Sildenafil increases the force of right atrial contractions in vitro via the NO-guanylyl cyclase pathway involving b-adrenoceptor linked mechanisms

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Sildenafil increases the force of right atrial

contractions in vitro via the NO-guanylyl cyclase pathway involving b-adrenoceptor linked

mechanisms

Sadhana Kanoo, Shripad B. Deshpande

Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221 005, India Correspondence:Shripad B. Deshpande, e-mail: desh48@yahoo.com

Abstract:

Sildenafil, a drug used in the treatment of erectile dysfunction, is a phosphodiesterase 5A inhibitor that increases cyclic guanosine monophosphate (cGMP) levels. In addition to its vascular actions, sildenafil is also known to alter cardiac functions. This study was undertaken to elucidate the effect of sildenafil on cardiac contractility and the underlying mechanisms. The experiments were con- ducted on spontaneously-beating right atria isolated from adult rats. The effect of sildenafil on the isometric contractionsin vitro was examined in the absence or presence of antagonists. Sildenafil (0.001–10 µM) produced a concentration-dependent increase in the atrial force of contraction without altering the atrial rate, even up to 10 µM. A concentration as low as 0.001 µM produced a significant increase (16%) in force and the increase was about 50% at 10 µM. Pretreatment with methylene blue (a guanylyl cyclase inhibitor) or N-w-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) blocked the force changes induced by sildenafil. Sildenafil-induced increase in force of contraction was also blocked by propranolol (ab-adrenoceptor antagonist) and diltiazem (an L-type Ca²⁺channel antagonist). The present results demonstrate that sildenafil increases the atrial force of contraction involving cGMP-b-adrenoceptor-Ca²⁺channel-dependent mechanisms.

Key words:

phosphodiesterase 5A inhibitor, cGMP, L-NAME, methylene blue, propranolol, Ca²⁺channels, diltiazem

Abbreviations: cAMP – cyclic adenosine monophosphate, cGMP – cyclic guanosine monophosphate, GTP – guanosine triphosphate, L-NAME – N-w-nitro-L-arginine methyl ester, NO – nitric oxide, PDE 5A – phosphodiesterase type 5A, PKA – protein kinase A, sGC – soluble guanylyl cyclase

Introduction

Sildenafil is a cyclic guanosine specific phosphodiesterase type 5A (PDE 5A) inhibitor that increases the

cyclic guanosine monophosphate (cGMP) levels by preventing its hydrolysis to 5´GMP [2]. Even though sildenafil is widely used in the treatment of erectile dysfunction, it was also found to be useful in the treatment of cardiac hypertrophy, myocardial ischemia/infarction, congestive cardiac failure, etc [7, 12, 14, 17, 18, 21, 22, 26]. Recently, sildenafil has been success- fully used in the treatment of pulmonary hypertension, where it improved hemodynamic parameters [5, 26]. The improvement of hemodynamic parameters was explained by decreased afterload leading to increased cardiac output. However, in a report else-

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where, no alterations are seen in the ejection fraction, the cardiac output or the ventricular contractility after sildenafil administration [8]. In contrast, activation of the sympathetic nervous system by sildenafil has been shown in one report [19] while suppression of isopre- naline/dobutamine-stimulated contractions of ventri- cles or myocytes has been reported in others [22–25].

Similarly, levels of cyclic adenosine monophosphate (cAMP) (which elevates cardiac intracellular Ca²⁺

concentration) in the heart after sildenafil administration, are also not in conformity with cardiac contractions [3, 27]. Thus, the effects of sildenafil on cardiac contractility are not consistent. Since the indications for the use of sildenafil are expanding to neurological diseases [26] in addition to cardiovascular pathologies (pulmonary hypertension, congestive cardiac failure, cardiac hypertrophy etc.), it is necessary to know its actions on cardiac activity. Even though G_i-guanylyl cyclase involvement via M2 receptors is known to modulate pacemaker activity [1, 28], the role of cGMP on cardiac contractility is not clear. Since sildenafil increases cGMP levels, it thus becomes per- tinent to examine its action on cardiac activity. The present study was therefore undertaken to investigate the effects of sildenafil on spontaneously beating rat right atrial preparationsin vitro. The in vitro prepara- tions are devoid of systemic influences originating from cardiovascular, nervous, respiratory and endo- crine systems that modulate atrial rate. Thus, this preparation allows us to examine the effect of sildenafil on atria so as to elucidate the underlying mechanisms.

Material and Methods

Animals

Male albino rats of Charles Foster strain weighing 180–220 g were used. The animals were housed in a temperature, humidity and light (12 h light and 12 h dark) controlled room withad libitum supply of food and water. The animal care and handling were carried out as per the guidelines given by the Ethical Clear- ance Committee of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.

Dissection and recording

The methods for the dissection and recording of rat right atrial contractions have been described earlier [1]. Briefly, animals were killed by cervical disloca- tion and exsanguination. The thorax was opened im- mediately, and the heart was dissected out and placed in a Petri dish containing chilled Krebs Ringer solution bubbled with 100% O₂. The right atrium was separated out and one end of the atrium was secured to the removable glass tissue holder. The tissue was then placed in an organ bath filled with Krebs Ringer solution maintained at 28 ± 1°C. The other end of atrium was fastened firmly by a fine thread to the force displacement transducer. The tissue was given an initial tension of 2 mN and was allowed to equili- brate for 30 min before taking the control recordings.

The atrial contractions were recorded on a chart re- corder (Biodevices, Ambala, India).

Drugs and solutions

Sildenafil citrate was procured from Mankind Pharma Pvt. Ltd., New Delhi, India. Propranolol, N-w-nitro- L-arginine methyl ester (L-NAME) and diltiazem were obtained from Sigma Chemical Co., St. Louis, MO, USA. Methylene blue was from BDH, England.

The stock (10^–2M) solution of all the drugs was pre- pared in distilled water and the final dilutions were made in Krebs Ringer solution. The Krebs Ringer solution had the following composition (mM): NaCl, 137;

KCl, 2.68; CaCl₂.2H₂O, 1.8; MgCl₂.6H₂O, 0.88;

NaH₂PO₄.2H₂O, 0.36; NaHCO₃, 7; and glucose, 11.

After bubbling with O₂, the pH of the solution was 7.4.

Experimental protocol

The animals were divided into two groups. In the first group (n = 8), after obtaining the initial recordings, the atrium was exposed to various concentrations of sildenafil (0.001–10 µM) in a cumulative manner while recording the contractions every 20 min, before expos- ing the preparation to the next higher concentration.

In the second group, after obtaining the initial recordings, the tissue was pretreated with methylene blue (100 µM; n = 6), L-NAME (10 µM; n = 4), propranolol (0.1 µM; n = 7) or diltiazem (0.1 µM; n = 4) for 15 min. Subsequently, the cumulative concentration- response relationship of sildenafil was determined in the presence of each antagonist.

Sildenafil increases atrial force of contraction

Sadhana Kanoo and Shripad B. Deshpande

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at various concentrations of sildenafil within a group were compared by using one-way analysis of variance (ANOVA) and between the groups by two-way ANOVA. Multiple comparisons were performed using the Student-Newman-Keuls test. A p value < 0.05 was considered significant.

Results

Sildenafil increased the force of atrial contrac- tions in a concentration-dependent manner

The response to various concentrations (0.001–10 µM) of sildenafil (n = 8) on atria isolated from male rats is shown in Figure 1. In these experiments, the initial atrial rate was 165± 8.8 beats per min (bpm). Silde- nafil did not alter the rate at any concentration, even when it was increased by 10,000 times.

On the other hand, sildenafil increased the force of contraction in a concentration-dependent manner (p < 0.05;

one-way ANOVA). A significant increase in force (16%) was observed even at a concentration of sildenafil as low as 0.001 µM (p < 0.05; Student-Newman- Keuls test; Fig. 1), and the increase was about 50% at 10 µM.

Fig. 1.Methylene blue blocked the sildenafil-induced force changes.

The upper panel depicts the original tracings of atrial contractions of an experiment at different concentrations of sildenafil. The initial (control) response is shown at 0. The horizontal line indicates 5 s and the vertical line indicates 0.5 mN. The mean ± SEM values are presented in the lower graphs. The values were obtained from 8 different experiments for sildenafil only and 6 different experiments for the methylene blue pretreated group (+ MB). The atria were pretreated with methylene blue (100 µM) for 15 min followed by cumulative concentrations of sildenafil. An asterisk (*) indicates a significant difference from the sildenafil alone response (p < 0.05; two-way ANOVA)

Tab. 1.Theper se effect of various antagonists on atrial contractions. The mean ± SEM values are from n number of observations. The force changes are expressed as a percentage of baseline

Antagonist n Rate (beats/min) Force (% of initial)

Before After Before After

Methylene blue (100 µM) 6 144.0 ± 4.4 204.7 ± 9.1* 100 56.0 ± 5.5*

L-NAME (10 µM) 4 129.0 ± 8.4 126.0 ± 7.3 100 96.0 ± 4.8

Propranolol (0.1 µM) 7 161.3 ± 8.5 160.0 ± 12.5 100 103.1 ± 6.1

Diltiazem (0.1 µM) 4 154.0 ± 19.2 94.0 ± 5.0* 100 119.1 ± 3.1*

* p < 0.05 as compared to before values (Studentst-test for paired observations)

Atrialresponse(%ofinitial)

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Methylene blue blocked the sildenafil-induced increase in force

The upstream pathway for the generation of cGMP was examined by using a guanylyl cyclase inhibitor (methylene blue; 100 µM). In these experiments (n = 6), pretreatment with methylene blue for 15 min increased the rate by 42% and decreased the force by 44% of initial (Tab. 1). After exposure to methylene blue, the sildenafil-induced increase in force was blocked at various concentrations (Fig. 1; p < 0.05, two-way ANOVA). There was a 20–40% decrease in contractile force in the methylene blue treated group with sildenafil (Fig. 1). There was no alteration in rate at various concentrations of sildenafil in this group.

L-NAME blocked the force changes induced by sildenafil

In this set of experiments (n = 4), nitric oxide (NO) involvement was ascertained by pretreating the atria with L-NAME (10 µM; 15 min). L-NAME per se did not show any significant change in the force of contraction (Tab. 1). Pretreatment with L-NAME blocked the sildenafil-induced increase in force of contraction (Fig. 2; p < 0.05, two-way ANOVA). There was a decrease in force by about 25% at 10 µM of sildenafil in contrast to the increase observed in sildenafil only group (Fig. 2; p < 0.05, Student-Newman-Keuls test for multiple comparisons). No rate changes were observed in this group.

Propranolol blocked the force changes induced by sildenafil

We examined the role of b-adrenoceptors for the in- creased force of contraction induced by sildenafil using propranolol. In this group (n = 7), propranololper se did not produce any significant alterations in rate and force (Tab. 1). Pretreatment with propranolol blocked the sildenafil-induced increase in force (Fig.

2, p < 0.05; two-way ANOVA). No significant alteration in rate was seen in this group.

Diltiazem blocked the force changes induced by sildenafil

The involvement of Ca²⁺channels was examined by using diltiazem. In this group (n = 4), diltiazem (0.1 µM) alone decreased the rate by 39% and increased the force by 19% (Tab. 1). In diltiazem-pretreated atria,

the sildenafil-induced increase in force was blocked at various concentrations (Fig. 2; p < 0.05, two-way ANOVA). No significant alteration in rate was observed in this group.

Discussion

The present findings demonstrate that sildenafil ex- hibits a positive inotropic effect on the spontaneously beating atria without influencing the rate. The increase in force was evident even at a very low concentration (0.001 µM) of sildenafil. Further, our observations provide evidence for the NO-guanylyl cyclase pathway involving b-adrenoceptor-Ca²⁺ chan-

Fig. 2.L-NAME, propranolol and diltiazem blocked the sildenafil (0.00110 µM)-induced changes in atrial force. The mean ± SEM values are presented in the graph. The atria were pretreated with L-NAME (10µM, n = 4), propranolol (0.1 µM, n = 7) and diltiazem (0.1 µM, n = 4) for 15 min and then were exposed to cumulative concentrations of sildenafil. The sildenafil only data are taken from Figure 1. An asterisk (*) indicates a significant difference from the sildenafil alone group (p < 0.05; two-way ANOVA)

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tion of cGMP to 5´GMP. This is the basis for the action of sildenafil, a drug widely used in the treatment of erectile dysfunction. It is reported that PDE 5A is expressed in various tissues including cardiac tissue [16, 25]. Exposure to a PDE 5A inhibitor thus increases the cGMP level under physiological conditions. In the present experiments, sildenafil produced a concentration-dependent increase in force of contraction that was blocked by methylene blue, a soluble guanylyl cyclase (sGC) inhibitor (Fig. 1). The increased force of contraction thus can be related to the cGMP levels. In a study elsewhere, cGMP is reported to possess biphasic activity depending upon the concentrations. At lower concentrations of cGMP, an increase in cardiac contractility is shown, while at higher concentrations, a decrease in contractility is shown [6]. The sildenafil-induced increased contractility observed in the present study represents the activity of cGMP at lower concentrations. It should be noted here that the basal physiological level of cGMP is necessary for cardiac contractility [6]. In our experiments and elsewhere, an inhibitor of guanylyl cyclase per se decreased the cardiac contractility, signi- fying the modulatory effect of cGMP on the contractile forces [6]. This evidence further substantiates that the physiological (lower) levels of cGMP are required for the basal cardiac contractility.

The sGC, an enzyme necessary for the conversion of GTP to cGMP, is activated by NO [4, 20]. It is shown that NO also exerts biphasic effects on the cardiac contractility similar to those observed with cGMP, where increased contractility at lower concentrations and decreased contractility at higher concentrations of NO has been reported [6, 10, 15, 21]. In our study, L-NAME blocked the sildenafil-induced increase in force of contraction (Fig. 2). Thus, it is presumed that in the present experimental conditions, the concentration of NO is in the lower range. The increase in NO in turn increases the cGMP production by sGC activation. Hence, our findings support the NO-cGMP mechanisms for the sildenafil-induced increase in atrial contractility, as reported elsewhere for NO and cGMP [24].

The increased force of contraction observed in this study can be explained on the basis of the following points. The concentrations of NO and cGMP generated in spontaneously beating atria are presumably

low in the physiological range. Lower concentrations of cGMP inhibit PDE 3 (an enzyme necessary for the catalysis of cAMP) activity, thus preventing the hydrolysis of cAMP increasing its accumulation [9, 11, 20]. In addition, lower concentrations of NO activate adenylyl cyclase directly (without involving cGMP) to enhance cAMP levels [20]. In addition, it has been shown that lower concentrations of cGMP activate the a-subunit of G-protein that is associated with the b- adrenoceptor [13]. In the heart, the b-adrenergic re- ceptors enhance adenylyl cyclase activity and cAMP production. All these mechanisms increase the cAMP levels, leading to positive inotropy. The present result with propranolol confirms the b-adrenoceptor in- volvement. The involvement of G_ahas been demon- strated in stimulated myocytes elsewhere [13]. Thus, the increased force of contraction in our experiments can be due to cGMP-G_a-dependent mechanisms, as shown in Figure 3. The accumulated cAMP in turn activates protein kinase A (PKA) and opens Ca²⁺channels which increases the sarcoplasmic Ca²⁺leading to increased contractile force. The blockade of a contractile response by diltiazem, an L-type Ca²⁺channel antagonist, signifies the involvement of the Ca²⁺channel.

The positive inotropic activity observed in atrial contractionsin vitro in this study provides insight into the underlying mechanisms that are involved in the

Fig. 3.Schematics showing the proposed pathway for the sildenafil- induced increase in atrial force of contractions. >-AR – >-adrenoceptors; AC adenylyl cyclase; sGC soluble guanylyl cyclase; =,>,C – G-protein subunits; EC-coupling excitation-contraction coupling;

PDE 5 phosphodiesterase type 5; PDE 3 phosphodiesterase type 3

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increased contractility induced by sildenafil. How- ever, extrapolation of these results to in vivo condi- tions should be considered in order to have the per- spective of the entire hemodynamic circulation. How- ever, the positive inotropic effect of sildenafil at such low concentrations observed in this study signifies its effect. Hence, sildenafil has to be administered care- fully when using the drug in newer indications such as pulmonary hypertension, pulmonary edema, and congestive cardiac failure.

The schematics shown in Figure 3 represent the proposed action of sildenafil on atrial tissue in this study. In a spontaneously beating atrial tissue, the NO-guanylyl cyclase mechanism produces basal cGMP levels. The cGMP thus generated is not hydro- lyzed because of the PDE 5 inhibitory actions of sildenafil leading to its accumulation. The increased levels of cGMP, in turn, may inhibit PDE 3 or activate G_a (associated with b-adrenoceptor) to increase cAMP levels. The cAMP, in turn, opens the L-type Ca²⁺channel, increasing the sarcoplasmic Ca²⁺leading to increased contractility.

In conclusion, the sildenafil-induced increase in force of atrial contraction in spontaneously beating atria is through cGMP generated by the NO-guanylyl cyclase pathway. The cGMP in turn activates b adre- noceptors leading to sarcoplasmic Ca²⁺elevationvia L-type Ca²⁺channels. Since the force changes were detected at very low concentrations of sildenafil, the consequences of the effects of increased cardiac contractility during the use of sildenafil have to be considered.

Acknowledgment:

SK wishes to thank University Grants Commission, New Delhi for financial support.

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Received:

January 27, 2009; in revised form: October 17, 2009.