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Substance P Is Released From theEndothelium of Normal and

Capsaicin-Treated Rat Hind-LimbVasculature, In Vivo, by Increased Flow

Vera Ralevic, Pamela Milner, Olga Hudlickai, Frantisek Kristek, and Geoffrey Burnstock

The rat hind-limb vasculature releases substance P when subjected to a rapid increase in flowthrough the vascular bed. This release also occurs during high flow after rats have beencapsaicinized, when loss of substance P-containing nerve fibers was verified by immunohis-tochemistry. Air treatment, a procedure shown by transmission electron microscopy to haveremoved endothelial cells from the arteries but not arterioles or capillaries of the hind-limbpreparations, eliminated this release. Thus, the substance P released is unlikely to arise fromperivascular nerves but rather from arterial endothelial cells. (Circulation Research1990;66:1178-1183)

Anumber of naturally occurring vasodilatorshave been shown to evoke their responses bystimulation of production of endothelium-

derived relaxing factor (EDRF) in endothelial cells.These substances include acetylcholine (ACh), sub-stance P (SP), ADP, ATP, bradykinin, thrombin, andhistamine (for reviews see Furchgottl and Vanhoutteet a12). Removal of the endothelium and the subse-quent abolition of a relaxant response to these sub-stances played a crucial part in early investigations,although these studies were largely confined to iso-lated vessels or vascular beds. More recently, gossy-pol, a selective and irreversible inhibitor of theEDRF-mediated vasodilation in isolated arteries, hasbeen used for demonstration of endothelium-dependent relaxations to ACh, SP, and ATP in therabbit hind limb in vivo.3-5 Of the naturally occurringvasodilators acting via the intimal surface, SP is oneof the most potent studied,1,6 and receptors for thispeptide have been demonstrated, by autoradio-graphic analysis, on the endothelium of the dogcarotid artery7 and renal artery.8 In view of thesestudies, there is strong evidence that SP may have a

From the Department of Anatomy and Developmental Biologyand Centre for Neuroscience, University College London, Lon-don, United Kindgom.

Supported by a Wellcome Trust Research Fellowship (F.K.), theScience and Engineering Research Council (V.R.), and the BritishHeart Foundation.Address for correspondence: G. Burnstock, Department of

Anatomy and Developmental Biology and Centre for Neuro-science, University College London, Gower Street, London WC1E6BT, UK.

Received April 24, 1989; accepted November 20, 1989.

physiological role in the regulation of vascular tone,and hence of blood flow, by an endothelium-dependent mechanism.A local endothelial source for vasoactive agents

was first suggested by the electron microscopicimmunocytochemical localization of choline acetyl-transferase (the ACh-synthesizing enzyme) in vascu-lar endothelial cells in the rat brain.9 Since then,further electron microscopic studies have shown thatendothelial cells may also be a source of SP and5-hydroxytryptamine (5-HT) in the rat femoral andmesenteric arteries,10 and of 5-HT in the rat coro-nary artery.'1

Insight into the physiological role of endothelialcells has come from studies demonstrating that incre-ments in flow through large conduit arteries in vitroor in situ12,13 and at the microvascular level14 induceactive vasodilatation of arterial smooth muscle that isabolished on removal of the endothelium. Further, ithas been shown that an increase in flow induces therelease of EDRF from the canine femoral artery1'516and the rabbit hind limb.3 This finding suggests thatendothelial cells play a role in blood flow regulationby releasing EDRF.

In the present study, we sought to determinewhether SP is released from endothelial cells of theperfused rat hind-limb preparation in response to anincrease in flow through the vascular bed.

Materials and MethodsPerfusion of Rat Hind-Limb Vasculature In Vivo

Male Wistar rats (300-420 g) were anesthetized byinjection with sodium pentobarbitone (60 mg/kg i.p.,

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Ralevic et al SP Release From Rat Hind-Limb Endothelial Cells 1179

diluted 1:1 with saline). The right jugular vein wascannulated for administration of heparin and addi-tional anesthetic if necessary, and the animal's bloodpressure was monitored by a pressure transducer(model P23, Gould, Cleveland, Ohio) from a cannulato the right carotid artery. The trachea was alsocannulated. The abdomen was opened, and the leftrenal artery and vein and the vessels supplying thebody wall, the scrotum, and all visible arterial andvenous side-branches in the pelvic and abdominalregion were ligated. After intravenous administrationof heparin (1,000 units), the aorta was cannulated toallow perfusion of the hindquarters. The vena cavawas cannulated and the outflow directed away fromthe animal via a fraction collector (model 201-202,Gilson, Worthington, Ohio). The hindquarters wereinitially flushed with 0.9% saline containing heparin(100 units/ml) and then perfused with a regulatedpulsatile flow, by use of a Watson-Marlow pump,with a Krebs-Ringer bicarbonate solution (gased with95% 02-5% CO2 and maintained at 370 C) contain-ing (mM) NaCl 118, KCl 4.7, KH2PO4 1.2, MgSO4.7H20 1.2, NaHCO3 25.0, D-glucose 5.5, CaCI2* 2H202.5, with added bovine serum albumin (10 g/l) andinsulin (10 units/l). Perfusion pressure was measuredby a pressure transducer (model P23, Gould) from aside arm of the inflow cannula and was matched so asnot to exceed the animal's blood pressure. Flow wasmeasured by timed collection in a graduated cylinder.Flow of the Krebs-Ringer bicarbonate solution

through the hindquarters was allowed to stabilize for15-20 minutes before collection. High flow wasachieved by rapidly increasing the output of thepump to approximately three times that of the basalflow for 2 minutes. Fractions (0.12 min/fraction) werecollected at basal flow immediately before high flowand over the period of high flow. This procedure wasrepeated to produce a second period of high flowafter 15-20 minutes of recovery at basal flow. Frac-tions were collected in polypropylene tubes, whichwere immediately placed on ice.

Endothelium RemovalAfter the first period of high flow, the endothelium

was removed, according to a protocol developed inthis laboratory,17 from the vasculature of five rats byintroduction of air into the perfusion line as a bubbleover a period of 5 seconds. This procedure wasrepeated at 10-second intervals for a total durationof 5 minutes. Transmission and scanning electronmicroscopic examination of the femoral artery, theartery supplying the extensor hallucis proprius mus-cle, and the extensor hallucis proprius muscle wasused to confirm that this procedure, together with thehigh flow, selectively removed the endothelium fromthe arteries but not from arterioles and capillaries(and without damage to the underlying smooth mus-cle) of the rat hind-limb preparation.

Capsaicinization of RatsFive 1- to 2-day-old rats were treated with capsa-

icin (50 mg/kg) administered subcutaneously.18These rats were allowed to reach maturity (about 3months) before use in SP release studies. Immuno-histochemical analysis of SP- and neuropeptide Y(NPY)-containing nerve fibers was performed on thefemoral artery and the artery supplying the hallucisproprius muscle from each of the capsaicin-treatedanimals for confirmation that specific depletion ofSP-containing fibers had occurred.The indirect fluorescence technique19 was used for

investigation of the presence of immunoreactivefibers to SP and NPY on stretch preparations offemoral artery, artery supplying the hallucis propriusmuscle, and arteries and capillaries within this mus-cle, as previously described.20

Substance P AssayFractions from the perfused hind limb were cen-

trifuged for removal of red blood cells, and thesupernatant was assayed for SP content by use of aninhibition enzyme-linked immunosorbent assay aspreviously described,20 with the following modifica-tions: The standards were prepared in the centri-fuged perfusate from the hind-limb preparationobtained before collection of fractions, and the anti-serum to SP was diluted in 0.1% Tween 20, 0.04%sodium azide in phosphate-buffered saline. Theresults were expressed as mean picomoles of SPreleased per minute +SEM and statistically evaluatedby use of a two-tailed Student's t test.

Electron MicroscopyFor transmission electron microscopic examination

of the vasculature, the femoral artery, the arterysupplying the extensor hallucis proprius muscle, andthe extensor hallucis proprius muscle were immersedin 3% glutaraldehyde in 0.1 M phosphate buffer.During fixation, arteries were cleaned and cut intoblocks. After washing in several changes of 0.1 Mphosphate buffer, the blocks were postfixed in 2%OS04, dehydrated in ascending grades of alcohol, andembedded in Spurr resin for electron microscopy.The ultrathin sections were stained with lead citrateand subsequently examined by use of an electronmicroscope (model 301, Philips Electronic Instru-ments, Mahwah, New Jersey).

ResultsThe basal flow through the hind-limb vasculature

was 9.5+±0.8 ml/min (n=15), with a perfusion pres-sure of 36.7+ 1.3 mm Hg (n= 15). High flow wasapproximately three times that of basal flow; the twoconsecutive periods of high flow were at rates of28.4+0.7 and 28.8±1.1 ml/min (n=15), with perfu-sion pressures of 69.1+ 1.8 and 75.4±5.8 mm Hg,respectively. In untreated rats (n =5) there was asignificant release of SP during both the first andsecond periods of high flow, from 4.4±+-3.4 to

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1180 Circulation Research Vol 66, No 5, May 1990

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3047.9+1.5 pmol/min (p<0.05), and from 4.4+1.8 to37.8-+ 11.7 pmol/min (p <0.002), respectively (Figure1). Repletionof SPpekdrapdlmitithefimarestr half

minue frm onetohihfo,rahdamxumtaprxmaey1miuefomost adte fl fgrdull t rtun o hebaalreeae evl efreth2-minute 40ido ig lwwsemntdCasiintetdrt n5shwdasml,siniicntevke rleseofSPduin bthth frs

FIGUR 1.plevelsof Subsomtane pr(SP), epssednsor pico-

rones of these animals was confirmed by immunohis-tochemical analysis of the femoral artery and theartery supplying the hallucis proprius muscle. In boththese arteries, there was almost total depletion ofSP-immunofluorescent fibers compared with thosefrom control animals, while NPY-containing fiberswere unaffected (Figure 2).The introduction of air into the rat hind limb was

shown by transmission and scanning electron micro-scopic examination to selectively remove the endo-thelium from the arteries of the hind limb (Figures 3aand 3b), leaving the endothelium of arterioles andcapillaries intact (Figure 3c). There was no signifi-cant difference in flow rate or perfusion pressure ofthe vascular bed after introduction of air; in these fiveanimals the flow rate before air was 9.0±0.5 ml/min,with perfusion pressure of 34.0±2.5 mm Hg, whileafter air the flow was 8.8±0.7 ml/min and perfusionpressure was 52.0±14.5 mm Hg. In air-treated ani-mals (n =5) the first increment of flow, that prior toair treatment, evoked a significant release of SP from1.5±0.3 to 21.4±6.4 pmol/min (p<0.02). After airtreatment, however, there was no significant releaseof SP, although the level (7.2 pmol/min) was slightlyhigher than basal values (2.2 pmol/min) (Figure 1).

DiscussionThe present results demonstrate that SP is re-

leased from the rat hind limb with increased flowonly when endothelial cells are present and that thesource of this SP is not periarterial nerves.

SP, one of the most potent of the naturally occur-ring vasodilators, has been shown to elicit relaxationof vascular smooth muscle by acting on receptors onthe luminal surface of endothelial cells. Its effectshave been demonstrated in isolated vessels such asthe rabbit and dog renal and celiac arteries,6 invessels in situ such as the canine femoral artery,2'and, more recently, in vivo in the rabbit hind-quarter.322 Together with the localization of SPreceptors on endothelial cells,7,8 there is overwhelm-ing evidence that SP can act as a regulator of vascularsmooth muscle tone, exerting its effects via theendothelium.Although pharmacological studies favor SP as a

mediator of vascular tone, it would be more likely tohave a physiological role if there was a local source ofthis agent. A supply of SP from within the endothelialcells themselves has already been proposed based onelectron microscopic immunocytochemical localiza-tion of SP in endothelial cells of the rat femoralartery.10 Thus, the endothelial cells are a likelysource of the SP released from the rat hind limb onincreased flow. However, release from alternativesources has to be considered. SP is not present inplatelets, and its concentration in plasma is very low.Although a neural origin of the SP released onperfusion is unlikely in view of the fact that it wouldhave to traverse the media, muscle coat, and elasticlamina to reach the lumen, SP has been shown torelax the canine femoral artery on adventitial appli-

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Ralevic et al SP Release From Rat Hind-Limb Endothelial Cells

FIGURE 2. Immunofluorescence micrographs showing SP-like immunoreactive nerve fibers in rat femoral artery (panel a) andartery supplying extensor hallucis proprius muscle (panel b). These fibers are almost completely absent in femoral artery (panel c)and in artery supplying extensor hallucis proprius muscle (panel d) after neonatal capsaicin treatment. (The faint SPimmunoreactive fiber in panel c was a rare example.) Calibration bar-SO gm.

cation, albeit at a potency some 50-100 times lessthan on luminal application.21 For elimination of SPfrom periarterial nerves as a contributor to therelease observed in high flow, animals were treatedwith capsaicin, a neurotoxin that depletes up to 91%of primary sensory neurones (for reviews seeFitzgerald23 and Nagy24). In these animals, the quan-tity of SP released during high flow was similar tothat observed in untreated animals, which suggeststhat the origin of this SP was independent of SPcontained within the primary sensory neuronesinnervating the hind limb.To ensure that the released SP was indeed of an

endothelial origin, we used air treatment, togetherwith the periods of high flow, to selectively removethe endothelium from the arteries of the rat hindlimb, as demonstrated by transmission electronmicroscopy. Before air treatment, high flow evoked asignificant release of SP. When these same animalswere subjected to a second period of high flow afterair treatment, there was no significant release of SP.The small amount of SP detected after air treatmentmay arise from the remaining intact endothelium ofarterioles and capillaries. Since there was no changein peripheral resistance of the vascular bed after air

treatment, it is unlikely that microembolizationplayed a significant part in the experiments. How-ever, since microembolization cannot be ruled outand could result in parts of the microcirculationbeing prevented from exposure to the perfusionmedium after air treatment, we cannot entirelyexclude nonneuronal cells of the microcirculation asa source of SP.

This study, together with the evidence for thelocalization of SP within endothelial cells of the ratcoronary artery25 and rat mesenteric and femoralarteries,10 strongly suggests an endothelial origin forSP. Our recent demonstration of release of SP fromcultures of human umbilical vein endothelial cellsgrown on microcarrier beads during increased flow(unpublished observation) is consistent with thishypothesis. The technique of increasing flow in thisstudy was employed as a stimulus to demonstrate thatSP may be released from endothelial cells in vivo; itis possible that a similar mechanism may occurphysiologically. It should be noted, however, thatconcomitant with an increase in flow rate there areoften changes in pressure and edema formation thatcould be related to the release of SP. In this study,bovine serum albumin was incorporated into the

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1182 Circulation Research Vol 66, No 5, May 1990

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FIGURE 3. Panel a: Transmission electron micrograph ofartery supplying extensor hallucis proprius muscle after air treatment. Notethat lamina elastica intema (I) is completely devoid of endothelial cells. M, smooth muscle cells of tunica media. Calibration bar-S,um. Panel b: Scanning electron micrograph of intimal suface of femoral artery after air treatment. Lamina elastica intema iscompletely devoid of endothelial cells. Calibration bar=5 pun. Panel c: Transmission electron micrograph of an arteriole of extensorhallucis proprius muscle after air treatment showing that intact endothelial cells (EC) are still present. Calibration bar= 1 pin.

perfusate to minimize edema formation. Therequirement of the endothelium for flow-dependentdilatation and the flow-induced release of EDRFhave been well documented.1345'16,26,27 In pharmaco-

logical studies, the production of EDRF occurs sec-

ondary to an initial stimulus. Increased flow alonemay be a sufficient initial stimulus to cause directrelease of EDRF. However, in the light of this and

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Ralevic et al SP Release From Rat Hind-Limb Endothelial Cells 1183

other studies, it is possible that EDRF release may

occur secondary to the interaction of a substancesuch as SP with its intimal receptor after the releaseof this substance from endothelial cells.

In conclusion, this study demonstrates that SP isreleased from the perfused rat hind limb with highflow. The source of release is likely to be the endo-thelial cells in arteries since a) capsaicin treatmentdestroyed SP-containing C fibers but did not affectrelease and b) removal of the endothelium from thearteries abolished release.

References1. Furchgott RF: Role of endothelium in responses of vascular

smooth muscle. Circ Res 1983;53:557-5732. Vanhoutte PM, Rubanyi GM, Miller VM, Houston DS: Mod-

ulation of vascular smooth muscle contraction by the endothe-lium. Annu Rev Physiol 1986;48:307-320

3. Fdrstermann U, Dudel C, Frdlich JC: Endothelium-derivedrelaxing factor is likely to modulate the tone of resistancearteries in rabbit hindlimb in vivo. J Pharmacol Exp Ther1987;243:1055-1061

4. Pohl U, Busse R, Deszi L, Bassenge E: Endothelium-mediateddilation in resistance vessels in vivo. Fed Proc 1987;46:360

5. Fdrstermann U, Dudel C: Endothelium-dependent dilation ofresistance arteries in vivo. Blood Vessels 1988;25:35

6. Zawadzki JV, Furchgott RF, Cherry P: The obligatory role ofendothelial cells in the relaxation of arterial smooth muscle.Fed Proc 1981;40:689

7. Stephenson JA, Burcher E, Summers RJ: Autoradiographicdemonstration of endothelium-dependent lnI-Bolton-Huntersubstance P binding to dog carotid artery. Eur J Pharmacol1986;124:377-378

8. Stephenson JA, Summers RJ: Autoradiographic analysis ofreceptors on vascular endothelium. Eur J Pharmacol 1987;134:35-43

9. Parnavelas JG, Kelly W, Burnstock G: Ultrastructural local-ization of choline acetyltransferase in vascular endothelialcells in rat brain. Nature 1985;316:724-725

10. Loesch A, Burnstock G: Ultrastructural localisation of sero-tonin and substance P in vascular endothelial cells of ratfemoral and mesenteric arteries. Anat Embryol (Berl) 1988;178:137-142

11. Burnstock G, Lincoln J, Feh6r E, Hopwood AM, KirkpatrickK, Milner P, Ralevic V: Serotonin is localized in endothelialcells of coronary arteries and released during hypoxia: Apossible new mechanism for hypoxia-induced vasodilatation ofthe rat heart. Experientia 1988;44:705-707

12. Smiegko V, Kozik J, Dolezel S: Role of endothelium in thecontrol of arterial diameter by blood flow. Blood Vessels1985;22:247-251

13. Hull SS, Kaiser L, Jaffe MD, Sparks HV: Endothelium-dependent flow-induced dilation of canine femoral and saphe-nous arteries. Blood Vessels 1986;23:183-198

14. Smiegko V, Lang DJ, Johnson PC: Dilator response of ratmesenteric arcading arteriole to increased flow. Fed Proc1987;46:643

15. Kaiser L, Hull SS Jr, Sparks HV Jr: Methylene blue andETYA block flow-dependent dilation in canine femoral artery.Am J Physiol 1986;250:H974-H981

16. Rubanyi GM, Romero JC, Vanhoutte PM: Flow-inducedrelease of endothelium-derived relaxing factor. Am J Physiol1986;250:H1145-H1149

17. Ralevic V, Kristek F, Hudlicka 0, Burnstock G: A newprotocol for removal of the endothelium from the perfused rathind-limb preparation. Circ Res 1989;64:1190-1196

18. Jancs6 G, Kiraly E, Jancs6-Gabor A: Pharmacologically-induced selective degeneration of chemoreceptive primarysensory neuron. Nature 1977;270:741-743

19. Coons AH, Leduc EH, Connolly JM: Studies on antibodyprotection. I. A method for the histochemical demonstrationof specific antibody and its application to a study of thehyperimmune rabbit. J Exp Med 1955;102:49-60

20. Belai A, Lincoln J, Milner P, Burnstock G: Progressivechanges in adrenergic, serotonergic, and peptidergic nerves inproximal colon of streptozotocin-diabetic rats. Gastroenterol-ogy 1988;95:1234-1241

21. Angus JA, Campbell GR, Cocks TM, Manderson JA: Vaso-dilation by acetylcholine is endothelium-dependent: A studyby sonomicrometry in canine femoral artery in vivo. J Physiol(Lond) 1983;344:209-222

22. Dudel C, Fdrstermann U: Gossypol attenuates selectively theblood pressure lowering effect of endothelium-dependentvasodilators in the rabbit in vivo. Eur J Pharmacol 1988;145:217-221

23. Fitzgerald M: Capsaicin and sensory neurones: A review. Pain1983;15:109-130

24. Nagy JI: Capsaicin's action on the nervous system. TrendsNeurosci 1982;5:362-365

25. Milner P, Ralevic V, Hopwood AM, Feh6r E, Lincoln J,Kirkpatrick KA, Burnstock G: Ultrastructural localisation ofsubstance P and choline acetyltransferase in endothelial cellsof rat coronary artery and release of substance P and acetyl-choline during hypoxia. Experientia 1989;45:121-125

26. Rubanyi GM: Role of endothelium in flow- and pressure-induced vascular responses. Blood Vessels 1988;25:47

27. Holtz J, Fdrstermann U, Pohl U, Giesler M, Bassenge E:Flow-dependent, endothelium-mediated dilation of epicardialcoronary arteries in conscious dogs: Effects of cyclooxygenaseinhibition. J Cardiovasc Pharmacol 1984;6:1161-1169

KEY WORDS * vascular endothelial cells * substance Pcapsaicin * increased flow

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V Ralevic, P Milner, O Hudlická, F Kristek and G Burnstockhind-limb vasculature, in vivo, by increased flow.

Substance P is released from the endothelium of normal and capsaicin-treated rat

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1990 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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