RESUMEN

Angiotensin II (Ang II), the main effector peptide of the renin-angiotensin system (RAS), acting on AT1 and AT2 receptors participates in the regulation of proliferation, differentiation and apoptosis in tumour cells. The peroxisome-proliferator activated receptor γ (PPARγ) and its ligands exert anti-tumour effects in various human cancer cell lines. The present study investigates the effects initiated by AT1- and AT2 receptor stimulation in SK-UT-1 cells, a human leiomyosarcoma cell line, and clarifies the role of the PPARγ in the AT2 receptor-induced differentiation and apoptosis.Selective stimulation of AT1- and AT2 receptors was achieved by incubation of the cells with Ang II (10-6 M) in the presence of the selective AT2 receptor antagonist, PD 123177 (10-6 M) and the AT1 receptor antagonist, losartan (10-5 M), respectively, the selective PPARγ antagonist, GW 9662, was used at concentration 10-6 M. The expression of smooth muscle cell differentiation markers, SM22α and calponin, was analysed at RNA- and protein levels using RT PCR and Western blot, which was also used to quantify Bcl-2-, Bax- and cleaved caspase-3 proteins. The translocation of the AT2-receptor interacting protein 1 (ATIP1) to the nuclei was studied by Western blot and immunofluorescence staining. The mitochondrial status and the metabolic activity in response to AT1- and AT2 receptor activation were assessed by the quantification of 99mTc - sestamibi and 2´-deoxy-2´-[18F]fluoro-D-glucose uptake.AT1 receptor stimulation did not exert any profound effects in quiescent SK-UT-1 cells. The effects induced by Ang II acting on AT2 receptors were time-dependent. A short, 3 - 6 h lasting stimulation promotes differentiation, i.e increases in the mRNA- and protein levels of SM22α and calponin, whereas a sustained stimulation for 48 h activates the intrinsic apoptotic pathway, as evidenced by reduced cell numbers, down-regulation of the anti-apoptotic Bcl-2 protein and increased levels of the Bax protein and cleaved caspase-3. The effects were reversed by the PPARγ antagonist, GW 9662, clearly implying a PPARγ-dependent mechanism. Our results also demonstrate a co-localisation of the AT2-receptor interacting protein, ATIP1, and the PPARγ in nuclei of SK-UT-1 cells and an accumulation of ATIP1 in the nuclear fraction in response to AT2 receptor stimulation. The regulation of the differentiation and apoptosis via the AT2 receptor favours an important functional role of this receptor in quiescent, slow-cycling SK-UT-1 cells and provides the rationale for the use of AT1 receptor antagonists for the treatment of human leiomyosarcomas.

Asunto(s)

Apoptosis , Diferenciación Celular , Leiomiosarcoma/metabolismo , PPAR gamma/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Anilidas/farmacología , Membrana Celular , Humanos , Imidazoles/farmacología , Losartán/farmacología , Piridinas/farmacología , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

AIM: The function of brain (neuronal) peroxisome proliferator-activated receptor(s) γ (PPARγ) in the delayed degeneration and loss of neurones in the substantia nigra (SN) was studied in rats after transient occlusion of the middle cerebral artery (MCAO). METHODS: The PPARγ agonist, pioglitazone, or vehicle was infused intracerebroventricularly over a 5-day period before, during and 5 days after MCAO (90 min). The neuronal degeneration in the SN pars reticularis (SNr) and pars compacta (SNc), the analysis of the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurones and the expression of the PPARγ in these neurones were studied by immunohistochemistry and immunofluorescence staining. The effects of PPARγ activation on excitotoxic and oxidative neuronal damage induced by glutamate and 6-hydroxydopamine were investigated in primary cortical neurones expressing PPARγ. RESULTS: Pioglitazone reduced the total and striatal infarct size, neuronal degeneration in both parts of the ipsilateral SN, the loss of TH-IR neurones in the SNc and increased the number of PPARγ-positive TH-IR neurones. Pioglitazone protected primary cortical neurones against oxidative and excitotoxic damage, prevented the loss of neurites and supported the formation of synaptic networks in neurones exposed to glutamate or 6-hydroxydopamine by a PPARγ-dependent mechanism. CONCLUSIONS: Activation of cerebral PPARγ confers neuroprotection after ischaemic stroke by preventing both, neuronal damage within the peri-infarct zone and delayed degeneration of neurones and neuronal death in areas remote from the site of ischaemic injury. Pioglitazone and other PPARγ agonists may be useful therapeutic agents to prevent progression of brain damage after cerebral ischaemia.

Asunto(s)

Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Ataque Isquémico Transitorio/tratamiento farmacológico , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , PPAR gamma/metabolismo , Sustancia Negra/efectos de los fármacos , Tiazolidinedionas/uso terapéutico , Animales , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/patología , Masculino , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Pioglitazona , Ratas , Ratas Wistar , Sustancia Negra/metabolismo , Sustancia Negra/patología , Tiazolidinedionas/farmacología

RESUMEN

Evidence from pharmacological studies has implicated substance P (SP), a natural ligand of tachykinin NK(1) receptors which can also interact with NK(2) receptors, in the generation of pressor and tachycardic responses to stress. Using selective blockade of brain NK(1) and NK(2) receptors, we tested in conscious rats the hypothesis that SP initiates, within the neuronal brain circuits, the sympathoadrenal, hypothalamic-pituitary-adrenal (HPA) and behavioural responses to noxious stimuli. Formalin injected s.c. through a chronically implanted catheter in the area of the lower leg was used as a pain stimulus. Rats were pretreated i.c.v. with vehicle or the selective, nonpeptide antagonists of tachykinin NK(1) and NK(2) receptors, RP 67580 and SR 48968, respectively. Ten minutes thereafter, formalin was injected s.c. and the cardiovascular responses were recorded, plasma concentrations of catecholamines, adrenocorticotrophic hormone (ACTH) and corticosterone were determined and the expression of the inducible transcription factor c-Fos in the paraventricular (PVN) and supraoptic nuclei was detected to identify neurones which were activated during pain stimulation. Blockade of NK(1) and NK(2) receptors attenuated the formalin-induced increases in mean arterial pressure and heart rate, adrenaline and ACTH concentrations in plasma, and completely abolished the pain-induced c-Fos expression in corticotrophin-releasing hormone neurones localised in the parvocellular division of the PVN. The results obtained provide pharmacological evidence that tachykinins, most probably SP, act as mediators within the neuronal circuits linked to the initiation and control of the cardiovascular, sympathoadrenal, HPA and behavioural responses to pain stimuli and provide an excitatory input to corticotrophin-releasing hormone neurones in the PVN to activate the HPA axis. Our data demonstrating the inhibition of the complex response pattern to noxious stimuli and stress are consistent with the proposed anxiolytic and antidepressant activity of NK(1) and NK(2) receptor antagonists.

Asunto(s)

Encéfalo/efectos de los fármacos , Hormona Liberadora de Corticotropina/metabolismo , Formaldehído/toxicidad , Neuronas/metabolismo , Dolor/fisiopatología , Núcleo Hipotalámico Paraventricular/metabolismo , Sistema Hipófiso-Suprarrenal , Receptores de Taquicininas/antagonistas & inhibidores , Animales , Presión Sanguínea , Frecuencia Cardíaca , Dolor/inducido químicamente , Ratas

RESUMEN

AIM: Both hyperbaric oxygenation (HBO) and inhibition of the c-Jun N-terminal kinases (JNKs) by the peptide inhibitor XG-102 (D-JNKI-1) are efficient protective strategies against ischaemia-induced neurodegeneration. The present study investigated whether the combination of HBO and JNK inhibitor, XG-102, provides additive neuroprotection against cerebral ischaemia. METHODS: Rat middle cerebral artery was occluded (MCAO) for 90 min. XG-102 [2 mg/kg, intraperitoneally] or HBO (3 ATA, 60 min) was applied 3 h after the onset of MCAO. For the combination treatment, HBO was started 10 min after the injection of XG-102. Twenty-four hours after MCAO, the infarct area, the neurological score and the immunohistochemistry staining in brain slices for cleaved-PARP, transferase-mediated biotinylated UTP nick end labelling, c-Jun and phosphorylated (activated) c-Jun were observed. RESULTS: XG-102 or HBO alone reduced the total infarct area by 43% and 63%, respectively. The combination diminished total infarct area by 78%, improved the neurological function and reduced brain oedema. Co-application of HBO and XG-102 also significantly reduced the cleavage of PARP, by 96% and 91% in cortical penumbra and ischaemic core, respectively. Moreover, cotreatment significantly attenuated the number of cells labelled with transferase-mediated biotinylated UTP nick end labelling and phosphorylated c-Jun. CONCLUSION: Our study demonstrates that HBO reinforces the efficiency of neuroprotective drugs such as XG-102 and vice versa. Both treatments, physical HBO and pharmacological XG-102, are already in phase I/II studies and promising strategies for clinical use.

Asunto(s)

Inhibidores Enzimáticos/uso terapéutico , Oxigenoterapia Hiperbárica/métodos , Infarto de la Arteria Cerebral Media/terapia , Péptidos/uso terapéutico , Envejecimiento , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/patología , Edema Encefálico/terapia , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Isquemia Encefálica/terapia , Inhibidores Enzimáticos/administración & dosificación , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Masculino , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/uso terapéutico , Péptidos/administración & dosificación , Fosforilación , Proteínas Proto-Oncogénicas c-jun/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factores de Tiempo

RESUMEN

Intracerebroventricular injections of oligonucleotide probes complementary to oxytocin mRNA are known to decrease systemic oxytocin levels. In this study we show that immunoreactive oxytocin in the magnocellular hypothalamic perikarya and in their neurohypophysial projections remains unaffected by intracerebroventricular injections with an oxytocin antisense probe in rats. Hybridization signal for oxytocin mRNA was increased in the supraoptic and paraventricular nuclei in these animals. Immunocytochemistry with a monoclonal antibody, raised against triple helical DNA resulted in an accumulation of cytoplasmic reaction product in many of the magnocellular oxytocin immunoreactive neurons and in a fraction of the Herring bodies inthe posterior pituitary lobe in the antisense treated rats. Such immunostaining could be abolished by pretreating sections with RNase H. Animals injected with a mismatch probe instead of the antisense probe were devoid of cytoplasmic or axonal triple helix immunostaining. Our findings indicate that oxytocinergic transcripts in magnocellular hypothalamic neurons form triple helix-like aggregates upon specific antisense targeting rather than being degraded by endogenous RNases. While de novo transcription of oxytocin is probably stimulated, systemic release of the nonapeptide may be impaired.

Asunto(s)

ADN sin Sentido/inmunología , ADN sin Sentido/metabolismo , ADN/inmunología , Hipotálamo/metabolismo , Neuronas/metabolismo , Oxitocina/genética , Oxitocina/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , ADN/genética , ADN sin Sentido/genética , Técnica del Anticuerpo Fluorescente , Hipotálamo/citología , Oxitocina/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Ribonucleasa H/metabolismo , Transcripción Genética

RESUMEN

Biochemical, physiological and functional studies suggest that the brain renin-angiotensin system (RAS) is regulated independently of the peripheral RAS. The classical actions of angiotensin II in the brain include blood pressure control, drinking behaviour, natriuresis and the release of vasopressin into the circulation. At least two subtypes of G-protein coupled receptors, the AT(1) and the AT(2) receptor, have been identified. Most of the classic actions of angiotensin II in the brain are mediated by AT(1) receptors. The AT(2) receptor is involved in brain development and neuronal regeneration and protection. Additionally, AT(2) receptors can modulate some of the classic angiotensin II actions in the brain. Selective non-peptide AT(1) receptor blockers, applied systemically, have been shown to inhibit both peripheral and brain AT(1)receptors. In genetically hypertensive rats, inhibition of brain AT(1) receptors may contribute to the blood pressure lowering effects of AT(1) receptor blockers. Animal studies have shown that AT(1) receptor antagonists enable endogenous angiotensin II to stimulate neuronal regeneration via activation of AT(2) receptors. In animal models, inhibition of the brain RAS proved to be beneficial with respect to stroke incidence and outcome. Blockade of brain and cerebrovascular AT(1) receptors by AT(1) receptor blockers prevents the reduction in blood flow during brain ischaemia, reduces the volume of ischaemic injury and improves neurological outcome after brain ischaemia. This paper reviews the actions of angiotensin II and its receptors in the brain, and discusses the possible consequences of AT(1) receptor blockade in neuroprotection, neuroregeneration, cerebral haemodynamics and ischaemia.

Asunto(s)

Antagonistas de Receptores de Angiotensina , Encéfalo/metabolismo , Sistema Renina-Angiotensina/fisiología , Animales , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/fisiopatología , Circulación Cerebrovascular/efectos de los fármacos , Receptor de Angiotensina Tipo 1

RESUMEN

In the brain, angiotensin II (Ang II) induces various effects such as blood pressure increase, the release of arginine vasopressin (AVP) and drinking behaviour. In the present study, we investigated the ability of the angiotensin II type-I (AT(1)) receptor antagonist, candesartan, administered peripherally, to block the central effects of Ang II. Experiments were performed in conscious rats instrumented with an intracerebroventricular (i.c.v.) cannula or a guide cannula into the paraventricular nucleus (PVN) and arterial and femoral catheters. Candesartan was administered intravenously (i.v.) at doses of 0.01, 0.1, 1 or 10 mg/kg. Controls received vehicle (0.05 N Na(2)CO(3)). The drinking response ( n=10-11 per group), the pressor response ( n=7-8) and the release of AVP into the circulation ( n=10-11) following i.c.v. Ang II (100 ng) were measured 0.5, 2, 4 and 24 h following i.v. drug application. Candesartan inhibited the central responses to i.c.v. injected Ang II dose- and time-dependently. At the highest dose (10 mg/kg), the drinking and pressor responses and the release of AVP in response to i.c.v. Ang II were completely blocked at 4 h and still markedly inhibited 24 h after the antagonist application (by 85%, 48% and 86%, respectively). The lowest dose of the antagonist was without effect. In a further experiment, the release of AVP induced by microinjection of Ang II (100 ng) into the PVN was determined before and 4 h after administration of vehicle or candesartan (1 mg/kg, i.v.). Candesartan completely blocked the AVP release into the circulation induced by Ang II microinjection into the PVN. Our results demonstrate that candesartan administered peripherally effectively inhibits responses mediated by AT(1) receptors localised in periventricular brain regions as well as inside the blood-brain-barrier.

Asunto(s)

Angiotensina II/antagonistas & inhibidores , Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina , Bencimidazoles/administración & dosificación , Tetrazoles/administración & dosificación , Animales , Arginina Vasopresina/antagonistas & inhibidores , Arginina Vasopresina/sangre , Compuestos de Bifenilo , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Relación Dosis-Respuesta a Droga , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Líquidos/fisiología , Masculino , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1 , Receptores de Angiotensina/fisiología , Vigilia/efectos de los fármacos , Vigilia/fisiología

RESUMEN

Angiotensin II (Ang II) acts as a neuromodulator/neurotransmitter in specific brain nuclei involved in the regulation of blood pressure and volume homeostasis. It also induces a highly differentiated transcription factor expression in these nuclei. We investigated whether adrenoceptors, which modulate other central actions of angiotensin II like the vasopressin release, also play a role in the AT1 receptor-mediated expression of the transcription factors (TF) c-Fos, c-Jun and Krox-24 in the rat brain. Ang II, injected intracerebroventricularly, induced the expression of c-Fos, c-Jun and Krox-24 in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Pretreatment with the alpha 1-adrenoceptor antagonist, prazosin, significantly inhibited the Ang II-induced transcription factor expression in the SON and PVN. The alpha 2-adrenoceptor antagonist, yohimbine, also reduced Ang II-stimulated transcription factors significantly in both nuclei. This inhibition was mainly localized in vasopressinergic magnocellular neurons in both nuclei. The beta-adrenoceptor antagonist, propranolol, did not influence the Ang II-induced expression of TF. Our results show that both, Ang II-induced vasopressin release and transcription factor expression, involve the same neuronal connections in the brain, implicating that the signal transduction pathways leading to the two different effects are at least to a certain degree convergent.

Asunto(s)

Angiotensina II/farmacología , Hipotálamo/metabolismo , Proteínas Inmediatas-Precoces , Prosencéfalo/metabolismo , Receptores Adrenérgicos/efectos de los fármacos , Factores de Transcripción/biosíntesis , Animales , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Genes fos/efectos de los fármacos , Genes fos/genética , Genes jun/efectos de los fármacos , Genes jun/genética , Hipotálamo/efectos de los fármacos , Inmunohistoquímica , Inyecciones Intraventriculares , Prosencéfalo/efectos de los fármacos , Ratas , Ratas Wistar , Factores de Transcripción/genética

RESUMEN

Substance P, the principal neurokinin peptide in the mammalian brain and the natural ligand for the NK(1) tachykinin receptor, plays an integrative role in the regulation of cardiovascular, neuroendocrine and behavioural responses to stress. In rats, stimulation of periventricular NK(1) receptors in the forebrain induces a distinct pattern of cardiovascular responses which is accompanied by intense grooming behaviour. Ligands for NK(3) receptors induce a different pattern of cardiovascular and behavioural responses which comprises an increased release of vasopressin from the posterior pituitary and wet-dog shakes behaviour. To define the brain areas in the rat which respond to stimulation of forebrain NK(1) and NK(3) receptors and participate in the generation of these responses, the induction of c-Fos immunoreactivity was examined in brains following intracerebroventricular injections of substance P and neurokinin B in conscious rats. Stimulation of central NK(1) receptors by substance P (25, 100 and 500 pmol) injected into the lateral ventricle elicited grooming behaviour (face washing and hind limb grooming) and resulted in a marked c-Fos expression in the paraventricular, dorsomedial and parabrachial nuclei and in the medial thalamus. At 25 pmol, substance P did not significantly increase c-Fos expression, at 100 pmol, maximal c-Fos activation was induced in all four brain regions which responded to the peptide. Intracerebroventricular pretreatment of rats with the selective and high-affinity, non-peptide NK(1) receptor antagonist, RP 67580 (500 pmol), but not with its inactive enantiomer, RP 68651, completely abolished the behavioural response to substance P and reduced the substance P-induced c-Fos expression in all brain areas to nearly control levels. Intracerebroventricular injection of the natural ligand for NK(3) receptors, neurokinin B (500 pmol), elicited wet-dog shakes behaviour and activated c-Fos expression in localized regions of the forebrain including the organum vasculosum laminae terminalis, subfornical organ, median preoptic nucleus, paraventricular, supraoptic and anterior hypothalamic nuclei, medial thalamus and in the ventral tegmental area. These results demonstrate that the neurokinins, substance P and neurokinin B, induce specific and different patterns of c-Fos expression in distinct regions of the rat brain. Brain areas which selectively responded to substance P have been traditionally linked to the central regulation of cardiovascular and neuroendocrine reactions to stress or involved in the processing of nociceptive responses. On the other side, brain areas activated by neurokinin B are known to be involved in the central regulation of blood pressure, water and salt homeostasis or control of behaviour.

Asunto(s)

Sistema Nervioso Autónomo/metabolismo , Encéfalo/metabolismo , Neuroquinina B/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Taquicininas/metabolismo , Estrés Fisiológico/metabolismo , Sustancia P/metabolismo , Analgésicos/farmacología , Animales , Sistema Nervioso Autónomo/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Encéfalo/efectos de los fármacos , Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inmunohistoquímica , Indoles/farmacología , Isoindoles , Masculino , Neuroquinina B/farmacología , Antagonistas del Receptor de Neuroquinina-1 , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Sistemas Neurosecretores/efectos de los fármacos , Sistemas Neurosecretores/fisiología , Dolor/metabolismo , Dolor/fisiopatología , Ratas , Ratas Wistar , Receptores de Neuroquinina-1/agonistas , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-3/agonistas , Receptores de Neuroquinina-3/antagonistas & inhibidores , Receptores de Neuroquinina-3/metabolismo , Receptores de Taquicininas/agonistas , Receptores de Taquicininas/antagonistas & inhibidores , Estrés Fisiológico/fisiopatología , Sustancia P/farmacología

RESUMEN

The effects of systemic treatment with the AT1 receptor antagonist telmisartan on central effects of angiotensin II (Ang II), namely, increase in blood pressure, vasopressin release into the circulation, and drinking response, were investigated in conscious, normotensive rats. The central responses to i.c.v. Ang II (30 ng/kg) were measured at 0.5, 2, 4, and 24 h following acute i.v. or acute and chronic oral telmisartan application. At a dose of 10 mg/kg i.v., the drinking response to i.c.v. Ang II was completely blocked over 4 h, while the pressor response and the release of vasopressin in response to i.c.v. Ang II were blocked by 60 to 80%. The inhibition of the centrally mediated pressor and drinking response to Ang II was sustained over 24 h. The lower doses of telmisartan (0.3 and 1 mg/kg) significantly inhibited the Ang II-induced actions over 4 h. A consistent 24-h inhibition of the central responses to i.c.v. Ang II was obtained after acute and chronic oral treatment with 30 mg/kg telmisartan. Oral treatment with 1 and 3 mg/kg telmisartan produced a slight but inconsistent inhibition of the central actions of Ang II. Telmisartan concentrations measured in the cerebrospinal fluid following 8 days of consecutive daily oral treatment (1-30 mg/kg) ranged from 0.87 +/- 0.27 ng/ml (1 mg/kg/day) to 46.5 +/- 11.6 ng/ml (30 mg/kg/day). Our results demonstrate that, following peripheral administration, the AT1 receptor antagonist telmisartan can penetrate the blood-brain barrier in a dose- and time-dependent manner to inhibit centrally mediated effects of Ang II.

Asunto(s)

Angiotensina II/antagonistas & inhibidores , Antagonistas de Receptores de Angiotensina , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Bencimidazoles/administración & dosificación , Benzoatos/administración & dosificación , Barrera Hematoencefálica/efectos de los fármacos , Vasoconstrictores/antagonistas & inhibidores , Angiotensina II/farmacología , Animales , Arginina Vasopresina/sangre , Arginina Vasopresina/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Barrera Hematoencefálica/fisiología , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Líquidos/fisiología , Masculino , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/metabolismo , Telmisartán , Vasoconstrictores/farmacología

RESUMEN

ANG II has been implicated in neuroplastic processes via stimulation of inducible transcription factors (ITF) in the brain. In the present study, we investigated the effects of acute vs. repetitive once daily intracerebroventricular injections of ANG II for 7 days on the expression of ITF and constitutive transcription factor (CTF) and the AT1 receptor in the median preoptic area (MnPO), the subfornical organ (SFO), and the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). After repetitive injections, the expression of c-Fos declined by approximately 50% in MnPO, SFO, PVN, and SON compared with controls injected once. The desensitization of c-Fos occurred on the transcriptional level as shown in the SON by RT-PCR. Apart from a novel expression of c-Jun in the SON, the ITF c-Jun, JunB, JunD, and Krox-24 did not change after repetitive stimulation. Neither were the CTF, calcium response element binding protein, activating transcription factor 2, and serum response factor altered after repetitive vs. single injections of ANG II. The AT1 receptor was coexpressed with c-Fos/c-Jun. Immunohistochemical stainings suggest an increase in AT1-receptor number in MnPO, SFO, PVN, and SON on chronic stimulation compared with once-injected controls. These findings demonstrate that repetitive periventricular stimulation with ANG II essentially alters the expression of transcription factors compared with acute stimulation and suggest c-Fos and c-Jun as major intermediates of the AT1-receptor transcription.

Asunto(s)

Angiotensina II/farmacología , Encéfalo/fisiología , Proteínas de Drosophila , Genes fos/efectos de los fármacos , Receptores de Angiotensina/genética , Factor de Transcripción Activador 2 , Angiotensina II/administración & dosificación , Animales , Encéfalo/efectos de los fármacos , Ventrículos Cerebrales/efectos de los fármacos , Ventrículos Cerebrales/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Esquema de Medicación , Genes jun/efectos de los fármacos , Inyecciones Intraventriculares , Masculino , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Especificidad de Órganos , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/fisiología , Área Preóptica/efectos de los fármacos , Área Preóptica/fisiología , Proteínas Proto-Oncogénicas c-fos/análisis , Proteínas Proto-Oncogénicas c-fos/genética , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Respuesta Sérica , Órgano Subfornical/efectos de los fármacos , Órgano Subfornical/fisiología , Núcleo Supraóptico/efectos de los fármacos , Núcleo Supraóptico/fisiología , Factores de Tiempo , Factores de Transcripción/metabolismo

Asunto(s)

Encéfalo/fisiología , Sistema Renina-Angiotensina/fisiología , Humanos

RESUMEN

Recent pharmacological evidence has implicated substance P and neurokinin A, natural ligands for neurokinin-1 and neurokinin-2 receptors, respectively, as neurotransmitters in brain neuronal circuits activated upon noxious stimulation. The expression of the inducible transcription factor, c-Fos, was used to identify areas in the brain activated by a noxious stimulus (the subcutaneous injection of formalin), and to investigate the effects of intracerebroventricular administration of selective, nonpeptide antagonists for neurokinin-1 and neurokinin-2 tachykinin receptors on the neural activity in these areas and on the behavioural response to formalin-induced pain. Formalin (5%, 50 microl), injected subcutaneously through a chronically implanted catheter in the region of the lower hindlimb, increased c-Fos expression in a number of brain areas related to nociceptive transmission or the integration of stress responses. Grooming behaviour, licking and biting directed to the injected site, was the most frequent behavioural response. Intracerebroventricular pretreatment of rats with either RP 67580 (500 pmol), the active enantiomer of a neurokinin-1 receptor antagonist, or with SR 48968 (500 pmol), the active enantiomer of a neurokinin-2 receptor antagonist, reduced the formalin-induced c-Fos staining in the prefrontal cortex, dorsomedial and ventromedial nuclei of the hypothalamus, the locus coeruleus and the periaqueductal gray. The neurokinin-1, but not the neurokinin-2, receptor antagonist attenuated the formalin-induced activation of c-Fos in the paraventricular nucleus of the hypothalamus. Simultaneous intracerebroventricular pretreatment with both neurokinin-1 and neurokinin-2 receptor antagonists did not produce any additional inhibitory effect on the post-formalin c-Fos expression. None of the tachykinin receptor antagonists had an effect on the formalin-induced c-Fos expression in the septohypothalamic nucleus, medial thalamus, parabrachial nucleus and central amygdaloid nucleus, indicating that neurotransmitters other than neurokinins are most probably responsible for the activation of these areas in response to noxious stimulation. While both tachykinin receptor antagonists reduced the grooming behaviour to formalin, the neurokinin-1 receptor antagonist was clearly more effective than the neurokinin-2 receptor antagonist. Intracerebroventricular pretreatment of rats with the inactive enantiomers of the tachykinin receptor antagonists, RP 68651 and SR 48965, was without effect. Our results show that (i) the modified formalin test elicited an intense grooming behaviour and expression of c-Fos in numerous forebrain and brainstem areas, (ii) both tachykinin receptor antagonists were able to attenuate the behavioural response to pain and to reduce the formalin-induced c-Fos expression in some, but not all, brain areas, and (iii) the neurokinin-1 antagonist, RP 67580, was more effective in inhibiting the behavioural response to formalin and the pain-induced activation of c-Fos than the antagonist for neurokinin-2 receptors, SR 48968, indicating that neurokinin-1 receptors are preferentially activated in neurokinin-containing pathways responding to noxious stimuli. Our results demonstrate that blockade of brain tachykinin receptors, especially of the neurokinin-1 receptor, reduces the behavioural response to pain and the pain-induced c-Fos activation in distinct brain areas which are intimately linked with nociceptive neurotransmission and the initiation and integration of central stress responses. Together with the previous findings of the inhibition of hypertensive and tachycardic responses to pain, the present data indicate that tachykinin receptor antagonists can effectively inhibit the generation of an integrated cardiovascular and behavioural response pattern to noxious stimuli.

Asunto(s)

Encéfalo/metabolismo , Dolor/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Taquicininas/antagonistas & inhibidores , Analgésicos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Benzamidas/farmacología , Encéfalo/efectos de los fármacos , Formaldehído , Aseo Animal/efectos de los fármacos , Indoles/farmacología , Inyecciones Intraventriculares , Isoindoles , Masculino , Dolor/inducido químicamente , Dolor/psicología , Piperidinas/farmacología , Ratas , Ratas Wistar

RESUMEN

In the past several years, the progress in the development of molecular biology and the elucidation of gene sequences has created new approaches for studying biological functions and developing new diagnostic and therapeutic strategies. One of the most exciting advances has been the development of antisense technology, which represents a new strategy allowing modulation of protein synthesis with high specificity by preventing protein expression at the level of RNA or DNA. Many classical pharmacological approaches in neurobiological research are often based on the inhibition of biologically active proteins, such as receptors for neurotransmitters, or enzymes involved in neurotransmitter synthesis or degradation. The use of antisense oligodeoxynucleotides offers an alternative tool to manipulate selectively the expression of neurotransmitters or their receptors in neuronal tissue. This approach is especially useful when selective, high-affinity antagonists are not available. As a result, this technology has gained acceptance in the study of cell signalling mechanisms and the molecular basis of neuronal function. This paper provides a brief background to the antisense technique and explores methodological aspects, particularly in the whole animal. The use of the antisense technology in studies focused on central mechanisms regulating the cardiovascular system is then discussed.

Asunto(s)

Encéfalo/fisiología , Fenómenos Fisiológicos Cardiovasculares , Oligonucleótidos Antisentido , Animales , Encéfalo/efectos de los fármacos , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/farmacología , ARN Mensajero/metabolismo

RESUMEN

BACKGROUND AND PURPOSE: Angiotensin-converting enzyme inhibitors have been shown to protect against stroke in hypertensive rats and to improve neurological outcome after cerebral ischemia in normotensive rats. The present study was designated to test the hypothesis that blockade of brain AT(1) receptors improves the recovery from focal cerebral ischemia and reduces expression of AP-1 transcription factors c-Fos and c-Jun, which have been associated with programmed cell death and neurodegeneration. METHODS: Experiments were carried out in normotensive male Wistar rats. Focal cerebral ischemia was induced by middle cerebral artery occlusion lasting for 90 minutes and followed by reperfusion. The selective AT(1) receptor antagonist irbesartan was infused intracerebroventricularly over a 5-day period before the induction of ischemia at a dose that inhibited brain but not vascular AT(1) receptors. Twenty-four hours after ischemia, neurological outcome was evaluated and expression of c-Fos and c-Jun proteins in the brain was studied immunocytochemically. RESULTS: Focal brain ischemia resulted in a strong induction of c-Fos and c-Jun proteins in the cortex, which positively correlated with the degree of neurological deficits. Treatment of rats with irbesartan significantly improved neurological outcome of focal cerebral ischemia when compared with the vehicle-treated group and markedly reduced the expression of c-Fos and c-Jun proteins in the cortex on the ligated side of the brain. Irbesartan pretreatment completely abolished the ischemia-induced c-Fos expression in the hippocampus. CONCLUSIONS: The present study shows a relationship between c-Fos and c-Jun expression and neurological outcome after focal brain ischemia. Our data indicate that long-term blockade of central AT(1) receptors improves the recovery from brain ischemia and reduces the expression of c-Fos and c-Jun proteins in the brain. Pretreatment with an AT(1) receptor antagonist has beneficial effects after cerebral ischemia.

Asunto(s)

Antagonistas de Receptores de Angiotensina , Compuestos de Bifenilo/uso terapéutico , Encéfalo/fisiopatología , Ataque Isquémico Transitorio/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Tetrazoles/uso terapéutico , Factor de Transcripción AP-1/antagonistas & inhibidores , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Arteriopatías Oclusivas/fisiopatología , Compuestos de Bifenilo/administración & dosificación , Encéfalo/efectos de los fármacos , Catéteres de Permanencia , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inmunohistoquímica , Bombas de Infusión , Inyecciones Intraventriculares , Irbesartán , Ataque Isquémico Transitorio/fisiopatología , Masculino , Arteria Cerebral Media/fisiopatología , Degeneración Nerviosa/fisiopatología , Examen Neurológico , Vehículos Farmacéuticos , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Ratas , Ratas Wistar , Recuperación de la Función , Tetrazoles/administración & dosificación , Factor de Transcripción AP-1/genética

RESUMEN

Angiotensin AT1 receptor antagonists represent a novel class of cardiovascular drugs. In conscious, normotensive rats, irbesartan ((2-n-butyl-3-[(2'-(1H-tetrazol-5-yl)-biphenyl-4-yl) methyl]-1,3-diaza-spiro[4,4]non) and losartan ((2 n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl -4-yl) methyl] imidazol), two specific, high- affinity angiotensin AT1 receptor antagonists administered intravenously (i.v.) at doses of 0.3, 1, 3 and 10 mg/kg body weight, or orally (p.o.) at doses of 1, 3, 10 and 30 mg/kg body weight, antagonized the pressor responses to i.v. angiotensin II (50 ng/kg body weight) in a dose-related manner and with similar potency. In the following sets of experiments, we tested the hypothesis that these angiotensin AT1 receptor antagonists, when applied systemically, can inhibit the effects of angiotensin AT1 receptor stimulation in the brain. Irbesartan and losartan were administered i.v. or p.o. at doses of 3, 10, 30 and 100 mg/kg body weight. The responses to 100 ng angiotensin II injected into the lateral brain ventricle (i.c.v.), namely blood pressure increase, vasopressin release into the circulation and drinking, were recorded for up to 3 h. While both angiotensin AT1 receptor antagonists dose-dependently attenuated the pressor responses to central angiotensin AT1 receptor stimulation to a similar degree (maximal inhibition, irbesartan: 62% i.v., 39% p.o.; losartan: 62% i.v., 46% p.o.; respectively), irbesartan was more effective with respect to the inhibition of vasopressin release (76% i.v., 65% p.o.) and drinking (63% i.v., 79% p.o.) than losartan (58% i.v., 33% p.o and 22% i.v., 56% p.o., respectively). We conclude that systemically administered angiotensin AT1 receptor antagonists have access to central angiotensin receptors. The degree of central angiotensin AT1 receptor blockade following peripheral application may vary between different representatives of this class of drugs.

Asunto(s)

Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina , Compuestos de Bifenilo/farmacología , Presión Sanguínea/efectos de los fármacos , Losartán/farmacología , Tetrazoles/farmacología , Vasopresinas/sangre , Administración Oral , Animales , Antihipertensivos/farmacología , Encéfalo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ingestión de Líquidos/efectos de los fármacos , Interacciones Farmacológicas , Inyecciones Intravenosas , Inyecciones Intraventriculares , Irbesartán , Masculino , Neurohipófisis/efectos de los fármacos , Ratas , Ratas Wistar , Vasopresinas/metabolismo

RESUMEN

BACKGROUND: The renin-angiotensin system is a widely studied hormonal system that comprises substrate-enzyme interactions, the end result of which is the production of the active peptide angiotensin II. Because angiotensin II affects blood pressure control, sodium and water homeostasis, and cardiovascular function and structure, a great deal of research effort has been directed toward blocking the renin-angiotensin system. Angiotensin II also may be involved in end-organ damage in hypertension, heart failure, and vascular disease. ANGIOTENSIN II RECEPTORS: At least two subtypes of angiotensin II receptors have been identified, angiotensin type 1 (AT)1 and type 2 (AT2). The AT1 receptor mediates all the known actions of angiotensin II on blood pressure control. Additionally, research has indicated that the AT1 receptor modulates cardiac contractility and glomerular filtration, increases renal tubular sodium reabsorption, and cardiac and vascular hypertrophy. Less is known about the function of the AT2 receptor. Evidence suggests that the AT2 receptor inhibits cell proliferation and reverses the AT1-induced hypertrophy. Indeed, these receptors are thought to exert opposing effects. ANGIOTENSIN RECEPTOR ANTAGONISTS: This newly introduced class of drugs is able to inhibit the renin-angiotensin system at the receptor level by specifically blocking the AT1 receptor subtype. These drugs induce a dose-dependent blockade of angiotensin II effects, resulting in reduced blood pressure, urinary protein, and glomerular sclerosis. It is postulated that AT1 receptor antagonists may provide end-organ protection by blocking angiotensin II effects via the AT1 receptor, leaving the AT2 receptor unopposed. Consequently, these agents may reduce the morbidity and mortality that result from myocardial infarction and other conditions resulting from structural alterations in the heart, kidney, and vasculature.

Asunto(s)

Antagonistas de Receptores de Angiotensina , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Enfermedades Cardiovasculares/tratamiento farmacológico , Sistema Renina-Angiotensina/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Vasos Sanguíneos/inervación , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Corazón/inervación , Humanos , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Contracción Miocárdica/efectos de los fármacos , Función Ventricular Izquierda/efectos de los fármacos

RESUMEN

The effects of moxonidine on blood pressure, heart rate and sympathetic tone were studied in conscious spontaneously hypertensive rats. Intravenous moxonidine (80 nmol) transiently increased blood pressure without affecting heart rate or splanchnic nerve activity. Moxonidine (20-80 nmol) given into the fourth cerebral ventricle dose-dependently lowered mean arterial pressure, heart rate and sympathetic outflow (maximally by 60 +/- 3 mm Hg, 148 +/- 10 beats min(-1) and 15 +/- 3 microV). Moxonidine was more effective by this route than after the injection into the lateral ventricle. Clonidine (20-80 nmol) produced an initial pressor response after both intracerebroventricular routes of administration. A decrease in blood pressure was observed only when clonidine was given into the fourth ventricle. Clonidine decreased heart rate and splanchnic nerve activity similarly like moxonidine when the substances were given into the fourth ventricle. The data imply that the hypotensive effect of moxonidine is related to central sympathoinhibition. The main site of this action appears to be in the brainstem region.

Asunto(s)

Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Hipertensión/fisiopatología , Imidazoles/farmacología , Sistema Nervioso Simpático/efectos de los fármacos , Animales , Catecolaminas/sangre , Clonidina/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Nervios Esplácnicos/efectos de los fármacos , Factores de Tiempo

RESUMEN

In the present study, we investigated the expression pattern of the inducible transcription factors (ITF) c-Fos, c-Jun, JunB, JunD, and Krox-24 following intracerebroventricular injections of hyperosmolar saline (0.2, 0.3, and 0.6 M NaCl) and its mediation via angiotensin and/or muscarinic receptors. c-Fos, c-Jun, and Krox-24 were differentially expressed in organum vasculosum laminae terminalis, median preoptic area, subfornical organ (SFO), and paraventricular and supraoptic nuclei. Expression of c-Fos and c-Jun was inhibited by pretreatment with the angiotensin AT1 receptor antagonist losartan (10 and 20 nmol icv) following 0.20 and 0.30 M saline. Pretreatment with atropine (15 nmol icv) inhibited the 0.30 and 0.60 M NaCl-induced expression of c-Fos, c-Jun, and Krox-24 in all areas except the SFO. Coexpression of the ITF with vasopressin and oxytocin, the major effector peptides in osmoregulation, was demonstrated, implying the corresponding genes as putative target genes of the ITF. The results show a highly differentiated ITF expression pattern in the brain mediated by angiotensinergic and muscarinergic pathways, suggesting a finely tuned regulation of target genes.

Asunto(s)

Ventrículos Cerebrales/fisiología , Proteínas de Unión al ADN/biosíntesis , Hipotálamo/metabolismo , Proteínas Inmediatas-Precoces , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Receptores de Angiotensina/fisiología , Receptores Muscarínicos/fisiología , Solución Salina Hipertónica/farmacología , Factores de Transcripción/biosíntesis , Animales , Atropina/farmacología , Proteína 1 de la Respuesta de Crecimiento Precoz , Regulación de la Expresión Génica/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Inyecciones Intraventriculares , Losartán/administración & dosificación , Losartán/farmacología , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Especificidad de Órganos , Oxitocina/biosíntesis , Núcleo Hipotalámico Paraventricular/metabolismo , Área Preóptica/metabolismo , Ratas , Ratas Wistar , Solución Salina Hipertónica/administración & dosificación , Núcleo Supraóptico/metabolismo , Vasopresinas/biosíntesis , Dedos de Zinc

RESUMEN

A substantial increase in [125I]Sar1, Ile-Angiotensin II binding activity can be observed 24 hours after treatment of R3T3 cells with AT2 receptor agonists and antagonists. An increase in the radioligand binding activity, although less profound, can also be observed 6 hours after AT2 receptor ligand treatment, on fetal human kidney cells expressing a recombinant human AT2 receptor. However, the increase in radioligand binding activity cannot be detected unless the ligands are removed from the cell surface by an acid-glycine (pH 3) wash, just prior to the binding assay. Interestingly, an acid-glycine wash 24 hours prior to the binding assay causes a dramatic decrease in the radioligand binding activity on untreated R3T3 cells. This decrease, which was prevented by angiotensin II treatment, suggests the existence of an unknown endogenous factor which, like the AT2 receptor ligands, seems to prevent AT2 receptor degradation.

Asunto(s)

Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Células 3T3 , Ácidos , Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Animales , Línea Celular , Glicina , Humanos , Ligandos , Ratones , Ensayo de Unión Radioligante , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/agonistas , Proteínas Recombinantes/metabolismo