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Effect of chronic administration of dexfenfluramine on blood pressure in salt-sensitive rats
Life Sciences, Vol. Printed in the USA
51, pp.
1021-1024
Pergamon
Press
EFFECT OF CHRONIC ADMINISTRATION OF DEXFENFLURAMINE ON BLOOD PRESSURE IN SALT-SENSITIVE RATS Neil E. Rowland, Melvin J. Fregly, Carolyn M. Poortman & Han Li Departments of Psychology and Physiology University of Florida, Gainesville, FL 32611 (Received
in final
form July
14,
1992)
Summary Chronic administration of dexfenfluramine, at a relatively low daily dose, slowed the development of salt-induced elevation of blood pressure in saltsensitive (Dahl DS and Rapp SS) rats. This effect could not be accounted for by drug-related anorexia alone. Elevated serotonin activity, possibly in the brain, may underlie this antihypertensive action of dexfenfluramine.
Dexfenfluramine (DFEN) is the active enantiomer of a substituted phenethylamine, fenfluramine [1]. Its neural effects, including action as an appetite suppressant, are dependent upon its actions both to release and to block the reuptake of serotonin (5HT) at nerve terminals [2,3]. All forms of experimental hypertension tested thus far are, in part at least, modulated by 5HT function. Systolic blood pressure in each of the renal, deoxycorticosterone-salt, spontaneously hypertensive (SHR) and Dahl salt-sensitive (DS) rat models of hypertension can be either prevented or reduced by peripheral administration of either L-tryptophan or L-5-hydroxytryptophan (5HTP) [4-6]. These agents are precursors of 5HT, and their exogenous administration increases the synthesis of 5HT in brain. Direct 5HT agonists also have antihypertensive action [7]. In both instances, a 5HT-noradrenergic interaction may be involved. Fenfluramine has been reported to both increase [8] and decrease [9] blood pressure, although most such studies have used high doses in anesthetized, normotensive animals. In an acute study in unanesthetized SHR [10], administration of d,l-fenfluramine produced a dose-dependent decrease in systolic pressure. This effect was prevented by the 5HT antagonist, metergoline, in a recent study in normotensive women, dexfenfluramine had a small hypotensive effect [11].
In the present experiments, DFEN was administered chronically to rats at dosages of either 2 or 6 mg/kg/day. The higher of these doses has sustained effects on body weight, and decreases brain 5HT levels [12, 13]. The lower dose has less marked effects on weight, but does not deplete brain 5HT. Preliminary experiments using female DS rats (abstract, [16]) we found that the higher dose of DFEN prevented the increases in blood pressure caused by high NaCI diet. The mean +SE increase in pressure was 18+6 mm Hg in the NaCI-only group, and -1+8 mm (p<0.05) in the NaCI + DFEN group, after 3 days of treatment. The mean body weight changes at this time were identical (+4g) in both groups suggesting that anorexia due to DFEN was minimal. However, because female DS rats do not become as hypertensive as males [14,15], we felt that a stronger demonstration of the effect of DFEN on development of high blood pressure would come from studies in males. In Study A, we use male DS rats and in Study B we use the genetically homozygous strain inbred by Rapp (JR/SS).
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1022
Dexfenfluramine
and Hypertension
Vol.
51, No. 13, 1992
Methods Animals and Housing: In Study A, eighteen 3 mo old male Dahl rats of the DS line, originating from the Brookhaven stock, were obtained from Harlan Labs and started in their experiments within 2 wk of arrival. In Study B, thirty 2 mo old Rapp salt sensitive (JR/SS) rats were obtained from Harlan Labs, and were held for 4 weeks prior to study. All rats were housed singly in stainless steel cages (19x26x18 cm) with tap water available ad libitum. Prior to the main experimental phase, Purina Rodent Chow (#5001) was available ad libitum. The vivarium was illuminated from 0800-2000 hr, and the average temperature was 23°C. Blood pressure measurements: Systolic blood pressure was recorded via a tail cuff and chart recorder (Narco Biosystems). The rats were warmed in group cages under an incandescent light, and were briefly restrained in a warmed plexiglas holder during the pressure recording. Rats were adapted several times to restraint and once to the tail cuff prior to the start of the experiment. Experimental treatments: In St.udy A, DS rats with mean body weight 427 g were divided into three groups of six. Two groups were fed moist (1:1, food:water) powdered Purina Chow to which NaCI was added (4% by weight of dry diet), and the control group received the moist diet without NaCI supplement. One of the high NaCI groups received a 14-day minipump (Alzet #2002) filled with DFEN to deliver 6 mg/kg/24 hr (dose as HCI salt; courtesy of Servier labs). Minipumps were implanted under the loose skin of the back using brief metofane anesthesia. Moist food was used in this study because we have found the moist high salt versions to be more palatable than equivalent dry diet: the rats spill less, lose less weight, and DS rats are less prone to acquiring an apparent learned aversion to the diet. Systolic blood pressure and body weight were recorded after 3, 7 and 14 days. Between-group statistical comparisons were made by 1-way ANOVA and Duncan post-hoc tests with significance level set at p<0.05. In Study B, which was designed to extend the findings of Study A to JR/SS rats, to use two dosages of DFEN, and to include a concurrent food restriction group, JR/SS rats with mean body weight 315 g were divided into five groups of six. The groups were: control (regular chow), 4% NaCI (moist chow + 4% NaCI), salt diet plus either low (2 mg/kg/day) or high (6 mg/kg/day) dosages of DFEN via osmotic minipump, and moist salt diet at 40 g/day (equal to 20 g dry food, or about 80% of normal intake) throughout the 14 day treatment phase. Systolic pressures were recorded after 3, 7 and 14 days, and again 14 days after the end of drug treatment. The high salt diet was continued in the 4 dietary groups during this phase. The previously restricted group was fed the high salt diet ad libitum during the final 14 days. Systolic blood pressures and body weights, as well as their changes from baseline values, were analyzed using 1- and 2-way ANOVA and post-hoc Duncan tests with significance level set at P<0.05. Results Study A: Male DS rats fed the 4% NaCI diet showed a rapid increase in blood pressure. This increase was completely prevented by concurrent treatment with DFEN (Figure 1). ANOVA main effects of group and days, and their interaction, were all highly significant (Ps<0.001). DFEN also induced body weight loss of 28 g during the first 7 days of treatment, significantly (P < 0.05) less than the weight change (-6 g) of the 4% NaCI only group. This is presumably a result of DFEN's anorectic action. In order to make an initial assessment of the extent to which decreased food intake might affect blood pressure, rats from the control (regular chow) group were subsequently placed on restricted access to moist 4% NaCI diet (40 g/day). After 7 days, they lost 65 g body weight and showed no increased in blood pressure. Thus, weight loss more severe than than that caused by DFEN prevents development of high blood pressure. Study B: The JR/SS rats showed a significant increase in blood pressure after after 14 days on the high salt diet (Table 1). This increase was completely abolished by treatment with either dose of DFEN, and without loss of body weight relative to the NaCI-only group. Food restriction also prevented development of high blood pressure, but in this case weight loss was substantial. During the 14 days after the minipumps expired, the pressures rose to the level of the NaCI-only group. The previously restricted group had regained some weight and their blood pressure increased during the 14 day post-restriction phase.
Vol. 51, NO. 13, 1992
Dexfenfluramine and Hypertension
1023
A
5o
o
~:
40
•
8
2o
~
10-
Z
MALE DS • NoCI+DFEN
Z~
o.
~ -10~ -20
0
3
7
days
1#
FIG. 1 Mean change in systolic blood pressure of male DS rats fed either standard chow or 4% NaCI-enriched diet, with or without administration of DFEN for 14 days. *P<0.05 NaCI diet differs from NaCI+DFEN. # P<0.05 vs control.
TABLE 1. Mean change from baseline in body weight and systolic blood pressure in JR/SS Rats (Study B) during (days 1-14) and after (day 28) treatments.
Group Chow control High NaCI NaCI + DFEN (6) NaCI + DFEN (2) NaCI/restricted
Body weight (g) 3 7 14 +7 20 +2 13 -14 2+ +2 13 -32 *+ -31 *+
40 32 29 33 -19*+
28 days
Blood pressure (mm Hg) 3 7 14 28 days
62 47 43 53 15*+
9 5 4 2 0
10 12 -3* 4 1
16* 40 5* 13" 4*
16* 43 53 62 28
*p<0.05 differs from high NaCI only group. +p<0.05 differs from high NaCI. Mean initial body weight = 315 g; mean initial blood pressure = 151 mm Hg (all groups). Dose of DFEN, in mg/kg/day, is shown in parentheses. Discussion In agreement with our preliminary study in female rats [16], these results confirm that DFEN prevents NaCI-induced elevation of blood pressure. This effect was fully and rapidly reversed after the treatment phase. Food restriction was also able to obtund elevation in blood pressure, but we do not believe that
1024
Dexfenfluramine
and Hypertension
vol.
51, No.
13, 1992
anorexia alone can fully account for the effect of DFEN on blood pressure. Thus, the weight loss due to DFEN occurred for only 3-7 days, while the effect on blood pressure was maximal for at least 14 days. Further, weight loss caused by food restriction was much more severe than that due to DFEN. At these doses, DFEN does not lower blood pressure of normotensive 'DR' rats (data not shown). Based on acute studies in vivo and in vitro, there is compelling evidence to suggest that the principal action of DFEN is to increase brain 5HT transmission by uptake inhibition and 5HT release [2,3,17]. The evidence that this effect is sustained over a 2 week treatment period, and/or whether biochemical tolerance develops, is less well-established. Thus, we speculate but do not prove that the sustained depressor effect of DFEN is due to increased 5HT transmission. In summary, chronic administration of DFEN may delay or prevent the onset of high blood pressure in unanesthetized, nonobese rats that are genetically predisposed to high blood pressure. The effect was sustained throughout a 2 week treatment period. These data complement results with DFEN in women [11 ]. Supported by grants from IRI Servier, and the American Heart Association, FL affiliate. We thank M. Garcia, J. Griffin and G. Smith for assistance. Address correspondence to N. Rowland, Department of Psychology, University of Florida, Gainesville, FL 32611-2065. References 1. 2. 3.
4. 5.
6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
J.C. LE DOUAREC, H. SCHMITT, AND M. LAUBIE, Arch. Int. Pharmacodyn. Therap. 161 206-232, (1966). S. GARATTINI, A. BIZZI, S. CACCIA, T. MENNINI, AND R SAMANIN, Clin. Neuropharmac., 1_[1Suppl. 1, $8-$33, (1988). N.E. ROWLAND AND J. CARLTON, ProD. Neurobiol., 27 13-62, (1986). M.J. FREGLY, O.E. LOCKLEY, J. VAN DEN VOORT, C. SUMNERS AND W,N. HENLEY, Can. J. Physiol. Pharmacol., 6,5 753-764, (1987). M.J. FREGLY, O.E. LOCKLEY, AND J.R. CADE, Pharmacology, 36 91-100, (1988). L.A. LARK, P.A. WILL, K.B. DECKER, W.M. STUDZINSKI AND J.A. WEYHENMEYER, Clin. Exper. Hyper: Theory & Prac., A12 1-13, (1990). P. VANHOUTTE, AND 21 OTHERS, Hypertension, 1.~1111-133, (1988). G.I. SIPES, RJ. ZIANCE AND J.P. BUCKLEY, J. Pharmac. Exp. Therap., 176 220-228, (1971). A.J. RAMIREZ, E. CHEMERINSKI & M.A.ENERO, Gen. Pharm., 13 427-432, (1982). RW. FULLER, T.T. YEN AND N.B. STAMM, Clin. Exp. Hypertension, 3 497-508, (1981). B. ANDERSSON, ME. ZIMMERMAN, T. HEDNER AND P. BJORNTORP, Eur. J. Clin. Pharmacol., 40 249-254 (1991). R. ROSEN, F. FUMERON, D. BETOULLE, F. BAIGTS, A. MANDENOFF, J. FRICKER AND M. APFELBAUM, Clinical Neuropharmacol., 1._[1(Suppl. 1) $105-$112, (1988). N.E. ROWLAND, Life Sciences, 3_.£92581-2586, (1986). L.K. DANE, D. KNUDSEN, E.V. OHANIAN, M. MUIRHEAD AND a. TUTHILL, J. Exp. Med. 14..~2,748759, (1975). N.E. ROWLAND AND MJ. FREGLY, Clin. Exper. Hyper: Theory & Prac., A1..~4367-375, (1992). C. POORTMAN, G. SMITH, M.J. FREGLY AND N.E. ROWLAND. FASEB J., 5 A851, (1991). M. GOBBI, E. FRITTOLI, T. MENNINI AND S. GARATTINI. Naunyn-Schmiedeberg's Arch. Pharmacol. 34.~51-6, (1992).
51, pp.
1021-1024
Pergamon
Press
EFFECT OF CHRONIC ADMINISTRATION OF DEXFENFLURAMINE ON BLOOD PRESSURE IN SALT-SENSITIVE RATS Neil E. Rowland, Melvin J. Fregly, Carolyn M. Poortman & Han Li Departments of Psychology and Physiology University of Florida, Gainesville, FL 32611 (Received
in final
form July
14,
1992)
Summary Chronic administration of dexfenfluramine, at a relatively low daily dose, slowed the development of salt-induced elevation of blood pressure in saltsensitive (Dahl DS and Rapp SS) rats. This effect could not be accounted for by drug-related anorexia alone. Elevated serotonin activity, possibly in the brain, may underlie this antihypertensive action of dexfenfluramine.
Dexfenfluramine (DFEN) is the active enantiomer of a substituted phenethylamine, fenfluramine [1]. Its neural effects, including action as an appetite suppressant, are dependent upon its actions both to release and to block the reuptake of serotonin (5HT) at nerve terminals [2,3]. All forms of experimental hypertension tested thus far are, in part at least, modulated by 5HT function. Systolic blood pressure in each of the renal, deoxycorticosterone-salt, spontaneously hypertensive (SHR) and Dahl salt-sensitive (DS) rat models of hypertension can be either prevented or reduced by peripheral administration of either L-tryptophan or L-5-hydroxytryptophan (5HTP) [4-6]. These agents are precursors of 5HT, and their exogenous administration increases the synthesis of 5HT in brain. Direct 5HT agonists also have antihypertensive action [7]. In both instances, a 5HT-noradrenergic interaction may be involved. Fenfluramine has been reported to both increase [8] and decrease [9] blood pressure, although most such studies have used high doses in anesthetized, normotensive animals. In an acute study in unanesthetized SHR [10], administration of d,l-fenfluramine produced a dose-dependent decrease in systolic pressure. This effect was prevented by the 5HT antagonist, metergoline, in a recent study in normotensive women, dexfenfluramine had a small hypotensive effect [11].
In the present experiments, DFEN was administered chronically to rats at dosages of either 2 or 6 mg/kg/day. The higher of these doses has sustained effects on body weight, and decreases brain 5HT levels [12, 13]. The lower dose has less marked effects on weight, but does not deplete brain 5HT. Preliminary experiments using female DS rats (abstract, [16]) we found that the higher dose of DFEN prevented the increases in blood pressure caused by high NaCI diet. The mean +SE increase in pressure was 18+6 mm Hg in the NaCI-only group, and -1+8 mm (p<0.05) in the NaCI + DFEN group, after 3 days of treatment. The mean body weight changes at this time were identical (+4g) in both groups suggesting that anorexia due to DFEN was minimal. However, because female DS rats do not become as hypertensive as males [14,15], we felt that a stronger demonstration of the effect of DFEN on development of high blood pressure would come from studies in males. In Study A, we use male DS rats and in Study B we use the genetically homozygous strain inbred by Rapp (JR/SS).
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1022
Dexfenfluramine
and Hypertension
Vol.
51, No. 13, 1992
Methods Animals and Housing: In Study A, eighteen 3 mo old male Dahl rats of the DS line, originating from the Brookhaven stock, were obtained from Harlan Labs and started in their experiments within 2 wk of arrival. In Study B, thirty 2 mo old Rapp salt sensitive (JR/SS) rats were obtained from Harlan Labs, and were held for 4 weeks prior to study. All rats were housed singly in stainless steel cages (19x26x18 cm) with tap water available ad libitum. Prior to the main experimental phase, Purina Rodent Chow (#5001) was available ad libitum. The vivarium was illuminated from 0800-2000 hr, and the average temperature was 23°C. Blood pressure measurements: Systolic blood pressure was recorded via a tail cuff and chart recorder (Narco Biosystems). The rats were warmed in group cages under an incandescent light, and were briefly restrained in a warmed plexiglas holder during the pressure recording. Rats were adapted several times to restraint and once to the tail cuff prior to the start of the experiment. Experimental treatments: In St.udy A, DS rats with mean body weight 427 g were divided into three groups of six. Two groups were fed moist (1:1, food:water) powdered Purina Chow to which NaCI was added (4% by weight of dry diet), and the control group received the moist diet without NaCI supplement. One of the high NaCI groups received a 14-day minipump (Alzet #2002) filled with DFEN to deliver 6 mg/kg/24 hr (dose as HCI salt; courtesy of Servier labs). Minipumps were implanted under the loose skin of the back using brief metofane anesthesia. Moist food was used in this study because we have found the moist high salt versions to be more palatable than equivalent dry diet: the rats spill less, lose less weight, and DS rats are less prone to acquiring an apparent learned aversion to the diet. Systolic blood pressure and body weight were recorded after 3, 7 and 14 days. Between-group statistical comparisons were made by 1-way ANOVA and Duncan post-hoc tests with significance level set at p<0.05. In Study B, which was designed to extend the findings of Study A to JR/SS rats, to use two dosages of DFEN, and to include a concurrent food restriction group, JR/SS rats with mean body weight 315 g were divided into five groups of six. The groups were: control (regular chow), 4% NaCI (moist chow + 4% NaCI), salt diet plus either low (2 mg/kg/day) or high (6 mg/kg/day) dosages of DFEN via osmotic minipump, and moist salt diet at 40 g/day (equal to 20 g dry food, or about 80% of normal intake) throughout the 14 day treatment phase. Systolic pressures were recorded after 3, 7 and 14 days, and again 14 days after the end of drug treatment. The high salt diet was continued in the 4 dietary groups during this phase. The previously restricted group was fed the high salt diet ad libitum during the final 14 days. Systolic blood pressures and body weights, as well as their changes from baseline values, were analyzed using 1- and 2-way ANOVA and post-hoc Duncan tests with significance level set at P<0.05. Results Study A: Male DS rats fed the 4% NaCI diet showed a rapid increase in blood pressure. This increase was completely prevented by concurrent treatment with DFEN (Figure 1). ANOVA main effects of group and days, and their interaction, were all highly significant (Ps<0.001). DFEN also induced body weight loss of 28 g during the first 7 days of treatment, significantly (P < 0.05) less than the weight change (-6 g) of the 4% NaCI only group. This is presumably a result of DFEN's anorectic action. In order to make an initial assessment of the extent to which decreased food intake might affect blood pressure, rats from the control (regular chow) group were subsequently placed on restricted access to moist 4% NaCI diet (40 g/day). After 7 days, they lost 65 g body weight and showed no increased in blood pressure. Thus, weight loss more severe than than that caused by DFEN prevents development of high blood pressure. Study B: The JR/SS rats showed a significant increase in blood pressure after after 14 days on the high salt diet (Table 1). This increase was completely abolished by treatment with either dose of DFEN, and without loss of body weight relative to the NaCI-only group. Food restriction also prevented development of high blood pressure, but in this case weight loss was substantial. During the 14 days after the minipumps expired, the pressures rose to the level of the NaCI-only group. The previously restricted group had regained some weight and their blood pressure increased during the 14 day post-restriction phase.
Vol. 51, NO. 13, 1992
Dexfenfluramine and Hypertension
1023
A
5o
o
~:
40
•
8
2o
~
10-
Z
MALE DS • NoCI+DFEN
Z~
o.
~ -10~ -20
0
3
7
days
1#
FIG. 1 Mean change in systolic blood pressure of male DS rats fed either standard chow or 4% NaCI-enriched diet, with or without administration of DFEN for 14 days. *P<0.05 NaCI diet differs from NaCI+DFEN. # P<0.05 vs control.
TABLE 1. Mean change from baseline in body weight and systolic blood pressure in JR/SS Rats (Study B) during (days 1-14) and after (day 28) treatments.
Group Chow control High NaCI NaCI + DFEN (6) NaCI + DFEN (2) NaCI/restricted
Body weight (g) 3 7 14 +7 20 +2 13 -14 2+ +2 13 -32 *+ -31 *+
40 32 29 33 -19*+
28 days
Blood pressure (mm Hg) 3 7 14 28 days
62 47 43 53 15*+
9 5 4 2 0
10 12 -3* 4 1
16* 40 5* 13" 4*
16* 43 53 62 28
*p<0.05 differs from high NaCI only group. +p<0.05 differs from high NaCI. Mean initial body weight = 315 g; mean initial blood pressure = 151 mm Hg (all groups). Dose of DFEN, in mg/kg/day, is shown in parentheses. Discussion In agreement with our preliminary study in female rats [16], these results confirm that DFEN prevents NaCI-induced elevation of blood pressure. This effect was fully and rapidly reversed after the treatment phase. Food restriction was also able to obtund elevation in blood pressure, but we do not believe that
1024
Dexfenfluramine
and Hypertension
vol.
51, No.
13, 1992
anorexia alone can fully account for the effect of DFEN on blood pressure. Thus, the weight loss due to DFEN occurred for only 3-7 days, while the effect on blood pressure was maximal for at least 14 days. Further, weight loss caused by food restriction was much more severe than that due to DFEN. At these doses, DFEN does not lower blood pressure of normotensive 'DR' rats (data not shown). Based on acute studies in vivo and in vitro, there is compelling evidence to suggest that the principal action of DFEN is to increase brain 5HT transmission by uptake inhibition and 5HT release [2,3,17]. The evidence that this effect is sustained over a 2 week treatment period, and/or whether biochemical tolerance develops, is less well-established. Thus, we speculate but do not prove that the sustained depressor effect of DFEN is due to increased 5HT transmission. In summary, chronic administration of DFEN may delay or prevent the onset of high blood pressure in unanesthetized, nonobese rats that are genetically predisposed to high blood pressure. The effect was sustained throughout a 2 week treatment period. These data complement results with DFEN in women [11 ]. Supported by grants from IRI Servier, and the American Heart Association, FL affiliate. We thank M. Garcia, J. Griffin and G. Smith for assistance. Address correspondence to N. Rowland, Department of Psychology, University of Florida, Gainesville, FL 32611-2065. References 1. 2. 3.
4. 5.
6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
J.C. LE DOUAREC, H. SCHMITT, AND M. LAUBIE, Arch. Int. Pharmacodyn. Therap. 161 206-232, (1966). S. GARATTINI, A. BIZZI, S. CACCIA, T. MENNINI, AND R SAMANIN, Clin. Neuropharmac., 1_[1Suppl. 1, $8-$33, (1988). N.E. ROWLAND AND J. CARLTON, ProD. Neurobiol., 27 13-62, (1986). M.J. FREGLY, O.E. LOCKLEY, J. VAN DEN VOORT, C. SUMNERS AND W,N. HENLEY, Can. J. Physiol. Pharmacol., 6,5 753-764, (1987). M.J. FREGLY, O.E. LOCKLEY, AND J.R. CADE, Pharmacology, 36 91-100, (1988). L.A. LARK, P.A. WILL, K.B. DECKER, W.M. STUDZINSKI AND J.A. WEYHENMEYER, Clin. Exper. Hyper: Theory & Prac., A12 1-13, (1990). P. VANHOUTTE, AND 21 OTHERS, Hypertension, 1.~1111-133, (1988). G.I. SIPES, RJ. ZIANCE AND J.P. BUCKLEY, J. Pharmac. Exp. Therap., 176 220-228, (1971). A.J. RAMIREZ, E. CHEMERINSKI & M.A.ENERO, Gen. Pharm., 13 427-432, (1982). RW. FULLER, T.T. YEN AND N.B. STAMM, Clin. Exp. Hypertension, 3 497-508, (1981). B. ANDERSSON, ME. ZIMMERMAN, T. HEDNER AND P. BJORNTORP, Eur. J. Clin. Pharmacol., 40 249-254 (1991). R. ROSEN, F. FUMERON, D. BETOULLE, F. BAIGTS, A. MANDENOFF, J. FRICKER AND M. APFELBAUM, Clinical Neuropharmacol., 1._[1(Suppl. 1) $105-$112, (1988). N.E. ROWLAND, Life Sciences, 3_.£92581-2586, (1986). L.K. DANE, D. KNUDSEN, E.V. OHANIAN, M. MUIRHEAD AND a. TUTHILL, J. Exp. Med. 14..~2,748759, (1975). N.E. ROWLAND AND MJ. FREGLY, Clin. Exper. Hyper: Theory & Prac., A1..~4367-375, (1992). C. POORTMAN, G. SMITH, M.J. FREGLY AND N.E. ROWLAND. FASEB J., 5 A851, (1991). M. GOBBI, E. FRITTOLI, T. MENNINI AND S. GARATTINI. Naunyn-Schmiedeberg's Arch. Pharmacol. 34.~51-6, (1992).