The NADPH oxidase inhibitor diphenyleneiodonium is also a potent inhibitor of cholinesterases and the internal Ca(2+) pump.

BACKGROUND AND PURPOSE: Diphenyleneiodonium (DPI) is often used as an NADPH oxidase inhibitor, but is increasingly being found to have unrelated side effects. We investigated its effects on smooth muscle contractions and the related mechanisms. EXPERIMENTAL APPROACH: We studied isometric contractions in smooth muscle strips from bovine trachea. Cholinesterase activity was measured using a spectrophotometric assay; internal Ca(2+) pump activity was assessed by Ca(2+) uptake into smooth muscle microsomes. KEY RESULTS: Contractions to acetylcholine were markedly enhanced by DPI (10(-4) M), whereas those to carbachol (CCh) were not, suggesting a possible inhibition of cholinesterase. DPI markedly suppressed contractions evoked by CCh, KCl and 5-HT, and also unmasked phasic activity in otherwise sustained responses. Direct biochemical assays confirmed that DPI was a potent inhibitor of acetylcholinesterase and butyrylcholinesterase (IC(50) approximately 8 x 10(-6) M and 6 x 10(-7) M, respectively), following a readily reversible, mixed non-competitive type of inhibition. The inhibitory effects of DPI on CCh contractions were not mimicked by another NADPH oxidase inhibitor (apocynin), nor the Src inhibitors PP1 or PP2, ruling out an action through the NADPH oxidase signalling pathway. Several features of the DPI-mediated suppression of agonist-evoked responses (i.e. suppression of peak magnitudes and unmasking of phasic activity) are similar to those of cyclopiazonic acid, an inhibitor of the internal Ca(2+) pump. Direct measurement of microsomal Ca(2+) uptake revealed that DPI modestly inhibits the internal Ca(2+) pump. CONCLUSIONS AND IMPLICATIONS: DPI inhibits cholinesterase activity and the internal Ca(2+) pump in tracheal smooth muscle.

Ryanodine receptors decant internal Ca2+ store in human and bovine airway smooth muscle.

Several putative roles for ryanodine receptors (RyR) were investigated in human and bovine airway smooth muscle. Changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current were investigated in single cells by confocal fluorimetry and patch-clamp electrophysiology, respectively, whereas mechanical activity was monitored in intact strips with force transducers. RyR released Ca2+ from the sarcoplasmic reticulum in a ryanodine- and chloroethyl phenol (CEP)-sensitive fashion. Neither ryanodine nor CEP inhibited responses to KCl, cholinergic agonists or serotonin, indicating no direct role for RyR in contraction; in fact, there was some augmentation of these responses. In tissues pre-contracted with carbachol, the concentration-response relationships for isoproterenol and salmeterol were unaffected by ryanodine; relaxations due to a nitric oxide donor were also largely unaffected. Finally, it was examined whether RyR were involved in regulating [Ca2+]i within the subplasmalemmal space using patch-clamp electrophysiology as well as Ca2+ fluorimetry: isoproterenol increased [Ca2+]i- and Ca2+-dependent K+ current activity in a ryanodine-sensitive fashion. In conclusion, ryanodine receptors in airway smooth muscle are not important in directly mediating contraction or relaxation. The current authors speculate instead that these allow the sarcoplasmic reticulum to release Ca2+ towards the plasmalemma (to unload an overly full Ca2+ store and/or increase the Ca2+-buffering capacity of the sarcoplasmic reticulum) without affecting bronchomotor tone.

Vasodilatory and electrophysiological actions of 8-iso-prostaglandin E2 in porcine coronary artery.

We examined the effects of several E-ring and F-ring isoprostanes on mechanical and electrophysiological activity in porcine coronary artery. Several isoprostanes evoked concentration-dependent contractions, with 8-iso-PGE2 being the most potent (-log EC50 of 6.9 +/- 0.1); this excitatory effect has been described in detail elsewhere and was not examined further here. 8-iso-PGE2 evoked dose-dependent relaxations in tissues preconstricted with the thromboxane A2-agonist U46619 (10(-6) M), with a negative log EC50 of 6.0 +/- 0.1 (n = 5). 8-iso-PGE1 and 8-iso-PGF2 beta also evoked relaxations (albeit with lower potency), whereas the other F-ring isoprostanes (8-iso-PGF1 alpha, 8-iso-PGF1 beta, and 8-iso-PGF2 alpha) were largely ineffective in this respect. The potency and efficacy of 8-iso-PGE2 in reversing tone were not dependent upon the concentration of U46619 used to preconstrict the tissues (10(-8) to 10(-6) M), indicating a lack of U46619-induced functional antagonism of these responses. 8-iso-PGE2 was able to completely relax tissues that had been denuded of endothelium (as indicated by loss of responsiveness to bradykinin). 8-iso-PGE2-evoked relaxations were markedly reduced by elevating the K+ equilibrium potential using 30 mM KCl and abolished by 60 mM KCl; they were also sensitive to charybdotoxin (10(-7) M) but not to 4-aminopyridine (1 mM). 8-iso-PGE2 also caused membrane hyperpolarization and augmentation of outward K+ current. We conclude that 8-iso-prostaglandin E2 acts directly on the smooth muscle to increase K+ conductance, leading to membrane hyperpolarization and vasodilation.

Mechanical and electrophysiological effects of isoprostanes on bronchial arterial smooth muscle.

We investigated the mechanical and electrophysiological responses of human and porcine bronchial arterial smooth muscle to isoprostanes (metabolites of membrane lipid peroxidation). These evoked a constrictor response which was sensitive to blockade of thromboxane receptors, as well as to a non-specific tyrosine kinase inhibitor and an inhibitor of Rho-kinase. The patch clamp technique was used to characterize the K+ and Ca2+ currents in these tissues, and to show that isoprostanes caused a brief enhancement of K+ currents followed by prolonged and marked suppression of the same.

Muscarinic excitation-contraction coupling mechanisms in tracheal and bronchial smooth muscles.

We investigated the mechanisms underlying muscarinic excitation-contraction coupling in canine airway smooth muscle using organ bath, fura 2 fluorimetric, and patch-clamp techniques. Cyclopiazonic acid (CPA) augmented the responses to submaximal muscarinic stimulation in both tracheal (TSM) and bronchial smooth muscles (BSM), consistent with disruption of the barrier function of the sarcoplasmic reticulum. During maximal stimulation, however, CPA evoked substantial relaxation in TSM but not BSM. CPA reversal of carbachol tone persisted in the presence of tetraethylammoium or high KCl, suggesting that hyperpolarization is not involved; CPA relaxations were absent in tissues preconstricted with KCl alone or by permeabilization with beta-escin, ruling out a nonspecific effect on the contractile apparatus. Peak contractions were sensitive to inhibitors of tyrosine kinase (genistein) or Rho kinase (Y-27632). Sustained responses were dependent on Ca(2+) influx in TSM but not BSM; this influx was sensitive to Ni(2+) but not La(3+). In conclusion, there are several mechanisms underlying excitation-contraction coupling in airway smooth muscle, the relative importance of which varies depending on tissue and degree of stimulation.