Inhibition of Contractility of Isolated Caprine Detrusor by the Rho Kinase Inhibitor Fasudil and Reversal by the Guanylyl Cyclase Inhibitor ODQ

Abstract

Background

Rho kinase (also called rho-associated protein kinase or ROCK), a serine/threonine kinase, is involved in muscle contractility since it phosphorylates myosin light chain phosphatase, hence inhibiting it. This leads to enhanced muscle contractility. Fasudil is an inhibitor of rho kinase that has been approved for the treatment of cerebral vasospasm in some Asian countries.

Objective

In this study, we tested the ability of fasudil to inhibit the contractility of the isolated caprine (goat) detrusor.

Methods

Twelve caprine detrusor strips were contracted using 80 mM KCl before and after the administration of 3 concentrations (10, 30, and 60 µM) of fasudil. Two reversal agents, the guanylyl cyclase inhibitor ODQ (10 µm) and the P2X receptor agonist diadenosine pentaphosphate (DAPP) (10 µM), were tested for their ability to reverse the inhibitory effect of fasudil on isolated detrusor contractility.

Results

Fasudil caused a dose-dependent inhibitory effect of KCl-induced detrusor contractility that was statistically significant at 30 and 60 µM concentrations. The inhibitory effect of 30 µM fasudil on detrusor contractility was significantly reversed by the addition of ODQ, but not by the addition of DAPP.

Conclusions

These results suggest that fasudil inhibits the contractility of the isolated detrusor. Fasudil could be investigated for use in clinical conditions such as overactive bladder that require detrusor muscle relaxation.

Introduction

The enzyme rho kinase (also called rho-associated protein kinase or ROCK), a serine/threonine kinase, is involved in muscle contractility since it phosphorylates myosin light chain phosphatase (MLCP), thereby inhibiting it. MLCP is responsible for the dephosphorylation of myosin, and so negatively regulates actin-myosin-based contractility, as illustrated by Joseph et al.¹ Hence, due to the action of rho kinase, muscle contraction is enhanced.¹ Drugs that inhibit rho kinase inhibit muscle contractility are available, and 2 such drugs, fasudil and ripasudil, have been approved for clinical use in Japan and China.² Fasudil was approved for the treatment of cerebral vasospasm and ripasudil for the treatment of glaucoma.² Since these drugs inhibit muscle contractility, they are potentially of importance in the treatment of disorders of the urinary bladder that require smooth muscle relaxation such as overactive bladder (OAB).^1,3 In this context, fasudil has been shown to inhibit the contractility of the isolated detrusor.^{4, 5, 6} In addition, it has recently been shown that fasudil can inhibit isolated detrusor muscle contractility by inhibiting the rho kinase pathway present in the urinary bladder urothelium and lamina propria.⁷

We conducted the present study to investigate the inhibitory effect of fasudil on the contractility of isolated caprine (goat) bladder and the possible reversal of the inhibitory effect using reversal agents that have to date not been used to reverse the inhibition caused by fasudil: the guanylyl cyclase inhibitor ODQ⁸ and the purinergic 2 (P2X) receptor agonist (diadenosine pentaphosphate, DAPP).⁹ The isolated caprine detrusor is an excellent substitute for the isolated human detrusor.^{8,10, 11, 12}

Methods

Tissue preparation

Twelve caprine urinary bladders were procured from a local butcher shop and brought to the pharmacology laboratory in Kreb’s solution. The sample size of 12 was chosen based on our previous studies on the inhibitory effects of drugs on isolated detrusor contractility^{8,10, 11, 12} and studies on the inhibitory effect of fasudil on isolated detrusor contractility.^6,7 The composition of Kreb’s solution was in millimoles: sodium chloride (NaCl) 111.5; potassium chloride (KCl) 4.6; magnesium sulfate (Mg₂SO₄) 1.16; sodium phosphate (Na₃P0₄): 1.16; calcium chloride (CaCl₂) 2.5; sodium bicarbonate (NaHCO₃)21.9; and glucose (C₆H₁₂O₆): 11.1.

Strips of detrusor muscle which measured 10×3 mm were dissected from the bladder as done previously in our laboratory.^{8,10, 11, 12} The urothelium was removed by gently scraping the strips. The strips were mounted in an organ bath containing sufficiently oxygenated Krebs solution having a volume of 25 mL and kept at a temperature of 37°C. A resting tension of 10 mN (about 2.5 g) was applied to the suspended strips. The study was sanctioned by the Institutional Review Board and Ethics Committee of our Institution (No. 15077 dated December 14, 2022).

Drugs

KCl was dissolved in 10 mL of double-distilled water to give a concentration of 3.2 M. This was made fresh at the beginning of every experiment. Fasudil (Tokyo Chemical Industry, Chennai, India) was dissolved in double-distilled water to give a stock solution of 3 mM. ODQ (Sigma Aldrich, St Louis, Missouri) was dissolved in dimethyl sulfoxide (DMSO) to give a concentration of 4 mM. At concentrations routinely used to dissolve drugs in our laboratory DMSO has been shown to not affect the contractility of the isolated detrusor muscle.^8,12 DAPP (Sigma Aldrich, St Louis, Missouri) was dissolved in double-distilled water to give a 500 µM stock solution.

Instruments used

To record the tissue’s contractile responses, an isometric force transducer (INCO, Ambala, India) was used. Changes in the tension of the mounted tissue were recorded by a verified digital data acquisition system, DAQ. The DAQ was connected to a computer using a USB port. The DAC and USB port work by converting the analogue signals from the sensors to digital signals readable by a computer.

Experimental procedure

Dose-response effects of fasudil

After stabilization of the isolated detrusor, the tension was readjusted to 25 mN. The response of the detrusor strips after administration of 80 mM KCl was studied. A concentration of 80 mM KCl is the standard concentration of KCl used in our laboratory to stimulate the isolated detrusor to contract.^8,12 After washing the bath, fasudil at a concentration of 10 µM was added to the bath and allowed to incubate for 10 minutes. Then 80 mM KCl was added again and the contractile response was got. During each tracing, after the administration of the drug, a contact time of 2 minutes was allowed, and then the tissue was washed until the baseline was reached. The procedure was then repeated with 2 higher concentrations of fasudil (30 and 60 µM).

Effects of reversal agents on inhibitory effect of fasudil on KCl-induced detrusor contractility

To confirm the inhibitory effect of fasudil on the isolated detrusor, the following procedure was used: the detrusor strips were made to contract with 80 mM KCl with 30 µM fasudil. After washing out of the KCl and fasudil and a period of rest, the reversal agent ODQ (10 µM), a guanylyl cyclase inhibitor, was incubated in the organ bath with 30 µM fasudil for10 minutes after which 80 mM KCl was added and the contraction of the detrusor was obtained. This process was repeated again with the agonist on the P2X purinergic receptor agonist, DAPP (10 µM). The concentrations of ODQ⁸ and DAPP⁹ that were used were based on previous studies.

Statistical analysis

Calculation of height of contraction and area under the contractile curve

The contractility of the detrusor was computed by measuring the maximum height of contraction and the area under the contractile curve (AUCC) of the tracings. We have computed smooth muscle contractility using these 2 parameters for a considerable period of time.^{8,10, 11, 12} Analyses were performed using the R Studio software (version 4.4.0), Posit PBC, Boston, MA, USA.

Calculation of percent inhibition of KCl-induced contractility of the detrusor by fasudil

The mean values of height of contraction and the AUCC after KCl administration were compared with mean values of these parameters after KCl administration along with the 3 concentrations of fasudil (10, 30, and 60 µM) to find the percent inhibition by the 3 concentrations of fasudil on KCl-induced contractility of the detrusor. The nonparametric test, the Wilcoxon signed rank test, was employed for statistical analysis.

Calculation of the percent inhibition of KCl-induced contractility by fasudil after administration of reversal agents

The mean percent inhibition of KCl-induced height of contraction and the AUCC due to 30 µM fasudil along with the 2 reversal agents (10 µM ODQ and 10 µM DAPP) were compared with the mean percent inhibition of these parameters due to KCl and 30 µM fasudil alone to determine whether these reversal agents reverse the inhibitory effect of fasudil or not. The nonparametric test, the Wilcoxon signed rank test, was employed for statistical analysis.

Results

Table 1 shows the effects of the 3 concentrations of fasudil (10, 30, and 60 µM) on KCl-induced detrusor muscle contractility. As shown, fasudil at concentrations of 30 and 60 µM significantly inhibited the KCl-induced height of contraction and the AUCC of the isolated detrusor. Table 2 shows the effects of the 2 reversal agents used in this study, ODQ and DAPP. As shown, ODQ significantly reversed the inhibitory effect of fasudil on the height of contraction and the AUCC since in the presence of ODQ, the percent inhibition became negative and statistically significant. As also shown, DAPP also reversed the inhibitory effects of fasudil, but not to a significant extent, unlike ODQ.

Table 1.

Inhibition by fasudil of potassium chloride (KCl)-induced contractility of isolated caprine detrusor (n = 12 for every drug administration).

Drug administered	% Inhibition of height*			% Inhibition of AUCC*
	Mean (SEM)	CI	P value	Mean (SEM)	CI	P value
80 mM KCl + 10 µM fasudil	17.78 (4.37)	8.16, 27.4	0.008	11.48 (5.95)	−1.61, 24.57	0.182
80 mM KCl + 30 µM fasudil	21.54 (3.27)	14.35, 28.73	0.002	22.23 (3.66)	14.18, 30.28	0.002
80 mM KCl + 60 µM fasudil	28.51 (4.01)	19.7, 37.32	0.002	28.12 (5.03)	17.06, 39.18	0.002

AUCC = area under the contractile curve.

^⁎Values of percent inhibition were determined by comparing values after the administration of fasudil and KCl with values after prior administration of KCl only.

Table 2.

Effects of reversal agents ODQ and DAPP on inhibition of potassium chloride (KCl)-induced contractility of isolated caprine detrusor by fasudil (n = 12 for every drug administration).

Drug administered	% Inhibition of height*			% Inhibition of AUCC*
	Mean (SEM)	CI	P value	Mean (SEM)	CI	P value
80 mM KCl + 30 µM fasudil + 10 µM ODQ	−21.54 (7.63)	−38.31, −4.77	0.034†	−33.0 (9.73)	−54.36, −11.6	0.004†
80 mM KCl + 30 µM fasudil + 10 µM DAPP	−1.23 (4.64)	−11.42, 8.96	0.388†	−0.98 (6.3)	−14.82,12.86	0.937†

AUCC = area under the contractile curve; DAPP = diadenosine pentaphosphate.

^⁎Values of percent inhibition were determined by comparing values after administration of KCL+ fasudil + reversal agent with values after administration of KCl + fasudil.

^†Values are negative because of reversal of inhibitory effect of fasudil by the reversal agent.

Discussion

This study has shown that the rho kinase inhibitor fasudil inhibits the 80 mM KCl-induced contractility of the isolated goat detrusor (Table 1; Figure A). As mentioned above, 80 mM KCl is the standard agonist we use to elicit contraction in the isolated detrusor in our laboratory.^8,12 KCl is a very effective smooth muscle contractile agent that crosses the muscle membrane into the cytosol via potassium channels to depolarize the cell membrane and thereby activate voltage-gated calcium channels. This leads to a rise in cytosolic free Ca²⁺ ions, leading to muscle contraction.¹² In addition, KCl can activate rhoA and rho kinase, actions that cause muscle contraction.¹³

Figure.

Representative traces from the study. (A) Contractile effect of 80 mM KCl before (on the left) and after (on the right) addition of 30 µM fasudil. (B) Contractile effect of 80 mM KCl + 30 µM fasudil before (on the left) and after (on the right) addition of 10 µM ODQ.

The results of the present study support the results of other studies which also found fasudil to inhibit the contractility of the isolated detrusor.^{4, 5, 6} However, the former studies^{4, 5, 6} did not use reversal agents to confirm the inhibitory effect of fasudil on detrusor contractility. In the present study, we used 2 reversal agents, the guanylyl cyclase inhibitor ODQ and the purinergic receptor (P2X) agonist DAPP, to reverse the inhibitory effect of fasudil on detrusor contractility (Table 2). ODQ reversed the inhibitory effect of fasudil on KCl-induced detrusor contractility (Figure B), since after its administration, the inhibitory effect of fasudil was abolished and even became negative. Hence, fasudil and ODQ have opposing effects on detrusor contractility. The reversal effect of ODQ occurs because ODQ inhibits the enzyme guanylyl cyclase, thereby raising cellular levels of cyclic guanosine monophosphate (cyclic GMP), which activates cyclic GMP-dependent protein kinase, also called protein kinase G, which in turn inhibits myosin light chain kinase, leading to muscle relaxation, as illustrated by Joseph et al.¹ ODQ has also been shown to reverse the inhibitory effect of fasudil on the contractility of the isolated human radial artery¹⁴ and the pregnant rat myometrium.¹⁵

In the present study, the P2X receptor agonist DAPP did not reverse the inhibitory effect on detrusor contractility by fasudil (Table 2). Purinergic (P2X) receptors are present in the detrusor¹⁶ and are known to mediate the contraction of the detrusor.¹⁶ A possible explanation for the lack of reversal effect of DAPP on the inhibitory effect of fasudil on detrusor contractility is that DAPP acts via P2X receptors which are independent of the rho kinase pathway.¹⁷ To our knowledge, no study to date has shown DAPP to reverse the inhibitory effect of fasudil on smooth muscle contractility.

A limitation of the present study is that we used the individual, noncumulative, dose- response isolated tissue study design and not the cumulative response type of study design. However, both of these study designs have their advantages and disadvantages.^18,19

OAB is a common clinical problem and is characterized by urinary urgency with or without urinary incontinence, often with increased frequency and nocturia, and with no local pathological features.¹ Currently, the mainstay of pharmacotherapy of OAB are anticholinergic drugs and selective β₃ agonists such as merabegron.¹ The currently used drugs have adverse effects¹² and moreover, their use can lead to tolerance.¹ Hence, new drugs for treating OAB will be useful.⁸ The results of the present study suggest that fasudil could be investigated for the treatment of OAB. Due to various factors such as increased longevity of individuals, the surge in the prevalence of obesity, and lifestyle changes such as increased stress and sedentary habits, there has been an increase in the global prevalence of OAB.²⁰ This makes the need for the development of novel drugs for treating OAB all the more urgent. Indeed, as discussed by Christ and Andersson²¹ and Andersson,²² rho kinase inhibitors are potentially a useful group of drugs for treating OAB. The bioavailability and safety profile of fasudil based on oral administration in normal healthy individuals has been found to be good.²³ Indeed, there is evidence that rho kinase is upregulated in the urinary bladder in patients with OAB.^24,25 However at present, the possible use of fasudil in OAB is somewhat premature. Before clinical trials of fasudil in patients with OAB are performed, more preclinical data, for instance, testing the effect of fasudil on detrusor contractility in vivo in an animal model may be useful.

Conclusions

This study has shown that at suitably low concentrations, which can be attained in the detrusor after oral administration of fasudil to patients with OAB, fasudil inhibits the contractility of the isolated detrusor. We have confirmed the inhibitory effect of fasudil using the reversal agent ODQ. The results, although somewhat premature, suggest that fasudil could be investigated for use in clinical conditions such as OAB that require detrusor muscle relaxation.

Declaration of competing interest

All authors indicate that they have no conflicts of interest financial or otherwise regarding the contents of this article.