Journal of Pharmacy & Pharmacognosy Research

Volume 4, Issue 6 (Nov-Dec), 2016 ISSN 0719-4250

Content:

Pages

IFC (Journal of Pharmacy & Pharmacognosy Research).

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1.- Original article Julio Alvarez-Collazo, Ana Iris López-Medina, Loipa Galán-Martínez, Julio L. Alvarez (2016) 2,32+ Butanedione monoxime attenuates the β-adrenergic response of the L-type Ca current in rat ventricular cardiomyocytes.

206-216

2.- Short Communication Julio Alvarez-Collazo, Loipa Galán-Martínez, Alicia Fleites-Vazquez, Alicia Sánchez-Linde, Karel Talavera-Pérez, Julio L. Alvarez (2016) Negative inotropic and dromotropic actions of SiO 2 + 2+ nanoparticles on isolated rat hearts: Effects on Na and Ca currents.

217-223

3.- Original article Billmary Z. Contreras-Moreno, Judith J. Velasco, Janne del C. Rojas, Lucero del C. Méndez, María T. Celis (2016) Antimicrobial activity of essential oil of Pimenta racemosa var. racemosa(Myrtaceae) leaves.

224-230

4. Original article Etagegnehu Assefa, Israel Alemayhu, Milkyas Endale, Fikre Mammo (2016) Iridoid glycosides from the root of Acanthus sennii.

J Pharm Pharmacogn Res JPPRes

231-237

© 2016 Journal of Pharmacy & Pharmacognosy Research

ISSN 0719-4250

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© 2016 Journal of Pharmacy & Pharmacognosy Research, 4 (6), 206-216, 2016 ISSN 0719-4250 http://jppres.com/jppres Original Article | Artículo Original

2,3-Butanedione monoxime attenuates the β-adrenergic response of the L-type Ca2+ current in rat ventricular cardiomyocytes [La 2,3-butanodiona monoxima atenúa la respuesta β-adrenérgica de la corriente de Ca2+ tipo L en cardiomiocitos ventriculares de rata] Julio Alvarez-Collazo1,2,#, Ana Iris López-Medina1,#, Loipa Galán-Martínez1, Julio L. Alvarez1* 1Laboratorio 2Laboratory

de Electrofisiología. Instituto de Cardiología y Cirugía Cardiovascular. Paseo y 17, Vedado, CP 10400, La Habana. Cuba. of Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. #Both authors contributed equally. *E-mail: [email protected]

Abstract

Resumen

Context: 2,3-Butanedione monoxime (BDM), an uncoupler of cardiac contraction, is commonly used in enzymatic dissociations to prevent hypercontraction of cardiomyocytes and in cardioplegic solutions to decrease oxygen demand during surgery. However, BDM affects multiple cellular systems including the L-type Ca2+ current (ICaL). If its phosphatase activity is the mechanism underlying the decrease ICaL in cardiomyocytes is a still unresolved question.

Contexto: La 2,3-butanodiona monoxima (BDM), un desacoplador de la contracción cardíaca, es comúnmente utilizada en la disociación enzimática para prevenir la hipercontracción de los cardiomiocitos y en soluciones de cardioplejia para reducir la demanda de oxígeno durante la cirugía. No obstante, la BDM reduce la corriente de Ca2+ tipo L (ICaL) pero su mecanismo de acción no ha sido dilucidado definitivamente.

Aims: To study the effects of BDM on ICaL of rat ventricular cardiomyocytes focusing our attention on the response of ICaL to βadrenergic stimulation. Methods: The whole-cell patch-clamp method was used to study ICaL in enzymatically dissociated rat ventricular cardiomyocytes. Results: Extracellular BDM (5 mM) decreased peak ICaL by ≈45%, slowed its fast inactivation but accelerated its slow inactivation. Cardiomyocytes incubated in BDM (≥ 30 min; 5 mM) perfused with normal extracellular solution, showed normal ICaL properties. However, extracellular BDM (in cardiomyocytes incubated in BDM or not) markedly reduced the response of ICaL to isoproterenol (1 µM). BDM also strongly attenuated the increase of ICaL in cardiomyocytes intracellularly perfused with cyclic AMP (50 µM). Conclusions: The decrease of basal ICaL by BDM is not related to its dephosphorylation action. Its effect on the Ca2+ channel occurs most probably in a site in the extracellular side or within the sarcolemmal membrane. Due to its phosphatase action, BDM strongly attenuates the response of ICaL to β-adrenergic stimulation. These actions of BDM must be taken into account both for its use in the dissociation of cardiomyocytes and in cardioplegic solutions and myocardial preservation. Keywords: 2,3-butanedione monoxime; calcium cardiomyocytes; myocardial preservation; patch-clamp.

channels;

Objetivos: Estudiar los efectos de la BDM sobre ICaL de cardiomiocitos ventriculares de rata, centrando la atención en la respuesta de ICaL al isoproterenol (ISO). Métodos: Se utilizó la técnica de patch-clamp para registrar ICaL en cardiomiocitos ventriculares de rata disociados enzimáticamente. Resultados: La BDM extracelular (5 mM) redujo ICaL en ≈45% y modificó su inactivación rápida. Los cardiomiocitos incubados en BDM (≥ 30 min; 5 mM) y perfundidos con solución extracelular normal, mostraron I CaL normales. No obstante, la BDM extracelular (en cardiomiocitos incubados en BDM o no incubados), redujo marcadamente la respuesta de ICaL al ISO (1 µM). La BDM atenuó fuertemente el aumento de ICaL en cardiomiocitos perfundidos intracelularmente con AMP cíclico. Conclusiones: La reducción de ICaL basal por BDM no está relacionada a su actividad desfosforiladora. Su efecto sobre el canal de Ca2+ ocurre probablemente en un sitio extracelular. Debido a su acción como fosfatasa, la BDM atenúa fuertemente la respuesta de ICaL al ISO. Estas acciones de la BDM deben ser consideradas tanto para su utilización en la disociación de cardiomiocitos como en las soluciones de cardioplejia y la preservación miocárdica.

Palabras Clave: 2,3-butanodiona monoxima; canales cardiomiocitos; patch-clamp; preservación miocárdica.

ARTICLE INFO Received | Recibido: August 12, 2016. Received in revised form | Recibido en forma corregida: September 23, 2016. Accepted | Aceptado: September 23, 2016. Available Online | Publicado en Línea: September 28, 2016. Declaration of interests | Declaración de Intereses: The authors declare no conflict of interest. Funding | Financiación: The study was supported by the Ministry of Public Health of Cuba (Research Project 1104012). Academic Editor | Editor Académico: Marisol Fernández.

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Alvarez-Collazo et al.

BDM attenuates β-adrenergic response of cardiac L-type Ca2+ current

INTRODUCTION Isolated adult cardiomyocytes stand for the experimental model of choice in studies of the biochemical, biophysical, electrical and contractile activities of normal and diseased myocardium and also in pharmacological studies (Bers, 2001). Dissociation methods of adult cardiomyocytes are quite diverse and almost every laboratory uses its own protocol. However, one feature in common to all methods is to try to prevent the well-known “calcium paradox phenomenon” (Daly et al. 1987) that results in hypercontraction and death of cardiomyocytes after calcium re-admission following perfusion of hearts will nominally calcium-free solutions. Because of its ability to uncouple cardiac (and skeletal) muscle contraction 2,3-butanedione monoxime (BDM), was profiled to have “cardioprotective” properties and has been used to prevent hypercontraction of Ca2+ -intolerant cardiomyocytes in many dissociation protocols (e. g. Kivistö et al., 1995; Chung et al., 2015; see for review Abi-Gerges et al., 2013). Originally developed as a reactivator of acetylcholinesterase (Wilson and Ginsburg, 1955), BDM has been considered as a “chemical phosphatase”. This compound affects serine/threonine protein phosphorylation (Stapleton et al., 1998) and has been shown to affect the activities of proteins like myosin-II light chain kinase (Siegman et al., 1994); BDM is known to increase the equilibrium constant for ATP hydrolysis inhibiting the rate of phosphate release and stabilizing the M.ADP.PI intermediate (Herrmann et al., 1992) whose overall effect is the inhibition of the myosin ATPase rate and a decrease in force production. Yet, BDM is not considered to be a general myosin ATPase inhibitor (Ostap, 2003). BDM has been also used in cardioplegic solutions to suppress contraction in order to preserve the myocardium by decreasing oxygen demand thus preserving energy (ATP) during surgery (Stringham et al., 1994; Vahl et al., 1995; Habazettl et al., 1998; Jayawant et al., 1999; Warnecke et al., 2002; Chambers, 2005; Reichert et al., 2013; Lee et al., 2016).

While these actions of BDM may be useful to obtain high yield of non-contracted myocytes after enzymatic digestion and even to preserve myocardium during cardioplegic arrest, BDM is known to affect other cellular mechanisms such as the activity of connexins (Verrechia and Hervé, 1997), to block the http://jppres.com/jppres

Na-Ca exchanger (Watanabe et al., 2006) and the expression (Borlak and Zwadlo; 2004) and the activity of ionic channels (e.g. Schlichter et al., 1992; Lopatin and Nichols, 1993). It has been shown that BDM promotes inhibition of mitochondrial respiration by acting directly on electron transport chain reducing cell viability (Hall and Hausenloy, 2016). BDM inhibits the Ltype Ca2+ (ICaL) in cardiac ventricular myocytes (Coulombe et al., 1990) and in guinea-pig taenia caeci (Lang and Paul, 1991); the effects on ICaL could be more marked in ventricular myocytes from spontaneously hypertensive rats (Xiao and McArdle, 1995). The decrease of ICaL was accompanied by an acceleration of its inactivation (Coulombe et al., 1990; Chapman, 1993; Allen and Chapman, 1995). Although Schwinger et al. (1994) suggested that BDM does not affect the βadrenergic response in human myocardium, Chapman (1993) and Allen and Chapman (1995) showed that, due to its phosphatase activity, BDM interfered with the β-adrenergic response of ICaL in ventricular myocytes. Nonetheless, this has been questioned by Eisfeld et al. (1997) and Allen et al. (1998) who demonstrated that BDM does not interfere with the interaction sites between PKA and cardiac Ca2+ channel expressed in HEK 293 cells and Xenopus oocytes, respectively. It can be argued, however, that heterologous expression systems do not exactly reproduce native cellular systems leaving open the question of whether BDM affects or not the β-adrenergic response of ICaL. Regarding the mechanism of the decrease of ICaL by BDM, the prevailing idea has been that due to its phosphatase like activity, this oxime affects the phosphorylated state of the L-type Ca2+ channel (Coulombe et al., 1990; Chapman, 1993; Allen and Chapman, 1995). Also, in murine DRG neurons, Huang and McArdle (1992) suggested that the decrease of an L-

type Ca2+ current by BDM could be related to alterations in PKA regulation of ICaL. On the other hand, Lang and Paul (1991) in guinea-pig taenia caeci cells suggested that blockade of ICaL by BDM could be related to the interactions of this oxime on resting and/or inactivated states of the Ca2+ channel. Ferreira et al. (1997) pointed out that the effects of BDM on ICaL and its increase in inactivation time constants could be mechanistically consistent with dephosphorylation but also with a dihydropyridinelike action; nevertheless, they ruled-out an open J Pharm Pharmacogn Res (2016) 4(6): 207



channel block as a mechanism of BDM on the Ltype Ca2+ current. A clear-cut mechanism of ICaL decrease by BDM has not been established. The aim of the present study was to re-evaluate the effects of BDM on ICaL of rat ventricular cardiomyocytes focusing our attention on the changes in the response of ICaL to β-adrenergic stimulation. Our results show that when cardiomyocytes were incubated in BDM, the β-adrenergic response of ICaL is greatly attenuated. MATERIAL AND METHODS Chemicals 2,3-Butanedione monoxime (C4H7NO2; PubChem CID: 6409633; CAS Number: 57-71-6; >98%) was purchased from Sigma Aldrich and was prepared in ethanol as stock solution. All other chemicals were also from Sigma Aldrich. Animals Experiments were performed using male adult Wistar (7 - 8 weeks) rats according to the procedures approved by the National Center for Laboratory Animal Reproduction (CENPALAB; Santiago de Las Vegas, La Habana, Cuba). Prior to experiment, animals were adapted for seven days to laboratory conditions (controlled temperature 25 ± 2°C, relative humidity 60 ± 10% and 12 h light/dark cycles). Tap water and standard diet for rodents supplied by CENPALAB were freely provided. All procedures were also conducted according to the European Commission guide-lines for the use and care of laboratory animals and approved by the Committee for Animal Care in Research of the Center. The minimum number of animals and duration of observation required to obtain consistent data were employed. Enzymatic isolation of ventricular cardiomyocytes Ventricular cardiomyocytes were isolated as previously described in detail (Alvarez-Collazo et al., 2012) and were kept in a K+-Tyrode solution containing 1 mM Ca2+ at room temperature (21 ± 2 °C) and used for experiments for 6 h. http://jppres.com/jppres

Patch-clamp recordings Aliquots of cardiomyocytes were transferred to a Petri dish (with the same K+-Tyrode solution) on the stage of an inverted microscope. Relaxed, noncontracting, cardiomyocytes exhibiting clear striated pattern were selected for patch-clamping. Whole-cell currents were recorded at room temperature (Alvarez-Collazo et al., 2012). Currents were filtered at 3 kHz and digitized at 50-μs intervals, stored on a computer and analyzed off-line with the ACQUIS1 software (version 2.0, CNRS License, France). To study L-type Ca2+ currents, K+ currents were blocked by substituting all potassium by cesium in extracellular and “intracellular” solutions. The extracellular solution contained (in millimolars): 117 NaCl, 20 CsCl, 10 HEPES, 2 CaCl2, 1.8 MgCl2, and 10 glucose, pH 7.4. The standard pipette (intracellular) solution contained (in millimolars): 130 CsCl, 0.4 Na2GTP, 5 Na2ATP, Na2-creatine phosphate, 2.0 MgCl2, 11 EGTA, 4.7 CaCl2 (free Ca2+ ≈108 nM), and 10 HEPES, with pH adjusted to 7.2 with CsOH. In the experiments, cells were first left to lie in Petri dishes filled with K+-Tyrode solution with 1 mM Ca2+. Cells attached to the micropipette could be positioned on the extremity of each of six microcapillaries (i.d. 250 μm) through which the different extracellular Cs+-containing solutions were perfused by gravity (≈15 μL/min), allowing rapid changes (≈1 s) of the extracellular medium. Pipette resistance was 1.0 - 1.2 MΩ. Membrane capacitance (Cm) and series resistance (Rs) were calculated on voltage-clamped cardiomyocytes as previously described (Alvarez et al., 2000). Average Cm and uncompensated Rs were 168 ± 8.6 pF and 3.3 ± 0.5 MΩ, respectively (n = 44). Rs could be electronically compensated up to 50% without ringing and was continually monitored during the experiment. Liquid junction potential was compensated before establishing the gigaseal. No leak or capacitance subtractions were performed in the recordings. For routine monitoring of the L-type Ca2+ current (ICaL) a double pulse voltage-clamp protocol was employed: from a holding potential (HP) of -80 mV every 4s the cell membrane was depolarized by a prepulse to -40 mV for 50 ms to inactivate the fast Na+ current. From this membrane potential, a 200ms test pulse to 0 mV evoked ICaL. Current-tovoltage relationships (I-V) and availability curves J Pharm Pharmacogn Res (2016) 4(6): 208



were obtained using the same prepulse protocol but interpolating 300-ms pulses from -40 to +50 mV between the pre- and test pulses. Pulses for I-V and availability curves were applied at 8 s intervals. The inactivation time course of ICaL was fitted to a double exponential using the fitting procedures of the ACQUIS1 software. Statistical analysis Results are expressed as means and standard errors of means. Statistical significance was evaluated by means of paired or unpaired Student’s t test according to the experimental situation. Differences were considered statistically significant for p < 0.05. RESULTS

tracellular solution (without BDM). Under these condition, ICaL density was 9.1 ± 1.4 pA/pF (n = 8), not significantly different from that of control cardiomyocytes. τfast was not different from that of control cardiomyocytes (5.9 ± 1.1 ms) and τslow was slightly decreased (46.0 ± 3.8 ms), but not statistically significant (Fig. 2A-B). In these BDMincubated cardiomyocytes, perfusion with an extracellular solution containing BDM (5 mM) had essentially the same “on - off” effect as in control (not incubated) cardiomyocytes; ICaL density at 0 mV was decreased by 43.8 ± 8.0% but τfast was markedly increased to 9.6 ± 1.0 ms (p < 0.05). Contrary to what occurred in control cardiomyocytes, τslow was increased by BDM to 57.2 ± 7.9 ms; however, this effect was not statistically significant (Fig. 2A-B).

Effects of BDM on basal ICaL Under control condition, peak ICaL density at 0 mV was 9.2 ± 0.6 pA/pF; its inactivation time course could be fitted to fast (τfast) and slow (τslow) exponentials whose values were 5.9 ± 0.3 ms and 53.2 ± 2.2 ms, respectively (n = 12). Extracellularly applied BDM decreased ICaL in a fast (2 - 3 pulses) tonic fashion (Fig. 1) with an IC50 near to 5 mM (5.4 ± 0.3 mM; n = 3), close to that reported by Chapman (1993) and Lang and Paul (1991) in cardiomyocytes and smooth muscle cells, respectively. From here on, in all the experiments reported, BDM was used at 5 mM concentration. At this concentration, BDM decreased peak ICaL by 43.1 ± 7.5% and significantly increased τfast to 7.6 ± 0.9 ms and decreased τslow to 44.7 ± 2.2 ms (Fig. 2A-B; p < 0.05). The action of BDM on ICaL was also rapidly reversed upon washout (“on - off” effect; Fig. 1). Basal ICaL in cardiomyocytes incubated in 5 mM BDM In a series of experiments, after enzymatic dissociation, cardiomyocytes were incubated in the same K+-Tyrode solution (1 mM Ca2+) supplemented with 5 mM BDM at room temperature (21 ± 2°C) for 30 minutes. Aliquots of cardiomyocytes were then transferred to the Petri dish containing K+-Tyrode solution without BDM. They were quickly patchclamped (time to achieve whole cell configuration was less than 2 min) and perfused with normal exhttp://jppres.com/jppres

Figure 1. Time course of the effect of extracellularly applied BDM on ICaL in a rat ventricular cardiomyocyte. BDM (5 mM) decreases ICaL by ≈50% in an “on - off” fashion. The action of BDM was fully reversible upon returning to control solution. The inset shows the current traces corresponding to the blue (Control) and red (stable BDM effect) arrows at different times during the experiment.

BDM does not change ICaL voltage-dependence Current-to-voltage relationships and availability curves of ICaL were obtained using standardized protocols. Availability curves of ICaL from -80 to 0 mV were fitted to a Boltzmann function (f∞ = 1 / 1 + exp [V0.5 / s]) to obtain the voltage for half inactivation (V0.5) and the slope factor (s). In control, not incubated, cardiomyocytes (n = 12) the effects of extracellular BDM (5 mM) were not voltage-dependent; voltage for maximal ICaL (0 mV) or its reversal poJ Pharm Pharmacogn Res (2016) 4(6): 209



tential (≈+50 mV) were not shifted (Fig. 3A). Consequently, availability curves were barely modified; V0.5 was -29.5 ± 0.3 mV in control and -31.7 ± 0.3 mV in the presence of BDM (Fig. 3B; n = 8). The slope factor, s, was not significantly changed (5.9 ± 0.4 mV vs 5.6 ± 0.3 mV). In BDM-incubated cardiomyocytes (n = 8) perfused with normal extracellular solution (as described above) voltage for maximal

ICaL and its reversal potential were similar to those of control cardiomyocytes (Fig. 3C). V0.5 and s were not significantly different from control cardiomyocytes (-30.9 ± 0.2 mV and 5.5 ± 0.2 mV, respectively; Fig. 3D). Perfusion of these cardiomyocytes with extracellular BDM (5 mM; Fig. 3D) produced no significant effects on V0.5 (-31.0 ± 0.2 mV) and s (5.3 ± 0.4 mV).

Figure 2. Extracellularly applied BDM decreases ICaL density and changes its inactivation time course.

Figure 3. Extracellular BDM did not change the voltage-dependence of ICaL.

A. In cardiomyocytes not incubated as well as in cardiomyocytes incubated in 5 mM BDM for at least 30 min, extracellularly applied BDM (5 mM) induced a similar decrease in ICaL density. B. Effects of extracellular BDM on fast (τfast) and slow (τslow) inactivation time constants. In both not incubated and incubated cardiomyocytes, extracellular BDM significantly increased τfast. However, in not incubated cardiomyocytes extracellular BDM decreased τslow but showed a tendency (not statistically significant) to increase τslow in incubated cardiomyocytes. It is to note that BDM incubation per se had no effect on ICaL density or its inactivation time constants when incubated cardiomyocytes are perfused with control extracellular solution. *p < 0.05 with respect to its control value.

A. Current-to voltage relationships, in cardiomyocytes not incubated in BDM, under control condition () and in the presence of 5 mM extracellular BDM (). B. Corresponding availability curves. V0.5 and s were 29.5 ± 1.7 mV and 31.7 ± 1.6 mV and 5.9 ± 1.1 mV and 5.6 ± 0.3 mV in control and BDM, respectively. C. Current-to voltage relationships, in cardiomyocytes incubated in BDM, under control condition () and in the presence of 5 mM extracellular BDM (). D. Corresponding availability curves. V0.5 and s were -30.9 ± 1.5 mV and 31.0 ± 1.5 mV and 5.5 ± 0.8 mV and 5.3 ± 0.4 mV in control and BDM, respectively.


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BDM attenuates the response of ICaL to β-adrenergic stimulation β-adrenergic stimulation increases ICaL via a wellcharacterized signaling cascade and is one of the most stable cardiomyocyte response to neuromediators (Bénitah et al., 2010). In order to investigate the possible effects of BDM (5 mM) on the response of ICaL to β-adrenergic stimulation by isoproterenol (ISO, 1 µM) we considered four experimental conditions: A.- Control, not incubated, cardiomyocytes on which ISO was applied (n = 6). B.- Cardiomyocytes incubated in BDM on which ISO was applied (n = 6). C.- Control, not incubated, cardiomyocytes on which BDM was applied and then ISO in the presence of BDM (n = 12). D.- Cardiomyocytes incubated in BDM on which BDM was applied and then ISO in the presence of BDM (n = 8). Cardiomyocytes included in “C” and “D” experimental conditions were the same already presented in the previous sections. Under control conditions (experimental condition “A”), 1 µM isoproterenol (ISO) induced an increase in ICaL, which was stable in 3 - 4 min. Mean increase of ICaL by ISO was 60.8 ± 4.1% (n = 6). τfast increased from 5.8 ± 0.3 ms to 6.7 ± 0.3 ms (p < 0.05) and τslow decreased from 56.6 ± 3.3 ms to 50.6 ± 2.6 ms (p < 0.05; Fig. 4A-B). These effects were similar to those described by our group under

similar experimental conditions (Alvarez et al., 2004). ISO did not prevent the decrease in ICaL by BDM. In three cardiomyocytes, BDM (5 mM) applied during the ISO effect still decreased ICaL by 44.3 ± 2.0%. When ISO was applied to BDM-incubated cardiomyocytes (experimental condition “B” as described above), mean increase of ICaL was only 7.4 ± 2.1% (n = 6; p < 0.05 with respect to condition “A”). Both τfast and τslow showed a tendency to decrease (from 5.9 ± 1.1 ms and 45.9 ± 3.8 ms to 5.5 ± 1.5 ms and 42.5 ± 2.7 ms, respectively) but without statistical significance (Fig. 4A-B). When BDM was applied before ISO in control cardiomyocytes, β-adrenergic response of ICaL was also greatly attenuated. In control cardiomyocytes (experimental condition “C”), ISO under the effect of extracellular BDM, increased ICaL by only 14.2 ± 4.3% (n = 12; p < 0.05 with respect to condition “A”); τfast was increased to 10.4 ± 1.4 ms and τslow was decreased to 36.6 ± 2.5 ms (p < 0.05; Fig. 4A-B). In BDM-incubated cardiomyocytes (experimental condition “D”), ISO was practically without effect under the action of extracellular BDM, ICaL was only increased by 2.8 ± 1.7%. Both τfast and τslow showed a tendency to decrease (from 9.6 ± 0.9 ms to 9.3 ± 1.3 ms and from 57.2 ± 7.9 ms to 53.6 ± 8.8 ms, respectively) but without statistical significance (n = 8; Fig. 4A-B).

Figure 4. BDM attenuates the response of ICaL to βadrenergic stimulation. A. In control cardiomyocytes, not incubated in BDM, isoproterenol (ISO, 1 µM) significantly (p < 0.05) increased ICaL density by ≈60%. If ISO was applied after ICaL was decreased by extracellular BDM (5 mM), then ICaL was increased by only ≈15%, but still statistically significant. In cardiomyocytes previously incubated in BDM (5 mM, the β-adrenergic increase in ICaL density was almost abolished. B. In control cardiomyocytes, not incubated in BDM, the characteristic response of ICaL inactivation to β-adrenergic stimulation is an increase in τfast and a decrease in τslow. This typical response was not changed when ISO was applied after ICaL was decreased by extracellular BDM (5 mM). However, in cardiomyocytes previously incubated in BDM there were no significant changes in τfast and τslow after βadrenergic stimulation. *p < 0.05 with respect to its control value.





From these results, it is clear that BDM attenuates β-adrenergic response of ICaL. We next studied whether BDM was also able to attenuate the response of ICaL to intracellular 3',5'-cyclic adenosine monophosphate (cAMP) a well-known activator of protein kinase A. Not incubated (n = 4) and BDMincubated (n = 4) cardiomyocytes were patchclamped with pipettes containing the normal “intracellular” solution but added with 50 µM cAMP. Immediately after patch rupture ICaL was continuously monitored. In not incubated cardiomyocytes, ICaL increased by 167.6 ± 22.0% from its initial value in about 2 min (Fig. 5A; see also, Alvarez-Collazo et al., 2012). In BDM-incubated cardiomyocytes, however, ICaL was barely increased by 10.0 ± 6.0% from its initial value (Fig. 5B). Moreover, in two control, not incubated, cardiomyocytes, application of extracellular BDM after the steady-state cAMP effect, still decreased ICaL by 42 and 48%. The effect was “on off” but after washout, ICaL never recovered its maximal attained value (Fig. 6).

Figure 6. Extracellularly applied BDM is able to decrease ICaL density in cardiomyocytes intracellularly perfused with cAMP. In a cardiomyocyte not incubated in BDM, after ICaL was maximally increased by intracellular cAMP, extracellular perfusion with 5 mM BDM is still able to decrease ICaL by ≈50%. Upon washout with normal extracellular solution, ICaL never returned to its maximal value previous to BDM. The inset shows the current traces corresponding to the blue (Control), red (stable BDM effect) and green (washout of BDM) arrows at different times during the experiment.

DISCUSSION

Figure 5. BDM attenuates the response of ICaL to intracellularly applied cyclic adenosine monophosphate (3', 5'-cAMP; 50 µM). A. Example of a control cardiomyocyte (not incubated in BDM) intracellularly perfused with cAMP in which there is a huge increase in I CaL density. B. In a cardiomyocyte previously incubated in 5 mM BDM, there was almost no effect of cAMP on ICaL.


The main outcome of the present investigation is that, in isolated rat ventricular cardiomyocytes, BDM attenuates the response of ICaL to β-adrenergic stimulation. Our results also suggest that BDM could inhibit the L-type Ca2+ channel by acting on a site in the external side of the sarcolemmal membrane. Our results show that extracellular BDM decrease ICaL in an “on - off” manner with an IC50 around 5 mM, similar to that commonly reported for cardiac myocytes (Coulombe et al., 1990; Chapman, 1993; but see Xiao and McArdle, 1995) and smooth muscle cells (Lang and Paul, 1991) but that is lower than the IC50 reported for the inhibition of an L-type Ca2+ current in neurons (Huang and McArdle, 1992) and of the human L-type Ca2+ channel expressed HEK 293 cells (Eisfeld et al., 1997) and Xenopus oocytes (Allen et al., 1998). The decrease of ICaL by BDM in the present J Pharm Pharmacogn Res (2016) 4(6): 212



experiments was not voltage-dependent since neither the I-V relationships nor the availability curves were modified by the oxime. This is in agreement with the results of Huang and McArdle (1992) in neurons, Lang and Paul (1991) in smooth muscle cells and Eisfled et al. (1997) in HEK-293 cells expressing the human L-type Ca2+ channel. Coulombe et al. (1990) and Ferreira et al. (1997) in rat and guinea-pig ventricular cardiac myocytes, respectively and Allen et al. (1998) in L-type Ca2+ channels expressed in Xenopus oocytes found a 4 - 6 mV leftward shift in ICaL availability but at much higher concentrations of BDM. In the present experiments, the inactivation time course of ICaL was affected by BDM; τfast was consistently increased while τslow was decreased. Similar results were reported by Allen and Chapman (1995) for the exponential and sustained phases of ICaL in guinea-pig ventricular cardiomyocytes using Ca2+ or Ba2+ as charge carriers. Although the effects of BDM on the fast inactivation of ICaL could be interpreted in terms of dephosphorylation or direct effects on channel gating (e.g. Allen and Chapman, 1995; Ferreira et al., 1997) it should be considered that τfast is related to the Ca2+-dependent inactivation (CDI; for review see Bénitah et al., 2010), which depends on the Ca2+ load of the sarcoplasmic reticulum (SR). It has been reported by Tripathi et al. (1999) that BDM is able to increase the open probability of SR Ca2+ channels. Additionally, BDM decreases peak ICaL. It is thus possible that under the action of BDM the SR Ca2+ load is decreased and CDI is diminished increasing τfast. The decrease we observed in τslow is most probably related to an effect of BDM on channel gating (e.g.; Ferreira et al., 1997). On the other hand, Eisfled et al. (1997) and Allen et al. (1998) reported accelerations in the inactivation time course of ICaL under the action of BDM. It is, however, difficult to explain such a discrepancy since those results were obtained measuring currents through L-type Ca2+ channels expressed in heterologous systems using Ba2+ as charge carrier. One important finding of the present results is that incubating cardiomyocytes in a BDM-containing solution did not affect ICaL density or its inactivation time course recorded when cardiomyocytes were perfused with control extracellular solution as described above. This is an expected result since the effect of extracellular BDM on ICaL was quickly eshttp://jppres.com/jppres

tablished an also rapidly washed out (“on - off”). Moreover, in those cardiomyocytes perfusion with an extracellular solution containing BDM produced essentially the same changes in ICaL properties as in not incubated cardiomyocytes. These results suggest that inhibition of basal ICaL by BDM is probably not related to its (intracellular) phosphatase activity (see Chapman, 1993; Allen and Chapman, 1995) but to a direct action on the L-type Ca2+ channel through a site located in the extracellular sarcolemmal interphase. Another possibility is that BDM, due to its lipophilicity, could penetrate the membrane and produce its ICaL blocking action either by directly interacting with the Ca2+ channel or by disturbing the lipid domains around the channel. Both possibilities (outside or within the membrane) are consistent with the fast decrease and washout (“on off”) of BDM effect on ICaL. Our results are also consistent with the idea that there is a minor role of PKA in the maintenance of basal ICaL (see for review Weiss et al., 2013). It should be noted here that the results of Eisfled et al. (1997) and Allen et al. (1998), using heterologous expression systems, supported the idea that BDM effects on basal ICaL were not mediated by dephosphorylation. However, as will be discussed below, the intracellular phosphatase activity of BDM, reflected in a decreased β-adrenergic response, is long lasting. The most important result of the present experiments is that BDM markedly attenuated the response of ICaL to β-adrenergic stimulation. In rat ventricular cardiomyocytes, ICaL usually respond to ISO (1 µM) with a ≥60% increase (see Alvarez et al., 2004; present results). However, our results show that when extracellular BDM was first applied to cardiomyocytes (basal ICaL is decreased) ISO was much less effective in increasing ICaL (≈15%). Furthermore, when cardiomyocytes incubated in BDM (at least 30 min) were patch-clamped and perfused with normal extracellular solution (time to achieve whole cell configuration was less than 2 min), the response of ICaL to ISO was greatly diminished or even abolished (see Fig. 4). These results are agreement with those of Lang and Paul (1991) in smooth muscle cells but are in contrast to those of Chapman (1993) and Allen and Chapman (1995) in cardiomyocytes and Huang and McArdle (1992) in neurons who found that cAMP-dependent phosphorylation could J Pharm Pharmacogn Res (2016) 4(6): 213



revert BDM effects on the L-type Ca2+ current. In order to clarify this aspect we conducted experiments in cardiomyocytes intracellularly perfused with cAMP to fully activate PKA and the results show that when cardiomyocytes were previously incubated in BDM, the cAMP-mediated increase in ICaL (>160% in control cardiomyocytes) was almost suppressed. Moreover, in cardiomyocytes not incubated in BDM and intracellularly perfused with cAMP, extracellular BDM was still able to decrease the stimulated ICaL by an amount similar to that observed in control conditions. CONCLUSIONS Overall the present results indicate that the decrease of basal ICaL by BDM is not related to the dephosphorylation action of this oxime and that this action of BDM on the L-type Ca2+ channel occurs most probably in a site in the extracellular side or within the sarcolemmal membrane. However, due to its phosphatase action, BDM strongly attenuates the response of ICaL to β-adrenergic stimulation. The experiments with BDM-incubated cardiomyocytes indicate that intracellular phosphataselike action of BDM could be long lasting. These actions of BDM must be taken into account both for its use in the dissociation and preservation of isolated myocytes, and for its utilization in cardioplegic solutions and myocardial preservation. We should remark that the concentrations of BDM used in the present experiments were lower than those commonly reported by other authors. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENT This work supported by the Ministry of Public Health of Cuba (Research Project 1104012).

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guinea-pig ventricular myocytes Pflügers Arch Eur J Physiol 430: 68-80. Allen TJ, Mikala G, Wu X, Dolphin AC (1998) Effects of 2,3butanedione monoxime (BDM) on calcium channels expressed in Xenopus oocytes. J Physiol (L) 508: 1-14. Alvarez-Collazo J, Díaz García CM, López Medina AI, Vassort G, Alvarez JL (2012) Zinc modulation of basal and βadrenergically stimulated L-type Ca2+ current in rat ventricular cardiomyocytes: consequences in cardiac diseases. Pflügers Archiv Eur J Physiol 464: 459-470. Alvarez JL, Aimond F, Lorente P, Vassort G (2000) Late postmyocardial infarction induces a tetrodotoxin-resistant Na+ current in rat cardiomyocytes. J Mol Cell Cardiol 32: 11691179. Alvarez JL, Hamplova J, Hohaus A, Morano I, Haase H, Vassort G (2004) Calcium current in rat cardiomyocytes is modulated by the carboxy-terminal ahnak domain. J Biol Chem 279: 12456-12461. Bénitah JP, Alvarez JL, Gómez AM (2010) L-type Ca2+ current in ventricular cardiomyocytes. J Mol Cell Cardiol 48: 26-36. Bers DM (2001) Excitation-contraction Coupling and Cardiac Contractile Force. Second edition Kluwer Academic Press, The Netherlands: Dordrecht. Borlak J, Zwadlo C (2004) The myosin ATPase inhibitor 2,3butanedione monoxime dictates transcriptional activation of ion channels and Ca2+-handling proteins. Mol Pharmacol 66: 708-717. Chambers DJ (2005) Mechanism of cardiac damage associated with cardiac surgery. Heart Metab 29: 5-9. Chapmann RA (1993) The effect of oximes on the dihydropyridine-sensitive Ca2+ current of isolated guineapig ventricular myocytes. Pflügers Arch Eur J Physiol 422: 325-331. Chung CS, Mechas C, Campbell KS (2015) Myocyte contractility can be maintained by storing cells with the myosin ATPase inhibitor 2,3-butanedione monoxime. Physiol Rep 3: e12445 doi: 1014814/phy212445. Coulombe A, Lefebvre IA, Deroubaix E, Thuringer D, Coraboeuf E (1990) Effect of 2,3-butanedione monoxime on slow inward and transient outward currents in rat ventricular myocytes. J Mol Cell Cardiol 22: 921-932. Daly MJ, Elys JS, Nayler WG (1987) Contracture and the calcium paradox in the rat heart. Circ Res 61: 560-569. Eisfeld J, Mikala G, Varadi G, Schwartz A, Klockner U (1997) Inhibition of cloned human L-type cardiac calcium channels by 2,3-butanedione monoxime does not require PKA-dependent phosphorylation sites. Biochem Biophys Res Comm 230: 489-492. Ferreira G, Artigas P, Pizarro G, Brum G (1997) Butanedione monoxime promotes voltage-dependent inactivation of Ltype calcium channels in heart. Effects on gating currents. J Mol Cell Cardiol 29: 777-787. Habazettl H, Voigtlander J, Leiderer R, Messmer K (1998) Efficacy of myocardial initial reperfusion with 2,3 butanedione monoxime after cardioplegic arrest is timedependent. Cardiovasc Res 37: 684-690. Hall AR, Hausenloy DJ (2016) Mitochondrial respiratory inhibition by 2,3-butanedione monoxime (BDM): Implications for culturing isolated mouse ventricular J Pharm Pharmacogn Res (2016) 4(6): 214



cardiomyocytes. Physiol Rep 4: e12606 doi: 1014814/phy212606. Herrmann C, Wray J, Travers F, Barman, T (1992) Effect of 2,3butanedione monoxime on myosin and myofibrillar ATPases: An example of an uncompetitive inhibitor. Biochemistry 31: 1222-12232. Huang GJ, McArdle JJ (1992) Novel suppression of an L-type calcium channel in neurones of murine dorsal root ganglia by 2,3-butanedione monoxime. J Physiol (L) 447: 257-274. Jayawant AM, Stephenson ES, Damiano RJ (1999) 2,3Butanedione monoxime cardioplegia: Advantages over hyperkalemia in blood-perfused isolated hearts. Ann Thorac Surg 67: 618-623. Kivistö T, Makiranta M, Oikarinen E-L, Karhu S, Weckström M, Sellin LC (1995) 2,3-Butanedione monoxime (BDM) increases initial yields and improves long-term survival of isolated cardiac myocytes. Jap J Physiol 45: 203-210. Lang RJ, Paul RJ (1991) Effects of 2,3-butanedione monoxime on whole-cell Ca2+ channel currents in single cells of the guinea-pig taenia caeci. J Physiol (L) 433: 1-24. Lee BK, Kim MJ, Jeung KW, Choi SS, Park SW, Yun SW, Lee SM, Lee DH, Min YI (2016) 2,3-Butanedione monoxime facilitates successful resuscitation in a dose-dependent fashion in a pig model of cardiac arrest. Am J Emerg Med 34: 1053-1058. Lopatin AN, Nichols CG (1993) Block of delayed rectifier (DRK1) K+ channels by internal 2,3-butanedione monoxime in Xenopus oocytes. Receptors Channels 1: 279-286. Ostap EM (2003) 2,3-Butanedione monoxime (BDM) as a myosin inhibitor. J Mus Res Cell Motil 23: 305-308. Reichert KL, Pereira do Carmo, HR, Lima F, Torina AG, de Souza Vilarinho KA, Martins de Oliveira PP, Silveira Filho LM, Barbosa de Oliveira Severino ES, Petrucci O (2013) Development of cardioplegic solution without potassium: Experimental study in rat. Rev Bras Cir Cardiovasc 28: 52430. Schlichter LC, Pahapill PA, Chung I (1992) Dual action of 2,3butanedione monoxime (BDM) on K+ current in human T lymphocytes. J Pharmacol Exp Ther 261: 438-46. Schwinger RH, Bohm M, Koch A, Morano I, Ruegg JC, Erdmann E (1994) Inotropic effect of the cardioprotective agent 2,3butanedione monoxime in failing and nonfailing human myocardium. J Pharmacol Exp Ther 269: 778-786.

Siegman MJ, Mooers SU, Warren TB, Warshaw DM, Ikebe M, Butler TM (1994) Comparison of the effects of 2,3butanedione monoxime on force production, myosin light chain phosphorylation and chemical energy usage in intact and permeabilized smooth and skeletal muscles. J Mus Res Cell Motil 15: 457-472. Stapleton MT, Fuchsbauer CM, Allshire AP (1998) BDM drives protein dephosphorylation and inhibits adenine nucleotide exchange in cardiomyocytes. Am J Physiol 275: H1260-H1266. Stringham JC, Paulsen KL, Southhard JA, Mentzer M, Belzer FO (1994) Prolonging myocardial preservation with a modified University of Wisconsin solution containing 2,3butanedione monoxime and Ca2+. J Thorac Cardiovasc Surg 107: 764-775. Tripathi A, Xu L, Pasek DA, Meissner G (1999) Effects of 2,3butanedione 2-monoxime on Ca2+ release channels (ryanodine receptors) of cardiac and skeletal muscle. J Membr Biol 169: 189-198. Vahl CF, Bonz A, Hagl C, Timek T, Herold U, Fuchs H, Kochsiek N, Hagl S (1995) Cardioplegia on the contractile apparatus level: evaluation of a new concept of myodardial preservation in perfused pig hearts. Thorac Cardiovasc Surg 43: 185-193. Verrechia F, Hervé JC (1997) Reversible blockade of gap junctional communication by 2,3-butanedione monoxime in rat cardiac myocytes. Am J Physiol 272: C875-C885. Warnecke G, Schulze B, Hagi C, Haverich A, Klima U (2002) Improved right heart function after myocardial preservation with 2,3-butanedione 2-monoxime in a porcine model of allogenic heart transplantation. J Thorac Cardiovasc Surg 123: 81-88. Watanabe Y, Koide Y, Kimura J (2006) Topics on the Na+/Ca2+ exchanger: pharmacological characterization of Na+/Ca2+ exchanger inhibitors. J Pharmacol Sci 102: 7-16. Weiss S, Oz S, Benmocha A, Dascal N (2013) Regulation of cardiac L-type Ca2+ channel CaV 1.2 via the β-adrenergiccAMP-protein kinase pathway: old dogmas, advances and new uncertainties. Circ Res 113: 617-631. Wilson IR, Ginsburg S (1955) A powerful reactivator of alkylphosphate-inhibited acetylcholinesterase. Biochim Biophys Acta 18: 168-170. Xiao YF, McArdle JJ (1995) Effects of 2,3-butanedione monoxime on blood pressure, myocardial Ca2+ currents, and action potentials of rats. Am J Hypertens 8: 1232-1240.

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Citation Format: Álvarez-Collazo J, López-Medina AI, Galán-Martínez L, Álvarez JL (2016) 2,3-Butanedione monoxime attenuates the βadrenergic response of the L-type Ca2+ current in rat ventricular cardiomyocytes. J Pharm Pharmacogn Res 4(6): 206-216.



© 2016 Journal of Pharmacy & Pharmacognosy Research, 4 (6), 217-223 ISSN 0719-4250 http://jppres.com/jppres Short Communication | Comunicación Corta

Negative inotropic and dromotropic actions of SiO2 nanoparticles on isolated rat hearts: Effects on Na+ and Ca2+ currents [Acciones inotropo y dromotropo negativas de nanopartículas de SiO 2 en corazones aislados de ratas: Efectos sobre las corrientes de Na+ y Ca2+] Julio Alvarez-Collazo1,2,#, Loipa Galán-Martínez1,#, Alicia Fleites-Vazquez1, Alicia Sánchez-Linde2, Karel Talavera-Pérez2, Julio L. Alvarez1* 1Laboratorio 2Laboratory

de Electrofisiología. Instituto de Cardiología y Cirugía Cardiovascular. Paseo y 17, Vedado, CP 10400, La Habana. Cuba. of Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. #Both authors contributed equally. *E-mail: [email protected]

Abstract

Resumen

Context: SiO2 nanoparticles (NP) are widely used in the industry and in varied biotechnological and medical applications. However, epidemiological studies suggest that pollution with fine particles (in which silica is an inorganic component) may increase morbidity and mortality from cardiovascular diseases, but little is known about their potential cardiovascular actions.

Contexto: Las nanopartículas de SiO2 (NP) se utilizan ampliamente en la industria y en variadas aplicaciones biotecnológicas y médicas. No obstante, hay estudios epidemiológicos que sugieren que la polución con partículas finas (en las que la sílica es un componente inorgánico) pueden aumentar la morbilidad y mortalidad por enfermedades cardiovasculares, pero poco se conoce sobre sus potenciales acciones cardiovasculares.

Aims: To study the actions of SiO2 nanoparticles on the electrical and contractile activity of rat hearts and to identify the possible underlying cellular mechanisms.

Objetivos: Estudiar las acciones de nanopartículas de SiO2 sobre las actividades eléctrica y contráctil de corazones de rata e identificar los posibles mecanismos subyacentes.

Methods: Surface electrogram (ECG) and force of contraction (FC) was recorded in isolated rat hearts. Na+ and Ca2+ currents (INa and ICaL, respectively) were recorded, with the patch-clamp technique, in enzymatically isolated rat ventricular cardiomyocytes.

Métodos: Se registró el electrograma de superficie (ECG) y la fuerza de contracción (FC) en corazones aislados de rata. Las corrientes de Na+ y Ca2+ (INa and ICaL, respectivamente) se registraron, con la técnica de patchclamp, en cardiomiocitos ventriculares de rata aislados enzimáticamente.

Results: SiO2 NP (1-30 µg/mL) decreased the FC and markedly increased QRS duration and QT interval in spontaneously beating hearts. Electric stimulation (RR = 400 ms) partially restored the FC. In patch-clamp experiments NP (30 µg/mL) decreased INa in a use-dependent manner and increased ICaL.

Resultados: Las NP de SiO2 (1-30 µg/mL) disminuyeron la FC y aumentaron marcadamente la duración del QRS y el QT en corazones espontáneos. La estimulación eléctrica (RR = 400 ms), restauró parcialmente la FC. En los experimentos con patch-clamp, las NP (30 µg/mL) disminuyeron INa de manera dependiente del uso e incrementaron ICaL.

Conclusions: SiO2 nanoparticles exert a negative inotropic action in rat hearts due, in part, to a decrease in the fast sodium current responsible for cardiac depolarization. SiO2 nanoparticles are also able to increase the L-type Ca2+ current. These actions should be taken into account when analyzing the toxic effects of these nanoparticles.

Keywords: calcium channels; heart; nanoparticles; patch-clamp; silica; sodium channels.

Conclusiones: Las nanopartículas de SiO2 ejercen una acción inotropo negativa en corazones de rata debido, en parte, a una reducción de la corriente rapida de sodio responsable de la despolarización cardíaca. Las NP de SiO2 también aumentaron la corriente de Ca2+ tipo L. Estas acciones deben ser tomadas en consideración al analizar los efectos tóxicos de estas nanopartículas. Palabras Clave: canales de calcio; nanopartículas; patch-clamp; silica.

canales

ARTICLE INFO Received | Recibido: September 5, 2016. Received in revised form | Recibido en forma corregida: September 23, 2016. Accepted | Aceptado: September 23, 2016. Available Online | Publicado en Línea: September 28, 2016. Declaration of interests | Declaración de Intereses: The authors declare no conflict of interest. Funding | Financiación: The study was supported by the Cuban Ministry of Public Health (Research Project 151045). Academic Editor | Editor Académico: Marisol Fernández.

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INTRODUCTION Nanoparticles (NP) of variable size and structure are widely used in industry, cosmetics as well as in medical and pharmaceutical products. Although there is a logarithmic increase in research on and applications of NP, including human health, it has been shown that NP may cause potential dangerous effects due to their high surface/volume ratio that makes them highly reactive or catalytic (Ying, 2001). NP can penetrate biologic membranes and cause toxic effects by interacting with different biological systems (Hanley et al., 2009). Yet, their biological actions are relatively not well understood even if nanotechnology foresee multiple applications of NP in medicine such as drug delivery (Hu et al., 2009), cancer therapy (Peer et al., 2007), biosensors (Lord and Kelley, 2009) and even nanoparticle drug-eluting stents (Yin et al., 2014). Thus, basic research is still required to evaluate potential toxicity issues related to the chemical properties of nanoparticle materials, as well as to their size and shape, but the wide variety of tissues, cells and cell membranes impose important hurdles to overcome in this promising field. Little is known about the mechanisms of action of NP on different biological systems (e.g. redox systems and metabolism; Fröhlich, 2013; Roy et al., 2014), as well as their action on voltage-dependent ionic channels (e.g. Liu et al., 2011). Moreover, the environmental impact of nanomaterials is still under study. In this sense silicon dioxide (SiO2; silica), a longstanding and widely used compound in industries, is known to be toxic and cause silicosis and bronchitis (see: Center for Construction Research and Training - Work Safely with Silica: “What are the Health Effects?” http://www.silica-safe.org).

However, SiO2 NP (10-15 nm diameter) are widely used in paints, rubbers, plastics, porcelain, batteries, adhesives, glass, steel, chemical fibers, plexiglass and aerogels (see: http://www.nanoparticlesmicrospheres.com/Products/). In addition SiO2 NP are also used in different applications in biotechnology and medicine, such as medical diagnostics, drug delivery, gene therapy, biomolecules detection and bioimaging (Kumar et al., 2010; Lee et al., 2011; Barandeh et al., 2012; Li et al., 2012). Epidemiological studies link air pollution with fine particles (silica is an inorganic component) to increases in morbidity and mortality http://jppres.com/jppres

Cardiac actions of silica nanoparticles

from cardiovascular diseases (Pope et al., 2004). However, there are only few studies of their potential cardiovascular actions (e.g. Duan et al., 2013). It was, thus, the purpose of the present investigation to study the actions of SiO2 nanoparticles on the electrical and contractile activity of rat hearts and to identify the possible underlying cellular mechanisms. MATERIAL AND METHODS SiO2 nanoparticles and chemicals SiO2 nanoparticles (LUDOX® TM-40 colloidal silica; CAS Number 7631-86-9; Molecular Weight 60.08; PubChem Substance ID 24866350) and all other chemicals were from Sigma Aldrich. Animals Male adult (7 - 8 weeks) Wistar rats were obtained from the National Center for Laboratory Animal Reproduction (CENPALAB; La Habana; Cuba). Prior to experiment, animals were adapted for seven days to laboratory conditions (controlled temperature 25 ± 2°C, relative humidity 60 ± 10% and 12 h light/dark cycles). Tap water and standard diet for rodents supplied by CENPALAB were freely provided. All procedures were conducted according to the guidelines for the use and care of laboratory animals approved by CENPALAB. Isolated hearts Rat hearts were mounted on a Langendorff column and perfused at constant flow (10 mL/min) with a Tyrode solution of the following composition (mmol/L): NaCl 140, KCl 2.5, MgCl2 0.5, CaCl2 2, Tris-hydroxymethylaminomethane 10, Glucose 10 (pH = 7.4, gassed with O2; T = 35°C). A bipolar platinum recording electrode was placed on the ventricular epicardium to record the surface electrocardiogram (ECG). Another bipolar platinum electrode was placed near the atrioventricular ring and was connected to an electronic stimulator. To record the force of contraction (FC), the cardiac apex was fixed to a force-displacement transducer with a surgical 6-0 silk thread. ECG and FC values were J Pharm Pharmacogn Res (2016) 4(6): 218



recorded at the spontaneous heart rate and at a fixed stimulus rate (400-ms RR interval).

by means of Student’s t test. Differences were considered statistically significant for p < 0.05.

Isolated ventricular cardiomyocytes

RESULTS AND DISCUSSION

Ventricular cardiomyocytes were isolated as previously described in detail (Alvarez-Collazo et al., 2012) and were kept in a K+-Tyrode solution containing 1 mM Ca2+ at room temperature (21 ± 2 °C) and used for experiments for 6 h. Patch-clamp recordings Cardiomyocytes were patch-clamped as previously described (Alvarez-Collazo et al., 2012). Whole cell currents were filtered at 3 kHz and digitized at 50μs intervals using the ACQUIS1 software (CNRS License). To study Na+ (INa) and L-type Ca2+ (ICaL) currents, K+ currents were blocked by substituting all potassium by cesium in extracellular and “intracellular” solutions. The extracellular solution contained (in millimolars): 117 NaCl, 20 CsCl, 10 HEPES, 2 CaCl2, 1.8 MgCl2, and 10 glucose, pH 7.4. The standard pipette (intracellular) solution contained (in millimolars): 130 CsCl, 0.4 Na2GTP, 5 Na2ATP, Na2-creatine phosphate, 2.0 MgCl2, 11 EGTA, 4.7 CaCl2 (free Ca2+ ≈ 108 nM), and 10 HEPES, pH 7.2 with CsOH. Pipette resistance was 1.0 - 1.2 MΩ. Membrane capacitance (Cm) and series resistance (Rs) were calculated as previously described (Alvarez-Collazo et al., 2012) and their average values were 154 ± 17 pF and 3.5 ± 0.3 MΩ, respectively (N = 10). Rs could be electronically compensated up to 50 %. Liquid junction potential was compensated before establishing the gigaseal. For routine monitoring of INa and ICaL a double pulse voltage-clamp protocol was used: from a holding potential (HP) of -80 mV every 4s the cell membrane was depolarized by a prepulse to -40 mV for 50 ms to activate INa. From this membrane potential a 200-ms test pulse to 0 mV evoked ICaL. The inactivation time courses of INa and ICaL were fitted to double exponentials using the fitting procedures of the ACQUIS1 software. Statistical analysis Results are expressed as means and standard errors of means. Statistical significance was evaluated http://jppres.com/jppres

Effects of SiO2 nanoparticles on electrical and mechanical activities of isolated hearts At concentrations of 1, 3 and 30 µg/mL, SiO2 nanoparticles (NP) induced a marked decrease in the force of contraction (FC) of isolated rat hearts; this effect was stable in ≈ 5 min (Fig. 1). The decrease in FC was variable and a concentration dependence could not be established. Pooled results of the three concentrations used in five hearts yielded, however, a statistically significant 64.1 ± 10.3% decrease in FC. The decrease in FC was accompanied by statistically significant increases in QRS and QT interval from 8.8 ± 0.4 ms and 58.8 ± 10.8 ms to 26.2 ± 6.2 ms and 127 ± 22.7 ms, respectively (Fig. 1). The RR interval showed a tendency to increase (423.3 ± 56.7 ms to 471.6 ± 55.8 ms) but without statistical significance. The marked increase in QRS duration strongly suggests that NP could be acting on the fast Na+ current (INa) responsible for the depolarization phase on the cardiac action potential. Although the negative inotropic action of the NP could be via their action on any of the mechanisms that lead to an increase intracellular Ca2+ during the excitationcontraction coupling (EC) such as Na+ and Ca2+ channels, the Na-Ca exchanger, the ryanodine receptor, the Ca-ATPase (Bers, 2001), the increase in QRS duration, and therefore the dispersion of the depolarization wave front (negative dromotropic effect) and desynchronization of the contraction in the whole heart, could be also responsible for the decrease of FC. Indeed, when hearts were electrically stimulated (~ 20 pulses, twice the threshold) at a 400 ms interval (not significantly greater than the control RR interval), the QRS was shortened and the FC could be partly restored (Fig. 1) indicating that the negative inotropic effects of NP was partially due to a dispersion of the depolarization (activation) wave front. However, effects on other major protagonists of the intracellular Ca2+ increase during the EC, specifically on the L-type Ca2+ current ICaL, cannot be ruled out. We must also point out that the NP seem to affect the repolarizing current system (mainly K+ currents) since the QT inJ Pharm Pharmacogn Res (2016) 4(6): 219


terval was significantly increased by an amount that cannot be only explained by the marked increase in the QRS duration. Two hearts developed arrhythmias such as extrasystoles with wide QRS complexes. The effects of NP on ECG and FC were only partially reversed upon returning to control solution. Effects of SiO2 nanoparticles on Na+ and Ca2+ currents of isolated ventricular cardiomyocytes Because in the experiments with isolated hearts we could not find a dependency of the NP action with the concentration, in the patch-clamp experiments we chose to work with the maximum concentration (30 µg/mL). Under control conditions INa and ICaL densities at -40 and 0 mV were 79.3 ± 2.7 pA/pF and 8.1 ± 0.5 pA/pF, respectively (frequency of 0.25 Hz; N = 6). The corresponding times to peak currents were 0.9 ± 0.1 ms and 4.3 ± 0.2 ms. The inactivation time course of both currents could be fitted to two exponentials (τfast and τslow) with values of 0.9 ± 0.04 ms and 5.7 ± 0.7 ms and 12.1 ± 1.4 ms and 50.3 ± 4.9 ms for INa and ICaL, respectively. As expected, both currents responded differently to the increase in stimulation frequency in control conditions (Fig. 2). Currents were stabilized at 0.25 Hz and stimulation stopped. After a rest period of one minute, the increase in the rate of voltage clamping to 1 Hz provoked no changes in INa. However, ICaL responded with a typical increase-decrease or “facilitation” (Fig. 2; see also Alvarez et al., 2004). When the same stimulation protocol was applied in the presence of NP at the maximal concentration of 30 µg/mL, INa showed a marked “use-dependence” decrease (pulse-to-pulse decrease after restoring stimulation) that could be fitted to one exponential with a time constant of 54.8 ± 2.1 sec. At the steadystate at high frequency, INa was significantly inhibited by 48.2 ± 9.2 % while at the steady-state at the control frequency (0.25 Hz) INa was also significantly inhibited by 34.0 ± 7.5 % (see Fig. 2). At 0.25 Hz NP significantly increased the time to peak INa to 1.9 ± 0.03 ms while τfast and τslow reached values of 0.94 ± 0.15 ms and 4.7 ± 0.8 ms, respectively (not statistically significant). The significant decrease in INa by NP at both rates predicts that the maximal rate of depolarization of the ventricular action potential will be reduced and the conduction of excitation, at the whole heart level, will be slower (Carmeliet and http://jppres.com/jppres


Vereecke, 2002).

This would create a greater dispersion of the depolarization wavefront and a desynchronization of the whole heart contraction giving as a result a negative inotropic action of NP. Indeed, as described above, when hearts were stimulated the FC was partially recovered. These results, however, do not rule out changes in intracellular Ca2+ load (via the Na+ - Ca2+ exchanger) (Bers, 2001) or even at the connexins level. One major protagonist of the cardiac excitationcontraction coupling and source of intracellular Ca2+ is the L-type Ca2+ current (Bers, 2001); therefore, another possible explanation for the negative inotropic effect of NP would be a decrease in ICaL. However, this was not the case. NP did not alter the frequency response of ICaL but rather increased ICaL at both control and high frequencies (Fig. 2). Peak ICaL at high frequency (during “facilitation”) was variably but significantly increased by 182 ± 90 % and steady-state ICaL at 0.25 Hz was significantly increased by 43.1 ± 5.9%. Time to peak ICaL, τfast and τslow were not significantly modified by NP reaching values of 4.3 ± 0.3 ms, 15.5 ± 1.8 ms and 50.3 ± 4.9 ms, respectively. It is to note that the effects of NP on both INa and ICaL were rapidly reversible (30-40 sec for INa and 10-15 sec for ICaL; “off” effect) upon washout with control extracellular solution. Due to the size of SiO2 particles (approximately 10 nm in diameter) it is difficult to foresee that a direct interaction between NP and specific aminoacid sequences (“sites”) within the Na+ and Ca2+ channels subunits (as with pharmacological agents), are responsible for the effects we describe here. One may hypothesize that by interacting with membrane lipids SiO2 NP may alter lipid microdomains (rafts) that are known to regulate ion channel function either by direct protein-lipid interaction or by modifying the lipid bilayer and ion channel environment (Maguy et al., 2006; Dart, 2010; Morris et al., 2012; Poveda et al., 2014). Other possibilities (Head et al., 2014) could be that NP modify the interaction of the lipid rafts with the cytoskeleton, a well-known modulator of ion channels activities (Calaghan et al., 2004), or any steps in intracellular signaling cascades such as the CaMKII-dependent phosphorylation of the L-type Ca2+ channel that determines ICaL “facilitation” (Bers and Morotti, 2014). However, due to the fast washout of NP actions on both INa (less than 40 J Pharm Pharmacogn Res (2016) 4(6): 220


sec) and ICaL (10-15 sec) effects on intracellular signaling pathways should be considered with caution. Use-dependent block of INa by local anesthetics or arrhythmic drugs has been interpreted in terms of “modulated” or “guarded receptor” hypotheses that consider state-dependent interaction of the drugs with specific sites within the Na+ channel (see


for review Wang and Strichartz, 2012).

Due to the size of SiO2 NP, it seems challenging to explain the usedependent action of NP in terms of specific site affinity according to Na+ channel conformation. Our results might suggest that other “non-specific” actions should be also considered to explain usedependent effects on INa. Figure 1. Effects of SiO2 nanoparticles (30 µg/mL) on the ECG (red lower trace) and force of contraction (black upper trace) of a spontaneously beating rat heart. Traces were selected in control conditions and at every minute under NP perfusion. After three minutes perfusion with NP, the heart was electrically stimulated (400 ms interval, ~20 pulses) with a suprathreshold stimulus (square pulse in blue).

Figure 2. Effects of SiO2 nanoparticles (30 µg/mL) on Na+ (INa) and Ca2+ (ICaL) currents simultaneously recorded on a single rat ventricular cardiomyocyte. The cell was patch-clamped with a double voltage pulse as indicated in Materials and Methods at 0.25 Hz (thin horizontal lines). At different times during the experiment (both in control and in the presence of NP) stimulation was stopped for one minute and reinitiated at 1 Hz (thick horizontal lines). After stabilization of current level, the rate of stimulation was returned to 0.25 Hz. The insets show INa and ICaL recordings at different times during the experiment marked with the corresponding colored symbols.




CONCLUSIONS SiO2 nanoparticles exert a negative inotropic action in rat hearts. A decrease in the fast sodium current responsible for cardiac depolarization partially explains this negative inotropism. SiO2 nanoparticles are also able to increase the L-type Ca2+ current. These actions should be taken into account when analyzing the toxic effects of these nanoparticles. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENT This work supported by the Ministry of Public Health of Cuba (Research Project 151045).

REFERENCES Alvarez-Collazo J, Díaz García CM, López Medina AI, Vassort G, Alvarez JL (2012) Zinc modulation of basal and βadrenergically stimulated L-type Ca2+ current in rat ventricular cardiomyocytes: consequences in cardiac diseases. Pflügers Archiv Eur J Physiol 464: 459-470. Alvarez JL, Hamplova J, Hohaus A, Morano I, Haase H, Vassort G (2004) L-type Ca2+current of rat cardiomyocytes is modulated by the carboxy-terminal ahnak domain. J Biol Chem 279: 12456-12461. Barandeh F, Nguyen PL, Kumar R, Iacobucci GJ, Kuznicki ML, Kosterman A, Bergey EJ, Prasad PN, Gunawardena S (2012) Organically modified silica nanoparticles are biocompatible and can be targeted to neurons in vivo. PLoS One 7: e29424. Bers DM (2001) Excitation-contraction Coupling and Cardiac Contractile Force. Second edition. Dordrecht, The Netherlands: Kluwer Academic Press. Bers DM, Morotti S (2014) Ca2+ current facilitation is CaMKIIdependent and has arrhythmogenic consequences. Front Pharm 5: 144. doi: 10.3389/fphar.2014.00144. Calaghan SC, Le Guennec J-Y, White E (2004) Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes. Prog Biophys Mol Biol 84: 29-59. Carmeliet E, Vereecke J (2002) Cardiac Cellular Electrophysiology. New York: Springer Science + Business Media. Dart C (2010) Lipid microdomains and the regulation of ion channel function. J Physiol 588: 3169-3178. Duan J, Yu Y, Li Y, Yu Y, Li Y, Zhou X, Huang P, Sun Z (2013) Toxic effect of silica nanoparticles on endothelial cells through DNA damage response via Chk1-dependent G2/M checkpoint. PLOS ONE 8 (4): e62087. Fröhlich E (2013) Cellular targets and mechanisms in the cytotoxic action of non-biodegradable engineered nanoparticles. Curr Drug Metab 14: 976-988.


Hanley C, Thurber A, Hanna C, Punnoose A, Zhang J, Wingett DG (2009) The influences of cell type and ZnO nanoparticle size on immune cell cytotoxicity and cytokine induction. Nanoscale Res Lett 4: 1409-1420. Head BP, Patel HH, Insel PA (2014) Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function. Membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling. Biochim Biophys Acta 1838: 532-545. Hu L, Mao ZW, Gao CY (2009) Colloidal particles for cellular uptake and delivery. J Mater Chem 19: 3108-3115. Kumar R, Roy I, Ohulchanskky TY, Vathy LA, Bergey EJ, Sajjad M, Prasad PN (2010) In vivo biodistribution and clearance studies using multimodal organically modified silica nanoparticles. ACS Nano 4: 699-708. Lee JE, Lee N, Kim T, Kim J, Hyeon T (2011) Multifunctional mesoporous silica nanocomposite nanoparticles for theranostic applications. Acc Chem Res 44: 893-902. Li Z, Barnes JC, Bosoy A, Stoddart JF, Zink JI (2012) Mesoporous silica nanoparticles in biomedical applications. Chem Soc Rev 41: 2590-2605. Liu Z, Ren G, Zhang T, Yang Z (2011) The inhibitory effects of nano-Ag on voltage-gated potassium currents of hippocampal CA1 neurons. Environ Toxicol 26: 552-558. Lord H, Kelley SO (2009) Nanomaterials for ultrasensitive electrochemical nucleic acids biosensing. J Mater Chem 19: 3127-3134. Maguy A, Hebert TE, Nattel S (2006) Involvement of lipid rafts and caveolae in cardiac ion channel function. Cardiovasc Res 69: 798-807. Morris CE, Juranka PF, Joós B (2012) Perturbed voltage-gated channel activity in perturbed bilayers: implications for ectopic arrhythmias arising from damaged membrane. Prog Biophys Mol Biol 110: 245-256. Peer D, Karp JM, Hong S, FaroKhzad OC, Margalit R, Langer R (2007) Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2: 751-760. Pope CA, Burnett RT, Thurston GD, Thun MJ, Calle EE, Krewski D, Godleski JJ (2004) Cardiovascular mortality and long-term exposure to particulate air pollution: epidemiological evidence of general pathophysiological pathways of disease. Circulation 109: 71-77. Poveda JA, Giudici AM, Renart ML, Molina ML, Montoya M, Fernández-Carvajal A, Fernández-Ballester G, Encinar JA, González-Ros JM (2014) Lipid modulation of ion channels through specific binding sites. Biochim Biophys Acta 1838: 1560-1567. Roy R, Kumar S, Tripathi A, Das M, Dwivedi PD (2014) Interactive threats of nanoparticles to the biological system. Immunol Lett 158: 79-87. Wang GK, Strichartz GR (2012) State-dependent inhibition of sodium channels by local anesthetics: A 40-year evolution. Biochem (Mosc) Suppl Ser A Membr Cell Biol 6: 120-127. Yin RX, Yang DZ, Wu JZ (2014) Nanoparticle drug- and geneeluting stents for the prevention and treatment of coronary restenosis. Theranostics 4: 175-200. Ying J (2001) Nanostructured Materials. New York: Academic Press.

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Author contributions: Contribution

Álvarez-Collazo J

Galán-Martínez L

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Álvarez JL

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Citation Format: Álvarez-Collazo J, Galán-Martínez L, Fleites-Vázquez A, Sánchez-Linde A, Talavera-Pérez K, Álvarez JL (2016) Negative inotropic and dromotropic actions of SiO2 nanoparticles on isolated rat hearts: Effects on Na+ and Ca2+ currents. J Pharm Pharmacogn Res 4(6): 217-223.



© 2016 Journal of Pharmacy & Pharmacognosy Research, 4 (6), 224-230, 2016 ISSN 0719-4250 http://jppres.com/jppres Original Article | Artículo Original

Antimicrobial activity of essential oil of Pimenta racemosa var. racemosa (Myrtaceae) leaves [Actividad antimicrobiana del aceite esencial de las hojas de Pimenta racemosa var. racemosa (Myrtaceae)] Billmary Z. Contreras-Moreno1,2,3*, Judith J. Velasco4, Janne del C. Rojas2,5, Lucero del C. Méndez1,2,3, María T. Celis1 1Laboratory

of Polymers and Colloids (POLYCOL), Faculty of Engineering, University of Los Andes (ULA), Ave. Don Tulio Febres Cordero, Mérida 5101, Venezuela. “C” of Natural Products, Research Institute, Faculty of Pharmacy and Bioanalysis, University of Los Andes (ULA), Mérida, Venezuela. Products Research Group (GIPRONA), Nucleus University Rafael Urdaneta (NURR), University of Los Andes (ULA), Trujillo, Venezuela. 4Microbiology and Parasitology Department, Faculty of Pharmacy and Bioanalysis, University of Los Andes (ULA), Mérida, Venezuela. 5Organic Biomolecular Research Group, Research Institute, Faculty of Pharmacy and Bioanalysis, University of Los Andes (ULA), Mérida, Venezuela. 2Laboratory

3Natural

*E-mail: [email protected]

Abstract

Resumen

Context: Essential oils represent a therapeutic alternative in natural products against pathogenic bacteria that have become resistant to antibiotics and threaten public health and individual health of patients.

Contexto: Los aceites esenciales representan una alternativa terapéutica en productos naturales contra bacterias patógenas que se han hecho resistentes a los antibióticos y que amenazan la salud pública y la salud individual de los pacientes.

Aims: To determine the antimicrobial activity of two essential oils of different densities, obtained by hydrodistillation of Pimenta racemosa var. racemosa fresh leaves collected from Táchira, Venezuela against different multirresistant bacterial strains of nosocomial origin. Methods: Disc diffusion agar method was carried out against seven reference strains: Candida albicans (CDC-B385), Candida krusei (ATCC 6258), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 23357), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923) and three different bacterial strains of nosocomial origin: Methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL) producing Escherichia coli and Enterobacter cloacae. Results: The essential oils of Pimenta racemosa var. racemosa inhibited the development of all microorganisms tested with minimum inhibitory concentration (MIC) values ranging from 20 to 400 μL/mL.

Objetivos: Evaluar la actividad antimicrobiana de dos aceites esenciales de diferentes densidades, obtenidos por hidrodestilación de las hojas frescas de Pimenta racemosa var. racemosa recolectadas en Táchira, Venezuela frente a diferentes cepas bacterianas multiresistentes de origen nosocomial. Métodos: Se usó el método de difusión en agar con disco para evaluar la actividad antimicrobiana de los aceites esenciales frente a siete cepas de referencia internacional: Candida albicans (CDC-B385), Candida krusei (ATCC 6258), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 23357), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923); y tres cepas bacterianas de origen nosocomial Staphylococcus aureus resistente a meticilina (SARM), Escherichia coli y Enterobacter cloacae productoras de β-lactamasa de espectro extenso (BLEE).

Conclusions: This is the first report concerning antimicrobial activity of essential oils obtained from Pimenta racemosa var. racemosa collected from Táchira, Venezuela with different densities. Furthermore, results showed the essential oils of this species might be an alternative as antimicrobial agent for the pharmaceutical industry.

Resultados: Los aceites esenciales de Pimenta racemosa var. racemosa evaluados inhibieron el desarrollo de todos los microorganismos ensayados con valores de concentración inhibitoria mínima (CIM) que oscilaron entre 20 y 400 µL/mL.

Keywords: antimicrobial activity; C. krusei; essential oil; MRSA; Pimenta racemosa.

Palabras Clave: aceite esencial; actividad antimicrobiana; C. krusei; Pimenta racemosa; SARM.

Conclusiones: Este es el primer reporte sobre actividad antimicrobiana de los aceites de diferentes densidades de esta especie colectada en Táchira, Venezuela. Además, los resultados revelaron que los aceites esenciales de esta especie pueden ser una alternativa como agente antimicrobiano para la industria farmacéutica.

ARTICLE INFO Received | Recibido: July 26, 2016. Received in revised form | Recibido en forma corregida: October 2, 2016. Accepted | Aceptado: October 10, 2016. Available Online | Publicado en Línea: October 12, 2016. Declaration of interests | Declaración de Intereses: The authors declare no conflict of interest. Funding | Financiación: The study was supported by Consejo de Desarrollo Científico, Humanístico, Tecnológico y de las Artes (CDCHTA), University of Los Andes (ULA), Mérida, Venezuela (Project Number I-1466-15-08-Ed). Academic Editor | Editor Académico: Gabino Garrido.

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Contreras-Moreno et al.

INTRODUCTION Pimenta racemosa var. racemosa (Mill.) J.W. Moore is an aromatic arboreal plant belonging to the genus Pimenta (Myrtaceae), native to the Caribbean and northwestern South America. However, it has been introduced successfully in the southeastern United States, Sri Lanka, East and West Africa and Indonesia (Dupont et al., 1954; Weiss, 2002; Contreras-Moreno et al., 2014a). In Venezuela, this genus is only represented by P. racemosa (Mill.) J.W. Moore (P. acris Kostel), and is distributed in Capital District, Falcón, Lara, Mérida, Nueva Esparta, Sucre, Táchira and Zulia states (Hokche et al., 2008). This species is cultivated as an ornamental, used in folk medicine, and commonly known as: Bay rum, Malagueta, Pepita and pepper species (Aristeguieta, 1973; Contreras-Moreno et al., 2014a; 2014b). Furthermore, it has been widely used due to the content of volatile essences, once distilled, are used in cosmetics, especially in formulations such as aftershave lotions, soaps, perfumes and hair treatments (Weiss, 2002; Boning, 2010; Contreras-Moreno et al.,. 2014a; 2014b). Regarding biological properties, the essential oil of this species has been studied for antioxidant (Jirovetz et al., 2007; Alitonou et al., 2012), insecticide (Leyva et al., 2007), antibacterial (Tajkarimi et al., 2010) and antifungal (Kim et al., 2008) activities. On the other hand, resistance to antibiotics has increased rapidly in recent years, causing a lot of concern, since it is becoming a major threat for patients, as the alternatives against infections caused by resistant pathogens is reduced. Methicillinresistant Staphylococcus aureus (MRSA) and enterobacteria producing extended-spectrum βlactamase (ESBL) as Escherichia coli and Enterobacter cloacae (Oteo et al., 2016) are the most dangerous microorganisms present in nosocomial infections, therefore the need for searching new alternatives in natural products. The aim of this investigation was to determine the antimicrobial activity of two essential oils of different densities, obtained by hydrodistillation of fresh leaves of Pimenta racemosa var. racemosa collected from Táchira, Venezuela against different multirresistant bacterial strains of nosocomial origin. Authors consider that P. racemosa var racemosa study is very important since it confirms, scihttp://jppres.com/jppres

Antimicrobial activity of Pimenta racemosa var. racemosa

entifically, its popular use of local people to aid skin infections produced by Gram positive microorganisms such as Staphylococcus. MATERIAL AND METHODS Botanical material Fresh leaves of Pimenta racemosa var. racemosa were collected in April 2012, near to Sector “Los Corredores de la Palmita”, Junín Municipality, Rubio town, located in southwestern Táchira state, Venezuela, altitude 859 m.a.s.l. Botanical identification was carried out by Dr Leslie R. Landrum, Herbarium Curator, School of Life Sciences, Arizona State University, USA. Specimens collected in the field are housed in the MERF Herbarium of the Faculty of Pharmacy and Bioanalysis, University of Los Andes (BC-01 code), Venezuela, and at the Herbarium of Arizona State University (ASU0075448 code), USA. Chemicals Müeller-Hinton Agar, Müeller-Hinton Broth and Sabouraud Dextrose Agar from BBLTM (BD, Maryland, USA); sodium chloride from Riedel-de-Haën (Hannover, Germany); dimethyl sulfoxide from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA); glucose from Merck (Darmstadt, Germany); azurfosin-methylene-blue from Lab-Line Chemical Product (LAB-LINE CA, Barquisimeto, Venezuela). Essential oils The essential oils of different densities used in this study, light (LO) and heavy (HO), were isolated by hydrodistillation of fresh leaves of Pimenta racemosa var. racemosa collected from Táchira, Venezuela and their chemical composition (Table 1) were previously reported by Contreras-Moreno et al. (2014a). Both oils, analyzed by GC/MS, revealed the presence of 17 and 13 components for LO and HO, respectively, being eugenol for both oils the major compound with 60.4% (LO) and 82.9% (HO) (Contreras-Moreno et al., 2014a).



Antimicrobial activity of Pimenta racemosa var. racemosa

Table 1. Volatile compounds (% total peak area) of LO and HO essential oil obtained from Pimenta racemosa var. racemosa leaves collected in Táchira, Venezuela. Compound

Essential oil (%) RI

LO*

HO*

3-Hexen-1-ol, (Z)

0.6

-

849

α-Pinene

0.5

-

936

1-Octen-3-ol

2.2

0.3

977

Myrcene

11.7

1.5

989

α-phellandrene

0.8

-

1003

p-cymene

1.0

0.2

1025

Limonene

5.4

0.9

1030

1,8-cineole

2.9

0.3

1033

β-ocimene

0.2

-

1049

Linalool

4.4

0.7

1100

4-Terpineol

0.9

0.2

1178

α-Terpineol

1.3

0.6

1190

Chavicol

6.0

9.3

1259

Eugenol

60.4

82.9

1364

α-Copaene

0.3

0.2

1377

Trans-(β)- caryophyllene

0.7

0.5

δ-cadinene

0.8

0.7

1417 1524

RI, retention indices relative to C6–C24 n-alkanes on the HP-5 MS column; MS, mass spectrum. * Taken from Contreras-Moreno et al. (2014a).

Antibiotics and fungicides Trimethoprin-Sulfamethoxazol® 10 μg, Vancomycin® 30 μg, Gentamycin® 10 μg, Aztreonam® 30 μg, Cefepime® 30 μg were purchased from BBLTM (BD, Maryland, USA), and Fluconazole® 100 μg from Liofilchem (Roseto degli Abruzzi, Italy). Vorcum® 200 mg (400 µg/mL Voriconazole) was adquired in Pfizer (Caracas, Venezuela). Microbiological analysis Bacterial strains MRSA (525), E. coli ESBL (7532) and E. cloacae ESBL (10221) were isolated from patients with nosocomial infections, hospitalized at the Neonatology Service (P28), Autonomous Institute University http://jppres.com/jppres

Hospital of Los Andes (Mérida, Venezuela). All yeasts and bacteria tested in this investigation are described in Table 2. Antimicrobial method The antibacterial activity was evaluated following the disc diffusion method described by Velasco et al. (2007). Antifungal assay was carried out according to the NCCLS (2004) disc diffusion methodology with some modifications, each yeast inoculum (2.5 mL) was incubated in Sabouraud's dextrose agar with chloramphenicol at 37°C for 18 h and the turbidity was adjusted to McFarland Nº 1 (3 x 108 CFU/mL) (NCCLS, 2004; Lozina et al., 2005; NarvaezFlorez et al., 2008). Twenty mL Müeller-Hinton agar (BBLTM) supplemented with methylene blue (0.5 μg/mL) and glucose (2%, w/v) were mixed with 1 mL of each yeast inoculum. The contents of Petri dishes were allowed to solidify at room temperature and kept at 4°C until analysis. A sterile control was also prepared (Pemán et al., 2006; CLSI, 2013; Buitrago et al., 2015). The minimum inhibitory concentration (MIC) was determined with all tested microorganisms by dilution of essential oil in dimethylsulphoxide (DMSO) within a range between 10-500 µL/mL concentrations. MIC was defined as the lowest concentration that inhibited bacterial growth visible (CLSI, 2013); a negative control using a saturated disc with DMSO to check the possible activity of this solvent against the microorganism tested was also included. The inhibitory zone around the disc was measured and expressed in mm. Experiments were performed by duplicate. Statistical analysis Statistical analyses were performed by using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA) to determine the statistical significance of differences (p < 0.05) in antimicrobial activity using the one-way analysis of variance (ANOVA) with two post-hoc analyses. The post-hoc Dunnett’s test was performed for the comparison of inhibition zones (Zi) between the control group (TrimethoprinSulfamethoxazol®, Vancomycin®, Gentamycin®, Aztreonam®, Cefepime®, Fluconazole® and Vorcum®) and the test groups (antimicrobial activities for both oils LO and HO) against test microorganisms J Pharm Pharmacogn Res (2016) 4(6): 226


(S. aureus, E. faecalis, E. coli, K. pneumonia, P. aeruginosa, C. albicans and C. krusei); while the posthoc Tukey test was performed for the comparisons of MIC between each microorganisms treated with oils (LO, HO, DMSO). All results (Table 2) were expressed as mean and standard deviation (SD) values of two parallel measurements. RESULTS Results observed in present investigation revealed that antimicrobial activity of essential oils (low-density oil, LO) and (high-density oil HO) of P. racemosa var. racemosa showed growth inhibition of all microorganisms tested with inhibition zones ranging between 14 to 32 mm and MIC values between 20-400 µL/mL. In general, there was a statistically significant difference between the Zi of both oils (for all reference bacterial strains) and their respective positive control (p