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An evaluation of vardenafil as a calcium channel blocker in pulmonary artery in rats.
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PMID:  24741191     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
OBJECTIVE: Vardenafil was reported to relax rat pulmonary artery through endothelium-dependent mechanisms. The aim of this in vitro study was to investigate other related mechanisms for this effect.
MATERIALS AND METHODS: Endothelium-intact and denuded artery rings were suspended in order to record isometric tension. In the rings with or without endothelium, the concentration-response curves for vardenafil were generated. In the rings without endothelium the contractile response induced by phenylephrine (Phe) or KCl was assessed in the presence or absence of vardenafil. In the last set of experiments, pulmonary artery rings were exposed to calcium-free isotonic depolarizing solution and the contractile response induced by the addition of calcium was evaluated in the presence or absence of vardenafil, nifedipine, verapamil or 1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one (ODQ).
RESULTS: Vardenafil attenuated pulmonary artery contraction induced by phenylephrine in the presence and absence of endothelium. In addition, vardenafil attenuated both Phe or KCl-induced contraction but, it's effect on the KCl dose-response curve was more significant. Vardenafil also inhibited the contractile response induced by calcium in a dose-dependent manner. Addition of nifedipine or verapamil did not significantly alter this effect while ODQ incubation significantly inhibited vardenafil-induced relaxation.
CONCLUSION: From these findings, it was proposed that vardenafil relaxed rat pulmonary artery through inhibiting calcium influx.
Authors:
Edibe Minareci; Gulay Sadan
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Type:  Journal Article    
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Title:  Indian journal of pharmacology     Volume:  46     ISSN:  1998-3751     ISO Abbreviation:  Indian J Pharmacol     Publication Date:  2014 Mar 
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Created Date:  2014-04-17     Completed Date:  -     Revised Date:  -    
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Nlm Unique ID:  7902477     Medline TA:  Indian J Pharmacol     Country:  India    
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Languages:  eng     Pagination:  185-90     Citation Subset:  IM    
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Journal Information
Journal ID (nlm-ta): Indian J Pharmacol
Journal ID (iso-abbrev): Indian J Pharmacol
Journal ID (publisher-id): IJPharm
ISSN: 0253-7613
ISSN: 1998-3751
Publisher: Medknow Publications & Media Pvt Ltd, India
Article Information
Copyright: © Indian Journal of Pharmacology
open-access:
Received Day: 11 Month: 4 Year: 2013
Revision Received Day: 14 Month: 6 Year: 2013
Accepted Day: 21 Month: 1 Year: 2014
Print publication date: Season: Mar-Apr Year: 2014
Volume: 46 Issue: 2
First Page: 185 Last Page: 190
PubMed Id: 24741191
ID: 3987188
Publisher Id: IJPharm-46-185
DOI: 10.4103/0253-7613.129315

An evaluation of vardenafil as a calcium channel blocker in pulmonary artery in rats
Edibe Minareciaff1
Gulay Sadanaff1
Department of Pharmacology, School of Medicine, Akdeniz University, Antalya 07070, Turkey
Correspondence: Correspondence to: Dr. Edibe Minareci, E-mail: edibekarasu@akdeniz.edu.tr

Introductıon

Intracellular calcium concentration ([Ca+2]i) has a pivotal role in determining vascular tone.[1] Calcium channels were identified in human pulmonary artery but, there is still much to be learned about the mechanisms for their effects in the pulmonary artery.[2] It has been demonstrated that an elevation in intracellular Ca+2 through Ca+2 channels, appeared to play a pivotal role in the development and perpetuation of pulmonary arterial hypertension.[3, 4, 5]

Pulmonary arterial hypertension (PAH) severity comes from its poor prognosis, high mortality rate and the absence of curative treatment.[6, 7] The median period of survival after diagnosis is about 2-3 years. Currently, clinicians use prostacyclin analogs, oral endothelin-receptor antagonists and oral phosphodiesterase type 5 (PDE-5) inhibitors for the treatment of PAH.[8, 9] A study by Pauvert et al., indicated that sildenafil, a specific PDE-5 inhibitor, was a potent pulmonary artery relaxant in chronic hypoxic rats and normalized the chronic hypoxia – induced increases in resting (Ca+2)i.[10] In monocrotaline-induced pulmonary hypertension model, we demonstrated that vardenafil, an another specific PDE-5 inhibitor, potently relaxed rat pulmonary artery rings through nitric oxide-dependent and – independent mechanisms.[11] In that study, vardenafil-induced relaxations were due to blockade of intracellular Ca+2 increase. Hence it was we suggested that vardenafil may be more effective than sildenafil or tadalafil for the treatment of pulmonary hypertension due to its Ca+2-related additional action. However, the relationship between vardenafil and Ca+2 influx in the pulmonary artery remains to be investigated. On the other hand, evidences about the presence of Ca+2 channels in human pulmonary artery suggest that their pharmacological manipulation may be considered in the treatment of PAH. If the mechanism depends only on the influx of extracellular calcium; then the vasodilator response to vardenafil should not be change by agents that reduce calcium entry across the cell membrane. This hypothesis inspired us whether or not calcium channel blockers could enhance vardenafil-induced relaxation response in rat pulmonary artery rings. Hence, this study was designed to evaluate an additional action involving calcium channels in relaxation rat pulmonary artery rings by vardenafil.


Materials and Methods
Experimental Animals

Male Wistar rats, with body weight ranging from 200 to 210 g, were used. Animals were obtained from Akdeniz University's Experimental Animals Unit. In document B.30.2.AKD.0.05.07.00-18 dated 6 June 2012, Akdeniz University Local Ethical Committee of Experimental Animals approved that all steps of this study were compliant with ethical rules. Prior to the experiments, animals were housed and fed at room temperature (22°C) for 1 week in groups.

Drugs

Phenylephrine (Phe), acetylcholine (ACh), ethylene glycol-bis (β-aminoethyl ether)-N, N, N’, N’- tetraacetic acid (EGTA), 1H-[1, 2, 4] oxadiazolo[4, 3-a] quinoxalin-1-one (ODQ), verapamil hydrochloride, cyclopiazonic acid and nifedipine were obtained from Sigma Chemical (St. Louis, MO, USA). Vardenafil was kindly provided by Bayer Health Care AG, Leverkusen, Germany. All drugs were dissolved in distilled water, except nifedipine, vardenafil, cyclopiazonic acid and ODQ in dimethyl sulfoxide (DMSO). Preliminary experiments showed that DMSO kept <0.2% (v/v) had no effect on tension development of isolated pulmonary artery rings.

Tissue Preparations and Vascular Reactivity Studies

Each animal was anesthetized with pentobarbital (45 mg/kg). The heart and lungs were removed en bloc; right and left main pulmonary arteries were dissected and harvested for vascular reactivity studies. Experiments were conducted on isolated main left and right pulmonary arteries. Main pulmonary artery branches were rapidly removed, gently cleaned of fat and connective tissues taking care not to damage the endothelium and cut into rings of about 3 mm then, carefully suspended by two-stainless-steel clips passed through the vessel lumen in a 20 ml organ bath containing Krebs-Henseleit solution (in mM: NaCl 118, KCl 5, NaHCO3 25, KH2PO4 1.0, MgSO4 1.2, CaCl2 2.5 and glucose 11.2) at 37°C and was continuously aerated with 95% O2 and 5% CO2 to obtain a pH of 7.4. Isometric tension was continuously measured with an isometric force transducer (FDI-05 force displacement transducer, BioPac), connected to a computer-based data acquisition system (MP35 transducer data acquisition system, BioPac). In some pulmonary artery rings, the endothelium was removed by inserting a plastic club into the lumen followed by gentle rubbing in rings for a few seconds. The tissues were equilibrated for 60 min under a resting tension of 1 g. During this time, Krebs solution was replaced every 15 min with fresh solution. After the 1 h equilibration period, pulmonary rings were challenged with 60 mM KCl (the same composition as Krebs’ solution with NaCl replaced by equimolar KCl) to check tissue viability. The integrity of the endothelium was confirmed by the addition of the endothelium-dependent vasodilator, ACh, 10−6 M, after contractions had been induced by Phe, 10−6 M. This was similarly made with the denuded vessels in order to exclude any endothelial function. The presence of functional endothelium was assessed by the ability of ACh to induce more than 80% relaxation of pulmonary artery rings precontracted with Phe. In endothelium-denuded rings, the relaxation to ACh was less than 10%.

Role of Endothelium in Vardenafil-induced Relaxation

After complete washout of ACh and Phe from the organ bath and the return of isometric tension to baseline values, to elucidate the role of endothelium in vardenafil-mediated relaxation, concentration-response to vardenafil (10−10-10−5 M) was studied in endothelium-intact and endothelium-denuded rings which were pre-contracted by Phe (10−6 M). Relaxation was expressed as the percentage of Phe-induced contraction.

Effect of Vardenafil on Contraction by Phe and KCl

The aim of these in vitro experiments was to determine the effect of vardenafil on the contractile responses induced by Phe or KCl in endothelium-denuded rings. Vardenafil (10−7-10−6 M) was added to the organ bath and after 20 min incubation, the concentration – response curve for Phe or KCl was gained by adding Phe (10−9-10−4 M) or KCl (10-60 mM) to the bath. Results were assessed by comparing the contractile response in the presence or absence of vardenafil in the endothelium-denuded rings. Tension was expressed as the percentage of 60 mM KCl-induced contraction.

Effect of Vardenafil on Extracellular Ca+2-induced Contraction

The denuded pulmonary artery rings were precontracted initially with 60 mM KCl. After washing, the Krebs solution was replaced by a Ca+2-free Krebs solution (Ca+2-free Krebs solution was prepared by the omission of CaCl2 and addition of 0.1 mM EGTA to chelate trace Ca+2) for 10 min. Next, Phe (10−6 M) and the Ca+2 ATPase inhibitor cyclopiazonic acid (10−5 M) were used to deplete intracellular Ca+2 stores and to prevent Ca+2 uptake to sarcoplasmic reticulum (SR), respectively. The Ca+2-free Krebs solution was then replaced with a Ca+2-free isotonic depolarizing solution containing a high KCl concentration (100 mmol KCl). Twenty minutes before Ca+2-induced contraction, vardenafil was added directly to organ bath. Finally, CaCl2 (3 × 10−5-3 × 10−2 M) was added cumulatively. The effect of vardenafil on the concentration-response curve for CaCl2 was examined by comparing the contractile response induced by the addition of CaCl2 in the presence or absence of vardenafil (10−7-10−6 M). We also investigated whether the calcium channel blocking action of vardenafil is yclic guanosine monophosphate [cGMP]-depent or independent. To clarify this mechanism, we used ODQ (guanylyl cyclase inhibitor, 10−4 M) incubation in the cumulative addition of CaCl2 (3 × 10−5-3 × 10−2 M) protocol. Then, to verify the contribution of L-type Ca+2 channels, concentration-response curves to CaCl2 ( 3 × 10−5-3 × 10−2 M) in arterial rings were also constructed in the presence of nifedipine (10−6 M) or verapamil (10−5 M).

The doses of these drugs in this study were chosen based on our pilot studies and in accordance with other reported.[4, 12, 13, 14]

Statistical Analysis

Experimental values of relaxation were calculated relative to the maximal changes from the contraction produced by Phe taken as 100% in each tissue. The contractile responses to Phe, KCl and CaCl2 are expressed as the percentage of the maximum contraction to the isotonic 60 mM KCl. Data are shown as the percentage of relaxation of n experiments, expressed as the mean ± standard error of the mean. Data were analyzed by two-way ANOVA for multiple comparisons followed by Bonferroni post-hoc test. P <0.05 was considered to indicate significance.


Results
Role of Endothelium in Vardenafil-induced Relaxation

The cumulative addition of the vardenafil (10−10-10−5 M) produced concentration-dependent relaxations in precontracted pulmonary artery rings with intact endothelium. Endothelium denudation significantly reduced the relaxation responses induced by vardenafil compared with responses obtained in endothelium-intact preparations for pulmonary artery. However, endothelium removal did not affect the relaxation responses when the highest concentration of vardenafil used (10−6 and 10−5 M) (Ema × 103.2 ± 2.7% and 103.2 ± 2.1%; pD2 8.3 ± 0.04 and 7.5 ± 0.03, n = 12), in endothelium intact and denuded groups, respectively) [Figure 1].

Effect of Vardenafil on Contraction by Phe and KCl

In endothelium-denuded rings, pre-incubation with various concentrations of vardenafil (10−7-10−6 M) inhibited the concentration-response contraction to Phe and depressed its maximal contraction to 80.78 ± 3.12% and 66.05 ± 3.23% respectively (vs. control group 95.14 ± 3.61%) [Figure 2a]. We also observed that 10−7 or 10−6 M vardenafil incubation inhibited the contractile response to KCl and depressed its maximal responses to 73.41 ± 3.23% and 59.27 ± 3.44% respectively (vs. control group 99.88 ± 2.63%) [Figure 2b].

Effect of Vardenafil on Extracellular Ca+2-induced Contraction

A separate set of experiments was performed in order to verify whether vardenafil-induced relaxations of the pulmonary artery involve Ca+2 entry blockade. Concentration-response curves to CaCl2 ( 3 × 10−5-3 × 10−2 M) in Ca+2-free Krebs solution were constructed in the absence or in presence of vardenafil (10−7-10−6 M). In the calcium-free isotonic depolarizing solution containing 100 mmol KCl, vardenafil significantly attenuated (P < 0.001) the contraction induced by the cumulative addition of CaCl2 ( 3 × 10−5-3 × 10−2 M) in dose-dependent manner in pulmonary artery preparations with denuded endothelium [Figure 3a]. To evaluate if attenuation in CaCl2-induced contraction due to vardenafil incubation was dependent to cGMP accumulation, we examined the effect of ODQ, a selective inhibitor of guanylyl cyclase, on the vardenafil-induced responses. It was found that artery relaxation induced by vardenafil incubation was significantly inhibited by ODQ [Figure 3b]. Further, concentration-response curves to CaCl2 were constructed in the presence of the L-type calcium channel blockers nifedipine (10−6 M), verapamil (10−5 M) alone or in combination with vardenafil (10−6 M) in endothelium-denuded pulmonary artery rings. Calcium channel blocker drugs partly reduced the CaCl2-induced contractions [Figure 4a]. Further, combination of either of these two calcium channel blockers with vardenafil had no additional inhibitory effect on CaCl2-induced contraction in comparison to these drugs alone [Figure 4b].


Discussion

It is widely known that endothelium-dependent vasorelaxation needs the presence of endothelium layer.[15] In this study, similar to our previous one, vardenafil relaxed the pulmonary artery rings despite mechanical removal of the endothelium, suggesting that vasorelaxant activity of vardenafil is also endothelium-independent.[11] The residual endothelium-independent pulmonary artery relaxation induced by vardenafil is still uncertain but may be related with Ca+2 entry mechanisms from the extracellular space and also other undefined direct effects in this tissue.[16] For an endothelium-independent vasodilator, major possible mechanism involved in its relaxant effect is the blockade of extracellular Ca+2 influx through transmembrane Ca+2 channels. Therefore, the present work was aimed to further understand the effects of vardenafil on vascular smooth muscle in order to shed more light on the mechanisms of the vasorelaxant action of this compound on isolated rat pulmonary artery rings.

The influx of extracellular Ca+2 is mainly through two kinds of transmembrane Ca+2 channels: receptor-operative Ca+2 channel (ROCC) and voltage-operative Ca+2 channel (VOCC).[17] The one induced by high-K+ solution was mainly due to the depolarization of smooth muscle cells and the influx of extracellular Ca+2 through VOCC. Phe, upon binding to α-adrenoceptor, stimulates the formation of inositol-1,4,5-triphosphate (IP3), which binds to and activates the specific IP3 receptor channel in SR membrane, induces the release of internal Ca+2 from SR and causes a transient contraction.[18] Simultaneously, Phe activates the ROCC and induces an influx of extracellular Ca+2, which causes a tonic contraction. In this in vitro study, vardenafil (10−7-10−6 M) attenuated both Phe and KCl-induced contraction in a dose-dependent manner, which is in agreement with previous reports.[19, 20] These results suggested that the vasorelaxant effect of vardenafil was due to the inhibition of (Ca+2)i increase in vascular smooth muscle cells. Nonetheless, vardenafil is more potent in relaxing the rat pulmonary artery that are precontracted with KCl than Phe. The magnitude of vardenafil-induced attenuation was greater in the KCl dose-response curve than in the Phe dose-response curve. This suggest that the VOCCs may be more sensitive to vardenafil than the ROCCs.

Pulmonary vasoconstriction is believed to be an early component of PAH.[21] Intracellular calcium concentration (Ca+2i) is a major trigger for pulmonary vasoconstriction, whereas depletion in the amount of (Ca+2)i tends to reduce contraction force. This is the basic mechanism of the clinical antihypertension agents being used in various hypertensions. Both extracellular Ca+2 and Ca+2 within intracellular stores contribute to the increase in cytosolic Ca+2 concentration.[22] Specifically, in vascular smooth muscle removal of extracellular Ca+2, the use of inhibitors of SR Ca+2 uptake and blockade of L-type calcium channels are important tools to distinguish which source is providing Ca+2 to the cell for functional responses. The rightward shift provoked by vardenafil in the contraction responses elicited by CaCl2 in pulmonary artery rings with denuded endothelium strongly suggests that blockage of Ca+2 influx through Ca+2 channels is an important mechanism by which vardenafil produces relaxation in precontracted arterial tissues.

In general, vardenafil-induced relaxation or other biological responses are mediated by two main mechanisms: (1) a NO-cGMP-dependent (2) NO-cGMP-independent.[11, 23] In addition, relaxation response induced by vardenafil has been proposed to be mediated by a reduction of intracellular Ca+2.[19, 24] Our survey of the literature on the interactions between these two mechanism failed to find any studies. Consequently, we studied the role of cGMP in the calcium channel blocking the action of vardenafil by studying the effects of ODQ incubation on CaCl2-induced contraction responses in the pulmonary artery. Our experimental results suggested that the calcium channel blocking the action of vardenafil is cGMP-dependent. Nitric oxide activates the synthesis of cGMP that, in turn, activates PDE5. Increased activity of PDE5 would result in cGMP degradation. The sustained relaxation of the pulmonary artery in the presence of PDE5 inhibitor is consistent with a continued elevation of cGMP. The effect of vardenafil on Ca+2-induced contraction was blocked by, ODQ a specific inhibitor of soluble guanylyl cyclase that synthezis cGMP and is activated by NO. As far our knowledge, this is the first study to report the contribution of cGMP on the calcium-channel blocking activity of vardenafil.

To further confirm our hypothesis that extracellular Ca+2 influx blockade contribute to the cellular responses to vardenafil, L-type calcium channel blockers nifedipine and verapamil were used in the present study. Nifedipine and verapamil significantly reduced the contractile response induced by CaCl2. The maximum contraction decreased from 110.22 ± 5.74% to 54.89 ± 5.34% and 56.11 ± 5.70%, respectively. To elucidate that the inhibitory effect of vardenafil on CaCl2-induced contractions was a result of L-type Ca+2 channel inhibition, its effect in combination with verapamil or nifedipine was also recorded. Addition of vardenafil did not alter the verapamil or nifedipine-induced inhibition of CaCl2 contraction responses, indicating that vardenafil relaxes pulmonary arteries primarily through inhibiting Ca+2 influx via L-type Ca+2 channels.

Vardenafil a PDE-5 inhibitor, is considered to be slightly more potent than sildenafil and tadalafil possibly due to its different chemical structure, which allows a slower dissociation rate from PDE-5.[25] Our results demonstrate that vardenafil has some other pharmalogical action in addition to PDE-5 inhibition. The first evidence came from our results showing that vardenafil continued to relax endothelium-denuded pulmonary arteries. The other finding that vardenafil markedly reduced CaCl2-induced contractions indicate that this additional mechanism could be related to blockade of Ca+2 channels. Our study is also in agreement with other reports demonstrating that vardenafil has an additional action of blocking Ca+2 channels, which enhances its vasorelaxant property in pulmonary artery and aorta.[14, 19]


Conclusions

It is known that vardenafil causes an accumulation of nitric oxide-driven cyclic guanosine monophosphate and subsequent endothelium-dependent relaxation in vascular smooth muscle.[26, 27] In the present study, vardenafil continued to relax endothelium-denuded pulmonary artery rings and significantly attenuated contraction responses elicited by CaCl2. Further, we aimed to explore the main mechanism by which vardenafil induced relaxation in rat isolated pulmonary artery we focused on the blockade of Ca+2 entry in modifying the contraction of arterial smooth muscle by using ODQ, nifedipine and verapamil. Combination of vardenafil with nifedipine or verapamil did not alter CaCl2-induced contraction responses but, ODQ incubation significantly attenuated vardenafil-induced relaxation. These data suggest that vardenafil-induced relaxation responses in endothelium-denuded pulmonary artery primarily depend on inhibiting Ca+2 influx through L-type Ca+2 channels and it's cGMP-dependent. Thus vardenafil is superior to other drugs that are used treatment of diseases with endothelial dysfunction such as pulmonary hypertension. However, many comprehensive studies with other techniques (electrophysiology, patch-clamping, etc.) are necessary support this finding.


Notes

Source of Support: Nil

Conflict of Interest: No

Acknowledgment

This study was supported by “The Scientific Research Projects Co-ordination Unit of Akdeniz University.”


References
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Figures

[Figure ID: F1]
Figure 1 

Concentration-dependent relaxant effect of vardenafil (10−10-10−5 M) in phenylephrine (10−6 M) precontracted pulmonary arteries with (•) or without (○) endothelium. Data are expressed as mean ± standard error of the mean of 12 rats



[Figure ID: F2]
Figure 2 

(a and b) Inhibitory effects of vardenafil (10−7-10−6 M) on the contraction induced by phenylephrine (10−9-10−4 M) or KCl (10-60 mM) in endothelium-denuded rat pulmonary artery rings. Results are presented as mean ± standard error of the mean of 12 rats. *P < 0.05 and **P < 0.01 versus control group



[Figure ID: F3]
Figure 3 

(a) Effect of vardenafil (10−7-10−6 M) on the cumulative-concentration curve induced by extracellular Ca+2 in endothelium-denuded pulmonary artery rings. Data are reported as mean ± standard error of the mean (SEM) of 12 rats. *P < 0.05 and **P < 0.01 versus control group. (b) Effect quinoxalin-1-one (ODQ) (10−4 M) incubation in the cumulative addition of CaCl2 (3 × 10−5-3 × 10−2 M). Results are presented as mean ± SEM of 12 rats. *P < 0.05 and **P < 0.01 versus group without ODQ incubation



[Figure ID: F4]
Figure 4 

(a and b) Concentration-response curves to CaCl2 in the absence or presence of nifedipine, verapamil and vardenafil in endotheliumdenuded pulmonary artery rings. Data are the mean ± standard error of the mean of 12 rats. *P < 0.01 versus control group. ○: Control, ■: Vardenafil alone, ▲: Nifedipine alone, ▼: Nifedipine + vardenafil, ♦: Verapamil alone, ◊: Verapamil + vardenafil



Article Categories:
  • Research Article

Keywords: KEY WORDS Calcium, phosphodiesterase type 5 inhibitors, pulmonary artery, vardenafil.

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