Cardiovascular Research 1992;26:26 1-264
26 1
Decreased hypothalamic and medullary GABA turnover in spontaneously hypertensive rats Susumu Sasaki, Tadao Kuwabara, Tatsuo Yoshitomi, Yuusuke Yoneda, Ken Takenaka, Toshiyuki Takesako, Motoo Tanaka, Masahiro Hirata, Shinichi Tanabe, Tetsuo Nakata, Kazuo Takeda, and Masao Nakagawa Objective: The aim was to assess whether Gamma-aminobutyric acid (GABA) neurone activities in the central nervous system, especially in the hypothalamus and medulla oblangata, are altered in hypertension. Methods: Central GABA content and turnover rate were measured in spontaneously hypertensive rats (SHR) and their normotensive Wistar Kyoto controls (WKY). GABA content was determined with high performance liquid chromatography, and in vivo GABA turnover rates were estimated by GABA accumulation after injection of amino-oxyacetic acid, a selective inhibitor of GABA degrading system. Two groups of nine week old male rats (32 SHR and 32 WKY) were used. Results: GABA concentrations in cerebrospinal fluid were lower in SHR than in WKY. Since hypothalamus and medulla oblongata are the possible active sites of this system, basal GABA contents and in vivo GABA turnover rates were measured in hypothalamus and medulla oblongata. Basal GABA content in the medulla oblongata and hypothalamus was almost equal in SHR and WKY. On the other hand, GABA turnover rates were significantly lower in SHR than in WKY in both the hypothalamus and the medulla. Conclusions: Since it is known that GABA is an inhibitory neurotransmitter in the central nervous system and that it controls autonomic and cardiovascular activities, the findings suggest that the decreased hypothalamic and medullary GABAergic activities may permit sympathetic hyperactivity to contribute to the increase in blood pressure in SHR.
Y
-Aminobutyric acid (GABA), an inhibitory neurotransmitter in the central nervous system, is known to control autonomic and cardiovascular activities.' We previously showed that acute intracerebroventricular (icv) injections of GABA reduced sympathetic nerve activity centrally, thereby decreasing blood pressure and heart rate in anaesthetised rats, and these reductions in sympathocardiovascular activities were larger in spontaneously hypertensiye rats (SHR) than in Wistar Kyoto (WKY) controls.- We also found that long term GABAergic stimulation attenuated the development of spontaneous hypertension in rats.4 These findings suggest that central GABAergic neurone activities were reduced in SHR and that this reduction in GABAergic activities would result in sympathetic hyperactivity and hypertension. Although the exact sites of GABAergic action in regulating sympathetic outflow remain undetermined, the medulla and the hypothalamus have been implicated since they contain GABA and its synthesising enzyme, glutamic acid decarboxylase,s and since they are major sites controlling sympathetic outflow.' ' In practice, blood pressure is altered by injection of either GABAergic agonists or antagonists directly into the medulla' as well as into the hypthalamus.9 Accordingly, for the first study, cerebrospinal fluid GABA concentrations in SHR were measured to determine whether overall central GABAergic activities are altered in hypertension; and for the second study, hypothalamic and medullary GABA contents and turnover rates were measured to determine whether GABAergic neurone activities in these
localised regions are altered in hypertension. We estimated GABA turnover rate by measuring the accumulation of the neurotransmitter after inhibition of its major catabolic pathway'" using amino-oxyacetic acid. This method is justified by the fact that GABA is metabolised almost exclusively into succinic semialdehyde by GABA-T (4-aminobutyrate:2-oxygIutamate aminotransferase, EC2.6.1.19), and therefore the amount of GABA accumulated after inhibition of GABA-T with amino-oxyacetic acid treatment is equal to the amount of GABA synthesised."
Methods Two groups of nine week old male rats (32 SHR and 32 WKY), from the strain bred originally by Okamoto and Aoki, were purchased from Charles River Breeding Laboratories (Kanagawa, Japan). All rats were first weighed and systolic blood pressures and heart rates were measured indirectly by a tail cuff method validated for use in awake rats. Animal care and procedures conformed to the position of the American Heart Association on research animal use. Experiment 1. CSF GABA coticentrcrtions in SHR Ten WKY and 10 SHR rats were used. After each rat was
anaesthetised with urethane (80 mg.100 g-', intraperitoneal), the atlanto-occipital membrane was exposed with the rat mounted on a stereotaxic apparatus (David Kopf), and the cisterna magna was punctured with a 27 gauge needle connected through polyethylene tubing to a 100 p.1
Second Department of Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan: S Sasaki, T Kuwabara, T Yoshitomi, Y Yoneda, K Takenaka, T Takesako, M Tanaka, M Hirata, S Tanabe, T Nakata, K Takeda, M Nakagawa. Correspondence to Dr Sasaki.
262
Sasaki, Kuwabara, Yoshitomi, Yoneda, Takenaka, Takesako, Tanaka. Hirata, Tanabe, Nakata, Takeda, Nakagawa
microsyringe. CSF was slowly withdrawn manually in a volume of 50 p I, and deproteinised immediately by adding 150 p1 of an aqueous 6.7% solution of sulphosalicylic acid. After removal of proteins by centrifugation (8 000 g, 5 rnin), the supernatant was kept at 4°C and analysed within 4 d. GABA concentrations were determined with the fully automatic high perfomance liquid chromatography technique described by Bohlem et al." Experiment 11. Hypothalamic and medullary GABA content in SHR Eight WKY and eight SHR were used. Each rat was killed by focused microwave irradiation (5 kW, 0.7 s)1° between 13.00 and 14.00 hours to avoid possible circadian variations in the endogenous level of neuroactive amino acids." The brain was immediately removed from the skull to a chilled plastic plate, and hypothalamus and medulla oblongata were separated according to the dissection procedure described by Glowinski and 1 ~ e r s e n . lThe ~ brain tissues were then weighed and ultrasonically homogenised with 5% sulphosalicylic acid (1.0 ml.100 mg-I). After standing at room temperature for 10 min, the homogenates were centrifuged (8000 g, 5 min) and the GABA concentrations of the supernatant were determined using the same methods as in experiment I. Experiment Ill. Hypothalamic and medullary GABA turnover in S H R Fourteen WKY and 14 SHR were used. Since a low dose of amino-oxyacetic acid has been known to inhibit the activity of GABA transaminase (a GABA degrading enzyme) to a greater extent than that of glutamate decarboxylase (a GABA synthesizing enzyme), the in vivo GABA turnover rate in the brain was estimated by measuring the accumulation of GABA following amino-oxyacetic acid injection.lO-" All rats were intraperitoneally injected with amino-oxyacetic acid hemihydrochloride (30 mg.kg-'), purchased from Sigma Chemical Company, in a volume of 100 p1, while the rats were awake. Half of the rats in each group were killed 60 min later and the other rats were killed 180 min later by microwave irradiation. Statistics Data are expressed as mean (SEM). Results from two groups were compared using a two tailed Student's t test for independent samples, and differences at a 5% level or less (pS0.05) were considered significant. GABA accumulation ratio (turnover) were compared using two way analysis of variance with repeated measures and interaction F ratio at a 5% level or less were considered significant.
Results Systolic blood pressures, measured with a tail cuff method while rats were awake, were already higher in SHR than in WKY, at 153(SEM 2) v 124(1) mm Hg, pc0.01, and heart rates were also higher in SHR than in WKY, at 380(18) v 316(8) beats-min-l, p
26 1
Decreased hypothalamic and medullary GABA turnover in spontaneously hypertensive rats Susumu Sasaki, Tadao Kuwabara, Tatsuo Yoshitomi, Yuusuke Yoneda, Ken Takenaka, Toshiyuki Takesako, Motoo Tanaka, Masahiro Hirata, Shinichi Tanabe, Tetsuo Nakata, Kazuo Takeda, and Masao Nakagawa Objective: The aim was to assess whether Gamma-aminobutyric acid (GABA) neurone activities in the central nervous system, especially in the hypothalamus and medulla oblangata, are altered in hypertension. Methods: Central GABA content and turnover rate were measured in spontaneously hypertensive rats (SHR) and their normotensive Wistar Kyoto controls (WKY). GABA content was determined with high performance liquid chromatography, and in vivo GABA turnover rates were estimated by GABA accumulation after injection of amino-oxyacetic acid, a selective inhibitor of GABA degrading system. Two groups of nine week old male rats (32 SHR and 32 WKY) were used. Results: GABA concentrations in cerebrospinal fluid were lower in SHR than in WKY. Since hypothalamus and medulla oblongata are the possible active sites of this system, basal GABA contents and in vivo GABA turnover rates were measured in hypothalamus and medulla oblongata. Basal GABA content in the medulla oblongata and hypothalamus was almost equal in SHR and WKY. On the other hand, GABA turnover rates were significantly lower in SHR than in WKY in both the hypothalamus and the medulla. Conclusions: Since it is known that GABA is an inhibitory neurotransmitter in the central nervous system and that it controls autonomic and cardiovascular activities, the findings suggest that the decreased hypothalamic and medullary GABAergic activities may permit sympathetic hyperactivity to contribute to the increase in blood pressure in SHR.
Y
-Aminobutyric acid (GABA), an inhibitory neurotransmitter in the central nervous system, is known to control autonomic and cardiovascular activities.' We previously showed that acute intracerebroventricular (icv) injections of GABA reduced sympathetic nerve activity centrally, thereby decreasing blood pressure and heart rate in anaesthetised rats, and these reductions in sympathocardiovascular activities were larger in spontaneously hypertensiye rats (SHR) than in Wistar Kyoto (WKY) controls.- We also found that long term GABAergic stimulation attenuated the development of spontaneous hypertension in rats.4 These findings suggest that central GABAergic neurone activities were reduced in SHR and that this reduction in GABAergic activities would result in sympathetic hyperactivity and hypertension. Although the exact sites of GABAergic action in regulating sympathetic outflow remain undetermined, the medulla and the hypothalamus have been implicated since they contain GABA and its synthesising enzyme, glutamic acid decarboxylase,s and since they are major sites controlling sympathetic outflow.' ' In practice, blood pressure is altered by injection of either GABAergic agonists or antagonists directly into the medulla' as well as into the hypthalamus.9 Accordingly, for the first study, cerebrospinal fluid GABA concentrations in SHR were measured to determine whether overall central GABAergic activities are altered in hypertension; and for the second study, hypothalamic and medullary GABA contents and turnover rates were measured to determine whether GABAergic neurone activities in these
localised regions are altered in hypertension. We estimated GABA turnover rate by measuring the accumulation of the neurotransmitter after inhibition of its major catabolic pathway'" using amino-oxyacetic acid. This method is justified by the fact that GABA is metabolised almost exclusively into succinic semialdehyde by GABA-T (4-aminobutyrate:2-oxygIutamate aminotransferase, EC2.6.1.19), and therefore the amount of GABA accumulated after inhibition of GABA-T with amino-oxyacetic acid treatment is equal to the amount of GABA synthesised."
Methods Two groups of nine week old male rats (32 SHR and 32 WKY), from the strain bred originally by Okamoto and Aoki, were purchased from Charles River Breeding Laboratories (Kanagawa, Japan). All rats were first weighed and systolic blood pressures and heart rates were measured indirectly by a tail cuff method validated for use in awake rats. Animal care and procedures conformed to the position of the American Heart Association on research animal use. Experiment 1. CSF GABA coticentrcrtions in SHR Ten WKY and 10 SHR rats were used. After each rat was
anaesthetised with urethane (80 mg.100 g-', intraperitoneal), the atlanto-occipital membrane was exposed with the rat mounted on a stereotaxic apparatus (David Kopf), and the cisterna magna was punctured with a 27 gauge needle connected through polyethylene tubing to a 100 p.1
Second Department of Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan: S Sasaki, T Kuwabara, T Yoshitomi, Y Yoneda, K Takenaka, T Takesako, M Tanaka, M Hirata, S Tanabe, T Nakata, K Takeda, M Nakagawa. Correspondence to Dr Sasaki.
262
Sasaki, Kuwabara, Yoshitomi, Yoneda, Takenaka, Takesako, Tanaka. Hirata, Tanabe, Nakata, Takeda, Nakagawa
microsyringe. CSF was slowly withdrawn manually in a volume of 50 p I, and deproteinised immediately by adding 150 p1 of an aqueous 6.7% solution of sulphosalicylic acid. After removal of proteins by centrifugation (8 000 g, 5 rnin), the supernatant was kept at 4°C and analysed within 4 d. GABA concentrations were determined with the fully automatic high perfomance liquid chromatography technique described by Bohlem et al." Experiment 11. Hypothalamic and medullary GABA content in SHR Eight WKY and eight SHR were used. Each rat was killed by focused microwave irradiation (5 kW, 0.7 s)1° between 13.00 and 14.00 hours to avoid possible circadian variations in the endogenous level of neuroactive amino acids." The brain was immediately removed from the skull to a chilled plastic plate, and hypothalamus and medulla oblongata were separated according to the dissection procedure described by Glowinski and 1 ~ e r s e n . lThe ~ brain tissues were then weighed and ultrasonically homogenised with 5% sulphosalicylic acid (1.0 ml.100 mg-I). After standing at room temperature for 10 min, the homogenates were centrifuged (8000 g, 5 min) and the GABA concentrations of the supernatant were determined using the same methods as in experiment I. Experiment Ill. Hypothalamic and medullary GABA turnover in S H R Fourteen WKY and 14 SHR were used. Since a low dose of amino-oxyacetic acid has been known to inhibit the activity of GABA transaminase (a GABA degrading enzyme) to a greater extent than that of glutamate decarboxylase (a GABA synthesizing enzyme), the in vivo GABA turnover rate in the brain was estimated by measuring the accumulation of GABA following amino-oxyacetic acid injection.lO-" All rats were intraperitoneally injected with amino-oxyacetic acid hemihydrochloride (30 mg.kg-'), purchased from Sigma Chemical Company, in a volume of 100 p1, while the rats were awake. Half of the rats in each group were killed 60 min later and the other rats were killed 180 min later by microwave irradiation. Statistics Data are expressed as mean (SEM). Results from two groups were compared using a two tailed Student's t test for independent samples, and differences at a 5% level or less (pS0.05) were considered significant. GABA accumulation ratio (turnover) were compared using two way analysis of variance with repeated measures and interaction F ratio at a 5% level or less were considered significant.
Results Systolic blood pressures, measured with a tail cuff method while rats were awake, were already higher in SHR than in WKY, at 153(SEM 2) v 124(1) mm Hg, pc0.01, and heart rates were also higher in SHR than in WKY, at 380(18) v 316(8) beats-min-l, p
