European Journal of Pharmacology 546 (2006) 127 – 133 www.elsevier.com/locate/ejphar
Differential expression of α1-adrenergic receptor subtypes in coronary microvascular endothelial cells in culture Enrique Mendez a,b , Claudia Calzada a , Esther Ocharan a , Alfredo Sierra a,d , Carlos Castillo a , Israel Ramirez a , Eduardo Meaney c , Alejandra Meaney c , Juan Asbun a , Angel Miliar a , Jorge Herrera a , Guillermo Ceballos a,⁎ a
Laboratorio Multidisciplinario, Sección de Posgrado, Escuela Superior de Medicina. Instituto Politécnico Nacional, México, D. F. 11340, México b Facultad de Ciencias Químicas, Universidad Veracruzana. Orizaba, Veracruz, 94340, Mexico c Hospital 1° de Octubre ISSSTE, México, D. F. 07300, Mexico d Instituto Nacional de Perinatologia, Mexico, D.F. 11340, Mexico Received 23 March 2006; received in revised form 27 June 2006; accepted 28 June 2006 Available online 5 July 2006
Abstract It has been postulated that in blood vessels under α1-related stimulation, the endothelial intracellular calcium concentration ([Ca2+]i) increases, which is necessary to induce nitric oxide synthesis, is the result of an increase in vascular smooth muscle, which subsequently, flows into the endothelial cells through gap junctions and it is not the result of a direct adrenergic stimulation of endothelial receptors. Others, however, postulate that endothelial α1Dadrenoceptors, have a direct effect on nitric oxide synthesis. In order to clarify this phenomena, in this work we analyzed the presence of α1receptor subtypes and their functional association with nitric oxide synthesis in rat coronary microvascular endothelial cells in culture, with pharmacological, immunological and reverse transcriptase polymerase chain reaction approaches. Our results show the presence and functional coupling with nitric oxide synthesis of α1A and α1D-adrenoceptor subtypes. α1B-adrenoceptor subtype is not coupled with nitric oxide production. © 2006 Elsevier B.V. All rights reserved. Keywords: Endothelium; α1-Adrenoceptor subtype; Nitric Oxide
1. Introduction The Sympathetic Nervous System and the vascular endothelium play an important role in the regulation of vascular tone, and consequently, of arterial pressure and blood flow in tissues. The activation of SNS leads to blood vessel contraction mediated by α1-adrenergic receptors in vascular smooth muscle, while VE activation is associated with a reduction in vascular smooth muscle tone (Gabella, 1984; Murphy, 1989). The regulatory systems of the body are not isolated entities. They are closely related; they feedback and modulate each other's activity. In this sense, activation of sympathetic nervous system ⁎ Corresponding author. Laboratorio Multidisciplinario, Escuela Superior de Medicina. Plan de San Luis y Diaz Mirón s/n, Col. Casco de Santo Tomas. México, D. F., C. P. 11340, Mexico. E-mail address:
[email protected] (G. Ceballos). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.06.070
can modulate vascular smooth muscle and vascular endothelium functions creating modulatory loops between these cell types. The vascular endothelium is strategically located for the mediation of the complex interaction between circulating blood and parenchymal cells in the different tissues and organs of the body. In blood vessels, endothelium generates vasoactive (relaxant and constrictor) factors including: Nitric Oxide (NO), Prostacyclin (PGI2), Endothelial Derived Hyperpolarizing Factor (EDHF), adenosine, Endothelin-1 (ET-1), Prostaglandin H2 (PGH2) and Thromboxane A2 (TXA2). The vascular tone depends on the balance between these factors, and in the ability of vascular smooth muscle to respond to them (Gonzalez et al., 1990; Griendling and Alexander, 1996; Vanhoutte and Mombouli, 1996; Pearson and Vanhoutte, 1993; Vanhoutte, 2000). Since Furchgott and Zawadzki (1980) described the essential participation of the endothelium in the relaxation of vascular smooth muscle cells through the release of endothelium derived
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releasing factor (EDRF), several studies revealed the ability of these cells to influence vascular tone and vasoactive responses when vessels are stimulated (Gonzalez et al., 1990; Tuttle and Falcone, 2001; Dora et al., 2000; Ibarra et al., 1995; Zschauer et al., 1997; Tran and Forster, 1996). However, controversy exists regarding direct endothelial stimulation by α1-adrenoceptor agonists. It has been postulated that in blood vessels under α1-related stimulation, the endothelial intracellular calcium concentration ([Ca2+]i) increases, which is necessary to induce NO synthesis, is the result of an increase in vascular smooth muscle, which subsequently flows into the endothelial cells through gap junctions (Yashiro and Duling, 2000) and it is not the result of a direct adrenergic stimulation of endothelial receptors. Others, however, postulate that endothelial α1D-receptors, have a direct effect on NO synthesis. (Filippi et al., 2001). In order to clarify this phenomena, in this work we analyzed the presence of α1-adrenoceptor subtypes and their functional association with nitric oxide synthesis in rat coronary microvascular endothelial cells in culture, with pharmacological, immunological and reverse transcriptase polymerase chain reaction (RT-PCR) approaches. 2. Material and methods The protocols for this study were approved by institutional ethical and research committees. 2.1. Isolation and culture of male rat coronary microvascular endothelial cells Cultures were obtained according to a previously described method (Nishida et al., 1993). Briefly, adult male Wistar rats (250–300 g) (10 rats for each primary culture) were anesthetized with pentobarbital sodium (50 mg/kg i.p.) and heparinazed (150 U/kg). Hearts were rapidly excised, washed in Hank's balanced salt solution, and dissected to discard atria, right ventricle, and connective and valvular tissues. Left ventricles were opened by the anterior wall, washed, and immersed in 70% ethanol for 40 s to devitalize epicardial mesothelial and endocardial endothelial cells. The remaining tissue was chopped and placed in types IA and IV collagenase solution (2 mg/ml) for 40 min at 37 °C. Digested tissue was passed through a 70-μm metallic mesh. Dissociated cells in the filtrate were centrifuged at 3000 rpm for 3 min in a 30% Percoll gradient. Finally, cells were resuspended in Dulbecco's Minimal Essential Medium (DMEM) supplemented with 20% Fetal Bovine Serum (FBS), endothelial cell growth factor (2 ml/ 100 ml), and an antibiotic/antimycotic solution (2 ml/100 ml; GIBCO-BRL). Cells were then plated in fibronectin-covered flasks and incubated in a humidified chamber at 37 °C in a 5% CO2 atmosphere. 2.2. Characterization Criteria for characterization of this cell type included identification of a typical “cobblestone” cell morphology and expres-
sion of factor VIII-related antigen using immunofluorescence cell staining (Rubio-Gayosso et al., 2000). More than 99% of cells expressed the von Willebrand factor. 2.3. Immunoexpression of α1-adrenoceptor subtypes To determine the expression of α1-adrenoceptor subtypes, we used an immunocytochemical process and fluorescence microscopy as follows: cells were washed with ice-cold Phosphate Buffer Solution (PBS) and fixed with methanol for 10 min at 4 °C. The cells were washed and incubated for 30 min at room temperature with blocking solution (2% bovine IgGfree albumin), then incubated for 24 h at 4 °C with α1A, α1B and α1D-adrenoceptor subtype antibodies (1:200 dilution, developed in goat; Santa Cruz Biotechnology). After this period, cells were washed and post incubated for 75 min at room temperature in a dark chamber with fluorescein, rhodamine or CY3conjugated secondary antibodies (Bovine anti-goat Ig –FITC and TRITC–, 1:200 dilution, Santa Cruz Biotechnology, and Rabbit anti goat Ig –CY3–1:200 dilution, Zymed Laboratories Inc). Finally, the relative expression of α1-adrenoceptor subtypes was evaluated by fluorescence microscopy by determining the density of fluorescent pixels/area (IPLab 3.61 software) in at least 100 cells per experiment (n = 4). 2.4. Immunoblotting Homogenates of cells were prepared via mechanical separation from plates with a cell scraper and low-speed centrifugation at room temperature for 5 min. The pellet was washed with PBS, added to ice-cold RIPA Buffer (containing, PBS, 1% Nonidet-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS)) with freshly added protease inhibitors (Roche), incubated on ice for 60 min, disrupted, and homogenized further by sonication (Bransonic 1210) (Sedmak and Grossberg, 1977). Homogenates were aliquoted and stored at − 80 °C until use for α1-adrenoceptor subtype immunoblotting assay. The cellular homogenates (150 μg) mixed with Laemmli sample buffer (containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris·HCl, pH = 6.8; Bio-Rad) were boiled for 5 min, and proteins were resolved by electrophoresis via SDSpolyacrylamide gel electrophoresis (PAGE) in a 10% gel and then transferred to 0.45 μm of nitrocellulose membranes (BioRad). The nonspecific binding was blocked by incubating the membranes in Blotto (containing PBS, 8% nonfat Milk, and 0.05% Tween 20), for 1 h at room temperature. Blocked membranes were incubated with primary antibody [polyclonal antibodies against α1A,1B and 1D adrenoceptor subtypes(Santa Cruz Biotechnology, Inc),] diluted with Blotto (1:200 dilution) for 2 h, washed, and incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Inc) diluted in blotto (1:500 dilution), washed three times with PBS 1X-0.05% Tween 20, and developed with diaminobenzidine substrate kit (Vector Laboratories). We used purified goat polyclonal antibody raised against the ribosomal protein S6 (RPS6) (molecular mass, ∼ 27.5 KDa) as control.
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2.5. Evaluation of nitric oxide synthesis. For detection of intracellular nitric oxide synthesis, cells were trypsinized and resuspended in culture medium DMEM supplemented with 2% FBS. A droplet of 25 μl (∼ 2.5 × 105 cells) was placed in the center of a non-coated coverslip. After the cells were attached, the nitric oxide-sensitive fluorescence dye DAF-2DA (Molecular probes, Inc) was added (Sugimoto et al., 2000) and incubated for 120 min in the dark at room temperature, washed with fresh buffer PBS and then incubated for an additional 60 min to allow complete de-esterification of intracellular diacetates. Coverslips were examined in an inverted epifluorescence microscope and computational system (Incyt2 systems inc.). Fluorescence was analyzed with an excitation and emission (λ) of 495 and 515, respectively (Leikert et al., 2001; Nakatsubo et al., 1998; Nofer et al., 2004). 2.6. Effects of bradykinin on nitric oxide synthesis As positive control, experiments were carried out in order to characterize the effects of bradykinin [1 μM] on nitric oxide synthesis in rat coronary microvascular endothelial cells in culture. The specificity of the assay was tested adding bradykinin while NO synthesis was inhibited with L-nitro arginine methylesther (L-NAME) [10 μM]. 2.7. Effects of phenylephrine on nitric oxide synthesis To characterize the effects of phenylephrine on nitric oxide synthesis in rat coronary microvascular endothelial cells in culture, we used phenylephrine [1 μM] (Castillo-Henkel et al., 2001) in the absence and presence of L-NAME [10 μM].
Fig. 2. Immunoblot analysis of α1-adrenceptor subtypes. Panel A. Protein blots of α1-adrenoceptor subtypes α1A, α1B and α1D. Reactivity was made evident in a single protein band with an apparent molecular mass of ∼ 80 kDa for each α1adrenoceptor subtype. RPS6 was used as control. Panel B. Values of relative expression of each α1-adrenoceptor subtypes α1A, α1B y α1D, standardized against RPS6 from 4 assays. *p b 0.05 α1A, α1D vs α1B.
2.8. Effects of prazosin, propranolol and rawolscine on phenylephrine response In order to determine the type of adrenoceptor involved in agonist-induced nitric oxide synthesis, experiments adding phenylephrine were carried out in the absence or presence of prazosin, propranolol, or rawolscine, selective antagonists of α1, β and α2, adrenoceptors, respectively. 2.9. Effects of 5-metilurapidil, ciclazosin and BMY-7378 on phenylephrine-induced response To determine the subtype of adrenoceptor inducing nitric oxide synthesis, experiments were carried out to evaluate the effects of phenylephrine in the absence or presence of 5-metilurapidil, ciclazosin and BMY-7378 (8-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride, selective of α1A, α1B and α1D-adrenoceptor antagonists, respectively. 2.10. RT-PCR of α1D-adrenoreceptor subtype
Fig. 1. A. Immunoexpression images of α1A (left panel), α1B (central panel), and α1D (right panel), in coronary microvascular endothelial cells. B. Relative density expressed as pixels/area count for α1-adrenoceptor subtypes. Data are expressed as means ± SE and were obtained in at least 100 cells per assay. *p b 0.05 control vs each receptor subtype.
Based on previous reports suggesting the presence α1A receptor subtype in endothelial cells (Boer et al., 1999; Kaneko and Sunano, 1993) and on our results showing no functional participation of α1B-adrenoceptors in nitric oxide synthesis, we decided only to analyze the presence of RNA-related α1D-adrenoceptor subtype. RNA was obtained according to a previously described method. Briefly, total RNA was extracted from cells using the Perfect RNA™, Eukaryotic, Mini (Eppendorf). Isolated RNA was quantitated using the GENESYS™ 10 Series (ThermoSpectronic). RNA (5 μg) was separated on a 1.0% agarose gel
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containing ethidium bromide in MOPS buffer. Running buffer and gel contained 0.2 M formaldehyde. To prevent trace amount of DNA contamination, RNA samples were treated with amplification grade DNase I (Invitrogen) before reverse transcription. All RNA samples were stored at − 70 °C in RNA elution solution until further use. 0.5 μg of RNA was reverse-transcribed using AMV reverse transcriptase and Taq DNA polymerase (Titan One Tube RT-PCR System, Roche). The protocol
followed was: 50 °C (30 min), 94 °C (2 min), 35 cycles of denaturation (94 °C; 10 seg), annealing (63 °C; 30 seg), and elongation (68 °C; 45 seg). Specific oligonucleotide primers were originally generated by using the Fast PCR program and checked by web program Primer3. Primer sequences and PCR size were: 5′-GTT CCC GCA GCT GAA ACC GTC-3′ (sense) and 5′-AGG CTG GCT TTC GAC TGC TGG-3′ (anti-sense), 349 bp. Each PCR product (10 μl)
Fig. 3. Fluorescence microscopy images of intracellular NO generation, cells were incubated with NO-sensitive fluorescence dye DAF-2DA. Fluorescence was analyzed with an excitation and emission (λ) of 495 and 515, respectively. Panel A, Bradykinin (1 μM) and phenylephrine (1 μM) effects, in absence and presence, of L-NAME (10 μM). For the analysis of the adrenergic receptor type, implicated in the response, we assay phenylephrine (1 μM), in absence and presence, of prazosin, propranolol and rawolscine (10 μM) and for adrenergic receptor subtype, phenylephrine (1 μM), in absence and presence, of 5-metilurapidil, ciclazosin and BMY-7378 (10 μM). Panel B. Graphs of pixels/area count, B1; effect of bradykinin (1 μM) and phenylephrine (1 μM) on nitric oxide generation, responses to each agonist were abolished in presence of L-NAME (10 μM), B2; effect of phenylephrine (1 μM), in absence and presence of adrenergic receptor types antagonists, and B3; effects of phenylephrine (1 μM) in absence and presence of α1-adrenoceptor subtypes antagonists. Values are expressed as means ± SE (n = 3), *p b 0.05 phenylephrine (1 μM) vs. B1, B2 and B3.
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was used for electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. 2.11. Statistical analysis Data are expressed as mean ± S.E.M. and were analyzed by one-way ANOVA. Data are compared vs. control experiments. Differences were considered significant at p b 0.05. 3. Results 3.1. Immunoexpression of α1-adrenergic receptor subtypes Our results show the presence of α1-adrenoceptor subtypes, α1A, α1B, and α1D in coronary microvascular endothelial cells. In Fig. 1A are shown, representative images of immunofluorescence expression of α1 adrenoceptors subtypes in this cell type. The relative expression of the receptor subtypes was obtained by an analysis of density of fluorescent intensity (pixels/area, IPLab 3.61 software), in at least 100 cells by experiment (n = 4). Our results show that the relative density order is α1D N α1A N α1B (Fig. 1B). 3.2. Immunoblotting With this approach, we also found the expression of α1A, α1B, and α1D-adrenoceptor subtypes in rat coronary microvascular endothelial cells. Immunoreactivity was made evident in a single protein band for each α1-adrenoceptor subtype with an apparent molecular mass of ∼ 80 kDa in homogenates of cells (Fig. 2A). The RPS6 was used as internal control (single band, ∼ 30 kDa). No bands were detected when the primary antibody was omitted (data not shown). Fig. 2B shows the relative expression of the receptor subtypes, the order of which is similar to that found in immunofluorescent studies (n = 4).
Fig. 4. RT-PCR of α1D receptor subtype. Primer sequences and PCR size were: 5′-GTT CCC GCA GCT GAA ACC GTC-3′ (sense) and 5′-AGG CTG GCT TTC GAC TGC TGG-3′ (anti-sense), 349 bp. specific mRNA transcripts were detected and shown as 349-pb fragment. Figure shows 2 repetitions.
partially diminished by 5-metilurapidil and BMY-7378, and abolished with a mix of both of them (Fig. 3B 3). No blockage of phenylephrine-induced effects was found in the presence of ciclazosin. We are aware of the possible toxic effects of [10 μM] antagonists employed. The cells in culture show no signals of toxicity, in fact they are still able to respond to stimuli (bradykinin or acetylcholine) in periods of time 3 times longer of experimental times employed (no longer times were assayed). 3.4. RT-PCR of α1D receptor subtypes Our results of RT-PCR analysis of α1D-adrenoceptor subtype show, specific mRNA transcripts, detected and shown as 349pb fragment (Fig. 4).
3.3. Nitric oxide synthesis. 4. Discussion To examine whether phenylephrine has a direct effect on nitric oxide generation in coronary microvascular endothelial cells, we incubated the cells with the NO-sensitive fluorescence dye DAF-2DA and stimulated with bradykinin (positive control) or phenylephrine (1 μM) for 60 min in the presence and absence of L-NAME. This resulted in a substantial increase in nitric oxide-dependent fluorescence intensity (Fig. 3A), which was abolished by pretreatment in both cases with L-NAME [10 μM] (Fig. 3B 1). On the other hand, to examine the adrenergic receptor subtype involved in phenylephrine-induced effects, we assayed phenylephrine in the presence and absence of prazosin, propranolol and rawolscine [10 μM], selective α1, β and α2 adrenoceptor antagonists respectively, and 5-metilurapidil, ciclazosin and BMY7378 [10 μM], selective of α1A, α1B and α1D adrenoceptor subtype antagonists respectively. Our results show that the phenylephrine-induced substantial increase in nitric oxide-dependent fluorescence intensity (Fig. 3A) was abolished by pretreatment with prazosin (Fig. 3B 2). It was
The function of organs and corporal tissues depends on adequate blood flow, leading to appropriate supply of oxygen and nutrients. The magnitude of blood flow is inversely proportional to blood vessel tone. The tone that a blood vessel maintains is influenced by the amount of the humoral factors released. The constant regulation exerted by sympathetic nervous system and vascular endothelium, maintains a relatively low activity (tonic activity) on vascular smooth muscle in basal conditions. It has been reported that vasoconstriction, induced by stimulation of sympathetic nerves or by sympathomimetic agents, is attenuated significantly by the presence of functional vascular endothelium. Vascular endothelium exerts its modulation through the release of vasodilator substances, whose activity opposes the vasoconstrictor effects of sympathomimetic agents (Nishina et al., 1999; Dora et al., 2000). Several explanations have been proposed for this inhibitory phenomenon exerted by endothelium on contractile agents. It has been suggested that it is the result of the basal activity of
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endothelium and/or of the direct or indirect stimulation of this tissue by the contractile agents (Dora et al., 1997). In support for the indirect stimulating action of the sympathomimetic drugs on the endothelium, it has been postulated (Dora et al., 1997; Dora et al., 2000) that phenylephrine, exclusively acting on smooth muscle of small mesenteric arteries of rat, induces an indirect release of NO and EDHF from endothelium, which in turn attenuates the phenylephrine contractile effect on vascular smooth muscle. In the same sense, it has been suggested (Yashiro and Duling, 2000) that indirect stimulation of endothelium can be the consequence of increases in [Ca2+]i in vascular smooth muscle, passing through mioendothelial gap junctions from smooth muscle to endothelium. This [Ca2+]i increase induces in turn, an increase in production and release of the endothelial relaxant factors. On the other hand, the possibility that inhibitory modulation of endothelium has relation with a direct action of sympathomimetic drugs on vascular endothelium, is possible because the presence of α1-adrenoceptors has been reported in several vascular tissues and cellular entities (Filippi et al., 2001; Boer et al., 1999), although only few works suggest the presence of α1-adrenoceptors in endothelium (Boer et al., 1999; Kaneko and Sunano, 1993; Jones et al., 1993; Filippi et al., 2001). Our results show that in coronary microvascular endothelial cells exist α1-adrenoceptors, whose activation is conducive to nitric oxide synthesis. We observed that in coronary microvascular endothelial cells in culture, phenylephrine, an α1-adrenoceptor agonist, induced an increase in nitric oxide synthesis. This effect was abolished by prazosin, a selective α1-adrenoceptors antagonist. The phenylephrine-induced effects seem to be specific and involve nitric oxide synthase activity, because it was not inhibited by β and α2 specific adrenoceptor antagonists and it was blocked in the presence of L-NAME, a competitive inhibitor of nitric oxide synthase (Docherty, 1998; Summers and McMartin, 1993; Varma and Deng, 2000; Bylund, 1992). These results show, functionally, the presence and activity of α1-adrenoceptors in coronary microvascular endothelium in culture. In order to analyze the adrenoceptor subtype expressed in these cells we used 3 approaches: 1) immunocytochemical, 2) immunoblotting and 3) pharmacological. Approaches 1 and 2 showed almost the same results; α1A, α1B and α1D adrenoceptor subtypes are expressed in this type of endothelium. The analysis suggested that α1D is expressed at a higher rate followed by α1A, with α1B being the subtype with the lesser expression. The third approach, using relatively selective of α1-adrenoceptor antagonists for each subtype, allowed us to suggest functional characteristics of each receptor. Thus, 5-metilurapidil, an α1A-adrenoceptors antagonist and BMY-7378, an α1Dadrenoceptors antagonist, partially blocked the phenylephrineinduced increase in nitric oxide. The presence of both antagonists, completely blocked the phenylephrine effects. On the other hand, Ciclazosine, an α1B-adrenoceptors antagonist, induced no change in phenylephrine-induced effects. Together, these effects suggest that only α1A and α1D-adrenoceptors are functionally related to the synthesis of nitric oxide, in coronary microvascular endo-
thelial cells and even though α1B-adrenoreceptors are present, they are not associated with nitric oxide synthesis. We do not have a feasible explanation for these phenomena, although it might represent a nonspecific binding of α1B-adrenoreceptor subtype antibody (crossover), and more work is necessary in order to clarify it. On the other hand, the presence of the α1D-adrenoceptor subtype in vascular endothelium has been previously suggested (Filippi et al., 2001). Our RT-PCR results demonstrate the expression of the α1D-adrenoceptor subtype in CMEC (349-pb fragment corresponding to the reported sequence). In conclusion, the present findings suggest the presence of α1A, α1B and α1D-adrenoceptor subtypes in the endothelium of coronary arteries from male rat, but only α1A and α1D activation leads to nitric oxide production. The main goal of this work was to show functional coupling between α1 adrenoceptor activation and nitric oxide synthesis. Our results suggest that α1B-adrenoceptor subtype is not coupled to nitric oxide synthesis, and its presence is questionable. These results may explain the endothelial-dependent modulation of α1-adrenergic agonist-induced effects on vascular smooth muscle. Acknowledgments This work was supported by a CONACyT grant #46187/A and I.P.N. References Boer, C., Scheffer, G., de Lange, J., Westerhof, N., Sipkema, P., 1999. Alpha-1adrenoceptor stimulation induces nitric oxide release in rat pulmonary arteries. J. Vasc. Res. 36, 79–81. Bylund, D.B., 1992. Subtypes of alpha 1- and alpha 2-adrenergic receptors. FASEB J. 832–839. Castillo-Henkel, C., Asbun, J., Ceballos, G., Castillo, M.C., Castillo, E., 2001. Relationship between extra and intracellular sources of calcium and the contractile effect of thiopental in rat aorta. Can. J. Physiol. Pharm. 79, 407–414. Docherty, J.R., 1998. Subtypes of functional alpha1- and alpha2-adrenoceptors. Eur. J. Pharmacol. 361, 1–15. Dora, K.A., Doyle, M.P., Duling, B.R., 1997. Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles. Proc. Natl. Acad. Sci. 94, 6529–6534. Dora, K.A., Hinton, J.M., Walker, S.D., Garland, C.J., 2000. An indirect influence of phenylephrine on the release of endothelium-derived vasodilators in rat small mesenteric artery. Br. J. Pharmacol. 129, 381–387. Filippi, S., Parenti, A., Donnini, S., Granger, H.J., Fazzini, A., Ledda, F., 2001. Alpha(1D)-adrenoceptors cause endothelium-dependent vasodilatation in the rat mesenteric vascular bed. J. Pharmacol. Exp. Ther. 296, 869–875. Furchgott, R.F., Zawadzki, J.V., 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373–376. Gabella, G., 1984. Structural apparatus for force transmission in smooth muscles. Physiol. Rev. 64, 455–477. Gonzalez, C., Martin, C., Hamel, E., Galea, E., Gomez, B., Lluch, S., Estrada, C., 1990. Endothelial cells inhibit the vascular response to adrenergic nerve stimulation by a receptor-mediated mechanism. Can. J. Physiol. Pharm. 68, 104–109. Griendling, K.K., Alexander, R.W., 1996. Endothelial control of the cardiovascular system: recent advances. FASEB J. 10, 283–292. Ibarra, M., Meneses, A., Ransanz, V., Castillo, C., Hong, E., 1995. Changes in endothelium-dependent vascular responses associated with spontaneous hypertension and age in rats. Arch. Med. Res. 26, S177–S183.
E. Mendez et al. / European Journal of Pharmacology 546 (2006) 127–133 Jones, C.J., DeFily, D.V., Patterson, J.L., Chilian, W.M., 1993. Endotheliumdependent relaxation competes with α1 and α2-adrenergic constriction in the canine epicardial coronary microcirculation. Circulation 87, 1264–1274. Kaneko, K., Sunano, S., 1993. Involvement of alpha-adrenoceptors in the endothelium-dependent depression of noradrenaline-induced contraction in rat aorta. Eur. J. Pharmacol. 240, 195–200. Leikert, J.F., Rathel, T.R., Muller, C., Vollmar, A.M., Dirsch, V.M., 2001. Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein. FEBS Lett. 50, 131–134. Murphy, R.A., 1989. Contraction in smooth muscle cells. Annu. Rev. Physiol. 51, 275–283. Nakatsubo, N., Kojima, H., Kikuchi, K., Nagoshi, H., Hirata, Y., Maeda, D., Imai, Y., Irimura, T., Nagano, T., 1998. Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Lett. 427, 263–266. Nishida, M., Carley, W.W., Gerritsen, M.E., Ellingsen, O., Kelly, R.A., Smith, TW., 1993. Isolation and characterization of human and rat cardiac microvascular endothelial cells. Am. J. Physiol. 264, H639–H652. Nishina, H., Ozaki, T., Hanson, M.A., Poston, L., 1999. Mechanisms of noradrenaline-induced vasorelaxation in isolated femoral arteries of the neonatal rat. Br. J. Pharmacol. 127, 809–812. Nofer, J.R., van der Giet, M., Tolle, M., Wolinska, I., von Wnuck Lipinski, K., Baba, H.A., Tietge, U.J., Godecke, A., Ishii, I., Kleuser, B., Schafers, M., Fobker, M., Zidek, W., Assmann, G., Chun, J., Levkau, B., 2004. HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J. Clin. Invest. 113, 569–581. Pearson, P.J., Vanhoutte, P.M., 1993. Vasodilator and vasoconstrictor substances produced by the endothelium. Rev. Physiol., Biochem. Pharmacol. 122, 1–67.
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Rubio-Gayosso, I., Sierra-Ramirez, A., Garcia-Vazquez, A., Martinez-Martinez, A., Munoz-Garcia, O., Morato, T., Ceballos-Reyes, G., 2000. 17Beta-estradiol increases intracellular calcium concentration through a short-term and nongenomic mechanism in rat vascular endothelium in culture. J. Cardiovasc. Pharmacol. 36, 196–202. Sedmak, J.J., Grossberg, S.E., 1977. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal. Biochem. 79, 544–552. Sugimoto, K., Fujii, S., Takemasa, T., Yamashita, K., 2000. Detection of intracellular nitric oxide using a combination of aldehyde fixatives with 4,5diaminofluorescein diacetate. Histochem. Cell Biol. 113, 341–347. Summers, R.J., McMartin, L.R., 1993. Adrenoceptors and their second messenger systems. J. Neurochem. 60, 10–23. Tran, Y., Forster, C., 1996. Effect of angiotensin receptor blockade in the rabbit aorta: influence of the endothelium. Can. J. Physiol. Pharm. 74, 1277–1286. Tuttle, J.L., Falcone, J.C., 2001. Nitric oxide release during alpha1-adrenoceptormediated constriction of arterioles. Am. J. Physiol. 281, H873–H881. Vanhoutte, P.M., 2000. Say NO to ET. J. Auton. Nerv. Syst. 81, 271–277. Vanhoutte, P.M., Mombouli, J.V., 1996. Vascular endothelium: vasoactive mediators. Prog. Cardiovasc. Dis. 39, 229–238. Varma, D.R., Deng, X.F., 2000. Cardiovascular alpha1-adrenoceptor subtypes: functions and signaling. Can. J. Physiol. Pharm. 78, 267–292. Yashiro, Y., Duling, B.R., 2000. Integrated Ca(2+) signaling between smooth muscle and endothelium of resistance vessels. Circ. Res. 87, 1048–1054. Zschauer, A.O., Sielczak, M.W., Smith, D.A., Wanner, A., 1997. Norepinephrineinduced contraction of isolated rabbit bronchial artery: role of alpha 1- and alpha 2-adrenoceptor activation. J. Appl. Physiol. 82, 1918–1925.
