Clinical Science (I992) 82, 5 13-5 I 9 (Printed in Great Britain)
513
Central bradykininergic system in normotensive and hypertensive rats Azucena L. ALVAREZ, Alejandro DELORENZI, Daniel SANTAJULIANA, Samuel FINKIELMAN, Victor E. NAHMOD and Carlos J. PIROLA Laboratorio de Sustancias Vasoactivas, lnstituto de lnvestigaciones Medicas 'A. Lanari', Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina (Received 26 March/4 October 1991; accepted 9 December 199 I )
cardiovascular changes [8]. The intracerebroventricular 1. The kinin antagonist des-Arg9-[Le~~lbradykinin, infusion of kinins increases blood pressure and heart rate injected into the lateral ventricle, caused a long-lasting, [8-101 as well as increasing the release of anti-diuretic dose-dependent reduction in arterial blood pressure and hormone [7]. The site of the hypertensive effect has been heart rate in spontaneously hypertensive rats but not in reported to be the lateral septa1 area [ l l ] , where an normotensive Wistar-Kyoto rats; the antagonist also activation of the cholinergic system could mediate this blocked the pressor response to ventricularly infused bradykinin-induced pressor response [12]. It is now clear bradykinin in both strains. that a central cholinergic activation can be demonstrated 2. Bradykinin content was increased in the hypoin spontaneously hypertensive rats (SHR) [13, 141. It is thalamus and septum and decreased in the dorsal medulla thus tempting to speculate that this phenomenon could, in of spontaneously hypertensive rats when compared with turn, depend on the activation of the kinin-kallikrein those of normotensive Wistar-Kyoto rats, whereas system. In fact, under certain experimental conditions, similar bradykinin contents were observed in the pineal SHR are known to present exaggerated responses to gland, hypophysis and rostroventrolateral medulla of both bradykinin [9]. rat strains. T h e purpose of this study was to assess in normo3. Increased concentrations of bradykinin and its pretensive Wistar-Kyoto rats (WKY) and SHR (1) the cursor kininogen were found in the cerebrospinal fluid of changes in blood pressure and heart rate induced by spontaneously hypertensive rats. intracerebroventricular administration of a kinin antagon4. Bradykinin receptor numbers, measured as the ist, des-Argy-[Leus]bradykinin, (2) the bradykinin content binding of ['251-Tyr']bradykinin to nervous tissue, were and bradykinin receptor number in areas of the central found to be increased in the dorsal medulla and hyponervous system involved in cardiovascular regulation, and physis, and to be decreased in the pineal gland, of spon(3) the bradykinin and kininogen concentrations in the taneously hypertensive rats. cerebrospinal fluid (CSF). Some of these observations 5. Therefore, the central kinin system may participate, have already been presented elsewhere [ 151. by both pre- and post-synaptic mechanisms, in the maintenance of hypertension in spontaneously hyperMATERIALS AND METHODS tensive rats.
Studies in vivo INTRODUCTION The components of the kinin-kallikrein system are present in the rat central nervous system. A bradykininlike immunoreactivity has been identified and a kinin-like biological activity has been found in brain extracts [l,21. Enzymes capable of generating and inactivating kinins were measured in brain tissue [2-41. Bradykinin produces a variety of effects when infused intracerebroventricularly, such as behavioural arousal and sedation [5], antinociceptive [6] and anti-diuretic [7] actions, and
Male SHR and WKY weighing 250-350 g and housed with a lightldarkness schedule of 1 2 h/12 h were used. Both SHR and WKY were anaesthetized with urethane (1.2 g/kg intraperitoneally). The trachea was cannulated and arterial blood pressure and heart rate were recorded throughout the experiment from a polyethylene cannula inserted into the left carotid artery and connected to a Statham transducer coupled to a Grass polygraph. A 25gauge stainless-steel cannula was directed to the lateral cerebral ventricle (intracerebroventricularly) through a burr hole in the skull using a stereotaxic apparatus (David ~~
Key words: bradykinin, central nervous system, cerebrospinal fluid, depressor effect, kinin antagonist, kinin receptor, spontaneously hypertensive rats. Abbreviations: CSF, cerebrospinal fluid; SHR. spontaneously hypertensive rats; WKY, Wistar-Kyoto rats. Correspondence: D r Carlos J.Pirola, Cardiology Department, Becker Building no. 210, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, U.S.A.
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A. L. Alvarez et al.
Kopf, Tujuna, CA, U.S.A.). Co-ordinates for implantation were 0.3 mm posterior to the bregma, 1.5 mm lateral to the midline and 3.5 mm below the dura [16]. At the end of each experiment the animals were killed and the position of the infusion cannula and the distribution of the infused volume were assessed by injection of 1 pl of 2% (w/v) Bromophenol Blue solution. The head was then perfused with saline (150 mmol/l NaCI) followed by 10% (w/v) formaldehyde, and sections were cut throughout the diencephallon for histological verification. Only data collected from experiments in which the correct insertion of the cannula was verified are reported. In fact, the dye injected into the lateral ventricle diffused through the entire ventricular system including the dorsolateral surface of the medulla. Saline and full doses of the bradykinin antagonist des-Arg9--[LeuX]bradykinin (0.1-10 pug) were infused (1 pl/min over 3 min) through the intracerebral guide cannula. Only one dose was applied per animal. ED,, values were estimated from five independent dose-response curves as the dose in the infused solution of the bradykinin antagonist that lowered blood pressure or heart rate by 50% of the maximum effect. CSF was obtained by cisterna magna puncture in anaesthetized animals placed in the stereotaxic apparatus. Samples (pool of three to five rats, 60-140 pI each, n = 6 per strain) used for the measurement of kinin concentration were collected in 0.5 ml of ice-cold 0.4 mol/l HCIO,. Insoluble components were separated by centrifugation at 17 000 g for 10 min. The supernatant was neutralized with 0.5 mol/l KOH and was freeze-dried. The residue was redissolved in the appropriate buffer. Bradykinin was measured by r.i.a. as previously described [ 171, using [ 12,1Tyr8]bradykinin (Dupont, Buenos Aires, Argentina). The r i a . was performed in 0.05 mol/l NaH,PO, buffer, pH 7.4 containing 0.1% BSA, 100 units of aprotinin/ml, 0.1 mmol/l phenylmethanesulphonyl fluoride and 0.1 mmol/l EDTA. The incubation mixture contained 0.1 ml of ['2sI-TyrX]bradykinin(5000-8000 c.p.m.), 0.1 ml of a 1:4000 dilution of antiserum and 0.1 ml of lysine bradykinin as standard ranging from 5 to 1000 pg or sample. The final volume was 0.3 ml. Tubes were incubated for 24 h at 4°C. Six milligrams of Dextran-charcoal(6.25 g of Norit A charcoal and 6.25 g of Dextran T70/1 in 50 mmol/l phosphate buffer, pH 7.4) was used to separate free from antibody-bound kinins. After centrifugation, the pellet was counted in an automatic y-counter (Alfa Nuclear, Buenos Aires, Argentina). Calculations were performed using a computerized linear least-square curve-fitting procedure from plots of c.p.m. versus log of standard amounts. Typical intra- and inter-assay coefficients of variation were 7 and 12%, respectively. The limit of detection of the assay was 7-12 pg. Portions of CSF (pool of three to five rats, 60-150 pl each, n = 11per strain) were kept at -20°C until determination of kininogen, which was performed by measuring kinins released with excess kallikrein (Sigma Co.) at 37°C in the presence of a cocktail of peptidase inhibitors comprising 3.0 mmol/l 1, 10-phenanthroline, 30 mmol/l EDTA and 30 prnolll N-ethylmaleimide. The reaction was stopped
by the addition of 0.5 ml of ice-cold 0.4 mol/l HCIO,, and samples were processed as described for the CSF kinin assay.
Studies in vitro The rats were killed by decapitation and the brain was rapidly removed. The dorsal and rostroventrolateral medulla, hypothalamus, septum, hypophysis and pineal gland were dissected and separated on ice-cold plates with the aid of a stereotaxic atlas [16]. For determination of kinin, the nervous tissue (pool of three to four rats) was submerged in 2 mol/l acetic acid acidified to pH 1 with concentrated HCI, boiled for 20 min, homogenized and centrifuged at 10 000 gfor 10 min. The pellet was stored at - 20°C until protein determination was performed. The supernatants were freeze-dried and the residue was dissolved in buffer for determination of kinin by r i a . as described above. In the procedure for the determination of the CSF concentration of kinin and the tissue content of kinin, the recovery of synthetic lysine bradykinin was higher than 80% and only 4-6% of the original amount of a purified dog kininogen, a possible interference in the assay [ 171, was detected. In addition, identification of the bradykininlike immunoreactivity was performed by using highperformance liquid chromatographic equipment (Waters Associates, Milford, MA, U.S.A.), which consisted of a radial compression module with a uBondapak C,, cartridge or a Lichrospher RP-8 (Merck, Darmstadt, Germany) and a model 440 fixed-wavelength U.V. detector. The analog detector outputs were recorded with an OmniScribe recorder. There were two mobile phases ( A and B): phase A consisted of 50 mmol/l triethylamine adjusted to pH 3 with phosphoric acid; phase B was methanol. After injection, the components were eluted from the column using a concave gradient from 0 to 30 min, and from 30 to 35 min the column was reverseprogrammed employing a Baseline 8 10 computer program (Waters Associates) installed in a APC IV NEC personal computer. Flow rates of 1-1.5 ml/min were reached with a Waters model 6000 and 590 solvent delivery system. Since more than 90% of the kinin-like immunoreactivity present in rat nervous tissue co-eluted with authentic bradykinin, we will refer to it as 'bradykinin'. To evaluate the parameters of bradykinin-binding sites, [12sI-Tyr']bradykinin was used as ligand [18]. [I2% Tyrllbradykinin (300-700 Ci/mmol) was used at concentrations of 0.025-20 nmol/l. The radiolabelled analogue was prepared by a modification of the chloramine-T method 1191 and was purified by using the chromatographic system described above. Fractions containing monoiodinated [Tyrllbradykinin were identified by its retention time using unlabelled monoiodinated [Tyr'lbradykinin prepared by the method of Odya et al. [181. The nervous tissue (pool of six to eight rats) was homogenized in 10 vol of 0.32 mol/l sucrose and homogenates were centrifuged at 1000 g for 10 min to
515
Bradykininergic system in spontaneously hypertensive rats
obtain a crude nuclear pellet, which was discarded. The resulting supernatant fraction was centrifuged at 17 000 g for 15 min to obtain a synaptosomal pellet. The pellet was resuspended in the original volume of 0.32 mol/l sucrose for the binding assay. Binding reactions were performed at 4°C in polypropylene test tubes. The tissue homogenate was added to each tube in 0.1 ml containing (mg of protein): rostroventrolateral and dorsal medulla, 0.7 and 1.4, respectively; hypothalamus, 0.8; septum, 0.4; hypophysis, 0.3; pineal gland, 0.02. The incubation mixture consisted of 0.1% BSA, 0.3 mmol/l bacitracin, 1 pmol/l SQ14225, 0.1 mmol/l EDTA, 0.1 mmol/l phenylmethanesulphonyl fluoride and 100 units of aprotinin/ ml in 0.05 mol/l phosphate buffer, pH 7.4. This protease-inhibitor mixture protects peptide integrity during incubation as assayed by the h.p.1.c. system described above. After incubation for 60 min, equilibrium was reached and 3 mi of ice-cold NaKHPO, buffer was added. Samples were passed through a glass filter (Whatman GF/ B) under vacuum. The filters were washed three times under vacuum with 3 ml of ice-cold buffer, then placed in tubes and the radioactivity bound was counted in a ycounter (Alfa Nuclear). The results are presented as specific [ 1251-Tyr1]bradykinin binding, which is defined as the difference between binding in the absence and presence of unlabelled bradykinin (1pmol/l). At a radioligand concentration of 5 nmol/l, specific binding accounted for 55-65% of the total binding. For calculation of the bradykinin receptor concentration (B,,,=,)and dissociation constant (&), analysis of the binding data by the non-linear least-square curve-fitting procedure using a generalized model for ligand-receptor systems [20] was used. Results
are expressed as means k SEM of five independent experiments. Protein concentrations were determined by the method of Lowry et al. [211. Statistical analysis was performed by using paired Student t-tests and analysis of variance with Tukey's test for individual differences. Results are expressed as means 5 SEM.
Drugs [Tyr'IBradykinin, lysine bradykinin, des-Argy[Leu*]bradykinin,BSA, bacitracin, EDTA (disodium salt), phenylmethanesulphonylfluoride and chloramine-T were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A). SQ14225 and aprotinin (Trasylol) were kindly provided by Squibb (Buenos Aires, Argentina) and Bayer (Buenos Aires, Argentina), respectively. All the reagents were of the best grade available. The polyclonal antiserum to lysine bradykinin was a generous gift from H. Nolly and 0.A. Cerretero, Henry Ford Hospital, Detroit, MI, U.S.A.
RESULTS The kinin antagonist des-Argy-[Leu*]bradykinin, infused at doses of 0.1-10 pg into the lateral ventricle, produced no change in arterial blood pressure and heart rate in WKY (Table 1),but induced a significant dosedependent long-lasting decrease in arterial blood pressure and heart rate in SHR. The same volume of vehicle produced no change in arterial blood pressure or heart rate in both strains of rats. The ED,, values for the hypotensive and bradycardic effects were 1.3k0.6 and
Table I. Effect of intracerebroventricular infusion of the kinin antagonist des-Arg9-[leu*]bradykininon arterial blood pressure and heart rate in WKY and SHR. The basal mean arterial blood pressure and heart rate were 166+4 mmHg and 398+5 beatslmin, respectively, for SHR and I15 +7 mmHg and 360+ 10 beatslmin, respectively, for WKY. Results were expressed as the meansfsm from 10 rats per group at each dose of antagonist. Only durations o f significant responses are included. Statistical significance: *P
513
Central bradykininergic system in normotensive and hypertensive rats Azucena L. ALVAREZ, Alejandro DELORENZI, Daniel SANTAJULIANA, Samuel FINKIELMAN, Victor E. NAHMOD and Carlos J. PIROLA Laboratorio de Sustancias Vasoactivas, lnstituto de lnvestigaciones Medicas 'A. Lanari', Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina (Received 26 March/4 October 1991; accepted 9 December 199 I )
cardiovascular changes [8]. The intracerebroventricular 1. The kinin antagonist des-Arg9-[Le~~lbradykinin, infusion of kinins increases blood pressure and heart rate injected into the lateral ventricle, caused a long-lasting, [8-101 as well as increasing the release of anti-diuretic dose-dependent reduction in arterial blood pressure and hormone [7]. The site of the hypertensive effect has been heart rate in spontaneously hypertensive rats but not in reported to be the lateral septa1 area [ l l ] , where an normotensive Wistar-Kyoto rats; the antagonist also activation of the cholinergic system could mediate this blocked the pressor response to ventricularly infused bradykinin-induced pressor response [12]. It is now clear bradykinin in both strains. that a central cholinergic activation can be demonstrated 2. Bradykinin content was increased in the hypoin spontaneously hypertensive rats (SHR) [13, 141. It is thalamus and septum and decreased in the dorsal medulla thus tempting to speculate that this phenomenon could, in of spontaneously hypertensive rats when compared with turn, depend on the activation of the kinin-kallikrein those of normotensive Wistar-Kyoto rats, whereas system. In fact, under certain experimental conditions, similar bradykinin contents were observed in the pineal SHR are known to present exaggerated responses to gland, hypophysis and rostroventrolateral medulla of both bradykinin [9]. rat strains. T h e purpose of this study was to assess in normo3. Increased concentrations of bradykinin and its pretensive Wistar-Kyoto rats (WKY) and SHR (1) the cursor kininogen were found in the cerebrospinal fluid of changes in blood pressure and heart rate induced by spontaneously hypertensive rats. intracerebroventricular administration of a kinin antagon4. Bradykinin receptor numbers, measured as the ist, des-Argy-[Leus]bradykinin, (2) the bradykinin content binding of ['251-Tyr']bradykinin to nervous tissue, were and bradykinin receptor number in areas of the central found to be increased in the dorsal medulla and hyponervous system involved in cardiovascular regulation, and physis, and to be decreased in the pineal gland, of spon(3) the bradykinin and kininogen concentrations in the taneously hypertensive rats. cerebrospinal fluid (CSF). Some of these observations 5. Therefore, the central kinin system may participate, have already been presented elsewhere [ 151. by both pre- and post-synaptic mechanisms, in the maintenance of hypertension in spontaneously hyperMATERIALS AND METHODS tensive rats.
Studies in vivo INTRODUCTION The components of the kinin-kallikrein system are present in the rat central nervous system. A bradykininlike immunoreactivity has been identified and a kinin-like biological activity has been found in brain extracts [l,21. Enzymes capable of generating and inactivating kinins were measured in brain tissue [2-41. Bradykinin produces a variety of effects when infused intracerebroventricularly, such as behavioural arousal and sedation [5], antinociceptive [6] and anti-diuretic [7] actions, and
Male SHR and WKY weighing 250-350 g and housed with a lightldarkness schedule of 1 2 h/12 h were used. Both SHR and WKY were anaesthetized with urethane (1.2 g/kg intraperitoneally). The trachea was cannulated and arterial blood pressure and heart rate were recorded throughout the experiment from a polyethylene cannula inserted into the left carotid artery and connected to a Statham transducer coupled to a Grass polygraph. A 25gauge stainless-steel cannula was directed to the lateral cerebral ventricle (intracerebroventricularly) through a burr hole in the skull using a stereotaxic apparatus (David ~~
Key words: bradykinin, central nervous system, cerebrospinal fluid, depressor effect, kinin antagonist, kinin receptor, spontaneously hypertensive rats. Abbreviations: CSF, cerebrospinal fluid; SHR. spontaneously hypertensive rats; WKY, Wistar-Kyoto rats. Correspondence: D r Carlos J.Pirola, Cardiology Department, Becker Building no. 210, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, U.S.A.
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A. L. Alvarez et al.
Kopf, Tujuna, CA, U.S.A.). Co-ordinates for implantation were 0.3 mm posterior to the bregma, 1.5 mm lateral to the midline and 3.5 mm below the dura [16]. At the end of each experiment the animals were killed and the position of the infusion cannula and the distribution of the infused volume were assessed by injection of 1 pl of 2% (w/v) Bromophenol Blue solution. The head was then perfused with saline (150 mmol/l NaCI) followed by 10% (w/v) formaldehyde, and sections were cut throughout the diencephallon for histological verification. Only data collected from experiments in which the correct insertion of the cannula was verified are reported. In fact, the dye injected into the lateral ventricle diffused through the entire ventricular system including the dorsolateral surface of the medulla. Saline and full doses of the bradykinin antagonist des-Arg9--[LeuX]bradykinin (0.1-10 pug) were infused (1 pl/min over 3 min) through the intracerebral guide cannula. Only one dose was applied per animal. ED,, values were estimated from five independent dose-response curves as the dose in the infused solution of the bradykinin antagonist that lowered blood pressure or heart rate by 50% of the maximum effect. CSF was obtained by cisterna magna puncture in anaesthetized animals placed in the stereotaxic apparatus. Samples (pool of three to five rats, 60-140 pI each, n = 6 per strain) used for the measurement of kinin concentration were collected in 0.5 ml of ice-cold 0.4 mol/l HCIO,. Insoluble components were separated by centrifugation at 17 000 g for 10 min. The supernatant was neutralized with 0.5 mol/l KOH and was freeze-dried. The residue was redissolved in the appropriate buffer. Bradykinin was measured by r.i.a. as previously described [ 171, using [ 12,1Tyr8]bradykinin (Dupont, Buenos Aires, Argentina). The r i a . was performed in 0.05 mol/l NaH,PO, buffer, pH 7.4 containing 0.1% BSA, 100 units of aprotinin/ml, 0.1 mmol/l phenylmethanesulphonyl fluoride and 0.1 mmol/l EDTA. The incubation mixture contained 0.1 ml of ['2sI-TyrX]bradykinin(5000-8000 c.p.m.), 0.1 ml of a 1:4000 dilution of antiserum and 0.1 ml of lysine bradykinin as standard ranging from 5 to 1000 pg or sample. The final volume was 0.3 ml. Tubes were incubated for 24 h at 4°C. Six milligrams of Dextran-charcoal(6.25 g of Norit A charcoal and 6.25 g of Dextran T70/1 in 50 mmol/l phosphate buffer, pH 7.4) was used to separate free from antibody-bound kinins. After centrifugation, the pellet was counted in an automatic y-counter (Alfa Nuclear, Buenos Aires, Argentina). Calculations were performed using a computerized linear least-square curve-fitting procedure from plots of c.p.m. versus log of standard amounts. Typical intra- and inter-assay coefficients of variation were 7 and 12%, respectively. The limit of detection of the assay was 7-12 pg. Portions of CSF (pool of three to five rats, 60-150 pl each, n = 11per strain) were kept at -20°C until determination of kininogen, which was performed by measuring kinins released with excess kallikrein (Sigma Co.) at 37°C in the presence of a cocktail of peptidase inhibitors comprising 3.0 mmol/l 1, 10-phenanthroline, 30 mmol/l EDTA and 30 prnolll N-ethylmaleimide. The reaction was stopped
by the addition of 0.5 ml of ice-cold 0.4 mol/l HCIO,, and samples were processed as described for the CSF kinin assay.
Studies in vitro The rats were killed by decapitation and the brain was rapidly removed. The dorsal and rostroventrolateral medulla, hypothalamus, septum, hypophysis and pineal gland were dissected and separated on ice-cold plates with the aid of a stereotaxic atlas [16]. For determination of kinin, the nervous tissue (pool of three to four rats) was submerged in 2 mol/l acetic acid acidified to pH 1 with concentrated HCI, boiled for 20 min, homogenized and centrifuged at 10 000 gfor 10 min. The pellet was stored at - 20°C until protein determination was performed. The supernatants were freeze-dried and the residue was dissolved in buffer for determination of kinin by r i a . as described above. In the procedure for the determination of the CSF concentration of kinin and the tissue content of kinin, the recovery of synthetic lysine bradykinin was higher than 80% and only 4-6% of the original amount of a purified dog kininogen, a possible interference in the assay [ 171, was detected. In addition, identification of the bradykininlike immunoreactivity was performed by using highperformance liquid chromatographic equipment (Waters Associates, Milford, MA, U.S.A.), which consisted of a radial compression module with a uBondapak C,, cartridge or a Lichrospher RP-8 (Merck, Darmstadt, Germany) and a model 440 fixed-wavelength U.V. detector. The analog detector outputs were recorded with an OmniScribe recorder. There were two mobile phases ( A and B): phase A consisted of 50 mmol/l triethylamine adjusted to pH 3 with phosphoric acid; phase B was methanol. After injection, the components were eluted from the column using a concave gradient from 0 to 30 min, and from 30 to 35 min the column was reverseprogrammed employing a Baseline 8 10 computer program (Waters Associates) installed in a APC IV NEC personal computer. Flow rates of 1-1.5 ml/min were reached with a Waters model 6000 and 590 solvent delivery system. Since more than 90% of the kinin-like immunoreactivity present in rat nervous tissue co-eluted with authentic bradykinin, we will refer to it as 'bradykinin'. To evaluate the parameters of bradykinin-binding sites, [12sI-Tyr']bradykinin was used as ligand [18]. [I2% Tyrllbradykinin (300-700 Ci/mmol) was used at concentrations of 0.025-20 nmol/l. The radiolabelled analogue was prepared by a modification of the chloramine-T method 1191 and was purified by using the chromatographic system described above. Fractions containing monoiodinated [Tyrllbradykinin were identified by its retention time using unlabelled monoiodinated [Tyr'lbradykinin prepared by the method of Odya et al. [181. The nervous tissue (pool of six to eight rats) was homogenized in 10 vol of 0.32 mol/l sucrose and homogenates were centrifuged at 1000 g for 10 min to
515
Bradykininergic system in spontaneously hypertensive rats
obtain a crude nuclear pellet, which was discarded. The resulting supernatant fraction was centrifuged at 17 000 g for 15 min to obtain a synaptosomal pellet. The pellet was resuspended in the original volume of 0.32 mol/l sucrose for the binding assay. Binding reactions were performed at 4°C in polypropylene test tubes. The tissue homogenate was added to each tube in 0.1 ml containing (mg of protein): rostroventrolateral and dorsal medulla, 0.7 and 1.4, respectively; hypothalamus, 0.8; septum, 0.4; hypophysis, 0.3; pineal gland, 0.02. The incubation mixture consisted of 0.1% BSA, 0.3 mmol/l bacitracin, 1 pmol/l SQ14225, 0.1 mmol/l EDTA, 0.1 mmol/l phenylmethanesulphonyl fluoride and 100 units of aprotinin/ ml in 0.05 mol/l phosphate buffer, pH 7.4. This protease-inhibitor mixture protects peptide integrity during incubation as assayed by the h.p.1.c. system described above. After incubation for 60 min, equilibrium was reached and 3 mi of ice-cold NaKHPO, buffer was added. Samples were passed through a glass filter (Whatman GF/ B) under vacuum. The filters were washed three times under vacuum with 3 ml of ice-cold buffer, then placed in tubes and the radioactivity bound was counted in a ycounter (Alfa Nuclear). The results are presented as specific [ 1251-Tyr1]bradykinin binding, which is defined as the difference between binding in the absence and presence of unlabelled bradykinin (1pmol/l). At a radioligand concentration of 5 nmol/l, specific binding accounted for 55-65% of the total binding. For calculation of the bradykinin receptor concentration (B,,,=,)and dissociation constant (&), analysis of the binding data by the non-linear least-square curve-fitting procedure using a generalized model for ligand-receptor systems [20] was used. Results
are expressed as means k SEM of five independent experiments. Protein concentrations were determined by the method of Lowry et al. [211. Statistical analysis was performed by using paired Student t-tests and analysis of variance with Tukey's test for individual differences. Results are expressed as means 5 SEM.
Drugs [Tyr'IBradykinin, lysine bradykinin, des-Argy[Leu*]bradykinin,BSA, bacitracin, EDTA (disodium salt), phenylmethanesulphonylfluoride and chloramine-T were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A). SQ14225 and aprotinin (Trasylol) were kindly provided by Squibb (Buenos Aires, Argentina) and Bayer (Buenos Aires, Argentina), respectively. All the reagents were of the best grade available. The polyclonal antiserum to lysine bradykinin was a generous gift from H. Nolly and 0.A. Cerretero, Henry Ford Hospital, Detroit, MI, U.S.A.
RESULTS The kinin antagonist des-Argy-[Leu*]bradykinin, infused at doses of 0.1-10 pg into the lateral ventricle, produced no change in arterial blood pressure and heart rate in WKY (Table 1),but induced a significant dosedependent long-lasting decrease in arterial blood pressure and heart rate in SHR. The same volume of vehicle produced no change in arterial blood pressure or heart rate in both strains of rats. The ED,, values for the hypotensive and bradycardic effects were 1.3k0.6 and
Table I. Effect of intracerebroventricular infusion of the kinin antagonist des-Arg9-[leu*]bradykininon arterial blood pressure and heart rate in WKY and SHR. The basal mean arterial blood pressure and heart rate were 166+4 mmHg and 398+5 beatslmin, respectively, for SHR and I15 +7 mmHg and 360+ 10 beatslmin, respectively, for WKY. Results were expressed as the meansfsm from 10 rats per group at each dose of antagonist. Only durations o f significant responses are included. Statistical significance: *P
