RESUMEN
Recently we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (oxidized form of Flavin Adenine Dinucleotide (FADH2,)) as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this study was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (>95% O2 for 48 hours) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, as compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change in mitochondrial redox state of hyperoxic lungs prior to histological changes characteristic of hyperoxia.
RESUMEN
The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.
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Alprostadil/farmacología , Membrana Corioalantoides/efectos de los fármacos , Dinoprostona/farmacología , Vasoconstrictores/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Alprostadil/antagonistas & inhibidores , Animales , Compuestos de Bifenilo/farmacología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/fisiología , Compuestos Bicíclicos Heterocíclicos con Puentes , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/metabolismo , Ácido Dibenzo(b,f)(1,4)oxazepina-10(11H)-carboxílico, 8-cloro-, 2-acetilhidrazida/farmacología , Dinoprostona/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Estradiol/farmacología , Estrógenos/farmacología , Ácidos Grasos Insaturados , Hidrazinas/farmacología , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Misoprostol/farmacología , Antagonistas de Prostaglandina/farmacología , Ratas , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inhibidores , Receptores de Prostaglandina/metabolismo , Receptores de Tromboxanos/agonistas , Receptores de Tromboxanos/antagonistas & inhibidores , Receptores de Tromboxanos/metabolismo , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/antagonistas & inhibidores , Xantonas/farmacología , Ácido alfa-Linolénico/farmacologíaRESUMEN
PURPOSE: To study vascular injury after whole thoracic irradiation with single sublethal doses of X-rays in the rat and to develop markers that might predict the severity of injury. METHODS AND MATERIALS: Rats that received 5- or 10-Gy thorax-only irradiation and age-matched controls were studied at 3 days, 2 weeks, and 1, 2, 5, and 12 months. Several pulmonary vascular parameters were evaluated, including hemodynamics, vessel density, total lung angiotensin-converting enzyme activity, and right ventricular hypertrophy. RESULTS: By 1 month, the rats in the 10-Gy group had pulmonary vascular dropout, right ventricular hypertrophy, increased pulmonary vascular resistance, increased dry lung weights, and decreases in total lung angiotensin-converting enzyme activity, as well as pulmonary artery distensibility. In contrast, irradiation with 5 Gy resulted in only a modest increase in right ventricular weight and a reduction in lung angiotensin-converting enzyme activity. CONCLUSION: In a previous investigation using the same model, we observed that recovery from radiation-induced attenuation of pulmonary vascular reactivity occurred. In the present study, we report that deterioration results in several vascular parameters for
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Pulmón/efectos de la radiación , Arteria Pulmonar/efectos de la radiación , Venas Pulmonares/efectos de la radiación , Traumatismos Experimentales por Radiación/patología , Animales , Peso Corporal/efectos de la radiación , Femenino , Hematócrito , Hipertrofia Ventricular Derecha/etiología , Pulmón/irrigación sanguínea , Pulmón/enzimología , Lesión Pulmonar , Dosis de Radiación , Traumatismos Experimentales por Radiación/enzimología , Ratas , Renina/metabolismo , Tórax/efectos de la radiación , Resistencia Vascular/efectos de la radiaciónRESUMEN
Brief hypoxia differentially regulates the activities of Ca(2+)-activated K(+) channels (K(Ca)) in a variety of cell types. We investigated the effects of hypoxia (<2% O(2)) on K(Ca) channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K(Ca) current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K(Ca) currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K(Ca) channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K(Ca) current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca(2+) external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K(Ca) channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K(Ca) currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K(Ca) single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K(Ca) currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.
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Astrocitos/efectos de los fármacos , Cicloparafinas/metabolismo , Hipocampo/citología , Canales de Potasio Calcio-Activados/fisiología , Animales , Animales Recién Nacidos , Astrocitos/fisiología , Western Blotting/métodos , Bloqueadores de los Canales de Calcio/farmacología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Células Cultivadas , Cromatografía Líquida de Alta Presión/métodos , Cicloparafinas/farmacología , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente/métodos , Proteína Ácida Fibrilar de la Glía/metabolismo , Indoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
Pharmacological blocking of serotonin (5-HT) 5A receptors abolishes aortic ventilatory chemosensitivity of carotid body denervated (CBD) piglets [J. Appl. Physiol. 92 (2002) 893]. Accordingly, the purpose of the present study was to determine whether 5-HT and 5-HT receptors exist at aortic sites that are chemosensitive after CBD. Aortas from CBD and sham CBD rats and piglets and from aortic denervated (AOD) and combined AOD+CBD piglets were harvested, sectioned and then studied using immunohistochemistry and western blot techniques. 5-HT immunoreactivity in piglets and rats was concentrated in the endothelium and sub-endothelial areas in several aortic regions studied, and in some areas also in the adventitia. At the aortic chemosensitive site (descending aorta in CBD piglets and the ascending aorta in CBD rats), the immunoreactivity was greater (P < 0.05) than in other aortic regions and greater than in other groups studied. The 5-HT(5a) receptor was expressed only at the chemosensitive sites and only in aortic innervated piglets. We conclude that the data from this and a previous study [J. Appl. Physiol. 92 (2002) 893] suggest that a serotonergic mechanism contributes to the aortic ventilatory chemoreflex after CBD.
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Aorta/metabolismo , Arterias Carótidas/metabolismo , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Aorta/inervación , Western Blotting , Arterias Carótidas/inervación , Desnervación , Inmunohistoquímica , Ratas , Ratas Sprague-Dawley , PorcinosRESUMEN
Arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathways have a variety of important lung functions. Recent observations indicate that cytochrome P-450 (P-450) monooxygenases are also expressed in the lung, localized to specific pulmonary cell types (e.g., epithelium, endothelium, and smooth muscle), and may modulate critical lung functions. This review summarizes recent data on the presence and biological activity of P-450-derived eicosanoids in the pulmonary vasculature and airways, including effects on pulmonary vascular and bronchial smooth muscle tone and airway epithelial ion transport. We hypothesize a number of potential functions of P-450-derived arachidonate metabolites in the lungs such as contribution to hypoxic pulmonary vasoconstriction, regulation of bronchomotor tone, control of the composition of airway lining fluid, and limitation of pulmonary inflammation. Finally, we describe a number of emerging technologies, including congenic and transgenic strains of experimental animals, P-450 isoform-specific inhibitors and inhibitory antibodies, eicosanoid analogs, and vectors for delivery of P-450 cDNAs and antisense oligonucleotides. These tools will facilitate further studies on the contribution of endogenously formed P-450 eicosanoid metabolites to lung function, under both normal and pathological conditions.
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Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Sistema Enzimático del Citocromo P-450/fisiología , Ácidos Hidroxieicosatetraenoicos/fisiología , Pulmón/fisiología , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Citocromo P-450 CYP4A , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Pulmón/enzimología , Pulmón/ultraestructura , Microsomas/enzimología , Microsomas/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Polimorfismo Genético , Receptores de Superficie Celular/fisiologíaRESUMEN
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.
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Sistema Enzimático del Citocromo P-450/metabolismo , Hipoxia/fisiopatología , Oxigenasas de Función Mixta/metabolismo , Circulación Pulmonar , Vasoconstricción , Amidas/farmacología , Animales , Caproatos/farmacología , Citocromo P-450 CYP4A , Femenino , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Técnicas In Vitro , Masculino , Fenilefrina/farmacología , Arteria Pulmonar/efectos de los fármacos , Conejos , Sulfonas/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
Aspiration of acid from the stomach and water from the mouth can cause significant lung injury. Animal experiments suggest that acid entering the lungs is normally neutralized by bicarbonate derived from the plasma. It is hypothesized that this process may be impaired in patients with cystic fibrosis and that some of the airway injury that they experience may be related to this defect. This disease is characterized by abnormalities in the cystic fibrosis transmembrane conductance regulator, which normally conducts bicarbonate and chloride exchange. Evidence is discussed regarding the role of water channels (aquaporins) in transporting water from the airspaces into the vasculature.
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Ácido Gástrico/metabolismo , Inhalación , Pulmón/metabolismo , Agua/metabolismo , Acuaporinas/metabolismo , Transporte Biológico , HumanosRESUMEN
Little information is available regarding the vasoactive effects of epoxyeicosatrienoic acids (EETs) in the lung. We demonstrate that 5, 6-, 8,9-, 11,12-, and 14,15-EETs contract pressurized rabbit pulmonary arteries in a concentration-dependent manner. Constriction to 5,6-EET methyl ester or 14,15-EET is blocked by indomethacin or ibuprofen (10(-5) M), SQ-29548, endothelial denuding, or submaximal preconstriction with the thromboxane mimetic U-46619. Constriction of pulmonary artery rings to phenylephrine is blunted by treatment with the epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide. Pulmonary arteries and peripheral lung microsomes metabolize arachidonate to products that comigrate on reverse-phrase HPLC with authentic regioisomers of 5,6-, 8,9-, 11,12-, and 14,15-EETs, but no cyclooxygenase products of EETs could be demonstrated. Proteins of the CYP2B, CYP2E, CYP2J, CYP1A, and CYP2C subfamilies are present in pulmonary artery and peripheral lung microsomes. Constriction of isolated rabbit pulmonary arteries to EETs is nonregioselective and depends on intact endothelium and cyclooxygenase, consistent with the formation of a pressor prostanoid compound. These data raise the possibility that EETs may contribute to regulation of pulmonary vascular tone.
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Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiología , Vasoconstricción , Vasoconstrictores/farmacología , Ácido 8,11,14-Eicosatrienoico/farmacología , Amidas/farmacología , Animales , Ácido Araquidónico/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Técnicas In Vitro , Masculino , Presión , Conejos , Sistema Vasomotor/efectos de los fármacosAsunto(s)
Pulmón/fisiopatología , Edema Pulmonar/fisiopatología , Animales , Perfusión , Presión , RatasRESUMEN
BACKGROUND: A model of shunt-induced pulmonary hypertension was used to study the effects of pulmonary overcirculation on endothelial nitric oxide synthase (eNOS) and cytochrome P450-4A (cP450-4A) vasodilatory mechanisms and related hemodynamic responses. METHODS: An aortopulmonary shunt was constructed in 6-week-old piglets (n = 7, sham-operated controls n = 8). Hemodynamic measurements were made 4 weeks later under serial experimental conditions: baseline (fractional concentration of oxygen, 0.4); inhaled nitric oxide, 25 ppm (INO); hypoxia (fractional concentration of oxygen, 0.14); hypoxia + INO; N(omega)-nitro-L-arginine methylester (L-NAME 30 mg/kg intravenously, competitive NOS inhibitor); and L-NAME + INO. Lung protein levels of eNOS and cP450-4A and NOS activity were compared between groups. RESULTS: Shunted animals had a higher baseline pulmonary artery pressure (p < 0.05). L-NAME resulted in a greater increase in pulmonary vascular resistance in shunted animals (150% +/- 26% shunt versus 69% +/- 14% control; p = 0.01). The INO administered during baseline conditions decreased pulmonary vascular resistance only in control animals (p < 0.05). Protein levels of eNOS and NOS activity were similar in both groups; however, cP450-4A protein levels were decreased in the shunted group (p = 0.02). CONCLUSIONS: The NO production was preserved in shunted animals but they demonstrated greater vasodilatory dependence on NO, evidenced by an exaggerated increase in pulmonary vascular resistance after NOS inhibition. Loss of the cP450-4A vasodilatory system may be the driving force for NO dependency in the shunted pulmonary circulation.
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Sistema Enzimático del Citocromo P-450/sangre , Endotelio Vascular/fisiopatología , Hipertensión Pulmonar/fisiopatología , Oxigenasas de Función Mixta/sangre , Óxido Nítrico Sintasa/sangre , Sistema Vasomotor/fisiopatología , Animales , Citocromo P-450 CYP4A , Endotelio Vascular/patología , Hemodinámica/fisiología , Hipertensión Pulmonar/patología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Presión Esfenoidal Pulmonar/fisiología , Porcinos , Resistencia Vascular/fisiología , Sistema Vasomotor/patologíaRESUMEN
Rabbit airway tissue is a particularly rich source of cytochrome P-4504A protein, but very little information regarding the effect(s) of 20-hydroxyeicosatetraenoic acid (20-HETE) on bronchial tone is available. Our studies examined the response of rabbit bronchial rings to 20-HETE and the metabolism of arachidonic acid and 20-HETE from airway microsomes. 20-HETE (10(-8) to 10(-6) M) produced a concentration-dependent relaxation of bronchial rings precontracted with KCl or histamine but not with carbachol. Relaxation to 20-HETE was blocked by indomethacin or epithelium removal, consistent with the conversion of 20-HETE to a bronchial relaxant by epithelial cyclooxygenase. A cyclooxygenase product of 20-HETE also elicited relaxation of bronchial rings. [14C]arachidonic acid was converted by airway microsomes to products that comigrated with authentic 20-HETE (confirmed by gas chromatography-mass spectrometry as 19- and 20-HETE) and to unidentified polar metabolites. [3H]20-HETE was metabolized to indomethacin-inhibitable products. These data suggest that 20-HETE is an endogenous product of rabbit airway tissue and may modulate airway resistance in a cyclooxygenase-dependent manner.
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Bronquios/metabolismo , Broncodilatadores/metabolismo , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Ácido Araquidónico/metabolismo , Bronquios/efectos de los fármacos , Broncoconstricción/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Ácidos Hidroxieicosatetraenoicos/farmacología , Técnicas In Vitro , Indometacina/farmacología , Masculino , Microsomas/metabolismo , ConejosRESUMEN
Stop-flow studies were used to characterize solute uptake in isolated rat lungs. These lungs were perfused at 8 or 34 ml/min for 10-28 s with solutions containing 125I-albumin and two or more of the following diffusible indicators: [3H]mannitol, [14C]urea, 3HOH, 201Tl+, or 86Rb+. After this loading period, flow was stopped for 10-300 s and then resumed to flush out the perfusate that remained in the pulmonary vasculature during the stop interval. Concentrations of 201Tl+ and 86Rb+ in the venous outflow decreased after the stop interval, indicating uptake from exchange vessels during the stop interval. The amount of these K+ analogs lost from the circulation during the stop interval was greater when the intervals were longer. However, losses of 201T1+ at 90 s approached those at 300 s. Because extraction continued after the vasculature had been flushed, vascular levels had presumably fallen to negligible levels during the stop interval. By 90 s of stop flow the vascular volume that was cleared of 201T1+ averaged 0.657 +/- 0.034 (SE) ml in the experiments perfused at 8 ml/min and 0.629 +/- 0.108 ml in those perfused at 34 ml/min. Increases in perfusate K+ decreased the cleared volumes of 201T1+ and 86Rb+. Uptake of [3H]mannitol, [14C]urea, and 3HOH during the stop intervals was observed only when the lungs were loaded at high flow for short intervals. Decreases in 201T1+ and 86Rb+ concentrations in the pulmonary outflow can be used to identify the fraction of the collected samples that were within exchange vessels of the lung during the stop interval and may help determine the distribution of solute and water exchange along the pulmonary vasculature.
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Pulmón/metabolismo , Animales , Óxido de Deuterio/farmacocinética , Agua Pulmonar Extravascular/fisiología , Manitol/farmacocinética , Modelos Biológicos , Tamaño de los Órganos/fisiología , Circulación Pulmonar/fisiología , Capacidad de Difusión Pulmonar/fisiología , Radiofármacos/farmacocinética , Ratas , Radioisótopos de Rubidio/farmacocinética , Albúmina Sérica Radioyodada/farmacocinética , Soluciones , Radioisótopos de Talio/farmacocinética , Urea/farmacocinéticaRESUMEN
We previously reported that 20-hydroxyeicosatetraenoic acid (20-HETE) is an endogenous cytochrome P450 (cP450) 4A metabolite of arachidonic acid (AA) in human lung tissue, and is a potent cyclooxygenase-dependent vasodilator of isolated pulmonary arteries. In the present investigations, we identified sources of cP450 4A immunospecific protein, messenger RNA (mRNA), and 20-HETE synthesis in rabbit lungs. Microsomes of peripheral lung tissue, airways, small and large vessels, and lysates of alveolar macrophages all express proteins of approximately 50 kD which cross-reacted with a primary antibody raised against rat liver cP450 4A1. Peripheral lung tissue, small and large pulmonary arteries, airways, and isolated vascular smooth muscle cells from small pulmonary arteries produced 20-HETE when incubated with AA. Expression of cP450 4A6/4A7 mRNA was readily detectable by reverse transcription-polymerase chain reaction using isoform-specific probes and 5 microg total RNA extracted from microdissected small pulmonary arteries. These data demonstrate that small pulmonary arteries express cP450 4A proteins and vascular smooth muscle cells derived from these arteries synthesize 20-HETE. Furthermore, cP450 4A appears to be widely distributed in rabbit tissue, raising the possibility that 20-HETE generated from nonvascular tissue could serve as a paracrine factor in the pulmonary circulation.
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Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Pulmón/metabolismo , Oxigenasas de Función Mixta/metabolismo , Arteria Pulmonar/metabolismo , Animales , Ácido Araquidónico/metabolismo , Citocromo P-450 CYP4A , Pulmón/irrigación sanguínea , Macrófagos Alveolares/metabolismo , Masculino , Microsomas/metabolismo , Músculo Liso Vascular/metabolismo , Reacción en Cadena de la Polimerasa , ConejosRESUMEN
1. Cerebral arteries express cytochrome P450 4A enzymes (P450 4A) and produce 20- hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of pial arterioles. It is not known which cell type in the vessel wall is responsible for the formation of 20-HETE. We examined whether freshly isolated cerebral arterial muscle cells (VSMCs) express P450 4A and produce 20-HETE. We also studied the effect of 20-HETE on pressurized cerebral arteries and on whole-cell L-type Ca2+current (ICa) recorded in cat cerebral VSMCs. 2. Cat cerebral VSMCs incubated with [14C]arachidonic acid ([14C]AA) produced 20-HETE (3.9 +/- 1.1 pmol min-1 (mg protein)-1). 3. Reverse transcription-polymerase chain reaction studies revealed that cat cerebral VSMCs express mRNA for P450 4A which metabolizes AA to 20-HETE. Cloning and sequencing of the cDNA amplified from mRNA isolated from VSMCs showed > 96 % amino acid homology to the rat and human P450 4A2 and 4A3. 4. 20-HETE (1-300 nM) induced a concentration-dependent constriction of cat cerebral arteries, which was inhibited by nifedipine. 5. Addition of 10 and 100 nM 20-HETE to the bath increased peak ICa by 50 +/- 3 and 100 +/- 10 %, respectively. This effect was not influenced by altering the frequency of depolarization. 20-HETE (100 nM) failed to increase ICa in the presence of nifedipine. 6. These results demonstrate that cat cerebral VSMCs express P450 4A enzyme, and produce 20-HETE which activates L-type Ca2+ channel current to promote cerebral vasoconstriction.
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Arterias Cerebrales/fisiología , Sistema Enzimático del Citocromo P-450/biosíntesis , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacología , Oxigenasas de Función Mixta/biosíntesis , Músculo Liso Vascular/fisiología , Transcripción Genética , Vasoconstricción/fisiología , Secuencia de Aminoácidos , Animales , Ácido Araquidónico/metabolismo , Gatos , Arterias Cerebrales/metabolismo , Clonación Molecular , Citocromo P-450 CYP4A , Sistema Enzimático del Citocromo P-450/química , Cartilla de ADN , Humanos , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Oxigenasas de Función Mixta/química , Datos de Secuencia Molecular , Músculo Liso Vascular/metabolismo , Nifedipino/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
Recent evidence suggests that water transport between the pulmonary vasculature and air spaces can be inhibited by HgCl2, an agent that inhibits water channels (aquaporin-1 and -5) of cell membranes. In the present study of isolated rat lungs, clearances of labeled (3HOH) and unlabeled water were compared after instillation of hypotonic or hypertonic solutions into the air spaces or injection of a hypotonic bolus into the pulmonary artery. The clearance of 3HOH between the air spaces and perfusate after intratracheal instillation and from the vasculature to the tissues after pulmonary arterial injections was invariably greater than that of unlabeled water, indicating that osmotically driven transport of water is limited by permeability of the tissue barriers rather than the rate of perfusion. Exposure to 0.5 mM HgCl2 in the perfusate and air-space solution reduced the product of the filtration coefficient and surface area (PfS) of water from the air spaces to the perfusate by 28% after instillation of water into the trachea. In contrast, perfusion of 0.5 mM HgCl2 in air-filled lungs reduced PfS of the endothelium by 86% after injections into the pulmonary artery, suggesting that much of the action of this inhibitor is on the endothelial surfaces. Confocal laser scanning microscopy demonstrated that aquaporin-1 is on mouse pulmonary endothelium. No aquaporin-1 was found on alveolar type I cells with immunogold transmission electron microscopy, but small amounts were present on some type II cells.
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Acuaporinas , Agua Corporal/fisiología , Canales Iónicos/metabolismo , Pulmón/metabolismo , Animales , Acuaporina 1 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente Directa , Inmunoquímica , Inyecciones Intraarteriales , Canales Iónicos/administración & dosificación , Pulmón/efectos de los fármacos , Pulmón/ultraestructura , Cloruro de Mercurio/farmacología , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Microscopía Electrónica , Concentración Osmolar , Arteria Pulmonar/fisiología , Conejos , Ratas , Ratas Sprague-DawleyRESUMEN
We investigated the effect of 20-hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite of the cytochrome P-450 (cP450) 4A pathway, on human pulmonary arterial tone. 20-HETE elicited a dose-dependent and indomethacin-inhibitable vasodilation of isolated small pulmonary arteries. Whole lung microsomes metabolized [24C]arachidonic acid into 20-HETE and a variety of leukotrienes, epoxyeicosatrienoic acids, and prostanoids. Indomethacin blocked formation of prostanoids without effects on the conversion of arachidonate into 20-HETE, 20-HETE was converted by lung microsomes into prostanoids, raising the possibility that 20-HETE may be metabolized by cyclooxygenase enzymes in vascular tissue to a vasodilatory compound. Western blots probed with a polyclonal antibody to cP450 4A identified a protein of approximately 50 kDa immunologically similar to the cP450 4A in rat liver. We conclude that small arteries from human lungs dilate upon exposure to 20-HETE in a cyclooxygenase-dependent manner and that the proteins and enzymatic activity required to synthesize this product are present in lungs. Our observations suggest that cP450 enzyme products could be endogenous modulators of pulmonary vascular tone.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/farmacología , Arteria Pulmonar/efectos de los fármacos , Vasodilatación , Animales , Ácido Araquidónico/metabolismo , Gatos , Bovinos , Relación Dosis-Respuesta a Droga , Eicosanoides/metabolismo , Femenino , Hurones , Humanos , Técnicas In Vitro , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Conejos , RatasRESUMEN
The present study examined the effects of 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) on the diameter of small renal arteries of the rat and assessed their action on K(+)-channel activity in vascular smooth muscle (VSM) cells isolated from these vessels. The R,S-isomer of 11,12-EET (1, 10, and 100 nM) increased the diameter of small renal arteries preconstricted with phenylephrine; however, the S,R-isomer was inactive. Both the R,S- and S,R-isomers of 14,15-EET had little effect on the diameter of these vessels even at a high concentration (100 nM). The vasodilator effect of 11(R),12(S)-EET was attenuated by tetraethylammonium (TEA, 1 mM) and iberiotoxin (100 nM), selective inhibitors of the large-conductance Ca(2+)-activated K+ (KCa) channel. In contrast, apamin (100 nM) and 4-aminopyridine (2 mM), which are inhibitors of other types of K+ channels, had no effect on the vasodilatory effect of 11,12-EET. In patch-clamp experiments, 100 nM racemic 11,12-EET increased outward K+ currents in VSM cells. Addition of the R,S-isomer or racemic 11,12-EET (1-100 nM), but not the S,R-isomer, increased the activity of KCa channel recorded from renal VSM cells with cell-attached patches. However, racemic EET had no effect on this channel when added to the internal (inside-out) or external (outside-out) face of excised membrane patches. These results suggest that 11,12-EET is a potent dilator of small renal arteries and that the R,S-isomer is the active enantiomer. The vasodilator effect of 11,12-EET appears to involve activation of KCa channel.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Canales de Potasio/efectos de los fármacos , Arteria Renal/efectos de los fármacos , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Calcio/farmacología , Conductividad Eléctrica , Tono Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio/fisiología , Ratas , Ratas Sprague-Dawley , Arteria Renal/fisiología , Estereoisomerismo , VasoconstricciónRESUMEN
The present study examined the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and 17-octadecynoic acid (17-ODYA), an inhibitor of the metabolism of arachidonic acid by P-450, on K(+)-channel activity in vascular smooth muscle cells (VSM) isolated from renal arterioles of the rat. Two types of K+ channels were characterized using inside-out excised membrane patches. One channel exhibited a large conductance (250.3 +/- 5 pS), was activated by membrane depolarization and elevations in cytoplasmic Ca2+ concentration, and was blocked by low concentrations (< 1 mM) of tetraethylammonium (TEA). The other K+ channel exhibited an intermediate conductance (46.3 +/- pS), was activated by membrane depolarization but not by changes in intracellular Ca2+ concentration, and was blocked by 4-aminopyridine (5 mM). Addition of 20-HETE to the bath (1-100 nM), reduced the frequency of opening of the large-conductance Ca(2+)-activated K+ channel recorded using cell-attached patches on VSM. It had no effect on the intermediate-conductance K+ channel: 17-ODYA (1 microM) increased the activity of the large-conductance Ca(2+)-activated K+ channel, and this effect was reversed by 20-HETE (10 nM). 20-HETE (1-1000 nM) reduced the diameter of isolated perfused small renal arteries of the rat by approximately 15% TEA (1 mM) blocked the vasoconstrictor response to 20-HETE (100 nM). These studies suggest that 20-HETE is an endogenously formed vasoconstrictor that acts in part by inhibiting the opening of the large-conductance Ca(2+)-activated K+ channel in renal arteriolar VSM.
Asunto(s)
Calcio/fisiología , Ácidos Hidroxieicosatetraenoicos/farmacología , Bloqueadores de los Canales de Potasio , Circulación Renal/efectos de los fármacos , Animales , Ácido Araquidónico/antagonistas & inhibidores , Arteriolas/citología , Arteriolas/efectos de los fármacos , Arteriolas/fisiología , Sistema Enzimático del Citocromo P-450/farmacología , Conductividad Eléctrica , Ácidos Grasos Insaturados/farmacología , Técnicas In Vitro , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Ratas , VasoconstricciónRESUMEN
Single-channel K+ currents were characterized in vascular smooth muscle cells freshly isolated from preglomerular arterioles (15-40 microns OD) of the rat. Under conditions of symmetrical K+ (145 mM), two types of single-channel K+ currents with unitary slope conductances of 68 +/- 2.6 and 251 +/- 4.9 pS were recorded from excised inside-out membrane patches. The open state probability (NPo) of these two types of K+ channels was voltage sensitive and the channels were highly selective for K+ over Na+. Elevation of intracellular calcium concentration ([Ca2+]i) from 0.1 to 0.5 microM on the cytoplasmic face of inside-out patches increased the frequency of opening and the NPo of both the 68-pS and the 251-pS K+ channels. Application of ATP (0.1-1 mM) to the internal surface of inside-out patches had no effect on the activities of both channel types. Internally applied Ba2+ (1 mM) blocked both of these channels. Externally applied tetraethylammonium (0.1-0.3 mM) or charybdotoxin (50 nM) blocked both the 68-pS and the 251-pS K+ channels. Externally applied apamin (50 nM), however, selectively blocked the 68-pS K+ channel but had no effect on the frequency of opening of the 251-pS K+ channel. Apamin also reduced macroscopic K+ current recorded from voltage-clamped rat renal arteriolar muscle cells by 25-30%. These results indicate the coexistence of two types of Ca(2+)-activated K+ channels in the membranes of vascular smooth muscle cell isolated from renal preglomerular arterioles of the rat that differ in unitary conductances and pharmacological properties.