- Email: [email protected]
Elevated tyrosine hydroxylase mRNA levels in medulla oblongata of spontaneously hypertensive rats
~: . . . . . :, ,,
-
MOLECULAR BRAIN RESEARCH
iZi,:u ELSEVIER
Molecular Brain Research 36 (1996) 197-199
Short communication
Elevated tyrosine hydroxylase mRNA levels in medulla oblongata of spontaneously hypertensive rats Toshio Kumai *, Masami Tanaka, Minoru Watanabe, Hironori Nakura, Tomonori Tateishi, Shinichi Kobayashi Department of Pharmacolo~', St. Marianna Unirersi~' School of Medicine, 2-16-I Sugao, Miyamae-ku, Kawa~aki 21h, Japan Accepted 31 October 1995
Abstract
The present study has investigated the expression of tyrosine hydroxylase (TH) mRNA and its activity in medulla oblongata of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). The TH mRNA levels were determined by Northern blot and dot blot analyses. The TH activity and the expression of TH mRNA in medulla oblongata of SHR were significantly higher than those of WKY. These results suggest that the hypertension of SHR may be related to the high activity of TH due to the high level of TH mRNA which increases norepinephrine levels in the medulla oblongata. Ke~vwords: Tyrosinc hydroxylase mRNA; Medulla oblongata; Spontaneously hypertensive rats
The sympathetic nervous system is thought to play an important role in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR) [17]. Howes demonstrated increased norepinephrine in several regions of the brain of SHR [8]. The medulla oblongata was seen as important for blood pressure regulation and synthesis of catecholamine [1]. Versteeg et al. [20] reported that norepinephrine in the brain stem, which includes the medulla oblongata, increased in SHR. However, the underlying mechanism has remained unsolved. Tyrosine hydroxylase (TH) is a rate-limiting enzyme in the synthetic pathway of catecholamine. We have previously reported that the expression of Ttt mRNA in adrenal medulla was increased in SHR [9]. The purpose of this study was to determine the TH mRNA level in the medulla oblongata of SHR. Male SHR and WKY (25 weeks old) were used. Systolic blood pressure was measured in conscious rats by the tail-cuff method using an electrosphygmomanometer (PSI00, Riken Kaihatsu). The medulla oblongata was dissected by the method of Glowinski and Iversen [7]. Norepinephrine levels were assayed as previously described [10]. The medulla oblongata was homogenized with 0.05 N
• Corresponding author. Fax: (81) (44) 975-050q. 0 1 6 9 - 3 2 8 X / 9 6 / $ 1 5 . ( X ) (t3 1996 Elsevier Science B.V. All rights reserved SSDI ( 1 1 6 9 - 3 2 8 X ( 9 5 ) 0 ( 1 2 9 6 - 0
perchloric acid in a glass tissue grinder. Norepinephrine was extracted with aluminum oxide. Norepinephrine was measured by HPLC-ECD (460 electrochemical detector, Waters). The mobile phase was a mixture of 50 mM sodium acetate, 20 mM citric acid, 3.75 mM sodium octyl sulphate, 1 mM di-n-butylamine, 0.134 mM EDTA, 5% ( v / v ) methanol. All separation was performed isocratically at a flow rate of 0.9 m l / m i n at 35°C. The detector potential was maintained at +0.65 V. TH activity was assayed by the method of Nagatsu ct al. [14]. The medulla oblongata was homogenized with 0.25 M sucrose in a glass tissue grinder. The homogenate was incubated with 1 mM L-tyrosine and 1 mM 6-methyl-5,6,7,8-tetrahydropterin at 37°C for 30 min. DOPA was extracted with aluminum oxide and Amberlite CG50. DOPA was measured by HPLC-ECD. The mobile phase was a mixture of 50 mM sodium acetate, 20 mM citric acid, 12.5 mM sodium octyl sulphate, 1 mM di-n-butylamine, 0.134 mM EDTA. All separation was performed isocratically at a flow rate of 0.7 m l / m i n at 27°C. The detector potential was maintained at + 0.65 V. TH activity was calculated as amount of DOPA synthesis from tyrosine. The methods of Northern blot and dot blot analyses for the TH mRNA level determination were previously described [11]. Total RNA fraction was extracted from medulla oblongata by the method of Chomczynski and Sacchi [2]. Northern
11 Kumai et al. / Molecular Brain Research 36 (1996) 197-199
198
blotting was performed as follows. Total RNA (20 p.g) was treated with glyoxal. After agarose gel electrophoresis, blotting to nitrocellulose membrane overnight. This membrane hybridized with the radiolabeled rat TH cDNA (containing the region from + 14 to + 1165 bp; a gift from Dr. D.M. Chikaraishi, Tufts University School of Medicine, Boston) [12] or human /3-actin cDNA (0.4 kb, Wako Junyaku Co.) [15]. These probes were labeled with [ot32p]dCTP. The membranes were washed and exposed to scientific imaging film (Eastman Kodak Co.) for 1 day. Dot blotting was carried out as follows. Total RNA (3 /.tg) was treated with glyoxal, and solution applied to nitrocellulose membrane. This membrane was baked and hybridized with the radiolabeled rat TH cDNA or human /3-actin cDNA. The membrane was washed and exposed to scientific imaging film (Eastman Kodak Co.) for 1 day. Statistical difference between mean values was calculated by Student's t-test. The TH m R N A expression in medulla oblongata of SHR was detected trough Northern blot analysis as a single band of 1.8 kb. This particular mRNA showed a significantly higher intensity than did that of W K Y (Fig. 1). As a matter of fact, when the [32 p] radioactivities of the dots were determined, the TH m R N A level in medulla oblongata of SHR was found to be as high as 2 times that of WKY. (SHR, 1.8-1-0.3 TH mRNA//3-actin mRNA, n = 3; WKY, 0.8 ___0.1 TH mRNA//3-actin mRNA, n = 3,
SHR
WKY
TH
/3 --Actin
Fig. l. The expression of TH mRNA and /3-actin in medulla oblongata of SHR and WKY by Northern blot analysis. Aliquots of total RNA (20/.tg) isolated from medulla oblongata of SHR (left lane) and WKY (right lane) were hybridized with radiolabeled rat TH cDNA or human /3-actin cDNA.
t-
~ 2.5
P<0.05
<, <
2
Z
E -rl. 5 _
tO
o ~0.5
= 0 r',¢'
0
I
SHR
(3)
WKY
(3)
Fig. 2. The ratio of expression of TH mRNA to fl-actin in medulla oblongata of SHR and WKY by dot blot analysis. Aliquots of total RNA (3 p.g) isolated from medulla oblongata of SHR (left column) and WKY (right column) were hybridized with radiolabeled rat TH eDNA or human /3-actin eDNA. The ratio of TH mRNA to /3-actin of medulla oblongata in SHR (left column) was significantly higher than that in WKY (right column) (P < 0.05). Mean + SEM.
P < 0.05) (Fig. 2). The higher TH mRNA level of SHR accompanied higher TH activities (SHR, 448.5 + 122.9 n g / g tissue/h, n = 7; WKY, 194.5 + 23.4 n g / g tissue/h, n = 6, P < 0.05) and a higher norepinephrine level (SHR, 444.9 + 30.0 n g / g tissue, n = 5; WKY, 349.7 + 30.4 n g / g tissue, n = 4, P < 0.05) in the medulla oblongata, as well as higher systolic blood pressure (SHR, 184.8 + 3.8 mm Hg, n = 6; WKY, 133.4 + 3.0 mm Hg, n = 5, P < 0.01). The major findings of the present study were that the increased expression of TH mRNA in SHR goes along with increased TH activity, norepinephrine level of medulla oblongata and hypertension. De Jong et al. demonstrated that norepinephrine levels were elevated in the ponsmedulla of SHR [5]. Feuerstein et al. reported that TH activity in medulla oblongata of a hypertensive strain of Sabra rats was significantly higher than that of normotensive strains [6]. Because TH is rate-limiting in catecholamine synthesis, an increase in its level would be reflected by a concomitant increase in the medulla oblongata norepinephrine level. Our data strongly suggest that the increased expression of TH mRNA in medulla oblongata results in the increases of both TH activity and norepinephrine level. Several regions in the medulla oblongata have been implicated in the control of blood pressure. A pressor zone in the rostral two-thirds of the medulla oblongata lying in the lateral reticular formation and a depressor zone in the caudal third of the medulla oblongata in the median position are two such [1]. The norepinephrine levels in the nucleus tractus solitarius, which contains the primary synapses of the afferents of the arterial baroreceptors, of
T. Kumai et al. / Molecular Brain Resear~:h 36 (1q961 107-199
S H R were l o w e r or similar to those o f W K Y [16,18]. Also, Luque et al. reported that the T H m R N A expression in the locus coeruleus o f S H R at the well-established hypertensive stage was similar to age matched W K Y [13]. On the other hand, V e r s t e e g et al. reported that the norepinephrine levels in the A1, A2, A5 and A7 regions o f the S H R brain stem w e r e higher than those of W K Y [9]. Smith and Barron indicated an increase in the level of activity of sympathetic neurons in the ventrolaterai medulla ( V L M ) o f S H R [19]. The c a t e c h o l a m i n e r g i c neurons in the V L M w e r e designated the A1 cell groups [4]. Further, the neurons in the V L M are i n v o l v e d in the m a i n t e n a n c e and reflex regulation of s y s t e m i c arterial pressure [3]. Therefore, our present data may suggest the elevation of noradrenergic m e c h a n i s m s in the maintenance regions of the v a s o m o t o r tone of S H R . In conclusion, the present results clearly indicate that the transcription of g e n o m i c D N A of T H is significantly increased in S H R . W h e t h e r the transcription rate of gen o m i c T H D N A is accelerated in the medulla oblongata of S H R still remains to be studied. The elevated activity of T H due to the high level of T H m R N A that results in increased norepinephrine levels in the medulla oblongata may play a crucial role in the pathogenesis o f hypertension in S H R .
[6]
[7] [81 [9]
[10]
[11]
[12]
[13]
[14]
Acknowledgements W e are grateful to Dr. D o n a M.Chikaraishi g e n e r o u s gift of rat T H e D N A .
[15] for the [16]
References [1] Alexander, R.S., Tonic and reflex functions of medullary sympathetic cardiovascular centers, J. Neurophysiol., 9 (1946) 205-218. [2] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162 (19871 156-159. [3] Ciriello, J., Caverson, M.M. and Polosa, C., Function of the ventrolateral medulla in the control of the circulation, Brain Res. Ret., 1 I (19861 359-391. [4] Dahlstrom, A. and Fuxc, K., Evidence for the existence of monoamine containing neurons in the central nervous system. 1. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62, Suppl. 232 (1964) 1-55. [5] De Jong, W., Nijkamp, F.P. and Bohus, B., Role of noradrenaline and serotonin in the central control of blood pressure in normoten-
[17]
[18]
[19]
[20]
lt~t~
sive and spontaneously hypertensive rats. Arch Int. Pharmaeodyn, 213 (1975) 272-284. Feuerstein, G., Zamir, N., Ben-lshay. D. and Gutman, Y., Inverse rclationship of medulla oblongata noradrenaline concentration and tyrosine hydroxylase activity in hypertensive-prone and -resistent rats, J. Neurochem., 33 (19791 393-395. Glowinski, J. and Iversen, I,.I.., Regional studies of catecholamines in the rat brain-I, J. Neurt~'hem., 13 (19661 655-669. Howes, L.G., Central catecholaminergic neurons and spontaneously hypertensive rats, J. Auton. Pharmacol., 4 (19841 207-217. Kumai, T., Tanaka, M., Watanabe, M. and Kobayashi, S., Elevated tyrosine hydroxylasc mRNA levels in the adrenal medulla of spontaneously hypertensive rats, Jpn. J. Pharmaeol., 65 (19941 367-369. Kumai, T., Tanaka, M., Watanabe, M., Matsumoto, ('. and Kobayashi, S., Possible involvement of androgen in increased norepinephrine synthesis in blood vessels of spontaneously hypertensive rats, Jpn. J. Pharmacol., 66 (19941 439-444. Kumai, T., Tanaka, M., Watanabe, M.. Nakura, tl. and Kobayashi. S., Influence of androgen on tyrosinc hydroxylase mRNA in adrenal medulla of spontaneously hypertensive rats. Hvpertenston, 26 (19951 208-212. Lewis, E.J., Tank, A.W., Weincr, N. and Chikaraishi, D.M., Regulation of tyrosine hydroxylase mRNA by glucocorticoid and cyclic AMP in a rat pheochromocytoma cell line, ,1. Biol. Chem.. 258 ( 19831 14632-14637. Luque, J.M., Guillamon, A. and Hwang, BH.. Quantitative autoradiographic study on tyrosine hydroxylase mRNA with in situ hybridization and a2 adrenergic receptor binding in the locus coeruleus of the spontaneously hypertensive rat. Neurosci. l.ett., 131 (1991) 163-166. Nagatsu, T., Oka, K. and Kato, T., Highly sensitive assay for tyrosine hydroxylasc activity by high-peffurmance liquid chromatography, J. Chromatogr.. 163 (1979) 247-252. Nakajima, I.S., Hamada, H., Reddy, P. and Kakunaga, T., Molecular structure of the human cytoplasmic /3-actin gene: interspecics homology of sequences in the intron. Proc. Natl. Acad. Sci. USA, 77 (1980) 5201-5205. Nomura, M., Ohtsuji, M. and Nagata, Y., Changes in thc alphaadrenoceptors in the medulla oblongata including nucleus tractus solitarius of spontaneously hypertensive rats. Neurochem. Res., 10 (19851 1143-1154. Okamoto, K., Nosaka, S., Yamori, Y. and Matsumoto. M., Participation of neural factor in the pathogenesis of hypertension in the spontaneously hypertensive rats, .Ipn. Heart J., 8 (19671 168-18(I. Saavedra, J.M., Grobecker, tt. and Axelrod, J., Changes in central catecholaminergic neurons in the spontaneously (genetic) hypertensive rat, Circ'. Res., 42 (19781 529-534. Smith, J.K. and Barton, K.W., Cardiovascular eflccts of L-glutamate and tetrodotoxin microinjected into the rostral and caudal ventrolateral medulla in normotensive and spontaneously hypertensive rats. Brain Res., 5(16 (1990) 1-8. Verstecg, D.H.G, Palkovits, M., van der Gugten, J., Wijnen, H.I..JM.. Smeets, G.W.M. and de Jong W., Catecholamine content of individual brain regions of spontaneously hypertensive rats (Stlrats), Brain Res'., 112 (19761 429-434.
-
MOLECULAR BRAIN RESEARCH
iZi,:u ELSEVIER
Molecular Brain Research 36 (1996) 197-199
Short communication
Elevated tyrosine hydroxylase mRNA levels in medulla oblongata of spontaneously hypertensive rats Toshio Kumai *, Masami Tanaka, Minoru Watanabe, Hironori Nakura, Tomonori Tateishi, Shinichi Kobayashi Department of Pharmacolo~', St. Marianna Unirersi~' School of Medicine, 2-16-I Sugao, Miyamae-ku, Kawa~aki 21h, Japan Accepted 31 October 1995
Abstract
The present study has investigated the expression of tyrosine hydroxylase (TH) mRNA and its activity in medulla oblongata of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). The TH mRNA levels were determined by Northern blot and dot blot analyses. The TH activity and the expression of TH mRNA in medulla oblongata of SHR were significantly higher than those of WKY. These results suggest that the hypertension of SHR may be related to the high activity of TH due to the high level of TH mRNA which increases norepinephrine levels in the medulla oblongata. Ke~vwords: Tyrosinc hydroxylase mRNA; Medulla oblongata; Spontaneously hypertensive rats
The sympathetic nervous system is thought to play an important role in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR) [17]. Howes demonstrated increased norepinephrine in several regions of the brain of SHR [8]. The medulla oblongata was seen as important for blood pressure regulation and synthesis of catecholamine [1]. Versteeg et al. [20] reported that norepinephrine in the brain stem, which includes the medulla oblongata, increased in SHR. However, the underlying mechanism has remained unsolved. Tyrosine hydroxylase (TH) is a rate-limiting enzyme in the synthetic pathway of catecholamine. We have previously reported that the expression of Ttt mRNA in adrenal medulla was increased in SHR [9]. The purpose of this study was to determine the TH mRNA level in the medulla oblongata of SHR. Male SHR and WKY (25 weeks old) were used. Systolic blood pressure was measured in conscious rats by the tail-cuff method using an electrosphygmomanometer (PSI00, Riken Kaihatsu). The medulla oblongata was dissected by the method of Glowinski and Iversen [7]. Norepinephrine levels were assayed as previously described [10]. The medulla oblongata was homogenized with 0.05 N
• Corresponding author. Fax: (81) (44) 975-050q. 0 1 6 9 - 3 2 8 X / 9 6 / $ 1 5 . ( X ) (t3 1996 Elsevier Science B.V. All rights reserved SSDI ( 1 1 6 9 - 3 2 8 X ( 9 5 ) 0 ( 1 2 9 6 - 0
perchloric acid in a glass tissue grinder. Norepinephrine was extracted with aluminum oxide. Norepinephrine was measured by HPLC-ECD (460 electrochemical detector, Waters). The mobile phase was a mixture of 50 mM sodium acetate, 20 mM citric acid, 3.75 mM sodium octyl sulphate, 1 mM di-n-butylamine, 0.134 mM EDTA, 5% ( v / v ) methanol. All separation was performed isocratically at a flow rate of 0.9 m l / m i n at 35°C. The detector potential was maintained at +0.65 V. TH activity was assayed by the method of Nagatsu ct al. [14]. The medulla oblongata was homogenized with 0.25 M sucrose in a glass tissue grinder. The homogenate was incubated with 1 mM L-tyrosine and 1 mM 6-methyl-5,6,7,8-tetrahydropterin at 37°C for 30 min. DOPA was extracted with aluminum oxide and Amberlite CG50. DOPA was measured by HPLC-ECD. The mobile phase was a mixture of 50 mM sodium acetate, 20 mM citric acid, 12.5 mM sodium octyl sulphate, 1 mM di-n-butylamine, 0.134 mM EDTA. All separation was performed isocratically at a flow rate of 0.7 m l / m i n at 27°C. The detector potential was maintained at + 0.65 V. TH activity was calculated as amount of DOPA synthesis from tyrosine. The methods of Northern blot and dot blot analyses for the TH mRNA level determination were previously described [11]. Total RNA fraction was extracted from medulla oblongata by the method of Chomczynski and Sacchi [2]. Northern
11 Kumai et al. / Molecular Brain Research 36 (1996) 197-199
198
blotting was performed as follows. Total RNA (20 p.g) was treated with glyoxal. After agarose gel electrophoresis, blotting to nitrocellulose membrane overnight. This membrane hybridized with the radiolabeled rat TH cDNA (containing the region from + 14 to + 1165 bp; a gift from Dr. D.M. Chikaraishi, Tufts University School of Medicine, Boston) [12] or human /3-actin cDNA (0.4 kb, Wako Junyaku Co.) [15]. These probes were labeled with [ot32p]dCTP. The membranes were washed and exposed to scientific imaging film (Eastman Kodak Co.) for 1 day. Dot blotting was carried out as follows. Total RNA (3 /.tg) was treated with glyoxal, and solution applied to nitrocellulose membrane. This membrane was baked and hybridized with the radiolabeled rat TH cDNA or human /3-actin cDNA. The membrane was washed and exposed to scientific imaging film (Eastman Kodak Co.) for 1 day. Statistical difference between mean values was calculated by Student's t-test. The TH m R N A expression in medulla oblongata of SHR was detected trough Northern blot analysis as a single band of 1.8 kb. This particular mRNA showed a significantly higher intensity than did that of W K Y (Fig. 1). As a matter of fact, when the [32 p] radioactivities of the dots were determined, the TH m R N A level in medulla oblongata of SHR was found to be as high as 2 times that of WKY. (SHR, 1.8-1-0.3 TH mRNA//3-actin mRNA, n = 3; WKY, 0.8 ___0.1 TH mRNA//3-actin mRNA, n = 3,
SHR
WKY
TH
/3 --Actin
Fig. l. The expression of TH mRNA and /3-actin in medulla oblongata of SHR and WKY by Northern blot analysis. Aliquots of total RNA (20/.tg) isolated from medulla oblongata of SHR (left lane) and WKY (right lane) were hybridized with radiolabeled rat TH cDNA or human /3-actin cDNA.
t-
~ 2.5
P<0.05
<, <
2
Z
E -rl. 5 _
tO
o ~0.5
= 0 r',¢'
0
I
SHR
(3)
WKY
(3)
Fig. 2. The ratio of expression of TH mRNA to fl-actin in medulla oblongata of SHR and WKY by dot blot analysis. Aliquots of total RNA (3 p.g) isolated from medulla oblongata of SHR (left column) and WKY (right column) were hybridized with radiolabeled rat TH eDNA or human /3-actin eDNA. The ratio of TH mRNA to /3-actin of medulla oblongata in SHR (left column) was significantly higher than that in WKY (right column) (P < 0.05). Mean + SEM.
P < 0.05) (Fig. 2). The higher TH mRNA level of SHR accompanied higher TH activities (SHR, 448.5 + 122.9 n g / g tissue/h, n = 7; WKY, 194.5 + 23.4 n g / g tissue/h, n = 6, P < 0.05) and a higher norepinephrine level (SHR, 444.9 + 30.0 n g / g tissue, n = 5; WKY, 349.7 + 30.4 n g / g tissue, n = 4, P < 0.05) in the medulla oblongata, as well as higher systolic blood pressure (SHR, 184.8 + 3.8 mm Hg, n = 6; WKY, 133.4 + 3.0 mm Hg, n = 5, P < 0.01). The major findings of the present study were that the increased expression of TH mRNA in SHR goes along with increased TH activity, norepinephrine level of medulla oblongata and hypertension. De Jong et al. demonstrated that norepinephrine levels were elevated in the ponsmedulla of SHR [5]. Feuerstein et al. reported that TH activity in medulla oblongata of a hypertensive strain of Sabra rats was significantly higher than that of normotensive strains [6]. Because TH is rate-limiting in catecholamine synthesis, an increase in its level would be reflected by a concomitant increase in the medulla oblongata norepinephrine level. Our data strongly suggest that the increased expression of TH mRNA in medulla oblongata results in the increases of both TH activity and norepinephrine level. Several regions in the medulla oblongata have been implicated in the control of blood pressure. A pressor zone in the rostral two-thirds of the medulla oblongata lying in the lateral reticular formation and a depressor zone in the caudal third of the medulla oblongata in the median position are two such [1]. The norepinephrine levels in the nucleus tractus solitarius, which contains the primary synapses of the afferents of the arterial baroreceptors, of
T. Kumai et al. / Molecular Brain Resear~:h 36 (1q961 107-199
S H R were l o w e r or similar to those o f W K Y [16,18]. Also, Luque et al. reported that the T H m R N A expression in the locus coeruleus o f S H R at the well-established hypertensive stage was similar to age matched W K Y [13]. On the other hand, V e r s t e e g et al. reported that the norepinephrine levels in the A1, A2, A5 and A7 regions o f the S H R brain stem w e r e higher than those of W K Y [9]. Smith and Barron indicated an increase in the level of activity of sympathetic neurons in the ventrolaterai medulla ( V L M ) o f S H R [19]. The c a t e c h o l a m i n e r g i c neurons in the V L M w e r e designated the A1 cell groups [4]. Further, the neurons in the V L M are i n v o l v e d in the m a i n t e n a n c e and reflex regulation of s y s t e m i c arterial pressure [3]. Therefore, our present data may suggest the elevation of noradrenergic m e c h a n i s m s in the maintenance regions of the v a s o m o t o r tone of S H R . In conclusion, the present results clearly indicate that the transcription of g e n o m i c D N A of T H is significantly increased in S H R . W h e t h e r the transcription rate of gen o m i c T H D N A is accelerated in the medulla oblongata of S H R still remains to be studied. The elevated activity of T H due to the high level of T H m R N A that results in increased norepinephrine levels in the medulla oblongata may play a crucial role in the pathogenesis o f hypertension in S H R .
[6]
[7] [81 [9]
[10]
[11]
[12]
[13]
[14]
Acknowledgements W e are grateful to Dr. D o n a M.Chikaraishi g e n e r o u s gift of rat T H e D N A .
[15] for the [16]
References [1] Alexander, R.S., Tonic and reflex functions of medullary sympathetic cardiovascular centers, J. Neurophysiol., 9 (1946) 205-218. [2] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162 (19871 156-159. [3] Ciriello, J., Caverson, M.M. and Polosa, C., Function of the ventrolateral medulla in the control of the circulation, Brain Res. Ret., 1 I (19861 359-391. [4] Dahlstrom, A. and Fuxc, K., Evidence for the existence of monoamine containing neurons in the central nervous system. 1. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62, Suppl. 232 (1964) 1-55. [5] De Jong, W., Nijkamp, F.P. and Bohus, B., Role of noradrenaline and serotonin in the central control of blood pressure in normoten-
[17]
[18]
[19]
[20]
lt~t~
sive and spontaneously hypertensive rats. Arch Int. Pharmaeodyn, 213 (1975) 272-284. Feuerstein, G., Zamir, N., Ben-lshay. D. and Gutman, Y., Inverse rclationship of medulla oblongata noradrenaline concentration and tyrosine hydroxylase activity in hypertensive-prone and -resistent rats, J. Neurochem., 33 (19791 393-395. Glowinski, J. and Iversen, I,.I.., Regional studies of catecholamines in the rat brain-I, J. Neurt~'hem., 13 (19661 655-669. Howes, L.G., Central catecholaminergic neurons and spontaneously hypertensive rats, J. Auton. Pharmacol., 4 (19841 207-217. Kumai, T., Tanaka, M., Watanabe, M. and Kobayashi, S., Elevated tyrosine hydroxylasc mRNA levels in the adrenal medulla of spontaneously hypertensive rats, Jpn. J. Pharmaeol., 65 (19941 367-369. Kumai, T., Tanaka, M., Watanabe, M., Matsumoto, ('. and Kobayashi, S., Possible involvement of androgen in increased norepinephrine synthesis in blood vessels of spontaneously hypertensive rats, Jpn. J. Pharmacol., 66 (19941 439-444. Kumai, T., Tanaka, M., Watanabe, M.. Nakura, tl. and Kobayashi. S., Influence of androgen on tyrosinc hydroxylase mRNA in adrenal medulla of spontaneously hypertensive rats. Hvpertenston, 26 (19951 208-212. Lewis, E.J., Tank, A.W., Weincr, N. and Chikaraishi, D.M., Regulation of tyrosine hydroxylase mRNA by glucocorticoid and cyclic AMP in a rat pheochromocytoma cell line, ,1. Biol. Chem.. 258 ( 19831 14632-14637. Luque, J.M., Guillamon, A. and Hwang, BH.. Quantitative autoradiographic study on tyrosine hydroxylase mRNA with in situ hybridization and a2 adrenergic receptor binding in the locus coeruleus of the spontaneously hypertensive rat. Neurosci. l.ett., 131 (1991) 163-166. Nagatsu, T., Oka, K. and Kato, T., Highly sensitive assay for tyrosine hydroxylasc activity by high-peffurmance liquid chromatography, J. Chromatogr.. 163 (1979) 247-252. Nakajima, I.S., Hamada, H., Reddy, P. and Kakunaga, T., Molecular structure of the human cytoplasmic /3-actin gene: interspecics homology of sequences in the intron. Proc. Natl. Acad. Sci. USA, 77 (1980) 5201-5205. Nomura, M., Ohtsuji, M. and Nagata, Y., Changes in thc alphaadrenoceptors in the medulla oblongata including nucleus tractus solitarius of spontaneously hypertensive rats. Neurochem. Res., 10 (19851 1143-1154. Okamoto, K., Nosaka, S., Yamori, Y. and Matsumoto. M., Participation of neural factor in the pathogenesis of hypertension in the spontaneously hypertensive rats, .Ipn. Heart J., 8 (19671 168-18(I. Saavedra, J.M., Grobecker, tt. and Axelrod, J., Changes in central catecholaminergic neurons in the spontaneously (genetic) hypertensive rat, Circ'. Res., 42 (19781 529-534. Smith, J.K. and Barton, K.W., Cardiovascular eflccts of L-glutamate and tetrodotoxin microinjected into the rostral and caudal ventrolateral medulla in normotensive and spontaneously hypertensive rats. Brain Res., 5(16 (1990) 1-8. Verstecg, D.H.G, Palkovits, M., van der Gugten, J., Wijnen, H.I..JM.. Smeets, G.W.M. and de Jong W., Catecholamine content of individual brain regions of spontaneously hypertensive rats (Stlrats), Brain Res'., 112 (19761 429-434.