Clinical and Experimental Pharmacology and Physiology

(2003)

30

,

528–531

EFFECT OF NITROGLYCERINE IN POPLITEAL PREPARATIONS FROM PATIENTS WITH PERIPHERAL OCCLUSIVE

ARTERIOPATHY PRECONTRACTED WITH KCl OR 5-HYDROXYTRYPTAMINE

Ana Ortega,* Martin Avellanal,

†

Gabriel España,

‡

Angel Flores

‡

and Amaya Aleixandre*

*Departamento de Farmacología, Facultad de Medicina, Universidad Complutense, Servicios de

†

Anestesiología y

‡

Cirugía Vascular del Hospital General Universitario Gregorio Marañón, Madrid, Spain

SUMMARY

1. At the present time, there are no studies in isolatedarteries from patients suffering from peripheral occlusivearteriopathy (POA). In the present study, we attempt tocharacterize the effect of nitroglycerine (GTN) in isolatedpopliteal preparations obtained after leg amputation in 60–90-year-old men and women suffering from POA.

2. After surgical operation, arterial samples were stored ina refrigerator at 4

�

C and, after 12–36 h, they were cut intorings and mounted in organ baths containing Krebs’–Henseleitsolution at 37

�

C and gassed constantly with 95% CO

2

and 5%O

2

. Because noradrenaline elicited very poor contractileresponses in these preparations, in the present study weevaluated the concentration-dependent contractions inducedby KCl (15–90 mmol/L) and 5-hydroxytryptamine (5-HT; 10

–7

to 10

–4

mol/L) in arteriopathic popliteal rings and, when thecorresponding maximum contractile effect had been obtained,we also evaluated the concentration-dependent relaxing effectproduced by GTN (10

–10

to 10

–5

mol/L) in all precontractedpreparations. As a reference, similar experiments were per-formed in popliteal preparations obtained following surgery onnon-arteriopathic vascular tissue where it was necessary toresect a certain percentage of healthy vessel.

3. The responses to KCl and 5-HT were greater in healthyvessel than in arteriopathic rings. The relaxing effect of GTNwas greater in preparations precontracted with 5-HT than inthose preparations precontracted with KCl. In addition,preparations precontracted with KCl relaxed even less whenthey were obtained from patients with POA.

4. The present data indicate that GTN is a vasodilator withlittle effect on depolarized arteries. The results also indicatethat the effect of this drug is even less in depolarized arteriesfrom patients with POA.

Key words: 5-hydroxytryptamine, KCl, nitroglycerine,peripheral occlusive arteriopathy, poplitea.

INTRODUCTION

Peripheral occlusive arteriopathy (POA) constitutes one of themain problems to be faced when dealing with cardiovasculardisease. Initial treatment of intermittent claudication is conserv-ative and includes physical exercise and the elimination of riskfactors. However, very few patients respond to these measures.Pharmacological treatments have also been used in patients withPOA, but there is much controversy about the possible use ofvasodilator drugs and, in particular, nitric oxide donors in patientswith POA.

1

Before now, no pharmacological studies have been conducted onisolated arteries from patients with POA. The present study wasperformed in order to characterize the responses to nitroglycerine(GTN) of isolated popliteal preparations, obtained after leg ampu-tation in patients suffereing from serious POA. A previous studyperformed by our research group established that noradrenalineelicited very poor contractile responses in these preparations.

2

Therefore, to evaluate the relaxing effect of GTN in the presentstudy, we have characterized the contractile effects of KCl (adepolarizing agent) and 5-hydroxytryptamine (5-HT; an agent that,like noradrenaline, activates vascular smooth muscle receptors) onpathological arterial tissue.

METHODS

Preparations and general protocol

To undertake the present study, popliteal samples were obtained after legamputation in 60–90-year-old men and women suffering from severe POA.After surgical operation, arterial samples were stored in a refrigerator at4

�

C. After 12–36 h, arterial samples were cut into rings (approximately6 mm in length), which were suspended between two stainless-steel hooksin organ baths containing Krebs’–Henseleit solution

3

of the followingcomposition (in mmol/L): NaCl 118.2; KCl 4.7; CaCl

2

2.5; KH

2

PO

4

1.2;MgSO

4

1.2; NaHCO

3

6.25; glucose 10.0. The solution was maintained at37

�

C and constantly bubbled with a mixture of 95% O

2

and 5% CO

2

. Thering preparations were mounted with a resting tension of 6 g and allowedto equilibrate for a 90 min period. During the equilibration period, thebathing solution was changed every 15 min (it was sucked out with a waterpump and then rapidly replaced in the organ bath) and the resting tensionwas frequently adjusted to 6 g.

After the equilibration period, we studied the concentration-dependentcontractions

induced

by

KCl

(15–90 mmol/L)

and

5-HT

(10

–7

to10

–4

mol/L)

in

the

popliteal

ring

preparations.

The

contractile

agentswere administered in increasing cumulative doses and we obtained only

Correspondence: Dr A Aleixandre, Dpto de Farmacología, Facultad deMedicina, Universidad Complutense, 28040 Madrid, Spain. Email:amaya@med.ucm.es

Received 16 July 2002; revision 22 October 2002; accepted17 November 2003.

GTN in arteriopathic popliteal rings

529

one KCl or 5-HT concentration–response curve for each popliteal ring. Wealso evaluated the concentration-dependent relaxing effect produced byGTN (10

–10

to 10

–5

mol/L) in all precontracted preparations by adding thisdrug in increasing cumulative doses when the corresponding maximumcontractile effect of KCl or 5-HT had been obtained.

During the experiments, isometric tension was recorded and measured bymeans of Letica SA transducers connected to the Letica SA PROTO5 data-acquisition program (Letica SA, Barcelona, Spain). The contractions elcitedby different doses of KCl and 5-HT were expressed in g and the relaxationof each dose of GTN was calculated and expressed as a percentage of themaximum contraction elicited.

As a reference, similar experiments were performed in popliteal prepar-ations obtained following surgery on non-arteriopathic vascular tissuewhere it was necessary to resect a certain percentage of healthy vessel.These popliteal samples were obtained from 60–90-year-old men andwomen suffering from aneurysm.

In all cases, samples were obtained with the patients’ consent and thepermission of the Hospital’s Clinical Research Ethics Committee.

Statistical analysis

Results are always expressed as the mean

±

SEM for 15–20 preparationsobtained from at least five patients (three to four preparations were obtainedfrom each patient). Comparisons were made between values obtained inexperiments performed with preparations from patients with POA and thoseobtained in the experiments performed with preparations of healthy pop-liteal tissue. Student’s

t

-test (unpaired data), with

P

�

0.05 indicatingstatistical significance, was used for these comparisons.

Drugs

The

following

compounds

and

drugs

were

used:

KCl

(Panreac

QuimicaSA, Barcelona, Spain), GTN (Almirall Prodesfarma, Barcelona, Spain) and5-HT (Sigma Chemical, St Louis, MO, USA). These drugs were dissolvedand diluted daily in distilled water. Drugs were added in cumulative dosesto the organ bath and the concentrations were always expressed as finalmolar levels in the bath solution.

RESULTS

The vasoconstrictor responses to KCl and 5-HT were significantlygreater in popliteal preparations obtained from healthy vessels thanin popliteal preparations obtained after leg amputation in patientssuffering from serious POA (see Fig. 1).

Nitroglycerine produced greater relaxations of popliteal prepar-ations precontracted with 5-HT than those precontracted with KCl.In addition, when GTN was added to popliteal rings precontractedwith 5-HT, the relaxing effect of this drug was very similar inhealthy tissue and in arterial tissue from patients with POANevertheless, high concentrations of GTN produced greater relax-ations of popliteal rings obtained from healthy vessels that hadbeen precontracted with KCl than those obtained from patientswith POA that had also been depolarized with this agent (seeFig. 2).

DISCUSSION

Treatment with vasodilatory drugs is usually not effective in vas-cular diseases that involve obstruction to circulation. Nevertheless,there is still interest in the possible use of specific pharmacologicaltreatments that manage to induce certain effective vasodilation inpatients suffering POA. Studies in isolated arteries are, in principle,orientative in the prediction of the actual

in vivo

effectiveness of

vasodilatory drugs; however, at the present time, relatively fewstudies exist using isolated preparations from human arteries and,as mentioned in the Introduction, there are no controlled studiesthat compare the pharmacological responses of isolated arteriesfrom patients suffering from POA with corresponding responses inhealthy arterial tissue. For these reasons, we undertook the presentresearch.

Fig. 1

Cumulative dose–response curves for (a) KCl and (b) 5-hydroxy-tryptamine (5-HT) in popliteal ring preparations from healthy vasculartissue (

�

) and from vascular tissue obtained after leg amputation in patientssuffering from serious peripheral occlusive arteriopathy (

�

). Data are themean

±

SEM of 15–20 preparations obtained from at least five patients.*

P

�

0.05, **

P

�

0.01 for differences between contractile effects.

530

A Ortega

et al.

In the present study, we have investigated the contractileresponses to KCl and 5-HT in isolated popliteal preparationsobtained after leg amputation in patients suffering serious POAand, after establishing the contractile responses, we demonstratedthe relaxing effect of GTN in these preparations. We performedsimilar experiments in popliteal preparations obtained followingsurgery where it was necessary to resect a certain percentage of

healthy vessel and, as expected, the responses to the contractileagents were smaller in preparations obtained from patients withPOA. We have also shown that the relaxing effect of GTN was verysimilar in all popliteal preparations when they were precontractedwith 5-HT but, in contrast, arterial tissue precontracted with KClshowed smaller relaxation responses to GTN and relaxed even lessin response to GTN when preparations were obtained from patientswith POA. Of course, the mechanisms of action by which contrac-tion is produced in response to KCl and 5-HT are very different.KCl is a depolarizing agent that facilitates the entry of extracellularcalcium through voltage-dependent channels, but the vascularactions of 5-HT are complex. 5-Hydroxytryptamine can even havea vasodilatory effect in some vascular beds because it is able torelease endothelial-derived relaxing factor and to inhibit the releaseof noradrenaline from sympathetic nerve terminals. When its effectin isolated arteries is studied, the contractile response is pre-dominant because 5-HT acts on post-synaptic 5-HT

2A

receptors.When these receptors are stimulated, phospholipase C is activatedand inositol 1,4,5-trisphosphate is formed, thereby releasingcalcium from the sarcoplasmic reticulum. These differences inmechanism probably explain why GTN differentially relaxesarterial tissue precontracted with KCl or 5-HT. However, the factthat both KCl and 5-HT contract the arteries of patients with POAto a lesser extent that the contraction seen for healthy arteries is, webelieve, linked to alterations in the vascular smooth muscle of thesepatients. We do not know why high doses of GTN had a greaterrelaxant effect in preparations of healthy tissue that had beenprecontracted with KCl compared with preparations obtained frompatients with POA, but perhaps this is also due to alterations in thevascular smooth muscle of the diseased arterial tissue. In fact, ananatomopathological study of popliteal arteries from patients withPOA revealed that this tissue has calcium deposits in atheromaplaques and sometimes also in the vascular smooth muscle.

4

Our results indicate, in particular, that GTN is a vasodilator withlittle effect on depolarized arteries. The present study also indicatesthat GTN is, above all, ineffective in depolarized arteries frompatients with POA. In this context, it is important to highlight thatpatients suffering from POA are usually hypertensive and someexperimental studies have indicated that the vascular smoothmuscle of hypertensive animals shows an increase in the numberand activation of voltage-dependent calcium channels.

5–7

We wouldlike to point out that, in 1996, Jones

et al

. indicated that isolatedtibial and peronea artery cells obtained following an amputationwere able to contract in response to angiotensin II and endothelin-1, but did not respond to isoproterenol and sodium nitroprusside.

8

Therefore, the results obtained by these researchers also indicatethat another nitric oxide donor, apart from GTN, has hardly anyeffect in producing a relaxation of arteries from patients with POA.However, in 1998, Boger

et al

. demonstrated that the adminis-tration of

L

-arginine restored nitric oxide formation and endo-thelium-dependent vasodilation in patients with POA, improvingtheir clinical symptons of intermittent claudication.

9

It is also truethat the results obtained in the present study when preparationswere precontracted with 5-HT suggest that GTN may produce avasodilatory effect in patients suffering POA when their arteries arecontracted with agonists acting on vascular smooth muscle recep-tors. This is, of course, important because it should be borne inmind that, when the condition is very serious, any therapy thatoffers some improvement for patients with POA is highly desirable

Fig. 2

Cumulative dose–response curve for nitroglycerine (GTN) inpopliteal ring preparations precontracted with (a) 90 mmol/L KCl and (b)10

–4

mol/L 5-hydroxytryptamine (5-HT) from healthy vascular tissue (

�

)and from vascular tissue obtained after leg amputation in patients sufferingfrom serious peripheral occlusive arteriopathy (

�

). Data are the mean

±

SEMof 15–20 preparations obtained from at least five patients. *

P

≤

0.05 fordifferences between the relaxing effects of GTN.

GTN in arteriopathic popliteal rings

531

because, in many cases, there is no other alternative to amputationof the limb. In fact, we believe that more studies in isolated arteriesfrom patients with POA are advisable.

ACKNOWLEDGEMENTS

This work was supported by a FIS (00/0925) grant.

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