Release Of Cyclooxygenase Products Research Articles

Endothelial Connexin37 and Connexin40 participate in basal but not agonist-induced NO release.

BackgroundConnexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown.ResultsCx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40+/− aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37−/− and Cx40−/− aortas but not in Cx40+/− aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40−/− and Cx40+/− aortas but not in Cx37−/− aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype.ConclusionsCx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0110-1) contains supplementary material, which is available to authorized users.

Analysis of erectile responses to bradykinin in the anesthetized rat.

The kallikrein-kinin system is expressed in the corpus cavernosa, and bradykinin (BK) relaxes isolated corpora cavernosal strips. However, erectile responses to BK in the rat have not been investigated in vivo. In the present study, responses to intracorporal (ic) injections of BK were investigated in the anesthetized rat. BK, in doses of 1-100 μg/kg ic, produced dose-related increases in intracavernosal pressure (ICP) and dose-related deceases in mean arterial pressure (MAP). When decreases in MAP were prevented by intravenous injections of angiotensin II (Ang II), increases in ICP, in response to BK, were enhanced. Increases in ICP, ICP/MAP ratio, and area under the curve and decreases in MAP in response to BK were inhibited by the kinin B2 receptor antagonist HOE-140 and enhanced by the angiotensin-converting enzyme (ACE) inhibitor captopril and by Ang-(1-7). Increases in ICP, in response to BK, were not attenuated by the nitric oxide (NO) synthase inhibitor (N(ω)-nitro-L-arginine methyl ester) or the soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but were attenuated by the cyclooxygenase inhibitor, sodium meclofenamate. Decreases in MAP were not attenuated by either inhibitor. These data suggest that erectile responses are mediated by kinin B2 receptors and modulated by decreases in MAP. These data indicate that ACE is important in the inactivation of BK and that erectile and hypotensive responses are independent of NO in the penis or the systemic vascular bed. Erectile responses to cavernosal nerve stimulation are not altered by BK or HOE-140, suggesting that BK and B2 receptors do not modulate nerve-mediated erectile responses under physiologic conditions. These data suggest that erectile responses to BK are mediated, in part, by the release of cyclooxygenase products.

Differential effects of losartan and candesartan on vasoconstrictor responses in the ratThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

Losartan has been reported to have inhibitory effects on thromboxane (TP) receptor-mediated responses. In the present study, the effects of 2 nonpeptide angiotensin II (AT1) receptor antagonists, losartan and candesartan, on responses to angiotensin II, the thromboxane A2 mimic, U46619, and norepinephrine were investigated and compared in the pulmonary and systemic vascular beds of the intact-chest rat. In this study, intravenous injections of angiotensin II, U46619, and norepinephrine produced dose-related increases in pulmonary and systemic arterial pressure. Losartan and candesartan, in the doses studied, decreased or abolished responses to angiotensin II. Losartan, but not candesartan, and only in a higher dose, produced small, but statistically significant, reductions in pressor responses to U46619 and to norepinephrine in the pulmonary and systemic vascular beds. Furthermore, losartan significantly reduced arachidonic acid-induced platelet aggregation, whereas candesartan had no effect. Pressor responses to angiotensin II were not changed by thromboxane and alpha-adrenergic receptor antagonists, or by cyclooxygenase and NO synthase inhibitors. These results show that losartan and candesartan are potent selective AT1 receptor antagonists in the pulmonary and systemic vascular beds and that losartan can attenuate thromboxane and alpha-adrenergic responses when administered at a high dose, whereas candesartan in the highest dose studied had no effect on responses to U46619 or to norepinephrine. The present data show that the effects of losartan and candesartan on vasoconstrictor responses are different and that pulmonary and systemic pressor responses to angiotensin II are not modulated or mediated by the release of cyclooxygenase products, activation of TP receptors, or the release of NO in the anesthetized rat.

Peroxynitrite does not impair pulmonary and systemic vascular responses.

The effects of peroxynitrite (ONOO-) on vascular responses were investigated in the systemic and hindquarters vascular bed and in the isolated perfused rat lung. Intravenous injections of ONOO- decreased systemic arterial pressure, and injections of ONOO- into the hindquarters decreased perfusion pressure in a dose-related manner. Injections of ONOO- into the lung perfusion circuit increased pulmonary arterial perfusion pressure. Responses to ONOO- were rapid in onset, short in duration, and repeatable without exhibiting tachyphylaxis. Repeated injections of ONOO- did not alter systemic, hindquarters, or pulmonary responses to endothelium-dependent vasodilators or other vasoactive agonists and did not alter the hypoxic pulmonary vasoconstrictor response. Injections of sodium nitrate or nitrite or decomposed ONOO- had little effect on vascular pressures. Pulmonary and hindquarters responses to ONOO- were not altered by a cyclooxygenase inhibitor in a dose that attenuated responses to arachidonic acid. These results demonstrate that ONOO- has significant pulmonary vasoconstrictor, systemic vasodepressor, and vasodilator activity; that short-term repeated exposure does impair vascular responsiveness; and that responses to ONOO- are not dependent on cyclooxygenase product release.

Vasodilator responses to adenosine and hyperemia are mediated by A(1) and A(2) receptors in the cat vascular bed.

Hemodynamic responses to adenosine, the A(1) receptor agonists N(6)-cyclopentyladenosine (CPA) and adenosine amine congener (ADAC), and the A(2) receptor agonist 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA) were investigated in the hindquarter vascular bed of the cat under constant-flow conditions. Injections of adenosine, CPA, ADAC, CPCA, ATP, and adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) into the perfusion circuit induced dose-related decreases in perfusion pressure. Vasodilator responses to the A(1) agonists were reduced by the A(1) receptor antagonists KW-3902 and CGS-15943, whereas responses to CPCA were reduced by the A(2) antagonist KF-17837. Vasodilator responses to adenosine were reduced by KW-3902, CGS-15943, and by KF-17837, suggesting a role for both A(1) and A(2) receptors. Vasodilator responses to ATP and the nonhydrolyzable ATP analog ATP gamma S were not attenuated by CGS-15943 or KF-17837. After treatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester, the cyclooxygenase inhibitor sodium meclofenamate, or the ATP-dependent K(+) (K) channel antagonists U-37883A or glibenclamide, responses to adenosine and ATP were not altered. Responses to adenosine, CPA, and CPCA were increased in duration by rolipram, a type 4 cAMP phosphodiesterase inhibitor, but were not altered by zaprinast, a type 5 cGMP phosphodiesterase inhibitor. When blood flow was interrupted for a 30-s period, the magnitude and duration of the reactive vasodilator response were reduced by A(1) and A(2) receptor antagonists. These data suggest that vasodilator responses to adenosine and the A(1) and A(2) agonists studied are not dependent on the release of cyclooxygenase products, nitric oxide, or the opening of K channels in the regional vascular bed of the cat. The present data suggest a role for cAMP in mediating responses to adenosine and suggest that vasodilator responses to adenosine and to reactive hyperemia are mediated in part by A(1) and A(2) receptors in the hindquarter vascular bed of the cat.

ATP-mediated release of arachidonic acid metabolites from venular endothelium causes arteriolar dilation.

This study was designed to test the hypothesis that venular administration of ATP resulted in endothelium-dependent dilation of adjacent arterioles through a mechanism involving cyclooxygenase products. Forty-three male golden hamsters were anesthetized with pentobarbital sodium (60 mg/kg ip), and the cremaster muscle was prepared for in vivo microscopy. ATP (100 microM) injected into venules dilated adjacent arterioles from a mean diameter of 51 +/- 4 to 76 +/- 6 microm (P < 0.05, n = 6). To remove the source of endothelial-derived relaxing factors, the venules were then perfused with air bubbles to disrupt the endothelium. Resting arteriolar diameter was not altered after disruption of the venular endothelium (51 +/- 5 microm), and the responses to venular ATP infusions were significantly attenuated (59 +/- 4 microm, P < 0.05). To determine whether the relaxing factor was a cyclooxygenase product, ATP infusion studies were repeated in the absence and presence of indomethacin (28 microM). Under control conditions, ATP (100 microM) infusion into the venule caused an increase in mean arteriolar diameter from 55 +/- 4 to 78 +/- 3 microm (P < 0.05, n = 6). In the presence of indomethacin, mean resting arteriolar tone was not significantly altered (49 +/- 4 microm), and the response to ATP was significantly attenuated (54 +/- 4 microm, P < 0.05, n = 6). These studies show that increases in venular ATP concentrations stimulate the release of cyclooxygenase products, possibly from the venular endothelium, to vasodilate the adjacent arteriole.

Nonadrenergic, noncholinergic relaxation of human isolated corpus cavernosum induced by scorpion venom

Objectives. To examine the effects of Tityus serrulatus scorpion venom (TSV) on human corpus cavernosum (HCC) using a bioassay cascade. Priapism is occasionally observed in scorpion envenomation, mostly in children. Methods. HCC strips were suspended in a cascade system and superfused with aerated and warmed Krebs’ solution at 5 mL/min. Noradrenaline (3 μmol/L) was infused to induce a submaximal contraction of the HCC strips. The release of cyclooxygenase products was prevented by infusing indomethacin (6 μmol/L). Results. N ω-nitro- l-arginine methyl ester (10 μmol/L; n = 10) increased the tone of the preparations and significantly reduced ( P <0.01) the acetylcholine (ACh) and TSV-induced relaxations. Subsequent infusion of l-arginine (300 μmol/L) partially reversed the increased tone and significantly restored the relaxations induced by TSV and ACh ( P <0.01). The soluble guanylyl cyclase inhibitor ODQ (10 μmol/L; n = 8) markedly reduced ( P <0.01) the relaxations induced by TSV, ACh, glyceryl trinitrate, and bradykinin. 7-Nitroindazole (10 μmol/L; n = 8) inhibited the relaxations induced by TSV by 84% ( P <0.01) and also caused small, but significant, reductions in the ACh and bradykinin-induced HCC relaxations ( P <0.05). Atropine (1 μmol/L; n = 6) abolished the relaxations evoked by ACh ( P <0.01), but had no effect on those elicited by TSV. Tetrodotoxin (1 μmol/L; n = 6) abolished the relaxations induced by TSV ( P <0.01) and also reversed the established TSV-induced relaxation (n = 4). Conclusions. Our results indicate that TSV relaxes HCC through the release of nitric oxide from nonadrenergic, noncholinergic (NANC) nerves. The elucidation of the mechanism responsible for the TSV-induced relaxations might be useful for a better understanding of the development of priapism in cases of scorpion envenomation.

Differential inhibition of functional dilation of small arterioles by indomethacin and glibenclamide.

Indomethacin or glibenclamide treatments attenuate functional dilation of larger-diameter "feed" arterioles paired with venules in hamster cremaster muscle. We tested the hypothesis that release of cyclooxygenase products from venules is important for functional dilation of third- and fourth-order arterioles. We also tested whether ATP-sensitive potassium channels are important during functional dilation of smaller arterioles. The microcirculation of hamster cremaster muscle was visualized with in vivo video microscopy. We measured diameter responses of third- and fourth-order arterioles paired and unpaired with venules in response to 2 minutes of muscle field stimulation (40 microseconds, 10 V, 1 Hz). Control diameters of vessels were 31+/-2 (n=19), 13+/-1 (n=12), 12+/-2 (n=12), and 10+/-1 (n=12) for paired and unpaired third-order and paired and unpaired fourth-order arterioles, respectively. In all groups, field stimulation resulted in increases in mean control diameter of >80%. Indomethacin (28 micromol/L) superfused on the preparation was used to inhibit cyclooxygenase metabolism, or glibenclamide (10 micromol/L) was used to block ATP-sensitive potassium channels. Indomethacin attenuated arteriolar vasodilations to electrical stimulation in paired third-order vessels only, whereas glibenclamide attenuated this vasodilation in all 4 groups. These results support a role for ATP-sensitive potassium channels in functional dilation of arterioles of all sizes regardless of whether or not they are paired with venules. Conversely, a role for cyclooxygenase products is limited to larger "feed arterioles" paired with venules. This study provides further evidence that venules may be the source of prostaglandin release during functional hyperemia.

NITRIC OXIDE AND PROSTANOID-DEPENDENT RELAXATION INDUCED BY ANGIOTENSIN II IN THE ISOLATED PRECONTRACTED MOUSE TRACHEAL MUSCLE AND THE ROLE OF POTASSIUM CHANNELS

In this study we examined the mechanism of the concentration-dependent relaxant effect of angiotensin II (A II) on mouse isolated tracheal rings precontracted by carbachol. The contractile effect of carbachol is increased while the relaxant effect of A II decreased by pretreatment with NG-nitro- l -arginine methyl ester (l -NAME). This finding suggests that the l -arginine nitric oxide (NO) pathway is activated by the peptide. The relaxing effect of A II was also mediated through the release of cyclo-oxygenase products of arachidonic acid, as evidenced by the inhibition of the response by lysine acetylsalicylic acid (ASA) pretreatment. A II also caused a relaxation in the isolated tracheal rings precontracted by K+(>60 m m). K+-channel blockers partially inhibited the relaxant effect of A II in carbachol-precontracted mouse tracheal rings. A non-selective blocker of K+channels, tetraethylammonium, completely abolished the relaxation induced by A II. Among the other channel blockers, the inhibitory effect of apamine and glibenclamide was higher than that of iberiotoxin, indicating the involvement of KATPand small conductance KCachannels in the relaxing response to the octapeptide. These results suggest that the mechanisms, involved in the relaxation induced by A II in the isolated mouse tracheal rings precontracted by carbachol, were firstly l -arginine NO and cyclo-oxygenase products of arachidonic acid, secondly K+channels

Involvement of platelet-activating factor in the modulation of vascular tone in the isolated perfused rabbit kidney.

The hypothesis that platelet-activating factor (PAF) plays a role in the modulation of the vasomotor tone and blood pressure was put forward by our group in previous in vivo studies in anaesthetised rabbits. The present study was undertaken to investigate the putative role of this lipid mediator in the vascular reactivity of the renal circulation, using the experimental model of the isolated perfused rabbit kidney. Dose-response curves to noradrenaline-induced vasoconstriction were performed before and after continuous infusions of two different PAF-receptor antagonists (WEB 2086 and yangambin) and of the phospholipase A2 inhibitor mepacrine. The increases in renal perfusion pressure elicited by noradrenaline were potentiated by all the above-mentioned treatments in a dose-dependent manner. Moreover, prostaglandin F2alpha-induced vasoconstriction was also potentiated by the administration of the PAF receptor antagonists and mepacrine. Furthermore, the administration of PAF into the renal circulation induced dose-related and long-lasting vasodilator responses, which were blocked by the PAF receptor antagonists. Nevertheless, PAF-induced renal vasodilation was also abolished by a pretreatment with mepacrine or with the cyclooxygenase inhibitor indomethacin, suggesting that it enhances the secondary formation of vasodilator arachidonic acid metabolites. The data indicate that PAF is involved in the modulation of the vasomotor tone in the renal circulation, through the release of cyclooxygenase products, constituting an additional mechanism of modulation of smooth muscle cell contractility to the ones exerted by well-known vasoactive substances of endothelial origin such as nitric oxide.

Coronary vasorelaxant effect of levosimendan, a new inodilator with calcium-sensitizing properties.

We examined the action of levosimendan, a new Ca2+-sensitizing inodilator, on isolated porcine coronary arteries. Vessel rings were studied in isometric myographs. Arterial cyclic adenosine monophosphate (cAMP) levels were determined by radioimmunoassay. Levosimendan (10(-7)-10(-3) M) completely relaxed arteries preconstricted by prostaglandin F2alpha (PGF2alpha) with a pD2 (-logEC50) value of 3.99 +/- 0.05 (n = 6-9 in all experiments). Pretreatment with levosimendan also prevented contraction induced by PGF2alpha. The vasorelaxation produced by levosimendan (10(-7)-10(-3) M) was not attenuated by removal of the endothelium. Levosimendan (10(-7)-10(-3) M) relaxed contractions induced by 30 mM K+ as well as 80 mM K+, whereas the K+ channel opener levcromakalim selectively relaxed contraction induced by 30 mM K+. Neither the cyclooxygenase inhibitor indomethacin nor the beta-adrenoceptor blocker propranolol influenced levosimendan-induced vasorelaxation. The Ca2+-entry blocker isradipine failed to relax arteries precontracted by endothelin-1 in Ca2+-free/EGTA medium. However, levosimendan (10(-7)-3 x 10(-3) M) completely relaxed endothelin-1-induced contractions in this medium. Levosimendan potentiated the relaxant effect of a cAMP-stimulating drug, isoprenaline, but also that of nitroglycerin and isradipine. At a maximal effective concentration, it increased arterial tissue contents of cAMP twofold. In conclusion, levosimendan produces coronary vasorelaxation by a mechanism that seems to be endothelium independent and not mediated by K+ channel opening, Ca2+-entry blockade, release of cyclooxygenase products, or beta-adrenoceptor stimulation. Accumulation of cAMP may possibly participate in vasorelaxation at high concentrations of levosimendan, but a cAMP-independent mechanism seems to be involved at lower concentrations.

Bradykinin-Induced Responses in a Coaxial Bioassay System Composed of Rat Anococcygeus Muscle and Guinea Pig Trachea

1. Epithelium-dependent effects of bradykinin (BK) were investigated in a coaxial bioassay system which consisted of guinea pig trachea as donor organ and rat anococcygeus muscle as test tissue. 2. BK (10 −9 to 10 −5 M) produced concentration-dependent relaxations on the phenylephrine (3×10 −6 M)-precontracted rat anococcygeus muscle mounted alone. Relaxations decreased significantly when muscle was mounted in epithelium-intact trachea. There was also a significant difference between the relaxations obtained in the muscle within epithelium-intact and epithelium-denuded trachea (at 10 −7 to 10 −5 M concentrations). 3. Capsaicin (10 −5 M) pretreatment did not change BK (10 −9 to 10 −5 M)-induced relaxations in each preparation compared with vehicle pretreatment. Indomethacin (10 −6 M) in combination with thiorphan (10 −5 M) and atropine (10 −6 M) did not affect the BK-induced relaxations of the muscle within capsaicin-pretreated epithelium-intact or denuded trachea. 4. CGS 8515 (a specific 5-lipoxygenase inhibitor, 10 −6 M) did not change BK (10 −5 M)-induced relaxation on the muscle alone, and caused an increase of BK-induced relaxation on the muscle within epithelium-intact trachea compared with that obtained without CGS 8515. 5. Results showed that epithelial or nonepithelial factors were capable of modulating the responsiveness of rat anococcygeus muscle to BK. The decreased relaxation by BK in anococcygeus muscle did not occur by the release of cyclooxygenase products or tachykinins from tracheal epithelium, but it may have occurred by the contractile action of lipoxygenase product secreted by nonepithelial sources. In addition, BK might stimulate the secretion of an epithelium-derived inhibitory factor from the trachea.

Pharmacological characterization of kinin-induced relaxation of human corpus cavernosum.

To characterize the kinin receptor subtype involved in the relaxation of human isolated corpus cavernosum (HCC) induced by bradykinin (BK), Lys-bradykinin (Lys-BK), Met-Lys-bradykinin (Met-Lys-BK) and des-Arg9-bradykinin, and to investigate whether the kinin-induced relaxation of HCC results from the stimulation of nonadrenergic, noncholinergic (NANC) neurons supplying the cavernosal tissue. Excised HCC tissues were immediately placed in Krebs solution and kept at 4 degrees C until use (never > 24 h after removal). HCC was cut in strips of approximately 2 cm, suspended in a cascade system and superfused with oxygenated and warmed Krebs solution at 5 mL/min. After equilibration for approximately 90 min, noradrenaline (3 micromol/L) was infused to induce a submaximal contraction of the HCC strips. The release of cyclo-oxygenase products was prevented by infusing indomethacin (6 micromol/L). HCC strips were calibrated by injecting a single bolus of the nitrovasodilator glyceryl trinitrate (GTN) and the sensitivity of the tissues adjusted electronically to be similar. The agonists (kinins, histamine and acetylcholine) were injected as a single bolus (up to 100 microL) and the relaxation of HCC expressed as a percentage of the submaximal relaxation induced by GTN. Bradykinin, Lys-BK and Met-Lys-BK significantly relaxed the HCC tissues; on a molar basis, there was no statistical difference among the degrees of relaxation induced by these peptides. The B1 kinin receptor agonist des-Arg9-bradykinin had no effect on the HCC. The infusion of the B2 kinin receptor antagonist Hoe 140 (50 nmol/L) virtually abolished the relaxation induced by BK, Lys-BK and Met-Lys-BK without affecting those induced by acetylcholine and histamine. The infusion of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester increased the tone of the HCC tissues and significantly reduced (P < 0.01) the relaxation induced by BK (74%), Lys-BK (90%), Met-Lys-BK (87%) and acetylcholine (89%) without affecting those induced by GTN. The subsequent infusion of L-arginine (300 micromol/L) partially reversed the increased tone and significantly (P < 0.01) restored the relaxation induced by BK, Lys-BK and Met-Lys-BK. The results were similar with the novel guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3,-alquinoxalin-1-one] which reduced by > 95% (P < 0.01) the relaxation induced by BK, Lys-BK, Met-Lys-BK, acetylcholine and GTN. The infusion of the sodium-channel blocker tetrodotoxin had no significant effect on the BK-, GTN- and acetylcholine-induced relaxation of HCC. This study clearly showed the existence of functional B2 kinin receptors in human erectile tissues that when activated lead to the release of NO and hence relaxation of the HCC tissues. As tetrodotoxin failed to affect the kinin-induced relaxation of HCC strips, it is likely that these peptides release NO from the endothelium of sinusoidal capillaries rather than from neuronal sources supplying the cavernosal tissue. Although tissue kallikreins and their components have been found in the male reproductive system, the physiopathological importance of these findings has yet to be elucidated.

Differential sensitivity of macrophages to bradykinin.

In this report, we investigated the responsiveness of subpopulations of elicited peritoneal macrophages between each other compared to resident tissue macrophages of alveoli of guinea pig to the action of bradykinin. Bradykinin stimulated the secretion of superoxide radical, arachidonic acid and prostaglandin E2 (PGE2) via the bradykinin B2 receptor subtype in peritoneal macrophages, indicated by complete inhibitory effect of the bradykinin B2 receptor antagonist HOE 140. The extent of the secretion, however, varied substantially between macrophages of different size. The highest level of the secretion was observed in the fraction containing the intermediate-size macrophages, while progressively lower level of the secretion was observed with decreasing size. In contrast, large macrophages obviously lost their secretory ability. Additionally, the bradykinin-stimulated release of cyclooxygenase products exerted an inhibitory action on NADPH-oxidase activity depending on size and stage of maturation/activation of macrophages, as judged by an increase in superoxide radical generation by indomethacin (100 microM) preincubation of cells. Furthermore, the investigation of resident tissue macrophages of alveoli has shown that these cells also express the bradykinin B2 receptor subtype. However, the receptor activity measured by bradykinin-induced increase in intracellular free calcium [Ca2+]i was very low compared to elicited peritoneal macrophages. These findings indicate that the stage of differentiation/maturation and activation of macrophages may be important for the ability of bradykinin to stimulate these cells to inflammatory responses in vivo.

An in vitro pharmacological examination of venom from the soldierfish gymnapistes marmoratus

The aim of the present study was to commence a characterisation of some of the basic pharmacological properties of venom from the soldierfish ( Gymnapistes marmoratus). Soldierfish venom was prepared by extraction into 10% glycerol and centrifugation to remove insoluble material. Protein content was determined and venom concentrations were expressed as μg venom protein. Soldierfish venom (0.5–15 μg/ml) produced concentration-dependent contractile responses in guinea-pig isolated ileum (GPI) and longitudinal smooth muscle (LSM) preparations. The muscarinic receptor antagonist atropine (10 nM) significantly inhibited responses of LSM to soldierfish venom (2.5 μg/ml). Responses to soldierfish venom (4–5 μg/ml) in GPI were not significantly affected by the ganglion-blocking drug mecamylamine (10 μM) or by incubation with blood cholinesterase. The cyclooxygenase inhibitor indomethacin (2 μM) significantly inhibited responses to soldierfish venom (2.5 μg/ml) in LSM. Neither the thromboxane A 2/prostaglandin H 2 receptor antagonist GR32191B (1 μM) nor the leukotriene receptor antagonist SB205312 (10 nM) significantly affected responses to soldierfish venom (5 μg/ml) in GPI. Responses to soldierfish venom (2.5-5 μg/ml) were not significantly inhibited by the histamine receptor antagonist mepyramine (0.5 μM), the angiotensin-converting enzyme inhibitor captopril (2 μM) or the neurokinin-1 receptor antagonist CP-99,994 (0.1 μM) in LSM. The angiotensin AT 1 receptor antagonist EXP3174 (0.1 μM) also failed to inhibit significantly the responses to soldierfish venom (5 μg/ml) in GPI. A fluorometric assay for the detection of 5-hydroxytryptamine (5-HT) and related compounds indicated a level in soldierfish venom of 1.60 ± 0.01 ng of 5-HT-like substance per μg venom protein. Soldierfish venom (0.5–10 μg/ml) produced concentration-dependent contractile responses in rat isolated stomach fundus strips, and these responses (2.5 μg/ml) were significantly inhibited by the 5-HT 1 5-HT 2 receptor antagonist methysergide (0.1 μM). These results suggest that soldierfish venom may stimulate the release of acetylcholine to act at muscarinic receptors on guinea-pig gastrointestinal smooth muscle. The venom also appears to be causing the release of cyclooxygenase products, such as prostaglandins, and contains 5-HT, or a 5-HT-like substance, that acts directly at 5-HT receptors.

Effect of NG-nitro-L-arginine-methyl-ester on cardiopulmonary function and biosynthesis of cyclooxygenase products during porcine endotoxemia.

To determine if inhibition of nitric oxide synthase with NG-nitro-L-arginine-methyl-ester (L-NAME) potentiates endotoxin-induced cardiopulmonary dysfunction and release of cyclooxygenase products in a porcine model of endotoxemia. Prospective, multiple group, controlled experimental study. Physiologic research laboratory at a veterinary medicine college. Fifty-seven domestic pigs (mean 28.7 +/- 0.8 [SEM] kg). Pentobarbital-anesthetized pigs were intubated and mechanically ventilated to normocapnia with room air. A ther-modilution cardiac output catheter was advanced into the pulmonary artery. Additional catheters were inserted into the jugular and femoral veins and femoral artery. The pigs received the following infusions: saline (control, n = 5); L-NAME (0.1, 0.5, 2.2, or 5.5 mg/ kg/hr, from -0.5 to 2 hrs, n = 16); Escherichia coli endotoxin (5 micrograms/ kg from 0 to 1 hr followed by 2 micrograms/kg from 1 to 2 hrs, i.v., n = 14); L-NAME plus endotoxin (n = 9); indomethacin plus endotoxin (n = 6); or L-NAME indomethacin plus endotoxin (n = 7). L-NAME significantly (p < .05) worsened endotoxin-induced hypoxemia and enhanced the increases in pulmonary vascular resistance index and systemic vascular resistance index at 30 to 60 mins. Endotoxin increased (p < .05) plasma concentrations of thromboxane B2 by seven- to eight-fold at 30 to 120 mins and 6-keto-prostaglandin F1 alpha by 16- to 24-fold at 60 to 120 mins. L-NAME enhanced (additive effect) endotoxin-induced increases in plasma concentrations of thromboxane B2 (60 mins) and significantly (p < .05) potentiated the increases in 6-keto-prostaglandin F1 alpha (120 mins). At 120 mins of endotoxemia, indomethacin (cyclooxygenase inhibitor) plus L-NAME markedly increased (p < .05, synergistic effect) systemic vascular resistance index compared with endotoxemic pigs pretreated with either L-NAME or indomethacin. During endotoxemia, inhibition of nitric oxide synthase with L-NAME may be deleterious to cardiopulmonary function, as evidence by potentiation of endotoxin-induced systemic and pulmonary vasoconstriction, impairment of gas exchange, and enhanced biosynthesis of cyclooxygenase products. Moreover, during endotoxemia, the concomitant inhibition of two important vasodilators (i.e., nitric oxide and prostacyclin) is associated with a potentiated (p < .05) increase in systemic vascular resistance index.

Release of calcitonin gene-related peptide in cardiac anaphylaxis.

We have investigated the antigen-stimulated release of calcitonin gene-related peptide (CGRP) from ovalbumin-sensitized guinea-pig isolated hearts and the interaction with other mediators of anaphylaxis released concomitantly. It was found that antigen challenge caused a significant increase of CGRP release (from basal 31.2 +/- 2.9 to 51.6 +/- 4.9 fmol/5 min). Anaphylactic CGRP release was significantly attenuated in the presence of the cyclooxygenase inhibitor indomethacin while the 5-lipoxygenase inhibitor Bay-X1005 ((R)-2-[4-quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) had no significant effect. Combined treatment with the histamine receptor (H1,H2) antagonists mepyramine and cimetidine also significantly attenuated anaphylactic release of CGRP. Under control conditions antigen injection increased release of cysteinyl-leukotrienes (LT), thromboxane (TXB2) and 6-keto-prostaglandin (PG)F1 alpha from basal values of 0.96 +/- 0.09, 2.7 +/- 0.7 and 3.4 +/- 0.28 ng/5 min respectively, to 5.9 +/- 0.9, 48.4 +/- 3.4 and 6.9 +/- 1.4 ng/5 min. Indomethacin abolished the release of cyclooxygenase products of arachidonate metabolism and simultaneously increased cysteinyl-LT release significantly (8.8 +/- 1.4 ng/5 min). Conversely Bay-X1005 completely abolished cysteinyl-LT release and had no significant effect on anaphylactic release of TXB2 and 6-keto-PGF1 alpha. Simultaneous blockade of H1 and H2 receptors abolished release of 6-keto-PGF1 alpha, while release of TXB2 and cysteinyl-LT was not significantly affected. The results indicate that CGRP is not a primary mediator of the immediate hypersensitivity reaction of the heart, but is in turn released by arachidonic acid metabolites of the cyclooxygenase pathway and histamine. In contrast, LT obviously do not contribute to anaphylactic CGRP release. CGRP is a potent coronary vasodilator and could act as endogenous functional antagonist of vasoconstrictor mediators also released during cardiac anaphylaxis such as cysteinyl-LT, platelet activating factor and TXA2.

Analysis of responses to pentoxifylline in the pulmonary vascular bed of the cat.

To test the hypothesis that pulmonary vasodilator responses to pentoxifylline are dependent on the synthesis of nitric oxide from L-arginine and are independent of the release of cyclooxygenase products. Prospective study. Research laboratory. Isolated lobar lung preparation, using mongrel cats. In separate experiments, the effects of NG-L-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, and the effects of a cyclooxygenase blocker, meclofenamate, were investigated on pulmonary arterial responses to pentoxifylline, acetylcholine, and isoproterenol during increased tone conditions induced by the thromboxane A2 mimic, U46619, in the pulmonary vascular bed of the cat. Lobar arterial perfusion pressure, systemic pressure, and left atrial pressure were continuously monitored, electronically averaged, and permanently recorded. Under increased tone conditions in the isolated left lower lobe vascular bed of the cat, NG-L-nitro-L-arginine methyl ester significantly reduced the vasodilator responses to pentoxifylline and to acetylcholine, whereas NG-L-nitro-L-arginine methyl ester had no significant effect on the vasodilator responses to isoproterenol. Vasodilator responses to pentoxifylline and acetylcholine were not significantly changed in the presence of meclofenamate, whereas meclofenamate markedly reduced the vasopressor effects of arachidonic acid. These data show that pentoxifylline has significant vasodilator activity in the pulmonary vascular bed of the cat. The present data also suggest that responses to pentoxifylline during increased tone conditions may, in part, be mediated by the release of nitric oxide and are independent of the release of cyclooxygenase products.

Prostaglandins play a role in memory consolidation in the chick

Previous studies showed that inhibitors of cyclooxygenases have amnesic effects in chicks in a passive avoidance task. The onset of amnesia has a delay of 2 h post-training. To investigate if this effect is due to the inhibition of induction of the enzyme during learning, the release of cyclooxygenase products into the extracellular fluid was measured at 1, 2 and 3 h post-training. A cyclooxygenase inhibitor, ibuprofen, inhibited the training-dependent increase of cyclooxygenase products only 2 h and 3 h after learning when injected pre-training, as did dexamethasone which prevents cyclooxygenase induction, and SC58125 (1,2-diarylcyclopentene), an inhibitor of inducible cyclooxygenase. Injections 30 min post-training showed the same effect with the exception of dexamethasone. Injecting SC58125, ibuprofen, indomethacin, or dexamethasone i.c. before training showed amnesic effects for training on a one-trial passive avoidance task at 2 h but not 1 h after training. Injections 30 min post-training produced the same effects with the exception of dexamethasone. I conclude that cyclooxygenases are induced during training and that cyclooxygenase products are of importance in memory formation of the chick.

Indomethacin blocks the febrile response induced by interleukin-8 in rabbits

Interleukin (IL)-8 induces fever in rats by a mechanism independent of the release of cyclooxygenase products. The purpose of this study was to investigate whether a similar mechanism is responsible for the pyrogenic effect of IL-8 in rabbits. Intravenous (0.31-5.0 ng/kg) or intracerebroventricular (15.6-250 pg) injections of IL-8 induced a dose-dependent increase in body temperature. The correlations between the doses of recombinant human IL-8 and the fever index were 0.98 and 0.99 for the intravenous and intracerebroventricular injections, respectively. The pyrogenic activity of IL-8 was not due to contamination with lipopolysaccharide (LPS), inasmuch as the Limulus amoebocyte lysate test showed < 10 pg endotoxin/micrograms IL-8, and boiled IL-8 lost its pyrogenic activity. Indomethacin (2 and 5 mg/kg i.p.) abolished the febrile response induced by the intravenous injection of LPS (5.0 ng/kg), IL-1 beta (5 ng/kg), and IL-8 (5 ng/kg). Indomethacin also abolished the fever induced by the intracerebroventricular injection of IL-8 (62.5 pg) but only partially reduced the response induced by the injection of IL-1 beta (25 pg icv). These results show that, different from rats, indomethacin blocks the febrile response induced by the central or peripheral administration of IL-8 in rabbits.