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Cardiovascular responses elicited by intrathecal kinins in the conscious rat

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Cardiovascular responses elicited by intrathecal kinins in the conscious rat
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  European Journal of Pharmacology, 210 (1992) 137-147 137 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/ 05.00 EJP 52218 ardiovascular responses elicited by intrathecal kinins in the conscious rat Paulo Lopes and R~jean Couture Groupe de Recherche sur le Systbrne Nert~eux Autonorne, D~Xpartement de Physiologie, Facultd de Mddecine, Unit ersitd de MontrtXal, C.P. 6128, Succursale A, Montr¢;al, Qudbec, Canada H3C 3.17 Received 6 June 1991, revised MS received 3 September 1991, accepted 15 October 1991 In the conscious, unrestrained rat, intrathccal (i.t.) injection of 0.81 pmol-81 nmol bradykinin (BK), kallidin (KD) and T-kinin at the T-9 spinal cord level produced transient (< 10 mini increases in mean arterial pressure (MAP) and longer lasting decreases in heart rate (HR). These effects were dose-dependent and similar with respect to intensity and time course for the three kinins. The des-Arg -BK fragment, a selective agonist for Bj receptors, was active only at 81 nmol. The pressor response induced by BK was enhanced by propranolol and by transection of the cervical spinal cord but was converted to a vasodcpressor effect by prazosin. The bradycardia was converted to tachycardia by prazosin, atropine, pentolinium, capsaicin and in spinal transccted rats. However, the cardiovascular responses to BK remained unaffected by diphenhydraminc plus cimetidine, morphine, indomethacin, adrenal medullectomy, i.t. idazoxan and after bulbospinal noradrenalinc deafferentation with 6-hy- droxydopaminc. These results suggest that the increase in MAP induced by i.t. BK is mediated by the sympathoadrcnal system while the decrease in HR is ascribable to a vagal reflex involving sensory C-fibers and a spinobulbar pathway. This pharmacological evidence therefore supports a role for kinins in cardiovascular regulation in the spinal cord. Kinins; Spinal cord; Cardiovascular system; Catecholamines; Vagal reflex; C-fibers 1 Introduction Kinins are biologically active peptides generated in plasma (bradykinin, BK) and peripheral tissues (kalli- din, KD) after cleavage of large protein precursors (kininogens) under the action of specific enzymes termed kallikreins. BK and KD are produced during tissue dammage and are involved in inflammatory reac- tions, causing pain by activating peripheral sensory endings, vasodilation and increased vascular permeabil- ity (Regoli and Barab6, 1980; Regoli, 1987). In addi- tion, rat plasma contains another kinin named T-kinin (Ile-Ser-BK) (Okamoto and Greenbaum, 1983a, b) which is apparently a mediator of the inflammatory response in the rat (Barlas et al., 1985; Adam et al., 1989). Several reports suggests that kinins may also be involved in central cardiovascular regulation (Diz, 1985). The presence of kinins, kininogen, kallikreins and kininase activity has been shown in the brain, the spinal cord and the cerebrospinal fluid of humans Correspondence to: R. Couture, Groupe de Recherche sur le Systbme Nerveux Autonome, D~partement de Physiologie, Facult~ de M6de- cine, Universit6 de Montreal, C.P. 6128, Succursale A, Montreal, Quebec, Canada H3C 3J7. Tel. 1.514.343 7060, fax 1.514.343 2111. (Scicli et al., 1984) and various other species (Perry and Snyder, 1984; Thomas et al., 1984; Kariya et al., 1985; Hermann et al., 1986; Madeddu et al., 1990; Yoshida and Nosaka, 1990). Autoradiographic studies (Steranka et al., 1988) and binding studies (Fujiwara et al., 1989; Sharif and Whiting, 1991) have revealed the presence of [3H]BK receptor binding sites (identified as B z re- ceptors) in the guinea pig spinal cord and other central areas of cardiovascular regulation. The intracere- broventricular (i.c.v.) injection of BK and/or KD pro- duces changes (decreases and/or increases) of blood pressure in cats, dogs, rabbits and rats (see Diz, 1985 for a review). The pressor effect is prevalent in the awake rat, indicating that anesthesia affects the blood pressure response to centrally administered BK (Lam- bert and Lang, 1970; Corr~a and Graeff, 1974; Hoff- man and Schmid, 1978; Buccafusco and Serra, 1985; Lindsey et al., 1989). Concomitant increases of blood pressure and levels of kinins in the cerebrospinal fluid have been reported in dogs (Thomas et al., 1984, 1987) and rats (Thomas and Hiley, 1988). In spite of these observations, no studies have attempted to determine whether the kinins can affect the cardiovascular system through an action in the spinal cord. The present study was therefore undertaken to de- termine the intrathecal (i.t.) action of BK, KD and T-kinin on mean arterial pressure (MAP) and heart   38 rate (HR). The cardiovascular response to des-Arg '~- BK, a metabolite of BK and a selective stimulant of the BI receptor, was also measured. The second objective was to investigate the spinal and peripheral mecha- nisms underlying the cardiovascular responses to BK. Intrathecal BK may affect the cardiovascular system through three possible mechanisms: (1) by direct acti- vation of preganglionic sympathetic fibres in the inter- mediolateral cell column, (2) indirectly, by facilitating the release of substance P or other transmitters from central sensory nerve terminals, as suggested by the presence of BK binding sites in the substantia gelati- nosa (Steranka et al., 1988) and by the behavioral nociceptives responses produced by i.t. BK in the rat (Laneuville and Couture, 1987; Laneuville et al., 1989), (3) indirectly, by activating bulbospinal catecholaminer- gic fibers which appear to play a role in central cardio- vascular regulation (Coote, 1988). A previous study from our laboratory had provided evidence that BK may inhibit spinal nociceptive sensory transmission and produce analgesia by activating bulbospinal noradren- ergic fibers (LaneuviIle et al., 1989). Thus, a possible interaction with sensory C-fibers and bulbospinal cate- cholaminergic fibers was examined. We also sought to examine the role played by the sympathoadrenal sys- tem, the vagus nerve, prostaglandins and other endoge- nous mediators in the spinal action of BK on the rat cardiovascular system. In order to exclude any influ- ence of anesthesia, all experiments were performed in conscious unrestrained rats. Some of these results have been presented as a preliminary communication (Lopes and Couture, 1991). 2 Materials and methods 2.1. Implantation of catheters and measurement of car- diot ascular parameters Male Wistar rats (Charles River, St-Constant, Qu6bec, Canada) weighing 200-250 g were implanted with three catheters under sodium pentobarbital anes- thesia (65 mg/kg intraperitoneally (i.p.)), as previously described (Hass6ssian and Couture, 1989; Hassdssian et al., 1990). Briefly, intraarterial (i.a.) and intravenous (i.v.) polyethylene tubes (Intramedic, Clay Adams, N J, U.S.A.; PE-50) were inserted into the medial tail artery and the right jugular vein while an i.t. catheter (PE-10) reached the 9th thoracic (T-9) segment. The experi- ments were performed in awake unrestrained rats, at least 24 h after catheter implantations, in a quiet room, with the cage covered by an opaque cloth to avoid visual cues to the rats. Only rats with no apparent motor deficit and showing normal behavior were used. The correct positioning of the i.t. catheter was verified by post-mortem examination at the end of each experi- ment. The arterial blood pressure was monitored via the i.a. catheter using a Statham pressure transducer (P231D) and the heart rate (HR) was measured with a cardiac tachometer (model 7P4) triggered by the arte- rial pressure pulses. The arterial blood pressure and the HR were recorded with a Grass T polygraph model 79 (Quincy, MA, U.S.A.). Each experiment was started with an i.t. injection of 30 ~1 of cerebrospinal fluid (CSF; for composition see Hassdsian and Couture, 1989, void volume of the i.t. catheter was 20 ~1) as a vehicle control. The arterial blood pressure and HR were then monitored for 30 min, after which the first test drug was injected i.t. in a volume of 10 1. The drug and the CSF (20 ~zl) flush were given within a total period of 60 s. Changes in the MAP and HR represent the difference between recordings obtained 30 s before the start of injection and the value at the designed time after injection. 2.2. Adrenal medtdlectomy and spinal chemical sympa- thectomy Adrenal medullectomy was performed 72 h prior to experiments in rats under pentobarbital anesthesia. The two adrenal glands were exposed through dorsal incisions and the medullas were extruded with gentle forceps pressure on the adrenal after slitting the cap- sule. The skin was sutured and rats were allowed to recover from anesthesia. Two days later, the rats were anesthetized again for catheter implantation as de- scribed above. The neurotoxin, 6-hydroxydopamine hy- drochloride (6-OHDA) (prepared in CSF containing 1% ascorbic acid), was administered in 50-~g (free base) doses at the T-2 and T-9 spinal cord levels through an i.t. catheter in pentobarbital-anesthetized rats. The i.t. catheter was removed thereafter, the surgical wound was sutured and the rats were returned to their cages for one week after which the chronic i.t. (T-9 level) and intravascular catheters were implanted. The noradrenaline content was reduced by around 60% in the spinal cord while serotonin, dopamine, adrenaline and their main metabolites were unaffected in rats that had received a single i.t. injection of 20/_Lg 6-OHDA one week earlier (Laneuville ct al., 1989). 2.3. capsaicin treatment The rats were treated with capsaicin according to the following protocol: 125 mg/kg given subcuta- neously (s.c.) over 3 days, with 25, 40 and 60 mg/kg on the first, second and third day, respectively. Capsaicin was made up in 10% ethanol and 10% Tween 80 in saline. Experiments were conducted four days after the last injection of capsaicin. When administered in adult rats, capsaicin produces a prolonged inactivation of  139 primary sensory C-fibers (Maggi and Meli, 1988). Rats pretreated with capsaicin failed to react to the applica- tion of a drop of solution containing 25 mg/ml cap- saicin in one eye. Similar treatment of control animals caused discomfort reactions. 2 4 Spinal cord transection Rats assigned to the spinal group had surgically implanted chronic i.t. and intravascular catheters. The spinal cord was transected at the C-6-C-7 vertebral level under halothane anesthesia (3-5 volume in a gas mixture of 95 O:-5 CO2). The dorsal cervical muscles were exposed; the intravertebral disc was cut with micro-scissors, and the spinal cord was transected while the i.t. cannula remained intact. Bleeding was minimal and usually stopped within 30 s with a ball of cotton wool inserted between the rostral and caudal segments of the transected spinal cord. The wound was closed and the animals recovered from anesthesia on inhaling a gas mixture containing 95 02-5 CO 2. The experiments were started 45 min after surgery in awake animals. 2 5 Experimental protocols In the first series of experiments, the time course effects of several doses of BK, KD, T-kinin and des- ArgLBK on MAP and HR was measured after i.t. injection at the T-9 spinal cord level. The experiments were started by the injection of CSF, followed 20 min later by increasing doses of peptide. A period of 30-45 min was allowed between doses so that the MAP and HR could return to pre-injection values. Only one peptide was given to each rat. The second series of experiments was designed to study the mechanism underlying the cardiovascular ef- fect of BK. The roles played by the sympathetic and parasympathetic components of the autonomic nervous system were examined using antagonists of al-adreno- ceptors (prazosin) and peripheral muscarinic acety[- choline receptors (methylnitrate atropine). The re- sponses to BK were also evaluated in rats following bilateral adrenal medullectomy. The participation of prostaglandins and histamine was determined with a cyclooxygenase inhibitor (indomethacin) and antihis- taminics for H~ (diphenhydramine) and H 2 (cimeti- dine) receptors. A ganglionic nicotinic receptor blocker (pentolinium) was administered to rats to determine whether the cardiovascular responses to BK were me- diated through a reflex mechanism. The rats received systemic capsaicin and i.t. 6-OHDA to assess the con- tribution of primary sensory C-fibers and bulbospinal catecholaminergic fibers on the effect of BK. The central contribution of catecholamines was also as- sessed with i.t. idazoxan, an a2-adrenoceptor antago- nist known to Mock the antinociceptive response in- duced by i.t. BK (Laneuville et al., 1989). A group of rats was treated with morphine (opioid agonist) to test for a nociceptive component of the cardiovascular re- sponse. The potential contribution of supraspinal cen- ters was studied in spinal rats. 2 6 Peptides and other compounds The peptides BK, KD, T-kinin and des-ArgKBK were purchased from Hfikabel Scientific Ltd. (Longueuil, Canada). Indomethacin, methylnitrate at- ropine, cimetidine, nicotine, pentolinium tartratc salt, diphenhydramine hydrochloride, propranolol hydro- chloride, capsaicin (8-methyl-N-vanillyl-6-nonenamide), heparin sodium salt (porcine, grade II) were all pur- chased from Sigma (St. I~uis, MO, U.S.A.), idazoxan was obtained from Reckitt and Colman (UK), 6-OHDA hydrochloride from Labkemi AB (Stockholm, Sweden), prazosin hydrochloride from Pfizer (Kirkland, Canada), L-ascorbic acid from Baker (Phillipsburg, N J, USA), morphine sulphate from BDH Pharmaceuticals (Toronto, Canada), sodium pentobarbital (somnotol) from MTC Pharmaceuticals (Missisauga, Canada), halothane (fluothane) from Ayerst (Montrdal, Canada) and Tween 80 from Fisher (Montrdal, Canada). The peptides were prepared in CSF as 1 mg/ml stock solutions, divided into aliquots of 100 /xl each, and stored at -20°C until used. Indomethacin was pre- pared in trizma base (0.2 M, Sigma)just before utiliza- tion, and the other drugs were prepared in saline. Doses of peptides are given in mo[ of the salt. 2 7 Statistical analysis of data The results are expressed as means _+ S.E.M. Statis- tical differences were evaluated with Student's t-test for paired or unpaired samples as required. A one-way analysis of variance (ANOVA) with a post-hoc Dun- nctt test was used for multiple comparisons with one control group. The time course of the effects was analysed with a two-way ANOVA in conjunction with Bonferroni confidence intervals. Only probability val- ues (P) smaller than 0.05 were considered to be statisti- cally significant. 3 Results 3 l Effects of i t kinins on MAP and HR An srcinal tracing showing the cardiovascular effect of 81 pmol BK given i.t. at T-9 to a freely moving rat is presented in fig. 1. A transient increase of blood pres- sure and decrease of HR were seen immediately after the peptide injection.  14 LU 200 l jS:~ o I 150 =~g 100 a_ 50 ~_~ 500 <_c E 400 < ~ 300 w~ I 200 CSF BK (81 pmol) 1 mm Fig. 1. Typical tracings of blood pressure and HR responses to i.t. injection of 81 pmol BK at the T-9 spinal thoracic level in a conscious rat. Note the lack of response to i.t. injection of CSF. The effects of four increasing doses of BK on MAP and HR are shown in fig. 2. Baseline MAP and HR were 91.4_+4.6 mm Hg and 365.5_+ 7.3 beats/min n = 22). BK produced dose-dependent increases in MAP and decreases in HR that peaked at 2 rain post-injection, The pressor response to BK was statisti- cally significant when compared to CSF values at 8.1 pmol 1-5 rain; F 4,42) - 4.40, P < 0.05), 81 pmol 1-10 rain; F 4,42)= 10.31, P < 0.005) and 810 pmol 1-10 rain; F 4,42)= 10.33, P < 0.005). The bradycardia in- duced by BK was also statistically significant at 8.1 15 10 0 -5 0 "I- E E 5 0_ < <~ t i i I i i 5 10 15 20 25 30 B 10 E -10 n- -20 -'r- -30 -40 0 Fig. 2. Time course of cardiovascular changes pmol BK at the T-9 spinal thoracic level. Changes in MAP A) and HR B) are shown. Each point represents the mean + S.E.M. for 22 rats. i i I i i 5 10 15 20 25 30 TIMI= ~'min~ m response to i.t. injection of CSF O) and increasing doses, 0.81 Co), 8.1 [] ), 81 •) and 810 ~ )  pmol 1-5 min; F 4,42) = 4.40, P < 0.05), 81 pmol 1-10 min; F 4,42)= 10.8, P < 0.005) and 810 pmol 1-10 rain; F 4,42) = 14.61, p < 0.005). The pressor response returned to baseline within 10 min while the bradycar- dia lasted longer and had not completely returned to pre-injection values 30 min after injection of 810 pmol BK. The cardiovascular responses elicited by the i.t. injection of 81 pmol BK during and 30 min after the assessment of the dose-response curve were not statis- tically different AMAP: F 1,28) = 2.74, P > 0.05; AHR: F 1,28) = 0.48, P > 0.05). Hence, no tachyphy- laxis was observed with BK under these experimental 141 conditions. The i.t. injection of CSF was without appre- ciable effect on MAP and HR. The i.t. injection of 100 ng BK 81 pmol), T-kinin 68.5 pmol) and KD 70 pmol) produced similar cardio- vascular responses with respect co the intensity and time course fig. 3). The pressor responses to T-kinin 1-5 min; F 3,29) = 8.89, P < 0.01) and KD 1-10 min; F 3,29) = 9.84, P < 0.005) were statistically significant when compared to CSF values. The bradycardia in- duced by T-kinin F 3,29)= 7.92, P <0.01) and KD F 3,29) = 6.45, P < 0.025) was also statistically signifi- cant between 1-10 min post-injection. Although a high 15 10 -1- E g 5 EL 5 i i T i i i 0 5 10 15 2 25 30 B 10 E -10 n- -20 "1- <1 -30 -40 p 5 i p i i i 1 15 2 25 3 TIME (mini Fig. 3. Time course of cardiovascular changes in response to i.t. injection of CSF ©, n = 22), 81 pmol BK e, n = 22), 70 pmol KD 11, n = 8), 68.5 pmol T-kinin D, n = 9) or 10.4 nmol des-Argg-BK A, n = 9) at T-9 spinal thoracic level. Changes in MAP A) and HR B) are shown. Each point represents the mean + S.E.M. for the number of animals indicated by n.
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