We investigated the mechanisms that underlie the responses to norepinephrine (NE) and thromboxane (Tx) A(2) (TxA2) in the canine pulmonary vasculature with fura 2 fluorimetric, intracellular microelectrode, and force transduction techniques. KCl, caffeine, and cyclopiazonic acid elevated intracellular Ca2+ concentration levels and tone, indicating that Ca2+ mobilization is sufficient to produce contraction. However, contractions evoked by NE or the TxA2 mimetic U-46619 were unaffected by nifedipine or by omitting external Ca2+ and were reduced only partially by depleting the internal Ca2+ store; furthermore, NE-evoked depolarization was subthreshold for voltage-dependent Ca2+ currents. Agonist-evoked contractions were insensitive to inhibitors of protein kinase C (calphostin C and chelerythrine), mitogen-activated protein kinase kinase (PD-98059), and p38 kinase (SB-203580) but were abolished by the tyrosine kinase inhibitor genistein and the Rho kinase inhibitor Y-27632. We conclude that, although Ca2+ influx and Ca2+ release are sufficient for contraction, they are not necessary for adrenergic or TxA2 contractions. Instead, excitation-contraction coupling involves the activation of tyrosine kinase and Rho kinase, leading to enhanced Ca2+ sensitivity of the contractile apparatus.

1 We examined the e ects of several E-ring and F-ring isoprostanes on mechanical activity in pulmonary artery and vein. 2 8-iso PGE 2 and 8-iso PGF 2a were powerful spasmogens in human vasculature and in canine pulmonary vein. 8-iso PGE 1 and 8-iso PGF 2b also exhibited moderate spasmogenic activity in canine pulmonary vein; 8-iso PGF 1a , 8-iso PGF 1b , and 8-iso PGF 3a were generally ine ective. Canine pulmonary arteries did not exhibit excitatory responses to any of the isoprostanes. 3 The spasmogenic e ects of 8-iso PGE 2 were markedly attenuated by the TP-receptor blocker ICI 192605 and by the EP-receptor blocker AH 6809 (7log K B =8.4 and 5.7, respectively). PGE 2 was a very weak agonist (&100 fold less so than 8-iso PGE 2 ). 4 In the presence of ICI 192605 (10 76 M), 8-iso PGE 1 evoked modest dose-dependent relaxations in human and canine pulmonary vein, and in canine pulmonary artery, but not in the human pulmonary artery. The other isoprostanes were generally ine ective as vasodilators in the pulmonary vasculature of both species. 5 The spasmogenic e ects of 8-iso PGE 2 and 8-iso PGF 2a did not involve elevation of [Ca 2+ ] i . 6 8-iso PGE 2 -evoked contractions were blocked by inhibitors of tyrosine kinase (genistein) and Rho kinase (Y 27632 and HA 1077), but not by inhibitors of protein kinase C (calphostin C or chelerythrine), mitogen-activated protein kinase kinase (PD 98059) or p38-kinase (SB 203580). 7 The actions of 8-isoprostanes in the lungs are compound-, species-and tissue-dependent. Several isoprostanes evoke vasoconstriction: in the case of 8-iso PGE 2 , this involves activation of TPreceptors, tyrosine kinases and Rho kinases. 8-iso PGE 1 is also able to cause vasodilation. British Journal of Pharmacology (2001)

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.

We compared the effects of two redox forms of nitric oxide, NO(+) [liberated by S-nitroso-N-acetyl-penicillamine (SNAP)] and NO. [liberated by 3-morpholinosydnonimine (SIN-1) in the presence of superoxide dismutase], on cytosolic concentration of Ca(2+) ([Ca(2+)](i); single cells) and tone (intact strips) obtained from human main stem bronchi and canine trachealis. SNAP evoked a rise in [Ca(2+)](i) that was unaffected by removing external Ca(2+) but was markedly reduced by depleting the internal Ca(2+) pool using cyclopiazonic acid (10(-5) M). Dithiothreitol (1 mM) also antagonized the Ca(2+) transient as well as the accompanying relaxation. SNAP attenuated responses to 15 and 30 mM KCl but not those to 60 mM KCl, suggesting the involvement of an electromechanical coupling mechanism rather than a direct effect on the contractile apparatus or on Ca(2+) channels. SNAP relaxations were sensitive to charybdotoxin (10(-7) M) or tetraethylammonium (30 mM) but not to 4-aminopyridine (1 mM). Neither SIN-1 nor 8-bromoguanosine 3',5'-cyclic monophosphate had any significant effect on resting [Ca(2+)](i), although both of these agents were able to completely reverse tone evoked by carbachol (10(-7) M). We conclude that NO(+) causes release of internal Ca(2+) in a cGMP-independent fashion, leading to activation of Ca(2+)-dependent K(+) channels and relaxation, whereas NO. relaxes the airways through a cGMP-dependent, Ca(2+)-independent pathway.