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Year : 2018  |  Volume : 8  |  Issue : 2  |  Page : 116-119  

Inhibition of spontaneous contractility of isolated caprine ureter by flupirtine


Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore, Tamil Nadu, India

Date of Submission09-May-2017
Date of Acceptance18-Jan-2018
Date of Web Publication19-Apr-2018

Correspondence Address:
Dr. Jacob Peedicayil
Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Vellore, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijabmr.IJABMR_159_17

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   Abstract 


Context: Kv7 potassium channels are expressed in several types of smooth muscles and could mediate physiological responses in the tissues expressed. Flupirtine is an analgesic that acts by opening Kv7 potassium channels. It has been shown to inhibit the contractility of several types of isolated smooth muscle. Aims: This study investigated the ability of flupirtine to inhibit the spontaneous contractility of isolated distal caprine (goat) ureter. Settings and Design: Spontaneous contractility of the isolated goat ureter was recorded using a physiograph. Materials and Methods: The ability of 1, 3, 10, 30, and 90 μM concentrations of flupirtine maleate to inhibit the spontaneous contractility of isolated distal goat ureter was investigated. The ability of the nonspecific potassium channel blocker 4-aminopyridine (4-AP; 1 mM) and the specific Kv7 channel blocker XE-991 (100 μM) to reverse the inhibitory effect of flupirtine on ureteric contractility was also investigated. Statistical Analysis Used: Both parametric and nonparametric statistical tests were used. Results: At 10, 30, and 90 μM concentrations, flupirtine significantly inhibited the spontaneous contractility of the isolated goat ureter. The EC50of flupirtine for a contact period of 10 min was 17.7 μM. The inhibitory effect of flupirtine on ureteric contractility was significantly reversed by 4-AP and XE-991. Conclusions: Flupirtine inhibits the spontaneous contractility of the isolated goat ureter by opening Kv7 channels.

Keywords: Contractility, flupirtine, isolated, Kv7channel, ureter


How to cite this article:
Naik GS, Kodagali R, Tyagi MG, Ernest K, Shanthi M, Mathew SK, Peedicayil J. Inhibition of spontaneous contractility of isolated caprine ureter by flupirtine. Int J App Basic Med Res 2018;8:116-9

How to cite this URL:
Naik GS, Kodagali R, Tyagi MG, Ernest K, Shanthi M, Mathew SK, Peedicayil J. Inhibition of spontaneous contractility of isolated caprine ureter by flupirtine. Int J App Basic Med Res [serial online] 2018 [cited 2019 Feb 21];8:116-9. Available from: http://www.ijabmr.org/text.asp?2018/8/2/116/230515




   Introduction Top


Flupirtine is a centrally acting analgesic available in many countries in Europe for treating various painful states.[1] It is an unusual analgesic in that it is thought to have a novel mechanism of action: opening of Kv7 potassium channels in neurons.[2],[3],[4] These channels were formally called KCNQ channels.[5] These channels are voltage-gated, 6-transmembrane channels involved in controlling cell excitability.[4] Flupirtine, in addition to acting as a Kv7 channel opener in neurons, also opens Kv7 channels in other body tissues such as smooth muscles. In this context, it has been shown to act as a Kv7 channel opener in vitro on isolated smooth muscles such as rat basilar artery,[6] rat pulmonary artery,[7] guinea pig detrusor,[8] and human detrusor.[9] To date, the ability of flupirtine to inhibit the contractility of the isolated ureter has not been studied. We recently showed that the calcium channel blocker (CCB) benidipine inhibits the contractility of the isolated caprine (goat) ureter.[10] Goat ureter is thought to be a suitable substitute for the human ureter for in vitro studies of the ureter.[11] The aim of this study was to investigate the inhibitory effect of flupirtine on the spontaneous contractility of isolated goat ureter, using a similar methodology as we used previously.[10]


   Materials and Methods Top


Tissue preparation

Ureteric tissues of the goat were obtained from a local slaughterhouse and transported to the Departmental Laboratory in oxygenated mammalian Ringer solution maintained at room temperature. The tissue specimen was dissected and the distal portion of the ureter was determined. Then, it was separated from the urinary bladder, and a 7-mm long specimen just above the vesicoureteric junction was used for the experiments after removing fat around the tissue. It was divided into strips 1.5–1.8 mm long, and the lumen was longitudinally cut open before mounting the strip as done previously.[10] The tissue was mounted in a 20 ml organ bath containing mammalian Ringer solution, adequately aerated with oxygen and kept at a temperature of 37°C. The Institutional Ethics and Research Review Board approved the study (IRB minute number: 7752, dated February 6, 2012).

Drugs and chemicals used

Flupirtine maleate was obtained from Santa Cruz Biotechnology, Dallas, TX, USA. The reversal agents, i.e., 4-aminopyridine (4-AP) and XE-991 were obtained from Santa Cruz Biotechnology, Dallas, TX, USA and Sigma Aldrich, Mumbai, India, respectively. The reversal agents were both dissolved in double distilled water. The vehicle used for dissolving flupirtine was 60% methanol to yield a clear solution and it was made sure that the maximum volume administered to the organ bath did not exceed 0.1 ml to avoid a vehicle effect. The composition of mammalian Ringer solution was as follows: NaCl: 154 mM; KCl: 5.6 mM; NaHCO3: 0.595 mM; dextrose: 5.5 mM; and anhydrous CaCl2: 2.2 mM per liter of double distilled water. These salts were obtained from Qualigens, Mumbai, India. The concentrations of flupirtine used in the study were 1, 3, 10, 30, and 90 μM. The concentrations of 4-AP and XE-991 that were used were 1 mM and 100 μM, respectively.

Experimental procedure

A maximum of 60 min was allowed for each tissue to exhibit spontaneous contractility. Tissues which failed to show spontaneous contractility within 60 min were discarded. Once the pattern of contractility was stable, control tracings of contractility were observed during two time periods. The first was 0–5 min and the second was 5–10 min. We validated this procedure.[12] Based on the validation, we found that tissues whose contractility were similar during the two time periods had consistent contractility for the entire study duration. After establishing this stable pattern, logarithmic doses of flupirtine were added to the organ bath. A contact period of 10 min was allowed to observe drug effects on tissue activity. The mechanism of action of flupirtine was studied using the reversal agents, 4-AP (1 mM) and XE-991 (100 μM). The reversal agent was added at the end of a 10-min incubation period with flupirtine. The tissue activity was observed for 15 min after adding the reversal agent. The concentrations of reversal agents were those used in previous studies.[13],[14]

Analysis of data

The tissue activity score, the product of the average height of contractility and the number of contractions over a specified period of time,[10],[15] was determined at each 5-min interval for the total study time of 35 min. Log-transformed activity scores, before and after flupirtine treatment, and the reversal agents were compared using repeated measures analysis of variance for each drug treatment including the vehicle. Greenhouse–Geisser correction was used to reduce the probability of type 1 error, if the Mauchly's test of sphericity was violated. P < 0.05 was considered statistically significant. The dose-response relationship was determined using the DRC package in R version 3.2.2, R Foundation for Statistical Computing, Vienna, Austria. For this, the percent inhibition of tissue activity score was used. The log-logistic model, Weibull type 1, and Weibull type 2 models were checked for fitting the data. Nonlinear regression was used to model and fit the dose-response curve for percent inhibition of tissue activity score and percent tissue activity score. The package DRC was used to perform this fit using R version 2.12.2. Weibull model 2.4 with lower and upper asymptote fixed at 0 and 100 produced the best fit for percent inhibition of the tissue activity score.


   Results Top


[Table 1] shows the effects of the vehicle used in the study (60% methanol) and flupirtine on the spontaneous contractility of the isolated goat ureter. The vehicle did not significantly inhibit ureteric contractility. Flupirtine at concentrations of 1 and 3 μM also did not significantly inhibit ureteric contractility. However, at concentrations of 10, 30, and 90 μM, flupirtine significantly inhibited ureteric contractility in a dose-dependent manner. The EC50 of flupirtine for a contact period of 10 min was 17.7 μM. As shown in [Table 2], the nonspecific potassium channel blocker 4-AP and the Kv7 channel blocker XE-991 significantly reversed the inhibitory effect of flupirtine on the contractility of the isolated ureter. A representative tracing of the reversal by XE-991 of the inhibition by flupirtine of the spontaneous contractility of the ureter is shown in [Figure 1].
Table 1: Inhibitory effect of flupirtine on spontaneous contractility of isolated caprine ureter (n=9 for each drug administration)

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Table 2: Reversal by 4-aminopyridine and XE-991 of inhibition by flupirtine of spontaneous contractility of isolated caprine ureter (n=9 for each drug administration)

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Figure 1: Representative tracing showing reversal by100 μ M XE-991of inhibition by 30 μM flupirtine of spontaneous contractility of isolated caprine ureter

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   Discussion Top


For the first time, this study has demonstrated that the Kv7 channel opener flupirtine inhibits the contractility of the isolated goat ureter. Flupirtine at concentrations ranging from 10 to 90 μM significantly inhibited spontaneous ureteric contractility [Table 1]. The inhibition by flupirtine was significantly reversed by the nonspecific potassium channel blocker 4-AP and the Kv7-specific channel blocker XE-991 [Table 2]. These results suggest that Kv7 channels are present in the ureter and that flupirtine exerts its inhibitory effect on spontaneous contractility of the ureter by opening Kv7 channels. Similar results have been found in studies on other isolated smooth muscles from the urinary tract. Thus, flupirtine has been found to inhibit the contractility of the isolated urinary bladder of guinea pigs [8],[16] and humans,[9] with subsequent reversal by XE-991.[9] 4-AP and XE991 when administered to isolated smooth muscle alone in the absence of an agonist are known to have a stimulant (contractile) effect. Thus, Huang [17] showed that at concentrations of 1-10 mM, 4-AP dose-dependently induced contractility of the isolated rat vas deferens. Yeung and Greenwood [18] showed that 10 μM XE-991 increased the spontaneous contractility of the isolated mouse portal vein.

An important problem seen in clinical urological practice is ureteric calculi. One type of therapy for ureteric calculi is the use of medical expulsion therapy (MET) - the use of drugs to reduce the associated pain and help expel the calculi. Drugs used for MET include CCB and α-adrenergic receptor blockers.[19] The aim of MET is to facilitate the spontaneous passage of ureteric stones. Meta-analyses have shown that patients with ureteric calculi treated with α-adrenergic receptor blockers and CCB are more likely to pass calculi with fewer colic episodes than those who do not receive MET.[19] The results of the current study suggest that potassium channel openers such as flupirtine can also be investigated for use in MET. To the best of our knowledge, to date, potassium channel openers have not been investigated in clinical trials for this purpose. Flupirtine is currently being used in a number of European countries for pain relief and is not known to have any major adverse effects.[20]


   Conclusions Top


The potassium channel opener flupirtine inhibits the spontaneous contractility of the isolated goat ureter. The inhibitory effect was reversed by the nonspecific potassium channel blocker 4-AP and the specific Kv7 channel blocker XE-991, suggesting that flupirtine inhibited ureteric contractility by opening Kv7 channels. Flupirtine could be investigated for clinical use as part of MET.

Acknowledgment

The authors acknowledge Drs A. Devasia, T. S. Vijayakumar, and S. M. Amirtham for their help.

Financial support and sponsorship

This study was funded by an intramural research grant, Christian Medical College, Vellore.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Türk C, Petřík A, Sarica K, Seitz C, Skolarikos A, Straub M, et al. EAU guidelines on diagnosis and conservative management of urolithiasis. Eur Urol 2016;69:468-74.  Back to cited text no. 19
    
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    Tables

  [Table 1], [Table 2]



 

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