Divergent behavioral roles of angiotensin receptor intracellular signaling cascades

Central injections of angiotensin II (AngII) increase both water and NaCl intake. These effects of AngII occur largely through stimulation of the AngII type 1 (AT(1)) receptor. Stimulation of the AT(1) receptor leads to a number of intracellular events, including phospholipase C (PLC) activation and the subsequent formation of diacylglycerol and inositol trisphosphate (IP(3)), which then activate protein kinase C (PKC) and increase intracellular calcium, respectively. In addition, AT(1) receptor stimulation leads to the activation of MAPK family members. Recent experiments using mutated AT(1) receptor constructs or the AngII analog Sar(1),Ile(4),Ile(8)-AngII (SII) revealed that MAPK activation can occur independent of PLC/PKC/IP(3) activation. The present experiments used in vitro and in vivo approaches to clarify the cellular and behavioral responses to SII. Specifically, SII mimicked AngII stimulation of MAPK in AT(1) receptor-transfected COS-1 cells and rat brain but blocked the effects of AngII in two distinct settings: in vitro stimulation of IP(3) and in vivo increases in water intake. Moreover, SII increased intake of 1.5% NaCl, despite the SII blockade of IP(3) formation and water intake. Examination of brain tissue showed increases in Fos expression in several AngII-sensitive brain areas after injection of AngII, but not SII. The lack of SII-induced IP(3) production, water intake, and Fos expression strongly suggest that the PLC/PKC/IP(3) pathway is required for water intake, but not NaCl consumption stimulated by AngII. Collectively, these results support the hypothesis that divergent intracellular signals from a single receptor type can give rise to separable behavioral phenomena.     

METACHROMATIC LEUKODYSTROPHY: ARYLSULFATASE-A DEFICIENCY IN SKIN FIBROBLAST CULTURES

Fibroblasts cultured from the skin of a patient with metachromatic leukodystrophy have been found to manifest the biochemical defect of this inborn error of metabolism, a deficiency of arylsulfatase A. Diseased cells had less than five per cent of normal arylsulfatase-A activity, while activities of other lysosomal enzymes-including arylsulfatase B, beta-galactosidase, beta-glucuronidase, and beta-N-acetylglucosaminidase-were comparable to those in control cells. The presence of dissociable inhibitors in extracts of the diseased cells was excluded by combination experiments. The deficiency of the enzyme in leukocytes was also confirmed and is comparable to that found in cultured fibroblasts. The finding that readily cultured fibroblasts from easily obtained skin biopsy specimens exhibit the enzymatic defect should prove valuable in the biochemical study of this disease.     

Crystal structure of saposin B reveals a dimeric shell for lipid binding

Saposin B is a small, nonenzymatic glycosphingolipid activator protein required for the breakdown of cerebroside sulfates (sulfatides) within the lysosome. The protein can extract target lipids from membranes, forming soluble protein-lipid complexes that are recognized by arylsulfatase A. The crystal structure of human saposin B reveals an unusual shell-like dimer consisting of a monolayer of alpha-helices enclosing a large hydrophobic cavity. Although the secondary structure of saposin B is similar to that of the known monomeric members of the saposin-like superfamily, the helices are repacked into a different tertiary arrangement to form the homodimer. A comparison of the two forms of the saposin B dimer suggests that extraction of target lipids from membranes involves a conformational change that facilitates access to the inner cavity.     

Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification

Angiotensin II (Ang-II) receptors were solubilized from differentiated N1E-115 neuroblastoma cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whereas other detergents, such as digitonin, sodium cholate, and Triton X-100, were much less effective. Binding of 125I-Ang-II or the antagonist 125I-Sar1,Ile8-Ang-II to 1% CHAPS-solubilized membranes was saturable and of high affinity. Moreover, these solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. Covalent cross-linking of 125I-Ang-II to either particulate or solubilized membrane fractions, with the homobifunctional cross-linker disuccinimidyl suberate, followed by size exclusion chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, resulted in the identification of the same two distinct 125I-Ang-II binding entities, with approximate molecular masses of 111 kDa and 68 kDa. The estimated molecular weights of the Ang-II binding sites in differentiated N1E-115 cells are in good agreement with the molecular weights obtained previously from solubilized rat brain membranes, suggesting that the N1E-115 Ang-II receptors are similar to those present in the brain. Finally, solubilized N1E-115 membranes could be purified by Ang-II affinity chromatography, resulting in only a single protein (66 kDa), which retained its ability to specifically bind 125I-Ang-II.     

Sodium appetite elicited by intracerebroventricular infusion of angiotensin II in the rat: I. Relation to urinary sodium excretion

Intracerebroventricular infusion of angiotensin II (Ang II) elicits a substantial sodium appetite in the rat. The present results demonstrate that this phenomenon consists of a small, early phase of sodium ingestion that is not the result of prior sodium loss but that thereafter urinary excretion of sodium exceeds intake and consequently the animals become hyponatremic and hypovolemic. The larger and more sustained bouts of sodium ingestion occurring 8-12 hr after the start of the Ang II infusion appear to represent a behavioral compensation for this incurred sodium deficit. These results confirm the arousal of a sodium appetite by action of Ang II on the brain but indicate the need for caution in assigning to it a direct and exclusive role in the neuroendocrine control of sodium intake.     

Up-regulation of angiotensin II receptors by in vitro differentiation of murine N1E-115 neuroblastoma cells

In vitro differentiation of murine neuroblastoma N1E-115 cells induced by low serum (0.5%) and dimethyl sulfoxide (1.5%) increased the uptake of 45Ca2+ as well as basal and forskolin-stimulated adenylate cyclase activity. Associated with these biochemical indices of differentiation was an increase in the density of binding sites for the angiotensin II (Ang II) receptor agonist 125I-[Sar1]-Ang II and the antagonist 125I-[Sar1,Ile8]-Ang II (125I-SARILE). This up-regulation was apparent within 24 hr and was maximal at 72 hr. Other manipulations that independently increased intracellular cAMP or Ca2+ levels produced a qualitatively similar up-regulation of Ang II receptors. In vitro differentiation did not diminish the specificity of these receptors for Ang-II related peptides. Sarcosine-substituted Ang II receptor antagonists such as [Sar1,Gly8]-Ang II, [Sar1,Thr8]-Ang II, or SARILE itself competed for 125I-SARILE in a monophasic fashion, whereas the competition displayed by the agonists Ang II, angiotensin III, and Crinia-Ang II for 125I-SARILE-labeled sites was biphasic, consisting of distinct high and low affinity components. Moreover, in vitro differentiation predominantly increased the density of high affinity sites for angiotensin III and Crinia-Ang II, but the lower affinity site for Ang II, and in all three cases the majority of this increased binding was insensitive to guanine nucleotides. Collectively, these results demonstrate that the expression of Ang II receptors on neuron-like cells is regulated by the biochemical events accompanying differentiation and suggest that the biphasic nature of the binding of some angiotensin agonists may be indicative of multiple receptor subtypes.     

The amygdala: site of genomic and nongenomic arousal of aldosterone-induced sodium intake

BACKGROUND.: Mineralocorticoids act on the brain to influence sodium intake, and they do so via intracellular type I receptors and possibly also via a direct membrane action, as they do in the kidney. One brain area implicated by lesion studies investigating the regulation of sodium appetite aroused by adrenal steroids is the amygdala. METHODS: To examine the mechanism by which mineralocorticoids act in the amygdala to arouse salt intake via a genomic and or membrane mode of action, rats were bilaterally fitted with cannulae directed to terminate in the amygdala. The genomic action of mineralocorticoids in arousing sodium intake was investigated by the administration of antisense oligodeoxynucleotides (ASDNs) against the mineralocorticoid receptor, and its effects on deoxycorticosterone (DOCA)-induced sodium intake over the course of several days was examined. The nongenomic action of mineralocorticoids on sodium intake was investigated by implantation into the amygdala of DOCA, aldosterone (ALDO), or their A-ring-reduced tetrahydro derivatives, 15 minutes prior to access to saline. Sodium intake was monitored immediately thereafter. RESULTS: Treatment of rats in the amygdala with ASDN against the mineralocorticoid receptor inhibited DOCA-induced sodium intake, whereas ASDN against the glucocorticoid receptor or sense/scrambled sequences had no effect. DOCA and ALDO increased saline intake within 15 minutes after steroid application. Similarly, the application of A-ring-reduced 3beta,5beta tetrahydroaldosterone and 5 alpha-tetrahydrodeoxycorticosterone produced the same increases in sodium intake. CONCLUSIONS: Together, the data imply that adrenal steroids, in addition to acting through classic cytosolic receptors, may also act on membrane receptor systems, producing rapid changes in behavior.     

Synthesis and characterization of NBD-PS: A fluorescent analog of cerebroside sulfate for diagnosis of arylsulfatase a deficiency disorders

N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]psychosine sulfate (NBDPS), a fluorescent analog of cerebroside sulfate (CS), was synthesized and tested as an alternative to the radiolabeled forms of CS used for assaying arylsulfatase A (ASA) in its physiological role as a cerebroside sulfate sulfohydrolase. NBD-PS simulates the natural substrate for ASA. Protocols have been developed for its use in differentiating low enzyme activities in diagnostic samples. Hydrolysis of NBD-PS is specific for ASA and optimal assay parameters were identical to those determined for CS. Differentiations between each of the major phenotypes for ASA activity were possible in the set of samples tested. One particular advantage was the ability to discriminate between individuals exhibiting arylsulfatase A pseudodeficiency and the truly deficient individuals with metachromatic leukodystrophy. Differential diagnosis was possible with fibroblast extracts by an assay that is more sensitive than procedures employing radioisotopes. Reaction products may be analyzed quantitatively by HPLC, or semiquantitatively with TLC. NBD-PS provides a simpler, safer, and more cost-effective means of performing natural substrate enzyme assays for ASA. Phenotyping with the fluorescence assay is an effective alternative to the laborious radioactive CS preparations and tissue culture loading studies that have previously been necessary.     

Genetic variants in TNFα, TGFB1, PTGS1 and PTGS2 genes are associated with diisocyanate-induced asthma

Diisocyanates are the most common cause of occupational asthma, but risk factors are not well defined. A case-control study was conducted to investigate whether genetic variants in inflammatory response genes (TNFα, IL1α, IL1β, IL1RN, IL10, TGFB1, ADAM33, ALOX-5, PTGS1, PTGS2 and NAG-1/GDF15) are associated with increased susceptibility to diisocyanate asthma (DA). These genes were selected based on their role in asthmatic inflammatory processes and previously reported associations with asthma phenotypes. The main study population consisted of 237 Caucasian French Canadians from among a larger sample of 280 diisocyanate-exposed workers in two groups: workers with specific inhalation challenge (SIC) confirmed DA (DA⁺, n?=?95) and asymptomatic exposed workers (AW, n?=?142). Genotyping was performed on genomic DNA, using a 5′ nuclease PCR assay. After adjusting for potentially confounding variables of age, smoking status and duration of exposure, the PTGS1 rs5788 and TGFB1 rs1800469 single nucleotide polymorphisms (SNP) showed a protective effect under a dominant model (OR?=?0.38; 95% CI?=?0.17, 0.89 and OR?=?0.38; 95% CI?=?0.18, 0.74, respectively) while the TNFα rs1800629 SNP was associated with an increased risk of DA (OR?=?2.08; 95% CI?=?1.03, 4.17). Additionally, the PTGS2 rs20417 variant showed an association with increased risk of DA in a recessive genetic model (OR?=?6.40; 95% CI?=?1.06, 38.75). These results suggest that genetic variations in TNFα, TGFB1, PTGS1 and PTGS2 genes contribute to DA susceptibility.