Tracy Tazzeo

Vasoconstrictor responses, and underlying mechanisms, to isoprostanes in human and porcine bronchial arterial smooth muscle.

We investigated the effects of five different isoprostanes (8‐iso PGE1, 8‐iso PGE2, 8‐iso PGF1α, 8‐iso PGF2α and 8‐iso PGF2β) on vasomotor tone in human and porcine bronchial arterial tissues. In the human bronchial arteries, 8‐iso PGE2 and 8‐iso PGF2α evoked powerful constrictions (magnitudes several fold greater than the responses to high millimolar KCl) with negative log concentration causing 50% excitation (EC50) values of 6.8 and 6.5, respectively; 8‐iso PGE1 was less potent (EC50 not calculated, since a clear peak contraction was not obtained), while the other isoprostanes were largely ineffective. In the porcine arteries, on the other hand, all three F‐ring isoprostanes as well as 8‐iso PGE2 evoked constrictor responses, although the peak magnitudes were approximately 50% of the KCl‐evoked response; 8‐iso PGE2 and 8‐iso PGF2α were the most potent, with negative log EC50 values of 6.5. We next sought to characterize the signaling pathways underlying the vasoconstrictor responses to 8‐iso PGE2, since this was the most potent of the isoprostanes we tested. These responses were largely reversed by the thromboxane A2‐selective (TP) prostanoid receptor antagonist ICI 192605 (10−8 M; 4(Z)‐6‐[(2,4,5 cis)2‐(2‐chlorophenyl)‐4‐(2‐hydroxy phenyl)1,3‐dioxan‐5‐yl]hexenoic acid) as well as by the nonspecific tyrosine kinase inhibitor genistein (10−5 and 10−4 M), and were reversed approximately 50% by the Rho‐kinase inhibitor Y27632 (10−5 M; (+)‐(R)‐trans‐4‐(1‐aminoethyl)‐N‐(pyridyl) cyclohexanecarboxamide dihydrochloride). We conclude, therefore, that 8‐iso PGE2 constricts bronchial vasculature through the activation of TP receptors, which in turn trigger tyrosine kinase and Rho‐kinase activities, resulting in powerful vasoconstriction. These findings are highly relevant to lung transplantation and to exercise‐induced asthma.

The NADPH oxidase inhibitor diphenyleneiodonium is also a potent inhibitor of cholinesterases and the internal Ca2+ 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.

Acute response of airway muscle to extreme temperature includes disruption of actin-myosin interaction.

Despite the emerging use of bronchial thermoplasty in asthma therapy, the response of airway smooth muscle (ASM) to extreme temperatures is unknown. We investigated the immediate effects of exposing ASM to supraphysiologic temperatures. Isometric contractions were studied in bovine ASM before and after exposure to various thermal loads and/or pharmacologic interventions. Actin-myosin interactions were investigated using a standard in vitro motility assay. We found steep thermal sensitivity for isometric contractions evoked by acetylcholine, with threshold and complete inhibition at less than 50°C and greater than 55°C, respectively. Contractile responses to serotonin or KCl were similarly affected, whereas isometric relaxations evoked by the nitric oxide donor S-nitrosyl-N-acetylpenicillamine or the β-agonist isoproterenol were unaffected. This thermal sensitivity developed within 15 minutes, but did not evolve further over the course of several days (such a rapid time-course rules out heat shock proteins, apoptosis, autophagy, and necrosis). Although heat-sensitive transient receptor potential (TRPV2) channels and the calmodulin-dependent (Cam) kinase-II-induced inactivation of myosin light chain kinase are both acutely thermally sensitive, with a temperature producing half-maximal effect (T(1/2)) of 52.5°C, the phenomenon we describe was not prevented by blockers of TRPV2 channels (e.g., ruthenium red, gadolinium, zero-Ca(2+) or zero-Na(+)/zero-Ca(2+) media, and cromakalim) or of Cam kinase-II (e.g., W7, trifluoperazine, and KN-93). However, direct measurements of actin-myosin interactions showed the same steep thermal profile. The functional changes preceded any histologic evidence of necrosis or apoptosis. We conclude that extreme temperatures (such as those used in bronchial thermoplasty) directly disrupt actin-myosin interactions, likely through a denaturation of the motor protein, leading to an immediate loss of ASM cell function.

Caffeine relaxes smooth muscle through actin depolymerization

Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using β-escin and depleting the internal Ca(2+) store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca(2+), suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle.

Role of tyrosine phosphorylation in U46619-induced vasoconstriction of pulmonary vasculature and its modulation by genistein, daidzein, and equol.

We compared the effects of genistein with its structural derivatives daidzein and equol on excitation of pulmonary artery and vein. The concentration of genistein necessary to inhibit contractions evoked by U46619 (1nM-100 uM) ranged from 10 to 100 μM. Genistein (55 μM) reduced KCl-responses by ∼50% and essentially abolished those evoked by U46619. Daidzein was much less effective against either agonist, and equol was ineffective against U46619. A23187-evoked contractions were markedly reduced by all 3 isoflavones, but caffeine-evoked contractions were not. Using the Western blot technique, we found many proteins were tyrosine phosphorylated within 30 seconds after stimulation with U46619, reaching a peak at 120 seconds and then falling at 300 seconds. One band at 110 kD was increased nearly 300% above baseline, while 3 others ranging from 60 to 80 kD were more than doubled in intensity. Genistein had little effect on baseline levels of phosphorylation but largely prevented the U46619-induced change; daidzein was much less effective in this respect, and equol did not significantly affect this phosphorylation. We conclude that these isoflavones provide powerful tools in the study of excitation-contraction coupling of pulmonary vasculature and that inhibition of tyrosine kinase activity may be useful clinically against pulmonary hypertension.