Studies on the cardiovascular effects produced by the spinal action of two substance P analogues in the rat: Evidence for a central catecholaminergic mechanism

European Journal of Pharmacology, 135 (1987) 345-354 Elsevier

345

EJP 00693

Studies on the cardiovascular effects produced by the spinal action of two substance P analogues in the rat: evidence for a central catecholaminergic mechanism R r j e a n C o u t u r e 1,., A l k a G u p t a

1 Ren6

K r r o u a c 1 a n d D o m e n i c o Regoli 2

l D~partement de Physiologic, CRSN, Faeult~ de M~deeine, Universit~ de Montreal, C.P. 6208, Succursale A, Montreal, Quebec, Canada H3C 3T8, and 2 D~partement de Physiologie et Pharmacologie, Faeult~ de M~decine, Universit~ de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4 Received 17 July 1986, revised MS received 8 December 1986, accepted 6 January 1987

The effects of two substance P (SP) analogues, [D-Trp7'9'I°]SP (anal) and [D-Pro4,Lys6,D-Trp7'9'a°,Phell]SP-(4-11) (ana2) on mean arterial pressure (MAP) and heart rate (HR) were measured following intrathecal administration at one of three spinal cord levels in rats anaesthetized with sodium pentobarbital. Following an initial increase, a profound and long-lasting fall in MAP and HR occurred when 6.5 nmol of either ana 1 or ana 2 was injected at T1-T3 or T8-T10. Only transient changes in MAP and a slight increase in HR was observed after injection of either peptide at L2-L4. The profound and long-lasting hypotension and bradycardia induced by ana 1 were not significantly altered after intravenous injection of hexamethonium, phentolamine, propranolol, atropine, diphenhydramine, cimetidine, methysergide, naloxone or morphine. However, the biphasic effect of ana I on MAP was prevented by the intrathecal administration of prazosin and yohimbine, suggesting that a central catecholaminergic mechanism including a 1- and a2-adrenergic receptors is involved. The latter treatment did not prevent the tachycardia which occurred when the bradycardia was blocked, indicating that different mechanisms mediate the spinal action of anal on MAP and HR. Finally, cervical transection of the spinal cord eliminated the profound and long-lasting depressor effect of ana 2, suggesting that a supraspinal mechanism is involved in this cardiovascular response. Substance P analogues; Spinal action; Blood pressure; Heart rate; Catecholaminergic mechanism; (Spinal cord transection)

1. I n t r o d u c t i o n

It was assumed on the basis of data obtained with smooth muscle preparations (Leander et al., 1981; Folkers et al., 1982; Regoli et al., 1984) that SP analogues containing D-amino acids will antagonize the spinal actions of tachykinins. It was soon realized that SP antagonists themselves exert effects on the rat spinal cord. Undeca- or C-terminal octapeptide analogues of SP containing D-Trp in positions 7 and 9 or 7, 9 and 10 * To whom all correspondence should be addressed.

cause an initial increase in blood pressure followed by a long-lasting fall to levels below 80 mm Hg within 20-30 min in rats anaesthetized with sodium pentobarbital or urethane. The hypotension was large enough in a number of experiments to cause the death of the animal (Loewy and Sawyer, 1982; Yashpal et al., 1985; Takano et al., 1985a). The present study served to analyze further the cardiovascular effects of two SP analogues [D-TrpT'9'I°]SP (ana.1) and [D-Pro4,Lys6,D Trp7'9'1°,Phe11]SP-(4-11) (ana2) given intrathecally at one of three spinal cord levels (T1-T3, T8-T10

0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

346 and L2-L4) in rats anaesthetized with sodium pentobarbital. Using the appropriate inhibitors we investigated the role played by the autonomic nervous system and the participation of some endogenous substances (e.g. histamine, 5-hydroxytryptamine, acetylcholine, opiates) in the spinal cord-mediated cardiovascular response induced by ana 1. In addition, we examined the effects of ana 2 after cervical transection of the spinal cord to determine whether supraspinal control mechanisms are involved in the long-lasting cardiodepressor effect of this analogue.

2. Materials and methods

2.1. Implantation of intrathecal catheters Male Wistar rats (250-300 g) were purchased from Charles River, St-Constant, Qurbec. A polyethylene catheter (PE-10) was inserted (under sodium pentobarbital anaesthesia, 65 m g / k g i.p.) through an incision made in the dura at the atlanto-occipital junction and extended so that the tip reached the vertebral T1-T3, T8-T10 or L2-L4 spinal cord levels. The correct positioning of the catheter was verified by post-mortem examination. A 7 day recovery period was allowed between implantation of the intrathecal catheter and the start of the experiments. Only rats not showing a motor deficit were used.

2.2. Spinal cord transection Transection of the spinal cord was performed after sodium pentobarbital anaesthesia of catheterized rats (n = 1 3 ) b y exposing the dorsal cervical muscles and sectioning the cord at the level of C6-C7 with a Birtcher (Model 753E) electrocutter. The experiments were conducted 30 min after surgery.

2.3. Experimental procedure The rats were tracheotomized under anaesthesia with sodium pentobarbital (65 m g / k g i.p.) and a cannula was inserted into the trachea to facilitate respiration. The systemic arterial blood pressure

was recorded from a catheter (PE-50) in the right carotid artery using a Statham pressure transducer (P23ID). The heart rate was measured with a cardiac tachometer (model 7P4) that was triggered by the arterial blood pressure pulses. Both signals were recorded on a Grass Polygraph (model 79). One jugular vein was cannulated with polyethylene tubing (PE-50) for intravenous (i.v.) injection of drugs. The rectal temperature of the rat was maintained at 37 ° C with a warming lamp.

2.4. Experimental protocols Arterial blood pressure and heart rate were recorded before each experiment for at least 15 min or until a stable tracing was obtained. At this time the animals were challenged with an intrathecal (i.th.) injection of 20/~1 of artificial cerebrospinal fluid (CSF; aqueous solution of 128.6 mM NaC1, 2.6 mM KC1, 2.0 mM MgC12 and 1.4 mM CaC12; pH adjusted to 7.2). Only those preparations showing no effect on both arterial blood pressure and heart rate were used in the experiments. Twenty minutes after the i.th. application of artificial CSF, ana I or ana 2 were given i.th. at a dose of 6.5 nmol at one of T1-T3, T8-T10 or L2-L4 spinal cord levels. The peptide, in 10/~1 of CSF, was delivered by means of a Hamilton microsyringe (50 ~tl) over a period of 20-30 s and the catheter was flushed with 10 /~1 of CSF over a period of 15-30 s (catheter volume, 6-8 /~1; total injection time for the peptide plus CSF, about 1 rain). Mean arterial blood pressure (MAP) and heart rate (HR) were recorded for a 2 h experimental period. In a second series of experiments the role played by the autonomic nervous system, opiates, endogenous neurotransmitters, autocoids or hormones in the cardiovascular effects of ana I was evaluated by using several inhibitory drugs which were given i.v. or i.th. 15-20 min prior to the i.th. injection of ana 1 at the T8-T10 spinal level. In a third series of experiments, ana 2 was given i.th. at the T8-T10 level in spinal rats and the effects on MAP and H R were compared with those obtained in intact animals.

347 2.5. Peptides and other drugs Anal, ana 2 and [Sarl,ValS]angiotensin II were supplied by Dr. D. Regoli, from Sherbrooke University. The drugs used in these experiments as well as their source are listed below. Noradrenaline hydrochloride, hexamethonium bromide, propranolol hydrochloride, phentolamine hydrochloride, yohimbine hydrochloride, atropine sulphate, diphenhydramine hydrochloride, cimetidine and naloxone hydrochloride were all purchased from Sigma, methysergide bimaleate from Sandoz, prazosin hydrochloride and Sotalol from Pfizer, morphine sulphate from Allen & Hanburys and sodium pentobarbital from M.T.C. Pharmaceuticals. Stock solutions (1 m g / m l ) of ana 2 were prepared in artificial cerebrospinal fluid (CSF). Ana 1 and [Sarl,ValS]angiotensin II were dissolved in pure acetic acid (BDH), the p H was subsequently adjusted to 7.2 with sodium hydroxide (10 N) and CSF was added to obtain the desired solution. The stock solutions of peptides were divided into aliquots of 100/~1 each and stored at - 2 0 ° C until used. The non-peptide compounds were prepared in saline (for i.v. administration) or in CSF (for i.th. administration). Doses of peptides are given in mol of the salt. 2.6. Statistical analysis The results are expressed as means + S.E.M. Unless stated, the significance of differences among groups was evaluated with the one-way analysis o f variance (Wallenstein et al., 1980) and the differences between untreated and inhibitortreated animals were compared by using the modified t statistic according to the method of Dunnett (1964). Probability values of P smaller than 0.05 were considered to be significant. 3. Results 3.1. Cardiovascular responses elicited by the i.th. administration of ana I and ana 2 A preliminary series of experiments concerned the cardiovascular effects of increasing doses of

ana 1 and ana 2 administered i.th. at the T8-T10 spinal level of rats anaesthetized with sodium pentobarbital. Few changes in M A P and H R were found at doses of peptide under 6.5 nmol while a triphasic M A P response and an initial increase followed by a drastic fall of H R were obtained with 6.5 nmol of ana 1 (fig. 1) and ana 2 (fig. 2). The cardiovascular effects produced by ana x and ana 2 described below were therefore measured only with 6.5 nmol of each peptide. Figures 1 and 2 illustrate the changes of MAP and H R induced by ana 1 and ana 2 applied at each of three spinal cord levels. The basal MAP and H R in rats anaesthetized with sodium pentobarbital were respectively 126.3 ___2.2 m m Hg (n = 39) and 405.0 + 13.6 b e a t s / m i n (n = 39). Ana 1 and ana 2 produced a biphasic response of MAP when given at the T1-T3 level. After an initial increase during the first 2-3 rain both analogues produced a marked vasodepressor effect which lasted for more than 45 min. The latter effect started sooner for ana 2 and the maximal hypotension ( - 100 m m Hg) was observed after 30 and 10 rain for ana I and ana 2, respectively. A transient tachycardia which had a higher amplitude for ana 2 appeared during the first 5 min; the H R then decreased drastically to reach - 2 0 0 b e a t s / rain after 25 rain (anal) or - 3 8 0 b e a t s / m i n after 10 rain (ana2). Similar effects were observed when anal and ana 2 were applied at T8-T10. A triphasic M A P response was observed: an initial hypotension of about - 20 m m Hg was followed 1 min later by an increase (20-30 m m Hg) in MAP with a maximum at 3-4 min then by a long-lasting hypotension ( - 8 0 to - 1 0 0 m m Hg for more than 45 min). The H R increased 2-3 rain after the injection of ana 1 or ana z. It reached a m a x i m u m at 4-6 min then decreased drastically to reach - 1 8 0 b e a t s / min (anal) and - 2 5 0 b e a t s / m i n (ana2) after 30 min. When applied at the L2-L4 level, ana I and ana 2 initially produced a fall in MAP. The maximal effect was observed after 1 min and averaged - 4 0 and - 1 8 m m Hg for ana~ and ana 2 respectively. The M A P returned to its pre-administration levels after 4-5 rain (anal) or 3 min (ana2) and thereafter continued to decrease (anax) or to

348

the large S.E.M. for the data recorded between 20-45 min (figs. 1 and 2). All animals survived the administration of the analogues at the L2-L4 level. Neither analogue significantly altered MAP or H R when applied intravenously in 10 intact rats anaesthetized with pentobarbital (results not shown).

increase (ana2) for the next 10 min. Finally, a slight reduction in MAP ( - 1 5 to - 2 0 mm Hg) persisted for 15-45 min after the administration of ana 1 or ana 2. A mild tachycardia was produced by both analogues 5 rain after their administration and lasted for more than 45 min. The magnitude of the cardiodepressor effects of ana 1 and ana 2 decreased in the following order: T1-T3 > TS-T10 > L2-L4 (figs. 1 and 2). Approximately 50% of the animals tested died 20-60 min after the administration of either analogue at T8-T10 and 75% after application of the same peptides at the T1-T3 spinal level. This explains

3.2. Characterization of the effects of anay Inhibitors of endogenous substances (catecholamines, histamine, acetylcholine, etc.) were used to further investigate the mechanism of action of

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349

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a n a 1. T h e effects of various inhibitors on the changes of M A P i n d u c e d b y the i.th. a d m i n i s t r a tion o f 6.5 n m o l o f a n a I at the T8-T10 spinal level were studied at 1, 5 a n d 30 min, which corres p o n d e d to the m a x i m a of the triphasic response of this analogue. T h e effects o f the i n h i b i t o r s on the changes of H R were s t u d i e d at 10 a n d 30 rain, which c o r r e s p o n d e d respectively to the m a x i m u m increase a n d decrease of H R . T h e results o b t a i n e d

in these e x p e r i m e n t s are s u m m a r i z e d in tables 1 a n d 2. H e x a m e t h o n i u m (a ganglionic blocker) a n d p h e n t o l a m i n e plus p r o p r a n o l o l (an a- a n d a Ba d r e n e r g i c i n h i b i t o r ) significantly (P < 0.001) red u c e d b a s a l M A P to 63.4 + 5.8 m m Hg, n = 6 a n d to 75.0 + 4.6 m m Hg, n = 6, respectively. M A P was also significantly r e d u c e d b y the i.th. a d m i n i s t r a t i o n of p r a z o s i n (an a l - a d r e n e r g i c inhibitor),

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yohimbine (an a2-adrenergic inhibitor) and propranolol (results not shown). The effects of these inhibitors on the changes of MAP and H R induced by ana 1 were therefore studied after an i.v. infusion of either noradrenaline (166 n g / m i n per rat) or an analogue of angiotensin II, [Sar 1, ValS]ATu (80 n g / m i n per rat) in order to raise basal MAP close to the values found in untreated animals. This had to be done to compare the effects of the SP analogue on MAP in inhibitortreated animals with those in untreated animals.

Hexamethonium failed to modify the effect of ana 1 on MAP and HR, which suggests that the autonomic nervous system does not take part in these cardiovascular responses. The cardiovascular changes induced by anal remained unaltered in animals treated i.v. with phentolamine plus propranolol. When phentolamine and propranolol were given separately, the cardiovascular effect of ana 1 also remained unchanged (results not shown). In addition, atropine (a muscarinic cholinergic inhibitor), a mixture of diphenhydramine, cimeti-

TABLE 1 Effects of various inhibitors on the changes of MAP induced by the i.th. administration of 6.5 nmol of anaI at Ts-T10 spinal level in pentibarbital-anaesthetized rats a Inhibitor

Dose

n

Baseline

~ MA P to ana 1

MAP

1 min b

5 rain b

30 rain b

Untreated Hexameth onium Phentolamine + propranolol Atropine Diphenhydramine + cimetidine + methysergide Naloxone Morphine

10 m g / k g i.v. 3mg/kgi.v. 3 m g / k g i.v. 3mg/kgi.v. 10, 10 and 5 m g / k g i.v.

6 6 6

130.35:7.5 121.3 + 10.5 ° 122.0+ 5.5 ~

-24.25:13.3 - 12.5 5 : 6 . 4 - 1 . 0 ± 6.1

19.45:15.6 8.85:2.5 -5.75:11.6

-83.05:19.2 -82.25:33.3 -89.85:18.5

5 7

113.05:5.0 113.95:4.3

- 2 3 . 7 + 7.4 -15.65:2,8

9.7± 5.1 32.65:7.5

-48.3±15.6 -82.95:17.4

10 m g / k g i.v. 5 m g / k g i.v.

10 5

109.5 5 : 6 . 9 140.8±10.6

- 25.0 + 4.5 -6.65:12.4

5.4± 9.9 22.85:8.7

-71.3±14.2 -126.45:11.5

Untreated Phentolamine + propranolol Prazosin Yohimbine Prazosin + yohimbine Sotalol Propranolol

100 Fgi-th100 # g i.th. 100 # g i.th. 100 # g i.th. 100 # g i.th. 100 # g i.th. 500 Fg i.th. 100#gi.th.

8 7

113.3+ 5.5 111.4+ 8.2

- 1 3 . 8 + 4.0 0.65:2.5 c

31.3± 2.6 7.35:3.1 a

-83.8±15.8 14.15:7.2

8 8 8

122.45:3.9 r 125.4_ 4.3 f 122.1 5 : 6 . 1 f

- 1 . 4 ± 0.8 - 0 . 8 + 1.3 c - 1.5 + 3.8

-5.95:5.7 d -3.15:3.6 d 2.45:2.9 d

-54.45:20.6 -43.55:18.6 -15.05:4.7

5 6

109,85:3.3 131,05:9.1 ~

7.4+ 7.8 d -4.75:4.4

18.2_+ 8.5 - 7 . 0 + 4.7 d

-97.45:10.0 -72.8+33.7

a The results are expressed as m e a n s + S.E.M. b Time after administration. Statistical significance of differences between the changes of M A P (mmHg) produced by ana 1 in untreated and in inhibitor-treated animals was calculated using the analysis of variance and the Dunnett's method: c p < 0.05, d p < 0.01; n = no. of experiments. Experiments in which MA P was elevated by an i.v. infusion of e noradrenaline (166 n g / m i n per rat) or f [Sarl,ValS]AT II (80 n g / m i n per rat) to baseline MA P are indicated. TABLE 2 Effects of various inhibitors on the changes of H R induced by the i.th. administration of 6.5 nmol of ana I at Ta-T10 spinal level in pentobarbital-anaesthetized rats. Inhibitor

Dose

n

Baseline

A H R to ana l

HR

10 min b

Untreated Hexamethonium Phentolamine + propranolol Atropine Diphenliydramine + cimetidine + methysergide Naloxone Morphine

10 m g / k g i.v. 3 m g / k g i.v. 3 m g / k g i.v. 3 mg/kgi.v. 10, 10 and 5 m g / k g i.v.

6 6 6

435.0+ 8.1 475.0_ 28.7 341.7 5:11.4 d

5 7

370.0+14.1 432.9 5:14.1

10 m g / k g i.v. 5mg,/kgi.v.

9 5

Untreated Phentolamine + propranolol Prazosin Yohimbine Prazosin + yohimbine Sotalol Propranolol

100 100 100 100 100 100 500 100

# g i.th. #g, i.th. # g i.th. #gi.th. # g i.th. # g i.th. # g i.th.

30 rain b

36.7+ 10.9 30.0 + 6.3 - 47.5 + 52.9 40.05: 22.9__+

-188.35:77.2 - 328.3 5:109.6 - 228.35:79.1

8.9 7.5

-108.05:68.2 - 270.0 5 : 7 2 . 2

407.0 + 18.0 463.35:7.6

- 24.4 5 : 5 0 . 1 -85.05:56.9

- 156.7 + 75.1 -365.05:92.1

8 7

440.6 + 15.7 407.1 5:21.8

- 59.4 + 77.7 21.45:13.5

- 265.6 5 : 8 2 . 4 54.3 5 : 2 3 . 7 d

8 8 8

471.95:14.5 440.05:20.4 436.3 5:16.3

17.1_+ 5.7 0.6+ 31.3 39.4 5: 9.4

- 110.7 5 : 8 6 . 9 -68.85:71.8 46.3 5: 6.3 a

5 6

444.05:19.4 365.05:18.8 c

- 143.05:117.5 21.75: 3.1

- 339.0+ 94.8 - 146.75:94.3

Statistical significance of differences between the changes of H R ( b e a t s / r a i n ) produced by ana 1 in untreated and in inhibitor-treated animals was calculated using the analysis of variance and Dunnett's method: c p < 0.05, d p < 0.01; n = no. of experiments. For more details see notes to table 1.

352 dine and methysergide (the inhibitors of histamine on the H 1 and the H 2 receptor and of 5-hydroxytryptamine on the 5-HT1 receptor, respectively), naloxone (an inhibitor of opioid receptors) and morphine failed to modify the cardiovascular effect of ana v The i.th. injection of a mixture of phentolamine and propranolol or of prazosin plus yohimbine significantly reduced the changes in MAP induced by ana~ while the effect of ana~ to decrease H R was abolished and replaced by tachycardia. However, when prazosin and yohimbine were given separately, only the increase in MAP induced by ana~ at 5 min was significantly reduced. The i.th. administration of two fl-adrenergic inhibitors, sotalol and propranolol, had no effect on the H R response induced by ana 1 while it reduced the MAP response at 1 min (sotalol) and 5 min (propranolol). 3. 3. Spinal cord transection The effects of cervical spinal cord transection on the cardiovascular responses elicited by the i.th. administration of ana 2 at T8-T10 spinal level are illustrated in fig. 3. The basal MAP and H R were 113.2 + 5.5 mm Hg and 440.6 +_ 15.7 b e a t s / min (n = 8) in intact animals and 78.6 + 8.4 mm Hg and 332.0 _+ 11.7 b e a t s / m i n (n -- 5; P < 0.001; calculated with Student's t-test for unpaired sampies) in spinal rats. The initial and secondary long-lasting hypotension and the profound bradycardia induced by ana 2 were abolished in spinal rats (P < 0.001; calculated with Student's t-test for unpaired samples). An increase of MAP began after 1 rain, reached a maximum at 3 min and returned slowly to the pre-administration level after 10 min. Tachycardia appeared 1 min after injection of ana 2, reached a maximum at 8 rain and lasted more than 45 min. The influence of the basal arterial blood pressure level on the cardiovascular effects of ana z in spinal rats was tested with noradrenaline (166 n g / m i n per rat) infused i.v. to increase the basal level of MAP and H R to 124.0 + 3.0 mm Hg and 389.4 +_ 19.6 beats/min (n = 8) respectively. Under these normotensive conditions, ana 2 had no significant effect on MAP and H R up to 45 min after administration.

Similar results were obtained with ana 1 after transection of the spinal cord (results not shown). However, not enough compound was available to examine any influence the drop in the basal blood pressure level in spinal rats might have had on the cardiovascular effects of anap

4. Discussion

Marked cardiovascular responses following the i.th. administration of two [D-Trp]-containing SP analogues in rats anaesthetized with sodium pentobarbital have been described. The results shown above suggest that a spinal rather than a peripheral site of action is responsible for these effects since no significant changes on MAP and H R were observed following intravenous injection of the two compounds. There were major differences in the cardiovascular responses depending on the site of administration in the spinal cord. Ana 1 and ana 2 showed both stimulatory and inhibitory effects on MAP and H R when applied at the T1-T3 and T8-T10 levels. The second cardiodepressor response started more rapidly after application at the T1-T3 level, while when given at the L2-L4 level the peptides induced only transient changes of MAP and a slight increase of HR. Thus, profound and long-lasting cardiovascular responses were produced only when the peptides were applied at the thoracic levels. The cardiodepressor effects of ana 1 were not significantly reduced in rats pretreated with the ganglionic blocker hexamethonium. It thus appears that the autonomic nervous system does not participate in the cardiodepressor effects induced by the i.th. administration of ana 1. In addition, the profound and long-lasting hypotension and bradycardia induced by anal were not modified by intravenously administered phentolamine, propranolol, atropine, diphenhydramine, cimetidine, methysergide, naloxone or morphine suggesting that peripheral catecholamines (a- and B-receptors), acetylcholine (muscarinic receptors), histamine (H 1 and H 2 receptors), 5-hydroxytryptamine (5-HT1 receptors) and opiate peptides are not involved in these cardiovascular responses. The changes in MAP and H R induced by ana 1

353 were significantly reduced in rats treated i.th. with phentolamine plus propranolol and also with prazosin plus yohimbine. However, the profound and long-lasting hypotension and bradycardia produced by ana 1 were not affected by either prazosin or yohimbine given separately or in presence of fl-adrenergic antagonists (propranolol and sotalol). It thus appears that fl-adrenergic mechanisms are not involved, while both a 1- and %adrenergic recepfors take part in the spinal depressive effects of ana 1 on MAP and HR. Whether the effect of a-adrenergic inhibitors on the cardiovascular responses to ana 1 implies the activation of adrenergic receptors by ana I remains to be elucidated. Further experiments are required to determine more precisely the site of action of this inhibition at the spinal level. In addition to prazosin and yohimbine, phentolamine and propranolol given i.th. prevented the increase of arterial pressure at 5 rain after administration of ana 1 but not the increase of HR at 10 and 30 min; the decreasing effect of ana I on HR was abolished and replaced by tachycardia. This finding points to the existence of different mechanisms in the spinal action of ana I on arterial blood pressure and heart rate and also suggests that the tachycardia induced by this analogue is not mediated by a central catecholaminergic mechanism. While a long-lasting fall in MAP and HR was observed in non-spinalized animals, spinal rats responded to ana 2 with an increase in MAP and HR. This suggests that a depressor centre, responsible for the inhibitory effects of ana 2 on cardiovascular functions, is located in a supraspinal area. Further experiments are under way to determine more precisely the central location of potential catecholaminergic neurons subserving the spinal action of [D-Trp]-containing SP analogues. The vasodepressor effect of [D-Trp]-containing SP analogues was attributed to inhibition of a bulbospinal SP pathway originating from the ventral medulla oblongata and possibly involved in the tonic excitation of vasomotor sympathetic preganglionic neurons (Loewy and Sawyer, 1982; Keeler and Helke, 1985; Takano et al., 1985a). This interpretation is based on the findings that SP antagonists inhibit both: (a) the cardiovascular

response elicited by kainic acid (glutamate agonist) and by bicuculline (7-aminobutyric acid receptor antagonist) applied to the ventral medulla in rats (Loewy and Sawyer, 1982; Keeler and Helke, 1985) and (b) the adrenal output of catecholamines produced by SP applied intrathecally at the T9 spinal level (Yashpal et al., 1985). However, the abovementioned interpretation appears to be no longer appropriate on the basis of the following considerations: (a) inhibition of ganglionic transmission by hexamethonium failed to prevent the vasodepressor effect of the SP analogue ana 1 when the basal blood pressure was maintained at a physiological level with noradrenaline; (b) inhibition of spinal a l- and a2-adrenergic receptors by prazosin and yohimbine blocked the vasodepressor effect of anap suggesting that a central catecholaminergic mechanism participates in the spinal action of [D-Trp]-containing SP analogues. Several other reports also questioned the antagonistic features of these SP analogues in the CNS. Jones et al. (1983), who used conventional electrophysiological techniques, reported that two SP antagonists [D-Pro4,D-TrpV'9]SP-(4-11) and [DPro4,D-TrpT,9'l°]SP-(4-11) (Mizrahi et al., 1982) failed to antagonize the excitatory action of SP following iontophoretic ejection in the cingulate cortex, the medial basal hypothalamus and the locus coeruleus of rats. Both compounds were themselves excitatory and enhanced the response to SP in the three brain areas studied. The same negative results were obtained with [D-Pro2,DTrp7'9'l°]SP and several similar analogues against the nociceptive effect of SP in the tail-frick test (Piercey, 1984; Couture et al., 1985). Salt et al. (1982) found that iontophoretically applied [DPro2,D-TrpT'93°]SP blocks the ability of L-glutamate but not that of SP to excite spinal neurons. The mechanism causing this blockade remains unknown. In addition, [D-Pro2,D-TrpT'9]Sp did not antagonize the depolarization of motoneurons evoked by SP in the isolated rat spinal cord (Salt et al., 1982). It has also been reported that intrathecal injections of the luteinizing hormone releasing hormone (LH-RH) analogue, [D-pGlul,D-Phe2,DTrp3'6]LH-RH will cause dose-dependent decreases in MAP in anaesthetized rats similar to

354 those caused by [D-Pro4,D-TrpT,9]SP-(4-11)

(Ta-

k a n o et al., 1 9 8 5 b ) . T h e p r e s e n c e o f a D - T r p residue in the peptides appears to be crucial for the spinal cardiovascular effects observed above. Caution must therefore be taken in interpreting results obtained with D-Trp-containing SP analogues.

Acknowledgements This work was supported by grants from the Medical Research Council of Canada (MRCC), The Kidney Foundation of Canada, The Quebec Heart Foundation and the University of Montreal (CAFIR). R.C. is a 'chercheur-boursier du Fonds de la Recherche en Sant6 du Qu6bec'; D.R. is a Career Investigator of the MRCC. We thank Jean-Guy Comeau and Daniel Courteau for technical assistance, Carole Champagne for the secretarial work and Giovanni Baptista Filosi for the photographs and illustrations.

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