遺伝子欠損マウスを用いた中枢レニン・アンジオテンシン系の機能解析

レニン-アンジオテンシン系(renin-angiotensin system; RAS)は、生体内の水・電解質平衡の調節、循環血液量および血圧の調節に重要な役割を担っており、高血圧の発症・維持に関与している。RASの唯一の基質であるアンジオテンシノーゲン(angiotensinogen; ATN)はレニンによりアンジオテンシンI(angiotensin I; AI)に変換されるが、AIは生理的に不活性である。AIはアンジオテンシン変換酵素(angiotensin converting enzyme; ACE)によりアンジオテンシンII(angiotensin II; AII)に変換され、細胞膜表面に存在する特異的なAII受容体に結合することにより生理作用を発現する。AII受容体にはタイプ1受容体(AT_1)およびタイプ2受容体(AT_2)の2種類のサブタイプが存在する。AT_1受容体はアンジオテンシンの主生理機能に関与していることが知られている。一方、AT_2受容体はAIIと結合するがその生理機能は不明な点が多い。　遺伝子工学の発達に伴いRASの各遺伝子の構造が明らかにされ、研究は急速に発展した。また、RASは循環血中のみで作用すると考えられてきたが、各構成因子が脳、心臓などの組織にも観察され血中RASとは独立した組織RASの概念が確立され、その生理機能の解明が注目されている。　現在、脳内でのRASの機能は、血圧制御の他、飲水行動の制御、疹痛閾値など知覚神経系の制御、カテコールアミン作動性神経系を介した運動機能の制御、学習記憶機能への関与、うつ不安症状の制御、脳血流量の制御、脳梗塞による遅発性神経細胞死への関与等が報告されている。これら報告の多くはアンジオテンシン自体を脳内へ直接注入した実験、ACE阻害剤(ACE inhibitor; ACEI)あるいはAII受容体サブタイプ選択的リガンドを用いた薬理試験で得られた知見であり、内因性の脳内RASを直接修飾した結果ではない。近年、発生工学的手法の導入により遺伝子欠損動物が作製され、RAS各構成因子の機能が個体レベルで解析できるようになった。ATN遺伝子欠損マウスは収縮期および拡張期の血液が低下しており、AT_2受容体遺伝子欠損マウスは収縮期および拡張期の血圧が上昇していることが報告されているが、中枢神経系の変化については検討されていない。　そこで、筆者は脳内RASの機能を解明する目的で、ATN遺伝子欠損マウス(筑波大学応用生物系村上和雄教授より提供)およびAT_2受容体遺伝子欠損マウス(バンダービルト大学医学部生化学稲上　正教授より提供)を用いて、行動薬理学的および神経化学的手法による機能解析を行った。なお、各実験における対照動物としては、ATN遺伝子欠損マウス(-/-)およびAT_2受容体遺伝子欠損マウス(-/y、-/-)ともそれぞれの野生型マウス(+/+ : +/y、+/+)を用いた。　中枢機能解析には以下の行動薬理学的解析手法を用いた。1. 自発運動量の測定(探索行動量および自発運動量に対する影響:ATN、AT_2)2. 強制水泳法(うつ状態の検討:ATN、AT_2)3. 明暗選択性試験(不安行動の検討:ATN、AT_2)4. 受動的回避反応(学習・記憶機能の検討:ATN、AT_2)5. 疼痛閾値に対する作用(知覚神経系の検討:ATN、AT_2)6. 脳浮腫形成に対する作用(遅発性神経細胞死への関与の検討:ATN、AT_2)7. オープンフィールド法(脱糞行動による不安状態の検討:AT_2)8. 高架式十字迷路法(不安状態の検討:AT_2)9. 脳内βエンドルフィン含量の測定(疼痛過敏の精査:AT_2)10. Hexobarbital誘発睡眠に対する作用(中枢神経の非特異的制御あるいは興奮性の検討:AT_2)　神経化学的機能解析としては、不安症状の発現に関与しているノルエピネフリン(Norepinephirine;NE)およびコルチコトロピン遊離因子(corticotropin releasing factor:CRF)、および疼痛閾値制御に関係しているβ-エンドルフィン作動性神経系の関与を検討した(AT_2)。　結果、ATN遺伝子欠損マウスは強制水泳法を用いた場合、うつ症状様行動の減弱が観察された。ATN遺伝子欠損マウスは自発運動量が低下していることより、ATN遺伝子欠損マウスのうつ症状様行動の減弱は運動量の増加に基づいた変化ではないと考えられる。また、ATN遺伝子欠損マウスは不安行動の増強あるいは減弱が認めれなかったことより、ATN遺伝子欠損マウスは特異的にうつ症状様行動の減弱が発現していると考えられる。　雄性AT_2受容体遺伝子欠損マウスは自発運動量には影響を受けず、探索行動量のみが減少していた。しかし、雄性AT_2受容体遺伝子欠損マウスの探索行動量および自発運動量は野生型マウスと比較して差がなかった。運動量に関しては性差が認められた。　AT_2受容体遺伝子欠損マウスは明暗選択性試験での明室滞在時間の短縮、高架式十字迷路でのオープンアーム滞在時間の短縮、オープンフィールド法での脱糞数の増加が認められ、野生型マウスと比較して、不安状態が強く発現していた。AT_2受容体遺伝子欠損マウスの強制水泳法の無動時間、hexobarbital誘発睡眠時間および自発運動量は野生型マウスと差がなかった。すなわち、AT_2受容体遺伝子欠損マウスは特異的に不安状態の増加を示し、この作用は非特異的な中枢抑制あるいは中枢興奮に起因するものではない。　ATN遺伝子欠損マウスおよびAT_2受容体遺伝子欠損マウスを用いた、受動的回避反応および疼痛閾値測定実験より、脳内のATNおよびAT_2は学習・記憶機能に関与している可能性は低いことが示唆された。　ATN遺伝子欠損マウスおよびAT_2受容体遺伝子欠損マウスの脳浮腫形成作用に対する検討結果より、ATNおよびAT_2受容体はグルタミン酸神経毒性発現には関係していないと考えられる。　AT_2受容体遺伝子欠損マウスは疼痛過敏になっていた。免疫組織学的に脳内β-エンドルフィン含有を測定した結果、β-エンドルフィン含有細胞が存在する視床下部弓状核において、AT_2受容体遺伝子欠損マウスは野生マウス型と比較してβ-エンドルフィン含量が減少していた。従って、AT_2受容体遺伝子欠損マウスの疼痛過敏には視床下部弓状核内でのβ-エンドルフィン含量の減少が関与していると考えられる。　行動薬理的検討で認められたAT_2受容体遺伝子欠損マウスの不安状態増加はα_1アドレナリン受容体アンタゴニストであるprazosinにより抑制され、α_1アドレナリン受容体作用薬(phenylephrine)α_2アドレナリン受容体拮抗薬(yohimbine)、α2アドレナリン受容体作用薬(clonidine)、β_1/β_2アドレナリン受容体拮抗薬(propranolol)、β_1/β_2アドレナリン受容体作用薬(isoprotenol)では影響がなかった。従って、AT_2受容体遺伝子欠損マウスは脳内でα_1アドレナリン受容体の感受性が高まっていることが不安状態を増強していると考えられる。AT_2受容体遺伝子欠損マウスにα_1アドレナリン受容体作用薬のphenylephrineを投与した場合、明室滞在時間はさらには短縮されることはなかった(不安状態の増強は認められなかった)。この点に関しては、AT_2受容体遺伝子欠損マウスはすでに強い不安状態を呈しており、さらなる増強作用は検出できなかったと考えられる。　AT_2受容体遺伝子欠損マウスの扁桃体での[^H]-prazosin結合は野生型マウスと比較して有意(PI］-CRF結合数にも変化はなかった。さらに、視床下部-下垂体-副腎系の活性化の指標である血中副腎皮質刺激ホルモンおよびコルチコステロン濃度は野生型マウスと差がなかった。従って、AT_2受容体遺伝子欠損マウスの不安状態状の増加にはCRF受容体は関与していないものと考えられる。　神経細胞においてAIIによる神経活動の修飾作用はAT_1およびAT_2受容体の相互作用が重要な働きをしていると推定される。AT_2受容体遺伝子欠損マウスはAT_2受容体が欠損した結果、AT_1受容体が活性化され不安を惹起したと考えられる。　以上、ATN遺伝子欠損マウスおよびAT_2受容体遺伝子欠損マウスを用いて、行動薬理学的および神経化学的手法を用いて機能解析を行った結果、脳内RASの機能として、現在解明さている中枢循環調節の他に、脳内ATNはうつ状態の制御に関与しており、脳内AT_2受容体はα_1アドレナリン受容体を介して不安状態を制御していることを明確にした。臨床上、ACEI(アンジオテンシン変換酵素阻害剤)は高血圧の治療に汎用されているが、同時に抗うつおよび抗不安作用を呈することが知られている。ACEIの抗うつおよび抗不安作用には脳内RASの関与が示唆されていたがその詳細は不明であった。本研究結果より、うつ不安症状と脳内RASの関係が明確になったと共に、ATN遺伝子欠損マウスおよびAT2受容体遺伝子欠損マウスは精神疾患病態モデルになりうる可能性が示唆された。Angiotensinogen (ATN) is the glycoprotein precursor of angiotensin II. Physiologically inactive decapetide angiotensin I (AI) is released from ATN by renin, and further hydrolyzed by angiotensin-converting enzyme (ACE) to produce the ocapeptide angiotensin II (AII). While the major source of plasma ATN is the liver, other tissues, including the brain, also produce ATN as part of the local renin-angiotensin systems (RAS). Although brain RAS appears to be independently regulated from peripheral RAS, circulating angiotensins do influence the central system via interaction with angiotensin receptors located in circumventricular organs. The presence of ATN in the adult brain has been determined immunohistochemically. The RAS has an important role in maintaining of blood pressure and electrolyte homeostasis. Although ATN has no known biological function, recent studies provide an interesting link of central ATN to the development of hypertension, the regulation of steroid hormone and cell growth. Specific functions of brain ATN are not well understood. Mutant mice lacking the gene encoding the ATN show decreases in blood pressure. However, specific central roles of ATN have yet to be established. There are two known major AII receptor subtypes, type 1 (AT_1) and type 2 (AT_2). The distribution of these receptor subtypes in the rat brain has been defined in binding studies using radioligands. Numerous studies were done on the effect of both AT_1 and AT_2 receptor antagonists on various responses to centrally administered AII. With regard to water drinking, salt appetite, vasopressin secretion and pressor responses to intracerebroventricular (i.c.v.) administration of AII, central administration of the AT_1 but not AT_2 ATN agonists, AT_1 receptor antibody or antisense oligonuceotide blocks these responses. AT_1 receptors (sensitive to losartan) may mediate anxiety, while either AT_1 receptors (sensitive to losartan) or AT_2 receptors (sensitive to PD123177) were reported to enhance cognition without influencing anxiety in laboratory animals. In contrast, both losartan and PD123177 were without significant effect in animal models of anxiety and memory. Thus, studies on the central function of AT_2 receptors yielded diverse and often conflicting results. Mutant mice lacking the gene encoding the AT_2 receptor resulted in increase in blood pressure, increased sensitivity to the presser action of ANG II, and attenuated exploratory behavior. To explore the role of ATN and AT_2 subtype in CNS, we searched for alterations in the behavioral responses of ATN-deficient mice and AT_2-deficient mice, respectively. No significant differences were found between male and female ATN-deficient mice in results of behavioral analysis. Our observations revealed that the absence of ATN induces the reduction of depressive-like behavior in the behavioral despair swim test. Immobility is reduced by clinically effective antidepressants and not by neuroleptics and anxiolytic drugs. The method has proven to be useful in identifying and evaluating potential antidepressive compounds. However, stimulants such as amphetamine and methamphetamine, which decrease immobility were reported to be false-positive compounds in the swim test. In the present studies, ATN-deficient mice had reduced spontaneous locomotor activity, thereby indicating that the reduction of depressive-like behavior in ATN-deficient mice did not result from alternations in motor activity. In addition, the time spent in the light area in the light/dark exploration task did not differ significantly between ATN-deficient and wild-type mice, thereby suggesting that ATN-deficient mice specifically exhibit the reduction of depressive-like behavior. Captopril, an angiotensin II converting enzyme inhibitor (ACEI), currently used as a clinical an antihypertensive drug, significantly reduced immobility in the force-swim-induced behavioral despair test in mice. In rats, ACEI, captopril and perinodopril, induced a reversal of escape deficits but did not affect motor activity in the learned helplessness paradigm. Clinically, captopril and other ACEIs have been reported to exhibit mood-elevating and antidepressant properties. Patients on captopril also had significantly better scores in tests of general well-being than seen in cases where reserpine, propranol, or alphamethyldopa had been prescribed. ATN-deficient mice were hypotensive, and exhibited the reduction of depressive-like behavior, as compared with findings in wild-type mice. Thus, the behavior in ATN-deficient mice was similar to that seen when ACEI was administered. These findings strongly suggest that a critical role for central endogenous ATN may regulate the depressant state in the brain Angiotensin may also be involved in learning and memory. Captopril, an AT_1 receptor antagonist losartan and an AT_2 receptor antagonist PD123177 show a cognitive enhancing action in rodents. However, the present studies assessing the role of ATN in modulating cognitive behavior by the passive avoidance task in STL of ATN-deficient mice showed no differences from findings in wild-type mice. In addition, no significant differences were found between ATN-deficient and wild-type mice concerning the pain threshold. Thus, the ATN may not be directly involved in mediating cognitive function. The transient upregulation of AT_2 receptors mRNA occurs in the cortex and hippocampus after ischemic injury and these changes may be related to molecular events, which lead to delayed neuronal cell death. ANG II inhibits N-methyl-D-aspartate (NMDA)-mediated nitric oxide (NO) and cGMP production through a mechanism involving an AT_2 receptor subtype. To study the role of ATN in post-traumatic neuronal injury, we examined the effects of cold injury on development of brain edema no significant differences were found between ATN-deficient and wild-type cerebra in the extent of edema after this injury. Cold-induced brain injury has been well established as a model of vasogenic brain edema. As the accumulation of glutamate was observed in interstitial fluids after vasogenic edema, it has been suggested that glutamate neurotoxicity may be involved in the tissue damage caused by trauma. Thus, ATN may not be involved in the glutamate neurotoxicity seen after traumatic cerebral injury. One of the currently accepted anxiety test is based on the animal\u27s exploratory behavior, either implicitly or explicitly, used as an index of anxiety. It was reported that male AT_2-deficient mice showed reduced exploratory behavior in the open field test. In the present study, we confirmed and extended it by demonstrating that male AT_2-deficient mice exhibited a selective reduction of exploratory but not locomotor activities. It can be difficult to assess an animal\u27s exploratory behavior, independently of other behavior such as the locomotion. In case of female AT_2-deficient mice, however, the locomotor activity did not differ significantly between AT_2-deficient mice and wild-type mice. The number of fecal boli in rodents is considered to be an index of their "emotionality" and reduction in the number of fecal boli rate diazepam administration, a drug widely prescribed for treatment of anxiety and related disorders were seen to be consistent with its anxiolytic action. Thus, an exaggerated response to a novel environment is consistent with an increase in anxiety-like state in AT_2-deficient mice. The elevated plus-maze task and the light/dark exploration task were used as a validated animal models of anxiety that are predictive of drug responses in humans. These tests, which are based on natural aversion of rodents to open or light spaces, are sensitive to the effects of both anxiolytic and anxiogenic agents in rodents. As in the novel environment, clear group differences were observed between AT_2-deficent mice and wild-type mice, using these test paradigms. The time spent in the light area and that in open arms area was significantly reduced in AT_2-deficient mice compared with findings in wild-type mice in the light/dark exploration task and the elevated plus-maze task, respectively. The reduction in time spent in the light area in the light/dark exploration task was reversed by anxiolytic diazepam. In addition, the hexobarbital-induced sleeping time and duration of immobility in the behavioral despair test did not differ significantly between AT_2-deficient and wild-type mice, suggesting that AT_2-deficient mice did not exhibit non-specific CNS depression and depressant-like behavior. These findings strongly suggest that AT_2-deficient mice specifically showed an anxiety-like state. Biochemical and electrophysioligical evidence suggests that AT_1 and AT_2 receptors have antagonistic effects. Activation of AT_1 and AT_2 receptors elicited stimulatory and inhibitory effects, respectively, on mitogen-activated protein (MAP) kinase in neurons. Within the brain, blockade of periventricular AT_2 receptors potentiated AT_1 receptor-mediated stimulation of drinking and vasopressin secretion. In catecholaminergic neurons, ANG II elicited an AT_1 receptor-mediated decrease in the delayed rectifier K^+ current (I_K) followed by an AT_2 receptor-mediated increase in I_K. These studies collectively suggest that functional interactions between the two receptor subtypes have a key role in ANG II-induced neuromodulatory actions in neurons. An AT_1 receptor antagonist losartan and an ACE inhibitor captopril, used to abolish endogenous ANG II, produced anxiolytic activity, which suggests that the anxiolytic action may reflect inhibition of AT_1 receptors. Thus, one possible explanation for the anxiety-like behavior we observed in AT_2-deficient mice may be activation of AT_1 receptors because of the absence of AT_2 receptors in the CNS. In ANG II-induced blood pressure responses in AT_2-deficient mice, AT_2 antagonized the AT_1-mediated pressor action of ANG II. The considerable body of evidence supports the view that anxiety disorders may be related to excessive norepinephrine (NE) activity. Moreover, functional interaction between AII and neuronal noradrenergic system may occur, The anxiety-like behavior seen in AT_2-deficient mice was suppressed only by the α_1 adrenoceptor antagonist prazosin and showed downregulation of the α_1 adrenoceptot in the amygdala, which may be induced by excessive NE release as a result of the absence of AT_2 receptors. This will result in the activation of the cerebral α_1 adrenoceptor. There is extensive evidence from animal work, that direct activation of the locus coeruleus which may reflect dysfunction of inhibitory α_2 adrenoceptor can produce signs of arousal that strongly resemble those seen in human anxiety states. However, the absence of AT_2 receptors in the locus coeruleus was observed in histochemical studies of both AT_2-dedicient and wild-type mice. The present results indicate that both the α_2 adrenoceptor antagonist yohimbine and the α_2 adrenoceptor agonist clonidine did not attenuate the anxiety-like behavior in AT_2-deficient mice. The amygdala appears to play a crucial role in conditioned fear and probably anxiety. Both AII (AT_1 and/or AT_2) and NE receptors are located in the amygdala. Thus, the anxiety-like behavior observed in AT_2-deficient mice may be mediated by interaction between the α_1 adrenoceptor and AII (AT_1 and/or AT_2) receptors in the amygdala. In fact, the stress-induced anxiety-like behavior may reflect excessive NE activity via central the α_1 adrenoceptor. CRF, a 41 amino acid residue peptide, that regulates the release of ACTH from the anterior pituitary, has been shown to mediate stress-induced changes in the autonomic nervous system, neuroendocrine functions, and behavior. The i.c.v. administration of CRF to laboratory animals produces anxiety-like behavior, such as altered locomotor activity and an increased anxiety in an elevated plus-maze. The CRF antagonist α-helical CRF_9-41 has no effect on the reduced time spent in the light area in AT_2-deficient mice. The number of [^I]-CRF binding sites in the amygdala was not significantly attenuated in AT_2-deficient mice, compared with wild-type mice. In addition, the plasma ACTH and corticosterone concentrations did not differ significantly between AT_2-deficient and wild-type mice indicating the endocrine abnormalities involving HPA was not observed. The anxiety-like behavior seen in AT_2-deficient mice may be related to abnormalities in the central noradrenergic neuronal system but not to the CRF neuronal system. Angiotensin may also be involved in learning and memory. However, the present studies assessing the role of the proposed AT_2 receptors in modulating cognitive behavior by the passive avoidance task, the step through latency of AT_2-deficient mice did not differ from that of wild-type mice. Thus, the AT_2 receptors may not be directly involved in mediating cognitive function. The pain threshold was significantly lower in AT_2-deficient mice, compared with findings in wild-type mice. The immunohistochemical distribution of β-endorphin in the brain was analyzed quantitatively in AT_2-deficient mice and wild-type mice, using microphotometry. The fluorescence intensity of β-endorphin in the arcuate nucleus of the medial basal hypothalamus (ARC) was significantly lower in AT_2-deficient mice, compared with findings in wild-type mice. We found that the AT_2 receptor does not influence learning behavior and brain edema formation. As AT_2-deficient mice have increased sensitivity to pain and decreased levels of brain β-endorphin, AT_2 receptors may perhaps mediate regulation of the pain threshold. To study the role of AT_2 receptors in post-traumatic neuronal injury, we examined the effects of cold injury on development of brain edema, no significant differences were found between AT_2-deficient and wild-type cerebra in the extent of edema after this injury. AT_2 receptors may be not related to the glutamate neurotoxicity seen after traumatic cerebral injury. For patients whose blood pressure is controlled by ACE inhibitors, these inhibitors show anxiolytic effects when the blood pressure is lowered. (mRen-2)27 transgenic rats, a hypertensive model dependent on increased expression of the renin angiotensin system, showed a greater anxiogenic profile than did control rats. Administration of captopril to (mRen-2)27 transgenic rats reversed the anxiety-like behavior. Blood pressure of AT_2-deficient mice is higher than that of wild-type mice. Captopril reversed the anxiety-like behavior in AT_2-deficient mice. Thus, the present studies uncovered a fact of the anxiety-like state seen in AT_2-deficient mice. AT_2-deficient mice can thus serve as a model reflecting the human clinical situation and may be appropriate for studies on effects of antihypertensive drugs in patients with elevated blood pressure.博士(獣医学)麻布大