Contraction Of Guinea Pig Gallbladder Research Articles

Polydatin Attenuates Carbachol-induced Contraction of Guinea Pig Gallbladder

The aim of this study was to determine whether polydatin, a glucoside of resveratrol isolated from the root of Polygonum cuspidatum, warranted development as a potential therapeutic for ameliorating the pain originating from gallbladder spasm disorders and the underlying mechanisms. Guinea pig gallbladder smooth muscles were treated with polydatin and specific inhibitors to explore the mechanisms underpinning polydatin-induced relaxation of carbachol-precontracted guinea pig gallbladder. Our results shown that polydatin relaxed carbachol-induced contraction in a dose-dependent manner through the nitric oxide/cyclic guanosine monophosphate/protein kinase G and the cyclic adenosine monophosphate/protein kinase A signaling pathways as well as the myosin light chain kinase and potassium channels. Our findings suggested that there was value in further exploring the potential therapeutic use of polydatin in gallbladder spasm disorders.

The role of TRPP2 in agonist-induced gallbladder smooth muscle contraction.

TRPP2 channel protein belongs to the superfamily of transient receptor potential (TRP) channels and is widely expressed in various tissues, including smooth muscle in digestive gut. Accumulating evidence has demonstrated that TRPP2 can mediate Ca(2+) release from Ca(2+) stores. However, the functional role of TRPP2 in gallbladder smooth muscle contraction still remains unclear. In this study, we used Ca(2+) imaging and tension measurements to test agonist-induced intracellular Ca(2+) concentration increase and smooth muscle contraction of guinea pig gallbladder, respectively. When TRPP2 protein was knocked down in gallbladder muscle strips from guinea pig, carbachol (CCh)-evoked Ca(2+) release and extracellular Ca(2+) influx were reduced significantly, and gallbladder contractions induced by endothelin 1 and cholecystokinin were suppressed markedly as well. CCh-induced gallbladder contraction was markedly suppressed by pretreatment with U73122, which inhibits phospholipase C to terminate inositol 1,4,5-trisphosphate receptor (IP3) production, and 2-aminoethoxydiphenyl borate (2APB), which inhibits IP3 recepor (IP3R) to abolish IP3R-mediated Ca(2+) release. To confirm the role of Ca(2+) release in CCh-induced gallbladder contraction, we used thapsigargin (TG)-to deplete Ca(2+) stores via inhibiting sarco/endoplasmic reticulum Ca(2+)-ATPase and eliminate the role of store-operated Ca(2+) entry on the CCh-induced gallbladder contraction. Preincubation with 2 μmol L(-1) TG significantly decreased the CCh-induced gallbladder contraction. In addition, pretreatments with U73122, 2APB or TG abolished the difference of the CCh-induced gallbladder contraction between TRPP2 knockdown and control groups. We conclude that TRPP2 mediates Ca(2+) release from intracellular Ca(2+) stores, and has an essential role in agonist-induced gallbladder muscle contraction.

In-vitro Bioassays for Cholecystokinin

Two in-vitro methods for the bioassay of cholecystokinin (CCK) are described. They should be useful for determinations of potencies of pharmaceutical formulations of CCK. Both are based on the hormone's ability to stimulate exocrine pancreas. The stimulatory effect on 45Ca efflux from preloaded suspended acinar cells or on release of amylase from pancreatic acini from the guinea-pig has been recorded. The assay based on stimulation of 45Ca outflux is more specific and precise than the method using measurements of amylase secretion. Therefore, the calcium assay was further validated against the conventional guinea-pig gall-bladder contraction assay. Both methods gave similar potency readings but the results from the in-vitro assay were more precise.

3-[5-(3,4-Dichloro-phenyl)-1-(4-methoxy-phenyl)-1H-pyrazol-3-yl]-2-m-tolyl-propionate (JNJ-17156516), a Novel, Potent, and Selective Cholecystokinin 1 Receptor Antagonist: In Vitro and in Vivo Pharmacological Comparison with Dexloxiglumide

3-[5-(3,4-Dichloro-phenyl)-1-(4-methoxy-phenyl)-1H-pyrazol-3-yl]-2-m-tolyl-propionate (JNJ-17156516) is a novel, potent, and selective cholecystokinin (CCK)1-receptor antagonist. In this study, the pharmacology of JNJ-17156516 was investigated both in vitro and in vivo, and the pharmacokinetic profile was evaluated in rats. JNJ-17156516 expressed high-affinity at the cloned human (pK(I) = 7.96 +/- 0.11), rat (pK(I) = 8.02 +/- 0.11), and canine (pK(I) = 7.98 +/- 0.04) CCK1 receptors, and it was also highly selective for the CCK1 receptor compared with the CCK2 receptor across the same species ( approximately 160-, approximately 230-, and approximately 75-fold, respectively). The high affinity of JNJ-17156516 at CCK1 receptors in vitro was confirmed in radioligand binding studies on fresh human gallbladder tissue (pK(I) = 8.22 +/- 0.05). In a functional in vitro assay of guinea pig gallbladder contraction, JNJ-17156516 behaved as a competitive antagonist, with a pK(B) value of 8.00 +/- 0.07. In vivo, JNJ-17156516 produced a parallel, rightward shift in the CCK-8S-evoked contraction of the guinea pig gallbladder. The dose required to shift the CCK-8S dose-response curve was 240 nmol kg(-1) i.v. In the anesthetized rat, JNJ-17156516 produced a dose-related decrease in the number of duodenal contractions evoked by infusion of CCK-8S, with an ED(50) = 484 nmol kg(-1). Pharmacokinetic analysis of JNJ-17156516 in rats, revealed that JNJ-17156516 had a half-life of 3.0 +/- 0.5 h and a very high bioavailability (108 +/- 10%) in this species. Overall, we have demonstrated that JNJ-17156516 is a high-affinity selective human CCK1 receptor antagonist with good pharmacokinetic properties in rats.

Role of PGE2 in maintenance of tonic contraction of guinea pig gallbladder

Role of PGE2 in maintenance of tonic contraction of guinea pig gallbladder

Role of prostaglandin E 2 and Ca 2+ in bradykinin induced contractions of guinea-pig gallbladder in vitro

In this study, we investigated the contribution of prostaglandin E 2 to bradykinin induced contractions of guinea-pig gallbladder in vitro and characterized the sources of activator Ca 2+ for the bradykinin mediated contractions. Contractions induced by bradykinin in guinea-pig gallbladder smooth muscle strips were significantly attenuated by the cyclooxygenase inhibitor piroxicam (10 μM). In the presence of piroxicam, a threshold concentration of prostaglandin E 2 (1 nM) significantly enhanced the contractile response to subsequent challenge with bradykinin. Contractile responses to bradykinin were abolished in a Ca 2+-free medium plus EDTA. The inhibitor of receptor mediated Ca 2+ entry, SK&F 96365 (1-[β-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl]-1 H-imidazole hydrochloride, 10–50 μM) dose dependently abolished the response to bradykinin, while this response was only partially attenuated by nifedipine (10–50 μM; a voltage-operated Ca 2+ channel antagonist). Thapsigargin (an inhibitor of the sarcoplasmic reticulum calcium ATP-ase pump, 1 μM) produced sustained contractions of guinea-pig gallbladder strips that were dependent on extracellular Ca 2+. After incubation of strips in a Ca 2+-free medium with thapsigargin, replacement of Ca 2+ caused a large sustained contraction. We conclude that the contractile response of guinea-pig gallbladder to bradykinin is modulated by prostaglandin E 2. Bradykinin induced contractions of guinea-pig gallbladder are highly dependent on extracellular Ca 2+ which enters through store-operated Ca 2+ channels and partially through voltage-operated Ca 2+ channels.

Influences of indomethacin on contractions induced by endothelins in guinea pig isolated gallbladder.

Endothelins (ETs) potently contract guinea pig gallbladder (GPGB) via endothelin-A and -B (ETA and ETB) receptors. This study assesses the possible participation of eicosanoids in the mediation of responses of the GPGB (Krebs' solution, 37 degrees C, 0.5 g load) triggered through each receptor type. Indomethacin (INDO; 5.6 microM) shifted the curve to endothelin-1 (ET-1) (0.1-100 nM) to the right, enhancing the CK50 (concentration causing 50% of response to KCl 80 mM) from 0.9 to 6.8 nM and reducing its EH (response to 100 nM) from 170 +/- 13 to 123 +/- 9 (% of response to 80mM KCl). INDO strongly depressed responses to sarafotoxin S6c (S6c; control CK50 0.9 nM), reducing its EH from 108 +/- 5 to 21 +/- 4. Neither BQ-123 nor BQ-788 (1 microM) changed responses to ET-1, but each markedly reduced responsiveness to ET-3 (control: CK50 of 9.7 nM and EH of 153 +/- 14; BQ-123: approximately = 100 nM and 44 +/- 12; BQ-788 approximately = 100 nM and 65 +/- 18). In the presence of BQ-123, INDO further depressed responses to ET-3 (EH 26 +/- 6), whereas in the presence of BQ-788, such responses were strongly enhanced (EH 126 +/- 9). These findings strongly suggest that contractions of GPGB caused via ETB receptors are mediated to a large extent by contractile eicosanoids, whereas those caused (at least by ET-3) via ETA receptors are limited by relaxant eicosanoids. The cellular sources and nature of the eicosanoids released by ETs remain to be established.

MUSCARINIC M2RECEPTORS ARE NOT PRIMARILY INVOLVED IN THE CONTRACTION OF GUINEA-PIG GALLBLADDER SMOOTH MUSCLE

The presence of M1–M4receptors in guinea-pig gallbladder smooth muscle cells has been reported recently. The majority of these receptors are said to be of M2subtype. However, there are controversial reports about the functional muscarinic receptors that mediate contraction in this tissue. Similar to gallbladder, it was claimed that M4receptors mediate guinea-pig uterine contractions, but these receptors have appeared to be of M2subtypes later. Therefore, the antagonistic affinities of three M2-selective muscarinic antagonists were determined in contraction and radioligand binding experiments in guinea-pig gallbladder in the present study. The antagonistic affinity values (p Ki) of gallamine, tripitramine and imperialine were as follows, respectively: 6.28±0.15, 8.65±0.10 and 6.55±0.07 against 0.250 n m [3H]QNB binding. All three antagonists displaced the concentration– response curves to carbachol to the right in parallel without affecting the maximum responses. The p A2values obtained from constrained Schild plots (−log KB) were 4.14±0.18 for gallamine, 6.79±0.09 for tripitramine, and 7.02±0.09 for imperialine. The antagonistic affinity values of gallamine, tripitramine and imperialine for M2receptors are reported to be 6.3, 9.6, 7.7, respectively. The pA2 values obtained in this study clearly indicate that the primary muscarinic receptors involved in carbachol-induced guinea-pig gallbladder contraction are not of M2subtype. The poor correlation between the antagonistic affinity values of these antagonists obtained at radioligand binding (p Ki) and contraction (pA2 ) experiments also support the conclusion that the majority of muscarinic receptors which have been reported to be of M2do not mediate the contractile responses.

Isolation and characterization of sulphated and nonsulphated forms of cholecystokinin-58 and their action on gallbladder contraction.

Cholecystokinin (CCK) exists in multiple molecular forms with different polypeptide lengths and the absence or presence of sulphation. We have isolated sulphated and nonsulphated forms of CCK-58 from porcine intestine and have determined their bioactivities in a guinea-pig gallbladder contraction assay. Both forms co-eluted in cation-exchange chromatography and in several rounds of reverse-phase (RP)-HPLC, but separated upon RP-HPLC using a water/acetonitrile system with heptafluorobutyric acid as counter ion. Nonsulphated CCK-58 was the form detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry because of desulphation in that process. The biological activity of CCK-58 and CCK-33 is equipotent, although the kinetics of the response differ. Sulphated CCK-58 was found to be 35 times more potent than nonsulphated CCK-58. In contrast, sulphated CCK-8 is 150 times more potent than nonsulphated CCK-8, and for sulphated and nonsulphated CCK-33, the activities differ by a factor of 100. This type of correlation indicates that the N-terminal end of CCK-58 partially compensates for the decrease in activity arising from the lack of sulphated tyrosine. Given its fairly high bioactivity, nonsulphated CCK-58 may have a physiological significance.

Evidence for the presence of muscarinic M2 and M4 receptors in guinea-pig gallbladder smooth muscle.

1. The affinities of 10 selective muscarinic receptor antagonists against [3H]-quinuclidinyl benzilate (QNB) binding were determined to characterize the muscarinic receptors present in guinea-pig gallbladder smooth muscle. The highest correlation was obtained for the comparison between the pKi values for the gallbladder smooth muscle and M2 sites. Pirenzepine revealed two binding sites with affinities indicating the presence of muscarinic M2 receptors in abundance and a minor population of an additional site(s). 2. Carbachol produced gallbladder contractions, stimulated phosphoinositide (PI) hydrolysis and inhibited cAMP formation concentration-dependently with pD2 values of 6.12 +/- 0.11, 5.18 +/- 0.33 and 7.19 +/- 0.15, respectively. 3. Pirenzepine, 4-DAMP, HHSiD, pF-HHSiD, AF-DX 116, methoctramine, AQ-RA 741, guanylpirenzepine and AF-DX 384 showed competitive antagonism against carbachol-induced gallbladder contractions. There was no correlation between the pA2 values for the gallbladder and pKi values for the M2 sites, whereas significant correlations were found for the M1, M3 and M4 sites, the best correlation being between the pA2 values for the gallbladder and M4 subtypes. 4. Finally, the presence of both m2 and m4 receptor proteins were demonstrated by Western blot analysis. It is concluded that guinea-pig gallbladder smooth muscle has both muscarinic M2 and M4 receptors, which are coupled to adenylate cyclase inhibition and PI hydrolysis. 5. Although it seems likely that M2 receptors do not play a primary role in carbachol-induced guinea-pig gallbladder contraction, the characterization of the muscarinic subtypes which mediate these contractile responses needs further evidence.

Preferential inhibition by ethanol of phorbol ester-induced smooth muscle contraction of guinea pig gallbladder

In order to investigate how ethanol inhibits the contractility of gallbladder smooth muscle, the effects of different ethanol concentrations were examined on gallbladder contractile responses to stimulants which act on the smooth muscle by different mechanisms. A low concentration (25 mM) of ethanol significantly inhibited the contractile response to phorbol-12,13-dibutyrate (PDBu), but not that to KCl. It also did not significantly affect contractile response to Ca 2+ ionophore A23187 in the presence of verapamil or that to Ba 2+ in a medium without Ca 2+. On the other hand, a higher concentration (200 mM) of ethanol significantly inhibited the contractile responses to PDBu, KC1, Ca 2+ ionophore A23187 and BaCl 2. These results together with our recent finding that PDBu-induced contraction of guinea pig gallbladder is completely dependent on activation of the voltage-dependent Ca 2+ channel suggest that low concentrations of ethanol selectively inhibit the signal transduction of gallbladder smooth muscle cells in the pathway from activation of protein kinase C to that of the voltage-dependent Ca 2+ channel, while high concentrations of ethanol inhibit the intracellular common pathway (probably in the cytoskeletal apparatus).

Characterization of des-Arg 9-bradykinin-induced contraction in guinea-pig gallbladder in vitro

We have reported that bradykinin induces graded contraction in guinea-pig gallbladder in vitro through activation of bradykinin B 2 receptors and prostanoid release, while des-Arg 9-bradykinin, a selective bradykinin B 1 receptor agonist, causes only a weak contraction, suggesting the presence of badykinin B 1 receptors in this tissue. In the present study, we attempted to characterise the receptor subtype and the possible mechanism by which des-Arg 9-bradykinin induces contraction in this preparation. Contractions induced by des-Arg 9-bradykinin in guinea-pig gallbladder (1 pM to 1 μM) increased significantly as a function of time elapsed after setting up of the preparation, reaching the maximum after 6 h of equilibration (EC 50 16.4 pM and E max 0.6±0.08 g). Des-Arg 9-bradykinin-induced contraction in guinea-pig gallbladder was totally prevented by cycloheximide (70 μM, an inhibitor of protein synthesis), indomethacin (3 μM), ibuprofen (30 μM), phenidone (30 μM) or Ca 2+-free medium plus EGTA, and was partially antagonised by MK 571 ((3-(3-(2-(7-chloro-2-quinolinyl) ethenyl) phenyl ((3-dimethyl amino-3-oxo-propyl) thio) methyl) propanoic acid, 0.1 μM) or by nicardipine (1 μM), but was not affected by dazoxiben (30 μM), staurosporine (100 nM) or L 655,240 (240 (3-[1-(4-clorobenzil)-5-fluoro-3-metilhyindol-2il] 2,2-dimetilpropanoic acid, 1 μM). Unexpectedly, des-Arg 9-bradykinin-induced contraction was unaffected by the selective bradykinin B 1 receptor antagonists, des-Arg 9-[Leu 8]-bradykinin and des-Arg 9-NPC 17761 (des-Arg 0- d-Arg [Hip 3, d-HipE (transtiofenil) 7, Oic 8]-des-Arg 9-bradykinin). However, the selective bradykinin B 2 receptor antagonists, HOE 140 ( d-Arg 0-[Hyp 3, Thi 5, d-Tic 7, Oic 8]-bradykinin) and NPC 17731 ( d-Arg 0 [Hyp 3, DHypE (transpropyl) 7, Oic 8]-bradykinin), completely blocked des-Arg 9-bradykinin-mediated contraction. Pre-treatment of the animals with Escherichia coli endotoxin (lipopolysaccharide, 30 μg/animal, i.v., 24 h) did not significantly change the response to des-Arg 9-bradykinin induction. It is concluded that des-Arg 9-bradykinin-induced contractions in guinea-pig gallbladder are mediated primarily by the release of proinflammatory eicosanoid(s) derived from the cyclo-oxygenase pathway. These effects are unrelated to thromboxane A 2 and do not seem to be coupled to activation of a protein kinase C-dependent mechanism. Response to des-Arg 9-bradykinin increases as a function of the equilibration period of the preparation by a mechanism dependent on protein synthesis and seems to be mediated by activation of bradykinin B 2 (but not B 1) receptors. Finally, in contrast to that observed for bradykinin, the contraction induced by des-Arg 9-bradykinin in guinea-pig galbladder is fully dependent on the influx of extracellular Ca 2+, partially through L-type Ca 2+ channels.

Contraction of guinea-pig gallbladder: muscarinic M 3 or M 4 receptors?

The muscarinic receptor mediating contraction of the guinea-pig isolated gallbladder, currently being disputed to belong either to the M 3 or M 4 subtype, was characterized by subtype-preferring agonists and discriminating antagonists. Highly significant correlations of agonist potencies to contract the gallbladder, e.g., arecaidine propargyl ester, oxotremorine, 5-methylfurtrethonium>arecoline, arecaidine 2-butyne-1,4-diyl bisester>( R)-nipecotic acid ethyl ester>4-[[ N-(4-chlorophenyl)carbamyl]oxy]-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343), ( S)-nipecotic acid ethyl ester>4-[[ N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyltrimethylammonium chloride (McN-A-343) were found with muscarinic M 3 receptors mediating contraction of the guinea-pig ileum and vasodilation in rat perfused kidney. Functional affinities at guinea-pig gallbladder muscarinic receptors of antagonists known to distinguish between native or cloned muscarinic M 3/m3 and M 4/m4 receptors, e.g., himbacine, methoctramine, mefurtramine, tripitramine, idaverine, zamifenacin and 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one (AQ-RA 741), were consistent with those at guinea-pig ileal muscarinic M 3 receptors but not with published data at recently defined muscarinic M 4 receptors in rabbit anococcygeus muscle or at muscarinic M 1 and M 2 receptors in rabbit vas deferens. Antagonist affinities at guinea-pig gallbladder correlated also best with published binding data on native or cloned muscarinic M 3/m3 receptors but not with those for muscarinic M 4/m4 receptors. The agonist potencies and antagonist affinities suggest that smooth muscle contraction elicited by muscarinic stimuli in guinea-pig gallbladder is mediated by functional muscarinic M 3 receptors.

Electric field stimulation-induced guinea pig gallbladder contractions: role of calcium channels in acetylcholine release.

Gallbladder motility is modulated by intrinsic cholinergic neurons. The aims of this study were to determine: (1) the effect of electric field stimulation (EFS) on guinea pig gallbladder smooth muscle, and (2) the role of calcium channels in mediating neurotransmitter release. Gallbladder muscle strips were studied isometrically in vitro. EFS (1-16 Hz, 100 V, 0.5-msec pulse width, 30-sec train duration) was used to activate the intrinsic nerves. Exogenous acetylcholine was also used to directly stimulate the smooth muscle. EFS produced a frequency-dependent contractile response that was completely abolished by tetrodotoxin. EFS-induced contractions at 16 Hz were suppressed by 84 +/- 4% with atropine, whereas hexamethonium had no effect. The L-type calcium channel blocker, nifedipine, reduced EFS contractions by 51 +/- 4%, whereas it reduced contractions to acetylcholine by only 11 +/- 5%. The N-type calcium channel blocker, omega-conotoxin GVIA, reduced EFS-induced contractions by 22 +/- 9%, but did not affect acetylcholine-induced contractions. EFS-induced contractions of the guinea pig gallbladder are primarily mediated by activation of postganglionic cholinergic neurons. The acetylcholine release from these cholinergic neurons is regulated by L- and N-type calcium channels. The inhibitory effect of calcium channel blockers on the gallbladder seen in vivo may be in part related to inhibition of acetylcholine release from the intrinsic cholinergic nerves of the gallbladder.

Effect of modulating voltage-dependent calcium channels on cholecystokinin and acetylcholine-induced contractions of the guinea pig gallbladder

The aim of this study was to investigate the role of extracellular Ca 2+ utilization in cholecystokinin (CCK) and acetylcholine-induced guinea pig gallbladder contractions by using agents that modulate influx of extracellular Ca 2+ through voltage-dependent calcium channels. METHODS: Guinea pig gallbladder muscle strips were studied isometrically at L max in vitro. Results: (1) Acetylcholine and CCK caused dose-dependent contractions, with ED max of 10 −4 to 10 −6 M, respectively. (2) Preventing influx of extracellular Ca 2+ by incubation in Ca 2+-free/0.1 mM EGTA solution inhibited the acetylcholine (10 −4 M)-induced contraction by 60 ± 3% compared to only 46 ± 5% ( P $ ̌ 0.05 ) for CCK (10 −6 M)-induced contraction. (3) Nifedipine (3 μM) inhibited the response to acetylcholine (10 −4 M) by 54 ± 3%, compared to only 34 ± 3% ( P < 0.01) for CCK (10 −6 M). (4) Bay K 8644 (10 −7 M) significantly increased ( P < 0.05) the contractile responses to low doses of each agonist: acetylcholine (10 −6 M) by 121 ± 44% and CCK (10 −9 M) by 94 ± 31%, but had no effect on the contraction to the ED max of each agonist. Conclusions: These studies demonstrate: (1) acetylcholine and CCK cause guinea pig gallbladder contraction by both intracellular Ca 2+ release and influx of extracellular Ca 2+ through voltage-dependent calcium channels; (2) the CCK-induced contraction is more dependent on intracellular Ca 2+ than is acetylcholine; and (3) acetylcholine and CCK-induced contractions can by modulated by manipulating influx of extracellular Ca 2+ through voltage-dependent calcium channels.

Effect of gastrin-releasing peptide (GRP) on guinea pig gallbladder contraction in vitro.

Few studies have reported the effects of gastrin-releasing peptide (GRP)/bombesin on the guinea pig gallbladder, and the results are contradictory. Because such contradictory results may, in part, be due to technical factors, we investigated the effect of GRP on guinea pig gallbladder smooth muscle, using an improved horizontal organ bath. The guinea pigs were killed and the gallbladder was removed. Four longitudinal muscle strips (2 x 12 mm) were suspended in Krebs-Ringer solution at 37 degrees C and aerated with 95% O2 and 5% CO2. The mechanical activity of the strips was recorded isotonically by displacement-voltage transducers, via L-arms, to which a piezoelectric element with a frequency of 100 Hz and movement of 50 microns was applied. GRP contracted gallbladder muscle strips dose dependently, but the calculated maximal response was 22.4% and 20.1% of the acetylcholine- and cholecystokinin octapeptide (CCK8)-induced responses, respectively. The GRP-induced contraction was unaffected by the muscarinic blocker, atropine, or by the CCK receptor antagonist, loxiglumide. It is concluded that GRP weakly, but apparently directly, stimulates guinea pig gallbladder contraction.

Mechanisms of bradykinin‐induced contraction of the guinea‐pig gallbladder in vitro

1. The mechanisms underlying bradykinin (BK)-mediated contractions in strips of guinea-pig gallbladder (GPG) were examined by use of selective bradykinin (BK) receptor agonists and antagonists. 2. Addition of BK and related kinins (0.1 pM-10 microM) after 2 h of equilibration of the preparation caused graded contractions characterized by two distinct phases: high affinity (0.1 pM-1 nM) and low affinity (3 nM-10 microM). The rank order of potency for the first phase (mean EC50, pM) was: BK (1.36) = Hyp3-BK (1.44) = Lys-BK (1.54) > Tyr8-BK (2.72) > Met-Lys-BK (4.30). The rank order of potency for the second phase (mean EC50, nM, at concentration producing 50% of the contraction caused by 80 mM KCl) was: Hyp3-BK (8.95) > Met-Lys-BK (12.78) > Tyr8-BK (33.75) > Lys-BK caused by 80 mM KCl) was: Hyp3-BK (8.95) > Met-Lys-BK (12.78) > Tyr8-BK (33.75) > Lys-BK (60.92) > BK (77.35). The contractile responses (g of tension) to 3 microM of BK (the highest concentration tested) were: Hyp3-BK, 1.76 +/- 0.09; BK, 1.65 +/- 0.12; Lys-BK, 1.45 +/- 0.13; Tyr8-BK, 1.36 +/- 0.15 and Met-Lys-BK, 1.36 +/- 0.15. The selective B1 agonist, des-Arg9-BK, caused only a weak contraction with maximal response (0.21 +/- 0.05 g), which corresponded to approximately 10% of that induced by BK. 3. BK-induced contraction in GPG was inhibited by indomethacin (3 microM) or ibuprofen (30 microM), and was partially reduced by phenidone (30 microM), but was not affected by atropine (1 JM), nicardipine (1 gM),Ca2+-free medium plus EGTA, dazoxiben (30 nM), L-655,240 (10 nM, a selective receptor antagonist ofthromboxane A2), MK-571 (0.1 microM, a selective leukotriene D4 receptor antagonist), tetrodotoxin(0.3microM), CP 96,345 (0.3 microM, a NK1 receptor antagonist), mepyramine (1 microM), glibenclamide (1 microM), H-7(3 microM), staurosporine (100 nM), or phorbol 12-myristate 13-acetate (1 microM). However, BK-induced contractions in GPG maintained in Ca2+-free medium were markedly attenuated by ryanodine (10microM).4. Prostaglandin E2, prostaglandin F2alpha or U46619 (0.1 nM to 100microM), caused concentration-dependent contractions in GPG with mean EC50s of 3.1 microM; 1.7 microM and 0.47 nM and maximal responses of1.36 +/-0.15; 1.32 +/- 0.20 and 0.96 +/- 0.09 g, respectively.5. The selective B2 receptor antagonists, Hoe 140, NPC 17731 and NPC 17761 (0.01 -1 microM), caused concentration-dependent displacements to the right of the contractile concentration-response curve for BK. The selective B1 receptor antagonist, des-Arg9-[Leu8]-BK (1 microM), did not affect BK-induced GPG contraction.6. These data suggest that both high and low affinity BK responses in GPG are mediated by activation of B2 receptors, and that BK-mediated contraction in GPG depends on the release of intracellular Ca2+sources sensitive to ryanodine. In addition, BK-induced contraction in GPG is mediated by release of proinflammatory eicosanoid(s) derived from the cyclo-oxygenase pathway from arachidonic acid metabolism unrelated to thromboxane A2, and seems not to be coupled to activation of a protein kinase C-dependent mechanism.

2-NAP: a selective, hydrophilic, non-peptide CCK A - receptor antagonist derived from the cholecystokinin C-terminal dipeptide.

Analogues of the cholecystokinin (CCK) C-terminal dipeptide (32-33, Asp-Phe-NH 2) have been prepared and the structure-activity relationships of this series are described. The sodium salt of 2-naphthalenesulphonyl L-aspartyl (2-phenethyl)amide, (2-NAP), displayed high affinity for CCK A receptors by its antagonism of CCK 8-stimulated guinea-pig gallbladder contraction. In addition, 2-NAP exhibits selectivity with respect to gastrin/CCK B receptors ( > 300-fold) and has a low log P (−0.91, chloroform/buffer).

Age-related changes in gallbladder contractility and cytoplasmic Ca2+ concentration in the guinea pig.

Gallbladder (GB) motility diminishes with aging. This study was performed to characterize mechanisms that are involved in changes in GB contractility that occur during aging. Cytoplasmic Ca2+ concentrations ([Ca2+]i) and the contractile force of guinea pig GB muscle strips were simultaneously measured using fura-2 and force-displacement transducers. The binding ability of the Ca2+ channel antagonist and GB muscle compliance were also examined. The COOH-terminal octapeptide of cholecystokinin (CCK-8) evoked a dose-dependent increase in force and [Ca2+]i. Changes of [Ca2+]i and contractile force of muscle strips in response to CCK-8 were significantly greater in young (2 mo old) compared with mature and aged (12 and 24 mo old) guinea pigs (changes in [Ca2+]i, ED50: 46.1 nM at 2 mo, 6.1 microM at 12 mo, and 2.8 mM at 24 mo; changes of contractile force, ED50: 24.8 microM at 2 mo, 2.1 mM at 12 mo, and 357 mM at 24 mo). However, the magnitude of the contraction at each percent change in [Ca2+]i was actually similar in young and aged guinea pigs. In a Ca(2+)-free buffer, the responses of [Ca2+]i and force to CCK-8 in both young and aged GB muscles decreased, but those were still dose and age dependent. Binding ability of the Ca2+ channel antagonist did not differ in the young and aged groups, but the compliance of the GB muscle strip decreased with aging. These results suggest that both a reduced mobilization of intracellular Ca2+ and a decreased muscle compliance are responsible, at least in part, for age-related reduced contraction of guinea pig GB in response to CCK.

Peripheral biological activity of SR 27897: a new potent non-peptide antagonist of CCK A receptors

SR 27897 is a new non-peptide antagonist of CCK A receptors: 1-[[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl] indolyl] acetic acid. This compound is a potent ligand for CCK A binding sites (rat pancreatic membranes, K i = 0.2 nM) and is highly selective (CCK B and gastrin/CCK A IC 50 ratios of 800 and 5000 respectively). In vitro, it is competitive antagonist of cholecystokinin (CCK)-stimulated amylase release in isolated rat pancreatic acini (pA 2 = 7.50) and of CCK-induced guinea pig gall bladder contractions (pA 2 = 9.57). In in vivo gastrointestinal models, SR 27897 confirmed the potency obtained in vitro: at 1 mg/kg (i.v.) it completely reversed the CCK-induced amylase secretion, at 3 μg/kg (p.o.) it antagonized by 50% the CCK-induced inhibition of gastric emptying of a charcoal meal in mice, and 72 μg/kg (p.o.) was the median effective dose for inhibiting CCK-induced gall bladder emptying in mice. SR 27897 was also very active (ED 50 = 27 μg/kg p.o.) in the gall bladder emptying protocol with egg yolk as an inducer of endogenous CCK release. SR 27897 had a long-lasting action in all the experiments, with no differences between oral and intravenous routes of administration. SR 27897 was more or less effective than L-364,718, depending on the model and the species. Both compounds increased the gall bladder volume of fasting mice, but the effect of SR 27897 was 10 times lower than that of L-364,718. In summary, SR 27897 is a selective antagonist of CCK A receptors, is highly potent in animal models whatever the route of administration and has a long duration of action. Thus this compound may be useful for studying the physiological or pathological role of CCK in humans.