http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2015; 53(4): 582–587 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.934964
ORIGINAL ARTICLE
Cardiovascular effects of a labdenic diterpene isolated from Moldenhawera nutans in conscious, spontaneously hypertensive rats
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Carlos Antoˆnio de Barros Correia Junior1, Rodrigo Jose´ Bezerra de Siqueira2, Leylliane Fa´tima Leal Interaminense1, Thayane Rebeca Alves-Santos1, Gloria Pinto Duarte1, Jorge Maurı´cio David3, Juceni Pereira David4, Pedro Jorge Caldas Magalha˜es2, and Saad Lahlou2 1
Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, PE, Brazil, 2Department of Physiology and Pharmacology, Federal University of Ceara´, Fortaleza, CE, Brazil, 3Institute of Chemistry, Federal University of Bahia, Salvador, BA, Brazil, and 4Pharmacy Faculty, Federal University of Bahia, BA, Brazil Abstract
Keywords
Context: The labdenic diterpene labd-8(17)-en-15-oic acid (labd-8) isolated from a methanolic extract of Moldenhawera nutans Queiroz & Alkin (Leguminosae) has hypotensive and tachycardiac properties in normotensive rats. A part of the hypotensive effect was due to a reduction in the sympathetic nerve drive to vessels, an event admittedly enhanced in spontaneously hypertensive rats (SHRs). Objectives: We assessed whether the cardiovascular effects induced by labd-8 could be enhanced in SHRs. Materials and methods: For in vivo experiments, arterial and venous catheters were implanted under anesthesia for blood pressure recording and drug administration, respectively. For in vitro experiments, thoracic aorta rings were suspended in organ baths containing warm (37 C) perfusion medium that was continuously bubbled with carbogen. Results: Intravenous injection of labd-8 (1, 3, 5, and 10 mg/kg) induced similar dose-dependent hypotension and tachycardia in both SHRs and Wistar–Kyoto rats (WKY). In SHRs, only the tachycardia response to labd-8 was significantly reduced by pretreatment with methylatropine or propranolol. However, both cardiovascular effects of labd-8 were reduced by hexamethonium while remained unchanged by L-NAME. In isolated aortic preparations from SHRs, labd-8 (1–1000 mg/mL) relaxed potassium-induced contractions with an IC50 (geometric mean [95% confidence interval]) value (536.5 [441.0–631.9] mg/mL) significantly greater than that (157.6 [99.1–250.5] mg/mL) obtained in preparations from WKY rats. Conclusion: In SHRs, the hypotension induced by labd-8 is associated with a reflex tachycardia and seems mediated partly through withdrawal of sympathetic vasomotor tone and partly through an active vasorelaxation. Its magnitude was not enhanced when compared with WKY rats likely because of impaired vasorelaxant effects of labd-8 in preparations from SHRs.
Autonomic nervous system, hypotension, isolated rat aorta, labd-8(17)-en-15-oic acid, relaxation, tachycardia
Introduction Diterpenes form a family of natural isoprenoid compounds derived from 2E,6E,10E-geranylgeranylpyrophosphate, a lead molecule that can suffer cyclization to generate two bicyclic enantiomer series, one nor-labdane that possesses chemical resemblance with steroids and another ent-labdane that differs from the former only by spatial configuration in a few carbons (Bruneton, 1995). In general, antimicrobial and antiproliferative properties have been attributed to labdane diterpenes (Pertino et al., 2013) and perhaps the most popular is forskolin (7b-acetoxy-8, 13-epoxy-1a,
Correspondence: Dr. Saad Lahlou, Department of Physiology and Pharmacology, Federal University of Ceara´, Rua Cel, Nunes de Melo 1127, 60431-270 Fortaleza, CE, Brazil. Tel: +55 85 3366 8334. Fax: +55 85 3366 8333. E-mail: [email protected]
History Received 18 February 2014 Revised 2 May 2014 Accepted 4 June 2014 Published online 9 December 2014
6b, 9a-trihydroxy-labd-14-ene-11-one), a well-known activator of adenylate cyclase (Sonoki et al., 1986). In the cardiovascular system, forskolin induced hypotension through its vasodilator effects upon vascular smooth muscle in laboratory animals (Dubey et al., 1981; Lindner et al., 1978). Several other labdane-type diterpenes have been reported to induce cardiovascular effects in rats. For instance, marrubenol was shown to induce vasorelaxant effects through blockade of L-type calcium channels (El-Bardai et al., 2003). The diterpene 8 (17),12E,14-labdatrien-18-oic acid decreased blood pressure in conscious normotensive rats, an effect mainly attributed to reduced peripheral vascular resistance through activation of the NO-pathway and blockage of L-type calcium channels (de Oliveira et al., 2006). Recently, it was shown that labdane ent-3-acetoxy-labda-8(17),13-dien-15-oic acid induced hypotension and vascular relaxation through the activation of the endothelial NO-cGMP pathway, the
Cardiovascular effects of labd-8 in SHRs
DOI: 10.3109/13880209.2014.934964
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of the autonomic nervous system in the mediation of cardiovascular responses to labd-8 was assessed in SHRs.
Materials and methods
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Plant material, extraction, and chemical analysis
Figure 1. Chemical structure of labd-8(17)-en-15-oic acid.
opening of potassium channels, and the alteration of calcium mobilization (Simplicio et al., 2014). Moldenhawera (Leguminosae: Caesalpinoideae) is a small neotropical genus represented by approximately 10 species (Queiroz et al., 1999). The taxonomy of this genus is confused, making difficult the classification of the species. Moldenhawera nutans Queiroz & Alkin (Leguminosae) is an endemic shrub of the coastal dunes of Salvador, State of Bahia, Brazil. Species of this genus are employed as a source for construction wood and the flowers of M. nutans are visited by bees for production of wild bee products (Viana et al., 2006). However, the former phytochemical study with the stems of this species was initiated to investigate the compounds responsible for the activity exhibited by an organic extract in the HIV-1 RT Assay (Roche, New York, NY) and the isolation and identification of a new dimeric diterpene were reported (David et al., 1998). Acid labdenic diterpenes from the ent-labdane class were previously described in the methanolic extract of M. nutans (David et al., 2007). The major labdenic diterpene found in this methanolic extract is labd-8(17)-en-15-oic acid (labd-8; Figure 1). We have first reported in conscious rats the dose-dependent decreases in mean arterial pressure (MAP) and increases in heart rate (HR) caused by the intravenous (i.v.) injection of labd-8 (Lahlou et al., 2007). As the hypotension was only reduced by blockade of ganglionic neurotransmission with hexamethonium, it was suggested that this response is partly due to withdrawal of sympathetic tone to the vasculature and partly to an active vascular relaxation (Lahlou et al., 2007). Indeed, labd-8 also relaxed rat-isolated aortic preparations contracted with KCl in a concentration-dependent manner. The present investigation was undertaken to give further support to these findings by assessing whether the magnitude of hypotensive effects of labd-8 is greater in conscious, spontaneously hypertensive rats (SHRs) in which the basal sympathetic activity is increased (Judy et al., 1976). Therefore, cardiovascular responses to labd-8 were studied in conscious SHRs and compared with those of normotensive Wistar–Kyoto (WKY) rats. Vascular effects of this diterpene were also compared with isolated thoracic aortic preparations obtained from SHRs and WKY rats. Furthermore, the role
Collection of stem parts (2.1 kg) of the plant occurred on November 2000 at the Metropolitan Park area of Abaete´, Salvador, State of Bahia, Brazil. Botanical identity of the species M. nutans was confirmed by Dr. Maria Lenise da Silva Guedes by comparison with a voucher specimen (#029057) registered at the Alexander Leal Costa Herbarium, Institute of Biology, Federal University of Bahia, Brazil. Extraction and chemical isolation were done from the dry raw material following the procedures previously reported (David et al., 1998; Lahlou et al., 2007), which achieved a purity grade of 99.5% w/w as determined by GC/MS analysis. Moreover, spectral analysis and NMR evaluation served to elucidate the molecular structure of labd-8 (David et al., 1998). The 1H, 13C NMR, and DEPT spectra were obtained on a Varian Gemini 2000 instrument (Varian, Palo Alto, CA) employing CDCl3 as both a solvent and a reference. Optical rotation was obtained in a Model 343 Perkin Elmer polarimeter (PerkinElmer, Akron, OH). Labd-8(17)-en-15-oic acid: [ ]D + 26.88 (c 0.8, MeOH); 1H NMR (300 MHz, pyridine-d6): -4.88 (s, 1H, H-17a), 4.55 (s, 1H, H-17b), 0.97 (d, J 6.5 Hz, 3H, Me-16), 0.84 (s, 3H, Me-19), 0.81 (s, 3H, Me-18), and 0.78 (s, 3H, Me-20); 13 C NMR (pyridine-d6): 35.9 (C-1), 19.3 (C-2), 41.9 (C-3), 33.7 (C-4), 56.9 (C-6), 24.4 (C-6), 42.2 (C-7), 148.6 (C-8), 55.5 (C-9), 39.7 (C-10), 21.0 (C-11), 39.2 (C-12), 33.6 (C-13), 38.3 (C-14), 179.8 (C-15), 19.7 (C-16), 106.4 (C-17), 21.8 (C-18), 30.7 (C-19), 14.6 (C-20). Solutions and drugs For in vivo experiments, the solvent employed to dissolve labd-8 was Tween 80 (2% v/v), which was brought to a given volume with sterile isotonic saline. Drugs were administered in a volume of 1 mL/kg body weight. Each i.v. injection was followed by a 60 mL (catheter volume) flush of physiological saline to ensure complete delivery of the dosage. For the experiments with isolated preparations, labd-8 in Tween 80 was dissolved directly in Tyrode’s solution, which was employed as the perfusion medium to maintain the viability of isolated preparations. The modified Tyrode’s solution was composed of (in mM): NaCl 136, KCl 5, MgCl2 0.98, CaCl2 2, NaH2PO4 0.36, NaHCO3 11.9, and glucose 5.5. All labd-8 solutions were sonicated just before use. Other drugs in this study were purchased from Sigma (St. Louis, MO) and were dissolved in saline just before use. Animals Adult male SHRs and WKY rats (age: 16–18 weeks) were used in this study. They were obtained from the institutional vivarium of the Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, Brazil. Animals were kept under controlled conditions of light (12 h lightdark cycle) and temperature (22 ± 1 C) with free access to water and standard rat chow. All animals were cared for in
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compliance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication 85-23, revised 1996). All procedures described here were reviewed by and had prior approval from local animal ethics committee.
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In vivo experiments Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg), and catheters (PE-10 fused to PE-50) were implanted in the abdominal aorta (for the recording of arterial blood pressure) and in the inferior vena cava (for drug administration) through the left femoral artery and vein, respectively, as described previously (Lahlou et al., 2007; de Siqueira et al., 2014). Twenty-four hours later, baseline MAP and HR were recorded on a Gilson model 5/6H polygraph (Medical Electronics Inc., Middletown, WI), as described previously (Lahlou et al., 2007). The maximal changes in MAP and HR caused by i.v. injections of increasing bolus (100 mL each) doses of labd-8 (1-10 mg/kg) were determined in conscious SHRs and WKY rats. In a separate set of animals, cardiovascular responses to labd-8 (1–10 mg/kg) were also studied in SHRs which have been pretreated intravenously 10 min earlier with either vehicle (1 mL/kg, n ¼ 5), hexamethonium (30 mg/kg, n ¼ 6), methylatropine (1 mg/kg, n ¼ 6), propranolol (2 mg/kg, n ¼ 5), or L-NAME (20 mg/kg, n ¼ 5). In vitro experiments In another set of experiments, SHRs and WKY rats were euthanized by stunning and exsanguination. After a median incision, segments of thoracic aorta were obtained as ring-like preparations that were immersed in bath chamber filled with a perfusion medium maintained at 37 C under constant bubbling with 95% O2 and 5% CO2 and pH 7.4. Resting tension was settled at 1 g through disposal of ring-like preparations between two points: one fixed in the bath and another in a force transducer disposed in a device appropriate for tension adjustment. Such set-up allowed the isometric tension recordings that were performed according to the previously described procedure (Interaminense et al., 2013; Lahlou et al., 2007). At the beginning of the experiment, each aortic ring was pre-contracted with phenylephrine (0.1 mM) and thereafter challenged by acetylcholine (1 mM) to evaluate the integrity of endothelium. Preparations were considered to possess an intact endothelium when the vasorelaxant response to acetylcholine was greater than 75%. Vasodilator effects were assessed only once in each thoracic aorta preparation by adding increasing concentrations of labd-8 (1–1000 mg/mL) on the steady state of sustained contractions induced by a high K+ (60 mM) concentration in endothelium-intact aortic preparations from SHRs (n ¼ 8) and WKY rats (n ¼ 5). A similar experiment was performed with increasing concentrations (0.04–4.1 mg/mL) of forskolin, a well-known activator of adenylate cyclase, used herein as a positive control. Statistical analysis Data are expressed as means ± standard error of the mean (S.E.M.). Maximal changes in MAP and HR after administration of a given dose of labd-8 were expressed
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as a percentage of baseline values. For in vitro data, peak deflections were used to measure the magnitude of the concentration–response curves, which were expressed as a percentage of KCl-induced contraction (in the absence of labd-8). The IC50 values were calculated by interpolation from semi-logarithmic plots, and expressed as geometric means [95% confidence interval]. The significance (p50.05) of the results was assessed by means of paired Student’s t-tests, Mann–Whitney U-test, and one- or two-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test when appropriate.
Results In vivo experiments Baseline values of MAP in conscious vehicle-pretreated SHRs were 150 ± 3 mmHg (n ¼ 5 rats) and were significantly (p50.001) higher than that measured in their control WKY rats (100 ± 4 mmHg, n ¼ 5). However, baseline HR values in SHRs (382 ± 28 beats/min) were not statistically different from those recorded in WKY rats (338 ± 13 beats/min). In both studied groups, there was no significant change in either baseline MAP or HR after i.v. administration of labd-8’s vehicle (Tween 80 at 2%) (data not shown) as well as labd-8 at 1 mg/kg (Figure 2A and B, respectively). However, increasing bolus doses of labd-8 (3, 5, and 10 mg/ kg) evoked immediate and dose-dependent decreases in MAP (p50.001, Figure 2A), an effect that was associated with a tachycardia which was also dose-dependent (p50.001,
Figure 2. Maximal changes in mean aortic pressure (MAP) (A) and heart rate (HR) (B) elicited by intravenous (i.v.) injections of increasing bolus doses (1–10 mg/kg) of labd-8(17)-en-15-oic acid (labd-8) in conscious SHRs (black columns) and WKY (white columns) rats. Values are means of changes expressed as a percentage of baseline values. Vertical bars indicate S.E.M. (5–6 rats per group). *p50.05 by Dunnett’s test with respect to basal values.
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DOI: 10.3109/13880209.2014.934964
Figure 2B). Hypotension and tachycardia responses to labd-8 occurred respectively within 2–4 and 6–8 s after the injection of labd-8 and both became significant at the dose of 3 mg/kg (Figure 2A and B, respectively). After all doses tested of labd8, pre-dose values of both MAP and HR were fully recovered within the first 1 min following labd-8 injection. Maximal percent decreases in MAP evoked by labd-8 in SHRs were not significantly (Figure 2A, p40.05) different from those recorded in WKY rats. The same was true for the maximal percent increases in HR although they tended to be increased in SHRs (Figure 2B, p40.05). In conscious SHRs, pretreatment with hexamethonium (30 mg/kg, i.v.) induced significant (p50.01) decreases in baseline MAP (113 ± 5 versus 155 ± 3 mmHg) without affecting significantly baseline HR (390 ± 13 versus 380 ± 12 beats/ min). However, pretreatment with methylatropine (1 mg/kg, i.v.) increased significantly (p50.01) the baseline HR (463 ± 18 versus 396 ± 11 beats/min) without affecting baseline MAP (147 ± 3 versus 150 ± 3 mmHg). Pretreatment with propranolol (2 mg/kg) decreased significantly (p50.01) baseline HR (363 ± 7 versus 434 ± 20 beats/min) but not baseline MAP (167 ± 6 versus 164 ± 2 mmHg) values. Pretreatment with hexamethonium significantly (p50.01) reduced both the hypotension (Figure 3C) and the tachycardia (Figure 3D) elicited by labd-8. The remaining hypotension (Figure 3C) and tachycardia (Figure 3D) were still significant (p50.05)
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only at the higher doses (5 and/or 10 mg/kg) of labd-8. In rats pretreated with methylatropine or propranolol, the tachycardia evoked by labd-8 was significantly reduced (p50.01, Figure 3B), whereas the hypotension remained unaltered (Figure 3A). In these animals, the remaining tachycardia elicited by labd-8 (5 and 10 mg/kg) was still statistically significant with respect to baseline values (p50.05, Figure 3B). Pretreatment with L-NAME (20 mg/ kg, i.v.) induced significant (p50.05) increases in MAP (178 ± 3 versus 152 ± 6 mmHg) and decreases in HR (327 ± 15 versus 434 ± 20 beats/min). Blockade of nitric oxide synthase by L-NAME did not alter the dose-dependent decreases in MAP (Figure 3A) and increases in HR (Figure 3B) evoked by labd-8. In vitro experiments In aortic rings with intact endothelium from WKY rats (n ¼ 5) and SHRs (n ¼ 8), KCl-induced contractions were significantly reduced by labd-8 (1–1000 mg/mL) in a concentrationdependent manner (p50.05); the first inhibitory effect of labd-8 became significant (Figure 4, p50.05) at a concentration of 3 and 300 mg/mL, respectively. The IC50 (geometric mean [95% confidence interval]) value for labd-8-induced vasorelaxant effects in preparations from SHRs was 536.5 [441.0–631.9] mg/mL, which was significantly (p50.01)
Figure 3. Maximal changes in mean aortic pressure (MAP) (A) and heart rate (HR) (B) elicited by intravenous (i.v.) injections of increasing bolus doses (1–10 mg/kg) of labd-8(17)-en-15-oic acid (labd-8) in conscious SHRs subjected to i.v. pretreatment with vehicle (Veh, 1 mL/kg), propranolol (Prop, 2 mg/kg), methylatropine (MA, 1 mg/kg), or L-NAME (20 mg/kg). The effect of pretreatment with i.v. hexamethonium was also investigated on the maximal changes in MAP (C) and HR (D) elicited by labd-8 (1–10 mg/kg). Values are means of changes expressed as a percentage of baseline values. Vertical bars indicate S.E.M. (5–6 rats per group). *p50.05 by Dunnett’s test with respect to basal values. #p50.01 by two-way ANOVA with respect to vehicle-pretreated SHRs.
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greater than that (157.6 [99.1–250.5] mg/mL) obtained in preparations from WKY rats. However, the maximal percent vasorelaxation value of labd-8 was of the same order of magnitude (p40.05) in preparations from SHRs and WKY (85.63 ± 2.55 versus 84.27 ± 1.05%, respectively). In preparations from both SHRs (n ¼ 3) and WKY (n ¼ 4) rats, forskolin (0.00004–4.1 mg/mL) inhibited the KCl-induced contraction in a concentration-dependent manner (p50.001, Figure 5), an effect that became significant at a concentration of 0.41 ng/mL (p50.05, Figure 5). The IC50 values for forskolin-induced reductions of KCl-induced contractions in preparations from SHRs and WKY rats were 32.1 [21.1–84.6] and 10.9 [3.6–103.6] ng/mL, respectively. Significant difference was observed between the two values (p50.05). In both studied groups, labd-8’s vehicle added to the bath at similar
Figure 4. Effects of increasing concentrations (1–1000 mg/mL) of labd8(17)-en-15-oic acid (labd-8) on the contraction induced by KCl (60 mM) in isolated thoracic aortic preparations with functional endothelium from WYK rats (n ¼ 5) and SHRs (n ¼ 8). The values for the pre-contraction induced by KCl in rings from WKY and SHRs were 2.35 ± 0.17 and 1.96 ± 0.16 g, respectively. No significant difference was observed between the two values. Vertical bars indicate S.E.M. *p50.05 by Dunnett’s test with respect to basal values.
Figure 5. Effects of increasing concentrations (0.00004–4.1 mg/mL) of the positive reference drug forskolin on the contraction induced by KCl (60 mM) in isolated thoracic aortic preparations with functional endothelium from WYK rats (n ¼ 4) and SHRs (n ¼ 3). Vertical bars indicate S.E.M. *p50.05 by Dunnett’s test with respect to basal values.
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volumes as those used for different tested concentrations of labd-8 was without significant effects on the potassium (60 mM)-induced contraction even at the highest concentration (0.4% v/v) of labd-8 vehicle used (data not shown).
Discussion In this study, the hypotensive and tachycardiac properties of the labdenic diterpene labd-8 were confirmed in conscious SHRs. Contrarily to its inert vehicle, i.v. administration of labd-8 induced brief tachycardia with an onset time of 6–8 s, which occurred subsequently to its hypotensive effect (maximum effect at 2–4 s). To produce such cardiovascular actions, a putative direct influence of labd-8 on b2-adrenergic or muscarinic M3 vascular receptors could be discarded considering that propranolol and methylatropine did not interfere with these phenomena. In contrast, a significant reduction in the labd-8-elicited hypotension was observed when ganglionic neurotransmission was impaired with hexamethonium, which could be indicative that an operational central sympathetic neural drive to the vascular system is partially necessary to the occurrence of this phenomenon. The dependency of a sympathetic neural drive was also reported for the effects caused by labd-8 in normotensive rats (Lahlou et al., 2007). Such sympathoinhibitory action induced by labd-8 may be mediated centrally or peripherally at sympathetic areas. It was also previously demonstrated in normotensive rats that hypotension induced by labd-8 could be resultant of its vasodilator effects since an important component of hypotension remained significant after blocking the autonomic traffic with hexamethonium (Lahlou et al., 2007). A similar profile regarding a component of hypotension not susceptible to hexamethonium was presently confirmed for the higher doses (5 and 10 mg/kg) of labd-8 in SHRs. To this compound, the ability of labd-8 in producing vasodilatation is attributed. Indeed, labd-8 induced a concentration-dependent relaxation in endothelium-containing aortic rings under sustained contraction evoked by KCl. The major finding of this study is that the sensitivity of this smooth muscle-relaxant activity of labd-8 was clearly reduced in preparations from SHRs when compared with that in WKY. It is noteworthy that the IC50 value for labd-8-induced vasorelaxant effects in preparations from WKY (157.6 [99.1– 250.5] mg/mL) was of the same order of magnitude than that (202.0 [92.0–443.7] mg/mL) previously reported in the same preparations from Wistar normotensive rats (Lahlou et al., 2007). The current study does not permit to draw a convincing explanation for this blunted vasorelaxant action of labd-8 in SHRs, but it is probable that its influence was enough to produce a similar level of hypotension when SHRs and normotensive rats are compared, which preclude the major hypothesis that justified the present study: although enhanced basal sympathetic activity is reported in SHRs (Judy et al., 1976), maximal percent decreases in MAP elicited by labd-8 in SHRs were not significantly enhanced with respect to WKY rats. It is possible that such enhancement occurred but was impaired by the blunted vasorelaxation of labd-8 in preparations from SHRs. It is noteworthy that the maximal percent decrease in MAP elicited by labd-8 in WKY (around
Cardiovascular effects of labd-8 in SHRs
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DOI: 10.3109/13880209.2014.934964
31%) was greater than that evoked by 8(17),12E,14-labdatrien-18-oic acid (around 18%) (de Oliveira et al., 2006) but seems minor than that recorded for labda-15-oic acid (around 43%) (Simplicio et al., 2014) in normotensive rats. Moreover, the maximal hypotension of labd-8 occurred at a dose (10 mg/ kg) three-fold higher than that (3 mg/kg) of labda-15-oic acid (Simplicio et al., 2014) but was two-fold lower than that (20 mg/kg) observed for 8(17),12E,14-labdatrien-18-oic (de Oliveira et al., 2006). It appears reasonable to reject a putative role of an endothelial dysfunction to explain the loss of vasorelaxant efficacy of labd-8 in aortic rings of SHRs. As a matter of fact, an apparent consensus concerning the existence of endothelial dysfunction in conduit and resistance arteries in various models of hypertension exists, which is characterized by decreased endothelium-dependent relaxations and increased contractions (Lu¨scher & Vanhoutte, 1986; Tesmafariam & Halpern, 1988). It should be considered that the vasorelaxant effects of labd-8 in isolated preparations of normotensive rats did not depend on the endothelial layer functional integrity because endothelium removal was deprived of effect on its relaxing action (Lahlou et al., 2007). Reinforcing this conclusion, the present findings revealed that L-NAME was inert to change the hypotensive profile of labd-8 in SHRs, which allow us to exclude the involvement of a putative NOrelated pathway in the establishment of the present effects. Like normotensive rats (Lahlou et al., 2007), labd-8 also caused pronounced tachycardia in SHRs which is dose dependent and appears from reflexogenic nature triggered by the decreased MAP in response to labd-8. Such conclusion is reasonable if we take into account the fact that maximum magnitude of tachycardia occurred within an ulterior onset time relative to hypotension as described above. Furthermore, labd-8-induced tachycardia requires functional cardiovascular efferents since it was partly reduced when blockade of ganglionic neurotransmission was achieved with hexamethonium. In this direction, both parasympathetic and sympathetic divisions of the autonomic nervous system appear to participate in mediation of this tachycardia, probably through reduction of vagal influence and increase of sympathetic drive to the heart. Such a hypothesis may be reinforced if we consider that tachycardia was reduced by either methylatropine or propranolol pretreatment.
Conclusion In summary, the present work reports hypotension and tachycardia in conscious SHRs treated intravenously with the labdenic diterpene labd-8. The tachycardia appears to be mediated reflexly through inhibition of vagal and activation of sympathetic drive to the heart while the hypotension is partly due to withdrawal of sympathetic tone to the vasculature and partly due to an active vascular relaxation. The major finding in this study is that the magnitude of labd-8elicited hypotension is not greater in SHRs in comparison with WKY rats. A likely explanation is a decrease in the vasodilator component of the labd-8-induced hypotension, conclusion supported by the evident impairment of labd-8 in
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producing vasorelaxant effects in preparations from SHRs. Thus, although labd-8 can be considered hypotensive, its efficacy as a vasodilator compound appears dependent on the cardiovascular status that vessels could be subjected.
Declaration of interest The authors report no conflicts of interest. We thank the ‘‘Fundac¸a˜o de Amparo a´ Pesquisa do Estado de Pernambuco’’ (FACEPE) and the ‘‘Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico’’ (CNPq) for funds and grants.
References Bruneton J. (1995). Pharmacognosy, Phytochemistry, Medicinal Plants. London, England: Lavoisier Technique & Documentation. David JP, David JM, Yang SW, Cordell GA. (1998). A bis-labdenic diterpene from Moldenhawera nutans. Phytochemistry 50:443–7. David JP, Ferrari J, David JM, et al. (2007). New triterpene and antibacterial labdenoic acid derivatives from Moldenhawere nutans. J Braz Chem Soc 18:1585–9. de Oliveira AP, Furtado FF, da Silva MS, et al. (2006). Calcium channel blockade as a target for the cardiovascular effects induced by the 8 (17), 12E, 14-labdatrien-18-oic acid (labdane-302). Vascul Pharmacol 44:338–44. de Siqueira RJ, Rodrigues KM, da Silva MT, et al. (2014). Linalool-rich rosewood oil induces vago-vagal bradycardic and depressor reflex in rats. Phytother Res 28:42–8. Dubey MP, Srimal RC, Nityanand S, Dhawan BN. (1981). Pharmacological studies on coleonol, a hypotensive diterpene from Coleus forskohlii. J Ethnopharmacol 3:1–13. El-Bardai S, Wibo M, Hamaide MC, et al. (2003). Characterisation of marrubenol, a diterpene extracted from Marrubium vulgare, as an L-type calcium channel blocker. Br J Pharmacol 140:1211–16. Interaminense LF, dos Ramos-Alves FE, de Siqueira RJ, et al. (2013). Vasorelaxant effects of 1-nitro-2-phenylethane, the main constituent of the essential oil of Aniba canelilla, in superior mesenteric arteries from spontaneously hypertensive rats. Eur J Pharm Sci 48:709–16. Judy WV, Watanabe AM, Henry DP, et al. (1976). Sympathetic nerve activity: Role in regulation of blood pressure in the spontaneously hypertensive rat. Circ Res 38:21–9. Lahlou S, de Barros Correia Jr CA, Vasconcelos dos Santos M, et al. (2007). Mechanisms underlying the cardiovascular effects of a labdenic diterpene isolated from Moldenhawera nutans in normotensive rats. Vascul Pharmacol 46:60–6. Lindner E, Dohadwalla AN, Bhattacharya BK. (1978). Positive inotropic and blood pressure lowering activity of a diterpene derivative isolated from Coleus forskohli: Forskolin. Arzneimittelforschung 28:284–9. Lu¨scher TF, Vanhoutte PM. (1986). Endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension 8:344–8. Pertino MW, Theoduloz C, Bastı´as M, Schmeda-Hirschmann G. (2013). Dimeric labdane diterpenes: Synthesis and antiproliferative effects. Molecules 18:5936–53. Queiroz LP, Lewis GP, Allkin R. (1999). A revision of the genus Moldenhawera Schrad. (Leguminosae-Caesalpinioideae). Kew Bull 54:817–52. Simplicio JA, Pernomian L, Sima˜o MR, et al. (2014). Mechanisms underlying the vascular and hypotensive actions of the labdane ent-3acetoxy-labda-8(17),13-dien-15-oic acid. Eur J Pharmacol 726C: 66–76. Sonoki H, Uchida Y, Masuo M, et al. (1986). Effects of forskolin on canine congestive heart failure. Nihon Yakurigaku Zasshi 88:389–94. Tesmafariam B, Halpern W. (1988). Endothelium-dependent and endothelium-independent vasodilators in resistance arteries from hypertensive rats. Hypertension 11:440–4. Viana BF, da Silva FO, Kleinert AMP. (2006). The bee flora of the sea coastal and dunes of Abaete´, Salvador, Bahia State, Brazil. Braz J Bot 29:13–25.
ORIGINAL ARTICLE
Cardiovascular effects of a labdenic diterpene isolated from Moldenhawera nutans in conscious, spontaneously hypertensive rats
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Carlos Antoˆnio de Barros Correia Junior1, Rodrigo Jose´ Bezerra de Siqueira2, Leylliane Fa´tima Leal Interaminense1, Thayane Rebeca Alves-Santos1, Gloria Pinto Duarte1, Jorge Maurı´cio David3, Juceni Pereira David4, Pedro Jorge Caldas Magalha˜es2, and Saad Lahlou2 1
Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, PE, Brazil, 2Department of Physiology and Pharmacology, Federal University of Ceara´, Fortaleza, CE, Brazil, 3Institute of Chemistry, Federal University of Bahia, Salvador, BA, Brazil, and 4Pharmacy Faculty, Federal University of Bahia, BA, Brazil Abstract
Keywords
Context: The labdenic diterpene labd-8(17)-en-15-oic acid (labd-8) isolated from a methanolic extract of Moldenhawera nutans Queiroz & Alkin (Leguminosae) has hypotensive and tachycardiac properties in normotensive rats. A part of the hypotensive effect was due to a reduction in the sympathetic nerve drive to vessels, an event admittedly enhanced in spontaneously hypertensive rats (SHRs). Objectives: We assessed whether the cardiovascular effects induced by labd-8 could be enhanced in SHRs. Materials and methods: For in vivo experiments, arterial and venous catheters were implanted under anesthesia for blood pressure recording and drug administration, respectively. For in vitro experiments, thoracic aorta rings were suspended in organ baths containing warm (37 C) perfusion medium that was continuously bubbled with carbogen. Results: Intravenous injection of labd-8 (1, 3, 5, and 10 mg/kg) induced similar dose-dependent hypotension and tachycardia in both SHRs and Wistar–Kyoto rats (WKY). In SHRs, only the tachycardia response to labd-8 was significantly reduced by pretreatment with methylatropine or propranolol. However, both cardiovascular effects of labd-8 were reduced by hexamethonium while remained unchanged by L-NAME. In isolated aortic preparations from SHRs, labd-8 (1–1000 mg/mL) relaxed potassium-induced contractions with an IC50 (geometric mean [95% confidence interval]) value (536.5 [441.0–631.9] mg/mL) significantly greater than that (157.6 [99.1–250.5] mg/mL) obtained in preparations from WKY rats. Conclusion: In SHRs, the hypotension induced by labd-8 is associated with a reflex tachycardia and seems mediated partly through withdrawal of sympathetic vasomotor tone and partly through an active vasorelaxation. Its magnitude was not enhanced when compared with WKY rats likely because of impaired vasorelaxant effects of labd-8 in preparations from SHRs.
Autonomic nervous system, hypotension, isolated rat aorta, labd-8(17)-en-15-oic acid, relaxation, tachycardia
Introduction Diterpenes form a family of natural isoprenoid compounds derived from 2E,6E,10E-geranylgeranylpyrophosphate, a lead molecule that can suffer cyclization to generate two bicyclic enantiomer series, one nor-labdane that possesses chemical resemblance with steroids and another ent-labdane that differs from the former only by spatial configuration in a few carbons (Bruneton, 1995). In general, antimicrobial and antiproliferative properties have been attributed to labdane diterpenes (Pertino et al., 2013) and perhaps the most popular is forskolin (7b-acetoxy-8, 13-epoxy-1a,
Correspondence: Dr. Saad Lahlou, Department of Physiology and Pharmacology, Federal University of Ceara´, Rua Cel, Nunes de Melo 1127, 60431-270 Fortaleza, CE, Brazil. Tel: +55 85 3366 8334. Fax: +55 85 3366 8333. E-mail: [email protected]
History Received 18 February 2014 Revised 2 May 2014 Accepted 4 June 2014 Published online 9 December 2014
6b, 9a-trihydroxy-labd-14-ene-11-one), a well-known activator of adenylate cyclase (Sonoki et al., 1986). In the cardiovascular system, forskolin induced hypotension through its vasodilator effects upon vascular smooth muscle in laboratory animals (Dubey et al., 1981; Lindner et al., 1978). Several other labdane-type diterpenes have been reported to induce cardiovascular effects in rats. For instance, marrubenol was shown to induce vasorelaxant effects through blockade of L-type calcium channels (El-Bardai et al., 2003). The diterpene 8 (17),12E,14-labdatrien-18-oic acid decreased blood pressure in conscious normotensive rats, an effect mainly attributed to reduced peripheral vascular resistance through activation of the NO-pathway and blockage of L-type calcium channels (de Oliveira et al., 2006). Recently, it was shown that labdane ent-3-acetoxy-labda-8(17),13-dien-15-oic acid induced hypotension and vascular relaxation through the activation of the endothelial NO-cGMP pathway, the
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of the autonomic nervous system in the mediation of cardiovascular responses to labd-8 was assessed in SHRs.
Materials and methods
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Plant material, extraction, and chemical analysis
Figure 1. Chemical structure of labd-8(17)-en-15-oic acid.
opening of potassium channels, and the alteration of calcium mobilization (Simplicio et al., 2014). Moldenhawera (Leguminosae: Caesalpinoideae) is a small neotropical genus represented by approximately 10 species (Queiroz et al., 1999). The taxonomy of this genus is confused, making difficult the classification of the species. Moldenhawera nutans Queiroz & Alkin (Leguminosae) is an endemic shrub of the coastal dunes of Salvador, State of Bahia, Brazil. Species of this genus are employed as a source for construction wood and the flowers of M. nutans are visited by bees for production of wild bee products (Viana et al., 2006). However, the former phytochemical study with the stems of this species was initiated to investigate the compounds responsible for the activity exhibited by an organic extract in the HIV-1 RT Assay (Roche, New York, NY) and the isolation and identification of a new dimeric diterpene were reported (David et al., 1998). Acid labdenic diterpenes from the ent-labdane class were previously described in the methanolic extract of M. nutans (David et al., 2007). The major labdenic diterpene found in this methanolic extract is labd-8(17)-en-15-oic acid (labd-8; Figure 1). We have first reported in conscious rats the dose-dependent decreases in mean arterial pressure (MAP) and increases in heart rate (HR) caused by the intravenous (i.v.) injection of labd-8 (Lahlou et al., 2007). As the hypotension was only reduced by blockade of ganglionic neurotransmission with hexamethonium, it was suggested that this response is partly due to withdrawal of sympathetic tone to the vasculature and partly to an active vascular relaxation (Lahlou et al., 2007). Indeed, labd-8 also relaxed rat-isolated aortic preparations contracted with KCl in a concentration-dependent manner. The present investigation was undertaken to give further support to these findings by assessing whether the magnitude of hypotensive effects of labd-8 is greater in conscious, spontaneously hypertensive rats (SHRs) in which the basal sympathetic activity is increased (Judy et al., 1976). Therefore, cardiovascular responses to labd-8 were studied in conscious SHRs and compared with those of normotensive Wistar–Kyoto (WKY) rats. Vascular effects of this diterpene were also compared with isolated thoracic aortic preparations obtained from SHRs and WKY rats. Furthermore, the role
Collection of stem parts (2.1 kg) of the plant occurred on November 2000 at the Metropolitan Park area of Abaete´, Salvador, State of Bahia, Brazil. Botanical identity of the species M. nutans was confirmed by Dr. Maria Lenise da Silva Guedes by comparison with a voucher specimen (#029057) registered at the Alexander Leal Costa Herbarium, Institute of Biology, Federal University of Bahia, Brazil. Extraction and chemical isolation were done from the dry raw material following the procedures previously reported (David et al., 1998; Lahlou et al., 2007), which achieved a purity grade of 99.5% w/w as determined by GC/MS analysis. Moreover, spectral analysis and NMR evaluation served to elucidate the molecular structure of labd-8 (David et al., 1998). The 1H, 13C NMR, and DEPT spectra were obtained on a Varian Gemini 2000 instrument (Varian, Palo Alto, CA) employing CDCl3 as both a solvent and a reference. Optical rotation was obtained in a Model 343 Perkin Elmer polarimeter (PerkinElmer, Akron, OH). Labd-8(17)-en-15-oic acid: [ ]D + 26.88 (c 0.8, MeOH); 1H NMR (300 MHz, pyridine-d6): -4.88 (s, 1H, H-17a), 4.55 (s, 1H, H-17b), 0.97 (d, J 6.5 Hz, 3H, Me-16), 0.84 (s, 3H, Me-19), 0.81 (s, 3H, Me-18), and 0.78 (s, 3H, Me-20); 13 C NMR (pyridine-d6): 35.9 (C-1), 19.3 (C-2), 41.9 (C-3), 33.7 (C-4), 56.9 (C-6), 24.4 (C-6), 42.2 (C-7), 148.6 (C-8), 55.5 (C-9), 39.7 (C-10), 21.0 (C-11), 39.2 (C-12), 33.6 (C-13), 38.3 (C-14), 179.8 (C-15), 19.7 (C-16), 106.4 (C-17), 21.8 (C-18), 30.7 (C-19), 14.6 (C-20). Solutions and drugs For in vivo experiments, the solvent employed to dissolve labd-8 was Tween 80 (2% v/v), which was brought to a given volume with sterile isotonic saline. Drugs were administered in a volume of 1 mL/kg body weight. Each i.v. injection was followed by a 60 mL (catheter volume) flush of physiological saline to ensure complete delivery of the dosage. For the experiments with isolated preparations, labd-8 in Tween 80 was dissolved directly in Tyrode’s solution, which was employed as the perfusion medium to maintain the viability of isolated preparations. The modified Tyrode’s solution was composed of (in mM): NaCl 136, KCl 5, MgCl2 0.98, CaCl2 2, NaH2PO4 0.36, NaHCO3 11.9, and glucose 5.5. All labd-8 solutions were sonicated just before use. Other drugs in this study were purchased from Sigma (St. Louis, MO) and were dissolved in saline just before use. Animals Adult male SHRs and WKY rats (age: 16–18 weeks) were used in this study. They were obtained from the institutional vivarium of the Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, Brazil. Animals were kept under controlled conditions of light (12 h lightdark cycle) and temperature (22 ± 1 C) with free access to water and standard rat chow. All animals were cared for in
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compliance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication 85-23, revised 1996). All procedures described here were reviewed by and had prior approval from local animal ethics committee.
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In vivo experiments Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg), and catheters (PE-10 fused to PE-50) were implanted in the abdominal aorta (for the recording of arterial blood pressure) and in the inferior vena cava (for drug administration) through the left femoral artery and vein, respectively, as described previously (Lahlou et al., 2007; de Siqueira et al., 2014). Twenty-four hours later, baseline MAP and HR were recorded on a Gilson model 5/6H polygraph (Medical Electronics Inc., Middletown, WI), as described previously (Lahlou et al., 2007). The maximal changes in MAP and HR caused by i.v. injections of increasing bolus (100 mL each) doses of labd-8 (1-10 mg/kg) were determined in conscious SHRs and WKY rats. In a separate set of animals, cardiovascular responses to labd-8 (1–10 mg/kg) were also studied in SHRs which have been pretreated intravenously 10 min earlier with either vehicle (1 mL/kg, n ¼ 5), hexamethonium (30 mg/kg, n ¼ 6), methylatropine (1 mg/kg, n ¼ 6), propranolol (2 mg/kg, n ¼ 5), or L-NAME (20 mg/kg, n ¼ 5). In vitro experiments In another set of experiments, SHRs and WKY rats were euthanized by stunning and exsanguination. After a median incision, segments of thoracic aorta were obtained as ring-like preparations that were immersed in bath chamber filled with a perfusion medium maintained at 37 C under constant bubbling with 95% O2 and 5% CO2 and pH 7.4. Resting tension was settled at 1 g through disposal of ring-like preparations between two points: one fixed in the bath and another in a force transducer disposed in a device appropriate for tension adjustment. Such set-up allowed the isometric tension recordings that were performed according to the previously described procedure (Interaminense et al., 2013; Lahlou et al., 2007). At the beginning of the experiment, each aortic ring was pre-contracted with phenylephrine (0.1 mM) and thereafter challenged by acetylcholine (1 mM) to evaluate the integrity of endothelium. Preparations were considered to possess an intact endothelium when the vasorelaxant response to acetylcholine was greater than 75%. Vasodilator effects were assessed only once in each thoracic aorta preparation by adding increasing concentrations of labd-8 (1–1000 mg/mL) on the steady state of sustained contractions induced by a high K+ (60 mM) concentration in endothelium-intact aortic preparations from SHRs (n ¼ 8) and WKY rats (n ¼ 5). A similar experiment was performed with increasing concentrations (0.04–4.1 mg/mL) of forskolin, a well-known activator of adenylate cyclase, used herein as a positive control. Statistical analysis Data are expressed as means ± standard error of the mean (S.E.M.). Maximal changes in MAP and HR after administration of a given dose of labd-8 were expressed
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as a percentage of baseline values. For in vitro data, peak deflections were used to measure the magnitude of the concentration–response curves, which were expressed as a percentage of KCl-induced contraction (in the absence of labd-8). The IC50 values were calculated by interpolation from semi-logarithmic plots, and expressed as geometric means [95% confidence interval]. The significance (p50.05) of the results was assessed by means of paired Student’s t-tests, Mann–Whitney U-test, and one- or two-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test when appropriate.
Results In vivo experiments Baseline values of MAP in conscious vehicle-pretreated SHRs were 150 ± 3 mmHg (n ¼ 5 rats) and were significantly (p50.001) higher than that measured in their control WKY rats (100 ± 4 mmHg, n ¼ 5). However, baseline HR values in SHRs (382 ± 28 beats/min) were not statistically different from those recorded in WKY rats (338 ± 13 beats/min). In both studied groups, there was no significant change in either baseline MAP or HR after i.v. administration of labd-8’s vehicle (Tween 80 at 2%) (data not shown) as well as labd-8 at 1 mg/kg (Figure 2A and B, respectively). However, increasing bolus doses of labd-8 (3, 5, and 10 mg/ kg) evoked immediate and dose-dependent decreases in MAP (p50.001, Figure 2A), an effect that was associated with a tachycardia which was also dose-dependent (p50.001,
Figure 2. Maximal changes in mean aortic pressure (MAP) (A) and heart rate (HR) (B) elicited by intravenous (i.v.) injections of increasing bolus doses (1–10 mg/kg) of labd-8(17)-en-15-oic acid (labd-8) in conscious SHRs (black columns) and WKY (white columns) rats. Values are means of changes expressed as a percentage of baseline values. Vertical bars indicate S.E.M. (5–6 rats per group). *p50.05 by Dunnett’s test with respect to basal values.
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DOI: 10.3109/13880209.2014.934964
Figure 2B). Hypotension and tachycardia responses to labd-8 occurred respectively within 2–4 and 6–8 s after the injection of labd-8 and both became significant at the dose of 3 mg/kg (Figure 2A and B, respectively). After all doses tested of labd8, pre-dose values of both MAP and HR were fully recovered within the first 1 min following labd-8 injection. Maximal percent decreases in MAP evoked by labd-8 in SHRs were not significantly (Figure 2A, p40.05) different from those recorded in WKY rats. The same was true for the maximal percent increases in HR although they tended to be increased in SHRs (Figure 2B, p40.05). In conscious SHRs, pretreatment with hexamethonium (30 mg/kg, i.v.) induced significant (p50.01) decreases in baseline MAP (113 ± 5 versus 155 ± 3 mmHg) without affecting significantly baseline HR (390 ± 13 versus 380 ± 12 beats/ min). However, pretreatment with methylatropine (1 mg/kg, i.v.) increased significantly (p50.01) the baseline HR (463 ± 18 versus 396 ± 11 beats/min) without affecting baseline MAP (147 ± 3 versus 150 ± 3 mmHg). Pretreatment with propranolol (2 mg/kg) decreased significantly (p50.01) baseline HR (363 ± 7 versus 434 ± 20 beats/min) but not baseline MAP (167 ± 6 versus 164 ± 2 mmHg) values. Pretreatment with hexamethonium significantly (p50.01) reduced both the hypotension (Figure 3C) and the tachycardia (Figure 3D) elicited by labd-8. The remaining hypotension (Figure 3C) and tachycardia (Figure 3D) were still significant (p50.05)
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only at the higher doses (5 and/or 10 mg/kg) of labd-8. In rats pretreated with methylatropine or propranolol, the tachycardia evoked by labd-8 was significantly reduced (p50.01, Figure 3B), whereas the hypotension remained unaltered (Figure 3A). In these animals, the remaining tachycardia elicited by labd-8 (5 and 10 mg/kg) was still statistically significant with respect to baseline values (p50.05, Figure 3B). Pretreatment with L-NAME (20 mg/ kg, i.v.) induced significant (p50.05) increases in MAP (178 ± 3 versus 152 ± 6 mmHg) and decreases in HR (327 ± 15 versus 434 ± 20 beats/min). Blockade of nitric oxide synthase by L-NAME did not alter the dose-dependent decreases in MAP (Figure 3A) and increases in HR (Figure 3B) evoked by labd-8. In vitro experiments In aortic rings with intact endothelium from WKY rats (n ¼ 5) and SHRs (n ¼ 8), KCl-induced contractions were significantly reduced by labd-8 (1–1000 mg/mL) in a concentrationdependent manner (p50.05); the first inhibitory effect of labd-8 became significant (Figure 4, p50.05) at a concentration of 3 and 300 mg/mL, respectively. The IC50 (geometric mean [95% confidence interval]) value for labd-8-induced vasorelaxant effects in preparations from SHRs was 536.5 [441.0–631.9] mg/mL, which was significantly (p50.01)
Figure 3. Maximal changes in mean aortic pressure (MAP) (A) and heart rate (HR) (B) elicited by intravenous (i.v.) injections of increasing bolus doses (1–10 mg/kg) of labd-8(17)-en-15-oic acid (labd-8) in conscious SHRs subjected to i.v. pretreatment with vehicle (Veh, 1 mL/kg), propranolol (Prop, 2 mg/kg), methylatropine (MA, 1 mg/kg), or L-NAME (20 mg/kg). The effect of pretreatment with i.v. hexamethonium was also investigated on the maximal changes in MAP (C) and HR (D) elicited by labd-8 (1–10 mg/kg). Values are means of changes expressed as a percentage of baseline values. Vertical bars indicate S.E.M. (5–6 rats per group). *p50.05 by Dunnett’s test with respect to basal values. #p50.01 by two-way ANOVA with respect to vehicle-pretreated SHRs.
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greater than that (157.6 [99.1–250.5] mg/mL) obtained in preparations from WKY rats. However, the maximal percent vasorelaxation value of labd-8 was of the same order of magnitude (p40.05) in preparations from SHRs and WKY (85.63 ± 2.55 versus 84.27 ± 1.05%, respectively). In preparations from both SHRs (n ¼ 3) and WKY (n ¼ 4) rats, forskolin (0.00004–4.1 mg/mL) inhibited the KCl-induced contraction in a concentration-dependent manner (p50.001, Figure 5), an effect that became significant at a concentration of 0.41 ng/mL (p50.05, Figure 5). The IC50 values for forskolin-induced reductions of KCl-induced contractions in preparations from SHRs and WKY rats were 32.1 [21.1–84.6] and 10.9 [3.6–103.6] ng/mL, respectively. Significant difference was observed between the two values (p50.05). In both studied groups, labd-8’s vehicle added to the bath at similar
Figure 4. Effects of increasing concentrations (1–1000 mg/mL) of labd8(17)-en-15-oic acid (labd-8) on the contraction induced by KCl (60 mM) in isolated thoracic aortic preparations with functional endothelium from WYK rats (n ¼ 5) and SHRs (n ¼ 8). The values for the pre-contraction induced by KCl in rings from WKY and SHRs were 2.35 ± 0.17 and 1.96 ± 0.16 g, respectively. No significant difference was observed between the two values. Vertical bars indicate S.E.M. *p50.05 by Dunnett’s test with respect to basal values.
Figure 5. Effects of increasing concentrations (0.00004–4.1 mg/mL) of the positive reference drug forskolin on the contraction induced by KCl (60 mM) in isolated thoracic aortic preparations with functional endothelium from WYK rats (n ¼ 4) and SHRs (n ¼ 3). Vertical bars indicate S.E.M. *p50.05 by Dunnett’s test with respect to basal values.
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volumes as those used for different tested concentrations of labd-8 was without significant effects on the potassium (60 mM)-induced contraction even at the highest concentration (0.4% v/v) of labd-8 vehicle used (data not shown).
Discussion In this study, the hypotensive and tachycardiac properties of the labdenic diterpene labd-8 were confirmed in conscious SHRs. Contrarily to its inert vehicle, i.v. administration of labd-8 induced brief tachycardia with an onset time of 6–8 s, which occurred subsequently to its hypotensive effect (maximum effect at 2–4 s). To produce such cardiovascular actions, a putative direct influence of labd-8 on b2-adrenergic or muscarinic M3 vascular receptors could be discarded considering that propranolol and methylatropine did not interfere with these phenomena. In contrast, a significant reduction in the labd-8-elicited hypotension was observed when ganglionic neurotransmission was impaired with hexamethonium, which could be indicative that an operational central sympathetic neural drive to the vascular system is partially necessary to the occurrence of this phenomenon. The dependency of a sympathetic neural drive was also reported for the effects caused by labd-8 in normotensive rats (Lahlou et al., 2007). Such sympathoinhibitory action induced by labd-8 may be mediated centrally or peripherally at sympathetic areas. It was also previously demonstrated in normotensive rats that hypotension induced by labd-8 could be resultant of its vasodilator effects since an important component of hypotension remained significant after blocking the autonomic traffic with hexamethonium (Lahlou et al., 2007). A similar profile regarding a component of hypotension not susceptible to hexamethonium was presently confirmed for the higher doses (5 and 10 mg/kg) of labd-8 in SHRs. To this compound, the ability of labd-8 in producing vasodilatation is attributed. Indeed, labd-8 induced a concentration-dependent relaxation in endothelium-containing aortic rings under sustained contraction evoked by KCl. The major finding of this study is that the sensitivity of this smooth muscle-relaxant activity of labd-8 was clearly reduced in preparations from SHRs when compared with that in WKY. It is noteworthy that the IC50 value for labd-8-induced vasorelaxant effects in preparations from WKY (157.6 [99.1– 250.5] mg/mL) was of the same order of magnitude than that (202.0 [92.0–443.7] mg/mL) previously reported in the same preparations from Wistar normotensive rats (Lahlou et al., 2007). The current study does not permit to draw a convincing explanation for this blunted vasorelaxant action of labd-8 in SHRs, but it is probable that its influence was enough to produce a similar level of hypotension when SHRs and normotensive rats are compared, which preclude the major hypothesis that justified the present study: although enhanced basal sympathetic activity is reported in SHRs (Judy et al., 1976), maximal percent decreases in MAP elicited by labd-8 in SHRs were not significantly enhanced with respect to WKY rats. It is possible that such enhancement occurred but was impaired by the blunted vasorelaxation of labd-8 in preparations from SHRs. It is noteworthy that the maximal percent decrease in MAP elicited by labd-8 in WKY (around
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DOI: 10.3109/13880209.2014.934964
31%) was greater than that evoked by 8(17),12E,14-labdatrien-18-oic acid (around 18%) (de Oliveira et al., 2006) but seems minor than that recorded for labda-15-oic acid (around 43%) (Simplicio et al., 2014) in normotensive rats. Moreover, the maximal hypotension of labd-8 occurred at a dose (10 mg/ kg) three-fold higher than that (3 mg/kg) of labda-15-oic acid (Simplicio et al., 2014) but was two-fold lower than that (20 mg/kg) observed for 8(17),12E,14-labdatrien-18-oic (de Oliveira et al., 2006). It appears reasonable to reject a putative role of an endothelial dysfunction to explain the loss of vasorelaxant efficacy of labd-8 in aortic rings of SHRs. As a matter of fact, an apparent consensus concerning the existence of endothelial dysfunction in conduit and resistance arteries in various models of hypertension exists, which is characterized by decreased endothelium-dependent relaxations and increased contractions (Lu¨scher & Vanhoutte, 1986; Tesmafariam & Halpern, 1988). It should be considered that the vasorelaxant effects of labd-8 in isolated preparations of normotensive rats did not depend on the endothelial layer functional integrity because endothelium removal was deprived of effect on its relaxing action (Lahlou et al., 2007). Reinforcing this conclusion, the present findings revealed that L-NAME was inert to change the hypotensive profile of labd-8 in SHRs, which allow us to exclude the involvement of a putative NOrelated pathway in the establishment of the present effects. Like normotensive rats (Lahlou et al., 2007), labd-8 also caused pronounced tachycardia in SHRs which is dose dependent and appears from reflexogenic nature triggered by the decreased MAP in response to labd-8. Such conclusion is reasonable if we take into account the fact that maximum magnitude of tachycardia occurred within an ulterior onset time relative to hypotension as described above. Furthermore, labd-8-induced tachycardia requires functional cardiovascular efferents since it was partly reduced when blockade of ganglionic neurotransmission was achieved with hexamethonium. In this direction, both parasympathetic and sympathetic divisions of the autonomic nervous system appear to participate in mediation of this tachycardia, probably through reduction of vagal influence and increase of sympathetic drive to the heart. Such a hypothesis may be reinforced if we consider that tachycardia was reduced by either methylatropine or propranolol pretreatment.
Conclusion In summary, the present work reports hypotension and tachycardia in conscious SHRs treated intravenously with the labdenic diterpene labd-8. The tachycardia appears to be mediated reflexly through inhibition of vagal and activation of sympathetic drive to the heart while the hypotension is partly due to withdrawal of sympathetic tone to the vasculature and partly due to an active vascular relaxation. The major finding in this study is that the magnitude of labd-8elicited hypotension is not greater in SHRs in comparison with WKY rats. A likely explanation is a decrease in the vasodilator component of the labd-8-induced hypotension, conclusion supported by the evident impairment of labd-8 in
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producing vasorelaxant effects in preparations from SHRs. Thus, although labd-8 can be considered hypotensive, its efficacy as a vasodilator compound appears dependent on the cardiovascular status that vessels could be subjected.
Declaration of interest The authors report no conflicts of interest. We thank the ‘‘Fundac¸a˜o de Amparo a´ Pesquisa do Estado de Pernambuco’’ (FACEPE) and the ‘‘Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico’’ (CNPq) for funds and grants.
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