Capsazepine, a vanilloid antagonist, abolishes tonic responses induced by 20-HETE on guinea pig airway smooth muscle

The aim of this study was to delineate the mode of action of 20-hydroxy-eicosatetraenoic acid (20-HETE) in airway smooth muscle (ASM) cells. ASM metabolizes arachidonic acid by various enzymatic pathways, including the cytochrome P-450 (CYP-450) omega-hydroxylase, which leads to the production of 20-HETE, a bronchoconstrictive eicosanoid. The present study demonstrated that 20-HETE induced concentration-dependent tonic responses in ASM, whereas transient responses were recorded in Ca2+-free solution, suggesting an intracellular Ca2+ release process. 20-HETE inotropic responses were abolished by 36 microM 2-aminoethoxydiphenyl borate or 1 microM thapsigargin but were insensitive to 10 microM ryanodine, indicating that inositol triphosphate receptors likely control the release of intracellular Ca2+. Sustained tension, which required Ca2+ entry, was partially blocked by 1 microM nifedipine (an L-type) and 100 microM Gd3+ (a nonselective cationic channel blocker). Moreover, in the absence of selective 20-HETE receptor antagonists, 20-HETE tonic responses were inhibited in a concentration-dependent manner (0.1-10 microM) by capsazepine, a well-characterized vanilloid receptor antagonist. Capsazepine was also observed to reverse cumulative responses to 20-HETE and capsaicin, a TRPV1 agonist. In addition, capsazepine pretreatment largely modified the sustained inotropic responses to 20-HETE, suggesting that 20-HETE cross-reacted with TRPV1 receptors with a low affinity (microM) or that its specific receptor was inhibited by the vanilloid antagonist. Data obtained using RHC-80267, ONO-RS-082, and eicosatetraynoic acid, respective inhibitors of diacylglycerol-lipase, phospholipase A2, and CYP-450 omega-hydroxylase, reveal that intracellular arachidonic acid production and its 20-HETE metabolite may be responsible for the activation of nonselective cationic channels and tonic responses.     

Effects of 5-oxo-ETE and 14,15-EET on reactivity and Ca2+ sensitivity in guinea pig bronchi

The reactivity and Ca2+ sensitivity of fresh as well as organ-cultured guinea pig bronchi challenged with 5-oxo-ETE and 14,15-EET were compared. Tension measurements, performed on fresh and 3-day cultured bronchi, revealed that the contractile responses to 5-oxo-ETE were largely increased in cultured explants, while 14,15-EET induced larger relaxations on Carbamylcholine (CCh) pre-contracted explants. In fresh bronchi, the contractile responses to 5-oxo-ETE were inhibited by 10 microM indomethacin whereas the relaxing responses induced by 14,15-EET were amplified in the presence of COX inhibitors. COX down expression resulted in a lack of indomethacin effect in cultured explants. One micromolar 5-oxo-ETE increased Ca2+ sensitivity in beta-escin-permeabilized cultured explants, while 1 microM Y-27632 abolished this hypersensitivity. In contrast, 1 microM 14,15-EET significantly reduced the Ca2+ hypersensitivity developed by cultured bronchi. In conclusion, pre-treatment of cultured guinea pig bronchi for 48 h with these eicosanoids modifies the pharmacological responsiveness and Ca2+ sensitivity of these cultured explants.     

Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.     

Platelet activation by tetraprenol via stimulation of phospholipase A2 action

Only tetraprenol (n = 4), among the (n)-polyprenols studied, induced activation of rabbit platelets. Tetraprenol-induced responses, including platelet aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release, were greatly inhibited by a thromboxane A2 (TXA2) receptor antagonist and a cyclooxygenase inhibitor, indicating an essential role for endogenously produced TXA2. The TXA2-mimetic agonist U46619 induced platelet aggregation, Ca2+ mobilization and phospholipase C action but did not induce arachidonic acid release. These results suggest that arachidonic acid is not released via phospholipase C but by phospholipase A2, and this is also supported by the finding that phospholipase C action was inhibited by depletion of extracellular Ca2+, while arachidonic acid release was not. Full arachidonic acid release was found to be induced by the synergistic action of U46619 and tetraprenol. Therefore, the initial, most essential response induced by tetraprenol is a small arachidonic acid release by phospholipase A2, which results in initial TXA2 formation. Further action of phospholipase C as well as Ca2+ mobilization and aggregation were induced by the initially formed TXA2 while further activation of phospholipase A2 required the synergistic action of tetraprenol and TXA2.     

Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets

The action of phospholipases A2 and C in the course of collagen-stimulated platelet activation and the effect of cytochalasins on the responses were studied. Stimulation of human platelets with collagen was accompanied by aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release. However, in the presence of a cyclooxygenase inhibitor or a thromboxane A2 (TXA2) receptor antagonist, collagen induced only weak arachidonic acid release and weak inositol phosphate formation. The TXA2 mimetic agonist U46619 induced all the responses except for arachidonic acid release, which was induced by synergistic action of collagen and U46619. The result that U46619 did not induce arachidonic acid release despite the activation of phospholipase C suggested that arachidonic acid was not released via phospholipase C but by phospholipase A2. These findings suggested that collagen initially induced weak activation of phospholipases A2 and C and that further activation of phospholipase C as well as Ca2+ mobilization and aggregation were induced by TXA2, whereas further activation of phospholipase A2 required the synergistic action of collagen and TXA2. Platelets pretreated with cytochalasins did not respond to collagen. Further analysis revealed that the initial activation of phospholipases A2 and C was specifically inhibited by cytochalasins, but the responses induced by U46619 or a synergistic action of collagen and U46619 were not inhibited. Therefore, we proposed that interaction of collagen receptor with actin filaments might have some roles in the collagen-induced initial activation of phospholipases.     

Relaxing effects of 5-oxo-ETE on human bronchi involve BK Ca channel activation

The present study investigated the ability of 5-oxo-EicosaTetraEnoic acid (5-oxo-ETE) for modulating airway smooth muscle (ASM) tone in human bronchi. 5-Oxo-ETE induced a concentration-dependent relaxing effect on human bronchi pre-contracted with methacholine (MCh) and arachidonic acid (AA). This relaxing response was highly sensitive to Iberiotoxin (IbTx), a large conducting Ca(2+)-activated K(+) channel (BK(Ca)) inhibitor. Furthermore, microelectrode measurements revealed that 5-oxo-ETE (0.1-10 microM) hyperpolarizes the membrane potential of human bronchial ASM cells. These hyperpolarizing effects were also inhibited in the presence of 10nM IbTx. Lastly, 5-oxo-ETE was shown to directly activate reconstituted BK(Ca) channels derived from human airway smooth muscles. In summary, the 5-oxo-ETE eicosanoid activates a specific K(+) conductance, involved in membrane hyperpolarization, which in turn reduces Ca(2+) entry and facilitates relaxation of smooth muscle cells.     

U-46619 but not serotonin increases endocannabinoid content in middle cerebral artery: evidence for functional relevance

Cerebral vascular smooth muscle cells express the CB(1) cannabinoid receptor, and CB(1) receptor agonists produce vasodilation of cerebral arteries. The purpose of this study was to determine whether vasoconstriction of rat middle cerebral artery (MCA) results in the local formation of endocannabinoids (eCBs), which, via activation of CB(1) receptors, oppose the vasoconstriction in a feedback manner. The thromboxane A(2) (TXA(2)) mimetic U-46619 significantly increased N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol (2-AG) content of isolated MCA, whereas 5-hydroxytrypamine (5-HT) decreased AEA and 2-AG content. If eCBs play a feedback role in the regulation of MCA tone, then CB(1) receptor antagonists should enhance the constriction of MCA produced by U-46619 but not 5-HT. U-46619 caused concentration-dependent constrictions of endothelium-denuded MCA. Two CB(1) receptor antagonists SR-141716 and AM-251 decreased the EC(50) value for U-46619 to constrict endothelium-denuded MCA without affecting the maximal effect. A low concentration of CB(1) receptor agonist Win-55212-2 (30 nM) produced vasodilation of MCAs constricted with low but not saturating concentrations of U-46619. SR-141716 had no effect on the 5-HT concentration-contraction relationship. These data suggest that TXA(2) receptor activation increases MCA eCB content, which, via activation of CB(1) receptors, reduces the constriction produced by moderate concentrations of the TXA(2) agonist. Although 5-HT-induced vasoconstriction is reduced by exogenous CB(1) receptor agonist, activation of 5-HT receptors does not increase eCB content. These results suggest that MCA production of eCBs is not regulated by constriction per se but likely via a signaling pathway that is specific for TXA(2) receptors and not 5-HT receptors.     

Pharmacological evaluation of both enantiomers of (R,S)-BM-591 as thromboxane A2 receptor antagonists and thromboxane synthase inhibitors

The aim of this work is to evaluate the anti-thromboxane activity of two pure enantiomers of (R,S)-BM-591, a nitrobenzene sulfonylurea chemically related to torasemide, a loop diuretic. The drug affinity for thromboxane A2 receptor (TP) of human washed platelets has been determined. In these experiments, (R)-BM-591 (IC50 = 2.4+/-0.1 nM) exhibited a significant higher affinity than (S)-BM-591 (IC50 = 4.2+/-0.15 nM) for human washed platelets TP receptors. Both enantiomers were stronger ligands than SQ-29548 (IC50 = 21.0+/-1.0 nM) and sulotroban (IC50 = 930+/-42 nM), two reference TXA2 receptor antagonists. Pharmacological characterisations of (S)-BM-591 and (R)-BM-591 were compared in several models. Thus, (R)-BM-591 strongly prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) and U-46619 (1 microM) while (S)-BM-591 showed a lower activity. On isolated tissues pre-contracted by U-46619, a stable TXA2 agonist, (S)-BM-591 was more potent in relaxing guinea-pig trachea (EC50 = 0.272+/-0.054 microM) and rat aorta (EC50 = 0.190+/-0.002 microM) than (R)-BM-591 (EC50 of 9.60+/-0.63 microM and 0.390+/-0.052 microM, respectively). Moreover, at 1 microM, (R)-BM-591 totally inhibited TXA2 synthase activity, expressed as TXB2 production from human platelets, while at the same concentration, (S)-BM-591 poorly reduced the TXB2 synthesis (22%). Finally, in rats, both enantiomers lost the diuretic activity of torasemide. In conclusion, (R)-BM-591 exhibits a higher affinity and antagonism on human platelet TP receptors than (S)-BM-591 as well as a better thromboxane synthase inhibitory potency. In contrast, (S)-BM-591 is more active than the (R)-enantiomer in relaxing smooth muscle contraction of rat aorta and trachea guinea pig. Consequently, (R)-BM-591 represents the best candidate for further development in the field of thrombosis disorders.     

20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 microM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 microM Gd3+ or 1 microM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 microM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 +/- 2mV(n = 7), as did 30 microM 1-oleoyl-2-acetyl-sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 microM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.     

Activity of a novel dual thromboxane A(2)receptor antagonist and thromboxane synthase inhibitor (BM-573) on platelet function and isolated smooth muscles.

The pharmacomodulation of sulfonylureas structurally related to torasemide and characterized by a TXA(2)antagonism led to the synthesis of BM-573. This original molecule showed a high affinity (IC(50)1.3 nM) for the TXA(2)receptor of human platelets in comparison with both reference compounds, SQ-29548 (IC(50)21 nM) and sulotroban (IC(50)930 nM). Moreover, this torasemide derivative was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid (ED(100)=0.13 microM) or by U-46619 (ED(50)=0.24 microM), a TXA(2)agonist. BM-573 relaxed the isolated rat thoracic aorta (ED(50)=28.4 nM) and guinea-pig trachea (ED(50)=17.7 nM) contracted by U-46619. BM-573 (1 microM) completely reduced the platelet production of TXB(2)induced by arachidonic acid. Finally, BM-573 (30 mg/kg, per os) lost the diuretic properties of torasemide in rats.     

Cellular mechanisms of thromboxane A2-mediated contraction in pulmonary veins

Our objectives were to identify the relative contributions of [Ca2+]i and myofilament Ca2+ sensitivity in the pulmonary venous smooth muscle (PVSM) contractile response to the thromboxane A2 mimetic U-46619 and to assess the roles of PKC, tyrosine kinases (TK), and Rho-kinase (ROK) in that response. We tested the hypothesis that U-46619-induced contraction in PVSM is mediated by both increases in [Ca2+]i and myofilament Ca2+ sensitivity and that the PKC, TK, and ROK signaling pathways are involved. Isometric tension was measured in isolated endothelium-denuded (E-) canine pulmonary venous (PV) rings. In addition, [Ca2+]i and tension were simultaneously measured in fura-2-loaded E- PVSM strips. U-46619 (0.1 nM-1 microM) caused dose-dependent (P < 0.001) contraction in PV rings. U-46619 contraction was attenuated by inhibitors of L-type voltage-operated Ca2+ channels (nifedipine, P < 0.001), inositol 1,4,5-trisphosphate-mediated Ca2+ release (2-aminoethoxydiphenylborate, P < 0.001), PKC (bisindolylmaleimide I, P < 0.001), TK (tyrphostin A-47, P = 0.014), and ROK (Y-27632, P = 0.008). In PV strips, U-46619 contraction was associated with increases in [Ca2+]i and myofilament Ca2+ sensitivity. Both Ca2+ influx and release mediated the early transient increase in [Ca2+]i, whereas the late sustained increase in [Ca2+]i only involved Ca2+ influx. Inhibition of both PKC and ROK (P = 0.006 and P = 0.002, respectively), but not TK, attenuated the U-46619-induced increase in myofilament Ca2+ sensitivity. These results suggest that U-46619 contraction is mediated by Ca2+ influx, Ca2+ release, and increased myofilament Ca2+ sensitivity. The PKC, TK, and ROK signaling pathways are involved in U-46619 contraction.     

Effects of U-46619 on pulmonary hemodynamics before and after administration of BM-573, a novel thromboxane A2 inhibitor

We studied the effects on pulmonary hemodynamics of U-46619, a thromboxane A2 (TXA2) agonist, before and after administration of a novel TXA2 receptor antagonist and synthase inhibitor (BM-573). Six anesthetized pigs (Ago group) received 6 consecutive injections of U-46619 at 30-min interval and were compared with six anesthetized pigs (Anta group) which received an increasing dosage regimen of BM-573 10 min before each U-46619 injection. Consecutive changes in pulmonary hemodynamics, including characteristic resistance, vascular compliance, and peripheral vascular resistance, were continuously assessed during the experimental protocol using a four-element Windkessel model. At 2 mg/kg, BM-573 completely blocked pulmonary hypertensive effects of U-46619 but pulmonary vascular compliance still decreased. This residual effect can probably be explained by a persistent increase in the tonus of the pulmonary vascular wall smooth muscles sufficient to decrease vascular compliance but not vessel lumen diameter. Such molecule could be a promising therapeutic approach in TXA2 mediated pulmonary hypertension as it is the case in pulmonary embolism, hyperacute lung rejection and endotoxinic shock.     

5,6-EET-induced contraction of intralobar pulmonary arteries depends on the activation of Rho-kinase.

The mechanism mediating epoxyeicosatrienoic acid (EET)-induced contraction of intralobar pulmonary arteries (PA) is currently unknown. EET-induced contraction of PA has been reported to require intact endothelium and activation of the thromboxane/endoperoxide (TP) receptor. Because TP receptor occupation with the thromboxane mimetic U-46619 contracts pulmonary artery via Rho-kinase activation, we examined the hypothesis that 5,6-EET-induced contraction of intralobar rabbit pulmonary arteries is mediated by a Rho-kinase-dependent signaling pathway. In isolated rings of second-order intralobar PA (1-2 mm OD) at basal tension, 5,6-EET (0.3-10 microM) induced increases in active tension that were inhibited by Y-27632 (1 microM) and HA-1077 (10 microM), selective inhibitors of Rho-kinase activity. In PA in which smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased with KCl (25 mM) to produce a submaximal contraction, 5,6-EET (1 microM) induced a contraction that was 7.0 +/- 1.6 times greater than without KCl. 5,6-EET (10 microM) also contracted beta-escin permeabilized PA in which [Ca(2+)](i) was clamped at a concentration resulting in a submaximal contraction. Y-27632 inhibited the 5,6-EET-induced contraction in permeabilized PA. 5,6-EET (10 microM) increased phosphorylation of myosin light chain (MLC), increasing the ratio of phosphorylated MLC/total MLC from 0.10 +/- 0.03 to 0.30 +/- 0.02. Y-27632 prevented this increase in MLC phosphorylation. These data suggest that 5,6-EET induces contraction in intralobar PA by increasing Rho-kinase activity, phosphorylating MLC, and increasing the Ca(2+) sensitivity of the contractile apparatus.     

Effects of a novel non-carboxylic thromboxane A2 receptor antagonist (BM-531) derived from torasemide on platelet function

In this study we examined the thromboxane A(2)(TXA(2)) receptor antagonist property of BM-531 (N-tert -butyl- N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on platelet function. The drug affinity for human washed platelet TXA(2)receptors labelled with [(3)H]SQ-29,548 has been determined (IC50: 0.0078 microM) and demonstrated to be higher than sulotroban (IC50: 0.93 microM) and SQ-29,548 (IC50: 0.021 microM). The antiaggregatory potency has been confirmed since we demonstrated that BM-531 prevented platelet aggregation in human citrated platelet-rich plasma induced by arachidonic acid (600 microM) (ED100: 0.125 microM), U-46619, a stable TXA(2)agonist (1 microM) (ED50: 0.482 microM) and collagen (1 microg mL(-1)) (% of inhibition: 42.9% at 10 microM) and inhibited the second wave of ADP (2 microM). Moreover, when BM-531 was incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100(trade mark)) was significantly prolonged. These results suggest that BM-531 can be regarded as a novel non-carboxylic TXA(2)antagonist with a powerful antiplatelet potency.     

Thromboxane receptor signalling in human myometrial cells.

We measured the effects of stable thromboxane A2 (TXA2) analogues on signalling in cultured human myometrial cells. U46619 and/or IBOP stimulated total inositol phosphates (IPs) and cAMP production, RhoA-associated protein kinase (ROK) activity and elevated intracellular calcium [Ca2+]i. Pretreatment of the cells with pertussis toxin did not inhibit IPs or [Ca2+]i production but the thromboxane receptor (TP) antagonist SQ-29548 did inhibit IPs and cAMP production, the elevation of [Ca2+]i, and the increase in ROK activity. Pretreatment with thapsigargin inhibited [Ca2+]i elevation. TP receptor-stimulated ROK activity was inhibited by the ROK inhibitor Y27632 while ROK activity was enhanced by the caspase 3 inhibitor, Z-DEVD-FMK. TP receptor-stimulated IPs production is additive to prostaglandin F2alpha (FP) or prostaglandin E (EP) receptor-stimulated IPs production and neither FP nor EP receptor-stimulated IPs production is inhibited by SQ29548. Thus cultured human myometrial cells express at least two functional TP receptor subtypes; TPalpha-like (cAMP-stimulating) and TPbeta-like (IPs, [Ca2+] and ROK-stimulating).     

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.     

Diabetes alters the reactivity of myocardium to a thromboxane analogue

Atrial contractile response to U-46619 was studied in auricles from normal and acutely diabetic rats. U-46619 induced an increment of dF/dt in diabetic atria, whereas nondiabetic auricles elicited a negative contractile effect. Blockers of arachidonic acid metabolism via cyclooxygenase inhibited the stimulatory action of U-46619. The stimulant action of the thromboxane A2 mimetic was attenuated when diabetic auricles were incubated with lipoxygenase(s) blocking agents. Results suggest that in diabetic atria, the abnormal inotropic effect induced by U-46619 may be associated with thromboxane formation and with lipoxygenase(s) metabolites.     

Extracellular signal-regulated kinase1/2 in contraction of vascular smooth muscle

Smooth muscle contractility is regulated by both intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ sensitivity of the contractile apparatus. Extracellular signal-regulated kinases1/2 (ERK1/2) have been implicated in modulating Ca2+ sensitivity of smooth muscle contraction but mechanisms of action remain elusive. This study investigated the roles of ERK1/2 in modulating [Ca2+]i, calcium sensitivity and the 20-kDa myosin light chain (MLC20) phosphorylation during contraction activated by alpha1-adrenoceptor agonist phenylephrine and thromboxane A2 mimetic U46619 in rat tail artery strips. A specific inhibitor for ERK1/2 activation, U0126, inhibited phenylephrine- and U46619-induced contraction, shifting both concentration-response curves rightward. During phenylephrine-stimulated contraction, U0126 exhibited concentration-dependent inhibition towards force but significant decreases in [Ca2+]i were detected only at higher concentration. Both phenylephrine and U46619 induced a transient activation of ERK1/2 which was abolished by U0126 but unaffected by a general tyrosine kinase inhibitor genistein or Rho kinase inhibitor Y27632 at concentrations inhibiting more than 50% force. Interestingly, U0126 had no effect on steady-state MLC20 phosphorylation levels stimulated by both receptor agonists. These results indicated that during contraction of rat tail artery smooth muscle activated by alpha1-adrenoceptor agonist or thromboxane A2 analogue, ERK1/2 increase Ca2+ sensitivity that does not involve the modulation of MLC20 phosphorylation.     

Mechanism of the negative inotropic effects of alpha 1-adrenoceptor agonists on mouse myocardium

This study was done to identify the mechanism of the alpha1-adrenoceptor (AR) mediated negative inotropic effects of phenylephrine (PE) on adult mouse myocardium. As reported by others, we also found that the nonselective alpha1AR agonist PE produced a negative inotropic effect on ventricular strips from adult mice that was inhibited by the alpha1AAR antagonist 5-methylurapidil (5MU) but not by the alpha1BAR antagonist chloroethylclonidine (CEC) or the alpha1DAR antagonist BMY 7378. The selective alpha1AAR agonist A61603 also produced a negative inotropic effect, which was antagonized by 5MU. Phorbol 12,13-dibutyrate (activator of all PKC isoforms) mimicked the negative inotropic responses to PE and A61603. The negative inotropic effects of PE were inhibited by bisindolylmaleimide (inhibitor of all PKC isoforms) but not by G? 6976 (inhibitor of Ca2+-dependent PKC). Rottlerin, an inhibitor of Ca2+-independent PKCdelta, antagonized the negative inotropic effects of PE and A61603. PE and A61603 increased the translocation of PKCdelta, which was prevented by rottlerin. These data suggest that the alpha1AR-mediated negative inotropy on adult mouse myocardium is signaled by Ca2+-independent PKCdelta.     

Agonist-induced cyclic ADP ribose production in airway smooth muscle

Cyclic ADP-ribose (cADPR) triggers sarcoplasmic reticulum (SR) Ca(2+) release in airway smooth muscle (ASM). SR Ca(2+) release is an important component of the intracellular Ca(2+) ([Ca(2+)](i)) response of ASM to agonists. Whether cADPR is endogenously produced in ASM during agonist stimulation has not been established. In this study, cADPR production was examined in acutely dissociated porcine ASM cells. ACh stimulation (> or = 1 microM) significantly increased cADPR levels, peaking between 30s and 1 min. This effect was inhibited by M(2) and M(3) muscarinic receptor antagonists. Histamine ((> or = 5 microM) increased cADPR levels to a greater extent than ACh, while diphenhydramine blocked histamine-induced cADPR elevation. Both bradykinin (100 nM) and endothelin-1 (100 nM) also increased cADPR levels to a greater extent than ACh or histamine. These results indicate that in porcine ASM, certain agonists acting via receptors increase cADPR levels. Furthermore, the extent of cADPR responses to agonist varies, possibly reflecting differences in G-protein coupling.