The nitric oxide/cyclic GMP pathway in organ transplantation:

Transcription

1 Pro. Nati. ad. Si. US Vol. 91, pp , Deember 1994 Pharmaology The nitri oxide/yli GMP pathway in organ transplantation: Critial role in suessful lung preservation (endothelium/leukoyte) DVD J. PNSKY*t, YOSHFUM NK$, NPL C. CHOWDHURY, Hui LOt, MHMT C. OZ, ROBRT. MCHLR, UGNUSZ KUBSZWSK 1, TDUSZ MLNSK, ND DVD M. STRNt Departments of *Mediine, Surgery, and tphysiology, Columbia University College of Physiians and Surgeons, New York, NY 132; and Department of Chemistry and 'nstitute of Biotehnology, Oakland University, Rohester, M 4863 Communiated by Robert F. Furhgott, June 14, 1994 BSTRCT Reestablishment of vasular homeostasis following ex vivo preservation is a ritial determinant of suessful organ transplantation. Beause the nitri oxide (NO) pathway modulates pulmonary vasular tone and leukoyte/endothelial interations, we hypothesied that reative oxygen intermediates would lead to dereased NO (and hene GMP) levels following pulmonary reperfusion, leading to inreased pulmonary vasular resistane and leukostasis. Using an orthotopi rat model of lung transplantation, a porphyrini mirosensor was used to make diret in vivo measurements of pulmonary NO. NO levels measured at the surfae of the transplanted lung plummeted immediately upon reperfusion, with levels moderately inreased by topial appliation of superoxide dismutase. Beause GMP levels delined in preserved lungs after reperfusion, this led us to buttress the NO pathway by adding a membrane-permeant GMP analog to the preservation solution. Compared with grafts stored in its absene, grafts stored with supplemental 8-Br-GMP and evaluated 3 min after reperfusion demonstrated lower pulmonary vasular resistanes with inreased graft blood flow, improved arterial oxygenation, dereased neutrophil inffiltration, and improved reipient survival. These benefiial effets were dose dependent, mimiked by the type V phosphodiesterase inhibitor 2-o-propoxyphenyl-8-aapurin-6-one, and inhibited by a GMP-dependent protein kinase antagonist, the R isomer of 8-(4-hlorophenylthio)guanosine 3',5'-yi monophosphorothioate. ugmenting the NO pathway at the level of GMP improves graft funtion and reipient survival following lung transplantation. One of the major impediments to the transplantation of vasular organs has been the short interval during whih the organ an survive in transit from donor to reipient. This is espeially problemati during linial lung transplantation, where the inability to preserve lungs beyond 4-6 hr does not allow suffiient time for immunologi ross-mathing and impedes efforts at multiple or distant organ prourement (1). Current lung preservation strategies have foused on preventing oxygen free radial damage to the pulmonary parenhyma as well as optimiing eletrolyte and solute onentrations of the preservation solution, but lungs still fail after transplantation, with elevated pulmonary vasular resistane, neutrophil infiltration, and poor gas exhange as ardinal features (1). Nitri oxide (NO) released from endothelial ells maintains vasular homeostati properties (2) by relaxing vasular smooth musle (3), inhibiting neutrophil adhesivity (4) and platelet aggregation (5), and maintaining endothelial barrier properties (6). n the lungs, endogenously produed NO The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U.S.C solely to indiate this fat. stimulates basal GMP prodution and regulates pulmonary vasular tone (7). We hypothesied that diminished NO availability during the immediate reperfusion period might ontribute to the elevated pulmonary vasular resistane and neutrophil reruitment that ours after lung transplantation. These experiments were designed to measure pulmonary NO and GMP levels after reperfusion and to determine whether augmentation of the NO pathway at the level of GMP would enhane lung preservation for transplantation. MTRLS ND MTHODS Lung Preservation/Transplantation. Preservation solutions inluded latated Ringer's (LR) solution (Baxter Health Care, Deerfield, L) or modified uro-collins (C) solution (kidney preservation solution; Baxter Health Care) to whih magnesium sulfate (1 ml of a 1% solution) and gluose (5 ml of a 5% solution) were added to eah liter before use. 8-Bromoguanosine 3',5'-yi monophosphate (8-Br-GMP) was purhased from Sigma. The R isomer of 8-(4- hlorophenylthio)guanosine 3',5 '-yli monophosphorothioate (Rp-8-pCPT-GMP[S]) was purhased from Biolog (La Jolla, C), and 2-o-propoxyphenyl-8-aapurin-6-one (M&B 22948) was a kind gift from Rhone Poulen Rorer (ssex, U.K.). Orthotopi rat left lung transplantation was performed as desribed (8) with inbred male Lewis rats (25-3 g) and uff tehniques for vasular anastomoses. Millar atheters and a transonis flow probe were used to measure pulmonary artery (P) pressure (mmhg), blood flow (ml/min), and left atrial (L) pressure (mmhg) (8). Pulmonary vasular resistane (PVR) was alulated as (mean P pressure minus L pressure)/p flow. rterial blood gas sampling was performed via the left femoral artery during inspiration of 1% oxygen. fter baseline measurements, the native (right) P was ligated, and serial measurements (representative solely of the transplanted lung) were taken every 5 min for 3 minutes. Reipient death was identified by the essation of ardia mehanial ativity. Diret Measurement with NO letrode. Piees of rat lung of equal weight (4 ± 4 mg) were obtained fresh, after preservation (6 hr, LR solution), or after preservation/ transplantation (3-min reperfusion) and plaed in a vial ontaining 2 ml of LR solution (37C under ambient onditions). Differential pulse voltammograms (urrent vs. potential) with a pulse amplitude of 4 mv were obtained with a porphyrini mirosensor (9) plaed on the tissue surfae. saturated alomel referene eletrode and platinum wire bbreviations: Rp-8-pCPT-GMP[S], R isomer of 8-(4-hlorophenylthio)guanosine 3',5'-yli monophosphorothioate; P, pulmonary artery; PVR, pulmonary vasular resistane; C, uro-collins; LR, latated Ringer's. tto whom reprint requests should be addressed at: Columbia University, P&S , 63 West 168th Street, New York, NY

2 Pharmaology: Pinsky et (auxiliary eletrode) were immersed in the LR solution. The sensor is a working eletrode at whih oxidation of NO ours, resulting in an eletri urrent (whih flows between working and auxiliary eletrodes). The referene eletrode is used to monitor the potential of the working eletrode. n aqueous NO solution prepared as desribed (1) was used to alibrate the sensor. n vivo measurement of NO was similarly performed with a porphyrini sensor with a flexible base lowered onto the exposed surfae of the lung with a miromanipulator. small pieoeletri signal was used to indiate that the sensor had made ontat with the tissue surfae. NO measurements were begun one the flexible sensor assumed an L-shaped onfiguration on the tissue surfae and the platinum wire was onneted to adjaent tissue. Continuous amperometri (urrent time) measurements were obtained at a onstant potential of.68 V during the period of warm ishemia as the vasular ross-lamp was released and as alium ionophore (23187, 9.5 tkm; Sigma) and superoxide dismutase (1 units/ml; Sigma) were topially applied. Myeloperoxidase, NO Synthase, and GMP ssays. For myeloperoxidase assays, transplanted lungs were exised 3 min after restoration of blood flow, rinsed briskly in physiologi saline, and snap froen in liquid nitrogen until the time of assay, whih was performed aording to standard hromogeni tehniques (11). GMP and NO synthase assays were performed on lung tissue that was preserved for 6 hr in C solution and then snap froen either immediately or after a 3-min reperfusion (37C; 3 ml/min) with oxygenated LR solution. Tissue was assayed for GMP as desribed for 2nM, b % C%' Potential, V D._ - Q. - CL ~ 2.5 2K 1.5 F 1.5 F B y 8- w o O' P P/R Pro. Natl. ad. Si. US 91 (1994) 1287 MP (11) by radioimmunoassay (New ngland Nulear). Protein ontent was determined after solubiliing trihloroaeti aid-preipitated protein with SDS (2%). Results are reported as pmol of GMP per mg of protein and are expressed as means ± SM of dupliate determinations. lternatively, tissue NO synthase ativity was quantified by measuring the onversion of L-[3H]arginine to L-[3H]itrulline by thin-layer hromatography (12). L-[3H]Citrulline prodution was expressed as the perentage of ounts in the L-itrulline spot divided by the total number of ounts (L-arginine + L-ornithine + L-itrulline). Statistis. Data were analyed by Student's t tests or by NOV. Reipient survival data were evaluated by ontingeny analysis with the x2 statisti. Results are expressed as means ± SM, with P <.5 onsidered statistially signifiant. RSULTS Differential pulse voltammograms with a porphyrini mirosensor were used to diretly measure NO prodution by same-sied biopsies of fresh lungs (Fig. 1, urve a), lungs that had been preserved for 6 hr at 4 C in LR solution (urve b), as well as lungs that had been similarly preserved but reimplanted into a reipient and reperfused for 1 min (urve ). Fresh lung tissue generated onsiderable NO (7 ± 8 nm; Fig. 1B, bar 1), and lung tissue subjeted to preservation alone (i.e., no reimplantation) also produed NO, although at redued levels (2 ± 7 nm; bar 2). n ontrast, negligible C _ 1!g 3 4._.3-1 O J, 1U w FG. 1. () Representative differential pulse voltammograms (urrent onentration vs. potential) of NO obtained with a porphyrini mirosensor. Curves: a, fresh lung; b, lung preserved for 6 hr in LR solution;, lung preserved for 6 hr in LR solution and then transplanted. Solid lines and dashed lines represent voltammograms obtained in the absene and presene of superoxide dismutase (1 units/ml), respetively. (B) Mean onentration of NO in fresh lung (bar 1) (n = 6), lung preserved for 6 hr in LR solution (bar 2) (n = 7), lung preserved for 6 hr in LR solution, after whih NO was measured in the presene of superoxide dismutase (bar 3) (n = 7), lung preserved for 6 hr in LR solution and then transplanted/reperfused (bar 4) (n = 5; P <.1), and lung preserved for 6 hr in LR solution and then transplanted/reperfused but NO was measured in the presene of superoxide dismutase (bar 5) (n = 5). (C) mperogram (urrent onentration vs. time) reorded in vivo with a porphyrini mirosensor plaed on the surfae of a transplanted lung. (D) GMP levels measured by radioimmunoassay in lungs preserved for 6 hr in C solution (P) or preserved for 6 hr in C solution followed by reperfusion (P/R) (n = 5 for eah ondition). () NO synthase ativity as measured by prodution of L-[3H]itrulline from [3H]arginine, with onditions as desribed in D (n = 5 for eah ondition). Values shown are means ± SM; *, P <.5.

3 1288 Pharmaology: Pinsky et al. Pro. Natl. ad. Si. US 91 (1994) NO was measured from tissue samples from lungs that were identially preserved and then reimplanted (1 ± 8 nm; P <.1; bar 4). ddition of superoxide dismutase to the solution in whih preserved/reimplanted lungs were immersed during NO measurement resulted in a large inrease in detetable NO levels (-25-fold inrease; bar 5), whereas only a 5% inrease (bar 3) was observed with the addition of superoxide dismutase during NO measurement of preserved but nonreperfused lungs. Continuous diret measurements of NO were made in vivo before and after the onset of reperfusion using the porphyrini mirosensor in an amperometri mode plaed on the transplanted (6 hr 4C LR preservation) lung surfae (Fig. 1C). NO was measured prior to reperfusion and reording ontinued as the vasular ross-lamp was released. NO levels were onstant at 16 ± 25 nm after preservation, but this level plummeted immediately after the onset of reperfusion. During ontinued reperfusion, topial addition of the alium ionophore did not alter the deline in NO, whereas superoxide dismutase inreased detetable NO levels (from =25 to =4 nm). Similar measurements performed in vivo on normal lung showed a loal NO onentration of 66 ± 1 nm, whih inreased 1% in the presene of superoxide dismutase (to 72 ± 11 nm). Beause NO levels were dereased, GMP levels were measured in lungs reperfused with oxygenated LR solution in order to measure pulmonary GMP levels without the onfounding influene of platelet-derived GMP. These studies demonstrated lower GMP ontent of preserved lung tissue after reperfusion (Fig. 1D) despite unaltered synthesis of NO (Fig. 1). Beause GMP levels deline after reperfusion, we investigated whether replenishing this intraellular seond messenger would enhane graft vasular funtion and reipient survival following lung transplantation. Using an orthotopi rat lung transplant model (8), in whih graft funtion was inversely related to the duration of hypothermi preservation (Fig. 2), survival inreased from <15% to 86% (P <.1) after supplementation of LR solution with the membranepermeable GMP analog 8-Br-GMP. mmediately after reperfusion, lungs that had been preserved for 4 hr in LR solution in the absene of 8-Br-GMP demonstrated a marked inrease in PVR and a deline in P flow and Po2, followed invariably by a drop in P pressure and rapid reipient death (Fig. 2B Left). When 8-Br-GMP was added to the preservation solution, these parameters were stabilied and reipients survived (Fig. 2B Right). To better understand the linial relevane of these findings, lungs were stored in C solution, the standard solution for human lung preservation (1). Supplemental 8-Br-GMP (.5 mm) inreased P flow (from 2.2 ± 1.4 to 26 ± 2.3 ml/min; P =.6; Fig. 3), inreased Po2 (from 192 ± 68!! B b-.o 3 22, 91? - 12 N to 474 ± 41 mmhg; P =.4; Fig. 3B), dereased PVR (from 7.4 ± 2. to 1.1 ±.14 Woods units x 1-3; P =.1; Fig. 3C), and improved reipient survival (from 19% to 1%; P =.6; Fig. 3D). These effets of 8-Br-GMP were dose dependent over a range of.5-.5 mm, with maximal benefiial effets seen by.5 mm (Fig. 3D). When the GMP-speifi (type V) phosphodiesterase inhibitor M&B (13) was used, similar preservation enhanement was observed (Fig. 3). To demonstrate that enhaned pulmonary preservation was the result of stimulation of the GMPdependent protein kinase rather than an effet mediated by purinergi reeptors, the relatively seletive GMPdependent protein kinase antagonist Rp-8-pCPT-GMP[S] (14) was shown to blok the benefiial effets of 8-Br-GMP, and 8-bromoguanosine 5'-monophosphate was without effet (Fig. 3). Leukostasis is an important ontributor to tissue damage in a range of ishemi states (15), leading us to assess the effet of 8-Br-GMP on the presene of leukoytes in the lung grafts after 6 hr of preservation in C solution followed by transplantation. n these experiments, myeloperoxidase ativity was signifiantly diminished in lung grafts stored in the presene (n = 5) vs. the absene (n = 6) of8-br-gmp (4 nm/m.rin was 1.75 ±.13 vs ±.27, respetively; P =.6). DSCUSSON These studies demonstrate that NO availability is sharply diminished after preservation and reperfusion of a rihly vasularied organ suh as the lung, resulting in a fall in tissue GMP levels and aompanying hanges in vasular funtion. The fall in detetable NO ours in the fae of no loss of NO synthase ativity, indiating that aelerated destrution may be the major mehanism aounting for the observed deline in NO. lthough superoxide dismutase inreased NO levels, it did not restore NO to prereperfusion levels. This is not surprising, for several reasons. There is likely to be a spetrum of reative oxygen intermediates formed within the lungs by multiple oxidant-generating systems within endothelial ells, resident alveolar marophages, and reruited neutrophils. n addition, the reation of NO with superoxide is rapid and thus may effetively ompete with the dismutation of superoxide (16), and the large sie of superoxide dismutase may restrit its aessibility to sites of superoxide formation. nd, finally, the porphyrini mirosensor (with a response time of -1 mse) is apable of deteting only the unreated portion of NO (9). While the levels of NO measured both in vivo and ex vivo are onsiderably higher than the minimal NO onentrations that relax vasular smooth musle (<15 nm) (1), NO onentrations in the present study were measured diretly on (-) 8-Br-GMP N 6 N~~~~~~~~". Redoent Death 8 K! (+) 8-Br-GMP r )-----D...G a 3-2 'a OD -1.1= n... < < v ( ) Preservation Duration (hrs) Time fter Reperfusion (min) Time fter Reperfusion (min) FG. 2. () Time dependene of lung transplant reipient survival after hypothermi preservation in LR solution, with effet of 8-Br-GMP (.5 mm) at 4 hr. Mean perentage survival is shown. Left to right, n = 5, 21, 5, 14, and 14 transplants; *, P <.5 and **, P <.1 vs. 1-hr preservation;!!, P <.1 vs. 4-hr preservation. (B) Representative hemodynami traing of a lung transplant after preservation for 4 hr in LR solution (Left) or in LR solution supplemented with 8-Br-GMP (.5 mm; Right): PP, mean P pressure (mmhg, diamonds); PF, mean P flow (ml/min; triangles); PVR (Woods units x 1-3; squares); and Po2 (mmhg; irles). Time orresponds to ligation of native P.

4 Pharmaology: Pinsky et al. Pro. Natl. ad. Si. US 91 (1994) 1289 C ~._ C D 1[ C + 8-Br-GMP B *t x - 11 to G C + Ur-CUMr 1 -) LO 8 t 6 n n 4 2 C C C + 8-Br-GMP 8- -.Zl o CD 6 4 C'a C2 en o u U.Ub U.UO 8-Br-GMP Conentration (mm; FG. 3. ffet of 8-Br-GMP after 6 hr of preservation in C solution, with measurements reorded 3 min after reperfusion. () P flow. (B) Po2. (C) PVR. Means ± SM are shown (n = 6 for C solution, n = 5 for C solution + 8-Br-GMP; *, P <.5; **, P <.1; ***, P <.1). (D) Dose-response of 8-Br-GMP, showing maximal ability to enhane lung preservation at.5 mm. () Reipient survival 3 min after reperfusion. Grafts were preserved in C solution alone (leftmost bar; n = 16), in the presene of the 8-Br-GMP (.5 mm; n = 5), the GMP-speifi phosphodiesterase inhibitor M&B (2 mm; n = 3), the ombination of 8-Br-GMP (.5 mm) and the GMP-dependent protein kinase inhibitor Rp-8-pCPT-GMP[S] (5 mm; n = 2), or with nonyli 8-bromoguanosine 5'-monophosphate (.5 mm; n = 2). the tissue surfae. Beause under normal physiologi onditions NO produed at the endothelial surfae is rapidly diluted and dissipated in blood and tissue, the highest onentrations exist diretly at sites of NO prodution, suh as endothelial ells, with lower onentrations in nearby effetor ells (suh as smooth musle) (17). s the surfae onentration of NO is not affeted by the volume of the stati solution around the sensor, surrounding edema fluid in the urrent experiments should not affet these measurements. The observed deline in NO levels in vivo after reperfusion annot be explained by an inrease in temperature of the mirosensor, beause the donor lung ahieves a temperature nearly equal to that of the reipient prior to the onset of reperfusion, and the diffusion-ontrolled eletrohemial proess on whih the porphyrini sensor is based (18) (as well as the measured temperature oeffiient) predits an -1.8% inrease in urrent per 1PC temperature inrement, whereas NO levels dereased after reperfusion. The observed deline in NO availability following pulmonary reperfusion parallels the piture that emerges from ardia models of reperfusion in whih endothelium-derived relaxation fator bioativity delines due to formation of reative oxygen intermediates (19). Beause oxygen free radials an quenh NO and form toxi intermediates suh as peroxynitrite (16), we hose to supplement the NO/GMP pathway at the level of GMP. lthough GMP may be formed by pathways that do not involve NO (suh as by stimulation of membrane-assoiated guanylyl ylase by atrial natriureti peptide, enterotoxin, or by alium/ reoverin in retinal rods) (2), stimulation of soluble guanylate ylase by NO appears to be the major pathway of pulmonary GMP prodution, based on experiments in whih inhibition of NO synthesis abrogates the pulmonary vasorelaxant effets of seletive GMP phosphodiesterase inhibition (13). Supplementing GMP levels with 8-Br-GMP has signifiant benefiial effets on graft vasular funtion and reipient survival, effets that appear to be mediated by the GMP moiety, as (i) they are mimiked by the GMP phosphodiesterase inhibitor M&B 22948, whih raises endogenous levels of GMP by preventing its degradation (13); (ii) 8-bromoguanosine 5'-monophosphate was without effet; and (iii) the GMP-dependent protein kinase inhibitor Rp-8-pCPTGMP[S] (14) abrogated the benefiial effets of 8-Br-GMP. These data also suggest that peroxynitrite formed during reperfusion by the ombination of NO with superoxide (16) is not linially signifiant, as this reation would not be altered by 8-Br-GMP supplementation. The benefiial ations of 8-Br-GMP are not limited to vasodilation but extend to diminishing leukostasis and improving gas exhange as well. This is onsistent with previous reports that NO bloks leukoyte/endothelial interations (4) and promotes maintenane of vasular barrier funtions (6) and that mehanial depletion of neutrophils from the perfusate improves the funtion of isolated/reperfused lungs (21). nitial inhibitory effets of GMP supplementation on neutrophil reruitment into the reperfused graft may be further magnified by inreases in loal NO onentrations, whih further redue leukoyte adhesion via interations of NO with superoxide (22). These observations are likely to be relevant to the neutrophil plugging of ishemi/reperfused vessels, whih ontributes to the no-reflow phenomenon (23). While other agents that augment loal adenosine release from ishemi myoardium also vasodilate and redue neutrophil infiltration (24), the ations of 8-Br-GMP on lung preservation annot be asribed to similar ativation of purinergi reeptors, as 8-bromoguanosine was without effet, and a speifi GMP phosphodiesterase inhibitor that inreases intraellular GMP levels mimiked the benefiial effets of 8-Br-GMP. Diret-ating vasodilators alone (suh

5 129 Pharmaology: Pinsky et al. as hydralaine) have not proven benefiial in lung preservation (25), suggesting that vasodilation alone is insuffiient to enhane preservation. Like GMP, MP is a vasodilator that an interfere with neutrophil adhesion to endothelium (26). lthough these studies do not exlude a role for MP in lung preservation, the importane of GMP omes from several lines of evidene. The GMP analog used in this study (8-Br-GMP) is 5 times more potent at stimulating the GMP-dependent protein kinase than the MP-dependent protein kinase (27). n addition, 8-Br-GMP does not interat with the allosteri binding sites on the GMP-regulated phosphodiesterases (28) and hene does not affet MP hydrolysis in intat ell studies. The GMP-speifi (type V) phosphodiesterase inhibitor M&B used in our study augments the pulmonary vasodilating effets of NO (GMP)-dependent vasodilators, but not those of a MP-dependent vasodilator suh as isoproterenol (13), suggesting that its benefiial vasular effets are GMP mediated. Finally, the Rp-8-pCPTGMP[S] ompound (14) that bloked preservation enhanement in our study is relatively seletive for the GMPdependent protein kinase (Ki =.5 um vs. 8.3,uM for the MP-dependent protein kinase; H.-G. Genieser, personal ommuniation). Our results with 8-Br-GMP are onsistent with previous studies demonstrating that NO donors blunt myoardial injury and neutrophil aumulation during reperfusion (12). The present studies show that there is a rapid fall in NO levels upon reperfusion and that augmenting the NO pathway at the level ofgmp provides a useful pharmaologial approah to normalie vasular funtion in the ritial early stages following pulmonary ishemia. This work was supported in part by grants from the Cysti Fibrosis Foundation, the merian Heart ssoiation, and the Publi Health Servie (HL5629). 1. Kirk,. J. B., Colquhoun,. W. & Dark, J. H. (1993) nn. Thora. Surg. 56, gnarro, L., Ross, G. & Tillish, J. (1991) West. J. Med. 154, Furhgott, R. F. & Zawadki, J. V. (198) Nature (London) 288, Kubes, P., Suuki, M. & Granger, D. (1991) Pro. Natl. ad. Si. US 88, Radomski, M., Palmer, R. & Monada, S. (1987) Lanet ih, Kubes, P. & Granger, D. (1992) m. J. Physiol. 262, H Pro. Natl. ad. Si. US 91 (1994) 7. Barer, G., mery, C., Stewart,., Bee, D. & Howard, P. (1993) J. Physiol. (London) 463, Chowdhury, N. C., Naka, Y., Pinsky, D. J., Yano,. J., Smith, C. R., Rose,.., Stem, D. M., Mihler, R.. & O, M. C. (1994) Surg. Forum J. 45, Malinski, T. & Taha, Z. (1992) Nature (London) 358, Jia, L. & Furhgott, R. F. (1993) J. Pharmaol. xp. Ther. 267, Pinsky, D., O, M., Liao, H., Brett, J., Siaa, R., Karakurum, M., Van Lookeren Campagne, M., Platt, J., Nowygrol, R., Koga, S. & Stem, D. (1993) J. Clin. nvest. 92, Pinsky, D. J., O, M. C., Koga, S., Taha, Z., Broekman, M. J., Marus,. J., Liao, H., Naka, Y., Brett, J., Cannon, P. J., Nowygrod, R., Malinski, T. & Stem, D. M. (1994) J. Clin. nvest. 93, Braner, D.. V., Fineman, J. R., Chang, R. & Soifer, S. J. (1993) m. J. Physiol. 264, H252-H Butt,., Van Bemmelen, M., Fisher, L. & Walter, U. (199) FBS Lett. 263, dkins, W. & Taylor,. (199) J. ppl. Physiol. 69, Hogg, N., Darley-Usmar, V. M., Wilson, M. T. & Monada, S. (1992) Biohem. J. 281, Malinski, T., Taha, Z., Patton, S., Kapturak, M. & Tomboulina, P. (1993) Biohem. Biophys. Res. Commun. 193, Kissinger, P. T., Preddy, C. R., Shoup, R.. & Heineman, W. R. (1984) in Laboratory Tehniques in letroanalytial Chemistry, eds. Kissinger, P. T. & Heineman, W. R. (Dekker, New York), pp Lefer,. M., Tsao, P. S., Lefer, D. J. & Ma, X.-L. (1991) FSB J. 5, Bentley, J. K. & Beavo, J.. (1992) Curr. Opin. Cell Biol. 4, Pillai, R., Bando, K., Shueler, S., Zebly, M., Reit, B. & Baumgartner, W. (199) nn. Thora. Surg. 5, Gaboury, J., Woodman, R. C., Granger, D. N., Reinhardt, P. & Kubes, P. (1993) m. J. Physiol. 265, H862-H Jerome, S. N., Smith, C. W. & Korthuis, R. J. (1993) m. J. Physiol. 264, H479-H Gruber, H.., Hoffer, M.., Mllister, D. R., Laikind, P. K., Lane, T.., Shmid-Shoenbein, G. W. & ngler, R. L. (1989) Cirulation 8, Hahida, M. & Morton, D. L. (1988) J. Thora. Cardiovas. Surg. 95, Boxer, L., llen, J., Baehner, R. & mik, V. (198) J. Clin. nvest. 66, Corbin, J., Ogreid, D., Miller, J., Suva, R., Jastorff, B. & Doskeland, S. (1986) J. Biol. Chem. 261, Thomas, M. K., Franis, S. H., Beebe, S. J., Gettys, T. W. & Corbin, J. D. (1992) dv. Seond Messenger Phosphoprotein Res. 25,