Colin Sumners

Angiotensin II in central nervous system physiology.

In summary, the prevailing concept is that brain Ang II increases blood pressure by activating AT1 receptors, and that these have a neuromodulating effect to increase the activity of autonomic nervous system. Pathways for Ang II stimulating thirst and blood pressure, increased vasopressin release and sympathetic activation have been outlined. Brain RAS synthesis, while incompletely understood, is active in the absence of a peripheral RAS. Angiotensin elicits specific receptor mediated signals in neurons, particularly in the hypothalamus and brainstem. These actions are due to neuronal membrane ionic currents and the regulation of transcription factors. The areas to be explored further are characterization and functional roles of the other AT receptor subtypes, such as AT4, AT(1-7) and nuclear AT-R. Their interactions with other peptides and transmitters, and their signaling pathways need to be investigated. The story that began 100 years ago with renin is certainly not ended and will continue to unfold as further investigations with new techniques progress.

Brain Microglial Cytokines in Neurogenic Hypertension

Accumulating evidence indicates a key role of inflammation in hypertension and cardiovascular disorders. However, the role of inflammatory processes in neurogenic hypertension remains to be determined. Thus, our objective in the present study was to test the hypothesis that activation of microglial cells and the generation of proinflammatory cytokines in the paraventricular nucleus (PVN) contribute to neurogenic hypertension. Intracerebroventricular infusion of minocycline, an anti-inflammatory antibiotic, caused a significant attenuation of mean arterial pressure, cardiac hypertrophy, and plasma norepinephrine induced by chronic angiotensin II infusion. This was associated with decreases in the numbers of activated microglia and mRNAs for interleukin (IL) 1&bgr;, IL-6, and tumor necrosis factor-&agr;, and an increase in the mRNA for IL-10 in the PVN. Overexpression of IL-10 induced by recombinant adenoassociated virus-mediated gene transfer in the PVN mimicked the antihypertensive effects of minocycline. Furthermore, acute application of a proinflammatory cytokine, IL-1&bgr;, into the left ventricle or the PVN in normal rats resulted in a significant increase in mean arterial pressure. Collectively, this indicates that angiotensin II induced hypertension involves activation of microglia and increases in proinflammatory cytokines in the PVN. These data have significant implications on the development of innovative therapeutic strategies for the control of neurogenic hypertension.

Chronic ethanol exposure potentiates NMDA excitotoxicity in cerebral cortical neurons

Abstract: The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined. Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA. Addition of NMDA (25–100 μM) to the culture dishes for 25 min in Mg2+‐free buffer resulted in a dose‐dependent increase in LDH accumulation. Phase‐contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes. Chronic exposure of neuronal cultures to ethanol (100 mM) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA‐stimulated LDH release by 36 and 22% in response to 25 μM and 50 μM NMDA, respectively. Neither basal LDH release nor that in response to maximal NMDA (100 μM) stimulation was altered by chronic alcohol exposure. In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 mM) only during the NMDA exposure period significantly reduced LDH release by ∼ 50% in both control and chronically treated dishes. This reduction by acute ethanol was also observed under phase‐contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes. These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.

Therapeutic Implications of the Vasoprotective Axis of the Renin-Angiotensin System in Cardiovascular Diseases

The recent discovery of angiotensin-converting enzyme 2 (ACE2) and the Mas receptor has resulted in the recognition of a counterregulatory, ACE2/Ang-(1-7)/Mas, axis within the renin-angiotensin system (RAS). Any disturbance in the balance between this and the ACE/AngII/AT1 receptor axis is suggested to lead to the development and progression of cardiovascular disease (CVD). Therefore, activation of the ACE2/Ang-(1-7)/Mas axis has been an obvious target for CVD therapeutics. In this review, we will focus on the current status of the RAS, highlight evidence for the existence of the ACE2/Ang-(1-7)/Mas axis, and discuss, the role of this axis in the pathophysiology of the cardiovascular, renal, pulmonary and central nervous systems and its potential for future CVD therapeutics.

Cytokine-stimulated inducible nitric oxide synthase expression in astroglia: Role of Erk mitogen-activated protein kinase and NF-κB

Expression of inducible nitric oxide synthase (iNOS), which leads to the production of nitric oxide (NO), is stimulated by proinflammatory cytokines such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α). Here we report on the roles of nuclear factor‐κB (NF‐κB) and mitogen‐activated protein (MAP) kinases in IL‐1β/TNF‐α–induced iNOS expression in adult rat astroglia. Cytokine‐induced increases in nitrite accumulation (an index of NO production) and iNOS expression were attenuated by inhibition of NF‐κB with pyrrolidine dithiocarbamate (PDTC). Similar attenuation of these cytokine‐induced responses was produced by inhibition of MAP kinase (MEK), the immediate upstream activator of Erk, using PD098,059. Combined treatment of astroglia with PDTC and PD098,059 completely abolished the cytokine‐induced increases in iNOS expression and nitrite accumulation. By contrast, the selective p38 kinase inhibitor SB203,580 amplified the effects of IL‐1β/TNF‐α on nitrite accumulation. In accordance with these findings, IL‐1β– and TNF‐α–induced a time‐dependent increase in Erk1/Erk2 activation. This cytokine action was completely abolished by PD098,059 but was not altered by PDTC. Finally, IL‐1β and TNF‐α induced degradation of NF‐κBs bound inhibitory protein, IκB‐α, leading to translocation of NF‐κB into the nucleus. IκB‐α expression was not restored to control levels by inhibition of MEK. Furthermore, inhibition of MEK with PD098,059 did not alter IL‐1β– and TNF‐α–induced expression of active NF‐κB. The results demonstrate that autonomous Erk and NF‐κB pathways mediate cytokine‐induced increases in iNOS expression in astroglia. GLIA 41:152–160, 2003.

NAD(P)H Oxidase Inhibition Attenuates Neuronal Chronotropic Actions of Angiotensin II

It is well established that the central cardiovascular effects of angiotensin II (Ang II) involve superoxide production. However, the intracellular mechanism by which reactive oxygen species (ROS) signaling regulates neuronal Ang II actions remains to be elucidated. In the present study, we have used neuronal cells in primary cultures from the hypothalamus and brain stem areas to study the role of ROS on the cellular actions of Ang II. Ang II increases neuronal firing rate, an effect mediated by the AT1 receptor subtype and involving inhibition of the delayed rectifier potassium current (IKv). This increase in neuronal activity was associated with increases in NADPH oxidase activity and ROS levels within neurons, the latter evidenced by an increase in ethidium fluorescence. The increases in NADPH oxidase activity and ethidium fluorescence were blocked by either the AT1 receptor antagonist losartan or by the selective NAD(P)H oxidase inhibitor gp91ds-tat. Extracellular application of the ROS scavenger, Tempol, attenuated the Ang II–induced increase in neuronal firing rate by 70%. In addition, gp91ds-tat treatment resulted in a 50% inhibition of Ang II–induced increase in firing rate. In contrast, the ROS generator Xanthine-Xanthine oxidase significantly increased neuronal firing rate. Finally, Ang II inhibited neuronal IKv, and this inhibition was abolished by gp91ds-tat treatment. These observations demonstrate, for the first time, that Ang II regulates neuronal activity via a series of events that includes ROS generation and inhibition of IKv. This signaling seems to be a critical cellular event in central Ang II regulation of cardiovascular function.

Mineralocorticoids modulate central angiotensin II receptors in rats

The effect of chronic administration of deoxycorticosterone acetate (DOCA) on the regulation of angiotensin II (AII) receptors in the brains of adult rats was compared with their drinking and pressor responsiveness to both peripheral and central administration of AII. Analysis of AII receptor binding in a block of tissue containing the hypothalamus, thalamus and septum (HTS) after treatment for 8 weeks with DOCA-salt (240 micrograms/kg/day) revealed a significant increase in the number of AII-binding sites compared to salt-loaded controls (Bmax 9.65 vs 6.80 fmol/mg protein) and no change in binding affinity (Kd). Significant increases in the drinking responses to peripheral (200 micrograms/kg) and central (10 ng) administration of AII were observed in these rats. Additional studies indicated that the pressor responses to either centrally (25 ng) or peripherally (20 micrograms/kg, s.c.) administered AII were augmented in DOCA-treated rats. The effect of mineralocorticoids on AII-binding sites was also investigated in primary neuronal cultures from the brains of one-day-old rats. Pretreatment of these cultures with either DOCA or aldosterone (ALDO) induced a time- and concentration-dependent increase in the specific binding of [125I]AII. Maximal increases in AII binding of 53 and 62% above control values were observed when cultures were treated with 500 pg of either ALDO or DOCA per milliliter of culture medium. Scatchard analysis of specific binding of [125I]AII in neuronal cultures treated with DOCA revealed a significant increase in Bmax but no change in Kd. Thus, mineralocorticoid hormones induce an increase in the number of AII-receptor binding sites in the HTS of rats which parallels physiological responses to both central and peripheral administration of AII. This relationship may be independent of the concentration of AII in the blood, since an increase in the number of AII binding sites was also observed in neurons cultured from the brains of one-day-old rats which had been treated with mineralocorticoid hormones.

Cerebroprotection by angiotensin‐(1–7) in endothelin‐1‐induced ischaemic stroke

Activation of angiotensin‐converting enzyme 2 (ACE2), production of angiotensin‐(1–7) [Ang‐(1–7)] and stimulation of the Ang‐(1–7) receptor Mas exert beneficial actions in various peripheral cardiovascular diseases, largely through opposition of the deleterious effects of angiotensin II via its type 1 receptor. Here we considered the possibility that Ang‐(1–7) may exert beneficial effects against CNS damage and neurological deficits produced by cerebral ischaemic stroke. We determined the effects of central administration of Ang‐(1–7) or pharmacological activation of ACE2 on the cerebral damage and behavioural deficits elicited by endothelin‐1 (ET‐1)‐induced middle cerebral artery occlusion (MCAO), a model of cerebral ischaemia. The results of the present study demonstrated that intracerebroventricular infusion of either Ang‐(1–7) or an ACE2 activator, diminazine aceturate (DIZE), prior to and following ET‐1‐induced MCAO significantly attenuated the cerebral infarct size and neurological deficits measured 72 h after the insult. These beneficial actions of Ang‐(1–7) and DIZE were reversed by co‐intracerebroventricular administration of the Mas receptor inhibitor, A‐779. Neither the Ang‐(1–7) nor the DIZE treatments altered the reduction in cerebral blood flow elicited by ET‐1. Lastly, intracerebroventricular administration of Ang‐(1–7) significantly reduced the increase in inducible nitric oxide synthase mRNA expression within the cerebral infarct that occurs following ET‐1‐induced MCAO. This is the first demonstration of cerebroprotective properties of the ACE2–Ang‐(1–7)–Mas axis during ischaemic stroke, and suggests that the mechanism of the Ang‐(1–7) protective action includes blunting of inducible nitric oxide synthase expression.

Angiotensin At1 Receptor Signalling Pathways In Neurons

1. The aim of the present article is to review the intracellular signal transduction pathways that are influenced by the peptide angiotensin (Ang) II, acting via its type 1 (AT1) receptor, in neurons.

Changes in skin angiotensin II receptors in rats during wound healing

Angiotensin (AII) is associated with increased vascular smooth muscle growth and we have found increased levels of tissue AII during healing of wounded skin. Here we have determined changes in skin AII receptors during wound healing in adult male Sprague-Dawley rats. An abdominal surgical incision was made under anesthesia and rats were sacrificed at different times after wounding. Specific binding of 125I-AII was significantly decreased at 12, 18 and 24 hours in the wounded tissue compared to control tissue from the same rat. By 3 days the binding had recovered to baseline levels. Receptors were mostly AT1, with a high and a low affinity site in the skin both in control and healing tissue. The Bmax of the high affinity site was significantly decreased in healing tissue but there was no significant change in Kd. Our results demonstrate that adult rat skin contains predominantly AT1 receptors and also that these receptors are downregulated for 12-24 hours after wounding.