Steven J. Fluharty

Sodium appetite elicited by intracerebroventricular infusion of angiotensin II in the rat: II. Synergistic interaction with systemic mineralocorticoids.

Angiotensin and mineralocorticoids, the hormones of sodium conservation, acted together to arouse a sodium appetite with shorter latency and greater magnitude than is produced by larger amounts of each acting alone. This potentiation was selective for sodium ingestion and occurred in the absence of significant changes in sodium balance. Therefore, because endogenous angiotensin and mineralocorticoids are concurrently elevated during sodium deficiency, sodium appetite may be aroused by a synergy of the peptide and the steroid.

Differential regulation of protein kinase C and (Na,K)-adenosine triphosphatase activities by elevated glucose levels in retinal capillary endothelial cells.

Elevated cellular sorbitol levels resulting from conversion of increased glucose by aldose reductase might deplete cellular myoinositol content, which could then lower inositol phosphates (InsPs) and diacylglycerol levels, key regulators of protein kinase C (PKC). Secondary to altered PKC activity, other cellular enzymes such as (Na,K)-ATPase could be affected. To test this hypothesis we examined the association between PKC activity, (Na,K)-ATPase activity, and sorbitol, myoinositol, and InsP levels in cultured bovine retinal capillary endothelial cells, a cell type prominently involved in diabetic retinopathy. Elevating glucose concentration in culture media from 100 to 400 mg/dl led to a 100% increase in sorbitol levels, which could be inhibited completely by sorbinil, an aldose reductase inhibitor. In contrast, no changes were observed in myoinositol or InsP levels. Subfractionated PKC activities showed a 100% increase in the membranous pool with a parallel decrease in the cytosolic fraction. Adding sorbinil did not affect PKC activity, whereas the PKC agonist, phorbol myristate acetate (PMA), stimulated translocation of PKC. Ouabain-inhibitable (Na,K)-ATPase activity was decreased 70% by elevated glucose levels. This decrease could be prevented by adding either PMA or sorbinil. Thus, in retinal capillary endothelial cells elevated glucose concentration can affect PKC and (Na,K)-ATPase activities, probably via different mechanisms.

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.

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.

Intracerebral Administration of Mineralocorticoid Receptor Antisense Oligonucleotides Attenuate Adrenal Steroid-Induced Salt Appetite in Rats

The amygdala contains mineralocorticoid (MR) and glucocorticoid receptors (GR) involved in the arousal of salt appetite. In the present investigation, MR antisense oligonucleotides injected into the amygdala inhibited salt appetite induced by systemic desoxycorticosterone (DOCA) but not adrenalectomy (ADX). In contrast, GR antisense or scrambled oligonucleotides had no effect on stimulated salt intake. MR antisense oligomers also decreased MR but not GR in amygdala, whereas GR antisense oligomers decreased GR but not MR. Immunocytochemical labelling of the biotinylated MR antisense revealed that distribution of the oligomer was restricted to the injection site, with incorporation in neurons and neighboring glial cells. Together, these data demonstrate the utility of receptor antisense oligonucleotides for investigating the central actions of adrenal steroids and the role of amygdala in MR in DOCA-induced sodium intake.

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.

Short- and long-term changes in adrenal tyrosine hydroxylase activity during insulin-induced hypoglycemia and cold stress.

Insulin-induced hypoglycemia increases the capacity for catecholamine biosynthesis in the rat adrenal medulla by two temporally distinct processes: a rapid increase in the affinity of tyrosine hydroxylase (TH) for cofactor and a more gradual increase in the maximal TH activity. Cold exposure leads to comparable long-term increases in adrenal TH activity, apparently without causing a prior activation of TH.

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

Abstract 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 ≈ 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.

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.

Salt appetite: a neurohormonal viewpoint

Sodium is a key component of virtually every mammalian physiological function. As such, many animals have evolved specialized mechanisms for detecting and ameliorating deficits in body sodium, including the development of a robust salt appetite, where normally aversive concentrations of salt are readily consumed during periods of sodium deprivation. Here, we review research spanning more than half a century focusing on the condition and detection of sodium deprivation, the important and unique function of taste in sodium homeostasis, as well as the neurohormonal interactions leading to behaviors aimed at the reversal of sodium deficits. Based on the present literature, we propose a model for the interaction of forebrain and brainstem systems for the mediating circuitry giving rise to salt appetite and discuss the remarkable parallel between what is known about the neurohormonal interactions that regulate salt appetite and those involved in energy homeostasis.