The new nitric oxide donor 2-nitrate-1,3-dibuthoxypropan alters autonomic function in spontaneously hypertensive rats

Autonomic Neuroscience: Basic and Clinical 171 (2012) 28–35

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The new nitric oxide donor 2-nitrate-1,3-dibuthoxypropan alters autonomic function in spontaneously hypertensive rats Maria S. França-Silva a, Matheus M.O. Monteiro a, Thyago M. Queiroz a, Alexsandro F. Santos b, Petrônio F. Athayde-Filho b, Valdir A. Braga a,⁎ a b

Biotechnology Center, Federal University of Paraíba, João Pessoa, PB, Brazil Department of Chemistry, Federal University of Paraíba, João Pessoa, PB, Brazil

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Article history: Received 22 June 2012 Received in revised form 11 September 2012 Accepted 12 October 2012 Keywords: Nitric oxide Hypertension Bradycardia Autonomic function

a b s t r a c t Previously, we found that the nitrate synthesized from glycerin, 2-nitrate-1,3-dibuthoxypropan (NDBP), increased NO levels in rat aortic smooth muscle cells, inducing vasorelaxation in mesenteric artery. However, its effects on blood pressure and heart rate as well as on autonomic function were not investigated. This study evaluated the action of NDBP on these cardiovascular parameters in spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats. We found that NDBP causes a biphasic response: hypotension and bradycardia followed by hypertension and tachycardia in WKY and SHR rats. Atropine (2 mg/kg) blunted the hypotension induced by NDBP (15 mg/kg) in WKY and SHR (−75± 9 vs −12±3 mm Hg, n=6; −101 ±6 vs −7± 2 bpm, n= 6; respectively, pb 0.05) and the pressor response to the compound was potentiated. Furthermore, vagotomy reduced the bradycardia in WKY and SHR (−136± 8 vs −17± 2, n= 4, p b 0.05; −141± 9 vs −8± 2, n=6, p b 0.05). Moreover, hexamethonium (30 mg/kg) reduced both bradycardia (−278± 23 vs −48±3 in WKY; −285±16 vs −27±19 in SHR, n=4; pb 0.05) and pressor response (28±8 vs −9± 5-WKY, n=6; 42±7 vs −19±8-SHR, n=5; pb 0.05). In addition, administration of methylene blue (4 mg/kg) attenuated the hypotensive and bradycardic responses to the NDBP in all groups. In conclusion, NDBP induces bradycardia by direct vagal stimulation and pressor response by increasing sympathetic outflow to the periphery. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Hypertension is a multifactorial disorder, which in a majority of cases is a result of changes in several mechanisms that regulate blood pressure. Studies suggest that changes in reflex control of blood pressure are involved in the development and maintenance of hypertension (Smith et al., 2004). Endothelial alterations with enhanced generation of contractile substances, reactive oxygen species (ROS) and reduced formation or activity of nitric oxide (NO) also may be involved in the pathogenesis of hypertension (Marín and Rodrigues-Martinez, 1997). NO is an important molecule in the modulation of cardiovascular function participating in the maintenance of normal vascular tone and as a neuromodulator, altering the sympathetic and parasympathetic outflow to the periphery (Zanzinger, 1999; Schmitt and Dirsch, 2009). The role of NO in the progress of changes caused by cardiovascular diseases is classically reported in the literature; therefore, NO is a potential therapeutic target in hypertension (Rajapakse and Mattson, 2009). Drugs which mimic the role of this molecule in the body such as NO donors are known to act in the treatment of hypertension. Therefore, a new NO donor, the 2-nitrate-1,3-dibuthoxypropan (NDBP), was

newly synthetized (Fig. 1). NDBP is a compound obtained from the synthetic route of glycerin, considered a “waste” in the biodiesel production process (Santos, 2009). Recently, our research group showed the vascular effects produced by NDBP (Franca-Silva et al., 2012). In the mesenteric artery from normotensive rats, NDBP evoked potent vasorelaxant effect through NO generation and activation of the sCG/ cGMP/PKG pathway. Furthermore, the release kinetic of NO was similar to other NO donors such as glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) (Franca-Silva et al., 2012). However the effects induced by NDBP on blood pressure, heart rate and autonomic function have not been investigated. The evidence that hypertension is associated with changes in autonomic nervous system and the fact that NO may exert a direct influence on autonomic control of blood pressure, reducing the deleterious effect of hypertension, led us to evaluate the effects of the NO donor, NDBP, on the autonomic balance of spontaneously hypertensive rats (SHR), and its control normotensive Wistar-Kyoto rats (WKY). 2. Material and methods 2.1. Animals

⁎ Corresponding author at: Biotechnology Center, Federal University of Paraiba, P.O. Box 5009, 58.051-970 João Pessoa, PB, Brazil. Tel.: +55 83 3216 7511. E-mail address: [email protected] (V.A. Braga). 1566-0702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.autneu.2012.10.002

Male Wistar-Kyoto and spontaneously hypertensive rats (250–300 g), housed under controlled conditions of temperature (21±1 °C) and

M.S. França-Silva et al. / Autonomic Neuroscience: Basic and Clinical 171 (2012) 28–35

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stabilization of the baseline parameters, NDBP was administered before and after (15 min) the bilateral section of the vagus nerve. The responses to the compound were evaluated before and after this surgical procedure.

NO2

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2.6. Evaluation of sympathetic activity in the NDBP-induced cardiovascular responses In order to investigate the effect of NDBP on the sympathetic activity, organic nitrate was administered 15 min before and after ganglionic blockade with hexamethonium (30 mg/kg, i.v.) in WKY and SHR conscious rats. 2.7. Evaluation of the participation of nitric oxide in the NDBP-mediated cardiovascular effects

H3C Fig 1. Structural formula of NDBP.

lighting cycle (lights on: 06:00–18:00 h) with water and food (Labina®, PURINA, Brazil) ad libitum were used in all experiments. All the experimental procedures were conducted in accordance with the Institutional Animal Care and Use Committee of the Federal University of Paraiba (CEPA 0209/10). 2.2. NDBP preparation NDBP was dissolved in a mixture of saline solution and chremophor and diluted to the desired concentrations with saline solution as previously described (Franca-Silva et al., 2012).

To investigate the effect of NDBP on NO availability, in vivo effects of NDBP was assessed blocking soluble guanylyl cyclase (sGC), an intermediate of the NO pathway, with intravenous administration of methylene blue (4 mg/kg, in bolus) in WKY and SHR conscious rats. 2.8. Statistical analysis Results are expressed as mean ± SEM. Data were analyzed by “one-way” analysis of variance (ANOVA) followed by a Bonferroni's post-test for multiple comparisons whenever appropriate. All statistical analyses were performed using GraphPad Prism (v. 5.0, GraphPad Software, Inc). Statistical significance was defined as p b 0.05. 3. Results 3.1. Effects of NDBP on blood pressure (BP) and heart rate (HR) in conscious rats

2.3. Surgical procedures and protocols Intra-aortic blood pressure was recorded using the technique described by Braga (2010). Briefly, under ketamine (75 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia, the rats were fitted with polyethylene catheters inserted into the lower abdominal aorta and inferior vena cava through left femoral artery and vein, respectively. Both catheters were filled with heparinized saline, tunneled subcutaneously, exteriorized and sutured at the dorsal surface of the neck. Twenty-four hours after the surgical procedures, experiments were performed in conscious rats. The arterial catheter was connected to a pre-calibrated pressure transducer coupled to an acquisition system (PowerLab, ADInstruments, Unit 13, 22 Lexington Drive, Bella Vista, NSW, Australia) connected to a computer installed with LabChart 5.0 software. 2.4. Assessment of cardiovascular effects of NDBP One day after the surgical procedures, blood pressure and heart rate were evaluated after the randomly administration of different doses of NDBP (1, 5, 10, 15 and 20 mg/kg, i.v.) in conscious rats. 2.5. Evaluation of the parasympathetic activity in NDBP-induced cardiovascular responses

Spontaneously hypertensive rats were significantly hypertensive compared with WKY rats (165.8 ± 5.6 vs 105.8 ± 7.8 mm Hg, n = 6, p b 0.05, respectively). Fig. 2 (A and C) shows representative tracings from spontaneously hypertensive and normotensive Wistar-Kyoto rats illustrating the changes in these parameters after administration of NDBP. From these results, the dose of NDBP (15 mg/kg) was chosen to perform the following protocols. Administration of NDBP (1, 5, 10, 15 and 20 mg/kg) elicited dose-dependent hypotension (− 4 ± 2, − 21 ± 6, − 54 ± 6, − 62 ± 10, − 76 ± 8 mm Hg, n = 6, p b 0.05) and bradycardia, except in the first dose (1 ± 5, − 70 ± 26, −248 ±10, −240 ± 25, −301± 29 bpm, p b 0.05) followed by hypertension (5± 2, 4 ± 1, 6 ± 2, 5 ±2, 12±3 mm Hg) and tachycardia (25 ± 4, 11±10, 55± 19, 40 ±9, 76 ± 16 bpm, p b 0.05) in normotensive Kyoto (WKY) rats as illustrated in Fig. 2B. Interestingly, in spontaneously hypertensive rats, hypotensive (−8 ± 1, −20±5, −86 ± 4, −95± 5, −118 ± 3 mm Hg, n = 6, pb0.05) and bradycardic (−9 ± 8, −125 ±37, −252 ±10, −269 ±16, −309 ± 18 bpm, n = 6, pb0.05) responses exhibited were significantly higher when compared to WKY rats. This increase was also found in hypertensive (9± 2, 15 ± 3, 50± 5, 57 ± 4, 60±8 mm Hg, p b 0.05) and tachycardic (30 ± 10, 45± 9, 49±11, 76± 15, 110 ± 19 bpm, p b 0.05) responses (Fig. 2D). 3.2. Effects of NDBP after parasympathetic blockade

In order to investigate the contribution of parasympathetic activity on hypotension promoted by NDBP, atropine (2 mg/kg, i.v.), a muscarinic blocker, was injected in normotensive and hypertensive conscious rats, and the effect of NDBP was evaluated before and after (15 min) the blockade. In other set of experiments, to assess whether NDBP induces bradycardia due to a central or peripheral action, WKY and SHR rats were submitted to vagotomy. Firstly, the rats were anesthetized with ketamine and xylazine (75 and 10 mg/kg, i.p., respectively) and after

The administration of NDBP (15 mg/kg) caused hypotension associated with profound bradycardia in all animals. The blockade of muscarinic receptors with atropine (2 mg/kg) abolished bradycardia induced by NDBP (15 mg/kg) in WKY (− 265 ± 25 vs. − 48 ± 7 bpm, n = 6, p b 0.05) and SHR (− 255 ± 9 vs − 25 ± 10 bpm, p b 0.05, n = 6). In addition, atropine blunted hypotension induced by NDBP in WKY (− 75 ± 9 mm Hg vs − 12 ± 3 mm Hg, n = 6, p b 0.05) as well as in SHR rats (− 101 ± 6 vs − 7 ± 2 bpm, n = 6, p b 0.05) and

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Fig. 2. Effect of NDBP on mean arterial pressure (MAP, mm Hg) and heart rate (HR, bpm) in normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Representative tracings from normotensive Wistar Kyoto (WKY) (A) and spontaneously hypertensive rat (SHR) (C) illustrating the changes in pulse arterial pressure (PAP, mm Hg), mean arterial pressure (MAP, mm Hg), and heart rate (HR, bpm) in response to administration of different doses of the NDBP (black arrows). Data from the group of WKY (B) and SHR (D) rats, illustrating the primary (black bars) and secondary responses (white bars) at different doses of NDBP. Values are means ± S.E.M. ⁎p b 0.05 versus baseline.

the pressor response to the compound was potentiated (35 ± 7 vs 65 ± 4 mm Hg-WKY; 33 ± 10 vs 82 ± 6 mm Hg-SHR; p b 0.05) as shown in Fig. 3. The acute administration of atropine induced a slight increase in heart rate without significantly altering blood pressure (data not shown). 3.3. Cardiovascular effects elicited by NDBP after vagotomy To further investigate the parasympathetic activity induced by NDBP, using a different experimental protocol, vagotomy was performed. Representative tracings from WKY and SHR rats show changes in blood pressure and heart rate elicited by NDBP before and after vagotomy as shown in Fig. 4 (A and C). Interestingly, vagotomy produced a large reduction in the NDBP-induced bradycardia in both WKY (−136 ± 8 vs −17± 2 bpm, n = 4, p b 0.05) and SHR rats (−141± 9 vs −8 ± 2 bpm, n = 6, p b 0.05). After vagotomy, the hypotensive response was attenuated in SHR (−66± 8 vs −34± 11 mm Hg, p b 0.05) as well as in WKY animals (−59 ± 11 vs −35± 9 mm Hg) as shown in Fig. 4 (B and D). 3.4. Effects of NDBP after ganglionic blockade The acute administration of hexamethonium (30 mg/kg, i.v.) produced inhibition of the NDBP-induced pressor response (28 ±8 vs

−9 ± 5 mm Hg-WKY, n =6; 42 ± 7 vs −19±8 mm Hg-SHR, n = 5; p b 0.05). In relation to HR, there was a reduction in bradycardia in both animal models (−278 ± 23 vs −48± 3 bpm-WKY; −285 ± 16 vs −27± 19 bpm-SHR, n = 4; p b 0.05). The remaining hypotension (−23± 3 and −13± 10 mm Hg, in WKY and SHR rats, respectively) can be attributed to the vascular effect elicited by NDBP (Fig. 5) as described previously by Franca-Silva et al. (2012).

3.5. Effects of NDBP after sGC blockade To evaluate nitric oxide release in the response induced by administration of NDBP (15 mg/kg, i.v.), hypotension and bradycardic responses were analyzed before and after acute infusion of soluble guanylyl cyclase inhibitor, methylene blue (4 mg/kg). Representative tracings are presented in Fig. 2 (A and C). In WKY rats both hypotension and bradycardic effects were reduced (− 82 ± 6 vs − 57 ± 7 mm Hg, n = 6 and − 322 ± 14 vs − 272 ± 11 bpm, n = 6, respectively, p b 0.05). Similar results were found in SHR regarding hypotensive (− 95 ± 8 vs − 70 ± 4 mm Hg, p b 0.05, n = 2) and bradycardic effects (− 291 ± 12 vs −235 ± 6 bpm, p b 0.05, n = 5) as illustrated in Fig. 6 (B and D). The administration of methylene blue caused a sudden and transient increase in blood pressure without changing heart rate (data not shown).

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Fig. 3. Effect of NDBP (15 mg/kg) before and after blockade with atropine (2 mg/kg, i.v.) in normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Representative tracings from WKY (A) and SHR (C) illustrating the changes in pulse arterial pressure (PAP, mm Hg), mean arterial pressure (MAP, mm Hg), and heart rate (HR, bpm) in response to administration of NDBP before and after atropine. Group data indicating the effect of NDBP (15 mg/kg) before (black bars) and after (white bars) muscarinic blockade in WKY (B) and SHR (D). Values are means ± S.E.M. ⁎p b 0.05 versus control hypotensive and bradycardic response. ≠p b 0.05 versus control pressor response.

4. Discussion The main findings of the present study were that acute intravenous infusion of NDBP, a NO donor, evoked short-lasting responses comprising initial hypotensive and bradycardic responses followed

by pressor and minor tachycardic responses. Bradycardia and hypotension were prevented by muscarinic blockade or by vagal transection. Ganglionic blockade substantially attenuated NDBP-induced changes in blood pressure and heart rate, and methylene blue, slightly reduced these changes. In WKY rats, there was no significant difference

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Fig. 4. Effect of NDBP (15 mg/kg) before and after vagotomy in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Representative tracings from WKY (A) and SHR (C) illustrating the changes in pulse arterial pressure (PAP, mm Hg), mean arterial pressure (MAP, mm Hg), and heart rate (HR, bpm) in response to administration of NDBP before and after vagotomy. Group data indicating the effect of NDBP (15 mg/kg) before (black bars) and after (white and streaked bars) vagotomy in WKY (B) and SHR (D). Values are means ± S.E.M. ⁎p b 0.05 versus control hypotensive and bradycardic responses.

in the secondary response induced by NDBP when compared to basal values, except in the tachycardia induced by higher dose administered. Spontaneously hypertensive (SHR) rats are widely used as animal model for studying essential hypertension in humans, because these animals have phenotypic characteristics similar to those observed in human essential hypertension (Pinto et al., 1998). This model is associated with increased peripheral vascular resistance, cardiac hypertrophy, heart failure, among other pathologies (Fazan et al., 2001). In addition, the sympathetic nerve activity is significantly increased in relation with their normotensive controls; this justifies, in part, the fact that the pressor and tachycardia responses were higher in SHR group. Furthermore, low levels of NO contribute to the development and maintenance of hypertension in this model (Wymann et al., 2003; Portaluppi et al., 2004). Thus, SHR were chosen as a model to investigate the effect of the NO donor NDBP on the autonomic nervous system. Initially, the administration of NDBP in SHR and WKY rats induced hypotension associated with bradycardia, instead of reflex tachycardia as observed in other NO donors such as sodium nitroprusside and nitroglycerin (Needleman, 1976; Caputi et al., 1980). In order to evaluate the involvement of parasympathetic activity in response induced by NDBP, we used atropine (2 mg/kg), which greatly reduced the NDBP-induced bradycardia. The reduction of bradycardia also attenuated the hypotension induced by our nitrate, suggesting

that NDBP reduced the mean blood pressure mainly by reducing the cardiac output in addition to its effect on the smooth muscle in the periphery as previously demonstrated by Franca-Silva et al. (2012). Contrary to these findings, previous reports have demonstrated that NO donors induce relaxation of the vascular smooth muscle and this effect is responsible for promoting hypotension in rats (Munhoz et al., 2012; Paulo et al., 2012).In accordance to this findings, Herring and Paterson (2001) show the direct influence of NO donors on cardiac vagal nerve endings, stimulating the release of acetylcholine (ACh). At the same time, evidence points to the role of NO in specific sites of the central nervous system (CNS), increasing parasympathetic activity directed to the heart with subsequent bradycardia, which in turn masks the reflex tachycardia to the hypotension (Kishi et al., 2001; Fletcher et al., 2006). In this context, in order to assess whether the NDBP-induced bradycardia occurred by a direct action of the compound in the heart or due to their actions in the CNS, vagotomy was performed in WKY and SHR animals. This surgical procedure could be visualized by instantaneous elevation in heart rate of the animals, since the parasympathetic activity directed to the heart was prevented. The vagotomy virtually abolished the NDBP-induced bradycardia, demonstrating that decrease in heart rate occurs due to central action of NDBP, stimulating the ACh release from vagus nerve. The bilateral

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Fig. 5. Effect of NDBP (15 mg/kg) before and after blockade with hexamethonium (30 mg/kg) in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Representative tracings from WKY (A) and SHR (C) illustrating the changes in pulse arterial pressure (PAP, mm Hg), mean arterial pressure (MAP, mm Hg), and heart rate (HR, bpm) in response to administration of NDBP before and after administration of hexamethonium. Data from the entire group, indicating the effect of NDBP (15 mg/kg) before (black bars) and after (white bars) ganglionic blockade in WKY (B) and SHR (D). Values are means ± S.E.M. ⁎p b 0.05 versus control hypotensive and bradycardic responses. ≠ p b 0.05 versus control pressor and tachycardic responses.

section of the vagus nerve also reduced the hypotension due to elimination of the bradycardia, confirming the ability of the nitrate to decline the cardiac output. The blockade of sGC with methylene blue resulted in a slight attenuation in hypotension and bradycardia induced by NDBP, suggesting that the vagomimetic properties of this substance are also mediated by NO-independent pathway; however, more studies are needed to clarify this fact. In a second moment, the NDBP caused hypertension and tachycardia and the ganglionic blockade substantially attenuated these drug-induced changes, showing that NDBP induces increases in sympathetic outflow to periphery while increases the parasympathetic discharge, confirming the ability of the compound to induce central effects. Several findings report that the NO acts in brain sites increasing the parasympathetic activity directly to heart with posterior bradycardia that masks the reflex tachycardia (Kishi et al., 2001; Fletcher et al., 2006).

Residual hypotensive and bradycardic responses were observed in the presence of hexamethonium (30 mg/kg), suggesting that there is a modest contribution of the vasorelaxation elicited by NDBP (Franca-Silva et al., 2012) to produce hypotension in both hypertensive and normotensive rats. The NO exerts vasorelaxation via activation of guanylate cyclase, increasing intracellular levels of cyclic guanosine monophosphate (cGMP), resulting in activation of protein kinase G (PKG), a peptide responsible for the phosphorylation of several proteins involved on vasorelaxation (Archer et al., 1994; McDonald and Murad, 1996). This effect can lead to a reduction in blood pressure, being attenuated by the blockage of soluble guanylyl cyclase with methylene blue. These data confirm the reports of Feelisch and Noack (1987) which stated that the release of NO by organic nitrates is suppressed by methylene blue, and this inhibition reduces vasodilation mediated by these compounds and consequently its hypotensive effect. Other studies have demonstrated that methylene blue is able to attenuate

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Fig. 6. Effect of NDBP (15 mg/kg) before and after acute infusion of methylene blue (4 mg/kg) in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Representative tracings from WKY (A) and SHR (C) illustrating the changes in pulse arterial pressure (PAP, mm Hg), mean arterial pressure (MAP, mm Hg), and heart rate (HR, bpm) in response to administration of NDBP before and after administration of methylene blue. Group data indicating the effect of NDBP (15 mg/kg) before (black bars) and after (white bars) sGC blockade in WKY (B) and SHR (D). Values are means ± S.E.M. ⁎p b 0.05 versus control hypotensive and bradycardic responses.

the vasorelaxation, as well as hypotension induced by NO donors such as SNP, confirming the results found in this study (Wei et al., 1992; Wang et al., 1995; Crestani, 2008). Here we report for the first time that the new NO donor, NDBP, induces hypotension due to a reduction in cardiac output by direct vagal stimulation, while causing pressor response by increasing sympathetic outflow to the periphery by stimulation of sympathetic outflow. These responses depend on the central action of the compound. There are few substances known to activate cardiac vagal neurons; therefore, our findings may encourage new studies to advance the understanding of the anti-hypertensive effects elicited by NDBP, which will help to advance the field towards clinical trials, considering that currently used organic nitrates on the clinic are effective in the treatment of hypertension but exhibit some undesirable effects. Acknowledgments The authors are grateful to José Crispim Duarte for technical assistance in experimental procedures. References Archer, S.L., Huang, J.M., Hamp, l V., Nelson, D.P., Shultz, P.J., Weir, K., 1994. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. U. S. A. 91, 7583–7587.

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