19,20-EpDPE, a bioactive CYP450 metabolite of DHA monoacyglyceride, decreases Ca²⁺ sensitivity in human pulmonary arteries

The aim of this study was to investigate the effect of docosahexaenoic acid monoacylglyceride (MAG-DHA) on human pulmonary arterial tone. Tension measurements on pulmonary arterial tissues demonstrated that MAG-DHA reduced U-46619-induced tone, which is highly sensitive to the H-1152 inhibitor. Results also showed that MAG-DHA treatments decreased RhoA activity levels, which in turn inactivated the Rho-kinase pathway, leading to a reduction in U-46619-induced Ca(2+) sensitivity of permeabilized pulmonary artery smooth muscle cells. According to the mechanical responses assessing U-46619-induced Ca(2+) sensitivity in the absence or presence of 3 μM MAG-DHA, MAG-DHA plus 1 μM N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH, a cytochrome P-450 epoxygenase inhibitor) and 300 nM 19,20-epoxydocosapentaenoic acid (a cytochrome P-450 epoxygenase-dependent DHA metabolite), our data suggest that the MAG-DHA is metabolized in a bioactive epoxymetabolite. This epoxyeicosanoid in turn decreases active tone and Ca(2+) sensitivity of smooth muscles cells through an inhibition of the Rho-kinase pathway. Together, these data provide primary evidence regarding the mode of action of MAG-DHA in human pulmonary arteries and suggest that this compound may be of pharmacological interest in patients with pulmonary hypertension to generate intracellular bioactive metabolites.     

Chest wall distortion and discharge of pulmonary slowly adapting receptors.

We assessed the effects of chest wall distortion, changes in lung volume, and abolition of airway smooth muscle tone on the discharge patterns of 92 pulmonary slowly adapting receptors (SAR) in decerebrate, spontaneously breathing cats. Distortion resulted from their inspiratory efforts against an occluded airway at functional residual capacity and at increased end-expiratory lung volumes. Approximately 40% of SAR increased discharge frequencies during occlusions. Modulation of SAR discharge during occlusions persisted after administration of atropine to eliminate airway smooth muscle tone. Phasic modulation of SAR discharge was eliminated during no-inflation tests after paralyzing the cats and ventilating them on a cycle-triggered pump. We conclude 1) parasympathetic modulation of airway smooth muscle tone makes no obvious contribution to SAR discharge in spontaneously breathing cats; 2) the no-inflation test (withholding of lung inflation during neural inspiration) in paralyzed and ventilated cats is a valid test for the presence of projections from SAR to medullary respiratory neurons; and 3) in the absence of tidal volume changes, distortion stimulates some SAR. Sensory feedback from receptors in the lung, not just those in the chest wall, may therefore provide information about abnormal chest wall configurations.     

Relaxing effects of 17(18)-EpETE on arterial and airway smooth muscles in human lung

Human cytochrome P-450 epoxygenase enzymes metabolize eicosapentaenoic acid (EPA), an omega-3-polyunsaturated fatty acid (PUFA), and leads to the production of 17(18)-epoxyeicosatetraenoic acid, or 17(18)-EpETE. The aim of the present study was to delineate the mode of action of 17(18)-EpETE on human pulmonary artery (HPA) and distal bronchi. Isometric tension measurements demonstrated that 17(18)-EpETE induced concentration-dependent relaxing effects in pulmonary artery and airway smooth muscles. Iberiotoxin (IbTx) and glyburide (Glyb), known BK(Ca) and K(ATP) channel inhibitors, respectively, reversed the relaxation induced by 17(18)-EpETE on both tissues types. Microelectrode measurements showed that exogenous addition of 17(18)-EpETE hyperpolarized the membrane potential of HPA and bronchial smooth muscle cells. These induced electrophysiological effects were reversed by the addition of 10 nM IbTx and 10 muM Glyb. Complementary experiments performed on human bronchi, using the planar lipid bilayer reconstitution technique, demonstrated that 17(18)-EpETE activated reconstituted BK(Ca) channels at low free Ca(2+) concentration. Moreover, in bronchi, the relaxing responses induced by 17(18)-EpETE were also related to reduced Ca(2+) sensitivity of the myofilaments, since free Ca(2+) concentration-response curves, performed on beta-escin-permeabilized cultured explants, were shifted toward higher Ca(2+). Together, these results provide new insight into the mode of action of 17(18)-EpETE in lung tissues and highlight this eicosanoid as a potent modulator of tone on both HPA and distal bronchi in vitro, which may be of clinical relevance in the pathophysiology of pulmonary hypertension and airway diseases.     

CYP4A mRNA, protein, and product in rat lungs: novel localization in vascular endothelium.

The vasodilatory effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on lung arteries is opposite to the constrictor effect seen in cerebral and renal vessels. These observations raise questions about the cellular localization of 20-HETE-forming isoforms in pulmonary arteries and other tissues. Using in situ hybridization, we demonstrate for the first time CYP4A (a family of cytochrome P-450 enzymes catalyzing formation of 20-HETE from the substrate arachidonic acid) mRNA in pulmonary arterial endothelial and smooth muscle cells, bronchial smooth muscle and bronchial epithelial cells, type I epithelial cells, and macrophages in adult male rat lungs. Moreover, we detect CYP4A protein in rat pulmonary arteries and bronchi as well as cultured endothelial cells. Finally, we identify endogenously formed 20-HETE by using fluorescent HPLC techniques, as well as the capacity to convert arachidonic acid into 20-HETE in pulmonary arteries, bronchi, and endothelium. These data show that 20-HETE is an endogenous product of several pulmonary cell types and is localized to tissues that optimally position it to modulate physiological functions such as smooth muscle tone or electrolyte flux.     

Contribution of cytochrome P-450 omega-hydroxylase to altered arteriolar reactivity with high-salt diet and hypertension

The present study evaluated the contribution of cytochrome P-450 omega-hydroxylase in modulating the reactivity of cremaster muscle arterioles in normotensive rats on high-salt (HS) and low-salt (LS) diet and in rats with reduced renal mass hypertension (RRM-HT). Changes in arteriolar diameter in response to ACh, sodium nitroprusside (SNP), ANG II, and elevated O(2) were measured via television microscopy under control conditions and following cytochrome P-450 omega-hydroxylase inhibition with 17-octadecynoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In normotensive rats on either LS or HS diet, resting tone was unaffected and arteriolar reactivity to ACh or SNP was minimally affected by cytochrome P-450 omega-hydroxylase inhibition. In RRM-HT rats, cytochrome P-450 omega-hydroxylase inhibition reduced resting tone and significantly enhanced arteriolar dilation to ACh and SNP. Treatment with 17-ODYA or DDMS inhibited arteriolar constriction to ANG II and O(2) in all the groups, although the degree of inhibition was greater in RRM-HT than in normotensive animals. These results suggest that metabolites of cytochrome P-450 omega-hydroxylase contribute to the altered reactivity of skeletal muscle arterioles to vasoconstrictor and vasodilator stimuli in RRM-HT.     

Formation of epoxyeicosatrienoic acids from arachidonic acid by cultured rat aortic smooth muscle cell microsomes.

The vasodilatory effect of epoxyeicosatrienoic acids (EpETrE), especially 5(6)-EpETrE, has been reported recently and a role of P-450-dependent arachidonic acid monooxygenase metabolites was suggested in vasoregulation. Accordingly, the presence of P-450-dependent arachidonic acid monooxygenase was investigated in rat aortic smooth muscle cells. Incubation of the microsomes of rat cultured aortic smooth muscle cells with 14C-arachidonic acid in the presence of 1 mM NADPH resulted in the formation of oxygenated metabolites. The metabolites were separated and purified by reverse phase and straight phase high performance liquid chromatography and identified by gas chromatography-mass spectrometry. Identified metabolites were 5(6)-EpETrE, 5,6-dihydroxyeicosatrienoic acid (DiHETrE), and 14,15-DiHETrE. The formation of these metabolites was totally dependent on the presence of NADPH, and inhibitors of cytochrome P-450-dependent enzymes, SKF-525A and metyrapone, reduced the formation of these metabolites. This is the first report that cytochrome P-450-dependent arachidonic acid metabolites, especially 5(6)-EpETrE and 14(15)-EpETrE, can be produced in the microsomes of vascular smooth muscle cells of rats.     

Effect of bronchomotor tone on work rate of breathing

We studied the optimal airway caliber for minimizing the work rate of breathing in the lung (W) with different bronchomotor tones in six normal subjects. The inhalation of methacholine contracted airway smooth muscle, and the inhalation of salbutamol relaxed it. To calculate W at a given alveolar ventilation (VA), anatomical dead space (VDanat), pulmonary resistance (RL), and dynamic compliance were measured simultaneously, breath by breath, during various breathing maneuvers. VDanat increased and RL decreased with both increased breathing frequency and tidal volume, even at a given airway tone. This suggests that the airway caliber varied even at a given bronchomotor tone. The minimum W at a given VA increased in constricted airways, but there was no significant difference between control airways after saline inhalation and relaxed airways. It has been suggested that airway smooth muscle tones at both control and relaxed conditions bring W to a minimum and that the airway smooth muscle tone existing in the control state acts to keep the airway caliber optimal in order to minimize the W and stabilize the airway mechanics.     

Pulmonary vasoactivity of lung endocrine cell-related peptides

Lung endocrine-like cells are believed to contain three immunohistochemically distinct peptides: bombesin, calcitonin, and Leu-enkephalin. Because these peptides exhibit smooth muscle stimulatory or inhibitory activity in some tissues, it has been suggested that their release from endocrine-like cells may influence airway or pulmonary vascular smooth muscle tone. To determine whether lung endocrine cell-related peptides could exert a regulatory influence in the pulmonary circulation, we evaluated their ability to constrict or dilate the vasculature of isolated perfused rat lungs. Neither bombesin nor calcitonin exhibited any pulmonary vascular effects. However, Leu-enkephalin provoked dose-dependent pulmonary vasoconstriction. These results suggest that Leu-enkephalin released from lung endocrine-like cells could be involved with regulation of pulmonary vascular tone.     

Hypoxic pulmonary vasoconstriction is modified by P-450 metabolites

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A (CYP4A) metabolite of arachidonic acid (AA) in human and rabbit lung microsomes and is a dilator of isolated human pulmonary arteries (PA). However, little is known regarding the contribution of P-450 metabolites to pulmonary vascular tone. We examined 1) the effect of two mechanistically distinct omega- and omega1-hydroxylase inhibitors on perfusion pressures in isolated rabbit lungs ventilated with normoxic or hypoxic gases, 2) changes in rabbit PA ring tone elicited by 20-HETE or omega- and omega1-hydroxylase inhibitors, and 3) expression of CYP4A protein in lung tissue. A modest increase in perfusion pressure (55 +/- 11% above normoxic conditions) was observed in isolated perfused lungs during ventilation with hypoxic gas (FI(O(2)) = 0.05). Inhibitors of 20-HETE synthesis, 17-oxydecanoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), increased baseline perfusion pressure above that of vehicle and amplified hypoxia-induced increases in perfusion pressures by 92 +/- 11% and 105 +/- 11% over baseline pressures, respectively. 20-HETE relaxed phenylephrine (PE)-constricted PA rings. Treatment with 17-ODYA enhanced PE-induced contraction of PA rings, consistent with inhibition of a product that promotes arterial relaxation, whereas 6-(20-propargyloxyphenyl)hexanoic acid (PPOH), an epoxygenase inhibitor, blunted contraction to PE. Conversion of AA into 20-HETE was blocked by 17-ODYA, DDMS, and hypoxia. CYP4A immunospecific protein confirms expression of CYP4A in male rabbit lung tissue. Our data suggest that endogenously produced 20-HETE could modify rabbit pulmonary vascular tone, particularly under hypoxic conditions.     

Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.     

Endothelium, arachidonic acid, and coronary vascular tone

Vasoactive mediators play an important role in the control of coronary vascular tone. Arachidonic acid (AA) metabolites and endothelium-derived vasoactive factors have been implicated in coronary vasoregulation. AA can be metabolized via three separate routes in blood vessels, mediated by cyclooxygenase, lipoxygenase, and cytochrome P-450-dependent monooxygenase enzymes. AA can evoke endothelium-dependent relaxations that are due in part to the formation of cytochrome P-450-dependent metabolites, inasmuch as drugs that modify cytochrome P-450 activity produce parallel changes in endothelium-dependent relaxations to AA. Moreover, some cytochrome P-450-derived metabolites formed biologically cause relaxations of isolated blood vessels. A cytochrome P-450-dependent pathway does not appear to contribute to endothelium-dependent relaxations induced by acetylcholine, which suggests that there may be a number of endothelium-derived relaxing factors (EDRFs). In addition, two endothelium-derived contractile factors have been described, including an unidentified cyclooxygenase metabolite of AA and a polypeptide isolated from cultured cells. As both prostaglandin I2 and acetylcholine-induced EDRF also inhibit platelet aggregation, endothelial injury and loss of these factors may predispose to vasospasm precipitated by release of platelet-derived mediators such as thromboxane A2 (TXA2) and 5-hydroxytryptamine. Unstable angina may be a clinical syndrome in which these events occur, which can be alleviated by inhibition of platelet activation and TXA2 formation with aspirin. Attenuation of endothelium-dependent relaxations can also occur without loss of endothelial cells. Neutrophil-endothelium interactions, precipitated by an ischemic episode, may initiate endothelial dysfunction and underlie the development of vasospasm in some conditions. Whether increased production of endothelium-derived contractile factors also occurs in vasospastic conditions remains to be determined.     

Effect of airway smooth muscle tone on airway distensibility measured by the forced oscillation technique in adults with asthma

Airway distensibility appears to be unaffected by airway smooth muscle (ASM) tone, despite the influence of ASM tone on the airway diameter-pressure relationship. This discrepancy may be because the greatest effect of ASM tone on airway diameter-pressure behavior occurs at low transpulmonary pressures, i.e., low lung volumes, which has not been investigated. Our study aimed to determine the contribution of ASM tone to airway distensibility, as assessed via the forced oscillation technique (FOT), across all lung volumes with a specific focus on low lung volumes. We also investigated the accompanying influence of ASM tone on peripheral airway closure and heterogeneity inferred from the reactance versus lung volume relationship. Respiratory system conductance and reactance were measured using FOT across the entire lung volume range in 22 asthma subjects and 19 healthy controls before and after bronchodilator. Airway distensibility (slope of conductance vs. lung volume) was calculated at residual volume (RV), functional residual capacity (FRC), and total lung capacity. At baseline, airway distensibility was significantly lower in subjects with asthma at all lung volumes. After bronchodilator, distensibility significantly increased at RV (64.8%, P < 0.001) and at FRC (61.8%, P < 0.01) in subjects with asthma but not in control subjects. The increased distensibility at RV and FRC in asthma were not associated with the accompanying changes in the reactance versus lung volume relationship. Our findings demonstrate that, at low lung volumes, ASM tone reduces airway distensibility in adults with asthma, independent of changes in airway closure and heterogeneity.     

S-nitrosoglutathione breakdown prevents airway smooth muscle relaxation in the guinea pig

Airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are low in children with near-fatal asthma. We hypothesized that GSNO could be broken down in the lung and that this catabolism could inhibit airway smooth muscle relaxation. In our experiments, GSNO was broken down by guinea pig lung homogenates, particularly after ovalbumin sensitization (OS). Two lung protein fractions had catabolic activity. One was NADPH dependent and was more active after OS. The other was NADPH independent and was partially inhibited by aurothioglucose. Guinea pig lung tissue protein fractions with GSNO catabolic activity inhibited GSNO-mediated guinea pig tracheal ring relaxation. The relaxant effect of GSNO was partially restored by aurothioglucose. These observations suggest that catabolism of GSNO in the guinea pig 1) is mediated by lung proteins, 2) is partially upregulated after OS, and 3) may contribute to increased airway smooth muscle tone. We speculate that enzymatic breakdown of GSNO in the lung could contribute to asthma pathophysiology by inhibiting the beneficial effects of GSNO, including its effect on airway smooth muscle tone.     

Possible mechanisms of oxygen sensing in the pulmonary circulation.

Oxygen tension is known to control the pulmonary vascular tone. We reviewed three hypotheses that try to explain the mechanism whereby hypoxia is sensed in the lung tissue. The first hypothesis concerns the role of the oxygen binding hemoprotein cytochrome P-450. Studies using various inhibitors and activators of cytochrome P-450 show that this enzyme affects pulmonary vascular tone. The data are, however, contradictory. The second hypothesis postulates that hypoxia reduces the synthesis of vasodilator oxygen radicals in the lung. This hypothesis is quite well supported by experimental data. The third hypothesis, similarly widely documented, states that slowing of the respiratory chain and altered cellular energetics is crucial for sensing of hypoxia. In this case, however, it is not exactly clear how changes in cellular energetics are connected with vascular tone. The possibility exists that changes in both the cytochrome P-450 activity and in the rate of electrons flow in the respiratory chain may alter the amount of oxygen radicals in the cells and, similarly as in the "oxygen radicals" hypothesis, govern calcium channels through the control of the redox status of these channels.     

Functional effects of 20-HETE on human bronchi: hyperpolarization and relaxation due to BKCa channel activation

Airway smooth muscle (ASM) metabolizes arachidonic acid (AA) through various enzymatic pathways, including cytochrome P-450 (CYP-450) omega-hydroxylase, which leads to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to delineate the mode of action of 20-HETE in human ASM cells. Isometric tension measurements demonstrated that 20-HETE induced a concentration-dependent relaxant effect in ASM on bronchi precontracted with either methacholine or AA. Relaxing effects of 20-HETE on resting tone were prevented by 10 nM iberiotoxin (IbTx), a BK(Ca) channel inhibitor. Microelectrode measurements showed that exogenous additions of 20-HETE (0.1-10 microM) hyperpolarized the membrane potential of human ASM cells. This concentration-dependent electrophysiological effect induced by the eicosanoid was prevented by 10 nM IbTx. Complementary experiments, using the planar lipid bilayer reconstitution technique, demonstrated that 20-HETE activated reconstituted BK(Ca) channels at low free Ca(2+) concentrations. Together, these results indicate that 20-HETE-dependent activation of BK(Ca) channels is responsible for the hyperpolarization and controlled relaxation of ASM in human distal bronchi.     

Hydrogen peroxide induces endothelium-dependent and -independent coronary arteriolar dilation: role of cyclooxygenase and potassium channels

Hydrogen peroxide, a relatively stable reactive oxygen species, is known to elicit vasodilation, but its underlying mechanism remains elusive. Here, we examined the role of endothelial nitric oxide (NO), prostaglandin, cytochrome P-450-derived metabolites, and smooth muscle potassium channels in coronary arteriolar dilation to abluminal H2O2. Pig subepicardial coronary arterioles (50-100 microm) were isolated and pressurized without flow for in vitro study. Arterioles developed basal tone and dilated dose dependently to H2O2 (1-100 microM). Disruption of th endothelium and inhibition of cyclooxygenase (COX) by indomethacin produced identical attenuation of vasodilation to H2O2. Conversely, the vasodilation to H2O2 was not affected by either the NO synthase inhibitor NG-nitro-l-arginine methyl ester or the cytochrome P-450 enzyme blocker miconazole. Inhibition of the COX-1, but not the COX-2 pathway, attenuated H2O2-induced dilation similarly to indomethacin. The production of prostaglandin E2 (PGE2), but not prostaglandin I2, from coronary arterioles was significantly increased by H2O2. Furthermore, inhibition of PGE2 receptors with AH-6809 attenuated vasodilation to H2O2 similar to that produced by indomethacin. In the absence of a functional endothelium, H2O2-induced dilation was attenuated, in an identical manner, by a depolarizing agent KCl and a calcium-activated potassium (KCa) channel inhibitor iberiotoxin. However, PGE2-induced dilation was not affected by iberiotoxin. The endothelium-independent dilation to H2O2 was also insensitive to the inhibition of guanylyl cyclase, lipoxygenase, ATP-sensitive potassium channels, and inward rectifier potassium channels. These results suggest that H2O2 induces endothelium-dependent vasodilation through COX-1-mediated release of PGE2 and also directly relaxes smooth muscle by hyperpolarization through KCa channel activation.     

Dextromethorphan Mediated Bitter Taste Receptor Activation in the Pulmonary Circuit Causes Vasoconstriction

Activation of bitter taste receptors (T2Rs) in human airway smooth muscle cells leads to muscle relaxation and bronchodilation. This finding led to our hypothesis that T2Rs are expressed in human pulmonary artery smooth muscle cells and might be involved in regulating the vascular tone. RT-PCR was performed to reveal the expression of T2Rs in human pulmonary artery smooth muscle cells. Of the 25 T2Rs, 21 were expressed in these cells. Functional characterization was done by calcium imaging after stimulating the cells with different bitter agonists. Increased calcium responses were observed with most of the agonists, the largest increase seen for dextromethorphan. Previously in site-directed mutational studies, we have characterized the response of T2R1 to dextromethorphan, therefore, T2R1 was selected for further analysis in this study. Knockdown with T2R1 specific shRNA decreased mRNA levels, protein levels and dextromethorphan-induced calcium responses in pulmonary artery smooth muscle cells by up to 50%. To analyze if T2Rs are involved in regulating the pulmonary vascular tone, ex vivo studies using pulmonary arterial and airway rings were pursued. Myographic studies using porcine pulmonary arterial and airway rings showed that stimulation with dextromethorphan led to contraction of the pulmonary arterial and relaxation of the airway rings. This study shows that dextromethorphan, acting through T2R1, causes vasoconstrictor responses in the pulmonary circuit and relaxation in the airways.     

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

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

Evidence against the involvement of a cytochrome P-450 mechanism in pulmonary hemodynamics in the newborn pig.

Control mechanisms operating through a cytochrome P-450 system have emerged lately as a possible important determinant of pulmonary hemodynamics. Their action may be expressed in the adjustment of vascular tone under both physiologic and pathophysiologic conditions. One such condition is the pulmonary constrictor response to hypoxia. The identity of the effector agent, or agents, is not known, though there are data implicating monooxygenase products of arachidonic acid. From this premise, we wanted to evaluate the effect of cytochrome P-450 inhibitors on basal pulmonary vascular tone during normoxia, and their effect upon hypoxic pulmonary vasoconstriction response. Experiments were performed in an isolated, perfused lung preparation from 1- and 7-day-old piglets, and the effects of two cytochrome P-450 inhibitors (metyrapone and ketoconazole) were tested on the perfusion pressure. At 10(-5) and 10(-4) M, metyrapone caused a modest, but significant, increase in pulmonary pressure (p less than 0.05) in 7-day-old preparations, while it was without effect in the 1-day-old preparation. Similarly, ketoconazole at concentrations from 10(-6) M upwards increased the perfusion pressure in the older animal (p less than 0.01). Responses to the inhibitors were not seen in preparations that had been pretreated with a cyclooxygenase inhibitor (indomethacin, 2.8 x 10(-6) M) or a dual cyclooxygenase-lipoxygenase inhibitor (BW755C, 10(-5) M). Hypoxic vasoconstriction was marginally enhanced by 10(-4) M metyrapone, while it was affected inconsistently by 10(-5) M ketoconazole. We conclude that vasoactive agents formed through cytochrome P-450 reactions have a minor role, or no role at all, in the control of pulmonary hemodynamics in the newborn pig.     

Cytochrome P-450 metabolites but not NO,PGI2, and H2O2 contribute to ACh-induced hyperpolarization of pressurized canine coronary microvessels

The endothelium-dependent hyperpolarization of cells has a crucial role in regulating vascular tone, especially in microvessels. Nitric oxide (NO) and prostacyclin (PGI2), in addition to endothelium-derived hyperpolarizing factor (EDHF), have been reported to hyperpolarize vascular smooth muscle in several organs. Studies have reported the hyperpolarizing effects of these factors are increased by a stretch in large coronary arteries. EDHF has not yet been identified and cytochrome P-450 metabolites and H2O2 are candidates for EDHF. With the use of the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethione oxonol [DiBAC4(3)], we examined whether NO, PGI2, cytochrome P-450 metabolites, and H2O2 contribute to ACh-induced hyperpolarization in pressurized coronary microvessels. Canine coronary arterial microvessels (60-356 mum internal diameter) were cannulated and pressurized at 60 cmH2O in a vessel chamber perfused with physiological salt solution containing DiBAC4(3). Fluorescence intensity and diameter were measured on a computer. There was a linear correlation between changes in the fluorescence intensity and membrane potential. ACh significantly decreased the fluorescence intensity (hyperpolarization) of the microvessels without any inhibitors. Endothelial damage caused by air perfusion abolished the ACh-induced decrease in fluorescence intensity. The inhibitors of NO synthase and cyclooxygenase did not affect the ACh-induced decreases in the fluorescence intensity. The addition of 17-octadecynoic acid, a cytochrome P-450 monooxygenase inhibitor, to those inhibitors significantly attenuated the ACh-induced decreases in fluorescence intensity, whereas catalase, an enzyme that dismutates H2O2 to form water and oxygen, did not. Furthermore, catalase did not affect the vasodilation produced by ACh. These results indicate that NO and PGI2 do not contribute to the ACh-induced hyperpolarization and that the cytochrome P-450 metabolites but not H2O2 are involved in EDHF-mediated hyperpolarization in canine coronary arterial microvessels.