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 ß-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.

Reduction of arterial graft smooth muscle mass by moderate heat therapy.

Radial artery (RA) graft spasm is a major cause of early graft failure in coronary artery bypass grafting surgeries. We explored the feasibility of thermal reduction of smooth muscle mass to attenuate vasoconstriction. Rat and rabbit femoral arteries were treated thermally in situ (45°C to 65°C; 0 s to 120 s) and then excised at various time points for histological and physiological study (pressure-diameter relationships). Human radial arteries were treated in vitro and studied in similar fashion. Weeks after thermal treatment, no overt indication was noted of vasospasm, thrombosis, or scarring in the arterial wall; however, this intervention led to a thermal dose-dependent reduction of vasoconstriction (to phenylephrine or potassium chloride) and to a conspicuous loss of smooth muscle. Pressure-diameter relationships showed no aneurismal dilation of these demuscularized arteries up to 200 mmHg. Qualitatively identical results were obtained in human radial arteries. Thermal ablation of RAs may provide a simple, safe, and effective solution to postsurgical vasospasm.

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 ß-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.

Isoprostanes constrict human radial artery by stimulation of thromboxane receptors, Ca2+ release, and RhoA activation.

OBJECTIVES: Radial artery vasospasm remains a potential cause of early graft failure after coronary bypass graft surgery, despite pretreatment with alpha-adrenergic or calcium channel blockers. We examined the roles of isoprostanes and prostanoid receptors selective for thromboxane A2 in the vasoconstriction of human radial arteries. METHODS: Human radial arterial segments were pretreated intraoperatively with verapamil/papaverine or nitroglycerine/phenoxybenzamine, or not treated. In the laboratory, we measured isometric contractions in ring segments, vasoconstriction in pressurized segments, and changes in [Ca2+] and K+ currents in single cells. RESULTS: Although phenoxybenzamine eliminated adrenergic responses, the isoprostane 15-F(2t)-IsoP and 2 closely related E-ring molecules (15-E(1t)-IsoP and 15-E(2t)-IsoP) still evoked powerful contractions; 15-E(2t)-IsoP was approximately 10-fold more potent than the other 2 agents. Responses were mediated through thromboxane receptors because they were sensitive to ICI-192605. Furthermore, they were sensitive to the Rho-kinase inhibitors Y-27632 or H-1152 (both 10(-5) mol/L) or to cyclopiazonic acid (which depletes the internal Ca2+ pool), but not to nifedipine. In single cells, 15-E(2t)-IsoP elevated [Ca2+]i and suppressed K+ current. CONCLUSIONS: Isoprostanes accumulate after coronary artery bypass graft surgery, yet none of the currently available antispasm treatments for radial artery grafts is effective against isoprostane-induced vasoconstriction. It is imperative that more specific treatment strategies be developed. We found that isoprostane responses in radial arteries are mediated by prostanoid receptors selective for thromboxane A2 with activation of Rho-kinase and release of Ca2+. Pretreatment of radial artery grafts with Rho-associated kinase inhibitors may potentially reduce postoperative graft spasm. Clinical studies to test this are indicated.

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 microM) ranged from 10 to 100 microM. Genistein (55 microM) reduced KCl-responses by approximately 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.

E-ring isoprostane augments cholinergic neurotransmission in bovine trachealis via FP prostanoid receptors.

Isoprostanes are prostaglandin-like molecules that accumulate in oxidative stress and also exert powerful biological effects on a wide variety of tissues. We investigated the effects of several different isoprostanes on contractions evoked by electrical field stimulation (EFS) in bovine trachealis, finding only 15-E2t-IsoP to augment those responses. Many others have shown that isoprostanes act on prostanoid receptors, usually those of the thromboxane-selective prostanoid receptor (TP) subtype, although some describe actions through prostaglandin E2-selective prostanoid receptor (EP) or less frequently through prostaglandin F2alpha-selective prostanoid receptors (FP). We used an extensive panel of highly selective agonists and antagonists of prostanoid receptors to characterize the ones through which 15-E2t-IsoP was acting here. Pretreatment with the FP-selective AL-8810 significantly inhibited the augmentation, whereas TP- and EP-selective blockers did not. On the other hand, the augmentation exerted by 15-E2t-IsoP was mimicked by submicromolar concentrations of the FP-selective agonists PGF2alpha and fluprostenol, as well as by micromolar concentrations of the TP-selective agonist U46619. The concentration-response relationship for exogenously added acetylcholine was not significantly affected by 15-E2t-IsoP, confirming that the effect of the latter on EFS-evoked responses was exerted prejunctionally (i.e., to enhance release of Ach from nerve endings), rather than a direct postjunctional effect via a receptor on the smooth muscle. Finally, we investigated whether the inhibitory (adrenergic) innervation was also modulated by 15-E2t-IsoP, finding EFS-evoked relaxations to be unaffected by the isoprostane. We conclude that 15-E2t-IsoP acts upon an FP receptor on the cholinergic nerve endings, leading to enhanced neurotransmission.

Pharmacological actions of isoprostane metabolites and phytoprostanes in human and bovine pulmonary smooth muscles.

We examined the responses to various isoprostane derivatives in bovine/human airway and pulmonary arteries. All biological activity of 15-F(2t)-IsoP was lost in its two major metabolites (15-keto-15-F(2t)-IsoP and 13,14-dihydro-15-keto-15-F(2t)-IsoP). We also examined the effects of several metabolites of 15-F(2t)-IsoP synthesized within our own laboratory-both epimers of 2,3-dinor-15-F(2t)-IsoP and of 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP, as well as 20-carboxy-2,3,4,5-tetranor-15 oxo-5,6,13,14-tetrahydro-15-F(2t)-isoP)-finding none of these to have any substantial excitatory effect. Finally, several plant-derived isoprostanes ("phytoprostanes") synthesized within our laboratory elicited little or no excitatory response in these three pulmonary smooth muscle preparations. We conclude that, although isoprostane exhibit powerful constrictor effects on airway and pulmonary vascular smooth muscles, metabolic processing of those isoprostanes essentially abolishes those biological actions; also, the phytoprostanes lack any appreciable pharmacological activity on those smooth muscle preparations.

Isoprostane-induced airway hyperresponsiveness is dependent on internal Ca2+ handling and Rho/ROCK signaling.

We previously reported the ability of isoprostanes to induce airway hyperresponsiveness (AHR). In this study, we examined the signaling mechanisms underlying that phenomenon with the standard muscle bath technique. Responses to a threshold concentration of carbachol (CCh, 3 x 10(-9) M) were significantly augmented by pretreatment for 20 min with 8-isoprostaglandin E(2) (15-E(2t)-IsoP, 10(-6) M): this AHR was obliterated in tissues pretreated with the selective Rho kinase (ROCK) inhibitor Y-27632 added 20 min before isoprostane, but not by cyclopiazonic acid (CPA). Increasing the CCh concentration to 3 x 10(-8) M (still considerably less than the half-maximally effective concentration of CCh) evoked larger contractions that were also augmented significantly by 15-E(2t)-IsoP: this AHR was completely abolished in tissues pretreated with CPA as well as those pretreated with Y-27632. We noted, however, that Y-27632 and CPA profoundly effect baseline tone and the cholinergic response per se, which confounds the interpretation of the data summarized above. We therefore modified the protocol by using combinations of CCh and blocker (CPA, Y-27632, or nifedipine) that were equieffective. In this way, we found that AHR could not be demonstrated under conditions in which Rho/ROCK signaling or Ca(2+) release was abolished (by Y-27632 and CPA, respectively). Likewise, other autacoids that act through G protein-coupled receptors via Rho/ROCK and Ca(2+) release (serotonin, histamine) mimicked this effect of isoprostane, whereas bradykinin did not. We conclude that isoprostane-induced AHR is mediated in part through an action on Rho/ROCK signaling. This novel finding may contribute to a better understanding of the mechanisms underlying AHR and asthma.

Membrane potassium currents in human radial artery and their regulation by nitric oxide donor.

OBJECTIVE: The human radial artery has demonstrated superior long-term results as a graft in coronary bypass surgery, but undesirable post-surgical spasm limits its clinical application. Few have examined its excitatory properties, especially the underlying ion channel mechanisms. In this study, we investigated the kinetic and pharmacological properties of the smooth muscle membrane potassium currents of this important artery. METHODS AND RESULTS: Using whole cell patch-clamp techniques, we found the K(+) current to be voltage-dependent and outwardly rectifying. Voltage-dependent inactivation was observed, being half-maximal at +28.0 mV but incomplete even at +40 mV. The K(+) currents were predominantly sensitive to the K(Ca) blocker tetraethylammonium (TEA; 63.9+/-12.1% inhibition, p<0.05), less sensitive to the Kv blocker 4-aminopyridine (4-AP; 32.8+/-4.4% inhibition, p<0.05), and the K(ATP) blocker glibenclamide (28.7+/-8.5% inhibition), at -20 mV testing potential. Resting membrane potential was -52.0+/-6.8 mV (n=5), and suppression of K(+) currents by TEA and iberiotoxin (IbTx) caused membrane depolarization. Western blot analysis with channel-specific antibodies confirmed the presence of K(Ca) and Kv channel proteins. TEA evoked 20.7+/-9.9% of the contractile response to 60 mM KCl, whereas IbTx caused about 10% of the above response at 10(-7) M. The nitric oxide donor SNAP augmented membrane K(+) currents in a concentration-dependent fashion; the augmentation was completely suppressed by TEA, but was relatively insensitive to the guanylate cyclase inhibitor ODQ. CONCLUSIONS: The radial artery manifests mainly Ca(2+)-dependent K(+) currents at rest; this current is augmented by nitric oxide through a cGMP- and protein kinase G-independent action. The relatively depolarized membrane potential, as well as its muscular structure, predisposes the radial artery to spasm. Agents that activate the Ca(2+)-dependent K(+) current could be of therapeutic value in preventing post-surgical vasospasm.

KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase.

Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 microM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.

Enhanced myosin phosphatase and Ca(2+)-uptake mediate adrenergic relaxation of airway smooth muscle.

We examined the mechanisms underlying relaxations evoked by isoproterenol (Iso) in isolated porcine, bovine, or human tracheal and bronchial tissues (TSM and BSM, respectively). Iso had little effect against contractions evoked by high KCl, indicating that it does not directly suppress voltage-dependent Ca(2+)-influx nor directly inhibit myosin light chain kinase. Furthermore, Iso was equally potent against carbachol (CCh) contractions in the presence versus absence of nifedipine (10(-6) M), establishing that the primary action of Iso is not through membrane hyperpolarization. However, Iso relaxations in porcine/bovine BSM were significantly suppressed by inhibitors of the internal Ca(2+) pump (cyclopiazonic acid; 10(-5) M) or of myosin light chain phosphatase (calyculin; 10(-6) M). Myosin light chain phosphatase activity was assayed directly (using (32)P-labeled myosin) and found to be enhanced in a time- and concentration-dependent fashion by Iso. Iso relaxations in human airway tissues, on the other hand, were not significantly affected by either calyculin or cyclopiazonic acid. Thus, we conclude that Iso acts largely in a voltage-independent fashion: in nonhuman airways, this involves enhanced Ca(2+) pump activity (to decrease [Ca(2+)](i)) and myosin light chain phosphatase activation (to decrease Ca(2+)-sensitivity of the contractile apparatus), whereas in human airways the underlying mechanisms are still unclear.

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 PGF1alpha, 8-iso PGF2alpha and 8-iso PGF2beta) on vasomotor tone in human and porcine bronchial arterial tissues. In the human bronchial arteries, 8-iso PGE2 and 8-iso PGF2alpha 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 PGF2alpha 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.

Involvement of TP and EP3 receptors in vasoconstrictor responses to isoprostanes in pulmonary vasculature.

Although isoprostanes generally act on smooth muscle via TXA(2)-selective prostanoid receptors (TPs), some suggest other prostanoid receptors or possibly even a novel isoprostane-selective receptor might be involved. We studied contractions to several isoprostanes in porcine pulmonary vasculature using organ bath techniques. 8-iso-prostaglandin E(2) (PGE(2)) was the most potent and efficacious of the isoprostanes, with a log EC(50) of -7.0 +/- 0.2 in the pulmonary artery and -6.8 +/- 0.2 in the pulmonary vein. The responses to all the isoprostanes were essentially completely blocked by the TP receptor antagonist ICI 192605 [4(Z)-6-[(2,4,5-cis)2-(2-chlorophenyl)-4-(2-hydroxyphenyl)1,3-dioxan-5-yl]hexenoic acid], and the equilibrium dissociation constants for ICI 192605 competing with U46619 or 8-iso-PGE(2) were both approximately 2 nM, indicating that isoprostane-evoked responses involve primarily TP receptors. Only 8-iso-PGE(2) was able to evoke substantial contractions in the presence of ICI 192605 and only in the pulmonary vein. The EC(50) of these ICI 192605-insensitive responses was -6.1 +/- 0.2. Using a variety of prostanoid agonists, we found the pulmonary vein lacked excitatory PGF(2alpha)-selective prostanoid receptor (FP) or PGD(2)-selective prostanoid receptor (DP) but expressed excitatory EP(3) receptors. The ICI 192605-insensitive responses to 8-iso-PGE(2) were unaffected by the EP(1) antagonist SC-19220 [8-chloro-debenz[b,f][1,4]oxazepine-10(11H)-carboxy-(2-acetyl) hydrazine; 10(-5) M] but were antagonized by the less selective DP/EP(1)/EP(2) antagonist AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; 10(-5) M) or by cyclopiazonic acid (10(-5) M; depletes the internal Ca(2+) store). Our data indicate that, whereas 8-iso-PGE(2) constricts pulmonary vasculature primarily through TP receptors, a substantial portion of this response is also directed through EP(3) receptors or possibly a novel isoprostane receptor.

Silk suture used in standard organ bath studies contracts upon exposure to Krebs buffer.

INTRODUCTION: Muscles of all types are routinely excised and studied under isometric conditions using force transducers in standard organ baths. In such studies, the muscle is stretched or "preloaded," as the magnitudes of the contractions evoked by various stimuli can vary markedly depending on this baseline parameter: many such studies refer to an optimal length and/or preload tension. While calibrating our equipment, we were surprised to find that the silk suture, which is commonly used in such studies, can generate considerable tension of its own, completely independent of any muscle tissue. METHODS: We compared two different types of silk suture in muscle baths using the standard organ bath technique, measuring baseline tension upon addition of various solvents/buffers. RESULTS: One type of silk suture was inert in that, upon stretching to a given degree, there was no important change in preload tension upon addition of bathing medium (Krebs buffer). The second type of silk suture, however, which is also widely available commercially, exhibited substantial contractile responses upon exposure to standard Krebs buffer solution, with magnitudes typically exceeding several grams force. This change developed over the first 30-60 min of exposure to Krebs, after which tension was stable. This change was not observed if the suture was presoaked for 60 min before hanging in the muscle bath, but was observed if the suture was allowed to dry again before use. Other solvents such as ethanol and DMSO did not alter tension. DISCUSSION: While this property of silk suture may be well known in the surgical setting, it is less well appreciated by other users of this material. This phenomenon is of major importance to any experimental study of muscle function, as it alters the preload tension under which such studies are carried out.