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Endothelium-dependent rat aortic relaxation to the aqueous leaf extract of Musanga cecropioides
Journul of Ethnophurmacology.
34 (1991) 283-286
283
Elsevier Scientific Publishers Ireland Ltd.
Short communication
Endothelium-dependent rat aortic relaxation to the aqueous leaf extract of Musanga cecropioides A. Kamanyia, M. Bopelet”, C.P. Aloamakab,
P.C.M. Obiefunab and A.B. EbeigbeC
‘Animal Physiology Laboratory, Faculty of Science, University of Yaounde, BP 812, Yaounde (Cameroon), bDepartment of Physiology, College of Medical Sciences, University of Benin, Benin City and L’Department of Physiology, Faculty of Basic Medical Science, Bendel State University, P.M. B. 14, Ekpoma, Bendel State (Nigeria)
(Accepted April 30. 1991)
Introduction An aqeuous leaf extract of Musanga cecropioides (family: Cecropiaceae) is used in traditional medical practice to induce labour due to its oxytocic effect (Bouquet, 1969); it has also been reported to have a blood pressure lowering effect in humans (Bouquet, 1969). Agents that lower blood pressure could act via one of several vasodilator mechanisms (Bolton, 1979; Ebeigbe and Aloamaka, 1985). In the present paper, the possibility that M. cecropioides could have a direct vasodilating effect has been examined using isolated rat aorta.
of Yaounde, Cameroon, and a voucher specimen deposited at the herbarium of the Faculty of Science, University of Yaounde. The leaves were sun-dried and ground into powder in a high-speed grinding mill. Three hundred grams of the tine powder were extracted in 1 1 of boiling water for 12 h, filtered and the residue discarded. About 850 ml of filtrate (MC) was recovered with a concentration equivalent to 350 mg/ml of the dry starting material.
Materials and Methods Extract preparation
Fresh leaves of M. cecropioides were collected from Moliwe in Fako Division of the Southwest Province of Cameroon in November 1989. The plant material was authenticated by Dr. Amougu Akoa of the Botany Department of the University Correspondence to: Professor A.B. Ebeigbe, Department of Physiology, Faculty of Basic Medical Science, Bendel State University, P.M.B. I4 Ekpoma, Bendel State, Nigeria.
0378-8741/$03.50 0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
Tissue preparation
Wistar strain rats weighing between 150-200 g were killed by cervical dislocation. The thoracic aorta was quickly removed, freed of connective tissues, cut into 2 mm ring segments and suspended on tine stainless steel rods in a 20-ml tissue bath and connected to a Grass FT.03 transducer for isometric tension measurement under a resting tension of 2 g. The bath contained physiological salt solution (PSS) of the following composition (mM): NaCl 119, KC1 4.7, MgS04 1.2, KH2P04 1.2, CaClz 1.6, NaHC03 24.9 and glucose 11.5. The PSS was bubbled continuously with a 95% O2 5% CO, gas mixture. The pH of the medium was 7.4 and temperature was maintained thermostatically at 37°C.
284
Protocol
Each tissue was precontracted with a bath concentration of lo-’ M noradrenahne (NA) and relaxations due to 3.5 x 10e3 g/ml of MC or 10m5 M acetylcholine (ACh) were tested on tissues with and without endothelium. Endothelium was removed by gently rubbing the inner lining of the rings. The influence of 1 PM methylene blue on the relaxation responses to Ach and MC was also examined. In other experiments, the effect of MC was also observed under baseline (non-contracted) tension where the dose response of the aortic rings to MC was tested in the presence and absence of endothelium. Values were expressed as percent of maximum response in the presence of endothelium. The source of the noradrenaline bitartrate, acetylcholine HCl and methylene blue was Sigma Chemical Co. (St. Louis, MO, U.S.A.). Statistics
Median effective concentration (EC&) values were computed using a programme for logit transformation of concentration-response curves. Comparison of mean values was effected using Student’s t-test. A P value of less than 0.05 was considered significant.
Fig.
I.
Representative
tracings
showing
responses of rat aortic rings to 3.5 x water extract of Musunga cwropioides acetylcholine (Ach) following IO-’ induced contractions in the presence endothelium.
the
relaxation
IO-’ g/ml of the boiling leaves (MC) and IO-’ M M noradrenaline (NA)(+E) or absence (-E) of
to the cumulative addition of MC. The maximal contractile response to MC was 1095 f 70 mg in the absence of endothelium and 890 f 55 mg in the presence of endothelium. The difference was statistically significant. The EC& values for MCinduced contractions in the absence and presence of endothelium were 9.37 (h1.51) x 10e4 g/ml and 2.63 (kO.14) x lo-” g/ml respectively. Discussion
Results The contractile response to lo-’ M NA was 1193 f 13 mg (mean f S.E.M.) in aortic rings with intact endothelium. Ach caused dosedependent relaxation of NA-induced contractions as previously reported from our laboratory (Ebeigbe and Aloamaka, 1987; Aloamaka et al., 1990; Obiefuna et al., 1991). Maximal Achinduced relaxation was 85.3 + 4.7X, n = 15 (Figs. 1 and 2); whereas MC elicited relaxation by 21.8 f 2.4%,, n = 8. Both Ach (n = 6) and MC (n = 4) failed to elicit relaxation in endothelium-denuded aortic rings (Figs. 1 and 2). Exposure of the aortic rings to lo-(’ M methylene blue (MB) prevented Ach-induced (n = 4) and MC-induced (n = 4) relaxations (Fig. 2). In baseline (non-precontracted) aortic rings, MC caused concentration-dependent contraction. Figure 3 shows results of the contractile responses
The results of the present study demonstrate that MC has a dual effect on the contractile state of rat aortic smooth muscle. When MC was applied under baseline (resting) condition, MC elicited
Fig. 2. Summary
of all results
obtained
using
the scheme
described in Fig. I. The influences of presence (+E) or absence (-E) of endothelium and exposure to 10-e M methylene blue (MB) on the relaxation responses of aortic rings to IO-’ M Ach and 3.5 x IO-’ g/ml MC following NA-precontraction are shown. Each bar represents experiments.
the mean
* S.E.M.
of 4 separate
285
0' I
I
4
3 -Log (g/ml)
Fig. 3. Concentration-response
M.
I
1
2
1
Cecropioides
curve for MC-induced
contrac-
tion of rat aortic rings. Values are expressed as percent of maximum
response
in
Endothelium-removal
the
presence
of
endothelium.
(n = 4) resulted in enhanced contractions
compared
to controls
indicated
for each point.
(n =
8); The
mean
&
S.E.M.
are
endothelium-dependent relaxations cause elevation of cyclic GMP levels within the smooth muscle (Rapoport and Murad, 1983) and methylene blue prevents the formation of cyclic GMP by inhibiting guanylate cyclase (Rapoport and Murad, 1983). Enhancement of contractile response following removal of the endothelium has been reported for a number of agonists e.g. clonidine (Egleme et al., 1984; White and Carrier, 1986) and noradrenaline and methoxamine (White and Carrier, 1986). In the present study, the observed enhancement of contractile response to MC in endotheliumdenuded rings suggests that MC induced the release of EDRF which dampens tone in the intact preparation. In conclusion, the present study shows that a crude aqueous leaf extract of MC elicits contractile response under baseline conditions, but induces endothelium-dependent relaxation of precontracted rat aortic smooth muscle. Acknowledgements
concentration-dependent contractions; however, following precontraction induced by NA, MC elicited endothelium-dependent relaxation. The magnitude of Ach-induced relaxation is significantly greater than that of MC, suggesting that MC is less effective in inducing the release of endothelium-derived relaxant factor (EDRF). EDRF is known to regulate vascular smooth muscle tone (Furchgott and Zawadzki, 1980; Ebeigbe et al., 1990). It is noteworthy that other vasodilators known to lower vascular resistance through the release of EDRF, e.g. histamine (Van de Voorde and Leusen, 1983; Obiefuna et al., 1991), ATP (De Mey and Vanhoutte, 1982) and hydralazine (Aloamaka and Ebeigbe, 1987), also elicit weaker relaxations than Ach. It is possible that different EDRFs mediate the vasorelaxant effect of MC and Ach. Evidence exists that the vascular endothelium may release more than one EDRF (Vanhoutte, 1987). The failure of both Ach and MC to elicit relaxation responses in the presence of methylene blue (Fig. 2) provides additional evidence that MC relaxation is mediated via the release of EDRF. Agents that elicit
The authors are grateful to Mr. R. Tatchum for assistance in the collection and processing of the plant materials. The assistance of Tony Oguogho is greatly appreciated. References Aloamaka,
C.P. and Ebeigbe. A.B.
(1987)
Influence
thelium on rat aortic smooth muscle relaxation hydralazine. Aloamaka, Ebeigbe,
Medical
Science Reseurch 15, 1491-1492.
C.P.,
Evbuomwan,
A.B.
(1990)
rat aortic relaxation ing inhibition
of endo-
to D600 and
M.1..
Attenuated
to acetylcholine
of nitric
oxide
Tosicology and Pharmacology Bolton. T.B. (1979) Mechanisms
Ighoroje,
A.D.A.
and histamine follow-
formation
from
t-arginine.
67, 266268. of action of transmitters
other substances on smooth muscle. Physiological 50, 6067
and
endothelium-dependent
and
Reviews
IS.
Bouquet. A. (1969) Feticheurs et Medicine rraditionelles du Congo. Orstrom, De
Mey,
J.G.
Brazaviile. and
pp. l-150.
Vanhoutte,
P.M.
(1982)
Heterogenous
behaviour of the canine arterial and venous wall: importance of the endothelium. Ebeigbe.
A.B.
hydralazine-induced Cardiovascular
Circulation
and Aloamaka,
Research 51, 439-447. C.P.
relaxation
(1985)
of arterial
Mechanisms smooth
of
muscle.
Research 19, 4-05.
Ebeigbe, A.B. and Aloamaka,
C.P. (1987) Role of endothelium
286 in magnesium-induced
relaxation of rat aorta.
Experimental
187, 25-31.
Medicine
Reseurch
in
Ebeigbe. A.B.. Cressier. F.. Konneh. M.K., Luu. D.T. and Criscione, L. (1990) Influence of N6-monomethyl-t-arginine on endothelium-dependent relaxations in the perfused messenteric vascular bed of the rat. Biochemicnl und Biophysicul Reseurch Communicutions 169. 873-879. Egleme. C.. Godfraind, T. and Miller, R.C. (1984) Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells. British Journul of Phurmucology 81. 1620.
Furchgott. R.F. and Zawadzki. J.V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288. 373-376. Obiefuna, P.C.M., Sofola. O.A. and Ebeigbe. A.B. (1991) Dietary salt-loading attenuates endothelium-dependent relaxa-
tion in response to histamine but not to acetylcholine in rat aortic rings Experimentul Physiology 76. 135-l 38. Rapoport, R.M. and Murad, F. (1983) Agonist-induced endothelium-dependent relaxation in rat thordcic aorta may be mediated through cGMP. Circulution Rcwurch 52. 351-357.
Van de Voorde. J. and Leusen. I. (1983) Role of the endotheliurn in the vasodilator response of rat thoracic aorta histamine. Europwn Journul of Phurmucology 87, I 13-l 18. Vanhoutte. P.M. (1987) The end of the quest? Nu/urc, 327. 459460. White, R.E. and Carrier, G.O. (1986) Alpha) and alpha? adrenoceptor agonist-induced contraction in rat mesenteric artery upon removal of endothelium. Europeun Journul o/ Phurmucology 122. 349-352.
34 (1991) 283-286
283
Elsevier Scientific Publishers Ireland Ltd.
Short communication
Endothelium-dependent rat aortic relaxation to the aqueous leaf extract of Musanga cecropioides A. Kamanyia, M. Bopelet”, C.P. Aloamakab,
P.C.M. Obiefunab and A.B. EbeigbeC
‘Animal Physiology Laboratory, Faculty of Science, University of Yaounde, BP 812, Yaounde (Cameroon), bDepartment of Physiology, College of Medical Sciences, University of Benin, Benin City and L’Department of Physiology, Faculty of Basic Medical Science, Bendel State University, P.M. B. 14, Ekpoma, Bendel State (Nigeria)
(Accepted April 30. 1991)
Introduction An aqeuous leaf extract of Musanga cecropioides (family: Cecropiaceae) is used in traditional medical practice to induce labour due to its oxytocic effect (Bouquet, 1969); it has also been reported to have a blood pressure lowering effect in humans (Bouquet, 1969). Agents that lower blood pressure could act via one of several vasodilator mechanisms (Bolton, 1979; Ebeigbe and Aloamaka, 1985). In the present paper, the possibility that M. cecropioides could have a direct vasodilating effect has been examined using isolated rat aorta.
of Yaounde, Cameroon, and a voucher specimen deposited at the herbarium of the Faculty of Science, University of Yaounde. The leaves were sun-dried and ground into powder in a high-speed grinding mill. Three hundred grams of the tine powder were extracted in 1 1 of boiling water for 12 h, filtered and the residue discarded. About 850 ml of filtrate (MC) was recovered with a concentration equivalent to 350 mg/ml of the dry starting material.
Materials and Methods Extract preparation
Fresh leaves of M. cecropioides were collected from Moliwe in Fako Division of the Southwest Province of Cameroon in November 1989. The plant material was authenticated by Dr. Amougu Akoa of the Botany Department of the University Correspondence to: Professor A.B. Ebeigbe, Department of Physiology, Faculty of Basic Medical Science, Bendel State University, P.M.B. I4 Ekpoma, Bendel State, Nigeria.
0378-8741/$03.50 0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
Tissue preparation
Wistar strain rats weighing between 150-200 g were killed by cervical dislocation. The thoracic aorta was quickly removed, freed of connective tissues, cut into 2 mm ring segments and suspended on tine stainless steel rods in a 20-ml tissue bath and connected to a Grass FT.03 transducer for isometric tension measurement under a resting tension of 2 g. The bath contained physiological salt solution (PSS) of the following composition (mM): NaCl 119, KC1 4.7, MgS04 1.2, KH2P04 1.2, CaClz 1.6, NaHC03 24.9 and glucose 11.5. The PSS was bubbled continuously with a 95% O2 5% CO, gas mixture. The pH of the medium was 7.4 and temperature was maintained thermostatically at 37°C.
284
Protocol
Each tissue was precontracted with a bath concentration of lo-’ M noradrenahne (NA) and relaxations due to 3.5 x 10e3 g/ml of MC or 10m5 M acetylcholine (ACh) were tested on tissues with and without endothelium. Endothelium was removed by gently rubbing the inner lining of the rings. The influence of 1 PM methylene blue on the relaxation responses to Ach and MC was also examined. In other experiments, the effect of MC was also observed under baseline (non-contracted) tension where the dose response of the aortic rings to MC was tested in the presence and absence of endothelium. Values were expressed as percent of maximum response in the presence of endothelium. The source of the noradrenaline bitartrate, acetylcholine HCl and methylene blue was Sigma Chemical Co. (St. Louis, MO, U.S.A.). Statistics
Median effective concentration (EC&) values were computed using a programme for logit transformation of concentration-response curves. Comparison of mean values was effected using Student’s t-test. A P value of less than 0.05 was considered significant.
Fig.
I.
Representative
tracings
showing
responses of rat aortic rings to 3.5 x water extract of Musunga cwropioides acetylcholine (Ach) following IO-’ induced contractions in the presence endothelium.
the
relaxation
IO-’ g/ml of the boiling leaves (MC) and IO-’ M M noradrenaline (NA)(+E) or absence (-E) of
to the cumulative addition of MC. The maximal contractile response to MC was 1095 f 70 mg in the absence of endothelium and 890 f 55 mg in the presence of endothelium. The difference was statistically significant. The EC& values for MCinduced contractions in the absence and presence of endothelium were 9.37 (h1.51) x 10e4 g/ml and 2.63 (kO.14) x lo-” g/ml respectively. Discussion
Results The contractile response to lo-’ M NA was 1193 f 13 mg (mean f S.E.M.) in aortic rings with intact endothelium. Ach caused dosedependent relaxation of NA-induced contractions as previously reported from our laboratory (Ebeigbe and Aloamaka, 1987; Aloamaka et al., 1990; Obiefuna et al., 1991). Maximal Achinduced relaxation was 85.3 + 4.7X, n = 15 (Figs. 1 and 2); whereas MC elicited relaxation by 21.8 f 2.4%,, n = 8. Both Ach (n = 6) and MC (n = 4) failed to elicit relaxation in endothelium-denuded aortic rings (Figs. 1 and 2). Exposure of the aortic rings to lo-(’ M methylene blue (MB) prevented Ach-induced (n = 4) and MC-induced (n = 4) relaxations (Fig. 2). In baseline (non-precontracted) aortic rings, MC caused concentration-dependent contraction. Figure 3 shows results of the contractile responses
The results of the present study demonstrate that MC has a dual effect on the contractile state of rat aortic smooth muscle. When MC was applied under baseline (resting) condition, MC elicited
Fig. 2. Summary
of all results
obtained
using
the scheme
described in Fig. I. The influences of presence (+E) or absence (-E) of endothelium and exposure to 10-e M methylene blue (MB) on the relaxation responses of aortic rings to IO-’ M Ach and 3.5 x IO-’ g/ml MC following NA-precontraction are shown. Each bar represents experiments.
the mean
* S.E.M.
of 4 separate
285
0' I
I
4
3 -Log (g/ml)
Fig. 3. Concentration-response
M.
I
1
2
1
Cecropioides
curve for MC-induced
contrac-
tion of rat aortic rings. Values are expressed as percent of maximum
response
in
Endothelium-removal
the
presence
of
endothelium.
(n = 4) resulted in enhanced contractions
compared
to controls
indicated
for each point.
(n =
8); The
mean
&
S.E.M.
are
endothelium-dependent relaxations cause elevation of cyclic GMP levels within the smooth muscle (Rapoport and Murad, 1983) and methylene blue prevents the formation of cyclic GMP by inhibiting guanylate cyclase (Rapoport and Murad, 1983). Enhancement of contractile response following removal of the endothelium has been reported for a number of agonists e.g. clonidine (Egleme et al., 1984; White and Carrier, 1986) and noradrenaline and methoxamine (White and Carrier, 1986). In the present study, the observed enhancement of contractile response to MC in endotheliumdenuded rings suggests that MC induced the release of EDRF which dampens tone in the intact preparation. In conclusion, the present study shows that a crude aqueous leaf extract of MC elicits contractile response under baseline conditions, but induces endothelium-dependent relaxation of precontracted rat aortic smooth muscle. Acknowledgements
concentration-dependent contractions; however, following precontraction induced by NA, MC elicited endothelium-dependent relaxation. The magnitude of Ach-induced relaxation is significantly greater than that of MC, suggesting that MC is less effective in inducing the release of endothelium-derived relaxant factor (EDRF). EDRF is known to regulate vascular smooth muscle tone (Furchgott and Zawadzki, 1980; Ebeigbe et al., 1990). It is noteworthy that other vasodilators known to lower vascular resistance through the release of EDRF, e.g. histamine (Van de Voorde and Leusen, 1983; Obiefuna et al., 1991), ATP (De Mey and Vanhoutte, 1982) and hydralazine (Aloamaka and Ebeigbe, 1987), also elicit weaker relaxations than Ach. It is possible that different EDRFs mediate the vasorelaxant effect of MC and Ach. Evidence exists that the vascular endothelium may release more than one EDRF (Vanhoutte, 1987). The failure of both Ach and MC to elicit relaxation responses in the presence of methylene blue (Fig. 2) provides additional evidence that MC relaxation is mediated via the release of EDRF. Agents that elicit
The authors are grateful to Mr. R. Tatchum for assistance in the collection and processing of the plant materials. The assistance of Tony Oguogho is greatly appreciated. References Aloamaka,
C.P. and Ebeigbe. A.B.
(1987)
Influence
thelium on rat aortic smooth muscle relaxation hydralazine. Aloamaka, Ebeigbe,
Medical
Science Reseurch 15, 1491-1492.
C.P.,
Evbuomwan,
A.B.
(1990)
rat aortic relaxation ing inhibition
of endo-
to D600 and
M.1..
Attenuated
to acetylcholine
of nitric
oxide
Tosicology and Pharmacology Bolton. T.B. (1979) Mechanisms
Ighoroje,
A.D.A.
and histamine follow-
formation
from
t-arginine.
67, 266268. of action of transmitters
other substances on smooth muscle. Physiological 50, 6067
and
endothelium-dependent
and
Reviews
IS.
Bouquet. A. (1969) Feticheurs et Medicine rraditionelles du Congo. Orstrom, De
Mey,
J.G.
Brazaviile. and
pp. l-150.
Vanhoutte,
P.M.
(1982)
Heterogenous
behaviour of the canine arterial and venous wall: importance of the endothelium. Ebeigbe.
A.B.
hydralazine-induced Cardiovascular
Circulation
and Aloamaka,
Research 51, 439-447. C.P.
relaxation
(1985)
of arterial
Mechanisms smooth
of
muscle.
Research 19, 4-05.
Ebeigbe, A.B. and Aloamaka,
C.P. (1987) Role of endothelium
286 in magnesium-induced
relaxation of rat aorta.
Experimental
187, 25-31.
Medicine
Reseurch
in
Ebeigbe. A.B.. Cressier. F.. Konneh. M.K., Luu. D.T. and Criscione, L. (1990) Influence of N6-monomethyl-t-arginine on endothelium-dependent relaxations in the perfused messenteric vascular bed of the rat. Biochemicnl und Biophysicul Reseurch Communicutions 169. 873-879. Egleme. C.. Godfraind, T. and Miller, R.C. (1984) Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells. British Journul of Phurmucology 81. 1620.
Furchgott. R.F. and Zawadzki. J.V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288. 373-376. Obiefuna, P.C.M., Sofola. O.A. and Ebeigbe. A.B. (1991) Dietary salt-loading attenuates endothelium-dependent relaxa-
tion in response to histamine but not to acetylcholine in rat aortic rings Experimentul Physiology 76. 135-l 38. Rapoport, R.M. and Murad, F. (1983) Agonist-induced endothelium-dependent relaxation in rat thordcic aorta may be mediated through cGMP. Circulution Rcwurch 52. 351-357.
Van de Voorde. J. and Leusen. I. (1983) Role of the endotheliurn in the vasodilator response of rat thoracic aorta histamine. Europwn Journul of Phurmucology 87, I 13-l 18. Vanhoutte. P.M. (1987) The end of the quest? Nu/urc, 327. 459460. White, R.E. and Carrier, G.O. (1986) Alpha) and alpha? adrenoceptor agonist-induced contraction in rat mesenteric artery upon removal of endothelium. Europeun Journul o/ Phurmucology 122. 349-352.