Biomedical subjects

S J Fluharty

Publications and source records attributed to S J Fluharty.

At least 19 recordsLinked to original sources

PubMed2001

Chimeric AT1/AT2 receptors reveal functional similarities despite key amino acid dissimilarities in the domains mediating agonist-dependent activation.

Chimeric AT1/AT2 angiotensin II (AngII) receptors in which the sixth and/or seventh transmembrane-spanning domains of the AT2 receptor were substituted into the AT1 receptor were used to investigate the activation mechanisms of the two receptor subtypes. Numerous reports have identified amino acid residues in the sixth and seventh transmembrane-spanning domains of the AT1 receptor involved in the intrareceptor activation mechanism following agonist binding. Many of these residues are not conserved in the AT2 receptor; the corresponding AT2 receptor residues are, in fact, disruptive of AngII-dependent activation when substituted into the AT1 receptor. Surprisingly, the chimeric AT1/AT2 receptors--which also lack these crucial AT1 residues--exhibited AngII-induced activation of phosphoinositide hydrolysis with efficacies and potencies similar to the wild-type AT1 receptor. Consistent with earlier reports, a AT1[Y292F] point mutant demonstrated greatly decreased agonist-induced activation of phosphoinositide hydrolysis. However, a AT1[Y292F/N295S] double-point mutant allowed for normal agonist-induced activation with a pharmacodynamic profile indistinguishable from the wild-type receptor. Despite amino acid dissimilarities, the same corresponding domains and even the same residue loci in both of the AngII receptor subtypes are equally able to mediate agonist-induced receptor activation. This suggests that these corresponding domains in the AT1 and the AT2 receptors are crucial to the activation mechanism, demonstrating greater structural flexibility than previously believed regarding AT1 receptor activation and supporting the possibility of a common activation mechanism for the two receptor subtypes.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed2001

Identification and function of disulfide bridges in the extracellular domains of the angiotensin II type 2 receptor.

The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop. The importance of these AT(1) disulfides in ligand binding is supported by the effect of dithiothreitol (DTT). DTT breaks disulfide bonds, thereby strongly inhibiting ligand binding in AT(1) receptors. Despite retaining the same cysteines, AT(2) receptor ligand binding is paradoxically enhanced by DTT. Thus, we constructed a series of AT(2) cysteine mutations, either individually or paired, to establish the role of the cysteines and the source of DTT's effects. The AT(2) cysteine mutants surprisingly confirmed that the cysteines form disulfide bonds in the same manner as in the AT(1) subtype. However, breaking the AT(2) disulfide bridges yielded two responses. As in AT(1) receptors, mutations disrupting the disulfide bond between the first and second extracellular loops reduced AT(2) binding by 4-fold. In contrast, mutations breaking the disulfide bridge between the amino terminus and the third extracellular loop increased AT(2) binding, mimicking DTT's effect on this subtype. Further analysis of AT(1)/AT(2) chimeric exchange mutants of these domains suggested that the AT(2) amino terminus and third extracellular loop may possess latent binding epitopes that are only uncovered after DTT exposure.

Angiotensin II↗

PubMed2001

Novel nuclear signaling pathway mediates activation of fibroblast growth factor-2 gene by type 1 and type 2 angiotensin II receptors.

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1',3'-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

Adrenal Medulla↗

PubMed2001

Identification of angiotensin II type 2 (AT2) receptor domains mediating high-affinity CGP 42112A binding and receptor activation.

Chimeric angiotensin II (AngII) receptors constructed of portions of the AT2 receptor substituted into the AT1 receptor revealed the AT2 third extracellular loop and seventh transmembrane-spanning domain as major determinants for the ability to bind and activate in response to the AT2 receptor-selective agonist CGP 42112A. Radioligand binding experiments showed that chimeric AngII receptors possessing the AT2 third extracellular loop and seventh transmembrane-spanning domain bound CGP 42112A with high affinity approaching that of the wild-type AT2 receptor. The presence of the AT2 third extracellular loop appeared sufficient for high-affinity CGP 42112A binding, which was further enhanced by the additional presence of the AT2 seventh transmembrane-spanning domain. Experiments with PD 123319, losartan, and [Sar1,Ile8]-AngII showed that increases in binding affinity associated with these domains were specific for CGP 42112A. Use of phosphoinositide hydrolysis as a functional index to measure activation of these chimeric AngII receptors further demonstrated that the AT2 seventh transmembrane-spanning domain was especially critical for CGP 42112A to act as an agonist. The absence of the AT2 seventh transmembrane-spanning domain prohibited CGP 42112A-induced activation of these receptors, even in the presence of high concentrations of CGP 42112A sufficient to saturate the binding sites. This study is the first to identify binding determinants of the AT2 receptor that are selective for CGP 42112A, and indicates that these determinants are at least partially distinct from those for the AT2-selective antagonist PD 123319. These differences may be a factor in the pharmacodynamic difference between these two ligands.

Animals↗

PubMed2000

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

UNLABELLED: 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.

Aldosterone↗

PubMed2000

Estrogen increases angiotensin II-induced c-Fos expression in the vasopressinergic neurons of the paraventricular nucleus in the female rat.

Previous studies in female rats have shown that estrogen treatment attenuates angiotensin II (AngII)-induced water intake. The mechanism underlying this attenuation may be decreased responsiveness to AngII, as revealed by a reduction in AngII binding to the angiotensin type 1 (AT1) receptor in the subfornical organ (SFO). It has not been determined whether these changes in receptor binding translate into changes in neuronal activity that, in turn, may influence behavior. Therefore, an estrogen-modulated change in neuronal pathways relevant to AngII-induced water intake was tested in ovariectomized (OVX) female rats using immunohistochemistry for the immediate early gene c-Fos as a marker for neuronal activation. Third cerebroventricular injection of AngII (6 ng) induced intense c-Fos immunoreactivity in forebrain regions associated with fluid intake, including the organum vasculosum of the lamina terminalis, the median preoptic nucleus, the SFO, the supraoptic nucleus and the paraventricular nucleus (PVN). Forty-eight-hour estradiol (10 microg) administration to OVX female rats increased AngII-induced c-Fos labeling in the lateral magnocellular neurons of the PVN by 30% as compared to vehicle-treated controls. Double labeling neurons in the PVN with c-Fos and either vasopressin or oxytocin antisera revealed that estrogen increased AngII-induced c-Fos expression by 28%, specifically in vasopressinergic neurons. Such changes in neuronal activation may explain the estrogen modulation of AngII-induced water intake that has been previously reported; it may be due to increased water retention to maintain plasma osmolality or to induction of a pressor response.

Angiotensin II↗

PubMed1999

The angiotensin AT(1) receptor antagonist irbesartan has near-peptide affinity and potently blocks receptor signaling.

The angiotensin II type 1 (AT(1)) receptor plays a pivotal role in the regulation of blood pressure and electrolyte balance, and is involved in the control of specific ingestive behaviours. Irbesartan (SR 47436/BMS 186295) is a recently developed angiotensin AT(1) receptor antagonist, chemically described as 2-butyl-3-([2'-¿1H-tetrazol-5-yl¿biphenyl-4-yl]methyl)-1, 3-diazaspiro (4,4)non-1-en-4-one. Irbesartan displays higher affinity for its target receptor than other similar antagonists. In radioligand binding assays performed on membranes from WB-Fischer 344 (WB) rat liver epithelial cells, irbesartan was able to displace [125I]angiotensin II with a K(i) of 4.05 nM as compared to losartan (DuP 753) and tasosartan (WAY 126756), which had K(i) values of 25.2 nM and 46.6 nM, respectively. Similarly, in functional assays, irbesartan exhibited the highest functional potency to block angiotensin II-induced inositol trisphosphate (IP(3)) turnover. The improved affinity of irbesartan for the angiotensin AT(1) receptor does not coincide with a concomitant increase in affinity for the angiotensin AT(2) receptor, as irbesartan and losartan exhibited the same low potency to displace [125I]angiotensin II in radioligand binding assays performed on membranes from PC-12w cells. In binding assays performed on peripheral tissues in rat, irbesartan bound to the angiotensin AT(1) receptor expressed in liver, adrenal, kidney and pituitary with an overall affinity closely approaching that of the high affinity peptidic antagonist [Sar(1), Ile(8)]angiotensin II. Due to the higher affinity of irbesartan over other similar antagonists for the angiotensin AT(1) receptor in many tissues and its greater potency to block receptor activation, irbesartan may be quite useful in the study of the angiotensin AT(1) receptor and its role in controlling ingestive behaviours and, furthermore, shows great potential to improve the treatment of hypertension and other cardiovascular disease states.

Adrenal Glands↗

PubMed1999

Estrogen decreases hypothalamic angiotensin II AT1 receptor binding and mRNA in the female rat.

Estrogen has been shown to modulate angiotensin II (AngII)-regulated behaviors, such as thirst, and may do so by influencing the central renin-angiotensin system (RAS). While numerous studies have attempted to correlate changes in AngII receptors or other components of the RAS with estrogen treatment, the low abundance of these genes has made comparisons difficult. Generally, such experiments have relied on traditional approaches to analyze gene expression that often restrict the experimenter to studying only a few mRNA species, whereas a behavior as complex as thirst may be influenced by changes in multiple genes. The present experiments utilized quantitative receptor autoradiography and mRNA expression profiling to identify and compare AngII receptors and their mRNA levels as well as other components of the RAS in female rat pituitary and hypothalamic-thalamic-septal (HTS) tissue samples. This relatively new approach to the study of gene expression permits the simultaneous comparison of multiple genes from a single tissue sample. These studies revealed that ovariectomized (OVX) female rats treated with estradiol benzoate (EB) had a 30%-40% reduction in the levels of AT(1) receptor mRNA in pituitary and HTS samples as compared to OVX, control animals. In the pituitary, the mRNA levels for angiotensinogen (AGT) were increased by 45% following estrogen administration. In addition, a reduction in [125I]-AngII binding to AT(1) receptors in the pituitary and the subfornical organ was measured following estrogen treatment. These results suggest that estrogen may modulate the pituitary and central RAS through a coordinate regulation of the angiotensin receptors and the levels of newly synthesized AngII.

Animals↗

PubMed1999

Ovarian steroid regulation of angiotensin II-induced water intake in the rat.

Spontaneous water intake as well as thirst elicited by ANG II has been shown to be influenced by the stage of the estrous cycle in the female rat. In these experiments, the contribution of each of the ovarian steroid hormones to the regulation of water intake was examined. Ovariectomized female rats were given replacement doses of estrogen, progesterone, or both, and their responsiveness to an intracerebroventricular injection of ANG II was tested. Forty-eight-hour treatment with estradiol benzoate attenuated ANG II-induced thirst by as much as 70% compared with control animals. The effect of estrogen on drinking was dose dependent and could be completely blocked with concurrent administration of the antiestrogen CI-628. In contrast, progesterone, given alone or after estrogen, did not significantly affect ANG II-induced water intake when animals were tested at 4 or 24 h after steroid administration. A central interaction between the peptide hormone ANG II and estrogen, involving a genomic mechanism, may underlie the cyclicity in water intake behavior observed in the rat.

Angiotensin II↗

PubMed1999

Mineralocorticoids and glucocorticoids cooperatively increase salt intake and angiotensin II receptor binding in rat brain.

Mineralocorticoids, such as deoxycorticosterone acetate (DOCA), and angiotensin II (AngII) act synergistically in the brain to elicit salt appetite. Glucocorticoids, such as dexamethasone (DEX), also may enhance the behavioral effects of DOCA and AngII. However, the brain regions involved in these behavioral interactions have not been elucidated. This study tested the hypothesis that DEX potentiates the effects of DOCA on AngII binding, especially at the AT1 receptor. We confirmed that DEX potentiated the effects of DOCA on salt appetite. Concomitantly, steroid-specific and region-specific changes in AT1 binding were noted. Specifically, in the hypothalamic paraventricular nucleus, treatment with DEX or DOCA + DEX increased AT1 binding. In the subfornical organ (SFO) and area postrema, there was an increase in AT1 binding when both steroids were combined, but not when given individually. However, there was no change in AT2 binding in any brain region studied and no change in AT1 or AT2 binding to either receptor subtype in the pituitary. The results indicate that DOCA and DEX may increase the sensitivity of the brain to the behavioral and physiological actions of AngII by upregulating AT1 receptors in the SFO and area postrema.

Angiotensin II↗

PubMed1999

Glucocorticoid and mineralocorticoid regulation of angiotensin II type 1 receptor binding and inositol triphosphate formation in WB cells.

Mineralocorticoids, glucocorticoids, and angiotensin II (AngII) act cooperatively to maintain body fluid homeostasis. Mineralocorticoids, such as aldosterone and deoxycorticosterone-acetate (DOCA), function synergistically with AngII in the brain to increase salt appetite and blood pressure. In addition, glucocorticoids increase AngII-induced drinking and pressor responses and may also facilitate the actions of aldosterone on salt appetite. The AngII Type 1 (AT1) receptor mediates many of the physiological and behavioral actions of AngII. This receptor is coupled to the G-protein Gq, which mediates AngII-induced inositol triphosphate (IP3) formation. The WB cell line, a liver epithelial cell line that expresses the AT1 receptor, was used to examine the cellular basis of glucocorticoid and mineralocorticoid regulation of AT1 function. In this study corticosterone and dexamethasone treatments increased the number of AT1 receptors by activating the glucocorticoid receptor (GR). This increase in AT1 binding resulted in enhanced AngII-stimulated IP3 formation. However, only supraphysiological doses of aldosterone or DOCA increased AT1 binding, and this effect also was mediated by GR activation. Furthermore, despite evidence that mineralocorticoids and glucocorticoids function together to increase AngII-stimulated actions in vivo, aldosterone and dexamethasone did not act synergistically to affect AT1 binding, Gq expression, or IP3 formation. These results indicate that GR activation, and the subsequent increases in AT1 binding and in AngII-stimulated IP3 formation, may represent a cellular mechanism underlying the synergy between adrenal steroids and AngII.

Aldosterone↗

PubMed1998

Adrenal steroid regulation of central angiotensin II receptor subtypes and oxytocin receptors in rat brain.

The neuropeptides angiotensin II (AngII) and oxytocin (OT) play important but opposing roles in the regulation of sodium appetite in the rat, AngII as a stimulatory peptide and OT as an inhibitory peptide. Adrenal steroids increase the density of AngII receptors in brain following in vivo administration, although the neuroanatomical and subtype specificity have not been thoroughly examined. Furthermore, previous studies demonstrate that adrenalectomy (ADX) leads to a reduction in OT receptors, although regions associated with sodium appetite remain to be examined. In the present study, quantitative receptor autoradiography was used to locate regions where perturbations in circulating adrenal steroids affect the density of oxytocin receptors and the angiotensin receptor subtypes AT1 and AT2. The results show that ADX results in a small, but significant decrease in AT1 expression in the paraventricular nucleus of the hypothalamus, subfornical organ, and the area postrema. That this effect is reversed by either aldosterone or low-dose corticosterone replacement suggests that occupancy of the mineralocorticoid receptor is responsible for the steroid effect. No changes were observed in AT2 or OT receptors in nuclei associated with sodium appetite, indicating that perturbations in adrenal steroids did not affect these receptors in brain regions implicated in the control of salt appetite.

Aldosterone↗

PubMed1998

Mutational analysis of the angiotensin type 2 receptor: contribution of conserved amino acids in the region of the sixth transmembrane domain.

Angiotensin II (AngII) mediates its physiological actions through two receptor subtypes: the Type 1 (AT1) and Type 2 (AT2) receptors. The subtypes have identical affinities for AngII, while sharing only 34% homology. Mutagenesis has focused mainly on the AT1 receptor, identifying residues important for AngII binding. In contrast, relatively little is known of the binding mechanism of the AT2 receptor. It has been hypothesized that residues that are conserved between the two subtypes that have been shown to be important in the AT1 receptor may also contribute to AngII binding in the AT2 receptor as well. To test this hypothesis, the role of two conserved residues in the sixth transmembrane domain of the AT2 receptor in ligand binding were investigated: tryptophan 269 and aspartate 279. In contrast to the AT1 receptor, mutation of Trp269 in the AT2 receptor to an alanine had no effect on AngII binding, while mutation of Asp279 to alanine similarly impaired AngII binding in both receptors. However, the more sterically conservative substitution of Asp279 to asparagine in the AT2 receptor showed near wild type affinity. Based on this finding, we mutated Asp263 in the AT1 receptor to asparagine. Subsequent studies indicated that this more conservative mutation had no effect on AngII binding to the AT1 receptor. Collectively, these results demonstrate that although there may be commonalities in ligand binding between the AT1 and AT2 AngII receptors, there are also clear differences.

Amino Acid Sequence↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

Analysis of angiotensin type 2 receptors in vasopressinergic neurons and pituitary in the rat.

Previous functional studies indicated that an angiotensin type 2 (AT2) receptor subtype may participate in the regulation of vasopressin release by angiotensin II (AngII). In the present study, AT2 receptor-directed antiserum immunohistochemically detected AT2 receptors within the hypothalamic paraventricular (PVN) and the supraoptic nuclei (SON) of the rat brain, more specifically, in identified vasopressinergic neurons. Considering the lack of AT2 binding in the PVN and the SON using receptor autoradiography, we tested the hypothesis that these AT2 receptors are transported to the posterior pituitary. Western blot analysis detected AT2 immunoreactivity in the posterior pituitary. However, no AT2 binding was detected in posterior pituitary membranes, and no AT2 binding was detected with quantitative receptor autoradiography in the neurohypophysis. Thus, if AT2 receptors are transported from the magnocellular vasopressin neurons to the posterior pituitary, their role in AngII regulation of vasopressin release at the neurohypophyseal terminals remains to be clarified.

Angiotensin II↗

PubMed1998

Infusion of the serotonin1B (5-HT1B) agonist CP-93,129 into the parabrachial nucleus potently and selectively reduces food intake in rats.

Unilateral infusion of the selective 5-HT1B agonist, CP-93,129 (3-(1,2,5,6-tetrahydropyrid-4-yl) pyrrolo[3,2-b]pyrid-5-one) into the parabrachial nucleus (PBN) of the pons reduced food consumption by rats. The hypophagia was dose-related (ED50 approximately 1 nmol) and associated with fewer observations of feeding and more periods of inactivity. Water intake, grooming and exploratory activity were unaffected. CP-93,129 also decreased food intake when injected into the hypothalamic paraventricular nucleus, but this action was 50-fold less potent than administration into the PBN. Autoradiography demonstrated 5-HT1B sites in the PBN; this binding was displaced by CP-93,129. The results implicate parabrachial 5-HT1B receptors in mediating serotonergic enhancement of satiation.

Animals↗

PubMed1998

Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries.

Antisense experiments are often complicated by the lack of reliable methods for selecting effective antisense sequences. Chimeric oligodeoxynucleotide (ODN) libraries and ribonuclease H (RNase H) were used to identify regions on the 1253 nucleotide angiotensin type-1 receptor (AT1) mRNA that are accessible to hybridization with antisense ODNs. Phosphorothioate antisense ODNs targeted against accessible sites reduced AT1 receptor levels by at least 50% in cell culture. ODNs to 4 sites produced a 70% to 80% reduction. In contrast, most sequences targeted between accessible sites were ineffective. When injected into the brains of rats, ODNs targeted to accessible sites reduced AT1 (by 65%) but not AT2 receptor levels. Additionally, AT1 receptor function as measured by agonist-induced water intake, was significantly attenuated in these rats. ODNs directed between accessible sites were ineffective at suppressing water intake. RNA mapping can be applied to any RNA target to facilitate selection of multiple, active antisense sequences for cell culture and in vivo experiments.

Animals↗

PubMed1997

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.

Alanine↗