Broilers are susceptible to pulmonary hypertension syndrome (PHS; ascites syndrome) when their pulmonary vascular capacity is anatomically or functionally inadequate to accommodate the requisite cardiac output without an excessive elevation in pulmonary arterial pressure. The consequences of an inadequate pulmonary vascular capacity have been demonstrated experimentally and include elevated pulmonary vascular resistance (PVR) attributable to noncompliant, fully engorged vascular channels; sustained pulmonary arterial hypertension (PAH); systemic hypoxemia and hypercapnia; specific right ventricular hypertrophy, and right atrioventricular valve failure (regurgitation), leading to central venous hypertension and hepatic cirrhosis. Pulmonary vascular capacity is broadly defined to encompass anatomical constraints related to the compliance and effective volume of blood vessels, as well as functional limitations related to the tone (degree of constriction) maintained by the primary resistance vessels (arterioles) within the lungs. Surgical occlusion of 1 pulmonary artery halves the anatomical pulmonary vascular capacity, doubles the PVR, triggers PAH, eliminates PHS-susceptible broilers, and reveals PHS-resistant survivors whose lungs are innately capable of handling sustained increases in pulmonary arterial pressure and cardiac output. We currently are using i.v. microparticle injections to increase the PVR and trigger PAH sufficient in magnitude to eliminate PHS-susceptible individuals while allowing PHS-resistant individuals to survive as progenitors of robust broiler lines. The microparticles obstruct pulmonary arterioles and cause local tissues and responding leukocytes to release vasoactive substances, including the vasodilator NO and the highly effective vasoconstrictors thromboxane A(2) and serotonin [5-hydroxytryptamine (5-HT)]. Nitric oxide is the principal vasodilator responsible for modulating (attenuating) the PAH response and ensuing mortality triggered by i.v. microparticle injections, whereas microparticle-induced increases in PVR can be attributed principally to 5-HT. Our observations support the hypothesis that susceptibility to PHS is a consequence of anatomically inadequate pulmonary vascular capacity combined with the functional predominance of the vasoconstrictor 5-HT over the vasodilator NO. The contribution of TxA(2) remains to be determined. Selecting broiler lines for resistance to PHS depends upon improving both anatomical and functional components of pulmonary vascular capacity.
The pulmonary hypertensive response to pulmonary vascular obstruction caused by intravenously injected microparticles is amplified by pretreatment with N(omega)nitro-L-arginine methyl ester (L-NAME). The L-NAME prevents the synthesis of the potent vasodilator nitric oxide (NO) by inhibiting both the constitutive [endothelial NO synthase (eNOS or NOS-3)] and inducible [inducible NO synthase (iNOS or NOS-2)] forms of NO synthase. In the present study we used the selective iNOS inhibitor aminoguanidine (AG) to evaluate the role of iNOS in modulating the pulmonary hypertension (PH) triggered by microparticle injections. Experiment 1 was conducted to confirm the ability of AG to inhibit NO synthesis by iNOS in broiler peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide (LPS, endotoxin). Mononuclear leukocytes treated with LPS produced 10-fold more NO than untreated (control) cells. The LPS-stimulated production of NO was partially inhibited by L-NAME and was fully inhibited by AG, thereby confirming that AG inhibits LPS-mediated iNOS activation in broilers. In Experiment 2 we evaluated the responses of male progeny from a base population (MP Base) and from a derivative line selected for one generation from the survivors of an LD50 microparticle injection (MP Select). The pulmonary arterial pressure (PAP) was lower in MP Select than in MP Base broilers. Both lines exhibited similar percentage increases in PAP after microparticles were injected, and AG modestly amplified the PH triggered by microparticles in both lines. In Experiment 3 we evaluated the responses of male progeny from a second base population (PAC Base) and from a derivative line selected for 3 generations using the unilateral pulmonary artery clamp technique (PAC Select). The PAP was lower in PAC Select than in PAC Base broilers, and both lines exhibited similar percentage increases in PAP in response to the microparticles. The PH triggered by microparticles was not amplified by AG but was doubled by L-NAME. These experiments demonstrate that during the 30 min following pulmonary vascular entrapment of microparticles, iNOS modulated the PH elicited in broilers derived from the MP pedigree line, but not in broilers from the PAC pedigree line. Different NOS-mediated responses among broiler populations may affect pulmonary hemodynamic characteristics of broiler lines selected using i.v. microparticle injections.
Nitric oxide is a potent vasodilator synthesized from l-Arg by NO synthase (NOS). Constitutive NOS in endothelial cells (eNOS) produces transient bursts of NO in low but physiologically effective levels. Activated monocytes and macrophages express inducible NOS (iNOS), which produces copious quantities of NO. Previous studies showed that NO attenuates pulmonary hypertensive responses induced by i.v. injections of lipopolysaccharide (LPS) or cellulose microparticles (MP). The present study determined whether changes in plasma NO concentrations could be used to assess the time course of NO production in response to LPS or MP injections. Broilers were injected i.v. with 1 mL of PBS (control), 1 mL of LPS (1 mg/mL), or 0.4 mL of MP (0.02 g/mL). Plasma samples were collected from 10 different broilers per group at 15, 30, 45, and 60 min and at 2, 3, 4, 5, 6, 8, 10, and 12 h postinjection. Total plasma NO concentrations were analyzed by nitrate + nitrite assay. After PBS or MP injection, plasma NO levels did not change throughout the 12-h period. Nitric oxide measured in the plasma increased in LPS-injected broilers from 4.8 +/- 0.8 microM at 15 min to 46.6 +/- 5.7 microM by 4 h postinjection, reached peak levels of 85.1 +/- 10.6 microM at 5 h, and returned to baseline levels similar to PBS-injected broilers by 12 h postinjection. We conclude that LPS triggered widespread iNOS expression by circulating monocytes and macrophages, resulting in copious NO production as reflected by significant increases in total plasma NO. Proportionally few monocytes and macrophages responded to MP entrapped in pulmonary arterioles. Consequently, NO produced by iNOS in activated leukocytes or by eNOS in the pulmonary vasculature had a minimal impact on total plasma NO. Total plasma NO from broilers did reflect the time course of massive iNOS activation in response to LPS, but biologically relevant quantities of NO produced by iNOS and eNOS activated during the local inflammatory response to entrapped MPs were too low to affect total plasma NO concentrations.
Nitric oxide (NO) is a potent pulmonary vasodilator that modulates the pulmonary vasoconstriction and pulmonary hypertension (PH) triggered by bacterial lipopolysaccharide (LPS) in broilers. The amplitude and duration of the LPS-induced PH are markedly enhanced following pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME), which inhibits NO synthesis by both the constitutive (endothelial) and inducible (inflammatory) forms of nitric oxide synthase (eNOS and iNOS, respectively). In the present study L-NAME and the selective iNOS inhibitor aminoguanidine (AG) were administered to differentiate between iNOS and eNOS as the primary source of NO that attenuates the pulmonary vascular response to LPS. Clinically healthy male progeny from ascites-susceptible and ascites-resistant lines were anesthetized, and their pulmonary artery was cannulated. The initial pulmonary arterial pressure (PAP) was recorded, then the broilers either remained untreated (control group) or were injected i.v. with AG. Ten minutes later all birds received an i.v. injection of LPS, followed 40 min later by an i.v. injection of L-NAME. When compared with untreated controls, AG neither increased the baseline PAP nor did it increase or prolong the PH response to LPS. The ascites-susceptible broilers maintained a higher PAP than the ascites-resistant broilers throughout the experiment, and the ascites-resistant broilers exhibited greater relative increases in PAP in response to LPS than did the ascites-susceptible broilers. Within 40 min after the LPS injection, PAP subsided to a level that did not differ from the respective preinjection value for each line. Injecting L-NAME reversed the decline in PAP, and within 5 min PAP returned to hypertensive levels approaching the maximum peak PH response to LPS. The absence of any impact of AG coupled with the profound response to L-NAME indicates that NO synthesized by eNOS rather than iNOS likely modulated the acute (within 1 h) PH elicited by LPS. Evidently eNOS is activated by the increased shear stress exerted on the endothelium during the PH response to LPS, whereas LPS-mediated up-regulation of iNOS expression may take longer than 1 h before biologically effective quantities of NO are produced.
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