Pig Pulmonary Artery Research Articles

Signaling Pathways Involved in Enhanced 5‐HT‐Induced Contraction of Pulmonary Artery Segments From Guinea‐Pigs With Hyperthyroidism

We have previously reported that hyperthyroidism in rats resulted in enhanced 5‐HT‐induced contractions of pulmonary artery segments by a mechanism involving activation of calcium‐activated chloride channels. We have investigated other possible pathways that might be involved in this response in this study. Pulmonary artery ring segments were set up in Krebs’ solution for isometric tension recording. 5‐HT‐induced contraction was significantly enhanced in main pulmonary artery segments from guinea‐pigs with hyperthyroidism compared with a similar preparation from control guinea pigs. Nifedipine (10−6M) and tempol (3×10−3M) significantly reduced 5‐HT‐induced contraction in artery segments from thyroxine‐treated while having no effect in artery segments from control guinea pigs. Y‐27632 (10−5M) and SB 202190 (10−5M) but not PD 98059 (10−5M) significantly reduced 5‐HT‐induced contractions in artery segments from control and thyroxine‐treated guinea pigs. However, the reduction was greater in artery segments from thyroxine‐treated guinea pigs. Genistein (10−5M) significantly reduced 5‐HT‐induced contractions in artery segments from control or thyroxine‐treated guinea pigs. Genistein was equipotent in both cases. These results would suggest that hyperthyroidism‐induced increased 5‐HT‐induced contraction of the guinea pig pulmonary artery is mediated by multiple signaling pathways including increased influx of extracellular calcium through L‐type dihydropyridine channels, tyrosine kinase pathway, Rho‐kinase pathway of calcium sensitization, increased generation of reactive oxygen species and p38 MAPK pathway.

The effect of urapidil, an alpha-1 adrenoceptor antagonist and a 5-HT1A agonist, on the vascular tone of the porcine coronary and pulmonary arteries, the rat aorta and the human pulmonary artery

Urapidil (Eupressyl®) an antihypertensive drug acting as an α1 antagonist and a 5-HT1A agonist, may be of special interest in the treatment of hypertension associated with preeclamptic toxaemia and hypoxia-induced pulmonary arterial vasoconstriction. However, the effect of urapidil on vascular tone has been poorly investigated. Vascular reactivity was evaluated using pulmonary and coronary arteries from 36 pigs, aortae from 22 rats and 9 human pulmonary artery samples suspended in organ chambers. Concentration-relaxation curves either to urapidil, 5-HT, or the 5-HT1A receptor agonist 8-OH-DPAT were constructed after pre-contraction of rings. Pig pulmonary and coronary artery rings were contracted with U46619, a thromboxane mimetic, rat aortic rings with either endothelin-1 or phenylephrine, and human pulmonary artery rings with U46619 or phenylephrine. Urapidil markedly inhibited phenylephrine-induced contractions in rat aortic rings with and without endothelium with a more pronounced effect observed in rings without endothelium. Both 5-HT and 8-OH-DPAT failed to induce relaxation in rat aortic rings with an intact endothelium. 5-HT, but not urapidil and 8-OH-DPAT, induced a concentration-dependent relaxation in the porcine coronary and pulmonary artery rings with an intact endothelium (P<0.05). 5-HT and phenylephrine but not urapidil caused concentration-dependent contractions in human pulmonary artery rings. The present findings, while confirming that urapidil is a potent inhibitor of α1-adrenoceptor-induced contraction, do not support the role of 5-HT1A receptor activation in the control of the vascular tone of the different types of arteries tested in response to urapidil. In addition, they indicate that urapidil seems to preferentially target arteries with endothelial dysfunction.

Experimental study of bioabsorbable iron-based pulmonary artery stent

Objective To monitor the degradation and bio-safety features of the bioabsorbable iron-based stent planted in the pig pulmonary artery. Methods A total of 19 miniature pigs were randomly assigned into 3 groups.Three cases were assigned as the control group; ten cases were assigned as stent implantation group A with one stent implanted in the pig pulmonary artery; the other six cases were assigned as stent implantation group B with two stents implanted in the pig pulmonary artery.Follow-up studies for 24 months were conducted in all cases.Repeated measures ANOVA were used to collect and analyze statistical processing data at multiple time points of the follow-up. Results After surgery, pulmonary artery blood flow velocity and right ventricular pressure were measured by echocardiography and cardiac catheterization.There was no significant difference in pulmonary artery blood flow velocity(Fgroup×time=1.06, P=0.40) and right ventricular pressure (Fgroup×time=0.58, P=0.86) among each group, and no vascular re-narrow circumstance was indicated.Pulmonary artery diameter expansion rate between stent group and control group was statistically different (Fgroup=3.57, P=0.05; Ftime=12.89, P<0.001; Fgroup×time=2.99, P=0.03), suggesting that the stent could maintain a good expansion of the role of vascular in the follow-up period even though in degradation.Difference in the level of serum iron concentrations among the groups was not statistically significant (Fgroup×time=0.94, P=0.52), suggesting that there was no evidence of iron overload.All animals survived to the follow-up endpoint, and no serious side effects caused by stent implantation were found. Conclusions Bioabsorbable iron-based stent planted in the pig pulmonary artery may experience a certain degree of degradation, and it is safe and stabile in animals. Key words: Bioabsorbable stent; Iron; Pulmonary artery; Congenital heart disease; Child

Selective glycolysis blockade in guinea pig pulmonary artery and aorta reverses contractile and electrical responses to acute hypoxia

The goal of this study was to clarify the mechanisms of hypoxic pulmonary vasoconstriction (HPV) reversal following selective glycolysis blockade and to assess possible contribution of endothelial electrogenesis to this phenomenon as a trigger mechanism.We compared smooth muscle (SM) contractility and endothelial cell (EC) membrane potential (MP) during acute hypoxia before and after glycolysis blockade. MPs were recorded from the endothelium of guinea pig pulmonary artery (GPPA) and thoracic aorta (GPTA) using the patch-clamp technique. Acute hypoxia caused hyperpolarization in GPTA EC, while EC from GPPA were depolarized. Also, acute hypoxia elicited constriction in isolated GPPA and dilatation in GPTA. Selective glycolysis inhibition always reversed both electrical and contractile responses in GPPA to hypoxia, but in GPTA this only occurred in 30% of experiments. It is likely that an unknown glycolysis-driven mechanism in EC mediates vascular tone regulation under hypoxia and underlies the paradoxical difference in the response of pulmonary and systemic arterial SM to hypoxia. Our data suggest that HPV development in GPPA might, at least partially, be driven by EC depolarization spreading to the underlying SM cells.

Influence of nanosized magnetite on the contractile activity of smooth muscle segments of the pulmonary artery of guinea pigs

This study investigates the contractile activity of smooth muscle segments of guinea pig pulmonary artery by the method of mechanografy and morphological picture of lungs, airways and blood vessels of the pulmonary circulation under the influence of nanosized magnetite. It is shown that chronic inhalation intake of magnetite nanoparticles has a cytotoxic effect, leading to inflammatory changes in the airways, lung parenchyma and pulmonary vessels, as well as a change in contractile activity of the pulmonary artery by the action of neurotransmitters and mediators of exercising their functions under physiological conditions or produced during the inflammatory the process.

Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1 receptor stimulation

Platinum-based anticancer drugs cause neurotoxicity. In particular, oxaliplatin produces early-developing, painful, and cold-exacerbated paresthesias. However, the mechanism underlying these bothersome and dose-limiting adverse effects is unknown. We hypothesized that the transient receptor potential ankyrin 1 (TRPA1), a cation channel activated by oxidative stress and cold temperature, contributes to mechanical and cold hypersensitivity caused by oxaliplatin and cisplatin. Oxaliplatin and cisplatin evoked glutathione-sensitive relaxation, mediated by TRPA1 stimulation and the release of calcitonin gene-related peptide from sensory nerve terminals in isolated guinea pig pulmonary arteries. No calcium response was observed in cultured mouse dorsal root ganglion neurons or in naïve Chinese hamster ovary (CHO) cells exposed to oxaliplatin or cisplatin. However, oxaliplatin, and with lower potency, cisplatin, evoked a glutathione-sensitive calcium response in CHO cells expressing mouse TRPA1. One single administration of oxaliplatin produced mechanical and cold hyperalgesia in rats, an effect selectively abated by the TRPA1 antagonist HC-030031. Oxaliplatin administration caused mechanical and cold allodynia in mice. Both responses were absent in TRPA1-deficient mice. Administration of cisplatin evoked mechanical allodynia, an effect that was reduced in TRPA1-deficient mice. TRPA1 is therefore required for oxaliplatin-evoked mechanical and cold hypersensitivity, and contributes to cisplatin-evoked mechanical allodynia. Channel activation is most likely caused by glutathione-sensitive molecules, including reactive oxygen species and their byproducts, which are generated after tissue exposure to platinum-based drugs from cells surrounding nociceptive nerve terminals.TRPA1 is necessary and sufficient for mechanical- and cold-hypersensitivity evoked by oxaliplatin/cisplatin. TRPA1 activation occurs through reactive molecules, after tissue exposure to platinum-based drugs.

Specific adrenergic responses of smooth muscles in the vascular wall of guinea pig pulmonary arteries during ovalbumin sensitization

The adrenergic contractile responses of smooth muscles in the vascular wall of guinea pig pulmonary arteries were studied during ovalbumin sensitization. Sensitization was followed by inhibition of contractile responses to an alpha-adrenoceptor agonist mesatone, prevented endothelium-derived relaxation, and potentiated the contractile response to isoproterenol. Administration of a beta-adrenoceptor agonist isoproterenol potentiated the increase in mechanical strain of smooth muscles in the pulmonary artery precontracted with high-potassium Krebs solution. Removal of the endothelium had no effect on the contractile response of smooth muscle segments from the pulmonary artery of intact and sensitized guinea pigs to b-adrenergic influences. The contractile responses of smooth muscles of the pulmonary artery are associated with activity of the cAMP-dependent signal system and play a role in the pathogenesis of ventilation-perfusion disturbances during atopic inflammation.

The role of myocardin in hypoxia-induced transdifferentiation of pulmonary artery endothelial cells into smooth muscle-like cells

To investigate the transdifferentiation of pulmonary artery endothelial cells (PAECs) into smooth muscle-like cells under hypoxia and the role of myocardin therein. Recombinant plasmid psimyocardin (pSi), capable of silencing the expression of myocardin gene, was constructed by RNAi to be used to transfect the PAECs. Endothelial cells of adult pig pulmonary small arteries were purified by immunomagnetic purification technique, and divided into 4 groups: normoxia group (to be cultured in normoxic cell culture box containing 21% O(2), 5% CO2, and 74% N(2)), normoxia + pSi group, hypoxia group (to be cultured in cell box containing 1% O(2), 5% CO2, and 74% N(2)), and hypoxia + pSi group to be cultured for 1, 4, and 7 days respectively. Indirect fluorescence technique and morphological examination were used to identify the smooth muscle (SM) -like cells and the alpha-SM-actin positive cell ratio. RT-PCR was used to detect the mRNA expression of myocardin. Alpha-SM-actin positive cell could not be seen in the normoxia group and hypoxia 1 d group. The alpha-SM-actin positive cell rate of the hypoxia 7 days group was 2.07% +/- 0.06%, significantly higher than that of the hypoxia 4 d group (0.96% +/- 0.08%, P < 0.01). mRNA expression of myocardin gene could not be seen in the normoxia, normoxia + pSi, hypoxia 1 days, and hypoxia + pSi 1 days groups. The mRNA expression of myocardin gene of the hypoxia 7 days group was 0.23 +/- 0.03, significantly higher than that of the hypoxia 4 days group (0.14 +/- 0.01, P < 0.01). The mRNA expression levels of myocardin gene of the hypoxia + pSi 4 days and 7 days groups 0.03 +/- 0.02 and 0.05 +/- 0.01, both significantly lower than those of the hypoxia 4 days and 7 days groups respectively (0.14 +/- 0.01 and 0.23 +/- 0.03, both P < 0.01). The SM-like cell transdifferentiation rates of the hypoxia + pSi 4 days and 7 days groups were 0.19% +/- 0.07% and 0.21% +/- 0.04% respectively, both significantly lower than those of the corresponding hypoxia groups (0.96% +/- 0.08 and 2.07% +/- 0.06% respectively, both P < 0.01). Some PAECs have the potential to transdifferentiate into SM-like cells and may be one of the resources of muscularization of peripheral small vessels. Hypoxia remarkably promotes this transdifferentiation and myocardin may play an important role in this process.

Inhibition of Nitric-Oxide Synthase Enhances Antigen-Induced Contractions and Increases Release of Cysteinyl-Leukotrienes in Guinea Pig Lung Parenchyma: Nitric Oxide as a Protective Factor

Nitric oxide (NO) in exhaled air is a biomarker of airway inflammation. However, the role of NO in the peripheral lung is not known. The aim of this study was to determine the role of endogenous NO in antigen-induced contractions of ovalbumin (OVA)-sensitized guinea pig lung parenchyma (GPLP). The contraction in this in vitro model of the peripheral lung closely resembles the corresponding response in human airways. Cumulatively increasing concentrations (10-10,000 microg/l) of OVA induced concentration-dependent contractions of the GPLP that were enhanced by the NO synthase (NOS) inhibitors N(omega)-nitro-L-arginine (L-NOARG; 100 microM), N(omega)-monomethyl-L-arginine (100 microM), N(omega)-nitro-L-arginine methyl ester (100 microM), and N-(3-(aminomethyl)benzyl)acetamidine (1400W; 1 microM). The enhancement induced by L-NOARG was reversed by coadministration with the 5-lipoxygenase inhibitor (R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid (BAY x1005; 3 microM), whereas coadministration of L-NOARG with the cyclooxygenase inhibitor indomethacin (10 microM) did not change the effect of L-NOARG alone. L-NOARG (100 microM) did not affect the cumulative concentration-response relations for either leukotriene (LT) D4 (0.1-100 nM) or histamine (1-30 microM). The NO donor NONOate (0.001-100 microM) was ineffective in GPLP but potently relaxed precontracted guinea pig pulmonary artery. Furthermore, L-NOARG enhanced the release of LTE4 and decreased the release of prostaglandin E2 induced by OVA. In conclusion, endogenous NO exerts an inhibitory effect on antigen-induced contractions in the peripheral lung. The action of NO apparently involves inhibition of the release of mediators rather than direct relaxation of airway smooth muscle. The findings support the belief that endogenous NO has a protective anti-inflammatory effect in the airways.

Prednisolone augments superoxide formation in porcine pulmonary artery endothelial cells through differential effects on the expression of nitric oxide synthase and NADPH oxidase.

1. Prednisolone, a potent anti-inflammatory drug, has proved ineffective in treating acute respiratory distress syndrome (ARDS). ARDS is associated with superoxide (O(2)(*-)) generation, which negates nitric oxide (NO). NO also downregulates NADPH oxidase and inhibits O(2)(*-) formation. A possible reason for the lack of effect of prednisolone may due to an inhibition of eNOS expression. In order to test this proposal, the effect of prednisolone on O(2)(*-) formation and the expression of gp91(phox) (catalytic subunit of NADPH oxidase) and eNOS in pig pulmonary artery (PA) segments and PA endothelial cells (PAECs) and PA vascular smooth muscle cells (PAVSMCs) was investigated. 2. PA segments and cells were incubated with prednisolone and tumour necrosis factor-alpha (TNF-alpha) for 16 h. O(2)(*-) formation was measured spectrophometrically and gp91(phox) and eNOS expression by Western blotting. The role of the NO-cGMP axis was studied using morpholinosydnonimine hydrochloride, the diethylamine/NO complex (DETA-NONOate), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ) and the stable cGMP analogues, 8-bromo cGMP and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP). NO release was studied using a fluorescence assay and O(2)(*-)-NO interactions with a nitrite/nitrate assay. 3. Prednisolone elicited significant increase in O(2)(*-) formation in intact PA segments and PAECs, but not PAVSMCs, in a concentration-dependent manner. In endothelium-denuded segments, prednisolone slightly enhanced O(2)(*-) release. TNF-alpha further increased prednisolone-enhanced O(2)(*-) formation in intact PA segments and PAECs. NADPH oxidase inhibitor, apocynin, inhibited O(2)(*-) formation. Increased O(2)(*-) release and gp91(phox) expression in PAECs elicited by prednisolone was blocked by SIN-1 (3-morpholinosydnonimine hydrochloride), DETA-NONOate, 8-pCPT-cGMP and 8-bromo cGMP. The effects of SIN-1 on gp91(phox) expression were reversed by ODQ. Finally, eNOS protein expression was significantly reduced by prednisolone. 4. Prednisolone increases O(2)(*-) in porcine PAECs through a downregulation of endogenous eNOS expression. Since the NO-cGMP axis inhibits gp91(phox) expression, the resultant decrease in endogenous NO formation then augments NADPH oxidase activity, which in turn results in increased O(2)(*-) formation. Since O(2)(*-) promotes inflammation, this mechanism may explain why prednisolone is ineffective in treating ARDS. Therapeutically, the coadministration of an NO donor may render prednisolone more effective in treating ARDS.

Inhaled prostacyclin is safe, effective, and affordable in patients with pulmonary hypertension, right-heart dysfunction, and refractory hypoxemia after cardiothoracic surgery

Inhaled prostacyclin is safe, effective, and affordable in patients with pulmonary hypertension, right-heart dysfunction, and refractory hypoxemia after cardiothoracic surgery

Pertussis toxin and N-ethylmaleimide inhibit histamine- but not calcium ionophore-induced endothelium-dependent relaxation

Endothelium-dependent relaxation of the guinea pig pulmonary artery induced by histamine was inhibited by preincubation of the tissue with 10 microM N-ethylmaleimide (NEM) for 10 min, whereas the endothelium-dependent relaxation induced by the calcium ionophore A 23187 was not affected by NEM. Pretreatment of the preparations with 0.2-1 microgram/ml pertussis toxin for 120 min inhibited concentration-dependently the histamine-induced relaxation. In contrast, endothelium-dependent relaxation in response to the calcium ionophore A 23187 was not affected by pertussis toxin. Since NEM and pertussis toxin are thought to interfere with membrane located GTP binding proteins, it is suggested that such a coupling protein is involved in the signal transduction of the histamine receptor leading to endothelium-dependent relaxation.

Lack of prejunctional modulation of noradrenaline release by endogenous nitric oxide in guinea pig pulmonary artery

The regulation of noradrenaline (NA) release by endogenous endothelium-derived compounds was investigated in the isolated guinea pig pulmonary artery preloaded with [ 3 H ]NA. The radioactivity uptake, the basal and electrical field stimulation (10 Hz, 2 ms, 360 shocks) evoked release of [ 3 H ]NA was similar in arteries with intact endothelium and after removal of the endothelium. The wide selectivity nitric oxide (NO) synthase inhibitor N-ω-nitro- l-arginine (100 μM) did not affect significantly the basal and stimulation-evoked release of [ 3 H ]NA in control and endothelium-denuded preparations. These results indicate that neither endogenous NO nor other compounds derived from the endothelium have substantial influence on the NA outflow from sympathetic nerves innervating the pulmonary artery.

Circulating nitrite anions are a directly acting vasodilator and are donors for nitric oxide

Inhaled nitric oxide (NO) is a pulmonary vasodilator, but also acts systemically, causing negative cardiac inotropic effects and a fall in systemic vascular resistance. Circulating metabolites of NO are presumed to be responsible. We questioned the role of nitrite anions and the manner in which they might contribute to these effects. Nitrite and nitrate anions coexist in blood, while circulating levels of dissolved NO are very low. Nitrate anions are not biologically active, but nitrite anions may have a biological role through the release of NO. In vitro, at 37 degrees C and in aerated Krebs bicarbonate solution, the steady-state concentration of dissolved NO was proportional to the concentration of NO in the gas. Nanomolar concentrations of dissolved NO coexisted with micromolar concentrations of nitrite anions. The idea of an equilibrium between the two in solution was also supported by the observed release of NO from nitrite anions in the absence of gas. With rings of precontracted pig pulmonary arteries (prostaglandin F(2alpha); 10 micromol/l), the steady-state concentration of dissolved NO causing 50% relaxation (EC(50)) was 0.84+/-0.25 nmol/l, corresponding to a gaseous concentration of 2.2 p.p.m. The EC(50) of nitrite was 4.5+/-0.7 micromol/l, a concentration normally found in plasma. The estimated concentration of dissolved NO derived from this nitrite was 4.5 pmol/l, some 100 times lower than would be needed to cause relaxation. The rate of exhalation of NO was increased and pulmonary vascular resistance was reduced by the addition of nitrite solution to the perfusate of isolated perfused and ventilated pig lungs, but only when millimolar concentrations were achieved. Thus circulating nitrite anions are a direct vasodilator, only being a carrier of effective amounts of "free" NO at higher than physiological concentrations.

Relationship of Hydraulic Impedance and Elasticity in the Pulmonary Artery of Maturing Newborn Pigs

The current study determined the dynamic stress–strain elastic moduli (EY) and characteristic impedances (Z0(2–7Hz)) of the main pulmonary artery in open-chest, anesthetized newborn pigs at 2 days, 2 weeks, and 3 months of age. EY and Z0(2–7Hz) were compared to those values derived from the Womersley and Moens–Korteweg equations (denoted EW-MK and Z0W-MK, respectively) to test the validity of these equations in describing the elasticity of the intact newborn pulmonary artery. EY was defined as the ratio of stress to strain. The current study hypothesized that: (1) EY and EW-MK are numerically similar, and therefore the Womersley and Moens–Korteweg equations accurately describe the viscoelastic properties of the main pulmonary artery of the newborn pig, and (2) that both EY and Z0 are elevated at birth and undergo a steady decline with maturation. EY was not significantly different from EW-MK, while Z0(2–7Hz) was nearly identical to Z0W-MK in all groups. The elastic modulus peaked (P < 0.001) in 2-week-old pigs compared with both younger and older animals, while Z0(2–7Hz) decreased with increasing age (48 h = 1237 ± 251 [SEM] dyn s cm−5, 2-week = 433 ± 95 dyn s cm−5, 3 month = 162 ± 17 dyn s cm−5, P < 0.001). These experiments validate the Womersley and Moens–Korteweg equations as accurately describing the elastic properties of the intact newborn pig pulmonary artery. These data also demonstrate that a diminution in Z0 may occur even with increased wall stiffness, as observed in our 2-week-old pigs.

Inhibition of endotoxin-induced vascular hyporeactivity by 4-amino-tetrahydrobiopterin.

The 4-amino analogue of tetrahydrobiopterin (4-ABH(4)) is a potent pterin-site inhibitor of nitric oxide synthases (NOS). Although 4-ABH(4) does not exhibit selectivity between purified NOS isoforms, a pronounced selectivity of the drug towards inducible NOS (iNOS) is apparent in intact cells. This work was carried out to investigate the potential iNOS selectivity of 4-ABH(4) in isolated pig pulmonary and coronary arteries. Endothelium-dependent relaxations of pig pulmonary and coronary artery strips to bradykinin or calcium ionophore A23187 were inhibited by 4-ABH(4) in a concentration-dependent manner. Half-maximal inhibition was observed at 60 - 65 microM (pulmonary artery) and 200 - 250 microM 4-ABH(4) (coronary artery). Pig coronary artery strips precontracted with 0.1 microM 9, 11-dideoxy-9, 11-methanoepoxy-prosta-glandin F(2alpha) (U46619) showed a time-dependent relaxation (monitored for up to 18 h) upon incubation with 1 microg ml(-1) lipopolysaccharide (LPS). Addition of 10 microM 4-ABH(4) 1 h after LPS led to a pronounced inhibition of the LPS-triggered relaxation, whereas the pterin antagonist had no effect when given> or =4 h after LPS. Incubation of pulmonary and coronary artery strips with 1 microg ml(-1) LPS attenuated contractile responses to norepinephrine (1 microM) and U46619 (0.1 microM). This hyporeactivity of the blood vessels to vasoconstrictor agents was inhibited by 4-ABH(4) in a concentration-dependent manner [IC(50)=17.5+/-5.9 microM (pulmonary artery) and 20.7+/-3 microM (coronary artery)]. The effect of 0.1 mM 4-ABH(4) was antagonized by coincubation with 0.1 mM sepiapterin, which is known to supply intracellular BH(4) via a salvage pathway. These results demonstrate that 4-ABH(4) is a fairly selective inhibitor of iNOS in an in vitro model of endotoxaemia, suggesting that this drug and/or related pterin-site NOS inhibitors may be useful to increase blood pressure in severe infections associated with a loss of vascular responsiveness to constrictor agents caused by endotoxin-triggered iNOS induction in the vasculature.

Cardiovascular effects of BRX-005: Comparison to bimoclomol

Concentration-dependent effects of BRX-005, the novel heat shock protein coinducer, cardioprotective and vasoprotective agent, on intracellular calcium transients and contractility were studied in Langendorff-perfused guinea pig hearts loaded with the fluorescent calcium indicator dye Fura-2. BRX-005 increased peak left ventricular pressure, the rate of force development and relaxation significantly in a concentration-dependent manner. The amplitude of [Ca 2+] i transients was left unaltered by the drug. In contrast to BRX-005, bimoclomol increased both contractility and the amplitude of [Ca 2+] i transients. In canine ventricular cardiomyocytes high concentrations of BRX-005 had no effect on depolarization, whereas bimoclomol suppressed action potential upstroke markedly. In guinea pig pulmonary artery preparations precontracted with phenylephrine, BRX-005 induced concentration-dependent relaxation. This effect of BRX-005 was independent of the integrity of endothelium indicating that vasorelaxant effect of the drug develops directly on vascular smooth muscle.

Further evidence that 5-HT-induced relaxation of pig pulmonary artery is mediated by endothelial 5-HT(2B) receptors.

The endothelial 5-HT receptor mediating relaxation of pig pulmonary artery has been characterized using the selective 5-HT(2B) receptor agonist BW 723C86 and a variety of structurally diverse 5-HT receptor antagonists. If arterial rings with intact endothelium were precontracted with prostaglandin F(2alpha) (3 microM), BW 723C86 caused concentration-dependent relaxation with a pEC(50)=8.21+/-0.03 and E(max)=89+/-4% relative to 5-HT. The relaxant responses to BW 723C86 were inhibited by the 5-HT(2B) receptor antagonist SB 204741, the 5-HT(2B/2C) receptor antagonist SB 206553 and the antimigraine drug pizotifen, yielding pA(2) values of 6.68, 7.20 and 8.32, respectively. The pA(2) values against BW 723C86 were similar to those determined against 5-HT. The relaxant effect of 5-HT was antagonized by a variety of 22 compounds of diverse chemical structures. Based on the calculated mean pA(2) values the order of the most potent antagonists was ritanserin (9.38) > methysergide (8. 86) > pizotifen (8.47) >/= methiothepin (8.32) > LY 53857 (7.84) >/= amoxapine (7.80) >/= loxapine (7.73) >/= metergoline (7.64) >/= mianserin (7.51) >/= rauwolscine (7.39). Compounds with weak blocking potency were yohimbine (6.37), spiperone (5.88) and ketanserin (5.85). Correlation analysis between the affinities of the antagonists in pig pulmonary artery and those from radioligand binding studies at human and rat 5-HT(2B) receptors showed a highly significant correlation (r=0.95 and 0.84, P<0.002 and <0.005). Correlation with 5-HT(2C) receptors was much lower (r=0.57, P=0.035), and no correlations were obtained with 5-ht(6) and 5-HT(7) receptors. It is concluded that the 5-HT receptor mediating endothelium-dependent relaxation of pig pulmonary artery is of the 5-HT(2B) subtype.

Endothelial reactivity to the immediate hypersensitivity reaction of guinea pig pulmonary artery

Ovalbumin at low doses (0.1 μg/ml) caused pronounced relaxations in the precontracted pulmonary arteries of sensitized guinea pigs but, at high doses, (1–100 μg/ml) the relaxations were blunted by the contractions. The relaxations in response to ovalbumin challenge were related to histamine, which is released during the immediate hypersensitivity reaction, because they were almost blocked by mepyramine (10 −5 M) plus cimetidine (10 −4 M) pretreatment and never observed in unsensitized animal arteries. Additionally, the inhibition of relaxations by endothelium removal or N G-nitro- l-arginine ( l-NOARG, 10 −4 M) treatment implies that the phenomenon requires endothelial nitric oxide synthesis. However, the contractions appear to depend on leukotriene production since they were markedly blocked in the presence of 2( S)-hydroxy-3( R)-[(2-carboxyethyl)thio]-3-[2-(8-phenyloctyl)phenyl]-propanoic acid (SKF 104353, 10 −5 M), a leukotriene receptor antagonist. These results indicate that ovalbumin-induced nitric oxide and histamine H 2 receptor dependent relaxations in pulmonary artery may have an important role in the recovery of the increased pulmonary vascular resistance during the hypersensitivity reaction.

Functional studies of leukotriene receptors in vascular tissues.

The paradoxical effects of cysteinyl-leukotrienes, namely contraction and relaxation, are now well documented in a number of vascular preparations from various species. The vascular smooth muscle contractions are associated with activation of a single receptor subtype and in some vascular smooth muscles with activation of two receptor subtypes. However, the receptors implicated in the contraction of vessels such as pig pulmonary arteries and veins, dog inferior vena cava, and dog splenic and mesenteric veins remain to be established. There are sufficient data concerning some vascular tissues to suggest that relaxations induced by cysteinyl-leukotrienes are via the stimulation of specific receptors present on the endothelium. The endothelium in human pulmonary arteries has one receptor (CysLT2) and activation induced the release of NO. However, in isolated human pulmonary veins two receptors are present, CysLT1 and CysLT2 (Figure 1). Activation of the former induced the release of a contractile factor whereas activation of the CysLT2 receptor released NO. In guinea pig pulmonary artery and guinea pig thoracic aorta, one receptor has been demonstrated since the relaxations are blocked by ICI-198615. These data suggest the presence of a CysLT1 receptor. Activation of this receptor leads to the release of a relaxant factor, namely, nitric oxide. In contrast, in human pulmonary arteries and veins activation of a receptor that is resistant to ICI-198615 is associated with NO release. These results suggest that there may be species differences even when analogous vascular preparations are examined. While the cysteinyl-leukotrienes are known to relax vascular smooth muscle in a variety of preparations from different species, there are presently two pathways known to be involved in this response. One involves the metabolites of arachidonic acid via the cyclooxygenase enzymatic pathway and the other implicates products of the L-arginine enzymatic pathway. Although both pathways may be present and active in the endothelium of the vascular preparations only one of these enzymatic pathways may be dominant and responsible for the relaxations observed. Ortiz and coworkers have demonstrated that in pulmonary veins the dominant pathway for cysteinyl-leukotriene relaxations is the NO pathway. There are some reports from animal studies that support a dominant role for NO in pulmonary veins. In contrast, Allen and co-workers demonstrated that the LTC4-induced relaxations in isolated human saphenous veins were not modified by treatment of tissues with an NO inhibitor but were significantly enhanced after treatment with indomethacin. These authors suggested that a contracting factor derived from the arachidonic acid pathway was released in preparations challenged with LTC4. In addition, these investigators demonstrated that the NO inhibitor had no effect on the LTC4 relaxations. Together, these results suggest that cysteinyl-leukotriene effects in human pulmonary veins are dominated by the NO pathway whereas in human systemic veins these mediator effects are modified by metabolites of the cyclooxygenase pathway. Unfortunately, most studies involving the actions of cysteinyl-leukotrienes on vessels have been performed in the presence of indomethacin, making interpretation of the relative contribution of the cyclooxygenase and NO pathways difficult. In any event, the cysteinyl-leukotrienes may have a prominent role in the activation of these pathways and the receptors involved have not been clearly established.