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ISSN: 1524-4571 Copyright 1991 American Heart Association. All rights reserved. Print ISSN: 0009-7330. Online

TX 72514Circulation Research is published by the American Heart Association. 7272 Greenville Avenue, Dallas,

1991;68;1340-1348 Circ. Res.MA Murray, DD Heistad and WG Mayhan

permeabilityRole of protein kinase C in bradykinin-induced increases in microvascular

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1340

Role of Protein Kinase C inBradykinin-Induced Increases in

Microvascular PermeabilityMargaret A. Murray, Donald D. Heistad, and William G. Mayhan

The goal of this study was to determine whether protein kinase C mediates bradykinin-inducedincreases in microvascular permeability. Permeability of the hamster cheek pouch wasevaluated using intravital fluorescent microscopy and fluorescein isothiocyanate (FITC)-dextran (MW 70,000). We examined effects of sphingosine, a protein kinase C inhibitor, onbradykinin-induced increases in permeability. Increases in permeability were quantitated bycounting the number of leaky sites and calculating the clearance of FITC-dextran. Duringbradykinin (10-6 M), leaky sites increased from 0 to 404 (mean+SEM) sites/0.11 cm2, andclearance increased from 1.7+1.0 to 229 ml/secxlO-6. The bradykinin type-2 receptorantagonist D-Arg, [Hyp3,Thi5,8,D-Phe7] -bradykinin virtually abolished formation of leaky sites inresponse to bradykinin. To determine whether changes in microvascular pressure contribute tothe increase in leaky sites, venular pressure was measured using a micropipette and survo-nulldevice. Increases in cheek pouch venular pressure were similar during application ofbradykinin and adenosine, which increased permeability, and isoproterenol, which did notincrease permeability in the cheek pouch. Thus, increases in permeability were not linked tochanges in microvascular pressure. The protein kinase C inhibitor, sphingosine (10-6 M),markedly attenuated responses to bradykinin. Leaky sites increased from 0 to only 21sites/0.11 cm2, and clearance increased from 3.91.4 to only 6.7+2.2 ml/secx 10-6. To test thespecificity of sphingosine, we examined effects of adenosine (10-6 M). Sphingosine did notsignificantly alter increases in microvascular permeability in responses to adenosine. We alsoexamined effects of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), another proteinkinase C inhibitor, on responses to bradykinin and adenosine. H-7 greatly attenuatedformation of leaky sites during stimulation with bradykinin and did not alter the number ofleaky sites produced during adenosine. The findings suggest that protein kinase C may mediateincreases in vascular permeability in response to bradykinin. (Circulation Research1991;68:1340-1348)

B radykinin and related kinins increase vascularpermeability.' Morphological evidence sug-gests that bradykinin increases macromolec-

ular efflux through the formation of intercellular gapsbetween endothelial cells23 that are located princi-pally in postcapillary venules.4 Responses to kininsare mediated by activation of specific bradykininreceptors.5,6From the Departments of Pharmacology and Internal Medicine,

Cardiovascular Center and Veterans Administration Medical Cen-ter, University of Iowa College of Medicine, Iowa City, Iowa.

Supported by a Medical Investigatorship and research funds fromthe Veterans Administration; by National Institutes of Healthgrants HL-16066, HL-14230, NS-24621, and HL-14230; and by aMedical Student Research Fellowship (M.A.M.) from the Ameri-can Heart Association and the Merck Company Foundation.Address for correspondence: Donald D. Heistad, MD, Depart-

ment of Internal Medicine, University of Iowa College of Medi-cine, Iowa City, IA 52242

Received April 24, 1990; accepted January 14, 1991.

In cultured endothelial cells, bradykinin activatesprotein kinase C through the formation of diacylglyc-erol, a product of phospholipase C hydrolysis of phos-phatidylinositol.7-13 Protein kinase C may be a mediatorof pulmonary edema, since the activation of this en-zyme with phorbol myristate acetate14-18 or H2O219produces increases in vascular permeability and/or cap-illary pressure in isolated lungs and whole animals. Thisfinding implicates protein kinase C as a possible medi-ator of increases in vascular permeability.

In previous studies, exogenous agonists (usuallyphorbol esters)14-18 or H20219 were used to activateprotein kinase C in vivo. There have been no studies, toour knowledge, that have tested the hypothesis thatendogenous stimuli increase vascular permeability byactivation of protein kinase C. The goal of this studywas to determine the role of protein kinase C inbradykinin-induced increases in permeability of the


Murray et al Protein Kinase C and Permeability 1341

microcirculation in vivo. We used two antagonists,sphingosine and 1-(5-isoquinolinylsulfonyl)-2-meth-ylpiperazine (H-7), that inhibit protein kinase Cthrough different mechanisms. Sphingosine inhibits ac-tivation of protein kinase C by competing with diacyl-glycerol for binding to the regulatory site.2021 In con-trast, H-7 apparently inhibits protein kinase C bycompeting with ATP for binding to the catalytic site onprotein kinase C.22,23 These inhibitors were used toelucidate a possible role of protein kinase C in micro-vascular responses.

Materials and MethodsAgents

Bradykinin, H-7, adenosine, D-Arg,[Hyp3,Thi5 8,D-Phe71-bradykinin, and des-Arg9-bradykinin were pur-chased from Sigma Chemical Co., St. Louis.Preparation ofAnimals

Adult, male Syrian hamsters weighing 130-180 gwere anesthetized with sodium pentobarbital (6 mg/100 g body wt, i.p.) and tracheotomized to facilitatespontaneous respiration. A femoral artery was can-nulated to obtain blood samples. A femoral vein wascannulated for injection of the intravascular tracerfluorescein isothiocyanate (FITC)-dextran (MW70,000, 50 mg/100 g body wt) and for injection ofsupplemental anesthesia (3 mg/100 g body wt/hr).The left cheek pouch was prepared for intravital

fluorescent microscopy and sampling of suffusate,using a technique similar to that described previous-ly.24 Briefly, the cheek pouch was spread gently overa small plastic baseplate, and an incision was made inthe outer skin to expose the cheek pouch membrane.A thick, vascular layer of connective tissue wascarefully incised and removed to expose the mi-crovasculature of the cheek pouch. The upper cham-ber was positioned over the baseplate and secured inplace by suturing the skin around the upper chamberwith a purse-string technique. The arrangementforms a triple-layered complex: the baseplate, upperchamber, and the cheek pouch membrane exposedbetween these two plates.24The hamster was then placed on a Lucite board

positioned on a heating mat regulated at 37C. Thechamber was connected to a reservoir containing abicarbonated buffer, which was continuously bubbledwith 95% N2-5% CO2. Throughout the experiment,the pouch was suffused with buffer, and the temper-ature in the chamber was maintained constant at37C. The chamber was also connected via a three-way valve to an infusion pump (Sage Instruments,Cambridge, Mass.) for administration of drugs intothe suffusate.The cheek pouch vasculature was epi-illuminated

with a vertical illuminator and viewed through anOlympus microscope (Jewelmont Microscope, Min-neapolis, Minn.). Fluorescent microscopy was accom-plished with filters that matched the spectral charac-teristics of FITC-dextran. Diameter of blood vessels

was measured by recording the image on videotapeand later measuring the diameter with an image-shearing device (model 908, Instrumentation forPhysiology & Medicine, Inc., San Diego).

Leukocyte rolling and adhesion in postcapillaryvenules in the hamster cheek pouch has been re-ported previously during superfusion with leuko-triene B4.25 In our studies, leukocyte rolling, oradhesion of cells to blood vessels, was not observed.Measurements of Perneability

Macromolecular leakage is manifested by extrava-sation of FITC-dextran, which appeared as fluores-cent spots or "leaky sites," as described previously.24After the administration of a drug, the number ofleaky sites was determined by visually counting threerandom fields. The location and number of leaky sitesin each field (0.11 cm2) were observed after eachintervention. To test the accuracy of counting leakysites, we compared the number of leaky sites countedby two different examiners in several studies (n= 12).The difference in number of leaky sites between thetwo observers varied by less than 1.5%.

In experiments in which clearance of FITC-dex-tran was calculated, the suffusion fluid was collectedat 5-minute intervals throughout the experiment,using a fraction collector (Micro-fractionator, GilsonMedical Electronics, Inc., Middleton, Wis.). Sampleswere collected in glass test tubes, and the concentra-tion of FITC-dextran (in nanograms per milliliter)was determined. Arterial blood samples were col-lected in heparinized capillary tubes (70-,l volume,Scientific Products, McGaw Park, Ill.), beginning 5minutes before and after injection of FITC-dextranand all successive experimental procedures. Duringthe course of an experiment, < 1 ml blood wasobtained through arterial sampling. The mean arte-rial pressure (105+2 mm Hg at the beginning of anexperiment and 90+2 mm Hg at the end) of thehamsters in which arterial samples were obtained wasnot significantly different from the hamsters in whicharterial samples were not obtained (109+2 mm Hg atthe beginning of the experiment and 933 at the end,p>0.05).To quantitate the concentration of FITC-dextran

in the plasma and suffusate, a standard curve forFITC concentration versus percentage transmissionwas completed on a fluorescence spectrophotometer(The Perkin-Elmer Corp., Norwalk, Conn.). Thestandard was FITC-dextran (MW 70,000), which wasprepared on a weight per volume basis. Bicarbonatedbuffer was used as a background, and a standardcurve was generated for each experiment, which wassubjected to linear regression analysis. The percent-age transmission for experimental samples (plasmaand suffusate) was measured on the spectrophoto-fluorometer, and the concentration was calculatedfrom the standard curve. It has been shown previ-ously that neutral FITC-dextran does not bind toplasma proteins.26 Thus, the standard curve wasappropriate for analysis of the plasma and suffusate


1342 Circulation Research Vol 68, No 5 May 1991

samples. In addition, minimal fluorescence signal(


TABLE 1. Effects of Bradykinin on Leaky Sites in HamsterCheek PouchesLeaky sites(sites/0.11 cm2) Bradykinin (M) n16+4 1x 10-7 6307 3x10` 6404 1 x 10-6 15648 3x 10-6 6656 1 xl0-5 10Values for leaky sites are mean SEM; n = number of hamsters.

There were no leaky sites under baseline conditions. The numberof leaky sites after suffusion of bradykinin for 5 minutes isreported.

sine (10-6 M) in the absence and presence of theB2-antagonist (10-7 M).We also examined the effect of a bradykinin B1

receptor agonist, des-Arg9-bradykinin, on the forma-tion of leaky sites in the cheek pouch. In six experi-ments, des-Arg9-bradykinin (10-6 M) was applied tothe pouch for 5 minutes. The number of leaky sitesand the diameter of a cheek pouch arteriole andvenule (n=4) were determined before and afterapplication of the B1-receptor agonist.Analysis of Data

Statistical analysis was performed using paired ttests to compare absolute changes in leaky sites,clearance, and vessel diameter during control condi-tions and experimental interventions. A value ofp


TABLE 2. Effects of Adenosine on Leaky Sites in HamsterCheek PouchesLeaky sites(sites/0.11 cm2) Adenosine (M) n15+3 1x - 7263 3 x 10-7 658+10 1x10-6 659+6 3 x 10-6 7688 1 x 10-5 4Values for leaky sites are mean+ SEM; n =number of hamsters.

There were no leaky sites under baseline conditions. The numberof leaky sites after suffusion of adenosine for 5 minutes is reported.

Sphingosine did not produce leaky sites and didnot significantly attenuate adenosine-induced forma-tion of leaky sites (Figure 3) or clearance of FITC-dextran (MW 70,000) (Figure 4).Effects of H-7

Application of H-7 did not produce leaky sites. H-7attenuated the number of leaky sites produced bybradykinin (Figure 5). Reapplication of bradykinin45-60 minutes after H-7 again increased leaky sites,from 00 to 5410 sites/0.1 1 cm2. Thus, inhibition ofleaky site formation after H-7 was not due to tachy-phylaxis to bradykinin. H-7 did not attenuate theformation of leaky sites in response to adenosine(Figure 6).

60-

Leaky Sites(#/0.11 cm2)

30-

0-

60.

30-

0CONT ADENO CONT ADENO

Sph

FIGURE 3. Bargraphs showing effect of 10]6Msphingosine(Sph) on leaky sites produced by 10`6 M adenosine(ADENO). Values are meanSEM in six hamsters. Undercontrol conditions (CONT), there were no leaky sites. Sph hadno significant effect on formation of leaky sites (p>0.05).

50

Cbarance(miUsec x 10-)25

s0-

25-

T

F.Thu

-

0.

CONT ADENO CONT ADENO.SphFIGURE 4. Bar graphs showing effect of sphingosine (Sph)on adenosine (ADENO)-induced clearance of fluoresceinisothiocyanate-dextran. Values are mean +SEM in six ham-sters. Sph had no significant effect on clearance (p>0.05).

60

Ly Sites(#0.11 cm2)

30

60-

30-

0~~~~~~~~~~~cONw BK COw BKE-7

FIGURE 5. Bar graphs showing effect of 10-10 M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on leaky sitesproduced by 10 6 M bradykinin (BK). Under control condi-tions (CONT), there were no leaky sites. Values aremean +SEM in seven hamsters. *p< 0. 05 vs. BK.

Microvascular Pressure and Vessel DiameterMicrovascular pressure increased during superfu-

sion of bradykinin, adenosine, and isoproterenol(Table 3). Arteriolar diameter generally increased,and venular diameter was not significantly altered.Superfusion of bradykinin and adenosine, but notisoproterenol, produced leaky sites.Bradykinin Receptor AntagonistsThe B2-receptor antagonist D-Arg,[Hyp3,Thi5'8,D-

Phe7]-bradykinin2930 significantly attenuated forma-tion of leaky sites in response to bradykinin (Figure7). Reduction of bradykinin-induced increases inleaky sites after the B2-receptor antagonist was notdue to tachyphylaxis, because the time interval be-tween each subsequent application of bradykinin wasat least 30 minutes27 and because we were able todemonstrate, in experiments examining effects ofsphingosine, that the number of bradykinin-inducedleaky sites was virtually identical after the first andthird applications of bradykinin (see above).The B2-antagonist (10-7 M) did not significantly

attenuate adenosine-induced formation of leakysites. The number of leaky sites in response toadenosine was 233 versus 222 sites/0.11 cm2 in

60-

Leaky Sites(#10.11 cm2)

30,

60 -

30.

T

O - CONT

H-7

FIGURE 6. Bar graphs showing effect of 10-10 M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on leaky sitesproduced by 10`6 M adenosine (ADENO). Under controlconditions (CONT), there were no leaky sites. Values aremean +SEM in five hamsters.

-



TABLE 3. Effects of Bradykinin, Adenosine, and Isoproterenol on Microvascular Pressure and Diameter in Hamster Cheek PouchesBradykinin study Adenosine study Isoproterenol study

Bradykinin Adenosine IsoproterenolControl (10-6 M) Control (10-6 M) Control (10~6 M) n

CPV pressure (mm Hg) 19.7+2.5 24.53.2* 21.21.6 261.9* 17.21.4 23.51.6* 6CPA diameter (,tm) 63.1 15.8 69.1 16.2 56.9 13.3 78.713.0* 55.5 12.8 74.9 14.5 4CPSV diameter (gm) 9313.4 96.413.4 92.211.5 89.18.5 94.411.8 98.521.7 4Leaky sites 00 476* 00 105 00 00 6Values are meanSEM; n=number of hamsters. CPV, cheek pouch venule (diameter = 34+3.1 gm); CPA, cheek pouch arteriole; CPSV,

cheek pouch small vein.*p0.05).The B1-receptor agonist des-Arg9-bradykinin5 did

not cause the formation of leaky sites (0+0 sites/0.11cm2 before and after des-Arg9-bradykinin). The B1-receptor agonist produced significant constriction ofarterioles in the cheek pouch (61 13 gm beforeversus 314.4 ,um after application of des-Arg9-bradykinin, p


between adjacent vascular endothelial cells may bethe result of endothelial receptors linked to contrac-tile proteins.We examined the possibility that the increase in

permeability during bradykinin and adenosine wasmediated in part by dilatation of precapillary resis-tance vessels. Bradykinin, adenosine, and isoprotere-nol all produced arteriolar dilatation, but only brady-kinin and adenosine increased permeability. Studiesin other tissues and preparations have in general alsodissociated vasodilatation from macromolecularleakage. In the rat cremaster muscle, histamine andnitroprusside produced vasodilation and proteinleakage, but isoproterenol produced vasodilationwithout an increase in permeability.37 In the hamstercheek pouch, isoproterenol inhibits increases in per-meability induced by bradykinin.4 These studies sug-gest that vasodilatation produced by stimulation of,3-adrenergic receptors does not produce an increasein vascular permeability.None of the vasodilators tested in this study pro-

duced venular dilation. Bradykinin, adenosine, andisoproterenol produced similar small increases invenular pressure, but only bradykinin and adenosineincreased permeability. These findings suggest thatincreases in permeability produced by bradykinin andadenosine are not the result of increases in hydro-static pressure.We examined receptor mechanisms by which

bradykinin increases permeability. B2-receptors ex-hibit much higher affinity for bradykinin than des-Arg9-bradykinin, whereas B1-receptors are more sen-sitive to the des-Arg9 metabolites.5 The relativepotencies of bradykinin and des-Arg9-bradykinin areoften used to classify responses as B2 or B1, respec-tively. Bradykinin increases vascular permeability,and it has been suggested that this response ismediated via activation of a B2-receptor.5 Our results,using a B2-receptor antagonist,29,30 are in agreementwith previous studies and indicate that the B2-recep-tor mediates the increase in permeability in thecheek pouch in response to bradykinin.

Application of the B1-receptor agonist des-Arg9-bradykinin did not increase permeability but didproduce significant constriction of arterioles. Con-striction of the cheek pouch arterioles indicated thatB1-receptors are present in cheek pouch vasculature,because B1-receptors have been reported to producevasoconstriction.38We considered the possibility that inhibition of

permeability by sphingosine and H-7 may be relatedto nonspecific effects on vascular permeability. Aden-osine, which increases permeability in the hamstercheek pouch,28 was used to test the specificity of theprotein kinase C inhibitors. Adenosine was chosenbecause, like bradykinin, its effects are receptormediated.39 Specific pathways by which adenosineincreases permeability have not been clearly estab-lished. In the presence of the inhibitors, responses toadenosine were preserved, which suggests that the

FIGURE 8. Diagram showing apparent sites of action of theprotein kinase C (PKC) inhibitors sphingosine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). Bradykinin(BK) stimulates receptor-mediated hydrolysis ofphosphatidyl-inositol 4,5-diphosphate (PIP2) by the phosphodiesterasephospholipase C (PLC), forming diacylglycerol (DAG) andinositol 1,4,5-trisphosphate (IP3). Sphingosine is a competitiveinhibitor of DAG, which binds to and activates PKC. H-7binds to the catalytic subunit ofPKC, inhibiting the activity ofPKC.

inhibitors were specific in blocking the effects ofbradykinin.

Other studies suggest that, at some concentrations,sphingosine is relatively selective in inhibiting severalprocesses that appear to be mediated by proteinkinase C activation. In platelets, sphingosine does notinhibit several other enzymes involved in signal trans-duction, including phospholipase C, calcium/calmod-ulin-dependent kinase, diacylglycerol kinase, andcAMP- and cGMP-dependent kinases.20,21 Sphin-gosine can inhibit calcium/calmodulin-dependent ki-nases, but the concentrations of sphingosine neededto inhibit calcium/calmodulin-dependent kinases aremuch higher than the concentration of sphingosineused in this study.40The results that we obtained with H-7, which also

is an inhibitor of protein kinase C, were similar tothose with sphingosine. H-7 inhibits protein kinases,such as cAMP- and cGMP-dependent kinases, aswell as myosin light chain kinase.22 However, theconcentrations of H-7 necessary to inhibit thesekinases are much higher than the concentrationsused in this study.22 The similar findings with the twoprotein kinase C inhibitors and the concentrationsthat were efficacious in blocking responses to brady-kinin suggest that protein kinase C activity wasinhibited relatively selectively.There was a large difference in concentration of

H-7 (10` M) and sphingosine (10-6 M) that wasnecessary to block effects of bradykinin, but notadenosine. This difference in potency may be ex-plained by the different sites of action of the inhibi-tors (Figure 8). Sphingosine interacts with the regu-



latory domain of protein kinase C,20,21 whereas H-7interacts at the catalytic domain.22,23 The effectiveconcentration of H-7 was far less than would beexpected if H-7 were a receptor antagonist. Thepotency of H-7 may be explained by its site of action,since H-7 competes with ATP for binding to thecatalytic site of protein kinase C. We speculate thatthe concentration of ATP in endothelial cells ofpostcapillary venules may be very low, so that a highconcentration of H-7 is not necessary for effectiveinhibition.

In summary, these experiments are the first at-tempt to determine the biochemical pathway ofbradykinin-stimulated increases in permeability invivo. The main conclusion is that protein kinase C isan integral part of the mechanism by which bradyki-nin increases microvascular permeability. This con-clusion is supported by the observations that sphin-gosine, which blocks the activation of protein kinaseC, and H-7, which blocks the catalytic site on acti-vated protein kinase C, greatly attenuate both forma-tion of microvascular leaky sites and clearance ofFITC-dextran (MW 70,000) in response to bradyki-nin stimulation. This finding, together with the find-ing that H-7 blocks pulmonary edema produced byhydrogen peroxide,'9 suggests that activation of pro-tein kinase C may mediate increases in permeabilityduring inflammation.

AcknowledgmentsWe thank Drs. Michael Shasby and Frank Faraci

for critical review of the manuscript. We thank Ms.Barbara Birchmier for typing the manuscript and Ms.Cynthia Lynch for technical assistance.

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KEY WORDS * hamsters * cheek pouch * H-7 sphingosine* adenosine * fluorescein isothiocyanate-dextran bradykinin


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