Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 56 issue 6 (june 2022) : 680-684

Relaxation of Goat Detrusor Muscle by L-arginine Involves NO-dependent but Guanylyl Cyclase Independent Activation of KCa Channels

Rousy K. Baruah2, Dilip K. Deka1
1Department of Pharmacology and Toxicology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India. 
2Office of the State Mission Director, Assam State Rural Livelihood Mission (ASRLM), Panjabari, Guwahati-781 037, Assam, India.
Cite article:- Baruah K. Rousy, Deka K. Dilip (2022). Relaxation of Goat Detrusor Muscle by L-arginine Involves NO-dependent but Guanylyl Cyclase Independent Activation of KCa Channels . Indian Journal of Animal Research. 56(6): 680-684. doi: 10.18805/IJAR.B-4292.
Background: Urinary incontinence is a major problem both in man and animals particularly dogs. L-arginine, the precursor of NO, relaxes coronary artery smooth muscle by opening of KATP channels. L-arginine has beneficial circulatory effects in patients with essential and secondary hypertension. However, not much is known about the role of L-arginine on bladder physiology. In view of this, the present work investigated the functional role of L- arginine on detrusor smooth muscle of goat. 

Methods: Detrusor strips of goat, collected from local abattoir were mounted in a thermostatically controlled (37° ± 0.5°C) organ bath (20 ml capacity) containing physiological solution. After 1 hr of equilibrium, carbachol (CCh) (10-5 M) was used to induce sub-maximal contraction. L-arginine (10-3 M) was added at the plateau of contraction to see any observable effect in absence and presence of modulators of NO and ion channels. 

Result: L- arginine (10-3 M) reversed the contractions induced by CCh (10-5 M) on detrusor tissues. Methylene blue (MB) (10-5 M), the non-specific guanylyl cyclase inhibitor, failed to attenuate the relaxant response of L-arginine but, the NO synthase inhibitor L-NAME (3 x 10-6 M) inhibited the relaxant response of L-arginine. The KATP channel blocker glibenclamide (10-6 M) failed to inhibit the relaxation induced by L-arginine while KCa channel blocker tetraethylammonium (TEA) (10-3 M) inhibited the relaxant response of L-arginine. The results of the present study suggest that L-arginine produces relaxation of goat detrusor muscle and the L-arginine-elicited relaxation is  NO-dependent but guanylyl cyclase independent which activates KCa channels.
The nitrergic mechanism on bladder physiology is paradoxical, yet NO is considered as one of the inhibitory mediators in the smooth muscle of many organs (Sneddon and Graham, 1992). NO produces relaxation of urinary bladder muscle in sheep (Thornbury et al., 1992), dog urethra (Hashimoto et al., 1993) and human urinary bladder (James et al., 1993). Photo-activated release of NO from a donor relaxes the detrusor in rat (Chung et al., 1996). Contrary to this, NO donors also produce excitatory response in bladder muscles of guinea pig (Moon, 2000) and mouse (Fujiwara et al., 2000). At the same time, NO donors evoke a complex response in pre-contracted human detrusor (Moon, 2002). In goat detrusor smooth muscle, sodium nitroprusside (SNP), an NO donor produces an inhibitory effect against Carbachol-induced contraction (Barua et al., 2010).
       
K+ channels play a vital role in regulating membrane potential and cellular excitability in smooth muscles of urinary tract (reviewed by Brading, 1992). Report also suggests that KATP channels are modulated by NO. In vascular smooth muscle, nitric oxide synthase inhibitors (L-arginine analogues) like L-NNA and L-NMMA inhibit ATP-sensitive K+ channels, an effect that could be reversed by L-arginine, an essential amino acid (Kontos and Wei, 1996). L-arginine-mediated vasodilatation has been reported to be independent of NO production both in vivo (Calver et al., 1990) and in vitro (Thomas et al., 1989 studies. In goat coronary artery, L-arginine mediated vasodilatation is through NO-independent activation of KATP channels (Deka et al., 2009).
       
L-arginine has beneficial circulatory effects in patients with essential and secondary hypertension (Nakaki et al., 1990; Nakaki and Kato, 1994). Exogenous administration of the amino acid markedly decreases blood pressure in these patients. It is generally believed that, the vascular effects of the amino acid are mediated by the vascular smooth muscle cells upon stimulation by inducible nitric oxide synthase (Moncada et al., 1991). However, it is not known if the amino acid would be of value in the treatment of urinary incontinence, which is a major problem both in man and animals particularly dogs. Therefore, we wanted to examine the role of L-arginine on detrusor muscle with goat as a model.
Goat urinary bladders were collected from a local abattoir in and around Khanapara within 20-30 minutes of slaughter and put in cold aerated modified Krebs’s solution [Composition in mM: NaCl 120, KCl 5.9, MgCl2 1.2, CaCl2.2H2O 2.5, NaHCO3 15, NaH2PO41.2, Glucose (Dextrose) 11] and brought to laboratory. The experiments were conducted between the period of August, 2007 and Sept, 2018, in the Department of Pharmacology and Toxicology, College of Veterinary Science, AAU, Khanapara, Guwahati, Assam.
       
The neck and dome parts of the bladders were removed and the bladders were cut open with the help of a fine scalpel in petri-dish containing aerated physiological solution. The fascia and urothelium of the urinary bladders were removed and detrusor smooth muscle (DSM) strips of about 2-3 mm breadth and 4-5 mm length were dissected out. The strips were tied on either ends with thread, mounted into a thermostatically controlled (37°±0.5°C) organ bath (20 ml capacity) containing physiological solution and were continuously aerated. The tissues were then equilibrated under a resting tension of 1.0 g for a period of 60 minutes. During this period the bathing fluid was changed every 15 minutes. Isometric contractions were recorded by means of a force displacement transducer connected to a single channel physiograph (Biodevices, India). After equilibrating the tissue strips for 1 hr, single dose of carbachol (CCh) (10-5 M) was used to produce sustained contraction. At the plateau of contraction, L-arginine (10-1 M) was added to see any observable effect of the amino acid on CCh - pre-contracted detrusor strips. Interestingly, L-arginine produced relaxation of the detrusor strips contracted with CCh.
 
Effect of methylene blue and L-NAME on L-arginine-elicited relaxation
 
Methylene blue (MB) is a known inhibitor of soluble guanylyl cyclase (GC), an enzyme activated by nitric oxide (Ignarro et al., 1986) and therefore, MB (10-5 M) was used to see the involvement of guanylyl cyclase on L-arginine-mediated relaxant response on goat DSM. After obtaining L-arginine (10-3 M) - elicited control relaxation response and following several washes of the tissues in the bath with PSS, tissues were incubated with MB for 5 min. Thereafter, CCh-induced contraction was induced followed by addition of L-arginine at the plateau of the contraction in the continued presence of MB.
       
L-NAME is an L-arginine analogue which by inhibiting endogenous NOS decreases the synthesis of NO (Moncada and Higgs, 1995). L-NAME (3 × 10-5 M) was used to see the influence of endogenous nitric oxide on L-arginine elicited relaxation of goat DSM. After obtaining L-arginine (10-3 M) - elicited control relaxation response and following several washes of the tissues in the bath with PSS, tissues were incubated with L-NAME for 5 min. Thereafter, CCh-induced contraction was induced followed by addition of L-arginine at the plateau of the contraction in the continued presence of L-NAME.
 
Effect of K+ channel blockers on L-arginine-elicited relaxation
 
TEA is a non-specific KCa channel blocker while glibenclamide is a KATP channel blocker. In order to examine the involvement of K+ channels, L-arginine elicited relaxation was obtained in the absence/presence of either TEA or glibenclamide. After obtaining L-arginine (10-3 M) - elicited control relaxation response and following several washes of the tissues in the bath with PSS, tissues were incubated with either TEA (10-3 M) or glibenclamide (10-6 M) for 20 minutes. Thereafter, CCh-induced contraction was induced followed by addition of L-arginine at the plateau of the contraction in the continued presence of either TEA or glibenclamide.
 
Statistics
 
The results (Absolute force/percent relaxation) are presented as means±S.E.M. (n). Students’ ‘t’ test (paired) was used to determine the level of significance.
Time matched control contraction response induced by CCh (10 µM) did not change the amplitude of contraction when the tissue remains contracted for 2 hrs (Fig 1). Amplitude of contraction was also not affected on repeated application of CCh (Fig 1b).
 

Fig 1a: Original traces showing time matched control contraction response induced by CCh (10 µM) in goat DSM. Note that there is no change in the amplitude of contraction when the tissue remains contracted for 2 hrs.



Fig 1b: Original traces showing that repeated application of CCh (10 µM) did not alter amplitudes of contraction in goat DSM.


       
Application of L-arginine (10-3 M) at the plateau of CCh-induced contraction elicited relaxation on goat DSM. The maximum relaxation produced by L-arginine was 53.03±0.99% (n=20, pooled data) when added at the steady state of CCh (10-5 M)-induced contraction (absolute force 0.85±0.02 g, n=20, pooled data).
 
Effect of methylene blue on L-arginine-elicited relaxation
 
Guanylyl cyclase inhibitor MB had no effect on L-arginine elicited relaxant response on goat DSM (Fig 2a). The per cent relaxation produced by L-arginine in absence and in presence of MB were 55.13±2.05% (n=4) and 57.75±1.29% (n=4), respectively. Interestingly, MB reduced the amplitudes of CCh-induced contractions (absolute force 0.66±0.06 g, n=4 in control and 0.47±0.04 g, n=4 in MB treated tissues) (Fig 2b).
 

Fig 2a: Original traces showing relaxant response induced by L-arginine (1 mM) on CCh (10-5 M) -contracted goat DSM in absence and in presence of MB (10 µM).



Fig 2b: Bar diagram showing CCh-induced absolute force of contraction (g) on goat DSM in absence (0.66±0.06 g) and in presence (0.47±0.04 g) of MB. Note that the absolute force of contraction was significantly (p<0.05) reduced in presence of MB.


       
MB has been shown to inhibit both NO mediated relaxation and an increase of cGMP in rat detrusor muscle (Chung et al., 1996). The fact that MB failed to attenuate L-arginine elicited relaxant response in goat DSM negates the involvement of guanylate cyclase in L-arginine elicited relaxation. But, present study fails to explain the inhibitory effect of MB on amplitude of CCh-induced contraction.
 
Effect of L-NAME on L-arginine-elicited relaxation
 
L- arginine failed to induce any relaxation when goat DSM were pre-treated with L-NAME (3×10-5 M) (Fig 3a). Thus, while the per cent relaxation produced by L-arginine was 45.87± 1.42 (n=4) in control, it was 0 (Zero) per cent (n=4) in L-NAME treated tissues (Fig 3b). Moreover, L-NAME had no significant effect on CCh (10-5 M) - induced absolute force of contraction.
 

Fig 3a: Original traces showing relaxant response of L-arginine (1 mM) on CCh-contracted detrusor tissues in absence and in presence of L-NAME (3´10-5 M).



Fig 3b: Bar diagram showing per cent relaxation of L-arginine in control and in presence of L-NAME on CCh-contracted tissues. L-arginine failed to induce relaxation in presence of L-NAME.


       
NO was not involved in L-arginine mediated KATP channel opening in goat coronary artery as L-arginine analogues could not inhibit L-arginine-elicited relaxation (Deka et al., 2009). Contrary to this, activation of KATP channel was inhibited by pretreatment of porcine coronary smooth muscle cells with L-arginine analogues and it was concluded that KATP channel activation was due to generation of NO by the essential amino acid (Miyoshi et al., 1994). Interestingly, L-NAME also could inhibit L-arginine elicited relaxation of CCh-contracted goat DSM implicating involvement of NO in L-arginine elicited relaxation in the present study.
 
Effect of K+ channel blockers on the relaxant responses of L-arginine in goat detrusor
 
L-arginine elicited relaxation was inhibited by pre-treatment of goat DSM with TEA (10-3 M) for 20 min (Fig 4a). Thus,
while L-arginine evoked a relaxation of 48.5±2.53% (n=4) in control, it failed to elicit relaxation in TEA treated tissues (Fig 4b). Further, there was a reduction in CCh-induced amplitude of contraction in presence of TEA (absolute force 0.93±0.05 g, n=4 in control and 0.43±0.03 g, n=4, in TEA treated tissues, p£0.001) (Fig 4c).
 

Fig 4a: Original traces showing L-arginine elicited relaxation in absence and in presence of TEA (10-3 M) and Glibenclamide (10-6 M) on CCh-contracted goat DSM.



Fig 4b: Bar diagram showing CCh-induced absolute force of contraction (g) on goat DSM in absence and in presence of TEA.



Fig 4c: Bar diagram showing L-arginine elicited relaxation in absence and in presence of TEA. Note that L-arginine failed to induce relaxation in presence of TEA on CCh-contracted tissues.


       
Contrary to this, pre-treatment of the tissues with glibenclamide (10-6 M) had no effect on either CCh-induced contraction or L-arginine-elicited relaxation. Rather, there was potentiation of L-arginine elicited relaxation in presence of glibenclamide (Fig 4d). The per cent relaxations elicited by L-arginine in absence and in presence of glibenclamide were 52.75±1.58% (n=4) and 58.75±1.56% (n=4), respectively. 

Fig 4d: Bar diagram showing L-arginine elicited relaxation in absence and in presence of glibenclamide. Note that glibenclamide potentiated the L-arginine-elicited relaxation in goat detrusor tissues (p<0.05).


       
TEA has been shown to inhibit L-arginine and bacterial lipopolysaccharide elicited large relaxation in rat isolated aorta (Hall et al, 1996) and a role for the involvement of Ca2+- activated K+ channels in the mechanism of NO- dependent vasodilator action of L-arginine was suggested in rat aorta. On the other hand, L-arginine elicited relaxation was inhibited by KATP channel blocker; glibenclamide in goat coronary artery suggesting a role for involvement KATP channels in L-arginine mediated relaxant response in coronary artery (Deka et al., 2009). However, in the present study, glibenclamide failed to attenuate L-arginine elicited relaxation and instead, TEA inhibited the L-arginine-elicited relaxation in CCh-contracted goat DSM. Therefore, L-arginine- elicited relaxation as observed in the present study is suggestive of mediating through opening of KCa channel in goat DSM. The observation that glibenclamide potentiated the L-arginine-elicited relaxation in detrusor tissue could be best explained by the fact that blocking of KATP channels would depolarize the smooth muscle tissues leading to an increase in the driving force of the outward K+ current. Glibenclamide has earlier been reported to cause small depolarizing effect in smooth muscle of rat aorta (Fauaz et al., 2000). K+ channel blockers like Ba2+, TEA and 4-AP have  been reported to cause an increase in the magnitude of hyperpolarisation to a KATP channel opener cromakalim in rabbit mesenteric artery (Murphy and Brayden, 1995). Similarly, blocking of KCa channel with TEA also augmented L-arginine elicited relaxation in goat coronary artery smooth muscle (Deka et al., 2009).
In conclusion, the results of the present study suggest that L-arginine produces relaxation in goat DSM. The L-arginine-elicited relaxation is NO dependent, but independent of guanylyl cyclase, which activates KCa channels.

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