Heterogeneous Behavior of the Canine Arterial and Venous Wall

Transcription

1 439 Hetergeneus Behavir f the Canine Arterial and Venus Wall Imprtance f the Endthelium J.G. De Mey and P.M. Vanhutte Frm the Divisin f Pharmaclgy, Faculty f Medicine, University f Antwerp, 261 Wilrijk, Belgium, and the Department f Physilgy and Biphysics, May Clinic and May Fundatin, Rchester, Minnesta SUMMARY. Experiments were designed t determine the cntributin f endthelial cells t the hetergeneus behavir f the arterial and venus wall. Rings f canine femral, pulmnary, saphenus, and splenic arteries and veins, with and withut endthelium, were munted fr ismetric tensin recrding in Krebs-Ringer bicarbnate slutin. Endthelium-dependent inhibitry respnses t acetylchline, adensine triphsphate, bvine thrmbin, and arachidnic acid were prminent in the arteries. In the veins, nly transient endthelium-dependent relaxatins t these substances were bserved. Remval f the endthelium decreased the augmentatin f the respnse t nrepinephrine caused by anxia in bth arteries and veins. In the veins, arachidnic acid and thrmbin caused endthelium-dependent increases in tensin during cntractins evked by nrepinephrine. The endthelium-independent inhibitry effects f isprterenl and adensine and the excitatry effects f acetylchline and ATP were mre prnunced in the veins than in the arteries. These experiments demnstrate that in the arterial and venus wall the endthelial cells can cntribute t bth inhibitry and excitatry respnses f the smth muscle cells f the media. Inhibitry endthelial respnses prevail in the arteries, and excitatry nes in the veins. (Ore Res 51: , 1982) BLOOD VESSELS f different anatmical rigin can respnd in a dissimilar manner t the same pharmaclgical r physilgical stimulus; this can als be the case in the same vascular bed fr the pre- and pstcapillary vessels (see Furchgtt, 1955; Bhr, 1965; Smly and Smly, 197; Shepherd and Vanhutte, 1975; Vanhutte, 1978, 198). Studies cmparing the reactivity f islated arteries with and withut endthelium have demnstrated that the endthelial cells play an bligatry rle in the relaxing effect f acetylchline, ATP and thrmbin (Furchgtt and Zawadzki, 198; De Mey and Vanhutte, 1981a, 1981b; Furchgtt et al., 1981; Chand and Altura, 1981; De Mey et al., 1982). Infrmatin is lacking cncerning the rle f endthelial cells in the respnse f smth muscle cells f the venus wall. Therefre, the present experiments were designed t cmpare the imprtance f the endthelium in the respnsiveness f different arteries and veins. Methds The experiments were perfrmed n ring preparatins f femral, intrapulmnary, saphenus, and splenic arteries and veins taken frm mngrel dgs f bth sexes (24-35 kg) anesthetized with sdium pentbarbital (3 mg/kg, iv). After excisin, the preparatins were placed in cld Krebs- Ringer bicarbnate slutin (cmpsitin ITIM: NaCl, 118.3; KC1, 4.7; CaCl 2, 2.5; MgSO 4-7H 2 O, 1.2; KH 2 PO 4, 1.2; NaHCO 3, 25.; CaEDTA,.26; glucse, 11.1) and carefully cleaned with special care being taken nt t tuch their luminal surface (cntrl rings). Sme rings were placed n filter paper, the bent tips f a watchmaker's frceps were inserted int their lumen, and the endthelial cell layer was remved by gently rlling the preparatins back and frth ver the filter paper fr 15 secnds. Ismetric Frce Recrding Each ring was attached t an ismetric frce transducer (Statham UC3) and suspended in an rgan chamber filled with 5 ml f Krebs-Ringer bicarbnate slutin (37 C) which was aerated with 95% O 2-5% CO 2. In certain experiments, the rgan chamber slutin was made anxic by aerating it with 95% N 2-5% CO 2. Earlier wrk has shwn that changing frm the gas mixture cntaining 95% O 2-5% CO 2 t the ne cntaining 95% N 2-5% CO 2 prduced a rapid decrease in bath P 2 frm 64 t belw 1 mm Hg withut a significant change in ph (Vanhutte, 1976; De Mey and Vanhutte, 198). Tw rectangular platinum electrdes were placed parallel t the preparatins and used t stimulate the adrenergic nerve endings in the bld vessel wall. Square wave (9 V, 2 msec) electrical impulses were prvided by a direct current supply and switching transistr (MBLE BD 139) triggered by a stimulatr (Janssen Scientific Instruments SUi) (Vanhutte et al., 1967, 1979). Befre the experiments were begun, the rings were placed at the ptimal pint f their length-tensin relatinship using a standard electrical stimulatin (15 Hz, 1 sec) r a standard cncentratin f nrepinephrine (5 X 1~ 7 M; Table 1). The preparatins were then allwed t equilibrate at their ptimal length fr 45 minutes. Histlgy T ascertain that the mechanical rubbing applied t the bld vessel rings had successfully remved the endthe-

2 Circulatin Research/V/. 51, N. 4, Octber TABLE 1 Respnses f Canine Arteries and Veins* Bld vessel Arteries Femral Pulmnary Saphenus Splenic Veins Femral Pulmnary Saphenus Splenic Electrical stimulatin Basal frce at ptimal lengthf (g) ED» (1-6 M) ED M (1-6 M) Maximal respnse (g) 8 Hz (% NE)J 15.3 ± ± ± ± ± ± ± ± ±.4.1 ±.4.54 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.1 ± 4.4 ± 6.2 ± 7.1 ± 1.2 ± 9.7 ± 8.4 ± ±.1 1. ± ± ± ± ± ± ±.1.19 ±.3.18 ±.3.13 ±.6.12 ±.5.48 ±.1.39 ±.9.22 ±.6.34 ±.7.58 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.1 ± 4.8 ± 7.3 ± 6.9 ± 6.3 ± 6.9 ± 7.4 ± 8.1 Endthelium Nrepinephrine 16 Hz (% NEtf Acetylchline ED5 6 ( 1 - M) Maximal respnse (% NE)t 6.4 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.8 * Data shwn as means ± SEM fr bld vessels frm different dgs; paired rings frm each vessel, with and withut endthelium, were studied in parallel. The values f basal frce are fr 36 rings in each grup; fr the ther data, six vessels were studied in each grup. t In the femral arteries and veins, the ptimal length was determined using 5 X 1~7 M nrepinephrine (7 min, every 2 min) and in all ther bld vessels using 25 Hz electrical stimulatin (1 sec, every 5 min). X Expressed as percent f the maximal respnse t nrepinephrine. In the pulmnary vein, maximal respnses were btained with 3 X 1~6 M, and in the ther preparatins with 1~4 M nrepinephrine. Difference between preparatins with and withut endthelium is statistically significant (P <.5). Hum, we incubated cntrl and rubbed rings fr 2 hurs in Krebs-Ringer slutin at 37 C. The rings were pened lngitudinally and stained in vitr with silver nitrate as described by Caplan et al. (1974). Briefly, the preparatins were immersed successively in the dark at rm tempera- ture in: (1) Hepes (2 ITIM) buffered (ph 7.4) slutin cntaining 4.6% glucse fr 15 secnds; (2).4% AgNO3 in 4.2% glucse slutin fr 6 secnds, and (3) 4.6% glucse slutin fr 6 secnds. The arteries then were fixed at rm temperature in.1 M sdium cacdylate cntaining E- FIGURE 1. Transverse sectins f tw preparatins f the same canine saphenus vein (X225). Upper: intima left as intact as pssible. Lwer: after mechanical nibbing f the intimal layers. Left: when the preparatin is transilluminated with white light, the silver nitrate grains shw up as black dts. Right: when the same preparatin is illuminated with green light, the silver cmplexes reflect the light. Nte that in the presence f the endthelial layer (E), the silver nitrate is strictly lcalized at the endthelial-iuminal brder (A and B), which, after mechanical rubbing f the intimal layer (C and D), diffuses tward the media (M).

3 De Mey and Vanhutre/Vascular Hetergeneity and Endthelium 7.5% sucrse. The vessels were examined under light and electrn micrscpy fllwing further fixatin with smium tetrxide and a plychrmatic staining (Van Reempts and Brgers, 1975). In the arteries and veins with endthelium, "en face" light micrscpic examinatin revealed a msaic pattern f silver lines, which are cnsidered t represent the brders f adjacent endthelial cells (Ple et al., 1958; Caplan et al., 1974); the study f transverse sectins revealed that the silver nitrate had accumulated at the intimal brder 441 f the bld vessel wall (Fig. 1). Electrn micrscpy cnfirmed the presence f endthelial cells in the cntrl rings (Fig. 2). By cntrast, in arteries and veins where the intimal layers had been rubbed mechanically, n msaic pattern was seen n "en face" light micrscpy; the transverse sectins revealed diffusin f the silver nitrate grains int the deeper layers f the bld vessel wall (Fig. 1). The absence f endthelial cells and medial diffusin f silver nitrate was cnfirmed by electrn micrscpy (Fig. 2). N FIGURE 2. Electrn micrgraph (X75) f the luminal part f tw preparatins f the same canine pulmnary vein. Upper: endthelium left as intact as pssible. Nte that the silver nitrate grains d nt penetrate beynd the endthelial cells (E). Lwer: after mechanical rubbing f the endthelium, n endthelial cells are seen, and the silver nitrate grains permeate int the media. SM = smth muscle cells.

4 442 Circulatin Research/ Vl. 51, N. 4, Octber 1982 structural damage was bvius in ther layers f the bld vessel wall. Drugs The drugs used were acetylchline chlride (Sigma), adensine (Sigma), adensine triphsphate (ATP; Sigma), arachidnic acid (Sigma), isprterenl hydrchlride (Aidrich Eurpe), /-nrepinephrine bitartrate (Fluka), phentlamine mesylate (Ciba), and thrmbin (bvine thrmbin, Rche). Stck slutins were prepared in distilled water, frm the stck slutins, /il were added t the rgan chamber slutin. All cncentratins are expressed as final mlar cncentratins (M). Statistical Analysis Each experimental grup cnsisted f six preparatins taken frm six dgs. Rings with and withut endthelium were prepared frm the same segment f bld vessel. The data are shwn as means ± SEM. The data were analyzed by use f analysis f variance. Differences were cnsidered significant at P <.5. Only significant differences will be mentined. When relaxatins were studied, the cmpunds were added during sustained cntractins evked by the ED3 (De Mey and Vanhutte, 198) f nrepinephrine, determined individually in each bld vessel (Table 1). The relaxatins are expressed as percent depressin f these cntractins. Results Nrepinephrine All bld vessels cntracted when expsed t increasing cncentratins (1CT 8 t 1~ 4 M) f nrepinephrine. T judge frm the ED5 values, the apparent sensitivity t nrepinephrine decreased in the fllwing rder: pulmnary vein, splenic vein, femral vein, femral artery, saphenus vein, pulmnary artery, splenic artery, saphenus artery. Remval f the endthelium did nt affect the sensitivity f the arteries and veins t nrepinephrine but reduced the maximal cntractile respnse t the catechlamine in all bld vessels studied, except the pulmnary vein (Table 1). Electrical Stimulatin Electrical stimulatin caused frequency-dependent cntractins f all bld vessels studied (Table 1). When expressed relative t the maximal respnse t exgeneus nrepinephrine, the largest cntractile respnses were btained in the saphenus artery and vein and in the splenic vein, fllwed, in descending rder, by the pulmnary artery, the splenic artery, the pulmnary artery, the pulmnary vein, the femral vein, and the femral artery. Remval f the endthelium did nt affect the respnsiveness f the arteries and veins t electrical stimulatin (Table 1). In all preparatins, phentlamine (3 X 1~ 6 M) ablished the cntractins evked by 8 Hz electrical stimulatin. Isprterenl The effect f increasing cncentratins f isprterenl was investigated during cntractins f the arteries and veins caused by nrepinephrine. In the veins, but nt in the arteries, isprterenl caused cmparable, cncentratin (1CT 8 t 5 X 1CT 6 M) dependent relaxatins; in all bld vessels, higher cncentratins (1~ 5 t 1CT 4 M) caused increases in tensin (Fig. 3). Remval f the endthelium did nt affect the respnsiveness f the arteries and veins t isprterenl. Adensine Arteries and veins, made t cntract with nrepinephrine, relaxed in a cncentratin-dependent manner when expsed t adensine (1CT 6 t 1~ 3 M); the veins were mre sensitive than the arteries (Fig. 3). Remval f the endthelium did nt affect the respnsiveness f the arteries and veins t adensine. Adensine Triphsphate In the fur arteries, ATP (1CT 6 t 1CT 4 M) caused cmparable, cncentratin-dependent relaxatins during cntractins induced by nrepinephrine; such relaxatins were bserved in endthelium-denuded arteries nly with cncentratins f ATP higher than Cfc SO 5 8 -LOG MOLAR CONCENTRATION ISOPROTERENOL 6 5 -LOG MOLAR CONCENTRATION ADENOSINE FIGURE 3. Effect f increasing cncentratins f isprterenl (upper) and adensine (lwer) n the cntractile respnses f femral (O), pulmnary (O), saphenus (A) and splenic (V) arteries (left) and veins (right) f the same six dgs t nrepinephrine. The bld vessel rings were made t cntract with the ED3 f nrepinephrine (see Table 1); when the cntractile respnse had stabilized, a cumulative cncentratin-respnse curve t either isprterenl r adensine was btained. Data expressed as percent depressin f the respnse t nrepinephrine, and shwn as means. Fr the sake f clarity, the SEM are mitted. Experiments perfrmed in parallel n rings withut endthelium (data nt shwn) revealed n significant differences frm cntrl rings.

5 De Mey and Vanhutte/Vascular Hetergeneity and Endthelium Lj z. 5 / ARTER/ES VE//VS LOG MOLAR CONCENTRATION ATP FIGURE 4. Effect f increasing cncentratins f adensine triphsphate (ATP) n the cntractile respnses f femral ( 2), pulmnary (O), saphenus (A) and splenic (V) arteries (left) and veins (right) f the same six dgs t nrepinephrine (ED3; Table 1). Rings with (full lines) and withut (dtted lines) endthelium were studied in parallel, the experimental prtcl is identical t that used in Figure 1. Data expressed as percent depressin f the respnse t nrepinephrine, and shwn as means. Fr the sake f clarity, the SEM are mitted. Full symbls indicate that the difference between preparatins with and withut endthelium is statistically significant. In veins with endthelium, ATP caused a biphasic respnse, with an initial relaxatin fllwed by cntractins; the initial relaxatry respnse t ATP is shwn. Relaxatins were nt bserved in the veins withut endthelium. The steady state further increases in tensin caused by ATP were cmparable in the preparatins with (data nt shwn) and withut endthelium (data shwn). Q) f «5. UJ 2 Q: 2 O Q> ( Ct Withut With Endthelium # LOG MOLAR CONCENTRATION ACETYLCHOLINE FIGURE 5. Direct effect f increasing cncentratins facetylchline in pulmnary veins with ( ) and withut ( ) endthelium. Data expressed as percent f the maximal respnse t nrepinephrine (see Table 1), and shwn as means ± SEM. Full symbls indicate that the difference between preparatins with and withut endthelium is statistically significant. 3 X 1 5 M (Fig. 4). In all veins, cncentratins f ATP higher than 1~ 6 M caused further increases in tensin during nrepinephrine-induced cntractins; these further increases in tensin were nt affected by the remval f the endthelium (Fig. 4). In the veins with endthelium, the increases in tensin caused by ATP were preceded by transient relaxatins. Acetylchline Acetylchline (3 X 1" 7 t 1CT 4 M) did nt significantly affect basal tensin in islated arteries, with and withut endthelium. All veins cntracted when expsed t acetylchline; remval f the endthelium did nt affect the cntractile respnses f the femral, saphenus, and splenic veins, but augmented thse f the pulmnary veins (Table 1; Fig. 5). In the fur arteries, made t cntract with nrepinephrine (ED3; Table 1), acetylchline caused cmparable, cncentratin-dependent (3 X 1CT 9 t 1CT 6 M) relaxatins, which were nt bserved in preparatins withut endthelium (Fig. 6). In the saphenus and femral veins, 3 X 1CT 8 t 3 X 1CT 7 M acetylchline, given during nrepinephrine-induced (ED3; Table 1) cntractins, caused transient relaxatins, which were nt seen in deendthelialized preparatins. In the splenic vein, bth with and withut endthelium, 1~ 8 t 3 X 1~ 7 M acetylchline caused relaxatins (Fig. 7). The same cncentratins f acetylchline did nt affect cntractile respnses f the O 3 X -j UJ t Z tfc Q_ 5 r«*e^ 'With endthelium Withut endthelium a Femral A Saphenus P.ulmnryvSplenic 8 -LOG MOLAR CONCENTRATION ACETYLCHOLINE FIGURE 6. Effect f increasing cncentratins f acetylchline n the cntractile respnses f femral (O), pulmnary (O), saphenus (A), and splenic (V) arteries f the same dgs t nrepinephrine (EDa; Table 1). Rings with (full lines) and withut (dtted lines) endthelium were studied in parallel. The experimental prtcl is identical t that used in Figure 1. Data expressed as percent depressin f the respnse t nrepinephrine, and shwn as means. Fr the sake f clarity, the SEM are mitted. Full symbls indicate that the difference between preparatins with and withut endthelium is statistically significant.

6 444 Circulatin Research/V/. 51, N. 4, Octber 1982 Z 5 FEMORAL VEIN SAPHENOUS VEIN SPLENIC VEIN B LOG MOLAR CONCENTRATION ACETYLCHOLINE FIGURE 7. Effect f increasing cncentratins f acetylchline n the cntractile respnses f femral (O, left), saphenus (A, middle), and splenic (S7, right) veins f the same dgs t nrepinephrine (ED3; Table 1). Rings with (full lines) and withut (dtted lines) endthelium were studied in parallel. The experimental prtcl is identical t that used in Figure 1. Data expressed as percent depressin f the respnse t nrepinephrine, and shwn as means. Fr the sake f clarity, the SEM are mitted. Full symbls indicate that the difference between preparatins with and withut endthelium is statistically significant. pulmnary veins, with and withut endthelium, t nrepinephrine. Higher cncentratins f acetylchline (1CT 6 t 1~ 4 M) caused increases in tensin during cntractins induced by nrepinephrine in the veins, but nt in the arteries; these increases were nt affected by the remval f the endthelium (Fig. 6 and 7). Thrmbin In the fur arteries made t cntract with nrepinephrine, bvine thrmbin (.1 t 1 U/ml) caused - r Z 2-5 Z Ui Q. 5.1 O.I 1..1 O.I BOVINE THROMBIN, Units /ml FIGURE 8. Effect f increasing cncentratins f bvine thrmbin n the cntractile respnse f femral (D), pulmnary (O), saphenus (A), and splenic (V) arteries (left) and veins (right) f the same six dgs t nrepinephrine (EDw, Table 1). Rings with (full lines) and withut (dtted lines) endthelium were studied in parallel. The experimental prtcl is identical t that used in Figure 1. Data expressed as percent depressin f the respnse t nrepinephrine, and shwn as means. Fr the sake f clarity, the SEM are mitted. Full symbls indicate that the difference between preparatins with and withut endthelium is statistically significant. 1. dse-dependent relaxatins which were absent in deendthelialized preparatins (Fig. 8). In the fur veins, cntracted with nrepinephrine, thrmbin caused slw further increases in tensin which stabilized within 1 minutes and were significantly larger in the presence f endthelium (Fig. 8); in the saphenus and splenic veins, the further increases in tensin were preceded by transient, endthelium-dependent relaxatins. Arachidnic Acid Arachidnic acid (1~ 7 t 3 X 1~ 5 M) caused cmparable, cncentratin-dependent relaxatins in the fur arteries cntracted with nrepinephrine. Remval f the endthelium shifted the dse-respnse curve fr the fatty acid t the right in the femral, pulmnary, and saphenus arteries and decreased its maximal effect in the femral and saphenus artery; in the splenic artery withut endthelium, arachidnic acid caused further increases in tensin (Fig. 9). In the pulmnary vein, 3 X 1~ 7 t 1~ 6 M arachidnic acid caused an endthelium-dependent decrease in tensin during the cntractile respnses t nrepinephrine; this was fllwed, at higher cncentratins, by an increase in tensin. In the ther veins, the nly significant effect f arachidnic acid was t augment the cntractile respnse t nrepinephrine. In all veins, the increases in tensin caused by arachidnic acid were smaller in the absence f endthelium (Fig. 9). Anxia Anxia, induced fr 1 minutes during cntractile respnses t nrepinephrine, caused a further increase in tensin in femral, pulmnary, saphenus, r 2 2 "5 x z LOG MOLAR CONCENTRATION ARACHIDONIC ACID FIGURE 9. Effect f increasing cncentratins f arachidnic acid n the cntractile respnses f femral (O), pulmnary (O), saphenus (A), and splenic (V) arteries (left) and veins (right) f the same dgs t nrepinephrine (ED3; Table 1). Rings with (full lines) and withut (dtted lines) endthelium were studied in parallel. The experimental prtcl is identical t that used in Figure 1. Data expressed as percent depressin f the respnse t nrepinephrine, and shwn as means. Fr the sake f clarity, the SEM are mitted, and when symbls verlap, nly ne is shwn. Full symbls indicate that the difference between preparatins with and withut endthelium is statistically significant.

7 De Mey and Vanhutte/Vascular Hetergeneity and Endthelium 445 ct Q: U. Z O I- UJ UJ. - rfi Wifh Endthelium Q Withut Endthelium FEMR4L PULMO/VAffV SAPHENOUS SPLENIC FIGURE 1. Effect f anxia (1 min) n the cntractile respnses t nrepinephrine (ED3; Table 1) in canine arteries (upper) and veins (lwer). Rings with (full bars) and withut (pen bars) endthelium were studied in parallel. Changes in tensin are expressed as percent f the cntractile respnse t nrepinephrine, and shwn as means ± sew. * the difference between rings with and withut endthelium is statistically significant. and splenic arteries. The anxic ptentiatin was larger in preparatins with, than in thse withut, endthelium; this difference was least prnunced in the pulmnary artery. Anxia did nt affect the cntractile respnsiveness t nrepinephrine in femral and saphenus veins, with r withut endthelium. In the presence f the endthelium, anxia augmented the cntractile respnse t nrepinephrine in the pulmnary and splenic veins; after remval f the intima, this increase was less prnunced in the splenic vein and reversed t a relaxatin in the pulmnary vein (Fig. 1). Discussin The present experiments cnfirm that the endthelium plays an bligatry rle in the relaxatins f islated arteries induced by acetylchline (Furchgtt and Zawadzki, 198; De Mey and Vanhutte, 1981a; Furchgtt et al., 1981; De Mey et al., 1982). The ability f the endthelial cells t cause relaxatin f the smth muscle cells f the media, when expsed t acetylchline, is cmparable in the three systemic and pulmnary arteries. Greater differences in sensitivity ccur fr the endthelium-dependent relaxatins f islated arteries caused by ATP (De Mey and Vanhutte, 1981a) and thrmbin (De Mey et al., 1982). The experiments with arachidnic acid cnfirm that, in islated arteries, the fatty acid can cause endthelium-dependent relaxatins (De Mey et al., 1982), which are remarkably similar in the different canine arteries tested. Thus, despite minr reginal differences in apparent sensitivity, the arterial wall appears relatively hmgeneus in its ability t exhibit endthelium-dependent relaxatins f the smth muscle cells f the media. This certainly is nt the case fr the veins studied. Only in the femral and saphenus vein culd mderate endthelium-dependent relaxatins be bserved during nrepinephrine cntractin. In the pulmnary vein, endthelium-mediated inhibitry respnses were suggested nly by the augmented respnsiveness t acetylchline after remval f the intima, whereas, in the splenic vein, n such evidence culd be btained. Obviusly, in the veins, the evaluatin f endthelium-dependent inhibitry respnses t acetylchline is cmplicated by the marked reginal differences in venus smth muscle respnsiveness t the direct stimulating prperties f the chlinergic transmitter (see Shepherd and Vanhutte, 1975; Vanhutte, 1978). ATP, thrmbin, and arachidnic acid caused nly transient relaxatins in certain islated veins. One lgical explanatin fr the transient character f these relaxatins is that ther actins f the vasactive substances mask the inhibitry endthelium-dependent effect that they have; the data with isprterenl and adensine demnstrate that it cannt be attributed t a relative inability f the venus smth muscle cells t relax. The cntact f nrepinephrine with endthelial cells may cause the smth muscle t cntract befre the amine has permeated the vascular wall (Bevan and Duckies, 1975; Pascual and Bevan, 198). Earlier wrk in the femral artery f the dg indicated that the intima may cntribute t the cntractile respnses t high K + and anxia (De Mey and Vanhutte, 1981a, 1981b). The present study demnstrates that endthelial cells can facilitate the cntractin f the vascular smth muscle cells f the media. This cnclusin is based n the bservatins that endthelium remval: (1) reduces the maximal cntractile respnse t nrepinephrine. This reductin is nt likely t be due t adverse effects f the prcedure t remve the endthelium n the viability f the preparatins. Indeed, the shape f the cncentratin-respnse curve was nt affected and the maximal respnse t the catechlamine was nly mderately, r nt significantly, reduced in sme bld vessels. Furthermre, the cntractile respnse t several ther interventins was enhanced by the prcedure, (2) reduces the increase in tensin caused by thrmbin in the veins, (3) reduces, r ablishes, the cntractins f the veins caused by arachidnic acid, and (4) reduces, r reverses, the increases in tensin caused by acute anxia during nrepinephrine-induced cntractin. The predminance f inhibitry endthelial respnses in arteries and f excitatry nes in the veins cannt be explained slely by differences in synthesis f prstacyclin, since the endthelium-dependent effects f acetylchline, ATP, thrmbin, and anxia n the canine femral artery are nt antagnized by inhibitrs f cyclxygenase (e.g., Furchgtt et al.,

8 446 Circulatin Research/Vi. 51, N. 4, Octber ; De Mey et al., 1982; De Mey and Vanhutte, 1981b) and since prstacyclin, if anything, depresses the cntractile respnse f the canine saphenus vein t nrepinephrine (Herman et al., 1978). Likewise, bth inhibitry and excitatry endthelial-dependent respnses were bserved in bld vessels independently f their embrylgical rigin (Tsuru et al., 1976; Bevan, 1979), their respnsiveness t sympathetic nerve stimulatin (Vanhutte, 1978; Bevan, 1979; Abel et al., 198; Vanhutte et al., 1981) r their chrnic expsure t arterial r venus bld (Vanhutte, 1978). Hwever, endthelium-dependent augmentatin f nrepinephrine-induced cntractins with thrmbin and arachidnic acid are seen nly in thse bld vessels studied which are lcated in the lw pressure side f the circulatin and are nt expsed t pulsatile bld flw. T judge frm the ED5 f nrepinephrine, the apparent sensitivity t the catechlamine varied amng the different arteries and veins reflecting the true hetergeneity in vascular pstjunctinal adrenergic respnsiveness (see Bevan et al., 198; Vanhutte et al., 1981). An imprtant difference between deendthelialized arterial and venus preparatins was the absence f cntractins induced by acetylchline, ATP, and thrmbin in the arteries. In the case f acetylchline, the present study cnfirms that the respnsiveness f venus smth muscle cells t the excitatry effect f acetylchline varies depending n their anatmical rigin (see Shepherd and Vanhutte, 1975; Vanhutte 1977, 1978). In the splenic vein cntracted with nrepinephrine, acetylchline causes a relaxatin which depends neither n prejunctinal inhibitin f nrepinephrine release (Vanhutte, 1977; Vanhutte et al., 1981) nr n generatin f inhibitry signals by the endthelial cells (Furchgtt and Zawadzki, 198; De Mey and Vanhutte, 1981a). This inhibitry effect ccurs at lwer cncentratins f acetylchline than thse required t activate the smth muscle cells, and is muscarinic in nature since it is ablished by atrpine (unpublished bservatins). The present study thus implies that, at least in certain vascular beds, the chlinergic transmitter may cause dilatin by virtue f a presumably direct inhibitry effect n the vascular smth muscle cells, in additin t any prejunctinal r endtheliummediated effects it may have (see Vanhutte 1977, 1978, 1981; Furchgtt et al., 1981 Vanhutte et al., 1981). Canine veins relax when expsed t isprterenl (e.g., Vanhutte and Shepherd, 197, 1973). These relaxatins d nt depend n the presence f functinal endthelial cells (Furchgtt et al., 1981) and are nt bserved in canine arteries, as already nted in earlier wrk, at least the splenic artery (e.g., Vanhutte and Shepherd, 197; Van Hee and Vanhutte, 1978). Other qualitative differences in respnsiveness, during expsure t nrepinephrine, between the arteries and veins were the presence f an inhibitry effect f arachidnic acid in certain deendthelialized arteries, and the absence f cntractin caused by anxia in certain deendthelialized veins (Vanhutte, 1976). The mst striking quantitative difference between arterial and venus preparatins was the greater respnse f the veins t the endtheliumindependent inhibitry effect f adensine (De Mey and Vanhutte, 1981a; Furchgtt et al., 1981). Whether r nt the hetergeneity f the smth muscle cells cntributes t the differences in sensitivity t endthelium-mediated phenmena will remain unknwn until the exact nature f the link between the endthelial and smth muscle cells is determined. Supprted in part by Grant HL Address fr reprints: Paul M. 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9 De Mey and Vanhulte/Vascular Hetergeneity and Endthelium 447 may be initiated frm the intima f the rabbit arta. In Vascular Neureffectr Mechanisms, edited by JA Bevan, T Gdfraind, RA Maxwell, PM Vanhutte. New Yrk, Raven Press, pp 3-34 Ple JCF, Sandus AG, Flrey HW (1958) The regeneratin f artic endthelium. ] Pathl Bacteril 75: Shepherd JT, Vanhutte PM (1975) Veins and Their Cntrl. Philadelphia, WB Saunders C, pp Smly AP, Smly AV (197) Vascular smth muscle. II. Pharmaclgy f nrmal and hypertensive vessels. Pharmacl Rev 22: Tsuru H, Ihikawa N, Shigei T (1976) Respnsiveness f islated dg veins t bradykinin and ther biactive peptides: Distributin f sensitivity t bradykinin and pssible crrelatin with genesis f the venus system. Bld Vessels 13: Van Hee RH, Vanhutte PM (1978) Chlinergic inhibitin f adrenergic neurtransmissin in the canine gastric artery. Gastrenterlgy 74: Vanhutte PM (1974) Inhibitin by acetylchline f adrenergic neurtransmissin in vascular smth muscle. Circ Res 34: Vanhutte PM (1976) Effect f anxia and glucse depletin n islated veins f the dg. Am J Physil 23: Vanhutte PM (1977) Chlinergic inhibitin f adrenergic transmissin. Fed Prc 36: Vanhutte PM (1978) Hetergeneity in vascular smth muscle. In Micrcirculatin, vl II, edited by G Kaley, BM Altura. Baltimre, University Park Press, pp Vanhutte PM (198) Physical factrs f regulatin. In Handbk f Physilgy, sec 2, The Cardivascular System, vl II, Vascular Smth Muscle, edited by DF Bhr, AP Smly, HV Sparks, Jr. Washingtn, D.C., American Physilgical Sciety, pp Vanhutte PM (1981) Why is acetylchline a vasdilatr? In Vasdilatatin, edited by PM Vanhutte, I Leusen. New Yrk, Raven Press, pp Vanhutte P, Shepherd JT (197) Effect f cling n beta-receptr mechanisms in islated cutaneus veins f the dg. Micrvasc Res 2: Vanhutte P, Clement D, Leusen I. (1967) The reactivity f islated veins t electrical stimulatin. Arch Int Physil Bichem 75: Vanhutte PM, Cen EP, De Ridder WJ, Verbeuren TJ (1979) Evked release f endgenus nrepinephrine in the canine saphenus vein. Inhibitin by acetylchline. Circ Res 45: Vanhutte PM, Verbeuren TJ, Webb RC (1981) Lcal mdulatin f adrenergic neureffectr interactin in the bld vessel wall. Physil Rev 61: Van Reempts, J, Brgers M (1975) A simple plychrme stain fr cnventinally fixed Epn-embedded tissue. Stain Technl 5: INDEX TERMS: Acetylchline Adensine triphsphate Arachidnic acid Arterial smth muscle Endthelium-dependent cntractins Endthelium-dependent relaxatin Thrmbin and venus smth muscle