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Division of Cardiovascular Pathophysiology (A.F., P.M., J.D.) and Metabolic Research Laboratory (A.R., J.G.-A., G.F.), University of Navarra School of Medicine, and Departments of Endocrinology (J.S., G.F.) and Cardiology and Cardiovascular Surgery (J.D.), Clínica Universitaria de Navarra, 31008 Pamplona, Spain
Address all correspondence and requests for reprints to: Dr. G. Frühbeck, Department of Endocrinology, Clínica Universitaria de Navarra, 31008 Pamplona, Spain. E-mail: gfruhbeck{at}unav.es.
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Abstract |
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Introduction |
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The vasoactive peptide angiotensin II (ANG II) is an important factor regulating blood pressure (11). Alterations in the synthesis of and responsiveness to ANG II have been implicated in the pathophysiology of arterial hypertension (12). In addition, Barton et al. (13) found that obesity is associated with activation of the renin-angiotensin system in nonadipose tissue. Besides, an adipose tissue-specific increase in the production of angiotensinogen during the onset of obesity has recently been reported (14). According to previous studies, the link between angiotensinogen production and the control of adipose mass involves ANG II, which has been implicated in the differentiation of adipose precursors by paracrine/autocrine mechanisms (15). Therefore, the present study explored the possibility that leptin might interfere with the vascular responses induced by ANG II through OB-Rb. To test this hypothesis, the in vitro effects of leptin on both ANG II-induced increases in the intracellular free calcium concentration ([Ca2+]i) transients in vascular smooth muscle cells (VSMCs) and isometric contraction of endothelium-denuded and intact aortic rings from Wistar rats as well as leptin receptor-deficient obese Zucker rats were assessed. In addition, the expression of OB-Rb in VSMCs from Wistar rats was investigated.
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Materials and Methods |
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Cell isolation and culture
Primary VSMCs were obtained from the thoracic aorta of Wistar and Zucker rats and cultured by the tissue explants method in accordance with previously published procedures (16). The culture was continued up to eight passages using 5% fetal bovine serum with an antibiotic-antimicotic product (Life Technologies, Inc., Gaithersburg, MD; 10,000 U/ml penicillin G sodium, 10,000 µg/ml streptomycin sulfate, and 25 µg/ml amphotericin B as Fungizone in 0.85% saline). Immunocytochemical characterization of isolated VSMCs was performed on primary cultures, using specific antibodies against smooth muscle-specific 
-actin (DAKO Corp., High Wycombe, UK). VSMCs were differently processed depending on the study protocol.
Detection of leptin receptors in VSMCs
Total RNA was extracted in TRIzol reagent (Life Technologies, Inc.) from a 75-mm2 flask of VSMCs, according to the manufacturer’s instructions. The yield and quality of the RNA were assessed by measuring absorbance at 260 and 280 nm and by electrophoresis on 1.5% agarose gels. One microgram of RNA was used to synthesize first strand cDNA after treatment for 60 min at 37 C with 10 U ribonuclease-free deoxyribonuclease I (Roche Molecular Biochemicals, Mannheim, Germany). The RT reaction was carried out in a volume of 20 µl containing 50 mmol/liter Tris-HCl (pH 8.3), 75 mmol/liter KCl, 3 mmol/liter MgCl2, 10 mmol/liter dithiothreitol, 100 ng random hexamers (Roche Molecular Biochemicals), 1 mmol/liter of each deoxy-NTP (Bioline, London, UK), 20 U ribonuclease inhibitor (Promega Corp., Madison, WI), and 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) and incubated at 37 C for 60 min. The enzyme was inactivated by heating at 95 C for 5 min. Four microliters from the RT reaction were amplified in a 50-µl reaction mixture containing 40 ng of each primer, 16 mmol/liter (NH4)2SO4, 67 mmol/liter Tris-HCl (pH 8.8), 2 mmol/liter MgCl2, 0.1% Tween 20, 0.2 mmol/liter of each deoxy-NTP, and 1 U BIOTAQ polymerase (Bioline). Primers used to amplify rat OB-Ra and OB-Rb cDNA were a common forward primer (5'-ACACTGTTAATTTCACACCAGAG-3') used in combination with a specific reverse primer either for the short (a) form (5'-AGTCATTCAAACCATAGTTTAGG-3') or the long (b) form (5'-TTCCAAAAGCTCATCCAACCC-3'). As a control, mock amplifications were carried out in the presence of RNA template and Taq polymerase, but in the absence of reverse transcriptase. As a control for RNA quality and quantity, ß-actin mRNA was amplified from all RNA samples using primers 5'-TCTACAATGAGCTGCGTGTG-3' and 5'-GGTCAGGATCTTCATGAGGT-3', based upon the sequence of rat ß-actin. cDNA was amplified for 40 (OB-Ra), 45 (OB-Rb), and 35 cycles (ß-actin) using the following parameters: 94 C for 30 sec, 55 C (OB-Ra and OB-Rb) and 59 C (ß-actin) for 30 sec, and 72 C for 30 sec, with a final extension step at 72 C for 7 min. Amplifications were carried out in a GeneAmp PCR System 2400 (Perkin-Elmer, Norwalk, CT). The amplified products were resolved in a 1.5% agarose gel with ethidium bromide. Images were captured with the Gel Doc 1000 UV fluorescent gel documentation system and Molecular Analyst 1.4.1 software (Bio-Rad Laboratories, Inc., Hercules, CA).
VSMCs from the aortas of Wistar rats were homogenized in ice-cold lysis buffer containing sacarose (0.32 mol/liter) and protease inhibitor cocktail (Roche). The protein content of the crude preparations was measured by the Bradford method (17), using lyophilized BSA (Bio-Rad Laboratories, Inc.) as standard. Equal amounts of proteins (30 µg) were run out in 8–12% SDS-PAGE, subsequently transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, UK) by electrotransfer, and blocked in 4% Tris-buffered saline-Tween 20 for 60 min at room temperature under agitation. The membranes were incubated overnight at 4 C under agitation with an N-terminal polyclonal antibody against OB-R (1:500) in 4% Tris-buffered saline-Tween 20. The antigen-antibody complexes were visualized using peroxidase-conjugated antigoat IgG antibody (1:10,000) and the enhanced chemiluminescence ECL detection system (Amersham Pharmacia Biotech).
The presence of OB-R immunoreactivity on intact VSMCs was studied by immunocytochemistry. Cells were grown on four-well plates, fixed with 4% paraformaldehyde, and washed with cold PBS. Fixed cells were treated with 5% dimethylsulfoxide and 0.25% Triton X-100 solution. After blocking with 4% fetal bovine serum for 1 h, cells were incubated overnight at 4 C with rat polyclonal antiserum specific for the N-terminal region of OB-R (1:100). After washing three times, cells were incubated with biotinylated goat antirat IgG antibody (1:100) in PBS for 1 h, washed, and reacted with avidin-fluorescein isothiocyanate conjugate (1:100; Sigma, St. Louis, MO) for 30 min. Finally, VSMCs were mounted in synthetic resin with 4',6-diamidino-2-phenylindole. All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Measurement of [Ca2+]i in VSMCs
Measurement of [Ca2+]i was performed as previously described (16). Briefly, nonconfluent cultures of VSMCs grown on round 22-mm coverslips were incubated in a humidified incubator for 45 min with fura-2/AM (4–7 µmol/liter) dissolved in dimethylsulfoxide with 0.02% pluronic acid. Fura-2-loaded VSMCs were placed in a thermostatically perfusion chamber on top of the stage of an inverted microscope equipped for epifluorescence (Diaphot, Nikon, Badhoevedorp, The Netherlands) and attached to an image analysis system (Magiscal Applied Imaging, Newcastle upon Tyne, UK). Upon binding Ca2+, fura-2 exhibits an absorption shift from 340 to 380 nm that can be monitored by measuring the emission at 510 nm. The [Ca2+]i was calculated as described by Grynkiewicz et al. (18).
Preparation of isolated aortic rings
The thoracic aorta was removed immediately after sacrifice, cleaned, and placed into Krebs-Heinseleit solution as previously described (16). The endothelium of the aorta was removed by gently rubbing the intimal surface with the tip of a small steel stick. In some experiments the endothelium of the aorta was preserved to analyze the effect of leptin on intact aortic rings. Both intact and endothelium-denuded rings were incubated for 60 min to equilibrate at a resting tension of 2.5 g, with buffer changes every 15 min to reach equilibrium conditions. The lack of a functional endothelium was confirmed by demonstrating the complete absence of relaxation induced by acetylcholine (10-5 mol/liter) in 80 mM KCl-precontracted aortic rings. The rings were then washed and stretched if necessary until a stable baseline force was obtained. The contraction with KCl was also used to test the contractile response of the tissue.
Study of vasoactive responses
Cumulative concentration-response curves to 10-9–10-6 mol/liter ANG II were obtained to determine the concentration needed for submaximal effects and the pD2 value. In a second group of experiments, VSMCs or aortic rings were previously incubated with different concentrations of leptin (10-10–10-7 mol/liter) before testing the ANG II-induced response. To study the specificity of the inhibitory effect of leptin on ANG II-mediated actions the response to another vasoactive substance was further tested. As leptin has been shown to induce a sympathoexcitation (1, 7, 19, 20, 21), and the sympathetic nerve activity represents one of the most important factors controlling ongoing vasoconstriction, the effect of leptin on the noradrenaline (NA; 10-7 mol/liter)-induced contractile response was examined. One sample per experiment was used to obtain control responses (100%) in the presence of the solvent. Repetitive use of experimental products on the same aortic ring preparations was avoided to stay clear of possible desensitization.
Statistical analysis
Values are given as the mean ± SEM. Statistical differences between mean values were determined using one-way ANOVA, followed by Dunnett’s t test. When the number of determinations were less than 10, statistical analysis was performed using the nonparametric Kruskal-Wallis test. P < 0.05 was considered statistically significant. Concentration-response curves were fitted by nonlinear regression, the concentration giving 50% of the maximal response (EC50) was determined, and the pD2 was calculated as -log EC50 (mol/liter).
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Results |
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). The receptor splice variants OB-Ra and OB-Rb were expressed in VSMCs, as demonstrated by PCR products of 237 and 612 bp, respectively. The easy detection of OB-R expression confirms the vascular smooth muscle as a potential target for leptin action.
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, a 130-kDa band was found in all experiments performed (n = 12). Due to the N-terminal nature of the polyclonal antibody used against OB-R, four additional bands of lower molecular weights were also observed. No immunoreactivity was found without the primary antibody (data not shown). The immunocytochemical analysis of the cellular distribution of OB-R indicates that all VSMCs were immunopositive, exhibiting a whole-cell staining pattern (Fig. 1C).
Effects of leptin on ANG II-induced [Ca2+]i responses in VSMCs
In the presence of 1.3 mmol/liter Ca2+ in the bathing medium, ANG II increased [Ca2+]i in VSMCs from Wistar rats in a concentration-dependent manner (pD2 = 8.6 ± 0.6). A concentration of 10-7 mol/liter ANG II, inducing a response of 728.2 ± 51.1 nmol/liter (basal [Ca2+]i, 64.8 ± 9.3 nmol/liter), was chosen for subsequent experiments.
Although no basal effects of leptin on [Ca2+]i in VSMCs were observed (data not shown), all leptin concentrations tested inhibited (P < 0.01) ANG II-induced [Ca2+]i stimulation in VSMCs from Wistar rats (Fig. 2A). The inhibitory effect was independent of cell passage. VSMCs exposed to ANG II showed a rapid rise in [Ca2+]i that reached a maximum in 10 sec, declined rapidly within 30–40 sec, and then gradually decreased in the next 1–2 min to near-resting values (Fig. 2B). In the presence of leptin (10-8 mol/liter) the maximum value reached was inhibited by approximately 75% (Fig. 2B).
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). However, the same leptin concentration was not able to inhibit ANG II (10-7 mol/liter)-induced [Ca2+]i stimulation in VSMCs obtained from Zucker rats (Fig. 3B).
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). No inhibitory effect of leptin was observed on ANG II-induced vasoconstriction in endothelium-denuded aortic rings from Zucker rats (Fig. 4B).
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Finally, to test the specificity of the inhibitory effect of leptin on ANG II, the response to a well known vasoactive substance such as NA was also studied. As expected in both intact and endothelium-denuded aortic rings, NA (10-7 mol/liter) produced a statistically significant (P < 0.001) vasoconstriction. As shown in Fig. 5, no significant effect of leptin (10-8 mol/liter) on the NA-induced contractile response was observed in either intact or endothelium-denuded aortic rings.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-nitro-L-arginine methyl ester administration. On the other hand, leptin has been shown to induce a clear vasodilation in both aortic and mesenteric arteries, which is abolished by endothelial denudation, suggesting that endothelium integrity is critical for leptin’s vascular relaxing action (8). Furthermore, functional leptin receptors have been identified in endothelial cells (22, 23, 24). Therefore, leptin seems to be able to stimulate endothelial NO-mediated vasodilation, although this fact has been questioned by Mitchell et al. (25) using the same physiological leptin concentrations as other research groups. Further support of the NO-mediated leptin actions has been provided by Vecchione et al. (26), showing that the effect of leptin on endothelial NO is mediated through the Akt-endothelial NOS phosphorylation pathway. The vasodilator effect has been shown to be dependent on the presence of the endothelium. Moreover, findings reported in the present study suggest that VSMCs may represent a target for the vasodilation actions of leptin by opposing ANG II-induced effects leading to vasoconstriction. The present study confirms that functional leptin receptors are expressed in VSMCs of Wistar rats. The band detected with a molecular mass of approximately 120–130 kDa probably corresponds to OB-Rb, which has been previously described in other animal and human cell types (27, 28, 29). This finding supports recent evidence of the expression of functional leptin receptors in calcifying vascular cells (30) and aortic smooth muscle cells (31). Together these results support the hypothesis that leptin plays a relevant role in modulating vasoregulatory function directly through OB-Rb on the vascular wall. Due to the fact that the antibody used to detect OB-R expression in Western blot analysis is raised against the N-terminal region, common to all five isoforms of the receptor, four additional bands of lower molecular weights, probably representing the different receptor isoforms, were also identified in VSMCs (32). The possibility that OB-Rb is functional in VSMCs is supported by other findings reported herein: 1) physiological concentrations of leptin block the ANG II-induced rapid increase in [Ca2+]i in cultured VSMCs of the aorta from Wistar rats; 2) leptin inhibits the ANG II-induced fast contraction in endothelium-denuded aortic rings from Wistar rats; and 3) no effects of leptin were found in the aorta and VSMCs obtained from Zucker rats with a mutation in the leptin receptor gene that results in the loss of its functional activity.
Central to the direct vasoconstrictor action of ANG II on smooth muscle is its capacity to increase [Ca2+]i. After the interaction with the AT1 receptor, ANG II mobilizes Ca2+ by releasing Ca2+ sequestered intracellularly and by increasing Ca2+ influx from the extracellular compartment. The former event may occur via phosphoinositide turnover, and the latter in association with voltage-sensitive Ca2+ channels (11, 33). As a consequence, [Ca2+]i increases and VSMC contraction ensue. Our finding that leptin retains the ability to inhibit ANG II-mediated stimulation of VSMC [Ca2+]i in Ca2+-free medium strongly suggests that the hormone blocks the ANG II-induced release of Ca2+ from the intracellular stores. Takekoshi et al. (34, 35) reported recently that leptin induces increases in cAMP levels in cultured porcine adrenal medullary chromaffin cells. As cAMP facilitates Ca2+ sequestration by the sarcoplasmic reticulum (36), we speculate that cAMP may mediate the ability of leptin to inhibit ANG II-induced stimulation of intracellular Ca2+ stores in VSMCs. Further studies are required to elucidate the ability of leptin to stimulate cAMP in these cells.
To improve our understanding of the physiological significance of leptin on ANG II-induced vasoconstriction, the effects were tested not only in denuded aortic rings, but also in intact vessels. Contrary to what was observed in endothelium-denuded preparations, in intact aortic rings no effect of leptin on ANG II-induced vasoconstriction was observed. Several plausible explanations may underlie this finding. Leptin may simultaneously induce the release of other vasoactive substances, with either a direct or indirect effect on the vascular tone of the ring. It is well known that the vasoconstrictor effect elicited by ANG II has a 2-fold component, a direct and an indirect effect, depending on the release of further endothelium-derived factors with a vasoconstrictor effect such as endothelin-1 (37). In addition, the incubation period used in our experiments was 30 min, and during this period the endothelium was exposed to the plentiful release of vasoactive factors such as NO and endothelin-1, among others. In fact, recombinant leptin has been shown to induce a slight decrease in passive wall tension (8). We have confirmed this finding in our study, showing that in intact aortic rings leptin produced a significant decrease in passive wall tension that was not observed in endothelium-denuded rings. The different molecular weights of ANG II and leptin (1 vs. 16 kDa, respectively) may also be responsible for the different transport possibilities and, consequently, the different abilities to reach the layer of VSMCs. Interestingly, it has been recently reported that leptin released by the fat depot produces a dose-dependent increase in plasma NO3-NO2 concentrations (38). In this sense, it may be speculated that the effects of leptin on the control of vascular tone may arise from two different sources. On the one hand, leptin present in the vascular lumen and, on the other hand, leptin released from perivascular adipocytes may participate in regulation of contraction of the vascular wall.
The lack of effect of leptin on vascular preparations from Zucker rats provides a potential mechanistic explanation for obesity-associated hypertension, i.e. the loss of antagonism of ANG II-mediated vasoconstriction. Two arguments add support to this possibility: 1) obesity-associated hypertension is characterized by both leptin resistance (19) and stimulation of the renin-angiotensin system (13); and 2) exaggerated vasoconstriction (39) and prolonged VSMC [Ca2+]i exposure (40) to ANG II have been reported in Zucker rats.
In conclusion, our results provide additional evidence that OB-Rb are expressed in VSMCs from the aorta of Wistar rats and, therefore, represent a target for leptin action. Our findings indicate that leptin inhibits ANG II-mediated vasoconstriction by blocking the release of Ca2+ from VSMC intracellular stores. The lack of effect of leptin on ANG II-mediated effects of vascular preparations obtained from leptin receptor-deficient Zucker rats suggests that OB-Rb are functional in the vascular wall of Wistar rats. Further studies are required to elucidate the pathophysiological relevance of this new vascular action of leptin in human obesity-associated arterial hypertension.
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Acknowledgments |
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Footnotes |
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Abbreviations: ANG II, Angiotensin II; [Ca2+]i, intracellular free calcium concentration; NA, noradrenaline; NO, nitric oxide; VSMC, vascular smooth muscle cell.
Received January 22, 2002.
Accepted for publication May 12, 2002.
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) and presence (
) of 10-8 mol/liter leptin. Maximal stimulated [Ca2+]i occurred at 1–3 sec, and the recovery time to basal concentrations was measured thereafter. Values are the mean ± SEM from 10–15 determinations/concentration. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. curve obtained in the absence of leptin).
) and endothelium-denuded (
) aortic rings in the absence of or after preincubation for 30 min with leptin (10-8 mol/liter). B, Histograms showing the ANG II (10-7 mol/liter)-induced changes in tension of endothelium-denuded aortic rings from adult obese Zucker rats in the absence or presence of leptin (10-8 mol/liter). Values are the mean ± SEM from at least 10 determinations/concentration. **, P < 0.01 vs. control response.