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Effects of indomethacin on venoconstrictor responses to bradykinin and norepinephrine
EFFECTS OF INDOMETHACIN ON VENOCONSTRICTOR RESPONSES TO BRADYKININ AND NOREPINEPHRINE I
MICHAEL R. GOLDBERG, PAUL D. JOINER, STANLEY GREENBERG, ALBERT L. HYMAN, PHILIP J, KADOWITZ 2
Department of Pharmacology Tulane University School o f Medicine New Orleans, La. 70112
ABSTRACT In order to determine whether the venoconstrictor response to BK was dependent on prostaglandin (PG) synthesis, effects ok Indomethacin (IN'DO) on responses to bradykinin (BK) and norepinephrine (NE) were studied in canine lateral saphenous vein. Cumulative dose-response curves (IQ-9-10-6M BK or NE) were done in the presence and absence of IN-DO (10-6M). In the presence of INDO, responses to BK were markedly enhanced while responses to NE were unchanged. After prolonged periods in the bath, responses to BK were enhanced in control strips while responses of strips which had been treated with INDO were depressed. These results suggest that BK does not normally cause venoconstriction by stimulating synthesis of a venoconstrictor PG and that the increase in response to BK after prolonged periods in the bath may be related to changes in PG synthetase.
I - Supported by USPHS grants HL-IIS02 and HL-15580 and a grant from the American Heart Association. 2 - Established Investigator of the American Heart Association.
PROSTAGLANDINS MARCH 1975
VOL. 9 NO. 3
385
PROSTAGLANDINS
Several authors have recently proposed that vasomotor effects of bradykiniD are due to the stimulation of prostaglandin (PG) synthesis. It has been suggested that arterial dilatation elicited by 5radykinln is due to increased synthesis of PGE 2 and that venoco~striction elicited by bradykinln is due to increased synthesis of P G F 2 ~ ( I ) . Evidence for this hypothesis are found in observations that bradykinin induced renal vasodilation is followed by an increase in PGE 2 in the renal venous blood (2) and that indomethacln, an inhibitor of PG synthesis attenuates the vascular effects of bradyklnln (3). Isolated canine veins have been shown to contract in response to 5radyklnln (4). If this venoconstrictlon is due to release (and therefore synthesis) of venoconstrictor PG's (e.g. PGF2~ ), then inhibition of PG synthesis should attenuate the response to bradykinin. We therefore studied the responses of canine isolated saphenous vein to bradykinin and norepinephrine in the presence and absence of indomethacin, an inhibitor of PG biosynthesis (5). METHODS Five mongrel dogs of either sex were anesthetlzedwlth pento5arbitol (35 mg/kg, i.v.). Lateral saphenous veins were excised, cleaned of excess connective tissue and cut into helical strips. Each strip was suspended under a 3-4 gram load in a muscle bath containing a physiologic salt solution (PSS). The composition of this solution was NaCI: 127 mM, CAC12:1.8 mM, MgCI2:0.5 mM, glucose: II mM, tris: 23.8 mM, pH 7.4. Temperature was maintained at 37°C and the solution was gassed with 100% 02. After a two hour equilibration period, cumulative doseresponse curves (6) for norepine~trine or bradykinin (I0-9-I0-6M) were determined. Two hours later, these were repeated in the presence and absence of indomethacin (10-6M). Dose-response-curves were determined a third time 2 hours after washout of indomethacin. Four strips were cut from the veins of each dog. Two of these were exposed to noreplnephrine and two to bradykinin. One of each pair of strips was exposed to indomethacin. Stock solutions of l-noreplnephrlne (Sigma Chemical Co.) and bradyklnin (Sandoz Pharmaceuticals) were diluted in PSS on the day of the experiment. Indomethacin (Merck, Sharp and Dohme, Inc.) and a molar equivalent of Na2CO 3 were dissolved in PSS on the day of the experiment. All concentrations are expressed as final bath concentration in moles/liter. RESULTS Norepinephrine and bradykinin both produced dose-related contractions of the venous strips. Force generated at a concentration of 10-6M for both agents, was 4.5 ~ 0.2 (SE) g/strip for norepinephrlne and 0.84 + 0.37 g/strip for bradykinln. Contractile responses to BK were not Fell maintained whereas the response to NE declined only after prolonged periods. The tachyphylaxis to bradykinin could be overcome by addition of higher concentrations.
386
MARCH 1975
VOL. 9 NO. 3
PROSTAGLANDINS
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MARCH 1975
VOL. 9 NO. 3
387
PROSTAGLANDINS
Indomethacln (10-6M) elicited a small, slowly developing increase in venous tone (0.36 g ~ 0.13 g/strlp). (Figure I and 2). Twenty minutes after addition of indomethacln to the bath, cumulative doseresponse curves for norepinephrine and bradykininwere again determined (Figure 1 and 2). In control strips (vehicle for indomethacin alone), responses to bradykinln and norepinephrlne were similar for the first and second dose-response curves. In strips exposed to indomethacin in the bath~ responses for norepinephrine were not altered (Figure I)~ whereas responses to bradykinln were markedly enhanced (0.84 ~ 0.37 g/strlp vs 2.04 ~ 0.60 g/strip) (Figure 2). After the strips were washed, dose-response curves were reported a third time. Control strips showed an enhanced response to bradykinln. Responses to bradyklnln for strips previously exposed to indomethacln were similar to the first dose-response curves for these strips. Responses to noreplnephrine were unaltered. DISCUSSION Results of the present study show that Indomethacin enhances the venoconstrlctor response to bradykinin in the absence of an effect on the response to norepinephrlne. Further, the sensitivity of control strips to bradyklnln tended to increase with time. This sensitization was not observed in strips which had been previously treated with Indomethacin. These data do not support the hypothesis that bradyklnln induced venoconstriction is mediated by synthesis of PGF2~(1). "On the contrary, enhanced responses to bradykinin by indomethacinmay indicate that bradykinin could have two opposing actions in the isolated venous preparation. A direct stimulating effect of bradyklnln on venous smooth muscle may therefore be modulated by the simultaneous productlon or release of venodilator PG (e.g. I~E2). Inhibition of PG synthesis by Indomethacln could inhibit this modulatory influence and potentiate responses to bradyklnin. Another possibility is that in the isolated preparation, indomethacin is interacting directly with bradykinin, perhaps at the receptor, to enhance the response of venous smooth muscle to bradykinin. At the concentration used, indomethacin has been shown to be a relatively specific inhibitor of PG synthesis in the absence of effects of other enzyme systems (5). Results with norepinephrine also suggest that the increased response to bradykinin do not represent a non-speciflc increase in sensitivity. In prelimlnary experiments with 5, 8, II, 14-eicosatetraynoic acid, an inhibitor of PG synthesis with a mechanism of action different from that of indomethacin (5), the pattern of responses to bradyklnin was similar so that it seems likely that the observed effects of indomethacin are due to inhibition of PG synthesis. Enhanced responsea to bradykinln after long periods (6 hours) in the tissue bath may result from changes which occur within the strip during this time. Attenuation of this enhancement by indomethacin suggests that PG synthesis may play a role in this late response. One
388
MARCH 1975
VOL. 9 NO. 3
PROSTAGLANDINS
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M A R C H 1975
VOL. 9 NO. 3
389
PROSTAGLANDINS
possible explanation is that changes in PG synthesis may occur which result in bradykinin stimulating production of a constrictor PC, the effects of which add to bradykinln induced contraction. Prolonged effects of indomethacin block this effect. Another possibility is that substrates for synthesis may be depleted so that a modulating dilator PG is no longer produced. In strips where PG synthesis was inhibited for a period of time, this substrate pool was not depleted and stimulation by bradykinin is again able to release dilator PG. Present results demonstrate that the mechanism of bradykinin action on canine superficial veins is different from that originally
postulated for isolated mesenteric veins (I). Many factors must be taken into consideration when studying the interactions of bradykinin with inhibition of PG synthesis in vitro. Among these factors are the possibility of other actions of the synthesis Inhlhitors not associated with inhibition of PG synthesis and biochemical changes which occur in an isolated vascular strip with time. In conclusion results of the present study show that bradykinin is a venoconstrictor agent but that this response is not the result of increased PC synthesis.
REFERENCES I.
Terragno, D.A., Terragno, N.A., McGiff, J.C.: Prostaglandin synthesis by arteries and veins. The Council for High Blood Pressure Research of the American Heart Association. Annual Conference, October, 1974.
2.
McGiff, J.C., Terragno, N.A., Malik, K.U., Lonigro, A.J.: Release of a prostaglandin E-like substance from canine kidney by bradykinin; Comparison with eledoisin. Circulation Research 31:3643, 1972.
3.
Collier, H.O.J., Dinneen, L.C., Perkins, A.C., Piper, P.J.: Curtailment by aspirin and meclofenamate of hypotension induced by bradykinin in the guinea pig. Naungn-Schmeideberg's Arch. Pharm. 259:159-160, 1967.
4.
DePasquale, N.P., Burch, G.E.: Influence of bradyklnin on isolated canine venous strips. American Heart Journal 75:630633, 1968.
5.
Flower, R.J.: Drugs which inhibit prostaglandin biosynthesis. Pharmacological Reviews. 26:33-66, 1974.
6.
Van Rossum, J.M.: Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch. int. Pharmacodyn. 143: 299-330, 1963.
390
M A R C H 1975
VOL. 9 NO. 3
MICHAEL R. GOLDBERG, PAUL D. JOINER, STANLEY GREENBERG, ALBERT L. HYMAN, PHILIP J, KADOWITZ 2
Department of Pharmacology Tulane University School o f Medicine New Orleans, La. 70112
ABSTRACT In order to determine whether the venoconstrictor response to BK was dependent on prostaglandin (PG) synthesis, effects ok Indomethacin (IN'DO) on responses to bradykinin (BK) and norepinephrine (NE) were studied in canine lateral saphenous vein. Cumulative dose-response curves (IQ-9-10-6M BK or NE) were done in the presence and absence of IN-DO (10-6M). In the presence of INDO, responses to BK were markedly enhanced while responses to NE were unchanged. After prolonged periods in the bath, responses to BK were enhanced in control strips while responses of strips which had been treated with INDO were depressed. These results suggest that BK does not normally cause venoconstriction by stimulating synthesis of a venoconstrictor PG and that the increase in response to BK after prolonged periods in the bath may be related to changes in PG synthetase.
I - Supported by USPHS grants HL-IIS02 and HL-15580 and a grant from the American Heart Association. 2 - Established Investigator of the American Heart Association.
PROSTAGLANDINS MARCH 1975
VOL. 9 NO. 3
385
PROSTAGLANDINS
Several authors have recently proposed that vasomotor effects of bradykiniD are due to the stimulation of prostaglandin (PG) synthesis. It has been suggested that arterial dilatation elicited by 5radykinln is due to increased synthesis of PGE 2 and that venoco~striction elicited by bradykinln is due to increased synthesis of P G F 2 ~ ( I ) . Evidence for this hypothesis are found in observations that bradykinin induced renal vasodilation is followed by an increase in PGE 2 in the renal venous blood (2) and that indomethacln, an inhibitor of PG synthesis attenuates the vascular effects of bradyklnln (3). Isolated canine veins have been shown to contract in response to 5radyklnln (4). If this venoconstrictlon is due to release (and therefore synthesis) of venoconstrictor PG's (e.g. PGF2~ ), then inhibition of PG synthesis should attenuate the response to bradykinin. We therefore studied the responses of canine isolated saphenous vein to bradykinin and norepinephrine in the presence and absence of indomethacin, an inhibitor of PG biosynthesis (5). METHODS Five mongrel dogs of either sex were anesthetlzedwlth pento5arbitol (35 mg/kg, i.v.). Lateral saphenous veins were excised, cleaned of excess connective tissue and cut into helical strips. Each strip was suspended under a 3-4 gram load in a muscle bath containing a physiologic salt solution (PSS). The composition of this solution was NaCI: 127 mM, CAC12:1.8 mM, MgCI2:0.5 mM, glucose: II mM, tris: 23.8 mM, pH 7.4. Temperature was maintained at 37°C and the solution was gassed with 100% 02. After a two hour equilibration period, cumulative doseresponse curves (6) for norepine~trine or bradykinin (I0-9-I0-6M) were determined. Two hours later, these were repeated in the presence and absence of indomethacin (10-6M). Dose-response-curves were determined a third time 2 hours after washout of indomethacin. Four strips were cut from the veins of each dog. Two of these were exposed to noreplnephrine and two to bradykinin. One of each pair of strips was exposed to indomethacin. Stock solutions of l-noreplnephrlne (Sigma Chemical Co.) and bradyklnin (Sandoz Pharmaceuticals) were diluted in PSS on the day of the experiment. Indomethacin (Merck, Sharp and Dohme, Inc.) and a molar equivalent of Na2CO 3 were dissolved in PSS on the day of the experiment. All concentrations are expressed as final bath concentration in moles/liter. RESULTS Norepinephrine and bradykinin both produced dose-related contractions of the venous strips. Force generated at a concentration of 10-6M for both agents, was 4.5 ~ 0.2 (SE) g/strip for norepinephrlne and 0.84 + 0.37 g/strip for bradykinln. Contractile responses to BK were not Fell maintained whereas the response to NE declined only after prolonged periods. The tachyphylaxis to bradykinin could be overcome by addition of higher concentrations.
386
MARCH 1975
VOL. 9 NO. 3
PROSTAGLANDINS
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MARCH 1975
VOL. 9 NO. 3
387
PROSTAGLANDINS
Indomethacln (10-6M) elicited a small, slowly developing increase in venous tone (0.36 g ~ 0.13 g/strlp). (Figure I and 2). Twenty minutes after addition of indomethacln to the bath, cumulative doseresponse curves for norepinephrine and bradykininwere again determined (Figure 1 and 2). In control strips (vehicle for indomethacin alone), responses to bradykinln and norepinephrlne were similar for the first and second dose-response curves. In strips exposed to indomethacin in the bath~ responses for norepinephrine were not altered (Figure I)~ whereas responses to bradykinln were markedly enhanced (0.84 ~ 0.37 g/strlp vs 2.04 ~ 0.60 g/strip) (Figure 2). After the strips were washed, dose-response curves were reported a third time. Control strips showed an enhanced response to bradykinln. Responses to bradyklnln for strips previously exposed to indomethacln were similar to the first dose-response curves for these strips. Responses to noreplnephrine were unaltered. DISCUSSION Results of the present study show that Indomethacin enhances the venoconstrlctor response to bradykinin in the absence of an effect on the response to norepinephrlne. Further, the sensitivity of control strips to bradyklnln tended to increase with time. This sensitization was not observed in strips which had been previously treated with Indomethacin. These data do not support the hypothesis that bradyklnln induced venoconstriction is mediated by synthesis of PGF2~(1). "On the contrary, enhanced responses to bradykinin by indomethacinmay indicate that bradykinin could have two opposing actions in the isolated venous preparation. A direct stimulating effect of bradyklnln on venous smooth muscle may therefore be modulated by the simultaneous productlon or release of venodilator PG (e.g. I~E2). Inhibition of PG synthesis by Indomethacln could inhibit this modulatory influence and potentiate responses to bradyklnin. Another possibility is that in the isolated preparation, indomethacin is interacting directly with bradykinin, perhaps at the receptor, to enhance the response of venous smooth muscle to bradykinin. At the concentration used, indomethacin has been shown to be a relatively specific inhibitor of PG synthesis in the absence of effects of other enzyme systems (5). Results with norepinephrine also suggest that the increased response to bradykinin do not represent a non-speciflc increase in sensitivity. In prelimlnary experiments with 5, 8, II, 14-eicosatetraynoic acid, an inhibitor of PG synthesis with a mechanism of action different from that of indomethacin (5), the pattern of responses to bradyklnin was similar so that it seems likely that the observed effects of indomethacin are due to inhibition of PG synthesis. Enhanced responsea to bradykinln after long periods (6 hours) in the tissue bath may result from changes which occur within the strip during this time. Attenuation of this enhancement by indomethacin suggests that PG synthesis may play a role in this late response. One
388
MARCH 1975
VOL. 9 NO. 3
PROSTAGLANDINS
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M A R C H 1975
VOL. 9 NO. 3
389
PROSTAGLANDINS
possible explanation is that changes in PG synthesis may occur which result in bradykinin stimulating production of a constrictor PC, the effects of which add to bradykinln induced contraction. Prolonged effects of indomethacin block this effect. Another possibility is that substrates for synthesis may be depleted so that a modulating dilator PG is no longer produced. In strips where PG synthesis was inhibited for a period of time, this substrate pool was not depleted and stimulation by bradykinin is again able to release dilator PG. Present results demonstrate that the mechanism of bradykinin action on canine superficial veins is different from that originally
postulated for isolated mesenteric veins (I). Many factors must be taken into consideration when studying the interactions of bradykinin with inhibition of PG synthesis in vitro. Among these factors are the possibility of other actions of the synthesis Inhlhitors not associated with inhibition of PG synthesis and biochemical changes which occur in an isolated vascular strip with time. In conclusion results of the present study show that bradykinin is a venoconstrictor agent but that this response is not the result of increased PC synthesis.
REFERENCES I.
Terragno, D.A., Terragno, N.A., McGiff, J.C.: Prostaglandin synthesis by arteries and veins. The Council for High Blood Pressure Research of the American Heart Association. Annual Conference, October, 1974.
2.
McGiff, J.C., Terragno, N.A., Malik, K.U., Lonigro, A.J.: Release of a prostaglandin E-like substance from canine kidney by bradykinin; Comparison with eledoisin. Circulation Research 31:3643, 1972.
3.
Collier, H.O.J., Dinneen, L.C., Perkins, A.C., Piper, P.J.: Curtailment by aspirin and meclofenamate of hypotension induced by bradykinin in the guinea pig. Naungn-Schmeideberg's Arch. Pharm. 259:159-160, 1967.
4.
DePasquale, N.P., Burch, G.E.: Influence of bradyklnin on isolated canine venous strips. American Heart Journal 75:630633, 1968.
5.
Flower, R.J.: Drugs which inhibit prostaglandin biosynthesis. Pharmacological Reviews. 26:33-66, 1974.
6.
Van Rossum, J.M.: Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch. int. Pharmacodyn. 143: 299-330, 1963.
390
M A R C H 1975
VOL. 9 NO. 3