Aging-related changes in the nigral angiotensin system enhances proinflammatory and pro-oxidative markers and 6-OHDA-induced dopaminergic degeneration

An age-related proinflammatory, pro-oxidant state in the nigra may increase the vulnerability of dopaminergic neurons to additional damage. Angiotensin II, via type 1 (AT1) receptors, is one of the most important known inflammation and oxidative stress inducers. However, it is not known if there are age-related changes in the nigral angiotensin system. In aged rats, we observed increased activation of the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) complex and increased levels of the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α, which indicate pro-oxidative, proinflammatory state in the nigra. We also observed enhanced 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell death in aged rats. This is associated with increased expression of AT1 receptors and decreased expression of AT2 receptors in aged rats, and is reduced by treatment with the AT1 antagonist candesartan. The present results indicate that brain angiotensin is involved in changes that may increase the risk of Parkinson's disease with aging. Furthermore, the results suggest that manipulation of the brain angiotensin system may constitute an effective neuroprotective strategy against aging-related risk of dopaminergic degeneration.     

Characterization of angiotensin receptors on human skin fibroblasts

 Angiotensin II is involved in blood pressure regulation, cell growth and angioneogenesis. The angiotensin receptors which mediate the intracellular effects of angiotensin II are expressed in numerous tissues and cell types. We studied the expression of angiotensin II receptors in cultured human skin fibroblasts derived from a skin biopsy. Angiotensin II binding characteristics were analyzed by radioligand binding assays. The DNA synthesis was assessed by [H]thymidine incorporation assays. Intracellular calcium concentrations were measured by fura-2 spectrofluorometry, and mRNA expression levels were analyzed by northern blot technology. Two distinct angiotensin receptors were detectable on human skin fibroblasts: the AT1 receptor with K _d=1.0± 0.7 nmol/l and B _max=17.9±0.9 fmol/mg protein, and an angiotensin_(1–7) binding site with K _d=26±6.6 nmol/l and B _max=80.4±3.5 fmol/mg protein, as shown by competition binding assays using selective angiotensin II receptor antagonists and the heptapeptide angiotensin_(1–7). The angiotensin AT1 receptor mRNA was substantially expressed in human skin fibroblasts and was subjected to homologous downregulation. In human skin fibroblasts angiotensin II caused a profound increase in intracellular calcium which was blocked by angiotensin AT1 receptor antagonists such as Exp-3174. Furthermore, both angiotensin II and angiotensin_(1–7) led to increased DNA synthesis in human skin fibroblasts. In conclusion, cultured human skin fibroblasts express angiotensin AT1 receptors and a putatively new angiotensin receptor activated by angiotensin_(1–7), both coupled to signaling pathways involved in DNA synthesis. Received: 5 July 1996 / Accepted: 29 November 1996     

Angiotensin II is a growth factor in the peri-implantation rat embryo

Angiotensin II (ANG II) is increasingly recognised as a growth factor, both in its own right and through interactions with other growth factors. There is a high density of ANG II receptors in the rat fetus, especially the AT2 receptor, the function of which is still uncertain. We have now studied the effects of ANG II on growth and development in the rat embryo in vitro between d 9.5 and 11.5, and characterised the receptor subtype mediating these effects. Embryos were cultured in whole rat serum, a high molecular weight retenate after ultrafiltration of whole rat serum, retenate with angiotensin II and retenate with ANG II and AT1 or AT2 receptor blockers. Growth and development were scored using conventional methods. Culture in retenate was associated with a marked reduction in growth and development by comparison with whole rat serum. This was partly, and significantly ( P <0.001), reversed by angiotensin II. The optimum concentration of angiotensin II was found to be angiotensin II 10⁻¹¹ M , within the physiological range. Angiotensin II had highly significant effects on both somatic ( P <0.001) and yolk sac/allantoic ( P <0.005) development. The latter effects suggest a role for angiotensin II in placentation. The effects of angiotensin II were blocked by PD123319, an AT2 blocker, but not by GR117289, an AT1 blocker. Interestingly, culture in retenate with GR117289 without added angiotensin II was also associated with some increase in growth ( P <0.05). Angiotensin II in low concentrations was measurable in the retenate, presumably arising from the action of endogenous renin on angiotensinogen. We therefore postulate that this effect of GR117289 was due to the action of endogenous angiotensin II on 'uncovered' AT2 receptors. This study has thus demonstrated a direct growth promoting effect of angiotensin II during organogenesis in the whole rat embryo in vitro. This effect is mediated through the AT2 receptors.     

Relationship between angiotensin II receptor expression and cardiovascular risk factors in Mexican patients with coronary occlusive disease

The density of Angiotensin II (Ang) receptors on tissue surfaces is regulated by multiple hormones, cytokines and metabolic factors and is profoundly affected by various pathological conditions, such as age, diet and environmental conditions. The participation of several cardiovascular risk factors in the regulation of Angiotensin II receptor expression has been incompletely studied. We performed an ex-vivo study with human aortic postsurgical specimens to test the hypothesis that Ang AT1 and AT2 receptor expression in human aortic arteries is associated with the presence of cardiovascular risk factors. We included 31 Mexican patients with coronary artery disease. We evaluated Angiotensin II receptor expression by immunostaining and angiotensin converting enzyme insertion/deletion (ACE I/D) polymorphisms by polymerase chain reaction. AT1 and AT2 receptor expression was increased in the aortic segments from the cardiovascular patients compared with control arteries and in patients with the DD genotype. There was a correlation between increased AT1 receptor expression and the number of cardiovascular risk factors present in the patient. Furthermore, reduction of AT1 expression correlated with the number of drug combinations used in the patients. These correlations were not present with respect to AT2 receptor expression. We suggest that increased AT1 receptor expression is associated with the DD genotype. Thus the presence of several cardiovascular risk factors as well as DD genotype, induce AT1 expression increasing the probability to develop coronary occlusive disease.     

Distribution of angiotensin II receptor subtypes in rat brain nuclei.

Angiotensin II (ANG II) receptor subtypes in rat brain were characterized and quantified by competitive radioligand binding using [125I]Sar1 Ile8 angiotensin II ([125I]sarilesin) as a tracer and ANG II, sarilesin and the subtype selective ligands DuP 753 (AT1) and CGP 42112A (AT2) as competitors. The distribution of AT1 and AT2 receptors was determined in midbrain, brainstem, hypothalamus as well as in individual hypothalamic and periventricular nuclei. Whereas in midbrain and brainstem the AT1: AT2 ratio was 40%: 60% and 70%: 30% respectively, the AT1 receptors were by far predominant in hypothalamus and in the nuclei investigated. Interestingly, we found that approximately 25% of the ANG II receptors in hypothalamus did not bind DuP 753 even at 0.1 mM. These sites which bind CGP 42112A, ANG II and sarilesin may represent a third ANG II receptor subtype.     

Effect of intranasal administration of CV-11974, a type 1 angiotensin II receptor antagonist,on airway hyperresponsiveness and airway inflammation induced by antigen inhalation in guinea pigs

BACKGROUND: Angiotensin II is a putative mediator in asthma, but the effect of topical administration of type 1 angiotensin II (AT1) receptor antagonists on allergic airway reactions is not known. OBJECTIVE: To investigate the effect of intranasal administration of CV-11974, an AT1 receptor antagonist, and of PD123319, a type 2 angiotensin II (AT2) receptor antagonist, on antigen-induced airway reactions in guinea pigs. METHODS: Thirty minutes after intranasal topical administration of CV-11974 (0.1 or 1.0 mg/ml) or PD123319 (10 mg/ml) into the airways, the animals were given an antigen challenge. Airway hyperresponsiveness and bronchoalveolar lavage fluid were analyzed 24 h after the antigen challenge. RESULTS: Although these compounds did not inhibit antigen-induced early-phase bronchoconstriction or late-phase airway eosinophilia, intranasal administration of CV-11974 (but not PD123319) inhibited antigen-induced airway hyperresponsiveness in a dose-dependent manner 24 h after the antigen challenge. CONCLUSION: Intranasal administration of an AT1 receptor antagonist reduces antigen-induced airway hyperresponsiveness.     

Overexpression of the human angiotensin II type 1 receptor in the rat heart augments load induced cardiac hypertrophy

Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT1 receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT1 receptor under the control of the alpha-myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT1 receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-gamma-S and increased sensitivity of intracellular Ca2+ [Ca2+]i to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT1 receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.     

Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor (AT) subtypes, in particular AT1 (AT1a and AT1b in the case of rodents). Although we and others have generated mutant mice in which the AT1a gene was disrupted, the function of mouse AT1 remains to be fully elucidated, due to the lack of effective tools involving antibodies against AT1 for detecting biological responses in cellular conditions. To avoid these problems, we constructed the hemagglutinin (HA)-tagged mouse AT1a, and stably introduced this recombinant receptor into human embryonic kidney 293-T cells. Radioligand binding of [(125)I] angiotensin II to AT1a was specific, saturable, and reversible. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.7 nM with a density of 1.2 x 10(5) sites/cells. Angiotensin II stimulated a rapid increase in cytosolic free calcium, and angiotensin II-induced phosphorylation of extracellular signal-regulated kinases (Erk) was found in a dose-dependent manner. After solubilization, Western blot analysis showed specific interactions between an anti-HA antibody and HA-tagged mouse AT1a. Furthermore, a significant proportion of HA-tagged mouse AT1a was specifically immunoprecipitated with this antibody. In the immunocytochemical and electronmicroscopic studies, treatment of this cell line with angiotensin II resulted in decrease in signals of the surface receptors. Based on these results, the cell line established here provides an excellent tool for studying angiotensin II actions mediated through mouse AT1a, at sub-nanomolar concentrations.     

Aging-related dysregulation of dopamine and angiotensin receptor interaction

It is not known whether the aging-related decrease in dopaminergic function leads to the aging-related higher vulnerability of dopaminergic neurons and risk for Parkinson's disease. The renin-angiotensin system (RAS) plays a major role in the inflammatory response, neuronal oxidative stress, and dopaminergic vulnerability via type 1 (AT1) receptors. In the present study, we observed a counterregulatory interaction between dopamine and angiotensin receptors. We observed overexpression of AT1 receptors in the striatum and substantia nigra of young adult dopamine D1 and D2 receptor-deficient mice and young dopamine-depleted rats, together with compensatory overexpression of AT2 receptors or compensatory downregulation of angiotensinogen and/or angiotensin. In aged rats, we observed downregulation of dopamine and dopamine receptors and overexpression of AT1 receptors in aged rats, without compensatory changes observed in young animals. L-Dopa therapy inhibited RAS overactivity in young dopamine-depleted rats, but was ineffective in aged rats. The results suggest that dopamine may play an important role in modulating oxidative stress and inflammation in the substantia nigra and striatum via the RAS, which is impaired by aging.     

A unilateral 6-hydroxydopamine lesion decreases the expression of metabotropic glutamate receptors in rat substantia nigra

Loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) in Parkinson's disease (PD) leads to augmentation of glutamatergic activity in the subthalamic nucleus (STN). Moreover, antagonizing excitotoxicity has yielded mostly symptomatic improvements in experimental animals of PD. Therefore, we used immunocytochemistry to examine the effect of unilateral 6-hydroxydopamine lesions of SNc on the expression of metabotropic glutamate receptors (mGluR1a, 2/3, 5, 4, 8) and tyrosine hydroxylase (TH). 6-Hydroxydopamine causes a fall in the number of mGluRs and TH in the lesioned lateral substantia nigra. Pharmacological activation of group II or III mGluRs or blockade of group I mGluRs for 1 week significantly increased the expression of the same group receptors. The alteration in the receptor expression may be a compensatory mechanism developed after mGluRs ligands neuroprotective treatment.     

Differences between squamous cell carcinoma and keratoacanthoma in angiotensin type-1 receptor expression

Angiotensin II receptors are the specific receptors of angiotensin II of the renin-angiotensin system. The existence and role of the receptors in the skin have not been determined. We immunohistochemically studied the expression of angiotensin receptors in the human skin. The results demonstrated the expression of angiotensin type 1 receptor (AT1) in the normal human suprabasal epidermis. The expression pattern suggests the role of AT1 in association with differentiation. In addition, we studied the expression of AT1 in squamous cell carcinoma (SCC) of the skin, SCC of the lip, and keratoacanthoma (KA). Our experiments showed that high, intermediate, and low levels of AT1 were observed in 37 (74.0%), 7 (14.0%), and 2 (4.0%) of 50 cases of SCC of the skin, respectively, and the negative periphery pattern was observed in 17 (77.3%) of 22 cases of KA. These observations suggest that the immunohistochemical study of AT1 is useful to distinguish SCC from KA. Studying the role and distribution of AT1 may help in understanding the pathophysiology of the skin.     

Losartan pretreatment reduces neurodegeneration and behavioural symptoms in 6-hydroxydopamine induced unilateral rat model of Parkinson's disease

Losartan is an angiotensin II receptor antagonist which is mainly used to treat hypertension. It has been shown that angiotensin II involves in NADPH-dependent oxidase activation. In this study, the effect of losartan in 6-hydroxydopamine (6-OHDA)-induced rat model of Parkinson's disease was investigated. The rats were daily pre-treated i.p. with losartan (90 mg/kg), for the duration of six days before the 6-OHDA injection in the left substantia nigra pars compacta (SNC), until one day afterwards. Losartan administration caused a significant decrease in the rotational and rigidity score in the lesioned rats after 2 weeks. Furthermore, the pretreatment with losartan significantly lowered the value of the markers of oxidative stress after 24 h. Moreover, losartan protected SNC dopaminergic neurons against toxicity of 6-OHDA. The results therefore suggested that losartan pretreatment attenuated the symptoms of Parkinson's disease probably by preventing 6-OHDA induced oxidative stress and neurodegeneration.     

Angiotensin AT1 receptor antagonist losartan and the defence reaction in the anaesthetised rat. Effect on the carotid chemoreflex

Modulation at the level of the nucleus tractus solitarii (NTS) appears to be an effective way of controlling cardiovascular reflexes. Angiotensin II acting on angiotensin AT1 receptors at the central nervous system appears to have an important role in these modulatory processes. The hypothalamic defence area (HDA) is a potential source of descending fibres containing angiotensin II that innervate the NTS. We investigated the effect of AT1 receptor blockade in the NTS on the response to stimulation of HDA in anaesthetised rats treated with the neuromuscular blocking agent pancuronium bromide. The characteristic increase in heart rate, blood pressure and phrenic nerve activity evoked by electrical stimulation of HDA is decreased by the microinjection of the AT1 receptor antagonist losartan into the NTS and the cardiovascular response to carotid body chemical stimulation is also reduced. These results support the hypothesis that AT1 receptors in the NTS play a role in the modulation of cardiovascular reflexes, and modify the influence exerted on the processing of these reflexes by other areas of the central nervous system.     

Interaction between Mas and the angiotensin AT1 receptor in the amygdala

The Mas-protooncogene is a maternally imprinted gene encoding an orphan G protein-coupled receptor expressed mainly in limbic structures of the rodent CNS. Because Mas and the product of the Mas-related gene enhance the effects of angiotensins on cells expressing angiotensin receptors of the AT1 subtype, we first compared the distribution of cells expressing AT1 receptors in different limbic and thalamic brain structures in Mas-knockout mice and in wildtype mice by an immunohistochemical approach. No significant differences could be found between the two strains. The Mas-protooncogene seems to be implicated in the signal transduction of angiotensin receptors and is expressed in the amygdala. Therefore we then analyzed whether field potentials are altered by angiotensin II in brain slices of the basolateral amygdala. An opposite action of angiotensin II was obtained in mice lacking the Mas-protooncogene in comparison to wildtype mice. The use of different angiotensin receptor antagonists provides the first in vitro evidence for a functional interaction between the Mas-protooncogene and the AT1 receptor.     

Expression of angiotensinogen and receptors for angiotensin and prorenin in the monkey and human substantia nigra: an intracellular renin–angiotensin system in the nigra

We have previously obtained in rodents a considerable amount of data suggesting a major role for the brain renin–angiotensin system (RAS) in dopaminergic neuron degeneration and potentially in Parkinson’s disease. However, the presence of a local RAS has not been demonstrated in the monkey or the human substantia nigra compacta (SNc). The present study demonstrates the presence of major RAS components in dopaminergic neurons, astrocytes and microglia in both the monkey and the human SNc. Angiotensin type 1 and 2 and renin–prorenin receptors were located at the surface of dopaminergic neurons and glial cells, as expected for a tissular RAS. However, angiotensinogen and receptors for angiotensin and renin–prorenin were also observed at the cytoplasm and nuclear level, which suggests the presence of an intracrine or intracellular RAS in monkey and human SNc. Although astrocytes and microglia were labeled for angiotensin and prorenin receptors in the normal SNc, most glial cells appeared less immunoreactive than the dopaminergic neurons. However, our previous studies in rodent models of PD and studies in other animal models of brain diseases suggest that the RAS activity is significantly upregulated in glial cells in pathological conditions. The present results together with our previous findings in rodents suggest a major role for the nigral RAS in the normal functioning of the dopaminergic neurons, and in the progression of the dopaminergic degeneration.     

Protective effects of urate against 6-OHDA-induced cell injury in PC12 cells through antioxidant action

There is evidence to support that oxidative stress is increased in Parkinson's disease (PD) and contributes to the degeneration of dopaminergic neurons. Recent research has shown that higher blood urate concentrations have now been linked to decreased risks and progression rates of PD. However, the mechanisms about urate to protect dopaminergic neurons are less clear. Our study investigated the effect of urate on oxidative stress induced by 6-hydroxydopamine (6-OHDA) in neuronal differentiated PC12 cells. We found that urate significantly reduced 6-OHDA-induced lactate dehydrogenas (LDH), malondialdehyde (MDA), and 8-hydroxy-deoxyguanosine (8-OHdG) generation but increased the superoxide dismutase (SOD) activity and glutathione (GSH) levels in the PC12 cells. These results suggested that urate can prevent PC12 cells from oxidative injury induced by 6-OHDA, which may play an important role in the mechanisms underlying the association of high plasma levels of urate with reduced risk and slower progression of PD. Urate treatment could be a potential therapeutic strategy for PD.     

Angiotensin receptor blockers for bipolar disorder

Studies have suggested that the brain renin angiotensin system (RAS) regulates cerebral flow, autonomic and hormonal systems, stress, innate immune response and behavior, being implicated in several brain disorders such as major depression, Parkinson’s and Alzheimer’s disease. The angiotensin II receptor subtype 1 (AT1R) is distributed in brain regions responsible for the control of stress response through peripheral and central sympathetic hyperactivation as well as in the hypothalamic paraventricular region, areas known for the release of several neurotransmitters related to inflammatory response facilitation. This relationship leads to the assumption that AT1R might be the receptor most related to the central deleterious actions of angiotensin II. New evidences from clinical studies have shown a possible role for RAS in the pathogenesis of bipolar disorder (BD), a multifactorial disorder with acknowledged presence of neuronal damage via oxidative stress in brain areas such as hippocampus, prefrontal cortex and striatum. Given the studies highlighting AT1R activation as a central pro-inflammatory pathway and, conversely, the involvement of inflammatory response in the pathogenesis of BD; this paper hypothesizes the use of AT1R antagonists for BD management and prevention of its neuroprogression, due to their anti-inflammatory and neuroprotective effects.     

Prostaglandin receptor EP2 protects dopaminergic neurons against 6-OHDA-mediated low oxidative stress

Dopaminergic neurons in the substantia nigra (SN) selectively die in Parkinson's disease (PD), but it is unclear how and why this occurs. Recent findings implicate prostaglandin E(2) (PGE(2)) and two of its four receptors, namely EP1 and EP2, as mediators of degenerative and protective events in situations of acute and chronic neuronal death. EP1 activation can exacerbate excitotoxic damage in stroke models and our recent study showed that EP1 activation may explain the selective sensitivity of dopaminergic neurons to oxidative stress. Conversely, EP2 activation may be neuroprotective, although toxic effects have also been demonstrated. Here we investigated if and how EP2 activation might alter the survival of dopaminergic neurons following selective low-level oxidative injury evoked by the neurotoxin 6-hydroxydopamine (6-OHDA) in primary neuronal cultures prepared from embryonic rat midbrain. We found that cultured dopaminergic neurons displayed EP2 receptors. Butaprost, a selective EP2 agonist, significantly reduced 6-OHDA neurotoxicity. EP2 receptors are coupled to stimulatory G-proteins (Gs), which activate adenylate cyclase, increasing cAMP synthesis, which then activates protein kinase A (PKA). Both dibutyryl cAMP and forskolin reduced dopaminergic cell loss after 6-OHDA exposure. Conversely, KT5720 and H-89, two structurally distinct high-affinity PKA inhibitors, abolished the protective effect of butaprost, implicating cAMP-dependent PKA activity in the neuroprotection by EP2 activation. Finally, we show that melanized dopaminergic neurons in the human SN express EP2. This pathway warrants consideration as a neuroprotective strategy for PD.