Long-term effects of losartan on structure and function of the thoracic aorta in a mouse model of Marfan syndrome

Background and purpose:

During development of thoracic aortic aneurysms in a mouse model of Marfan syndrome, upregulation of matrix metalloproteinase (MMP)-2 and -9 was accompanied by compromised aortic constriction and endothelium-dependent relaxation. Losartan has been proposed for the prevention of thoracic aortic aneurysm. We hypothesized that losartan would suppress MMP-2/-9 activation and improve aortic vasomotor function in this model.

Experimental approach:

A well-characterized mouse model of Marfan syndrome (Fbn1^C1039G/+) was used. Starting at 6 weeks old, Marfan mice were untreated or given losartan (0.6 g·L⁻¹ in drinking water, n= 30). The littermate Fbn1^+/+ mice served as control. Thoracic aortas were studied at 3, 6 and 9 months by histology and by contractility assays in isolated segments in vitro.

Key results:

Losartan improved elastic fibre organization and increased aortic breaking stress. Losartan reduced the activity and protein expression of MMP-2 and MMP-9 at all ages. Aortic constriction in response to membrane depolarization or phenylephrine was increased by losartan at 3 and 9 months by 100–200%. Active force of aortic smooth muscle was also increased at 6 and 9 months. Acetylcholine-induced endothelium-dependent relaxation was improved by 30% after 3 months of losartan treatment, but such improvement disappeared with longer duration of treatment, accompanied by reduced phosphorylation of endothelial nitric oxide (NO) synthase^Ser1177, Akt^Thr308 and Akt^Ser473, compared with the control.

Conclusions and implications:

Losartan improved the contractile function of aorta and reduced MMP activation. However, the endothelial NO pathway remained suppressed in the thoracic aorta during losartan treatment, which might limit its long-term benefits in Marfan syndrome.     

Inhibition by picotamide of thromboxane production in vitro and ex vivo

Summary The effect of picotamide on platelet function has been studied in vitro and ex vivo.Picotamide at micromolar concentrations inhibited platelet aggregation induced by ADP, arachidonic acid and collagen, and it also inhibited the production of thromboxane A₂ (TxA₂). Unlike aspirin, picotamide did not affect the synthesis of prostacyclin by blood vessels.In eight healthy subjects who took picotamide 1200 mg/d platelet aggregation and TxA₂ production were inhibited.Picotamide appears to be an antiplatelet drug that reduces TxA₂ synthesis without affecting cyclooxygenase activity.     

NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone, a novel antithrombotic agent with dual inhibition of thromboxane A(2) synthesis and calcium entry

BACKGROUND AND PURPOSE

1,4-Naphthoquinones exhibit antiplatelet activity both in vivo and in vitro. In the present study, we investigated the antiplatelet effect of a novel naphthoquinone derivative NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone and its mechanism of action.

EXPERIMENTAL APPROACH

We measured platelet aggregation, Ca²⁺ mobilization, thromboxane B2 formation and P-selectin expression and examined several enzymatic activities. Furthermore, we used the irradiated mesenteric venules in fluorescein sodium–treated mice to monitor the antithrombotic effect of NP-313 in vivo.

KEY RESULTS

NP-313 concentration-dependently inhibited human platelet aggregation induced by collagen, arachidonic acid, thapsigargin, thrombin and A23187. NP-313 also inhibited P-selectin expression, thromboxane B₂ formation and [Ca²⁺]_i elevation in platelets stimulated by thrombin and collagen. NP-313 at 10 µM inhibited cyclooxygenase, thromboxane A₂ synthase, and protein kinase Cα, whereas it did not affect phospholipase A₂ or phospholipase C activity. In the presence of indomethacin and an adenosine 5-diphosphate scavenger, NP-313 concentration-dependently inhibited thrombin- and A23187-induced [Ca²⁺]_i increase through its inhibitory effects on Ca²⁺ influx, rather than blocking Ca²⁺ release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca²⁺ influx through store-operated calcium channel but had no effect on Ca²⁺ influx through store-independent calcium channel evoked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol. Nevertheless, it had little effect on cyclic AMP and cyclic GMP levels. Also, intravenously administered NP-313 dose-dependently inhibited the thrombus occlusion of the irradiated mesenteric vessels of fluorescein-pretreated mice.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results indicate that NP-313 exerts its antithrombotic activity through dual inhibition of thromboxane A₂ synthesis and Ca²⁺ influx through SOCC.     

Stretch-induced contraction of rabbit isolated pulmonary artery and the involvement of endothelium-derived thromboxane A2

1. The mechanism of stretch-induced contraction of the intrapulmonary artery of rabbit was studied with special regard to the endothelium-dependence and production of prostanoids.
2. Isolated intrapulmonary artery of rabbits in ring form produced contraction when stretched slowly up to 180% of its initial muscle length (=100%) at a rate of 0.44 mm s⁻¹, with a stimulus period of 5 min.
3. The stretch-induced contraction was attenuated by the mechanical removal of the endothelium, inhibitors of cyclo-oxygenase such as aspirin and indomethacin, [1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino) carbonyl] hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ 29,548), which is a thromboxane A₂/prostaglandin H₂ receptor antagonist, or by ozagrel, an inhibitor of thromboxane A₂ synthase.
4. Biochemical assay indicated that the production of thromboxane B₂, a stable metabolite of thromboxane A₂, was increased 17 times in response to stretch only when the endothelium was intact. The production of thromboxane B₂ was also inhibited by aspirin or ozagrel.
5. The production of 6-keto prostaglandin F_1α, a stable metabolite of prostacyclin, was also increased in response to stretch in the preparation with intact endothelium. However, ozagrel showed no apparent effect on the production of 6-keto prostaglandin F_1α.
6. These results suggest that a mechanical stimulus like stretch can act on endothelial cells of rabbit pulmonary artery to cause contraction by activation of arachidonic acid metabolism via the cyclo-oxygenase pathway and subsequent release of thromboxane A₂ and/or an increase in the ratio of thromboxane A₂/prostacyclin.

     

Effects of KP-496, a novel dual antagonist at the cysteinyl leukotriene receptor 1 and the thromboxane A(2) receptor, on airway obstruction in guinea pigs

BACKGROUND AND PURPOSE: KP-496 is a novel dual antagonist for cysteinyl leukotriene receptor 1 (CysLT(1)) and thromboxane A(2) (TXA(2)) receptor (TP). The aim of this study was to evaluate the pharmacological profile of inhaled KP-496 and its effects on airway obstruction.EXPERIMENTAL APPROACH: Antagonist activities of inhaled KP-496 were investigated using bronchoconstriction induced in guinea pigs by LTD(4) or U46619, a stable TXA(2) mimetic. Guinea pigs sensitized with injections of ovalbumin were used to assess the effects of inhaled KP-496 on bronchoconstriction induced by antigen (i.v.). Another set of guinea pigs were sensitized and challenged with ovalbumin by inhalation and the effects of inhaled KP-496 on immediate and late airway responses and airway hyperresponsiveness were investigated.KEY RESULTS: KP-496 significantly inhibited LTD(4)- and U46619-induced bronchoconstriction in a dose-dependent manner. The inhibitory effects of KP-496 (1%) were comparable to those of montelukast (a CysLT(1) antagonist, p.o., 0.3 mg kg(-1)) or seratrodast (a TP antagonist, p.o., 3 mg kg(-1)). KP-496 (1%) and oral co-administration of montelukast (10 mg kg(-1)) and seratrodast (20 mg kg(-1)) significantly inhibited antigen-induced bronchoconstriction, whereas administration of montelukast or seratrodast separately did not inhibit antigen-induced bronchoconstriction. KP-496 exhibited dose-dependent and significant inhibitory effects on the immediate and late airway responses and airway hyperresponsiveness following antigen challenge.CONCLUSIONS AND IMPLICATIONS: KP-496 exerts effects in guinea pigs which could be beneficial in asthma. These effects of KP-496 were greater than those of a CysLT(1) antagonist or a TP antagonist, in preventing antigen-induced airway obstruction.     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate

Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver.     

Involvement of endothelial thromboxane A2 in the vasoconstrictor response induced by 15-E2t-isoprostane in isolated human umbilical vein

The present study was undertaken to evaluate the contractile response of several E- and F-ring isoprostanes (IsoP) in human umbilical vein (HUV) and to investigate the role of the endothelium on the effect of 15-E_2t-IsoP, the most potent vasoconstrictor isoprostane, in human vessels. HUV rings with or without endothelium were suspended in an organ bath for recording the isometric tension in response to different agonists. The inhibitors to be evaluated were applied 30 min before the addition of the agonist. All of the compounds tested produced concentration-dependent contractions when tested on HUV rings with endothelium. Although these compounds were equieffective, significant differences were observed in their potency, with U46619 being the most potent followed by 15-E_2t-IsoP > 15-E_1t-IsoP = 15-F_2t-IsoP > 15-F_1t-IsoP = 9-epi-15-F_2t-IsoP in descending rank order of potency. 15-E_2t-IsoP was the most potent of the isoprostanes evaluated and, therefore, the one employed in the present study. When intact endothelium HUV rings were used, 15-E_2t-IsoP-induced contraction was unaffected by the endothelin-converting enzyme inhibitor, phosphoramidon (10 μM), suggesting that short-term endothelin-1 release is not involved in this response. However, the non-selective cyclooxygenase (COX) inhibitor, indomethacin (10 and 30 μM), and the COX-2 selective inhibitor, NS-398 (3, 10 and 30 μM) produced inhibitory effects on 15-E_2t-IsoP-induced contraction of HUV rings with endothelium. These results indicate that COX-derived contractile prostanoids are involved in this effect. Furthermore, the apparent pK _b values estimated for indomethacin (5.5) and NS-398 (5.4) suggest that the prostanoids involved are derived from the COX-2 isoenzyme pathway. On HUV rings with endothelium, the phospholipase A₂ inhibitor, oleyloxyethyl phosphorylcholine (30 and 100 μM), induced an inhibitory effect on 15-E_2t-IsoP-induced contraction, suggesting that the phospholipase A₂ pathway is also involved in this effect. In addition, the thromboxane A₂ synthase inhibitor furegrelate (10 and 30 μM) also inhibited 15-E_2t-IsoP-induced contraction of HUV rings with endothelium, indicating that thromboxane A₂ is one of the contractile prostanoids involved in this response. Endothelium denudation clearly diminished the vasoconstrictor potency of 15-E_2t-IsoP, demonstrating that the endothelium releases a vasoconstrictor factor in response to 15-E_2t-IsoP. The absence of an inhibitory effect at the highest concentration of furegrelate (30 μM) on 15-E_2t-IsoP-induced contraction of HUV rings without endothelium suggested that endothelium is the source of thromboxane A₂. We conclude that prostanoids derived from the COX-2 isoenzyme pathway participate in 15-E_2t-IsoP-induced vasoconstriction of isolated HUV rings. Our results also indicate that endothelial thromboxane A₂ is one of the prostanoids involved in this effect.     

Single dose pharmacokinetics and effects on platelet function of the thromboxane receptor blocker BM 13.177

Summary The pharmacokinetics and pharmacodynamic effect on platelet activation of a single 800 mg oral dose of BM 13.177 have been investigated in 8 male volunteers. BM 13.177 disappeared from plasma with a terminal elimination half-life of 0.85 h. 52% of the dose was excreted unchanged in urine. Assuming complete absorption, total clearance was calculated to be 741.3 ml/min and renal celearance to range from 310.4 to 396.9 ml/min. The pharmacodynamic studies were performed ex vivo/in vitro in platelets stimulated either with methyl mercury chloride or with U 46619. Methyl mercury chloride is a platelet activator that requires TXA₂ formation from endogenous arachidonic acid, whereas U 46619 is a stable PGH₂ analogue and thromboxane mimetic at the platelet TXA₂/PGH₂ receptor. A close correlation between the plasma concentration-time profile of BM 13.177 and inhibition of platelet shape change or aggregation was demonstrated.     

C2-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta: role of RhoA/Rho-kinase and intracellular Ca2+ concentration

It is known that ceramide may play an important regulatory role in vascular tone although its effect on vascular tone and the mechanisms involved are controversial. The present study was designed to investigate the effects of ceramide and its key initial regulators, TNF-α and neutral sphingomyelinase (SMase), on vascular tone of isolated rat thoracic aortic rings and elucidate the mechanisms involved in the changes in vascular tone induced by ceramide. Contractile responses and Fura-2 Ca²⁺ signals were measured in rat thoracic aortic rings or strips. 10⁻⁵ M C₂-ceramide, 0.1 U/ml neutral sphingomyelinase (SMase), and 5×10⁻⁷ g/ml TNF-α had no effect on resting tone in rat thoracic aortic rings. However, in phenylephrine-induced pre-contracted rings, treatment with ceramide, SMase, and TNF-α evoked a gradual but sustained vasodilation. Vasodilation effect in response to 10⁻⁵ M C₂-ceramide was not significantly changed by the absence or presence of endothelium, a cyclooxygenase pathway inhibitor (10⁻⁶ M indomethacin), or PKC inhibitors (10⁻⁵ M H-7 & 5×10⁻⁷ M calphostin-C). Pretreatment with 1 μM Y-27632, a RhoA/Rho-kinase inhibitor, significantly inhibited the phenylephrine-induced contraction itself as well as the C₂-ceramide-induced vasodilation. Pre-treatment with 10⁻⁵ M C₂-ceramide had no effect on phasic rise in [Ca²⁺]_i and tension evoked by stimulation with 10⁻⁸ M phenylephrine, but post-treatment of C₂-ceramide significantly reduced the phenylephrine-induced secondary tonic [Ca²⁺]_i and tension plateau. Our results indicate that C₂-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta. Furthermore, inhibition of phenylephrine-induced activation of RhoA/Rho-kinase pathway as well as phenylephrine-induced elevations in [Ca²⁺]_i are clearly a key factors in C₂-ceramide-induced vasodilation. G.-J. Jang and D.-S. Ahn contributed equally to this work     

Requirement of Pretone by Thromboxane A(2) for Hypoxic Pulmonary Vasoconstriction in Precision-cut Lung Slices of Rat

Hypoxic pulmonary vasoconstriction (HPV) is physiologically important response for preventing mismatching between ventilation and perfusion in lungs. The HPV of isolated pulmonary arteries (HPV-PA) usually require a partial pretone by thromboxane agonist (U46619). Because the HPV of ventilated/perfused lungs (HPV-lung) can be triggered without pretone conditioning, we suspected that a putative tissue factor might be responsible for the pretone of HPV. Here we investigated whether HPV can be also observed in precision-cut lung slices (PCLS) from rats. The HPV in PCLS also required partial contraction by U46619. In addition, K(+) channel blockers (4AP and TEA) required U46619-pretone to induce significant contraction of PA in PCLS. In contrast, the airways in PCLS showed reversible contraction in response to the K(+) channel blockers without pretone conditioning. Also, the airways showed no hypoxic constriction but a relaxation under the partial pretone by U46619. The airways in PCLS showed reliable, concentration-dependent contraction by metacholine (EC(50), ~210 nM). In summary, the HPV in PCLS is more similar to isolated PA than V/P lungs. The metacholine-induced constriction of bronchioles suggested that the PLCS might be also useful for studying airway physiology in situ.     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Investigation of the different adrenoceptor targets of nebivolol enantiomers in rat thoracic aorta

Background and purpose:

Nebivolol is a highly selective β₁-adrenoceptor antagonist with β₃-adrenoceptor agonist properties and is a racemate mixture of D- and L-enantiomers. However, the cellular mechanisms of the effects of each enantiomer are not yet clear and are a matter for debate. The aim of the present experiments was to determine the adrenoceptors involved in the vascular effects of D- and L-enantiomers of nebivolol in rat thoracic aorta.

Experimental approach:

Responses to nebivolol enantiomers were evaluated in rings of thoracic aorta from male Sprague-Dawley rats.

Key results:

D-nebivolol (0.1–10 µmol·L⁻¹), but not L-nebivolol, significantly shifted to the right the concentration-response curve to phenylephrine, an α₁-adrenoceptor agonist, in a concentration-dependent manner. For the following experiments, aortic rings were constricted with endothelin 1 and now both enantiomers produced an endothelium-dependent relaxation of the rings involving the nitric oxide pathway. This relaxation was not modified by 1 µmol·L⁻¹ CGP 20,712A (β₁-adrenoceptor antagonist), but significantly blunted by 7 µmol·L⁻¹ L-74,8337 (β₃-adrenoceptor antagonist). However, only the vasorelaxation induced by D-nebivolol was significantly reduced by 1 µmol·L⁻¹ ICI 118,551 (β₂-adrenoceptor antagonist).

Conclusions and implications:

Our results suggest that the nebivolol enantiomers act on different targets. D-nebivolol induced vasorelaxation by activating β₂- and β₃-adrenoceptors and antagonizing α₁-adrenoceptors. L-nebivolol induced vasorelaxation by activating only β₃-adrenoceptors in our model. Our results emphasize that nebivolol is a β₁-adrenoceptor antagonist with several important pharmacological differences from other β₁-adrenoceptor antagonists.     

Effect of treatment at night with S-1452, a thromboxane A2 receptor antagonist,on the morning rise in platelet aggregation

Summary It is well known that platelet aggregation shows a morning rise, which may contribute to the increase in the onset of ischaemic heart diseases during the morning period. The present study was undertaken to determine whether nocturnal dosage with S-1452, a thromboxane AZ receptor antagonist, would blunt the morning rise in platelet aggregability.S-1452 50 mg or placebo were given orally to 8 healthy subjects at 10.00 h (day trial) or 22.00 h (night trial) according to a cross-over design. Plasma concentrations of S-1452 and its metabolites, bisnor-( + )-S-145 and tetranor-(+ )-S-145, and platelet aggregation were determined during the 12-hour period following the dose. Mean plasma concentrations of S-1452, bisnor-( + )-S-145 and tetranor-(+ )-S-145 during the absorption phase were lower after the nocturnal dose than after the morning dose. The maximum plasma concentration and area under the plasma concentration-time curve of the compounds were also lower and the time to the maximum concentration were delayed after the treatment at night. A morning rise in platelet aggregation was observed following placebo treatment. The inhibitory effect of S-1452 on platelet aggregation was observed at 3 hours and persisted for up to 9 h in both trials.The results suggest that S-1452 is absorbed more slowly after the nocturnal dose than after the morning dose. However nocturnal treatment with 50 mg S-1452 may blunt the morning rise in platelet aggregability.     

Pathophysiology of isoprostanes in the cardiovascular system: implications of isoprostane-mediated thromboxane A2 receptor activation

Isoprostanes are free radical-catalysed PG-like products of unsaturated fatty acids, such as arachidonic acid, which are widely recognized as reliable markers of systemic lipid peroxidation and oxidative stress in vivo. Moreover, activation of enzymes, such as COX-2, may contribute to isoprostane formation. Indeed, formation of isoprostanes is considerably increased in various diseases which have been linked to oxidative stress, such as cardiovascular disease (CVD), and may predict the atherosclerotic burden and the risk of cardiovascular complications in the latter patients. In addition, several isoprostanes may directly contribute to the functional consequences of oxidant stress via activation of the TxA₂ prostanoid receptor (TP), for example, by affecting endothelial cell function and regeneration, vascular tone, haemostasis and ischaemia/reperfusion injury. In this context, experimental and clinical data suggest that selected isoprostanes may represent important alternative activators of the TP receptor when endogenous TxA₂ levels are low, for example, in aspirin-treated individuals with CVD. In this review, we will summarize the current understanding of isoprostane formation, biochemistry and (patho) physiology in the cardiovascular context.     

Involvement of protein kinase C in reduced relaxant responses to the NO/cyclic GMP pathway in piglet pulmonary arteries contracted by the thromboxane A2-mimetic U46619

1. Impairment of nitric oxide (NO)/cyclic GMP production and/or increased activities of thromboxane A₂ (TXA₂) and endothelin-1 (ET-1) have been associated with pulmonary hypertension. We have analysed the interactions of noradrenaline (NA), the TXA₂-mimetic U46619 and ET-1 with the relaxation induced via cyclic GMP in isolated piglet intrapulmonary arteries.
2. The contractions induced by NA were augmented by endothelium removal or by methylene blue and pre-contracted rings were fully relaxed by acetylcholine, sodium nitroprusside (SNP), atrial natriuretic peptide and 8-bromo-cyclic GMP. In contrast, U46619- and ET-1 induced contractions were endothelium-independent and only partially relaxed by the latter vasodilators. Whereas the reduced responses to SNP in arteries contracted by U46619 were independent of the U46619-induced tone, a higher concentration of ET-1 (tone higher than that induced by NA) was required to reduce the vasodilator responses to SNP. NA, U46619 and ET-1 had no effect on the SNP-induced increases in cyclic GMP.
3. The reduced relaxant responses to SNP in arteries pre-contracted by U46619 were specific for piglet pulmonary arteries since they were not observed in piglet mesenteric or coronary arteries or in rat pulmonary arteries. Furthermore, there were no differences in the relaxant response to the adenylate cyclase activator forskolin in piglet pulmonary arteries pre-contracted by either NA, U46619 or ET-1.
4. SNP-induced relaxation was inhibited by thapsigargin (but not by inhibition of the membrane Na⁺/K⁺ ATPase nor K⁺ channels) indicating a role for Ca²⁺ sequestration by the Ca²⁺ ATPase in the effects of SNP.
5. The phorbol ester 12-myristate, 13-acetate inhibited the relaxant response to SNP. The inhibitory effect of U46619 on SNP-induced relaxation was abolished by the protein kinase C inhibitor (PKC) staurosporine suggesting that PKC may be a part of the signal transduction mechanism.
6. In summary, piglet pulmonary arteries when activated by a TXA₂-mimetic show abnormally reduced relaxant responses to the NO/cyclicGMP pathway. This effect appears to be mediated by activation of PKC.

     

Effects of prostaglandin I2 synthesized in the endothelium and in the smooth muscle on mechanical properties of the canine thoracic aorta

Summary In circular-cut strips prepared from canine thoracic aorta, acetylcholine (ACh) and A23187 relaxed endothelium-intact tissues [E(+) preparations] pre-contracted with noradrenaline or excess concentrations of K. These relaxations were associated with marked increases in the amount of 6-keto PGF₁. After removal of the endothelium [E(–) preparations] the relaxation ceased, and the amounts of 6-keto PGF₁ were markedly reduced. In E(+) preparations, application of indomethacin attenuated the increase in 6-keto PGF₁ induced by ACh or A23187 in the presence of noradrenaline or high K, but not the endothelium-dependent relaxations. In E(–) preparations, ACh (0.1–10 M) neither increased the amount of 6-keto PGF₁ nor produced a contraction. In dispersed single endothelial cells, A23187 markedly increased but 118 mM K did not modify the amount of 6-keto PGF₁. Both noradrenaline and high K increased the production of 6-keto PGF₁ in the E(–) preparations but to a lesser extent than that in the E(+) preparations. This action was attenuated by indomethacin. The amplitude of the noradrenaline-and K-induced contractions was enhanced with indomethacin pretreatment in both E(+) and E(–) tissues. PGI₂-Na (10 nM), reduced the amplitude of noradrenaline-induced contractions, concentration dependently and to the same extent in both E(+) and E(–) preparations. These results indicate that synthesis of PGI₂ in the endothelium is not causally related to the endothelium dependent relaxation. PGI₂ synthesized in the endothelium may not act directly on the muscle tissue, but PGI₂ synthesized in the smooth muscle tissue may produce an inhibition of contraction.     

The effect of inhibition of Ca2+-independent phospholipase A2 on chemotherapeutic-induced death and phospholipid profiles in renal cells

We demonstrate that cells derived from primary cultures of rabbit proximal tubules (RPTC), human embryonic kidney (HEK293) and human kidney carcinomas (Caki-1) express microsomal Ca(2+)-independent phospholipase A(2) (iPLA(2)gamma) and cytosolic Ca(2+)-independent phospholipase A(2) (iPLA(2)beta). Inhibition of iPLA(2) activity in these cells using the iPLA(2) inhibitor bromoenol lactone (BEL) (0-5.0microM) for 24h did not induce cell death as determined by annexin V and propidium iodide (PI) staining. However, BEL treatment prior to cisplatin (50muM) or vincristine (2microM) exposure reduced apoptosis 30-50% in all cells tested (RPTC, HEK293 and Caki-1 cells). To identify the phospholipids altered during cell death electrospray ionization-mass spectrometry and lipidomic analysis of HEK293 and Caki-1 cells was performed. Cisplatin treatment reduced 14:0-16:0 and 16:0-16:0 phosphatidylcholine (PtdCho) 50% and 35%, respectively, in both cell lines, 16:0-18:2 PtdCho in Caki-1 cells and increased 16:1-22:6 plasmenylcholine (PlsCho). BEL treatment prior to cisplatin exposure further decreased 14:0-16:0 PtdCho, 16:0-16:1 PlsCho and 16:0-18:1 PlsCho in HEK293 cells, and inhibited cisplatin-induced increases in 16:1-22:6 PlsCho in Caki-1 cells. Treatment of cells with BEL prior to cisplatin exposure also increased the levels of several arachidonic containing phospholipids including 16:0-20:4, 18:1-20:4, and 18:0-20:4 PtdCho, compared to cisplatin only treated cells. These data demonstrate that inhibition of iPLA(2) protects against chemotherapeutic-induced cell death in multiple human renal cell models, identifies specific phospholipids whose levels are altered during cell death, and demonstrates that alterations in these phospholipids correlate to the protection against cell death in the presence of iPLA(2) inhibitors.     

Adenosine negatively regulates duodenal motility in mice: role of A(1) and A(2A) receptors

BACKGROUND AND PURPOSE

Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved.

EXPERIMENTAL APPROACH

RT-PCR was used to investigate the expression of mRNA encoding for A₁, A_2A, A_2B and A₃ receptors. Contractile activity was examined in vitro as changes in isometric tension.

KEY RESULTS

In mouse duodenum, all four classes of adenosine receptors were expressed, with the A_2B receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; this was antagonized by the A₁ receptor antagonist and mimicked by N⁶-cyclopentyladenosine (CPA), selective A₁ agonist. The relaxation induced by A₁ receptor activation was insensitive to tetrodotoxin (TTX) or N^ω-nitro-l-arginine methyl ester (l-NAME). Adenosine also inhibited cholinergic contractions evoked by neural stimulation, effect reversed by the A₁ receptor antagonist, but not myogenic contractions induced by carbachol. CPA and 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680), A_2A receptor agonist, both inhibited the nerve-evoked cholinergic contractions. l-NAME prevented only the CGS-21680-induced effects. S-(4-Nitrobenzyl)-6-thioinosine, a nucleoside uptake inhibitor, reduced the amplitude of nerve-evoked cholinergic contractions, an effect reversed by an A_2A receptor antagonist or l-NAME.

CONCLUSIONS AND IMPLICATIONS

Adenosine can negatively regulate mouse duodenal motility either by activating A₁ inhibitory receptors located post-junctionally or controlling neurotransmitter release via A₁ or A_2A receptors. Both receptors are available for pharmacological recruitment, even if only A_2A receptors appear to be preferentially stimulated by endogenous adenosine.

LINKED ARTICLE

This article is commented on by Antonioli et al., pp. 1577–1579 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01529.x