Thrombin generation in vascular tissue.

BACKGROUND: Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. METHODS AND RESULTS: In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pM) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. CONCLUSION: Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC.

Differential inducible nitric oxide synthase expression in systemic and pulmonary vessels after endotoxin.

Inducible nitric oxide synthase (iNOS) is associated with vascular hypocontractility in systemic vessels after endotoxin lipopolysaccharide (LPS) administration. Although lung iNOS is increased after LPS, its role in the pulmonary circulation is unclear. We hypothesized that whereas iNOS upregulation is responsible for LPS-induced vascular dysfunction in systemic vessels, iNOS does not play a significant role in the pulmonary artery (PA). Using isolated aorta (AO) and PA rings, we examined the effect of nonselective NOS inhibition [N(G)-monomethyl-L-arginine (L-NMMA); 100 micromol/l] and selective iNOS inhibition (aminoguanidine, AG; 100 micromol/l) on alpha(1)-adrenergic-mediated vasoconstriction (phenylephrine; 10(-9) to 10(-3) M) after LPS (Salmonella typhimurium, 20 mg/kg ip). We also determined the presence of iNOS using Western blot and immunohistochemistry. LPS markedly impaired AO contractility (maximal control tension 1,076 +/- 33 mg vs. LPS 412 +/- 39 mg, P < 0.05), but PA contractility was unchanged (control 466 +/- 29 mg vs. LPS 455 +/- 27 mg, P > 0.05). Selective iNOS inhibition restored the AO's response to vasoconstriction (LPS + AG 1,135 +/- 54 mg, P > 0.05 vs. control and P < 0.05 vs. LPS), but had no effect on the PA (LPS + AG 422 +/- 38 mg, P > 0.05 vs. control and LPS). Western blot and immunohistochemistry revealed increased iNOS expression in the AO after LPS, but iNOS was not detected in the PA. Our results suggest that differential iNOS expression after LPS in systemic and pulmonary vessels contributes to the phenomenon of sepsis/endotoxemia-induced systemic hypotension and pulmonary hypertension.

Interleukin-11 attenuates pulmonary inflammation and vasomotor dysfunction in endotoxin-induced lung injury.

Interleukin (IL)-11, like other members of the gp130 receptor class, possesses anti-inflammatory properties. We hypothesized that IL-11 pretreatment would attenuate endotoxin [lipopolysaccharide (LPS)]-induced lung inflammation and diminish injury to endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation that require cGMP in Sprague-Dawley rats. LPS (20 mg/kg ip) increased lung tumor necrosis factor (TNF)-alpha compared with the saline control (0.7 +/- 0.15 ng/g lung wet wt for control vs. 3.5 +/- 0.09 ng/g lung wet wt for LPS; P < 0.05). IL-11 (200 mg/kg ip) injected 10 min before LPS administration attenuated the LPS-induced lung TNF-alpha levels (1.6 +/- 0.91 ng/g lung wet wt; P < 0.05 vs. LPS). IL-11 also diminished LPS-induced lung neutrophil sequestration as assessed by myeloperoxidase units (2.1 +/- 0.25 U/g lung wet wt for saline and 15.6 +/- 2.02 U/g lung wet wt for LPS vs. 7.07 +/- 1.65 U/g lung wet wt for LPS plus IL-11; P < 0.05). Similarly, TNF-alpha binding protein (175 mg/kg) attenuated LPS-induced myeloperoxidase activity (6.04 +/- 0.14 U/g lung wet wt; P < 0.05). Both IL-11 and TNF-alpha binding protein similarly attenuated LPS-induced endothelium-dependent vasomotor dysfunction with improved relaxation responses to 10(-7) and 10(-6) M acetylcholine and A-23187 in phenylephrine-preconstricted isolated pulmonary artery rings (P < 0.05 vs. LPS). Endothelium-independent relaxation responses to sodium nitroprusside were also improved after LPS at 10(-6) M (P < 0.05 vs. LPS). Moreover, IL-11 decreased endotoxin-induced mortality in CF1 mice from 90 to 50% (P

Independent and combined effects of inhaled nitric oxide, liquid perfluorochemical, and high-frequency oscillatory ventilation in premature lambs with respiratory distress syndrome.

Acute lung injury caused by tidal volume ventilation in the premature lamb with respiratory distress syndrome (RDS) is characterized by progessive deterioration in gas exchange and lung inflammation. Inhaled nitric oxide (iNO) improves gas exchange and decreases lung neutrophil accumulation in premature lambs with RDS. Mechanical lung recruitment techniques such as high-frequency oscillatory ventilation (HFOV) and partial liquid ventilation (PLV) also decrease lung injury and improve gas exchange in experimental models of neonatal respiratory failure. We hypothesized that two lung recruitment strategies (HFOV and PLV) would have similar effects on gas exchange and lung inflammation, and would augment the response to iNO. We studied the individual and combined effects of iNO, HFOV, and PLV (perflubron) in 31 extremely premature lambs (115 d, 0.78 term) using seven mechanical ventilation protocols. Four groups were treated with conventional ventilation (control CV, CV + iNO, CV + PLV, and CV + PLV + iNO). Three groups were treated with HFOV (control HFOV, HFOV + iNO, HFOV + PLV). Control CV animals had progressive deterioration in gas exchange over the 4-h study period (a/AO2 at 4 h = 0.06 +/- 0.01). In contrast, both HFOV and CV + PLV caused sustained improvements in oxygenation at 4 h (HFOV a/AO2 = 0. 27 +/- 0.06, CV + PLV a/AO2 = 0.25 +/- 0.04; p < 0.01 versus CV). Both lung recruitment strategies improved oxygenation when combined with iNO (5 ppm). Lung neutrophil accumulation was reduced by HFOV, PLV, and iNO compared to CV. We conclude that HFOV and PLV with perflubron cause similar improvements in gas exchange and lung inflammation in the premature lamb with severe RDS, and both strategies augment the oxygenation response to iNO.

L-arginine attenuates endothelial dysfunction in endotoxin-induced lung injury.

BACKGROUND: Pulmonary vasorelaxation to endothelium-dependent and independent agonists is dysfunctional in endotoxin-induced acute lung injury. L-arginine is the precursor to endothelial production of nitric oxide (NO), suggesting that arginine and NO are intimately linked. We hypothesized that L-arginine would attenuate endotoxin-induced dysfunction of guanosine 3',5'-cyclic monophosphate-mediated pulmonary vasorelaxation. METHODS: Concentration-response curves were generated for acetylcholine, calcium ionophore A23187, and sodium nitroprusside (SNP) in isolated phenylepherine-preconstricted pulmonary artery rings (10(-9) to 10(-6) mol/L) 4 hours after endotoxin (500 mg/kg intraperitoneal) or saline injection. The effect of L-arginine in vitro was determined with L- or D-arginine (50 mmol/L) 30 minutes before dose response. RESULTS: Endothelium-dependent pulmonary vasorelaxation was dysfunctional after endotoxin injection as demonstrated by impaired responses to acetylcholine and A23187 (P < .05 vs control). Endotoxin-induced dysfunction of these endothelium-dependent responses was attenuated by L-arginine (P < .05 vs endotoxin). Endothelium-independent vasorelaxation (SNP) was also dysfunctional after endotoxin treatment (P < .05 vs control). L-arginine failed to attenuate the endotoxin-induced dysfunction of the response to SNP. The concentration responses for endothelium-dependent and independent vasorelaxing agonists in endotoxin-treated rats were not influenced by D-arginine. CONCLUSION: L-arginine supplementation attenuates endotoxin-induced dysfunction of endothelium-dependent pulmonary vasorelaxation.

Tissue-specific protein kinase C isoforms differentially mediate macrophage TNFalpha and IL-1beta production.

UNLABELLED: Macrophage subpopulations are differentially activated during sepsis, shock, or trauma; however, it is unknown whether inherent mechanistic and phenotypic differences exist between macrophage subpopulations that may account for region-specific inflammation. We hypothesized that macrophage expression/function of protein kinase C (PKC) isoforms is tissue specific (alveolar versus peritoneal). Rat alveolar and peritoneal macrophages were each probed for the expression of PKC isoforms alpha, beta1, beta2, gamma, delta, epsilon, zeta, and theta by immunoblot. PKC isoforms alpha, beta1, beta2, and zeta were detected in both populations; however, isoforms epsilon, gamma, and eta were found in alveolar macrophages only. To investigate the functional role of the Ca2+-dependent PKC (cPKC) versus Ca2+-independent PKC (nPKC) isoforms, pan-PKC isoform inhibition (cPKC and nPKC), or cPKC isoform selective inhibition (alpha, beta1, beta2, gamma) was performed before endotoxin (lipopolysaccharide, Salmonella minnesota, 100 ng/mL) stimulation in vitro. Pan-PKC isoform inhibition attenuated TNFalpha and IL-1beta production by each population; however, selective cPKC (alpha, beta1, beta2, gamma) inhibition decreased peritoneal, but not alveolar, macrophage TNFalpha production. IL-1beta production was not affected by cPKC inhibition in either population. CONCLUSIONS: 1) alveolar and peritoneal macrophages constitutively express different PKC isoforms; 2) alveolar macrophages uniquely express isoforms epsilon, gamma, eta; 3) TNFalpha production is regulated by cPKCs in peritoneal macrophages, but by nPKCs in alveolar macrophages; 4) nPKCs regulate IL-1beta production in both populations. These results suggest that tissue-specific PKC isoforms differentially mediate macrophage function, which may have important regulatory implications in the compartmentalization of immune function. Further understanding may allow region-specific manipulation of inflammation.

L-arginine prevents lung neutrophil accumulation and preserves pulmonary endothelial function after endotoxin.

L-Arginine supplementation has been shown to restore endothelium-derived nitric oxide production in several pathological states. The purpose of this study was to examine the effect of administration of exogenous L-arginine on the endotoxin-induced lung neutrophil accumulation and impairment of endothelium-dependent guanosine 3',5'-cyclic monophosphate (cGMP)-mediated pulmonary vasorelaxation in rats. Endothelium-dependent relaxation was tested by receptor-dependent [acetylcholine (ACh)] and receptor-independent (A-23187) pathways. Endothelium-independent relaxation was tested with sodium nitroprusside (SNP). In isolated pulmonary arterial rings, concentration-response curves were generated with ACh, A-23187, and SNP (10(-9) to 10(-6) M) 4 h after endotoxin (500 micrograms/kg i.p.) with and without prior administration of L-arginine (300 mg/kg i.p.). Lung neutrophil accumulation was determined by myeloperoxidase (MPO) assay. After endotoxin, lung neutrophil accumulation was significantly increased (MPO activity, 3.8 +/- 0.4 vs. 0.8 +/- 0.1 units/g lung weight in control cells; P < 0.05), which was prevented by L-arginine treatment (MPO activity, 1.3 +/- 0.3 units/g lung weight; P < 0.05 vs. endotoxin). Endotoxin produced a significant impairment of endothelium-dependent cGMP-mediated pulmonary vasorelaxation by receptor-dependent (ACh) and -independent (A-23187) pathways as well as of endothelium-independent relaxation (SNP). Prior treatment with L-arginine, but not with D-arginine, preserved endothelium-dependent vasorelaxation. Neither L- nor D-arginine influenced endotoxin-induced impairment of endothelium-independent, cGMP-mediated pulmonary vasorelaxation. We conclude that administration of exogenous L-arginine prevents endotoxin-induced lung neutrophil accumulation and attenuates its associated impairment of endothelium-dependent, cGMP-mediated pulmonary vasorelaxation.

Acute and chronic effects of bilateral lung transplantation without cardiopulmonary bypass on the first transplanted lung.

BACKGROUND: Bilateral lung transplantation (BLT) without cardiopulmonary bypass (CPB) may exacerbate reperfusion injury to the initially engrafted lung because of increases in pulmonary flow during implantation of the second graft. METHODS: In a retrospective review of 23 BLT patients, we hypothesized that BLT without CPB injures the first transplanted lung measured by acute and late graft dysfunction compared to the second transplanted lung. Of the 23 BLT, 19 underwent transplantation without CPB while 4 patients were placed on CPB secondary to hemodynamic instability. RESULTS: Acute graft function was assessed by radiographic scoring of lung quadrants (blinded radiologist; 0 = no infiltrate; 1 = infiltrate; maximum = 2 per lung) and by arterial/alveolar oxygen tension ratios (PaO2/ FiO2) ratios. Late graft function was evaluated by quantitative perfusion scan. Lung perfusion was graded as abnormal if less than 50% on the right or less than 45% on the left (Fisher's exact). Radiographic scores were not different between first and second implanted lungs at 1 and 24 hours, PaO2/FiO2 ratios at 1 and 24 hours were 273+/-26 and 312+/-23, respectively, and perfusion scans at 3 and 12 months revealed normal differential blood flow. CONCLUSIONS: These findings suggest no acute or chronic differences occur between the first or second transplanted lung completed without CPB.

KATP channels contribute to beta- and adenosine receptor-mediated pulmonary vasorelaxation.

ATP-sensitive K+ (KATP) channels have been implicated in the regulation of vasomotor tone in aortic, mesenteric, and pulmonary vascular smooth muscle. Several investigators have described an association between KATP channels and isoproterenol (Iso)-stimulated relaxation responses. To study the relationship between receptor-dependent pulmonary vasorelaxation and KATP channels, we examined the response to agonists that generate adenosine 3',5'-cyclic monophosphate at two distinct levels of the signal transduction pathway after inhibition or activation of KATP channels in isolated rat pulmonary artery rings. Cumulative concentration responses to beta-adrenergic receptor stimulation (Iso), purinergic receptor stimulation [adenosine (Ado)], and direct stimulation of adenylate cyclase [forskolin (FSK)] were studied with and without concurrent inhibition of KATP channels (glibenclamide or tolbutamide). In addition, the effect of direct KATP channel activation (cromakalim) on the response to beta-adrenergic and purinergic receptor stimulation was determined. Last, we investigated the influence of KATP channel inhibition on endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation linked to guanosine 3',5'-cyclic monophosphate production. KATP channel inhibition impaired the response to Iso and Ado. Activation of KATP channels caused a leftward shift in the dose responses of Iso and Ado, with a significant decrease in the 50% effective concentration for each agent. KATP channel inhibition did not impair the pulmonary arterial vasorelaxation response to FSK, acetylcholine, or sodium nitroprusside. KATP channels appear to contribute to beta-adrenergic and purinergic receptor-stimulated vasorelaxation in rat pulmonary arteries.

Hemorrhage activates myocardial NFkappaB and increases TNF-alpha in the heart.

The heart is a tumor necrosis factor (TNFalpha) producing organ. Locally (v systemically)-produced TNFalpha likely contributes to myocardial dysfunction via direct suppression of myocardial contractile function, the induction of myocardial apoptosis, and the genesis of cardiac hypertrophy. Although recent studies have demonstrated increased myocardial TNFalpha following endotoxemia, it remains unknown whether shock, in the absence of sepsis, activates myocardial nuclear factor kappa B (NFkappaB, a TNFalpha transcription factor) and/or increases TNFalpha in the heart. To study this, rats were hemorrhaged and resuscitated, after which hearts were harvested and analysed for evidence of NFkappaB activation (electrophoretic mobility shift assay) and assayed for TNFalpha levels. Hemorrhage and resuscitation activated NFkappaB and resulted in a dramatic increase in myocardial TNFalpha. This study constitutes the initial demonstration that hemorrhagic shock activates the signaling mechanisms which culminate in increased myocardial TNFalpha. Indeed, this may have important clinical implications, since hemorrhage is a frequent complication of both iatrogenic and accidental trauma, as well as a potent instigator of multiple organ failure.

LPS induces late cardiac functional protection against ischemia independent of cardiac and circulating TNF-alpha.

Lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha independently induce cardioprotection against ischemia in the rat at 24 h after administration, suggesting that endogenously synthesized TNF-alpha may play a role in LPS-induced protection. The purposes of this study were 1) to delineate the time course of LPS-induced cardiac functional protection against ischemia and its relation with myocardial and circulating TNF-alpha profile, 2) to examine whether prior protein synthesis inhibition abrogates the protection, and 3) to assess the effects of TNF-alpha inhibition and neutralization on the protection. Rats were treated with LPS (0.5 mg/kg i.p.). Cardiac functional resistance to normothermic global ischemia-reperfusion was examined at sequential time points after LPS treatment in isolated hearts by the Langendorff technique. Myocardial and circulating TNF-alpha was determined by enzyme-linked immunosorbent assay at 1-24 h after LPS treatment. Protection was apparent at 24 h, 3 days, and 7 days but not at 2 or 12 h. Maximal protection at 3 days was abolished by cycloheximide pretreatment (0.5 mg/kg i.p. 3 h before LPS treatment). Increases in myocardial and circulating TNF-alpha preceded the acquisition of protection. Dexamethasone pretreatment (4.0 or 8.0 mg/kg i.p. 30 min before LPS treatment) abolished peak increase in myocardial TNF-alpha and substantially suppressed circulating TNF-alpha (54.3 and 85.9% inhibition, respectively) without an influence on the maximal protection. Similarly, maximal protection was not affected by TNF binding protein (40 or 80 microg/kg i.v. immediately after LPS treatment). The results suggest that LPS-induced cardiac functional protection against ischemia is a delayed and long-lasting protective response that may involve de novo protein synthesis. Although LPS-induced increase in myocardial and circulating TNF-alpha precedes the delayed protection, it may not be required for the delayed protection.

Endotoxin stuns cGMP-mediated pulmonary vasorelaxation.

Net pulmonary vascular tone is determined by the balance of pulmonary vasorelaxation and vasoconstriction. In endotoxemic rats, cGMP-mediated pulmonary vasorelaxation is impaired through neutrophil-dependent mechanisms, yet agonist stimulated vasoconstriction remains intact. Endotoxin-induced lung neutrophil accumulation is a transient response. In models of myocardial ischemia-reperfusion injury, "stunning" or reversible cardiac dysfunction is also associated with a reversible neutrophil presence. We hypothesized that lung neutrophil accumulation and dysfunction of cGMP-mediated pulmonary vasorelaxation is reversible after an endotoxin challenge. Our purpose was to examine lung neutrophil accumulation and endothelium-dependent and -independent mechanisms of cGMP-mediated pulmonary vasorelaxation 4 and 48 h after endotoxin challenge. Rats (n = 5 per group) were studied 4 and 48 h after injection of saline or endotoxin (500 micrograms/kg, intraperitoneal). Endothelium-dependent relaxation by receptor-dependent (acetylcholine) and -independent (A23187) mechanisms and endothelium-independent (sodium nitroprusside) relaxation were studied in isolated pulmonary artery rings preconstricted with phenylephrine. Lung neutrophil accumulation was examined by lung myeloperoxidase assay. Lung neutrophil accumulation was increased at 4 h (p < .05 vs. control) and was attenuated by 48 h (p < .05 vs. endotoxin x 4 h) following endotoxin challenge. Similarly, the endotoxin-induced dysfunction of endothelium-dependent and -independent cGMP-mediated pulmonary vasorelaxation at 4 h normalized by 48 h. Endotoxin appears to induce reversible dysfunction of pulmonary vasorelaxation through stunning of vascular endothelial and smooth muscle cells.

Phosphodiesterase inhibition overcomes pulmonary vasomotor dysfunction in acute lung injury.

Production of cGMP is impaired in endotoxin-induced acute lung injury. This results in dysfunction of endothelium-dependent and -independent cGMP-mediated pulmonary vasorelaxation and, therefore, pulmonary hypertension. We hypothesized that cyclic nucleotide phosphodiesterase (PDE) inhibition would attenuate endotoxin-induced impairment to cGMP-mediated mechanisms of pulmonary vasorelaxation. The purpose was to examine the effect of stimulating cGMP production with concurrent inhibition of cGMP catabolism by PDE inhibition following endotoxin-induced acute lung injury. Isolated pulmonary arterial rings from rats (n = 5) were studied 6 hrs after endotoxin (20 mg/kg ip) or saline. In a third group (n = 5), PDE inhibition was accomplished with in vitro 3-isobutyl-1-methylxanthine (IBMX, 1 microM for 30 min). Cyclic GMP-mediated relaxation was interrogated by stimulating (1) endothelium-dependent mechanisms with the receptor-dependent agonist acetylcholine and the receptor-independent agonist A23187, a calcium ionophore, and an (2) endothelium-independent mechanism with sodium nitroprusside. PDE inhibition attenuated endotoxin-induced vasomotor dysfunction. A two-pronged approach-stimulating cGMP production and preventing cGMP catabolism with PDE inhibition-may offer a therapeutically accessible mechanism to overcome vasomotor dysfunction in acute lung injury.

Pentoxifylline treatment attenuates pulmonary vasomotor dysfunction in acute lung injury.

Acute lung injury (ALI) is characterized by pulmonary hypertension. Although the pathophysiology of ALI is complex, cytokine production, especially tumor necrosis factor-alpha (TNF-alpha), is known to mediate histologic lung injury. Pentoxifylline (PTX) is known to inhibit the expression of many cytokines, including TNF-alpha. The purpose of this study was to determine the effect of PTX treatment on endotoxin-induced impairment of endothelium-dependent mechanisms of pulmonary vasorelaxation. Mechanisms of endothelium-dependent relaxation were studied with the muscarinic receptor agonist, acetylcholine (ACh), and the receptor-independent calcium ionophore, A23187. Endothelium-independent pulmonary vasorelaxation was examined by direct stimulation of smooth muscle guanylate cyclase with the nitric oxide donor, sodium nitroprusside (SNP). Five rats received PTX (50 mg/kg) and endotoxin (20 mg/kg), endotoxin alone, or saline ip. After 6 hr, dose-response curves to ACh, A23187, and SNP were determined in isolated pulmonary artery rings preconstricted with phenylephrine (PE). PTX attenuated but did not eliminate endotoxin-induced impairment of endothelium-dependent and -independent pulmonary vasorelaxation. These data suggest that PTX may offer a therapeutic modality for the treatment of pulmonary hypertension in ALI.

Alpha-adrenergic activation of myocardial NF kappa B during hemorrhage.

Hemorrhage and resuscitation has been recognized as an exclusively destructive process which results in multiple organ dysfunction. Although it is well established that endogenous adaptation (preconditioning) mechanisms exist, it is unknown whether hemorrhage and resuscitation induces endogenous adaptive/protective mechanisms in the heart. Furthermore, alpha 1-adrenoceptors and nuclear factor kappa B (NF kappa B) have each been implicated in stress-induced signal transduction; however, whether they might be involved in hemorrhage-induced adaptive signal transduction remains unknown. This study tests the hypothesis that H/R activates myocardial NF kappa B and results in myocardial adaptation via alpha 1-adrenoceptors. Rats were briefly (10 min) hemorrhaged to 35 mmHg and resuscitated, sham operated, or neither, with and without prior alpha 1-adrenoceptor inhibition (prazosin). Hearts were then isolated and either probed for NF kappa B activation or subjected to a second insult consisting of global normothermic I/R (20 min/40 min). Antecedent hemorrhage and resuscitation activated myocardial NF kappa B and improved left ventricular developed pressure, coronary flow, and end diastolic pressure following ischemia-reperfusion (P < 0.05, ANOVA with Bonferroni-Dunn). Hemorrhage-induced adaptation was abolished by prior alpha 1-adrenoceptor blockade. This study constitutes the initial demonstration that H/R activates myocardial NF kappa B and induces adaptive signal transduction against ischemia-reperfusion injury.

Effects of inhaled nitric oxide on pulmonary edema and lung neutrophil accumulation in severe experimental hyaline membrane disease.

To determine the effects of inhaled NO (iNO) on pulmonary edema and lung inflammation in experimental hyaline membrane disease (HMD), we measured the effects of iNO on pulmonary hemodynamics, gas exchange, pulmonary edema, and lung myeloperoxidase (MPO) activity in extremely premature lambs (115 d of gestation, 0.78 term). In protocol 1, we measured the effects of iNO (20 ppm) on lung vascular endothelial permeability to 125I-labeled albumin (indexed to blood volume using 57Cr-tagged red blood cells) during 1 h (n = 10) and 3 h (n = 14) of conventional mechanical ventilation with FiO2 = 1.00. In comparison with controls, iNO improved pulmonary hemodynamics and gas exchange, but did not alter lung weight-to-dry weight ratio or vascular permeability to albumin after 1 or 3 h of mechanical ventilation. To determine whether low dose iNO (5 ppm) would decrease lung neutrophil accumulation in severe HMD, we measured lung MPO activity after 4 h of mechanical ventilation with or without iNO (protocol 2). Low dose iNO improved gas exchange during 4 h of mechanical ventilation (PaO2 at 4 h: 119 +/- 35 mm Hg iNO versus 41 +/- 7 mm Hg control, p < 0.05), and reduced MPO activity by 79% (p < 0.05). We conclude that low dose iNO increases pulmonary blood flow, without worsening pulmonary edema, and decreases lung neutrophil accumulation in severe experimental HMD. We speculate that in addition to its hemodynamic effects, low dose iNO decreases early neutrophil recruitment and may attenuate lung injury in severe HMD.

Protein kinase C isoform diversity in preconditioning.

Protein kinase C (PKC) appears to be a common intracellular effector and signal collector during cardiac preconditioning; however, it remains unknown whether agonists that activate different PKC isoforms are also linked to select aspects of myocardial protection. Using agonists that are known to activate unique combinations of PKC isoforms, we interrogated the relationship between isoform activation and the different aspects (pH, function, and viability) of endogenous myocardial protection. To study this, isolated rat hearts were subjected to ischemia-reperfusion (I/R) (20 min/40 min), without (control = Ctrl) or with receptor-dependent [phenylephrine (PE), 50 microM; adenosine (ADO), 125 microM] or -independent [phorbol myristate acetate (PMA), 100 nM] activation of PKC. Function, pH, and viability were assessed by rate pressure product (%RPP) and coronary flow (CF; ml/min), by 31P NMR, and by CF creatine kinase (CK; U/liter) leak, respectively. PMA, which activates PKC delta but not eta, resulted in intracellular pH (pHi) and viability protection, but did not protect against postischemic myocardial stunning. ADO, which activates PKC eta but not delta, protects against stunning, but not acidosis or necrosis. PE, which activates PKC delta and eta, provided global myocardial protection against necrosis, acidosis, and stunning. Different PKC isoforms may be linked to distinct aspects of myocardial protection. Targeted activation of PKC isoforms may allow precise mechanistic application of preconditioning-like myocardial protection.