Reactive oxygen species are messengers in maintenance of human and guinea pig gallbladder tonic contraction

The tonic contraction of human and guinea pig gallbladder (GB) is dependent on basal levels of PGE(2) and thromboxane A(2) (TxA(2)). The pathway involved in the genesis of these prostaglandins has not been elucidated. We aimed to examine the source of reactive oxygen species (ROS) and whether they contribute to the genesis of GB tonic contraction by generating basal prostaglandin levels. Tonic contraction was studied in human and guinea pig GB muscle strips treated with ROS scavengers (Tiron and catalase), apocynin (an inhibitor of NADPH oxidase), and NOX-1 small interference RNA (siRNA). The subunits of NADPH oxidase and their functional roles were determined with specific antibodies in GB muscle cells. ROS scavengers reduced the GB tonic contraction and H(2)O(2) and PGE(2) levels. Apocynin also inhibited the tonic contraction. Antibodies against subunits of NADPH oxidase present in GB muscle cells lowered H(2)O(2) and PGE(2) levels. NOX-1 siRNA transfection reduced the tonic contraction, NOX-1 expression, and levels of H(2)O(2) and PGE(2). Tiron and apocynin inhibited the expected increase in tension and H(2)O(2) levels induced by stretching of muscle strips. H(2)O(2) increased the levels of PGE(2) and TxA(2) by increasing platelet-activating factor-like lipids that phosphorylate p38 and cPLA(2) sequentially. H(2)O(2) generated by NADPH oxidase participates in a signal transduction pathway that maintains the GB tonic contraction by activating PAF, p38, and cPLA(2) to generate prostaglandins.     

Calcitonin gene-related peptide: an inhibitor of guinea pig gallbladder contraction.

Calcitonin gene-related peptide (CGRP) relaxes vascular and intestinal smooth muscle. This study localized CGRP in the guinea pig gallbladder, examined the effects of CGRP on KCl- and ACh-induced contraction, and determined CGRPs site of action in the gallbladder. The gallbladder of male Hartley guinea pigs was used in in vitro tension studies, radioimmunoassay, or immunocytochemical studies. Radioimmunoassay showed that 8.0 +/- 0.5 pmol/g of immunoreactive CGRP was present. Immunocytochemistry demonstrated that immunoreactive-CGRP nerve fibers occurred around blood vessels, in gallbladder smooth muscle layers, and were associated with ganglia. No immunoreactive cell bodies were observed, even after colchicine treatment. The in vitro tension studies showed that CGRP inhibits either KCl- or acetylcholine-stimulated contraction. CGRP may in part act directly on the gallbladder smooth muscle to inhibit contraction.     

Calcitonin gene related peptide relaxes cholecystokinin-induced contraction in guinea pig gallbladder strips in vitro.

Calcitonin gene related peptide has been shown to relax vascular and intestinal smooth muscle. This study examines the effects of calcitonin gene related peptide on cholecystokinin-induced contraction of guinea pig gallbladder strips in vitro. Calcitonin gene related peptide was found to cause a dose-dependent relaxation of cholecystokinin-induced tension, which was blocked by the calcitonin gene related peptide receptor antagonist human calcitonin gene related peptide. Previous studies demonstrated that calcitonin gene related peptide acted directly on guinea pig gallbladder smooth muscle to inhibit acetylcholine- or KCl-induced contraction. The present results further confirm that calcitonin gene related peptide acts directly on the smooth muscle. In addition, the use of L-NG-nitroarginine methyl ester, glibenclamide, and other agents strongly suggests that calcitonin gene related peptide also acts by way of the nonadrenergic noncholinergic nervous system, to induce the relaxation of cholecystokinin-induced contraction observed in the guinea pig gallbladder strips.     

Prostaglandins do not mediate the actions of cholera toxin on pancreatic acini or gastric chief cells from the guinea pig

Recent reports suggest that prostaglandins, rather than cAMP, play a major role in mediating cholera toxin-induced water and electrolyte secretion from rabbit intestinal loops. We examined the role of prostaglandins in mediating toxin-induced pancreatic and gastric exocrine secretion. In these tissues, indomethacin, a potent inhibitor of prostaglandin synthesis, did not alter the stimulatory effects of cholera toxin on increases in cellular cAMP or enzyme secretion. Moreover, the addition of cholera toxin did not alter prostaglandin E2 release from either tissue. In contrast to their effects in rabbit intestinal loops, prostaglandins do not regulate cholera toxin-induced enzyme secretion from the guinea pig pancreas or stomach.     

Prostaglandins and pacemaker activity in isolated guinea pig SA node

Prostaglandins PGE₂, PGE₁, PGF_2α, and PGA₁ substantially increase automaticity in SA-nodal, right atrial preparations excised from guinea pigs. This natural pacemaker tissue is sensitive to nanomolar doses of PG with, for example, 10⁻⁸ M PGE₂, increasing SA rate by about 20%. If these preparations are pretreated with 2 μM indomethacin, a blocker of endogenous prostaglandin synthesis, then spontaneous rate drops and subsequent rate increases due to PGE₂ administration can be more easily demonstrated. Guinea pig pacemaker tissue differs from similar rabbit tissue not only in that it is directly responsive to PGE₂, but also in that PGE₂ does not depress the absolute response to transmural stimulation (adrenergically mediated rate increase). The positive chronotropic responses to PGE₂ also occur when the guinea pig tissue is pretreated in 0.6 μM propranolol, which causes blockade of beta-adrenergic receptors.The pacemaker myocardium in the guinea pigs thus appears to be directly stimulated by exogenous PGE₂ at very low doses. The observation that 2 μM indomethacin reduces SA-nodal rate suggests the presence of a very sensitive, functionally important, PGE-like system which modulates heart rate in this mammalian species.     

Interrelations between cyclic AMP, cyclic GMP and contraction in guinea pig gallbladder stimulated by cholecystokinin.

The changes in isometric tension and in concentrations of cyclic AMP and cyclic GMP in guinea pig gallbladder muscle induced by the C-terminal octapeptide of cholecystokinin (C8-CCK) were studied before and after the addition of indomethacin 3 × 10^?6 g/ml. The contractile response to the hormone was not affected by indomethacin, nor was the associated decrease in cyclic AMP concentration. However, indomethacin completely prevented the increase in cyclic GMP following addition of C8-CCK. The results suggest that in isolated guinea pig gallbladder, cyclic GMP is not essential for the C8-CCK-induced decrease in cyclic AMP concentration, and that the contractile response induced by the hormone is independent of this nucleotide.     

Cooling-induced contraction of guinea pig tracheal smooth muscle

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.     

Absence of effect of somatostatin on the guinea pig gallbladder

The effect of somatostatin (GH-RIH) on cholecystokinin octapeptide (OP-CCK) or acetylcholine (ACh) induced contraction of the guinea pig gallbladder was evaluated in vitro. GH-RIH failed to inhibit the muscle contraction induced by OP-CCK or ACh. To correlate with the in vitro results, the effect of GH-RIH on OP-CCK induced contraction of the gallbladder was evaluated in the guinea pig in vivo. GH-RIH did not affect the OP-CCK induced contraction of the gallbladder. Our results suggest that GH-RIH does not have direct inhibitory effect on the contraction of the guinea pig gallbladder induced by OP-CCK or ACh.     

Tensiometric study of muscular strips of guinea pig gallbladder

The tensiometric properties of smooth muscle strips from 10 male guinea pig gallbladders were evaluated following acetylcholine (ACH), cholecystokinin octapeptide (CCK-OP), cerulein (CRL) and histamine (HIS) administration. All agonists induced dose-dependent tonic contractions with the maximum effect caused by the octapeptide. CRL showed a 9-folds higher relative potency when compared to CCK-OP. ED50s of agonists were: ACH 1.36 +/- 0.28 SEM microM (n = 14; range 0.20-3.60); HIST, 5.7 +/- 1.9 microM (n = 12; range 1-23); CRL 0.72 +/- 0.15 nM (n = 8; range 0.35-1.07); CCK-OP, 6.77 +/- 1.80 nM (n = 12; range 0.44-20.32); For the same strips, max tension (g), was: 1.97 (SEM 0.12) for ACH; 1.5 (0.18) for HIST; 1.81 (0.18) for CRL; 2.44 (0.14) for CCK-OP. Pretreatment of the strips with atropine (1 microM) completely abolished ACh-induced contractions, without affecting either CCK-OP or CRL responses. The model represents a valid "in vitro" study of different molecules whose action might stimulate, enhance or inhibit the physiological hormonal and non-hormonal effect of the agonists at the level of animal and human gallbladder smooth muscle.     

Catecholamine-containing cells in the nerve plexus of the guinea pig gallbladder

A population of catecholamine-containing cells, broadly belonging to the class of small intensely fluorescent (SIF) cells, was observed in the ganglionated plexus and around blood vessels of the guinea pig gallbladder. Their morphological features were studied by fluorescence and electron microscopy. Some cells were closely associated with ganglion neurons within the ganglionated plexus. Others were clustered into small groups located along blood vessels. Counts carried out on the whole gallbladder showed that these cells varied greatly in number between individuals and that they were most numerous shortly after birth (on average 230 cells). In the adult, their average number was about 30.     

In vitro effect of calcitonin on guinea pig gallbladder

Calcitonin (CT) is a 32 amino acidic polypeptide hormone which has been found in almost all species and whose effects are mainly concerned with calcium and phosphorous homeostasis. Three preparations are employed for therapeutic uses: salmon (sCT), porcine (pCT) and human CT (hCT). The sCT is the most powerful one and in human volunteers a strong relaxing effect has been shown on gallbladder (GB) basal volume and emptying in response to a meal, intraduodenal instillation of a liquid meal and i.v. cholecystokinin (CCK) infusion. Our study was aimed at investigating if a direct sCT effect could be demonstrated on smooth muscle strips from guinea pig GBs "in vitro" (organ bath). Isometric contractions were measured in response to maximal doses of acetylcholine (ACh: 10(-4) M), KCl (80 mM) and cholecystokinin octapeptide (CCK-OP: 10(-6) M), in absence and in presence of four doses of sCT (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) and 1 x 10(-6) M). sCT did not affect the initial strip basal tone. ACh, CCK-OP and KCl caused, as expected, a powerful contraction of the strips, but no effect was shown when each of the sCT doses was administered before ACh (1.28+ 0.69 SEM without sCT vs 1.28g+ 0.69 with sCT; n = 6) and CCK-OP (1.46g+ 0.19 without sCT vs 1.46g+ 0.19 with sCT; n = 8) or 5 min after the induced KCl contraction. On the basis of these preliminary results, we conclude that no evidence of a direct sCT effect was found on guinea pig GBs when considering either basal smooth muscle tone or isometric contraction in response to ACh, KCl and CCK-OP. Further studies are therefore required to clarify the influence of CT on GB dynamics in vivo and to elucidate its the physiological significance.     

Two components of contraction in guinea pig papillary muscle

Biphasic contractions were produced in guinea pig papillary muscle by inducing partial depolarization in a K+ -rich solution (22 mM) containing 10(-6) M isoproterenol. However, when the same conditions were applied to frog and rat, monophasic contractions were obtained. In the case of guinea pig, an increase in the beating frequency produced an increase in amplitude of the first component and a reduction of the second, while in frog and rat, only a decrease in the amplitude of contractions was recorded. Caffeine (10(-3) M) eliminated the first component and increased the second in guinea pig, while in the case of rat and frog it decreased the amplitude of contractions. Procaine (10(-3) M) suppressed the first component and decreased the second one. The contraction in frog appears to be similar to the second component of contraction in guinea pig, while in rat, the contraction is comparable with the first component in guinea pig. It is suggested that the calcium ions which activate the two components of contraction in guinea pig under the given experimental conditions may arise from two different sources.     

Actions of histamine on muscle and ganglia of the guinea pig gallbladder

Histamine is an inflammatory mediator present in mast cells, which are abundant in the wall of the gallbladder. We examined the electrical properties of gallbladder smooth muscle and nerve associated with histamine-induced changes in gallbladder tone. Recordings were made from gallbladder smooth muscle and neurons, and responses to histamine and receptor subtype-specific compounds were tested. Histamine application to intact smooth muscle produced a concentration-dependent membrane depolarization and increased excitability. In the presence of the H(2) antagonist ranitidine, the response to histamine was potentiated. Activation of H(2) receptors caused membrane hyperpolarization and elimination of spontaneous action potentials. The H(2) response was attenuated by the ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide in intact and isolated smooth muscle. Histamine had no effect on the resting membrane potential or excitability of gallbladder neurons. Furthermore, neither histamine nor the H(3) agonist R-alpha-methylhistamine altered the amplitude of the fast excitatory postsynaptic potential in gallbladder ganglia. The mast cell degranulator compound 48/80 caused a smooth muscle depolarization that was inhibited by the H(1) antagonist mepyramine, indicating that histamine released from mast cells can activate gallbladder smooth muscle. In conclusion, histamine released from mast cells can act on gallbladder smooth muscle, but not in ganglia. The depolarization and associated contraction of gallbladder smooth muscle represent the net effect of activation of both H(1) (excitatory) and H(2) (inhibitory) receptors, with the H(2) receptor-mediated response involving the activation of K(ATP) channels.     

Inhibition of arachidonic acid-induced contraction of guinea pig lung strips

The effects of several enzyme inhibitors on arachidonic acid-induced contractions of guinea pig lung strips were studied. Varying concentrations of indomethacin, an inhibitor of cyclooxygenase, produced only a limited effect on contraction of tissue strips. By contrast, nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and phenidone, which inhibit either lipoxygenase, or both lipoxygenase and cyclooxygenase, caused a dose-related antgonism of the arachidonic acid-induced contraction. The effects of these latter agents were similar to that of FPL 55712. Results indicate that the products of cyclooxygenase are predominantly involved in the early phase and the products of lipoxygenase are predominantly related to the late phase of arachidonic acid-induced contraction.     

Effects of bile acids on the muscle functions of guinea pig gallbladder

Hydrophobic bile acids impair gallbladder emptying in vivo and inhibit gallbladder muscle contraction in response to CCK-8 in vitro. This study was aimed at determining the mechanisms of muscle cell dysfunction caused by bile acids in guinea pig gallbladders. Muscle cells were obtained by enzymatic digestion. Taurochenodeoxycholic acid (TCDC), a hydrophobic bile acid, caused a contraction of up to 15% and blocked CCK-induced contraction. Indomethacin abolished the TCDC-induced contraction. Hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had no effect on muscle contraction but prevented the TCDC-induced contraction and its inhibition on CCK-induced contraction. Pretreatment with NADPH oxidase inhibitor PH2I, xanthine oxidase inhibitor allopurinol, and free-radical scavenger catalase also prevented TCDC-induced contraction and its inhibition of the CCK-induced contraction. TCDC caused H2O2 production, lipid peroxidation, and increased PGE2 synthesis and activities of catalase and SOD. These changes were significantly inhibited by pretreatment of PH2I or allopurinol. Inhibitors of cytosolic phospholipase A2 (cPLA2), protein kinase C (PKC), and mitogen-activating protein kinase (MAPK) also blocked the TCDC-induced contraction. It is concluded that hydrophobic bile acids cause muscle cell dysfunction by stimulating the formation of H2O2 via activation of NADPH and xanthine oxidase. H2O2 causes lipid peroxidation and activates cPLA2 to increase PGE2 production, which, in turn, stimulates the synthesis of free-radical scavengers through the PKC-MAPK pathway.