Comparative changes in the blood-brain barrier and cerebral infarction of SHR and WKY rats.

Hypertension is involved in the exacerbation of stroke. It is unclear how blood-brain barrier (BBB) tight-junction (TJ) and ion transporter proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. In the present study, we investigated cerebral infarct volume following permanent 4-h middle cerebral artery occlusion (MCAO) and characterized the expression of BBB TJ and ion transporter proteins in brain microvessels of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats at 5 wk (prehypertension), 10 wk (early-stage hypertension), and 15 wk (later-stage hypertension) of age. Hypertensive SHR show increased infarct volume following MCAO compared with WKY control rats. BBB TJ and ion transporter proteins, known to contribute to edema and fluid volume changes in the brain, show differential protein expression patterns during hypertension development. Western blot analysis of TJ protein zonula occludens-2 (ZO-2) showed decreased expression, while ion transporter, Na(+)/H(+) exchanger 1 (NHE-1), was markedly increased in hypertensive SHR. Expression of TJ proteins ZO-1, occludin, actin, claudin-5, and Na(+)-K(+)-2Cl(-) cotransporter remain unaffected in SHR compared with control. Selective inhibition of NHE-1 using dimethylamiloride significantly attenuated ischemia-induced infarct volume in hypertensive SHR following MCAO, suggesting a novel role for NHE-1 in the brain in the regulation of ischemia-induced infarct volume in SHR.

Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation.

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-betaII, PKC-gamma, PKC-eta, PKC-mu, and PKC-lambda also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-epsilon and PKC-zeta were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 microM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-gamma and PKC-theta in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.

Angiotensin II induction of AP-1 in neurons requires stimulation of PI3-K and JNK.

Angiotensin II (Ang II) acts via its type 1 (AT(1)) receptor in neurons to regulate the activity of multiple intracellular signaling molecules, including intracellular Ca(2+), protein kinase C, phosphatidylinositol 3-kinase (PI3-K), and c-Jun NH(2)-terminal kinase (JNK). The present studies investigated the upstream signaling molecules involved in the Ang II stimulation of activator protein-1 (AP-1) DNA binding in neurons. Treatment of neurons cultured from neonatal rat hypothalamus and brainstem with Ang II (100 nM) showed a time-dependent increase in AP-1 DNA binding and this effect was inhibited by the AT(1) receptor antagonist, losartan (1 microM), the PI3-K inhibitor, LY294002 (10 microM), and the JNK inhibitor, JNK inhibitor II (100 nM). Furthermore, Ang II (100 nM) causes a time-dependent increase in JNK activity which was attenuated by PI3-K inhibition. These data establish, for the first time, a signaling cascade involved in the Ang II activation of AP-1 DNA binding in neurons.

Drinking behavior elicited by central injection of angiotensin II: roles for protein kinase C and Ca2+/calmodulin-dependent protein kinase II.

Prior studies utilizing neurons cultured from the hypothalamus and brain stem of newborn rats have demonstrated that ANG II-induced modulation of neuronal firing involves activation of both protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). The present studies were performed to determine whether these signaling molecules are also involved in physiological responses elicited by ANG II in the brain in vivo. Central injection of ANG II (10 ng/2 microl) into the lateral cerebroventricle (icv) of Sprague-Dawley rats increased water intake in a time-dependent manner. This ANG II-mediated dipsogenic response was attenuated by central injection of the PKC inhibitors chelerythrine chloride (0.5-50 microM, 2 microl) and Go-6976 (2.3 nM, 2 microl) and by the CaMKII inhibitor KN-93 (10 microM, 2 microl). Conversely, icv injection of chelerythrine chloride (50 microM, 2 microl) and KN-93 (10 microM, 2 microl) had no effect on the dipsogenic response elicited by central injection of carbachol (200 ng/2 microl). Furthermore, injection of ANG II (10 ng/2 microl) icv increases the activity of both PKC-alpha and CaMKII in rat septum and hypothalamus. These data suggest that signaling molecules involved in ANG II-induced responses in vitro are also relevant in physiological responses elicited by ANG II in the whole animal model.

Cytokine-stimulated inducible nitric oxide synthase expression in astroglia: role of Erk mitogen-activated protein kinase and NF-kappaB.

Expression of inducible nitric oxide synthase (iNOS), which leads to the production of nitric oxide (NO), is stimulated by proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Here we report on the roles of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein (MAP) kinases in IL-1beta/TNF-alpha-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-kappaB 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-1beta/TNF-alpha on nitrite accumulation. In accordance with these findings, IL-1beta- and TNF-alpha-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-1beta and TNF-alpha induced degradation of NF-kappaB's bound inhibitory protein, IkappaB-alpha, leading to translocation of NF-kappaB into the nucleus. IkappaB-alpha expression was not restored to control levels by inhibition of MEK. Furthermore, inhibition of MEK with PD098,059 did not alter IL-1beta- and TNF-alpha-induced expression of active NF-kappaB. The results demonstrate that autonomous Erk and NF-kappaB pathways mediate cytokine-induced increases in iNOS expression in astroglia.

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. 2. The AT1 receptors couple to a wide variety of signalling pathways in peripheral tissues, such as kidney, heart and vascular smooth muscle. A similar diversity of signalling mechanisms exists for AT1 receptors in neurons. 3. We outline the known neuronal AT1 receptor signalling pathways as they relate to function. Pathways that couple activation of AT1 receptors to short-term changes in neuronal membrane ionic currents and firing rate will be reviewed. These are different from the pathways that elicit longer-term changes in enzyme activity and gene expression and, ultimately, increases in noradrenaline synthesis. 4. Novel AT1 receptor signalling pathways discovered through gene expression profiling and their potential functional significance have been discussed.