- Email: [email protected]
Arterial endothelium-derived relaxing factor (AEDRF) does not suppress papillary muscle or portal vein contractions
European Journal of Pharmacology, 142 (1987) 471-474
471
Elsevier EJP 20014 Short communication
Arterial endothelium-derived relaxing factor (AEDRF) does not suppress papillary muscle or portal vein contractions Y u r i P. V e d e r n i k o v *, A n a t o l i M. Vihcrt a n d H a r a l d L c i s n e r a All-Union Cardiology Research Centre, U.S.S.R. Academy of Medical Sciences, 3 a Cherepkovskaja st. 15 a, Moscow 121552, U.S.S.R. and 1 Institute o/ Pathophysiology, Medical Academy Erfurt, G.D.R.
Received 6 July 1987, accepted 1 September 1987
10 - 6 M EDRF released from perfused arterial donor segments in a superfusion bioassay system, either spontaneously or stimulated by acetylcholine, did not inhibit the contractility of electrically stimulated right ventricle papillary muscle of pig, rabbit and rat. EDRF also did not inhibit spontaneously contracting rat portal vein and thus relaxed precontracted arterial rings denuded of endothelium. The results suggest that EDRF released by arterial endothelial cells is a local modulator of arterial smooth muscle and is not important for extraarterial tissue regulation.
Endothelial relaxing factor; Smooth muscle; Papillary muscle
1. Introduction Furchgott and Zawadski (1980) discovered a new substance by which endothelium could modulate vascular smooth muscle. Many reports on the involvement of endothelium-derived relaxing factor (EDRF) in endogenous and pharmacological agent actions have appeared since 1980 (for reviews see Furchgott, 1983; 1984; Busse et al., 1985; Vanhoutte, 1986; Vanhoutte et al., 1986). Busse et al. (1985) recently described the abluminal release of E D R F . This suggested that E D R F was a modulator of tissues adjacent to the arterial wall. We could not confirm the abluminal release of E D R F in arterial preparations of different species in our superfusion bioassay experiments (Vedernikov et al., 1987). Such a discrepancy from the results of Busse et al. (1985) could be due to differences in bioassay systems and in oxygen * To whom all correspondence should be addressed: AllUnion Cardiology Research Centre, U.S.S.R. Academy of Medical Sciences, 3d Cherepkovskaja st. 15a, Moscow 121552, U.S.S.R.
tension of perfusion solutions. In the present study we used a bioassay system similar to that described by Vedernikov et al. (1987) for the study of the direct effect of arterial E D R F ( A E D R F ) on papillary muscle of various species and on rat portal vein.
2. Materials and methods The animals (pig, rabbit and rat) were exsanguinated through the carotid arteries. The hearts were perfused with cardioplegic solution (St. Thomas Hospital), removed and placed in the same medium. Papillary muscle was excised from the right ventricle, ligated from both sides and placed under the superfusion line between two parallel platinum electrodes. The vessels to be studied were removed, trimmed of excess fatty and connective tissue in modified physiological salt solution (PSS) at room temperature. The side-branches of donor vessels (4 cm in length) were carefully ligated. The endothelium was removed from arterial recipient ring segments (5
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
472
Hxec°ht~~ ange 37"C
III
~
oter cket
Corbogen
Ill
Donor vessel
Position
I 23 4
Recipient tissues
Fig. 1. Superfusion system for bioassay of EDRF release. Bioassay artery ring with endothelium removed and papillary muscle/portal vein are equilibrated under constant superfusion with PSS in position 1 and 4 respectively. Moving recipient preparations under donor vessel, position 3, allow the study of A E D R F effects after its spontaneous or stimulated release. A E D R F activator (ACH) is infused above the heat exchanger. RP, roller pump; FT, isometric force transducer; PSS, modified physiological salt solution.
femoral artery ring (n = 11); rat aorta-papillary muscle-aorta ring ( n = 9); pig femoral arterypapillary muscle-coronary artery ring (n = 2); r a t / rabbit aorta-rabbit femoral artery ring-rat portal vein (n = 2). After an equilibration period (60-90 min) with PSS superfusion and stabilization of the passive tension (6 g for coronary ring, 2 g for femoral artery ring, 0.5 g for papillary muscles and portal vein) the preparations were superfused with PSS containing indomethacin, 10 -5 M (Sigma, USA), phenylephrine, 10 .6 M (Serva, FRG) or prostaglandin F2,, 2 x 10 -6 M (Serva, FRG) for pig tissues. The completeness of endothelium removal in recipient rings was confirmed if acetylcholine (ACH, Sigma, USA), 10 - 6 M final concentration, applied at the influsion site with a perfusor-V1 (B. Broun, FRG) did not produce relaxation. Recipient tissues were moved in turn to position 3 to check the influence of spontaneously released EDRF. ACH was then infused through perfusion line 3 and the action of stimulated EDRF release on tissues could be compared.
3. Results
mm in width) by gentle rubbing of the luminal surface with a wooden applicator. The PSS composition was (in mM): NaC1 118; KC1 4.7; MgSO 4 1.16; KH2PO 4 1.18; NaHCO 3 24.88; CaC12 2.52; Na pyruvate 2; EDTA-Na salt 0.026; glucose 11. PSS was saturated with carbogen, pH 7.4 at 37 ° C. The bioassay system shown in fig. 1 was used. A segment of donor artery was perfused with PSS, t = 37°C intraluminally and externally. Electrically stimulated (1 Hz, 1 ms, 4-10 V) papillary muscle or rat portal vein were superfused through channel 4. E D R F release was monitored by superfusion of a ring of vessel with mechanically removed endothelium through channel 1. PSS was delivered through channels 1-4 at a constant flow rate of 2 m l / m i n with a roller pump (Gilson MP-2, France). The change in tension of the recipient tissues was registered with isometric force transducers (FT-03 C, Grass Instruments, USA) on an ink-writing recorder (Goetz Metrawatt, Model SE 460, Austria). The following bioassay systems were used: rabbit aorta-papillary muscle-
Papillary muscles of the species studied did not respond with a decrease in contraction amplitude to placement under perfused donor vessel segments or after ACH-stimulated release of AEDRF. Artery rings denuded of endothelium showed a slight relaxation in response to spontaneously released E D R F (fig. 2A) and a pronounced relaxation after E D R F release stimulated by ACH (fig. 2A,B). ACH applied to papillary muscle produced a slight decrease (abolished after atropine, 10-6 M (Sigma, USA) pretreatment) or a negligible change in contraction amplitude (fig. 2B). When tonic tension of rabbit papillary muscle was produced by an increase in stimulation frequency (5-10 Hz) ACH infused directly or through the donor vessel produced equal decreases in tension (not shown). Spontaneously contracting rat portal vein with A C H applied directly or through donor rabbit or rat aorta showed a transient increase in frequency of the phasic and tonic components of contraction (fig. 2C). These effects of ACH were abolished after portal vein pretreatment with atropine, 10 - 6
473
M. When the muscarinic receptors of the portal vein were blocked, the ACH-stimulated release of A E D R F did not inhibit contractile activity while A
• °ACH 10"6M
B 3
.,,:.,o'%,
5 m~in 3
!
i_~
-'-e 5
5rain
/ ACH 10"6M
ACH 10"6M
Imin
I_
Fig. 2. A E D R F effect on contractile activity of pig (A), rabbit (B) papillary muscles and rat portal vein (C). (A) Pig coronary artery ring put under perfused femoral artery (position 3) showed a slight relaxation due to the spontaneous release of EDRF. Pronounced relaxation was seen after ACH-stimulated release of EDRF. No inhibitory action of A E D R F was seen when pig papillary muscle was under a femoral artery still stimulated by ACH. The order of recipient tissue positioning under a perfused donor artery did not change the quality of the reactions. Coronary rings developed 4 g tension after PGF2,~, 2 × 1 0 -6 M. (B) Rabbit papillary muscle superfused through rabbit aorta (position 3) was inhibited by ACH infused into the donor vessel similarly to the direct ACH effect (abolished by atropine, 10 -6 M pretreatment (not shown)). When the rabbit femoral artery ring instead of papillary muscle was in position 3 pronounced relaxation was evident. Femoral artery rings developed 8 g tension after phenylephrine, 10 -6 M. (C) ACH infused through the rabbit aorta produced changes in phasic and tonic contractions of rat portal vein before (left) but not after atropine pretreatment (right). Note the absence of inhibitory effect of E D R F release stimulated by ACH in portal vein (right) but the pronounced relaxation of rabbit femoral artery ring (below) when the artery instead of portal vein was in position 3. Femoral artery rings developed a tension of 10 g after phenylephrine, 10 - 6 M. Dots indicate ACH infusion or change in perfusion channels: 1 and 4, direct superfusion; 3, superfusion through the donor vessel.
it strongly relaxed rabbit femoral artery or rat aorta ring preparations (fig. 2C).
4. Discussion The results of the present study show the lack of inhibitory effect of A E D R F released from rabbit or rat aorta and pig femoral artery on the contractility of papillary muscles of the corresponding species. A E D R F released from rat and rabbit aorta did not influence the contractility of rat portal vein. In spite of the intriguing possibility that A E D R F could be a modulator of extravascular tissues that was suggested by the experiments of Busse et al. (1985) but not confirmed by our experiments (Vedernikov et al., 1987) we could not show a direct effect of A E D R F on non-arterial tissues, thus supporting the view that A E D R F is a local modulator. It is of interest to find whether myocardial endothelium can produce an EDRF-like factor to influence vessel smooth muscle. PSS superfused through the left ventricle of spontaneously beating rat Langendorff hearts did not inhibit rat aorta rings precontracted with phenylephrine or prostaglandin F2~ and with their endothelium removed. We conclude that arterial tone and reactivity are modulated locally by spontaneous or stimulated release of EDRF. If E D R F penetrates the vessel wall or reaches extravascular tissues through the vasa vasorum it does not produce an inhibitory effect on non-arterial tissues. Myocardial endothelial factor, if it exists, does not modulate arterial contractility.
References Busse, G., G. Trogisch and E. Bassange, 1985, The role of endothelium in the control of vascular tone, Basic Res. Cardiol. 80, 475. Furchgott, R.F., 1983, Role of endothelium in response of vascular smooth muscle, Circ. Res. 53, 557. Furchgott, R.F., 1984, The role of endothelium in response of vascular smooth muscle to drugs, Ann. Rev. Pharmacol. Toxicol. 24, 175. Furchgott, R.F. and J.V. Zawadski, 1980, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373.
474
Vanhoutte, P.M., 1986, Could the absence or malfunction of vascular endothelium precipitate the occurrence of vasospasm?, J. Mol. Cell Cardiol. 18, 679. Vanhoutte, P.M., G.M. Rubanyi, V.M. Miller and D.S. Houston, 1986, Modulation of vascular smooth muscle contraction by the endothelium, Ann. Rev. Physiol. 48, 307.
Vedemikov, Y.P., T. Gr~er and N. Tiedt, 1987, Is there an abluminal release of endothelium-derived relaxing factor (EDRF)?, Basic Res. Cardiol. 82, 172.
471
Elsevier EJP 20014 Short communication
Arterial endothelium-derived relaxing factor (AEDRF) does not suppress papillary muscle or portal vein contractions Y u r i P. V e d e r n i k o v *, A n a t o l i M. Vihcrt a n d H a r a l d L c i s n e r a All-Union Cardiology Research Centre, U.S.S.R. Academy of Medical Sciences, 3 a Cherepkovskaja st. 15 a, Moscow 121552, U.S.S.R. and 1 Institute o/ Pathophysiology, Medical Academy Erfurt, G.D.R.
Received 6 July 1987, accepted 1 September 1987
10 - 6 M EDRF released from perfused arterial donor segments in a superfusion bioassay system, either spontaneously or stimulated by acetylcholine, did not inhibit the contractility of electrically stimulated right ventricle papillary muscle of pig, rabbit and rat. EDRF also did not inhibit spontaneously contracting rat portal vein and thus relaxed precontracted arterial rings denuded of endothelium. The results suggest that EDRF released by arterial endothelial cells is a local modulator of arterial smooth muscle and is not important for extraarterial tissue regulation.
Endothelial relaxing factor; Smooth muscle; Papillary muscle
1. Introduction Furchgott and Zawadski (1980) discovered a new substance by which endothelium could modulate vascular smooth muscle. Many reports on the involvement of endothelium-derived relaxing factor (EDRF) in endogenous and pharmacological agent actions have appeared since 1980 (for reviews see Furchgott, 1983; 1984; Busse et al., 1985; Vanhoutte, 1986; Vanhoutte et al., 1986). Busse et al. (1985) recently described the abluminal release of E D R F . This suggested that E D R F was a modulator of tissues adjacent to the arterial wall. We could not confirm the abluminal release of E D R F in arterial preparations of different species in our superfusion bioassay experiments (Vedernikov et al., 1987). Such a discrepancy from the results of Busse et al. (1985) could be due to differences in bioassay systems and in oxygen * To whom all correspondence should be addressed: AllUnion Cardiology Research Centre, U.S.S.R. Academy of Medical Sciences, 3d Cherepkovskaja st. 15a, Moscow 121552, U.S.S.R.
tension of perfusion solutions. In the present study we used a bioassay system similar to that described by Vedernikov et al. (1987) for the study of the direct effect of arterial E D R F ( A E D R F ) on papillary muscle of various species and on rat portal vein.
2. Materials and methods The animals (pig, rabbit and rat) were exsanguinated through the carotid arteries. The hearts were perfused with cardioplegic solution (St. Thomas Hospital), removed and placed in the same medium. Papillary muscle was excised from the right ventricle, ligated from both sides and placed under the superfusion line between two parallel platinum electrodes. The vessels to be studied were removed, trimmed of excess fatty and connective tissue in modified physiological salt solution (PSS) at room temperature. The side-branches of donor vessels (4 cm in length) were carefully ligated. The endothelium was removed from arterial recipient ring segments (5
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
472
Hxec°ht~~ ange 37"C
III
~
oter cket
Corbogen
Ill
Donor vessel
Position
I 23 4
Recipient tissues
Fig. 1. Superfusion system for bioassay of EDRF release. Bioassay artery ring with endothelium removed and papillary muscle/portal vein are equilibrated under constant superfusion with PSS in position 1 and 4 respectively. Moving recipient preparations under donor vessel, position 3, allow the study of A E D R F effects after its spontaneous or stimulated release. A E D R F activator (ACH) is infused above the heat exchanger. RP, roller pump; FT, isometric force transducer; PSS, modified physiological salt solution.
femoral artery ring (n = 11); rat aorta-papillary muscle-aorta ring ( n = 9); pig femoral arterypapillary muscle-coronary artery ring (n = 2); r a t / rabbit aorta-rabbit femoral artery ring-rat portal vein (n = 2). After an equilibration period (60-90 min) with PSS superfusion and stabilization of the passive tension (6 g for coronary ring, 2 g for femoral artery ring, 0.5 g for papillary muscles and portal vein) the preparations were superfused with PSS containing indomethacin, 10 -5 M (Sigma, USA), phenylephrine, 10 .6 M (Serva, FRG) or prostaglandin F2,, 2 x 10 -6 M (Serva, FRG) for pig tissues. The completeness of endothelium removal in recipient rings was confirmed if acetylcholine (ACH, Sigma, USA), 10 - 6 M final concentration, applied at the influsion site with a perfusor-V1 (B. Broun, FRG) did not produce relaxation. Recipient tissues were moved in turn to position 3 to check the influence of spontaneously released EDRF. ACH was then infused through perfusion line 3 and the action of stimulated EDRF release on tissues could be compared.
3. Results
mm in width) by gentle rubbing of the luminal surface with a wooden applicator. The PSS composition was (in mM): NaC1 118; KC1 4.7; MgSO 4 1.16; KH2PO 4 1.18; NaHCO 3 24.88; CaC12 2.52; Na pyruvate 2; EDTA-Na salt 0.026; glucose 11. PSS was saturated with carbogen, pH 7.4 at 37 ° C. The bioassay system shown in fig. 1 was used. A segment of donor artery was perfused with PSS, t = 37°C intraluminally and externally. Electrically stimulated (1 Hz, 1 ms, 4-10 V) papillary muscle or rat portal vein were superfused through channel 4. E D R F release was monitored by superfusion of a ring of vessel with mechanically removed endothelium through channel 1. PSS was delivered through channels 1-4 at a constant flow rate of 2 m l / m i n with a roller pump (Gilson MP-2, France). The change in tension of the recipient tissues was registered with isometric force transducers (FT-03 C, Grass Instruments, USA) on an ink-writing recorder (Goetz Metrawatt, Model SE 460, Austria). The following bioassay systems were used: rabbit aorta-papillary muscle-
Papillary muscles of the species studied did not respond with a decrease in contraction amplitude to placement under perfused donor vessel segments or after ACH-stimulated release of AEDRF. Artery rings denuded of endothelium showed a slight relaxation in response to spontaneously released E D R F (fig. 2A) and a pronounced relaxation after E D R F release stimulated by ACH (fig. 2A,B). ACH applied to papillary muscle produced a slight decrease (abolished after atropine, 10-6 M (Sigma, USA) pretreatment) or a negligible change in contraction amplitude (fig. 2B). When tonic tension of rabbit papillary muscle was produced by an increase in stimulation frequency (5-10 Hz) ACH infused directly or through the donor vessel produced equal decreases in tension (not shown). Spontaneously contracting rat portal vein with A C H applied directly or through donor rabbit or rat aorta showed a transient increase in frequency of the phasic and tonic components of contraction (fig. 2C). These effects of ACH were abolished after portal vein pretreatment with atropine, 10 - 6
473
M. When the muscarinic receptors of the portal vein were blocked, the ACH-stimulated release of A E D R F did not inhibit contractile activity while A
• °ACH 10"6M
B 3
.,,:.,o'%,
5 m~in 3
!
i_~
-'-e 5
5rain
/ ACH 10"6M
ACH 10"6M
Imin
I_
Fig. 2. A E D R F effect on contractile activity of pig (A), rabbit (B) papillary muscles and rat portal vein (C). (A) Pig coronary artery ring put under perfused femoral artery (position 3) showed a slight relaxation due to the spontaneous release of EDRF. Pronounced relaxation was seen after ACH-stimulated release of EDRF. No inhibitory action of A E D R F was seen when pig papillary muscle was under a femoral artery still stimulated by ACH. The order of recipient tissue positioning under a perfused donor artery did not change the quality of the reactions. Coronary rings developed 4 g tension after PGF2,~, 2 × 1 0 -6 M. (B) Rabbit papillary muscle superfused through rabbit aorta (position 3) was inhibited by ACH infused into the donor vessel similarly to the direct ACH effect (abolished by atropine, 10 -6 M pretreatment (not shown)). When the rabbit femoral artery ring instead of papillary muscle was in position 3 pronounced relaxation was evident. Femoral artery rings developed 8 g tension after phenylephrine, 10 -6 M. (C) ACH infused through the rabbit aorta produced changes in phasic and tonic contractions of rat portal vein before (left) but not after atropine pretreatment (right). Note the absence of inhibitory effect of E D R F release stimulated by ACH in portal vein (right) but the pronounced relaxation of rabbit femoral artery ring (below) when the artery instead of portal vein was in position 3. Femoral artery rings developed a tension of 10 g after phenylephrine, 10 - 6 M. Dots indicate ACH infusion or change in perfusion channels: 1 and 4, direct superfusion; 3, superfusion through the donor vessel.
it strongly relaxed rabbit femoral artery or rat aorta ring preparations (fig. 2C).
4. Discussion The results of the present study show the lack of inhibitory effect of A E D R F released from rabbit or rat aorta and pig femoral artery on the contractility of papillary muscles of the corresponding species. A E D R F released from rat and rabbit aorta did not influence the contractility of rat portal vein. In spite of the intriguing possibility that A E D R F could be a modulator of extravascular tissues that was suggested by the experiments of Busse et al. (1985) but not confirmed by our experiments (Vedernikov et al., 1987) we could not show a direct effect of A E D R F on non-arterial tissues, thus supporting the view that A E D R F is a local modulator. It is of interest to find whether myocardial endothelium can produce an EDRF-like factor to influence vessel smooth muscle. PSS superfused through the left ventricle of spontaneously beating rat Langendorff hearts did not inhibit rat aorta rings precontracted with phenylephrine or prostaglandin F2~ and with their endothelium removed. We conclude that arterial tone and reactivity are modulated locally by spontaneous or stimulated release of EDRF. If E D R F penetrates the vessel wall or reaches extravascular tissues through the vasa vasorum it does not produce an inhibitory effect on non-arterial tissues. Myocardial endothelial factor, if it exists, does not modulate arterial contractility.
References Busse, G., G. Trogisch and E. Bassange, 1985, The role of endothelium in the control of vascular tone, Basic Res. Cardiol. 80, 475. Furchgott, R.F., 1983, Role of endothelium in response of vascular smooth muscle, Circ. Res. 53, 557. Furchgott, R.F., 1984, The role of endothelium in response of vascular smooth muscle to drugs, Ann. Rev. Pharmacol. Toxicol. 24, 175. Furchgott, R.F. and J.V. Zawadski, 1980, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373.
474
Vanhoutte, P.M., 1986, Could the absence or malfunction of vascular endothelium precipitate the occurrence of vasospasm?, J. Mol. Cell Cardiol. 18, 679. Vanhoutte, P.M., G.M. Rubanyi, V.M. Miller and D.S. Houston, 1986, Modulation of vascular smooth muscle contraction by the endothelium, Ann. Rev. Physiol. 48, 307.
Vedemikov, Y.P., T. Gr~er and N. Tiedt, 1987, Is there an abluminal release of endothelium-derived relaxing factor (EDRF)?, Basic Res. Cardiol. 82, 172.