J O U R N A L O F A P P L I E D P H Y S I O L O G Y

DECEMBER 1994/Volume 77, Number 6

CONTENTS

THIS MONTH IN T H E J O U R N A L 2515

INVITED E D I T O R I A L

Invited Editor ial on " N i t r i c oxide release is present from incubated skeletal muscle preparations"

S. S. Segal 2517

ENVIRONMENT

Effect of heat stress on muscle energy metabolism during exercise M. A. Febbraio, R. J . Snow, C. G. Stathis, M. Hargreaues, and M. F. Carey 2827

Effect of acclimatization to high altitude (5,050 m) on motor uni t activation pattern and muscle performance

C. Orizio, F. Esposito, and A. Veicsteinas 2840

E X E R C I S E AND M U S C L E

Enhanced adipose tissue lipoprotein lipase activity in detrained rats: independent of changes in food intake

Ε. V. Lambert, G. Wooding, Μ. I . Lambert, J . H. Koeslag, and T. D. Noakes 2564 Cardiorespiratory kinetics and femoral artery blood velocity during dynamic knee extension exercise

J . K. Shoemaker, L . Hodge, and R. L . Hughson 2625 Training-related enhancement i n the control of motor output in elderly humans

D. A. Keen, G. H. Yue, and R. M. Enoka 2648 Evaluation of a symmetrically disposed Pitot tube flowmeter for measuring gas flow during exercise

J . Porszasz, T. J . Barstow, and K. Wasserman 2659 Muscle chemoreflex alters vascular conductance in nonischemic exercising skeletal muscle

S. W. Mittelstadt, L . B. Bell, K. P. O'Hagan, and P. S. Clifford 2761

( Continued)

Cover: A geometric model explores the role of symmetry in distributed control of respiration. Pertur­bations to symmetry (e.g., using vagal stimulation) induce chaotic dynamics i n breathing pattern. (See Sammon. J . Appl. Physiol 77: 2481-2495, 1994.)

This Journal is pr inted on "acid-free" paper.

(Contents continued)

Recovery of medial gastrocnemius muscle grafts in rats: implications for the plantar flexor group

S. W. Miller, C. A. Hassett, T. P. White, and J . A. Faulkner Exercise, potassium, and muscle deconditioning post-thoracic organ transplantation

M. J . Hall, G. I. Snell, E. A. Side, D. S. Esmore, Ε. H. Walters, and T. J . Williams

M E T A B O L I S M

Nitr i c oxide release is present from incubated skeletal muscle preparations T. W. Bahn and J . L . Nadler

C a 2 + and lipoiysis in adipocytes from exercise-trained rats T. Izawa and T. Komabayashi

Preload release increases blood flow and decreases fatigue during repetitive isotonic muscle contractions

Β. T. Ameredes, W. F. Brechue, and W. N. Stainsby Pentoxifylline inhibits basal glucose production in humans

Ε. P. M. Corssmit, J . A. Romijn, E. Endert, and H. P. Sauerwein Dopamine and mucosal oxygenation in the porcine je junum

R. Germann, M. Haisjackl, W. Hasibeder, H. Sparr, G. Luz, R. Plattner, Η. Pernthaler, Β. Friesenecker, and Μ. Falk

The need to scale for differences in body size and mass: an explanation of Kleiber's 0.75 mass exponent ( M O D E L I N G I N P H Y S I O L O G Y )

A. M. Nevill

C O N T R O L O F B R E A T H I N G , C I R C U L A T I O N , AND T E M P E R A T U R E

Sympathetic and parasympathetic indicators of heart rate control at altitude studied by spectral analysis

R. L. Hughson, Y. Yamamoto, R. E. McCullough, J . R. Sutton, and J . T. Reeves Cisternal N a + transport inhibit ion and the ventilatory response to C 0 2

M. P. Sullivan and J . M. Adams Soft palate muscle activity in response to hypoxic hypercapnia

T. Van der Touw, N. O'Neill, T. Amis, J . Wheatley, and A. Brancatisano Effect of N a + and K + channel blockade on baseline and anoxia-induced catecholamine release from rat carotid body

T. P. Doyle and D. F. Donnelly Forearm compression during exercise increases sympathetic nerve traffic

J . McClain, J . C. Hardy, and L . I. Sinoway Pulmonary congestion enhances responses of lung rapidly adapting receptors to cigarette smoke in rabbit

K. Ravi, C. T. Kappagoda, and A. C. Bonham Dose dependency of perceived breathlessness on hypoventilation during exercise in normal subjects

H. R. Η arty and L . Adams Hypoxic inhibit ion of breathing i n fetal sheep: relationship to brain adenosine concentrations

B. J. Koos, Β. A. Mason, Ö. Punla, and A. M. Adinolfi Hamsters vs. rats: metabolic and ventilatory response to development in chronic hypoxia

P. B. Frappell and J . P. Mortola Enhancement of parasympathetic cardiac activity during activation of muscle metaboreflex in humans

T. Nishiyasu, N. Tan, Κ Morimoto, M. Nishiyasu, Y. Yamaguchi, and N. Murakami

(Contents continued)

Effect of hypercapnia on laryngeal airway resistance in normal adult humans S. T. Kuna, C. R. Vanoye, J . R. Griffin, and J . D. Updegrove

Cerebral hemodynamics during sensorimotor activation in humans M. Sitzer, U. Knorr, and R. J . Seitz

Effects of head-down bed rest on complex heart rate variability: response to L B N P testing A. L . Goldberger, J . E. Mietus, D. R. Rigney, M. L . Wood, and S. M. Fortney

Assessment of frequency shifts in R-R interval variability and respiration wi th complex demodulation ( M O D E L I N G I N P H Y S I O L O G Y )

J . Hayano, J . A. Taylor, S. Mukai, A. Okada, Y. Watanabe, K. Takata, and T. Fujinami

C E L L U L A R A S P E C T S O F LUNG FUNCTION

Quantitation of neutrophil migration in acute bacterial pneumonia in rabbits C. M. Doerschuk, J . Markos, Η. Ο. Coxson, D. English, and J . C. Hogg

Comparative age-related acute and chronic pulmonary oxygen tolerance in rats S. Laudert, D. W. Thibeault, M. M. Rezaiekhaligh, S. M. Mabry, and M. Huntrakoon

Rapid neutrophil accumulation and protein oxidation in irradiated rat lungs H. Fliss and M. Menard

Role of nonenzymatically generated prostanoid, 8-iso-PGF 2 a , in pulmonary oxygen toxicity ( R A P I D C O M M U N I C A T I O N )

C. A. Vacchiano and G. E. Tempel

S Y S T E M I C C I R C U L A T I O N AND FLUID B A L A N C E

^-Adrenergic effects on left ventricular f i l l ing: influence of aging and exercise t ra in ing J . R. Stratton, W. C. Levy, R. S. Schwartz, I. B. Abrass, and M. D. Cerqueira

Regulation of bioenergetics in 0 2 - l i m i t e d isolated rat hearts M. Samaja, S. Casalini, S. Allibardi, A. Oorao, and S. I,. Chierchia

Contribution of potassium to exercise-induced vasodilation in humans J . R. Wilson, S. C. Kapoor, and G. G. Krishna

Dextran sulfate activates contact system and mediates arterial hypotension via B 2 k in in receptors

M. Siebeck, J . C. Cheronis, E. Fink, J . Kohl, B. Spies, M. Spannagl, M. Jochum, and H. Fritz

Quantification of adult cerebral hemodynamics by near-infrared spectroscopy C. E. Elwell, M. Cope, A. D. Edwards, J . S. Wyatt, D. Τ Delpy, and E. 0. R. Reynolds

Volume-homeostatic mechanisms in humans during graded water immersion A. S. Larsen, L . B. Johansen, C. Stadeager, J . Warberg, N. J . Christensen, and P. Norsk

Evaluation of the needle technique for producing an arteriovenous fistula ( S P E C I A L C O M M U N I C A T I O N )

M. Huang, Μ. H. LeBlanc, and R. L . Hester

G A S E X C H A N G E , M E C H A N I C S , AND A I R W A Y S

Altered mechanical properties of lung parenchyma in postobstructive pulmonary vasculopathy

S. M. Kelly, J . Η. Τ Bates, and R. P. Michel

(Contents continued)

Artifacts associated w i t h acoustic rhinometric assessment of infants and young children: a model study

J . E. Buenting, R. M. Dalston, T. L . Smith, and A. F. Drake 2558 Effect of inhaled budesonide on ozone-induced airway hyperresponsiveness and bronchoalveolar lavage cells in dogs

W. Η M. Stevens, E. Ädelroth, J . Wattie, M. J . Woolley, R. Ellis, M. Dahlbäck, and P. M. O'Byrne 2578

Longitudinal distribution of ozone absorption in the lung: comparison of nasal and oral quiet breathing

J . R. Kabel, A. Ben-Jebria, and J . S. Ultman 2584

Dynamic surface tension of surfactant T A : experiments and theory D. R, Otis, J r . , E. P. Ingenito, R. D. Kamm, and M. Johnson 2681

Effects of acute lung injury on dynamic tissue properties E. P. Ingenito, L . Mark, and B. Davison 2689

Effects of airway parasympathetic tone on responses to intravenous bronchoconstrictor agonists in rats

R. Sorkness, J . Clough, and R. F. Lemanske, J r . 2698 Relationship between Cr and breathing pattern in mechanically ventilated patients

H. Burnet, M. Bascou-Bussac, C. Martin, and Y. Jammes 2703 Validation of near-infrared spectroscopy i n humans

D. M. Mancini, L . Bolinger, H. L i , K. Kendrick, B. Chance, and J . R. Wilson 2740 Regional diaphragm shortening measured by sonomicrometry

Y. Wakai, A. M. Leevers, and J . D. Road 2791 Effect of sternothyrohyoid myectomy on upper airway mechanics in normal horses

S. J . Holcombe, W. L . Beard, Κ W. Hinchcliff, and J . T. Robertson 2812 Oxygen transport to tissue by persistent bubbles: theory and simulations ( M O D E L I N G I N P H Y S I O L O G Y )

Μ. E. Burkard and H. D. Van Liew 2874 One-dimensional simulation of aerosol transport and deposition in the human lung ( M O D E L I N G I N P H Y S I O L O G Y )

C. Darquenne and M. Paiva 2889 Influence of site of tracheal pressure measurement on in situ estimation of endotracheal tube resistance ( S P E C I A L C O M M U N I C A T I O N )

P. Navalesi, P. Hernandez, D. Laporta, J . S. Landry, F. Maltais, D. Navajas, and S. B. Gottfried 2899

P U L M O N A R Y C I R C U L A T I O N AND L U N G FLUID B A L A N C E

Time course of blood volume changes in an isolated lung lobe after venous pressure elevation

Μ. B. Maron and S. M. Lane 2720 T N F potentiates PAF-induced pulmonary vasconstriction in the rat: role of neutrophils and thromboxane A 2

S.-W. Chang 2817 Effect of chronic hypoxia on pulmonary vascular pressures i n isolated lungs of newborn pigs

C. D. Fike and M. R. Kaplowitz 2853

Subject Index to Volume 77 Author Index to Volume 77

2919 2935

Dextran sulfate activates contact system and mediates arterial hypotension via B 2 kinin receptors

M A T T H I A S S I E B E C K , J O H N C . C H E R O N I S , E D W I N F I N K , J A C E K K O H L , B E R N D S P I E S , M I C H A E L S P A N N A G L , M A R I A N N E J O C H U M , A N D H A N S F R I T Z Departments of Surgery, Medicine, and Clinical Chemistry and Clinical Biochemistry, Ludwig-Maximilians University, Klinikum Innenstadt, D-80336 Munich, Germany

Siebeck, Matthias , J o h n C . Cheronis , E d w i n F i n k , J a -cek K o h l , B e r n d Spies , Michael Spannagl , Marianne J o -chum, and H a n s F r i t z . Dextran sulfate activates contact sys­tem and mediates arterial hypotension via B 2 k i n i n receptors. J . Appi Physiol. 77(6): 2675-2680, 1994.—To define some of the mechanisms underlying dextran sulfate (DXS)-induced hy­potension, we investigated the effects of either the plasma ka l ­l ikrein inhibitor des-Pro 2 - [Arg 1 5 ] aprotinin (BAY χ 4620) or the specific bradykinin B 2-receptor antagonist Hoe-140 on the hy­potensive response to DXS . I n the f irst study, anesthetized miniature pigs were given D X S alone, D X S plus B A Y χ 4620 i n various doses, or saline. As expected, D X S alone produced a profound but transient systemic arterial hypotension wi th a concomitant reduction i n kininogen. Circulating k in in levels, complement fragment des-Arg-C3a, and fibrin monomer were all increased. Treatment wi th B A Y χ 4620 produced a dose-de­pendent attenuation of these effects wi th complete blockade of the hypotension as well as the observed biochemical changes at the highest dose (360 mg). I n a second study, two groups of pigs were given either D X S alone or DXS plus Hoe-140. D X S - i n -duced hypotension was completely blocked by Hoe-140 pre-treatment; however, kininogen was again depleted. We con­clude, therefore, that DXS-induced hypotension is produced by activation of plasma kal l ikrein that results in the production of bradykinin and that liberation of bradykinin and its action on B 2 receptors in the vasculature are both necessary and suffi­cient to produce the observed effects on circulatory pressure.

plasma kal l ikrein inhibitor ; pigs; serine protease

D E X T R A N S U L F A T E ( D X S ) is a negatively charged macro-molecule that has been shown to activate prekal l ikrein (15). A s such, it is one of a variety of compounds that are able to activate the contact phase of blood coagulation, a multifunctional system of proteases that controls vaso-regulation (via the formation of the potent vasodilator bradykinin) and amplifies the humoral inflammatory re­sponse (via activation of the coagulation, fibrinolysis, and complement pathways) (2).

T h e essential components of the contact system are the zymogen factor X I I (Hageman factor), a negatively charged surface or macromolecule that induces a change in the conformation of factor X I I , high-molecular-mass kininogen, prekal l ikrein , and factor X L I n this complex, factor X I I and prekal l ikrein can activate each other in the presence of kininogen, resulting in the cleavage of kininogen by kal l ikrein and the release of bradykinin (2). I n addition, factor X l l a converts factor X I to its active form, factor X I a , thereby activating the coagulation cas­cade. B o t h the H a g e m a n factor fragment (8) and throm­bin (13) are able to activate the classical pathway, whereas p lasma kal l ikrein can activate the alternative

pathway (6) of the complement system. Interaction of plasma kal l ikrein , factor X I a , and factor X l l a (3) also leads to activation of single-chain urokinase-type plas­minogen activator and of a contact system-dependent plasminogen proactivator (1). I n their activated form, these proteases convert plasminogen to p lasmin and are considered to be the primary agents involved in intrinsic fibrinolysis. F ina l ly , bradykinin and thrombin induce the release of tissue-type plasminogen activator from endo­thelial cells, thereby triggering extrinsic fibrinolysis.

Because the contact system combines the activation of the p lasma cascade systems with the release of the hypo­tensive mediator bradykinin, the system has long been implicated in the pathophysiology of septic shock, dis­seminated intravascular coagulation, and multiple organ failure including adult respiratory distress syndrome (14, 17). I n addition, bacterial lipopolysaccharide has been shown to activate the contact system in vitro (18), and in humans plasma kal l ikrein has been shown to be acti ­vated during endotoxemia (5). F inal ly , p lasma kal l ikrein inhibitors such as aprotinin (22) or a 1 - a n t i t r y p s i n P i t t s ­burgh (4) can attenuate the effects of bacterial - or endo-toxin-induced experimental shock.

I n a previous investigation we found that infusion of D X S decreased the amount of uncleaved kininogen (kinin-containing kininogen) in pig plasma and reduced systemic arterial pressure. Blood pressure returned to normal only after kinin-containing kininogen had been depleted. B o t h the cleavage of kininogen and hypoten­sion were attenuated or blocked by C r e s t e r a s e inhibitor and high doses of aprotinin (9), both of which inhibit p lasma kal l ikrein, suggesting a role for bradykinin in this effect. I n rabbits, however, D X S - i n d u c e d arterial hypo­tension has been shown to be mediated via the release of serotonin from platelets (25), which can be stimulated by thrombin, another mediator of the activated contact system.

T h e present investigation was conducted to clarify the mechanism of D X S - i n d u c e d hypotension in pigs. I t was our hypothesis that D X S - i n d u c e d hypotension is p r i ­marily mediated by bradykinin and that other mediators such as serotonin, platelet-activating factor, and metabo­lites of arachidonic acid, i f involved, are dependent on bradykinin. T w o studies were performed, one with the plasma kal l ikrein inhibitor d e s - P r o 2 - [Arg 1 5 ]aprot inin ( B A Y χ 4620) (7) and the second with the dekapeptide D -Arg° [Hyp 3 ,Thi 5 ,D -Tic 7 ,Oic 8 ]bradykinin (Hoe-140), a bradykinin B 2 - receptor antagonist (20). B o t h com­pounds were able to completely block D X S - i n d u c e d hy­potension, whereas only the former compound prevented the decrease of uncleaved kininogen.

2675 0161-7567/94 $3.00 Copyright © 1994 the American Physiological Society

2676 K I N I N G E N E R A T I O N I N V I V O BY D E X T R A N S U L F A T E I N F U S I O N

M A T E R I A L S A N D M E T H O D S

DXS and BAY χ 4620

Recombinant BAY χ 4620 (7) was generously provided by P. Stadler, D. Hörlein, and F. Schumann (Bayer AG, Wuppertal , Germany). For these studies, the compound was dissolved in sterile physiological saline and infused intravenously.

DXS, wi th a molecular mass of — 500,000 Da (Pharmacia L K B , Uppsala, Sweden), was dissolved in 30 m l of sterile physi­ological saline 1 h before use and infused intravenously in a dose of 5 mg/kg for 1 h. The dose of D X S used i n this study was based on previous experimentation (9). Animals were observed for 2 h from the start of the D X S infusion.

On the basis of prel iminary pharmacokinetic experiments (data not shown), the entire dose administered was divided into a bolus dose (one-third of the total) injected 15 m i n before the start of the D X S infusion, followed by a continuous infusion over 135 min during which t ime the remaining two-thirds of the dose was delivered. The total dose of B A Y χ 4620 was varied from 45 to 360 mg. Three control groups, 1) D X S plus saline, 2) saline alone, and 3) high-dose BAY χ 4620 (360 mg) alone, were also included in this study.

A l l animal procedures were approved by the Regierung von Oberbayern and complied w i t h the Tierschutzgesetz in der Fas-sung of August 18, 1986. The animals, 35 miniature pigs, were fasted overnight but had free access to water. Sedation was achieved by azaperone (3 mg/kg im) and metomidate (3.75 mg / kg ip) . After induction of anesthesia w i t h pentobarbital sodium (24 mg/kg iv) , a 6.5-mm orotracheal tube was placed and vent i ­lation was maintained by use of a 900C ventilator (Siemens Elema AB) . Vascular catheters (arterial and central venous, Swan-Ganz) and a short large-bore cannula i n the external jug­ular vein were inserted through groin and neck incisions, re­spectively, for blood sampling. After the preparation, the ani ­mals rested for 1 h.

Blood pressure was recorded every 5 min w i t h other physio­logical measurements [e.g., cardiac output (CO) and pulmo­nary arterial pressure (PAP)] being made every 30 min . Blood was collected for biochemical analysis and k in in extraction every 15 min.

Blood sampling and extraction for the k in in measurement was based on previously reported procedures (19, 21). Briefly, a 10-ml plastic syringe was fil led w i t h 3 m l of 0.8 Μ H C l and cooled on ice. Through the large-bore cannula in the external jugular vein, 5 ml of blood were drawn into the syringe. Care was taken to achieve maximum aspiration speed by avoiding contact between the t ip of the syringe and the vessel wall . Dur ­ing the aspiration the syringe was rotated for the rapid mixing of H C l and blood. The sample was then injected immediately into a second plastic syringe containing 2 m l of ice-cooled 0.8 Ν H C l , shaken vigorously, and placed on ice. The final p H of the sample was adjusted to 2.0 by the addition of 1.15 ml of a 2 Ν N a O H solution. After 3.68 g of NaCl were added to the sample, i t was mixed in an ice-cooled kinematic inversion shaker (Tur -bola, Bachofen, Basel, Switzerland) for 10 min.

For the k in in extraction, 3 m l of n-butanol were added to each syringe, and the sample was mixed again for 4 m i n and then centrifuged for 4 min at 1,800 g. The butanol phase was separated into an ice-cooled test tube, and the process was re­peated two more times wi th the centrifugation period extended to 6 and 8 min , respectively. The three butanol fractions were pooled, and the cellular debris and residual plasma proteins were discarded. For the removal of lipids and simultaneous reextraction of kinins into an aqueous medium, 1.5 ml of petro­leum ether and 2 ml of distilled water were added to the butanol extracts, and the solution was mixed i n the shaker for 10 min and then centrifuged at 800 g for 14 min . The aqueous phase was then transferred into Eppendorf tubes w i t h a glass Pasteur pipette, frozen, and stored at - 8 0 ° C for subsequent assay.

Plasma levels of uncleaved kininogen were measured by a l ­lowing trypsin to degrade any high- and low-molecular-mass kininogen contained i n the sample, w i t h subsequent measure­ment of the released k i n i n (22, 24).

The assay of the k i n i n levels i n the blood as well as the mea­surement of k i n i n produced from intact kininogen i n the plasma (see above) utilized a double-antibody radioimmunoas­say (21). The ant ik in in antibody was from K. Shimamoto (Sap­poro, Japan). The second antibody, anti-rabbit globulin f rom the donkey, was purchased from IDS (Tyne and Wear, U K ) . The detection l i m i t for k i n i n using this technique is 30 f m o l / m l .

The anaphylatoxin C3a was measured as des-Arg-C3a, a stable and biologically inactive degradation product of C3a pro ­duced by carboxypeptidase B. A modified radioimmunoassay was used (22) based on a method described for human des-Arg-C3a (12) w i th the antiserum against des-Arg-C3a raised in rab­bits. des-Arg-C3a and antiserum against des-Arg-C3a were gifts from the late B. Damerau (Göttingen, Germany). The f i ­br in monomer i n plasma was measured wi th an enzyme i m m u ­noassay based on a monoclonal antibody (23). This antibody binds to the N H 2 - t e r m i n a l end of the α-chain of porcine f i b r in . Thrombin cleavage of known amounts of fibrinogen was used to prepare the standard curve used i n the assay.

DXS and Hoe-140

Six weaned domestic pigs w i t h a body weight ranging from 26 to 34 kg were used in this study. Anesthesia, venti lation, and surgical procedure were the same as described above. A l l an i ­mals received D X S (2 mg/kg) as a continuous infusion over 60 min . Three animals were randomized to receive a bolus injec­t ion of Hoe-140 (30 Mg/kg body wt) 10 min before the start of the D X S infusion. Hoe-140 was a gift from B. A. Schoelkens and K. J . W i r t h , Hoechst AG, Frankfurt , Germany. Three con­tro l animals received a saline injection instead of Hoe-140. The dose of Hoe-140 was chosen on the basis of preliminary inh ib i ­t ion experiments w i t h bolus injections of bradykinin as well as pilot experiments w i t h D X S (data not shown). The dose chosen for this study was found to completely block the hypotensive response to bolus injections of bradykinin up to 2,000 ng/injec-t ion; the threshold dose needed to produce a decrease of 3-5 m m H g in diastolic blood pressure was 10-20 ng of bradykinin before the administration of Hoe-140. CO was measured every 5 min w i t h the thermodilution method; arterial and venous pressures in the systemic and pulmonary circulations were re­corded simultaneously. Systemic vascular resistance (SVR) was calculated by using a standard formula {79.9[mean arterial blood pressure ( M A B P ) - central venous pressure]/CO}. To ­tal protein was measured w i t h the Biuret method.

Statistical Analysis

Unless otherwise noted, data are presented as means ± SE. The level of significance was chosen as a = 0.05.

The evaluation of the blood pressure changes produced by DXS had to take into account that administration of the lower doses of the inhibitor only delayed the time of onset of D X S - i n -duced hypotension and that complete inhibit ion would occur only at higher doses. Therefore, for each individual experi­ment, we measured the t ime after the start of the D X S infusion at which the blood pressure had fallen to <60% of the baseline value. These data were analyzed wi th the Mantel-Haenszel test. The software program that was used (TEST, Ins t i tut für Datenanalyse und Versuchsplanung, Gauting, Germany) simul­taneously performed multiple Scheffe-like comparisons be­tween the groups.

Furthermore, the distribution of the measurements of un­cleaved kininogen was not normal and the baseline levels were not equal in all groups. Here, the time from start of the DXS infusion to a 50% decrease of the uncleaved kininogen level was

K I N I N G E N E R A T I O N I N V I V O B Y D E X T R A N S U L F A T E I N F U S I O N 2677

Α , α. 100 -

5 ~ 50· :

Β Λ Β 4 0 -

ί I 3 0 -

~ 2 0 -

<= C q S > ~ 1 0 0 -8 # : .ε ~ 5 0 -

π "

DXS+NaCI DXS+45 mg

Y\ DXS+90 mg

Y 1 DXS+180 mg DXS+360 mg NaCI

Α , α. 100 -

5 ~ 50· :

Β Λ Β 4 0 -

ί I 3 0 -

~ 2 0 -

<= C q S > ~ 1 0 0 -8 # : .ε ~ 5 0 -

π "

I ι ι ι I ι ι ι 1

A ] I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1

••VHtrf**̂ -« 1

Α , α. 100 -

5 ~ 50· :

Β Λ Β 4 0 -

ί I 3 0 -

~ 2 0 -

<= C q S > ~ 1 0 0 -8 # : .ε ~ 5 0 -

π "

I ι ι ι I ι ι ι 1

\ . 1 \ 1 I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1

v j I ι ι ι I 1 ι ι 1 ] ι ι ι I ι ι ι 1

υ - J

= 1 4 i I i 2 ^

Q. 500 η

<S? ^ 300 -Ο ~

1 0 0 -

F _ 4 0 -

5 I 2 0 -

~ 0 -

1 ! 1 1 1 1 1 . 1

i 1 I ι ι ι I ι ι ι 1

k i I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1 υ - J

= 1 4 i I i 2 ^

Q. 500 η

<S? ^ 300 -Ο ~

1 0 0 -

F _ 4 0 -

5 I 2 0 -

~ 0 -

I ι ι ι I ι ι ι 1

A : I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1

-I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1

- K - K • . <

υ - J

= 1 4 i I i 2 ^

Q. 500 η

<S? ^ 300 -Ο ~

1 0 0 -

F _ 4 0 -

5 I 2 0 -

~ 0 -

I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1 I ι ι ι I ι ι ι 1

I 1 1 ' I ' 1 1 1 Ί ι ι ι ι ι ι ι ι Ί ι ι ι ι ι ι ι ι Ί ι • ι ι ι ι ι ι Ί I ι ι ι ι ι ι ι Ί ι ι ι ι ι ι ι ι 0 60 120 0 60 120 0 60 120 0 60 120 0 60 120 0 60 120 Time (min) Time (min) Time (min) Time (min) Time (min) Time (min)

FIG. 1. Effects of dextran sulfate (DXS) and plasma kal l ikre in inhibitor B A Y χ 4620 over t ime. Min iature pigs received D X S infusion (5 mg/kg iv) for 1 h plus either saline solution or BAY χ 4620 i n doses of 45, 90,180, or 360 mg over 2 h (5 groups) or received saline solution alone (1 group). A: early decrease of mean arterial blood pressure ( M A B P ; % of baseline value) in control group was delayed by low doses of inh ib i tor , attenuated by higher dose, and completely prevented by highest dose (P < 0.001). B: D X S alone produced biphasic rise i n pulmonary arterial pressure (PAP) at 10 and 60 m i n . Secondary rise i n PAP was attenuated by high doses of inh ib i tor (P = 0.0017). C: k in in-con­taining kininogen i n plasma is given as % of baseline value. Early decrease i n kininogen i n control group was delayed by low and higher doses of inhibi tor and blocked by highest dose (P < 0.001). D: release of k i n i n was attenuated w i t h increasing doses of inhib i tor and completely blocked by highest dose (P < 0.0015). E: anaphylatoxin des-Arg-C3a (C3a) in plasma is given as % of baseline value. C3a in plasma rose to reach maximum at 1 h i n control group. Formation of C3a was attenuated w i t h increased doses of inhib i tor (P < 0.05). F: in group t h a t received D X S alone, f i b r in rose substantially. Higher doses of inhib i tor produced part ia l inhib i t ion , w i t h highest dose producing complete inhib i t ion of f ibr in formation (P < 0.05).

measured i n each animal and analyzed w i t h the aforemen­tioned procedure (Mantel-Haenszel test).

The distribution of the measurements of k in in also was not normal, but, i n this case, the Kruskal l -Wal l is analysis of ranks was used. For a global test, the average of all repeated measure­ments during D X S infusion i n each animal was formed. M u l t i ­ple tests at each t ime were decided according to the closure principle (16).

For a global test of PAP, the average of the repeated measure­ments during D X S infusion i n each animal was formed. These values were analyzed by analysis of covariance, w i t h the base­line measurement as covariate. Mult ip le comparisons between groups were made w i t h Tukey's test. Because of their distr ibu­t ion , results of des-Arg-C3a and f ibr in were analyzed w i t h Kruskal l -Wal l is analysis. A global value could not be formed because of missing values. Therefore, the multiple niveau alpha

was ascertained w i t h a modified Bonferroni procedure (10). For the data involving D X S and Hoe-140, the statistical analysis was l imi ted to a comparison at 25 min , the time of maximum blood pressure decrease. The Mann-Whitney U test was used wi th Ρ < 0.05 considered significant.

R E S U L T S

DXS and BAY x 4620

MABP (Fig. 1). D X S (5 mg/kg) induced a rapid decline in M A B P that reached a nadir of 44.5 ± 2.6% of baseline value at 15 min in the group D X S + N a C I . M A B P re­turned to normal levels by 30 min after the D X S infusion was started. I n the group that received D X S and the low­est dose of B A Y χ 4620 ( D X S + 45 mg of B A Y χ 4620),

2678 K I N I N G E N E R A T I O N I N V I V O B Y D E X T R A N S U L F A T E I N F U S I O N

0 60 120 Time (min)

0 60 120 Time (min)

F I G . 2. Effects of D X S and Hoe-140 over time. Six weaned pigs re­ceived D X S infusion iv (2 mg/kg) for 1 h. I n addition, 3 pigs received 30 Mg / kg of Hoe-140 ( · ) 15 min before D X S and 3 pigs received saline solution (O). T ime course in each group is presented as mean ± SE. x-Axis, time after onset of D X S infusion. A: control animals had sharp decrease in M A B P from 15 to 35 min after onset of D X S infusion, whereas Hoe-140-treated animals had completely stable blood pres­sure; their M A B P was significantly higher at 25 min . Β: systemic vascu­lar resistance (SVR) is given as % of baseline value. Ar ter ia l hypoten­sion in control animals was paralleled by systemic vasodilation, whereas Hoe-140-treated animals had stable SVR; their SVR was sig­nificantly higher at 25 min . C: cardiac output (CO) is given as % of baseline value. From 5 to 30 m i n after onset of D X S infusion, CO was elevated in control animals. Hoe-140-treated animals had unchanged CO (NS). D: heart rate (HR) is given as % of baseline value. Control animals responded w i t h rise i n H R to D X S infusion. Hoe-140-treated animals had no change i n H R (NS). E: values of uncleaved kininogen declined to 18 ± 1 % of baseline without any difference between groups. F: values of total protein remained stable.

M A B P remained stable unti l 20 min, decreased to its nadir of 45 ± 3.5% after 30 min, and then returned to baseline level. T h e augmented dose of the inhibitor ( D X S + 90 mg of B A Y χ 4620) led to a further delay in the onset of hypotension that reached its nadir of 46 ± 4.8% at 45 min. T h e next inhibitor dose level ( D X S + 180 of mg B A Y χ 4620) not only delayed the onset but also reduced the magnitude of hypotension to 78 ± 10% at 55 min. M A B P remained constant during the entire observation period in the group with D X S and the highest inhibitor dose ( D X S + 360 mg of B A Y χ 4620) and in the control group without D X S . T h e time to 60% hypotension dif­fered significantly between the groups (Mantel -Haenszel test, Ρ < 0.001), and in the multiple comparison test, the difference between D X S + N a C l and D X S + 360 mg of B A Y χ 4620 was statistically significant (P < 0.001).

PAP (Fig. 1). P A P displayed a biphasic rise to 118 ± 11% at 10 min and 198 ± 13% at 55 min in response to the D X S infusion. Administration of B A Y χ 4620 delayed or attenuated only the second P A P increase, with the group receiving D X S + 360 mg of B A Y χ 4620 snowing an early rise to 120% of baseline value but no secondary peak. T h e

groups were significantly different in the global test (mean value during D X S infusion, analysis of covari -ance, Ρ = 0.0017), but multiple between-group compari ­sons did not reveal a significant difference between D X S + N a C l and D X S + 360 mg of B A Y χ 4620. A l l multiple tests over time were significant from 35 to 90 m i n .

Circulating kininogen (Fig. 1). I n the group D X S + N a C l , uncleaved kininogen in plasma decreased to 54 ± 19% at 15 min a n d to 21 ± 1% at 30 min and remained around 20 ± 1% of baseline value for the remainder of the study. T h i s decrease in uncleaved kininogen was delayed by the simultaneous administration of B A Y χ 4620; in the group D X S + 180 mg of B A Y χ 4620, kininogen de­creased to 30 ± 10% at 60 min. I n the group with the highest dose of the inhibitor, D X S + 360 mg of B A Y χ 4620, and in the group without D X S , no decrease i n k i n ­inogen was observed. T h e time to 50% kininogen de­crease differed significantly between the groups ( M a n ­te l -Haenszel test, Ρ < 0.001). T h e difference between D X S + N a C l a n d D X S + 360 mg of B A Y χ 4620 was statistically significant (multiple Scheffe-like compari ­sons, Ρ < 0.02).

Free kinin (Fig. 1). K i n i n levels i n blood as a function of time corresponded closely with the changes seen in M A B P . B A Y χ 4620 produced a dose-dependant delay in the release of k i n i n with increasing doses while also pro­gressively reducing the amplitude of k inin release. I n the group with the highest inhibitor dose, D X S + 360 mg of B A Y χ 4620, only two of five animals had kinin levels above the detection limit, the highest being 47 fmol /ml . T h e group without D X S had no detectable kinin release. T h e global test as well as all multiple tests for repeated measurements during the infusion of D X S revealed a statistically significant difference between the groups ( P < 0.002). T h e comparison between the groups D X S f N a C l and D X S + 360 mg of B A Y χ 4620 was significant (P < 0.02).

Complement activation (Fig. 1). T h e plasma concentra­tion of des -Arg -C3a rose to three times baseline value at 60 m i n in the group D X S + N a C l . A l l groups except the control group without D X S showed such an increase, with a peak at 60 min followed by a slow decline. T h e peak of des -Arg -C3a flattened when the dose of the inhib­itor increased. E x c e p t for baseline measurements, at each time the difference between the groups was statisti­cally significant, and the global test was significant (Hommel 's multiple test, Ρ < 0.05).

Fibrin monomer (Fig. 1). F i b r i n monomer in plasma increased steadily during the 2-h observation period, with peak levels of 39 ± 7 μg/ml being reached at 2 h in the group D X S + N a C l . E x c e p t for the control group without D X S administration, all groups had an increase in fibrin monomer that showed a dose-dependent reduc­tion based on the amount of inhibitor. T h e difference between the groups was statistically significant at each time and in the global test (P < 0.05). I t must be noted, however, that at the baseline value there was a marginal difference between the groups (P = 0.051).

DXS and Hoe-140 (Fig. 2)

T h e control animals in this study experienced severe arterial hypotension similar to that seen in Fig . 1A. D X S

K I N I N G E N E R A T I O N I N V I V O BY D E X T R A N S U L F A T E I N F U S I O N 2679

(2 mg/kg) induced a rapid decline in M A B P that reached a nadir of 51 ± 8% of baseline value at 25 min . M A B P returned to baseline values at 40 min . I n the Hoe-140-treated group M A B P remained completely stable. T h e s e groups were significantly different ( P < 0.05).

T h e arterial hypotension of the control animals was paralleled by a decrease i n S V R . S V R decreased to 40 ± 6% at 25 min and was restored to baseline levels at 40 min . T h e Hoe-140-treated group did not experience this vasodilation, with the difference being statistically signif­icant ( P < 0.05).

Hypotension and vasodilation were accompanied by rises in cardiac output and heart rate ( H R ) . I n the con­trol animals, C O rose to 134 ± 2 5 % at 25 m i n and re­turned to baseline values at 40 min, and H R rose to 150 ± 40% at 25 min and returned to baseline values at 45 min . B o t h effects appeared to be prevented in the Hoe-140-treated group, but the differences were not statistically significant.

Uncleaved kininogen decreased to 18% with no appar­ent difference between the groups, whereas total protein remained stable i n both groups.

D I S C U S S I O N

T h e principal finding in this study was that both B A Y χ 4620, a p lasma kal l ikrein inhibitor, and Hoe-140, a b r a ­dykinin B 2 - receptor antagonist, prevented D X S - i n d u c e d hypotension in pigs.

D X S infusion produced all the effects that would be predicted, assuming that it is an activator of the contact system. At 15 min , very high kinin concentrations in blood were found, which closely correlated with a brisk decrease in arterial blood pressure. Uncleaved kininogen decreased and reached a steady-state level at 30 min , whereas anaphylatoxin C 3 a increased, reaching a peak at 60 min. F i b r i n monomer also increased continuously during the observation period. T h e s e data corroborate our assumption that activation of the contact system had taken place as a consequence of the infusion of D X S .

D X S infusion also produced alterations in lung func­tion, particularly a transient increase in mean P A P . Other parameters of lung function, such as extravascular lung water and airway pressure, however, showed only minor changes (data not shown). One possible explana­tion for the increase in P A P is the activation of thrombin because thrombin is a potent pulmonary vasoconstrictor (11). However, our data do not rule out other causes of the P A P rise.

Administration of B A Y χ 4620 before and during D X S infusion delayed or attenuated the D X S - i n d u c e d changes, with the highest dose producing a complete blockade of the D X S - i n d u c e d changes measured in this system.

Compared with natural aprotinin from bovine lungs, B A Y χ 4620, the mutant that was employed in our study, is a relatively potent and selective p lasma kal l ikrein i n ­hibitor with an inhibitor constant for h u m a n plasma k a l ­l ikrein roughly 100-fold lower than that of natural apro­t inin . T h e inhibitor constants of B A Y χ 4620 for h u m a n plasma kal l ikrein and h u m a n plasmin are 5 X 10~ 1 0 and 2 X 10~ 1 0 M , respectively, whereas the corresponding v a l ­

ues of wild-type aprotinin are 3 X 10~ 8 and 8 Χ 1 0 " 1 1 Μ (W. Schröder, B a y e r A G Pharmaceut ica l Research , Wuppertal , Germany, personal communication) .

I n this regard, it is important to note that p lasma lev­els of B A Y χ 4620 were in the range of 1.5 μΜ when a complete blockade of D X S - i n d u c e d changes was achieved (data not shown). I n previous experiments, a complete blockade of D X S - i n d u c e d hypotension was only achieved with p lasma concentrations of wild-type aprotinin ~ 1 2 . 5 μΜ (9). T h i s indicates that B A Y χ 4620 is also a far better inhibitor of p lasma kal l ikrein than natural aprotinin when used i n vivo. A s a result, this compound may be useful as a probe to investigate patho­logical conditions, such as hypotension and bleeding dur­ing extracorporeal circulation and hemodialysis and he­reditary angioedema, that are based on activation of the contact system.

T h e parallels between in vitro and in vivo potencies of the two inhibitors aprotinin and B A Y χ 4620 support the notion that D X S - i n d u c e d hypotension was mediated by bradykinin, a peptide that is released after the specific cleavage of high-molecular-weight kininogen by plasma kal l ikrein .

B r a d y k i n i n is one of the most potent hypotensive agents yet described. T h e finding that the effect of D X S infusion, namely hypotension, elevated k inin levels in the blood and that the decrease of the precursor of bra ­dykinin, uncleaved kininogen, was prevented by the plasma kal l ikrein inhibitor B A Y χ 4620 gives support to the notion that the decrease in blood pressure was me­diated by the release of bradykinin . However, other va ­soactive mediators such as prostacyclin, platelet-activat­ing factor, or serotonin can be released either directly by D X S or indirectly via thrombin or bradykinin and could also be responsible for all or part of the response. W e therefore wanted to determine whether the blockade of bradykinin B 2 receptors alone would prevent D X S - i n ­duced hypotension.

I n the second part of this study, we studied the effect of the B 2 - receptor antagonist Hoe-140 on D X S - i n d u c e d hy­potension. I n this study, D X S infusion also led t r a n ­siently to severe arterial hypotension that was accompa­nied by vasodilation and increases in H R and C O i n the control animals, whereas Hoe-140 completely blocked these effects i n the treated group. However, serial mea­surements of uncleaved kininogen demonstrate that ef­fective activation of the contact system had taken place equally in both groups. T o t a l protein measurements were unchanged in both groups, making specific cleavage the likely cause for the decrease in kininogen. T h e rela­tively small sample size i n this study was selected on the basis of pilot experiments that indicated that the actual dose of Hoe-140 used in this study would completely block bradykinin- induced hypotension. T h e finding that the bradykinin receptor blockade with Hoe-140 com­pletely abolished D X S - i n d u c e d hypotension once more points to bradykinin as the primary causative mediator.

I n summary, we have shown that D X S infusion in pigs produces activation of the contact system of blood coagu­lation with the following consequences: the appearance of high kinin levels in blood; a decrease i n uncleaved k i n ­inogen in plasma; severe transient arterial hypotension

2680 K I N I N G E N E R A T I O N I N V I V O BY D E X T R A N S U L F A T E I N F U S I O N

accompanied by vasodilation and a compensatory rise in C O and H R ; and activation of the coagulation, comple­ment, and fibrinolysis cascades. B o t h the p lasma k a l ­likrein inhibitor B A Y χ 4620 and the bradykinin B 2 re­ceptor antagonist Hoe-140 were able to block D X S -induced hypotension. Only the former compound prevented the decrease of uncleaved kininogen, whereas the latter compound prevented hypotension during on­going kininogen cleavage. W e conclude that D X S infu­sion led to the release of bradykinin from kininogen via activation of the contact system and led to arterial hypo­tension via stimulation of the B 2 k inin receptor.

Astrid Braune and Tanja Hennes provided ski l l fu l technical assis­tance.

Part of this work was supported by the Deutsche Forschungsge­meinschaft (SFB 207).

Address for reprint requests: M . Siebeck, Dept. of Surgery, Univ . of Munich , K l i n i k u m Innenstadt, Nussbaumstrasse 20, D-80336 Munich , Germany.

Received 31 January 1994; accepted in f inal form 28 July 1994.

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