(Hypertension. 1996;28:224-227.)
© 1996 American Heart Association, Inc.
Articles |
Angiotensin II Increases Norepinephrine Turnover in the Anteroventral Third Ventricle of Spontaneously Hypertensive Rats
the Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka City (A.T., T.T., I.A., M.F.), and Hizen National Mental Hospital, Center for Emotional and Behavioral Disorder (K.N., H.U.), Kanzaki, Saga, Japan.
Correspondence to Akira Tsukashima, MD, Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka City 812, Japan.
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Abstract |
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We evaluated the effect of angiotensin II (Ang II) administered by intracerebroventricular injection on norepinephrine turnover in the anteroventral third ventricle in adult spontaneously hypertensive rats (SHR, n=35) and age-matched Wistar-Kyoto rats (WKY, n=38). Ang II (100 ng) or saline (vehicle control) was administered into the cerebral ventricle 30 minutes after injection of
-methyl-p-tyrosine (250 mg/kg IP). Norepinephrine turnover was assessed by evaluation of the norepinephrine concentration before and 1 hour after such administration. The pressor response to Ang II administration was significantly greater in SHR than in WKY (+43±3 versus +23±2 mm Hg, P<.01). Baseline norepinephrine turnover (response to saline) was reduced in the ventral median preoptic nucleus of SHR. Ang II significantly increased norepinephrine turnover in the organum vasculosum lamina terminalis and ventral median preoptic nucleus of SHR (organum vasculosum lamina terminalis: 40±5% by Ang II versus 18±6% by saline, P<.05; ventral median preoptic nucleus: 32±3% by Ang II versus 21±2% by saline, P<.05) but not of WKY (37±5% versus 29±5%, P=NS, and 30±2% versus 32±3%, P=NS, respectively). Thus, norepinephrine turnover in the anteroventral third ventricle region induced by intracerebroventricular administration of Ang II was increased in SHR. This effect may contribute to the enhanced pressor response to central Ang II seen in this model.
Key Words: angiotensin II • brain • hypertension, genetic • norepinephrine • nervous system • rats, inbred SHR
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Introduction |
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The role of the brain renin-angiotensin system in the development and maintenance of hypertension in SHR has been well delineated. For instance, brain Ang II levels are higher,1 and the pressor response to intracerebroventricular administration of Ang II is augmented2 in SHR. In addition, blood pressure reduction was observed by central but not by peripheral administration of Ang II antagonists3 4 or angiotensin-converting enzyme inhibitors.5 6 More recently, the antisense inhibition of Ang II subtype 1 receptor mRNA or angiotensinogen mRNA in the brain resulted in the significant reduction of blood pressure in SHR.7 8
The region of the AV3V is considered essential to the pressor and dipsogenic responses produced by intracerebroventricular administration of Ang II, as such responses are attenuated by induction of an electrolytic lesion of the AV3V.9 More specifically, local injection of the neurotoxin 6-hydroxydopamine into the AV3V region attenuates the effect of intracerebroventricular Ang II,10 11 suggesting that the norepinephrine systems present in the AV3V may be involved in the central action of Ang II. The altered response of AV3V neurons to Ang II in SHR is suggested by the evidence that Ang II–induced excitation of the neurons in the OVLT occurs at significantly lower thresholds in SHR than in normotensive WKY.12 These observations may suggest the possible alteration of norepinephrine neuronal activity in the nuclei of the AV3V region in response to Ang II in SHR. To test this hypothesis, we investigated whether norepinephrine turnover in response to intracerebroventricular injection of Ang II would be altered in discrete nuclei within the AV3V region of SHR.
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Methods |
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Animals
A total of 35 male SHR (332±3 g) and 38 male WKY (349±3 g) (14 weeks old) were obtained from Charles River Japan Co. They were housed in our animal colony with controlled temperature, humidity, and lighting. Standard rat chow and tap water were provided ad libitum.
Surgical Procedures
All procedures were carried out according to the institutional guidelines for animal experimentation. Three days before the experiment, rats were anesthetized with pentobarbital (50 mg/kg IP), and a stainless steel cannula was implanted stereotaxically into the right lateral cerebroventricle. It was fixed in place with dental cement and stainless steel screws. One day before the experiment, the rats were anesthetized with ether, and a catheter was inserted into a femoral artery for blood pressure measurement. The opposite end of the catheter was exited through the dorsal skin of the back. Catheter patency was maintained with heparinized 0.9% saline. Blood pressure was determined with a transducer attached to the catheter and was recorded with a polygraph.
Experimental Protocol
On the day of the experiment, an intra-arterial catheter was connected to the transducer, and arterial pressure and heart rate were monitored continuously in conscious rats. Rats were injected with intraperitoneal -MT (250 mg/kg, Sigma Chemical Co) followed in 30 minutes by intracerebroventricular Ang II (100 ng/2 µL saline, Sigma) or saline (2 µL) with a Hamilton syringe. Rats in the control group (SHR, n=12; WKY, n=11) were decapitated 30 minutes after -MT injection for measurement of norepinephrine concentration before intracerebroventricular injection of Ang II or saline. The rats in the saline group (SHR, n=10; WKY, n=11) and Ang II group (SHR, n=13; WKY, n=16) were decapitated 60 minutes after injection of either saline or Ang II. The brain was quickly removed and frozen at -80°C until the assay of norepinephrine.
Tissue Preparation
Sections of brain 300 µm thick were cut with a cryostat and the following three nuclei were dissected from the AV3V: the OVLT, the ventral MnPO, and the dorsal MnPO. Dissection was done by hand with a micro knife under a stereomicroscope on a cold plate (-12°C),13 with reference to the atlas of Paxinos and Watson.14 The OVLT was dissected from one slice just rostral to the anterior commissure; the ventral MnPO (ventral to the anterior commissure) was dissected from three slices caudal to the OVLT; and the dorsal MnPO (dorsal to the anterior commissure) was dissected from two slices just caudal to the OVLT.
Assay of Catecholamine and Protein
Tissue was homogenized in 100 µL of 0.1 mol/L formic acid containing 400 µmol/L NaHSO3, 50 µmol/L ascorbic acid, and isoproterenol (internal standard). A volume of 20 µL homogenate was used for protein assay. The remainder was centrifuged at 10 000 rpm for 10 minutes. Supernatants were assayed for catecholamines by high-performance liquid chromatography. The chromatographic system used Neurochem (ESA Inc) that had 16 coulometric electrochemical detectors in series and was set to increase at a constant voltage. Proteins in the homogenates were determined by the method of Lowry et al.15 Tissue norepinephrine concentration was assessed as norepinephrine (picograms)/protein (micrograms).
Catecholamine Turnover
Catecholamine utilization was evaluated with the -MT method.16 -MT blocks catecholamine synthesis so that norepinephrine turnover can be measured without interference by newly synthesized catecholamines. Norepinephrine concentration was measured before (control) and after intracerebroventricular injection of Ang II or saline and calculated as follows: [1-(Norepinephrine Concentration After Intracerebroventricular Ang II or Saline/Norepinephrine Concentration Before Intracerebroventricular Injection)]x100 (percent).
Statistical Analysis
Results are expressed as mean±SE. Statistical analysis was performed with one-way ANOVA followed by multiple comparisons with Scheffe's test. A value of P<.05 was considered statistically significant.
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Results |
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Blood Pressure and Heart Rate Responses
Mean arterial pressure (MAP) and heart rate (HR) at baseline and after intracerebroventricular injection of Ang II are shown in the Table
. Baseline MAP was significantly higher in SHR than in WKY (P<.001), and baseline HR did not differ between the strains. Intraperitoneal injection of -MT by itself did not affect MAP or HR in both strains. The increase in MAP evoked by intracerebroventricular injection of Ang II was significantly greater in SHR than in WKY (P<.01). HR of both strains was unaffected by intracerebroventricular Ang II.
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Effects of Ang II on Norepinephrine Utilization in AV3V Regions
As indicated in the top panel of the Figure, baseline norepinephrine turnover (response to saline) in the OVLT did not differ between SHR and WKY (18±6% versus 29±5%, respectively; P=NS). Intracerebroventricular Ang II significantly increased norepinephrine turnover only in SHR (40±5% by Ang II versus 18±6% by saline, P<.05). The middle panel of the Figure shows norepinephrine turnover in the ventral MnPO. Baseline norepinephrine turnover was significantly lower in SHR compared with WKY (21±2% versus 32±3%, P<.05). Intracerebroventricular injection of Ang II significantly increased norepinephrine turnover in SHR (32±3% by Ang II versus 21±2% by saline, P<.05) but not in WKY (30±2% versus 32±3%, P=NS). The bottom panel of the Figure shows norepinephrine turnover in dorsal MnPO. Baseline norepinephrine turnover did not differ significantly between SHR and WKY. In addition, norepinephrine turnover was unaffected by Ang II in both strains.
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Discussion |
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In the present study, we evaluated the involvement of norepinephrine neurons within the AV3V region in the central action of Ang II and found that norepinephrine turnover was increased by intracerebroventricular Ang II in SHR but not in WKY.
The role of norepinephrine neurons of the central nervous system in the development and maintenance of hypertension has been investigated either by measurement of tissue norepinephrine content or the activity of catecholamine synthesizing enzyme or by evaluation of norepinephrine turnover of the various nuclei. Norepinephrine content is either increased17 or decreased13 18 in the brain stem or hypothalamus of SHR. On the other hand, the activity of the norepinephrine-forming enzyme dopamine ß-hydroxylase is decreased in hypothalamic nuclei of SHR. As regards norepinephrine turnover in hypertensive animals, Patel et al19 reported that norepinephrine turnover was decreased in the cortex of young SHR and increased in the hypothalamus of adult SHR. They also showed that neurogenic hypertension induced by the transection of the aortic depressor nerve was associated with increased norepinephrine turnover in the neurons of the hypothalamus.20 In the present study, baseline norepinephrine turnover in the ventral MnPO was decreased in SHR compared with WKY, whereas norepinephrine turnover in either the dorsal MnPO or OVLT did not differ between the strains. Thus, norepinephrine turnover of SHR may differ according to the region of the brain.
The effect of central and peripheral Ang II on norepinephrine turnover has been investigated in various brain tissues. For instance, intravenous administration of Ang II increases norepinephrine turnover of the locus coeruleus in rabbits.21 Intracerebroventricular Ang II also increases norepinephrine turnover of the locus coeruleus and hypothalamus in rats.16 22 Sumners and Phillips16 investigated catecholamine utilization after central injection of Ang II in the areas including the subfornical organ and OVLT of normotensive rats and found no substantial change in norepinephrine turnover of these nuclei. This finding is in accordance with the present observation that norepinephrine turnover remains unchanged after intracerebroventricular Ang II in the OVLT and MnPO of WKY. In contrast, the present study demonstrates that norepinephrine turnover in the OVLT and ventral MnPO is increased by central injection of Ang II in SHR. The difference between SHR and WKY in Ang II–induced facilitation of norepinephrine release is also reported in the anterior hypothalamus.23 These observations may suggest an alteration of the relationship between central Ang II and norepinephrine neuronal activities in the various brain areas of SHR. One can argue that the increase in norepinephrine turnover in SHR might be due to the greater increase in blood pressure after Ang II injection. However, considering the evidence that baseline norepinephrine turnover is not increased in the OVLT and dorsal MnPO but is decreased in the ventral MnPO of SHR in the present study and that norepinephrine turnover in various brain areas including hypothalamus is reported to be unchanged by the pressor stimulus induced by intravenous injection of hypertonic saline,16 the change in norepinephrine turnover seems to be attributable to the action of Ang II rather than the increase in blood pressure. It is possible that baseline or Ang II–induced norepinephrine turnover may be influenced by the age or level of blood pressure in SHR. Patel et al19 investigated norepinephrine turnover in the brain areas of SHR and normotensive rats at various ages and found that norepinephrine turnover of hypothalamus was increased in 18-week-old SHR compared with age-matched WKY. This difference, however, was not observed in younger rats. Since we used 14-week-old SHR and WKY in the present study, the role of norepinephrine neurons within the AV3V region in the pressor response to intracerebroventricular Ang II in younger or prehypertensive SHR needs to be clarified in future studies.
The efferent system from AV3V nuclei that mediates the central pressor response to Ang II has been vigorously investigated. Since an anterior hypothalamic knife cut eliminates the pressor response to centrally administered Ang II, Hartle and Brody24 hypothesize that AV3V nuclei send the efferent pressor pathway that projects to the brain stem or to the neurohypophysis through the anterior hypothalamus. We have focused on the AV3V nuclei as sites of actions of Ang II and norepinephrine, but the role of Ang II and norepinephrine turnover within the anterior hypothalamus in blood pressure regulation has also been reported in the salt-sensitive model of SHR.25 26 27 Since ascending noradrenergic pathways from brain stem project to the AV3V region and participate in cardiovascular regulation,28 we speculate that the increased norepinephrine turnover in the AV3V nuclei may have contributed to the augmented pressor response to intracerebroventricular Ang II in SHR. Although the magnitude of blood pressure increase was smaller than in SHR, a pressor response to Ang II did occur in WKY without a significant change in norepinephrine turnover. This observation may suggest that an increase of norepinephrine turnover per se may not be essential to the pressor response elicited by intracerebroventricular Ang II. However, it seems reasonable to hypothesize that in SHR the Ang II–induced increase in norepinephrine turnover in specific nuclei of the AV3V would reinforce the action of Ang II on the efferent pressor pathway from these nuclei. The hypothesis that the catecholamine projections into the AV3V region modulate the central effect of Ang II is also supported by the previous report by Bellin et al11 that injection of 6-hydroxydopamine into the MnPO or OVLT attenuates the pressor response elicited by intracerebroventricular Ang II.
In conclusion, norepinephrine turnover in the nuclei of the AV3V differed between SHR and WKY. The increase in norepinephrine turnover induced by the central administration of Ang II in some nuclei within the AV3V of SHR may be related to the increased pressor response observed in this strain.
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Selected Abbreviations and Acronyms |
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Received October 6, 1995; first decision December 5, 1995; accepted March 26, 1996.
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