Enhanced Norepinephrine Release in Hypothalamus from Locus Coeruleus in SHR

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1 Enhanced Norepinephrine Release in Hypothalamus from Locus Coeruleus in SHR Shingo KAWASAKI, M.D., Kazuo TAKEDA, M.D., Motoo TANAKA, M.D., Hiroshi ITOH, M.D., Masahiro HIRATA, M.D., Tetsuo NAKATA, M.D., Junko HAYASHI, M.D., Minako OGURO, M.D., Susumu SASAKI, M.D., and Masao NAKAGAWA, M.D. SUMMARY The hypothesis that a functional projection from the locus coeruleus (LC) to the posterior hypothalamus contributes to the development of hypertension in SHR, was tested by measuring norepinephrine (NE) in the posterior hypothalamus by brain dialysis after injections of L-glutamate (L-glu) into LC. L-glu elicited a prolonged elevation of blood pressure in both SHR and WKY. Pressor effects were significantly larger in SHR than in WKY. Extracellular NE in the posterior hypothalamus increased after LC stimulation; NE release was significantly higher in SHR than in WKY. Injections of 6-hydroxydopamine (6-OHDA) into posterior hypothalamus lowered the resting blood pressure and attenuated the pressor responses to L-glu injections into the LC in SHR. These findings suggest that the LC projects functionally to the posterior hypothalamus and that the projection can contribute to the development of hypertension in SHR. Additional Indexing Words: Hypertension Brain dialysis L-glutamate 6-Hydroxydopamine Microinjection Radio-enzymatic assay T has been reported that sympathetic hyperactivity may contribute to the pathogenesis of hypertension in SHR.1) Part of such hyperactivity may be a consequence of central alterations of the noradrenergic system.2) This hypothesis is supported by several studies. For example, depletion of central catecholamines by administration of 6-OHDA reversed the hypertension in several animal models.3) In addition, transection of the aortic depressor nerve in rats produces an elevated arterial blood pressure and increased NE turnover in the hypothalamus.4) As previously demonstrated, the elimina- From the Second Department of Medicine, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kyoto, Japan. Address correspondence to: Kazuo Takeda, M.D., Second Department of Medicine, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602, Japan. Received for publication February 5, Accepted September 6,

2 256 KAWASAKI, ET AL. Jpn. Heart J. March 1991 tion of baroreflex by sinoaortic denervation can produce both elevated blood pressure, and increased extracellular NE in the posterior hypothalamus.5) These facts suggest that the central noradrenergic system has an important role in the regulation of arterial blood pressure. The locus coeruleus contains almost half of the total number of noradrenaline-synthesizing neurons in the brain.6) It innervates many levels of the central nervous system,7) and anatomical connections between the locus coeruleus and the sympathetic nervous system have been suggested.8) This study examines whether functional projections of the locus coeruleus to the posterior hypothalamus can contribute to the development of hypertension in SHR. Two types of experiments were performed. First extracellular NE was measured in the posterior hypothalamus by brain dialysis after injections of L-glutamate into the locus coeruleus. Second, blood pressure responses to L-glu injections into the locus coeruleus were measured in rats that received injections of 6-hydroxydopamine (6-OHDA) in the posterior hypothalamus. The experimental results support the role of an LC-posterior hypothalamus circuit in hypertension. MATERIALS AND METHODS Eight week old WKY and SHR rats (WKY, n=12; SHR, n=11) were used for 6-OHDA treatment study; 9 week old rats (WKY, n=6; SHR, n= 5) were used for brain dialysis. Systolic blood pressure and heart rate were measured in conscious rats by a tail cuff method (Narco-Biosystem Inc.) before and after 6-OHDA treatment. Under urethane (1.2g/kg) anesthesia, a cannula was inserted into the right femoral artery for blood pressure measurements with a strain gauge transducer (MPU-290, NEC-Sanei Sokki). The heart rate was calculated from the blood pressure pulse. Microinjection of L-glutamate: Under urethane anesthesia, the rats were mounted on a stereotaxic apparatus (David-Kopf). A stainless steel tube (30 gauge) was inserted in the left locus coeruleus at coordinates (anteroposterior -1.5mm, lateral 1.2mm, dorsoventral 2.0mm)9) and L-glutamate (2M, 1ƒÊl) was injected from a microsyringe connected to the stainless steel tube. 6-OHDA treatment: A stainless tube was inserted bilaterally into the posterior hypothalamus at coordinates (anteroposterior 4.6mm, lateral }1.0mm, dorsoventral 2.5 mm)9) under pentobarbital (30mg/kg, i.p.) anesthesia. In one group 6-

3 Vol.32 No.2 NOREPINEPHRINE RELEASE IN HYPOTHALAMUS 257 OHDA (5ƒÊg/2ƒÊ1) was injected (6-OHDA group), and in another group a 2ƒÊl saline vehicle injection was performed (vehicle group). Brain dialysis: Under urethane anesthesia, rats were placed on the stereotaxic apparatus and a brain dialysis probe (Carnegie-Medicine Co.)10) was placed in the left posterior hypothalamus using a standard stereotaxic technique at coordinates (anteroposterior 4.6mm, lateral 1.0mm, dorsoventral 2.5mm),9) and fixed with dental cement. The brain dialysis probe was connected to the microinfusion pump (IP-2, B.R.C. Co.) with a silicon tube, and perfused for 90min with artificial cerebrospinal fluid11) at the rate of 1ƒÊl/min. Catecholamine assay: The perfusate (90ƒÊl) obtained by brain dialysis was collected in the sampling tube and frozen in the refrigerator. Later, a portion of the sample (50ƒÊl) was analyzed for catecholamine concentration with Peuler and Johnson's radio-enzymatic assay.12) Drugs: L-glutamate (dissolved in sodium chloride (2mol/l) and adjusted to ph 8 with sodium hydroxide) was used for microinjections into the locus coeruleus. Five ƒêg of 6-OHDA were dissolved in 2.5ƒÊl of isotonic sodium chloride solution containing 1mg/ml of ascorbic acid. It was adjusted to ph 5.5 with sodium hydroxide. Statistical analysis: In each experiment, data from groups were analyzed using Student's t-test for comparing means of independent samples. Differences at a 5% level (p<0.05) were considered to be statistically significant. RESULTS Increased responses of blood pressure and extracellular norepinephrine of posterior hypothalamus to microinjection of L-glutamate into the locus coeruleus: L-glu elicited a prolonged elevation of blood pressure in both SHR and WKY over 20min (Fig. 1). Pressor effects were significantly larger in SHR than in WKY at 15min (+17.8 }2.0% vs }1.3%; p<0.05) (Fig. 2). Extracellular norepinephrine release in the posterior hypothalamus was also significantly increased after injection of L-glu in the locus coeruleus. Before injections, the extracellular NE was significantly larger in SHR than in WKY

4 258 KAWASAKI, ET AL. Jpn. Heart J. March 1991 Fig. 1. Time course of blood pressure responses to microinjections of L-glu into LC in WKY and SHR. Values indicate mean }SEM. Fig. 2 (left). Pressor responses to microinjections of L-glu after vehicle and 6-OHDA treatment of the posterior hypothalamus. Values indicate mean } SEM. Fig. 3 (right). Increases of norepinephrine content of posterior hypothalamus after microinjection of L-glu after vehicle and 6-OHDA injections in the hypothalamus. Values indicate mean }SEM. (275 }37 vs. 162 }19pg/ml, p<0.05). The release of NE was also elevated significantly in SHR (+77.4 }34.0% in SHR vs }15.6% in WKY, p<0.01) (Fig. 3).

5 Vol.32 No.2 NOREPINEPHRINE RELEASE IN HYPOTHALAMUS OHDA treatment and pressor response to L-glu: One week after the injections of 6-OHDA or vehicle into the posterior hypothalamus, the systolic blood pressure of the SHR 6-OHDA group was significantly reduced from the level in the SHR vehicle group (vehicle: 171 }1 vs. 6-OHDA: 148 }5mmHg, p<0.01). However, there were no significant differences between the 6-OHDA and vehicle group of WKY rats (6-OHDA: 124 }4 vs. vehicle: 114 }2mmHg, NS) (Fig. 4). Pressor effects to L-glu were significantly larger in vehicle-shr than in vehicle-wky. However, the pressor responses of the 6-OHDA group were significantly smaller than those of the vehicle group (SHR-vehicle 22.4 }3.6, SHR-6-OHDA 10.4 }2.1%, p<0.01). There were no significant differences in pressor responses of the 6-OHDA and vehicle groups of WKY rats (WKY-vehicle 16.1 }2.6, WKY- 6-OHDA 12.0 }2.4%, NS) (Fig. 5). DISCUSSION Present results show that microinjection of L-glu into the locus coeruleus elicits pressor responses in both WKY and SHR, while the pressor effects of microinjections of L-glu into the locus coeruleus were significantly larger in SHR than in WKY rats. Previous studies reported that electrical stimulation of the locus coeruleus evokes a rise in arterial blood pressure13) and that the pressor responses to electrical stimulation of the locus coeruleus are greater Fig. 4. Blood pressure one week after injection of 6-OHDA or vehicle into the posterior hypothalamus both in WKY and SHR. Values indicate mean }SEM.

6 260 KAWASAKI, ET AL. Jan. Heart J. March 1991 Fig. 5. Pressor response to microinjection of L-glu into LC in WKY and SHR. Values indicate mean }SEM. in SHR.14) In our study, L-glu may chemically stimulate the noradrenergic cell bodies without affecting fibers of passage, resulting in a pressor response similar to that of electrical stimulation. These findings suggest that the locus coeruleus plays a role in the regulation of arterial blood pressure. The locus coeruleus contains almost half of the noradrenergic neurons in the brain.6),15) It innervates many sites in the neuraxis, including the lateral tegmentoreticular tract,16) nucleus tractus solitarius,17) and the A1 and A2 areas,18) which are important in the regulation of blood pressure. The increases in the NE content of the posterior hypothalamus during elevated blood pressure were significantly higher in SHR than in WKY. This suggests that noradrenergic neurons in the locus coeruleus may project to the posterior hypothalamus and supply NE, resulting in the elevation of blood pressure. Furthermore, the results also raise the possibility that the activity of locus coeruleus neurons may be enhanced in SHR compared with WKY. In order to confirm whether NE in the posterior hypothalamus may contribute to the rise of blood pressure, we studied blood pressure after the inhibition of NE synthesis in the posterior hypothalamus by 6-OHDA. In accordance with a previous study,3) the injections of 6-OHDA into the posterior hypothalamus significantly lowered blood pressure in SHR. This indicates that a depletion of hypothalamic noradrenergic neurons results in a lower arterial blood pressure in SHR. This concept is supported by the

7 Vol.32 No.2 NOREPINEPHRINE RELEASE IN HYPOTHALAMUS 261 previous result that the rise in blood pressure during stimulation of the posterior hypothalamus is mediated by noradrenergic neurons.19) However, noradrenergic depletion induced by 6-OHDA did not affect blood pressure in normotensive rats. It is possible that the 6-OHDA injection did not deplete all the norepinephrine in the posterior hypothalamus, and that a small, residual portion of norepinephrine may have some effect on the blood pressure of normotensive rats. However, it may also suggest that posterior hypothalamic NE is not engaged in the maintenance of baseline blood pressure in normotensive rats. Przuntek and Philippu found that lesions of the posterior hypothalamus diminished the pressor response to electrical stimulation of the locus coeruleus in normotensive cats.20) This result indicates that projections from the locus coeruleus may elevate blood pressure through a relay in the posterior hypothalamus. This effect is similar to the finding that 6-OHDA injections into the posterior hypothalamus attenuate the pressor responses to LC microinjection of L-glu in SHR, but not in WKY. These findings imply that the noradrenergic activity of the locus coeruleus is facilitated in SHR compared to WKY. REFERENCES 1. Takeda K, Bunag RD: Sympathetic hyperactivity during hypothalamic stimulation in spontaneously hypertensive rats. J Clin Invest 62: 642, Winternitz SR, Wyss JM, Oparil S: The role of posterior hypothalamic area in the pathogenesis of hypertension in the spontaneously hypertensive rat. Brain Res 324: 51, Charmers JP: Brain amines and models of experimental hypertension. Cite Res 36: 469, Patel KP, Ciriello J, Kline RL: Noradrenergic mechanisms in brain and peripheral organs after aortic nerve transection. Am J Physiol 244: H481, Kawasaki S, Takeda K, Tanabe S, Nakata T, Hayashi J, Oguro M, Suzuka T, Nakamura Y, Lee LC, Sasaki S, Nakagawa M: Increased extracellular concentration of norepinephrine in the hypothalamus by sinoaortic denervation. Am J Hypertens 1: 291, Swanson LW, Hartmann BK: The central adrenergic system: an immunofluorescence study of the location of cell bodies and their efferent connection in the rat utilizing dopamineĈ-hydroxylase as a marker. J Comp Neurol 163: 467, Palkovits M, Jacobowitz D: Topographic atlas of catecholamine and acetylcholine esterase containing neurons in rat brain. I. Forebrain (Telencephalon, Diencephalon). J Comp Neurol 157: 13, Bunag RD, Inoue A: Differentiated pressor and sympathetic responses to dual brain stimulation: Ventromedial hypothalamus versus locus coeruleus. Proc Suc Exp Biol Med 178: 91, Pellegrino LJ, Pellegrino AA: A Stereotaxic Atlas of the Rat Brain, Appleton-Century-Crofts, New York, Tossman U, Ungerstedt U: Neuroleptic action on putative amino acid neurotransmitters in the brain studied with a new technique of brain dialysis. Neurosci Lett Suppl 7, S479, 1981

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