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Effect of the neurotensin receptor antagonist SR48692 on rat blood pressure modulation by neurotensin
Life Sciences, Vol. 58, No. 8, pp. 665-674, 19% CopyTight0 19% Elscvier !Jcience Inc. Printed in the USA. All rights mewed 0024-32051% $15.00 t .oo
ELSEVIER
00243205(95)02335-6
EFFECT OF THE NEUROTENSIN RECEPTOR ANTAGONIST SR48692 ON BAT BLOOD PRESSURE MODULATION BY NEUROTENSIN D. Gully, L. Lespy, M. Canton, W. Rostene, P. Kitabgi, G. Le Fur* and J.P. Matiand Sanofi Recherche, 195 route d’Espagne, 3 1036 Toulouse CCdex and *32-34, rue Marbeuf, 75008 Paris, France (Received in final form December 4, 1995)
When administered as an intravenous injection in the pentobarbitoneanaesthetized rat, neurotensin (NT) elicits a biphasic depressor-pressor effect that can be evaluated by the mean arterial blood pressure (MABP). The first hypotensive phase elicited by low doses of NT is dependent on the interaction of NT with its specific receptors and may be mediated by the release of histamine, since it is prevented by oral pretreatment with the selective NT receptor antagonist SR 48692 and by intravenous pretreatment with a selective H1 receptor antagonist mepyramine. The hypertensive effect evoked by higher doses of NT is histamine-independent but remains NT receptor-mediated. The prevention of the biphasic effect on MABP by oral administration of the NT receptor antagonist SR 48692 validates the implication of NT receptors in the histamine release phenomenon. Key Words: neurotensin, vasopressor effects, histamine, SR 48692
NT is a tridecapeptide found in numerous areas of the central nervous system, in the gastrointestinal tract and in circulating blood of animals and also in man (5). It possesses a wide range of effects on both the central and peripheral nervous systems and on peripheral tissues in particular in the cardiovascular system (5, 3, 4). In vitro, NT contracts some isolated blood vessels such as the rat portal vein (19) and the coronary vasculature of rat heart (16). When given intravenously to the rat, NT dilates cutaneous vessels and increases vascular permeability (8). Administered at low doses, NT evokes hypotension in anaesthetized rats and at intermediate doses a transient hypotension preceding a slight hypertension. At higher doses, only hypertension is seen (5, 18). In contrast with the effects observed in rats, intravenous NT injection in guinea pigs reveals only a hypertensive response (17). The hypotension induced at low and medium doses of NT in anaesthetized rats is completely abolished when the endogenous histamine stores of animals are depleted by pretreatment with the compound 48/80 (a histamine and serotonin releaser from mast cells, 18) or with disodium cromoglycate (an antiallergic drug which is believed to stabilize mast cells membranes, 18) or when histamine H 1 receptors were blocked by mepyramine pretreatment (18) proving an evident role of endogenous mediators such as histamine and serotonin (6, 8). In addition, NT has been described earlier as a potent histamine releaser increasing histamine plasma level after intravenous injection (6). Repeated intravenous injections of NT leads to a prolonged unresponsiviness that became entirely refractory to the hypotensive effects of compound 48/80, suggesting a common site of action for NT and this compound. In fact, the presence of NT receptors on mast cells (13) argues for a direct effect of NT (6).
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In summary, NT administered intravenously in anaesthetized rats induces a severe decrease or a slight increase in blood pressure or both effects depending on the dose. The intervention of a release of histamine in the hypotensive effect is demonstrated in rats. However the implication of specific NT receptors in this event remains yet to be elucidated. The recent discovery of a potent and selective non-peptide antagonist of NT receptor (10) that was described to antagonize the cardiovascular effect elicited by NT in guinea pigs (16) provides a useful tool for determining the possible role of the NT receptor in the vasopressor effect of this peptide in anaesthetized rats (10).
Chemicals. SR 48692 [{[l-(7-chloroquinolein-4-y1)-5-(2,6-dimethoxypheny1)-1H-pyr~ole-3carbonyll-amino}tricyclo(3.3.1.1 .3.7) decan-2-carboxylic acid] was synthesized at Sanofi Recherche (Montpellier, France) and was suspended with Tween 80 in an aqueous solution of 0.6 % methylcellulose for oral administration. NT, histamine, acetylcholine hydrochloride and NG-nitro-L-arginine methyl ester (L-NAME) were obtained from Sigma Chemical Co (L’isle d’Abeau, France), mepyramine was from Research Biomedical Inc. (Natick MA, USA), indomethacin as “indocid injectable” was provided from M.S.D. Laboratories and substance P was from Bachem. Experimental protocol. The experiments were performed on male Sprague-Dawley rats (weighing 250-300 g) anaesthetized with sodium pentobarbitone (30 mg.kg-1, intraperitoneal, supplemented as required) and kept warm at 37°C. Catheters were implanted into the left jugular vein (for the administration of drugs) and into the right carotid artery (for blood pressure measurement). This latter catheter was connected to a Statham P 10 EZ pressure transducer coupled to a Gould RS 3400 recorder (Oxnard, CA, USA) for continous monitoring of mean arterial blood pressure (MABP) and heart rate (HR). Both catheters were tilled with a 2% saline solution of heparin 2500 UI (Roche, France). When the diastolic blood pressure and heart rate were stabilized (60-90 mm Hg ; 320-480 beats.min-*, respectively), exogenous NT was injected intravenously (1 ml.kg-l) in a wide range of doses (0.2 to 10 nmol.kg-l). Only one dose of peptide could be tested in each animal because of tachyphylaxis. SR 48692 was always administered per OS as a suspension in a 0.6 % methyl cellulose saline solution, one, two or three hours prior to the bolus injection of NT. Mepyramine, L-NAME and indomethacin were administered intravenously as a bolus at the dose of 3 or 10 mg.kg-1 to groups of anaesthetized rats fifteen minutes before NT injection. Measurements were made before, during and for 15 minutes following Histamine (0.1 mg.kg’l), substance P (2.5 pg.kg-1) and acetylcholine (10 also injected intravenously in bolus after one hour oral treatment with 1 mg.kgAnalysis of data. Variations in MABP were expressed as means f S.E.M., in millimeters of Hg or as a percentage of variation versus the maxima1 level obtained after NT administration (n is the number of animals). Statistical analysis of the data was performed by ANOVA followed by a Dunnett’s t test for multiple comparisons.
MABP in control animals receiving the corresponding vehicles remained stable throughout the study period and was unaffected by the volumes of vehicle administered (Table 1). Bolus intravenous injection of NT produced two kinds of effect in MABP of the anaesthetized rat as
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revealed in Table 1 by the MABP values measured one minute after NT injection. A dosedependent and significant decrease in blood pressure developed within the first minute after injection at the lowest NT dose 0.4 nmol.kg-l and the MABP level returned quickly to preinjection levels in about five minutes (Fig. 1). TABLE 1 Effect of NT on the mean arterial blood pressure of anaesthetized rats
MABP Control (mm Hg*S.E.M.)
101 f 4 102*4 103* 4 109* 6 109*5
MABP 1 min after Injection of Indicated Dose of NT (nmol.kg-l) 0.2 0.4 1.0 4.0 10.0
76 f 5**
Animals number
4
50 f 8***
6 27 f 4***
6 120* 7
4 120*6
5
Intravenous injection ofNT was performed in the range of 0.2 to 10 nmol.kg-l(one animal per dose), in pentobarbitone-anaesthetized rats. MABP values measured one minute after the NT injection, were expressed in mmHg as mean values f S.E.M.. Statistical significance was expressed versus corresponding basal values, ** p < 0.01 ; *** p < 0.001. The maximum decrease in MABP that could be evoked by NT injections was important, from 103 f 4 to 27 f 4 mm Hg (n = 6) at 1 nmol.kg-1 (Table 1). At this dose, results obtained during time-course studies of the NT effect were different, the peak effect was always within the minute, but the depressor effect was sustained and still significant fitly minutes after NT injection (Fig 1). With increasing doses of NT, the depressor effect disappeared and a slight, but reproducible increase in MABP became evident for the two tested doses (4 or 10 nmolkg-l, Table 1 and Fig. 1). Oral administration of SR 48692 (0.1 - 10 mg.kg-1) did not alter basal levels of MABP (data not shown). As shown in Table 2, oral doses of SR 48692 (0.1 - 1 mg. kg-I) administered 1 hr prior to NT injection, dose-dependently inhibited (32 to 95 %) the depressor effect of 0.4 nmolkg-I NT. However, ten to a hundred fold higher doses of SR 48692 (l-10 mg.kg-I) were necessary to only partially inhibit (< 53 Oh) the more prolonged depressor effect obtained with a 2.5 fold higher amount of NT (1 nmol.kg-1, Table 2). Similar experiments, performed using a 4 nmol.kg-I NT stimulus revealed that SR 48692 also dose-dependently inhibited the hypertensive response (100 % inhibition at 10 mg. kg-1 SR 48692). Time-course study of the inhibitory effect of SR 48692 (1 mg.kg-1) tested against 0.4 nmol.kg-I NT, demonstrate a shorter duration of action (lower than 3 hours, Fig. 2) than previously reported in neuropharmacological models on conscious animals (5 to 6 hours, 10).
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-60
I
1
I
I
0
5
10
15
20
limes (mid
FIG. 1 Kinetic profile of the modulation of MABP by increasing doses of NT. NT was injected as intravenous bolus in the rat at the doses of (0) 0.4 (n = 14) ; (a), 1.0 (n = 19) ; (A), 4 (n = 4) and (V), 10 nmol.kg-l (n = 5). Results were expressed as the means f S.E.M. of the MABP variations measured in mm Hg.
TABLE 2 SR 48692 antagonism of the biphasic pressor effects of NT in the anaesthetized rat NT Injection (nmokkg-1) 0.4 1.0 4.0
0.0 - 50.2 (f 11.1) - 75.2 (f 6.9) + 10.8 (f 1.7)
Changes in MABP after NT in Rats Pretreated with SR 48692 at Indicated Dose (mg.kg-1) 0.01 0.1 10 1.0 - 33.6 (* 6.9) ND ND
+ 4.6*** (* 2.4) (Z.9) + 9.4 (A 2.0)
+ 2.6*** (’ 1.9) - 53.0” (f 5.4) + 6.6 (f 1.4)
ND - 35.0*** - 0.6** (* 2.5)
Results were expressed as variations of MAE3P in mmHg with S.E.M. in brackets. ND means not determined. Statistical significance p versus NT controls for each group : * p
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SR 48692
NT -60
FIG. 2 Time-course study of the inhibition by SR 48692 of NT-induced h potension. The experiments were performed in rats pretreated orally by 1 mg.kg- Y SR 48692 at various times (-1 H, -2 H, -3 H) prior the injection of 0.4 nmol.kg-1NT (5 to 6 animals per group). Results were expressed as MABP variations in mm Hg obtained 1 minute after NT injection. Statistical significance versus NT controls * p < 0.05 ; ** p < 0.01. NW) mepfl0)
20
0
mepf31
NT(0.4)
j NT(l)
FIG. 3 Inhibition by mepyramine of the biphasic effects of NT on the rat MABP. Results of experiments performed in the presence of injections of 0.4, 1.0 and 10 nmol.kg-l of NT were expressed as MABP variations in mm Hg obtained 1 min after NT injection, Mepyramine (hatched plots) was injected intravenously, 15 min prior to NT. Respective doses of NT (NT, nmokkg-1) and mepyramine (mep, mg.kg-lj were noted in brackets. Statistical significance versus NT controls ** p < 0.01 ; *** p < 0.001.
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Acute intravenous pretreatment with 3 mg.kg-l(1 ml.kg-1) mepyramine, a potent and selective histamine Hl receptor antagonist, fifteen minutes prior to NT completely abolished the vasodepressor effect of a low dose of NT (0.4 nmol.kg-l), partially but significantly prevented the maximum effect obtained with 1 nmol.kg-1 of peptide but was unable to mod@ the hypertensive effect-induced by 4 nmol.kg-l of NT, even at the highest dose of mepyramine 10 mg.kg-1 (Fig 3). This last dose of me yramine inhibits significantly the hypotension elicited by the medium dose of NT (1 nmol.kg- P, Fig. 4). In the same model, mepyramine is able to block the hypotension induced by bolus injection of 0.1 mg.kg-1 histamine (results expressed in MABP means decrease : - 48 f 8 mm Hg for histamine value and - 6 f 3 mm Hg for mepyramine values obtained at both 3 and 10 mg.kg-l). 20 ,
-60
I 0
I 5
I
I
10
15
20
Times (min) FIG. 4 Kinetics of the partial prevention by mepyramine (W, n = 10) pretreatment of the severe hypotension induced by 1 nmol.kg-l NT (alone,.). Mepyramine was administered intravenously 15 min prior to NT injection at the dose of 10 mg.kg-1. Results were expressed as MABP variations calculated in mm Hg and measured continuously until 15 minutes after the NT injection. Statistical significance versus corresponding basal values : * p < 0.05 ; ** p < 0.01. The uncomplete prevention of NT-induced hypotension suggests that vasomodulators other than histamine may mediate part of the considerable depressor effect of NT in the anaesthetized rat and encouraged us to perform complementary experiments. Using a similar protocol, we tested compounds able to antagonize the effects of other well-known vasomodulatory agents, such as NG-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor) and indomethacin (cyclooxygenase inhibitor). Intravenous injections of these inhibitors were performed at the dose of 10 mg.kg-l fifteen minutes prior to the NT injection. Neither L-NAME nor indomethacin were capable of completely reversing the NT effect even if both compounds partially prevented the acute hypotension obtained within the first minutes (Fig. 5). In fact, they were devoid of action on the sustained pressor effect measured ten minutes later, suggesting that neither nitric oxide nor cyclooxygenase pathways were implicated.
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0
-20
-40
-60
’
I
I
I
I
0
5
10
15
Times
20
(mid
FIG. 5 Lack of effect of L-NAME (W, n ~10) and indomethacin (A, n = 10) in reversing the sustained hypotension induced by the NT dose of 1 nmol. kg-l (alone, 0). Both compounds were administered intravenously at the dose of 10 mg/kg-I, 15 minutes prior to NT injection. Results were expressed as MAEIP variations calculated in mm Hg and measured continuously until 15 minutes after the NT injection. Statistical significance versus corresponding basal values : * p < 0.05 ; ** p < 0.01. TABLE Selectivity
of the inhibitory
3
effect of SR 48692 on NT-induced hypotension
MABP variations (mm Hg f S.E.M.) Histamine
Control
-41*5
SR 48692
-40*5
(0.1 mg.kg-l)
Control
-42~6
Substance P (2.5 pg.kg-1) SR 48692 Control
-41*
7
- 61 f 10
Acetylcholine (10 pg.kg-l) SR 48692
-54*
7
Animals were pretreated by oral administration of 1 mg.kg-1 SR 48692, 1 hour prior to bolus intravenous injection of histamine, substance P or acetylcholine (the respective doses are indicated in brackets). MABP values measured one minute after the mediator injection, were expressed in mm Hg as mean values * S.E.M..
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The specificity of the inhibition obtained with SR 48692 was demonstrated by the lack of MAFJP variation measured in similar experiment using oral pretreatment with SR 48692 (1 mg.kg-*, one hour prior to NT) and bolus intravenous injection of various compounds capable to induce a comparable hypotension, such as histamine (0.1 mg.kg-I), substance P (2.5 ug.kg-*) and acetylcholine (10 ug.kg -l)(Table 3).
Discussion In the pentobarbitone anaesthetized rat, intravenous bolus injections of small doses (from 0.2 to 0.4 nmol.kg-1) of NT induced a significant and dose-dependent hypotension characterized by a rapid tachyphylaxis that justified the use of only one animal per dose. The amplitude of this hypotension varied between individual animals whatever the level of basal MABP values. The depressor effect evoked by low doses of NT was completely abolished by intravenous mepyramine pretreatment. These results were in agreement with previous reports describing that histamine release from mast cells was implicated in these hypotensive effects of NT. They were also in agreement with the presence of specific binding sites for NT on rat mast cells (2) and with the capacity for NT to induce rat mast cell degranulation in vitro (6, 20). In addition, increasing histamine levels were measured in plasma after intravenous NT injections (6) and the histamine release by NT was inhibited by pretreating the animals with the mast cell degranulating agent compound 48/80 or with disodium cromoglycate (18). It is unknown whether the histamine release induced by NT is obtained by a direct effect of the peptide on G proteins as suggested by Mousli (15) and by Aridor et al (1) or is receptor-mediated as concluded by many researchers (22, 11). However results obtained in inhibiting mast cell secretion of histamine by peptide antagonists (14) or by the non peptide compound SR 48692 (7) validate more precisely the receptor-mediated hypothesis. The next step in our studies demonstrates that although the NT-induced hypotension was mediated by release of histamine, it could be prevented for more than two hours by oral pretreatment with the selective non peptide antagonist of NT receptors, SR 48692 at dose as low as 1 mg.kg-l. Obviously, the histamine release involved in the hypotensive effect of NT was NT receptor-mediated because SR 48692, at micromolar concentrations, was devoid of affinity for the histamine HI receptor (data not shown). The efficacy of SR 48692 could signify that the first event is an interaction of NT with specific receptors in the vicinity of vessels subsequently causing a release of histamine responsible for the hypotension. In addition, SR 48692 was unable to modifL hypotension induced by other vasomodulators such as histamine, substance P or acetylcholine, confirming the specificity previously demonstrated in binding assays for this non-peptide NT antagonist (10). Nevertheless, intermediate doses of NT (1.0 nmol.kg-1) evoked an important decrease in pressure parameters differing in its kinetics of onset and duration from the decrease obtained at the dose of 0.4 nmol.kg-1 (more than five minutes of duration of action). This sustained hypotension was only partially prevented (60 %) by a hundred fold higher doses of SR 48692 (10 mg.kg-l), suggesting the involvement at higher doses of NT, of another system besides histamine in example mast cells serotonin (17). The part of the hypotension not reversed by the antagonist may be caused by other mediators, including serotonin, endothelium-derived relaxing factor/nitric oxide and cyclooxygenase products, This hypothesis could be verified by investigating whether or not the hypotension was decreased after blockade of the nitric oxide synthesis by L-NAME (24) or after blockade of the cyclooxygenase pathway by indomethacin. In fact, kinetics recording established that both compounds reduced the transient phase of hypotension but not the sustained one.
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It was interesting to note that the injection of higher doses (10 nmol.kg-l ) of NT induced a slight but significant pressor effect which was also NT receptor-dependent because prevented by SR 48692 oral pretreatment with a till antagonism at 10 nmolkg-1 and it remained completely insensitive to histamine H1 blockade by mepyramine pretreatment, This hyperpressor effect was previously described by Di Paolo et al, 1990 (9) and was probably mediated by an activation of the sympathetic nervous system, for instance by catecholamines released from adrenal medulla as in the case of NT-evoked hypertension in guinea pigs (20). In conclusion, utilization of the orally potent and selective non peptide antagonist of NT receptors SR 48692 has provided pharmacological evidence for the role of these NT receptors in the biphasic depressor and pressor effects induced by intravenous injection of NT in the pentobarbitone-anaesthetized rat. Moreover, the stimulation of NT receptors by lower doses of the peptide elicited a release of histamine responsible for rapid hypotension (prevented by Hl receptor blockade). At higher doses of NT, may be causing the release of catecholamines leading to the slight hypertension. The effect of intermediate doses of NT could be separated into transient and sustained hypotensions, the former partially histamine release-dependent and the latter related to other mechanisms (independent of nitric oxide synthesis or release of cyclooxygenase derivatives), may be mediated by serotonin release and not yet well understood. The biphasic characteristic of the NT response was a species-dependent phenomenon. For instance, in rabbits, pigs, goats and dogs, NT was found to produce a decrease in blood pressure while in guinea pigs, woodchucks, sheep and cats, only pressor responses were observed (20). However it is necessary to keep in mind that, as in numerous pharmacological models, NT produces biphasic effects depending on the level of dose administered. In fact, the NT vasopressor response is related to the sensitivity of the species to release histamine, rats being perhaps the most sensitive species. It is interesting to speculate that the presence of mast cells and peptidergic neurons containing NT in numerous tissues is responsible for the possible role of this peptide in as yet undetermined pathophysiological states.
Acknowledgement$ We would like to thank A.J. Patacchini for her help&l comments Lasserre and M. Wanner for their technical assistance.
on the manuscript,
D.
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M. ARIDOR, L. TRAUB and R. SAGI-EISENBERG, J. Cell. Biol. u909-917 (1990). Z. BAR-SHAVIT, S. TERRY, S. BLUMBERG and R. GOLDMAN, Neuropeptides 2 325-335 (1982). 3. G. BISSETTE, B. MANBERG, C. NEMEROFF and A. PRANGE, Life Sci. 23 21732183 (1978). 4. D. BROWN and R. MILLER, Br. Med. Bull. 38 239-245 (1982). 5. R. CARRAWAY and S. LEEMAN, J. Biol. Chem. 248 6854-6861 (1973). 6. R. CARRAWAY, D. COCHRANE, J. LANSMAN, S. LEEMAN, B. PATERSON and H WELCH, J. Physiol. m 404-4 14 (1982). 7. D. COCHRANE, R. FELDBERG, L. MILLER, R. CARRAWAY and A. BARROCAS, FASEB J. 8 Al494 (1994). 8. L. CHAHL, Naunyn-Schmiedeberg’s Arch. Pharmacol. 309 159-163 (1979). 9. E. DI PAOLO and E. RICHELSON, Eur. J. Pharmacol. 175 279-283 (1990). 10. D. GULLY, M. CANTON, R. BOIGEGRAIN, F. JEANJEAN, J.C. MOLIMARD, M. PONCELET, C. GUEUDET, M. HEAULME, R. LEYRIS, A. BROUARD, D.
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PELAPRAT, C. LABBE-JUILLE, J. MAZELLA, P. SOUBRIE, J.P. MAFFRAND, W. ROSTENE, P. KITABGI and G. LE FUR, Proc. Natl. Acad. Sci. .%J65-69 (1993). R. KEROUAC, S. St-PIERRE and F. RIOUX, Peptides 5 695-699 (1984). M. KUROSE and K. SAEKI, Eur. J. Pharmacol.l4 129-136 (1981). L. LAZARUS, M. PERRIN, M. BROWN and J. RIVIER, Biochem and Biophys.Res. Comm. 76 1079-1085 (1977). L. MILLER, D. COCHRANE, R. CARRAWAY and R. FELDBERG, Agents and actions 3 l-7 (1993). M. MOUSLI, J.L. BUEB, C. BRONNER, R. ROUOT and Y. LANDRY, TIPS 1135% 362 (1990). D. NISATO, P. GUIRAUDOU, G. BARTHELEMY, G. GULLY and G. LE FUR, Life Sci.% 95-100 (1994). R. QUIRION, F. RIOUX, S. ST-PIERRE and D. REGOLI, Eur. J. Pharmaco1.s 221-223 (1979). R. QUIRION, F. RIOUX, D. REGOLI and S. St-PIERRE, Life Sci. 27 1889-1895 (1980). F. RIOUX, R. QUIRION, D. REGOLI, M.A. LEBLANC and S. St-PIERRE, Eur. J. Pharmacol. &,j 273-279 (1980). F. RIOUX, R. KEROUAC, R. QUIRION and S. St-PIERRE, Annals of the New York Acad. of Sci. 400 56-74 (1982) F. RIOUX, R. KEROUAC and S. St-PIERRE, Peptides 121-125 (1985). R. SAGI-EISENBERG, Z. BEN-NERIAH, I. PECHT, S. TERRY and S. BLUMBERG, Neuropharmacol. 22 197-20 1 (1983). SCHAEFFER P., LAPLACE M.C., SAVI P., PFLIEGER A.M., GULLY D. and J.M. HERBERT, The J. of Biol. Chem.270, 3409-3413 (1995). C. WALDER, C. TI-IIEMERMANN and J. VANE, Br. J. Pharmacol. 107 476-480 (1992).
ELSEVIER
00243205(95)02335-6
EFFECT OF THE NEUROTENSIN RECEPTOR ANTAGONIST SR48692 ON BAT BLOOD PRESSURE MODULATION BY NEUROTENSIN D. Gully, L. Lespy, M. Canton, W. Rostene, P. Kitabgi, G. Le Fur* and J.P. Matiand Sanofi Recherche, 195 route d’Espagne, 3 1036 Toulouse CCdex and *32-34, rue Marbeuf, 75008 Paris, France (Received in final form December 4, 1995)
When administered as an intravenous injection in the pentobarbitoneanaesthetized rat, neurotensin (NT) elicits a biphasic depressor-pressor effect that can be evaluated by the mean arterial blood pressure (MABP). The first hypotensive phase elicited by low doses of NT is dependent on the interaction of NT with its specific receptors and may be mediated by the release of histamine, since it is prevented by oral pretreatment with the selective NT receptor antagonist SR 48692 and by intravenous pretreatment with a selective H1 receptor antagonist mepyramine. The hypertensive effect evoked by higher doses of NT is histamine-independent but remains NT receptor-mediated. The prevention of the biphasic effect on MABP by oral administration of the NT receptor antagonist SR 48692 validates the implication of NT receptors in the histamine release phenomenon. Key Words: neurotensin, vasopressor effects, histamine, SR 48692
NT is a tridecapeptide found in numerous areas of the central nervous system, in the gastrointestinal tract and in circulating blood of animals and also in man (5). It possesses a wide range of effects on both the central and peripheral nervous systems and on peripheral tissues in particular in the cardiovascular system (5, 3, 4). In vitro, NT contracts some isolated blood vessels such as the rat portal vein (19) and the coronary vasculature of rat heart (16). When given intravenously to the rat, NT dilates cutaneous vessels and increases vascular permeability (8). Administered at low doses, NT evokes hypotension in anaesthetized rats and at intermediate doses a transient hypotension preceding a slight hypertension. At higher doses, only hypertension is seen (5, 18). In contrast with the effects observed in rats, intravenous NT injection in guinea pigs reveals only a hypertensive response (17). The hypotension induced at low and medium doses of NT in anaesthetized rats is completely abolished when the endogenous histamine stores of animals are depleted by pretreatment with the compound 48/80 (a histamine and serotonin releaser from mast cells, 18) or with disodium cromoglycate (an antiallergic drug which is believed to stabilize mast cells membranes, 18) or when histamine H 1 receptors were blocked by mepyramine pretreatment (18) proving an evident role of endogenous mediators such as histamine and serotonin (6, 8). In addition, NT has been described earlier as a potent histamine releaser increasing histamine plasma level after intravenous injection (6). Repeated intravenous injections of NT leads to a prolonged unresponsiviness that became entirely refractory to the hypotensive effects of compound 48/80, suggesting a common site of action for NT and this compound. In fact, the presence of NT receptors on mast cells (13) argues for a direct effect of NT (6).
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In summary, NT administered intravenously in anaesthetized rats induces a severe decrease or a slight increase in blood pressure or both effects depending on the dose. The intervention of a release of histamine in the hypotensive effect is demonstrated in rats. However the implication of specific NT receptors in this event remains yet to be elucidated. The recent discovery of a potent and selective non-peptide antagonist of NT receptor (10) that was described to antagonize the cardiovascular effect elicited by NT in guinea pigs (16) provides a useful tool for determining the possible role of the NT receptor in the vasopressor effect of this peptide in anaesthetized rats (10).
Chemicals. SR 48692 [{[l-(7-chloroquinolein-4-y1)-5-(2,6-dimethoxypheny1)-1H-pyr~ole-3carbonyll-amino}tricyclo(3.3.1.1 .3.7) decan-2-carboxylic acid] was synthesized at Sanofi Recherche (Montpellier, France) and was suspended with Tween 80 in an aqueous solution of 0.6 % methylcellulose for oral administration. NT, histamine, acetylcholine hydrochloride and NG-nitro-L-arginine methyl ester (L-NAME) were obtained from Sigma Chemical Co (L’isle d’Abeau, France), mepyramine was from Research Biomedical Inc. (Natick MA, USA), indomethacin as “indocid injectable” was provided from M.S.D. Laboratories and substance P was from Bachem. Experimental protocol. The experiments were performed on male Sprague-Dawley rats (weighing 250-300 g) anaesthetized with sodium pentobarbitone (30 mg.kg-1, intraperitoneal, supplemented as required) and kept warm at 37°C. Catheters were implanted into the left jugular vein (for the administration of drugs) and into the right carotid artery (for blood pressure measurement). This latter catheter was connected to a Statham P 10 EZ pressure transducer coupled to a Gould RS 3400 recorder (Oxnard, CA, USA) for continous monitoring of mean arterial blood pressure (MABP) and heart rate (HR). Both catheters were tilled with a 2% saline solution of heparin 2500 UI (Roche, France). When the diastolic blood pressure and heart rate were stabilized (60-90 mm Hg ; 320-480 beats.min-*, respectively), exogenous NT was injected intravenously (1 ml.kg-l) in a wide range of doses (0.2 to 10 nmol.kg-l). Only one dose of peptide could be tested in each animal because of tachyphylaxis. SR 48692 was always administered per OS as a suspension in a 0.6 % methyl cellulose saline solution, one, two or three hours prior to the bolus injection of NT. Mepyramine, L-NAME and indomethacin were administered intravenously as a bolus at the dose of 3 or 10 mg.kg-1 to groups of anaesthetized rats fifteen minutes before NT injection. Measurements were made before, during and for 15 minutes following Histamine (0.1 mg.kg’l), substance P (2.5 pg.kg-1) and acetylcholine (10 also injected intravenously in bolus after one hour oral treatment with 1 mg.kgAnalysis of data. Variations in MABP were expressed as means f S.E.M., in millimeters of Hg or as a percentage of variation versus the maxima1 level obtained after NT administration (n is the number of animals). Statistical analysis of the data was performed by ANOVA followed by a Dunnett’s t test for multiple comparisons.
MABP in control animals receiving the corresponding vehicles remained stable throughout the study period and was unaffected by the volumes of vehicle administered (Table 1). Bolus intravenous injection of NT produced two kinds of effect in MABP of the anaesthetized rat as
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revealed in Table 1 by the MABP values measured one minute after NT injection. A dosedependent and significant decrease in blood pressure developed within the first minute after injection at the lowest NT dose 0.4 nmol.kg-l and the MABP level returned quickly to preinjection levels in about five minutes (Fig. 1). TABLE 1 Effect of NT on the mean arterial blood pressure of anaesthetized rats
MABP Control (mm Hg*S.E.M.)
101 f 4 102*4 103* 4 109* 6 109*5
MABP 1 min after Injection of Indicated Dose of NT (nmol.kg-l) 0.2 0.4 1.0 4.0 10.0
76 f 5**
Animals number
4
50 f 8***
6 27 f 4***
6 120* 7
4 120*6
5
Intravenous injection ofNT was performed in the range of 0.2 to 10 nmol.kg-l(one animal per dose), in pentobarbitone-anaesthetized rats. MABP values measured one minute after the NT injection, were expressed in mmHg as mean values f S.E.M.. Statistical significance was expressed versus corresponding basal values, ** p < 0.01 ; *** p < 0.001. The maximum decrease in MABP that could be evoked by NT injections was important, from 103 f 4 to 27 f 4 mm Hg (n = 6) at 1 nmol.kg-1 (Table 1). At this dose, results obtained during time-course studies of the NT effect were different, the peak effect was always within the minute, but the depressor effect was sustained and still significant fitly minutes after NT injection (Fig 1). With increasing doses of NT, the depressor effect disappeared and a slight, but reproducible increase in MABP became evident for the two tested doses (4 or 10 nmolkg-l, Table 1 and Fig. 1). Oral administration of SR 48692 (0.1 - 10 mg.kg-1) did not alter basal levels of MABP (data not shown). As shown in Table 2, oral doses of SR 48692 (0.1 - 1 mg. kg-I) administered 1 hr prior to NT injection, dose-dependently inhibited (32 to 95 %) the depressor effect of 0.4 nmolkg-I NT. However, ten to a hundred fold higher doses of SR 48692 (l-10 mg.kg-I) were necessary to only partially inhibit (< 53 Oh) the more prolonged depressor effect obtained with a 2.5 fold higher amount of NT (1 nmol.kg-1, Table 2). Similar experiments, performed using a 4 nmol.kg-I NT stimulus revealed that SR 48692 also dose-dependently inhibited the hypertensive response (100 % inhibition at 10 mg. kg-1 SR 48692). Time-course study of the inhibitory effect of SR 48692 (1 mg.kg-1) tested against 0.4 nmol.kg-I NT, demonstrate a shorter duration of action (lower than 3 hours, Fig. 2) than previously reported in neuropharmacological models on conscious animals (5 to 6 hours, 10).
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-60
I
1
I
I
0
5
10
15
20
limes (mid
FIG. 1 Kinetic profile of the modulation of MABP by increasing doses of NT. NT was injected as intravenous bolus in the rat at the doses of (0) 0.4 (n = 14) ; (a), 1.0 (n = 19) ; (A), 4 (n = 4) and (V), 10 nmol.kg-l (n = 5). Results were expressed as the means f S.E.M. of the MABP variations measured in mm Hg.
TABLE 2 SR 48692 antagonism of the biphasic pressor effects of NT in the anaesthetized rat NT Injection (nmokkg-1) 0.4 1.0 4.0
0.0 - 50.2 (f 11.1) - 75.2 (f 6.9) + 10.8 (f 1.7)
Changes in MABP after NT in Rats Pretreated with SR 48692 at Indicated Dose (mg.kg-1) 0.01 0.1 10 1.0 - 33.6 (* 6.9) ND ND
+ 4.6*** (* 2.4) (Z.9) + 9.4 (A 2.0)
+ 2.6*** (’ 1.9) - 53.0” (f 5.4) + 6.6 (f 1.4)
ND - 35.0*** - 0.6** (* 2.5)
Results were expressed as variations of MAE3P in mmHg with S.E.M. in brackets. ND means not determined. Statistical significance p versus NT controls for each group : * p
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SR 48692
NT -60
FIG. 2 Time-course study of the inhibition by SR 48692 of NT-induced h potension. The experiments were performed in rats pretreated orally by 1 mg.kg- Y SR 48692 at various times (-1 H, -2 H, -3 H) prior the injection of 0.4 nmol.kg-1NT (5 to 6 animals per group). Results were expressed as MABP variations in mm Hg obtained 1 minute after NT injection. Statistical significance versus NT controls * p < 0.05 ; ** p < 0.01. NW) mepfl0)
20
0
mepf31
NT(0.4)
j NT(l)
FIG. 3 Inhibition by mepyramine of the biphasic effects of NT on the rat MABP. Results of experiments performed in the presence of injections of 0.4, 1.0 and 10 nmol.kg-l of NT were expressed as MABP variations in mm Hg obtained 1 min after NT injection, Mepyramine (hatched plots) was injected intravenously, 15 min prior to NT. Respective doses of NT (NT, nmokkg-1) and mepyramine (mep, mg.kg-lj were noted in brackets. Statistical significance versus NT controls ** p < 0.01 ; *** p < 0.001.
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Acute intravenous pretreatment with 3 mg.kg-l(1 ml.kg-1) mepyramine, a potent and selective histamine Hl receptor antagonist, fifteen minutes prior to NT completely abolished the vasodepressor effect of a low dose of NT (0.4 nmol.kg-l), partially but significantly prevented the maximum effect obtained with 1 nmol.kg-1 of peptide but was unable to mod@ the hypertensive effect-induced by 4 nmol.kg-l of NT, even at the highest dose of mepyramine 10 mg.kg-1 (Fig 3). This last dose of me yramine inhibits significantly the hypotension elicited by the medium dose of NT (1 nmol.kg- P, Fig. 4). In the same model, mepyramine is able to block the hypotension induced by bolus injection of 0.1 mg.kg-1 histamine (results expressed in MABP means decrease : - 48 f 8 mm Hg for histamine value and - 6 f 3 mm Hg for mepyramine values obtained at both 3 and 10 mg.kg-l). 20 ,
-60
I 0
I 5
I
I
10
15
20
Times (min) FIG. 4 Kinetics of the partial prevention by mepyramine (W, n = 10) pretreatment of the severe hypotension induced by 1 nmol.kg-l NT (alone,.). Mepyramine was administered intravenously 15 min prior to NT injection at the dose of 10 mg.kg-1. Results were expressed as MABP variations calculated in mm Hg and measured continuously until 15 minutes after the NT injection. Statistical significance versus corresponding basal values : * p < 0.05 ; ** p < 0.01. The uncomplete prevention of NT-induced hypotension suggests that vasomodulators other than histamine may mediate part of the considerable depressor effect of NT in the anaesthetized rat and encouraged us to perform complementary experiments. Using a similar protocol, we tested compounds able to antagonize the effects of other well-known vasomodulatory agents, such as NG-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor) and indomethacin (cyclooxygenase inhibitor). Intravenous injections of these inhibitors were performed at the dose of 10 mg.kg-l fifteen minutes prior to the NT injection. Neither L-NAME nor indomethacin were capable of completely reversing the NT effect even if both compounds partially prevented the acute hypotension obtained within the first minutes (Fig. 5). In fact, they were devoid of action on the sustained pressor effect measured ten minutes later, suggesting that neither nitric oxide nor cyclooxygenase pathways were implicated.
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0
-20
-40
-60
’
I
I
I
I
0
5
10
15
Times
20
(mid
FIG. 5 Lack of effect of L-NAME (W, n ~10) and indomethacin (A, n = 10) in reversing the sustained hypotension induced by the NT dose of 1 nmol. kg-l (alone, 0). Both compounds were administered intravenously at the dose of 10 mg/kg-I, 15 minutes prior to NT injection. Results were expressed as MAEIP variations calculated in mm Hg and measured continuously until 15 minutes after the NT injection. Statistical significance versus corresponding basal values : * p < 0.05 ; ** p < 0.01. TABLE Selectivity
of the inhibitory
3
effect of SR 48692 on NT-induced hypotension
MABP variations (mm Hg f S.E.M.) Histamine
Control
-41*5
SR 48692
-40*5
(0.1 mg.kg-l)
Control
-42~6
Substance P (2.5 pg.kg-1) SR 48692 Control
-41*
7
- 61 f 10
Acetylcholine (10 pg.kg-l) SR 48692
-54*
7
Animals were pretreated by oral administration of 1 mg.kg-1 SR 48692, 1 hour prior to bolus intravenous injection of histamine, substance P or acetylcholine (the respective doses are indicated in brackets). MABP values measured one minute after the mediator injection, were expressed in mm Hg as mean values * S.E.M..
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The specificity of the inhibition obtained with SR 48692 was demonstrated by the lack of MAFJP variation measured in similar experiment using oral pretreatment with SR 48692 (1 mg.kg-*, one hour prior to NT) and bolus intravenous injection of various compounds capable to induce a comparable hypotension, such as histamine (0.1 mg.kg-I), substance P (2.5 ug.kg-*) and acetylcholine (10 ug.kg -l)(Table 3).
Discussion In the pentobarbitone anaesthetized rat, intravenous bolus injections of small doses (from 0.2 to 0.4 nmol.kg-1) of NT induced a significant and dose-dependent hypotension characterized by a rapid tachyphylaxis that justified the use of only one animal per dose. The amplitude of this hypotension varied between individual animals whatever the level of basal MABP values. The depressor effect evoked by low doses of NT was completely abolished by intravenous mepyramine pretreatment. These results were in agreement with previous reports describing that histamine release from mast cells was implicated in these hypotensive effects of NT. They were also in agreement with the presence of specific binding sites for NT on rat mast cells (2) and with the capacity for NT to induce rat mast cell degranulation in vitro (6, 20). In addition, increasing histamine levels were measured in plasma after intravenous NT injections (6) and the histamine release by NT was inhibited by pretreating the animals with the mast cell degranulating agent compound 48/80 or with disodium cromoglycate (18). It is unknown whether the histamine release induced by NT is obtained by a direct effect of the peptide on G proteins as suggested by Mousli (15) and by Aridor et al (1) or is receptor-mediated as concluded by many researchers (22, 11). However results obtained in inhibiting mast cell secretion of histamine by peptide antagonists (14) or by the non peptide compound SR 48692 (7) validate more precisely the receptor-mediated hypothesis. The next step in our studies demonstrates that although the NT-induced hypotension was mediated by release of histamine, it could be prevented for more than two hours by oral pretreatment with the selective non peptide antagonist of NT receptors, SR 48692 at dose as low as 1 mg.kg-l. Obviously, the histamine release involved in the hypotensive effect of NT was NT receptor-mediated because SR 48692, at micromolar concentrations, was devoid of affinity for the histamine HI receptor (data not shown). The efficacy of SR 48692 could signify that the first event is an interaction of NT with specific receptors in the vicinity of vessels subsequently causing a release of histamine responsible for the hypotension. In addition, SR 48692 was unable to modifL hypotension induced by other vasomodulators such as histamine, substance P or acetylcholine, confirming the specificity previously demonstrated in binding assays for this non-peptide NT antagonist (10). Nevertheless, intermediate doses of NT (1.0 nmol.kg-1) evoked an important decrease in pressure parameters differing in its kinetics of onset and duration from the decrease obtained at the dose of 0.4 nmol.kg-1 (more than five minutes of duration of action). This sustained hypotension was only partially prevented (60 %) by a hundred fold higher doses of SR 48692 (10 mg.kg-l), suggesting the involvement at higher doses of NT, of another system besides histamine in example mast cells serotonin (17). The part of the hypotension not reversed by the antagonist may be caused by other mediators, including serotonin, endothelium-derived relaxing factor/nitric oxide and cyclooxygenase products, This hypothesis could be verified by investigating whether or not the hypotension was decreased after blockade of the nitric oxide synthesis by L-NAME (24) or after blockade of the cyclooxygenase pathway by indomethacin. In fact, kinetics recording established that both compounds reduced the transient phase of hypotension but not the sustained one.
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It was interesting to note that the injection of higher doses (10 nmol.kg-l ) of NT induced a slight but significant pressor effect which was also NT receptor-dependent because prevented by SR 48692 oral pretreatment with a till antagonism at 10 nmolkg-1 and it remained completely insensitive to histamine H1 blockade by mepyramine pretreatment, This hyperpressor effect was previously described by Di Paolo et al, 1990 (9) and was probably mediated by an activation of the sympathetic nervous system, for instance by catecholamines released from adrenal medulla as in the case of NT-evoked hypertension in guinea pigs (20). In conclusion, utilization of the orally potent and selective non peptide antagonist of NT receptors SR 48692 has provided pharmacological evidence for the role of these NT receptors in the biphasic depressor and pressor effects induced by intravenous injection of NT in the pentobarbitone-anaesthetized rat. Moreover, the stimulation of NT receptors by lower doses of the peptide elicited a release of histamine responsible for rapid hypotension (prevented by Hl receptor blockade). At higher doses of NT, may be causing the release of catecholamines leading to the slight hypertension. The effect of intermediate doses of NT could be separated into transient and sustained hypotensions, the former partially histamine release-dependent and the latter related to other mechanisms (independent of nitric oxide synthesis or release of cyclooxygenase derivatives), may be mediated by serotonin release and not yet well understood. The biphasic characteristic of the NT response was a species-dependent phenomenon. For instance, in rabbits, pigs, goats and dogs, NT was found to produce a decrease in blood pressure while in guinea pigs, woodchucks, sheep and cats, only pressor responses were observed (20). However it is necessary to keep in mind that, as in numerous pharmacological models, NT produces biphasic effects depending on the level of dose administered. In fact, the NT vasopressor response is related to the sensitivity of the species to release histamine, rats being perhaps the most sensitive species. It is interesting to speculate that the presence of mast cells and peptidergic neurons containing NT in numerous tissues is responsible for the possible role of this peptide in as yet undetermined pathophysiological states.
Acknowledgement$ We would like to thank A.J. Patacchini for her help&l comments Lasserre and M. Wanner for their technical assistance.
on the manuscript,
D.
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