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Phorbol Dibutyrate Stimulates the Release ofDiffusible Endothelium-DerivedVasoconstrictor Factor(s) From

Canine Femoral ArteriesGabor M. Rubanyi, Allan Luisi, and Anthony Johns

The purpose of this study was to analyze the effect of the tumor-promoting phorbol ester12,13-dibutyrate (PDBu) on the synthesis/release of nonprostanoid endothelium-derivedvasoactive factors. In bioassay experiments (in the presence of 10`- M indomethacin), infusionof PDBu (10-9-10-7 M) through a femoral artery (donor) segment with endothelium evokedfurther, concentration-dependent contraction of superfused canine coronary artery bioassayrings without endothelium (already contracted with lo- M PDBu). Removal of the endothe-lium from the donor segment abolished further contractions of the bioassay ring to 10-9 MPDBu and significantly depressed the contractile responses to 1o-8 and 10-7 M PDBu infusedthrough the donor segment. The inactive phorbol ester 4ca'phorbol 12,13-didecanoate had no

effect on vascular preparations mounted in the bioassay system. Selective exposure of thebioassay tissue to 10- M PDBu completely inhibited its responsiveness to basally releasedendothelium-derived relaxing factor. These data indicate that PDBu stimulates the release of adiffusible and bioassayable vasoconstrictor mediator(s) from the endothelium of caninefemoral arteries. (Circulation Research 1991;68:1527-1531)

T he vascular endothelium produces nonpros-tanoid vasoactive substances that relax (en-dothelium-derived relaxing factor[s] [EDRF],

see References 1 and 2) or contract (endothelium-derived contracting factor[s] [EDCF]3) the underly-ing vascular smooth muscle. The intracellular mech-anisms that control the biosynthesis of thesemediators in endothelial cells is poorly understood.Although an increase in cytosolic concentration offree Ca'+ was reported to be a common signal for thesynthesis of both EDRF and EDCF,1-4 several con-ditions (e.g., hypoxia, shear stress, and elevatedtransmural pressure) seem to modulate the biosyn-thesis of these two mediators in an opposite way.4'5-11Recent studies indicate that many cell functions are

controlled by the activity of protein kinase C.12 Theseindications were based on observations made largelywith tumor-promoting phorbol esters whose onlyknown receptor is protein kinase C.13 Several stimu-lants of the release of EDRF (e.g., acetylcholine,histamine, bradykinin, and 5-hydroxytryptamine)

were reported to activate phosphatidylinositol hydrol-ysis (one product of which is diacylglycerol, a naturalactivator of protein kinase C12). However, prolongedexposure to phorbol 12,13-dibutyrate (PDBu) inhib-ited endothelium-dependent relaxation evoked by his-tamine in the guinea pig pulmonary artery,14 by sub-stance P in rabbit aorta,15 and by acetylcholine incanine femoral and coronary arteries.'6 These resultssuggested that PDBu suppressed receptor-mediatedprocesses linked to the synthesis/release of EDRF. Itis not known, however, whether phorbol esters cantrigger the synthesis or release of endothelium-derivedvasoconstrictor factor(s).The purpose of the present study was to analyze

the effect of PDBu and the inactive phorbol ester4a-phorbol 12,13-didecanoate (4a-PDD) on the syn-thesis or release of nonprostanoid endothelium-de-rived vasoactive factors. Experiments were designedusing isolated canine femoral and coronary arteriesmounted in a bioassay system, which allows separateanalysis of the synthesis/release and action of endo-thelium-derived vasoactive factors.1""17

From the Department of Pharmacology, Berlex Laboratories,Inc., Cedar Knolls, N.J.Address for correspondence: Gabor M. Rubanyi, MD, PhD,

Schering AG, Research Center, Mullerstrasse 170-178, D-1000Berlin 65, FRG.

Received July 6, 1989; accepted January 23, 1991.

Materials and MethodsExperiments were performed on femoral and left

circumflex coronary arteries isolated from mongreldogs weighing 15-25 kg. The blood vessels were

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1528 Circulation Research Vol 68, No 6 June 1991

cleaned of loose adipose and connective tissue andwere studied in Krebs-Ringer bicarbonate solutionwith the following composition (mM): NaCl 118, KCl4.7, CaCI2 2.5, MgSO4 1.2, KH2P04 1.2, NaHCO3 25.0,edetate calcium disodium 0.026, and glucose 11.1(control solution). All experiments were performed inthe presence of 10-5 M indomethacin to inhibit thesynthesis of prostanoids in these blood vessels.9.18

Bioassay StudiesA bioassay technique described in detail earlier17

was used. Side branches of segments (3.0-3.5 cmlong) of the left and right femoral artery were tied(donor segments). The segments were fixed to stain-less steel cannulas (1.5 mm i.d.) and placed into anorgan chamber maintained at 37°C and filled with 20ml of aerated (95% 02-5% CO2) control solution.The segments were perfused at constant flow (2ml/min) by means of a multichannel roller pump(Minipuls 2, Gilson Co., Inc., Worthington, Ohio)with control solution maintained at 37°C. A stainlesssteel tube was also placed in the organ chamberthrough which control solution was pumped at thesame rate. A ring of coronary artery in which theendothelium had been removed (bioassay ring) wassuspended directly below the organ chamber by twostainless steel stirrups passed through its lumen. Onestirrup was connected to a force transducer (modelFTO3D, Grass Instrument Co., Quincy, Mass.) forrecording changes in isometric force. The assembly ofbioassay ring, stirrups, and force transducer could bemoved freely below the organ chamber, allowing thepreparation to be superfused with the perfusatepassing through the femoral artery segment or withthe stainless steel tube (direct superfusion). Thetransit time between the donor segment and thebioassay ring was 1 second. Drugs were infused intothe perfusate with infusion pumps (model 901, Har-vard Apparatus, South Natick, Mass.) either up-stream of the femoral artery (site 1, allowing contactwith the donor segment) or below it (site 2, avoidingcontact with the donor segment).

Experimental ProtocolTwo femoral artery segments and two coronary

artery bioassay rings were prepared from the sameanimal, mounted in two identical bioassay appara-tuses, and studied in parallel. In one donor segment,the endothelium was removed by gentle mechanicalrubbing of the intimal surface17 before mounting.The bioassay rings were superfused directly withcontrol solution for 60 minutes. During this intervalthe rings were stretched in a stepwise manner untilthe basal tension reached approximately 8 g, theoptimal tension for rings of isolated canine coronaryarteries.19To evoke sustained contraction of the bioassay

rings (n=7), the endoperoxide analogue U46619(5 x 108 M) was added to the perfusate. After thecontraction reached steady level (approximately10-15 minutes), 10-6 M acetylcholine was infused

through the direct line to check the absence offunctional endothelium on the rubbed bioassay ring.Then the bioassay ring was moved below the outletfrom the donor segment, 10`6 M acetylcholine wasinfused into the perfusate upstream of the perfusedsegment (at site 1), to determine the presence andabsence of functional endothelium in the intact andrubbed donor segments, respectively. Sixty minutesafter washout of U46619, the bioassay ring wascontracted with 10` M PDBu by infusing the drugdownstream of the perfused segments (at site 2). and30-45 minutes later, increasing concentrations(10-9-i0-' M) of PDBu were infused upstream of thedonor segment (site 1) while the infusion of 10` MPDBu was maintained at site 2. Control experimentswere also performed using the same protocol butsuperfusing the bioassay ring through the stainlesssteel tube.

In separate experiments (n=3), the effect of selec-tive exposure of the bioassay tissue to 10`7 M PDBuon its reactivity to basally released EDRF was stud-ied. First, the bioassay rings were contracted with10`8 M U46619, and the effect of basal EDRF wastested by moving the assay ring from the directperfusion line to the outlet from a donor segmentwith endothelium. Thirty minutes after washout ofU46619, the bioassay tissue was contracted by 10-7 MPDBu infused at site 2, and the reactivity to basalEDRF was tested in a similar way to that described inthe U46619-contracted bioassay rings.

DrugsAcetylcholine hydrochloride, indomethacin, PDBu,

and 4a-PDD were all purchased from Sigma ChemicalCo., St. Louis. U46619 was purchased from TheUpjohn Co., Kalamazoo, Mich. Drugs were dissolvedin distilled water (acetylcholine and U46619), in 96%ethanol (PDBu and 4a-PDD), or in 1.5 x 10` MNa2C03 (indomethacin). Stock solutions were pre-pared daily and kept on ice during the experiments.

Calculations and Statistical AnalysisChanges in isometric force induced by drugs are

expressed either as grams or as a percentage of theinitial contractile responses. Data are shown asmean±SEM; n represents the number of dogs fromwhich blood vessels were isolated.

Statistical comparisons were performed by Stu-dent's t test for paired or unpaired observations.Significance was accepted at p <0.05.

ResultsAcetylcholine (10-6 M) relaxed the bioassay rings

contracted by U46619 when infused upstream of thedonor femoral artery segment with endothelium(-85.4±5.1%; n=7) (Figure 1, lower left trace), butnot when infused directly on the bioassay ring(-1.2±5.6%; n=8) or upstream of the donor segmentwithout endothelium (2.6±3.2%; n =7) (Figure 1, up-per left trace). Infusion of 10`7 M PDBu downstreamof the donor segment (at site 2, see "Materials and

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Rubanyi et al Phorbol Esters and EDCF 1529

Donor ArteryWithout Endothelium

ACh

4 7-A 10-9 M 106M 10-'M

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Methods") contracted the bioassay ring to a similarextent (5.0±0.3 g; n=7) than did 5x108 M U46619(4.1±0.6 g; n=7). Infusion of increasing concentra-tions (10-9-10` M) of PDBu into the perfusate up-stream of the donor segments (at site 1, see "Materialsand Methods") caused further, concentration-depen-dent increase in isometric force of the bioassay ringwhen the donor segment had endothelium (Figure 1,lower trace, and Figure 2). These responses startedseveral minutes after the infusion of PDBu started,developed slowly, and were only partially reversibleafter the infusion of PDBu was stopped. Infusion of10-6 M 4a-PDD had no effect (data not shown; n =3).Removal of the endothelium from the donor segmentprevented the contractions evoked by 10-9 M PDBuand significantly depressed the contractions in re-sponse to infusion of 108 and 10 7M PDBu (Figure 1,upper trace, and Figure 2). During direct superfusion

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FIGURE 1. Tracings from an original bioassayexperiment showing the change of isometric force incanine coronary artery rings without endothelium(bioassay tissue) superfused with the effluent fromperfused donor canine femoral artery segments with(lower trace) or without (upper trace) endothelium.The absence (top) and presence (bottom) offunc-tional endothelium in the donor segments was dem-onstrated by the absence and presence, respectively,of relaxations of the bioassay ring contracted by5x10 8M U46619 in response to infusion of10'6Macetylcholine (ACh) through the donor segment(left). Infusion of 10` Mphorbol 12,13-dibultrate(PDBu) downstream ofthe donor segment (at site 2)evoked slowly developing contractions in the bioas-say rings. Infusion of increasing concentrations ofPDBu (10-9-10-7M) through the donor segment (atsite 1) evoked further contraction of the superfusedbioassay ring only if the endothelium was present inthe donor artery.

(through the stainless steel tube), infusion of 10-10`' M PDBu at site 1 caused no further contractionof the bioassay rings already contracted by 10` MPDBu (site 2) (10-` M, 0%; 10 8 M, 2.3%; 10` M,-1.7%). Selective exposure of the bioassay tissue toPDBu (10-7 M) completely abolished the responsive-ness of the bioassay vascular smooth muscle to basallyreleased EDRF (Figure 3).

DiscussionThe important and novel finding of the present

bioassay study is that infusion of increasing concen-trations of PDBu (l0-9-10`- M) through the donorfemoral artery segments with endothelium evokedconcentration-dependent increases of isometric forceof superfused bioassay coronary artery rings alreadyexposed to i0` M PDBu. The contractions of thebioassay ring induced by infusion of PDBu through

FIGURE 2. Effect ofinfusion ofincreasing con-centrations (10-9-10-7 M) of phorbol 12,13-dibutyrate (PDBu) through the donor femoralartery segment with (0) or without (o) endothe-lium on isometric force of bioassay coronaryartery rings without endothelium contracted by107M PDBu (for experimental details see "Ma-terials and Methods" and Figure 1). The change(increase) of isometric force of bioassay rings isexpressed as grams (left) or percent of initialcontraction (right) evoked by 10`" M PDBu(100%: donor with endothelium, 5.0±0.3 g;donor without endothelium, 4.1+0.5 g). Datashown are mean+±SEM of seven experiments.*p<0.05.

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the donor segment must be due to the modulation ofthe release of vasoactive factor(s) from the endothe-lium, since removal of the endothelium from thedonor segment significantly depressed the contrac-tions. The experiments were carried out in the pres-ence of indomethacin; thus the contribution of pros-tanoids to the response can be ruled out.9The observed phenomenon can be explained by

two equally possible mechanisms: 1) PDBu inhibitsthe synthesis or release of EDRFs, and 2) PDBufacilitates (or triggers) the production of EDCFs.Recent studies on the guinea pig pulmonary artery,14rabbit aorta,15 and canine femoral and coronaryarteries16 showed that PDBu inhibited endothelium-dependent relaxations to a variety of agonists. Previ-ous bioassay studies revealed that selective exposureof the donor tissue to PDBu depresses the produc-tion of EDRF released under basal conditions or inresponse to acetylcholine.16 Thus, inhibition of therelease of EDRF from femoral arteries may explainthe observed phenomenon.However, inhibition of basal release of EDRF is an

unlikely explanation for the contraction of the bioas-say tissue in response to stimulation of the donorsegment with 10-9 and 10 8 M PDBu (Figures 1 and2) since responsiveness of the bioassay ring to basalEDRF was inhibited by its selective exposure to 10`7M PDBu (Figure 3). Thus, the most likely explana-tion is that PDBu triggers the production of a diffus-ible and bioassayable endothelium-derived va-soconstrictor factor(s). Apparently this mechanismseems to be fully responsible for contractions evokedby low concentrations of PDBu (10` M). At higherPDBu concentrations (10 8 and 10`7 M), this mech-

FIGURE 3. Effect ofselective exposure of thebioassay ring to 10` M U46619 (top) or to10' Mphorbol 12,13-dibutyrate (PDBu; bot-tom) on its responsiveness to basally releasedendothelium-derived relaxing factor. Leftpanel: Tracings fron an original bioassayexperiment. Right panel: Relaxation to ba-sally released endothelium-derived relaxingfactor of bioassay rings contracted by U46619and its inhibition by contraction with PDBu.Data are expressed as percent inhibition ofinitial contraction (100%: U46619, 8.0 0.5 g;PDBu, 8.0±0.8 g) and shown as mean SEMof three experiments. * The effect ofPDBu wasstatistically significant (p <0.05).

anism facilitates the direct contractile effect of thephorbol ester on vascular smooth muscle.

Endothelin is a recently discovered potent endo-thelium-derived vasoconstrictor polypeptide4 whosegene was shown to have a phorbol ester-sensitiveregulatory element (t-RA) in the 5' flanking region.20Indeed, phorbol esters stimulate the synthesis andrelease of endothelin in cultured endothelial cells.21Although it is possible that PDBu stimulates thesynthesis and release of endothelin from the endo-thelium of canine femoral artery, the present studywas not designed to characterize the nature of thediffusible vasoconstrictor factor(s); it requires furtherinvestigations.PDBu is a tumor-promoting phorbol ester, which

activates protein kinase C.13 Although non-specificphorbol effect cannot be ruled out completely, it islikely that the effects of PDBu are mediated bystimulation of protein kinase C. This conclusion isbased on the finding that the inactive phorbol ester4a-PDD (which does not activate protein kinase C13)was devoid of any contractile activity under the sameexperimental conditions. Protein kinase C is presentin endothelial cells2223 and is activated by PDBu andother active phorbol esters, but not by the inactivephorbol ester 4a-PDD.24-26The present observations in combination with re-

cent studies14-16 indicate that the phorbol esterPDBu depresses the production of EDRF and facil-itates the synthesis/release of EDCF(s). The PDBu-induced disturbed balance between the production ofrelaxing and contracting endothelium-derived vaso-active factors mimic pathological conditions (e.g.,hypoxia and hypertension) where a shift from EDRFto EDCF production were described.8,10,11,27 Thus,

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Rubanyi et al Phorbol Esters and EDCF 1531

phorbol esters may be a useful tool for furthercharacterization of the cellular events of endothelialdysfunction, which can lead to (or are the conse-

quences of) various cardiovascular disorders.

AcknowledgmentsThe authors wish to thank Mrs. S. Packie for her

excellent secretarial assistance. The Animal Facilityat Berlex is accredited by the American Associationfor the Accreditation of Laboratory Animal Care.

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3. Rubanyi GM: Endothelium-derived vasoconstrictor factors, inRyan US (ed): Endothelial Cells, Vol III. Boca Raton, Fla,CRC Press, Inc, 1988, pp 62-74

4. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, KobayashiM, Mitsui Y, Yazaki Y, Katsutoshi G, Masaki T: A novelpotent vasoconstrictor peptide produced by vascular endothe-lial cells. Nature (Lond) 1988;332:411-415

5. Furchgott RF, Zawadzki JV: The obligatory role of endothe-lial cells in the relaxation of arterial smooth muscle byacetylcholine. Nature (Lond) 1980;288:373-376

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7. Winquist RJ, Bunting PB, Baskin EP, Wallace AA: Decreasedendothelium-dependent relaxation in New Zealand genetichypertensive rats. J Hypertension 1984;2:541-545

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9. Rubanyi GM, Romero JC, Vanhoutte PM: Flow-inducedrelease of endothelium-derived relaxing factor. Am J Physiol1986;250(Heart Circ Physiol):H1145-H1149

10. Harder DR: Pressure-induced myogenic activation of catcerebral arteries is dependent on intact endothelium. Circ Res1987;60:102-107

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traction of isolated canine carotid arteries. Am J Physiol1988;255(Heart Circ Physiol):H783-H788

12. Nishizuka Y: Turnover of inositol phospholipids and signaltransduction. Science 1984;225:1365-1370

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14. Weinheimer G, Wagner B, Osswald H: Interference of phor-bolesters with endothelium-dependent vascular smooth mus-

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of endothelium-derived relaxing factor. EurJPharmacol 1987;137:167-171

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18. DeMey JG, Claeys M, Vanhoutte PM: Endothelium-dependent inhibitory effects of acetylcholine, adenosine tri-phosphate, thrombin and arachidonic acid in the caninefemoral artery. J Pharmacol Exp Ther 1982;222:166-173

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21. Emori T, Hirata Y, Ohta K, Shichiri M, Marumo F: Secretorymechanism of immunoreactive endothelin in cultured bovineendothelial cells. Biochem Biophys Res Comm 1989;160:93-100

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KEY WORDS * endothelium * endothelium-derived contractingfactors * endothelium-derived relaxing factor * phorbol esters* vascular smooth muscle

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G M Rubanyi, A Luisi and A Johnsvasoconstrictor factor(s) from canine femoral arteries.

Phorbol dibutyrate stimulates the release of diffusible endothelium-derived

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1991 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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