Daniel K. Yee

Oxidative stress and HNE conjugation of GLUT3 are increased in the hippocampus of diabetic rats subjected to stress

Recent studies demonstrate that cellular, molecular and morphological changes induced by stress in rats are accelerated when there is a pre-existing strain upon their already compromised adaptive responses to internal or external stimuli, such as may occur with uncontrolled diabetes mellitus. The deleterious actions of diabetes and stress may increase oxidative stress in the brain, leading to increases in neuronal vulnerability. In an attempt to determine if stress, diabetes or stress+diabetes increases oxidative stress in the hippocampus, radioimmunocytochemistry was performed using polyclonal antisera that recognize proteins conjugated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). Radioimmunocytochemistry revealed that HNE protein conjugation is increased in all subregions of the hippocampus of streptozotocin (STZ) diabetic rats, rats subjected to restraint stress and STZ diabetic rats subjected to stress. Such increases were not significant in the cortex. Because increases in oxidative stress may contribute to stress- and diabetes-mediated decreases in hippocampal neuronal glucose utilization, we examined the stress/diabetes mediated HNE protein conjugation of the neuron specific glucose transporter, GLUT3. GLUT3 immunoprecipitated from hippocampal membranes of diabetic rats subjected to stress exhibited significant increases in HNE immunolabeling compared to control rats, suggesting that HNE protein conjugation of GLUT3 contributes to decreases in neuronal glucose utilization observed during diabetes and exposure to stress. Collectively, these results demonstrate that the hippocampus is vulnerable to increases in oxidative stress produced by diabetes and stress. In addition, increases in HNE protein conjugation of GLUT3 provide a potential mechanism for stress- and diabetes-mediated decreases in hippocampal neuronal glucose utilization.

Melanocortin receptor signaling through mitogen-activated protein kinase in vitro and in rat hypothalamus.

The central melanocortin system has emerged as a potential regulator of food intake. This action of melanocortins appears to occur through intrahypothalamic, melanocortin-containing projections, including those from the arcuate to the paraventricular nucleus (PVN). Although the complexity of feeding behavior and the long duration of the effects of melanocortins on food intake suggest changes in gene expression, the mechanism by which such changes occur has been elusive. In the present report, we describe experiments using in vitro and in vivo approaches to demonstrate melanocortin-induced phosphorylation (activation) of members of the mitogen-activated protein kinase (MAPK) family of transcription factors. First, application of the melanocortin agonist MTII to COS-1 cells resulted in an increase in phosphorylated MAPK after the cells were transfected with the melanocortin type 4 receptor (MC4-R), but not the type 3 receptor. Formation of cAMP, however, was observed when either receptor subtype was transfected. Subsequent experiments revealed that the effect of MTII on MAPK activation in MC4-R-transfected cells was dose-dependent and was maximal after 10 min of MTII exposure. Second, central injections of MTII increased the number of phospho-MAPK-immunoreactive cells in the rat PVN compared to vehicle-injected animals. When coupled with immunohistochemical identification of PVN neurons containing oxytocin, a clear segregation was apparent, allowing for a precise anatomical description of the pattern of activated MAPK within the PVN. These data are the first to suggest a differential coupling of MC4-R and may describe a mechanism through which the long-term and persistent behavioral actions of melanocortins are mediated.

Immunohistochemical mapping of angiotensin type 2 (AT2) receptors in rat brain.

Recently developed antisera selective for angiotensin Type 2 (AT2) receptors were used to localize AT2 receptors in rat brain by immunohistochemistry. While the results from these experiments were largely consistent with previous autoradiographic and radioligand binding analyses of AT2 receptor populations in brain, there were also some notable differences in the distribution of immunoreactivity. More specifically, in agreement with previous studies, AT2 antisera detected apparent receptor populations in the locus coeruleus and the bed nucleus of the accessory olfactory tract, whereas AT2 receptor-immunoreactivity in the cerebellum was primarily associated with the Purkinje cell layer and the deep cerebellar nuclei rather than the molecular layer as has been previously reported in autoradiographic studies. Other regions with prominent immune-staining included all subfields of the hippocampus, which had been previously reported to contain exclusively AT1 receptors. Limbic structures such as the amygdala, thalamic areas such as the rhomboid thalamic nucleus, the paraventricular thalamic nucleus, hypothalamic areas such as the paraventricular hypothalamic nucleus, and the supraoptic nucleus also exhibited prominent AT2-immunoreactivity. In the paraventricular hypothalamic nucleus, AT2 receptor staining appeared to be associated primarily with the magnocellular neurons. In all regions examined, AT2 receptor immunoreactivity was associated with the cytoplasm and cell membrane and was not localized within the nucleus. Collectively, these results confirm and extend the neuroanatomical resolution of previous autoradiographic studies as well as identify new AT2 receptor populations in rat brain.

Angiotensin II receptor signalling

Angiotensin II plays a key role in the regulation of body fluid homeostasis. To correct body fluid deficits that occur during hypovolaemia, an animal needs to ingest both water and electrolytes. Thus, it is not surprising that angiotensin II, which is synthesized in response to hypovolaemia, acts centrally to increase both water and NaCl intake. Here, we review findings relating to the properties of angiotensin II receptors that give rise to changes in behaviour. Data are described to suggest that divergent signal transduction pathways are responsible for separable behavioural responses to angiotensin II, and a hypothesis is proposed to explain how this divergence may map onto neural circuits in the brain.

Structural determinants for the activation mechanism of the angiotensin II type 1 receptor differ for phosphoinositide hydrolysis and mitogen-activated protein kinase pathways

While the mechanism whereby the angiotensin II type 1 receptor (AT(1) receptor) activates its classical effector phospholipase C-beta (PLC-beta) has largely been elucidated, there is little consensus on how this receptor activates a more recently identified effector, the p42/44 mitogen-activated protein kinases (p42/44(MAPK)). Using transfected COS-1 cells, we investigated the activation of this signaling pathway at the receptor level itself. Previous mutational studies that relied on phosphoinositide turnover as an index of receptor activation have indicated that key residues in the second and seventh transmembrane domains participate in AT(1) receptor activation mechanisms. Thus, we introduced a variety of mutations-AT(1)[D74N], AT(1)[Y292F], AT(1)[N295S], and AT(1)[AT(2) TM7], which is composed of a chimeric substitution of the AT(1) seventh transmembrane domain with its AT(2) counterpart. These mutations that strongly diminished the receptors ability to activate PLC-beta had little to no effect on its ability to activate p42/44(MAPK), which not only suggests that p42/44(MAPK) does not exclusively lie downstream of the G-protein G(q)/PLC-beta pathway but also indicates that more than one activation state may exist for the AT(1) receptor. The failure of a protein kinase C inhibitor to block AT(1) receptor activation of p42/44(MAPK) further corroborated evidence that the receptors activation of p42/44(MAPK) is largely independent of the G(q)/PLC-beta/PKC pathway. Taken together, the experimental evidence strongly suggests that the mechanism whereby the AT(1) receptor activates p42/44(MAPK) is fundamentally different from that for PLC-beta, even at the level of the receptor itself.

Biochemical Characterization of Two Distinct Angiotensin AT2 Receptor Populations in Murine Neuroblastoma N1E-115 Cells

Abstract: The murine neuroblastoma N1E‐115 cell line possesses a high density of angiotensin II (Angll) receptors that can be solubilized with the zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate. These solubilized binding sites exhibited high affinity for CGP‐42112A and not Losartan, indicating that they were of the AT2 subtype. However, displacement of 125I‐Angll with the AT2 nonpeptide antagonist PD‐123319 resulted in a biphasic curve, suggesting heterogeneity of the AT2 receptor population in N1E‐115 cells. In support of this view, separation of two receptor populations was accomplished with heparin‐Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin‐Sepharose column, two of which bound 125I‐Angll with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented ∼80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCI for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT2 receptors insofar as they exhibited high affinity for CGP‐42112A and little or no affinity for the AT1‐selective antagonist Losartan. However, whereas the nonpeptidic AT2‐selective antagonist PD‐123319 completely displaced the binding of 126I‐Angll from peak I in a monophasic fashion (IC50= 9.1 ± 4.1 nM; mean ± SEM; n = 3), PD‐123319 was much less effective in displacing 125I‐Angll from peak III (IC50= 196 β 27 nM; mean β SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in 125I‐Angll specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTPγS significantly reduced high‐affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT2 receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated 125I‐Angll binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin‐Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT2 receptors.

Mutational analysis of the angiotensin II type 2 receptor: contribution of conserved extracellular amino acids

While much work has been done examining the ligand-binding characteristics of the AT1 receptor, very little attention has been focused on the AT2 receptor. Both receptors bind angiotensin II (AngII) with identical affinities, but share only 34% homology. Although it is tempting to assume that conserved residues between the two subtypes are responsible for the binding of AngII, there is little data to support this view. To determine the commonalities in ligand binding of the AT1 and AT2 receptors, we have chosen several conserved extracellular amino acids which have been shown to be important in AngII binding [1,2] to the AT1 receptor for mutational studies of the AT2 receptor. Specifically, we have mutated tyrosine108 in extracellular loop 1 (ECL1), arginine182 in ECL2, and aspartate297 in ECL3 of the AT2 receptor in order to determine their contribution to AngII binding. In the AT2 receptor, mutation of tyrosine108 to an alanine resulted in a receptor with wild-type binding for AngII, while mutation of either arginine182 or aspartate297 drastically impaired AngII binding ( > 100 nM). These results demonstrate both similarities as well as clear differences between receptor subtypes in the contributions to AngII binding of several conserved extracellular amino acid residues.

Endogenous angiotensin II-induced p44/42 mitogen-activated protein kinase activation mediates sodium appetite but not thirst or neurohypophysial secretion in male rats.

The renin–angiotensin–aldosterone system makes a critical contribution to body fluid homeostasis, and abnormalities in this endocrine system have been implicated in certain forms of hypertension. The peptide hormone angiotensin II (AngII) regulates hydromineral homeostasis and blood pressure by acting on both peripheral and brain targets. In the brain, AngII binds to the angiotensin type 1 receptor (AT1R) to stimulate thirst, sodium appetite and both arginine vasopressin (AVP) and oxytocin (OT) secretion. The present study used an experimental model of endogenous AngII to examine the role of p44/42 mitogen‐activated protein kinase (MAPK) as a signalling mechanism to mediate these responses. Animals were given a combined treatment of furosemide and a low dose of captopril (furo/cap), comprising a diuretic and an angiotensin‐converting enzyme inhibitor, respectively, to elevate endogenous AngII levels in the brain. Furo/cap induced p44/42 MAPK activation in key brain areas that express AT1R, and this effect was reduced with either a centrally administered AT1R antagonist (irbesartan) or a p44/42 MAPK inhibitor (U0126). Additionally, furo/cap treatment elicited water and sodium intake, and irbesartan markedly reduced both of these behaviours. Central injection of U0126 markedly attenuated furo/cap‐induced sodium intake but not water intake. Furthermore, p44/42 MAPK signalling was not necessary for either furo/cap‐ or exogenous AngII‐induced AVP or OT release. Taken together, these results indicate that p44/42 MAPK is required for AngII‐induced sodium appetite but not thirst or neurohypophysial secretion. This result may allow for the discovery of more specific downstream targets of p44/42 MAPK to curb sodium appetite, known to exacerbate hypertension, at the same time as leaving thirst and neurohypophysial hormone secretion undisturbed.

Role of the amino terminus in ligand binding for the angiotensin II type 2 receptor.

Key amino terminal residues in type 1 (AT1) angiotensin II (AngII) receptors are not conserved within type 2 (AT2) receptors. We therefore characterized amino terminal mutants that are transiently expressed in COS-3 membranes. AT2 amino terminal deletion drastically reduced affinity for AngII, suggesting its importance for this subtype. AT1-AT2 amino terminal exchanges retained wild type AngII affinities (Kd ranging from 3-5 nM), indicating compensation despite substantial sequence dissimilarities. Finally, binding of AT2 selective ligands (CGP42112A and PD123319) was not dependent on the amino terminus.

Cloning and expression of angiotensin II type 2 (AT2) receptors from murine neuroblastoma N1E-115 cells: evidence for AT2 receptor heterogeneity

Homology-based PCR was used to isolate angiotensin II type 2 (AT2) receptor cDNA from murine neuroblastoma N1E-115 cells. Despite subtle differences in the nucleotide sequence (the N1E-115 clone coded for Phe133 as TTC and Gln326 as CAG; base substitutions are in bold-italics), the AT2 receptor protein was identical to other reported murine AT2 clones. When transfected into COS-1 cells, the expressed AT2 receptor displayed high affinity for AngII and for AT2-selective compounds, GTP gamma S-insensitive agonist binding and enhanced agonist binding by dithiothreitol. Previously, we have demonstrated that N1E-115 cells possess two distinct subpopulations of AT2 receptors, defined as peak I and peak III receptors, that can be separated by heparin-sepharose chromatography. The two subpopulations differ pharmacologically, biochemically and immunologically. The binding properties of the cloned AT2 receptor closely resembled that of peak III receptors. Moreover, antisera raised against peak I AT2 receptors failed to immunoreact to either peak III receptors or cloned AT2 receptors expressed in COS-1 cells. Collectively, these data suggest that the cloned AT2 receptor is identical to peak III receptors from N1E-115 cells and that a novel AT2 receptor (peak I) remains to be cloned.