93
European Journal of Pharmacology, 198 (1991) 93-95 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100411H EJP 20829
Snort communication
Bradykinin and angiotensin II inhibit neurotransmission in rabbit ear artery by releasing prostanoids Andrfis Z. R 6 n a i Institute of Organic Chemistry, L. E6tc6s Unicersity, P.O. Box 32, H-1518 Budapest, Hungary Received 19 March 1991, accepted 2 April 1991
In isolated, perfused rabbit ear artery bradykinin, like [MetS]enkephalin and angiotensin II, inhibited neurogenic constrictions in concentrations lower than 10-SM without affecting the responses to exogenous norepinephrine. The IC50 value of bradykinin was 1.95 × 10-1° M. The inhibitory action of bradykinin and angiotensin II but not of [MetS]enkephalin was reduced or even reversed by indomethacin pretreatment, suggesting that endogenous prostanoids are involved. Ear artery (rabbit); Bradykinin; Angiotensin II; [MetS]enkephalin; Indomethacin
1. Introduction
It was shown recently that, in contrast to their actions in the majority of vascular beds, both angiotensin I and angiotensin II potently inhibit neurotransmission in the rabbit isolated ear artery (R6nai, 1990). As a possible mechanism for this inhibition, the stimulation of a local prostanoid-generating system has been suggested, since, on the one hand, angiotensin II has been reported to release a prostaglandin I2 (PGI2)-like substance and, to a lesser extent, PGE 2 from rat mesenteric vasculature (Dusting et al., 1981) and, on the other hand, prostanoids such as PGE t and PGI 2 have been shown to inhibit neurotransmission in the rabbit isolated ear artery (Hadhfizy and Nfidor, 1976; Hadhfizy et al., 1984). To elucidate this possible mechanism, the effect of angiotensin II was tested in the absence and presence of an inhibitor of cyclooxygenase, namely indomethacin. The action of another known prostaglandin-releasing vasoactive peptide, bradykinin, (for review see Nasjletti and Malik, 1979) was also investigated under similar conditions.
2. Materials and methods
2.1. Drugs The following drugs were used: angiotensin II (Sigma), bradykinin triacetate (Reanal, Hungary), in-
Correspondence to: A.Z. R6nai, Institute of Organic Chemistry, L. E6tv6s University, P.O. Box 32, H-1518 Budapest, Hungary.
domethacin (Chinoin, Hungary), noradrenaline bitartarate (Sigma) and [MetS]enkephalin (J. Hepp, L. E6tvfs University, Hungary).
Z2. Methods 2.2.1. Experimental Ear arteries, prepared from male New Zealand rabbits (4-5 kg body weight), were perfused and stimulated as described previously (R6nai, 1990). In brief, artery segments, cannulated at both ends, were bathed and perfused at a steady rate of 2.4-2.5 ml/min with carbogen (95% 02-5% CO2)-saturated Krebs solution at 37 °C. Field electrical stimulation was used; stimulation parameters were as follows: square-wave pulses of 1 ms duration, supramaximal ,~oltage (9 V/cm), trains of five pulses at 5 Hz frequency were delivered every 60 s, and intra-arterial pressure changes were recorded. The cyclooxygenase inhibitor was added to both the bathing and perfusion fluid, whereas all the other substances were added only to the bath, i.e. to the adventitial side of preparations. 2.2.Z Evah~ation As less than three doses of bradvkinin could be tested with good reproducibility under identical conditions in the same preparation, the parameters of the dose-response curve were calculated as described in a textbook by Tallarida and Jacobs (1979) from 11 points obtained in 6 independent experiments (3-4 points at 3 dose levels). Statistical evaluation of the data was done with
Student's t-test for paired or unpaired samples as necessary.
3. Results
R6nai et al., 1983) was not affected significantly by indomethacin (fig. 1).
4. Discussion
Bradykinin, in concentrations below 10-8M, inhibited the contractile responses (detected as pressure changes) elicited by electrical stimulation in a dose-dependent manner without affecting the responses to exogenously administered norepinephrine. Bradykinin was at least as potent an inhibitor of neurotransmission in the rabbit isolated ear artery as angiotensin II (R6nai, 1990), the IC5o value being 1.95 + 0.3 × 10- ~o M (mean + S.E.). At 10 -8 M, the inhibition of electrically induced contractions was 97.0 _+ 3.0% (arithmetic mean + S.E.M., n = 4)whereas the responses to 10 - 6 M norepinephrine were only moderately, though significantly, reduced (17.4 + 3.8%, n = 4, P < 0.01, paired t-test). Indomethacin at 3 × 10 - 6 M, a concentration recommended by Hadhfizy et al. (1984) in this preparation, slightly but significantly increased basal pressure (by 14.2 + 2.7%, n = 4, P < 0.05 paired t-test) and enhanced the contractile response to both electrical stimulation (by 130.6 + 27.7%, n = 4, P < 0.02) and exogenous norepinephrine (by 61.7 + 17.5%, n - 4, P < 0.05). These changes developed fully within 15 min and were present throughout the experiment. The inhibitory effect of 10 -8 M bradykinin (which is approximately 50 times the IC50 value) was significantly reduced but not abolished by 60 min preincubation with indomethacin (see fig. 1). The effect of 10 -8 M angiotensin II (approximately 30 times the IC50, R6nai, 1990) was not only abolished but even reversed under the same conditions (fig. 1). The inhibitory action o f 10 - 6 M [MetS]enkephalin (approximately 10 times the IC5o,
BK
.~ 100-
It appears that angiotensin and, at least in part, bradykinin exert their inhibitory effects in the rabbit isolated ear artery by promoting the generation of prostanoid(s), products of the cyclooxygenase pathway, whereas the opioid peptide [MetS]enkephalin acts by a different mechanism. It is known that angiotensip and bradykinin receptors (at least certain type(s) thereof) fall into the group of receptors that are coupled to p h o s p h o l i p a s e A 2 in a stimulatory fashion (for review see Axelrod et al., 1988). It should be mentioned that Barab6 et al. (1979) have found that bradykinin has a vasoconstricting rather than dilating effect in isolated, perfused rabbit ear. However, the experimental conditions (whole ear, resting perfusion) as well as the concentration range (detectable changes above 10 -8 M) used were considerably different from the ones used in the present experiments. Furthermore, the effects found in the lower concentration range may have a different, and perhaps more relevant, physiological significance. Since (i) the rabbit ear artery is used as a vascular migraine model, (ii) bradykinin and angiotensin II rank among the most potent endogenous substances known as inhibitors of vasoconstriction in this preparation (the IC50 values being 1.95 x 10-1° and 3.60 × 10-lo M, respectively, present paper and R6nai, 1990), and (iii) cyclooxygenase inhibitors constitute a significant part of the anti-migraine therapeutic armoury, the contribution of these endogenous peptides to the pathomechanism of migraine should be taken into consideration.
ME
AT-II
References
'
._ c
50
.-.
o
:: 5o
° o .
Q.
Fig. 1. The effect of 10 -s M bradykinin (BK), 10 -8 M angiotensin II (AT-II) and 10 -6 M [MetSlenkephalin (ME) on neurotransmission in rabbit isolated ear artery in the absence (open columns) and presence (hatched columns) of 3× 10 -6 M indomethacin. Mean_+ S.E.M. values are plotted; n = 4 for each column. * * * P < 0.001 (unpaired t-test).
Axelrod, J., R.M. Burch and C.L. Jelsema, 1988, Receptor-mediated activation of phospholipase A 2 via GTP-binding proteins: arachidonic acid and its metabolites as second messengers, Trends Neurosci. I1, 117. Barab6, J., F. Marceau, B. Th6riault, J.-N. Drouin and D. Regoli, 1979, Cardiovascular actions of kinins in the rabbit, Can. J. Physiol. Pharmacol. 57, 78. Dusting, G.J., E.M. Mullins and R.D. Nolan. 1981, Prostacyclin (PGI 2) release accompanying angiotensin conversion in rat mesenteric vasculature, European J. Pharmacol. 70, 129. Hadhfizy, P., B. Malomv61gyi and K. Magyar, 1984, Differential contractile responsiveness of isolated rabbit arteries from different vascular beds to cyclooxygenase inhibitors and PGI 2, European J. Pharmacol. 98, 323. Hadhfizy, P. and 'r. Nfidor, 1976, Effects of indomett, acin and PGE t on the vasoconstrictor responses of the rabbit ear artery to nerve stimulation, Prostaglandins 11,241.
95 Nasjletti, A. and K.V. Malik, 1979, Relationships between the kallikrein-kinin and prostaglandin system, Life Sci. 25, 99. R6nai, A.Z., 1990, Inhibition of neurotransmission by angiotensin l and II in rabbit isolated ear artery, European J. Pharmacol. 179, 281.
R6nai, A.Z., P. Serf6z6, L.G. H~sing, Jr. and E.S. Vizi, 1983, The conversion of met-enkephalin-Arg6, Phe 7 to met-enkephalin in rabbit ear artery, Life Sci. 33 (Suppl. I), 101. Tallarida, R.J. and L.S. Jacob, 1979, The Dose-Response Relation in Pharmacology (Springer, New York) p. 98.
European Journal of Pharmacology, 198 (1991) 93-95 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100411H EJP 20829
Snort communication
Bradykinin and angiotensin II inhibit neurotransmission in rabbit ear artery by releasing prostanoids Andrfis Z. R 6 n a i Institute of Organic Chemistry, L. E6tc6s Unicersity, P.O. Box 32, H-1518 Budapest, Hungary Received 19 March 1991, accepted 2 April 1991
In isolated, perfused rabbit ear artery bradykinin, like [MetS]enkephalin and angiotensin II, inhibited neurogenic constrictions in concentrations lower than 10-SM without affecting the responses to exogenous norepinephrine. The IC50 value of bradykinin was 1.95 × 10-1° M. The inhibitory action of bradykinin and angiotensin II but not of [MetS]enkephalin was reduced or even reversed by indomethacin pretreatment, suggesting that endogenous prostanoids are involved. Ear artery (rabbit); Bradykinin; Angiotensin II; [MetS]enkephalin; Indomethacin
1. Introduction
It was shown recently that, in contrast to their actions in the majority of vascular beds, both angiotensin I and angiotensin II potently inhibit neurotransmission in the rabbit isolated ear artery (R6nai, 1990). As a possible mechanism for this inhibition, the stimulation of a local prostanoid-generating system has been suggested, since, on the one hand, angiotensin II has been reported to release a prostaglandin I2 (PGI2)-like substance and, to a lesser extent, PGE 2 from rat mesenteric vasculature (Dusting et al., 1981) and, on the other hand, prostanoids such as PGE t and PGI 2 have been shown to inhibit neurotransmission in the rabbit isolated ear artery (Hadhfizy and Nfidor, 1976; Hadhfizy et al., 1984). To elucidate this possible mechanism, the effect of angiotensin II was tested in the absence and presence of an inhibitor of cyclooxygenase, namely indomethacin. The action of another known prostaglandin-releasing vasoactive peptide, bradykinin, (for review see Nasjletti and Malik, 1979) was also investigated under similar conditions.
2. Materials and methods
2.1. Drugs The following drugs were used: angiotensin II (Sigma), bradykinin triacetate (Reanal, Hungary), in-
Correspondence to: A.Z. R6nai, Institute of Organic Chemistry, L. E6tv6s University, P.O. Box 32, H-1518 Budapest, Hungary.
domethacin (Chinoin, Hungary), noradrenaline bitartarate (Sigma) and [MetS]enkephalin (J. Hepp, L. E6tvfs University, Hungary).
Z2. Methods 2.2.1. Experimental Ear arteries, prepared from male New Zealand rabbits (4-5 kg body weight), were perfused and stimulated as described previously (R6nai, 1990). In brief, artery segments, cannulated at both ends, were bathed and perfused at a steady rate of 2.4-2.5 ml/min with carbogen (95% 02-5% CO2)-saturated Krebs solution at 37 °C. Field electrical stimulation was used; stimulation parameters were as follows: square-wave pulses of 1 ms duration, supramaximal ,~oltage (9 V/cm), trains of five pulses at 5 Hz frequency were delivered every 60 s, and intra-arterial pressure changes were recorded. The cyclooxygenase inhibitor was added to both the bathing and perfusion fluid, whereas all the other substances were added only to the bath, i.e. to the adventitial side of preparations. 2.2.Z Evah~ation As less than three doses of bradvkinin could be tested with good reproducibility under identical conditions in the same preparation, the parameters of the dose-response curve were calculated as described in a textbook by Tallarida and Jacobs (1979) from 11 points obtained in 6 independent experiments (3-4 points at 3 dose levels). Statistical evaluation of the data was done with
Student's t-test for paired or unpaired samples as necessary.
3. Results
R6nai et al., 1983) was not affected significantly by indomethacin (fig. 1).
4. Discussion
Bradykinin, in concentrations below 10-8M, inhibited the contractile responses (detected as pressure changes) elicited by electrical stimulation in a dose-dependent manner without affecting the responses to exogenously administered norepinephrine. Bradykinin was at least as potent an inhibitor of neurotransmission in the rabbit isolated ear artery as angiotensin II (R6nai, 1990), the IC5o value being 1.95 + 0.3 × 10- ~o M (mean + S.E.). At 10 -8 M, the inhibition of electrically induced contractions was 97.0 _+ 3.0% (arithmetic mean + S.E.M., n = 4)whereas the responses to 10 - 6 M norepinephrine were only moderately, though significantly, reduced (17.4 + 3.8%, n = 4, P < 0.01, paired t-test). Indomethacin at 3 × 10 - 6 M, a concentration recommended by Hadhfizy et al. (1984) in this preparation, slightly but significantly increased basal pressure (by 14.2 + 2.7%, n = 4, P < 0.05 paired t-test) and enhanced the contractile response to both electrical stimulation (by 130.6 + 27.7%, n = 4, P < 0.02) and exogenous norepinephrine (by 61.7 + 17.5%, n - 4, P < 0.05). These changes developed fully within 15 min and were present throughout the experiment. The inhibitory effect of 10 -8 M bradykinin (which is approximately 50 times the IC50 value) was significantly reduced but not abolished by 60 min preincubation with indomethacin (see fig. 1). The effect of 10 -8 M angiotensin II (approximately 30 times the IC50, R6nai, 1990) was not only abolished but even reversed under the same conditions (fig. 1). The inhibitory action o f 10 - 6 M [MetS]enkephalin (approximately 10 times the IC5o,
BK
.~ 100-
It appears that angiotensin and, at least in part, bradykinin exert their inhibitory effects in the rabbit isolated ear artery by promoting the generation of prostanoid(s), products of the cyclooxygenase pathway, whereas the opioid peptide [MetS]enkephalin acts by a different mechanism. It is known that angiotensip and bradykinin receptors (at least certain type(s) thereof) fall into the group of receptors that are coupled to p h o s p h o l i p a s e A 2 in a stimulatory fashion (for review see Axelrod et al., 1988). It should be mentioned that Barab6 et al. (1979) have found that bradykinin has a vasoconstricting rather than dilating effect in isolated, perfused rabbit ear. However, the experimental conditions (whole ear, resting perfusion) as well as the concentration range (detectable changes above 10 -8 M) used were considerably different from the ones used in the present experiments. Furthermore, the effects found in the lower concentration range may have a different, and perhaps more relevant, physiological significance. Since (i) the rabbit ear artery is used as a vascular migraine model, (ii) bradykinin and angiotensin II rank among the most potent endogenous substances known as inhibitors of vasoconstriction in this preparation (the IC50 values being 1.95 x 10-1° and 3.60 × 10-lo M, respectively, present paper and R6nai, 1990), and (iii) cyclooxygenase inhibitors constitute a significant part of the anti-migraine therapeutic armoury, the contribution of these endogenous peptides to the pathomechanism of migraine should be taken into consideration.
ME
AT-II
References
'
._ c
50
.-.
o
:: 5o
° o .
Q.
Fig. 1. The effect of 10 -s M bradykinin (BK), 10 -8 M angiotensin II (AT-II) and 10 -6 M [MetSlenkephalin (ME) on neurotransmission in rabbit isolated ear artery in the absence (open columns) and presence (hatched columns) of 3× 10 -6 M indomethacin. Mean_+ S.E.M. values are plotted; n = 4 for each column. * * * P < 0.001 (unpaired t-test).
Axelrod, J., R.M. Burch and C.L. Jelsema, 1988, Receptor-mediated activation of phospholipase A 2 via GTP-binding proteins: arachidonic acid and its metabolites as second messengers, Trends Neurosci. I1, 117. Barab6, J., F. Marceau, B. Th6riault, J.-N. Drouin and D. Regoli, 1979, Cardiovascular actions of kinins in the rabbit, Can. J. Physiol. Pharmacol. 57, 78. Dusting, G.J., E.M. Mullins and R.D. Nolan. 1981, Prostacyclin (PGI 2) release accompanying angiotensin conversion in rat mesenteric vasculature, European J. Pharmacol. 70, 129. Hadhfizy, P., B. Malomv61gyi and K. Magyar, 1984, Differential contractile responsiveness of isolated rabbit arteries from different vascular beds to cyclooxygenase inhibitors and PGI 2, European J. Pharmacol. 98, 323. Hadhfizy, P. and 'r. Nfidor, 1976, Effects of indomett, acin and PGE t on the vasoconstrictor responses of the rabbit ear artery to nerve stimulation, Prostaglandins 11,241.
95 Nasjletti, A. and K.V. Malik, 1979, Relationships between the kallikrein-kinin and prostaglandin system, Life Sci. 25, 99. R6nai, A.Z., 1990, Inhibition of neurotransmission by angiotensin l and II in rabbit isolated ear artery, European J. Pharmacol. 179, 281.
R6nai, A.Z., P. Serf6z6, L.G. H~sing, Jr. and E.S. Vizi, 1983, The conversion of met-enkephalin-Arg6, Phe 7 to met-enkephalin in rabbit ear artery, Life Sci. 33 (Suppl. I), 101. Tallarida, R.J. and L.S. Jacob, 1979, The Dose-Response Relation in Pharmacology (Springer, New York) p. 98.
