Guinea Pig Gallbladder Research Articles

The effect of phosphatase inhibitors on myosin light chain phosphorylation and relaxation in guinea pig gallbladder and urinary bladder smooth muscle

The effect of phosphatase inhibitors on myosin light chain phosphorylation and relaxation in guinea pig gallbladder and urinary bladder smooth muscle

Regulation of PI hydrolysis and cAMP formation by muscarinic M3 receptor in guinea pig gallbladder.

Carbachol (10(-8)-10(-3) M) produced two distinct biochemical responses in the guinea pig gallbladder smooth muscle: simulation of phosphoinositide (PI) hydrolysis and inhibition of forskolin-mediated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The mean effective dose (ED50) concentration (1.6 x 10(-5) M) of carbachol-mediated stimulation of PI hydrolysis was 145 times greater than the ED50 concentration (1.1 x 10(-7) M) of carbachol mediated inhibition of cAMP formation. The inhibitory effect of carbachol on cAMP formation was antagonized by the pretreatment of pertussis toxin. To determine whether these two biochemical responses were mediated by the same or different subtypes of muscarinic receptors, the relative potencies of muscarinic receptor antagonists were calculated by Schild analysis. The M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) exhibited inhibitory constant (Ki) values at 0.3 and 1.2 nM in antagonizing the stimulation of PI hydrolysis and the inhibition of cAMP formation, respectively. The corresponding Ki values for pirenzepine, a muscarinic M1 antagonist, were 11 and 130 nM. The corresponding Ki values for AF-DX 116, a muscarinic M2 antagonist, were 34 and 450 nM. Thus 4-DAMP was 37x and 108x more potent than pirenzepine in antagonizing the stimulation of PI hydrolysis and the inhibition of cAMP formation, respectively. In addition, compared with AF-DX 116, 4-DAMP was 113x and 375x more potent in reducing stimulation of PI hydrolysis and inhibition of cAMP formation. Cholecystokinin (CCK) octapeptide (10(-10)-(10-6) M) caused a significant increase of PI hydrolysis but had no inhibitory effects on cAMP formation evoked by forskolin (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological analysis of receptors for bombesin-related peptides on guinea pig gallbladder smooth muscle

The aim of this study was to characterize the receptor(s) for bombesin (BN) and its homologues (gastrin releasing peptide, GRP; neuromedin B, NMB; neuromedin C, NMC) in guinea pig gallbladder muscle strips. Dose-dependent contractions were induced by all peptides tested (potency: BN = GRP > NMC > NMB, but with similar efficacy: BN = GRP = NMC = NMB). The contractions were resistant to tetrodotoxin, atropine, phentolamine, and propranolol. BN tachyphylaxis (1 μM) abolished subsequent contractile responses to BN, GRP and NMC; and partially antagonized the response to NMB (66 ± 7% inhibition). NMB tachyphylaxis (10 μM) markedly inhibited subsequent contractile responses to NMB (78 ± 5%); and partially antagonized the contractile response to BN (36 ± 4%), GRP (31 ± 12%) and NMC (22 ± 2%). At 1 μM, both [ d-Phe 6, Des-Met 14]-BN(6–14) ethylamide and ICI 216,140, two BN receptor antagonists, reduced the contractile actions of BN (82 ± 4% and 59 ± 8% inhibition, respectively), GRP (75 ± 11% and 45 ± 5%), and NMC (73 ± 9% and 51 ± 6%) while having no marked effect on NMB contractions. Our pharmacological approaches (receptor tachyphylaxis and differential antagonism) provide support for two types of receptors for BN-like peptides on guinea pig gallbladder smooth muscle: a GRP-preferring receptor and a NMB-preferring receptor.

Functional investigation of muscarinic receptor subtype mediating ACh-induced contractions in guinea-pig gallbladder

The antimuscarinic agents nuvenzepine and pirenzepine were tested on four guinea pig isolated smooth muscle preparations in order to better investigate the existence of differences in the functional activities of such antagonists, as suggested by previous reports. The effects of both compounds were compared to those of atropine. Nuvenzepine showed a four-fold higher affinity than pirenzepine in competitively antagonizing acetylcholine-induced contractions on isolated ileal musculature (pA2=7.08±0.15) and on longitudinal ileum dispersed cells (pA2=7.11±0.19). By contrast, unlike pirenzepine which was ineffective, nuvenzepine inhibited histamine-induced ileal motor activity in a dualistic manner, behaving as an irreversible competitive H, antagonist (pA2=5.02±0.11).Nuvenzepine was almost equipotent to pirenzepine in competitively preventing bethanechol-induced gall-bladder contractions (pA2=7.23±0.16) and it displayed a four-fold higher potency than pirenzepine in blocking vagal-stimulated tracheal constrictions (pIC50=6.77±0.06). Both compounds were definitely less potent than atropine.On the whole, these findings indicate that, on the selected preparations, nuvenzepine substantially shares the antimuscarinic properties of pirenzepine but it is also endowed with a (weak) H1-blocking action. Furthermore, based on some observations, the presence in gallbladder smooth muscle of muscarinic receptors distinguishable from those of ileum could be speculated.

A developmental study of the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder

A histochemical investigation of age-related changes that occur with respect to the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder was carried out. In all age groups examined (embryonic stages day 34 and 52, 2 to 4-day old, 6-month old and 2-year old), the mean percentage of NADPH-diaphorase-positive neurons per ganglion was obtained by taking the number of neurons that were immunoreactive to protein gene product 9.5 (a general neuronal marker) as 100%. In addition, the possible co-existence of NADPH-diaphorase and nitric oxide synthase in the ganglionated plexus of 2 to 4-day old and 6-month old guinea-pig gallbladder was investigated. NADPH-diaphorase was not present in the ganglionated plexus of the gallbladder at embryonic day 34. At embryonic day 52, all the protein gene product 9.5-immunoreactive neurons showed positive staining to NADPH-diaphorase; this dropped to a minimum at 2-4 days (26.7%), rose slightly at 6 months (33.6%), and finally returned close to the 100% value at 2 years. In the gallbladders of 2-year old guinea-pigs, some (3 out of 10) ganglia were devoid of protein gene product 9.5-immunoreactive neurons, but NADPH-diaphorase-stained granules were found within the ganglia. However, all those neurons that were immunopositive to protein gene product 9.5 also expressed NADPH-diaphorase. Moreover, NADPH-diaphorase-positive neurons in the gallbladder of 2 to 4-day-old and 6-month-old guinea-pigs were found to express nitric oxide synthase.

Signal transduction pathway of the muscarinic receptors mediating gallbladder contraction.

In gallbladder smooth muscle, carbachol interacts with M3 receptors to mediate contraction. To examine components of the intracellular second messenger system that is coupled to these receptors we have tested whether carbachol stimulates the formation of inositol phosphates (IP) to cause contraction. Guinea pig gallbladder muscle strips were prelabeled with [3H]inositol and were incubated with 0.1 mmol/l carbachol, a concentration causing maximal contraction. [3H]inositol monophosphates, [3H]inositol bisphosphates and [3H]inositol trisphosphates and contraction were measured at various times (0-90 s). To examine whether a pertussis toxin-sensitive guanine nucleotide binding protein is coupled to the muscarinic receptors, guinea pigs were pretreated with pertussis toxin (180 micrograms/kg i.v./24 h). The effectiveness of pertussis toxin treatment was determined by measuring [32P]ADP-ribosylation of a approximately 40/41 kDa protein from gallbladder homogenates. Carbachol caused a significant time-dependent increase in the formation of [3H]inositol monophosphates, [3H]inositol bisphosphates and [3H]inositol trisphosphates. The time course of [3H]inositol trisphosphate turnover caused by carbachol was biphasic, and was detectable at 15 s and maximal at 60 s; at 75 s and 90 s formation of [3H]inositol trisphosphates decreased, whereas the time course of carbachol-induced contraction of the gallbladder smooth muscle strips reached a plateau after 90 s. The effects of carbachol on [3H]inositol trisphosphates and on contraction were abolished by atropine.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of myosin light-chain phosphorylation in guinea pig gallbladder smooth muscle contraction.

In acetylcholine (ACh)-stimulated gallbladder smooth muscle, we have previously shown that phosphorylation of the 20,000-Da myosin light chains is necessary for the initiation of contraction, that myosin is stably phosphorylated at steady state, and that dephosphorylation of cross bridges is not necessary for the slowing of cross-bridge cycling rates during the period of steady-state isometric stress. The present studies were undertaken to determine whether 1) K+ (60 or 80 mM) or cholecystokinin (CCK, 10(-8) M) stimulation is accompanied by changes in myosin light-chain phosphorylation in gallbladder smooth muscle and 2) dephosphorylated noncycling cross bridges exist in K(+)- or CCK-stimulated gallbladder smooth muscle. Isometric stress, isotonic shortening velocity, and myosin light-chain phosphorylation were determined during contraction with K+ or CCK. Steady-state isometric stress was reached within 2.5 min of stimulation with K+ or CCK and was maintained for the duration of the stimulation. Stimulation with K+ or CCK was associated with rapid increases in myosin light-chain phosphorylation and maintenance of myosin light-chain phosphorylation during the stimulation. In contrast, isotonic shortening velocity was maximal at 1 min of stimulation with either K+ or CCK and then declined significantly to values that were only 26-32% of the peak velocity. These data, along with data from previous experiments with ACh, suggest that myosin light-chain phosphorylation is essential in the initiation of contraction in gallbladder smooth muscle, regardless of the source of Ca2+ or of the contractile agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide modulates the calcitonin gene-related peptide-induced relaxation in guinea pig gallbladder strips in vitro.

Calcitonin gene-related peptide (CGRP) induces a dose-dependent relaxation in cholecystokinin-induced tension in guinea pig gallbladder strips in vitro. This effect seems to be modulated, in part, via activation of the nonadrenergic noncholinergic nervous system. Blockers of nitric oxide synthesis significantly reduced the CGRP effect. Increases in nitric oxide (NO) after treatment with CGRP suggested the involvement of NO in the observed relaxation.

Ionic basis of the action potential of guinea pig gallbladder smooth muscle cells.

Smooth muscle cells in the intact guinea pig gallbladder had a resting membrane potential of about -45 mV and had spontaneous action potentials that consisted of a rapid depolarization, a transient repolarization, a plateau phase, and a complete repolarization. These action potentials lasted approximately 570 ms and occurred at a frequency of approximately 0.4 Hz. Action potentials were abolished by the dihydropyridine (DHP)-sensitive Ca2+ channel blocker nifedipine (1.0 microM) and were enhanced by the DHP-sensitive Ca2+ channel agonist BAY K 8644 (0.5 microM). The K+ channel blockers tetraethylammonium chloride (5.0 mM) and 4-aminopyridine (4-AP; 2.0 mM) prolonged the action potential, whereas charybdotoxin (100 nM), a blocker of calcium-activated potassium channels, had no effect. Whole cell currents were characterized in enzymatically isolated smooth muscle cells from the same preparation. 4-AP, a blocker of voltage-dependent K+ channels, suppressed 70% of the outward current at 0 mV. Charybdotoxin (100 nM) reduced an additional 15% of the current at 0 mV. Single calcium-activated potassium channels were identified. The potential for half-activation of these channels, at a cytosolic Ca2+ concentration of 100 nM, was 66.8 mV. A fivefold increase in cytosolic Ca2+ resulted in a shift of the activation curve by -53 mV. External tetraethylammonium chloride (200 microM) reduced the mean single channel current by 48% at 0 mV. The whole cell outward current was abolished by replacement of intracellular K+ for Cs+. Ca2+ currents were inhibited by nifedipine and were increased by BAY K 8644. We conclude that DHP-sensitive voltage-dependent Ca2+ channels are responsible for the depolarization of the action potentials and that the repolarization is due to primarily 4-AP-sensitive K+ current.

Characterization of muscarinic receptors on guinea pig gallbladder smooth muscle

Background : Cholinergic agonists are of major importance for the regulation of gallbladder motility. However, the gallbladder muscarinic receptors have not been localized or characterized directly using radioligands, and it has not been clearly established which subtype of muscarinic receptor mediates contraction. The aim of the present study was to characterize the gallbladder muscarinic receptors. Methods : Binding studies to guinea pig gallbladder sections were performed using 1-[ N-methyl- 3H] scopolamine methyl chloride. Carbachol-induced contraction was measured using muscle strips. Results : Binding of 1-[ N-methyl- 3H] scopolamine methyl chloride was reversible, dependent on time, temperature, and pH. Autoradiography showed binding only over the smooth muscle. Binding and carbachol-induced contractions were inhibited by muscarinic receptor antagonists with the following potencies: atropine > N-methyl-scopolamine > silahexocyclium-methylsulfate > AF-DX 384 [(±)-5,11-dihydro-11-{[(2-{2-[(dipropylamino)-methyl]-1-piperidinyl}ethyl)amino]carbonyl}-6 H-pyrido (2,3b) (1,4)-benzodiazepine-6-one] > hexahydro-sila-difenidol hydrochloride > AF-DX 116 [(±)-11-({2-[(diethylamino)methyl]-1-piperidinyl}-acetyl)-5,11-dihydro -6 H-pyrido (2,3b)(1,4)benzodiazepine-6-one] > pirenzepine. Carbachol inhibited binding to gallbladder sections over the same range of concentrations that caused contractions. The concentration-contraction curves for carbachol were not altered by tetrodotoxin. Conclusions : Gallbladder smooth muscle cells possess muscarinic receptors of the M3 type. These receptors mediate carbachol-induced contraction.

Effect of loxiglumide (CR 1505) on CCK-induced contractions and 3H-acetylcholine release from guinea-pig gallbladder

Release of [ 3H]-acetylcholine ( 3H-ACh) and muscle contractions in response to cholecystokinin (CCK) were measured and recorded simultaneously from isolated guinea-pig gallbladder. Cholecystokinin octapeptide (CCK8) (10 −10–10 −7 M) enhanced the release of [ 3H]ACh and the contractions of the muscle. TTX (10 −6 M) inhibited the CCK-induced release of 3H-ACh by only 30%. In Ca 2+-free medium CCK8 had no effect. Loxiglumide, (CR 1505), a newly synthesized nonpeptide CCK-A-receptor antagonist, D.L.- (3,4-dichlorbenzoilamino) -5 - / N - (3-methoxypropyl)-pentylamino-5-oxo-pentanoic acid, antagonized both the ACh-releasing effect of CCK and the contractions in a dose-dependent manner. The affinity (pA 2) of CR 1505 to CCK-receptors, determined by the shift of the concentration-response curves for CCK8 was 8.36. It was 5 logarithmic orders higher than the pA 2 of proglumide. The IC 50 value of CR 1505 calculated by the CCK-induced release of 3H-ACh was 10 nM. The results suggest the existence not only of muscular CCK receptors but also neuronal receptors for CCK probably located on cholinergic nerves.

Electrogenic bicarbonate secretion in gallbladder: induction by barium via neuronal, possibly VIP-ergic pathways.

In guinea-pig gallbladder epithelium, cAMP converts electroneutral HCO3- secretion into an electrogenic process. The effects of blood side Ba2+ (5 mmol/l) on HCO3- transport were investigated in vitro, using pH-stat and voltage clamp techniques to determine unidirectional fluxes of HCO3- and transepithelial electrical characteristics. Serosal, not mucosal addition of Ba2+ elevated short-circuit current (Isc), transepithelial potential difference, and tissue conductance; it inhibited the absorptive HCO3- flux while leaving the secretory flux unchanged. The Isc effect of Ba2+ was inhibited or prevented by tetrodotoxin; D- and L-propranolol; the Cl- channel blocker 4-N-methyl-N-phenylaminothiophene-3-carboxylic acid; the intracellular Ca2+ antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)ocytl ester; noradrenaline, by a yohimbine-sensitive action; somatostatin; HCO3(-)-free solutions. Thus Ba2+ appeared to release a neurotransmitter that gives rise to cAMP synthesis sufficient to turn part of electroneutral HCO3- secretion electrogenic. In a search for the involved signalling pathways, the H1-receptor antagonist, cetirizine, largely and hexamethonium, atropine, atenolol, indomethacin, and trifluoperazine entirely failed to antagonize the Isc effect of Ba2+. Similarly, carbachol, dobutamine, salbutamol, and serotonin were unable to mimic the action of Ba2+ and Isc effects of histamine were small and short-lived. By contrast, vasoactive intestinal peptide (VIP; 3 x 10(-7) mol/l) completely transformed HCO3- secretion into an electrogenic process. The VIP receptor antagonist (4Cl-DPhe6, Leu17) VIP, delayed and reduced the Isc responses to Ba2+ and VIP. As guinea-pig gallbladder epithelial cells possess cAMP-coupled VIP receptors close to VIPergic neurons, Ba2+ is likely to act by releasing VIP from neural terminals.

Effect of neurotensin on the canine gallbladder motility: in vivo and in vitro experiments

Neurotensin (NT) (10 −8–10 −6) exerted a dose-dependent increase in the tone and release of [ 3H]ACh in the guinea-pig gallbladder muscle strips but was inefficient in the canine gallbladder muscle strips. However, in conscious dogs NT (2.5–20 ng/kg intravenously (i.v.)) dose-dependently increased the gallbladder pressure. Similar was the effect of CCK8 (1–10 ng/kg i.v.) and carbachol (0.5–2 μg/kg i.v.). The NT- or CCK8-induced gallbladder pressure was inhibited by atropine (10–50 μg/kg i.v.) or hexamethonium (0.5–3 mg/kg i.v.). Somatostatin (1–2 μg/kg i.v.) or VIP (0.5–1 μg/kg i.v.) also reduced or even abolished the NT- or CCK8-induced gallbladder pressure. The NT-induced increase of the tone of guinea-pig gallbladder preparations was accompanied by an increase of [ 3H]ACh release, suggesting the involvement of cholinergic innervation.

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).

Voltage-dependent calcium channel current in isolated gallbladder smooth muscle cells of guinea pig

The voltage-dependent Ca2+ current was studied in enzymatically dispersed guinea pig gallbladder smooth muscle cells using the whole cell patch-clamp technique. Depolarizing voltage (V) steps induced an inward current (I) that was carried by Ca2+. The threshold potential was -40 to -30 mV, the maximal current was observed at +10 to +20 mV, and the reversal potential was around +80 mV. I-V curves obtained with holding potentials of -80 and -40 mV were not significantly different. This current had a high sensitivity to dihydropyridine drugs, and the Ba2+ or Sr2+ current was larger than the Ca2+ current. Activation was accelerated by increasing the membrane potential. In general, the time course of decay was well fitted by the sum of two exponentials, but consideration of a third (ultra-slow) decay component was also necessary when the current generated by a 2-s command pulse was analyzed. Superimposition of activation and inactivation curves showed the presence of a significant window current. Carbachol suppressed the Ca2+ current only when the pipette contained a low concentration of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. These results show that the L-type Ca2+ current is dominant in gallbladder smooth muscle cells and may contribute to excitation-contraction coupling.

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.

The non-adrenergic/non-cholinergic (NANC) innervation of the guinea pig gallbladder

The non-adrenergic/non-cholinergic (NANC) innervation of the guinea pig gallbladder

2‐Naphthalenesulphonyl l‐aspartyl‐(2‐phenethyl)amide (2‐NAP)‐a selective cholecystokinin CCKA‐receptor antagonist

1. The in vitro pharmacological characterization of the sodium salt of 2-naphthalenesulphonyl 1-aspartyl-(2-phenethyl)amide [2-NAP], a hydrophilic compound derived from the C-terminal aspartate-phenylalanine dipeptide of cholecystokinin (CCK), is described. 2. 2-NAP behaved as a competitive antagonist of sulphated cholecystokinin octapeptide (CCK-8) at CCKA-receptors in both intact tissue bioassays (guinea-pig gall bladder, pancreas and ileum, human and rabbit gall bladder) and a radioligand displacement assay (guinea-pig pancreatic cells). The mean pKB, over assays, was 6.5. 3. Compared to the other assays, the rabbit gall bladder assay gave a significantly higher pKB estimate [7.0] for 2-NAP and a significantly lower estimate [8.9] for devazepide (formerly L-364,718 and MK-329), a well-characterized CCKA-receptor antagonist; these anomalous results suggest that a different class of CCKA-receptors may be involved. 4. 2-NAP, was found to be highly selective, having at least 300 fold greater affinity for CCKA-receptors than for 50 other pharmacological loci, including gastrin/CCKB, as estimated by bioassay or radioligand displacement.

Effects of Ca 2+ channel antagonists in guinea-pig normal and skinned gall bladder

CaCl 2 (0.01–50 mM, in K +-depolarized tissues), KCl (0.1–100 mM) and acetylcholine (1 nM–10 mM) produced concentration-dependent contractions of guinea-pig isolated gall bladder. Nifedipine (1–100 μM), verapamil (1–100 μM), diltiazem (1–100 μM), cinnarizine (1–100 μM), and flunarizine (1–100 μM) each produced a concentration-related inhibition of the log concentration-effect curve for CaCl 2. The rank order of potencies of these antagonists, measured as the IC 50 against Ca 2+ (50 mM)-induced contraction of depolarized gall bladder, was diltiazem (0.25 μM) ⩾ verapamil (0.8 μM)≈ nifedipine (1.2 μM) > cinnarizine (25 μM) ≈ flunarizine (80 μM). Verapamil and diltiazem depressed KCl-induced contraction with an effectiveness and potency similar to those displayed against CaCl 2 but nifedipine, cinnarizine and flunarizine were less effective against contractions in response to KCl compared to CaCl 2. Verapamil and diltiazem, but not the other Ca 2+ channel antagonists tested, had a specific inhibitory effect on the contractions due to KCl when compared to acetylcholine-induced contractions. Cinnarizine (10–100 μM) and flunarizine (10–100 μM), but not the other antagonists tested, depressed Ca 2+ (20 μM)-evoked contraction of skinned guinea-pig gall bladder preparations. It is concluded that distinct differences exist between the Ca 2+ channel antagonists examined. The action of nifedipine, verapamil and diltiazem is restricted to the plasmalemma whereas cinnarizine and flunarizine also act on the intracellular contractile apparatus.

Characterization of leukotriene-induced contraction of the guinea-pig gallbladder in vitro.

Metabolites of arachidonic acid like prostaglandins have an established role in the pathogenesis of gallstone formation and cholecystitis, but any contribution by leukotrienes is less clear. Leukotrienes might contribute to the disease process by contracting the inflamed and (or) obstructed gallbladder, resulting in further inflammatory damage and biliary pain. To better define the role of leukotrienes, we assessed their effects on gallbladder contractility in vitro. Both leukotriene C4 (LTC4) and D4 (LTD4) had a concentration-dependent excitatory effect on guinea-pig gallbladder smooth muscle. The LTD4-receptor antagonist MK-571 (1 microM) competitively depressed the contractile response, to both LTD4 and LTC4. The source of calcium was defined using ryanodine to deplete intracellular calcium stores and nifedinine to block extracellular entry. Ryanodine (10 microM) antagonized gallbladder contraction at low concentrations of LTD4 (10(-10) and 10(-9) M). Nifedipine (1 microM) had a greater inhibitory effect on the contractile response at high concentrations of LTD4 (10(-8)-10(-6) M). LTD4-induced contractions were unaffected in tissues pretreated with the neural blocker tetrodotoxin or the muscarinic antagonist atropine. Thus, leukotrienes act directly on the gallbladder smooth muscle, causing contraction at concentrations found in models of cholecystitis, suggesting that these inflammatory mediators contribute to the symptoms and morbidity associated with gallbladder disease.