ET-1-induced pulmonary vasoconstriction shifts from ET(A)- to ET(B)-receptor-mediated reaction after preconstriction.

Endothelin-1 (ET-1) has been reported to induce pulmonary vasoconstriction via either ET(A) or ET(B) receptors, and vasorelaxation after ET-1 injection has been observed. Our study investigated the effects of ET-1 in isolated rabbit lungs, which were studied at basal tone (part I) and after preconstriction (U-46619; part II). Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. In part I, ET-1 (10(-8) M; n = 6; control) was injected after pretreatment with the ET(A)-receptor antagonist BQ-123 (10(-6) M; n = 6) or the ET(B)-receptor antagonist BQ-788 (10(-6) M; n = 6). The same protocol was carried out in part II after elevation of pulmonary vascular tone. ET-1 induced an immediate PAP increase (DeltaPAP 4.3 +/- 0.4 mmHg at 10 min) that was attenuated by pretreatment with BQ-123 (P < 0.05 at 10 min and P < 0.01 thereafter) and that was more pronounced after BQ-788 (P < 0.01 at 10 min and P < 0.001 thereafter). In part II, ET-1 induced an immediate rise in PAP with a maximum after 5 min (DeltaPAP 6.3 +/- 1.4 mmHg), leveling off at DeltaPAP 3.2 +/- 0.2 mmHg after 15 min. Pretreatment with BQ-123 failed to attenuate the increase. BQ-788 significantly reduced the peak pressure at 5 min (0.75 +/- 0.4 mmHg; P < 0.001) as well as the plateau pressure thereafter (P < 0.01). We conclude that ET-1 administration causes pulmonary vasoconstriction independent of basal vascular tone, and, at normal vascular tone, the vasoconstriction seems to be mediated via ET(A) receptors. BQ-788 treatment resulted in even more pronounced vasoconstriction. After pulmonary preconstriction, ET(A) antagonism exerted no effects on PAP, whereas ET(B) antagonism blocked the PAP increase. Therefore, ET-1-induced pulmonary vasoconstriction is shifted from an ET(A)-related to an ET(B)-mediated mechanism after pulmonary vascular preconstriction.     

Late-phase bronchial vascular responses in allergic sheep

Sheep were classified on the basis of their airway response to Ascaris suum antigen aerosols as allergic or nonsensitive. Allergic sheep were classed as acute or dual responders. Acute responders had only an immediate increase in mean airflow resistance after antigen, whereas dual responders had an immediate and late-phase (6-8 h after antigen challenge) increase in mean airflow resistance; nonsensitive sheep had minimal airway responses to antigen (less than 30% increase from base line). The sheep were anesthetized 2 wk later and, after a left thoracotomy, were challenged with antigen to determine bronchial vascular responses; bronchial artery blood flow was measured with an electromagnetic flow probe. Airway responses to antigen aerosol challenge were similar in the anesthetized and conscious animals. The mean fall in bronchial vascular resistance (BVR) immediately after antigen challenge was similar in acute and dual responders (41 +/- 7 and 47 +/- 9% of base line, respectively). In dual responders, late-phase airway responses were preceded by a significant increase from base line in Qbr and a fall in bronchovascular resistance (BVR). The mean fall in BVR 6-8 h after antigen challenge in documented dual responders was significantly different from bronchial vascular responses in acute responders (59 +/- 3 vs. 89 +/- 10%, respectively). Sheep without airway responses to A. suum had no significant changes in bronchial hemodynamics or airways mechanics. Late-phase-associated changes in BVR are a specific response to antigen challenge and may be a sensitive index of mediators being released.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of bronchial arterial blood flow and bronchovascular resistance in sheep

We studied the bronchial arterial blood flow (Qbr) and bronchial vascular resistance (BVR) in sheep prepared with carotid-bronchial artery shunt. Nine adult sheep were anesthetized, and through a left thoracotomy a heparinized Teflon-tipped Silastic catheter was introduced into the bronchial artery. The other end of the catheter was brought out through the chest wall and through a neck incision was introduced into the carotid artery. A reservoir filled with warm heparinized blood was connected to this shunt. The height of blood column in the reservoir was kept constant at 150 cm by adding more blood. Qbr was measured, after interrupting the carotid-bronchial artery flow, by the changes in the reservoir volume. The bronchial arterial back pressure (Pbr) was measured through the shunt when both carotid-bronchial artery and reservoir Qbr had been temporarily interrupted. The mean Qbr was 34.1 +/- 2.9 (SE) ml/min, Pbr = 17.5 +/- 3.3 cmH2O, BVR = 3.9 +/- 0.5 cmH2O X ml-1 X min, mean pulmonary arterial pressure = 21.5 +/- 3.6 cmH2O, and pulmonary capillary wedge pressure (Ppcw) = 14.3 +/- 3.7 cmH2O. We further studied the effect of increased left atrial pressure on these parameters by inflating a balloon in the left atrium. The left atrial balloon inflation increased Ppcw to 25.3 +/- 3.1 cmH2O, Qbr decreased to 21.8 +/- 2.4 ml/min (P less than 0.05), and BVR increased to 5.5 +/- 1.0 cmH2O.ml-1.min (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hypoxia on bronchial circulation

We studied the effect of systemic hypoxia on the bronchial vascular pressure-flow relationship in anesthetized ventilated sheep. The bronchial artery, a branch of the bronchoesophageal artery, was cannulated and perfused with a pump with blood from a femoral artery. Bronchial blood flow was set so bronchial arterial pressure approximated systemic arterial pressure. For the group of 25 sheep, control bronchial blood flow was 22 ml/min or 0.7 ml.min-1.kg-1. During the hypoxic exposure, animals were ventilated with a mixture of N2 and air to achieve an arterial PO2 (PaO2) of 30 or 45 Torr. For the more severe hypoxic challenge, bronchial vascular resistance (BVR), as determined by the slope of the linearized pressure-flow curve, decreased acutely from 3.8 +/- 0.4 mmHg.ml-1.min to 2.9 +/- 0.3 mmHg.ml-1.min after 5 min of hypoxia. However, this vasodilation was not sustained, and BVR measured at 30 min of hypoxia was 4.2 +/- 0.8 mmHg.ml-1.min. The zero flow intercept, an index of downstream pressure, remained unaltered during the hypoxic exposure. Under conditions of moderate hypoxia (PaO2 = 45 Torr), BVR decreased from 4.6 +/- 0.3 to 3.8 +/- 0.4 mmHg.ml-1.min at 5 min and remained dilated at 30 min (3.6 +/- 0.5 mmHg.ml-1.min). To determine whether dilator prostaglandins were responsible for the initial bronchial vascular dilation under conditions of severe hypoxia (PaO2 approximately equal to 30 Torr), we studied an additional group of animals with pretreatment with the cyclooxygenase inhibitors indomethacin (2 mg/kg) and ibuprofen (12.5 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)     

An endothelin receptor antagonist,SB-217242, inhibits airway hyperresponsiveness in allergic mice

Within the airways, endothelin-1 (ET-1) can exert a range of prominent effects, including airway smooth muscle contraction, bronchial obstruction, airway wall edema, and airway remodeling. ET-1 also possesses proinflammatory properties and contributes to the late-phase response in allergic airways. However, there is no direct evidence for the contribution of endogenous ET-1 to airway hyperresponsiveness in allergic airways. Allergic inflammation induced in mice by sensitization and challenge with the house dust mite allergen Der P1 was associated with elevated levels of ET-1 within the lung, increased numbers of eosinophils within bronchoalveolar lavage fluid and tissue sections, and development of airway hyperresponsiveness to methacholine (P < 0.05, n = 6 mice per group). Treatment of allergic mice with an endothelin receptor antagonist, SB-217242 (30 mg x kg(-1) x day(-1)), during allergen challenge markedly inhibited airway eosinophilia (bronchoalveolar lavage fluid and tissue) and development of airway hyperresponsiveness. These findings provide direct evidence for a mediator role for ET-1 in development of airway hyperresponsiveness and airway eosinophilia in Der P1-sensitized mice after antigen challenge.     

Effect of left atrial pressure on bronchial vascular hemodynamics

We studied the bronchial vascular response to downstream pressure elevation by increasing left atrial pressure (Pla) and mean airway pressure (Paw) with positive end-expiratory pressure (PEEP). In seven pentobarbital-anesthetized ventilated sheep, we cannulated and perfused the bronchial branch of the bronchoesophageal artery. Steady-state bronchial artery pressure- (Pba) flow (Qba) relationships were obtained as Pla was increased by inflating a balloon catheter in the left atrium. Bronchial vascular resistance (BVR), determined by the inverse slope of the Pba-Qba relationship, increased significantly from 3.2 +/- 0.3 (SE) mmHg.ml-1.min-1 at a Pla of 2.9 +/- 0.7 mmHg to 5.1 +/- 0.5 mmHg.ml-1.min-1 at a Pla of 20.1 +/- 2.0 mmHg (P = 0.0007). Under control Qba (23.3 +/- 1.2 ml/min), these changes in BVR represent a 3.6 +/- 0.7-mmHg increase in Pba per mmHg increase in Pla. The zero-flow pressure increased 1.3 +/- 0.2 mmHg/mmHg increase in Pla. After infusion of papaverine, a smooth muscle paralytic agent, directly into the bronchial artery, BVR decreased significantly to 1.3 +/- 0.7 mmHg.ml-1.min-1 (P = 0.0004). Under these dilated conditions, BVR was unaltered by increases in Pla. After papaverine administration, Pba increased 0.9 +/- 0.1 and 1.2 +/- 0.1 mmHg/mmHg increase in Pla during control and zero-flow conditions, respectively. Thus the effect of Pla elevation on BVR appears to be dependent on active smooth muscle responses. Paw elevation had similar effects on Pba. Under control Qba, Pba increased 2.2 +/- 0.4 mmHg/mmHg increase in Paw.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interdependence of bronchial circulation and clearance of 99mTc-DTPA from the airway surface.

The extent to which the systemic vasculature is involved in soluble-particle uptake in the conducting airways has not been studied extensively. In anesthetized, ventilated sheep, 6-10 microl of technetium-99m-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA) was delivered through a microspray nozzle to a fourth-generation airway. Perfusion of the cannulated bronchial artery was varied between control flow (0.6 ml x min(-1) x kg(-1)), high flow (1.8 ml x min(-1) x kg(-1)) or no flow (the infusion pump was stopped). Airway retention of the radioactive tracer was monitored using gamma camera imaging, and venous blood was sampled. During control perfusion, tracer retention at the site of deposition at 30 min averaged 20 +/- 6% (n = 7). With no flow, retention was significantly elevated to 32 +/- 8% (P = 0.03). In another group of sheep (n = 5) with a control retention of 13 +/- 4%, high flow resulted in an increase in tracer (25 +/- 4%; P = 0.04). Maximum blood uptake of tracer was calculated by estimating circulating blood volume and averaged 16% of total activity during control flow. Only during high-flow conditions was 99mTc-DTPA in the blood decreased (10%; P = 0.04). Most of the tracer was cleared by mucociliary clearance as visualized by imaging. This component was substantially decreased during no flow. The results demonstrate that both decreased and increased airway perfusion limit removal of soluble tracer applied to the conducting airways.     

Paradoxical coronary microcirculatory constriction during ischemia: a synergic function for nitric oxide and endothelin

A paradoxical microcirculatory constriction has been observed in hearts of patients with ischemia, secondary to coronary stenosis. Here, using the isolated mouse heart (Langendorff), we examined the mechanism of this response, assuming involvement of nitric oxide (NO) and endothelin-1 (ET-1) systems. Perfusion pressure was maintained at 65 mmHg for 70 min (protocol 1), or it was reduced to 30 mmHg over two intervals, between the 20- and 40-min marks (protocol 2) or from the 20-min mark onward (protocol 3). In protocol 1, coronary resistance (CR) remained steady in untreated heart, whereas it progressively increased during treatment with the NO synthesis inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) (2.7-fold) or the ET(A) antagonist BQ-610 (2.8 fold). The ET(B) antagonist BQ-788 had instead no effect by itself but curtailed vasoconstriction to BQ-610. In protocol 2, hypotension raised CR by 2.2-fold. This response was blunted by reactive oxygen species (ROS) scavengers (mannitol and superoxide dismutase plus catalase) and was converted into vasodilation by l-NAME, BQ-610, or BQ-788. Restoration of normal pressure was followed by vasodilation and vasoconstriction, respectively, in untreated and treated preparations. In protocol 3, CR progressively increased with hypotension in the absence but not presence of L-NAME or BQ-610. We conclude that the coronary vasculature is normally relaxed by two concerted processes, a direct action of NO and ET-1 curtailing an ET(B2)-mediated tonic vasoconstriction through ET(A) activation. The negative feedback mechanism on ET(B2) subsides during hypotension, and the ensuing vasoconstriction is ascribed to ET-1 activating ET(A) and ET(B2) and reactive nitrogen oxide species originating from ROS-NO interaction.     

Interleukin-1beta and tumor necrosis factor-alpha mediation of endotoxin action on growth hormone

In humans and sheep, endotoxin (LPS) administration results in increased growth hormone (GH) concentrations. To determine the role of cytokines in the effect of LPS on GH, sheep were challenged with IL-1beta or TNF-alpha. GH data were compared with results with LH, where the major effects of LPS are known to act via the hypothalamus. Intracerebroventricular (icv) administration of IL-1beta or TNF-alpha did not alter plasma concentrations of GH. Endotoxin was then administered intravenously (iv) in combination with icv injection of IL-1 receptor antagonist (IL-1RA), TNF antagonist (sTNF-R1), or saline. Administration of LPS increased GH (P < 0.0001), although coadministration of IL-1ra or sTNF-R1 icv did not alter GH response to LPS. In contrast, plasma concentrations of LH were profoundly inhibited by icv administration of either cytokine (P < 0.03), but the LH response to LPS was not altered by cytokine antagonists. Intravenous administration of either IL-1beta or TNF-alpha increased plasma concentrations of GH (P < 0.0001). Administration of IL-1RA and sTNF-R1 iv prevented LPS-induced increases in GH. Although LH was suppressed by high iv doses of IL-1beta (P = 0.0063), the antagonists did not alter the LH response to LPS. To determine whether LPS might directly activate GH release, confocal microscopy revealed colocalization of CD14, the LPS receptor, with GH and, to a lesser extent, LH and some prolactin (PRL)-containing cells, but not ACTH or TSH. These data are consistent with the effects of LPS on GH secretion originating through peripheral cytokine presentation to the pituitary, as well as a potential to act directly on selective populations of pituitary cells via CD14.     

Effect of nephrectomy and of adrenergic receptor blockers on the cardiorenal actions of endothelin

The cardiorenal actions of endothelin-1 (ET-1) were evaluated in rats following nephrectomy, in rats during alpha-adrenergic blockade with phentolamine, and in rats during beta-adrenergic blockade with propranolol. Female rats were anesthetized with pentobarbital and, following surgery, were allowed 60 min to stabilize before 3 x 20 min-control clearances were collected. ET-1 was then infused at a rate of 100 ng kg-1 min-1 for 30 min, the infusion was stopped, and three additional clearances were collected. Four groups of rats were studied: in Group 1 (n = 10), ET-1 was infused; in Group 2 (n = 5), a bilateral nephrectomy was performed 120 min before infusing ET-1; in Group 3 (n = 5), ET-1 was infused into rats treated with phentolamine (0.015 mg kg-1 min-1); and in Group 4 (n = 5), ET-1 was infused into rats treated with propranolol (0.015 mg kg-1 min-1). At 30 min during infusion of ET-1 into Group 1 rats, mean arterial blood pressure had increased (P less than 0.01) by 27 +/- 2% (SE) and the glomerular filtration rate had decreased (P less than 0.01) by 71 +/- 6% of baseline values. Nephrectomy potentiated and prolonged the ET-1-induced systemic vasoconstriction. Phentolamine had no effect on the cardiorenal actions of ET-1 whereas propranolol enhanced ET-1-induced changes in mean arterial blood pressure; mean arterial blood pressure increased 38 +/- 2% at 30 min during ET-1 + propranolol infusion (P less than 0.01 versus value with ET-1 alone). These data indicate that the kidney affects ET-1-induced systemic vasoconstriction and that beta-adrenergic (but not alpha-adrenergic) receptors are activated during infusion of ET-1 with a resultant attenuation of ET-1-induced changes in systemic blood pressure.     

Effect of endothelin antagonism on the production of cytokines in eosinophilic airway inflammation

Endothelin (ET)-1 has been launched as an important mediator in bronchial asthma, which is an eosinophilic airway inflammation. However, the interplay between ET-1 and other proinflammatory mediators during the development of airway inflammation has not been elucidated. We wanted to study 1) whether the production of ET-1 precedes the production of other proinflammatory mediators and 2) whether ET-1 stimulates the production of these mediators within the airways. These hypotheses were studied during the development of an eosinophilic airway inflammation in rats. The increase in ET-1 mRNA level in lung tissue preceded the increase in mRNA levels of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-8. Treatment of the animals with the ET receptor antagonist bosentan resulted in a substantial decrease in the concentrations of tumor necrosis factor-alpha, IL-4, IL-1beta, interferon-gamma, and ET-1 in bronchoalveolar lavage fluid. In conclusion, the synthesis of ET-1 as measured by increased mRNA level precedes the synthesis of other proinflammatory cytokines of importance for the development of an eosinophilic airway inflammation, and ET antagonism inhibits the production of these mediators within the airways. Whether treatment with ET antagonists will prove beneficial for patients with eosinophilic airway inflammations like bronchial asthma is not yet known.     

Insulin sensitivity and big ET-1 conversion to ET-1 after ETA- or ETB-receptor blockade in humans.

Cardiovascular diseases are characterized by insulin resistance and elevated endothelin (ET)-1 levels. Furthermore, ET-1 induces insulin resistance. To elucidate this mechanism, six healthy subjects were studied during a hyperinsulinemic euglycemic clamp during infusion of (the ET-1 precursor) big ET-1 alone or after ET(A)- or ET(B)-receptor blockade. Insulin levels rose after big ET-1 with or without the ET(B) antagonist BQ-788 (P < 0.05) but were unchanged after the ET(A) antagonist BQ-123 + big ET-1. Infused glucose divided by insulin fell after big ET-1 with or without BQ-788 (P < 0.05). Insulin and infused glucose divided by insulin values were normalized by ET(A) blockade. Mean arterial blood pressure rose during big ET-1 with or without BQ-788 (P < 0.001) but was unchanged after BQ-123. Skeletal muscle, splanchnic, and renal blood flow responses to big ET-1 were abolished by BQ-123. ET-1 levels rose after big ET-1 (P < 0.01) in a similar way after BQ-123 or BQ-788, despite higher elimination capacity after ET(A) blockade. In conclusion, ET-1-induced reduction in insulin sensitivity and clearance as well as splanchnic and renal vasoconstriction are ET(A) mediated. ET(A)-receptor stimulation seems to inhibit the conversion of big ET-1 to ET-1.     

Effect of bronchial artery blood flow on cardiopulmonary bypass-induced lung injury

Cardiovascular surgery requiring cardiopulmonary bypass (CPB) is frequently complicated by postoperative lung injury. Bronchial artery (BA) blood flow has been hypothesized to attenuate this injury. The purpose of the present study was to determine the effect of BA blood flow on CPB-induced lung injury in anesthetized pigs. In eight pigs (BA ligated) the BA was ligated, whereas in six pigs (BA patent) the BA was identified but left intact. Warm (37 degrees C) CPB was then performed in all pigs with complete occlusion of the pulmonary artery and deflated lungs to maximize lung injury. BA ligation significantly exacerbated nearly all aspects of pulmonary function beginning at 5 min post-CPB. At 25 min, BA-ligated pigs had a lower arterial Po(2) at a fraction of inspired oxygen of 1.0 (52 +/- 5 vs. 312 +/- 58 mmHg) and greater peak tracheal pressure (39 +/- 6 vs. 15 +/- 4 mmHg), pulmonary vascular resistance (11 +/- 1 vs. 6 +/- 1 mmHg x l(-1) x min), plasma TNF-alpha (1.2 +/- 0.60 vs. 0.59 +/- 0.092 ng/ml), extravascular lung water (11.7 +/- 1.2 vs. 7.7 +/- 0.5 ml/g blood-free dry weight), and pulmonary vascular protein permeability, as assessed by a decreased reflection coefficient for albumin (sigma(alb); 0.53 +/- 0.1 vs. 0.82 +/- 0.05). There was a negative correlation (R = 0.95, P < 0.001) between sigma(alb) and the 25-min plasma TNF-alpha concentration. These results suggest that a severe decrease in BA blood flow during and after warm CPB causes increased pulmonary vascular permeability, edema formation, cytokine production, and severe arterial hypoxemia secondary to intrapulmonary shunt.     

Endothelin in the perinatal circulation

During the fetal period, blood is oxygenated through the placenta, and most of the cardiac output bypasses the lung through the ductus arteriosus. At birth, pulmonary vascular resistance falls with the initiation of ventilation. Coincidentally, the ductus arteriosus constricts. Endothelin-1 (ET-1) appears to play an important role during that transition period and postnatally. ET-1 can dramatically increase resistance in the placental microcirculation and may be involved in blood flow redistribution with hypoxia. At birth, the increase in oxygen tension is important in triggering ductus vasoconstriction. It is proposed that oxygen triggers closure of the ductus arteriosus by activating a specific, cytochrome P450-linked reaction, which in turn stimulates the synthesis of ET-1. On the neonatal heart, ET-1 has a positive chronotropic but negative inotropic effect. In the newborn piglet and the fetal lamb, both term and preterm, ET-1 causes a potent, long-lasting pulmonary vasoconstriction. Furthermore, a transient dilator response has been identified, and it is ascribed to nitric oxide formation. ET receptors are abundant in the piglet pulmonary vasculature. They are predominantly of the ETA constrictor subtype, though ETB2 constrictor receptors may also be present in certain species. The dilator response is linked to the ETB1 receptor, and the number of ETB1 receptors is reduced in hypoxia-induced pulmonary hypertension. ET-1 appears to be a causative agent in the pathogenesis of hypoxia- and hyperoxia-induced pulmonary hypertension as demonstrated by reversal of hemodynamic and morphological changes with treatment with an ETA receptor antagonist. Findings are amenable to practical applications in the management of infants with pulmonary hypertension or requiring persistent patency of the ductus arteriosus.     

Effect of in vivo fetal infusion of dexamethasone at 0.75 GA on fetal ovine resistance artery responses to ET-1

At 110-111 days gestation, instrumented fetal sheep were administered saline or dexamethasone (2.2 microgram. kg(-1). h(-1) iv) for 48 h. Measurement of fetal blood pressure showed a greater increase in dexamethasone-treated (n = 6) compared with control (n = 5) fetuses (7.3 +/- 2.3 vs. 0.6 +/- 2.3 mmHg, P < 0.05). Fetuses were delivered by cesarean section, and the femoral muscle and brain were obtained under halothane anesthesia. Femoral and middle cerebral arteries (approximately 320-micrometer internal diameter) were evaluated using wire myography. Sensitivity to KCl (2.5-125 mM) and the magnitude of the maximal vasoconstriction to 125 mM K(+) were similar in femoral and middle cerebral arteries from dexamethasone-treated vs. control fetuses. Acetylcholine-induced vasorelaxation was similar in femoral arteries from control and dexamethasone-treated fetuses. Middle cerebral arteries did not relax to acetylcholine. Sensitivity to endothelin-1 (ET-1; 0.1 pM-0.1 microM) and magnitude of the ET-1-induced vasoconstriction were greater in femoral arteries from dexamethasone-treated vs. control fetuses (P < 0.05). Autoradiographical studies with receptor-specific ligands demonstrated increased ET(A)-receptor binding, the principal receptor subtype, in femoral muscle vessels (P < 0.001) but decreased ET(A)-receptor binding in middle cerebral arteries (P < 0.01) from dexamethasone-treated compared with control fetuses. Relatively little ET(B)-receptor binding was evident in all tissues examined. We conclude that hyperreactivity to ET-1, due to increased ET(A)-receptor binding, may be involved in the dexamethasone-induced increase in peripheral vascular resistance in fetal sheep in vivo.     

Response of the bronchial circulation to acute hypoxemia and hypercarbia in the dog

We have examined the effect of acute hypoxemia and hypercarbia on bronchial blood flow (Qbr) in 10 anesthetized, ventilated, open-chest dogs using a modification of the radioactive microsphere technique. After surgery, dogs were divided into two groups of five. Group 1 was ventilated for 30 min with each of the following gas mixtures: 1) room air; 2) 15% O2-85% N2; 3) 10% O2-90% N2, and group 2 with 1) room air; 2) 5% CO2-30% O2-65% N2; 3) 10% CO2-30% O2-60% N2. Measurements of pulmonary arterial, left atrial and aortic pressures, cardiac output, and blood gases were made before injection of 46Sc-, 153Gd-, and 103Ru-labeled microspheres into the left atrium as a marker of Qbr. After the final measurements, dogs were killed and the lungs removed and the parenchyma stripped off the large and small airways of the left lung. Knowing the radioactivity in the trachea, bronchi, parenchyma, and in the blood from the reference-flow sample and also the aortic and left atrial pressures, total and regional Qbr, and bronchovascular resistance (BVR) were calculated. Results showed that acute hypoxemia (10% O2) caused a significant (P less than 0.05) decrease in Qbr and increase in BVR and acute hypercarbia (10% CO2) caused a significant (P less than 0.05) increase in Qbr and decrease in BVR.     

Hyperinsulinemia fails to augment ET-1 action in the skeletal muscle vascular bed in vivo in humans

Endogenous endothelin action is augmented in human obesity and type 2 diabetes and contributes to endothelial dysfunction and impairs insulin-mediated vasodilation in humans. We hypothesized that insulin resistance-associated hyperinsulinemia could preferentially drive endothelin-mediated vasoconstriction. We applied hyperinsulinemic-euglycemic clamps with higher insulin dosing in obese subjects than lean subjects (30 vs. 10 mU.m(-2).min(-1), respectively), with the goal of matching insulin's nitric oxide (NO)-mediated vascular effects. We predicted that, under these circumstances, insulin-stimulated endothelin-1 (ET-1) action (assessed with the type A endothelin receptor antagonist BQ-123) would be augmented in proportion to hyperinsulinemia. NO bioactivity was assessed using the nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine. Insulin-mediated vasodilation and insulin-stimulated NO bioavailability were well matched across groups by this approach. As expected, steady-state insulin levels were approximately threefold higher in obese than lean subjects (109.2 +/- 10.2 pmol/l vs. 518.4 +/- 84.0, P = 0.03). Despite this, the augmentation of insulin-mediated vasodilation by BQ-123 was not different between groups. ET-1 flux across the leg was not augmented by insulin alone but was increased with the addition of BQ-123 to insulin (P = 0.01 BQ-123 effect, P = not significant comparing groups). Endothelin antagonism augmented insulin-stimulated NO bioavailability and NOx flux, but not differently between groups and not proportional to hyperinsulinemia. These findings do not support the hypothesis that insulin resistance-associated hyperinsulinemia preferentially drives endothelin-mediated vasoconstriction.     

Endothelin-1 and endothelin-converting enzyme-1 in human granulomatous pathology of eyelid: an immunohistochemical and in situ hybridization study in chalazia

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in several functions of eye pathophysiology, such as regulation of intraocular tension and retinal reactive vasoconstriction. As ET-1 pro-inflammatory and fibrosing activity is emerging in different fields of pathology, we investigated the expression of ET-1 and endothelin-converting enzyme-1 (ECE-1) in chalazia, granulomatous lesions of the eyelid. ET-1 and ECE-1 were analyzed by immunohistochemistry (IHC) in twenty surgically removed chalazia, with regard to expression in eyelid structures and inflammatory infiltrate. Phenotype of ET-1 expressing inflammatory cells was established by double immunofluorescence. The cellular localization of prepro-ET-1 (pp-ET-1) mRNA and ECE-1 mRNA was studied by nonisotopic in situ hybridization (ISH). Neutrophils (PMNs), macrophages and T-lymphocytes were scattered in stroma, around alveoli and grouped in lipogranulomas. PMNs, macrophages, basal epithelium of meibomian adenomers and central ducts immunostained for ET-1. ECE-1 protein was found in meibomian adenomers, conjunctival epithelium, tarsal mucous glands and in inflammatory cells. Hybridization signals for pp-ET-1 mRNA and ECE-1 mRNA were recognized in healthy and degenerating meibomian ducts, adenomers, inflammatory cells, as well as in vessel walls. ECE-1 mRNA was also present in conjunctival epithelium and Henle's crypts. Our findings suggest that the multifunctional peptide ET-1 may have a role in molecular genesis of tissue damage in chalazia. ET-1 cytokine activity is likely to support the migration of inflammatory cells and the setting of lipogranulomas; ET-1 stimulation might contribute to proliferation of fibroblasts and collagen synthesis. ET-1 upregulation on meibomian adenomers and ducts may further enhance granulomas formation by stimulating lipid release.     

Minimal lung and systemic responses to TNF-alpha in preterm sheep

TNF-alpha has been associated with chorioamnionitis and the subsequent development of bronchopulmonary dysplasia in preterm infants. We asked whether bioactive recombinant ovine TNF-alpha could induce chorioamnionitis, lung inflammation, lung maturation, and systemic effects in fetal sheep. We compared the responses to IL-1alpha, a cytokine known to induce these responses in preterm sheep. Intra-amniotic TNF-alpha caused no chorioamnionitis, no lung maturation, and a very small increase in inflammatory cells in the fetal lung after 5 h, 2 days (d), and 7 d. In contrast, IL-1alpha induced inflammation and lung maturation. TNF-alpha given into the airways at birth increased granulocytes in the bronchoalveolar lavage fluid of ventilated preterm lungs and decreased the mRNA for surfactant protein C but did not adversely effect postnatal lung function. An intravascular injection of IL-1alpha caused a systemic inflammatory response in fetal sheep, whereas there was no fetal response to intravascular TNF-alpha. Fetal and newborn preterm sheep are minimally responsive to TNF-alpha. Therefore, the presence of a mediator such as TNF-alpha in a developing animal does not necessarily mean that it is causing the responses anticipated from previous results in adult animals.     

Endothelin-3-dependent pulmonary vasoconstriction in monocrotaline-induced pulmonary arterial hypertension

Blockade of the endothelin (ET) system is beneficial in pulmonary arterial hypertension (PAH). The contribution of ET-3 and its interactions with ET receptors have never been evaluated in the monocrotaline (MCT)-induced model of PAH. Vasoreactivity of pulmonary arteries was investigated; ET-3 localization was determined by confocal imaging and gene expression of prepro-ET-3 quantified using RT-PCR. ET-3 plasma levels tended to increase in PAH. ET-3 localized in the media of pulmonary arteries, where gene expression of prepro-ET-3 was reduced in PAH. ET-3 induced similar pulmonary vasoconstrictions in sham and PAH rats. In sham rats, the ET(A) antagonist A-147627 (10nmol/l) significantly reduced the maximal response to ET-3 (E(max) 77+/-1 to 46+/-2%, mean+/-S.E.M., P<0.001), while the ET(B) antagonist A-192621 (1mumol/l) reduced the sensitivity (EC(50) 21+/-7 to 59+/-16nmol/l, P<0.05) without affecting E(max). The combination of both antagonists completely abolished ET-3-induced pulmonary vasoconstriction. In PAH, the ET(A) antagonist further reduced the maximal response to ET-3 and shifted the EC(50) (E(max) 23+/-2%, P<0.001, EC(50) 104+/-24nmol/l, P<0.05), while the ET(B) antagonist only shifted the EC(50) (123+/-36nmol/l, P<0.05) without affecting the E(max). In PAH, dual ET receptor inhibition did not further reduce constriction compared to selective ET(A) inhibition. ET-3 significantly contributes to pulmonary vasoconstriction by activating the ET(B) at low concentration, and the ET(A) at high concentration. The increased inhibitory effect of the ET(A) antagonist in PAH suggests that the contribution of ET(B) to ET-3-induced vasoconstriction is reduced. Although ET-3 is a potent pulmonary vasoconstrictor in PAH, its potential pathophysiologic contribution remains uncertain.