Serum Irisin Level is Higher and Related with Insulin in Acanthosis Nigricans-related Obesity.

OBJECTIVE: Acanthosis nigricans (AN) is proved to be a skin phenotype of hyperinsulinemia especially in obese patients. Irisin is a new myokine which plays an important role in metabolic disorders, such as obesity, insulin resistance, and type 2 diabetes. The role of irisin in the development of AN-related obesity is not yet understood. In this study, we aimed to investigate the relationship between irisin and AN-related obesity. Patients & Measurements: 30 obese patients without AN (OB group), 30 obese patients with AN (AN group), and 20 age-matched healthy volunteers (control group, CON) were included in this study. Weight, BMI, lipid profile, FFA, UA, and CRP were measured in all participants. Oral Glucose Tolerance tests (OGTT) were performed and serum glucose and plasma insulin were measured at 0, 30, 60,120 and 180 min. The AUC (area under curve) of glucose and insulin was calculated. Serum irisin was measured by ELISA. RESULTS: Hyperinsulinemia is found in both AN and OB groups. The AN group had higher levels of insulin but better blood glucose tolerance and insulin response. The difference in irisin levels between the 3 groups was statistically significant, with the AN group showing the highest serum level of irisin. Serum irisin levels were positively correlated with BMI, and fasting insulin. CONCLUSION: AN is a state of hyperinsulinmia and has better insulin response and glucose tolerance compared to obese patients without AN. Serum irisin may be a protective factor against impaired beta cell function in obesity with AN.

Increased progesterone receptor A expression in labouring human myometrium is associated with decreased promoter occupancy by the histone demethylase JARID1A.

Progesterone regulates female reproductive function predominantly through two nuclear progesterone receptors (PRs), PR-A and PR-B. During human parturition myometrial PR expression is altered to favour PR-A, which activates pro-labour genes. We have previously identified histone H3 lysine 4 trimethylation (H3K4me3) as an activator of myometrial PR-A expression at labour. To further elucidate the mechanisms regulating PR isoform expression in the human uterus at labour, we have (i) determined the methylation profile of the cytosine-guanine dinucleotides (CpG) island in the promoter region of the PR gene and (ii) identified the histone-modifying enzymes that target the H3K4me3 mark at the PR promoters in term and preterm human myometrial tissues obtained before and after labour onset. Bisulphite sequencing showed that despite overall low levels of PR CpG island methylation, there was a significant decrease in methylated CpGs with labour in both preterm (P < 0.05) and term (P < 0.01) groups downstream of the PR-B transcription start site. This methylation change was not associated with altered PR-B expression, but may contribute to the increase in PR-A expression with labour. Chromatin immunoprecipitation revealed that the histone methyltransferase, SET and MYND domain-containing protein 3 (SMYD3), bound to the PR gene at significantly higher levels at the PR-A promoter compared with the PR-B promoter (P < 0.010), with no labour-associated changes observed. The H3K4 demethylase, Jumonji AT-rich interactive domain 1A (JARID1A), also bound to the PR-A, but not to the PR-B promoter prior to term labour, and decreased significantly at the onset of labour (P = 0.014), providing a mechanism for the previously reported increase in H3K4me3 level and PR-A expression with labour. Our studies suggest that epigenetic changes mediated by JARID1A, SMYD3 and DNA methylation may be responsible, at least in part, for the functional progesterone withdrawal that precipitates human labour.

Identification of the DDAH2 protein in pig reproductive tract mucus: a putative oestrus detection marker.

Precisely detecting oestrus is important for artificial insemination. The aims of this study were to identify oestrus-specific sow mucus proteins to determine the optimal time for artificial insemination. The proestrous- and oestrous-stage mucus proteins were purified and analysed with proteomic tools such as two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight analyses. Among the differentially expressed proteins, the dimethylarginine dimethylaminohydrolase 2 (DDAH2) protein showed a 3.6-fold increase during the proestrous stage compared to that during the oestrous stage. A western immunoblot study revealed that two of three sow mucus samples clearly showed negative anti-DDAH2 antibody activity during the oestrous stage. This study demonstrated that the pig DDAH2 mucus protein exists during the proestrous stage, but not during the oestrous stage, suggesting that mucus DDAH2 could be useful as an oestrus detection marker.

Term myometrium is characterized by increased activating epigenetic modifications at the progesterone receptor-A promoter.

Term human myometrial expression of progesterone receptor (PR)-A is increased relative to PR-B, and as PR-A is a repressor of progesterone action mediated through PR-B, this increase may mediate the withdrawal of progesterone action and precipitate the onset of labour. PR-A and PR-B expression is regulated by two separate promoters of the PR gene. We hypothesized that epigenetic histone modifications at the two promoters contribute to the labour-associated regulation of PR-A and PR-B expression in term myometrium. PR total, PR-B and PR-A mRNA levels were determined using quantitative real-time PCR, and chromatin immunoprecipitation was used to determine the levels of activating and repressive histone modifications at the PR-A and PR-B promoters in human myometrial samples not in labour (n = 4) and in labour (n = 4). Chromatin extracts were immunoprecipitated with antibodies against activating (histone H3 and H4 acetylation and histone H3 lysine 4 trimethylation), and repressive (histone H3 lysine 9 trimethylation, histone H3 lysine 27 trimethylation and asymmetrical histone H3 arginine 2 dimethylation) histone modifications. PR-A mRNA levels increased during labour, while PR-B mRNA levels remained constant resulting in an increase of PR-A/PR-B mRNA ratio, as expected. Regardless of labour status, significantly higher levels of the activating histone modifications were found at the PR-A promoter compared with the PR-B promoter (P <0.001). H3K4me3 increased significantly at both promoters with labour onset (P =0.001). Low levels of the repressive histone modifications were also present at both promoters, with no labour-associated changes observed. Our data indicate that the PR-A promoter is epigenetically marked for activation in term myometrium more extensively than the PR-B promoter, and that labour is associated with an increase in H3K4me3 activating modification, consistent with the previously described increase in PR protein at this time.

Deletion of insulin-regulated aminopeptidase in mice decreases susceptibility to pentylenetetrazol-induced generalized seizures.

The peptide angiotensin IV (Ang IV) influences seizure susceptibility in rat and mouse models. Indeed, Ang IV has been shown to protect rats from limbic seizures in the focal pilocarpine model. Moreover, both anticonvulsive and antiepileptogenic effects of Ang IV have been reported in the acute pentylenetetrazol (PTZ) and kindling model of generalized seizures in mice. It has been hypothesized that the latter effects on seizures could be established via a modulatory effect on dopamine receptors in the basal ganglia or via an indirect interaction between Ang IV and adenosine A1 receptors. However, a possible role for insulin-regulated aminopeptidase (IRAP), the high affinity binding site for Ang IV, has not been studied yet. To unequivocally unravel the involvement of IRAP in generalized seizure generation, we investigated the susceptibility of male IRAP wild-type (IRAP(+/+)) and knock-out (IRAP(-/-)) mice to PTZ-induced seizures. Challenging these mice intravenously with PTZ resulted in significantly increased thresholds for myoclonic twitch and generalized clonic seizures with loss of righting reflexes in IRAP(-/-) mice compared to their IRAP(+/+) littermates. These behavioural data were confirmed by video-electrocorticography monitoring. Our study shows that IRAP(-/-) mice are less sensitive to the development of PTZ-induced seizures and suggests that IRAP is involved in generalized seizure generation.

Involvement of cannabinoid-1 and cannabinoid-2 receptors in septic ileus.

BACKGROUND Cannabinoid (CB) receptors are involved in the regulation of gastrointestinal (GI) motility under physiological and pathophysiological conditions. We aimed to characterize the possible influence of CB(1) and CB(2) receptors on motility impairment in a model of septic ileus. METHODS Lipopolysaccharide (LPS) injections were used to mimic pathophysiological features of septic ileus. Spontaneous jejunal myoelectrical activity was measured in rats in vivo, and upper GI transit was measured in vivo by gavaging of a charcoal marker into the stomach of mice, in absence or presence of LPS, and CB(1) and CB(2) receptor agonists and antagonists. Tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 levels were measured using enzyme-linked immunosorbent assay. Histology was performed with haematoxylin-eosin staining. KEY RESULTS Lipopolysaccharide treatment significantly reduced amplitude and frequency of myoelectric spiking activity and GI transit in vivo in a dose-dependent manner. TNF-alpha and IL-6 were increased in LPS-treated animals and histology showed oedema and cell infiltration. Both, the CB(1) agonist HU210 and the CB(2) agonist JWH133 reduced myoelectrical activity whereas the CB(1) antagonist AM251 caused an increase of myoelectrical activity. Pretreatment with AM251 or AM630 prevented against LPS-induced reduction of myoelectrical activity, and also against the delay of GI transit during septic ileus in vivo. CONCLUSIONS & INFERENCES The LPS model of septic ileus impairs jejunal myoelectrical activity and delays GI transit in vivo. Antagonists at the CB(1) receptor or the CB(2) receptor prevent the delay of GI transit and thus may be powerful tools in the future treatment of septic ileus.

The angiotensin IV/AT4 receptor.

The angiotensin AT(4) receptor was originally defined as the specific, high-affinity binding site for the hexapeptide angiotensin IV (Ang IV). Subsequently, the peptide LVV-hemorphin 7 was also demonstrated to be a bioactive ligand of the AT(4) receptor. Central administration of Ang IV, its analogues or LVV-hemorphin 7 markedly enhance learning and memory in normal rodents and reverse memory deficits observed in animal models of amnesia. The AT(4) receptor has a broad distribution and is found in a range of tissues, including the adrenal gland, kidney, lung and heart. In the kidney Ang IV increases renal cortical blood flow and decreases Na(+) transport in isolated renal proximal tubules. The AT(4) receptor has recently been identified as the transmembrane enzyme, insulin-regulated membrane aminopeptidase (IRAP). IRAP is a type II integral membrane spanning protein belonging to the M1 family of aminopeptidases and is predominantly found in GLUT4 vesicles in insulin-responsive cells. Three hypotheses for the memory-potentiating effects of the AT(4) receptor/IRAP ligands, Ang IV and LVV-hemorphin 7, are proposed: (i) acting as potent inhibitors of IRAP, they may prolong the action of endogenous promnestic peptides; (ii) they may modulate glucose uptake by modulating trafficking of GLUT4; (iii) IRAP may act as a receptor, transducing the signal initiated by ligand binding to its C-terminal domain to the intracellular domain that interacts with several cytoplasmic proteins.

In vitro autoradiographic localization of calcitonin and amylin binding sites in monkey brain.

Calcitonin (CT) and amylin are related peptides with potent central actions, including suppression of appetite and gastric acid secretion. Little is known about the distribution and binding characteristics of amylin receptors in species other than rat; therefore, in this study, by using in vitro autoradiography, we have mapped the distribution of 125I-rat amylin binding sites in the monkey brain and compared this distribution to that of binding sites for 125I-salmon CT (125I-sCT). Highest densities of 125I-amylin binding were in the hypothalamus, including the arcuate nucleus and parts of the ventromedial hypothalamic nuclei, and the solitary nucleus. Rostrally, moderate to high density binding was present in parts of the preoptic area, bed nucleus of the stria terminalis, amygdala and accumbens nucleus (Acb). Caudally, binding of amylin was more restricted, with moderate to high density binding present only in dorsal raphe, and area postrema. The primary visual cortex displayed strong and periodic CT binding in layer 4. The subcortical pattern of distribution of amylin and CT receptors in the monkey was similar to that seen previously in the rat, although the relative densities of binding to different brain structures were not always conserved. As with rat, monkey amylin receptors were a subset of the sites labeled with 125I-sCT. Analysis of receptor specificity indicated a greater relative potency of CT peptides in competing for 125I-amylin binding in monkey, when compared to rat, while, there was a decrease in the relative potency of CT gene-related peptides, potentially due to differences the level of receptor activity modifying proteins (RAMPs) in monkey versus rat brain. Amylin receptors in primates are likely to perform a similar role to those in rats; however, the interaction of the receptors with related peptides may differ.

Effect of I.C.V. injection of AT4 receptor ligands, NLE1-angiotensin IV and LVV-hemorphin 7, on spatial learning in rats.

Central administration of angiotensin IV (Ang IV) or its analogues enhance performance of rats in passive avoidance and spatial memory paradigms. The purpose of this study was to examine the effect of a single bolus injection of two distinct AT4 ligands, Nle1-Ang IV or LVV-haemorphin-7, on spatial learning in the Barnes circular maze. Mean number of days for rats treated with either Nle1-Ang IV or LVV-haemorphin-7 to achieve learner criterion is significantly reduced compared with controls (P < 0.001 and P < 0.05 respectively). This is due to enhanced ability of the peptide-treated rats to adopt a spatial strategy for finding the escape hatch. In all three measures of learning performance, (1) the number of errors made, (2) the distance travelled and (3) the latency in finding the escape hatch, rats treated with either 100 pmol or 1 nmol of Nle1-Ang IV or 100 pmol LVV-haemorphin-7 performed significantly better than the control groups. As early as the first day of testing, the rats treated with the lower dose of Nle1-Ang IV or LVV-haemorphin-7 made fewer errors (P < 0.01 and P < 0.05 respectively) and travelled shorter distances (P < 0.05 for both groups) than the control animals. The enhanced spatial learning induced by Nle1-Ang IV (100 pmol) was attenuated by the co-administration of the AT4 receptor antagonist, divalinal-Ang IV (10 nmol). Thus, administration of AT4 ligands results in an immediate potentiation of learning, which may be associated with facilitation of synaptic transmission and/or enhancement of acetylcholine release.

The brain renin-angiotensin system: location and physiological roles.

Angiotensinogen, the precursor molecule for angiotensins I, II and III, and the enzymes renin, angiotensin-converting enzyme (ACE), and aminopeptidases A and N may all be synthesised within the brain. Angiotensin (Ang) AT(1), AT(2) and AT(4) receptors are also plentiful in the brain. AT(1) receptors are found in several brain regions, such as the hypothalamic paraventricular and supraoptic nuclei, the lamina terminalis, lateral parabrachial nucleus, ventrolateral medulla and nucleus of the solitary tract (NTS), which are known to have roles in the regulation of the cardiovascular system and/or body fluid and electrolyte balance. Immunohistochemical and neuropharmacological studies suggest that angiotensinergic neural pathways utilise Ang II and/or Ang III as a neurotransmitter or neuromodulator in the aforementioned brain regions. Angiotensinogen is synthesised predominantly in astrocytes, but the processes by which Ang II is generated or incorporated in neurons for utilisation as a neurotransmitter is unknown. Centrally administered AT(1) receptor antagonists or angiotensinogen antisense oligonucleotides inhibit sympathetic activity and reduce arterial blood pressure in certain physiological or pathophysiological conditions, as well as disrupting water drinking and sodium appetite, vasopressin secretion, sodium excretion, renin release and thermoregulation. The AT(4) receptor is identical to insulin-regulated aminopeptidase (IRAP) and plays a role in memory mechanisms. In conclusion, angiotensinergic neural pathways and angiotensin peptides are important in neural function and may have important homeostatic roles, particularly related to cardiovascular function, osmoregulation and thermoregulation.

Evidence that the angiotensin IV (AT(4)) receptor is the enzyme insulin-regulated aminopeptidase.

Central infusion of angiotensin IV or its more stable analogues facilitates memory retention and retrieval in normal animals and reverses amnesia induced by scopolamine or by bilateral perforant pathway lesions. These peptides bind with high affinity and specificity to a novel binding site designated the angiotensin AT(4) receptor. Until now, the AT(4) receptor has eluded molecular characterization. Here we identify the AT(4) receptor, by protein purification and peptide sequencing, to be insulin-regulated aminopeptidase (IRAP). HEK 293T cells transfected with IRAP exhibit typical AT(4) receptor binding characteristics; the AT(4) receptor ligands, angiotensin IV and LVV-hemorphin 7, compete for the binding of [(125)I]Nle(1)-angiotensin IV with IC(50) values of 32 and 140 nm, respectively. The distribution of IRAP and its mRNA in the brain, determined by immunohistochemistry and hybridization histochemistry, parallels that of the AT(4) receptor determined by radioligand binding. We also show that AT(4) receptor ligands dose-dependently inhibit the catalytic activity of IRAP. We have therefore demonstrated that the AT(4) receptor is IRAP and propose that AT(4) receptor ligands may exert their effects by inhibiting the catalytic activity of IRAP thereby extending the half-life of its neuropeptide substrates.

Characterization of the AT(4) receptor in a human neuroblastoma cell line (SK-N-MC).

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II (Ang II), binds to a distinct receptor designated the AT(4) receptor. The peptide elicits a range of vascular and central actions including facilitation of memory retention and retrieval in several learning paradigms. The aim of this study was to characterize the AT(4) receptor in a human cell line of neural origin. Receptor binding studies indicate that the human neuroblastoma cell line SK-N-MC cells express a high-affinity Ang IV binding site with a pharmacological profile similar to the AT(4) receptor: (125)I]-Ang IV and (125)I]-Nle(1)-Ang IV bind specifically to the SK-N-MC cell membranes (K(d) = 0.6 and 0.1 nM) in a saturable manner (B(max) = 1.2 pmol/mg of protein). AT(4) receptor ligands, Nle(1)-Ang IV, Ang IV and LVV-haemorphin 7 (LVV-H7), compete for the binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to the SK-N-MC cell membranes with rank order potencies of Nle(1)-Ang IV > Ang IV > LVV-H7 with IC(50) values of 1.4, 8.7 and 59 nM ([(125)I]-Ang IV) and 1.8, 20 and 168 nM ([(125)I]-Nle(1)-Ang IV), respectively. The binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to SK-N-MC cell membranes was not affected by the presence of GTP gamma S. Both Ang IV and LVV-H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT(4) receptor in the SK-N-MC cells is a 180-kDa glycoprotein; under non-reducing conditions a 250-kDa band was also observed. In summary, the human neuroblastoma cell line, SK-N-MC, expresses functional AT(4) receptors that are responsive to Ang IV and LVV-H7, as indicated by an increase in DNA synthesis. This is the first human cell line of neural origin shown to express the AT(4) receptor.

Potentiation of cholinergic transmission in the rat hippocampus by angiotensin IV and LVV-hemorphin-7.

Recent evidence demonstrates that the fragment of angiotensin II, angiotensin II (3-8) termed angiotensin IV, binds with high affinity to a specific binding site, the AT(4) receptor. Intracerebroventricular injection of AT(4) receptor agonists improves the performance of rats in passive avoidance and spatial learning paradigms. AT(4) receptors and cholinergic neurons are closely associated in regions involved in cognitive processing, such as the hippocampus and neocortex. We therefore postulated that AT(4) receptors affect cognitive processing by modulating cholinergic neurotransmission. To test this, we examined the effect of AT(4) receptor ligands, angiotensin IV and LVV-hemorphin-7, on potassium-evoked [(3)H]acetylcholine ([(3)H]ACh) release from rat hippocampal slices. Hippocampal slices from male Sprague--Dawley rats were incubated with [(3)H]choline chloride, perfused with Krebs--Henseleit solution and [(3)H]ACh release was determined. Angiotensin IV and LVV-hemorphin-7 both potentiated depolarisation-induced [(3)H]ACh release from the rat hippocampus in a concentration-dependent manner with the maximal dose (10(-7)M) of each inducing an increase of 45+/-7.5% (P<0.01) and 95.8+/-19% (P<0.01) above control, respectively. Potentiation of release by both agonists was attenuated by the AT(4) receptor antagonist, divalinal-Ang IV. Angiotensin IV-induced potentiation was not affected by AT(1) and AT(2) receptor antagonists. These results indicate that stimulation of AT(4) receptors can potentiate depolarisation-induced release of ACh from hippocampal slices and suggest that potentiation of cholinergic transmission may be a mechanism by which AT(4) receptor ligands enhance cognition.

Design and synthesis of tricyclic derivatives as high density lipoprotein cholesterol enhancers.

A pharmacophore for increasing HDLC was proposed based on common structural features of non-thio-containing compounds with HDLC enhancing properties. A search of the compound database identified various series of these non-thio-containing compounds, including a novel tricyclic imidazoisoquinolone. Preparation of 1-aryl-3-oxo-1,3-dihydro-2-benzofuran-1-carboxamides using a novel and widely applicable one-step process from 2-acyl benzoic acids is reported. Reaction of diamines with 1-aryl-3-oxo-1,3-dihydro-2-benzofuran-1-carboxamides and related aza-analogues proceeded with regio-control to furnish imidazoisoquinolones, pyrimidoisoquinolones, and imidazonaphthyridines. NMR studies and X-ray crystallography confirmed the regiochemistry of the products. Compounds of these series increased concentrations of HDLC in test animals following oral administration.

Autoradiographic localization and quantification of components of the Renin-Angiotensin system in tissues.

In situ radioligand binding with autoradiography allows localization and quantification of bound radiolabeled ligands in tissues. This is a very sensitive technique that enables the characterization of binding kinetics and ligand specificity and the quantification of the amount of radioligand bound in different structures within the tissue. This technique is complementary to the higher resolution of immunohistochemical localization of proteins or binding sites on fixed tissue sections and in situ hybridization histochemical localization of mRNA.

The angiotensin converting enzyme (ACE) inhibitor, perindopril, modifies the clinical features of Parkinson's disease.

BACKGROUND: Animal studies have demonstrated an interaction within the striatum between the angiotensin and dopaminergic systems. In rats, the angiotensin converting enzyme (ACE) inhibitor, perindopril, crosses the blood brain barrier and increases striatal dopamine synthesis and release. In humans, angiotensin type 1 receptors have been found on dopaminergic neurons in the substantia nigra and striatum. In Parkinson's disease, there is a marked reduction of these receptors associated with the nigrostriatal dopaminergic neuron loss. AIMS: We performed a double blind placebo controlled crossover pilot study in seven patients to investigate the effect of the ACE inhibitor, perindopril on the clinical features of moderately severe Parkinson's disease. RESULTS: After a four week treatment period with perindopril, patients had a faster onset in their motor response to L-dopa and a reduction in 'on phase' peak dyskinesia, p=0.021 and p=0.014 respectively. Patients also reported more 'on' periods during their waking day in their movement diary, p=0.007. Perindopril was well tolerated without any significant postural hypotension or renal dysfunction. CONCLUSIONS: These results suggest that ACE inhibitors such as perindopril may have a place in the management of motor fluctuations and dyskinesia in Parkinson's disease and justify further study.

Distribution of angiotensin IV binding sites (AT4 receptor) in the human forebrain, midbrain and pons as visualised by in vitro receptor autoradiography.

Angiotensin IV and other AT4 receptor agonists, improve memory retention and retrieval in the passive avoidance and swim maze learning paradigms. Angiotensin IV binding sites (also known as the AT4 receptors) are widely distributed in guinea pig and monkey (Macaca fascicularis) brains where high densities of the binding sites have been detected in the hippocampus, neocortex and motor nuclei. However, the distribution of the binding sites in the human brain is not known. We have recently localised the angiotensin IV binding sites (AT4 receptors) in post-mortem human brain using iodinated Nle-angiotensin IV, a higher affinity and more stable analogue of angiotensin IV. This radioligand bound with relatively high affinity and specificity to angiotensin IV binding sites. In competition studies on consecutive sections through the prefrontal cortex and claustrum, angiotensin IV, Nle-angiotensin IV and LVV-hemorphin 7 competed for the binding of 125I[Nle]-angiotensin IV with nanomolar affinities. Angiotensin II and the AT1 and AT2 receptor antagonists were ineffective in competing for the binding at concentrations of up to 10 microM. We found high densities of 125I[Nle]-angiotensin IV binding sites throughout the cerebral cortex including the insular, entorhinal, prefrontal and cingulate cortices. Very high densities of the binding sites were observed in the claustrum, choroid plexus, hippocampus and pontine nucleus. Some thalamic nuclei displayed high densities of binding including the anteroprincipal, ventroanterior, anteromedial, medial dorsal and ventrolateral nuclei. The caudate nucleus, putamen, many amygdaloid nuclei and the red nucleus all displayed moderate densities of binding with a higher level detected in the substantia nigra pars compacta. In the hypothalamus, high densities binding sites were found in the ventromedial nucleus with lower levels in the dorsomedial and paraventricular nuclei. The distribution of 125I[Nle]-angiotensin IV binding sites in the human brain is similar to that found in other species and supports multiple roles for the binding sites in the central nervous system, including facilitation of memory retention and retrieval.

Up regulation of AT4 receptor levels in carotid arteries following balloon injury.

Angiotensin IV, (V-Y-I-H-P-F), binds to AT4 receptors in blood vessels to induce vasodilatation and proliferation of cultured bovine endothelial cells. This latter effect may be important not only in developing tissues but also in injured vessels undergoing remodelling. In the present study, using normal rabbit carotid arteries, we detected AT4 receptors in vascular smooth muscle cells and in the vasa vasorum of the adventitia. Very low receptor levels were observed in the endothelial cells. In keeping with the described binding specificity of AT4 receptors, unlabelled angiotensin IV competed for [125I]angiotensin IV binding in the arteries, with an IC50 of 1.4 nM, whereas angiotensin II and angiotensin III were weaker competitors. Within the first week following endothelial denudation of the carotid artery by balloon catheter, AT4 receptor binding in the media increased to approximately 150% of control tissue. AT4 receptor binding further increased in the media, large neointima and re-endothelialized cell layer to 223% at 20 weeks after injury. In view of the known trophic effects of angiotensin IV, the elevated expression of AT4 receptors, in both the neointima and media of arteries, following balloon injury to the endothelium, suggests a role for the peptide in the adaptive response and remodelling of the vascular wall following damage.

Effect of chronic angiotensin-converting enzyme inhibition on striatal dopamine content in the MPTP-treated mouse.

We have previously shown that chronic treatment with the angiotensin-converting enzyme inhibitor perindopril increased striatal dopamine levels by 2.5-fold in normal Sprague-Dawley rats, possibly via modulation of the striatal opioid or tachykinin levels. In the present study, we investigated if this effect of perindopril persists in an animal model of Parkinson's disease, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse. C57BL/6 mice were treated with the neurotoxin (30 mg/kg/day intraperitoneally) for 4 days and then left for 3 weeks to allow the degeneration of striatal dopaminergic terminals. At this time, the mice exhibited a 40% decrease in striatal dopamine content and an accompanying 46% increase in dopamine D2 receptor levels compared with control untreated mice. The dopamine content returned to control levels, and the increase in dopamine D2 receptor levels was attenuated in mice treated with perindopril (5 mg/kg/day orally for 7 days) 2 weeks after the last dose of MPTP. When the angiotensin-converting enzyme inhibitor was administered (5 mg/kg/day for 7 days) immediately after the cessation of the MPTP treatment, there was no reversal of the effect of the neurotoxin in decreasing striatal dopamine content. Our results demonstrate that perindopril is an effective agent in increasing striatal dopamine content in an animal model of Parkinson's disease.