Brain Research, 112 (1976) 429--434 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
429
Catecholamine content of individual brain regions of spontaneously hypertensive rats (SH-rats)
DIRK H. G. VERSTEEG, MIKL6S PALKOVITS*, JAN VAN DER GUGTEN, HENK L. J. M. WIJNEN, GERARD W. M. SMEETS and WYBREN DE JONG Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Utrecht (The Netherlands)
(Accepted May 10th, 1976)
Many reports have contributed to the notion that catecholamine neurons in the brain stem participate in the central regulation of arterial blood pressure (for reviews see refs. 2, 6). In recent years considerable effort has been made to relate the increase in blood pressure of spontaneously hypertensive rats (SH-rats 5) to catecholamine metabolism in the brain. Initially, a lower noradrenaline level and aromatic amino acid decarboxylase activity 2a, and also a decreased noradrenaline synthesis 9, were observed in the brain stem of SH-rats compared to that of normotensive controls. However, in later studies, in which SH-rats were compared with rats of the genetically related normotensive Wistar-Kyoto strain (W/K-rats), these findings were not corroborated2L Moreover, considerable differences in the activity of catecholamine synthesizing enzymes occurred in the brain stem of various strains of rats with substantial differences in bloodpressure level10, whereas no correlation was evident between the activity of any of these enzymes and blood pressure 10. Although the latter observations seem to argue against the existence of obvious correlates, it might be that the occurrence of changes in catecholamine content and metabolism in relatively small brain regions escaped detection in these studies, a possibility which was also suggested by Yamabe et al.2L In fact, it was observed in our laboratory that noradrenaline levels were slightly elevated in the pons-medulla of recent generation SH-rats compared to those of W/K-rats 7 and 10 weeks after birthL The present study was undertaken to investigate this phenomenon in more detail. Using a sensitive radiometric method for the simultaneous assay of noradrenaline, dopamine and adrenaline 20, we measured the catecholamine content of individual nuclei and brain regions of SH-rats and W/K-rats. Male Wistar-Kyoto-NIH Cpb (F6) and SHR-NIH Cpb (F32) were obtained from TNO Zeist (The Netherlands) at an age of 14 weeks (for genealogy see ref. 5). Systolic blood pressure measurements were carried out with a tail-plethysmographic method on trained conscious rats s. The rats were killed by decapitation at an age of * 1st Department of Anatomy, Semmelweis University Medical School, Budapest.
430 16 weeks. Systolic blood pressure of the W/K-rats was 125 + 1 mm Hg (mean S.E.M., n = 18); that of the SH-rats was 196 zk 2 mm Hg (n .... 18). Following decapitation the brains were rapidly taken out and frozen on dry ice. Serial sections of 300/~m werecut in a cryostat at --10°C. Individual brain nuclei and regions were removed with small punches lz. A total of 27 regions were punched from telencephalon, diencephalon, mesencephalon, pons and medulla oblongata as previously described 1215,21. Tissue pellets of 3 rats were pooled. Estimated weights of the pooled tissue ranged from 0.3 to 5 mg. The pellets were homogenized in 100/zl 0.1 N HC104. Protein was assayed in 5-25 #1 samples of the homogenates according to Lowry et al. la. The homogenates were subsequently centrifuged for 15 min at 10,000 >~ g. Noradrenaline, dopamine and adrenaline were assayed in 20/~1 samples of the supernatants according to Van der Gugten et al. 2°. Data are expressed as the percentage of means of the catecholamine content (in pg/#g protein) of the W/K controls. A significantly elevated dopamine content was observed in the frontal cortex, but not in the cingulate cortex, and in the nucleus interstitialis striae terminalis of SHrats compared to the controls (Fig. 1, telencephalon). No differences were measured in the noradrenaline concentrations of the telencephalic regions. Whereas noradrenaline and dopamine levels were similar in the diencephalic regions of the SH- and W/K-rats, a significantly elevated adrenaline concentration was apparent in the nucleus paraventricularis of SH-rats (Fig. 1, diencephalon). In the brain stem elevations of noradrenaline were measured in the nucleus cuneiformis (A7 region), the A5 region, the nucleus raphe magnus, the reticular formation, the A1 region and the A2 region. In the latter region elevated adrenaline and dopamine levels were observed as welt (Fig. 1, brain stem). The observed elevations in the brain levels of noradrenaline, dopamine and
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429
Catecholamine content of individual brain regions of spontaneously hypertensive rats (SH-rats)
DIRK H. G. VERSTEEG, MIKL6S PALKOVITS*, JAN VAN DER GUGTEN, HENK L. J. M. WIJNEN, GERARD W. M. SMEETS and WYBREN DE JONG Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Utrecht (The Netherlands)
(Accepted May 10th, 1976)
Many reports have contributed to the notion that catecholamine neurons in the brain stem participate in the central regulation of arterial blood pressure (for reviews see refs. 2, 6). In recent years considerable effort has been made to relate the increase in blood pressure of spontaneously hypertensive rats (SH-rats 5) to catecholamine metabolism in the brain. Initially, a lower noradrenaline level and aromatic amino acid decarboxylase activity 2a, and also a decreased noradrenaline synthesis 9, were observed in the brain stem of SH-rats compared to that of normotensive controls. However, in later studies, in which SH-rats were compared with rats of the genetically related normotensive Wistar-Kyoto strain (W/K-rats), these findings were not corroborated2L Moreover, considerable differences in the activity of catecholamine synthesizing enzymes occurred in the brain stem of various strains of rats with substantial differences in bloodpressure level10, whereas no correlation was evident between the activity of any of these enzymes and blood pressure 10. Although the latter observations seem to argue against the existence of obvious correlates, it might be that the occurrence of changes in catecholamine content and metabolism in relatively small brain regions escaped detection in these studies, a possibility which was also suggested by Yamabe et al.2L In fact, it was observed in our laboratory that noradrenaline levels were slightly elevated in the pons-medulla of recent generation SH-rats compared to those of W/K-rats 7 and 10 weeks after birthL The present study was undertaken to investigate this phenomenon in more detail. Using a sensitive radiometric method for the simultaneous assay of noradrenaline, dopamine and adrenaline 20, we measured the catecholamine content of individual nuclei and brain regions of SH-rats and W/K-rats. Male Wistar-Kyoto-NIH Cpb (F6) and SHR-NIH Cpb (F32) were obtained from TNO Zeist (The Netherlands) at an age of 14 weeks (for genealogy see ref. 5). Systolic blood pressure measurements were carried out with a tail-plethysmographic method on trained conscious rats s. The rats were killed by decapitation at an age of * 1st Department of Anatomy, Semmelweis University Medical School, Budapest.
430 16 weeks. Systolic blood pressure of the W/K-rats was 125 + 1 mm Hg (mean S.E.M., n = 18); that of the SH-rats was 196 zk 2 mm Hg (n .... 18). Following decapitation the brains were rapidly taken out and frozen on dry ice. Serial sections of 300/~m werecut in a cryostat at --10°C. Individual brain nuclei and regions were removed with small punches lz. A total of 27 regions were punched from telencephalon, diencephalon, mesencephalon, pons and medulla oblongata as previously described 1215,21. Tissue pellets of 3 rats were pooled. Estimated weights of the pooled tissue ranged from 0.3 to 5 mg. The pellets were homogenized in 100/zl 0.1 N HC104. Protein was assayed in 5-25 #1 samples of the homogenates according to Lowry et al. la. The homogenates were subsequently centrifuged for 15 min at 10,000 >~ g. Noradrenaline, dopamine and adrenaline were assayed in 20/~1 samples of the supernatants according to Van der Gugten et al. 2°. Data are expressed as the percentage of means of the catecholamine content (in pg/#g protein) of the W/K controls. A significantly elevated dopamine content was observed in the frontal cortex, but not in the cingulate cortex, and in the nucleus interstitialis striae terminalis of SHrats compared to the controls (Fig. 1, telencephalon). No differences were measured in the noradrenaline concentrations of the telencephalic regions. Whereas noradrenaline and dopamine levels were similar in the diencephalic regions of the SH- and W/K-rats, a significantly elevated adrenaline concentration was apparent in the nucleus paraventricularis of SH-rats (Fig. 1, diencephalon). In the brain stem elevations of noradrenaline were measured in the nucleus cuneiformis (A7 region), the A5 region, the nucleus raphe magnus, the reticular formation, the A1 region and the A2 region. In the latter region elevated adrenaline and dopamine levels were observed as welt (Fig. 1, brain stem). The observed elevations in the brain levels of noradrenaline, dopamine and
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A
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431 ~OFof
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N^ l"1WlI~
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