Gen. Pharmac. Vol. 22, No. 1, pp. 137-142, 1991 Printed in Great Britain.All rights reserved
0306-3623/91 $3.00+ 0.00 Copyright © 1991PergamonPresspie
MODULATION OF ~ - A D R E N E R G I C - I N D U C E D CONTRACTIONS BY ENDOTHELIUM-DERIVED RELAXING FACTOR IN RAT AORTA P,. VINET2, C. BRIEVAl, G. PINARDI1and M. PENNA1. ~Department of Pharmacology, Faculty of Medicine, University of Chile, P.O. Box 70.000, Santiago 7, Chile and 2Department of Pharmaceutical Sciences, University of Valparaiso, Chile (Received 25 April 1990) Abstract--1. In rat thoracic aorta, endothelium removal produced a significant increase of the maximal contraction (Er,~) and of the pD2 value (-log EDs0) induced by norepinephrine, phenylephrine and clonidine, and did not affect the maximal contractile response to 70 mM KCI. 2. Clonidine did not induce a contraction in aorta with intact endothellum, but after endothelium removal, the contractile response was 94.8% of the Em~ produced by norepinephrine in aorta with endothelium. 3. Pre-incubation with methylene blue (10-5 M) and hemoglobin (0.02%), which inhibit EDRF effects, produced the same effects as the mechanical removal of endothelium on the contractile responses to ~-adrenergic agonists. 4. These results suggest that EDRF formation and release is an important factor in the modulation of ~-adrenerglc-induced vasoconstriction.
INTRODUCTION Endothelium-derived relaxing factor (EDRF) is a potent vasodilator and inhibitor of platelet adhesion and aggregation. It is likely that EDRF is nitric oxide (NO), or a labile nitroso species which induces arterial relaxation by binding to the heme group of soluble guanylate cyclase causing activation of the enzyme and increasing cyclic GMP (cGMP) formarion. Elevated intracellular cGMP levels could then cause vascular smooth muscle relaxation and inhibition of platelet function (Rapaport et al., 1983; Palmer et al., 1987; Moncada et al., 1987; Amezcua et al., 1988; Ignarro et al., 1988; Ignarro, 1989). EDRF can be released from endothelial cells after stimulation with a variety of chemically different substances, including amines, peptides, polyunsaturated fatty acids and adenine nucleotides, that interact with selective receptors on the endothelial cell surface associated with the formation and/or release of EDRF. Notably, endothelial cells have an obligatory role in the vascular relaxation induced by acetylcholine (Furchgott et al., 1980), substance P (Zawadski et aL, 1981) and other vasodilators (Cherry et al,, 1982; Spokas et al., 1983; Van de Voorde et al., 1983). Thus, a functional endothelium may have a protective role in the prevention of vasospasm and thrombus formation at sites where platelets are activated. There has been an exponential growth in knowledge on the role of endothelial cells in the modulation of underlying smooth muscle tone in response to pharmacological agents, physiological stimuli and disease. Functional endothelial cells can release both relaxing and contracting factors (Furehgott, 1984). *To whom correspondence should be addressed.
The influence of endothelial cells in the modulation of vasoconstriction induced by ~ - and ~2-adrenergic agonists is not completely understood. Carrier et al. (1984) reported that endothelium removal in rat aorta does not affect significantly the maximal contractile response to norepinephrine, but increases the maximal response to phenylephrine and clonidine. In the present work, the influence of functional endothelial cells on ~-adrenergic-induced vasoconstriction and the effects of known EDRF or NO inhibitors like methylene blue and hemoglobin (Martin et al., 1985) has been studied in rat isolated thoracic aorta. MATERIALS AND METHODS Rats of UCHA strain (University of Chile, Santiago) weighing 200 to 300 g were killed by a blow to the head, and a section of the thoracic aorta was rapidly removed and carefully cleaned of all fat and connective tissue, taking special care to avoid endothelial damage. Rings (5 to 6 mm long) were prepared and mounted on stainless steel hooks in 30 ml tissue baths. The rings were attached to Grass FT-03 force-displacement transducers to record isometric contractions on a 7D Grass polygraph (Grass Instruments Co., Quincy, MA). The baths were filled with 30 ml of a modified Krebs--Henseleitsolution of the followingcomposition (raM): NaC1 122.0, KCI 4.7, CaC12 2.0, MgC12 1.2, KH2PO4 1.2, NaHCO3 15.0, glucose 11.5 and EDTA 0.026. The solution was maintained at 37°C and bubbled with a 95% 02 and 5% CO2 gas mixture. The pH of the gasified solution was 7.4. From each thoracic aorta, two rings were prepared. The endothelial lining of one of the rings was removed by inserting the points of a small forceps and rolling gently the ring over filter paper during 15 sec (Carrier et al., 1984). The effectiveness of this procedure of endothelium removal has been demonstrated by electron microscopy, silver staining and the unresponsiveness to acetylcholine (Furchgott, 1984). The paired rings were allowed to equilibrate in the 137
R. VINET et al.
138
tissue bath for 60 min under an optimal resting tension of 1.5 g. During the equilibration period, the bath solution was changed every 15 min to prevent metabolite accumulation (Altura et aL, 1970). To test the integrity of the smooth muscle and to establish a reference maximal response, the preparations were contracted at least three times with a depolarizing high K + solution of the following composition (mM): NaC1 56.7, KC1 70.0, CaCI2 2.0, MgCI2 1.2, KH2PO 4 1.2, NaHCO 3 15.5, glucose 11.5 and EDTA 0.026 at pH 7.4. When the K +-induced contraction reached a steady maximal response, the rings were repeatedly washed and allowed to re-equilibrate during 20 additional min before testing the agonists. The functional integrity of the endotbelium was assessed at the end of each experiment by recording the relaxation induced by acetylcholine (10-5-10 -4 M) in the tings previously maximally contracted by norepinephrine (10 -7 M). The arterial segments in which the endothelial lining was effectively removed did not relax under the influence of acetylcholine, as opposed to the segments with intact endothelium, in which a relaxation of 70 to 80% was observed. Cumulative dose-response curves were obtained by a stepwise increase in the concentration of agonists; additions were made as soon as a steady response was obtained from the preceding dose. The adrenergic agonists used were norepinephrine (NE; aj- and a2-, unselective), phenylephrine (Ph; at-selective) and clonidine (C; a2-selective). Each pair of rings was exposed to only one agonist. Concentrations are expressed as the log of the final molar concentration of the drug in the bath. In order to study the eventual role of EDRF as a modulator of a-adrenergic-induced contractions, arterial segments were incubated with methylene blue (10 -5 M) for 20 min before the addition of agonists. Methylene blue is a well known inhibitor of EDRF effects, and the results of these experiments were compared to those obtained in the presence of human hemoglobin (0.02%), which also inhibits EDRF (Martin et aL, 1985). The contractile tension developed was expressed as the percentage of the maximal contraction (Emax) obtained by K+-depolarization in each aortic ring. In every dose-
Table 1. Maximal contraction induced by 70 mM KCI solution in rat aortic rings with and without endothelium With endothelium Without endothelium 1466.6 ± 80.7 1429.0 + 95.8 Values are mean + SEM of mg of developed tension/mg of wet tissue weight in 14 experiments.
response curve, the dose causing the 50% of the maximal effect (EDs0) was determined according to the method of Fleming et aL (1972) and was expressed as pD 2 ( - log EDs0). Statistical analysis of the data was carried out using mean values __+SEM. The significance was assessed by Student's "t" test for paired and unpaired data. Differences were considered significant at P < 0.05. All drugs used in this work were obtained from Sigma Chemical (St Louis, MO) and were dissolved in fresh Krebs-Henseleit solution before each experiment. RESULTS
Effects o f K +-depolarization In paired experiments, the effects o f the mechanical removal o f the e n d o t h e l i u m o n the c o n t r a c t i o n induced b y a 70 m M KCI depolarizing solution was studied. Table 1 shows t h a t the m a x i m a l K + - i n d u c e d c o n t r a c t i o n (expressed as m g o f developed tension/mg o f wet tissue weight) was n o t statistically different in aortic rings with or w i t h o u t endothelium. Therefore, e n d o t h e l i u m removal did n o t affect the ability o f the vascular s m o o t h muscle to c o n t r a c t maximally with K+-depolarization. This Em~ was used as a reference for each arterial ring. W e f o u n d t h a t expressing the drug-induced c o n t r a c t i o n as a percentage o f this reference m a x i m a l K + - c o n t r a c t i o n represents a valuable a n d confident criteria for such measurements.
100
100
90-
90.
80-
AvI
70-
~70-
60SO.
_~-
40.~
-9.5 -9.0
•
•
30.
':i,o.f J 0 ~--'~L~,
•
20. 10. ,
,
-8.5 -8.0 -7.5 -7'.0 -6'.5 -6.0 Nor~ii-it~w-ine (log 14)
0q
-9.0 -8:s -e'.o -7'.s -i.o -6.5 -6.0 -s'.s -s.o Ptlmylel~'¢Jne flog H)
Fig. 1. The effect of endothelium removal on the norepinephrine (A) and phenylephrine (B) dose-response curves in rat aortic rings. The developed tension of each cumulative dose is expressed as percentage of the maximal contractile response achieved by a 70 mM KC1 depolarizing solution in each ring. Doses are expressed as the log of the final molar concentration in the bath. Values are mean _+ SEM of 12 paired experiments. *Significant difference. Rings with intact endothelium (@); rings without endothelium (C)).
EDRF and #-adrenergic vasoconstriction
139
Table 2. Effect of endothelium (E) removal on Emax and pD2 values for norepinephrine (NE), phenylephrine (Ph) and clonidine (C) in rat aortic tings Em,x pD2 Drug (n) With E WithoutE With E Without E NE (12) 40.9 _+3.6 98.6 _+1.3j 7.70 _+0.05 8.10 ± 0.07' Ph (12) 44.5 _ 5.0 98.8 -+ 1.51 6.91 -+0.03 7.55 -+0.03' C (8) 0.0 38.8 -+3.3' -6.31 _+0.151 Emx = maximal contraction with agonist x 100/maximal contraction with 70 mM KCI solution. pD2= -log EDs0. ~P < 0.001 between tings with and without endothelium.
endotbelium removal, Ph induced a highly significant increase both in En~x and pD2 (Table 2). As shown in Fig. 2, aortic rings with intact endothelium did not contract in the presence of C. However, the removal of the endothelial lining induced a significant Em~ of 38.8 _+ 3.3% of the maximal K +induced contraction (Table 2).
9O 90-
~-
Effects of EDRF inhibition
10 O_ -8.0
-;.s -7:o -6-.s -6-.o -s:s -s'.0 Clonidim
(.log H)
Fig. 2. The effect of endothelium removal on the clonidine dose-response curve in rat aortic rings. The developed tension of each cumulative dose is expressed as percentage of the maximal contractile response achieved by a 70 mM KCI depolarizing solution in each ring. Doses are expressed as the log of the final molar concentration in the bath. Values are mean _+SEM of 8 paired experiments. Rings with intact endothelium did not contract. *Significant difference. Rings with intact endothelium (@); rings without endothelium (O).
Role of endothelium in the vasoconstrictor response to ct-adrenergic agonists Figure 1 shows the dose-response curve to N E in aortic rings with and without endothelium. Endothelium removal induced a highly significant increase o f Em~ as well as P D 2 values (Table 2). The dose-response curve to Ph was similar in all respects to the N E curve (Fig. 1). In fact, after
Figure 3 illustrates the effect of methylene blue (10 -5 M) on the dose-response curve to N E in aortic rings with and without endothelium. The presence of this E D R F inhibitor produced a significant increase (P < 0.001) of the Em~ recorded in aortic rings with intact endothelium (Table 3). Em~ values of aortic rings with and without endothelium exposed to methylene blue showed no significant differences. The pD2 values in the vessels with intact endothelium exposed to methylene blue increased significantly (P < 0.001, Table 3). Furthermore, methylene blue did not affect significantly the dose-response curve to N E in aortic rings without endothelium (Fig. 3, Table 3). In the presence of methylene blue, the d o s e response curves to Ph and C behaved as the N E curve (Figs 3 and 4). Table 3 shows that Em~xvalues for Ph and C in vessels with intact endothelium increased significantly (P < 0.001) with exposure to methylene blue. In vessels without endothelium, no significant change was observed in the presence of methylene blue. The pD2 value for Ph in vessels with intact endothelium exposed to methylene blue was significantly different (P < 0.02) to the value in control vessels exposed only to Ph. In aortic rings with intact endothelium exposed to methylene blue, C induced a contraction similar to that observed in rings without endothelium (Fig. 4, Table 3). The incubation of the vessels with methylene blue was similar in all respects to the mechanical removal of the endothelium.
Table 3. Effect of methylene blue (MB) on Er~x and pD 2 values for norepinephrine (NE), phenylephtine (Ph) and clonidine (C) in rat aortic rings with and without endothelium (E) E~
Drug (n = 6)
pD2
With E
Without E
With E
Without E
N E control
53.5 _+ 6.7
98.6 ± 1.6'
7.74 ± 0.06
8.06 ± 0.052
NE+MB Ph control Ph+MB
107.7_+3.73 41.3 _+4.3 109.4_+2.13
99.2±1.2 99.4 ± 1.41 104.2_+1.8
8.19±0.043
6.87 _+0.09 7.48_+0.094
7.55±0.03 7.46 ± 0.042 7.60±0.05
C control C+MB
0.0 40.9+2.9 ~
39.7 _+ 4.31 38.7_+3.7
-6.77±0.09
6.21 _+ 0.17 6.63±0.104
Emx and pD2 as in Table 2. 'P < 0.001 between vessels with and without E. 2p < 0.01 between vessels with and without E.' 3p < 0.001 between control and MB-treated vessels. 4p < 0.05 between control and MB-treated vessels.
R. VINETet al.
140 A
120
:::T
,,o,,
"9.5 "gO -8L$ -8'.0 "7'.$ -7LO -iS -6.0
I
'2-9.0 "8.5 -8.0 -7.5 -7iO "6L5 -gO "5T5 -5.0
Noropimptrim (log H)
Pl~onyl(~phr'im (log M)
Fig. 3. The effect of methylene blue (MB) on the norepinephrine (A) and phenylephrine (B) dose-response curves in rat aortic rings with and without endothelium. MB (10 -5 M) was added to the medium 20 rain before the addition of the agonists. The developed tension of each cumulative dose is expressed as percentage of the maximal contractile response achieved by a 70 mM KC1 depolarizing solution in each ring. Doses are expressed as the log of the final molar concentration in the bath. Values are mean + SEM of 6 paired experiments. Curves of rings without endothelium + MB are not significantly different from control curve without endothelium and without MB. Curves of rings with endothelium + MB are significantly different from control curve with endothelium and without MB. Control with intact endothelium (0); control without endothelium (C)); MB with intact endothelium (m); MB without endothelium (F-I). 100
The incubation of aortic tings with and without endotbelium with human hemoglobin (0.02%) induced in the tings with intact endotbelium a significant enhancement of the Em~x induced by NE. These results were similar to those obtained using methylene blue.
90 80
70,
50. DISCUSSION
10.
0,. -8.0
-7.5
-7.0 -6.5 -6.0 Clonidine (log if)
-5.5
-5.0
Fig. 4. The effect of methylene blue (MB) on the clonidine dose-response curve in rat aortic rings with and without endothelium. MB (10-SM) was added to the medium 20 min before the addition of clonidine. The developed tension of each cumulative dose is expressed as percentage of the maximal contractile response to a 70 mM KC1 depolarizing solution in each ring. Doses are expressed as the log of the final molar concentration in the bath. Values are mean + SEM of 6 paired experiments. Curves of rings without endothelium + MB are not significantly different from control curve without endothelium and without MB. Curves of rings with endothelium + MB are significantly different from control curve with endothelium and without MB. Control with intact endothelium (0); control without endothelium ((3); MB with intact endothelium (m); MB without endothelium (Fl).
The results obtained in the present work clearly indicate the importance o f a functional endothelium in the modulation of ~-adrenerglc-induced vasoconstriction. Endothelium removal increased the pD2 values and the Em~ induced by all the ~-adrenergic agonists tested. The effect of endotbelium removal appears to be more important for C, because this ~2-selective agonist induced a contraction only in aortic rings without endothelium (Fig. 2, Table 2). Our results do not completely agree with those reported by Cartier et al. (1984) first, because they did not find a significant difference in the Em~ induced by N E in vessels with or without endothelium, and second, because they reported that C was able to contract aortic rings with intact endothelium. These discrepancies might be due to the fact that Carrier et al. (1984) expressed the contractile responses as the percentage of the Era,x for each agonist, while our results are expressed as the percentage of the Em,x obtained by K÷-depolarization. It is important to point out that the maximal response to K÷-depolar ization, used as a reference expressing the maximal ability of each vessel to contract, was usually
EDRF and ~-adrenergic vasoconstriction obtained in the third consecutive exposure to the 70 mM KC1 solution. Carrier et al. (1984) used as a reference the Emaxobserved with a single exposure to a maximal concentration of the agonist being studied. Our experience indicates that a single exposure of the vessel to KCI or to a drug is not enough to ensure that the system achieves the maximal response. In fact, when expressed as mg of developed tension/mg of wet tissue weight, the contractions observed in our experiments were up to 326% higher than the contractions reported by Carrier et al. (1984). Furthermore, the Emax to drugs in our experiments was always higher. Apparently, the endothelial lining of our vessels was preserved in a better functional condition, a fact confirmed by the lack of response we observed in intact aortas exposed to clonidine. There has been conflictive reports, showing that a selective ~2adrenergic agonist like clonidine can induce contractile responses going from 13% to about 64% of the Ema~ produced by norepinephrine (Medgett et al., 1978; Weiss et al., 1983; Carrier et al., 1984). These discrepancies might reflect different degrees of endothelial damage, since in our work, aortic rings with intact endothelium did not contract in the presence of high doses of clonidine (10 -s M). However, after the physical removal of the endothelial lining the maximal clonidine-induced contraction was 94.8% of the NE contraction in aorta with endothelium (Table 2), indicating that ~2-adrenoceptors can produce a significant vasoconstriction when the endothelium is not present. The presence of an intact endothelium appears to exert an inhibitory effect upon the ability of the vessels to contract in response to ~-adrenergic stimulation. The mechanism of the modulatory effect of endothelium on ~-adrenergic-induced contractions is unknown, but EDRF seems to be one of the factors involved in this process. It has been postulated that ~-adrenergic agonists could activate an endothelial receptor-mediated releasing of EDRF that could counteract the agonist-induced vasoconstriction; the removal of the endothelial layer would eliminate this mechanism and enhance the constrictor effect. In addition, the endothelium could be acting as a diffusion barrier and as an extraneuronal re-uptake site; removal of the endothelial layer would allow an increase in the effective concentration of the adrenergic agonist at the receptor sites on the smooth muscle cell membrane, producing a greater receptor occupancy and enhancing the resulting contraction. Methylene blue (10 -5 M) and human hemoglobin (0.02%) inhibit EDRF-mediated relaxation, and also the relaxation induced by vasodilator substances acting through the releasing or formation of nitric oxide (NO). In fact, methylene blue is an inhibitor of guanylate cyclase, while hemoglobin binds to NO to produce a nitrosil-hemoprotein complex that is unable to enter the cell membrane (Furchgott, 1984; Ignarro et al., 1985; Martin et al., 1985; Vanhoutte et al., 1986; Moncada et al., 1987; Ignarro, 1989). The incubation of aortic rings with methylene blue (Figs 3 and 4, Table 3) and with hemoglobin produced an effect comparable to the mechanical removal of endothelium and can be considered as a chemical removal of this lining. Since methylene blue inhibits soluble guanylate cyclase and hemoglobin seems to inhibit
141
EDRF directly, this endothelial factor must be considered as one of the modulating factors in the action of a-adrenergic-induced contractions in rat thoracic aorta. Acknowledgements--This work was supported by Project 1174-89, Fondecyt, and Project B 2680-8935, DTI, Univer-
sity of Chile. REFERENCES
Altura B. M. and Altura B. T. (1970) Differential effects of substrate depletion on drug-induced contractions of rabbit aorta. Am. J. Physiol. 219, 1698-1705. Amezcua J, L., Dusting G. J., Palmer R, M. J. and Moncada S. (1988) Acetylcholine induces vasodilatation in the rabbit isolated heart through the release of nitric oxide, the endogenous nitrovasodilator. Br. J. Pharmac. 95, 830-834. Carrier G. O. and White R. E. (1984) Enhancement of alpha-I and alpha-2 adrenergic agonist-induced vasoconstriction by removal of endothelium in rat aorta. J. Pharmac. Exp. Ther. 232, 682-687. Cherry P. D., Furchgott R. F., Zawadski J. V. and Jothianandan D. (1982) Role of endothelial cells in relaxation of isolated arteries by bradykinin. Proc. NatL Acad. Sci. USA 79, 2106-2110. Fleming W. W., Westfall D. P., de la Lande I. S., Jellet L. B. (1972) Log-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues. J. Pharmac. Exp. Ther. lgl, 339-345. Furchgott R. F. (1984) The role of endothelium in the response of vascular smooth muscle to drugs. Ann. Rev. Pharmac. Toxicol. 24, 175-197. Furchgott R. F. and Zawadski J. V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 372376. Ignarro L. J. (1989) Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ. Res. 65, 1-21. Ignarro L. J. and Kadowitz P. J. (1985) The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation. Ann. Rev. Pharmac. Toxicol. 25, 171-191. Martin W., Villani G. M., Jothianandan D. and Furchgott R. F. (1985) Selectiveblockade of endothelium-dependent and giyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta. J. Pharrnac. Exp. Ther. 232, 708-716. Medgett J. C., McMulloch M. W. and Rand M. J. (1978) Partial agonist action of clonidine on prejunctional and postjunctional alpha adrenoceptors. Naunyn-Schmiedeberg's Arch. Pharmac. 304, 215-221. Moncada S., Herman A. S. and Vanhoutte P. M. (1987) Endothelium-derived relaxing factor is identified as nitric oxide. Trends Pharmac. Sci. 8, 365-368. Palmer R. M. J., Ferrige A. G. and Moncada S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524--526. Rapaport R. M. and Murad F. (1984) Mechanisms of adenosine triphosphate-, thrombin- and trypsin-induced relaxation of rat thoracic aorta. Circ. Res. 52, 352357. Spokas E. G., Folco G., Quilley J., Chander P. and McGiff J. C. (1983) Endothelial mechanism in the vascular action of hydralazine. Hypertension 5, 107-111. Van de Voorde J. and Leusen I. (1983) Role of the endothelium in the vasodilator response of rat thoracic aorta to histamine. Eur. J. Pharmac. 87, 113-120.
142
R. VINET et al.
Vanhoutte P. M., Rubanyi G. M., Miller V. M. and Houston D. S. (1986) Modulation of vascular smooth muscle contraction by the endothelium. Ann. Rev. Physiol. 48, 307-320. Weiss R. J., Webb C. R. and Smith C. B. (1983) Alpha-2 adrenoceptors on arterial smooth muscle: selective label-
ing by [3H] clonidine. J. Pharmac, Exp. Ther. 225, 599~505. Zawadski J. V., Furchgott R. F. and Cherry P. (1981) The obligatory role of endothelial ceils in the relaxation of arterial smooth muscle by substance P. Fed. Proc. 40, 689.
0306-3623/91 $3.00+ 0.00 Copyright © 1991PergamonPresspie
MODULATION OF ~ - A D R E N E R G I C - I N D U C E D CONTRACTIONS BY ENDOTHELIUM-DERIVED RELAXING FACTOR IN RAT AORTA P,. VINET2, C. BRIEVAl, G. PINARDI1and M. PENNA1. ~Department of Pharmacology, Faculty of Medicine, University of Chile, P.O. Box 70.000, Santiago 7, Chile and 2Department of Pharmaceutical Sciences, University of Valparaiso, Chile (Received 25 April 1990) Abstract--1. In rat thoracic aorta, endothelium removal produced a significant increase of the maximal contraction (Er,~) and of the pD2 value (-log EDs0) induced by norepinephrine, phenylephrine and clonidine, and did not affect the maximal contractile response to 70 mM KCI. 2. Clonidine did not induce a contraction in aorta with intact endothellum, but after endothelium removal, the contractile response was 94.8% of the Em~ produced by norepinephrine in aorta with endothelium. 3. Pre-incubation with methylene blue (10-5 M) and hemoglobin (0.02%), which inhibit EDRF effects, produced the same effects as the mechanical removal of endothelium on the contractile responses to ~-adrenergic agonists. 4. These results suggest that EDRF formation and release is an important factor in the modulation of ~-adrenerglc-induced vasoconstriction.
INTRODUCTION Endothelium-derived relaxing factor (EDRF) is a potent vasodilator and inhibitor of platelet adhesion and aggregation. It is likely that EDRF is nitric oxide (NO), or a labile nitroso species which induces arterial relaxation by binding to the heme group of soluble guanylate cyclase causing activation of the enzyme and increasing cyclic GMP (cGMP) formarion. Elevated intracellular cGMP levels could then cause vascular smooth muscle relaxation and inhibition of platelet function (Rapaport et al., 1983; Palmer et al., 1987; Moncada et al., 1987; Amezcua et al., 1988; Ignarro et al., 1988; Ignarro, 1989). EDRF can be released from endothelial cells after stimulation with a variety of chemically different substances, including amines, peptides, polyunsaturated fatty acids and adenine nucleotides, that interact with selective receptors on the endothelial cell surface associated with the formation and/or release of EDRF. Notably, endothelial cells have an obligatory role in the vascular relaxation induced by acetylcholine (Furchgott et al., 1980), substance P (Zawadski et aL, 1981) and other vasodilators (Cherry et al,, 1982; Spokas et al., 1983; Van de Voorde et al., 1983). Thus, a functional endothelium may have a protective role in the prevention of vasospasm and thrombus formation at sites where platelets are activated. There has been an exponential growth in knowledge on the role of endothelial cells in the modulation of underlying smooth muscle tone in response to pharmacological agents, physiological stimuli and disease. Functional endothelial cells can release both relaxing and contracting factors (Furehgott, 1984). *To whom correspondence should be addressed.
The influence of endothelial cells in the modulation of vasoconstriction induced by ~ - and ~2-adrenergic agonists is not completely understood. Carrier et al. (1984) reported that endothelium removal in rat aorta does not affect significantly the maximal contractile response to norepinephrine, but increases the maximal response to phenylephrine and clonidine. In the present work, the influence of functional endothelial cells on ~-adrenergic-induced vasoconstriction and the effects of known EDRF or NO inhibitors like methylene blue and hemoglobin (Martin et al., 1985) has been studied in rat isolated thoracic aorta. MATERIALS AND METHODS Rats of UCHA strain (University of Chile, Santiago) weighing 200 to 300 g were killed by a blow to the head, and a section of the thoracic aorta was rapidly removed and carefully cleaned of all fat and connective tissue, taking special care to avoid endothelial damage. Rings (5 to 6 mm long) were prepared and mounted on stainless steel hooks in 30 ml tissue baths. The rings were attached to Grass FT-03 force-displacement transducers to record isometric contractions on a 7D Grass polygraph (Grass Instruments Co., Quincy, MA). The baths were filled with 30 ml of a modified Krebs--Henseleitsolution of the followingcomposition (raM): NaC1 122.0, KCI 4.7, CaC12 2.0, MgC12 1.2, KH2PO4 1.2, NaHCO3 15.0, glucose 11.5 and EDTA 0.026. The solution was maintained at 37°C and bubbled with a 95% 02 and 5% CO2 gas mixture. The pH of the gasified solution was 7.4. From each thoracic aorta, two rings were prepared. The endothelial lining of one of the rings was removed by inserting the points of a small forceps and rolling gently the ring over filter paper during 15 sec (Carrier et al., 1984). The effectiveness of this procedure of endothelium removal has been demonstrated by electron microscopy, silver staining and the unresponsiveness to acetylcholine (Furchgott, 1984). The paired rings were allowed to equilibrate in the 137
R. VINET et al.
138
tissue bath for 60 min under an optimal resting tension of 1.5 g. During the equilibration period, the bath solution was changed every 15 min to prevent metabolite accumulation (Altura et aL, 1970). To test the integrity of the smooth muscle and to establish a reference maximal response, the preparations were contracted at least three times with a depolarizing high K + solution of the following composition (mM): NaC1 56.7, KC1 70.0, CaCI2 2.0, MgCI2 1.2, KH2PO 4 1.2, NaHCO 3 15.5, glucose 11.5 and EDTA 0.026 at pH 7.4. When the K +-induced contraction reached a steady maximal response, the rings were repeatedly washed and allowed to re-equilibrate during 20 additional min before testing the agonists. The functional integrity of the endotbelium was assessed at the end of each experiment by recording the relaxation induced by acetylcholine (10-5-10 -4 M) in the tings previously maximally contracted by norepinephrine (10 -7 M). The arterial segments in which the endothelial lining was effectively removed did not relax under the influence of acetylcholine, as opposed to the segments with intact endothelium, in which a relaxation of 70 to 80% was observed. Cumulative dose-response curves were obtained by a stepwise increase in the concentration of agonists; additions were made as soon as a steady response was obtained from the preceding dose. The adrenergic agonists used were norepinephrine (NE; aj- and a2-, unselective), phenylephrine (Ph; at-selective) and clonidine (C; a2-selective). Each pair of rings was exposed to only one agonist. Concentrations are expressed as the log of the final molar concentration of the drug in the bath. In order to study the eventual role of EDRF as a modulator of a-adrenergic-induced contractions, arterial segments were incubated with methylene blue (10 -5 M) for 20 min before the addition of agonists. Methylene blue is a well known inhibitor of EDRF effects, and the results of these experiments were compared to those obtained in the presence of human hemoglobin (0.02%), which also inhibits EDRF (Martin et aL, 1985). The contractile tension developed was expressed as the percentage of the maximal contraction (Emax) obtained by K+-depolarization in each aortic ring. In every dose-
Table 1. Maximal contraction induced by 70 mM KCI solution in rat aortic rings with and without endothelium With endothelium Without endothelium 1466.6 ± 80.7 1429.0 + 95.8 Values are mean + SEM of mg of developed tension/mg of wet tissue weight in 14 experiments.
response curve, the dose causing the 50% of the maximal effect (EDs0) was determined according to the method of Fleming et aL (1972) and was expressed as pD 2 ( - log EDs0). Statistical analysis of the data was carried out using mean values __+SEM. The significance was assessed by Student's "t" test for paired and unpaired data. Differences were considered significant at P < 0.05. All drugs used in this work were obtained from Sigma Chemical (St Louis, MO) and were dissolved in fresh Krebs-Henseleit solution before each experiment. RESULTS
Effects o f K +-depolarization In paired experiments, the effects o f the mechanical removal o f the e n d o t h e l i u m o n the c o n t r a c t i o n induced b y a 70 m M KCI depolarizing solution was studied. Table 1 shows t h a t the m a x i m a l K + - i n d u c e d c o n t r a c t i o n (expressed as m g o f developed tension/mg o f wet tissue weight) was n o t statistically different in aortic rings with or w i t h o u t endothelium. Therefore, e n d o t h e l i u m removal did n o t affect the ability o f the vascular s m o o t h muscle to c o n t r a c t maximally with K+-depolarization. This Em~ was used as a reference for each arterial ring. W e f o u n d t h a t expressing the drug-induced c o n t r a c t i o n as a percentage o f this reference m a x i m a l K + - c o n t r a c t i o n represents a valuable a n d confident criteria for such measurements.
100
100
90-
90.
80-
AvI
70-
~70-
60SO.
_~-
40.~
-9.5 -9.0
•
•
30.
':i,o.f J 0 ~--'~L~,
•
20. 10. ,
,
-8.5 -8.0 -7.5 -7'.0 -6'.5 -6.0 Nor~ii-it~w-ine (log 14)
0q
-9.0 -8:s -e'.o -7'.s -i.o -6.5 -6.0 -s'.s -s.o Ptlmylel~'¢Jne flog H)
Fig. 1. The effect of endothelium removal on the norepinephrine (A) and phenylephrine (B) dose-response curves in rat aortic rings. The developed tension of each cumulative dose is expressed as percentage of the maximal contractile response achieved by a 70 mM KC1 depolarizing solution in each ring. Doses are expressed as the log of the final molar concentration in the bath. Values are mean _+ SEM of 12 paired experiments. *Significant difference. Rings with intact endothelium (@); rings without endothelium (C)).
EDRF and #-adrenergic vasoconstriction
139
Table 2. Effect of endothelium (E) removal on Emax and pD2 values for norepinephrine (NE), phenylephrine (Ph) and clonidine (C) in rat aortic tings Em,x pD2 Drug (n) With E WithoutE With E Without E NE (12) 40.9 _+3.6 98.6 _+1.3j 7.70 _+0.05 8.10 ± 0.07' Ph (12) 44.5 _ 5.0 98.8 -+ 1.51 6.91 -+0.03 7.55 -+0.03' C (8) 0.0 38.8 -+3.3' -6.31 _+0.151 Emx = maximal contraction with agonist x 100/maximal contraction with 70 mM KCI solution. pD2= -log EDs0. ~P < 0.001 between tings with and without endothelium.
endotbelium removal, Ph induced a highly significant increase both in En~x and pD2 (Table 2). As shown in Fig. 2, aortic rings with intact endothelium did not contract in the presence of C. However, the removal of the endothelial lining induced a significant Em~ of 38.8 _+ 3.3% of the maximal K +induced contraction (Table 2).
9O 90-
~-
Effects of EDRF inhibition
10 O_ -8.0
-;.s -7:o -6-.s -6-.o -s:s -s'.0 Clonidim
(.log H)
Fig. 2. The effect of endothelium removal on the clonidine dose-response curve in rat aortic rings. The developed tension of each cumulative dose is expressed as percentage of the maximal contractile response achieved by a 70 mM KCI depolarizing solution in each ring. Doses are expressed as the log of the final molar concentration in the bath. Values are mean _+SEM of 8 paired experiments. Rings with intact endothelium did not contract. *Significant difference. Rings with intact endothelium (@); rings without endothelium (O).
Role of endothelium in the vasoconstrictor response to ct-adrenergic agonists Figure 1 shows the dose-response curve to N E in aortic rings with and without endothelium. Endothelium removal induced a highly significant increase o f Em~ as well as P D 2 values (Table 2). The dose-response curve to Ph was similar in all respects to the N E curve (Fig. 1). In fact, after
Figure 3 illustrates the effect of methylene blue (10 -5 M) on the dose-response curve to N E in aortic rings with and without endothelium. The presence of this E D R F inhibitor produced a significant increase (P < 0.001) of the Em~ recorded in aortic rings with intact endothelium (Table 3). Em~ values of aortic rings with and without endothelium exposed to methylene blue showed no significant differences. The pD2 values in the vessels with intact endothelium exposed to methylene blue increased significantly (P < 0.001, Table 3). Furthermore, methylene blue did not affect significantly the dose-response curve to N E in aortic rings without endothelium (Fig. 3, Table 3). In the presence of methylene blue, the d o s e response curves to Ph and C behaved as the N E curve (Figs 3 and 4). Table 3 shows that Em~xvalues for Ph and C in vessels with intact endothelium increased significantly (P < 0.001) with exposure to methylene blue. In vessels without endothelium, no significant change was observed in the presence of methylene blue. The pD2 value for Ph in vessels with intact endothelium exposed to methylene blue was significantly different (P < 0.02) to the value in control vessels exposed only to Ph. In aortic rings with intact endothelium exposed to methylene blue, C induced a contraction similar to that observed in rings without endothelium (Fig. 4, Table 3). The incubation of the vessels with methylene blue was similar in all respects to the mechanical removal of the endothelium.
Table 3. Effect of methylene blue (MB) on Er~x and pD 2 values for norepinephrine (NE), phenylephtine (Ph) and clonidine (C) in rat aortic rings with and without endothelium (E) E~
Drug (n = 6)
pD2
With E
Without E
With E
Without E
N E control
53.5 _+ 6.7
98.6 ± 1.6'
7.74 ± 0.06
8.06 ± 0.052
NE+MB Ph control Ph+MB
107.7_+3.73 41.3 _+4.3 109.4_+2.13
99.2±1.2 99.4 ± 1.41 104.2_+1.8
8.19±0.043
6.87 _+0.09 7.48_+0.094
7.55±0.03 7.46 ± 0.042 7.60±0.05
C control C+MB
0.0 40.9+2.9 ~
39.7 _+ 4.31 38.7_+3.7
-6.77±0.09
6.21 _+ 0.17 6.63±0.104
Emx and pD2 as in Table 2. 'P < 0.001 between vessels with and without E. 2p < 0.01 between vessels with and without E.' 3p < 0.001 between control and MB-treated vessels. 4p < 0.05 between control and MB-treated vessels.
R. VINETet al.
140 A
120
:::T
,,o,,
"9.5 "gO -8L$ -8'.0 "7'.$ -7LO -iS -6.0
I
'2-9.0 "8.5 -8.0 -7.5 -7iO "6L5 -gO "5T5 -5.0
Noropimptrim (log H)
Pl~onyl(~phr'im (log M)
Fig. 3. The effect of methylene blue (MB) on the norepinephrine (A) and phenylephrine (B) dose-response curves in rat aortic rings with and without endothelium. MB (10 -5 M) was added to the medium 20 rain before the addition of the agonists. The developed tension of each cumulative dose is expressed as percentage of the maximal contractile response achieved by a 70 mM KC1 depolarizing solution in each ring. Doses are expressed as the log of the final molar concentration in the bath. Values are mean + SEM of 6 paired experiments. Curves of rings without endothelium + MB are not significantly different from control curve without endothelium and without MB. Curves of rings with endothelium + MB are significantly different from control curve with endothelium and without MB. Control with intact endothelium (0); control without endothelium (C)); MB with intact endothelium (m); MB without endothelium (F-I). 100
The incubation of aortic tings with and without endotbelium with human hemoglobin (0.02%) induced in the tings with intact endotbelium a significant enhancement of the Em~x induced by NE. These results were similar to those obtained using methylene blue.
90 80
70,
50. DISCUSSION
10.
0,. -8.0
-7.5
-7.0 -6.5 -6.0 Clonidine (log if)
-5.5
-5.0
Fig. 4. The effect of methylene blue (MB) on the clonidine dose-response curve in rat aortic rings with and without endothelium. MB (10-SM) was added to the medium 20 min before the addition of clonidine. The developed tension of each cumulative dose is expressed as percentage of the maximal contractile response to a 70 mM KC1 depolarizing solution in each ring. Doses are expressed as the log of the final molar concentration in the bath. Values are mean + SEM of 6 paired experiments. Curves of rings without endothelium + MB are not significantly different from control curve without endothelium and without MB. Curves of rings with endothelium + MB are significantly different from control curve with endothelium and without MB. Control with intact endothelium (0); control without endothelium ((3); MB with intact endothelium (m); MB without endothelium (Fl).
The results obtained in the present work clearly indicate the importance o f a functional endothelium in the modulation of ~-adrenerglc-induced vasoconstriction. Endothelium removal increased the pD2 values and the Em~ induced by all the ~-adrenergic agonists tested. The effect of endotbelium removal appears to be more important for C, because this ~2-selective agonist induced a contraction only in aortic rings without endothelium (Fig. 2, Table 2). Our results do not completely agree with those reported by Cartier et al. (1984) first, because they did not find a significant difference in the Em~ induced by N E in vessels with or without endothelium, and second, because they reported that C was able to contract aortic rings with intact endothelium. These discrepancies might be due to the fact that Carrier et al. (1984) expressed the contractile responses as the percentage of the Era,x for each agonist, while our results are expressed as the percentage of the Em,x obtained by K÷-depolarization. It is important to point out that the maximal response to K÷-depolar ization, used as a reference expressing the maximal ability of each vessel to contract, was usually
EDRF and ~-adrenergic vasoconstriction obtained in the third consecutive exposure to the 70 mM KC1 solution. Carrier et al. (1984) used as a reference the Emaxobserved with a single exposure to a maximal concentration of the agonist being studied. Our experience indicates that a single exposure of the vessel to KCI or to a drug is not enough to ensure that the system achieves the maximal response. In fact, when expressed as mg of developed tension/mg of wet tissue weight, the contractions observed in our experiments were up to 326% higher than the contractions reported by Carrier et al. (1984). Furthermore, the Emax to drugs in our experiments was always higher. Apparently, the endothelial lining of our vessels was preserved in a better functional condition, a fact confirmed by the lack of response we observed in intact aortas exposed to clonidine. There has been conflictive reports, showing that a selective ~2adrenergic agonist like clonidine can induce contractile responses going from 13% to about 64% of the Ema~ produced by norepinephrine (Medgett et al., 1978; Weiss et al., 1983; Carrier et al., 1984). These discrepancies might reflect different degrees of endothelial damage, since in our work, aortic rings with intact endothelium did not contract in the presence of high doses of clonidine (10 -s M). However, after the physical removal of the endothelial lining the maximal clonidine-induced contraction was 94.8% of the NE contraction in aorta with endothelium (Table 2), indicating that ~2-adrenoceptors can produce a significant vasoconstriction when the endothelium is not present. The presence of an intact endothelium appears to exert an inhibitory effect upon the ability of the vessels to contract in response to ~-adrenergic stimulation. The mechanism of the modulatory effect of endothelium on ~-adrenergic-induced contractions is unknown, but EDRF seems to be one of the factors involved in this process. It has been postulated that ~-adrenergic agonists could activate an endothelial receptor-mediated releasing of EDRF that could counteract the agonist-induced vasoconstriction; the removal of the endothelial layer would eliminate this mechanism and enhance the constrictor effect. In addition, the endothelium could be acting as a diffusion barrier and as an extraneuronal re-uptake site; removal of the endothelial layer would allow an increase in the effective concentration of the adrenergic agonist at the receptor sites on the smooth muscle cell membrane, producing a greater receptor occupancy and enhancing the resulting contraction. Methylene blue (10 -5 M) and human hemoglobin (0.02%) inhibit EDRF-mediated relaxation, and also the relaxation induced by vasodilator substances acting through the releasing or formation of nitric oxide (NO). In fact, methylene blue is an inhibitor of guanylate cyclase, while hemoglobin binds to NO to produce a nitrosil-hemoprotein complex that is unable to enter the cell membrane (Furchgott, 1984; Ignarro et al., 1985; Martin et al., 1985; Vanhoutte et al., 1986; Moncada et al., 1987; Ignarro, 1989). The incubation of aortic rings with methylene blue (Figs 3 and 4, Table 3) and with hemoglobin produced an effect comparable to the mechanical removal of endothelium and can be considered as a chemical removal of this lining. Since methylene blue inhibits soluble guanylate cyclase and hemoglobin seems to inhibit
141
EDRF directly, this endothelial factor must be considered as one of the modulating factors in the action of a-adrenergic-induced contractions in rat thoracic aorta. Acknowledgements--This work was supported by Project 1174-89, Fondecyt, and Project B 2680-8935, DTI, Univer-
sity of Chile. REFERENCES
Altura B. M. and Altura B. T. (1970) Differential effects of substrate depletion on drug-induced contractions of rabbit aorta. Am. J. Physiol. 219, 1698-1705. Amezcua J, L., Dusting G. J., Palmer R, M. J. and Moncada S. (1988) Acetylcholine induces vasodilatation in the rabbit isolated heart through the release of nitric oxide, the endogenous nitrovasodilator. Br. J. Pharmac. 95, 830-834. Carrier G. O. and White R. E. (1984) Enhancement of alpha-I and alpha-2 adrenergic agonist-induced vasoconstriction by removal of endothelium in rat aorta. J. Pharmac. Exp. Ther. 232, 682-687. Cherry P. D., Furchgott R. F., Zawadski J. V. and Jothianandan D. (1982) Role of endothelial cells in relaxation of isolated arteries by bradykinin. Proc. NatL Acad. Sci. USA 79, 2106-2110. Fleming W. W., Westfall D. P., de la Lande I. S., Jellet L. B. (1972) Log-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues. J. Pharmac. Exp. Ther. lgl, 339-345. Furchgott R. F. (1984) The role of endothelium in the response of vascular smooth muscle to drugs. Ann. Rev. Pharmac. Toxicol. 24, 175-197. Furchgott R. F. and Zawadski J. V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 372376. Ignarro L. J. (1989) Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ. Res. 65, 1-21. Ignarro L. J. and Kadowitz P. J. (1985) The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation. Ann. Rev. Pharmac. Toxicol. 25, 171-191. Martin W., Villani G. M., Jothianandan D. and Furchgott R. F. (1985) Selectiveblockade of endothelium-dependent and giyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta. J. Pharrnac. Exp. Ther. 232, 708-716. Medgett J. C., McMulloch M. W. and Rand M. J. (1978) Partial agonist action of clonidine on prejunctional and postjunctional alpha adrenoceptors. Naunyn-Schmiedeberg's Arch. Pharmac. 304, 215-221. Moncada S., Herman A. S. and Vanhoutte P. M. (1987) Endothelium-derived relaxing factor is identified as nitric oxide. Trends Pharmac. Sci. 8, 365-368. Palmer R. M. J., Ferrige A. G. and Moncada S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524--526. Rapaport R. M. and Murad F. (1984) Mechanisms of adenosine triphosphate-, thrombin- and trypsin-induced relaxation of rat thoracic aorta. Circ. Res. 52, 352357. Spokas E. G., Folco G., Quilley J., Chander P. and McGiff J. C. (1983) Endothelial mechanism in the vascular action of hydralazine. Hypertension 5, 107-111. Van de Voorde J. and Leusen I. (1983) Role of the endothelium in the vasodilator response of rat thoracic aorta to histamine. Eur. J. Pharmac. 87, 113-120.
142
R. VINET et al.
Vanhoutte P. M., Rubanyi G. M., Miller V. M. and Houston D. S. (1986) Modulation of vascular smooth muscle contraction by the endothelium. Ann. Rev. Physiol. 48, 307-320. Weiss R. J., Webb C. R. and Smith C. B. (1983) Alpha-2 adrenoceptors on arterial smooth muscle: selective label-
ing by [3H] clonidine. J. Pharmac, Exp. Ther. 225, 599~505. Zawadski J. V., Furchgott R. F. and Cherry P. (1981) The obligatory role of endothelial ceils in the relaxation of arterial smooth muscle by substance P. Fed. Proc. 40, 689.
