Sara Morales

A redox‐based mechanism for the contractile and relaxing effects of NO in the guinea‐pig gall bladder

1 The purpose of this study was to determine the effects of sodium nitroprusside (SNP), 2,2′‐(hydroxynitrosohydrazino)bis‐ethanamine (DETA/NO) and 3‐morpholinosydnonimine (SIN‐1), NO donors which yield different NO reactive species (NO+, NO. and peroxynitrite, respectively), as well as exogenous peroxynitrite, on gall bladder contractility. 2 Under resting tone conditions, SNP induced a dose‐dependent contraction with a maximal effect (10.3 ± 0.7 mN, s.e.m.) at 1 mm. Consistent with these findings, SNP caused a concentration‐dependent depolarization of gall bladder smooth muscle. The excitatory effects of SNP were dependent on extracellular calcium entry through L‐type Ca2+ channels. Furthermore, the contraction and depolarization were sensitive to tyrosine kinase blockade, and an associated increase in tyrosine phosphorylation was detected in Western blot studies. 3 DETA/NO induced dose‐dependent relaxing effects. These relaxations were sensitive to the guanylyl cyclase inhibitor 1H‐[1,2,4]oxidiazolo[4,3‐a]quinoxaline‐1‐one (ODQ, 2 μm) but they were not altered by treatment with the potassium channel blockers tetraethylammoniun (TEA, 5 mm) and 4‐aminopyridine (4‐AP, 5 mm). 4 When tested in a reducing environment (created by 2.5 mm 1,4‐dithiothreitol, DTT), SNP caused a relaxation of gall bladder muscle strips. Similarly, the SNP‐induced contraction was converted to a relaxation, and associated hyperpolarization, when DTT was added during the steady state of an SNP‐induced response. 5 SIN‐1 (0.1 mm), which has been shown to release peroxynitrite, induced relaxing effects that were enhanced by superoxide dismutase (SOD, 50 U ml−1). The relaxations induced by either SIN‐1 alone or SIN‐1 in the presence of SOD were strengthened by catalase (1000 U ml−1) and abolished by ODQ pretreatment. However, exogenous peroxynitrite induced a concentration‐dependent contraction, which was dependent on activation of leukotriene (LT) metabolism and extracellular calcium. The peroxynitrite‐induced contraction was abolished in the presence of the peroxynitrite scavenger melatonin. These results suggest that SIN‐1 behaves as an NO. rather than a peroxynitrite source. 6 We conclude that, depending on the redox state, NO has opposing effects on the motility of the gall bladder, being a relaxing agent when in NO. form and a contracting agent when in NO+ or peroxynitrite redox species form. Knowledge of the contrasting effects of the different redox forms of NO can clarify our understanding of the effects of NO donors on gall bladder and other smooth muscle cell types.

Cyclic AMP-mediated inhibition of gallbladder contractility: role of K+ channel activation and Ca2+ signaling.

We have examined the mechanisms of cAMP‐induced gallbladder relaxation by recording isometric tension and membrane potential in the intact tissue, and global intracellular calcium concentrations ([Ca2+]i) and F‐actin content in isolated myocytes. Both the phosphodiesterase (PDE) inhibitor, IBMX (100 μM) and the adenylate cyclase activator, forskolin (2 μM) caused decreases in basal tone that exhibited similar kinetics. IBMX and forskolin both caused concentration dependent, right‐downward shifts in the concentration–response curves of KCl and cholecystokinin (CCK). IBMX and forskolin elicited a membrane hyperpolarization that was almost completely inhibited by the ATP‐sensitive K+ channel (KATP) channel blocker, glibenclamide (10 μM). IBMX also induced an increase in large‐conductance Ca2+‐dependent K+ (BK) channel currents, although the simultaneous blockade of BK and KATP channels did not block IBMX‐ and forskolin‐induced relaxations. Ca2+ influx activated by L‐type Ca2+ channel activation or store depletion was also impaired by IBMX and forskolin, indicating a general impairment in Ca2+ entry mechanisms. IBMX also decreases [Ca2+]i transients activated by CCK and 3,6‐Di‐O‐Bt‐IP4‐PM, a membrane permeable analog of inositol triphosphate, indicating an impairment in Ca2+ release through IP3 receptors. Ionomycin‐induced [Ca2+]i transients were not altered by IBMX, but the contractile effects of the Ca2+ ionophore were reduced in the presence of IBMX, suggesting that cAMP can decrease Ca2+ sensitivity of the contractile apparatus. A depolymerization of the thin filament could be reason for this change, as forskolin induced a decrease in F‐actin content. In conclusion, these findings suggest that multiple, redundant intracellular processes are affected by cAMP to induce gallbladder relaxation.

Relaxation of canine gallbladder to nerve stimulation involves adrenergic and non‐adrenergic non‐cholinergic mechanisms

Electrical field stimulation (EFS) of dog gallbladder strips induced a frequency‐dependent contractile response followed by an off‐relaxation that was turned into a pure inhibitory response after atropine pretreatment. Guanethidine reduced the atropine‐induced relaxing responses, so an adrenergic mechanism can partially account for the nerve‐mediated gallbladder relaxation. However, guanethidine pretreatment also revealed a nonadrenergic noncholinergic (NANC) relaxation induced by EFS, which was frequency independent. NANC relaxations were reduced by L‐arginine methyl ester (L‐NAME, 100 μmol L–1), a nitric oxide synthase inhibitor (D‐p‐Cl‐Phe6, Leul7; 10 μmol L–1), a vasoactive intestinal peptide (VIP) receptor antagonist, and an inhibitor of haem oxygenase, (copper protoporphyrin IX; CuPP‐IX; 10 μmol L–1), suggesting that nitric oxide (NO), VIP and carbon monoxide (CO), respectively, are released in response to EFS. Immunoreactivities for haem oxygenase‐2 (HO‐2) and VIP, and histochemical staining for NADPH diaphorase were observed in nerve cell bodies and fibres, demonstrating the presence of CO, VIP and NO as putative NANC neurotransmitters in dog gallbladder. These data support the hypothesis that NO, VIP and CO contribute to NANC relaxation of the canine gallbladder.

Contribution of different phospholipases and arachidonic acid metabolites in the response of gallbladder smooth muscle to cholecystokinin

Guinea pig gallbladder muscle strips were used to investigate the contribution of different sources of diacylglicerol (DAG) in the cholecystokinin (CCK)-induced contraction. The involvement of arachidonic acid (AA) in this response was also investigated. Three distinct pathways for DAG production were investigated with specific phospholipase (PL) inhibitors. U-73122 (10 microM) was used for inhibition of phosphoinositide-specific-PLC (PI-PLC), D-609 (100 microM) for phosphatidylcholine specific-PLC (PC-PLC), and propranolol (100 microM) for phospholipase D (PLD). Separate or combined inhibition of each of these enzymes showed that the CCK-induced output of DAG involves the parallel activation of each of these phospholipases. Thus, after inhibition of a PL subtype, the remaining subtypes were able to functionally compensate in mediating CCK-induced contraction. Inhibition of AA production via DAG-lipase or phospholipase A(2) (PLA(2)) was accomplished using RHC-80267 (40 microM), mepacrine (100 microM) and 4-BPB (100 microM). These inhibitors diminished contractile response, indicating that AA is an important modulator of CCK-induced contraction. Indomethacin (10 microM) and nordihydroguaiaretic acid (NDGA, 100 microM), which inhibit subsequent steps in AA metabolism through the cyclooxygenase and 5-lipooxygenase pathways, also inhibited contractions. Taken together, these results show that CCK redundantly activates PC-PLC, PI-PLC and PLD, to produce DAG, which in turn stimulates PKC and provides a substrate for the generation of AA. sPLA(2) is also a source of AA, whose metabolites are, in part, responsible for determining the magnitude of the CCK-evoked contraction.