List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Distant effects of nitric oxide inhalation in endotoxemic pigs

Manja C. A. Nilsson, Kristina Hambraeus-Jonzon, Marco Lattuada, Luni Chen, Ren Li, Kjell Alving, Peter Wiklund, Göran Hedenstier-na, Filip Fredén Critical Care Medicine 2010; 38:242–248

II Distant effects of nitric oxide inhalation in lavage induced lung in-

jury in anaesthetised pigs Manja C. A Nilsson, Kristina Hambraeus-Jonzon, Maria Bergquist, Kjell Alving, Peter Wiklund, Filip Fredén Manuscript

III NO effect of metabolic acidosis on nitric oxide production in hypox-

ic and hyperoxic lung regions in pigs Manja C. A Nilsson, Filip Fredén, Peter Wiklund, Kristina Hambra-eus-Jonzon Acta Physiologica Scand 2011; 202(1):59-68

IV Hypercapnic acidosis transiently weakens hypoxic pulmonary vaso-

constriction in anesthetized pigs, without affecting the endogenous pulmonary nitric oxide production. Manja C. A Nilsson, Filip Fredén, Anders Larsson, Maria Bergquist, Peter Wiklund, Kristina Hambraeus-Jonzon Submitted

Reprints were made with permission from the respective publishers.

Contents

Introduction ..................................................................................................... 9 Nitric oxide ............................................................................................... 10 Pulmonary blood flow .............................................................................. 11 Hypoxic pulmonary vasoconstriction (HPV) ........................................... 12 Nitric oxide inhalation .............................................................................. 12 Distant effects of nitric oxide inhalation .................................................. 13 Acidosis, NO and HPV ............................................................................ 14

Aims .............................................................................................................. 16

Material and Methods ................................................................................... 17 Subjects .................................................................................................... 17 Anesthesia ................................................................................................ 17 Fluid therapy ............................................................................................ 17 Ventilation ................................................................................................ 18 Monitoring and Measurements ................................................................. 18 Blood gases .............................................................................................. 19 Cross circulation ....................................................................................... 19 Nitric oxide ............................................................................................... 19 Nitric oxide synthase activity ................................................................... 20 Endothelin-1 in plasma ............................................................................. 21 ETA and ETB receptor immunoreactivity: Immunohistochemical Staining .................................................................................................... 21 Nitrite and nitrate in plasma ..................................................................... 22 cGMP Enzyme Immunoassay (EIA) ........................................................ 22 Calculations .............................................................................................. 22 Protocols ................................................................................................... 23 Statistical analyses .................................................................................... 26

Results ........................................................................................................... 27 Paper I: Distant effects of INO in endotoxemic pigs ............................... 27 Paper II: Distant effects of INO in lavage-injured pigs ........................... 28 Paper III: Effects of metabolic acidosis on endogenous nitric oxide production and pulmonary blood flow ..................................................... 30 Paper IV: Effects of hypercapnic acidosis on endogenous nitric oxide production and pulmonary blood flow ..................................................... 32

Discussion ..................................................................................................... 35 Main findings: .......................................................................................... 35 Possible mechanisms for the distant effect of INO in endotoxemia ......... 35 Distant effect of INO in lavage-induced lung injury ................................ 37 Metabolic acidosis and endogenous NO production ................................ 38 Hypercapnic acidosis and endogenous NO production ............................ 39 Nitric oxide synthase activity ................................................................... 40 Exhaled nitric oxide ................................................................................. 40 Nitrate and Nitrite ..................................................................................... 41 Acidosis, HPV, and pulmonary vascular resistance ................................. 42 Acidosis and oxygenation ........................................................................ 44 Rationale for INO dose ............................................................................ 44 Rationale for HCl administration ............................................................. 44

Conclusions ................................................................................................... 45

Future perspectives ....................................................................................... 46

Acknowledgements ....................................................................................... 47

References ..................................................................................................... 48

Abbreviations

ARDS Adult Respiratory Distress Syndrome cNOS constitutive calcium-dependent Nitric Oxide Synthase CETO2 Concentration of end tidal Oxygen cGMP cyclic Guanosine MonoPhosphate CVP Central Venous Pressure DO2 Delivery of Oxygen ENO Exhaled Nitric Oxide ET-1 Endothelin 1 ETA, ETB rec Endothelin A and Endothelin B receptor HL Hyperoxic Lung regions HPV Hypoxic Pulmonary Vasoconstriction INO Inhalation of Nitric Oxide iNOS calcium-independent Nitric Oxide Synthase (inducible) LLL Left Lower Lobe L-NAME Nw-nitro-L-arginine methyl ester MaP Mean systemic arterial Pressure MPaP Mean Pulmonary arterial Pressure NO Nitric Oxide NOS Nitric Oxide Synthase PaO2 Partial pressure of Oxygen in arterial blood PAO2 Alveolar Partial pressure of Oxygen PcwP Pulmonary capillary wedge Pressure PvO2 Partial pressure of Oxygen in mixed venous blood PVR Pulmonary Vascular Resistance QT Cardiac output QLLL Regional pulmonary blood flow to the Left Lower Lobe QLLL/QT Relative blood flow to Left Lower Lobe SNO´s Serum Nitrosothiols SNO-alb Serum Nitroso-albumin SNO-Hb Serum Nitroso-hemoglobin SVR Systemic Vascular Resistance v-a diff Veno-arterial difference VILI Ventilator-induced lung injury V/Q Ventilation/perfusion ratio

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Introduction

The delivery of oxygen (DO2) to cells within the body is crucial for all ad-vanced forms of life. The optimization of DO2 is a central issue in intensive care medicine and often presents a major challenge for critically ill patients. DO2 is dependent on cardiac output (QT) and arterial content of oxygen, which is influenced by the match between ventilation and perfusion (V/Q) in the lung.

In 1992, and based on studies by Sjöstrand et al [1] Lachmann introduced the concept “open up the lung and keep the lung open” [2]. Since then, the importance of a protective strategy during mechanical ventilation has been the focus. The goals of protective ventilation are to maintain plateau pres-sures and tidal volumes as low as possible while simultaneously applying adequate positive end-expiratory pressure (PEEP) to prevent atelectasis [3]. This protective strategy is associated with reduced acute lung injury (ALI) and improved outcome in ARDS [3, 4], presumably due to reduced mechan-ical stretch. A side effect is alveolar hypoventilation with hypercapnia and acidosis, so called “permissive hypercapnia”.

Beside ventilator settings causing acidosis, critically ill patients with sep-tic ARDS usually have a pathological gas exchange that causes hypercapnic acidosis, and metabolic acidosis frequently occurs due to circulatory and renal failure.

Acidosis affects both the systemic and pulmonary vascular tone and cir-culation, which in turn affects V/Q and DO2. The way acidosis affects nitric oxide (NO), an important regulator of pulmonary blood flow, is unclear;

however, sepsis causes both acidosis and increased NO production, which

suggests a link between the two events. Inhaled NO has been used in patients with septic ARDS to cause vasodi-

latation in ventilated lung regions and thus, improve V/Q and DO2 [5, 6]. However, in many of the patients, arterial oxygenation or pulmonary arterial pressures do not improve: these patients are classed as “non-responders”, and the mechanism for “non response” is unknown.

There is evidence inhalation of nitric oxide (INO) affects remote lung re-gions, other organs and endogenous NO production [7-9], and these distant effects might contribute to explaining “non response” to INO.

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The focus of this thesis is on perfusion of the lung and endogenous NO production in the lung dur-

ing metabolic and hypercapnic acidosis, while considering NO is an important regulator of the pulmonary blood flow, and

distant effects of INO in two different lung injury models: endotox-emia and lavage injury.

Nitric oxide NO, an atmospheric gas and one of the toxic components of air pollution, is also enzymatically synthesized in a number of tissues and cell types by NO synthase (NOS). There are three different isoenzymes: two constitutive cal-cium (Ca2+) -dependent NOS, neuronal NOS and endothelial NOS (eNOS), and, a Ca2+ -independent inducible enzyme (iNOS). NOS oxidize L-arginine to produce NO and L-citrulline in equimolar amounts. In the lungs, NOS is found in many cell types, including vascular endothelial cells, airway epi-thelial cells, macrophages, bacteria and neurons [10-12].

Small amounts of NO are generated in a tightly regulated manner by the two Ca2+ dependent NOS, and although this activation is short-lived, the NO produced serves as a diffusible signaling molecule mediating numerous phy-siological processes, including vasodilatation and neurotransmission. If the cells are induced by certain cytokines, microbes, or microbial products, iNOS expression results in sustained production of NO, which exerts cyto-static/cytotoxic and cytoprotective actions in mammalian tissues and antimi-crobial activity toward certain pathogens. Although resting un-stimulated cells do not express iNOS, the capacity to express this enzyme exists in near-ly every tissue in the body. After stimulation of iNOS, the increase in NO production is delayed several hours [13].

NO can be generated by the reduction of nitrite, which occurs via several routes involving enzymes, proteins, vitamins, or even simple protons. All pathways for generating NO from nitrite are potentiated during hypoxia and acidosis [14]: these pathways may serve as a backup system for NO genera-tion in conditions such as hypoxia, in which the NOS/L-arginine system is compromised.

NO produced in the endothelium may diffuse into the underlying smooth muscle cell to activate guanylate cyclase which catalyzes the conversion of cyclic guanosine triphosphate (cGTP) to cyclic guanosine monophosphate (cGMP). cGMP causes vasodilatation by decreasing intracellular free cal-cium and activating potassium channels leading to hyperpolarization of the smooth muscle cell [15, 16]. NO may also diffuse into the vascular lumen and either enter the erythrocyte or react with plasma constituents [17].

NO is an unstable free radical with an approximate half-life in blood of 1-2 milliseconds [18]. A majority of NO will react rapidly with oxyhemoglo-

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bin in the erythrocyte to produce nitrate and methemoglobin. In plasma, NO can be oxidized to nitrite, a process catalyzed by ceruloplasmin [14], or react with plasma thiols to form nitrosothiols of which s-nitroso-albumin (SNOAlb) is considered to represent the major reaction product [19, 20].

Pulmonary blood flow Pulmonary blood flow is controlled by both passive and active factors. Pas-sive factors include changes in cardiac output, left atrial pressure, airway and interstitial pressures, gravitational force and vascular obstruction or recruit-ment [21]. Active factors alter pulmonary vascular resistance and tone by causing contraction or relaxation of vascular smooth muscle. Normally va-sodilators have little or no effect on pulmonary vascular pressures, indicating there is little or no resting tone.

Gravity increases blood pressure in pulmonary vessels with distance down the lung, but as the pulmonary arterial and pulmonary venous pres-sures increase by the same amount, the “driving pressure” is the same. Blood flow increases because of the characteristics of the pulmonary circulation: the greater intravascular pressures lead to more recruitment and distention of vessels, thus, the resistance to blood flow decreases with distance down the lung [22].

There is greater lung perfusion heterogeneity within isogravitational planes than across gravitational planes; this is a consistent finding in a varie-ty of animal species, both quadrupeds and bipeds [23]. Hence, gravitational influences account for only 1–25% of the variability in regional perfusion, with the remainder being attributed to the influences of the individual’s vas-cular structure [24, 25].

Some apparent vertical perfusion gradient in vivo is due to compression of the dependent lung increasing local lung density, and therefore, perfu-sion/volume, and there are vertical gradients in lung density. In the depen-dent portion of the lung, a given volume of lung will contain more lung tis-sue (capillaries) and less air; thus, having a greater density than in nonde-pendent regions [23].

In humans and pigs, Ca2+-dependent NOS activity is higher in dorsal than in ventral lung regions [11, 26], which causes lower vascular tone in dorsal lung regions and could contribute to more homogenous perfusion when pa-tients are placed in prone position. Vasodilatory NO has an active role in the regulation of pulmonary perfusion, making the pulmonary circulation uni-form despite gravity and is probably an evolutionary phenomenon since the quadruped stage [27].

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Hypoxic pulmonary vasoconstriction (HPV) Hypoxic pulmonary vasoconstriction (HPV) is a powerful endogenous me-chanism within the lung, by which ventilation perfusion matching is achieved, and was first reported in 1946 by von Euler and Liljestrand [28]. A weakened HPV may result in life-threatening hypoxemia in patients with respiratory failure [29].

HPV is caused by constriction of the precapillary vessels [29] that supply blood to areas of the lung with poor alveolar ventilation or hypoxia. The main determinant of HPV is alveolar PO2 (PAO2), but mixed venous PO2

(PVO2) may contribute up to one-fifth of the response [29]. The core me-chanism of HPV resides within pulmonary artery smooth muscle cells, and HPV is initiated, at least in part, by the inhibition of a family of O2-sensitive voltage dependent potassium (Kv) channels leading to membrane depolari-zation, the opening of voltage dependent Ca2+ channels and vasoconstriction [30].

HPV has rapid onset, reaches its maximum within minutes, can be sus-tained for many hours with no fading or potentiation over time, and reverses within minutes on reoxygenation [31]. The magnitude of HPV is modulated by circulating mediators and the autonomic nervous system [30]. NO is an important modulator of pulmonary blood flow and attenuates HPV [32], whereas, nitric oxide synthase (NOS) blockade augments HPV [33-36].

The hypoxic vasoconstrictor response is effective in diverting blood flow and reducing hypoxemia when the hypoxic region is a small fraction of the whole lung. In these circumstances, a large increase of local vascular resis-tance has little influence on total vascular resistance. As the hypoxic lung segment becomes larger, its activity is manifest as substantial increases in transmural pulmonary arterial and perfusion pressures, and the response becomes less effective in diverting blood flow [37]. When pulmonary hy-poxia is global, there will be no effective blood flow distribution, but a sub-stantial increase in pulmonary arterial pressure. The resulting pulmonary hypertension can cause right ventricular failure in patients with chronic ob-structive lung disease and pulmonary edema at high altitude.

Nitric oxide inhalation In 1991, Frostell et al demonstrated inhalation of nitric oxide (INO) acts as a selective pulmonary vasodilator [32]. INO reverses pulmonary hypertension induced by hypoxia or thromboxane without any effect on systemic circula-tion, as NO is rapidly bound to hemoglobin and inactivated: INO has no effect on non-constricted pulmonary vascular bed. In patients with healthy lungs separately ventilated with unilateral INO to hyperoxic lung during contra-lateral hypoxia, and during bilateral normoxia or hyperoxia, INO has

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no effect on pulmonary blood flow distribution during bilateral normoxia or hyperoxia [38]. Conversely, INO to a hyperoxic lung increases perfusion to this lung by redistributing the blood flow, if the opposite lung is hypoxic [38], indicating INO potentiates HPV in healthy lungs.

INO was considered a perfect selective pulmonary vasodilator with great potential in the treatment of ARDS [39]. As NO is delivered to ventilated lung regions, blood flow to the oxygenated lung regions increases and V/Q improves. Simultaneously, INO relieves pulmonary hypertension, reduces right ventricular after-load and increases the right ventricular ejection frac-tion. However, in the clinical setting, 40 to 60% of patients with septic ARDS are non-responders, that is, INO does not increase arterial oxygena-tion or decrease pulmonary arterial pressures [5, 6]. The exact mechanism for non-response is unclear. There is also the drawback of rebound response, occasionally observed on withdrawal of INO, with life-threatening pulmo-nary hypertension, reduced oxygenation, and even death. Rebound response may be caused by an INO-induced down regulation of NOS activity (nega-tive feedback mechanism) [40] and an up-regulation of vasoconstrictors, mainly Endothelin-1 (ET-1) [41] and cyclooxygenase derived products [42].

INO is approved by the Food and Drug Administration (FDA) for persis-tent pulmonary hypertension in the newborn, treatment of term and near-term neonates with hypoxic respiratory failure and for patients in all ages for pulmonary hypertension after cardiac surgery. In adult patients with ALI/ARDS, randomized controlled trials confirm a marked increase in ar-terial oxygenation without any sign of clinically relevant side effects [39]; however, the increase in PaO2 is only transient and there is no improvement in important outcome parameters such as mortality and ventilator free days [43-45]. Therefore, INO is considered a rescue treatment in adult patients with severe refractory hypoxemia.

Distant effects of nitric oxide inhalation INO has distant effects both within the lungs and in other organs [7-9], thus INO prolongs bleeding time [46], has an effect on microcirculation in Sickle cell disease [47, 48], improves peripheral circulation during reperfusion of the splanchnic organs [8], and reduces inflammation in lower extremity ischemia reperfusion in humans [9]. In healthy pigs with regional hypoxia, INO causes vasodilatation in ventilated lung regions and vasoconstriction and decreased NO-production in lung regions not reached by INO. This is more pronounced in hypoxic regions [7], i.e. INO augments HPV, as ob-served in healthy patients [38]. The distant down-regulation of NO produc-tion is caused by a blood-borne mediator or mediators [7]. This was demon-strated by reproducing these findings in an experimental model where blood was cross-circulated between two pigs, with INO being administered to one

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pig and endogenous NO production (exhaled NO and NOS activity) and regional pulmonary blood flow being measured in the hyperoxic and hypox-ic lung regions in the other pig [7].

Whether INO has distant effects in pigs with lung injury is unknown. If endogenous NO production is reduced in non-ventilated lung regions, as in healthy pigs, HPV would be potentiated, thereby reducing the pulmonary shunt. A reduction in endogenous NO production could also contribute to the severe rebound effect sometimes observed on withdrawal of INO [40]. How-ever, an increase in endogenous NO production in non-ventilated lung re-gions would reduce pulmonary vascular tone in these regions, and increase the shunt. This could contribute to the non-response to INO frequently oc-curring in patients with septic ARDS treated with INO.

Acidosis, NO and HPV ARDS is associated with hypoxia and respiratory acidosis due to pathologi-cal gas exchange in the lung and the use of permissive hypercapnia during mechanical ventilation to prevent pulmonary hyperinflation and ventilator-induced lung injury (VILI) [3, 49]. Metabolic acidosis is common in septic ARDS patients due to circulatory and/or renal failure.

However, the effect of acidosis on pulmonary NO production during hyperoxia and hypoxia remains unclear. Metabolic acidosis increases NO concentrations in exhaled gas (ENO) in anesthetized normoxic rats [50, 51], but decreases constitutive NO synthase (cNOS) activity in endothelial cells [52, 53]. Likewise, hypercapnic acidosis both increases [54] and decreases endogenous NO production [55].

If production of NO in lung tissue is enhanced during hypoxic and acidot-ic conditions in the severely diseased lung [56], there are important clinical implications. Increased NO in hypoxic lung regions modulates HPV [33-36] with potentially both beneficial and damaging effects. Increased production of NO during hypoxia maintains active vasorelaxation and prevents exces-sive pulmonary hypertension and edema formation. Thus, the small thin-walled pulmonary arteries are protected from damaging high pressures [57, 58]. However, increased NO production in hypoxic lung regions attenuates HPV [32] and increases shunt [50, 51] which could be detrimental for pa-tients with acute respiratory failure [29].

Metabolic acidosis causes vasoconstriction in the pulmonary circulation [59] and increases HPV[60, 61]. Hypercapnic acidosis also causes vasoconstric-tion in the pulmonary circulation [59, 62, 63] but the effects on HPV vary [64-66] . The CO2 molecule causes vasodilatation [67, 68] and in an isolated rat lung model, the CO2-mediated vasodilator effect on pulmonary vascular tone is more pronounced when pulmonary hypertension is induced by Endo-

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thelin 1(ET-1) or hypoxia [68]. With higher pulmonary arterial pressure, the vasodilatory effect becomes stronger. Furthermore, the pulmonary vasodila-tor effects of CO2 are generally unaffected by L-NAME (NOS blocker), suggesting NO is not involved in hypercapnic vasodilatation [68]. However, extrapolating these results to a clinical situation is difficult, as the perfused lung is denervated and isolated from the systemic circulation with particular concerns being related to the lack of pulmonary–systemic interaction.

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Aims

The aims of this thesis were to investigate

if INO had distant effects on pulmonary endogenous NO production in pigs with acute endotoxin-induced lung injury (Paper I ).

if INO had distant effects on pulmonary endogenous NO production in pigs with acute lavage-induced lung injury (Paper II).

if metabolic acidosis affected NOS dependent and/or NOS independent NO production in hypoxic and hyperoxic lung regions (Paper III).

if hypercapnic acidosis affected endogenous NO production in hypoxic and hyperoxic lung regions, and whether hypercapnic acidosis affected HPV (Paper IV).

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Material and Methods

Subjects Eighty-two (n=82) anesthetized and mechanically ventilated Swedish coun-try breed pigs, of both sexes, were included in the studies. Thirty-six (n=36) in Paper I, twelve (n=12) in Paper II, eighteen (n=18) in Paper III and six-teen (n=16) in Paper IV. The pigs were 12-14 weeks old and weighted be-tween 25 and 35 kg. The studies were approved by the Animal Research Ethics Committee of Uppsala University, Uppsala, Sweden.

Anesthesia The pigs were pre-medicated with Pentobarbital 540 mg and atropine 0.5 mg intraperitoneally thirty minutes before induction of anesthesia (Papers I, II and III), or, Soletil Forte (Tiletamin and Zolazepam) 6 mg/kg and Rompun (Xylazin chloride) 2.2 mg/kg intramuscularly (i.m.) three minutes before induction of anesthesia (Papers I and IV).

Anesthesia was induced with pentobarbital 8-10 mg/kg (Papers I, II and III) or propofol 40-100 mg intravenously (i.v.) (Paper IV). Anesthesia was main-tained by infusions of: midazolam 20-70 mg/hour, fentanyl 0.4-1.4 mg/hour and pancuronium bromide 4-13 mg/hour i.v. (Papers I and II); chlormetia-zole (0.8-1.6 g/h), fentanyl (0.2-0.4 mg/h), and pancuronium bromide (4-8 mg/h) i.v. (Paper III); or, with propofol infusion 3 mg/kg/hr and an infusion of ketamin vet. 5g, fentanyl 1 mg and pancuronium 60 mg in 1000 ml of buffered glucose 25 mg/ml, 4 ml/kg/hr (Paper IV).

Fluid therapy A large bore catheter was inserted in the external jugular vein for infusion of warm isotonic buffered Ringer’s solution at a rate of: 20-30 ml/kg/h (Paper I); 15-25 ml/kg/h (Paper II); or, 10-15 ml/kg/h (Papers III and IV). The pigs in Paper I also received a bolus of 20 ml low molecular dextran (Promiten) and 500 ml of dextran 70 (Macrodex).

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All pigs received the anesthetic drugs mixed in buffered 25 mg/ml glu-cose solution.

Ventilation Tracheotomies were performed on all pigs and a cuffed endotracheal tube (inner diameter 6.0) was inserted. A second cuffed endotracheal tube (inner diameter 4.5 mm) was inserted through the tracheostoma and positioned in the bronchus to the left lower lobe (LLL), while temporarily deflating the cuff of the tube in the trachea (Papers II, III and IV). A medial sternotomy allowed inspection of the lungs to ensure the tubes were guided into a posi-tion that separated the LLL from the rest of the lung. The different and per-sistent fractions of the concentration of end tidal Oxygen (CETO2) during LLL hypoxia, and normoxia or hyperoxia to the other lung regions were considered additional proof of separation. All pigs were mechanically ventilated by Ser-vo 900 C ventilators (Siemens Elema, Lund, Sweden), set at volume-controlled ventilation: two ventilators were synchronized in Papers II, III and IV. The ventilation was adjusted to obtain equal end-inspiratory plateau pressures within the LLL and in other parts of the lungs. A PEEP of 7 cm H20 (Paper I), or 5 cm H2O (Papers II, III, IV) was applied. The fraction of inspired oxygen (FiO2) was 0.9 (Papers I, II) or 0.8 to hyperoxic lung re-gions and 0.05 to hypoxic lung regions (Papers III, IV).

The minute ventilation was adjusted by changing respiratory frequency, aiming at an end-tidal CO2 concentration (EtCO2) of 33-49 torr (4.4-6.5 kPa) in the initial control situation: this was then kept constant throughout the experiments.

Hypercapnia (Paper IV) was induced by inhalation of 10% CO2 in 5% O2, balance nitrogen to the LLL and 10% CO2 in 80% O2, balance nitrogen to the rest of the lung.

Monitoring and Measurements All pigs had pulmonary artery catheter (CriticathTM No 7 F, Ohmeda, Pte Ltd, Singapore), a central venous catheter via the external jugular vein, and an arterial catheter in the carotid artery inserted: the position of all catheters was confirmed by pressure tracing. Arterial blood pressures, pulmonary ar-tery pressures, PcwP and temperature were recorded (7010 monitor, Mar-quette Electronics, Milwaukee, WI, USA). Cardiac output was measured by thermodilution (Paper I) or continuously by enclosing the pulmonary artery in an ultrasonic flow probe (12 SB) (Papers II, III and IV).

For measurements of Qt by thermodilution, a thermal indicator of 10 ml of saline 8-10°C was injected into the right atrium. The first measurement

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was ignored, and Qt was derived from the mean of three consecutive mea-surements. QLLL was measured continuously by in an ultrasonic flow probe (4, 6 or 8 SB) enclosing the artery to the LLL (Papers II, III and IV). The flow probes were connected to flow meters (T208, Transonic volume flow meter, Transonic Systems Inc, Ithaca, NY, USA). Different flow probes were used for the LLL artery depending on the size of the vessel.

Blood gases Mixed venous and arterial blood samples were collected for analysis of O2 tensions (PvO2 and PaO2), PaCO2, pH (ABL 625, Radiometer, Copenhagen, Denmark), SaO2 and methemoglobin (OSM 3, Radiometer, Copenhagen).

Cross circulation Eighteen pairs of pigs (n=36) were cross circulated and included in (Paper I): each pair consisted of siblings. A double-lumen dialysis catheter (15 cm, 11.5 F medcomp; Duoflow, Harleysville, PA, USA) was introduced via the right external jugular vein of each pig, and placed with the distal opening in the right atrium, as judged by pressure curves. The catheters were hepari-nized and heparin (300 U/kg) was administered intravenously in each pig before cross circulation started. Blood was drawn from the proximal lumen and pumped through polyvinyl chloride tubing by two separate non-pulsatile roller-pumps (PMO 10–220 Type; Gambro, Lund, Sweden) at a rate of 300 to 500 mL/min to the other pig in the pair, where the blood was delivered through the distal lumen of the double-lumen catheter. When the flow was increased above 500 mL/min, the catheter tended to adhere to the wall of the right atrium.

Nitric oxide NO in a mixture of 1000 parts per million (ppm) NO, balance nitrogen (AGA, special gas AGA Gas AB, Sundbyberg, Sweden), was administered through a volumetrically calibrated flow meter connected by a y-piece to the low flow inlet of the servo ventilator (Papers I and II). The inspired gas was passed through a canister containing soda lime to absorb NO2. The concen-trations of inspired NO and NO2 were measured by chemiluminescence (ana-lyzer M9841 NOX Lear Siegler Measurement Controls Corporations, En-glewood, CO, USA) in the inspiratory limb of the ventilator tubing, more than 50 cm from the y-piece to avoid contamination by expired gas.

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The NO concentration in exhaled air (ENO) from the lung regions not re-ceiving NO were measured continuously by chemiluminescence (Papers I and II) (analyzer model 42, Thermo Environmental Instruments Inc., Frank-lin, MA, USA): ENO was measured alternately from the hypoxic LLL and the hyperoxic lung regions (Papers III and IV). To ensure complete mixing and avoid contamination by inspired gas, NO was measured in the expiratory limb of the ventilator tubing more than 100 cm from the endotracheal tubes: the average concentration (mean expired values) over ten breaths was used for statistical analysis.

Nitric oxide synthase activity Nitric oxide synthase (NOS) activity was analyzed in lung tissue in all pigs, and tissues from both hypoxic and hyperoxic lung regions (Papers III and IV) were analyzed.

NOS activity was measured by the conversion of L-[U-14C] arginine to L-[U-14C] citrulline. For the citrulline formation assay, tissues were homoge-nized in ice-cold homogenization buffer containing 320 mM sucrose, 10 mM HEPES, 0.1 mM EGTA, 1mM DL-dithiothreitol, 10 g ml-1 trypsin inhibitor, 10 g ml-1 leupeptin, 100 g ml-1 phenylmethylsulfonyl fluoride, and 2 g ml-1 aprotinin (adjusted to pH 7.2 at 20 C with 1 M HCl). The ho-mogenate was centrifuged at 10,000 g for 30 min at 4 C, and the soluble fraction was used for the measurement of NOS activity. The tissue extract was added to pre-warmed (37 C) tubes containing 100 l of a buffer con-sisting of 50 mM potassium phosphate, pH 7.2, 50mM L-valine, 100 M NADPH, 1 mM L-citrulline, 20 M L-arginine, and L-[U-14C] arginine (Amersham, UK; 150000 dpm), and 1.2 mM MgCl2. For each sample, dupli-cate incubations (10 min at 37 C) were performed in the presence or absence of either EGTA (2mM) or EGTA plus N monomethyl-L-arginine (2mM each), to determine the level of the Ca2+-dependent, cNOS, (neuronal, nNOS and endothelial, eNOS) and the Ca2+-independent (inducible, iNOS) NOS activity. The method does not distinguish between the two isoforms of Ca2+-dependent NOS. The reaction was terminated by removal of the substrate, and dilution was by adding 1.5 ml of 1:1 (v/v) H2O/Dowex AF 50W-X8 at pH 7.5. Five ml (5 ml) of H2O was added to the incubation mix, and 2 ml of the supernatant was removed and examined by liquid scintillation counting for the presence of L-[U-14C] citrulline: the level of citrulline was expressed as picomoles per gram of tissue (wet weight) per minute ( mol/g/min). NOS-activity, as measured by citrulline formation, measures the expression of NOS in the tissue, or any post-translational modification of NOS affecting NO formation, and is irreversible under the conditions of the assay.

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Endothelin-1 in plasma ET-1 in arterial and mixed venous blood was analyzed (Papers I and II). The ET-1 levels in plasma were quantified by radioimmunoassay, as previously described [69], with antiserum E1 and iodinated ET-1 (Amersham Pharma-cia Biotech) as a tracer. The detection limit for the assay was 3.9 pmol/tube: the intra-assay variation was 6% and inter-assay variation 14%, cross-reactivity of the E1 antiserum was 100% for ET-1, 27% for ET-2, and 8% for ET-3..

ETA and ETB receptor immunoreactivity: Immunohistochemical Staining The ETA and ETB receptor immunoreactivity were analyzed (Paper I). From each tissue specimen, 10 m-thick cryostat sections were cut and attached to poly-lysine-coated glass slides. The sections were fixed in acetone for 10 mins at room temperature, air-dried, and rinsed in 0.01% phosphate-buffered saline (pH 7.4) for 30 mins. The sections on one glass slide were stained with hematoxylin-eosin as a survey stain. The sections were treated with 0.3% hydrogen peroxide in phosphate-buffered saline buffer for 30 mins, and then incubated with 5% bovine serum albumin in phosphate-buffered saline for 30 mins. Between each of the staining steps, the sections were washed carefully with phosphate-buffered saline. The sections were immu-nostained with the streptavidin-biotin complex (ABC) technique, incubated overnight at 4°C with primary antibodies (ETA receptor: Santa Cruz Bio-technology, Inc., Product No.sc-21193 U.S., dilution 1:150; ETB receptor: Santa Cruz Biotechnology, Inc., Product No.sc-21196 U.S., dilution, 1:400; Santa Cruz, CA, USA), and then, incubated for 1 hr with the respective bio-tinylated secondary antibodies and ABComplex/horseradish peroxidase complex. The binding of avidin and biotin was demonstrated by 3-amino-9-ethylcarbazole. As a negative control, the primary antibody was replaced by phosphate-buffered saline, and the sections were then treated in the same way as described. The sections were examined under the microscope by three trained laboratory personnel blinded to each other and to treatment group assignment. All areas in the sections were systematically examined by dividing the areas into a grid: the square grid was placed on one of the ocu-lars. In the two sections from each pig, the vessels were divided into positive or negative for immunostaining, and the immuno-reactivity was calculated as the number of positive immunostained vessels compared to the total num-ber of vessels in the section.

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Nitrite and nitrate in plasma Nitrite and nitrate in arterial and mixed venous blood were measured in plasma by electrophoresis (Paper III). All plastics, filters and vials were washed with de-ionized water and allowed to dry before use. Plasma sam-ples were diluted in de-ionized water, centrifuged (Ultrafree-MC filters; Millipore, Billerica, MA, USA) at 5000 x g for 30 min, and analyzed imme-diately with an HP 3D capillary electrophoresis system (Hewlett Packard, Waldbronn, Germany). Samples were injected (-6 kV for 20 s) onto an ex-tended light path capillary 104 cm in length and with 0.75 mm internal di-ameter. Ultra violet detection of nitrite and nitrate was at 214 nm. The work-ing electrolyte consisted of 25 mm sodium sulfate (Merck, Darmstadt, Ger-many) and 5% NICE-Pak OFM Anion-BT (osmotic flow modifier; Waters, Sollentuna, Sweden) dissolved in de-ionized water. Analysis was with an applied negative potential of 300 V/ cm. Data were acquired with an HP 3D Chem Station data system at a response time of 0.1 s, with 0.01 peak width selection.

cGMP Enzyme Immunoassay (EIA) For quantification of cGMP, lung tissue lysates were analyzed (Papers II and IV) with a commercially available EIA kit (Detect X® Direct Cyclic GMP, Arbor Assays, Michigan, USA), according to manufacturer’s instruc-tions. Optical density was determined by a Tecan Sunrise instrument (Tecan Nordic AB, Mölndal, Sweden), with the Magellan software, at 450 nm with a correction at 570 nm. For normalization, the protein concentration in the tissue lysates was determined with a commercially available Protein Assay Kit based on the Bradford Assay (Coomassie Plus Assay Kit, Thermo Scien-tific, Illinois, USA).

Calculations When calculating global and regional PVR, it was assumed the whole lung was perfused under zone 3 conditions; hence, perfusion pressure through the lung equaled MPaP – PcwP. PVR was calculated as (MPaP – PcwP)/QT. The vascular resistance of the LLL, (PVRLLL) (Papers II, III and IV) was calcu-lated as (MPaP – PcwP)/QLLL. The vascular resistance of the rest of the lung, PVRRL (Paper II) or PVRHL (Papers III and IV) was calculated as (MPaP – PcwP)/(QT -QLLL). Systemic vascular resistance (SVR) was calculated as (MaP –CVP)/QT (Paper IV).The calculated values for vascular resistances in mmHg /l/min were multiplied by 80 to get the unit dyne sec/cm 5. The rela-tive perfusion of the LLL was calculated as QLLL/QT.

23

Protocols

Paper I Eighteen pairs of pigs were cross-circulated (Figure 1) and an intravenous endotoxin infusion (LPS, E.coli 0111:B4, Sigma Chemicals, St Louis, Mo, USA) of 10 microgram kg –1 hour-1 per pig was administered. Baseline val-ues were registered and blood samples taken after 2.5 hours of endotoxin infusion. One pig in each of nine pairs of cross-circulated pigs then received INO 80 ppm (NO-blood donor pigs). The NO blood recipient group com-prised 9 pigs that were to be cross circulated and who received endotoxin and NO blood. The control group comprised 9 pigs that were to be cross circulated and received endotoxin. New measurements were taken 1, 2 and 3 hours after the start of INO (Figure 2).

endotoxin endotoxin

Cross circulation

80 ppm

+/- INO

Figure 1. Experimental design in Paper I.

24

NO donor group

Cross circulationEndotoxinInhaled NO: 80 ppm

NO blood recipient group

Cross circulationEndotoxinBlood with NO

Control group

Cross circulationEndotoxin

Protocol

Measurements

Endotoxin

INO

2.5h 4.5h 0h 3.5h 5.5h

Cross Circulation

Figure 2. Protocol in Paper I.

Paper II Twelve pigs were anesthetized and the LLL was ventilated separately. Base-line measurements were obtained 30 minutes after preparation. In all pigs, lavage injury was induced in the whole lung, except LLL, by 20ml/kg of warm saline. The lavage procedure was repeated 10 times, whereupon, the fluid was clear. In six pigs, INO 40 ppm was given to the LLL after the la-vage was completed (INO group): six pigs served as the control group. Data were collected one hour after the lavage procedure ended, when the lung injury was considered stable, and thereafter, every 30 minutes for 3.5 hours.

Paper III Eighteen healthy anesthetized pigs were separately ventilated with hypoxic gas (FIO2 0.05, balance nitrogen) to the LLL and hyperoxic gas (FIO2 0.8, balance nitrogen) to the rest of the lung. The pigs were studied in three groups (Figure 3):

Control group (n=6), with data collection at 90 min (baseline) and 180 min of LLL hypoxia

HCl group (n=6), with data collection at 90 minutes of LLL hypoxia (baseline), after which an infusion of HCl (5 mmol/ml) 15 ml/h was started. New data were collected after 90 minutes of HCl infusion (approximately 180 min of LLL hypoxia).

25

L-NAME+HCl group (n=6): L-NAME 5 mg/kg (supplied by Sigma-Aldrich Sweden AB, Stockholm, Sweden) was administered intra-venously and ENO was measured continuously. When there was no further decrease in ENO, a second dose of L-NAME 5 mg/kg was administered, this was followed by an infusion of L-NAME 2.5 mg/kg/h throughout the experiment, to ensure the enzymatic produc-tion of NO was completely inhibited. Data were collected after the second bolus dose of L-NAME at 90 minutes of regional LLL hy-poxia (baseline). Then an infusion of HCl (5 mmol/ml) 15 ml/h was started and new data were collected after 90 minutes of HCL infu-sion (approximately 180 min of LLL hypoxia).

The HCl infusion was administered in a central venous catheter together with isotonic buffered Ringer’s solution. The total amount of infused fluid was similar in all pigs.

Control group

LLL hypoxiaMeasurements at 90 min and 180 min

HCl group

LLL hypoxia

Measurements every 30 min 90 min = before HCl inf.180 min = 90 min of HCl inf.

L-NAME+HCl group

LLL hypoxia

Measurements every 30 min 90 min = after L-NAME, before HCl inf.180 min = 90 min of HCl inf.+L-NAME

Measurements

Surgery + LLL hypoxia all groups

L-NAME+HCl group: L-NAME 5 mg/kg x 2 + inf. L-NAME

0h

HCl and L-NAME+HCl groups: HCl inf. 75 mmol/h

90 min of LLL hypoxia

180 min of LLL hypoxia(= 90 min of HCl infusion )

Protocol

Figure 3. Protocol in Paper III.

Paper IV Sixteen healthy pigs were anesthetized and the LLL was separately venti-lated. Baseline measurements were taken in all pigs 30 min after preparation and ventilation with hyperoxic gas (FIO2 0.8, balance nitrogen) to both lungs. The inhaled gas was then changed from hyperoxic to hypoxic (FIO2 0.05, balance nitrogen) to the LLL, and data were collected after 30 min of re-

26

gional LLL hypoxia. The pigs were then randomized into a control group (n = 8), or hypercapnia group (n = 8). In the control group, regional LLL hy-poxia was continued throughout the experiment. In the hypercapnia group, the LLL was ventilated with 10% CO2 in 5% O2, balance nitrogen, and the rest of the lung was ventilated with 10% CO2 in 80% O2, balance nitrogen throughout the experiment. In both groups, data were collected every 30 min for 3.5 hours.

At the end of each experiment, with the pig anesthetized, alive and venti-lated, lung tissue was excised and immediately frozen in liquid nitrogen for analysis of NOS activity (Papers I-IV) and cGMP (Papers II and IV): pieces from both the hypoxic LLL and the HL were excised (Papers III and IV). Finally, the pigs were euthanized by an intravenous injection of potassium chloride (KCl).

Statistical analyses Data in the text and tables are presented as mean (SD). A two-way analysis of variance (ANOVA) for repeated measures on one factor was applied to disclose any interaction effects (pAB), or differences within or between groups (pA). Data collection periods (fixed) and pigs (random) comprised the two block factors for comparisons within the groups. Groups (fixed) and pigs (random) comprised the two block factors for comparisons between the groups. Scheffe’s (Paper I) and Tukey’s tests (Paper II-IV) were used as posthoc tests. A probability of <0.05 was accepted as significant. A one-way ANOVA was used for analysis of NOS activity and cGMP. Analyses were performed with the SOLO Statistical System (version 4.0; BMDP Statistical Software Inc., Los Angeles, CA, USA) (Paper I) and with Statistica (version 8, Statsoft.Inc., Tulsa, OK, USA) (Paper II-IV).

27

Results

Paper I: Distant effects of INO in endotoxemic pigs After 30 minutes of INO, ENO started to increase in the NO-blood recipient group, as compared to the control group. At the end of the experiment, the in-crease in ENO in the NO-blood recipient group had reached 300% (Figure 4).

-50

0

50

100

150

200

250

300

350

400

0 30 60 90 120 150 180

Ch

ange

NO

E(%

)

Time (min)

p 0.03

Figure 4. Change in exhaled NO (ENO). = Control group (mean). =NO-blood recipient group (mean). Whiskers =SEM. Endotoxemia and cross-circulation caused a 100 % increase in ENO ( ), whereas, endotoxemia, and cross-circulation with NO inhaled pigs ( ) caused a 300 % increase in ENO.

The cNOS activity was higher (p 0.02) in the NO-blood recipient group than the control group (Figure 5). In the NO-blood recipient group, 86% +/- 9% were positive for ETB receptor immunoreactivity, compared to 60%+/- 16% in the control group (p< 0.01). There were no significant differences between the groups in plasma ET-1 levels in arterial and mixed venous blood. QT, PcwP, MaP, MPaP, PaO2 and PaCO2 did not differ between the groups.

28

0

25

50

75

100

Control NO-bloodrec Donator

NOS-activity

cNOS

iNOS

p 0.02

p 0.0004

pmol/g/ min

Figure 5. NOS activity in lung tissue. Mean values. Whiskers =SEM. cNOS (cal-cium dependent). iNOS (calcium independent). Donator = pigs receiving INO, “do-nating NO-blood”. There was an increase in cNOS activity in the NO-blood reci-pient pigs. iNOS activity decreased in the pigs receiving NO inhalation

Paper II: Distant effects of INO in lavage-injured pigs cNOS activity decreased in lavage-injured lung regions (RL) in the INO group, compared to the control group (Figure 6).

cGMP in RL was 1.1 (0.4) pmol/mg in the control group and 1.1 (0.9) pmol/mg in the INO group (p 0.9). In the control group, cGMP in LLL was 1.4 (0.8) pmol/mg and 2.3 (0.6) pmol/mg in the INO group (p=0.06).

ENO from RL increased from 2.4 (1.8) ppb to 3.7 (2.1) ppb in the control group and from 2.4 (0.9) ppb to 4.7 (1.7) ppb in the INO group (p=1.0). No difference was observed between the groups, and ENO levels returned to baseline in both groups at the end of the experiment.

29

0

50

100

150

200

250

cNOS HL

NOS activity

Control

INO

iNOS HL cNOS LLL iNOS LLL

pmol/g/min

p 0.04

p 0.24p 0.09

p 0.9

iNOS HL cNOS LLL iNOS LLL

pmol/g/min

p 0.04

p 0.24p 0.09

p 0.9

Figure 6. NOS activity. Mean values. Whiskers =SEM. RL: lavage-injured lung regions. LLL: healthy lung region. INO 40 ppm to the LLL (INO group) caused a decrease in cNOS activity in the lavage-injured lung regions.

QLLL/QT increased from 20 (7) [mean (SD)] to 43 (14) % in the control group and from 27 (12) % to 53 (18) % in the INO group (p= 0.8) (Figure 7). MPaP increased in both groups after lavage, and no differences were seen between the groups. There were no differences between the groups regarding PVR, PVRRL or PVRLLL. QT decreased (p< 0.05) in the control group, com-pared to the INO group, although there were no differences in PcwP (p= 0.8), heart rate (p= 0.6), MaP (p= 0.9) or airway plateau pressures (p> 0.05). PaO2 decreased from 69 (4.5) kPa to 21 (18) kPa in the control group and from 68 (3) kPa to 33 (15) kPa in the INO group, without any significant differences between the groups. There were no differences between the groups regarding ET-1 in mixed venous or in arterial blood, and the lavage procedure did not increase ET-1.

30

0

10

20

30

40

50

60

70

80

Baseline

Control

INO

QLLL/QT

%

60 90 120 150 180 210 240 270

NO inhalation

Minutes postlavage

Lavage

Figure 7. QLLL/QT. Mean values. Whiskers =SEM. Lavage was induced in all pigs after baseline measurements. The relative blood flow to healthy LLL increased by 100% in both groups after lavage injury, and there were no differences between the groups.

Paper III: Effects of metabolic acidosis on endogenous nitric oxide production and pulmonary blood flow There was no statistical difference in MaP, MPaP, PcwP, or QT among the groups over time. After 90 min of HCl-infusion, pH did not differ between the HCl group (7.20 [0.05]) and the L-NAME+HCl group (7.13 [0.09]) (p> 0.05): the pH in both groups was lower than the pH in the control group (p< 0.001).

Acidosis induced by infusion of HCl did not change ENO from the hypoxic LLL or the hyperoxic regions of the lung (HCl group) (Figure 8) either over time or when compared to the control group (p> 0.05). ENO from the hypox-ic LLL and the hyperoxic lung regions in the L-NAME+HCl group was con-sistently low during the study (< 0.5 ppb), with no significant change over time during infusion of HCl.

31

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

07.43

307.29

607.26

907.20

1207.19

1507.12

1807.08

2106.98

Time (min)pH

ENO (ppb)

HCl-inf

= Hypoxia

= Hyperoxia

Figure 8. ENO from hyperoxic and hypoxic lung regions during HCl infusion and decreasing pH. Mean values. Whiskers =SEM

There were no significant differences between the Control and HCl groups in iNOS or cNOS activity, neither in hypoxic nor in hyperoxic lung regions (Figure 9). There were no differences in veno-arterial nitrite and nitrate be-tween, or within, the groups. Nitrate in arterial and mixed venous blood de-creased in the L-NAME+HCl group (p< 0.05).

0

50

100

150

200

250

300

Hyperoxia Hypoxia Hyperoxia HypoxiaCa2+ dependent

NOS activityCa2+ independent

NOS activity

p 0.14 p 0.87 p 0.26 p 0.10

Control groupHCl group

pmol/g/min

Figure 9. NOS activity in lung tissue. Mean values. Whiskers =SEM

In the control group, QLLL/QT was 7 (3) % after 90 min of LLL hypoxia, with no further redistribution of blood flow, or change in PVRLLL over the subse-quent 90 min (p> 0.05). The 90 min HCl infusion caused a further redistribu-

32

tion of pulmonary blood flow, resulting in a decrease in QLLL/QT from 7 (3) % to 3 (1) % (p< 0.01) in the HCl group. In the L-NAME+HCl group, QLLL/QT was 4 (1) % after 90 min of hypoxia during NOS blockade by L-NAME, and then decreased to 1 (1) % (p< 0.05) during 90 minutes of HCl infusion (Figure 10). Despite PVRLLL being high at the start of HCl infusion due to the NOS-blockade, after 90 minutes of HCl infusion, PVRLLL in-creased by 380 (212) % (p< 0.05) in the HCl group, and 384 (254) % (p= 0.01) in the L-NAME+HCl group. In the HCl group, PVRHL increased by 58 (28) % (p< 0.05), but did not change during HCl infusion in the L-NAME+HCl group.

PaO2 increased in the HCl group but not in the control group (p< 0.05). In the L-NAME+HCl group, PaO2 was already higher during NOS-blockade than in the control group, with no further changes over time during HCl-infusion.

0

12345678

QLLL/QT

(%)

0 min HClBaseline

90 min HCl

Control group

HCl group

L-NAME + HClgroup

**

*

Figure 10. QLLL/QT. Mean values. Whiskers =SEM

Paper IV: Effects of hypercapnic acidosis on endogenous nitric oxide production and pulmonary blood flow Hypercapnia rapidly decreased pH from 7.38 (0.03) to 7.10 (0.02) (p< 0.01): the pH then slowly decreased to 7.01 (0.04). In the control group, pH was normal throughout the experiment (7.40 (0.03) -7.37 (0.03)).

33

There were no differences between the groups for ENO from the hypoxic LLL (p= 0.99), or ENO from the hyperoxic lung regions (p= 1.0), and there were no differences in iNOS or cNOS activity in either the hypoxic or the hyperoxic lung regions (Figure 11). In hyperoxic lung, cGMP was 8.2 (6.5) pmol/mg in the control group and 4.1 (1.6) pmol/mg in the hypercapnia group (p= 0.1). In hypoxic lung, cGMP was 3.7 (1.5) pmol/mg in the control group and 2.6 (1.4) pmol/mg in the hypercapnia group (p= 0.2).

Figure 11. NOS activity in lung tissue. Mean values. Whiskers =SEM

QLLL/QT decreased by 72 (5) % during LLL hypoxia in the hypercapnia group and by 68 (9) % in the control group. In the control group, there was no further change in QLLL/QT throughout the experiment. Inhalation of CO2

transiently increased QLLL/QT from 6 (1) % to 9 (2) % (p< 0.01). QLLL/QT

remained elevated for 1.5 hours and then declined to the same level as in the control group after 3.5 hours (Figure 12).

There were no differences between the groups in PVR, PVRLLL or PVRHL. However, pulmonary arterial end diastolic – PcwP gradient (Ppadiast-PcwP gradient) and MPaP were higher in the hypercapnia group than in the control group (p< 0.01).

PaO2 decreased in both groups with LLL hypoxia, and a further decrease was observed in the hypercapnia group (p< 0.01): PaO2 remained unchanged in the control group. Oxygen delivery and PvO2 increased in the hypercapnia group (p< 0.01). In the hypercapnia group, QT increased by 45-50% after introduction of CO2 inhalation (p< 0.01) and remained elevated throughout

34

the experiment: QT did not change over time in the control group. CVP and PcwP did not differ between the groups, however, hypercapnia decreased SVR (p< 0.01) and increased the heart rate (HR) (p< 0.01).

Figure 12. QLLL/QT. Mean values. Whiskers =SEM

35

Discussion

Main findings: INO to endotoxemic pigs caused an increase in endogenous NO produc-

tion in lung regions not reached by INO: both cNOS activity and ENO increased (Paper I).

INO in lavage-induced lung injury did not affect endogenous NO pro-duction in lung regions not reached by INO, as opposed to findings in endotoxemic pigs (Paper II).

Metabolic acidosis did not affect endogenous NO production, in hypoxic or hyperoxic lung regions, as indicated by the lack of change in ENO, plasma nitrite/nitrate and NOS activity. There were no indications of in-creased NO production via alternative pathways during acidosis, as there were no changes in ENO or nitrite, and a decrease in nitrate during NOS inhibition (Paper III).

Metabolic acidosis increased HPV independently of endogenous nitric oxide, even though NOS blockade eliminated NO production (Paper III).

Hypercapnic acidosis did not affect endogenous NO production in hy-poxic or hyperoxic lung regions, as demonstrated by the lack of change in ENO, NOS activity and cGMP (Paper IV).

Hypercapnic acidosis did not potentiate HPV, contrary to metabolic aci-dosis; instead, a transient reduction of HPV was observed (Paper IV).

Possible mechanisms for the distant effect of INO in endotoxemia After INO the transformation of NO in the lung into longer-lived bioactive NO species has distant effects [70]. NO can react with thiols in proteins, such as albumin (SNO-alb) and hemoglobin (SNO-Hb) and form long-lived S-nitrosothiols (SNO´s) [19]. Thus, INO can be transported to remote tissues

36

and exert a vasodilating effect [71]. In this study (Paper I), blood exposed to INO passed through the systemic circulation of the INO-treated animal be-fore reaching the pulmonary circulation of the “NO blood receiver”, thus, SNO´s might have been transported a long way before entering the lungs of the receiving pig.

SNO-Hb is formed when Hb in its R (oxygenated) structure binds NO at the reactive and highly conserved Cys 93 residues [72]. The NO group is then released from Hb coincident with allosteric transition to the T-structure of Hb, as triggered by deoxygenation [72]. In arterial blood, SNO-Hb is do-minant, whereas, in venous blood, HbFeNO is prominent, as it cannot re-lease NO [73]. This means NO delivery from Hb probably takes place along the vascular tree in the donating pig and the receiving pig receives venous blood with mostly HbFeNO. The observed increase in ENO in the NO blood recipient group was unlikely to be due to NO delivery from SNO-Hb. In experimental models with ischemia/reperfusion injury and NO inhalation, SNO-alb releases NO and vasodilates vessels in the gut [71]. However, whether release of NO from SNO-alb could occur under the present study conditions is unknown. The role of SNO´s in human biology is unclear [74, 75]: the reason for this uncertainty is the occurrence of extremely low levels of SNO´s in human blood and the absence of an arterio-venous gradient for SNO´s [75].

NO can be stabilized in blood and tissues through oxidation to nitrate and nitrite. These substances can be considered endocrine molecules that are transported by the blood, accumulate in tissues, and have the potential to be converted back to NO under physiological and pathological conditions [14]. Under normoxic conditions, plasma nitrite is stable and abundant (several hundred nanomolar in plasma and a T1/2 of 1-5 min [18]).

There are numerous pathways for generating NO from nitrite in the tissue, such as, xanthine oxidoreductase (XOR), deoxygenated myoglobin (deoxy-Mb), enzymes of the mitochondrial chain, and protons. All these pathways are potentiated during hypoxia and acidosis [14]. In the blood vessels, nitrite reacts with ferrous deoxyhemoglobin (HbFe2+) and a proton (H+) to generate NO and methemoglobin (HbFe3+) [14]. Deoxyhemoglobin reductase reduces nitrite to NO, and maximal reduction is when Hb is 50% saturated with oxy-gen [76].

As the pigs in this study (Paper I) received high FiO2, none of the pigs were hypoxemic, despite massive endotoxemic capillary leakage and a high degree of intrapulmonary shunt: even the mixed venous blood did not reach saturation as low as 50%. Thus, NO production from nitrite was unlikely to be the main mechanism for the observed increase in ENO.

Increased shear stress induced by increased blood flow, vasoconstriction and/or increased blood viscosity increases eNOS and NO production [77,

37

78]. However, there were no differences in QT, pulmonary pressures, or PVR among the groups; thus, in the present study (Paper I), there was no evi-dence of shear stress inducing an increase in NO production.

The injection of ET-1 causes greater increase in ENO than endogenous en-dothelium-dependent vasodilators and exogenous NO donors [79]. Endotox-emia and INO both increase ET-1[41, 80, 81], and the levels of ET-1 ob-served in this study (Paper I) were comparable with levels in other endotox-in pig models [41, 82]. The effect of ET-1 is mediated by the ETA and ETB receptors. There are two subtypes of the ETB receptors: the ETB1 receptor is located on the endothelium, and when stimulated the receptor causes vasodi-latation by releasing prostacyclin and NO [83]; and, the ETB2 and ETA recep-tors, both located on smooth muscle cells, mediate contraction [84]. ET-1 is mainly produced by vascular endothelium and acts locally in a paracrine fashion in many organs, e.g. lung, kidney and intestine. As 80% of ET-1 passes abluminally [85], the plasma levels of ET-1 might not reflect actual ET-1 activity in the lung.

The analysis of ETB and ETA receptor immunoreactivity in lung tissue re-vealed ETB immunoreactivity was higher in NO-blood recipient pigs, espe-cially in endothelial and smooth muscle cells; However, the immunoreactiv-ity of the staining detects the presence of the receptors, but not whether they are active or not. The findings indicated the mechanism for increased NO production in lung regions not reached by INO could be stimulation of the ETB1 receptor by ET-1.

The increased NO production did not affect global pulmonary vascular re-sistance. Endotoxin administration causes release of numerous vasoactive factors, including endothelin [41, 80, 81]. The vasodilator effect of increased NO concentrations may be counteracted by the vasoconstrictive effect of high levels of endothelin. Thus, the net result might be the absence of effect on pulmonary vascular resistance and pulmonary artery pressures, as was observed in this study (Paper I).

Distant effect of INO in lavage-induced lung injury In pigs with lavage-induced lung injury, cNOS activity decreased in lung regions not reached by INO. However, there was no decrease in ENO or cGMP in the same lavage-injured lung regions and no change in pulmonary vascular tone. Lavage induced a redistribution of pulmonary blood flow, increasing the blood flow to the healthy LLL by 100% in both groups. INO to the LLL did not appear to cause any additional redistribution of blood flow, possibly because the vessels were already maximally dilated.

Lavage-induced acute pulmonary injury is characterized by hypoxemia, an increased right-to-left shunt, decreased lung compliance and pulmonary

38

hypertension, all similar to the characteristics of ARDS [86]. The major cause of impairment of gas exchange is reduced lung surfactant activity [86]. Saline lavage by itself has minor consequences for permeability changes or inflammation [86, 87], in contrast to endotoxin-induced lung injury [86]. Unlike endotoxin models, lavage-induced injury did not increase ET-1, and this might explain the different findings in the two lung injury models. There were no signs of increased NO production in lavage-injured lung regions not reached by INO, as cGMP and ENO were unchanged and there was a de-crease in cNOS activity.

Metabolic acidosis and endogenous NO production Metabolic acidosis did not affect endogenous NO production in the pigs’ lungs. These findings differed from previous studies with HCl infusion in rats [50, 51]. During infusion with HCl, ENO increases after 2 only hours in anesthetized normoxic rats [50]: during the whole infusion plasma ni-trite/nitrate increases and arterial blood pressure decreases [50]. In one study [51], iNOS activity in lung tissue increased and cNOS activity decreased. In another study [50], there were indirect signs of increased iNOS-induced NO production, as the increase in ENO, nitrite/nitrate and hypotension could be prevented by a selective iNOS blocker. A two-fold increase in HCl, resulting in the same pH, blunted the increase in ENO, whereas nitrite/nitrate produc-tion was comparable to the lower dose of HCl [50]. Arterial blood pressure did not decrease with a higher dose of HCl, despite systemic NO production (nitrite/nitrate levels) being the same [50]. ENO is influenced by pulmonary blood flow, as decreased blood flow results in less scavenging of NO, and more NO escapes into the exhaled air. Lower blood pressure might have contributed to the increase in ENO, despite the amount of systemic NO pro-duction (nitrite/nitrate levels) being the same. In the present pig model of metabolic acidosis (Paper III), the systemic hemodynamics was stable throughout the experiments, eliminating effects of pulmonary blood flow on ENO.

The difference in results between the pig and rat models could be ex-plained by different responses in the two species. In pigs, iNOS induction appears of less magnitude [88]. Rodent macrophages produce large amounts of NO, whereas macrophages of pigs are more akin to human macrophages and generate little NO [86].

There are two aspects of NO production in the lung: the effects on pul-monary blood flow distribution, and the possibility high levels of NO may become proinflammatory and cytotoxic, because of its reactive nature [89]. In studies with metabolic acidosis [50, 51], there are indications of lung in-flammation, such as increased intrapulmonary IL-6 levels and inflammatory cells in lung tissue [51], increased myeloperoxidase activity, and increased

39

lung injury score [50]. Oxygenation is impaired [51] or unaffected [50]. In the present study (Paper III), the effects of HCl-infusion on NO-production and regional pulmonary blood flow were examined in hypoxic lung regions, while the rest of the animal was well oxygenated and had stable systemic hemodynamics. An increased NO production in hypoxic lung regions would attenuate HPV and impair oxygenation. Metabolic acidosis increased HPV and PaO2, but ENO from hyperoxic and hypoxic lung regions did not change with decreasing pH.

Hypercapnic acidosis and endogenous NO production Hypercapnic acidosis did not affect endogenous NO production in hypoxic or hyperoxic pig lungs. Inhalation of CO2 causes a decrease in ENO mea-surements because of a quenching effect on the chemiluminescence process [90]. The average reduction rate of NO measurement, as a function of CO2, is estimated to be 0.38% per 1% CO2 [90]. Even with the quenching effect, hypercapnia did not induce changes in ENO in the present study (Paper IV).

The results from previous studies on the effect of hypercapnia on endo-genous NO production are inconclusive, and the discordance between stu-dies is probably due to interspecies differences and different experimental models. In anesthetized rabbits [91] and buffer-perfused rabbit lungs [55, 92], hypercapnia decreases ENO, whereas intravascular NO release is un-changed [92]. In stimulated fetal rat alveolar epithelial cells exposed to 15% CO2 (hypercapnia), there is a marked increase in NO production and NOS activity [54].

Hypercapnia induced reduction of NO appears physiologically meaning-ful. In poorly ventilated areas of the lung, CO2 accumulates, oxygen concen-trations are low, and the small airways and alveoli collapse. These conditions can cause a deficiency in local pulmonary NO production that may enhance HPV, reduce shunt, thus, improving ventilation/perfusion matching [55].

In the systemic circulation, hypercapnia causes vasodilatation through increased NO production [93-96]. The mechanism responsible may be in-creased shear stress [97], due to increased blood flow. Hypercapnia is asso-ciated with increased metabolism, and the subsequent need for increased blood flow, which renders increased NO production meaningful.

The effect of alveolar CO2 exposure and intravascular CO2 exposure on en-dogenous NO production has been studied in isolated rabbit lungs, in which alveolar and intravascular CO2 levels were separately controlled by a mem-brane oxygenator. Inhalation of CO2 suppresses both ENO and perfusate nitrite/nitrate, whereas intravascular hypercapnia does not change ENO or perfusate nitrite/nitrate [90]. The greater suppression of epithelial, rather than endothelial, NO production is explained by differential regulation of

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intracellular pH (pHi) between the epithelium and the endothelium [90]. The pulmonary capillary bed has an intravascular pH equilibrium system, due to the action of membrane-bound carbonic anhydrase [98]. CO2 freely crosses cell membranes, whereas, hydrogen ions do not. When CO2 crosses cell membranes, pHi declines and NOS activity is inhibited. These findings were supported by the results of the present study (Paper IV). Even though hyper-capnia was induced by 10% CO2 inhalation, this resulted in intravascular hypercapnia.

Nitric oxide synthase activity In general, enzymes operate at an optimal pHi, which can differ depending on the cell type. Endothelial NOS activity increases during alkalosis via in-flux of extracellular calcium, and decreases during acidosis [53]. Alkalosis favors cNOS activity, whereas, acidosis favors iNOS activity in cultured cells [53, 99]. In all pigs, iNOS activity was generally lower than cNOS ac-tivity, which supported findings from previous studies on healthy and endo-toxemic pigs [26, 88]. Endotoxin is a potent stimulator of iNOS activity, but pigs appear to respond differently to, for example, rats, with minimal iNOS induction on LPS exposure [88].

Exhaled nitric oxide NO in the gas phase is fairly stable, and if this highly diffusible gas is pro-duced in hollow organs, it should be detectable in luminal air [100]. ENO is dependent on expiratory flow rate, due to ENO arising from both the alveo-lar region and the airways, with local production of NO in the airways and diffusion of NO away from the airways. At low exhalation flow rates, ENO will predominantly be of airway origin: this is especially important in the spontaneously breathing individual and in the case of obstructive lung dis-ease. However, the pigs were paralyzed and mechanically ventilated at con-stant settings throughout the experiment and these factors minimize variation in expiratory flow rate.

ENO measured from a tracheostoma, as in the present studies, reflects NO production from the lower airways, provided other factors influencing ENO remain constant. Several factors, such as changes in ventilation, PEEP, FIO2, pH, and regional and total blood flow influence ENO [101]: ventilation, PEEP and FIO2 were kept constant in all studies.

QT increased after the introduction of CO2 inhalation. An increased pul-monary blood flow increases shear stress, stimulates endothelial NOS (eNOS) activity, and increases ENO [77, 78]. ENO might also decrease due to increased pulmonary blood flow, as NO is scavenged by the blood and

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less escapes into exhaled air [101]. However, during hypercapnic acidosis, no change in ENO from hypoxic or hyperoxic lung regions was observed.

NO can be converted to NO2/NO3 in the gas phase and this conversion in-creases with increasing FIO2 and NO concentration [102]. The expired con-centration of NO2/NO3 was not measured, but assumed extremely low and stable, as ENO was low and FIO2 was kept constant.

Nitrate and Nitrite Nitrate is mainly derived from the diet (for example green leafy vegetables) and endogenously from metabolism of NO (NO + oxyHb Nitrate + MetHb). As the content of nitrate in the diet can be high and the plasma half-life is long (5-6 hours) [103], nitrate measurement is unreliable as an ade-quate index of systemic NO generation.

Nitrite is a better measurement of ongoing NO production, as 70% of plasma nitrite is derived from cNOS activity in the endothelium [104] and plasma half-life is 1-5 min. Ingestion of nitrate can influence plasma nitrite [105]. When nitrate is ingested, it is actively taken up by the salivary glands and secreted into saliva, where commensal bacteria reduce nitrate to nitrite [103]. Nitrite enters the circulation when saliva is swallowed. Although it is assumed the reduction of nitrate to nitrite cannot be catalyzed by mammalian enzymes, inorganic nitrate is reduced to nitrite and then NO in mammalian tissues (rodents and humans) via the involvement of xanthine oxidoreductase [106]. The nitrite ion can be recycled back to bioactive NO in blood and tissues, and all pathways for the generation of NO from nitrite are poten-tiated during hypoxia and acidosis [14].

Although the pigs with metabolic acidosis (Paper III) were exposed to re-gional pulmonary hypoxia, they were all well oxygenated. This was due to high FIO2 to hyperoxic lung regions and augmented HPV, and probably ex-plained the absence of NO generation from nitrite. Nitrate and nitrite in blood reflect NO production in the whole body and not just the lungs. There-fore, the veno-arterial differences in nitrite and nitrate were considered the best indicator of NO production in the lung. A decrease in nitrite over the pulmonary vascular bed reflects NO production, as nitrite is consumed when NO is produced. An increase in nitrate over the pulmonary vascular bed reflects NO production, as nitrate is produced when NO reacts with oxyge-nated hemoglobin. There were no significant veno-arterial differences in either nitrite or nitrate in pigs with metabolic acidosis with, or without, NOS blockade. However, because of high background levels of nitrate, small changes in plasma nitrate concentrations may not sensitively reflect acute changes in NOS activity [104]. Nitrate circulates in micromolar levels in contrast to the picomolar levels of NO. The nitrate decrease over time in

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arterial and venous blood in the L-NAME+HCl group was probably due to NOS blockade and metabolism of dietary nitrate (t1/2 5 hours).

Acidosis, HPV, and pulmonary vascular resistance Metabolic acidosis, such as L-NAME, potentiates HPV [33-36], and this was supported by the findings in the present study (Paper III). When metabolic acidosis was added to NOS-blockade, HPV was further augmented, and the blood flow to the hypoxic lung region was almost eliminated.

The mechanism responsible for the acidosis-induced vasoconstriction was not elucidated. In vivo studies indicate the effects of acidosis on arterial muscle tone occur independently of muscarinic, -adrenergic, or sympathetic nervous system innervations, and may be mediated by mechanisms inherent to the blood vessel wall [107, 108]. pH may act at an intracellular site to differentially regulate voltage-gated potassium channels in pulmonary and systemic vascular smooth muscle cells; thus, modulating vascular reactivity [108]. A decrease in pHi potently dilates coronary resistance arteries, but constricts small pulmonary arteries [108], therefore, implicating the voltage-gated potassium channels in the HPV response [109-112].

Metabolic acidosis preferentially caused vasoconstriction in hypoxic lung regions, resulting in further redistribution of pulmonary blood flow from hypoxic to hyperoxic lung regions, which was consistent with a study by Lejeune et al [60]. PVR increased by almost 400% in hypoxic lung regions, both with and without ongoing NOS-blockade, whereas, the change in PVR in hyperoxic lung regions was smaller (HCl group) or insignificant (L-NAME+HCl group) (Paper III).

Conversely, hypercapnic acidosis did not potentiate HPV. Hypercapnic aci-dosis increased QT and the relative blood flow to the hypoxic LLL (QLLL/QT) during the first 90 minutes. HPV was transiently reduced. There are several mechanisms related to hypercapnia-induced QT increase: first, hypercapnia can initiate a sympathetically mediated release of catecholamines due to neuroadrenal stimulation [113, 114]; second, hypercapnic acidosis induces ATP-sensitive K+ channel–mediated vasodilatation [115]; and, third, preload may be increased via venoconstriction in acidemia [116]. Increased heart rate and decreased after load were the most obvious mechanisms observed in this study (Paper IV).

The intrapulmonary shunt (QS/QT) varies directly with QT. This is tenta-tively explained by decreased HPV, due to a combination of increased PvO2 [117, 118], Ppa [119], and pulmonary blood flow [120, 121]. These factors probably contributed to a transient decrease in HPV; thus, explaining the observed transient increase in QLLL. The increase in QT and PvO2 persisted, whereas, QLLL/QT slowly declined towards the values in the control group.

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Simultaneously (60 – 90 min of LLL hypoxia) with the transient increase in QLLL, there was a decrease in PVRLLL, but the decrease did not reach statis-tical significance (p= 0.07).

PVRHL did not increase in hypercapnic pigs, indicating the transient redi-stribution of pulmonary blood flow to the LLL was not explained by a higher impedance of hyperoxic lung regions. There was no change in PVR in the hypercapnic pigs, but as QT increases, the calculation of PVR might be diffi-cult to interpret. Therefore, the Ppadiast-PcwP gradient may be more accu-rate for expressing resistance to flow through the pulmonary vascular bed [122]. The Ppadiast-PcwP gradient increased in the hypercapnia group after 90 min of CO2 inhalation, indicating increased pulmonary vascular tone. The pulmonary hypertension in the hypercapnic pigs was probably explained by increased pulmonary blood flow and an increased pulmonary vascular tone over time.

In classical textbooks, as Nunns applied respiratory physiology [123], hyper-capnia is said to “reinforce hypoxic vasoconstriction”, as observed in nu-merous experiments on different species [92, 124]. Concurrently, in an iso-lated rat lung model [68], the CO2-mediated vasodilator effect on pulmonary vascular tone is more evident when pulmonary hypertension is induced by hypoxia or ET-1, and the vasodilatory effect of CO2 is pH-independent [68]. The vasoactive action of CO2 is dependent on the initial pulmonary vascular resistance; during basal tone condition, CO2 is a mild vasoconstrictor, while, at high pulmonary vascular resistance, as in hypoxia, CO2 is a potent vasodi-lator [64, 67, 68, 125]. It has been proposed that the effect of CO2 dilatation is a direct action on smooth muscle, and constriction is caused by increasing concentration of intracellular hydrogen ions [68]. Hence, depending on the balance between the dilating effect of the CO2 molecule and the vasoconstric-tive effect of the hydrogen ion, hypercapnia causes both decreased and in-creased HPV.

In the present study (Paper IV), hypercapnia caused a transient decrease in HPV, which could be explained by an initial dilation effect of CO2 com-bined with an increase in QT. As intracellular pH decreased, the vasotonus in hypoxic lung regions increased and HPV approached normal values.

Extracellular pH decreased rapidly to 7.10, and then slowly decreased to pH 7.01. However, the rate of change in pHi might have been different from the rate of change in extracellular pH. The pHi in the pulmonary capillary bed is regulated by membrane-bound carbonic anhydrase [98]. CO2 freely crosses cell membranes, whereas hydrogen ions do not.

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Acidosis and oxygenation Metabolic acidosis increased PaO2 due to augmented HPV, but as the he-moglobin was fully saturated, and there was no change in QT, the effect on DO2 was minimal. Hypercapnic acidosis decreased PaO2. The hypercapnia-induced increase in PvO2, QT and intravascular pulmonary pressure probably lead to the recruitment of pulmonary vessels in poorly ventilated lung re-gions and increased shunt and venous admixture.

A lower PAO2, due to the increase in the inspiratory CO2 partial pressure, was considered an additional explanation for the decrease in PaO2. However, there were no differences between the groups in the calculation of the PAO2. Although PaO2 decreased, the net effect of hypercapnic acidosis on tissue oxygenation was increased DO2 caused by increased QT.

Rationale for INO dose In clinical practice, the recommended dose of INO for achieving local ef-fects in the lung is 20 ppm or less [126]. To be able to demonstrate distant effects, 80 ppm was used in the endotoxin study (Paper I) and 40 ppm in the lavage-induced lung injury study (Paper II). The cross-circulated blood from the pigs exposed to INO was diluted in the receiving pig’s blood volume. It was only possible to cross-circulate 300 to 500 mL/min of blood correspond-ing to 17% of cardiac output. The high dose used was in accordance with other studies investigating distant effects of INO [8, 9].

Rationale for HCl administration Lactate may be a more physiological inducer of acidosis than HCl, as lactate often results in acidosis clinically. However, HCl induced acidosis is a well described model, and used in Pedoto [50] and Hagues [51] studies on NO production and acidosis. The magnitude of the pulmonary arterial pressure response to acidosis is related to the change in the blood pH, rather than to the associated anions of the acids. At the same pH, both lactate and HCl have the same effect of decreasing pH, however, to achieve the same drop in pH as HCl, lactate needs to be given in higher doses [127]

The HCl infusion was administered at a fixed rate and not titrated to a target pH, this resulted in a pH of 7.13 in the L-NAME+HCl group and a pH of 7.2 in the HCl group at data collection. However, the more pronounced acidosis (p> 0.05) in the L-NAME+HCl pigs did not account for the stronger HPV response. When the HCl pigs reached a pH of 7.11, they still had a QLLL/QT of 2.3%, compared to 1% in the L-NAME+HCl group.

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Conclusions

INO in endotoxemic pigs causes an increase in ENO from lung regions not reached by INO, this corresponds to non-ventilated lung regions in single pigs. The effect was caused by increased endogenous NO produc-tion in these lung regions, and could potentially increase shunt and coun-teract the expected increase in arterial oxygenation caused by INO. This finding may contribute to explaining the non-response often seen in sep-tic patients treated with INO (Paper I).

INO in pigs with lavage-induced lung injury does not affect endogenous NO production in lung regions not reached by INO (Paper II).

Metabolic acidosis has no effect on NOS-dependent or NOS-independent NO production in either hypoxic or hyperoxic lung regions in pigs (Paper III).

Both metabolic acidosis and NOS blockade increase HPV, but the ef-fects are independent of each other (Paper III).

Further work is required to determine if the positive effects of mild me-tabolic acidosis on oxygenation i.e. augmented HPV and reduced shunt, is beneficial for severely ill intensive care patients.

Hypercapnic acidosis does not affect endogenous pulmonary NO pro-duction in hypoxic or hyperoxic lung regions, as demonstrated by a lack of change in ENO, NOS activity and cGMP (Paper IV).

Hypercapnic acidosis does not potentiate HPV, as opposed to metabolic acidosis, instead, there is a transient decrease in HPV (Paper IV).

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Future perspectives

Further exploration of the mechanisms of non-response and rebound re-sponse in INO therapy would provide the clinician with tools for rendering more efficient and safe therapy for ARDS. A study of INO in an endotox-emic single pig model with separate ventilation of the LLL would determine if the results from the cross circulation study are reproducible in the single pig model: this means increased NO-production in the LLL would be ex-pected when the rest of the lung receives INO. There was a strong indication NO production depended on ETB stimulation. If endogenous NO production could be blocked by inhalation of an ETB receptor antagonist to the LLL, this would strengthen the evidence ETB receptors mediate increased NO produc-tion in lung regions not reached by INO in endotoxemic pigs.

Inhalation of a low dose of ET-A receptor antagonist results in selective pulmonary vasodilatation, increased PaO2, and decreased intrapulmonary shunt in lavage-induced lung injury [128]. The effect is similar to that caused by INO, but is probably mediated through a different mechanism. Rebound response at withdrawal of INO is probably caused by a combination of down-regulation of endogenous NO-production and persistently increased ET-1 levels due to exogenous NO [41], and intravenous administration of an ET-A receptor antagonist protects against rebound response [129].

With this background, a combination of ET-A receptor antagonist-inhalation and INO in experimental septic ARDS could determine the occur-rence of any potentiation, and if this combination prevents rebound response.

Although hypercapnia transiently decreased HPV, and then normalized the HPV without potentiation over 3.5 hours, it is unclear what happened after this. Thus, further studies to elucidate the longer term effects of pro-longed hypercapnia on HPV and arterial oxygenation would be relevant, especially a clinical setting, which involves longer time perspectives than were covered in this study.

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Acknowledgements

There are many who directly or indirectly have contributed to this thesis. I would like to express my sincere gratitude to:

Filip Fredén, my tutor for introducing me to scientific work. For having the courage to take on a PhD student in the child bearing phase of life. For many amusing hours in the lab and for clever thoughts.

Kristina Hambraeus Jonzon, my cotutor for your never ending enthusiasm and encouragement. For your “hawk eye“ and insightful comments and thoughts.

Göran Hedenstierna for letting me use the lab facilities and for scientifically fruitful discussions.

Lars Wiklund, for creating an inspiring research environment at the depart-ment of Anaesthesiology and Intensive Care.

Anders Larsson for “lending out your scientific brain”.

Agneta Ronéus, Karin Fagerbrink and Maria Lundqvist for their skilled technical assistance in the laboratory, positive attitude and always trying to help with all matter of problems.

Peter Wiklund, Kjell Alving and Maria Bergquist, coauthors for all help with analyses.

Colleagues at the department of Anaesthesiology and Intensive Care in Upp-sala and Hudiksvall for making my every day at work educational and joy-ful.

Torbjörn Karlsson, head of the department of Anaesthesiology and Intensive Care, for creating conditions for research and for allowing me precious time off from clinical work. My family and friends. Especially my husband Pelle and our children Arild, Josefine and Freja. You all make me happy!

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