 760 and 1943 in the bronchi, and 234 and 3638 in the pancreas. Arginine injection also affects a number of amino acids and related compounds in the plasma and tissues most notably ornithine, isoleucine, phosphoserine, leucine and ethanolamine. However, plasma level changes do not predict tissue level changes which are highly specific for an individual compound and tissue. There was no general indication that arginine injection stimulates nitric oxide (NO) formation in any tissue. Thus, arginine is rapidly absorbed from the abdominal cavity into the blood stream, is quickly taken up by the tissues studied and disappears after about 2 to 3 hours. The effects seen after arginine administration could be caused by arginine per se and/or changes in one or more of the related amino compounds but not by NO.

To target the treatment of small points of cancer, Beriplast P, already used clinically as a physiological tissue adherent drug carrier, was mixed with the anticancer drug, mitomycin C (MMC). In this in vitro study, MMC did not release quickly from the clot of MMC/Beriplast P. The antitumor effect of this mixture was examined for its effect on cancer growth. In one series of experiments, tumor tissues were inoculated with MMC/100 microliters Beriplast P and in another series, MMC/100 microliters Beriplast P was injected into tumors at a weight of 300 mg. In the first series of experiments, tumor tissue treated with 0.3 mg MMC/100 microliters Beriplast P was replaced with plasma cells and lymphocytes, and no viable cancer cells could be found. In the second series, MMC/100 microliters Beriplast P delayed tumor growth, and the survival of Balb/c mice injected with 0.08 mg MMC/100 microliters Beriplast P was significantly longer than that of mice injected with 0.08 mg MMC/100 microliters saline solution (P = 0.026). In addition, the abdominal aorta, vena cava, and intestine around the area of treatment with 1.6 mg MMC/100 microliters Beriplast P were not damaged. These results indicate that the mixture of Beriplast P and MMC is more effective than MMC solution alone in the local treatment of residual cancer.

Rat aorta smooth muscle cells which express all three alpha 1-adrenoreceptors (alpha 1A, alpha 1B and alpha 1D) were used to determine the effect of stimulation of alpha 1-adrenergic receptor subtypes on cell growth. "Combined" alpha 1-adrenoreceptor subtype stimulation with norepinephrine alone caused a concentration-dependent, prazosin-sensitive increase in protein content and synthesis: 48 h of stimulation at 1 microM increased cell protein to 216 +/- 40% of time-matched controls (p = 0.008) and RNA to 140 +/- 13% (p = 0.03); protein synthesis increased to 167 +/- 13% (p < 0.01) after 24 h. Stimulation with norepinephrine plus the selective alpha 1A/alpha 1D antagonist 5-methylurapidil produced greater increases in alpha-actin mRNA (270 +/- 40% at 8 h; p = 0.007), total cell protein (220 +/- 45% at 24 h; p = 0.004), and RNA (135 +/- 8% at 24 h; p = 0.01). These effects were prevented by pretreatment with the selective alpha 1B antagonist chloroethylclonidine. Comparable results were obtained for intact aortae. Stimulation with norepinephrine plus 5-methylurapidil increased (p < 0.05) tissue protein, RNA, dry weight, and alpha-actin mRNA; and as in culture cells, combined stimulation with norepinephrine alone attenuated these responses. By comparison, adventitia (fibroblasts) was unaffected. Removal of endothelial cells had no effect. alpha 1B mRNA decreased by 42 +/- 12% (p = 0.01) in cultured cells during combined alpha 1-adrenoreceptor stimulation and by 23 +/- 8% (p = 0.03) for intact aorta. alpha 1D and beta-actin mRNA were unchanged in cultured cells, aorta media, and adventitia. These findings suggest that prolonged stimulation of chloroethylclonidine-sensitive, possibly alpha 1B-adrenoceptors induces hypertrophy of arterial smooth muscle cells and that stimulation of 5-methylurapidil-sensitive, non-alpha 1B-adrenoreceptors attenuates this growth response.

The ability of the novel, non-peptide, neuropeptide Y Y1 receptor antagonist, BIBP 3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininami de), to antagonize neuropeptide Y- and sympathetic-mediated vasoconstriction was examined in isolated segments of the thoracic vena cava of guinea-pigs. Increasing concentrations (10(-9) - 10(-6) M) of BIBP 3226 caused a parallel and rightward shift in the neuropeptide Y dose-response curve but did not significantly change the effect of noradrenaline. The calculated pA2 value for BIBP 3226 was 8.0 +/- 0.08, a value fully compatible with the reported affinity at rodent and human neuronal Y1 receptors. BIBP 3226 (10(-6) M) also readily reversed the established vasocontraction induced by neuropeptide Y. BIBP 3226 (10(-6) M) markedly inhibited the slow long-lasting contraction evoked by high frequency electrical field stimulation, leaving a rapid component which was abolished by phentolamine. Its enantiomer, BIBP 3435 ((S)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininami de), which exerts a much weaker action on neuropeptide Y Y1 receptors, had no such inhibitory effect. In propranolol-pretreated vessels, the vasoconstriction evoked by nerve stimulation was enhanced; then BIBP 3226 inhibited the peak response by 44%, and the integrated contractile effect by 90%. We conclude that BIBP 3226 is a potent and competitive antagonist of neuropeptide Y Y1 receptor-mediated vasoconstriction in guinea-pig vena cava and that endogenous neuropeptide Y acting on the neuropeptide Y Y1 receptor is likely to account for the long-lasting component of the sympathetic vasoconstriction in response to high-frequency stimulation in this vessel.

Graded caval occlusion in conscious rabbits caused a biphasic response. Phase I was characterized by a fall in conductance so that arterial pressure was maintained. When cardiac output had fallen to 71 +/- 4% of its baseline level, phase II supervened. During phase II, conductance rose abruptly and arterial pressure fell to a life threatening level (< 40 mm Hg). When administered into the fourth ventricle, the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester prevented the onset of phase II. The mean threshold dose for this effect was 4 mumol (range: 0.4-11). When administered intravenously, a dose of 275 mumol N-nitro-L-arginine methyl ester prevented the onset of phase II in only one out of six rabbits. It is concluded that a central brainstem nitrergic mechanism is involved in the onset of the decompensatory phase II of the haemodynamic response to hypovolaemia.

Neurokinin receptors have been characterized by biological assays using naturally occurring and selective agonists as well as peptide and non peptide antagonists. Six preparations have been used: the rabbit vena cava and the rat urinary bladder, treated with a NK-2 receptor antagonist for the NK-1 receptor, the rabbit pulmonary artery and the hamster urinary bladder for the NK-2, the rat portal vein and the guinea pig ileum, treated with a NK-1 receptor antagonist, for the NK-3. Treatment with antagonists was required because of the presence (in some preparations) of two functional sites contributing to the biological effect. Differences in the order of potency of agonists between each couple of receptors have been demonstrated, especially with tachykinins and the selective agonists. Such differences are even more evident with antagonists, some of which show apparent affinity (pA2) values 1.5 to 3 log units higher in one than in the other member of each couple. Based on data obtained in pharmacological experiments, it is concluded that NK-1, NK-2 and NK-3 receptors show differences strong enough to justify the assumption that their coding and/or expression diverge among species.

We examined which subtypes of alpha 1-adrenoceptors are expressed in rat vena cava by using both functional and [3H]prazosin binding experiments. Pretreatment with chloroethylclonidine inactivated about 80% of the specific [3H]prazosin binding sites and reduced the maximal noradrenaline-induced contraction to the same extent. Competition with subtype-selective agonists and antagonists showed primarily the alpha 1B-adrenoceptor subtype in vena cava. The number of alpha 1-adrenoceptors estimated with [3H]prazosin binding and the maximal noradrenaline-induced contraction were dose-dependently inhibited by phenoxybenzamine, indicating the absence of receptor reserve for noradrenaline in vena cava. As the noradrenaline-induced contraction was largely inhibited in Ca(2+)-free solution, these results suggest that alpha 1B-adrenoceptors can be mainly linked to Ca2+ influx in rat vena cava.

In superfused pig retina discs, preincubated with 3H-noradrenaline, the electrically (3 Hz) evoked tritium overflow was inhibited by gamma-aminobutyric acid (GABA) and the GABAB receptor agonist R-(-)-baclofen but was not affected by the GABAA receptor agonist 3-amino-1-propanesulphonic acid. The effect of GABA was counteracted by the GABAB receptor antagonist CGP 35348 [P-(3-aminopropyl)-P-diethoxymethylphosphinic acid] but was not influenced by the GABAA receptor antagonist SR 95531 [2-(3-carboxypropyl)-3-amino-6-paramethoxyphenylpyridazinium bromide]. Furthermore, CGP 35348 antagonized the inhibitory effect of R-(-)-baclofen on the electrically (0.66 Hz) evoked tritium overflow in superfused rat vena cava segments, preincubated with 3H-noradrenaline, and on the electrically (0.5 Hz) induced rise in diastolic blood pressure in pithed rats. The present results suggest that GABA is capable of inhibiting noradrenaline release (most likely from vascular sympathetic nerve endings) in the pig retina via GABAB receptors and that CGP 35348 is a valuable tool for the characterization of GABAB receptors in vitro and in situ.

To reveal a potential modulating effect of vasoactive pharmacological agents on the prostanoid production of the venous wall, prostacyclin and thromboxane release from venous tissue slices was studied. Aortic and caval vein samples from 20 rats as well as from 21 cats were studied. Prostacyclin and thromboxane productions were determined by radioimmunoassay as 6-keto-PGF1 alpha and TxB2 released into the incubation medium. Venous tissue produced significantly less prostacyclin per unit weight than arterial tissue in rats (30.7 +/- 4.6 vs. 52.1 +/- 8.2 pg/mg/min), while in cats an opposite situation was found (16.6 +/- 3.2 vs. 7.06 +/- 1.9 pg/mg/min). Thromboxane production of venous tissue was consequently higher than corresponding values for aortic tissue (3.72 +/- 0.46 vs. 1.54 +/- 0.14 in rats and 3.4 +/- 0.6 vs. 1.33 +/- 0.19 in cats, all values in pg/mg/min). Norepinephrine and dopamine significantly increased both the prostacyclin and the thromboxane release from venous tissue, while isoproterenol had no effect. Vasopressin significantly increased thromboxane release and decreased the ratio of prostacyclin vs. thromboxane production (from 10.4 +/- 1.6 to 7.5 +/- 1.6, in acetylsalicylic acid pretreated cats). Angiotensin and thrombin had no significant effects. Bradykinin (0.5 microgram/ml) significantly augmented prostacyclin release from venous tissue (14.4 +/- 2.6 from 10.9 +/- 2.4 pg/mg/min) and decreased thromboxane release (0.65 +/- 0.18 from 1.35 +/- 0.22 pg/mg/min). Methionine-enkephalin (5 micrograms/ml) significantly reduced the thromboxane release from venous tissue slices. The presented material demonstrates that several vasoactive agents modulate the vasoactive prostanoid release of the venous wall. In some cases, the prostacyclin and the thromboxane productions are influenced separately, which in turn will have its impact on smooth muscle activity and thrombocyte aggregation.

Endothelial damage in saphenous vein harvested before coronary artery and peripheral vascular surgery has been well documented. Autogenous saphenous vein grafts are subject to early thrombotic occlusion, a process that is related to injury of this endothelial monolayer. A monoclonal antibody that binds to areas of endothelial damage (P14G11) and a non-specific immunoglobulin G (IgG) have been linked to urokinase. These conjugates were investigated in vivo using a rat vena cava model. The P14G11-urokinase conjugate significantly reduced thrombus formation compared with controls and non-conjugated urokinase (P < 0.02). No reduction in thrombus formation was seen with the IgG-urokinase conjugate. This shows that thrombus formation after endothelial damage in an in vivo model can be reduced with a targeted thrombolytic agent. Conjugates such as this may have a role in preventing early thrombotic occlusion in vein grafts.

To investigate biochemical and biological parameters involved in preproendothelin-1 (preproET-1) maturation we infected Spodoptera frugiperda (Sf21) cells with a suitable engineered baculovirus vector carrying the cDNA encoding the entire human 212 amino acids precursor. Culture supernatants were tested by RIA using an anti-ET-1 serum, ET-1-like immunoreactive material (IRM) was detected in the infected Sf21 cells but not in control, wild-type or mock-infected cells. Fractionation of the culture supernatant by RP-HPLC coupled to an ET-1 specific RIA yielded two main peaks corresponding to the retention times of human bigET-1 and ET-1. Furthermore, culture supernatant of preproET-1 expressing Sf21 cells elicited a characteristic dose-response vasoconstrictive activity on rabbit vena cava, consistent with the amount of ET-1 as estimated by RP-HPLC coupled to RIA. These results suggest that insect cells possess the enzymatic activities necessary for human preproET-1 full maturation even though no such peptide has ever been found in insect cells.

1. Whole-cell patch-clamp method was applied to single smooth muscle cells freshly isolated from the rat inferior vena cava. 2. Depolarizing pulses, applied from a holding potential of -90 mV, activated both Na+ and Ca2+ channels. The fast Na+ current was inhibited by nanomolar concentrations of tetrodotoxin (TTX). The slow Ba2+ current (measured in 5 mM Ba2+ solution) was inhibited by Cd2+ and modulated by dihydropyridine derivatives. When the cells were held at a holding potential of -80 mV, racemic Bay K 8644 increased the Ba2+ current (ED50 = 10 nM) while racemic isradipine inhibited the current (IC50 = 21 nM). 3. The voltage-dependency of isradipine blockade was assessed by determining the steady-state availability of the Ca2+ channels. From the shift of the inactivation curve in the presence of isradipine, we calculated a dissociation constant of 1.11 nM for inactivated Ca2+ channels. Scatchard plots of the specific binding of (+)-[3H]-isradipine obtained in intact strips incubated in 5.6 mM or 135 mM K+ solutions confirmed the voltage-dependency of isradipine binding. 4. Specific binding of (+)-[3H]-isradipine was completely displaced by unlabelled (+/-)-isradipine, with an IC50 of 15.1 nM. This value is similar to the IC50 for inhibition of the Ba2+ current (21 nM) in cells maintained at a holding potential of -80 mV. 5. Bay K 8644 had no effects on the Ba2+ current kinetics during a depolarizing test pulse. The steady-state inactivation-activation curves of Ba2+ current were not significantly shifted along the voltage axis.6. The present data suggest the existence of two distinct dihydropyridine binding sites which can be bound preferentially by agonist or antagonist derivatives.

Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.

1. 5-Hydroxytryptamine (5-HT) relaxes rings of neonatal porcine isolated vena cava by both an endothelium-dependent and an endothelium-independent mechanism. The receptor mediating the latter response has been shown to be a 5-HT1-like receptor (positively coupled to adenylyl cyclase) located on the vascular smooth muscle. The features of the endothelium-dependent response to 5-HT in this preparation are now described. 2. In ring preparations contracted with the stable thromboxane-A2-mimetic, U-46619 (10 nM), and in the presence of the 5-HT2 receptor antagonist ketanserin (1 microM), low concentrations of 5-HT (1-100 nM) evoked an endothelium-dependent, rapid, 'spike-like' relaxation. Higher concentrations of 5-HT (0.1-10 microM) elicited a more sustained, but endothelium-independent relaxation. 3. Relaxation induced by low concentrations (1-100 nM) of 5-HT was abolished by endothelium removal, and was markedly (but not totally) inhibited by the guanylate cyclase inhibitor, methylene blue (10 microM) or by the inhibitor of endothelium-derived nitric oxide (NO) synthesis, L-NG-monomethylarginine (L-NMMA; 100-500 microM). 4. The endothelium-dependent response to 5-HT was mimicked by alpha-methyl-5-HT, 5-methoxytryptamine, tryptamine and 2-methyl-5-HT, but not by sumatriptan or 8-hydroxy-di-n-propylaminotetralin (8-OH-DPAT) at concentrations up to 10 microM. In contrast, relaxation evoked by 5-carboxamidotryptamine (5-CT) was endothelium-independent. 5. The endothelium-dependent relaxation induced by 5-HT or alpha-methyl-5-HT was antagonized by methysergide, methiothepin, cyproheptadine and metergoline, but not by ketanserin, spiperone, ondansetron, verapamil, cyanopindolol, mesulergine, ICS 205-930, or indomethacin. 6. These results suggest that the endothelium-dependent relaxation of porcine vena cava induced by 5-HT is largely mediated by the release of NO (although other endothelium-derived relaxing factors may also be involved) and that 5-HT is acting at a receptor which is not '5-HT1-like', 5-HT2, 5-HT3 or 5-HT4 and is not comparable to recognised 5-HT receptor ligand binding sites. The characteristics of this receptor are discussed in relation to the endothelial 5-HT receptor types in other blood vessels.

1 Cells of the aortic endothelium isolated from the guinea-pig and bathed at 37 degrees C with a calcium-free superfusion fluid had membrane potentials of minus 41 plus or minus 7 mV (mean plus or minus s.e mean). 2 Depolarization was produced by addition of potassium (50-200 mM) or certain other monovalent metal cations to the superfusion fluid. Depolarization was rapidly reversed on return to the original superfusate. 3 Several divalent metal cations, notably calcium (16 mM), caused depolarization which was only slowly and incompletely reversed on return to the original calcium-free superfusate. 4 Repolarization after exposure to calcium was accelerated and made more complete by addition of indomethacin (0.25 mM) to the superfusate, 5 The trivalent cations of lanthanum, aluminium or iron (0.1 mM) inhibited the depolarizing effect of calcium (16 mM). 6 Exposure to histamine (100 mug/ml) or heating to 45 degrees C for 1 h caused depolarization in the presence but not in the absence of calcium. Subsequent removal of histamine or cooling again to 37 degrees C in the continued presence of calcium permitted only slow and partial repolarization. However, repolarization was more rapid and complete in the presence of indomethacin (0.25 mM). 7 Heating to 45 degrees C for 5 h in the presence of calcium caused progressive and almost complete depolarization. Lanthanum, cinchocaine, indomethacin, flufenamic, meclofenamic and salicylic acids, phenylbutazone and aminopyrine each reduced the depolarization, but hydrocortisone, chloroquine, benzindamine, isoprenaline and aminophylline did not.

The fast component of the rabbit vena cava contraction after repeated stimulation by NA was investigated. Experimental conditions which are known to increase the cellular calcium content led to an increase in contraction amplitude. Transient incubation between stimulations in a solution with high [Ca2+], in a solution with high [K], in a K+-free solution or in a solution containing ouabain is an example of such conditions. When the duration of a singly stimulus in a train of NA stimuli was prolonged the following contraction decreased. With increasing incubation time in Ca2+-free solution the maximum of contraction amplitude decreased far more after repeated stimulation than after a single stimulus. The results allow the interpretation that the fast component of the rabbit vena cava contraction which is induced by NA is related to the cellular calcium content. Moreover the results suggest that only a fraction of the cellular calcium is releasable by NA and that Ca2+-free conditions and/or NA exposure deplete this fraction.