Ece İ s k e n d e r ! 1), M .S c .* / H ü ly a Ç a b a d a k i 1), P h .D .* * / A h m e t A k ı c ı , M . D . * M . Z a f e r G ö r e n , M . D . , P h .D .* / A t i l a K a r a a l p , M . D . * N e f i s e B . U lu s o y , M . D . * * * / B e k i K a n , P h .D .* * E s a m E. E l - F a k a h a n y , P h . D . * * * * / Ş u le O k t a y , M . D . , P h .D .* * D e p a r t m e n t o f F h a r m a c o lo g y a n d C lin ic a l P h a rm a c o lo g y , S c h o o l o f M e d ic in e , M a r m a r a U n iv e rs ity , Is ta n b u l, T u rk e y . ’ * D e p a r t m e n t o f B io p h y s ic s , S c h o o l o f M e d ic in e , M a r m a r a U n iv e rs ity , Is ta n b u l, T u rk e y . * * * D e p a r t m e n t o f In t e r n a l M e d ic in e , S c h o o l o f M e d ic in e , M a r m a r a U n iv e rs ity , Is ta n b u l, T u rk e y .
* * * * D e p a r t m e n t o f P s y c h ia try , S c h o o l o f M e d ic in e , U n iv e rs ity o f M in n e s o ta , M in n e a p o lis , U .S .A .
A B S T R A C T
Objective: Acetylcholine is one of the major
contractile transmitters in gallbladder; whereas, it is also innervated by non-adrenergic non- cholinergic nerves which mediate relaxation. It was postulated that nitric oxide (NO) which activates soluble guanylate cyclase to increase cyclic GMP (cGMP) levels in the target cells may be involved in these processes. This study was designed to investigate whether muscarinic receptor stimulation via carbachol (CCh) induces NO-mediated cGMP synthesis in guinea-pig gallbladder.
M ethods: cGMP levels were measured via
radioimmunoassay in gallbladder slices
incubated with carbachol (CCh) (10 6-10 3 mol/l) in the presence and absence of NO synthase
(NOS) inhibitor L-NAME or muscarinic antagonist scopolamine. The effect of L-NAME and another NOS inhibitor L-NMMA on carbachol-induced
contractions were also investigated in an in vitro
organ bath.
Results: Carbachol stimulated cGMP formation
in guinea-pig gallbladder slices significantly. CCh-induced cGMP formation was abolished by both L-NAME (1mmol/l) and scopolamine (108- 106 mol/l). In contraction experiments, L-NAME (3x10-4 mol/l) did not produce any change in the resting tension of the strips and the concentration-response curves to carbachol, whereas L-NMMA (3x10-4 mol/l) induced a slight, but significant contraction (6.3+1.9 % of carbachol (106 mol/l)-induced response). When carbachol was added to the bath after L-NMMA- induced increase in tension reached its
0 ) These authors equally deserve to be the first author.
( A c c e p t e d 16 J a n u a r y , 2 0 0 2 ) M a r m a r a M e d ic a l J o u r n a l 2 0 0 2 ; 1 5 (1 ) : 7 - 1 4
Correspondance to: Şube Oktay, M.D, - Department of Pharmacology, School of Medicine, Marmara University, Haydarpaşa 81326 İstanbul, Turkey,
maximum, the amplitude of the contractile response at the end was 108.0+4.0 % of the control.
Conclusion: Therefore, it may be concluded that
muscarinic receptor activation by carbachol
stimulates NO production in guinea-pig
gallbladder and the subsequent increase in cGMP counteracts with carbachol-induced contractions. However, it seems likely that an intact enteric nervous system is required for such an interaction to be prominent. The muscarinic receptor subtype(s) involved in this interaction remains to be elucidated.
K e y W o r d s : cGMP, NO, Guinea-pig,
gallbladder, L-NAME, L-NMMA
IN T R O D U C T IO N
Generation of nitric oxide was first described in the vascular endothelium in response to acetylcholine or other vasorelaxants (1-4). It is now well established that NO stimulates guanylate cyclase with a subsequent increase in intracellular cGMP, leading to the relaxation of the smooth muscle (5-7).
In gallbladder smooth muscle, accumulation of intracellular cGMP and muscle contraction are inversely related events (8). Acetylcholine and cholecystokinin (CCK) are major contractile transmitters, whereas gallbladder is innervated by non-adrenergic non-cholinergic nerves which mediate relaxation (9). It was postulated that nitric oxide (NO) might be involved in this relaxation process and was reported that inhibition of NO synthesis potentiated the contractile responses of guinea-pig gallbladder in
response to both CCK and bethanechol in v iv o
(10). A similar potentiation was also claimed for acetylcholine-induced contractions of sphincter of Oddi of guinea-pigs (11).
The present study was designed to investigate whether carbachol stimulates NO formation in guinea-pig gallbladder slices. We also aimed to demonstrate the effect of NO synthase inhibitors on the concentration-contraction curves of carbachol in gallbladder muscle strips.
M A T E R IA L A N D M E T H O D S
The experimental protocol was approved by Marmara University Institutional Animal Care and Use Committee.
1. cGMP Assay
1.1. Preparation of G allbladder Tissue Slices
Guinea-pigs (300-350 g) of both sexes were killed after overnight fasting. Gallbladders were
dissected and rinsed immediately in
physiological buffer solution (PBS; composition in mmol/l: NaCI, 110; KCI, 5; CaCI2, 1.8; MgS04, 1; NaH2P04, 1; Na2HP04, 1; glucose, 25; sucrose, 50) at pH 7.4. Tissues were sliced in ice-cold buffer, and incubated with 25 pi [2-3H]- adenine in 2 ml fresh PBS gassed with mixture of 95% 0 2 and 5% C02 at 37°C, in order to standardize data for the number of viable cells. At the end of the 1h incubation period, tissues were washed with fresh PBS to remove the unbound [2-3H]-adenine. Tissue slices were suspended with 2 ml PBS. Fifty pi of slice suspension and sufficient volume of PBS were added to each tube to a total incubation volume of 500 pi after the addition of carbachol, scopolamine, sodium nitroprusside and/or L- NAME. Stock solutions of drugs were prepared in distilled water and dilutions were with PBS.
1.2. Study Protocol
a) The reaction was started by adding 50 pi of sodium nitroprusside (103 mol/l) or carbachol (10 6-10 3 mol/l) for 5 min and then, stopped by keeping the tubes in boiling water for 10 minutes. Subsequently, the slices were homogenized on ice with an Ultra-Turrax homogenizer and centrifuged at 1000xg, 4°C for 10 minutes. (2- 3H)-Adenine incorporation was measured in 100 pi of the supernatant via a liquid scintillation counter (Tricarb 2000, Packard, USA). The remaining supernatants were stored at -70°C until the day of cGMP assay.
b) Gallbladder slices were pretreated with either L-NAME (10 3 mol/l) or scopolamine (10 9-106 mol/l) for 10 minutes followed by carbachol addition and then, the above procedure was applied. The effect of L-NAME and scopolamine on basal cGMP levels were also tested.
1.3. Radioimmunassay
cGMP levels in the supernatants were determined according to the radioimmunassay (RIA) procedure described by Wotta et al (12). Briefly, each sample was diluted about five times to establish the measurements within the calibration curve calculation limits (cGMP concentration in standard solutions: 1.9-500 fmol/100 pi). Then, anti-cGMP IgG (rabbit) and [,25l]-cGMP were added to each tube. Samples and standards were acetylated with 2 pi of 2:1 triethylamine:acetic anhydride. After an overnight incubation of the antigen/antibody complex at 4°C, samples were precipitated with 2 ml of ice- cold ethanol and centrifuged. The pellet was used to measure the radioactivity via a gamma counter (QC Gamma Bioscan, Bioscan Inc. Washington DC, USA).
2. Contraction Experim ents
Longitudinal strips of gallbladders of guinea-pigs (300-350 g) of both sexes were prepared and mounted in an organ bath containing Krebs solution (composition in mmol/l: NaCI, 118.4; KCI, 4.7; CaCI2, 2.5; MgSO„, 1.2; NaHC03, 25.0; KH2P04, 1.2; glucose, 11.1) at 37°C bubbled with a mixture of 95% 0 2 and 5%C02 under a resting tension of 1.0 g and were allowed to equilibrate for 3 h. Isometric contractions were recorded on a polygraph (Grass Model 7, MA, USA) via a force- displacement transducer (Grass FT03, MA, USA). Cumulative concentration-response curves were constructed to carbachol (3x10 8 - 3x104 mol/l) using incremental increases in concentration spaced at 0.5 log intervals. Concentrations were added once a sustained response to the previous concentration was reached. An interval of 45 min was then allowed during which the tissues were washed with Krebs solution. The gallbladder strips were incubated with L-NAME (3x10 4 mol/l) for the last 15 min and a second concentration- response curve to carbachol was constructed in the presence of the inhibitor.
Single concentration experiments: Three
consecutive contractions were induced by 10'6 mol/l carbachol, the last one being in the presence of L-NMMA (3x104 mol/l) incubated for 10-15 min.
3. Statistical Analysis
cGMP assay: Data were expressed as the ratio of cGMP (fmol/100 pi) and [8H]cAMP(cpmx10 5).
Contraction experiments: The EC50 values and the slopes of concentration-response curves
were calculated by means of logistic
transformation with individual maximal responses accepted as 100%, and linear regression analysis. In single-concentration experiments, the amplitude of contraction in the presence of the antagonists was expressed as % of the corresponding control response in the absence of the inhibitor.
Since always there has been great variability between the basal levels of slices obtained from different animals, the results were compared with their own basal cGMP concentration for even experiment. Student's t-test and Wilcoxon matched pairs signed-ranks test were used to test the statistical significance of the differences between groups when appropriate. The level of significance was considered to be P<0.05. Data were expressed as mean + S.E.M.
4. Chemicals
(-)- Scopolamine HBr was purchased from RBI Chemicals (USA), anti-cGMP IgG (rabbit) and cGMP standards from Calbiochem-Novabiochem Corporation (San Diego, CA), [2-3H]-Adenine and Guanosine 3', 5' cyclic phosphoric acid, 2'- O'succinyl 3- [125l] iodotyrosine methyl ester from
Amersham (UK), carbamoylcholine chloride
(carbachol), N-nitro-L-arginine methyl ester (L-
NAME), N-nitro-monomethyl-L-arginine (L-
NMMA), sodium nitroprusside and all other chemicals from Sigma Chemical (St. Louis, MO, USA).
RESULTS
1. cGMP Assay
1.1. cGMP form ation induced by carbachol and sodium nitroprusside
The basal cGMP level in guinea-pig gallbladder slices was 335.6+107.4 [(fmol/100 |.il)/cpmx10-5] (n=11). The NO donor sodium nitroprusside (10 3 mol/l) produced a 16.8 ± 6.1 fold increase in cGMP production, indicating that the assay system is functioning, (data not shown). Carbachol induced a concentration-dependent increase in cGMP levels up to 2.9 + 0.6 fold at 10'4 mol/l (range: 1.2-7.1; n=11).
1.2. Effect of L-NAME and scopolam ine on carbachol-induced increase in cGMP levels
The NOS inhibitor, L-NAME (10-3 mol/l), completely abolished the increase in cGMP production in response to carbachol (10 6-10 4 mol/l) (n=7; Fig. 1). The muscarinic receptor antagonist scopolamine (10 8-10 6 mol/l) also
prevented carbachol (104 mol/l)-induced
stimulation in cGMP production, indicating the involvement of muscarinic receptors (n=4; Fig. 2). Scopolamine alone did not show any significant effect on cGMP levels. Although cGMP concentration in the presence of L-NAME were lower than basal levels, the difference was not statistically significant.
■ Basal I---» L-NAME (1 O'3 mol/l) 3 CCh C = 3 CCh + L-NAME (1 0 * mol/l) X £
İ
o-*— Ta
n
n
KT6 10^ 104 [C C h ] (m o l/l) 10 -F i g . l : Effect of L-NAME on carbachol (CCh)-induced cGMPformation in guinea-pig gallbladder slices. Data were expressed as mean+S.E.M. of 7 independent experiments. 'p<0.05 vs corresponding control value with CCh. + p<0.05 vs corresponding CCh response in the absence of L-NAME.
F i g .2 : Effect of scopolamine (Scop.) on carbachol (CCh)- induced cGMP formation in guinea-pig gallbladder slices. Data were expressed as mean±S.E.M. of 4 independent experiments. *p<0.05 vs corresponding control value with CCh.
2. Effect of NOS Inhibitors on the Contractile Responses to Carbachol
Carbachol contracted gallbladder smooth muscle strips in a concentration-dependent manner with a pD2 value of 6.15+0.30. The addition of L- NAME (3x10-4 mol/l) into the organ bath did not cause any change in the tension of the strips. The concentration-response curves to carbachol were almost identical in the absence and presence of L-NAME (pD2= 6.31+0.27) (Fig. 3). On the other hand, L-NMMA alone (3x104 mol/l) induced a slight, but significant (p<0.05) contraction which achieved 6.3+1.9% of contraction related to that elicited by a half- maximal carbachol concentration (106 mol/l). When carbachol (106 mol/l) was added to the bath after L-NMMA-induced increase in the tension reached its maximum, it induced a contraction of 108.0+4.0% of the control response (Fig. 4). Therefore, the resulting contractile response to carbachol (10 6 mol/l) in the presence of L-NMMA (3x104 mol/l) was siqnificantly higher than that to carbachol alone (p<0.05).
F ig .3 : Carbachol (CCh)-induced conlractions of guinea-pig gallbladder strips in the absence and presence of L- NAME (3x10'4 mol/l). Data were expressed as mean±S.E.M. of 3 independent experiments.
CONTRACTION (%)
3 o
F i g .4 : Carbachol (CCh; 1x10 6 mol/l) -induced contractions of guinea-pig gallbladder strips in the absence and presence of L-NMMA (3x104 mol/l). Data were expressed as mean±S.E.M. of 6 independent experiments.
D IS C U S S IO N
Data presented here indicate that carbachol stimulates NO generation via muscarinic receptors in guinea-pig gallbladder slices. However, the contribution of NO to gallbladder
contractions under in v it r o conditions does not
seem to be crucial.
The gallbladder is unique within the
gastrointestinal tract because it functions as a storage organ as well as an organ with contraction capability. Maximal gallbladder emptying occurs postprandially in response to CCK and vagal stimulation. On the other hand the resting tone is an important factor which facilitates relaxation (13). Relaxation of the Oddi sphincter must coincide with gallbladder contraction so that gallbladder bile can enter the doudenum, and these simultaneous events appear to be regulated by CCK, NO and the enteric nerves (14). NO-synthesizing nerve cell bodies and axons have been found along the biliary tract of guinea-pigs and more densely located in the distal common bile duct near the sphincter of Oddi (15).
Previous studies have suggested a functional role for NO in both contraction and relaxation of guinea-pig sphincter of Oddi (11, 14, 16). On the other hand, little was known of the role of NO on gallbladder motility until Salomons et al (17) reported that the prairie dog gallbladder contains constitutive NOS and synthesizes NO. These
investigators claimed that NO is important for the maintenance of basal gallbladder tone and is an inhibitor of the contractile response of the gallbladder to agonists such as CCK and bethanechol. Another report presenting similar functional data for guinea-pig gallbladder has been published by Mourelle et al (10). In accordance with these reports, the present study revealed that carbachol produced an increase in intracellular cGMP levels that was antagonized by the NOS inhibitor, L-NAME, indicating stimulation of NOS by carbachol. The intracellular effects of NO include increased guanylate cyclase activity with the generation of cGMP and inhibition of inositol 1, 4, 5- triphosphate (IP3)-mediated Ca2+ release (18-20). The presence of M,, M2, M3 and M4 receptors in guinea-pig gallbladder has been claimed by several groups depending on receptor binding and contraction experiments, Western blot analysis and also on second messenger assays (21-29). It was demonstrated that carbachol stimulated phosphoinositide hydrolysis and inhibited cAMP formation in this tissue (24, 27). Mt, M3 and M5 receptors predominantly increase IP3 generation, and M2 and M4 receptors inhibit adenylate cyclase as second messenger systems which mediate their actions. Cell
transfection experiments, however, have
demonstrated that individual muscarinic receptor subtypes can also regulate multiple effector pathways (30, 31).
Cholinergic agonists have been reported to be coupled to NOS in several tissues of various species. Acetylcholine has been shown to relax vascular smooth muscle through the production of NO and subsequent production of cGMP (32, 33). Cholinergic agonists also increase cGMP levels in the heart and the negative inotropic and chronotropic effects linked to muscarinic receptor activation are inhibited, at least in part, by NOS inhibitors (34, 35). Activation of muscarinic receptors in the brain has been shown to result in stimulation of the activity of guanylate cyclase which was attenuated by NOS inhibition (36). The involvement of M, receptors in the stimulation of NOS and cGMP generation in N1E-115 mouse neuroblastoma cells was suggested since pirenzepine was found to antagonize these responses with high potency
pharmacological Investigation of muscarinic receptors coupled to NO generation suggested involvement of the M3 muscarinic receptor subtype (38). In the majority of the tissues studied to date, muscarinic receptors of the M3
subtype mediate endothelium-dependent
relaxation of the vasculature (39). The findings of the present study revealed that carbachol- induced increase in intracellular cGMP was mediated via muscarinic receptors In guinea-pig gallbladder since scopolamine antagonized this effect. However, further studies are needed to demonstrate the muscarinic subtype(s) involved in this process.
The second part of this study was performed to elucidate the contribution of NO production as a second messenger system to carbachol-induced contraction of guinea-pig gallbladder. Salomon et al. (17) have found that NOS inhibition by L- NAME contracts gallbladders of the prairie dogs
both in v iv o and in v itr o , concluding that NO was
important for the maintenance of basal gallbladder tone. Similar findings have been
reported by Mourelle et al. (10) for guinea-pigs in
v iv o . These investigators have measured
intraluminal gallbladder pressure which was significantly Increased by systemic application of L-NAME, L-NMMA and L-NA. They have concluded that intraluminal gallbladder pressure may reflect changes in the intrinsic tone of its musculoelastic wall or in the resistance of the sphincter of Oddi to bile flow (9,40).
Indeed, a significant increment in muscular tension of Oddi sphincter after NOS inhibition has been reported previously (11, 41). There was little information about the influence of NO production in gallbladder contraction in response to the cholinergic agonist, bethanechol in vitro. Mourelle et al (10) have claimed a potentiation (approximately 1.5 fold) of bethanechol-induced contraction by 27 pg/ml of L-NAME tested with a single concentration (2pg/ml = app. 1x105 mol/l) of the agonist. On the other hand, no increase in the resting tone due to L-NAME was reported. In the present study, L-NAME did not show a contractile effect alone, similar to the above study, but unlike Mourelle et al. (10), we were unable to demonstrate a potentiation at the concentration-contraction curve of carbachol. This discrepancy may be due to the great
variability of the responses. Furthermore, in the above mentioned report, the standard errors of the mean amplitude of contraction in the absence and presence of L-NAME were very high (10).
The lack of potentiation under in v it r o conditions
might indicate that an intact enteric neuronal system is required for a tonic regulation of gallbladder tone and its cholinergic control by NO.
L-NAME has been demonstrated to have a weak antagonistic affinity at M, and M, receptors in rat
brain in v it r o (42), and it was suggested that alkyl
esters of L-arginine such as L-NAME are poor choices as NOS inhibitors in studies of coupling of muscarinic receptors to NO generation (43). Therefore, it may be speculated that antagonistic activity of L-NAME may counteract a possible potentiation of carbachol-induced contractions by L-NAME. Supporting this conclusion, L-NMMA which is a NOS inhibitor without any antagonistic affinity at muscarinic receptors (43) induced a small contractile response which was 6.3+1.9 % of the amplitude induced by 106 mol/l carbachol (p<0.05). However, L-NMMA failed to further potentiate carbachol-induced contractions. This may be because 3x104 mol/l L-NMMA abolishes NO-mediated inhibitory tone with a consequent slight contraction, and no further increase in the contractile response to carbachol was observed. In conclusion, muscarinic receptor activation by carbachol stimulates NO production in guinea- pig gallbladder smooth muscle cells. The subsequent increase in cGMP may counteract carbachol-induced contraction. However, it seems likely that an intact enteric nervous system is required for such an interaction, since the potentiating effect of NOS inhibitors on cholinergic gallbladder contraction could not be demonstrated functionally. The muscarinic receptor subtype(s) involved in this interaction remains to be elucidated.
A c k n o w le d g e m e n t s
This work was supported by the National Institute of Health, USA (FIRCA grant R03-W00615 and NS-30454) and Marmara University Research Fund (SB/DYD-154).
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